TREATMENT OF INDUSTRIAL AND AGRO-INDUSTRIAL WASTEWATER … · Treatment of Industrial and Agro-Industrial Wastewater Using Constructed Wetlands Mar-Yam Sultana Acknowledgements iv

LABORATORY OF ENVIRONMENTAL SYSTEMS

DEPARTMENT OF ENVIRONMENTAL AND NATURAL RESOURCES

MANAGEMENT

SCHOOL OF ENGINEERING

UNIVERSITY OF PATRAS

TREATMENT OF INDUSTRIAL AND AGRO-INDUSTRIAL

WASTEWATER USING CONSTRUCTED WETLANDS

Ph.D. THESIS

BY

MAR-YAM SULTANA

SUPERVISOR:

Dr. CHRISTOS S. AKRATOS ASSISTANT PROFESSOR

AGRINIO, GREECE

NOVEMBER, 2014

LABORATORY OF ENVIRONMENTAL

SYSTEMS

DEPARTMENT OF ENVIRONMENTAL

AND NATURAL RESOURCES

MANAGEMENT

SCHOOL OF ENGINEERING

UNIVERSITY OF PATRAS

TREATMENT OF INDUSTRIAL AND AGRO-INDUSTRIAL

WASTEWATER USING CONSTRUCTED WETLANDS

“This thesis is submitted in fulfillment of the requirements for the degree of

Doctor of Philosophy”

By

MAR-YAM SULTANA

SUPERVISOR:

Dr. CHRISTOS S. AKRATOS ASSISTANT PROFESSOR

AGRINIO, GREECE

NOVEMBER, 2014

Treatment of Industrial and Agro-Industrial Wastewater Using Constructed Wetlands Mar-Yam Sultana

i

The thesis of Mar-Yam Sultana entitled "Treatment of industrial and agro-industrial wastewater

using constructed wetlands" examined and approved as to content and presentation.

The Examination Committee:

Chairman of the examination committee Members

Dr. Christos S. Akratos Dr. Dimitrios Vayenas

Professor

Assistant Professor (Member of the Advisory Committee)

(Supervisor)

Dr. Stavros Pavlou

Professor

(Member of the Advisory Committee)

Dr. Vassilios Tsihrintzis

Professor

(Member of the Examination Committee)

Dr. Vagelis Papadakis

Associate Professor


Dr. Ioannis Kalavrouziotis

Associate Professor


Dr. Athanasia Tekerlekopoulou

Lecturer


Date of Presentation: 18 November, 2014


ii

Dedicated to my family


iii

This Ph.D. was funded by the Greek State Scholarships Foundation (IKY).


Acknowledgements iv

Acknowledgements

All praises are due to “Almighty Allah” who enables me to pursue higher study in my life as

well as to complete successfully the PhD research work and writing up of this PhD dissertation.

I would firstly like to acknowledge my supervisors, Prof. Dr. Dimitrios Vayenas and Assistant

Professor Dr. Christos S. Akratos, Laboratory of Environmental Systems, Department of

Environmental and Natural Resources Management, School of Engineering, University of Patras

for their excellent academic thoughtful guidance, advice, critical comments, warm

encouragement and continuous support throughout the PhD. It was a pleasure to learn from and

cooperate with two experts in their respective fields. I especially appreciate their trust in me by

giving me the opportunity to carry out my PhD research. I’m very grateful for their valuable

suggestions and directions from the start of the research work for its successful completion and

finally to write this dissertation. They significantly helped me to better structure and sharpen my

writing of manuscripts. Without their continuous and valuable supervision, this thesis would not

have been possible. There is no way to express how much it meant to me to have been a member

of the Environmental Systems Laboratory. I express my deepest gratitude to Prof. Dr. Stavros

Pavlou, Department of Chemical Engineering, University of Patras, for being a member of the

advisory committee and for also providing me encouragement and constructive feedback.

I would also like to thank my other examiners, Professor Dr. Vassilios Tsihrintzis, Dr. Vagelis

Papadakis, Associate Professor, Dr. Ioannis Kalavrouziotis, Associate Professor, Hellenic Open

University, for being members of the examination committee and their valuable comments and

suggestions on my thesis.

I want to thank all the respective teachers of the Department of Environmental and Natural

Resources Management, University of Patras for their inspiration during my research period.

Special thank to Dr. Vagelis Papadakis, Associate Professor, Dr. Maria Panitsa, Assistant

Professor and Dr. Dimitris Vlastos, Assistant Professor, Department of Environmental and

Natural Resources Management, University of Patras for providing conveyance from Patras to

Agrinio and vice versa at the beginning of the research period.


Acknowledgements v

I would like to acknowledge Dr. Athanasia Tekerlekopoulou, Lecturer, Department of

Environmental and Natural Resources Management, University of Patras, for her valuable help in

conducting the experiments and chemical analyses as well as her constructive comments and

suggestions. I would also like to thank her for participating in the examination committee of this

doctoral dissertation. Additionally, during my research period her enormous moral support made

me more determined to conduct my PhD work. I also want to express my deep gratitude to her

entire family for making me feel at home.

I would like to acknowledge the support of Dr. Christina Oikonomou, Dr. Konstantinos

Karanasios, Michael Michailides, PhD student, Triantafyllos Tatoulis, PhD student, Olga

Tsolcha, PhD student of the Laboratory of Environmental Systems, Department of

Environmental and Natural Resources Management. Three persons of the Laboratory of

Environmental Systems in particular deserve the proverbial flowers: Dr. Kostas Karanasios,

Michael Michailides and Triantafyllos Tatoulis for all their support. Their contribution cannot be

expressed in words. It was a pleasure to work together with them and I also feel very fortunate to

have friends like them.

I’m very much thankful to the undergraduate students Giorgos Kefalides, Christina Mourti and

Ilias Kanakakis for their contribution and time during the research work. Evaggelia Tasoula, who

was a Master student of the Laboratory of Environmental Systems, Department of Environmental

and Natural Resources Management, deserves a special word of thanks - without her support it

would have been very difficult for me to continue research and manuscript writing.

My heartfelt thanks to Areti Gianni, Artemis Damati and Miltiadis Zamparas, Department of

Environmental and Natural Resources Management. It was wonderful to share office space with

them. Their congenial behavior made my research period pleasant. I also thanks my present and

ex- colleagues from other laboratories with whom I have enjoyed my past four years.

Solicitous thanks to Ms. Sandy Coles, for English correction of my all manuscripts and this

dissertation. Her amicable attitudes made me more comfortable and relaxed during my living

time in Greece.


Acknowledgements vi

I want also to thank all the official staff members of the Department of Environmental and

Natural Resources Management, for making all the official documents for this dissertation.

My sincere thanks to the Foreign Language Unit, University of Patras for giving the taught

Modern Greek language Course. Especially I want to express my gratitude to Ms. Nikolitsa

Vasiliou, because of her hard work during the Greek Language Course, which has helped me to

overcome the language barrier.

I want to express my deep gratitude to Dr. Dimitra Kritikou (wife of Prof. Vayenas). Her

affectionate mind always makes me feel that I’m very close to my family. Her hospitality made

my stay in Greece lively. Her psychological support at the very beginning gave me the strength to

stay here. I wish to extend my earnest thanks to the whole family of Prof. Vayenas for making me

feel like a member of their family.

I’m also very thankful to Mrs. Anastasia Diamantidou (wife of Dr. Akratos) for helping us in

different circumstances and also for her cordial relationship.

I warmly express my deepest sense of gratitude and heartfelt thanks to my parents and parents-in-

law for their material and spiritual support, appreciated in all aspects of my life. Especially my

most gratitude to my mother for her willingness and encouragement I attained this moment. Also

I’m very much grateful to my father; his every single advice motivates me to move forward in my

life. My hearty gratitude and thanks to my elder sister, brother-in-law and my little nephew, as

they also inspired me all the time during my PhD.

Lastly, but certainly not least, many thanks to my husband, Abu Chowdhury, for his

encouragement, patience and support during the PhD, for which I am very appreciative. I can just

say thanks for everything and may Allah give him all the best in return.


Abstract vii

Treatment of Industrial and Agro-Industrial Wastewater Using Constructed

Wetlands

Mar-Yam Sultana

Laboratory of Environmental Systems

Department of Environmental and Natural Resources Management

School of Engineering

University of Patras

Supervisor: Dr. Christos S. Akratos, Assistant Professor

ABSTRACT

Environmental pollution from untreated wastewater disposal is one of the most serious

environmental issues. Hexavalent chromium, Cr(VI), is known to be a very toxic compound,

frequently found in polluted industrial wastewaters, and causes major environmental problems.

On the other hand, among the agro-industrial wastewaters, dairy wastewaters can also cause

serious environmental pollution due to their high organic loads. Specifically, when untreated

dairy wastewater is deposited into surface water bodies it can cause eutrophication and

environmental toxicity.

The use of constructed wetlands began 40 years ago in North America and Europe. The idea

arose from the use of wetlands as final recipients to treat effluent wastewaters. After studies on

their construction and improved operations, today constructed wetlands are used as a processing

technology in many countries for the treatment of municipal wastewater, industrial wastewater,

landfill leachates, etc. Due to their simplicity and low operational cost, constructed wetlands are

becoming more prevalent in wastewater treatment all over the world. Their range of applications

is no longer limited to municipal wastewater or industrial wastewater but has expanded to the

treatment of heavily polluted wastewaters such as agro-industrial effluents. Constructed wetlands

can tolerate high pollutant loads and toxic substances without reducing their removal ability, thus

these systems are very effective bio-reactors even in hostile environments. The potential

application of constructed wetlands in the treatment of chromium-bearing wastewaters has been

reported recently. Additionally, secondary cheese whey, a nutrient-rich wastewater which has


Abstract viii

high potential of polluting surface and/or groundwater, is now being treated either by

conventional or biological treatment processes. However, limited research has been conducted on

the treatment of secondary cheese whey using constructed wetlands.

The objectives of this PhD research were to evaluate a) the effect of different parameters (HRT,

temperature, physiochemical parameters) on the treatment of wastewater containing Cr(VI) and

secondary cheese whey, using pilot-scale horizontal subsurface flow (HSF) constructed wetlands,

b) a sustainable disposal technique of chromium treated reed biomass and c) the treatment

efficiency of undiluted secondary cheese whey using pilot-scale HSF constructed wetland at very

low HRT and removal of Cr(VI) by providing cheese whey as source of carbon.

In the 1st experimental period of this dissertation, the research focused on the study of integrated

chromium removal from aqueous solutions in HSF constructed wetlands. Two pilot-scale HSF

constructed wetlands (CWs) units were built and operated. One unit was planted with common

reeds (Phragmites australis) and one was kept unplanted. Influent concentrations of Cr(VI)

ranged from 0.5 to 10 mg/L. The effects of temperature and hydraulic residence time (8 - 0.5

days) on Cr(VI) removal were studied. Temperature proved to affect Cr(VI) removal in both

units. In the planted unit, maximum Cr(VI) removal efficiencies of 100% were recorded at

HRT’s of 1 day with Cr(VI) concentrations of 5, 2.5 and 1 mg/L, while a significantly lower

removal rate was recorded in the unplanted unit. Harvested reed biomass from the CWs was co-

composted with olive mill wastes. The final product had excellent physicochemical

characteristics (C/N: 14.1-14.7, germination index (GI): 145-157%, Cr: 8-10 mg/kg dry mass),

fulfills EU requirements, and can be used as a fertilizer in organic farming.

In the 2nd

experiment of the first experimental period of this research, two horizontal subsurface

flow pilot-scale constructed wetlands were built and operated for almost two years to treat

secondary cheese whey. One unit was planted with common reeds (Phragmites australis) and one

was kept unplanted. The pilot-scale wetlands operated under various hydraulic residence times

(8, 4, 2 and 1 day), temperatures (2.4 to 32.90C) and COD influent concentrations (1200 to 7200

mg/L) in order to examine their effect on secondary cheese whey treatment efficiency. Both units

successfully removed organic matter, as COD removal efficiencies of 91% and 77.23% were


Abstract ix

recorded for the planted and unplanted unit, respectively. Hydraulic residence time affected COD

removal efficiency only when limited to 1 day. Temperature significantly affected COD removal

only in the unplanted unit, while the planted unit's efficiency was affected only by the annual

plant growth cycle. It should be noted that COD effluent concentrations were below EU

legislation units (120 mg/L) even when the CWs operated under the shortest hydraulic residence

time ever reported in the literature (2 days) with COD influent concentrations ranging from 1200

to 3500 mg/L.

In the 2rd

experimental period, a mixed solution of cheese whey and hexavalent chromium was

treated using pilot-scale horizontal subsurface flow constructed wetlands. This study was

performed in order to assess the effect of hydraulic residence time, the initial concentrations of

both substances (i.e., Cr(VI) and cheese whey), the presence of vegetation, and surface load

throughout the treatment process. Two hydraulic residence times (HRT) (8 and 4 days) were

applied. The average electrical conductivity did not show any significance and the average pH

values also did not fluctuate. COD concentrations varied between 2000 to 3000 mg/L, and Cr(VI)

concentrations were between 0.5 and 5 mg/L. Regarding the removal of organic matter, the

planted pilot units had the highest removal rates of around 70%, compared to the unplanted units

with around 50%. The vegetation does not affect the removal of Cr(VI) whereas for COD

removal, the vegetation does not perform its proper function which leads us to conclude that

Cr(VI) influences the removal of COD.

The overall outcome of this research is a significant contribution to the treatment of Cr(VI) and

secondary cheese whey using constructed wetland technology. It could also be concluded that,

constructed wetlands can potentially remove both Cr(VI) and COD at very low HRTs (1 and 2

days, respectively), when receiving moderate pollutant concentrations (5 mg Cr(VI)/L and >5000

mg COD/L), without any seasonal effect. Moreover, by using cheese whey as the carbon source,

Cr(VI) can be successfully removed in constructed wetland systems with 4 days of HRT.

Key Words: Hexavalent chromium, cheese whey, HSF constructed wetlands, vegetation, HRT,

temperature, common reeds, composting.


Περίληψη x

Επεξεργασία Βιομηχανικών και Αγρο-Βιομηχανικών Λυμάτων με τη Χρήση

Τεχνητών Υγροβιότοπων

Mar-Yam Sultana

Εργαστήριο Περιβαλλοντικών Συστημάτων

Τμήμα Διαχείρισης Περιβάλλοντος και Φυσικών Πόρων

Πολυτεχνική Σχολή

Πανεπιστήμιο Πατρών

Επιβλέπων: Χρήστος Σ. Ακράτος, Επίκουρος Καθηγητής

ΠΕΡΙΛΗΨΗ

Η ρύπανση του περιβάλλοντος από τα ανεπεξέργαστα λύματα αποτελεί ένα από τα

σημαντικότερα περιβαλλοντικά ζητήματα. Το εξασθενές χρώμιο (Cr(VI)), που είναι γνωστό για

την τοξική του δράση, εντοπίζεται συχνά σε βιομηχανικά υγρά απόβλητα και προκαλεί

σημαντικά περιβαλλοντικά προβλήματα. Από την άλλη τα υγρά απόβλητα τυροκομικών

μονάδων επίσης αποτελούν σημαντική περιβαλλοντική απειλή, λόγω του υψηλού οργανικού

τους φορτίου. Ειδικότερα όταν ανεπεξέργαστα τυροκομικά υγρά απόβλητα καταλήγουν σε

επιφανειακά υδάτινα σώματα μπορούν να προκαλέσουν ευτροφισμό και τοξικά φαινόμενα.

Η χρήση των τεχνητών υγροβιότοπων ξεκίνησε πριν από περίπου 40 χρόνια στη Βόρεια Αμερική

και την Ευρώπη. Η ιδέα προήλθε από τη χρήση φυσικών υγροβιότοπων ως τελικών αποδεκτών

επεξεργασμένων υγρών αποβλήτων. Μετά από εκτεταμένη έρευνα σήμερα οι τεχνητοί

υγροβιότοποι χρησιμοποιούνται ευρέως ως τεχνολογία επεξεργασίας διαφόρων ειδών υγρών

αποβλήτων και απορροών (π.χ. αστικά, βιομηχανικά, δισταλλάγματα κλπ.). Λόγω της απλότητας

τους και του χαμηλού λειτουργικού κόστους οι τεχνητοί υγροβιότοποι αποτελούν πλέον μια

ανταγωνιστική τεχνολογία. Το εύρος των εφαρμογών τους δεν περιορίζεται πλέον μόνο στην

επεξεργασία αστικών υγρών αποβλήτων, αλλά έχει επεκταθεί και στην επεξεργασία ισχυρών

υγρών αποβλήτων, όπως των αγροτοβιομηχανικών. Οι τεχνητοί υγροβιότοποι είναι ανθεκτικοί

σε υψηλά ρυπαντικά φορτία και σε τοξικές ουσίες χωρίς να επηρεάζεται σημαντικά η λειτουργία

τους. Συνεπώς οι τεχνητοί υγροβιότοποι είναι ιδιαιτέρως αποτελεσματικοί βίο-αντιδραστήρες


Περίληψη xi

ακόμα και ιδιαίτερα εχθρικά περιβάλλοντα. Η δυνατότητα χρήσης τεχνητών υγροβιότοπων για

την επεξεργασία υγρών αποβλήτων με χρώμιο, μόλις πρόσφατα έχει αρχίσει να μελετάται. Επί

πλέον ο δευτερογενής ορρός γάλακτος (τυρόγαλα), που είναι ένα υγρό απόβλητο με υψηλό

περιεχόμενο θρεπτικών, κυρίως επεξεργάζεται με τη χρήση φυσικοχημικών και βιολογικών

μεθόδων, ενώ η χρήση τεχνητών υγροβιότοπων είναι περιορισμένη.

Ο κύριος σκοπός της παρούσας διατριβής ήταν η αξιολόγηση της επίδρασης διαφόρων

παραμέτρων (υδραυλικού χρόνου παραμονής-HRT, θερμοκρασίας, φυσικοχημικών παραμέτρων)

στην επεξεργασία αποβλήτων που περιέχουν Cr(VI) καθώς και του δευτερογενούς ορρού

γάλακτος με τη χρήση πιλοτικών μονάδων τεχνητών υγροβιότοπων οριζόντιας υπόγειας ροής.

Επιπλέον η παρούσα διατριβή στόχευε και στην εξεύρεση μιας βιώσιμης τεχνικής για την

επεξεργασία της φυτικής βιομάζας και στην χρήση του δευτερογενούς ορρού γάλακτος, ως πηγή

άνθρακα στην επεξεργασία του Cr(VI).

Στη διάρκεια της 1ης

πειραματικής περιόδου της παρούσας διατριβής, η έρευνα επικεντρώθηκε

στη μελέτη της ολοκληρωμένης απομάκρυνσης του χρωμίου από υδατικά διαλύματα και στην

επεξεργασία δευτερογενή ορρού γάλακτος από πιλοτικές μονάδες τεχνητών υγροβιότοπων

οριζόντιας υπόγειας ροής. Για την ολοκληρωμένη απομάκρυνση του Cr(VI) χρησιμοποιηθήκαν

δύο πιλοτικές μονάδες τεχνητών υγροβιότοπων οριζόντιας υπόγειας ροής. Η μία πιλοτική

μονάδα ήταν φυτεμένη με κοινό καλάμι (Phragmites australis), ενώ η άλλη παρέμεινε αφύτευτη.

Οι συγκεντρώσεις του Cr(VI) στα υδατικά διαλύματα κυμάνθηκαν από 0.5 έως 10 mg/L. Επίσης

εξετάστηκε η επίδραση της θερμοκρασίας και του HRT (8 - 0.5 ημέρες) στην αφαίρεση του

Cr(VI). Η θερμοκρασία αποδείχτηκε να επηρεάζει την αφαίρεση του Cr(VI) και στις 2 πιλοτικές

μονάδες. Οι αποδόσεις απομάκρυνσης του Cr(VI) στην φυτεμένη πιλοτική μονάδα έφθασαν το

100% ακόμα και για HRT της 1 ημέρας, με συγκεντρώσεις εισόδου Cr(VI) 5, 2.5 και 1 mg/L. Σε

αντίθεση, η αφύτευτη πιλοτική μονάδα κατέγραψε σημαντικά χαμηλότερες αποδόσεις

απομάκρυνσης Cr(VI). Η φυτική βιομάζα που συλλέχθηκε από την φυτεμένη πιλοτική μονάδα

κομποστοποιήθηκε μαζί με στερεά απόβλητα ελαιοτριβείου. Το τελικό προϊόν της

κομποστοποιήσης είχε εξαιρετικά φυσικοχημικά χαρακτηριστικά (C/N: 14.1-14.7, δείκτης

βλαστικότητας (GI): 145-157%, Cr: 8-10 mg/kg dry mass), τα οποία πληρούν τα όρια της

Ευρωπαϊκής Ένωσης για τη χρήση του ως λίπασμα σε οργανικές καλλιέργειες.


Περίληψη xii

Η δεύτερη πειραματική διάταξη της 1ης

πειραματικής περιόδου περιελάμβανε δύο όμοιες

πιλοτικές μονάδες με τις παραπάνω, που ωστόσο χρησιμοποιήθηκαν για την επεξεργασία

δευτερογενούς ορρού τυρογάλακτος. Οι πιλοτικές μονάδες λειτούργησαν υπό διαφόρους

χρόνους παραμονής (8, 4, 2 και 1 ημέρα), θερμοκρασίες (από 2.4 έως 32.90C) και συγκεντρώσεις

εισόδου COD (από 1200 έως 7200 mg/L) Οι δύο μονάδες επεξεργάστηκαν επιτυχώς το

δευτερογενή ορρό γάλακτος, αφού καταγράφηκαν για την φυτεμένη και την αφύτευτη πιλοτική

μονάδα, αποδόσεις αφαίρεσης COD της τάξης του 91% και 77.23%, αντίστοιχα. Ο υδραυλικός

χρόνος παραμονής επηρέασε την απόδοση τω δύο πιλοτικών μονάδων μόνο όταν μειώθηκε στην

1 ημέρα. Αντιθέτως, η θερμοκρασία επηρέασε μόνο την αφύτευτη πιλοτική μονάδα, ενώ η

απόδοση της φυτεμένης επηρεάστηκε μόνο από τον ετήσιο κύκλο ανάπτυξης των φυτών. Πρέπει

να τονιστεί ότι οι συγκεντρώσεις εξόδου του COD ήταν χαμηλότερες των ορίων της Ε.Ε., ακόμα

και για χρόνους παραμονής 2 ημερών (ο χαμηλότερος που έχει αναφερθεί μέχρι τώρα στη

βιβλιογραφία) με αρχικές συγκεντρώσεις εισόδου COD από 1200 έως 3500 mg/L.

Στη διάρκεια της 2ης

πειραματικής περιόδου οι τέσσερεις συνολικά πιλοτικές μονάδες που

χρησιμοποιήθηκαν στην 1η περίοδο, χρησιμοποιήθηκαν επίσης και για την επεξεργασία ενός

μεικτού διαλύματος δευτερογενή ορρού γάλακτος και Cr(VI). Στόχος των πειραμάτων που

πραγματοποιήθηκαν ήταν η αξιολόγηση της επίδρασης του χρόνου παραμονής (8 και 4 ημέρες),

των συγκεντρώσεων εισόδου του Cr(VI) (από 0.5 έως 5 mg/L) και του COD (από 2000 έως 3000

mg/L), του φυτού και του επιφανειακού φορτίου στην απόδοση των πιλοτικών μονάδων. Όσον

αφόρα την αφαίρεση της οργανικής ύλης, οι φυτεμένες πιλοτικές μονάδες κατέγραψαν

υψηλότερα ποσοστά απομάκρυνσης (περίπου 70%) σε σύγκριση με τις αφύτευτες (περίπου

50%). Σε αντίθεση, η απομάκρυνση του Cr(VI) έδειξε να μην επηρεάζεται από την παρουσία

φυτών. Τέλος, παρατηρήθηκε ότι η ύπαρξη του Cr(VI) επηρεάζει την απομάκρυνση του

οργανικού φορτίου.

Τα τελικά συμπεράσματα της παρούσας διατριβής αποτελούν μια σημαντική συνεισφορά στην

επεξεργασία υγρών αποβλήτων που περιέχουν Cr(VI) καθώς και του δευτερογενή ορρού

γάλακτος από τεχνητούς υγροβιότοπους. Επίσης μπορεί να συμπεραθεί ότι η χρήση των

τεχνητών υγροβιότοπων για την αφαίρεση Cr(VI) και COD μπορεί να επιτευχθεί ακόμη και σε

πολύ χαμηλούς χρόνους παραμονής (1 και 2 ημερών, αντίστοιχα), καθώς και σε υψηλές αρχικές


Περίληψη xiii

συγκεντρώσεις (5 mg Cr(VI)/L και >5000 mg COD/L, αντίστοιχα). Τέλος, η χρήση του

δευτερογενή ορρού γάλακτος ως πηγή άνθρακα στην αφαίρεση του Cr(VI), ήταν πλήρως

επιτυχημένη.

Λέξεις κλειδιά: Εξασθενές χρώμιο, ορρός γάλακτος, οριζόντιας υπόγειας ροής τεχνητοί

υγροβιότοποι, υδραυλικός χρόνος παραμονής, φύτευση, θερμοκρασία κοινά καλάμια,

κομποστοποιήση.


Content xiv

CONTENTS

Chapter 1: Introduction ............................................................................................................... 1

1.1) Background ......................................................................................................................... 1

1.2) Research objectives and thesis relevance ............................................................................ 6

1.3) Novelty ................................................................................................................................ 9

Chapter 2: Industrial and Agro-Industrial Wastewaters ....................................................... 11

2.1) Industrial Wastewater .................................................................................................... 11

2.1.1) Textile wastewater ..................................................................................................... 12

2.1.2) Tannery wastewater ................................................................................................... 13

2.1.3) Acid mine .................................................................................................................. 14

2.1.4) Landfill leachate ........................................................................................................ 15

2.2) Agro-Industrial Wastewater .......................................................................................... 16

2.2.1) Dairy wastewater ....................................................................................................... 16

2.2.2) Animal farm ............................................................................................................... 17

2.2.3) Olive mill wastewater ................................................................................................ 17

2.2.4) Winery wastewater .................................................................................................... 18

2.3) Chromium (Occurrence and Uses) ................................................................................ 18

2.3.1) Environmental chemistry of Cr ................................................................................. 22

2.3.2) Physical and chemical characteristics ....................................................................... 24

2.3.3) Nutrition and toxicity of Cr: Health risk to humans .................................................. 26

2.3.3.1) Toxicity of Cr to animals ................................................................................... 27

2.3.3.2) Toxicity of Cr to plants and algae ..................................................................... 28

2.3.4) Remediation or treatment .......................................................................................... 29

2.4) Cheese Manufacturing .................................................................................................... 29

2.4.1) Cheese whey .............................................................................................................. 31

2.4.2) Sources ...................................................................................................................... 32

2.4.3) Characteristics and composition of cheese whey ...................................................... 32

2.4.4) Cheese whey – pollutant characteristics .................................................................... 33


Chapter 3: Constructed Wetlands............................................................................................. 35

3.1) Components of Constructed Wetlands .......................................................................... 37


Content xv

3.1.1) Water ......................................................................................................................... 37

3.1.2) Soil substrate/porous media ....................................................................................... 37

3.1.3) Wetland vegetation/macrophytes .............................................................................. 39

3.1.3.1) Role of macrophytes ............................................................................................... 40

i) Physical appearence ............................................................................................... 40

ii) Surface area for attached microbial growth .......................................................... 41

iii) Root release/creation of aerobic environment ..................................................... 42

iv) Organic compound release ................................................................................... 43

v) Nutrient uptake and storage ................................................................................... 44

3.1.4) Microorganisms ......................................................................................................... 44

3.2) Types of Constructed Wetlands ..................................................................................... 45

3.2.1) Surface Flow/ Free Water Surface flow .................................................................... 46

3.2.2) Horizontal Sub-Surface Flow .................................................................................... 48

3.2.3) Vertical Flow ............................................................................................................. 46

3.3) Design Factors ................................................................................................................. 52

3.3.1) Pretreatment ............................................................................................................... 52

3.3.2) Hydrology .................................................................................................................. 52

3.3.2.1) Hydraulic loading rate (HLR) ............................................................................ 53

3.3.2.2) Hydraulic Retention Time (HRT) ...................................................................... 53

2.3.3) Design of bed/bed area .............................................................................................. 55

3.3.4) Selection of wetland plants ........................................................................................ 56

3.3.5) Inlet structure ............................................................................................................. 59

3.3.6) Outlet Structure ......................................................................................................... 59

3.4) Removal Mechanisms of Constructed Wetlands .......................................................... 59

3.4.1) Organic matter removal ............................................................................................. 60

3.4.2) Nitrogen removal ....................................................................................................... 62

3.4.3) Phosphorus removal .................................................................................................. 64

3.4.4) Total suspended solids removal ................................................................................. 65

3.4.5) Heavy metals (Cr) removal ....................................................................................... 65

3.5) Application for Cr(VI) treatment .................................................................................. 66


Content xvi

3.6) Application of CWs in Agro-Industrial wastewaters Treatment .................................... 74

3.6.1) Pre-treatment stages in treatment of Dairy Wastewaters .......................................... 74

3.6.2) CW types in treatment of Dairy Wastewaters .......................................................... 74

3.6.3) Vegetation for Dairy Wastewater treatment in CW systems ..................................... 78

3.6.4) Pre-treatment stages in treatment of Animal Farm Wastewater ................................ 79

3.6.5) CW types in treatment of Animal Farm Wastewater ................................................ 79

3.6.6) Vegetation for Animal Farm Wastewater in CW systems ....................................... 82

3.6.7) Pre-treatment stages in treatment of OMW wastewater ............................................ 83

3.6.8) CW types in treatment of OMW wastewater ............................................................ 83

Chapter 4: Materials and Methods ........................................................................................... 90

4.1) First Experimental Period .............................................................................................. 90

4.1.1) Description of pilot-scale CW unit ............................................................................ 90

4.1.2) Wastewater preparation for Cr(VI) treatment ........................................................... 90

4.1.3) Wastewater preparation for secondary cheese whey ................................................. 93

4.1.4) Water quality monitoring .......................................................................................... 94

4.1.5) Assessment of evapotranspiration ............................................................................. 94

4.1.6) Reed biomass in composting ..................................................................................... 97

4. 1.6.1) Composting materials and process ................................................................... 97

4.1.6.2) Physicochemical analyses ................................................................................. 97

4.2) Second Experimental Period ........................................................................................ 100

4.2.1) Description of pilot-scale CW unit .......................................................................... 100

4.2.2) Description of the experimental setup ..................................................................... 100

4.2.3) Wastewater preparation ........................................................................................... 101

Chapter 5: Pilot-Scale HSF Constructed Wetlands Treating Cr(VI) .................................. 104

5.1) Effects of Physicochemical Parameters of Wastewater ............................................. 104

5.1.1) Temperature ............................................................................................................. 104

5.1.2) pH ............................................................................................................................ 105

5.1.3) Electrical Conductivity (EC) ................................................................................... 109

5.1.4) Dissolve Oxygen (DO) ............................................................................................ 112

5.2) Treatment Efficiency of pilot-Scale HSF Constructed Wetlands ............................. 112

5.3) Effect of HRT ................................................................................................................. 117


Content xvii

5.4) Cr Mass Balance ............................................................................................................ 121

5.5) Composting of Plant Biomass ...................................................................................... 124

Chapter 6: Pilot-Scale HSF Constructed Wetlands Treating Cheese Whey ....................... 127

6.1) Physicochemical Parameters ........................................................................................ 127

6.1.1) pH ............................................................................................................................ 127

6.1.2) EC ............................................................................................................................ 128

6.1.3) DO ........................................................................................................................... 128

6.2) Treatment Efficiency of Pilot-Scale HSF Constructed Wetlands ............................. 128

6.3) Effect of HRT ................................................................................................................. 132

6.4) Effect of Temperature ................................................................................................... 136

6.5) Effects of Vegetation ..................................................................................................... 137

Chapter 7: Combined Treatment of Cr(VI) and Cheese Whey in Pilot-Scale HSF

Constructed Wetlands ............................................................................................................... 139

7.1) Physicochemical Parameters ........................................................................................ 139

7.1.1) pH ............................................................................................................................ 139

7.1.2) EC ............................................................................................................................ 141

7.2) Removal Efficiency of Cr(VI) in Four Pilot-Scale HSF Constructed Wetlands with

Co-Treated Wastewaters ..................................................................................................... 141

7.2.1) Effect of vegetation on Cr(VI) removal .................................................................. 147

7.2.2) Effect of HRTs on Cr(VI) removal ......................................................................... 153

7.2.3) Comparison with experimental results of the first operational period .................... 154

7.3) Removal Efficiency of COD in Four Pilot-Scale HSF Constructed Wetlands with Co-

Treated Wastewaters ............................................................................................................ 155

7.3.1) Effect of vegetation on Cr(VI) removal .................................................................. 160

7.3.2) Effect of HRTs on Cr(VI) removal ......................................................................... 162

7.3.3) Comparison with experimental results of the first operational period .................... 167

Chapter 8: General Discussion ................................................................................................ 169

8.1) Overall discussion .......................................................................................................... 169

8.2) Application ..................................................................................................................... 174

Chapter 9: Conclusion and Future Research ......................................................................... 175

9.1) Overall Conclusions ...................................................................................................... 175

9.2) Recommendations for Future Research ...................................................................... 177


Content xviii

References .................................................................................................................................. 178

Appendix .................................................................................................................................... 236

Curriculum Vitae ...................................................................................................................... 246


List of Tables xix

List of Tables

Table 2.1 Chromium compounds with different oxidation states ............................................ 21

Table 2.2 Amount of cheese whey obtained from different type of cheese productions. ....... 32

Table 2.3 General composition of fresh whey .......................................................................... 33

Table 3.1 The major roles of macrophytes in constructed treatment wetlands ........................ 42

Table 3.2 HLR for different types of CWs ............................................................................... 54

Table 3.3 Key design and operational specifications for HF and VF CWs ............................. 56

Table 3.4 Free water surface constructed wetland (FWS CW) ................................................ 68

Table 3.5 Horizontal surface flow constructed wetland (HSF CW)......................................... 69

Table 3.6 Vertical flow constructed wetland (VF CW) ............................................................ 69

Table 3.7 Treatment of chromium-polluted wastewaters using hybrid constructed wetland

systems ..................................................................................................................................... 73

Table 3.8 Studies of dairy wastewater treatment by CWs ................................................... 76-77

Table 3.9 Studies of animal farm wastewater treatment by CWs ....................................... 80-81

Table 3.10 Studies of olive mill wastewater treatment by CWs .............................................. 84

Table 3.11 Pollutant influent and effluent concentrations compared with EU standards (EU

Directive 1991/271/EEC) ......................................................................................................... 89

Table 4.1 Description of experimental set-ups of composting ................................................. 97

Table 4.2 Concentrations of Cr(VI) and COD used in the second experimental period ........ 101

Table 5.1 Comparisons of Cr (VI) removal between different HRTs and temperature ......... 118

Table 5.2 Cr concentrations recorded in different parts of reed plants .................................. 123

Table 5.3 Physicochemical parameters during the composting period .................................. 126

Table 6.1 Physicochemical characteristics of SCW wastewater ............................................ 127

Table 6.2 Treatment of dairy wastewaters using different types of CWs .............................. 135

Table 6.3 Comparison of COD removal rates with different HRTs ....................................... 136

Table 7.1 Physicochemical characteristics of co-treated wastewaters (mixed of cheese whey

and Cr(VI) solution) ............................................................................................................... 139

Table 7.2 Comparisons of Cr(VI) removal during the two operational periods..................... 154

Table 7.3 Comparisons of COD removal in the two experimental sessions .......................... 167


List of Figures xx

List of Figures

Fiugre 1.1 Worldwide available water resources ...................................................................... 1

Fiugre 1.2 Global water withdrawal and waste over time ......................................................... 2

Fiugre 1.3 Ratio of treated to untreated wastewater reaching water bodies for 10 regions ...... 3

Fiugre 1.4 Generation of hazardous waste throughout the world .............................................. 5

Fiugre 2.1 Chromium speciation in water ............................................................................... 19

Fiugre 2.2 Pure chromium ....................................................................................................... 20

Fiugre 2.3 Chromium(VI) oxide .............................................................................................. 21

Fiugre 2.4 Use of Chromium and its compounds .................................................................... 22

Fiugre 2.5 Chromium circulation in the environment .............................................................. 24

Fiugre 2.6 Possible forms of Cr(VI) in solution in the presence of iron particles.................... 24

Fiugre 2.7 Distribution of Cr(VI) species as a (a) function of pH, and (b) fraction of HCrO4-

and Cr2O7-2

at pH 4 as a function of total Cr(VI) ..................................................................... 26

Fiugre 2.8 Flow sheet for the manufacture of cheese ............................................................... 30

Fiugre 2.9 Simplified cheese production inputs and outputs ................................................... 32

Fiugre 3.1 Schematic diagram of a constructed wetland and its mechanisms ......................... 36

Fiugre 3.2 Possible interactions in the root zone of wetlands for wastewater treatment ......... 36

Fiugre 3.3 Different types of substrates for CWs (a) gravel, (b) course gravel, (c) fine gravel,

(d) sands, (e) soil ...................................................................................................................... 38

Fiugre 3.4 Micrograph of gas spaces in a Typha latifolia root (scale bar, 1 mm) .................... 40

Fiugre 3.5 Biofilm on submerged parts of wetland plants ....................................................... 41

Fiugre 3.6 Release of oxygen from roots of Phragmites australis. The blue colour around the

roots is formed by radical oxygen release from the roots that oxidized the reduced form of

methylen blue ........................................................................................................................... 43

Fiugre 3.7 Classification of CWs for wastewater treatment ..................................................... 46

Fiugre 3.8 Schematic diagram of free water surface constructed wetland ............................... 47

Fiugre 3.9 Schematic diagram of horizontal surface flow constructed wetland ...................... 48

Fiugre 3.10 Substrate structure of HSF CWs ........................................................................... 49

Fiugre 3.11 Vertical flow constructed wetland ........................................................................ 51

Fiugre 3.12 Different types of wetland plants .......................................................................... 58

Fiugre 3.13 Processes occurring in a wetland .......................................................................... 60


List of Figures xxi

Fiugre 4.1 Pilot-scale HSF CWs for the treatment of Cr(VI) ................................................... 91

Fiugre 4.2 Pilot-scale HSF CWs for the treatment of SCW ..................................................... 91

Fiugre 4.3 Inflow perforated plastic pipe (diffuser) for both units ........................................... 92

Fiugre 4.4 Outlet structure of the CWs (a) Tube arranged at the same height as the filler material)

(b) Effluent collected point (c) 35L plastic tank for overflow wastewater collection ..................... 92

Fiugre 4.5 Influent storage tank with adjusted valve................................................................ 93

Fiugre 4.6 Time series chart for air temperature (2011-2012) ................................................. 95

Fiugre 4.7 Time series chart for precipitation (2011-2012) ..................................................... 96

Fiugre 4.8a Composting materials in Bin 1 at day 1 ................................................................ 98

Fiugre 4.8b Composting materials in Bin 2 at day 1 ................................................................ 98

Fiugre 4.9a Mature compost in Bin 1 at 102 days .................................................................... 99

Fiugre 4.9b Matured compost in Bin 2 at 102 days ................................................................. 99

Fiugre 4.10 Integrated treatment of cheese whey and Cr(VI) in HSF CWs ........................... 100

Fiugre 4.11 Time series chart for air temperature, 0C (2013) ................................................. 102

Fiugre 4.12 Time series chart for precipitation (2013) ........................................................... 103

Fiugre 5.1a pH values of influent and effluent wastewaters in the Cr-U unit ........................ 107

Fiugre 5.1b pH values of influent and effluent wastewaters in the Cr-P unit ........................ 108

Fiugre 5.2a EC value of influent and effluent wastewaters in the Cr-U unit ......................... 110

Fiugre 5.2b EC values of influent and effluent wastewaters in the Cr-P unit ........................ 111

Fiugre 5.3a Time series charts of Cr(VI) influent and effluent concentrations for Cr-U ....... 115

Fiugre 5.3b Time series charts of Cr(VI) influent and effluent concentrations for Cr-P ....... 116

Fiugre 5.4a Effect of surface load on Cr(VI) removal for Cr-U............................................. 119

Fiugre 5.4b Effect of surface load on Cr(VI) removal for Cr-P ............................................. 120

Fiugre 5.5 Harvested reed plants treated with Cr(VI) solutions ............................................. 122

Fiugre 5.6 Time series of compost and ambient temperature during composting period ...... 125

Fiugre 6.1a Time series charts for COD removal with influent and effluent concentrations in

the SCW–U ............................................................................................................................. 130

Fiugre 6.1b Time series charts for COD removal with influent and effluent concentrations in

the SCW-P .............................................................................................................................. 131

Fiugre 6.2a Correlation of surface load and removal rates for COD in the SCW-U.............. 133

Fiugre 6.2b Correlation of surface load and removal rates for COD in the SCW-P .............. 134


List of Figures xxii

Fiugre 7.1a Time series charts for Cr(VI) removal with influent and effluent concentrations in

the SCW-U unit ...................................................................................................................... 142

Fiugre 7.1b Time series charts for Cr(VI) removal with influent and effluent concentrations in

the SCW-P unit ....................................................................................................................... 143

Fiugre 7.2a T Time series charts for Cr(VI) removal with influent and effluent concentrations

in the Cr-U unit ....................................................................................................................... 144

Fiugre 7.2b Time series charts for Cr(VI) removal with influent and effluent concentrations in

the Cr-P unit ........................................................................................................................... 145

Fiugre 7.3 Belowground parts of reed plants ......................................................................... 148

Fiugre 7.4a Effect of surface load on Cr(VI) removal in the SCW-U unit ............................ 149

Fiugre 7.4b Effect of surface load on Cr(VI) removal in the SCW-P unit ............................. 150

Fiugre 7.5a Effect of surface load on Cr(VI) removal in the Cr-U unit ................................. 151

Fiugre 7.5b Effect of surface load on Cr(VI) removal in the Cr-P unit ................................. 152


the SCW-U unit ...................................................................................................................... 156


the SCW-P unit ....................................................................................................................... 157


the Cr-U unit ........................................................................................................................... 158


the Cr-P unit ........................................................................................................................... 159

Fiugre 7.8a Correlation charts of surface load and removal rates of COD in the SCW-U unit

................................................................................................................................................ 163

Fiugre 7.8b Correlation charts of surface load and removal rates of COD in the SCW-P unit

................................................................................................................................................ 164

Fiugre 7.9a Correlation charts of surface load and removal rates of COD in the Cr-U unit .. 165

Fiugre 7.9b Correlation charts of surface load and removal rates for COD in the Cr-P unit 166


Abbreviation and Acronyms xxiii

Abbreviation and Acronyms

CW Constructed wetland

Cr Chromium

Cr(VI) Hexavalent Chromium

Cr(III) Trivalent Chromium

SCW Secondary Cheese whey

FWS Free Water Surface

HSF Horizontal Surface Flow

BOD Biochemical Oxygen Demand

COD Chemical Oxygen Demand

TSS Total Suspended Solids

HRT Hydraulic Residence Time

HLR Hydraulic Loading Rate

TP Total Phosphorus

Τ Temperature

ΤΚΝ Total Kjedahl Nitrogen

EPA Environmental Protection Agency

ACD Acid Mine Drainage

OMW Olive mill Wastewater


Chapter 1: Introduction

CHAPTER 1: GENERAL INTRODUCTION

1.1) Background ....................................................................................................................... 1

1.2) Research objectives and thesis relevance ........................................................................ 6

1.3) Novelty ............................................................................................................................... 9


Chapter 1: Introduction 1

CHAPTER 1: GENERAL INTRODUCTION

1.1) Background

The environment is composed of atmosphere, earth, water and space. Without pollution, it

remains clean and enjoyable. The complex nature of the environment gets changed by

different activities of man, including industrialization, construction, transportation, etc. Such

activities, although desirable for human development and welfare, lead to environmental

degradation and the release of objectionable materials into the environment leading to loss of

quality of life.

Over half of the world’s population faces water scarcity. Almost 900 million people in the

world still do not have access to safe water and some 2.6 billion, almost half the population of

the developing world, do not have access to adequate sanitation. At least 1.8 million children

under five years old die every year due to water related diseases. Additionally, 1.2 billion

people (one fifth of the world’s population) live in areas of water scarcity (Corcoran et al.,

2010; WWDR, 2014). Figure 1.1 shows the world distribution of renewable water resources.

Figure1.1: Worldwide available water resources (WWDR, 2014).



Among the different sectors of water uses, presently agriculture uses most water; between

70% and 90% of all water in most regions (Grobicki, 2007; Corcoran et al., 2010). About 70-

90% of the world’s available fresh water is used during food production. Figure 1.2 shows the

global withdrawal, consumption and waste of water for the different sectors. Approximately

20% and 10% of total freshwater withdrawals are used by the industrial and domestic sectors,

respectively (WWDR, 2014). Of these two sectors, industry generates a substantial proportion

of wastewaters, although these figures vary considerably between countries (WWAP, 2009).

Nearly all human activities result in the production of wastewater. Globally, about 80% of

wastewater from human settlements and industrial sources is discharged into the environment

without treatment (Corcoran et al., 2010).

Figure 1.2: Global water withdrawal and waste over time (Corcoran et al., 2010).

In many developing countries more than 70% of industrial wastes are disposed untreated into

free surface water (WWAP, 2009). In many cases, industrial wastewaters not only drain

directly into rivers and lakes but also contaminate groundwater aquifers and wells. These

wastewaters also contaminate coastal ecosystems and contribute to growing marine dead

zones. 90% of all untreated wastewater is discharged directly into rivers, lakes or oceans in



many developing countries (UN Water, 2008). Figure 1.3 shows the ratio of treated and

untreated wastewaters around the world.

Figure 1.3: Ratio of treated to untreated wastewater reaching water bodies for 10 regions

(Corcoran et al., 2010).

Industrial discharge contains a wide range of contaminants and originates from numerous

sources. Untreated industrial wastewater has the potential to be a highly toxic source of

pollution. A vast array of complex organic compounds and heavy metals are used in modern

industrial processes and if they are released into the environment can cause both human health

and environmental disasters. Toxic industrial waste is generated by mining, pulp mills,

tanneries, sugar refineries, and pharmaceutical production, among others.

Normally industrial wastewaters are categorized into two types: inorganic industrial

wastewater and organic industrial wastewater. Coal and steel industries produce inorganic

wastewaters along with non-metallic minerals industries, commercial enterprises and

industries for the surface processing of metals (iron picking works and electroplating plants)

that also generate inorganic wastewaters. These wastewaters contain a large proportion of

suspended matter, which could be eliminated by sedimentation, together with chemical



flocculation through the addition of iron or aluminum salts, flocculation agents and various

organic polymers. Chemical industries and large-scale chemical works, which mainly use

organic substances for chemical reactions, generate organic industrial wastewaters.

Since the Stone Age, about 1150 million tones of heavy metals (copper, lead, cobalt, zinc,

cadmium and chromium) have been mined from the earth. During the period 1959-1990, the

production of six major non-ferrous metals (zinc, copper, lead, tin, nickel and aluminum)

increased eightfold (Sheoran and Sheoran, 2006). Nowadays, the effluents from metal-

working industries contain hazardous waste compounds (chromium, nickel, zinc, cadmium,

lead, iron and titanium). Of these, the electroplating industry is an important pollution

distributor, dry cleaning and car repair shops generate solvent waste, and printing plants

release inks and dyes. The pulp and paper industry used heavily chlorine-based substances,

and generates chloride organics and dioxins, as well as suspended solids and organic matter

containing wastes. The petrochemical industry discharges large amounts of phenols and

mineral oils. Also wastewater from different food and dairy processing plants contain high

proportion of suspended solids and organic materials. Due to the various characteristics of

industrial wastewaters, their treatment must be designed specifically for the particular type of

effluent produced.

The sludge remaining after the treatment of industrial wastewaters contains many hazardous

wastes (including heavy metals) and these are often found in lake sediments and can degrade

planktonic communities and aquatic ecosystems. Heavy metals can impact human health and

wildlife in various ways. For example, lead interferes with the nervous system which leads to

disabilities in children, and heavy metal accumulation leads to the damage of human and

animal organs. Figure 1.4 shows the recent scenario of hazardous waste generation throughout

the world (UNSD/UNEP Questionnaires on Environment Statistics, Waste section; last update

March 2011).

When wastewater is mixed with fresh water, bacteria use the pollutants as an energy source

and decompose them. This pollutant decomposition occurs in a natural environment where

dissolved oxygen content is high and aerobic (oxygen-using) bacteria decompose pollutants

and produce new bacterial cells. In environments where dissolved oxygen content is low,

anaerobic (non-oxygen-using) bacteria decompose the organic waste material and release

odorous gases such as hydrogen sulphide and methane (Ferguson, 2003). Moreover, if there is



an overwhelming amount wastewater and all the oxygen will be used up, the anaerobic

bacteria will take over the place. Finally, the water will become anaerobic and all forms of

oxygen-dependent life will die. Therefore, insufficient wastewater management directly

impacts the biological diversity of aquatic ecosystems and disrupts the fundamental integrity

of human life.

Figure 1.4: Generation of hazardous waste throughout the world.

(http://epp.eurostat.ec.europa.eu/portal/page/portal/waste/key_waste_streams/hazardous_waste)

There is also a relationship between wastewater and climate change because the changes in

climatic conditions change the volume and quality of water availability which ultimately

influence water usage practices. Changes in climate will also require adaptation, in terms of

how the wastewater is treated. Because the treatment practice of industrial wastewaters has an

impact on climate change as same treatment processes of wastewater generate methane,

nitrous oxide and carbon dioxide (Corcoran et al., 2010).



Water is a limited resource, the demand for which is growing with the rise in global

population. Therefore, the treatment and reuse of wastewaters is essential. To be successful

and sustainable, wastewater management must be an integral part of rural and urban

development planning, across all sectors.

1.2) Research objectives and thesis relevance

Cr(VI) is one of the most hazardous metals and it is known to a 100-fold more toxicity than

Cr(III), for both acute and chronic exposures, because of its high water solubility, mobility

and easy reduction. Due to its toxicity, stringent regulations are imposed on the discharge of

total Cr into surface waters to below 0.05 mg/L by the EPA (Baral and Engelken, 2002) and

the EU (EC, 1998). A wide range of technologies is available for the reduction/removal of

Cr(VI) from aqueous solutions, some of which are well-established methods that have been

practiced for decades such as precipitation, filtration, chemical oxidation or reduction, ion

exchange, reverse osmosis, electrochemical treatment, membrane technologies, evaporative

recovery, and activated carbon adsorption (Owlad et al., 2009). However, these approaches

are costly and can themselves produce other waste problems. Therefore, there is a need to

develop cost-effective and more environment-friendly techniques. Biological systems, such as

bioreduction, bio-accumulation or bio-sorption using living cells, as well as aquatic plants,

have been examined for their chromium removal abilities (Fude et al., 1994; Aguilar et al.,

2008; Calheiros et al., 2009). These systems offer potential alternatives to existing

technologies that remove Cr(VI) from water and wastewater. Advances in the field of

environmental microbiology and biotechnology indicate that bacteria (Stasinakis et al., 2002;

Zouboulis et al., 2004), fungi (Sanghi et al., 2009), yeast (Chen and Wang, 2007), and algae

(Bankar et al., 2009), either as pure or mixed cultures, can remove chromium (VI) from

aqueous solutions. Until present, most microbial Cr(VI) removal was obtained by pure culture

techniques under either anaerobic or aerobic conditions depending on the species or

consortium in question (Shen and Wang, 1993). However, the main disadvantage of using

pure cultures to remove chromium compounds is related to the use of sterile conditions to

prevent external microbial contamination, thus increasing treatment costs and hindering their

wide applicability. Additionally, most of the biological methods for treatment of Cr(VI)-

containing wastewater are operated in batch mode (Chirwa and Wang, 2004) probably due to

the eventual loss of the active biomass. It seems impossible to continuously remove Cr(VI) on



a long-term basis using bioreactors, without intermittently reseeding the biomass (Chen and

Hao, 1997).

Like other agro-industries, the dairy industry produces large quantities of wastewaters which

are characterized by high organic content, i.e., high Chemical Oxygen Demand (COD) and

Biological Oxygen Demand (BOD) (Demirel et al., 2005). The dairy industry processes large

volumes of milk, and the major waste produced from processing is water. Raw milk produces

yogurt, butter, cheese, etc., by means of different processes (pasteurization, coagulation,

filtration, centrifugation, chilling, etc) (Rivas et al., 2010). Cheese production is an important

portion of the dairy industry in the European Union as more than 40% its annual milk

production is processed into cheese (EC, 2009). More than 45 million tons of cheese whey

(CW) is produced in the EU each year (Jasko, 2011). Cheese whey is the liquid by-product of

the cheese making process (Rajeshwari et al., 2000).

Every year 160 million tons of cheese whey are produced worldwide and half of the total

cheese whey production is disposed directly into the environment (Smithers, 2008; Guimaraes

et al., 2010). The disposal of cheese whey has always been a major problem for the dairy

industry. As dairy wastewaters contain large amounts of proteins, when they are discharged

directly into the environment, not only is there a significant loss of resources but also major

environmental pollution. Its direct disposal can affect the physical and chemical structure of

soil, for which crop yields decrease. In addition, when cheese whey is discharged into water

resources, it affects the aquatic ecosystems by consuming excessive oxygen, (Siso, 1996;

Panesar et al., 2007), and the nutrients it contains lead to eutrophication of the receiving

waters.

Dairy wastewaters are generally treated by using different biological methods such as

activated sludge processes, aerated lagoons, trickling filters, sequencing batch reactor (SBR),

anaerobic sludge blanket (UASB) reactor, anaerobic filters, etc. (Demirel et al., 2005;

Prazeres et al., 2012). Frequently, the post-treatment of dairy wastewaters is also done using

physicochemical treatment methods consisting of coagulation/flocculation by various

inorganic and organic natural coagulants, and membrane processes such as nanofiltration (NF)

and/or reverse osmosis (RO) (Kushwaha et al., 2011).



Recently constructed wetlands (CWs) are used to treat different kinds of wastewaters

including agro-industrial (Dunne et al., 2005; Vymazal, 2010). CWs are known from the

decade 80's and low-cost technology that has been used to treat various types of wastewaters

for nearly twenty years (Ghosh and Gopal, 2010; Hunt et al., 2003). They are an attractive

treatment option because they use solar energy, are simple to construct and operate, have low

maintenance cost and are inexpensive and sustainable compared to conventional treatment

methods (Jamieson et al., 2007). They have grown in popularity since the early 1980s (Reed

et al., 1995). In addition, due to their high pollutant removal efficiency, they can be used to

treat a wide variety of wastewaters (Sultana et al., 2014b). These systems are very cost

effective compared to the physicochemical wastewater treatment methods (Knox et al., 2006).

Various applications/experiments using CWs for Cr removal have been recorded in the last

decade, at both laboratory-scale and full-scale (Sultana et al., 2014b). These applications

either examine Cr removal from various types of wastewaters and activated sludge, or from

Cr-containing aqueous solutions. The extremely high Cr removal efficiencies (up to 100%)

achieved in these studies together with their low capital and operational costs, make CWs an

attractive alternative for Cr removal. CW is a flexible treatment method, as it can be used as a

single treatment step or as a polishing stage in an existing treatment facility depending on the

exact specification of the wastewaters concerned. Horizontal subsurface flow (HSF) CWs

were tested (Aguilar et al., 2008; Calheiros et al., 2009) and appeared rather efficient at

removing Cr(VI) as removal rates reached 100%. Other researchers have studied horizontal

subsurface flow (HSF) CWs for removing Cr.

One of the main issues concerning Cr(VI) removal using CWs is the post-treatment

management of plant biomass that contains high Cr concentrations. For the most effective

pollutant removal, the plants should be harvested at the end of each vegetation cycle. After

harvesting, the contaminated plants can either be discarded into a bare field which adds to the

environmental risk, or they can be used for some other purpose. A compatible method of

disposal for this biomass is needed and for this reason, in this dissertation, harvested CW reed

biomass was co-composted with olive mill wastes. Previous studies have examined the role of

plant species and porous media on Cr(VI) removal, but no comparative studies have been

conducted on the effects of temperature and hydraulic retention time (HRT).

Recently, CWs have also been more frequently used to treat different kinds of agro-industrial

wastewaters, including dairy. The use of CWs for treating dairy wastewaters is a relatively



recent application of biological treatments (Schaafsma et al., 2000) and a significant number

of studies have been conducted. Some of these mixed dairy wastewater with other wastewater

types such as dirty farmyard waters (Dunne et al., 2005) and municipal wastewaters (Mantovi

et al., 2002; 2003). Only two studies have used CWs to treat secondary cheese whey (SCW)

wastewater (Comino et al., 2011; Mantovi et al., 2011). HSF CWs appear to be more efficient

at treating dairy wastewater as they achieve extremely high organic matter removal

efficiencies (up to 95%). However, only a few studies have examined systematically either

the effect of temperature (Sharma et al., 2013a) or the effect of the pollutant loading rate

(Sharma et al., 2013b) on CW performance.

The objectives of this PhD research were:

Using pilot-scale HSF CWs to assess the removal of different concentrations of Cr(VI)

with different hydraulic retention times (HRT) and determine the HRT with highest

the removal rate through a series of experiments.

To propose a viable and cost effective treatment for the harvested reed biomass that is

contaminated with Cr(VI).

To examine secondary cheese whey treatment using pilot-scale HSF CWs. To assess

the effect of different COD influent concentrations and HRTs, at various temperatures

and pollutant loads, on COD removal.

To examine the use of cheese whey as a carbon source for Cr(VI) removal from HSF

CWs.

To compare the removal rates of Cr(VI) and COD during two experimental periods

and also to compare the effects of HRTs, the influent concentrations of Cr(VI) and

COD, and vegetation.

1.3) Novelty

i) Various studies have been conducted on Cr(VI) removal using CWs. These studies

examined different parameters including the role of the plant species and porous media, but to

date no comparative studies have been conducted on the effects of temperature and HRT on

Cr(VI) removal. One of the major issues regarding Cr(VI) removal using CWs is the disposal

of plant biomass that contains high Cr concentrations as there are no studies in the literature



on the compatible method of disposal of this plant material disposal. This dissertation presents

the experimental results of pilot-scale HSF CWs that removed Cr(VI) from wastewater.

Specifically, this work discusses the effect of HRT and temperature on the removal of Cr(VI)

and the disposal of the contaminated biomass by composting. For the first time, a viable and

cost effective solution for treating the contaminated reed biomass is proposed. Reed biomass

was co-composted with olive mill wastes and produced high quality compost.

ii) Published experiments using different CWs to treat dairy wastewater have achieved high

organic matter removal rates (up to 99%), although they operated with high HRTs (11 days)

and low influent COD concentrations. However, in most of these experiments the dairy

wastewaters were mixed, and thus diluted, with different dairy plant cleaning wastewaters and

pure cheese whey was not treated. Only a few studies have examined systematically either the

effect of temperature or the effect of the pollutant loading rate on CW performance for dairy

wastewater treatment but not for the treatment of pure SCW. The second part of this PhD

work presents the experimental results of HSF CWs for cheese whey wastewater treatment

and specifically the effect of temperature, vegetation, organic load and HRT on the removal

of COD were examined for the first time in pilot-scale CWs treating pure cheese whey

solution. In this research, two pilot-scale HSF CWs successfully operated under extremely

low HRTs of 2 days. In addition, an initial design guideline is proposed for CW units treating

secondary cheese whey.

iii) The final experimental results of this PhD research present an integrated treatment of

cheese whey and Cr(VI) wastewaters in CWs for the first time. HSF CWs can successfully

remove Cr(VI) with 4 days HRT and in the presence of COD.


Chapter 2: Industrial and Agro-Industrial Wastewaters

CHAPTER 2: INDUSTRIAL AND AGRO-INDUSTRIAL

WASTEWATERS

2.1) Industrial Wastewater .................................................................................................... 11

2.1.1) Textile Wastewaters .................................................................................................. 12

2.1.2) Tannery Wastewaters ................................................................................................ 13

2.1.3) Acid mine .................................................................................................................. 14

2.1.4) Landfill leachate ........................................................................................................ 15

2.2) Agro-Industrial Wastewaters ........................................................................................ 16

2.2.1) Dairy wastewaters ..................................................................................................... 16

2.2.2) Animal farm .............................................................................................................. 17

2.2.3) Olive mill wastewater................................................................................................ 17

2.2.4) Winery wastewater .................................................................................................... 18

2.3) Chromium (Occurrence and Uses) ................................................................................ 18

2.3.1) Environmental chemistry of Cr ................................................................................. 22

2.3.2) Physical and chemical characteristics ....................................................................... 24

2.3.3) Nutrition and toxicity of Cr: health risks to humans ................................................. 26

2.3.3.1) Toxicity of Cr to animals ..................................................................................... 27

2.3.3.2) Toxicity of Cr to plants and algae ....................................................................... 28


2.4) Cheese Manufacturing ................................................................................................... 29

2.4.1) Cheese whey .............................................................................................................. 31

2.4.2) Sources ...................................................................................................................... 32

2.4.3) Characteristics and composition of cheese whey ...................................................... 32

2.4.4) Cheese whey – pollutant characteristics.................................................................... 33



Chapter 2: Industrial and Agro-Industrial Wastewaters 11

CHAPTER 2: INDUSTRIAL AND AGRO-INDUSTRIAL

WASTEWATER

Water pollution is one of the most serious environmental problems and it is caused by a

variety of human activities such as industrial, agricultural and domestic. Untreated or

improperly treated wastewaters are the major source of surface water body pollution. Many

water born diseases and other health problems are caused by polluted waters. Sediments

carried out by runoff water from agricultural fields and discharge of untreated or partially

treated sewage and industrial effluents, disposal of fly ash or other solid wastes into a water

body all cause severe water pollution. Water pollution can be caused by pathogens, inorganic

compounds, organic material and macroscopic pollutants. Worldwide, some 780 million

people still lack access to clean drinking water sources (WHO, 2012). Over time, a series of

treatment technologies have been developed to treat wastewaters.

2.1) Industrial Wastewater

The production and use of chemical compounds has increased tremendously worldwide and

many of these compounds are biologically non-degradable. Therefore, the major concern is to

treat the wastewater before it is discharged into the environment. However, wastewaters are

not properly treated and this affects water quality (UNESCAP, 2000). Effluents discharged

from industrial and agriculture activities are high in volume and also include high

concentrations of heavy metals and organic compounds. In most of cases those effluents are

drained without proper treatment (UNESCAP, 1999).

Wastewater is categorized and defined according to its sources of origin. Wastewaters

discharged principally from residential sources and generated by such activities as food

preparation, laundry, cleaning and personal hygiene, are termed as “domestic wastewater”.

Industrial/commercial wastewaters originate and are released from manufacturing processing

industries and commercial activities such as printing, textile, steel, food and beverage

processing (Medcities and ISR EWC, 2003).

Various industries are major sources of pollution. Based on the type of industry, various

levels of pollutants can be discharged into the environment directly or indirectly through



public sewer lines. Industrial wastewater can also include sanitary wastes, manufacturing

wastes and relatively uncontaminated water from heating and cooling operations (Emongor et

al., 2005). One of the major problems of wastewater is their high content of heavy metals,

solvents, dyes, pesticides, etc (Oller et al., 2011), which can have toxic effects (Nies, 1999;

Duncan et al., 2003). The main types of industrial and agro-industrial wastewaters include:

o Textile

o Tannery

o Acid mine

o Landfill leachate

o Dairy

o Animal farm

o Olive mill

o Winery

2.1.1) Textile Wastewaters

Among the waste producing industries, textile dyeing processes are the most environmental

unfriendly industrial processes, because they produce colored wastewaters that are heavily

polluted with dyes, textile auxiliaries and chemicals (Roussy et al., 2005). In the textile

industry, the presence of color in discharges is one of the major problems. The characteristics

of textile wastewater depend on the production, technology and chemicals used (Awomeso et

al., 2010). Usually textile industries consume huge quantities of water and generate large

volumes of wastewater through various steps in dyeing and finishing processes and the

released wastewater is a complex mixture of different polluting substances such as inorganic,

organic, elemental and polymeric products (Brown and Laboureur, 1983). Dye wastes are the

most predominant substances in textile wastewater and these substances not only toxic to the

biological world but also the dark color of those substances blocks sunlight that creates acute

problems in ecosystems (Choi et al., 2004).

A wide range of chemicals and dyestuffs, are required for dyeing and finishing and these are

generally organic compounds of complex structure. All of these compounds are not contained

in the final product but are found in waste and cause disposal problems (Savin and Butnaru,

2008). Major pollutants in textile wastewaters are high suspended solids (SS) (26200 mg/L),



chemical oxygen demand (COD) (12000-50000 mg/L), chromium (Cr) (50 mg/L), heat,

colour (4750 mg/L), acidity, and other soluble substances; (Bisschops and Spanjers 2003;

Venceslau et al., 1994; World Bank, 2007). In addition, only 47%-87% of dyestuffs are

biodegradable (Pagga and Brown, 1986). More than 100,000 dyes are available today (of

which azo dyes represent about 70% on weight basis), and over one million tons dyes are

produced per year, of which 50% are textile dyes (Boyter, 2007).

2.1.2) Tannery Wastewaters

Tanning is the chemical process through which animal hides and skin are converted into

leather and related products. The conversion of hides into leather is usually done by many

tanning agents and the process generates a highly turbid, colored and foul smelling

wastewater. The major polluting substances of tannery effluents include sulfide, chromium

(Cr), volatile organic compounds, large quantities of solid waste and suspended solids like

animal hair and trimmings (EC, 2003). The presence of these substances in the effluent affects

humans, agricultural fields and livestock. The toxins can cause diseases in tannery workers

such as eye diseases, skin irritations, kidney failure and gastrointestinal problems (Midha and

Dey, 2008). Also, certain streams from the production process of tannery industries contain

high salinity which is a concerning factor for the environment and can jeopardize the

biological wastewater treatment (Lefebvre and Moletta, 2006).

Untreated tannery wastewaters contain high COD, Biochemical Oxygen Demand (BOD)

levels, trivalent chromium (Cr(III)), sulfides, sodium chloride (NaCl), Calcium (Ca),

Magnesium (Mg), organics and other toxic ingredients and when these untreated wastewaters

are directly released into natural water bodies, they effect ecosystem flora and fauna and

increase the health risk to human beings (Kolomaznik et al., 2008; Chattopadhyay et al.,

1999). These wastewaters contain high loads of organic matter as well as high concentrations

of sulphate, sulphide, chloride and Cr which are very difficult to treat (Reemtsma and Jekel,

1997; Reemtsma et al., 2002). In the tanning process, about 300 kg chemicals are added per

ton of hides (Verheijen et al., 1996) and after processing, about 30-35 L of processed

wastewaters are generated per kg of skin with variable pH values and high concentrations of

SS, BOD, COD, including Cr (Nandy et al., 1999).



During the tanning process chromium salts are used and these generate two forms of Cr;

hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)). Of these two forms, Cr(VI)

is highly toxic to living organisms even at low concentrations causing carcinogenic effects. It

also creates toxicity in anaerobic digestion by accumulating metal in the intracellular fraction

of biomass. Environmental protection regulations have imposed strict limits for Cr emission.

Due to the high toxicity of Cr(VI), the U.S. Environmental Protection Agency (EPA) and the

European Union (EU) regulate total Cr concentrations in surface waters to below 0.05 mg/L,

including Cr(III), Cr(VI). Thus, removal of chromium from the wastewater is very important.

2.1.3) Acid mine

Acid Mine Drainage (AMD) occurs when sulfide-bearing material is exposed to oxygen and

water, and creates hydroxide, sulfate and hydrogen ions (Durkin & Hermann 1994). The

waste rocks which contain sulfides are the main source of AMD. Pyrite (FeS2) is responsible

for the generation of acid and metal dissolution in coal and hard rock (Wildeman et al. 1991).

Mining activities expose this mineral to water and cause increased acidity, concentrations of

heavy metals, sulfate, and other total dissolved solids. AMD is a major source of water

pollution in coal mining regions. It normally contains high levels of iron, aluminum, and acid

(Hadley and Snow, 1974). Acid mine waters also contain high concentrations of dissolved

heavy metals and sulphate with a high turbidity and low pH values (around 2) (Feng et al.,

2004). Arsenic and chromium are often abundant constituents of AMD where arsenic

concentrations range from 100-500 mg/L in highly acidic AMD and 70−80% of chromium is

present as Cr(VI) (Wang et al., 2013). Due to these characteristics (low pH and high

concentrations of heavy metals and other toxic elements), surface and groundwater as well as

soils can be severely contaminated by AMD (Peppas et al., 2000).

The amount of acid generated depends on environmental parameters such as pH, temperature,

oxygen concentration of the air (if saturation <than 100%), and water phase, degree of

saturation with water, chemical activity of Fe3+

, surface area of exposed metal sulfide,

chemical activation energy for acid generation, and bacterial activity. Apart from, these the

permeability of the waste rock dump is particularly important because dumps with high

permeability have high oxygen ingress which contributes to higher chemical reaction (Akcil

and Koldas, 2006).



Wastes from acid mine factories are often reddish-brown in color, which indicates high levels

of oxidized iron (Hadley and Snow, 1974). Acid generation and metal dissolution are the

primary problems associated with pollution due to mining activities. Debris from waste piles

can be transported by the wind which results in contamination of surrounding areas with

metals. Silt and sediments may run-off into nearby streams and become obstacles to water

flow. In underground mines, alkaline drainage is more common than in surface mines and it is

not as environmentally damaging as AMD (Akcil and Koldas, 2006). One feature of AMD is

that after the closure of a mine its sources may remain active for decades or even centuries

(Modis et al., 1998).

2.1.4) Landfill leachate

With the worldwide increase of population urbanization, the disposal of solid waste has

become a major issue. Despite significant progress in reducing waste volume by reuse and

recycling, the bulk of municipal waste is still disposed of in landfill sites. The most

economical method of disposing of municipal solid waste is landfilling (Ustohalova et al.,

2006). Leachates are defined as the aqueous effluent generated as a result of rainwater

percolation through wastes, biochemical processes, and the dormant water content of wastes.

When exposed to the oxygen-free (anaerobic) conditions of a landfill, biodegradable wastes,

such as paper, card and waste food, are decomposed by bacteria and produce gas and soluble

chemicals. These soluble chemicals mix with liquids in the waste (e.g. rainwater) to form

landfill leachate (URL 1). Toxicity analyses carried out using different organisms have

confirmed that landfill leachates contain potentially toxic substances (Renou et al., 2008) and

therefore, it should undergo treatment before being discharged into receiving waters.

Landfill leachate contains large amounts of organic and inorganic compounds (Bodzek et al.,

2006). Usually this waste has high BOD (e.g. 9500 mg/L) values and even higher COD

(14000 mg/L) content (Kjeldsen et al., 2002). Major inorganic compounds of leachate are:

calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), ammonium (NH4+), iron (Fe),

manganese (Mn), chloride (Cl-), sulphates (SO4

-2) and bicarbonates (HCO3

-) (Kjeldsen et al.

2002).

Leachate flow rate is closely linked to precipitation, surface run-off and infiltration or

intrusion of groundwater percolating through the landfill (Renou et al., 2008). When the waste



is compacted at lower depth leachate production is greater because compaction reduces the

filtration rate (Lema et al., 1988). Many factors affect the quality of leachates such as age,

precipitation, seasonal weather variation, waste type and composition (depending on the

standard of living of the surrounding population). Moreover, the composition of landfill

leachates varies depending on the age of the landfill (Baig et al., 1999).

2.2) Agro-Industrial Wastewaters

Agro-industries are one of the major contributors to the world industrial wastewater pollution

problem. Wastewaters of agro-food industries contain proteins, sugars, oils and greases and

are predominantly loaded with organic wastes and are rich in organic contents. The disposal

of these effluents into the environment contributes to surface and groundwater contamination,

causing eutrophication, ecosystem imbalance and human health risks. Environmental

regulatory authorities of worldwide are imposing stricter criteria for the discharge of

wastewaters from industries. As regulations become stricter, obviously these wastewaters

should be treated efficiently.

2.2.1) Dairy wastewaters

The dairy industry generates large quantities of wastewater, as per liter of processed milk, 0.2

to 10 L of wastewater are produced (Vourch et al., 2008). Dairy wastewaters contain high

concentrations of organic matter (e.g. fat, milk, protein, lactose, lactic acid), minerals and

detergents. Typical dairy wastewater characteristics include 4000-59000 mg/L COD, 70-800

mg/L TSS, 100-1400 mg/L TN and 25-450mg/L TP (Vourch et al., 2008).

Dairy wastewaters are mainly generated by the milk processing units where the

pasteurization, homogenization of fluid milk are processed. Large amounts of wastewaters are

also created from the production of dairy products such as butter, cheese, milk powder etc.

Large volume of water are used in cleaning processing units, resulting in wastewater which

also contains detergent, sanitizers, base, salts and organic matter, depending upon sources

(Belyea et al., 1990). Differences in production widely change the volume and strength of the

wastewater. Thus, effluent characteristics rely on the production of wastewater volume per



unit (litres waste water/kg product), concentration (mg/L), and weight of waste generated per

unit of g waste/kg product (Carawan et al., 1979).

2.2.2) Animal farm

Animal farming is one of the main agriculture components worldwide and a major

environmental concern (Hunt and Poach, 2001). Animal farms as medium or small

enterprises, lack manpower, finance and technical competence in order to efficiently treat

their wastewater. Animal farm’s wastewater treatment problem is also reinforced by the fact

that these wastewaters have high organic matter and phosphorus loads (Babatunde and Zhao,

2010). Animal farm wastewaters are usually characterized by high concentrations of organic

matter (COD: 38000-85000 mg/L), nitrogen (TKN 1400-3300 mg/L; NH4+-N: 500-1300

mg/L) and TSS (28500-61000 16 mg/L) (Kim and Kwon, 2006).

2.2.3) Olive mill wastewater

Olive mills are scattered all over the Mediterranean region and produce large volumes of olive

mill wastewater (OMW) over a relatively short period of time (usually from November to

February). Wastewater from olive processing is one of the strongest industrial effluents with

high organic loads (Paraskeva and Diamadopoulos, 2006). Annually 11 million tons of olives

are processed in olive mills producing about 9 million tons of OMW (Aktas, et al., 2001).

OMW is an aqueous, dark, bad-smelling and turbid suspension having a very high organic

load (COD=15–390 g L-1

), an acidic pH (4.6–5.8) and a significant content of phytotoxic and

antibacterial phenol substances (total phenols=1.5–14 g/L) (Bubba et al., 2004). The main

problem of this wastewater is that olive oil production is seasonal, so the treatment process

should be flexible enough to operate in a non-continuous mode, otherwise the wastewater

needs to stored. Moreover, most olive mills are small enterprises, mainly family businesses,

dispersed around the olive production areas, making individual on-site treatment options

costly. If the untreated effluent of olive mill wastes is deposited, it may cause, plant leaf and

fruit abscission and inhibition of seed germination. Discharge into water bodies increases

their phosphorous content, causes colour alteration and bad odour. Finally, the disposal of

olive mill wastewater in open spaces enhances microbial fermentation with the production of

methane and a wide array of harmful or simply bad-smelling gases.



2.2.4) Winery wastewater

Another agro-industrial waste is winery wastewater. Wineries, distilleries and other grape

processing industries annually generate large volumes of strongly organic wastewater

(Petruccioli et al., 2000). Winery wastewater is created from the various processes and

operations which are carried out in wine production as well as the water used to wash

equipment and bottles, and from cooling processes (Serrano et al., 2011). Large volumes of

polluted water are produced in winemaking and it varies from one winery to another,

depending on the production period and the unique style of winemaking of the different

wineries (Agustina et al., 2007).

Wastes from wineries, in addition to water, also include unspent grapes and juice, wine and

remnants from winemaking such as alcohol and sugars, and cleaning agents (Petruccioli et al.,

2002). Large suspended matter of winery wastewater contains grape skins, leaves, stems, and

seeds, while smaller suspended particles are mainly dead yeast cells and cell fragments (lees),

grit, dirt, diatomaceous earth, and bentonite (Vlyssides et al., 2005). Maximum organic

loading usually occurs from early September through early November (harvest season).

Different winemaking operations – such as cleaning of the crusher, pomace conveyors,

presses, and fermentation tanks may also produce high strength wastewaters (Conradie et al.,

2014). Winery wastewater is characterized with low pH (because of organic acids produced in

the fermentation process), with variable salinity and nutrient levels, and these characteristics

indicate that the wastewater has the potential components for environmental pollution (Mosse

et al., 2011). Winery wastewater also contains high concentrations of readily biodegradable

soluble organic matter and a variable content of suspended solids. COD concentrations vary

from 500 to 45,000 mg/L and TSS from 12 to 7300 mg/L, while BOD is about 0.4–0.9 of the

COD value (Shepherd et al., 2001; Petruccioli et al., 2002; Masi et al., 2002; Mosteo et al.,

2008). Nitrogen and phosphorus concentrations in winery wastewaters are usually low and the

pH values vary from 3.5 to 7. The main biodegradable fractions in winery wastewater are

ethanol, glucose, fructose and tartaric acid (Serrano et al., 2011).

2.3) Chromium (Occurrence and Uses)

Chromium (Cr) has an atomic number of 24 and a mass number is 51.9961 and belongs to the

first series of transition metals. Among the world’s most strategic and critical materials, Cr



has a wide range of uses in the metals and chemical industries. Cr alloys increase metal

endurance to impact, corrosion, and oxidation (Jacobs and Testa, 2004). Cr can exist in

various oxidation states which ranging from 0 to 6+. The speciation and other activities of Cr

compounds depend on the various chemical and biological changes that occur in the

environment. The solubility and adsorption of Cr in soil and sediments depend on the

different forms of Cr species (Sharma, 2002). Cr can exist in different oxidation states (Fig.

2.1), but depending on the ranges of redox potentials and pH that are commonly found in

soils, the Cr(III) and Cr(VI) states prevail (Ščančar and Milačič, 2014). The two common

stable oxidation states of Cr present in the environment, i.e. trivalent chromium, Cr(III) and

hexavalent chromium, Cr(VI), have different charges, physicochemical properties as well as

chemical and biochemical reactivity (Jacobs and Testa, 2004). Cr(III) is a trace element

essential for living organisms. Moreover, Cr(III) is responsible for the control of glucose and

lipid metabolism in mammals (Anderson, 1989).

While Cr(III) is an essential microelement, Cr(VI) is considered as a carcinogenic agent

(Flora et al., 1990; IARC, 1990) and the carcinogenicity of Cr(VI)-containing compounds has

been documented by governmental organizations (EPA, 1984) and international agencies

(IARC, 1990). The toxic and carcinogenic properties of Cr(VI) compounds appear due to the

possible free diffusion of chromate (CrO42−

) ions over living cell membranes and their action

as an oxidizing agent, as well as the formation of free radicals during the reduction of Cr(VI)

to Cr(III) inside the cell (O’Brein et al., 2003; Gad, 1989).

Figure 2.1: Chromium speciation in water (Ščančar and Milačič, 2014).

http://pubs.rsc.org/en/results?searchtext=Author%3AJanez%20%C5%A0%C4%8Dan%C4%8Dar


http://pubs.rsc.org/en/results?searchtext=Author%3ARadmila%20Mila%C4%8Di%C4%8D



Three thermodynamic stable forms of chromium, Cr(0), Cr(III) and Cr(VI), are used and

available in the environment. Cr(0) (Fig. 2.2) is exclusively found in metallic form and is used

in commonly iron-based alloy components such as stainless steel (Zhitkovich, 2011).

Figure 2.2: Pure chromium

Cr(VI) has strong oxidizing capacity and exists only as pH-dependent soluble oxygenated

species governed by the following equation (Nieboer and Jusys, 1988):

H2CrO4↔ H+

+ HCrO4− log (K a1) = 0.6………………… (2.1)

HCrO4−↔ H

+ + CrO4

2-

log (K a2) = -5.9...………….….. (2.2)

HCrO4− and CrO4

2− (Fig. 2.3) are the dominant species of Cr(VI) in natural systems because

environmental pH varies only between 3 and 10 (Jacobs and Testa, 2004). The species also

depend on the solubility of Cr(VI) in solution (EPA, 2006). Different compounds of Cr(VI)

are usually soluble in water because of its great mobility through soil and water bodies,

whereas Cr(III) is insoluble and therefore less mobile. Example of Cr compounds with

different oxidation states are listed in Table 2.1.



Figure 2.3: Chromium(VI) oxide

Table 2.1: Chromium compounds with different oxidation states (Zayed and Terry, 2003).

Chemical species Oxidation state Examples Remarks Elemental Cr

Cr(0)

Does not occur naturally

Divalent Cr

Cr(II)

CrBr2,CrCl2,CrF2, CrSe, Cr2Si Relatively unstable and is readily

oxidized to the trivalent state

Trivalent Cr

Cr(III)

CrB, CrB2

,CrBr3, CrCl.6H2O, CrCl3

, CrF3, CrN,

KCr(SO4)2.12H2O

Forms stable compounds and occurs

in nature in ores, such as

ferrochromite (FeCr2O4)

Tetravalent Cr Cr(IV)

Cr dioxide CrO2, Cr tetrafluoride

CrF4

Does not occur naturally and

represents an important intermediate

that influences the rate of reduction

of the Cr(V) form.

Chromium (IV) compounds are less

common.

The Cr(IV) ion and its compounds

are not very stable and because of

short half-lives, defy detection as

reaction intermediates between

Cr(VI) and Cr(III).

Pentavalent Cr Cr(V)

Tetraper–oxochromate

CrO43−, potassium perchromate

Does not occur naturally and

represents an important intermediate

that influences the rate of reduction

of the Cr(VI) form.

Chromium (V) species are derived

from the anion CrO43− and are long

lived enough to be observed directly.

However, there are relatively few

stable compounds containing Cr(V).

Hexavalent Cr Cr(VI) (NH4)2CrO4 ,BaCrO4, CaCrO4

,K2CrO4, K2Cr2O7

The second most stable state of Cr.

However, Cr(VI) rarely occurs

naturally, but is produced from

anthropogenic sources. It occurs

naturally in the rare mineral crocoite

(PbCrO4).



Chromium is used in many human activities (e.g. chemical industry, production of dyes,

wood preservation, leather tanning, chrome plating, manufacturing of various alloys, and

many other applications and products) (Fig. 2.4) and causes environmental pollution.

Stainless steel is the product containing the highest Cr content (up to 20% Cr by weight)

(Zhitkovich, 2011). Cast irons may contain from 0.5% to 30% of Cr, which adds strength,

toughness and corrosion and wear resistance. Cr is also used in nonferrous alloys (nickel,

iron-nickel, cobalt, aluminum, titanium and copper). The pigmentations in paints and inks

contain large amounts of Cr. Other usages of Cr include leather tanning, metal corrosion

inhibition, drilling muds, textile dyes, catalysts, and wood water treatment. In the refractory

industry chromite is used to make bricks, mortar, and ramming and gunning mixes, which

enhances their thermal shock and slag resistance, volume stability, and strength (Bielicka et

al., 2005). Therefore, the use of Cr compounds in these industries has resulted in increased

amount of Cr in the environment (Gode and Pehlivan, 2007). These industries, release great

volumes of effluents which contain Cr ranging from 0.5 to 270,000 mg/L (Patterson, 1985).

Figure 2.4: Use of Chromium and its compounds (Bielicka et al., 2005).

2.3.1) Environmental chemistry of Cr

Chromium is the earth’s seventh most abundant element, 21st most abundant metal in the

earth’s crust and is mined as chromite (FeCr2O4) (Cervantes and Campos-Garcia, 2007,

Barnhart, 1997). Due to its acute colored compounds, it was named according to Greek word

“Chroma” (χρώμα) which means color. It is a silver coloured hard metal with a melting point

Metal corrosion

inhibitors

Catalyst

s

Inks

Medicine

s

Textile dyes

Pigments

Chemical

s Paints

In wood treatment In leather tanning

Ferrous and nonferrous alloys

Chromium

and its

compounds



of 19070C. It was discovered by Louis Nicolas Vauquelin in 1797 when he produced

chromium oxide (CrO3) was produced by mixing mineral crocoite (lead chromate) with

hydrochloric acid. In 1798 metallic Cr was isolated by heating is oxide in a charcoal oven

(Vauquelin, 1798). Chromium normally is solid at room temperature, but at high temperatures

of 4000C with pressures of 200 to 300 atm, it reacts with halogen gases such as fluorine. Cr

also reacts with the other halogen gases such as chlorine, bromine, and iodine, to form a

variety of brightly colored compounds. It also dissolves in dilute hydrochloric acid and

sulfuric acid (Jacobs and Testa, 2004).

Chromium is widely diffused in nature in rocks, fresh water and seawater (Krishnamurthy and

Wilkens, 1994). The recommended guidelines for Cr(VI) in freshwater are 1 µg/L and for

Cr(III) 8 µg/L. For marine life the recommended guidelines are 1 µg/L for Cr(VI) and 50

µg/L for Cr(III), for irrigation water 8 µg/L for Cr(VI) and 5 µg/L for Cr(III) and for drinking

water is 50 µg/L for Cr(VI) (Krishnamurthy and Wilkens, 1994; Pawlisz et al., 1997).

The atmosphere assists long-range transfer of Cr to various ecosystems. Cr-containing

particles are carried by the wind for different distances before they fall, or one washed out

from the air and are deposited onto terrestrial and water surfaces. How much area is covered

by the metal depends on the meteorological factors, topography and vegetation. Wet

precipitation and dry fallout of Cr from the atmosphere also depend on the particle sizes.

Conversely, Cr transportation within the terrestrial and water systems depends highly on the

chemical forms of Cr and their affinity to chemical transformation, precipitation and

absorption/adsorption processes occurring in the environment. Cr speciation forms occurring

in soil, plants and water are shown in Fig. 2.5.



Figure 2.5: Chromium circulation in the environment (Bielicka et al., 2005).

2.3.2) Physical and chemical characteristics

Chromium is extracted from chromite ore [(Fe,Mg)O(Cr, Al, Fe)2O3] and both stable species;

Cr(III) and Cr(VI) are strongly absorbed to iron hydroxide solids forming particulates of

Cr(III) and Cr(VI) in water (Matthews and Morning, 1980; Dzombak and Morel, 1990) (Fig.

2.6).

Figure 2.6: Possible forms of Cr(VI) in solution in the presence of iron particles (Parks 2005)

Soil Ocean & Sea

Cr(III) Cr(VI)

Atmosphere Cr(III), Cr(VI)

Industry

Soil

Cr(III) Cr(VI)

Plants

Cr(III) Cr(VI)

People and Animals

Cr(III) Cr(VI)

OH

O-

Cr(VI) + OH -

(Sorbed) Cr(VI) (Soluble)

OH2+

Fe(OH)3

Cr(VI)

(Fixed)



Parks (2005) described the three fractions of Cr that are in association with iron hydroxide as

follows:

(1)"Soluble"; this form of Cr can pass through a 0.45 μm pore size filter,

(2)"Sorbed"; this fraction is chemically sorbed to the iron hydroxide, but that might be

rescued either in acidic or basic solution without complete dissolution of the iron hydroxide

solid,

(3) "Fixed" this fraction is associated with iron hydroxide and not released until the solid is

completely dissolved.

These fractions can occur for Cr(III) and/or Cr(VI), but Cr(VI) is more likely to remain

soluble (Parks 2005).

Cr(III) is a stable oxidation state and reacts slowly to form complexes because of its low

kinetic potential energy, but it is not a strong oxidizer (Jacobs and Testa, 2004). Although

CrO42−

is relatively stable, its high positive reduction potentiality makes it a strong oxidizing

agent. Therefore, it remains unstable in acid solutions in the presence of electron donors such

as Fe2+,

H3AsO3, and HSO3−, or organic molecules with oxidizable groups (alkanes, alkenes,

alcohols, aldehydes, ketones, carboxylic acids, mercaptans, etc.) (Wiberg, 1965; Beattie and

Haight, 1972).

The pH and oxidation-reduction (redox) potential are the two measurable parameters which

determine Cr speciation forms and their mobility. Cr(VI) generally exists in water in the form

of monomeric state (HCrO4- and CrO4

-2) or bimeric state (Cr2O7

-2). Monomeric species

deliver a yellow color to water as Cr2O7-2

is orange in color. The relative concentrations of

these species are depend on both pH and concentration of Cr(VI) (Jacobs and Testa, 2004).

Fig. 2.7 presents an example of the relation to pH and Cr(VI) concentration. Cr(VI) is a strong

oxidant and is reduced in the presence of electron donors which are generally found in a

reduced subsurface environment where ferrous iron, reduced sulfur, and some organic

materials occur. Dichromate can react with soil organic carbon to produce water, Cr(III), and

CO2, with Cr(III) likely to precipitate as a hydroxide (Eq. 2.3) (Palmer and Puls, 1994).

2 Cr2O7-2

+ 3C0 + 16H

+ -- 4Cr

3+ + 3C02 + 8H2O……………………. (2.3)



(a)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

Frac

tion

of C

r(V

I)T

otal

pH

(b)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Frac

tion

of C

r(V

I)T

otal

Cr(VI) Total(M)

10-4

10-3

10-2

10-1

100

101

102

103

104

Figure 2.7: Distribution of Cr(VI) species (a) as a function of pH and (b) Fraction of HCrO4-

and Cr2O7-2

at pH 4 as a function of total Cr(VI) (Hsu, 2011).

2.3.3) Nutrition and toxicity of Cr: health risks to humans

Chromium is a highly pollutant metal that can cause genetic mutations and cancer. As is

already stated, the toxicity of Cr depends highly on its existing form, and Cr(VI) is more toxic

than Cr(III) for plants, animals and microorganisms (Kimbrough et al., 1999).

The reduction of Cr(VI) is considered as a detoxification process when it occurs at a distance

from the target site, for toxic or genotoxic effect. However, when the reduction of Cr(VI)

occurs near the cell nucleus of target organs, it may activate chromium toxicity (Dayan and

Paine, 2001). If Cr(VI) is reduced to Cr(III) extracellularly, it is not readily transported into

H2CrO4

HCrO4-

CrO42-

Cr2O72-



cells and no toxicity is observed. The balance which exists between extracellular Cr(VI) and

intracellular Cr(III), determines the ultimate amounts and rates at which Cr(VI) can enter cells

and impart its toxic effects (Cohen et al., 1993).

High Cr(VI) concentrations have significant harmful effects on human health such as lung

cancer, as well as kidney, liver, neurological and gastric damage (Kimbrough et al., 1999,

Nethercott et al., 1994; Wang et al., 1997; EPA 1998). The respiratory tract is the primary

organ which is affected by inhalation of Cr following acute exposure. Dermal exposure to

Cr(VI) can lead to dermal ulcers owing to the corrosive nature of Cr(VI) compounds (EPA,

2007). On the contrary, Cr(III) is less toxic, less harmful, and even an essential nutrient for

humans (Anderson, 1997; Katz and Salam, 1994). However excess quantities of Cr(III) also

cause damage to aquatic organisms, disrupting the food chain (Bosnic et al., 2000).

Considering the toxicity effects of Cr, the permitted discharge limit of Cr into surface and

potable waters has been set to below 0.05 mg/L by the EPA (Baral and Engelken, 2002) and

the EU (EC, 1998).

2.3.3.1) Toxicity of Cr to animals

Cr(III) is found in animal tissues (including liver, kidney and muscle) as Cr(III) may increase

insulin. Other biological effects are reported less frequency (e.g. on immune response and

lipid metabolism) (EFSA, 2009). Sometimes a trace amount of Cr is essential for human and

animal nutrition (Jeejeebhoy et al., 1977; Mertz, 1969) and it is an integral component of the

glucose tolerance factor (GTF) which is required to maintain normal glucose tolerance

(Mertz, 1969). For fat metabolism in animals Cr is considered as an important substance

(Anderson, 1989). The supplementation of Cr(III) in farm animals can decrease serum cortisol

levels in several species (Lindemann, 2007). Cr(III) might be the (or one of the) extreme

intracellular toxic form(s) of Cr(VI) (Stearns, 2000). Nguyen et al., (2008) reported that

oxidation from Cr(III) to Cr(VI) may occur in biological systems under certain circumstances.

For a long time Cr(VI) has been known as the most dangerous form of the metal because of

its high ability to enter cell membranes and its powerful oxidising properties (EFSA, 2009).

After entering the cells, Cr(VI) can be readily reduced by glutathione (GSH), cysteine and

other cellular reductants, as well as cytochrome b5 (Plant, 2003).



The aquatic environment is considered as a major sink for metal pollutants. Generally surface

water is polluted after discharging of metal containing wastewaters from manufacturing

processes of different industries (Elwood, 1980). However, in water, Cr compounds are very

stable and accumulate in sediments and that become a source for Cr accumulation in aquatic

life. Cr toxicity to aquatic life depends on both biotic and abiotic factors. The biotic factors

include the genus, age and developmental stage, while the abiotic factors include temperature,

Cr concentration, Cr oxidation states, pH, alkalinity, salinity, and hardness of the water

(Velma et al., 2009). Acute toxicity of Cr(VI) for both marine and fresh water organisms

appears to range between 1 and 330 mg/L but actual values depend on species, salinity, pH,

alkalinity, and temperature of water (SCCWRP, 1974). The toxicity effects of Cr(VI) to

aquatic life including marine and freshwater fish have been recorded by various researchers

(Mishra and Mohanty, 2008; Velma et al., 2009; Svecevicius, 2006). The growth and survival

of fish in Cr-containing water depend on the dose and exposure time to Cr (Patton et al.,

2007; Farag et al., 2006).

2.3.3.2) Toxicity of Cr to plants and algae

It was reported by Pratt (1966), that plant growth might be stimulated at low concentrations of

chromium but it is not an essential element for plants (Huffman and Allaway, 1973). Yet the

solubility of Cr(VI) in water is a threat for plants (Nieboer and Richardson, 1980). Although

Cr has little influence on growth of certain plant species at lower concentrations (Shanker et

al., 2009), it is highly toxic at higher concentrations and inhibits various activities in plants

and sometimes causes total damage (Shanker et al., 2005, 2009; Vajpayee et al., 1999; Dube

et al., 2003). Toxicity symptoms of Cr(III) and Cr(VI) in plants include inhibition of

germination, root growth, seedling growth and development, induction of leaf chlorosis and

necrosis besides physiological and biochemical alterations (Oliveira, 2012; Shankar et al.,

2005). Studies have reported that Cr has toxic effects in different species, including cereals,

pulses, vegetables, forages and trees (Haas and Brusca, 1961; Shanker et al., 2004, 2005;

Ganesh et al., 2006; Jun et al., 2009; Shanker et al., 2009). Both terrestrial plants and the

aquatic plants have found to be affected by Cr(VI); however, great variation is observed in

their response (Chandra and Kulshreshtha, 2004). When soils contain 5 to 60 mg/kg

concentration of Cr(VI), plant growth is inhibited due to root damage (Anon, 1974). Cr(VI)

also causes toxicity in algae by affecting growth, photosynthesis, morphology, and enzyme



activities (Gorbi and Corradi, 1993). The amount of Cr(VI) that causes toxicity in algae varies

within the range of 20 to 10,000 mg/L (Sharma, 2002).

2.3.4) Remediation or treatment

Metal compounds are not biodegradable and therefore can be regarded as long-term

environmental features. Since they can also have accumulative properties, they are the subject

of close attention.

Various treatment technologies have been developed for the removal of heavy metals

including Cr(VI) from water and wastewater. Several physicochemical methods are used to

treat wastewater to remove heavy metals, including ion exchange (Pansini et al., 1991,

Rengaraj et al., 2001), activated carbon (Perez-Candela M. et al., 1995; Mohan and Pittman,

2006; Chingombe et al., 2005; Owlad et al., 2009), chemical precipitation (Kongsricharoern

and Polprasert, 1996; Ramakrishnaiah and Prathima, 2012), adsorption (Hashem et al., 2007;

Ravikumar et al., 2005), reverse osmosis (Pérez Padilla and Tavani, 1999) and membrane

technologies (Pugazhenthi et al., 2005; Muthukrishnan and Guha, 2008; Aroua et al., 2007).

In some cases these physicchemical processes are extremely expensive especially when the

metal concentrations in the solution range from 1 to 100 mg/L (Nourbakhsh et al., 1994).

Furthermore these methods usually produce large quantities of toxic chemical sludge of which

disposal is a major problem (Benjamin, 1983; Mandi et al., 1996). On the other hand, Cr(VI)

interacts with microorganisms through enzymatic reduction, biosorption and bioaccumulation

(Tekerlekopoulou et al., 2013). Cr(VI) is mainly accumulated by bacteria, as it uses the

sulfate pathway (Vaiopoulou and Gikas, 2012). Many researchers have examined biological

Cr(VI) removal using bacteria (Stasinakis et al., 2002; Zouboulis et al., 2004), fungi (Sanghi

et al., 2009), yeast (Chen and Wang, 2007), and algae (Bankar et al., 2009). Biological

treatment of Cr(VI) is gaining ground due to its zero requirement of chemicals and its low

operating cost (Srivastava and Majumder, 2008).

2.4) Cheese Manufacturing

Cheese production is one of the most classical examples of food preservation which started in

c. 6000-7000 B.C. (Scott, 1986). Throughout the world cheeses are common in all cultures

and one of the oldest food items. In ancient times, milk was a seasonal food and cheese-



making was a method of milk conservation process to avoid the waste of surplus production.

Bedouins carried milk in bags on their trips into the desert. The heat of the desert caused

acidification and coagulation of this milk with acid-producing bacteria, which undoubtedly

led to curdling and the subsequent motion in the bags produced curds which resulted in an

acidic liquid (whey) forming on top of the milk as curd sediment (Wit, 2001).

Cheese is a nutritious foods made by processing milk from cows, goats, sheep, buffalos,

camels and yaks. Cheese is produced from processed milk by coagulation of the milk protein

known as casein (Scott, 1986) and acidification (Fox et al., 2000). The common steps

followed in all cheese manufacturing include milk acidification, milk coagulation, whey

removal, packaging and storage. There are many types of cheese which depend on different

processing methods (Fox, 1999). A flowchart of cheese manufacturing and the waste flows

from each step is presented in Fig. 2.8.

Figure 2.8: Flow sheet for the manufacture of cheese (Wit, 2001).

Raw Milk

Cream

Starter

Rennet

Hot

Water

Pasteurization

Whey

Cream

Separation

Whey

Draining Curd

Brining

Resting

Pressing

Moulding

Ripening

Cheese

Whey Standardization

Mixing

Pasteurization

Setting

Draining off

(1/3 of whey)

Cutting

Stirring

Scalding



At first, starter cultures are added to the milk to produce lactic acid. Then rennet is used to

coagulate the milk protein. Standardization and pasteurization takes place by using a separator

and pasteurizer in line. The curds and whey are separated and the curds are washed out and

cut into cubes. The cubes then precipitate as a curd, leaving whey as the supernatant liquid.

One third of this whey is subsequently drained out and the same amount of hot water (± 40°C)

is added as replacement. In this process (scalding process) the curd particles shrink and as a

consequence whey is squeezed during stirring. After stirring, the curd is pressed together to

separate it from the remaining whey. Then the curd blocks are milled, salt is added, and the

curds are pressed. Pressed cheese is wrapped to protect it from moisture loss and growth of

mould during storage period. Cheese is maturated in temperature and humidity controlled

stores to develop flavor and texture through regular turning, and salting or brine washing of

the cheese surface (EPA, 2000a).

2.4.1) Cheese whey

After the production of cheese or the removal of fat and casein (80% of the proteins) from

milk, the remaining green-yellowish liquid is known as whey (Smithers, 2008). It is estimated

that the worldwide production of whey is over 100 billion (100 thousand million) kilograms

per year (Smithers, 2008; OECD-FAO 2012). Approximately half of this total whey

production is produced in the European Union (EU) (EC, 2009). During the production of

casein or fresh-cultured cheese, only 8% of the total produced whey is produced directly as a

by-product from skimmed milk. Whey represents 85–95% of the milk volume and contains

about 55% of the milk nutrients comprising milk sugar (lactose), serum proteins (whey

proteins), minerals, a small amount of fat, and most of the water soluble minor nutrients from

milk such as vitamins (Farizoglu et al., 2004; Wit, 2001). The liquid effluents of dairy

industries and cheese dairies are major contributors to the worldwide industrial pollution

problem (Papachristou and Lafazanis, 1997).

Normally two types of cheese whey are produced through the processing techniques and these

are sweet whey and acid whey according to pH value. Sweet whey is produced at

approximately pH 6.5 when rennet is introduced at coagulation time of casein. The acid whey

production procedure depends on fermentation or addition of organic or mineral acids to

coagulate the casein at pH <5 (Panesar et al., 2007).



2.4.2) Sources

Approximately 1 kg of cheese and 9 kg of sweet whey are obtained from 10 litres of milk

(Fig. 2.9). To produce different types of cheese for example, mozzarella, semi-hard and hard

cheeses, two different starter cultures are used (mesophilic and thermophilic) and

approximately 3-13 L of whey per kg cheese production is drained out depending on the

cheese production (Table 2.2). When the cheese whey is drained is either refined, or

processed into different products, or disposed of. Small cheese particles which are present in

the whey have to be removed by filtering.

Figure 2.9: Simplified cheese production inputs and outputs.

Table 2.2: Amount of whey drained from different type of cheese productions (Atamer et al.,

2013).

Hard cheese Semi-hard

cheese

Soft cheese Sour milk

cheese

Fresh cheese

Milk (L) (for 1

kg Cheese)

13–14 12–13 8–9 5–6 4–5

Drained whey

(L)

12–13 11–12 7–8 4–5 3–4

Drained whey

(%)

92–93 91–92 86–89 80–83 75–80

2.4.3) Characteristics and composition of cheese whey

Cheese whey is a liquid with a total solid content of about 6,5% depending on the cheese type

and the amount of water added during production. About 85-95% of the milk volume is

cheese whey and about 55% of the milk nutrients remain in this potential wastewater. It’s a

Whey

8.5 Kg

Cream

0.5 Kg

Milk

10 Kg

Cheese

1 Kg



nutritious liquid, containing proteins, lactose, vitamins and minerals, but also enzymes,

hormones and growth factors (Wit, 2001). Around 39000-60000 mg/L of lactose is contained

in cheese whey and this forms the main fraction of organic loads (Ghaly and Kamal, 2004).

Aside from lactose, it also contains fats (0.99-10.58 kg/m3, 990-10580 mg/L), soluble protein

(1.4-8 kg/m3, 1400-8000mg/l), lipids (4-5 kg/m, 4000-5000 mg/l) and mineral salts (8-10% of

the dry extract) (Prazere et al., 2012; Chatzipaschali and Stamatis, 2012). The main

components of the whey protein are true proteins, peptides and non-protein (NPN)

components. Whey proteins are determined as the components which are soluble at pH 4.6 in

their native form (Fox, 2003). The main composition of fresh whey is presented in Table 2.3.

Characteristics of cheese whey also depend on the quality and type of the milk (cow, goat,

sheep and buffalo) used for the cheese making (Wit, 2001) and on the evaluated milk portion

and other parameters such as mechanisms of cheese making, the acid used for coagulation,

time and temperature of coagulation (Chatzipaschali and Stamatis, 2012).

Table 2.3: General composition of fresh whey.

S.No Constituent Unit Sweet whey Acid whey

1 Water % 93-94 94-95

2 Dry matter % 6-6.5 5-6

3 Lactose % 4.5-5 3.8-4.3

4 Lactic acid % traces up to 0.8

5 Total protein % 0.8-1.0 0.8-1.0

6 Whey protein % 0.6-0.65 0.6-0.65

7 Citric acid % 0.1 0.1

8 Minerals % 0.5-0.7 0.5-0.7

9 pH 6.2-6.4 4.6-5.0

10 SH Value about 4 20-25

(Source: http://www.dairyforall.com/whey.php)

2.4.4) Cheese whey – pollutant characteristics

Cheese whey is a protein and lactose rich by-product which is very biodegradable (~99%)

with high organic content (~ 70 g COD/L) and low alkalinity (Mawson, 1994). Cheese-whey

production is increasing annually worldwide and new bio-productions are seeking through

biotechnology to fully exploit the whey produced (Siso, 1996). Every year 160 million tons of

cheese whey is produced worldwide. However, approximately half this amount is not treated

and is discharged directly into the environment (Smithers, 2008; Guimaraes et al., 2010;

http://www.dairyforall.com/whey.php



OECD-FAO, 2012). The disposal of cheese whey during the production of cheese has always

been a main problem in the dairy industry. When this potential wastewater is discharged

directly into the environment, this causes a significant loss of resources and serious

environmental pollution. Thus, cheese whey represents an important environmental problem

because of the high volumes produced and its high organic matter content (Prazere et al.,

2012; Chatzipaschali and Stamatis, 2012; Mawson, 1994).

Whey characterized by its high organic content, i.e. high concentration of BOD and COD of

between 27000-60000 mg/L and 50000-102000 mg/L, respectively (Prazere et al., 2012).

Lactose represents the majority of the organic load (over 90% of BOD and 97.7% of the total

COD) (Chatzipaschali and Stamatis, 2012). If protein is extracted from the whey, COD is

reduced to 10000 mg/L. Thus, cheese whey is a complex substrate that is ultimately a mixture

of easily biodegradable carbohydrates, mostly lactose, proteins and lipids (Dermirel et al.,

2005).

Its direct disposal can affect the physical and chemical structure of soils, for which crop yields

decrease. In addition, when cheese whey is discharged into water resources, it affects aquatic

life by causing eutrophication of the receiving waters (Siso, 1996, Panesar et al., 2007). The

biological treatment of cheese whey depends on its organic load. If the ratio of BOD and

COD in whey is higher than 0.5 then it is suitable to treat by biological processes. The

temperature and reaction of the wastewater also determine the efficiency of biodegradation.

2.4.5) Remediation or treatment

Dairy wastewaters including cheese whey are generally treated using various physicochemical

methods including coagulation/flocculation by various inorganic and organic natural

coagulants, and membrane processes like nanofiltration (NF) and/or reverse osmosis (RO)

(Kushwaha et al., 2011). Various biological methods are also used to treat cheese whey

including activated sludge processes, aerated lagoons, trickling filters, sequencing batch

reactors (SBR), anaerobic sludge blanket reactors (UASB), anaerobic filters, etc. (Demirel et

al., 2005; Prazeres et al., 2012; Carvalho et al., 2013; Tatoulis et al., 2014b).

Treatment of Industrial and Agro-Industrial Wastewater using Constructed Wetlands Mar-Yam Sultana

Chapter 3: Constructed Wetlands

CHAPTER 3: CONSTRUCTED WETLANDS

3.1) Components of Constructed Wetlands ......................................................................... 37

3.1.1) Water .......................................................................................................................... 37

3.1.2) Soil substrate/porous media ....................................................................................... 37

3.1.3) Wetland vegetation/macrophytes ............................................................................... 39

3.1.3.1) Role of macrophytes ............................................................................................ 40

i) Physical appearance .............................................................................................. 40

ii) Surface area for attached microbial growth .......................................................... 41

iii) Root release/creation of aerobic environment ....................................................... 42

iv) Organic compound release .................................................................................... 43

v) Nutrient uptake and storage ................................................................................... 44

3.1.4) Microorganisms ........................................................................................................ 44

3.2) Types of Constructed Wetlands ..................................................................................... 45

3.2.1) Surface Flow/ Free Water Surface Flow .................................................................... 46

3.2.2) Horizontal Sub-Surface Flow ..................................................................................... 48

3.2.3) Vertical flow ............................................................................................................... 50

3.3) Design Factors ................................................................................................................. 52

3.3.1) Pretreatment ............................................................................................................... 52

3.3.2) Hydrology................................................................................................................... 52

3.3.2.1) Hydraulic Loading Rate (HLR) ........................................................................... 53

3.3.2.2) Hydraulic Retention Time (HRT) ........................................................................ 54

3.3.3) Design of Bed/Bed Area ............................................................................................ 55

3.3.4) Selection of wetland plants ........................................................................................ 56

3.3.5) Inlet structure .............................................................................................................. 59

3.3.6) Outlet Structure .......................................................................................................... 59

3.4) Removal Mechanism of Constructed Wetlands ........................................................... 59

3.4.1) Organic matter removal .............................................................................................. 60

3.4.2) Nitrogen removal ........................................................................................................ 62

3.4.3) Phosphorus removal ................................................................................................... 64

3.4.4) Total suspended solids removal ................................................................................. 65

3.4.5) Heavy metal (Cr) removal ......................................................................................... 65

3.5) Application of CWs in Cr(VI) treatment ...................................................................... 66

3.6) Application of CWs in Agro-Industrial wastewaters Treatment ............................... 74


Chapter 3: Constructed Wetlands

3.6.1) Pre-treatment stages in treatment of Dairy Wastewaters ........................................... 74

3.6.2) CW types in treatment of Dairy Wastewaters ............................................................ 74

3.6.3) Vegetation for Dairy Wastewater treatment in CW systems ..................................... 78

3.6.4) Pre-treatment stages in treatment of Animal Farm Wastewater ................................ 79

3.6.5) CW types in treatment of Animal Farm Wastewater ................................................. 79

3.6.6) Vegetation for Animal Farm Wastewater in CW systems ......................................... 82

3.6.7) Pre-treatment stages in treatment of OMW wastewater............................................. 83

3.6.8) CW types in treatment of OMW wastewater ............................................................. 83


Chapter 3: Constructed Wetlands 35

CHAPTER 3: CONSTRUCTED WETLANDS

Constructed wetlands (CWs) are a low–cost technology which has been used to treat various

types of wastewaters for more than thirty years (Vymazal, 2010; Hunt et al., 2003; Kadlec and

Wallace, 2009). Compared to conventional systems, CWs appear to be rather attractive as

they use solar energy and are easily operated (Jamieson et al., 2007). Their treatment

processes can purify many wastewaters (Hunt et al., 2006). Modern CWs have been designed

to emphasize specific characteristics of wetland ecosystems for improved treatment capacity,

using various physical, chemical and biological processes for pollutant removal (Fig. 3.1).

CW removal performance depends upon the wetland design, microbial community, and the

different kinds of plants involved (Ibekwe et al., 2003). Usually the main role in pollutant

removal is attributed to the microorganisms which are attached either onto the roots of

wetland plants or on the soil substrate where they catalyze chemical changes and conduct

desirable modifications of nutrients, metallic ions and other substances. The region

surrounding each root is aerobic and called the rhizosphere. The main pollutant removal

mechanisms in CWs are three: physical, chemical and biological processes, including

filtration, sedimentation, adsorption, volatilization and bioaccumulation by plants (plant

uptake) or microorganisms. The rhizosphere is the most efficient reaction zone in CWs where

all the processes are taken place by the interaction of plants, microorganisms and pollutants

(Fig. 3.2) (DeBusk, 1999; Vymazal et al., 2006; Stottmeister et al., 2003). The treatment

process of CWs is based on the biological processes occurring in the natural ecological

system, which unite the saturated substrates and vegetation. A population of microbes

develops within the root system and the substrate, and decomposes the various pollutants to

yield stable compounds.

Kadlec and Knight (1996) describe the historical background of the use of natural and CWs

for wastewater treatment and disposal. Research studies on the use of CWs for wastewater

treatment started in Europe in the 1950s and in the United States of America (USA) in the late

1960s. Research efforts were increased in the USA throughout the 1970s and 1980s, with

significant federal involvement by the Tennessee Valley Authority (TVA) and the US

Department of Agriculture in the late 1980s and early 1990s.



Figure 3.1: Schematic diagram of a constructed wetland and its mechanisms.

Figure 3.2: Possible interactions in the root zone of wetlands for wastewater treatment

(Stottmeister et al. 2003).

CWs have the potential to treat different types of wastewaters including municipal wastewater

(Vymazal, 2005), acid mine drainage (Mays and Edwards, 2001; Machemer et al., 1993;

Yang et al., 2006), industrial wastewater (Di Luka et al., 2011; Khan et al., 2009; Maine et al.,

2006; Kongroy et al., 2012), agricultural wastewater (Bubba et al., 2004; Grafias et al., 2010;

Nahlik and Mitsch, 2006), tannery wastewater (Calheiros et al., 2007, 2008a,b, 2010, 2012;

Kucuk et al., 2003; Dotro et al., 2011b). These wastewaters usually contain high

concentrations of organic matter, nutrients and heavy metals and CWs are very cost effective



compared to conventional methods of wastewater treatment (Kadlec and Knight, 1996; Knox

et al., 2006).

3.1) Components of Constructed Wetlands

CWs are usually comprised of water, soil substrate/porous media and vegetation (EPA, 1998).

However, endemic microorganisms also play a vital role in the degradation of contaminants in

the wetland. Normally water is present on the surface of the wetland or within the roots of the

vegetation for a long period of time.

3.1.1) Water

Water is the most important component of CWs because it links all of the functions in a

wetland and the treatment efficiency of a CW depends on its characteristics (EPA, 1998).

Slight changes of hydrology (surface area and water depth) greatly influence the treatment

efficiency of the wetland because wetland sharply interacts with the atmosphere through

rainfall and evapotranspiration (the combined loss of water through evaporation from the

water surface and loss through transpiration by plants). Density of the plants growing in CWs

affects the water flow path through the wetland by creating obstacles. Water can find its

sinuous way through the plant parts; stems, leaves, roots, and rhizomes in the wetland (EPA,

2000b). When the substrates are flooded, the physical and chemical characteristics are altered.

In a saturated substrate, water replaces the atmospheric gases in the pore spaces and microbial

metabolism consumes the available oxygen. Since oxygen is consumed more rapidly,

substrates become anoxic (without oxygen). This reducing environment is important in the

removal of pollutants such as nitrogen and metals.

3.1.2) Soil substrate/porous media

The soil substrate or the porous media is an essential component of any CWs. It supports the

growth of emergent plants and microbes on their surfaces and regulates hydraulic

conductivity and nutrient adsorption (Kadlec and Knight, 1996; Calheiros et al., 2008a). The

biological and chemical transformations of the pollutants take place in the soil

substrate/porous media with the help of microorganisms and plants (EPA, 1995). Therefore,

the CW acts as primary sink for pollutants (Vymazal, 2003; Lesage et al., 2007b; Allende et

al., 2012; Vymazal and Krasa, 2003). Sometimes the permeability of different substrates



affects the movement of water in the wetland (EPA, 1998). Various types of substrates are

used in CWs such as gravel (fine and course), sand and soil (Fig. 3.3) (EPA, 1995; Korkusuz,

2005). However, mixtures of sand and gravel may improve hydraulic conditions and the

removal of contaminants (IWA specialist group, 2000; Stottmeister et al., 2003). In vertical

flow constructed wetlands (VF CWs), a grain size of 0.06 to 0.1 mm was found to be the most

effective, whereas, that for horizontal-flow systems the most effective grain size was 0.1 mm

(Stottmeister et al., 2003). In subsurface CWs the typical effective sizes of the media vary

between 2 and 128 mm and the porosity varies from 28% to 45% (EPA 2000b). The accurate

or proper use of substrate in CWs increases pollutant removal efficiency (Calheiros et al.,

2008a).

Figure 3.3: Different types of CWs substrates for (A) gravel, (B) course gravels, (C) fine

gravels, (D) sands, (E) soil.

A B

C D

E



3.1.3) Wetland vegetation/macrophytes

Plants are important components of CWs because of their significant roles in the wastewater

treatment processes (Brix, 2003). In relation to wastewater treatment, the most significant

functions of wetland plants are the physical effects associated with the presence of dense

vegetation and the increased surface area for attachment and growth of microbes (Sim, 2003).

Vascular plants (the higher plants) and non-vascular plants (algae) that have tissues are

normally used in CWs (Brix, 2003). Like all other photoautotrophic organisms, these

macrophytes use the solar energy to assimilate inorganic carbon from the atmosphere to

produce organic matter, which thereby provides the energy source for heterotrophs (bacteria

and fungi) (Brix, 1997). Photosynthesis by algae increases the dissolved oxygen content of

the water which is very significant for the treatment of pollutants in CWs. The major

anatomical appearance of wetland plants is the presence of air spaces in different parts of the

leaves, stems, rhizomes and roots (Gopal and Masing, 1990; Brix, 1998). The presence of

aerenchyma (air-filled) tissue (Fig. 3.4) and lacunae in many wetlands plants helps these

plants to grow in anaerobic or anoxic environments. Their hollow vessels are capable to

transfer oxygen from the leaves to the root zone and the surrounding root area (Armstrong et

al., 1990; Brix and Schierup, 1990). This enhances the active microbial aerobic

decomposition process and the uptake of pollutants from water. Gopal and Masing (1990)

summarized that these air spaces have been considered important reservoirs of oxygen which

is transported through interconnected channels down to the roots and other submerged organs

(Williams and Barber, 1961; Kawase and Whitmoyer, 1980).

The oxygen supply capacity of plants differs from species to species due to the differences in

vascular tissues, metabolism, and root distribution (Steinberg and Coonrod, 1994). During

low temperatures, the potential oxygen release into the root zone may decrease, because root

and rhizome respiration consumes relatively large proportions of oxygen, which diffuses

through plant shoots, and the oxygen demand for root and rhizome respiration declines at low

temperatures (Callaway and King, 1996). The metabolism function of the indigenous

microflora is a combined execution of the availability of light, oxygen, temperature, nitrogen

and phosphorus (Atlas, 1981).



Figure 3.4: Micrograph of gas spaces in a Typha latifolia root (scale bar, 1 mm)

(Vymazal and Kropfelova 2008).

3.1.3.1) Role of macrophytes

The existence of macrophytes is one of the most conspicuous features of wetlands and their

appearance distinguishes CWs from unplanted soil filters or lagoons. The macrophytes

growing in CWs have several properties in regards to the treatment process that make them an

essential component of the wetlands (Brix, 1997). The metabolism of the macrophytes (plant

uptake, oxygen release, etc.) affects the treatment processes to different extents depending on

design. The roles of wetland plants in CW systems can be divided into 5 categories:

i) Physical appearance

The physical parts of the plants stabilize the surface of the beds and slow down the water flow

which aids sediment settling and trapping substances from the water. Their presence also

provides good conditions for physical filtration, and large surface areas for attached microbial

growth. Growth of macrophytes distributes and reduces water velocity, allowing for

sedimentation of suspended solids, reduces the risk of erosion and re-suspension, and

increases contact time between effluent and plant surface area, thus, resulting in increased

pollutant removal (Vymazal and Kröpfelová, 2008; Sim, 2003). In vertical flow (VF) CWs,

the presence of macrophytes, together with an intermittent loading regime, helps to prevent

clogging of the medium (Bahlo and Wach, 1990). The movements of the plants as a resultant

of wind currents keep the surface of bed open, and the growth of roots within the medium of



wetlands helps to decompose organic matter and prevents clogging (Brix, 2003). The major

roles of macrophytes in constructed treatment wetlands are summarized in Table 3.1. Another

important effect of vegetation in wetlands is the insulation that provides by covering the

surface area during winter especially in temperate climates (Smith et al., 1996). When the

standing litter is covered by snow it provides a perfect insulation and helps keep the soil free

of frost. The litter layer helps protect the soil from freezing during winter, and also keeps the

soil cooler during spring (Haslam, 1971a, b; Brix, 1994, 1998).

ii) Surface area for attached microbial growth

The above-and below-ground biomass of macrophytes provide large surface areas for growth

of microbial biofilms (Gumbricht, 1993a, b; Chappell and Goulder, 1994). The plant tissues

are inhabited by dense communities of photosynthetic algae as well as bacteria and protozoa.

Likewise, the roots and rhizomes that are buried in the wetland also provide a substrate for

attached growth of microorganisms (Brix, 2003). Thus, biofilms occur on both the above and

below ground tissue of the macrophytes (Fig. 3.5).

As plants grow and die, the dead leaves and stems fall to the surface of the substrate and form

multiple layers of organic debris (the litter/humus component). This accumulation of partially

decomposed biomass creates highly porous substrate layers and provides large attachment

surfaces for microbial organisms. Thus, the biofilms, as well as the biofilms on all other

immersed solid surfaces in the wetland system, including dead macrophyte tissues, are

responsible for the majority of the microbial processing that occurs in wetlands and for

nitrogen reduction (Brix, 1997).

Figure 3.5: Biofilm on submerged parts of wetland plants (Brix, 2003).



Table 3.1: The major roles of macrophytes in constructed treatment wetlands.

Aerial parts of plant

• Light attenuation > reduced growth of phytoplankton

• Influence on microclimate

• Reduced wind velocity

• Aesthetic pleasing appearance of system

• Insulation of the filtration beds

Below-ground parts

Plant tissue in water Substrate for bacterial growth

• Filtering effect > filter out large debris

• Flow velocity reduction

• Promotion of sedimentation

• Decreased re-suspension

• Prevention of medium clogging

• Provide surface area for attached biofilms

• Source of carbon compounds through root exudates

• Micro-aerobic environment via root oxygen release

Roots and rhizomes in the

sediment

• Stabilize the sediment surface > less erosion

• Prevents the medium from clogging in vertical flow systems

• Release/leakage of oxygen and exudates to root

• Released oxygen:

— increased aerobic degradation

— assist to precipitation of heavy metals

— increased nitrification

— increase degradation (and nitrification)

• Uptake of nutrients

Other functions of plants in the CW

• Removal of pathogens

• Production of ornamental plants

• Production of fiber materials

• Bioenergy crops

• Increased wildlife diversity

iii) Root release/creation of aerobic environment

It is well documented that aquatic macrophytes release oxygen from roots into the rhizosphere

which influences the biogeochemical cycles in the sediments through the effects on the redox

status of the sediments (Barko et al., 1991; Sorrell and Boon, 1992). Qualitatively this is

easily visualized by the reddish colour associated with oxidized forms of iron on the surface

of the roots and experimentally by submerging a root system into a reduced solution

containing methylen blue (Fig. 3.6). Plants transfer this oxygen through the hollow plant

tissue and release it from the root systems into the rhizosphere, where aerobic degradation of

organic matter and nitrification takes place (Brix, 1997).

Phragmites spp. allocates 50% of its plant biomass to root and rhizome systems and increased

root biomass allows for greater oxygen transport into the substrate, creating a more aerobic

environment favoring nitrification reactions which account for the high nitrogen removal by



Phragmites in wetlands (Sim, 2003). For the nitrification process to occur maximum rate, a

minimum of 2 mg O2 /L is required. It is evident that the rate of nitrification is most likely the

rate limiting factor for overall nitrogen removal from a CW system (Sikora et al., 1995).

Figure 3.6: Release of oxygen from roots of Phragmites australis. The blue colour around the

roots is formed by radical oxygen release from the roots that oxidized the reduced form of

methylen blue (Brix 2003).

The rates of oxygen from roots depend on the internal oxygen concentration, the oxygen

demand of the surrounding medium and the permeability of the root-walls of the plants

(Sorrell and Armstrong, 1994). Wetland plants preserve internal oxygen through the suberized

and lignified layers in the hypodermis and outer cortex (Armstrong and Armstrong, 1988).

These layers close the radial leakage of oxygen which allows more oxygen to reach the apical

meristem (the tissue which contains meristematic cells and found in zones of the plant where

growth can take place). Thus, wetland plants attempt to limit their oxygen losses to the

rhizosphere. The oxygen leakage at the root-tips serves to oxidize and detoxify potentially

harmful reducing substances in the rhizosphere (Brix, 2003).

iv) Organic compound release

Plants release a wide variety of organic compounds from their root systems, at rates up to

25% of the total photosynthetically fixed carbon. This carbon release could be a source of



food for denitrifying microbes (Brix, 1997). Decomposing plant biomass also provides a

durable, readily available carbon source for microbial populations.

v) Nutrient uptake and storage

Aquatic plants or macrophytes of wetlands require nutrients for their growth and reproduction

and they can uptake nutrients (macro or micro-nutrients) through roots during their active

growing stage, and these nutrients are translocated to the rhizomes (Mitsch and Gosselink,

1993). As wetland plants are very productive, considerable amounts of nutrients can be bound

in the biomass. Phosphorus and nitrogen uptake capacity of emergent macrophytes is roughly

in the range 30 to 150 kg P/ha/year and 200 to 2500 kg N/ha/year (Brix and Schierup, 1989;

Gumbricht, 1993a; b; Brix, 1994). However, the amounts of nutrients which are removed by

harvesting are generally insignificant compared to the loads in wastewaters (Brix, 1994).

If the plants of wetlands are not harvested, the vast majority of the nutrients that have been

incorporated into the plant tissue will be returned to the water by decomposition processes

(Vymazal and Kropfelova 2008). Long-term storage of nutrients in the wetland systems

results from the undecomposed fraction of the litter. The huge plant biomass will also create a

deposition of refractory nutrient-containing compounds in wetlands (Kadlec and Knight,

1996).

3.1.4) Microorganisms

A fundamental characteristic of wetlands is that its functions are largely controlled by

microorganisms and their metabolism (Wetzel, 1993). The ecological food web in wetlands

requires microbes for the transformation of energy. Influent wastewater is the major food

source in a CW and provides energy, stored in organic molecules. Microbial activity is

particularly important in the transformations of nutrients into various biologically useful

forms (EPA, 2000b). Microorganisms include different bacteria, yeasts, fungi, protozoa,

algae, etc. The microbial biomass is also a major sink for organic carbon and many nutrients.

Microorganisms which naturally live in water, soil, and on the roots of wetland plants feed on

organic materials and/or nutrients, then reduce, break down or completely remove a wide

variety of contaminants from wastewaters.



Widely distributed microorganisms in nature have the capacity to utilize hydrocarbons as the

sole source of carbon (energy) for their metabolism. The microbial community on the plant

rhizosphere creates an environment which enhances the degradation of many volatile organic

compounds (Pardue et al., 2000). Microbial populations can adapt to changing conditions and

can grow rapidly in suitable environments with energy-containing materials. In addition,

when environmental conditions are no longer suitable, many microorganisms become

dormant and can remain so for years (Hilton, 1993).

Microorganisms not only contribute to the transformation of organic and inorganic

substances, but they also can alter the reduction/oxidation (redox) conditions of the substrate

and thus affect the processing capacity of the wetland (EPA, 2000b). Many microbes are

capable of functioning under both aerobic and anaerobic conditions (facultative anaerobes) in

response to changing environmental conditions. In aerobic respiration, microbes use O2 to

oxidize part of the carbon in the contaminants to carbon dioxide (CO2). The rest of the carbon

is used to produce new cell mass. Thus, the major by-products of aerobic respiration are CO2,

water, and an increased population of microorganisms (Francis, 1996; Riser-Roberts, 1992).

Transformation of organic contaminants by microbes takes place since the organisms can use

the contaminants for their own growth and reproduction. Organic contaminants give two

major benefits to organisms: they provide a source of carbon, which is one of the basic

building blocks of new cell constituents, and they provide electrons, which the organisms can

extract to obtain energy (Christensen and Elton, 1996). Energy for growth and reproduction of

microorganisms comes by catalyzing energy-producing chemical reactions that involve

breaking chemical bonds and transferring electrons away from the contaminant. The energy

obtained from these electron transfers is used along with electrons and carbon from the

contaminant to produce more cells (Christensen and Elton, 1996; Riser-Roberts, 1992).

3.2) Types of Constructed Wetlands

According to Kadlec and Knight (1996) CWs can be classified by various parameters but the

two most important criteria are the water flow regime (free water surface and sub-surface) and

the type of macrophytic growth (emergent, submerged, free floating and rooted with floating

leaves) (Fig. 3.7). Different types of CWs can be combined to create a hybrid or combined

system that combines the specific advantages of each system.



Figure 3.7: Classification of CWs for wastewater treatment.

3.2.1) Surface Flow/ Free Water Surface Flow

The surface flow (SF) also termed as Free Water Surface (FWS) (Fig. 3.7) normally consists

of basins or channels, with soil or another porous media to support the rooted vegetation, and

water of relatively shallow depth flows through the unit. The wetland is submerged and water

flows horizontally on top of the wetland soil, infiltrates the soil or is evaporated as the surface

of water is exposed to the atmosphere (EPA, 1993). The water is allowed to flow on top of the

ground surface until collected at the outlet of the wetland. This system is designed to allow a

permanent depth of water that flows horizontally across the wetland bed surface with

landscape similar to a natural wetland. The common characteristic of FWS wetlands are either

a shallow basin of soil or other medium with a controlled water level to ensure that the

sediment, aquatic plants and soil are always submerged. Only the plant stems occur above the

water level. The surface area of FWS CWs can be quite large and they are similar to swamps

and marshes with diverse ecology (Gray, 2004). FWS CWs are also a combination of open

water areas with some floating vegetation as well as emergent plants rooted in the soil (Fig.

3.8). It is quite ensure about plug-flow conditions in FWS CWs because of the shallow water

depth, low flow velocity, and presence of the plant stems (Reed et al., 1988).

Constructed

wetland

Emergent Plant

Submerged

Free floating

Rooted with

floating leaves

Sub-surface flow flow

Surface flow

Vertical Horizontal

Hybrid System



Figure 3.8: Schematic diagram of a Free Water Surface constructed wetland.

A typical FWS constructed wetland with emergent macrophytes normally contains 10 to 15

cm of flowing water in the horizontal dimension of the main basin. The depth of the basins

ranges from 40 to 60 cm and usually more than 50% of the surface is covered by densed

emergent vegetation (Vymazal, 2010). Sometimes naturally occurring species may be present

in this wetland (Kadlec, 1994). Planted macrophytes are usually not harvested and the litter

provides organic carbon for denitrification which leads to anaerobic pockets within the litter

layer (Vymazal, 2010). Aeration of the sediment in FWS takes place by halophytic plants

which act as a source of oxygen providing dissolved oxygen with their roots to a wide variety

of microorganisms.

FWS are land-intensive treatment systems. The water which enters the wetland contains

particulate and dissolved pollutants and spreads out over the large area of shallow water with

emergent or submerged vegetation. Settlable organics are rapidly removed through the

deposition and filtration process. Suspended microbial growth and their attachments are

responsible for the removal of soluble organics. These wetlands are very effective in

removing suspended solids through filtration and sedimentation (Kadlec and Knight, 1996).

Nitrogen is effectively removed by nitrification (in the water column) and subsequent

denitrification (in the litter layer). Ammonium is oxidized by nitrifying bacteria in aerobic

zones, and nitrate is converted to free nitrogen or nitrous oxide in the anoxic zones by

denitrifying bacteria. These systems are also efficient for the sustainable removal of



phosphorus, but at relatively slow rates because of limited contact between the water column

and the soil (Kadlec et al., 2000).

3.2.2) Horizontal Sub-Surface Flow

Sub-surface systems CWs may be classified according to the direction of their water flow into

horizontal (HSF) and vertical (VF) (Fig. 3.9) types. The technology of wastewater treatment

with horizontal surface flow (HSF) CWs started in Germany based on coarse media by Kathe

Seidel commencing in the 1950s and by Reinhold Kickuth in the 1970s with soil media and

high clay content (Vymazal, 2009). Since then, this technology has spread into many

European countries and is now used worldwide for treatment of different types of

wastewaters. The design typically consists of a rectangular planted bed with different species

(Phragmites spp.) and gravel or rock beds, sealed by an impermeable layer. Among the use of

treatment wetlands, HSF CWs are a widely applied concept (Vymazal, 2010).

Figure 3.9: Schematic diagram of a Horizontal Sub-Surface flow constructed wetland.

HSF CWs are shallow beds with depths of 40-60 cm, normally filled with gravel in which

different macrophytes are planted (Fig. 3.9). The flow depth through the HSF constructed

wetland is generally between 0.6 to 0.3 meters (Cooper, 1993). This system is called

horizontal sub-surface flow because the wastewater is fed into the inlet zone and flows

horizontally (i.e., the inlet and outlet are horizontally opposed) through the porous medium

under the surface of the bed, until it reaches the outlet zone where it is collected. Precise inlet



and outlet structures ensure uniform horizontal and vertical flow patterns through the gravel

bed. The water level is maintained under the surface of the gravel which is kept dry. No odour

is produced, insects cannot breed and there is no danger of human contamination from

wastewater being treated.

HSF CWs have different oxidation zones (i.e., aerobic, anoxic and anaerobic zones). The

aerobic zones occur around roots and rhizomes where oxygen leaks into the substrate (Brix,

1987; Cooper et al., 1996). The gravel bed provides a suitable environment for microbial

growth and attachment. Gravel can provide surface areas of about 225m2/m

3

(http://wholewater.com). Uniform gravels are used for the gravel bed and the size range varies

between 3 to 10 millimeters (Cooper, 1993). As gravel is used as the porous media,

subsurface flow wetlands act as filters which have larger surface areas for biofilm growth, and

pollutant removal rates are higher than any in other types of CWs. Biofilm grows to a certain

thickness which then stabilizes, but does not obstruct the wastewater flow (Cooper, 1993). In

this system, wastewaters stay beneath the surface and are in contact with the roots and

rhizomes of the plants (Fig. 3.10).

Figure 3.10: Substrate structure of HSF CWs.



The beds are normally sealed with either clay or a plastic liner/membrane to prevent leakage

at all sides. Plants play the most important roles in HSF constructed wetland by providing

substrate (roots and rhizomes) for the growth of attached bacteria, radial oxygen loss (oxygen

diffusion from roots to the rhizosphere), by nutrient uptake and insulation of the bed surface

in cold and temperate regions (Brix, 1994). The efficient reaction zone in CWs is the root

zone area (rhizosphere) where physicochemical and biological processes take place by the

interaction of plants, microorganisms and pollutants (Stottmeister et al., 2003). The plant root

zone has the most suitable environment for microbial growth as oxygen concentrations are

higher around the roots, therefore an extensive plant root zone will lead to higher biofilm

development, chemical precipitation and metal binding (Brix, 1997).

In HSF CWs bed, pollutants are removed by microbial degradation and chemical and physical

processes in a network of aerobic, anoxic, anaerobic zones and this purification process takes

place in the aerobic zone, which is situated in the rhizosphere (Vymazal 2010). In this system

water is not exposed during the treatment process therefore, minimizing energy losses through

evaporation and convection, and for this reason HSF CWs are more suitable for winter

application (Wallace et al., 2000). The main advantage of HSF CWs is that they require

smaller spaces because the contact area of water, microorganisms and substrate is large. As

the water level is lower and not exposed to the atmosphere, the risk of spread of vector

disease and odour is lower compared to the surface flow type wetlands in which wastewater is

exposed to the atmosphere.

Mainly domestic and municipal wastewaters are treated in HSF CWs around the world.

However, many other types of wastewaters, including industrial and agricultural, landfill

leachate, runoff waters and are treated by HSF CWs systems (Vymazal, 2010). Properly

designed HSF CWs have several advantages and provide high and consistent levels of

treatment efficiencies (Hammer, 1992; USDA et al., 1995a, b; Cooper et al., 1996; Kadlec

and Knight, 1996; IWA, 2000).

3.2.3) Vertical flow

Vertical Flow CWs (VF) were first developed by Seidel in Germany in the 1970s (Vymazal

and Kropfelova, 2008) and then operated in Europe (Burk and Lawrence, 1990). Brix (1992)

provides an overview of Seidel’s systems in Europe. VF CWs consist of sand or gravel beds



with planted emergent macrophytes. The wastewater is distributed on the surface of the bed

and it percolates through the porous media down to the outlet zone usually placed on the

bottom of the bed (Fig. 3.11). The bed drains completely free to allow air to refill the bed

which leads to better oxygen transfer for nitrification (Cooper et al., 1996). The wetland can

also be operated by an up-flow pattern, by inverting the position of the inlet and outlet pipes,

or in a tidal fashion, with fill and drain cycles. The temporarily saturated (in the case of tidal

flow or fill and drain wetlands) or unsaturated states of VF CWs favour oxygen regeneration

in all areas of the wetland while reduction processes such as denitrification are limited. Less-

degradable pollutants require a combination of anaerobic and aerobic processes for

biodegradation. VF CW systems are efficient at treating industrial wastewaters because

anaerobic and aerobic processes take place in these systems sequentially (Yamagiwa and Ong,

2007).

Figure 3.11: Vertical flow constructed wetland

VF CWs can efficiently remove BOD5, COD and bacteria because of their higher ability to

transform. They are considered smaller (1-2 m2/ person equivalent - PE) than HSF CWs

which need 5-10 m2/PE for secondary treatment (Cooper, 1999). VF CWs have more equal

root distribution and water-root contact, and fewer problems of bad odour and proliferation of

insects since they do not have a free water surface (Haberl et al., 1995; Cooper, 1999). The

main purpose of vegetation in VF constructed wetlands is to help maintain the hydraulic

conductivity of the bed (Vymazal and Kropfelova, 2008).



Phosphorus removal in VF CWs is very restricted, because it is not possible for the sand bed

medium to bind phosphorus for a prolonged period (Arias et al., 2001, Del Bubba et al.,

2003; Arias and Brix, 2005). This system is very effective in removing suspended solids and

organics and they can also nitrify at high loading rates even during cold winters (Brix et al.,

2002; Vymazal, 2010). VF CWs are primarily used to treat domestic or municipal

wastewaters. However, in recent years VF systems have also been used to treat other types of

wastewaters (Vymazal, 2010).

3.3) Design Factors

The successful use of constructed wetland technology for the efficient treatment of

wastewater depends mainly upon proper design and operational specifications. Quantifying

the hydraulics of CWs is crucial to understand their function and assess their efficiency in

removing pollutants. The criteria for wetland design include hydrology, substrates, plant

selection, and maintenance procedures (Kusler and Kentula, 1996).

3.3.1) Pretreatment

Pretreatment of effluents before they enter the CW system is necessary to remove coarse and

heavy solids. This is a critical first step that removes most solids (measured as Total

Suspended Solids - TSS) which settle on the bottom of the CW and are degraded by anaerobic

bacteria. Usually a simple septic tank or settling tank is used for small domestic sewage

volumes. Brix and Arias (2005) suggested using two- or three-chambered sedimentation tank

for domestic wastewater pretreatment. For municipal sewage pretreatment a combination of

screening and Imhoff tank is used and when storm water runoff is also to be treated, a grit

chamber is included. When oils and grease are contained in the wastewater, normally grease

filters are used. According to Cooper et al. (1996), a properly sized, built and maintained

septic or settling tank is expected to reduce BOD load by up to 20-30%. Pretreatment also

helps to minimize the possible problem of media clogging.

3.3.2) Hydrology

Hydrology is one of the primary factors in controlling wetland functions (Hammer, 1989).

Flow rate should be regulated to achieve satisfactory treatment. Sufficient water supply is



crucial to establish an efficient CW system (Sim, 2003). Hydrology influences plant species

composition (Bunce et al., 1999), soil characteristics, and nutrient cycles (Kadlec and Knight,

1996; DeBusk, 1999). The flow and storage volume determine the time that water spends in

the wetland system as well as the degree of mixing, thus influencing the interactions between

pollutants and the wetland ecosystem; consequently determining pollutant removal and gas

emissions. Hydrological factors in wetland design belong to the hydroperiod, hydraulic

residence time, hydraulic loading rate, losses to the atmosphere (evapotranspiration), and

overall water balance.

3.3.2.1) Hydraulic Loading Rate (HLR)

The hydraulic loading rate (HLR) refers to the loading on a water volume per unit area over a

specified time interval. Typically, hydraulic loading rates are specified in cm/d (ITRC, 2003).

HLR expressed as:

HLR = Qi/A………………… (3.1)

Where, Qi: wastewater inflow (m3/ d), and A is the wetland top surface area (m

2)

The pollutant loading rate at the inlet (LRi, kg/ m2/d

1) is defined as:

LRi= HLR×Ci…………… (3.2)

Where: Ci is the inlet concentration (mg/L).

Appropriate HLRs and HRTs are very significant parameters for wastewater treatment in

CWs. The HLR depends on the soil material (a critical parameter for subsurface flow

wetlands), flow rate, area-size and hydraulic residence time. Finally, the HLR is determined

by the required pollutant removal efficiency (WPCF, 1990). Lower HLRs or long HRTs result

in better pollutant removal. Table 3.2 presents typical loading rate for the different types of

CWs.



Table 3.2: HLRs for different types of CWs (Vymazal, 2010).

Recommended Loading Rates for FWS CWs

Parameter Loading rates Reference

BOD5 11.2 g/m2d

Wallace and Knight 2006;

EPA, 2000b TSS 7 g/m

2d

TKN 1.5 g/m2d

Recommended Loading Rates for HSF CWs

Parameter Loading rates Ref

BOD5 6 g/m2d

EPA, 2000b TSS 20 g/m

2d

Recommended rates for VF CWs

Parameter Loading rates Ref

TKN 6.5 g/m2d Platzer, 1998

BOD/COD 0.05 g/m2d Stefanakis et al., 2014

3.3.2.2) Hydraulic Retention Time (HRT)

Hydraulic residence time (HRT) is one of the key design parameters which control the

removal efficiency of contaminants and nutrients in CWs (Walker, 1998; Conn and Fiedler,

2006). HRT is determined by the mean volume of the wetland system and mean out flow

(Qo), and is expressed as:

HRT= V/Qo………………… (3.3)

Where: V is the mean volume of water in the wetland (m3), and Qo is the mean outflow

(m3/d).

From the literature it is apparent that the treatment efficiency of pollutants in a CW system is

usually improved by decreasing the hydraulic loading; the longer the HRT, the greater the

nutrient removal (Sakadevan and Bavor, 1999). Metcalf and Eddy Inc., (1991) and Watson



and Hobson (1989) suggested that the most effective HRT range is 4 to 15 days. Gersberg et

al., (1989) found that a short HRT of 3–6 days was effective in removing disease-causing

bacteria and viruses. Significantly HRT can differ depending on whether secondary or tertiary

treatment is intended. Generally, tertiary treatment requires longer retention times. Higher

reduction efficiency for mass balances of N and P can be achieved by Phragmites if water

retention time is more than 5 days (Sim, 2003).

3.3.3) Design of Bed/Bed Area

CWs are usually designed to be of minimum size and cost in order to perform the required

level of pollutant removal. Generally, HSF CWS should have a 3-4: 1 length to width ratio

and be rectangular in shape if minimal treatment area is available. A long length-width ratio is

required to ensure plug flow hydraulics (Miller and Black, 1985). The size of wetland beds

has been designed according to many models beginning with the simple “rule of thumb”, or

complex dynamic, compartmental models for the design spectrum. A simple formula (Eq. 3.4)

is given by Vymazal (2002) to determine surface area for the wetland cells. This formula has

resulted in a general “rule of thumb” for a total area of cells of 5m2/PE.

………………… 3.4

Where,

Ah = Surface area of bed, m2

Qd = Average flow, m3/d

Co = Influent BOD5, mg/L

C = Effluent BOD5, mg/L

KBOD = Rate constant, m/d

Table 3.3 summaries key design and operational specifications for the efficient performance

of HSF and VF CWs. Normally, VF systems require less land (1-3 m2/PE) compared to HF

systems (5-10 m2/PE). The dimensions of vertical flow systems vary between 1-2 m

2/PE; 1

m2/PE for BOD removal only, and 2 m

2/PE for BOD removal followed by nitrification

(Cooper and Green, 1995).



In addition to the wetland bed, the following criteria also contribute to the design of

horizontal subsurface flow wetlands:

Aspect ratio (Width: Length): 1:4

Slope of Wetland Bed: 0.5 - 1%

Minimum substrate bed depth: 0.6m

Hydraulic conductivity of substrate: 10-3

m/s to 10-2

m/s (Ellis et al., 2003).

Table 3.3: Key design and operational specifications for HF and VF CWs.

Parameter

Subsurface flow

HF VF

Flow

Horizontal, continuous

Vertical, intermittent by

batch

Bed design equation

*Ah= Qd (ln Ce-Ci)/ KBOD

**A1=5.25 P0.35

+0.9P

A = the area of a second

bed, estimated at 50%

of A1

A2= the area of a second

bed

Specific area (m2/PE)

5 -10

1 – 2

Recommended organic

loading

***8 – 12g BOD5 m2/d

**** 25g COD m2/d

Prevailing condition Anaerobic Aerobic

(*Cooper et al., 1996, **Grant and Griggs, 2001, ***Kadlec and Knight, 1996, ****Platzer,

1999.)

Where Ce (mg/L) is effluent concentration, Ci (mg/L) is influent concentration, K (-1

day) is a

temperature-dependent first order reaction rate constant, Ah (m2) is the surface area of the bed,

and KBOD (m/d) is the BOD rate constant. A1 and A2 are the area of the first and second bed

in a vertical configuration respectively.

3.3.4) Selection of wetland plants

Availability of suitable plants, expected water quality, normal and extreme water depths,

climate, maintenance requirements and ultimate goals, are among the variables that determine

the selection of plant species for CWs (Stottmeister et al., 2003). For wetland vegetation

common reed plants (Phragmites australis) are more popular in Europe (Cooper and Green,



1995). The choice of plants for treatment of wastewater in CWs is an important issue because

they must survive the potentially toxic effects of the effluent and its variability. Aquatic plants

can absorb inorganic (nutrients, metals, etc), and organic pollutants from wastewater by

mixing them into their own structure (Haberl et al., 2003), so they can be released throughout

the plant growth cycle or stored (Shetty et al., 2005). Further, they amplify the nourishment of

the residuals in the wetland (Vymazal, 2007).

Wetland plants have special features that allow them to grow in environments where water

presence is continuous. They must tolerate high organic and nutrient loadings and have well-

developed underground organs that provide large surfaces for microbial growth (Vymazal,

2011). There are variations in pollutant removal and accumulations between species but most

accumulate pollutants mainly in their belowground tissues (Peverly et al., 1995; Stoltz and

Greger, 2002; Weis and Weis, 2004). Plants which are used for CWs, can be categorized into

two types: rooted and floating forms. The rooted plants are classified into emergent, floating

and submerged (Hammer, 1989). Some studies refer that pollutant uptake depends on the

plant’s form and chemical speciation. Rooted submerged plants are more efficient for

phytoremediation due to their soil binding roots and rhizomes which help the algae, microbes

and invertebrates to colonize (Qian et al., 1999).

According to Brix and Schierup (1989), Wetzel (2001) and Cronk and Fennessy (2001),

emergent macrophytes are the dominating life form in wetlands and they can grow within a

water table range of 50 to 150 cm below the soil surface. The characteristics of these plants

include their aerial parts (i.e. leaves, stems) and extensive root and rhizome system, and they

are morphologically adapted to grow in a submerged substrate due to their large internal air

spaces that allow the movement of oxygen to the roots and rhizomes. The species of this form

include Phragmites australis (common reed), Glyceria spp. (mannagrasses), Eleocharis spp.

(spikerushes), Typha spp. (cattails), Scirpus spp. (bulrushes), Iris spp. (blue and yellow flags)

and Zizania aquatica (wild rice) (Fig. 3.12) (Kadlec and Knight, 1996). This group of plants

produces huge amount of biomass both aerial and belowground parts. These plants produce an

extensive root matrix and have a large surface area for nutrient and ion uptake due to their

horizontal and vertical growth of plant tissues.



Figure 3.12: Different types of wetland plants.

(Source: http://www.intechopen.com/books)

Submerged aquatic plants have their photosynthetic tissues entirely submerged but the

flowers of the plants are exposed to the atmosphere. Examples of these types of macrophytes

are Elodea, Myriophyllum, Ceratophyllum, Isoetes, Littorella, Lobelia (Brix, 2003). These

types of aquatic plants grow very fast. Some species rootless and float under the surface of the

water (e.g. Ceratophyllym demersum). This submerged aquatic plant can grow quickly in

medium-hard to hard water at a certain temperature and it can take up nutrients for its quick

growth (URL 2).

Many researchers have investigated the effectiveness of floating plants in treatment wetlands

(Boutwell and Hutchings, 1999; Hart et al. 2003; Hubbard et al., 2004). The floating form of

macrophytes includes free-floating and floating-leaved plants. Free-floating plants take their

nutrients from water. Examples of floating plants are Nymphaea spp. and Nuphar spp.

(waterlilies), Potamogeton natans (pondweed), and Hydrocotyle vulgaris (pennyworth),

Eichhornia crassipes (water hyacinth), Pistia stratiotes (water lettuce) and Lemna spp. and

Spirodella spp. (duckweed) (Brix and Schierup, 1990, Kadlec and Knight, 1996). Some free

floating plants have well developed submerged roots (e.g. Eichhornia, Trapa, Hydrocharis)

and some surface-floating plants have no roots (e.g. Lemnaceae, Azolla, Salvinia) (Brix,

2003).



3.3.5) Inlet structure

In CW systems it is essential to have an even distribution of wastewater over the whole bed

area. For this reason some systems have use a series of pipe with holes or gutters to distribute

the flow evenly. An open trench perpendicular to the flow direction, and simple, single point

weir boxes can be used for the inlet devices. In some cases perforated pipe is used for both

surface and subsurface CWs. Another method is to completely flood the bed as part of the

intermittent dosing cycle (Cooper and Green, 1995).

3.3.6) Outlet Structure

Outlet structures of CWs help to control uniform flow through the wetland as well as the

operating depth. The design of subsurface flow wetlands should allow controlled flooding at

15 cm to foster desirable plant growth and to control weeds. The use of an adjustable outlet

which is recommended to maintain an adequate hydraulic gradient in the bed and can also

have significant benefits in operating and maintaining the wetland.

3.4) Removal Mechanism of Constructed Wetlands

The mechanism of pollutant removal in CWs is a complex combination of physicochemical

and biological process including sedimentation, binding to substrate, plant uptake and

precipitation as insoluble forms of substances (Fig. 3.13) (Kadlec and Knight, 1996). A CW

system complex as its behaviour depends on both external (e.g. flow-rate, wastewater

composition and temperature) and internal factors (e.g. bacteria growth and development)

(Samso and Garcia, 2013). The system can efficiently remove different pollutants including

organic matter, suspended solids, nitrogen, phosphorus, trace metals and nutrients (Vymazal

et al., 1998; Hammer, 1989).

Physical processes: Sedimentation and filtration are the main physical processes occurring in

CW systems and are related to the water flow. Longer retention times increase pollutant

removal, as suspended particles are removed by sedimentation, On the other hand too long a

retention time lead to detrimental effects. Nutrients, such as phosphorous, nitrogen and other

chemicals, are also removed through sedimentation, as they attach to the setting sediments.



Figure 3.13: Processes occurring in a Constructed Wetland (ITRC, 2003).

Chemical processes: This process occurs by the precipitation of wastewater pollutants as

insoluble forms and their reaction with oxygen, soil minerals or other substrates in the

wetland. When wetlands contain ferrous iron components, a chemical transformation can

occur between the iron and incoming sulfides, and insoluble ferrous sulfide is formed which

then settles to the wetland bed. These degradation and transformation processes are caused by

the aerobic and anaerobic environment of the wetlands’ substrate.

Biological processes: Six major biological processes define CWs performance:

photosynthesis, respiration, fermentation, nitrification, denitrification and microbial removal

(Tousignant et al., 1999). Photosynthesis is performed by wetland plants and algae, adding

carbon and oxygen to the wetland. Both carbon and oxygen accelerate the nitrification process

in CWs.

3.4.1) Organic matter removal

The main processes of organic matter removal in CWs include volatilization, photochemical

oxidation, sedimentation, sorption and biodegradation (ITRC, 2003). Organic matter removal

efficiency in CWs is generally high (Kadlec and Brix, 1995; Vymazal et al., 1998). Puigagut

et al. (2007) reported that BOD5 and COD removal efficiencies vary between 75 and 93% and

64 and 82%, respectively. Particulate organic matter is removed through filtration and



sorption followed by biodegradation occurring in the sediment layer. In a CW system soluble

organic matter could be biodegraded either aerobically or anaerobically depending on the

oxygen concentration of the bed (Kadlec, 2000). The residual BOD5, which is in colloidal and

soluble form, is then continuously removed by microbs, which is attached to the substrate and

the plant roots (Reed et al., 1995, Kadlec and Knight, 1996). Heterotrophic microorganisms

attached to the roots, rhizomes and soil particles are known to be the major contributor to both

BOD and COD reduction. Organic matter is made up of about 50% carbon which

microorganisms use an energy source for the cell synthesis. Near the surface of the surface

flow CW systems, aerobic microorganisms consume oxygen to breakdown organics which

provide energy and biomass for the microorganism. In sub-surface flow systems, this process

occurs near the root zone area where microbial biofilm develops. However, in the other CW

types anoxic decomposition prevails and anaerobic bacteria breakdown organic matter to

produce methane. The alterations of carbon cycle which take place through chemical reactions

in CWs are listed below (Kadlec and Knight,1996):

The degradation processes of organic matter in the aerobic zone:

C6H12O6 → 3CH3COOH (acetic acid) + H2………… (3.5)

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O…………... (3.6)

The degradation processes of organic matter in anaerobic zones:

C6H12O6→2CH3CHOHCOOH (lactic acid)…………… (3.7)

C6H12O6→2CH3CH2OH (ethanol) + 2CO2…………………… (3.8)

Acetic acid, lactic acid, ethanol, H2 and CO2 are the primary end products of fermantatoin by

microorganisms. Then, either the primary end products are mineralized to CO2 and CH4, or

they undergo secondary fermentation to volatile fatty acids (Vymazal and Kröpfelová, 2009).

Anaerobic sulfate-reducing bacteria and methane-forming bacteria utilize the end products of

fermentation (Eqs. 3.9, 3.10 and 3.11) depending on their metabolic activities.

CH3COOH + H2SO4→2CO2 + 2H2O + H2S…………… (3.9)



CH3COOH + 4H2→2CH4 + 2H2O…………………… (3.10)

4H2 + CO2→2CH4 + 2H2O…………………… (3.11)

Elimination by the decomposition of plant mass and other natural organics in a CW system

may be limited. Therefore, it is quite difficult to achieve zero BOD5 in effluent of CW

systems. Moreover, temperature influences the concentration of BOD5 (Reed et al., 1995) and

due to seasonal plant growth carbon compounds can be added to the root zone in the form of

plant detritus and root exudates, and this may be an important carbon source in treatment

wetlands (Pinney et al., 2000).

3.4.2) Nitrogen removal

The nitrogen entering wetland systems can be measured as organic nitrogen and ammonia

(expressed as TKN), or as nitrite and nitrate (Tanner et al., 2002). Nitrogen is an essential

nutrient for all living organisms being present in different forms (Hagopian and Riley, 1998),

nevertheless nitrogen compounds are among the main pollutants of concern in wastewaters

because of their role in contributing to eutrophication, algal blooms, and depreciation of

dissolved oxygen levels in receiving water bodies. Furthermore, unionized ammonia (NH3)

and nitrite (NO2-) are toxic to fish and other aquatic organisms in low concentrations.

Nitrogen removal through CWs is mainly performed by the successive microbial pathways of

ammonification, nitrification and denitrification. In wetland systems, the initial removal of

organic nitrogen as TSS is usually rapid. Microorganisms play a crucial role in the

transformation and removal of nitrogen from wastewaters. Microbial processes convert

particulate organic nitrogen by decomposition into new biomass and ammonium. The removal

mechanisms for N in CWs include volatilization, ammonification, nitrification/denitrification,

plant uptake and matrix adsorption (Brix, 1993). However, the major removal mechanism in

most CWs is microbial nitrification and denitrification (Vymazal, 1998; Moshiri, 1993;

Cooper et al., 1996). Organic nitrogen is transformed into NH4+-N through a complex

biochemical process called ammonification. The rate of ammonification is related to the

organic matter degradation proportion and it occurs quickly in an aquatic environment.

Ammonia nitrogen exists in aqueous solution as either unionised ammonia (NH3) or the

ammonium ion depending on the pH (Vymazal, 1998) as described below:

NH3+ H2O ↔ NH4+ + OH

- ……………… (3.12)



Un-ionised ammonia is relatively volatile and can be removed from solution to the

atmosphere via diffusion in a process called ammonia volatilisation. However, the loss of

ammonia through volatilization from flooded soils and sediments is insignificant if the pH

value is below 7.5. At pH values of 9.3, loss via volatilization is significant (Vymazal, 1998).

NH4+-N produced by ammonification can then be converted into NO2

--N (nitrite nitrogen) and

NO3--N by microbial nitrification. NH4

+-N is transformed first into NO2

--N, which is unstable

and then into the chemically stable NO3--N. Nitrate nitrogen can be used as a nutrient for

plants and may play a role in eutrophication.

Biological nitrification followed by denitrification is believed to be the major pathway for

ammonia removal in CW systems. Nitrification is an aerobic process carried out by

heterotrophic nitrifying bacteria (Keeney, 1973; Paul and Clark, 1996). It is a biological

oxidation of ammonium to nitrate with nitrite as an intermediate in the reaction sequences.

The nitrifying bacteria obtain energy from the oxidation of ammonia or nitrite and carbon

dioxide is used as a carbon source for the synthesis of new cells. Plant roots provide oxygen

which is very important for the oxidation of ammonia to nitrite and oxidation of nitrite-N to

nitrate-N. The oxidation process occurs following the equations below (Wallace and

Nicholas, 1969):

NH+4

+ 1.5 O2 →NO2- + 2H

+ + H2O……………… (3.13)

NO2- + 0.5 O2→ NO3

-………………………………(3.14)

_________________________________

NH+

4 + 2 O2→ NO3- + 2H

+ + H2O………………….(3.15)

Vymazal (1995), summarized that the nitrification process is affected by temperature, pH of

wastewater, the inorganic carbon source, the microbial population and the concentrations of

ammonium–N and dissolved oxygen. According to Cooper et al. (1996), the optimum pH

value ranges from 7.0 to 9.5 for bacterial function while maximum activity is obtained at pH

of about 8.5. When pH values are below 7.0, adverse effects on ammonia oxidation become

pronounced (Lee and Lin, 1999).

Denitrification is achieved in anoxic conditions in which nitrate or nitrite serves a respiratory

electron acceptor for denitrifying bacteria to carry out the oxidation of carbonaceous organic

substrates, with a pH range of 6.5 to 7.5 (Gersberg et al., 1984; Kadlec and Knight, 1996;



Brix et al., 2002). According to Cooper et al. (1996), the presence of dissolved oxygen

inhibits the enzymatic activity required for denitrification. The denitrification process can be

described as:

6 (CH2O) + 4 NO3- → 6CO2 + 2N2 +6H2O…………… (3.16)

This reaction occurs in the presence of organic substrate only under anaerobic or anoxic

conditions when nitrogen is used as an electron acceptor instead of oxygen. The

denitrification process is also dependent on temperature and pH (Vymazal, 1998). This

process proceeds very slowly at temperatures below 5°C, and the optimum pH ranges from 7

to 8. In acidic environments, nitrogen oxides such as NO (nitrogen monoxide) or N2O

(nitrogen dioxide) could also be formed (Vymazal, 1995).

3.4.3) Phosphorus removal

Phosphorus (P) in wastewater is typically present as orthophosphate, dehydrated

orthophosphate (polyphosphate) and organic phosphorus compounds (Vymazal, 1998). It is

highly mobile and present in solution as particles and detritus, or in the cells of aquatic

organisms. Like nitrogen, phosphorus is an essential macronutrient for the growth of plants

and other organisms but it is also responsible for eutrophication processes. Free

orthophosphate is a major link between organic and inorganic phosphorus cycling in wetlands

as it is the only form of phosphorus thought to be utilized directly by algae and macrophytes

(Vymazal, 1995). It occurs mainly from the use of mineral fertilizers and from manures or

slurries washed off land/soil during rainy periods.

Phosphorus removal mechanisms in CWs are adsorption, complexation and precipitation.

High removal rates are anticipated in submerged bed designs when appropriate soils are

selected as the media. A significant clay content with iron, aluminum, and calcium will

enhance phosphorus removal. Phosphorus is removed through precipitation by iron and

aluminum in acidic conditions (pH <6), and by calcium and magnesium in alkaline conditions

(pH >8) (Polprasert and Veenstra, 2000). Phosphorus removal is low in surface flow wetlands

due to limited contact of wastewater with the soil and root zones (Hammer, 1989; Vymazal et

al., 1998). CWs with gravel media have high conductivity and permit water to flow

throughout the bed. For this reason, the impermeable nature of the bed provides a limited



surface area for adsorption, ion exchange and/or chemical reaction to take place and once the

active sites are utilized, phosphorus removal ceases (Newman et al., 2000).

Some CW systems in Europe use sand instead of gravel to increase the phosphorus retention

capacity, but selecting this media results in a larger system because of the reduced hydraulic

conductivity of sand compared to gravel. If significant phosphorus removal is a requirement,

then very large land areas or alternative treatment methods will probably be required.

Phosphorus removal by wetland vegetation is low as only a small fraction of phosphorus is

removed. In order to have efficient removal of phosphorus through macrophytes it is

necessary to harvest the macrophyte biomass (Vymazal et al., 1998). Plant uptake may be

significant only in systems where the surface loading rate is low.

3.4.4) Total suspended solids removal

Suspended solids are mainly removed by physical processes such as sedimentation, filtration,

and flocculation. Suspended solids removal is very effective in SF CWs. Most of the removal

probably occurs within the first few meters of travel distance from the inlet zone. All

settleable and floatable solids are removed in wetland systems due to long hydraulic residence

times. Non settleable and colloidal solids are removed by bacterial decomposition and

adsorption to the wetland media and plant root system (Stowell et al., 1981). Filtration occurs

by impaction of particles onto the roots and stems of the macrophytes or onto the soil/gravel

particles in SSF systems. The extensive root system adds surface area to the wetland media,

which reduces water velocity and reinforces settling and filtration in the root network. For

FWS systems, most of the suspended solids are removed within the first meters, giving rise to

a ‘bank’ of sludge that can hinder the water flow. Subsurface-flow systems can clog when too

many pores become filled with particulates.

3.4.5) Heavy metal (Cr) removal 1

The removal mechanism of heavy metals in CWs is a complex combination of

physicochemical and biological process including sedimentation, binding to substrate, plant

uptake, and precipitation as insoluble forms (mainly sulphides and (oxy-) hydroxides) (Kadlec

1 Sutlana et al., (2014b)



and Knight, 1996). Normally plants receive elements in their ionic forms. Most of the metal

ions penetrate into the plant cell through specific metal ion carriers or channels. In the cells of

plants, Cr ions can form chelating compounds with chelators (e.g., organic acids, glutathione,

phytochelatin and metallothionein protein) in order to reduce metal toxicity within the cells

(Cobbett and Goldsbrough, 2000; Pilon-Smits, 2005). These chelating compounds can

conduct further sequestration in cell vascular of the roots (Pilon-Smits, 2005). Once the metal

ions enter into the roots, they are either stored in the roots or translocated into the shoots

through xylem tissue (Jabeen et al., 2009). Then metals can pass through the root cell wall or

root xylem and be transported into stem xylem with the help of proteins or chelators (Pilon-

Smits, 2005). They can then translocate from stem xylem to leaf tissue where they can

combine with chelators (Cobbett and Goldsbrough, 2000).

The organic acids of root extracts form complexes with Cr compounds and increase their

availability for plant uptake (Bartlett and James, 1988). The presence of organic acids

influences the Cr uptake in roots (Srivastava et al., 1998). The channel of Cr transportation is

a mechanism which involves essential anions carriers such as sulfate (Cervantes et al., 2001).

Both forms of Cr(VI and III) enter the root cells by the symplast method where Cr(VI) is

decreased and stored in the cortex region (Shanker et al., 2005). Though Cr transportation is

very weak in the aerial parts, its movement and accumulation depend on its chemical structure

inside the tissue (James and Barlett, 1983). Root membranes of plants can be damaged by the

hexavalent form of chromium due to its high oxidation power. Moreover, some essential

elements such as Fe, K, Mg, Mn, P and Ca, are compressed by the Cr(VI) for cells uptake

because of their similarity in ionic forms (Gardea-Torresdey et al., 2005, Pandey and Sharma,

2003).

3.5) Application of CWs in Cr(VI) treatment

Table 3.4 presents previous experiments/applications of FWS CWs for Cr (VI and III)

removal. Different types of wastewaters such as metallurgic, industrial, tannery sewage etc.,

were treated. The majority of these experiments/applications used floating plants, as their

expanded root area enhances the growth of microorganisms. In FWS CWs emergent plants

can also accumulate the contaminants in their tissues and also can provide carbon the source

for degrading the bacteria (Dunbabin and Bowner, 1992). Furthermore, they favor suspended



solid settlement and oxygen transfer into the rhizosphere (Brix, 1994, 1997; Kadlec et al.,

2000). Sen and Bhattacharyya (1994), Maine et al. (2004) and Hadad et al. (2006), have stated

that metal concentrations were higher in roots than leaves of floating plants. Maine et al.

(2009), performed experiments with three phases of vegetation in free water surface wetlands.

They found that when Eichhornia crassipes was dominant in the vegetation, contaminants

remained in the macrophytic biomass, but when both E. crassipes and T. domingensis were

used, contaminants remained in the sediments.

Table 3.5 summarizes previous experiments/applications of HSF CWs treating Cr (VI and

III). The most commonly used plants were Phragmites australis and Typha latifolia. The

roots of Phragmites australis can grow horizontally and vertically and provide a substantial

surface area for the growth of microorganisms. Fibbi et al. (2012), found that Cr accumulated

in underground and aboveground tissues but most accumulated in roots. Although restricted

amounts of Cr pass into shoots and a higher amount of Cr accumulates in roots, the affinity of

Cr to bind to the root cell walls is the cause of shoot translocation (Pendias and Pendias,

2001). Lesage et al. (2007a), used Phragmites australis for the treatment of domestic

wastewater. They pointed out that the position of the plants in the wetland has a significant

effect on pollutant removal except for Cr and Zn metals. Vymazal et al. (2007) performed

experiments in HSF CWs with two macrophytes: Phragmites australis and Phalaris

arundinacea to treat municipal sewage. They found that a negligible amount of metals are

stored in the biomass of both plants. However, emergent macrophytes accumulate relatively

small amounts of Cr compounds to submerged or free-floating vegetation. They also found

that metal concentrations were higher in the roots than leaves. Calheiros et al., (2008b) also

observed that Cr accumulation occurred chiefly in the rhizomes of Phragmites australis and a

small translocation occurred to the shoots. The phytoextraction process can be effective if a

substantial amount of Cr is translocated by the root medium to the plant shoot (Zayed and

Terry, 2003). Plants that can assemble 1000 mg Cr/kg are considered as hyper accumulators

plants (Lasat, 2002).



Table 3.4: Free water surface constructed wetlands (FWS CW) used for Cr removal.

Reference CW area,

(m2)

Wastewater

type

HRT,

(days) Porous media

Inlet concentration

of Cr (mg/L)

Plants

Removal %

Baker et al.,

2012 720

Petroleum

coke - Oil sands - Chara spp (Algae) -

Di Luca et al.,

2011 2000 Industrial 7-12

Sediment (5

layers bentonite) 0.4 Typha domingensis 90

Hadad et al,

2010 2000

Sewage and

Industrial 7-12

Muddy

sediment 0.033 Typha domingensis 85

Khan et al.,

2009 4145.71 Industrial 1.7 Sediment 0.07-0.35

T. latifolia, S. cyperniu ,

C. aquatilis , P. australis, J.

articulatus,C. demersum, L. gibba L.,

E. cressipes, P. glabrum , A. plantago

aquatica, P. stratiotes

89

Maine et al.,

2009 2000 Tool factory 7-12 Sediment 0.018

E. crassipes 88

E. crassipes + T. domingensis 7

T. domingensis 30

Maine et al.,

2006 18 Industrial 7 Soil 0.022

E. crassipes, T. domingensis,

Pontederia cordata L. 86

Aguilar et al.,

2008 450 Tannery 2 Sediment 22-31 Typhs spp., Scirpus americanus 99

Maine et al.,

2007 2000 Tool factory - Sediment 0.014

E. crassipes 62

T. domingensis 58

Srisatit and

Sengsai, 2003 5

Chromium

solution 10 Soil -

Vetiveria zizanioides (Linn) 89.29

Vetiveria nemoralis 86.30



Table 3.5: Horizontal surface flow constructed wetlands (HSF CW) used for Cr removal.

Reference CW area,

(m2)

Wastewater

type

HRT,

(days)

Porous

media

Inlet

concentration of

Cr (mg/L)

Plants

Removal %

Arroyo et al. 2010 210 Domestic 10.5 - 5.64 Typha Latifolia, Iris pseudacorus,

Salix atrocinera 55

Dotro et al., 2012 4.5 Tannery 5, 2.4 Granitic

rock 1.1, 0.08-5.9 Typha Latifolia 50-95

Dotro et al., 2011a 0.31 Tannery 1 Pea gravel 5 Typha spp. 90-99

Fibbi et al., 2012 180 Textile 2.1 Gravel 0.0008-0.0158 Phragmites Australis 72

Lesage et al., 2007a 65 Domestic 6.7 Gravel 0.113 Phragmites Australis 87

Vymazal et al.,

2007 3520

Municipal

sewage - Soil 0.0005

Phragmites Australis and Phalaris

arumdinacea 92.8

Kelvin and Tole,

2011 1750 Domestic 11 Gravel 0.5 Macro-Hydroplants 60

Kucuk et al., 2003 378 Tannery 5-11 Gravel 0.2 Phragmites Australis 43-55

Dorman et al.,

2009 568L

Coal

combustion

waste

5 Quartz Sand 0.048 Ceriodaphnia dubia 69.28

Sultana et al.,

2014a

0.86

Synthetic

8, 4, 2, 1

Fine gravel

5, 2.5, 1, 0.5

Phragmites australis

100

Table 3.6: Vertical flow constructed wetland (VF CW) used for Cr removal

Reference CW area

(m2)

Wastewater

types HRT Porous media

Inlet

concentration of

Cr (mg/L)

Plants

Removal %

Mant et al., 2006 30 L tank Tannery 6h Gravel 10-20

Penisetum purpureum 78.1

Brachiaria decumbens 68.5

Phragmites Australis 56.7

Yadav et al., 2010 0.035 m2 Synthetic solution 6-48hrs Gravel 10, 15, 20 C. indica L. 98.3



The most important parameter to achieve high Cr removal efficiencies is the selection of the

optimal CW type. While all CW types present high removal efficiencies up to 100% (Tables

3.4-3.6), VF CWs have not often been applied for Cr removal (Mant et al., 2006; Yadav et

al., 2010) and only in laboratory-scale experiments under operating conditions that do not

correspond to real-life conditions (e.g., continuous recirculation and fill and draw operation).

Therefore, it is not presently possible to safely evaluate their Cr removal efficiency. On the

other hand, FWS and HSF CWs have been extensively used in laboratory-scale experiments

and full-scale applications (Vymazal, 2005; Maine et al., 2007; Khan 2009; Hadad et al.,

2010; Di Luca et al., 2011). These two types of CWs present similar Cr removal efficiencies

(7-99% for FWS and 50-100% for HSF) for similar feed Cr concentrations (0.014 to 31 mg/L

for FWS and 0.008 to 20 mg/L for HSF), while they operated with HRTs from 1 to 12 days.

Thus, both these CW types can be successfully used for Cr removal from wastewaters.

All researchers conclude that plant vegetation plays a significant role in Cr removal; however

it is not clear if different vegetation types affect CW efficiencies. The comparative study of

Cr removal by different types of constructed wetlands in Tables 3.4 to 3.7 reveals that

Phragmites and Typha are the two most commonly used plant species. CWs planted with

both Phragmites and Typha appear to achieve high Cr removal efficiencies (43 to 100% for

Phragmites and 85 to 99% for Typha), while both plants appear to operate successfully in a

wide range of feed Cr concentrations (0.005 to 20 mg/L for Phragmites and 0.033 to 31 mg/L

for Typha). CWs planted with C. indica (Yadav et al., 2010) and Scirpus (Aguilar et al.,

2008) also achieved high removal efficiencies (up to 99%), while they received high feed Cr

concentration (22 to 31 mg/L for Scirpus and 10 to 20 mg/L for C. indica). Furthermore,

Phragmites australis presents higher Cr accumulation rates than ten other plant taxa (Table

3.4) (Khan et al., 2009). Phragmites australis was found to be more tolerant to Cr and more

effective at Cr removal than Typha latifolia in CWs treating activated sludge (Stefanakis and

Tsihrintzis 2012a). On the other hand, Mant et al. (2006) report that Penisetum purpureum

and Brachiaria decumbens are more efficient at Cr removal than Phragmites australis. The

well-developed root system of the two former plants provides greater surface area for biofilm

development, chemical precipitation and metal binding. The four plant species mentioned

above appear to tolerate high feed Cr concentrations and affect positively Cr removal. While

a number of other plant species were also used in CWs treating Cr, they cannot be safely

evaluated as feed Cr concentrations in these experiments were low, ranging from 0.014 to 1

mg/L. Based on the above findings it seems that although vegetation plays a significant role



in Cr removal in CWs, the type of vegetation used is not a crucial parameter. Therefore,

vegetation should be selected based only on availability (i.e., indigenous plants should be

prefered) and cost.

The design of constructed wetlands can influence removal efficiency. Yadav et al. (2012),

concluded that Cr removal was enhanced when CW depth was increased, due to the CW's

increased effective area and the appearance of anaerobic conditions, which also enhance Cr

removal. In another study of Yadav et al. (2010), the authors refer that deeper CWs can

accumulate more Cr than swallower CWs.

Although HRT is a significant operational parameter of CWs, its effect on Cr removal has not

been fully investigated. Concerning VF CWs, only Yadav et al. (2010) examined the effect of

three different HRTs (12, 24 and 48 hr), concluding that increased HRT leads to increased Cr

removal. However, in their experiments CWs were operated under draw and fill mode, which

does not correspond to real operational conditions. Two studies discuss the effect of HRT on

HSF CWs (Kucuk et al., 2003; Sultana et al., 2014a). Although Kucuk et al. (2003) present

experimental results using different HRTs, their discussion is mainly focused on organic

matter and nutrient removal and less on Cr removal. On the other hand, Sultana et al. (2014a)

conducted a series of comparative experiments with five different HRTs (8, 4, 2, 1 and 0.5

days), where HRTs of 1 day were proved not to limit Cr removal.

While FWS and HSF CWs have been extensively used in Cr removal, VF and hybrid (VF

combined with HSF) CW systems are less studied (Tables 3.6 and 3.7). Mant et al. (2006),

performed experiments with tannery wastewater in a 30 L tank which was planted with three

macrophytes: Penisetum purpureum, Brachiaria decumbens and Phragmites australis. It was

found that Phragmites australis accumulated a significant amount of Cr in its leaves and

stems compared to the other two species. However, some toxicity effects of Cr have been

exhibited on this plant. Penisetum purpureum also accumulated a significant amount of Cr in

its leaves and stems.

Although a significant amount of research on CWs treating Cr wastewaters has been recorded

in the literature, several issues have not been fully covered:



1. Data on the effect of HRT on CW performance is limited and exists only for VF and

HSF CWs. Furthermore, the effect of HRT should be examined simultaneously with

different influent concentrations to assess maximum Cr loads that can be treated by

each CW.

2. Cr toxicity on common plant species used in CWs has not been examined. Therefore

maximum Cr influent concentrations cannot yet be defined.

3. Another key point that has not been fully investigated is the precise contribution of

microbial activity to Cr removal. While different microbial species have been

identified in CWs treating Cr, which all need an external carbon source, no

comparative studies examining the effect of different carbon sources and wastewater

types are available. For example, co-treatment of Cr with other types of wastewaters

could enhance or limit Cr removal.



Table 3.7: Treatment of chromium polluted wastewaters using hybrid constructed wetland systems

Reference CW area (m2) Wastewater

type

HRT Porous media Inlet

concentration of

Cr (mg/L)

Plants Removal

%

Kongroy et

al., 2012

CNU SSF-1500

FWS-2300

Municipal and

Industrial

Gravel and

soil

0.016 SSF-Phragmites australis, Typha orientalis,

Vetiveria zizanioides, Cyperus alternifolius,

Eleocharis dulcis

FWS- P. australis, T. orientalis, C. alternifolius,

Ludwigia adscendens and Myriophyllum

aquaticum

44

MPI FWS- 1020 0.039 Ipomoea aquatica, Eichhornia crassi, V.

zizanioides, T. orientalis, C. zlternifolius,

Nymphaea spp.

96

Soda et al.,

2012

Main CW- 520

Experimental CW-

25

Metallurgic Main CW- 3 hrs

Experimental

CW- 1.5 hrs

0.0108 Main CW- Acorus gramineus, Cyperus

alternifolius L., Iris pseudacorus, Lythrum

anceps, Myosotis scorpioides, Phyla nodiflora

L. and Zantedeschia aethiopica

Experimental CW- Acorus gramineus, Cyperus

alternifolius L.

12



3.6) Application of CWs in Agro-Industrial wastewaters Treatment

CWs are mainly used to treat domestic and municipal wastewaters but more recent

applications of CWs include treatment of other types of wastewater, such as industrial and

agricultural wastewaters, various runoff waters and landfill leachate (Kadlec and Wallace,

2008). Agro-industry includes post-harvest activities involved in the transformation,

preservation and preparation of agricultural produce for intermediary states or final

consumption. Agro-industrial wastewaters are usually characterized by their high organic

load and their quantity and quality variations over a year (Awady and Wahaab, 1999). In this

section, the main CW applications for agro-industrial wastewater, including dairy and animal

farm wastewater wastewaters. Furthermore, special discussion is made to olive mill

wastewater (OMW) due its toxicity from high phenol concentrations. CWs are also used for

other agro-industrial wastewater treatment, including coffee processing (Rossmann et al.,

2012), winery (Grismer et al., 2003; Masi et al., 2002), vinegar (Justin et al., 2009), fish

farms (Sindilariu et al., 2009; Vymazal, 2009), molasses production (Olguın et al., 2008) and

the sea food industry (Sohsalam et al., 2008).

3.6.1) Pre-treatment stages in treatment of Dairy Wastewaters

In most cases of dairy wastewater treatment by CWs, the pre-treatment stages aim mainly at

removing suspended solids. Therefore, in the majority of experiments/applications, the pre-

treatment stages included either simple settling basins (Browne and Jenssen, 2005; Gasiunas

et al., 2005a; Munoz et al., 2006; Newman et al., 2000) or a settling stage combined with

biological treatment, such as aerobic and anaerobic lagoons (Moreira et al., 2010; Shamir et

al., 2001), or oxidation ponds (Tanner et al., 1995) (Table 3.8).

3.6.2) CW types in treatment of Dairy Wastewaters

The type of CW seems to be crucial for dairy wastewater treatment. As shown in Table 3.8,

only four research groups have focused on dairy wastewater treatment using FWS CWs

(Dunne et al., 2005; Jamieson et al., 2007; Schaafsma et al., 2000; Shamir et al., 2001), as

these wastewaters have high pollutant loads and are therefore difficult to treat. As mentioned

previously, dairy wastewater is characterized by high concentrations of organic matter. Thus



a FWS CW cannot achieve efficient pollutant removal performance, as the high organic

matter concentration creates anoxic or anaerobic conditions in the water column and reduces

the amount of oxygen available for microbial organic matter oxidization. As shown in Table

3.8, when pollutant surface loads are low in FWS CWs (Newman et al., 2000; Shamir et al.,

2001), removal efficiencies are high for organic matter (91-98%), nitrogen (80%),

phosphorus (89-92%) and TSS (96-99%). According to Schaafsma et al. (2000), organic

matter and nutrient removal efficiencies are also significant, but in some cases, nitrate and

nitrite concentrations increase in the effluent. In most experiments/applications using VF

CWs, it was observed that these wetland systems were poor at phosphorus removal. Dunne et

al. (2005), report that their VF CW system did not show any significant reduction in pollutant

concentration between the wetland’s inlet and outlet, although BOD reduction was

significant.

HSF CW systems, however, appear to be more efficient than the other two systems when

treating dairy wastewater, and 17 experiments/applications employing HSF CWs for diary

wastewater treatment have been reported. HSF CWs are far more efficient bio-reactors than

FWS CWs, as the removal efficiencies of the former are in the range of 28-99% for COD, 21-

99% for nitrogen, 2-98% for phosphorus, and 45-95% for TSS. It should be mentioned that

these removal efficiencies were achieved with pollutant surface loads 10 times higher than

those applied in FWS CWs. The most efficient CW system for dairy wastewater treatment

appears to be a hybrid system of both VF and HSF stages. Hybrid systems have been tested

by three research groups (Browne and Jenssen, 2005; Lee et al., 2010; Rousseau et al., 2004),

and each demonstrated high removal efficiencies for all pollutants (83-96% for COD, 65-

92% for nitrogen, 52-99% for phosphorus and 83-99% for TSS), even with higher pollutant

surface loads than those applied to HSF systems.



Table 3.8: Studies of dairy wastewater treatment by CWs.

Reference CW Surface Area

(m2)

Plant Species Pre-treatment Surface Load (gr/m2/day) Removed Surface Load (gr/m2/day)

C TKN TP TSS C TKN TP TSS

FWS

Dunne et al., 2005 4265 Carex riparia, Typha latifolia, Phragmites australis, Sparganium erectum, Glyceria fluitans,

Iris pseudacorus, Phalaris arundinaceae, Alisma

plantago-aquatica

Three-chambered tank

6.84 _ 0.05 2.55 6.22 _ 0.04 2.52

Jamieson et al.,

2007

100 Typha latifolia, Lemna spp. Storage tank 6.5 0.8 0.12 4.8 6.37 0.64 0.11 4.61

Schaafsma et al.,

2000

500 Typha latifolia, Scripus tabernaemontani, Litaneutria minor

Settling basin - - - - - - - -

Shamir et al.,

2001

630 Typha domingensis, Scirpus validus, Phragmites

australis

Solid separators,

anaerobic lagoons, aerobic ponds

20.36 23.2 _ 67.36 0.61 6.0 _ 24.2

Hybrid

Browne and

Jenssen, 2005

1990 Phragmites australis, Senecio sylvaticus, Urtica dioica, Typha latifolia, Sparganium erectum,

Butomus umbellatus, Cucurbita maxima

Settling tanks 1.28 0.74 0.10 1.96 1.2 0.68 0.09 1.92

Lee et al., 2010 1.87 Sotalia fluviatilis _ 173.5 _ 2 22.3 156 _ 1.6 20.1

Rousseau et al.,

2004

80 Phragmites australis _ 50-1500 50-

1500

1.5-40 20-400 45-

1350

32.5-

975

0.8-21 18.8-376

Farnet et al., 2009 160 Phragmites australis - _ _ _ _ - -

Gasiunas et al.,

2005

50-1900 Phragmites australis Septic tank, settling

in ponds

5.7-8.3 0.56-

1.97

0.17-

1.71

_ 4.5-6.6 0.2-0.8 0.12-

1.2

_

Ghosh and

Gopal, 2010

1.63 Typha angustata Secondary treatment in storage tank

8.6-34.5 1.3-4.9 OP: 0.3-1.1

0.2-0.7 5.2-20.7

0.78-2.94

OP: 0.09-

0.33

0.16-0.56

Ibekwe et al.,

2003

600 Phragmites communis, Scirpus validus Storage lagoons _ _ _ _ - - - -

Kern et al., 2000 Two beds (15 m2

and 10 m2)

Phragmites australis, Stuckenia pectinata, Carex

acutiformis, Glyceria maxima, Typha latifolia

Settling tank 26.8-45.3 _ _ _ 24.1-

40.8

_ _ _

Mantovi et al.,

2003

72 Phragmites australis Imhoff tank 110.1 5.84 1.15 62.3 101.3 2.83 0.70 56.6

Moir et al., 2005 20 - Batch reactor (SBR) - - - - - - - -

Moreira et al.,

2010

398 Lemna sp., Pontederia cordata Anaerobic/Facultative lagoon, Aerobic

lagoon

29.4 3.4 2 8.4 11.2 1.45 0.5 4.9

Munoz et al.,

2006

892 Sotalia fluviatilis Settling tank 68.5 _ 0.6 16 _ _ _ _

* data not sufficient to calculate pollutant loads



Table 3.8: continued

Reference CW Surface

Area (m2)

Plant Species Pre-treatment Surface Load (gr/m2/day) Removed Surface Load (gr/m2/day)


HSF

Farnet et al.,

2009

160 Phragmites australis - _ _ _ _ - -

Gasiunas et al.,

2005b

50-1900 Phragmites australis Septic tank, settling in ponds

5.7-8.3 0.56-1.97

0.17-1.71

_ 4.5-6.6 0.2-0.8 0.12-1.2

_

Ghosh and

Gopal, 2010

1.63 Typha angustata Secondary treatment in storage tank

8.6-34.5 1.3-4.9 OP: 0.3-1.1

0.2-0.7 5.2-20.7

0.78-2.94

OP: 0.09-

0.33

0.16-0.56

Ibekwe et al.,

2003

600 Phragmites communis, Scirpus validus Storage lagoons _ _ _ _ - - - -

Kern et al., 2000 Two beds (15 m2

and 10 m2)

Phragmites australis, Stuckenia pectinata, Carex

acutiformis, Glyceria maxima, Typha latifolia

Settling tank 26.8-45.3 _ _ _ 24.1-

40.8

_ _ _

Mantovi et al.,

2003

72 Phragmites australis Imhoff tank 110.1 5.84 1.15 62.3 101.3 2.83 0.70 56.6

Moir et al., 2005 20 - Batch reactor (SBR) - - - - - - - -

Moreira et al.,

2010

398 Lemna sp., Pontederia cordata Anaerobic/Facultati

ve lagoon, Aerobic lagoon

29.4 3.4 2 8.4 11.2 1.45 0.5 4.9

Munoz et al.,

2006

892 Sotalia fluviatilis Settling tank 68.5 _ 0.6 16 _ _ _ _

Mustafa et al.,

2009

7600 Typha latifolia, Carex riparia, Glyceria maxima,

Carex riparia, Glyceria maxima, Phalaris

arundinacea, Carex riparia

_ 1.2 _ _ 0.21 1.14 _ _ 0.2

Newman and

Clausen, 1997

138.6 Typha angustifolia, Phragmites australis, Suillus

pungens, Typha latifolia, Lythrum salicaria,

Eleocharis obtusa, Litaneutria minor

Bulk tank 52.6 1.9 0.514 14.8 14.7 0.5 0.14 6.7

Newman et al.,

2000

138.6 (each cell) Typha angustifolia, Phragmites australis, Suillus

pungens

Settling basin 51.3 1.96 0.5 24.6 38.9 0.55 0.23 22.1

Smith et al., 2005 100 Typha latifolia Heated storage tank 17 _ _ _ 16.7 - - -

Tanner et al.,

1995

19 Scripus validus Oxidation pond 0.9-4.1 0.6-2.7 0.2-0.8 1.9-8.5 0.76-

3.5

0.4-1.8 0.12-

0.48

1.5-6.5



3.6.3) Vegetation for Dairy Wastewater treatment in CW systems

One of the main issues in CW treatment systems is to identify the role of the plants in

pollutant removal and to define their toxicity boundaries. In dairy wastewater treatment,

where organic loads are very high, a variety of different plant species have been used (Table

3.8). As reported by Browne and Jenssen (2005) Phragmites australis, Scirpus sylvaticus and

Urtica dioica were able to grow in CWs treating dairy wastewater and did not exhibit any

toxic effect. The exact contribution of plants in nutrient removal is a controversial issue as

almost all related studies give different removal efficiencies. Gottschall et al. (2007) report

that nutrient removal due to plant uptake was significantly lower in their study compared to

previous studies (Comin et al., 1997; Greenway and Woolley, 2000), which reported that

plant uptake is responsible for 27-66% of nitrogen removal and 47-65% of phosphorus

removal. In addition, Newman et al. (2000) report that only 3% of nitrogen removal could be

attributed to plant uptake. Mantovi et al. (2003) also attribute most nutrient removal to

biofilm biochemical oxidation and plant uptake. Tanner et al. (1995) reported that planted

wetlands showed greater removal efficiencies of N and P from dairy farm wastewaters than

unplanted wetlands. They recorded higher TP removal in the summer months due to higher

plant biomass growth and temperatures. The unplanted wetland proved to be less efficient at

removing both N and P with higher loading rates. Percentage removal of NH4+-N increased

with retention time in the planted HSF CW, whereas the unplanted wetland showed lower

performance. Plant rhizosphere aeration may stimulate aerobic decomposition processes by

increasing nitrification and subsequent gaseous losses of N through denitrification, (Hansen

and Anderson, 1981; Reddy et al., 1989) and by decreasing the relative levels of dissimilatory

nitrate reduction to ammonium (Tiedje, 1988).

Dipu et al. (2011) examined the effect of different plant species (Typha sp., Eichhornia sp.,

Salvinia sp., Pistia sp., Azolla sp. and Lemna sp.) in CW removal efficiency. Generally, the

plant vegetation appeared to neutralize pH from the initial alkaline values recorded in diary

wastewaters. Concerning organic matter removal, Dipu et al. (2011) found that Azolla sp. and

Eichhornia sp.-based CWs (83.07%) were more efficient at pollutant removal, followed by

Typha sp.-based CWs (80%), however these differences were insignificant. Ghosh and Gopal

(2010) examined plant tolerance to dairy wastewater and found that young Typha plants

yellowed when wastewater with high EC values was applied to the CWs. Ghosh and Gopal

(2010) also mentioned that plant density and height were maximum near the CW’s inlet and



attributed this to the higher nutrient concentrations, which could promote plant growth.

Ghosh and Gopal (2010) found that at 4 days HRT, plant growth was higher and pollutant

removal efficiencies were also high. According to Ibekwe et al., (2003) treated dairy

wastewater can be recycled and used for irrigation. The authors observed the existence of a

diverse bacterial community (especially Nitrosospira sp.) that oxidizes ammonia and which

could also be involved in the nitrification process in the wetlands and influence the final

quality of effluent water.

Munoz et al. (2006) suggest that artificial aeration is needed to enhance removal efficiency

and pore volumes. Schaafsma et al. (2000) propose that increasing plant density and

wastewater recirculation will promote denitrification.

3.6.4) Pre-treatment stages in treatment of Animal Farm Wastewater

Similar to dairy wastewater treatment, most of the experiments/applications on animal farm

wastewater treatment using CWs contained pre-treatment stages mainly to remove suspended

solids. Suspended solid removal is essential before the wastewaters enter the CW stage,

because high suspended solid concentrations can cause clogging of the porous media.

Additionally, their reduction also reduces the organic load (Table 3.9).

3.6.5) CW types in treatment of Animal Farm Wastewater

Experiments/applications on animal farm wastewater treatment have tested all three CW

types (Table 3.9). When pollutant surface loads were low (0.95-1.62 and 0.33-0.34 gr/m2 day

for TKN and TP, respectively) in FWS CWs (Gottschall et al., 2007; Hunt et al., 2003; Stone

et al., 2004; Yeh et al., 2009) removal efficiencies for TKN and TP were 35.8-46.7 and 8.8-

23.5% (Table 3.9). Gottschall et al. (2007) showed that TKN removal was lower than NH4+

removal. The wetland was NH4+-dominated and showed greater uptake of NH4

+ than NO3

-.

The FWS CW cells received various surface loads of COD, TN and TSS, from 8.13-87.1

gr/m2 day, 0.4-3.5 gr/m

2 day and 1299-35109 gr/m

2 day, and the removal efficiencies

recorded were 36.7-48.5%, 30-47.8% and 26.3-49.3%, respectively (Hunt et al., 2006). HSF

and VF CWs appear to be more efficient at treating animal farm wastewater as their pollutant

removal efficiencies were higher than those of FWS CWs (Table 3.9).



Table 3.9: Studies of animal farms wastewater treatment by CWs.

Reference CW area

(m2) Plant Species Pre-treatment

Surface Load (gr/m2/day) Removed Surface Load (gr/m2/day)


FWS

Gottschall et al.,

2007 327 Typha latifolia, Typha angustifolia Facultative pond _

0.95-

1.62

0.33-

0.34 _ _

0.38-

0.65 0.05 _

Hunt et al., 2003 120.6 Juncus effusus, Scirpus tabernaemontani, Solanum americanum, Typha latifolia, Typha angustifolia

Anaerobic lagoon 2.74 3.5 _ _ _ _ _ _

Hunt et al., 2006 440 Typha latifolia, Schoenoplectus americanus

Two-stage anaerobic

lagoon system, storage tank

8.13-87.1 0.4-3.5 _ 3425-

35109

3.3-

34.5 0.2-1.6 _ 1370-14044

Hunt et al., 2009 241.2

Scirpus tabernaemontani, Schoenoplectus americanus,

Scirpus cyperinus, Juncus effusus, Typha latifolia,

Solanum americanum

Anaerobic lagoon 10.7-12 3.7-4.4 1-1.6 11.5-18.8

6.4-7.2 2.9-3.5 0.3-0.5 10.6-17.3

Kantawanichkul

and Somprasert,

2005

0.5 Cacomantis flabelliformis Storage tank 51.7 22.5 1.84 14 31.0 15.8 1.1 13.3

Poach et al., 2007 220 Typha latifolia, Schoenoplectus americanus Lagoon, storage

tank 7.14 1.23 1.1 24.2 4.2 0.7 0.32 15.0

Stone et al., 2004 440 Typha latifolia, Schoenoplectus americanus _ _ 2.3 1.2 _ _ 0.9 0.09 _

Trias et al., 2004 4000 _ Concrete storage,

anaerobic lagoon

1709-

1775

1068-

1533 _

1947-

2270

1542-

1633

982-

1410 _ 1900-2200

Yeh et al., 2009 440 Typha latifolia, Schoenoplectus americanus Lagoon, storage

tank 1.9-:6.1 0.7-4.0 0.8-1.6 4.4-8.2 0.9-2.7 0.3-1.8

0.16-

0.32 1.8-3.3

Zhao et al., 2004 0.09 Phragmites australis Diluted feed tank 259.4 _ OP:

12.7 949.2 192 _ OP: 6.5 132

Hybrid

Poach et al., 2003 120 Schoenoplectus spp., Typha spp., Sparganium spp.

Anaerobic lagoon,

holding tank, nitrification unit

_ 1.4-1.5 _ _ _ 0.98-

1.05 _ _



Table 3.9: Continued

Reference CW area

(m2) Plant Species Pre-treatment

Surface load (gr/m2/day) Removed surface load (gr/m2/day)


HSF

Hathaway et al.

(2010) 4500

Juncus sp., Nuphar lutea, Pontederia cordata, Typha

sp., Scirpus tabernaemontani _ _ 0.007 0.004 _ - 0.005 0.001 _

Kadlec et al. (2005)

18 Scirpus tabernaemontani Settlement tank 9.7-47.3 0.7-4.54 _ _

4.85-

23.6

0.42-2.7

_ _

Knight et al. (2000)

6000 Typha spp., Scirpus spp., Phragmites australis Settling basin,

anaerobic lagoon 0.17-0.26

0.15-0.68

_ 0.21-1.85

0.12-

0.18

0.09-0.48 0.07-0.93

Lee et al., 2004 31.1 Eichhornia crassipes

Solid separation, anaerobic digestion

and aerobic oxidation

39-137

6.9- 26.2

1.5-4.7 30.3-

62.4

31.2-

110

1.03-3.9

0.7-2.1 29.4-60.5

Meers et al., 2005 0.25 Phragmites australis Sludge reactor 3.2 0.104 0.265 _ 2.24 0.08 0.21 _

Meers et al., 2008 4500

Phragmites australis, Typha latifolia, Carex

pseudocyperus, Carex acutiformis, Scirpus lacustris, Scirpus maritimus, Filipendula ulmaria, Iris

pseudacorus, Mentha aquatica, Acorus calamus,

Sparganium erectum, Alisma plantago-aquatica, Lythrum salicaria

Centrifugation,

composting, sludge reactor

0.14 0.03 0.005 _ 0.13 0.03 0.005 _

Zhao et al., 2011 0.9 Typha latifolia, Phragmites australis Secondary treated 4.2 _ 0.2016 6.1 _ _ _ _

VF

Aktas et al., 2001 n.a. Phragmites australis _ 36-474 46-79 8-174

73- 1836

21.6-

284

20.7-35.6 6.4-139

_

Babatunde and Zhao, 2010

575 Phragmites australis _ 575 _ 35 491 _ 32.8



Table 3.9 shows that removal efficiencies of organic matter and nutrients are greater in HSF

than FWS CWs. Specifically, removal efficiencies are 78-90% for COD, 22-90% for BOD,

71-98% for TP, 22-90% for TN, and 22-51% for TSS. The applied surface loads for these

pollutants were 3.2-47.3 gr/m2

day, 0.17-0.26 gr/m2

day, 0.104 gr/m2

day, 0.15-0.68 gr/m2

day, and 0.21-1.85 gr/m2

day, respectively. According to Lee et al. (2004), removal

efficiencies are higher when surface loads are lower. Meers et al. (2005), suggested that by

increasing the HRT and the plant root depth zone in HSF CWs, removal efficiencies would

increase. Results from other studies indicate that HSF CWs can effectively remove pollutants

from various animal wastewaters (Hathaway et al., 2010; Kadlec et al., 2005; Knight et al.,

2000). Babatunde and Zhao (2010), used VF CWs with tidal flow strategy, which can

promote oxygen supply. When treating wastewaters containing high organic loads, where the

oxygen supply from the plant root zone is not sufficient, tidal flow VF CWs increase DO

concentration in the biofilm and thus enhance organic matter degradation.

3.6.6) Vegetation for Animal Farm Wastewater in CW systems

A variety of plants were used in CWs treating animal farm wastewater (Table 3.9). The

selection of plant species was mainly based on the availability of the plant in the area and not

on other criteria. While planted CWs showed higher removal efficiencies, no significant

variations in removal efficiencies were recorder between the different plant species.

The experimental data of Hunt et al. (2006) showed that the increase of nitrogen loading

caused the increase of denitrification enzyme activity (DEA), thus resulting in increased

nitrogen removal efficiency. Another study of Hunt et al., (2009) demonstrated that, when

surface load was high, pollutant removal efficiencies increased in the treatment of swine

wastewaters. Hunt et al., (2009) also found that a layer, which is formed above the soil layer

by a combination of decayed plant material, heterotrophic bacteria and the suspended solids

of the swine wastewater, is characterized by very high DEA. A study by Stone et al. (2004)

revealed that FWS CWs can significantly treat TN but not TP. Poach et al. (2007) observed

that organic matter, TSS and TP removal efficiencies were not influenced by the presence of

aerobic conditions in the FWS CWs. However, aerobic conditions accelerated the

nitrification process thus increasing the removal efficiency of TN. Concerning CW soil,

Babatunde and Zhao (2010) used alum sludge, which proved to enhance treatment efficiency,

specifically phosphorus removal.



3.6.7) Pre-treatment stages in treatment of OMW wastewater

Pre-treatment stages for OMW involve advanced treatment methods including coagulation

(Bubba et al., 2004), electrochemical oxidation (Grafias et al., 2010), and biological trickling

filters (Herouvim et al., 2011) (Table 3.10). The only two experiments/applications on OMW

treatment using CWs lacking a pre-treatment stage (Kapellakis et al., 2009; Yalcuk et al.,

2010) diluted the OMW with tap water before introducing it into the CW system. Although

CWs have been used to treat a variety of wastewaters, only few attempts to treat OMW have

been published (Table 3.10). The main characteristic of all these experiments/applications is

that a pre-treatment stage is imperative to reduce the high organic and phenol loads which

can be toxic to CW vegetation.

3.6.8) CW types in treatment of OMW wastewater

OMW treatment by CWs appears to be sufficient when a HSF system, a VF system, or a

combination of them is used (Table 3.10). The only attempt to treat OMW using FWS

(Kapellakis et al., 2009) showed a significant removal efficiency for organic matter (86%),

however the organic surface load applied was the lowest reported in the literature (5-15 gr/m2

day). HSF CWs appear to be more efficient at treating OMW (Bubba et al., 2004), as removal

efficiencies were 69% for COD, 12% for nitrogen, 55% for phosphorus, 50% for TSS, and

79% for phenols. It should be mentioned that these removal efficiencies were achieved with

pollutant surface loads higher than those applied to FWS systems, but lower than those

applied to VF systems. The most efficient CW system for OMW treatment appears to be the

VF system (Bubba et al., 2004; Grafias et al., 2010; Herouvim et al., 2011; Yalcuk et al.,

2010). VF CWs show high removal efficiencies for all pollutants (72-86% for COD, 75% for

nitrogen, 88-95% for phosphorus, 79% for phenols), while the pollutant surface loads applied

were among the highest reported for all industrial and agro-industrial wastewaters. It appears

from the literature that VF CWs are more efficient at organic matter and phenol removal, but

attention should be paid to the existence of SS, which can cause porous media to clog and

thus damage the CW system. Yalcuk et al. (2010), attributes VF CW treatment ability to

more efficient oxygen transport in the porous media. The higher oxygen concentration in VF

CWs leads to increased organic matter oxidation and ammonia nitrification.



Table 3.10: Studies of olive mill wastewater treatment by CWs.

Reference CW

CW

area

(m2)

Plant Species Pre-treatment

Surface load (gr/m2/day) Removed surface load (gr/m

2/day)

C TKN TP TSS Phenols C TKN TP TSS Phenols

Aktas et

al., 2001 HSF 0.85

Phragmites

australis Coagulation 77.03 1.08 0.42 9.32 16.85 53.1 0.13 0.23 4.6 13.2

Bubba et

al., 2004 VF 0.24 -

Electrochemical

oxidation 15 _ _ _ _ 12.9 _ _ _ _

Grafias et

al., 2010 VF 0.1256

Typha

latifolia C.

alternatifolius

Diluted with tap

water 114.71 _

OP:

2.74 _ _ 82.5 _

OP:

2.6 _ _

Herouvim

et al., 2011 VF

Phragmites

australis Biological filter 6589 175 20.0 - 997 4810 131 17.6 - 748

Kapellakis

et al., 2012 FWS

Phragmites

australis Dilution 5-15 - - - -

4.3-

12.9 - - - -



As agro-industrial wastewaters have common characteristics (i.e., high organic loads, low pH

values, toxic effects) the CWs treating them present the same problems. Thus, general

suggestions can be made on the design and operation of CWs treating agro-industrial

wastewater. The following suggestions concern issues such as the pre-treatment stage, chosen

vegetation, porous media, and CW plain view, and are made based on the previous discussion

and the experimental results of published research.

Regarding the effect of hydraulic loading rates on CW performance, Ghosh and Gopal (2010)

state that it is negatively correlated with organic matter and nitrogen. The effect of hydraulic

load was significant as it could increase threefold the effluent concentration of organic matter

and nitrogen, when it was tripled. Kantawanichkul and Somprasert (2005) also found that

hydraulic load negatively affects nitrogen removal, due to DO decrease. Temperature usually

affects CWs performance, as the main pollutant mechanism of biological degradation, is

temperature dependant. Lee et al. (2010), who examined organic matter and TSS temporal

variations, found that temperature did not significantly affect their removal. This was also

observed by Akratos and Tsihrintzis (2007), who state that organic matter degradation is not

affected by temperature, as aerobic and anaerobic bacteria responsible for organic matter

degradation can function even at low temperatures (50C). Contrary to these observations,

Mustafa et al., (2009) and Newman et al. (2000), report that nitrogen and phosphorus removal

has a strong dependence on temperature. Akratos and Tsihrintzis (2007), who also reported a

strong correlation between nitrogen removal and temperature, claim that this phenomenon

could be attributed to plant uptake and to the fact that nitrified bacteria increase their

performance in temperatures above 150C.

The majority of the experiments/applications presented in this discussion either included a

pre-treatment stage or used diluted wastewater in order to eliminate toxic effects on CW

vegetation. From the published research it is deduced that CWs were used mainly as

polishing treatment stages for agro-industrial wastewater treatment. Bearing in mind that the

main advantage of CWs is their low operational cost, they should be coupled with other low-

cost treatment technologies (e.g., biological trickling filters, coagulation-floculation).

Biological trickling filters have been successfully used in combination with CWs for OMW

treatment (Herouvim et al., 2011; Michailides et al., 2011). The necessity for a pre-treatment

stage is apparent as effluent pollutant concentrations remain above EU recommended limits

(Table 3.11), thus prohibiting its direct disposal or use for irrigation. Effluent concentrations



below EU limits were reported only in cases where influent concentrations were very low

(Gasiunas et al., 2005; Ghosh and Gopal, 2010; Hunt et al., 2009; Jamieson et al., 2007; Kern

et al., 2000; Mantovi et al., 2003; Mustafa et al., 2009; Schaafsma et al., 2000). This

treatment inefficiency could be overcome if CWs were coupled with advanced pre-treatment

stages, which would not only remove SS but would also reduce organic matter and other

pollutant loads. A variety of low cost physicochemical and biological treatment methods have

been successfully tested in agro-industrial wastewater treatment, so the combination of one of

these methods with CWs could be an integrated and viable solution for agro-industrial

wastewater treatment.

Concerning vegetation in CW systems treating agro-industrial wastewater, although

numerous plant species have been tested, no specific species has been proved to be superior

over the others for pollutant removal. The main concern is to select a species resistant to the

toxic wastewaters involved and which is indigenous to the geographical area. Application of

different species indicate that the most tolerant plant species is Phragmites sp. (the common

reed), as it appears resistant to the toxic effects of diary, animal farm and olive mill

wastewaters and demonstrates high pollution removal efficiencies. Furthermore, it can be

found free-growing in most areas. Another issue concerning vegetation is the density of the

initial plants sown. Research results show that increased plant density increases pollutant

removal in the treatment of agro-industrial wastewaters. Initially dense vegetation could also

minimize the adjustment period and possible toxic effects. Therefore, in CWs treating agro-

industrial wastewater, the initial planting density should increase from 4 plants/m2

(Akratos

and Tsihrintzis, 2007) to 6 or 8 plants/m2 depending on the toxicity of the wastewater

concerned. As the plants themselves are responsible for only a small percentage of nutrient

removal (around 3%) (Gottschall et al., 2007; Newman et al., 2000), there is no need for their

periodic removal. On the contrary, this action would cause a decrease in pollutant removal

efficiency, as the oxygen levels in the CW would decrease. The CW should only be replanted

when the initial vegetation turns yellow and dies. Removal of dead plants avoids increasing

nutrient concentrations in the CW caused by the deposition of decaying plant biomass.

During the operation of many CWs treating agro-industrial wastewater, plant density was

recorded to be higher near the CW inlet. This is attributed to higher nutrient concentrations at

and around this point. This phenomenon confirms that horizontal flow CWs (FWS and HSF)

function as plug-flow reactors and therefore the biochemical processes occurring are more

intense near the inlet. This phenomenon could be exploited to increase CW efficiency in two



ways. The first is to change the design of the CW plain view from the common rectangular

shape to trapezoidal. This change would increase the CW area near the inlet where nutrient

availability is higher, plant density is higher and biochemical processes are more intense.

This trapezoidal design was tested by Kotti et al. (2010), who used FWS CWs for municipal

wastewater treatment and found that the trapezoidal CW was c. 8% more efficient at pollutant

removal than the rectangular CW. The second way is to use multiple inlet points along the

units. In this way nutrient availability increases along the length of CW, therefore increasing

both plant density and pollutant removal. This scenario was tested by Stefanakis et al. (2011),

who used three different inlet points and two different schemes (33:33:33 and 60:25:15).

Their results showed that a gradual wastewater inflow from multiple inlet points (60:25:15)

increased pollutant removal efficiencies.

Another CW design issue is the origin of the soil (FWS) or porous media (HSF and VF) used

in the treatment of agro-industrial wastewaters. Most studies have not examined thoroughly

the effect of CW substrate; however one attempt was made to use zeolite as the substrate

(Stefanakis and Tsihrintzis, 2012a). Zeolite, which is a natural absorbent, has also been used

as a substrate of post-treatment filters (Stefanakis and Tsihrintzis, 2012a; b) that increase

organic matter and ammonia removal. Other substrate materials having been tested include

bauxite, flying ash, river gravel, and quarry gravel (Akratos and Tsihrintzis, 2007; Arias et

al., 2001; Drizo et al., 1999; Stefanakis and Tsihrintzis, 2012a; b). From the literature it can

be concluded that substrate origin and chemical composition is critical, as minerals with

reactive Fe and Al or calcareous materials, which promote Ca phosphate precipitation are

rather efficient at phosphorus removal, and materials with high cation-exchange ability will

promote ammonium removal. The major problem in CW application is porous media

clogging, especially when treating wastewaters containing high TSS concentrations, such as

agro-industrial wastewaters. Future research should focus on testing materials that would

increase CW porosity and thus limit the clogging effect. These materials could include the

plastic materials which are already used in biological trickling filters (Michailides et al.,

2011), where biofilm density is higher than in CWs.

Finally the selection of the optimal CW type is a crucial issue for agro-industrial wastewater

treatment. From the previous discussion it appears that the use of subsurface flow CWs (HSF

and VF) is preferable for such heavily polluted wastewaters, since these systems are more

efficient bioreactors. Thus, the most effective treatment systems include both HSF and VF



treatment stages. Although dissolved oxygen concentration levels in FWS CWs are higher

than those in HSF and VF systems, its use in treating wastewaters with high pollutant loads is

not common, as it requires higher HRTs than HSF and VF CWs. This problem was overcome

by Stefanakis and Tsihrintzis (2009) who combined FWS and HSF only by raising the water

level in a HSF system. This new CW type achieved similar removal rates to the HSF system

despite receiving 15-20% increased pollutant loads.



Table 3.11: Pollutant influent and effluent concentrations compared with EU standards (EU Directive 1991/271/EEC).

Influent concentration (mg/L) Effluent concentration (mg/L)

Reference

N-

NH4+

N-

NO3-

N-

NH4+

N-

NO3-

COD TKN TP COD TKN TP

EU standards 120 TN:

10 2

Dairy

Jamieson et al., 2007 1747 237 188 3.7 37 34 19 14 0.6 17

Schaafsma et al., 2000 1900 164 72 5.5 53 53 3.3 32 9.9 2.7

Shamir et al., 2001 285 296 196 <2 - 277 247 128 <2 -

Ghosh and Gopal, 150 20 40 4.5 4 15 3.2 0.2 0.14 2.2 2010

Kern et al., 2000 682 97 74 0.6 10.4 89 63 25 32 4.7

Mantovi et al., 2003 1219 65 22 0.5 13 98 33 25 0.5 5

Moir et al., 2005 2000 - 0.3 - 5.3 210 - 0.1 2.3 6.4

Moreira et al., 2010 254 29 18 - 17 158 17 11 - 13

Mustafa et al., 2009 1500 - 40 3.4 - 75 - 0.4 1 -

Newman et al., 2000 2700 102 7.7 0.3 26 611 74 52 0.1 14

Tanner et al., 1995 - 38 - - 11 - 23 - - 7.5

Animal farm

Hunt et al., 2006 796 171 139 0.6 - 471 87 66 0.6 -

Kantawanichkul and 1034 - 448 - - 310 - 134 - - Somprasert, 2005

Poach et al., 2007 445 66 - - 71 280 30 - - 65

Stone et al., 2004 - 116 86 - 56 - 70 53 - 48

Trias et al., 2004 3220 1333 1072 - - 2200 333 313 - -

Zhao et al., 2004 2500 - 90 2 40 625 - 60 - 19

Hathaway et al., 2010 - 20-120 - - 20-50 - 5-15 - - 10-30

Kadlec et al., 2005 2240 135 118 0 - 658 34 14 7.5 -

Knight et al., 2000 - 907 366 - - - 248 221 - -

Lee et al., 2004 1160 200 185 3.7 40 190 156 144 1.7 21

Meers et al., 2005 3167 104 4.4 101 265 950 26 0.6 9.3 53

Meers et al., 2008 1700 360 - - 58 115 7.8 - - 0.8

OMW

Bubba et al., 2004 129100 90 - - - 95100 560 - - -

Herouvim et al., 2011 14000 506 123 - 95 3500 99 36 - 12

Yalcuk et al., 2010 2880 - 0.9 - 68 750 - 0.5 - 3.5


Chapter 4: Materials and Methods

CHAPTER 4: MATERIALS AND METHODS

4.1) First Experimental Period ............................................................................. 90

4.1.1) Description of pilot-scale CW unit.................................................................................. 90

4.1.2) Wastewater preparation for Cr(VI) treatment ................................................................. 90

4.1.3) Wastewater preparation for secondary cheese whey ....................................................... 93

4.1.4) Monitoring of Wastewater Parameters ............................................................................ 94

4.1.5) Assessment of evapotranspiration ................................................................................... 94

4.1.6) Reed biomass in composting ........................................................................................... 97

4. 1.6.1) Composting materials and process .................................................................. 97

4.1.6.2) Physicochemical analyses ................................................................................. 97

4.2) Second Experimental Period ....................................................................... 100

4.2.1) Description of pilot-scale CW unit................................................................................ 100

4.2.2) Description of the experimental setup ........................................................................... 100

4.2.3) Wastewater preparation ................................................................................................. 101


Chapter 4: Materials and Methods 90

CHAPTER 4: MATERIALS AND METHODS

4.1) First Experimental Period

4.1.1) Description of pilot-scale CW unit

Four pilot-scale HSF CWs were constructed using plastic trapezoidal tanks (units) with

dimensions 1.26 m long, 0.68 m wide (upper base), 0.73 m deep, and a total volume of 0.62

m3. The experiment was carried out in the Department of Environmental and Natural

Resources Management, located in Agrinio, Greece. Two units were used for Cr(VI) removal

(Fig. 4.1) and the other two for SCW treatment (Fig. 4.2). All units were filled with fine

gravel (D50=6mm) and placed in an open-air facility. Two pilot-scale units were planted (Cr-

P, SCW-P) with common reeds (Phragmites australis) and the other two were kept unplanted

(Cr-U, SCW-U). Cr-P and SCW-P were planted at the beginning of the experiments using

reeds obtained from local streams. Six stems of Phragmites australis (7 reeds/m2) were

planted in each unit. Wastewater inflow was through a perforated plastic pipe (diffuser)

placed across the width of the tank at the upstream side (Fig. 4.3). The outlet structure of the

units was an orifice (1/4 inch diameter) at the base of the downstream end of each unit, which

was connected to a U pipe (Fig. 4.4a, b). Overflow wastewater was collected in a 35L plastic

tank for proper disposal (Fig. 4.4c).

4.1.2) Wastewater preparation for Cr(VI) treatment

Tap water enriched with Cr(VI) was introduced into two CW units (Cr-P and Cr-U). The

Cr(VI) concentrations ranged from 0.5 to 10 mg/L. Five different HRTs (i.e., 8, 4, 2, 1 and 0.5

days) were applied to examine the effect of HRT on CW pollutant removal efficiency. The

void volume of clean tanks was estimated by draining the tanks and measuring the water

volume of each tank. Water volume and mean porosity for Cr-P were 108 L and 28%,

respectively, and for Cr-U, 112 L and 29%, respectively. Wastewater was introduced into the

units from the influent storage tanks using valves that were properly adjusted to give the

desired discharge (Fig. 4.5).



Figure 4.1: Pilot-scale HSF CWs for the treatment of Cr(VI).

Figure 4.2: Pilot-scale HSF CWs for the treatment of SCW.



Figure 4.3: Perforated inflow pipes (diffuser) in both units (Cr-U and Cr-P).

Figure 4.4: Outlet of the CWs: (a) Tube positioned at the same height as the filler material),

(b) Effluent collection point, (c) 35L plastic tank for overflow wastewater collection.

a b

c



Figure 4.5: Influent storage tank with adjustable valve.

4.1.3) Wastewater preparation for secondary cheese whey

Void volume was estimated by draining the tanks and measuring the water volume of each

tank. Water volume and mean porosity for SCW-P were 104 L and 25%, respectively, and for

SCW-U 114 L and 27%, respectively. A series of HRTs (i.e., 8, 4, 2 and 1 day) were studied

to examine the effect of HRT on the pilot-scale HSF CWs in removing COD. Influent COD

concentrations ranged from 1200 to 7200 mg/L in order to examine the effect of pollutant

load on COD removal.

Cheese whey: The secondary cheese whey used as the influent source in this experiment was

obtained from the factory of Papathanasiou S.A., ―Dairy and Cheese products of Trixonidas

highland, Agrinio, Greece. Fresh secondary cheese whey was collected regularly from the

factory and transported to the laboratory where it was refrigerated at 4°C to avoid any

acidification or change in its chemical composition. The COD concentrations of the

secondary cheese whey ranged from 40,000 to 50,000 g/L. A full-scale biological filter was

recently constructed and operated in order to treat the factory’s wastewaters (Tatoulis et al.,

2014a). The pilot-scale CWs were examined as a post-treatment stage to the effluent from the

biological filter. Therefore, the secondary cheese whey was diluted with tap water before

being introduced into the pilot-scale CW units, in order to adjust COD influent concentrations

to the biological filter’s effluent concentrations (1000 to 5000 mg/L).



4.1.4) Monitoring of Wastewater Parameters

Samples were collected with a frequency equal to the HRT and were taken from the influent

and the effluent points of each unit. Physicochemical parameters such as pH, electrical

conductivity (EC) and dissolved oxygen (DO) were measured in-situ for both points. pH and

EC were measured using a CONSORT C 835 multi-parameter analyzer, and DO was

measured using a HANNA HI-9143 Microprocessor D.O. meter. In the laboratory the

samples were analyzed for Cr(VI) following the 3500-Cr D Colorimetric method with

detection limit of 0.013 mg/L (APHA, 1998). COD was monitored by the absorbance of the

sample after dichromate digestion at 1500C for 2 h in the presence of silver and mercury

sulfates (closed reflux method, APHA et al., 1989). The absorbances were measured using a

HANNA C99 Multiparameter Bench Photometer (digested in HANNA instruments C9800

REACTOR). Meteorological data were obtained from the National Observatory of Athens

meteorological station in Agrinio. For the two-year duration of the experiment (2011-2012),

the mean experimental temperature was 20.6oC and the mean precipitation was 1390 mm

(Figs. 4.6 and 4.7).

4.1.5) Assessment of evapotranspiration

Evapotranspiration (ET) was assessed on a daily basis. Each morning the outlet storage tanks

were emptied and the volume of the treated wastewater was measured. Additionally, the

volume of the wastewater introduced into the units was also measured on a daily basis.

Influent and effluent volumes as well as precipitation levels were then used to assess ET.

During days with high temperatures and solar radiation, ET exceeded wastewater influent

volumes, leading to a reduction in the units’ water level, as ET values reached 15.5 L/d.



0 45 90 135

180

225

270

315

360

405

450

495

540

585

630

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

Tem

per

atu

re (

oC

)

Days

Figure 4.6: Time series chart for air temperature (2011-2012).



0 45 90 135

180

225

270

315

360

405

450

495

540

585

630

0

5

10

15

20

25

30

35

40

45

50

55

60

R

ain

fall

Dep

th (

mm

)

Days

Figure 4.7: Time series chart for precipitation (2011-2012).



4.1.6) Reed biomass in composting

4. 1.6.1) Composting materials and process

Olive pomace (OP), olive leaves (OL) and Cr treated reed plants (RP) were used for this

study. Two trapezoidal bins with dimensions 1.26 m long, 0.68 m wide (upper base), 0.73 m

deep and a total volume of 0.62 m3 were used for the composting experiments. Table 4.1

presents the experimental set-ups, where in bin 1, RP mass was higher than in bin 2. Figures

4.8a & b show the first day of composting after mixing of all the materials in both bins.

During composting, aeration was achieved by mechanical turning, which took place daily for

the first three days, every four days during the thermophillic phase, and once a week during

the maturation phase. Moisture contents were maintained above 45% as moisture contents

between 45-60%, by weight, are ideal for the composting process (Gajalakshmi and Abbasi

2008). The composting mixture was kept in the bins for 66 days. It was then removed from

the bins and stored in a protected area for up to 102 days to mature (Fig. 4.9).

Table 4.1: Description of experimental set-ups of composting.

Bin

(B)

Olive

pomace

Olive

leaf

Reed

plant

Seed

compost

(kg)

Wetting

agent

Initial

mass

(kg)

Initial

vol

(L)

Final

mass

(kg)

Final

vol (L)

B1 3.5 2.0 0.5 35.66 Water 194.49 435 139.72 263

B2 3.75 2.0 0.25 36.23 Water 204.78 435 144.83 270

4.1.6.2) Physicochemical analyses

During the experiments both compost and ambient temperature were monitored daily.

Compost temperature was measured using a temperature probe positioned in the middle of

each bin at a depth of 0.25 m. Compost samples were taken from each bin every six days and

analyzed for moisture content, pH, electrical conductivity (EC) and volatile solids (VS).

Moisture content was determined by drying the sample at 1050C for 24 h in an oven. To

measure volatile solids, the oven dried sample was burned at 6000C for four hrs (APHA et al.,

1998). pH and EC were measured in aqueous extract (using 1:10w/v). The remaining sample

portion was air dried, ground, sieved (0.5 mm), and then used to determine nitrogen (TKN),

total organic carbon (TOC), total phosphorus (TP), potassium (K), and sodium (Na), and

perform phytotoxicity tests. TKN was measured following the methods of Bremner and



Mulvaney (1982), and TP was measured following Murphy and Riley (1962). Na and K were

measured using a flame photometer (Model FP902). Phytotoxicity was estimated using the

germination index (GI) as described by Zucconi et al. (1981) using Lepidium sativum seeds.

Total Cr was measured according to wet digestion method (Allen, 1974) and the resulting

extracts were analyzed by atomic absorption spectrometry (AAS) to determine the

concentration of Cr.

Figure 4.8a: Composting materials in Bin 1 at day 1.

Figure 4.8b: Composting materials in Bin 2 at day 1.



Figure 4.9a: Matured compost in Bin 1 at 102 days.

Figure 4.9b: Matured compost in Bin 2 at 102 days.



4.2) Second Experimental Period

4.2.1) Description of pilot-scale CW unit

The four pilot-scale CWs that were used in the first experimental period were also used in the

second experimental period (Fig. 4.10). At the end of the first experimental period, when reed

plants were regenerating, the second experimental period started. The hydraulic structure and

the outlet design of the pilot units remained unchanged.

Figure 4.10: Integrated treatment of cheese whey and Cr(VI) in pilot-scale HSF CWs.

4.2.2) Description of the experimental setup

The four pilot-scale CWs are shown in Fig. 4.10. Two units were used for the treatment of

cheese whey wastewater and two for Cr(VI) solution wastewater. In the first experimental

period, the pilot-scale CWs operated at different hydraulic residence times (HRT) and

received different concentrations of pollutants in order to examine the effect of all operating



parameters on the removal rates of COD and Cr(VI). In the second experimental session, the

four pilot-scale CWs were used to co-treat mixed cheese whey and Cr(VI) wastewaters.

Section 4.1 describes the first experimental period, i.e. residence times and inlet

concentrations of COD in the pilot units (SCW-P - planted and SCW-U - unplanted).

Similarly, hydraulic residence times and inlet concentrations of Cr(VI) in the pilot-scale units

(Cr-P - planted and Cr-U - unplanted) were discussed in Section 4.1.

In the second experimental period, co-treatment of cheese whey and Cr(VI) wastewaters was

performed in the four pilot-scale constructed wetlands. Cheese whey was used as the carbon

source in the treatment of Cr(VI). Inlet concentrations of COD and Cr(VI) are presented in

Table 4.2. The same HRTs and inlet concentrations were used in both experiments to allow

comparison of all results. For the second experimental period (i.e. 2013), the mean

temperature was 22.4oC and total precipitation was 667 mm (Figs. 4.11 and 4.12).

Table 4.2: Concentrations of Cr(VI) and COD used in the second experimental period.

HRT (days) Inlet concentration of

COD mg/L

Inlet concentration of

Cr mg/L

SCW-P, SCW-U

8 3000 5

4 2500 2.5, 0.5

Cr-P, Cr-U

8 2500 5

4 2000 2.5

4.2.3) Wastewater preparation

Fresh SCW was collected regularly from Papathanasiou S.A. It was transported to the

laboratory and refrigerated at 4°C. The COD concentration in the SCW ranged between

45000 to 50000 mg/L. The SCW was then diluted with tap water and Cr(VI) was added to

prepare a combined wastewater. This mixed wastewater was introduced into the pilot-scale

CW units as influent by adjusting the different concentrations of COD and Cr(VI) (COD

ranged from 2500 to 3000 mg/L and the concentrations of Cr(VI) were 0.5, 2.5 and 5 mg/L.



0 30 60 90 120 150 180 210 240

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

T

emp

erat

ure

(o C

)

Days

Figure 4.11: Time series chart for air temperature (2013).



0 30 60 90 120 150 180 210 240

0

5

10

15

20

25

30

35

40

45

50

55

60

R

ain

fall

Dep

th (

mm

)

Days

Figure 4.12: Time series chart for precipitation (2013).


Chapter 5: Pilot-Scale HSF Constructed Wetlands Treating Cr(VI)

CHAPTER 5: PILOT-SCALE HSF CONSTRUCTED WETLANDS

TREATING Cr(VI)

5.1) Effects of Physicochemical Parameters of Wastewater ................................................... 104

5.1.1) Temperature .................................................................................................................. 104

5.1.2) pH .................................................................................................................................. 105

5.1.3) Electrical Conductivity (EC) ......................................................................................... 109

5.1.4) Dissolve Oxygen (DO) .................................................................................................. 112

5.2) Treatment Efficiency of pilot-Scale HSF Constructed Wetlands .................................. 112

5.3) Effect of HRT ...................................................................................................................... 117

5.4) Cr Mass Balance ................................................................................................................. 121

5.5) Composting of Plant Biomass ............................................................................................ 124


Chapter 5: Pilot-Scale HSF Constructed Wetlands Treating Cr(VI) 104


TREATING Cr(VI)

5.1) Effects of Physicochemical Parameters of Wastewater

Two pilot scale units (Cr-P and Cr-U) were used for Cr(VI) treatment and operated continuously

for two years under different HRTs, and different Cr(VI) influent concentrations. During the

operation period, physicochemical parameters (i.e., temperature, pH, Electrical Conductivity-EC

and Dissolved Oxygen-DO) were measured at the influent and effluent points of both pilot-scale

units. Although the parameters did not show any significant variations between the units, their

effects are briefly discussed in the following section.

5.1.1) Temperature

Temperature variations followed seasonal changes. As the Cr(VI) solutions used to feed Cr-P and

Cr-U were prepared using only tap water, temperatures at the inlet points of both units did not

present great temporal variations and ranged from 20 to 250C. On the other hand, wastewater

temperature at the outlet points ranged between 2.5 to 32.9oC, showing seasonal dependence.

However, temperature did not show significant variation between the units (p value=0.095).

Srisatit and Sengsai (2003) reported lower effluent temperature in planted wetlands than

unplanted wetlands and argued that the vegetations of the planted wetland blocked the sunlight.

During the first year of operation of the pilot-scale units, with an HRT of 8 days, Cr(VI) removal

efficiency did not show great variations between the two units (Michailides et al., 2013).

Moreover, with the exception of the initial commissioning phase, Cr-U showed high

concentrations of Cr(VI) in the effluent (mean value 3.84 mg/L) which were not affected by

temperature fluctuations.

To statistically assess the effect of temperature on Cr(VI) removal efficiency with HRTs of 8

days, the data were divided into two sets, one above and one below 150C. This temperature value



was selected because below it neither the bacteria responsible for nitrogen removal nor the

vegetation functions properly (Akratos and Tsihrintzis, 2007). Cr-U showed extremely low

removal efficiencies (15% for temperatures >15oC and 7% for temperatures <15

0C) in this

period, which proved significantly different (p value = 0.0<0.05). Although the Cr-P units

showed higher removal efficiencies in the same period (95% for temperatures >150C and 80% for

temperatures <150C) they were also affected by temperature (p value = 0.004<0.05). Although

temperature affected Cr(VI) removal efficiency in both units, it could not be attributed to the

same phenomenon. Cr(VI) in Cr-P is mainly removed, as already mentioned, through plant

accumulation. Thus, the effect of temperature on Cr-P performance is mainly caused by the

reeds’ annual growth cycle, as in low temperatures common reeds limit their growth and usually

decay. Nevertheless, even at low temperatures Cr(VI) removal efficiencies remained high (80%).

On the other hand, temperature affects Cr-U performance as low temperatures limit microbial

activity.

5.1.2) pH

Hydrogen ion activity (i.e. pH) is a key factor that influences metal chemistry and mobility and

has a significant impact on the uptake of heavy metals (Zeng et al., 2011). The solubility,

mobility and bioavailability of metals increase when the pH decreases (Gambrell, 1994).

Hydroxide minerals of metals are less soluble under pH conditions of natural water. As

hydroxide ion activity is directly related to pH, the solubility of metal hydroxide minerals

increases with decreasing pH, and they become potentially available for biological processes as

pH decreases (Salomons, 1995). During the operational period pH values were monitored at the

inlet and outlet points of both the Cr-P and Cr-U units. Figure 5.1a presents the pH time series

chart for the Cr-U unit. pH does not present any significant variations during the operational

period for the Cr-U unit. The mean influent and effluent pH values in the Cr-U unit were

identical (pH 7.46), while maximum and minimum influent values of pH in Cr-U were 7.96 and

7.04, respectively and at the Cr-U outlet pH maximum and minimum values were 7.91 and 5.77,

respectively. As stated above, metal mobility is influenced by pH value. Heavy metals are co-

precipitated in ferrous-iron oxides and the oxiferric hydroxide surfaces are positively charged in

acidic pH conditions and negatively charged in alkaline pH conditions (Sheoran and Sheoran,



2006). Thus to enhance the adsorption and removal of metal oxyanions, iron-coprecipitation

should occur under acidic pH conditions (Brix, 1993). Furthermore, as pH increases there is

competition between OH- and chromate ions (CrO4

2-) and these become the dominant species at

higher pH values (<6), however adsorption ceases at pH >9 (Baral et al., 2006). Kongroy et al.

(2012) stated that Cr accumulation in sediment could be due to precipitation under high pH

condition (pH= 8-9). Lesage et al. (2007b) reported that too low and too high pH value is

unfavourable for metal sorption. Mant et al. (2006) came to same conclusion about pH though

they did not mention the pH value. As in this study the mean pH value of the Cr-U unit was

found to be 7.46, it could be concluded that this pH value may enhance the precipitation of

Cr(VI) in the unplanted unit.

Figure 5.1b presents the pH time series chart for the Cr-P unit. The mean influent pH value was

7.49 and the mean effluent pH value was 6.76. pH values showed no dependence on HRT as they

remained stable throughout the operation period. At the effluent point of CW-P unit, the

maximum and minimum values of pH were recorded as 7.38 and 5.32, respectively.

Peng et al. (2009) stated that lower pH values can increase the bioavailability of metals in

sediment. It is also reported that metal mobility is high when pH ranges between 5 to 6.7, and

moderate when pH values reach 7 (Valerie et al., 2004). pH not only influences metal

bioavailability (Zeng et al., 2011) but also influences plant growth in substrate (Calheiros et al.,

2007). According to EPA (2000b), the optimum pH range is 3.7 to 8 for reed plant development

in CWs. However, in this study, the pH value of the Cr-P unit varied between 7.38 and 5.32

which was suitable for plant growth and also for bioavailability of Cr(VI). At this pH range the

ionic forms of Cr(VI) are more soluble and mobile in water (Baral et al., 2006) and when these

ionic forms become soluble, they become more available for bioaccumulation (Cooper et al.,

1996). The same pH range was also recorded by other authors (Qian et al., 2012; Dotro et al.,

2011a;b; Fibbi et al., 2012; Kongroy et al., 2012). Furthermore, the pH data were statistically

analyzed to assess variations between the planted and unplanted units. The results indicates that

there were no significant variations of pH value between the units as the p value was 0.100 >

0.05.



0 45 90 135 180 225 270 315 360 405 450 495 540 585 630

6,0

6,5

7,0

7,5

8,0

8,5

9,0

9,5

10,0

Influent

Effluent

HRT alterations

Plant Harvesting

Win

ter

AutumnSummerSpringWinterAutumnSummerS

pri

ng

1 d

ay

HR

T

12

h H

RT

1 d

ay

HR

T

2 day HRT

p

H

Days

8 days HRT

4 d

ay

s H

RT

2 d

ay

HR

T

1 d

ay

HR

T1

2 h

HR

T

Figure 5.1a: pH values of influent and effluent wastewaters in the Cr-U unit.



0 45 90 135 180 225 270 315 360 405 450 495 540 585 630

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

9,5

10,0

10,5

11,0

11,5

12,0

Influent

Effluent

HRT alterations

Plant Harvesting

p

H

Days

8 days HRT

4 d

ay

s H

RT

2 d

ay

HR

T

1 d

ay

HR

T

12

h H

RT

2 day HRT

1 d

ay

HR

T1

2 h

HR

T

1 d

ay

HR

TW

inte

r

AutumnSummerSpringWinterAutumnSummer

Sp

rin

g

Figure 5.1b: pH values of influent and effluent wastewaters in the Cr-P unit.



5.1.3) Electrical Conductivity (EC)

Electrical conductivity is an essential parameter for wetlands because it measures a solution’s

ability to carry an electrical current and quantifies the amount of dissolved salts in the

wastewaters. This value was monitored for both pilot-scale units to assess its role in Cr(VI)

removal. Figure 5.2a shows the EC chart for the Cr-U unit with different HRTs during the

operational period. The mean EC in the CW-U unit was almost same for both influent and

effluent points with the value 357 of µS/cm. Maximum and minimum values of EC at influent of

CW-U unit were 685 and 265 µS/cm, respectively. At the effluent point, the maximum and

minimum values of EC were 784 and 235 µS/cm, respectively. However, yearly performance of

EC in the CW-U unit did not show any variation. The EC values recorded were rather low,

because of the use of aqueous solutions and not real wastewaters. The EC chart for the Cr-P unit

is presented in Fig. 5.2b. Mean EC values showed an increasing trend in the Cr-P unit with a

mean influent value of 356 µS/cm and a mean effluent of 442 µS/cm.

Many authors (Dotro et al., 2012; Kongroy et al., 2012; Kelvin and Tole 2011; Maine et al.,

2007) have reported high EC values in the influents as all of them used real wastewaters

including tannery, metallurgic factory, tool factory and storm runoff wastewaters. However,

Kelvin and Tole (2011) also observed an increased trend of EC in their study. Overall EC values

of both units were assessed statistically and the results indicate that there was no significant

variation of EC concentrations between the units (p value= 0.111> 0.050).



0 45 90 135 180 225 270 315 360 405 450 495 540 585 630

200

300

400

500

600

700

800

Influent

Effluent

HRT alterations

Plant Harvesting

1 d

ay

HR

T

EC

(µ

S/c

m)

Days

8 days HRT

4 d

ay

s H

RT

2 d

ay

s H

RT

1 d

ay

HR

T1

2 h

HR

T

2 days HRT

12

h H

RT

1 d

ay

HR

T

Sp

rin

g

Summer Autumn Winter Spring Summer Autumn

Win

ter

Figure 5.2a: EC value of influent and effluent wastewaters in the Cr-U unit.



0 45 90 135 180 225 270 315 360 405 450 495 540 585 630

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400E

C (

µS

/cm

)

Days

8 days HRT

4 d

ay

s H

RT

2 d

ay

s H

RT

1 d

ay

HR

T

12

h H

RT

2 day HRT

1 d

ay

HR

T1

2 h

HR

T

1 d

ay

HR

T

Sp

rin

g

Summer Autumn Winter Spring Summer Autumn

Win

ter

Influent

Effluent

HRT alterations

Plant Harvesting

Figure 5.2b: EC values of influent and effluent wastewaters in the Cr-P unit.



5.1.4) Dissolve Oxygen (DO)

Dissolves oxygen (DO) is an important parameter to in CWs since the amount of dissolved

oxygen in the water is directly related to aerobic bacteria in the system. Sufficient DO is essential

to support plant, aquatic and microbial life in CWs. Very low or very high concentrations of DO

can be harmful to aquatic life. The DO concentration at the inlet and outlet of both wetlands did

not show any significant variation in this study. The mean DO values recorded at the inlet and

outlet point of CW-U unit were around 6.22 and 4.22, respectively. In the CW-P unit, the mean

influent and effluent concentrations of DO were 6.43 and 4.33 mg/L, respectively. The overall

variations of DO concentration in the pilot-scale units were analyzed statistically and the results

showed that the variations of DO concentration between the units were not significant (p

value=0.435 >0.05).

DO concentrations have no significant influence on Cr(VI) reduction (Jakobs, 2012; Han, 1999).

Dotro et al. (2011a), stated that oxygen availability has no impact on Cr removal in an abiotic

system when the DO remains at 7.4 mg/L. Willow and Cohen (2003), also state that DO in water

has little influence on heavy metal removal. However, Maine et al. (2006) also recorded a similar

DO value to the one recorded here.

5.2) Treatment Efficiency of pilot-Scale HSF Constructed Wetlands 1

Figures 5.3a % b present time series charts for influent and effluent concentrations of Cr(VI) for

both the planted and unplanted units. Effluent concentrations were corrected for rainfall and ET

volumes have been removed. Effluent concentration correction was necessary as on days with

high rainfall, the effluent concentrations were lower due to dilution by rainwater, leading to an

overestimation of removal efficiency. During the treatment periods with high temperatures and

high solar radiation (e.g. summer), the water level decreased due to high ET rates, leading to

underestimation of removal efficiency.

1 Data presented in Sultana et al., 2014a



During the first year of operation, the two CW units showed significant differences in Cr(VI)

removal, with the exception of the first one hundred days. During these first 100 days, both units

showed a similar fluctuation in removal efficiency that was attributed to the CW commissioning

phase (Michailides et al., 2013). In the first year of treatment, the wetlands were treated with

mean Cr(VI) influent concentrations of 5.61 and 5.47 mg/L for CW-P and CW-U, respectively

and an HRT of 8 days was used. The mean Cr(VI) effluent concentrations were recorded 0.86

and 3.84 mg/L for CW-P and CW-U, respectively and the mean removal efficiency of Cr was

recorded as 85% and 26% for CW-P and CW-U, respectively. From the results of the first year, it

is clear that Cr(VI) removal in CW-P was higher than in CW-U with 8 days of HRT. Therefore,

the difference in removal efficiencies between the two units could only be attributed to the

presence of the reed vegetation.

For the most effective pollutant removal from the wetlands, the plants should be harvested in

winter when the life cycle of reed is complete. Following plant harvesting (Fig. 5.3b), the effluent

concentrations of the CW-P increased, reinforcing the assumption that the main Cr(VI) removal

mechanism is plant biomass accumulation. With the exception of the initial 100 days, the Cr-U

showed high effluent concentrations most of the year. These high effluent concentrations were

not affected by temperature fluctuations thus implying that microbial accumulation of Cr(VI) is

minimal and that during the initial 50 days of operation, Cr(VI) was mainly removed via porous

media absorption. The ability of reeds to accumulate Cr(VI) in their roots and transfer it to stems

and leaves has been proved in previous experiments by Aguilar et al. (2008).

After the regeneration of reed plants, in the second year, both the wetlands were treated with

different concentrations of Cr(VI) with different HRTs. Throughout the operation period the two

units showed significant differences in Cr(VI) removal, with the exception of the first 40 days of

the second year as the plants were regrowing (Fig. 5.3b). During these 40 days both units showed

a similar fluctuation in removal efficiency. These differences in Cr(VI) removal efficiencies are

not due to temperature fluctuations, as the environmental temperature rose in the same period.

However, both Cr-P and Cr-U present clear differences in Cr(VI) removal, as the mean removal

rate in Cr-P was 87.47%, but only 21% in Cr-U.



Figure 5.3a shows that Cr(VI) concentrations decreased slightly in the Cr-U unit. In this unit, the

main mechanisms of Cr(VI) removal were probably accumulation by microorganisms and

absorption by the porous media. Although the removal rate was low, it may also be explained as

some chromium hydroxide precipitation is dependent on pH. When pH is decreases precipitation

of chromium hydroxide increases within a certain range of pH (5.5-8) (Karale et al., 2007; Alves

et al., 1993). This limited Cr(VI) removal in the Cr-U unit can also be attributed to the fact that

microorganisms received water contaminated with Cr(VI) and not wastewater. The contaminated

water has lower concentrations of organic carbon and nutrients and therefore limits the growth of

microorganisms due to the lack of sufficient carbon and nutrients. Furthermore, the porous media

used in these pilot-scale units was river gravel which does not have high absorption ability.

On the contrary, the Cr-P unit removed almost all the Cr(VI), as its removal efficiency was close

to 100% for most of the operating period (Fig. 5.3b). In this unit, Cr(VI) removal efficiency

showed variations in specific time periods. In the winter period when the plants were

regenerating, Cr(VI) removal efficiency in the Cr-P unit showed a fluctuation but still showed

better performance than Cr-U. This reinforces the argument that the main removal mechanism of

Cr(VI) is accumulation in reed plants throughout the year. The other period where CW-P showed

lower Cr(VI) removal efficiency, was when the HRTs were changed. This is because the Cr-P

unit required an adjustment period when higher Cr(VI) loads were introduced. Otherwise a high

removal efficiency of Cr(VI) was observed throughout the experimental period in the Cr-P unit

which suggests that the Cr(VI) removal was performed by the plants. In addition to this,

hydroponic gravel bed systems contain an extensive variety of micro-environments near the plant

root surfaces and within the biofilms that form on root surfaces and gravel (Williams, 1993).

Nevertheless, if no biofilm develops on the plant roots, roots still serve as ground for metal

binding, due to the presence of organic ligands such as carboxyl, hydroxyl, and phenolic

functional groups (Mant et al., 2006). Although the Cr-P was subjected to several variations of

HRT and Cr(VI) feed concentration, the plants did not show any signs of injury and continued to

grow normally.



0 45 90 135 180 225 270 315 360 405 450 495 540 585 630

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Con

cen

trat

ion

(m

g/L

)

Influent

Effluent

HRT alterations

Plant Harvesting

Days

Sp

rin

gSummer Autumn Winter Spring Summer Autumn

Win

ter

8 days HRT

4 d

ays

HR

T

2 d

ays

HR

T

1 d

ay H

RT

12 h

HR

T

2 days HRT

1 d

ay H

RT 12

h H

RT

1 d

ay H

RT

Figure 5.3a: Time series charts of Cr(VI) influent and effluent concentrations for Cr-U.



0 45 90 135 180 225 270 315 360 405 450 495 540 585 630

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Influent

Effluent

HRT alterations

Plant Harvesting

C

once

ntr

atio

n (

mg/

L)

Days

Sp

rin

gSummer Autumn Winter Spring Summer Autumn

Win

ter

8 days HRT

4 d

ays

HR

T

2 d

ay

s H

RT

1 d

ay

HR

T 12

h H

RT

2 days HRT

1 d

ay H

RT 12

h H

RT

1 d

ay

HR

T

Figure 5.3b: Time series charts of Cr(VI) influent and effluent concentrations for Cr-P.



A number of studies have been done on the removal of Cr from different wastewaters using HSF

CWs planted with reeds. Different authors used different Cr concentrations either with a single

species or with mixed vegetation. Concerning the use of different chromium concentrations,

Arroyo et al. (2010) used the highest concentration (5.64 mg/L) with a long HRT and different

plant species, where they recorded 55% Cr removal efficiency. Whereas, using a single species,

with almost the same Cr concentrations and a much lower HRT (1 day), the highest Cr removal

efficiency was recorded by Sultana et al., (2014a). Dotro et al. (2011b) also used the same

concentration with a longer HRT and Typha spp., and achieved 90-99% removal rates of Cr(VI).

Fibbi et al. (2012), Lesage et al. (2007a) and Kucuk et al. (2003), used single vegetation of

common reeds but with very low concentrations of Cr (0.0008-0.0158, 0.113, 0.2 mg/L,

respectively) and HRTs of not less than 2 days. Among these research groups, Lesage et al.

(2007a), recorded the highest removal efficiency of 87%. However, they did not discuss effects

of HRT on Cr removal.

5.3) Effect of HRT

Four different HRTs were applied to both CW units throughout the operational period (Figs. 5.3a

& b). During the first year an HRT of 8 days was used to avoid plant stress from the high Cr(VI)

loads. After the first year of operation, the HRT was adjusted from 8 days to 4, then to 2, and

finally to 1 day. HRTs of 12hrs were also tested however, removal efficiencies of both units

showed great variations probably due to the diurnal cycles of the plant vegetation. Pollutant

surface loading rate (SLR) and HRT are very significant parameters for wastewater treatment

efficiency in CWs. The mean influent SLR ranged from 0.03 to 0.55 gr/m2/d for Cr-U and from

0.03 to 0.52 gr/m2/d for Cr-P. Figures 5.4a & b presents correlation charts for influent Cr(VI)

surface load and Cr(VI) removal efficiency. Cr-P showed a slight increase in Cr(VI) removal

efficiency with SLR, while Cr(VI) removal efficiency in Cr-U decreased slightly with SLR.

Nevertheless, removal efficiency in both units did not vary significantly with SLR when influent

concentrations ranged from 0.5 to 10 mg/L.

Figure 5.3a & b present the effect of different HRTs on influent (5, 2.5, 1 and 0.5 mg/L) and

effluent concentration of Cr(VI). Maximum Cr(VI) removal in CW-P was found to be 100 % (i.e.

effluent concentration was 0 mg/L) with HRT of 8, 4, 2 and 1 day and Cr(VI) concentration of 5



mg/L, while the lowest removal rate of 44.78% was observed in the Cr-P unit with HRTs of 8

days. In the Cr-U unit, the maximum removal rate was recorded 50.24% at 8 days of HRT

(during the regeneration period). The removal rate of Cr(VI) in Cr-P at the lowest HRT (1 day)

was also 100% with the highest Cr(VI) concentration of 5 mg/L. However, the Cr-U unit showed

only 9.17 % of removal of Cr(VI) with the same initial influent concentration. Only a slight

difference was found between inlet and outlet of the Cr-U. From this description, it is clear that

there was a significant variation between two pilot-scale wetlands due to the HRT.

One-way ANOVA statistical analyses were performed to examine if the Cr(VI) removal

efficiencies showed statistically significant differences with different HRTs. Statistical analysis

was not performed on the data of the first operational period, however the differences between

the planted and unplanted units were obvious. The data of the second year used for the ANOVA

analysis were taken from periods during which temperatures were not significantly different (p

value =0.095>0.05) and Cr(VI) feed concentrations were almost identical, thus exhibiting only

the effect of HRT (Table 5.1). Mean removal values (under steady state conditions) and

temperatures for HRTs of 8, 4, 2 and 1 days at higher (21.8-28oC) and lower (16.7-18.3

oC)

temperatures are presented in Table 5.1. It should be noted that effluent concentrations were

always below the permitted limits of 0.05 mg/L (EC, 1998).

Table 5.1: Comparisons of Cr (VI) removal between different HRTs and temperatures.

Time period HRT

(d)

Cr(VI) Feed

concentration

(mg/L)

Temperature 0C

Cr(VI) removal (%)

CW-U unit CW-P unit

Mean SD Mean SD Mean SD

High temperatures

29/4-9/5/12 8 4.8 21.8 1.0 16.8 17.7 100 0

22/5-10/6/12 4 5.1 23.3 2.3 16.9 10.9 100 0

11/6-24/6/12 2 4.9 28.0 1.5 31.9 14.2 100 0

27/6-4/7/12 1 5.0 27.9 0.4 37.1 10.9 100 0

Low temperatures

25/10-2/11/12 2 1.0 18.3 2.6 9.2 29.9 100 0

3/11-8/11/12 1 0.9 16.7 2.3 45.9 2.5 100 0



0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

-80

-70

-60

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

R

emo

va

l (%

)

Surface load (gr/m2/day)

Figure 5.4a: Effect of surface load on Cr(VI) removal for Cr-U



0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

10

20

30

40

50

60

70

80

90

100

110

Rem

oval

(%

)


Figure 5.4b: Effect of surface load on Cr(VI) removal for Cr-P.



For the high temperature season, Cr(VI) removal did not differ significantly in the Cr-U (p value

= 0.061>0.05) and Cr-P units (all removal rates were 100%). For the low temperature period,

Cr(VI) removal efficiencies for CW-U were significantly different (p value =0.009>0.05), while

for the Cr-P unit there were no significant differences as all removal rates of Cr(VI) were 100%.

It is clear that the Cr-P units have stable Cr(VI) removal efficiencies that depend only on plant

function and decrease only for a short period of the year (from plant harvesting to regeneration

period). The stable Cr(VI) removal efficiency demonstrated by the CWs throughout most of the

year even though the planted unit operated with low HRTs (of 1 day), makes CWs a promising

treatment technology for Cr(VI) removal. As Cr-P removal efficiencies with HRTs of 1 day were

high, an HRT of 12 hours was also tested. However, results were not satisfactory as Cr(VI)

removal efficiency exhibited significant variations during the same day. After the summer when

the reed plants had aged, the Cr(VI) influent concentration was decreased to 2.5 mg/L, and HRTs

of 2, 1 and 0.5 days were tested. In this experiment the CW-P unit also showed 100% efficiency

for HRTs of 2 and 1 day with Cr(VI) concentrations of 2.5 mg/L, while the CW-U unit showed

only 69.23% and 12.04% removal efficiency for HRTs of 2 and 1 day, respectively. As the life

cycle of reeds completes at the end of the autumn the same experiment was also carried out with

a low concentration of Cr(VI) (1 mg/L) for HRTs of 2 and 1 day. In this case, the Cr-P unit again

demonstrated 100% removal efficiency, whereas the Cr-U unit showed very low removal

efficiencies.

5.4) Cr Mass Balance

During the two year operation of the pilot-scale units, plant biomass was removed from the Cr-P

unit twice. Specifically, above ground plant biomass was harvested in January (Fig. 5.5). After

harvesting, the leaves and stems were separated and dried in order to estimate their Cr content.

The total dry mass of leaves and stems was 3 and 4 kg, respectively. To ensure plant regeneration

during the spring root biomass was not removed. Nevertheless, in several studies it is reported

that for common reeds the ratio of root mass to above ground mass ranges from 1:1.8-1:9.9

(EPA, 1998; Cronk and Fennessy, 2001). Thus, root mass should range from 7.2 kg to 39.6 kg.

To estimate total Cr concentration in the roots, a single reed root was removed in each harvest.

Table 5.2 presents the Cr concentrations recorded in different parts of the reed plants from the



first and second years of experiments and from the literature. It can be seen that Cr concentrations

show great variations in common reeds; however the Cr concentrations in the present study are

among the highest reported to date. The accumulation of Cr(VI) in the different plant parts was,

in decreasing order, root> leaves> stems (Table 5.2) as also reported in previous studies (Lesage

et al., 2007b; Mant et al., 2006; Fibbi et al., 2012). Upward translocation depends on the metal

pollutant, the plant species and other environmental factors (Yadav et al., 2010; Kongroy et al.,

2012). Wetland plants, including common reeds, are capable of reducing Cr(VI) to Cr(III), either

inside their tissue after digestion of Cr(VI) or outside by the release of root exudates (Fibbi et al.,

2012). The mechanism of metal partitioning is a strategy plants use to manage the effects of toxic

ions in their roots and prevent any effects on leaves, photosynthesis or other metabolic activities

(Sune et al., 2007). In this study, the Cr(VI) concentrations in reeds were found to be 820 mg/kg

in the roots, 750 mg/kg in the leaves, and 540 mg/kg in the stems.

Figure: 5.5 Harvested reed plants treated with Cr(VI) solutions.



Table 5.2: Cr concentrations recorded in different parts of reed plants.

Reference

Cr concentration (mg/kg d.m.)

Roots Leaves Stems Shoots Whole

plant

Khan et al., 2009 2.3 - - 1 -

Lesage et al., 2007b 17.75 0.725 1.075 - -

Calheiros et al., 2008b 1647-4825 109-627 369-883 - -

Mant et al., 2006 406.2 12.863 15.505 - -

Liu et al., 2010 22.2 - - 12.7 -

Fibbi et al., 2012 - 0.66-4.62 1.12-2.83 - -

Kongoy et al., 2012 - - - - 11.3

Laing et al., 2009 - 0.28-1.21 0.29-0.72 - -

Vymazal et al., 2007 - - - 0.4-15 -

Sultana et al., 2014a

(Present experimental

results)

820 750 540 - -

During the two years of operation, the Cr-P and Cr-U units received 24 and 25.6 m3 wastewater,

respectively. The total Cr(VI) mass introduced into these units was 77.6 gr for Cr-P and 84.5 gr

for Cr-U. This difference occurs due to the different volumes of wastewater introduced into each

unit. Cr(VI) removal varied in the two units as Cr-P achieved higher removal efficiencies

removing 69 gr of Cr, while the Cr-U removed only 21.5 gr of Cr. Thus, the Cr-P unit removed

70% more Cr(VI) than the Cr-U unit. In the Cr-U Cr(VI) was removed either by absorption by

porous media or by biological processes. On the other hand, Cr-P removal mechanisms include

porous media absorption and biological accumulation together with plant accumulation. As the

exact Cr mass removed in Cr-P by porous media absorption and biological accumulation could

not be assessed, it can be assumed that this value is at least equal to the Cr mass removed in the

Cr-U. Thus, plant accumulation in CW-P removed a maximum of 47.5 gr Cr. Total Cr content in

above ground biomass was estimated at 4.5 gr /kg dry matter. On the other hand, total Cr content

in roots should range from 5.9 gr/kg d.m to 32.5 gr/kg dry matter (EPA, 1998; Cronk and

Fennessy, 2001). As Cr(III) effluent concentrations in the pilot-scale operation were below

detectable units, it can be assumed that the remaining Cr mass removed from the Cr-P, was either

absorbed into the porous media or removed through biological accumulation.



5.5) Composting of Plant Biomass

The only by-product produced by the CWs is plant biomass, which is usually used either as

fodder or as fuel. Plant biomass from CWs treating wastewaters with Cr(VI) should be handled as

a hazardous material. Until now no viable solution for the treatment of this plant biomass has

been proposed. In the present study plant biomass was co-composted with olive pomace and

olive leaves.

Temperature evolution describes the bio-chemical activities of compost (Ryckeboer et al., 2003).

Figure 5.6 presents a time series chart of both compost and ambient temperature where both

pilot-scale compost bins showed a very typical initial temperature rise during the composting

process. The thermophillic phase (over 450C) established after 5 days. This short time period was

due to the generation of heat as a result of aerobic microbial activities on the readily available

organic materials within the compost.

Initial pH values of 5.44 and 5.25 were recorded in compost Bins 1 and 2, respectively (See

Chapter 4; Table 4.1). In the final compost product, pH values were higher (8.17 and 8.18 for

Bins 1 and 2, respectively). Electrical conductivity (EC) values increased during the composting

process from 590 µS/cm to around 700 µS/cm. It is notable that the EC values of the mature

compost in this study were low and far below the Hellenic standard upper limits (4 S/cm)

(Lasaridi et al., 2006). Thus, the product is of sufficient quality to be applied on a wide variety of

sustainable crop cultivations. During the composting process, minor organic matter (OM) loss

was observed in all the experiments resulting in a decrease in VS and TOC throughout the

process, especially during the maturation phase (Table 5.3). Total Kjeldahl nitrogen (TKN)

increased during the composting process in both bins from 1.71% to 3.5%. In general, the

increasing trend of organic nitrogen can be characterized as a concentration effect resulting from

the degradation of organic compounds which decreased the dry matter content. According to

Italian legislation (Legislativo, 2010), if mature compost consists of more than 3% nitrogen it can

be used as a nitrogenous fertilizer. The carbon to nitrogen (C/N) ratio, i.e., the total organic

carbon (TOC) to TKN, has been widely referred (Chowdhury et al., 2013) as an index of compost

stability and maturity. The final C/N ratio for both bins was around 14. Notably, C/N ratios

below 20 characterize mature compost (Chowdhury et al., 2013).



0 20 40 60 80 100

10

15

20

25

30

35

40

45

50

55

60

65

T

emp

era

ture

(0C

)

Days

Watering

Turning

BIN_1

BIN_2

Air Temp

Figure 5.6: Time series of compost and ambient temperature during composting period.



Total Cr concentration in both bins did not show fluctuations during the composting process. The

limited differences between Cr concentrations at day 0 and at the end of thermophillic and

maturation stage could be attributed to the fact that the samples at day 0 were not well

homogenized. Although the two bins received different amounts of reed biomass, Cr

concentrations in the compost did not present great differences. Furthermore, in final compost

total Cr content was (10 mg/kg dry mass) well below the EU limits (70 mg/kg dry mass) for

organic farming applications (EU, 2004). The mature compost produced in both bins was also

rich in other nutrients (Table 5.3). Due to the presence of high nutrient content these composts

could be used to replenish soils that have been exhausted by intensive cultivation as well as soils

suitable for greenhouse cultivations (Hachicha et al., 2006).

One of the key factors used to assess compost maturity is the phytotoxicity test. The GI values

(Table 5.3) obtained from both bins at the initial stage (day 1) indicated that the initial

composting materials were phytotoxic in nature (72 and 75%, for bins 1 and 2, respectively).

During the composting process a clear decreasing trend in phytotoxicity was observed, as GI

values in the final compost were 157% and 144% in Bins 1 and 2, respectively. The GI values

indicate that the mature compost of the present study is not phytotoxic and therefore it could be

used as a high quality organic soil amender.

Table 5.3: Physicochemical parameters of compost during the composting period.

Initial day End of thermophillic phase End of maturation

Bin(B) 1 2 1 2 1 2

MC (%) 51.9 52.6 50.8 48.7 50.45 48.12

pH 5.44 5.25 7.91 7.81 8.17 8.16

EC (µS/cm) 595 589 625 645 714 637

VS (%) 96.7 97 94.7 94.7 94 94

OM (%) 95.82 96.26 90.99 91.05 88.8 88.1

TOC (%) 55.58 55.83 52.78 52.81 51.5 51.1

TKN (%) 1.71 1.71 3.16 3.02 3.6 3.5

C/N 32.54 32.69 16.68 17.46 14.1 14.7

P (%) 0.09 0.07 0.15 0.16 0.17 0.2

Na (%) 0.07 0.07 0.11 0.14 0.13 0.14

K (%) 0.021 0.02 0.032 0.033 0.037 0.036

GI (%) 75.21 72.09 119.42 129.79 157.48 144.69

MC: Moisture Content, EC: Electrical Conductivity, VS: Volatile Solid, TOC: Total Organic Carbon, TKN: Total

Kjedhal Nitrogen, C/N: Carbon Nitrogen ratio, P: Phosphorus, Na: Sodium, K: Potassium, GI: Germination Index


Chapter 6: Pilot-Scale HSF Constructed Wetlands Treating Cheese Whey


TREATING CHEESE WHEY

6.1) Physicochemical Parameters ............................................................................................. 127

6.1.1) pH .................................................................................................................................. 127

6.1.2) EC .................................................................................................................................. 128

6.1.3) DO ................................................................................................................................. 128

6.2) Treatment Efficiency of Pilot-Scale HSF Constructed Wetlands .................................. 128

6.3) Effect of HRT ...................................................................................................................... 132

6.4) Effect of Temperature ........................................................................................................ 136

6.5) Effects of Vegetation ........................................................................................................... 137


Chapter 6: Pilot-Scale HSF Constructed Wetlands Treating Cheese Whey 127


TREATING CHEESE WHEY1

6.1) Physicochemical Parameters

Two pilot-scale units (SCW-U and SCW-P) were used to treat secondary cheese-whey (SCW) at

different concentrations of COD with different HRTs. Two identical pilot-scale constructed

wetlands were used and the experiment was run for two years. The main physicochemical

characteristics of diluted cheese whey as influent and the effluents of wastewater (temperature,

pH, electrical conductivity-EC and dissolved oxygen-DO) were monitored and analyzed during

the experiment. Apart from EC, the other parameters did not show any significant variations

between the units, as briefly discussed below. Table 6.1 shows the physicochemical

characteristics of the SCW wastewater.

Table 6.1: Physicochemical characteristics of the SCW wastewater.

CW Position pH EC DO

Mean SD Max Min Mean SD Max Min Mean SD Max Min

CW-U Inlet 5.41 1.1 7.03 3.4 901 321.3 2051 463 4.3 0.3 4.97 3.85

Outlet 6.35 0.3 6.85 4.2 1885 621.1 4070 683 4.25 0.27 4.98 4

CW-P Inlet 5.29 1.1 7.04 3.5 894 315.7 2073 452 4.35 0.46 5.74 4.01

Outlet 6.26 0.2 6.71 5.9 1907 382.7 2090 723 4.6 0.38 5.17 4

6.1.1) pH

Casein precipitation in cheese processing leads to the formation of two types of whey depending

on the pH: acidic whey (pH<5) and sweet whey (pH=6–7) (Panesar et al., 2007). Acidic pH helps

maintain a filamentous biomass (Ghaly, 1996). The low buffering capacity of cheese whey is the

main reason for rapid acidification in biological treatments (Castelló et al., 2009). Secondary

cheese whey has acidic characteristics with pH values within the range of 3–6 (Carvalho et al.,

2013). In this study the pH was found to be almost neutral due to the use of tap water during the

1 Data presented in Sultana et al. (2014c)



dilution of SCW. The average influent pH values in the SCW-U and SCW-P units were 5.41 and

5.29, respectively. Mean effluent pH values were 6.35 and 6.26 for SCW-U and SCW-P,

respectively (Table 6.1).

6.1.2) EC

Highly acidic solutions usually have has high EC values (Chegini et al., 2014). For this study, the

mean influent EC values were 901 and 894 µS/cm for SCW-U and SCW-P, respectively, and the

mean effluent EC values were 1885 μS/cm and 1907 μS/cm in SCW-U and SCW-P, respectively.

This slight increase of EC values in the SCW-P could be attributed to the dilution of salts

contained either in the porous media or in plant detritus (Mashauri et al., 2000). Additionally,

warm water holds more ions in solution than cold water. Therefore, the seasonal increase in

ambient temperature increases ET rates, resulting in increased ion concentrations and increased

EC.

6.1.3) DO

A sufficient amount of available free oxygen is essential for good water quality. When

wastewater contains high amounts of organic compounds, microorganisms use more oxygen and

decrease the DO concentration. In this study, DO concentrations did not show any significant

variations between the pilot-scale CW units, as the mean influent concentrations recorded were

4.35 and 4.25 mg/L for SCW-P and SCW-U, and the mean effluent concentrations were 4.35 and

4.60 mg/L for SCW-P and SCW-U, respectively. However, the presence of plants in the wetlands

leads to high DO concentrations during the day as photosynthesis occurs. It should be mentioned

that DO concentrations were always above 3 mg/L in both CW units, indicating the existence of

aerobic conditions.

6.2) Treatment Efficiency of Pilot-Scale HSF Constructed Wetlands

Figure 6.1 presents time series charts for influent and effluent concentrations of COD for both

units. Figure 6.1 also shows plant harvesting events and changes in HRT (vertical solid and



dashed lines, respectively). Effluent concentrations were corrected as rainfall and ET volumes

were removed. During the first year, the pilot-scale CWs were operated with HRTs of 8 days, to

avoid plant stress and ensure CW adjustment and mean influent COD concentrations were 2487

and 2650 mg/L for the SCW-P and SCW-U units respectively. Mean COD removal efficiencies

were 76.4% and 71% for SCW-P and SCW-U, respectively. Both SCW-P and SCW-U units

showed similar fluctuations in COD removal efficiency at the beginning of the experiment due to

the CWs commissioning phase (Kadlec and Wallace, 2009), and also during autumn and winter,

when ambient temperatures decreased. Intense fluctuations of COD removal efficiencies were

observed in both units when HRTs were altered. Nevertheless, steady-state conditions quickly

reestablished in both units.

During the second year of operation both CW units were operated under different HRTs (i.e., 8,

4, 2 and 1 day) to assess the effect of HRT on COD removal. Furthermore, the effect of COD

influent concentration was also investigated by using two different influent concentrations (2500

and 5000 mg/L). Mean COD removal efficiencies of 91% and 76% were recorded in SCW-P and

SCW-U, respectively, and both units achieved COD removal efficiencies of up to 100%. SCW-U

presented the highest COD removal efficiencies when the COD influent concentration was 2500

mg/L and HRT was 4 days (94.24%) and 8 days (96.84%) (Fig. 6.1a). SCW-U effluent

concentrations increased mainly when ambient temperatures were low and at low HRTs (2 and 1

day) (Fig. 6.1a).

In the SCW-P unit, COD removal remained high for the majority of the operation period and

decreased only after plant harvesting (Fig. 6.1b). However, the reed plants re-generated quickly

after harvesting and the removal efficiency in the planted unit increased to 100% in less than two

months (Fig 6.1a). SCW-P presented high COD removal efficiencies for almost all the HRTs

applied, while it decreased only when HRT was limited to 1 day (Fig. 6.1b). It should be

mentioned that under steady-state conditions the effluent values always satisfied the permitted

EU discharge limit (120 mg/L) for surface waters (Directive 91/271/EEC). SCW-P performance

was also affected by influent concentration, as COD removal efficiency decreased when COD

influent concentration exceeded 3500 mg/L, resulting in effluent concentrations that exceeded

legislation limits.



45 90 135 180 225 270 315 360 405 450 495 540 585 630

0

1000

2000

3000

4000

5000

6000

7000

8000C

once

ntr

atio

n (

mg/

L) Influent

Effluent

HRT changes

Harvesting

Days

8 days HRT

4 d

ays

HR

T

2 d

ay

s H

RT

1 d

ay

HR

T 4 days HRT

8 d

ay

s H

RT

Sp

rin

g

Summer Autumn Winter Spring Summer Autumn Winter

Figure 6.1a: Time series charts for COD removal with influent and effluent concentrations in the SCW–U.



45 90 135 180 225 270 315 360 405 450 495 540 585 630

0

1000

2000

3000

4000

5000

6000

7000

8000C

once

ntr

atio

n (

mg/

L) Influent

Effluent

HRT changes

Harvesting

Days

8 days HRT

4 d

ay

s H

RT

2 d

ay

s H

RT

1 d

ay

HR

T

4 days HRT

8 d

ay

s H

RT

WinterAutumnSummerSpringWinterAutumnSummer

Sp

rin

g

Figure 6.1b: Time series charts for COD removal with influent and effluent concentrations in the SCW-P.



Organic matter in CWs is removed either by settlement and filtration (particulate organic matter)

or by aerobic and anaerobic degradation (soluble organic matter) (Akratos and Tsihrintzis, 2007;

Cooper et al., 1996; Stefanakis et al., 2014). As the SCW was diluted with tap water before being

introduced into the pilot-scale units, the particulate organic matter fraction was limited.

Therefore, the removal of organic matter by suspended solids settlement was also limited. It can

thus be concluded that organic matter in both pilot-scale CWs was removed through microbial

activity. Since DO concentrations were constantly above 3 mg/L (Table 6.1) aerobic degradation

was the main removal mechanism for organic matter in the present pilot-scale CWs.

6.3) Effect of HRT

To assess the effects of HRT on COD removal, four different HRTs were applied in the pilot-

scale units (8, 4, 2 and 1 day). Furthermore, COD influent concentrations varied from 1200 to

7200 mg/L and also affected COD removal efficiencies. The use of different HRTs and COD

influent concentrations affected pollutant surface load rate (SLR), which ranged from 19.39 to

770 gr/m2/d for SCW-U and from 11.34 to 620 gr/m

2/d for SCW-P. The removed organic load

ranged from 5.63 to 357.8 gr/m2/d (mean value 92.8 gr/m

2/d) for SCW-U and from 2.19 to

362.63 gr/m2/d (mean value: 120.1 gr/m

2/d) for SCW-P. Figure 6.2 presents correlation charts for

influent COD surface load and removal efficiency. SCW-P showed a slight decrease in COD

removal efficiency with increased SLR, while COD removal efficiency in SCW-U increased

slightly with SLR (Fig. 6.2a). However, in both units, removal efficiency did not vary

significantly with SLR when influent concentrations ranged 1200 to 7200 mg/L. Regardless of

the SLR, more than 60% of COD was removed in the SCW-U, while in SCW-P, COD removal

was very high (>90%) with same load. It should be noted that the CWs units used in this

experiment achieved extremely high COD removal efficiencies (up to 100%), while receiving

among the highest COD influent concentrations (from 1200 to 7200 mg/L) and operating under

the lowest HRT (2 days) ever reported in the literature (Table 6.2).



0 100 200 300 400 500 600 700 800

-60

-40

-20

0

20

40

60

80

100

120

R

emov

al %


Figure 6.2a: Correlation of surface load and removal rates for COD in the SCW-U.



0 100 200 300 400 500 600 700 800

0

20

40

60

80

100

120

Rem

oval

(%

)




Figure 6.2b: Correlation of surface load and removal rates for COD in the SCW-P.

Table 6.2: Treatment of dairy wastewaters using different types of CWs.

Reference CW area

(m2)

Wastewater

type

HRT

(days)

CW

type

Inlet concentration of

organic matter, mg/L

Plants

Removal %

COD BOD COD BOD

Dipu et al., 2010 - Dairy 5-15 FWS 2920 1300 Typha spp., Eichhornia sp., Salvinia

sp., Pistia sp.

72 77

Idris et al., 2012 1.28 Dairy

processing

factory

- HSF - 24.4 Arundo donax, Phragmites australis - 65

Mantovi et al., 2003 75 Dairy and

domestic

sewage

11 HSF 1200 450 Phragmites australis >90 >90

Jamieson et al.,

2007

100 Livestock 90 FWS - 1747 Typha latifolia, Lemna spp. - 98

Tanner et al., 1995 19 Farm 7, 5.5, 3, 2 HSF - 57-200 Schoenoplectus validus - 50-80

Schaafsma et al.,

2000

500 Dairy farm 42 FWS - 1913.9 Typha latifolia, Scirpus

tabernaemontani,

Litaneutria minor

- 97

Dunne et al., 2005 4265 Dairy farmyard - FWS - 2500 Carex riparia, Typha latifolia,

Phragmites australis, Sparganium

erectum, Glyceria fluitans,

Iris pseudacorus, Phalaris

arundinaceae, Alisma plantago-

aquatica

- 99

Sharma et al., 2013a 500 Milking parlor - Hybrid SSF 1395-1637 3749-4988 Phragmites australis >89 >89

Comino et al., 2011 703.9 Cheese factory 4-15 Hybrid 2000 1000 Phragmites australis 80 80

Mantovi et al., 2011 2700 Cheese factory - HSF 938 595 Typha latifolia >95 >95

Sultana et al., 2014c

(Present study)

0.86 Cheese whey 2-8 HSF 1200-7200 - Phragmites australis 91



Table 6.3: Comparison of COD removal rates with different HRTs.

Time period HRT

(days)

COD feed

concentration

(mg/L)

Ambient

temperature

(oC)

COD removal (%)

SCW-U unit SCW-P unit

Mean SD Mean SD Mean SD

26/04/12-09/05/12 8 2900 21.8 1.0 100 1.57 100 2.99

23/05/12-10/06/12 4 2950 23.3 2.3 94.24 17.85 100 1.22

13/06/12-25/06/12 2 2925 28.0 1.5 85.6 13.1 100 1.02

27/06/12-04/07/12 1 3250 27.9 0.4 76.28 23.89 76 11.38

One-way ANOVA analysis was performed to statistically assess the effect of HRT on CW

efficiency. The data used for this comparison were taken from periods where temperature values

were not significantly different (p value = 0.876>0.05). The concentration of COD feed was

almost identical, thus indicating only the effect of HRT (Table 6.3). One-way ANOVA results

showed that for HRTs of 8 to 2 days no significant differences in COD removal were observed

for SCW-P (p value ranged from 0.986 to 0.999>0.05) and SCW-U (p ranged from 0.215 to

0.969>0.05) units. However, HRTs of 1 day significantly affect COD removal efficiency as “p”

values were below 0.05 for SCW-P (p value = 0.000) and SCW-U (p value = 0.003-0.009). Thus,

an HRT of 1 day limits organic matter removal as COD removal efficiency was decreased to

approximately 70% in both pilot-scale units. Nevertheless, the CW pilot-scale units proved to be

rather efficient at organic matter removal with very low HRTs (down to 2 days).

6.4) Effect of Temperature

Temperature has been shown to affect organic matter removal in CWs as it influences both

microbial activity and plant function (Akratos and Tsihrintzis, 2007; Taylor et al., 2011). The

effect of temperature on COD removal was assessed statistically as experimental data for HRTs

of 4 days were divided into two groups: one above and one below 150C, as this temperature value

is reported to be crucial for microorganism and vegetation performance (Akratos and Tsihrintzis,

2007). Both units showed different COD removal efficiencies for temperatures above (95% for

SCW-U and 97.6% for SCW-P) and below (60.8% for SCW-U and 90.3 for SCW-P) 15oC. One-

way ANOVA analysis showed that although organic matter removal was significantly affected by



temperature in SCW-U (p value =0.00<0.05), it was not significantly affected in the SCW-P unit

(p value =0.378>0.05).

The significant effect of temperature on COD removal in the SCW-U unit occurred because

microbial activity was limited in this unit. On the other hand, COD removal efficiency in SCW-P

was not significantly affected by temperature, proving that plants have the capacity to enhance

COD removal all year round as the rhizosphere not only stimulates the microbial community but

also provides increased surface area for microbial growth and activity (Gerberg et al., 1986).

Comino et al. (2011) used constructed wetlands to treat cheese whey wastewater and reported

that low temperatures did not affect organic matter removal. On the contrary, other studies (Kern

2003; Sharma et al., 2013a) report that COD removal efficiency was significantly affected by

temperature. However, it should be noted that these studies were conducted in areas with cold

climates (below 5oC). In the present research, COD removal in SCW-P was limited only

following plant harvesting, thus indicating that the reed vegetation was crucial for efficient CW

operation. Therefore, the experimental results revealed that plants play a key role in maintaining

high COD removal rates even at low temperatures or in cold seasons.

6.5) Effects of Vegetation

Vegetation plays an important role on CW performance as it provides large surface areas for

microbial growth and transports oxygen from the leaves to the roots, which is then used by the

microorganisms to oxidize organic compounds (Vymazal and Kropfelova, 2009). Although

vegetation effect on COD removal in HSF CW has been studied by many research groups

(Tanner, 2001; Vymazal and Kropfelova, 2009; Kaseva 2004; Akratos and Tsihrintzis, 2007;

Brisson and Chazarenc, 2009), the precise contribution of plant vegetation remains a

controversial issue.

The planted and unplanted pilot-scale units presented different organic matter removal

performances, as mean COD removal efficiencies were 83% for SCW-P and 70% for SCW-U.

To examine the effect of vegetation on COD removal, a paired t-test was performed to determine

statistical significance at the 95% confidence level. Results revealed that vegetation significantly



affected COD removal as the confidence interval was (7, 18), indicating that COD removal

efficiencies between SCW-P and SCW-U were significantly different. The effects of vegetation

were also observed during the autumn of the second year, when the units were operated again

under 4 days HRT receiving wastewater with COD influent concentrations of approximately

2500 mg/L. During this period, COD removal efficiency in SCW-P was limited to 80% compared

to the 100% removal efficiency achieved in the spring of the same year. This phenomenon could

only be attributed to plant senescence. The effect of vegetation on COD removal could be

attributed to enhanced oxygen release from the root zone, as secondary cheese whey mainly

contains easily biodegradable organic substances.

Based on the above results, some initial design guidelines can be given for CW units treating

secondary cheese whey: a) constructed wetlands treating secondary cheese whey should operate

with HRTs not less than 2 days in order to successfully remove organic matter, b) COD influent

concentrations also should be taken into consideration and should not exceed 3500 mg/L in order

to avoid effluent concentrations above legislation limits. If the constructed wetland receives

higher COD influent concentrations, then it should be operated with HRTs greater than 8 days,

and c) a CW treating secondary cheese whey could safely remove 120 gr/m2/d of COD. The use

of the removed organic matter load as design guideline is safer, as it avoids the limitation of the

different characteristics of secondary cheese whey.


Chapter 7: Combined Treatment of Cr(VI) and Cheese Whey in Pilot-Scale HSF Constructed Wetlands

CHAPTER 7: COMBINED TREATMENT OF CR(VI) AND CHEESE

WHEY IN PILOT-SCALE HSF CONSTRUCTED WETLANDS

7.1) Physicochemical Parameters ............................................................................................. 139

7.1.1) pH .................................................................................................................................. 139

7.1.2) EC .................................................................................................................................. 141

7.2) Removal Efficiency of Cr(VI) in Four Pilot-Scale HSF Constructed Wetlands with Co-

Treated Wastewaters ................................................................................................................. 141

7.2.1) Effect of vegetation on Cr(VI) removal ........................................................................ 147

7.2.2) Effect of HRTs on Cr(VI) removal ............................................................................... 153

7.2.3) Comparison with experimental results of the first operational period .......................... 154

7.3) Removal Efficiency of COD in Four Pilot-Scale HSF Constructed Wetlands with Co-

Treated Wastewaters ................................................................................................................. 155

7.3.1) Effect of vegetation on COD removal ........................................................................... 160

7.3.2) Effect of HRTs on COD removal .................................................................................. 162

7.3.3) Comparison with experimental results of the first operational period .......................... 167


Chapter 7: Combined Treatment of Cr(VI) and Cheese Whey in Pilot-Scale HSF Constructed Wetlands 139

CHAPTER 7: COMBINED TREATMENT OF CR (VI) AND CHEESE

WHEY IN PILOT-SCALE HSF CONSTRUCTED WETLANDS

7.1) Physicochemical Parameters

Integrated treatment of cheese whey and Cr(VI) wastewaters was performed in four pilot-scale

constructed wetlands. The physicochemical parameters of all the units did not show any

significant variations (Table 7.1). The temperature of the effluent ranged from 5 to 33°C,

depending on the season.

Table 7.1: Physicochemical characteristics of co-treated wastewaters (mixed) cheese whey and

Cr(VI) solution.

pH EC

Mean SD Max. Min. Mean SD Max. Min.

SCW-U Inlet 6.95 0.519 7.66 4.39 660 69 790 527

Outlet 6.50 0.45 7.40 6.15 1054 422 1969 370

SCW-P Inlet 6.90 0.53 7.39 4.16 651 80 810 517

Outlet 6.39 0.49 7.45 6.00 724.5 456 1820 208

Cr-U Inlet 7.04 0.45 7.53 6.25 618 410 1320 331

Outlet 6.98 0.42 7.62 5.74 470.5 351 1708 205

Cr-P Inlet 7.04 0.44 7.60 6.05 612 426 1369 340

Outlet 6.66 0.469 7.59 6.02 564 452.44 1886 450

7.1.1) pH

pH is the most important factor governing metal speciation, solubility, transport and

bioavailability of metals in aqueous solutions. The effect of pH on heavy metal availability in

plants has been reported by many researchers and it is accepted that when pH decreases, the

solubility of cationic forms of metals in the soil increases and they become more readily available



to plants (Evans et al., 1995). pH affects both solubility of metal hydroxide minerals and

adsorption-desorption processes. Presence of organic matter in aqueous solutions plays an

important role in metal binding, as organic carbon compounds influence metal leaching. Fotovat

et al. (1996), reported that heavy metals solubility may be influenced by the presence of OC.

However, the pilot units used to treat SCW during the first operational period, did not show any

significant variation in pH in the second experimental period with an average value of pH 6.9 in

the influent and 6.39 in the effluent for the SCW-P unit. The mean pH was 6.95 in the influent

and 6.5 in the effluent of the SCW-U unit (Table 7.1). During the first experimental period, these

two units were treated only with SCW and the mean pH value recorded was almost near to

neutral. In the second experimental period, the pH values of SCW-U and SCW-P were also found

to be near neutral as tap water was used to dilute the SCW. No significant variations were

recorded in the Cr-P with average influent and effluent values of 7.04 at influent and 6.66

respectively. For Cr-U, the mean pH was 7.04 in the influent and 6.98 in the effluent (Table 7.1).

In the first experimental period, the latter two units were used to treat only Cr(VI) solution and

the pH was found near to neutral (around 7). However, in the second experimental period,

influent and effluent pH values were also found to be near neutral. These pH values could

promote Cr(VI) adsorption/precipitation on the gravel bed system, as when pH increases OH-

compete with chromate ions (CrO42-

). OH- ions become the dominant specie at higher pH values

(>6), but adsorption ceases at pH >9 (Baral et al., 2006). Moreover, Kongroy et al. (2012)

reported that Cr accumulation in sediment could be due to precipitation under high pH condition

(pH= 8-9).

Several researchers have reported that there is no significant effect of pH in the presence of

organic matter on the adsorption of Cr(VI) (Rai et al., 1989; Zachara et al., 1989). Organic matter

has a relatively high proportion of negatively charged binding sites. pH can influence the

effectiveness of the cation exchange capacity of sediments because the negatively charged

binding sites will be occupied by a high number of sodium or hydrogen cations (Knight et al.,

1999). Moreover, when organic matter mineralization occurs, it increases CO2 levels in water

which in turn decreases the pH value at effluent (Maine et al., 2007). Abdulla et al. (2010) found

that COD removal was optimum when the pH ranged 6.6 to 8. Song et al. (2003) also observed

that COD removal rates decrease when pH is lower than 5.0 or higher than 8.5. Maine et al.



(2006), Qian et al. (2012) and Calheiros et al. (2007), also found similar pH ranges as these found

in this study. Therefore, may be concluded that this pH value also enhanced the removal rate of

COD in CWs.

7.1.2) EC

The pilot-scale CWs units that were used to treat cheese whey during the first experiment, did not

show any significant variation in EC in the second experiment. The mean influent value of EC

was 651 µS/cm and the mean effluent value was 724.5 µS/cm in the planted pilot-scale

constructed wetland (SCW-P). The average electrical conductivity in the un-planted pilot-scale

constructed wetland (SCW-U) was 660 µS/cm at the influent and 1054 µS/cm at the effluent

point. This variation in EC value is occurred probably because of high ET, which leads to

wastewater condensation.

The Cr-P pilot units used to treat Cr(VI) in the first experimental period, did not show any

significant variation in EC. In the second operation period, the same unit (Cr-P) also did not show

any significant variation in EC as a mean influent value of 612 µS/cm was recorded and the

average effluent value was 564 µS/cm. The average EC recorded in the Cr-U was 618 µS/cm at

the influent and 470.5 µS/cm at the effluent point in the second experimental period (Table 7.1).

The EC values recorded here were lower than those of other studies as a synthetic wastewater

solution was used which was diluted with tap water, whereas in other studies (Dotro et al., 2012;

Maine et al., 2006) real wastewater was used.

7.2) Removal Efficiency of Cr(VI) in Four Pilot-Scale HSF Constructed

Wetlands with Co-Treated Wastewaters

Figures 7.1a and b present time series charts of influent and effluent concentrations of Cr(VI) for

SCW-U and SCW-P. The effluent concentrations were corrected by removing rainfall and ET

(See Section 4.1.5). The SCW-U and SCW-P units, did not show any difference in Cr(VI)

removal as the average removal rate is 100% almost all year round. It should be noted that during

the first operational period, these two pilot-scale units treated only diluted SCW. Therefore, they

showed great adaptability to co-treating both cheese whey and Cr(VI).



0 30 60 90 120 150 180 210 240

0

1

2

3

4

5

6

C

on

cen

tra

tio

n (

mg

/L)

Days

Influent

Effluent

HRT alteration

Spring Summer Autumn

8 days HRT

4 days HRT

Figure 7.1a: Time series charts for Cr(VI) removal with influent and effluent concentrations in the SCW-U unit.



0 30 60 90 120 150 180 210 240

0

1

2

3

4

5

6

Influent

Effluent

HRT alteration

Co

nce

ntr

ati

on

(m

g/L

)

Days


8 days HRT

4 days HRT

Figure: 7.1b Time series charts for Cr(VI) removal with influent and effluent concentrations in the SCW-P unit.



0 30 60 90 120 150 180 210 240

0

1

2

3

4

5

6

Influent

Effluent

HRT alteration

Co

nce

ntr

ati

on

(m

g/L

)

Days

8 days HRT

4 days HRT

AutumnSummerSpring

Figure 7.2a: Time series charts for Cr(VI) removal with influent and effluent concentrations in the Cr-U unit.



0 30 60 90 120 150 180 210 240

0

1

2

3

4

5

6

Influent

Effluent

HRT alteration

Co

nce

ntr

ati

on

(m

g/L

)

Days


8 days HRT

4 days HRT

Figure 7.2b: Time series charts for Cr(VI) removal with influent and effluent concentrations in the Cr-P unit.



The performance of a CW depends on the complete development of the root-rhizome area and the

microbial activity in the system. Plants emit oxygen from their roots into the rhizosphere which is

very significant in CWs as it permits the aerobic degradation of substances and nitrification

(Brix, 1994). Different retention mechanisms depend on the oxygen concentration of the wetland

but metals are retained in its vegetation (Maine et al., 2006). Maine et al. (2005), reported that

when oxygen concentration ranged between 0-10.6 mg/L in their pilot-scale study, metals co-

precipitated with iron and were retained in the wetland’s sediment. On the other hand, in their

full-scale study (Maine et al., 2006) observed that when the oxygen concentration ranged

between 0-7.1 mg/L, metal precipitation was prevented. The extensive root systems of

macrophytes release oxygen into the rhizosphere and enhance the precipitation of Fe to form

iron-plaque (Otte et al., 1995). Metal accumulation in the iron-plaque is due to the metal binding

affinity to iron oxyhydroxides (Otte et al., 1995). Therefore, metals like Cr(VI) may either be

directly sorbed by the plants from the wastewater solution or co-precipitated with Fe onto plants

roots.

According to above description, the similar behavior was also observed in the other two pilot

units (Cr-P and Cr-U) in removing Cr(VI) (Figs. 7.2a & b), where the average removal rate was

100% for both units. In the first experimental period, the Cr-P unit showed the high efficiency

while the removal rate in Cr-U was lower. In the second experimental period when SCW was

introduced into the Cr-U, the removal efficiency of Cr(VI) was almost 100% all the year round.

However, Cr(VI) removal was not only due to absorption by plant biomass but also due to

microbial activity, since the microorganisms developed adequately due to the presence of

nutrients in the SCW. Moreover the acidic nature of the SCW (Carvalho et al., 2013) promoted

the growth of filamentous biomass (Ghaly, 1996). Most microorganisms grow best at neutral pH

(7.0) (Hamad, 2012). Bacterial metabolic activity increases when pH range is between 3.5 and

5.5 (Cimino and Caristi, 1990) but Beaubien & Jolicoeur (1984) reported that bacterial growth of

generally occurs in neutral to slightly acidic conditions. In this study the mean pH value was

found to be near neutral, therefore the pH promoted microbial growth and participated in Cr(VI)

removal mechanisms. The gravel bed system also assisted Cr(VI) removal by various

mechanisms including co-precipitation, sorption and ion exchange (Marchand et al., 2010).



Additionally, Dotro et al. (2009) state that gravel media dominates Cr removal through sorption

to iron oxides and co-precipitation of iron phases with available Cr.

7.2.1) Effect of vegetation on Cr(VI) removal

CW vegetation plays an important role in Cr removal as it (a) releases root exudates which may

directly or indirectly impact metal mobility and toxicity; (b) provides large surface area for

microbial growth, (c) accumulates Cr in its tissues (Cheng et al., 2002; Southichak et al., 2006;

Zhang et al., 2010). The presence of vegetation in CWs is very important for the treatment of

Cr(VI) because it can also slow down the flow rates in the wetland, so that metals have more time

to be absorbed and precipitated (Maine et al., 2007). The main removal processes of Cr(VI) by

plants are adsorption, chelation and ion exchange. Zayed and Terry (2003) and Yadav et al.

(2005), stated that Cr ions have the ability to bind or precipitate in the cell walls of plant tissue

which also limits their translocation. Hence, vegetation is essential for the treatment of Cr in

CWs.

Of the four pilot-scale HSF CWs used in this experiment, Cr-U and Cr-P had already been used

to treat Cr(VI), while SCW-U and SCW-P had been used for SCW treatment. During the second

operational period the CWs were used to co-treat Cr(VI) and SCW. SCW was used due to its

nutritional value as it contains lactose, proteins, calcium, nitrogen and phosphorus

(Chatzipaschali and Stamatis 2012; Prazeres et al., 2012). Therefore, due to the use of this mixed

wastewater of SCW and Cr(VI), rapid growth of plants and increased microbial activity were

anticipated. As the Cr(VI) removal rate was 100% in both planted units all year round, reed

plants seemed the best vegetation for this integrated experiment since they survived until the end

of the studied period without showing any symptoms of toxicity. Choo et al. (2006), reported that

metal ions can be strongly absorbed by root cells via plasmalemma and also adsorbed by cell

walls via passive diffusion in the roots of aquatic plants. Organic acids deriving from root

extracts also form complexes with Cr and could lead to increased plant uptake (Bartlett and

James 1988; Srivastava et al., 1998). The channel of Cr transportation is a working mechanism

which involves essential anion carriers, such as sulfate (Cervantes et al., 2001).



As microorganisms are also an imperative part of CW function, it can be assumed that they had

an affect on the mobilization and bioavailability of Cr(VI) throughout the experiment period. As

the aerial parts of the reed plants grew vigorously during the treatment period, the belowground

part also grew and filled the wetland units (Fig. 7.3). The root zone is the most active microbe-

metal interaction zone in CWs. Extensive root systems also support symbiotic root-colonizing

bacteria and fungi and ultimately play a protective role by mitigating metal toxicity (Marchand et

al., 2010). They also increase the efficiency of phytoremediation by accumulating metals in the

plant tissues (de Souza et al., 1999; Marchand et al., 2010). Moreover, the continuous feeding of

the pilot units with mixed wastewater boosted plant growth and ratified the microorganisms for

their proper function.

Figure 7.3: Belowground parts of reed plants.



0,00 0,05 0,10 0,15 0,20

0

10

20

30

40

50

60

70

80

90

100

110

120

R

emo

va

l (%

)


Figure 7.4a: Effect of surface load on Cr(VI) removal in the SCW-U unit.



0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09 0,10 0,11 0,12 0,13 0,14 0,15

0

10

20

30

40

50

60

70

80

90

100

110R

emo

va

l (%

)


Figure: 7.4b Effect of surface load on Cr(VI) removal in the SCW-P unit.



0,05 0,06 0,07 0,08 0,09 0,10 0,11 0,12 0,13 0,14

0

10

20

30

40

50

60

70

80

90

100

110R

emo

va

l (%

)


Figure 7.5a: Effect of surface load on Cr(VI) removal in the Cr-U unit.



0,05 0,06 0,07 0,08 0,09 0,10 0,11 0,12 0,13 0,14 0,15

0

10

20

30

40

50

60

70

80

90

100

110R

emo

va

l (%

)


Figure 7.5b: Effect of surface load on Cr(VI) removal in the Cr-P unit



7.2.2) Effect of HRTs on Cr(VI) removal

Figures 7.4 and 7.5 present correlation charts between Cr(VI) surface loads and Cr(VI) removal

rates in the four pilot units. The removal rates of Cr(VI) in the four pilot units were not affected

by the different surface loads and remained stable (100%) throughout the second experiment.

Furthermore, mixing cheese whey with the Cr(VI) solution of may help plants to remove the

Cr(VI) and promote the degradation of Cr(VI) by microorganisms.

Hydraulic loading rate (HLR) and HRTs are important parameters for wastewater treatment in

CWs because when the loading rate is low, HRTs are high. If HLR is high, the effluent passes

rapidly through the wetland which reduces the available time (HRT) for the effective degradation

processes of pollutants. However, Figures 7.4 and 7.5 show that Cr(VI) removal rates were high

for all units with increased surface load. Even in the two unplanted units (Cr-U and SCW-U), the

removal of Cr(VI) was 100% with increased pollutants load. Thus, there was no significant

relationship between the efficiency of Cr(VI) removal and surface load.

The above observation helps to reinforce that bioaccumulation is the main action of Cr(VI)

removal in CWs. During the second operational period, the influent surface load was 0.0085-0.15

gr/m2/d for SCW-U, 0.014-0.14 gr/m

2/d for SCW-P, 0.06–0.114 gr/m

2/d for the Cr-U, and 0.05-

0.13 gr/m2/d for Cr-P. With HRTs of four days, the lower removal rate of Cr(VI) remained

constant in all seasons in this experiment. Theoretically, higher HRT means lower loading rate

and there will be stable performance of removal efficiency. However, in this study with a

reasonable surface load, removal of Cr(VI) showed a higher efficiency in the four pilot units with

8 and 4 days of HRTs. Therefore, it can be concluded that removal of Cr(VI) is not dependent on

loading rate, but on the optimum functions of the plant vegetation. In addition, cheese whey

effects Cr(VI) removal as it contains nutrient and organic elements which help microorganisms to

colonize and degrade pollutants.



7.2.3) Comparison with experimental results of the first operational period

To compare the removal rates of Cr(VI) of the two experimental sessions, the time periods were

selected when the average temperature (p value = 0.229> 0.05), HRTs, and influent

concentrations of Cr(VI) were almost identical. One-way ANOVA analysis was performed for

the Cr-P and Cr-U pilot units, to investigate whether the removal efficiencies of Cr(VI) in the

units show statistically significant differences between the two experimental sessions. Table 7.2

presents means and standard deviations for temperature and Cr(VI) removal rates, and the other

operational parameters (HRTs and influent concentrations) for both units (Cr-P and Cr-U) in both

sessions.

Table 7.2: Comparisons of Cr(VI) removal during the two operational periods.

Time periods Temperature

(0C)

HRTs

(Days)

Influent

concentrations

(mg/L)

Removal (%)

Cr-P

28/4 -15/6/2011 19.46 ± 3.31 8 5 77 ± 14.96

20/4 – 15/6/2013 20.17 ± 3.23 8 5 100 ± 0

Cr-U

28/4 -15/6/2011 19.46 ± 3.31 8 5 35.18 ± 10.51

20/4 – 15/6/2013 20.17 ± 3.23 8 5 99.29 ± 1.16

One-way ANOVA results showed that the removal rate of Cr(VI) between the two sessions is

statistically significant (p value = 0.000 <0.05) for the Cr-P. This difference may be because the

time period chosen for the first operational period was the initial period (first 90 days) of the

operation. Usually during these early days of treatment in CWs, the performance is not stable.

After this adaptation stage, the Cr-P consistently showed 100% Cr(VI) removal efficiency. The

Cr-U unit also showed statistically significant differences in Cr (VI) removal between the two

sessions (p value = 0.000 <0.05). This supports the view that the main removal mechanism of

Cr(VI) during the second operational period in the Cr-U unit was microbial activity and not

adsorption by plant biomass.



7.3) Removal Efficiency of COD in Four Pilot-Scale HSF Constructed

Wetlands with Co-Treated Wastewaters

Figure 7.6a &b present time series charts for COD influent and effluent concentrations in the

SCW-U and SCW-P units. The effluent concentrations were corrected by removing precipitation

and ET rates from the original effluent concentrations. The average COD removal efficiency was

70.7% for the SCW-P and 48.27% for the SCW-U. SCW-P showed over 50% removal rate of

COD when the input COD concentration ranged between 2000 mg/L and 5000 mg/L for both

HRTs (8 and 4 days) (Figure 7.6a). Both the SCW-P and SCW-U units, presented slightly higher

COD removal rates (74.8% for the SCW-P and 51.5% for the SCW-U) during the first

operational period. These differences in COD removal could be attributed to the possible toxic

effects of Cr(VI) to the microorganisms that removed the organic matter.

Figures 7.7a & b present time series charts for COD influent and effluent concentration in the Cr-

P and Cr-U units. The mean COD removal efficiency was 65.6% for the Cr-P and 58.2% for the

Cr-U. Cr-P and Cr-U presented lower COD removal rates compared to SCW-P and SCW-U. The

Cr-P unit showed high Cr(VI) removal efficiency in the first experimental period with high

concentrations of Cr(VI) and plant growth, however removal efficiencies were higher in the

second experimental period. In the second period, when SCW was mixed with the Cr(VI)

solution, the Cr-P unit showed a slight improvement in growth of vegetation and moderate COD

removal. In the first experimental period the Cr-U and Cr-P units received only the Cr(VI)

without nutrients or any other substances. In the second experimental period when these two

units received SCW-containing wastewater, the Cr-P unit showed slight improvement in plant

growth (by visual observation) which boosted microorganism growth and in the pollutant

degradation process. The microorganisms used the organic carbons in the SCW as a source of

energy to remove COD from the wastewater.



0 30 60 90 120 150 180 210 240

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Co

nce

ntr

ati

on

(m

g/L

)

Days

Influent

Effluent

HRT alteration


8 days HRT 4 days HRT

Figure 7.6a: Time series charts for COD removal with influent and effluent concentrations in the SCW-U unit.



0 30 60 90 120 150 180 210 240

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Influent CW

Effluent CW

HRT alteration

Co

nce

ntr

ati

on

(m

g/L

)

Days



Figure 7.6b: Time series charts for COD removal with influent and effluent concentrations in the SCW-P unit.



0 30 60 90 120 150 180 210 240

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Influent

Effluent

HRT alteration

Co

nce

ntr

ati

on

(m

g/L

)

Days



Figure 7.7a: Time series charts for COD removal with influent and effluent concentrations in the Cr-U unit.



0 30 60 90 120 150 180 210 240

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

Influent

Effluent

HRT alteration

Co

nce

ntr

ati

on

(m

g/L

)

Days



Figure 7.7b: Time series charts for COD removal with influent and effluent concentrations in the Cr-P unit.



However, in the Cr-U unit which was treated only with Cr(VI) in the first experimental period, a

moderate removal of COD (58.2%) was observed. One possible explanation for this could be

that, here SCW was introduced for the first time and it favoured the growth of microorganisms in

the gravel bed which increased COD removal. It has been reported that organic matter

concentrations at wetland inlet points are involved with enhanced biofilm growth because of the

greater abundance of nutrients at this point (Caselles-Osorio and García, 2006). The addition of

Cr containing solution and cheese whey in wetlands, microorganisms needed to adapt the mixed

wastewater as the media became an organic source of carbon. Moreover, Cr(VI) produced

different toxic substances with organic exudates of plant roots for bioaccumulation which

affect the growth of bacteria and increased the organic matter in the wetland. Several studies have

reported that when the concentration of wastewater, and consequently of Cr(VI) increases (=5

mg/L and >5 mg/L), the maximum biomass concentration of microorganisms decreases, thus

demonstrating the toxic effect of this metal on cell metabolism (Magro et al., 2012; URL 3;

Stasinakis et al., 2003). According to the literature, the critical concentration of Cr(VI) that

affects substrate removal, ranges from 5 to 50 mg/L (Stasinakis et al., 2003). In this experiment,

the shock load of Cr(VI) (5 mg/L) was introduced at the beginning of the experiment and was

later reduced. However, Magro et al. (2012) and Stasinakis et al. (2003) started their experiments

with low concentrations of Cr(VI) (around 1 mg/L) that they gradually increased. Thus, the

results of this experiment could be explained by the dependency of Cr toxicity (Stasinakis et al.,

2003).

7.3.1) Effect of vegetation on COD removal

The removal mechanism of COD in the CW system was the biofilm attached to the support

media (gravel and rhizomes). The vegetation in the wetland transports oxygen from the leaves to

the roots, which is then used by the microorganisms to oxidize organic compounds (Vymazal and

Kropfelova, 2009). Depending on the dissolved oxygen availability within the wetland bed,

organic matter degradation takes place under anaerobic conditions. The presence of vegetation

contributes to the aerobic degradation of organic matter through increasing the oxygen

concentration in the root zone area.



To assess the effect of vegetation on COD removal, one-way ANOVA statistical analysis was

performed where the concentration of COD was high with different concentration of Cr(VI) for

all units. The removal rate of COD for the SCW-P and SCW-U units was not significantly

different as the p value was 0.353>0.05 while the mean removal rates were 65.53 ± 23.78% and

70.66 ± 18.31% for SCW-P and SCW-U, respectively. For Cr-P and Cr-U units, a one-way

ANOVA statistical analysis also was performed to evaluate the removal rate of COD. The result

for these two units also did not show any statistically significant difference in COD removal (p

value =0.189>0.05). The mean removal rate of COD was 58.20 ± 23.94% for Cr-U and 65.60 ±

18.86% for Cr-P. Analysis of the results showed that there was no significant variation in COD

removal between the planted and unplanted units even in the presence of Cr(VI), as COD

removal in all units was around 65%. However, in the first experimental period, the SCW-P and

SCW-U units showed significantly high COD removal rates (almost 100%) when treated only

with SCW wastewater. In the second experimental period, Cr(VI) was added to the SCW

wastewater and the SCW-P and SCW-U units showed lower COD removal rates even with

moderate Cr(VI) loadings. In the Cr-P and Cr-U units, a reasonable COD removal rate was

observed when SCW was first introduced into these two units. The presence of reed plants might

significantly favour the removal of organic matter in the two planted units, although the main

mechanism of organic matter removal is the activity of aerobic and anaerobic bacteria (Vymazal,

2002).

Plants enhance the treatment processes of wastewater in CWs in a number of ways, such as

settlement of suspended solids, providing large surface area for microorganism growth,

increasing uptake of nutrients and trace elements and providing oxygen release (Kadlec et al.,

2000; EPA, 2000b; Brix, 1994, 1997). However, the superior role of plants in CWs is not always

evident, and seems to depend on several parameters, such as the operational period, type of

vegetation and characteristics of the wastewater. Calheiros et al. (2007) found no difference

between planted and unplanted units in the removal of COD from tannery wastewater. Dotro et

al. (2012) and Maine et al. (2006), achieved a significant COD removal rate of around 70% when

treating tannery wastewater. The findings from this study showed that the application of CW

systems to the treatment of integrated wastewater might be an attractive approach but long-term

operation is required to ascertain for the role of vegetation in this system.



7.3.2) Effect of HRTs on COD removal

To assess the effect of HRT on COD removal, two different HRTs were applied in the pilot units,

i.e. 8 and 4 days. In addition, two different COD concentrations were used as influent (2000 to

3000 mg/L) in order to examine the removal of COD. The different HRTs and influent

concentrations of COD affected the rate of surface load (SLR), which ranged from 34.2 to 128.3

gr/m2/d for SCW-P, from 38.5 to 120.2 gr/m

2/d SCW-U, from 31.6 to 122.8 gr/m

2/d for SCW-P

and 24.2 to 87.8 gr/m2/d SCW-U.

Figures 7.8 and 7.9 show the correlation charts of surface load and COD removal performance

for the four pilot units. COD removal rates showed an increasing trend with COD surface loading

in the units. However, the removal efficiency of COD did not differ significantly when the

concentration range of influent was 2000 to 2500 mg/L. In the presence of Cr(VI) in both pilot

units (Cr-P and Cr-U), significant amount of COD removal was observed with moderate surface

loading (Fig. 7.9a & b). On the other hand, with the same influent range, above 60% COD

removal was observed in the SCW-P and SCW-U pilot units (Fig. 7.8a & b). The removal of

COD in planted wetlands occurred because of the presence of vegetation, whereas in the

unplanted wetlands the removal of COD is thought to be due to microbial degradation. In this

experiment, Cr(VI) was used in combination with SCW. In the presence of Cr(VI), significant

amount of COD removal was recorded in all units with moderate surface loading of COD at both

HRTs (8 and 4 days). Thus, it could be concluded that HSF CWs can treat the combined

wastewater by removing organic matter at low residence times, i.e. up to 4 days, even in the

presence of heavy metals such as Cr(VI). Sin et al. (2000), reported that up to 2 mg/L of Cr ions

had no significant effect on the COD removal efficiency of a sequential batch reactor, but may

have a significant effect on the COD removal efficiency of an activated sludge process operated

with a short HRT (2 days). They also observed a reduction in COD adsorption in the presence of

heavy metal ions at HRTs of 2.5 days and less but at 5 days of HRTs, the retention of organic

matters within the reactor was sufficient for biological assimilation and organic adsorption.



0 20 40 60 80 100 120 140

-20

-10

0

10

20

30

40

50

60

70

80

90

100R

emo

va

l (%

)


Figure 7.8a: Correlation charts of surface load and removal rates of COD in the SCW-U unit.



-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

-10

0

10

20

30

40

50

60

70

80

90

100R

emo

va

l (%

)


Figure 7.8b: Correlation charts of surface load and removal rates of COD in the SCW-P unit.



-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

-20

-10

0

10

20

30

40

50

60

70

80

90

100R

emo

va

l (%

)


Figure 7.9a: Correlation charts of surface load and removal rates of COD in the Cr-U unit.



-10 0 10 20 30 40 50 60 70 80 90 100 110

-10

0

10

20

30

40

50

60

70

80

90

100R

emo

va

l (%

)


Figure: 7.9b Correlation charts of surface load and removal rates for COD in the Cr-P unit



7.3.3) Comparison with experimental results of the first operational period

To compare COD removal rates between the two operational periods, specific time periods were

selected when the average temperature (p value = 0.229> 0.05), the hydraulic residence time and

the inlet concentration of COD were almost identical. One-way ANOVA statistical analysis was

performed for SCW-P and SCW-U, to investigate whether removal efficiencies of COD in the

two pilot units show statistically significant differences between the two operational periods.

Table 7.3 presents means and standard deviations for temperature and COD removal rates and the

values of the other operational parameters (residence time, inlet concentration) for the two units

in both sessions.

Table 7.3: Comparison of COD removal in the two experimental sessions.

Time period Temperature

(0C)

HRTs

(Days)

Influent

concentrations

(mg/L)

Removal

(%)

SCW-P

28/4 -15/6/2011 19.46 ± 3.31 8 2500 49.33 ± 10.06

20/4 – 15/6/2013 20.17 ± 3.23 8 2500 55.75 ± 27.31

SCW-U

28/4 -15/6/2011 19.46 ± 3.31 8 2500 48.57 ± 29.55

20/4 – 15/6/2013 20.17 ± 3.23 8 2500 49.03 ± 22.58

The ANOVA results showed that the COD removal between the two sessions did not show

statistically significant differences (p value =0.576> 0.05) for SCW-P pilot unit. Similarly, SCW-

U also did not show any statistical significant differences in COD removal between the two

sessions (p value = 0.976> 0.05). This result suggests that the combined wastewater of cheese

whey and Cr(VI) can be treated in CWs and could that removal rates of organic matter are

substantial even in the presence of Cr(VI).

Statistically, there was no significant variation between the two experimental periods, but the

removal rate of COD reduced from 91% to 70% in the SCW-P unit and remained the same (65%)

in the SCW-U. This reduction in COD removal rate might be due to the addition of Cr(VI) to the



SCW solution. As a result, the effluent COD values were relatively high but the removal rates

remained satisfactory in the systems fed with integrated wastewater. However, during the first

experimental period, these two units were very efficient in treating diluted (Sultana et al., 2014c).

The toxicity of Cr(VI) could affect the COD removal efficiency. An increased concentration of

Cr(VI) ≥5 mg/L decreases the COD removal efficiency according to Stasinakis et al. (2003).

Stasinakis et al. (2003), also reported that 1 to 3 mg/L of Cr(VI) did not have any significant

impact on COD removal, but reductions in removal were observed when Cr(VI) concentrations

were greater than 5 mg/L. In this case, the reduction in COD removal was less than 10 % at

concentrations of 5 mg Cr(VI)/L. No change on the COD removal was observed under shock

loading conditions with a concentration of 5 mg/L of Cr(VI) (Stasinakis et al., 2003). Ertugrul et

al. (2006), also observed little to no impact on microorganisms during the first day of shock

loading with 5 mg/L and 10 mg/L Cr(VI). Shock loading of 5 mg/L and 10 mg/L Cr(VI) also

decreased COD removal efficiency in the second day, and the CWs required 3 and 5 days,

respectively, to recover to a steady state condition (Ertugrul et al., 2006). As the concentration of

Cr(VI) increased, a more pronounced effect of Cr(VI) on microorganisms was observed in their

study.

Heavy metals like Cr(VI) are highly toxic substances and have toxic effects on microorganisms,

because they tend to adhere to microorganisms and produce complexes with bacteria (Sin et al.,

2000). Metal ion complexion with heavy metals effects the growth of bacteria and can lead to

their death. Suthirak and Sherrard (1981) and Dilek et al. (1998), observed that Cr and Ni inhibit

microbial activity and negatively affect COD removal efficiency at concentrations above 10

mg/L. Thus, results showed that wastewater containing trace levels of heavy metal ions within

the effluent discharge limits may affect COD removal efficiency at short HRTs. Moreover, at

trace amounts, heavy metal ions act as a stronger competitor and reduce COD adsorption on the

bioflocs (Sin et al., 2000).


Chapter 8: General Discussion and Applications

CHAPTER 8: GENERAL DISCUSSION AND APPLICATION

8.1) Overall discussion ......................................................................................................... 169

8.2) Application ..................................................................................................................... 174


Chapter 8: General Discussion and Applications 169

CHAPTER 8: GENERAL DISCUSSION AND APPLICATIONS

8.1) Overall discussion

Over the last years, constructed wetlands (CWs) have been shown to successfully treat both

Cr(VI) and SCW. Although significant research has been conducted certain important issues

such as the effects of HRT, vegetation and temperature have not been thoroughly studied. The

main objectives of this PhD included the investigation of the effect of these parameters (i.e.

HRT, vegetation and temperature) on Cr(VI) and COD removal from industrial and agro-

industrial wastewater. The optimum goal was to operate CWs with the lowest possible HRTs,

while maintaining Cr(VI) and COD concentrations under legislation limits, and to provide a

sustainable treatment solution for the toxic by-products (i.e. reed biomass).

Based on the experimental results it can be concluded that Cr(VI) uptake by the plant biomass

was the main removal mechanism as the planted unit (Cr-P) recorded full Cr(VI) removal

(100%) for most of the operation period. These high removal efficiencies of Cr-P decreased

only when the plant biomass was harvested. Although the ability of common reeds to remove

Cr(VI) has been reported previously (Khan et al., 2009; Mant et al., 2006; Fibbi et al., 2012;

Maine et al., 2009; Calheiros et al., 2008b), for the first time planted and unplanted CW units

showed such high differences in Cr(VI) removal with the highest Cr(VI) influent

concentrations. As previously reported (Khan et al., 2009; Lesage et al., 2007b; Calheiros et

al., 2008b; Mant et al., 2006; Liu et al., 2010), Cr(VI) was found to accumulate mainly in

reed’s root zone. The high Cr(VI) accumulation by reed roots is attributed to the mechanism

responsible for metal uptake, by which plants receive the ionic forms of metals through their

root cells and the metal ions then enter into the root system. The ion can be stored in the root

tissue or translocated into the shoots through xylem tissue (Jabeen et al., 2009).

To maintain stable Cr(VI) removal throughout the year, plant biomass should be only

harvested once every year. This plant biomass is considered a toxic waste due to its high Cr

content. Until now, no viable, cost-effective treatment solutions for this reed biomass have

been recorded. In the frames of this research, harvested plant biomass was co-composted with

olive mill wastes leading to a final compost with high quality physicochemical characteristics,

which meets the EU limits for organic farming use.



On the other hand, the main Cr(VI) removal mechanisms in the unplanted unit (Cr-U) were

probably the accumulation of Cr ions by microorganisms, adsorption to the porous media or

the precipitation of Cr hydroxide. Cr(VI) precipitation was probably limited due to the neutral

pH values. Baral et al. (2006) and Dotro et al. (2011b), mentioned that with increased pH

values (above acidic limits), the solubility and precipitation of chromium hydroxide

decreased.

The effect of HRT on Cr(VI) removal was thoroughly investigated for the first time in the

present dissertation. Kucuk et al. (2003), used different HRTs in HSF CWs treating

wastewater containing Cr (VI), although the discussion on the effect of HRT on Cr(VI)

removal is limited. While a wide range of HRTs (i.e. from 8 days to 1 day) were used in this

study, HRT seems to not have a significant effect on Cr(VI) especially in the planted unit (Cr-

P) where the main removal mechanism was plant accumulation. Cr(VI) removal was only

limited when an HRT of 12hr was applied, probably affected by the plants diurnal cycle.

However, it is notable that planted CWs in this study achieved complete Cr(VI) removal

when operated under the lowest HRT (1 day) ever reported. Thus, the main drawback of CWs

(i.e. high area requirements) can be overcomed. The present CWs not only operated

successfully under low HRTs but also while receiving high Cr(VI) feed concentrations. Thus,

pollutant loading rates of the presented study were among the highest reported in the

literature, and extremely high Cr(VI) removal rates (up to 100%) were achieved.

CWs used for secondary cheese whey (SCW) treatment in the present study proved to be

rather successful at COD removal, as the planted unit (SCW-P) recorded the highest COD

removal rates (up to 100%) during the whole operation period. COD removal was limited

only after plant harvesting. However, the plants re-generated quickly after harvesting and the

COD removal efficiency in the planted unit increased to 100%. The main mechanism of COD

removal was microbiological degradation, as also reported in previous studies (Liu et al,

2006; Kadlec and Wallace, 2009, Vymazal and Kröpfelová, 2009). The huge surface area of

the plant root system helped the microorganisms to flourish and they used the organic carbon

for their energy source. Aerobic microorganisms near the rhizosphere consumed the plant-

released oxygen and used it to breakdown organic compounds (Kadlec and Knight 1996;

Vymazal and Kröpfelová, 2009). Moreover, anaerobic bacteria also contributed to the

degradation of organic compounds by the multi-step process (Vymazal et al., 1998; Vymazal

and Kröpfelová, 2009). The removal of COD in the SCW-U unit was probably caused by the



combination of aerobic and anaerobic degradation (Kadlec and Brix, 1995; Vymazal et al.,

1998; Vymazal and Kröpfelová, 2009).

To assess effects of HRT on COD removal, four different HRTs were applied in both pilot-

scale units and SCW-P presented high COD removal efficiencies for all applied HRTs (8, 4

and 2 days). However, when HRT was limited to 1 day and the removal efficiency of COD in

both units was significantly affected. Thus, it can be concluded that an HRT of 1 day does

limit organic matter removal in a CW. SCW-U presented the highest COD removal

efficiencies with moderate influent COD concentrations at 4 and 8 days of HRT, but removal

decreased when ambient temperatures were low and at low HRTs (2 and 1 day). There is a

lack of scientific data regarding the effect of HRT on the removal of COD from SCW in

CWs. However, general studies on COD removal in CWs and effects of HRT have been

conducted (Akratos and Tsihrintzis 2007; Ghosh and Gopal 2010; Toet et al., 2005). These

studies have demonstrated the effect of HRT in HSF CWs and revealed that higher HRTs

improve effluent quality. Toet et al. (2005), reported that HRTs have no significant influence

on organic matter removal, while Akratos and Tsihrintzis (2007) reported that HRTs above 8

days are adequate for relatively high organic matter removal efficiencies. In this study, the

HRT of 2 days resulted in maximum removal of COD in the SCW-P unit and maintained the

stability of the treatment efficiency throughout the monitoring period. Normally, higher HRT

implies lower loading rate and more contact time, thus more stability in efficiency. However,

in this study, the lower HRT supported the development of aerobic conditions in the planted

unit and the removal of COD from the CW system was accomplished by a successful

combination of physical and microbial processes (IWA, 2000).

The effect of temperature on COD removal was assessed statistically for HRTs of 4 days and

both units showed different COD removal efficiencies for temperatures above and below

15oC. The significant effect of temperature on COD removal in the SCW-U was due to the

limited microbial activity at low ambient temperature. On the other hand, COD removal

efficiency in the SCW-P was not significantly affected by temperature which proved that

plants had the capacity to enhance COD removal all year round as the rhizosphere enhances

microbial growth and activity (Gerberg et al., 1986). Comino et al. (2011) reported that low

temperatures did not affect organic matter removal. On the contrary, Kern (2003) and Sharma

et al. (2013a) reported that COD removal efficiency was significantly affected by

temperature. In this research, COD removal in the SCW-P only decreased when plants were



harvested which indicates that the annual growth cycles of the reeds were crucial for CW

operation, and also revealed that plants played the key role in maintaining high COD removal

rates even at low temperatures or in cold seasons.

After two years of operation (first experimental period), the four pilot-scale CW units were

used to co-treat (second experimental period) Cr(VI) and SCW for approximately six months.

In this second experimental period, the Cr(VI) removal efficiency in all planted and unplanted

pilot-scale HSF CW units (SCW-U, SCW-P, Cr-U and Cr-P) was 100% all year round, and

did not show any significant differences in Cr(VI) removal. Thus, the main removal

mechanism of Cr(VI) was not plant accumulation but microbial activity. During the first

operational period, the SCW-U, SCW-P units only treated diluted SCW, therefore, in second

experimental period they showed great adaptability to co-treat the cheese whey and Cr(VI).

However, Cr-U and Cr-P had already been used to treat Cr(VI) in the first experimental

period, and SCW was introduced in the second. Due to the use of this mixed wastewater of

SCW and Cr(VI), rapid plant growth enhanced microbial activity and hence Cr(VI) removal.

The second experimental period was designed to investigate the possible use of SCW as a

carbon source for Cr(VI) removal. The results showed that SCW proved to be beneficial for

the treatment of Cr(VI) in HSF CWs. In the first experimental period, the Cr(VI) removal

efficiency in Cr-U was not as high as in the Cr-P unit. However, when SCW was introduced,

both units showed the highest (100%) possible removal efficiencies. In the first experimental

period, tap water was used to dilute the wastewater. Tap water does not contain organic

carbon and thus limited the growth of microorganisms. In the second experimental period,

cheese whey was mixed with Cr(VI) solution in all the units and the microorganisms obtained

energy from the organic carbon contained in the SCW to remove Cr(VI) from all the units.

For the two different HRTs (8 and 4 days), the removal rates of Cr(VI) in the four pilot units

were not affected by the different surface loads, and remained stable (100%) removal

throughout the experiment. This is the first time that SCW was used in a CW system to treat

Cr(VI), therefore comparisons with other studies cannot be made. However, landfill leachate

and activated sludge which are also very good potential sources of organic carbon, have been

used in the treatment of heavy metals in different studies (Kamarudzaman et al., 2011;

Stasinakis et al., 2003; URL 3) and they also recorded a high removal efficiency of heavy

metals including Cr(VI). Sochacki et al. (2014), also used peat as an internal carbon source in

a CW system and achieved high removal of heavy metals.



The high Cr(VI) removal efficiencies observed in all units suggest that Cr(VI) removal

mechanisms should be associated with microbial populations and the CW media composition

because a sustainable amount of COD was removed which proves that all units contained

significant populations of microorganisms on the gravel bed. It has been reported that bacteria

growing in biofilms produce exopolymers that can act as molecular sieves or adsorbents

(Ragusa et al., 2004). Moreover, Dotro et al. (2009) reported that iron and organic-bound

phases dominate the Cr(VI) removal mechanisms. Therefore, it can be concluded that removal

of Cr(VI) in the unplanted units were most likely due to chemical precipitation or sorption by

microorganisms or to the gravel bed.

The COD removal efficiencies in the four HSF CW units differed between the units. The low

removal efficiency of COD might be due to the sub-lethal effect of Cr(VI) on

microorganisms. Chua et al. (1999), studied the sub-lethal effects of heavy metals on

activated sludge microorganisms and found that in the presence of Cr and at 5 days of HRT,

the overall COD removal efficiency showed no significant change, rather the sorption

capacity of biomass for dissolved COD which was reduced by 12%. They also described that

heavy metals affected the COD adsorption capacity of the activated sludge system.

Additionally, metal ions acted as a strong competitor against the organic compounds for

active sites on the bioflocs instead of acting as a toxic microbial inhibitor which finally

hampers the organic adsorption and affects the COD removal efficiency under shorter HRTs.

The decreased COD removal rate of SCW-P in the second experimental period compared to

the first experimental period could be attributed to: (a) when pilot units were fed with SCW

and Cr(VI) mixed wastewater, the microorganisms had to adapt to the mixed wastewater; (b)

microorganisms have the affinity to adhere with metal ions, so the bioflocs of microbes might

be occupied by Cr ions and these ions acted as a strong competitor against organic

compounds which finally hampered the adsorption of organic substances and affected the

COD removal particularly at shorter HRTs (Chua et al., 1999; Sin et al., 2000); (c) Cr(VI)

produced different toxic substances or metal ion complexes with organic exudates of plant

roots for bioaccumulation that affect the growth of bacteria and increase organic matter in the

wetland (Cervantes et al., 2001).



Although statistical analyses did not show any significant variations between the two

experimental periods, COD removal rates decreased from 91% to 70% in the SCW-P unit,

while for the SCW-U, the removal rate was similar (65%). Sochacki et al. (2014), referred

that the disadvantage of using external carbon sources is that they increase effluent COD

values. It has been observed in other studies that Cr and Ni inhibit microbial activity and

negatively affect COD removal efficiency at concentrations above 10 mg/L (Suthirak and

Sherrard, 1981; Dilek et al., 1998). Therefore, it could be concluded that wastewaters

containing trace levels of heavy metal ions within the effluent discharge limits, may have an

effect on COD removal efficiency at short HRTs.

8.2) Application

The application of a constructed wetland system for the treatment of Cr(VI) and SCW was

shown to be feasible. Performance of the pilot-scale HSF CWs at the end of the research

revealed that it can be successfully applied to treat any industrial wastewater containing

Cr(VI), and the treatment would be efficient even with HRTs of 1 day and with optimum

load. Based on the present experimental results a CW treating Cr(VI) could receive a

hydraulic load up to 0.13 m3/d while a CW treating SCW could receive up to 0.06 m

3/d,

which corresponds to 80 kg of processed milk.

Common reeds (Phragmites australis) proved to be efficient vegetation for the treatment of

Cr(VI) in a CW system and mainly contributed to the removal of Cr(VI) and COD from

wastewater. They also showed no toxic effect from Cr(VI), even at the highest concentrations.

Composting of Cr(VI) treated reed plant biomass proved to be a suitable solution for the

disposal of plant biomass from a CW system. The compost was stable and therefore safe and

could be applied to organic food crops.

Cheese whey could be used as a carbon source in the treatment of Cr(VI) using a CW system.

Practically, it might be possible by collecting the two wastewaters from different sources and

mixing them in a tank before introducing them into the CW treatment system. Moreover,

transport of the liquids to the mixing tank and into the CW area may be an issue.


Chapter 9: Conclusions and Recommendations

CHAPTER 9: CONCLUSIONS AND RECOMMENDATIONS

9.1) Overall Conclusions ...................................................................................................... 175

9.2) Recommendations for Future Research ..................................................................... 177


Chapter 9: Conclusions and Recommendations 175

CHAPTER 9: CONCLUSIONS AND RECOMMENDATIONS

9.1) Overall Conclusions

HSF CWs proved to be successful in treating aqueous Cr(VI) solution, SCW wastewater and

a combined wastewater of Cr(VI) and SCW, as they achieved high removal efficiencies for

both Cr(VI) and COD.

Concerning the treatment of aqueous Cr(VI) solutions by HSF CWs, based on two years of

experiments, it can be concluded that:

The planted CW unit removed Cr(VI) rather efficiently with mean removal rates of 88%.

The unplanted unit showed significantly lower Cr(VI)removal efficiencies (21%). These

differences in Cr(VI) removal can be attributed to the role of the plant vegetation.

Under steady state conditions, effluent concentrations in the Cr-P unit were always below

the permitted legislation limit of 0.05 mg/L.

The only period during which Cr(VI) removal efficiency of the planted units decreased

was that following plant harvesting. This decrease was temporary and Cr(VI) removal

efficiency rose again when plant biomass increased.

Removal efficiency in the planted unit was not affected by HRT, as Cr(VI) removal

efficiencies were high and rather stable even with HRTs as low as 1 day.

HRTs below 1 day cannot be applied successfully in CWs, as CW pollutant removal

efficiency is affected by diurnal cycles of the plant vegetation.

A viable disposal method for plant biomass obtained from CWs treating Cr(VI)

wastewaters was presented for the first time. Plant biomass was co-composted with olive

mill wastes and produced a final product that meets EU regulations for organic farming

applications.



Secondary cheese whey (SCW) was treated using HSF CWs. Following two years

experiments it could be concluded that:

Both pilot-scale CW units (planted and unplanted) proved to be rather effective in SCW

treatment, as they achieved high COD removal efficiencies of (91% and 77.23% for SCW-P

and SCW-U, respectively. These high COD removal rates, even in the SCW-U unit, reveal

that organic matter was mainly removed by biological processes. Nevertheless, the plant

vegetation significantly affected COD removal, proving the positive effect of vegetation on

CW operation.

Under steady-state conditions effluent concentrations in SCW-P were always below the

permitted legislation limits (120 mg/L), when COD influent concentrations were below 3500

mg/L.

Ambient temperature negatively affected only the SCW-U unit, while the SCW-P appeared

to be affected only by the annual plant growth cycle.

Both CW units demonstrated satisfactory COD removal efficiencies even when operated

under HRTs of 2 days, which is at present the lowest HRT reported in the literature. HRTs of

1 day proved to be insufficient for COD removal in both units.

In the second experimental period, the four HSF pilot-scale CWs treated a mixed wastewater

of Cr(VI) and SCW for six months. From these experiments it can be concluded that:

In all four pilot-scale CWs units, Cr(VI) was removed with a success rate of 100%

with an HRT of 4 days.

The average COD removal rate was 77% in the planted units, indicating the important

role of vegetation in the wetland. The average removal rate of COD was 56% in the

unplanted unit.

HRTs of 4 and 8 days were sufficient to remove Cr(VI) and COD from the combined

wastewater of Cr(VI) and SCW.



Comparing the data from two experimental periods, results indicate that removal of

Cr(VI) was not only due to the adsorption/absorption by the plant biomass but also the

action of microorganisms.

Regarding COD removal, the results were not affected by the two different period

which may suggests that that the combined wastewater of cheese whey and Cr(VI) can

be treated in CWs and could that removal rates of organic matter are substantial even

in the presence of Cr(VI).

9.2) Recommendations for Future Research

Although significant results were drawn from the pilot-scale HSF CW operation, a series of

factors should also be examined in order to safely use HSF CWs to treat Cr(VI) and SCW.

Therefore, future research should focus on:

Evaluating the toxicity of chromium on plant species which are commonly used in

CWs.

Assessing the role of microorganisms on Cr(VI) and COD removal in CWs with

different HRTs.

Conducting additional experiments for the co-treatment of Cr(VI) and SCW to

determine optimal operational conditions (i.e., HRT, influent concentrations), as in

this research, COD effluent concentration exceeded legislation limits.

To further improve CW performance on agro-industrial wastewater treatment. Specific

design and operation parameters should be examined, including vegetation density,

step feeding, and special porous media.


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WEBSITES

URL 1: http://www.aquastress.net

URL 2: http://www.aquaticplantcentral.com

URL 3: home.eng.iastate.edu/~tge/ce421-521/jenny.pdf

http://www.aquastress.net/

http://www.aquaticplantcentral.com/

http://home.eng.iastate.edu/~tge/ce421-521/jenny.pdf


Appendix

APPENDIX


Appendix 236

APPENDIX

Measurement Procedure of Hexavalent Chromium, Cr(VI):

The concentration of Cr(VI) was calculated by «3500- Cr D Colorimetric method» according

to Standard Methods for the Examination of Water and Wastewater (APHA, 1989).

Instruments:

1. Spectrophotometer (BOECO, Germany, S-20 Spectrophotometer) (Figure 1.)

2. Pipettes.

3. Cups boiling.

4. Magnets 4.

Figure 1: Spectrophotometer (BOECO, Germany, S-20 Spectrophotometer).

Solutions - Reagents

1. Concentrated H2SO4.

2. Diphenyl Carbazide [50ml pure acetone (assay> 99.5%) and 0.25g dust Diphenyl

Carbazide, poured in the bottle that wrapped well in foil, and stirred until the powder

was dissolved (the solution should be clear). The date of reagent making have been

written and kept in the refrigerator for 4 days only)


Appendix 237

Procedure of Analysis:

Samples were filtered in vacuum pump with filters of 0.45mm Milliporefilters (GN-6

Metricel Grid 47mm, Pall Corporation). Then 1mL of filtered samples was added in a beaker

that contained 49ml of deionized water for making of 1:50 dilution ratio. Placed in stirring

and thereafter 1ml diphenyl-carbazide was added and 2-3 drops of H2SO4. The sample

covered with aluminum foil and stirred for 5-7 min for the development and configuration of

color. The spectrophotometric measurement of the absorbance of the sample became at

540nm.

Manufacture standard calibration curve for measurement of the Cr(VI):

Stock Solution of Cr(VI) for Concentration of 500mg/L

500mg/L = 500mg/1L H2O= 0.5gr/1L H2O

Atomic weight of K2Cr2O7 = 294.1846 gr.

In, 294.1846gr of K2Cr2O7 → 103.9922gr of Cr X=1.4145gr K2Cr2O7

X; gr of K2Cr2O7 → 0.5 gr of Cr in K2Cr2O7

Therefore, In 1L → 0.5gr Cr Z=0.05 gr Cr

0.1L → Z; Cr

In, 294.1846gr of K2Cr2O7 → 103.9922gr of Cr X=0.14145gr K2Cr2O7

X; gr of K2Cr2O7 → 0.05 gr of Cr in K2Cr2O7

So, the stock solution was prepared by adding, 0.14145gr of K2Cr2O7 in 100ml Milli- Qwater.

Standard Solution of Cr(VI) for Concentration of 5mg/L:

500 mg/L 5 mg/L

5 mg/L


Appendix 238

C1× V1 = C2× V2

500mg/L ×V1 = 5 mg/L ×100mL

V1 = 1mL Cr (100mL – 1mL = 99mL H2O)

Therefore, the standard solution was prepared by adding 1mL of the stock in 100mL Milli-

Qwater.

After preparing the Stock and the Standard solution and the corresponding selected range of

concentrations of the standard solutions were prepared, the range was from 0.04mg/L to 4

mg/L.

The Equation was for the preparation of standard solutions:

C1× V1 = C2× V2

Where, C1= 5 mg/L

V1= 50 mL

For, C2 = 0.04 mg/L

C1 × V1 = C2 ×V2

5 × V1 = 0.04 × 50 V1 = 0.4mL

Therefore, 0.4ml of the Standard solution was added add in 49.6mL Milli- Qwater and stirred.

For, C2 = 0.08 mg/L

C1 × V1 = C2 ×V2

5 × V1 = 0.08 × 50 V1 = 0.8mL

Therefore, 0.8mL of the Standard solution was added in 49.6mL Milli- Qwater and stirred.

For, C2 = 0.16mg/L

C1 × V1 = C2 ×V2

5 × V1 = 0.16 × 50 V1 = 1.6mL

Therefore,, 1.6mL of the Standard solution was added in 48.4mL Milli- Qwater.

For, C2 = 0.2mg/L


Appendix 239

C1 × V1 = C2 ×V2

5 × V1 = 0.2 × 50 V1 = 2mL

Therefore, 2mL of the Standard solution was added in 48mL Milli- Qwater.

For, C2 = 0.32mg/L

C1 × V1 = C2 ×V2

5 × V1 = 0.32 × 50 V1 = 3.2mL

Therefore, 3.2mL of the Standard solution was added in 46.8mL Milli- Qwater.

For, C2 = 0.5mg/L

C1 × V1 = C2 ×V2

5 × V1 = 0.5 × 50 V1 = 5mL

Therefore, 5mL of the Standard solution was added in 45mL Milli- Qwater.

For, C2 = 0.64mg/L

C1 × V1 = C2 ×V2

5 × V1 = 0.64 × 50 V1 = 6.4mL

Therefore, 6.4mL of the Standard solution was added in 43.6mL Milli- Qwater.

For, C2 = 0.9mg/L

C1 × V1 = C2 ×V2

5 * V1 = 0.9 * 50 V1 = 9mL

Therefore, 9ml of the Standard solution was added in 41mL Milli- Qwater.

For, C2 = 1mg/L

C1 × V1 = C2 ×V2

5 × V1 = 1 × 50 V1 = 10mL


For, C2 = 1.3mg/L


Appendix 240

C1 × V1 = C2 ×V2

5 × V1 = 1.3 × 50 V1 = 13mL


For, C2 = 1.4mg/L

C1 × V1 = C2 ×V2

5 × V1 = 1.4 × 50 V1 = 14mL


For, C2 = 1.6mg/L

C1 × V1 = C2 ×V2

5 × V1 = 1.6 × 50 V1 = 16mL


For, C2 = 2.24mg/L

C1 × V1 = C2 ×V2

5 × V1 = 2.24 × 50 V1 = 22.4mL

Therefore, 22.4ml of the Standard solution was added in 27.6mL Milli- Qwater.

For, C2 = 3.52mg/L

C1 × V1 = C2 ×V2

5 × V1 = 3.52 × 50 V1 = 35.2mL

Therefore, 35.2ml of the Standard solution was added in 14.8mL Milli- Qwater.

For, C2 = 4mg/L

C1 × V1 = C2 ×V2

5 × V1 = 4 × 50 V1 = 40ml


Then the graph was constructed where, in “y” axis represented the concentrations and the “x”

axis represented the absorbance.


Appendix 241

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1 1,2

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

1,3

1,4

1,5

1,6

1,7

C

r(V

I) C

on

cen

tra

tio

n, m

g/L

Absorbance (540nm)

Equation y = a + b*x

Adj. R-Squa 0,99983

Value Standard Err

mg/L Intercept 0 --

mg/L Slope 1,3199 0,005

Preparation of blank for spectrophotometer:

50 mL distilled water was taken in a beaker.

The beaker was put in stirring

1mL Diphenyl Carbazide was added and after that 2-3 drops of concentrated

sulfuric acid (H2SO4) was added

Then it measured in the spectrophotometer at 540nm with a plastic cuvette.

Sample preparation:

1-1.5mL sample was taken with pipette and filtered.

49mL of distilled water and 1ml of the filtered sample were taken in a beaker of 50

mL for making the dilution ratio of 1:50.

Then the beaker was put in stirring

1mL Diphenyl Carbazide was added and after that 2-3 drops of concentrated

sulfuric acid (H2SO4) was added.


Appendix 242

Covered the beaker with foil and waited for 5-7 minutes.

When the sample was turned in purple colour then it measured in the

spectrophotometer at 540nm with a plastic cuvette.

Calculation:

The concentrations of samples were calculated by following equation:

Cr (VI) (mg/L) = Absorption × Dilution ratio × Slope


Appendix 243

Measurement Procedure of dissolved Chemical Oxygen Demand (d-COD):

The determination of chemical oxygen demand, was performed according to the method

"Closed Reflux" described in Standard Methods for the examination of water and wastewater

(APHA et al. 1989).

The COD represents the amount of oxygen consumed in the chemical oxidation of organic

compounds, which contained both the wastewater and natural waters. The oxidation is done

with potassium dichromate in an acidic environment and the results are expressed in mg/L of

water.

Apparatus Required:

Instruments:

1. Digester (HANNA instruments C9800 REACTOR).

2. Photometer (HANNA C99 Multiparameter Bench Photometer for Laboratories).

3. Bottles for digestion.

Reagents:

1. Digest Solution of K2Cr2O7*

2. Catalyst**(concentrated sulfuric acid 96%, H2SO4 with Silver sulfate,Ag2SO4).

According to the Standard Methods for the examination of water and wastewater (APHA et

al. 1989), the digestion solution for “Closed Reflux” method was prepared by following

procedure:

*Digest solution:

• 500 mL distilled water.

• 10.216 gr K2Cr2O7 (having been dried at 150 ° C for 2h).

• 167 mL H2SO4.

• 33.3 gr HgSO4.

• Diluted with distilled water to a volume of 1000 mL.


Appendix 244

**Catalyst

• In concentrated H2SO4, Ag2SO4 was added in ratio of 5.5 gr Ag2SO4 / kg of H2SO4 and

stirred for 1day to dissolve. stirred for 1day to dissolve.

Figure 2: COD Photometer (ΗΑΝΝΑ C99 Multiparameter Bench Photometer for

Laboratories) –Digester of COD (ΗΑΝΝ Αinstruments C9800 REACTOR).

22 gr Ag2SO4 4Kg H2SO4 dH2SO4=1.83g/mL

d=m/ V V =m/d=4000 gr/1.83gr/ mL =2185.79 mL = 2.186L

22grAg in 2.186L H2SO4

X 1 L

X=10.064gr AgSO4/ L


Appendix 245

Analysis procedure:

• 2 mL of solution at appropriate dilution was added in two vials and a third vial was filled

with distilled water.

• Then in the three vials, 1.2mL of digestion solution and 2.8 mL catalyst were added.

• The vials were then put in vortex.

• After vortex, the vials placed in the digester (150 ° C) for 2 hours.

• After two hours, the bottles removed and opened the lids to do "chuff", reclosed, and waited

for 10 min. to cool.

• Again the vials were put for vortex.

• After 2 hour the samples were measured in absorption photometer of COD (Figure 2),

Programme 31 (3 consecutive measurements around, read direct).

Calculation:

The COD concentrations of samples were calculated by following equation:

Dissolved COD, (mg/L) = Absorbance × Dilution ratio

Treatment of Industrial and Agro-industrial Wastewater Using Constructed Wetlands Mar-Yam Sultana

Short Curriculum Vitae 246

Short Curriculum Vitae of

Mar-Yam Sultana

Father’s Name : Prof. Dr. M. Abdul Karim

Mother’s Name : Sayeeda Begum

Date of Birth : 10 September, 1984

Nationality : Bangladeshi

Permanent Address : Village: Parialpur, Post Office: Setabganj-5216,

Police Station: Bochaganj, District: Dinajpur,

BANGLADESH

Present Address : Laboratory of Environmental Systems,

School of Engineering,

Department of Environmental and Natural

Resources Management, University of Patras,

G. Seferi 2, Agrinio, T.K. 30100, GREECE.

Email: [email protected],

[email protected]

EDUCATIONAL QUALIFICATION:

PhD Candidate (2011-2014) : Laboratory of Environmental Systems,

School of Engineering,

Department of Environmental and Natural

Resources Management, University of Patras,

G. Seferi 2, Agrinio, T.K. 30100, GREECE

Thesis title: Treatment of Industrial and Agro-Industrial Wastewater Using Constructed

Wetlands

Master of Science (Jan 2007-June 2008) : MS in Irrigation and Water Management*

Department of Irrigation and Water Management,

Faculty of Agricultural Engineering

Bangladesh Agricultural University,

Mymensingh-2202, BANGLADESH

mailto:[email protected]




* MS Thesis Title: Performance Improvement of a Traditional Pedal Pump.

Bachelor of Science (July 2002-June 2006) : B.Sc. in Agricultural Engineering

Faculty of Agricultural Engineering

Bangladesh Agricultural University,

Mymensingh-2202, BANGLADESH

PUBLICATION IN INTERNATIONAL JOURNALS

1. M.K. Michailides, M.Y. Sultana, A.G. Tekerlekopoulou, C.S. Akratos, D.V. Vayenas,

2013. Biological Cr(VI) removal using bio-filters and constructed wetlands, Water

Science Technology, 68: 2228-2233.

2. M.Y. Sultana, A.K.M.M.B. Chowdhury, M.K. Michailides, C.S. Akratos, A.G.,

Tekerlekopoulou, D.V., Vayenas, 2014. Integrated Cr(VI) removal using constructed

wetlands and composting, Journal of Hazardous Materials, In press.

3. M.Y. Sultana, C.S. Akratos, S. Pavlou and D. V. Vayenas, 2014. Chromium removal in

constructed wetlands: A review. International Biodeterioration and Biodegradation, 96:

181-190.

4. M. K. Michailides, T. Tatoulis, M.Y. Sultana, A. Tekerlekopoulou, I. Konstantinou, C.

S. Akratos, S. Pavlou, D.V. Vayenas. 2014. Start-up of a free water surface constructed

wetland for treating olive mill wastewater. Journal of Chemical Industry (Hemijska

industrija). doi:10.2298/HEMIND140820076M.

5. M.Y. Sultana, C. Mourti, T. Tatoulis, C.S., Akratos, A.G. Tekerlekopoulou, D.V.,

Vayenas, 2014. Effect of hydraulic retention time, temperature, and organic load on a

horizontal subsurface flow constructed wetland treating cheese whey wastewater.

Submitted.



PUBLICATION IN NATIONAL JOURNALS

M. Y. Sultana; A. Khair and K. M. Hasanuzzaman 2008. Improvement of Performances of the

Traditional Pedal Pump. Journal of Environmental Science and Natural Resources, 1: 73-78.

PUBLICATION IN INTERNATIONAL CONFERENCES

1. M.K. Michailides, M.Y. Sultana, A.G. Tekerlekopoulou, C.S. Akratos and D.V.

Vayenas. (2013). Biological Cr(VI) removal using bio-filters and constructed

wetlands, Proceedings of International conference on Asset management for

enhancing energy efficiency in water and wastewater systems, 24-26 April, 2013,

Marbella, Spain.

2. M.Y. Sultana, M.K. Michailides, C.S. Akratos, A.G. Tekerlekopoulou and D.V.

Vayenas (2013). Effect of hydraulic residence time on Cr(VI) removal using

constructed wetlands. Proceedings of the 13th International Conference on

Environmental Science and Technology, 5-7 September 2013, Athens, Greece.

3. M.Y. Sultana, M.K. Michailides, C.S. Akratos, A.G. Tekerlekopoulou and D.V.

Vayenas (2013). Constructed wetlands pilot-scale units for the co-treatment of Cr(VI)

and secondary cheese-whey. Proceedings 4th

Conference of Decentralized Wastewater

Treatment Plants, 25-27 October 2013, Volos.

4. M.K. Michailides, M.Y. Sultana, C.S. Akratos, A.G. Tekerlekopoulou and D.V.

Vayenas (2014). Integrated chromium waste treatment. Proceedings of 4th

Environmental Conference of Macedonia, 14-16 March, Thessaloniki.

5. M.Y. Sultana, T. Tatoulis, C. S. Akratos, A.G. Tekerlekopoulou and D.V. Vayenas

2014. Effect of operational parameters on the performance of a horizontal subsurface

flow constructed wetland treating secondary cheese whey and Cr(VI) wastewater.

Proceedings of 2nd International Conference on Advances in Civil, Structural and

Environmental Engineering - ACSEE’ 14, Zurich 25-26 October, Switzerland.



Research Experience:

Worked as Scientific Officer under the approval of Bangladesh Agricultural University

Research System (BAURES) funded by Japanese Government (NIAES), research project

entitled “Rice Paddy Flux Observation in Bengal Lowland (Project#2005/29/NIAES)”

Department of Environment Science, Bangladesh Agricultural University, Mymensingh from

August, 2008-July, 2010.

References:

1. Professor Dr. Dimtrios V. Vayenas

Environmental Systems Laboratory


University of Patras, G. Seferi 2, Agrinio 30100

GREECE

Email: [email protected]

Tel: +302641074117

2. Dr. Christos S. Akratos

Assistant Professor

Environmental Systems Laboratory


University of Patras, G. Seferi 2, Agrinio 30100

GREECE

Email: [email protected]

Tel: +302641074209



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TREATMENT OF INDUSTRIAL AND AGRO-INDUSTRIAL WASTEWATER … · Treatment of Industrial and Agro-Industrial Wastewater Using Constructed Wetlands Mar-Yam Sultana Acknowledgements iv