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UNIVERSITY OF HAWAII LIBRARY

DESIGN AND COMPARISON OF DIN REMOVAL RATES BETWEEN FIVE 'LOW-TECH' FIXED FILM BIOLOGICAL REACTORS TREATING

AQUACUL TURE WASTEWATER ON COCONUT ISLAND

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

•

MASTER OF SCIENCE -

IN

OCEANOGRAPHY

DECEMBER 2005

By

Lauren Carter Roth

Thesis Committee:

Edward Laws, Chairman David Karl

Barry Raleigh

. ...

10 002605469 UNIVE.RSITY OF HAWAII

HAWN Qlll

.H3 no. 4039

We certi fy that we have read thi s thesis and that it is satisfactory in the scope and quality as a thesis for the Master of Science degree in Oceanography_

T HESIS COMM ITTEE

Chairman

•

,

--II

© Copyright 2005

By

Lauren C. Roth

111

ACKNOWLEDGEMENTS

The work written in this thesis is the accumulation of education I have

obtained from both the Department of Oceanography at the University of Hawaii and

that of my work with John Todd of Ocean Arks International. This thesis is dedicated

to my parents, who have provide"d much support and interest in my chosen directions

in life, and to Pacific islanders, of whom I hope will benefit from this work.

I first want to thank my committee, Drs. Edward Laws, David Karl, and Barry

Raleigh who have invested much time and effort in helping shape this scope of work

and for their insights and critique of the thesis itself. I also would like to thank Grieg

Steward, who served on my academic committee who also supported me with advice

and insight. Also, I would like to give much thanks to my funders: SeaGrant Hawaii,

Center for Sustainable Future and the Hawaii Board of Water Supply. This project

would also not have occurred without the support of Hawaii Institute of Marine

Biology (HIMB) and I extend a warm thank you to Jo-Ann Leong, Jane Ball and the

HIMB staff.

I would also like to express my gratitude to David Hashimoto who analyzed

my water samples and to Jane Schoonmaker, who performed the X-ray diffraction

analysis. I particularly want to thank Mikael Schneider, a Global Environmental

Science graduate of the University of Hawaii, who helped build and operate both the

systems on Coconut Island and MSB, aided with sampling, media analysis, and

research contribution for this report.

IV

There are many volunteers I would also like to acknowledge who helped

construct the remediation system at Coconut Island: Rob Norris, Michael Cerrea,

Stephen Kubota, Roseanne and family, Meredith Grinimer, Orion Stambro, Fred

Nakumura and the Sierra Club hiking club, Yves, Jason Silverstein, Jim Lakey and

the HIMB maintenance crew. The commencement of this project was truly a

community effort! Additionally, I would like to thank Kai Fox, who has volunteered

to aid in the continuance of maintaining the system.

Lastly, I would like to thank the entire Ocean, GeoChem, Bio, and HIMB

offices who have helped to keep me on track and have provided the necessary tools

for me to complete my thesis studies. Mahala nui loa!

v

ABSTRACT

Over the past century, the human needs for the Earth's resources have

increased and the consequences of these actions are becoming more apparent as

ecological diversity and functionality are in decline. An approach to better service

our continuance and future generations' livelihoods is being explored by

implementing natural systems teclmologies. Ecological engineering is a mechanism

that can provide simple, 'low-tech' design and infrastructure while encouraging the

growth and development of nature's complexity as the primary operating system.

Ecologically engineered fixed film biological reactors were designed and

implemented to remediate freshwater, aquaculture wastewater on Coconut Island,

Oahu. The biological reactors principally operate by utilizing the inherent,

multifaceted biochemical development ofbiofilm ecologies. The primary objective of

this research was to design 'low-tech' systems that compared naturally occurring

media commonly found on Pacific Islands for their use in fixed film biological

reactor technology to remove dissolved inorganic nitrogen from the wastewater. The

media researched for the comparison study included: coral rubble, lava rock

(pumice), bluestone (hydropressurized basalt), coconut fiber, and a synthetic

geotextile material. The fixed film filters were connected to one freshwater

aquaculture tank containing Mozambique tilapia (Oreochromis mossambicus). Both

the influent (fish tank) and the effluents from the five filters were analyzed for total

suspended solids, ammonium, nitrate/nitrite, and phosphate concentrations. A

replicate system was also designed on a small scale to test for data reproducibility and

VI

was compared to the Coconut Island system. Overall, ammonium and total

suspended solids reduction was marked in all the systems. Therefore in order to

compare the media, the removal rate of ammonium + nitrate/nitrite (DIN) was

calculated for all the systems and statistical analysis was conducted to determine

differences between DIN removal rate efficiencies.

VI!

TABLE OF CONTENTS

ACKNOWLEDGEMENTS....................................................... IV

ABSTRACT..................................................................... ..... VI

LIST OF TABLES ................................................................... x

LIST OF FIGURES................................................................. xi

SECTION 1. Introduction................................................. I

SECTION 2.

SECTION 3.

SECTION 4.

Objectives.. ................................................ 4

Background.. ............................................... 7

1. Nitrification and Total Suspended Solids...... ..... 7

n. Phosphorus.............. .............. ................ 10

III. Fixed film reactors.. .. .. .. .. .... .. .. .. .... ...... .... . 11

IV. Biofilms ............................................... 12

Methods....... ......... ......... ............... ............ 15

1. Experimental Methods.... ................ ........... 15

I. Study Area.......... ............ .............. 16

2. Materials.... ............ ...................... 16

3. Small Scale Replication................ ..... 24

4. Sampling...................................... 26

5. FlowlHydraulic Residence Time.......... 26

6. Media .......................................... 27

Vlll

SECTION 5.

SECTION 6.

SECTION 7.

7. X-Ray Diffraction ........................... 28

8. Void Ratio and Specific Surface Area... 28

II. Laboratory Methods ...... , . .. . .. . .. .. .. .. . .. .. . . .. . .. .. .. .. . .. 32

1. TSS ............................................. 32

2. Nutrient Analysis........................... 32

III. Statistical Analysis......................................... ... 33

Results.......................................................... ..... 36

1. Media Results............................................... ... 36

II. Flow and Hydraulic Residence Time....................... 38

1. Flow....... .... ... ........... ....... ........... 38

2. Hydraulic Residence Time............... ... 39

III. Water Chemistry Results.. .................................. 40

I. Dissolved Oxygen and pH.............. .... 40

2. Nutrient and TSS Results................... 43

3. Statistical Results.. .................. ....... 46

4. Operations................................ .... 56"

Discussion............................................................................. 58

Future of Remediation System.................................. 63

REFERENCES ........................................ -....................................... 64

IX

LIST OF TABLES

Table Page

1 (Egli 2003)............................................................................ 9

2 Properties ofbilfilter media........................................................ 31

3a Hydraulic Residence Time (HRT) of the Coconut Island system....... ..... 39

3b Hydraulic Residence Time (HRT) of the MSB system........................ 39

4a Coconut Island O2 and pH values................................................. 41

4b MSB O2 and pH values............................................................. 42

5a Fixed Film Bioreactor Performances at Coconut Island........................ 44

5b Fixed Film Bioreactor Performances at MSB.................................... 45

6 Comparison ofC! and MSB biofilter systems based on Kruskal-Wallis test. 47

x

LIST OF FIGURES

Figure

1......................................... ....................................... ................. 18

2 .............................................................. :.................................. 21

3 ................................................................................................. 21

4 ................................................................................................. 22

5 ................................................................................................. 23

6 ................................................................................................. 25

7 ................................................................................................. 48

8 ................................................................................................. 49

9 ................................................................................................. 50

10 ................................................................................................ 51

11 ................................................................................................ 52

12 ................................................................................................ 53

13 ................................................................................................ 55

Xl

SECTION 1

Introduction

The Living Planet Report notes that in the past three decades the world's species

diversity has declined by 33% (WWF 2002). During the same time, the human need for

renewable resources (human footprint) has increased by 50%. Of the Earth's ecosystems,

freshwater species are cited as the most vulnerable with the least amount of regenerative

capacity. The Fresh Water Index in the same report describes a 50% decline of 194

freshwater aquatic species between 1970 and 1999 (WWF 2002). The United Nations

(UN) reports that 1.1 billion people around the world still lack access to safe drinking

water; 2.4 billion people live in areas with chronic water shortages; and contaminated

water and poor sanitation cause an estimated 80% of all diseases in the developing world

(UN, 2002). These global statistics are alarming. After attending the UN International

Meeting for Small Island Developing States (SIDS) in Mauritius, January 2005, I realized

that suitable technologies for correcting these problems are only in their infancy.

Hawken et al. (1999) point out that industrialized nations dominate technological

advancement yet deplete nonrenewable supplies and seek more. A more enlightened

approach would involve the use of sustainable technologies appropriately scaled to

address societal needs in a particular socio-politicallenvironmental milieu (Hawken et. al.

1999). Furthermore, advanced technologies that work well in first -world nations may be

associated with problems that become major obstacles to their widespread application in

second- and third-world countries (Weisman 1998). Examples include reliance on

I

sophisticated fanning equipment in countries that lack equipment maintenance

infrastructure and the use of agricultural pesticides in countries where there is no

satisfactory mechanism to train fanners about safety issues associated with their use.

Such use of inappropriate technologies can cascade into a 'tech' dependency so that

"when the developed word sneezes the rest of the (developing) world catches its cold"

(UN 2005). Weisman (1998) argues that addressing the growing needs of the global

human society will require an appropriate balance between nature and technology, e.g.,

the use of integrated pest management in agriculture. Sustainable development requires

that the relationship between dynamic human economic systems and the dynamics of

ecologic systems be such that human life can continue indefinitely without destroying the

diversity, complexity, and function of the environment (World Commission on

Environment and Development, 1987).

In order to implement sustainable practices, tools need to be provided to m.ake the

transition to alternative, ecologically sound practices for energy, agriculture, building

design, resource use, and water management. Ecological design/engineering is one

approach that provides a bridge towards more sustainable practices. Ecological design

I

incorporates lessons learned from natural selection processes that have been controlling

evolution for billions of years (Todd and Todd, 1994). Ecological design is intended to

support the long-term welfare of both humans and the environment by weaving together

nature and culture. Ecologically designed technologies provide and promote diversity in

their development through the incorporation oflocal materials, both living and nonliving.

The research described here was intended to approach aquatic food production and

wastewater remediation holistically by connecting these "industrial" ecological systems.

2

The research plan was to design, implement, and analyze a pilot bioremediation system to

treat aquaculture wastewater generated by fish culture facilities at the Hawaii Institute of

Marine Biology (Coconut Island, Oahu, Hawaiian Islands). The intention was to

scrutinize the potential application of such systems on other Pacific islands that share

common natural resources with the Hawaiian Islands.

3

SECTION 2

Objectives

In numerous parts ofthe world, freshwater aquaculture suffers from the constraint

oflimited freshwater supply. Recirculating aquaculture systems have therefore become

more common, since they not only conserve water but also reduce the land requirement

to farm fish (Bovendeur et al. 1987). There are a variety of recirculation filters available.

However, because of differences in local farming and operating conditions, lack of

expertise, and costs, imported aquaculture wastewater treatment systems sometimes fail

to provide adequate treatment and/or to be cost-effective and environmentally viable (Al­

Hafedh et al. 2003). A major concern in recirculation systems is accumulation of

ammonia. EPA water quality criteria with respect to ammonia/ammonium are based

entirely on the toxicity of ammonia, since the amm<\nium ion is essentially benign. Since

the equilibrium between ammonia and ammonium shifts toward ammonia as the pH is

raised, the criterion for total ammonia (i.e., ammonium plus ammonia) becomes stricter

as pH increases. There is also a positive correlation between temperature and toxicity.

For the protection of sensitive fish species, the four-day average total annnonia

concentration should not exceed 4.7 IlM at a pH of 9 and temperature of 30°C (EPA

1986). At a pH of7 and temperature of 25°C the corresponding criterion is 61 J.!M.

Ammonia/ammonium results from the release of nitrogenous waste products by

cultured animals. To achieve satisfactory control over ammonia concentrations in a cost­

effective manner, aquaculture recirculation systems in third-world countries need to be

4

tuned to local environmental conditions while being simple to operate. The experimental

aquaculture wastewater treatment systems at Coconut Island consisted of trickling filters,

which experience with conventional wastewater treatment systems has shown to be

relatively simple and inexpensive to operate (Laws, 2000). The filters contained readily

available media as substrates for microbial growth. The trickling filters were expected to

provide habitats for (a) ammonia oxidizing bacteria that would convert ammonia to

nitrate and (b) denitrifying bacteria that would convert the nitrate to N2. The objectives of

this study were fivefold:

I. Design "low-tech" fixed-film biological reactors that contain either a

commercially available synthetic medium or one or another of several

media that are commonly found on Hawaiian and other Pacific Islands and

that, to the best of my knowledge, had not previously been studied for

their utility in fixed-film biological reactors.

II. Engineer the bioreactors· as flow-through treatment systems that receive

equal fluxes of the aquaculture wastewater.

III. Compare ammonium and nitrate/nitrite (dissolved inorganic nitrogen

(DIN), and phosphate removal efficiencies of the trickling filters as

influenced by substrate type.

IV. Determine factors that may influence nitrogen removal.

v. Compare the reproducibility ofbioreactor performance with small-scale

replicas.

5

The following sections of this thesis will address these objectives in the following

manner. I begin in Section 3 with some background information regarding the basic

biochemical transformations involved in the conversion of ammonia to nitrogen gas

(nitrification and denitrification), history ofbiofilm reactors, and biofilm development.

Section 4 discusses the experimental methods including the location ofthe study,

materials that were used, design of the small-scale replica, sampling methods, discussion

of the media that were chosen, laboratory methods, and statistical methods used for this

study. Section 5 discusses the results of the study, which includes analysis of the media,

flow and hydraulic residence time, water chemistry, and statistical results. Section 6 is

the discussion. Lastly in Section 7, there is a discussion on the future of the remediation

project.

6

SECTION 3

Background

I. Nitrification and Total Suspended Solids

One of the primary purposes of wastewater bioreactor systems is to remove

inorganic nitrogen; in particular ammonium (Malone and Beecher 2000, Metcalf and

Eddy 1991). Hence, comparing the removal ofammonium"and inorganic nitrogen in the

biofilm reactors as a function of substrate type was the primary focus of this study.

Nitrification is a process carried out by groups of nitrifying bacteria, namely

Nitrosomonas and Nitrobacter, that oxidize ammonia (or equivalently ammonium) to

nitrite, and nitrite to nitrate (Schlesinger, 1997). Nitrification is commonly achieved in

biofilm reactors (Lekang et ai, 2000, Tseng et ai, 2004, Zhu et ai, 1999). Interestingly,

Giesake et al. 200 I found that different species of Nitrosomonas tended to colonize the

biofilm at different depths in the reactor. This observation suggests some fonn of niche

differentiation between Nitrosomonas species; alt!'lOugh the oxidation of ammonia to

nitrate is a common biogeochemical trait. The two steps in the nitrification process are

typically carried out as follows:

Ammonium oxidation to nitrite by Nitrosomonas:

2NH/ + 302 -> 2 N02' + 4H+ + 2H20

Conversion of nitrite to nitrate by Nitrobacter:

2N02' + O2 ----'> 2 N03'

7

(I)

(2)

The energy released from the reactions shown in is coupled with the fixation of

carbon by these chemoautotrophic bacteria (Schlesinger, 1997). Consequently, the two

reactions essentially supply the energy needed by bacteria for growth. At this point, the

nitrogen has just changed in form, and has not been removed. Aerobic oxidation of

organic matter is associated with a slightly more negative standard free energy change

than denitrification, and empirically one finds that the relative importance of aerobic

respiration and denitrification is sensitive to dissolved oxygen concentrations. The

denitrification process reduces the nitrate (from the nitrification process) into nitrogen

gas. A large group of denitrifying bacteria allows this process to occur. Denitrification

typically occurs under anoxic or anaerobic conditions by a variety of bacteria that use

nitrate as an electron acceptor in the oxidation of organic matter. The denitrification

reaction can be expressed as follows:

(3)

However, both Pastorelli et al. (1999) and Wik (1999) demonstrated that within

biofihn reactors nitrification and denitrification can occur simultaneously during aerobic

phases since biofilms innately can harbor both aerobic and anoxic conditions within the

fihn. Furthermore, Egli (2003) showed that Annamox can nitrify and denitrify in one

step. (Table 1, Eq. 6).

8

Table 1. (Egli 2003)

RI::tctiol1 I!JH.'rg~' BIOmass "icld (kJ/mnl{' . )hi gdllo

hl·1 1\'1l3 + 150: --> ~O~· + 1120 + Ir -::~7:'i 1.3-2.6

1'".2 NI b + (>;, + ~ ~r + ~ c- --> "II~()JI + 11:0 -I -lfI.6

1-:".:; NIl1UIl + IbU --> 1\'O~' + 4c' ~ 5H' 22.5

E".4 NO.::- + 0.5 02 ~ NO,.- -7~ 11')- I.g

Eq' .' 4 "03+ 51CH~01 +'I~t..,. ~N, + 5('0: + ilhO -IIS0'·' 27U'

1:".6 NH, +~O:!~~N.!+2H.!O -J57 1.5

~ -a) Gtbbs In:c (.'ncrgy change (pll f. 2) ~L): b) PCI' mol of oxidized ammonium or nitrite: (Chapter 1. Egli ;.'1 aL 200 I: EglL 20f)O: Jettell <,I aI., ~()O2). c) \Vith :.lel"tatc as c:lC\:trPll donor d) For growth of "arliCOCC1H d(~nifr!fical1.\" with glllttlm:1tl.:' as cmholl sllhslralc.

Factors that influence nitrification and denitrification rates include dissolved

oxygen concentrations and pH. According to the EPA, the DO concentration required for

the growth of nitrifying bacteria must be less than 2.0 mglL (EPA.2000). The best pH

range for performance of nitrifying bacteria is between 7.0 and 9.0. For Nitrosomonas,

the optimum pH varies from 7.9 to 8.2, whereas for Nitrobacter the optimum range is

from 7.2 to 7.6 (Alleman, 1984). Villaverde et al. (1997) found that within the range of

5.0-9.0, a pH increase of one unit produces a 13% increase of the nitrification rate in

submerged biofilters, which is correlated with increasing alkalinity. Relevant to this point

is the fact that nitrification is an acid-producing process and consumes alkalinity

(Schlesinger, 1997). Below a pH of 6.8, nitrifying bacteria can become inhibited and will

not oxidize ammonia (Michael et aI., 1995).

9

Wastewater generally contains large quantities of particulate matter (suspended

solids), which usually becomes trapped within the substrate. Total suspended solids are

typically correlated to biological oxygen demand (BOD) (Malone and Beecher 2000).

Before microbial digestion can take place, particulate materials typically need to be

hydrolyzed (Levine et al. 1985). Studies have shown that bacteria can utilize particulate

matter as a carbon source by releasing extra-cellular enzymes that presumably initiate the

hydrolysis process (Larsen and Harremoes 1994). The breakdown ofthe suspended

solids via hydrolysis provides a carbon source, which has been shown to increase

denitrification rates (Janning et aI1996).

II. Phosphorus

The removal of phosphorus as phosphate was not the focus of this study because

phosphate is not directly toxic to biological organisms. Phosphorus is nevertheless an

essential nutrient and is coupled with transformations of inorganic nitrogen via biomass

production and respiration. Biological phosphorus removal is associated with nitrification

since nitrification is the energy source for chemosynthetic bacteria. Since denitrification

is associated with the oxidation of organic matter, it is logically linked with the

transformation of organic phosphorus to phosphate. Under anaerobic conditions

phosphorus is released, while during aerobic stages phosphorus has been found to

accumulate in the biomass of active phosphorus accumulating organisms (PAOs) as

polyphosphates as long as there is an external carbon source available (Pastorelli et al.

1999). The incorporation of nitrogen and phosphorus into organic matter typically occurs

10

in a ratio that is more-or-Iess constant among organisms within rather broad taxonomic

categories. For marine microalgae, for example, the N:P ratio is roughly 16 by atoms

(Redfield et. a!., 1963). For macroalgae, the corresponding ratio is 30 (Atkinson and

Smith 1983). Marine bacteria have a N:P ratio of about 7 by atoms (Gundersen et al.

2002). Deviations by more than a factor of two from these ratios are uncommon.

III. Fixed film reactors

Over the years, technologies have been developed to address concerns related to

excess loading of anthropogenic nutrients into biological systems. In the next segment I

will provide some background on some of the "low tech" biological technologies that

have been utilized to remove nutrients from human waste streams.

The first common biofilm reactors consisted of trickling filters. These fixed

biomass or biofilm reactors have been used to remediate anthropogenic wastes for over

one hundred years (Karamanev and Nikolov 1991, Mendez et aI, 1992). During the early

1970's, fixed-film media filters for treatment of aquaculture wastewater began to emerge

(Wagener et a!. 2002). Other biofilters that have evolved for waste treatment include

submerged fixed-film filters, rotating biological contactors, fluidized bed reactors, and

recycled beds (Boller et ai, 1994). Due to the robust nature ofbiofilms, bioreactors have

been applied to remediate numerous high-strength wastewaters including municipal

sewage (Pastorelli et aI1999), pulp and paper industry wastes (Rusten et a!. 1994a),

grease (lipid) wastewater (Liu et a!. 1999), and industrial chemical plant waste (Rusten et

al 1999). Biofilm reactors have been compared to freecsuspended cells in a ferrnenter for

11

waste treatment. However, the processes occurring in biofilm reactors have been shown

'to be significantly more stable than in fermentation systems containing free-suspended

cells (Karamanev and Nikolov 1991). In biofilm reactors the medium becomes coated

with thin films of bacteria (biofilms) that extract nutrients from the water passing through

the bed.

IV. Biofilms

An important characteristic ofbiofilm wastewater treatment systems is the

diversity ofbac!eria and other microorganisms that constitute the biofilm community.

Biofilms were first discovered in the 17th century when Anton von Leewnhoek (the

inventor ofthe microscope) saw microbial aggregates from scrapings of plaque from his

teeth (Schachter 2003). Through time microbiologists have assumed that most microbes

lived as free-floating cells and have staged most microbial experiments under this

assumption. More recently, researchers have found that bacteria and other microbial life

live in communities as biofilms (Schachter 2003). Zobell (1943) observed layers of

microbial cells adhering to bottle walls, and the addition of surfaces within the bottle

increased biological activity of the suspended culture. Since these initial findings,

biofilm microbial communities has become of great interest to many facets of science;

from medical studies to engineering to ecology and evolution. Biofilms are classically

defined as a collection of microorganisms, predominantly bacteria, enmeshed within a

three-dimensional gelatinous matrix of extra-cellular polymers secreted by the

microorganisms. Within each three-dimensional biofilm, a complex microniche is

12

<

defined by the behavior of a diversity of microbial components. These mushroom­

shaped microcolonies presuppose a measure of growth controlled by complex cell-to-cell

communication and often display coordinated activities that mimic those of multicellular

life forms (Costerton 1995).

Biofilms develop on virtually all surfaces submerged in water and have important

implications for wastewater engineering. There are several orderly processes that occur

as the biofilm reaches "maturity": conditioning the surface, bacterial adhesion, contact,

adsorption, growth, exo-polysacharride production, attachment, re-entrainment

(sloughing), and grazing (Costerton 1995). Heterotrophic and nitrifying bacteria co-exist

in the filters (Malone and Beecher 2000). The heterotrophs that form the biofilm are

necessary for nitrifying bacteria to attach since it is the heterotrophs that oxidize the

inorganic carbon. Without the heterotrophs' biomass, it is difficult for nitrifying bacteria

to adhere and multiply. However, at a high BOD concentration, the heterotrophs tend to

multiply at a higher rate than the nitrifying bacteria (Metcalf and Eddy 1991).

The choice of a bioreactor medium for robust biofilm development depends on

factors such as the surface area, durability, mechanical strength and cost. Some basic

requirements for an ideal medium are the same for aquaculture, industrial and domestic

'wastewater treatment (Chipperfiled, 1967). One important property is void space.

Adequate void space is needed to supply oxygen and permit free passage of the

wastewater. Additionally, the substrate should promote the uniform spread ofliquid to all

parts ofthe filter.

Typically, industrial-scale systems to treat aquaculture wastewater use plastic

substrates as media for biofilm development. Since 1961, the evolution of plastic media

13

has become widespread, and researchers continue to test various plastic substrates to find

the ideal shape for remediation of wastewater (Escritt, 1984). Sophisticated shape and

design can result in a high specific surface area (surface area per unit volume) and large

void spaces to support the growth of bacterial biomass. Another advantage of plastic is its

low density, which is desirable when constructing the filter. The basic materials for

plastic media are polypropylene, polyethylene, polyvinylchloride, polystyrene, and

polyvinylidene (Kruner et ai, 1983). However, these plastic media are often not the most

enviromnentally friendly or cost effective choices, especially for remote communities.

For Pacific island communities, plastic media are not readily available and would need to

be shipped over large distances. Additionally, once they no longer serve their purpose,

plastic media can create a burden on local landfills. Many Pacific islands are plagued

with inadequate landfill space for trash disposal. Plastics and other "wastes" are often

disposed of in local streams or coastal waters.

Although there has been great interest in the use of plastic media for wastewater

treatment systems, the popUlarity of plastic does not mean that other media are

ineffective. On the contrary, studies show that media that contain minerals compare

favorably with plastic with respect to the development ofbiofilm communities, even

when differences in surface area are taken into account (Bruce 1970). For attached

bacterial communities naturally occurring media may potentially contribute minerals and

surface "roughness" that plastic media may lack. This study was intended to compare

, "naturally" occurring substrates locally available on Pacific Islands for their use in fixed-

film reactors for treating wastewater from small-scale aquaculture systems.

14

SECTION 4

Methods

I. Experimental Methods

Five experimental biofilm reactors were constructed on Coconut Island (Cl) to

treat wastewater from an experimental tank containing approximately 300 Mozambique

tilapia (Oreochromis mossambicus), which are native to the Zambezi river system. Each

reactor contained an experimental substrate to be evaluated with respect to its ability to

facilitate ammonium and inorganic nitrogen removal. The substrates consisted of(1) lava

rock (pumice), (2) bluestone (hydropressurized basalt), (3) coral rubble, (4) coconut fiber,

and (5) geotextile material (Texel 090903.50M PPWH). The last of these is a

commercially available synthetic product. The size of the tanks, volume of media in the

tanks, design of the inflow and outflow, and flow rates were controlled for each of the

bioreactors. The experimental design involved weekly sampling to measure total

suspended solids (TSS), nitrate/nitrite, ammonium, and phosphate concentration in the

fish tank effluent and the effluents from the five treatment systems. Dissolved oxygen,

temperature, and pH were also monitored on sampling days. Temperature and dissolved

oxygen were recorded using a DO meter (YSI model 58). pH was recorded using a hand­

held meter (IQ Scientific model 3000) with precision measurements recorded to O.lpH

unit (pH was standardized by analyzing replicate samples in the laboratory using

colorimetric methods). Both the DO and pH meters were calibrated before use.

15

1. Study Area

Coconut Island is located off the windward (east) side of Oahu at 21° 25'56.77N,

1570 47' 14.50W. The Hawaii Institute of Marine Biology currently uses the land for

marine research and aquaculture. Freshwater on Oahu comes from aquifers and is routed

to Coconut Island through a system of pipes that distribute the water to buildings and

research facilities. Tilapia aquaculture tanks account for by far the greatest freshwater use

on Coconut Island. Roughly 15.9 million liters of freshwater are used per month for the

aquaculture facility. Historically the tanks have been operated in a once-through ~ow

mode, with the overflow being discharged into Kaneohe Bay. Motivation for treating and

recycling the tank overflow comes from two sources: (1) the cost of the water, and (2) the

fact that the water contains elevated nutrient concentrations coming from the fish tanks.

2. Materials

The use of sustainable practices was a priority for this research project. Hence

most of the materials, including tanks and plumbing fixtures, were recycled items that

could be reconfigured and redesigned for durable and suitable reuse. Many of the system

components were discarded items obtained from the Coconut Island "landfill". One

fiberglass tank 1.55m long, 0.89m wide, and 0.61m height was used as a holding tank to

store the fish tank effluent before it was pumped to the bioreactors. Five cylindrical tanks

made from roughly one-meter-long sea pipes were chosen and reconfigured into biofilm

reactors. Each tank had a diameter of 107 em and height of approximately 91 cm. Each

tank was filled with 58 cm of media and the water level adjusted to 62 cm (Figure I).

Some recycled PVC pipes and fixtures were used to make plumbing connections. All "

16

recycled materials were thoroughly washed with freshwater before being incorporated

into the treatment system.

17

Figure 1

Fix.ed Fi lm Reactor Design

M la

PVC Pipe

1 .5"

2J

Efflv<"nt

(To WetlamlfToro)

Influent

18

One, 8,700-Iiter fish tank containing approximately 300 tilapia was connected to

the holding tank. The effluent from the fish tank flowed into the holding tank via a 7.5-

em diameter overflow pipe. The fish tank used in this study was chosen because it was

the only tank that consistently remained filled with freshwater.! The overflow from the

fish tank was piped into a 38-mm opening in the holding tank. A 0.25-horsepower sump

pump with a float-activated switch moved the water from the holding tank (when the

water level reached approximately 0.2m) to the study area, where the flow was

partitioned amongst the five fixed-film reactors. Figure 2 is a flow diagram of the study

system on Coconut Island. Each of the five fixed-film reactors had similar plumbing to

receive the inflow and discharge the effluent. The inflow entered through a 76-mm

opening on the bottom of the tank and was then routed through a 19-mm diameter pipe to

increase pressure. A ball valve was connected to the 19-mm diameter pipe to facilitate

adjustment of the incoming flow rate to each tank. Rotating spray bars purchased from

Aquatic Ecosystems (RB8) were secured onto the 19 mm inlet. The two spray bars that

came with the RB8 kit were replaced with new PVC spray bars designed to better fit the

dimensions of the tanks. The modified spray bars were each 46 cm long. Holes were

drilled into the bars with a 1/3 cm drill bit. Two bars were connected to each of the

fluidized bearings, and caps were placed on the ends. The bars rotated as the influent

rose and flowed through the spray holes, allowing for a fairly even distribution of water

over the media (Figure 3).' The influent was sprayed over the media to increase the

dissolved oxygen concentration in the water without the use of an aerator. Once the·

influent entered the tanks, the flow moved downward through the media and out the

! Other research fish tanks were sporadically switched back and forth from a freshwater to saltwater medium.

19

bottom via a 5-cm diameter "infiltrator" constructed from PVC pipe with 13-mm holes to

minimize clogging (Figure 4). The flow through the 5-cm diameter pipe was reduced and

routed through a 0.19 mm section of pipe just prior to leaving the tan1e Outside the tank

was a standpipe with a snorkel to maintain the water level and a faucet to take samples

from prior to discharge (Figure 5). Water quality was monitored in the effluent from the

fish tank and the effluent from the five bioreactors prior to discharge.

20

Figure 2

Holding t nkJ pu m p statJ'On

To outf. 1I

Figure 3

fish T

21

r GEote.lil .. • Coral Rubbl ~ • SJues one lava Rock Influent Efflu n.t

• •

Figure 4

•

• •

22

Figure 5

23

3. Small Scale Replication

A small-scale replica of the study system on Coconut Island was used to

determine whether the performance of the treatment systems was reproducible when

appropriately scaled according to residence time. The replica was located on the roof of

the Marine Science Building on the University of Hawaii Campus and will be referred to

as MSB. Overflow from the CI fish tank was collected twice weekly and placed in the

holding tank of the MSB system. Approximately 225 liters oftilapia wastewater were

brought to the MSB each week. The holding tank was elevated in order to provide

gravitational flow to five 4.5-liter bioreactors scaled in proportion to the CI

bioremediation system. Faucets permitted dosing the flow equally among the trickling

filters. Because of small differences in the flow due to faucet imprecision, measuring

containers placed under the filters were used to quantity flow rates. A total of 75 liters of

wastewater were collected from Coconut Island and put in the system on Tuesday and

Friday, and 38 liters were added every Monday and Wednesday. A diagram of the replica

system is shown in Figure 6.

24

Figure 6

HOLDING TANK

• •

25

SMALL-SCALE BIOFILM REACTORS

MEASURING TANKS

4. Sampling

The MSB study system was activated on April 2, 2004 and continued through

June 9, 2004. Sampling of the inflow and effiuent from the bioreactors was made on a

weekly basis throughout the study period. Weekly sampling of the CI system commenced

June 16, 2004 and continued until the beginning of December 2004. Water samples were

taken a total often times. All water samples were collected in 800-mL plastic bottles.

Temperature, pH, and dissolved oxygen concentration (with the same equipment

• mentioned above) were recorded immediately. The samples were then placed in a cooler

filled with ice and taken to MSB 519 for further processing (see below).

5. FlowlHydraulic Residence Time

Flow rate can influence the formation ofbiofilms and their structural

characteristics (Casey et al. 1999) including homogeneity (Costerton et al. 1995). Thus,

flow rate along with residence time need to be considered in analyzing the performance

of a biofilm reactor. From July through November 2004 the CI treatment system

remained "open", i.e., the effiuent from the treatment system was discharged at a

designated outfall. Flow (liters per day) was estimated (1) using a flow meter and (2)

using the "bucket stopwatch" method? In the latter case empty carboys were placed at

the outflow of the biofilm reactors and the time required to fill the carboys recorded with

a stopwatch.

2 The bucket stopwatch method involves recording the amount of time required to fill a container of known volume.

26

The hydraulic residence time (HRT) within the bioreactor contributes to the

treatment performance. HRT is defined as the ratio of volume of water in the system

(m3) divided by the flow rate (m3 d· l

) (Campbell and Ogden 1999). The volume of water

in the trickling filters is equal to the volume of the trickling filters multiplied by the void

ratio. 3

6. Media

With the exception of the geotextile fabric, the media that were chosen for the

bioreactors were materials that are commonly found on Pacific Islands. The media

consisted of lava rocks, bluestone (commonly referred as gravel, a hydropressurized

basalt), coral rubble, coconut fiber husk, and a geotextile fabric.

Three size categories were chosen for the rock media. Type I rocks were those

greater than 7.5 cm in diameter. Type II rocks were 5.0-7.5 cm in diameter. Type III

were 1.3-2.5 cm diameter rocks. The three size categories were chosen so that the rock

bioreactors could be filled with graded bedding. The smallest rocks were placed near the

top and the largest rocks near the bottom of the reactor. This design tends to prevent

clogging and facilitates removal ofbiofilms that slough off, thus minimizing the need for

back-flushing. Type II lava rocks (pumice) were selected by hand from the area adjacent

to the CI parking lot. Type III lava rocks were collected from inshore areas near the

project site on Coconut Island. Type I lava rocks were purchased at a local gardening

store. Type II bluestone was purchased from Ameron, Kailua, Oahu, and Type I and

3 The void ratio is by definition the volume of water required to fill a container filled with a porous medium divided by the volume of the container.

27

Type III were collected on Coconut Island. Coral rubble Type I and II were purchased

from the Sand Island Quarry, Honolulu, Oahu, and Type III were collected from inland

sites on Coconut Island. The geotextile material was donated by Ocean Arks

International and came in only one size. All of the coconuts were collected on Coconut

Island, and theii- coir was husked on the island. Like the geotextile material, the coconut

fiber had only one size class.

7. X-Ray Diffraction

X-Ray diffraction is a method utilized by geologists to characterize the major

mineral contents within rocks by analyzing mineral structures at various angles. Dr. Jane

Schoonmaker of the Department of Oceanography, University of Hawaii at Manoa

conducted this analysis.

8. Void Ratio and Specific Surface Area

Since biofilms are attached microbial communities, their activity is very

dependent on physical characteristics of the medium such as porosity and specific surface

area. Increasing the surface area of the medium, for example, has been shown to increase

the level of treatment (Bellelo et aI. In Press). In the work described here, porosity was

measured using the void ratio for all the substrates used in the fixed film reactors. The

void ratio is defined as the volume that remains filled with water after the medium has

been placed in the filter housing divided by the total filter volume (Timmons et al. 1997),

28

1.e.,

where cp is the porosity, Vp is the volume ofliquid that can be accommodated with the

medium in place, and Vrnis the total volume of the filter housing. It is common to express

porosity as a percentage by mUltiplying the above ratio by 100. cp was calculated by

filling a one-liter beaker to the mark (V m) with dry medium. Water was then poured into

the beaker until the water just reached the mark. The volume of water required to fill the

beaker to the mark was recorded (Vp). This exercise was repeated for each of the five

media and, in the case ofthe rock media, for each of the size classes.

The surface area available for microbial attachment and the surface "stickiness"

are also characteristics that have been shown to influence the efficiency of trickling filter

systems (Cooksey, 1995). In the work reported here the specific surface areas of the

inorganic media were determined by sampling 500 mL of Type I objects and 150 mL of

Type II and III objects. Although mercury absorption is a more refined was to estimate

surface area, a "paper" method was chosen to provide ballpark estimates of the media's

surface areas. Each object was covered with a fine layer of paper, with care taken to

ensure that adjacent pieces of paper did not overlap. The area of paper required to cover

the object surface was then measured, and the surface area was calculated. This process

was repeated for each substrate.

For lava rocks and coral, cavities were neglected. The cavities are not uniform,

and because of their small size, their geometry was difficult to determine. The specific

29

surface areas for the lava rocks and coral should therefore be considered lower bounds to

the true specific surface areas. Specific surface area measurements for the coconut fibers

were not made. Since coconut fibers are organic substrates, this measurement is quite

difficult because there are surfaces that exist on the macro and micro levels. In addition

to the complexity of making this measurement, this medium is degradable with time, and

its surface area therefore has a temporal dependence. Table 2 shows the results of the

porosity and specific surface area measurements for the media according to the different

size classes (Types I-III) and their total arrangement in the fixed film reactors (Total).

30

Table 2. Properties ofbilfilter media

Specific Surface area

MEDIA Type Porosity (%) (m'll)

I 53.0 0.30

II 48.0 0.37 Lava Rock

III 52.0 0.43

Total 52.0 >0.33

I 68.0 0.17

II 48.0 0.47 Coral Rubble

III 43.0 0.88

Total 56.0 >0.45

I 55.0 0.15

Bluestone n 38.5 0.76

Total 48.0 0.38

Coconut Fibers Total 75.0 -

Geotextile Total 85.0 0.20 /

31

\

II. Laboratory Methods

The following laboratory procedures are similar to the methods used in Laws and

Roth (2004).

1. TSS

Once the samples were taken, they were immediately stored on ice in a cooler.

Once the samples were brought to the laboratory, they were either filtered that day or

stored in the laboratory refrigerator for up to one week. Although refrigeration does

impede biochemical processes, it does not necessarily bring them to a halt. Therefore,

whenever possible, the samples were immediately analyzed. The TSS analysis method

followed American Public Health Association et al. (1995). The water samples were

filtered through preweighed glass fiber filters (Whatman GFF) with a nominal porosity of

0.7).lm. Approximately IL of the water sample was filtered. The filters were dried in a

drying oven at 105°C to constant weight. The filters were weighed on an analytical

balance (Metler model H20T) to the nearest 0.01 mg. Blanks were run by filtering 250

ml of distilled water through a filter. The concentration of the total suspended solids was

calculated as the difference in the weights of the filter before and after filtering.

32

2. Nutrient Analysis

Filtrate from the TSS analysis was transferred to plastic bottles to be processed

for nutrient analysis. Concentrations of nitrite/nitrate, ammonium and phosphate were

measured using colorimetric techniques on a Technicon Instruments AutoAnalyzer. The

methods used for the nutrient analysis were Technican (1977) for nitrite/nitrate,

ammonium EPA 350.1, and standard colorimetric methods (American Public Health

Association et a!., 1995) for phosphate. Limits of detection were 0.10 11M for

nitrite/nitrate, 0.13 11M for ammonium, and 0.05 11M for phosphate. Dissolved inorganic

nitrogen (DIN) was equated to the sum of nitrate/nitrite and ammonium.

III. Statistical Analysis

Nutrient removal rates (k) were calculated from the equation

C = c'e-kt , t (4)

Where Ce is the nutrient concentration in the effluent from the bioreactor, C j is the

nutrient concentration in the inflow, k is the rate of nutrient removal, and t is the

residence time of the water in the bioreactor. Solving equation 4 for k gives

k = In(C j /C,) t

33

(5)

Nutrient removal rates calculated for the CI and MSB systems were compared

statistically using either Kruskal-Wallis (KW) or one-way analysis of variance

(ANOV A). Initial comparisons were made between identical treatments from the CI and

MSB systems to determine if there was a significant difference between the performance

of the CI and MSB systems. The null hypothesis was that the CI and MSB systems

performed in a similar manner when removal rates were scaled to residence time

(equation 5). The null hypothesis was rejected if the difference in nutrient removal rates

was significant by a two-tailed test at p = 0.05. If there was no reason to reject the null

hypothesis, the nutrient removal rates from the MSB and CI systems were pooled and a

KW or one-way ANOV A run to determine whether there were significant differences in

nutrient removal rates between the five treatments.

ANOVA calculations assume that (1) all sample populations are normally

distributed, (2) all sample populations have equal variance, and (3) all observations are

mutually independent. The Kruskal-Wallis test is a nonparametric or distribution-free

analogue of ANOV A that is commonly used when the first and/or second assumptions of

ANOVA are not met. The ANOVA test is known to be robust to modest violations of the

first two assumptions, and in cases where these assumptions hold entirely or even

approximately, the ANOV A is generally the more efficient statistical procedure for

detecting departures from the null hypothesis (Sokal and Rohlf, 1981, p. 429). A simple

test for normality that.is particularly useful when sample sizes are sma1l4 is the Lilliefors

modification of the Kolmogorov-Smirnov test for goodness of fit (Sokal and Rohlf, 1995,

p. 711). The test is based on differences in two cumulative relative frequency

4 For the MSB data sets, n = 9.

34

distributions and is available in the MA TLAB statistical toolbox. I used the Lilliefors test

to decide whether to analyze the nutrient removal rate data using KW or ANOV A.

If the assumption that the CI and MSB bioreactors were similar could not be

rejected at p = 0.05, the data from the two systems were pooled, giving a combined'data

set with 27 replicates for each ofthe five treatments. The rationale for pooling the data

was to increase the sample size and hence the probability of detecting differences

between treatments. Given the larger number of sample size in the combined data sets, it

seemed reasonable to examine the data for outliers, the rationale again being to increase

the power of the KW or ANOV A. The MATLAB BOXPLOT routine was used to detect

outliers. A datum is considered to be an outlier by BOXPLOT if it lies outside 1.5 times

the interquartile range. For a normally distributed variate, the interquartile range

corresponds to 1.35 normal deviates, and the BOXPLOT criterion therefore corresponds

to a deviation by more than 2.0 normal deviates. The two-tailed probability that an

observation wil1lie outside this range is 4%. Data identified by BOXPLOT as outliers

were excluded from subsequent analysis.

35

SECTIONS

Results

I. Media Results

Coral Rubble. As expected, the X-ray diffraction results for the coral powder detected

calcite and aragonite, two fonns of calcium carbonate (CaC03). CaC03 is susceptible to

dissolution by microbial processes that produce CO2 (e.g., respiration) andlor H+ (e.g.,

equation I). The relevant equation is

This is obviously a concern in a trickling filter that utilizes coral rubble as a substrate for

bacterial activity (respiration and nitrification).

Lava Rock (Pumice). The X-ray diffrac!ion results for the pumice powder indicated that

the lava rock (basalt) is composed of plagioclase feldspar, olivine, and pyroxene.

Plagioclase minerals were also found in the bluestone. These minerals are also subject to

attack by C02 and acid. Products of the weathering of olivine and pyroxene include

calcium, iron, silicon and magnesium (Schlesinger, 1997).

36

Bluestone. The X-ray diffraction results for the bluestone powder (another form of

basalt) contained the same minerals as the lava rock pumice. Additional minerals found

in the bluestone included quartz and chlorite, which are not commonly found in basalt.

The presence of the quartz and chlorite suggest that the bluestone underwent

hydrothermal alteration.

Non-rock materials

Geotextile. The geotextile fabric used is a woven material made from polypropylene.

Coconut fiber. Coconut fiber is an organic substrate and hence contains carbon. This is

potentially advantageous, since organic carbon is typically added (e.g., acetate) in tertiary

wastewater treatment systems to stimulate nutrient removal (Casey et aI1999). A

concern with the use of a natural, organic medium for filtration is fiber degradation over

time. All natural fibers will eventually degrade, but the rate of degradation will vary

according to the chemical composition. Luzton (1996) showed that natural fibers could

be used successfully as media in slow sand filtration and that coconut fiber was the most

favorable medium with respect to degradation, with a weight loss of only 2% after four

weeks. Coconut fiber, commonly referred to as coir, is one of the hardest natural fibers

because of its high lignin content, 46% (Sudhakaran, 2003). Interestingly, lignin has

become the subject of recent research for its anti-algal activity. The Center for aquatic

Plant Management in England has discovered and developed the use of natural fibers as a

method of algal control. They have concluded that when natural fibers are placed in

37

water, the soluble components (carbohydrates and hemicelluloses) are washed out,

leaving lignin to slowly decompose. The decomposition process leads to the production

of fulvic and humic acids via bacterial and fungal enzymes activity. These substances, in

the presence of dissolved oxygen, form hydrogen peroxide, which inhibits the growth of

algae (IACR-Center for Aquatic Plant Management, 1999).

II. Flow and Hydraulic Residence Time

1. Flow

On Coconut Island the flow through the system was restricted by the output range

ofthe 0.25 horsepower sump pump and the approximately a three-meter head between

the fish tank and the holding tan1e The maximum and minimum flow rates were

approximately 21 m3 and 4 m3 per day, respectively. Geometric mean flow rates through

the treatment system when the system was "open" and "closed" were 14.6 m3 and 11.8

m l per day, respectively.

The flow on the MSB' system was restricted by the total amount of wastewater

delivered to the system weekly. Although the CI and MSB systems were intended to

have similar hydraulic residence times, the average residence time in the MSB systems

was about 3.5 times longer.

38

2. Hydraulic Residence Time

Table (3) shows the void ratio, volume ofliquid in each tank, and HRT for the study

system at Coconut Island and MSB based on average flow.

Table 3a: Hydraulic Residence Time (HRT) of the Coconut Island system

Volume of media Volume ofliquid Hydraulic Residence

Void Ratio (liters) (liters) Time (days)

Lava 0.52 603 314 0.11

Coral 0.56 603 339 0.12

Bluestone 0.48 603 290 0.10

Coconut 0.75 603 453 0.16

Geotextile 0.85 603 514 0.18

Table 3b: Hydraulic Residence Time (HRT) of the MSB system

Volume of media Volume ofliquid Hydraulic Residence

Void Ratio (liters) (liters) Time (days)

Lava 0.52 4.53 2.35 0.38

Coral 0.56 4.53 2.54 0.41

Bluestone 0.48 4.53 2.16 0.35

Coconut 0.75 4.53 3.37 0.55

Geotextile 0.85 4.53 3.82 0.62

39

III. Water Chemistry Results

1. Dissolved Oxygen and pH

Dissolved oxygen remained greater than 2mgIL in the effluent from all five

biofilm reactors, and effluent pH in aU cases was in the range 7.0-9.0. Due to some

technical problems with the DO and pH meters only 67% and 50% of the sampling days

were recorded for DO and pH respectively at Coconut Island. DO and pH measurements

were conducted at MSB on the sampling days and were measured randomly between

sampling dates. Tables 4a and 4b show the mean and standard deviation for each filter

for CI and MSB respectively.

40

Table 4a Coconut Island 0, and pH yalues

0, (mgIL) n=12 pHn=9

Coral mean 8.0 8.7 stdev 0.9 0.3

Bluestone mean 7.5 8.3 stdey 0.3 1.2

Lava mean 7.7 8.5 stdey 0.8 0.4

Geotextil. mean 7.7 8.4 stdev 1.0 0.4

Coconut mean 5.9 7.8 stdey 0.4 0.1

Fisb Tank mean 7.3 8.3 stdev 0.6 0.3

41

Table 4b

MSB 0, and pH values 0, (mg/L) (n~19) pH (n~19)

Coral mean 4.9 8.6 stdev 1.3 0.2

Bluestone mean 5.1 8.5

stdev 1.2 0.3

Lava mean 4.9 8.4 stdev 1.4 0.3

Geotexlile mean 5.0 8.1

stdev 1.3 0.4

Coconut mean 4.6 7.8 stdev 1.3 0.3

Fish Tank mean 7.0 8.3 stdev 0.8 0.4

42

2. Nutrient and TSS Results

Table 5 summarizes characteristics of nutrient and TSS concentrations in the

inflow and effluent of the CI and MSB biofilter systems. Average concentrations and

percent removal have been calculated using both geometric means and median values. In

. • the case of ammonia, effluent concentrations were in many cases below the limit of

detection «0.13 11M). As long as the median value in the data set is above the limit of

detection, the median value is an unbiased measure ofthe true median. However, in four

often cases, the median ammonia concentration in the effluent was below the limit of

detection. The geometric mean of the data set is a biased estimator of the true geometric

mean as long as any of the measured values are below the limit of detection.5 The same

logic applies to an arithmetic mean. For nitrate/nitrite and phosphate analytical

sensitivity was not a problem, and because nitrate/nitrite concentrations were much

higher than ammonia concentrations, analytical sensitivity had virtually no effect on DIN

characteristics.

5 The limit of detection was substituted for values below the limit of detection.

43

Table 5a: Fixed Film Bioreactor Performances at Coconut Island. In all cases, n = 18.

TSS (mg NH, NO,INO, DIN PO, drywt/L) (J.lM) (j.tM) (j.tM) (J.lM) DIN:P

Lava Geometric mean 0.02 0.29 13.92 14.34 1.82 7.9 Median 0.05 0.26 14.68 14.99 1.96 7.4

% Reduction Geometric mean 99 92 -84 6 -18 Median 97 91 -47 -2 -21

Bluestone Geometric mean 0 0.37 9.42 10.97 1.56 7.0 Median 0.06 0.38 16.56 16.87 1.68 8.7

% Reduction Geometric mean 100 89 -25 28 -2 Median 96 90 -38 -9 -10

Coral Geometric mean 0.23 0.31 11.74 12.55 1.44 8.7 Median 0.35 0.30 13.33 13.46 1.55 8.1

% Reduction Geometric mean 89 91 -56 18 6 Median 75 90 -21 13 4

Geotextile Geometric mean 0.08 0.71 7.99 9.72 1.54 6.3 Median 0.23 1.07 13.93 15.01 1.95 7.3

% Reduction Geometric mean 96 79 -6 36 0 Median 84 72 -29 -6 -15

Cocoout Fiber Geometric mean 0.17 0.37 5.54 7.28 1.50 4.9

Median 0.36 0.13 8.94 10.39 1.48 6.4 % Reduction Geometric mean 91 89 26 52 2

Median 74 91 4 38 7

(Fish tank) Geometric mean 2.02 3.45 7.55 15.29 1.54 10.0 Median 1.39 3.41 9.73 14.05 1.52 9.2

44

Table 5b: Fixed Film Rioreactor PerfoTIllances at MSB. In all cases, n ~ 9.

TSS (mg dry NH, N02IN03

DIN PO, wtlL) (f1M) (flM) (11M) (f1M) N:P

Lava Geometric mean 0.41 0.24 11.56 12.01 1.54 7.8 Median 0.37 0.13 10.15 10.48 1.43 7.7

% Reduction Geometric mean 93 85 4 19 -45 Median 97 90 10'" 27 -30

Blnestone Geometric mean 0.31 0.24 8.42 8.86 1.16 7.7 Median 0.42 0.13 10.53 11.03 0.98 10.1

% Reduction Geometric Mean 95 85 30 40 -9 Median 97 82 10 26 -5

Coral Geometric mean 0.5 0.64 8.40 9.45 0.61 15.5 Median 0.52 0.86 7.33 8.18 0.62 13.2

% Reduction Geometric Mean 91 60 30 36 42 Median 96 45 36 35 42

Geotextile Geometric mean 0.19 0.19 8.31 8.56 0.82 10.4 Median 0.15 0.13 7.54 7.67 0.76 12.0

% Reduction Geometric Mean 97 88 31 42 23 Median 95 92 32 41 32

Coconut Fiber Geometric mean 0.6 1.29 1.46 5.21 0.47 11.0

Median 0.57 1.55 2.10 4.27 0.44 7.5 % Reduction Geometric mean 90 20 88 65 55

Median 95 23 79 70 49

(Fish tank) Geometric mean 5.83 1.61 12.02 14.81 1.06 13.9 Median 12.21 2.81 11.53 14.31 1.06 13.8

45

3. Statistical Results

It was impossible to carry out a meaningful comparison of anunonia removal rates

calculated from equation 5 for the CI and MSB systems since effluent concentrations

were so frequently below the limit of analytical detection. The CI systems, with

residence times of roughly three hours, removed 70-90% of the anunonia and reduced

effluent concentrations one micromolar or less. The MSB systems, with the exception of

the coconut fiber treatment, produced comparable effluent concentrations. The fact that

the residence times in the MSB systems were substantially longer (eleven hours) and the

influent anunonia concentrations lower suggests that there is a threshold concentration

below which ammonia removal either ceases or proceeds at a very slow rate. The MSB

coconut fiber treatment was an unusual both in tenns of its anunonia removal, which was

anomalously low, and its nitrate removal, which was higher than that of any other

treatment. A likely explanation is that the environment in the MSB coconut fiber

treatment was more reducing and hence facilitated nitrate reduction at the expense of

nitrification.

To compare removal rates of DIN between the CI and MSB systems, the

Lilliefors test was first used to detennine if the data for each bioreactor were normally

distributed. In some cases we could not accept the null hypothesis, and we therefore used

a Kruskal-Wallis (KW) test to compare the CI and MSB DIN removal rates. Results of

the KW comparison are summarized in Table 6 and Figs. 7-12. In aJl cases the p-values

were greater than 0.05 and the 'null hypothesis that the CI and MSB systems removed

DIN at comparable rates, i.e., k values calculated from equation 5 was not rejected.

46

Table 6

Comparison ofCI and MSB biofilter systems based on Kruskal-Wallis test.

Media 'X.' P-value

Coconut Fiber 0.04 0.84

Lava 0.76 0.38

Bluestone 2.71 0.10

Geotextile 1.52 0.22

Coral Rubble 0.07 0.80

47

~ ::l -co >

Figure 7: Kruskal-Wallis analysis of coconut fiber bioreactors ar MSB and CI

2

o

-2

-4

-6

-8

-10

-12

-14

-16

-18 ~--------~-----------------------L--------~ 1 2

MSB (Group 1) CI (Group 2)

48

<J)

~ -'" >

8

6

4

2

o

-2

-4

-6

-8

-10

Figure 8: Kruskal-Wallis analysis of lava rock bioreactors ar MSB and CI

-

-

-

-

I ..l -

,

1 2 MSB (Group 1) CI (Group 2)

49

---------------- - ---

Figure 9: Kruskal-Wallis analys is of bluestone bioreactors ar MSB and CI

<J)

CD :::> -'" >

4

2

o

-2

-4

-6

-8

-10

-12

,

""5 ~

~

+

1 2 MSB (Group 1) CI (Group 2)

50

-

•

., OJ :J iU >

Figure 10: Kruskal-Wallis analysis of geotextile bioreactors ar MSS and CI 6

5

4

3

2

1

0

-1

-2

-3

-4

1 MSS (Group1)

51

T I

I

I

-.L 2

CI (Group 2)

Figure 11 : Kruskal-Wallis analysis of coral rubble bioreactors from MSB and CI

6

4

2 -

o -

'" -2 -(])

'" -'" > -4 -

-6 I -8 I

-10 -

-12 -, 1 2

MSB (Group 1) CI (Group 2)

52

6

4

2

o

'" -2 OJ ::l

'" > -4

-6

-8

-10

-12 ' r

Figure 12: One-way ANOVA analysis (combined data from CI and MSB) of the media in the bioreactors

,

+ + -I

I I

I

I l

~ ~

~ ~

--

~

-1 2 3 4 5

-

-

-

-

-

-

-

Coconut Fiber Lale Rock Bluestone Geotextile Coral Rubble •

53

Given the similarity of the CI and MSB results, the data were pooled, resulting in

five data sets each containing 27 k values. Given the large value ofn (i.e., 5x27 = 135),

we elected to discard outliers on the assumption that there were probably some spurious

data whose presence in the data set would confound identification of treatment effects.

BOXPLOT identified a total of 15 outliers, i.e., 11% ofthe data. Since the criterion used

by BOXPLOT should exclude about 4% of the data if the underlying distribution is

nonnal (see above), this result suggests that roughly 7% of the data may have been

spurious. With the outliers removed, the Lilliefors test was repeated on the combined CI

and MSB data. This time the null hypothesis was rejected that the data were not

nonnally distributed for all five treatments. Therefore the treatment effects were tested

using a one-way ANOV A. Treatment effects were judged to be significant at p = 0.0003.

To determine the cause of the treatment effect, we carried out a multiple comparisons test

using the MATLAB program MULTCOMPARE with a = 0.05. Based on this test, the

coconut fiber treatment, which had the highest DIN removal rate, was judged to be

significantly different from the other four treatments (Fig. 13). There were no significant

differences between the other four treatments with respect to DIN removal.

54

Figure 13: Multiple comparison of means between 5 media types

1 o

2- o

3 o

4 o

5 o

-5 -4 -3 -2 -1 o 1 2 The 4 bioreactors haw means significantly different from Group 1 (Coconut Fiber)

55

Phosphate removal rates were compared in the same manner as the DIN. The

Lilliefors test indicated that not all the data sets were normally distributed, and a KW test

was therefore used to test the null hypothesis that the CI and MSB systems were similar

with respect to rates of phosphate removal. This null hypothesis could be accepted for all

treatments except coconut fibers. The phosphate removal rate for coconut fibers was

about 3.5 times faster in the MSB system. For the other treatments the CI and MSB data

sets were combined and outliers discarded. There were a total of 8 outliers among the n =

4x27 = 108 data. The Lilliefors test indicated that we could accept the null hypothesis

that the data were normally distributed for all four treatments at p < 0.05. We therefore

tested for treatment effects using a one-way ANOV A. Treatment effects were judged to

be significant at p = 5xIO·6. Coral rubble had the highest phosphate removal rate and was

judged to be significantly different from both lava rock and bluestone by

MULTCOMPARE at p < 0.05. Lava rock was significantly different from the other three

treatments (p < 0.05) and was associated with an increase in phosphate concentration.

Although not a focus of this research, the geometric mean reduction ofTSS for all

the treatments was 89% or greater.

4. Operations

I would like to make a few comments relative to maintenance of the biofilter

systems and their potential for use elsewhere. Knowledge of the average nutrient loads

and their temporal variability are crucial to the informed design of a bioreactor

wastewater treatment system. Water quality data collected before the research

56

commenced indicated a low nitrogen burden. However, water quality analyses carried out

during this project revealed there was significant temporal variability in ammonium

concentrations coming from the fish tanle This could be attributed to times offeeding

and types of feed.

Generally, the bioreactor systems were fairly easy to operate. The pump needed

to be cleaned occasionally with a hose (once per week). The rotating trickling spray bars

needed to be cleaned often (more than once per week) since algae can build up within the

pipe and restrict flow. No back flushing was found to be necessary in the fixed film

reactors during the study periods. During times of light or no rainfall, especially during

the summer months on Coconut Island, evaporative losses were considered and some

replacement water was added.

57

'.

SECTION 6

Discussion

EPA water quality criterion, with respect to ammoniaiannnoniurn, are based

entirely on the toxicity of annnonia, since the ammonium ion is essentially benign.

However, this criterion ranges since the concentration of annnoniaiammoniurn is entirely

based upon temperature and pH. One obvious concern with a biological filter is the

possibility that removal efficiency becomes substrate-limited at substrate concentrations

that are potentially dangerous to cultured species. The results indicate that this did not

happen with respect to annnoniaiannnonium removal in the systems studied here. Within

a few hours total annnoniurn concentrations were reduced from 3.5 /-1M to less than 1 /-1M

in the CI biofilters. Although the coconut fiber treatment achieved the best overall rate of

DIN removal, the CI and MSB systems behaved rather differently, in contrast to the

consistent behavior displayed by the other treatments. The CI coconut fiber system

removed about 16% of the DIN per hour primarily by removing total annnonia and a

much smaller fraction of the nitrate .. The MSB coconut fiber system removed about 8.5%

of the DIN per hour primarily by removing nitrate and a smaller fraction of the total

ammonia. Redox conditions within the bioreactors probably account for these

differences, which are implied by the differences in mean DO concentrations between the

two sites. The implication is that a properly engineered coconut fiber bioreactor can be

very effective in reducing total annnonia and DIN concentrations, but empirical studies

will probably be needed to scale the reactor design to the flow rate and concentration of

inorganic nitrogen species in the wastewater.

58

Perhaps one conclusion from this work is that a single biofilter design may not be

the most efficient way to nitrify and denitrify. This is not a particularly surprising

conclusion, since the redox conditions that facilitate these two processes are quite

different. While the environment within the biofilter is undoubtedly heterogeneous in

some respects, insofar as inorganic nitrogen transformations are concerned, the filters

seemed to behave as ifthe environment within them was either reducing (denitrification)

or oxidizing (nitrification). All but the MSB coconut fiber system appeared to be

oxidizing environments. Given the fact that the CI and MSB coconut fiber biofilters

were intended to function in a similar manner, it is surprising that they behaved so

differently, despite the fact that their overall removal rate of DIN was similar. One

explanation may come from the DO analysis; as the retention time increases, more

oxygen is consumed. Although the effluents from the MSB reactors remained

significantly above 2.0mg/L, the DO was noticeably less than the CI reactor effluents,

indicating that the environment within the filters becomes more reducing as retention

time increases.

The phosphate results varied between the filters. The coral rubble and coconut

fiber reactors appear to have removed phosphate most efficiently, while the basalt rock

media (lava and bluestone) reactors tended to have higher concentrations of phosphate in

their effluents compared to the influent. One explanation for this occurrence may again

be attributed to the rock media itself. Basalts can have the mineral apatite which is

typically mined for phosphorus. Furthermore, Felitsyn (2002) found that alkaline basic

volcanic rocks can serve as the continental source for the formation of phosphates.

59

There is no mechanism analogous to denitrification that can remove phosphate

from a biofilter. Possible mechanisms ofremoval from the dissolved phase include

precipitation, adsorption, and biological uptake. The fact that the coral rubble treatment

achieved the highest rate of phosphate removal probably reflects the fact (noted above)

that the rubble contains calcium carbonate. Calcium can effectively bind phosphate

under basic conditions. The fact that phosphate concentrations actually increased in the

effiuent from the lava rock biofilter is provocative, since the minerals in the rock contain

calcium, iron, and aluminum, all of which are capable of binding phosphate under

appropriate pH conditions. Uptake or release of phosphate is associated with the

synthesis or decomposition of organic matter, respectively. To the extent that these

processes were occurring, nitrogen would also be taken up or released. Assuming that

much of the biological activity in the biofilters is due to bacteria, the N:P ratio associated

with that activity would probably be about 7 by atoms (see above). Since phosphate

concentrations in the lava rock biofilters increased by about 0.3-0.4 f.LM", the implied

increase in DIN would be about 2-3 I!M if bacterial respiration were responsible for the

increase in phosphate. Since DIN concentrations in the lava rock biofilters either

decreased or remained more-or-less constant, the implication is that any increases in DIN

due to respiration were more than offset by losses caused by denitrification. Without

knowing the rates of denitrification in the biofilters, it is impossible to pursue this line of

. reasoning much further. In almost all cases the ratio of DIN to phosphate decreased

between the inflow and outflow of the bioreactors, a result very likely reflecting the role

of denitrification/nitrification and the lack of an analogous mechanism for removing

phosphate. The only exception was the MSB coral rubble system, where the long

60

residence time (9.8 hours) may have facilitated phosphate removal via chemical reactions

with calcium.

Although TSS were significantly reduced in all the bioreactors (89-100%), there

was no need for removal of solids via backflushing during the course ofthis study. It is

likely that the reduction in TSS can be attributed to particles being trapped within the

media. Once trapped, the particles may be consumed by metazoans or broken down

through the action of extracellular enzymes released by bacteria. The potential impact of

such remineralization on inorganic nutrient concentrations in the effiuent depends on the

composition of the TSS. Assuming that the TSS consists entirely of organic matter and

that the organic matter contains roughly 45% carbon by weight (Laws and Berning 1991),

the concentration of particulate organic carbon in the fish tank effiuent would be, in

round numbers, 100-200 fLM. The C:N ratio in the TSS is unknown but is likely in the

range 5-10 by atoms. Hence remineralization ofthe TSS could release anywhere

between 10 and 40 fLM DIN. In other words, the nitrogen associated with the TSS is

present in the fish tank effiuent at a concentration comparable to the DIN in the same

effluent. The same logic applies to phosphorus. Given the fact that the biofilters removed

roughly 90% of the TSS and that no backflushing of the filters to remove solids was

necessary (nor has been necessary up to the present time of October 2005), the

implication is that the biofilters may have been removing substantially more nitrogen

than is implied by the DIN budget in Table 3.

Although this study has answered some important questions, as in often the case

in scientific research, it has generated even more questions. Issues that were not

addressed in this work include the composition, heterogeneity, and spatial distribution of

61

the microbial communities within the biofilters, the stoichiometry of the microbial

communities with respect to the uptake and regeneration of nutrients, and the impact of

abiotic processes such as precipitation, adsorption, and weathering of media on nutrient

budgets. The bioreactors are microcosms of rather intense microbial activity.

Scrutinizing the biogeochemical interactions that take place in the biofilters may further

our understanding of the dynamic processes occurring both within these systems and

within nature.

62

SECTION 7

Future of Remediation System

Since the remediation system was put into operation, the facility has hosted a

variety of educational groups from the local community, NGOs, Hawaii Pacific

University, Windward Community College, and the University of Hawaii at Manoa.

Persons visiting the site have been interested in integrated food production and waste

treatment systems that conserve water and utilize excess nutrients. It is hoped that this

project will receive additional funding for the continuation of its educational mission and

will provide training for individuals from the community and/or from local educational

institutions. I am hopeful that the continued operation of the biofilter system will provide

an opportunity for additional research aimed at advancing the design of sustainable,

engineered ecosystems that convert wastes and conserve water on Coconut Island.

63

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73