CO2-supplemented brewery wastewater treatment bymicroalgae and biomass upgrading for bioenergy

production

Alice Maria Garcia Ferreira

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisors: Dr. Luısa Maria Gouveia da SilvaProf. Dr. Helena Maria Rodrigues Vasconcelos Pinheiro

Examination Committee

Chairperson: Prof. Dr. Jorge Humberto Gomes LeitaoSupervisor: Dr. Luısa Maria Gouveia da Silva

Members of the Committee: Prof. Dr. Ana Paula Vieira Soares Pereira Dias

Outubro 2016

Acknowledgments

Upon completion of this thesis, I have to thank all those who supported me and helped me, not only

along this last semester but also throughout my personal and academic life.

First, I would like to thank my supervisors, Dr. Luısa Gouveia and professor Helena Pinheiro for

having accepted to guide this thesis. Their constant guidance, sharing of scientific knowledge, writing

tips, encouragement and motivation were very important for the success of this work.

To Dr. Alberto Reis and Dr. Paula Marques, whom not being my official supervisors, were always

available for helping me and sharing knowledge. I would specially like to thank Dr. Alberto for his

constant guidance in the wastewater treatment trials and Dr. Paula Marques for her immense help with

the dark fermentation assays and further analysis of the results.

To Dr. Paula Passarinho for helping me in the experimental work necessary for the determination of

protein and total sugars content; to Dr. Cristina Oliveira for all her help in the analysis of the microalgae

fatty acid content and the interpretation of the chromatograms; and to Dr. Belina Ribeiro for performing

the phosphorus determination.

To the entire team of the LNEG Bioenergy Unit at building G, who warmly welcomed me during

my time at LNEG. A special thanks to Graca Conceicao and Natercia de Sousa for all their tips and

laboratory support.

To my colleagues from building G, Diana Francisco, Ricardo Galrica, Paula Assemany, Henrique

Mendonca and Leonilde Marchao for their knowledge sharing and laboratory assistance. You all made

my time at LNEG better with all the conversations, laughs and companionship.

To Dr. Ana Filipa Ferreira and professor Ana Paula Dias for conducting the pyrolysis experiments at

Instituto Superior Tecnico (IST). I would specially like to thank Dr. Ana Filipa Ferreira for her kindness

and sharing of scientific knowledge with the analysis of the pyrolysis data.

To the project ERANETLAC/0001/2014 (ELAC2014/BEE0357) GREENBIOREFINERY - Processing

of brewery wastes with microalgae for producing valuable compounds, for the financial support and to

Sociedade Central de Cervejas e Bebidas S.A. for allowing the collection of the brewery effluent.

I would also like to thank my friends and colleagues from IST (Amigos Fitsches) for always being

there for me during the good and bad times and helping me grow as an engineer and as a person.

You made my years at IST the best ones yet and I wouldn’t have reached so far without your constant

support and motivation. I definitely made friends for life.

Last but not least, to my parents, Julia and Urbano, and my brother Luis Paulo for their love, en-

couragement and caring throughout all my life, for always being there for me through thick and thin and

whom without I would not be who I am today. I would also like to thank my boyfriend Pedro, for his love

and encouragement in all the aspects of my life and for all the tips at LaTeX.

This thesis is dedicated to my late grandma Alice, who raised me since I was little and who I am sure

is very proud.

ii

Abstract

The ability of microalgae to grow in nutrient-rich environments and to accumulate nutrients from the

wastewater, make them attractive for sustainable and low-cost wastewater treatment coupled with the

production of potentially valuable biomass, which can be used for the production of bioenergy, food,

feed, fertilizers, among others.

The first objective of this work was to treat the brewery wastewater from Sociedade Central de

Cervejas e Bebidas, S.A., using bubble-column photobioreactors (PBRs), using air with 10% (v/v) CO2

supplementation. The PBRs were inoculated with the microalga Scenedesmus obliquus and several

residence times (HRT) were tested: 2.1±0.5, 3.5±0.4, 5.3±0.3, 6.5±0.4, 8.7±0.2 and 10.4±0.2 days.

The experiments achieved a maximum volumetric productivity of 0.2 gAFDW L-1 d-1 for a dilution rate of

0.29 d-1, corresponding to a HRT of 3.5 days. The maximum removal efficiencies attained were: 93 and

89% for ammonia and total nitrogen, respectively; 41% for phosphorus; and 62% for COD. Except for

the 0.48 d-1 test, all the treated waters are in accordance with the Portuguese environmental legislation

(Decree-Law 236/98). Considering the volumetric productivity, treatment efficiency and lower residence

time, 0.29 d-1 represents the optimal dilution rate.

The potential of S. obliquus biomass produced in different wastewaters (poultry, swine, cattle, do-

mestic, brewery and dairy) was evaluated for the production of bioH2 through dark fermentation with

Enterobacter aerogenes, and of bio-oil through pyrolysis. The results for bioH2 were 390 mL H2/gVS,

with a purity (H2/CO2) of 7, and for bio-oil production was 83%, both for S. obliquus grown in swine

wastewater.

Keywords: Brewery wastewater treatment; Scenedesmus obliquus; Bio-hydrogen; Pyrolysis; Bio-oil;

Dark Fermentation

iii

Resumo

As microalgas tem a capacidade de crescer em meios ricos em nutrientes, o que as torna opcoes

interessantes para o tratamento de aguas residuais, de um modo mais sustentavel e a baixo custo,

com a producao de biomassa que pode ser utilizada para a producao de bioenergia, alimentos, racao

animal, fertilizantes, entre outros.

Um dos objectivos desta dissertacao foi o tratamento de aguas residuais da industria cervejeira,

sob arejamento com suplementacao de 10% (v/v) CO2, utilizando fotobiorreactores tipo coluna de bol-

has. Os fotobiorreactores foram inoculados com a microalga Scenedesmus obliquus e foram testados

varios tempos de residencia: 2,1±0,5, 3,5±0,4, 5,3±0,3, 6,5±0,4, 8,7±0,2 and 10,4±0,2 dias. A pro-

ductividade volumetrica maxima foi 0,2 gAFDW L-1 d-1 para uma taxa de diluicao de 0,48 d-1, que cor-

responde a um tempo de residencia de 3,5 dias. As taxas de remocao maximas obtidas foram: 93 e

89% para os azotos amoniacal e total, respectivamente; 41% para o fosforo; e 62% para a CQO. Com

excepcao do ensaio a 0,48 d-1, todas as aguas tratadas estao de acordo com a legislacao ambiental

portuguesa (Decreto-Lei 236/98). Considerando a productividade volumetrica, eficacia de tratamento e

menor tempo de residencia, 0,29 d-1 representa a taxa de diluicao optima.

Foi ainda avaliado o potencial de S. obliquus, cultivada em diferentes resıduos (avicultura, suinicul-

tura, vacaria, domestico, cervejas e lacticınios), para a producao de bioH2 por dark fermentation com

Enterobacter aerogenes e producao de bio-oleo por pirolise. O maior rendimento de producao de bioH2

foi 390 mL H2/gSV, com um grau de pureza (H2/CO2) de 7, e para o bio-oleo resultante da pirolise foi

83%, ambos para a biomassa cultivada no efluente da suinicultura.

Palavras Chave: Tratamento de aguas da industria cervejeira; Scenedesmus obliquus; Bio-hidrogenio;

Pirolise; Dark Fermentation

v

Contents

1 Literature Review 1

1.1 Wastewater treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1.2 Overview of the brewing industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1.3 Wastewater treatment in the brewing industry . . . . . . . . . . . . . . . . . . . . . 4

1.1.3.1 General characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.1.3.2 Conventional wastewater treatment . . . . . . . . . . . . . . . . . . . . . 5

1.1.3.3 Microalgae-based wastewater treatment . . . . . . . . . . . . . . . . . . 7

1.2 Microalgae production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.2.1 Biology and biodiversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.2.2 Microalgae biomass production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.2.2.1 General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.2.2.2 Nutritional modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.2.2.3 Cultivation parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.2.2.4 Cultivation systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.2.2.4.1 Open systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.2.2.4.2 Closed systems . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.2.2.4.3 Operational mode: continuous . . . . . . . . . . . . . . . . . . . 15

1.2.3 Harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1.2.4 Scenedesmus obliquus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1.2.4.1 Biology and general characteristics . . . . . . . . . . . . . . . . . . . . . 17

1.2.4.2 Environmental applications . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1.3 Microalgae-based biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1.3.1 Biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1.3.2 Conversion technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1.3.2.1 Thermochemical conversion . . . . . . . . . . . . . . . . . . . . . . . . . 20

1.3.2.1.1 General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

vii

1.3.2.1.2 Pyrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

1.3.2.1.3 Other thermochemical conversion technologies . . . . . . . . . 22

1.3.2.2 Biochemical conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1.3.2.2.1 Biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . 23

1.3.2.2.2 Other biochemical conversion technologies . . . . . . . . . . . . 24

1.4 The Biorefinery strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2 Background and Goals 29

2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.2 Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3 Materials and Methods 33

3.1 Brewery wastewater treatment by Scenedesmus obliquus . . . . . . . . . . . . . . . . . . 35

3.1.1 Brewery wastewater collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.1.2 Microalgae culture and acclimatization . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.1.3 Photobioreactor operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.1.4 Microalgae growth monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.1.4.1 Determination of dry biomass weight and ash-free dry weight . . . . . . . 37

3.1.5 Biomass and supernatant recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.1.6 Brewery wastewater and supernatants characterization . . . . . . . . . . . . . . . 38

3.1.6.1 Chemical Oxygen Demand . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.1.6.2 Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.1.6.3 Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.1.7 Microalgae biomass characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.1.7.1 Protein content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3.1.7.2 Chlorophyll content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.1.7.3 Total sugar content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.1.7.4 Oil characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.1.7.4.1 Fatty Acid content . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.1.7.4.2 Iodine value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3.1.8 Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3.1.8.1 Determination of biomass productivity . . . . . . . . . . . . . . . . . . . . 42

3.2 Biohydrogen production from the microalgal biomass . . . . . . . . . . . . . . . . . . . . . 42

3.2.1 Fermentative bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3.2.1.1 Bacteria culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3.2.1.2 Bacterial growth profile and optical density (OD) versus dry biomass

weight (DBW) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

viii

3.2.2 Microalgae biomass characterization - Volatile Solids (volatile solids (VS)) content 43

3.2.3 Dark fermentation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.2.4 Analysis of the gas phase (headspace) composition . . . . . . . . . . . . . . . . . 44

3.3 Bio-oil production from the microalgal biomass . . . . . . . . . . . . . . . . . . . . . . . . 45

3.3.1 Microalgae biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3.3.2 TGA (thermogravimetric analysis) . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3.3.3 HATR-FTIR (infrared spectroscopy) . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3.3.4 Pyrolysis system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3.3.5 Pyrolysis process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3.3.6 Characterization of the pyrolysis products . . . . . . . . . . . . . . . . . . . . . . . 46

4 Results and Discussion 47

4.1 Brewery wastewater treatment by Scenedesmus obliquus . . . . . . . . . . . . . . . . . . 49

4.1.1 Wastewater characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

4.1.2 Microalgae growth evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

4.1.3 Photobioreactor performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

4.1.4 Brewery wastewater treatment evaluation . . . . . . . . . . . . . . . . . . . . . . . 54

4.1.5 Biomass characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

4.1.6 Effect of CO2 supplementation on microalgae growth and wastewater treatment

performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4.2 Biohydrogen production from the microalgal biomass . . . . . . . . . . . . . . . . . . . . . 60

4.2.1 Fermentative bacteria growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

4.2.2 Scenedesmus obliquus biomass characterization . . . . . . . . . . . . . . . . . . . 62

4.2.3 Scenedesmus obliquus as feedstock for biohydrogen production by Enterobacter

aerogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

4.3 Bio-oil production from the microalgal biomass . . . . . . . . . . . . . . . . . . . . . . . . 64

4.3.1 Scenedesmus obliquus biomass characterization . . . . . . . . . . . . . . . . . . . 64

4.3.1.1 Thermogravimetric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 64

4.3.1.2 HATR-FTIR (infrared spectroscopy) . . . . . . . . . . . . . . . . . . . . . 65

4.3.2 Characterization of the bio-oil and bio-char . . . . . . . . . . . . . . . . . . . . . . 66

4.4 BioH2 by dark fermentation and bio-oil through pyrolysis production from S. obliquus

grown in brewery wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

5 Conclusions and Future Work 69

5.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

5.2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

ix

A Bio-mitigation of CO2 emissions 83

A.1 Bio-mitigation of CO2 emissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

B Auxiliary tables for FTIR analysis 89

x

List of Figures

1.1 Brewing process and main waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Schematic overview of the most widely applied wastewater treatment systems: anaerobic

(A) and aerobic (B) treatment systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.3 Schematic diagram of two basic types of photobioreactors: flat-plate and tubular . . . . . 14

1.4 Schematic diagram of vertical bioreactors: A) Bubble column reactor; B) Air-lift column

reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.5 Algal biomass conversion processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1.6 Simplified process flow diagram envisioned for algae wastewater treatment with CO2 mit-

igation and biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.1 Schematic representation of the PBR system for the continuous assays . . . . . . . . . . 36

3.2 Fermentative bio-hydrogen production by Enterobacter aerogenes, using S. obliquus as

a substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.3 Schematic diagram of the fixed bed pyrolysis apparatus . . . . . . . . . . . . . . . . . . . 46

4.1 Variation of ln ODλ=540nm during the cultivation period of time for the continuous assays . 50

4.2 Biomass concentration (AFDW) and volumetric productivity (PX) of the different Scenedesmus

obliquus cultures at the steady-state as a function of dilution rate in the 6 PBR . . . . . . 51

4.3 Fed-batch acclimatization phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

4.4 Continuous photobioreactor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.5 Microscopic observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.6 Chl a/AFDW ratio of steady-state cultures of Scenedesmus obliquus grown in brewery

effluent at different dilution rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

4.7 Fatty acids profile for Scenedesmus obliquus grown in brewery wastewater at different

retention times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.8 The growth curve of E. aerogenes, in liquid medium, at 33 °C and orbital shaking of 200

rpm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

xi

4.9 The calibration line between OD measured at λ=640 nm and DBW, in relation to the

growth of E. aerogenes, in liquid medium, at 33 °C and orbital shaking of 200 rpm . . . . 61

4.10 Hydrogen production yields and gas purity obtained from the fermentation of dried Scenedesmus

obliquus, grown in different wastewaters and synthetic medium, by a strain of the fermen-

tative bacteria Enterobacter aerogenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4.11 TG (%) and DTG (%/min) curves for the microalga Scenedesmus obliquus biomass grown

in different wastewaters (domestic, swine, poultry, brewery, dairy, AD cattle) resulting from

the thermogravimetric analysis in N2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

4.12 FTIR spectra of the microalga Scenedesmus obliquus biomass grown in different wastew-

aters (domestic, swine, poultry, brewery, dairy, AD cattle) . . . . . . . . . . . . . . . . . . . 66

4.13 Production yields of bio-oil, biochar and bio-gas from the pyrolysis of Scenedesmus obliquus

biomass grown in different wastewaters (domestic, swine, poultry, brewery, dairy, AD cattle) 67

B.1 FTIR spectra for microalgae in general . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

xii

List of Tables

1.1 Characteristics of the brewery wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2 Conventional wastewater treatment steps, objectives and conventional technologies . . . 6

1.3 General composition of different microalgae in percentage of dry weight basis . . . . . . . 10

1.4 Desired characteristics of algae for mass culturing . . . . . . . . . . . . . . . . . . . . . . 11

3.1 Average composition of the different waste effluents used as culture medium for microalgae. 43

4.1 Brewery wastewater average composition of the different collections . . . . . . . . . . . . 49

4.2 Summary of growth related values for Scenedesmus obliquus grown in brewery wastew-

ater at different hydraulic retention time (HRT) . . . . . . . . . . . . . . . . . . . . . . . . . 49

4.3 Characterization of the brewery wastewater after biological treatment with Scenedesmus

obliquus for all 6 continuous mode trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.4 Nutrient maximum removal rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.5 Microalgae biomass composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

4.6 Fatty acids composition of Scenedesmus obliquus grown in brewery wastewater at differ-

ent retention times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.7 Iodine values of the oil obtained from Scendesmus obliquus grown in brewery wastewater

at different retention times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4.8 Total VS content (% on a dry basis) for each Scenedesmus obliquus dried biomass culti-

vated in various types of wastewater media . . . . . . . . . . . . . . . . . . . . . . . . . . 62

4.9 Evaluation of the H2 produced by dark fermentation with Eneterobacter aerogenes of

Scenedesmus obliquus biomass cultivated in various types of wastewater media in terms

of yield and purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4.10 Comparison between dark fermentation and pyrolysis process results . . . . . . . . . . . 68

B.1 FTIR band assignments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

xiii

xiv

Acronyms

A absorbance

AFDW ash-free dry weight

ATP adenosine triphosphate

BOD biological oxygen demand

BSA bovine serum albumin

BWW brewery wastewater

COD chemical oxygen demand

D dilution rate

DBW dry biomass weight

EC electrocoagulation

EVLs emission value limits

FAME fatty acids methyl esters

FM fermentation medium

FTIR Fourier transform infrared

GC gas chromatography

GHG greenhouse gases

HATR horizontal total attenuated reflection accessory

HRT hydraulic retention time

LCA Life Cycle Analysis

xv

LHV Lower Heating Value

MAP microwave-assisted pyrolysis

OD optical density

PBR photobioreactor

PET polyethylene terephthalate

PUFAs polyunsaturated fatty acids

SCC Sociedade Central de Cervejas e Bebidas, S.A.

TCD thermal conductivity detector

TDS total dissolved solids

TGA thermogravimetric analysis

TKN total kjehldahl nitrogen

TOC total organic carbon

TS total solids

TSS total suspended solids

VFA volatile fatty acids

VS volatile solids

WWTP Wastewater Treatment Plant

xvi

1Literature Review

Contents

1.1 Wastewater treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.2 Microalgae production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.3 Microalgae-based biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1.4 The Biorefinery strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

1

2

1.1 Wastewater treatment

1.1.1 Introduction

Today, although it is universally recognized the major importance of a sustainable water manage-

ment, water resources still face serious threats regarding quantity and quality. The exponential human

population growth, the rapid industrialization and economic development since the mid-20th century,

with the consequent pollution increase, affect negatively the availability and quality of water resources,

in many areas worldwide (Posadas et al., 2015; Abdel-Raouf et al., 2012).

The water scarcity together with water quality degradation is becoming a major issue worldwide.

Organic and inorganic substances that are released into the environment as a result of the discharge

of effluents from domestic, agricultural and industrial activities lead to severe pollution problems. To

avoid this, a previous treatment is required to ensure a clean effluent to be discharged into natural water

bodies without the risk of contamination or pollution (Abdel-Raouf et al., 2012).

The conventional primary and secondary treatments of wastewaters should be able to eliminate the

easily settled materials and to oxidize the organic material present in the wastewater. They produce

an apparently clean effluent which may be released into natural water bodies. However, this secondary

effluent is loaded with inorganic nitrogen and phosphorus and causes eutrophication and more long-term

problems when refractory organics and heavy metals are present (Abdel-Raouf et al., 2012).

The traditional wastewater treatment requires a large amount of chemicals and the processes are

very high energy-demanding. The use of algae guarantees remarkable advantages, such as the reduc-

tion of energy consumption, emissions, costs, hazardous sludge formation, as the algae provides the O2

used by heterotrophic and autotrophic microorganisms to oxidize and/or assimilate the organic carbon,

as well as the nitrogen and phosphorous. In addition, due to their capacity to remove heavy metals and

others toxic organic compounds, it does not lead to secondary pollution (Abdel-Raouf et al., 2012).

1.1.2 Overview of the brewing industry

The high organic load of effluents resulting from different industries, namely the agro-food ones, is

a major environmental problem. In particular, the effluents derived from the dairy and brewery sectors

are rich in organic compounds, such as proteins, phosphates, ammonia and/or nitrate that need to be

removed from the residual waters before they can be discharge into the environment (Raposo et al.,

2010).

The brewing industry holds a strategic economic position with the annual world production reaching

almost 2 billion hectoliters in 2014 (Statista Inc., 2016). In fact, beer is the fifth most consumed beverage

worldwide. The brewing process involves two main steps - brewing and packaging of the finished product

3

- which generate by-products (e.g., spent grains from mashing, yeast surplus, etc.) responsible for

pollution when mixed with effluents. In addition, breweries use water for heating and cooling, cleaning

packaging vessels, production machinery and process areas and sanitary water, which also generates

high quantities of polluted water. It is widely estimated that for every liter of beer, approximately 3 - 10

liters of waste effluent is generated, namely in brewing, rising, and cooling processes (Olajire, 2012;

Simate et al., 2011).

The most significant environmental issues associated with the operation of breweries include water

consumption, wastewater, solid waste and by-products, energy use and emissions. The brewing process

often generates large amounts of wastewater and solid wastes (Fig. 1.1). These wastes must be dis-

posed off or treated accordingly to discharge regulations set by government entities, in order to protect

both human and animal lifes, and the environment. Cleaner production is continuously advocated for in

brewing industry in order to reduce consumption and emissions from the production process, products

and services. Although it has an ancient tradition, the brewery industry is a dynamic sector open to new

developments in technology and scientific progress (Olajire, 2012; Fillaudeau et al., 2006).

Figure 1.1: Brewing process and main waste (Fillaudeau et al., 2006).

1.1.3 Wastewater treatment in the brewing industry

1.1.3.1 General characteristics

Wastewater is one of the most significant waste products of brewery operations, so water manage-

ment and waste disposal have become a relevant cost factor and a critical aspect in the operation of

4

brewery plants. All brewery plants try to minimize the waste disposal costs while the legislation regard-

ing this becomes stricter (Fillaudeau et al., 2006).

Brewery wastes are large in bulk and generally high in moisture. This wastewater usually has a high

chemical oxygen demand (COD) from all the organic components present, including sugars, soluble

starch, ethanol, volatile fatty acids, among others, which are easily biodegradable. It is not toxic and

does not usually contain appreciable quantities of heavy metals. The temperature ranges from 25 to

38 °C, occasionally reaching higher temperatures. The pH levels can vary depending on the amount

and type of chemicals used in cleaning and sanitizing (e.g. caustic soda, phosphoric acid, nitric acid,

etc.). Nitrogen (N) and phosphorus (P) levels depend especially on the handling of raw material and

the amount of yeast present in the effluent (Simate et al., 2011). Table 1.1 summarizes the common

composition of the brewery effluents.

Table 1.1: Characteristics of the brewery wastewater (Simate et al., 2011).

Parameter ValuepH 3 - 12Temperature (°C) 18 - 40COD (mg L-1) 2000 - 6000BOD (mg L-1) 1200 - 3600Phosphates as PO4 (mg L-1) 10 - 50TKN (mg L-1) 25 - 80TS (mg L-1) 5100 - 8750TSS (mg L-1) 2901 - 3000TDS (mg L-1) 2020 - 5940VFA (mg L-1) 1000 - 2500

BOD: Biochemical Oxygen Demand, TKN: Total Kjehldahl Nitrogen, TS: Total Solids,TSS: Total Suspended Solids, TDS: Total Dissolved Solids, VFA: Volatile Fatty Scids

One of the main reasons for removing nutrients from wastewater effluents is to control eutrophication

which corresponds to the uncontrollable growth of algae or higher hydrophytes triggered by the addition

of wastewater-borne nutrients (Hammouda et al., 1995). The disposal of untreated, or partially treated

wastewater into water bodies can generate severe pollution problems because the effluents contain a

high amount of organic compounds that require oxygen for degradation (Simate et al., 2011). The neg-

ative impacts of such nutrient overloading include nuisance algae, low dissolved oxygen concentrations

and fish kills, undesirable pH shifts, and cyanotoxin production (Christenson and Sims, 2011).

1.1.3.2 Conventional wastewater treatment

Nowadays, there are several technologies available for the treatment of effluents from agro-food in-

dustries, such as physical, chemical, or biological, which allow the removal of the organic and inorganic

nutrients, specially nitrogen (ammonia and/or nitrate) and phosphorus (Raposo et al., 2010). Table 1.2

summarizes the main steps, objectives and techniques traditionally used for wastewater treatment. The

most important criterion to select the treatment system is its flexibility to overcome the constant fluctua-

tions of the organic load while maintaining the process economically viable (Mata et al., 2012).

5

Table 1.2: Conventional wastewater treatment steps, objectives and conventional technologies. Adapted from Si-mate et al. (2011) .

WASTEWATER OBJECTIVE CONVENTIONAL TECHNOLOGIESTREATMENT STEPPRIMARY Removal of the largest particles that can cause

operational problems during the wastewatertreatment and removal of a fraction of the sus-pended solids and organic matter content in theraw wastewater

- Screening- Sedimentation- Flotation- Filtration- Chemical precipitation

SECONDARY Removal of biodegradable organic matter (dis-solved or in suspension) and the remaining sus-pended solids

- Lagoons- Aerated lagoons- Activated sludge- Trickling filters- Anaerobic digesters

TERTIARY Removal of nutrients (mainly nitrogen and phos-phorus) and pathogen disinfection

- Nitrification/Denitrification- Biological Phosphate Removal- Chemical precipitation- Chlorination- Ozonation- UV-treatment

POLISHING Removal of toxic compounds, heavy metals andnon-biodegradable suspended materials

- Activated Carbon- Chemical Flocculation- Osmosis- Distillation- Electrodialysis

The first treatment steps consist mainly on physical unit operations, where physical forces are applied

to remove contaminants. These types of methods can remove coarse solid matter, but not dissolved pol-

lutants. In general, these methods often result in incomplete contaminant removal and/or separation. In

chemical methods, different chemicals can be added to the brewery wastewater to alter its chemistry.

These may involve pH adjustment and/or coagulation/flocculation (Simate et al., 2011). Chemical treat-

ment often leads to secondary contamination of the sludge product, which results in additional problems

of safe disposal (Christenson and Sims, 2011).

Yet, while chemical and physical based technologies are available to remove nutrients, such as ni-

trogen, phosphorus, and metals from wastewater, they consume significant amounts of energy and

chemicals, which makes them costly processes. The energy and cost associated to the current wastew-

ater tertiary treatment processes are still a problem for industries and municipalities (Christenson and

Sims, 2011).

Biological methods are based on the activity of a wide range a microorganisms, converting the

biodegradable organic pollutants present in the wastewaters. Biological treatment of wastewater can

be divided in aerobic or anaerobic. Anaerobic treatment is characterized by biological conversion of

organic compounds (COD) into biogas (mainly methane and carbon dioxide, with traces of hydrogen

sulphide), while aerobic treatment requires air (oxygen) to oxidize the biological oxygen demand (BOD)

into CO2 and H2O (Driessen and Vereijken, 2003).

Since brewery effluents have both chemical (high organic content) and microbial contaminants they

are generally treated by biological methods. Anaerobic digestion constitutes a common option to treat

6

the brewery effluent, when it is being discharged into a municipal sewer, due to its ability to remove

COD, BOD and suspended solids at low hydraulic residence time ranges and the fact that there is

virtually no biological sludge production. Moreover, direct utilization of the biogas generated in boilers is

a current solution for breweries to become more independent of external fuel supply (Simate et al., 2011;

Fillaudeau et al., 2006). Nonetheless, breweries face more stringent wastewater quality limits, which can

not be achieved by anaerobic treatment only. So, there is a need to follow anaerobic treatment with an

aerobic post-treatment. Activated sludge is the most frequent and widely applied aerobic system for

the treatment of industrial effluents. Other aerobic system is the airlift reactor. The integration of airlift

reactors with an denitrification unit have been developed and applied to brewery effluents. The several

types of anaerobic and aerobic reactors that can be applied to brewery wastewater treatment are shown

in Figure1.2 (Driessen and Vereijken, 2003).

Figure 1.2: Schematic overview of the most widely applied wastewater treatment systems: anaerobic (A) and aer-obic (B) treatment systems. Adapted from Driessen & Vereijken (2003) .

Compared to physicochemical or chemical methods, biological treatment processes present three

major advantages: (1) the treatment technology is mature, (2) high efficiency in COD and BOD removal

(80-90%), and (3) low investment cost. However, despite being quite effective for wastewater treatment,

these methods require a high energy input. Conventional aerobic treatment methods, such as activated

slugde processes, involve oxygen supply through mechanical aeration, which is energy demanding.

Aeration accounts for 45-75% of a wastewater treatment plant’s total energy costs (Razzak et al., 2013).

1.1.3.3 Microalgae-based wastewater treatment

Although most of the biological treatment processes involve the utilization of bacteria, microalgae

are already applied for effluent treament, either as single species (e.g. Chlorella, Scenedesmus or

Arthrospira) or as mixed cultures/consortia (Raposo et al., 2010). In fact, the removal of nutrients from

wastewater with the aid of microalgae has been done for years since it compares favorably with the

7

traditional treatment systems such as aerobic and anaerobic reactors (Mata et al., 2012). Moreover, the

interest in this concept has been revived in recent years due to the fact that the conventional treatment

processes suffer from some important disadvantages: (1) variable efficiency depending upon the nutri-

ent to be removed; (2) costly to operate; (3) the chemical processes often lead to secondary pollution;

(4) the sludge generated has almost no value; (5) the impossibility to reuse water; and (6) loss of valu-

able potential nutrients (N and P). The last disadvantage is especially serious, because conventional

treatment processes lead to incomplete utilization of natural resources (de la Noue et al., 1992). There-

fore, microalgae-based treatment seems to be quite promising for combining the biomass growth with

the biological removal of the wastewater contaminants in a less expensive and ecologically safer way

with the added benefits of resource recovery and recycling (Christenson and Sims, 2011).

In conventional wastewater treatment, N and P are removed from wastewater in two separate pro-

cesses: N is converted to N2 gas through bacterial nitrification-denitrification while P is precipitated with

metal salts (de Bashan and Bashan, 2004). Microalgae, on the other hand, remove these nutrients in

a single process by incorporating them in the produced biomass. Because both nutrients are essential

for microalgal growth, the removal of one nutrient strongly depends on the availability of the other, which

means that microalgae cannot remove nitrogen if there is no phosphorus available in the wastewater, or

vice versa. The concentrations of N and P in wastewater are quite variable, and because their sources

are different, their concentrations often vary independently from each other, which poses a challenge

when engineering nutrient removal from wastewater using microalgae (Beuckels et al., 2015).

The treatment of industrial effluents occurs via photosynthesis and through nutrients recycling with N

and P assimilation into the algal-bacterial biomass. Thus, when irradiated with natural light, microalgae

produce O2, which is then used by aerobic bacteria to mineralize organic matter and oxidize NH4+. In

turn, microalgae consume the CO2 released by bacteria (Razzak et al., 2013).

Microalgae are efficient species in removing nitrogen, phosphorus and other toxic materials from

wastewater and have a disinfecting effect due to increase in pH during photosynthesis. Hence, mi-

croalgae can play an interesting phytoremediation role during the final steps of wastewater treatment

(Abdel-Raouf et al., 2012). By removing nitrogen, phosphorus, and carbon from water, microalgae can

also help reduce eutrophication in the aquatic environment and sequester CO2, minimizing the green-

house effect. Moreover, they can grow in extreme conditions, using water unfit for human consumption

and in land not adequated for food production (Mata et al., 2012).

Regarding the potential of using microalgae for brewery wastewaters treatment some studies already

exist. For example, Raposo et al. (2010) studied the possibility of reducing the amount of nitrogen and

phosphates, as well as the COD and BOD, by using Chlorella vulgaris as a monoculture as well as using

a consortium of microalgae with cyanobacteria and bacteria. They concluded that the consortium was

more effective in the reduction of the contaminants. In addition, Travieso et al. (2008) studied the efficacy

8

of a distillery effluent treatment, also using C. vulgaris. They obtained more than 98% reduction of COD

and BOD5 and an effluent with a final quality which allowed it to be discharged into the environment.

More recent studies developed by Mata et al. (2012) aimed to use the microalga Scenedesmus obliquus

for a brewery wastewater treatment coupled to the production of algae biomass as potential feedstock

for biofuels. Other study by Mata et al. (2014) used the microalga Chlamydomonas sp. grown in

20% (v/v) of brewery wastewater, blended with pentose sugars (xylose, arabinose or ribose resulting

from the hydrolysis of brewers spent grains), to produce biodiesel. They concluded that the culture

without pentose addition presented the best sustainability results. Kong et al. (2012) also suggested

that coupling the cultivation of mixotrophic microalgae and brewery wastewater treatment is a potential

way to produce microalgae biomass, accumulate hydrocarbon and remove organic and inorganic salts.

In conclusion, the use of microalgae for wastewater treatment presents major advantages since

it rests on the principles of natural exosystems, with the O2 needed for the bacteria being provided

through microalgae photosynthesis, minimizing the hazardous solid sludge formation and greenhouse

gases (GHG) emissions, coupled to the production of useful algal biomass-energy with recycling of the

nutrients present in the wastewater (Gouveia et al., 2016a). The CO2 emission bio-mitigation aspects are

further discussed in Appendix A. However, some progress has to be made regarding biotechnological

processes, which are, in general, still expensive, suffer from engineering difficulties and are unstable.

More specifically, algal systems have some drawbacks: (1) generation times are long (hours to days);

(2) harvesting is difficult and costly; (3) light is required; and (4) the biomass concentration is rather low

(de la Noue et al., 1992).

1.2 Microalgae production

1.2.1 Biology and biodiversity

Microalgae are aquatic microscopic and single-cell organisms that live in fresh water and marine en-

vironments. Thus, they are at the bottom of the food chain and many living organisms depend upon them

(AL-Rajhia et al., 2012). Over 36,000 different species of algae can be found in the natural ecosystems.

They can be classified as red algae (Rhodophyta), green algae (Chlorophyta), brown algae, diatoms

(Bacillariophyta), blue green algae (prokaryotes) or dinoflagellates (Razzak et al., 2013).

Microalgae are driven by the same photosynthetic process adopted by higher plants, but, unlike

them, algae do not require a vascular system to transport nutrients. In the adequate conditions, microal-

gae utilize photosynthetic energy, carbon and nutrients to grow biomass. They store the cellular material

compounds such as carbohydrates (mainly starch), polymers, cellulose, hemicellulose, proteins and en-

ergy reserves in the form of lipids (including fatty acids and glycerides), among others, that can be used

as raw material in many applications of commercial interest. For example, microalgae have the potential

9

to be a major source of biofuels worldwide, or to be used for water treatment and pollution control, as

feed in aquaculture, as fertilizer in agriculture or even, as a source of other high value chemicals, includ-

ing pharmaceutical drugs production (Mata et al., 2012) . It can also be used for dyes, agar manufacture

or to produce bioplastics materials (Razzak et al., 2013).

Table 1.3 shows the general composition of different microalgae species that is reported in the liter-

ature.

Table 1.3: General composition of different microalgae in percentage of dry weight basis (Becker, 2007).

Microalgae species Protein Carbohydrates LipidsChlamydomonas rheinhardii 48 17 21Chlorella vulgaris 51-58 12-17 14-22Dunaliella salina 57 32 6Scenedesmus obliquus 50-56 10-17 12-14Arthrospira maxima 60-71 13-16 6-7Nannochloropsis sp. n.d. 17 31-68n.d.: not defined.

From the values shown in Table 1.3, it’s clear that most of the algae contain a large amount of protein,

dominantly enzymatic or crude proteins, providing nutrients similar to those which can be usually found

in foods and feeds. Many algae species under general cultivation conditions commonly have a lipid

content between 10 and 30%, however, this value can double or triple under stressed conditions (e.g.

nutrional, light source and salts), and reach up to 80% by weight of dry biomass (Li, 2012).

1.2.2 Microalgae biomass production

1.2.2.1 General aspects

Microalgae culture consists of a single specific strain selected for production of biomass or of a

biomass-derived product. For the optimal growth of microalgae, the required culture conditions are:

(a) water media at the adequate pH and temperature, (b) necessary nutrients, (c) dosed CO2 and (d)

the presence of sunlight. Growth factors can be classified as (i) carbon source from CO2, (ii) energy

source from light, (iii) nitrogen source (e.g. ammonia, nitrates) and phosphorus (e.g. phosphates) from

wastewater or other culture media, (iv) other minerals and (v) potentially added vitamins (Razzak et al.,

2013). Table 1.4 shows the characteristics of algae and their advantages and disadvantages for mass

culturing.

1.2.2.2 Nutritional modes

Microalgae can assume many types of nutritional modes and are capable of shifting modes de-

pending on the environmental conditions. Microalgae can be autotrophic, if they require only inorganic

compounds, such as CO2, salts and light energy source for growth. For autotrophic algae, photosynthe-

sis is the key for their survival, by which they convert solar radiation and CO2 absorbed by chloroplasts

10

Table 1.4: Desired characteristics of algae for mass culturing (Razzak et al., 2013).

Algae characteristic Advantages Disadvantages(1) Growth in extreme envi-ronments

Reduced problems with competingspecies and predators

Only limited numbers of species available. Culturedifficult to maintain on a large scale under extremeenvironments

(2) Rapid growth rate Provides competitive advantage overcompeting species and predators. It hasa reduced pond area required

Growth rate is usually inversely related to cell size(i.e. fast growing cells are normally very small insize)

(3) Large cell size, colonialor filamentous morphology

Reduces harvesting costs Large cells usually grow slower than smaller sizecells

(4) Wide tolerance of envi-ronmental conditions

Less control of culture conditions re-quired for reliable culture

(5) Tolerance of shear force Allows cheaper pumping and mixingmethods

(6) High cell product content Higher value of biomass Products are usually secondary metabolites (notdirectly involved in normal growth). High concen-trations of secondary metabolites normally meanslower growth

into adenosine triphosphate (ATP) and O2, which can be used, in the absence of light, in respiration

to produce energy to support their growth. Microalgae can also be heterotrophic. These are non-

photosynthetic, which means they require an external source of organic compounds and nutrients in

substituion of light as an energy source (Brennan and Owende, 2010).

Some photosynthetic algae are mixotrophic, i.e. they have the ability to combine both autotrophic

photosynthesis and heterotrophic assimilation of exogenous organic nutrients. Therefore, microalgae

are not strictly dependent on photosynthesis, as either light or organic carbon substrates can support cell

growth. One benefit of mixotrophic cultures is the reduction of photoinhibition, which improves growth

rates. Successful mixotrophic production of algae allows the integration of both photosynthetic and

heterotrophic components during the diurnal cycle, that reduces the biomass loss during dark respiration

and decreases the amount of organic substances assimilated during growth (Brennan and Owende,

2010).

1.2.2.3 Cultivation parameters

Carbon is the most important element for microalgae growth since it constitutes over 50% in most

microalgae. In aquatic environments, carbon sources existent are usually in oxidized forms (inorganic),

combined with molecular oxygen, including carbon dioxide (CO2), bicarbonate (HCO3-) or carbonate

(CO32-). Algae can directly utilize the first two, but generally not carbonate (Christenson and Sims,

2011). Simple organic carbon sources like acetate and glucose are usually preferred by microalgae, yet

they can also utilize other organic compounds such as eutrophic compounds containing nitrogen and

phosphorus (Mata et al., 2012).

Nitrogen is other essential nutrient for the microalgae growth because it contributes for the formation

of proteins that are composed of amino acids chains linked by peptide bonds. Microalgae contain 5-

10% nitrogen and the most common compounds assimilated by microalgae are ammonium (NH4+) and

11

nitrate (NO3-). Phosphorus is also an important macronutrient for growth, which is absorbed by algae as

inorganic orthophosphate (PO43-) in an active process that requires energy. Phosphorus, stored in the

cells, is assimilated in the form of polyphosphate granules by microalgae (Rasoul-Amini et al., 2014).

Because the atmosphere provides a near infinite, although slowly transferred, source of carbon, there

is a bigger concern regarding nitrogen and phosphorus as the limiting factors for microalgae growth

when analyzing a water source. The N and P requirements of microalgae have been an active research

field in microalgal ecology and physiology for over half a century. In 1958, Redfield studied the nutrient

concentration in marine microalgae and found a ratio of 106:16:1 for C:N:P (molar ratio) in the biomass.

This ratio, known as Redfield ratio represents an average of specific N to P ratios that vary from 8 to

45. However, it became clear that this is not a fixed ratio and that nutrients concentrations in microalgal

biomass can be quite variable, especially in freshwater species (Beuckels et al., 2015; Christenson and

Sims, 2011).

Microalgae have the ability to adjust to the nutrients concentration of their biomass according to the

supply of nutrients in the medium. The microalgal P and N concentrations can range from 0.03 to over

3%, and from 3 up to 12% of dry biomass, respectively. Because N and P are used by microalgae to

produce many chemical compounds, changes in the concentrations of these nutrients will influence the

biochemical composition of the biomass. As said before, nitrogen is mainly used for synthesis of proteins

while P is incorporated into ribossomal RNA, so when either one of these nutrients is limiting, the protein

content decreases and cell division slows down, giving space for cells to accumulate C-rich metabolites

such as carbohydrates or lipids by C acquisition through photosynthesis. It is known that microalgae first

accumulate carbohydrates and start accumulating lipids only when nutrient stress is more advanced.

This way, nutrient starvation can be intentionally designed into the process as a method of increasing

the value of the algae biomass (Beuckels et al., 2015).

The effect of pH on growth varies according to the strains of microalgae. In general, a pH of 7 for

freshwater microalgae and 8 for marine microalgae is the optimum for growth, while at pH below 4 most

strains cease to grow (Farrelly et al., 2013).

One of the methods to reduce costs of nutrients and water is to integrate algae biomass production

with wastewater treatment, which was first suggested in 1960s by Oswald and Glueke, since some

wastewater streams contain nutrients and water sources readily available for algae cells (Li, 2012).

1.2.2.4 Cultivation systems

The culture systems are generally classified according to their design as open or closed systems.

The open systems are normally outdoor facilities that include ponds, lagoons, deep channels, shallow

circulating units and others, while the closed systems are vessels or tubes with walls made of trans-

parent materials located in outdoor facilities under sunlight irradiation or indoor facilities under artificial

12

irradiation (Razzak et al., 2013).

1.2.2.4.1 Open systems

Open ponds have been used for large scale microalgae cultivation due to their simple construction

and easy operation. These systems can be classified as natural water systems such as lakes, lagoons,

ponds and artificial water systems such as artificial pond, tanks and containers (Razzak et al., 2013).

The most popular open culture system being currently used is the raceway pond. A raceway pond is

made of a closed loop recirculation channel that is typically about 0.3 m deep. Mixing and circulation are

produced by a paddlewheel that operates continuously, preventing algae sedimentation and enhancing

light utilization. Flow is guided around bends by baffles placed in the flow channel. Raceway channels

are usually built in concrete, or compacted earth, and may be lined with white plastic. During daylight,

the culture can be fed continuously in front of the paddlewheel where the flow begins. Broth is harvested

behind the paddlewheel, on completion of the circulation loop (Chisti, 2007). The productivity of the

biomass has been shown to be as high as 60-100 mg dw L-1 d-1 (Razzak et al., 2013).

The major limitations in open systems include: (a) poor light utilization by the cells, (b) significant

evaporation losses, (c) temperature and light irradiation fluctuations within a diurnal cycle and seasonally,

(d) limited diffusion of CO2 due to significant losses to the atmosphere, (e) larges areas of land required,

and (f) higher risk of contamination with unwanted algae and other microorganisms such as mold, fungi

yeast and bacteria. To overcome these limitations, simple plastic covers or green houses over the open

ponds have been proposed to allow the extension of the growing period. Additionally, a permeable

plastic cover facilitates the transfer and supply of CO2 and the maintenance of mild temperatures during

the night. It has been reported that the covering of open ponds improves biomass productivity, but

contamination remains an unsolved problem (Razzak et al., 2013).

1.2.2.4.2 Closed systems

Closed systems, mainly known as photobioreactors PBRs were developed to overcome some of

the problems associated with open pond systems. The major advantages of the closed systems are

the minimization of water evaporation and the reduction of contamination from competitive microalgae,

predators and pathogens that can kill the desired microalga (Razzak et al., 2013). Given this, photo-

bioreactors allow the culture of single-species of microalgae for longer periods of time with lower risk of

contamination, making these systems more appropriate for sensitive strains as the closed configuration

facilitates the control of potential contaminants. However, the costs of closed systems are substantially

higher than open pond systems (Brennan and Owende, 2010).

Flat-plate photobioreactors (Fig. 1.3a) have received much attention due to their large surface area

13

exposed to light and high cellular density. These bioreactors are made of transparent materials for

maximum energy capture, and a thin layer of dense culture flows across the flat-plate. Mixing and

deoxygenation processes of the culture suspension are affected by continuously bubbling air at the

bottom of the reactor. Flat-plate bioreactors are suitable for outdoor mass cultures because of their

low accumulation of dissolved O2 and their high photosynthetic efficiency. They can be accomodated

in 1000-2000 L volume capacity that can operate successfully for long periods of time (Razzak et al.,

2013; Brennan and Owende, 2010).

Another type of closed PBRs is the tubular photobioreactor (Fig. 1.3b) that consists of an array of

straight transparent tubes made of plastic or glass, where light crosses and it is captured by the algae

cells. The tubes are generally less than 0.1 m in diameter to ensure light penetration and are commonly

oriented to maximize sunlight capture (Chisti, 2007). The microalgal broth is circulated from a gas

exchange vessel (i.e. the degassing column), connected to the main reactor, through the tubes, where

it is exposed to light for photosynthesis and back to the reservoir with the help of a mechanical pump

or an airlift pump (Razzak et al., 2013). Biomass sedimentation in tubes is prevented by maintaining a

highly turbulent flow. Photosynthesis generates oxygen, which in higher levels inhibits photosynthesis;

furthermore, when combined with intense sunlight it produces photo-oxidative damage to algal cells. To

remove the excess of O2, the culture must periodically return to the degassing column that is bubbled

with air or CO2 to strip out the accumulated O2 (Chisti, 2007).

Figure 1.3: Schematic diagram of two basic types of photobioreactors: flat-plate and tubular (Posten, 2009).

Column photobioreactors (Fig. 1.4) offer the most efficient mixing, with lower shear stress, the high-

est volumetric mass transfer rates and the best controllable growth conditions. Additionally, they are

low-cost, have lower energy requirements, and are compact and easy to operate. The vertical columns

are aerated through a gas sparger system, place at the bottom of the reactor, and illuminated through

transparent walls or internally (Brennan and Owende, 2010). Column PBRs can be categorized into bub-

ble column and air-lift photobioreactors based on their liquid flow patterns inside the photobioreactors.

Bubble-column reactors consist of cylindrical vessels that are less expensive and have high surface-

area-to-volume ratio. These reactors were designed to promote the contact between both gaseous and

14

liquid phases, with mass transfer being maintained by gas bubbling upwards from the sparger. Light is

supplied from outside the column. Airlift reactors consists of a vessel with two interconnecting zones.

The gas mixture flows upward to the surface from the sparger in one tube, called gas riser and the

medium then flows down toward the bottom in a region called the down comer, circulating between both

regions. In the upper section, the bubble release combined with the liquid flow promotes an effective

mixture, comparable to stirred tank reactors (Sharma et al., 2015; Teixeira et al., 2007).

Figure 1.4: Schematic diagram of vertical bioreactor: A) Bubble column reactor; B) Air-lift column reactor (Sharmaet al., 2015).

1.2.2.4.3 Operational mode: continuous

According to their feeding rate, photobioreactors can be operated in batch, fed-batch or continuous

mode. In continuous mode operation (chemostat), a flow of fresh medium, containing the required nutri-

ents, is fed into the culture at a given rate, while the culture liquid is continuously removed at the same

rate, in order to maintain the culture volume constant. One of the most interesting features of chemostat

is the possibility to grow microorganisms under constant environmental conditions, at a constant specific

growth rate. At the steady-state, the dilution rate (D) equals the specific growth rate (µ), when the death

rate is negligible (Teixeira et al., 2007).

There are several advantages of using continuous bioreactors instead of the batch mode such as:

a higher degree of control; the growth rates can be regulated and maintained for longer periods of

time; biomass concentration can be controlled by varying the dilution rate; results are more reliable and

easily reproducible, due to the steady-state operation regime; and increased opportunities for system

investigation and analysis. There are, however, some inherent disadvantages: the relatively high and

complexity of this process; wall growth and cell aggregation can also cause wash-out or prevent optimum

steady-state growth; the original product strain can be lost over time, if it is overtaken by a faster-growing

one; higher contamination risk due to longer growth periods (Mata et al., 2010).

15

1.2.3 Harvesting

Harvesting is the main bottleneck toward an effective application in the energy sector and it is com-

monly mentioned as one of the major limitations to the economic viability of large scale microalgae

cultivation. It has been estimated to contribute 20-40% of the total cost of producing the biomass. Sep-

arating the algae from water remains a major hurdle to industrial scale processing partly because of

the small size of the algal cells (typically 3-30 µm for unicellular eukaryotic algae), the negative surface

charge, the relatively low cell density (typically 200-600 mg/L) coupled with the need to handle large

volumes of water (Olguın, 2012; Christenson and Sims, 2011).

Current harvesting methods include chemical and mechanical based operations. Biological based

methods, specially considering autoflocculation verified in some algae strains without the addition of

chemicals, are also being investigated as an alternative to reduce the associated costs (Christenson

and Sims, 2011). Presently, the major conventional technologies for harvesting microalgae are centrifu-

gation, filtration, coagulation-flocculation and flotation.

Flocculation is usually the first step of the bulk harvesting process and its aim is to aggregate the

microalgal cells to increase their effective particle size and thus facilitate the following steps. The compo-

sition of microalgae gives them a negatively charged surface, so they can be aggregated and separated

using metal cations or other flocculating agents. Other possibility to improve flocculation is to adjust the

pH value to a range of 10-11 with NaOH (Olguın, 2012). However, the addition of metals and chemicals

to the microalgae culture can pollute the environment when directly discharged with no further treatment.

Gravity sedimentation is the most common technique used in wastewater treatment because of the

large volumes treated and low value of the biomass generated. However, due to the small size of the

microalgae and their low specific gravity they present very slow settling rates for routine algal harvesting

and, therefore, centrifugation is better for the recovery of biomass. Additionally, it is more adequate for

the harvesting of high-value metabolites (Pittman et al., 2011; Brennan and Owende, 2010). Nonethe-

less, centrifugation has an high energy demand.

A non-conventional method that seems to be an attractive future trend, as it allows a decrease on

energy and the absence of the use of chemicals as flocculants, is the electrocoagulation (EC). EC is

based on an electrical current applied through two reactive electrodes (e.g. aliminium electrodes) sub-

merged in the microalgae suspension. The anode electrode suffers an electrolytic oxidation, releasing

metal ions that will act as coagulant agents for the formation of microalgae flocs. Additionally, oxygen

and hydrogen microbubbles are generated due to the water electrolysis (Matos et al., 2013). In studies

performed by Matos et al. (2013) and Gouveia et al. (2016) , EC allowed the recovery of more than

97 and 80% of the marine Nannochloropsis sp. and the freshwater Chlorella vulgaris, respectively, with

a reduction on energy consumption of 87 and 89%, respectively, when compared with centrifugation

alone.

16

1.2.4 Scenedesmus obliquus

1.2.4.1 Biology and general characteristics

Scenedesmus obliquus is a robust freshwater photosynthetic microalga that easily grows in large

scale under almost inexpensive culture conditions. It can grow in wastewaters of different origins show-

ing good adaptation capacity and small nutrient requirements (Gouveia and Oliveira, 2009).

This microalga belongs to the family Scenedesmaceae and it is commonly found in colonies as

multiples of two arranged linearly or slighty in a zigzag manner, with two or four cells being the most

common. However, S. obliquus may produce unicells as well. It is able to withstand harsh conditions,

adopting a protective phenotype such as eight-celled colonies (Singh and Singh, 2014; Lurling, 2003).

The optimum temperature range for the S. obliquus growth is relatively wider, between 14 and 30 °C,

which is a relevant aspect for the outdoor cultivation, as temperature is one of the major environmental

limiting factors for the microalgae productivity.

Another important aspect of S.obliquus is the possibility of doubling the biomass productivity by

operating the cultivation and harvesting in continuous mode instead of batch. This increase can be

explained because the biomass is kept in the exponential growth phase due to the continuous addition

of new medium, with fresh nutrients, to the cultivation reactor (Mata et al., 2013).

1.2.4.2 Environmental applications

This green microalga is very versatile as raw material for biofuels production. It contains approxi-

mately 12-14% of oil and 10-17% of sugar and is therefore a good source for biodiesel, bioethanol and

biohydrogen production (Ferreira et al., 2013a). S. obliquus is able to accumulate glucose-based carbo-

hydrates at a very high level and with high productivities, which makes it very attractive for its use as a

fermentation substrate (Ortigueira et al., 2015). This microalgae is also one of the best candidates for

biodiesel production, with a lipid content ranging between 18.8 and 29.3% on a dry basis for a nutrient-

replete medium and up to 42% on a dry basis for a nutrient-deficient medium (Gouveia and Oliveira,

2009).

In microalgae-based wastewater treatments, there are two genera that are most frequently present:

Chlorella and Scenedesmus. Regarding the application of S. obliquus to wastewater treatments there

are a few studies. For example, Hodaifa et al. (2008) used rinse water from the olive-oil extraction

industry, with no dilution, for growing microalgae S. obliquus, showing that although this wastewater is

N deficient, the highest percentage of mono and polyunsaturated fatty acids (PUFAs) in the biomass’

lipids fraction was reached. This microalga also showed promising results in nutrient removal from urban

wastewaters (Batista et al., 2015), registering growth rates similar to those reported for a complete syn-

thetic medium (Batista et al., 2014). McGinn et al. (2012) also experimented the use of Scenedesmus

17

sp. for a municipal wastewater treatment in batch and continuous mode, and obtained a near complete

removal of nutrients from the wastewater. Other work by Martinez et al. (2000) studied the removal

of P and N by S. obliquus, cultured in urban wastewater, previously submitted to secondary sewage

treatment, under different conditions of stirring and temperature. They achieved higher specific growth

rates for the stirred cultures, and higher removal rates were achieved with stirring at 25 °C. The protein

content was notably low (around 11.8% by weight), and the PUFAs content was high. Shen et al. (2015)

studied the effect of different concentrations of Total Organic Carbon (TOC) and feeding CO2 on algal

growth and nutrient removal on S. obliquus cultured with secondary municipal wastewater. This work

showed that this microalga could be used for simultaneous organic pollutants reduction, N, P removal

and lipid accumulation.

S. obliquus also showed potential for brewery wastewater treatment, allowing removal rates of 57.5,

20.8, 56.9% for COD, total N and total C, respectively (Mata et al., 2012). Scenedesmus sp. were also

characterized as high-CO2-tolerant species, being able to tolerate until a maximum of 80% (v/v) of CO2

(Solovchenko and Khozin-Goldberg, 2013). This observation suggests that these species have potential

for CO2 fixation since flue gases usually contain up to 15% CO2 (Wang et al., 2008).

1.3 Microalgae-based biofuel production

1.3.1 Biofuel production

Nowadays, fossil fuels are cheaper and readily available. However, due to their increasing prices and

negative long-term impact on the environment and sustainable development, there is the need to find

alternatives. Of the many options available, microalgae are seen as one of the best potential feedstocks

for producing sustainable fuels for transportation, provided that one can improve its production cost to

a point competitive for fossil fuels (Mata et al., 2014). Microalgae can grow and be harvested almost

continuously, reducing the seasonal problems of raw materials supply for the biofuel industry. In addition,

their high biomass and lipid productivities, and the possibility of using nutrients from waste streams (e.g.

wastewaters and/or CO2 flue gas emissions) can help reduce the environmental impacts and costs of

cultivating them for biofuel applications (Mata et al., 2013).

Pittman et al. (2011) recently found that dual-use microalgae cultivation for wastewater treatment

coupled with biofuel generation is an attractive option for reducing energy, fertilizer and freshwater con-

sumption costs, as well as reducing greenhouse gas emissions. They concluded that, based on current

technologies, algae cultivation for biofuels without the use of wastewater is unlikely to be economically

viable or provide a positive energy return. Lundquist et al. (2010) analyzed different scenarios of

algae-based wastewater treatment coupled with biofuel production and found that only those cases that

emphasized wastewater treatment were able to produce cost competitive biofuels. The authors con-

18

cluded that large scale algae biofuels production is not viable without wastewater treatment as the main

goal. Park et al. (2011) also showed that the costs of algal production and harvesting using wastewater

treatment in High Rate Aeration Ponds are essentially covered by the wastewater treatment plant capital

and operation costs. These systems are economically more sustainable and have less environmental

impacts, in terms of their water footprint, energy and fertilizer use, when compared to the cultivation

systems that use freshwater and fertilizers. Additionally, Kumar et al. (2010) noted that microalgae-

mediated CO2 fixation and biofuel production can become more sustainable by coupling microalgal

biomass production with existing power generation and wastewater treatment infrastructures.

Although there are some who argue that using wastewater as a source of nutrients poses contam-

ination risks and should not be preferred over using fertilizer and freshawater, some recently published

Life Cycle Analysis (LCA) studies have confirmed the use of wastewater for microalgae-based biofuel

production as a very useful approach to ensure the economic viability and the sustainability of the whole

production process (Olguın, 2012).

Significant obstacles still need to be overcome before microalgae-based biofuel production becomes

cost-effective and can impact the world’s supply of transport fuel. Some recommendations have been

made recently to overcome the economic constraints of microalgae production on a large scale such

as: a) to recover the nutrients found in wastewater to cultivate the microalgae at a low cost with the

additional benefit of eliminating pollutants from the environment, integrating microalgae cultivation with

fishfarms, food processing facilities and wastewater treatment plants; b) to combine the production of

microalgae for biofuels with the production of bulk chemicals, food and feed ingredients; c) to use a

biorefinery-based production strategies; d) to improve the capabilities of microalgae through genetic en-

gineering and advances in engineering of photobioreactors; e) to carry out more research to understand

and potentially manipulate algal lipid metabolism; f) to consider anaerobic fermentation for biogas pro-

duction as a final step in future microalgae-based biorefinery strategies; g) to integrate the co-digestion

of microalgae with wastewater sludge for biogas production; h) to significantly improve the efficiency,

cost structure and ability to scale up algal biomass production, lipid extraction, and biofuel production;

and, finally, i) to apply a multidisciplinary approach in which systems biology, metabolic modeling, strain

development, photobioreactor design and operation, scale-up, biorefining, integrated production chain,

and the whole system design (including logistics) are considered (Olguın, 2012).

1.3.2 Conversion technologies

Although the culturing of microalgae at an industrial scale can be expensive, is has a huge potential

in producing fuel. The conversion technologies can be separated into two basic categories of thermo-

chemical and biochemical conversion (Fig. 1.5). The choice of conversion process depends on different

factors such as the type and quantity of biomass feedstock, the desired form of the energy, economic

19

considerations, project specific aspects, and the desired end form of the product.

Figure 1.5: Algal biomass conversion processes (Brennan and Owende, 2010)

The waste from brewer fermentation broth is a potential resource for the biofuel production since this

kind of waste is rich in proteins, water, dead yeast cells, nutrients such as nitrogen and phosphorus and

enzymes that can supply enough nutrients to algae growth (Axelsson, 2012).

1.3.2.1 Thermochemical conversion

1.3.2.1.1 General aspects

Thermochemical conversion consists on the thermal decomposition of organic components in the

biomass to yield fuel products (Brennan and Owende, 2010), which can be achieved through three

different thermochemical conversion routes. According to the oxygen content, there is direct combus-

tion (complete oxidation), gasification (partial oxidation) and pyrolysis (absence of oxygen) (Silva et al.,

2016). The thermochemical conversion techniques applied to biomass are a promising pathway to pro-

cess the microalgae and separate different compounds (Ferreira et al., 2015). These types of processes

have various benefits and advantages which are: (1) a small footprint; (2) an efficient nutrient recov-

20

ery; (3) no fugitive gas emissions; (4) short processing time in the order of minutes; (5) capability of

handling a variety feedstocks and blends; and (6) high-temperature elimination of pathogens and phar-

maceutically active compounds. After conversion, only minor residues are left such as ash for disposal.

Ash can also be used as a fertilizer, which reduces disposal fees associated with fuel, dumping and

transportation costs. While utilising microalgal biomass as a feedstock for thermal processing in energy

generation still releases CO2 back to the atmosphere, there would be an overall reduction in emissions

per unit energy as the CO2 is being recycled by the algae (Farrelly et al., 2013).

1.3.2.1.2 Pyrolysis

Pyrolysis is a promising thermochemical conversion technique for energy recovery, waste manage-

ment, and converting biomass into useful energy products, which has attracted considerable attention

during the past decade (Silva et al., 2016). This technology consists in the thermal decomposition of

materials at medium to high temperatures (350-700 °C), in the absence of oxygen or lower than stoichio-

metric oxygen for complete combustion to convert them into different valuable products: biochar, bio-oil

and biogas (Silva et al., 2016; Razzak et al., 2013).

Conventional pyrolysis consists of the slow, irreversible, thermal decomposition of the organic com-

ponents present in the biomass. Slow pyrolysis has traditionally been used for the production of char.

Short residence time pyrolysis (fast, flash, rapid) of biomass at moderate temperatures has generally

been used to obtain high yield of liquid products (Silva et al., 2016).

The pyrolysis method has been used for the commercial production of a wide range of fuels, solvents,

chemicals, and other products from biomass feedstocks (Razzak et al., 2013). Biochar is used as carbon

source for producing different carbon based materials, and bio-oil as chemical feedstock for valuable

chemical products (Ferreira et al., 2014). Pyrolysis is of great interest since the product selectivity

can be controlled and tuned to suite end-use interests by regulating the various process parameters

(Bordoloi et al., 2016).

Major research on characterization of pyrolysis products is focused on lignocellulosic biomass, such

as woody and herbaceous plants because it is renewable and abundant. However, microalgae are

pointed out as a promising feedstock for pyrolysis processes (Ferreira et al., 2014). Microalgae produce

a large variety of lipids that can be extracted before their conversion into biofuels or be used directly in

pyrolysis processes (Ferreira et al., 2016). Bio-oil similar to fossil oil can be obtained from microalgae,

and it may be used directly as a liquid fuel, added to petroleum refinery feedstocks, or catalytically

upgraded into transport grade fuels (Razzak et al., 2013).

Biochar can be used in various ways such as a soil amendment, energy carrier, adsorbents and

catalyst support. According to Lehmannm (2007), biochar application in soil improves the fertility, en-

hances water holding capacity, increases the pH and nutrient retention capacity of soil, reducing the

21

need for fertilisers on agriculture. In addition, it helps to decrease problems related to the CO2 emis-

sions through long term carbon sequestration and it is a safer method of organic waste management

(Bordoloi et al., 2016). Biochar derived from the pyrolysis of algae is seen as more beneficial than other

forms of biomass due to its high nitrogen and phosphorus contents (Farrelly et al., 2013).

The thermogravimetric analysis (TGA) is the usual technique to first characterize the microalgae be-

havior in pyrolysis atmosphere. This technique generates a TG curve which represents the evolution of

the mass (weight loss) as a function of the temperature. The derivative of this curve (DTG) represents

the curve rate of weight variation (%/min) and allows for a qualitative analysis of the biomass compo-

sition. The further deconvolution of the DTG curves can provide the biomass components’ contents

(quantitative analysis) (Ferreira et al., 2016).

To date, various thermochemical conversion routes of microalgae have been investigated and there

are a few reports on pyrolysis of microalgae, including the conversion of microalgae species Oscil-

latoria tenuis, Chlorella protothecoides, Spirulina platensis, Microcystis aeruginosa, Chlorella vulgaris

and Synechococcus. Miao and Wu (2004) performed fast pyrolysis of autotrophic and heterotrophic

Chlorella protothecoides and reported bio-oil yields of 16.6 and 57.2%, respectively. In addition, the

bio-oils obtained had a better quality than those from wood in terms of viscosity, density and heating

value. Pan et al. (2010) also investigated the pyrolysis of Nannochloropsis sp. residue with and with-

out the presence of HZSM-5 catalyst and obtained bio-oil rich in aromatic hydrocarbons from catalytic

pyrolysis. Du et al. (2011) performed a microwave-assisted pyrolysis (MAP) of Chlorella sp., reaching

the maximum bio-oil yield of 28.6% under the microwave power of 750 W. Other study done by Ferreira

et al. (2014) reported the production of bio-oil from microalgae (Chlorella vulgaris and Scenedesmus

obliquus) through pyrolysis in a fixed bed reactor with and without catalyst, obtaining significant differ-

ences in bio-oil yields in the range of 26-38 wt% and 28-50 wt%, respectively.

1.3.2.1.3 Other thermochemical conversion technologies

In a direct combustion process, biomass is burnt in the presence of air, usually in a furnace, boiler,

or steam turbine at temperatures above 800 °C, to convert the stored chemical energy in the biomass

into hot gases. Any type of biomass can be burned, however combustion is only feasible for the biomass

with moisture content lower than 50% on a dry basis. The heat produce has to be immediately used

because storage is not possible (Brennan and Owende, 2010).

Gasification involves the partial oxidation of biomass into a combustible gas mixture at high temper-

atures (800-1000 °C). In this process, the biomass reacts with O2 and water (steam) to produce syngas,

a mixture of CO, H2, CO2, N, and traces of CH4. Syngas has low calorific value (typically 4-6 MJ m-3)

that can be burnt directly or used as a fuel for gas engines or turbines (Brennan and Owende, 2010).

Thermochemical liquefaction processes can convert wet algal biomass into liquid fuel through a

22

complex sequence of physical structure and chemical changes. It is a low-temperature (300-500 °C)

and high pressure (5-20 MPa) process aided by a catalyst in the presence of hydrogen to generate

bio-oil (Brennan and Owende, 2010).

1.3.2.2 Biochemical conversion

1.3.2.2.1 Biohydrogen production

Hydrogen (H2) is a naturally occuring molecule, that is a clean and efficient energy carrier. It is now

universally accepted as an environmentally safe, renewable energy resource and the most promising

alternative in the succession of fossil fuels, with several technical, socio-economic and environmental

benefits. It has the highest energy content per unit weight of any known fuel (142 kJ/kg) and can be

transported for domestic/industrial consumption through conventional means, being safer to handle than

domestic natural gas. Moreover, H2 is the only carbon-free fuel, since its oxidation process produces

only water (Das and Veziroglu, 2008).

Presently, 40% of H2 is produced from natural gas, 30% from heavy oils and naphtha, 18% from

coal, 4% from electrolysis and about 1% is produced from biomass. Traditionally, large-scale hydrogen

is produced through steam reforming of natural gas, electrolysis of water and thermocatalytic reforming

of other H2 containing organic compounds. However, these processes are polluting and expensive. In

this context, biological hydrogen (bioH2) production processes are gaining more attention mainly due to

the fact that they are catalyzed by microorganisms in an aqueous environment at ambient temperature

and atmospheric pressure (Brennan and Owende, 2010; Das and Veziroglu, 2008).

BioH2 can be produced mainly by two routes: photobiologically - biophotolysis of water using green

algae and cyanobacteria and photodecomposition of organic compounds by photosynthetic bacteria

(Das and Veziroglu, 2008) - and by fermentative processes such as dark fermentation. The first one

is based on the uptake of CO2 or other organic substrates and water by photosynthetic organisms,

which means that it requires a constant light source supply that, in addition to low yields, are the major

drawbacks of the process (Ortigueira et al., 2015). In recent years, bioH2 production through dark

fermentation has received increased attention due to its many advantages, such as the high hydrogen

production rates, the potential to convert biomass or bio-wastes into hydrogen, and the feasibility of the

process design and control (Batista et al., 2014). Dark fermentation consists in the conversion of sugars

into H2, CO2 and organic acids by microorganisms, through the acidogenic pathway. In theory, any

sugar-containing biomass can be used as feedstock, and its appeal increases if the chosen biomass

is readily available and low-cost. Microalgae are attractive feedstocks for dark fermentation, since they

are able to absorb solar energy and CO2 and convert it into chemicals, which can be stored as carbon

compounds, such as starch, a fermentable substrate for bioH2 production (Ortigueira et al., 2015). This

23

process is even more attractive when the microalgae biomass could be produced using wastewaters,

which is a win-win process, treatment and energy production.

BioH2 can be produced by microbial consortia, where the feedstock are unsterilized waste mate-

rials, or through more controlled fermentation conditions by specific and highly H2-producing strains.

Good H2-producers include mesophiles, such as Enterobacter aerogenes. This is an anaerobic facul-

tative bacterium that has been described as a good H2 producer in fermentations with biomass from

cyanobacteria such as Anabaena (Ferreira et al., 2012) and microalgae such as Nannochloropsis (No-

bre et al., 2013), and Scenedesmus obliquus, grown in Bristol medium (Batista et al., 2014) and urban

wastewater (Batista et al., 2015), biomass.

The culture of microalgae has several advantages. Regarding their role as a feedstock for H2 pro-

duction, the microalgae present additional advantages: they are a good source of sugars, with starch

as their storage carbohydrate; the microalgal cell wall polysaccharides may also contribute as a car-

bohydrate substrate for bioH2 fermentations; the cell walls of microalgae are also, in general, rich in

cellulose, but lack hemicellulose and lignin, which facilitates the breakdown of biomass into fermentable

sugars, making microalgal biomass more prone to milder pre-treatment (Batista et al., 2014); it does not

generate toxic or environmentally harmful products; and the possibility to extract high-value co-products

from the biomass prior to be submitted to fermentation (e.g. pigments, amino acids or proteins), which

may help for the economic feasibility of producing these types of biofuels (Ortigueira et al., 2015).

1.3.2.2.2 Other biochemical conversion technologies

Other possible way to use the microalgae sugars for energy purposes is through alcoholic fermenta-

tion. Alcoholic fermentation consists in the conversion of biomass materials that contain sugars, starch

or cellulose into ethanol. In this process, the biomass is mashed and the starch is converted to sugars

which are then mixed with water and yeast (commonly Saccharomyces cerevisiae) in fermenters at a

given temperature. The yeast breaks down the sugars and converts them into ethanol. Following this,

a purification process (distillation) is used to remove the water and other impurities present in the di-

luted alcohol product (10-15% ethanol). The concentrated ethanol (95% volume for one distillation) is

removed and condensed into liquid form, which can be used as a supplement or substitute for petrol in

cars. The remaining solid residue can be used for cattle-feed or for gasification (Brennan and Owende,

2010).

A number of microalgae species have high lipid contents, which may vary from 1-70%. The produced

lipids are chemically similar to common vegetable oils and are therefore a potential source of biodiesel

(Farrelly et al., 2013). Biodiesel is a derivative of oil crops and biomass which can be used directly

in conventional diesel engines. It is a mixture of monoalkyl esters of long chain fatty acids (FAME)

derived from a renewable lipid feedstock such as algal oil. After the extraction process, the resulting oil

24

can be converted into biodiesel by a transesterification process, which consists in a chemical reaction

between triglycerides and alcohol (usually methanol) in the presence of a catalyst to produce biodiesel.

In order to decrease energy demand, time and costs from lipid extraction and subsequently conversion

into biodiesel, several works have been done on the development of an in situ transesterification directly

from the biomass (Gouveia et al., 2016b; Bharathiraja et al., 2016; Jazzar et al., 2015). This emerging

technique consists in a single step method, where the extraction of the lipids and their transesterification

occurs simultaneously in one reactor. The alcohol acts as a solvent as well as a transesterification agent.

This methodology allows the reduction in size of biodiesel production units and thus the production costs,

in addition to better yields (Gouveia et al., 2016b).

Algal biodiesel presents major advantages over petroleum diesel because it is renewable, biodegrad-

able, and quasi-carbon neutral under sustainable production. In addition, it is non-toxic and contains

reduced levels of particulates, carbon monoxide, soot, hydrocarbons and SOx, and reduces emissions

of CO2 of up to 78% when compared to emissions from petroleum diesel (Brennan and Owende, 2010).

Algal biodiesel was also found to be in accordance with the standard values imposed by the Interna-

tional Biodiesel Standard for Vehicles (EN14214) (EN, 2004), with lower heating value, lower viscosity

and higher density, when compared to typical properties of fossil oils (Brennan and Owende, 2010).

Anaerobic digestion is many times preferred over other conversion processes because it is appro-

priate for high moisture content (80-90% moisture) organic wastes, which can be useful for wet algal

biomass, avoiding the energy demanding drying step. Anaerobic digestion is the conversion of organic

wastes into a biogas, which is mainly composed by methane (55-75%) and carbon dioxide (25-45%),

with traces of other gases such as hydrogen sulphide (Molinuevo-Salces et al., 2016). Because mi-

croalgae usually have a high proportion of proteins, resulting in low C/N ratios, the performance of the

anaerobic digester is affected. Also, a high protein content can result in an increased ammonium pro-

duction which can reached inhibitory levels to anaerobic microorganisms (Brennan and Owende, 2010).

1.4 The Biorefinery strategy

As biofuel-based microalgae remains expensive, the biorefinery is a way to overcome this bottle-

neck, taking advantage of all biomass components (Ferreira et al., 2016). According to the International

Energy Agency, a Biorefinery has been defined as ”the sustainable processing of biomass into a spec-

trum of marketable products and energy”. Also, a biorefinery may be defined as a facility that integrates

biomass conversion processes and equipment to produce fuels, power, materials and/or chemicals from

biomass (Olguın, 2012).

This concept of biorefinery meets environmental concerns to reduce greenhouse gas emissions

through the growth of photosynthetic microorganisms, converting CO2 into highly valuable compounds

such as carotenoids and polyunsaturated fatty acids (Chagas et al., 2015).

25

Figure 1.6: Simplified process flow diagram envisioned for algae wastewater treatment with CO2 mitigation andbiofuel production (Razzak et al., 2013).

Figure 1.6 represents an integrated microalgae culture-wastewater treatment-biofuel production pro-

cess. It shows that microalgae grow utilizing flue gas CO2 with the input of nutrients from a wastewater

treatment plant and solar energy, generating biomass that can be used for the production of biofuels and

value added by-products. The algae utilize the nitrogen and phosphorus in wastewater, with sufficient

light condition, and convert the CO2 into biomass through photosynthesis. The produced biomass is

harvested periodically and may then be used in various applications such as co-firing, biofuels, biochem-

icals, animal feed and food supplements (Farrelly et al., 2013). Thus, one can notice that commercial

scale of CO2 capture with wastewater treatment and biofuel production using microalgae culturing en-

tails a holistic approach, which requires various process steps to be integrated effectively in the context

of a single industrial facility (Razzak et al., 2013). This method of co-processing creates an efficient, cost

effective system for wastewater treatment, coupled with the reduction of carbon emissions and biomass

valorization (Farrelly et al., 2013). Gouveia (2014) gives a great review on some examples of microalgae

biorefineries (Gouveia, 2014).

The integration of microalgae-based biofuels and bioproducts production with wastewater treatment

has major advantages for both industries. However, major challenges to the implementation of an in-

tegrated system include the large-scale production of algae and the harvesting of microalgae in a way

that allows for downstream processing to produce biofuels and other bioproducts of value. Selected

media will be required to have a high CO2 fixation rate, a rapid growth rate, while being easily cultivated

on a large scale in order to generate a large biomass yield and produce valuable by-products. These

26

useful by-products provide revenue to finance the production of microalgae and the carbon mitigation

processes (Christenson and Sims, 2011). A cost and energy balance shows that energy production

from microalgae is an attainable target with the currently available technologies. In general, the biofuels

produced are too expensive when compared to fossil fuel prices. However, with the introduction of car-

bon taxes and the ever increasing price of oil, the cost energy balance will become more economically

favourable (Farrelly et al., 2013).

Careful management of the carbon cycle would ensure that biomass could be utilized for various

commercial applications while ensuring that sufficient carbon is stored in biological media, thereby

maintaining safe levels of CO2 in the atmosphere. The organic matter produced by microalgae can

be transformed into a wide range of valuable products, with many possible commercial applications. In

fact, microalgae can potentially revolutionize a large number of biotechnology areas including biofuels,

cosmetics, pharmaceuticals, nutrition and food additives, aquaculture, and pollution prevention (Mata

et al., 2010). Some high value molecules that can be produced from microalgae cultivation include fatty

acids, polysaccharides, triglycerides, antioxidants, vitamins, pigments and stable isotope biochemicals.

Carotenoids, phycobiliproteins, and chlorophyll are used as pigments in food and feed production (Far-

relly et al., 2013) and cosmetic additives against UV light, or also as pharmacological agents due to their

wound healing and anticancer properties (Miazek et al., 2014).

27

28

2Background and Goals

Contents

2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.2 Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

29

30

2.1 Background

Urban and industrial wastes with high nutrient loads are daily discharged into the environment, caus-

ing severe pollution problems (Posadas et al., 2015). Therefore it becomes mandatory to develop more

efficient processes capable of achieving the quality imposed for discharged waters by European Regu-

lations, while consuming less energy and enhancing nutrient recovery from the wastewater.

Unfortunately, conventional wastewater treatment technologies present some techno-economic limi-

tations. For instance, activated sludge processes need to supply oxygen in an adequate concentration to

the various microbes that will degrade the pollutants in the wastewater stream. This is achieved through

mechanical aeration in the form of paddles or blowers, which represents 45–75% of the total operation

costs in a Wastewater Treatment Plant (WWTP) (Posadas et al., 2015). In this context, microalgae-

based wastewater treatment offers several advantages over conventional treatment processes. First,

they have lower cost and energy demands because oxygen is supplied directly through photosynthe-

sis rather than energy-intensive electromechanical blowers. Second, microalgae recycle nutrients from

wastewater into a biomass, which can be further used as a biofertilizer and/or as a feedstock for bio-

fuel production or other high value compounds with interesting cosmetic, pharmaceutical and nutritional

applications (Beuckels et al., 2015). Lastly, they can incorporate carbon through photosynthesis to pro-

duce biomass, making them attractive means for CO2 bio-mitigation strategies, which contributes to the

reduction of GHG emissions from fossil fuel power plants.

The current work is a part of the European project ”GREENBIOREFINERY”. This project aims to

develop new strategies to generate valuable bioproducts by integrating the treatment of brewery wastes

with the production of microalgae biomass and other products generated by its further processing, re-

ducing their environmental impact.

The Sociedade Central de Cervejas e Bebidas, S.A. (SCC) is a portuguese brewery. This facility

generates domestic wastewater, which includes sanitary wastewater from toilets and kitchens and in-

dustrial wastewater, from the brewing and the washing processes. The brewery has its own Wastewater

Treatment Plant where the domestic and industrial wastewaters are treated before being discharged into

”Ribeira da Alfarrobeira” (Watershed of Tagus River) (Agencia Portuguesa do Ambiente (APA), 2013).

The wastewater treatment starts with physical removal of coarse solids and chemical neutralization of

the effluent, followed by primary settling of suspended solids. This primary effluent is then sent to

an anaerobic bioreactor (BIOPAQ IC) where anaerobic bacteria decompose organic matter to produce

methane biogas. After that, the secondary effluent undergoes activated sludge treatment and can then

be discharged.

Additionally, SCC has a tertiary treatment system by reverse osmosis that allows the reuse of part

of the treated effluent for other purposes in the brewery and, consequently, results in a reduction of

31

water consumption (Agencia Portuguesa do Ambiente (APA), 2013). This system was installed under

the ”Brewing a Better World” Programme where the Company commited to protect water resources and

reduce CO2 emissions, among others (Sociedade Central de Cervejas e Bebidas (SCC), 2014). In this

context, there is the potential for implementing microalgae-based systems in the brewery wastewater

treatment, replacing the activated sludge process, which would allow the brewery to reduce their eco-

logical footprint in terms of fossil fuel use, CO2 emissions and water consumption, with savings in energy

and other costs.

2.2 Goals

The biotechnological challenges described above have led to the three major goals of the present

work:

1) Study the potential of microalgae culture for treating brewery wastes, including liquid and gaseous

residues;

2) Analyze the influence of CO2 addition on the growth of Scenedesmus obliquus in brewery wastew-

ater;

3) Evaluate the potential of the biomass produced for the generation of biofuels, particularly bio-

hydrogen by dark fermentation and bio-oil by pyrolysis.

With this in mind, the implementation of a biotechnological process to transform waste organic matter,

previously considered as a problem, into products with higher commercial value, represents an important

challenge for the improvement of the sustainability and competitiveness of beer producers. Thus, as

a result of this effort, it is expect to contribute for the development of a waste-to-product biorefinery

technology flexible and customizable enough to be used at different breweries according to the local

needs and possibilities.

32

3Materials and Methods

Contents

3.1 Brewery wastewater treatment by Scenedesmus obliquus . . . . . . . . . . . . . . . 35

3.2 Biohydrogen production from the microalgal biomass . . . . . . . . . . . . . . . . . . 42

3.3 Bio-oil production from the microalgal biomass . . . . . . . . . . . . . . . . . . . . . 45

33

34

3.1 Brewery wastewater treatment by Scenedesmus obliquus

3.1.1 Brewery wastewater collection

The microalgae culture medium was collected from the brewery SCC at Vialonga, after the prelim-

inary screening, primary sedimentation and secondary treatment by anaerobic digestion. This effluent

is riched in nutrients needed for microalgae growth, in particular nitrogen and phosphorus. The effluent

was stored in a refrigeration chamber at 10 °C. Due to large amounts of slurry present in the effluent,

it was left to settle for 24 hours at room temperature. Only the supernatant was used as the algal cul-

ture medium, to avoid clogging problems in the feeding tubes. The brewery wastewater (BWW) was

characterized at the time of use. The characterization results are presented in section 4.1.1.

3.1.2 Microalgae culture and acclimatization

The microalga used in this work was Scenedesmus obliquus (ACOI 204/07) from the Coimbra Uni-

versity Algotec, Portugal. S. obliquus was first grown in a 250 mL Erlenmeyer flask with the appropriate

culture medium (Bristol), at room temperature, under orbital shaking (150 rpm) and continuous artificial

light of 72 W (4 lamp × 18 W/865), up to an optical density (ODλ=540 nm) of approximately 1.2. Bristol

medium contains NaNO3 (0.250 g/L), KH2PO4 (0.175 g/L), K2HPO4 (0.075 g/L), MgSO4.7H2O (0.075

g/L), Fe-EDTA (0.060 g/L), CaCl2.2H2O (0.033 g/L), NaCl2 (0.025 g/L) and Chu’s trace elements solution

(1 mL/L) (Vonshak, 1986). The medium was autoclaved at 121 °C for 15 min before inoculation.

After 9 days growing in Bristol medium, the entire volume of microalgae culture was used as inocu-

lum and equally divided (120 mL each) between five 6 L polyethylene terephthalate (PET) bubble-column

photobioreactors (14 cm in diameter and 40 cm in height) with the brewery wastewater, agitated by fil-

tered compressed air at room temperature (average temperature of 26°C). The culture was continuously

illuminated by 3 fluorescent lamps (Philips 36 W) assembled at one lateral side of the PBR, with an aver-

age light intensity of 3.2 klux (measured with a Phywe Lux-meter). They were grown in fed-batch mode

for biomass acclimatization. The initial volume of brewery wastewater was 2 L, which was increased up

to a working volume of 5 L after 6 days of cultivation.

3.1.3 Photobioreactor operation

Once the culture reached the stationary phase, the operational mode was switched from fed-batch

to continuous, by feeding a source of brewery wastewater to the culture in a continuous mode, and the

filtered compressed air was replaced by air enriched in CO2 (10% (v/v)). Air agitation was adjusted

manually. Different values of HRT were studied (2, 3.5, 5, 6.5, 8, 9.5 days) and the microalgae growth

was monitored along the time of the assay. The continuous photobioreactor system was composed

35

by five PBRs operating in parallel, that were continuously fed with brewery wastewater from a 20 L

polycarbonate carboy containing the feed effluent, through a series of silicone rubber tubes attached to

glass tubes at both ends, one immersed in the effluent in the carboy and the other end at each PBR. A

working volume of 5 L was maintained by overflow at a fixed level and the outlet streams were collected

in plastic containers, through silicone rubber tubing. The air supplied was mixed with CO2 through a ”Y”

connection, reaching the culture at a flow rate of 0.1 vvm (measured with an American Meter Company

flow meter). The air reached the culture through an aquarium Elite air diffuser (Hagen), placed at the

center bottom of the PBR. The CO2 was supplied from bottles provided by the brewery SCC, without

further purification. The CO2 feeding was adjusted manually according to the pH of the culture, which

should be maintained between 7 and 8. The culture was continuously illuminated as it was already

mentioned in section 3.1.2. Figure 3.1 represents the scheme of the system described above.

Figure 3.1: Schematic representation of the PBR system for the continuous assays. Adapted from Marchao (2016).

In order to test the six different HRT values mentioned, different feeding rates were imposed in

each PBR, using Hoffman tubing clamps. These flow rates were controlled manually by adjusting the

clamps and adding fresh effluent every day to maintain the liquid level in the carboy and minimize

fluctuations. On week days, the flow rates were measured several times a day. However, on weekends,

these values were estimated by measuring the volume of the outlet stream collected during that period of

time (assuming that evaporation and liquid level variations were negligible). Finally, the final values of the

feeding flow rate were calculated as the mean value of all measured values during the operational time.

Nevertheless, due to the manual control technique used, some variations occurred and the final values

of HRT studied were 2.08±0.47, 3.51±0.44, 5.27±0.29, 6.50±0.42, 8.74±0.18 and 10.4±0.2 days.

These residence time values were calculated by equation 3.1, where Vworking is the photobioreactor

working volume (L) and F i (L day-1) is the mean value of flow rate in the photobioreactor i.

36

HRT (d) =Vworking

Fi(3.1)

The continuous assays ended when the algal growth reached the stationary phase. The biomass

and the effluent were then collected for further analysis.

3.1.4 Microalgae growth monitoring

Microalgae growth was monitored 2 times a day, during week days, by measuring the optical density

at 540 nm (ODλ=540nm) against distilled water, using a Hitachi U-2000 spectrophotometer. pH was also

measured on the same schedule using a laboratory pH meter (InoLab WTW) in order to check whether

a pH value between 7 and 8 was maintained.

Culture samples were observed, once a week, under an optical microscope (Olympus BX 60) with

100x magnification to verify the presence of Scenedesmus and other microalgae from the effluent.

The dry biomass weight (DBW) and ash-free dry weight (AFDW) were determined at the end of

the assay, when the culture reached the steady-state, according to ODλ=540nm measurements. The

procedure of these two measures is described below, in section 3.1.4.1.

3.1.4.1 Determination of dry biomass weight and ash-free dry weight

The microalgal growth was evaluated by measuring the DBW and AFDW of the 6 cultures. This was

done by filtering 5 mL of microalgal culture through a pre-weighed 25 mm Whatman GF/C 45 µm filters.

The empty filters were first placed in porcelain crucibles and weighted after being incinerated for 1h at

550 °C in a muffle furnace (winitial). After filtering the samples, the filters were left to dry overnight at

105 °C and then weighted after cooling in a desiccator (woven). DBW was calculated by dividing the

weight of microalgae biomass retained in the filter by the filtered culture volume (Vsample), according to

equation 3.2.

DBW (gDBW /Lsuspension) =woven − winitial

Vsample(3.2)

For the AFDW measurement, the filters were further incinerated in the same initial conditions in

the muffle furnace (1h at 550 °C) and finally weighted. The AFDW value was calculated by dividing the

difference between the filter weights before (woven) and after (wmuffle) incineration at 550 °C by the filtered

culture volume (Vsample), as it is presented on equation 3.3 below.

AFDW (gAFDW /Lsuspension) =woven − wmuffle

Vsample(3.3)

All the weightings of the filters were done in a Mettler Toledo Ab204-5 analytical balance.

37

3.1.5 Biomass and supernatant recovery

At the end of the assay, the collected culture volume was left to settle for 48 hours at room tem-

perature in graduated cylinders for a primary concentration of the biomass through decantation. The

supernatant was collected and filtered through 47 mm Whatman 0.45 mm filters. The concentrated

biomass was recovered by centrifugation at 10 000 rpm for 10 min at 15 °C (Heraeus multiuge 3SR+

centrifuge, Thermo Scientific) and freeze-dried (Heto Power Dry LL3000, Thermo Scientific). Both fil-

tered supernatant and freeze-dried biomass were stored at -18 °C, for further characterization.

3.1.6 Brewery wastewater and supernatants characterization

The effluent from SCC was first characterized in terms of pH, COD, nitrogen (ammonia and Kjeldahl

nitrogen) and phosphorus, before and after being settled for 24 hours, respectively, raw and decanted

effluent. In order to evaluate the efficiency of the wastewater treatment by microalgae, the same anal-

yses were performed for the different filtered supernatants collected after the cultivation runs. All the

procedures done for these analyses are described below, from sections 3.1.6.1 to 3.1.6.3. In the end,

the removal efficiency of COD, nitrogen and phosphorus were determined according to equation 3.4,

where Ce and Cs are the concentrations of the measured parameter in the effluent and in the filtered

supernatant, respectively.

Removal efficiency (%) =Ce − CsCe

× 100 (3.4)

3.1.6.1 Chemical Oxygen Demand

Chemical oxygen demand (COD) was determined according to the standard method 5220 B (APHA,

1998). A 20 mL sample was mixed with the proper reagents (∼0.5 g of HgSO4, 30 mL of concentrated

H2SO4 and 10 mL of potassium dichromate solution) and left to reflux for 2 h at 150 °C in a Bloc digester

20 P-Selecta. After cooling, the mixture was diluted with distilled water up to a volume of 400 mL and

titrated with standard ferrous ammonium sulphate (0.25 N), using 2-3 drops of ferroin indicator solution.

3.1.6.2 Nitrogen

Ammonia nitrogen was quantified by titration after a preliminary distillation step based on standard

methods 4500-NH3 B and C (APHA, 1998). In a Kjeldahl tube, a 150 mL sample buffered at pH 9.5

with 25 mL of borate buffer was distilled in a Buchi Distillation Unit K-350 for 6 min. The distillate was

collected in an Erlenmeyer flask containing 50 mL of boric acid indicator solution and was titrated with a

stock solution of H2SO4 0.02 N.

38

The Kjeldahl nitrogen was determined according to the modified Kjeldahl method adapted from the

standard method 4500-Norg B (APHA, 1998). In a Kjeldahl tube, 5 mL of sample and 50 mL of digestion

solution (K2SO4 (134 g/L), H2O (650 mL/L), concentrated H2SO4 (200 mL/L) and 2 g HgO/25 mL H2SO4

6N) were mixed together and digested in a Buchi Digestor Unit K-424 for 4–4.5 h. After cooling, it was

diluted with 100 mL of distilled water and distilled in the distillation unit for 6 min after the addition of 50

mL of reagent sodium hydroxide-sodium thiosulfate (NaOH (500 g/L) and Na2S2O3.5H2O (25 g/L)). The

distillate was collected in an Erlenmeyer flask containing 50 mL of boric acid indicator solution and was

titrated with a stock solution of H2SO4 0.02 N.

3.1.6.3 Phosphorus

A commercial kit for phosphorus determination was used with the Phosver 3 (ascorbic acid) method

using Powder Pillows (Spectrophotometer HACH DR/2010). This tests provides the following results,

in mg/L: PO43- (phosphate), P-PO4

3- (phosphorus presented in the phosphate) and P2O5 (phosphorus

pentoxide) (Gouveia et al., 2016a).

3.1.7 Microalgae biomass characterization

The microalgal biomass was characterized in terms of crude protein, chlorophyll, total sugars and

fatty acid contents. The analyses were done in duplicate (triplicate for total sugars analysis). All the pro-

cedures for these characterization processes are described in the sections below, from 3.1.7.1 to 3.1.7.4.

3.1.7.1 Protein content

The Lowry method (Lowry et al., 1951) was used to measure the protein content of the pretreated

biomass. For this method, 2 main solution were prepared: Reagent A (50 mL of Na2CO3 3% (w/v in

NaOH 0.1 M), 0.5 mL of CuSO4.5H2O 1% (w/v) and 0.5 mL of potassium sodium tartrate 4% (w/v)) and

Reagent B (Folin-Ciocalteu reagent diluted 1:2 in Millipore water). First, a standard absorbance versus

protein concentration curve was established. Several solutions of bovine serum albumin (BSA) were

prepared in a concentration range of 25-500 mg L-1. Then, 0.5 mL of the prepared BSA solutions were

collected into a test tube. 2.5 mL of Reagent A were added to the test tube, the mixture was stirred in the

vortex for 5 s and left to rest for 10 min. Following, 0.5 mL of Reagent B was added and mixed again in

the vortex, being left to rest for 30 min. Lastly, the absorbance of the mixtures was measured at 750 nm in

a spectrophotometer (Hitachi U-2000), against distilled water that was submitted to the same procedure.

For the pretreatment of the samples, 100-200 mg of the freeze-dried microalgae were added to a test

tube with 1 mL of NaOH 0.1 M and boiled in a water bath for 5 min. After cooling in running water, 0.5 mL

of the pretreatead biomass were collected and the Reagents A and B were added in the same procedure

described above. The absorbance (A) of the samples at 750 nm was measured against a NaOH (0.1

39

M) solution that was submitted to the same pretreatment and procedure. Finally, the absorbance was

converted to protein concentration using the calibration curve previously obtained. The protein content

of the biomass was calculated using the equation 3.5, where C is the protein concentration (mg L-1), V

is the volume (L) of the NaOH solution used to pretreat the biomass, D is the dilution factor and mbiomass

is the amount of biomass (mg).

Protein content (%w/w) =C × V ×D

mbiomass× 100 (3.5)

3.1.7.2 Chlorophyll content

Microalga culture samples from the PBR (2 mL) were first concentrated by centrifugation for 10

min at 3900 rpm (Sigma 2-6E, Sartorius). Then, 2 mL of acetone (99.5%, Sigma-Aldrich) and glass

beads were added to the tube and the extraction of chlorophyll was performed by submitting the mixture

to vortex during 2 min followed by 2 min in an ice bath. This step was repeated three times. The

mixture was then centrifuged at 3900 rpm for 20 minutes, and the supernatant collected. The extraction

procedure was repeated until a colourless supernatant was obtained. All the tubes were covered with

aluminum foil to prevent pigment degradation by light exposure. The total volume of the extract phases

collected was quantified (Vextracts). Chlorophylls a (Ca) and b (Cb) in the extracts were quantified by

spectrophotometry (Hitachi U-2000), measuring the absorbance at 630, 647, 664 and 691 nm, against

acetone. The calculations were performed using equations 3.6 and 3.7 (Ritchie, 2008).

Ca (mg/L) = −0.3319×A630 − 1.7485×A647 + 11.9442×A664 − 1.4306×A691 (3.6)

Cb (mg/L) = −1.2825×A630 − 19.8839×A647 + 4.8860×A664 − 2.3416×A691 (3.7)

The chlorophyll concentrations in the culture samples (Chl a and Chl b) were then calculated using

equation 3.8.

Chl (mg/L) =C × VextractsVsample

(3.8)

Finally, the chlorophyll contents (Chl a and Chl b) in the algal cells (mg/g) were calculated by dividing

the concentration of the chlorophylls (mg/L) by the cell dry weight (g/L) in the culture samples and total

chlorophyll content was obtained by summing the values for Chl a and Chl b.

40

3.1.7.3 Total sugar content

The sugars present in microalgal cells were first extracted by quantitative acid hydrolysis (Hoebler

et al., 1989). For this, 300 mg of freeze-dried biomass were measured to each test tube and 3 mL

of H2SO4 (72% (w/w)) were added. The tubes were then placed in a thermostatic bath at 30 °C for 1

hour. After this, the mixtures were transferred to 250 mL capped flasks, the H2SO4 was diluted up to

a concentration of 4% (w/w) and the flasks were autoclaved at 121 °C during 1 h. Finally, the samples

were filtered through an Acrodisc GHP, Pall filter with 3 mm of diameter and 0.45 µm of pore size.

Following extraction, the total sugars content was determined by the colorimetric method of the

phenol-sulfuric reagent (DuBois et al., 1956). First, two aqueous solutions were prepared: phenol so-

lution 5% (w/v), and a standard glucose solution 100 mg/L. From this standard solution, five dilutions

were made (10, 20, 40, 60 and 80 mg/L) to build the calibration line. Next, 1 mL of each standard sugar

solution and 1 mL of the samples were transferred to test tubes, in triplicate, and 1 mL of the phenol so-

lution was added to each tube. A volume of 5 mL of H2SO4 96% (m/m) was added to each tube and the

mixture was agitated in a vortex mixer. The tubes were left to rest during 10 min without external heat-

ing, allowing the reaction to occur. After cooling the tubes in a cold water bath, the absorbances of the

samples and the standard sugar solutions were measured at 490 nm (Aλ=490 nm), against Millipore water.

Using standard sugar solutions, it was possible to build a calibration curve that was used to calculate

the total sugar content of the various samples through their absorbance at the given wavelength.

3.1.7.4 Oil characterization

3.1.7.4.1 Fatty Acid content

Fatty acid composition of the biomass samples was analysed, in duplicate, by gas chromatography

(GC). For this, the fatty acids were first transesterified by the method of Lepage & Roy (1986) with

modifications. 100 mg of freeze-dried microalgae were added to Pyrex tubes with Teflon-sealed screw

caps. Then, 2 mL of a methanol/acetyl chloride (95:5 v/v) mixture and 0.2 mL of heptadecanoic acid

in petroleum benzin 60-80 °C (5 mg/mL) as internal standard solution were also added. The mixture

was heated at 80 °C for 1 h and was cooled to room temperature before being diluted with 1 mL of

water and 2 mL of n-heptane. The tube contents were left to stand until phase separation. The upper

layer was recovered, dried over anhydrous Na2SO4, filtered, and collected in vials, containing the fatty

acids methyl esters (FAME) derivatives ready for GC or to be stored at -18 °C until use. FAMEs were

analysed in a CP-3800 GC (Varian, USA) equipped with a 30-m SUPELCOWAX 10 capillary column

(film 0.32 µm) with helium as carrier gas at a constant flow rate of 3.5 mL/min. Injector and detector

(flame ionization) temperatures were 250 °C and 280 °C, respectively. The split ratio was 1:50 for the

first 5 min and 1:10 for the remaining time. The column temperature programme started at 200 °C for 8

min, increased up to 240 °C at a rate of 4 °C/min, and was held at that value for 16 min. Individual fatty

41

acid contents were calculated as a percentage of the total fatty acids present in the sample, determined

from the chromatographic peak areas.

3.1.7.4.2 Iodine value

Oils extracted from S. obliquus biomass were characterized in terms of iodine value according to the

European Standard EN 14111 (EN, 2004).

3.1.8 Calculations

3.1.8.1 Determination of biomass productivity

In continuous culture at steady-state (if biomass decay is negligible), µ equals the D value, which is

defined by equation 3.9, where F i is the mean value of flow rate in the photobioreactor i, Vworking is the

photobioreactor working volume (L) and HRT is the hydraulic retention time.

D (d−1) =Fi

Vworking=

1

HRT(3.9)

Due to this fact, volumetric biomass productivity (PX, g L-1) day-1) can be determined by equa-

tion 3.10, where X (gAFDW L-1) is the biomass concentration at steady-state.

PX (gAFDWL−1d−1) = D ×X (3.10)

3.2 Biohydrogen production from the microalgal biomass

3.2.1 Fermentative bacteria

3.2.1.1 Bacteria culture conditions

The fermentative bacteria Enterobacter aerogenes, ATCC 13408 Sputum (American Type Culture

Collection, Manassas, USA) was used for the production of hydrogen by dark fermentation (bioH2).

The bacteria culture was kept at 4 °C in solid CASO Agar (from MERCK: 15 g/L peptone from casein,

5 g/L peptone from soymeal, 5 g/L sodium chloride and 15 g/L agar–agar) and grown in a synthetic

growth medium (20 g/L peptone (CULTIMED) solution with 5 g/L NaCl). Bacteria were harvested from

the exponential growth phase. The fermentation medium (FM) for the bioH2 production assays (basal

fermentation medium) contained K2HPO4 (7.0 g/L), KH2PO4 (5.5 g/L), tryptone (5.0 g/L, BactoTM), yeast

extract (5.0 g/L, BactoTM), (NH4)2SO4 (1.0 g/L), MgSO4.7H2O (0.25 g/L), Na2MoO4.2H2O (0.12 g/L),

42

CaCl2.2H2O (21 mg/L), nicotinic acid (20 mg/L), NiCl2 (20 mg/L), Na2SeO3 (0.172 mg/L), with a pH of

around 6.8. Both media were autoclaved before use (Batista et al., 2015).

3.2.1.2 Bacterial growth profile and OD versus DBW

The profile growth of the E. aerogenes was obtained previously. For this purpose, the bacteria were

freshly cultured in a synthetic growth media and the evolution of the bacterial culture was registered over

24 h through the measurement of the OD at 640 nm (ODλ=640nm), against Millipore water, using an a

UV/Visible spectrophotometer (Hitachi U-2000).

In order to determine the initial concentration of the fermentative bacteria inoculated, in each experi-

ment, the relationship between the OD and the correspondent DBW (gDBW/LSuspension) was also achieved

by centrifuging the cellular suspension at 10 000 rpm, for 10 min, and at 4 °C. Then, the pellet was

ressuspended in water and several different dilutions were prepared (namely, 1:2, 1:5, 1:10, 1:20, 1:40

and 1:50) and their corresponding ODλ=640nm was registered. Simultaneously, a certain volume of the

each cellular suspension (diluted solution) was filtered, under vacuum conditions, using Sartorius cellu-

lose acetate filters (0.45 µm of porosity and 47 mm of diameter) and the DBW was calculated according

to the equation 3.2 (section 3.1.4.1).

3.2.2 Microalgae biomass characterization - Volatile Solids (VS) content

In this work, the microalga used as substrate - Scenedesmus obliquus – was obtained by growing

in several different effluents as culture medium, such as poultry, swine, cattle, domestic, brewery and

dairy. The cattle wastewater was pre-treated by anaerobic digestion (AD cattle waste). All these mi-

croalgae biomass were obtained from collaboration works at LNEG. Table 3.1 presents the chemical

characterization of each effluent.

Table 3.1: Average composition of the different waste effluents used as culture medium for microalgae.

Medium N-NH3 TKN PO43- P-PO4

3- COD Reference(mg N/L) (mg N/L) (mg/L) (mg/L) (g O2/L)Poultry waste 122.71±1.91 - - 27.90±1.62 3.70±0.72 (Barata, 2016)Swine waste 2472.4±1.98 3171 - 6.98±0.63 14.16±1.25 (Barata, 2016)AD Cattle waste 498 618 23.5 - 0.00291 (Mendonca et al., 2016)Brewery wastewater 4.11 28.00 20.00 6.50 0.24 (Assemany, 2017)Dairy wastewater 204 312 18 - 3.00 (Mendonca et al., 2016)

In this work, the different microalgal biomass was characterized in terms of its volatile solids (VS)

content (APHA, 1998). So, approximately 1 g of freeze-dried S. obliquus (wsample) was dried in an oven

at 105 °C for 24 h. The DBW of the microalgal biomass was determined (woven). Then, total ash was

determined by incinerating the sample at 550 °C in a muffle furnace for 1 h (wmuffle). The VS content

was calculated according the equation 3.11. The analyses were performed in duplicate.

43

V S (gV S/gbiomass) =woven − wmuffle

wsample(3.11)

3.2.3 Dark fermentation assays

Batch fermentation assays were performed in 159 mL serum bottles which were closed with butyl rub-

ber stoppers and crimped with aluminum seals. Each contained 26 mL of basal fermentation medium

(see section 3.2.1) and microalgal biomass (Headspace volume/liquid phase volume = 5). The biore-

actors containing the fermentation medium and the substrate (S. obliquus) were previously submitted

to autoclave sterilization at 121 °C for 15 min. The sterilized bioreactors were then aseptically purged

by bubbling N2 through them to eliminate O2, before inoculation with exponentially grown E. aerogenes

at 1% (v/v). The fermentation was carried out under orbital shaking (150 rpm) for 6 h at 30 °C. The

initial concentration of the substrate (microalgal biomass) was 2.5 gVS/LFM (Batista et al., 2015). All the

experiments were performed in triplicate and the results are expressed as average ± standard deviation.

Control fermentation assays, without microalgal biomass, were also prepared for comparison. Figure 3.2

exemplifies the process described above.

Figure 3.2: Fermentative bio-hydrogen production by Enterobacter aerogenes, using S. obliquus as a substrate.Adapted from Ferreira et al. (2012) .

3.2.4 Analysis of the gas phase (headspace) composition

The content of H2 and CO2 in the fermentation headspace was analyzed by GC, at atmospheric pres-

sure, in a Varian 430-GC equipped with thermal conductivity detector (TCD) and a fused silica column

(Select Permanent Gases/CO2-Molsieve 5A/Borabound Q Tandem #CP 7430). Injector and column

were operated at 80 °C and the detector at 120 °C. Helium was the carrier gas. Previously, calibration

curves were obtained, in the range of the expected H2 and CO2 concentrations, using standard mixtures,

in order to determine the composition of the gaseous phase. Specific hydrogen productions yields – mL

H2/gVS and mL H2/LFM – were calculated by dividing the total volume of produced hydrogen by the initial

amount (in terms of VS) of S. obliquus and by the fermentation medium volume in the bioreactor (serum

bottle), at 6 h of the process.

44

3.3 Bio-oil production from the microalgal biomass

3.3.1 Microalgae biomass

The microalgae used for pyrolysis assays were the same used for dark fermentation assays, and its

characterization and informations were already described above, in section 3.2.2.

3.3.2 TGA (thermogravimetric analysis)

The microalgae biomass was characterized by TGA. This technique allows the understanding of the

behaviour of biomass in pyrolysis conditions, requiring only a small amount of biomass (around 20 - 30

mg). The pyrolysis temperature was also selected by TGA.

Thermogravimetric analysis was performed in a TGA apparatus (NETZSCH STA 409 PC/PG), sim-

ulating pyrolysis conditions, with a weighting precision of ±0.01% and sensitivity in the mass measure-

ments of 0.1 µg. The samples were heated from 30 to 1100 °C at a heating rate of 25 °C/min in a

nitrogen (99.996%) atmosphere. Temperature was measured with an experimental uncertainty of ± 1

°C.

The TG curve represents the evolution of the mass (weight loss) as a function of the temperature.

The DTG is the TG derivative and represents the curve rate of weight variation (%/min).

3.3.3 HATR-FTIR (infrared spectroscopy)

The microalgal biomass was also characterized by Fourier transform infrared (FTIR) spectroscopy

using reflectance mode. Spectra were obtained with a resolution of 16 cm-1, using a spectrophotometer

from BOMEN (FTLA200-100, ABB). This equipment has a horizontal total attenuated reflection acces-

sory (HATR), from PIKE Technologies, with a ZnSe crystal. The infrared spectra were recorded at room

temperature in the range of 3725-725 cm-1. Sixty-four scans were accumulated for each spectrum to

obtain an acceptable signal-to-noise ratio. Additionally the reflectance signal (R) was corrected using

the Kubelka-Munk (KM) function in eq. 3.12.

KM =(1−R)2

2R(3.12)

3.3.4 Pyrolysis system

The pyrolysis experiments were all performed at Instituto Superior Tecnico, in collaboration with the

Mechanical Engineering Department (IDMEC). All tests were done in a quartz fixed bed reactor (16 mm

in internal diameter and 150 mm in length) in nitrogen atmosphere. The quartz reactor was filled with

carborundum and it was externally heated using a Termolab circular electric furnace equipped with PID

45

controller to ensure complete pyrolysis (Silva et al., 2016). Figure 3.3 shows a schematic diagram of the

pyrolysis apparatus used.

Figure 3.3: Schematic diagram of the fixed bed pyrolysis apparatus (Silva et al., 2016).

3.3.5 Pyrolysis process

The biomass pyrolysis temperature was select based on a previous thermogravimetry analysis, as it

was already mentioned in section 3.3.2. The main weight loss occurred in the range 300 °C and 500 °C.

Thus, the pyrolysis reference temperature chosen was 475 °C.

The pyrolysis test was started by placing a certain amount of fresh microalgae (about 2.5 g) into the

quartz reactor. Nitrogen was used as the carrier gas and its reference flow was monitored using a mass

flow controller. The pyrolysis was set to 475 °C and N2 flow to 200 mL/min.

After the 15 min of the pyrolysis process, the reactor was removed of the oven and the biochar was

removed. The bio-oil was recovered by washing the reactor with acetone followed by rotating evaporation

of acetone under a reduced pressure.

3.3.6 Characterization of the pyrolysis products

The yields of pyrolysis products (biochar, bio-oil and biogas) were quantified. The weighted mass of

biochar and bio-oil enable the possibility of calculating the yield of such products. The yield of biogas

was determined from the difference of the biochar and bio-oil yield.

46

4Results and Discussion

Contents

4.1 Brewery wastewater treatment by Scenedesmus obliquus . . . . . . . . . . . . . . . 49

4.2 Biohydrogen production from the microalgal biomass . . . . . . . . . . . . . . . . . . 60

4.3 Bio-oil production from the microalgal biomass . . . . . . . . . . . . . . . . . . . . . 64

4.4 BioH2 by dark fermentation and bio-oil through pyrolysis production from S. obliquus

grown in brewery wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

47

48

4.1 Brewery wastewater treatment by Scenedesmus obliquus

In the continuous mode trials, 6 mean HRT were tested: 2.08±0.47, 3.51±0.44, 5.27±0.29, 6.50±0.42,

8.74±0.18 and 10.4±0.2 days. The trial for each one ended when the steady state was established.

4.1.1 Wastewater characterization

The composition of the wastewater used in the continuous mode assays is shown in table 4.1. As it

was already said in section 3.1.1, to avoid clogging problems in the feeding tubes, the brewery effluent

was left to settle for 24 h to remove most of the suspended solids. This is known as the decanted effluent.

In Table 4.1 it is also depicted the composition of the effluent as it comes from the brewery - raw effluent.

Comparing both effluents it is clear the difference between their composition, the most notable being

the total suspended solids (TSS) and the organic load (COD), which are lower in the decanted effluent

because most of them were removed through sedimentation.

Table 4.1: Brewery wastewater average composition of the different collections. Mean values and their standarddeviation are shown for at least two replicates.

pH TSS N-NH3 TKN PO43- P-PO4

3- P2O5 COD(mg TSS/L) (mg N/L) (mg N/L) (mg/L) (mg/L) (mg/L) (mg O2/L)

Raw effluent 7.2 515±55 24.5±2.1 120±8 41.25 13.50 30.75 628±18Decanted effluent 8.9 17.5±7.5 29.4±1.4 72.8±0.0 37.75 12.25 28.25 226±0

4.1.2 Microalgae growth evaluation

The efficiency of the wastewater treatment by microalgae is directly or indirectly controlled by their

growth rate, depending on their metabolism and availability of nutrients, on the operating conditions, and

on the harvesting of the produced biomass for the effective nutrient removal (Mata et al., 2012).

The microalgal growth was indirectly evaluated by the biomass dry weight determined at the end of

the assays, and consequently their daily productivity can be calculated through equation 3.10. All the

values determined for the different PBRs are shown in Table 4.2.

Table 4.2: Summary of growth related values for Scenedesmus obliquus grown in brewery wastewater at differentHRT. The values in bold correspond to the PBR that obtained the highest volumetric productivity amongall the PBRs. For AFDW and PX, mean values and their standard deviation are shown for at least twodata points at the steady-state condition.

PBR HRT (d) D (d-1) tsteady-state (d) AFDW (g/L) PX (mg AFDW L-1 d-1) pHI 2.1 0.48 9 0.36±0.00 173±0 7.09II 3.5 0.29 14 0.76±0.02 217±6 8.22III 5.3 0.19 24 0.88±0.14 167±27 7.93IV 6.5 0.15 22 0.58±0.08 83.9±11.6 7.17V 8.7 0.11 29 0.95±0.07 109±8 6.85VI 10.4 0.10 32 0.81±0.05 77.9±4.8 7.08

49

The microalgae growth was indirectly followed by measuring the ODλ=640nm throughout the time of

the 6 different continuous assays, and this evolution is shown in Figure 4.1. In this figure, it can be

observed the evolution of the optical density of the different cultures until they reached its steady-state.

Significant fluctuations of the OD in short periods of time are clearly observed in the beginning of the

assays. Those variations attenuate throughout the cultivation time until they become more constant

and reach a plateau, which indicates the end of the trial, where cultures are assumed to be in their

steady-state.

Figure 4.1: Variation of ln ODλ=540nm during the cultivation period of time for the continuous assays. PBR I: 2.08 d,PBR II: 3.51 d, PBR III: 5.27 d, PBR IV: 6.50 d, PBR V: 8.74 d, PBR VI: 10.4 d.

The steady-state times for all the cultures are in accordance to the literature, which states that a

microalgal culture reaches its steady-state after 3-5 residence times (Teixeira et al., 2007). The three

trials with lower HRT (PBR I, II and III) had higher turnover times, around 4.5, and the other three with

higher HRT (PBR IV, V and VI) had turnover times around 3. This may suggest that for lower dilution

rates the microalgal culture tends to reach the steady-state earlier, ie, after a minimum of fresh medium

renovations.

At the end of the trial, the biomass concentration and volumetric productivity were calculated, and

they are represented as a function of the dilution rate in Figure 4.2. According to this figure, AFDW

is very dependent on the dilution rate. For higher dilution rates, the AFDW value presents a drastic

reduction (from 0.88±0.14 to 0.51±0.01 gAFDW L-1). For lower D there is a fluctuation in the values of

AFDW. This reduction of biomass concentration is reported in literature for general chemostat operation

50

(Teixeira et al., 2007). The highest biomass concentration was obtained for a dilution rate of 0.11 d-1

(0.95±0.07 gAFDW L-1).

Figure 4.2: Biomass concentration (AFDW) and volumetric productivity (PX) of the different Scenedesmus obliquuscultures at the steady-state as a function of dilution rate in the 6 PBRs. Mean values and their standarddeviation (error bars) are shown for at least two data points at the steady-state condition.

Regarding biomass productivity, it is possible to observe an increase in its value up to a maximum at

D=0.29 d-1 (217±6 mgAFDW L-1 d-1) after which it decreases. This means that D=0.29 d-1 is the optimal

dilution rate since it corresponds to the maximum biomass productivity. This behaviour is also reported

in Tang et al. (2012), where it is possible to observe a decrease in biomass productivity for dilution rates

both lower and higher than the optimal value.

Consulting the literature, the results obtained, although lower, are comparable to volumetric produc-

tivities achieved by McGinn et al. (2012) when cultivating Scenedesmus in secondary municipal wastew-

ater in continuous mode at a dilution rate of 0.75 d-1 (234 and 267 mg L-1 d-1). Also, the present results

are comparable to the ones obtained by Marchao (2016) (between 80.5±3.0 and 224±23 mgAFDW L-1

d-1).

4.1.3 Photobioreactor performance

To start the continuous trials, some previous preparation work was required. First the microalgae

were grown in synthetic medium (Bristol) for 9 days, until a significantly high density was achieved, to

ensure the culture was robust enough to survive to potential risks and stresses associated to wastewa-

ters. Since Bristol fulfills all the nutrient requirements of Scenedesmus, the lag phase in this medium is

negligible.

The culture volume was then equally divided between the 5 reactors and an initial volume of brewery

effluent was added (2 L). The volume was gradually increased to ensure a milder acclimatization of

the cells to the new medium. Since wastewaters usually present their own consortium of microalgae

51

and other organisms, but also some harmful compounds, they impose more extreme conditions to the

microalgae. This means they need more time to get accustomed to the new conditions, until they are

strong enough to overgrow the other organisms.

Initially, the culture presented a yellow color from the brewery effluent and a visible amount of foam,

which indicated the presence of bacteria and cell lysis. With time the foam disappeared and the cultures

gradually changed their color to green as result of the microalgae dominance. This fed-batch phase

lasted for 17 days and some photos of it are shown in Figure 4.3.

Figure 4.3: Fed-batch acclimatization phase. A: Day 1; B: Day 6 (initial volume of 2 L); C: Day 6, after adding moreeffluent to make up the final volume of 5 L; D: Day 13: Color change to green.

The continuous trials (Figure 4.4) started with a culture OD around 1 by feeding decanted fresh

effluent to the culture in a imposed feeding rate to achieve the desired retention times, and by adding

CO2 to the air flow. However, this did not happen initially because the air mixing system was not working

properly. To solve the problem a direct connection was made using a ”Y” connection. The entrance of

CO2 was manually regulated by maintaining the culture at a pH range of 7-8. The pH of the effluent was

initially 7, but it tended to stabilize around 9 because of the microalgae growth. With the addition of CO2,

it was possible to maintain the culture in the pH range already mentioned.

The reactors used were bubble-column PBRs, so the mixing was also ensured by the aeration sys-

tem. Regardless of the efficiency of this system in guaranteeing the homogenization of the culture, there

was some algae accumulation in the grooves, due to the design of the carboys. To ensure an homoge-

neous sample, the PBRs were subjected to vigorous mixing before the collection of the samples. Other

issue observed during the experience was the agglomeration of microalgae on the wall of the PBRs

52

Figure 4.4: Continuous photobioreactor system. First day of the continuous trials.

more exposed to light, which resulted in overshadowing of the culture. To minimize this problem, the

PBRs were turned around throughout the day to allow a higher exposure of microalgal cells to light.

Near the end of the trials, the compressor broke down and the aeration and mixing were then provided

by an aquarium air compressor (Sera Precision air 550 R plus).

Because there was not a sufficient number of carboys available, the PBR II trial was performed in

one of the carboys already in use after the end of the previous trial. To do this, most of the volume was

withdrawn and more fresh effluent and microalgae culture were added for fed-batch acclimatization of

the microalgae; the air flow feed contained CO2 in the fed-batch acclimatization phase. The same can

be said about PBR I, since this trial had to be repeated.

As it was mentioned previously, the wastewater comes with its own consortium of microalgae and

bacteria. The presence of other microalgae, rather than Scenedesmus, was evidenced by observing the

culture under an optical microscope. Photos taken with the microscope are available in Figure 4.5. In

all microscope observations, the majority of S. obliquus cells were grouped together in chains of 2 or 4

cells, showing membrane integrity.

Figure 4.5: Microscopic observations. A: Presence of Scenedesmus obliquus and Chlorella. In this photo, it ispossible to see colonies of Scenedesmus, which is a commonly found form, B: Clear dominance ofScenedesmus cells, C: Flocculated biomass.

53

4.1.4 Brewery wastewater treatment evaluation

As it was one of the main goals of this thesis, the biological treatment performance by Scenedesmus

was evaluated by measuring the nutrient (N and P) contents and organic load (COD) present in the

brewery effluent at the end of the trials. The results are shown in Table 4.3. Following this, it was possible

to compare the values obtained for the 6 treated effluents to the emission value limits (EVLs) depicted

in the Portuguese legislation (Decree-Law 236/98), in order to assess the possibility of discharging

them into a natural water body without potential harmful consequences for the environment (Decree-

Law, 1998). Maximum removal efficiency was also calculated for ammonia and total Kjeldahl nitrogen,

phosphorus and COD and are presented in Table 4.4.

Table 4.3: Characterization of the brewery wastewater after biological treatment with Scenedesmus obliquus for all6 continuous mode trials. The HRT equal to zero corresponds to the initial effluent (decanted). Thevalues in bold correspond to the lowest HRT, which ensures that the treated water is within the limitsdepicted at the Decree-Law 236/98. For all the parameters, mean values and their standard deviationare shown for at least two data points at the steady-state condition.

HRT (d) D (d-1) N-NH3 TKN PO43- P-PO4

3- P2O5 COD pH(mg N/L) (mg N/L) (mg P/L) (mg P/L) (mg P/L) (mg O2/L)0 0 29.4±1.4 72.8±0.0 37.75 12.25 28.25 226±0 8.85

2.1 0.48 8.4±1.4 18.2±1.40 35.00 11.50 26.25 100±4 8.743.51 0.29 2.8±0.0 8.4±1.4 29.25 9.50 21.75 86.2±0.0 8.725.3 0.19 4.2±0.0 11.2±0.0 28.75 9.50 21.50 88.4±2.2 8.816.5 0.15 5.6±1.4 14.0±0.0 30.75 10.00 23.00 94.8±0.0 8.628.7 0.11 2.8±1.4 14.0±2.8 23.50 7.75 17.75 100±4 8.5510.4 0.10 2.1±0.7 8.4±2.8 22.25 7.25 16.75 113±0 8.60

Legislation 10 15 - 10 - 150 6-9(Decree-law 236/98)

According to Tables 4.3 and 4.4, the PBR with the lower D (0.10 d-1) provided the best conditions for

the treatment of the brewery effluent because it allowed the highest removal of most pollutants. On the

other hand, it had the lowest removal of organic load (COD) with 50.0% removal. However, because the

purpose of an efficient wastewater treatment comprises not only a high nutrient removal efficiency, but

must also take into account optimal biomass production, we can conclude that D=0.29 d-1 represents the

better compromise for both requirements, since it has the biggest volumetric productivity (217.09±5.71

mgAFDW L-1 d-1), it allows the second best conditions for the wastewater treatment (90.5 and 84.6% for

ammonia and total Kjeldahl nitrogen, respectively), and it presents the highest COD removal efficiency

(61.9%).

Table 4.4: Nutrient maximum removal efficiency.

HRT (d) D (d-1) Maximum removal efficiency (%)N-NH3 TKN P COD

2.1 0.48 71.4 73.1 6.1 55.83.51 0.29 90.5 88.5 22.4 61.95.3 0.19 85.7 84.6 22.4 60.96.5 0.15 81.0 80.8 18.4 58.18.7 0.11 90.5 80.8 36.7 55.8

10.4 0.10 92.9 88.5 40.8 50.0

54

For all the dilution rates, there was a high removal of both forms of nitrogen, with efficiency values

ranging from 71.4 to 92.9% and 73.1 to 88.5%, respectively for ammonia and total Kjeldahl nitrogen. This

suggests that this type of biological treatment was very efficient for nitrogen removal. On the contrary,

P had the lowest removal rates, which reveals that the treatment was not very efficient in the removal of

this nutrient. However, it should be noted that 40.8% P removal for D=0.10 d-1 is very relevant.

Comparing the results obtained in the present work with the values reported in literature, one can find

different ranges of nutrient removal efficiency. Mata et al. (2012) obtained a removal efficiency around

11-24.4% for N, while Raposo et al. (2010) and Darpito et al. (2015) both achieved a removal efficiency

above 85%, in batch mode brewery wastewater treatment using Scenedesmus and Chlorella. The range

of values obtained (73.1-88.5%) are, therefore, comparable to these results. Concerning P removal, the

values determined are significantly lower than those obtained by the same studies, 54-66% and about

90%, respectively. For continuous trials performed by McGinn et al. (2012), near complete removal

of these nutrients was achieved, even for a very short retention time (0.75 d-1 = 1.33 d). Regarding

the results for COD removal, they are comparable to Mata et al. (2012), which values range from 13.3

to 66.8%. The removal efficiency achieved are also comparable to the ones obtained by Gouveia et

al. (2016) for domestic wastewater treatment using Scenedesmus (95 and 63%, respectively for N

and COD removal). However, they are very much lower than the removal efficiency for P (92%). Also

comparing the results obtained with the ones presented by Marchao (2016), it is clear that a higher

removal efficiency was obtained for total nitrogen, but not for COD. It is also important to note that in

general the present work was able to achieve a higher removal efficiency for phosphorus. Nonetheless

there was not a significant difference between the removal rates obtained by both.

Finally, the results presented in Table 4.3 show that, excluding D = 0.48 d-1, all the other treated

effluents can be discharged into natural water bodies with no harmful consequences for the environment,

since they meet the legal discharge requirements. This means that the biological treatment performed

on the brewery wastewater by Scenedesmus was effective. The higher dilution rate corresponds to the

lowest retention time, which may explain the lower removal efficiency. This means that extending the

cultivation period promotes higher removal efficiency values and allows better results. However, when

developing a technology for treating large amounts of effluent, which are continuously being generated,

it is important to treat a great amount of wastewater, to achieved the EVLs imposed by law, in the least

time possible. Nowadays, one major obstacle that still hinders the widespread application of the algal

treatment process is its relatively long HRT to obtain an efficient nutrient removal when compared to

traditional activated sludge processes, which can achieve efficient overall reduction of nutrients within a

much sorter time (4-6 h) (Wang et al., 2010). This said, D=0.29 d-1, which corresponds to HRT=3.5 days,

represents the lowest HRT, where is possible to obtain a treated water within the legal levels imposed,

being another reason for it to be the optimal dilution rate.

55

4.1.5 Biomass characterization

The results of the microalgal biomass characterization of S. obliquus grown in brewery wastewater

with different residence times are available in Table 4.5.

Table 4.5: Microalgae biomass composition. For all the parameters, mean values and their standard deviation areshown for at least two data points at the steady-state condition.

HRT (d) D (d-1) Chlorophyll a (mg/L) Chlorophyll a+b (mg/L) Crude protein (%) Total sugars (%)2.1 0.48 11.8±0.4 31.1±2.8 39.3±0.6 23.4±0.13.5 0.29 21.9±0.7 36.7±1.2 34.2±2.0 24.0±3.25.3 0.19 19.9±2.8 28.8±3.4 40.4±4.5 20.1±0.86.5 0.15 18.6±1.4 40.0±2.0 37.7±1.8 23.1±0.78.7 0.11 17.6±0.0 27.0±0.7 37.6±0.7 26.1±1.3

10.4 0.10 22.2±0.5 36.0±1.0 38.9±2.5 23.2±0.3

From Table 4.5, we can conclude that protein content values are below the known range of values

for S. obliquus, presented in Becker et al. (2007) (50-56%). Searching protein content values already

reported in literature, we find 20.4 and 32.7%, respectively, for S.obliquus grown in Bristol medium

(Batista et al., 2014) and in domestic wastewater (Gouveia et al., 2016a). The present values are in

accordance with the reported values and are slightly higher than those. This can be explained by the

higher amount of nitrogen present in the brewery wastewater, when compared to Bristol and domestic

wastewater, which is predominantly used for the synthesis of proteins (Beuckels et al., 2015).

Regarding total sugars content, the values obtained are above 20%, which are higher than the ones

reported by Becker et al. (2007) (10-17%). In literature, we also find contents of 30.7 and 31.8%

for S. obliquus grown, respectively, in Bristol medium (Batista et al., 2014; Miranda et al., 2012) and

11.7% for S. obliquus used for domestic wastewater treatment (Batista et al., 2015). The present values

are then contained between these values, which means that S. obliquus grown in brewery wastewater

has a great potential as a fermentative substrate for the production of biohydrogen, when compared

to domestic wastewater, despite presenting a lower sugar content than microalgae grown in synthetic

medium.

Relatively to chlorophyll contents, all values are in a range between 26 and 40 mg/g (or 18 - 32 mg

L-1). The values determined, although lower, are comparable to the ones achieved by Raposo et al.

(2010) for Chlorella in brewery wastewater (around 45 mg L-1).

According to Veloso et al. (1991), the Chla/AFDW ratio is a clear indicator of the physiological state

of algal cells, since it is extremely sensitive to the health of the culture. A value lower than 1% means the

population is at risk of crashing, which can be due to predators or the lack of nutrients. In this context,

the values shown in Figure 4.6 are significantly higher than 1%, which suggests that there is no risks for

the algal cells, with nutrient requirements being fulfilled by the waste medium. These high ratio values

can also be explained by the inclusion of CO2 in the nutrient mixture (Veloso et al., 1991).

Lastly, comparing the present biomass characterization with the one presented by Marchao (2016),

56

Figure 4.6: Chl a/AFDW ratio of steady-state cultures of Scenedesmus obliquus grown in brewery effluent at differ-ent dilution rates. Mean values and their standard deviation (error bars) are shown for at least two datapoints at the steady-state condition.

using S. obliquus for brewery wastewater treatment, but without CO2 addition, it is notable that the

present values for protein content are lower. However, a direct comparison cannot be made, since these

values were determined by different methods, in particular the present values were calculated using

the Lowry method which provides a more accurate estimative of protein content. Likewise, comparing

the chlorophyll contents, the majority of the present chlorophyll contents are much higher than those

reported by Marchao (2016). Additionally, comparing the Chla/AFDW ratios, we can conclude that the

supplementation of the culture with CO2 provides an additional feed of nutrients, which has a positive

effect on the stability of the microalgae culture.

Fatty acid profile was also determined for the microalgal biomass in study and the results are pre-

sented in Figure 4.7. The categorization of the fatty acids is also presented in Table 4.6, along with the

methyl esters content. All microalgal lipids are mainly composed of unsaturated fatty acids (46-66%),

but a significant percentage of palmitic acid (C16:0) is also present (14-25%). In general, the dominant

fatty acids are palmitic and linoleic acids, which is in accordance with the results achieved by Gouveia

et al. (2008).

The results presented in Table 4.6 show that microalgae-derived biodiesel from S. obliquus grown in

brewery wastewater is mostly composed by unsaturated fatty acids, with contents of 46 to 66%. Among

the unsaturated fatty acids, there are some important aspects that should be taken into account for

a quality biodiesel, namely the linolenic acid (C18:3) and PUFAs (≥4 double bonds) contents, which,

according to the EN 14214 (EN, 2004), should be lower than 12 and 1%, respectively. According to this,

only the oils extracted from S. obliquus grown at a dilution rate of 0.11 d-1 present linolenic acid content

within the specifications. Regarding PUFAs, all the oils have lower contents than the value depicted by

the European standard, since they are not even detected. Despite this, the oils produced in all dilution

rates may be used for good quality biodiesel if associated with other oils, or without restrictions as raw

57

Figure 4.7: Fatty acids profile for Scenedesmus obliquus grown in brewery wastewater at different retention times.

material for other biofuels production processes (Gouveia and Oliveira, 2009).

Table 4.6: Fatty acids composition of Scenedesmus obliquus grown in brewery wastewater at different retentiontimes. The percentage of saturated, unsaturated and monounsaturated fatty acids corresponds to theproportion of each one relatively to the total amount of fatty acids.

HRT (d) D (d-1) Saturated (% w/w) Unsaturated (% w/w) Monounsaturated (% w/w) Esters (% westers/wbiomass)2.1 0.48 33.0 46.3 13.2 9.43.5 0.29 31.4 48.1 14.9 6.05.3 0.19 27.7 64.3 21.2 3.56.5 0.15 30.2 57.4 18.4 6.48.7 0.11 27.0 65.7 40.9 6.110.4 0.10 24.3 63.3 15.7 7.9

The oils obtained from the biomasses were also characterized in terms of iodine value, the results of

which are shown in Table 4.7. The results are in accordance with the European standard specifications

(<120 gI2 /100 g), which makes the present microalgal oils competitive with some traditional vegetable

oils for biodiesel production, such as soy or sunflower (Gouveia and Oliveira, 2009). However, consider-

ing that only the saponifiable lipids can be used to produce biodiesel, which corresponds to 30-50 wt%

of lipids (Veillette et al., 2015), and that Olkiewicz et al. (2015) obtained 39-65% of saponifiable lipids,

we can assume that 50% of total lipids are fatty acids transesterified into biodiesel. The oil contents of

the microalga grown in brewery wastewater are then between 7 and 19%, which is relatively low.

Finally, the biochemical composition presented above suggest that S. obliquus grown in brewery

wastewater is more appropriate for food and feed production since they are very rich in protein and

chlorophyll contents, which were pointed out as important commercial aspects in section 1.4. However,

dried microalgae has a dark green colour, a slight fishy smell and undesirable powder like consistency

which affects the texture of food, limiting their incorporation into food products in large scale (Farrelly

et al., 2013). Moreover, the food safety regulation rules are very strict and there is not sufficient data

58

Table 4.7: Iodine values of the oil obtained from Scenedesmus obliquus grown in brewery wastewater at differentretention times.

HRT (d) D (d-1) Iodine value2.08 0.48 863.51 0.29 845.27 0.19 1086.50 0.15 1028.74 0.11 8710.4 0.10 117

to support the safety of using microalgae grown in wastewaters for food and feed production, not to

mention the public perception on eating algae that was cultivated in waste. On the other hand, the

sugar contents are slightly relevant, which means there is also the potential to use these microalgae

for bioH2, or even, bioethanol production, after a preliminary extraction of pigments like chlorophyll.

Regarding biodiesel, the studied microalgae do not contain a significant amount of oil, which discards

them as an option for biodiesel production. However, according to Sialve et al. (2009), when the cell lipid

content does not exceed 40%, the anaerobic digestion process of the whole biomass appears to be the

optimal strategy on an energy balance basis, for the energetic recovery of cell biomass. Nonetheless,

experiments performed for this microalga (see section 4.3) show potential for thermochemical pyrolysis,

which should be further investigated.

4.1.6 Effect of CO2 supplementation on microalgae growth and wastewater treat-

ment performance

Since the C:N ratio in wastewater, such as brewery wastewater, is lower than for microalgal biomass,

the microalgae production and wastewater treatment can be enhanced by supplying more CO2 (Park

and Craggs, 2011). In fact, an elevation of the CO2 levels in the cell culture is known to improve the

specific growth rate and photosynthetic activity of microalgae. The present work and the work developed

by Marchao (2016) both studied the growth of S. obliquus in brewery wastewater, respectively, with and

without CO2 supplementation. The comparison between the two result sets enables the conclusions

regarding the effect of CO2 in the microalgae culture.

Looking into the performance of the microalgae in removing the nutrients from the brewery effluent,

it is possible to observe that similar removal efficiencies were achieved by both studies. However, it is

important to mention that the effluents used for the experiments presented significant differences be-

tween their physicochemical composition. The brewery effluent used for the present work had, visibly,

more suspended solids and presented higher amounts of contaminants. This mean that, while Marchao

(2016) obtained similar removal rates, the amount of nutrients removed was less than in the present

work, since it started from a ”cleaner” effluent. Seeing that the experiments studied equivalent HRT and

took about the same amount of time, this observation supports the fact that CO2 supplementation had

59

an positive effect of the wastewater treatment, allowing the removal of higher amounts of nutrients in the

same time frame, when compared to microalgae treatment with no CO2 supplementation. Additionally,

observing the pH ranges shown in Marchao (2016) (around 9), it is important to take into account pH

mediated nutrient removal processes. According to Heubeck et al. (2006), for pH values higher than 8,

most nutrient removal occurs by pH mediated processes such as phosphate precipitation and ammonia

volatilisation. These processes are more rapid than microalgal assimilation, and so they are the ones

to act first on the removal of nutrients. The addition of CO2 promotes the decrease in the culture’s pH

down to values at which the pH mediated processes are inhibited. This can be seen has a advantage,

since both phosphate precipitation and ammonia volatilisation represent losses of important nutrients,

which, in microalgae-based wastewater treatment, can be incorporated into microalgae biomass and

enhance their biochemical value. These informations suggest that CO2 addition ensures that more nu-

trients are available for microalgae growth. Moreover, the feeding of CO2 to the culture overcomes both

carbon limitation and pH inhibition of microalgal growth (Heubeck et al., 2007). Nonetheless, because

microalgae assimilation most likely does not act as quickly as pH mediated processes, this can explain

the fact that for the lowest retention time studied (HRT = 2.08 d), the treated water did not meet the legal

requirements, since for a pH range between 7-8, the only nutrient removal is by microalgae assimilation,

which can not eliminate the nutrients at a rate comparable to the residence time.

Regarding the microalgae growth, it was expected to achieve higher biomass productivities with CO2

supplementation since this increases the levels of dissolved carbon in the culture, ensuring no risk of

carbon limitation. However, the results do not show enough evidences to support this observation,

since not all PBR achieved higher volumetric productivities than the ones obtained by Marchao (2016).

This was probably due to some inhibition of the microalgal photosynthesis at low pH and non-optimal

environmental conditions as a result of the acidification of the stromal compartment of the chloroplast

(Solovchenko and Khozin-Goldberg, 2013). Regarding chlorophyll contents, it is clear that, for the mi-

croalgae grown in the medium supplemented with CO2, there was a higher synthesis of this pigment.

This observation may be explained by the enhancement of the photosynthesis process due to the CO2

supplementation. Now, this was also verified by Sutherland et al. (2015), who studied the effect of CO2

addition in microalgae used for wastewater treatment. This studied revealed that for higher percentages

of CO2 fed to the culture, and consequently lower pH, a higher content of Chl a was achieved.

4.2 Biohydrogen production from the microalgal biomass

4.2.1 Fermentative bacteria growth

Initially, the growth profile of the bacteria E. aerogenes used in this work was studied, in order to

identify the exponential growth phase, in which the cell viability is the highest, which is crucial because

60

it is in these conditions that bacteria performance is more efficient. So, it is at this phase that the

bacteria must be collected and inoculated in the fermentation medium, aiming to obtain a higher bioH2

production. The growth curve obtained is represented in Figure 4.8. It follows a sigmoidal profile where

3 distinct phases are evident: between 0-1.25 h (lag phase), between 1.25-9 h (exponential phase) and

between 9-15 h (stationary phase).

Figure 4.8: The growth curve of E. aerogenes, in liquid medium, at 33 °C and orbital shaking of 200 rpm.

Also, the relationship OD vs DBW of the cellular suspension (section 3.2.1.2), was studied and it is

shown in the Figure 4.9. A linear relationship between the OD and the DBW was observed (R2=0.9906),

which can be represented by the equation 4.1.

ODλ=640nm = 2.0169×DBW (gDW /Lsuspension)− 0.0997 (4.1)

Figure 4.9: The calibration line between OD measured at λ=640 nm and DBW, in relation to the growth of E.aerogenes, in liquid medium, at 33 °C and orbital shaking of 200 rpm.

61

4.2.2 Scenedesmus obliquus biomass characterization

In order to quantify the initial concentration of substrate (S. obliquus), the organic content of the

microalga was determined, in terms of its VS value (APHA, 1998), and the values are presented in

Table 4.8. Thus, the initial substrate concentration, in all fermentations, was 2.5 gVS/LFM aiming to

compare the results with those observed for the microalgae grown in synthetic medium (Bristol). For the

dark fermentation assay, it was first necessary to determine the total VS for each biomass in order to

calculate the bioH2 yield. The values determined are present in Table 4.8.

Table 4.8: Total VS content (% on a dry basis) for each Scenedesmus obliquus dried biomass cultivated in varioustypes of wastewater media.

Growth medium VS (%) ReferenceDairy wastewater 84.7Brewery wastewater 81.4Swine waste 92.1 Present workPoultry waste 93.1Cattle waste 77.2Domestic wastewater 76.6 (Batista et al., 2015)Bristol 75.5 (Batista et al., 2014)

In Table 4.8 it can be observed that the values registered are very similar and comparable to the

values reported in the literature for S. obliquus growing in synthetic medium (Bristol medium, 75.5%)

(Batista et al., 2014) and in domestic wastewater (76.6%) (Batista et al., 2015). In addition, the high

contents of VS registered (between 77.2-93.1%) may suggest that all the S. obliquus studied in this

work have potential as feedstock for hydrogen production by dark fermentation, stating the objectives of

this work.

4.2.3 Scenedesmus obliquus as feedstock for biohydrogen production by En-

terobacter aerogenes

In order to evaluate the potential of the different S. obliquus as substrate for bioH2 by dark fermen-

tation, experiments were undertaken, according to the procedure described in section 3.2.3 and an

initial substrate concentration of 2.5 gVS/LFM. The processes yields (mL H2/gVS and mL H2/LFM) and

purity (H2/CO2) were evaluated and compared with those obtained for the microalgae grown in synthetic

medium (Batista et al., 2014). These results are shown in Table 4.9 and in Figure 4.10.

The best results were clearly obtained for the S. obliquus grown in swine and poultry wastes, with

values considerably higher of process yields (around 7-fold) and gas purity (around 6-fold) (Figure 4.10;

Table 4.9). This behavior may be associated with the fact that the microalgal biomass separation, after

culturing, was performed by centrifugation without washing the microalga. This leads us to predict that

the chemical composition of each culture medium (wastewater) (Table 3.1) had an influence on the

compounds which were adsorbed to the surface of microalgae, being more available to be metabolised

62

Table 4.9: Evaluation of the H2 produced by dark fermentation with Enterobacter aerogenes of Scenedesmusobliquus biomass cultivated in various types of wastewater media in terms of yield and purity.

Growth medium ηbioH2 (mL H2/gVS) ηbioH2 (mL H2/gFM) PuritybioH2 (H2/CO2) ReferenceDairy wastewater 56.4±7.0 141±17 1.04±0.09Brewery wastewater 67.1±6.2 168±15 1.02±0.07Swine waste 390±22 976±54 7.12±0.44 Present workPoultry waste 378±7 946±18 6.82±0.16AD Cattle waste 50.1±1.7 125±4 1.56±0.08Domestic wastewater 56.8 111.6 1.0* (Batista et al., 2015)Bristol 57.6 135.6 1.2 (Batista et al., 2014)*Data not shown.

by the bacteria and influencing the process results. An important point to highlight is the high purity

of the produced biogas (CO2/H2 volume ratio) - 7 and 6.7, respectively (Table 4.9) - which is crucial

to facilitate the hydrogen purification process. Exception to this behavior was observed when using

the S. obliquus grown on pre-treated cattle wastewater, which can be attributed to a significantly lower

concentration of organic load (COD) of the effluent (Table 3.1). Nevertheless, the results obtained were

promising, when compared with the others (Figure 4.10; Table 4.9). For the microalga grown in the other

effluents, lower results were attained, however all of them are very similar to those registered when using

as substrate S. obliquus grown in synthetic medium (Batista et al., 2014), which is very interesting on

the environmental (pollution control, renewable energy) and economical (resources recovery, low total

cost waste management) standpoints.

Figure 4.10: Hydrogen production yields and gas purity obtained from the fermentation of dried Scenedesmusobliquus, grown in different wastewaters and synthetic medium, by a strain of the fermentative bacteriaEnterobacter aerogenes.

The S. obliquus (substrate) was sterilized with the fermentation medium, because the autoclaving

conditions (121 °C, 2 bar for 15 min) acts as a thermal treatment, promoting the breakage of microalgae

cells and the release of sugars and other compounds (e.g. storage polysaccharides) with potential as

feedstock to produce bioH2. So, in this work the thermal treatment of the substrate (S. obliquus) was

63

associated with the fermentation medium sterilization step (essential to a fermentative process with pure

bacteria), which is an advantage from the energetic point of view (Batista et al., 2014). Moreover, the

fermentation was carried out under orbital shaking (150 rpm) for 6 h at 30 °C, which is an advantage

considering the energy costs since the process time is short (6 h) and occurs at a temperature similar

to room temperature (30 °C).

In conclusion, the results presented in this study undoubtedly demonstrated the potential of the

S. obliquus biomass as feedstock for hydrogen production by dark fermentation, even if it is grown in

industrial effluents. This is particularly valuable from the economic point of view since it allows both the

treatment of an industrial effluent with simultaneous microalgal growth and the subsequent production

of an efficient energy carrier (H2). Lastly, this work proved to be possible to integrate two sustainable

bioprocesses. The conversion of microalgal biomass into an energy carrier (H2) results in a closed loop

system that allows an efficient nutrient recycle (wastes) and a more complete use of the energy content

of algal biomass, contributing to a real circular bioeconomy (Lakaniemi et al., 2013).

4.3 Bio-oil production from the microalgal biomass

4.3.1 Scenedesmus obliquus biomass characterization

4.3.1.1 Thermogravimetric analysis

The TGA studies were performed for all the different microalgae biomass. Figure 4.11 shows the

resulting TG and DTG curves for the microalgae in the pyrolysis conditions (N2).

Figure 4.11: TG (%) and DTG (%/min) curves for the microalga Scenedesmus obliquus biomass grown in differentwastewaters (domestic, swine, poultry, brewery, dairy, AD cattle) resulting from the thermogravimetricanalysis in N2.

From Fig. 4.11, it is possible to observe different stages of decomposition in the DTG curves. Stage 1,

which corresponds to the removal of moisture, occurs up to a temperature of 180 °C. It is also important

to point out that S. obliquus grown in AD cattle and dairy wastewater present a higher moisture content,

64

when compared to the others. Stage 2 includes three devolatization zones, ranging from 210 to 450 °C:

the first zone refers to the intrinsic lipid decomposition (e.g. aldeydes and ketones) at 200 to 250 °C,

which is more perceptible for the S. obliquus grown in swine, AD cattle and brewery wastewaters; the

second zone corresponds to the decomposition of proteins and carbohydrates between 250 and 380 °C

- the decomposition of carbohydrates starts previously at 200-250 °C, merging with the previous zone -

for most microalgae, but for the S. obliquus grown in dairy wastewater, this zone seems to end at a lower

temperature (around 330 °C), since this zone merges with the following one; the last zone is related to

lipids decomposition, mainly associated to the break-down of hydrocarbon chains of fatty acids, taking

place at temperatures from 380 to 450 °C. However, for the S. obliquus cultivated in dairy wastewater

this zone starts at 330 °C. Finally, stage 3, which corresponds to the oxidation of the bio-char, is not

evident for any microalgae. These observations are in agreement with the studies reported by Ferreira

et al. (2015), Lopez-Gonzalez et al. (2014) and Kebelmann et al. (2013). Moreover, for the microalga

grown in dairy wastewater, a final decomposition stage is observed between 600 and 1050 °C, which

it can be assumed to be related to the volatile metal loss and carbonate decomposition (Ferreira et al.,

2015).

Scenedesmus grown in different media shows the same behaviour in inert conditions pyrolysis. Each

DTG curve presents two main peaks, which correspond, respectively, to the loss of moisture and the

three devolatization zones. The latter peak has the maximum located between 270 and 370 °C. This

maximum is associated to the carbohydrates and proteins decomposition. The microalgal biomass

cultivated in swine waste presents the highest peak, while the ones cultivated in domestic and AD cattle

wastewater have the lowest ones. This means that they present the highest and the lowest contents

in protein and carbohydrates, respectively. These results are in accordance with the values obtained

in section 4.2 for the production of bioH2, in which the microalga grown in swine waste produced more

bioH2, while the ones grown domestic and AD cattle both produced the lowest yields. Since the bioH2

production is directly related to the carbohydrate content of the microalgal biomass, microalgae with

higher carbohydrates normally generate higher bioH2 yields, and vice-versa.

4.3.1.2 HATR-FTIR (infrared spectroscopy)

S. obliquus grown in different waste media was also characterized by infrared spectroscopy. Fig-

ure 4.12 shows the FTIR spectra of the microalgae in study.

The FTIR spectrum peak at 3600-3000 cm-1 observed for all the S. obliquus biomasses corresponds

to the moisture content. It can be seen that all microalgae show small peaks at 3000-2800 cm-1 and

around 1740 cm-1. The more significant peaks appear at 1700-1500 cm-1 and 1150-950 cm-1. The S.

obliquus grown in dairy wastwater presents a considerably higher maxima for the first mentioned range of

wavenumbers, when compared to the other cultures; while the S. obliquus grown in domestic wastewater

65

Figure 4.12: FTIR spectra of the microalga Scenedesmus obliquus biomass grown in different wastewaters (do-mestic, swine, poultry, brewery, dairy, AD cattle).

presents a significantly lower maxima, for the latter mentioned range of wavenumbers. Table B.1 and

Figure B.1 in Appendix B show, respectively, the FTIR band assignments and common wavenumbers

for each component (Ferreira et al., 2015). The peaks located at 2925 and 1740 cm-1 correspond to

the lipids, the peak located at 1025 cm-1 corresponds to the carbohydrates and the peaks located at

1650 and 1540 cm-1 correspond to the proteins. For the microalga cultivated in domestic wastewater,

there is also a peak around 800 cm-1, which according to Table B.1 corresponds to nucleotides and

chlorophyll pigments. For all the microalgae biomass there is also a very high peak at 700-600 cm-1

which represents the S-O stretching vibration of sulphonic components (Pugazhendy, 2012). Finally,

according to the FTIR spectra of each microalga (Fig. 4.12), it is possible to conclude that the S. obliquus

cultivated in dairy wastewater is the most protein rich microalga, while the one cultivated in domestic

wastewater presents a significantly lower content in carbohydrates. On the other hand, the microalga

grown in swine, poultry and brewery waste all present very high carbohydrate contents. This observation

is also in accordance with the results obtained from the TGA study and the bioH2, since these three

microalgae achieved the highest production yields.

4.3.2 Characterization of the bio-oil and bio-char

Figure 4.13 presents the yields obtained for each pyrolysis product (bio-oil, bio-char and bio-gas).

From the results obtained, it is possible to conclude that the microalga grown in swine wastewater has

a greater potential for bio-oil production with a yield of 82.8%, followed by the S. obliquus grown in

brewery wastewater (64.1%). However, all the other cultures studied presented bio-oil yields lower than

66

the S. obliquus grown in synthetic medium. On the other hand, the microalga cultivated in poultry waste

achieved the highest yield of bio-gas production (46.5%). The bio-char yields do not present significant

changes, raging from 18.1 to 37.2%. The yields obtained for the S. obliquus grown in swine wastewater

were significantly higher when compared to the others, with bio-oil and bio-char production yields already

making 100%. This may be due to the composition of this biomass that enhances the bio-oil production,

namely the carbohydrate content, which according to TGA and FTIR data, is very high for this microalga.

No reference to this was found in literature, however. Other explanation for this result may also be the

low amounts of microalga available (less than 2 g), which is susceptible to a higher error.

Figure 4.13: Production yields of bio-oil, biochar and bio-gas from the pyrolysis of Scenedesmus obliquus biomassgrown in different wastewaters (domestic, swine, poultry, brewery, dairy, AD cattle). The values corre-spondents to Bristol were obtained from Silva et al. (2016).

4.4 BioH2 by dark fermentation and bio-oil through pyrolysis pro-

duction from S. obliquus grown in brewery wastewater

Finally, comparing the results achieved for both the bioH2 production and pyrolysis assays, it is

possible to conclude which route is more effective and brings more benefits for the valorization of the

biomass produced. For this, both sets of results were converted to energy units based on their Lower

Heating Value (LHV), which for bioH2 is 120 MJ/kg (Heywood et al., 1988) and for bio-oil from pyrolysis

of S. obliquus grown in synthetic medium is 23.6 MJ/kg (Ferreira et al., 2013b). This last value was

assumed for all the culture conditions, considering that any potential differences are negligible. For

bioH2, it was also necessary to convert the volume of H2 produced to mass weight, which was calculated

considering the ideal gas law, at atmospheric pressure and 30 °C (temperature of the bioH2 assays),

and the molar mass of H2 (2 g mol-1). The energy was then divided by the microalga mass in order to

determine the energy yields for each produced product (bioH2 and bio-oil, kJ kgbiomass-1) that are shown

67

in Table 4.10.

Table 4.10: Comparison between dark fermentation and pyrolysis process results. The values in bold correspondto the highest energy yields for each biofuel (bioH2 and bio-oil).

Medium Energy yield from bioH2 (kJ/kgbiomass-1) Energy yield from bio-oil (kJ kgbiomass

-1)Dairy wastewater 546 10699

Brewery wastewater 528 15131Swine waste 2885 19543Poultry waste 3410 7603

AD Cattle waste 450 -Domestic wastewater 428 9054

Bristol - 13592

According to these results, we can conclude that bio-oil gives higher energy yields (1 to 2 orders of

magnitude higher), when compared to bioH2, which may suggest this would be a better route since it

has a greater energy potential for each kg of S. obliquus biomass produced. However, this conclusion

must be further investigated taking into account the energy required for the production of both biofuels,

in order to correctly assess their economic potential and sustainability.

68

5Conclusions and Future Work

Contents

5.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

5.2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

69

70

5.1 Conclusions

The integrated microalgae-based approach studied in this work achieves simultaneous goals. First,

the combination of the microalgae growth with the brewery and CO2 effluents treatment, proved to be

possible, achieving removal rates up to 90% for nitrogen, 22 and 62% for phosphorus and organic load,

respectively, for a residence time of 3.5 days. Moreover, it corresponded to the maximum volumetric

productivity (0.2 gAFDW L-1 d-1). This concomitant process of treatment and production of valuable mi-

croalgae biomass enables the reduction in the cost of acquisition of nutrients, the environmental stress

of their synthetic production and the use of potable water for microalgae cultivation and, at the same

time opens the possibility for a new type of brewery effluent treatment. Second, using microalgae for

wastewater treatment is an advanced technology based on natural ecosystems, which presents no risk

for the environment, and allows the recovery and valorization of the biomass produced towards biofuel

production.

The supplementation of the culture medium with CO2 presents benefits for the microalgae growth and

the brewery wastewater treatment, since it prevents nutrient losses through pH mediated processes such

as phosphate precipitation and ammonia volatilisation. This ensures that most nutrients are available for

microalgal assimilation, preventing both carbon limitation and pH inhibition of the microalgal growth.

Regarding the bioH2 production by dark fermentation process, the hydrogen yields attained for S.

obliquus grown in wastewater media (390 and 378 mL H2/gVS, respectively, for biomass grown in swine

and poultry waste) were considerably higher than the ones using synthetic medium, with lower costs,

energy input, GHG emissions and minimal impact on freshwater supplies. Moreover, the biogas pro-

duced achieved high purity levels (the H2/CO2 ratio was up to 7 for swine waste), which is crucial for

reducing costs associated with purification. For the pyrolysis experiments, higher yields of bio-oil were

achieved for all the wastewaters in study (32-83%).

In conclusion, the possibility of producing biofuels while simultaneously achieving nutrient remedia-

tion from wastewaters has a great potential in improving the sustainability and profitability of the whole

system, representing one of the best strategies for the bioenergy future.

5.2 Future Work

Based on the results achieved by the present work, there is some aspects that need to be further

investigated. Regarding the growth of microalgae in brewery wastewater with CO2 supplementation,

it is important to further study the influence of different concentrations of CO2 on both the volumetric

productivity and treatment performance, specifically for lower residence times. To reduce cultivation and

harvesting costs, outdoor experiments must be performed in order to take advantage of solar irradiance,

71

and alternative harvesting methods such as electrocoagulation should be tested, respectively.

For bioH2 production, alternative pre-treatment processes can be researched and extraction of high-

value metabolites can be done prior to dark fermentation. In addition, the remaining liquid phase of the

fermentation trials can be characterized for further applications.

According to the results achieved by pyrolysis experiments, studies should be performed for further

reduction in the pyrolysis process duration. Moreover, the possibility of extracting high-value components

prior to pyrolysis should also be studied.

Furthermore, a LCA study should be performed for the integrated process of wastewater treatment

and biofuel generation to asses the environmental impacts of each stage of the process and evaluate

the sustainability and the profitability of the whole system.

72

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ABio-mitigation of CO2 emissions

Contents

A.1 Bio-mitigation of CO2 emissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

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A.1 Bio-mitigation of CO2 emissions

The emission of greenhouse gases is dramatically increasing in the atmosphere as a result of hu-

man activities and industrialization, reaching the highest value ever recorded in 2014. CO2 is the main

GHG, currently accounting for 60% of these emissions (Chagas et al., 2015). A large fraction of the an-

thropogenic CO2 emissions results from the combustion of fossil fuels for energy production, which are

expected to rise considerably in the coming years due to the increasing energy demand, especially in

the emerging economies of the developing world. The International Energy Agency predicts that global

energy related emissions of CO2 will reach 38 billion tonnes per annum in 2030, which corresponds

to 70% above 2002 levels, with two-thirds of the emissions coming from developing countries (Farrelly

et al., 2013).

Nowadays, it is widely recognised that high levels of CO2 in the atmosphere have detrimental effects

on the environment, being a positive driver of the greenhouse effect causing atmospheric temperatures

to rise. This leads to the melting of polar ice, rising sea levels, climate change and extreme weather

conditions (Farrelly et al., 2013).

CO2 emissions rates depend on world economic activity, the energy efficiency of the processes, and

the carbon content of the source of CO2 emissions. The Kyoto protocol required countries to reduce

their CO2 emissions, by decreasing fossil fuel consumption or by increasing net carbon sequestration in

terrestrial carbon sinks (Farrelly et al., 2013).

Beer is the main alcoholic beverage consumed in the world. During beer production, in addition to

nitrogen and phosphorous compounds present in liquid waste currents, CO2 is the release product from

the fermentative steps. Also flue gases are released from breweries from the production of steams for

the facilities. It can be estimated that beer production releases about 140 Mt of CO2 every year, almost

three times the amount of CO2 released from the production of biofuels, and its purity reaches 99%

(Chagas et al., 2015).

Currently, 55 - 65% of all anthropogenic CO2 emissions are removed from the atmosphere by natural

sinks, which includes interactions with the ocean as well as photosynthesis in vegetation on the land.

The ocean absorbs over 25% of the annual release of anthropogenic CO2, making it the principal natural

carbon sink for emitted CO2. However, increased levels of CO2 in sea water leads to the formation of

carbonic acid, in a process called ocean acidification. This process affects the marine organisms, such

as zooplankton, bacteria and benthos, at depths of 1000 m or more. Though the long term effects are

not fully predictable, it it believed that further increases in ocean acidity will lead to the demise of marine

life (Farrelly et al., 2013).

Global warming and carbon emissions have raised interest in strategies for sequestering carbon

released through the burning of fossil fuels. Human activities affect, directly and indirectly, almost half

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of the terrestrial biological carbon cycle, accounting for an estimated 9 GtC/yr emissions. As the rate of

release of CO2 exceeds the rate of natural carbon sequestration, there is an increase of its concentration

in the atmosphere, being estimated at a rate of 4.2 GtC/yr (Farrelly et al., 2013).

CO2 capture and storage technologies are considered as an integral part of the measures for GHG

emissions abatement. They include: (1) chemical reactions such as washing emissions with alkaline

solutions; (2) deep injection of sequestered gas (underground, in the ocean depths); and (3) biological

processes, using autotrophic organisms. However, the first two methods usually require the use of

chemicals and high energy costs, and the captured CO2 needs to be stored to prevent its return to the

atmosphere. To aggravate this situation, there is the risk of the occurrence of leaks from the reservoirs,

which can result in the release of large volumes of CO2. The last one has attracted much attention as

an alternative strategy with biological mitigation of CO2 being carried out by plants and photosynthetic

organisms (Arbib et al., 2014).

In this context, the use of microalgae seems to be an effective and environmental-friendly alternative

to reduce this gas in the atmosphere through photochemical reactions. Microalgae are able to grow

much faster than the terrestial plants and have the ability to efficiently uptake and use dissolved inor-

ganic forms of carbon in an aqueous environment. This process resultes in a CO2 conversion efficiency

about 10-50 times higher than terrestrial plants(Chagas et al., 2015; Arbib et al., 2014). However, rather

than being used strictly for CO2 capture and storage, algae have potential for CO2 capture and subse-

quent utilization. Carbon fixed by microalgae, through photosynthesis and other metabolic pathways, is

incorporated into biomass, particularly carbohydrates and lipids (Bhakta et al., 2015).

Photosynthesis is the process by which plants and algae utilize anthropogenic carbon to produce

biomass. Through this process, flue gases containing high concentrations of CO2 can be used to culti-

vate large amounts of biomass, assuming that microalgae consume an average of 2 g of CO2 for every

1 g of biomass produced (Chagas et al., 2015; Farrelly et al., 2013). They can be used to capture CO2

from three different sources: from the atmosphere, from power plants and industrial processes, and

from soluble carbonate. The capture of atmospheric CO2 is probably the most basic method to sink

carbon, relying on the mass transfer from the air to the microalgae during the process of photosynthesis.

However, because the atmospheric CO2 concentration is low (260 ppm), its capture is economically un-

feasible. On the other hand, CO2 capture from flue gas emissions from fossil fuel usage in power plants

that burn fossil fuels achieves better recovery due to the higher CO2 of up to 20%, and adaptability of

this process for both photobioreactor and raceway pond systems for microalgae production (Brennan

and Owende, 2010).

The selection of suitable microalgae strains for CO2 bio-mitigation is very important for its efficacy

and cost competitiveness. The desirable attributes for high CO2 fixation are: high growth and CO2

utilization rates; high tolerance of trace constituents of flue gases such as SOx and NO2; possibility for

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valuable by-products and co-products; ease of harvesting associated with spontaneous settling or bio-

flocculation characteristics; high water temperature tolerance to minimize cost of cooling exhaust flue

gases; possibility of conjugating this process with wastewater treatment (Brennan and Owende, 2010).

There are many advantages associated with utilizing microalgae to capture CO2 from flue gases of

power plants. For a flow rate of 0.3 L/min of air with 4% CO2 concentration, a carbon fixation rate of 14.6

g C m-2 day-1 can be achieved. This makes microalgae very well suited to carbon mitigation, since their

growth rates are able to keep up with the continuous flow of CO2 from the power plants (Farrelly et al.,

2013).

Fossil fueled plant flue gases typically contain varying levels of CO2, CO, NOx, SOx, N2, H2O and

also excess of O2, not used in combustion. This means that, before CO2 can be sequestered, it must be

removed from the gas stream to be purified. Biological carbon mitigation offers great potential as some

strains of microalgae are able to grow using flue gases evading the purification costs. The removal of

CO2 can occur before or after combustion, but both means of separation can be expected to reduce

overall plant efficiency by 8-12%. However, while direct use of flue gas can reduce the cost of pretreat-

ment, it imposes extreme conditions on microalgae and many strains are known to be critically inhibited

by high levels of these gases. Additionally, many studies have shown that elevated levels of CO2 in the

air streams increase productivity of microalgae till a certain limit (above 20% for many strains), from

which their growth is inhibited (Farrelly et al., 2013).

Bubbling carbon dioxide into the microalgae culture may not be effective enough, as bubble residence

time may be too short, and much can be lost in the atmosphere. Moreover, good sources of highly

concentrated CO2, such as from flue gas, are not always near enough to wastewater sources to justify

the cost of transfer and use. Therefore, the optimization of CO2 delivery through directly bubbling or

other means remains an engineering challenge (Christenson and Sims, 2011).

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BAuxiliary tables for FTIR analysis

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Table B.1: FTIR band assignments (Ferreira et al., 2015).

Figure B.1: FTIR spectra for microalgae in general.

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