Assessing and enhacing methane productivity from anaerobic digestion using cyanothece bg0011 as feedstock

ASSESSING AND ENHANCING METHANE PRODUCTIVITY FROM ANAEROBIC DIGESTION USING CYANOTHECE BG0011 AS FEEDSTOCK By NGUYET T.. Abstract of Dissertation Presented to the Graduate School of the

ASSESSING AND ENHANCING METHANE PRODUCTIVITY FROM ANAEROBIC

DIGESTION USING CYANOTHECE BG0011 AS FEEDSTOCK

By

NGUYET T M DOAN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

2017

© 2017 Nguyet T M Doan

To my beloved parents Luc Huu Doan and Tuyet Thi Bach Tu, my mother in-law Du Thi Tran, husband Tanh Tran Nhan Nguyen and my son Phuc Huu Doan Nguyen

ACKNOWLEDGMENTS

I would like to express the great gratitude to my academic supervisor Dr Pratap

C Pullammanappallil for advising, supporting, and encouraging me during my PhD study at University of Florida I admired him by his knowledge, gentleness, and

kindness The next person I would like to appreciate is my co-advisor, Dr Spyros A Svoronos for insightful ideas Both of them gave the best supports and led me to have the right way to accomplish my study

I would like to give special thanks to my PhD committee members for helping me

to enhance my knowledge and have a good vision for my PhD project Thanks to Dr Ray A Bucklin who supported me all the time for my PhD program Dr Edward J

Phlips promoted me to study for Cyanobacteria BG0011 and Dr Ben Koopman’s

flexibility advices were useful for my research

I would like to thank Dr Melanie J Correll, Dr Bruce A Welt, Dr Eric McLamore,

Dr Bin Gao from Department of Agricultural and Biological Engineering, UF for

supporting me in using centrifuge, lab space, gas detector, microscope, and nano and distilled water; Dr Mark Brenner, Department of Geological Science at University of Florida, who showed me how to collect soil samples and gave me sediment samples;

Dr Keelnatham T Shanmugam, Microbiology and Cell science at University of Florida, for helping me for using High Performance Liquid Chromatography (HPLC) to test

volatile fatty acid; Dr Kien Pham, Biomedical Science at University of Florida, for

helping me in using centrifuge and microscope I would like to send my thanks to my colleagues and my friends who spent time with me during my study and research at University of Florida: Yingxiu Zhang, Tung Chen, Caesar Moreira, Ziynet Boz,

Samriddhi Sinha, Sunchang Yang, Wen Ji, Na Wu, Wei Wu, Patrick Dube, Bailey

Slagle, Samuel Sammy, Nusheng Chen, Lidwine Hyppolite, Arnoldo Poveda, Steve

Miller, Saman Souri, Rubi Raymundo, Daniel Preston, Lane Paul, and Robin Snyder I

will also never forget the support and comradery of undergraduate students as we

worked together in a team: Kavir Maharaj, Kevin Gilroy, Bianca Gouvea, Nicole Giatti,

Quan Nguyen, Jennifer Jackson, Logan McCoy, Christian Svetics, Calvin Heimburg,

Raquel Bradley, James Clover, Dylan Wald, and Alexson Joseph I would like to thank

my Gainesville families for supports and treating me as their family member: Dr Khe

Chau’ family, Dr Dolores Krausche and grandma, Mr Lac’s family, Ms Lien Duong’s

family, Mr Phuoc’s family, Mr Roger Sedlacek’s family, and Mr Donny Dillon’s family I

would like send my beloved thanks to Vietnamese students in University of Florida

I would like to thank Dr Dorota Z Haman who gave me the best environment in

the ABE department for study I am proud to be part of the family of Department of

Agricultural and Biological Engineering, University of Florida, USA

Many thanks are given to An Giang University, Vietnam; Department of

Agricultural and Biological Engineering, University of Florida, USA for PhD research

assistantship; Office of Energy, Florida Department of Agricultural and Consumer

Services, USA; PhD Fellowship Program of Vietnam’s Ministry of Agriculture and Rural

Development; Vietnam’s Ministry of Education and Training

Finally, a personal record I would like to express all my gratitude to my mom, my

dad, my son, and my beloved husband who are always with me all the time and all their

life to support and encourage me on all my ways I would like to appreciate my friends

and my relatives in my hometown (An Giang) who supported my parents and my son

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS 4

TABLE OF CONTENTS 6

LIST OF TABLES 10

LIST OF FIGURES 11

ABSTRACT 15

CHAPTER 1 INTRODUCTION 18

Motivation 18

Problem Statement 23

Objectives 25

Research Approach 25

2 METHODOLOGY 28

Measurement of Optical Density of Microalgal Cell Culture Suspensions 28

Salinity Adjustment 28

Cell and Supernatant Dry Weight 29

Volatile Solid Measurement 33

Cell Specific Growth Rate Assessment 35

Extraction of Exopolysaccharide 35

Determination of Reducing Sugar 38

Prepare Dinitrosalicylic Acid Reagent 38

Calibration Curve for Reducing Sugar in Microalgae 38

Total Volatile Fatty Acid Measurement 42

Biochemical Methane Potential (BMP) Measurement 44

3 LONG-TERM SEMI-CONTINUOUS CULTIVATION OF CYANOTHECE BG0011 47

Rationale 47

Methods 49

Experimental Apparatus 49

Results and Discussions 52

Cell Mass, EPS, and pH Variation 52

BG0011 Growth 57

Summary 63

4 EFFECT OF NITROGEN SOURCES ON GROWTH OF CYANOTHECE BG0011 64 Rationale 64

Methods 67

Microalgal Strain and Medium Conditions 67

Experimental Design and Materials 67

Measurement and Analysis 69

Cell Growth Assessment 69

Results and Discussions 70

Cell Dry Weight and Specific Growth Rate 70

Monod Kinetics 74

Summary 80

5 EFFECT OF LIGHT:DARK DURATION ON CYANOTHECE BG0011 GROWTH AND EPS PRODUCTION 82

Rationale 82

Methods 83

Experimental Apparatus 83

Measurement 84

Data Analysis 85

Results and Discussions 85

Cell Growth 85

EPS 91

Summary 96

6 ANAEROBIC DIGESTION IN HIGH SALINITY CONDITIONS 97

Rationale 97

Methods 99

Anaerobic Digesters 99

Inoculum 99

Operation 102

Measurement 104

Results and Discussions 104

Salt-acclimation and Methane Production 104

Salt-acclimation Mechanism 113

Volatile Fatty Acids (VFAs) 114

Summary 119

7 BIOCHEMICAL METHANE POTENTIAL OF CYANOTHECE BG0011 CULTURES

120

Rationale 120

Methods 120

Results and Discussion 123

BMP Assays Using Inoculum from Salt Acclimated Mesophilic Digester 123

BMP of Assays Using Inoculum from Mesophilic Dairy Manure Digester 126

Summary 131

8 ASSESSING THE EFFICIENCY OF HYDROLYSIS AND BIOGASIFICATION OF CYANOTHECE BG0011 CULTURES 132

Rationale 132

Methods 134

Experimental Apparatus and Materials 134

Measurements 137

Results and Discussions 137

Temperature and pH 137

Enzymes 139

TiO2 and UV 143

BMP after Hydrolysis with TiO2 and UV 144

Summary 149

CONCLUSIONS 150

APPENDIX 153

A VFA CALCULATION 153

B LIGHT DARK DURATION 155

C MEDIUM FORMULA FOR CYANOTHECE BG0011 164

E EQUIPMENTS 167

F MEETING 185

LIST OF REFERENCES 186

BIOGRAPHICAL SKETCH 198

LIST OF TABLES

2-1 Absorbance substrates for the level of 1.5 mg/L 40

2-2 Absorbance substrates for the level of 0.75 mg/L 40

2-3 Absorbance substrates for the level of 0.45 mg/L 41

2-4 Absorbance substrates for the level of 0.15 mg/L 41

4-1 Effect of concentration of Nitrate (NO3-N) and Ammonium (NH4-N) on BG0011 cell growth 77

5-1 Experimental design 84

5-2 Specific growth rate and EPS 95

6-1 Sample site description 100

6-2 Salt acclimation for mesophilic and thermophilic digesters 105

7-1 Various concentrations of molasses and sugar as substrate for BMP 122

7-2 BMP assays using mesophilic dairy-manure digester inoculum 123

7-3 Methane yield in experiments with mesophilic dairy-manure digester inoculum

128

8-1 Photocatalytic experiments with TiO2 136

8-2 BMP assays for pretreatments with TiO2 and UV 136

8-3 Enzymatic hydrolysis with samples (5 mL EPS), phosphoric acid 2N (0.1 mL or 0.08 mL), heating for 30 min 142

8-4 Effects of UV and TiO2 exposure for 30 min on hydrolysis 143

8-5 VFA results from treatments 149

LIST OF FIGURES

1-1 A diagram of biogas production using saline microalgae 22

2-1 BG0011 suspension in centrifuge tubes 30

2-2 BG0011 suspension after centrifuge to separate cell pellet and supernatant 31

2-3 Cell pellet with different dilutions 31

2-4 BG0011 cell in cuvette to measure the optical density 32

2-5 BG0011 cell in aluminum dishes for dry weight 32

2-6 Correlation between cell dry weight and OD 33

2-7 Freeze dryer for drying EPS 36

2-8 Wet EPS of BG0011 after extraction by ethanol (200 proof) 37

2-9 BG0011 EPS after freeze-drying 37

2-10 Determination reducing sugars by DNS reagent solution 39

2-11 Calibration curve of sugar concentration (mg/mL) and OD 42

2-12 Determination of total volatile fatty acid concentration from anaerobic effluent using titration 44

3-1 Cyanothece BG0011 photo-bioreactor 50

3-2 Dynamic cell growth and EPS in the semi-continuous photo-bioreactor 51

3-3 Cyanothece BG0011 divide cell under the microscope 400X 53

3-4 Cyanothece BG0011 under microscope 400X 53

3-5 Cell mass and EPS dry weight from semi-continuous photo reactor 54

3-6 Exopolysaccharide extraction from Cyanothece BG0011 in a plane view 56

3-7 Exopolysaccharide extraction from Cyanothece BG0011 in a vertical view 57

3-8 Cell mass product in the first six days and specific growth rate at the batch photobioreactor 58

3-9 Biomass product in between 6th-19th days in the batch stage 58

3-10 Specific growth rate after the withdrawal of 3 L at 170th day 60

3-11 Specific growth rate after the withdrawal of 2 L at the 296th day 61

4-1 Schematic of experimental apparatus to test effects of nitrogenous compounds on BG0011 growth 68

4-2 Change in cell dry weight at different nitrate concentrations during a 6-day incubation period 71

4-3 Cell dry weight at different ammonium- concentrations during a six-day incubation period 73

4-4 Cell dry weight when grown on dinitrogen gas in the presence and absence of oxygen during a six-day incubation period 73

4-5 Effect of nitrate concentration of specific growth rate of BG0011 cells 75

4-6 Effect of ammonium concentration of specific growth rate of BG0011 cells 77

5-1 Cell dry weight during growth under light:dark of 3:21 hours 86

5-2 Cell dry weight during growth under light:dark of 6:18 hours 86

5-3 Cell dry weight during growth under light:dark of 13:11 hours 87

5-4 Cell dry weight during growth under light:dark of 18:6 hours 87

5-5 Cell dry weight during growth under light:dark of 21:3 hours 88

5-6 Cell dry weight during growth under light:dark of 24:0 hours 88

5-7 Analysis of variance of specific growth rate for various light:dark periods 90

5-8 Effect of light:dark duration on growth 91

5-9 Differences of mean EPS after 12 days of incubation 92

5-10 Tukey test to compare mean EPS after 12 days of incubation 92

5-11 Differences in mean EPS after 22 days of incubation 93

5-12 Tukey test to compare mean EPS after 22 days of incubation 94

6-1 Sample locations in North of Shell Mound Dredge, Levy County, Florida 100

6-2 Marine sediment samples and inoculum 102

6-3 Anaerobic digester D-1M, D-2M and D-3T set up 104

6-4 Salinity and methane yield versus time for the mesophilic digester D-1M 107

6-5 Methane accumulation over time in mesophilic digester 108

6-6 Methane accumulation over time in thermophilic digester D-3T 109

6-7 Salinity and methane yield per sugar versus time at thermophilic conditions 110

6-8 Salinity and methane yield versus time at sediment mesophilic conditions 112

6-9 Methane accumulation over time in sediment mesophilic digester D-2M 113

6-10 Volatile fatty acid in the mesophilic digester D-1M 115

6-11 Volatile fatty acid in the thermophilic digester D-3T 116

6-12 Volatile fatty acid in the sediment inoculated mesophilic digester D-2M 116

6-13 Volatile fatty acid accumulation in mesophilic fermenter 118

6-14 Volatile fatty acid in the maintained thermophilic fermenter 119

7-1 Methane accumulation in BMP assays using mesophilic salt acclimated anaerobic digester inoculum 125

7-2 Methane accumulation in BMP assays using dairy-manure digester inoculum 127 8-1 Hydrolysis efficiency by varying pH and temperature condition 138

8-2 Hydrolysis efficiency of enzymes with pH=4.5 139

8-3 Hydrolysis efficiency of enzymes with pH=5 141

8-4 Effect of UVB, UVC without TiO2, and UVC with TiO2 on hydrolysis 144

8-5 BMP for BG0011 after pretreatment with UV and TiO2 145

8-6 Pairwise comparison of BMP means among experiments 146

8-7 Differences of BMP means from hydrolysis experiments 147

8-8 Methane accumulation (mL at STP) for BG0011 after pretreatment with UV and TiO2 147

8-9 Methane yield (%) of treatments compared to theoretical estimates 148

8-10 Methane yield in treatments 148

B-1 Cell dry weight with 6:18 of light:dark hours in experiment B1 155

B-2 Cell dry weight with 6:18 of light:dark hours in experiment B2 155

B-3 Cell dry weight with 6:18 of light:dark hours in experiment B3 156

B-4 Cell dry weight with 16:8 of light:dark hours in experiment A1 156

B-5 Cell dry weight with 18:8 of light:dark hours in experiment A2 157

B-6 Cell dry weight with 18:6 of light:dark hours in experiment A3 157

B-8 Cell dry weight with 21:3 of light:dark hours in experiment C2 158

B-9 Cell dry weight with 21:3 of light:dark hours in experiment C3 159

B-10 Cell dry weight with 13:11 of light:dark hours in experiment D1 159

B-11 Cell dry weight with 13:11 of light:dark hours in experiment D2 160

B-12 Cell dry weight with 13:11 of light:dark hours in experiment D3 160

B-13 Cell dry weight with 24:0 of light:dark hours in experiment E1 161

B-14 Cell dry weight with 24:0 of light:dark hours in experiment E2 161

B-15 Cell dry weight with 24:0 of light:dark hours in experiment E3 162

B-16 Cell dry weight with 3:21 of light:dark hours in experiment F1 162

B-17 Cell dry weight with 3:21 of light:dark hours in experiment F2 163

B-18 Cell dry weight with 3:21 of light:dark hours in experiment F3 163

D-1 Glucose concentrations in experiments 166

D-2 Normality test for BMP from algae feedstock pretreatment by UV and TiO2 166

E-1 Prepare samples to test Volatile fatty acid using Syringe 1 mL and Sterile Syringe Filter 0.2 microliter Coring Incorporate 167

E-2 A demonstration of how to use the core to collect sediment samples 167

E-3 HLPC to measure Volatile fatty solid 168

E-4 Cyanothece BG0011 taken under the microscope 168

E-5 Different dilutions of Cyanothece BG0011 169

E-6 Calibration of optical density and mass dry weight with different dilution concentrations 169

E-7 Measure of Optical density of different dilution time of Cyanothece BG0011 170

E-8 Measure biogas from the BMP bottles 170

E-9 Cyanothece BG0011potocatalysis under UV light and TiO2 84911924 171

E-10 Spectrophotometer Fisher 171

E-11 Autoclave for sterilizing equipment 172

E-12 Centrifuge Beckman Model J2-21 172

E-13 Vortex Genie 2 Fisherbrand 173

E-14 Light intensity measurement 173

E-15 Gas bag 10L RESTEK catalog 22953 174

E-16 Equipment to seal the metal cap of BMP bottle 174

E-17 Cyanothece BG0011 growth on different nitrogen sources 175

E-18 Cyanothece BG0011 on the experiment at the light:dark cycle is 18:6 and 6/18 hours 175

E-19 Centrifuge 5415D at 10 rpm in 15 min 176

E-20 Cyanothece BG0011 on the experiment at the light:dark cycle is 21:3 hours 176

E-21 Cyanothece BG0011 growth on different nitrogen sources 177

E-22 Cyanothece BG0011 on the experiment at the light:dark cycle is 21:3 and 13:11 hours 177

E-23 Cyanothece BG0011 in the experiment light:dark cycle 18:6 hours 178

E-24 Cyanothece BG0011 growth on the different nitrogenous sources 178

E-25 Exopolysaccharide (EPS) freeze dry weight under VIRTIS lyotroll 179

E-26 Centrifuge to separation of Pellet and supernatant of Cyanothece BG0011 179

E-27 Dry weight Cell mass and EPS at 105oC, 24 hours 180

E-28 Exopolysaccharide of Cyanothece BG0011 after freeze dry 181

E-29 Determination of gas composition by Gas Chromatography Gow-Mac Instruments series 580 182

E-30 Microscope Digital Camera DP80 – Olympus 182

E-31 Gas detector CO2, O2 and CO, Bridge analyzers, Inc 183

E-32 Pretreatment BG0011 at 121oC, 15 min using metal bottles 183

E-33 Thermometer to measure temperature in the anaerobic digesters champers Omega 184

F-1 Supervisory committee on the day of dissertation defense 185

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

ASSESSING AND ENHANCING METHANE PRODUCTIVITY FROM ANAEROBIC

DIGESTION USING CYANOTHECE BG0011 AS FEEDSTOCK

By Nguyet T M Doan April 2017

Chair: Pratap C Pullammanappallil

Cochair: Spyros A Svoronos

Major: Agricultural and Biological Engineering

Seeking renewable and sustainable energy to replace depleting fossil fuels as well as addressing climate change is a global challenge Biofuel production from blue green algae (cyanobacteria) has gained much attention recently Cyanobacteria can be

not only used as biocatalyst but also as feedstock for biofuel production Cyanothece BG0011, the microalga used in this research, was isolated from a shallow lake in the

Florida Keys It is a saline, atmospheric nitrogen-fixing microalga, and produces

extracellular polysaccharide (EPS) The EPS can be potentially used as feedstock for biofuel and bio-products Anaerobic digestion is a mixed culture, energy efficient

process that can convert organic substrates to biogas fuel BG0011 culture solution may

be directly fed into anaerobic digester for biogas production without separating or

dewatering the cells and EPS Thus, this study was conducted to further establish

BG0011 as feedstock for mixed culture fermentation

Objectives of this study were to (1) investigate the effect of nitrogen sources

ABSTRACT

light:dark duration on BG0011 growth and EPS production, (3) determine the

biochemical methane production potential of BG0011 cultures, and (4) assess the

efficiency of enzymatic and a novel photocatalysis treatment on hydrolysis and

saccharification of BG0011 cultures

Cyanothece BG0011 was cultivated and harvested for over a year in an

air-sparged, artificially-illuminated (average light intensity 122 µmol photon m-2 s-1), continuous, 10 L closed photo-bioreactor (6- liter working volume).Cell density ranged between 1 and 1.5 g ash free dry weight (afdw)/L and EPS concentration between 2 and 3.0 g afdw/L

semi-BG0011 can utilize ammonia and nitrate as nitrogen sources for growth

Maximum specific growth rate on ammonia was 0.56 day-1 and nitrate 0.58 day-1 Cell biomass concentration reached the highest value of 1.8 g afdw/L when grown on 0.9

mM NO3-N Specific growth rate was lower at 10mM of NO3- There was low or no cell growth at 10 mM NH4 Specific growth rate was between 0.34 and 0.39 day-1 when grown on dinitrogen gas in the absence or presence of oxygen

Specific growth rate was found to be positively correlated to light duration

increasing from 0.021 day-1 when exposed to 3-hour light (and 21-hour dark) to 0.36 day-1 for 24-hour light Highest cell density (2.35 g afdw/L) and EPS concentration (2.6 g afdw/L) was recorded after 22 days of incubation under 18-hour light (and 6-hour dark) duration Specific growth rate under these conditions was 0.33 day-1

From biochemical methane potential studies, using entire BG0011 culture as feedstock, an average methane yield of 217.16 ± 37.3 mL at STP (g VS-1) was obtained

inoculum Compare this value to a theoretical value of 370 mL of methane at STP (g glucose-1) Assays that were incubated with conventional inoculum from a mesophilic dairy manure digester did not produce any methane even after 332 days of incubation

It was confirmed that inhibition of conventional inoculum was not due to salinity

Enzymatic treatment with a combination of amylase, hemicellulase, and cellulase enzymes produced 0.74 g reducing sugars per g ash free dry weight of supernatant, which was the highest yield obtained from treatment using enzymes individually and in combination Algae culture samples were exposed to UVB and UVC radiation for 30 minutes in the presence of titanium dioxide (TiO2) as photocatalyst, and to UVC for 30 minutes without TiO2 UVC exposure without TiO2 yielded 0.91-0.95 g sugar/L which was better than UVB (0.42 g sugar/L) and UVC (0.80 g sugar/L) exposure in the

presence of TiO2 Biochemical methane potential assays on UV treated samples

showed that treatment of BG0011 with UVC without TiO2 generated higher average methane of 292 ± 45.9 mL at STP (g VS-1) than other UV treatments

Results from this study indicated that it is applicable and feasible to feed BG0011 directly to salt-acclimated anaerobic digesters to produce biogas UVC treatment of BG0011 cultures enhances methane yield The EPS produced by BG0011 can be enzymatically saccharified using commercial enzymes Additionally, BG0011 can be grown in nitrate and ammonium

CHAPTER 1 INTRODUCTION

Motivation

Fossil fuel sources including oil, coal, and natural gas supply about 85% of

energy demand worldwide (Quintana et al., 2011) However, these sources are

gradually depleting Another serious problem is that using fossil fuel can cause air

pollution and green-house gas emissions Thus, new energy sources that are

sustainable and producing less air pollution are needed As alternatives, biofuels such

as biogas, bio-ethanol, bio-methanol, bio-butanol, biodiesel, and bio-hydrogen can help

mitigate the energy problems These biofuels can be produced from renewable biomass

resources as plants and organic residuals and can potentially help reduce the fossil fuel

usage and net CO2 emission Recently, the first generation feedstocks from food crops

such as corn, sugar beet, sugarcane soybean, and other edible oil seeds have been

considered for biofuel production However, these feedstocks compete with food

production and consequently can lead to rising food prices and affect food security

Recognizing limitations of the first generation feedstocks, conversion processes were

developed to utilize the second generation feedstocks like agricultural residuals, food

wastes, forest residuals, and energy crops (wood/grasses) Nevertheless, biofuel

production using these feedstocks faces technical and economic challenges due to lack

of sufficient arable land to grow energy crops as well as high costs for pretreatments

and low efficiency of conversion during processing

Overcoming limitations of 1st and 2nd generation feedstocks, third generation

feedstocks that use microalgae as feedstock source or biocatalyst for biofuel conversion

biofuel industry is that they can be easily cultivated using simple, low-cost systems; exhibit rapid growth potential; and can be cultivated in saline/brackish water and

wastewater Microalgae’s cell wall has a no lignin and low cellulose content, which makes it more easily degradable than woody/grassy feedstocks in thermal gasification, pyrolysis, and anaerobic digestion processes (Harun et al., 2010; Vergara-Fernández et al., 2008; Zhu et al., 2014) Microalgae can also be used as biocatalysts to produce various biofuels including hydrogen, diesel, ethanol, and bio-products like lipids,

nutraceuticals, pigments, and polysaccharides (Jajesniak et al., 2014; Parmar et al., 2011) Among microalgae, cyanobacterial strains are promising candidates for biofuel or bio-product production because they can grow under saline conditions and are able to fix nitrogen (Zehr, 2011) and produce extracellular products like polysaccharides (De

Philippis and Vincenzini, 1998; Hays and Ducat, 2014; Kaushik et al., 2013)

As a contribution to clean energy field, this study focuses on the use of

Cyanothece BG0011 as a biocatalyst for extracellular polysaccharide (EPS) production,

and the utilization of the cellular biomass together with EPS as feedstock for biogas

production in an anaerobic digester Cyanothece BG0011 was isolated from a shallow

lake in the Florida Keys by Dr Edward Phlips, School of Fisheries and Aquatic

Sciences, University of Florida It is a saline microorganism, capable of atmospheric nitrogen (N2) fixation, and produces extracellular polysaccharide (Phlips et al., 1989)

Previously, this microorganism was classified as a Synechococcus strain, but genomic

sequencing and phylogenetic analysis recently identified it as a Cyanothece (Slagle, 2016) Recent investigations on this microorganism has focused on the effect of salinity, light, carbon dioxide, and phosphorus abundance on its growth and extracellular

polysaccharide production in batch cultures (Moreira, 2014; Phlips et al., 2016; Slagle, 2016; Zhang, 2014) Importantly these investigations showed that the cells could grow under salinity levels as high as 75 psu (Slagle, 2016) The extracellular polysaccharide produced with air-sparging and air enriched with 1% CO2 sparging was shown to be made up of primarily glucose, mannose and xylose with lesser amounts of rhamnose, fucose, galactose, galacturonic acid, glucuronic acid and arabinose (Moreira, 2014)

These studies show that Cyanothece BG0011 is a promising candidate for biofuel and

bio-product production

The research theme presented in this dissertation is to investigate the feasibility of

utilizing the Cyanothece cultures along with the EPS as feedstock in anaerobic

digesters for biogas production or for production of organic acids Anaerobic digestion is

a biochemical process that converts organic compounds (like carbohydrates, fats, and proteins) to biogas in an oxygen-free environment through the concerted, syntrophic action of mixed culture of naturally occurring microorganisms Biogas produced from anaerobic digestion process is primarily a mixture of CH4 (50-60%) and CO2 (40-50%) The benefit of anaerobic digestion is that methane may be produced from various

feedstocks like organic wastes (agricultural residues, and livestock, industrial and

municipal wastes) and/or dedicated energy crops It is an attractive option for waste management as it reduces odors, flies, and pathogens, in addition to producing valuable by-products like biogas and bio-fertilizer (Wilkie, 2005) The complete mineralization of organic compounds in an anaerobic digester proceeds through a sequence of

biochemical steps: (1) hydrolysis (de-polymerization of large macromolecules to soluble monomers by extracellular enzymes), (2) acidogenesis (production of mixtures of

organic acids like acetic, propionic, and butyric acids from solubilized monomers), (3) acetogenesis (conversion higher chain organic acids to acetic acid), and (4)

methanogenesis processes (Garcı́a-Ochoa et al., 1999; Park et al., 2005) It is

conceivable to terminate the process at acidogenesis step so as to produce organic acids Organic acids are higher-value products compared to biogas Figure 1-1 below is

a schematic diagram depicting the utilization of Cyanothece cultures in an anaerobic

digester to produce biogas-biofuel The biogas may be used on site for electricity

production or for heat, or it can be cleaned and scrubbed-off its carbon dioxide content and injected into natural gas pipelines for utilization elsewhere The residue from

digestion process can be land applied Since the cells are capable of atmospheric nitrogen fixation, the residue which will be primarily cell debris may be considered to be

a nitrogen fertilizer It may be also possible to grow the Cyanothece in wastewater to

utilize pollutants like ammonia, nitrate and phosphates, thereby treating the wastewater

Figure 1-1 A diagram of biogas production using saline microalgae.

Problem Statement

Further research is needed to establish this saline microalga as a feedstock for biofuel production Thus, this research study addresses the following questions

1 To utilize this microalga for wastewater treatment or to culture the

microorganism in wastewater, among other requirements it is important that the cells should possess the ability to grow in the presence of ammonia and nitrate So the question is: how do other nitrogen sources (like ammonia and

nitrate) affect the growth of Cyanothece?

2 Even though the species was isolated from Florida Keys where the light:dark duration is almost equal throughout the year, utilization of the species in Northern Latitudes requires growth under both reduced as well as extended light duration So the second question is: what is the effect of duration of light:dark cycle on the growth and EPS production capabilities?

3 It is proposed to feed the BG0011 cultures together with the EPS directly into

an anaerobic digester for biogas production, which means it is necessary to determine the methane potential of this feed Anaerobic digestion can be carried out at two temperature regimes namely mesophilic (~38°C) and thermophilic (~55°C) and the microbial populations at these temperatures is different, so it is also necessary to investigate biogasification efficiency at these temperatures Furthermore, as natural gas (methane) prices are low, it may be more economical to produce other products instead of methane The complete mineralization of organic compounds in an anaerobic digester goes

through an intermediate volatile organic acid (acetic, propionic and butyric acid) step It may be more economical to stop the anaerobic digestion

process at mixed acid fermentation step and use the organic acids to

produce other high value products Therefore, the next question is what is the biochemical methane potential (BMP) and organic acid potential of BG0011 feedstock? Is the efficiency and yield of these potentials affected by anaerobic digestion temperature (mesophilic vs thermophilic)?

4 Several biobased products and fuels can be produced starting with sugar (glucose) as raw material Processes have been developed for saccharifying terrestrial ligno-cellulosic biomass A common method is thermal treatment followed by enzymatic saccharification Commercially available enzyme cocktails are employed in this step It is possible that the EPS in BG0011 may also be saccharified using commercial enzymes More recently it was shown that a photocatalytic pretreatment process enhanced biogas

production, implying that this pretreatment may have enhanced hydrolysis or saccharification So the next question addressed is: can the EPS be

hydrolyzed and saccharified using enzymatic or other processes?

Objectives

Experiments were conducted to answer the questions above with specific

objectives as outlined below

Objective 1: To assess the effect of ammonia- and nitrate- nitrogen sources on growth

of Cyanothece BG0011 and compare it to growth on atmospheric

nitrogen in the presence and absence of oxygen

Objective 2: To assess the effect of light:dark duration over a 24-hour period on

growth of Cyanothece BG0011 and EPS production

Objective 3: To determine the biochemical methane and organic acid production

potential of Cyanothece BG0011 cultures containing EPS at mesophilic

temperature using inoculum from a conventional anaerobic digester, using inoculum adapted to seawater salinity

Objective 4: To assess the efficiency of thermal, enzymatic and photocatalytic

hydrolysis and saccharification treatment of Cyanothece BG0011

Research Approach

In order to achieve the above objectives, this study used the following

approaches to conduct the research Methods are described in subsequent chapters

 To address all objectives, it was essential that BG0011 cultures be readily

available for use as inoculum for cell growth experiments as well as in large quantities for use as feedstock anaerobic digestion, mixed-acid fermentation,

hydrolysis, and saccharification experiments Therefore, Cyanothece BG0011

was cultured semi-continuously in a 6-L working volume (10 L total volume)

photobioreactor by sparging air and using a 13:11 hour light:dark cycle A

specified volume of mixed liquor was withdrawn and the same volume of growth medium was added Optical density and the EPS content of mixed liquor were measured The withdrawal volume and frequency were varied

 To address objective 1, Cyanothece BG0011 was cultured in sealed 500 mL

flasks with gas bags attached and placed on a shaker The gas bag contained a mixture of CO2 and argon when ammonia and nitrate were used as nitrogen sources, and contained N2 and CO2 when dinitrogen gas served as nitrogen source Optical density and cell dry weight were measured Each treatment was carried out in triplicate

 To address objective 2, Cyanothece BG0011 was cultured in 500 mL bottles

sparged with air The light:dark duration was varied, and optical density and cell dry weight were monitored Each treatment was carried out in triplicate

Microbial ecology of anaerobic digester inocula is dependent on operating temperature regime and moreover since the saline BG0011 culture would be used as directly as feedstock for anaerobic digestion, it was necessary to

develop salt-acclimatized digesters at both mesophilic and thermophilic

temperatures Two five-liter digesters were started up at mesophilic and

thermophilic temperature using mixed liquor from a dairy manure digester and food waste digester respectively These were fed table sugar dissolved in a nutrient solution containing sodium chloride along with other micro and macro nutrients The amount of salt (sodium chloride) was gradually increased as the microbial cultures became acclimated The methane production rate, pH and

volatile organic acids were monitored in the digesters Once the salinity reached 3.8%, it was maintained at this value A third five-liter digester, which was started

up using marine sediment, was acclimated for methanogenesis at mesophilic temperature and monitored like other two digesters Two other salt acclimatized digesters were deliberately allowed to become acidic by feed overloading These were operated at mesophilic and thermophilic temperatures respectively Mixed liquor from these digesters was inoculum for organic acid potential assays

 To address objective 3, the biochemical methane potential of the culture was measured in sealed 280 mL serum bottles that were incubated with aliquot of BG0011 culture and mixed liquor from salt acclimated digesters and marine sediment digester BMP at both thermophilic and mesophilic temperatures was measured CH4 and CO2 composition of biogas produced in assays was

analyzed Similar experiment was carried out to determine organic acid

production potential except that inoculum from a deliberately acidified five-liter salt acclimated digester was used in these experiments The volatile acid was measured at the end of the incubation period

 To address objective 4, Cyanothece BG0011 was pretreated using conventional

methods like heat and enzyme addition and a combination of the two The

efficiency of pretreatment was compared by measuring the sugar concentration

in the treated mixture A combination of enzymes like cellulase, hemicellulase, and amylase were used for pretreatment In addition, a photocatalytic method that was previously shown to be beneficial for improving anaerobic digestibility of microalgae biomass was also tested

CHAPTER 2 METHODOLOGY

Some common experimental and analytical procedures employed for research

presented in this thesis is described in this Chapter Description of experimental

apparatus used in specific investigations is described in later chapters

Measurement of Optical Density of Microalgal Cell Culture Suspensions

- 1 mL of cell suspension withdrawn from photo-bioreactor was placed in a 2.0 mL

Eppendorf tube and centrifuged for 15 min at 10,000 rpm Cell pellet was

separated from supernatant Supernatant was poured into another cuvette to

measure pH and salinity

- 1 mL DI water was added to cell pellet and re-suspended Optical density (OD)

for the cell resuspension was measured by a Fisher Scientific Educational

spectrophotometer (λ=540 nm) If cell OD was higher than 0.6, then sample was

diluted to make the measurement more accurate Optical density was recorded

daily or every two days The equation for cell concentration estimation is

𝑂𝑝𝑡𝑖𝑐𝑎𝑙 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ∗ 𝑂𝐷 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑚𝑒𝑛𝑡 (2-1)

Salinity Adjustment

Salinity of the supernatant was measured using salinity refractometer First, rinse

the glass lens of salinity refractometer with DI water, then test blank with DI water Put

few drops of supernatant on the glass lens, close the clear plastic cover and look

through the eyepiece to read salinity in the internal scale The internal scale ranges

from 0 to 100% salinity Rinse glass lens after use with DI water

Due to evaporative losses, the salinity increased gradually in photo-bioreactors and was maintained around 3.5% by adding autoclaved DI water

𝑊𝑎𝑡𝑒𝑟 𝑎𝑑𝑑𝑒𝑑 =(𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑠𝑎𝑙𝑖𝑛𝑖𝑡𝑦− 3.5)∗ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑐𝑢𝑙𝑡𝑢𝑟𝑒 𝑖𝑛 𝑝ℎ𝑜𝑡𝑜𝑏𝑖𝑜𝑟𝑒𝑎𝑡𝑜𝑟

Cell and Supernatant Dry Weight

After a growth period, BG0011 suspension and supernatant were dried to

measure cell dry weight and identify the co-efficient of correlation between OD and cell dry weight

1 100 mL BG0011 was centrifuged for 30 min at 12,000 x g Pellet and supernatant was separated

2 Supernatant was poured into another tube

3 Rinse cell pellet with deionized water (DI), centrifuge and remove supernatant until salinity of rinse water (supernatant) was zero Figures 2-1, 2-2, 2-3, 2-4, and 2-5 show BG0011 suspension in centrifuge tubes, separation of cell pellet and supernatant, cell pellet, measurement of optical density, and dry weight

measurement, respectively

4 Then, re-suspend cell pellet in 100 mL DI water and vortex well

5 Dilute the BG0011 suspension to different appropriate concentrations as needed, and absorbance of the suspensions was measured with a spectrophotometer at

540 nm wavelength

6 Dry empty 50 mm diameter aluminum dishes in an oven, weigh and store them in

a desiccator containing drierite (anhydrous CaSO4)

7 20 mL of different concentrations of BG0011 was placed in the empty aluminum dishes and dried at 105oC until constant weight (usually around 24 to 48 hours)

8 The dried samples were placed in the desiccator for 10 min to cool down and then weighed

9 Steps 5 to 8 were carried out for the supernatant separated in step 2

Figure 2-1 BG0011 suspension in centrifuge tubes

Credits: photo courtesy of author

Figure 2-2 BG0011 suspension after centrifuge to separate cell pellet and supernatant

Credits: photo courtesy of author

Figure 2-3 Cell pellet with different dilutions

Credits: photo courtesy of author

Figure 2-4 BG0011 cell in cuvette to measure the optical density

Credits: photo courtesy of author

Figure 2-5 BG0011 cell in aluminum dishes for dry weight

Credits: photo courtesy of author

Volatile Solid Measurement

After dry weight measurement, dried BG0011 cell biomass and supernatant samples were burned in a muffle furnace at 550oC and the ash residue weighed

Calculations to determine volatile solids (VS) cell dry weight (CDW), EPS dry weight (EPS DW) and the correlation between OD and CDW are given below (Figure 2-6)

Figure 2-6 Correlation between cell dry weight and OD

The ash free cell dry weight (CDW), also afdw, was calculated by equation:

𝐶𝐷𝑊 =[(𝑊2−𝑊1)−(𝑊3− 𝑊1]∗ 3.5

where

W1: weight of empty aluminum dish

W2: weight of Aluminum dish + dried 20 mL BG0011 cell culture

y = 0.5517xR² = 0.9919

W3: weight of Aluminum dish + ash residue after burning dry sample (W2 above)

in muffle furnace at 550°C

S: the salinity of BG0011 sample Salinity from BG0011 suspension was

corrected to 3.5% salinity

V: volume of initial sample that was dried (20 mL)

Unit of cell dry weight is g/L

- Dry weight of EPS was calculated by the following equation

𝐸𝑃𝑆 𝐷𝑊 = [(𝑊4−𝑊1)−(𝑊5−𝑊1)]∗ 3.5

𝑉×𝑆 × 100 − 𝑊6

𝑉 ×1000 (2-4) where

W1: weight of empty aluminum dish

W4: weight of aluminum dish + dry 20 mL BG0011 supernatant (EPS)

W5: weight of aluminum dish + ash residue remaining after dry sample (W2 above) was burned in muffle furnace at 550°C

W6: the weight loss when dried 20 mL volume culture media was burned in muffle furnace at 550°C (accounted for minerals in media that may hydrate when dried)

V: volume of initial sample that was dried (20 mL)

S: the salinity of BG0011 supernatant

Salinity from BG0011 suspension was corrected to 3.5% salinity

Unit of EPS is g/L

Cell Specific Growth Rate Assessment

Computation for cell growth used the following equations

where X(t) is the BG0011 cell dry weight at the time t, X0 is the dry weight at the

beginning of the experiment, and μ is the specific growth rate The Monod equation was used to estimate maximum specific growth rate and half-saturation constants for the limiting substrate

𝜇 =𝜇𝑚𝑎𝑥 ∗𝑆

where μ is the specific growth rate, μmax is the maximum specific growth rate, S is

substrate, and KS is the substrate half-saturation coefficient (corresponding to 1/2 µmax, constant of substrate uptake) The components of the exponential growth and Monod equation were estimated using the trend line and Solver function in MS-Excel

Extraction of Exopolysaccharide

Exopolysaccharides (EPS) extraction uses an alcohol extraction method (Chi et al., 2007; Patel et al., 2013) as follows

1 Ethanol 200 proof (100%) was stored in a refrigerator at 4°C overnight

2 BG0011 suspension was centrifuged at 10000 rpm for 30 min to separate supernatant and cell pellet

3 Ethanol was mixed with supernatant (500 mL) in the ratio 1:1 by volume in a

2000 mL beaker placed in an ice bowl containing ice and using glass rod to stir the mixture EPS precipitated as wet gel

4 The gel was taken out the beaker and was dissolved in 250 mL DI water to remove the salt remaining in the gel

5 Then 500 mL ethanol 200 proof was added again to precipitate wet gel The solution was stored in the refrigerator at 4°C overnight

6 Precipitated EPS gel was removed, placed in the glass tray, and covered by plastic food wrap with holes to release moisture

7 Then the precipitated EPS was freeze-dried under VIRTIS lyotroll for 4 days

at -80°C (Figure 2-7)

Figure 2-7 Freeze dryer for drying EPS

Credits: photo courtesy of author

8 The remaining alcohol and water was discarded to obtain EPS (Figures 2-8 and 2-9)

Figure 2-8 Wet EPS of BG0011 after extraction by ethanol (200 proof)

Credits: photo courtesy of author

Figure 2-9 BG0011 EPS after freeze-drying

Credits: photo courtesy of author

Determination of Reducing Sugar

Reducing sugar from Cyanothece BG0011 was determined by DinitrosaIicyIic Acid

Reagent (DNS) method (Lindsay, 1973; Miller, 1959; Saqib and Whitney, 2011), which

is shown in Figure 2-10 Dinitrosalicylic acid reagent for reducing sugar (Miller, 1959) was composed of dinitrosalicylic acid, Rochelle salt, phenol, sodium bisulfite, and

sodium hydroxide

Prepare Dinitrosalicylic Acid Reagent

Dissolve 10 g of 3,5-dinitrosalicylic acid in 200 mL of 2 M NaOH solution and 500

mL DI water in a brown bottle (to avoid the light), at room temperature Mixture liquid was stirred by a magnetic stir bar and 300 g Rochelle Salt (Na-K-tartrate) was added and solution made up to 1000 mL with DI water Protected this solution from CO2 in well-filled bottles (Lindsay, 1973)

Calibration Curve for Reducing Sugar in Microalgae

Calibration curve for reducing sugar is a standard curve of sugar that is used for determining unknown concentration of sugar in the substrate Three kinds of sugar consisting of glucose, arabinose, and galacturonic acid were used as a standard for calibration curve at four concentrations of 0.15, 0.45, 0.75, and 1.5 mg/mL The sugar solutions were combined to give 13 different combinations of sugar as shown in Figure 2-3 and Tables 2-1, 2-2, 2-3, and 2-4 Procedure for analysis was as follows

1 1 mL of sample or 1 mL DI water (blank) was placed in the 10 mL glass tube

2 2 mL DNS reagent was added

3 Close the top of tube by stopper and vortex liquid mixture well Then, tubes were heated in a boiling water beaker for 4-5 minutes and cooled to room temperature

4 After that, 2 mL DI water was added and vortexed to mix the solution well

5 1 mL of mixture solution was placed in the cuvette and optical density measured

at 570 mm on the colorimeter by a Fisher Scientific Educational

Spectrophotometer

6 The DI water and DNS reagent solution was first set at zero for the blank

solution Then, mean of combinations of each concentration of sugar was used to draw a curve to show the relationship between color (absorbance) and

concentration of sugar (Figure 2-11) The correlation coefficient of OD and sugar concentration from calibration was y = 0.462x, R2= 0.9966

Figure 2-10 Determination reducing sugars by DNS reagent solution

Credits: photo courtesy of author

Table 2-1 Absorbance substrates for the level of 1.5 mg/L

Types Glucose Arabinose Galacturonic

acid

Absorbance Spectrophotometer

Table 2-2 Absorbance substrates for the level of 0.75 mg/L

Types Glucose Arabinose Galacturonic

acid

Absorbance spectrophotometer