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The Growth of Microalgae on Anaerobic Digestate for Wastewater Treatment and

Biofuel Production

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The Growth of Microalgae on Anaerobic Digestate for Wastewater Treatment and Biofuel
Production

By

MATTHEW BRIAN PADDOCK

THESIS

Submitted in partial satisfaction of the requirements for the degree of

MASTERS OF SCIENCE

in

Biological Systems Engineering

in the

OFFICE OF GRADUATE STUDIES

of the

UNIVERSITY OF CALIFORNIA

DAVIS

Approved:

_______________________
Jean S. VanderGheynst, Chair

_______________________
Ruihong Zhang

_______________________
Annaliese K. Franz

Committee in Charge

2019

i




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Table of Contents
Abstract............................................................................................................................................v
Acknowledgments ......................................................................................................................... vii
List of Figures ................................................................................................................................ viii
List of Tables ................................................................................................................................... xi
1. Chapter 1 - Introduction and Research Objectives ................................................................... 1
1.1. Introduction ........................................................................................................................ 1
1.1.1. Microalgae .................................................................................................................... 5
1.1.2 History of Microalgae Biofuels ................................................................................... 11
1.1.2. History of Microalgal Wastewater Treatment .......................................................... 22
1.2. Research Goal and Objectives .......................................................................................... 29
Objective 1: (Chapter 2) Quantify Microalgal Growth on Wastewater ............................... 30
Objective 2: (Chapter 3) Identify the Microbiome in the Microalgae/Wastewater System30
Objective 3: (Chapter 4) Identify Way to Induce Lipid Production in Microalgae ............... 31
1.3. Hypothesis......................................................................................................................... 32
2. Chapter 2 - Growing Microalgae on Stripped Food Waste Permeate .................................... 34
2.1. Background ....................................................................................................................... 34
2.2. Materials and Methods .................................................................................................... 42
2.2.1. Microalgae Strain Selection ....................................................................................... 42
2.2.2. Wastewater Collection and Preparation ................................................................... 42
2.2.3. Microalgae Cultivation ............................................................................................... 43
2.2.4. Biomass Quantification .............................................................................................. 46
2.2.5. Nutrient Quantification ............................................................................................. 46
2.2.6. Growth Models .......................................................................................................... 47
2.2.7. Data Analysis .............................................................................................................. 48
2.3. Results ............................................................................................................................... 49
2.3.1. Summary .................................................................................................................... 49
2.3.2. Growth on Raw SFWP ................................................................................................ 50
2.3.2.1. Algae Growth ....................................................................................................... 50
2.3.2.2. Nutrient Removal ................................................................................................ 55
2.3.2.3. Analysis ................................................................................................................ 56
2.3.3. Growth on VSF-SFWP ................................................................................................. 58
2.3.3.1. Algae Growth ....................................................................................................... 58

ii
 2.3.3.2. Nutrient Removal ................................................................................................ 63
2.3.3.3. Analysis ................................................................................................................ 65
2.3.4. Growth on SSF-SFWP ................................................................................................. 67
2.3.4.1. Algae Growth ....................................................................................................... 67
2.3.4.2. Nutrient Removal ................................................................................................ 70
2.3.4.3. Analysis ................................................................................................................ 81
2.3.5. Discussion....................................................................................................................... 82
3. Chapter 3 - Identifying the Microbiome in the Microalgae-Wastewater System ................. 85
3.1. Background ....................................................................................................................... 85
3.2. Materials and Methods .................................................................................................... 93
3.2.1. Microalgae Strain Selection ....................................................................................... 93
3.2.2. Wastewater Collection and Preparation ................................................................... 93
3.2.3. Microalgae Cultivation ............................................................................................... 93
3.2.4. Biomass Quantification .............................................................................................. 95
3.2.5. Nutrient Quantification ............................................................................................. 95
3.2.6. DNA Extraction, Quantification, and Verification..................................................... 96
3.2.7. Microbiome Analysis.................................................................................................. 96
3.2.8. Quantitative PCR ........................................................................................................ 96
3.2.9. Predictive Metagenomics .......................................................................................... 98
3.2.10. Data Analysis ............................................................................................................ 98
3.3. Results ............................................................................................................................... 99
3.3.1. Microalgae Growth .................................................................................................... 99
3.3.2. Nutrient Removal ..................................................................................................... 104
3.3.3. Microbial Community Analysis ................................................................................ 109
3.3.4. Predictive Metagenomics ........................................................................................ 138
3.4. Discussion........................................................................................................................ 146
3.5 Conclusion ........................................................................................................................ 158
4. Chapter 4 - Optimizing Lipid Production of Microalgae in Wastewater .............................. 161
4.1. Background ..................................................................................................................... 161
4.2. Materials and Methods .................................................................................................. 167
4.2.1. Strain Selection ........................................................................................................ 167
4.2.2. Wastewater Collection and Preparation ................................................................. 167
4.2.3. Microalgae Cultivation Sodium Acetate Induction Experiments ........................... 167

iii
 4.2.4. Microalgae Cultivation for Salt Stress Experiments ............................................... 169
4.2.5. Synthetic Seawater .................................................................................................. 169
4.2.6. Biomass Quantification ............................................................................................ 170
4.2.7. Total and Neutral Lipid Quantification ................................................................... 170
4.3. Results ............................................................................................................................. 172
4.3.1. Lipid Induction with Sodium Acetate ...................................................................... 172
4.3.1.1. Lipid Accumulation ............................................................................................ 172
4.3.1.2. Analysis .............................................................................................................. 173
4.3.2. Lipid Induction Through Salt Stress ......................................................................... 174
4.3.2.1. Lipid Accumulation ............................................................................................ 174
4.3.2.2. Analysis .............................................................................................................. 176
4.4. Discussion........................................................................................................................ 177
5. Chapter 5 - Conclusion ........................................................................................................... 180
6. Chapter 6 - Future Work ........................................................................................................ 184
7. Chapter 7 - References ........................................................................................................... 187
8. Chapter 8 - Appendix.............................................................................................................. 208

iv
Abstract

The use of microalgae for combined wastewater treatment and biofuel production has shown

promise as a viable method for reducing oleaginous biomass production costs. Little work has

been done to characterize the complex interactions that exist between microalgae,

wastewater, and bacteria contained in these environments. This thesis focuses on three key

areas: quantifying how microalgae grow and treat wastewater in semi‐continuous conditions,

identifying the microbial community associated with microalgae in wastewater, and

determining methods of lipid induction in microalgae growing on wastewater for biofuel

production.

The results suggest that (1) microalgae offer an efficient method of wastewater

treatment in semi‐continuous conditions, (2) biomass production rates can be influenced by the

microbial community that develops in the wastewater, and (3) lipid induction processes can

radically increase the biofuel potential of this system in a short period. Microalgae grown on

sterilized wastewater had maximum biomass productivity of 600 mg/L/d in 50% (v/v) semi-

continuous conditions while removing 100% of N-NH4, 92.2% of TN, and 88.1% of sCOD. The

microbial community that grew alongside microalgae in wastewater was found to be

dominated by Proteobacteria. Despite this, each microalgae-based community that developed

was unique and a large variation in the final microalgae biomass concentration (0.114 g/L to

0.890 g/L) was seen across the nine bioreactors. Specific OTUs were found to correlate with

different parameters such as microalgae biomass concentration, bacteria biomass

concentration, and nutrient removal rates of TN, N-NH4, and sCOD. Induced lipid accumulation

in microalgae growing in wastewater showed that a two-stage process was effective. The

v
addition of both sodium acetate and sodium chloride increased neutral lipid content by 73.97%

in three days and 188.87% in six hours, respectively.

These results show the potential for utilizing microalgae for a combined wastewater

treatment-biofuel production system, but additional research needs to be performed to

understand the microbial community and identified the ways this community affects the

growth of microalgae and removal of nutrients from wastewater.

vi
Acknowledgments

I want to thank my dissertation committee for all their guidance in analyzing, writing, and

editing the information contained in this thesis. In particular, I am grateful to Jean

VanderGheynst for being my advisor and mentor for the last four years from undergraduate to

graduate school. She has helped me tremendously by providing the guidance, knowledge,

opportunities, and motivation needed to appreciate research in this field.

I would also like to thank Brendan Higgins of Auburn University for mentoring me

extensively with microalgae bioreactors. With his guidance, I was able to understand these

microalgae systems and give me a solid background in this area before starting this degree.

I am also thankful to other fellow researchers within the Bioprocess Engineering Lab. I

would like to recognize some critical individuals for their help: these include Jesus Fernández-

Bayo for his help using Qiime 2 and analyzing microbiome data, Lauren Jabusch for her help

setting up and using microalgae bioreactors, Shannon Ceballos for teaching biology techniques

and helping me troubleshoot when things inevitably went wrong.

I want to also thank members of the labs of Annaliese Franz and Ruihong Zhang for

collaboration on the CEC project. I am also thankful for the lending of support and equipment

from the labs of Tina Jeoh Zicari, Karen McDonald, and Tien-Chieh Hung.

Lastly, I would like to thank my family: my parents Mark and Linley, as well as my

grandma Margaret, my partner Anna, and her dog Zoe Michelle for all their support in helping

me achieve this milestone.

Funding for the studies contained in this thesis came from California Energy Commission

grant ARV-15-008 and the National Science Foundation grant #1438211.

vii
List of Figures
Figure 1.1: Global Fossil Fuel Consumption (1800-2016) .............................................................. 1
Figure 1.2: Different Types of Microalgae Metabolisms ............................................................... 5
Figure 1.3: Typical Microalgae Cell Structure ................................................................................ 6
Figure 1.4: Typical Cyanobacteria Cell Structure ........................................................................... 8
Figure 1.5: Metabolic Pathways for Lipid Synthesis in Microalgae .............................................. 9
Figure 1.6: Transesterification of Triglycerides for Biofuel Production ..................................... 10
Figure 1.7: First Large-Scale Microalgae Production Pilot Plant at MIT (1951).......................... 12
Figure 1.8: Closed Photobioreactors and Open Pond Systems for Growing Microalgae .......... 14
Figure 1.9: US Oil Prices in 2017 Adjusted Inflation Dollar ......................................................... 16
Figure 1.10: A Brief Chronology of the Research Activities in the Aquatic Species Program .... 17
Figure 1.11: Outdoor Raceway Ponds at The Pacific Northwest National Laboratory (2010) .. 20
Figure 1.12: Testing the Oxygen Regeneration Abilities of Microalgae (1962).......................... 23
Figure 1.13: Artist’s Conception of Incorporation of Algatrons into a Space Station ................ 25
Figure 1.14: Algae Turf Scrubber Design...................................................................................... 26
Figure 1.15: The St. Helena Wastewater Treatment Facility in Operation (2016) ..................... 28
Figure 2.1: Nutrients Utilized by Microalgae During Growth on Wastewater ........................... 34
Figure 2.2: UC Davis Renewable Energy Anaerobic Digester (READ) High Rate Digester System
....................................................................................................................................................... 36
Figure 2.3: Idealized Microalgae Wastewater Treatment and Biofuel Production Process ...... 41
Figure 2.4: Glass Bottle Algae Bioreactor .................................................................................... 44
Figure 2.5: Hybrid Tube Algae Bioreactor .................................................................................... 44
Figure 2.6: Hybridization Tube Bioreactor Systems for Growing Microalgae ............................ 45
Figure 2.7: Bottle Photobioreactors Growing Microalgae on Raw SFWP .................................. 50
Figure 2.8: Microalgae Optical Density (750 nm) of Batch Raw SFWP and BG-11..................... 51
Figure 2.9: Microalgae Biomass Concentration of in Batch Raw SFWP ..................................... 51
Figure 2.10: Comparison of Microalgae Biomass Concentration and Optical Density .............. 52
Figure 2.11: Logistic Growth Model of Microalgae in Batch Raw SFWP .................................... 53
Figure 2.12: Logistic Growth Models of Microalgae Optical Density in Batch ........................... 54
Figure 2.13: Concentration and Removal Efficiencies of (A., C.) N-NH4 and (B., D.) sCOD in
Batch Raw SFWP with Microalgae ............................................................................................... 55
Figure 2.14: Sterile and Intentionally Contaminated Microalgae in VSF-SFWP at Day 11 ........ 58
Figure 2.15: Sterile and Intentionally Contaminated Microalgae Optical Density (750 nm) of in
Batch and Semi-Continuous VSF-SFWP ....................................................................................... 60
Figure 2.16: Sterile and Intentionally Contaminated Microalgae (A.) Biomass Concentration
and (B.) Total Biomass in Batch and Semi-Continuous Conditions VSF- SFWP ......................... 61
Figure 2.17: Logistic Growth Model of Sterile and Intentionally Contaminated Microalgae
Optical Density (750) nm in Batch VSF-SFWP ............................................................................. 62

viii
Figure 2.18: Sterile and Intentionally Contaminated Microalgae Final Concentrations and
Removal Efficiencies of (A., C.) N-NH4 and (B., C.) sCOD in Batch and Semi-Continuous VSF-
SFWP ............................................................................................................................................. 64
Figure 2.19: Microalgae in Batch and Semi-Continuous Conditions on SSF-SFWP at Day 21 ... 68
Figure 2.20: Microalgae (A.) Optical Density (750 nm), (B.) Biomass Concentration, and (C.)
Total Biomass Produced on SSF-SFWP ........................................................................................ 69
Figure 2.21: Microalgae Removal Dynamics of (A.) sCOD, (B.) TN, (C.) N-NH4, and (D.) TON in
Semi-Continuous SSF-SFWP ......................................................................................................... 72
Figure 2.22: Microalgae Removal Efficiency and Sequential Removal Efficiency of (A., B.)
sCOD, (C., D.) TN, (E., F.) N-NH4, and (G., H.) TON in Semi-Continuous SSF-SFWP .................... 75
Figure 2.23: TN Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP ....... 76
Figure 2.24: N-NH4 Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP . 78
Figure 2.25: sCOD Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP ... 80
Figure 3.1: Potential Interactions Between Bacteria and Microalgae in Wastewater .............. 87
Figure 3.2: Microbial Community Analysis of Microalgae Microbial Community ..................... 89
Figure 3.3: Hybrid Tube Bioreactors with Microalgae (Left to Right: Algae 1-9) on SFWP after
166 Hours .................................................................................................................................... 100
Figure 3.4: Microalgae-Bacteria (A.) Optical Density (750 nm) and (B.) Biomass Concentration
on SFWP ...................................................................................................................................... 101
Figure 3.5: Hybrid Tube Reactor of SFWP (Left to Right: SFWP 1-3) After 166 Hours ............. 102
Figure 3.6: (A.) Optical Density and (B.) Biomass Concentration of SFWP .............................. 102
Figure 3.7: Microalgae and Bacteria Biomass Concentrations from SFWP .............................. 103
Figure 3.8: Final Concentration of (A) sCOD, (B) TN, and (C) N-NH4 of Microalgae on SFWP . 106
Figure 3.9: Removal Efficiencies of (A) sCOD, (B) TN, and (C) N-NH4 of Microalgae on SFWP 107
Figure 3.10: Correlation Between Microalgae Biomass Concentration and TN Removal ....... 108
Figure 3.11: NDMS of Microbial Community in SFWP .............................................................. 111
Figure 3.12: PCA with 95% CI of Microbial Community in SFWP .............................................. 112
Figure 3.13: 3D PCA with 95% CI of Microbial Community in SFWP ........................................ 113
Figure 3.14: Correlation Loading Plot of the Microbial Community in SFWP .......................... 114
Figure 3.15: Dendrogram of Microbial Community Similarity on SFWP. ................................. 115
Figure 3.16: Relative Abundance of Bacterial Phyla in the SFWP Microbiota ......................... 120
Figure 3.17: Relative Abundance of Bacterial Classes in the SFWP Microbiota ...................... 121
Figure 3.18: Relative Abundance of Bacterial Order in the SFWP Microbiota ......................... 122
Figure 3.19: Relative Abundance of Bacterial Families in the SFWP Microbiota ..................... 123
Figure 3.20: Relative Abundance of Bacterial Genus in the SFWP Microbiota ........................ 124
Figure 3.21: Relative Abundance Heat Map of OTUs in Initial SFWP ....................................... 125
Figure 3.22: Relative Abundance Heat Map of OTUs in SFWP After 166 Hours ...................... 126

ix
Figure 3.23: Relative Abundance Heat Map of OTUs in SFWP with Microalgae After 166 Hours
..................................................................................................................................................... 127
Figure 3.24: Biplot of Phylum Level OTUs in SFWP with Microalgae After 166 Hours ............ 129
Figure 3.25: (A.) Shannon Diversity Index and (B.) Pielou’s Evenness Index of Microbial
Communities in SFWP ................................................................................................................ 130
Figure 3.26: OTU Correlation with N-NH4 Removal in Microalgae-SFWP Community ............ 132
Figure 3.27: OTU Correlation with Microalgae Biomass Concentration in SFWP .................... 133
Figure 3.28: OTU Correlation with Bacteria Concentration in Microalgae-SFWP Community 134
Figure 3.29: OTU Correlation with Low Microalgae Biomass Concentration in SFWP (A.)
Volcano Plot (B.) Original Abundance and (C.) Normalized Abundance .................................. 135
Figure 3.30: (A., B.) Clusters of Co-Occurring OTUs and (C.) Their Relative Abundance in the
SFWP Microbial Community ...................................................................................................... 137
Figure 3.31: Oxygen Requirements of Bacteria Present in SFWP ............................................. 139
Figure 3.32: Bacterial Metabolisms Identified in SFWP ............................................................ 141
Figure 3.33: Heat Map of the Relative Abundance of Bacterial Metabolisms in SFWP .......... 142
Figure 3.34: Abundance of Bacteria Associated with Plants in SFWP ...................................... 143
Figure 3.35: Characteristics that Correlate with N-NH4 Removal in Microalgae-SFWP
Community ................................................................................................................................. 144
Figure 3.36: Characteristics that Correlate with sCOD Removal in Microalgae-SFWP
Community …………………………………………………….……………………………………………….………………..…145
Figure 4.1: Idealized 2-Stage System for SFWP Grown Microalgae Lipid Accumulation ......... 166
Figure 4.2: Schematic of Microalgae Biomass Lipid Analysis .................................................... 171
Figure 4.3: Sodium Acetate Induced C. sorokiniana Total Lipid Content ................................. 172
Figure 4.4: Sodium Acetate Induced C. sorokiniana (A.) Neutral Lipid Content and (B.) Percent
Change in Neutral Lipid Content................................................................................................ 173
Figure 4.5: Salt-Induced C. sorokiniana Total Lipid Accumulation ........................................... 174
Figure 4.6: Salt-Induced C. sorokiniana Neutral Lipid Accumulation ....................................... 175
Figure 4.7: Normalized Percent Change of Salt-Induced C. sorokiniana Neutral Lipid
Accumulation.............................................................................................................................. 176
Figure 6.1: Artist’s Concept of Futuristic Microalgae Fuel Farm in the American Southwest . 186

x
List of Tables
Table 1.1: Oil Content and Productivity of Traditional Agricultural Crops and Microalgae ........ 3
Table 2.1: Stripped Food Waste Permeate (SFWP) Composition ............................................... 37
Table 2.2: Prior Research of Microalgae Grown on Food Waste Anaerobic Digestate Effluents
....................................................................................................................................................... 38
Table 2.3: Prior Research of C. sorokiniana Grown on Anaerobic Digestate Effluents ............. 40
Table 2.4: Nutrient Composition of Medias ................................................................................ 43
Table 2.5: Set-Ups of Microalgae Growing on SFWP .................................................................. 49
Table 2.6: Logistic Growth Model of Microalgae in Batch Raw SFWP ....................................... 53
Table 2.7: Logistic Growth Models of Microalgae Optical Density in Batch .............................. 54
Table 2.8: Logistic Growth Model of Sterile and Intentionally Contaminated Microalgae
Optical Density (750 nm) in Batch VSF-SFWP ............................................................................. 62
Table 2.9: TN Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP .......... 76
Table 2.10: N-NH4 Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP... 78
Table 2.11: sCOD Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP .... 80
Table 2.12: Results of Microalgae Grown on SFWP .................................................................... 84
Table 3.1: Prior Research on the Microalgae-Wastewater Microbial Community .................... 92
Table 3.2: Nutrient Composition of 30% Raw SFWP ................................................................... 93
Table 3.3: Correlation Between Microalgae Biomass Concentration and TN Removal .......... 108
Table 3.4: OTU Correlation with N-NH4 Removal in Microalgae-SFWP Community ............... 132
Table 3.5: OTU Correlation with Microalgae Biomass Concentration in SFWP ....................... 133
Table 3.6: OTU Correlation with Bacteria Concentration in Microalgae-SFWP Community ... 134
Table 3.7: OTU Abundance Difference in High/Low Biomass Concentrations in SFWP .......... 135
Table 3.8: Characteristics that Correlate with N-NH4 Removal in Microalgae-SFWP Community
..................................................................................................................................................... 144
Table 3.9: Characteristics that Correlate with sCOD Removal in Microalgae-SFWP Community
..................................................................................................................................................... 145
Table 4.1: Methods of Lipid Induction in C. sorokiniana .......................................................... 162
Table 4.2: Carbon Source Lipid Accumulation Setup ................................................................ 168
Table 4.3: Salt Stress Lipid Accumulation Setup………………………………………….…………………………169

xi
1. Chapter 1 - Introduction and Research Objectives

1.1. Introduction

The recent drive for renewable biofuels has been led, in part, by the problems associated with

traditional fossil fuels: the increasing carbon dioxide concentrations that are leading to global

warming, increasing environmental destruction caused from collecting and burning of these

fuels, and lack of energy security within developed countries whose infrastructure depend on

this source of energy [1]. The three most widely used fossil fuels: crude oil, natural gas, and

coal, are inherently limited and expected to only last for another 50.2 years, 52.6 years, and

134 years, respectively [2]. Despite this, much of the world has become dependent on these

non-renewable fuel sources for their energy production. As of 2016, crude oil made up most of

the global energy production (Figure 1.1).

120000
Energy Production (TWh)

80000

40000

0
1800 1840 1880 1920 1960 2000
Year

Figure 1.1: Global Fossil Fuel Consumption (1800-2016)

◼ Natural Gas, ◼ Coal, ◼ Crude Oil
Data based on total yearly energy production [3]. Energy production in terawatt hours (TWh)

1
 This growing need for alternative energy has prompted the United States to invest in

cleaner, more sustainable fuel sources. A primary focus of this effort was toward the

production of biofuels from traditional crops, specifical ethanol from corn [4]. While this effort

has been successful at increasing energy independence, it has also produced more greenhouse

gases than traditional fossil fuels. Much of this increase is due to a land-use change from

agriculture for food production to agriculture for fuel production [5]. This inflates the prices of

goods by causing competition between food and fuel [6]. One alternative fuel that avoids the

problems produced with corn ethanol are biofuels derived from microalgae.

Microalgae has had a long history of being used as an alternative fuel product. Much of

the crude oil present on earth is the product of billions of years of geological activity on ancient

photosynthetic organisms, many of which were microalgae [7]. Today, these organisms still

contain traits that make them good candidates for biofuel production. Microalgae can utilize

non-arable water, land, and gas, reproduce quickly, and produce high concentrations of lipids

that can be directly converted into biofuels [8].

Unlike traditional biofuel crops, microalgae can readily grow on and utilize water, land,

and gas that is unsuitable for agriculture or human use. Water sources like seawater and

wastewater have been investigated extensively as microalgae growth mediums [9]. Wastewater

is of particular interest since it contains high concentrations of soluble nutrients that can be

utilized by microalgae for combined wastewater treatment and biofuel production [10, 11]. In

addition, microalgae can grow on land that would be unable for traditional crops. Microalgal

ponds and growth systems can be set up on any area as microalgae are not dependent on the

soil beneath them for nutrients or structure [12]. Since microalgae utilize carbon dioxide, they

2
can often be grown with waste gases high in carbon dioxide to increase their growth rate [1].

These advantages are of particular importance economically since they ensure that fuel and

food production are not in competition with one another as was with corn ethanol [13].

Some species of microalgae are fast-growing and can accumulate high concentrations of

lipids, making them ideal for biofuel production [14]. Oil production from microalgae is at least

10 times more productive than oil production from traditional crops (Table 1.1). Lipid

concentration in microalgae has been reported to reach total dry weight fractions as high as

80% [15]. Note that oil content and lipid content will be used interchangeably. If microalgae

were to produce a lipid content of 30%, microalgae farms could, in theory, produce 58,700 L

oil/ha/year. In comparison corn can realistically produce 2,053 L ethanol/ha/year [16]. Energy-

wise, microalgae oil would produce an equivalent of 2,054,500 MJ/ha/year [17], while corn

ethanol would produce and the equivalent of 49,272 MJ/ha/year [18], a difference of nearly 42

fold.

Table 1.1: Oil Content and Productivity of Traditional Agricultural Crops and Microalgae
Biological Oil Source Oil content Oil productivity Biodiesel productivity
(% weight) (L/ha/year) (kg/ha/year)
Corn/Maize 44 172 152
Hemp 33 363 321
Soybean 18 636 562
Jatropha 28 741 656
Camelina 42 915 809
Canola/Rapeseed 41 974 862
Sunflower 40 1070 946
Castor 48 1307 1156
Palm 36 5366 4747
Microalgae (low productivity) 30 58,700 51,927
Microalgae (high productivity) 70 136,900 121,104
Data from: [8]. ha = hectare

3
 Although microalgae biofuels have shown great potential, no commercial-scale

microalgae biofuel production facilities exist. This problem is because microalgae biofuels cost

significantly more than traditional fuels to produce. As of 2011, microalgae biofuel production

costs were estimated to be between $10.87/gal to $13.32/gal [19]. Funding given to researches

to identify cost-reducing measures for microalga biofuels have been minimal and closely tied

with socio-economic events. Currently, the most realistic and direct method of microalgal

biofuel production comes from integration with wastewater treatment as a means to combine

multiple services together [1, 9]. Research to better understand the engineering and biological

factors that impact the microalgae-wastewater interactions as they relate to water treatment

and lipid productivity can help to reduce these costs and make microalgae-based biofuels more

economical.

This thesis focuses on understanding the wastewater treatment performance and the

productivity of biomass and lipids from microalgae grown on wastewater. It is divided into

three chapters that focus on understanding microalgae-wastewater interaction. These areas

include investigating the growth of microalgae in both batch and semi-continuous operation on

raw and sterilized wastewater (Chapter 2), identifying and quantifying the microbial community

and the effects that this puts on the growth of microalgae and treatment of wastewater

(Chapter 3), and identifying approaches to enhance lipid production in microalgae grown on

wastewater (Chapter 4).

4
1.1.1. Microalgae

Microalgae are eukaryotic microorganisms typically found in bodies of water, such as lakes,

ponds, and oceans. Most microalgae are photoautotrophic, meaning they use carbon dioxide

(CO2 ) and water (H2 O) from the environment to produce organic carbon compounds (often

depicted as glucose: C6 H12 O6 ) in the presence of light (𝑒𝑣) (eq. 1). This, combined with the

uptake of nitrogen, phosphorous, potassium, and micronutrients from their surroundings,

enable them to act as the primary producers in most ecosystems [20, 21].

6CO2 + 12H2 O + 𝑒𝑣 → C6 H12 O6 + 6H2 O eq. 1

Some types of microalgae possess the ability to use organic carbon directly as an energy

and carbon source (heterotrophic). Others can utilize both inorganic and organic carbon

together by combining their autotrophic and heterotrophic metabolisms (mixotrophic) [20, 21].

Diagrams showing these different metabolisms in microalgae are shown in Figure 1.2.

Figure 1.2: Different Types of Microalgae Metabolisms

In autotrophic growth, light acts as the energy source for microalgae to convert CO 2 and other nutrients into
biomolecules. This process is similar to heterotrophic growth, except in this case the breaking down of organic
materials acts as the energy and carbon source, so light is not necessary. If both light and organic carbon are
present, both processes can occur within the same system in a process known as mixotrophic growth.

5
 Microalgae cells are structurally similar to plant cells in that they contain chloroplasts

and a cell wall. Some species even maintain large vacuoles. The chloroplasts found within

photosynthetic organisms are the result of endosymbiosis of cyanobacteria that occurred

billions of years ago [1, 20, 21]. The diagram of a generic microalgae structure is shown in

Figure 1.3.

Figure 1.3: Typical Microalgae Cell Structure

The Plasma membrane and Cell wall acts to protect the cell and control what enters; the Cytoskeleton and
Cytoplasm bind the cell together and allow for transport; the Ribosomes bound to the rough endoplasmic perform
protein synthesis; Smooth endoplasmic reticulum synthesizes lipids; the Golgi body uses vesicles to transport
materials throughout the cell; Peroxisomes breakdown organic matter; the Mitochondria create energy by
breaking down compounds; Chloroplasts help to sequester light into carbon compounds in the starch grains using
the thylakoid; the Nucleus holds the genetic material of the cell and is surrounded by a nuclear envelope; Nuclear
pores allow for transport of genetic material out of the nucleus; the nucleolus synthesizes ribosome [1, 20, 21].
Image credit: [22]

6
 The two most commonly studied types of eukaryotic microalgae are diatoms

(Bacillariophyceae) and green microalgae (Chlorophyceae). Diatoms are infamous for being the

dominant makeup of phytoplankton in the ocean. Unlike other microalgae, diatoms can

polymerize silica to produce robust and crystalline cell walls. There exist about 100,000 species

in this group. Green microalgae are the ancestors of modern plants and are abundant in

freshwater systems. The widely studied Chlorella genus has been used as nutritional

supplements and food. Other green microalgae genus including Dunaliella, Haematoccus, and

Crypthecodinium, are grown commercially to produce chemicals for pharmaceuticals and

nutraceuticals. There exist about 2000 species classified as green microalgae [20, 21, 23].

In defining microalgae as eukaryotic photoautotrophic microorganisms, it is important

also to recognize cyanobacteria (sometimes referred to as blue-green algae). While these

organisms are not technically microalgae, they are often grouped with eukaryotic microalgae

due to their similarities. Their structure is shown in Figure 1.4. Unlike eukaryotic microalgae,

they do not have a nucleus or complex organelles like a chloroplast or mitochondria. Instead,

they rely on phycobilisomes within the thylakoid membrane to harvest light energy for

chemical conversion. Some cyanobacteria are specialized to fix nitrogen gas from the

atmosphere, making them a key part in the nitrogen cycle. Spirulina (renamed Arthrospira) and

Nostoc are a common genus of cyanobacteria that are used for food or as nutritional

supplements by many cultures throughout the world. Their simple structures allow them to

grow and evolve quickly. Like other bacteria, cyanobacteria can be genetically modified with

relative ease. As such, they are used as model organisms for studying photosynthesis [20, 21,

23].

7
 Figure 1.4: Typical Cyanobacteria Cell Structure
The cyanobacteria cell is a simple capsule surrounded by a slime coat. A mucoid sheath separates the cell wall from
the slime coat. The cell wall is made up of an outer membrane, peptidoglycan layer, and a cell membrane on the
innermost part. Photosynthetic pigments, phycobilins, line the internal lamellae (thylakoid) for photosynthesis.
Carboxysome uses enzyme RuBisCo for carbon dioxide fixation. Ribosomes are found throughout the internal fluid,
or cytoplasm. Genetic material is held within the cell in the nucleoid region. Image credit: [24]

While both eukaryotic microalgae and cyanobacteria have similar requirements for

growth, they do differ characteristically. The most substantial difference that makes eukaryotic

microalgae popular for industrial biofuel production is that they can accumulate large

quantities of lipids [1, 20, 21]. Microalgae can produce two families of lipids: polar lipids

(phospholipids and glycolipids) and neutral lipids (monoglycerides, diglycerides, triglycerides,

isoprenoids, and sterols) [17, 25].

The metabolic pathway to produce lipids in microalgae is similar to that seen in plants

(Figure 1.5). Sequestered carbon that gets synthesized into pyruvate can be hydrolyzed into

various lipids from Acetyl-CoA (formed in the Krebs cycle) and Glycerol-3P (formed in glycolysis)

within the chloroplast. Acetyl-CoA can be converted to phosphatidic acid using fatty acid

synthase before being transported to the endoplasmic reticulum (ER) and transformed into
8
either phospholipids or diglycerides (DAG). Cells can use these lipids or further convert them to

triglycerides (TAG) and store them within lipid bodies. Glycerol can go on to produce glycolipids

in the chloroplast. These glycolipids can be broken down through hydrolysis to form

phosphatidic acid, or transported to the ER to be converted into TAG [26]. Understanding this

metabolism is key to understanding microalgae lipid accumulation.

Figure 1.5: Metabolic Pathways for Lipid Synthesis in Microalgae

ER: Endoplasmic Reticulum; 3-PGA: 3-phosphoglycerate; Accase: acetyl CoA carboxylase; ACP: acyl carrier protein;
AGPPase: ADP glucose pyrophosphorylase; ER: Endoplasmic reticulum; GDAT: putative glycolipids: DAG
acyltransferase; Glc6P: glucose-6-phosphate; KAS: 3-ketoacyl-ACP; PDAT: Phospholipids: DAG acyltransferase; PDH:
pyruvate dehydrogenase. Putative pathways were proposed using dashed lines. Image credit: U.S. Department of
Energy [27].

9
 Of the various types of lipids microalgae can produce, it is the neutral lipids that are

most useful for biofuel production. Most of these occur in the form of TAGs. Unlike other lipids,

TAGs can be directly converted to fatty acid methyl esters (FAME) through transesterification

(Figure 1.6). These molecules are the primary components of biodiesel [28]. TAG production in

microalgae is highly variable between different species of microalgae and is sensitive to

different environmental conditions (i.e., nutrients, temperature, salinity, etc.). In some cases,

microalgae can be induced to increase overall lipid content through alterations of their

environment [8].

Figure 1.6: Transesterification of Triglycerides for Biofuel Production

Triglyceride can react with three methanol molecules to form glycerol and three methyl esters. These methyl
esters can be used a fuel directly.

10
1.1.2 History of Microalgae Biofuels

German scientists first proposed largescale microalgae production during World War II (WWII)

as a means to produce lipids for food [29, 30]. Unfortunately, this research was largely

overlooked at the time, as their system was economical due to low overall lipid productivities.

After the war, though, many researchers saw a newfound potential in this idea.

The postwar economic boom caused a surge in scientific research throughout the world

[31]. At this time there was a growing fear of a ‘Malthusian catastrophe,’ or a major food

shortage caused by the exponential growth of the population. This was predicted to lead to

widespread famine across the globe [32]. Estimates from the United Nations (UN) at the time

suggested that half of the world was already hungry or malnourished and that food production

would need to be increased by 25-35% in the next 25 years to keep up with the growing

population [33]. Countries around the world became interested in finding ways to conserve,

utilize, and produce resources to meet this growing demand [34]. The US government began to

make sizable investments in non-military projects as an effort to increase the production of

resources [35]. This funding spurred interest in new areas of engineering and science, one such

being the mass cultivation of microalgae.

Early experiments from microalgae mass cultivation suggested it would become the

food source that could end world hunger and support the growing population. Early

experiments confirmed microalgae’s fast growth rate, high protein content, and the ability to

use non-arable resources for growth [32, 36, 37]. Studies of mass microalgae production of

Chlorella sp. began in Stanford, California with funding from the Carnegie Institution of

Washington in 1948. This research was followed by mass microalgae cultivation in Essen,

11
German (1949) and the development of the first microalgae pilot plant (Figure 1.7) in

Cambridge, Massachusetts (1951) [38]. Spurred by growing potential around mass microalgae

cultivation, some researchers looked towards using organisms as a source of lipids for foods

and bioproducts, as spurred on by the work of scientists during WWII [39]. One study in this

area showed that microalgae Chlorella pyrenoidosa could accumulate up to 70% of its dry

weight as lipid [40]. This species would later go on to become popular for biofuel production

due to these findings [41, 42]. Other scientists took this interest to investigate microalgae for

use in wastewater treatment (see section 1.1.3).

Figure 1.7: First Large-Scale Microalgae Production Pilot Plant at MIT (1951)
This system, funded by the Carnegie Institute for Science, was part of a large research project into using
microalgae for food built atop the Massachusetts Institute of Technology (MIT). It was the first study of its kind and
paved the way for future mass algae culturing projects. Image credit: Carnegie Institution for Science [38].

12
 Growth systems for microalgae mass cultures were developed throughout the 1950’s.

Two different types of growth systems quickly predominant during this time. They were closed

photobioreactors [43] and open ponds [44, 45].

Closed photobioreactors are systems that utilize transparent materials to grow

microalgae within while minimizing contact from the outside world. They are often tubular to

maximize surface area to volume ratios such that microalgae cells get ample light. These

systems offer excellent control over microalgae in use since they can maintain cultures in a

sterile environment [46].

Open ponds are systems that are not protected from their environment. They are often

little more than a hole in the ground with mechanisms to keep the culture mixed and

suspended. They take on four simple forms: circular central-pivot ponds, flat/shallow ponds,

inclined ponds, and raceway ponds [44]. Circular central-pivot ponds were first developed in

Asia to feed the growing demand for microalgae as a health food [47, 48]. They have a similar

design to that of a clarifier used in wastewater treatment. Flat/shallow ponds are one of the

simplest models of open ponds and are often just a shallow trough to retain microalgae

growing in water. The design of these systems was based on the observational growth of

microalgae growing in natural pond systems and stabilization ponds [49]. Similarly, inclined

ponds use a shallow system to grow microalgae, but with a slight incline to force microalgae to

flow down as they grow [50]. This downward flow aids in mixing and aeration of the culture.

The last design is the raceway pond, with the most popular being the High Rate Algal Pond

(HRAP). These ponds were initially developed for wastewater treatment with microalgae [51].

They are the most widely used for largescale growth as they have lower operational costs than

13
other systems and are easy to design and build [52]. Figure 1.8 shows examples of these two

types of systems.

Figure 1.8: Closed Photobioreactors and Open Pond Systems for Growing Microalgae
Tubular photobioreactors show closed photobioreactors pitched at an incline to maximize light exposure. Two
HRAPs represent the open pond system. Image credit: U.S. Department of Energy [27].

Energy production from microalgae was first experimentally investigated in 1955. During

that time, microalgae cultures were proposed as a feedstock for methane production from

anaerobic digestion [53]. Early tests at the University of California, Berkeley confirmed the

ability of microalgae to be used for natural gas production [54, 55]. A closed-loop system was

developed for the large-scale growth of microalgae for gas production utilizing digestate from

the anaerobic digester as the nutrient source [51, 56].

14
 The investigation into microalgae for direct electricity production through use in

biochemical fuel cells was briefly investigated as well. This research looked into both the direct

polarization of cathodes through oxygen production [57, 58] as well as the polarization of

anodes through the degradation of dead microalgae by bacteria [59, 60]. Due to low current

outputs of these systems, they were primarily abandoned until their revitalization in recent

years due to advancements in technology.

The consideration for using microalgae as a food source still dominated over its use for

energy production, due to the relatively inexpensive gas and oil prices at the time. Throughout

other parts of the world, microalgae started becoming a popular food source, so companies

began growing these organisms for commercial sale [31]. In both Japan and Taiwan, microalgae

became well accepted as a health food and companies (dubbed “Chlorella Factories”) focused

on microalgae for food production [31, 61]. It was at this time that other genus of microalgae

began in mass cultivation in Europe and Asia: Arthrospira (Spirulina), Dunaliella, and

Haematococcus. The idea of mass microalgae production in the US started losing steam as

people were less inclined to consume it as a foodstuff. In addition, the idea of a ‘Malthusian

catastrophe’ was greatly lessened as the Green Revolution dismissed the need for novel food

sources and birth control helped curb unchecked population growth [32].

The 1970’s oil crisis (Figure 1.9) prompted the need for the US to develop alternative

energy. In the earlier part of this decade, microalgae were grown for use as a feedstock in

methane production [62] as well as direct hydrogen production for hydrogen fuel cells [63, 64].

By 1978, the US government had formally developed the Aquatic Species Program (ASP) within

the US Department of Energy (DOE) Solar Energy Research Institute (SERI). The primary goal of

15
this program was to produce fuels from microalgae while utilizing waste carbon dioxide from

coal-fired power plants. Initially, the project geared toward growing microalgae for hydrogen,

methane, and ethanol production, but quickly shifted gears to growing microalgae for lipids to

be used for biodiesel production.

150
Cost per barrel ($/barrel)

100

50

0
1940 1955 1970 1985 2000 2015
Year

Figure 1.9: US Oil Prices in 2017 Adjusted Inflation Dollar

The energy crisis throughout the 1970’s cause spikes in the cost of oil in 1973 and 1979. Another energy crisis
was seen in the 2000’s, beginning in 2003 and peaking in 2008 before returning to pre-crisis levels in 2016.
Data compiled from 1940-1944 US Average, 1945-1983 Arabian Light, 1984-2017 Brent Dated [2].

The project was split into laboratory studies and outdoor studies/system analysis. The

laboratory studies consisted of collecting, screening, and characterizing microalgae strains as

well as developing strains through the study of biochemistry and physiology of lipid production;

this later shifted towards the genetic engineering of microalgae. Outdoor studies and system

analysis originally consisted of assessing microalgae production for combined wastewater

treatment and biomass production but switched to growing microalgae purely for lipids. This

16
involved constructing and testing both small pond (< 100 m3) and large pond (1000 m3) studies

as well as developing resource assessment of these systems [1]. The landmarks of this study are

summarized in Figure 1.10.

Figure 1.10: A Brief Chronology of the Research Activities in the Aquatic Species Program
Studies in the ASP investigated lab studies and outdoor culture studies and system analysis. This study lasted from
the late 1970’s to 1996. Image Credit: U.S. Department of Energy [1].

The project lasted for over 20 years and was fundamental to the current understanding

of microalgae biofuel production. The result of this project included the screening and

characterising over 3,000 new microalgae strains, developing large-scale screening methods to

identify highly productive microalgae, identifying ways to induce lipid accumulation in

microalgae (through nitrogen and silica deficiency), understanding of the metabolic role of

17
Acetyl-CoA carboxylase in lipid production, developing tools for genetically engineering

microalgae, and methods of scaling up the growth of microalgae for biofuel production [1].

By the end of the project, two major conclusions were reached: no apparent

engineering or economic issues were hindering the feasibility of large-scale microalgae

cultivation, and microalgae biomass and lipid productivities were below their theoretical

potential. The project researchers suggested that the only near- to mid-term application of

microalgae for biofuel production would be to integrate it with wastewater treatment. Future

work would need to increase the photosynthetic efficiency and lipid conversion in microalgae

through genetic engineering [1].

Throughout the 1990’s, public concern rose over the environmental impacts caused by

human activity. In particular, the public became alarmed with the idea that fossil fuels were

negatively impacting the earth through global warming. Photosynthetic microorganisms offered

a solution as they could sequester this atmospheric carbon dioxide directly. The main driver for

the development of mass microalgae cultivation was no longer solely a means to increase fuel

production; it was one to reduce the harmful effects of global warming. The Japanese

government responded in 1990 and launched the Research Institute of Innovative Technology

for the Earth (RITE). That primary goal of this project was to develop effective methods for

biological fixation of carbon dioxide from the atmosphere using microalgae. This project

focused on the screening of microalgae best suited for carbon sequestration, developing

engineering systems to grow microalgae and maximize carbon dioxide fixation and solar

irradiance collection, and utilizing microalgae for energy and bioproducts [65]. The project was

able to identify and characterize microalgae that could tolerate high carbon dioxide

18
concentrations as well as other extreme conditions [23]. One species, Chlorococcum littorale,

was found to grow under elevated carbon dioxide concentrations and ferment organics to

produce ethanol, a potential fuel source [66]. Studies from this project also showed the ability

of microalgae grow on untreated flue gas from power stations [67, 68].

In the early 2000’s, the world faced another energy crisis, driven again by rising oil

prices. This crisis was due to the geopolitical conflicts in the middle east and became further

aggravated by Hurricane Katrina in 2005 followed by the start Great Recession from the

collapse of the housing market in 2007 [69]. During this time, oil prices increased by over 600%

in just under ten years (Figure 1.9). The volatility in oil prices, along with the well documented

environmental dangers associated with petroleum fuels led the public to push for the

development of alternative energy sources.

This US government responded to this demand with the Energy Independence and

Security Act of 2007 which promoted the use of renewable energy and set new fuel economy

standards in cars [70]. Following that, additional investment in microalgae biofuels came from

the American Recovery and Reinvestment Act of 2009, the Office of Energy Efficiency and

Renewable Energy and Office of Fossil Energy Small Business Innovative Research (SBIR) of the

DOE, state funding through universities and national labs (Figure 1.12), and private investments

[71]. The Air Force Office of Scientific Research (AFOSR) started the algal biojet program to

produce jet fuel from microalgae. A sudden boom in microalgae related fuel endeavors took

over, and various microalgae biofuel startups like Solazyme (now TerraVia) and Sapphire Energy

were founded [72]. The research on microalgae made during this time was outlined in the 2010

National Algal Biofuels Technology Roadmap published by the DOE [27]. This publication also

19
acted as a guide to show researchers where advancements were needed to reduce the cost of

algae biofuels.

Figure 1.11: Outdoor Raceway Ponds at The Pacific Northwest National Laboratory (2010)
HRAPs pictured at PNNL. The photo was taken with the QuickBird satellite. Image Courtesy of Pacific Northwest
National Laboratory (PNNL), U.S. Department of Energy [73, 74].

Since the 2000’s, numerous studies have been carried out to understand microalgae

biofuel production. While most of this research has focused on the conversion of microalgae to

biodiesel [8, 75], other energy conversion methods have been explored: bioethanol [76], other

bio-alcohols [77], electricity production through microbial fuel cells [78], methane production

through anaerobic digestion [79], and electricity production through biomass combustion [80].

Despite the long history and newfound interest in microalgae biofuels, a commercially

available algal biofuel does not exist. This absence is due, in part, to the relatively low cost of

petroleum-based fuels, which have made microalgae biofuel production largely uneconomical

20
[81]. Research to reduce production costs is critical for this technology to become prevalent [1].

One such method that has shown the potential to do this is the combination of microalgae

biofuel production with wastewater treatment [9, 10, 82, 83]. Section 1.1.3 will cover the

history of microalgae wastewater treatment in more detail.

21
1.1.2. History of Microalgal Wastewater Treatment

Microalgae have long been recognized as critical microorganism in wastewater and sewage

treatment [84, 85]. Early studies of these systems identified that microalgae played an essential

role in wastewater treatment directly through the uptake of nutrients from waste [86, 87] and

indirectly through the oxygenation of wastewater for aerobic microbes which break down the

waste [88-90]. This idea was used as the basis to study and develop the first microalgae

wastewater treatment facilities in the 1950’s [91].

This interest in microalgae wastewater treatment became popular during the post-war

boom, led in part by William J. Oswald at the University of California, Berkeley [9]. Studies were

done there to understand better the ability of microalgae to aerate [92, 93] and treat [94]

wastewater. Specialized pond systems to grow microalgae for this sole purpose were

developed (HRAPs) [51]. During this time many other researchers were studying the use of

microalgae as a potential food source. The idea of combining wastewater treatment and food

production using microalgae was investigated but quickly abandoned for a variety of reasons

[95-97]. The proposed use of wastewater-grown microalgae for energy production began

through the use of these organisms as a feedstock for methane production [53, 55, 56] and as

electron acceptors in biochemical fuel cells [57, 58]. (See section 1.1.2 for more information on

microalgae biofuels).

With the increasing threat of nuclear war after the end of WWII, the US and Soviet

Union (USSR) began a nuclear arms race. During this conflict, the need for methods to sustain

humans in controlled environments, like nuclear submarines [98, 99] and space capsules [100,

22
101], became a significant engineering problem. Microalgae showed the potential to close this

loop.

These environmental control systems (later named controlled ecological life-support

systems [CELSS]) would require constant recycling of waste to regenerate needed supplies for

days, months, or even years at a time [102]. Methods to close the loop of these cycles used

microalgae’s regeneration properties (i.e., scrubbing carbon dioxide from the air while

replenishing oxygen to the cabin [Figure 1.12]) [103, 104], nutritional properties to feed crew

members of the vessel [105], and the ability to recycle human waste products [106-108].

Figure 1.12: Testing the Oxygen Regeneration Abilities of Microalgae (1962)

This project, contracted to the Bioastronautics Section of the Boeing Company, was an effort to see if large-
scale microalgae cultures could maintain oxygen supply for humans in space. Image credit: U.S. Navy Aerospace
Medical Research Labs [109]

23
 Implementation of this microalgae system in the US took the form of a semi-continuous

photobioreactor dubbed the ‘Algatron’ [110-112]. Early tests of this system with mice showed

that the system could successfully act as a photosynthetic gas exchanger over a long duration of

time [113, 114]. Later experimentation showed microalgae could be grown on the excrement

from mice [115]. Additional studies examined these systems for their ability to treat human

waste with the goal of implementation in spacecraft (Figure 1.13) to replace fecal bags [116,

117]. Microalgae strain C. pyrenoidosa TX 71105, now classified as C. sorokiniana UTEX 1230

[118, 119] showed great promise in use for CELSS and was used almost exclusively by NASA

(Figure 1.14) due to its fast specific growth rate, high heat tolerance, and ability to grow on a

variety of substrates [120]. This strain later became a popular organism for use in wastewater

treatment (Appendix 8.1).

The USSR also experimented the use of microalgae for air regeneration [121, 122] as

well as for food and waste removal [31]. In the 1970’s, tests using the CELSS Bios-3 showed that

humans could live with plants and microalgae in a closed system and have water, air, and

partial nutrient regeneration of human waste through biological systems [123, 124].

While microalgae life-support systems were constructed and tested on earth, the idea

of using them for spacecraft never took-off; these systems were not as reliable or robust as the

chemical or physical processes later used by both the US and USSR space agencies [111]. As the

space race ended, research into using microalgae in wastewater treatment geared towards

utilization on the municipal scale [125].

24
 Figure 1.13: Artist’s Conception of Incorporation of Algatrons into a Space Station
Algatrons (white circles with the black circle inside them) would face the sun and concentrate light onto the
algae through a cone-shaped reflective chamber. Such systems would be fed with human waste for growth
and aid in air regeneration for a crew [126].

By the early 1970’s, the environmentalist movement had grown to that point that the

public became vocally concerned over the effects of pollution on water systems [127].

Researchers started analyzing different areas of water quality. One of these areas was the

impact of human activity on phytoplankton, the primary producers of the ocean [128-131].

During the same time, the aquaculture industry saw rapid advancements due to the

development of new equipment like polyvinyl chloride (PVC) plumbing and fiberglass tanks

[132]. These newly developed materials allowed more control over natural systems. The Woods

25
Hole Oceanographic Institution even proposed the idea of using wastewater to grow

phytoplankton for combined aquaculture and wastewater treatment [133-136].

Researchers at the Smithsonian Institute’s Marine Systems Laboratory began developing

microcosms and mesocosms of water ecosystems for research [137]. One researcher, Walter

Adey, developed a system to remove or “scrub” key nutrients from the water using “turfs” of

macro and microalgae to replenish the system with cleaned, oxygenated water (Figure 1.14).

Dubbed the Algae Turf Scrubber (ATFTM )[138], this devices became a popular tool for scientists,

aquaculturists, and hobbyist [139] before later being considered to be a powerful tool for

wastewater treatment [140-142].

Figure 1.14: Algae Turf Scrubber Design

This system sprays water from the top of a screen where algal biofilms form. A large light is placed on the
front to allow the growth and nutrient removal by such organisms. Water then leaves through a drain. Image
credit: [143].

26
 The energy crisis of the 1970’s prompted the research of alternative energy sources.

Microalgae grown in wastewater was reinvestigated for methane generation [62, 144, 145].

Following this, The US government ordered the development of the Aquatic Species Program

(see section 1.1.2) which studied the growth of microalgae on wastewater. The program later

shifted its focus into the growth of microalgae for biofuels on less complicated media sources.

One of the significant conclusions made by this project was that the only near- to mid-term

application of microalgae for biofuel production would be to integrate it with wastewater

treatment [1]. This conclusion was substantial, as it promoted an economical solution to large-

scale microalgae production.

Multiple HRAPs for treating wastewater were constructed (Figure 1.15) in California [51,

146, 147] and Florida [148]. Also, the first fully functional combined wastewater treatment and

biomass production pond system was constructed in Singapore [149]. Since then, other new

microalgae wastewater treatment systems have been implemented throughout the world

utilizing the HRAP system [11].

Most of the research into microalgae wastewater treatment during the turn of the

century became coupled with biofuel production due to the volatility in oil prices (see 1.1.2).

The energy crisis in the early 2000’s spurred this drastic increase in oil prices. Many notable

projects were developed during that time to show the potential of wastewater grown

microalgae for biofuel production. One of which was NASA’s Offshore Membrane Enclosures

for Growing Algae (OMEGA) project, which utilized microalgae grown in semi-permeable

membranes to treat wastewater and release clean water to the ocean [150]. An additional

small-scale study showed the ability to grow native microalgae for biofuel production in dairy

27
and municipal wastewater [151]. Efforts to scale up these process have also been

demonstrated in HRAPs [82].

Figure 1.15: The St. Helena Wastewater Treatment Facility in Operation (2016)
This facility used an HRAP to treat municipal waste through an innovative process developed by Dr. William
Oswald. Image credit: City of St. Helena [152].

Since then, international efforts have backed in-depth research of microalgae for

wastewater treatment, using different types of wastewater [153-155] as well as immobilized

systems [156-158]. Most of this research has focused on the use of wastewater as a nutrient

source for microalgae to later be used as a biofuel [82, 83, 159] as suggested by the ASP in 1998

[1] as well as other, more recent assessments [9, 142, 160]. Despite this, few large-scale

processes currently exist. The lack of these large-scale microalgae facilities is mainly due to the

absence of research to understand these complex systems.

28
1.2. Research Goal and Objectives

Current research devoted to growing microalgae for biofuel production needs to focus on

making this process more economical. One such method is to combine microalgae biofuel

production with wastewater treatment [1]. Many types of wastewater have high

concentrations of macronutrients (N, P, K) that are readily utilized by microalgae [11]. Supplying

wastewater as a nutrient source for microalgae can reduce the overall operating costs for both

biofuel production and wastewater treatment [9, 142, 160].

This project hopes to study the growth of a well-documented wastewater-growing

microalgae strain, Chlorella sorokiniana, in stripped food waste permeate (SFWP) collected

from the University of California, Davis Renewable Energy Anaerobic Digester (READ). The

grown microalgae biomass is intended to be used for biofuel application, and as such, methods

of promoting the accumulation of lipids to be used as precursors for biodiesel will need to be

identified and tested.

While other studies have identified how microalgae grow in different types of

wastewater, they often neglect the microbial communities that affect the growth of such

systems. The complex interactions between microalgae and other microorganisms cannot be

overlooked, as they directly affect the replicability and robustness of large-scale systems. An

investigation into the microbiota produced through the introduction of microalgae in SFWP will

also be studied.

29
To better understand this, three main objectives have been identified:

Objective 1: (Chapter 2) Quantify Microalgal Growth on Wastewater

The growth of microalgae in wastewater will be quantified in batch and semi-continuous

operation for biomass accumulation and nutrient removal. The process for growth in semi-

continuous operation will be controlled through sterilization of the wastewater to exclude the

interactions from other microorganisms. The different feed rates in semi-continuous operation

will be compared with the microalgae grown in batch conditions to determine if semi-

continuous operation improve performance. Such continuous systems would more realistically

represent the commercial growth of microalgae on wastewater systems.

Objective 2: (Chapter 3) Identify the Microbiome in the Microalgae/Wastewater System

The microbial community that was already existing in wastewater source could influence the

growth of microalgae as well as the nutrient composition of the material itself. Understanding

this community and the changes it experiences with the introduction of microalgae will be

identified, quantified, and compared to wastewater without microalgae through microbiome

analysis of the extracted DNA from the wastewater. The effect of these different microbial

communities against the microalgae biomass production, as well as rates of nutrient removal,

will be assessed. Predictive metagenomic analysis will be done to identify the different

characteristic selected for by microalgae growth.

30
Objective 3: (Chapter 4) Identify Way to Induce Lipid Production in Microalgae

Methods to induce lipid accumulation in microalgae will be identified from the literature. The

most promising of these methods will be tested in microalgae growing on wastewater and

quantified to evaluate the overall biodiesel fuel potential for this system. The processes

identified will be such that they are compatible in a two-stage system utilizing wastewater.

The research presented in this thesis will help to increase the knowledge around

growing microalgae for combined wastewater treatment and biofuel production. Through the

characterizing of the direct interactions between microalgae and wastewater using batch and

semi-continuous conditions, identifying the microbial community and inferring interactions,

and quantifying the interaction between microalgae and stressors to increase lipid yield, I hope

to add to the current understanding how these systems interact. The goal of this research is to

better distinguish microalgae as a candidate for this type of combined operation. While this

thesis cannot hope to make these fuels single-handedly economically viable, it should cast light

on key developments that can help increase the potential of these systems to be successful.

31
1.3. Hypothesis

It is hypothesized that C. sorokiniana would grow to higher biomass concentration on SFWP

than on synthetic media. This is based on both the high nutrient content of the wastewater, the

addition of carbon sources in the wastewater water that would allow for mixotrophic growth

(which has shown higher biomass and lipid content that fully autotrophic growth [161]), and

prior research which pointed to high growth yields on a similar material (Appendix 8.1). In

addition, it was thought that this microalga would significantly alter the microbial community

surrounding them and would benefit from such microbes around them for metabolic

synergism.

Since this wastewater does not regularly interact with microalgae or plants, it is

hypothesized that such pathogens would not exist in the wastewater. Lastly, it was thought that

since algae lipid production was deeply tied to nutrients and stress, there would be ways to

increase overall lipid content in microalgae through such methods.

While there are many reports of microalgae that do not grow well on wastewater, the

microalgae selected, C. sorokiniana, has long been documented to grow on wastewater [162]. It

is suspected that the microbial community would change with the addition of microalgae

because the addition of oxygen into the water would substantially change the metabolic

pathways that can be used by bacteria in such conditions. While there are many instances of

bacteria negatively affecting the growth of microalgae [163], there are also many reports of

algae benefitting from a robust microbial community. Prior research has shown that nonsterile

wastewater has supported the growth of microalgae, often to concentrations higher than that

of many synthetic media (Appendix 8.1). This increase in growth would imply that a

32
wastewater-based microbial community may only serve to benefit the microalgae. Lastly, it has

been shown that lipid induction is possible in microalgae and that high lipid concentration can

be achieved in a variety of species, including C. sorokiniana. Success in utilizing the same types

of lipid induction techniques used in large-scale facilities [164] are hypothesized to apply to this

system.

33
2. Chapter 2 - Growing Microalgae on Stripped Food Waste Permeate

2.1. Background

The growth of microalgae on wastewater has long been considered the only viable method of

mass microalgae production for biofuels [1]. Many of these wastes contain the nutrients (Figure

2.1) essential for the growth of microalgae [165].

Figure 2.1: Nutrients Utilized by Microalgae During Growth on Wastewater

Wastewater contains high concentrations of nitrogen (N), phosphorus (P), potassium (K), organic carbons,
and carbon dioxide that are directly used by microalgae.

One such wastewater that has shown great potential for supporting algae growth is

anaerobic digestate effluent (ADE). ADE is produced through the degradation of organic waste

by methanogenic bacteria to produce methane and carbon dioxide, with the former being

combusted for energy production [166]. Leftover waste products (solid digestate and digestate

effluent) from this process are typically removed and discarded. Utilizing such waste for

microalgae growth would serve a dual purpose of treating wastewater and growing microalgae.

34
 The idea of using anaerobic digestate to grow microalgae was purposed in the early

1950’s as a method to continuously produce and breakdown microalgae for energy production

through anaerobic digestion [51]. Since then, a handful of studies have been published

describing the growth of different strains of microalgae on forms of digestate. These studies are

summarized in Appendix 8.1. The table summarizes a variety of microalgae and wastewater

factors.

The ADE for this study came from the University of California, Davis Renewable Energy

Anaerobic Digester (READ). This facility takes in campus food waste and generates natural gas,

which is subsequently used to power this process as well as resupply energy to the grid [167].

Figure 2.2 summarizes this process. The final significant products alongside electricity from the

combustion of biogas include flue gas, solid digestate, and permeate. This permeate is high in

nutrients like nitrogen, phosphorus, and potassium, that are essential to growing crops [26,

168]. Flue gas from this system is high in carbon dioxide, which is also a vital nutrient for

photosynthesis [26]. The ADE from this system is further processed by CleanWorld (not shown)

using a proprietary stripping process to remove the high levels of ammonia. This process results

in higher levels of sodium ions in the wastewater. The final effluent is dubbed stripped food

waste permeate (SFWP). The nutrient properties of this waste are displayed in Table: 2.1.

35
36

Figure 2.2: UC Davis Renewable Energy Anaerobic Digester (READ) High Rate Digester System
This system uses a three-stage anaerobic digester process to convert waste to biogas for direct conversion to electricity on the UC Davis campus. Solid feed is
ground up and fed into the hydrolysis tank. During this stage, most biogas produced is in the form of CO2. Material from this tank is then fed into the
biogasification tank, where methanogenic microorganisms break down organic acids into methane, CO 2, water, and other gases. The solid waste is stabilized in
the biostabilization tank to reduce the. Solid and liquid wastes are separated. Liquid is filtered to produce a permeate, which is a precursor to the material used
in this study. Biogas produced earlier is then scrubbed and dewatered before being used to power turbines. Any excess biogas is flared to produces flue gas.
[167, 169-172].

36
 Table 2.1: Stripped Food Waste Permeate (SFWP) Composition
30% SFWP 100% SFWP
(calculated)
Parameter Concentration (mg/L)
TKN 83 ± 4 278 ± 12
N-NH4 62 ± 5 207 ± 16
sCOD 1230 ± 12 4100 ± 40
TP 14.0 ± 0.5 46.6 ± 1.7
K 347 ± 2 1155 ± 7
Ca <2 <5
Mg 0.8 ± 0.2 2.7 ± 0.6
B 0.3 ± 0 0.9 ± 0.1
Na 1002 ± 13 3340 ± 42
Acetate 212 708
(SFWP Collected on 8/15-16/2016) Data from: [173]. TKN = total Kjeldahl Nitrogen, N-NH4 = total ammonia-
nitrogen, sCOD = soluble chemical oxygen demand, TP = total phosphorus, K = elemental potassium, Ca =
elemental calcium, Mg = elemental magnesium, B = elemental boron, Na = elemental sodium

Prior studies have shown the ability of microalgae to grow on different types of

anaerobic digestate from food waste for wastewater treatment and/or biofuel production

(Table 2.2). These wastes included kitchen waste digestate effluent (KWDE), food waste

digestate effluent (FWDE), and electro coagulated food and dairy waste digestate effluent (EC-

FDWDE). Multiple strains of both Chlorella and Scenedesmus have been tested, with C. vulgaris

producing the highest biomass and lipid productivity at 220 mg/L/d and 77.0 mg/L/d,

respectively. The highest lipid content was created by C. sorokiniana at a value of 60%. Nutrient

removal efficiencies varied between samples, but those with lower initial nutrient

concentrations showed higher removal efficiencies of total nitrogen (TN), ammonia nitrogen (N-

NH4), total phosphate (TP), and chemical oxygen demand (COD). These conditions also

promoted higher biomass productivities as well and follow with other research that shows

certain organic compounds in wastewater can hinder algae growth [174].

37
Table 2.2: Prior Research of Microalgae Grown on Food Waste Anaerobic Digestate Effluents
Organism Media Growth Lipid Nutrients Nutrient Source
Final density, % of dry weight, (mg/L) Removal (%)
Productivity, Productivity
Specific growth
rate
Chlorella 6.67% KWDE 0.3 g/L TN = 138 TN = 13a [175]
ellipsoidea 21 mg/L/db N-NH4 = 105 N-NH4 = 36a
0.12 d-1 TP = 2 TP = 81a
sCOD = 390 sCOD = 44a
C. 3% KWDE 0.26 g/L 60% [176]
sorokiniana (Gauze Filtered)
22.8 mg/L/d 15.0 mg/L/d
in Seawater 0.35 d-1
C. EC-FDWDE 1.71 g/L 35% TN = 43 TN = 82 [177]
vulgaris + 5% (v/v) CO2 220 mg/L/d 77.0 mg/L/dc TP = 1 TP = 88
1.03 d-1 sCOD = 77
Scenedesmus 5% FWDE 1.49 g/L 30.8% TN = 70 TN = 87 [178]
bijuga (1.2 μm Filtered + 50.8 mg/L/d 15.6 mg/L/d TP = 4 N-NH4 = 100
Autoclaved) sCOD = 343 TP = 91
sCOD = 66
S. 6.67% KWDE 0.22 g/L TN = 138 TN = 32a [175]
quadricauda 86.0 mg/L/db N-NH4 = 105 N-NH4 = 41a
0.18 d-1 TP = 2 TP = 81a
sCOD = 390 sCOD = 46a
TN = total nitrogen, N-NH4 = total ammonia-nitrogen, sCOD = soluble chemical oxygen demand, TP = total
phosphorus
KWDE = kitchen waste digestate effluent, EC-FDWDE = electro coagulated food and dairy waste digestate effluent,
FWDE = food waste digestate effluent
a
= estimated from graphs
b
= calculated by dividing the total biomass concentration (g/L) by the time (d)
c
= calculated by multiplying lipid content (%) by biomass productivity (g/L/d )

Initial screening (following a standardized process [179]) of different strains of

microalgae in various dilutions of SFWP revealed that two strains of Chlorella sorokiniana were

good candidates for this system: UTEX 1230 and UTEX 2805. Optimal conditions were then

determined to be at a 30% dilution of the wastewater [180]. Both strains have had a long

history of growing on wastewater. UTEX 1230 has been widely studied to grown on waste,

starting with use by NASA for bioregenerative life support systems in the 1960’s (section 1.1.3).

38
UTEX 2805 is a more recently characterized strain that was isolated directly from wastewater

[162]. Many studies produced by researchers at the University of California, Davis Bioprocess

Engineering Laboratory, have utilized this organism for growth on wastewater with success

[181].

Other studies have grown C. sorokiniana on anaerobic digestates and permeates (Table

2.3). Biomass and lipid productivities ranged from 9 mg/L/d to 33 mg/L/d and 2.3 mg/L/d to

15.0 mg/L/d, respectively. While these values are lower than that of other organisms grown on

anaerobic digestate (Appendix 8.1), many of those other microalgae were unable to grow on

the SFWP [180].

It is well documented that supplemental carbon dioxide yields higher microalgae

biomass [182] and productivities [183] than atmospherically aerated counterparts when

maintained at or below an organisms specific threshold for toxicity. Systems that utilized waste

carbon dioxide (in the form of flue gas, which is 6-8% CO2 typically) reported higher yields of

biomass than on methods that used atmospheric concentrations [184]. This supplementation

approach acts to increase microalgae growth while sequestering waste carbon dioxide that

would otherwise seep into the atmosphere [185]. Implementation of such systems has shown

great potential and has been tested during both the Aquatic Species Program (ASP) [1] and

Research Institute of Innovative Technology for the Earth (RITE) program [67, 68]. These

projects showed the potential of using the nutrients supply from wastewater and carbon

dioxide from flue gas for increasing algae biomass and lipid productivity. In this study, a high

carbon dioxide concentration will be used to simulate the effluent from flue gas. This proposed

process is summarized in Figure 2.3.

39
 Table 2.3: Prior Research of C. sorokiniana Grown on Anaerobic Digestate Effluents
Media Growth Lipid Nutrients Nutrients Study
Final density, % of dry weight, (mg/L) Removal
Productivity, Productivity (%)
Specific growth rate
3% KWDE (Gauze Filtered) 0.26 g/L 60% [176]
in Seawater 23 mg/L/d 15.0 mg/L/d
0.35 d-1
6% PLDE (Centrifuged) 0.39 g/L 8.2% TN = 152 [186]
33 mg/L/db 2.7 mg/L/dc TP = 9
6% PLDE (Centrifuged) 0.63 g/L 15.7% TN = 96 TKN = 45 [186]
53 mg/L/d 8.2 mg/L/dc TP = 7 TP = 71
10% CWDE (Centrifuged) 0.27 g/L 26.8 TN = 231 TN = 87 [187]
13 mg/L/d c
3.4 mg/L/d N-NH4 = 89 N-NH4 = 65
TP = 112 TP = 65
P-PO4 = 58
10% CWDE (Centrifuged) 0.15 g/L 24.2 g/L TN = 231 TN = 87 [187]
9 mg/L/d c
2.3 mg/L/d N-NH4 = 89 N-NH4 = 72
TP = 112 TP = 64
P-PO4 = 56
10% CWDE (Centrifuged) 0.28 g/L 24.0 TN = 231 TN = 86 [187]
16 mg/L/d 3.9 mg/L/dc N-NH4 = 89 N-NH4 = 75
TP = 112 TP = 61
TN is total nitrogen, N-NH4 is total ammonia-nitrogen, TP is total phosphorus, P-PO4 is total orthophosphate
KWDE = kitchen waste digestate effluent, PLDE = Poultry Litter Digestate Effluent, CWDE = Cattle Waste Digestate
Effluent
b
= calculated by dividing the total biomass concentration (g/L) by the time (d)
c
= calculated by multiplying lipid content (%) by biomass productivity (g/L/d)

40
41

Figure 2.3: Idealized Microalgae Wastewater Treatment and Biofuel Production Process
The proposed process involves the inoculation of wastewater with algae. Light and supplemental carbon dioxide are to be added to the media to support
growth. This process is intended to have two primary products: algae biomass and treated/partially-treated water. The algae biomass can then be used for
biofuel production through the extraction of lipids within the cells.

41
2.2. Materials and Methods

2.2.1. Microalgae Strain Selection

The microalgae strain selected for use in this study was Chlorella sorokiniana UTEX 2805 from

the University of Texas Culture Collection of Algae. UTEX 2805 is a more recently characterized

microalgae strain that was isolated directly from wastewater [162]. Many previous studies have

utilized this organism for growth on wastewater with success [181].

2.2.2. Wastewater Collection and Preparation

The permeate for this study came from the University of California, Davis Renewable

Energy Anaerobic Digester (READ) facility. It is the result of liquid digestate that was further

processed through ultrafiltration to produce a the permeate, that was then stripped to remove

additional nitrogen. The final product, stripped food waste permeate (SFWP), was stored in

non-sterile containers at 4°C until experimentation.

Two different sterilization techniques were used to remove microbial contaminants

from the SFWP. Due to the high ammonia and sCOD concentration in the digestate, autoclaving

was avoided so as not to volatilize the ammonia or degrade the organic molecules. Vacuum

Sterile Filtration of the SFWP (VSF-SFWP) was performed using a progression of 1.2µm (GC/F

Whatman, Buckinghamshire, United Kingdom), 0.45 µm (Whatman), and 0.2 µm (Whatman)

filters through a vacuum filtration system. The final collected effluent was rerun through a 0.2

µm (Whatman) filter in sterile conditions and stored at 4°C until experimentation. The Syringe

Sterile Filtration of SFWP (SSF-SFWP) utilized a progression of 0.45 µm (Whatman), and 0.2 µm

(Whatman) filters through 15 ml syringes. The final effluent was sterilized and stored in the

42
same conditions as the VSF-SFWP. Additionally, controls for the experiments were run on both

BG-11 (MilliporeSigma, Rocklin, CA) and N8-NH4 media [188].

The wastewater was diluted using autoclaved distilled water and the microalgae

inoculum to a 30% dilution. The properties of the 30% SFWP used in this study before and after

sterilization as well as the control mediums are shown in Table 2.4.

Table 2.4: Nutrient Composition of Medias

BG-11* N8-NH4* 30% Raw SFWP 30% VSF-SFWP 30% SSF-SFWP
Nutrients Concentration (mg/L)
TN 247.3 200.1 133.3 ± 28.8 36.7 ± 1.4 103.1 ± 3.2
N-NH4 0 200.1 57.3 ± 3.3 19.4 ± 0.5 42.2 ± 1.3
N-NO3 247.3 0 1.0 ± 0.2 0.7 ± 0.3 0.4
N-ONa 0 0 75.0 ± 29.4 16.6 ± 1.5 60.5
TP 7.1 221.9 56.0 ± 1.0 11.7 ± 0.0 73.0
sCOD - - 424 ± 12 144 ± 10 960 ± 4
TN = total nitrogen, N-NH4 = total ammonia-nitrogen, N-NO3 = total nitrate/nitrite, N-ON = total organic nitrogen,
TP = total phosphorus, sCOD = soluble chemical oxygen demand
VSF = Vacuum Sterile Filtered, SSF = Syringe Sterile Filtered
*
Values calculated stoichiometrically
a
Calculated from other nitrogen measurements

2.2.3. Microalgae Cultivation

Stock strains of C. sorokiniana were grown on N8-NH4 media [188] under the same light

and carbon dioxide conditions as used in the experimentation. The Raw SFWP experiments

were set up in 500 ml glass bottles with 75 mL of Raw SFWP and 150 ml of autoclaved distilled

water (Figure 2.4). The control was grown in 225 ml of BG-11. The inoculum consisted of 25 ml

of sterile microalgae grown in N8-NH4. The final microalgae biomass inoculum concentration in

the bottles was ~13 mg/L.

This setup was switched to a hybridization tube bioreactor system (Figure 2.5) to

increase light availability for the two filtered SFWP systems. In these systems, bioreactors were

inoculated with 20 ml of microalgae culture grown on N8-NH4 into 60 ml of 33.3% SFWP mixed

43
with 100 ml of autoclaved distilled water to a volume of 200 ml. The final microalgae density of

the inoculated cultures was ~5 mg/L for growth on both VSF-SFWP and SSF-SFWP. The hybrid

tubes were randomly organized in sets of four within 20-gallon fish tanks. These tanks were

filled with water to moderate temperature.

Figure 2.4: Glass Bottle Algae Bioreactor Figure 2.5: Hybrid Tube Algae Bioreactor
This reactor has 0.2 µm filters on top of the orange This reactor is set up similar to the Glass Bottle Algae
cap to ensure sterility from inflow and outflow of air. Bioreactor, but with a smaller reactor diameter to
A tube extends to the bottom of the reactor to increase the surface area to volume ratio over the
aerate such that bubbles move upwards through the glass, increasing light availability. Image used with
entire algae reactor. A stir bar is at the bottom to permission from C. W. Simmons. Originally published
mix through magnetic stirring. Image used with in [181].
permission from B. T. Higgins. Originally published in
[181].

Both types of bioreactors were used indoors under ~10,000 LUX light illumination from

T5 growth lamps (16∶8 ratio light-dark cycle). In experiments using filtered wastewater, carbon

dioxide was added to the air to produce ~6% v/v CO2 concentration to mimic that of flue gas.

Air and air/CO2 mixtures were humidified and bubbled into each reactor at a rate of ~50

44
ml/minute. Media in each reactor was stirred from the bottom using a ½ inch stir bar at a rate

of ~100 rpm. This setup is shown in Figure 2.6.

Figure 2.6: Hybridization Tube Bioreactor Systems for Growing Microalgae

Hybrid tube reactors are indicated with orange caps. Stir bars within the reactors are activated by the magnetic
stirrers below. Airflow into the reactors flows through 0.2 µm filters on the top of the caps for air in from the
airflow meters shown on the left. The growth light was placed behind the tank. The heater shown on the right was
located within the container, but not used in these experiments. Image used with permission from C. W. Simmons.
Originally published in [181].

Samples from each experiment were taken every 1-3 days for optical density

measurement (680 nm and 750 nm) using a microplate reader (Spectramax M2, Molecular

Devices, Sunnyvale, CA). Correlations were developed for comparing the different optical

densities to cell biomass density (Appendix 8.2-8.3). Larger samples were taken at specific time

points for later biomass dry weight concentration and nutrient composition: this was 5 ml of

45
sample from the batch, 60 ml from the 30% (v/v) semi-continuous operation, and 100 ml from

the 50% (v/v) semi-continuous operation. Samples were stored in 50 ml plastic centrifuge

tubes. Microalga cells were separated from solution via centrifugation at 5000 g for 5 minutes

(IEC Multi RF, Thermo Electron Corporation, Waltham, MA). Decantation of the supernatant

followed this. Samples were immediately frozen at -20°C for later analysis.

2.2.4. Biomass Quantification

Biomass samples were thawed from storage at -20°C, washed with distilled water, and

concentrated into pre-weighed plastic 15 ml tubes before being pelleted at 5000 g for 5

minutes (IEC Multi RF, Thermo Electron Corporation, Waltham, MA). Excess water was

decanted. The pellet was lyophilized at −45°C for two days using a freeze drier (Freezone4.5,

Labconco, Kansas City, MO). Samples were subsequently weighed on an analytical balance. The

biomass concentration was calculated from this dry weight and the volume of algae sampled.

Correlations between optical density (750 nm) and biomass concentration were developed

(Appendix 8.2-8.3).

2.2.5. Nutrient Quantification

Fresh and spent media was thawed from storage at -20°C for each of the SFWP samples. These

samples were homogenized before being analyzed for nutrient content using colorimetric

assays (HACH, Loveland, CO). These tests included total nitrogen (Nitrogen-Total Reagent Set

TNT Persulfate Digestion Method HR), ammonia nitrogen (Nitrogen-Ammonia Reagent Set TNT

AmVer (Salicylate) HR), total phosphorus (Phosphorus-Total TNT Reagent Set LR), nitrate/nitrite

46
nitrogen (NitraVer X Nitrogen-Nitrate Reagent Set HR), and soluble chemical oxygen demand

(COD Digestion Vials HR). The assays were analyzed using a spectrophotometer (DR 2500,

HACH, Loveland, CO). Total organic nitrogen was calculated using the difference between total

nitrogen and the sum of ammonia nitrogen and nitrate/nitrite nitrogen [189].

The standards used for each assay had their reported error: 2% for the total nitrogen

test, 2.8% for the ammonia nitrogen test, 2.4% for total phosphorus, 5% for the nitrate/nitrite

nitrogen, and 1.9% for chemical oxygen demand. These errors were not included in the analysis

of such nutrients.

2.2.6. Growth Models

Growth models were determined for biomass concentration and optical density by using the

logistic growth formula as described by Pierre-François Verhulst (eq. 2). These values were fit

using the nlinfit, a nonlinear regression tool in MATLAB. In this formula, N is the concentration

of algae, r is the growth rate, Kmax is the carrying capacity, and t is time. The solution to this

differential equation (eq. 3) was used to estimate the concertation of the microalgae over time.

For modeling purposes K was the maximum algae concentration and N0 was the initial

concentration of microalgae inoculated into the bottle (Appendix 8.4). Regression model

statistics for the coefficient of determination and root mean squared error (RMSE) [190] were

also calculated using MATLAB.

𝑑𝑁 𝐾𝑚𝑎𝑥 −𝑁
= rN (eq. 2)
𝑑𝑡 𝐾𝑚𝑎𝑥

𝐾𝑚𝑎𝑥 𝑁0 𝑒 𝑟𝑡
N= (eq. 3)
𝐾𝑚𝑎𝑥+𝑁0 (𝑒 𝑟𝑡 −1)

47
2.2.7. Data Analysis

Data analysis and statistical calculations were performed using JMP v.14 (SAS Institute, Cary,

NC) for regression [190], ANOVA (analysis of variance) [191], and Tukey-Kramer Honest

Significant Difference tests [190]. Data were graphed using Microsoft Excel 2016 (Microsoft

Corporation, Redmond, WA) and R version 3.5.1 (Vienna, Austria).

48
2.3. Results

2.3.1. Summary

A total of three separate experiments were performed to analyze the growth of microalgae C.

sorokiniana on unsterilized (Raw) and sterilized SFWP. In the first experiment, the microalgae

culture was grown on unfiltered SFWP using ambient carbon dioxide concentrations in batch

operation. The second experiment utilized vacuum sterile filtered SFWP (VSF-SFWP) aerated

with 6% CO2 to simulate the addition of carbon-rich flue gases. The microalga was grown in

batch and 30% (v/v) semi-continuous operation with and without the introduction of

contamination from 1 ml of Raw SFWP. The last experiment tested syringe sterile filtered SFWP

(SSF-SFWP) in batch and semi-continuous growth of algae under 30% (v/v) and 50% (v/v) semi-

continuous operation. These experiments are summarized in Table 2.5.

Table 2.5: Set-Ups of Microalgae Growing on SFWP

Section Media CO2 Reactor Operation Media Replacements Days
2.3.2 30% Raw SFWP atm Batch on BG-11 (n=1) 32
Batch (n=3)
2.3.3 30% VSF-SFWP 6% Batch (n=3) 11
30% v/v SC (n=3) 7
C* Batch (n=3)
C* 30% v/v SC (n=3) 7
2.3.4 30% SSF-SFWP 6% Batch (n=3) 21
30% v/v SC (n=3) 6
50% v/v SC (n=3) 6
SC = Semi-continuous, C* = Introduced contamination, atm = atmospheric CO2 concentration (~0.04%)

49
2.3.2. Growth on Raw SFWP

2.3.2.1. Algae Growth

This experiment documented the growth kinetics of microalgae C. sorokiniana growing on 30%

diluted Raw SFWP using atmospheric carbon dioxide concentrations (setup seen in Figure 2.7).

Figure 2.7: Bottle Photobioreactors Growing Microalgae on Raw SFWP

The microalgae cultures achieved an optical density (750 nm) of 4.66 ± 0.20 OD750 on

30% SFWP and 3.23 OD750 on BG-11 (Figure 2.8). When grown on 30% Raw SFWP, the culture

reached a maximum culture density of 1.79 ± 0.14 g/L which equates to biomass productivity of

64.9 mg/L/d after 28 days (Figure 2.9). From day 28 to 32, the culture biomass concentration

decreased to 1.47 ± 0.14 g/L. This peak in culture density at day 28 is seen in the optical density

reading as well as biomass concentration results (Figure 2.10).

50
 4
Optical Density (750 nm)

0
0 5 10 15 20 25 30 35
Days

Figure 2.8: Microalgae Optical Density (750 nm) of Batch Raw SFWP and BG-11
 30% SFWP (n=3),BG-11 (n=1)
2
Biomass Concentration (g/L)

1.5

0.5

0
0 5 10 15 20 25 30 35
Days

Figure 2.9: Microalgae Biomass Concentration of in Batch Raw SFWP

n=3 for biomass concentration.

51
 2 4
Biomass Concentration (g/L)

Optical Density (750 nm)

1.5 3

1 2

0.5 1

0 0
0 5 10 15 20 25 30 35
Days

Figure 2.10: Comparison of Microalgae Biomass Concentration and Optical Density

 Optical Density (750 nm),  Biomass Concentration
n=3 for both measurements.

Nonlinear regression was performed to fit microalgae growth to the logistic model. Both

the microalgae biomass concentration (Table 2.6, Figure 2.11) and optical density (750 nm) of

microalgae grown on both Raw SFWP and BG-11 (Table 2.7, Figure 2.12) were used for

modeling. Results indicated that the microalgae grown on 30% Raw SFWP had a specific growth

rate of 0.33 d-1 and a maximum concentration of 1.61 g/L with an R2 of 0.964. Optical density

data showed that microalgae grown on BG-11 displayed a specific growth rate of 0.65 d-1 and a

maximum concentration of 1.74 OD750 with an R2 of 0.892. In comparison microalgae grown on

Raw SFWP at 30% dilution showed a lower specific growth rate of 0.42 d-1 but a higher

maximum concentration of 2.17 OD750 with a lower R2 value of 0.687.

52
 Table 2.6: Logistic Growth Model of Microalgae in Batch Raw SFWP
Kmax rmax R2 RMSE
-1
1.61 ± 0.19 g/L 0.33 ± 0.07 day 0.9693 0.11 g/L

2
Biomass Concentration (g/L)

1.5

0.5

0
0 5 10 15 20 25 30 35
Days

Figure 2.11: Logistic Growth Model of Microalgae in Batch Raw SFWP

 Biomass Concentration,  Model

53
 Table 2.7: Logistic Growth Models of Microalgae Optical Density in Batch
Kmax rmax R2 RMSE
-1
30% SFWP 2.16 ± 0.42 OD750 0.41 ± 0.13 day 0.7123 0.48 OD750
BG-11 1.73 ± 0.15 OD750 0.65 ± 0.12 day-1 0.8927 0.20 OD750

4
Optical Density (750 nm)

0
0 5 10 15 20 25 30 35

3
Optical Density (750 nm)

0
0 5 10 15 20 25 30 35
Days

Figure 2.12: Logistic Growth Models of Microalgae Optical Density in Batch

 30% SFWP, BG-11, Model

54
2.3.2.2. Nutrient Removal

Reactors that had microalgae grown on 30% Raw SFWP showed a significant decrease in

the concentration of N-NH4 by 98.2% after 32 days from an initial concentration of 57.3 ± 3.3

mg/L (Figure 2.13 A., Figure 2.13 C.). In addition, the concentration of sCOD also showed a large

decrease initially, but steadily increased after that for a net increase from the initial

concentration of 424 ± 12 mg/L by 2.4 ± 1.1% (Figure 2.13 B., Figure 2.13 D.). The maximum

sCOD removal was achieved at day five at 41.6 ± 1.4%.

A. 100 B. 600
Concentration (mg/L)

75 Concentration (mg/L)
400

50

200
25

0 0
0 10 20 30 0 10 20 30

100 100
C. D.
N-NH4 Removal (%)

sCOD Change (%)

75 75

50 50

25
25

0
0 0 10 20 30
0 10 20 30
-25
Days Days

Figure 2.13: Concentration and Removal Efficiencies of (A., C.) N-NH4 and (B., D.) sCOD in
Batch Raw SFWP with Microalgae
Graph A. shows the concentration of N-NH4 over time, B. shows the concentration of sCOD over time, C. shows the
Percent Removal of N-NH4, and D. shows the Percent Removal of sCOD. n=3 for all measurements.

55
2.3.2.3. Analysis

The results indicated that the microalgae grown on 30% Raw SFWP had a specific growth rate

of 0.33 ± 0.07 day-1 and maximum concentration of 1.61 ± 0.19 g/L with an RMSE of 0.11 g/L

and an R2 of 0.9693. Nonlinear regression of the optical density data showed that microalgae

grown on BG-11 displayed a specific growth rate of 0.65 ± 0.12 day-1 and maximum

concentration of 1.73 ± 0.15 OD750 with an RMSE of 0.20 and R2 of 0.8927. In comparison

microalgae grown on 30%, Raw SFWP showed a lower specific growth rate of 0.41 ± 0.13 day-1

but a higher maximum concentration of 2.16 ± 0.42 OD750 with a higher RMSE of 0.48 OD750 and

a lower R2 value of 0.7123.

The optical density of microalgae grown on Raw SFWP showed higher delineation from

the logistic growth equation than microalgae grown on BG-11. This delineation is speculated to

be related to biofilm formation and potential mixing problems when sampling. However,

microalgae grown on wastewater showed a higher optical density than its counterpart on BG-

11, which was demonstrated by the higher maximum concentration as calculated through the

growth equation.

Interestingly, the culture continued to grow slowly in the stationary phase. This is likely

due to the utilization of more complex forms of nutrients. It is well known that microalgae will

preferentially use ammonia before nitrates [192]. In this case, ammonia was likely consumed

first before other forms of nitrogen such as nitrate/nitrite and organic nitrogen were

consumed. Also, the microbial community may aid in breaking down other organic molecules

that could later be utilized by the microalgae.

56
 The N-NH4 removal efficiency was significant within the culture and reached levels as

high as 98.1% removal. Most of this removal is speculated to be from the growth of organisms,

although some of the ammonia was lost in aeration by stripping.

Using the empirically derived microalgae stoichiometry to resent the total nitrogen in

the microalgae biomass: C106H263O110N16P [193], an estimated 84.5% of removed nitrogen was

taken up into the microalgae biomass. Since measurements of total nitrogen and

nitrate/nitrate, were not done, it is unclear how much nitrogen was lost through stripping. This

result does suggest that no more than 14% was lost through stripping.

Reactors containing microalgae initially showed a decrease in sCOD concentration

followed by an increase later in growth. This is likely due to the first utilization of simple organic

molecules such as acetate by algae and bacteria, followed by the secretion of metabolic

compounds by algae later in the process [103].

57
2.3.3. Growth on VSF-SFWP

2.3.3.1. Algae Growth

This experiment tested the growth of C. sorokiniana on sterile SFWP to eliminate any influence

the microbial community would have on the overall growth of microalgae. Supplemental

carbon dioxide was added at 6% (v/v) to simulate the addition from flue gas. These tests were

done in both batch and semi-continuous operation. Simulated “contamination” of the cultures

was done by the addition of 1 ml of Raw SFWP into the final 200 ml of algae culture.

Growth of C. sorokiniana on 30% VSF-SFWP was performed with 7 consecutive feeds of

30% v/v/d using both batch and semi-continuous growth on VSF-SFWP. Setup is shown in

Figure 2.14.

Sterile Intentionally Contaminated

Batch 30% v/v SC Batch 30% v/v SC

Figure 2.14: Sterile and Intentionally Contaminated Microalgae in VSF-SFWP at Day 11

(from left to right) Batch: 1, 2, 3; 30% v/v SC (Semi-Continuous): 1, 2, 3; Intentionally Contaminated Batch: 1, 2, 3;
Intentionally Contaminated 30% v/v SC (Semi-Continuous): 1, 2, 3.

Optical density (750 nm) data showed close similarities between the sterile and

contaminated cultures (Figure 2.15) in both batch and semi-continuous conditions. Both batch

58
growth treatments had similar final biomass concentrations (Figure 2.16 A.) of 0.99 ± 0.06 g/L

and 0.92 ± 0.03 g/L, respectively. In semi-continuous conditions, the final biomass

concentration was measured at 0.47 ± 0.03 g/L and 0.45 ± 0.03 g/L for sterile and contaminated

cultures, respectively (Figure 2.16 B.). There was no significant difference between sterile and

contaminated batch conditions and sterile and contaminated semi-continuous conditions for

both optical density (750 nm) and biomass concentration (Tukey-Kramer HSD, 0.95). In semi-

continuous operations, the microalgae biomass content was 0.037 g/day in sterile SFWP and

0.035 g/day in contaminated SFWP.

Nonlinear regression was performed to fit microalgae growth to the logistic model. The

optical density (750 nm) of 30% VSF-SFWP for sterile and cultures fit to a specific growth rate of

1.27 ± 0.05 day-1, carrying capacity of 0.73 ± 0.02 OD750, root mean squared error (RMSE) of

0.01 OD750 , and a least-squared regression value of 0.9979 (Table 2.8, Figure 2.17). Similarly,

the contaminated microalgae had a specific growth rate of 1.34 ± 0.09 day-1, carrying capacity

of 0.69 ± 0.03 OD750, an RMSE of 0.02 OD750, and a least-squared regression value of 0.9945.

59
 0.8

0.6
Optical Density (750 nm)

0.4

0.2

0
0 2 4 6 8 10
Days

Figure 2.15: Sterile and Intentionally Contaminated Microalgae Optical Density (750 nm) of
Batch and Semi-Continuous VSF-SFWP
 Sterile 30% Semi-Continuous Growth,Contaminated 30% Semi-Continuous Growth,  Sterile Batch Growth,
 Contaminated Batch Growth
n=3 for all measurements.

60
A.
Biomass Concentration (g/L) 1

0.8

0.6

0.4

0.2

0
0 2 4 6 8 10

B. 0.3
y = 0.037x - 0.094
R² = 0.9954
p<0.0001
Total Biomass (g)

0.2
y = 0.035x - 0.095
R² = 0.9983
p<0.0001
0.1

0
0 2 4 6 8 10
Days

Figure 2.16: Sterile and Intentionally Contaminated Microalgae (A.) Biomass Concentration
and (B.) Total Biomass in Batch and Semi-Continuous Conditions VSF- SFWP
 Sterile 30% Semi-Continuous Growth,Contaminated 30% Semi-Continuous Growth,  Sterile Batch Growth,
 Contaminated Batch Growth
Graph A. is the biomass concentration over time while graph B. is the total biomass produced. n=3 for all
measurements.

61
 Table 2.8: Logistic Growth Model of Sterile and Intentionally Contaminated Microalgae
Optical Density (750 nm) in Batch VSF-SFWP
Kmax rmax R2 RMSE
Sterile Batch -1
0.73 ± 0.02 OD750 1.27 ± 0.05 day 0.9979 0.01 OD750
Contaminated Batch 0.69 ± 0.03 OD750 1.34 ± 0.09 day-1 0.9945 0.02 OD750

0.8
Optical Density (750 nm)

0.6

0.4

0.2

0
0 2 4 6 8 10 12

0.8
Optical Density (750 nm)

0.6

0.4

0.2

0
0 2 4 6 8 10 12
Days

Figure 2.17: Logistic Growth Model of Sterile and Intentionally Contaminated Microalgae
Optical Density (750) nm in Batch VSF-SFWP
 Sterile Batch,Intentionally Contaminated Batch, Model

62
2.3.3.2. Nutrient Removal

C. sorokiniana grown on 30% VSF-SFWP showed a significant decrease in the

concentration of N-NH4 from an initial concentration of 19.4 ± 0.5 mg/L (Figure 2.18 A.). The

decrease in overall N-NH4 was 96.6 ± 3.2% and 98.1 ± 2.5% for sterile and contaminated batch,

respectively, and 97.5 ± 2.5% and 97.7 ± 3.0% for sterile and contaminated semi-continuous

conditions, respectively (Figure 2.18 C.). The empirically derived stoichiometric formula

(C106H263O110N16P [193]) was used to estimate total nitrogen accumulation in microalgae

biomass. Based on the results from the batch experiments, the percent of nitrogen sequestered

into microalgae biomass was 169.9% for sterile conditions and 158.7% for intentionally

contaminated conditions, suggesting that these cultures were nitrogen starved and utilized

most or all of the available nitrogen quickly.

Concentrations of sCOD were converse to those of N-NH4. C. sorokiniana increased

sCOD from an initial concentration of 144 ± 14 mg/L (Figure 2.18) by 391 ± 35%, 275 ± 28%, 243

± 64%, and 131 ± 34% for sterile and contaminated batch and sterile and contaminated semi-

continuous conditions, respectively (Figure 2.18 D.).

63
A. 20 a** B. 800 b*

b*
Concentration (mg/L)

Concentration (mg/L)
b*
15 600

c*
10 400

5 200 a*

b** b** b** b**

0 Initial Batch 30% v/v SC C* Batch C* 30% v/v SC
0 Initial Batch 30% v/v SC C* Batch C* 30% v/v SC

C. 100 D. 500

Percent Produced (%)

Percent Removal (%)

375
75

50 250

25 125

0 Sterile Batch Sterile 30% v/v Semi-Continious Contaimnated Batch Contaimnated 30 % v/v Semi-Continious
0 Sterile Batch Sterile 30% v/v Semi-Continious Contaimnated Batch Contaimnated 30 % v/v Semi-Continious

Figure 2.18: Sterile and Intentionally Contaminated Microalgae Final Concentrations and
Removal Efficiencies of (A., C.) N-NH4 and (B., C.) sCOD in Batch and Semi-Continuous VSF-
SFWP
◼ Initial Conditions, ◼ Sterile Batch, ◼ Sterile 30% v/v Semi-Continuous, ◼ Intentionally Contaminated Batch, ◼
Intentionally Contaminated 30% v/v Semi-Continuous.
Graph A. is the final N-NH4 concentrations, B. is the final sCOD concentrations, C. is the percent N-NH4 removal,
and D. is the percent production of sCOD. Groups a, b, and c are compared against one another using a Tukey-
Kramer HSD at 95%. n=3 for all measurements. P value: **<0.0001, *<0.05

64
2.3.3.3. Analysis

Overall, C. sorokiniana was able to grow and accumulate biomass when grown on a 30%

dilution of VSF-SFWP in 30% (v/v) semi-continuous conditions. Both batch and semi-continuous

conditions showed similar concentrations of biomass. As such, no significant difference could

be attributed to the microbes that grew within the environment of the contaminated culture

(Tukey-Kramer HSD, 0.95). This suggests that the microbial community did not have a

significant impact on the overall growth of the microalgae in these conditions. This may be

because such a small concentration of bacterial inoculum was added (1 ml of Raw SFWP per

200 ml) or that the sterilized wastewater could not harbor the same microbes as Raw SFWP.

Similarly, microalgae growth on Raw SFWP, this culture continued to grow slowly in the

stationary phase. Both sterile and intentionally contaminated batch growth of C. sorokiniana

showed very similar specific growth rate and carrying capacity, with the sterile treatment being

1.27 ± 0.05 day-1 and 0.73 ± 0.02 OD750 and the contaminated treatment being 1.34 ± 0.09 day-1

and 0.69 ± 0.03 OD750, respectively.

Final N-NH4 removal efficiencies were above 95% across all cultures and did not differ

significantly between treatments and sCOD increased over time as the microalgae grew. This

was likely due to excretions from C. sorokiniana into the wastewater. Both sterile conditions

and the contaminated batch conditions produced similar amounts of sCOD (580 ± 109 mg/L)

while contaminated semi-continuous growth produced a significantly different concentration

(333 ± 35 mg/L, Tukey-Kramer HSD, 0.95). All of these were significantly different than the

initial concentration (480 ± 34 mg/L, Tukey-Kramer HSD, 0.95). In the cases of contaminated

65
cultures, the sCOD concentration was lower due to the possible utilization of these metabolites

by bacteria which likely proliferated in semi-continuous conditions.

The VSF-SFWP was challenging to vacuum filter due to the high concentration of

particulate matter in the waste. This required wastewater to be left out in room temperature

conditions for long periods of time while slowly trickling through the filter. Additional

movement through these filters caused bubbling and foaming as the sterilized water percolated

down into the container. These factors may have led to the reduction in both N-NH4 and sCOD

through the volatilization of ammonia and fatty acids as well as chemical transformation by

bacteria before the material was filtered into a sterile container using a 0.2 μm filter. This

reduction in essential nutrients made the VSF-SFWP different in composition to Raw SFWP. As

such, it does not provide an ideal representation of how microalgae would grow on a sterile

media source similar in composition to Raw SFWP.

66
2.3.4. Growth on SSF-SFWP

2.3.4.1. Algae Growth

While C. sorokiniana growing on VSF-SFWP in semi-continuous conditions was able to produce

high biomass concentrations and significantly reduce the nutrient content of the wastewater,

the media used was characteristically different from the Raw SFWP reported. Another method,

sterile syringe filtering (SSF), was used to sterilize the SFWP without significantly altering the

composition of the waste. The newly sterilized waste, SSF-SFWP, was more similar to that of

Raw SFWP (Table 2.4).

C. sorokiniana grown on SSF-SFWP (Figure 2.19) showed a maximum optical density of

0.922 ± 0.281 OD750 in batch conditions after 11.1 days. In semi-continuous conditions, this

optical density was higher, at a value of 1.28 ± 0.02 OD750 and 1.23 ± 0.02 OD750 for 30% v/v and

50% v/v after 6 media replacements, respectively (Figure 2.20 A.). The biomass concentration

harvested at the end of the experiment for batch growth was 0.61 ± 0.08 g/L while semi-

continuous growth produced 0.98 ± 0.01 g/L and 0.94 ± 0.02 g/L for 30% v/v semi-continuous

growth and 50% v/v semi-continuous growth, respectively (Figure 2.20 B.). This equated to

biomass productivities of 350 mg/feed for 30% (v/v) operation and 600 mg/feed for 50% (v/v)

operation.

67
 Batch 30% v/v SC 50% v/v SC

Figure 2.19: Microalgae in Batch and Semi-Continuous Conditions on SSF-SFWP at Day 21

(from left to right) Batch: 1, 2, 3; 30% v/v SC (Semi-Continuous): 1, 2, 3; 50% v/v SC: 1, 2, 3.

There was no significant difference in the final biomass concentration or optical density

at 750 nm between 30% (v/v) or 50% (v/v) semi-continuous operation (Tukey-Kramer HSD,

0.95). There was a significant difference between the final biomass concentration and optical

density between semi-continuous growth and batch growth (Tukey-Kramer HSD, 0.95). Total

Biomass produced was, on average, 0.021 g/day for 30% (v/v) semi-continuous growth and

0.036 g/day for 50% (v/v) semi-continuous growth (Figure 2.20 C). These values were

determined through linear regression of the biomass harvested.

68
 A. 1.5
Optical Density (750 nm)

0.5

0
0 5 10 15 20 25

B. 1.2
Concentration (g/L)

0.8

0.4

0
0 5 10 15 20 25

C. 0.6
y = 0.036x - 0.269
Biomass Collected (g)

R² = 0.9865
0.4
p<0.0001

0.2 y = 0.021x - 0.166

R² = 0.9753
p<0.0001
0
0 5 10 15 20 25
Days

Figure 2.20: Microalgae (A.) Optical Density (750 nm), (B.) Biomass Concentration, and (C.)
Total Biomass Produced on SSF-SFWP
 30% Semi-Continuous Growth,50% Semi-Continuous Growth,  Batch Growth Graph
A. the optical density over time, B. the biomass concentration over time, and C. shows the total biomass produced.
n=3 for all measurements.

69
2.3.4.2. Nutrient Removal

The nutrient removal rates showed a reduction in sCOD, TN, N-NH4, and TON across all

treatments (Figure 2.21). C. sorokiniana reduced sCOD levels in 30% SSF-SFWP from the initial

2879 ± 12 mg/L to 972 ± 13 mg/L in batch conditions. Both 30% (v/v) and 50% (v/v) semi-

continuous conditions showed lower final concentrations of sCOD than batch conditions. The

30% (v/v) semi-continuous treatment had a final sCOD range between 590 ± 61 mg/L to 892 ±

74 mg/L with an average final sCOD removal concentration of 716 mg/L. Meanwhile, the 50%

(v/v) semi-continuous conditions had a range of final concentrations between 540 ± 8 mg/L to

753 ± 6 mg/L with an average final sCOD removal concentration of 658 mg/L (Figure 2.21 A.).

Nitrogen in the form of TN, N-NH4, and TON was reduced similarly to sCOD. TN started

at an initial concentration of 309.4 ± 9.5 mg/L and was reduced to 39.1 ± 5.6 mg/L in batch

conditions. A higher level of reduction was found in semi-continuous conditions with 30% (v/v)

reducing TN to a final average value of 25.5 mg/L and 50% v/v semi-continuous conditions

showing a reduction to a final average value of 23.4 mg/L (Figure 2.21 B.).

Final N-NH4 concentrations showed complete, or near complete removal, of ammonium

starting from an initial concentration of 126.4 ± 1.3 mg/L. A final concentration of 0.4 ± 0.2

mg/L N-NH4 was achieved in batch conditions. Semi-continuous conditions showed a reduction

further than that of batch to an average value between feeds of 0.1 mg/L. 30% (v/v) had a

range of final concentrations from 0.0 ± 0.4 mg/L to 0.4 ± 1.0 mg/L while 50% (v/v) had values

between 0.0 ± 0.3 mg/L to 0.2 ± 1.0 mg/L (Figure 2.21 C.).

TON was also reduced significantly from the initial concentration of 182.5 mg/L to 38.1 ±

0.1 mg/L in batch conditions. An average reduction to 25.0 mg/L was seen for 30% (v/v) semi-

70
continuous growth with a range from 14.3 ± 5.2 mg/L to 33.4 ± 2.5 mg/L. The average reduction

was lower in 50% (v/v) semi-continuous conditions at 23.6 mg/L with a range from 10.9 ± 0.1

mg/L to 34.0 ± 0.3 mg/L (Figure 2.21 D.).

71
A. 4000
Concentration (mg/L)
3000

2000

1000

0
0 5 10 15 20 25

B. 400
Concentration (mg/L)

300

200

100

0
0 5 10 15 20 25

C. 160
Concentration (mg/L)

120

80

40

0
0 5 10 15 20 25

D. 180
Concentration (mg/L)

120

60

0
0 5 10 15 20 25
Days

Figure 2.21: Microalgae Removal Dynamics of (A.) sCOD, (B.) TN, (C.) N-NH4, and (D.) TON in
Semi-Continuous SSF-SFWP
 30% Semi-Continuous Growth,50% Semi-Continuous Growth,  Batch Growth
Graph A. shows the sCOD dynamics, graph B. shows the sCOD dynamics, graph C. shows the N-NH4 dynamics, and
graph D. shows the TON dynamics. n=3 for all measurements.

72
 Microalgae treatments showed high rates of removal of sCOD, TN, N-NH4, and TON in

30% SSF-SFWP (Figure 2.22). While batch conditions showed a reduction in sCOD by 66.2%

overall, a higher final reduction was seen in semi-continuous conditions with a 75.7% reduction

for 30% (v/v) and 78.0% for 50% (v/v) (Figure 2.22 A.). The efficiency of sCOD removal

decreased over time, with the initial removal efficiency in both semi-continuous conditions

being the highest (69.0% and 73.8%, respectively) before decreasing to an average value of

18.8% for 30% (v/v) and 54.0% for 50% (v/v) between each media replacement. 50% (v/v)

showed lower variability in final sCOD concentrations across media replacements than those at

30% (v/v) (Figure 2.22 B.).

Final TN removal was calculated at 87.4% for batch conditions, 92.7% for 30% (v/v), and

92.2% for 50% (v/v) semi-continuous operation (Figure 2.22 C.). Removal efficiency decreased

through subsequent feeds to an average removal of 73.1% for 30% (v/v) and 79.2% for 50%

(v/v) semi-continuous operation (Figure 2.22 D.).

The removal of N-NH4 in was found to be 99.7% for batch conditions, and 99.9% for 30%

(v/v), and 100% for 50% (v/v) semi-continuous operation (Figure 2.22 E.). Near complete

removal rates were seen until the end of the second media replacement at day 14.1 to 57.8% in

50% (v/v) semi-continuous conditions and by the end of the fourth feed at day 16.1 to 74.1%

for 30% v/v semi-continuous conditions. The 50% (v/v) system fully recovered back to 100%

removal efficiencies before being decreased again before the last media replacement, where

sCOD removal efficiencies dropped to 64.4%. The 30% (v/v) treatment partially recovered and

an increase in the removal efficiencies to 88.9% was observed before decreasing to 55.6% at

the final measurement (Figure 2.22 F.)

73
 TON removal was calculated to be 79.1% for batch conditions, 87.5% for 30% (v/v), and

86.5% for 50% (v/v) semi-continuous operation (Figure 2.22 G.). The removal efficiency

decreased more in the 30% (v/v) treatment than in the 50% (v/v) treatment after the first

measurement (81.7% to 44.2% vs 82.3% to 70.2%, respectively). Removal efficiencies then

recovered completely and even increased for 50% (v/v) to 88.0%, but only went as high as

73.9% for 30% (v/v) semi-continuous operation (Figure 2.22 H.).

1 1
The rates of TN removal at the three different retention times (3/day, 2/day, and 1/day)

were 12.5 mg/day, 19.3 mg/day, and 36.1 mg/day in 30% v/v semi-continuous growth

treatments and 20.7 mg/day, 32.6 mg/day, and 60.1 mg/day in 50% (v/v) semi-continuous

growth treatments, respectively. Batch growth shows an average removal rate of 6.0 mg/day of

TN (Figure 2.23). The total quantity of TN removed between feeds for the three retention times

was 37.4 mg/feed, 38.6 mg/feed, and 36.1 mg/feed for 30% v/v semi-continuous growth and

62.0 mg/feed, 65.3 mg/feed, and 60.0 mg/feed for 50% v/v semi-continuous growth,

respectively. A total of 126.1 mg was removed during batch growth after 21 days (Table 2.9).

These relationships were developed with regression lines with an R2 value greater than 0.9997.

These removal rates were maximum at 37.4 ± 1.3 mg for 30% (v/v) semi-continuous operation

and 62.5 ± 2.6 mg for 50% (v/v) semi-continuous operation. The 50% (v/v) semi-continuous

operation removed 40.1% more nitrogen compared to the 30% (v/v) semi-continuous

operation. This is directly proportional to the 40% increase in volume removed using the 50%

(v/v) semi-continuous operation method.

74
 A. 100 100
B.
Percent Removal 75 75

50 50
(%)

25 25

0 0
5 15 25 5 15 25

C. 100 D. 100
Percent Removal

75 75

50 50
(%)

25 25

0 0
5 15 25 5 15 25

E. 100 100
F.
Percent Removal

75 75

50 50
(%)

25 25

0 0
5 15 25 5 15 25

G. 100 100
H.
Percent Removal

75 75

50 50
(%)

25 25

0 0
5 15 25 5 15 25
Days Days

Figure 2.22: Microalgae Removal Efficiency and Sequential Removal Efficiency of (A., B.)
sCOD, (C., D.) TN, (E., F.) N-NH4, and (G., H.) TON in Semi-Continuous SSF-SFWP
 30% Semi-Continuous Growth,50% Semi-Continuous Growth
Graph A. shows the total percent sCOD removal, B. shows the sequential sCOD removal, C. shows the percent TN
removal, D. shows the sequential TN removal, E. shows the total N-NH4 removal, F. shows the sequential N-NH4
removal, G. shows the percent removal of TON, H. shows the sequential TON removal. n=3 for all measurements.

75
 600

500
60.1 mg/day
TN Removed (mg)

400

32.6 mg/day
300
20.7 mg/day 36.1 mg/day
200
19.3 mg/day
12.5 mg/day
100
6.0 mg/day

0
0 5 10 15 20 25
Days

Figure 2.23: TN Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP
 30% Semi-Continuous Growth,50% Semi-Continuous Growth,  Batch Growth
n = 3 for all measurements.

Table 2.9: TN Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP
Feeding Rate 30% (v/v) Semi-Continuous 50% (v/v) Semi-Continuous
𝟏 12.5 20.7
/day (mg/day)
𝟑
Total/feed (mg) 37.4 62.0
R2 1.0000 1.0000
𝟏 19.3 32.6
/day (mg/day)
𝟐
Total/feed (mg) 38.6 65.3
R2 N/A N/A
1/day (mg/day) 36.1 60.1
Total/feed (mg) 36.1 60.1
R2 0.9997 0.9997

76
 The rate of the quantity of N-NH4 removed at the three different retention times of

1 1
/day, 2 /day, and 1/day had showed rates of 4.1 mg/day, 7.6 mg/day, and 14.7 mg/day for
3

30% (v/v) semi-continuous growth and 8.7 mg/day, 14.1 mg/day, and 27.1 for 50% (v/v) semi-

continuous growth. Batch growth showed an average removal rate of 2.6 mg/day (Figure 2.24).

The quantity of N-NH4 removed between feeds for the three retention times was 12.2 mg/feed,

15.2 mg/feed, and 14.7 mg/feed for 30% (v/v) semi-continuous operation and 26.0 mg/feed,

28.2 mg/feed, and 27.1 mg/feed for 50% (v/v) semi-continuous operation, respectively. A total

of 54.1 mg was removed during batch growth after 21 days (Table 2.10). These relationships

were developed with regression lines with an R2 value greater than 0.9986. N-NH4 removal

rates topped out at 14.1 ± 1.6 mg for 30% (v/v) semi-continuous growth and 27.1 ± 1.1 mg for

50% (v/v) semi-continuous growth. In this case, 50% (v/v) semi-continuous growth removed

48.1% more ammonia than 30% (v/v) semi-continuous growth despite being only an increase in

loading of only 40%.

77
 250

200
27.1 mg/day
N-NH4 Removed (mg)

150
14.1 mg/day

8.7 mg/day 14.7 mg/day

100
7.6 mg/day
50 4.1 mg/day
2.7 mg/day

0
0 5 10 15 20 25
Days

Figure 2.24: N-NH4 Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP
 30% Semi-Continuous Growth,50% Semi-Continuous Growth,  Batch Growth
n = 3 for all measurements.

Table 2.10: N-NH4 Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP
Feeding Rate 30% (v/v) Semi-Continuous 50% (v/v) Semi-Continuous
𝟏 4.1 8.7
/day (mg/day) 𝟑
Total/feed (mg) 12.2 26.0
R2 0.9997 0.9998
𝟏 7.6 14.1
/day (mg/day)
𝟐
Total/feed (mg) 15.2 28.2
R2 N/A N/A
1/day (mg/day) 14.7 27.1
Total/feed (mg) 14.7 27.1
R2 0.9994 0.9986

78
 The rate of sCOD removal by C. sorokiniana at the three different retention times was

18.5 mg/day, -0.5 mg/day, and 31.9 mg/day for 30% (v/v) semi-continuous operation and 53.2

mg/day, 79.1 mg/day, and 31.9 mg/day for 50% (v/v) semi-continuous operation. These rates

are compared with the average removal rate of 18.08 mg/day for batch growth (Figure 2.25).

The total amount of sCOD removed between feeds for the three retention times above was

55.6 mg/feed, -1.0 mg/feed, and 31.9 mg/feed for 30% (v/v) semi-continuous operation and

159.5 mg/feed, 158.2 mg/feed, and 147.9 mg/feed for 50% v/v semi-continuous operation,

respectively. A total of 381.6 mg was removed during batch growth after 21 days (Table 2.11).

The coefficient of regression for those of the 30% (v/v) semi-continuous growth showed a

larger deviation from the mean than those of the 50% (v/v) semi-continuous. R2 values for 30%

1
(v/v) semi-continuous growth at the 3 /day feeding rate was 0.9387, but at 1/day were 0.7341.

The values for those in 50% (v/v) semi-continuous operation had R2 values of 0.9993 and

0.9992, respectively. The sCOD removal rates in 30% (v/v) semi-continuous growth were highly

dependent on the feeding rate. Values for these rates were not consistent, and those of the 1/d

feeding rate showed a low R2 value. In addition, 30% (v/v) semi-continuous growth showed an

1
increase in sCOD for /day feeding rate. In this case, only one such feed was done, so no
2

statistical analysis could be computed to analyze the replication of this result. 50% (v/v) semi-

continuous growth, on the other hand, reached a maximum removal of 155.2 ± 6.4 mg for all

three feeding rates and appeared more robust than its 30% (v/v) counterpart. Across all three

1
feeding rates, the 50% (v/v) counterpart removed 319.8% more sCOD. When comparing the
3

/day feeding rate, which showed the highest removal of sCOD for the 30% (v/v) operation, the

50% (v/v) still removed over 186.8% more sCOD despite being a loading increase of only 40%.
79
 1600

1200 147.9 mg/day

sCOD Removed (mg)

79.1 mg/day
800
53.2 mg/day
-0.5 mg/day
31.9 mg/day
400
18.5 mg/day

18.08 mg/day
0
0 5 10 15 20 25
Days

Figure 2.25: sCOD Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP
 30% Semi-Continuous Growth,50% Semi-Continuous Growth,  Batch Growth
n = 3 for all measurements.

Table 2.11: sCOD Removal Rates for Microalgae in Batch and Semi-Continuous SSF-SFWP
Feeding Rate 30% (v/v) Semi-Continuous 50% (v/v) Semi-Continuous
𝟏 18.5 53.2
/day (mg/day) 𝟑
Total/feed (mg) 55.6 159.5
R2 0.9387 0.9993
𝟏 -0.5 79.1
𝟐
/day (mg/day)
Total/feed (mg) -1.0 158.2
R2 N/A N/A
1/day (mg/day) 31.9 147.9
Total/feed (mg) 31.9 147.9
R2 0.7341 0.9992

80
2.3.4.3. Analysis

Overall, the nutrient composition of SSF-SFWP was more similar to that of the Raw

SFWP characterized than VSF-SFWP. As such, results from this experiment could represent what

would be expected for the growth and nutrient removal capabilities of C. sorokiniana on Raw

SFWP without the influence of other microorganisms.

The 50% (v/v) semi-continuous growth produced higher removal rates of sCOD, TN, and

TON, while having the same removal efficiencies for N-NH4 as 30% (v/v) semi-continuous

growth. In addition, 50% (v/v) semi-continuous growth showed to be more robust as it had

higher removal rates between feeds. Analysis of the total nutrient removal showed that 50%

(v/v) semi-continuous growth removed 20.3% more N-NH4 per liter, and 320.0% more sCOD per

liter compared to 30% (v/v) semi-continuous growth. Such a phenomenon where higher

nutrient loads lead to higher nutrient removal efficiencies has been observed before [194].

Semi-continuous growth also reached higher biomass productivities than batch growth

with an average productivity of 350 mg/L/day for 30% (v/v) semi-continuous growth and 600

mg/L/day for 50% (v/v) semi-continuous growth vs 28.8 mg/L/d in batch growth. These results

suggest that semi-continuous growth could be a potential method of growing algae for large-

scale production as it led to higher productivities over traditional batch-grown microalgae in

this study.

Overall, 50% (v/v) semi-continuous operation was seen to be better at producing high

biomass productivities while also removing nutrients at rates similar to those, or higher than

those of 30% (v/v) semi-continuous operation.

81
2.3.5. Discussion

The growth of C. sorokiniana on 30% Raw SFWP, VSF-SFWP, and SSF-SFWP showed high

biomass productivities as well as efficient nutrient removal capabilities. These are summarized

in Table 2.12. The highest biomass concentration (1.79 ± 0.14 g/L) was achieved in the batch

growth of C. sorokiniana on Raw SFWP after 28 days. Such high concentrations were never

achieved on sterilized wastewaters. The highest biomass productivity for batch conditions was

89.8 ± 5.0 mg/L/d and was executed on VSF-SFWP with 6% CO2. The highest biomass

productivity in semi-continuous conditions was 616.7 mg/L/d which was obtained using 30%

VSF-SFWP. When grown on VSF-SFWP, microalgae showed a biomass production rate of ~600

mg/L/d despite the introduction of contaminants. In SSF-SFWP, the same productivity (600

mg/L/d) was achieved at higher media replacement rates of 50% (v/v). In all conditions, N-NH4

removal was >98%. In cases where sCOD concentration was higher than 1000 mg/L, favorable

removal efficiency was achieved. In other instances, microalgae produced more sCOD than

consumed. While nitrogen losses due to stripping were not measured, estimates using

stoichiometry suggest that this loss was minimal. These low stripping rates were confirmed in

Chapter 3.

These results show that microalgae can significantly reduce the nutrients from SFWP

while growing to high biomass densities in both batch and semi-continuous conditions. The

nutrient content of SSF-SFWP was the most similar to the Raw-SFWP of the wastewater

sterilized. Results showed that microalgae grew almost equivalently in 30% (v/v) semi-

continuous conditions on VSF-SFWP as 50% (v/v) semi-conditions conditions on SSF-SFWP. In

82
VSF-SFWP, biomass concentrations accumulated were higher than that of batch growth, likely

because semi-continuous conditions kept the microalgae in the exponential growth phase.

Batch growth of microalgae on Raw SFWP reached a higher biomass concentration than

that on sterile wastewater. This higher biomass concentration was hypothesized to be due to

the presence of microorganisms, namely bacteria, were able to break down organic compounds

and release carbon, nitrogen, phosphate, as well as other bound minerals that would aid in the

growth of microalgae. Some of these organic compounds found in wastewater act to hinder the

growth of microalgae on wastewater, so the breakdown would reduce such stress on

microalgae [195]. This constant release of nutrients into the wastewater prevented the culture

from reaching a stationary phase.

Overall the results suggest that higher loading rates (50% v/v) can be used to effectively

remove nutrients at a higher efficiency while producing high biomass productivities of

microalgae. Any commercial application of this approach would require this system to be

robust in non-sterile, open conditions. It is essential to understand how microalgae would grow

in the presence of different microbial communities as bacteria and microalgae can have

complex interactions. An in-depth study of mixed microbial communities produced with C.

sorokiniana growing in SFWP will be examined in the next chapter. Understanding this

interaction will help to assess the impact such microbes have on the total biomass

concentration as well as nutrient removal efficiencies from microalgae.

83
 Table 2.12: Results of Microalgae Grown on SFWP
Media CO2 Reactor Setup Days Growth Nutrients Removal (%)
30% Raw-SFWP atm Batch 28 1.79 g/L N-NH4 = 98.2
63.9 mg/L/d sCOD = 5.8
0.33 d -1

30% VSF-SFWP 6% Batch 11 0.99 g/L N-NH4 = 96.6

89.8 mg/L/d sCOD = -391.9
30% v/v SC 616.7 mg/L/d N-NH4 = 99.2
sCOD = -242.6
C* Batch 0.92 g/L N-NH4 = 98.1
83.7 mg/L/d sCOD = -274.6
C* 30%v/v SC 583.3 mg/L/d N-NH4 = 99.2
sCOD = -131.1
30% SSF-SFWP 6% Batch 21 0.61 g/L TN = 87.4
28.8 mg/L/d N-NH4 = 99.7
TON = 79.1
sCOD = 66.2
30% v/v SC 350 mg/L/d TN = 92.7
N-NH4 = 100
TON = 89.3
sCOD = 75.8
50% v/v SC 600 mg/L/d TN = 92.2
N-NH4 = 100
TON = 88.1
sCOD = 78.0
SC = Semi-continuous, C* = Contaminated, atm = atmospheric CO2 concentration (~0.04%)
TN = total nitrogen, N-NH4 = total ammonia nitrogen, N-ON = total organic nitrogen, TP = total phosphorus,
sCOD = soluble chemical oxygen demand

84
3. Chapter 3 - Identify the Microbiome in the Microalgae-Wastewater System

3.1. Background

The microalgae microbiome has long been of interest due to its importance in ecological

systems [163]. These complicated and sophisticated bacteria-microalgae interactions are very

poorly understood, yet vital to the understanding of how aquatic ecosystems operate.

Microalgae are unique in that they stimulate their microbiomes. These are often referred to as

phycospheres.

Phychospheres are mucus regions surrounding microalgae cells that are often inhabited

by other microorganisms [163]. It is analogous to the rhizosphere in plants. These coexisting

bacteria interact with microalgae in many different relationships including mutualism,

parasitism, commensalism, amensalism, synergism, predation, and competition. In many cases,

complex carbon compounds from microalgae can be utilized by bacteria, while bacteria can act

to convert micro and macronutrients into usable forms for microalgae as well as excrete

vitamins and other co-factors that are required for growth by some microalgae [163]. Other

bacteria may also produce auxins, or plant growth hormones, which help microalgae grow and

divide more quickly [196]. Certain species of bacteria that grow in the rhizosphere in plants can

interact with microalgae in a similar way [197]. Metabolically, microalgae produce O2 which can

be utilized by bacteria for respiration, and, in return bacteria produce CO2 which can be taken

up by microalgae. At the same time, they both compete for macronutrients, micronutrients, O2,

and space. In some cases, these organisms may produce inhibitory products (i.e., algae can

produce bactericides while bacteria can produce algicides). Some types of microalgae have also

been documented to prey on other microalgae for growth [198].

85
 The other way microalgae and bacteria interact through the macroenvironment where

these organisms will alter the larger ecosystem as a result of their growth. An excellent example

of this is eutrophication, in which microalgae grow at unsustainable rates in an environment

due to a substantial increase of nutrients into water systems. This process results in oxygen

depletion from the water, shading, and in some cases, the release of large quantities of toxins

into the environment [199]. All of these factors can have significant impacts on the microbial

community. On the other hand, certain species of bacteria can alter the environment similarly

and affect the growth of microalgae. These changes include depletion of O2 in the water,

utilization of inorganic nutrients, high CO2 production which decreases the pH of the water, the

release of inhibitory products to the water, and predation [163]. A summary of possible

bacterial and microalgae interactions in wastewater is shown in Figure 3.1.

In either of these cases, it is hard to tell which bacteria are beneficial or harmful to

microalgae since they can affect each other’s growth in many ways. One method to find

relationships is to develop correlations between different environmental or biological

conditions and the specific organisms of interest. Obtaining this information requires the

identification of microorganisms in these microbial communities.

Early techniques for identifying microorganisms utilized dichotomous and diagnostic

keys that would identify specific traits of organisms that could be observed or tested for [200].

These techniques included microscopy, organism isolation, staining, and metabolite

identification. While this process was appropriate for identifying specific organisms, it was

insufficient for mapping out the entire microbial community.

86
 Figure 3.1: Potential Interactions Between Bacteria and Microalgae in Wastewater
Bacteria and microalgae can utilize nutrients from wastewater and interact with one another. Competition is over
simple organic compounds, O2, N, P, micronutrients, and space.

In recent years, new technology has allowed for the mass identification of organisms by

examining genes on their DNA, rather than biological traits [201]. This system allows for

accurate, high-throughput organism identification and classification. Also, identification using

nucleic acid approaches works regardless of any divergent traits that are seen between

organisms of different lineages. It will also work even if the organism in question has never

been identified before or if it cannot be cultured. Unlike dichotomous keys, identification is not

87
prone to error from human interpretation of physical traits such as structure. The main reason

nucleic acid approaches have become more popular in recent years is that they can be done

relatively inexpensively and with high accuracy [202], which allows for the identification of

organisms in a sample through microbial community analysis (MCA).

MCA is accomplished through the amplification of genes on highly conserved regions of

DNA. In most cases, the gene encoding the 16S rRNA (the portion of the DNA that codes for

ribosomal RNA) is used as an indicator of phylogenetic identification in bacteria [203]. A part of

the DNA sequence is amplified through PCR (polymerase chain reaction) to create measurable

quantities of the conserved genetic sequence [204]. DNA sequencing is performed on the PCR

amplified regions of DNA, and those reads are compared to a database of known sequences to

identify the OTU (operational taxonomic unit) [205], based on some threshold of identification.

This process is carried out for one to millions of DNA sequences within a sample. After all the

reads are complete, data is compiled and analyzed to determine the relative abundance of

OTUs based on the amplified sequence reads per total number of reads.

In some cases, predictive metagenomics may be performed to identify the physical or

genetic characteristics of the organisms that have the potential to be present in the microbial

community [206]. While this data is just an inference of genes within the community, the

analysis provides another tool to be used to understand the complex interactions within the

community.

This process has allowed for the large-scale identification of many different microbial

communities in a short period. A general overview of this process for the application of

88
identifying the microbial community associated with the growth of microalgae on wastewater is

shown in Figure 3.2.

A.

B.

D. C.

Figure 3.2: Microbial Community Analysis of Microalgae Microbial Community

Steps include the A. extraction of DNA from the wastewater sample, B. sequencing and quantification of DNA, C.
identification of the DNA through a database, and D. mapping and analysis of identified OTUs.

Past studies have identified distinct OTUs within the microalgae-wastewater

microbiome. One investigation that utilized anaerobic digestate produced from piggery waste

identified four unique Bacteria OTUs and 3 Archaeal OTUs through DGGE (denaturing gradient

gel electrophoresis). Of the bacteria, three taxa were dominant: Bacteria (1), Proteobacteria (2),

and Betaproteobacteria (3). All Archaea OTUs were only identified down to the domain level

[207]. A study of municipal wastewater with cyanobacteria dominated microalgae culture

found eight dominant OTUs using DGGE. The final consortium was 50% Bacteroidia, 25%

Flavobacteria, 12.5% Betaproteobacteria, and 12.5% Gammaproteobacteria [208]. Following up

89
on this study, the authors tested different concentrations of microalgae biomass mixed with

sludge to identify which mixture produced the highest nutrient removal efficiency. A 5:1 ratio of

microalgae biomass to activated sludge system removed 91.0% of nitrogen, 93.5% of total

phosphate, and >91.2% sCOD after 10 days from an initial concentration of 47.6 mg/L, 8.6 mg/L,

and 380.0 mg/L, respectively. The bacterial community was dominated by Alphaproteobacteria,

Gammaproteobacteria, Flavobacteria, and Sphingobacteria [209].

Analysis of the microbiome from the OMEGA project (see section 1.1.2) showed that a

Desmodesmus and Scenedesmus dominated microalgae culture initially had a microbial

community dominated by Proteobacteria, specifically Gammaproteobacteria. As wastewater

was added as a form of nutrients over time, the microbial community shifted to being

dominated by Bacteroides (Flavobacteriia, and Sphingobacteriia) and Cytophagia after 13 days

[210]. One study looked to grow a mixed microalgae culture dominated by Scenedesmus on

municipal wastewater mixed with lake water in lab-scale photobioreactors. The microbiota was

found to be made up mainly by Proteobacteria and Bacteroides (Sphingobacteriaceae,

Comamonadaceae, and Planctomycetaceae). Nutrient removal efficiencies of TN, N-NH4, and

TP were 49.3%, 100%, and 100%, respectively, after 16 days [211]. A similar study looked at the

microbiota that developed in a semi-continuous anoxic-aerobic alga-bacterial photobioreactor

grown with synthetic wastewater in continuous operation with an HRT (hydraulic retention

time) of 2 days. Over 80% of the organisms were identified to belong to the phylum

Proteobacteria. Removal efficiencies of TOC and TN were approximately 80%, while N-NH4

removal efficiency achieved 95% [212]. A study growing microalgae Picochlorum sp. biofilms on

synthetic textiles fed with saline agricultural wastewater effluents in a fixed-bed

90
photobioreactor found that such a system was able to remove as much as 95% of the TN and

sCOD within 5 hours of wastewater addition. Gammaproteobacteria dominated the microbial

community with the majority within this taxon belonging to the genus Pseudomonas [213].

Another study demonstrated that Chlorella sp. that grew in shaking flasks with CO2 air bubbling

bioreactors fed with swine lagoon wastewater removed high concentrations (>90%) of sCOD, N-

NH4, and TP in 3 days. The microbial community in the shaking flasks was dominated by

Cytophagia (62.7%), Trebouxiophyceae (23.2%), and Alphaproteobacteria (7.6%). In the CO2

bubbling reactors, the microbial community was dominated by Trebouxiophyceae (35.0%),

Alphaproteobacteria (20.7%), Betaproteobacteria (18.8%), Cytophagia (9.8%), and

Flavobacteriia (7.3%) [214]. In a more recent study testing microalgae Auxenochlorella

protothecoides and Chlorella sorokiniana growing on winery wastewater, researchers found

both species of microalgae to grow better on wastewater than on control media [215]. The

microbial community did not increase the microalgae biomass productivity, but high rates of TN

and N-NH4 removal were observed (~100%). Table 3.1 summarizes these studies.

The goal of the experiment in this chapter was to identify the microbial community that

co-exists with microalgae in Raw SFWP. It was expected that different biomass concentrations

and nutrient removal efficiencies would be observed and potentially correlated with specific

OTUs identified through the process described above. Such results were supposed to be similar

to those achieved in previous studies in that Proteobacteria would dominate the community.

91
 Table 3.1: Prior Research on the Microalgae-Wastewater Microbial Community
Culture Wastewater Dominant Organisms Nutrient Source
Removal (%)
Mixed microalgae Piggery Bacteria, Proteobacteria, [207]
Anaerobic Betaproteobacteria, Archaea
Digestate
Mixed microalgae Municipal Bacteroidia, Flavobacteria, sCOD = 98.2 [208]
cyanobacteria Wastewater Betaproteobacteria, TKN = 88.3
dominant Gammaproteobacteria TP = 64.8

Mixed microalgae + Municipal Alphaproteobacteria, sCOD > 91.2 [209]

active sewage Wastewater Gammaproteobacteria, TN = 91.0
sludge (5:1 ratio) Flavobacteria, Sphingobacteria TP = 93.5

Mixed microalgae Municipal Gammaproteobacteria to [210]

Desmodesmus/ Wastewater Bacteroides, Cytophagia
Scenedesmus
dominant

Mixed microalgae Municipal Proteobacteria, Bacteroides TN = 49.3 [211]

Scenedesmus Wastewater N-NH4 = 100
dominant + Lake Water TP = 100

Mixed microalgae Synthetic Proteobacteria TOC = 88 [212]

Chlorella vulgaris Wastewater TN = 76
and N-NH4 = 95
Pseudanabaena
dominant

Picochlorum sp. Saline Gammaproteobacteria sCOD >95 [213]

Agricultural TN >85
Effluents
Mixed microalgae Swine Alphaproteobacteria, sCOD = 88 [214]
Chlorella sp. Lagoon Betaproteobacteria, N-NH4 = 98
dominant Wastewater Cytophagia, Flavobacteria, TP = 93
Trebouxiophyceae
Auxenochlorella Winery Proteobacteria, Bacteroidetes sCOD = -38 [215]
protothecoides Wastewater TN = 100
N-NH4 = 100
Chlorella Winery Proteobacteria, Bacteroidetes sCOD = -70 [215]
sorokiniana Wastewater TN = 100
N-NH4 = 100
TN = total nitrogen, TKN = total Kjeldahl nitrogen, N-NH4 = total ammonia nitrogen, TP = total phosphate, sCOD =
soluble chemical oxygen demand, TOC = total organic carbon

92
3.2. Materials and Methods

3.2.1. Microalgae Strain Selection

Similar to Chapter 2, the microalgae strain selected for use in this study was Chlorella

sorokiniana UTEX 2805 from the University of Texas Culture Collection of Algae. See 2.2.1 for

more information.

3.2.2. Wastewater Collection and Preparation

SFWP used in this study was collected from the UC Davis Renewable Energy Anaerobic Digester

(READ) facility. This liquid digestate was further processed through ultrafiltration to produce a

permeate, that was then stripped to remove additional nitrogen. The final product, stripped

food waste permeate (SFWP), was stored at 4°C for approximately two months before the

experiment. The properties of this Raw SFWP used in this study are shown in Table 3.2.

Table 3.2: Nutrient Composition of 30% Raw SFWP

Parameter Concentration (mg/L)
TN 52.0 ± 10.1
N-NH4 18.4 ± 0.6
N-NO3 0.7 ± 0.1
N-ON 32.9 ± 10.1
sCOD 387 ± 58
TN = total nitrogen, N-NH4 = total ammonia nitrogen, N-NO3 = total nitrate/nitrite nitrogen, N-ON = total organic
nitrogen, TP = total phosphate, sCOD = soluble chemical oxygen demand
n=3 for all measurements.

3.2.3. Microalgae Cultivation

Pre-cultures were grown in N8-NH4 media [188] to an optical density (750 nm) of 0.350 OD750

(equivalent to 0.24 g/L of microalgae). 20 ml of this algae stock was inoculated in sterilized

hybridization tubes, 9 with 180 ml of 33% SFWP (diluted with autoclaved distilled water) to

93
produce a 30% SFWP culture. Three additional Hybrid Bottles were set up with 200 ml of 30%

SFWP to monitor the change in the microbial community and nutrients without microalgae.

Hybrid Bottles were randomly organized in sets of 4 and placed in 20-gallon fish tanks filled

with water to moderate temperature.

Bioreactors were grown inside under approximately 10,000 LUX light illumination from

T5 growth lamps (16∶8 ratio light-dark cycle). Caps were modified to allow for air flow in and

out of the system through 0.22 um sterile syringe filters. Humidified air with 6% CO2 was

supplied to each bioreactor at a rate of approximately ~50 ml/minute. Reactor contents were

stirred at the bottom of each bottle at ~100 rpm using a ½ inch stir bar.

Samples for optical density were taken after hours 73 and 166 and measured at 750 nm

and 680 nm using a microplate reader (Spectramax M2, Molecular Devices, Sunnyvale, CA).

Correlations were developed for each optical density against the other (Appendix 8.5). At the

last time point, ~190 ml of sample was removed for biomass dry weight measurements and

nutrient composition. Algae cells were washed 3 times and concentrated into a single 50 ml

tube and separated from solution through centrifugation at 5000 g for 5 minutes (IEC Multi RF,

Thermo Electron Corporation, Waltham, MA) before the separation of effluent and cell

contrate. Samples were immediately frozen at -20°C for later biomass and nutrient analysis.

Similarly, E. coli DH5α [216] was grown on 200 ml LB broth [217] for 19 hours at 37°C at

~200 RPM. The culture was washed 3 times and concentrated into a single preweighed plastic

15 ml tube and subsequently centrifuged at 5000 g for 5 minutes (IEC Multi RF, Thermo

Electron Corporation, Waltham, MA) to remove the effluent from cell concentrate. Samples

were frozen at -20°C for later biomass analysis.

94
3.2.4. Biomass Quantification

SFWP biomass samples were thawed and concentrated into preweighed plastic 15 ml tubes

before being pelleted at 5000 g for 5 minutes (IEC Multi RF, Thermo Electron Corporation,

Waltham, MA). The pellets were lyophilized at −45°C for two days (Freezone4.5, Labconco,

Kansas City, MO) and weighed on an analytical balance. The weight of the reweighed tubes was

subtracted from that of the tube with biomass for final dry weight calculations. Biomass

concentration was calculated from this. Correlations were made for optical density (750 nm)

against the biomass concentrations (Appendix 8.5).

The E. coli pellet was lyophilized at −45°C at the California Processing Tomato Industry

Pilot Plant within the August A. Busch III Brewing and Food Science Laboratory (University of

California Davis, Davis, CA). Final weight was measured, and the weight of the reweighed tubes

was subtracted from that of the tube with biomass for final dry weight calculations.

3.2.5. Nutrient Quantification

Fresh and spent media were thawed for each of the SFWP samples and homogenized before

being analyzed for nutrient content using a variety of colorimetric assays (HACH, Loveland, CO).

These tests included total nitrogen (Nitrogen-Total Reagent Set TNT Persulfate Digestion

Method HR), ammonia nitrogen (Nitrogen-Ammonia Reagent Set TNT AmVer Salicylate HR),

and soluble chemical oxygen demand (COD Digestion Vials HR). The colorimetric changes were

analyzed using a scanning spectrophotometer (DR 2500, HACH, Loveland, CO).

95
3.2.6. DNA Extraction, Quantification, and Verification

DNA was extracted from lyophilized biomass (DNeasy PowerSoil Kit, Qiagen, Hilden, Germany).

Quality was analyzed using fluorometric quantitation assay (Qubit dsDNA BR Assay Kit,

Invitrogen, Carlsbad, CA) on a fluorometer (Qubit Fluorometer, Invitrogen, Carlsbad, CA) and

through spectrophotometry (NanoDrop One, Thermo Scientific, Waltham, MA). DNA quality

and concentration data are shown in Appendix 6.

3.2.7. Microbiome Analysis

Sequencing of the extracted bacterial DNA in the microalgae/wastewater system was done

using a MiSeq amplicon metagenomic sequencing machine (Illumina, San Diego, CA) through

RTL Genomics (Lubbock, TX). The V4-V5 region of the 16S rDNA was amplified using primers

515F-Y (5′ GTGYCAGCMGCCGCGGTAA) and 926R (5′ CCGYCAATTYMTTTRAGTTT).

Sequences were analyzed using Qiime 2 [205] where they were they were processed

following the “Moving Pictures” tutorial (https://docs.qiime2.org/2018.8/tutorials/moving-

pictures/) using the Greengenes [218] database of 16S rDNA sequences. Analysis of the data

was performed through R version 3.5.1 [219] using the jtclaypool/microbiome

(https://github.com/jtclaypool/microbiome) package and VEGAN [220]. Figures were

produced using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA).

3.2.8. Quantitative PCR

Quantitative PCR (qPCR) was performed on extracted DNA to measure bacterial concentrations

indirectly. DNA from model organism E. coli DH5α was used as a standard for qPCR. DNA

96
concentrations were normalized with a biomass concentration of SFWP 3 as the SFWP sample

had a microbial community most similar to that of the algae cultures (Figure 3.11, Figure 3.12,

Figure 3.13). This normalization was used as the standard for DNA per unit of bacterial biomass.

The accuracy between the actual and predicted biomass concentrations of the Initial samples

using this method were 62.88%, 100.91%, and 108.96%. In the other SFWP samples, this

accuracy was calculated at 30.07% for SFWP 1 and 12.83% for SFWP 3. This process assumes

little variability in DNA per unit biomass between samples with and without microalgae. Since

this is a clear distinction between the accuracy of this standard for the SFWP samples, this value

given for bacteria biomass concentration can only be considered an estimate since it is not a

direct measure.

Samples were amplified using the 799F-mod3 (5′-CMGGATTAGATACCCKGG-3′) and

926 R (5′-CCGTCAATTCMTTTRAGTTT-3′) primers. The 799F-mod3 primer has been shown to

avoid amplification (>99% in a 50:50 microalgae-bacteria mix) of chloroplast 16S rRNA genes

[221].

The DNA was amplified using a StepOnePlus Instrument (Applied Biosystems, Foster

City, CA) using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Each

reaction well had a total of 25 μl, with 2 μl of template DNA, 5 μl of 0.5 μM of each primer,

0.5μl of 5 μg/ml BSA, and 12.5 μl of 2X SYBR Green. E. coli DNA was serially diluted as a

standard 1:10 to achieve five concentrations. The reaction included 40 cycles of 95 °C for 15

s, 47 °C for 15 s, and 72 °C for 45 s.

97
3.2.9. Predictive Metagenomics

Predictive metagenomics was performed using a taxonomic-to-phenotypic prediction program

to identify the putative functional behaviors of the bacteria in the SFWP. The web-based

metagenomic program, METAGENassist [222], was used in coordination with the

METAGENassist Tutorial [223] to utilize the taxonomic identification output from Qiime 2. Data

were normalized, as per the tutorial, through row-wise normalization through normalization by

sum and column-wise normalization through Pareto scaling. No data filtering was performed.

The OTUs were matched using KEGG [224] orthology to identify characteristics of the organisms

in the community.

3.2.10. Data Analysis

Data analysis, statistical comparisons, and transformations were performed using JMP v.14 (SAS

Institute, Cary, NC) for regression [190], ANOVA (analysis of variance) [191], Tukey-Kramer

Honest Significant Difference tests [190], and Box-Cox Transformations [225]. Additional

statistical calculations were performed using R version 3.5.1 (Vienna, Austria) [226]. These

included non-metric multidimensional scaling (NMDS) using Bray-Curtis dissimilarity [227],

Principal Component Analysis (PCA) [228], Hierarchical cluster analysis using Ward’s selection

criteria [229] and Pearson’s correlation [230]. The latter two statistical methods, as well as t-

tests, were normalized using the method described in 3.2.9. Data were graphed using Microsoft

Excel 2016 (Microsoft Corporation, Redmond, WA) and R version 3.5.1 (Vienna, Austria).

98
3.3. Results

3.3.1. Microalgae Growth

C. sorokiniana showed highly variable growth (Figure 3.3) on unsterilized SFWP as

demonstrated by both optical density measurements taken at 750 nm and in final biomass

concentration (Figure 3.4) in all nine reactors (Algae 1-9). At 166 hours, Algae 2, 3, and 5

continued to show growth through optical density measurements, while Algae 1, 4, 6, 7, 8, and

9 showed a decline in growth from hour 73. Of these reactors, the highest biomass

concentration after 166 hours was 0.901 g/L, and the lowest was 0.142 g/L. The average

concentration of the freeze-dried algae-bacteria biomass was 0.563 ± 0.305 g/L. Each

bioreactor has biomass concentrations higher than that found in the Initial biomass

concentration (0.0688 g/L).

Final biomass concentrations for the three bioreactors with just unsterilized SFWP

(SFWP 1-3) showed little change from the initial optical densities at 750 nm Figure 3.5. The final

biomass concentrations were 0.019 g/L, 0.029 g/L, and 0.041 g/L for samples 1, 2, and 3,

respectively. These values were slightly lower than the initial concentration (Figure 3.6).

Bacterial biomass concentrations estimated using qPCR (Figure 3.7) show that the

overall concentration of bacteria biomass decreased from that of the Initial (66.3 mg/L) samples

in all cases except in Algae 5. In seven of the nine bioreactors, no more than 3% of the total

biomass was estimated to be bacterial, except Algae 4 (19.6%) and Algae 5 (50.7%).

99
Figure 3.3: Hybrid Tube Bioreactors with Microalgae (Left to Right: Algae 1-9) on SFWP after
166 Hours

100
A. 2
Optical Density (750 nm)

1.5

0.5

0
0 2 4 6
Days

B. 1

0.8
Biomass Concentration (g/L)

0.6

0.4

0.2

0
Initial Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9
Figure 3.4: Microalgae-Bacteria (A.) Optical Density (750 nm) and (B.) Biomass Concentration
on SFWP
 Algae 1,  Algae 2,  Algae 3, - Algae 4, -- Algae 5,  Algae 6, Algae 7,  Algae 8,  Algae 9,  Initial
n = 3 for optical density measurements, n = 1 for biomass concentrations.

101
 Figure 3.5: Hybrid Tube Reactor of SFWP (Left to Right: SFWP 1-3) After 166 Hours

A. B.
0.12 0.12
Biomass Concentration (g/L)
Optical Density (750 nm)

0.06 0.09

0 0.06
0 2 4 6

-0.06 0.03

-0.12 0
Days Initial 1 2 3

Figure 3.6: (A.) Optical Density and (B.) Biomass Concentration of SFWP
 SFWP1,  SFWP2,SFWP3, Initial
n = 3 for optical density measurements, n = 1 for biomass concentrations.

102
 1

0.8
Biomass Concentration (mg/L)

0.6

0.4

0.2

0
Initial Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9
Figure 3.7: Microalgae and Bacteria Biomass Concentrations from SFWP
Bacteria, C. sorokiniana
n=1 for all measurements.

103
3.3.2. Nutrient Removal

Nutrient removal differences between the nine algae bioreactors were highly variable.

Final sCOD concentrations for algae samples ranged from 332 mg/L to 620 mg/L with an

average value across the algae samples of 448 ± 101 mg/L (Figure 3.8 A.). sCOD removal rate

efficiencies ranged from -60.3 to 14.0% with an average removal rate efficiency of -15.8 ±

26.2% (Figure 3.8 B.). Only three reactors showed a reduction of sCOD from the initial values

(387 ± 58 mg/L): Algae 4 (332 mg/L), Algae 9 (362 mg/L), and Algae 3 (385 mg/L). These removal

efficiencies were calculated at 14.0%, 6.4%, and 0.5%, respectively. Both Algae 1 (598 mg/L)

and Algae 2 (620 mg/L) showed much higher concentrations of sCOD than those of the initial

SFWP, which equated to production efficiencies of 54.7% and 60.3% in total sCOD. The rest of

the microalgae bioreactor samples had values slightly higher than that of the initial

concentration.

The SFWP samples without any microalgae had final sCOD concentrations that ranged

from 403 mg/L to 469 mg/L with an average value of 436 ± 33 mg/L and sCOD removal rates

efficiencies between -21.2% to -4.2% with an average of -12.6 ± 8.5%. These were all higher

than the initial value of sCOD in SFWP (387 ± 58 mg/L).

The final concentrations of TN were also highly variable amongst microalgae samples

but lower than the initial concentration (52.0 ± 10.01mg/L). Final concentrations for TN in

microalgae bioreactors ranged from 16.7 mg/L to 29.3 mg/L, with an average final TN

concentration of 27.3 ± 5.2 mg/L (Figure 3.8 B.). Removal rate efficiencies ranged from 33.6% to

66.8% with an average removal efficiency of 45.7 ± 10.4% (Figure 3.9 B.). This TN removal

correlated significantly with microalgae biomass concentration (g/L) (Figure 3.10, Table 3.3). In

104
this case, data points were normalized using a Box-Cox transformation on microalgae biomass

(λ = 0.621).

SFWP without microalgae showed a final TN concentration that ranged from 39.3 mg/L

to 44.0 mg/L at an average value of 42.0 ± 5.2 mg/L. The removal efficiency was between 12.2%

and 21.6% with an average of 16.3 ± 4.8%.

Approximately 35% of the TN was in the form of N-NH4, which occurred at an initial

concentration of 18.4 ± 0.6 mg/L. In the microalgae bioreactors, the final N-NH4 concentration

ranged from 0.5 mg/L to 6.5 mg/L with an average value of 3.0 ± 1.6 mg/L (Figure 3.8 C.). These

final concentrations equated to a TN removal efficiency that ranged from 80.1% to 92.3% with

an average of 84.0 ± 8.9% (Figure 3.9 C.).

In SFWP without microalgae, the final TN concentrations ranged from 18.4 mg/L to 19.5

mg/L with an average value of 16.7 ± 2.9 mg/L. Removal efficiencies ranged from -6.0% to

25.7% with the average being 9.4 ± 15.9%.

Total nitrate/nitrite was measured in a randomly selected sample (Algae 6) to determine

if nitrate/nitrite was a significant contributor to the total nitrogen concentration. A

concentration of 1.11 g/L of nitrate/nitrite was measured, suggesting that nitrate played a small

role in the total nitrogen. As such, no additional measurements were taken for nitrate/nitrite

values on other samples.

105
 A. 800
Concentration (mg/L)

600

400

200

0 Initial SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9

B. 60
Concentration (mg/L)

40

20

0 Initial SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9

C. 20
Concentration (mg/L)

15

10

Figure 3.8: Final Concentration of (A) sCOD, (B) TN, and (C) N-NH4 of Microalgae on SFWP
◼ Final sCOD Concentration, ◼ Final TN Concentration, ◼ Final N-NH4 Concentration
A. is the final sCOD concentration, B. is the final TN concentration, and C. is the final N-NH4 concentration. n=1 for
all measurements.

106
 A.
Percent Removal (%) 100

50

0 SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9

-50

-100

B. 100
Percent Removal (%)

75

50

25

0 SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9

C. 100
Percent Removal (%)

75

50

25

-25
Figure 3.9: Removal Efficiencies of (A) sCOD, (B) TN, and (C) N-NH4 of Microalgae on SFWP
◼ Removal Efficiency of sCOD, ◼ Removal Efficiency of TN, ◼ Removal Efficiency of N-NH4
A. is the sCOD removal efficiency, B. is the TN removal efficiency, and C. is the N-NH4 removal efficiency. n=1 for all
measurements.

107
Table 3.3: Correlation Between Microalgae Biomass Concentration and TN Removal
Organisms Formula R2 Radj2 RMSE P-Value

Microalgae -13.068x + 14.474 0.4813 0.4072 4.029 0.0382*

*Significant at 95%
40
Percent TN Removal (%)

30

20

10
0.2 0.4 0.6 0.8 1
Box-Cox Transformed Microalgae Biomass
Concentration (λ = 0.621)

Figure 3.10: Correlation Between Microalgae Biomass Concentration and TN Removal

108
3.3.3. Microbial Community Analysis

Analysis of the microbial community showed that there were quantifiable differences between

the microbial communities that evolved in the microalgae bioreactors compared to the

bioreactors without microalgae. Both of these treatments were also found to be different from

the community that was initially found in the SFWP. This difference can be seen using non-

metric multidimensional scaling with Bray-Curtis dissimilarity (Figure 3.11). Similarly, Principal

Component Analysis (PCA) using the loadings PC 1 (34.9%) and PC 2 (18.5%) with a 95%

confidence interval showed that there was a similarity between SFWP samples and Algae

samples, but both of these samples were significantly different from the initial samples (Figure

3.12). Three dimensional PCA with loadings of PC 1, PC 2, and PC 3 (14.0%), show that there is a

clear distinction between Algae and SFWP with Initial treatments (Figure 3.13). The SFWP

samples show little distribution on PC 2 and PC 3, while Algae samples show different levels of

distribution across all three PCs. Both NDMS and PC confirm there is a difference between

microbial communities before and after the experimentation.

Loading scores from PCA were then used to analyze the OTUs across all samples (Figure

3.14). The OTU with the highest Loading 1 values were Aquamicrobium, Rhizobiales,

Xanthobacteraceae, Pseudomonadaceae, and Alcaligenaceae while the lowest values in Loading

1 were Halomonas, Petrimonas, Bacteroidales, Firmicutes, and Desulfurispirillum. Other notable

OTUs, as well as those with high values in Loading 2, are shown in Figure 3.14, and all OTUs and

their corresponding loading values are in Appendix 8.6. Differences between the treatments

were also analyzed using hierarchical cluster analysis (HCA) with Ward’s method for selection

criteria [229] and Pearson’s correlation [230] for similarity distance (height) (Figure 3.15). The

109
results from this method identified two groups: Initial samples and those with and without

microalgae (SFWP and Algae). This method showed that there was some overlap between the

Algae and SFWP treatments as well. Specifically, Algae 4, Algae 1, Algae 2, and SFWP 3 were

very similar to each other and Algae 3, Algae 5-9, and SFWP 1-2 were similar to each other. This

similarity suggests that the microbial communities that develop were very different from those

present in the initial conditions of the wastewater, but not different between those with and

without microalgae.

110
 1

Algae8

0.5
Algae9

Algae5 Initial1
Algae3
NDMS2

Initial3

Algae7 0
Algae2
-2 -1 Algae6 Algae1 0 1 2
Initial2

Algae4
SFWP1 SFWP3
-0.5
SFWP2

-1
NDMS1

Figure 3.11: NDMS of Microbial Community in SFWP

 Initial,  SFWP, Algae

111
Figure 3.12: PCA with 95% CI of Microbial Community in SFWP
 Initial,  SFWP, Algae

112
Figure 3.13: 3D PCA with 95% CI of Microbial Community in SFWP
 Initial,  SFWP, Algae

113
 0.4

Aquamicrobium

Bacteria
0.2 Xanthobacteraceae
Rhodobacteraceae Rhizobiales
Microbacteriaceae
Petrimonas Phyllobacteriaceae
Bacteroidales
Firmicutes Desulfurispirillum

Halomonas Vibrio 0
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4
PC 2 (18.5%)

-0.2 Flavobacteriaceae

Alcaligenaceae
-0.4

-0.6

Pseudomonadaceae

-0.8
PC 1 (34.9%)

Figure 3.14: Correlation Loading Plot of the Microbial Community in SFWP

PCA loading values correspond to PC1 (34.9%) for Loadings 1 and PC2 (18.5%) for Loadings 2. Only highly
influential OTUs are labeled on the plot.

114
 Figure 3.15: Dendrogram of Microbial Community Similarity on SFWP
Selection criteria made using Ward’s method, and height measured using Pearson’s correlation.

115
 Further analysis of the microbial community showed that the phylum Proteobacteria

dominated the microbial community across all treatments (Figure 3.16), making up at least

51.64% of the total OTU count. Of the Proteobacteria, three classes were abundant across all

samples: Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria (Figure 3.17).

These OTUs were further identified down into order, family, and genus (Figure 3.18, Figure

3.19, Figure 3.20).

The SFWP microbial community found in the Initial SFWP (Initial 1-3) was made up of

the following: Proteobacteria of the class Gammaproteobacteria (58.00 ± 6.57%), Bacteroides

of the class Bacteroidia (16.59 ± 4.04%); Firmicutes (12.67 ± 10.06%) in the classes Clostridia

(4.47 ± 1.32%) and unknown Firmicutes (8.20 ± 8.87%); Chrysiogenetes of class Chrysiogenetes

(5.49 ± 2.72%), unknown bacteria (4.60 ± 3.56%), Lentisphaerae of unknown class (1.29 ±

1.16%), and Tenericutes from the class Mollicutes (1.37 ± 0.11%) (Figure 3.20) . Of the

Gammaproteobacteria, the genus Halomonas was found in a relative abundance of 44.63 ±

5.12%, and the relative abundance of family Pseudomonadaceae was 7.90 ± 1.00% with genus

Pseudomonas at 3.41 ± 1.90%. The relative abundance of the genus Vibrio was found between

3-4% in Initial 1 and 2, and 9.28% in Initial 3. In the order Bacteroidales, the relative abundance

of genus Petrimonas was between 9.91% and 10.63% in Initial 1 and 2, and 5.78% in Initial 3.

Also, in order Chrysiogenales, the relative abundance of genus Desulfurispirillum was 5.50 ±

2.72%. No Actinobacteria were identified in any of these cultures.

After SFWP was aerated for 166 hours at room temperature in bioreactors (SFWP 1-3),

the microbial community shifted to one being dominated by three phyla: Proteobacteria (73.36

± 11.84%) in classes Deltaproteobacteria (25.28 ± 24.51%), Alphaproteobacteria (27.44 ±

116
15.01%), and Betaproteobacteria (20.63 ± 4.24%); Actinobacteria that were all in the class

Actinobacteria (17.94 ± 14.33%); Bacteroides (8.24 ± 2.88%) in the class Flavobacteriia, with

SFWP 3 having some in an unknown class (4.88%) (Figure 3.21). SFWP 3 also had remnants of

Firmicutes of class Erysipelotrichi (0.94%). Within Proteobacteria, the class Alphaproteobacteria

was dominated by family Phyllobacteriaceae (18.26 ± 9.15%) within the Rhizobiales order. The

relative abundance of the genus Aquamicrobium of this family was 12.34%, 17.31%, and 5.64%,

in SFWP 1, 2, 3, respectively. In Betaproteobacteria, the relative abundance of family

Alcaligenaceae within the order Burkholderiales was 20.00 ± 3.44%. Genus Halomonas of the

class Pseudomonadaceae was found in SFWP 2 and 3 at relative abundances of 6.84% and

10.99%. Gammaproteobacteria was only dominant in SFWP 3 (40.31%) with 5.23% being made

up of Pseudomonas. Within the same class, Actinobacteria of order Actinomycetes had a high

relative abundance of family Dietziaceae of 8.53% and Microbacteriaceae of 18.31% in SFWP 1

and 5.10% and 7.71%, respectively in SFWP 2. The family Flavobacteriales in the order

Flavobacteriales showed a relative abundance of 5.23% and 10.96% in SFWP 1 and SFWP 2,

respectively.

Bacteria growing with microalgae in SFWP (Algae 1-9) showed similar microbial

communities to those of just SFWP alone. In this case, these microalgae samples had a higher

relative abundance of unknown bacteria cultures (Figure 3.22). The microalgae bioreactors

were dominated by Proteobacteria (78.74 ± 6.65%) of the classes Gammaproteobacteria (39.01

± 19.94%), Alphaproteobacteria (25.69 ± 18.07%), and Betaproteobacteria (14.04 ± 8.44%). Of

these Gammaproteobacteria, the most dominant was from the family Pseudomonadaceae

117
(24.84 ± 15.05%) which ranged in abundance from 0% to 50.10% of the total OTU count. Within

the same class, family Xanthomonadaceae was found at a relative abundance of 28.18% in

Algae 3, and 12.45% and 14.60% in Algae 8 and 9, respectively. Another Proteobacteria that

was seen in high relative abundance was from class Betaproteobacteria, order Burkholderiales,

and family Alcaligenaceae which was found at a relative abundance of 14.04 ± 8.44% with all

microalgae bioreactors having relative abundance above 5%. Algae 4, 6, and 8 all had relative

abundances around 25%. Within the family Alcaligenaceae, the genus Aquamicrobium showed

high relative abundance (11.11 ± 10.75%) and ranged from 0% to 33.69%. Aquamicrobium was

found in reactors 1-7 and 9. These were at relative abundances over 10% in Algae 5, 7, and 9.

Other genera from the same family were seen in high concentrations in some microalgae

bioreactors: Kerstersia was found at 19.83% relative abundance in Algae 8; Pusillimonas was

found at relative abundance greater than 5% in Algae bioreactor 4, 5, and 6; Achromobacter

was found as high as 28.18% in Algae 3, and relative abundances above 10% in Algae 8 and 9;

Bordetella was identified at a relative abundance of 5.20% in Algae 9. Within

Alphaproteobacteria, family Phyllobacteriaceae from order Rhizobiales produced high

abundances in Algae 5 (39.49%), Algae 9 (25.34%), Algae 7 (16.42%), and Algae 6 (12.68%).

Algae 1, 3, and 4 had relative abundances over 5%. Genus Halomonas, from order

Gammaproteobacteria, had relative abundance values above 10% in Algae 1, 2, 4, and 7. All

118
cultures except culture 8 had Actinobacteria of the class Actinobacteria (6.67 ± 5.56%). Genus

Dietzia of family Dietziaceace showed abundance over 5% in Algae 5 and 6. All reactors except

Algae 4 had bacteria that were unidentified and listed as Unknown (12.13 ± 6.32%). Algae 2, 3,

4, and 7 displayed the occurrence of Bacteroides in the family Flavobacteriaceae (10.04 ±

5.58%). The highest relative abundance of these OTUs was found in Algae 2 at 18.18%. Algae 3

showed the occurrence of Firmicutes in the family Bacillaceae (0.81%).

Within these samples, some organisms were identified to ~100% similarity to those in

the Greengenes database. This threshold has been calculated to be the optimal similarity

required between the OTU and database to assign species identification when using the V4

region of16S rDNA [231]. OTUs identified to meet this threshold include Desulfurispirillum

alkaliphilum (99.98%) and Anaerorhabdus furcosa (99.61%). Using the 97% identity threshold

[232], which has long been the standard set for species identification, additional OTUs were

identified down to the species level: Pseudomonas caeni (99.07%), Aquamicrobium defluvii

(98.96%), Petrimonas sulfuriphila (98.67%), Paenchrobactrum glaciei (97.98%), Pusillimonas

terrae (97.66%), and Denitromonas indolicum (97.58%).

119
 100

75
Percent Relative Abundance (%)

50

25

Figure 3.16: Relative Abundance of Bacterial Phyla in the SFWP Microbiota

◼ Proteobacteria, ◼ Actinobacteria, ◼ Bacteroidetes, ◼ Unknown, ◼ Firmicutes, ◼ Chrysiogenetes, ◼
Lentisphaerae, ◼ Tenericutes

120
 100

75
Percent Relative Abundance (%)

50

25

Figure 3.17: Relative Abundance of Bacterial Classes in the SFWP Microbiota

◼ Gammaproteobacteria, ◼ Alphaproteobacteria, ◼ Betaproteobacteria, ◼ Deltaproteobacteria, ◼ Unknown, ◼
Actinobacteria, ◼ Bacteroidia, ◼ Flavobacteriia, ◼ Chrysiogenetes, ◼ Bacilli, ◼ Clostridia, ◼ Erysipelotrichi, ◼
Mollicutes, ◼ Unknown Bacteroidetes, ◼ Unknown Firmicutes, ◼ Unknown Lentisphaerae

121
 100

75
Relative Abundance (%)

50

25

Figure 3.18: Relative Abundance of Bacterial Order in the SFWP Microbiota

◼ Pseudomonadales, ◼ Rhizobiales, ◼ Oceanospirillales, ◼ Burkholderiales, ◼ Unknown, ◼ Actinomycetales, ◼
Rhodobacteriales, ◼ Unknown Bacteroidetes, ◼ Bacteroidales, ◼ Flavobacteriales, ◼ Chrysiogenales, ◼ Unknown
Firmicutes, ◼ Bacillales, ◼ Clostridiales, ◼ Erysipelotrichales, ◼ Unknown Lentisphaerae, ◼ Unknown
Alphaproteobacteria, ◼ Caulobacterales, ◼ Rhodocyclales, ◼ Desulfuromonadales, ◼ Alteromonadales, ◼
Vibrionales, ◼ Xanthomonadales, ◼ Acholeplasmatales

122
 100

75
Relative Abundance (%)

50

25

Figure 3.19: Relative Abundance of Bacterial Families in the SFWP Microbiota

◼ Pseudomonadaceae, ◼ Halomonadaceae, ◼ Alcaligenaceae, ◼ Phyllobacteriaceae, ◼ Unknown, ◼ Unknown
Rhizobiales, ◼ Xanthobacteraceae, ◼ Dietziaceae, ◼ Microbacteriaceae, ◼ Rhodobacteraceae, ◼ Unknown
Actinomycetales, ◼ Nocardiaceae, ◼ Unknown Bacteroidetes, ◼ Unknown Bacteroidales, ◼ Porphyromonadaceae,
◼ Flavobacteriaceae, ◼ Chrysiogenaceae, ◼ Unknown Firmicutes, ◼ Bacillaceae, ◼ Unknown Clostridiales, ◼
Eubacteriaceae, ◼ Peptococcaceae, ◼ Erysipelotrichaceae, ◼ Unknown Lentisphaerae, ◼ Unknown
Alphaproteobacteria, ◼ Caulobacteraceae, ◼ Brucellaceae, ◼ Rhodocyclaceae, ◼ Pelobacteraceae, ◼
Colwelliaceae, ◼ Unknown Vibrionales, ◼ Vibrionaceae, ◼ Xanthomonadaceae, ◼ Acholeplasmataceae

123
 100

75
Relative Abundance (%)

50

25

Figure 3.20: Relative Abundance of Bacterial Genus in the SFWP Microbiota

◼ Unknown Pseudomonadaceae, ◼ Halomonas, ◼ Unknown Alcaligenaceae, ◼ Unknown, ◼ Aquamicrobium, ◼
Unknown Rhizobiales, ◼ Unknown Phyllobacteriaceae, ◼ Unknown Xanthobacteraceae, ◼ Dietzia, ◼ Unknown
Microbacteriaceae, ◼ Unknown Actinomycetales, ◼ Unknown Actinomycetales, ◼ Unknown Dietziaceae, ◼
Frondihabitans ◼ Rhodococcus, ◼ Nocardioides, ◼ Unknown Bacteroidetes, ◼ Unknown Bacteroidales, ◼
Petrimonas, ◼ Unknown Flavobacteriaceae, ◼ Desulfurispirillum, ◼ Unknown Firmicutes, ◼ Unknown Bacillaceae,
◼ Unknown Clostridiales, ◼ Garciella, ◼ Desulfitispora, ◼ Anaerorhabdus, ◼ Unknown Lentisphaerae, ◼ Unknown
Alphaproteobacteria, ◼ Unknown Caulobacteraceae, ◼ Paenochrobactrum, ◼ Bordetella, ◼ Kerstersia, ◼
Pigmentiphaga, ◼ Pusillimonas, ◼ Rhodopseudomonas, ◼ Denitromonas, ◼ Desulfuromonas, ◼ Thalassomonas, ◼
Unknown Halomonadaceae, ◼ Pseudomonas, ◼ Unknown Vibrionales, ◼ Vibrio, ◼ Unknown Xanthomonadaceae,
◼ Unknown Vibrionales

124
 OTU ID Initial1 Initial2 Initial3
k_Bacteria 6.40 0.50 6.91
o_Bacteroidales 7.38 9.81 6.25
g_Petrimonas 10.63 9.91 5.78
g_Desulfurispirillum 7.38 2.38 6.72
p_Firmicutes 2.76 18.43 3.41
o_Clostridiales 2.48
g_Garciella 1.38 1.29
g_Desulfitispora 2.95 2.08 3.22
p_Lentisphaerae 2.26 1.61
g_Halomonas 46.46 38.85 48.58
f_Pseudomonadaceae 5.31 6.84 1.33
g_Pseudomonas 2.56 2.08 5.59
g_Vibrio 3.25 3.87 9.28
g_Acholeplasma 1.28 1.49 1.33
Figure 3.21: Relative Abundance Heat Map of OTUs in Initial SFWP
k: Kingdom, p: Phylum, c: Class, o: Order, f: Family, g: Genus.
Percent Abundance
0% 25% 50%

125
 OTU ID SFWP1 SFWP2 SFWP3
k_Bacteria 0.47
o_Actinomycetales 0.78 0.35
f_Dietziaceae 2.56
f_Dietziaceae 0.94
g_Dietzia 5.02 5.10 2.64
f_Microbacteriaceae 17.47 7.54 0.65
g_Frondihabitans 0.84 0.17
g_Rhodococcus 2.93 1.59
g_Nocardioides 2.72
2.51
p_Bacteroidetes 4.88
f_Flavobacteriaceae 5.23 10.96 3.64
g_Anaerorhabdus 0.94
c_Alphaproteobacteria 0.98 0.71
f_Caulobacteraceae 0.42 0.77
o_Rhizobiales 5.60 2.72
g_Paenochrobactrum 4.05 1.65
f_Phyllobacteriaceae 12.13 5.24 2.29
g_Aquamicrobium 12.34 17.31 5.46
f_Xanthobacteraceae 3.82 1.40
f_Rhodobacteraceae 1.67 3.77
f_Alcaligenaceae 5.39 9.91 13.75
g_Bordetella 3.71 4.33
g_Kerstersia 3.32
g_Pusillimonas 4.86 4.75 7.81
g_Rhodopseudomonas 2.04
g_Denitromonas 2.02
g_Desulfuromonas 4.65 0.91 1.65
g_Thalassomonas 0.52
g_Halomonas 6.84 10.99
f_Pseudomonadaceae 4.86 3.66 35.08
g_Pseudomonas 0.87 5.23
o_Vibrionales 0.59
Figure 3.22: Relative Abundance Heat Map of OTUs in SFWP After 166 Hours
k: Kingdom, p: Phylum, c: Class, o: Order, f: Family, g: Genus.
Percent Abundance
0% 25% 50%

126
 Algae Algae Algae Algae Algae Algae Algae Algae Algae
OTU ID
1 2 3 4 5 6 7 8 9
k_Bacteria 5.74 5.63 5.40 17.38 9.67 14.34 21.1 17.79
o_Actinomycetales 0.64 0.74
g_Dietzia 2.04 0.46 4.36 7.51 9.02
f_Microbacteriaceae 1.85 0.76 1.55 2.52 5.37 3.06 7.05
g_Rhodococcus 2.51 3.87
f_Flavobacteriaceae 18.17 8.51 7.96 5.51
f_Bacillaceae 0.81
f_Caulobacteraceae 1.90
o_Rhizobiales 3.99 3.12 3.77 6.65 8.16 10.66 4.01 9.56
g_Paenochrobactrum 2.52
f_Phyllobacteriaceae 3.79 0.99 1.42 5.79 2.90 5.03
g_Aquamicrobium 5.35 0.99 5.70 7.72 33.69 9.77 16.42 20.30
f_Xanthobacteraceae 2.33 1.98 4.22 6.44 2.36 4.04 4.64 10.40
f_Rhodobacteraceae 0.49 2.00 1.18 3.87 2.94 10.07
f_Alcaligenaceae 9.24 5.55 11.69 18.12 13.53 10.17
g_Bordetella 5.06
g_Kerstersia 19.83 5.20
g_Pigmentiphaga 1.04
g_Pusillimonas 0.99 7.25 7.73 10.96
g_Thalassomonas 0.61
f_Halomonadaceae 0.30
g_Halomonas 15.08 14.60 3.85 11.43 12.25
f_Pseudomonadaceae 50.10 36.65 16.42 36.56 14.81 19.87 18.14 31.01
g_Pseudomonas 3.31
o_Vibrionales 1.72
g_Vibrio 4.79
f_Xanthomonadaceae 1.51
g_Achromobacter 4.41 28.18 12.45 13.09
Figure 3.23: Relative Abundance Heat Map of OTUs in SFWP with Microalgae After 166 Hours
k: Kingdom, p: Phylum, c: Class, o: Order, f: Family, g: Genus.
Percent Abundance
0% 25% 50%

127
 Analysis at the phylum level of OTUs identified has been scaled using PC 1 (59.1%) and

PC 2 (29.0%) to identify the phylum most responsible for changes in the microbial communities

(Figure 3.24). The Initial samples were seen to be in communities that had higher abundances

of Bacteroidetes, Firmicutes, Chrysiogenetes, Tenericutes, and Lentisphaerae. Actinobacteria

were seen to affect those in Algae 6, SFWP 2, and Algae 3 the most while Proteobacteria were

seen to change those in Algae 8, Algae 5, Algae 1, Algae 8, Algae 9, SFWP 3. Algae 2 was also

seen to be affected by Proteobacteria as well as with the communities seen in the Initial

samples.

The Shannon Diversity Index (Figure 2.25 A.) was calculated for the three treatments. It

showed an average value (1.90 ± 0.05) for Initial 1-3, which is lower than that of the microbial

community that formed in SFWP after 166 hours of organism growth (2.57 ± 0.31). These values

were higher than the average diversity index value for the microbiome associated with

microalgae growth (2.03 ± 0.23). Pielou’s Evenness Index (Figure 2.25 B.) showed a similar trend

across the treatments, where the evenness for the initial conditions (0.75 ± 0.01) was lower

than the SFWP (0.85 ± 0.07). The average evenness of the treatment with microalgae growth

(0.84 ± 0.08) was approximately the same as the SFWP.

128
Figure 3.24: Biplot of Phylum Level OTUs in SFWP with Microalgae After 166 Hours
PC 1 is 59.1% and PC 2 is 29.0%

129
A. 3
Shannon Diversity Index

0 Initial1 Initial2 Initial3 SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9

B. 1
Pielou's Evenness Index

0.8

0.6

0.4

0.2

Figure 3.25: (A.) Shannon Diversity Index and (B.) Pielou’s Evenness Index of Microbial
Communities in SFWP
◼ SFWP, ◼ Algae, ◼ Initial.
A. is the Shannon Diversity Index and B. is Pielou’s Evenness
n = 1 for all measurements.

130
 Correlations were developed for the relative abundance of specific OTUs against

nutrient removal and biomass production of both microalgae and bacteria. Box-Cox

transformations of the conditions were performed in an attempt to normalize the distribution

of the parameter. The transformation values were as follows: λ = -0.322 for estimated bacteria

biomass (g/L), λ = 0.621 for estimated microalgae C. sorokiniana biomass (g/L), λ = -1.372 for TN

removal (%), and λ = 2 for N-NH4 removal (%). No such transformation could be calculated for

sCOD removal (%).

In the case of N-NH4 removal, only the OTU associated with the genus Achromobacter

was identified to correlate with N-NH4 removal significantly (Table 3.4, Figure 3.26). No OTUs

were recognized to have significant correlations with TN or sCOD. OTUs of the genus

Halomonas correlated with TN removal, and family Alcaligenaceae showed a relationship with

sCOD removal at 90% significance but are not at the threshold of being significant (Appendix

8.6).

Multiple OTUs showed a high correlation with biomass concentration in these

experiments. Both genus Pusillimonas and family Alcaligenaceae (Table 3.5, Figure 3.27)

showed relative abundances inversely proportional to the total biomass concentration of

microalgae.

Also, family Rhodobacteraceae showed a positive correlation with bacteria biomass that

increased when added with the genus Aquamicrobium (Table 3.7, Figure 3.29). These

relationships were all significant.

High and low Algae biomass concentrations were grouped together and compared to

determine the contributing factors to this difference. OTUs were normalized and ran in a

131
Student’s t-test with equal grouped variance at a threshold at p< 0.05. Only the OTU of the

genus Pusillimonas was seen significant at a p-value of 0.0179 (Table 3.6, Figure 3.29 A.). This

was a significant fold change (FC) at a log2(FC) of -5.0351. The difference is also seen in the

original abundances (Figure 3.29 B.) and normalized abundances (Figure 3.29 C.).

Table 3.4: OTU Correlation with N-NH4 Removal in Microalgae-SFWP Community

OTU Formula R2 Radj2 RMSE P-Value

Achromobacter -0.1449x + 42.385 0.5464 0.4826 7.038 0.0229*

*Significant at 95%

30
Relative Abundance (%)

20

10

0
140 190 240 290
Box-Cox Transformed N-NH4
Removal (λ = 2 )

Figure 3.26: OTU Correlation with N-NH4 Removal in Microalgae-SFWP Community

132
 Table 3.5: OTU Correlation with Microalgae Biomass Concentration in SFWP
OTUs Formula R2 Radj2 RMSE P-Value
Pusillimonas -13.249x + 11.541 0.7096 0.6681 2.517 0.0044*
Alcaligenaceae -20.608x + 27.339 0.4604 0.3833 6.625 0.0445*
*Significant at 95%

15 30
Relative Abundance (%)

Relative Abundance (%)

10 20

5 10

0 0
0.2 0.4 0.6 0.8 1 0.2 0.4 0.6 0.8 1
Box-Cox Transformed Algae Biomass Box-Cox Transformed Algae Biomass
Concentration (λ = 0.621) Concentration (λ = 0.621)

Figure 3.27: OTU Correlation with Microalgae Biomass Concentration in SFWP

133
 Table 3.6: OTU Correlation with Bacteria Concentration in Microalgae-SFWP Community
OTUs Formula R2 Radj2 RMSE P-Value
Rhodobacteraceae 1.153x – 3.546 0.4630 0.3863 2.534 0.0437*
Aquamicrobium + -0.1047xA + 0.5081xR + 5.0554 0.7779 0.7039 1.0382 0.0109*
Rhodobacteraceae
*Significant at 95%

6 8
Relative Abundance (%)

Relative Abundance (%)

6
4

2
2

0 0
0 2 4 6 8 0 2 4 6 8
Box-Cox Transformed Bacteria Box-Cox Transformed Bacteria
Biomass Concentration (λ = -0.322) Biomass Concentration (λ = -0.322)

Figure 3.28: OTU Correlation with Bacteria Concentration in Microalgae-SFWP Community

134
 Table 3.7: OTU Abundance Difference in High/Low Biomass Concentrations in SFWP
OTU FC log2(FC) P-Value -log(P)

Pusillimonas 0.0350 -5.035 0.0179* 1.7467

*Significant at 95%

A.

B. C.

Figure 3.29: OTU Correlation with Low Microalgae Biomass Concentration in SFWP (A.)
Volcano Plot (B.) Original Abundance and (C.) Normalized Abundance

135
 A total of four separate clusters were identified with co-occurrence of OTUs in at least

20% of the samples (Figure 3.30 A.). The clusters were identified as follows: Cluster 1 has OTUs

from families Microbacteriaceae and Alcaligenaceae; Cluster 2 has OTUs from genus

Paenochrobactrum and family Rhodobacteraceae; Cluster 3 has OTUs from order Rhizobiales

and genus Kerstersia; Cluster 4 has OTUs from order Bacteroidales, families Bacillaceae and

Xanthomonadaceae, and genera Petrimonas, Desulfurispirillum, Anaerorhabdus, Acholeplasma.

Interactions between organisms are shown in Figure 3.30 B. The relative abundance of these

co-occurring OTUs is shown in Figure 3.30 C. Cluster 4 showed high OTU abundance in the initial

SFWP and had an average relative abundance of 39.86 ± 7.02% with a maximum in Initial 2 of

47.97%. This cluster was not seen in any of the other treatments, except for one in the

microalgae bioreactors (Algae 2, 4.79%). Clusters 1, 2, and 3 were seen in SFWP after 166 hours

of the experiment in SFWP 1 (19.14%, 9.41%, 4.13%) and SFWP 2 (11.31%, 4.12%, 5.10%). Only

Cluster 1 was seen in SFWP 3, and at a low relative abundance (0.65%). Cluster 3 was only seen

in one microalgae culture: Algae 8 (6.96%). Microalgae samples showed difference abundances

of Clusters 1 and 2, with the two exceptions mentioned above as well as Algae 4, which showed

no abundance of Cluster 2, and Algae 5 and 8 which had no OTUs from Custer 1. Relative

abundance values for Cluster 1 and 2 in those cultures that had them ranged from 0.76% to

17.11% and 4.12% to 19.97%, respectively.

136
A. B.
OUT ID Lowest Phylogenetic Level
A25
\a Cluster 1
A6 Microbacteriaceae A30
A30 Alcaligenaceae
.. A6 A29
Cluster 2
A25 Paenochrobactrum
A29 Rhodobacteraceae A11
Cluster 3
A24 Rhizobiales A32 A12
A16
A32 Kerstersia
Cluster 4
A11 Bacteroidales A14 A48
A12 Petrimonas
A14 Desulfurispirillum A45
A24 A20
A16 Bacillaceae
A20 Anaerorhabdus
A48 Acholeplasma
A45 Xanthomonadaceae
C.
50
Releative Abundance (%)

40

30

20

10

Figure 3.30: (A., B.) Clusters of Co-Occurring OTUs and (C.) Their Relative Abundance in the
SFWP Microbial Community
◼ Cluster 1, ◼ Cluster 2, ◼ Cluster 3, ◼ Cluster 4
A. shows the Co-occurrence OTUs, B. is the interactions within each cluster, and C. is the relative abundance of the
members of each cluster in the sample. n=1 for all measurements.

137
3.3.4. Predictive Metagenomics

The predicted metagenomes suggest that there were distinct differences in the characteristics

of the OTUs identified in SFWP. In the majority of treatments, the microbial community was

made up mainly by organisms with predicted aerobic metabolic pathways (Figure 3.31). The

Initial wastewater sample had an average of 52.53 ± 4.16% of OTUs predicted to have aerobic

oxygen metabolisms, 25.09 ± 4.36% anaerobic pathways, and 22.38 ± 5.10% were unknown.

After 166 hours of growth, the SFWP showed a similar abundance of predicted aerobic

pathways (50.44 ± 12.82%), anaerobic pathways (19.91 ± 4.36%), and OTUs with unknown

oxygen demand (29.64 ± 8.54%). The abundance of organisms with predicted aerobic activities

in the microalgae samples ranged from 28.76% (Algae 5) to 69.60% (Algae 3). In seven of the

nine reactors, the organisms with aerobic activities made up the majority of OTUs identified

based on oxygen metabolisms. Algae 5 had a majority of organisms with predicted anaerobic

pathways, and Algae 9 had a majority of unknown microorganisms. Algae 8 had no known

organism with predicted anaerobic activities present.

138
 100

80
Relative Abundance (%)

60

40

20

Figure 3.31: Oxygen Requirements of Bacteria Present in SFWP

◼ Aerobic, ◼ Anaerobic, ◼ Unknown

139
 Analysis of the identified metabolisms revealed that predicted metabolic pathways for

ammonia oxidation, nitrate reduction, dehalogenation, and sulfate reduction made up at least

half of the identified pathways in all the samples (Figure 3.32). The average relative abundances

of these metabolisms in Initial samples 1-3 were 65.81 ± 4.84%, 58.03 ± 6.82%, 65.81 ± 4.84%,

63.49 ± 8.83%, respectively (Figure 3.33). In the SFWP bioreactors, these metabolisms shifted to

relative abundances of 53.91 ± 10.89%, 44.35 ± 15.39%, 49.68 ± 18.10, and 36.39 ± 21.56%,

respectively. In the microalgae bioreactors, the most common metabolisms were found at

abundances of 55.37 ± 17.90%, 50.55 ± 12.99%, 55.15 ± 17.01%, and 49.11 ± 14.04%,

respectively.

There were no major differences between the relative abundance of the metabolisms

found in the initial SFWP, but after 166 hours, the SFWP samples did show a difference in these

major metabolisms. In this case, SFWP 1 and SFWP 2 showed lower abundances of these major

metabolisms with a range from 23.33% to 53.66%. In comparison, SFWP 3 had a range of

relative abundances across these four key metabolisms between 61.28% and 70.51%.

In the microalgae bioreactors, Algae 2 showed the highest relative abundance of OTUs

with ammonia oxidization (82.66%) and dehalogenation (82.89%) metabolisms. In comparison

Algae 5 showed the lowest relative abundance of those metabolisms (27.04% and 34.76%,

respectively). This discrepancy can be seen through the higher unknown relative abundance in

OTU metabolism between Algae 5 and Algae 2 (65.24% vs. 16.35%). This variability between

samples within the microalgae treatment was also seen with nitrate reduction and sulfate

reduction. Algae 1-4 contained the most OTUs with identified metabolisms (<32% unknown)

while samples Algae 5-9 showed the least OTU with identified metabolisms (≥ 50% unknown).

140
 80

70

60
Relative Abundance (%)

50

40
141

30

20

10

0
Initial1 Initial2 Initial3 SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9
Figure 3.32: Bacterial Metabolisms Identified in SFWP
◼ Ammonia oxidizer, ◼ Nitrate reducer, ◼ Dehalogenation, ◼ Sulfate reducer, ◼ Chitin degradation, ◼ Xylan degrader, ◼ Sulfate oxidizer, ◼ Atrazine
metabolism, ◼ Nitrogen fixation, ◼ Sulfur metabolizing, ◼ Naphthalene degrading, ◼ Syntrophic, ◼ Lignin degrader, ◼ Dinitrogen-fixing, ◼ Carbon fixation, ◼
Sulfur oxidizer, ◼ Denitrifying, ◼ Selenate reducer, ◼ Chlorophenol degrading, ◼ Cellobiose degrading, ◼ Gramicidin producer, ◼ Sulfur reducer, ◼ Unknown

141
 Ammonia oxidation Nitrite reduction Dehalogenation Sulfate reduction Unknown
Initial1 64.96 61.71 64.96 64.96 25.39
Initial2 61.45 50.15 61.45 54.01 36.17
Initial3 71.02 62.22 71.02 71.50 20.64
SFWP1 53.66 40.38 37.76 23.33 36.40
SFWP2 43.14 31.34 40.77 24.57 50.44
SFWP3 64.92 61.34 70.51 61.28 25.62
Algae1 73.64 69.84 71.79 68.00 26.36
Algae2 82.66 59.70 82.89 63.73 16.35
Algae3 68.05 58.73 63.17 57.99 31.95
Algae4 64.38 62.25 69.11 59.73 28.37
142

Algae5 27.04 28.97 34.76 28.97 65.24

Algae6 38.88 46.29 39.96 40.92 50.16
Algae7 48.41 40.44 44.61 39.09 51.59
Algae8 48.10 48.10 50.00 50.00 50.00
Algae9 47.15 40.60 40.10 33.56 52.85
Figure 3.33: Heat Map of the Relative Abundance of Bacterial Metabolisms in SFWP
Percent Abundance
0 50 100

142
 Further analysis of the predicted metagenomes showed that the SFWP initially

contained some bacteria that utilize plants as a host. The output from the metagenomic

analysis showed that the initial SFWP had bacteria with this characteristic ranging in relative

abundances of 2.29% to 12.13% (Figure 3.34). In the SFWP bioreactors, only two of the three

bioreactors showed any presence of these organisms after 166 hours. The relative abundances

of these organisms were also lower than the mean from the initial SFWP (2.39% < 6.55%).

Throughout the microalgae bioreactor samples, only reactors 1, 2, 3, and 6 had bacteria

identified that use plants as hosts.

15
Relative Abundance (%)

10

Figure 3.34: Abundance of Bacteria Associated with Plants in SFWP

Metagenomic classification of organisms showed that some characteristics correlated

(>95%) with nutrient removal. Box-Cox transformed N-NH4 removal correlated significantly with

three separate characteristics: sulfur-oxidizing, sporulating organisms, and identified plant

hosts (Table 3.8, Figure 3.35). The sCOD removal showed a significant correlation with the

Pielou’s Evenness Index of the microalgae bioreactors (Table 3.9, Figure 3.36). Also, Atrazine

metabolisms and nonsporulating organisms correlated with sCOD removal at 90%, but these

were not deemed significant at the threshold used for this study (Appendix 8.7).

143
Table 3.8: Characteristics that Correlate with N-NH4 Removal in Microalgae-SFWP Community
Organisms Formula R2 Radj2 RMSE P-Value

Sulfur Oxidizing -0.234x + 68.225 0.7886 0.7584 6.290 0.0014*

Sporulating -0.187x + 78.804 0.7512 0.7157 5.586 0.0025*
Identified Plant Hosts 0.033x – 5.798 0.5120 0.4423 1.682 0.0302*
*Significant at 95%

60 60

Relative Abundance (%)

Relative Abundance (%)

40 40

20 20

0 0
100 200 300 100 200 300
Box-Cox Transformed N-NH4 Box-Cox Transformed N-NH4
Removal (λ = 2) Removal (λ = 2)

6
Relative Abundance (%)

0
100 200 300
Box-Cox Transformed N-NH4
Removal (λ = 2)

Figure 3.35: Characteristics that Correlate with N-NH4 Removal in Microalgae-SFWP

Community

144
Table 3.9: Characteristics that Correlate with sCOD Removal in Microalgae-SFWP Community
Organisms Formula R2 Radj2 RMSE P-Value

Pielou’s Evenness Index -0.002x + 0.873 0.4964 0.4245 0.06 0.0341*

*Significant at 95%

Pielou's Evenness Index 0.9

0.8

0.7

0.6
-20 30 80
sCOD Removal (%)

Figure 3.36: Characteristics that Correlate with sCOD Removal in Microalgae-SFWP

Community

145
3.4. Discussion

Overall, the total biomass concentrations across all microalgae samples varied to a high

degree. These estimated concentrations ranged from 0.142 g/L to 0.901 g/L. In addition,

nutrient removal efficiencies (TN, N-NH4, sCOD) varied between treatments and across samples

to a high degree. Since conditions were held constant (media, temperature, light, CO 2, etc.), the

cause of this discrepancy was the difference in the microbiomes that developed.

Box-Cox transformed microalgae biomass concentrations showed a significant

correlation with TN removal. This result is consistent with the previous results (Chapter 2). Both

N-NH4 removal and sCOD removal did not show any significant trend in removal with algae

biomass, suggesting that the microbial community was altering these levels as previous results

showed microalgae was able to reduce both in sterile culture (Chapter 2). This decrease seen

could be through the transformation and utilization of nitrogen sources in the case of N-NH4 or

the production/excretion of organic compounds for sCOD [20, 21].

Quantitative PCR showed that eight of the nine microalgae bioreactors had estimated

bacteria biomass concentrations less than that of microalgae biomass concentrations. The

sample, Algae 5, had bacteria biomass representing 50.75% of the total biomass (0.364 g/L).

The second highest bacterial biomass abundance was found in Algae 4 at 19.60% (0.142 g/L). All

other algae cultures had estimated algae biomass concentrations at less than 3% of the total

biomass. Microalgae can be separated into those with low biomass concentration (Algae 4,

Algae 5, Algae 6, Algae 8 at 0.114 g/L to 0.321 g/L), and high biomass concentration (Algae 1,

Algae 2, Algae 3, Algae 7, Algae 9 at 0.672 g/L to 0.890 g/L). While there was no correlation

between bacteria biomass concentration and microalgae biomass concentration, the two

146
cultures with the lowest biomass concentrations had the highest bacteria concentrations. This

result suggests that crucial microorganisms, rather than overall high bacterial biomass

concentration might alter the microalgae biomass concentration.

Analysis of the dissimilarity of the bacterial cultures showed that there were distinct

differences between the microbiome found in the initial SFWP and the community that later

developed in the aerated SFWP and microalgae microbiomes. While the NDMS shows a

distinction between the SFWP and Algae cultures, both PCA and hierarchical clustering with

Ward’s Method showed that the microbial community of the SFWP was similar to that of the

microalgae cultures, with SFWP 3 being the most similar to Algae (1-9). These microbial

communities were most different in key OTUs with Halomonas and Pseudomonadaceae having

the greatest differences.

Phylogenetic identification of the OTUs showed that Proteobacteria dominated all of the

cultures, but those that were aerated had higher abundances of Actinobacteria than the initial

SFWP. Also, OTUs of the phyla Firmicutes, Chrysiogenetes, Lentisphaerae, and Tenericutes were

reduced to a high degree within the bioreactors with or without microalgae. Aerated samples

were made up mainly by those from Gammaproteobacteria, Alphaproteobacteria,

Betaproteobacteria, and Actinobacteria, while the initial SFWP was made up mostly by

Gammaproteobacteria and Clostridia. Heat maps showed that OTUs in Halomonas were

dominant within the initial samples, but not in the aerated samples. OTUs from

Pseudomonadaceae, Aquamicrobium, and Microbacteriaceae dominated aerated treatments.

Algae showed similar shifts to that of just SFWP, but with some cultures showing high relative

147
abundances of OTUs not found in those of just SFWP, such as Flavobacteriaceae and

Achromobacter.

Microalgae bioreactors (Algae) showed a much higher relative abundance of unknown

bacteria (10.78 ± 7.16%) than those seen in the SFWP cultures (0.16 ± 0.27%). This increase in

unknown reads might have been caused by the high relative abundance of microalgae DNA to

bacterial DNA, which made sequencing more difficult. It could also have been from other

material produced by the microalga that was present in the DNA extract. This excess DNA

would have made it more difficult for the sequencing machine to read. Another possibility is

that chloroplast DNA that was unable to be identified as such. It is also possible that these were

organisms that associated themselves with microalgae but have not yet been classified.

While the cultures did differ in their microbial communities, there was no significant

difference between the Shannon Diversity or Pielou’s Evenness indices across treatments, but

cultures with aeration showed slightly higher values on average. Despite this, at the phylum

level, there was a clear distinction between those phyla that evolved in the Initial samples and

those that evolved between the Algae and SFWP samples. Within the Algae and SFWP samples,

Actinobacteria and Proteobacteria were seen to alter those communities between the two

treatments.

Within the microalgae cultures, some abnormalities and trends were observed that

might have contributed to lower microalgae biomass concentrations. Algae 5 showed the

highest concentration of bacteria across all the treatments and samples tested. A unique

feature of these samples was that33.69% of the OTUs were associated with the genus

Aquamicrobium. This genus has been identified as nitrifying bacteria and reported to grow in

148
high abundances in microalgae photobioreactors utilizing domestic wastewater as a media

[233]. It is unclear if this genus has any effect on the growth of microalgae, but it might be the

cause of the high estimated bacteria biomass and low algae biomass concentration. Algae 9 and

Algae 7 also contained this bacterium, but at lower relative abundances of 20.30% and 16.42%,

respectively. Algae 8 also showed a low concentration of microalgae and was the only

microalgae sample with the family Caulobacteraceae and genus Bordetella at any abundance.

This samples also had an abundance of Kerstersia nearly four times that of Algae 9, the only

other bioreactor with that OTU. No known interactions exist between microalgae and the two

genera described above. Algae 4 showed no reads of OTUs of unknown bacteria and was the

only culture to have the genus Paenochrobactrum and Pseudomonas, as well being the sample

with the highest abundance of Alcaligenaceae at 18.12%. It is unclear if this culture was the

only one to have the two genera described above, or if the extracted DNA was of a quality to be

sequenced down to the genus level in just this case. Little is known about the genus

Paenochrobactrum, and no interactions between it and microalgae have been reported. Some

species of Pseudomonas have been documented to produce algicides [234] while others have

found that the nutrient removal from wastewater by microalgae was enhanced by the presence

of Pseudomonas putida [235]. It is possible that this genus may have caused a decrease in

biomass, but it is unclear. Since the OTU identified as Alcaligenaceae was only identified down

to the family level, little can be said about possible interactions between it and microalgae

without knowing a more specific phylogenetic level. In addition, other cultures had

Alcaligenaceae, but with relative abundances that were almost half that found in this culture.

149
Algae 4, 5, and 6 all had an abundance of Pusillimonas from 7.25% to 10.96%. The only other

culture with this genus was Algae 2 with a relative abundance less than 1%.

The linear correlation between Box-Cox transformed microalgae biomass concentrations

also showed inverse proportionality to the relative abundance of Pusillimonas as well as

Alcaligenaceae at 95% significance. In addition, OTUs of the genus Pusillimonas were seen to be

significantly different between Algae samples grouped with high and low biomass

concentrations. Organisms from the Pusillimonas genus have been identified to grow in biofilm

formations during wastewater treatment systems [236, 237]. The only species-level

identification within these microalgae cultures identified was Pusillimonas terrae (97.66%),

suggesting that this species might be one of those present, if not the only species of this genus

present in this system. Little characterization has been done for this genus, but some species

have been documented to denitrify nitrate [238] (in the case of Pusillimonas terrae specifically

[239]) and use complex hydrocarbons as a carbon source through organotrophic metabolism

[240]. It is unclear how this organism interacts with microalgae.

Similarly, little can be said about those in the family Alcaligenaceae unless they were to

be identified down to a specific genus. It is important to point out that Pusillimonas is in the

Alcaligenaceae family, but the abundance of Alcaligenaceae include other organisms such as

Kerstersia, Rhodopseudomonas, Bordetella, Pigmentiphaga, as well as an unknown genus,

which suggests that these multiple genera may be the cause of the lower biomass

concentration, but no conclusions of this effect can be made.

Specific differences in the microbial communities also appeared in those cultures with

high concentrations of microalgae biomass. Algae 2 was the only bioreactor with

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Thalassomonas (0.61%) and Vibrio (4.79%) as well as the only bioreactor with high yields with

any Pusillimonas (0.99%). The Thalassomonas genus is well documented as an abundant

nanoplankton in the ocean that lives among microalgae [241]. The genus Vibrio of the order

Vibrionales has been found in the wild growing in the psychosphere of diatoms in estuaries

[242]. Some species were also found that this genus is often attributed to coral bleaching and

have adverse effects on the growth of coralline algae [243], suggesting that some interaction

may exist between it and the microalgae in this experiment, but it is unclear if it is entirely

beneficial. Algae 3 was the only culture with an abundance of Bacillaceae (0.81%) and

Pigmentiphaga (1.04%). The family Bacillaceae contains a variety of bacteria that grow with

microalgae [244], but unless the OTU is identified down a more specific level, little can be said

about the interaction with the microalgae in this case. Pigmentiphaga have been found to grow

in direct contact within the Chlamydomonas phycosphere in co-cultures and protect the cells

from exogenous hydrogen peroxide [245]. Algae 7 showed OTUs of the order Vibrionales

(1.72%). In Algae 9, there was also a detectable abundance of Kerstersia at 5.20%, which is only

seen in Algae 8 at a low relative abundance. Also, this culture had organisms of family

Xanthomonadaceae (1.51%). This family has been documented to grow in amongst microalgae

in photobioreactors as a key element in biofilm association [246]. Across all sample, there were

four cultures with Halomonas, while the low biomass cultures only had one. This genus has

been shown to produce vitamin cobalamin that can directly be utilized by some microalgae

[247] as well as produce algicides [248]. The microalgae in this study, C. sorokiniana, does not

require cobalamin for growth [118], but might benefit in other ways from interacting with

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Halomonas. Also, three of the cultures showed any abundance of Achromobacter, where the

low biomass cultures only had one.

After applying Box-Cox transformed linear correlations on the total N-NH4 removal

efficiency, Achromobacter showed a significant correlation with the decrease of this nutrient

across the microalgae bioreactors. In addition, this genus was only seen in cultures that

contained microalgae. Characterization of this genus shows it as an obligate aerobe that breaks

down a variety of organic acids and amino acids for carbon but is usually unable to utilize

carbohydrates. Some species have been found to be facultative lithoautotrophs that use a

hydrogen-oxidation pathway. Others can use nitrate/nitrite for respiration and perform nitrate

reduction [249]. Studies have shown that wastewater isolated Achromobacter xylosoxidans can

remove ammonia, nitrate, and nitrite effectively using a variety of organic compounds [250].

Another species, Achromobacter piechaudii, had been isolated growing in a consortium with

microalgae Botryococcus braunii, suggesting that this organism might develop a symbiotic

relationship based on biotin formation [251]. C. sorokiniana does not have a biotin requirement

which implies another relationship, if any, exists between this organism and the microalgae

[118].

Two significant correlations were developed against microalgae biomass

concentrations. Both Pusillimonas and Alcaligenaceae OTUs shows a negative relationship with

this biomass concentration. Bacteria biomass concentrations showed there was a significant

correlation with Rhodobacteraceae bacteria and the combination of OTUs from

Rhodobacteraceae and genus Aquamicrobium. Without any further specification into which

organisms within the family were identified, it is hard to determine the relationship.

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 While this analysis shows the potential interactions between specific bacteria and

microalgae, it is unable to determine if these interactions do exist and if they alter the

microalgae biomass. Showing such a relationship between the bacteria and microalgae would

require an exhaustive study of isolating each unique bacterium growing amongst microalgae

and regrowing them individually in a co-culture with microalgae to tell if these bacteria alter

the microalgae biomass concentrations. Even this might not be all-telling since some bacteria

will change their ecological role upon the presence of other organisms or require other

organisms to be present to grow. Results did indicate that four distinct clusters of OTUs were

found amongst samples tested, with Cluster 4 being dominant in the Initial conditions and

Clusters 1-3 being found in the aerated conditions. These results suggest that significant shifts

that occur in the microbial communities upon aeration may help to pinpoint organisms that

require one another for growth, but it is by no means all the possible co-interactions. Given this

information, the OTUs identified in this chapter point to possible beneficial and harmful

bacteria present in the microalgae-wastewater microbiome.

It should be noted, that while these microalgae biomass changes are likely caused by

the differences in the bacteria present within the microbial community, these changes could

also have been caused by other microorganisms that were not picked up by 16S rDNA

sequencing. These include viruses and other eukaryotes. These domains do infect microalgae

and have a significant impact on largescale culturing systems [252]. Unfortunately, less work

has been done to characterize, sequence, and catalog DNA from these domains of life. As such,

less information exists on databases for these organisms. In particular with viruses, no known

homologous genetic region can be used as a basis for identification [253].

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 One source of error in any 16S rDNA analysis is the potential for amplification bias. The

primers selected for this analysis have been well documented and utilized by organizations like

the Joint Genome Institute for Microbiome Studies. It is reported that these primers present

less bias than other primers used for this type of sequencing [254]. Despite this, bias still exists,

but it is unclear if it has affected the results of this study to any degree.

Predictive metagenomic analysis showed that more identified anaerobic organisms

appeared after aeration and that four metabolisms became dominant across all the cultures:

ammonia oxidation, nitrite reduction, dehalogenation, and sulfate reduction. Also, the removal

of N-NH4 was shown to be significantly correlated with three metagenomic characteristics:

sulfur oxidation metabolisms, sporulating organisms, and organisms that use plants as hosts.

The first two being negatively correlated with N-NH4 removal percentages and the latter being

positive.

Sulfur oxidation is a chemolithotroph metabolism. Those samples with high ammonia

removal efficiencies might have also had high removal efficiencies of sulfur-based compounds

due to a more active bacterial community [255]. It is also possible that high sulfur

concentrations prevent the growth of nitrification organisms from removing ammonia, so

cultures with higher sulfur oxidation organisms inhibited the growth of those with nitrification

metabolisms initially. As conditions improved from sulfur oxidation, it may have become more

hospitable, and those ammonia oxidizing bacteria then took over as sulfide concentrations

decreased. Such inhibition of nitrification from sulfide has been documented [256]. Another

possibility is that these sulfur-oxidizing organisms utilized ammonia as a nitrogen source for

growth or depend on an organism that converts ammonia to forms they can use [255]. As

154
ammonia concentrations decrease, so do these nutrients, they rely on for growth, thus

preventing them from growing.

It can be hypothesized that at lower concentrations of N-NH4, fewer organisms would

possess the ability to sporulate or form an endospore for protection during metabolic

dormancy. Those bacteria that were metabolically stressed would form these encapsulations

and stop reproducing [257]. This protective measure would cause them to be outcompeted by

organisms that could more efficiently use lower ammonia concentrations or other nitrogen

sources, which resulted in this correlation.

Many plant hosts are specialized in that they can fix nitrogen into various forms, one of

these forms is ammonia. It is not surprising that as ammonia concentration decreased, more of

these organisms became present. In this case, nitrogen fixation takes N2 and converts it directly

to NH3 [258]. Nutrient stress may have facilitated symbiosis [259].

It was found that sCOD removal was also significantly correlated with the Pielou’s

Evenness Index. This negative correlation suggests that communities are more evenly

distributed at higher sCOD concentrations (i.e., lower sCOD removal rates). One study has

shown that higher species’ evenness results in higher sCOD removal rates, though they

concluded that higher sCOD concentrations resulted in this higher evenness [260]. This study

follows on the idea that at higher sCOD concentrations, more organic compounds are available

for a wider variety of organisms to utilize. At low sCOD, it is likely some specific carbon sources

are not available while others are more abundant, creating an imbalance in evenness.

While sulfur concentrations were not measured in the waste, a large portion of bacteria

growing in the wastewater possessed metabolisms indicative of a plentiful source of sulfur

155
compounds. Studies utilizing anaerobically digested food waste showed elemental sulfur

concentration to be around 0.25% of the mass on a wet basis [261]. This concentration is

significant enough that it would influence the microbial community. Identifying correlations

between bacterial OTUs and sulfur removal efficiencies might better explain microbiome

characteristics in future studies.

While qPCR has been used to estimate the overall biomass concentration of bacterial

samples mixed with microalgae [215], such measurement can only be used as an estimate. In

the case of this experiment, SFWP 3 was selected as the biomass concentration to be compared

against. Originally E. coli was to be used for this, but the high concentration of amplified DNA to

biomass gave unreasonable numbers. This result is likely because the wastewater had

suspended solids and bacterial excretions like biofilms that would add mass but not more DNA

specifically, further diluting the DNA in these samples. Unfortunately, the microbial

communities between all the SFWP cultures were different. These different bacteria

contributed to different biomass concentrations. In addition, this microbiome was similar, but

not the same to those seen in microalgae. Utilizing this method assumes that all bacteria are

the same size and shape across samples. It also assumes that there is no potential bias in

amplification between bacteria or any inhibitory compounds in the extracted DNA. Utilization

of SFWP 1 or 2 instead of SFWP 3 would result in estimated biomass concentrations higher than

those achieved in the experiment. This discrepancy is due to them having higher ratios of

biomass to amplified DNA than that of SFWP 3. Due to this, qPCR does not seem to be an

accurate way to measure the bacterial biomass concentration of microalgae-bacteria

156
wastewater samples. Despite this, it does give a robust relative estimate of which cultures

contained more bacteria than others.

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3.5 Conclusion

Overall, the microbial community in microalgae cultures has a strong influence on the total

algae biomass (0.114 g/L to 0.890 g/L) as well as the removal of nutrients (TN: 33.55% - 66.78%,

N-NH4: 64.67% - 97.25%, sCOD: -60.34% - 14.04%), within the systems. While some of these

cultures were seen to be similar regarding diversity to those of aerated SFWP, others were not,

suggesting no clear trend in the development of the microbial communities between replicates

in the same conditions. The only correlations across samples that were significant were that

microalgae biomass concentrations increased with increasing removals of TN. Since the same

cannot be said for N-NH4 or sCOD, it is clear that the bacteria add confounding factors in more

ways than just affecting microalgae biomass.

Certain bacterial OTUs were seen to dominate different samples. The initial samples

showed high relative abundances of Halamonas (38.85% to 48.58%) which decreased in both

the SFWP and Algae samples after aeration. Across both the aerated samples there was no OTU

that was seen to be dominant across all replicates. Microbacteriaceae, Aquamicrobium, and

Pseudomonadaceae made up the majority of the OTUs seen high in SFWP. In microalgae

samples, the dominant organisms were unidentified bacteria, Flavobacteriaceae,

Aquamicrobium, Alcaligenaceae, Pseudomonadaceae, and Achromobacter.

Some OTUs correlated with different parameters including microalgae biomass

concentration (Pusillimonas and Alcaligenaceae). Family Rhodobacteraceae as well as the

combination of family Rhodobacteraceae and genus Aquamicrobium correlated with bacterial

biomass concentration while N-NH4 removal efficiency was correlated with the genus

Achromobacter. It cannot be concluded if these organisms cause a change in the

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microenvironment or vice versa. Regardless, the analysis revealed the OTUs that might warrant

further investigation.

Co-occurring organisms were seen across all samples, with four distinct clusters found.

The largest cluster of 7 different OTUs was seen in abundances nearing 50% in the initial

wastewater before dropping to unmeasurable levels in SFWP and Algae samples.

Predictive metagenomics was able to show what types of characteristics were present

amongst the bacteria between samples. This helped to identify traits that the microalgae-

wastewater environment has selected for. While this method helps to add additional

classifications for the OTUs, it is highly dependent on the genome sequence available for the

associated organisms. Since not all units had been sequenced to specific taxonomic groups,

there was a high relative abundance of unknowns, which made it challenging to take significant

conclusions away from the analysis. Despite this, three traits did show significant correlated

with N-NH4 removal: sulfur oxidation metabolisms, sporulating organism, and organisms

associated with plants as hosts. In addition, Pielou’s Evenness Index was seen to correlate to a

significant level with sCOD removal.

This chapter shows the importance of understanding the interactions between

microalgae and bacteria. It is unclear what led to the shift in these microbial communities, but if

such a change occurred in lab conditions, it could only be expected to happen in large scale or

industrial systems. In these cases, the effects would happen in a more destructive fashion.

The biomass can only be profitable if it has a high enough concentration of lipids that

can be subsequently used for biofuel production. The next chapter focuses on ways to induce

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lipid accumulation of microalgae growing in wastewater to make these biofuels more

economical.

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4. Chapter 4 - Optimizing Lipid Production of Microalgae in Wastewater

4.1. Background

The microalgae C. sorokiniana is well adaptable to growing on wastewater (Chapter 2).

However, a high concentration of neutral lipids needs to be produced during cultivation for

them to be economical. The analysis suggests that the breakeven point for wastewater grown

microalgae biofuels exists at $2.23 per gallon when the microalgae culture is grown to a lipid

content of 28.6% [262]. These lipid contents are directly tied with the cost of microalgae

biofuels. One study found that the cost of microalgae-wastewater facilities could be reduced by

39% by doubling the lipid content of microalgae from 25% to 50% total dry weight [263].

Increasing the microalgae lipid content would help to make this fuel more economical and

potentially cost competitive with traditional fossil fuels.

Experiments have been done to boost the lipid content and lipid productivity in

microalgae through the addition of chemical stimulants at the initialization of growth, as well as

through a separate, 2-stage system after growth. A collection of previous experiments done on

C. sorokiniana to stimulate lipid accumulation are summarized in Table 4.1.

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 Table 4.1: Methods of Lipid Induction in C. sorokiniana
Chemical Lipid Biomass Lipid Study
Content Concentration Productivity
(%) (g/L) (mg/L/d)a
Growth
Mixotrophic Sodium Acetate (15 g/L) 3.5 58b [264]
Sodium Acetate (1.6 g/L) 29 1.0 117 [265]
Glucose (10 g/L) 51 1.3 67 [266]
Glucose (6 g/L) 32 4.6 244 [161]
Glycerol (10 g/L) 2.3 7b [264]
Nitrogen-Limiting N (2.5 mg/L) 22 0.1 1 [267]
Salt Stress Sodium chloride (30 g/L) 36 1.0 36 [268]
Sodium chloride (20 g/L) 54 0.4 17 [269]
Sodium chloride (15 g/L) 1.6 15b [264]
Sodium chloride (0.6 g/L) 19 1.7 14 [270]
Potassium chloride (1.1 g/L) 29 1.7 23 [270]
Magnesium chloride (1.9 g/L) 19 1.4 12 [270]
Magnesium chloride (0.15 g/L) 1.4 3b [264]
Calcium chloride (2.8 g/L) 40 1.3 26 [270]
Calcium chloride (0.15 g/L) 2.1 9b [264]
Iron (5.6 mg/L) 33 0.5 7 [271]
Ferric Chloride (0.15 g/L) 1.9 6b [264]
Trisodium citrate (0.2 g/L) 1.6 3b [264]
Additional NaNO3 (1.5 g/L) + Final effluent 40 0.2 3 [272]
Nitrogen Urea (1.5 g/L) + Final effluent 48 0.2 5 [272]
KNO3 (1.5 g/L) + Final effluent 23 0.2 2 [272]
NH4NO3 (1.5 g/L) + Final 26 0.1 1 [272]
effluent
Urea (1.75 g/L) + Final effluent 62 0.2 6 [272]
NOx (8 mg/L) 3 0.3 2 [273]
NaNO3+ NOx (34 mg/L) 13 0.3 11 [273]
Seawater Deep Seawater (20%) 77 2.9 189 [274]
Deep Seawater (50%) 45 2.2 109 [274]

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Combined Seawater + Anaerobic 60 0.3 15 [176]
Methods Digestate (3 g/L)
Sodium chloride (10 g/L) + 29 2.7 77b [264]
Sodium acetate (10 g/L)
Other Methods Hydrogen peroxide (0.15 g/L) 0.8 2b [264]
Bovine serum albumin (0.1 g/L) 2.0 15b [264]
2-Stage
Nitrogen Transferred to N-free media 28 3.2 448 [161]
Starvation
Salt Shock Added NaCl (60 g/L) + 1 g/L 38 106 [275]
sodium bicarbonate
Added NaCl (100-200 g/L) 48 0.7 175 [268]
a = Calculated for all data except those published by [264]. The calculation was done by multiplying the biomass
concentration by the lipid content and dividing by the number of days of the study (not shown).
b = This value is neutral lipid content rather than total lipid content.

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 One of the most successful methods of inducing lipid accumulation in microalgae was

done by adding an organic carbon source to the media at the beginning of growth. One study

found that glucose added at 6 g/L at the beginning of growth produced a biomass

concentration of 4.6 g/L and overall lipid productivity of 244 mg/L/d [161]. Since microalgae

cultivation on wastewater would be operated in non-sterile conditions at large scale, such

carbon sources would likely be consumed, in no small degree, by heterotrophic bacteria, which

could outcompete microalgae. This problem would be most prominent during inoculation

before the microalgae would be at a high biomass density. The use of nitrogen depletion to

trigger lipid accumulation has also been discounted for wastewater grown microalgae since

such an approach significantly reduces the overall growth of microalgae. In the literature, deep

seawater was reported to induce C. sorokiniana to accumulate 77% of its mass as lipids. This

high lipid content combined with biomass accumulation to 2.9 g/L suggests benefits to

commercial microalgae production. The combination of seawater and anaerobic digestate, on

the other hand, did not seem to benefit microalgae in the same way as deep seawater alone

[176]. This result was also confirmed through experimentation from our collaborators [180].

A two-stage method of inducing lipid accumulation by the transfer of microalgae to

nitrogen-free media has shown success in reports from literature (Table 4.1) and collaborators

[180]. Such methods require a long holding period of multiple days to stress the microalgae.

Methods of lipid accumulation through the addition of NaCl increased the overall lipid content

in a shorter period than most other ways (48% final lipid content after 6 hours) [268].

Based on these previous results, as well as the limitations put in place by utilizing

wastewater as a nutrient stream, two methods of lipid accumulation were attempted: carbon

164
source addition and salt shock. The carbon source addition would be done through the addition

of sodium acetate (since acetate is a typical byproduct of anaerobic digestion as well as an

inexpensive carbon source). This chemical has been successful at increasing lipid content when

added at the beginning of growth for microalgae [264, 265]. Despite this, adding it in at the

beginning of growth for microalgae on wastewater could cause problems with growth as

outlined in the paragraph above. It was decided to add sodium acetate as part of a 2-stage

system to prevent this. The second method tested was salt shock through the addition of NaCl

and seawater as part of a 2-stage system. This method was selected from successful prior

experimentation [268, 275]. The idealized 2-stage system for lipid accumulation is summarized

in Figure 4.1.

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 A B C D

Figure 4.1: Idealized 2-Stage System for SFWP Grown Microalgae Lipid Accumulation
The 2-stage system starts with the inoculation of algae into SFWP (A), where it can grow until it reaches the
stationary phase (B). At this point, the microalga has some set lipid content. Addition of some chemical (C), in this
case, sodium acetate, sodium chloride, or seawater can induce the algae to accumulate lipids (D) due to one of the
multiple factors outlined above. This entire process will be done using light and CO2 supplementation for growth
and lipid sequestration.

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4.2. Materials and Methods

4.2.1. Strain Selection

Similar to the two previous chapters, the microalgae strain selected for use in this study was

Chlorella sorokiniana UTEX 2805 from the University of Texas Culture Collection of Algae. See

section 2.2.1 for more information.

4.2.2. Wastewater Collection and Preparation

The wastewater used was Stripped Food Waste Permeate (SFWP) collected from the UC Davis

Renewable Energy Anaerobic Digester (READ) which take in campus food waste from the dining

commons for biogas production. This wastewater was stored in non-sterile containers at 4°C

before being subsequently sterilized using sterile syringe filtration. See section 2.2.2 for more

information about the sterilization process. Before use, the wastewater was diluted using

autoclaved distilled water and microalgae inoculum to a 30% dilution. The properties of the

30% SSF-SFWP used in this study are seen in Table 2.4

4.2.3. Microalgae Cultivation Sodium Acetate Induction Experiments

Microalgae cultures were grown in 500 ml glass bottles inoculated with 100 ml of C. sorokiniana

culture grown on N8-NH4 into 150 ml of SSF-SFWP mixed with 300 ml of autoclaved distilled

water to a final volume of 500 ml. The bottles were randomly organized and illuminated.

Lighting for this was similar to previous experiments (10,000 LUX light illumination with

T5 lamps, a ratio of 16∶8 light-dark cycle). Caps included fittings to allow for airflow in and out

of the system through 0.22 µm sterile syringe filters. Carbon dioxide was added to the air to

167
produce a v/v concentration of approximately 6% CO2 in the gas phase to mimic the

concentration of carbon dioxide from flue gas. The Air/CO2 mixture was humidified by flowing

through water before being bubbled into each reactor at a rate of ~50 ml/minute. Media in

each reactor stirred from the bottom of each bottle at ~100 rpm using a ½ inch stir bar.

Experiments were started after cultures reached the stationary phase of growth.

In the sodium acetate (NaAc) induction tests, five reactors were set up with three of

them having concentrations of 10 g/L of sodium acetate (equivalent to 7.2 g/L Acetate). This

concentration was selected as 10-15 g/L was seen as optimal for increasing lipid productivity in

C. sorokiniana [264]. This setup is summarized in Table 4.2.

Table 4.2: Carbon Source Lipid Accumulation Setup

Bioreactor Variable
1 Control (t = 0 days)
2 Control (t = 3 days)
3 10 g/L NaAc (t = 1 days)
4 10 g/L NaAc (t = 2 days)
5 10 g/L NaAc (t = 3 days)

Sodium acetate was dissolved in distilled water, and the solution was sterile filtered

through a 0.2 µm filter to produce a stock solution of 400 g/L. A total of 8 ml of solution was

added to each of the three reactors to create a solution of approximately 10 g/L NaAc.

Before the addition of acetate, one of the reactors was sampled for biomass

concentration at the initial time point (t = 0 days). Each day, for three days, 100 ml of culture

was removed from a single reactor and frozen to be later dried for biomass and lipid analysis.

After three days, the final NaAc reactor, as well as a control reactor, was harvested.

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4.2.4. Microalgae Cultivation for Salt Stress Experiments

Microalgae for salt stress experiments were grown in the same manner as for sodium acetate

induction but in 1 L glass bottles inoculated with 100 ml of C. sorokiniana culture grown on N8-

NH4 into 300 ml of SSF-SFWP mixed with 600 ml of autoclaved distilled water to a final volume

of 1 L.

In the salt-stress induction tests, five reactors were set up with four separate salt

stressor variables: 16.7 g/L NaCl, 39 g/L artificial seawater, 27.8 g/L NaCl, and 55.6 g/L NaCl.

These salts were added in their dry form directly into the solution where they were mixed until

they were dissolved. This setup is shown in Table 4.3.

100 ml of each sample was taken every 2 hours for 6 hours. The samples were frozen for

later biomass and lipid concentration analysis. Such sampling was done in non-sterile

conditions.

Table 4.3: Salt Stress Lipid Accumulation Setup

Bioreactor Variable
1 None
2 16.7 g/L NaCl
3 39 g/L Synthetic Seawater
4 27.8 g/L NaCl
5 55.6 g/L NaCl

4.2.5. Synthetic Seawater

The synthetic seawater used was based off a popular artificial seawater mixture described by

Kester, et al., 1967 [276]. The mixture only included major ions that were identified in

concentrations greater than 0.1 g/kg, as those were predicted to be most impactful for the lipid

169
accumulation in microalgae. The final mixture consisted of 23.93 g/L NaCl, 4.01 g/L Na2SO4, 0.71

g/L KCl, 0.25 g/L Na2CO3, 5.07 g/L MgCl2, 1.52 g/L CaCl22H2O.

4.2.6. Biomass Quantification

Biomass samples were thawed, washed three times, and concentrated into pre-weighed plastic

15 ml tubes before being pelleted at 5000 g for 5 minutes (IEC Multi RF, Thermo Electron

Corporation, Waltham, MA). Tubes were subsequently frozen at -85°C. The pellet of sodium

acetate induced lipid algae cells were lyophilized at −45°C for two days (Freezone4.5, Labconco,

Kansas City, MO) before being weighed. All other samples were lyophilized at the California

Processing Tomato Industry Pilot Plant within the August A. Busch III Brewing and Food Science

Laboratory (University of California Davis, Davis, CA). The final weight was measured, and

biomass concentration calculated from this dry weight and the volume of algae sampled.

4.2.7. Total and Neutral Lipid Quantification

Lipid was extracted from microalgae samples using a method described by Floch et al. 1956

[277]. Samples were stored at -20°C before use.

Total lipid quantification was done using the method outlined by Cheng et al., 2011

[278] in which a colorimetric assay using the sulfo-phospho-vanillin (SPV) reaction produced

quantifiable measurements of lipid concentration. The standard used for this process was

culinary algae oil (Thrive, San Francisco, CA). No difference was seen between the use of

culinary algae oil and pure soybean oil (Better Living Brands LLC, Pleasanton, CA) in the SPV

assay.

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 Neutral lipid quantification was done using a method similar to that outlined by Higgins,

et al., 2014 [279]. The neutral lipid standard was pure soybean oil (Better Living Brands LLC,

Pleasanton, CA).

Extracts from the acetate samples showed higher rates of chlorophyll degradation,

potentially from free-thaw cycles during repeated testing. This degradation of chlorophyll

resulted in less interference for these assays and higher lipid content in total lipid quantification

and neutral lipid quantification. Due to this, a different approach was taken for measuring the

neutral lipid content of microalgae induced with salts. After the extraction of lipids and

measurement of Nile-Red fluorescence as described above, the samples were normalized to

that of the sodium acetate control as opposed to utilizing the neutral lipid standard. The initial

concentration values were adjusted such that they averaged to initial neutral lipid

concentrations seen in the sodium acetate control conditions. This adjustment was due to the

limitation put in place by the four-month wait to further degrade pigments.

The process of lipid extraction and quantification using the total lipid assay and neutral

lipid assay are outlined in Figure 4.2.

Figure 4.2: Schematic of Microalgae Biomass Lipid Analysis

The illustration shows the process of harvesting algae through centrifugation, freeze-drying (lyophilizing), lipid
extraction, and the colorimetric assay. Image used with permission from B. T. Higgins. Originally published in [181].

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4.3. Results

4.3.1. Lipid Induction with Sodium Acetate

4.3.1.1. Lipid Accumulation

The initial total lipid content was measured at 36.29% (Figure 4.3) with the neutral lipid

content at 6.55% (Figure 4.4) in the control at t = 0 days. One day after sodium acetate addition

there was no substantial change in the total lipid content. During that time a 47.5% increase in

neutral lipids was observed within the microalgae. Up until day three, the total lipid content

over that time decreased about 20% for both the control and for the experimental. During this

time there was a slight drop in neutral lipid content by day two to 8.72 % that rose to 11.40% by

day three, which equated to a total increase of 74.0% from the initial value at a final lipid

concentration in the reactor of 0.12 g/L neutral lipids. Over these three days, the control

neutral lipid content rose just 6.8%. Biomass concentrations were constant across the samples

tested (~1 g/L).

50
Total Lipid Content (%)

40

30

20

10

0
1 2 3 4
Day

Figure 4.3: Sodium Acetate Induced C. sorokiniana Total Lipid Content

◼ Control (0 g/L NaAc), ◼ 10 g/L NaAc
The standard deviation is based on the assay (n=4). The experiment itself was run in single reactors (n=1).

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 A. 12 B. 80

Change in Neutral Lipid (%)

Neutral Lipid Content (%)

60
8

40

4
20

0 0
0 1 2 3 1 2 3
Day Day

Figure 4.4: Sodium Acetate Induced C. sorokiniana (A.) Neutral Lipid Content and (B.) Percent
Change in Neutral Lipid Content
◼ Control (0 g/L NaAc), ◼ 10 g/L NaAc
A. is the neutral lipid content and B. is the change in neutral lipid. The standard deviation is based on the
assay (n=4). The experiment itself was run in single reactors (n=1).

4.3.1.2. Analysis

Results from the treatment of microalgae with 10 g/L of sodium acetate addition indicated

there was a moderate decrease in total lipids over time for both the control and the

experimental setups. A large accumulation of neutral lipids was noted within the first day after

inoculation (an increase of 47.5% from the initial lipid content of 6.55%). A second increase was

observed at day 3, where neutral lipid accumulation further increased to a total accumulation

change of 74.0%. Throughout this time, the biomass concentration did not change significantly,

and the neutral lipid shift could not be accounted for by time alone.

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4.3.2. Lipid Induction Through Salt Stress

4.3.2.1. Lipid Accumulation

The total lipid content and neutral lipid content across all samples before salt addition (t = 0

hours) were 23.16 ± 1.96 % and 6.55 ± 1.20%, respectively (Figure 4.4, Figure 4.5). There were

no major changes in total lipid content in any of the samples during the first 6 hours of

induction with salts. The largest change was a ~20% decrease in total lipids at hours 6 in the

55.6 g/L NaCl sample. Despite the consistent total lipid contents, there were notable changes in

the neutral lipid content. After two hours of salt stress, a slight decrease was seen in neutral

lipid content across samples 1-3 and 5, with a small increase in sample 4. When normalized

against the control and the initial concentration at each treatment, there was a slight increase

in lipid content that was 9.45%. This was an increase of 31.14 ± 24.49% after normalization

(Figure 4.6). At hour four, all samples showed a substantial increase in neutral lipid

accumulation with the treatment of 39 g/L synthetic seawater being the lowest at a 44.49%

increase and the treatment of 27.8 g/L NaCl being the highest value at 164.48%. By hour six, the

treatment with 55.6 g/L NaCl produced the highest neutral lipid concentration increase at

188.87%. The treatments with 16.7 g/L NaCl, 39 g/L synthetic seawater, and 27.8 g/L NaCl all

showed a decrease in lipid accumulation from hour four (27.37%, 41.54%, and 18.34%,

respectively).

The highest neutral lipid increase was found in the treatment with 55.6 g/L NaCl at

18.82% neutral lipid after 6 hours. The second highest neutral lipid content overall was at hour

four by the treatment with 27.8 g/L NaCl at 14.19% followed by a 13.51% at hour four by the

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treatment with 55.6 g/L NaCl. Biomass concentrations were constant across the samples tested

(~0.6 g/L).

30

25
Total Lipid Content (%)

20

15

10

0
0 2 4 6
Hour

Figure 4.5: Salt-Induced C. sorokiniana Total Lipid Accumulation

◼ 0 g/L NaCl, ◼ 16.7 g/L NaCl, ◼ 39 g/L Synthetic Seawater, ◼ 27.8 g/L NaCl, ◼ 55.6 g/L NaCl,
The standard deviation is based on the assay (n=4). The experiment itself was run in single reactors (n=1).

30
Neutral Lipid Content (%)

25

20

15

10

0
0 2 4 6
Hour

Figure 4.6: Salt-Induced C. sorokiniana Neutral Lipid Accumulation

◼ 0 g/L NaCl, ◼ 16.7 g/L NaCl, ◼ 39 g/L Synthetic Seawater, ◼ 27.8 g/L NaCl, ◼ 55.6 g/L NaCl,
The standard deviation is based on the assay (n=4). The experiment itself was run in single reactors (n=1).

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 200

160
Percent Change (%)

120

80

40

0
2 4 6
Hour

Figure 4.7: Normalized Percent Change of Salt-Induced C. sorokiniana Neutral Lipid

Accumulation
◼ 16.7 g/L NaCl, ◼ 39 g/L Synthetic Seawater, ◼ 27.8 g/L NaCl, ◼ 55.6 g/L NaCl,
Data were normalized against the initial lipid concentration and against the percent change of lipid at 0 g/L NaCl to
account for sampling error.

4.3.2.2. Analysis

Results from salt stress-induced lipid accumulation indicate that salts can cause a large increase

in neutral lipid content algae biomass, but no significant changes in overall lipid content. All

four salt concentrations tested caused a rise in neutral lipids within six hours of addition. The

maximum increase was seen at the highest concentration of NaCl tested: 18.82% neutral lipid

content from an initial content of 6.97%. This change was a total increase from the normalized

control by 188.87% and accounted for all of the lipid measured in the total lipid assay. Even at

the lowest salt concentration, 16.7 g/L, a 50% increase in neutral content was seen in 6 hours.

The lowest three salt concentrations tested showed a peak before hour 6 while lipid

concentrations continued to increase by hour 6 at the highest NaCl concentration of 55.6 g/L.

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4.4. Discussion

The addition of sodium acetate, sodium chloride, and synthetic seawater as part of a 2-stage

system increased neutral lipid content while not increasing total lipid content in microalgae C.

sorokiniana growing on 30% SSF-SFWP. This suggests a transformation of existing lipids to

neutral lipids upon induction with these chemicals.

Sodium acetate yielded an increase in neutral lipid concentrations of 47.47% after one

day and 73.97% after three days of contact with the chemical. Despite the substantial increase

in neutral lipid content from sodium acetate, the final concentration of neutral lipids was

measured at 11.40%. A neutral lipid content this low would not be useful for large-scale

microalgae growth for biofuel production. Also, the three days required to induce these lipids

would make this method impractical. As noted, during these days there was a decrease in total

lipids by about 20%, making this process no better than other long-term lipid induction

methods.

Other studies have shown that when sodium acetate was added initially, microalgae

accumulated much higher lipid content than seen in this experiment [264, 265]. It is possible

that C. sorokiniana cannot effectively metabolize this carbon source during the stationary phase

of growth on SSF-SFWP due to other limiting nutrients in the wastewater.

Results from this study also suggest that C. sorokiniana will initially increase neutral lipid

content when incubated with sodium acetate after one day and three days. This result indicates

that there is either a stress factor caused by the addition of such a large concentration of

sodium acetate or that some of the acetate is quickly utilized and used to convert lipids to

neutral initially. The second neutral lipid accumulation seen at day three might be due to the

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slow utilization of acetate or a lag period to fully utilize acetate effectively. Additionally,

depletion of acetate or other nutrients might also cause nutrient stress that leads to more lipid

accumulation.

The addition of salts (NaCl and synthetic seawater) to C. sorokiniana showed a higher

neutral lipid accumulation in a shorter duration than that caused by sodium acetate addition

with no substantial change in total lipid content. Within 6 hours of being incubated with 55.6

g/L NaCl, the neutral lipid content in microalgae increased by 189% to a final content of 18.82%

which was nearly three times higher than the initial lipid concentration. Despite this substantial

increase, this result is still less than the 48% lipid content produced by C. sorokiniana HS1

incubated with 100-200 g/L NaCl after 6 hours [268] as well as the 38% lipid content produced

in the same strain after 2 days in 60 g/L NaCl with 1 g/L sodium bicarbonate. The initial lipid

content in this strain was 25% when grown on BG-11, similar to the 23.16% reported in this

study.

The final biomass concentration for the salt-stressed microalgae was lower than that

used in the sodium acetate induced neutral lipid experiments by ~0.4 g/L. This result may be

because the salt stress experiments were performed after the sodium acetate experiment, and

some nitrogen had volatilized from the stored wastewater during that time. Another possibility

is that some of the microalgae biomass was lost through freeze-drying or the samples were

dried to lower moisture content than what could be done with the machine used for sodium

acetate samples. This discrepancy is possible since the UC Davis California Processing Tomato

Industry Pilot Plant within the August A. Busch III Brewing and Food Science Laboratory was

used for the salt stress samples while the Freezone4.5 was used for the sodium acetate induced

178
samples. It doesn’t alter the efficacy of the method of lipid accumulation since these 2-stage

experiments were done after the microalgae reached the stationary phase of growth.

Results from the lipid assays indicated variation in lipid content across the different

sampling times in the salt stress conditions. This variation may have been caused by sampling or

mixing error. In this experiment, lipid content would fluctuate up or down between different

time points for all the treatments. This difference is most obviously seen by the decrease in

lipid content at time 2 hours by the control and three of the four treatments. This error was

accounted for in the percent change calculations, where treatments were normalized

concerning their initial treatment as well with the control at that time point. Due to this,

percent changes do not directly reflect the percent change in the treatment alone, but also

include the control as a method to normalize for this fluctuation.

One major problem encountered in this study was that there was no standard method

of measuring lipid content in microalgae. While multiple methods exist, there are problems

with each that cause measuring biases [279]. In this study, the measured total lipid content was

less in the salt stress samples (23.16%) than the sodium acetate samples (36.29%). Notably,

there were higher levels of green pigmentation in the lipid extract samples from the salt stress

experiments than those of the sodium acetate samples. Over approximately four months,

pigmentation in the sodium acetate samples seemed to degrade after being stored at -20 °C

whereas little to no degradation was seen in the salt stress samples. Due to this pigment

degradation, there was less interference in colors when using the colorimetric assays. This

might explain the higher total lipid content observed in the assay.

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 This problem was carried over during measurement of the neutral lipid content using

the Nile red based lipid assay. This assay was hindered by having excitation and absorbance

wavelengths that overlap with those seen in chlorophyll absorption spectra. Because of this

only relative fluorescence analysis could be used on the salt-stressed samples as they produced

fluorescence values well below that of the solvent blank. It is hypothesized that this low

fluorescence value to be due to absorption of the excitation and fluorescence wavelengths. The

use of bleach to remove pigmentation helped increase fluorescence but was also observed to

oxidize lipids in the process. The method of breaking down these pigments through prolonged

freezing, as used in sodium acetate induced samples, helped to reduce levels of these pigments

and give a higher fluorescence value.

Despite this, the lipid content measured through both methods were likely errored on

the conservative side since pigmentation was still present during measurement and, for the

neutral lipid assay, the added bleach may have oxidized neutral lipids, reducing the content

measured. Knowing the limitation of these assays suggest that the viability of the lipid induction

methods tested might be higher than what was reported in this experiment, as the lipid values

measured were likely lower than the actual values.

All in all, both sodium acetate and salt addition to microalgae cultures were shown to

increase neutral lipid content. The most successful accumulation was seen after 6 hours after

the addition of 55.6 g/L NaCl when neutral lipids increased 189% to a final content of 18.82%.

180
5. Chapter 5 - Conclusion

Combining microalgae production with wastewater treatment is considered the only

economically viable way to produce algae biofuels using current technology [1]. This conclusion

was due, in part, to the ability of microalgae to utilize a waste material for nutrients and

produce two high-value products: algae biomass and partially treated wastewater.

Unfortunately, not enough research has been done to understand the operation of this

combined system. As a result, no such facility currently exists.

This thesis has covered three key areas necessary for combining microalgae wastewater

treatment with biofuel production: growing microalgae in both batch and semi-continuous

conditions on wastewater, characterizing the microbial community that associates itself with

microalgae in these conditions, and identifying methods of lipid accumulation that would be

viable in wastewater conditions. The results of the experiments conducted in this thesis suggest

that such a combined process can be developed to significantly reduce the nutrient content in

wastewater while increasing total neutral lipid content for biofuel production. Also, this study

found that the microbial community has a great influence on microalgae biomass production

and specific bacteria may play a vital role in the productivity of these systems.

Microalgae C. sorokiniana UTEX 2805 grown on a 30% dilution of unsterilized SFWP

produced high biomass concentrations of 1.79 g/L after 28 days (biomass productivity = 63.9

mg/L/d). When utilizing sterilized wastewater and supplemental CO2, the microalgae biomass

concentration topped out at 0.99 g/L, but after a shorter period of 11 days (biomass

productivity = 89.8 mg/L/d). It was hypothesized this was due in part to beneficial interactions

with microbes present in the wastewater.

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 In sterile conditions, microalgae produced high biomass productivities in semi-

continuous growth. The biomass productivity between feeds was ~600 mg/L/d for 30% (v/v/d)

on 30% VSF-SFWP, ~350 mg/L/d for 30% v/v/d on 30% SSF-SFWP and 600 mg/L/d for 50% v/v/d

on 30% diluted VSF-SFWP. This high productivity is the result of cultures being maintained in

the exponential growth phase. Microalgae cultures that were intentionally contaminated did

not have biomass productivity rates significantly different than those of sterile culture (616.7

mg/L/d and 583.3 mg/L/d, respectively). This result suggests that in low concentrations, the

microbial community doesn’t affect microalgae production greatly. Nutrient removal conditions

on sterile wastewater (SSF-SFWP) showed that both 30% v/v/d and 50% v/v/d feed rates

removed high concentrations of TN (~92%), N-NH4 (~100%), and sCOD (~77%). The 50% v/v

semi-continuous feeding showed higher removal rates and concentrations of nutrients

removed (TN: 60.1 mg/day, N-NH4: 27.1 mg/day, sCOD: 147.9 mg/day) than the 30% v/v (TN:

36.1 mg/day, 14.7 mg/day, 31.9 mg/day). This result suggests higher nutrient removal rates

might be accomplished at higher volume semi-continuous media replacement rates.

Microalgae inoculated at low densities into unsterilized SFWP at a 30% dilution

displayed variable biomass concentrations after seven days of growth (0.114 g/L to 0.890 g/L).

This discrepancy was due to the microbial communities that developed as all other conditions

were consistent between samples. This variability produced discrepancies in the removal of TN,

N-NH4, and sCOD. Across the samples, it was seen that taxa from Pseudomonadaceae and

Halomonas caused the most significant differences in the community structure between

incubated cultures and the initial wastewater. OTUs associated with the genus Achrombacter

showed a significant correlation with the removal of N-NH4 while Pusillimonas and

182
Alcaligenaceae showed a significant correlation with microalgae biomass concentration. The

family Rhodobacteraceae alone showed a significant correlation with the bacteria biomass

concentration which increased even more when combined with the genus Aquamicrobium.

Additional characterization of the OTUs was made using predictive metagenomics to

characterize the microbial community more extensively. It is not clear if the changes in

microalgae biomass are the result of these organisms growing in the wastewater, or if

environmental changes (i.e., decreased microalgae biomass concentration) caused them to

grow. The results illustrate the importance of understanding the community dynamics between

microalgae and bacteria.

Neutral lipid transformation in microalgae was shown to be inducible through a 2-stage

process by using both organic carbon addition and salt stress without changing total lipid

content substantially in the process. Results from the addition of sodium acetate at 10 g/L

produced a 43.47% increase in neutral lipids after one day for a final neutral lipid content of

9.66%. Waiting an additional three days from inoculation increased the neutral lipid by 73.97%

to a content of 11.40%. The addition of 55.6 g/L of NaCl to microalgae increased neutral lipid

content by 188.87% to content of 18.82% after just six hours of addition. High rates of neutral

lipid accumulations were also seen at lower NaCl concentrations (27.8 g/L NaCl) after 4 hours

(164.47% increase to a content of 14.19%). This result suggests that this method can induce

high levels of neutral lipids in a short period.

Overall, this thesis shows that there is potential in combining wastewater treatment

with microalgae production for future biofuel production. Microalgae are highly adaptable to

both batch and semi-continuous conditions for treating wastewater and producing biomass,

183
but the microbial community heavily influences such concentrations. Microalgae biomass can

be induced to produce high quantities of neutral lipids in a short period with simple chemicals,

increasing its biofuel potential. These key chapters further promote the feasibility of developing

such a combined system.

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6. Chapter 6 - Future Work

Currently, algae-based biofuels lack the infrastructure and funding to be economical.

The biggest obstacle to this is the low price of petroleum-based fuels. Studies suggest that

there is considerable variability between assessments of the current cost of algae biofuels

[262]. Key factors within algae biofuels production will need to be addressed to encourage

future investment into this enterprise.

Biological problems exist in the culturing of microalgae, as with any agricultural crop,

that decrease total biomass yield. These include grazing by pathogens, infections from viruses,

and competition from other organisms [280]. Few economic methods exist to prevent or treat

such problems, despite an increase in research in this area in recent years [281]. In the case of

wastewater, existing chemical properties of this waste hinder the growth of microalgae and

reduce the biomass production rates of combined systems [195]. These factors act as

deterrents to those looking to grow microalgae for a stable source of income.

In addition, large infrastructure needs to be developed to grow microalgae for biofuels.

Large areas of land will need to be devoted to growing microalgae if it is to compete directly

with petroleum-based fuels (Figure 6.1). While the idea of combining wastewater treatment

and biofuel production will help to reduce the cost of growing microalgae, it still required the

design and construction of very costly facilities. Retrofitting existing wastewater facilities would

require drastic modifications that would be costly. There also lacks any universal methods for

harvesting microalgae [282]. Most of the existing methods are expensive as they either require

significant energy or expensive equipment or chemicals. Any method to improve the harvesting

of microalgae would most significantly reduce the cost of microalgae biofuels and increase the

185
feasibility of using these organisms for fuel production. Processing equipment to convert

microalgae to fuels will also need to be constructed. Such systems will differ in initial processing

steps to traditional petroleum-based fuels. This further increases the costs of algae-based

biofuels.

Figure 6.1: Artist’s Concept of Futuristic Microalgae Fuel Farm in the American Southwest
Microalgae systems use HRAPs for algae growth and square ponds for settling for harvesting. It has been
hypothesized that such systems would be built in deserts or badlands, where there is a high concentration of solar
energy and inexpensive land that is unusable for agriculture. Image credit: [283]

Advances in biotechnology have helped produce organisms that can accumulate more

high-quality lipids and other fuel stuff than wild microorganisms [284]. Modifications have been

proposed to increase the photosynthetic rate and breadth of light used for photosynthesis,

increase resistance to pathogens, and modify cells that are easier to harvest [285]. Such

186
manipulations of biological organisms face significant public backlash for fear of dangers

associated with GMOs (genetically modified organisms) [286]. Despite this, the final report of

the Aquatic Species Program listed metabolic engineering as a significant area of interest to

make these fuels viable in the future [1].

In comparison to other energy sources, algae fuels have received very little financial

investment [81]. Despite this, research has come a long way into developing systems to grow

and process microalgae. These innovations have also helped the private industry produce new

microalgae-based products. Most of these new products take on the form of health foods and

nutraceuticals like culinary oils [287], butter substitutes [288], and dietary supplements [289,

290] to name a few.

While algae-based biofuels will not be at the gas station any time soon, there is a good

chance that one day may change. Approaches such as genetic engineering and combined

biofuel production and wastewater treatment will help to increase the yield of algae while

reducing its cost, but high capital costs for growth ponds, harvesting mechanisms, and

processing equipment act as a significant deterrent to such endeavors. Finding ways to reduce

these costs through research and innovation will help lead the world to a newer, more

sustainable fuel source. Without significant investment in these fuels, such endeavors will be

impossible. Despite these setbacks, many have high hopes that someday there will be

largescale microalgae production facilities for fuel production.

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2010, 101(14):5144-5149.
341. Marcilhac C, Sialve B, Pourcher A-M, Ziebal C, Bernet N, Béline F: Control of nitrogen
behaviour by phosphate concentration during microalgal-bacterial cultivation using
digestate. Bioresource Technology 2015, 175:224-230.

207
 8. Chapter 8 - Appendix

8.1. Prior Studies of Microalgae Growing on Anaerobic Digestate Effluents

Table A1: Previous Studies of Growing Microalgae on Anaerobic Digestate Effluents for Biomass Production, Lipid Accumulation, or
Wastewater Treatment
Organism Media Pretreatment CO2 (%) Days Biomass Lipid Nutrient Initial Nutrients Study
(concentration, (% dry weight, Removal
productivity, productivity)
specific growth
rate)
Acutodesmus
A. obliquus 10% Agro- Centrifuged + atm 25 1.0 g/L 19.5% TP = 84% TN = 399 mg/L [291]
(Dairy) DE 1.2 μm 40 mg/L/d* 7.8 mg/L/d** COD = 39% N-NH4 = 318 mg/L
Filtered TP = 11 mg/L
COD = 1038mg/L
208

Anabaena
A. spiroides 16.7% 1.2 μm biogas 7 0.64 g/L/d TN = 58% TN = 33 mg/L [292]
Domestic DE Filtered + upgrade 79 mg/L/d TP = 37% TP = 3 mg/L
UV Sterilizer (32.57% 0.32 d-1 COD = 62% COD = 132 mg/L
CO2)
Ankistrodesmus
A. gracilis 5% Piggery 0.2 μm 5 4.7 1.3 g/La TN = 85%a TN = 115 mg/La [293]
Waste DE Filtered 283 mg/L/d* N-NH4 = 87%a N-NH4 = 70 mg/La
TP = 85%a TP = 3 mg/La
Arthrospira
A. platensis 2% Pig atm 12 0.77 g/L 18% TN = 80% TN = 17 mg/L [294]
Waste DE + 58 mg/L/d* 10.5 mg/L/d
NaHCO3

208
 A. platensis 10% Swine Sanitized with atm N-NH4 = 92% N-NH4 = 7-21 mg/L [295]
Waste DE + 0.2 g/L P-PO4 = 67% P-PO4 = 26-68 mg/L
NaHCO3 and Na2S2O5 at COD = 23% COD = 120-180 mg/L
NaNO3 for a day
A. platensis Sago Starch Gauze atm 14 0.65 g/L N-NH4 = 99% TN = 3 mg/L [296]
Factory DE Filtered 46 mg/L/d* P-PO4 = 89% N-NH4 = 3 mg/L
0.54 d-1 COD = 95% P-PO4 = 21 mg/L
COD = 1340 mg/L
Botryococcus
B. braunii 5% Piggery 0.2 μm 5 4.7 1.39 g/L a TN = 89%a TN = 115 mg/La [293]
Waste DE Filtered 295 mg/L/d* N-NH4 = 91%a N-NH4 = 70 mg/La
TP = 85%a TP = 3 mg/La
B. braunii Struvite atm 7 N-NH4 = 19% N-NH4 = 34 mg/L [297]
Stripped P-PO4 = 76% TP = 7 mg/L
Piggery 0.29 d-1 COD = 31%
Waste DE
209

Chlorella
C. ellipsoidea 6.7% Kitchen atm 14 0.3 g/L TN = 13% TN = 138 mg/L [175]
Waste DE 21 mg/L/d* N-NH4 = 36% NH4-N = 105 mg/L
0.12 d-1 TP = 81% TP = 2 mg/L
COD = 44% COD = 390 mg/L
C. minutissima 6% Poultry Centrifuged atm 12 0.65 g/L 12.2% TN = 54% TN = 99 mg/L [186]
Litter DE 54 mg/L/d 6.6 mg/L/d TP = 71% TP = 7 mg/L
C. minutissima 6% Poultry Centrifuged atm 12 0.39 g/L 9.9% TN = 152 mg/L [186]
Litter DE 24 mg/L/d 2.4 mg/L/d TP = 9 mg/L
C. pyrenoidosa Swine Autoclaved 14 13 4.26 g/L N-NH4 = 66% TN = 1135 mg/L [298]
Manure DE 285 mg/L/d TP = 78% N-NH4 = 1093 mg/L
COD = 63% TP = 25 mg/L
COD = 1135 mg/L

209
 C. pyrenoidosa Swine Autoclaved atm 8 4.81 g/L 19.46 N-NH4 = 52% TN = 1135 mg/L [298]
Manure DE + 601 mg/L/d TP = 88% N-NH4 = 1093 mg/L
Phosphate COD = 74% TP = 25 mg/L
COD = 1135 mg/L
C. pyrenoidosa Swine Autoclaved atm 13 3.98 g/L 15.61 N-NH4 = 67% TN = 1135 mg/L [298]
Manure DE 130 mg/L/d TP = 96% N-NH4 = 1093 mg/L
COD = 49% TP = 25 mg/L
COD = 1135 mg/L
C. pyrenoidosa Struvite atm 7 N-NH4 = 49% N-NH4 = 34 mg/L [297]
Stripped TP = 64% TP = 7 mg/L
Piggery 0.29 d-1 COD = 29%
Waste DE
C. pyrenoidosa 5.3% Starch 0.45 μm atm 9 3.01 g/L 25.4% TN = 92% TN = 281 mg/L [299]
Processing Filtered 58 mg/L/d 14.7 mg/L/d** P-PO4 = 91% P-PO4 = 27 mg/L
and Alcohol 0.56 d-1 COD = 76% COD = 4000 mg/La
Wastewater
210

DE
C. pyrenoidosa Starch 0.45 μm 5-9% 10 2.05 g/L 7.3% TN = 83%* TN = 240.3–382.7 mg/L [300]
Processing Filtered 37 mg/L/d 24.2 mg/L/d TP = 97%* N-NH4 = 217.6–334.7
Wastewater Autoclaved 1.02 d-1 COD = 66%* mg/L
DE TP = 22.7–40.2 mg/L
COD = 702.4–1026.2
mg/L

C. regularis Struvite atm 7 N-NH4 = 58 mg/L [301]

Stripped P-PO4 = 9 mg/L
Piggery 0.10 d-1
Waste DE
C. regularis Struvite atm 7 N-NH4 = 58% N-NH4 = 34 mg/L [297]
Stripped P-PO4 = 87% P-PO4 = 7 mg/L
Piggery 0.31 d-1 COD = 35%
Waste DE

210
 C. regularis var. Struvite atm 7 N-NH4 = 57% N-NH4 = 34 mg/L [297]
minima Stripped P-PO4 = 66% P-PO4 = 7 mg/L
Piggery 0.15 d-1 COD = 21%
Waste DE
C. sorokiniana 3% Kitchen Gauze atm 11 0.26 g/L 60% [176]
Waste DE + Filtered 23 mg/L/d 15.0 mg/L/d
Seawater 0.35 d-1
C. sorokiniana 6% Poultry Centrifuged atm 12 0.39 g/L 8.2% TN = 152 mg/L [186]
Litter DE 33 mg/L/d* 2.7 mg/L/d** TP = 9 mg/L
C. sorokiniana 6% Poultry Centrifuged atm 12 0.63 g/L 15.7% TKN = 45% TN = 96 mg/L [186]
Litter DE 53 mg/L/d 8.2 mg/L/d** TP = 71% TP = 7 mg/L
C. sorokiniana 10% Cattle Centrifuged atm 24 0.27 g/L 26.8 TN = 87% TN = 231 mg/L [187]
UTEX 1230 Waste DE 13 mg/L/d 3.4 mg/L/d** N-NH4 = 65% N-NH4 = 89 mg/L
TP = 65% TP = 112 mg/L
P-PO43 = 58%
C. sorokiniana 10% Cattle Centrifuged atm 24 0.15 g/L 24.2 g/L TN = 87% TN = 231 mg/L [187]
211

UTEX 2714 Waste DE 9 mg/L/d 2.3 mg/L/d** N-NH4 = 72% N-NH4 = 89 mg/L
TP = 64% TP = 112 mg/L
P-PO4 = 56%
C. sorokiniana 10% Cattle Centrifuged atm 24 0.28 g/L 24.0 TN = 86% TN = 231 mg/L [187]
UTEX CS-01 Waste DE 16 mg/L/d 3.9 mg/L/d** N-NH4 = 75% N-NH4 = 89 mg/L
TP = 61% TP = 112 mg/L
P-PO4 = 47%
C. vulgaris 5% Animal atm 10 TN = 60 mg/L [302]
Waste DE N-NH4 = 55 mg/L
0.73 d-1 TP = 2 mg/L
COD = 100 mg/L
C. vulgaris 6.7% Agro- Centrifuged 3 N=NH4 = 94%a N-NH4 < 1 mg/L [303]
Zootechnical 26 mg/L/d P-PO4 = 96%a COD = 2193 mg/L
Waste DE 0.5 d-1

211
 C. vulgaris 6.7% Agro- Centrifuged 3 14 TN > 90%a TN = 159 mg/L [303]
Zootechnical N-NH4 = 98%a N-NH4 = 152 mg/L
Waste DE TP = 7 mg/L
C. vulgaris 6.7% Agro- Centrifuged, 3 14 TN > 85%a TN = 69 mg/L [303]
Zootechnical Autoclave N-NH4 = 100%a N-NH4 = 61 mg/L
Waste DE TP = 10 mg/L
C. vulgaris 5% Piggery 0.2 μm 5 4.7 1.0 g/La TN = 93%a TN = 115 mg/La [293]
Waste DE Filtered 212 mg/L/d* N-NH4 = 100% N-NH4 = 70 mg/La
a

TP = 3 mg/La
C. vulgaris 3.3% Agro- Centrifuged + atm 30 0.74 g/La TN = 174 mg/L [304]
(Dairy) 1.2 μm 25 mg/L/d* N-NH4 = 98 mg/L
Waste DE Filtered TP = 162 mg/L
COD = 2 mg/L
C. vulgaris 16.7% 1.2 μm biogas 7 1.04 g/L TN = 56% TN = 33 mg/L [292]
Domestic DE Filtered + upgrade 139 mg/L/d TP = 54% TP = 3 mg/L
UV Sterilized (32.57% 0.39 d-1 COD = 65% COD = 132 mg/L
212

CO2)
C. vulgaris 16.7% 1.2 μm biogas 6 0.36 g/L TN = 51% TN = 51 mg/L [305]
Domestic DE Filtered + upgrade 112 mg/L/d TP = 63% TP = 2 mg/L
UV Sterilized (34.45% 0.36 d-1 COD = 59% COD = 169 mg/L
CO2)
C. vulgaris 20% Autoclaved atm 30 2.01 g/La TN = 98 mg/L [304]
Municipal 67 mg/L/d* N-NH4 = 97 mg/L
Wastewater TP = 350 mg/L
DE + HS COD < 1 mg/L
Media
C. vulgaris 20% Sewage Autoclaved atm 30 0.48 g/La TN = 550 mg/L [304]
Sludge DE 16 mg/L/d* N-NH4 = 290 mg/L
TP = 96 mg/L
COD = 4 mg/L

212
 C. vulgaris Electro- 5 6 1.71 g/L 35% TN = 82% TN = 43 mg/L [177]
Coagulated 220 mg/L/d 77.0 mg/L/d** TP = 88% TP = 1 mg/L
Dairy and 1.03 d-1 COD = 77%
Food Waste
DE
C. zofingiensis Two-Phase Autoclave atm 11 COD = 37% TN = 14 mg/L [306]
Olive Mill
Waste DE + 0.49 d-1
Glucose
C. sp. 5% Dairy 1.5 μm atm 21 1.71 g/L 13.6% TN = 78% TN = 173 mg/L [307]
Manure DE Filtered 81 mg/L/d* 11.1 mg/L/d** N-NH4 = 100% N-NH4 = 112 mg/L
0.35 d-1 TP = 76% TP = 12 mg/L
COD = 34% COD = 1188 mg/L
C. sp. 30% atm 12 1.29 g/L N-NH4 = 66 mg/L [308]
Chroococcus 108 mg/L/d TP = 32 mg/L
sp. Biomass
213

DE +
Domestic
Wastewater
C. sp. 2% Swine atm 12 0.58 g/L N-NH4 = 100% N-NH4 = 66 mg/L [309]
Manure DE 41 mg/L/d P-PO4 = 91% P-PO4 = 6 mg/L
COD = 55% COD = 154 mg/L
C. sp. 16.7% 1.2 μm biogas 7 0.25 g/La TN = 56% TN = 70 mg/L [310]
Domestic DE Filtered + upgrade 36 mg/L/d* TP = 55% TP = 2 mg/L
UV Sterilized (35.3% COD = 60% COD = 216 mg/L
CO2)
C. sp. 16.7% 1.2 μm biogas 6 0.49 g/L TN = 73% TN = 61 mg/L [311]
Domestic DE Filtered + upgrade 82 mg/L/d* TP = 68% TP = 2 mg/L
UV Sterilized COD = 79% COD = 180 mg/L

213
 C. sp. 16.7% 1.2 μm biogas 6 0.62 g/L TN = 84% TN = 60 mg/L [312]
Domestic DE Filtered + upgrade 103 mg/L/d TP = 80% TP = 6 mg/L
UV Sterilized (26.14% COD = 89% COD = 164 mg/L
CO2)
C. sp. 16.7% 1.2 μm biogas 8 0.56 g/L TN = 55% TN = 80 mg/L [313]
Domestic DE Filtered + upgrade 70 mg/L/d* TP = 51% TP = 9 mg/L
UV Sterilized (28.4% COD = 64% COD = 201 mg/L
CO2)
C. sp. 40% Sludge 3 2.11 g/L TN = 47% TN = 532 mg/L [314]
Liquor DE 45 mg/L/d TP = 94% TP = 12 mg/L
COD = 68% COD = 1508 mg/L
C. sp. 30% atm 12 1.42 g/L [308]
Chroococcus 118 mg/L/d*
sp. Biomass
DE + BG-11
C. sp. 10% 1.2 μm 1 5 3.01 g/L TN = 99%a TN = 250 mg/La [315]
214

Municipal Filtered 602 mg/L/d* TP = 91%a TP = 11 mg/La

Wastewater COD = -147% a COD = 33 mg/La
DE +
Wastewater
Reject
Chroococcus
C. sp. 30% atm 12 0.79 g/L TN = 85% N-NH4 = 59 mg/L [308]
Chroococcus 66 mg/L/d* TP = 89% TP = 14 mg/L
sp. Biomass COD = 70% COD = 578 mg/L
DE
Desmodesmus
D. sp. 10% Piggery 1.2 μm atm 14 0.41 g/L TN = 76%a TN = 83 mg/La [316]
Wastewater Filtered 29 mg/L/d N-NH4 = 100%a N-NH4 = 70 mg/La
DE TP = 100%a TP = 2 mg/La

214
 D. sp. 5% Piggery 1.2 μm atm 10 0.32 g/L 25.7% TN = 76% TN = 44 mg/L [317]
Wastewater Filtered 32 mg/L/d 8.2 mg/L/d** N-NH4 = 93% N-NH4 = 40 mg/L
DE TP = 100% P-PO4 = 1 mg/L
Dictyosphaerium
D. Struvite atm 7 1.61mg/L* 34.3% N-NH4 = 58 mg/L [301]
ehrenbergianum Stripped 161 mg/L/d 55.3 mg/L/d P-PO4 = 9 mg/L
Piggery 0.14 d-1
Waste DE
Dunaliella
D. salina Struvite atm 7 0.09 d-1 N-NH4 = 12% N-NH4 = 34 mg/L [297]
Stripped P-PO4 = 81% P-PO4 = 7 mg/L
Piggery COD = 19%
Waste DE
D. tertiolecta 6% Centerfuged atm 19 0.99 g/L 12% [318]
Municipal 275 mg/L/d 33 mg/L/d
215

Wastewater
DE +
Flowback
Water
Ettlia
E. oleoabundans 2% Soy atm 22 2.93 g/L 9.8% N-NH4 = 32 mg/L [319]
Waste DE 133 mg/L/d* 13.1 mg/L/d TP = 7 mg/L
Euglena
E. gracilis 5% Animal atm 10 TN = 60 mg/L [302]
Waste DE N-NH4 = 55 mg/L
0.86 d-1 TP = 2 mg/L
COD = 100 mg/L
Micractinium

215
 M. pusillum 20% Cheese atm 8 0.14 g/L N-NH4 = 100% N-NH4 = 25 mg/L [320]
Factory 18 mg/L/d* TP = 44% TP = 16 mg/L
Waste DE 0.35 d^-1
Microcystis
M. aeruginosa 5% Animal atm 10 TN = 60 mg/L [302]
Waste DE N-NH4 = 55 mg/L
0.43 d-1 TP = 2 mg/L
COD = 100 mg/L
Muriellopsis
M. sp. 50% atm 5 TN = 97% TN = 158 mg/L [321]
Digestate 1020 mg/L/d N-NH4 = 97% N-NH4 = 158 mg/L
Centrate TP = 30% TP = 18 mg/L
COD = -22% COD = 67 mg/L
Nannochloropsis
N. salina 6% atm 19 0.72 g/L 30.2% [318]
216

Municipal 225 mg/L/d 68.0 mg/L/d

Wastewater
DE +
Flowback
Water
N. salina 3% atm 19.9% TN = 100% TN = 80 mg/L [322]
Municipal 83 mg/L/d 29.2 mg/L/d N-NH4 = 100% N-NH4 = 68 mg/L
Wastewater TP = 100% TP = 11 mg/L
DE + COD = 80 mg/L
Seawater
N. salina 3% atm 10 0.82 g/L 36% TN = 100% TN = 80 mg/L [194]
Municipal 73.3 mg/L/d 26.4 mg/L/d N-NH4 = 100% N-NH4 = 68 mg/L
Wastewater TP = 100% TP = 11 mg/L
DE COD = 80 mg/L
Neochloris

216
 N. oleoabundans 0.5% Dairy 10 µm 2-3 11 0.4 g/L 9.5% TN = 15 mg/L [323]
Manure DE Filtered 40 mg/L/d 3.8 mg/L/d N-NH4 = 10 mg/L
TP = 2 mg/L
N. oleoabundans 6.7% Agro- Centrifuged 3 N-NH4 = 100% N-NH4 < 1 mg/L [303]
Zootechnical 24 mg/L/d P-PO4 = 96% COD = 2193 mg/L
Waste DE 0.37 d-1
N. oleoabundans 16.7% 1.2 μm biogas 6 0.33 g/L TN = 52% TN = 51 mg/L [305]
Domestic DE Filtered + upgrade 85 mg/L/d TP = 54% TP = 2 mg/L
UV Sterilized (34.45% 0.33 d-1 COD = 61% COD = 169 mg/L
CO2)
Nitzschia
N. palea 16.7% 1.2 μm biogas 7 0.94 g/L TN = 59% TN = 33 mg/L [292]
Domestic DE Filtered + upgrade 107 mg/L/d TP = 60% TP = 3 mg/L
UV Sterilized (32.57% 0.37 d-1 COD = 68% COD = 132 mg/L
CO2)
217

Parachlorella
P. kessleri 10% Agro- Centrifuged + atm 25 1.10 g/L 22.7% TP = 84% TN = 399 mg/L [291]
(Dairy) 1.2 μm 44 mg/L/d* 10 mg/L/d** COD = 45% N-NH4 = 318 mg/L
Waste Filtered TP = 11 mg/L
Effluent COD = 1038 mg/L
Phormidium
P. bohneri 2% Swine atm 12 0.48 g/L N-NH4 = 100% N-NH4 = 66 mg/L [309]
Manure DE 34 mg/L/d P-PO4 = 92% P-PO4 = 6 mg/L
COD = 71% COD = 154 mg/L
P. bohneri 20% Cheese atm 8 0.33 g/L N-NH4 = 100% N-NH4 = 25 mg/L [320]
Factory 41 mg/L/d* TP = 88% TP = 16 mg/L
Waste DE 0.62 d-1
P. bohneri Dairy Waste atm 16 0.57 g/L N-NH4 = 100% N-NH4 = 30 mg/L [324]
DE 33 mg/L/d P-PO4 = 6 mg/L
0.58 d-1

217
 P. sp. 25% Swine atm 5 0.44 g/L N-NH4 = 100% N-NH4 = 54 mg/L [325]
Manure DE 89 mg/L/d* TP = 6% TP = 21 mg/L
Pseudokirchneriella
P. subcapitata 40% atm 5 1130 mg/L/d TN = 98% TN = 126 mg/L [321]
Digestate N-NH4 = 57% N-NH4 = 126 mg/L
Centrate TP = 82% TP = 14 mg/L
COD = -211% COD = 54 mg/L
Scenedesmus
S. accuminatus 20% 0.2 μm 5 4.7 [293]
Livestock Filtered
Waste DE
S. accuminatus 5% Piggery 0.2 μm 5 12 2.68 g/La TN = 86%a TN = 115 mg/La [293]
Waste DE Filtered 810 mg/L/d* N-NH4 = 100% N-NH4 = 70 mg/La
a

TP = 100%a TP = 3 mg/La
S. accuminatus 10% Piggery 1.2 μm atm 5 TN = 122 mg/L [326]
218

Waste DE Filtered + 118 mg/L/d N-NH4 = 120 mg/L

Autoclaved TP = 8 mg/L
COD = 104 mg/L
S. bijuga 6% Poultry Centrifuged atm 12 0.38 g/L 9.5% TN = 152 mg/L [186]
Litter DE 32 mg/L/d* 3.0 mg/L/d** TP = 9 mg/L
S. bijuga 6% Poultry Centrifuged atm 0.68 g/L 10% TN = 53% TN = 85 mg/L [186]
Litter DE 57 mg/L/d* 5.7 mg/L/d** TP = 71% TP = 7 mg/L
S. bijuga 5% Food 1.2 μm atm 28 1.49 g/L 30.8 % TN = 87% TN = 70 mg/L [178]
Waste DE Filtered + 51 mg/L/d 15.6 mg/L/d N-NH4 = 100% TP = 4 mg/L
Autoclaved TP = 91% COD = 343 mg/L
COD = 66%
S. capricornutum 16.7% 1.2 μm biogas 7 0.71 g/L TN = 46% TN = 33 mg/L [292]
Domestic DE Filtered + upgrade 94 mg/L/d TP = 41% TP = 3 mg/L
UV Sterilized (32.57% 0.34 d-1 COD = 55% COD = 132 mg/L
CO2)

218
 S. obliquus Struvite atm 7 N-NH4 = 58 mg/L [301]
Stripped P-PO4 = 9 mg/L
Piggery 0.09 d-1
Waste DE
S. obliquus Struvite atm 7 N-NH4 = 52% N-NH4 = 34 mg/L [297]
Stripped TP = 77% TP = 7 mg/L
Piggery 0.30 d-1 COD = 31%
Waste DE
S. obliquus 47% Piggery Cloth Filtered Biogas in 7 2.18 g/L TN = 75% TN = 57 mg/L [327]
Waste DE + Headspa 311 mg/L/d TP = 82% TP = 61 mg/L
Autoclaved ce 0.38 d-1 COD = 75% COD = 1504 mg/L
(32.6%
CO2)
S. obliquus 4% Agro- Centrifuged 3.00 N-NH4 = 100% N-NH4 < 1 mg/L [303]
Zootechnical 26.0 mg/L/d COD = 1645 mg/L
Waste DE 0.23 d-1
219

S. obliquus 2% Swine atm 12 0.75 g/L N-NH4 = 100% N-NH4 = 66 mg/L [309]
Manure DE 57 mg/L/d P-PO4 = 95% P-PO4 = 6 mg/L
COD = 85% COD = 154 mg/L
S. obliquus 5% Piggery 0.2 μm atm 14 1.20 g/L 31.6% TN = 83% TN = 112 mg/L [328]
Waste DE Filtered 85 mg/L/d 27.0 mg/L/d TP = 89% TP = 13 mg/L
S. obliquus 16.7% 1.2 μm biogas 6 0.45 g/L TN = 53% TN = 51 mg/L [305]
Domestic DE Filtered + upgrade 217 mg/L/d TP = 59% TP = 2 mg/L
UV Sterilized (34.45% 0.45 d-1 COD = 63% COD = 169 mg/L
CO2)
S. obliquus 16.7% 1.2 μm biogas 7 0.47 g/L TN = 67% TN = 70 mg/L [310]
Domestic DE Filtered + upgrade 67.1 mg/L/d* TP = 67% TP = 2 mg/L
UV Sterilized (35.3% COD = 71% COD = 216 mg/L
CO2)

219
 S. obliquus 16.7% 1.2 μm biogas 7 1.09 g/L TN = 57% TN = 33 mg/L Wang,
Domestic DE Filtered + upgrade 151 mg/L/d TP = 53% TP = 3 mg/L 2016
UV Sterilized (32.57% 0.40 d-1 COD = 60% COD = 132 mg/L #756}
CO2)
S. quadricauda 6.7% Kitchen atm 14 0.22 g/L TN = 32% TN = 105 mg/L [175]
Waste DE 86 mg/L/d* N-NH4 = 41% N-NH4 = 138 mg/L
0.18 d-1 TP = 81% TP = 2 mg/L
COD = 46% COD = 390 mg/L
S. quadricauda 5% Piggery 0.2 μm 5 4.7 2.95 g/La TN = 95%a TN = 115 mg/L a [293]
a
Waste DE Filtered 820 mg/L/d* N-NH4 = 100% N-NH4 = 70 mg/L a
TP = 100%a TP = 3 mg/L a
S. subsalsa Struvite atm 7 N-NH4 = 58 mg/L [301]
Stripped P-PO4 = 9 mg/L
Piggery 0.07 d-1
Waste DE
S. sp. (1-6.5%) 0.22 μm atm 6 0.42 g/L N-NH4 = 100% N-NH4 = 22 mg/L [329]
220

Swine Filtered 70 mg/L/d* P-PO4 = 92% P-PO4 = 6 mg/L

Manure/ 1.62 d-1
Algae
Biomass DE
S. sp. (1-6.5%) 0.22 μm atm 6 0.14 g/L N-NH4 = 58% N-NH4 = 21 mg/L [329]
Swine Filtered 23 mg/L/d* P-PO4 = 46% P-PO4 = 6 mg/L
Manure DE 1.34 d-1
S. sp. (1-6.5%) 0.22 μm atm 6 0.11 g/L N-NH4 = 47% N-NH4 = 21 mg/L [329]
Cow Manure Filtered 18 mg/L/d* P-PO4 = 69% P-PO4 = 3 mg/L
DE 1.38 d-1
S. sp. (1-6.5%) 0.22 μm atm 6 0.33 g/L N-NH4 = 100% N-NH4 = 23 mg/L [329]
Swine Filtered 55 mg/L/d* P-PO4 = 65% P-PO4 = 7 mg/L
Manure DE + 1.59 d-1
MgSO4

220
 S. sp. (1-6.5%) 0.22 μm atm 6 0.4 g/L N-NH4 = 92% N-NH4 = 23 mg/L [329]
Swine Filtered 67 mg/L/d* P-PO4 = 83% P-PO4 = 5 mg/L
Manure DE + 1.40 d-1
MgCl
S. sp. (1-6.5%) 0.22 μm atm 6 0.55 g/L N-NH4 = 100% N-NH4 = 23 mg/L [329]
Swine Filtered 92 mg/L/d* P-PO4 = 100% P-PO4 = 7 mg/L
Manure DE +
Lake Water
S. sp. (1-6.5%) 0.22-μm atm 6 0.09 g/L N-NH4 = 41% N-NH4 = 22 mg/L [329]
Swine Filtered 15 mg/L/d* P-PO4 = 25% P-PO4 = 7 mg/L
Manure DE + 0.98 d-1
Trace
Vitamins and
Minerals
S. sp. (1-6.5%) 0.22-μm atm 6 0.07 g/L N-NH4 = 23% N-NH4 = 20 mg/L [329]
Swine Filtered 12 mg/L/d* P-PO4 = 14% P-PO4 = 7 mg/L
221

Manure DE + 0.85 d-1

EDTA-Fe
S. sp. 6% Swine DE atm 13 0.48 g/L TN = 54-90 mg/L [330]
37 mg/L/d* N-NH4 = 90-180 mg/L
0.25 d-1 TP = 2.7-3.5 mg/L
S. sp. 6% Swine DE Biogas in 13 0.53 g/L [330]
headspa 41 mg/L/d*
ce 0.5 d-1
(26.1%
CO2)
S. sp. 3.3% Agro- Centrifuged atm 30 0.85 g/La TN =174 mg/L [304]
(Dairy) 1.2 μm 28 mg/L/d* N-NH4 = 98 mg/L
Waste DE Filtered TP = 162 mg/L
COD = 2 mg/L

221
 S. sp. (1-6.5%) 0.22 μm atm 6 0.42 g/L N-NH4 = 100% TKN = 22 mg/L [329]
Swine Filtered 70 mg/L/d* P-PO4 = 92% N-NH4 = 6 mg/L
Manure/ 1.62 d-1
Algae
Biomass DE
S. sp. 20% Autoclaved atm 30 1.85 g/La TN = 98 mg/L [304]
Municipal 61.7 mg/L/d* N-NH4 = 97 mg/L
Wastewater TP = 350 mg/L
DE + HS COD < 1 mg/L
Media
S. sp. (1-6.5%) 0.22 μm atm 6 0.45 g/L N-NH4 = 47% TKN = 87 mg/L [329]
Algae Filtered 75 mg/L/d* P-PO4 = 69% N-NH4 = 25 mg/L
Biomass DE 1.38 d-1
S. sp. 20% Sewage Autoclaved atm 30 0.61 g/L TN = 550 mg/L [304]
Sludge DE 20 mg/L/d N-NH4 = 290 mg/L
TP = 96 mg/L
222

COD = 4 mg/L
S. sp. 25% Energy Autoclaved 1-2 16 4.0 g/L 15% TN = 83% TN = 303 mg/L [331]
Grass DE + 250 mg/L/d* 37.5 mg/L/d** N-NH4 = 100% N-NH4 = 101 mg/L
BG-11 TP = 96% TP = 1 mg/L
COD = 18% COD = 348 mg/L
S. sp. 25% Energy 1-2 16 4.3 g/L 18% TN = 70% TN = 385 mg/L [331]
Grass DE + 269 mg/L/d* 48.4 mg/L** N-NH4 = 100% N-NH4 = 167 mg/L
BG-11 TP = 98% TP = 1 mg/L
COD = 22% COD = 408 mg/L
S. sp. 25% Energy Autoclaved 1-2 16 3.2 g/L 34% TN = 92% TN = 108 mg/L [331]
Grass DE 200 mg/L/d* 68.0 mg/L/d** N-NH4 = 100% N-NH4 = 89 mg/L
TP = 94% TP < 1 mg/L
COD = 6% COD = 427 mg/L

222
 S. sp. 25% Energy 1-2 16 3.2 g/L 25% TN = 89% TN = 178 mg/L [331]
Grass DE 200 mg/L/d* 50.0 mg/L/d** N-NH4 = 85% N-NH4 = 160 mg/L
TP = 98% TP = 1 mg/L
COD = 9% COD = 418 mg/L
S. sp. 25% Autoclaved 1-2 16 1.9 g/L 16% TN = 49% TN = 385 mg/L [331]
Molasses 120 mg/L/d* 19.0 mg/L/d** N-NH4 = 97% N-NH4 = 167 mg/L
Wastewater TP = 93% TP = 1 mg/L
DE + BG-11 COD = 49% COD = 1345 mg/L
S. sp. 25% 1-2 16 2.7 g/L 19% TN = 58% TN = 448 mg/L [331]
Molasses 169 mg/L/d* 32.1 mg/L/d** N-NH4 = 100% N-NH4 = 226 mg/L
Wastewater TP = 96% TP = 1 mg/L
DE + BG-11 COD = 20% COD = 1380 mg/L
S. sp. 25% Autoclaved 1-2 16 1.4 g/L 23% TN = 59% TN = 115mg/L [331]
Molasses 88 mg/L/d* 20.1 mg/L/d** N-NH4 = 81% N-NH4 = 82 mg/L
Wastewater TP = 89% TP < 1 mg/L
DE COD = -19% COD = 634 mg/L
223

S. sp. 25% 1-2 16 1.9 g/L 20% TN = 64% TN = 270 mg/L [331]
Molasses 119 mg/L/d* 23.8 mg/L/d** N-NH4 = 74% N-NH4 = 202 mg/L
Wastewater TP = 96% TP < 1 mg/L
DE COD = 14% COD = 1290 mg/L
S sp. microalgae 27.3% 1.6 µm 7 1.0 g/L TKN = 93% N-NH4 = 259 mg/L [332]
consortium Municipal/In Filtered 143 mg/L/d* TP = 113 mg/L
dustrial 0.90 d-1 COD = 57 mg/L
Waste DE
Selenastrum
S. bibraianum 16.7% 1.2 μm biogas 7 0.35 g/L a TN = 61% TN = 70 mg/L [310]
Domestic DE Filtered + upgrade 50 mg/L/d* TP = 64% TP = 2 mg/L
UV Sterilized (35.3% COD = 6% COD = 216 mg/L
CO2)
Spirulina

223
 S. maxima Struvite atm 7 N-NH4 = 58 mg/L [301]
Stripped P-PO4 = 9 mg/L
Piggery 0.08 d-1
Waste DE
S. maxima Struvite atm 7 N-NH4 = 7% N-NH4 = 34 mg/L [301]
Stripped P-PO4 = 42% P-PO4 = 7 mg/L
Piggery 0.23 d-1 COD = 25%
Waste DE
S. maxima 2% Pig Slurry atm 0.65 g/L TN = 76% TN = 76 mg/L [333]
DE + N-NH4 = 100% N-NH4 = 25 mg/L
Seawater 0.08 d-1 P-PO4 = 99% P-PO4 = 13.6 mg/L
S. sp. 20% atm 19 0.30 g/L 5 g/L [334]
Digested 16 mg/L/d* 78.0 mg/L/d**
Algae 0.35 d-1
Biomass DE
+ Zarrouk
224

Medium
Spongiochloris
S. sp. Abattoir 0.45 μm atm 42 1.85 g/L [335]
Waste DE + Filtrated 44 mg/L/d*
Minerals
Synechocystis
S sp. 3% atm 13.2% TN = 100% TN = 80 mg/L [322]
Municipal 151 mg/L/d 19.9 mg/L/d N-NH4 = 100% N-NH4 = 68 mg/L
Wastewater TP = 100% TP = 11 mg/L
DE + COD = 80 mg/L
Seawater

Tetraselmis

224
 T. sp. 2.4% atm 10 48% N-NH4 = 20 mg/L [336]
Tetraselmis TP < 0 mg/L
Biomass DE
+ Red Sea
Salt
Mixed Culture
C. vulgaris 90% 50% atm 7 0.75 g/L N-NH4 = 89% [337]
+ S. acutus 10% Municipal 106.71 P-PO4 = 95%
Wastewater mg/L/d*
DE 0.3 d-1
Chlorella sp. and 25.2% Dairy atm 30 1.54 g/L TN = 72% TN = 300 g/m3 [338]
Scenedesmus sp. Manure DE TP = 58% TP = 74.6 g/m3
dominant 0.05 d-1
Oocystis sp., Diluted atm 25 1.4-3.1% N-NH4 = 80- [339]
Scenedesmus, Swine 100%a
Chlorella sp., Manure DE COD = 42-
225

Protoderma sp., 73.5%

Chlamydomonas
sp.
Chlorella sp. and Diluted Centrifuged atm 25 N-NH4 = 84- N-NH4 = 1664 mg/L [340]
Scenedesmus sp. Swine 0.05–0.33 100% COD = 3858 mg/L
dominant Manure DE g/L/d TP = 54-79%
COD = 42-73%
Chlorella sp., 10% Centrifuged atm 14 [341]
Scenedesmus Livestock 0.84 g/L/d
sp., and Waste DE + 0.78 d-1
Synechocyctis nutrients
sp. dominant
DE= digestate effluent, TN = total nitrogen, N-NH4 = total ammonia nitrogen, N-NO3 = total nitrate/nitrite nitrogen, N-ON = total organic nitrogen, TP = total
phosphate, COD = total chemical oxygen demand (in most cases this is dissolved COD)

225
8.2. Growth of C. sorokiniana on Raw SFWP

4
y = 0.7134x - 0.044
Optical Density (750 nm)

R² = 0.9836
3

2
y = 0.6373x - 0.0091
R² = 0.9683
1

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Optical Density (680 nm)

Batch 30% Raw SFWP Batch BG-11

Figure A1: C. sorokiniana Optical Density (680 nm) vs. Optical Density (750 nm) of in Batch
Raw SFWP

2
Biomass Concentration (g/L)

1.5

1 y = 0.4907x + 0.3496
R² = 0.8073

0.5

0
0 0.5 1 1.5 2 2.5 3 3.5
Optical Density (750 nm)

Figure A2: C. sorokiniana Optical Density (750 nm) vs. Biomass Concentration in Batch Raw SFWP

226
Figure A3: Hybrid Tube Photobioreactors Growing C. sorokiniana on VSF-SFWP

227
8.3. Growth of C. sorokiniana on VSF-SFWP and SSF-SFWP

Figure A4: C. sorokiniana Growing in Hybrid Tube Reactors on 30% VSF-SFWP Before Semi-Continuous
Operation

0.8
y = 0.8744x - 0.0081
Optical Density (750 nm)

0.7 R² = 0.9833

0.6
y = 0.7092x + 0.0889
R² = 0.9958 y = 0.7694x + 0.0209
0.5 R² = 0.9853

0.4 y = 0.2496x + 0.3227

R² = 0.6586
0.3
0.4 0.5 0.6 0.7 0.8 0.9 1
Optical Density (680 nm)

Sterile Batch Sterile 30% v/v Semi-Continuous

Contaimnated Batch Contaimnated 30% v/v Semi-Continuous

Figure A5: Correlations of Optical Density (680 nm) vs. Optical Density (750 nm) of C. sorokiniana
Growing in Batch and Semi-Continuous Conditions on Sterile and Intentionally Contaminated VSF-
SFWP

228
 0.8
Biomass Concentration (g/L)
y = 1.8577x - 0.1934
0.7
R² = 0.8123

0.6

y = 5.3512x - 1.831
0.5 R² = 0.86

0.4
0.3 0.4 0.5 0.6
Optical Density (750 nm)

Sterile 30% v/v Semi-Continuous Contaimnated 30% v/v Semi-Continuous

Figure A6: Optical Density at 750 nm vs. Biomass Concentration of C. sorokiniana Growing in Semi-
Continuous Conditions on Sterile and Intentionally Contaminated VSF-SFWP

30% v/v SC 50% v/v SC

Figure A7: C. sorokiniana Growing on SSF-SFWP at Day 11

(from left to right) 30% v/v SC (Semi-Continuous): 1, 2, 3; 50% v/v SC (Semi-Continuous): 1, 2, 3

229
 50% v/v SC Batch Batch 30% v/v SC

Figure A8: C. sorokiniana Growing on SSF-SFWP at Day 14

(from left to right) Batch: 1, 2, 3; 50% v/v SC (Semi-Continuous): 1, 2, 3; Batch (unused): 4, 5, 6; 50% v/v SC (Semi-
Continuous): 1, 2, 3

Batch 30% v/v SC Batch 50% v/v SC

Figure A9: C. sorokiniana Growing on SSF-SFWP at Day 16

(from left to right) Batch: 1, 2, 3; 30% v/v SC (Semi-Continuous): 1, 2, 3; Batch (unused): 4, 5, 6; 50% v/v SC (Semi-
Continuous): 1, 2, 3

230
 Batch 50% v/v SC 30% v/v SC Batch

Figure A10: C. sorokiniana Growing on SSF-SFWP at Day 17

(from left to right) Batch: 1, 2, 3; 50% v/v SC (Semi-Continuous): 1, 2, 3; 30% v/v SC (Semi-Continuous): 1, 2, 3;
Batch (unused): 4, 5, 6

Batch Batch 30% v/v SC 50% v/v SC

Figure A11: C. sorokiniana Growing on SSF-SFWP at Day 18

(from left to right) Batch (unused): 4, 5, 6; Batch: 1, 2, 3; 30% v/v SC (Semi-Continuous): 1, 2, 3; 50% v/v SC (Semi-
Continuous): 1, 2, 3

231
 1.5
Optical Density (750 nm) y = 1.0054x - 0.1382
1.2 R² = 0.9974
y = 0.9486x - 0.0992
R² = 0.9341
0.9
y = 1.0099x - 0.1714
0.6 R² = 0.9944

0.3

0
0 0.3 0.6 0.9 1.2 1.5
Optical Density (680 nm)

Batch 30% v/v Semi-Continuous 50% v/v Semi-Continuous

Figure A12: Correlations of Optical Density (680 nm) vs. Optical Density (750 nm) of C. sorokiniana
Growing in Batch and Semi-Continuous SSF-SFWP

0.6
Biomass Concentration (g/L)

y = 0.3403x + 0.0202
R² = 0.8492
0.4

0.2
y = 0.1785x + 0.0448
R² = 0.8623
0
0 0.3 0.6 0.9 1.2 1.5
Optical Density (750 nm)

30% v/v Semi-Continuous 50% v/v Semi-Continuous

Figure A13: C. sorokiniana Optical Density (750 nm) vs. Biomass Concentration in Batch and Semi-
Continuous Conditions SSF-SFWP

232
8.4. MATLAB Verhult Equation Model

%%*************************************************************************
%***MATLAB Script to Model Microalgae Growth Using the Verhulst Equation***
%***********************Written by Matthew Paddock*************************
%****************************Updated 9/10/2018*****************************
%**************************************************************************
%% Clear Workspace
close all
clear all
clc

%% Input Data
global N0
data = [xlsread('C:\Users\mpadd\Dropbox\Matt\Algae\SFWP CEC\MATLAB_Data.xls',
'Batch')]; % index file of data in excel .xls file
t = data(39,3:10); % time (days)
N = data(40,3:10); % microalgae exponential growth data (g/L)
N0= N(1,1); % initial concentration of microalgae (g/L)

% Initial Guesses for nlinfit (can be anything)

rmaxGuess= .2; % predicted growth rate (day^-1)
NmaxGuess= 0.6; % predicted maximum algae concentration, defined as K (g/L)
beta=[rmaxGuess, NmaxGuess];

[betahat,f,J]=nlinfit(t,N,@Logisticfuncalgae,beta);

% Predicted Values of Nmax and rmax

Nmax=betahat(2); % select values from function
rmax=betahat(1);

% 95% Confidence Intervals for Parameters

betaci=nlparci(betahat,f,J);

%% Print Output
fprintf('\n NLINFIT output:\n')
fprintf('\n Nmax (g/L) = %8.4f , CI =(%8.4f,%8.4f)', Nmax,
betaci(2,1),betaci(2,2))
fprintf('\n rmax (day^-1) = %8.4f, CI = (%8.4f,%8.4f)',rmax,
betaci(1,1),betaci(1,2))

%% Plot
% Plot Experiment and Model Data
tmod=linspace(0,35);
Nmod= N0*Nmax.*(exp(rmax.*tmod))./(Nmax-N0.*(1-exp(rmax.*tmod)));
plot(t, N, 'ko', tmod, Nmod, 'k-')
xlabel('Days')
ylabel('Biomass Concentration (g/L)')
legend('Experimental', 'Model')

%% Statistical Analysis
% Find the Expected Value at Certain Time Points
Nmodex= N0*Nmax.*(exp(rmax.*t))./(Nmax-N0.*(1-exp(rmax.*t)));
% Find the Correlation Coefficients Between the Model and Data
R = corrcoef(N,Nmodex);
R = R(2,1); % correlation coefficient

233
Rsquared = R^2; % correlation of determination
fprintf('\n\n Coefficient of Determination = %8.4f\n',Rsquared)

% Find the Root Mean Squared Error

errorsquared=(N - Nmodex).^2; % residuals between model and data squared
mse=mean(errorsquared); % mean Squared Error
RMSE = sqrt(mse); % root Mean Squared Error
fprintf('\n Root Mean Square Error (g/L) = %8.4f\n\n', RMSE)

%% Logistic Growth Function

% function
function [ yhat ] = Logisticfuncalgae(beta,t)
% Logistic function for algae growth as first described by Verhulst
global N0
b = beta(2); % Nmax
c = beta(1); % rmax

yhat = b.*N0.*(exp(c.*t))./(b-N0.*(1-exp(c.*t)));

end

234
8.5. Growth of C. sorokiniana for Microbial Community Analysis

1.4

y = 0.8731x - 0.0297
Optical Density (750 nm)

1.1
R² = 0.9928

0.8

0.5

0.2

-0.1
-0.1 0.3 0.7 1.1 1.5
Optical Density (680 nm)

Figure A14: Correlations of Optical Density (680 nm) vs. Optical Density (750 nm) of C.
sorokiniana in Raw SFWP Batch

1
Biomass Concentration (g/L)

0.8 y = 0.7019x + 0.0275

R² = 0.93
0.6

0.4

0.2

0
-0.1 0.2 0.5 0.8 1.1 1.4
Optical Density (750 nm)

Figure A15: Optical Density at 750 nm vs. Biomass Concentration of C. sorokiniana in Raw
SFWP Batch

235
 8.6. Microbiome Analysis Results

Table A2: DNA Quality for Bacteria Grown in SFWP With and Without C. sorokiniana after 166 Hours
Label Q-bit Concentration (ug/ml) Nanodrop Concentration (μg/mL) A260 A260/A280
Initial 1 17 231.0 0.578 1.81
Initial 2 17.4 269.6 0.674 1.78
Initial 3 17.3 246.1 0.615 1.79
SFWP 1 15.5 14.4 0.036 1.87
SFWP 2 13.7 11.6 0.029 2.04
SFWP 3 19.6 110.5 0.276 1.88
Algae 1 20 178.6 0.446 1.84
Algae 2 21 273.2 0.683 1.81
Algae 3 19.6 123.7 0.309 1.85
Algae 4 20 179.1 0.448 1.85
Algae 5 18.7 190.5 0.476 1.83
236

Algae 6 19.2 181.2 0.453 1.84

Algae 7 17 187.2 0.468 1.83
Algae 8 17.2 175.6 0.439 1.84
Algae 9 17.2 197.7 0.494 1.82
blank <0.5 1.1 0.003 0
E. coli DH5α 47.3 - - -

236
 Table A3: Microalgae and SFWP Results of Bacteria Grown in SFWP With and Without C. sorokiniana after 166 Hours
Label Biomass Productivity N-NH4 TN sCOD N-NH4 Removal TN Removal sCOD
(g/L) (mg/L/d) (mg/L) (mg/L) (mg/L) (%) (%) Removal (%)
Initial1 0.0688 18.4 50.2 387
Initial2 0.0688 18.4 50.2 387
Initial3 0.0688 18.4 50.2 387
SFWP1 0.0194 -7.14 16.8 39.3 469 8.51 21.59 21.18
SFWP2 0.0286 -5.81 19.5 42.7 403 -5.98 14.95 4.19
SFWP3 0.0412 -3.99 13.7 44.0 435 25.72 12.29 12.56
Algae1 0.9013 130.30 1.8 26.7 598 90.04 54.68 54.68
Algae2 0.6755 97.66 3.5 24.7 620 80.98 50.83 60.34
Algae3 0.8069 116.66 6.5 16.7 385 64.67 66.78 -0.49
Algae4 0.1419 20.51 3.7 31.3 332 80.07 37.54 -14.04
Algae5 0.3641 52.64 0.5 29.3 462 97.28 41.53 19.46
Algae6 0.1931 27.92 2.2 31.3 389 88.22 37.54 0.49
Algae7 0.8870 128.25 3.0 22.7 466 83.70 54.82 20.44
237

Algae8 0.3251 47.00 2.8 33.3 415 84.60 33.55 7.39

Algae9 0.7702 111.36 2.8 29.3 362 84.60 41.53 -6.40

237
 Table A4: OTU count of Bacteria Grown in SFWP With and Without C. sorokiniana after 166 Hours
OTU Initial Initial Initial SFWP SFWP SFWP Algae Algae Algae Algae Algae Algae Algae Algae Algae
ID 1 2 3 1 2 3 1 2 3 4 5 6 7 8 9
A1 65 5 73 0 0 8 59 74 73 0 81 90 117 100 106
A2 0 0 0 15 10 0 0 0 0 0 0 6 6 0 0
A3 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0
A4 0 0 0 18 0 0 0 0 0 0 0 0 0 0 0
A5 0 0 0 96 146 45 21 6 59 0 35 84 0 0 0
A6 0 0 0 334 216 11 19 10 21 32 0 50 25 0 42
A7 0 0 0 16 5 0 0 0 0 0 0 0 0 0 0
A8 0 0 0 56 0 27 0 0 34 0 0 36 0 0 0
A9 0 0 0 52 72 0 0 0 0 0 0 0 0 0 0
A10 0 0 0 0 0 83 0 0 0 0 0 0 0 0 0
A11 75 99 66 0 0 0 0 0 0 0 0 0 0 0 0
A12 108 100 61 0 0 0 0 0 0 0 0 0 0 0 0
A13 0 0 0 100 314 62 0 239 115 101 0 0 45 0 0
238

A14 75 24 71 0 0 0 0 0 0 0 0 0 0 0 0
Chloro 0 0 0 0 0 0 1053 1229 541 1110 1900 3297 1165 3477 2946
-plast
A15 28 186 36 0 0 0 0 0 0 0 0 0 0 0 0
A16 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0
A17 0 25 0 0 0 0 0 0 0 0 0 0 0 0 0
A18 14 13 0 0 0 0 0 0 0 0 0 0 0 0 0
A19 30 21 34 0 0 0 0 0 0 0 0 0 0 0 0
A20 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0
A21 23 0 17 0 0 0 0 0 0 0 0 0 0 0 0
A22 0 0 0 0 28 12 0 0 0 0 0 0 0 0 0
A23 0 0 0 8 22 0 0 0 0 0 0 0 0 9 0
A24 0 0 0 107 78 0 41 41 51 0 31 76 87 19 57
A25 0 0 0 0 116 28 0 0 0 32 0 0 0 0 0
A26 0 0 0 232 150 39 39 13 0 18 27 27 0 0 30
A27 0 0 0 236 496 93 55 13 77 98 157 91 134 0 121
238
 A28 0 0 0 73 40 0 24 26 57 0 30 22 33 22 62
A29 0 0 0 32 108 0 5 0 27 15 0 36 24 0 60
A30 0 0 0 103 284 234 95 73 158 230 0 126 83 0 0
A31 0 0 0 71 124 0 0 0 0 0 0 0 0 24 0
A32 0 0 0 0 95 0 0 0 0 0 0 0 0 94 31
A33 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0
A34 0 0 0 93 136 133 0 13 0 92 36 102 0 0 0
A35 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0
A36 0 0 0 0 58 0 0 0 0 0 0 0 0 0 0
A37 0 0 0 89 26 28 0 0 0 0 0 0 0 0 0
A38 0 0 0 0 15 0 0 8 0 0 0 0 0 0 0
A39 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0
A40 472 392 513 0 196 187 155 192 52 145 0 0 100 0 0
A41 54 69 14 93 105 597 515 482 222 464 69 185 148 147 0
A42 26 21 59 0 25 89 0 0 0 42 0 0 0 0 0
A43 0 0 0 0 0 10 0 0 0 0 0 0 14 0 0
239

A44 33 39 98 0 0 0 0 63 0 0 0 0 0 0 0
A45 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9
A46 0 0 0 0 0 0 0 58 381 0 0 0 0 59 78
A47 13 15 14 0 0 0 0 0 0 0 0 0 0 0 0

239
 Table A5: OTU Identification of Bacterial Grown in SFWP With and Without C. sorokiniana after 166 Hours
OUT ID Kingdom Phylum Class Order Family Genus
A1 Bacteria - - - - -
A2 Bacteria Actinobacteria Actinobacteria Actinomycetales - -
A3 Bacteria Actinobacteria Actinobacteria Actinomycetales Dietziaceae -
A4 Bacteria Actinobacteria Actinobacteria Actinomycetales Dietziaceae -
A5 Bacteria Actinobacteria Actinobacteria Actinomycetales Dietziaceae Dietzia
A6 Bacteria Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae -
A7 Bacteria Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae Frondihabitans
A8 Bacteria Actinobacteria Actinobacteria Actinomycetales Nocardiaceae Rhodococcus
A9 Bacteria Actinobacteria Actinobacteria Actinomycetales Nocardioidaceae Nocardioides
A10 Bacteria Bacteroidetes - - - -
A11 Bacteria Bacteroidetes Bacteroidia Bacteroidales - -
A12 Bacteria Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Petrimonas
A13 Bacteria Bacteroidetes Flavobacteriia Flavobacteriales Flavobacteriaceae
A14 Bacteria Chrysiogenetes Chrysiogenetes Chrysiogenales Chrysiogenaceae Desulfurispirillum
240

Chloro Bacteria Cyanobacteria Chloroplast Chlorophyta - -

-plast
A15 Bacteria Firmicutes - - - -
A16 Bacteria Firmicutes Bacilli Bacillales Bacillaceae -
A17 Bacteria Firmicutes Clostridia Clostridiales - -
A18 Bacteria Firmicutes Clostridia Clostridiales Eubacteriaceae Garciella
A19 Bacteria Firmicutes Clostridia Clostridiales Peptococcaceae Desulfitispora
A20 Bacteria Firmicutes Erysipelotrichi Erysipelotrichales Erysipelotrichaceae Anaerorhabdus
A21 Bacteria Lentisphaerae - - - -
A22 Bacteria Proteobacteria Alphaproteobacteria - - -
A23 Bacteria Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae -
A24 Bacteria Proteobacteria Alphaproteobacteria Rhizobiales - -
A25 Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae Paenochrobactrum
A26 Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae -
A27 Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae Aquamicrobium
A28 Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Xanthobacteraceae -

240
 A29 Bacteria Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae -
A30 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae -
A31 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Bordetella
A32 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Kerstersia
A33 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Pigmentiphaga
A34 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Pusillimonas
A35 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Rhodopseudomonas
A36 Bacteria Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae Denitromonas
A37 Bacteria Proteobacteria Deltaproteobacteria Desulfuromonadales Pelobacteraceae Desulfuromonas
A38 Bacteria Proteobacteria Gammaproteobacteria Alteromonadales Colwelliaceae Thalassomonas
A39 Bacteria Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae -
A40 Bacteria Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae Halomonas
A41 Bacteria Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae -
A42 Bacteria Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas
A43 Bacteria Proteobacteria Gammaproteobacteria Vibrionales - -
A44 Bacteria Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae Vibrio
241

A45 Bacteria Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae -

A46 Bacteria Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae Achromobacter
A47 Bacteria Tenericutes Mollicutes Acholeplasmatales Acholeplasmataceae Acholeplasma

241
 Table A6: Relative Abundance of Bacteria Grown on 30% Raw SFWP With and Without C. sorokiniana after 166 Hours
OTU ID Initial1 Initial2 Initial 3 SFWP1 SFWP2 SFWP3 Algae1 Algae2 Algae3 Algae4 Algae5 Algae6 Algae7 Algae8 Algae9
A1 6.40 0.50 6.91 0.47 5.74 5.63 5.40 17.38 9.67 14.34 21.10 17.79
A2 0.78 0.35 0.64 0.74
A3 2.56
A4 0.94
A5 5.02 5.10 2.64 2.04 0.46 4.36 7.51 9.02
A6 17.47 7.54 0.65 1.85 0.76 1.55 2.52 5.37 3.06 7.05
A7 0.84 0.17
A8 2.93 1.59 2.51 3.87
A9 2.72 2.51
A10 4.88
A11 7.38 9.81 6.25
A12 10.63 9.91 5.78
A13 5.23 10.96 3.64 18.17 8.51 7.96 5.51
242

A14 7.38 2.38 6.72

A15 2.76 18.43 3.41
A16 0.81
A17 2.48
A18 1.38 1.29
A19 2.95 2.08 3.22
A20 0.94
A21 2.26 1.61
A22 0.98 0.71
A23 0.42 0.77 1.90
A24 5.60 2.72 3.99 3.12 3.77 6.65 8.16 10.66 4.01 9.56
A25 4.05 1.65 2.52
A26 12.13 5.24 2.29 3.79 0.99 1.42 5.79 2.90 5.03
A27 12.34 17.31 5.46 5.35 0.99 5.70 7.72 33.69 9.77 16.42 20.30
A28 3.82 1.4 2.33 1.98 4.22 6.44 2.36 4.04 4.64 10.40
242
 A29 1.67 3.77 0.49 2.00 1.18 3.87 2.94 10.07
A30 5.39 9.91 13.75 9.24 5.55 11.69 18.12 13.53 10.17
A31 3.71 4.33 5.06
A32 3.32 19.83 5.20
A33 1.04
A34 4.86 4.75 7.81 0.99 7.25 7.73 10.96
A35 2.04
A36 2.02
A37 4.65 0.91 1.65
A38 0.52 0.61
A39 0.30
A40 46.46 38.85 48.58 6.84 10.99 15.08 14.6 3.85 11.43 12.25
A41 5.31 6.84 1.33 4.86 3.66 35.08 50.1 36.65 16.42 36.56 14.81 19.87 18.14 31.01
A42 2.56 2.08 5.59 0.87 5.23 3.31
A43 0.59 1.72
243

A44 3.25 3.87 9.28 4.79

A45 1.51
A46 4.41 28.18 12.45 13.09
A47 1.28 1.49 1.33

Percent Abundance
0% 25% 50%

243
Table A7: Shannon Diversity Index and Pielou’s Evenness Index of Bacteria Grown in SFWP With and
Without C. sorokiniana after 166 Hours
Shannon Diversity Pielou’s Evenness
Initial 1 1.92 0.75
Initial 2 1.93 0.75
Initial 3 1.84 0.74
SFWP 1 2.71 0.89
SFWP 2 2.78 0.88
SFWP 3 2.21 0.77
Algae 1 1.69 0.71
Algae 2 2.00 0.72
Algae 3 2.28 0.84
Algae 4 1.93 0.80
Algae 5 1.87 0.90
Algae 6 2.34 0.91
Algae 7 2.22 0.89
Algae 8 1.77 0.85
Algae 9 2.14 0.93

244
Figure A16: Principal Component Loading Values for Microalgae Microbiome Analysis
 Initial,  SFWP, Algae

245
 Table A8: OTU Loadings for Microbiome Analysis
OTUs Loading 1 Loading 2 OTUs Loading 1 Loading 2
Aquamicrobium 0.2857 0.2965 Frondihabitans 0.0166 0.0246
Rhizobiales 0.1887 0.1703 Dietziaceae 0.0156 0.0240
Xanthobacteraceae 0.1589 0.2035 Denitromonas 0.0145 0.0154
Pseudomonadaceae 0.1505 -0.6966 Vibrionales 0.0114 -0.0101
Alcaligenaceae 0.1480 -0.3371 Alphaproteobacteria 0.0108 -0.0171
Microbacteriaceae 0.1454 0.1543 Pigmentiphaga 0.0101 -0.0050
Phyllobacteriaceae 0.1412 0.1261 Bacillaceae 0.0090 -0.0044
Dietzia 0.1363 0.0450 Thalassomonas 0.0056 -0.0171
Pusillimonas 0.1260 -0.0766 Bacteroidetes 0.0052 -0.0908
Bacteria 0.1218 0.2339 Anaerorhabdus 0.0023 -0.0399
Achromobacter 0.1114 0.0692 Halomonadaceae -0.0003 -0.0187
Rhodobacteraceae 0.1078 0.1696 Clostridiales -0.0598 0.0174
Flavobacteriaceae 0.0766 -0.1892 Garciella -0.0747 0.0246
Kerstersia 0.0703 0.0623 Lentisphaerae -0.0900 0.0342
Rhodococcus 0.0607 -0.0060 Acholeplasma -0.1053 0.0368
Bordetella 0.0575 0.0490 Pseudomonas -0.1290 -0.0734
Desulfuromonas 0.0401 0.0261 Desulfitispora -0.1485 0.0534
Nocardioides 0.0367 0.0501 Vibrio -0.1941 0.0209
Actinomycetales 0.0299 0.0244 Desulfurispirillum -0.2021 0.0745
Dietziaceae 0.0258 0.0396 Firmicutes -0.2178 0.0684
Paenochrobactrum 0.0251 -0.0609 Bacteroidales -0.2494 0.0856
Caulobacteraceae 0.0233 0.0110 Petrimonas -0.2619 0.0903
Rhodopseudomonas 0.0230 0.0354 Halomonas -0.5909 -0.0064
Xanthomonadaceae 0.0211 0.0654

246
 Table A9: OTUs that Correlate with TN Removal in SFWP With C. sorokiniana
Organisms Formula R2 Radj2 RMSE P-Value
Alcligenaceae 1359.785x -5.756 0.4196 0.3366 6.872 0.0593

Alcaligenaceae
Relative Abundance (%) 32

24

16

0
0 0.005 0.01 0.015 0.02 0.025
Box-Cox Transformed TN

Figure A17: OTUs that Correlate with TN Removal in SFWP With C. sorokiniana

247
 Table A10: OTUs that Correlate with sCOD Removal in SFWP With C. sorokiniana
Organisms Formula R2 Radj2 RMSE P-Value
Halomonas 0.165x + 3.787 0.3960 0.3097 5.71 0.0594

Halomonas
16
Relative Abundance (%)
12

0
-20 0 20 40 60 80
sCOD Removal (%)

Figure A18: OTUs that Correlate with sCOD Removal in SFWP With C. sorokiniana

248
 8.7. Predictive Metagenomic Analysis

Table A11: Predictive Oxygen Requirements and Energy Trophs OTUs on SFWP With and Without C. sorokiniana
Parameter Initial Initial Initial SFWP SFWP SFWP Algae Algae Algae Algae Algae Algae Algae Algae Algae
1 2 3 1 2 3 1 2 3 4 5 6 7 8 9
Oxygen Anaerobic 295 261 215 418 658 254 55 26 77 190 193 193 134 0 121
Aerobic 552 482 586 895 1141 1101 734 1013 941 784 134 377 351 237 191
unknown 169 266 255 599 1066 347 239 276 334 295 139 361 331 237 284
Energy Hetero- 23 0 17 399 642 138 76 19 136 98 192 175 134 0 121
(troph) Organo- 0 0 0 52 72 0 0 0 0 0 0 0 0 0 0
Auto- 0 0 0 56 0 27 0 0 34 0 0 36 0 0 0
unknown 993 1009 1039 1405 2151 1537 952 1296 1182 1171 274 720 682 474 475
249

249
 Table A12: Predicted Metabolisms OTU Count of Bacterial Grown on 30% Raw SFWP With and Without C. sorokiniana
Parameter Initial Initial Initial SFWP SFWP SFWP Algae Algae Algae Algae Algae Algae Algae Algae Algae
1 2 3 1 2 3 1 2 3 4 5 6 7 8 9
Nitrogen fixation 75 24 71 112 40 0 24 26 57 0 30 22 33 22 62
Atrazine metabolism 552 482 586 149 326 900 670 674 308 651 69 221 248 147 0
Lignin degrader 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0
Dinitrogen-fixing 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0
Carbon fixation 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0
Nitrite reducer 627 506 657 772 898 1044 718 785 794 790 135 431 330 228 242
Dehalogenation 660 620 750 722 1168 1200 738 1090 854 877 162 372 364 237 239
Xylan degrader 0 0 0 342 238 11 19 10 21 32 0 50 25 9 51
Sulfur oxidizer 0 0 0 39 0 0 0 58 381 0 0 0 0 59 78
Selenate reducer 75 24 71 0 0 0 0 0 0 0 0 0 0 0 0
Chlorophenol 0 0 0 0 0 0 0 58 381 0 0 0 0 59 78
degrading
Syntrophic 0 0 0 89 26 28 0 0 0 0 0 0 0 0 0
250

Ammonia oxidizer 660 620 750 1026 1236 1105 757 1087 920 817 126 362 395 228 281
Sulfate reducer 660 545 755 446 704 1043 699 838 784 758 135 381 319 237 200
Cellobiose degrading 23 0 17 0 0 0 0 0 0 0 0 0 0 0 0
Gramicidin producer 0 0 0 0 0 0 0 58 381 0 0 0 0 59 78
Naphthalene 0 0 0 56 0 27 0 58 415 0 0 36 0 59 78
degrading
Chitin degradation 59 60 157 373 241 110 19 73 21 74 0 50 39 0 42
Sulfur metabolizing 0 0 0 95 0 27 0 0 34 0 0 36 0 0 0
Sulfur oxidizer 0 0 0 208 386 172 0 297 541 101 0 36 45 59 78
Sulfur reducer 0 0 0 0 0 0 0 58 381 0 0 0 0 59 78
Denitrifying 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0
unknown 258 365 218 696 1445 436 271 215 432 360 304 467 421 237 315

250
 Table A13: Predicted Cell Physiology OTU Count of Bacterial on 30% Raw SFWP With and Without C. sorokiniana
Parameter Initial Initial Initial SFWP SFWP SFWP Algae Algae Algae Algae Algae Algae Algae Algae Algae
1 2 3 1 2 3 1 2 3 4 5 6 7 8 9
Flagella No 865 910 839 1687 2583 1676 987 1211 1301 1269 435 855 715 422 539
unknown 151 99 217 225 282 26 41 104 51 0 31 76 101 52 57
Motility Yes 202 166 242 558 810 950 570 629 691 696 262 378 296 215 199
No 131 100 78 335 581 245 21 245 208 133 35 120 45 0 0
unknown 683 743 736 1019 1474 507 437 441 453 440 169 433 475 259 397
Number 1 14 13 0 220 223 72 21 6 93 0 35 120 0 0 0
of Mem- 2 580 492 574 171 750 277 155 431 181 278 0 0 145 24 0
branes unknown 199 114 301 592 1213 680 214 302 716 477 274 445 358 253 405
Gram Negative 866 744 899 1610 2574 1606 948 1235 1175 1269 350 715 693 374 490
Stain Positive 42 199 36 271 218 72 21 6 104 0 35 120 0 0 0
unknown 108 66 121 31 73 24 59 74 73 0 81 96 123 100 106
Shape Cocci 0 0 0 56 0 27 0 0 34 0 0 36 0 0 0
Spirilla 75 24 71 0 0 0 0 0 0 0 0 0 0 0 0
251

Bacilli 643 526 650 1290 1881 735 333 617 779 560 281 368 438 109 399
unknown 298 459 335 566 984 940 695 698 539 709 185 527 378 365 197
Sporu- Sporulatin 580 492 574 726 1163 565 293 376 694 499 66 300 241 199 222
lation g
Non- 263 252 308 560 724 907 559 797 398 640 96 284 231 156 90
sporulatin
g
unknown 173 265 174 626 978 230 176 142 260 130 304 347 344 119 284
Cell Chains 0 0 0 71 124 0 0 0 0 0 0 0 0 24 0
Arrange- Clusters 472 392 513 447 564 271 215 269 492 163 62 111 100 59 108
ment
Filaments 14 13 0 0 0 0 0 0 0 0 0 0 0 0 0
unknown 530 604 543 1394 2177 1431 813 1046 860 1106 404 820 716 391 488

251
 Table A14: Predicted DNA Characteristics OTU Count on 30% Raw SFWP With and Without C. sorokiniana
Parameter Initial Initial Initial SFWP SFWP SFWP Algae Algae Algae Algae Algae Algae Algae Algae Algae
1 2 3 1 2 3 1 2 3 4 5 6 7 8 9
Genome 2M-3M 75 24 71 0 0 0 0 0 0 0 0 0 0 0 0
Size 3M-4M 33 39 98 93 136 133 0 76 0 92 36 102 0 0 0
4M-5M 472 392 513 0 196 187 155 192 52 145 0 0 100 0 0
5M-6M 26 21 59 91 97 89 0 0 0 42 0 0 0 0 0
7M-8M 0 0 0 0 0 0 0 58 381 0 0 0 0 59 78
unknown 65 30 73 103 260 20 64 74 100 15 81 126 141 124 175
GC 45-50% 33 39 98 0 0 0 0 63 0 0 0 0 0 0 0
55-60% 75 24 71 93 136 133 0 13 0 92 36 102 0 0 0
60-65% 498 413 572 0 221 276 155 192 52 187 0 0 100 0 0
65-70% 0 0 0 95 0 27 0 58 415 0 0 36 0 59 78
70-75% 0 0 0 52 72 0 0 0 0 0 0 0 0 0 0
unknown 159 260 47 64 74 134 15 81 162 141 124 175 65 30 73
252

252
 Table A15: Growth Condition OTU Count of Bacteria Grown on 30% Raw SFWP With and Without C. sorokiniana
Parameter Initial Initial Initial SFWP SFWP SFWP Algae Algae Algae Algae Algae Algae Algae Algae Algae
1 2 3 1 2 3 1 2 3 4 5 6 7 8 9
Temp Thermophilic 14 13 0 0 0 0 0 0 0 0 0 0 0 0 0
Range Mesophilic 314 262 313 668 825 300 175 248 638 248 58 265 170 205 196
Psychrophilic 472 392 513 263 671 294 176 449 226 246 35 84 145 0 0
unknown 216 342 230 981 1369 1108 677 618 488 775 373 582 501 269 400
Habitat Soil 0 0 0 16 5 0 0 0 0 0 0 0 0 0 0
Wastewater 0 0 0 93 136 133 0 13 0 92 36 102 0 0 0
Air 14 13 0 0 116 28 0 0 0 32 0 0 0 0 0
Host 0 0 0 334 216 11 19 10 21 32 0 50 25 0 42
Associated
Aquatic 23 0 17 0 0 0 0 0 0 0 0 0 0 0 0
unknown 979 996 1039 1469 2392 1530 1009 1292 1331 1113 430 779 791 474 554
Human No 75 24 71 191 386 62 0 297 496 101 0 0 45 59 78
Path-
ogen Yes 531 452 670 167 491 321 176 261 111 187 35 84 100 24 0
unknown
253

65 30 73 88 136 47 64 74 134 15 81 162 141 100 175

Host Humans 304 444 363 658 764 877 593 872 476 639 150 367 341 256 157
Plants 0 0 0 232 150 39 39 13 0 18 27 27 0 0 30
unknown 712 565 693 1022 1951 786 396 430 876 612 289 537 475 218 409

253
 Table A16: Characteristics that Correlate with sCOD Removal in SFWP With C. sorokiniana
Organisms Formula R2 Radj2 RMSE P-Value

Atrazine Metabolism 0.455x + 25.097 0.3424 0.2485 17.66 0.0979

Nonsporulating 0.359x + 30.150 0.3593 0.2678 13.42 0.0880

Atrazine Metabolism Nonsporulating

Relative Abundance (%)

Relative Abundance (%)

80 70
y = 0.455x + 25.097
60 R² = 0.3424
50
40
y = 0.359x + 30.150
30
20 R² = 0.3593

0 10
-20 0 20 40 60 80 -20 0 20 40 60 80
sCOD Removal (%) sCOD Removal (%)

Figure A19: Characteristics that Correlate with sCOD Removal in SFWP With C. sorokiniana

254
 8.8. Lipid Accumulation Setup
255

Figure A20: Photobioreactor Set-Up for Lipid Induction of C. sorokinana on SSF-SFWP

255

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