© 2018

SUO XIAO

ALL RIGHTS RESERVED PHAGOTROPHIC ALGAE BASED APPROACHES FOR

ADVANCED WASTEWATER TREATMENT

A Dissertation Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Suo Xiao

December, 2018 PHAGOTROPHIC ALGAE BASED APPROACHES FOR

ADVANCED WASTEWATER TREATMENT

Suo Xiao

Dissertation

Approved: Accepted:

Advisor Department Chair Dr. Lu-Kwang Ju Dr. Michael Cheung

Committee Member Interim Dean of the College Dr. Nic D. Leipzig Dr. Craig Menzemer

Committee Member Dean of the Graduate School Dr. Christopher M. Miller Dr. Chand Midha

Committee Member Date Dr. John M. Senko

Committee Member Dr. Zhenmeng Peng

ii ABSTRACT

Municipal wastewater treatment plants (WWTPs) generate huge quantities of organic solids such as waste activated sludge (WAS) and waste grease (WG). Large volumes of organics-rich wastewater are also generated by the food industry. The increasing wastewater production and more stringent environmental regulations have created an urgent need in sustainable management of wastewater and organic solids.

The microalga Ochromonas danica has unique phagotrophic capability, grows faster than common photosynthetic algae, and produces polyunsaturated fatty acids (PUFA).

These properties make O. danica an excellent candidate as the new microbial agent for resource recovery from wastewater by direct ingestion of particulates. In this research, three new phagotrophic algae-based processes were studied for carbon recovery from different wastewater organics including waste activated sludge (WAS), waste grease (WG) and high-strength food wastewaters.

WAS contains concentrated bacteria and particulate organics. Currently, its disposal requires costly treatment. Ultrasonication was studied to release WAS particulates and bacteria for direct ingestion by O. danica. Destruction/lysis of strong bacterial cell wall was unnecessary, thus minimizing energy-requirement. Effects of sonication power, duration, and WAS volume were studied with a 3 × 6 × 3 factorial design. Quantitative correlations describing the extent of particulate organics (as iii Volatile Solids VS) release were established. By proper increase of initial WAS pH, the VS release by sonication could be further improved with lower energy consumption. O. danica growth on the released WAS VS was found to follow the

Monod-type kinetics but, unlike the typical Monod dependency for soluble substrates, the specific cell growth rate, , correlated with the prey-to-predator ratio, i.e., the ratio of (fed VS concentration)-to-(initial O. danica concentration), significantly better than

-1 with the VS alone. The best-fit kinetics had the following parameters: μmax = 0.198 h

and KM = 1.056 (g-VS/g-algae). Batch cultivations in fermentors at pH 5 confirmed algae production under nonsterile conditions using this new technology, giving a high volumetric algae productivity of 2.8 g/L-day with 38% VS reduction and 44.5% O. danica VS yield. Aerobic digestion of the remaining WAS from the sonication step was also compared with digestion of the non-sonicated WAS. The total oxygen

uptake required (in mg O2/L) for the remaining WAS to reach the Class B Biosolids requirement in specific oxygen uptake rate was approximately 80% lower, indicating substantially reduced aeration cost. Compared to the conventional aerobic digestion, the new ultrasonication-phagotrophic algae process could offer enhanced overall

WAS digestion with lower energy consumption, while producing algae biomass and products.

O. danica cultivation was also studied with waste grease (WG, collected from a municipal WWTP) and two types of industrial food wastewaters (from cheese- and apple juice-making plants). The O. danica growing on WG synthesized and iv accumulated PUFAs, mainly C18:2n6, C18:3n3, C18:3n6, C20:4n6 and C22:5n6, to up to 67% of intracellular FAs, from the WG with only 15% PUFA. The study showed feasibility of converting WG to PUFA-rich O. danica algae culture, possibly as aquaculture/animal feed. The two industrial food wastewaters were tested for toxicity to O. danica, analyzed for compositions, and identified for major issues to address for the algal production. New process designs were proposed and evaluated.

The growth kinetics and yield of O. danica in these food wastewaters were determined.

Overall, the research provided fundamental insights of phagotrophic algal growth on different particulate organics including bacteria-sized particles from WAS,

WG, and the bacteria grown from food wastewaters. The processes developed will find applications in bioconversion of organics solids for microalgae production and sustainable wastewater treatment.

v ACKNOWLEDGEMENTS

I want to thank my advisor Dr. Lu-Kwang Ju for his passion in research that motivated me, for his critical analysis that influenced my view of different challenging scientific and engineering questions, for his patience in revising my manuscripts that encouraged me to complete my research work, and for his efforts to fund my PhD study. Learning his diligence and his attitude towards science can help me face whatever challenges in my future life and career.

I would also like to thank Dr. Nic D. Leipzig, Dr. Christopher M. Miller, Dr.

John M. Senko and Dr. Zhenmeng Peng as my committee members for giving valuable comments on my research for the past years.

I would like to thank those anonymous reviewers for giving me suggestions on my submitted manuscripts to help me finally publish my work. I would also thank

Chemosphere, Water Research and Applied Microbiology and Biotechnology for publishing my research work.

I would like to thank all my lab mates: Dr. Nicholas Callow, Dr. Yajie Chen,

Dr. S. M. Mahfuzul Islam, Dr. Qian Li, Dr. Abdullah Al Loman, Dr. Shida Miao, Dr.

Soroosh Soltani Dashtbozorg, Dr. Cong Li, Ms. Krutika Invally, Mr. Ashwin

Sancheti, Ms. Napaporn Vongpanish and Mr. Jacob Kohl.

vi I would like to thank Mr. Gilbert Stadler for assistance in waste sludge and waste grease sampling at Akron WRF. I also want to thank Ovivo USA LLC for funding research in sludge digestion and food wastewater projects for two years.

I would like to thank my parents Yu Xiao and Xiangyuan Li for their constant support of me while encouraging me with their best wishes always.

Finally, I would like to thank my wife Weixiu Zeng who has always been there cheering me up and onward while standing by me in unwavering support.

vii TABLE OF CONTENTS

LIST OF TABLES ...... xv

LIST OF FIGURES ...... xvi

CHAPTER

I. INTRODUCTION ...... 1

II. BACKGROUND ...... 6

2.1. Sustainable Wastewater Treatment ...... 6

2.2. Waste Activated Sludge (WAS) ...... 8

2.3. Waste Grease (WG) ...... 13

2.4. Phagotrophic Algae ...... 15

2.5. Ultrasonication and its application in WAS management ...... 19

2.6. Cheese wastewater and fruit juice wastewater ...... 21

2.6.1. Cheese wastewater ...... 22

2.6.2. Fruit juice wastewater ...... 23

III. PRELIMINARY OBSERVATIONS ...... 25

Summary ...... 25

3.1. Introduction ...... 25

3.2. Materials and Methods ...... 27

3.2.1. Materials ...... 27

viii 3.2.2. Sludge as substrate for O. danica cultivation ...... 28

3.2.3. Aerobic digestion of Akron sludge ...... 29

3.2.4. Effect of O. danica on aerobic digestion of Akron sludge...... 30

3.2.5. Analytical methods ...... 30

3.3. Results and Discussion ...... 30

3.3.1. Feasibility of using sludge to grow phagotrophic alga Ochromonas

danica 30

3.3.2. Aerobic digestion of WAS ...... 32

3.3.3. Inoculating O. danica in WAS aerobic digestion at pH 5 ...... 34

3.4. Conclusion ...... 36

IV. ENERGY-EFFICIENT ULTRASONIC RELEASE OF BACTERIA AND PARTICULATES TO FACILITATE INGESTION BY PHAGOTROPHIC ALGAE FOR WASTE SLUDGE TREATMENT AND ALGAL BIOMASS AND LIPID PRODUCTION ...... 38

Summary ...... 38

4.1. Introduction ...... 39

4.2. Materials and Methods ...... 44

4.2.1. Algae culture and medium ...... 44

4.2.2. Waste activated sludge ...... 45

4.2.3. WAS sonication and collection of supernatant containing released

small particles and organics ...... 46

ix 4.2.4. Preliminary algae growth and lipid production from sonicated

WAS...... 48

4.2.5. Analytical methods ...... 48

4.3. Results and Discussion ...... 49

4.3.1. Phagotrophic algal growth and lipid production in sonication-

generated supernatant...... 49

4.3.2. Size distributions of released particles ...... 52

4.3.3. Correlations between OD610 and derived count rate as concentration

indicators of released particles ...... 55

4.3.4. Effects of sonication factors ...... 56

4.3.5. Energy efficiency consideration and comparison ...... 65

4.4. Conclusion ...... 68

4.5. Acknowledgements ...... 69

V. PHAGOTROPHIC MICROALGAE PRODUCTION FROM WASTE

ACTIVATE SLUDGE UNDER NON-STERILE CONDITIONS ...... 70

Summary ...... 70

5.1. Introduction ...... 71

5.2. Materials and Methods ...... 74

5.2.1. Algae culture and medium ...... 74

5.2.2. WAS, WAS sonication and supernatant collection ...... 75

5.2.3. Determination of growth kinetics of O. danica in WAS supernatant 76

x 5.2.4. Reflocculation of bacterial particles in supernatant collected after

alkaline sonication ...... 78

5.2.5. Batch cultivation of O. danica in fermentor ...... 79

5.2.6. Analytical methods ...... 80

5.3. Results and Discussion ...... 81

5.3.1. Alkaline sonication ...... 81

5.3.2. Growth kinetics and yield of O. danica in WAS supernatant from

alkaline sonication ...... 86

5.3.3. Reflocculation and endogenous digestion of WAS VS in sonication-

generated supernatants ...... 90

5.4. Conclusions ...... 98

5.5. Acknowledgment ...... 99

VI. ULTRASONIC-PHAGOTROPHIC PROCESS FOR WASTE ACTIVATED

SLUDGE CONVERSION AND MICROALGAE PRODUCTION ...... 100

Summary ...... 100

6.1. Introduction ...... 101

6.2. Materials and Methods ...... 104

6.2.1. Phagotrophic algae and waste activated sludge ...... 104

6.2.2. Sludge sonication and collection of supernatant containing released

small particles and organics ...... 104

6.2.3. Analytical methods ...... 105

xi 6.2.4. Statistical analysis ...... 106

6.3. Results and Discussion ...... 106

6.3.1. Sludge selection: RAS vs. WAS for algae production ...... 106

6.3.2. Effect of sonication on bacteria-sized volatile solids release ...... 108

6.3.3. Algal growth on supernatant generated from subsequent sonication

treatments ...... 109

6.3.4. SOUR, OUR and PIB in remaining solids generated from subsequent

sonication-centrifugation treatment ...... 111

6.3.5. Compare ultrasonic-phagotrophic process with aerobic digestion .. 115

6.4. Conclusion ...... 117

6.5. Acknowledgments...... 117

VII. CONVERSION OF WASTEWATER-ORIGINATED WASTE GREASE TO POLYUNSATURATED FATTY ACIDS-RICH ALGAE WITH PHAGOTROPHIC CAPABILITY ...... 118

Summary ...... 118

7.1. Introduction ...... 119

7.2. Materials and methods ...... 122

7.2.1. Culture and media ...... 122

7.2.2. Waste grease collection and preparation ...... 123

7.2.3. O. danica cultivation on WG ...... 124

7.2.4. Analytical methods ...... 125

xii 7.3. Results ...... 129

7.3.1. Waste grease compositions ...... 129

7.3.2. O. danica growth on WG ...... 130

7.3.3. Algal lipid analysis ...... 137

7.5. Discussion ...... 142

7.6. Acknowledgment ...... 147

VIII. ALGAE PRODUCTION FROM INDUSTRIAL FOOD

WASTEWATERS ...... 148

Summary ...... 148

8.1. Introduction ...... 149

8.2. Materials and Methods ...... 153

8.2.1. Materials ...... 153

8.2.2. Marine Ochromonas cultivation...... 153

8.2.3. Cheese wastewater study ...... 154

8.2.4. Apple juice wastewater study ...... 157

8.2.5. Analytical methods ...... 161

8.3. Results and Discussion ...... 163

8.3.1. Cheese wastewater study ...... 163

8.3.2. Apple juice wastewater study ...... 173

8.4. Conclusion ...... 180

xiii IX. CONCLUSIONS AND RECOMMENDATIONS ...... 182

9.1. Conclusions ...... 183

9.2. Recommendations ...... 185

9.2.1. WAS project ...... 185

9.2.2. WG project ...... 188

9.2.3. Food wastewater project...... 188

REFERENCES ...... 189

APPENDIX

PUBLICATIONS ...... 213

xiv LIST OF TABLES

Table 2. 1 Typical Properties of primary and secondary sludge [15] ...... 10

Table 2. 2 Microbial content in wastewater sludge [25]...... 12

Table 4. 1 Comparison of specific ultrasonic (US) energy inputs for WAS pretreatment ...... 68

Table 6. 1 Microbial content in wastewater sludge [25]...... 101

Table 6. 2 Fecal coliform density in raw RAS, supernatant1+2 and solid2 ...... 114

Table 7. 1 Fatty acid compositions of UWG and LWG (in weight %) ...... 130

Table 7. 2 Comparison of O. danica with other microalgae ...... 146

Table 8. 1 Synthetic apple juice wastewater with N&P supplementation ...... 159

Table 8. 2 Synthetic apple juice wastewater with N&P supplementation ...... 161

Table 8. 3 Characteristics of cheese wastewater ...... 164

Table 8. 4 Wastewater from apple juice manufacturing company ...... 175

xv LIST OF FIGURES

Figure 1. 1 Overall application of phagotrophic algae for resource recovery in WWTPs. (WG: waste grease. RAS: return activated sludge. WAS: waste activated sludge. PUFAs: polyunsaturated fatty acids.) ...... 3

Figure 2. 1 Municipal wastewater treatment process (WWTP)...... 7

Figure 2. 2 Microscopic picture of sludge (taken by Suo)...... 10

Figure 2. 3 Microscopic pictures of O. danica grow on glucose and oleic acid (scale bar: 20 μm)...... 18

Figure 2. 4 Metabolic versality of phagotrophic algae...... 18

Figure 2. 5 Acoustic cavitation process. [84]...... 20

Figure 2. 6 Effect of sonication on WAS flocs...... 21

Figure 3. 1 Algae growth on untreated whole sludge, sonicated whole sludge and sonication-generated supernatant...... 32

Figure 3. 2 Aerobic digestion of WAS: (a) VS reduction and (b) SOUR...... 34

Figure 3. 3 Effect of O. danica on WAS aerobic digestion: ...... 36

Figure 4. 1 Profiles of O. danica number concentration and VS in centrifugation- separated supernatant and solids, respectively, during the phagotrophic growth in sonication-generated WAS supernatant ...... 51

Figure 4. 2 Microscopic pictures showing increasing algal cell concentration and diminishing concentration of small particles/bacteria, taken at 0 h and 34 h, respectively (scale bar: 20 μm)...... 52

Figure 4. 3 Volume-average particle size distributions in supernatants collected by centrifugation (500 g, 10 min) from 100 mL WAS sonicated at 120 W for up to 600 s ...... 54

xvi Figure 4. 4 Effects of sonication time (120 W, 100 mL) on particles found in centrifugation-collected WAS supernatants, in terms of (primary y-axis) summed volume percentages of the 3 size groups of particles (shown in Fig. 4.3) and (secondary y-axis) mean particle size of the dominant group ...... 55

Figure 4. 5 Correlation of OD610 with derived count rate (DCR) for supernatants collected at different sonication time from two sets of sonication experiments with different TS (18.0 and 31.8 g L-1) and same power (120 W) and WAS volume (100 mL) ...... 56

Figure 4. 6 Effects of sonicator probe-tip depth on OD610/TS and VS/TS released into supernatants; sonication done at 120 W and 500 mL WAS with probe tip at either 2 or 4 cm below WAS surface (labeled as “-2 cm” and “-4 cm” systems; total WAS depth ~ 10 cm) ...... 57

Figure 4. 7 Time profiles for release of small particles, indicated as OD610/TS, by sonication of different volumes (100, 300, and 500 mL) of WAS at different powers (90, 120 and 180 W) ...... 59

Figure 4. 8 Correlation between released concentrations of organics (VS) and small particles (detected as OD610) in supernatants collected from experiments of 3 × 3 × 6 factorial design for P, V and t effects (standard deviations shown only for 180 W-100 mL systems as examples) ...... 61

Figure 4. 9 Comparison of model fitting for OD610/TS data in sonication-generated supernatants using the 180 W-300 mL system as an example; regression compared: multiple linear, power-law, and saturation-type models ...... 63

Figure 4. 10 Effect of power and volume on energy efficiency, (OD610/TS)/Ev, estimated at 240 s of sonication; Ev referring to sonication energy input per unit volume...... 67

Figure 5. 1 OD610 in supernatants made from 90 W sonication of WAS and RAS samples adjusted to different initial pH by alkaline addition. (WAS, 120 mL with 27 g/L TS, was sonicated for 4 min; RAS, 45 mL with 9 g/L TS, for 3 min. These preliminary experiments were done without replicates; OD variations determined from later experiments with at least duplicates were found to average at 3.8% ± 2.1%.) ...... 82

Figure 5. 2 WAS sonication at 120 W: (A) pH decrease with alkaline sonication time (300 mL WAS of 31.5 g/L TS); (B) micro-particle release profiles plotted as OD610/TS against sonication time/volume (t/V) for two alkaline-sonicated WAS, one with 300 mL at 31.5 g/L TS and the other with 100 mL at 26.8 g/L TS; and (C) comparison of particle release (OD610/TS) and organics release (VS %) profiles in xvii neutral versus alkaline sonication, as a function of specific sonication energy inputs

(kJ/kg-TSo), where the neutral sonication results were from a previous study [163]. . 86

Figure 5. 3 Specific growth rates, μ, of O. danica growing in WAS supernatants prepared by alkaline sonication plotted against the initial VS/XOd ratio (Left) and VS concentration (Right), respectively, for two sets of cultures seeded at different initial O. danica concentrations (0.1 and 0.3 g/L). Each data point was obtained from one culture flask; duplicate samples were taken at 0 and 6 h, respectively, to determine the initial growth rate μ and its associated standard deviation (plotted as an error bar here). The dashed line represented the correlation described by the equation given in figure, obtained by best-fitting the equation with data from both sets of cultures. Clearly, the equation in the left figure described both sets of data much better than the equation in the right figure...... 89

Figure 5. 4 Effects of (A) pH and (B) initial VS (at pH 5.0 ± 0.1) on VS reflocculation in WAS supernatants generated by alkaline sonication. The same sonicated WAS supernatant was used in both sets, (A) and (B), of experiments. Each data point represents the average and standard deviation (error bar) obtained from duplicate samples with pH and/or initial VS adjustments...... 92

Figure 5. 5 Microscopic pictures at 0 and 20 h of O. danica batch cultivation in WAS supernatant from alkaline sonication (scale bar: 20 μm)...... 96

Figure 5. 6 Results from two batches of O. danica cultivation in supernatants generated from alkaline sonication of WAS; supernatants had 7 and 11 g/L initial VS, respectively: (A) profiles of O. danica growth (in cell number concentration CN) and ammonium generation (NH4+-N); for example, the legend “7 g/L-CN” denotes the O. danica cell number concentration data from the batch of cultivation with WAS supernatant of 7 g/L initial VS, and (B) VS distribution among O. danica cells (VS- algae), non-algal solids (non-algal VS) and supernatant (VS-sup) during the batch cultivations...... 97

Figure 6. 1 Effect of initial sludge-generated supernatant on algae number yield. .. 107

Figure 6. 2 Bacteria-sized VS release as a function of specific energy input...... 109

Figure 6. 3 Algal number concentration during growth on different supernatant generated from subsequent sonication, (b) Algae yield from VSfed...... 111

Figure 6. 4 (a) SOUR profile of remaining solids; (b) OUR profile of remaining solids...... 113

Figure 6. 5 VS reduction in raw RAS. Error bars from replicate systems...... 115

xviii Figure 6. 6 Overall ultrasonic-phagotrophic process design...... 117

Figure 7. 1 Effect of pH on growth of O. danica in medium with 0.42% (v/v) LWG as sole organic C source ...... 132

Figure 7. 2 Comparison of O. danica growth properties on 1.3% (v/v) UWG and LWG...... 135

Figure 7. 3 O. danica cells with high lipid content collected after growing on UWG for 168 h (scale bar: 20 μm) ...... 136

Figure 7. 4 Effect of waste grease (UWG) concentration on O. danica growth ...... 137

Figure 7. 5 TLC results for lipids after primuline staining: (left to right) oleic acid and vegetable oil included to indicate respective positions for free fatty acids (FFAs) and triglycerides (TGs); center lane for UWG; and right 2 lanes for remaining extracellualr WG and intracellualr lipid extracts, respectively, collected after 135-h cultivation. The algal-lipid lane showed also non-fluorescent black spots (indicated by arrows for 2 clearest ones)...... 138

Figure 7. 6 Changes of O. danica culture properties during WG-supported stir-flask cultivation: (A) time profiles of cell growth (CDW, g/L), total intracellular FA production (mg/L), and total PUFA production (mg/L); and (B) time profiles of intracellular FA content (g FAs per g dry cell biomass), PUFA content (g PUFAs per g dry cell biomass), and the % PUFA in total FAs...... 141

Figure 7. 7 Changes of FA composition in O. danica cells with cultivation time, where the seed culture at 0 h was prepared in a glucose-based medium while the cultivation was done in a medium with 1.3% (v/v) WG. The percentages of FAs present in the WG are labeled...... 142

Figure 8. 1 Original marine culture...... 165

Figure 8. 2 Salinity tolerance of O. danica under gradually increasing NaCl concentrations ...... 168

Figure 8. 3 Algal growth in increasing salinity starting at 15 g/L NaCl...... 169

Figure 8. 4 Growth of lactose-consuming bacteria (selected from wastewater RAS) on diluted cheese wastewaters of 1/6 and 1/3 strengths (i.e., with 6- and 3-fold dilutions) ...... 171

Figure 8. 5 O. danica growth on dilute cheese wastewater...... 173

Figure 8. 6 Algae mixed with apple juice wastewater (scale bar: 50 μm) ...... 174 xix Figure 8. 7 Effect of nitrogen and phosphorus supplementation on O. danica growth on synthetic AJWW...... 177

Figure 8. 8 Bacteria batch growth on synthetic AJWW...... 178

Figure 8. 9 Maximal O. danica concentration in 12 ± 2 h fill-and-draw processes. 179

Figure 8. 10 Algae tank in fill-and-draw process (left: 0 h and right: 12 h)...... 180

xx CHAPTER I

INTRODUCTION

Traditional wastewater treatment is to remove the soluble pollutants and separate solid pollutants from water. Almost all municipal wastewaters in the US are treated by biological processes [1] and more than 8 million dry tons of waste activated sludge (WAS) are generated annually [2]. WAS must be stabilized to reduce volatile solids and pathogen level before its disposal and land application. WAS treatment is however expensive in both capital and operating costs, accounting for up to 50% of the total cost of wastewater treatment processes [3]. Waste grease (WG) is also a byproduct collected from wastewater treatment process (WWTP). As reported by

National Renewable Energy Laboratory (NREL), an average of 6 kg/person/year and

4 kg/person/year are generated as brown and yellow grease, respectively [4]. The disposal of WG collected from wastewater influent may account for up to 10% of the total sludge disposal cost [5]. Food industry also generate large quantity of high- strength wastewaters which require onsite treatment before discharged into sewer lines. Sustainable wastewater treatment aims recovery of all useful resources such as chemicals, nutrients, energy, and water itself [6]. The abovementioned WAS, WG and organics in food wastewater are concentrated wastes which can be appropriately utilized to offset the cost of WWTP and improve the sustainability of overall WWTP. 1 Phagotrophic algae can not only assimilate CO2, light and soluble organics, but also directly ingest and digest small particulate organics including bacteria [7], starch [8] and oil droplets [9]. For example, the phagotrophic alga Ochromonas danica can grow on sugar-rich ketchup and Escherichia coli with a doubling time of

10 h and 7 h, respectively [10, 11].

As shown in Figure 1.1, the overall objective of my research is to use phagotrophic alga, Ochromonas danica to provide several alternative solutions for sustainable wastewater treatment by recovering wastewater organics into algal biomass. WAS and WG collected from local wastewater treatment plant will be chosen as representative byproducts from municipal wastewater treatment process while cheese and apple juice wastewaters will be chosen as representative high- strength industrial food wastewater.

2 Figure 1. 1 Overall application of phagotrophic algae for resource recovery in

WWTPs. (WG: waste grease. RAS: return activated sludge. WAS: waste activated sludge. PUFAs: polyunsaturated fatty acids.)

Recently WAS is now being considered as organic resource rather than hazardous waste [12, 13]. WAS is composed of microbial cells (predominantly bacteria), extracellular polymeric substances (EPS) and inorganic materials [14]. WAS is a potential bacteria source for phagotrophic algal cultivation. However, as bacteria, in the form of individual or colony, are attached to or trapped in WAS flocs, phagotrophic algae have no accessibility to bacteria in WAS. The first aim is to design pretreatment to release bacteria from WAS. It is hypothesized that ultrasonication can release bacteria-sized particles (BSP) from WAS flocs for subsequent phagotrophic algal growth. Ultrasonication will be designed to selectively release bacteria-sized particles from WAS flocs with minimal cell lysis so that substantial operational

3 energy can be saved. It is also hypothesized that various operational factors, including power, volume and time can significantly affect the release of BSP. Accordingly, experiments will be designed to sort out the effect of abovementioned factors on ultrasonic release. Ultrasonic energy will be calculated as specific energy (kJ/kg of total solids) and compare with other ultrasonic energy cost for aerobic or anaerobic digestion pretreatment.

The second aim is to produce microalgae O. danica from WAS under non- sterile condition. So far no one has studied the kinetics of phagotrophic algal growth on BSP released from WAS. The understanding of the growth kinetics is critical for not only the algae production but also WAS stabilization. It is hypothesized that phagotrophic algal growth depends on BSP-to-O. danica ratio. Monod equation (or other saturation type equations) will be adopted to describe the dependence of phagotrophic algal growth rate on BSP-to-O. danica ratio. Reflocculation can take place in sonicated WAS. It is hypothesized that BSP concentration and pH have significant effect on reflocculation ratio.

The third aim is to develop ultrasonic-phagotrophic process for algae production and sludge treatment. It is hypothesized that ultrasonication can selectively release active biomass (bacteria) from WAS for phagotrophic algal cultivation so that the digestion of remaining solids requires substantially reduced time and aeration to achieve USEPA Class B biosolids.

The fourth aim is to produce algae from wastewater-originated waste grease. 4 Waste grease (WG) collected from nearby wastewater plant will be simply concentrated through a melt-screening process. The hypothesis is that O. danica can grow on WG and produce PUFA. The pretreated WG will be used as major carbon source for algae production through fermentation process. The raw WG is characterized by high free fatty acid content and solid state at room. This will be the first ever study of the conversion of solid waste grease by phagotrophic algae.

Phagotrophic growth kinetics will be characterized in terms of specific growth rate

-1 (μg, h ) or doubling time (τd, h). The conversion of fatty acids from WG into algal fatty acids will be analyzed and the value of the process will be evaluated.

The fifth aim is to produce algae from industrial food wastewaters through two-stage process under non-sterile condition. The high-strength food wastewaters are potentially good source for microalgae production, however, the complexity of food wastewaters has not been evaluated. It is hypothesized that microalgae can be produced from food wastewaters with certain treatments. Two stage bacteria-algae process can be used to recover organics in food wastewater to algal biomass without sterilization requirement. Retention time in first stage will be limited so that dispersed fast-growing bacteria can deplete soluble waste organics and outcompete other microorganisms. Phagotrophic algae can consume those bacteria in the second stage thus algae can be produced. It is expected that high-cell-density algae be produced, and the effluent can meet sewage discharge regulations.

5 CHAPTER II

BACKGROUND

2.1. Sustainable Wastewater Treatment

The objectives of wastewater treatment are to reduce the level of soluble pollutants and separate solids from wastewater, to meet regulatory limits [15]. As shown in Figure 2.1, generally, municipal wastewater treatment processes (WWTP) include preliminary, primary secondary and tertiary treatment.

Preliminary treatment uses screens, grit chambers and comminutors to remove leaves, grits and large particles. Primary treatment applies gravity (sedimentation) to remove large/heavy organics and light organics (fat, oil and grease, FOG). The removal efficiency of biochemical oxidation demand (BOD), suspended solids and

FOG are: 25-50%, 50-70%, and 65%, respectively [16]. Secondary treatment consists of biological treatment followed by secondary clarifier [17]. Bacteria-dominated microbial cells rapidly convert the soluble organics into their biomass, aggregate into flocs and settle in the following clarifier. The secondary effluent may be further treated through disinfection (by chlorine, ultraviolet or ozone) to reduce pathogen before discharged into receiving waters [18]. In addition to pathogen reduction, nitrogen and phosphorus should be removed in advanced treatment to reduce eutrophication potential [19]. Wastewater treatment is energy consuming as about 3% 6 of electricity produced in US is used for wastewater treatment [20]. The United States spends over $25 billion on domestic wastewater annually and almost all these wastewaters are treated by biological processes [1]. In addition, more stringent environmental regulations on emerging contaminants also call out for extensive researches in water purification [21, 22].

Figure 2. 1 Municipal wastewater treatment process (WWTP). (WG: waste grease. WAS: waste activated sludge. RAS: return activated sludge.)

Sustainable wastewater treatment aims to recover whatever useful materials including water itself simultaneously with water treatment [6]. Membrane bioreactors, combining activated sludge and membrane filtration processes, are used to treat wastewater for reuse in irrigation [22]. Full scale anaerobic ammonia oxidation

(Anammox) process has been used for NH3 removal with considerable reduction in cost [23]. Stabilized waste activated sludge and precipitated phosphorus (in the form of struvite) can be used as fertilizers [24].

Resource and energy recovery can be achieved either through direct wastewater treatment or wastewater-generated solids treatment. Food wastewaters

7 with high organic load, especially high soluble organics, may be directly used as source for production of fertilizer, biogas, biohydrogen and bioelectricity (Section

2.6) through bioprocesses. In contrast, for municipal wastewaters with lower strength, resource and energy recovery can be achieved through conversion of solids collected from WWTPs. Large quantity of organic solids is separated from water after WWTPs.

The main drawback of activated sludge process is the waste activated sludge (WAS) generation. Approximately 1% volume of sludge, with 1 to 7% (w/v) solids content, is generated during wastewater treatment process [25]. Both WAS and waste grease

(WG) are attracting more attention as renewable feedstocks for resource and energy recovery from wastewater treatment processes. Researchers are developing more robust and sustainable processes to offset wastewater treatment cost and improve water quality.

2.2. Waste Activated Sludge (WAS)

Wastewater treatment is an essential component in modern society for minimization of environmental impacts and improvement of sustainability of human activities. Since 1914, activated sludge process has been the most employed biological wastewater treatment process [17] due to its high efficiency, stable performance and low cost [26-28]. In activated sludge process, biodegradable organics are consumed by microorganisms, predominately bacteria in wastewater, to support their growth and energy demand. About 0.5 kg of dry sludge is produced after 8 treating 1 kg COD in wastewater by aerobic process [24]. Certain fraction of sludge can be recycled to the bioreactor to improve process efficiency and stability while others need to be removed [29].

The main drawback of the activated sludge process is the generation of byproduct, waste activated sludge (WAS) during the treatment process. Currently about 45 million dry ton sludge is being produced worldwide per year [30]. WAS treatment is however expensive in both capital and operating costs, accounting for up to 50% of the total cost of wastewater treatment processes [3].

As shown in Figure 2.2, WAS comprises microbial cells and other organic and inorganic matter that are generally agglomerated together in a network formed by microbial extracellular polymeric substances (EPS) and cations [14, 30, 31]. Typical properties of sludge are listed in Table 2.1.

9

Figure 2. 2 Microscopic picture of sludge (taken by Suo).

Table 2. 1 Typical Properties of primary and secondary sludge [15]

Secondary sludge Parameter Primary sludge 6.6-8.0 pH 5.5-8.0 600-1500 550-1200 Alkalinity (mg/L as CaCO3) 0.6-1.2 Total solids % (TS) 4-9 60-85 Volatile solids (% of TS) 65-80 30-40 Protein (% of TS) 18-30 - Cellulose (% of TS) 8-16 2.5-5.0 Nitrogen (N, % of TS) 1.4-4.2 0.6-2.9 3-10 Phosphorus (P2O5, % of TS) 18,000-23,000 Energy content, kJ/kg TS 24,000-28,000

10

WAS management typically includes thickening, stabilization, dewatering and disposal [15]. The first step is to concentrate sludge solids and reduce water content.

The second step is to treat sludge to reduce organic content and pathogen level. The third step is dewatering to further reduce water content. The final step is disposal which includes landfill, land application and incineration.

As sludge generation is increasing due to population increase, urbanization, and advanced nutrient removal in wastewater treatment [24, 32], and regulations on sludge disposal are more stringent, reducing sludge production in WWTPs has become an attractive field for both engineers and researchers [31]. Sludge production reduction can be achieved either in wastewater or waste sludge handling units [33]. In wastewater treatment line, sludge reduction can be achieved by: treating return activated sludge (RAS) with various technologies including chemical, mechanical, thermal, electrical, adding chemical un-coupler, and introducing predators such as protozoa and metazoan [31]. In sludge treatment line, sludge reduction is achieved through the enhanced sludge digestion with additional different pretreatments including mechanical, enzymatic, thermal, chemical, electrical processes [33].

WAS is not allowed to be directly released into environment without proper treatment since it may contain human pathogenic organisms including bacteria, viruses, protozoa, and helminths that all may be originally present in domestic sewage and then concentrated in sewage sludge during wastewater treatment [34, 35]. Table

2.2 shows the typical pathogen indicator content in sludge. Pathogens must be 11 destroyed or reduced to an acceptable level at the wastewater treatment plant before land application [36].

Traditionally, both aerobic and anaerobic digestion can be used for WAS stabilization. Aerobic digestion has advantages of lower capital cost and easier operation but it requires large energy input [37]. Anaerobic digestion can recover energy from WAS in the form of biogas (with 60-70% methane). The main obstacle in anaerobic digestion is the slow digestion rate [38] and it can be resolved by various pretreatment processes to disintegrate WAS and (partially) disrupt bacterial cells [14,

39, 40]. High temperature [41] or long retention time [34] is required to further reduce pathogen level in WAS during digestion processes. Recently, novel technologies such as electro-dewatering [42], microwave irradiation [43], alkaline treatment [44], sonication [45] and et al. have been applied for enhanced pathogen reduction in WAS.

Processes vary significantly in their effectiveness. Moreover, the effectiveness of a particular process also depends on the operating conditions [35].

Table 2. 2 Microbial content in wastewater sludge [25].

Value Fecal Coliforms Salmonella sp. Helminth Ova Log (MPN/g TS) Log (MPN/g TS) Ova/g TS Viable Total

7.6 ± 1.0 5.2 ± 1.3 72 ± 38 77 ± 43 Mean 9.0 7.4 160 178 Max 5.4 2.9 4 4 Min

(MPN: most probable number. TS: total solids)

12 On the other hand, WAS can also be viewed as organic and inorganic nutrients produced and/or gathered from wastewater if effective approaches and processes can be developed to capture and convert these nutrients into value-added products while facilitating partial or complete WAS treatment for safe disposal of the remainders [13,

46]. Recycling and reuse for agricultural land are recognized as reasonably practicable options for final disposal of sewage sludge. In the U.S., the majority of biosolids

(stabilized sewage sludge) are applied to agricultural land and approximately 75% of these are of class B status [47]. Utilization of waste sludge as a renewable resource for energy recovery is the appropriate solution to manage the continuously increasing waste sludge generation effectively to meet stringent environmental quality standards and to sustain the supply of reliable and affordable energy for our future generations and ourselves [13]. Recent utilization of WAS for production of: biogas [3], hydrogen

[48], biodiesel [49-51], fatty acids [52], protein [53], enzyme [54-56], bio-flocculants

[57-59] , biochar [60] have been studied.

2.3. Waste Grease (WG)

Waste grease are collected from grease separators (i.e. grease traps and interceptors) which are installed between wastewater effluent points and the sewer system to allow waste grease to be trapped in a chamber, while grease-free water exits to the sewer [4]. In wastewater treatment process, WG are separated from water

13 surface in primary treatment by sedimentation [16].

WG consists of glycerides and free fatty acids (FFAs). FFAs are generated from food processing (i.e. frying) or microbial degradation (i.e. lipase-catalyzed hydrolysis) [61]. Some fraction of fatty acids bind with metals (mainly calcium) to form soap [5]. The calcium bind saturated soaps are responsible for the deposit that cause overflow in sanitary sewer lines [62].

Huge volumes of WG are generated every year. It is estimated by National

Renewable Energy Laboratory (NREL) (from 30 metropolitan areas in US) that an average of 6 kg/person/year and 4 kg/person/year are generated as brown and yellow grease, respectively [4]. Another rough estimation by Pastore et al. [5], about 1,600–

3,800 kg of waste grease per day might be collected in urban wastewater plant sized for 100,000 of equivalent inhabitants. The disposal of WG collected from wastewater influent may account for up to 10% of the total sludge disposal cost [5].

The recovery of grease from wastewater is not only for successive wastewater treatment but also facilitate the sustainability of overall waste treatment [4]. The WG utilization methods include: anaerobic co-digestion, biopolymer/biochemical production and biodiesel [63]. WG can be used as cheap and renewable feedstock for biodiesel production as it does not compete with land use. However, the high moisture and free fatty acid content retarded the application of commercial cheap base- catalyzed transesterification thus increase the process cost of production [4, 61].

Moreover, due to the high saturation level in fatty acids, the cold-flow property of the 14 biodiesel produced from WG is inferior to traditional diesel [64].

Recently biogas has been qualified as a transportation biofuel by USEPA [38].

Biogas from anaerobic digestion can generate electricity to compensate for partial or complete operational cost at wastewater treatment facilities. The addition of WG may substantially increase biogas production from anaerobic digesters at wastewater treatment facilities. Nevertheless, currently the biogas utilization at US wastewater treatment plants (WWTPs) is less than 10%. Most biogas is flared while some is used for onsite process heat and power production [38, 65, 66]. Moreover, the presence of

WG can cause fatty acid inhibition to bacteria and sludge floating [67].

WG utilization still needs much more investigations, though its over-complex composition has impeded the process development.

2.4. Phagotrophic Algae

Ochromonas as other phagotrophic flagellates, are the primary bacteria consumers in lakes and oceans [68]. Phagotrophy can act as a means of incorporating nitrogen, phosphorus or essential vitamins [68]. Three modes of nutrition, phototrophic, organo-osmotrophic and phagotrophic, are combined in Ochromonas

(Figure 2.4) [8].

Ochromonas danica is 4-7 µm × 5-13 µm (observed in this study) with a cell volume of 52-207 µm3 [69-71]. O. danica can grow osmotrophically in chemostat at

15 dilution rates ranging between 0.03 and 0.10 h-1 at temperature ranging between 15 and 28 °C [70]. Compared to photoautotrophy, mixotrophic growth can result in higher growth rate and cell concentration in a shorter time. Fed-batch can be a highly efficient way for mass cultivation of the flagellate Ochromonas [72]. An ingestion rate of 182 bacteria/cell-h was observed by Holen [73] using O. danica as predator. An ingestible size limit of about 7 µm has been suggested in a study of predation on bacterial communities by an Ochromonas sp. [74]. Within the ingestible limit,

Chrzanowski and Šimek [75] reported, according to results of their own study with

Pseudomonas sp. of different cell sizes and other cited studies, that O. danica preferentially ingested cells with volumes corresponding to cocci of 1.1-1.3 µm in diameter. Ochromonas respond to increased concentrations of bacteria by increased growth rate, and the effect of light on the net growth rate was negligible [76]. The rates of bacteria ingestion by O. danica were affected by both food quality (as carbon: element ratio) and cell size. Cells of high food quality (low carbon: element ratio) were ingested at higher rates than cells of low food quality. Cell size also influenced ingestion rate but to a much lesser extent than did food quality [77].

Recently, the versatile metabolic capability of phagotrophic algae such as

Ochromonas danica has been explored to produce algal biomass and lipids. The microscopic pictures of O. danica cells are shown in Figure 2.3. O. danica can grow on smaller microorganisms [69, 77, 78], particles [79] and oil droplets [9] as well as dissolved organics [7, 70, 80], without dependency on light availability (Figure 2.4). 16

For example, O. danica can grow on ketchup (sugar-rich) with an approximately 10 h doubling time, to produce algal biomass at 40% cell yield and algal lipids at 18% intracellular lipid yield [10]. O. danica can also grow (~ 12 h doubling time) by ingesting droplets of waste cooking oil in aqueous media [9]. The process can also be used to reduce the acid value of waste cooking oil, to make it a better feedstock for biodiesel production. O. danica has also been shown to grow on washed cells of pregrown Escherichia coli, a common bacterium, in a wide pH range of 4 to 7 [11].

The doubling time varies from 7 to 20 h, depending on the fed E. coli-to-O. danica ratio. The bacterial cells supported O. danica biomass yield averages about 25%

(w/w), with intracellular lipid content of 27% ± 8%.

17

Figure 2. 3 Microscopic pictures of O. danica grow on glucose and oleic acid (scale bar: 20 μm).

Figure 2. 4 Metabolic versality of phagotrophic algae.

18

2.5. Ultrasonication and its application in WAS management

Ultrasound refers to sound waves with frequencies exceeding 20 kHz that travel in a medium as a sequence of compressions and rarefactions. As shown in

Figure 2.5, acoustic cavitation involves nucleation, bubble growth and implosive collapse. As the waves move across the areas of low pressure (rarefactions), gas and vapor bubbles are formed and subsequently increase in size until a critical diameter when they implode, creating a phenomenon known as cavitation [81, 82]. Strong turbulent eddies of size 5–100 mm is induced around the collapsing bubbles. High temperature and pressure developed inside the collapsing bubbles could induce many physico-chemical effects [45]. These localized hot spots have temperatures of roughly

5000 °C, pressure about 500 atmospheres, and lifetime of a few microseconds [82].

The violent collapse produces very powerful hydromechanical shear forces in the bulk liquid surrounding the bubble [83]. These physical effects are even more efficient in multiphase systems and especially on solid surfaces due to asymmetrical collapse with projection of a very fast jet toward the solid close to cavitation bubbles. which is reason for choosing ultrasonic cleaning and also of most of ultrasonic solid processing, such as sludge disintegration [84].

19

Figure 2. 5 Acoustic cavitation process. [84]

Ultrasonication is an emerging and promising mechanical disruption technique for sludge disintegration [85]. Hydro-mechanical shear forces are predominant effect on ultrasonic activated sludge disintegration [86]. Small sludge particles and gas bubbles may act as cavitation nuclei during sonication so the cavitation threshold is reduced [83]. As illustrated in Figure 2.6, during ultrasonication the sludge flocs are disintegrated, resulting in release of small particles which including extracellular polymeric substance (EPS) and bacteria into aqueous phase. Further sonication can cause significant cell lysis and increase in soluble compounds in water [87-89]. The effect of frequency, from 41 kHz to 3217, on sludge disintegration was studied by

Tiehm et al. [83] and it found that sludge disintegration was most significant at low frequencies in which large cavitation bubbles are generated. Wang et al. [90] found that the effect of various factors on ultrasonic sludge disintegration followed: sludge 20 pH > sludge concentration > ultrasonic intensity > ultrasonic density.

Figure 2. 6 Effect of sonication on WAS flocs.

Ultrasonication has been widely used in sludge management such as bacteria recovery [91, 92], EPS extraction [93], enzyme recovery [54], sludge reduction [31,

33, 94], aerobic digestion [95, 96], anaerobic digestion [83, 97-100], sludge dewatering [101] and et al.

2.6. Cheese wastewater and fruit juice wastewater

Food industry uses water extensively for preparing raw materials, generating vapor, cleaning packing materials, and washing equipment and floors. High load of organic materials, such as sugars, carbohydrates, and fermented products is recognized as the main characteristics of the food wastewaters [102]. Compared to municipal wastewater, industrial food wastewater has higher strength (COD level) and biodegradability (BOD/COD ratio). Due to the high organic load, direct discharge can 21 lead to excessive burden or even failure in local wastewater treatment operation so on-site treatment or pretreatment are required to remove major pollutants [15, 103,

104].

2.6.1. Cheese wastewater

Cheese is a worldwide main agricultural product. Cheese effluents can be divided into three types: cheese whey from cheese production, second cheese whey from cottage cheese production and the cheese whey wastewater from washing of pipelines, storage and tanks. Cheese wastewater is characterized by high BOD caused by lactose, protein and fat [105], high salinity and composition variation [106].

Various processes, including physicochemical, biological and integrated, have been studied for cheese wastewater treatment. Compared to biological processes, physicochemical processes are less sensitive to high salinity in cheese wastewater but have high operational cost [106, 107]. High dilution ratios, long HRTs, and sludge production are recognized as main drawbacks in aerobic treatment [104]. Anaerobic process can treat higher strength cheese wastewater with less sludge generation, however, sludge flotation and potential active biomass washout due to presence of fat in cheese wastewater is identified as a problem in anaerobic treatment process [108].

Integrated processes are considered as better approach for cheese wastewater treatment. Anaerobic-aerobic process can be applied for complete treatment of cheese wastewater via two steps: the main fraction of organic matter destroyed in anaerobic 22 process and then a polishing step by aerobic treatment to further reduce the organics in effluent [109, 110]. Physicochemical pretreatment can reduce 50% time needed for aerobic biodegradation of cheese wastewater [108]. The coagulation/flocculation and precipitation pretreatment followed by aerobic digestion [111] or Fenton-like process

[105] can achieve better treatment efficiency up to ~100% COD removal.

Sustainable wastewater treatment aims to recover the waste materials and reuse the treated wastewater [46]. The organic waste in cheese wastewater (mainly lactose and proteins) can be valorized through: anaerobic digestion to produce biogas

[112], lactose hydrolysis to produce glucose and galactose [113], fermentation to generate hydrogen [114], and microbial fuel cells to produce bioelectricity [115].

Prazeres, Rivas [116] used NaOH precipitation treated cheese wastewater for tomato production and the sludge collected by precipitation could be used as fertilizer.

2.6.2. Fruit juice wastewater

As cheese industries, concentrated fruit juice industries also produce a huge volume of wastewater [117, 118]. Fruit juice wastewater is mainly generated from the various processes including soaking, washing, rinsing, fluming, blanching, scalding, heating, pasteurizing, chilling, cooling and general cleaning purposes [119]. The strength and composition in effluents from the fruit juice industry depends on the specific fruit processing operations [120].

Various processes, including biological, chemical and electrochemical, have 23 been studied for treating fruit juice wastewaters. 90-95% soluble COD removal can be achieved in sequencing batch reactor (SBR) as well as in activated sludge reactor

(ASR) [121]. However, aerobic treatments are criticized for high energy costs, sludge generation and no valuable products [118]. Zerrouki, Rihani [120] obtained improved biogas yield and COD removal by anaerobic digestion with pH adjustment and nutrient supplementation. El-Kamah, Tawfik [122] developed integrated UASR-

UASR-AS system (two upflow anaerobic sponge reactor followed by activated sludge reactor) for fruit juice wastewater treatment and achieved high removal efficiency with a total HRT of 23 h. Low pH values, nutrients imbalance, and fluctuations in the amount of effluent are major problems in the biological treatment of raw [121]. Can

[118] studied three electrochemical processes: electrocoagulation, electrooxidation and electro-Fenton for treating high COD (20.7 g/L) fruit juice wastewater and achieved 52.4~84.4% COD removal. It was concluded that the electro-Fenton process was most effective in COD removal. Homogeneous photo-Fenton oxidation process can be used to pretreat or mineralize apple juice wastewater in a short time [123].

Fruit juice wastewater can be used for energy recovery in the form of biogas

[124], hydrogen [125], and electricity [126], Noronha, Britz [127] reused fruit juice wastewater treated by membrane-ultrafiltration-UV disinfection hybrid process as cooling or boiler make-up water and bottle washing. Blöcher, Britz [128] further upgraded the membrane treated wastewater by nanofiltration/low pressure reverse osmosis to produce water of drinking quality. 24

CHAPTER III

PRELIMINARY OBSERVATIONS

Summary

In this chapter, preliminary observations were obtained for detailed studies in the following three waste activated sludge-algae chapter work. First, the feasibility of phagotrophic algal growth on untreated WAS, ultrasonication treated WAS and supernatant from ultrasonication treated WAS were tested. Second, the aerobic digestion of Akron WAS was characterized in terms of VS and SOUR reduction.

Third, the application of O. danica in WAS aerobic digestion was tested. Important observations were obtained for future researches on: ultrasonic release of small particles for phagotrophic algal growth, high density algae cultivation and WAS treatment.

3.1. Introduction

The activated sludge process, as the most widely used biological wastewater treatment process, has been used for more than 100 years for their high efficiency, stable performance and low cost [26-28]. However, the main drawback is the production of waste activated sludge (WAS) and its disposal costs up to 50% of

25 overall wastewater treatment process [3, 31]. WAS, in the form of aggregated flocs, consist of microbial cells (predominantly bacteria), extracellular biopolymers and inert solids [14, 26, 30]. WAS management including thickening, stabilization, dewatering and disposal/land application [15]. WAS stabilization aims to reduce sludge amount, pathogen level and vector attraction potential. WAS can be treated by various processes including aerobic digestion, anaerobic digestion, composting, lime stabilization and air drying [35].

On the other hand, WAS is being considered as a resource rather than a waste as various compounds can be utilized with or without transformation of WAS [13, 24,

32]. There have also been studies on WAS utilization for production of biogas [3], hydrogen [48], biodiesel [49-51], fatty acids [52], protein [53], enzyme [54-56], bio- flocculants [57-59], biochar [60], and fertilizer [129-131].

Microalgae cultivation can be used for production of biofuels [132], aquaculture [133], nutrition [134], and wastewater treatment [135]. Phagotrophic algae have been discovered by microbiologist 100 years ago and their extraordinary versatile nutrition modes include phototrophy, osmo-organotrophy and phagotrophy

[8]. Phagotrophic algae, together with other phagotrophic protists, are the major consumers of bacteria in food webs [136]. Phagotrophic microalga, Ochromonas danica, can capture bacteria by whipping their flagella to create current to attract bacteria towards cell surface for ingestion [137]. High ingestion rate of 182 bacteria/cell-h was observed when feeding E. coli to O. danica [73]. High density O.

26 danica cultivation has been demonstrated in fermentation [10]. O. danica can synthesize lipids of various fatty acids such as myristic (C14:0), palmitic (C16:0), stearic (C18:0), oleic (C18:1n9), linoleic (C18:2n6), α-linolenic (C18:3n3), γ- linolenic (C18:3n6), eicosatrienoic (C20:3n3) and arachidonic (C20:4n6) acids [138-

141]. PUFAs such as C18:3n6, C20:4n6, C20:5n3 and C22:6n3 have been reported to offer significant health benefits and have been included in infant formulae, nutritional supplements and aquaculture feed [142].

As bacteria is the major microorganism in WAS flocs and phagotrophic algae is naturally the predator of bacteria, is it possible to use phagotrophic algae to convert bacteria, including pathogens in WAS to achieve simultaneous microalgae production and WAS treatment? The main objective is to demonstrate the feasibility of producing microalgae from WAS through phagotrophic process. The second objective is to see if phagotrophic algae can destruct bacteria in WAS to reduce overall specific oxygen uptake rate (SOUR) in aerobic digestion process.

3.2. Materials and Methods

3.2.1. Materials

3.2.1.1.Waste activate sludge

Gravity-belt thickened WAS (thereafter referred to as WAS for simplicity), including 0.5% thickening agent (CLARIFLOC NE483 from POLYDYNE, Inc.), was 27 collected from the nearby wastewater treatment plant of the City of Akron (Ohio). It had 40-45 g/L TS, containing around 75% VS.

Returned activated sludge (RAS) collected after the secondary clarifiers from the same plant were also used in some early experiments. RAS samples, having 9-15 g/L TS with about 70% VS.

3.2.1.2.Phagotrophic microalga Ochromonas danica

The phagotrophic algae used was O. danica (ATCC #30004). The pure O. danica culture was maintained with regular subculturing at room temperature in 1-L

Erlenmeyer flask with 450 mL culture under magnetic stirring. The medium composition was as follows: 8 g/L glucose, 0.45 g/L yeast extract, 0.45 g/L peptone,

0.5 g/L NH4Cl, 0.4 g/L MgCO3, 0.3 g/L KH2PO4, 0.2 g/L nitrilotriacetic acid, 0.1 g/L

MgSO47H2O, 0.05 g/L CaCO3, 4.4 mg/L Na2EDTA (disodium

ethylenediaminetetraacetate), 3.15 mg/L FeCl36H2O, 0.97 mg/L H3BO3, 0.25 mg/L

thiamine, 0.18 mg/L MnCl24H2O, 0.02 mg/L ZnSO47H2O, 0.01 mg/L CoCl26H2O, 6

μg/L Na2MoO42H2O, and 2.5 μg/L biotin [10].

3.2.2. Sludge as substrate for O. danica cultivation

Three systems were evaluated for algal growth. using a 20 kHz Misonix

Sonicator® (Model XL2020) (Farmingdale, NY, USA) with maximum power of 600

28

W. (The ultrasonic power delivered to the horn was indicated by the instrument and reported in this study.) The tip of the probe was placed in the center of glass beakers with a submerged depth of electrode of 1-1.5 cm.

System1: 30 mL untreated RAS was diluted by 20 mL deionized water. 10 mL centrifuged algae seed was inoculated.

System2: 40 mL WAS in 50 mL centrifuge tube was sonicated at 120 W for 3 min. 20 mL of the sonicated WAS was diluted by 200 mL tap water and 40 mL of algae seed was inoculated.

System3: 150 mL RAS in 250 mL beaker was sonicated at 120 W for 10 min.

The sonicated RAS was centrifuged at 350 g for 10 min to collect supernatant with released small particles. 10 mL of the centrifuged algae seed was inoculated into 50 mL supernatant.

3.2.3. Aerobic digestion of Akron sludge

300 mL dilute WAS with VS=12-19 g/L was digested for 5 to 9 days. The dissolved oxygen (DO) was controlled >1%, >5% and >10% by high magnetic mixing and aeration rate in the first 24h, 48 h and after 48 h, respectively. Total solids (TS), volatile solids (VS) concentration and specific oxygen uptake rate (SOUR) were followed.

29

3.2.4. Effect of O. danica on aerobic digestion of Akron sludge

Different volumes of algae seed were inoculated into 300 mL dilute WAS.

Aerobic digestion was done at pH 5 and room temperature. Total solids (TS), volatile solids (VS) concentration and specific oxygen uptake rate (SOUR) were followed.

3.2.5. Analytical methods

TS and VS were determined according to the standard methods [143]. For tracking phagotrophic algae growth quantitatively, the sample taken was added with an equal volume of 2% glutaraldehyde solution to fix the cells and stop their motility.

The cell number could then be counted using a Petroff-Hausser counting chamber

(Hausser Scientific, Horsham, PA, USA) under a microscope. Microscopic pictures were obtained using a DP71 digital camera (Olympus America, Center Valley, PA,

USA) coupled to the microscope. SOUR in supernatant and remaining solid were measured by USEPA Method 1683.

3.3.Results and Discussion

3.3.1. Feasibility of using sludge to grow phagotrophic alga Ochromonas danica

Three different sludges, untreated whole sludge, sonicated whole sludge and sonication-generated supernatant were fed to O. danica to evaluate the algal growth

(Figure 3.1). The VS-to-initial algae number ratios were 15 g VS/1010 cell, 10 g 30

VS/1010 cell and 7 g VS/1010 cell, respectively. Within 20-24 h, algal number concentration increased from 0.6 × 1010/L, 0.2 × 1010/L and 0.6 × 1010/L to 1.8 ×

1010/L, 1.4 × 1010/L and 5.5 × 1010/L, respectively. Algal number increase was seen in all three systems. However, robust algal growth was only observed in O. danica fed with sonication-generated supernatant at lower food-to-algae ratio compared to other two systems.

A separation operation, either by centrifugation or sedimentation to collect potentially ingestible VS which are smaller than O. danica, is necessary as the incorporation of large solids may consume DO, interact with O. danica, and complicate downstream algae collection process.

Ultrasonication can release small particles from sludge to boost phagotrophic algal growth. The operational parameters including power, volume and time may have significant effect on release. Moreover, since ultrasonication is energy-intensive, energy efficiency should be considered. The ultrasonic release of potentially ingestible particles (including bacteria) from WAS with considering energy cost will be thoroughly studied in Chapter IV.

The kinetics of phagotrophic algal growth on small particles released from sludge can be further studied so that process for algae production can be designed.

The stability of released particles needs further study as their reflocculation can reduce algal yield and ultrasonic energy efficiency in microalgae production. These will be investigated in detail in Chapter V. 31

7.0

6.0

5.0 untreated whole /L) 4.0

10 sludge 3.0 sonicated whole

(x 10 (x sludge 2.0 sonication-generated

1.0 supernatant Algae number concentration concentration number Algae 0.0 0 12 24 36 48 60 Time (h)

Figure 3. 1 Algae growth on untreated whole sludge, sonicated whole sludge and sonication-generated supernatant.

3.3.2. Aerobic digestion of WAS

Aerobic digestion of Akron thickened WAS (VS = 15.1 ± 2.4 g/L, n = 7) is shown in Figure 3.2. Only 24 ± 4 % reduction in VS was achieved within 161 ± 36 h, which is consistent with previously reported low VS reduction in Akron sludge [144].

This low reduction could be due to low C: N ratio and long SRT (13 ± 2 day) operation in Akron WRF. Given that the sludge may be already partially digested due to long SRT, which makes 38% VS reduction difficult to achieve, SOUR can be used as alternative criterium for aerobic digestion at 20 °C [35]. SOUR in sludge should be

reduced to lower than or equal to 1.5 mg O2/g TS-h to be considered as Class B biosolids. For Akron sludge, 126 ± 33 h is required before SOUR drop below 1.5 mg

O2/g TS-h. Accordingly, the slow decrease in SOUR, which requires longer treatment

32 time, is identified as the main issue in Akron sludge digestion.

35 (a) 30

25

13.55 g/L 20 18.53 g/L pH6.6 15.52 g/L 15 13.8 g/L

13.29 g/L pH5 VS Reduction (%) Reduction VS 10 12.31 g/L pH5 18.66 g/L

5

0 0 24 48 72 96 120 144 168 192 216 240 Time (h)

33

8 7.5 (b) 7 6.5 6

h) 5.5 -

5 13.55 g/L /g TS /g

2 4.5 18.53 g/L pH6.6 4 15.52 g/L 3.5 13.8 g/L 3 13.29 g/L pH5 2.5

SOUR (mg O SOUR(mg 12.31 g/L pH5 2 18.66 g/L 1.5 1 0.5 0 0 24 48 72 96 120 144 168 192 216 240 Time (h)

Figure 3. 2 Aerobic digestion of WAS: (a) VS reduction and (b) SOUR.

3.3.3. Inoculating O. danica in WAS aerobic digestion at pH 5

The effect of O. danica on WAS aerobic digestion is shown in Figure 3.3.

~16.2 g/L TS (or 12.5 g/L VS) was used for aerobic digestion at pH 5. About 25.8%

VS reduction was achieved after 206 h among all 5 systems and there was no difference between control and algae system. Nevertheless, significant reduction in

SOUR was observed in O. danica systems. The SOUR dropped almost immediately after the inoculation of O. danica. Figure 3.3 also indicates that to maintain the DO at the same level as control, much less aeration was needed in algae systems, which

34 means the addition of O. danica can reduce the operation cost in aerobic digestion. It is presumably that O. danica ingested and digested bacteria, especially aerobic bacteria in out layer WAS floc [145], released by magnetic stirring. Since most bacteria are trapped in WAS flocs pretreatment is required to release more bacteria including pathogens from WAS flocs.

It is also necessary to collect bacteria-sized particles released from WAS flocs so that the supernatant with release bacteria can be fed to O. danica for algae cultivation, VS reduction and pathogen reduction. The remaining solids may or may not need further digestion. These will be studied in Chapter VI.

30 (a) 25

20

15

10 VS Reduction (%) Reduction VS 5

0 0 24 48 72 96 120 144 168 192 216 Time (h)

0.1% Algae 0.2% Algae 0.9% Algae 2.2% Algae Control

35

5 (b)

4

h) -

3

/g TS /g 2

2 SOUR (mg O SOUR(mg 1

0 0 24 48 72 96 120 144 168 192 216 Time (h) 0.1% Algae 0.2% Algae 0.9% Algae 2.2% Algae Control

Figure 3. 3 Effect of O. danica on WAS aerobic digestion: (a) VS reduction and (b) SOUR.

3.4. Conclusion

The above preliminary observations demonstrated feasibility of converting waste activated sludge by phagotrophic microalga Ochromonas danica. Pretreatment is necessary to release small, ingestible particles from WAS to support robust phagotrophic algal growth. Ultrasonication may be able to disperse bacteria including pathogens into aqueous phase for O. danica to digest (to produce algae) and destruct

(to reduce pathogen). The treatment time required for remaining solids stabilization may be much reduced as the active and pathogenic microorganisms are transferred.

36

The phagotrophic growth kinetics need further study to design algae production process. Both algae production and WAS stabilization can be achieved through optimization of ultrasonic release and phagotrophic growth. These details will be presented in the following three chapters.

37

CHAPTER IV

ENERGY-EFFICIENT ULTRASONIC RELEASE OF BACTERIA AND

PARTICULATES TO FACILITATE INGESTION BY PHAGOTROPHIC ALGAE

FOR WASTE SLUDGE TREATMENT AND ALGAL BIOMASS AND LIPID

PRODUCTION

Summary

Wastewater treatment generates large amounts of waste activated sludge

(WAS) that contains concentrated bacteria and particulate organics and requires costly treatment prior to disposal. This study develops an approach to harness the unique capability of oleaginous phagotrophic microalgae for treating WAS and producing algal biomass and lipids. WAS ultrasonication is studied for releasing particulates and bacteria suitable for direct ingestion by phagotrophic microalgae, without bacterial destruction/lysis, and thus minimizing energy-requirement. Particle release into supernatant was followed by optical density at 610 nm (OD610) and volatile solid concentration (VS); OD610 correlated well with micron-size particle count rates measured by dynamic light scattering. Microalgae (Ochromonas danica) grew with

7.6-h doubling time in sonication-generated WAS supernatant alone, giving approximately 66% (w/w) cell yield from consumed VS and ~30% intracellular lipids.

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Effects of sonication power (P in W), WAS volume (V in mL) and sonication duration (t in s) were studied with a 3 × 3 × 6 factorial design. Supernatant OD610 increased with increasing P and t and decreasing V. Multiple linear regression gave

푂퐷610 the following equation with only significant terms: = −0.0536 + 푇푆

푃×푡 0.000592푃 − 0.000213푡 + 0.000003푃 × 푡 + 0.000274 (R2 = 0.94). Sonicating 푉

500-mL WAS at 180 W for 240 s was selected for giving high particulate release

(~29% VS) with maximal energy efficiency, corresponding to a specific energy input of 4,320 kJ (kg TS)-1, which was much lower than the range (15,000-250,000 kJ (kg

TS)-1) reported previously for WAS ultrasonication. The results supported development of new ultrasonication-phagotrophic algae process for WAS treatment and algae production.

Keywords: Waste activated sludge, ultrasonication, phagotrophic algae, sludge digestion, sludge utilization, sustainability

4.1. Introduction

Wastewater treatment is an essential component in minimization of environmental impacts and improvement of sustainability of human activities.

Activated sludge processes are commonly employed for wastewater treatment. Large amounts of waste activated sludge (WAS) are generated from these processes [146]. 39

WAS comprises microbial cells and other organic and inorganic matter that are generally agglomerated together in a network formed by microbial extracellular polymeric substances (EPS) and cations [14]. The concentrated aggregates of microorganisms and organics cannot be released into the environment without proper treatment or digestion. WAS treatment is however expensive in both capital and operating costs, accounting for up to 50% of the total cost of wastewater treatment processes [3]. Greater than 8 million dry tons of biosolids are generated from WAS treatment in the U.S. annually [24].

On the other hand, WAS can also be viewed as organic and inorganic nutrients produced and/or gathered from wastewater, if effective approaches and processes can be developed to capture and convert these nutrients into value-added products [13, 46,

147, 148]. It would be even more desirable if such approaches/processes can facilitate

WAS treatment for safe disposal of the residues after nutrient recovery. Recently, the versatile metabolic capability of phagotrophic algae such as Ochromonas danica has been explored to produce algal biomass and lipids. O. danica can grow on smaller microorganisms [69, 77, 78], particles [79] and oil droplets [9] as well as dissolved organics [7, 70, 80], without dependency on light availability. For example, O. danica can grow on ketchup (sugar-rich) with an approximately 10 h doubling time, to produce algal biomass at 40% cell yield and algal lipids at 18% intracellular lipid yield [10]. O. danica can also grow (~ 12 h doubling time) by ingesting droplets of waste cooking oil in aqueous media [9]. The process can also be used to reduce the 40 acid value of waste cooking oil, to make it a better feedstock for biodiesel production.

O. danica has also been shown to grow on washed cells of pregrown Escherichia coli, a common bacterium, in a wide pH range of 4 to 7 [11]. The doubling time varies from 7 to 20 h, depending on the fed E. coli-to-O. danica ratio. The bacterial cells supported O. danica biomass yield averages about 25% (w/w), with intracellular lipid content of 27% ± 8%.

Results of these studies support the possibility of using phagotrophic algae to extract value/nutrients and remove harmful agents from WAS. However, WAS is commonly present as large flocs [149] that are too large to ingest by phagotrophic algae like O. danica, which is 4-7 µm × 5-13 µm (observed in this study) with a cell volume of 52-207 µm3 [69-71]. An ingestible size limit of about 7 µm has been suggested in a study of predation on bacterial communities by an Ochromonas sp.

[74]. Within the ingestible limit, Chrzanowski and Šimek [75] reported, according to results of their own study with Pseudomonas sp. of different cell sizes and other cited studies, that O. danica preferentially ingested cells with volumes corresponding to cocci of 1.1-1.3 µm in diameter. Therefore, breaking WAS into ingestible cells/particles can significantly improve the feasibility of this new phagotrophic algae- based approach.

Sonication is a clean mechanical way of achieving WAS floc disintegration

[85, 150]. Sonication of WAS to improve its aerobic or anaerobic digestion has been

41 studied [83, 95, 97, 151]. However, high energy intensity was used in these studies because the purpose there included rupturing the microbial cell wall to kill them and release intracellular materials for easier digestion. For example, the sonication energy used was 105 kJ per kg total solids for enhancing anaerobic digestion of sonicated

WAS [97]. Utilizing high energy sonication can incur high costs in construction of the sonication devices and in the electricity required for running the sonication treatment

[1]. Excessive disintegration of WAS flocs is also known to significantly worsen the sludge dewaterability [45, 101, 152].

Unlike the WAS sonication treatments studied so far, the approach pursued in this study is to use suitably low sonication energy to cause release of intact, individual microorganisms and small particles from WAS flocs, without requirement of high energy to rupture the tough bacterial cell walls. Phagotrophic algae have evolved to lyse cells and digest particles effectively inside their bodies [7, 8, 79]. This distinction is very important. The use of low sonication energy not only saves the sonication device and energy cost but also preserves more nutrients as enclosed in intact bacterial cells and small particles. The latter is advantageous because phagotrophic algae can consume these bacteria and small particles to produce algal biomass and lipids, instead of releasing dissolved nutrients to be wasted (consumed) by osmotrophic microorganisms in WAS like in the conventional digestion processes.

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The new approach envisioned involves a two-step process of WAS sonication followed by phagotrophic algae growth in the supernatant containing released particulate and dissolved organics. The algae grown in the supernatant can be harvested without otherwise more challenging separation of the algal biomass from the remaining WAS flocs (if the entire sonicated WAS is fed for phagotrophic algae growth). A preliminary experiment indicated successful O. danica growth and lipid production from the supernatant containing bacterial cells and small particles collected after WAS sonication.

The focus of this study is to investigate the nature and factors that affect the efficiency of releasing small particles (including individual bacterial cells) and organics from WAS flocs by sonication. Particle release from WAS flocs by sonication has not been examined in previous sonication studies, which have been more concerned with use of sonication for disinfection purpose or for release of soluble organics (e.g., soluble COD) to enhance digestion. For example, Hua and

Thompson [153] investigated ultrasonic inactivation (not lysis) of Escherichia coli by plate-counting colonies of remaining viable cells developed in Petri dishes. The effect of ultrasound on viability of WAS bacteria has been well studied by Foladori et al.

[154] using dilute WAS. Chu et al. [45] thoroughly examined the effects of sonication at different intensities and time on WAS floc disintegration, microbial inactivation and organic dissolution. However, the floc disintegration was followed with the mean floc size, without information on released individual cells. 43

In this study, sonication-effected releases of small particles and organics from

WAS flocs (in terms of optical density measured at 610 nm and volatile solids, i.e.,

OD610 and VS) are examined. The study results also allow comparison of estimated sonication energy consumption between this approach of releasing small particulate

VS and the previous approach of rupturing cell wall for enhanced WAS digestion.

This study lays the necessary foundation for realizing the envisioned process of combined WAS sonication and phagotrophic algae growth, as a novel value-capturing approach to convert the nutrients in WAS into potentially value-added algal biomass and lipids while facilitating, if not completing, the WAS treatment.

4.2. Materials and Methods

4.2.1. Algae culture and medium

The phagotrophic algae used was O. danica, originally obtained from

American Type Culture Collection (ATCC® 30004TM, ATCC, Manassas, VA, USA).

The pure O. danica culture was maintained with regular subculturing at room temperature in 1-L Erlenmeyer flask with 450-500 mL culture under magnetic stirring. The medium composition was as follows: 8 g L-1 glucose (Fisher Scientific,

Fairlawn, NJ, USA), 0.45 g L-1 yeast extract (Fisher Scientific), 0.45 g L-1 tryptone

-1 - (Fisher Scientific), 0.5 g L NH4Cl (EMD Chemicals, Gibbstown, NJ, USA), 0.4 g L

1 -1 MgCO3 (Sigma-Aldrich, St. Louis, MO, USA), 0.3 g L KH2PO4 (Sigma-Aldrich), 44

-1 -1 0.2 g L nitrilotriacetic acid (Sigma-Aldrich), 0.1 g L MgSO47H2O (Sigma-

-1 -1 Aldrich), 0.05 g L CaCO3 (Sigma-Aldrich), 4.4 mg L Na2EDTA (disodium

-1 ethylenediaminetetraacetate) (Sigma-Aldrich), 3.15 mg L FeCl36H2O (Fisher

-1 -1 Scientific), 0.97 mg L H3BO3 (Sigma-Aldrich), 0.25 mg L thiamine hydrochloride

-1 -1 (Sigma-Aldrich), 0.18 mg L MnCl24H2O (Sigma-Aldrich), 0.02 mg L

-1 -1 ZnSO47H2O (Sigma-Aldrich), 0.01 mg L CoCl26H2O (Sigma-Aldrich), 6 μg L

-1 Na2MoO42H2O (Sigma-Aldrich), and 2.5 μg L biotin (Sigma-Aldrich) [10].

4.2.2. Waste activated sludge

The gravity-belt thickened WAS (thereafter referred to as WAS for simplicity), with 0.5% Polydyne Brand ClariflocTM NE483 (Riceboro, GA, USA) added as thickening agent, was collected from the nearby wastewater treatment plant of the City of Akron (Ohio). The total solids concentration (TS) of WAS was 40 to 45 g L-1, with around 75% volatile solids (VS). Fresh WAS samples were used in most experiments; in some cases, the samples were stored at 4 °C in a refrigerator before use but never for more than 48 h.

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4.2.3. WAS sonication and collection of supernatant containing released small

particles and organics

Original WAS was diluted by equal volume of de-ionized water to have 20 ± 1 g L-1 TS. Sonication was done using a 20 kHz Misonix Sonicator® (Model XL2020)

(Farmingdale, NY, USA) with maximum power of 600 W. (The ultrasonic power delivered to the horn was indicated by the instrument and reported in this study.) The tip of the probe was placed in the center of glass beakers with a submerged depth of electrode of 2 cm. Low-speed magnetic stirring was used to facilitate mixing, allowing WAS to move more homogeneously through the active sonicating zone near the sonicator probe. The magnetic stirring itself contributed minimally to the particle release into supernatants. For example, in a 500 mL WAS under the same magnetic stirring with sonication probe in place but not turned on, the supernatant OD610 was found to increase from 0.009 ± 0.004 at 0 min to only 0.276 ± 0.028 after 10 min, compared negligibly to, e.g., the OD610 (5.36 ± 0.40) observed in its 120 W sonicated counterparts.

Sonication experiments were designed to study the effects of 3 independent factors, i.e., sonication power (P), sludge volume (V) and sonication time/duration (t), on 2 response variables measured in the supernatants generated by sonication, i.e.,

OD610/TS and VS/TS, where OD610 was optical density at 610 nm, correlating with the particulate concentration released (shown later in Results and Discussion), and VS

46 was the organic concentration released (including both particulate and dissolved organics). Both OD610 and VS were normalized by TS as these two released concentrations were expected to change with the TS of initial sludge sample sonicated. P, V and t were varied according to a 3 × 3 × 6 factorial design: P at 90,

120 and 180 W; V at 100, 300 and 500 mL; and t at 0, 60, 120, 240, 420 and 600 s. At the end of each designed sonication experiment, multiple samples of 40-mL sonicated sludge were centrifuged (Sorvall Legend X1R, Thermo Scientific, Osterode,

Germany) at 500 g for 10 min and the supernatants were collected for measurements of OD610 and VS. Longer-time sonication (>10 min) was not studied because it might cause seriously deteriorated dewaterability of sonicated WAS flocs [152].

Previous researchers reported substantial bacterial cell lysis after 10 min sonication while bulk floc disintegration was the dominant phenomenon during the first 10 min

[87, 88]. Data reported were the means and standard deviations from at least duplicate samples. Multiple linear regression using stepwise method (α = 0.05 for adding or removing terms) (Minitab version 18, Minitab Inc., State College, PA, USA) was used to investigate and model the response variable OD610/TS as a function of P, V, t and their two-way and three-way interactions in the sonication experiments.

Nonlinear regression with power-law and saturation-type models were also done with

Minitab for best-fit equations of OD610/TS data.

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4.2.4. Preliminary algae growth and lipid production from sonicated WAS

Sonication was done by the same setup and procedure as described in the previous section. 300 mL diluted WAS was sonicated at 120 W for 5 min. After centrifugation, the supernatant collected was used for phagotrophic algal cultivation.

4.2.5. Analytical methods

Optical density at 610 nm (OD610) were measured by a UV-vis spectrometer

(Model UV-1601, Shimadzu Corporation, Columbia, MD, USA) after proper sample dilution to ensure measured value to be < 0.7, to avoid nonlinear correlation with particle concentration. TS and VS were determined according to the standard methods

[143]. For tracking phagotrophic algae growth quantitatively, the sample taken was added with an equal volume of 2% glutaraldehyde solution to fix the cells and stop their motility. The cell number could then be counted using a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA, USA) under a microscope.

Microscopic pictures were obtained using a DP71 digital camera (Olympus America,

Center Valley, PA, USA) coupled to the microscope. Particle size distribution in supernatant was determined by dynamic light scattering in a Zetasizer Nano ZS instrument (Malvern Instruments, UK), using a 633-nm laser as light source and measuring the photon count rate by a detector at 175°. The measurement position and attenuation index for laser power could be automatically adjusted by the instrument to 48 ensure suitable count rate for the detector and the best signal-to-noise ratio. The measured photon count rates could be converted to derived count rates (DCR) according to the attenuation indices used in actual measurements; DCR represented the “theoretical” scattering intensity in absence of the laser light attenuation filter

[155]. Algal lipid content was determined by the chloroform-methanol extraction method. Briefly, 20 mL algae sample was centrifuged at 500 g for 10 min to remove supernatant. The remaining algal cells were added with 7 mL HPLC grade chloroform

(Fisher Scientific)-methanol (Fisher Scientific) solution (2:1, v/v), 2 drops of hydrochloric acid (Fisher Scientific), and 0.1 mL de-ionized water. The mixture was vortexed for 5 min and then centrifuged at 2000 g for 10 min to separate cell debris from the solvent phase containing dissolved lipids. The solvent phase was collected and dried by filtered air for 3 h and the weight of lipids was measured.

4.3. Results and Discussion

4.3.1. Phagotrophic algal growth and lipid production in sonication-generated

supernatant

The growth profile, in cell number concentration, of O. danica in a supernatant generated by 5-min sonication at 120 W is shown in Fig. 4.1. Clearly, the phagotrophic algae could grow on the small particles released from WAS flocs by sonication, with a doubling time of 7.6 h (during the first 22 h). Microscopic pictures 49 taken for the initial (0 h) and final (34 h) samples, as given in Fig. 4.2, show increased algal cell population and disappearance of small particles/bacteria during the process.

Comparison of initial and final (at 34 h) total VS, including algal cells and other organics in both supernatant and solids collected by centrifugation (Fig. 4.2), showed a 37% ± 2% overall VS reduction in the algal growth experiment. The final algal mass produced was estimated by multiplying the increased (final – initial) cell number concentration, (1.84 × 1010 – 3.43 × 109 cells L-1), by the average single-cell weight determined from O. danica seed culture, i.e., 2.82 × 10-11 g cell-1. Direct measurement of algal mass weight in this experiment was not possible because some supernatant organics reflocculated during the experiment (visible in Fig. 4.2 for the 34-h sample) and they could not be easily separated from algal cells. The maximum algal mass yield, = (algal mass produced)/(VS consumed), was estimated to be 66%, occurring at

22 h. The lipid content in the total VS collected by centrifugation was 18.3% ± 0.2%, corresponding to about 30% lipid content in the algal cells. The results of this preliminary experiment supported the feasibility of incorporating sonication- facilitated phagotrophic algae growth on WAS for purposes of WAS treatment and value capturing.

The reflocculation observed during the algal growth experiment could be caused by the interactions between multivalent cations like Ca2+ and Mg2+ and the negatively charged functional groups on bacterial surfaces and other biopolymers released by sonication into the supernatant [156]. The WAS used was thickened at the 50 wastewater treatment plant with addition of polymeric flocculant. The effect of this flocculant on the reflocculation observed here needs further study to determine and understand. Future study with fed-batch or continuous feeding of sonicated WAS supernatant to minimize reflocculation and increase algae yield is also warranted.

Figure 4. 1 Profiles of O. danica number concentration and VS in centrifugation- separated supernatant and solids, respectively, during the phagotrophic growth in sonication-generated WAS supernatant

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Figure 4. 2 Microscopic pictures showing increasing algal cell concentration and diminishing concentration of small particles/bacteria, taken at 0 h and 34 h, respectively (scale bar: 20 μm).

4.3.2. Size distributions of released particles

It was reported that during sonication, the average WAS particle size could reduce from hundreds of μm down to several μm [45, 150]. In this study, only micron-sized small particles are of interest because they are potentially suitable food for ingestion by O. danica: for more rigid microbial cells, the reported size limit was about 7 μm [74]; for oil (and potentially more compressible flocs/aggregates), ingestion of droplets larger than 10 µm was seen under microscope [9]. These small particles were separated from remaining larger flocs by collecting only the supernatant after 10 min centrifugation at 500 g. The (volume-averaged) size distributions of particles in supernatants thus collected, measured by Dynamic Light

Scattering (DLS), are shown in Fig. 4.3 for sonication of 100 mL WAS (TS = 31.8 g

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L-1) at 120 W for up to 10 min. It was clear that without sonication (0 s), the WAS supernatant was dominated by nanoparticles of 30 ± 2.5 nm. The absence of more amounts of larger (micron-sized) particles was presumably caused by the use of chemical flocculant for WAS thickening. Micron-sized particles were quickly released by sonication, seen after just 60 s here. In these (and other) supernatants collected, up to 3 particle-size peaks were generally detected: the smallest group

(subcellular colloids) peaked at 30-100 nm, the middle group (bacteria-like) peaked around 1 μm, and the largest group (aggregates) peaked around 5-6 μm. Particles larger than 10 μm were not found, presumably because they were removed to the settled pellets by centrifugation. As shown in Fig. 3, the larger aggregates of 4-8 μm were first generated and then further broken into smaller particles after longer (600 s here) or higher-power (data not shown) sonication. The changes with sonication time for summed volume percentages of these 3 size groups of particles and for the average particle size (and standard deviation) of the dominant group are shown in Fig. 4.4.

With 1-10 min sonication, predominant majority (86-98 vol%) of the particles in supernatants were in the middle size group, with mean particle sizes in the range of

0.7-1 μm, which were comparable to sizes of common bacteria such as Escherichia coli, about 1 μm measured by DLS equipment [157, 158]. Therefore, the ultrasonic pretreatment coupled with centrifugal separation used in this study was appropriate for releasing and collecting desired small particles from WAS flocs for subsequent phagotrophic consumption and growth of the algae O. danica. 53

Figure 4. 3 Volume-average particle size distributions in supernatants collected by centrifugation (500 g, 10 min) from 100 mL WAS sonicated at 120 W for up to 600 s

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Figure 4. 4 Effects of sonication time (120 W, 100 mL) on particles found in centrifugation-collected WAS supernatants, in terms of (primary y-axis) summed volume percentages of the 3 size groups of particles (shown in Fig. 4.3) and (secondary y-axis) mean particle size of the dominant group

4.3.3. Correlations between OD610 and derived count rate as concentration

indicators of released particles

In DLS particle size analysis, derived count rates (DCR) correlate with the number concentrations of particles [155]. In this study, OD610 was more routinely measured for indicating concentrations of the desired micron-sized particles released by sonication into supernatants. As an example, DCR and OD610 values are plotted in Fig. 4.5 for supernatants collected at different sonication times (up to 10 min) from two sets of experiments made with different TS (18.0 and 31.8 g L-1) but same WAS volume (100 mL) and sonication power (120 W). A good linear relationship is shown

55 between OD610 and DCR, suggesting OD610 as a suitable indicator of the particle concentrations of interest to this current study.

Figure 4. 5 Correlation of OD610 with derived count rate (DCR) for supernatants collected at different sonication time from two sets of sonication experiments with different TS (18.0 and 31.8 g L-1) and same power (120 W) and WAS volume (100 mL)

4.3.4. Effects of sonication factors

4.3.4.1. Sonication probe tip depth

An initial set of experiments were done to evaluate the sensitivity of sonication probe position on outcomes of OD610/TS and VS/TS, where OD610 and

VS are values measured in the supernatants collected after sonication and TS is the initial TS of WAS sonicated. Use of OD610/TS and VS/TS is to normalize against the 56 small variations of TS (20 ± 1 g L-1) of the WAS samples in different experiments. In

Fig. 4.6, results of OD610/TS and VS/TS are compared for two sonication experiments made at the same power (120 W) and WAS volume (500 mL) but different probe positions: one with probe tip at 2 cm below WAS surface, the other at

4 cm below surface (the total WAS depth was about 10 cm). The results showed that the sonication outcomes were not significantly sensitive to probe tip depths; only the two OD610/TS values were significantly different, presumably due to experimental errors because the two VS/TS values were similar. Nevertheless, the probe tip was placed at 2 cm below WAS sample surface in all of the latter experiments.

Figure 4. 6 Effects of sonicator probe-tip depth on OD610/TS and VS/TS released into supernatants; sonication done at 120 W and 500 mL WAS with probe tip at either 57

2 or 4 cm below WAS surface (labeled as “-2 cm” and “-4 cm” systems; total WAS depth ~ 10 cm)

4.3.4.2.Power, volume and time

OD610/TS results from the experiments of 3 × 3 × 6 factorial design for P, V and t effects are shown in Fig. 4.7. Generally, particle release increased with increasing sonication power and decreasing WAS volume but in nonlinear and complex relationships. There have been very few studies on the effect of volume on sludge sonication. Kidak et al. [159] observed a slight decrease of sludge disintegration at 500 mL compared to 300 mL. In this current study, the volume effect on OD610/TS profiles was found to be very complex; the effect was much stronger at

120 W than at 90 and 180 W, and the 300 and 500 mL volumes had almost negligible effect at 90 W. With increasing sonication time, OD610/TS for the higher power- lower volume systems (specifically, all 180 W system and the 120 W-100 mL system) showed approaching-to-plateau profiles except for the final time period of 420-600 s, while the plateauing trends were not/less apparent for the lower power systems.

Sonication disintegrates sludge floc and releases small particles into supernatant mainly by hydro-mechanical shear forces produced by cavitation bubbles [85]. The different plateau levels might correlate with the maximum percentages of micron- sized particles that could be generated from floc solids by the cavitation bubbles created at different powers. The profile-deviating increases of OD610/TS during the

58 final period of 420-600 s were most apparent in systems of smallest volume (100 mL) and higher powers. These were believed to be caused by the temperature increases along with sonication; for example, in the 180 W-100 mL system, WAS temperature increased to 38, 47 and 60 C after 240, 420 and 600 s, respectively, while in the lower power-larger volume systems, temperature never rose above to 40 C.

Figure 4. 7 Time profiles for release of small particles, indicated as OD610/TS, by sonication of different volumes (100, 300, and 500 mL) of WAS at different powers (90, 120 and 180 W)

The heating effects over longer sonication in smaller WAS volumes also affected the release of non-particulate (or OD610-undetectable) VS. VS results are plotted against OD610 in Fig. 4.8 for all these examined systems. VS correlated well 59 with OD610 in all but the 180 W-100 mL systems, which had the most significant temperature increases. Linear regression of all data with OD610 < 4 (excluding 2 points from the 180 W-100 mL systems) gives the following best-fit equation (R2 =

0.965):

VS = 0.825 × OD610 + 0.042

The slope of VS/OD610 = 0.825 likely corresponds to the organic content (VS, in g L-

1) of released micron-sized particles per unit OD610. In systems of OD > 4 and all

180 W-100 mL systems, VS were larger than the values predicted by the above equation, presumably from release of soluble or OD610-undetectable VS due to higher temperature, higher power, smaller volume and/or longer sonication time.

Except for the 180 W-100 mL systems, the largest contribution of non-particulate VS in the total released VS was no more than 15%. Future studies are necessary to evaluate how the generation of different concentrations or percentages of non- particulate VS, in addition to the particulate VS, may affect (positively or negatively) the subsequent algal growth in supernatant and the safe/efficient disposal/treatment of remaining WAS solids. Nevertheless, the current study results confirmed that most of the systems investigated here supported the desired outcome of producing particulate

VS ingestible by O. danica without extensive destruction of bacterial cells.

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Figure 4. 8 Correlation between released concentrations of organics (VS) and small particles (detected as OD610) in supernatants collected from experiments of 3 × 3 × 6 factorial design for P, V and t effects (standard deviations shown only for 180 W-100 mL systems as examples)

4.3.4.3.Regressions for power, volume and time effects

The complex non-linear plateauing and heating effects described above make developing a single quantitative model for results from all systems difficult. Three types of modeling were attempted in this study. First, a standard stepwise multiple linear regression was used to evaluate the effects of P (W), 1/V (mL-1), t (s) and their interactions; OD610/TS was used as the response and P, 1/V, t, P/V, t/V, P×t, and

P×t/V were all included as potential predictors. The final regression equation (R2 =

0.94) with only significant terms is

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푂퐷610 = −0.0536 + 0.000592푃 − 0.000213푡 + 0.000003푃 × 푡 푇푆 푃 × 푡 + 0.000274 푉

P, t, P×t and P×t/V all have p < 0.001. According to the model, OD610/TS increases with power, total energy input (P×t) and volumetric energy input (P×t/V) increases.

The negative regression coefficient for t term is probably due to multicollinearity, as indicated by the very large variation inflation factor (VIF) value of 13.07. Although the model can explain approximately 94.4% (R2) of the variations in the response, the model predicts a linearly increasing trend of OD610/TS with time for every P-V system. This is fine for the lower P-larger V systems, namely, all 90 W systems and

120 W-300 mL and 120 W-500 mL systems, but fundamentally misrepresents the clearly plateauing/saturating trends in the other higher P-smaller V systems.

Comparison of data with model-predicted values is shown in Fig. 4.9 using the 180

W-300 mL system as an example.

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Figure 4. 9 Comparison of model fitting for OD610/TS data in sonication-generated supernatants using the 180 W-300 mL system as an example; regression compared: multiple linear, power-law, and saturation-type models

Power-law regression was an attempt to account for the nonlinear release trends of bacteria-sized particles. The best-fit equation obtained by using Minitab, R2

= 0.96, is

푂퐷610 = (2.6647 × 10−7)푃2.2555푉−0.3813푡0.7537 푇푆

The equation shows that, among P, V and t, P is the most dominant factor (with the largest exponent), followed by t and then V. The negative exponent for V indicates the decreasing effect of sonication in an increasing volume of sonicated WAS. R2 value increased to 96%, from 94.4% for the above multiple linear regression.

Nevertheless, the power-law model still does not describe well the plateauing profiles seen in the 180 W systems, as shown in Fig. 4.9 for the 180 W-300 mL system.

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These plateauing profiles can be better described by a saturation-type model, i.e.,

푂퐷610 푂퐷610 ( ⁄푇푆) ∙ 푡 = 푚푎푥 푇푆 퐾 + 푡 where (푂퐷610⁄ ) is the maximum value (plateau) of OD610/TS and 퐾 is the 푇푆 푚푎푥 half-maximum constant (time required to reach half of the maximum value). For each

180 W system, the time profile of OD610/TS is fitted to this 2-parameter model for best-fit values of (푂퐷610⁄ ) and 퐾 (excluding the obvious outlier at 600 s in 푇푆 푚푎푥 the 180 W-100 mL system). For all three 180 W systems, R2 values are found to be larger than 96.4%. The plateau values of the three 180 W systems are similar, = 0.46

± 0.02, suggesting the maximally releasable concentrations of micron-sized particles depend more strongly on the local sonication intensity near the probe tip and is rather independent of the total WAS volume used. The larger WAS volume just requires longer sonication time for increasing the bulk concentration of released small particles. This is correctly indicated by the best-fit 퐾 values obtained for these systems, 153 ± 28 s for 100 mL system, 243 ± 30 s for 300 mL, and 277 ± 30 s for

500 mL, which clearly increase with increasing V. As an example, the much improved fitting by this model is also shown in Fig. 4.9 for the 180 W-300 mL system. This model however cannot be used to get meaningful values of

(푂퐷610⁄ ) and 퐾 for the lower power systems that did not show clear 푇푆 푚푎푥

64 plateauing profiles of OD610/TS. Further experiments are needed to determine how these parameters may change with P and V.

4.3.5. Energy efficiency consideration and comparison

While the sonication operation can benefit from further optimization, feasibility of the proposed process of combined sonication and phagotrophic algae growth is supported by the results obtained. More importantly, this process promises the potential of lower sonication energy consumption than the other sonication processes designed for enhancing sludge digestion. Energy efficiency is therefore considered here. Ultimate optimization requires precise knowledge of O. danica growth rates and biomass and lipid yields in sonication-generated supernatants with different OD610/TS and knowledge of disposal and further treatment of remaining

WAS solids. Without them, the energy efficiency is only considered here by comparison among the systems examined in this study and then with values from other literature reports.

The time profiles of OD610/TS shown in Fig. 4.7 already indicate the faster particle release rates during the initial periods in higher P, smaller V systems. The released OD610 (and thus VS) percentages in short initial periods are however too low to support more concentrated algal growth in supernatants and to render remaining WAS solids safe or easier to treat for disposal (e.g., meeting Class B 65 biosolids requirements). On the other hand, the particle release (OD610) in 180 W systems is seen to significantly slow down after about 240 s sonication (Fig. 4.7). A reasonable time for comparing and considering sonication energy efficiency is therefore chosen at 240 s here. Energy efficiency is defined here as the increase of

OD610/TS (corresponding to % release of WAS TS as micron-sized particles) per unit volumetric energy input (P×t/V). At 240 s, this is calculated as

(푂퐷610/푇푆) −(푂퐷610/푇푆) 240푠 0푠 × 푉. The calculated value has physical meaning of % 푃×240 release per, e.g., kJ L-1. The values calculated for different P-V systems are summarized in Fig. 4.10. Energy efficiency compared at this chosen time (240 s) increased with increasing V and P, except for the P increase from 120 W-100 mL to

180 W-100 mL presumably because for this smallest WAS volume investigated, 240 s sonication at 120 W already releases majority of the releasable bacteria-sized particles and a further P increase to 180 W can no longer give a larger-than-proportional increase in % particle release. Among the evaluated systems, the 180 W-500 mL system gives the highest energy efficient. At 240 s, the % VS release into the supernatant (= measured VS in supernatant divided by initial VS) in this system reached (28.9 ± 0.6)%.

66

Figure 4. 10 Effect of power and volume on energy efficiency, (OD610/TS)/Ev, estimated at 240 s of sonication; Ev referring to sonication energy input per unit volume

The ultrasonic energy used per kg total solids for the most energy-efficient system (180 W-500 mL) in this study is compared in Table 4.1 with several literature values used for ultrasonic pretreatment of WAS for subsequent anaerobic or aerobic digestion processes. The system in this study indeed has the lowest energy consumption, because it is designed to only release O. danica-ingestible particles without bacteria destruction and solids solubilization. As shown in Fig. 4.1, algae concentration peaked after 1 day growth in a sonication-generated supernatant. The combined ultrasonic-algal process for algal production is unlikely to be longer than

2~3 days. With (28.9 ± 0.6)% VS or more already released for algae consumption, the 67

remaining WAS solids may require much shorter/simpler treatment for reaching

biosolids disposal requirements compared to conventional aerobic and anaerobic

WAS digestion. The proposed ultrasonic-algae process may not only produce algae

from waste sludge but also reduce the subsequent treatment time required, reducing

the overall wastewater treatment cost. The algae biomass and particularly lipids

produced may be more valuable products than the biogas from anaerobic digestion.

Further studies to develop and optimize this new WAS conversion/treatment approach

are warranted.

Table 4. 1 Comparison of specific ultrasonic (US) energy inputs for WAS pretreatment

P t V TS Specific Energy No. -1 Process Reference (W) (s) (mL) (g L-1) (kJ (kg TS) )

1 200 1800 100 36 100,000 Anaerobic digestion [97]

2 225 600 500 18.5 14,600 Anaerobic digestion [160]

3 480 1800 200 17 254,000 Aerobic digestion [95]

Both anaerobic and 4 50 2850 50 14.26 200,000 [161] aerobic digestion

5 180 240 500 20 4,320 US-algae growth This study

4.4. Conclusion

68

With proper sonication, bacteria-rich WAS could be used as sole substrate for phagotrophic alga O. danica cultivation. The sonication effectively released bacteria- sized organic particles with minimal organic solubilization to support WAS organic conversion by O. danica to algal biomass and lipids. The approach also achieved VS reduction to meet at least partial requirement of WAS treatment. Without the need of bacterial wall destruction, the required ultrasonic energy was significantly lower than previously reported. Further optimization of ultrasonic particle release from WAS is warranted; for example, by adjusting WAS pH and by expanding the investigation range of sonication power and volume. More study is also desirable to improve O. danica cell and lipid yields by minimizing particle reflocculation during the algal cultivation stage. The process of short-time ultrasonication coupled with phagotrophic algae growth on released bacteria/particulates can be very competitive to conventional aerobic and anaerobic digestion processes for WAS management.

4.5. Acknowledgements

This work was partially supported by grants from Ovivo USA LLC. We would also like to thank Mr. Gilbert Stadler of the Akron Water Reclamation Facility

(Akron, OH) for assistance in sludge sample collection.

69

CHAPTER V

PHAGOTROPHIC MICROALGAE PRODUCTION FROM WASTE ACTIVATE

SLUDGE UNDER NON-STERILE CONDITIONS

Summary

In this study waste activated sludge (WAS) was sonicated to release bacteria- sized volatile solids (VS) from flocs, after initial pH adjustment to 10 for higher energy efficiency. The released VS supported growth of phagotrophic alga

Ochromonas danica. Initial-rate growth experiments confirmed the Monod-type kinetics but the specific cell growth rate, , correlated with the prey-to-predator ratio, i.e., the ratio of (fed VS concentration)-to-(initial O. danica concentration), significantly better than with the VS alone, as the typical Monod dependency on

-1 soluble substrates. The best-fit kinetics had the following parameters: μmax = 0.198 h

and KM = 1.056 (g-VS/g-algae). Post-sonication reflocculation could render particles too large to ingest by O. danica; therefore, pH and VS effects on reflocculation were investigated. Batch cultivations were then conducted in fermentors at pH 5, under nonsterile conditions. Algae number reached 8.86 × 1010 L-1 after 20 h, corresponding to ~2.3 g/L dry-weight and volumetric algae productivity of 2.8 g/L-day. VS

70 reduction was 38%, giving an O. danica VS yield of 44.5%. The ultrasonication-algae process can be used to produce algae while achieving at least partial WAS treatment.

Keywords:

Microalgae production, phagotrophic algae, waste activated sludge, ultrasonication, alkaline treatment, nutrient recovery

5.1. Introduction

Current wastewater treatment plants commonly employ activated sludge processes [1]. These operations generate, on dry weight basis, more than eight million tons of waste activated sludge (WAS) in the U.S. every year [2]. WAS needs to be properly treated prior to disposal, typically by aerobic or anaerobic digestion [95].

These digestion processes incur high capital and operating costs because of the long treatment times generally required; for examples, more than 20 days for aerobic digestion [162] and 20-30 days for anaerobic digestion [14]. From the resource recovery point of view, WAS however retains the largest portion of resources originally present in the influent wastewater. WAS is therefore also the most important stream to work on for nutrient recovery, to improve the sustainability of human activities that generate the wastewater in the first place [13]. Recycling and reuse for agricultural land are recognized as reasonably practicable options for final

71 disposal of sewage sludge. In the U.S., the majority of treated sludge (Class B biosolids) are applied to agricultural land [47]. There have also been studies on WAS utilization for production of biogas [3], hydrogen [48], biodiesel [49-51], fatty acids

[52], protein [53], enzyme [54-56], bio-flocculants [57-59], biochar [60], and fertilizer

[129-131].

Recently, we have proposed a new approach of combining low-energy sonication and growth of phagotrophic algae to recover resources from WAS [163]. A unique feature of this approach is the low sonication energy required to release individual bacterial cells from WAS for their ready ingestion as food by phagotrophic algae. Using Ochromonas danica as an example, this phagotrophic algae can ingest whole bacterial cells and other particulates smaller than about 7 m [74]. This differs fundamentally from the higher sonication energy needed to rupture tough bacterial cell walls and, perhaps, even to break down macromolecules if to ultrasonically promote aerobic or anaerobic WAS digestion by common osmotrophic microorganisms. It has been reported that low-energy sonication can disaggregate

WAS flocs without substantial reduction of bacterial viability while high-energy sonication can further disintegrate the released cells [89]. In the previous study [163] it was concluded that sonication could release bacteria-sized organic particles with minimal organic solubilization and that O. danica could grow in the supernatants thus generated. The approach also reduced volatile solids (VS) concentration to help meet the WAS treatment requirement. Most importantly, the work proved the sonication 72 energy required for releasing micron-sized particles from WAS, optimized at 4,320 kJ/kg total solids (TS), was indeed much lower than those used in other studies of sonication-enhanced WAS digestion processes, i.e., 15,000-250,000 kJ/kg TS [95, 97,

161, 164]. This new approach may therefore be very competitive to the conventional aerobic and anaerobic digestion processes for WAS management.

The overall economics of this new approach can be substantially improved by maximizing production and value of the phagotrophic algae biomass. One value- added product is algal lipid/oil which may be extracted as fuel or chemical feedstock

[165] or may increase the nutritional/energy value of algae biomass as animal or aquaculture feed [166]. The phagotrophic algae O. danica chosen for this study is oleogenic and metabolically versatile, with photosynthetic, osmotrophic and phagotrophic capabilities [8]. Some fatty acids found in O. danica lipids included myristic, palmitic, stearic, oleic, linoleic, α- linolenic and the particularly high-value

γ-linolenic and eicosatetraenoic acids, including arachidonic acid (ARA) [138-141].

We have previously shown that O. danica can synthesize and accumulate lipids/oil to reach high intracellular lipid contents (30-80%), depending on culture and substrate conditions [9, 10, 167].

However, the growth kinetics and yield were never studied for O. danica in non-sterilized supernatants generated from WAS by the low-energy sonication.

Further, increasing the algae concentration produced can substantially reduce the

73 downstream processing cost. Lin et al. [10] used sugar-rich ketchup to achieve high density culture (> 30 g/L) of O. danica under sterile fermentation conditions.

Producing O. danica (or any other algae) at high volumetric productivity under nonsterile, phagotrophic conditions would be far more challenging. Here we hypothesized that the phagotrophic capability of O. danica can ingest the sonication- dispersed bacteria effectively and be harnessed in a proper process design to achieve higher O. danica concentrations and productivity while keeping interference and contamination issues from the released bacteria and other microorganisms controlled even under nonsterile open conditions. In this study, we aimed to gain the above sets of new knowledge and test the hypothesis of achieving O. danica cultivation under nonsterile conditions using WAS bacteria/particles generated by sonication. These results are very important to further development and economics of the new approach of recovering nutrients from WAS by combining low-energy sonication with phagotrophic algae growth.

5.2. Materials and Methods

5.2.1. Algae culture and medium

The phagotrophic algae used was O. danica (ATCC #30004). The pure O. danica culture was maintained with regular subculturing at room temperature in 1-L

Erlenmeyer flask with 450 mL culture under magnetic stirring. The medium

74 composition was as follows: 8 g/L glucose, 0.45 g/L yeast extract, 0.45 g/L peptone,

0.5 g/L NH4Cl, 0.4 g/L MgCO3, 0.3 g/L KH2PO4, 0.2 g/L nitrilotriacetic acid, 0.1 g/L

MgSO47H2O, 0.05 g/L CaCO3, 4.4 mg/L Na2EDTA (disodium

ethylenediaminetetraacetate), 3.15 mg/L FeCl36H2O, 0.97 mg/L H3BO3, 0.25 mg/L

thiamine, 0.18 mg/L MnCl24H2O, 0.02 mg/L ZnSO47H2O, 0.01 mg/L CoCl26H2O,

6 μg/L Na2MoO42H2O, and 2.5 μg/L biotin [10].

5.2.2. WAS, WAS sonication and supernatant collection

Gravity-belt thickened WAS (thereafter referred to as WAS for simplicity), including 0.5% thickening agent (CLARIFLOC NE483 from POLYDYNE, Inc.), was collected from the nearby wastewater treatment plant of the City of Akron (Ohio). It had 40-45 g/L TS, containing around 75% VS. Fresh WAS sample was diluted to 28.8

± 2.3 g/L TS with deionized water to reduce viscosity for more homogeneous sonication. Returned activated sludge (RAS) collected after the secondary clarifiers from the same plant were also used in some early experiments. RAS samples, having

9-15 g/L TS with about 70% VS, were sonicated without dilution. Sonication was done using a 20 kHz sonicator (Misonix Sonicator®, Model XL2020) at 90, 120 and

180 W (with 30, 45, and 60 μm amplitude). The sonication time was 7 min for WAS samples used in the early study on growth kinetics of O. danica in WAS supernatant

(described in section 2.3) and reflocculation (described in section 2.4) and was 10 min

75 for batch cultivation of O. danica in fermentor (described in section 2.5). Immediately before sonication, pH was adjusted to 10 using 3 M NaOH for most experiments reported here. The tip of sonication probe was placed 2 cm below sample surface in the center of beaker. Low-speed (200 rpm) magnetic mixing was used throughout the sonication duration to further promote homogeneous sonication. The magnetic stirring itself was determined to contribute minimally to the particle release into supernatants

[163]. The sonicated sample was centrifuged (Sorvall Legend X1R, Thermo

Scientific, Osterode, Germany) at 500 g for 10 min to collect the supernatant containing released bacteria and particles. Dynamic light scattering particle-size measurements were done previously to ensure that these sonication and centrifugation conditions collected only particles smaller than about 8 m and the particles were predominantly (90 %) in the size range of 0.4-1.1 m [163].

5.2.3. Determination of growth kinetics of O. danica in WAS supernatant

The substrate dependency of microbial specific growth rate (μ, in h-1) is commonly described in the saturation-type Monod equation, i.e.,

휇 푆 휇 = 푚푎푥 퐾푀 + 푆 where 휇푚푎푥 is the maximum rate, 퐾푀 is the half-maximum Monod constant, and S is the substrate concentration, say, in g/L. However, for phagotrophic growth of O. danica cells by preying on WAS bacteria and organic particles, the substrates are in a 76 discrete finite-size form not as soluble molecules continuously distributed

(considering the relative scale of cells to molecules) in the medium surrounding the growing cells. Results of a previous study in this laboratory suggested that the phagotrophic growth rate of O. danica correlated better with the bacteria-to-O. danica concentration ratio than with the bacteria concentration itself [11]. In other words, the growth dependency might be better described in the following equation:

푉푆 휇푚푎푥 (푋 ) 휇 = 푂푑 푉푆 퐾푀 + ( ) 푋푂푑 where VS, in g/L, corresponds to the combined concentration of ingestible bacteria

and organic particles, and 푋푂푑, in g/L, is the O. danica cell dry-weight concentration.

This hypothesis, however, had not been carefully examined in the previous study; it was tested in this current study.

Sonication was done by the same setup and procedure as described in the previous section. 500 mL diluted WAS was sonicated at 180 W for 7 min. After centrifugation at 500 g for 10 min, the supernatant collected, after pH adjustment to

5.0 ± 0.1, was used for the O. danica growth study. For testing the above hypothesis, two initial algae concentrations were used. For systems inoculated with 0.1 g/L O. danica seeds, the VS concentration was varied from 0 to 1.95 g/L, corresponding to

the initial VS/XOd ratio of 0:1 to 19.5:1. For systems with 0.3 g/L O. danica seeds, the

VS concentration was changed from 0.05 to 1.05 g/L, for the initial VS/XOd ratio of

77

0.17:1 to 3.5:1. All of the 11 systems were grown in a shaker (Thermo Scientific

MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville, NC) at 250 rpm and 20

°C. Initial specific growth rates were determined from growth data during the first 6

푁 h, by μ = ln( 푡⁄ )⁄훥푡, where N is the algal number concentration, t is the growth 푁0 period (6 h), and subscripts 0 and t denote the initial (t = 0) and time t (= 6 h). The μ

® values from all systems were used to get the best-fit 휇푚푎푥 and 퐾푀 using Minitab

(Minitab version 18, Minitab Inc., US). The correlation strength of μ with VS versus

that with VS/XOd was also compared.

5.2.4. Reflocculation of bacterial particles in supernatant collected after alkaline

sonication

Reflocculation of sonication-released WAS bacteria/particles to large sizes would prevent O. danica ingestion. Conditions unfavorable to reflocculation were important to identify for design of high-density cultivation. Effects of pH and VS on reflocculation were observed. For pH effect, the freshly prepared supernatant (pH ~

8.8) was adjusted, with addition of 1 M H2SO4, to pH 7, 6 and 5, respectively, under magnetic mixing. pH adjusted samples were centrifuged again (10 min at 500 g) and

the remaining VS (VSr) in supernatants were measured together with the original VS

(VSo) prior to pH adjustment. The reflocculated fraction, causing VS removal by the

centrifugation, was calculated as 1 - (VSr/VSo). Similar procedures were used for

78 comparing the VS effect (at pH 5) at 1.7, 5.2 and 10.4 g/L, respectively, with dilution by deionized water.

5.2.5. Batch cultivation of O. danica in fermentor

O. danica fermentations with aforementioned sonication-generated WAS supernatant (1 L) as the sole medium (without any nutrient supplementation) were made in 3-L fermentors (BioFlo 110, New Brunswick Scientific). An O. danica seed culture, 160 mL at ~ 1 g/L, was centrifuged (500 g, 10 min); the supernatant was discarded (to avoid introduction of remaining nutrients in seed culture medium) and cells resuspended in deionized water were used as inoculum. A pH probe (Mettler

Toledo P0720-5584) was used to control pH at 5.0 ± 0.1, throughout the fermentation

by automatic acid/base (0.2 N H2SO4/NaOH) addition. A dissolved oxygen (DO) probe (Mettler Toledo P0720-6282) was used to maintain DO above 10% air

saturation by adjusting O2 flowrate. Agitation was provided with a 6-blade turbine impeller at 300 rpm. The working volume varied in the range of 0.7-1.1 L due to sample taking and acid/base addition for pH adjustment.

79

5.2.6. Analytical methods

Supernatant optical density at 610 nm (OD610) was measured using a UV-vis spectrometer (Model UV-1601, Shimadzu Corporation, Columbia, MD) after proper dilution (to < 0.7) to avoid nonlinear correlation with particle concentration. TS and

VS were determined according to the standard methods [143]. O. danica cell dry-

weight concentrations (XOd) were measured for the seed cultures used as inocula for experiments, by centrifuging culture samples at 500 g for 10 min, drying the collected cells at 103 °C for 12 h in an oven, and then weighing the dried cells to determine

XOd. NH3-N concentrations in samples from high-density O. danica fermentation were measured with centrifugation-collected supernatants (7,441 g, 10 min) using an ammonia selective electrode (Orion 9512HPBNWP, Thermal Fisher Scientific). For measurement of O. danica cell number concentration, the sample taken was added with an equal volume of 2% glutaraldehyde (to fix the cells and stop their motility) and then cell number counted using a Petroff–Hausser counting chamber (# 3900) under a microscope. Microscopic pictures were taken using a DP71 digital camera

(Olympus America, Center Valley, PA) coupled to the microscope.

80

5.3. Results and Discussion

5.3.1. Alkaline sonication

Alkaline addition prior to WAS sonication was used to improve sonication effectiveness. While a systematic study was not done for the potentially complex effects of multiple factors involved (pH, sonication power and time, WAS TS and volume, etc.), preliminary experiments were made to assess the suitable pH to use.

For example, the supernatant OD610 results from 90-W sonication of WAS and RAS

(returned activated sludge) samples adjusted to different initial pH values are shown in Figure 5.1. While the OD610 dependency on initial pH differed at pH > 10 for

WAS versus RAS, the dependency was similar at lower pH and showed a larger marginal increase of OD610 when the pH used was increased from about 9 to 10. The pH effects observed here were largely consistent with previous reports although stronger sonication was typically used in the earlier studies [168-171]. High pH renders extracellular polymeric substances (EPS) and bacterial surfaces in WAS flocs more negatively charged, with stronger electrostatic repulsion to favor de- flocculation, and it promotes EPS hydrolysis including breakage of disulfide bonds in proteins [172]. It was also reported that pH ≥ 10 caused significant organic solubilization and damaged cell structures including cell wall, membrane and nuclei, compared to lower pH [173]. The high pH-induced cell damages might be responsible for the OD610 decrease seen in Figure 5.1 for the RAS samples sonicated at initial

81 pH > 10. According to results of these preliminary experiments, alkaline was added to raise initial WAS pH to 10 prior to sonication in later experiments in this study.

WAS RAS 4 1.2

3.5 1.1 3 1 2.5 0.9

2

OD610 (RAS) OD610 OD610 (WAS) OD610 1.5 0.8

1 0.7 6 7 8 9 10 11 12 13 Initial pH

Figure 5. 1 OD610 in supernatants made from 90 W sonication of WAS and RAS samples adjusted to different initial pH by alkaline addition. (WAS, 120 mL with 27 g/L TS, was sonicated for 4 min; RAS, 45 mL with 9 g/L TS, for 3 min. These preliminary experiments were done without replicates; OD variations determined from later experiments with at least duplicates were found to average at 3.8% ± 2.1%.)

During the sonication of WAS with initial pH 10, pH would drop presumably due to neutralization by acidic groups generated from EPS hydrolysis; an example is shown in Figure 5.2a for a 300-mL WAS (31.5 g/L TS) sonicated at 120 W, where pH dropped to 8.1 in 12 min. Processwise, it is not easy to control pH of viscous thickened WAS to be relatively homogeneous at 10 throughout sonication, and allowing pH to drop without control has the advantages of minimizing base dose, 82 bacterial cell lysis, later neutralization to acidic condition (pH ≤ 5.5) good for O. danica cultivation, and wastewater salinity increase due to base/acid addition.

As sonication time increased, more particles were released leading to increasing OD610 in the supernatant. This phenomenon is shown in Figure 5.2b for two WAS samples, both sonicated at 120 W but with different TS and sonication volume; one was the same WAS shown in Figure 5.2a, the other had smaller TS

(26.8 g/L) and volume (100 mL). It appears that the two systems can be represented

by a same correlation of normalized OD610/TSo (proportional to fractional particle

release from TSo, original TS in the WAS sample) with the normalized sonication

time/volume (t/V). The profiles of increasing OD610/TSo with t/V show slight but clear plateauing curvatures, and the results can be well described by the following 2nd- order polynomial equation (R2 = 0.99):

푂퐷610 푡 2 푡 = −0.0105 ( ) + 0.1492 + 0.0133 Eq. (1) 푇푆표 푉 푉

The most important reason of using alkaline sonication is to increase the sonication energy efficiency. This effect is shown in Figure 5.2c by comparing the

particle release (OD610/TSo) and associated organics (VS) release (as % of VSo in the

푃×푡 WAS sample) at different specific sonication energy inputs (= , in kJ/kg-TS, 푇푆×푉 where P is the sonication power) for WAS sonicated without (pH ~7) and with

alkaline addition (pHo = 10). Alkaline sonication was significantly more energy

efficient for particle and organics releases; for example, with 9,000 kJ/kg-TSo

83 sonication energy, OD610/TSo reached 0.26 and about 45% total VS was released into the supernatant in the alkaline sonicated WAS samples, while for the sonication

without pH adjustment, OD610/TSo and VS release (estimated by interpolation) were only about 0.17 and 18%, respectively. In the previous study with only neutral WAS sonication experiments [163], VS release was found to parallel the OD610 release at an essentially constant ratio (VS/OD610) of 0.825, presumably corresponding to the organic content (VS, in g/L) of released micron-sized particles per unit OD610. In the alkaline WAS sonication experiments done in the current study, the VS/OD610 ratio was consistently larger than 0.825, starting at about 4-6 and decreasing to about 1.3 after 7+ min sonication. These larger VS/OD610 ratios and their decreasing trends with time suggested that alkaline addition caused EPS hydrolysis and soluble organics release at the early stage when pH was still high, in addition to release of the OD610-

detectable micro-particles (including bacterial cells). Take the 9,000 kJ/kg-TSo sonication of WAS of 50 g/L TS, containing 37.5 g/L VS, as an example for estimation. The VS released was 45%, i.e., 16.9 g/L. The VS portion due to released

micro-particles could be estimated as (OD610/TSo) × TSo × (VS/OD610) = 0.26 × 50

× 0.825 = 10.7 g/L, representing approximately 64% of the total VS released.

A preliminary cost comparison can be made between the sonication with and without the alkaline addition. The base was added here only to raise the “initial” WAS pH to promote effectiveness of a short sonication operation, not for long-term alkaline sludge treatment. The NaOH amount required was only about 1.5 wt% of WAS TS, 84 adding a cost of $6/ton-TS at a price of $400/ton for industrial 99% NaOH (caustic soda) flakes. This small amount of alkaline could, however, lower the sonication energy cost substantially. For example, according to Figure 5.2c, for releasing 20%

WAS VS into the supernatant, the specific energy required by alkaline sonication

(~1,000 kJ/kg-TS) was an order of magnitude lower than that by neutral sonication

(~10,000 kJ/kg-TS). At an electricity cost of $70/MWh in the US, these energy requirements correspond to, per ton TS, $19.5 and $195 for the alkaline sonication and neutral sonication, respectively. So, the combined NaOH and energy costs for alkaline sonication would be $25.5/ton-TS, far cheaper than the $195 energy cost required for the sonication without alkaline addition. Moreover, because the sludge pH already dropped to ~8 along the alkaline sonication, minimal or no acid addition may be needed to correct/adjust the sludge pH before the subsequent treatments for final disposal. Alkaline sonication is expected to be much more cost-effective than the neutral sonication for releasing WAS bacteria/organic particles into supernatant to support the phagotrophic algae growth.

10 (A)

9 pH 8

7 0 200 400 600 Sonication time (s)

85

0.3

TSo 31.5 g/L, 300 mL (B) L/g)

- TSo 26.8 g/L, 100 mL 0.2

0.1 y = -0.0105x2 + 0.1492x + 0.0133

OD610/TSo (# OD610/TSo R² = 0.989 0 0 0.5 1 1.5 2 2.5 3 Sonication time/volume (s/ml)

0.5 (C) 0.4

0.3

0.2

OD/TSo or VS % VS or OD/TSo 0.1

0 0 2000 4000 6000 8000 10000 12000 Specific sonication energy (kJ/kg-TSo) VS% pHo 10 VS% pHo 10 OD/TSo pHo 10 OD/TSo pHo 10 VS% pHo 7 OD/TSo pHo 7

Figure 5. 2 WAS sonication at 120 W: (A) pH decrease with alkaline sonication time (300 mL WAS of 31.5 g/L TS); (B) micro-particle release profiles plotted as OD610/TS against sonication time/volume (t/V) for two alkaline-sonicated WAS, one with 300 mL at 31.5 g/L TS and the other with 100 mL at 26.8 g/L TS; and (C) comparison of particle release (OD610/TS) and organics release (VS %) profiles in neutral versus alkaline sonication, as a function of specific sonication energy inputs

(kJ/kg-TSo), where the neutral sonication results were from a previous study [163].

5.3.2. Growth kinetics and yield of O. danica in WAS supernatant from alkaline

sonication

86

Specific growth rates, μ, of O. danica growing in WAS supernatants prepared

by alkaline sonication were plotted in Figure 5.3, against the initial VS/XOd ratio and

VS concentration, respectively, for two sets of cultures seeded at different initial O. danica concentrations (0.1 and 0.3 g/L). As expected, μ exhibited the saturation-like

(Monod-type) dependency on “food availability”; however, a single correlation could describe the dependency for both sets of cultures much better if the “food availability” was quantitated by the ratio of food particles-to-phagotrophic cells, i.e.,

푉푆 0.198 ( ) 푋푂푑 2 휇 = 푉푆 (R = 0.88), Eq. (2) 1.056+( ) 푋푂푑 than if the “food availability” was quantitated simply by VS, i.e.,

0.186 푉푆 휇 = (R2 = 0.78) Eq. (3). 0.139+푉푆

This finding confirms the fundamental difference of cell growth dependency on discrete food particles from that on soluble molecular substrates.

Predator-prey dynamics has been considered in other fields dealing with organisms of much larger sizes. For example, Arditi and Ginzburg [174] discussed the situations when prey dependency (analogous to the VS dependency in this case) and

prey-predator ratio dependence (analogous to VS/XOd dependency) may apply, with examples such as rates of barnacle Balanus balanoides eaten per snail Urosalpinx cinerea and spider mite Tetranychus urtieae eaten per predatory mite Phytoseiulus persimilis. They argued that prey dependency must fail at sufficiently high predator

87 densities when predators inevitably interfere with each other’s food search or capture, and the ratio dependency must become irrelevant at sufficiently low predator densities when interference cannot occur due to their distant separation. By similar reasoning, in this study concerning only microorganisms, the number of discrete bacteria/food particles is far smaller and the distance between them far larger, compared to the case of soluble molecular substrates on the same weight basis. The large distance between bacteria/food particles requires O. danica cells to travel farther for food ingestion and heightens their possible interferences/competition for food. The specific growth rate of O. danica in this situation would not only depend on VS but also on O. danica

concentration, and thus the VS/XOd ratio dependency observed. As expected from this

reasoning, at very high VS (> 1 g/L) or VS/XOd (> ca. 4), the ratio dependency appeared to be less significant in Figure 5.3.

-1 According to Eq. (2), the maximum specific growth rate µmax was 0.198 h , which corresponded to a doubling time of 3.5 h. This was faster than the doubling time of 7 h reported for O. danica growing on washed E. coli cells in deionized water

[11]. It is unknown if the presence of hydrolyzed/released EPS organics from the alkaline-sonicated WAS played any beneficial roles in O. danica growth in the current study. Holen et al. could achieve steady state in a two-stage continuous process at dilution rate (D) of up to 0.1 h-1 (for both stages), with E. coli growing in the first stage on soluble nutrients and O. danica growing in the second stage on the effluent from the first stage (containing E. coli cells). In addition, they found that at 88 this condition, each O. danica cell ingested, on average, 182 bacterial cells per h [73].

Eq. (2) also indicates the requirement of relatively low VS/XOd ratios to support high

O. danica growth rates because the half-maximum constant (Monod-type Ks) was

only 1.056 (g/g VS/XOd). The fast growth rate and low Ks value indicate that O. danica can be used for fast conversion of WAS particles/organics released by alkaline sonication and for reduction of bacteria and digestible particles to low concentrations.

0.25 Xo = 0.1 g/L Xo = 0.3 g/L 0.25 Xo = 0.1 g/L Xo = 0.3 g/L

0.2 0.2

0.15 0.15

)

)

1

- 1

푉푆 - 0.1 0.198 ( )

(h 0.1

푋푂푑 μ (h μ

휇 = μ 푉푆 0.186 푉푆 1.056 + ( ) 휇 = 푋 0.139 + 푉푆 0.05 푂푑 0.05

0 0 0 5 10 15 20 0 0.5 1 1.5 2

-0.05 -0.05 푉푆/푋푂푑 (g/g) VS (g/L)

Figure 5. 3 Specific growth rates, μ, of O. danica growing in WAS supernatants prepared by alkaline sonication plotted against the initial VS/XOd ratio (Left) and VS concentration (Right), respectively, for two sets of cultures seeded at different initial O. danica concentrations (0.1 and 0.3 g/L). Each data point was obtained from one culture flask; duplicate samples were taken at 0 and 6 h, respectively, to determine the initial growth rate μ and its associated standard deviation (plotted as an error bar here). The dashed line represented the correlation described by the equation given in figure, obtained by best-fitting the equation with data from both sets of cultures. Clearly, the equation in the left figure described both sets of data much better than the equation in the right figure. 89

5.3.3. Reflocculation and endogenous digestion of WAS VS in sonication-generated

supernatants

Sonicated WAS can reflocculate; some possible reasons have been proposed, which include the interactions between released multivalent cations (Ca2+ and Mg2+) and EPS and the hydrogen bonding between EPS biopolymers [164]. In this study, reflocculation of WAS VS in sonication-generated supernatants can reduce the algal yield from this coupled sonication and phagotrophic algae cultivation process. The examined effects of pH and VS concentration on reflocculation are shown in Figures

5.4(A) and 5.4(B), respectively. The fractions of VS reflocculated in supernatants after pH adjustment to 5, 6, 7 and 8.8 (unadjusted), measured within 15 min

(including 10 min centrifugation), were found to be (3.1 ± 4.5)%, (11.8 ± 0.4)%, (20.4

± 5.7)% and (0 ± 3.8)%, respectively. One-way ANOVA confirmed the statistical significance of pH effect on reflocculation with p = .025. It is unknown if the high reflocculation fractions at pH 6 and 7 were related with the pH dependency of polymeric flocculant used by the plant for WAS thickening. Nonetheless, pH 5 gave lower reflocculation and was in the ideal pH range for phagotrophic cultivation of O. danica in open systems [11]. The effect of VS on reflocculation was examined only at this pH (5.0 ± 0.1), with initial VS of 1.7, 5.2 and 10.4 g/L, respectively. Under magnetic stirring, the sonication-generated supernatant VS decreased clearly in systems of all 3 VS concentrations during the first h; the VS then remained essentially 90 constant during the next 7 h in the two systems of lower initial VS (1.7 and 5.2 g/L).

In the system of highest initial supernatant VS (10.4 g/L), the VS further decreased slightly during 1-4 h and then remained constant. Results of the reflocculated VS fractions are plotted against time in Figure 5.4(B). Regardless of the initial VS, (8.1 ±

5.1)% VS might become too large to ingest by O. danica due to reflocculation within the first hour of feeding. VS had a mild effect on reflocculation only at the later stage and only at rather high VS (> 5.2 g/L).

0.3 (A)

0.2

0.1

0.0

4 5 6 7 8 9 Reflocculated VS fraction VS Reflocculated -0.1 pH

91

0.20 (B)

0.15

0.10

0.05

1.7 g/L 5.2 g/L 10.4 g/L Reflocculated VS fraction VS Reflocculated

0.00 0 2 4 6 8 Time (h)

Figure 5. 4 Effects of (A) pH and (B) initial VS (at pH 5.0 ± 0.1) on VS reflocculation in WAS supernatants generated by alkaline sonication. The same sonicated WAS supernatant was used in both sets, (A) and (B), of experiments. Each data point represents the average and standard deviation (error bar) obtained from duplicate samples with pH and/or initial VS adjustments.

5.3.4. Nonsterile O. danica cultivation in fermentor

Previous study showed that there was no difference whether phagotrophic growth was done at pH 4-7 [11]; however, as described above, pH had a significant effect on reflocculation. pH 5 was chosen for the batch cultivation to suppress not only reflocculation but also the bacterial respiration, to reduce demand on oxygen supply. Microscopic pictures taken at 0 h and 20 h are shown in Figure 5.5. O. danica number increase and bacteria/particle reduction are evident. Time profiles of O. danica growth and ammonium release, the latter occurred because bacterial biomass was a C-limiting, N-excessive food source, are shown in Figure 5.6(A) for two batch

92 cultivations with different initial supernatant VS, i.e., 7 and 11 g/L, respectively. The

-1 specific O. danica growth rate (8-20h) was 0.15 ± 0.10 h from both growth curves, corresponding to a doubling time of 4.6 h. The  value found here agreed reasonably

well with the model established in Eq. (2), corresponding to an average VS/XOd ratio of 3.3 (g/g) over the long growth period considered here.

It should be mentioned that during the cultivation, the morphology of O. danica was observed to change. The cells from seed culture (at 0 h) were in a teardrop shape. They became almost spherical at 8 h, when adjusted to the condition of high

prey/predator VS/XOd ratios. They returned to the teardrop shape at 20 h, as the

VS/XOd ratio became limiting. The morphology change was accompanied by observed motility change (not quantitated). The percentage of moving O. danica cells and their swimming speed were low during the first 8 h or so but clearly increased later when the O. danica concentration became higher and the bacteria/particle availability decreased. Others have also observed the increasing swimming speed of aquatic phagotrophic organisms at low prey levels [175].

Ammonia was produced during the phagotrophic digestion of bacterial particles. From the two batches of cultivation, the maximum ammonium released per

+ initial VS provided was consistently found as 20.5 ± 0.9 and 21.0 ± 0.2 mg NH4 -N/g

VS for the 7 g/L and 11 g/L systems, respectively. In the batch with higher initial VS

+ (11 g/L), the ammonium release reached about 260 mg/L NH4 -N. In a set of previous

93 tests for O. danica tolerance of ammonium concentration by inoculating O. danica

seeds to WAS with different NH4Cl concentrations added as N source, the growth at

+ pH 5 was not affected by up to 250 mg/L NH4 -N but became negatively affected at

+ about 300 mg/L NH4 -N (detailed data not shown; after 21 h at this higher ammonium concentration, the percentage of motile cells clearly dropped from about 40% to 10% under microscopic observation). This factor was considered when designing these two batch cultivations. Accordingly, this inhibitory ammonium accumulation due to the C- limiting nature of WAS biomass would limit the maximum O. danica concentration achievable in the current process. In the future, one possible solution to this upper limit is to supplement C-rich waste source to balance the C:N ratio and reduce ammonium accumulation in the process [11, 176].

The VS distribution among O. danica cells, non-algae solids, and supernatant is also followed during the cultivation, as shown in Figure 5.6(B). The algal VS concentration was estimated by multiplying its number concentration by the algal VS per cell measured in the seed culture free of other solids (~2.4 × 10-11 g/cell); the VS concentration in non-algal solids was obtained by subtracting the estimated algal VS from total VS in solids collected by centrifugation. The VS distribution roughly remained the same after 20 h, when O. danica growth already stopped. For the cultivation with 11 g/L initial VS, at 20 h the algal VS and non-algal solids VS were approximately 2.1 g/L and 1.5 g/L, respectively. (Note that VS was separately measured to be approximately 90% of TS for O. danica cells.) The non-algal solids 94

VS was about 15% of the original VS provided, which compared reasonably well with the average of 13.5% reflocculated VS shown in Figure 5.4(B) after 8 h mixing of the system with a similar initial VS. About 50% VS remained in the supernatant after 20 h, which could be mainly debris excreted by O. danica after ingestion and metabolic processing since O. danica could no longer use them for growth. The O. danica number yields from the two batch cultivations averaged at (8.4 ± 0.6) × 109 cells/g-VS and (7.8 ± 0.2) × 109 cells/g-VS, respectively. O. danica mass yield from the consumed VS was estimated at 44% and that from the total VS provided was approximately 18.5%.

95

Figure 5. 5 Microscopic pictures at 0 and 20 h of O. danica batch cultivation in WAS supernatant from alkaline sonication (scale bar: 20 μm).

(A) 10 300 9 250

8 /L)

10 7 200 6

5 150

N (mg/L) N

- +

4 4 100

3 NH

Cell number (x 10 number Cell 2 50 1 0 0 0 5 10 15 20 25 30 35 Time (h)

+ + 7 g/L-CN 11 g/L-CN 77 g/L-NH4-N g/L-NH4 -N 1111 g/L-NH4-Ng/L-NH4 -N

96

(B) VS-sup non-algal VS VS-algae 120%

100%

80%

60%

40% VS distribution VS

20%

0% 0 20 24 30 Time (h)

Figure 5. 6 Results from two batches of O. danica cultivation in supernatants generated from alkaline sonication of WAS; supernatants had 7 and 11 g/L initial VS, respectively: (A) profiles of O. danica growth (in cell number concentration CN) and ammonium generation (NH4+-N); for example, the legend “7 g/L-CN” denotes the O. danica cell number concentration data from the batch of cultivation with WAS supernatant of 7 g/L initial VS, and (B) VS distribution among O. danica cells (VS- algae), non-algal solids (non-algal VS) and supernatant (VS-sup) during the batch cultivations.

An attempt was made to test if the non-algal (reflocculated) solids were indeed non-metabolizable to O. danica. There was no easy way to separate all non-algal solids from the O. danica cells; so, the whole culture was filtered through a 270 mesh

(53 μm) screen to collect the larger non-algal solids. After deionized water wash, thus collected solids were re-sonicated into a fine-particle suspension and re-inoculated

97 with O. danica seeds at a suitably large VS/XOd ratio of 5. It was confirmed that the solids even after resonication could not support detectable O. danica growth.

5.4. Conclusions

Alkaline WAS ultrasonication was more energy efficient to release ingestible food sources for phagotrophic microalgae. With 9,000 kJ/kg-TS sonication, 45% VS was released, containing mainly (64%) bacteria/microparticles. The released organics supported rapid O. danica growth under nonsterile conditions, showing growth-rate

-1 dependency on (VS/O. danica) ratio with μmax = 0.198 h and KM = 1.056 (g/g).

Fermentor cultivations were studied at pH 5 to minimize reflocculation, with feed VS at concentrations avoiding ammonium inhibition (due to C-limited WAS-VS). High microalgae productivity (2.8 g TS/L-day) was achieved. Ultrasonication improved feasibility of microalgae production from WAS, serving purposes of nutrient recovery and WAS treatment. For large-scale applications, continuous-flow processes may be more suitable. Given the fast O. danica growth rate in sonicated WAS supernatants

(doubling time of 3.5-4.6 h at pH 7-5), long retention time may not be necessary.

Further, carbon-rich waste organics such as waste grease collected at wastewater

treatment plants may be co-processed to improve algal yield and avoid NH3 release from algal consumption of only WAS organics. Future studies are warranted to determine effects of such co-processing of sonicated WAS supernatant and carbon-

98 rich waste organics and to optimize conditions for stable and high-productivity continuous-flow processes.

5.5. Acknowledgment

This work was partially supported by grants from Ovivo USA LLC. We would also like to thank Mr. Gilbert Stadler of the Akron Water Reclamation Facility

(Akron, OH) for assistance in sludge sample collection.

99

CHAPTER VI

ULTRASONIC-PHAGOTROPHIC PROCESS FOR WASTE ACTIVATED

SLUDGE CONVERSION AND MICROALGAE PRODUCTION

Summary

In this study I developed an ultrasonic-phagotrophic process for waste activated sludge (WAS) conversion by phagotrophic alga Ochromonas danica.

Ultrasonic pretreatment was applied to release active biomass including bacteria from

WAS floc and O. danica was used for subsequent conversion and volatile solids (VS)

reduction. The yield of O. danica on released VS was 0.3 g O. danica/g VSfed. The

푡 estimated total oxygen uptake 푇푂푈 = (푂푈푅)푑푡 in remaining solids dropped by ∫0

65-92%, indicating substantially reduced aeration cost in aerobic digestion. Compared to traditional aerobic digestion, the integrated ultrasonic-phagotrophic process showed enhanced overall WAS digestion with reduced energy cost. The novel process can be used for microalgae production with partial WAS stabilization.

Keywords:

Waste activated sludge (WAS), phagotrophic algae, ultrasonication, aerobic digestion, pathogen reduction, specific oxygen uptake rate (SOUR).

100

6.1. Introduction

Almost all domestic wastewater in US are treated by biological processes in which activated sludge process has been a central component for more than a century since 1914 [1, 26]. The main drawback is the production of waste activated sludge

(WAS). Greater than 8 million dry tons of biosolids are generated in the US annually

[2]. Pathogens entering wastewater are often concentrated in wastewater biosolids that represent potential risks to human and animal health thus must be appropriately treated to reduce pathogen level and vector attraction potential before its disposal and land application [34-36]. Table 6.1 shows the typical pathogen indicator content in sludge. The disposal of waste sludge represents up to 50% of the operating costs of a wastewater treatment plant [15]. Recycling and reuse for agricultural land are recognized as reasonably practicable options for final disposal of sewage sludge. In the U.S., the majority of biosolids are applied to agricultural land and approximately

75% of these are of class B status [47].

Table 6. 1 Microbial content in wastewater sludge [25].

Fecal Coliforms Salmonella sp. Helminth Ova (Ova/g TS) Value Log (MPN/g TS) Log (MPN/g TS) Viable Total

77 ± 43 Mean 7.6 ± 1.0 5.2 ± 1.3 72 ± 38 178 Max 9.0 7.4 160 4 Min 5.4 2.9 4

101

Activated sludge flocs are comprised of a conglomerate of materials including; microorganisms, extracellular polymeric substances (EPS), inert particulates, endogenous residues, unbiodegradable organics and water [92]. Microorganisms, mainly bacteria, are glued together in a polymeric network formed by microbial extracellular polymeric substances (EPS) and cations [14]. It is expected that the most active aerobic bacteria are on the outer layer of sludge flocs due to better access to substrate and dissolved oxygen (DO) in aqueous phase [145].

Reduction in sludge production will certainly save the cost of downstream sludge management including thickening, stabilization, dewatering and disposal [15].

Sludge reduction can be achieved either in wastewater or sludge treatment line [31].

Different physical (ultrasonication, microwave, thermal), chemical (alkaline, ozonation) and biological treatment (microbial predation, endogenous metabolism) can be used. Traditionally, both aerobic and anaerobic digestion can be used for WAS reduction and stabilization. However, at least 20 days of retention time is required to convert sludge to USEPA Class B or Class A biosolids [14]. High temperature [41], long retention time [34] or base addition [177] is required to further reduce pathogen level in WAS during digestion processes. Anaerobic digestion can convert sludge to biogas as valuable product. However, less than 10% of the produced biogas is currently being properly used [38]. Compared to anaerobic digestion, aerobic digestion is a better option for small plant due to its lower capital cost and simpler operation. However, high energy cost and lower pathogen inactivation are considered 102 as the main disadvantages of aerobic digestion [161].

Sludge production can also be reduced through microbial predation [31], however, long-term stable predation process is difficult to maintain [33]. As in modern society sustainable wastewater treatment is more desirable, researchers are seeking alternative solutions to sludge reduction with production of valuable products.

Recently phagotrophic algae are being employed for microalgae production with reclamation of waste organics such as waste ketchup [10], waste cooking oil [9], solid waste grease [178], wastewater [167] and waste activated sludge [179]. Phagotrophic algae can grow actively on small organics including bacteria, oil droplet and casein

[8, 79]. Ochromonas sp. can acquire nutrients via ingestion of bacteria [176]. Lin and

Ju [11] studied the phagotrophic growth of Ochromonas danica on pure bacteria

E.coli with doubling time of 7 h with 30% (w/w) lipid content. Hourly ingestion of

182 E. coli per O. danica was observed by Holen [73] in sterile two-stage chemostat.

Pretreatment is needed to release bacteria-sized particles from sludge flocs for following phagotrophic algal growth [163, 180]. Ultrasonication has been used for sludge disintegration before digestion [95, 97], bacteria recovery [91, 92], sludge dewatering [152], enzyme extraction [181] and etc. Hydro-mechanical shear forces are predominantly responsible for ultrasonic activated sludge disintegration [86].

Sludge disintegration efficiency decreases with increasing ultrasound frequency [83].

During ultrasonication the sludge flocs are initially disintegrated with release of small particles which including EPS and bacteria into aqueous phase. Further sonication can 103 cause significant cell lysis and soluble organic increase [87, 88].

The objective of this research is to determine the feasibility of using ultrasonication to release active biomass (e.g. bacteria) into aqueous phase for phagotrophic algal cultivation with enhanced volatile solids (VS) digestion. It is hypothesized that ultrasonic pretreatment can release major active aerobic bacteria on the out layer of sludge flocs. It is also hypothesized that the released bacteria together with other small particles can support phagotrophic algal growth. Moreover, the time and energy required for stabilizing remaining solids by aerobic digestion may be much reduced as the transfer of bacteria from sludge flocs to aqueous phase. Both algal yield on released particulate VS and digestion in remaining solids will be considered for designing novel ultrasonic-phagotrophic process.

6.2. Materials and Methods

6.2.1. Phagotrophic algae and waste activated sludge

The Ochromonas danica culture, cultivation method and waste activated sludge sample are described in previous papers [163, 179].

6.2.2. Sludge sonication and collection of supernatant containing released small

particles and organics

Original sludge was used directly after collected from Akron plant. Sonication 104 was done using a 20 kHz Misonix Sonicator® (Model XL2020) (Farmingdale, NY,

USA) with maximum power of 600 W. (The ultrasonic power delivered to the horn was indicated by the instrument and reported in this study.) The tip of the probe was placed in the center of glass beakers with a submerged depth of electrode of 1-1.5 cm.

Low-speed magnetic stirring was used to facilitate mixing, allowing sludge solids to move more homogeneously through the active sonicating zone near the sonicator probe. The magnetic stirring itself contributed minimally to the particle release into supernatants.16 Immediately after sonication, RAS were centrifuged at 350 g for 10 min while WAS were centrifuged at 500 g for 10 min, to separate supernatant from remaining large particulate solids.

6.2.3. Analytical methods

TS and VS were determined according to the standard methods [143]. For tracking phagotrophic algae growth quantitatively, the sample taken was added with an equal volume of 2% glutaraldehyde solution to fix the cells and stop their motility.

The cell number could then be counted using a Petroff-Hausser counting chamber

(Hausser Scientific, Horsham, PA, USA) under a microscope. Microscopic pictures were obtained using a DP71 digital camera (Olympus America, Center Valley, PA,

USA) coupled to the microscope. The specific oxygen uptake rate, SOUR in supernatant and remaining solid were measured by USEPA Method 1683. Sonicated

105 sludge samples were centrifuged at 500 g for 10 min to separate supernatant from remaining solids. 50 mL supernatant was added by 450 mL water to measure DO decrease with time. Pathogen indicator density (total coliform and fecal coliform) were determined by USEPA Method 1680, Class B dilution.

6.2.4. Statistical analysis

Student’s t-test was done using Microsoft Excel® to determine if algal yield from RAS (n=5) is significantly different from WAS (n=6).

One-way ANOVA was done using Microsoft Excel® to test if subsequent sonication has significant effect on specific oxygen uptake rate (SOUR) in solids

One-way ANOVA was done using Microsoft Excel® to compare if subsequent sonication has significant effect on time (in days) required to achieve Class B biosolids in SOUR.

6.3. Results and Discussion

6.3.1. Sludge selection: RAS vs. WAS for algae production

Two types of activated sludge, return activated sludge (RAS) and gravity belt thickened waste activated sludge (WAS), were compared for their support for O. danica growth. The algae grown, calculated by the difference between the initial and

106 maximal cell number concentration within 20 ± 4 h, was plotted against initial VSsup

concentration (VS0, g/L) as shown in Figure 6.1. It is notably that the algal yield

10 (slope value) from RAS-generated supernatant (1.4 × 10 cell/g VS0) was about 2-

10 fold of that from WAS-generated supernatant (0.7 × 10 cell/g VS0), indicating more algal digestible VS were released during ultrasonic pretreatment. Student’s t-test

(unpaired, equal variance, p-value = 0.000145 < 0.05) indicated that algal yield from

RAS was significantly higher than that from WAS.

90 80

70 yRAS = 14.087x R² = 0.8631

60

/L) 9

50 RAS (x 10 (x

0 40 WAS

A -

m Linear (RAS) 30 yWAS = 6.8892x A R² = 0.886 Linear (WAS) 20 10 0 0 2 4 6 8 10 12

VS0 (g/L)

Figure 6. 1 Effect of initial sludge-generated supernatant on algae number yield.

RAS flocs are formed by filamentous bacteria and extracellular biopolymers

[149] while WAS flocs are gravity belt thickened from RAS with addition of 0.5% polymeric flocculant. The microbial composition of RAS and WAS, in terms of the 107 percentage of bacteria, cell debris, EPS and inorganic materials should be almost the same. However, bacterial distribution may change significantly in the thickening process as certain fraction of bacteria at the out layer of original RAS floc are embedded during the thickening process. When ultrasonication was exerted on RAS, as active bacteria are located on the outer layer of flocs, more active bacteria can be released. On the contrary, when WAS is exposed to ultrasound waves, more non-cell fractions, including EPS and polymeric flocculants can be released. Accordingly, RAS was used for following studies. Although RAS has lower solids concentration compared to WAS, it can be further concentrated by increasing settling time in the secondary clarifier.

6.3.2. Effect of sonication on bacteria-sized volatile solids release

As shown in Figure 6.2, the VS release ratio is plotted against specific energy

(in kJ/L) to evaluate the energy efficiency. It was found that at low power level 60 W

(or 0.4 W/mL), the VS release ratio increased linearly with specific energy input (R2 =

0.995). At higher power levels (120 W and 180 W), the VS release first increased quickly and then subsided and plateaued. At 0.8 W/mL, with the same ultrasonic energy input of 960 kJ/L, the multi-sonication system showed much higher VS release

(0.80 vs 0.59), which indicated that ultrasonic release efficiency could be affected by the presence of already released small particles.

108

1.00

) 0

0.80

/VS sup

0.60 0.4 W/mL 0.8 W/mL 0.40 1.2 W/mL 0.8 multi 0.20 Linear (0.4 W/mL) y = 0.0002x + 0.0186 VS release ratio (VS release VS R² = 0.9948 0.00 0 300 600 900 1200 1500 Specific energy (kJ/L)

Figure 6. 2 Bacteria-sized VS release as a function of specific energy input.

6.3.3. Algal growth on supernatant generated from subsequent sonication treatments

Multi-sonication was done at 120 W (or 0.8 W/mL) to evaluate following algal growth on each batch of supernatant generated by subsequent sonication and status of remaining solids after centrifugal separation. As shown in Figure 6.3a, whole WAS without sonication could not support robust O. danica growth. Both supernatants generated from 1st and 2nd 10min sonication could support robust growth while VS generated from the 3rd batch of sonication couldn’t, presumably due to fast reflocculation in 3rd sonication-generated supernatant as observed under microscope.

Assuming individual algal cell weight to be 2.4 x 10-11 g/cell [179], about 30 ± 2% (g

109

st nd cell/g VSfed) yield could be achieved from 1 and 2 sonication-generated supernatants (Figure 6.3b). In addition, 49.8 ± 0.8% VS reduction was achieved, which contributed to 40% of overall VS reduction in raw RAS.

0min 1st 10min 2nd 10min 3rd 10min 7.0 (a) 6.0

5.0

/L) 4.0 10

3.0

( x 10 x ( number concentration concentration number 2.0

1.0

O. danica O. 0.0 0 5 10 15 20 25 30 Time (h)

110

control 1st 10min 2nd 10min 3rd 10min (b) 0.35

0.30 )

fed 0.25 VS

- 0.20 cell/g - 0.15

0.10 Yield (g Yield

0.05

0.00

Figure 6. 3 Algal number concentration during growth on different supernatant generated from subsequent sonication, (b) Algae yield from VSfed.

6.3.4. SOUR, OUR and pathogen in remaining solids generated from subsequent

sonication-centrifugation treatment

It is important to see if the remaining solids can meet USEPA biosolids regulations for disposal or land application. The SOUR profile of remaining solids generated from subsequent sonication treatments are shown in Figure 6.4a. The

st initial SOUR in raw sludge was 13.5 mg O2/g TS-h. After the 1 10 min of sonication and centrifugal separation, the SOUR was still as high as that of raw sludge, presumably due to the released organics, especially soluble fractions, trapped in

remaining solids. The SOUR rapidly dropped to 7.7 mg O2/g TS-h and 5.8 mg O2/g

TS-h after 2nd and 3rd sonication-centrifugation treatment, respectively. One-way

111

ANOVA analysis (p-value = 0.0017 < 0.05) indicated a significant effect of sonication on SOUR in remaining solids. The reduction in SOUR in remaining solids indicated the release of active biomass (bacteria) into supernatant after ultrasonic pretreatment [92].

It was not easy to achieve Class B in remaining solids by sonication alone

(Figure 6.4a), nontheless, the time for remaining solids to meet USEPA Class B biosolids regulations was 2 d, 3 d and 3 d for solid1, solid2 and solid3, respectively, compared to 6-7 d required for raw RAS digestion. Sonication also showed a significant effect on digestion time (in days) required to achieve Class B biosolids in

SOUR (one-way ANOVA, p-value = 0.0027 < 0.05).

The major cost of aerobic digestion is energy cost for the long-time aeration due to high OUR in sludge [161]. The OUR profile of raw sludge, solid1, solid2 and soild3 are shown in Figure 6.4b. There was a significant decrease in OUR right after each sonication-centrifugation treatment. As sludge digestion is a cell decay process

[144], each OUR profile can be described by a 1st order exponential equation 푂푈푅 =

푡 푂푈푅 푒−푘푑푡. The total oxygen uptake 푇푂푈 = (푂푈푅)푑푡, calculated by integrating 0 ∫0

OUR with time (t) required before reaching Class B Biosolids are 35%, 19%, and 8% for solid1, solid2 and solid3 compared to control (raw RAS), respectively. The significant reduction in TOU indicated substantial aeration reduction during digestion process which can save operational cost.

112

16 (a) 14

12

h) -

10 /g TS /g 2 control 8 solid1 6 solid2 solid3 SOUR (mg O SOUR(mg 4

2

0 0 24 48 72 96 120 144 168 192 Time (h)

160 (b) 140 -0.012x ycontrol = 79.61e R² = 0.9037 120

control h)

- -0.025x ysolid1 = 59.248e

/L 100 solid1 2 R² = 0.8612 solid2 80 -0.023x ysolid2 = 30.366e solid3 R² = 0.8624 60 Expon. (control) -0.018x OUR (mg (mg OUR O y = 14.126e Expon. (solid1) 40 solid3 R² = 0.9653 Expon. (solid2) 20 Expon. (solid3)

0 0 24 48 72 96 120 144 168 192 Time (h)

Figure 6. 4 (a) SOUR profile of remaining solids; (b) OUR profile of remaining solids. 113

Fecal coliforms (FC), as pathogen indicator bacteria (PIB) were measured to evaluate effect of ultrasonic pretreatment on pathogen distribution (Table 6.2). The

FC density in raw RAS was 4.18 million MPN/g TS and higher than 2 million MPN/g

TS as Class B biosolids regulated by EPA. In contrast, both FC density in Sup1+2 and

Solid2 were lower than 2 million MPN/g TS, indicating the Class B pathogen density were met after ultrasonic pretreatment.

Table 6. 2 Fecal coliform density in raw RAS, supernatant1+2 and solid2

Sample raw RAS Sup1+2 Solid2

Fecal coliforms 4.18 1.60 1.98 (million MPN/g TS)

It has been reported that in activated sludge samples, low levels of specific energy (in kJ/L) produced a prevalent disaggregation of flocs releasing single cells in the bulk liquid, while disruption of bacteria was induced only by very high levels of specific energy [154]. Chu et al [45] found that the survival ratios of heterotrophic bacteria and of total coliform decreased with sonication power density and time. In this study, each 10min subsequent sonication could first disperse bacteria from RAS flocs and inactivate cells at higher energy levels. The overall effect of ultrasonic pretreatment can be the redistribution of bacteria in supernatant and solids and 114 inactivation of certain fraction of bacteria including pathogens.

6.3.5. Compare ultrasonic-phagotrophic process with aerobic digestion

Only 27 ± 1 % VS reduction was achieved after 10 days of aerobic digestion of RAS (Figure 6.5), which is much lower than 40% VS reduction digested by O. danica within 1 day (section 6.3.3). The low VS reduction in Akron sludge had been reported in the literature before [144], probably due to the low C: N ratio in raw wastewater and long solids retention time (SRT) in activated sludge process for nitrification as well as reduction in WAS production.

0.30

0.25

0.20 ratio)

0.15

0.10 VS reduction ( reduction VS 0.05

0.00 0 24 48 72 96 120 144 168 192 216 240 264 Time (h)

Figure 6. 5 VS reduction in raw RAS. Error bars from replicate systems.

115

The proposed ultrasonic-phagotrophic process is shown in Figure 6.6.

Sonication and centrifugation/sedimentation will be done to release and collect bacteria from RAS. The collected dispersed bacteria, as supernatant will be fed to O. danica in bioreactor for algal cultivation and VS reduction under non-sterile condition. The O. danica grown from sonication-generated supernatant (after 0.5-1 days) can be collected as solids by centrifugation at low speed (300-500 g, 5-10 min).

The O. danica may be used as feedstock for biofuels or feed for aquaculture with future applicability test. The supernatant can further go through municipal wastewater treatment process to remove nitrogen and other nutrients. The remaining solids may be concentrated by mixing different batches of solid2 (for example, 3X). 3 days digestion process may be needed before used as Class B biosolids, depending on the

SOUR level. The overall ultrasonic-phagotrophic process may only need 2-3 days which is much shorter compared to 6-7 days of traditional aerobic digestion process.

One potential concern can be the interactions between O. danica and protozoa, the phagotrophic competitors. Protozoa and their cysts embed in WAS flocs as bacteria. Protozoan germination, development and outbreak can take place under favorable conditions (e.g. food level as free bacteria concentration increase). Further studies on the factors (e.g. sonication, retention time, pH and etc) controlling the interaction/competition between O. danica and wastewater protozoa in continuous systems are needed.

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Figure 6. 6 Overall ultrasonic-phagotrophic process design.

6.4. Conclusion

In this study, ultrasonic-phagotrophic process was developed for waste sludge conversion and microalgae production. Active biomass including bacteria were released from sludge and collected as sole substrate for phagotrophic algal cultivation.

Two 10-min subsequent sonication can release 80% VS to support robust O. danica growth with a yield of 30% (g/g) and 40% VS reduction which is much higher than

27% in control aerobic digestion. Moreover, the remaining solids required less than

50% digestion time with 80% reduced total oxygen uptake to achieve Class B biosolids. Produced O. danica biomass may be used as biofuel feedstock and/or aquaculture feed, which need further study.

6.5. Acknowledgments

This study was partially funded by Ovivo USA LLC. We thank Mr. Gilbert

Stadler at Akron water reclamation facility (Akron WRF) for assistance in sludge sampling and collecting plant operation information.

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CHAPTER VII

CONVERSION OF WASTEWATER-ORIGINATED WASTE GREASE TO

POLYUNSATURATED FATTY ACIDS-RICH ALGAE WITH PHAGOTROPHIC

CAPABILITY

Summary

Grease balls collected from a municipal wastewater treatment plant were melt- screened and used for cultivation of microalga Ochromonas danica, which could phagocytize droplets and particles as food. After autoclaving, the waste grease WG separated into two (upper and lower) phases. O. danica grew well on both, accumulating 48-79% intracellular lipids. Initial WG contained approximately 50:50 triglycerides and free fatty acids (FFAs); over time almost only FFAs remained in extracellular WG presumably due to lipase. PUFAs, mainly C18:2n6, C18:3n3,

C18:3n6, C20:4n6 and C22:5n6, were synthesized and enriched to up to 67% of intracellular FAs, from the original 15% PUFA content in WG. The study showed feasibility of converting wastewater-originated WG to PUFA-rich O. danica algae culture, possibly as aquaculture/animal feed. WG dispersion is identified as a major processing factor to further improve for optimal WG conversion rate and cell and FA yields.

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Keywords:

Waste grease, wastewater treatment plant, phagotrophic algae, polyunsaturated fatty acids, sustainability.

7.1. Introduction

Waste grease (WG) is generated in large quantities every year. Each person in the United States generates, on average, 6 kg brown grease and 4 kg yellow grease per year, estimated by the National Renewable Energy Laboratory (NREL) of the US

Department of Energy from 30 metropolitan areas in the US [4]. Much of the waste grease ended up in the municipal wastewater treatment plants. Pastore et al. [5] estimated that 1,600–3,800 kg/day WG can be collected in an urban wastewater treatment plant sized for a population of 100,000 and that the WG disposal accounts for up to 10% of the total sludge disposal cost. The removal and use of WG for value- added applications can lower the biological oxygen demand (BOD) loading and thus treatment cost on the wastewater treatment facilities, reduce problems associated with clogging of pipes and pumps, and improve the environmental and economical sustainability of overall waste treatment [4].

WG has been considered as cheap and renewable feedstock for biodiesel production [63]. However, WG contains high contents of moisture and free fatty acids

(FFA) and is therefore difficult to use in the common base-catalyzed commercial 119 biodiesel production processes [4, 61]. Moreover, the cold-flow properties of the WG derived biodiesel, due to high saturation level, are generally unacceptably worse than the traditional diesel [64]. WG has also been considered for use in anaerobic co- digestion with waste activated sludge [63]. Biogas produced from anaerobic digestion can offset the costs at wastewater treatment facilities and WG co-digestion can increase the biogas production. However, the biogas utilization for onsite process heat and power production at wastewater treatment plants in the US is less than 10%; most biogas is flared [38, 65, 66]. There is a critical need for developing other possible value-added uses of WG, particularly for small- and medium-sized wastewater treatment plants where the high capital costs associated with biodiesel production or anaerobic digestion are economically infeasible.

In this study we investigated the feasibility of converting WG to algal biomass containing high content of polyunsaturated fatty acids (PUFAs). Algal PUFA production has been reported for both photosynthetic processes using Isochrysis galbana [182], Nannochloropsis oculata, Pavlova lutheri, and Isochrysis sp. [183],

Nannochloropsis limnetica [184], Phaeodactylum tricornutum [185] and Fistulifera solaris [186] and organotrophic processes using Chlorella saccharophila [187],

Nitzschia laevis [188], Crypthecodinium cohnii [189] and Porphyridium cruentum

[190]. Organotrophic processes support much faster rates of algal growth and algal lipids including PUFA production. For example, the growth rate of Chlorella zofingiensis on glucose was 227% higher than the rate under photoautotrophic growth 120

[191] and Chlorella vulgaris growing on glucose produced lipids at rates of 35 (in dark) to 54 (with light) mg/L-d but only at 4 mg/L-d under photoautotrophic growth

[192]. For WG conversion, organotrophic algae are obviously more suited. However, the PUFA production by organotrophic algae has so far used dissolved organic substrates such as glucose [187, 189], acetate [193] and glycerol [194], relying on the osmotrophic uptake of soluble nutrients through the algal cell membrane. WG is practically insoluble in aqueous media and WG from wastewater treatment plants is generally semisolid at room temperature. Using microalgae capable of only osmotrophic and/or photosynthetic growth for WG conversion does not appear to be the most effective approach. In this study the microalga selected for evaluation is

Ochromonas danica, which is capable of phagotrophic ingestion of droplets and particles including bacteria [9, 167], in addition to photosynthetic and osmo- organotrophic metabolism [8]. The growth rate of O. danica SAG 933-7 preying on bacterial cells (Pseudomonas fluorescens strain Pf 0-1) was reported at ~0.4 d-1, 4-fold faster than its photoautotrophic growth rate (~0.1 d-1) under otherwise similar conditions [69]. Recently, 30 g/L high-cell-density osmo-organotrophic cultivation of

O. danica on waste ketchup has been achieved [10]. O. danica can synthesize lipids of various fatty acids such as myristic (C14:0), palmitic (C16:0), stearic (C18:0), oleic

(C18:1n9), linoleic (C18:2n6), α-linolenic (C18:3n3), γ-linolenic (C18:3n6), eicosatrienoic (C20:3n3) and arachidonic (C20:4n6) acids [138-141]. PUFAs such as

C18:3n6, C20:4n6, C20:5n3 and C22:6n3 have been reported to offer significant 121 health benefits and have been included in infant formulae, nutritional supplements and aquaculture feed [142]. Owing to the wastewater-related sources, it is unlikely and not recommended to use WG-derived algal PUFAs for direct human consumption.

Nonetheless, it is feasible to include algal PUFAs or the algae biomass rich in PUFA content in aquaculture [195, 196] or other animal feed [197].

The main questions to answer in this work were whether O. danica can grow effectively on the semisolid WG from wastewater treatment plant and synthesize and accumulate PUFAs-rich intracellular lipids. Development of simple procedures for

WG preparation to support the O. danica cultivation was an important early-stage objective. Properties of WG-based cell growth and lipid conversion and accumulation were investigated. Fatty acid composition, including PUFAs, in the algal lipids produced was analyzed. There are no previous reports on algae cultivation or PUFA production using the semisolid waste grease from wastewater treatment plants as substrate.

7.2. Materials and methods

7.2.1. Culture and media

The phagotrophic algae used was Ochromonas danica (ATCC® 30004TM,

ATCC, Manassas, VA, USA). The seed culture was maintained with regular subculturing at room temperature in 1-L Erlenmeyer flask with 450-500 mL culture 122 under magnetic stirring. The medium composition was as follows: 8 g/L glucose

(Fisher Scientific, Fairlawn, NJ, USA), 0.45 g/L yeast extract (Fisher Scientific), 0.45

g/L tryptone (Fisher Scientific), 0.5 g/L NH4Cl (EMD Chemicals, Gibbstown, NJ,

USA), 0.4 g/L MgCO3 (Sigma-Aldrich, St. Louis, MO, USA), 0.3 g/L KH2PO4

(Sigma-Aldrich), 0.2 g/L nitrilotriacetic acid (Sigma-Aldrich), 0.1 g/L MgSO47H2O

(Sigma-Aldrich), 0.05 g/L CaCO3 (Sigma-Aldrich), 4.4 mg/L Na2EDTA (disodium

ethylenediaminetetraacetate) (Sigma-Aldrich), 3.15 mg/L FeCl36H2O (Fisher

Scientific), 0.97 mg/L H3BO3 (Sigma-Aldrich), 0.25 mg/L thiamine hydrochloride

(Sigma-Aldrich), 0.18 mg/L MnCl24H2O (Sigma-Aldrich), 0.02 mg/L ZnSO47H2O

(Sigma-Aldrich), 0.01 mg/L CoCl26H2O (Sigma-Aldrich), 6 μg/L Na2MoO42H2O

(Sigma-Aldrich), and 2.5 μg/L biotin (Sigma-Aldrich) [10].

7.2.2. Waste grease collection and preparation

Waste grease (WG) samples were taken from the nearby Akron Water

Reclamation Facility (WRF) (Akron, Ohio). WG, floating on the water surface after primary treatment, were collected as grease balls with diameters of 3-15 cm. The collected WG balls contained leaves, plastics and other non-fat matters that should be removed. Raw WG balls were melt at 80-90 °C and filtered through a screen with 1 mm pore size. The filtered WG was then autoclaved at 120 °C for 20 min. The autoclaved WG formed two distinct phases, termed as upper waste grease (UWG) and

123 lower waste grease (LWG) hereafter. Both were analyzed for compositions, i.e., contents of moisture, “oil”, “soap”, and impurities. Greases typically contain both glycerides and free fatty acids (FFAs). FFAs are generated from the food processing

(e.g., frying) or microbial degradation (e.g., lipase-catalyzed hydrolysis). Mineral ions such as calcium are commonly present to bind with FFAs to form soap [5].

7.2.3. O. danica cultivation on WG

All experiments were done at room temperature, with 10 mL seed culture inoculated into 225 mL autoclaved medium in 500 mL Erlenmeyer flasks under magnetic stirring (250-300 rpm). Experiments were first done to evaluate the pH effect on O. danica growth in (L)WG-based medium. Media were prepared with pH adjusted to 4.5 and 6.5, respectively, prior to autoclaving. pH increased during autoclaving and the actual initial culture pH was 5.8 and 6.9. The lower pH system

was later (29 h) added with 5 mL filter-sterilized 0.2 M NaH2PO4 to drop the pH to

5.3, in an attempt to widen the pH difference for the study purpose. Medium used in the pH effect experiments was the same as that given above for seed culture, except that 1 mL LWG was used as the sole organic C source in place of glucose, yeast extract and tryptone in the seed culture medium. Later experiments were all done with

initial pH of 7.0  0.1 and with additional buffer capacity (1.0 g/L NaH2PO4 and 0.06 g/L Na2HPO4) in the media (since the growth was found clearly better at higher pH,

124 as described more in the Results and Discussion.) A set of experiments was done in such media but with different amounts (0.5-5 mL) of UWG, to examine the effects of

WG amounts on O. danica growth and WG dispersion/agglomeration situation.

Another set of experiments was done with media containing 3 mL LWG or UWG as organic C source to compare the substrate effects of these two phases formed in

autoclaved WG. The media used in these experiments had 1.0 g/L NaH2PO4 and 0.06

g/L Na2HPO4 as the additional pH buffer but only 50% concentrations of all other components. The larger WG amount and halved N-source concentration gave larger

C:N ratios in the media, enabling evaluation of their effects on intracellular lipid accumulation. Medium with such a higher C:N ratio was also used in the final experiments for analysis of production and compositions of intracellular fatty acids, including PUFAs, in O. danica cells grown on WG.

7.2.4. Analytical methods

7.2.4.1.Waste grease composition

Moisture content was analyzed by weighing WG samples, each about 1 g, before and after drying overnight at 103 °C. Oil content was analyzed by mixing 15 mL hexane with 2 mL of melt WG and vortexing the mixture for 2 h. After centrifugation at 1,300 g for 5 min (Sorvall® RC 5C Plus, Thermo Scientific,

Asheville, NC, USA), the hexane phase was collected, air dried and weighed to 125 determine the amount of hexane extracted materials, hereafter referred to as the “oil” fraction in sample. The remaining solids were added with 5 drops of 12 M HCl

(Fisher Scientific) and again hexane-extracted. This group of hexane-extracted materials after acidification was referred to as the “soap” fraction. The oil fraction was brownish and melt at 45 °C while the soap fraction was white and did not melt at

45 °C. The impurities amount was calculated by subtracting the weights of oil, soap and moisture fractions from the total weight of WG sample.

7.2.4.2.Cell concentration

Algae cell dry weight concentration (CDW) was measured by centrifuging 10 mL sample at 500 g for 10 min, washing the cell pellet once with de-ionized water, drying the washed cells at 103 °C overnight, and weighing for the cell dry weight.

Algae cell cumber concentration was also generally measured for tracking cell growth. Sample was added with an equal volume of 2% glutaraldehyde to fix the cells and stop their motility. The cell number was then counted using a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA, USA) under a microscope.

Microscopic pictures were obtained using a DP71 digital camera (Olympus America,

Center Valley, PA, USA) coupled to the microscope.

7.2.4.3.Intracellular lipid content 126

Algal lipid content was determined using the typical chloroform-methanol extraction method [163]. Briefly, the cell pellet collected by centrifuging a 20 mL culture sample as above was added with 7 mL HPLC-grade chloroform (Fisher

Scientific)-methanol (Fisher Scientific) solution (2:1, v/v), 2 drops HCl (12 M) and

0.1 mL de-ionized water. The mixture was vortexed for 5 min and then centrifuged at

2,000 g for 10 min to separate cell debris from the solvent phase containing dissolved lipids. The solvent phase collected was dried under filtered air for 3 h and then weighed to determine the intracellular lipid content, = (lipid weight)/(cell dry weight).

7.2.4.4.Thin-layer chromatography

Lipid samples (5 or 10 μL per spot) were applied on TLC silica gel plates (200

× 200 mm, pH 5, MF254, alumina back) (Agela Technologies, PA, USA) and developed in a glass chamber in three steps with in-between plate drying. Diethyl ether (100%, Fisher Scientific) was the eluent used in the first 2 repeated steps, allowed to rise for 3 cm from the original spot (which was placed at 2 cm above the edge of TLC plate). The eluent used in the third step was a hexane-diethyl ether-acetic acid mixture (70:30:1, by volume), allowed to develop to the full height of 15 cm from the original spot. Lipids were visualized as fluorescent spots in a UV box after spraying the plate with a solution of 5% primuline (Tokyo Chemical Industry, Tokyo,

Japan) in acetone (Sigma-Aldrich).

127

7.2.4.5.Fatty acid composition analysis

Detailed fatty acids (FAs) analysis was done after converting FAs in the algal lipids to fatty acid methyl esters (FAMEs) according to the National Renewable

Energy Laboratory (NREL) standard method (TP-5100-60958): determination of total lipids as fatty acid methyl esters by in situ transesterification [198]. The FAME sample analysis was carried out using a Shimadzu GC-17A (version 3) gas chromatograph equipped with a flame ionization detector (GC-FID) (Shimadzu,

Australia). The column used was a FAMEWAX capillary column (Restek, Bellefonte,

PA, USA) with dimensions: 30 m length × 0.25 mm inner diameter and 0.25 µm film thickness. Helium was used as carrier gas and the sample injection volume was 2 µL.

Samples were analyzed with a constant injector temperature of 230 °C running with split ratio of 10:1. The GC oven temperature was programmed at an initial temperature of 130 °C, held for 1 min, then ramped to 220 °C at 4 °C/min and held for 20 min. The FID was held at 230 °C for the duration of analysis. FAME identification was done using a 37-component FAMEs standard mixture (C4 - C24)

(18919-1AMP, Supelco) with methyl tridecanoate (C13:0 FAME) as the internal standard. The minor compounds having weight percentage of less than 1% were not reported and were neglected in estimating the FAME yields (%). The FA and PUFA quantities reported in this work are all given in terms of quantities of their

128 corresponding FAMEs formed by this NREL standard method used.

7.3. Results

7.3.1. Waste grease compositions

UWG and LWG formed after autoclaving of melt-screened WG were found to differ significantly in moisture contents: (3.3 ± 3.3)% in UWG and (17.2 ± 0.9)% in

LWG; however, on the dry weight basis, they were very similar in all other fractions; oil – (76.8 ± 3.2)% in UWG and (77.8 ± 3.2)% in LWG, soap – (17.7 ± 2.9)% in

UWG and (15.6 ± 0.2)% in LWG, and impurities – (5.4 ± 0.3)% in UWG and (6.6 ±

3.5)% in LWG. Even their fatty acid compositions, shown in Table 7.1, did not differ significantly. It appears that the two phases differed only in minor surface-active components, which created a more stable water-in-oil emulsion to retain more moisture in LWG. The major fatty acids in WG were found to be palmitic (C16:0), oleic (C18:1) and linoleic (C18:2) acids, making up about 90% of total FAs. The high palmitic acid content (~40%) can be responsible for their semisolid state. Converting these common FAs to PUFAs can significantly improve the value of WG.

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Table 7. 1 Fatty acid compositions of UWG and LWG (in weight %)

Fatty acid UWG (%) LWG (%)

C14:0 2.6 ± 0.4 2.6 ± 0.4

C16:0 39.7 ± 0.1 39.8 ± 0.2

C18:0 2.8 ± 1.7 2.3 ± 1.3

C18:1n9 37.6 ± 1.3 38.2 ± 0.7

C18:2n6 13.9 ± 0.1 13.8 ± 0.4

C18:3n3 1.3 ± 0.1 1.1 ± 0.1

unidentified 2.2 ± 0.2 2.2 ± 0.3

7.3.2. O. danica growth on WG

7.3.2.1.pH effect

Profiles of pH changes and O. danica growth on 0.42% LWG (v/v), containing

~2.9 g/L oil and soap fractions, with different initial pH (5.8 and 6.9) are shown in

Fig. 7.1. The pH decreased as cells grew, mainly due to consumption of ammonia as

N source. Cell growth stopped after 51 and 71 h, respectively, corresponding to the onsets of pH increase. Maximum CDW reached was comparable in the two systems, i.e., 0.55  0.07 g/L. Growth was not limited by the available N source because, as shown later, other systems with same or half N-source concentrations could reach much higher CDW. Growth was also not stopped by oxygen transfer limitation because other systems, under same agitation and aeration conditions, could reach higher CDW. Growth was therefore probably limited by C-source availability. 130

Accordingly, the algal cell yield from combined oil and soap in WG (not all consumed) was estimated at 18.7% (w/w). This was lower than the O. danica cell yield, up to about 40%, from waste ketchup (sugars-rich) as substrate [10]. While this might be partially attributed to presence of non-metabolizable materials in WG oil and soap, a more serious limitation was in the rate of WG availability for O. danica consumption under the rather gentle magnetic stirring (see Discussion). This growth impediment by WG availability rate supports the observed faster growth at higher pH in Fig. 7.1. The majority of more water soluble and surface-active WG components are likely FFAs. At higher pH, more of them were anionic with higher solubility and surface-activity, thus providing more dissolved substrate and finer WG particles for O. danica. Later experiments were therefore made with higher initial pH (near neutrality).

131

Figure 7. 1 Effect of pH on growth of O. danica in medium with 0.42% (v/v) LWG as sole organic C source

7.3.2.2.UWG vs. LWG as substrate

At the same initial pH (7.0  0.1), O. danica grew faster with LWG than UWG as substrate (Fig. 7.2). LWG, with much higher water content, dispersed better than

UWG in the medium. Plausibly, the better dispersion facilitated formation of smaller

132 ingestible WG particles/droplets to support faster cell growth. With LWG, cell number grew to (4.2  0.6)  1010 cells/L at 144 h before declining to (2.6  0.8)  1010 cells/L at 168 h; the CDW profile was almost parallel. pH increased between 120 and 168 h

(not measured at 144 h), consistent with the dominant endogenous metabolism leading to declining cell concentrations. With UWG, declines of cell number and

CDW were not yet apparent till the end of experiment. Cell number reached only 2.6-

2.9  1010 cells/L at 144 – 168 h, much lower than the maximum concentration in the

LWG system. CDW was also lower in the UWG system, except for the final point at

168 h when it sharply increased to 2.98  0.05 g/L (from 0.70  0.03 g/L at 120 h).

This cell weight increase was found to be partially caused by accumulation of intracellular lipids: the measured lipid content of final culture (at 168 h) collected from the UWG system was very high, 78.8%  3.2%, much higher than the 48.0% 

4.4% measured for the LWG system (Fig. 7.2). The high lipid accumulation also gave a much higher individual-cell dry weight 1  10-9 g/cell for the final culture harvested from the UWG system, compared to (3-7)  10-10 g/cell measured for all other samples from both UWG and LWG systems (Fig. 7.2).

Initial media for the two systems had the same N concentration, 0.055 g/L.

They were also added with the same volume (3 mL) of UWG and LWG. However, due to very different moisture contents, UWG provided 18% more fat (oil and soap portions) than LWG: 10.48 versus 8.60 g/L in media, corresponding to fat-to-N ratios of 190 and 156, respectively. Presumably, growth in the UWG system became N- 133 limited near the end (after 144 h as the cell number maintained similar) and the stationary-phase cells ingested the excess fat and stored lipids at the very high content

(78.8%) as food reserve (see Fig. 7.3 for a microscopic picture of the lipid-laden cells). The final cell yield was 28.4%, higher than the 18.7  2.3% yield for the C- limited systems described in the previous section (pH effect). Growth in the LWG system was also slightly N-limited. At 120 h, cell yield in this system was already

19.4%. With a further CDW increase during 120-144 h, as expected from the large increase of cell number, the maximum cell yield (at 144 h) would be larger than the

C-limited 18.7  2.3% yield. Under this operating condition, the balanced fat-to-N ratio for O. danica growth on WG was near 156. More importantly, the results showed fat-to-N ratio as a critical design factor that can significantly affect the lipid content in processes converting WG to algal lipids.

134

7 UWG

pH 6

5 0 24 48 72 96 120 144 168 192 3.5 Time (h) 5E+10 UWG-CN LWG-CN 3 4E+10 UWG-CDW LWG-CDW 2.5

3E+10 2 1.5 2E+10 1 (g/L) CDW 1E+10 Cell number (#/L) number Cell 0.5 0 0 12 0 24 48 72 96 120 144 168 192 Time (h) Intracellular 10 UWG LWG lipid: 78.8% 

cells) 8 11 6 Intracellular 4 lipid: 48.0% 

2 0 24 48 72 96 120 144 168 192

CDW/CN (g/10 CDW/CN Time (h)

Figure 7. 2 Comparison of O. danica growth properties on 1.3% (v/v) UWG and LWG.

135

Figure 7. 3 O. danica cells with high lipid content collected after growing on UWG for 168 h (scale bar: 20 μm)

7.3.2.3.Effect of UWG concentration

Growth on UWG was slower than on LWG (Fig. 7.2). Experiments were done to see if the situation could be improved by providing more UWG to the O. danica culture. Growth profiles on different volume % UWG are shown in Fig. 7.4. Cell concentration measurements in later stages of cultivation (after ~ 3 days) scattered substantially because WG agglomeration made sampling more challenging and less homogeneous. Cell growth rates did not differ substantially in the wide range of

UWG amount compared, 0.2-2.0%. With higher percentages added, UWG appeared to agglomerate earlier into large particles. This might have not allowed the available rate of consumable UWG to increase with higher UWG additions to support faster cell growth. Improving WG dispersion is critically important in future studies and process design.

136

3.E+10

UWG vol% 2.E+10 0.2% 0.4% 1.2%

Cell number (#/L) numberCell 1.E+10 2.0%

0.E+00 0 50 100 150 200 250 Time (h)

Figure 7. 4 Effect of waste grease (UWG) concentration on O. danica growth

7.3.3. Algal lipid analysis

7.3.3.1.Thin-layer chromatography (TLC)

A representative set of TLC results are shown in Fig. 7.5, with oleic acid and vegetable oil included to indicate the positions of free fatty acids and triglycerides.

The results showed that UWG consisted mainly of FFAs (> 50%) and triglycerides, consistent with the literature reports of high FFA content in WG [61]. The remaining extracellular WG, collected as agglomerated particles after 135-h cultivation, was found to contain practically only FFAs. The last TLC lane in Fig. 7.5 is for the intracellular O. danica lipids. It showed that cells harvested (at 135 h) had roughly

137 equal amounts of triglycerides and FFAs, in addition to other polar materials present as non-fluorescent black spots below the FFAs spot. The intracellular lipid content of this sample was 54.4 ± 14.3%.

Figure 7. 5 TLC results for lipids after primuline staining: (left to right) oleic acid and vegetable oil included to indicate respective positions for free fatty acids (FFAs) and triglycerides (TGs); center lane for UWG; and right 2 lanes for remaining extracellualr WG and intracellualr lipid extracts, respectively, collected after 135-h cultivation. The algal-lipid lane showed also non-fluorescent black spots (indicated by arrows for 2 clearest ones).

138

7.4.3.2.Fatty acid analysis and PUFA production

Intracellular fatty acid compositions were examined in a cultivation experiment with 1.3% WG (1:1 UWG and LWG). The profiles of cell growth (CDW, g/L), total intracellular FA production (mg/L), and total PUFA production (mg/L) are shown in Fig. 7.6A and the profiles of intracellular FA content (g FAs per g dry cell biomass), PUFA content (g PUFAs per g dry cell biomass), and the % PUFA in total

FAs are shown in Fig. 7.6B. Along the cell growth, not only more FA and PUFA

(mg/L) were produced but the FA and PUFA contents inside cells (g/g dry cells) were higher. The FA content appeared to increase only after cell growth stopped or slowed down, from 0.17 to 0.32 g/g during 96-144 h; on the other hand, PUFA content accumulated continuously from the initial 0.06 g/g in glucose-grown seed culture to

0.12 g/g at 144 h. Hypothetically, the WG agglomeration in stir-flasks limited the ingestion rate of WG, which was prioritized for cell mass synthesis during active growth and caused the relatively constant FA content (about 0.15 g/g) during 0-96 h; the cells could store more lipids (FAs) as food reserve only after the demand to support active growth eased or ceased. PUFAs, on the other hand, served different or additional purposes beyond food reserve and were separately regulated, giving the profile of continuous increase. Corresponding to these different profiles, the PUFA percentage in total FAs had an increase-then-decrease trend, varying in the range of

37-67%. Note that the fed WG contained only 15.2% PUFAs. O. danica growing on

WG could indeed significantly enrich the PUFA content in intracellular lipids. 139

Detailed intracellular FA compositions are summarized in Fig. 7.7, showing the changes of individual FAs with cultivation time. The percentages of FAs in the

WG used as feed (Table 7.1) are labeled in the figure. For all these FAs, the provision with WG caused their intracellular contents to be higher at the end of cultivation (144 h) than at 0 h. The increases were particularly larger for the three major FAs in WG: palmitic acid (C16:0, 39.7% in WG), oleic acid (C18:1n9, 37.6%) and linoleic acid

(C18:2n6, 13.9%). The trends of their transient intracellular contents were however different: contents of myristic acid (C14:0), stearic acid (C18:0) and oleic acid clearly decreased during active cell growth but increased sharply when growth slowed or stopped; contents of palmitic and linoleic acids continued to increase during the cultivation; and α-linolenic acid (C18:3n3), while undetectable in the glucose-grown seed culture, increased and maintained around 0.6% throughout 48-144 h. The different trends presumably reflected their net rates of ingestion and consumption/conversion at different culture stages. Different transient trends were also found for FAs that were not originally present in WG. Contents of the longest- chain FAs including erucic acid (C22:1n9), docosatetraenoic acid (C22:4n6) and docosapentaenoic acid (C22:5n6) generally increased during the cultivation. The main

C20 PUFA in O. danica lipids, i.e., arachidonic acid (C20:4n6), appeared to increase slightly during active cell growth on WG but decreased in the later stage. The largest

PUFA content increase was clearly from linoleic acid synthesis/accumulation.

Metabolic flux models can be developed in the future to better understand these 140 dynamic FA profiles.

CDW (g/L) Algal FA (mg/L) PUFA (mg/L) 0.5 (A) 160 0.4 120 0.3 80

0.2 CDW (g/L) CDW 0.1 40

0.0 0

0 20 40 60 80 100 120 140 (mg/L) PUFA or FA Intracellular Time (h)

FA content (g/g) PUFA content (g/g) % PUFA in FA 0.4 80 (B)

0.3 60

0.2 40

0.1 20

0.0 0 FA or PUFA CDW) (g/g content PUFA or FA 0 20 40 60 80 100 120 140 (%) FA intracellular in PUFA % Time (h)

Figure 7. 6 Changes of O. danica culture properties during WG-supported stir-flask cultivation: (A) time profiles of cell growth (CDW, g/L), total intracellular FA production (mg/L), and total PUFA production (mg/L); and (B) time profiles of intracellular FA content (g FAs per g dry cell biomass), PUFA content (g PUFAs per g dry cell biomass), and the % PUFA in total FAs.

141

1.3)%

12%

± 0 h 48 h

0.1)%

10% ± WG: (37.6 WG:

8% (13.9 WG:

0.1)%

6%

±

0.4)%

1.7)%

±

4% ±

FA content content CDW) (g/g FA 0.1)%

WG: (39.7 WG:

± WG: (2.6 WG:

2% (2.8 WG: WG: (1.3 WG: 0%

Fatty acids

Figure 7. 7 Changes of FA composition in O. danica cells with cultivation time, where the seed culture at 0 h was prepared in a glucose-based medium while the cultivation was done in a medium with 1.3% (v/v) WG. The percentages of FAs present in the WG are labeled.

7.5. Discussion

While the prepared WG would form two distinct phases after autoclaving, the composition analysis showed they did not differ noticeably in major components other than the much higher moisture content (17.2 ± 0.9)% in LWG, compared to the

(3.3 ± 3.3)% in UWG. Consisting mainly of FFAs (> 50% on dry basis) and triglycerides (by TLC in Fig. 7.5), both were confirmed to support growth of

142 phagotrophic O. danica. The growth was faster at pH nearer neutrality than at pH <

5.8 (Fig. 7.1). However, cell growth stopped while substantial amounts of WG were still present, giving relatively low cell yields on the basis of combined amount of oil and soap fractions in the fed (not consumed) WG, which ranged from 19% (w/w) to

28% depending on the fat-to-N ratios in the feed. The suboptimal dispersion of WG offered by magnetic stir-bars used in this study is believed to be primarily responsible for the lower cell yield. O. danica could grow by consuming dissolved and ingestible

(micron and submicron sized) oil droplets/particles [9]. To support cell growth, the required rate of substrate availability = 휇푋/푌푋/푆, where 휇 is the specific growth rate

-1 (h ), 푋 is cell concentration (e.g., g/L) and 푌푋/푆 is cell yield (e.g., g cells produced per g WG consumed). So, the availability rate of WG in ingestible forms/particle- sizes needed to increase with time to match the increasing cell concentration.

However, the more hydrophilic surface-active components in WG would decrease with time as they dissolved and got consumed, causing the observation that WG was better dispersed initially but gradually agglomerated into larger, more wax-like particles (over 1-3 days depending on the WG amount). With time both the more soluble consumable components and small, ingestible particles became less available to match the increasing substrate consumption rate from increasing cell concentration; further cell growth could no longer be supported even when a substantial amount of

WG remained. Improving and sustaining the WG availability rate for O. danica’s phagotrophic consumption is identified as an important objective for future study. 143

The fat-to-N ratio in feed was found to be another important factor that affected the lipid content of O. danica grown on WG. Under the operating condition used in this study, the balanced fat-to-N ratio was estimated to be near 156 (w/w). The O. danica culture grown at this fat-to-N ratio (156) had an intracellular lipid content of about

48% while the stationary-phase culture grown in an N-limited medium (fat-to-N ratio

= 190) accumulated lipids to a very high content of about 79% (Fig. 7.2). The intracellular algal lipids contained both triglycerides and FFAs, in roughly equal amounts for a culture sample with 54% intracellular lipids (Fig. 7.5), and some polar materials that did not fluoresce on the TLC plate sprayed with primuline dye. On the other hand, the unconsumed WG remaining in the broth was shown to be practically all FFAs. Because triglycerides and FFAs would form one single phase, there were no separate triglyceride particles in the medium for O. danica to preferentially phagocytize and leave behind only FFA particles. Also, because FFAs generally had slightly higher solubility than triglycerides at near neutral pH, it is unlikely that triglycerides preferentially dissolved (over FFAs) for uptake through the cell membrane. The finding of only FFAs in the remaining WG was probably the result of triglyceride hydrolysis by extracellular lipase, at rates faster than the WG consumption by O. danica. The capability of an Ochromonas species to produce lipase was reported [8].

More importantly, the intracellular lipids in WG-supported O. danica culture were found to be rich in PUFAs, constituting up to 67% of all FAs (Fig. 7.6), with 144

C18:2n6, C20:4n6, C22:5n6, C18:3n3 and C18:3n6 being the main PUFAs (Fig. 7.7).

O. danica was reported as rich in arachidonic acid (C20:4n6) [138, 140] which is the precursor of a family of biologically and clinically important eicosanoids including prostaglandins, thromboxanes, and leukotrienes [199]. O. danica is also rather unique in its capability of synthesizing both vegetal α-linolenic acid (C18:3n3) and animal γ- linolenic (C18:3n6) [139], as confirmed in this study (Fig. 7.7).

The FA content (g FAs/g CDW) and % PUFAs in total FAs observed in this study for O. danica grown on WG are compared in Table 7.2 with those values reported for several FA/PUFA-rich microalgae cultivated in phototrophic and/or osmo-organotrophic (with soluble organic sources) processes for different intended applications. While the values varied with culture conditions and stages, both FA content and PUFA percentage in the WG-supported O. danica culture compared favorably over the others. The O. danica culture could have the highest PUFA content to support further development for value-added uses, e.g., as aquaculture and other animal feed.

145

Table 7. 2 Comparison of O. danica with other microalgae

% Cultivation Total FAs Species PUFAs Ref. (Purpose) (mg/g CDW) in FAs

Isochrysis Phototrophic 103-201 34-62 [182] galbana (Aquaculture)

Phaeodactylum Phototrophic 49-146 31-45 [200] tricornutum (Aquaculture)

Chlorella Osmo-organotrophic 90-110 18-56 [201] zofingiensis (Wastewater/biodiesel)

Crypthecodinium Osmo-organotrophic 76-152 41-52 [202] cohnii (DHA production)

Ochromonas Phagotrophic This 130-320 37-67 danica (WG/aquaculture?) study

In conclusion, this study demonstrated feasibility of converting wastewater- originated WG to PUFA-rich algae culture by using a phagotrophic alga O. danica that could phagocytize droplets and particles as food, in addition to its photosynthetic and osmo-organotrophic capabilities. The alga grew well on easily prepared WG

(15% PUFAs) and accumulated 48-79% intracellular lipids, including up to 67%

PUFAs in total FAs. Main PUFAs in cells were (in descending %) C18:2n6, C20:4n6,

C22:5n6, C18:3n3 and C18:3n6 (similar % of the last two). WG conversion rate and yield would depend on WG dispersion into ingestible sizes, an important factor for further process design. 146

7.6. Acknowledgment

The authors thank Mr. Gilbert Stadler of the Akron Water Reclamation Facility

(Akron, OH) for assistance in grease ball sample collection. The authors also acknowledge the assistance of Dr. Nicholas Callow and Mr. Jacob Kohl in fatty acid analysis.

147

CHAPTER VIII

ALGAE PRODUCTION FROM INDUSTRIAL FOOD WASTEWATERS

Summary

Making use of the unique phagotrophic capability of the microalga

Ochromonas danica, a two-stage (bacteria-algal) continuous process for algal production from unsterilized synthetic glucose-based wastewater under open conditions has been demonstrated in our previous work. However, real wastewaters from different sources can have widely varying compositions and characteristics and these effects on the growth of O. danica or the applicability of the new algal production process have not been investigated. In this chapter, two types of industrial food wastewaters, i.e., wastewaters from cheese- and apple juice-making plants, were studied. Both represented high-strength industrial food wastewaters containing high concentrations of biodegradable organics that may support significant algae production. But they had very different and potentially challenging properties to the process employing phagotrophic algae. In this study these wastewaters were tested for toxicity to O. danica, analyzed for compositions, and identified for major issues to address for the algal production. The growth kinetics and yield of O. danica in these food wastewaters were determined. 148

8.1. Introduction

Huge quantities of water are used in the food industry for food manufacturing and cleaning process, resulting in the production of large volumes of wastewater [123].

Food wastewaters, due to their high organic contents, generally must be treated on-site or pretreated before discharge to alleviate the burden of municipal wastewater treatment, avoid eutrophication and minimize odor generation [106, 111, 118, 120, 122]. The management of food wastewater becomes a major problem as the food industry is required to comply to increasingly more stringent discharge regulations. Research into developing sustainable wastewater treatment is further promoted to recover nutrients and energy from waste streams [46] for higher energy efficiency and cleaner production in a more sustainable society [26]. In this study we examined two high-strength industrial food wastewaters for their use to support production of algae biomass and lipids in a new wastewater treatment process employing the phagotrophic microalgae

Ochromonas danica.

Cheese is an important agricultural product. In cheese-making, roughly 4 L of wastewater are generated for processing 1 L of milk. The cheese wastewater had high biological oxygen demand (BOD), salinity, and composition variation [106]. The organic matters in cheese wastewater (mainly lactose and proteins) have been studied for valorization through anaerobic digestion to produce biogas [112], lactose hydrolysis to produce glucose and galactose [113], fermentation to generate hydrogen [114], and

149 microbial fuel cells to produce bioelectricity [115]. More recently, Prazeres et al. [116] treated cheese wastewater by NaOH precipitation and studied the use of the treated wastewater for tomato production and the precipitation-collected sludge as the fertilizer.

Fruit juice wastewater is mainly produced from various processes such as soaking, washing, rinsing, fluming, blanching, scalding, heating, pasteurizing, chilling, cooling and general cleaning purposes [119]. Low pH values, nutrients imbalance, and high fluctuations in strength and composition are considered as major problems in treating fruit juice wastewaters [121]. Like the cheese wastewater, fruit juice wastewaters have also be studied for energy recovery in the form of biogas [124], hydrogen [125], and electricity [126]. In addition, Noronha et al. [127] studied the reuse of fruit juice wastewater treated by a membrane ultrafiltration-UV disinfection hybrid process for cooling, boiling and washing. Blöcher et al. [128] further upgraded the membrane treated juice wastewater by nanofiltration (low pressure reverse osmosis) to produce water of drinking quality.

Microalgae cultivation has been studied for removing nitrogen, phosphorus, toxic metals and emerging organic contaminants from wastewater [203-205]. Algal biomass collected can be used for the production of biodiesel [206], biogas [207], bio- crude oil [208], biochar [209] and acetone, butanol, and ethanol [210]. Compared to photo-autotrophic algae, heterotrophic algae cultivation has the advantages of higher biomass and lipid productivity, simpler bio-reactor design and the potential to

150 simultaneously remove organic carbon and other nitrogen and phosphorus compounds from wastewater [211]. Using wastewater as substrate to grow algae is a promising way to produce algal biomass with low cost [212]. The high BOD food wastewater is particularly suitable for algae production due to their high biodegradability and expected high algae concentration which can reduce the downstream costs in algae product collection. Algae processes have been investigated for algal biomass production as well as treatment of wastewater from municipal [213] and industrial sources such as dairy production [214], meat-processing [212], poultry litter leachate

[215], olive mill [216], aquaculture [217], soy bean processing [218] and piggery wastewaters [219]. However, in open systems the algae can be easily displaced by bacteria or other microorganisms that are faster growing and/or more adapted to the specific wastewater; on the other hand, wastewater sterilization inevitably increases the cost and diminishes the feasibility of algae production [203].

Microalgae Ochromonas is capable of three metabolic modes: phototrophic, heterotrophic on dissolved organic compounds, and phagotrophic [8]. Phagotrophy can act as a means of incorporating nitrogen, phosphorus or essential vitamins [68]. The versatile nutrition modes enable Ochromonas to serve for multiple purposes of wastewater and activated sludge treatments and algae production. Ochromonas danica can grow organo-osmotrophically in chemostat at dilution rates ranging between 0.03 and 0.10 h-1 at temperatures ranging between 15 and 28 °C [70]. O. danica can grow on sugar-rich ketchup with a 10 h doubling time, 40% cell yield and 18% intracellular 151 lipid yield, respectively [10]. O. danica can engulf waste cooking oil droplets, grow rapidly and reduce acid value of waste cooking oil, which becomes a better feedstock for biodiesel production [9]. O. danica can grow on Escherichia coli, a common bacterium, with a doubling time of 7~20 h average yield of about 25% (w/w) and a lipid content of 27% ± 8%. Growth rate depends on the E. coli-to-O. danica ratios [220]. O. danica can quickly consume organics released from waste activated sludge flocs. 41% and 20% of the reduced volatile solids were converted into algal biomass and lipids, respectively [221]. Recently high productivity (2.8 g/L-d) of production O. danica from waste activated sludge under non-sterile condition has been demonstrated [179].

Previous work in our group demonstrated the steady state operation of two-stage process (bacteria-then-O. danica), using high C: N ratio glucose-based simulated wastewater [167]. The operation conditions including dilution rate, pH, and dissolved oxygen level were further studied [222]. It was concluded that, under certain conditions, steady state could be achieved. However, so far the complexity of real wastewater has not been investigated. For example, the salinity, inhibitory compounds, and C: N ratio in wastewaters have not been studied. So far there are no studies on microalgae production from industrial food wastewaters under non-sterile condition. In this study

I chose two representative high-strength food wastewaters: cheese wastewater and apple juice wastewater as substrates for algae production.

152

8.2. Materials and Methods

8.2.1. Materials

The freshwater Ochromonas danica culture was maintained as described in earlier chapters. The marine Ochromonas culture used in this study was kindly provided by Dr. Robert Andersen of the University of Washington (Seattle, WA).

The cheese wastewater sample was obtained from a cheese-making company in Wisconsin. The apple juice wastewater sample was obtained from a juice-making company in Washington. These wastewaters were frozen in plastic containers at -20

°C before use in the study.

8.2.2. Marine Ochromonas cultivation

The marine algae investigated in this study was not an axenic culture of

Ochromonas; some micron-sized non-algae cells could be seen under a microscope.

Standard L1 marine culture medium [223] was used for its cultivation. The marine algae was cultured under the ambient light and temperature of the laboratory. Per Dr.

Andersen’s instructions, a single grain of dry rice was sterilized in a dry oven at 107

°C for three hours and then added to 5 mL autoclaved culture medium in 10 mL glass tubes. Presumably the dry grain would slowly release sugar (and other organics) to

153 maintain a steady supply of bacteria for the Ochromonas to consume, which grew faster in presence of the bacteria.

8.2.3. Cheese wastewater study

8.2.3.1.Toxicity test

Preliminary toxicity test was done by mixing the algae seed culture with the cheese wastewater or apple juice wastewater samples. The viability of algae cells, in terms of cell motility and integrity, was observed under a microscope (Olympus

America, Center Valley, PA, USA) for 24 h. Toxicity of diluted wastewater samples was further examined if the sample at full strength showed inhibition to algae.

8.2.3.2.Salinity effect and adaptation

The medium composition was as follows: 200 mL deionized water, 0.08 g/L

yeast extract (Fisher Scientific), 0.08 g/L tryptone (Fisher Scientific), 0.25 g/L NH4Cl

(EMD Chemicals, Gibbstown, NJ, USA), 0.2 g/L MgCO3 (Sigma-Aldrich, St. Louis,

MO, USA), 0.15 g/L KH2PO4 (Sigma-Aldrich), 0.1 g/L nitrilotriacetic acid (Sigma-

Aldrich), 0.05 g/L MgSO47H2O (Sigma-Aldrich), 0.025 g/L CaCO3 (Sigma-Aldrich),

4.4 mg/L Na2EDTA (disodium ethylenediaminetetraacetate) (Sigma-Aldrich), 3.15

mg/L FeCl36H2O (Fisher Scientific), 0.97 mg/L H3BO3 (Sigma-Aldrich), 0.25 mg/L

154 thiamine hydrochloride (Sigma-Aldrich), 0.18 mg/L MnCl24H2O (Sigma-Aldrich),

0.02 mg/L ZnSO47H2O (Sigma-Aldrich), 0.01 mg/L CoCl26H2O (Sigma-Aldrich), 6

μg/L Na2MoO42H2O (Sigma-Aldrich), and 2.5 μg/L biotin (Sigma-Aldrich).

Cheese wastewater had a very high salinity, adaptation experiments were therefore designed to gradually increase the NaCl concentration in the algae growth medium and monitor the profiles of cell growth (as optical density OD at 680 nm) and pH change. Media with two types of main carbon source were used: Medium 1 had 4 g/L glucose and Medium 2 had 2.8 g/L glucose plus 2.5 g/L glycerol. In an experiment, the NaCl concentration was increased stepwise and the observed OD680 and pH profiles were recorded. For comparison, a control system with the algae growing in the regular glucose-based (10 g/L) medium without any NaCl addition was also included in the experiment. The motility of algae cells was also observed under a microscope.

Another experiment was done using same medium as described above but started with 10 g/L glucose and 15 g/L NaCl. Algae seeds used have been adapted to this salinity by gradually increasing NaCl concentration from 0 to 15 g/L within 48 h.

NaCl concentration was further increased from 15 g/L to 21 g/L. Cell concentration

(as OD680), pH and NaCl concentration were recorded.

8.2.3.3.Lactose effects: O. danica growth on lactose, sludge bacteria enrichment for 155

growth on lactose, and growth of enriched bacterial culture in diluted cheese

wastewater

An 80 h batch growth experiment was done to examine if the pure O. danica culture can grow on lactose (same medium as in section 8.2.3.2 but with 10 g/L lactose as carbon source) and, if so, to compare the growth on lactose with that on glucose. A bacterial culture that can grow on lactose, presumably with lactase production, was enriched from a sample of returned activated sludge (RAS) taken from a nearby wastewater treatment plant (Akron, OH), by sequential cultivation in a lactose-based medium using microbial seeds collected by centrifugation from the previous batch growth. The medium included 130 mL deionized water, 50 mL RAS, 5

mL 0.2 mol/L NaH2PO4 and 0.5 mL 0.2 mol/L Na2HPO4. 30 mL cheese wastewater was gradually added within 2 h. The final salinity was about 14 g/L inorganics.

Bacteria that could grow on lactose were collected as pellet by 1st centrifugation at

500 g for 10 min to remove sludge flocs and then 2nd centrifugation at 8000 rpm for

10 min. The growth of this enriched microbial culture was then determined, by

OD600 measurements, in cheese wastewater after 3- and 6-times dilutions with deionized water.

156

8.2.3.4.O. danica growth in diluted cheese wastewater

Based on the salinity adaptation results, the decision was made to dilute the cheese wastewater with 5-fold volume of deionized water (i.e., at a 6-fold dilution) and then evaluated the O. danica growth in the diluted cheese wastewater. The diluted cheese wastewater contained 16-17 g/L inorganics and 5 g/L organics. The algae was grown in a shaker (Thermo Scientific MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville, NC) at 250 rpm and 20 °C. The shake-flask algae growth was determined in 4 (2 x 2) types of diluted cheese wastewater to evaluate two effects

(with and without): sterilization (by autoclaving) and glucose (3 g/L) addition. O. danica cell number concentrations were followed for up to 6 days and the cells were observed under a microscope to observe if non-algae contamination occurred during the cultivation.

8.2.4. Apple juice wastewater study

8.2.4.1.Toxicity test

Toxicity test was done using raw apple juice wastewater mixed with 10-20%

(v/v) of the O. danica seed culture. Cell viability was observed in terms of cell motility and integrity.

157

8.2.4.2.O. danica growth on synthetic apple juice wastewater with nutrient

supplementation

A synthetic wastewater, with the composition shown in Table 8.1, was used.

The composition was created to simulate the following measured properties of the real sample received from state of Washington. Using the synthetic wastewater avoided complications from varying composition of real apple juice wastewater samples and negated the need of shipping and storing huge volumes of wastewater samples from the far away state of Washington.

158

Table 8. 1 Synthetic apple juice wastewater with N&P supplementation

Composition Quantity (mL)

Deionized H2O 100

Mixed sugars solution1 10

Salts solution2 15

Trace elements solution3 2

Vitamins solution4 0.2

NaH2PO4 (0.2 M) 9

Na2HPO4 (0.2 M) 1

Note:

1. Mixed sugars solution contained 40 g/L sugars including 80% fructose (Sigma- Aldrich), 13% glucose (Fisher Scientific) and 7% sucrose (Fisher Scientific).

2. Salts solution contained 5 g/L NH4Cl (EMD Chemicals, Gibbstown, NJ, USA), 4 g/L

MgCO3 (Sigma-Aldrich, St. Louis, MO, USA), 3 g/L KH2PO4 (Sigma-Aldrich), 2 g/L

nitrilotriacetic acid (Sigma-Aldrich), 1 g/L MgSO47H2O (Sigma-Aldrich), and 0.5 g/L CaCO3 (Sigma-Aldrich).

3. Trace elements solution contained 440 mg/L Na2EDTA (disodium

ethylenediaminetetraacetate) (Sigma-Aldrich), 315 mg/L FeCl36H2O (Fisher

Scientific), 97 mg/L H3BO3 (Sigma-Aldrich), 18 mg/L MnCl24H2O (Sigma-Aldrich),

2 mg/L ZnSO47H2O (Sigma-Aldrich), 1 mg/L CoCl26H2O (Sigma-Aldrich), and 600

μg/L Na2MoO42H2O (Sigma-Aldrich). 4. Vitamins solution included 2500 μg/L biotin (Sigma-Aldrich) and 250 mg/L thiamine hydrochloride (Sigma-Aldrich).

8.2.4.3.Two-stage fed-batch process for algae production

A two-stage fed-batch process was designed, after some preliminary

159 investigations, to develop processes for stable algae production. The composition of the synthetic apple juice wastewater used in this study is shown in Table 8.2. Only fructose was used here as it was the major sugar component (80%) in the real apple juice wastewater sample. Tap water, instead of deionized water (Table 8.1), was used to prepare the wastewater, potentially providing some more trace elements. Bacteria, selected from the supernatant of Akron waste activated sludge, were grown in the 1st tank for 12 h, typically reaching an OD610 of approximately 2.0. 300 mL of the mixed bacteria thus grown in the 1st tank were transferred to the 2nd tank for O. danica growth. For the first batch, 300 mL O. danica seed culture was inoculated and the algae was allowed to grow for 12 h by consuming the bacterial culture. After 12 h, another 300 mL of the bacterial culture from the 1st tank was fed, making the 2nd tank to have a total volume of 600 mL. After another 12 h of phagotrophic growth of O. danica in the 2nd tank, 300 mL of the grown culture was removed (as the produced algae culture) and a new batch of 300 mL bacterial culture was fed. This fed-batch process was subsequently repeated in 12-h cycles.

160

Table 8. 2 Synthetic apple juice wastewater with N&P supplementation used for two-stage fill-and-draw process

Composition Concentration (g/L)

Fructose 2

Nitrilotriacetic acid 0.2

NH4Cl 0.5

KH2PO4 0.3

MgCO3 0.4

CaCO3 0.05

MgSO47H2O 0.1

Tap water 300 mL

8.2.5. Analytical methods

Total solids (TS) and volatile solids (VS) concentrations were measured according to the standard wastewater analysis methods [143]. Optical density (OD) was measured using a UV-vis spectrometer (Model UV-1601, Shimadzu Corporation,

Columbia, MD) after proper dilution (to have a value < 0.7) to avoid nonlinear correlation with the particle concentration. For tracking phagotrophic algae growth quantitatively, the sample taken was added with an equal volume of 2% glutaraldehyde solution to fix the cells and stop their motility. The cell number could

161 then be counted using a Petroff-Hausser counting chamber (Hausser Scientific,

Horsham, PA, USA) under a microscope. Microscopic pictures were obtained using a

DP71 digital camera (Olympus America, Center Valley, PA, USA) coupled to the microscope. O. danica cell dry-weight (CDW) concentrations were measured for the seed cultures used as the inocula for experiments, by centrifuging culture samples at

500 g for 10 min, drying the collected cells at 103 °C for 12 h in an oven, and then

weighing the dried cells to determine CDW. NH3-N concentrations were measured with centrifugation-collected supernatants (7,441 g, 10 min) using an ammonia selective electrode (Orion 9512HPBNWP, Thermal Fisher Scientific). Algal lipid content was determined by the chloroform-methanol extraction method. Briefly, 20 mL algae sample was centrifuged at 500 g for 10 min to remove supernatant. The settled algae cells were added with 7 mL HPLC grade chloroform (Fisher Scientific)- methanol (Fisher Scientific) solution (2:1, v/v), 2 drops of hydrochloric acid (Fisher

Scientific), and 0.1 mL de-ionized water. The mixture was vortexed for 5 min and then centrifuged at 2000 g for 10 min to separate cell debris from the solvent phase containing dissolved lipids. The solvent phase was collected and dried by filtered air for 3 h and the weight of lipids was measured. Sugars including glucose, fructose, sucrose and lactose were analyzed by HPLC method [224].

162

8.3. Results and Discussion

8.3.1. Cheese wastewater study

8.3.1.1.Cheese wastewater inhibition test and composition

Cells of the inoculated O. danica seed culture lysed immediately in the cheese wastewaters of 100% and 50% strengths. Even in the 10%-strength cheese wastewater, the algae cells were apparently “shocked” and most cells lost their motility. The cheese wastewater sample had a pH of 5.6. The inhibition effects could not be removed by pH adjustments, neither by increasing the wastewater pH to 6.6

(potentially to alleviate inhibition by compounds like short-chain fatty acids) nor by decreasing the pH to 4.3 (potentially to alleviate ammonia inhibition).

The characteristics and compositions observed/measured for the cheese wastewater sample are summarized in Table 8.3. The cheese wastewater sample had a very high soluble inorganics concentration (104 g/L), much higher than the 35 g/L salinity of sea water. The high salinity could cause the observed inhibition to O. danica cells in the above toxicity test. The cheese wastewater needed to be diluted prior to use for supporting the algae growth. The extent of dilution should however be kept at a minimum so that the algae produced could be at a higher concentration and cheaper to harvest. Studies on the salinity tolerance of this phagotrophic algae and the possibility of adapting it to higher salinity were necessary. Further, the cheese wastewater contained a high lactose concentration (19.2 g/L), which accounted for 163 about 2/3 of all soluble organics (VS), consistent with the literature report that lactose was responsible for the high COD in cheese wastewater [106]. Lactose was however reported to be ineffectively utilized by Ochromonas [8]. Studies were done to evaluate ways for lactose utilization in the O. danica based processes. Results of these salinity tolerance and lactose utilization studies are described in the following sections.

Table 8. 3 Characteristics of cheese wastewater

Properties Descriptions

Appearance White emulsion

Smell Like milk or cream, slightly acidic

Microscopic observation Dispersed fat droplets and other particles pH 5.6

Lactose 19.2 g/L

Soluble organics Protein 7.2 g/L

Fat 3.3 g/L

Soluble inorganics 104 g/L

Volatile Suspended Solids 1.4 g/L

Nonvolatile Suspended Solids 0 g/L

Total Solids 135.8 g/L

Total Volatile Solids 31.7 g/L (23.3%)

164

8.3.1.2.Salinity tolerance study

The salinity issue may be addressed by two steps: 1) dilution of cheese wastewater to a suitable salinity range and 2) use of either a phagotrophic marine algal species or the freshwater O. danica adapted to a higher salinity environment.

Through a literature survey, a marine Ochromonas strain [223] reported to have phagotrophic capability was identified and obtained from Dr. Robert Andersen at the University of Washington (Seattle) (Figure 8.1). After a 2-month evaluation following the suggested protocol, the strain could be maintained but grew very slowly, unlikely to be suitable for the intended purpose of algae production in this project.

Figure 8. 1 Original marine culture. 165

The salinity tolerance of the freshwater O. danica was next evaluated with a gradual stepwise increase of NaCl concentration in two media: one had 4 g/L glucose as the carbon source, the other had 2.8 g/L glucose and 2.5 g/L glycerol. Glycerol production and/or accumulation is a well-known mechanism for cells to cope with conditions of high osmotic pressures [225, 226]. The inclusion of glycerol in one of the media was therefore to explore the possibility of using glycerol addition to help O. danica tolerate the wastewater salinity. The observed profiles of cell growth (by

OD680 measurement) and pH change under the increasing NaCl concentration are shown in Figure 8.2. For comparison, results from a control in the glucose-based (10 g/L) medium without any NaCl addition are also shown in the figure. The control system was not done simultaneously with the two NaCl-added systems.

Including glycerol in the medium did not improve O. danica’s growth in the rising salinity condition. Instead, the glycerol-containing medium supported slower cell growth and lower maximum algal concentration. Under the stepwise NaCl addition, O. danica growth in both media was exponential up to approximately 32 h, when 21 g/L NaCl had been added. At even higher NaCl concentrations, specific cell growth rates decreased and then the decline phase started to appear. Further, when observing the culture samples taken after the NaCl concentration was increased to 21 g/L and beyond, most of the cells in both media stopped moving and changed their shapes from the normal tear-drop shape to more spherical shapes reflecting their loss of capability to maintain morphology. The study showed that the algae could tolerate 166 the salinity corresponding to at least 21 g/L NaCl. An adaptation and/or subpopulation selection for growth in higher salinity should be evaluated for minimizing the necessary dilution of the high-strength WW in future studies.

Algal growth on increasing salinity was further studied using acclimatized cells at 15 g/L NaCl. NaCl concentration was then started to increase from 15 g/L. As shown in Figure 8.3, algal concentration (OD680) increased throughout 144 h of experiment from 0.1 to 1.9. The doubling time was 14.1 h in the first 44 h when NaCl increased from 15 to 18 g/L. The doubling time (or growth rate) was comparable to previously reported 10-18 h at low salinity conditions [10]. Therefore further studies used 1/6 dilute cheese wastewater to keep inorganic concentration to be ~17 g/L.

167

40

30

20

NaCl (g/L)NaCl 10

0 0 20 40 60 80

7 4 g/L glucose

6 2.8 g/L glucose + 2.5 g/L glycerol

5 pH 4

3

2 0 20 40 60 80 2.5 4 g/L glucose

2 2.8 g/L glucose + 2.5 g/L glycerol

1.5

1

Cell growth (OD680) growthCell 0.5

0 0 20 40 60 80 Time (h)

Figure 8. 2 Salinity tolerance of O. danica under gradually increasing NaCl concentrations

168

OD 680 pH NaCl (g/L) 8.0 25

7.0 20 6.0

5.0 15 4.0

10 (g/L)NaCl

OD680 or pHorOD680 3.0

2.0 5 1.0

0.0 0 0 20 40 60 80 100 120 140 160 Time (h)

Figure 8. 3 Algal growth in increasing salinity starting at 15 g/L NaCl.

8.3.1.3.Lactose utilization study

The batch cultivation of O. danica in a lactose-based medium showed that O. danica could not use lactose as a C source. According to the HPLC analysis, no lactose was consumed after 80 h of cultivation although some initial cell growth was observed, which was attributed to the growth supported by the 0.45 g/L yeast extract and 0.45 g/L tryptone in the medium.

Taking advantage of the phagotrophic ability of O. danica, a process can be designed that involves bacterial growth on lactose and then algal growth on the

169 bacteria generated. Only few bacteria can grow on lactose by producing lactase to first hydrolyze lactose into galactose and glucose [106]. Lactase-producing bacteria (LPB) was first selected from a RAS sample taken from the City of Akron wastewater treatment plant, as described in the Materials and Methods section. For evaluation of the LPB’s salinity tolerance and growth in cheese wastewater, the selected LPB was inoculated in 3- and 6-fold diluted cheese wastewater, respectively, and the cell concentration in OD600 and pH were measured as shown in Figure 8.4. Note that with 6-fold dilution, the wastewater had a salinity of 17.3 g/L, the same as the tolerable threshold for insignificantly affected O. danica growth as determined in the salinity tolerance study. A long lag phase of bacterial growth was observed, 24 h in the wastewater of 1/6 strength and 34 h in 1/3 strength, which could be due to the initial salinity shock. Nonetheless, the bacteria could grow reasonably well afterwards with the shortest (observed) doubling time (in OD600) of 2.73 h and 4.56 h in the 6- and 3-fold diluted cheese wastewaters, respectively.

170

1/6 strength--OD600 1/3 strength--OD600 1/6 strength--pH 1/3 strength--pH 5.0 7

4.0 6.5 6 3.0

5.5 pH

OD600 2.0 5 1.0 4.5 0.0 4 0 10 20 30 40 50 60 Time (h)

Figure 8. 4 Growth of lactose-consuming bacteria (selected from wastewater RAS) on diluted cheese wastewaters of 1/6 and 1/3 strengths (i.e., with 6- and 3-fold dilutions)

8.3.1.4.O. danica growth on diluted cheese wastewater

As O. danica could grow at NaCl = 15-18 g/L with doubling time of 14 h. 1/6

strength (17 g/L inorganics and 5 g/L VS0) of cheese wastewater was used for O. danica batch growth (Figure 8.5). Lactose were not consumed in sterilized systems, indicating ineffective utilization of lactose by O. danica, which is consistent as other

Ochromonas species [8]. The doubling time (0-44 h) for sterilized no glucose, sterilized with glucose, nonsterilized no glucose and nonsterilized with glucose were

11.0 h, 11.8 h, 13.9 h and 13.5 h, respectively. Two-way ANOVA with replication was done in Excel® and it was found that sterilization had significant effect on algal growth rate within 44 h while glucose addition and its interaction with sterilization 171 were not significant. Glucose addition increased growth and algae yield from 2.3 x

1010 to 3.0 x 1010 cell per g non-lactose VS (fat & protein). In non-sterilized systems algae also dominated with about 20% yeast and less than 5% bacteria. Lactose was consumed. Algae consumed majority of bacteria but not yeast and algae yield was about 1.6 x 1010 cell/g non-lactose VS.

The above results have proved the feasibility of producing algae from cheese wastewater. As the sterilization of cheese wastewater can incur high cost, producing algae in open system will be more economically desirable. Our previously developed two-stage process using synthetic wastewater (glucose-based), a 7 h dispersed bacterial growth for consuming soluble organics in wastewater and outcompeting other microorganisms, followed by 50 h phagotrophic algal growth for consuming bacteria grown from wastewater could successfully produce algae from synthetic wastewater [222]. In current study, as O. danica can adapt to higher salinity (18 g/L as

NaCl) and can grow on bacteria that consumed lactose and other organics in cheese wastewater, bacteria-then-algae (2-stage) continuous process may be feasible for producing algae from cheese wastewater in open condition, which deserves further studies.

172

Sterilized no glucose Sterilized + glucose nonsterilized no glucose nonsterilized + glucose 7.0 6.0 5.0

/L) 4.0 10

3.0 (x 10 (x 2.0 1.0

Cell number concentration numberCell 0.0 0 24 48 72 96 120 144 168 Time (h)

Figure 8. 5 O. danica growth on dilute cheese wastewater.

8.3.2. Apple juice wastewater study

8.3.2.1.Toxicity test

The toxicity of apple juice wastewater should be tested before batch study.

20% Algae Seeds were inoculated into apple juice wastewater. Algae were not inhibited after mixing with the apple juice wastewater. Large solids, probably pectin material, were seen under microscope (Figure 8.6). As they are too large to consume, in later growth studies these easily settleable solids are removed before inoculating algae seeds.

173

Figure 8. 6 Algae mixed with apple juice wastewater (scale bar: 50 μm)

8.3.2.2.Apple juice wastewater (AJWW) analysis

Table 8.4 shows the composition of apple juice wastewater. The AJWW is slightly acidic and the major solids (80%) are soluble. Three sugars including fructose, glucose and sucrose were found and fructose is the dominant (80% of total sugar). O. danica can consume all these sugars [10]. However, the C:N:P ratio in

AJWW was about 1636:16:1, indicating nitrogen (N) and phosphorus (P) deficiency for microbial cultivation. Specifically for O. danica, the C:N:P ratio in osmotrophically growing cells varied from 56:8:1 to 180:18:1 as a function of growth rate and temperature [70], while the ratio in phagotrophically growing cells ranged between 31:5:1 and 187:16:1 which was affected by the ratio in preys [71] therefore N and P supplementation was necessary for balanced algal growth.

174

Table 8. 4 Wastewater from apple juice manufacturing company

Property Value

pH 6.1

Total Solids (TS) 6.1 ± 0.3 g/L

Volatile Solids (VS) 5.3 ± 1.0 g/L

Suspended Solids (SS) 1.2 ± 0.1 g/L

Volatile SS (VSS) 1.2 ± 0.1 g/L

Total Dissolved Solids (TDS) 4.9 ± 0.2 g/L

Dissolved Volatile Solids (DVS) 3.6 ± 0.5 g/L

Sucrose 0.3 ± 0.1 g/L

Glucose 0.6 ± 0.1 g/L

Fructose 3.6 ± 0.8 g/L

Soluble Inorganics 1.2 ± 0.3 g/L

NH3-N 4.2 ± 1.2 mg/L

Total Kjeldahl Nitrogen (TKN) 17.5 ± 14.8 mg/L

PO4-P 1.1 ± 0.1 mg/L

8.3.2.3.Batch study with N and P supplementation

Algal growth on sugars in AJWW was evaluated in four sterile systems

(Figure 8.7). In authentic-AJWW-88:7:1 system and authentic-AJWW-88:12:1

175 system, algae grew to 4.4 × 1010 cells/L and 4.0 × 1010 cells/L, respectively. The average cell number yield was 1.0 × 1010 cell/g sugar. In the simulated-AJWW-8:1:4 system, algae grew to (3.47 ± 0.01) × 1010 cells/L after 51 h with a cell dry weight of

1.27 ± 0.48 g/L. In the simulated-AJWW-4:1:2 system, algae grew to (3.90 ± 0.72) ×

1010 cells/L after 42 h with a cell dry weight of 1.20 ± 0.46 g/L. Within 42 h, algae grew faster in lower C:N system. The average cell number yield was 1.3 × 1010 cell/g sugar. Cell yields were 0.48 and 0.46 for the 8:1:4 and 4:1:2 systems, respectively. All sugars (sucrose, glucose and fructose) in AJWW were consumed.

The faster growth rate in authentic AJWW was presumably due to the presence of yeast extract and tryptone as source of amino acids.

Algae could grow well on sugars in AJWW with the addition of N&P.

Simulated AJWW was used for further studies.

176

5.0

4.0

3.0

/L) 10

2.0 (x 10 (x

1.0 Algae number concentration concentration number Algae 0.0 0 12 24 36 48 60 Time (h)

authentic AJWW, 4 g/L sugar, 88:7:1 authentic AJWW, 4g/L sugar, 88:12:1 simulated AJWW, 2.6 g/L sugar, 8:1:4 simulated AJWW, 2.6 g/L sugar, 4:1:2

Figure 8. 7 Effect of nitrogen and phosphorus supplementation on O. danica growth on synthetic AJWW.

8.3.2.4.Bacterial growth on synthetic AJWW

Bacteria batch growth on synthetic AJWW is shown in Figure 8.8. Bacteria could grow on AJWW with a doubling time of ~ 2 h. The slower growth rate compared to that (~0.5 h doubling time) in normal rich medium (i.e. with yeast extract) could be due to the lack of organic nitrogen (i.e. amino acids) or growth factor in the synthetic AJWW.

177

Bacterial growth on synthetic AJWW 3 y = 0.1091e0.3207x 2.5 R² = 0.9952 2 1.5

OD610 1 0.5 0 0 2 4 6 8 10 12 Time (h)

Figure 8. 8 Bacteria batch growth on synthetic AJWW.

8.3.2.5.Two-stage fill-and-draw process

The results of two-stage fill-and-draw process are shown in Figure 8.8. A simulated AJWW with C:N:P ratio of 7:1:1 was used (Table 8.2). Within each fed- batch cycle, the culture in the algae tank changed from a more turbid, whitish color at the beginning (right after addition of the new batch of bacteria grown in the bacteria tank) to a greener and clearer appearance (closer to the algae seed culture appearance) at the end of the cycle (Figure 8.9). Two runs of 8 and 10 cycles, respectively, were carried out in the 1st and 2nd runs. The O. danica cell number concentrations harvested at the end of each cycle are shown in Figure 8.8. Run 1 was started at a lower initial algae concentration (0.16 ± 0.02 x 1010 cells/L) and the harvested algae number concentration increased gradually to approach 1.3-1.5 x 1010 cells/L after 6-8 cycles.

178

With this finding, Run 2 was started at an algae concentration slightly higher than this

1.3-1.5 x 1010 cells/L range. Throughout the 2nd run, the harvested algae concentration remained relatively constant, averaging at (1.46 ± 0.14) × 1010 cells/L. Despite the different initial algae concentrations, both Runs 1 and 2 appeared to approach the similar pseudo-steady state algae number concentration. The average specific growth rate (μ) in the first run was 0.08 ± 0.02 h-1. Assuming the dry weight of a single O. danica cell is about 2.82 × 10-11 g [163], the O. danica yield from the 2 g/L fructose in the wastewater was 20.6 ± 2.0% for the and 2nd run.

2nd run-Algae number/L 2nd run-OD610 1st run-Algae number/L 2nd run-Floc number/L 2.0 1.0

1.6 0.8

1.2 0.6

/L) /L) 10

/L) or OD610or /L) 0.8 0.4

(x 10 (x 10

(x 10 (x 0.4 0.2

Floc number concentration Flocnumber Algal number concentration number Algal 0.0 0.0 0 1 2 3 4 5 6 7 8 9 10 11 Fill-and-draw cycle

Figure 8. 9 Maximal O. danica concentration in 12 ± 2 h fill-and-draw processes.

179

Figure 8. 10 Algae tank in fill-and-draw process (left: 0 h and right: 12 h).

8.4. Conclusion

Both cheese wastewater and apple juice wastewater have been tested for their toxicity to phagotrophic alga O. danica. High salinity in cheese wastewater was identified as primary inhibitory factor to O. danica. High salinity issue could be solved by diluting raw cheese wastewater and adapting freshwater O. danica to higher salinity level (15-20 g/L NaCl). Lactose in cheese wastewater couldn’t be digested by

O. danica so a bacterial stage was necessary for lactose conversion. Apple juice wastewater showed no inhibitory effect on O. danica. However, nitrogen and phosphorus deficiency were identified. O. danica could grow well on apple juice wastewater with N&P supplementation. Two-stage fill-and-draw process 180 demonstrated feasibility of producing algae from apple juice wastewater under non- sterile condition.

181

CHAPTER IX

CONCLUSIONS AND RECOMMENDATIONS

In this research, I developed different phagotrophic algae-based processes for conversion of waste activated sludge (WAS) and waste grease (WG) from a municipal wastewater treatment plant, and organics in cheese and apple juice wastewaters, for microalgae production and organic recovery from wastewaters. Energy-efficient ultrasonication was applied to WAS to release bacteria-sized particles for O. danica to ingest and grow. High productivity phagotrophic algal cultivation was achieved using

WAS as whole medium under non-sterile condition. Remaining solids required significantly lower operational cost for aerobic digestion to reach USEPA Class B biosolids regulations. Simply prepared WG can be used as primary carbon source for

O. danica cultivation and polyunsaturated fatty acid (PUFA) production. Two-stage process can be designed for algae production from food wastewaters after the potential toxicity, digestibility and nutrient balance issues are identified and (partially) resolved.

182

9.1. Conclusions

Preliminary observations demonstrated feasibility of converting waste activated sludge by phagotrophic microalga Ochromonas danica. Pretreatment is necessary to release small, ingestible particles from WAS to support robust phagotrophic algal growth. Ultrasonication may be able to disperse bacteria including pathogens into aqueous phase for O. danica to digest (to produce algae) and destruct

(to reduce pathogen).

With proper sonication, bacteria-rich WAS could be used as sole substrate for phagotrophic alga O. danica cultivation. The sonication effectively released bacteria- sized organic particles with minimal organic solubilization to support WAS organic conversion by O. danica to algal biomass and lipids. Without the need of bacterial wall destruction, the required ultrasonic energy was significantly lower than previously reported.

Alkaline WAS ultrasonication was more energy efficient to release ingestible food sources for phagotrophic microalgae. With 9,000 kJ/kg-TS sonication, 45% VS was released, containing mainly (64%) bacteria/microparticles. The released organics supported rapid O. danica growth under nonsterile conditions, showing growth-rate

-1 dependency on (VS/O. danica) ratio with μmax = 0.198 h and KM = 1.056 (g/g).

Fermentor cultivations were studied at pH 5 to minimize reflocculation, with feed VS

183 at concentrations avoiding ammonium inhibition (due to C-limited WAS-VS). High microalgae productivity (2.8 g TS/L-day) was achieved.

Ultrasonic-phagotrophic process was developed for waste sludge conversion and microalgae production. Active biomass including bacteria were released from sludge and collected as food for phagotrophic algal cultivation. Compared to raw sludge aerobic digestion, more than 50% digestion time can be saved with 80% reduced energy cost to achieve Class B biosolids. Produced O. danica biomass may be used as biofuel feedstock and/or aquaculture feed, which need further study.

WG study demonstrated feasibility of converting wastewater-originated WG to PUFA-rich algae culture by using a phagotrophic alga O. danica that could phagocytize droplets and particles as food. The alga grew well on easily prepared WG

(15% PUFAs) and accumulated 48-79% intracellular lipids, including up to 67%

PUFAs in total FAs. Main PUFAs in cells were (in descending %) C18:2n6, C20:4n6,

C22:5n6, C18:3n3 and C18:3n6 (similar % of the last two).

Both cheese wastewater and apple juice wastewater have been tested for their toxicity to phagotrophic alga O. danica. High salinity in cheese wastewater was identified as primary inhibitory factor to O. danica. High salinity issue could be solved by diluting raw cheese wastewater and adapting freshwater O. danica to higher salinity level (15-20 g/L NaCl). Lactose in cheese wastewater could not be consumed by O. danica so a bacterial stage was necessary for lactose conversion. Apple juice

184 wastewater showed no inhibitory effect on O. danica. However, nitrogen and phosphorus deficiency were identified. O. danica could grow well on apple juice wastewater with N and P supplementation. Two-stage fill-and-draw process demonstrated feasibility of producing algae from apple juice wastewater under non- sterile condition.

9.2. Recommendations

9.2.1. WAS project

9.2.1.1.Ultrasonic pretreatment scale-up considerations

Energy-efficient sonication has been proved in laboratory scales; however, demonstration in a larger scale is important as the sonication energy requirement can be quite different from that at the full scale. Ultrasonic release of bacteria-sized particles can use lower power (120-200 W) than that for ultrasonic hydrolysis used prior to conventional digestion. Power is therefore not an obstacle in large-scale implementation. Higher release efficiency may be achieved through optimizing volume, reactor configuration (deeper reactor for ultrasonic probes), and multiple sequential sonication stages. These deserve more studies.

185

9.2.1.2.Pathogen destruction by O. danica.

The capability of O. danica to destroy pathogens has not been thoroughly studied. The decay kinetics of dispersed pathogens, in the presence and absence of O. danica, can be compared. Moreover, the effects of various factors such as pH, DO, reflocculation and carbon-source supplementation on O. danica’s ingestion and destruction of pathogens warrant future investigation. The results may substantially increase the value of the phagotrophic algae processes and the products produced.

9.2.1.3.Large-scale applications

Continuous-flow processes may be more suitable, given the fast O. danica growth rate in sonicated WAS supernatants (doubling time of 3.5-4.6 h at pH 7-5).

Long retention time may not be necessary. The stability of continuous production of

O. danica should also be tested. The stability can be evaluated by observing the interaction between O. danica and other phagotrophic/bacterivory species. The interaction/competition may be observed through either extending retention time in phagotrophic algal stage for protozoan (oo)cysts to germinate and develop, or inoculating active protozoan cells.

186

9.2.1.4.Carbon supplementation to improve algae yield and reduce NH3 remained in

water

Carbon-rich waste organic solids such as waste grease collected at wastewater

treatment plants may be co-processed to improve algal yield and avoid NH3 release from algal consumption of only WAS organics. Future studies are warranted to determine effects of such co-processing of sonicated WAS supernatant and carbon- rich waste organics and to optimize conditions for stable and high-productivity continuous-flow processes.

9.2.1.5.Sludge effect

Sludge effect was not studied. Sludge may differ in the way they are collected, thickened and co-thickened. The effect of sludge on ultrasonic release can be very different as the concentration, composition (C: N ratio and bacteria community) and flocculation of WAS can vary from place to place with different temperature, wastewater source, and wastewater treatment process including sludge handling.

Different sonicated sludge can be tested for O. danica cultivation for wider geographical applications of phagotrophic algae for WAS conversion.

187

9.2.2. WG project

As WG conversion rate and yield depend on the WG dispersion into ingestible sizes, pretreatment to obtain WG microemulsion can be expected to improve both

WG conversion and algal growth rate.

9.2.3. Food wastewater project

The effect of salinity on phagotrophic algal growth on the bacteria growing on cheese wastewater can be further studied and then the two-stage continuous process can be tested for microalgae production from cheese wastewater.

A two-stage process has been demonstrated for algal production from apple juice wastewater with nitrogen and phosphorus supplementation. Further process improvement may be obtainable by studying the possibility of adding certain fractions of raw wastewater directly into the algal stage, bypassing the bacterial stage, to

achieve higher algal yield with less NH3 in the effluent.

188

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APPENDIX

PUBLICATIONS AND CONFERENCE PRESENTATIONS

Lu-Kwang Ju, Cong Li, Suo Xiao. (2018). Producing algal biomass and products from organic solid material. US Patent: US 10,000,402 B2

Cong Li, Suo Xiao, Lu-Kwang Ju*. (2016). Cultivation of phagotrophic algae with waste activated sludge as a fast approach to reclaim waste organics. Water Research, 91, 195-202.

Suo Xiao, Napaporn Vongpanish, Jacob Kohl, Lu-Kwang Ju*. Conversion of waste grease by phagotrophic algae. 2016. Presented at the 107th AOCS Annual Meeting & Expo, Salt Lake City, Utah, USA.

Krutika Invally, Suo Xiao, Lu-Kwang Ju* Effects of Rhamnolipid on Phagotrophic Algae as Sensitive Ecologically Important Model Organism. 109th AOCS Annual Meeting & Expo, Minneapolis, Minnesota, USA

Suo Xiao & Lu-Kwang Ju*. (2018). Energy-efficient ultrasonic release of bacteria and particulates to facilitate ingestion by phagotrophic algae for waste sludge treatment and algal biomass and lipid production. Chemosphere, 209, 588-598.

Suo Xiao & Lu-Kwang Ju*. (2018). Phagotrophic microalgae production from waste activated sludge under non-sterile conditions. Water Research, 145, 190-197.

Suo Xiao & Lu-Kwang Ju*. (2018). Conversion of wastewater-originated waste grease to polyunsaturated fatty acids-rich algae with phagotrophic capability. Accepted. Applied Microbiology and Biotechnology.

Suo Xiao & Lu-Kwang Ju*. (2019). Algae production from industrial food wastewaters. In preparation for 2018 submission.

Suo Xiao & Lu-Kwang Ju*. (2019). Ultrasonic-phagotrophic process for waste activated sludge conversion and microalgae production. In preparation for 2018 submission

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