SHEETS2013_Cultivation of Nannochloropsis Salina in Open Raceway Ponds

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Cultivation of Nannochloropsis Salina in Diluted Anaerobic Digester Effluent under

Simulated Seasonal Climatic Conditions and in Open Raceway Ponds

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in theGraduate School of The Ohio State University

By

Johnathon Sheets, B.S

Graduate Program in Food, Agricultural and Biological Engineering

The Ohio State University

2013

Thesis Committee:

Dr. Yebo Li, Advisor

Dr. Peter Ling

Dr. Brian McSpadden Gardener


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cultivations. However, some factors encountered in outdoor systems are difficult to

simulate in laboratory settings. Evaluation of the effects of predator contamination on

biomass, lipid, and fatty acid production is required for prediction of biofuel yield.

Finally, economic analyses are critical to evaluate future research opportunities that can

improve the microalgae to biofuel process.

In this study, specific growth rate, biomass productivity, total nitrogen (TN)

removal, lipid and fatty acid content of Nannochloropsis salina were analyzed for their

response to simulated seasonal climatic conditions using municipal wastewater AD

effluent as a nutrient source. Results were compared with cultures using commercial

under equivalent TN levels for proper comparison.Data collected from simulated

conditions were used to predict biomass growth in open raceway ponds during July,

August, October and November culture periods in Wooster, Ohio, USA (lat. 40.8050°N).

Preliminary experiments showed the favorable AD effluent loading ratio was 7% (v/v),

with a maximum specific growth rate of 0.327 ± 0.016 d -1

, biomass productivity of 204 ±

12 mg L-1 d -1 and TN removal of 97%. Under simulated seasonal conditions, N. salina

specific growth rate was significantly affected (p


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acid profile was significantly affected by lighting conditions, with a 29% increase in

C20:5 (eicosapentaenoic acid, EPA) and 43% increase in C20:4 content for varied

illumination culture over constant illumination cultures.

Using the quadratic models attained under simulated seasonal conditions, accurate

prediction (22% error) was attained for specific growth rate of N. salina cultured in an

850-L open raceway pond using AD effluent as a nutrient source and July culture period

with highest average light exposure, highest average media temperature, fed batch

cultivation mode and no predator contamination. Cultures in August with fed batch

operating mode and 1060-L volume were exposed to predator contamination and showed

inadequate prediction. October and November cultures with predators and semi-

continuous operating mode (1060-L) also showed inadequate prediction, indicating

models must account for additional operating conditions. When contaminated with

predators, N. salina lipid content declined from 0.37 ± 0.02 to 0.26 ± 0.01 (g g-1

dry

biomass weight), total fatty acid content declined from 0.671 ± 0.070 to 0.335 ± 0.029 (g

g-1 lipids) and EPA content declined from 0.272 ± 0.03 to 0.084 ± 0.011 (g g-1 lipids),

drastically reducing the net value of N. salina biomass.

The results from this study demonstrated the effects of realistic seasonal

conditions on N. salina culture using AD effluent as a nutrient source. While limited light

and predators can diminish biomass lipid and fatty acid productivity, AD effluent was

successfully shown to be a suitable nutrient replacement for commercial nutrients under

simulated seasonal climatic conditions. Data attained will be used to improve modeling

of large-scale open raceway ponds in a seasonal climate.


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Acknowledgements

I would like to first thank my advisor, Dr. Yebo Li, for providing guidance,

support and countless learning opportunities throughout this thesis research. I would like

to thank the members of my thesis committee: Dr. Peter Ling and Dr. Brian McSpadden

Gardener for offering their time, support, comments, questions and guidance.

I would like to thank the following members of the Department of Food,

Agricultural and Biological Engineering: Mike Klingman for assistance in experimental

set-up and equipment repair, Mary Wicks for careful proofreading of proposals, thesis

documents and journal articles, Peggy Christman for administrative assistance and Candy

McBride for her support mediating between Columbus and Wooster.

I am extremely thankful to my fellow laboratory colleagues. Thanks to Stephen

Park for being a wonderful teacher of lab equipment/protocols, a patient responder to my

questions about algae and for his assistance maintaining my experiments. I would also

like to thank Dr. Xumeng Ge for his invaluable support refining thesis documents,

assistance maintaining experiments and insight to improve my research. Thanks to Ting

Cai for providing the baseline research, without which this thesis could not have been

possible. I would also like to thank Jia Zhao, Shengjun Hu, Fuquing Xu, Dr. Xiaolan

Luo, Dr. Cong Li, Dr. Yi Zheng, Dr. Jiying Zhu, Siam Racharaks, Juliana Vasco and

Xinjie Tong for making my Master’s research both fun and enlightening.


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I would also like to thank the members of Touchstone Research Laboratory for

giving me the opportunity to conduct open raceway pond research. Thanks to Dr. Phil

Lane for allowing me to use the ponds and laboratory equipment. Thanks to Doug Amie

for his timely assistance in equipment maintenance. Special thanks to Dr. Xueyan Liu for

patiently teaching and allowing me to use the laboratory equipment.

Finally, I would like to thank my family and friends for their positive support

throughout this process. Thanks to Mom and Dad for listening, providing advice and

helping improve my scientific writing. I especially want to thank Megan Jones for being

patient, understanding and incredibly supportive of my goals.


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Vita

March 4, 1989 ................................................Born, Columbus, Ohio

May, 2011 .....................................................B.S. Engineering Management-

Environmental, Miami University

May, 2011-January 2012 ..............................Associate Environmental Consultant,

ENVIRON International Corporation

January 2012-June 2012 ...............................Graduate Teaching Associate, Department

of Food, Agricultural and Biological

Engineering, The Ohio State University

June 2012-Present .........................................Graduate Research Associate, Department

of Food, Agricultural and Biological

Engineering, The Ohio State University

Fields of Study

Major Field: Food, Agricultural and Biological Engineering


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Table of Contents

Abstract ............................................................................................................................... ii

Acknowledgements ............................................................................................................. v

Vita .................................................................................................................................... vii

Table of Contents ............................................................................................................. viii

List of Tables ..................................................................................................................... xi

List of Figures ................................................................................................................... xii

Chapter 1: Introduction ....................................................................................................... 1

Chapter 2: Literature Review .............................................................................................. 5

2.1 Microalgae Cultivation Systems ............................................................................... 6

2.1.1 Mechanism of microalgae growth ...................................................................... 6

2.1.2 Photobioreactors ................................................................................................. 8

2.1.3 Open raceway ponds ........................................................................................... 9

2.1.3.1 Brief introduction of open raceway ponds .................................................. 9

2.1.3.2 Issues in open raceway ponds ................................................................... 11

2.1.3.3 Performances of open raceway pond cultivations .................................... 13

2.2 Wastewater Nutrients for Microalgae Biomass Production .................................... 17

2.2.1 Wastewater use and biomass synthesis by microalgae ..................................... 17

2.2.2 Anaerobic digester effluent as microalgae nutrient source .............................. 23

2.3 Effects of Operational and Climatic Conditions on Microalgae Productivity ........ 28

2.3.1 Effect of cultivation parameters on microalgae productivity ........................... 28

2.3.2 Effect of light availability on microalgae productivity .................................... 30

Chapter 3: Cultivation of Nannochloropsis salina in Diluted Anaerobic Digester Effluentunder Simulated Seasonal Climatic Conditions ................................................................ 36

3.1 Introduction ............................................................................................................. 37

3.2 Materials and Methods ............................................................................................ 38

3.2.1 Microalgae strain and seed culture ................................................................... 38

3.2.2 AD effluent ....................................................................................................... 39


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3.2.3 Cultivation of N. salina at different loading ratios of AD effluent .................. 39

3.2.4 Cultivation of N. salina in diluted AD effluent under varied illumination andtemperatures............................................................................................................... 40

3.2.5 Analytical methods ........................................................................................... 42

3.3 Results and Discussion ............................................................................................ 48

3.3.1 AD effluent composition .................................................................................. 48

3.3.2 Effect of AD effluent loading ratio on cultivation of N. salina ........................ 49

3.3.3 Effects of simulated seasonal illumination and temperature on biomass production and nitrogen removal ............................................................................... 52

3.3.3.1 Biomass production .................................................................................. 52

3.3.3.2 Effect of temperature and seasonal illumination on total nitrogen removal............................................................................................................................... 60

3.3.4 Effects of nutrient source and culture system on lipid and fatty acid production

................................................................................................................................... 61

3.4 Conclusion ............................................................................................................... 64

Chapter 4: Analysis of Nannochloropsis salina Cultivation using Anaerobic DigesterEffluent as a nutrient source in Open Raceway Ponds ..................................................... 65

4.1 Introduction ............................................................................................................. 66

4.2 Materials and Methods ............................................................................................ 67

4.2.1 Light exposure model for biomass productivity prediction.............................. 67

4.2.2 Model validation experiments .......................................................................... 69

4.2.2.1 AD effluent ............................................................................................... 69

4.2.2.2 Cultivation of N. salina in open raceway ponds ....................................... 70

4.2.3 Analytical procedures ....................................................................................... 72

4.2.3.1 AD effluent composition........................................................................... 72

4.2.3.2 Biomass and lipid/fatty acid productivity ................................................. 72

4.2.3.3 Predator contamination ............................................................................. 74

4.2.3.4 Economic analysis .................................................................................... 74

4.3 Results and Discussion ............................................................................................ 75

4.3.1 Groundwater at Cedar Lane Farms ................................................................... 75

4.3.2 Accumulated biomass in each culture period ................................................... 76

4.3.3 Predicted biomass productivity v. actual open raceway pond results .............. 78

4.3.4 Effects of predator contamination on biomass, lipid, and fatty acid productivity ................................................................................................................................... 80


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4.3.5 Economic analysis ............................................................................................ 84

4.3.5.1 Operating costs.......................................................................................... 86

4.3.5.2 Biodiesel or EPA production .................................................................... 87

4.4 Conclusion ............................................................................................................... 88

Chapter 5: Conclusions and Recommendations ............................................................... 90

References ......................................................................................................................... 92


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List of Tables

Table 1: Status of microalgae cultivations in open raceway ponds .................................. 14 Table 2: Key differences between open raceway ponds and photobioreactors ................ 17 Table 3: Status of wastewater nutrient removal and biomass production by microalgae . 19 Table 4: Status of microalgae cultivations in anaerobic digester effluent ........................ 25 Table 5: Operational parameters effects on photoautotrophic microalgae growth ........... 28 Table 6: Previous experiments evaluating the effects of light availability on microalgae production ......................................................................................................................... 32 Table 7: Experimental design for the study of loading ratio effects (n=2) ....................... 40 Table 8: Experimental design for the study of simulated seasonal climatic conditions

(n=2) .................................................................................................................................. 42 Table 9: Anaerobic digester effluent characteristics ......................................................... 49

Table 10: Simulated seasonal climatic conditions ANOVA parameter effects analysis .. 53 Table 11: Effect of lighting conditions and nutrient sources on lipid and fatty acid content(n=2) .................................................................................................................................. 62 Table 12: Open raceway pond cultivation dates ............................................................... 68 Table 13: Cedar Lane Farm groundwater quality analysis ............................................... 75 Table 14: Assumptions used in theoretical economic analysis of N. salina biomass production ......................................................................................................................... 85


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List of Figures

Figure 1: Nitrogen assimilation in microalgae cells (adapted from Cai et al., (2013a)) ..... 7 Figure 2: Open raceway pond schematic (adapted from Oswald, (1995)) ....................... 10 Figure 3: Anaerobic digestion process schematic (adapted from Li et al., (2011a)) ........ 23 Figure 4: Experimental set-up for simulated seasonal climatic conditions ...................... 41 Figure 5: Effect of added TN concentration on N. salina specific growth rate ................ 50 Figure 6: Effect of temperature on specific growth rate/light exposure ........................... 54 Figure 7: Effect of light exposure on N. salina specific growth rate cultured incommercial nutrients ......................................................................................................... 56 Figure 8: Effect of light exposure on N. salina specific growth rate cultured in diluted

anaerobic digester effluent ................................................................................................ 57 Figure 9: Effects of light availability and nutrient source on biomass productivity ......... 58

Figure 10: Effects of temperature and light availability on TN removal by N. salina cultured in diluted anaerobic digester effluent .................................................................. 60 Figure 11: Open raceway pond ......................................................................................... 70 Figure 12: Accumulated N. salina biomass in open raceway ponds cultured in dilutedanaerobic digester effluent ................................................................................................ 76 Figure 13: Effect of season on average light exposure and temperature .......................... 77 Figure 14: Predicted versus actual specific growth rate cultured in diluted anaerobicdigester effluent in open raceway ponds ........................................................................... 78 Figure 15: Predicted versus actual biomass productivity in open raceway ponds ............ 80

Figure 16: Accumulated predator concentration for N. salina cultured in diluted anaerobicdigester effluent in open raceway ponds ........................................................................... 81 Figure 17: Effect of predators on lipid and total fatty acid content in open raceway ponds........................................................................................................................................... 82 Figure 18: Effect of predators on fatty acid constituents in open raceway ponds ............ 83 Figure 19: Annual operating costs for commercial nutrient media requirements ($/year) 86 Figure 20: Net value of harvestable dry N. salina biomass .............................................. 87


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Chapter 1: Introduction

Diminishing fossil fuel reserves and concerns over climate change due to

greenhouse gas emissions have stimulated research aimed at sustainable fuel production

(Sander and Murthy, 2010). According to the U.S. Department of Energy, biofuels from

biomass represent the only renewable resource that can supplement petroleum-based

liquid transportation fuels and simultaneously decrease greenhouse gas emissions (EERE,

2013). Although conventional biofuels like corn ethanol and soybean diesel have been

produced as alternatives to fossil fuels, there are concerns about potential conflicts with

food supplies and land protection. Microalgae-based biodiesel has attracted great

attention due to high potential productivity and noncompetition against food production

systems (Campbell et al., 2011).

Mass cultivation of microalgae biomass requires large amounts of nutrients.

Addition of commercial fertilizer can dramatically increase production costs, limiting

progress of large scale endeavors (Davis et al., 2011, Yang et al., 2011). As a result,

wastewater streams containing primary nutrients required for microalgae growth are

highly attractive, since the nutrients are available at no cost (Jiang et al., 2011, Mcginn et

al., 2011, Pittman et al., 2011). Anaerobic digestion (AD) is a process that has been

widely used to decompose organic waste and produce biogas (Khanal et al., 2010). The

effluent from the AD process contains high concentrations of nitrogen and phosphorus,

and has been recognized as a suitable nutrient source for microalgae cultures (Anderson


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et al., 2002, Cai et al., 2013b, Chen et al., 2012c, Levine et al., 2011, Park et al., 2010a,

Woertz et al., 2009, Yuan et al., 2012). To date, AD effluent has mainly been used for

culturing freshwater microalgae and is usually sterilized and diluted prior to use to

eliminate growth inhibiting compounds. Concerns about high sterilization costs and

massive freshwater requirements in large scale cultivation systems have necessitated

research on marine microalgae. Culturing marine microalgae with diluted AD effluent

could alleviate massive freshwater and commercial fertilizer requirements (Batan et al.,

2010). Recently, a marine microalgae strain, Nannochloropsis salina, was evaluated by

Cai et al. (2013b) for nutrient removal and lipid production using municipal wastewater

AD effluent as a nutrient source. High biomass and lipid productivities were achieved in

2-L bioreactors under multiple dilutions of AD effluent, constant illumination and

temperature (200 µmol m-2 s-1 light intensity, 24-h light availability, 25 ± 1°C), indicating

a promising microalgae strain for biofuel production. However, there are several

important issues that need to be addressed for the scale up of this technology.

Firstly, data obtained in ideal laboratory settings cannot be used to directly predict

outdoor production due to dramatically varied weather conditions. In order to reduce

operating costs, commercial-scale microalgae cultivation would most likely be conducted

in open raceway ponds under outdoor conditions without light or temperature control

(Oswald, 1995). Light intensity, light availability and temperature will change depending

on different geographical locations, climates and seasons, and these factors could affect

the growth performance of microalgae in open raceway ponds (Singh and Dhar, 2011).

Therefore, prior to scale up of any microalgae cultivation technology, it is essential to


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evaluate biomass growth at light exposure and temperature levels comparable to outdoor

conditions in the geographical region of cultivation (Flynn et al., 1993).

Secondly, adequate comparison between commercial nutrients and diluted AD

effluent is crucial for economic analysis of scale-up operations. According to the

nitrogen-to-phosphorus (N/P) ratio in AD effluent and the average N/P ratio of marine

plankton, nitrogen would be the major limiting nutrient for N. salina (Cai et al., 2013b).

Therefore, productivity in diluted AD effluent needs to be compared with productivity in

commercial nutrients at similar nitrogen levels.

Thirdly, predator-prey oscillations may occur in outdoor open raceway ponds,

which can lead to algal biomass crashes, causing productivity to decline by two orders of

magnitude (Smith et al., 2010). While there are publications describing the biomass

productivity of microalgae monocultures open raceway ponds using commercial nutrients

(Bellou and Aggelis, 2012, Braden Crowe, 2012, Ashokkumar and Rengasamy, 2012, Lin

and Lin, 2011), very few used nutrients from wastewater or outline the adverse effects

predators have on lipid production in microalgae biomass. Analysis of predators on

marine strains is also very limited (Bartley et al., 2013). A research gap exists in

quantitative analysis of the effects of predators on microalgae cultivation (Day et al.,

2012a).


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Chapter 2: Literature Review

Economic, resource, and life cycle assessments on microalgae biodiesel

production systems have revealed high costs due to large water, nutrient, and energy

requirements (Amer et al., 2011, Batan et al., 2010, de Vries et al., 2010, Pate et al.,

2011, Pittman et al., 2011, Sander and Murthy, 2010, Yang et al., 2011). Improvements

in cultivation, harvesting and downstream processing are essential for microalgae

biofuels to become cost competitive and environmentally friendly. Genetic engineering

of highly productive strains, low energy harvesting methods and advanced downstream

processing have garnered much interest (Amer et al., 2011, Zeng et al., 2011). However,

sustainable biomass production in large-scale cultivation systems remains a challenge due

to massive freshwater and commercial fertilizer requirements. Idealized cultivation

systems in laboratory settings have revealed many capable strains and wastewater

streams for biomass production and nutrient removal. However, little research has

analyzed the effects of a changing climate in wastewater fed-microalgae cultures. Proper

prediction of product yields in high volume open raceway ponds requires quantitative

analysis of the effects of dynamic seasonal conditions (light/temperature) and predators

on microalgae biomass, lipid and fatty acid productivity. Economic analyses including

the effect of predators provide data that can determine existing research opportunities

prior to the large scale development of microalgae biofuels.


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2.1 Microalgae Cultivation Systems

2.1.1 Mechanism of microalgae growth

Microalgae are photosynthetic microorganisms that utilize photosynthetic active

radiation (PAR), carbon dioxide (CO2) and nutrients to grow rapidly while suspended in

water (Sheehan, 1998). Light is captured by pigment-protein antenna systems, is

transferred as excitation energy to photosynthetic reaction centers and converted to

chemical energy, driving the formation of adenosine triphosphate, or ATP (Stephenson et

al., 2011). Nicotinamide adenine dinucleotide phosphate (NAPDH), typically formed by

water splitting, and ATP are used to fix CO2 into carbohydrates with the catalyst ribulse-

1,5-biphosphate carboxylase oxygenase (RUBISCO) (Stephenson et al., 2011).

Carbohydrates are used for all cellular functions, including biomass generation and lipid

synthesis (Stephenson et al., 2011). Lipids in the form of triacylglycerides (TAGs) are the

precursors for biodiesel. Besides CO2, the primary nutrients for microalgae are nitrogen

and phosphorus (Demirbas and Fatih Demirbas, 2011). Nitrogen and phosphorus are

found in peptides, proteins, enzymes, chlorophylls, genetic materials, nucleic acids, lipids

and intermediate compounds of the carbohydrate metabolism (Cai et al., 2013a).

Microalgae uptake nitrogen in the forms of ammonium (NH4+) and nitrate (NO3

-) for the

production of several metabolites. The enzyme nitrate reductase reduces nitrate to nitrite

(NO2-), followed by the reduction of NO2

- to NH4

+ by nitrite reductase. Nitrate reductase

uses the reduced form of nicotinamide adenine dinucleotide (NADH) while nitrite

reductase uses ferredoxin (Moberg et al., 2012, Cai et al., 2013a). Ammonium is


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transported across cellular membranes by ammonium transporter proteins (Perez-Garcia

et al., 2011). Ammonium transporters can be either high affinity (regulated by nitrogen

status of cells) or low affinity (respond to linear increase in ammonium concentration).

Ammonium is transported to the chloroplast, where the enzyme glutamine synthesase,

with glutamate and ATP, converts NH4+ to the amino acid glutamine (Cai et al., 2013a,

Inokuchi et al., 2002). The nitrogen assimilation pathway is shown visually in Figure 1.

Figure 1: Nitrogen assimilation in microalgae cells (adapted from Cai et al., (2013a))

Phosphorylation, the generation of ATP and adenosine diphosphate (ADP) from

phosphorus with an accompanied energy source, is described as the primary phosphorus

uptake mechanism in algae species (Cai et al., 2013a, Jansson, 1988). The assimilation of

nitrogen is considered the rate determining nutrient affecting microalgae growth when

adequate light and inorganic carbon sources are present in aqueous growth media (Cai et

al., 2013a). Secondary nutrients in minimal amounts (Fe, K, Mg, Ca, Na) are integral to

the photosynthetic process (Cai et al., 2013a). Cultivation systems require adequate light,

CO2, nitrogen, phosphorus and trace elements to maintain biomass production.

http://www.sciencedirect.com/science/article/pii/S1364032112006429


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The majority of microalgae biofuel studies use the specific growth rate to

determine biomass accumulation. Denoted by the symbol µ (d -1), specific growth rate is

defined by the function

= 1

, where X is the biomass concentration (g L-1), and t is

the time period of exponential growth (d) (Michael L.Shuler and Fikret Kargi, 2002).

Specific growth rate is affected by light intercepted by microalgae, CO2, nutrient

concentration, pH and media temperature. Each parameter effect is described in Section

2.3.1. Biomass productivity (mg L-1 d -1) can be determined by multiplication of the

specific growth rate (d -1) and the final biomass concentration in exponential growth phase

(Xt, g L-1). Multiplication of the biomass productivity (mg L-1 d -1) by the lipid content (g

g-1 dry biomass) can determine the lipid productivity (mg lipids L-1 d -1). The total fatty

acids (g g-1

total lipids) multiplied by lipid productivity can be used to represent the

biodiesel production potential of a microalgae cultivation system. The lipid content (g g-1

of TVS) represents the amount of biofuel intermediates that can be produced in a

microalgae biomass production system. The amount and type of fatty acids represents the

quality of algae biomass. The cetane number reflects the readiness of biodiesel to auto-

ignite when it is injected into a combustion chamber. Fatty acids are reported in terms of

total fatty acids, total saturated fatty acids, and total unsaturated fatty acids.

2.1.2 Photobioreactors

Photobioreactors have many designs and applications, and are the most used

cultivation system for research practices. Many design aspects, including lighting,

mixing, water, CO2 flow, nutrient supply, temperature and pH can be controlled in closed


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photobioreactors (Kunjapur and Eldridge, 2010). At high biomass density, microalgae

accumulation causes minimal light penetration and declined growth. Internal or external

light sources can help maintain light penetration when this self-shading effect occurs

(Ogbonna et al., 1996), but this application greatly increases cost. CO 2 utilization in

photobioreactors appears to be a strain specific and bubbling apparatuses are not

technologically capable for full carbon capture (Mata et al., 2010, Murray et al., 2012).

New technologies for more efficient gas systems are being researched, yet scalability still

remains an issue for gas exchange (Bentley and Melis, 2012, Pilon et al., 2011, Sheldon,

2011). There appears to be more control in combatting predator contaminants in

photobioreactors. However capital and operating costs are significantly high for

photobioreactors (Shen et al., 2009).

2.1.3 Open raceway ponds

2.1.3.1 Brief introduction of open raceway ponds

Open raceway ponds have been used for large scale algae biomass production

since the 1950’s (W.J.Oswald, 1957). The systems are called “raceway” because of their

geometry, coupled with a paddlewheel that mixes and circulates microalgae growth

media (Demirbas and Demirbas, 2010). Open raceway ponds are made from poured

concrete or dug into the earth and lined with plastic. These systems are semi-continuously

operated, in which complete mixing takes place in a batch setting followed by controlled

harvesting and nutrient repletion (Craggs et al., 2012). A controlled semi-continuous

system could allow daily harvesting and nutrient addition to maintain steady biomass


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concentrations. In order to minimize the self-shading effect caused by biomass

accumulation, open raceway ponds are most effective when operated at shallow heights

and high surface areas, allowing maximum light penetration through the pond surface

(Terry and Raymond, 1985). A basic design of an open raceway pond is shown in Figure

2.

A network of open raceway ponds over a large surface area is called an “algae

farm.” Large-scale outdoor culture of a few microalgae and cyanobacteria strains in open

raceway ponds is established in the United States, Asia, Australia, and the Middle East

(Demirbas, 2009). These systems are primarily in use to produce high value products

including nutritional supplements, aquaculture feed and fertilizer. Large scale production

of microalgae for biodiesel is limited due to the introduction of new companies, patent

protected strains and limited proof of concept. The challenges affecting large scale algae

production in open raceway ponds include evaporative water losses, limited light

CO2, Nutrients Harvest

Paddlewheel

Figure 2: Open raceway pond schematic (adapted from Oswald, (1995))


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availability, temperature control, inefficient CO2 mass transfer and predator

contamination (Craggs et al., 2011).

2.1.3.2 Issues in open raceway ponds

Culturing freshwater microalgae strains in open raceway ponds require vast

volumes of freshwater, a resource that is becoming increasingly scarce and valuable

(Yang et al., 2011). Desert regions with intense, available sunlight and high temperatures

have been proposed as ideal conditions for vast microalgae farms (Vigon et al., 1982).

However, freshwater is expensive in desert regions, so water refill due to evaporative

losses represents a significant operating cost and environmental concern (Sheehan, 1998).

Inversely, regions with available and low cost freshwater are known for seasonal climatic

conditions with fluxes in temperature, light intensity and light availability. Climatic

variation allows less operational control of maintaining ideal microalgae growth media.

Microalgae require CO2 to provide the necessary inorganic carbon to produce biomass

via photosynthesis. Efficient CO2 mass transfer is a challenge due to shallow depths, and

significant losses have been shown for traditional diffuser systems (Langley, 2012).

Maintaining high throughput systems could require large investment in freshwater,

temperature, light and CO2 regulation schemes to maintain optimal monoculture

conditions (Williams and Laurens, 2010).

Control of predator contamination is the most glaring issue facing the scale-up of

open raceway ponds. Spirulina, Arthrospira, and Dunaliella are the only successful

strains of algae cultured in open raceway ponds, due to adaptation in extreme salinity, pH


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and temperatures (Day et al., 2012a). Predators cannot easily survive in these

environments and monocultures can be readily attained. However, most of the lipid-rich

microalgae strains cannot survive in extreme environments. In fact, the status of research

and development for predator effects on microalgae biofuels is limited (Day et al.,

2012a). Contamination by other microalgae species in an open raceway pond

environment is inevitable (Day et al., 2012a). Contamination by alkaliphilic Oocystis has

even been shown to negatively impact the resistant Spirulina strain (Belay, 1997). Dense

cultures of microalgae in open raceway ponds have been shown to leak nearly 75% of the

photosynthetic fixed carbon out of their cells (Wolfstein et al., 2002). This can lead to

bacterial contamination that can cause competition for nutrients, limited light availability

and microalgae cell death (Day et al., 2012a). Fungal chytrids have also been shown to

impact mass monocultures. Paraphysoderma sedebokerensis has been shown to

negatively impact the growth of 13 unicellular Chlorophyta (Gutman et al., 2009).

Grazing by zooplankton (ciliates, amoeba, rotifers) have significant impact on the open

raceway pond ecosystem (Day et al., 2012a). Although traditionally resistant in highly

saline environments, Dunaliella cultivations have even been decimated by ciliate

predators (Post et al., 1983). While zooplankton predators are a problem facing all open

raceway pond systems, little is published on the subject with respect to biomass, lipid and

fatty acid production (Day et al., 2012a).

Because predator-prey oscillations can lead to declined productivity by two orders

of magnitude (Smith et al., 2010), there is much interest in control and detection methods.

Rapid detection of predators in Nannochloropsis and Scenedesmus cultures has been


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Table 1: Status of microalgae cultivations in open raceway ponds

Microalgae

Strain

Volume and Location Nutrient Source Operating

Mode

Retention Time

(days)

Biomass

Productivity

(mg L-1-d-1)

Lipid Content

(% of dry

weight)

Reference

Chlorella sp. 5 L indoor Modified artificialseawater

Batch 25 47.0a 24.0-38.1 (Bellou and Aggelis, 2012)

Nannochloropsis

salina5 L indoor Modified artificial

seawaterBatch 25 101.4a 10.0-26.6 (Bellou and Aggelis, 2012)

Chlorella sp. 20 L indoor Pretreated ADeffluent

Semi-continuous

35 6.83 b 9.34-10.78 (Chen et al., 2012c)

Pleurochrysis

carterae

160e L outdoor Modified f/2 media Semi-

continuous

varied 170-230 21-24 (Moheimani and Borowitzka,

2011)

Neochloris

oleoabundans

375 Le outdoor Bristol medium Semi-continuous

59 NR 11 (da Silva et al., 2009)

Nannochloropsis

salina

780 L outdoor f/2 media Batch 90 13 15-25 (Braden Crowe et al., 2012)

Botryococcus

mahabali.1000 L outdoor CHU 13 medium Batch 14 90c 20.6d (Dayananda et al., 2010)

Botryococcus

bruanii

2000 L outdoor CHU 13 medium Batch 15 114 18.02-19.98 (Ashokkumar and Rengasamy,2012)

Scenedesmus

rubescens

9000 Le outdoor Ammonium

Nitrogen

Batch 20 3.16-4.56 b 11.3-15.3f (Lin and Lin, 2011)

Mixed Speciesof algae/bacteria

2,375,000 L outdoor MunicipalWastewater

Effluent

Batch 365 4.4-11.5 b NR (Craggs et al., 2012)

NR=not reporteda. calculated from specific growth rate (d -1)*max biomass density (mg L-1)

b. areal productivity (g m-2 d -1)c. calculated from: (max biomass density-initial biomass density)/(batch growth period (days))d. fat content + hydrocarbon contente. calculated from pond dimensionsf. FAME content

1 4


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Bellou and Aggelis (2012) developed a custom laboratory scale reactor (5 L

working volume) resembling open raceway ponds to test the two highly cited fatty acid

producing strains Chlorella sp. and Nannochloropsis salina. Under ideal lighting

conditions, maximum attainable biomass productivities were 47 mg L-1 d -1 (Chlorella sp.)

and 101 mg L-1

d -1

( N. salina), respectively. Adaptation of cultures to a continuous

harvesting mode caused a significant decline in N. salina biomass productivity (19 mg L-1

d -1). Using constant illumination and pretreated, diluted AD effluent for nutrients,

Chlorella sp. isolates maintained high areal biomass productivity (6.83 g m-2

d -1

) in in a

small 20 L open raceway pond in semi-continuous mode (Chen et al., 2012c). Moheimani

and Borowitzka (2011) studied the effect of natural diurnal light on Pleurochrysis

carterae biomass production in 160 L open raceway ponds. Their results showed a

significant increase culture media pH during the light phase, indicating open raceway

ponds may require acid buffering techniques to maintain optimum culture conditions.

Neochloris oleoabundans was shown to be a promising biofuel strain under semi-

continuous conditions (da Silva et al., 2009). While biomass and lipid productivity was

lower than previously reported values (Chisti, 2007), the extrapolated areal oil yield

(21,523 L ha-1

) outperformed first generation biofuels. da Silva et al. (2009) also stated

that environmental variables (light/temperature) and operational variables

(nutrients/mixing) controlled the effectiveness of open raceway pond algae cultures.

Braden Crowe et al. (2012) cultured Nannochloropsis salina in an outdoor desert

environment for 90 days in the spring and summer of 2011. Their results showed low

volumetric productivity (13 mg L-1

d -1

) and lipid content (15-25%). In their study, solar


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radiation and temperature fluctuated dramatically during the spring growing season, and

microscopy showed presence of motile invasive species. Dayananda et al. (2010) and

Ashokkumar and Rengasamy (2012) showed the effects of outdoor open raceway pond

scale-up on biomass productivity of Botryoccoccus mahabali and Botryoccoccus braunii,

respectively. Dayananda et al. (2010)’s results demonstrated a high Botryococcus

mahabali final biomass concentration in 1000 L open raceway ponds (2 g L-1), and

identified this strain to be useful for biofuel production. Ashokkumar and Rengasamy

(2012)’s results showed high biomass productivity in open raceway ponds (114 mg L-1

d -

1

), with similar lipid content (17-19%) to laboratory results, but indicated further research

is needed to overcome the high cost of commercial nutrients. Under a similar operating

mode, Lin and Lin (2011) studied the effects of ammonium concentration on the growth

of Scenedesmus rubescens. High areal biomass productivity (3.16-4.56 g m-2 d -1) was

established; indicating ammonium based wastewaters may be a suitable nutrient source

for Scenedesmus rubescens. Craggs et al. (2012) cultivated a mixed algal-bacterial

consortium at massive scale. Their results were aimed at municipal wastewater treatment;

yet high biomass production (4.4-11.5 g m-2 d -1) and nutrient removal rate (65% of

ammonia nitrogen) were achieved.

Because capital costs for photobioreactors are so high, these systems will likely

not be used for scale-up of microalgae for biofuels. Nevertheless, poor light and

temperature control will cause diminished productivity in open raceway ponds compared

to laboratory settings. The majority of cultivation research using open raceway ponds

used commercial nutrients and idealized temperature and lighting conditions. Analysis of


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microalgae cultivation with diluted wastewater in open raceway ponds exposed to natural

conditions is critical to predicting realistic large scale production. Table 2 displays a

summary of the key differences between open raceway ponds and photobioreactors.

Table 2: Key differences between open raceway ponds and photobioreactors

Item Open raceway pond Photobioreactor

Capital Costs Low HighOperating Cost Low High

Biomass Productivity Low HighCarbon Dioxide Utilization Low High

Contamination Risk High LowLight Source Natural Natural/Artificial

Large Scale Cultivation? Yes No

2.2 Wastewater Nutrients for Microalgae Biomass Production

2.2.1 Wastewater use and biomass synthesis by microalgae

There is concern about harmful blue-green algae blooms due to eutrophication

caused by nitrogen and phosphorus runoff from agricultural activities (Anderson et al.,

2002). However, under controlled conditions, microalgae have the potential to eliminate

these nutrients from industrial, agricultural, and municipal wastewaters (Benemann and

Oswald, 1979). In order to meet economic and regulatory requirements, facilities usually

treat wastewater for reduction of chemical oxygen demand (COD), biochemical oxygen

demand (BOD), heavy metals, solids and nutrients (Abou-Shanab et al., 2011). The

traditional municipal wastewater treatment process consists of: 1. primary treatment of

suspended solids by sedimentation, 2. secondary treatment by bacterial uptake of


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dissolved and suspended organic material, and 3. tertiary treatment of nutrients and

metals to reach discharge requirements set by a regulatory body. Nutrient remediation in

the tertiary phase involves two separate bacterial species with specificity towards uptake

of nitrogen and phosphorus (Metcalf and Eddy, 2003). However, conventional tertiary

wastewater treatment by biological nitrification and de-nitrification has shown economic

limitations due to organic carbon requirement (Metcalf and Eddy, 2003). The goal to

reduce costs of tertiary treatment processes has led to research the efficiency of

microalgae to consume nutrients from a multitude of wastewaters while simultaneously

producing lipid-rich biomass (Mcginn et al., 2011). Table 3 displays selected research

articles focusing on microalgae systems for nutrient removal, biomass and lipid

production.


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Table 3: Status of wastewater nutrient removal and biomass production by microalgae

Microalgae Strain Wastewater Type Nutrient Nutrient Removal

(% of original)

Biomass Productivity

(mg L-1 d-1)

Lipid Content

(% of dry weight)

Reference

Botryococcus braunii Agricultural NO3-N 80 708a 11.2 b (An et al., 2003)

Botryococcus braunii Agricultural (livestock) TN

TP

88

98

84.8 19.8c (Shen et al., 2008)

Chlorella vulgaris Agro-industrial

(dairy and piggery)

NH3-N

PO4-P

90

60

NR NR (Gonzalez et al., 1997)

Chlorella sp. Municipal TN

PO4-P

68.4-82.8

83.2-90.6

NR NR (Wang et al., 2010b)

Chlorella sorokiniana Synthetic NH3-N

NO3-N

100

41

400a NR (Ogbonna et al., 2000)

Chlorella zofingiensis Agricultural (dairy) TN

PO4-P

97.5

51.7

NR 17.9 (Huo et al., 2012)

Scenedesmus dimorphus Agro-industrial

(dairy and piggery)

NH4-N

PO4-P

90

55

NR NR (Gonzalez et al., 1997)

Scenedesmus obliquus Municipal

(secondary treated)

NH4-N

TP

100

98

24.1d 16e (Martinez et al., 2000)

Nannochloropsis sp. Seawater/Municipal TN

PO4-P

NR

NR

25a 27.8 (Jiang et al., 2011)

Nannochloropsis sp. F&M- M24

Industrial

(syngas quench effluent)

NR NR 330a NR (Biondi et al., 2012)

Native Species Industrial (carpet mill) NO3-N

PO4-P

99.7-99.8

96.6-99.1

39 6.82 (Chinnasamy et al.,

2010)

Native Species Municipal Wastewater NH4-N 64.2 4.4-11.5f NR (Craggs et al., 2012) NH3-N=ammonia nitrogen, NH4-N=ammonium nitrogen, NO3-N=nitrate nitrogen, TN=total nitrogen, PO4-P=phosphate phosphorus, TP=total phosphorus, NR=not reported

a. estimated from: (max biomass density-initial biomass density)/ batch growth period (days) b. determined from final hydrocarbon concentration divided by final biomass concentrationc. calculated from oil productivity/biomass productivityd. calculated from hourly biomass productivitye. average hydrocarbon contentf. areal productivity (g m-2 d -1)

1 9


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Table 3 shows the dearth of microalgae strains shown to be capable of nutrient

removal and lipid synthesis in diluted wastewater. An et al. (2003) and Shen et al. (2008)

separately studied the growth and nutrient remediation potential of freshwater

Botryococcus braunii cultured in diluted agricultural wastewaters. An et al. (2003) used

pretreated diluted piggery wastewater and determined B. braunii is resistant to high NO3-

concentration (788 mg L-1) and low NH4+ concentration (4 mg L-1) while maintaining

high biomass productivity (708 mg L-1 d -1) and moderate hydrocarbon content (11.2%).

An et al. (2003) indicated low NH4+ concentrations of this wastewater were beneficial in

keeping NH3 toxicity at a minimum. In diluted livestock wastewater, B.braunii showed

lower biomass productivity (84.8 mg L-1 d -1), higher lipid content (19%) while

maintaining high removal efficiencies for both TN (88%) and TP (98%) (Shen et al.,

2008). Diluted livestock wastewater attracted the contaminant green algae Chlorella sp.,

but Shen et al. (2008) observed the two strains could coexist.

Freshwater Chlorella strains are highly studied for nutrient removal and biofuel

production due to their relative robustness in a variety of culture conditions (Gonzalez et

al., 1997, Ogbonna et al., 2000). Chlorella sp. growth was stable in multiple effluents

from the municipal wastewater treatment process (Wang et al., 2010b). Chlorella sp. had

high nutrient removal efficiency (68.4-82.8%) in dilutions of influent wastewater,

wastewater after primary settling, effluent from secondary treatment, and the aqueous

portion of centrifuged sludge from secondary treatment. Chlorella zofingiensis grown in

100-L open ponds accumulated moderate lipid content (17.9%) and performed high

nutrient removal efficiency (97.5% of TN) (Huo et al., 2012). Natural light and


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temperature variance greatly affected the growth rate in their experiments. Gonzalez et al.

(1997) and Martinez et al. (2000) separately showed how Scenedesmus sp. cultures could

remove nutrients from diluted wastewater and produce valuable biomass. Gonzalez et al.

(1997) showed Scenedesmus dimorphus cultures caused nearly complete ammonium

nitrogen removal (90%) in diluted agro-industrial wastewater while Martinez et al. (2000)

showed Scenedesmus obliquus could accumulate up to 16% hydrocarbons when cultured

in diluted urban wastewater.

Marine strains are gaining interest due to the concerns of massive freshwater

requirements for large scale biomass cultivation. For example, Jiang et al. (2011)

cultivated Nannochloropsis sp. in sterilized seawater mixed with municipal wastewater at

multiple dilutions. Biomass productivity was higher in most dilutions than in traditional

f/2 media fertilizers (Guillard and Ryther, 1962). Addition of CO2 improved growth in

their seawater/municipal wastewater media. However, culture growth was not sustained

in dilutions of 80% and 100%, indicating toxic inhibition. Cultured in diluted syngas

wastewater and a novel panel bioreactor, Nannochloropsis sp. F&M-M24 productivity

was approximately 330 mg L-1 d -1 (Biondi et al., 2012). Further exploration of wastewater

nutrient removal by marine strains will be beneficial to decreasing nutrient and water

requirements in the microalgae cultivation process.

Attempts at outdoor cultivation of monocultures with wastewater have inherent

disadvantages due to predator contamination (Woertz et al., 2009). Combatting this

disadvantage is the use of native species of an environment, especially those born in

wastewater conditions. (Chinnasamy et al., 2010) showed a consortium of freshwater and


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marine strains grew favorably (39 mg L-1 d -1) in untreated diluted carpet mill wastewater.

Cultured in High Rate Algal Ponds and municipal wastewater, a microalgae-bacterial

consortium showed long term productivity and efficient wastewater treatment at massive

scale (>2 ML) (Craggs et al., 2012). While Table 3 focuses on nitrogen and phosphorus,

microalgae have also been known to uptake heavy metals (Wilde and Benemann, 1993)

and COD (Lau et al., 1995).

High NH4+ levels encountered in wastewaters can inhibit growth due to

dissociation to toxic free ammonia (NH3). Azov and Goldman (1982) tested the effect of

free ammonia (NH3) in several species of microalgae by adding various concentrations of

ammonium chloride (NH4Cl). Their results show the dissociation reaction between

ammonium and toxic ammonia is likely pH, temperature and strain dependent. They

explained marine and freshwater species were inhibited in a similar fashion by free NH3,

although different strains are more sensitive to NH3 than others. Chen et al. (2012a)

tested the effects of ammonia on settling of freshwater and marine strains. Their results

showed that NH3 concentrations between 38.37 and 57.31 mmol L-1 caused high settling

efficiencies in marine strains. Chen et al. (2012a) explained aqueous ammonia damaged

cellular structure and increased cell volume, leading to settling. Chlorella vulgaris

maintained stable growth at a total ammonia nitrogen concentration range of 20 to 250

mg L-1

(Tam and Wong, 1996). Additionally, Scenedesumus sp. showed inhibition in

NH4-N concentrations greater than 100 ppm (Park et al., 2010). Due to the concerns of

ammonia inhibition, proper dilution must be conducted to determine maximum loading

ratios for the specific strain of study.


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2.2.2 Anaerobic digester effluent as microalgae nutrient source

Anaerobic digestion (AD) is a fermentation process used for sludge stabilization

and degradation (Demirbas and Demirbas, 2010). AD uses biological organisms to break

down complex organic materials into simpler compounds using exo-enzymes without the

presence of oxygen. Carbohydrates, lipids, and proteins are converted into simpler

monomers, which are then converted to biogas (CH4 and CO2) by methanogenic

organisms (Khanal et al., 2010). A diagram of the AD process is shown in Figure 3.

Figure 3: Anaerobic digestion process schematic (adapted from Li et al., (2011a))

Many substrates have been used in the AD process to degrade organic matter and

produce biogas for useful energy. The most common feedstock for AD is municipal

waste sludge (Chandler et al., 1980), but the adaptation of digesters for degradation of

food wastes, animal wastes and lignocellulosic biomasses have been developed (Brown et

al., 2012). Additionally, biogas production using microalgae biomass after lipid

http://www.sciencedirect.com/science/article/pii/S1364032110002224


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extraction as a co-digestion substrate has been evaluated (Park and Li, 2012).

Nitrogenous compounds, typically in the form of ammonium, are used to maintain

adequate carbon to nitrogen ratio in anaerobic digesters. The reduction of ammonium is

not complete in anaerobic digestion because microorganisms in AD lack sufficient

autotrophic metabolism for inorganic nitrogen (Park et al., 2010). The digestate in AD is

defined as anaerobic digestion effluent (AD effluent). AD effluent is high in nitrogen and

phosphorus and is typically used as fertilizer for agricultural farmland (Li et al., 2011a).

However, concerns about eutrophication of public waterways (Anderson et al., 2002) has

increased regulation on direct fertilizer application and necessitated further treatment of

AD effluent. Traditional bacterial nitrification-denitrification is an option for this

wastewater stream, although costly organic carbon is required for most operating

conditions (Park et al., 2010). Utilization of AD effluent as a nutrient source for

microalgae has been researched as an alternative and selected literature are summarized

in Table 4.


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Table 4: Status of microalgae cultivations in anaerobic digester effluent

Microalgae

strain

AD feedstock Retention

Time

(days)

Total Nitrogen Total Phosphorus Biomass productivity

(mg L-1 d-1)

Lipid Content

(% dry weight)

Reference

InitialConcentration (mg/L)

Removal(%)

InitialConcentration

(mg/L)

Removal(%)

Chlorella sp. MunicipalSludge

12 225a 88.89 b 120a 16.67 b 6.8c NR (Yuan et al., 2012)

Chlorophyta MunicipalSludge

2d 42.6-81.4e 47.9-91.5 b 5.1-10.5 97.8±3.9 234±32 NR (Ruiz-Martinez et al.,2012)

Nannochloropsis

salina

Municipalwastewater

10 160 100 22.86 100 92 26-30 (Cai et al., 2013b)

Chlorella sp. LiquidManure

1d 200 72 1.52±0.04 57.9 6.83c 10.78±2.94 (Chen et al., 2012c)

Neochloris

oleoabundans

Dairy Manure 11 NR 90-95 NR NR 88.3±8 1.6±0.4f (Levine et al., 2011)

Chlorella sp. Dairy Manure 20 100-245 75.7-82.5g 15-30 62.5-70.1 70-90h 9.0-13.7f (Wang et al., 2010a))

Mixed Culture Dairy Manure 12 30e 96 2.6 99 2.8c 10-29 (Woertz et al., 2009)

Chlorella sp. Liquid SwineManure

7 105a 12.99 30a 79.08 32.9-182.9i 28-32 (Hu et al., 2012)

Scenedesmus sp. Piggery Waste 5c 90-100ae 88.9a NR NR 213 NR (Park et al., 2010)

Chlorella

minutissima

Poultry Litter 12 NR 60 NR 80 76


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A variety of AD effluent has been studied for nutrient removal and biomass

synthesis by microalgae. Yuan et al. (2012) cultured a wild strain of Chlorella sp. in

filtered, autoclaved and diluted AD effluent from small scale municipal activated sludge

digesters. Under 12/12 h light-dark cycle, maximum biomass productivity was 6.8 g m-2

d -1

with TN removal rate of 36.5 g m-3

d -1

and a TP removal rate of 6.5 g m-3

d -1

.

Constant illumination cultures showed more than double biomass productivity (15.6 g m-2

d -1). In Yuan et al. (2012)’s experiments, Chlorella sp. showed preference for ammonium

over nitrate, and biomass from these experiments was determined to be a valuable co-

digestion product for volatile solids reduction in an anaerobic digester. Polycultures with

multiple strains of Chlorophyta showed stable nitrogen (43-81%) and phosphorus (48-

92%) removal in diluted municipal sludge membrane digester AD effluent (Ruiz-

Martinez et al., 2012). The only marine strain tested was Nannochloropsis salina, which

showed complete nitrogen and phosphorus removal, high biomass productivity (92 mg L-

1 d

-1) and stable lipid composition in the optimal dilution (160 mg L

-1 TN) of municipal

wastewater AD effluent (Cai et al., 2013b).

Animal manure is the most common of agricultural AD feedstock. Chen et al.

(2012c) cultured wild Chlorella sp. in diluted manure AD effluent in a small open

raceway pond (20-L) and demonstrated similar productivity (6.83 g m-2 d -1) to Yuan et al.

(2012) in a 35 day semi continuous cycle (1 d HRT). Levine et al. (2011) tested multiple

dilutions of AD effluent on the growth and lipid productivity of Neochloris

oleoabundans, and also examined the effects of sterilizing AD effluent prior to use. Their

results showed that a 1:50 (v/v) sterilized AD effluent dilution provided low microalgae


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productivity (0.088 mg L-1 d -1), low FAME productivity (1.6 %) and high TN removal

(90-95%). The biomass productivity in their study was comparable to microalgae

cultured in commercial nutrients. However, non-sterilized diluted AD effluent increased

ciliate and bacteria contamination, which likely limited lipid productivity (Levine et al.,

2011). Chlorella sp. showed similar results at multiple dilutions of dairy manure AD

effluent, with 100% ammonia nitrogen, nearly 80% TN, and 70% TP depleted over a 22

day batch cycle (Wang et al., 2010a). Wang et al. (2010a) speculated that algal growth in

diluted AD effluent is directly affected by the availability of nutrients, stability of pH,

temperature, and initial inoculation density. Woertz et al. (2009) used low concentrations

of AD effluent in a mixed microalgae culture. Under these dilutions, the mixed culture

showed nearly complete removal of ammonia nitrogen (96%) and orthophosphate (96%),

while producing maximum algae biomass at 0.3 g L-1 and a large range of lipid contents

(10-29 %). Chlorella and Scenedesmus showed similar biomass productivity and lipid

content to each other in diluted piggery waste AD effluent (Park et al., 2010, Singh et al.,

2011b). Additionally, a consortium of several freshwater strains had sustained growth (79

mg L-1 d -1) in small volume (200 mL) reactor using piggery waste AD effluent and 12/12

h light-dark cycle (Singh et al., 2011b). Compositional analysis of biomass showed Singh

et al. (2011b)’s consortium had high protein and carbohydrate content, indicating its use

as a fertilizer may be more useful than for biofuel. The previous research in this area

typically use systems exposed to idealized culture conditions. Analysis of the operational

parameters affecting outdoor cultivation systems is critical to obtaining realistic

prediction of biomass productivity using diluted AD effluent at large scale.


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2.3 Effects of Operational and Climatic Conditions on Microalgae Productivity

2.3.1 Effect of cultivation parameters on microalgae productivity

Table 5 describes the general effects of a specific cultivation parameter on

microalgae specific growth rate and lipid productivity when holding other parameters

constant. The symbol (+) indicates an operational parameter causes has a direct positive

effect while (-) indicates a negative relationship. Strain-by-strain differences may exist,

but the reviewed studies give general insight to the effect of each parameter on

microalgae production. While these operational conditions can be controlled in

photobioreactors, there is limited control in open raceway ponds.

Table 5: Operational parameters effects on photoautotrophic microalgae growth

Parameter Effect on specific growth rate Effect on lipid

production

Reference (s)

Temperature (+) to optimum Strain dependent (Geider, 1987)(Roessler, 1990)

Light Intensity (+) to a saturation level Strain dependent (Sorokin and Krauss, 1958)(Shifrin and Chisholm, 1981)

Nitrogenconcentration

(+) to a saturation level (-) with increasingnutrient concentration

(Roessler, 1990)(Shifrin and Chisholm, 1981)

Salinity Strain dependent Strain dependent (McLachlan, 1961)(Cohen et al., 1988)

pH (-) pH >8.8 Strain dependent(Chen and Durbin, 1994)

(Cohen et al., 1988)

Nitrogen Source (-) with increasing NH4+

Strain dependent (Lourenco et al., 2002)

Carbon Dioxide (+) to a saturation level Strain dependent(Clark and Flynn, 2000)

(Tsuzuki et al., 1990)


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acid and oleic acid content increased when elevated from low CO2 concentration to high

CO2 concentration, a variety of microalgae strains showed varying lipid and fatty acid

production under similar growth conditions (Tsuzuki et al., 1990).

Besides inorganic carbon in the form of CO2, nitrogen is quantitatively the most

important nutrient contributing to microalgae biomass, with nearly 1% to 10%

constituting the dry weight (Perez-Garcia et al., 2011). While many reports have

identified nitrogen and phosphorus limitation as a potential strategy for lipid

accumulation (Shifrin and Chisholm, 1981, Dean et al., 2010, Hsieh and Wu, 2009, Li et

al., 2008), these nutrients are important for maintaining cellular nitrogen to phosphorus

(N/P) ratio. There exists a trade-off between lipid accumulation and biomass production,

and optimization of cultivation parameters is required for desired biomass productivity

and composition (Wang and Lan, 2011). Light intensity, typically reported in terms of

photosynthetic active radiation (PAR), has the most significant impact on the growth rate

of most photosynthetic strains. It is simple to measure but difficult to control in outdoor

cultivation systems.

2.3.2 Effect of light availability on microalgae productivity

Light intensity is a limited parameter in large scale cultivation (Ogbonna et al.,

1995). Light intensity is not homogeneous throughout a growth system, especially when

high biomass density causes the “self-shading effect.” Open raceway ponds rely on

available sunlight, meaning seasonal variation likely has significant impact on microalgae

biomass and lipid productivity. Addition of wastewater will increase turbidity and likely


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decrease light penetration efficiency. Variation of light cycles, or limited light

availability, attempts to provide realistic estimates of large scale production under natural

conditions. Many studies use a 12/12, 14/10, 16/8 and 18/6 h light-dark cycles in order to

determine the effects of natural lighting on microalgae biomass and lipid production A

summary of selected literature studying the impact of light availability on microalgae

productivities for multiple strains are shown in Table 6.


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Table 6: Previous experiments evaluating the effects of light availability on microalgae production

Microalgae

strain

Nitrogen

source

Operating

Mode

Retention Time

(days)

Light Availability

(h light/h dark)

Temperature

(°C)

Biomass

Productivity

(g L-1d-1)

Lipid Content

(% of dry

weight)

Reference

Marine Strains f/2 media Batch 1-2 12/12 23 Varied Varied (Shifrin and

Chisholm, 1981)Chlorella kessleri Sludgecentrate

Batch 11 4/20 19-21 0.043-0.058 8.04-10.63 (Li et al., 2011b)

Chlorella

prothecoides

Sludgecentrate

Batch 11 4/20 19-21 0.040-0.168a 7.36-24.06 (Li et al., 2011b)

Scenedesmus

obliquus

Jaworskiformulation

Semi-continuous

5-7 b Seasonal Variance Seasonal Variance 0.0089-0.0143c 13-14d (Hulatt andThomas, 2011)

Nannochloropsis

gaditana

Commercialnutrients

Semi-Continuous

2.5f 12/12 25 75 ± 3e 1.5-2.5g (Fabregas et al.,2002)

Scenedesmus

obliquus

Artificialwastewater

Semi-Continuous

1 14/10 17-25.5 0.100-0.150 NR (Voltolina et al.,2005)

Chlamydomonas

reinhardtiiHarris media Batch NR 16/8 15-25 0.10-0.25 j NR (Janssen et al.,

2000) Nannochloropsis

oculataf/2 media Batch 28 12/12 19-21 0.0204 150-200h (Lee et al., 2011)

Haematococcus

pluvialiis

Bristol media

with NaNO3

Continuous 1.7 Seasonal Variance Seasonal Variance 13.2c NR (Chaumont and

Thepenier, 1995) NR=not reporteda. calculated from: (max biomass density-initial biomass density)/(batch growth period (days)

b. kept biomass density between 1.5 and 3.5 g L-1

c. areal productivity in g m -2 day-1 d. lipid content calculated from areal productivity/biomass productivitye. steady state cell density (*10^6)f. 40% per day renewal rateg. pg of lipids per cell (40%)h. grams of FAME per g oili. light duration in square-wave light/dark cycle

j. g microalgae per mol of light intensity

3 2


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Shifrin and Chisholm (1981) studied the effects of nitrogen deprivation, silicon

depravation, and a 12/12 h light-dark cycle on the growth of several species of

microalgae. The light-dark cycle caused cell productivity to follow a distinct sinusoidal

pattern. Both cell mass per volume and lipid content decreased as soon as light was

unavailable and increased during the light period. This was attributed to the consumption

of lipids for energy in the dark period. When light was available and nutrient conditions

constant, lipid content was unaffected. Therefore, in the context of light cycles, biomass

productivity is likely the critical metric of importance for open cultivation systems.

Li et al. (2011b) cultured two Chlorella strains in dilutions of the liquid portion of

waste activated sludge under a 20/4 h light-dark cycle and under constant illumination.

This group also tested the effect of CO2 supplementation on growth productivities and

FAME content. Their results showed that decreasing light availability under both air

supplementation and 5% CO2 supplementation caused a decline in biomass productivity

when compared to continuous illumination cultures. Increased light intensity caused an

increase in biomass concentration for both strains. FAME content and TN uptake were

unaffected by the decrease in light availability. Chlorella prothecoides showed higher

biomass and lipid productivity, likely due to the heterotrophic nature of this strain, in

which organic carbon can be utilized for microalgae metabolism in the dark (Perez-

Garcia et al., 2011).

Hulatt and Thomas (2011) cultured Scenedesmus obliquus during the spring and

summer months in a horizontal tubular photobioreactor in the United Kingdom

(lat.53.3827°N). This strain showed a mean monthly biomass productivity of 14.26 g m-2


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each day. However, they noted a decrease in productivity during the highest light

intensity period (9:00-12:00) followed by an increase until 18:00. It was indicated that

photo-oxidation, or high O2 concentration, caused photosynthetic inhibition during the

high light intensity period. Biomass concentration declined from 1.3 g L-1 to 0.9 g L-1

during the dark phase, indicating no growth during non-lit periods.

Open raceway ponds will face fluxes in both light intensity and light availability

during a growing season. Therefore, further research on the effects of light availability on

microalgae production will improve the predictability of outdoor cultivations.

Additionally, temperature effects are critical for outdoor systems in seasonal climates.

Although some outdoor cultivation was evaluated, further research that varies light

intensity and light availability is required. Culturing marine strains with wastewater

nutrients in open raceway ponds is a limited area of research and provides an opportunity

to reduce both freshwater and nutrient costs for large scale endeavors.

Nannochloropsis salina is a promising marine strain for nutrient removal and

biofuel synthesis. Laboratory results have shown extremely high lipid content (70% of

dry weight) under commercial nutrient starvation (Shifrin and Chisholm, 1981). In open

ponds and commercial nutrients, productivity of N. salina has reached 24.5 g m-2

d -1

(Boussiba et al., 1987). N. salina has also been shown to be highly productive in

municipal AD effluent (Cai et al., 2013b). Cultivation of N. salina in diluted AD effluent

under simulated seasonal conditions and in open raceway ponds could improve prediction

of large-scale productivity. Eicosapentaenoic acid (EPA, C20:5) is the characteristic fatty

acid of N. salina (Van Wagenen et al., 2012).


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Chapter 3: Cultivation of Nannochloropsis salina in Diluted Anaerobic Digester

Effluent under Simulated Seasonal Climatic Conditions

In this chapter, specific growth rate, lipid, fatty acid production and nitrogen

removal of Nannochloropsis salina were evaluated using municipal wastewater anaerobic

digester effluent as a nutrient source under simulated seasonal light exposure and

temperature conditions. Under constant light intensity and 24-h light availability, the

favorable AD effluent loading ratio was 7% (v/v), corresponding to a total nitrogen (TN)

concentration of 200 mg L-1. At this TN concentration, specific growth rate was 0.327 ±

0.016 d -1, biomass productivity was 204 ± 12 mg L-1 d -1 and TN removal was 97%.

Introduction of N. salina to varied light intensity (39-116 µmol m-2

s-1

) and 24 h light

availability cultures caused a decline in biomass productivity (204 to 48 mg L-1d -1), 29%

increase in eicosapentaenoic acid (C20:5) and 43% increase in C20:4 content. Light

exposure significantly affected specific growth rate (p


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3.1 Introduction

Microalgae has been proposed as a promising biofuel feedstock due to high oil

content, high growth rates and non-competition with food production (Sheehan, 1998).

Based on life cycle environmental assessments and techno-economic analyses, massive

nutrient requirement and freshwater use are two primary issues that hinder this process

from reaching commercial development (Amer et al., 2011, Singh et al., 2011a, Singh et

al., 2012, Yang et al., 2011).

Anaerobic digester effluent (AD effluent) is a nutrient-rich wastewater stream

produced in the anaerobic digestion process, and has great potential to meet the nutrient

demand for microalgae cultivation (Chen et al., 2012c, Cordoba et al., 2008, Levine et al.,

2011, Park et al., 2010b). Since AD effluent usually contains high concentration of

ammonium, dilution is needed to mitigate the inhibitory effect of ammonia on microalgal

growth (Azov and Goldman, 1982, Park et al., 2010b). In most of the previous studies,

sterilized AD effluent was diluted in freshwater for cultivating freshwater microalgae.

Freshwater strains have shown high risk of bacterial contamination, and concerns about

depletion of freshwater resources and the high cost of sterilization hinder this process. An

alternative and more promising approach is to grow marine microalgae using nutrients

from AD effluent, so seawater or brackish water could be used to reduce the consumption

of freshwater, and decrease the risk of bacterial contamination by high osmotic pressures

(Batan et al., 2010).

Cai et al. (2013b) investigated cultivation of a marine microalgae strain,

Nannochloropsis salina, in artificial sea salt media using AD effluent as a nutrient source


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under constant illumination and temperature, and observed relatively high biomass

productivities. However, it is unclear if this technology is appropriate for commercial-

scale cultivation under outdoor conditions with seasonal climatic variations in

temperature, light intensity and light availability. Additionally, in order to assess the

suitability of replacing commercial nutrients with AD effluent, the performance of

microalgae cultivation using diluted AD effluent needs to be compared to commercial

nutrients at equivalent nutrient concentrations.

In this chapter, specific growth rate, nutrient removal, lipid and fatty acid

production of Nannochloropsis salina were evaluated using AD effluent as a nutrient

source under simulated seasonal temperature, light intensity and light availability

conditions. For the purpose of comparison, all experiments were also conducted with

commercial nutrients under equivalent total nitrogen (TN) levels.

3.2 Materials and Methods

3.2.1 Microalgae strain and seed culture

The marine microalgae N. salina (849/6) was obtained from the Culture

Collection of Algae and Protozoa (CCAP, Oban, Scotland). Seed cultures were cultivated

in 2-L reactors (1-L working volume) in f/2 media (Guillard and Ryther, 1962)

containing the following ingredients: 0.075 g L-1 NaNO3, 0.00565 g L-1 NaH2PO4⋅2H2O,

1 ml L-1 trace elements stock solution, and 1 mL L-1 vitamin mix stock solution. The

minor ingredients in the trace element stock solution included Na2EDTA, FeCl3⋅6H2O,CuSO4⋅5H2O, ZnSO4⋅7H2O, CoCl2⋅6H2O, MnCl2⋅4H2O, Na2MoO4⋅2H2O, and biotin,


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while the vitamin stock solution contained cyanocobalamin (vitamin B12), and thiamine

HCl (vitamin B1). Seed cultures were cultivated under constant illumination (200 µmol

m-2

s-1

, 24 h light availability) at 20°C and salinity of 20 parts per thousand (ppt) and air

flow rate of 450 mL min-1.

3.2.2 AD effluent

AD effluent was collected from a commercial-scale liquid anaerobic digester (KB

Compost Services, Akron, OH, USA) which uses municipal wastewater from the city of

Akron, Ohio as feedstock. The digester is coupled with a D5LL solid bowl decanter

centrifuge (ANDRITZ AG, Graz, Austria) that runs continuously at 3200 rpm. About

1030 L of AD effluent, the liquid portion separated by the centrifuge, was collected and

stored at 4°C before use.

3.2.3 Cultivation of N. salina at different loading ratios of AD effluent

Each reactor consisted of a 2-L glass bottle (1-L working volume) equipped with

a rubber stopper and stainless steel tubing with 4.76 mm diameter for air inlet/outlet to

the reactor. Reactors were placed in a chamber coated white to thoroughly distribute light

illumination. The 32-W GE F32T8-SPX50 fluorescent lamps (GE Lighting, Ravenna,

OH, USA) provided average photosynthetic photon flux of 200 µmol m -2 s-1 measured by

BQM quantum meter (Apogee Instruments, Logan, UT, USA). Ambient air (0.039%

CO2) was provided at a pressure of 69 kPa (10 psi) to provide an average air flow of 450

mL min-1. The reactors were filled to 1-L with AD effluent according to loading ratio,

deionized water, and seed culture to reach an initial optical density of 0.5 at 440nm.


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Commercial nutrients (Proline f/2 algae feed, Pentair Aquatic Eco-Systems, FL, USA)

were used in equivalent reactor systems at similar added TN concentration and served as

the experimental control.

Instant Ocean Sea Salt® was added to each reactor to reach a final salinity of 20

parts per thousand (ppt). Each reactor was cultivated under constant illumination (200

µmol m-2 s-1, 24-h light availability) at 20°C in a ten-day batch cycle. DI water was added

daily to replenish the original working volume of each reactor. Table 7 displays the

loading ratios and equivalent TN concentrations added. Each experimental trial was

performed in duplicate.

Table 7: Experimental design for the study of loading ratio effects (n=2)

Treatment AD effluent

Loading

(% by volume)

Commercial nutrients

Loading

(% by volume)

TN Concentration

Added

(mg L-1

)

1 0 0 0

2 2.1 0.12 60*, 50+

3 4.2 0.21 120*, 100+

4 7.0 0.42 200

5 10.5 0.62 300

6 14.0 0.84 400

*=AD effluent +=commercial nutrients

3.2.4 Cultivation of N. salina in diluted AD effluent under varied illumination and

temperatures

The favorable nutrient loading from the loading ratio experiment corresponded to

an AD effluent loading of 7.0 % (v/v), commercial nutrients loading of 0.42% (v/v) and


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TN concentration of 200 mg L-1. Six 2-L reactors (1-L working volume) were filled with

70 mL AD effluent, deionized water, and seed culture (in exponential growth) to bring

reactors to an initial volume of 1-L, initial optical density of 0.5 (@440nm), and initial

TN concentration of 200 mg L-1. Instant Ocean Sea Salt was added to reach a final

salinity of 20 ppt. Two reactors were exposed to 24-h light availability, two reactors were

exposed to 12-h light availability, and two reactors were exposed to 6-h light availability.

Six additional reactors were exposed to the same light availability conditions with

commercial f/2 media as the source of nutrients (TN=200 mg L-1

).

Each reactor was placed in custom polyvinyl chloride (PVC) cylindrical holders

with removable caps in order to limit light penetration solely through the top of each

reactor. Figure 4 describes the reactor system used for the study of simulated seasonal

climatic conditions.

Figure 4: Experimental set-up for simulated seasonal climatic cond

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