Cultivation of Nannochloropsis Salina and Synechocystis Sp. PCC6803 in Anaerobic Digestion Effluent for Nutrient Removal and Lipid Production

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Cultivation of Nannochloropsis salina and Synechocystis sp. PCC6803 in Anaerobic Digestion Effluent for Nutrient Removal and Lipid Production

THESIS

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

Ting Cai

Graduate Program in Food, Agricultural and Biological Engineering

The Ohio State University

2012

Master's Examination Committee:

Dr. Yebo Li, Advisor

Dr. Jiyoung Lee

Dr. Jay Martin

Ting Cai

2012

Abstract

Energy demands and climate change are among the major issues that challenge the development and sustainability of our society. Biofuels are one of the most promising substitute energy resources for fossil fuels because they are renewable and help reduce greenhouse gas emissions. Microalgae are a group of organisms living in a wide range of environments and have the potential to fix carbon dioxide and produce biomass more efficiently than other plants. Thus, microalgal biomass is believed to be one of the most promising feedstocks for biofuel production. The water and nutrients required for the growth of microalgae could be supplied from wastewater. Open ponds and photobioreactors are the two main types of algae cultivation systems, with the former often being more suitable for large scale biomass production due to low capital costs. However, contaminants and water loss through evaporation are two major concerns for open pond systems.

Anaerobic digestion (AD) is used worldwide to treat organic waste and produce biogas as a renewable energy. The digester liquor (AD effluent) is rich in nitrogen and phosphorus and is usually land applied as fertilizer. When there is not enough land within an economic transportation range, AD effluent needs specific treatment before being discharged. The abundant nutrients in AD effluent make it a good nutrient supplement for

ii algae cultivation. Most of previous research about using AD effluent to grow algae has focused on freshwater algal species in digested wastewater from livestock production systems. However, there are no reports on the study of seawater algae Nannochloropsis salina and Synechocystis sp. PCC6803 in digested municipal wastewater.

The approach of cultivating marine algae N. salina in AD effluent has several advantages. Firstly, the high salinity growth conditions for N. salina could help reduce the invasion of other species. Secondly, the lipid content of N. salina is in the range of

21-36%, making it a good candidate for lipid production. In addition, the use of AD effluent could not only provide nutrients required by N. salina, but also make up the water loss due to daily evaporation. Cyanobacteria Synechocystis sp. PCC6803 is a robust strain which could survive in a wide range of salinities. Its potential for producing large amounts of biomass for bioenergy production is also attractive because of its high growth rate.

In this study, the growth rate, nutrient removal efficiency, and biomass and lipid productivity of N. salina and Synechocystis sp. PCC6803 were evaluated in both batch and semi-continuous cultivation systems using digested municipal wastewater as nutrient supplement. Batch results showed that N. salina grew well at AD effluent loadings of 3,

6, 12, and 18%. In contrast, the growth of Synechocystis sp. PCC6803 was gradually inhibited as the effluent loading increased from 3% to 24%, with a highest growth rate of

0.717 d-1 obtained at 3% loading. For both N. salina and Synechocystis sp. PCC6803, the nutrient removal efficiency decreased as the effluent loading increased, while N. salina was more efficient than Synechocystis sp. PCC6803 for nutrient removal. Although the

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highest biomass yield of Synechocystis sp. PCC6803 was 1.6 times that of N. salina, its

lower lipid content resulted in lower lipid production. The optimal growth rate of N.

salina and Synechocystis sp. PCC6803 was obtained at 6% and 3% effluent loading,

respectively.

The semi-continuous studies were carried out for 18 days using these optimal

loading rates at different harvesting frequencies (1-, 2-, 3-day intervals) and harvesting

ratios (25% and 50%). Compared to the batch study, both biomass and lipid productivity

of N. salina were improved in the semi-continuous study at the same effluent loading.

The highest lipid productivity of N. salina was 38.7 mg L-1 d-1 with 35.3 mg L-1 d-1

nitrogen removal and 3.8 mg L-1 d-1 phosphorus removal, when harvested every two days

at a 50% harvesting ratio. The biomass productivity of Synechocystis sp. PCC6803 was

40% higher than that of N. salina, but its maximum lipid productivity (29.1 mg L-1 d-1), obtained at a 50% harvesting ratio and daily harvesting, was lower than that of N. salina.

This study demonstrated the feasibility of cultivating N. salina and

Synechocystis sp. PCC6803 in AD effluent for nutrient removal and for biomass and lipid

production. The results obtained from this study will be scaled up in pilot-scale and

commercial scale raceway ponds to evaluate the commercial potential of algae culture

with AD effluent.

Dedication

This document is dedicated to my family.

Acknowledgments

I want to give my deepest gratitude and appreciation to my advisor, Dr. Yebo Li, for his constant encouragement, tremendous support and guidance throughout my graduate study. I would like to thank professors serving on my committee: Dr. Jay Martin and Dr. Jiyoung Lee for their time, suggestions and encouragement. Thank Dr. Jiyoung

Lee for her precious advice and help with my research project. I also want to thank

OSU/OARDC for providing me with two honorable scholarships for my graduate study at OSU.

I would like to thank the staff members of Food, Agricultural and Biological

Engineering Department: Mr. Mike Klingmans for his great technical support for my experiment setup, Mrs. Mary Wicks for her time and extreme patience in correcting my writing and improving my thesis, Mrs. Peggy Christman and Mrs. Candy McBride for their kind help and administrative support.

I feel lucky to have been working with these excellent members in our lab.

Special thanks go to Mr. Stephen Y. Park for his generous help and enthusiastic support for my study. I want to thank Dr. Zhongjiang Wang, Dr. Xiaolan Luo, Ms. Fuqing Xu,

Ms. Lo Niee Liew, Mr. Shengjun Hu, Mr. Dan Brown, Mr. Phil Cherosky, and Mr. Jia

Zhao for their help and encouragement during my stay in Wooster.

Especially, I want to thank my dearest parents, my brother and my family for their deep understanding and infinite support for my study. Finally, I would like to thank my husband, Ye Yuan, for always standing by my side through the good and bad times. I would not have been able to finish this thesis without his love and support.

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Vita

May 1987 ...... Born - Yan’an, China

July, 2010 ...... B.S. Bioengineering, China Agricultural

University, Beijing, China

September, 2010 to present ...... Graduate Fellow, 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 Acknowledgments...... vi Vita ...... viii List of Tables ...... xi List of Figures ...... xii Chapter 1 Introduction ...... 1 Chapter 2 Literature Review ...... 6 2.1 Wastewater Characteristics and Eutrophication due to Nutrients Runoff ...... 6 2.1.1 Characteristics of different wastewater streams ...... 6 2.1.2 Eutrophication due to nutrients runoff ...... 11 2.2 Cultivation of Cyanobacteria for Nutrient Removal and Biofuel Production ...... 14 2.2.1 Use of cyanobacteria for nutrients removal from wastewater ...... 14 2.2.2 Bioenergy products from cyanobacteria ...... 18 2.3 Cultivation of Microalgae in Wastewater for Nutrient Removal and Biofuel Production ...... 27 2.3.1 Nutrients requirement of microalgae growth ...... 29 2.3.2 Cultivation of microalgae in wastewater for nutrients removal and lipid production ...... 32 Chapter 3 Cultivation of Nannochloropsis salina in AD Effluent for Nutrient Removal and Lipid Production...... 38 3.1 Introduction ...... 38 3.2 Material and Methods ...... 41 3.2.1 AD effluent and algae inoculum cultures ...... 41 3.2.2 Batch and semi-continuous cultivation of N. salina in AD effluent ...... 42 3.2.3 Analytical methods ...... 44 ix

3.3 Results and Discussion ...... 46 3.3.1 Characteristics of AD effluent ...... 46 3.3.2 Batch study ...... 47 3.3.2.1 The effects of effluent loading on microalgae growth and biomass yield 47 3.3.2.2 The effects of effluent loading on nutrient removal and lipid production 51 3.3.3 Semi-continuous study ...... 56 3.3.3.1 The effects of harvesting frequency and harvesting ratio on algae growth and biomass yield ...... 56 3.3.3.2 The effects of harvesting frequency and harvesting ratio on nutrient removal and lipid productivity ...... 59 3.4 Conclusion ...... 62 Chapter 4 Cultivation of Synechocystis sp. PCC6803 in AD Effluent for Nutrient Removal and Lipid Production ...... 63 4.1 Introduction ...... 63 4.2 Material and Methods ...... 66 4.2.1 AD effluent and inoculum cultures ...... 66 4.2.2 Batch and semi-continuous cultivation of Synechocystis sp. PCC6803 in AD effluent ...... 67 4.2.3 Analytical methods ...... 68 4.3 Results and Discussion ...... 68 4.3.1 Characterization of AD Effluent ...... 69 4.3.2 Growth curve and biomass yield of Synechocystis sp. PCC6803 in batch cultivation ...... 69 4.3.3 Nutrient removal efficiency and lipid production of Synechocystis sp. PCC6803 ...... 73 4.3.4 Performance of Synechocystis sp. PCC6803 in semi-continuous cultivation .. 77 4.4 Conclusion ...... 82 Chapter 5 Conclusions and Recommendations ...... 83 References ...... 85

List of Tables

Table 1 Total nitrogen (TN) and total phosphorus (TP) content of different wastewater streams ...... 8 Table 2 Nutrient removal, biomass productivity and lipid content of various genera of cyanobacteria and microalgae in different waste streams ...... 16 Table 3 Bioenergy productivity of various species of cyanobacteria ...... 20 Table 4 Nitrogen and phosphorus levels at different effluent loading ratios ...... 43 Table 5 Experimental design of semi-continuous experiment ...... 44 Table 6 Characteristics of the effluent ...... 47 Table 7 Growth rate of N. salina at different effluent loadings ...... 49 Table 8 Nutrient removal rate and lipid productivity of N. salina at different harvesting frequencies and ratios ...... 61 Table 9 Growth rate of Synechocystis sp. PCC6803 at different effluent loadings ...... 71 Table 10 Nutrient removal efficiency of Synechocystis sp. PCC6803 and N. salina in batch cultivation ...... 74 Table 11 Growth rate, biomass and lipid productivity of Synechocystis sp. PCC6803 and N. salina at different harvesting frequencies ...... 81

List of Figures

Figure 1 Algae cultivation in wastewater for bioremediation and biofuel production ..... 28 Figure 2 Algae growth curves at different effluent loadings ...... 49 Figure 3 Biomass yield of N. salina at different effluent loadings ...... 51 Figure 4 TAN, TN and TP removal rates at different effluent loadings ...... 53 Figure 5 Algal lipid contents at different effluent loadings ...... 54 Figure 6 Algal lipid yields at different effluent loadings ...... 55 Figure 7 Algal growth curves at different harvesting frequencies and harvesting ratios: (a) harvested every day with 25% or 50% culture removed; (b) harvested every 2 days with 25% or 50% culture removed; (c) harvested every 3 days with 25% or 50% culture removed ...... 57 Figure 8 Accumulated algal biomass yield at different harvesting frequencies and harvesting ratios in 18-day semi-continuous cultivation ...... 59 Figure 9 Blast analysis for strain identification ...... 67 Figure 10 The growth curve of Synechocystis sp. PCC6803 at different effluent loadings ...... 71 Figure 11 The biomass yield of Synechocystis sp. PCC6803 at different effluent loadings ...... 72 Figure 12 The lipid content and lipid yields of Synechocystis sp. PCC6803 at different effluent loadings ...... 76 Figure 13 Growth curve of Synechocystis sp. PCC6803 at different harvesting intervals: (a) 1-day; (b) 2-day; (c) 3-day ...... 78

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Chapter 1 Introduction

With increasing population and rapid developing industries, the world primary energy consumption, which was latest reported 12 billion tonnes of oil equivalent per year, continues to increase ( BP p.l.c., 2011). Fossil fuel accounted for more than 85% of the primary energy consumption, with oil (34%), coal (30%), and natural gas (24%) as the major energy sources, while nuclear energy, hydroelectricity and renewable energy accounted for 5%, 6% and 1.3% of the total primary energy consumption, respectively

(BP, 2011). Though still widely used, the conventional fossil fuels such as coal, petroleum and natural gas have caused serious environmental problems and at the same time they are depleted. It is one of the major challenges for current society to find sustainable and renewable energy resources for the future because the potential energy crisis may lead to global instability, economic recession, and may also affect people’s quality of life. Biofuel is among the top choices in renewable energy list.

Microalgae, which are environmentally friendly biofuel feedstock, have attracted increasing interest for commercial production. The cultivation of microalgae is favored because of the following advantages: (1) microalgae do not compete with crops for arable land and freshwater because they can be cultivated in brackish water and non-arable land;

(2) microalgae can grow rapidly and have high oil contents of 20–50% on a dry weight basis (Chisti, 2007); (3) microalgae have the ability to fix carbon dioxide, thus reducing

greenhouse gas emissions; (4) microalgae can utilize nutrients from wastewaters,

providing an alternative method for wastewater treatment; and (5) byproducts of

microalgae cultivation after lipid extraction, namely algae biomass residue, can be used

as a nitrogen source, such as a protein-rich animal feed or fertilizer for crops (Spolaore et

al., 2006). In summary, microalgae cultivation combines biofuel production, carbon

dioxide mitigation, and wastewater treatment, consequently displaying great application

potential. However, mass cultivation of microalgae requires large amounts of

freshwater/seawater and nutrients, which are among the major parts of costs for algal biofuel production.

Besides microalgae, cyanobacteria (blue green algae) are another group of

efficient photosynthetic microorganisms and have been the subjected to studying for

bioenergy production in recent years. Cyanobacteria could produce diverse types of

bioenergy including hydrogen, ethanol, diesel, and methane (Hallenbeck, 2012; Stal and

Moezelaar, 1997; Varel et al., 1988). Compared with microalgae or crops, less attention

has been placed on cyanobacteria for production of lipid-based biofuels because their

lipid content is usually lower than most microalgae (Parmar et al., 2011). However, the

fast growth rate of cyanobacteria during exponential phase could also lead to high lipid

productivity (Sheehan et al., 1998). Genetic modification of cyanobacteria has been

attempted to increase their lipid productivity (Liu et al., 2010). Nutrient removal from

wastewater by cyanobacteria was investigated due to their robustness and simple growth

needs and some strains of cyanobacteria were shown to be efficient for nitrogen and

phosphorus removal.

Large amount of water used for agricultural, municipal, and industrial purposes

result in issues due to superfluous wastewater generation. Excessive nutrients, such as

nitrogen and phosphorus, in the wastewater may cause eutrophication and upset the

balance of the ecosystem. Eutrophication as a serious environmental pollution problem

has become more widespread since the mid-20th century (Smith and Schindler, 2009; Le

et al., 2010; Dodds et al., 2009; Smith, 2003; Rodhe, 1969). The adverse impacts of eutrophication have brought severe damages to the environment, threatened human health, and added extra financial burden to the government and society (Dodds et al.,

2009). These facts reflect the urgent need of an economical approach for reducing nutrient runoff and recycling nutrient in wastewaters. An integrated microalgae cultivation and wastewater treatment system will help remove the nutrients in wastewater while producing useful products. Algal biofuel is to be economically viable if combined with wastewater treatment. Microalgae have been proven to be efficient in removing nitrogen, phosphorus, and toxic metals from a wide variety of wastewaters (Ahluwalia and Goyal, 2007; Mallick, 2002).

Anaerobic digestion (AD) is a mature technology used to decompose organic matter under oxygen-free conditions and produce biogas (Abbasi and Abbasi, 2010). It is applied worldwide for the treatment of agricultural wastes, industrial wastewater, and domestic wastewater, etc (Verstraete et al., 2005). Issues arise in the treatment of digester effluent. The digester liquor, which contains high amounts of nutrients such as ammonium, phosphate, etc., is typically disposed in agricultural fields and used as a

fertilizer (Salminen et al., 2001). However, it is concerned that the excessive application

of digested effluent in agricultural lands will become a potential source of nitrogen

pollution in farming areas (Salminen et al., 2001; Woli et al., 2004; Lei et al., 2007).

Therefore, an effective process for nutrients removal is necessary for the post-treatment

of the anaerobic digester effluent. AD effluent biological treatment by algae cultivation

is an appealing option, but the potential of using AD effluent as algae culture medium is

not yet demonstrated as an economic approach to recycle the nutrients and produce biomass.

A majority of algae strains that have been studied for the removal of nutrients from wastewater were freshwater algae (Hongyang et al., 2011; Min et al., 2011; Zhen-

Feng et al., 2011; Chaiklahan et al., 2010; Kong et al., 2010). However, the supply of freshwater for algae cultivation is a big issue where freshwater is lacking. Marine microalgae Nannochloropsis salina is a promising candidate for biofuel production due to high lipid content and they can grow in brackish water or seawater (Hoffmann et al.,

2010; Emdadi and Berland, 1989; Boussiba et al., 1987). Limited information about removal of nutrients by N. salina from AD effluent is available. Blue-green algae

Synechocystis sp. PCC6803 is found to be a very robust strain that lives in a wide range of environment and is able endure high salt stress. Its simple structure and well-known

genome information make it a popular subject for bioenergy production via genetic

modification and regulation (Huang et al., 2002; Schubert et al., 1993). The broad

existence of Synechocystis sp. PCC6803 in natural environment such as lakes and

reservoirs could also provide large amount of biomass feedstock for bioenergy

production. So far, no research on growing Synechocystis sp. PCC6803 in AD effluent

has been reported.

The first purpose of this study is to investigate the feasibility of cultivating

microalgae N. salina for nutrients removal and lipid production in AD effluent from a

commercial scale anaerobic digester fed with municipal wastewater. The digested

municipal wastewater had high concentrations of carbon, nitrogen and phosphorus, which

could support the growth of microalgae. However, high solids content and large bacteria

population in the original effluent may inhibit algal growth. Pre-treated by centrifugation to removal large particles and bacteria, the AD effluent was then diluted at different ratios before fed to algae. Batch cultivation was conducted to find the optimal effluent loading with which higher growth rate could reach. Semi-continuous experiment was aimed at finding the proper harvesting frequency and ratio in order to provide information for operation of four large algae ponds (one acre area) on a local farm to facilitate the commercialization of algae cultivation in AD effluent.

This study also compared the nutrients removal efficiency and lipid productivity

of N. salina and Synechocystis sp. PCC6803 for bioenergy production and treatment of

AD effluent. Although the lipid content of Synechocystis sp. PCC6803 is lower than N.

salina as reported, its high biomass productivity is attractive and may lead to high lipid

production. The robustness of Synechocystis sp. PCC6803 makes it less susceptible to

invasive species and reduces the risk of contamination in large ponds, thus easy to

maintain. The results from this study could also provide base-line data for growing

Synechocystis sp. PCC6803 in polluted water bodies in a controlled manner to help clean

the water and produce biomass at the same time.

Chapter 2 Literature Review

The increasing scarcity of water, rapid population growth, and increasing urbanization calls for efficient wastewater treatment and recycle in the world. Anaerobic digestion as a mature waste treatment process generates large amounts of nutrient-rich effluent. Excessive nutrients discharge to the environment will cause eutrophication, which promotes the growth of harmful algae. Algal fuel as a promising biofuel is to be economically viable if combined with wastewater bioremediation. Interests have been raised in the use of blue-green algae (cyanobacteria) for bioenergy production due to their high growth rate. Green algae are commonly studied for biofuel production and have been proved to be effective in removing nitrogen, phosphorus, and even heavy metals from various streams of wastewater.

2.1 Wastewater Characteristics and Eutrophication due to Nutrients Runoff

2.1.1 Characteristics of different wastewater streams

Wastewater refers to liquid waste discharged by domestic residences, commercial properties, industry, and agriculture and has a wide range of potential contaminants and concentrations. According to the 2008 Clean Watersheds Needs Survey, the amount of wastewater generated in the United States is 12 million t d-1 (U.S. Environmental

Protection Agency, 2002). The composition of wastewaters varies with sources: municipal wastewater, agricultural wastewater, industrial wastewater.

The increasing urbanization and expansion of urban populations results in

increased amount of municipal wastewater. A city with a population of 500,000 and

water consumption of 0.2 t d-1 per capita would produce approximately 85,000 t d-1 of wastewater (Pescod, 1992). Municipal wastewater contains small concentrations of suspended and dissolved organic and inorganic solids. Table 1 shows the levels of the nitrogen and phosphorus in different sources of wastewater. Compared with animal wastewater, municipal wastewater has less nitrogen and phosphorus. However, there are often considerable amounts of heavy metals such as lead, zinc, and copper in raw municipal sewage. Municipal wastewater treatment processes include three stages: primary treatment, secondary treatment, and advanced treatment. Buoyant and non- buoyant materials are separated in the first stage using physical or chemical methods.

Dissolved organics and colloidal materials are removed during the second stage by biological or chemical treatments. The removal of dissolved inorganic components, including nitrogen and phosphorus, takes place in the advanced treatment process through a number of different unit operations including ponds, post-aeration, filtration, carbon adsorption, and membrane separation. Microalgae cultivation for nitrogen and phosphorus removal from municipal wastewater has been most extensively studied

(Bhatnagar et al., 2010; Ruiz-Marin et al., 2010).

Table 1 Total nitrogen (TN) and total phosphorus (TP) content of different wastewater streams

Wastewater category Description TN TP N/P Reference(s) (mg L-1) (mg L-1) Municipal wastewater Sewage 15-90 5-20 3.3 (Bennette D. Burks and Minnis, 1994) Animal wastewater Dairy 185-2636 30-727 3.6-7.2 (Bradford et al., 2008; Barker et al., 2001) Poultry 802-1825 50-446 4-16 (Bradford et al., 2008; Yetilmezsoy and Sakar, 2008a) Swine 1110*-3213 310-987 3.0-7.8 (Barker et al., 2001; Millmier et al., 2000) Beef feedlot 63-4165 14-1195 2.0-4.5 (Bradford et al., 2008; Barker et al., 2001) Industrial wastewater Textile 21-57* 1.0-9.7† 2.0-4.1 (Sen and Demirer, 2003), (Chinnasamy et al., 2010) Winery 110* 52 2.1 (Mosse et al., 2011) Tannery 273* 21† 13.0 (Durai and Rajasimman, 2011) Paper mill 1.1-10.9 0.6-5.8 3.0-4.3 (Slade et al., 2004) Olive mill 532 182 2.9 (Ammary, 2004) Anaerobic digestion effluent Dairy manure 125-3456 18-250 7.0-13.8 (Wang et al., 2010a; Cho et al., 2011) 8 Poultry manure 1380-1580 370-382 3.6-4.3 (Yetilmezsoy and Sapci-Zengin, 2009; Yetilmezsoy and Sakar, 2008b) Sewage sludge 427-467 134-321 - (Montusiewicz et al., 2010) Food waste and 1640-1885* 296-302 - (El-Mashad and Zhang, 2007) dairy manure 3- * Total Kjeldahl nitrogen (TKN), † Total orthophosphates (PO4 -P)

It was reported that an annual average of 665 billion ton of water was used by

industries around the world between 1987 and 2003. The total water usage of the U.S.

was reported to be approximately a third of the world total (Newman, 2011). Although it varies depending on the source, most industrial wastewater normally contains more heavy metal pollutants and less nitrogen or phosphorus than other types of wastewater

(Ahluwalia and Goyal, 2007). Due to the limited amount of nitrogen and phosphorus, it is hard to achieve large-scale microalgae cultivation in industrial wastewater. Most research of microalgae cultivation in industrial wastewater is focused on the bioremediation of heavy metals (Ahluwalia and Goyal, 2007). Selection of strains with high metal sorption capacity is crucial to the achievement of high metal removal efficiency. So far, only a few algal species have been studied for metal sorption ability. There are several reports evaluating the nitrogen, phosphorus and heavy metal removal from industrial wastewater by algae, such as those from carpet industries (Chinnasamy et al., 2010).

Agricultural wastewater, which is mainly produced from livestock production, is another major source of wastewater. Current animal feeding operations in the U.S. generate more than 450 million ton of manure and manure-contaminated runoff water annually (Kellogg et al., 2000). Livestock operations have shifted from small and medium scale to large scale during the past decade. As a result, nutrients have become spatially concentrated in high livestock production areas (Kellogg et al., 2000). The wastewater produced from animal farms is often rich in nitrogen and phosphorus as shown in Table 1. Approximately half of the nitrogen in animal waste is in the form of ammonium, and half is in the form of organic nitrogen. Factors such as animal diet, age, usage, productivity, management, and location will significantly affect the actual nutrient

content in animal wastewater. The nitrogen-to-phosphorus (N/P) ratio is typically 2–8 for

dairy, swine and beef feedlot wastewater. The traditional treatment of animal manure is

land application as fertilizer. However, the nutrients in manure cannot be totally taken up

by crops due to different N/P ratio requirements of the plants and nutrient availability.

Therefore the excess nutrients accumulate in the soil, which can increase nutrient loss

through runoff, resulting in eutrophication in receiving waters. Other than manure,

agricultural runoff can also contain herbicides, fungicides, insecticides, and nitrate and

phosphate components from agricultural operations. Agricultural runoff may bring

particles, dissolved ions and molecules, and living microorganisms into receiving waters

and reduces the water quality.

Anaerobic digestion (AD) is a mature technology which uses microorganisms to

decompose organic waste and produce biogas. Many AD systems have been built all over

the world for municipal, industrial, and agricultural waste treatment (Verstraete et al.,

2005). Efforts have been focused on the optimization of biogas yield and degradation of the volatile solids (Mayer et al., 2009); one neglected aspect is the post-treatment of the

AD effluent. Most of the AD effluent is separated by a dewatering system into liquid and solid fractions. The solid portion is usually composted then marketed as potting media or soil amendment, while the liquid portion is traditionally used as fertilizer for land application (Mayer et al., 2009). Excessive land application of AD effluent can result in nitrogen and phosphorus runoff, and may contribute to eutrophication. Efficient and cost- effective nutrient recovery methods should be considered in order to reduce the risk of nitrogen and phosphorus pollution from AD. Compared with typical agricultural, municipal, and industrial wastewater, AD effluent contains relatively more nitrogen and

10 phosphorus and less carbon because the microbial activity during the digestion converts the carbon to methane (Wang et al., 2010a). The nitrogen in AD effluent is mainly in the form of ammonium (Singh et al., 2011).

2.1.2 Eutrophication due to nutrients runoff

Eutrophication, which can be human-caused or natural, is defined as an increase in the rate of supply of organic matter in an ecosystem (Nixon, 1995). Humans now have significant influence on almost every major aquatic ecosystem, and their activities have greatly changed the nutrients fluxes from the landscape to receiving waters. Agricultural run-off carrying fertilizers and untreated sewage effluent will cause human-induced eutrophication. Rapid agricultural intensification leads to application of vast quantities of fertilizer each year (Matson et al., 1997). It was estimated that the global production of nitrogen fertilizer will increase from current 80 million metric tonnes per year to 134 million metric tonnes per year by 2020 (Levy and Kashibhatla, 1994).

The application of animal manures as nitrogen fertilizer for croplands is another big source of nitrogen input to the ecosystem (Carpenter et al., 1998). Phosphorus (P) is an essential nutrient element for crop production and livestock agriculture. The balance in

P inputs in feed and fertilizer and P output in farm produce has been disrupted due to the rapid growth and intensification of crop and livestock farming in many areas. Surface runoff with P was accelerated by soil P building up due to excessive use of P fertilizer

(Sharpley et al., 2003). With the rapid development of AD technology, large amount of

AD effluent are being produced and the traditional disposal of AD effluent as agricultural fertilizer will also cause human-induced eutrophication.

Eutrophication as a serious environmental pollution problem has become more

widespread since the mid-20th century (Rodhe, 1969). According to the survey

conducted by the International Lake Environment Committee, 48% of lakes and

reservoirs in North America are eutrophic; in Asia & the Pacific, 54%; in Europe, 53%;

in South America, 41%; and in Africa, 28% (ILEC and Lake Biwa Research Institute,

1993). The adverse ecological impacts caused by eutrophication can be categorized based

on three aspects: (1) reducing the biodiversity and replacing the dominant species, (2)

increasing the water’s toxicity, and (3) increasing the turbidity of the water and

decreasing the lifespan of the lakes. The economic loss due to eutrophication in U.S.

freshwaters was estimated to be $2.2 billion per year (Dodds et al., 2009). One specific example of freshwater eutrophication is Ohio’s largest inland lake, Grand Lake St. Marys

(GLSM). Animal manure applied to the land in the lake’s watershed is one of the primary causes for nutrient overloading, while wastewater treatment plants surrounding the lake

contribute 5–10% of the phosphorus load to the lake (the Ohio Departments of Natural

Resources, 2010). The algal blooms in GLSM generate health concerns for the surrounding residents and directly affect the quality of drinking water, fishing and tourism industries which play a significant role in the local economy.

Eutrophication generally promotes the rapid growth of algae and plankton, which is known as algae blooms. Algae blooms are problematic in natural waters because they cause a severe reduction in water quality. Dissolved oxygen content and pH of the water are two of the major influences of algal blooms. The mass growth of algae during the day produces excessive oxygen and the water becomes supersaturated. While, at night, the algae consume most of the oxygen in the water and cause low dissolved oxygen levels, which may kill fish and other organisms in the water (Reid et al., 1990). Large algal

blooms can raise the pH of the water as high as 9.5 and many natural processes occurring

in the water are affected as a result (Moore et al., 1997). Algal bloom takes place when

one or more species of phytoplankton reproduces at a rapid rate, multiplying quickly in a

short time, which leads to visible discoloration (most often yellow, green, blue-green, red

or brown) of the water. Phytoplanktons are photosynthetic microscopic organisms that

live beneath the surface of almost all oceans and bodies of fresh water. Phytoplanktons

obtain energy from sunlight through the process of photosynthesis. Blue-green bacteria

(called cyanobacteria) is one group of organisms that belong to the phytoplankton

community and it is commonly found in algal blooms (Diersing, 2009). Many species of the bloom-forming cyanobacteria produce toxic secondary metabolites (cyanotoxins) and pose a potential risk to public health (Paerl et al., 2001). Therefore, research has been

focused on the study of cyanotoxins. With the world-wide occurrence of harmful

cyanobacterial blooms, there are more and more studied of cyanotoxins going on in

universities or research institute (Jaiswal et al., 2008). Cyanotoxins can be categorized

into three groups: hepatotoxin, neurotoxin and dermatoxin. Microcystins and nodularins,

which belong to hepatotoxin, are two most common cyanotoxins and they have many

structural variants (Rinehart et al., 1994).

There are about 2,000 species of cyanobacteria known and they are divided into

150 genera (Pulz and Gross, 2004). Not all cyanobacteria are toxic. Some species of

cyanobacteria can produce very high value bio-products and have been used in food

supplements, pharmaceuticals and cosmetics (Gantar and Svircev, 2008). Spirulina, also

known as Arthrospira, contains a high amount of essential amino acids, vitamins, and

fatty acids, and thus can be used as nutritional products or animal feed.

Immunomodulator produced by Lyngbya majuscule has potential use in pharmaceuticals

(Skulberg, 2000). Many bioactive compounds isolated from cyanobacteria have been used to substitute synthetic chemicals in cosmetics (Singh et al., 2005). Another unique feature of cyanobacteria is their simple growth needs, which makes it a viable option for

biofuel production. Biotechnologists have been working on the culture, screening, and genetic engineering techniques to exploit cyanobacteria for the cost-effective production

of high-value products (Balasubramanian et al., 2012; Hallenbeck, 2012; Quintana et al.,

2011; Brooks, 2010).

2.2 Cultivation of Cyanobacteria for Nutrient Removal and Biofuel Production

2.2.1 Use of cyanobacteria for nutrients removal from wastewater

Previous experiments showed that cyanobacteria tend to grow better with high concentrations of phosphorus and low N/P ratio (Havens et al., 2003). According to data collected from lakes with various N/P ratios, Smith et al. drawn a conclusion that N: P mass ratio lower than 22: 1 will more probably stimulate cyanobacteria dominance

(Smith, 1983; Smith et al., 1995). The reason for this phenomenon is that cyanobacteria are better at competing for nitrogen than other phytoplankton when N is scarce. Thus, when the supply of P increases, the concentration of N is relatively lower and N the limiting factor, which will make cyanobacteria the dominant species (Smith and Bennett,

1999). When grown in controlled environment such as ponds and reactors, many strains of cyanobacteria are shown ability to remove nitrogen and phosphorus from wastewater.

Extensive research has been done on cyanobacteria due to their robustness and simple growth needs.

Spirulina (Arthrospira) was cultivated in outdoor raceways under tropical

conditions to evaluate its ability to remove nutrients from anaerobic effluents of pig

wastewater (Olguin et al., 2003). The results of semi-continuous test showed that the

average annual biomass productivity of Spirulina is 11.8 g m-2 d-1, the ammonium

nitrogen removal rate is 13.6 mg L−1 d−1, with removal efficiency ranging from 84% to

93%, and total phosphate removal rate were 72%-85%. Since the culture system is

outdoor raceways, temperature and the depth of raceway ponds are two factors that have

major influences on the productivity. Average productivity in semi-continuous cultures was higher in summer than in autumn or winter. The productivity was 14.4 g m-2 d-1 and

15.1 g m-2 d-1 when the depth was 0.15 m and 0.20 m, respectively. The protein content of

biomass was not affect by season in batch test, but was affected by the time when the

biomass was harvested during each season in semi-continuous test. The average protein

content of the semi-continuous cultures was 48.9% ash-free dry weight. The ability of

nutrients removal of cyanobacteria Sprirulina was demonstrated in another study in olive

oil mill wastewater (OMWW). The OMWW was pretreated with sodium hypochlorite to

decrease the phenol concentration and turbidity. Maximum biomass production (1.7 g L-

1) was obtained when the OMWW is loaded at a rate of 10% of total volume with the

-1 -1 supplementation of 1 g L NaNO3 and 5 g L NaHCO3. It was observed that phenols, phosphorus and nitrogen were totally removed in 18-day batch test.

Table 2 Nutrient removal, biomass productivity and lipid content of various genera of cyanobacteria and microalgae in different waste streams

TN TP Lipid Biomass Category Genus and Waste Reactor type Retention Initial conc. Removal Initial Removal content productivity References species stream time (mg L-1) efficiency (%) conc. efficiency (% dry cell (g L-1 d-1) (d) (mg L-1) (%) weight) Cyanobacteria Oscillatoria sp. c Raceway 14 498 100 76 100 - - (Craggs et al., 1997) Phormidium c Phototbioreactor 2-3 12-17 53-62* 3-18 100 0.023-0.057 - (Laliberte et al., 1997) bohneri P. bohneri a Phototbioreactor 30 0.9-1.1 82* 0.08-0.15 85 - - (Dumas et al., 1998) Planktothrix c Phototbioreactor 9 43-59 26-40* 7.5 100 0.027-0.05 - (Margarita Silva-Benavides and isothrix Torzillo, 2012) Spirulina sp. d Raceway - - 84-96* - 72-87 1.44-1.51 - (Olguin et al., 2003) Spirulina sp. b Photobioreactor 28 167 100 20 100 0.1 7.1-16.9 (Markou et al., 2012) Spirulina b Raceway 15 2-3 96-100* 18-21 87-99 5.32-7.42 8.1-11.2 (Phang et al., 2000) platensis Synechococcus e Phototbioreactor 8 25.5 29-54** 6.7 77-88 - - (Aguilar-May and del Pilar elongatus Sanchez-Saavedra, 2009) (Garbisu et al., 1992) Chlorophyte Botryococcus c Flask 10 4.48-7.67 100** 0.04-0.39 100 - - (Sawayama et al., 1992) 16 braunii, B.braunii, c Photobioreactor 35 5.5 27.3** 0.08 62.5 0.4 - (Sawayama et al., 1994) Chlorella sp. c Photobioreactor 13-21 290 61† 530 61 3.46 4.7–6.3 (Min et al., 2011) Chlorella sp. c Flask 10 33-71 75-82* 6-201 83-91 - - (Wang et al., 2010b) Chlorella sp. d Flask 21 100-240 76-83 15-30 63-75 - 9-13.7 (Wang et al., 2010a) C. vulgaris a Flask 30 1074 89.5 180 92 1-1.38 - (Wang et al., 2010c) C. vulgaris d Flask 8 1722 93.6 111.6 89.2 0.4-0.76 - (Heredia-Arroyo et al., 2011) C. vulgaris a Photobioreactor 9 36.3 90-95* 111.8 10-60 - - (Gonzalez et al., 1997) C. vulgaris b Raceway 12 0.47-50.83 4.4-45.1* 0.07-4.01 33.1-33.3 0.1-0.2 40 (Lim et al., 2010) C. kessleri e Flask 12 129.6 81.1* - - - - (Lee and Lee, 2002) C. kessleri e Flask 3 168 8-19** 10-12 8-20 - - (Lee and Lee, 2001) Chlamydomonas c Photobioreactor 31 128.6 55.8† 120.6 17.4 2.0 25.25 (Kong et al., 2010) reinhardtii Scenedesmus sp. e Photobioreactor 0.2-4.5 14-44 30-100 1.4-6 30-100 - - (Zhang et al., 2008) Scenedesmus sp. d Photobioreactor 5 100 90* - - 0.2 - (Park et al., 2010) Scenedesmus sp. e Photobioreactor 14 5-15 83-99 0.2-1 99 0.15-0.65 30-53 (Xin et al., 2010b) Scenedesmus sp. c Flask 15 15.5 98.5 0.5 98 0.11 31-33 (Xin et al., 2010a) S. obliquus c Photobioreactor 0.2-8 27 79-100* 12 47-98 - - (Ruiz-Marin et al., 2010) * S. obliquus c Photobioreactor 8 27.4 79-100 11.8 55-98 0.024 27-34 (Martı́nez et al., 2000) + - - † * Ammonia nitrogen (NH4 -N), ** Nitrate (NO3 -N), Nitrite (NO2 -N), Total Kjeldahl nitrogen a. Animal wastewater; b. Industrial wastewater; c. Municipal wastewater; d. AD effluent; e. Artificial wastewater 16

The nitrogen and phosphorus removal ability of Planktothrix isothrix was studied in municipal wastewater (Margarita Silva-Benavides and Torzillo, 2012). The effects of mixing condition (shaking or not) and irradiance (20 and 60 μmol photons m−2 s−1) on growth of Planktothrix isothrix were tested. Planktothrix isothrix co-cultured with

Chlorella had the highest growth under unshaken condition, which corresponded to 80% nitrogen removal and 100% phosphorus removal. Due to the small cell size of the microalgae, harvesting technique is one of the bottlenecks for large scale production of algal biofuel (Singh and Dhar, 2011). Filamentous cyanobacteria with the ability of self- aggregation and sedimentation could help reduce the cost of harvesting process and offer an attractive option for large-scale cultivation of cyanobacteria for biomass production and wastewater bioremediation (Noüe and Proulx, 1988). Co-cultivation of the

Planktothrix and the unicellular Chlorella under unshaken condition could take advantage of the self-aggregation ability of filamentous Planktothrix and also make full use of the space in wastewater column because Planktothrix and Chlorella inhabit in different depth of wastewater.

Oscillatoria is a genus of marine filamentous cyanobacterium that inhabiting near-surface waters of tropical and subtropical seas and it is capable of fixing atmo- atmospheric nitrogen (Carpenter and Price, 1976). Cultivation of Oscillatoria in municipal wastewater diluted with seawater was performed in corrugated raceway ponds by Craggs et al.(1997) to evaluate its ability to removal ammonium nitrogen and orthophosphate in both batch and continuous experiments. Complete removal of ammonium and orthophosphate from wastewater diluted by seawater with 1:1 ratio was obtained in continuous running. No extra mixing was needed in corrugated raceway

system. The adherent property of Oscillatoria also simplified the biomass harvesting

mechanism.

Synechococcus is a widespread unicellular cyanobacterium in the marine

environment and makes important contributions to global carbon fixation (Scanlan,

2003). Synechococcus has been extensively studied since its discovery and identification

because of its beneficial features: 1) it is non-toxic; 2) it is able to removal nitrogen,

phosphorus and even heavy metals very efficiently; 3) it has high tolerance to a wide

range of temperature; and 4) it produces high-value products such as glutamate and

hydrogen (Sasikala and Ramana, 1994; Matsunaga et al., 1988; Ikeya et al., 1997).

Aguilar-May et al compared the growth rate and nutrients removal rate of chitosan-

immobilized and free cells of Synechococcus elongatus in artificial wastewater (Aguilar-

May and del Pilar Sanchez-Saavedra, 2009). It appeared that immobilized cell cultures

had a lag phase of growth due to the immobilization method, but their growth rate was

similar to that of free-living cell cultures. Free cells were more efficient in ammonia and

phosphorus removal than immobilized cells, but immobilized cells has higher protein

content.

2.2.2 Bioenergy products from cyanobacteria

Cyanobacteria are a special group of photosynthetic bacteria that are able to

convert sunlight, H2O and CO2 into carbohydrates, lipids and proteins, all of which are potential feedstock for bioenergy production. The lipids can be converted into fuels by chemical, biochemical, and thermochemical processes, or a combination of these approaches. Carbohydrates may be fermented under dark and anaerobic conditions to produce ethanol, with carbon dioxide as byproducts (Stal and Moezelaar, 1997).

Cyanobacteria may possess nitrogenase or hydrogenase that enables them to generate

hydrogen gas, which is a potential alternative to limited fossil fuels (Hallenbeck, 2012).

Methane is another bioenergy source that can be produced by anaerobic digestion of

cyanobacterial cellular biomass (Varel et al., 1988). Four types of bioenergy (hydrogen,

ethanol, diesel, and methane) that can be produced from cyanobacteria biomass are

discussed in the following paragraphs.

Hydrogen (H2) is a promising candidate as an ideal fuel because: first of all, it is

clean, the combustion of hydrogen only produces water, no any carbon dioxide; secondly,

it has high energy yield (122kJ g-1) (Lay et al., 1999). Besides energy carrier, hydrogen

also plays various roles in chemical synthesis and electrical storage. H2 is used as a

hydrogenating agent to increase the level of saturation of unsaturated fats and oils, and

also used in the production of ammonia and methanol (Das and Veziroglu, 2001).

Currently, nearly 90% of hydrogen is produced from natural gas or light oil fractions with

steam at high temperatures. Biological hydrogen production by photosynthetic

microorganisms has several advantages over hydrogen production by water electrolysis,

thermochemical processes and radiolytic processes. Biological hydrogen production can utilize renewable energy resources, such as the organic fraction of solid waste (Lay et al.,

1999), and it is normally operated at ambient temperature and atmosphere pressure. A

wide variety of species and strains have been examined for hydrogen production.

Bandyopadhyay et al. (2010) proved that Cyanothece sp. ATCC 51142, a unicellular,

diazotrophic cyanobacterium with the capacity to generate high levels of hydrogen under

aerobic conditions, can produce hydrogen at rates as high as 465 μmol per mg of

chlorophyll per hour in the presence of glycerol. Further research showed that

Table 3 Bioenergy productivity of various species of cyanobacteria

Fuel Species Productivity References

Ethanol Oscillatoria sp. 400 μmol L-1 d-1 (Heyer and Krumbein, 1991)

Synechococcus sp. 54 nmol L-1 d-1 (Deng and Coleman, 1999)

Synechocystis PCC6803 5.2 mmol L-1 d-1 (Dexter and Fu, 2009)

Hydrogen Anabaena variabilis 6.91 nmol μg-1 of protein h-1 (Sveshnikov et al., 1997)

Cyanothece sp. 465 μmol mg-1 of chlorophyll h-1 (Bandyopadhyay et al., 2010)

Spirulina maxima 400 μmol L-1 h-1 (Ananyev et al., 2008)

Lipids Synechococcus sp. 75 mg L-1 d-1 (Griffiths and Harrison, 2009)

Nostoc paludosum 1.9 g m2 d-1 (Vargas et al., 1998)

Synechocystis PCC6803 0.133 g L-1 d-1 (Liu et al., 2010)

Synechocystis PCC6803 6.4 nmol L-1 d-1 (Kaczmarzyk and Fulda,

Synechococcus sp. 8.4 nmol L-1 d-1 (Kaczmarzyk and Fulda,

Methane Spirulina maxima 0.33 L CH4/g VS (Varel et al., 1988)

S. maxima 0.09 L CH4/g VS (Inglesby and Fisher, 2012)

S. maxima 0.63–0.74 L CH4/g VS (Sialve et al., 2009)

S. platensis 0.47–0.69 L CH4/g VS (Sialve et al., 2009)

Cyanothece 51142 is able to use high concentrations of CO2 or glycerol for enhanced H2

production, making biohydrogen production by Cyanothece 51142 an attractive option because both of these carbon sources are abundantly available as industrial waste products. Anabaena species and strains also have high rate of hydrogen production according to Sveshnikov et al. (1997). Two mutants of Anabaena variabilis ATCC 19413 were studied and the results showed that mutants deficient in uptake and reversible hydrogenases have higher rates of H2 production compared with wild-type strains.

Mutant A. variabilis PK84 can produce H2 by rate of 6.91 nmol nmol/μg of protein/h in

350 ml cultures, which is more than 4 times of that of the wild type. Nitrogen and CO2

starvation could improve the H2 production by 1.8-1.9 and 1.4-1.5 times, respectively.

Bioethanol produced from renewable resources is another promising biofuel and it

has been used as vehicle fuel to reduce the use of fossil fuels in many countries. The

amount of ethanol fuel produced from sugarcane in Brazil and corn in U.S. accounts for

87.1% of the world’s total ethanol fuel production in 2011(Cuellar, 2012). However, the

use of agricultural crops such as sugarcane and corn for ethanol production remains

controversial as it competes with world’s food supply (Rittmann, 2008). Cyanobacteria

can secrete glucose and sucrose to the media and the anaerobic fermentation of these

sugars under dark conditions produces ethanol (Parmar et al., 2011). The advantage of

using cyanobacteria for ethanol production is that the fermentation process can be

performed by cyanobacteria themselves without addition of yeast as that in the

fermentation of traditional agricultural crops. Various strains have been screened and

evaluated for ethanol production. Heyer and Krumbein (1991) examined 37

21 cyanobacterial strains to test their ability of fermentation and secretion of fermentation products. Each strain was able produce at least one fermentation product, but only 16 strains could produce ethanol and only two Oscillatoria strains could secrete remarkable amounts of ethanol (>10 μmol/sample). Normally, cyanobacteria only produce a small amount of energy through fermentation to maintain cell activities. Genetic modification has been applied in order to increase the ethanol production of cyanobacteria.

Synechococcus sp. strain PCC 7942, a unicellular freshwater cyanobacterium, is one of the few cyanobacterial strains that have been relatively well-characterized in terms of physiology, biochemistry, and genetics. Deng and Coleman (1999) successfully transformed Synechococcus sp. strain PCC7942 with bacterial genes in order to create a novel pathway for ethanol production in this cyanobacterium. Ethanol is usually excreted to the medium by algae and cyanobacteria under dark and anaerobic conditions (Heyer and Krumbein, 1991). The new pathway created for ethanol synthesis in Synechococcus sp. strain PCC7942 functions during oxygenic photosynthesis and does not require anaerobic environment. It was anticipated that ethanol production at industrial scale could be achieved with the optimization of growth conditions and the development of ethanol retrieval or sequestering technologies. Besides sugars, cellulosic material is another substrate for ethanol production. Some cyanobacteria can also excrete cellulose to the media and provide feedstock for ethanol production. For example, Crinalium epipsammum, a filamentous cyanobacteria isolated from the surface layer of sandy soil of coastal dunes in the Netherlands was observed to produce cellulose of more than 25% of its dry cell weight, which is unusual for cyanobacteria (De Winder et al., 1990). Genetic

modification with the cellulose synthase genes from Gluconobacter xylinus enabled

Synchococcus sp. PCC 7942 to produce extracellular non-crystalline cellulose, which is an ideal feedstock for ethanol production (Nobles and Brown, 2008).

Compared with microalgae or crops, less attention has been placed on cyanobacteria for production of lipid-based biofuels because their lipid content is usually lower than most microalgae (Parmar et al., 2011). However, the fast growth rate of cyanobacteria during exponential phase could also lead to high lipid productivity

(Sheehan et al., 1998). Among the 2000 species of cyanobacteria identified, only a few have been examined for biodiesel production and limited information regarding the lipid content and production, or lipids profile is available (Miao and Wu, 2006). The lipid content of cyanobacteria is usually between 5%-15% (Quintana et al., 2011). Griffiths and Harrison (2009) reviewed the information about growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 chlorophyta and 5 cyanobacteria as well as other taxa and found that although the lipid content of most microalgae can be increased under nitrogen starvation, the lipid content of cyanobacteria was hardly increased, with Synechococcus showing a relatively high lipid content (11% of dry cell

weight) and lipid productivity (75 mg L−1 d−1). Vargas et al. (1998) analysed the

biochemical composition and fatty acid content of twelve strains of filamentous,

heterocystous, nitrogen-fixing cyanobacteria, which were all isolated from natural

environments. The data showed that the lipid content of the 12 strains varied between

8% to 11% of the dry weight, and the protein and lipid level reached the highest during

stationary phase. According to lipid profile analysis by gas chromatography–mass

spectrometry (GC-MS), total fatty acid levels in the strains assayed ranged between 3 and

5.7% of the dry weight. All strains showed high levels of polyunsaturated fatty acids

(PUFAs) and saturated fatty acids (SAFAs), with values of 24–45% and 31–52% of total

fatty acids, respectively.

Synechocystis sp. PCC6803, a freshwater cyanobacterium, is one of the most

extensively studied model organisms for the analysis of photosynthetic processes. Its

genomic, biochemical and physiological date have been thoughtfully reported, offering valuable information for genetic modification for this strain (Huang et al., 2002). Liu et

al. (2010) successfully applied a fatty acid secretion strategy in Synechocystis sp.

PCC6803 by introduced acyl-acyl carrier protein thioesterases into the cell. High levels of fatty acids could be produced and secreted into the medium by the mutant strains at an efficiency of up to 133 mg L-1 of culture per day at a cell density of 1.5 × 108 cells ml-1

(0.23 g of dry weight/liter). The effects of growth temperature on fatty acid composition

Synechocystis PCC6803 were studied by Wada and Murata (1990). They found that the

composition of the fatty acid did not change when the temperature kept constant.

However, a shift from higher temperature to lower temperature will stimulate the

desaturase activities of C18 and C16 acids, which is related to the photosynthetic electron

transport. Many biotechnology companies are working on the gene transformation of

cyanobacteria to increase their lipid content. It was reported that biologists from a

Seattle-based bioscience firm Target Growth Inc. (TGI) increased the lipid content of

cyanobacteria by about 400 percent (Roberts et al., 2011). However, they did not disclose

the lipid content of the genetic modified cyanobacterium, which made it hard to compare

this cyanobacterium with other lipid-producing algae. ExxonMobil invested $600 million

in Synthetic Geonomics for cyanobacteria based fuel development. BP Oil, the world’s

third-largest energy company, also tries to explore biofuel from cyanobacteria with $500

million investment to support the cyanobacteria research in the Arizona State University's

BioDesign Institute (Brooks, 2010). Joule Unlimited, a Massachusetts biotechnology

company, also holds a key patent about genetically-engineered cyanobacteria that can

simply secrete diesel fuel or ethanol when it is fed with sunlight, water and carbon

dioxide (Woods et al., 2001). It is said that the cyanobacteria will produce ethanol in

recoverable quantities of at least 1.7 mole ethanol per mg of chlorophyll per hour. The

company claimed that they are able to produce 75,708 liters of fuel per acre per year and

the product will be cost competitive with crude oil at $50 a barrel (Totty, 2012).

Methane, as a major component of biogas from anaerobic digestion (AD), is

produced by the biological breakdown of organic matter. The biomass or biomass residue after lipids extraction from microalgae and cyanobacteria is another source of feedstock for anaerobic digestion. The conversion of microalgal and cyanobacterial biomass into

methane also increases the energy recovery from the cellular biomass, enhancing their economic value. The methane yield and production rates of anaerobic digestion are affected by the feedstock characteristics, reactor design and operation conditions (Zhang et al., 2007). The biochemical composition (lipids, carbohydrates, protein) of the feedstock is a major factor that affects the microbial populations (hydrolytic, acetogenic, and methanogeneic bacteria) in the AD system. However, it was confirmed that cyanobacterial biomass alone is not favorable for supporting methanogenic activity

(Chellapandi et al., 2010). Varel et al. (1988) evaluated the methane production from fermentation of the cyanobacterium, Spirulina maxima, as the sole substrate. The ultimate methane yield of 0.33 L CH4/g VS fed was obtained after 105 days of batch fermentation, which is significantly lower than that of cattle or swine wastes. The biomass of Spirulina

maxima as the sole feedstock for an advanced flow-through anaerobic reactor was

investigated for production of methane and the maximum methane yield is 90±19 ml CH4 per g VS with a hydraulic retention time (HRT) of 10 days and an organic loading rate

(OLR) of 0.5 g L-1d-1 (Inglesby and Fisher, 2012) . There are mainly two factors that

influence the methane yield of the anaerobic digestion of cyanobacterial biomass. First of

all, the high protein content of algal and cyanobacterial biomass often results in the

release of high amount of ammonia into the reactor, which negatively inhibits the

microbial activities. The other factor is the high sodium concentrations brought by the

marine species may have negative impact on the AD reactors (Sialve et al., 2009).

Maintaining a proper C/N ratio by combining different substrates will increase the

performance of the digester and improve the biogas productivity (Mata-Alvarez et al.,

2000). Co-digestion of microalgal and cyanobacteria biomass with other feedstock is

necessary to improve the health of the AD reactor and improve the methane yield

consequently, making the microalgal biodiesel economically sustainable. When the C/N

ratio of the substrate is lower than 20, Excessive nitrogen released in the form of

ammonia will cause an accumulation of volatile fatty acids, inhibiting the microbial

activities (Sialve et al., 2009). Park and Li (2012) evaluated the methane yield of the co- digestion of algae biomass residue (ABR) with lipid-rich fat, oil, and grease waste

(FOG). The optimal methane yield of 0.54 L CH4/g VS d was observed when the algae

biomass residue was loaded at 50% loading rate, which also resulted in greater lipid

degradation than that on the digestion of 100% FOG or 100% ABR. Yen and Brune

(2007) studied the do-digestion of waste paper and algal sludge. The maximum methane yield (1.6L CH4/L d) was obtained when the algal sludge was mixed with waste paper at a ratio of 2:3. The optimal C/N ratio for co-digestion of algal sludge and waste water appeared to be 20-25 based on the results. Co-digestion of sewage sludge and Spirulina maxima was reported to increase the methane yield of digester by 2.1 fold (0.36L CH4/g

VS) when the mixing ratio is 1:1(Samson and Leduy, 1983) . It was proved that the addition of sewage sludge to algal biomass helped balanced the C/N ratio and significantly increased the activity of the methanogenic bacteria, leading to the improvement of methane production.

2.3 Cultivation of Microalgae in Wastewater for Nutrient Removal and Biofuel

Production

Compared with cyanobacteria, microalgae have gained more interests in the past few decades because of the dual benefits of cultivating microalgae for both nutrients removal and biofuel production. From 1978 to 1996, the Aquatic Species Program (ASP) funded by the U.S. Department of Energy (DOE) studies about 300 species, mostly green algae and diatoms, focusing on their ability to produce oils under normal and severe conditions such as extreme temperature, salinity or pH, in the meanwhile, utilizing waste

CO2 from coal fired power plants (Sheehan et al., 1998). The feasibility of large-scale algae production in open ponds was also tested in this program. Later, research focus

27 shifted to both biodiesel production and wastewater bioremediation by microalgae to enhance the sustainability of microalgae cultivation system (Figure 1). Various species of microalgae have been studies in different sources of wastewater and their nutrients removal efficiency was also reviewed by numerous researchers. So far, the bottlenecks of large-scale algae cultivation system lie in the downstream processing such as harvesting, lipids extraction process and biodiesel conversion process. The energy cost for those processes need to be balanced with the energy output of algae biofuel to make it economically viable. A complete life cycle analysis of the algae cultivation system is still needed to evaluate the sustainability of algal biofuel production.

Figure 1 Algae cultivation in wastewater for bioremediation and biofuel production

2.3.1 Nutrients requirement of microalgae growth

The growth of microalgae needs a variety of micro-/macro- elements which not

only constitute algae cells, but also help maintain their metabolism system. Except the

four basic elements, i.e., carbon, nitrogen, phosphorus and sulfur, ionic components such

as sodium, potassium, iron, magnesium, calcium, and trace elements must be provided in

algae cultivation (Richmond, 1986). Silicon is required for the cultivation of certain algae

strains (Andersen, 2005). The concentration of any nutrient element could be a limiting

factor for algae growth. Carbon is always supplied to the cells during cultivation by

pumping air or pure CO2, or flue gas into the cultures. Nitrogen to phosphorus ratio is a

mainly concerned factor when analyzing the nutrients compositions of the culture medium. It has been pointed out that the optimal nitrogen to phosphorus ratio for algae is

16:1 (Stumm and Morgan, 1970). Therefore, adjustment of nutrients concentrations may be needed in order to match the proper ratio. Most of the wastewaters contain the major nutrient components for algae growth. According to different sources, the composition of wastewater will vary significantly.

According to their ability of using organic or inorganic carbon source, microalgae are divided into three categories: autotrophic, heterotrophic, and mixotrophic. Currently, all of the three types of microalgae are being studied for biofuel production. Mixotrophic microalgae are preferred because they can utilize both organic carbons in the wastewater and CO2 from different resources. Microalgae are able to fix CO2 from atmosphere,

- 2- industrial exhaust gases, and soluble carbonates (HCO3 , CO3 ) (Wang et al., 2008). 29

Mitigation of CO2 from flue gas is another research focus of algae which would bring dual benefits to the environment and biofuel production (Zeiler et al., 1995).

Nitrogen is the second most important nutrient after carbon for microalgae and a number of nitrogen compounds are available for algae to assimilate: nitrate, nitrite, ammonium, and organic nitrogen including amino acid, peptides, and proteins. Among them, ammonium is the most preferable chemical form because it takes less energy for algae to assimilate into amino acid (Y. Collos, 1986). Ammonium tolerances of different algae species varies from 25 µmol N/L to 1000 µmol N/L (Y. Collos, 1986). Studies show that nitrogen concentration in the culture medium affects the speed of lipids accumulation by microalgae. Nitrogen starvation will increase lipids content of microalgae. However, the total lipids yield is usually offset by the low biomass yield under nitrogen starvation condition (Sheehan et al., 1998). Therefore, the nitrogen concentration in the medium is critical for optimal lipids production. Ammonia stripping is an undesirable phenomenon in algae cultivation ponds using wastewater. Studies showed that significant amount of ammonia will escape to the atmosphere when the pH is higher than 9 (Konig et al., 1987). Temperature is also a key factor affecting the speed of ammonia stripping. Huge surfaces of algae ponds exposing to warm weather will accelerate ammonia release even when the pH is below 9 (Green et al., 1996).

Inorganic phosphates play a significant role in algae cell growth and metabolism

- 2- (Theodorou et al., 1991). P in the forms of H2PO4 and HPO4 are preferred inorganic phosphates for algae growth. The consumption rate of P is affected by not only the concentration of P in the medium and the growth phase of algae, but also the pH, the 30

concentrations of other ions such as Ca2+, Mg2+, and temperature (Martínez et al., 1999).

In most cases, addition of P is needed for algae growth because of the precipitation of

phosphates in culture medium. Excessive phosphates added that were not utilized may

cause eutrophication if not processed properly before discharged, which is a big issue for

microalgae cultivation in wastewater (Yun et al., 1997).

Excessive P is the most common cause of eutrophication in freshwaters (Correll,

1998). The amount of P is always more limiting than other essential elements because C,

N can be obtained from the atmosphere. However, human activities contribute a lot to the

increase of P concentration in receiving water streams. The concentrations of P higher

than 20µg/L will be big problem for most waters (Correll, 1998).

Silicon is generally considered as one kind of macronutrients needed for growth

of diatoms, silicoflagellates, and some chrysophytes (Andersen, 2005). Silicate is added

as Na2SiO3·9H2O. However, it is better to omit it from the medium if the species do not

require silicate because silicic acid will enhance precipitation of some other essential elements. Microalgae growth depends not only on an adequate supply of essential

+ + 2+ 2+ − 2− macronutrients (C, N, P, Si) and major ions (Na , K , Mg , Ca , Cl , SO4 ) but also on

a number of micronutrients (iron, manganese, zinc, cobalt, copper, molybdenum,

selenium, etc.). But, many of the micronutrients are toxic to most algae species at high

concentrations. Some of them also form precipitations with other essential elements and

reduce their availability. However, some algae strains are particularly tolerant to heavy

metals and their potential to absorb metals was demonstrated in many studies (Mehta and

Gaur, 2005). 31

2.3.2 Cultivation of microalgae in wastewater for nutrients removal and lipid production

As reviewed in the first section, most wastewater contains carbon, nitrogen,

phosphorus and other essential elements that support the growth of microalgae.

Tremendous efforts have been put on research of microalgae cultivation in wastewaters in

the past decades. Studies showed positive results regarding the potential of utilizing

microalgae to remove nitrogen, phosphorus as well as heavy metals from wastewaters.

However, different species varied in the ability of nutrients removal and lipid production.

Chlorella is a genus of single-cell green algae which belongs to the phylum

Chlorophyta and it is one of the most extensively studied species for wastewater

bioremediation and lipid production (Kessler, 1976). Wang et al. (2010b) studied the

growth of a wild-type Chlorella sp. in municipal wastewaters collected from different

points of the treatment process flow of a municipal wastewater treatment plant. It was

found that this algae strain grew best in the centrate because of its much higher levels of

nitrogen, phosphorus, and COD than the other three wastewaters. A specific growth rate

of 0.948 d-1, total nitrogen removal rate of 82.8% and total phosphorus removal rate of

85.6% were observed in centrate during 10-day batch cultivation. Min et al. (2011)

further investigated the growth of this wild strain Chlorella sp. in centrate in a pilot-scale

photobioreactor (PBR). The effects of different harvesting ratio and exogenous CO2 levels on the biomass productivity of Chlorella sp. were tested in the pilot-scale test. The system was able to produce algal biomass at a rate of 34.6 g m-2 d-1 under low light

conditions (25 μmol·m-2s-1) and a one fourth harvesting rate, which corresponded to 70%

chemical oxygen demand, 61% total Kjeldahl nitrogen, and 61% soluble total phosphorus

reduction. However, the addition of CO2 to the system did not significantly increase the

biomass productivity or nutrient removal in centrate because its organic carbon

concentration was already enough for the growth of Chlorella sp.. Wang et al. (2010a) also tested anaerobic digested dairy manure as culture medium for microalgae Chlorella

sp. The growth rate of Chlorella sp. was greatly affected by the dilution times of digested manure, with the highest growth rate of 0.409 d-1 at 25 times dilution. Ammonia was

completely removed in all of the four diluted samples during the 21-day batch test.

However, the removal rate of total nitrogen, total phosphorus and COD varied with

different diluted manures. The total fatty acid content of the dry biomass increased from

9% to 13.7% as the dilution multiples increased from 10 to 25. Based on the results of

these batch tests, Wang et al. (2010c) moved on to the lab-scale semi-continuous

cultivation of Chlorella vulgaris in undigested and digested dairy manure to further

exploit the potential of Chlorella vulgaris for nutrient reduction and biomass production.

It was shown that in order to achieve the same removal rate of ammonium nitrogen,

Chlorella sp. grown in undigested dairy manure required a shorter hydraulic retention

time (5 day) than that grown in digested dairy manure due to the fact that the undigested

dairy manure had much higher organic carbon content which could support the growth of

mixotrophic Chlorella sp..Algae grown in digested dairy manure performed better in

terms of CO2 sequestration per milligram of harvested dried biomass (1.68 mg CO2/mg dry weight (DW) than that grown in undigested dairy manure (0.99 mg CO2/mg DW).

However, the higher biomass productivity was obtained with the undigested one. The

advantage of mixotrophic cultivation of Chlorella vulgaris was also demonstrated by

other researchers. Heredia-Arroyo et al. (2011) studied the biomass and lipid production

of Chlorella vulgaris under autotrophic, heterotrophic, and mixotrophic conditions with

different substrates. The results showed that although the lipid content of Chlorella

vulgaris grown under autotrophic and heterotrophic conditions are higher than that grown

under mixotrophic condition, the higher biomass productivity of mixotrophic cultivation

resulted in higher total lipid production. This distinct feature of Chlorella vulgaris makes

it favorable for the treatment of carbon-rich wastewater for nutrients recovery and lipid

production.

Scenedesmus is another green alga that has been widely used in wastewater bio-

treatment. Scenedesmus sp is often single celled or colonial, forming 2- to 32-celled,

usually 4- or 8-celled colonies. A parallel comparison study of the efficiency of ammonia

and phosphorus removal by Chlorella vulgaris and Scenedesmus dimorphus was

conducted using the secondary effluent from an agroindustrial wastewater of a dairy

industry and pig farming (Gonzalez et al., 1997). S. dimorphus was shown to be more

efficient in removing ammonia than C. vulgaris. However, they both were able to remove

all ammonia from the wastewater and showed similar phosphorus removal efficiency by

the end of 9-day batch experiment. The growth rate and nutrients removal efficiency of

Scenedesmus sp. LX1 in liquor prepared from the effluent of an electronic device factory

in batch and continuous mode was investigated by Zhen-Feng et al. (2011). The nitrogen

concentration reduced from 35 mg L-1 to 10 mg L-1 and the phosphorus concentration reduced from 2.0 mg L-1 to 0.25 mg L-1, with a specific algal growth rate of 0.09 d-1.

However, the algal growth rate decreased to 0.02 d-1 under continuous inflow of 60 L h-1,

which also led to lower nitrogen and phosphorus removal efficiency. It was noticed that

the accumulated metal ions in the effluent inhibited the algal growth, thus reducing the bio-treatment efficiency of wastewater.

Although N to P atomic ratio (N: P) of natural phytoplankton is about 15, it was reported that Scenedesmus sp. requires an N/P ratio of approximately 30 to grow without

limitation by either nutrient (Rhee, 1978). In the study of effects of nitrogen and

phosphorus concentrations on growth, nutrient uptake, and lipid accumulation of a

Scenedesmus sp. LX1 by Li et al. (2010b), it was also proved that the nutrient uptake

ratios (N/P) is not consistent with the N/P ratio in empirical elementary composition in

microalgal cells. The authors inferred that Scenedesmus sp. LX1 has the ability to over-

uptake nitrogen or phosphorus, when the N/P ratio in growth medium is the atomic ratio

of algae cell. For example, at N/P ratio of 2:1 and 4:1 in growth medium, the nutrient

uptake ratios (N/P) were around 2:1 and 4:1, respectively, after 13 days of cultivation,

indicating an over-uptake of phosphorus in microalgal cells. At N/P ratio of 12:1 and

20:1, the nutrient uptake ratios (N/P) were about 10:1 and 9:1, respectively, indicating the

over-uptake of nitrogen. The results showed that when Scenedesmus sp. LX1 was grown

in an environment with N/P ratio of 5:1–12:1, 83–99% nitrogen and 99% phosphorus

could be removed. Although unbalanced N/P ratio will cause insufficient nutrient

removal from medium, the lipid content of algal biomass could increase under nutrient

limitation, which has been testified for many algae species, including Scenedesmus sp..

The lipid content of Scenedesmus sp. LX1 could reach 53% of dry cell weight under

phosphorus limitation (0.1 mg L-1) (Xin et al., 2010b).

There are many other microalgae species which are able to remove nutrients from

wastewater effectively. Chlamydomonas is a unicellular green alga that is typically

spherical to subspherical with two flagella and it is widespread in freshwater

(Algaebase,). Kong et al. (2010) cultivated Chlamydomonas reinhardtii in municipal

wastewater for nutrients removal and lipid production in biocoil photobioreactor. The

maximum algal biomass yield was 2.0 g L−1 d−1 in the 10-day batch cultivation,

corresponding to reduction of 55.8 mg nitrogen and 17.4 mg phosphorus per liter per day.

The optimal growth pH for C. reinhardtii is found to be around 7.5 and the lipid content

of dry cell weight is about 25%. It was demonstrated that a proper amount of external

inorganic carbon supply through injection of CO2 could boost algae biomass production.

Sawayama et al. (1992) found that Botryococcus braunii, a hydrocarbon-rich green

microalga, was able to remove phosphorus, nitrate and nitrite effectively, but not

ammonium, when grown in secondarily treated sewage from domestic waste-water in

batch experiment. Ammonium was reported to be inhibitory to cell growth in this

particular culture. Later in the continuous cultivation system, the growth rate of

Botryococcus braunii was maintained at 196 mg dry weight/L per week for one month.

However, the nitrate removal rate (27%) and phosphorus removal rate (62.5%) was lower

compared with that in batch system (both are close to 100%).

Nannochloropsis is a genus of yellow green algae of the family Monodopsidaceae that is less than 5 μm in its maximum dimension (Hibberd, 1981). Members of this genus include 6 species: N. gaditana, N. granulate, N. limnetica, N. oceanica, N. oculata, N.

salina. (Hibberd, 1981; Andersen et al., 1998). Among these various species,

Nannochloropsis salina is attractive to us because it is marine algae and has a relatively

high biomass and lipid productivity (Emdadi and Berland, 1989; Boussiba et al., 1987).

Therefore, it is a promising candidate for biofuel production. Additionally,

Nannochloropsis salina is mixotrophic which means it can not only uptake the organic

carbon in the ADE, but also perform photosynthesis to sequence carbon oxide in the air

or flue gas (Huerlimann et al., 2010; GonenZurgil et al., 1996; Zittelli et al., 1999), which

is helpful to reduce greenhouse gas in the air. Most of previous studies on

Nannochloropsis focused on lipid content and compositions to evaluate its nutritional

components such as EPA, DHA and GLA (Emdadi and Berland, 1989; Zou et al., 2000;

Zou and Richmond, 1999; Hu et al., 1997; Christian et al., 2009). For N. salina, studies showed that there is no lipid accumulation during the exponential growth phase. Instead, the lipid content could be as high as 50% of dry weight during the stationary phase

(Emdadi and Berland, 1989). The biomass productivity of N. salina could reach 24.5 g m-

2 d-1 in outdoor ponds (Boussiba et al., 1987) and its lipid content could be enhanced up to

70% of dry weight by nitrogen starvation (Shifrin and Chisholm, 1981). However, high

lipid content is often offset by low growth rate, which leads to low overall oil

productivity (Huerlimann et al., 2010; Rodolfi et al., 2009). Therefore, for mass

continuous algae cultivation, it is important to determine an efficient feeding frequency

and harvesting ratio according to the growth rate and lipid content of algae in order to

achieve maximum overall lipids production.

Chapter 3 Cultivation of Nannochloropsis salina in AD

Effluent for Nutrient Removal and Lipid Production

The growth, lipid productivity, and nutrient removal of Nannochloropsis salina in

anaerobic digested municipal wastewater were evaluated in this study. Results from batch

reactors showed that N. salina was able to grow well under 3, 6, 12, and 18% AD effluent

loading with the highest growth rate being 0.645 d-1. The growth of N. salina was

inhibited when the effluent loading increased to 24%. The nutrient removal efficiency

and lipid content of N. salina decreased as the effluent loading increased. The highest

biomass yield (0.92 g L-1) was obtained with 6% effluent loading, while the highest lipid

productivity (29.2 mg L-1 d-1) occurred in 3% effluent loading. Three harvesting

frequencies (1-, 2-, and 3-day interval) and two harvesting ratios (25%, 50%) were tested

in semi-continuous reactors and the highest lipid productivity (38.7 mg L-1 d-1) was

achieved with a 2-day harvesting interval and 50% harvesting ratio, where nitrogen and phosphorus were removed at a rate of 35.3 mg L-1 d-1 and 3.8 mg L-1 d-1, respectively.

3.1 Introduction

Anaerobic digestion (AD) is a technology to decompose organic matter under

oxygen-free conditions and involves a variety of anaerobic microorganisms. The final

products of AD are biogas (a mixture of methane (40-70% of total biogas volume) and

CO2) and liquid effluent (rich in nitrogen and phosphorous with total solids less than

10%). The technology is implemented in the treatment of agricultural wastes, food

wastes, and wastewater sludge to reduce the pollution and produce bioenergy. Due to its

nitrogen and phosphorus content, the effluent is typically used as a fertilizer or

composted and used as a soil amendment. However, the use of the effluent as a fertilizer

may be limited due to an insufficient amount of farmland within the economic transportation distance for the amount of effluent produced. Thus, the need for cost effective technologies for removing nitrogen and phosphorus from the effluent or for alternative uses remains a challenge for the AD industry.

Researchers have been working on nutrient removal and recovery from AD effluent by chemical and physical methods. Lei et al. (2007) successfully applied

+ ammonium (NH4 ) stripping on AD effluent and the removal rates for nitrogen, phosphorus, chemical oxygen demand (COD), and suspended solids were 78%, 99.9%,

82.1%, and 91%, respectively. This method requires a large amount of calcium hydroxide

+ and it is suggested that the remained precipitates after NH4 stripping be composted and

can be used as a fertilizer. The method of struvite (MgNH4PO4) crystallization is

effective for phosphorus removal from wastewater but not for other nutrients (Battistoni

et al., 1997; Song et al., 2011). Maekawa et al. (1995) investigated nutrient removal and recovery from wastewater using an intermittent aeration batch reactor followed by an

+ ammonium (NH4 ) crystallization process and obtained a removal efficiency of 91% for

total nitrogen (TN) and 99% for total ammonium nitrogen (TAN). Membrane filtration is

one of the most important and commonly employed technologies for the purification of

wastewater and effluent streams because of its high removal efficiency and water

recovery capacity (Ahmad et al., 2005; Fan et al., 2008). Suspended solids,

microorganisms, and macromolecules in AD effluent can be removed by microfiltration

or ultrafiltration, while nanofiltration and reverse osmosis can be applied for the removal

of smaller organic molecules (Waeger et al., 2010).

The use of microalgae to remove nutrients from AD effluent has been recently

studied and is argued to be effective (Wang et al., 2010a; Phang et al., 2000; Sooknah and

Wilkie, 2004; Kebede-Westhead et al., 2003). The capability of microalgae to remove

nitrogen and phosphorus from the AD effluent depends on a variety of factors, such as

the species, feed concentration, temperature, and pH. Wang et al. (2010a) reported the

effectiveness of using digested dairy manure as a nutrient supplement for the cultivation

of microalgae Chlorella sp. The digested manure was diluted before being fed to the

microalgae. The removal rates of TAN, TN, and total phosphorus (TP) varied with the

dilution ratio. Biomass yield and total fatty acid content of the dry algal biomass also changed with the increasing dilution ratio. Park et al. (2010) used Scenedesmus sp. to

+ remove NH4 from AD effluent of livestock waste. Their study showed that Scenedesmus

-1 + sp. can tolerate up to 100 mg L NH4 .

Previous studies on Nannochloropsis mostly focused on the lipid content and

composition related to its nutritional components such as eicosapentaenoic acid (EPA),

docosahexaenoic acid (DHA) and gamma-linolenic acid (GLA) (Emdadi and Berland,

1989; Zou et al., 2000; Zou and Richmond, 1999; Hu et al., 1997; Christian et al., 2009).

For Nannochloropsis salina, studies showed that the lipid content could be as high as

50% of the dry weight during the stationary phase (Emdadi and Berland, 1989). The

biomass productivity of N. salina reached up to 24.5 g m-2 d-1 in outdoor ponds (Boussiba

et al., 1987) and its lipid content was enhanced up to 70% of its dry weight by nitrogen

starvation (Shifrin and Chisholm, 1981). However, high lipid content is often offset by

low growth rate, which leads to low overall lipid productivity (Huerlimann et al., 2010;

Rodolfi et al., 2009). Therefore, for continuous microalgae cultivation, it is important to

determine an optimal feeding frequency and harvesting ratio according to the growth rate

and lipid content of the microalgae in order to achieve maximum overall lipid production.

The purpose of this study was to test the feasibility of growing N. salina in AD

effluent. The AD effluent was collected from a commercial anaerobic digester after the

removal of solids using a centrifuge. The performance of N. salina was evaluated by

comparing its growth rate and nutrient removal efficiency at different effluent loadings.

Batch experiments were conducted to find the optimal effluent loading. The optimal harvesting ratio and frequency were determined via semi-continuous laboratory experiments in order to provide basic reference data for future pilot scale production.

3.2 Material and Methods

3.2.1 AD effluent and algae inoculum cultures

Four liter AD effluent was collected from a commercial-scale wet anaerobic digester (KB Compost Services, Akron, OH, USA) coupled with a D5LL solid bowl decanter centrifuge (ANDRITZ AG, Graz, Austria). The feedstock for this digester was the municipal wastewater from the city of Akron. The centrifuge ran continuously at

3200 rpm. The effluent was kept in 4°C before use.

The marine microalga N. salina (CCAP 849/6) was obtained from Culture

Collection of Algae and Protozoa (CCAP, Oban, Scotland). Cultures were cultivated in f/2 medium (Guillard and Ryther, 1962) containing the following ingredients: 0.075 g L-1

-1 -1 NaNO3, 0.00565 g L NaH2PO4⋅2H2O, 1 ml L 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, while the vitamin stock solution contained

cyanocobalamin (vitamin B12), and thiamine HCl (vitamin B1). These solutions were used

in the media following recipes provided in the CCAP website (CCAP). Seed cultures

were cultured under continuous light (200 µmol m-2 s-1) at 25±1°C.

3.2.2 Batch and semi-continuous cultivation of N. salina in AD effluent

Each reactor consisted of a 2-L glass bottle capped with a rubber stopper and

stainless steel tubing with a 4.76 mm (0.188 in) inner diameter inserted as an air inlet for

the reactor. A polypropylene tee barb was connected at the bottom of the reactor to

evenly disperse the incoming air in a horizontal direction and prevent the microalgae from adhering to the reactor wall. The walls of the chamber were coated white to

thoroughly distribute the illumination within. The 32-W GE F32T8-SPX50 fluorescent

lamps (GE Lighting, Ravenna, OH, USA) were used to provide a constant photosynthetic

photon flux of approximately 200 µmol m-2 s-1, measured by a BQM quantum meter

(Apogee Instruments, Logan, UT, USA). Ambient air was provided at a pressure of 27.6

kPa (4.0 psi) through a custom-made polyvinyl chloride (PVC) manifold, which provided

a constant air flow of 175 ml min-1 to each reactor through clear PVC tubing.

The AD effluent was diluted before being fed to the microalgae. As shown in

Table 4, loading ratios ranging from 3% to 24% were tested to find the optimal loading of

effluent for microalgal growth. Deionized (DI) water was added to adjust the final

working volume of the reactor to 1 L. The salinity of each reactor was adjusted to 20‰

with Instant Ocean® sea salt (Spectrum Brands, Madison, WI, USA). Microalgae were

inoculated to each reactor to have an initial optical density of 0.1. DI water was added

daily to make up the water lost through evaporation before taking samples for analysis.

By the end of each batch experiment, the microalgal biomass was obtained using a

Sorvall RC 6 Plus centrifuge (Thermo Scientific, Waltham, MA, USA). The supernatant

remaining after centrifugation was analyzed for the nitrogen and phosphorus content.

TAN, TN, and TP removal efficiencies during the 10-day cultivation were calculated

based on the initial and final concentrations of TAN, TN, and TP in the supernatant.

Table 4 Nitrogen and phosphorus levels at different effluent loading ratios

AD effluent TAN TN TP loading ratio (%) (mg L-1) (mg L-1) (mg L-1) 3 68 80 11.43

6 137 160 22.86

12 273 320 45.72

18 409 480 68.58

24 546 640 91.44

In the semi-continuous experiments, the effects of harvesting frequency (1-, 2-

and 3-day intervals) and harvesting ratio (25% and 50% of total volume) on algal

biomass yield and nutrient removal were studied at a 6% effluent loading ratio (TN loading level of 160 mg L-1). Table 5 shows the experimental design of the semi- continuous experiment. The reactors were replenished with fresh effluent medium after

harvesting. For each reactor, the optical density of the culture and the nitrogen and

43 phosphorus concentration in the medium was analyzed daily. All experiments were carried out in duplicate and average values were reported.

Table 5 Experimental design of semi-continuous experiment

Harvesting Hydraulic Harvesting ratio Treatment frequency retention time (%) (d) (d) 1 1 25 4

2 1 50 2

3 2 25 8

4 2 50 4

5 3 25 12

6 3 50 6

3.2.3 Analytical methods

The optical density peak of 440 nm was used to establish a relationship with cell concentration. A Biomate 3 spectrophotometer (Thermo Fisher Scientific, Madison, WI,

USA) was used to measure the absorbance of cells at 440 nm. Microalgal biomass was determined gravimetrically and was reported as its ash-free dry weight (AFDW). A 50 g sample was placed in a clean centrifuge tube and centrifuged at 10,000 rpm for 15 min.

The supernatant was used for determining nutrient removal efficiencies. The precipitate was washed with 25 ml of 0.5 M NH4HCO3 solution and then centrifuged at 10,000 rpm for 15 min. The precipitate was washed again with 10 ml of 0.5 M NH4HCO3 and transferred to a clean, ignited, and tared porcelain crucible and dried in a Thelco Model

18 oven (Precision Scientific, Chennai, India) at 95°C for 12 hours. After the sample was dried to constant weight, it was cooled in a desiccator and weighed. Ash weight was obtained by igniting the sample in an Isotemp muffle furnace (Fisher Scientific,

Dubuque, IA, USA) at 500°C for 4 hours. The AFDW was calculated as the difference between the dry weight and the weight of the residual ash.

The concentrations of 13 elements (Na, Mg, Al, P, K, Ca, Fe, Mn, Ni, Cd, Cu, Zn, and Mo) in the culture medium were measured using an inductively coupled plasma-mass spectrometry (ICP-MS) unit 7500cx (Agilent Technologies, Santa Clara, CA, USA) following US EPA method 6062A (EPA, 2007). Prior to ICP-MS analysis, the sample was digested in a microwave accelerated reaction system (CEM Corporation, Matthews,

NC, USA) at 190°C for 10 minutes. The digested sample was diluted 50-fold and analyzed via ICP-MS. TAN, TN, total phosphorus (TP), and chemical oxygen demand

(COD) were determined using a 3900 spectrophotometer (Hach Company, Düsseldorf,

Germany) coupled with a DRB200 dual block reactor (Hach Company, Düsseldorf,

Germany).

Lipid content of the algal biomass was analyzed using a modified version of Bligh and Dyer’s method (Bligh and Dyer, 1959). Before extraction, the cells were disrupted

with a UP400S ultrasonic processor (Hielscher Ultrasonics, Teltow, Germany) and an

H22 titanium sonotrode with a 22 mm tip. The frequency of the ultrasound was 24 kHz

and the output was set at 100 W. Lipids were extracted by mixing chloroform-methanol

(2:1 v/v) with the samples in a proportion of 1:1. The samples were placed in a stoppered

test tube to which solvents were added. The tube was closed and shaken manually for 10 min. Water and methanol were added to give a final solvent ratio of chloroform:

methanol: water of 1:1:0.9. The mixture was mixed for 5 min and then left to settle

overnight. The top layer of the mixture (water and methanol) was removed with a pipette.

The biomass layer and the chloroform layer were filtered using Whatman No. 1 filter

paper (GE Healthcare, Maidstone, UK). The tube and filter paper were washed with 30

ml of chloroform. The chloroform was evaporated using a rotary evaporator. The weight

of the crude lipid obtained from each sample was measured using an electronic scale.

3.2.4 Statistical analysis

Statistical significance was determined by analysis of variance (ANOVA) using

SAS software (Version 8.1, SAS Institute Inc., Cary, NC, USA) with a threshold p-value

of 0.05.

3.3 Results and Discussion

3.3.1 Characteristics of AD effluent

The chemical composition of the AD effluent is shown in Table 6. The AD

effluent mainly contained carbon (2014 mg L-1), nitrogen (2930 mg L-1), and phosphorus

(381 mg L-1), which were higher concentrations than those in the original municipal

+ wastewater. About 85% of the nitrogen was in the form of NH4 , which was readily

available for the microalgae to use. The nitrogen-to-phosphorus (N/P) ratio of the effluent

was 7, which was lower than the atomic ratio of 16 for N. salina, indicating a nitrogen limitation for microalgal growth (Hecky and Kilham, 1988). The relatively low total solids (0.287%) of the effluent indicated a low turbidity (EPA, 2006), which was beneficial to the photosynthesis of N. salina. The effluent also contained other essential ions for microalgal growth, such as Na+, K+, Mg2+, Ca2+, Fe2+, and Al3+.

Table 6 Characteristics of the effluent

Characteristic Unit Concentration TS % 0.287±0.005 TVS % 0.208±0.002 Total carbon mg L-1 2014±65 TN mg L-1 2667±30 -1 NH4-N mg L 2276±45 TP mg L-1 381±6 COD mg L-1 2661±75 Na mg L-1 89±4 K mg L-1 121.7±3.5 Ca mg L-1 32.95±1.95 Mg μg L-1 680±26 Al μg L-1 1166±43 Fe μg L-1 4141±58 Mn μg L-1 151.4±19.5 Ni μg L-1 25.69±3.64 Co μg L-1 <0.000 Cu μg L-1 26.75±2.39 Zn μg L-1 105.7±12.3 Mo μg L-1 20.93±0.56

3.3.2 Batch study

3.3.2.1 The effects of effluent loading on microalgae growth and biomass yield

Five effluent loadings (3%, 6%, 12%, 18% and 24%) were tested and the nitrogen levels at each loading are shown in Table 4. The microalgal growth curves in terms of optical density at different effluent loadings are shown in Figure 2. N. salina survived at all five effluent loadings. A 1-d lag phase was observed at effluent loadings from 3%-

18%. It took 4 d for N. salina to adapt at the highest effluent loading of 24%. After the

sixth day, the microalgal growth curve at 3% loading started to level off due to

exhaustion of nutrients.

Microalgal growth rate (d-1) was calculated by plugging the OD values in the

following equation:

ln ln/ = ln /)]/

where OD0 is the optical density at the beginning day of cultivation, and ODt is the

optical density measured on the fifth day of cultivation. The average specific growth rates

at loadings of 3%, 6%, 12%, 18% and 24% were 0.624, 0.645, 0.643, 0.591 and 0.334 d-1, respectively (Table 7). These growth rates were higher than that of N. oculata (0.14 d-1)

-1 and Nannochloropsis sp. (0.28 d ) grown in f/2 medium using NaNO3 as the sole source

of nitrogen (Converti et al., 2009; Brown et al., 1998). However, the growth rates in this

-1 study are comparable to that of N. oculata (0.571 d ) with 2% CO2 aeration (Chiu et al.,

2009) and that of Nannochloropsis sp. (0.54 d-1) grown in 50% municipal wastewater

(Jiang et al., 2011).

5 3% 4 6% 12% 18% 3 24% 440nm

Absorbance 1

0 012345678910 Cultivation time (day)

Figure 2 Algae growth curves at different effluent loadings

Table 7 Growth rate of N. salina at different effluent loadings

Effluent Growth rate loading (%) (d-1) 3 0.624±0.006 6 0.645±0.001 12 0.643±0.001 18 0.591±0.006 24 0.334±0.019

As shown in Figure 3, the final microalgal biomass concentrations at each effluent loading varied in the range of 0.68-0.92 g L-1, where the highest concentration of 0.92 g

L-1 was obtained at an effluent loading of 6%. The biomass concentration dropped from

0.82 g L-1 to 0.68g L-1 when the effluent loading increased from 18% to 24%, suggesting an inhibition of microalgal growth in high effluent concentrations. Other studies have found likewise results. The growth of Nannochloropsis sp. was inhibited when the loading of municipal wastewater increased from 50% to 80% (Jiang et al., 2011). The maximum biomass concentration of Nannochlorophsis sp. cultivated in municipal wastewater was 0.212 g L-1 (Jiang et al., 2011) which was lower than that obtained in this

study. However, Gouveia and Oliveira (2009) reported an average biomass concentration

of 1.6 g L-1 for Nannochlorophsis sp. grown in commercial medium. The biomass

concentration of N. oculata was greatly increased from 0.268 g L-1 to 1.277 g L-1 when aerated with 2% CO2 instead of air (Chiu et al., 2009).

1.0 (g/L) 0.5 yield

Biomass 0.0 3 6 12 18 24 Effluent loading (%)

Figure 3 Biomass yield of N. salina at different effluent loadings

3.3.2.2 The effects of effluent loading on nutrient removal and lipid production

– – + It has been shown that nitrate (NO3 ), nitrite (NO2 ), and NH4 are the main forms of inorganic nitrogen that are directly available for microalgae (Barsanti and Gualtieri,

– + 2006). Although NO3 is the nitrogen source commonly used in culture media, NH4 is actually the preferential form for many types of microalgae because it does not need to be

+ reduced before amino acid synthesis. However, it was noted that NH4 at concentrations greater than 450 mg L-1 is often toxic to microalgae (Barsanti and Gualtieri, 2006). Most

+ of the nitrogen in the effluent used in this study was in the form of NH4 (Table 6). As

+ shown in Figure 4, N.salina was able to remove the NH4 completely at all effluent

+ loadings studied. Therefore, the toxicity of NH4 to microalgae (Abeliovich and Azov,

+ -1 1976) was not observed for N. salina at NH4 levels less than 546 mg L . This result is also in agreement with a study by Hii et al. (2011) in which Nannochloropsis sp. was able

+ to grow in f/2 medium enriched with 400-900 µM NH4 during 10-day cultivation. A

complete TN removal was observed at 3% and 6% effluent loadings. TN removal

efficiency decreased to 87% as the effluent loading increased from 6% to 24%, indicating

that there was still some organic nitrogen not consumed by N. salina. The nitrogen removal efficiency of N. salina in this study was much higher than that from other studies. For example, Wang et al. (2010a) studied the nutrient removal efficiency of microalgae Chlorella sp. in anaerobically digested dairy manure. The highest TKN removal efficiency was 82.5% with an initial TKN of 250 mg L-1 after 22 days of

cultivation. Another study of Chlorella sp. grown in municipal wastewater for 15 days

showed a TN removal rate of 89.1% with an initial TN of 116 mg L-1 (Li et al., 2011).

Microalga Chlamydomonas reinhardtii grown in municipal wastewater was only able to

removal 55% TKN (initial concentration was 128.6 mg L-1) after 10 d of culture (Kong et

al., 2010).

As shown in Figure 4, more than 99% of the phosphorus in the medium was removed in the 10-d batch study. However, the N/P ratio of natural phytoplankton is about 15, and the initial N/P ratio of the effluent was 7. As the uptake of phosphorus was higher than expected, based on N/P ratios, N. salina might have the ability to uptake excess phosphorus. At N/P ratios of 2 and 4 in the growth medium, the nutrient uptake ratios (N: P) of Scenedesmus sp. LX1 were around 2:1 and 4:1, respectively, after 13 days of cultivation, indicating an over-uptake of phosphorus in microalgal cells (Xin et al.,

2010b). Another explanation of the high phosphorus removal might be attributed to an

increase in the pH of the culture medium from 7 to more than 10 during the cultivation

period. High pH (>10) may have caused the precipitation of phosphorus with other

elements such as calcium, thus facilitating the phosphorus removal (Wang et al., 2010b).

105 TN NH4‐N TP 100

(%) 95

90 efficiency

85 Removal 80 3 6 12 18 24 Effluent loading (%)

Figure 4 TAN, TN, TP removal rates at different effluent loadings

Figure 5 shows the effects of effluent loading on lipid content of N. salina. The

lipid content reached up to 35% of dry cell weight and then decreased as the AD effluent

loading in the culture medium increased from 3% to 18%, which is consistent with other

research showing that the lipid content of microalgae cells could be increased as the nutrient loading decreased (Emdadi and Berland, 1989; Converti et al., 2009; Suen et al.,

1987). The lipid content of N. oculata and Chlorella vulgaris increased from 7.9% to

15.9% and from 5.9% to 16.4%, respectively, when the NaNO3 concentration decreased

from 300 mg L-1 to 75 mg L-1 (Converti et al., 2009). The lipid content of Chlorella sp.

was also enhanced from 9% to 13.7% as the loading ratio of the anaerobic digested

manure decreased from 10% to 4% (Wang et al., 2010a). The lipid content of

Nannochloropsis sp. is usually in the range of 21 – 36% of dry cell weight (Gong and

Jiang, 2011), which is in agreement with this study.

30 AFDW)

20 content

10 Lipid

0 3 6 12 18 24 Effluent loading (%)

Figure 5 Algal lipid contents at different effluent loadings

The lipid productivities at different effluent loadings are shown in Figure 6. As

the effluent loading increased, the lipid productivity dropped from 29.2 mg L-1 d-1 to 14

mg L-1∙d-1. However, the lipid productivities at 3% and 6% effluent loading were not significantly different (p>0.05) because the biomass yield at 6% loading was slightly higher than that at 3% loading. The lipid productivity obtained in this study was much higher than for N. salina fed with artificial sea water in outdoor ponds which produced lipids at a rate of 0.03 mg L-1 d-1 (Boussiba et al., 1987). It was reported by Hu and Gao

-1 (2006) that Nannochloropsis sp. aerated with 2,800 µl CO2 L in f/2-enriched artificial

seawater had a highest lipid production of 13.64 mg L-1 d-1 in 10-d cultivation. The lipid

content and lipid productivity of microalgae were not only affected by nutrient loading,

but also by other factors such as temperature, irradiance, and salinity (Converti et al.,

2009; Su et al., 2011). An increase of irradiance from 100 to 500 µmol photons m-2 s-1 led to an increase in the lipid content of N. oculata from 22.5% to 44.5%, resulting in an increase in the lipid productivity from 103 mg L-1 d-1 to 324 mg L-1 d-1 (Su et al., 2011).

The lipid content of C. vulgaris decreased from 20% to 8.2% when the temperature

increased from 25 to 35 ˚C, causing a decrease in lipid productivity as well (Converti et

al., 2009).

30 (mg/L/day)

10 productivity

Lipid 0 3 6 12 18 24 Effluent loading (%)

Figure 6 Algal lipid yields at different effluent loadings

3.3.3 Semi-continuous study

3.3.3.1 The effects of harvesting frequency and harvesting ratio on algae growth and

biomass yield

The batch study showed that N. salina could adapt to effluent loadings between

3% and 18%. The effluent loading of 6% was chosen for the semi-continuous study

because of the relatively high growth rate and biomass yield of N. salina under this

condition. Different harvesting frequencies (1-, 2-, and 3-day intervals) and harvesting

ratios (25% and 50%) were tested for an 18-d culture of N. salina (Table 5). As shown in

Figure 7, at different harvesting frequencies, when half of the culture was harvested each time, the cultures were able to reach steady state earlier that those with 25% of the culture harvested. At a harvesting interval of 1 d, the reactors reached steady state on the second day when half of the culture was harvested; whereas, for those with 25% culture harvested, the reactors did not reach steady state until the sixth day. Both harvesting interval and ratio affected the accumulated biomass yield. According to Figure 8, at a

25% harvesting ratio, the biomass yield decreased as the harvesting interval increased from 1 d to 3 d, as the hydraulic retention time (HRT) increased from 4 d to 12 d.

However, at a 50% harvesting ratio, the highest biomass yield was obtained with a 2-d harvesting frequency at a HRT of 4 days. Although lower harvesting ratios resulted in higher algal cell density, the highest biomass yield during the 18-d continuous cultivation was obtained at a harvesting ratio of 50% and a harvesting interval of 2 d. Similar results were also presented by Min et al. (2011) in a study of Chlorella sp. using municipals wastewater as a nutrient supplement. The biomass productivity of Chlorella sp. was

increased by 22.8–66.7% when the harvesting ratio increased from 25% to 50%.

(a) 5 1d_25% 1d_50% 4

OD440 2

0 0123456789101112131415161718 Cultivation time (day)

(b) 5 2d_25% 4 2d_50%

OD440 2

0 024681012141618 Cultivation time (day)

Figure 7 Algal growth curves at different harvesting frequencies and harvesting ratios: (a) 1-d harvesting interval with 25% or 50% culture removed; (b) 2-d harvesting interval with 25% or 50% culture removed; (c) 3-d harvesting interval with 25% or 50% culture removed

Figure 7 continued

OD440 2

0 0369121518 Cultivation time (day)

3 20% 50% (g/L)

2 biomass

1 Accumulated 0 123 Harvesting interval (day)

Figure 8 Accumulated algal biomass yield at different harvesting frequencies and harvesting ratios in 18-day semi-continuous cultivation

3.3.3.2 The effects of harvesting frequency and harvesting ratio on nutrient removal and lipid productivity

As shown in Table 8, at the same harvesting ratio, the TN removal rate decreased as the HRT increased. Higher harvesting ratios led to higher TN removal rates at each harvesting frequency. The TN removal efficiency was significantly increased as the culture was harvested less frequently (p < 0.05) because less frequency resulted in a longer HRT during which more algae biomass would be accumulated and more nutrients could be assimilated. The highest biomass productivity (155.3 mg L-1 d-1), lipid content

(24.9% AFDW), and lipid productivity (38.7 mg L-1 d-1) were obtained at a harvesting ratio of 50% and harvest interval of 2 d (Table 8). The maximum lipid productivity of

38.7 mg L-1 d-1 obtained in the semi-continuous study was 1.33 times of that in the batch study, showing an advantage of semi-continuous cultivation for N. salina. The average

lipid productivity of Nannochloropsis sp. has been reported to be 37.6–90 mg L-1 d-1

(Gong and Jiang, 2011; Mata et al., 2010). Thus, this study demonstrated the technical feasibility of growing N. salina in AD effluent for both nutrient removal and lipid production.

Table 8 Nutrient removal rate and lipid productivity of N. salina at different harvesting frequencies and ratios

Harvesting TN removal TN removal TP removal TP removal Biomass Harvesting Lipid content Lipid productivity frequency rate efficiency rate efficiency productivity ratio (%) (% AFDW) (mg L-1 d-1) (d) (mg L-1 d-1) (%) (mg L-1 d-1) (%) (mg L-1 d-1) 1 25 32.9±3.0 53.5±4.3 4.1±0.3 33.6±2.8 132.1±8.9 18.0±0.6 23.7±2.4 1 50 56.5±4.8 55.2±4.6 4.3±0.4 22.9±2.2 143.0±4.8 19.0±0.4 27.1+1.5 2 25 19.0±2.9 73.4±4.5 3.4±1.1 67.6±3.1 104.3±2.7 19.8±0.9 20.6±1.5 2 50 35.3±4.2 77.8±4.0 3.8±1.2 45.7±6.5 155.3±1.8 24.9±0.5 38.7±2.4 3 25 13.4±2.5 84.7±2.0 2.3±1.6 72.5±5.7 87.4±1.9 18.1±1.1 15.9±1.3 3 50 26.2±2.5 89.0±1.7 3.9±1.3 82.8±1.8 121.4±4.1 22.7±0.4 27.6±1.4

61 Data are the means ± SE

3.4 Conclusion

It is feasible to culture microalgae N. salina in AD effluent for nutrient removal

and lipid production. However, proper dilution was necessary to mitigate the negative

effects of toxic components on microalgal growth. N. salina was able to tolerate up to

480 mg L-1 TN (18% effluent loading). N. salina fed with 6% effluent had the highest biomass yield, along with 29% lipid content and 100% nitrogen and phosphorus removal efficiencies. The biomass and lipid productivity of N. salina was further improved in semi-continuous cultivation due to frequent replenishment of culture medium. The optimal operation parameters for semi-continuous cultivation are: 2-d harvesting interval and 50% harvesting ratio.

Chapter 4 Cultivation of Synechocystis sp. PCC6803 in AD Effluent for

Nutrient Removal and Lipid Production

This study tested the growth of cyanobacteria Synechocystis sp. PCC6803 in AD

effluent for nutrient removal, lipid and biomass production and compared its performance

with that of N. salina presented in previous chapter. The results from batch study showed

that Synechocystis sp. PCC6803 produced biomass at a higher rate (150 mg L-1 d-1) than

N. salina with 3% effluent loading. However, the lipid productivity of Synechocystis sp.

PCC6803 was lower due to its low lipid content. It was noted that Synechocystis sp.

PCC6803 was less efficient than N. salina in removing nitrogen and phosphorus. The

growth rate of Synechocystis sp. PCC6803 was also inhibited by high effluent loading.

The biomass productivity of Synechocystis sp. PCC6803 was further improved to 212.4

mg L-1 d-1 in semi-continuous study when 50% culture was harvested every day, resulting a lipid productivity of 29.1 mg L-1 d-1.

4.1 Introduction

In order to reduce dependence on fossil fuel and to mitigate global warming,

researchers have been working to develop biofuels and biomaterials as renewable

substitutes (Rittmann, 2008; Chisti, 2008; Farrell et al., 2006). Efficient photosynthetic

organisms such as algae and cyanobacteria are among the most popular research subjects

for production of biofuels and biomaterials. Cyanobacteria (blue green algae) are a group

of the oldest forms of organisms on the earth and play important roles in biogeochemical

63 cycles of nitrogen, carbon, and oxygen (Rippka et al., 1979; Gademann and Portmann,

2008). Cyanobacteria contribute to 30% of the annual oxygen production on the earth

(Sharma et al., 2011). Some cyanobacteria have unique metabolic pathways that can fix atmospheric nitrogen through photosynthesis (Karl et al., 2002). Most of the secondary metabolites of cyanobacteria are useful chemical compounds that have biological activities, such as antibacterial, antifungal, antitumoral, anti-HIV, antiviral, and anti- inflammatory; thus, they have been widely applied in clinical therapies (Sharma et al.,

2011; Abed et al., 2009). Although a major focus on cyanobacteria in previous decades has been on the negative roles they have played in the eutrophication of lakes, oceans, rivers, and other water bodies, interest has shifted to biofuel production from cyanobacteria due to their fast growth rate, high biomass yield, and simple growth needs

(Parmar et al., 2011; Heisler et al., 2008; Smith, 2003; Anderson et al., 2002).

Synechocystis sp. PCC6803 is a promising cyanobacteria strain which is attractive because of its high biomass yield and robustness. Synechocystis sp. PCC6803 is able to survive in a wide range of temperature, salinity, and pH conditions (Schubert et al., 1993;

Antal and Lindblad, 2005; Anderson and Mcintosh, 1991; Ohkawa et al., 2000; Ogawa and Kaplan, 2003; Kanesaki et al., 2002). Its genomic, biochemical, and physiological data have been extensively reported, offering valuable information for genetic modification of this strain to improve its potential for bioenergy production (Huang et al.,

2002). The high biomass yield of Synechocystis sp. PCC6803 could provide a large volume of feedstock for production of bioenergy, such as bio-ethanol, methane, biodiesel, or hydrogen. Unlike most microalgae, the lipid content of cyanobacteria will not increase under nitrogen starvation. Instead, Synechocystis sp. PCC6803 has the highest lipid

64 content during the exponential phase when its growth rate is the highest (Kim et al.,

2011; Hu et al., 2008). Similarly, the nutrient (nitrogen and phosphorus) uptake rates of

Synechocystis sp. PCC6803 are also the highest during this optimal growth phase.

Therefore, theoretically, optimal lipid production and nutrient removal rates could be achieved at the same time when cultivating Synechocystis sp. PCC6803.

Cyanobacteria are able to utilize both organic and inorganic carbon.

Synechocystis sp. PCC6803 can fix CO2 and perform photosynthesis in sunlight. It is also capable of heterotrophic growth with supply of organic carbon. Although nitrogen can be utilized in the forms of ammonium, nitrite, nitrate, or N2, cyanobacteria prefer using ammonium nitrogen because it is in a less reduced form and takes less energy to consume

(Converti et al., 2006). Phosphorus is another essential element for growth of cyanobacteria although, compared to nitrogen, a smaller amount is required. The uptake rate of phosphorus is affected by the pH of the medium and the existence of other elements such as calcium (Ritchie et al., 1997). Lack of ions such as Mg2+, K+, Na+ will also decrease the phosphorus uptake rate (Ritchie et al., 1997; Seale et al., 1987).

The effectiveness of Synechocystis sp. PCC6803 for optimal uptake of nutrients concurrent with optimal lipid production, suggests that it could be a cost effective treatment for wastewater, such as the effluent from an anaerobic digestion (AD). AD effluent has a sufficient amount of nutrients (nitrogen, phosphorus, organic carbon, and other trace elements) that could support the growth of Synechocystis sp. PCC6803 (Table

6) at a relatively low cost. According to analysis, more than 80% of the nitrogen in AD effluent is ammonium. The nitrogen/phosphorus ratio of 7 indicates excessive

65 phosphorus supply in the medium. Therefore, the growth of Synechocystis sp. PCC6803 will not be limited by phosphorus.

The objectives of this study were to: 1) evaluate the growth of Synechocystis sp.

PCC6803 in AD effluent; 2) compare the biomass yield, nitrogen and phosphorus removal efficiencies, and lipid productivity of Synechocystis sp. PCC6803 with those of

N. salina in batch and semi-continuous cultivation.

4.2 Material and Methods

4.2.1 AD effluent and inoculum cultures

The AD effluent used in this study was the same as the one used in the study discussed in Section 3.2.1 of Chapter 3. It was collected from a commercial-scale wet anaerobic digester (KB Compost Services, Akron, OH, USA) coupled with a D5LL solid bowl decanter centrifuge (ANDRITZ AG, Graz, Austria). The feedstock for this digester was the municipal wastewater from the city of Akron. The centrifuge ran continuously at

3200 rpm. The effluent was kept in 4°C before use.

The cyanobacteria strain used in this study was obtained from Touchstone

Research Laboratories (Tridelphia, WV). DNA sequencing was used to identify the strain and it was identified as Synechocystis sp. PCC6803. The identification results are shown in Figure 9 (This part of work was done by Dr. Jiyoung Lee’s lab in OSU, Columbus,

OH).

Figure 9 BLAST analysis for strain identification

4.2.2 Batch and semi-continuous cultivation of Synechocystis sp. PCC6803 in AD effluent

The reactor configuration used to grow the Synechocystis sp. PCC6803 in the AD effluent was the same as the configuration described in Section 3.2.2 of Chapter 3.

In batch cultivation, different dilutions were applied to the AD effluent before being fed to Synechocystis sp. PCC6803 (Table 5). Deionized (DI) water was added to adjust the final working volume of the reactor to 1 L. The salinity of each reactor was adjusted to 20‰ with Instant Ocean® sea salt (Spectrum Brands, Madison, WI, USA).

Seed cultures were inoculated to each reactor to have an initial optical density of 0.1. DI water was added daily to make up the water lost through evaporation before taking samples for analysis. By the end of each batch experiment, the biomass was obtained using a Sorvall RC 6 Plus centrifuge (Thermo Scientific, Waltham, MA, USA). The supernatant remaining after centrifugation was analyzed for the nitrogen and phosphorus content. TAN, TN, and TP removal efficiencies during the 10-day cultivation were

calculated based on the initial and final concentrations of TAN, TN, and TP in the

supernatant.

According to the study of N. salina in semi-continuous cultivation in Chapter 3,

the harvesting ratio of 50% led to higher biomass and lipid productivity than the ratio of

25%. Thus, only a 50% harvesting ratio was used for the semi-continuous cultivation of

Synechocystis sp. PCC6803 and the harvesting interval varied between 1-3 days.

4.2.3 Analytical methods

The optical density peak of 440 nm was used to establish a relationship with cell

concentration. Biomass was determined gravimetrically and is reported on an ash-free dry

weight (AFDW) basis. The concentrations of 13 elements (Na, Mg, Al, P, K, Ca, Fe, Mn,

Ni, Cd, Cu, Zn, and Mo) in the culture medium were measured using an ICP-MS unit

7500cx (Agilent Technologies, Santa Clara, CA) following US EPA method 6062A.

TAN, TN, and TP and COD were determined using a HACH 3900 spectrophotometer

(Düsseldorf, Germany) coupled with a HACH DRB200 dual block reactor (Düsseldorf,

Germany) following the manufacturer’s instruction manual. Lipid content of the algal biomass was analyzed using a slightly modified version of Bligh and Dyer’s method

(Bligh and Dyer, 1959). The procedures used for determination of the above parameters were the same as those described in Section 3.2.3 of Chapter 3.

4.2.4 Statistical analysis

Statistical significance was determined by analysis of variance (ANOVA) using

SAS software (Version 8.1, SAS Institute Inc., Cary, NC, USA) with a threshold p-value of 0.05.

4.3 Results and Discussion

4.3.1 Characterization of AD Effluent

The chemical composition of the AD effluent is shown in Table 6. The AD effluent mainly contained nitrogen and phosphorous, which are both required for growth of cyanobacteria. There were also various kinds of metal ions in the AD effluent because the feedstock for the digester was municipal wastewater which contained the ions. The effects of N/P ratio on growth of cyanobacteria have been widely evaluated. It has been shown that an N/P ratio of less than 29 tends to trigger a large population of cyanobacteria (Smith, 1983). The N/P ratio of AD effluent used in this study was about 7, which was favored by Synechocystis sp. PCC6803 (Kim et al., 2011).

4.3.2 Growth curve and biomass yield of Synechocystis sp. PCC6803 in batch cultivation

Figure 10 illustrates the growth curves of Synechocystis sp. PCC6803 at five effluent loadings (3%, 6%, 12%, 18% and 24%). As the effluent loading increased, the inhibition on the growth of Synechocystis sp. PCC6803 gradually increased. No obvious lag phase was observed at effluent loadings of 3%-12%. A 4-day and 7-day lag phase was observed for effluent loadings of 18% and 24%, respectively. N. salina had a shorter lag phase (2 days) when grown with 18% effluent loading. Although the rate slowed down,

Synechocystis sp. PCC6803 grown in 3% effluent continued to grow after the 6th day; whereas, N. salina began to go into the stationary phase on the 6th day (Chapter 3, Figure

2) due to nutrient depletion. The average specific growth rates of Synechocystis sp.

PCC6803 at the five effluent loadings are shown in Table 9. The growth rate of

Synechocystis sp. PCC6803 decreased significantly (p<0.05) as the effluent loading increased. However, N. salina was able to grow fast in effluent loading 3%-18% with the growth rate decreased only by 0.054 d-1 (Table 7). The growth rate of Synechocystis sp.

PCC6803 was higher than that of N. salina at an effluent loading of 3%, but was lower at all other four effluent loadings. This result demonstrated that Synechocystis sp. PCC6803 is more sensitive than N. salina to the toxic components in the effluent. Synechocystis sp.

PCC6803 tends to grow better in low nutrient loading conditions. The growth rates of

Synechocystis sp. PCC6803 in effluent loadings of 3% - 12% were higher than those of

Synechocystis sp. PCC6803 (0.41 d-1) cultivated in BG-11 medium in photobioreactors

Kim et al. (2010). It was reported that the maximum specific growth rates of PCC6803 could be 2.0-2.5 d-1, but the specific growth rate of Synechocystis sp. PCC 6803 was always lower than 1 d-1 in less favorable conditions (Ohkawa et al., 2000; Zhang et al.,

1994). The specific growth rate of Synechocystis sp. PCC6803 depends on many factors such as temperature, pH, concentration of nitrogen and phosphorus, etc. In order to maintain high growth rate, the nutrient supply rate must match the rate of biomass synthesis (Zhang et al., 2008). Thus, semi-continuous cultivation could offer the benefit of continuous nutrient supply and help maintain the robust growth of Synechocystis sp.

PCC6803.

6 3% 5 6% 12% 4 18% 440nm

24% at

1 Absorbance 0 012345678910 Cultivation time (day)

Figure 10 The growth curve of Synechocystis sp. PCC6803 at different effluent loadings

Table 9 Growth rate of Synechocystis sp. PCC6803 at different effluent loadings

Effluent Growth rate loading (%) (d-1) 3 0.717±0.004 6 0.611±0.002 12 0.539±0.003 18 0.272±0.006 24 0.077±0.008

The final biomass concentrations by the end of the 10-day cultivation are

presented in Figure 11. The biomass concentration decreased from 1.51 g L-1 to 0.41 g L-1 as the effluent loading increased from 3% to 24%, indicating an inhibition on growth at high effluent loading. The highest biomass yield 1.51 g L-1 was obtained at an effluent

loading of 3% and it was 1.64 times the highest biomass yield of N. salina which was

achieved at 6% effluent loading in batch experiments. The biomass concentration of

Synechocystis sp. PCC6803 grown in BG-11 medium varied between 0.43-1.2 g L-1 as

reported by Kim et al. (2010). Phang et al. (2000) cultivated another cyanobacteria,

Spirulina platensis, in digested sago starch factory wastewater and observed a biomass

concentration of 0.528-0.610 g L-1, which is much lower than that obtained in this study.

The growth of Synechocystis sp. was also found to be closely related to light irradiance

(Kim et al., 2011; Martinez et al., 2012). In a study of the effect of light on Synechocystis

sp., the highest biomass concentration of 4.53 g L-1 was achieved at an irradiance of 1600

µmol photons m-2 s-1 after 6 days of cultivation (Martinez et al., 2012).

1.8

1.2 (g/L)

yield

0.6 Biomass 0.0 3 6 12 18 24 Effluent loading (%)

Figure 11 The biomass yield of Synechocystis sp. PCC6803 at different effluent loadings

4.3.3 Nutrient removal efficiency and lipid production of Synechocystis sp. PCC6803

The removal efficiencies of TAN, TN and TP during the 10-day cultivation were investigated and the results are shown in Table 10. Ammonium, which is the preferential nitrogen form, removal was 100% at effluent loadings of 3-18%, and was 83% at 24% a loading ratio. Both the TN and TP removal efficiency of Synechocystis sp. PCC6803 decreased as the effluent loading increased. Compared with Synechocystis sp. PCC6803,

N. salina was more efficient in nutrient removal because the ammonium removal efficiency of N. salina was 100% at all loading ratios. N. salina also showed higher utilization of nitrogen and phosphorus. The uptake ratio (N/P) for Synechocystis sp.

PCC6803 is about 6, close to the nitrogen to phosphorus ratio in biomass of

Synechocystis sp. PCC6803 which is 8:1 (Kim et al., 2011). However, it has been noted that ammonium could be removed not only by active uptake during growth of organisms, but also by ammonia stripping into the atmosphere (Chaiklahan et al., 2010). Analysis of the cellular nitrogen content of Synechocystis sp. PCC6803 and N. salina is needed in order to determine the amount of nitrogen in the effluent that contributed to the their growth. Kim et al. (2011) studied the nitrogen uptake rate of Synechocystis sp. PCC6803 supplied with BG-11 medium and 2.5% CO2 in photo-bioreactors. The results showed

-1 -1 that the nitrogen uptake rate was 0.46 g N gdry weight d . It has also been suggested that stoichiometry be used to assess the necessary supply rate of nutrients for photosynthetic microorganisms to produce biomass. However, this method may not apply to using algae to remove nitrogen and phosphorus from wastewater because many factors can affect the nutrient removal such as ammonia stripping, phosphorus precipitation, etc. Therefore, the

73 nutrient loading should be determined based on the growth rate of microalgae, biomass yield, or apparent nutrient removal efficiency.

Table 10 Nutrient removal efficiency of algae culture system with Synechocystis sp. PCC6803 and N. salina in batch cultivation

Effluent loading Synechocystis sp. (%) PCC6803 N. salina TAN removal 3 100±0 100±0 6 100±0 100±0 12 100±0 100±0 18 100±0 100±0 24 82.54±2.51 100±0 TN removal 3 100±0 100±0 6 93.42±5.52 100±0 12 89.45±6.34 97.32±0.21 18 84.91±3.60 91.43±0.54 24 71.20±4.30 87.59±0.61 TP removal 3 100±0 100±0 6 100±0 100±0 12 97.47±0.51 99.8±0.13 18 94.32±1.75 99.4±0.14 24 83.57±5.00 99.3±0.23

As presented in Figure 12, the lipid content of Synechocystis sp. PCC6803 did not change significantly (p>0.05) as the effluent loading varied. In contrast, for N. salina, a decrease in lipid content was observed as effluent loading increased. This result is not surprising because many researchers have noticed that the reactions of cyanobacteria and microalgae to nutrient deficiency are quite different. Piorreck and Pohl (1984) studied and compared the effects of nitrogen starvation on the lipid metabolism of green algae

(Chlorella vulgaris and Scenedesmus obliquus) and cyanobacteria (Anacystis nidulans,

Microcystis aeruginosa, Oscillatoria rubescens, and Spirulina platensis). It was found that the lipid content and composition in the green algae changed during batch cultivation and was dependent on the nitrogen concentration in the media, but this effect was not observed in the cyanobacteria. Piorreck and Pohl (1984) used “endosymbiont theory” to explain these differences between blue green algae (cyanobacteria) and green algae. It is well-known that blue-green algae are prokaryotes and green algae are eukaryotes.

According to the “endosymbiont theory”, the chloroplasts of eukaryotic organisms are considered to have originated from cyanobacteria through endosymbiosis. Blue-green algae and chloroplasts both contain the same kind of polar lipids and polyunsaturated fatty acids but little or no neutral lipids such as triacylglycerols, which are localized in the cytoplasm of eukaryotic algae. Nitrogen deficiency stimulates a decrease in the chlorophyll of green algae which results in a reduction in the chloroplast apparatus and in the polar lipids and polyunsaturated fatty acids in the chloroplast membranes, bringing an increase in the percentage of cytoplasmic neutral lipids and fatty acids in eukaryotic algae. However, the blue-green algae cell and its components are reduced evenly as the nitrogen supply decreases.

Although the lipid content of Synechocystis sp. PCC6803 was not altered significantly (p>0.05, Figure 12), its lipid yield decreased significantly (p<0.05) as the effluent loading increased because of the significant decrease in the biomass yield

(p<0.05). The highest lipid yield of 20 mg L-1d-1 is, however, lower than that of N. salina

(29 mg L-1 d-1) because the highest lipid content of N. salina was 35.38%, which was 2.68 times the lipid content of Synechocystis sp. PCC6803 at 3% effluent loading. Even though the biomass yield of Synechocystis sp. PCC6803 could be as high as 1.64 times that of N. salina, it is still not as competitive as N. salina for lipid production.

Nonetheless, Synechocystis sp. PCC6803 is still a very good candidate for biomass production as a feedstock for biofuel because of its high biomass yield.

20 24 Lipid content Lipid yield 16 18 ）

12 (%)

12 (mg/L/day

8 content yield

6 Lipid 4 Lipid

0 0 3 6 12 18 24 Effluent loading (%)

Figure 12 The lipid content and lipid yields of Synechocystis sp. PCC6803 at different effluent loadings

4.3.4 Performance of Synechocystis sp. PCC6803 in semi-continuous cultivation

It has been stated in previous sections that the growth rate of Synechocystis sp.

PCC6803 was greatly affected by effluent loading, a result of the nutrient concentration.

The highest growth rate, biomass yield, and lipid yield were all attained at the loading of

3%. Therefore, a semi-continuous experiment for Synechocystis sp. PCC6803 was conducted with 3% effluent loading with 1-, 2-, and 3-day harvesting intervals. As shown in Figure 13, the growth rate of Synechocystis sp. PCC6803 reached steady state within four days after inoculation, which was also observed in the study of N. salina. The optical density was maintained between 0.7-1.6, 1.2-3, and 2-4 for harvesting intervals of 1, 2, and 3 days, respectively. Longer harvesting intervals led to longer hydraulic retention times (HRT), allowing cells to grow more and reach higher cell densities. However, as the cell density increased, the self-shading effects became stronger and reduced the cell growth rate (Shigesada and Okubo, 1981). Therefore, the growth rate of Synechocystis sp.

PCC6803 and N. salina decreased as the HRT increased (Table 11).

(a) 5

3 440

OD 2

0 0123456789101112131415161718 Cultivation time (day)

(b) 5

OD440 2

0 024681012141618 Cultivation time (day)

Figure 13 Growth curve of Synechocystis sp. PCC6803 at different harvesting intervals: (a) 1-day; (b) 2-day; (c) 3-day

Figure 13 continued

OD440 2

0 0 3 6 9 12 15 18 Cultivation time (day)

Nitrogen and phosphorus removal rate (mg L-1 d-1) decreased as the harvesting interval increased. The nutrient removal efficiency of Synechocystis sp. PCC6803 was more than 89%. Nitrogen and phosphorus were totally removed at 2 and 3 days harvesting interval, indicating that nutrient starvation might take place during cultivation.

The effluent loading could be increased in the future in order to achieve higher nutrient removal rate. The nutrient removal rates of Synechocystis sp. PCC6803 obtained here was comparable with other cyanobacteria, too. Ratana et al. (2010) used digested pig wastewater to cultivate Spirulina platensis in semi-continuous process and the average removal rates for TN and TP were 34 mg L-1 d-1 and 4 mg L-1 d-1, respectively. Another group of researchers studied the nutrient removal capability of Spirulina sp. in outdoor

raceways treating digested pig wastewater and reported that the NH4-N removal

efficiency was 84-96% with a rate of 13.6 mg L-1 d-1 (Olguin et al., 2003).

The growth rate, biomass productivity, and lipid content and productivity of

Synechocystis sp. PCC6803 and N. salina are summarized in Table 11. The biomass

productivity of Synechocystis sp. PCC6803 decreased as the harvesting interval

increased. The lipid content and lipid productivity did not change significantly (p>0.05).

This result was different for N. salina, where the highest lipid productivity was obtained

with a 2-day harvesting interval, probably due to the high biomass productivity and lipid

content under these conditions. In spite of its higher biomass productivity, Synechocystis

sp. PCC6803 had lower lipid productivity because its lipid content was much lower than

that of N. salina’s. The biomass productivity of Synechocystis sp. PCC6803 obtained in

semi-continuous cultivation was much higher than for other cyanobacteria strains

reported in the literature. Laliberte et al. (1997) investigated the growth and nutrient

removal efficiency of Phormidium bohneri in domestic wastewater and recorded biomass

productivities of 23-57 mg L-1 d-1, along with ammonium and phosphorus removal rates of up to 20 mg L-1 d-1. Monoculture of Planktothrix isothrix in municipal wastewater

produced biomass at rates of 27-50 mg L-1 d-1 in one laboratory study (Margarita Silva-

Benavides and Torzillo, 2012). The potential of cultivating cyanobacteria Synechocystis

sp. PCC6803 in AD effluent for nutrient removal and biomass production was

demonstrated in this study.

Table 11 Growth rate, biomass and lipid productivity of Synechocystis sp. PCC6803 and N. salina at different harvesting frequencies

Specific Biomass Lipid Lipid Treatment growth rate productivity content productivity (d-1) (mg L-1 d-1) (%) (mg L-1 d-1)

Synechocystis sp. PCC6803 1d_50% 0.720±0.004 212.4±5.6 13.7±1.8 29.1±2.3 2d_50% 0.346±0.002 204.6±2.5 13.6±2.4 27.7±1.7 3d_50% 0.237±0.007 186.4±4.3 13.5±1.5 25.1±1.6

N. salina 1d_50% 0.688±0.005 143.0±2.7 19.0±0.4 27.1±1.5 2d_50% 0.353±0.004 155.3±1.8 24.9±0.5 38.7±2.4 3d_50% 0.233±0.002 121.4±4.1 22.7±0.4 27.6±1.4

4.4 Conclusion

The results from this study demonstrated the potential of growing

Synechocystis sp. PCC6803in AD effluent for biomass and lipid production. The growth of Synechocystis sp. PCC6803 was affected by effluent loading, thus proper dilution was needed to maintain optimal growth rate. The highest biomass productivity of

Synechocystis sp. PCC6803 in the batch (150 mg L-1 d-1) and semi-continuous (212.4 mg

L-1 d-1) studies was 1.6 times and 1.4 times, respectively, higher than that of N. salina in similar batch and semi-continuous cultivation studies. However, the lipid productivity of

Synechocystis sp. PCC6803 became lower as the harvesting interval increased because of its relatively low lipid content. The lipid productivity of Synechocystis sp. PCC6803 in batch cultivation was 20 mg L-1 d-1, and increased by 45.5% in semi-continuous cultivation. The nitrogen and phosphorus removal rates of 34.5 mg L-1 d-1and 5.7 mg L-1 d-1 were obtained at a 1-day harvesting frequency and 50% harvesting ratio.

Chapter 5 Conclusions and Recommendations

Anaerobic digested municipal wastewater was shown to be a suitable culture medium for both Nannochloropsis salina and Synechocystis sp. PCC6803. Proper dilution is necessary to mitigate the negative effects of toxic components in effluent on algae growth. The growth rate and biomass productivity of N. salina and

Synechocystis sp. PCC6803 were substantially affected by effluent loading rate. N. salina had higher tolerance to ammonium and grew well under effluent loading of 3-18%, while the growth rate of Synechocystis sp. PCC6803 gradually decreased as the effluent loading increased. N. salina and Synechocystis sp. PCC6803 removed nitrogen and phosphorus effectively (>90% removal efficiency) under effluent loading of 3% and 6%, respectively. The highest biomass yield of Synechocystis sp. PCC6803 in 10-day batch study (1.5 g L-1) was 1.6 times of that of N. salina. But the lipid productivity of N. salina was higher than Synechocystis sp. PCC6803 due to higher lipid content of N. salina.

The biomass and lipid productivity of N. salina and Synechocystis sp. PCC6803 were both improved in semi-continuous cultivation at their optimal effluent loading, 6% and 3%, respectively. The nutrient removal rate was also increased due to frequent medium replenishment. The optimal conditions recommended for N. salina and

Synechocystis sp. PCC6803 were 2-day harvesting interval with 50% harvesting ratio and: 1-day harvesting interval with 50% harvesting ratio, respectively, where the highest biomass and lipid productivity were obtained. Although the lipid productivity of

Synechocystis sp. PCC6803 was lower than N. salina, its higher biomass yield still makes it a good candidate for biomass production and wastewater bio-treatment.

This study demonstrated the feasibility of growing N. salina and Synechocystis sp.

PCC6803 in AD effluent under high salinity (20‰) for bioenergy production. The optimal operation parameters for semi-continuous cultivation attained in this study need to be tested in a large pond which is subjected to changes of many factors such as temperature fluctuation, limited light, larger surface area, poor mixing. The downstream processes such as harvesting and lipid extraction are also important for the large scale algal biofuel production system. Further study and improvement are required to make algal biofuel economically viable.

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