Accepted Manuscript Review Cultivation of Microalgal Chlorella for Biomass and Lipid Production Using Wastewater as Nutrient Resource Sheng-Yi Chiu, Chien-Ya Kao, Tsai-Yu Chen, Yu-Bin Chang, Chiu-Mei Kuo, Chih-Sheng Lin PII: DOI: Reference:

S0960-8524(14)01692-7 http://dx.doi.org/10.1016/j.biortech.2014.11.080 BITE 14286

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

30 August 2014 7 November 2014 9 November 2014

Please cite this article as: Chiu, S-Y., Kao, C-Y., Chen, T-Y., Chang, Y-B., Kuo, C-M., Lin, C-S., Cultivation of Microalgal Chlorella for Biomass and Lipid Production Using Wastewater as Nutrient Resource, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.11.080

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Cultivation of Microalgal Chlorella for Biomass and Lipid Production Using Wastewater as Nutrient Resource

Sheng-Yi Chiu a,b,†, Chien-Ya Kaoa,c,†, Tsai-Yu Chena, Yu-Bin Changa, Chiu-Mei Kuoa, Chih-Sheng Lina,* a

Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan

b

Water Technology Division, Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan

c

†

Agricultural Technology Research Institute, Hsinchu, Taiwan These authors equally contributed to this work

* Author for correspondence: Chih-Sheng Lin, Ph.D. Department of Biological Science and Technology, National Chiao Tung University, No.75 Po-Ai Street, Hsinchu 300, Taiwan Tel: 886-3-5131338 E-mail: [email protected]

1

Abstract

2

Using wastewater for microalgal cultures is beneficial for minimizing the use of

3

freshwater, reducing the cost of nutrient addition, removing nitrogen and phosphorus

4

from wastewater and producing microalgal biomass as bioresources for biofuel or

5

high-value by-products. There are three main sources of wastewater, municipal

6

(domestic), agricultural and industrial wastewater, which contain a variety of

7

ingredients. Some components in the wastewater, such as nitrogen and phosphorus, are

8

useful ingredients for microalgal cultures. In this review, the effects on the biomass and

9

lipid production of microalgal Chlorella cultures using different kinds of wastewater

10

were summarized. The use of the nutrients resource in wastewater for microalgal

11

cultures was also reviewed. The effect of ammonium in wastewater on microalgal

12

Chlorella growth was intensively discussed. In the end, limitations of

13

wastewater-based of microalgal culture were commented in this review article.

14 15 16 17

1. Introduction As the fast growing world population and economic developments, the demand for

18

energy use of fossil fuel will continue to rise. But, the fossil fuels are not sustainable

19

energy resource in the long term. The burning of fossil fuels can lead to increase in

20

greenhouse gas (GHG) emissions and the environmental impact on global warming

21

(Hill et al., 2006). Renewable energy resources offer clean alternatives to fossil fuels.

22

They produce little pollution or GHG, and they will never run out. Currently, the main

23

of commercially available biofuel, kinds of renewable energy, are bioethanol and

24

biodiesel. Bioethanol is almost derived from sugar cane or corn starch and biodiesel is

25

derived from oil crops including oilseed rape and soybean. However, these crop-based 1

26

biofuels are economically competition with the production and the price of food (Hill et

27

al., 2006).

28

Biofuels derived from microalgae have been proposed as an alternative approach

29

that does not impact on agriculture. Microalgae have been estimated to produce higher

30

biomass productivity than plant crops in terms of land area required for cultivation, are

31

predicted to have lower cost per yield, and have the potential to reduce GHG emissions

32

through the replacement of fossil fuels. The main inputs required for microalgae growth

33

are sunlight, water, carbon dioxide (CO2), and nutrients. Water and inorganic nutrients

34

are identified as important limiting resources for microalgae culture. The nutrients for

35

microalgae cultivation (mainly nitrogen and phosphorus) can be obtained from liquid

36

effluent wastewater; therefore, besides providing microalgae growth environment, there

37

is the potential possibility of waste effluents treatment. This could be explored by

38

microalgae farms as a source of income in a way of wastewater treatment and obtain the

39

nutrients for microalgae growth (Cantrell et al., 2008).

40

1.1. Microalgae for CO2 mitigation

41

Microalgae are one of the earth's most important natural resources. They

42

contribute to approximately 50% of global photosynthetic activity. For autotrophic

43

algae, photosynthesis is a key component of their survival, whereby they convert solar

44

radiation and CO2 absorbed by chloroplasts and used in respiration to produce energy

45

to support growth.

46

During the recent decades, a number of post-combustion CO2 capture methods

47

have been developed using chemical, physical and biological methods (Kumar et al.,

48

2011). In recent years, the bio-regenerative methods using microalgae via

49

photosynthesis have been significant potential made to reduce the atmospheric CO2 to

50

ensure a safe and reliable living environment. In biological methods, particularly 2

51

microalgal photosynthesis, have several merits, such as higher CO2 fixation rates than

52

terrestrial plants and no requirement for further disposal of the trapped CO2.

53

Microalgae-based CO2 biological fixation is regarded as a potential way to not only

54

mitigates CO2 emissions but also shows the potential to produce lipid-rich microalgal

55

biomass as a regenerative energy-source (Ho et al., 2011). One of the most

56

understudied methods of CO2 reduction is the use of microalgae that convert CO2 from

57

a point source into biomass. Microalgae use CO2 efficiently because they can grow

58

rapidly and can be readily incorporated into engineered systems, such as

59

photobioreactors. The CO2 reduction by microalgal photosynthesis and biomass

60

conversion into health food, food additives, feed supplements, and biofuel is

61

considered a simple and appropriate process for CO2 circulation on Earth (Ho et al.,

62

2011). The incorporation of CO2 into energy-reserve components in biomass, such as

63

carbohydrates and lipids, by photosynthesis-driven microalgal fixation of CO2 is the

64

most promising route for CO2 sequestration from flue gas (Chiu et al., 2011; Kao et al.,

65

2014; Kumar et al., 2014) and biogas (Kao et al., 2012a; 2012b).

66

1.2. Microalgae for biofuel production

67

Many microalgae at or near optimal conditions potentially providing the benefits of

68

well-controlling are exceedingly rich in oil (Chisti et al., 2007; Ho et al., 2010), which

69

can be converted to many products such as renewable fuels, such as biodiesel, by

70

transesterification (Chen et al., 2011). The biodiesel produced from algal oil has

71

physical and chemical properties similar to diesel from petroleum, to biodiesel

72

produced from crops of 1st generation and compares favorably with the International

73

Biodiesel Standard for Vehicles (EN14214) (Brennan and Owende, 2010). Biodiesel

74

from microalgae seems to be the only renewable biofuel that has the potential to

75

completely displace petroleum-derived transport fuels without adversely affecting the 3

76

food supply and other crop products. According to some estimates, the yield of oil

77

from algae is over 200 times the yield from the best performing plant/vegetable oils

78

(Singh and Gu, 2010). The production of microalgae to harvest oil for biodiesel has not

79

been undertaken on a commercial scale, but working feasibility studies have been

80

conducted to arrive.

81

Despite microalgae inherent potential as a biofuel resource, many challenges have

82

impeded the development of algal biofuel technology to commercial viability that

83

could allow for sustainable production and utilization. They majorly include: (1)

84

species selection must balance requirements for biofuel production and extraction of

85

valuable co-products; (2) attaining higher photosynthetic efficiencies through the

86

continued development of production systems (Chen et al., 2011); (3) potential for

87

negative energy balance after accounting for requirements in water pumping, CO2

88

transfer, harvesting and extraction; (4) few commercial plants in operation. Therefore,

89

there is a lack of data for large scale plants.

90

1.3. Microalgal biorefinery

91

Microalgal biomass is one of the most potential feedstock for biorefinery, because it

92

can be converted into biofuels and various co-products (Vanthoor-Koopmans et al.,

93

2013). Microalgal biomass can used to produce biodiesel by extracting lipids to

94

transesterification into fatty acid methyl ester (FAME) as biodiesel. Some oleaginous

95

microalgae as nonedible biodiesel feedstock shows great potential due to its high oil

96

yield (5,000-100,000 L ha-1 year-1). The non-lipid fraction of the algal biomass,

97

consisting mainly of protein and carbohydrate, can also be processed to various biofuels,

98

including methane and alcoholic fuels (McGinn et al., 2011). Some microalgal strains

99

are rich in carbohydrates which is a potential feedstock for bioethanol production. The

100

carbohydrates in microalgae mainly come from starch in chloroplasts and 4

101

cellulose/polysaccharides on cell walls. The cellulose/polysaccharides of microalgae

102

should be hydrolyzed to fermentable sugar for bioethanol production. From the point of

103

biofuels, the microalgal cultivation integrated with biorefinery can increase the energy

104

productivity and recycle the CO2 for fuel production to achieve the sustainable

105

development. To achieve high biomass productivity per unit area, higher biomass

106

densities would be needed. Though high biomass density could be achieved in thin-plate

107

photobioreactors and fermenters, the downstream processes, such as dewatering and

108

dying facilities, remain cost and energy intensive processes. Thus, the new

109

cost-effective downstream process should be developed and introduced for biofuel

110

production.

111

In addition to produce energy by microalgal biomass, microalgal biomass can be

112

applied in high valuable products such as food supplements, cosmetics and animal

113

feeds, mainly containing polyunsaturated fatty acids (e.g., DHA and EPA),

114

chlorophylls, carotenoids and phycobilins, among other (Ho et al., 2013; Yen et al.,

115

2013). However, many developed processes focused on obtaining one specific product

116

from microalgal biomass without complete utilization. In other words, the product

117

extraction and conversion processes were developed for one specific product. This

118

mostly means that the other available and valuable components in the microalgae were

119

lost as waste. Thus, integrated processes for complete utilization of microalgal biomass

120

are still need to be developed for enhancement of microalgal biomass utilization

121

sustainability.

122

1.4. Microalgae for wastewater treatment

123

Inorganic nitrogen and phosphorus are is particularly difficult to remove from

124

wastewater. Due to the ability of microalgae to use both wastewater pollutions for their

125

growth, microalgae are particularly useful to reduce the concentration of inorganic 5

126

nitrogen and phosphorus in wastewater (Ahluwalia and Goyal, 2007). Many species of

127

microalgae are able to effectively grow in wastewater conditions through their ability to

128

utilize abundant inorganic nitrogen and phosphorus in the wastewater. Therefore, the

129

mass culture of microalgae can be potentially used for wastewater treatment as a tertiary

130

process (Martin et al., 1985).

131

A complete tertiary process in the wastewater treatment aimed at removing

132

nitrogen and phosphorus is estimated to be about four times more expensive than

133

primary treatment (de la Noüe et al., 1992). It is identical that microalgal cultures offer

134

an elegant solution to tertiary treatment due to the ability of microalgae to use inorganic

135

nitrogen and phosphorus for their growth (Oswald, 1988a; Tam and Wong, 1996). And

136

also, their capacity to remove heavy metals, as well as some toxic organic compounds,

137

therefore, does not lead to secondary pollution. In conclusion, microalgae cultures by

138

wastewater can significantly contribute to the management of water ecosystems by

139

providing an inexpensively environment-friendly system for wastewater treatment.

140

The major advantages of mass cultured microalgae having over conventional

141

aerobic wastewater treatment systems are reduced cost due to the decrease in energy

142

input, low initial capital cost, and low operational cost (Wong and Tam, 1998).

143

However, there are also lot disadvantages, e.g., microalgae cultivation is the space

144

requirement. Since microalgae rely on photosynthesis, the availability of sunlight to

145

reach the microalgae is critical. Therefore, microalgae-based wastewater treatment

146

systems should be performed in low land-cost areas where sunlight and warm

147

temperatures. The potential microalgal wastewater process was shown as the simplified

148

process flow diagram envisioned for typical wastewater treatment, algal wastewater

149

treatment with flue gas mitigation and production of various algal biomass products.

150

The integrated process of using wastewater for microalgae production can be shown as 6

151

the flow diagram envisioned for typical wastewater treatment, microalgal wastewater

152

treatment with flue gas mitigation, and production of various algal biomass products.

153

The typical wastewater treatment can be simply organized into three steps of

154

solid-liquid separation, horizontal anaerobic fermentation and an activated sludge

155

process in aerobic treatment. After the three-step wastewater treatment, the biochemical

156

oxygen demand (BOD) and suspended solids (SS) of treated wastewater were both

157

markedly reduced (Su et al., 1997). The effluent contains nitrogen, phosphorus and

158

other nutrients, and the flue gas consists CO2, both provide the nutrition, including

159

nitrogen, phosphorus and carbon sources, for the cultivation of microalgae. The

160

integrated system of microalgal wastewater treatment incorporated with flue gas was

161

using wastewater and flue gas process; that is not only an environmental-friendly but

162

also a sustainable process for wastewater treatment and CO2 mitigation (Rawat et al.,

163

2011; Razzak et al., 2013).

164 165

2. Wastewater used for microalgae cultures

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In the past two decades, remarkable efforts have been put into research of

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microalgae cultivation using wastewater. Some of studies indicated that utilizing

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microalgae could efficiently remove the nitrogen, phosphorus, and heavy metal

169

elements from wastewater (Cabanelas et al., 2013; Cho et al., 2013; Min et al., 2011;

170

Sun et al., 2013; Wang et al., 2010; Zhu et al., 2013b). Additionally, the nutrient

171

components in wastewater significantly affect the microalgae growth, and their biomass

172

and lipid production (Cai et al., 2013). The nutrient components of different wastewater

173

streams that have been used for microalgal cultivation are discussed as follows.

174

2.1. Wastewater characterization

7

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Depending on the origin, wastewater used in microalgal cultivations can be divided

176

into three main types as municipal, agriculture and industrial liquid waste products. The

177

wastewater treatment generally consists two or three phases as follows: a primary

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treatment phase for the sedimentation and/or floatation of solid materials, a secondary

179

treatment phase in which suspended and dissolved organic materials are removed using

180

by physical, chemical and biological processes, and a tertiary treatment phase, such as

181

disinfection and filtration, in which final treatment of the water is performed prior to

182

discharge into the environment (Ramalho, 1977). The treated water discharged from a

183

process of wastewater treatment is known as effluent.

184

There are several common wastes in the effluents of wastewater, including sludge

185

and scum, organic waste, inorganic waste, nutrients, toxins, pathogenic organisms, etc..

186

For microalgal growth, nutrients in wastewater are extremely interest for the more

187

discussion in this review article. Excessive nutrients in the wastewater, such as

188

nitrogen and phosphorus, may cause eutrophication in lakes and upset the balance of

189

the ecosystem (Cai et al., 2013). Nutrients are also the substances that are required for

190

the growth of microalgae. The major nutrients of interest are nitrogen and phosphorous.

191

Nitrogen may be present in effluent as ammonia (NH4 +), organically bound nitrogen or

192

even nitrite (NO2-) and nitrate (NO3-). Phosphorous is presented in influent and effluent

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primarily in the form of phosphates (PO43-).

194

2.2. Nutrients from wastewater for microalgal cultures

195

The total nitrogen (TN) and total phosphorus (TP) concentrations of wastewater

196

would differ significantly depending on the wastewater type (Table 1). Nitrogen in the

197

form of ammonia and nitrates in wastewater is the most commonly found nitrogen

198

containing chemicals. Ammonium is among the most common chemical forms of

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nitrogen that can be readily absorbed by most microalgal species and strains. In this 8

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respect, a cheap source of nitrogen in wastewater or effluent of can be used for

201

microalgal cultivation (Razzak et al., 2013). It may imply that some studies showed

202

wastewater-based microalgal cultures are inhibited with the high concentration of TN,

203

especially high ammonium. However, when the pH of the culture and other growth

204

conditions are controlled, ammonium is a reliable nitrogen source (de-Bashan et al.,

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2005). In conclusion, regardless of the negative effects on microalgal growth in

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ammonium-supplementation, it is still the preferred nitrogen source if the

207

environmental parameters for proper development of the culture are controlled (Razzak

208

et al., 2013).

209

Phosphorus is another important element required for microalgae growth and

210

metabolism. Phosphorus is an essential element contributing as ATP in microalgal

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cells. Therefore, phosphorus availability has a large impact in microalgae growth as it

212

is considerably affected in photosynthesis (Razzak et al., 2013). Phosphorus is usually

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available in the wastewater as inorganic anions species such as H2PO4- and HPO42-

214

(Martinez et al., 1999).

215

The concentrations of TN and TP which are relatively low (TN: about 15-90 mg

216

L-1; TP: about 5-20 mg L-1) in domestic secondary effluent. The lower TN and TP are

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the typical water quality of domestic wastewater. The concentrations of TN and TP in

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the wastewater from livestock breeding and agriculture are usually about 185-3,213 mg

219

L-1 of TN and about 30-987 mg L-1 of TP, such as anaerobic digested poultry litter

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effluent, swine or dairy manure wastewater. However, these kinds of wastewater

221

always contained nutrients of an extremely high concentration, and thus had to be

222

diluted before they used for microalgal cultivation.

223

2.2.1. Municipal wastewater

9

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The increasing urbanization and expansion of urban populations has resulted in

225

greater quantities of municipal wastewater, or defined as domestic wastewater. The

226

composition of municipal wastewater varies significantly from one location to another.

227

Municipal wastewater effluent typically contains human and other organic waste,

228

nutrients, microorganisms and household and industrial chemicals. Compared with

229

industrial and agricultural wastewater, there is less nitrogen and phosphorus in

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municipal wastewater. However, because of the activities of some localized

231

small-scale factories, a significant level of heavy metals such as lead, zinc, and copper

232

will also present in raw municipal sewage. Microalgae cultivation using municipal

233

wastewater as the nutrient sources has been most extensively studied as the main topic

234

of nitrogen and phosphorus removal (Li et al., 2011; Ruiz-Marin et al., 2010).

235

Although, the nitrogen and phosphorus contained in municipal wastewater would be a

236

potential nutrient sources, the complexity, variety and toxicity of the municipal

237

wastewater should be considered for distinct stages of the treatment in order to fit the

238

wastewater discharge standard. Thus, it is no doubt the study of microalgal cultivation

239

using wastewater, the performance of wastewater treatment that remove most toxic

240

components should be also considered (Cabanelas et al., 2013).

241

2.2.2. Agricultural wastewater

242

In general, agriculture is the largest user of water in the world except few regions

243

(Abdel-Raouf et al., 2012). The water carrying waste material from agricultural

244

activities, including animal manure, plant stalks, hulls and leaves, etc.. The

245

large livestock and poultry operations would be a major source of point source

246

wastewater. During the past decades, livestock operations have intensively increasing

247

from small-scale to large-scale. The nitrogen and phosphorus are the main component

248

in the wastewater produced from animal farms (Zhu et al., 2013a). Ammonium and 10

249

organic nitrogen are the main components present in agriculture wastewater. The main

250

form of nitrogen waste in animal waste is ammonium that is almost half of total

251

nitrogen source. Because the animal wastewater produced from the agricultural

252

activities such as animal diet, usage, productivity, and location will significantly affect

253

the nutrient content in animal wastewater (An et al., 2003; de Godos et al., 2009; Wang

254

et al., 2010; Wilkie and Mulbry, 2002; Zhu et al., 2013b).

255

2.2.3. Industrial wastewater

256

There are many types of industrial wastewater based on different industries and

257

contaminants; each sector produces its own particular combination of pollutants.

258

Compared to agricultural wastewater, industrial wastewater varies depending on the

259

source operations. Heavy metal pollutants present in most industrial wastewater;

260

furthermore, nitrogen or phosphorus in industrial wastewater is usually less than

261

municipal or agricultural wastewater (Chinnasamy et al., 2010). The amount of

262

wastewater depends on the technical level of process in each industry sector and will

263

be gradually reduced with the improvement of industrial technologies. The increasing

264

rates of industrial wastewater in developing countries are thought to be much higher

265

than those in developed countries.

266

Due to the complexity of industrial wastewater, the screening and isolation of high

267

tolerance of metal and organic material microalgae species and strains is crucial to

268

achieve high growth efficiency. Nevertheless, only a few literatures studied the

269

microalgal cultivation with high metal tolerance as well as the nitrogen and phosphorus

270

removal (Ahluwalia and Goyal, 2007).

271 272 273

3. Efficiency of microalgal Chlorella growth in wastewater The efficient growth of microalgae in wastewater is dependent on many variables. 11

274

For example, the obviously differ depending on the types and the sources of

275

wastewater. In addition, the variation is also shown in the tolerant capacity of different

276

microalgal species to a particular wastewater condition. Chlorophytes is one of the

277

largest phyla of microalgae, with a variety of species and a wide geographical

278

distribution. Chlorophytic microalgae have been shown to be potential used to a

279

variety of wastewater conditions and very efficiency at nutrients removal from

280

wastewater. The potential microalga strains of Chlorophytes, Chlorella, have been

281

studied enormously and also shown to be effective in nitrogen and phosphorus removal

282

from wastewater with a wide range of initial concentrations (Cai et al., 2013) and the

283

growth of Chlorella species cultured in wastewater have examined in most studies (Wu

284

et al., 2014).

285

For the comparison of different wastewater effects on the microalgal growth, the

286

three different types of wastewater, the nutrient contents and the microalgal Chlorella

287

species and strains used in the recent literatures were summarized in Table 1.

288

Furthermore, the effects of different wastewaters on microalgal Chlorella cultures were

289

organized in Table 2. Then, the efficiency of Chlorella growth in different types of

290

wastewater is reviewed in this section.

291

3.1. Cultures in municipal wastewater

292

In the published literatures, most studies were using municipal wastewater for

293

microalgal cultivation. One of the main reasons is that the municipal wastewater

294

treatment is relatively mature in developed countries. However, the chemical

295

concentrations in municipal wastewater are complex because some municipal

296

wastewater treatment would mix with low concentrations of industrial wastewater that

297

produced in local small factories. Some municipal wastewater would contain a small

298

amount of heavy metal or chemicals, which were diluted by domestic wastewater to fit 12

299

in with ease for biological treatment and wastewater discharge standard (Gizgis et al.,

300

2006). Thus, the effects on the growth of microalgal culture would vary. In this review,

301

we summarized the microalgal biomass production versus different sources of

302

municipal wastewater mainly on the contents of phosphorus and nitrogen.

303

Microalgal Chlorella usually performed a high nutrient pollutions removal and

304

utilized efficiency when the algal cells cultured in domestic wastewater. Lau et al. (1996)

305

studied the ability of C. vulgaris in nutrients removal and reported a nutrient removal

306

efficiency of 86% for inorganic nitrogen and 78% for inorganic phosphorus. It is

307

recently reported that C. vulgaris cultivated by several streams from municipal

308

wastewater yields microalgal biomass from 39 to 195 mg L-1 d-1 (Cabanelas et al., 2013).

309

Cho et al. (2013) also revealed that Chlorella sp. yield the highest biomass production

310

approximately 3.0 g L-1 using 10% anaerobic digestion tanks, and conflux line of the

311

90% wastewaters combined wastewater as nutrients for microalgal cultivation. Li et al.

312

(2011) showed that the biomass productivity of Chlorella sp. growing in the centrate, a

313

highly concentrated municipal wastewater stream generated from activated sludge

314

thickening process, was 0.9 g L-1 d -1. Thus, using municipal wastewater for microalgal

315

culture could be a useful and practical strategy as an advance green treatment process.

316

3.2. Cultures in agricultural wastewater

317

Compared to municipal wastewater, there can be very high in nitrogen,

318

phosphorus, and organic matter in both soluble and solid particle forms in the effluent

319

from agricultural wastewater that is often derived from manure or animal waste. In

320

spite of the high nutrient concentration, studies have demonstrated that the microalgae

321

would be the potential candidate with efficient growth on agricultural waste.

322

Furthermore, microalgae are efficient nitrogen and phosphorus remover from

323

manure-based wastewater (An et al., 2003; Wilkie and Mulbry, 2002). The high 13

324

concentrations of nitrogen and phosphorus of agriculture wastewater, e.g., piggery

325

wastewater, is a potential nutrient source for microalgal growth (de Godos et al., 2009).

326

The higher availability of carbon and nitrogen in piggery wastewater, together with the

327

favorable temperatures and solar irradiations supported higher microalgal biomass

328

productivity. However, the extremely high concentration of NH4+-N in livestock

329

wastewater may be a growth reducing factor. The use of piggery wastewater

330

containing volatile fatty acids, like acetic acid, propanoic acid and butric acid, which

331

provided carbon source in the wastewater, could also be the pH regulator. Huo et al.

332

(2012) used acetic acid as the pH regulator, higher oil content and specific growth rate

333

of C. zofingiensis culture using dairy wastewater can be obtained. In fact, organic

334

carbon source contained in piggery wastewater could provide the mixotrophic

335

condition for microalgae. Lot of the studies showed that the biomass and lipid

336

production significantly enhanced by heterotrophic or mixotrophic cultivation

337

conditions (Perez-Garcia et al., 2011).

338

As we summarized literatures, the studies used agricultural wastewater are not as

339

many as municipal wastewater literatures (Table 1 and Table 2). That may be due to

340

the maturation of well-controlled municipal wastewater treatment and the availability

341

of sewage piping system. However, the properties of agricultural wastewater show a

342

great potential for microalgal biomass and lipid production. This could be the benefit

343

for the countries with intensive pig farming. This is a kind of industrial animal

344

agriculture and the localized wastewater treatment could provide for microalgal

345

cultivation. Zhu et al. (2013a; 2013b) have demonstrated that using microalga C.

346

zofingiensis cultivation combined with piggery wastewater treatment showed the

347

advantage of nutrient removal and the highest productivities of biomass and lipid. The

348

microalgal biomass production could achieve 2.9 g L-1 in a 10-day cultivation. It is 14

349

worthy of note that the diluted piggery wastewater strategy provided an optimal

350

nutrient concentration for C. zofingiensis cultivation. It was also mentioned that the

351

high dilution multiples applied to the digested manure for microalgal growth can be

352

proposed as an effective way to convert high strength dairy manure into profitable

353

byproducts (Wang et al., 2010).

354

3.3. Cultures in industrial wastewater

355

There are few studies using industrial wastewater for microalgal cultivation

356

because the complexity of the contents of industrial wastewater (Table 1 and Table 2).

357

However, there is also many of interest in the use of microalgae as bioremediation for

358

industrial-derived wastewaters, mostly for the heavy metal removal and the

359

biodegradation of organic chemical toxins (hydrocarbons and biocides), rather than

360

nitrogen and phosphorus (Ahluwalia and Goyal, 2007; de-Bashan and Bashan, 2010).

361

In general, microalgal growth rates are lower in many industrial wastewaters because

362

of the low nitrogen and phosphorus concentration and high concentration of toxic

363

compounds. Consequently, there is less potential for utilizing industrial wastewaters

364

for large-scale microalgal mass production (Pittman et al., 2011). But, a huge amount

365

of specifically industrial wastewater is potential resources for microalgae cultivations.

366

Part of the studies focused on the treatment of the industrial wastewater, like

367

textile wastewater decoloring by microalgae as bioremediation. The potential source of

368

industrial wastewater for microalgal cultivation is food processing wastewater, such as

369

soybean processing, brewery and chemical fermentation wastewater (Farooq et al.,

370

2013; Su et al., 2011; Sun et al., 2013). The contents in these industrial wastewaters

371

would be simpler than that of other industrial wastewater, such as carpet mill effluents,

372

iron and steel industrial wastewater, petrochemical industrial wastewater. These

373

wastewaters contain high contents of metals, phenols and chlorinated organic 15

374

compounds those are toxic to microalgal cells as inhibitors. For the further using

375

industrial wastewater with higher concentration of growth inhibitory elements for

376

microalgal cultivation, the adaptive process for microalgal cells or the dilutions of

377

wastewater are needed. In the study, Chinnasamy et al. (2010) used the carpet mill

378

effluent from the city of Dalton, USA, makes up 100-115 million L of wastewater per

379

day. This wastewater was shown to be low enough in toxins and enough phosphorus

380

and nitrogen to support microalgae growth. The data showed that microalge C.

381

saccharophila is able to grow particularly well on the untreated wastewater.

382

Additionally, the contents of food processing wastewater are mainly biological

383

available compounds, such as ethanol, acetic acid and propionic acid, which provided a

384

potential organic carbon source for microalgal cultivation by heterotrophic or

385

mixotrophic cultivation mode.

386 387 388

4. Effects of ammonium and C:N:P ratios in wastewater on Chlorella cultivation We used 3-dimensional reaction surface plots and contour plots to demonstrate the

389

biomass production and productivity of microalgal Chlorella versus a variety of

390

concentrations of NH4+-N and TP in the wastewater used for Chlorella cultures (Fig. 1).

391

In Fig. 1A, the optimal concentration of NH4+-N and TP for biomass production of

392

the Chlorella cultures were between 200 to 400 mg L-1 for NH4+-N and 100 to 200 mg

393

L-1 for TP. However, the NH4 +-N concentration in typical municipal wastewater is just

394

between 10 to 80 mg L-1 (as TN between 16 to 250 mg L-1 in Table 1). The maximum

395

biomass production obtained via municipal wastewater cultivation was approximate 1.5

396

g L-1. This municipal wastewater was obtained as centrate III with 466 mg L-1 of

397

NH4+-N (Cabanelas et al., 2013). However, a higher NH4+-N of the centrate IV (905 mg

398

L-1) contributed to lower biomass production. In addition, Cabanelas et al. (2013) 16

399

showed that as the municipal concentrate with higher NH4+-N above 100 mg L-1, the

400

lower TN removal. However, there is no obvious linear relationship between NH4 +-N

401

removal and biomass production, but it showed a bell-shaped curve pattern for the

402

NH4+-N removal and biomass productivity. The high NH4+-N content in wastewater is

403

as growth inhibitory factor for microalgae because that may contribute to the correlation

404

with pH value. Tam and Wong (1996) showed that the growth of Chlorella was reduced

405

as NH4 +-N > 700 mg L-1 with a pH level below 7. The growth of C. vulgaris in cultures

406

containing NH4+-N was also accompanied with the change of pH value

407

(Przytocka-Jusiak et al., 1977). The dissociation of NH4+ to NH3 occurs at pH level > 8.

408

The lower pH value reduces the formation of NH3 and the nitrogen exists in NH4 + ion

409

form, which is less toxic to microalgal cells. If the nitrogen source as the form of NH3,

410

it would dissipate the pH level within microalgal cells because of high membrane

411

permeability of NH3 (Taiz and Zeiger, 2006). Therefore, cellular mechanism of pH

412

regulation may help the assimilation of NH3 in the wastewater used by microalgae.

413

The optimal condition for biomass productivity of the Chlorella cultures versus

414

NH4+-N is 200 to 400 mg L-1. The values are similar to that of biomass production. The

415

TP concentration for biomass productivity is < 100 mg L-1, this value is little less than

416

that of biomass production (Fig. 1B). However, it can be concluded that the effects of

417

NH4+-N and TP concentrations in wastewater on the Chlorella biomass production and

418

productivity are similar.

419

The carbon:nitrogen (C:N) and carbon:phosphorus (C:P) ratios could be considered

420

when the wastewater is as the nutrient source for the wastewater-based microalgal

421

cultivation. The C:N and C:P ratios in municipal wastewater, such as domestic sewage

422

(C:N is 3.5:1 and C:P is 20:1) and dairy lagoon water (C:N, 3:1 and C:P, 10:1) are low

423

compared to the typical ratios for optimal conditions for microalgae biomass production 17

424

(C:N, 6:1 and C:P, 48:1) (USDA, 1992). This deficient carbon in wastewater would be

425

the restriction for microalgal biomass production; as a result, incomplete assimilation of

426

wastewater nutrients by microalgal cultures occurred. In addition, some published

427

literatures also mentioned that carbon and nitrogen metabolism are linked in microalgae

428

(Fernandez and Galvan, 2007; Perez-Garcia et al., 2011). The reason is that the carbon

429

generated directly from respiration shared each other and the energy was also generated

430

in the TCA cycle (Fernandez and Galvan, 2007). These carbon limitations of C:N and

431

C:P ratios in wastewater could be overcome by waste CO2 addition, like flue gas.

432

C. kessleri culture could successfully remove high concentrations of nitrogen (NH4 +-N

433

or nitrate) from the glucose supplemented wastewater (Lee and Lee, 2002). Thus,

434

sufficient supply of carbon source would be benefit to nitrogen and phosphorus

435

utilization. However, cheaper organic carbon sources need to be looked for, because the

436

cost of the organic carbon substrate is estimated to be about 80% of the total cost of the

437

cultivation medium. Cheap carbon sources, such as crude glycerol from biodiesel

438

industry, sugars from industrial and agricultural waste, acetate from anaerobic digestion,

439

cellulosic materials and cane molasses can be used to replace the expensive pure

440

glucose for the cultivation of mixotrophic algae (Abreu et al., 2012).

441

The typical N/P ratio for optimal conditions for microalgae biomass production was

442

8:1 (USDA, 1992); however, the N/P ratios in most sources of wastewater are close to

443

1:1 (Table 1). It means that the phosphorus source in wastewater is sufficient rather

444

than nitrogen source. This result may also imply that nitrogen source in wastewater

445

would be the limiting factor for microalgal growth.

446 447 448

5. Lipid production of Chlorella cultures in wastewater Although the main focus in the field of using wastewater for microalgae culture is 18

449

to evaluate the viability of microalgae cultivated in wastewater for the inorganic

450

nutrients removal, as the view of sustainable process, it is no doubt that utilization of

451

the produced biomass after wastewater treatment is important to put into consideration.

452

Therefore, the recent studies focused on maximizing the biomass and lipid yield of

453

microalgae has shifted the interest from nutrient removal (Cai et al., 2013).

454

In many studies, the lipid content and production of microalgae cultivated in

455

wastewater was not determined. Table 2 and Fig. 2A demonstrate that the higher lipid

456

contents could be achieved under the wastewater cultivation of lower concentrations of

457

NH4+-N and phosphorus. It is reasonable because that low concentration of NH4+-N

458

during microalgal cultivation is a trigger factor that low available nitrogen (nitrogen

459

deficient condition) contributes to lipid accumulation. Generally, the average lipid

460

productivity of microalgae in the effluent of municipal wastewater was less than

461

10 mg L-1 d -1 (< 10% lipid content). Compared with that, many microalgal species

462

cultured in agriculture wastewater could produce average lipid productivity ranging

463

from 20 to 100 mg L-1 d -1 (Wu et al., 2014). Fig. 2B and 2C show the lipid production

464

and lipid productivity of the microalgal cells under wastewater cultivation.

465

Nevertheless, comparing to the respond pattern of lipid content data in Fig. 2A, these

466

data show a contradict result that higher NH4 +-N in wastewater could generate higher

467

lipid production and lipid productivity of microalgae. However, it is not surprising

468

because the optimal condition range of NH4+-N and phosphorus of respond patterns for

469

lipid production and lipid productivity consist with the response pattern for biomass

470

production and biomass productivity (Fig. 1). This may imply that biomass production

471

and productivity are the dominant factor for the results of lipid production and

472

productivity.

473

However, the lipid production is also dependent on the microalgal species used. 19

474

C. ellipsoidea YJ1 isolated by Yang et al. (2011a) could achieve a higher lipid content

475

of about 40% in domestic secondary effluent. In the study reported by Su et al. (2011),

476

C. pyrenoidosa achieved an average lipid productivity of 236 mg L-1 d-1 under the

477

condition of fed-batch culture using soybean processing wastewater. Additionally,

478

Abreu et al. (2012) reported that C. vulgaris was cultivated in hydrolyzed cheese whey

479

(5 g L-1 glucose and 5 g L-1 galactose), and a lipid productivity about 250 mg L-1 d -1

480

was obtained.

481

Wang et al. (2010) evaluated a batch culture-grown Chlorella sp. on anaerobically

482

digested dairy manure. The total fatty acid content ranged from 9.0% to 13.7% DW

483

(0.141 to 0.233 g L-1) depending on the wastewater concentration used. C. vulgaris

484

grown in an artificial wastewater medium resulted in 20-42% of lipid content (Feng et

485

al., 2011). C. pyrenoidosa was also shown to adapt and utilize high concentrations of

486

organic components in wastewater, resulting in a significant growth potential and lipid

487

production (Su et al., 2011). Farooq et al. (2013) reported that using the two-stage

488

autotrophic-heterotrophic cultivation for C. vulgaris, the lipid productivity was

489

increased from 31 to 108 mg L-1 day. It was also reported that C. pyrenoidosa could

490

attain an average lipid content of 37%, and a high lipid productivity of approximate 0.4

491

g L-1 d -1 using soybean processing wastewater without a supply of additional nutrients

492

(Su et al., 2011).

493

The properties of a biodiesel fuel, including its ignition quality, combustion heat,

494

cold filter plugging point (CFPP), oxidative stability, viscosity and lubricity, are

495

determined by the structure of its component fatty esters. High levels of saturated fatty

496

acids tend to increase the stability of biodiesel because unsaturated fatty acids result in

497

poor oxidative stability. Thus, the biodiesel qualities are dependent on fatty acid

498

composition. The typical fatty acid composition of Chlorella under conditions of 20

499

photoautotrophic and heterotrophic cultivations, nitrogen starvation, and outdoor in a

500

photobioreactor were C16:0, C16:1, C16:2, C16:3, C18:0, C18:1, C18:2, C18:3 (Kao et

501

al. 2012a; Petkov and Garcia, 2007). Among of them, C16:0, C18:0, C18:1 and C18:2

502

are suitable fatty acids for biodiesel production, and these are the most abundant fatty

503

acids in microalgae.

504

The fatty acid profile was shown in Li et al. (2011), for Chlorella sp. cultivated in

505

the centrate, a highly concentrated municipal wastewater, the most abundant fatty acids

506

obtained from algae were C18:2 (14.4-24.4% in total lipid) and C16:0 (15.2-19.1%). C.

507

vulgaris cultivated in the pretreatment of dairy wastewater by UV and NaClO, C16:0

508

was the most abundant fatty acid, ranging from 36.9 to 45.3% of the total fatty acids

509

(Qin et al., 2014). The Chlorella cultures in mixtrotrophic growth with acetic acid

510

regulation having a higher C18:1 (32-35%) which is good for biodiesel production was

511

observed (Huo et al., 2012; Liu et al., 2011). Farooq et al. (2013) reported the main

512

constituents of the lipid extracted from C. vulgaris (UTEX-265) cultivation in brewery

513

wastewater under two-stage photoautotrophic–mixotrophic mode utilizing glucose

514

were C16:0 (20%), C18:1 (44%), and C18:2 (~14%). Above of all studies, the

515

C16–C18 percentage of total fatty acid methyl ester of the Chlorella species

516

wastewater cultivation in heterotrophic or mixotrophic conditions was over 80%,

517

which is an ideal lipid source for biodiesel production (Liu et al., 2011).

518 519 520

6. Limitations of microalgal biomass productivity by wastewater The high biomass production and in some cases of high lipid productivity that have

521

been demonstrated in many of the reviewed studies of wastewater-grown microalgae

522

suggests that there is real potential in the utilization of these high nutrient resources for

523

cost-effective biofuel production (Pittman et al., 2011; Razzak et al., 2013; Singh and 21

524

Gu, 2010; Wu et al., 2014). However, there are some limitations that need to be

525

addressed when wastewater used as the nutrients for microalgae cultures.

526

6.1. Microalgal growth in unsterilized wastewater

527

Microalgae can effectively grow and utilize nutrients in wastewater, providing an

528

attractive means to cultivate microalgae low cost. But, microalgal resistance to biotic

529

pollution is necessary to be considered as an important issue for microalgal growth in

530

wastewater. The biotic pollution is existed or produced by various bacteria, fungi and

531

zooplanktons in wastewater. The microbial existence is the most important

532

characteristic of wastewater (U.S. DOE, 2010; Wang et al., 2013), and also one of the

533

most significant difference between wastewater and culture medium for microalgae

534

culture. However, the growth of microalgae is sometimes restricted by the presentation

535

of bacteria and protozoa in wastewater. Thus, most of the reported data was obtained

536

under sterilized conditions, i.e., the wastewater was treated by autoclave sterilization

537

and/or centrifugation before it was applied for microalgae culture. In laboratory scale, it

538

is easy to sterilize wastewater as microalgal culture broth. But the cost and difficulty

539

would increase exponentially in large-scale microalgal cultivation using wastewater. In

540

order to guarantee stable microalgal growth using wastewater against possible biotic

541

contamination, it is necessary to develop the control techniques of biotic pollution

542

(Wang et al., 2013).

543

The literature showed that C. zofingiensis cultivated in piggery wastewater

544

pretreated by autoclaving and NaClO had no evident difference in the performance of

545

nutrient removal, i.e., microalgal growth and lipid production (Zhu et al., 2013b). The

546

pretreatment of dairy wastewater by UV and NaClO which were feasible for large-scale

547

cultivation was also investigated. The results showed that the highest biomass and lipid

22

548

productivity of C. vulgaris could be obtained by the NaClO-pretreated dairy

549

wastewater.

550

The stability of mass culture is an important issue for wastewater-based microalgal

551

cultures. Zhu et al. (2013b) and Qin et al. (2014) have mentioned that NaClO pretreated

552

wastewater was feasible for microalgal culture, but the hydraulic retention time for

553

NaClO pretreatment need around 15 to 30 min. That means the extra area or volume for

554

the wastewater pretreatment was needed. UV disinfection can instantaneously neutralize

555

microorganisms as they pass by UV lamps submerged in the effluent. The process adds

556

nothing to the water but UV light, therefore there is no impact on the chemical

557

composition or the dissolved oxygen content of the water. If an integrated microalgal

558

cultivation system with wastewater was applied as a continuous flow process, the UV

559

disinfection would be a promising method for wastewater sterilization.

560

6.2. Microalgal cultivation in outdoor system with a large scale

561

The cultivation scale of most recent studies was in the range from several liters to

562

several cubic meters, which means in order to achieve microalgal biomass production

563

under industrial scale it is necessary to enlarge the recent cultivation scale by tens of

564

thousands of times. This, not surprisingly, would bring along a host of problems

565

including political, social and economic as well as scientific (Williams and Laurens,

566

2010).

567

The most important question is what kind of cultivation system could provide

568

microalgal biomass production in a huge scale economically and

569

environmental-friendly. Due to the complexity of construction and the requirement of

570

huge covering area, most of the photobioreactors and microalgal ponds used in recent

571

studies are not possibly suitable for economically industrial-scale microalgal biomass

572

production. Therefore, the R&D efforts on novel cultivation system are very important 23

573

in the future research. The most important limitation is actually caused by light

574

attenuation in the systems of microalgal culture. Therefore, using microalgal species

575

that have the ability to use organic matters in wastewater for heterotrophic growth may

576

be a novel alternative approach to overcome the light limitation (Perez-Garcia et al.,

577

2011).

578

6.3. Life cycle analysis of wastewater-derived microalgal biofuel

579

A critical determinant as to the true potential of algal biofuel production using

580

wastewater resources is whether the process will provide a positive energy output

581

which will also determine the economic viability of the process. It was extremely

582

discussed on the need of life cycle analysis of wastewater-derived microalgal biofuel

583

by Pittman et al. (2011).

584

To date, microalgae-based biofuel production has not yet been commercialized to

585

large-scale. Debates exist regarding life-cycle impacts of large-scale microalgae-based

586

biofuel production, especially the impact on water usage. Yang et al. (2011a) have

587

examined that the life-cycle water and nutrients usage of microalgae-based biodiesel

588

production. Large-scale microalgae biodiesel production has been criticized for the

589

significant amount of freshwater usage (U.S. DOE, 2009). However, microalgae-based

590

biodiesel production may consume much less water than conventional feedstock-based

591

biodiesel production if microalgae are cultivated in wastewater. Without recycling

592

harvested water, the water footprint is approximately 3,726 kg-water/kg-biodiesel, and

593

around 0.33 kg nitrogen and 0.71 kg phosphorus is required (Yang et al., 2011b).

594

Recycling harvest water reduces the water by 84% and nutrients usage by 55%. Using

595

wastewater as microalgae culture medium decreases 90% water requirement, and

596

eliminates the need of all the nutrients except phosphorus. The results confirm the

597

competitiveness of microalgae-based biofuels and highlight the necessity of recycling 24

598

harvested water and using wastewater as water source (Yang et al., 2011b).

599

6.4. Need of efficient techniques for the processes of microalgae following

600

cultivation

601

Besides to the limitations mentioned above, an important issue to be resolved in

602

the applications with microalgae cultivation for biofuel productions is the need for

603

efficient and cost-effective harvesting and processing of microalgae following

604

cultivation. Recovery has been estimated to contribute 20-30% of the total cost of

605

producing the microalgal biomass (Molina Grima et al., 2003). The initial harvesting

606

step is not only costly, but also affects any later processes downstream. The lack of

607

efficient microalgal harvesting systems is the major reason why wastewater-derived

608

microalgal biofuels is not used extensively by the wastewater industry (de-Bashan and

609

Bashan, 2010).

610

Following the microalgal harvesting, the microalgae have to go through a number

611

of further processes. The need for improvements in these further processes still also

612

remains to be largely developed (Brennan and Owende, 2010). Microalgal biomass can

613

be utilized for the production of various biofuels such as bioethanol, biodiesel,

614

biomethane and biohydrogen. However, significant improvements in the efficiency, cost

615

structure and ability to scale up microalgal growth must be made to produce

616

commercially viable biofuels (Pittman et al., 2011). For this purpose, a defined set of

617

technology breakthroughs will be required to develop the optimum utilization of

618

microalgal biomass for the commercial production of biofuels (Singh and Olsen, 2011).

619

In conclusion, lowering the cost of harvesting microalgae and harvesting in a way

620

that allows for the creation of bioproducts remains a large challenge. The choice of a

621

particular harvesting or dewatering method will dictate what upstream conditions must

622

be met (Christenson and Sims, 2011). Thus, the total solution for using wastewater into 25

623

microalgal biofuel production should be concerned. The technologies process evaluation

624

of integrated process systems is still to be carefully investigated.

625 626 627

7. Conclusion Based on current microalgal technologies for biofuel production alone is unlikely

628

to be economically viable or provide a positive energy return. The high biomass and

629

lipid productivity of wastewater-grown microalgae suggests that this microalgal

630

cultivation strategy offers real potential for biofuel generation. Among of municipal,

631

agricultural and industrial wastewater, agricultural wastewater shows a great potential

632

for microalgal Chlorella growth. The ammonium concentration in wastewater would

633

be the dominant factor for the results of microalgal Chlorella biomass and lipid

634

production. However, more efforts should be devoted before the economical and

635

sustainable microalgal biomass and lipid production using wastewater as nutrients

636

resource.

637 638

Acknowledgements

639

The work was financially supported by grants MOST 103-3113-E-006-006 from

640

the Ministry of Science and Technology. This work was also supported in part by the

641

Aim for the Top University Program of the National Chiao Tung University and

642

Ministry of Education, Taiwan.

26

643

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644

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849

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35

850

Tables and Figures captions

851

Table 1. Nutrients of nitrogen and phosphorus in the wastewater used for the

852

microalgae Chlorella cultivations.

853

Table 2. Biomass and lipid production and productivity of the microalgae Chlorella

854

cultures using wastewater.

855

Fig. 1. Three-dimensional reaction surface plots and contour plots for the biomass

856

production (A) and productivity (B) of the microalgal Chlorella cultured in the

857

wastewater with different NH4+-N and total phosphorous (TP) concentrations. The detail

858

collected data were indicated in Table 1 and Table 2.

859

Fig. 2. Three-dimensional reaction surface plots and contour plots for the lipid content

860

(A), production (B) and productivity (C) of microalgal Chlorella cultured in wastewater

861

with different NH4 +-N and total phosphorus (TP) concentrations. The detail collected

862

data were indicated in Table 1 and Table 2.

36

Figure 1

(A) Biomass production

(B) Biomass productivity

6000 4000 2000 0 -2000

200

500 400 300 200 100

400

500 400 300 200 100

200

400

600

NH4+‐N (mg/L)

800

1500

1000

500

0

-500

0 2000 4000 6000 8000

600

Total phosphorus (mg/L)

800 600

200

500 400 300 200 100

400

600

800

-200 0 200 400 600 800 1000 1200 1400

600

Total phosphorus (mg/L)

Biomass production (mg/L)

8000

-500 0 500 1000 1500 2000

2000

Biomass productivity (mg/L/day)

-2000 0 2000 4000 6000 8000 10000

10000

500 400 300 200 100

200

400

600

NH4+‐N (mg/L)

800

Figure 2

(B) Lipid production

Lipid production (mg/L)

16 14 12 10 8 6 200

150

100

50

200 150 100

3000 2000 1000 0 -1000

150

50

10 12 14 16

200

Total phosphorus (mg/L)

4000

-2000

150

100

50

100

50

200 150 100

400 300 200 100 0 -100 -200

0 1000 2000 3000 4000

150

100

50

-200 -100 0 100 200 300 400 500

500

50

200

Total phosphorus (mg/L)

Lipid content (%)

18

-2000 -1000 0 1000 2000 3000 4000 5000

5000

Lipid productivity (mg/L/day)

6 8 10 12 14 22

20

(C) Lipid productivity

Total phosphorus (mg/L)

(A) Lipid content

50

140 120 100 80 60 40 20

200 150 100

-100 0 100 200 300 400

140 120 100 80 60 40 20

50

100 150 NH4+‐N (mg/L)

200

50

100

150

NH4+‐N (mg/L)

200

50

100

150

NH4+‐N (mg/L)

200

Table 1. Nutrients of nitrogen and phosphorus in the wastewater used for the microalgae Chlorella cultivations Wastewater type

Municipal wastewater

Wastewater source

Microalgae

NH4+-N (mg/L)

TN content (mg/L)

TP content (mg/L)

TN/TP

Centrate Metropolitan wastewater treatment plant

Chlorella sp.

86

132

215

0.6

Li et al., 2011

Centrate Metropolitan wastewater treatment plant

Chlorella sp.

83

116

212

0.5

Li et al., 2011

Pretreated urban wastewater WWTP1

Chlorella sp.

81

84

6

14.0

Cabanelas et al., 2013

Pretreated urban wastewater WWTP2

Chlorella sp.

40

42

6

7.0

Cabanelas et al., 2013

Effluent from primary settler

Chlorella sp.

31

36

3

12.0

Cabanelas et al., 2013

Centrate I

Chlorella sp.

125

130

175

0.7

Cabanelas et al., 2013

Centrate II

Chlorella sp.

125

130

55

2.4

Cabanelas et al., 2013

Centrate III

Chlorella sp.

466

471

55

8.6

Cabanelas et al., 2013

Centrate IV

Chlorella sp.

905

909

55

16.5

Cabanelas et al., 2013

Metro plant centrate

Chlorella sp.

264

290

530

0.5

Min et al., 2011

10% anaerobic digestion tanks and the conflux line of the 90% wastewaters

Chlorella sp. 227

220

250

17

14.7

Cho et al., 2013

Conventional secondary-treatment

Chlorella vulgaris (SAG 211-12)

24

223

330

0.7

Ruiz et al., 2011

Reference

Agricultural wastewater

Industrial wastewater

Conventional secondary-treatment

Chlorella vulgaris (SAG 211-12)

50% piggery wastewater

205

223

330

0.7

Ruiz et al., 2011

Chlorella zofingiensis

148

156

0.9

Zhu et al., 2013

50% piggery wastewater

Chlorella zofingiensis

139

146

1.0

Zhu et al., 2013

50% piggery wastewater

Chlorella zofingiensis

139

146

1.0

Zhu et al., 2013

Dairy wastewater

Chlorella vulgaris

70

71

61

1.2

Qin et al., 2014

10% dairy wastewater

Chlorella zofingiensis G1

5

12

15

0.8

Huo et al., 2012

Digested dairy manure (20x dilution)

Chlorella sp.

112

173

13

13.3

Wang et al., 2010

Digested dairy manure

Chlorella sp.

100

130

18

7.2

Wang et al., 2010

Soybean processing wastewater

Chlorella pyrenoidosa

170

190

46

4.1

Su et al., 2011

Riboflavin manufacturing wastewater

Chlorella pyrenoidosa

885

120

7.4

Sun et al., 2013

Brewery wastewater

Chlorella vulgaris

90

18

5.0

Farooq et al., 2013

Table 2. Biomass and lipid production and productivity of the microalgae Chlorella cultures using wastewater Wastewater type

Municipal wastewater

Wastewater source

Microalgae

Biomass Biomass Lipid Lipid Lipid production productivity content production productivity (mg L-1) (mg L-1 day-1) (%) (mg L-1) (mg L-1 day-1)

Reference

Centrate Metropolitan Chlorella sp. wastewater treatment plant

1175

313

11

120

35.6

Li et al., 2011

Centrate Metropolitan Chlorella sp. wastewater treatment plant

1060

283

11

120

31.2

Li et al., 2011

Pretreated urban wastewater WWTP1

Chlorella sp.

1500

116

Cabanelas et al., 2013

Pretreated urban wastewater WWTP2

Chlorella sp.

1340

117

Cabanelas et al., 2013

Effluent from primary settler

Chlorella sp.

1160

56

Cabanelas et al., 2013

Centrate I

Chlorella sp.

1180

125

Cabanelas et al., 2013

Centrate II

Chlorella sp.

1320

195

Cabanelas et al., 2013

Centrate III

Chlorella sp.

1520

194

Cabanelas et al., 2013

Centrate IV

Chlorella sp.

1180

138

Cabanelas et al., 2013

Metro plant centrate

Chlorella sp.

600

50

Min et al., 2011

Agricultural wastewater

Industrial wastewater

10% anaerobic digestion tanks and the conflux line of the 90% wastewaters

Chlorella sp. 227

3010

400

Conventional secondary-treatment

Chlorella vulgaris (SAG 211-12)

717

95

Ruiz et al., 2011

Conventional secondary-treatment

Chlorella vulgaris (SAG 211-12)

1228

88

Ruiz et al., 2011

50% piggery wastewater

Chlorella zofingiensis

2962

296

37.3

1105

111

Zhu et al., 2013a, b

50% piggery wastewater

Chlorella zofingiensis

2860

286

33.3

987

99

Zhu et al., 2013b

50% piggery wastewater

Chlorella zofingiensis

2005

201

34.8

698

70

Zhu et al., 2013b

Dairy wastewater

Chlorella vulgaris

1870

450

10.3

193

48

Qin et al., 2014

10% dairy wastewater

Chlorella zofingiensis G1

144

29

17.9

26

5

Huo et al., 2012

Digested dairy manure (20x dilution)

Chlorella sp.

1710

81

13.4

233

11

Wang et al., 2010

Digested dairy manure Soybean processing wastewater Riboflavin manufacturing wastewater

Chlorella sp. Chlorella pyrenoidosa Chlorella pyrenoidosa

1391

69

10.1

141

7

Wang et al., 2010

2150

640

37

806

240

Su et al., 2011

1250

254

39.3

491

99

Sun et al., 2013

Brewery wastewater

Chlorella vulgaris

2266

227

26.7

605

61

Farooq et al., 2013

10.8

325

65

Cho et al., 2013

Highlights 1. Agricultural wastewater shows a great potential for microalgal Chlorella growth 2. NH4 +-N in wastewater is the key factor for microalgal biomass and lipid production 3. Optimal N and P concentrations in wastewater for Chlorella cultivation were indicated 4. Limitations of microalgal biomass productivity by wastewater were detail discussed

Cultivation of microalgal Chlorella for biomass and lipid production using wastewater as nutrient resource.

Using wastewater for microalgal cultures is beneficial for minimizing the use of freshwater, reducing the cost of nutrient addition, removing nitrogen...
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