Heterotrophic cultivation of microalgae using aquaculture wastewater: A biorefinery concept for biomass production and nutrient remediation

Ecological Engineering 99 (2017) 47–53

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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng

Heterotrophic cultivation of microalgae using aquaculture wastewater: A biorefinery concept for biomass production and nutrient remediation Abhishek Guldhe, Faiz A. Ansari, Poonam Singh, Faizal Bux ∗ Institute for Water and Wastewater Technology, Durban University of Technology, P.O. Box 1334, Durban, 4000, South Africa

a r t i c l e

i n f o

Article history: Received 13 July 2016 Received in revised form 21 September 2016 Accepted 13 November 2016 Keywords: Microalgae Aquaculture wastewater Heterotrophic Biomass Biorefinery

a b s t r a c t Cultivation of microalgae utilizing wastewater substrate could form a sustainable biorefinery with double benefit of biomass generation and nutrient remediation. In this study potential of aquaculture wastewater is evaluated for cultivation of Chlorella sorokiniana in heterotrophic mode for generation of high value biomass. Nutrient removal potential is also assessed. Aquaculture wastewater with 400 mgL−1 sodium nitrate supplementation resulted in biomass productivity of 498.14 mgL−1 d−1 . The biomass generated showed lipid productivity of 150.19 mgL−1 d−1 , carbohydrate productivity of 172.91 mgL−1 d−1 and protein productivity of 141.57 mgL−1 d−1 . The nutrient removal efficiencies were 75.56% for ammonium, 84.51% for nitrates, 73.35% for phosphates and 71.88% for COD (chemical oxygen demand). The findings of this study underline the potential of aquaculture wastewater for production of valuable microalgal biomass which can be utilized for biofuels or feed application. This biorefinery concept also polished aquaculture wastewater which can be effectively reused. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Microalgal biomass has been proven as a sustainable feedstock for biofuels, feed and numerous value added products pertinent to nutraceuticals and therapeutic industry. Microalgae can be effectively grown using wastewater for biomass generation and nutrient remediation (Ma et al., 2016; Rawat et al., 2011). Heterotrophic cultivation of microalgae is advantageous in terms of eliminating light dependency and higher biomass yields over phototrophic cultivation (Kim et al., 2013; Venkata Mohan et al., 2015). Microalgal biomass generated is rich in lipids, carbohydrates and proteins. The microalgal biomass can be utilized primarily for biofuels generation (biodiesel, biomethane, biohydrogen etc.) and for feed applications (aquaculture and animal) (Singh et al., 2015a). Alternatively, microalgal biomass can also be utilized for value added products such as pigments, nutraceuticals, bioplastic etc. (Suganya et al., 2016). Use of synthetic medium makes the commercial scale microalgal biomass generation unfeasible. Heterotrophic cultivation using different waste streams have been gaining interest from researchers as it reduces the production cost by dropping the usage

∗ Corresponding author. E-mail address: [email protected] (F. Bux). http://dx.doi.org/10.1016/j.ecoleng.2016.11.013 0925-8574/© 2016 Elsevier B.V. All rights reserved.

of expensive inorganic chemicals (Medeiros et al., 2015; Rawat et al., 2011). Biorefinery concept where different industries are integrated together for various products and mutual benefits could prove as a sustainable and economical approach for microalgal biomass generation. Aquaculture is one of the fastest growing food industries. This growing industry generates wastewater rich in nutrients such as ammonia, nitrates, phosphates and organic load (Gao et al., 2016; Lananan et al., 2014). Wastewater generated in aquaculture industry needs treatment prior to its reuse or release in environment to avoid the eutrophication. This wastewater treatment step adds to the production cost of aquaculture produce. Existing wastewater treatment processes used in aquaculture are denitrification process to release nitrogenous compounds in atmosphere and chemical precipitation using ferrous chloride to remove phosphorous compounds. These processes are not only adding to the cost but also lead to toxic by-products (Mook et al., 2012; Nasir et al., 2015). Microalgal cultivation using aquaculture could prove itself as a promising biorefinery for economical biomass generation and sustainable wastewater remediation. Microalgae can utilize the nutrients and organic load present in aquaculture wastewater to produce valuable biomass which can be subsequently utilized for biofuels or feed applications. Microalgae have been studied as a feed supplement and also as a whole feed for aquaculture industry. Microalgae contain proteins, long chain fatty acids, pigments which

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are essential for fish nutrition. Whole microalgae or lipid extracted microalgae can thus be utilized in fish feed production (Ju et al., 2012). There are several studies on utilization of wastewater for microalgal cultivation (Caporgno et al., 2015; Ramanna et al., 2014). However, using aquaculture wastewater for this purpose is not well explored area. There are very few studies which report the use of aquaculture wastewater for biomass production, but most of these studies mainly focus on phototrophic cultivation and nutrient removal. Very few studies report the biomass composition, which is important for its applications. Thus it is important to investigate the potential of aquaculture wastewater for heterotrophic cultivation of microalgae and evaluate the biomass composition. In this study, aquaculture wastewater has been used to cultivate Chlorella sorokiniana heterotrophically. Microalgal growth, nutrient removal and biomass composition were thoroughly investigated and compared with synthetic media cultivation. Sodium nitrate supplementation strategy has also been developed to achieve possible sealing biomass and primary metabolite productivities. 2. Materials and methods 2.1. Wastewater collection and characterization The aquaculture wastewater was collected from aquaculture research facility, Durban, South Africa. At this facility Nile tilapia are reared in 5000 L tanks in controlled temperature (27–32 ◦ C) with continuous aeration. A biofiltration system was used to remove nutrients and organic load from recycled water at regular interval. For this study wastewater was sourced from the collection tank before the treatment. Aquaculture wastewater (AW) was collected in 25 L containers and brought to laboratory immediately after collection. The pH, electrical conductivity, temperature, salinity, dissolve oxygen (DO), dissolve oxygen percentage were measured at the time of sample collection by YSI MP – AES. The total solid and total dissolve solid were calculated by standard methods of APHA 2005 (APHA-AWA-WEF, 2005). The chemical oxygen demand (COD) was determined by closed refluxed method. Centrifuged sample were used to analyze ammonia (NH4 + ), nitrates (NO3 − ), nitrites (NO2 − ) and phosphates (PO4 3− ) concentration by GalleryTM Automated Photometric analyzer (Thermo Scientific, USA). For the heavy metals analysis, sample were digested in microwave (Milestone S.R.L., Italy, output power 1200 W) at 180 ◦ C for 20 min at 1000 W using acid mixture (15 mL HNO3 and 4 mL HClO4 ). After cooling the solution was allowed to evaporate from digested samples until the volume reduced to 5 mL. The samples were filtered through filter paper and further diluted using deionized water to 50 mL for heavy metals analysis using microwave plasma atomic emission spectrometry (Agilent Technologies 4200 MP-AES). Bacterial count was determined by determined by heterotrophic plate count method. 2.2. Microalgae cultivation Chlorella sorokiniana strain was used for the heterotrophic cultivation using aquaculture wastewater (AW). The AW was first filtered using glass fiber filter papers and then autoclaved prior to microalgal inoculation (Nasir et al., 2015). The microalgal cultures were maintain in 1 L conical flask with 500 mL working volume. The cultivation conditions were: temperature 25 ◦ C, shaking at 110 rpm in complete dark phase. Microalgal culture flasks were wrapped in aluminum foil and were kept in dark conditions. These conditions were kept constant for all the experiments. Microalgae were also cultivated in BG11 nutrient medium supplemented with glucose for comparative analysis. Microalgae were also grown in BG11

medium and AW under phototrophic condition for comparative analysis. Each set of experiment was done in duplicate. Supplementation experiments were carried out by adding 200, 400, 600 and 1500 mgL−1 sodium nitrate in AW. 2.3. Analytical methods 2.3.1. Growth and biomass analysis Microalgal growth was monitored daily by determining optical density at 680 nm using spectrophotometric method. Biomass was estimated gravimetrically at initial, middle and late log phases of growth. Biomass productivity (mgL−1 d−1 ) was calculated at late log phase gravimetrically (Singh et al., 2015b). Biomass was harvested using centrifuge and freeze dried using lyophilizer (Mini lyotrap, LTE scientific Ltd., United Kingdom) for further analysis. 2.3.2. Nutrient removal The nutrients removal efficiency was determined on every alternate day. For analysis 10 mL of sample were collected from culture flask and centrifuged. The samples were then filtered using 0.45 ␮m syringes filters. These samples were analyzed for nitrates (NO3 − ), nitrites (NO2 − ), TON, ammonia (NH4 + ) and phosphates (PO4 3− ) using GalleryTM Automated Photometric analyzer (Thermo Scientific, USA) (Gupta et al., 2016). The chemical oxygen demand (COD) was analyzed by closed refluxed method. The removal efficiency in percentage was determined by using following equation Percentage removal =

Initial concentration − Final concentration × 100 Initial concentration

(1)

2.3.3. Biochemical composition of biomass Microalgal biomass collected from each experiment was analyzed for lipids, carbohydrates and proteins. Lipids were extracted from the harvested biomass using microwave assisted solvent extraction. Dried biomass was mixed with chloroform and methanol (2:1 v/v) and then subjected to microwave treatment (100 ◦ C for 10 min at 1000 W) for cell disruption (Guldhe et al., 2014). Biomass residues were removed by filtration. The organic layer was collected and oven dried at 70 ◦ C for lipid recovery. The lipids obtained were measured gravimetrically and the percentage lipid content was determined based on lipid recovered from known weight of dry biomass. Lipids productivity was calculated according to equation described by (Singh et al., 2015b) Lipid productivity = biomass productivity ×

lipid content 100

(2)

Where, biomass productivity is in mgL−1 d−1 , lipids content is in percentage per dry biomass weight. Proteins extraction was done following the method given by Lopez et al. (Lopez et al., 2010). The quantitative analysis of proteins was done by Lowry’s method. A spectrophotometer (SpectroquantPharo 300, Merck) was used to measure the absorbance of the extraction mixture at 750 nm. Standards for calibration were prepared by using bovine serum albumin (BSA) in lysis buffer. The standard calibration curve prepared using BSA was used for proteins quantification. Proteins productivity was determined using equation 3. Protein productivity = biomass productivity ×

protein content (3) 100

Where, biomass productivity is in mgL−1 d−1 , proteins content is in percentage per dry biomass weight. Total carbohydrates were quantified using the phenol-sulfuric acid method (Prajapati et al., 2013). Dried biomass was mixed with sulfuric acid (2% v/v) and autoclaved for 30 min at 121 ◦ C for hydrolysis. The mixture was then neutralized with 1 M NaOH/H2 SO4 . Supernatant was collected by centrifugation at 1509 × g for 10 min.

A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53

Biomass productivity

600

Biomass productivity (mg/l/day)

49

500

400

300

200

100

0 BG11 H

AW H

AW H200

AW H400

AW H600

BG11 P

AW P

Fig. 1. Biomass productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different concentration of sodium nitrate supplementation. BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1 sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

240

Lipid productivity

Lipid productivity (mg/l/day)

200

160

120

80

40

0 BG11 H

AW H

AW H200

AW H400

AW H600

BG11 P

AW P

Fig. 2. Lipid productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different sodium nitrate supplementation. BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1 sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

0.1 mL of supernatant was diluted to 1 mL, and then mixed with 1 mL of phenol (5% w/v) and 5 mL of 96% H2 SO4 . After cooling to 25–30 ◦ C, the absorbance of this solution was measured at 490 nm using a spectrophotometer (Spectroquant Pharo 300, Merck). Total carbohydrates were quantified from a calibration curve prepared using glucose as a standard (Prajapati et al., 2013). Carbohydrates productivity was determined by the Eq. (4). Carbohydrate productivity = biomass productivity ×

carbohydrate content 100

(4)

Where, biomass productivity is in mgL−1 d−1 , carbohydrate content is in percentage per dry biomass weight.

3. Results and discussion 3.1. Wastewater characterization Aquaculture wastewater (AW) was analyzed for presence of nitrates, ammonia and phosphates required for microalgal cultivation. The physicochemical parameters for collected AWW are depicted in Table 1. The collected wastewater from aquaculture facility showed ammonia 5.32 mgL−1 , nitrates 40.67 mgL−1 , phosphate 8.82 mgL−1 and COD 96 mgL−1 (Table 1). Guo et al., 2013 used aquaculture wastewater for growth of microalgae Platymonas subcordiformis. In their study aquaculture wastewater showed presence of 47.8 mgL−1 nitrates and 8.87 mgL−1 phosphates, which is similar to aquaculture used in this study. The pH of the collected wastewater was 7.28. The bacterial load in AWW was found to be

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A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53

Carbohydrate productivity (mg/l/day)

Carbohydrate productivity 200

150

100

50

0 BG11 H

AW H

AW H200

AW H400

AW H600

BG11 P

AW P

Fig. 3. Carbohydrate productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different sodium nitrate supplementation. BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1 sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

200

Protein productivity

Protein productivity (mg/l/day)

180 160 140 120 100 80 60 40 20 0 BG11 H

AW H

AW H200

AW H400

AW H600

BG11 P

AW P

Fig. 4. Protein productivities of C. sorokiniana in BG11 medium and aquaculture wastewater with different sodium nitrate supplementation. BG11 H- Blue green 11 media heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200- Aquaculture wastewater heterotrophic supplemented with 200 mgL−1 sodium nitrate, AW H 400- Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600- Aquaculture wastewater heterotrophic supplemented with 600 mgL−1 sodium nitrate, BG11 P- Blue green 11 media phototrophic, and AW P- Aquaculture wastewater phototrophic.

1.79 × 103 cfu mL−1 (Table 1). The other nutrients present in the AWW are depicted in Table 1. AWW also showed presence of other micronutrients such as iron, magnesium, zinc, molybdenum which are required for various physiological activities of microalgae. 3.2. Microalgae growth and biomass production using aquaculture wastewater The heterotrophic cultivation of C. sorokiniana showed biomass yields of 2.47 gL−1 in AW and 4.02 gL−1 in BG11 medium (Table 2). Higher biomass yield in synthetic medium (BG11) is because of the higher nutrient concentration compared to that in AW. Similarly, biomass productivity of 353.36 mgL−1 d−1 in AW was found to be lower than the BG11 medium (573.79 mgL−1 d−1 ). Li et al. (2011) cultivated chlorella sp. in sludge centrate and observed

high biomass productivity of 920 mgL−1 d−1 . In their study sludge centrate was composed of higher nutrient concentration (COD: 2304 mgL−1 ; ammonia: 82.5 mgL−1 ; phosphate 212 mgL−1 ) than the aquaculture wastewater used in this study. The lower biomass productivities suggest the need of nutrient supplement for microalgal cultivation in AW to achieve comparable biomass yields to that of the synthetic medium. Phototrophic cultivation of C. sorokiniana using AW resulted in biomass productivity of 107.86 mgL−1 d−1 (Fig. 1). The biomass productivity in heterotrophic cultivation was 3 times higher than the phototrophic cultivation. Kim et al. (2013) in their study found that growth rate of C. sorokiniana in heterotrophic mode was 0.53 d−1 while it was 0.24 d−1 in autotrophic mode. This result clearly demonstrates that the heterotrophic mode of nutrition is the most suitable cultivation strategy for using aquaculture wastewater for microalgal biomass generation.

A. Guldhe et al. / Ecological Engineering 99 (2017) 47–53 Table 1 Characterization of aquaculture wastewater. Parameter

Unit

Value

pH Temperature Total solid Total dissolve solid Salinity DO COD NH4 + -N NO2 − -N NO3 − -N TON PO4 3− -P Fe Mo Zn Na Ni Mg K Bacterial density

– ◦ C gL−1 gL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 mgL−1 cfu mL−1

7.28 27.3 0.42 0.35 0.26 4.17 96 5.32 5.52 40.67 38.80 8.82 4.05 3 0.85 66.25 0.3 8.45 5.9 1.795 × 103

51

ammonium may be volatilized by increased pH and temperature. Similarly, phosphorous could get precipitated by increase in pH and dissolved oxygen. In heterotrophic mode for algal cultivation the organic carbon present in the medium is the primary source of energy. Initial COD observed in AW was 96 mgL−1 , which was reduced to 27 mgL−1 after 7 days of cultivation of C. sorokiniana in AW. The percentage removal efficiency of C. sorokiniana in AW for COD was 71.89%. The nutrient removal efficiencies depend upon the initial concentration of nutrients in the wastewater and microalgal strains used. In a study by Zhou et al. (2012) the initial concentration of ammonia was 91 mg L−1 , COD was 2324 mg L−1 and phosphates was 212 mg L−1 in wastewater and they cultivated Auxenchlorella protothecoides which showed high removal efficiency (Table 4). Zhang et al. (2013) cultivated Chlorella sp. ZTY4 using domestic wastewater in heterotrophic mode. The removal efficiencies found in their study were 40.8% for COD, 15.8% for total nitrogen and 49.9% for total phosphorous respectively. Microalgal cultivation thus can be implemented as a polishing step for aquaculture wastewater before its reuse or release into the environment. 3.4. Nutrient supplementation

Table 2 Biomass, lipid, protein and carbohydrates yields in C. sorokiniana under different cultivation condition in synthetic media and aquaculture wastewater. Units

Biomass gL−1

Lipid content %

Protein content %

Carbohydrate content%

BG11 H AW H AW H200 AW H400 AW H600 BG11 P AW P

4.02 2.47 2.9 3.49 3.54 2.54 1.51

35.75 39.1 35.3 30.15 26.1 26 33.45

28.64 24.57 26.38 28.42 29.46 29.46 29.46

33.64 36.1 35.97 34.71 32.79 29.74 35.43

BG11 H- Blue green 11 heterotrophic, AW H- Aquaculture wastewater heterotrophic, AW H 200-Aquaculture wastewater heterotrophic supplemented with 200 mgL−1 sodium nitrate, AW H 400-Aquaculture wastewater heterotrophic supplemented with 400 mgL−1 sodium nitrate, AW H 600-Aquaculture wastewater heterotrophic supplemented with 600 mgL−1 sodium nitrate, BG11 P-Blue green 11 phototrophic, AW P-Aquaculture wastewater phototrophic.

Biomass productivities of C. sorokiniana in AW were lower than the synthetic media. Nitrogen is major nutrient which plays role in algal growth, in synthetic media nitrogen is supplied in forms of nitrates. Furthermore, removal rate for nitrates found in this study was 84.51% which clearly demonstrates the high uptake of this nutrient. Thus sodium nitrate supplementation in AW was investigated in this study. Increasing biomass productivities were observed with increasing concentrations of sodium nitrate supplementation. Highest biomass productivity of 505.71 mgL−1 d−1 was observed with 600 mgL−1 sodium nitrate in AW. At 400 mgL−1 sodium nitrate supplementation in AW 498.12 mgL−1 d−1 biomass productivity was observed which is comparable to highest biomass productivity at 600 mgL−1 sodium nitrate supplementation (Fig. 1). It is economical to use minimum chemical supplementation while using wastewater as a nutrient medium. Thus 400 mmgL−1 d−1 of sodium nitrate supplementation was determined to be optimum for C. sorokiniana cultivation in AW.

3.3. Nutrient removal from aquaculture wastewater 3.5. Biochemical composition of biomass Microalgal C. sorokiniana utilized the nutrients present in the aquaculture wastewater for its growth. The nutrient removal efficiencies were determined after the 7 day cultivation period. The removal efficiencies were found to be 84.51% for nitrates, 96.38% for nitrites, 75.56% for ammonia, and 73.35% for phosphorus respectively (Table 3). These nutrients are utilized by microalgal cells for various physiological processes to generate biomass rich in lipids, carbohydrates and proteins. Nitrogen is important for protein and genetic material synthesis while phosphorous is major constituent of adenosine triphosphate (ATP) required for short term energy storage and transfer (Kim et al., 2016). Microalgae utilize inorganic nitrogen in form of ammonium, nitrate and nitrite by assimilation process where nitrate and nitrite undergo reduction to form ammonium which is then incorporated in amino acid glutamine (Cai et al., 2013). The removal of these inorganic nutrients is not solely governed by the uptake via microalgal cells. Some amount of

Lipid, carbohydrate and protein content and productivities were determined for biomass harvested from all the experiments. Lipid content of C. sorokiniana cultivated using AW was 39.1% DCW, while it was 35.75% DCW for synthetic medium. Similarly, carbohydrate content in C. sorokiniana grown using AW was 36.1% DCW, while it was 33.64% DCW for synthetic medium (Table 2). In previous studies, Auxenchlorella protothecoides grown on wastewater showed lipid content of 33.22% (Zhou et al., 2012); Chlorococcum sp. RAP13 grown on dairy effluent showed lipid content of 39–42% (Ummalyma and Sukumaran, 2014) and Scenedesmus sp. LX1 grown using secondary effluent from domestic treatment plant showed lipid content of 31–33% (Xin et al., 2010) (Table 4). C. sorokiniana when grown in raw sewage showed lipid content of 22.74% (Gupta et al., 2016). Very few studies on microalgal cultivation using wastewaters report the biochemical constituents of the biomass.

Table 3 Nutrients and COD removal efficiency of C. sorokiniana cultivated in aquaculture wastewater.

Initial Final Removal efficiency Removal rate

Units

COD

NO3 −1 -N

NO2 −1 -N

NH4 + -N

PO4 3− -P

mgL−1 mgL−1 % mgL−1 d−1

96 27 71.88 8.63

40.67 6.3 84.51 4.3

5.52 0.2 96.38 0.67

5.32 1.3 75.56 0.5

8.82 2.35 73.35 0.8

Current study 71.88 73.35 84.51 75.56 C = 37.1P = 131.1 C = 24.87P = 87.86 B = 2.47 P = 353.36 Aquaculture wastewater

C = 39.1P = 138.17

Gao et al. (2016) – 82.7 – – –

Chlorella sp. (Phototrophic) C. vulgaris (Phototrophic) C. sorokiniana

– P = 42.6 Aquaculture wastewater

–

Nasir et al. (2015) – 63.1 − 92.2 – –

–

–

–

–

– 98 – – – C = 31–33 P = 0.11

Secondary effluent from domestic treatment plant Aquaculture wastewater

–

93 – – – – – B = 1.48–1.94 Dairy effluent

C = 39–42

83 90.8 79.9 85.5 80.9 98.48 – – – 78.3 93.9 100 – – –

Chlorella sp. Chlorella sp. Auxenchlorella protothecoides Chlorococcum sp. RAP13 Scenedesmus sp. LX1

– – C = 33.22 – P = 920 B = 1.16 Wastewater centrate Autoclaved centrate Wastewater

P = Productivity mg L−1 d−1 , C=Content in% P = Productivity mg L−1 d−1 , C=Content in% P = Productivity mg L−1 d−1 , C=Content in%

– – –

Refs. COD removal % PO4 −3 -P removal % NO3 −1 -N removal % NH4 + -N removal % Carbohydrate Protein Lipid

Biomass productivity P = mgL−1 d−1 B = Biomass production g L−1 Type of wastewater Microalgae

Table 4 Previous studies reporting microalgae cultivation in different wastewaters.

Ummalyma and Sukumaran (2014) Xin et al. (2010)

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Wang et al. (2010) Li et al. (2011) Zhou et al. (2012)

52

Microalgae are known to accumulate more lipid and carbohydrates in stressed conditions as a defense mechanism. The nitrogen and phosphorous content in the AW was lower than the BG11 medium. Thus the cultivation of microalgae in AW is under a nutrient stress environment which results in higher lipid and carbohydrate contents. Protein content in the microalgal cells is directly related to the growth. Reduced growth is observed in microalgae grown on AW compared to synthetic medium. This has been reflected in the protein content also, the protein content for C. sorokiniana grown in AW was 24.57% DCW which was lower than the protein content (28.64% DCW) of biomass generated using synthetic medium (Table 2). Generally, in microalgal cultivation a tradeoff is observed for biomass and lipid productivities with nitrogen stress. With increasing concentration of nitrogen in medium biomass productivities increases while lipid content decreases. In this study with sodium nitrate supplementation lipid productivity increased upto 400 mgL−1 supplementation after that lower lipid productivity was observed with 600 mgL−1 . Lipid productivity with 400 mgL−1 sodium nitrate supplementation in AW was 150.19 mgL−1 d−1 . Lipid productivity with 600 mgL−1 supplementation was found to be 131.98 mgL−1 d−1 (Fig. 2). Similar trend was observed for carbohydrate productivity; highest carbohydrate productivity of 172.91 mgL−1 d−1 was observed in C. sorokiniana grown in AW supplemented with 400 mgL−1 sodium nitrate (Fig. 3). Protein productivity was found to be gradually increasing with the increasing concentration of sodium nitrate in AW. Highest protein productivity of 148.98 mgL−1 d−1 was observed with 600 mgL−1 sodium nitrate in AW. The 400 mgL−1 supplementation selected as optimum for biomass production resulted in protein productivity of 141.57 mgL−1 d−1 which is slightly lower than the highest (Fig. 4). These results highlight the promising potential of cultivation of microalgae in AW for production of high quality biomass. Supplementation strategy has been found to be very effective to enhance the biomass productivity with adequate primary metabolites composition. The lipid and carbohydrates in microalgal biomass can be used for various biofuels synthesis such as biodiesel, biomethane and bioethanol. Protein component of biomass makes it suitable for aquaculture or animal feed application. There are very few studies which report the heterotrophic cultivation of microalgae in wastewater. Table 4 depicts the comparison of biomass yields, biochemical composition and nutrient removal efficiencies observed in this study to the previous reports on heterotrophic microalgal cultivation in different wastewaters. The biomass productivity for C. sorokiniana observed in this study using AW was higher compared to the previous reported studies of heterotrophic and phototrophic cultivation of microalgae in different wastewater streams (Table 4). The nutrient removal efficiencies for COD, ammonium, nitrate, nitrite and phosphates were comparable to the previous reports of heterotrophic cultivation of microalgae in various wastewater substrates. Most of the previous studies only focus on lipid productivities of microalgae for biodiesel synthesis. In this study biomass was also evaluated for protein and carbohydrate productivities for its application in other biofuels and feed applications.

4. Conclusion The findings of this study have clearly highlighted the potential of aquaculture wastewater as a nutrient substrate for cultivation of microalgae. Heterotrophic mode of cultivation of microalgae had shown better biomass and metabolites productivities than the phototrophic mode. Nutrient supplementation strategy improves the biomass growth as well as lipid, carbohydrate and protein productivities. Microalgal biomass generated using aquaculture

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