Mixotrophic cultivation of a microalga Scenedesmus obliquus in municipal wastewater supplemented with food wastewater and flue gas CO2 for biomass production

Journal of Environmental Management 159 (2015) 115e120

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Research paper

Mixotrophic cultivation of a microalga Scenedesmus obliquus in municipal wastewater supplemented with food wastewater and flue gas CO2 for biomass production Min-Kyu Ji a, Hyun-Shik Yun a, Young-Tae Park a, Akhil N. Kabra b, In-Hwan Oh a, Jaeyoung Choi a, * a b

Green City Technology Institute, Korea Institute of Science and Technology, Seoul 136-791, South Korea Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul 133-791, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 March 2015 Received in revised form 26 May 2015 Accepted 29 May 2015 Available online 7 June 2015

The biomass and lipid/carbohydrate production by a green microalga Scenedesmus obliquus under mixotrophic condition using food wastewater and flue gas CO2 with municipal wastewater was investigated. Different dilution ratios (0.5e2%) of municipal wastewater with food wastewater were evaluated in the presence of 5, 10 and 14.1% CO2. The food wastewater (0.5e1%) with 10e14.1% CO2 supported the highest growth (0.42e0.44 g L
1), nutrient removal (21e22 mg TN L
1), lipid productivity (10 e11 mg L
1 day
1) and carbohydrate productivity (13e16 mg L
1 day
1) by S. obliquus after 6 days of cultivation. Food wastewater increased the palmitic and oleic acid contents up to 8 and 6%, respectively. Thus, application of food wastewater and flue gas CO2 can be employed for enhancement of growth, lipid/carbohydrate productivity and wastewater treatment efficiency of S. obliquus under mixotrophic condition, which can lead to development of a cost effective strategy for microalgal biomass production. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Scenedesmus obliquus Biomass Food wastewater Flue gas CO2 Bio-energy

1. Introduction Microalgae have gained much attention in recent years and have been utilized for various applications including wastewater treatment, CO2 mitigation and as feedstock for biofuel production (Lizzul et al., 2014). Integration of algal systems with the existing industrial or waste treatment activity can emerge as an effective dual strategy for cost effective biomass production with simultaneous wastewater utilization (Chisti, 2007). Application of biological system for CO2 sequestration from flue gas is an eco-friendly and cost effective strategy (Jiang et al., 2011). Microalgae serve multiple advantages for CO2 sequestration including higher photosynthetic efficiency compared to terrestrial plants, source of renewable energy and utilization of their biomass to obtain high value products (Chisti, 2007). Exhaust flue-gases with high concentrations of CO2 (~15%, v/v) that are released from different industrial sectors including thermal power, cement, steel and incineration, can be used as an economical CO2 source for

* Corresponding author. E-mail address: [email protected] (J. Choi). http://dx.doi.org/10.1016/j.jenvman.2015.05.037 0301-4797/© 2015 Elsevier Ltd. All rights reserved.

photosynthetic microalgal biomass production (Chiu et al., 2011; Praveenkumar et al., 2014a). However, NOx, SOx and H2S, and particulate matters in flue gas can cause acidification to the medium and pose environmental stress to algae (Chiu et al., 2011; Jiang et al., 2013; Kumar et al., 2014). Therefore, microalgal species tolerant to industrial flue gas should be secured for effective microalgae biomass production; few microalgal species such as Chlorella sp., Scenedesmus dimorphus have been identified to sustain and grow in the presence flue gas (Kumar et al., 2014; Praveenkumar et al., 2014a). Microalgae biomass production can be performed through autotrophic cultivation in a photobioreactor by using solar energy for fixing CO2 (Jiang et al., 2011; Ji et al., 2013a). Heterotrophic (Liu et al., 2011) or mixotrophic (Bhatnagar et al., 2011; Praveenkumar et al., 2014a) cultivation of microalgae using organic compounds (i.e., glucose and acetate) as energy and carbon sources can also be employed for biomass production. Among the aforementioned strategies, mixotrophic operation possess several advantages including elimination of light requirement, higher microalgae growth, cost effective biomass harvesting and substrate degradation (Chisti, 2007; Brennan and Owende, 2010). Under mixotrophic conditions microalgae can simultaneously assimilate

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M.-K. Ji et al. / Journal of Environmental Management 159 (2015) 115e120

the inorganic and organic substrates through concurrent respiratory and photosynthesis processes, which are sum of the photoautotrophic and heterotrophic growth (Bhatnagar et al., 2011). Cultivation of microalgae using different organic wastewaters to attain simultaneous biomass production and wastewater treatment has been demonstrated (Abreu et al., 2012; Ji et al., 2013b). Food effluent is one of the most highly produced wastewaters throughout the world that can be used as an efficient system to achieve high microalgae biomass yield and lipid productivity for biofuel production, as it is rich in nutrients including nitrogen, phosphorus, calcium, iron, aluminum and total organic carbon (mainly acetic acid) (Shin et al., 2014). Therefore, a mixotrophic cultivation system using an organic substrate waste and high concentrations of CO2, especially from industrial-exhaust-gas sources, can be a practical approach for cost-effective microalgae biomass production (Cheirsilp and Torpee, 2012; Praveenkumar et al., 2014a). Most of the previous studies have been focused on the feasibility of mixotrophic cultures and isolation of potent microalgae using synthetic CO2-enriched air with municipal wastewater for biofuel feedstock generation, and efforts to apply actual industrial fluegases with food wastewater are very limited (Praveenkumar et al., 2014a; Shin et al., 2014). The present study investigated the combined effect of food wastewater dose and real coal-fired flue gas CO2 concentration on biomass production, lipid/carbohydrate productivity, fatty acid composition and nutrient removal efficiency of Scenedesmus obliquus under mixotrophic cultivation using municipal wastewater. 2. Materials and methods 2.1. Algal strain, culture conditions and inoculum preparation Nephroselmis sp. and S. obliquus were isolated from acid mine drainage (Heungil mining station, Gangneung and Hyupsung mining station, Taebaek, South Korea) and was registered in GenBank under Accession No. HE861879 and HE861884, respectively. The microalga was inoculated into 2 L circular column containing 1.7 L bold's basal medium at 10% (Vinoculum/Vmedia) (Bischoff and Bold, 1963). The microalgal cells were incubated under white fluorescent light illumination at 120 mmol photon m
2 s
1 at 25  C for 10 days while aerated with filter-sterilized air at a flow rate of 0.5 L min
1. The microalgal suspension in the BBM was adjusted to an absorbance of 1.0 at an optical density (OD) of 680 nm as measured using a spectrophotometer (Hach DR/2800, Loveland, Colorado, USA). Three milliliters of microalgae were used as initial inoculums for further experiments. 2.2. Wastewater sampling and analysis Secondary municipal wastewater (MWW) was collected from municipal wastewater treatment plant at Gangneung, South Korea, and food wastewater from food wastewater (FW) treatment plant at Dangjin, South Korea. Wastewaters were immediately filtered using 0.2 mm nylon micro filters to remove the microorganisms and suspended solid particles. Many research reported that wastewater pretreatment using membrane filtration technology are economical method with production of optimal microalgal medium (Cho et al., 2011; Cicci et al., 2013). The physicochemical properties of the MWW and FW were analyzed (Table 1). The standard methods 4500-N C, 4500-NH3 G and 4500 P. B. 5 were used to determine the T-N, NH4-N and T-P in water samples, respectively (APHA, 1998). Metal ions were analyzed using an inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian 730-ES, USA) after acidification with 1% (v/v) nitric acid. Total organic carbon (TOC) concentration was determined using a Shimadzu TOC-VCPH

Table 1 Physico-chemical characteristics of (a) municipal, and (b) food wastewater. Parameter

(a) MW

(b) FW

pH T-N (mg L
1) T-P (mg L
1) TOC (mg L
1) Salinity (%) Metallic ions Al3þ (mg L
1) B3þ (mg L
1) Ca2þ (mg L
1) Cd2þ (mg L
1) Co2þ (mg L
1) Cr6þ (mg L
1) Cu2þ (mg L
1) Fetotal (mg L
1) Mg2þ (mg L
1) Mn2þ (mg L
1) Pb2þ (mg L
1) S (mg L
1) Zn2þ (mg L
1)

7.3 21 ± 0.4 2.3 ± 0.2 18.6 ± 0.2 0.3

6.0a 725 ± 1.6 62.5 ± 1.1 10,173 ± 2.3 3.7

0.096 ± 0.002 1.27 ± 0.03 33.2 ± 0.1 NDb NDb NDb 0.031 ± 0.003 0.008 ± 0.001 38.66 ± 0.06 0.019 ± 0.002 0.112 ± 0.001 28.97 ± 0.05 NDb

329.9 ± 2.7 0.57 ± 0.009 1077 ± 1.0 NDb NDb 0.015 ± 0.003 0.045 ± 0.003 26.68 ± 0.07 93.37 ± 1.2 1.89 ± 0.04 0.304 ± 0.008 114.5 ± 1.7 1.15 ± 0.009

a b

After adjusted of pH with NaOH. ND: Not detected.

analyzer (Tokyo, Japan). The solution pH was measured using an Orion 5-Star pH/ORP/Cond./DO Meter (Thermo Scientific, USA). Experiments were performed in triplicates.

2.3. Growth of microalgae and consequent removal of nutrients from the wastewater The experiments were carried out in two phases. The first phase was conducted to select FW tolerant and mixotrophically growing microalgal species based on their biomass yield after 6 days of cultivation in Bold's Basal Medium supplemented with 5% synthetic CO2. The second phase was focused on the determination of optimum massive biomass production using selected microalga in batch experiments. The MWW filtrate was diluted with FW in the presence of flue gas CO2, to give three different FW (0.5, 1 and 2%, v/v) and CO2 (5, 10 and 14.1%) concentrations, i.e., a total of twelve wastewater solutions including undiluted wastewater were prepared, and the volume ratio of the wastewater to each of the diluents were as follows: 100:0 (undiluted wastewater at 5, 10 and 14.1% CO2), 99.5:0.5 (FW0.5 at 5, 10 and 14.1% CO2), 99:1 (FW1 at 5, 10 and 14.1% CO2) and 97:2 (FW2 at 5, 10 and 14.1% CO2). The batch experiments were conducted using 500 mL aluminum crimp sealed serum bottles containing 300 mL filter sterilized MW, inoculated with 3% (Vinoculum/Vwastewater) and supplemented with FW and flue gas CO2. The bottles were incubated in a shaker incubator at 150 rpm, 27  C, under white fluorescent light illumination at intensity of 120 mmol photon m
2 s
1 for 6 days. The flue-gas used in this study was collected from a 325 MW demonstration-scale coal-burning power plant located at Yeongdong Thermal Power Plantin Gangneung, South Korea. The typical composition of the flue gas was CO2, 14.1%; CO, 300 ppm; NOx, 207 ppm; and SOx, 53 ppm. During incubation, 6 mL of mixed liquor was periodically collected from each serum bottle to measure T-N, TP and TOC. The OD680 was converted to dry cell weight (DCW) using a linear relationship between OD680 and DCW (g L
1) (APHA, 1998). The relationship between optical density and DCW of was obtained after an extensive data analysis and is given by Eqs. (1) and (2) for Nephroselmis sp. and S. obliquus, respectively;

    Dry weight g L
1 ¼ 0:3541  OD680
0:0089 R2 ¼ 0:9918 (1)

M.-K. Ji et al. / Journal of Environmental Management 159 (2015) 115e120

    Dry weight g L
1 ¼ 0:3834  OD680
0:0122 R2 ¼ 0:9923 (2) The experiments were performed in triplicates and the average values were reported. 2.4. Analysis of lipid, carbohydrate content, and fatty acid composition Total lipids were extracted from freeze-dried algal biomass using the modified method described by Bligh and Dyer (1959). The fatty acids methyl esters (FAMEs) were analyzed using a method modified by Lepage and Roy (1984). The carbohydrate concentration was determined using a phenol-sulfuric acid method (Rao and Pattabiraman, 1989). The analytical methods are detailed by Salama et al. (2014). Each experiment was carried out in triplicate and the average values were reported. 2.5. Statistical analysis One-way analysis of variance (ANOVA) was used to examine the differences among average values. SigmaPlot version 10.0 for Windows (Systat Software, San Jose, CA, USA) was used for all statistical analyses, and differences in the variables were considered significant at the p < 0.05 level of confidence. 3. Results and discussion 3.1. Growth assessment of S. obliquus The growth of two microalgal species cultivated in BBM at 1, 2 and 3% FW supplemented with 5% synthetic CO2 was assessed using dry cell weight value (Supplementary Fig. 1). S. obliquus accounted for higher dry cell weight and exhibited no growth inhibition (
52%) at 2% FW compared to Nephroselmis sp. Several studies have reported the tolerance of S. obliquus to wastewaters (i.e., piggery wastewater and municipal wastewater) and flue gas CO2 (Ji et al., 2013b; Gentili, 2014), and was thus further investigated for biomass production using municipal wastewater in the presence of food wastewater/flue gas CO2. The growth of S. obliquus in different dilutions of MWW with FW in the presence of 5, 10 and 14.1% flue gas CO2 has been illustrated in Fig. 1. FW supported higher microalga growth with CO2 than the undiluted MWW after 6 days of cultivation. FW1 with 10% CO2 showed the highest dry cell weight (0.44 g dwt L
1), which was 2.1 times higher compared to the undiluted MWW with 5% CO2. FW0.5 and FW1% contained favorable TOC concentrations (TOC ¼ 56e103 mg L
1) for the microalgal growth. Few algal species can utilize the TOC (83% acetate) from FW (Table 1) for rapid growth under mixotrophic conditions in the presence of light. Acetic acid (or acetate) is initially transformed to acetyl coenzyme A (acetyl-CoA) and is further assimilated into algal carbohydrates via glyoxylate cycle in cooperation with tricarboxylic acid cycle (TCA cycle). Shin et al. (2014) reported that food wastewater with 78 mg L
1 of acetate supported the optimal microalgal growth, while acetate above 400 mg L
1 was toxic for the growth as reported in literatures. In addition, 20 times diluted anaerobically digested food wastewater showed the highest growth of Scenedesmus bijuga, but the biomass production was similar to that with synthetic media (BG-11) due lack of inorganic carbon. The growth of Chlorella sp. was significantly increased (2 times)

117

with shortest lag phase (1day) in swine wastewater with acetate compared to control (Hu et al., 2012). Bhatnagar et al. (2011) reported that acetate was a better organic carbon supplement compared to glycerol and methanol for C hlorellaglobosa, C hlorella minutissima and S. bijuga, as the presence of acetate increased the chlorophyll content by 56e91% than observed under phototrophic growth conditions. The growth of Chlorella sp. was higher in mixotrophic (0.8 g cell/L d) culture with 10% flue gas CO2 and glucose compared to autotrophic (0.56 g cell/L d) culture (Praveenkumar et al., 2014b). The microalga growth was decreased at concentrations higher than FW1, which can be attributed to the toxicity of salinity, TOC and aluminum from FW (Liang et al., 2009; Salama et al., 2013). Increase of CO2 concentration from 5 to 14.1% in the MWW slightly increased the microalga growth, which might be due to sufficient inorganic carbon source during autotrophic and mixotrophic culture mode. The solution pH in different dilutions of FW was increased due to microalga growth, and the highest pH (10.1e10.4) was observed in 0.5e1% FW due to high algal growth. The presence of calcium, magnesium, manganese and iron in FW also contributed for the higher microalgal growth compared to undiluted MWW (Table 1) (Ji et al., 2014).

3.2. Nutrient removal as a result of algal growth The removal of T-N, T-P and TOC was determined at intervals during cultivation of S. obliquus (Figs. 2e4). T-N uptake by the microalga increased with the increase in concentration of FW up to 1%, and strongly correlated with the microalgal growth (Fig. 2). Amount of T-N removal by the microalga ranged between 13.9e17.1 (63e88%), 19.8e21.8 (83e97%), 21.4e21.8(78e90%) and 18.3e18.9 (54e62%) mg L
1 in MWW, 0.5% FW, 1% FW and 2% FW supplemented with 5e14.1% CO2, respectively. The highest T-N removal (21.1e21.8 mg L
1) was observed with 0.5 and 1% FW with 10 and 14.1% CO2. The highest T-P (2.85 mg L
1) uptake was observed in 2% FW, which might be due to the formation of inorganic precipitates (such as calcium phosphate) at an alkaline pH (Fig. 3) (Ji et al., 2014). Insoluble precipitates (i.e., hydroxyapatite and struvite) were obþ 2þ tained in the presence of ions such as Ca2þ, PO3
4 , NH4 and Mg between pH 8 to11 (Cohen and Kirchmann, 2004). Amount of T-P removal by the microalga ranged between 1.77e1.98 (77e85%), 1.86e2.0 (80e86%), 2.15e2.31 (82e89%) and 2.30e2.52 (73e84%) mg L
1 in MWW, 0.5% FW, 1% FW and 2% FW supplemented with 5e14.1% CO2, respectively. The removal of TOC ranged between 58 and 74% in the presence of FW (Fig. 4), while it increased (11e31%) in undiluted MWW. Most of the strains belonging to genus Scenedesmus sp. are known for assimilation of organic carbon by mixotrophic or heterotrophic ways since there is enough inorganic carbon in the culture solution. However, the bacterial cultures residing in the aeration tanks perform the extensive degradation of the organic carbon sources in the effluent discharge from municipal WWTPs to mostly inert carbon matters that are recalcitrant to be utilized easily by microalgae (Wang et al., 2010). Shin et al. (2014) reported that 66% of COD was removed from 20 times diluted food wastewater under mixotrophic condition for 4 days. Hu et al. (2012) also reported that 62% of COD was removed when Chlorella sp. was cultivated in swine wastewater with acetate for 5 days. FW 1% supplemented with 10 and 14.1% CO2 showed the highest TOC removal (74%). Volatile organic compound (VOC) stripping might also contribute for notable removal of TOC from the medium with FW in the absence of microalga. Zhang and Jahng (2010) reported a removal of approximately 20% COD from the wastewater effluent due to VOCs stripping.

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M.-K. Ji et al. / Journal of Environmental Management 159 (2015) 115e120

(b) 0.5

5% CO2 10% CO2

0.4

Dry cell weight (g L )

14.1% CO2

-1

-1

Dry cell weight (g L )

(a) 0.5

0.3 0.2 0.1

5% CO2 10% CO2

0.4

14.1% CO2

0.3 0.2 0.1 0.0

0.0 0

2

4

0

6

(d) 0.5

5% CO2 10% CO2

0.4

Dry cell weight (g L )

14.1% CO2

-1

-1

Dry cell weight (g L )

(c) 0.5

2

4

6

Cultivation time (days)

Cultivation time (days)

0.3 0.2 0.1 0.0

5% CO2 10% CO2

0.4

14.1% CO2

0.3 0.2 0.1 0.0

0

2

4

6

Cultivation time (days)

0

2

4

6

Cultivation time (days)

Fig. 1. Growth of S. obliquus in different dilutions of FW supplemented with 5, 10 and 14.1% flue gas CO2 (a-MWW, p < 0.0349; b-0.5% FW, p < 0.0347; c-1% FW, p < 0.0369 and d-2% FW, p < 0.0357).

3.3. Total lipid and FAME profile Lipids are substances related biosynthetically or functionally to fatty acids and their derivatives (Chisti, 2007). Table 2 shows the total lipid content/productivity of microalgal biomass harvested after 6 days. S. obliquus cultivated in MWW in the presence of FW supplemented with 5, 10 and 14.1% CO2 showed a total lipid content ranging from 18.4 to 22.9%, 18.4 to 23.3 and 19.7e22.5%, respectively, based on their dry biomass. The lipid content was slightly lower in FW than undiluted MWW except with 2% FW, which might be due to different cultivation conditions. The lipid content of microalgae under photoautotrophic condition was higher than under mixotrophic condition (Liang et al., 2009; Cheirsilp and Torpee, 2012). Heredia-Arroyo et al. (2011) also reported that the lipid content was lower (14e17%) under mixotrophic condition with high biomass production (1.4 g L
1) compared with autotrophic (0.4 g L
1) and heterotrophic (0.75 g L
1) conditions. Highest lipid content was found in 2% FW, which might be due to TOC and salinity induced stress. Microalgae cellular lipid content was increased under high salinity due to formation of triacylglycerols (TAG) in response to osmotic pressure (Zhila et al., 2010). The lipid productivity of the microalga cultivated in 0.5% FW supplemented with 10 and 14.1% CO2 was higher (11 mg L day
1) than that of undiluted MWW and 1e2% FW, respectively, due to higher biomass production with relatively high lipid content (Table 2). Praveenkumar et al. (2014b) reported that lipid productivity was higher (2.7 time) under mixotrophic condition in the presence of 10% flue gas CO2 and glucose compared to autotrophic condition. The content and composition of fatty acids in microalgae are species-specific (Chisti, 2007). Palmitic acid (C16:0), oleic acid (C18:1n9c), linolelaidic acid (C18:2n6t) and g-linolenic acid (C18:3) were the major fatty acids accumulated by S. obliquus, such that they accounted for approximately 80e85% of the total fatty acids (Fig. 4 and Supplementary Table 1). The amount of saturated (SFA),

mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty acids in the microalga grown in FW supplemented with 5, 10 and 14.1% CO2 accounted for 29e36%/21e27%/37e48%, 34e38%/21e27%/ 37e43% and 35e39%/22e29/36e40 of the total fatty acids, respectively (Fig. 4). SFA (mainly, palmitic acid) and MUFA (mainly, oleic acid) contents increased with increasing CO2 concentration up to 14.1%. Specifically, relatively high fractions of C16:0 (palmitic acid) and C18:1 (oleic acid) fatty acids were observed under mixotrophic condition compared with MWW, which are desirable for the production of biodiesel (Liu et al., 2011). The fatty acids of microalga cultivated in 1e2% FW supplemented with 10 and 14.1% CO2 showed a highest concentration of palmitic acid (36e38%), while the highest oleic acid (22e24%) content was observed with 0.5e1% FW supplemented with 10 and 14.1% (Supplementary Table 1). Praveenkumar et al. (2014b) reported that palmitic and oleic acid contents were increased (2 and 7%) in mixotrophic culture compared to autotrophic culture. Cultivation of S. bijuga in 20 times diluted food wastewater increased the oleic acid content by 13% compared to BG-11 media (Shin et al., 2014). High oleic acid content renders the oils with a reasonable balance of fuel properties including ignition quality, combustion heat, cold filter plugging point (CFPP), oxidative stability, viscosity and lubricity (Liu et al., 2011), while palmitic acid imparts a higher oxidative stability and cetane number, and a lower NOX emissions (Cheirsilp and Torpee, 2012). 3.4. Carbohydrate content Microalgae carbohydrates mainly consist of starch (storage component in chloroplasts) and cellulose/polysaccharides (structural components in cell walls). Carbohydrates (storage compounds) serve as the energy source for the metabolic processes of the organisms (Carrieri et al., 2010). Total carbohydrate content/ productivity of the microalgal biomass harvested after 6 days was

M.-K. Ji et al. / Journal of Environmental Management 159 (2015) 115e120

40

Total nitrogen (mg L-1)

(d)

30

(c) (b)

(a)

20

FAME composition (%)

100

Initial Final

SFA MUFA PUFA

80

60

40

20

10 0

(a) (b) (c) (d)

5 0

5 10 14

5 10 14

5 10 14

5 10 14

CO2 concentration (%)

Total phosphorous (mg L-1)

Initial Final

(a) (b) (c) (d)

10 14 CO2 concentration (%)

(d)

3

(c) (b)

(a)

2

1

0

(a) (b) (c) (d)

Fig. 4. Changes in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) of S. obliquus cultivated in different dilutions of FW supplemented with 5, 10 and 14.1% flue gas CO2 for 6 days (a-MWW, b-0.5% FW, c-1% FW and d-2% FW).

4

5 10 14

5 10 14

5 10 14

5 10 14

CO2 concentration (%) Fig. 2. Residual T-N and T-P after 6 days cultivation of S. obliquus in different dilutions of FW supplemented with 5, 10 and 14.1% flue gas CO2 (a-MWW, b-0.5% FW, c-1% FW, and d-2% FW).

250

Total organic carbon (mg L -1)

119

Initial Final

(d)

200

150

determined (Table 2). The total carbohydrate content of the S. obliquus cultivated in MWW in the presence of FW supplemented with 5, 10 and 14.1% CO2 ranged from 20.5 to 26.5%, 23.8 to 25.5 and 25.5e28.8%, respectively, based on their dry biomass. Carbohydrate content was increased with increasing CO2 concentration except with 2% FW condition. Thus, presence of CO2 in sufficient amount significantly influences the accumulation of carbohydrate in microalgae. Some studies found that carbohydrate accumulation in microalgae was improved under increased CO2 concentration (Giordano, 2001; Xia and Gao, 2005). Increased dissolved CO2 concentration from 3 to 186 mmol/L during cultivation of Chlorella pyrenoidosa and Chlorella reinhardtii elevated the carbohydrate content from 9.30 to 21.0% and 3.19e7.40% (w/w), respectively (Xia and Gao, 2005). The carbohydrate productivity of the microalga cultivated with 0.5% FW supplemented with 14.1% CO2 was higher (15.8 mg L day
1) than that other conditions (Table 2). Specially, it was 3 times higher compared to undiluted MWW supplemented with 5% CO2. It should be noted that in the present results, the intermittent FW and flue gas CO2 could significantly improve algal carbohydrate productivity by increasing both the carbohydrate content and the biomass productivity. Under mixotrophic conditions the yield of biomass was observed to be higher over ATP than with autotrophic mode. In addition, under optimal mixotrophic conditions, the excess energy left out after cellular maintenance during dark phase, could be used for energy storage either in form of starch or lipid (Yang et al., 2000). This elucidates the possibility of obtaining increased biomass and carbohydrate content in microalgae when grown under an optimal mixotrophic condition.

(c)

4. Conclusions

100 (b)

50

0

(a)

5 10 14

5 10 14

5 10 14

5 10 14

CO2 concentration (%) Fig. 3. Residual TOC after 6 days cultivation of S. obliquus in different dilutions of FW supplemented with 5, 10 and 14.1% flue gas CO2 (a-MWW, b-0.5% FW, c-1% FW and d2% FW).

The biomass, lipid/carbohydrate productivity and nutrient removal efficiency of S. obliquus cultivated under mixotrophic condition in municipal wastewater was enhanced in the presence of food wastewater and flue gas CO2 (in/organic carbon and major/ trace elements). The FW (0.5e1%) with 10 and 14.1% CO2 lead to production of microalga feedstock suitable for obtaining highefficiency biodiesel (60e64% of saturated and monounsaturated fatty acid). Application of FW and flue gas CO2 for mixotrophic cultivation of S. obliquus in municipal wastewater with simultaneous wastewater treatment could lead to development of a costeffective and environmentally sustainable strategy.

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M.-K. Ji et al. / Journal of Environmental Management 159 (2015) 115e120

Table 2 Lipid/carbohydrate content and productivity of S. obliquus cultivated in different dilutions of FW and flue gas CO2 concentration. Parameters

Lipid (%) Lipid productivity (mg L
1 day
1) Carbohydrate (%) Carbohydrate productivity (mg L
1 day
1)

10% CO2

5% CO2

14.1% CO2

MWW

0.5% FW

1% FW

2% FW

MWW

0.5% FW

1% FW

2% FW

MWW

0.5% FW

1% FW

2% FW

19.7 5.80 17.5 5.15

18.4 8.85 20.5 9.85

18.7 9.95 21.5 11.6

22.9 8.59 26.5 9.92

20.2 8.24 19.2 7.85

19.7 10.5 23.8 12.8

18.4 9.80 26.4 14.1

23.3 9.11 25.5 9.95

20.3 9.02 22.9 10.1

20.0 11.2 28.8 15.8

19.7 10.0 25.5 13.0

22.5 8.93 25.9 10.3

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