Bioresource Technology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation Min-Kyu Ji a, Hyun-Shik Yun a, Sanghyun Park a, Hongkyun Lee a, Young-Tae Park a, Sunyoung Bae b, Jungyeob Ham c, Jaeyoung Choi a,⇑ a b c
Environmental Remediation Research Group, Korea Institute of Science and Technology (KIST), Gangneung Institute, Gangneung 210-340, South Korea Department of Chemistry, Seoul Women’s University, Seoul 139-774, South Korea Natural Medicine Center, Korea Institute of Science and Technology (KIST), Gangneung Institute, Gangneung 210-340, South Korea
h i g h l i g h t s A dual strategy for wastewater reuse and biofuel feedstock production. Food wastewater improved the growth and lipid productivity of S. obliquus. Food wastewater supported the algal autoflocculation.
a r t i c l e
i n f o
Article history: Received 15 October 2014 Received in revised form 12 December 2014 Accepted 15 December 2014 Available online xxxx Keywords: Scenedesmus obliquus Biomass Food wastewater Bioenergy Autoflocculation
a b s t r a c t Effect of food wastewater (FW) on the biomass, lipid and carbohydrate production by a green microalga Scenedesmus obliquus cultivated in Bold’s Basal Medium (BBM) was investigated. Different dilution ratios (0.5–10%) of BBM either with FW or salt solution (NaCl) or sea water (SW) were evaluated. S. obliquus showed the highest growth (0.41 g L 1), lipid productivity (13.3 mg L 1 day L 1), carbohydrate productivity (14.7 mg L 1 day L 1) and nutrient removal (38.9 mg TN L 1 and 12.1 mg TP L 1) with 1% FW after 6 days of cultivation. The FW promoted algal autoflocculation due to formation of inorganic precipitates at an alkali pH. Fatty acid methyl ester analysis revealed that the palmitic and oleic acid contents were increased up to 8% with FW. Application of FW improved the growth, lipid/carbohydrate productivity and biomass recovery efficiency of S. obliquus, which can be exploited for cost effective production of microalgae biomass. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgal biomass production is significantly influenced by the presence of inorganic and organic carbon sources in the cultivation medium, which affect the biochemical properties of microalgae (Cheirsilp and Torpee, 2012). Autotrophic microalgal growth for biodiesel production has gained much attention, but insufficiency of light at high cell concentrations limits its application for high cell density biomass production (Liang et al., 2009; Hu et al., 2012). Mixotrophic growth occurs when the microalgae are provided with both inorganic (CO2) and organic carbon (e.g., glucose and acetate) sources (Wang et al., 2012). This can significantly reduce the dependence of microalgae growth on light, which is observed under pure photoautotrophic conditions, and stimulate ⇑ Corresponding author. Tel.: +82 33 6503701; fax: +82 33 6503729. E-mail address: [email protected] (J. Choi).
the algal growth and increase the cell density (Wang et al., 2012). However, the additional cost of organic carbon will make the mixotrophic cultivation economically unfeasible (Bhatnagar et al., 2011), which will further increase the cost of biofuel production and impede its commercialization. Microalgae cultivation using organic wastewaters for simultaneous biomass production and wastewater treatment has been reported as a cost effective strategy (Bhatnagar et al., 2011; Ji et al., 2014a). Food effluent is one of the most highly produced wastewaters throughout the world, and its treatment has emerged as a social issue by the law of London Dumping Convention in South Korea. Food wastewater (FW) is rich in nutrients including nitrogen, phosphorus, calcium, iron, aluminum and total organic carbon, which might be an efficient wastewater feedstock for mixotrophic microalgal cultivation to achieve a reasonably high biomass yield and lipid productivity for energy generation (Shin et al., 2014).
http://dx.doi.org/10.1016/j.biortech.2014.12.053 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
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M.-K. Ji et al. / Bioresource Technology xxx (2014) xxx–xxx
Several studies have reported the application of municipal and piggery wastewaters with synthetic CO2 for biomass and bioenergy production (Zhou et al., 2011; Wang et al., 2012). FW has not been investigated extensively for microalgal cultivation. The present study investigated the effect of food wastewater on biomass production, lipid productivity and fatty acid composition of Scenedesmus obliquus cultivated in Bold’s Basal Medium (BBM) supplemented with CO2 as flue gas.
Table 1 Physico-chemical characteristics of (a) NaCl, (b) sea water and (c) food wastewater.
2. Methods 2.1. Algal strain, culture conditions and inoculum preparation S. obliquus was isolated from acid mine drainage (AMD) (Donghae, South Korea) and was registered in GenBank under Accession No. HE861884. The microalga was inoculated in 2 L reactor containing 1.7 L Bold’s Basal Medium (BBM) at 10% concentration (Vinoculum/Vmedia) (Bischoff and Bold, 1963). The microalgal cells were incubated under white fluorescent light illumination at a light intensity of 120 lmol photon m 2 s 1 at 25 °C for 10 days. During the incubation, each column was aerated with filter-sterilized air at a flow rate of 0.5 L min 1 to agitate the suspension and to simultaneously supply CO2 (0.03%). Three milliliters (OD680 = 1.5) of microalgae were used as initial inoculums for further experiments.
a b c
Parameter
(a) NaCl
(b) SW
(c) FW
pH T-N (mg L 1) NH4-N (mg L 1) NO3-N (mg L 1) NO2-N (mg L 1) T-P (mg L 1) PO4-P (mg L 1) TOC (mg L 1) Salinity (%)
6.7 NDb ND ND ND ND ND 0.1 ± 0.03 3.7c
7.83 0.27 ± 0.02 ND 0.25 ± 0.01 ND 0.03 ± 0.01 0.04 ± 0.01 20.3 ± 0.12 3.7
6.0a 1385 ± 1.9 1197 ± 1.1 105.4 ± 0.9 45.2 ± 2.2 108 ± 0.6 91.6 ± 0.73 14,898 ± 0.54 3.7c
Metallic ions Al3+ (mg L 1) B3+ (mg L 1) Ca2+ (mg L 1) Cd2+ (mg L 1) Co2+ (mg L 1) Cr6+ (mg L 1) Cu2+ (mg L 1) Fe (total dissolved) (mg L Mg2+ (mg L 1) Mn2+ (mg L 1) Pb2+ (mg L 1) S (mg L 1) Zn2+ (mg L 1)
ND 0.016 ± 0.007 ND ND ND ND ND ND ND ND ND ND ND
0.018 ± 0.002 4.49 ± 0.10 387.4 ± 1.17 ND ND ND ND ND 1118.3 ± 1.95 ND 0.03 ± 0.01 947.95 ± 0.88 ND
316.4 ± 0.13 0.511 ± 0.007 1055 ± 2.01 ND ND ND ND 24.7 ± 0.06 97.01 ± 1.24 1.89 ± 0.05 0.05 ± 0.01 122.07 ± 0.91 1.29 ± 0.08
1
)
After adjusted of pH with NaOH. ND: not detected. After adjusted of salinity.
2.2. Algae cultivation and growth The BBM was diluted with each of food wastewater (FW) and NaCl/sea water (SW) (control), to give five different concentrations (0.5%, 1%, 3%, 5% and 10%), i.e., a total of sixteen solutions including undiluted BBM were prepared, and the volume ratio of the BBM to each of the diluents were as follows: 100:0 (undiluted wastewater), 99.5:0.5 (BBM or FW/NaCl/SW), 99:1 (BBM or FW/NaCl/SW), 97:3 (BBM or FW/NaCl/SW), 95:5 (BBM or FW/NaCl/SW) and 90:10 (BBM or FW/NaCl/SW). SW and NaCl were used to compare the effect of total organic carbon from the FW on the microalgae growth based on concentration of same salinity. The batch experiments were conducted using 500 mL aluminum crimp sealed serum bottles containing 300 mL BBM, inoculated with 1.5% (Vinoculum/Vwastewater) of microalga solution and supplemented with 5.1% flue gas CO2. The bottles were incubated in a shaker incubator at 150 rpm, 27 °C, under white fluorescent light illumination (alternate light/dark periods of 16 h/8 h) at an intensity of 120 lmol photon m 2 s 1 for 6 days. 2.3. Analytical methods FW was collected from a food wastewater treatment plant at Dangjin, South Korea and sea water (SW) from gyeongpodae at Gangneung, South Korea. Wastewaters were immediately filtered using 0.2 lm nylon micro filters to remove the microorganisms and suspended solid particles. The physicochemical properties of the FW were analyzed (Table 1). The analytical methods of TN, NH4-N, TP, anions (i.e., NO2 , NO3 and PO34 ), metal ions (i.e., Ca2+, Mg2+ and Fetotal), total organic carbon (TOC) and pH were described by Ji et al. (2014a). Other analytical methods are detailed in Supplementary Information. 3. Results and discussion 3.1. Growth assessment of S. obliquus FW supported higher microalga growth than the undiluted BBM and NaCl/SW diluted BBM after 6 days of cultivation
(Fig. 1). The highest dry cell weight (0.41 g dwt L 1) was observed for FW 1, which was 2.8 times higher compared to the undiluted BBM due to highest removal of nutrient (38.9 mg L 1 T-N and 12.1 mg L 1 T-P) (Supplementary Fig. 1). The TOC concentrations (TOC = 75–149 mg L 1) in FW 0.5 and FW 1 were favourable for the microalgal growth. Shin et al. (2014) reported that the TOC (83% acetate, 15% lactate and 2% propionate) from FW (Table 1) could be utilized by some algae species for rapid growth under mixotrophic or photoheterotrophic mode in light. Acetic acid (or acetate) is one of the most commonly used organic carbon substrate by the mixotrophic or heterotrophic cultures of microalgae (Hu et al., 2012; Liu et al., 2013). Shin et al. (2014) reported that optimal microalgal growth was observed in 78 mg L 1 of acetate from food wastewater, and acetate above 400 mg L 1 might be toxic for microalgal growth as reported in literatures (Chen and Johns, 1996). The mixotrophic microalgal culture can simultaneously assimilate the inorganic and organic substrates through concurrent respiratory and photosynthesis processes (Bhatnagar et al., 2011). The growth rate of mixotrophic culture is the sum of the photoautotrophic and heterotrophic growth (Liang et al., 2009). The algae cultures exhibit a higher growth under CO2 conditions in the presence of organic carbon substrates (i.e., acetate and glucose) (Liang et al., 2009; Hu et al., 2012). Shin et al. (2014) reported that the highest growth of Scenedesmus bijuga was observed in 20 times diluted anaerobically digested food wastewater. But, the biomass production was similar to that observed in synthetic media (BG-11), which might be due to lack of inorganic carbon for mixotrophic cultivation. Concentrations higher than FW 1 decreased the microalgal growth, which might be due to toxicity of salinity, TOC and aluminium from FW (Liang et al., 2009; Salama et al., 2013). The formation of inorganic precipitate occurred during cultivation in FW 0.5 to FW 10, which might coat the microalgae cell surface and decrease the nutrient uptake at above FW 3 (Ji et al., 2014a). Increase of NaCl and SW concentration in the culture media from 0% to 3% (NaCl/SW) slightly increased the microalga growth. Sodium ion facilitates photosynthesis in microalgae through inorganic nutrients uptake, intracellular pH regulation, and
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
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3.2. Effect of FW on surface charge and floc formation of microalgal biomass Zeta potential (ZP) of microalgal cells is often used as an indicator of cell stability. Zeta potential of S. obliquus shifted from a negative ( 5.51 mV) to a positive (+0.41 mV) charge with the FW 1 at day 6, which might be due to the formation of inorganic precipitates (such as struvite, calcium phosphate and calcium carbonate) at an alkaline pH (Ji et al., 2014a). While the zeta potential was maintained from 5.51 to 5.46, 4.39 and 4.42 in undiluted BBM, NaCl 1 and SW 1, respectively. Ions such as Ca2+, PO34 , NH+4 and Mg2+ in the aqueous phase can produce insoluble precipitates such as hydroxyapatite and struvite between pH 8 to 11 (Cohen and Kirchmann, 2004). Supplementary Fig. 2 shows the SEM image and energy dispersive X-ray spectrum of the precipitates collected after 6 days incubation of undiluted BBM and FW 1 with S. obliquus. Carbon, nitrogen and oxygen were the dominant elements of the precipitates detected by EDX spectroscopy. EDS analysis showed 54.04% C, 9.61% N, 24.64% O, 3.61% Na, 5.17% Si and 2.94% P for undiluted BBM (Supplementary Fig. 2a), while the composition of elements changed and/or appeared as 38.48% C, 7.01% N, 33.27% O, 1.00% Na, 2.12% Si, 4.59% P, 1.02% Mg, 1.52% Al, 9.01% Ca and 1.98% Fe for FW 1 (all values in wt%) in the presence of algae (Supplementary Fig. 2b). The EDS spectra of centrate municipal wastewater precipitate also showed the presence of calcium phosphate, calcium carbonate, and/or struvite (Ji et al., 2014a). FW properties promoted the formation of visible microalgae flocs due to attachment of microalgal cells, which settled down as clusters due to formation of bridges between the flocs. Supplementary Fig. 3 shows the microscopic images of the flocs formed with undiluted BBM, NaCl 1, SW 1 and FW 1. Floc density was higher in FW 1 than other conditions, which is supported by the zeta potential data. The enhanced flocculation with FW was presumably due to presence of large number of ionic species in FW, which decreased the double-layer repulsion between the colloidal particles at high ionic strength. Under favorable conditions, the precipitates carry positive surface charges and induce flocculation through charge neutralization and/or sweeping flocculation (Chisti, 2007). 3.3. Total lipid FAME and carbohydrate profile
Fig. 1. Growth of S. obliquus in different dilutions of (a) NaCl, (b) SW and (c) FW.
alkalotolerance (Zhila et al., 2010). Concentrations higher than 5% (NaCl /SW) decreased the microalgal growth, which might be due to toxicity of salinity (Salama et al., 2013). Microalgal growth in different dilutions of NaCl, SW and FW increased the solution pH, and 0.5–1% FW showed the highest pH (10.8 and 11.1) due to high algal growth (Chisti, 2007). In addition, presence of calcium, magnesium, manganese and iron in FW promoted higher microalgal growth compared to undiluted BBM and NaCl/SW (Fig. 1c) (Ji et al., 2014a).
Total lipid content/productivity of microalgal biomass harvested after 6 days was determined (Table 2). The total lipid content of S. obliquus cultivated in BBM in the presence of NaCl, SW and FW ranged between 21.9% to 24.1%, 21.5% to 24.3% and 19.7% to 20.7%, respectively, based on their dry biomass. The lipid content increased slightly with increasing NaCl, SW and FW dose from 0.5% to 10%. Zhila et al. (2010) reported that a growth medium with high salinity increased the microalgae cellular lipid content by improving the formation of triacylglycerols (TAG). The lipid content was lower in FW than with NaCl and SW conditions, which might be due to different trophic culture modes. The lipid content of microalgae under photoautotrophic condition was higher than those under mixotrophic condition (Liang et al., 2009; Cheirsilp and Torpee, 2012; Wang et al., 2012). The aforementioned trend was consistent with the results in this study. The lipid productivity of the microalga cultivated with FW 1 was higher (13.3 mg L day 1) than that of undiluted BBM, NaCl and SW, respectively, due to highest biomass production (Table 2). The fatty acids accumulated in S. obliquus majorly possessed palmitic acid (C16:0), oleic acid (C18:1n9c), linolelaidic acid (C18:2n6t), linoleic acid (C18:2n6c) and c-linolenic acid (C18:3), such that the sum of these four fatty acids accounted for approximately 75–85% of the total fatty acids (Fig. 2). The amount of saturated, mono-unsaturated and poly-unsaturated fatty acids in the
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
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Table 2 Lipid/carbohydrate content and productivity of S. obliquus cultivated in different dilutions of NaCl, SW and FW. Parameters
Undiluted BBM
Lipid (%) Lipid productivity (mg L 1 day 1) Carbohydrate (%) Carbohydrate productivity (mg L 1 day
22.3 5.57 21.0 5.24
1
)
BBM + NaCl%
BBM + SW%
BBM + FW%
0.5
1
3
5
10
0.5
1
3
5
10
0.5
1
3
5
10
22.3 6.11 21.2 5.80
21.9 5.87 22.5 6.01
22.9 6.43 23.2 6.49
22.5 5.92 21.9 5.75
24.1 6.15 22.2 5.65
22.5 5.29 21.1 5.89
21.5 6.00 22.8 6.32
22.7 6.25 23.2 6.38
23.1 6.42 23.3 6.46
24.3 6.21 21.7 5.54
19.7 9.86 24.5 12.0
19.7 13.3 21.8 14.7
20.8 3.57 22.1 3.80
– – – –
– – – –
Fig. 2. Changes in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) of S. obliquus cultivated in different dilutions of NaCl, SW and FW.
microalga grown with different NaCl, SW and FW dilutions accounted for 25–28%/18–21%/52–55%, 25–28%/17–20%/52–55% and 26–33%/21–26%/43–53% of the total fatty acids, respectively. Palmitic acid and oleic acid contents increased with increasing FW dose. In particular, relatively high ratios of C16:0 (palmitic acid) and C18:1 (oleic acid) fatty acids were observed under mixotrophic growth, which are desirable for the production of biodiesel (Liu et al., 2011). The fatty acids of microalga cultivated in FW 3 showed a high concentration of palmitic (27%) acid and oleic acid (16%). Ji et al. (2014b) reported that oleic acid content was increased (4– 19%) under mixotrophic culture condition using monosodium glutamate wastewater (MSGW) for 16 days compared to BG11 media. Also, Shin et al. (2014) reported the oleic acid content was increased (13%) in 20 diluted digested food wastewater effluent compared to synthetic media (BG11). The fatty acid methyl esters (FAMEs) of S. obliquus cultivated in FW possessed more saturated (16:0) and mono-unsaturated fats (C18:1n9c), which are more suitable for use in hot and cold weather environments due to their typically high oxidative stability and lower melting point. Carbohydrates serve two main purposes for algae; they act as structural components in the cell walls and as storage components inside the cell. The most frequently reported cultivation factors that affect the carbohydrate content are nutrient starvation/limitation, salt stress, light intensity and temperature (Ji et al., 2014b). In addition, the metabolic mode such as autotrophic, heterotrophic or mixotrophic affects the biomass composition (Liang et al., 2009). The total carbohydrate content of S. obliquus after 6 days of cultivation in BBM with NaCl, SW and FW ranged from 21.2% to 23.2%, 21.1% to 23.2% and 21.1% to 24.0%, respectively, based on their dry biomass (Table 2). The carbohydrate content was slightly high in FW 0.5%. The carbohydrate productivity of the microalga cultivated with FW 1% was higher (14.7 mg L day 1) than that of undiluted BBM, NaCl and SW, respectively, due to higher biomass production (Table 2).
4. Conclusions Application of food wastewater as a nutrient source (organic carbon and trace elements) improved the biomass, lipid productivity and nutrient removal efficiency of S. obliquus cultivated in BBM. The microalga grown with 0.5–1% FW was promising for the generation of high-efficiency biodiesel (19–23% palmitic acid and 10% oleic acid). Abiotic precipitation also supported the removal of nutrients and autoflocculation of algal cells. Utilization of S. obliquus for the generation of biofuel feedstock with simultaneous reuse of FW in the presence of flue gas CO2 can be a cost-effective and environmentally sustainable strategy. Acknowledgement The project was financially supported by the Korea Institute of Science and Technology (Gangneung) (Grant 2Z04230). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 12.053. References Bhatnagar, A., Chinnasamy, S., Singh, M., Das, K.C., 2011. Renewable biomass production by mixotrophic algae in the presence of various carbon sources and wastewaters. Appl. Energy 88, 3425–3431. Bischoff, H.W., Bold, H.C., 1963. Phycological Studies IV. Some Soil Algae from Enchanted Rock and Related Algal Species. University of Texas Publication 6318, pp. 1–95. Cheirsilp, B., Torpee, S., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol. 110, 510–516.
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
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Liu, J., Huang, J., Sun, Z., Zhong, Y., Jiang, Y., Chen, F., 2011. Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production. Bioresour. Technol. 102, 106–110. Salama, E.-S., Kim, H.-C., Abou-Shanab, R.A.I., Ji, M.-K., Oh, Y.-K., Kim, S.-H., Jeon, B.H., 2013. Biomass, lipid content, and fatty acid composition of freshwater C. mexicana and Scenedesmus obliquus grown under salt stress. Bioprocess. Biosyst. Eng. 36, 827–833. Shin, D.Y., Cho, H.U., Utomo, J.C., Choi, Y.N., Xu, X., Park, J.M., 2014. Biodiesel production from Scenedesmus bijuga grown in anaerobically digested food wastewater effluent. Bioresour. Technol. (in press). doi:10.1016/ j.biortech.2014.10.090. Wang, H.Y., Xiong, H.R., Hui, Z.L., Zeng, X.B., 2012. Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids. Bioresour. Technol. 104, 215–220. Zhila, N.O., Kalacheva, G.S., Volova, T.G., 2010. Effect of salinity on the biochemical composition of the alga Botryococcus braunii Kütz IPPAS H-252. J. Appl. Phycol. 23, 47–52. Zhou, W., Li, Y., Min, M., Hu, B., Chen, P., Ruan, R., 2011. Local bioprospecting for high-lipid producing microalgal strains to be grown on concentrated municipal wastewater for biofuel production. Bioresour. Technol. 102, 6909–6919.
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation Min-Kyu Ji a, Hyun-Shik Yun a, Sanghyun Park a, Hongkyun Lee a, Young-Tae Park a, Sunyoung Bae b, Jungyeob Ham c, Jaeyoung Choi a,⇑ a b c
Environmental Remediation Research Group, Korea Institute of Science and Technology (KIST), Gangneung Institute, Gangneung 210-340, South Korea Department of Chemistry, Seoul Women’s University, Seoul 139-774, South Korea Natural Medicine Center, Korea Institute of Science and Technology (KIST), Gangneung Institute, Gangneung 210-340, South Korea
h i g h l i g h t s A dual strategy for wastewater reuse and biofuel feedstock production. Food wastewater improved the growth and lipid productivity of S. obliquus. Food wastewater supported the algal autoflocculation.
a r t i c l e
i n f o
Article history: Received 15 October 2014 Received in revised form 12 December 2014 Accepted 15 December 2014 Available online xxxx Keywords: Scenedesmus obliquus Biomass Food wastewater Bioenergy Autoflocculation
a b s t r a c t Effect of food wastewater (FW) on the biomass, lipid and carbohydrate production by a green microalga Scenedesmus obliquus cultivated in Bold’s Basal Medium (BBM) was investigated. Different dilution ratios (0.5–10%) of BBM either with FW or salt solution (NaCl) or sea water (SW) were evaluated. S. obliquus showed the highest growth (0.41 g L 1), lipid productivity (13.3 mg L 1 day L 1), carbohydrate productivity (14.7 mg L 1 day L 1) and nutrient removal (38.9 mg TN L 1 and 12.1 mg TP L 1) with 1% FW after 6 days of cultivation. The FW promoted algal autoflocculation due to formation of inorganic precipitates at an alkali pH. Fatty acid methyl ester analysis revealed that the palmitic and oleic acid contents were increased up to 8% with FW. Application of FW improved the growth, lipid/carbohydrate productivity and biomass recovery efficiency of S. obliquus, which can be exploited for cost effective production of microalgae biomass. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Microalgal biomass production is significantly influenced by the presence of inorganic and organic carbon sources in the cultivation medium, which affect the biochemical properties of microalgae (Cheirsilp and Torpee, 2012). Autotrophic microalgal growth for biodiesel production has gained much attention, but insufficiency of light at high cell concentrations limits its application for high cell density biomass production (Liang et al., 2009; Hu et al., 2012). Mixotrophic growth occurs when the microalgae are provided with both inorganic (CO2) and organic carbon (e.g., glucose and acetate) sources (Wang et al., 2012). This can significantly reduce the dependence of microalgae growth on light, which is observed under pure photoautotrophic conditions, and stimulate ⇑ Corresponding author. Tel.: +82 33 6503701; fax: +82 33 6503729. E-mail address: [email protected] (J. Choi).
the algal growth and increase the cell density (Wang et al., 2012). However, the additional cost of organic carbon will make the mixotrophic cultivation economically unfeasible (Bhatnagar et al., 2011), which will further increase the cost of biofuel production and impede its commercialization. Microalgae cultivation using organic wastewaters for simultaneous biomass production and wastewater treatment has been reported as a cost effective strategy (Bhatnagar et al., 2011; Ji et al., 2014a). Food effluent is one of the most highly produced wastewaters throughout the world, and its treatment has emerged as a social issue by the law of London Dumping Convention in South Korea. Food wastewater (FW) is rich in nutrients including nitrogen, phosphorus, calcium, iron, aluminum and total organic carbon, which might be an efficient wastewater feedstock for mixotrophic microalgal cultivation to achieve a reasonably high biomass yield and lipid productivity for energy generation (Shin et al., 2014).
http://dx.doi.org/10.1016/j.biortech.2014.12.053 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
2
M.-K. Ji et al. / Bioresource Technology xxx (2014) xxx–xxx
Several studies have reported the application of municipal and piggery wastewaters with synthetic CO2 for biomass and bioenergy production (Zhou et al., 2011; Wang et al., 2012). FW has not been investigated extensively for microalgal cultivation. The present study investigated the effect of food wastewater on biomass production, lipid productivity and fatty acid composition of Scenedesmus obliquus cultivated in Bold’s Basal Medium (BBM) supplemented with CO2 as flue gas.
Table 1 Physico-chemical characteristics of (a) NaCl, (b) sea water and (c) food wastewater.
2. Methods 2.1. Algal strain, culture conditions and inoculum preparation S. obliquus was isolated from acid mine drainage (AMD) (Donghae, South Korea) and was registered in GenBank under Accession No. HE861884. The microalga was inoculated in 2 L reactor containing 1.7 L Bold’s Basal Medium (BBM) at 10% concentration (Vinoculum/Vmedia) (Bischoff and Bold, 1963). The microalgal cells were incubated under white fluorescent light illumination at a light intensity of 120 lmol photon m 2 s 1 at 25 °C for 10 days. During the incubation, each column was aerated with filter-sterilized air at a flow rate of 0.5 L min 1 to agitate the suspension and to simultaneously supply CO2 (0.03%). Three milliliters (OD680 = 1.5) of microalgae were used as initial inoculums for further experiments.
a b c
Parameter
(a) NaCl
(b) SW
(c) FW
pH T-N (mg L 1) NH4-N (mg L 1) NO3-N (mg L 1) NO2-N (mg L 1) T-P (mg L 1) PO4-P (mg L 1) TOC (mg L 1) Salinity (%)
6.7 NDb ND ND ND ND ND 0.1 ± 0.03 3.7c
7.83 0.27 ± 0.02 ND 0.25 ± 0.01 ND 0.03 ± 0.01 0.04 ± 0.01 20.3 ± 0.12 3.7
6.0a 1385 ± 1.9 1197 ± 1.1 105.4 ± 0.9 45.2 ± 2.2 108 ± 0.6 91.6 ± 0.73 14,898 ± 0.54 3.7c
Metallic ions Al3+ (mg L 1) B3+ (mg L 1) Ca2+ (mg L 1) Cd2+ (mg L 1) Co2+ (mg L 1) Cr6+ (mg L 1) Cu2+ (mg L 1) Fe (total dissolved) (mg L Mg2+ (mg L 1) Mn2+ (mg L 1) Pb2+ (mg L 1) S (mg L 1) Zn2+ (mg L 1)
ND 0.016 ± 0.007 ND ND ND ND ND ND ND ND ND ND ND
0.018 ± 0.002 4.49 ± 0.10 387.4 ± 1.17 ND ND ND ND ND 1118.3 ± 1.95 ND 0.03 ± 0.01 947.95 ± 0.88 ND
316.4 ± 0.13 0.511 ± 0.007 1055 ± 2.01 ND ND ND ND 24.7 ± 0.06 97.01 ± 1.24 1.89 ± 0.05 0.05 ± 0.01 122.07 ± 0.91 1.29 ± 0.08
1
)
After adjusted of pH with NaOH. ND: not detected. After adjusted of salinity.
2.2. Algae cultivation and growth The BBM was diluted with each of food wastewater (FW) and NaCl/sea water (SW) (control), to give five different concentrations (0.5%, 1%, 3%, 5% and 10%), i.e., a total of sixteen solutions including undiluted BBM were prepared, and the volume ratio of the BBM to each of the diluents were as follows: 100:0 (undiluted wastewater), 99.5:0.5 (BBM or FW/NaCl/SW), 99:1 (BBM or FW/NaCl/SW), 97:3 (BBM or FW/NaCl/SW), 95:5 (BBM or FW/NaCl/SW) and 90:10 (BBM or FW/NaCl/SW). SW and NaCl were used to compare the effect of total organic carbon from the FW on the microalgae growth based on concentration of same salinity. The batch experiments were conducted using 500 mL aluminum crimp sealed serum bottles containing 300 mL BBM, inoculated with 1.5% (Vinoculum/Vwastewater) of microalga solution and supplemented with 5.1% flue gas CO2. The bottles were incubated in a shaker incubator at 150 rpm, 27 °C, under white fluorescent light illumination (alternate light/dark periods of 16 h/8 h) at an intensity of 120 lmol photon m 2 s 1 for 6 days. 2.3. Analytical methods FW was collected from a food wastewater treatment plant at Dangjin, South Korea and sea water (SW) from gyeongpodae at Gangneung, South Korea. Wastewaters were immediately filtered using 0.2 lm nylon micro filters to remove the microorganisms and suspended solid particles. The physicochemical properties of the FW were analyzed (Table 1). The analytical methods of TN, NH4-N, TP, anions (i.e., NO2 , NO3 and PO34 ), metal ions (i.e., Ca2+, Mg2+ and Fetotal), total organic carbon (TOC) and pH were described by Ji et al. (2014a). Other analytical methods are detailed in Supplementary Information. 3. Results and discussion 3.1. Growth assessment of S. obliquus FW supported higher microalga growth than the undiluted BBM and NaCl/SW diluted BBM after 6 days of cultivation
(Fig. 1). The highest dry cell weight (0.41 g dwt L 1) was observed for FW 1, which was 2.8 times higher compared to the undiluted BBM due to highest removal of nutrient (38.9 mg L 1 T-N and 12.1 mg L 1 T-P) (Supplementary Fig. 1). The TOC concentrations (TOC = 75–149 mg L 1) in FW 0.5 and FW 1 were favourable for the microalgal growth. Shin et al. (2014) reported that the TOC (83% acetate, 15% lactate and 2% propionate) from FW (Table 1) could be utilized by some algae species for rapid growth under mixotrophic or photoheterotrophic mode in light. Acetic acid (or acetate) is one of the most commonly used organic carbon substrate by the mixotrophic or heterotrophic cultures of microalgae (Hu et al., 2012; Liu et al., 2013). Shin et al. (2014) reported that optimal microalgal growth was observed in 78 mg L 1 of acetate from food wastewater, and acetate above 400 mg L 1 might be toxic for microalgal growth as reported in literatures (Chen and Johns, 1996). The mixotrophic microalgal culture can simultaneously assimilate the inorganic and organic substrates through concurrent respiratory and photosynthesis processes (Bhatnagar et al., 2011). The growth rate of mixotrophic culture is the sum of the photoautotrophic and heterotrophic growth (Liang et al., 2009). The algae cultures exhibit a higher growth under CO2 conditions in the presence of organic carbon substrates (i.e., acetate and glucose) (Liang et al., 2009; Hu et al., 2012). Shin et al. (2014) reported that the highest growth of Scenedesmus bijuga was observed in 20 times diluted anaerobically digested food wastewater. But, the biomass production was similar to that observed in synthetic media (BG-11), which might be due to lack of inorganic carbon for mixotrophic cultivation. Concentrations higher than FW 1 decreased the microalgal growth, which might be due to toxicity of salinity, TOC and aluminium from FW (Liang et al., 2009; Salama et al., 2013). The formation of inorganic precipitate occurred during cultivation in FW 0.5 to FW 10, which might coat the microalgae cell surface and decrease the nutrient uptake at above FW 3 (Ji et al., 2014a). Increase of NaCl and SW concentration in the culture media from 0% to 3% (NaCl/SW) slightly increased the microalga growth. Sodium ion facilitates photosynthesis in microalgae through inorganic nutrients uptake, intracellular pH regulation, and
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
M.-K. Ji et al. / Bioresource Technology xxx (2014) xxx–xxx
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3.2. Effect of FW on surface charge and floc formation of microalgal biomass Zeta potential (ZP) of microalgal cells is often used as an indicator of cell stability. Zeta potential of S. obliquus shifted from a negative ( 5.51 mV) to a positive (+0.41 mV) charge with the FW 1 at day 6, which might be due to the formation of inorganic precipitates (such as struvite, calcium phosphate and calcium carbonate) at an alkaline pH (Ji et al., 2014a). While the zeta potential was maintained from 5.51 to 5.46, 4.39 and 4.42 in undiluted BBM, NaCl 1 and SW 1, respectively. Ions such as Ca2+, PO34 , NH+4 and Mg2+ in the aqueous phase can produce insoluble precipitates such as hydroxyapatite and struvite between pH 8 to 11 (Cohen and Kirchmann, 2004). Supplementary Fig. 2 shows the SEM image and energy dispersive X-ray spectrum of the precipitates collected after 6 days incubation of undiluted BBM and FW 1 with S. obliquus. Carbon, nitrogen and oxygen were the dominant elements of the precipitates detected by EDX spectroscopy. EDS analysis showed 54.04% C, 9.61% N, 24.64% O, 3.61% Na, 5.17% Si and 2.94% P for undiluted BBM (Supplementary Fig. 2a), while the composition of elements changed and/or appeared as 38.48% C, 7.01% N, 33.27% O, 1.00% Na, 2.12% Si, 4.59% P, 1.02% Mg, 1.52% Al, 9.01% Ca and 1.98% Fe for FW 1 (all values in wt%) in the presence of algae (Supplementary Fig. 2b). The EDS spectra of centrate municipal wastewater precipitate also showed the presence of calcium phosphate, calcium carbonate, and/or struvite (Ji et al., 2014a). FW properties promoted the formation of visible microalgae flocs due to attachment of microalgal cells, which settled down as clusters due to formation of bridges between the flocs. Supplementary Fig. 3 shows the microscopic images of the flocs formed with undiluted BBM, NaCl 1, SW 1 and FW 1. Floc density was higher in FW 1 than other conditions, which is supported by the zeta potential data. The enhanced flocculation with FW was presumably due to presence of large number of ionic species in FW, which decreased the double-layer repulsion between the colloidal particles at high ionic strength. Under favorable conditions, the precipitates carry positive surface charges and induce flocculation through charge neutralization and/or sweeping flocculation (Chisti, 2007). 3.3. Total lipid FAME and carbohydrate profile
Fig. 1. Growth of S. obliquus in different dilutions of (a) NaCl, (b) SW and (c) FW.
alkalotolerance (Zhila et al., 2010). Concentrations higher than 5% (NaCl /SW) decreased the microalgal growth, which might be due to toxicity of salinity (Salama et al., 2013). Microalgal growth in different dilutions of NaCl, SW and FW increased the solution pH, and 0.5–1% FW showed the highest pH (10.8 and 11.1) due to high algal growth (Chisti, 2007). In addition, presence of calcium, magnesium, manganese and iron in FW promoted higher microalgal growth compared to undiluted BBM and NaCl/SW (Fig. 1c) (Ji et al., 2014a).
Total lipid content/productivity of microalgal biomass harvested after 6 days was determined (Table 2). The total lipid content of S. obliquus cultivated in BBM in the presence of NaCl, SW and FW ranged between 21.9% to 24.1%, 21.5% to 24.3% and 19.7% to 20.7%, respectively, based on their dry biomass. The lipid content increased slightly with increasing NaCl, SW and FW dose from 0.5% to 10%. Zhila et al. (2010) reported that a growth medium with high salinity increased the microalgae cellular lipid content by improving the formation of triacylglycerols (TAG). The lipid content was lower in FW than with NaCl and SW conditions, which might be due to different trophic culture modes. The lipid content of microalgae under photoautotrophic condition was higher than those under mixotrophic condition (Liang et al., 2009; Cheirsilp and Torpee, 2012; Wang et al., 2012). The aforementioned trend was consistent with the results in this study. The lipid productivity of the microalga cultivated with FW 1 was higher (13.3 mg L day 1) than that of undiluted BBM, NaCl and SW, respectively, due to highest biomass production (Table 2). The fatty acids accumulated in S. obliquus majorly possessed palmitic acid (C16:0), oleic acid (C18:1n9c), linolelaidic acid (C18:2n6t), linoleic acid (C18:2n6c) and c-linolenic acid (C18:3), such that the sum of these four fatty acids accounted for approximately 75–85% of the total fatty acids (Fig. 2). The amount of saturated, mono-unsaturated and poly-unsaturated fatty acids in the
Please cite this article in press as: Ji, M.-K., et al. Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.12.053
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Table 2 Lipid/carbohydrate content and productivity of S. obliquus cultivated in different dilutions of NaCl, SW and FW. Parameters
Undiluted BBM
Lipid (%) Lipid productivity (mg L 1 day 1) Carbohydrate (%) Carbohydrate productivity (mg L 1 day
22.3 5.57 21.0 5.24
1
)
BBM + NaCl%
BBM + SW%
BBM + FW%
0.5
1
3
5
10
0.5
1
3
5
10
0.5
1
3
5
10
22.3 6.11 21.2 5.80
21.9 5.87 22.5 6.01
22.9 6.43 23.2 6.49
22.5 5.92 21.9 5.75
24.1 6.15 22.2 5.65
22.5 5.29 21.1 5.89
21.5 6.00 22.8 6.32
22.7 6.25 23.2 6.38
23.1 6.42 23.3 6.46
24.3 6.21 21.7 5.54
19.7 9.86 24.5 12.0
19.7 13.3 21.8 14.7
20.8 3.57 22.1 3.80
– – – –
– – – –
Fig. 2. Changes in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) of S. obliquus cultivated in different dilutions of NaCl, SW and FW.
microalga grown with different NaCl, SW and FW dilutions accounted for 25–28%/18–21%/52–55%, 25–28%/17–20%/52–55% and 26–33%/21–26%/43–53% of the total fatty acids, respectively. Palmitic acid and oleic acid contents increased with increasing FW dose. In particular, relatively high ratios of C16:0 (palmitic acid) and C18:1 (oleic acid) fatty acids were observed under mixotrophic growth, which are desirable for the production of biodiesel (Liu et al., 2011). The fatty acids of microalga cultivated in FW 3 showed a high concentration of palmitic (27%) acid and oleic acid (16%). Ji et al. (2014b) reported that oleic acid content was increased (4– 19%) under mixotrophic culture condition using monosodium glutamate wastewater (MSGW) for 16 days compared to BG11 media. Also, Shin et al. (2014) reported the oleic acid content was increased (13%) in 20 diluted digested food wastewater effluent compared to synthetic media (BG11). The fatty acid methyl esters (FAMEs) of S. obliquus cultivated in FW possessed more saturated (16:0) and mono-unsaturated fats (C18:1n9c), which are more suitable for use in hot and cold weather environments due to their typically high oxidative stability and lower melting point. Carbohydrates serve two main purposes for algae; they act as structural components in the cell walls and as storage components inside the cell. The most frequently reported cultivation factors that affect the carbohydrate content are nutrient starvation/limitation, salt stress, light intensity and temperature (Ji et al., 2014b). In addition, the metabolic mode such as autotrophic, heterotrophic or mixotrophic affects the biomass composition (Liang et al., 2009). The total carbohydrate content of S. obliquus after 6 days of cultivation in BBM with NaCl, SW and FW ranged from 21.2% to 23.2%, 21.1% to 23.2% and 21.1% to 24.0%, respectively, based on their dry biomass (Table 2). The carbohydrate content was slightly high in FW 0.5%. The carbohydrate productivity of the microalga cultivated with FW 1% was higher (14.7 mg L day 1) than that of undiluted BBM, NaCl and SW, respectively, due to higher biomass production (Table 2).
4. Conclusions Application of food wastewater as a nutrient source (organic carbon and trace elements) improved the biomass, lipid productivity and nutrient removal efficiency of S. obliquus cultivated in BBM. The microalga grown with 0.5–1% FW was promising for the generation of high-efficiency biodiesel (19–23% palmitic acid and 10% oleic acid). Abiotic precipitation also supported the removal of nutrients and autoflocculation of algal cells. Utilization of S. obliquus for the generation of biofuel feedstock with simultaneous reuse of FW in the presence of flue gas CO2 can be a cost-effective and environmentally sustainable strategy. Acknowledgement The project was financially supported by the Korea Institute of Science and Technology (Gangneung) (Grant 2Z04230). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 12.053. References Bhatnagar, A., Chinnasamy, S., Singh, M., Das, K.C., 2011. Renewable biomass production by mixotrophic algae in the presence of various carbon sources and wastewaters. Appl. Energy 88, 3425–3431. Bischoff, H.W., Bold, H.C., 1963. Phycological Studies IV. Some Soil Algae from Enchanted Rock and Related Algal Species. University of Texas Publication 6318, pp. 1–95. Cheirsilp, B., Torpee, S., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol. 110, 510–516.
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