Abstract：Production of biofuel and mass cultivation of microalgae mainly depends on the nature of microalgae strain. The aim of this study was to screen microalgae strains for swine manure wastewater treatment and lipid production from natural water area. Through multi-step screening, three experimental protocols including streak plate method, single-cell isolated offspring and capillary theory of separation, were used for separating and purifying the algal strains from 51 sites. As a result, 118 microalgaes were obtained, among which 71 facultative heterotrophic microalgae strains were screened including 33 strains which were proved to be tolerant to swine manure wastewater, among which 17 strains were adapted well in swine manure wastewater without further acclimation. Some algae strains were preliminarily identified by morphological observation as Chlorella sp. and Scenedesmus sp.. All candidate strains were grown in swine manure wastewater for 7 days to evaluate their specific growth rate and lipid content. The 13-6, 19-4, 20-6 and 34-2 strains showed faster specific growth rates than other candidate strains during 7 days with the average specific growth rates of 0.147, 0.162, 0.177 and 0.154 d-1, respectively. The total lipid content of the candidate algae which grew in swine manure wastewater ranged from 7.4% to 29.2%. Among them, the 19-4, 24-1 and 34-2 strains showed higher lipid content for 19.7%, 22.9% and 28.8%, respectively, which suggested that these candidates could accumulate higher lipid content than other strains. Two strains (C. sorokinlana 19-4 (GenBank No. KU948990) and Chlorella sp. 34-2 (GenBank No. KU948991)) were chosen for other studies because of their ability to adapt to swine manure wastewater for high growth rates (0.162 and 0.154 d-1) and high lipid content (19.7% and 28.8%). The maximal biomass concentrations of C. sorokinlana 19-4 and Chlorella sp. 34-2 in swine manure wastewater in 30 L photobioreactor reached 0.78 and 1.12 g/L, and algal growth rate reached 0.153 and 0.149 d-1, and lipid content reached 18.73% and 29.27%, respectively. Total nitrogen and total phosphorus were reduced during the cultivation period of the inoculation treatments, whereas no obvious variation was observed in the corresponding negative control. Total nitrogen was reduced 60.24% from 90.81 to 36.11 mg/L for Chlorella sp. 34-2, and 70.56% from 90.81 to 26.73 mg/L for C. sorokinlana 19-4. Total phosphorus was reduced 85.07% from 4.02 to 0.60 mg/L for Chlorella sp. 34-2, 90.98% from 4.02 to 0.36 for C. sorokinlana 19-4 during the 7 days. The production of biodiesel made from microalgae mainly influence by the fatty acid component. The fatty acid compositions of Chlorella sp. 34-2 cultured in swine manure wastewater and BG11 medium were investigated. It was found that C16:0, C18:2n6c and C18:3n3 were the major fatty acids, which accounted for more than 83.97% of the total fatty acids. Their fatty acid composition met the requirements of standards of the raw materials for biodiesel production. This species has great potential for use in the treatment of swine manure wastewater and biodiesel production.
Boelee N C, Temmink H, Janssen M, et al. 2011. Nitrogen and phosphorus removal from municipal wastewater effluent using microalgal biofilms[J].Water Resesrch, 45: 5925-5933. de Godos I, Blanco S, Garcia-Encina P A, et al. 2009. Long-termoperation of high rate algal ponds for the bioremediation of piggery wastewaters at high loading rates[J]. Bioresource Technology, 100: 4332-4339. Farooq W, Lee Y C, Ryu B G, et al. 2013. Two-stage cultivation of two Chlorella sp. strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity[J]. Bioresource Technology, 132: 230-238. Grzebyk D, Sako Y, Berland B. 1998. Phylogenetic analysis of nine species of Prorocentrum (Dinophyceae) inferred from 18S ribosomal DNA sequences, morphological comparisons, and description of Prorocentrum panamensis sp. nov[J]. Journal of Phycology, 34: 1055-1068. Hu B, Min M, Zhou W G, et al. 2012. Enhanced mixotrophic growth of microalga Chlorella sp. on pretreated swine manure for simultaneous biofuel feedstock production and nutrient removal[J]. Bioresource Technology, 126: 71-79 Indarti E, Majid M I A, Hashim R, et al. 2005. Direct FAME synthesis for rapid total lipid analysis from fish oil and cod liver oil[J]. Journal of Food Composition and Analysis, 18: 161-170. Ji Y, Hu W, Li X, et al. 2013. Mixotrophic growth and biochemical analysis of Chlorella vulgaris cultivated with diluted monosodium glutamate waste water[J]. Bioresource Technology, 152: 471-476. Ji Y, Hu W, Li X, et al. 2014. Mixotrophic growth and biochemical analysis of Chlorella vulgaris cultivated with diluted monosodium glutamatewastewater[J]. Bioresource Technology, 152: 471-476. Liu C H, Chang C Y, Liao Q, et al. 2013. Photoheterotrophic growth of Chlorella vulgaris ESP6 on organic acids from dark hydrogen fermentation effluents[J]. Bioresource Technology, 145: 331-336. Logan C, Ronald S. 2011. Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts[J]. Biotechnology Advances, 29: 686-702. Manuel S A, Víctor M L-P, Erasmo C, et al. 2013. Green microalga Scenedesmus acutus grown on municipal wastewater to couple nutrient removal with lipid accumulation for biodiesel production[J]. Bioresource Technology, 146: 744-748. Pienkos P T and Darzins A. 2009. The promise and chanllenges of microalgal-derived biofuels[J]. Biofuels Bioproducts and Biorefining, 3: 431-440. Rippka R, Deruelles J, Waterbury J B, et al. 1979. Genetic assignments, strain histories and properties of pure cultures of cyanobacteria[J]. Journal of General Microbiology, 111: 1-61. Scott S A, Davey M P, Dennis J S, et al. 2010. Biodiesel from algae: challenges and prospects. Current Opinion in Biotechnology, 21(3): 277-286. Singh A, Nigam P S, Murphy J D. 2011. Mechanism and challenges in commercialisation of algal biofuels[J]. Bioresource Technology, 102(1): 26-34. Tam N F Y, Wong Y S. 1990. The comparison of growth and nutrient removal efficiency of Chlorella pyrenoidosain settled and activated sewages[J]. Environmental Pollution, 65: 93-108. van Beilen J B. 2010. Why microalgal biofuels won’t save the internal combustion machine[J]. Biofuels Bioproducts&Biorefining, 4: 41-52. Volker A R H, Tan D, et al. 2013. A novel species in the genus Heveochlorella (Trebouxiophyceae, Chlorophyta) witnesses the evolution from an epiphytic into an endophytic lifestyle in tree-dwelling green algae[J]. European Journal of Phycology 48: 200-209. Wang H, Xiong H, Hui Z, et al. 2012. Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids[J]. Bioresource Technology, 104: 215-220. Wang L, Li Y C, Chen P, et al. 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp[J]. Bioresource Technology, 101: 2623-2628. Wang L, Li Y, Chen P, et al. 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp[J]. Bioresource Technology, 101: 2623-2628. Wijffels R H and Barbosa M J. 2010. An outlook on microalgal biofuels[J]. Science, 329(5993): 796-799. Woertz I, Feffer A, Lundquist T, et al. 2010. Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel feedstock[J]. Journal of Environmental Engineering, 135(11): 1115-1122.
