小球藻与固氮菌Mesorhizobium sp.共培养对小球藻生长和油脂积累的促进效果
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摘要
微藻油脂不仅可以作为功能油脂,同时也是生产生物柴油的重要原料之一。为解决微藻生长与油脂积累之间的矛盾,利用藻菌共培养技术在缺氮条件下将无菌小球藻与细菌以不同初始比例进行共培养,通过测定藻细胞生物量、油脂含量和脂肪酸比例等来研究藻菌共培养对小球藻生长和油脂积累的影响。结果表明,在小球藻与固氮菌B2. 3 70∶1(V/V)共培养体系中,小球藻的生物量和油脂含量较同样条件下单独培养小球藻有了显著提高。其生物量最高可达1. 68g/L、总脂含量为45. 2%、总脂产率为75. 94 mg/(L·d)、中性脂含量为23. 0%及中性脂产率为38. 65mg/(L·d),其生物量和油脂含量分别较单独小球藻培养时提高了66. 3%和47. 7%。同时细菌的加入显著提高了藻细胞内C18∶1脂肪酸的比例。结论表明,通过藻菌共培养技术能够有效提高微藻生物油脂的质量和产量,具有较好的实际利用价值。
Microalgal oil is an important functional oil,but also one of the major sources for bio-diesel production. In order to solve the contradiction of algae growth and lipid accumulation,the Chlorella vulgaris were co-cultured with azotobacter at different initial ratio under nitrogen source deficiency conditions,and the impact of consortium system growth,lipid accumulation based on the dry weight,lipid content,and fatty acids composition in the incubation period were evaluated. The results showed that the biomass and lipid content of algae were significantly higher than that of the pure culture of axenic C. vulgaris when C. vulgaris and B2. 3 strains were inoculated at the ratio of 70∶ 1. The biomass,lipid content,lipid productivity,neutral lipid content and neutral lipid productivity of algae in the co-cultures were 1. 68 g/L,45. 2%,75. 94 mg/(L·d),23. 0% and 38. 65 mg/(L·d),respectively. And the biomass concentration and lipid content in the co-culture system were 66. 3%and 47. 7% higher than that of the axenic pure algal cultures,respectively. Furthermore,the consortium system significantly increased the proportion of C18 ∶ 1 fatty acids. It was indicated that the co-cultivation of algaebacteria system can effectively contribute to the quality and quantity of microalgal bio-oil,and has great potential for production of bio-diesel.
Microalgal oil is an important functional oil,but also one of the major sources for bio-diesel production. In order to solve the contradiction of algae growth and lipid accumulation,the Chlorella vulgaris were co-cultured with azotobacter at different initial ratio under nitrogen source deficiency conditions,and the impact of consortium system growth,lipid accumulation based on the dry weight,lipid content,and fatty acids composition in the incubation period were evaluated. The results showed that the biomass and lipid content of algae were significantly higher than that of the pure culture of axenic C. vulgaris when C. vulgaris and B2. 3 strains were inoculated at the ratio of 70∶ 1. The biomass,lipid content,lipid productivity,neutral lipid content and neutral lipid productivity of algae in the co-cultures were 1. 68 g/L,45. 2%,75. 94 mg/(L·d),23. 0% and 38. 65 mg/(L·d),respectively. And the biomass concentration and lipid content in the co-culture system were 66. 3%and 47. 7% higher than that of the axenic pure algal cultures,respectively. Furthermore,the consortium system significantly increased the proportion of C18 ∶ 1 fatty acids. It was indicated that the co-cultivation of algaebacteria system can effectively contribute to the quality and quantity of microalgal bio-oil,and has great potential for production of bio-diesel.
引文
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[24]Courchesne N M D,Parisien A,Wang B,et al.Enhancement of lipid production using biochemical,genetic and transcription factor engineering approaches.Journal of Biotechnology,2009,141(1-2):31-41.
[25]Casadevall E,Dif D,Largeau C,et al.Studies on batch and continuous cultures of Botryococcus braunii:hydrocarbon production in relation to physiological state,cell ultrastructure,and phosphate nutrition.Biotechnology and Bioengineering,1985,27(3):286-295.
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[27]Yamaberi K,Takagi M,Yoshida T.Nitrogen depletion for intracellular triglyceride accumulation to enhance liquefaction yield of marine microalgal cells into a fuel oil.Journal of Marine Biotechnology,1998,6:44-48.
[28]Tang D,Han W,Li P,et al.CO2biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2levels.Bioresource Technology,2011,102(3):3071-3076.
[29]Wang R,Xue S,Zhang D,et al.construction and characteristics of artificial consortia of Scenedesmus obliquus-bacteria for S.obliquus growth and lipid production.Algal Research,2015,12:436-445.
[30]Gui M M,Lee K T,Bhatia S.feasibility of edible oil vs.nonedible oil vs.waste edible oil as biodiesel feedstock.Energy,2008,33(11):1646-1653.
[31]Knothe G.will biodiesel derived from algal oils live up to its promise?A fuel property assessment.Lipid Technology,2011,23(11):247-249.
[32]Munoz R,Kollner C,Guieysse B.biofilm photobioreactors for the treatment of industrial wastewaters.Journal of Hazardous Materials,2009,161(1):29-34.
[33]Gardes A,Iversen M H,Grossart H P,et al.diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii.International Society for Microbial Ecology,2011,5(3):436-445.
[2]Wang S,Wang X,Tao H,et al.Heterotrophic culture of Chlorella pyrenoidosa using sucrose as the sole carbon source by co-culture with immobilized yeast.Bioresource Technology,2018,249:425-430.
[3]宋东辉,侯李君,施定基.生物柴油原料资源高油脂微藻的开发利用.生物工程学报,2008,24(3):341-348.Song D H,Hou L J,Shi D J.Exploitation and utilization of rich lipids-microalgae,as new lipids feedstock for biodiesel production-a review.Chinese Journal of Biotechnology,2008,24(3):341-348.
[4]Brennan L,Owende P.Biofuels from microalgae-A review of technologies for production,processing,and extractions of biofuels and co-products.Renewable and Sustainable Energy Reviews,2010,14(2):557-577.
[5]Georgianna D R,Mayfield S P.Exploiting diversity and synthetic biology for the production of algal biofuels.Nature,2012,488(7411):329-335.
[6]Sayre R.Microalgae:the potential for carbon capture.Bioscience,2010,60(9):722-727.
[7]Shu C,Tsai C,Chen K,et al.Enhancing high quality oil accumulation and carbon dioxide fixation by a mixed culture of Chlorella sp.and Saccharomyces cerevisiae.Journal of The Taiwan Institute of Chemical Engineers,2013,44(6):936-942.
[8]姜进举,苗凤萍,冯大伟,等.微藻生物柴油技术的研究现状及展望.中国生物工程杂志,2010,30(2):134-140.Jiang J J,Miao F P,Feng D W,et al.Research situation and prospect of microalgae biodiesel.China Biotechnology,2010,30(2):134-140.
[9]Dash A,Banerjee R.Enhanced biodiesel production through phyco-myco co-cultivation of Chlorella minutissima and Aspergillus awamori:an integrated approach.Bioresource Technology,2017,238:502-509.
[10]刘金丽,王俊峰,刘天中,等.缺氮条件对栅藻油脂积累与光合作用的影响.海洋科学,2013,37(7):13-19.Liu J L,Wang J F,Liu T Z,et al.The effects of nitrogen starvation on lipid accumulation and photosynthesis of Scenedesmus dimorphus.Marine Sciences,2013,37(7):13-19.
[11]左正三,孙小曼,任路静,等.微藻生产油脂培养新技术.中国生物工程杂志,2018,38(7):102-109.Zuo Z S,Sun X M,Ren L J,et al.Improvement of lipid accumulation in microalgae by novel cultivation strategies.China Biotechnology,2018,38(7):102-109.
[12]刘天中,张维,王俊峰,等.微藻规模培养技术研究进展.生命科学,2014,26(5):509-522.Liu T Z,Zhang W,Wang J F,et al.A review of mass cultivation technology for microalgae.Chinese Bulletin of Life Sciences,2014,26(5):509-522.
[13]Wang R,Tian Y,Xue S,et al.Enhanced microalgal biomass and lipid production via co‐culture of Scenedesmus obliquus and Candida tropicalis in an autotrophic system.Journal of Chemical Technology and Biotechnology,2016,91(5):1387-1396.
[14]Ramanan R,Kim B H,Cho D H,et al.Algae-bacteria interactions:evolution,ecology and emerging applications.Biotechnology Advances,2016,34(1):14-29.
[15]Yang L,Tan X,Li D,et al.Nutrients removal and lipids production by Chlorella pyrenoidosa cultivation using anaerobic digested starch wastewater and alcohol wastewater.Bioresource Technology,2015,181:54-61.
[16]Guan S,Chen W,Wang E,et al.Mesorhizobium caraganae sp.nov,a novel rhizobial species nodulated with Caragana spp.in China.International Journal of Systematic and Evolutionary Microbiology,2008,58(11):2646-2653.
[17]Porra R J,Thompson W A,Kriedemann P E.Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents:verification of the concentration of chlorophyll standards by atomic absorption spectroscopy.Biochimicaet Biophysica Acta(BBA)-Bioenergetics,1989,975(3):384-394.
[18]Bligh E G,Dyer W J.A rapid method of total lipid extraction and purification.Canadian Journal of Biochemistry and Physiology,1959,37(8):911-917.
[19]宋琪,李泉,邴欣,等.尼罗红-荧光光谱法测定亚心形扁藻油脂含量.中国油脂,2016,41(10):98-101.Song Q,Li Q,Bing X,et al.Determination of lipid content in Platymonas subcordiformis by Nile red-fluorescence spectrometry.China Oils and Fats,2016,41(10):98-101.
[20]Klock J H,Wieland A,Seifert R,et al.Extracellular polymeric substances(EPS)from cyanobacterial mats:characterisation and isolation method optimisation.Marine Biology,2007,152(5):1077-1085.
[21]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding.Analytical Biochemistry,1976,72(1-2):248-254.
[22]Dubois M,Gilles K A,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances.Analytical Chemistry,1956,28(3):350-356.
[23]Xu L,Cheng X,Wang Q.Enhanced lipid production in Chlamydomonas reinhardtii by co-culturing with Azotobacter chroococcum.Frontiers in Plant Science,2018,9:741-753.
[24]Courchesne N M D,Parisien A,Wang B,et al.Enhancement of lipid production using biochemical,genetic and transcription factor engineering approaches.Journal of Biotechnology,2009,141(1-2):31-41.
[25]Casadevall E,Dif D,Largeau C,et al.Studies on batch and continuous cultures of Botryococcus braunii:hydrocarbon production in relation to physiological state,cell ultrastructure,and phosphate nutrition.Biotechnology and Bioengineering,1985,27(3):286-295.
[26]Papanikolaou S,Komaitis M,Aggelis G.single cell oil(SCO)production by Mortierella isabellina grown on high-sugar content media.Bioresource Technology,2004,95(3):287-291.
[27]Yamaberi K,Takagi M,Yoshida T.Nitrogen depletion for intracellular triglyceride accumulation to enhance liquefaction yield of marine microalgal cells into a fuel oil.Journal of Marine Biotechnology,1998,6:44-48.
[28]Tang D,Han W,Li P,et al.CO2biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2levels.Bioresource Technology,2011,102(3):3071-3076.
[29]Wang R,Xue S,Zhang D,et al.construction and characteristics of artificial consortia of Scenedesmus obliquus-bacteria for S.obliquus growth and lipid production.Algal Research,2015,12:436-445.
[30]Gui M M,Lee K T,Bhatia S.feasibility of edible oil vs.nonedible oil vs.waste edible oil as biodiesel feedstock.Energy,2008,33(11):1646-1653.
[31]Knothe G.will biodiesel derived from algal oils live up to its promise?A fuel property assessment.Lipid Technology,2011,23(11):247-249.
[32]Munoz R,Kollner C,Guieysse B.biofilm photobioreactors for the treatment of industrial wastewaters.Journal of Hazardous Materials,2009,161(1):29-34.
[33]Gardes A,Iversen M H,Grossart H P,et al.diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii.International Society for Microbial Ecology,2011,5(3):436-445.