利用蔗渣半纤维素水解液产油酵母的筛选、鉴定和发酵实验
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摘要
油脂既是构成和维持生命活动的基本物质之一，也是重要的工业原料，具有广泛的应用价值。目前油脂资源主要来源于动植物，随着人们对微生物油脂的研究不断深入，利用微生物发酵法获得油脂，不但可以缓解动植物油脂资源紧缺的局面，而且产油微生物可以进行废弃物的资源化再利用，为油脂资源的获得提供了一条新的途径。
本论文以此为立题依据。木糖是自然界中含量最大的五碳糖。采集广西自治区十种不同区域和生态类型的土壤和植物体作为样品，经过甘油富集培养、木糖筛选平板分离，筛选出40株酵母，再经过苏丹黑染色法初筛得到4株脂肪粒密度较高的菌株，经过摇瓶复筛，得到两株油脂得率系数较高的酵母菌H2-1和J2-2，分别为14．56和10．73。根据常规生理生化特征和26S rDNA序列分析结果，确认H2-1为小红酵母(Rhodotorula minuta)，J2-2为粘红酵母(Rhodotorula glutinis)。
通过摇瓶发酵，考察了不同浓度金属离子(Ca~(2+)、Mn~(2+)、Cu~(2+)、Co~(2+))对菌株J2-2发酵产油的影响。研究结果表明：培养基中添加适量的Mn~(2+)、Cu~(2+)有利于细胞积累油脂，但Ca~(2+)、Co~(2+)不利于油脂的积累。
对J2-2菌株发酵产油培养条件进行研究，发现最佳产油条件为：20％接种量，培养时间6d，26℃，摇床转速200r／min，起始pH10．0。在此发酵条件下，该菌的油脂产量为3．85 g／L，是优化前的1．16倍；生物量为13 g／L，是优化前的1．04倍。
初步研究了J2-2利用蔗渣半纤维素水解液发酵产油的情况。J2-2利用蔗渣半纤维素水解液和利用纯木糖发酵产油的油脂得率系数分别为10．23，10．73，两者的产物转化率比较接近。菌株J2-2利用蔗渣半纤维素水解液发酵油脂的脂肪酸组成和植物油类似。
Lipid is one of the basic material and life-sustaining activities, as well as important industrial raw materials, a broad application value. Currently fats resources mainly come from animal and plant, with about microbial lipid in-depth study, using microbial fermentation fats, not only will it ease the animal and plant fats scarce resources situation, and oil could be abandoned microbial resources of the use of resources for fats provided a new way.
This paper is based on these points. D-xylose is one of the most abundant pentose sugars in nature. To isolate oleaginous yeasts that can utilize xylose from diverse soil samples, we collected soils from a litchi orchard, a longan orchard, a carambola orchard, and woods, and isolated yeasts by the glycerol enrichment and measured their xylose assimilating capacity. A subset of these isolates was grown in nitrogen-limited media and then screened for their lipid contents by the Sudan Black B staining. Four colonies were selected. Then by fermentation xylose in shake flask, two oleaginous yeasts named H2-1 and J2-2 which had high performance in utilizing xylose were selected from four oleaginous yeasts. The lipid coefficient of two yeasts reached 14.56 and 10.73, respectively. H2-1 was identified as Rhodotorula minuta and J2-2 was identified as Rhodotorula glutinis based on physiological and biochemical characteristics and 26S rDNA sequence analysis.
The effects of metal ions (Ca~(2+)、Mn~(2+)、Cu~(2+)、Co~(2+)) on lipid accumulation in the cells of strain J2-2 were investigated by fermenting in shaking flask. It revealed that certain quantity of Mn~(2+) and Cu~(2+) benefited lipid production, but Ca~(2+) and Co~(2+) inhibited accumulation of the lipid.
With different culture conditions investigated in shake flasks, the results of experiments indicated that strain J2-2 was cultivated at 26℃for 144 hours with 200r/min at initial pH 10.0, the lipid production reached 3.85 g/L just 1.16 times as before, and the biomass reached 13 g/L just 1.04 times as before.
The strain J2-2 producing lipids in utilizing sugar cane bagasse hemicellulose hydrolysates was preliminarily studied by fermenting in shaking flask. The lipid coefficients of J2-2 in utilizing sugar cane bagasse hemicellulose hydrolysates and xylose reached 10.23 and 10.73, respectively, showed similar transformation rate. Fatty acids analysis of microbial oil from sugar cane bagasse hemicellulose hydrolysates had fatty acid composition and proportion similar to normal vegetable oils.
本论文以此为立题依据。木糖是自然界中含量最大的五碳糖。采集广西自治区十种不同区域和生态类型的土壤和植物体作为样品，经过甘油富集培养、木糖筛选平板分离，筛选出40株酵母，再经过苏丹黑染色法初筛得到4株脂肪粒密度较高的菌株，经过摇瓶复筛，得到两株油脂得率系数较高的酵母菌H2-1和J2-2，分别为14．56和10．73。根据常规生理生化特征和26S rDNA序列分析结果，确认H2-1为小红酵母(Rhodotorula minuta)，J2-2为粘红酵母(Rhodotorula glutinis)。
通过摇瓶发酵，考察了不同浓度金属离子(Ca~(2+)、Mn~(2+)、Cu~(2+)、Co~(2+))对菌株J2-2发酵产油的影响。研究结果表明：培养基中添加适量的Mn~(2+)、Cu~(2+)有利于细胞积累油脂，但Ca~(2+)、Co~(2+)不利于油脂的积累。
对J2-2菌株发酵产油培养条件进行研究，发现最佳产油条件为：20％接种量，培养时间6d，26℃，摇床转速200r／min，起始pH10．0。在此发酵条件下，该菌的油脂产量为3．85 g／L，是优化前的1．16倍；生物量为13 g／L，是优化前的1．04倍。
初步研究了J2-2利用蔗渣半纤维素水解液发酵产油的情况。J2-2利用蔗渣半纤维素水解液和利用纯木糖发酵产油的油脂得率系数分别为10．23，10．73，两者的产物转化率比较接近。菌株J2-2利用蔗渣半纤维素水解液发酵油脂的脂肪酸组成和植物油类似。
Lipid is one of the basic material and life-sustaining activities, as well as important industrial raw materials, a broad application value. Currently fats resources mainly come from animal and plant, with about microbial lipid in-depth study, using microbial fermentation fats, not only will it ease the animal and plant fats scarce resources situation, and oil could be abandoned microbial resources of the use of resources for fats provided a new way.
This paper is based on these points. D-xylose is one of the most abundant pentose sugars in nature. To isolate oleaginous yeasts that can utilize xylose from diverse soil samples, we collected soils from a litchi orchard, a longan orchard, a carambola orchard, and woods, and isolated yeasts by the glycerol enrichment and measured their xylose assimilating capacity. A subset of these isolates was grown in nitrogen-limited media and then screened for their lipid contents by the Sudan Black B staining. Four colonies were selected. Then by fermentation xylose in shake flask, two oleaginous yeasts named H2-1 and J2-2 which had high performance in utilizing xylose were selected from four oleaginous yeasts. The lipid coefficient of two yeasts reached 14.56 and 10.73, respectively. H2-1 was identified as Rhodotorula minuta and J2-2 was identified as Rhodotorula glutinis based on physiological and biochemical characteristics and 26S rDNA sequence analysis.
The effects of metal ions (Ca~(2+)、Mn~(2+)、Cu~(2+)、Co~(2+)) on lipid accumulation in the cells of strain J2-2 were investigated by fermenting in shaking flask. It revealed that certain quantity of Mn~(2+) and Cu~(2+) benefited lipid production, but Ca~(2+) and Co~(2+) inhibited accumulation of the lipid.
With different culture conditions investigated in shake flasks, the results of experiments indicated that strain J2-2 was cultivated at 26℃for 144 hours with 200r/min at initial pH 10.0, the lipid production reached 3.85 g/L just 1.16 times as before, and the biomass reached 13 g/L just 1.04 times as before.
The strain J2-2 producing lipids in utilizing sugar cane bagasse hemicellulose hydrolysates was preliminarily studied by fermenting in shaking flask. The lipid coefficients of J2-2 in utilizing sugar cane bagasse hemicellulose hydrolysates and xylose reached 10.23 and 10.73, respectively, showed similar transformation rate. Fatty acids analysis of microbial oil from sugar cane bagasse hemicellulose hydrolysates had fatty acid composition and proportion similar to normal vegetable oils.
引文
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[12]．刘波，孙艳，李永红，等．产油微生物油脂生物合成与代谢调控研究进展．微生物学报， 2005，1：153-156
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[21].dos Santos MM, Raghevendran V , Kotter P , et al . Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments. Metab Eng, 2004, 6 : 352-363.
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[25].Oelkers P , Cromley D , Padamsee M, et al . The DAG1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem, 2002 ,277 : 8877-8881.
[26].Sandager L , Gustavsson M, Stahl U , et al . Storage lipid synthesis is non-essential in yeast. J Biol Chem , 2002 ,277 : 6478-6482.
[27].Sorger D , Daum G Synthesis of triacylglycerols by the acyl coenzyme A: diacyl-glycerol acyltransferase Dgalp in lipid particles of the yeast Saccharomyces cerevisia. J Bacteriol, 2002,184:519-524.
[28].Gangar A , Karande A , Rajasekharan R. Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast . J Biol Chem , 2001 , 276 : 10290-0298.
[29].Raychaudhuri S , Reddy M, Rajkumar N R , et al. Cytosolic iron superoxide dismutase is a part of the triacylglycerol biosynthetic complex in oleaginous yeast. Biochem J , 2003 , 372 : 587-594.
[30].Gangar A , Raychaudhuri S , Rajasekharan R. Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast. Biochem J, 2002 , 365 : 577-589.
[31].http://www.bioconversion.dicp.ac.cn/WEB/03%20yjfx.htm
[32].Evans C T , Scragg A H , Ratledge C. Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by adenine nucleotides. Eur J Biochem , 1983 ,132 : 609-615.
[33].Botham P A , Ratledge C. A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous microorganisms. J Gen Microbiol, 1979 ,114 : 361-375.
[34].Palmieri L , Palmieri F , Runswick M J , et al. Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein. FEBS Lett, 1996 , 399 : 299-302.
[35].Wynn J P , Hamid A A, Li Y, et al. Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiology, 2001 ,147 : 2857-2864.
[36].Papanikolaou S , Sarantou S , Komaitis M, et al . Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media. J Appl Microbiol ,2004 ,97 : 867-875.
[37]．石天虹，刘雪兰，刘辉，等．微生物发酵的影响因素及其控制．家禽科学，2005，2：4 5-4 8．
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[2]．郭平银，肖爱军，郑现和，等．能源植物的研究现状与发展前景．山东农业科学， 2007,4：126-129
[3]．金青哲，刘元法，岳琨．生物柴油发展现状和趋势．粮油加工与食品机械,2006,1：57-60
[4].RATL EDGE C. Lipid biotechnology , a wonderland for the microbial physiologist [J ] . J Am Oil Chen Soc , 1987 ,64 :1647-1656.
[5]．李魁等．利用食品工业废弃物生产真菌油脂．食品工业科技，1997(6)：65-69．
[6].Sorger D , Daum G Triacylglycerol biosynthesis in yeast . Appl Microbiol Biotechnol , 2003 ,61 : 289-2991
[7].Ratledge C, Wynn J P. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv App 1 Microbiol, 2002, 51:1-51
[8].S. Papanilolaou, I. Chevalou, M. Komaitis, et al. Single cell oil-production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures. Appl Microbiol Biotechnol, 2002, 58: 308-312
[9]．刘淑君，杨文博，施安辉．酵母菌HL的批次发酵及发酵动力学初探．微生物学通报，2000， 27(1)：8-11．
[10]．施安辉，谷劲松，刘淑君，等．高产油脂酵母菌株的选育、发酵条件的优化及油脂成 分分析冲国酿造，1997,4：10-14
[11]．李永红，刘波，孙艳，等．广谱碳源产油酵母菌的筛选．中国生物工程杂志，2005，25 (12)：39-44．
[12]．刘波，孙艳，李永红，等．产油微生物油脂生物合成与代谢调控研究进展．微生物学报， 2005，1：153-156
[13].Tyson K S, Bozell J, Wallace R, et al. Biomass oil analysis:research needs and recommendations. NREL /TP - 510 -34796. June 2004
[14].Papanikolaou S , Komaitis M, Aggelis G Single cell oil ( SCO) production by Mortierella isabellin grown on high-sugar content media. Bioresource Technol, 2004 , 95 : 287-291.
[15]．颜治，陈晶．徽生物油脂及其开发利用研究进展．粮食与油脂，2003，7：13-15．
[16]．薛照辉，吴谋成，尉万聪，等．生物技术在油脂工业中的应用．食品技术，2002，3：8-10．
[17].Fall R, Phelps P, Spindler D. Bioconversion of xylan to triglycerides by oil-rich yeasts. Appl Environ Microbiol, 1984,47: 1130-1134
[18].Tyson K S, Bozell J, Wallace R. Biomass oil analysis: Research needs and recommendations. NREL Technical Report, NREL/TP510-34796,2004
[19]. Adams IP , Dack S , Dickinson F M, et al. The distinctiveness of ATP xitrate lyase from Aspergillus nidulans . Biochim Biophys Acta ,2002 ,1597 : 36-41.
[20].Ratledge C. Regulation of lipid accumulation in oleaginous microorganisms. Biochem Soc Trans, 2002, 30 : 1047-1050.
[21].dos Santos MM, Raghevendran V , Kotter P , et al . Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments. Metab Eng, 2004, 6 : 352-363.
[22]．董文宾，梁西爱，代春吉，等．微生物油脂及其新的应用研究．粮油加工与食品机械，2006， 3：54-55
[23].Gill C O, Hall M J, Ratledge C. Lipid accumulation in an oleaginous yeast(Candida 107)growing on glucose in single-stage continuous culture. Appl Environ Microbiol, 1977,33:231-239
[24].Sorger D , Daum G. Triacylglycerol biosynthesis in yeast . Appl Microbiol Biotechnol , 2003 , 61 : 289-299.
[25].Oelkers P , Cromley D , Padamsee M, et al . The DAG1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem, 2002 ,277 : 8877-8881.
[26].Sandager L , Gustavsson M, Stahl U , et al . Storage lipid synthesis is non-essential in yeast. J Biol Chem , 2002 ,277 : 6478-6482.
[27].Sorger D , Daum G Synthesis of triacylglycerols by the acyl coenzyme A: diacyl-glycerol acyltransferase Dgalp in lipid particles of the yeast Saccharomyces cerevisia. J Bacteriol, 2002,184:519-524.
[28].Gangar A , Karande A , Rajasekharan R. Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast . J Biol Chem , 2001 , 276 : 10290-0298.
[29].Raychaudhuri S , Reddy M, Rajkumar N R , et al. Cytosolic iron superoxide dismutase is a part of the triacylglycerol biosynthetic complex in oleaginous yeast. Biochem J , 2003 , 372 : 587-594.
[30].Gangar A , Raychaudhuri S , Rajasekharan R. Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast. Biochem J, 2002 , 365 : 577-589.
[31].http://www.bioconversion.dicp.ac.cn/WEB/03%20yjfx.htm
[32].Evans C T , Scragg A H , Ratledge C. Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by adenine nucleotides. Eur J Biochem , 1983 ,132 : 609-615.
[33].Botham P A , Ratledge C. A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous microorganisms. J Gen Microbiol, 1979 ,114 : 361-375.
[34].Palmieri L , Palmieri F , Runswick M J , et al. Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein. FEBS Lett, 1996 , 399 : 299-302.
[35].Wynn J P , Hamid A A, Li Y, et al. Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiology, 2001 ,147 : 2857-2864.
[36].Papanikolaou S , Sarantou S , Komaitis M, et al . Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media. J Appl Microbiol ,2004 ,97 : 867-875.
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