由于丁醇对生产菌的抑制甚至毒害作用导致丙酮丁醇发酵过程中出现低产物浓度、低产率现象是目前生物法获取丁醇过程中亟待解决的一个关键科学问题。为获得高丁醇耐受性及高丁醇产量生产的菌株,该文采用自行设计的“三明治”筛选方法,从土壤中筛选出4株高丁醇耐受性菌株,其中菌株a914的发酵性能最佳,经P2培养基和木薯粉培养基发酵后其丁醇的产量分别为5.44和3.02 g/L。菌株a914经16S rDNA鉴定其与拜氏梭菌(Clostridium beijerinckii) 的同源性高达99%,以及结合菌株a914的生理生化特性最终确定菌株a914为拜氏梭菌(Clostridium beijerinckii)。同时还对菌株a914的发酵性能进行了初步研究,试验结果表明,其最适的木薯粉浓度为80.0 g/L、酵母浸粉浓度为3.0 g/L、碳酸钙添加量为3.0 g/L,在此条件下丁醇和总溶剂产量分别达到6.73和9.83 g/L。
As butanol has toxic and inhibitory effects on its producing microorganisms, the yield and concentration of biobutanol are low, which is a key scientific challenge for the acetone, butanol and ethanol (ABE) fermentation process. In order to obtain strains with high butanol tolerance and high butanol yield, four strains with high butanol tolerance were selected from soil samples using a new “sandwich” isolation method, in which the strain a914 had the best fermentation performance. When it was used for ABE fermentation in P2 or cassava flour medium, the amounts of butanol produced were 5.44 g/L and 3.02 g/L, respectively. According to 16S rDNA sequence, the strain a914 was 99% homologous to Clostridium beijerinckii, and it was eventually identified as C. beijerinckii combining with its physiological and biochemical characteristics. Besides, the fermentation conditions of C. beijerinckii were optimized. Butanol and total solvent produced reached 6.73 g/L and 9.83 g/L, respectively, under the optimized condition with 80 g/L cassava flour, 3 g/L yeast extract, and 3 g/L calcium carbonate.
[1] DRRE P. New insights and novel developments in clostridialacetone/butanol/isopropanol fermentation [J]. Applied Microbiology and Biotechnology, 1998, 49(6):639-648.
[2] QURESHI N, EZEJI T C, EBENER J, et al. Butanol production by Clostridium beijerinckii. Part Ⅰ: use of acid and enzyme hydrolysed cornfiber [J]. Bioresource Technology, 2008, 99(13): 5 915-5 922.
[3] EZJI T C, QURESHI N, BLASCHEK H P, et al. Bioproduction ofbutanol from biomass: From genes to bioreactors [J]. Current Opinion Biotechnology, 2007, 18 (3): 220-227.
[4] GUO Ting, TANG Yan, ZHANG Qiu-yan, et al. Clostridium beijerinckii mutant with high inhibitor tolerance obtained by low-energy ion implantation [J]. Journal of Industrial Microbiology & Biotechnology, 2012, 39(3):401-407.
[5] LEEL S Y, PARK J H, JANG S H, et al. Fermentative butanol production by Clostridia [J]. Biotecbnology & Bioengineering, 2008, 101(2): 209-228.
[6] GHOLIZADEH L. Enhanced butanol production by free and immobilized Clostridium sp. ells using butyric acid as co-substrate [D]. Boras:University College of Boras, 2009.
[7] JONES D T, WOODS D R. Acetone-butanol fermentation revisited [J]. Microbiological Reviews, 1986, 50(4): 484-524.
[8] KEIS S, SHAHEEN R, JONES D T. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii and descriptions of Clostridium saccharoperbutylacetonicum sp. Nov. and Clostridium saccharobutylicm sp. nov [J]. International Journal of Systematic & Evolutionary Microbiology, 2001(51): 2 095-2 100.
[9] QURESHI N, LOLAS A, BLASCHEK H P. Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101 [J]. Journal of Industrial Microbiology & Biotechnology, 2001, 26(5): 290.
[10] SIKKEMA J, BONT J A D, POOLMAN B. Mechanisms of membrane toxicity of hydrocarbons [J].Micobiology an Molecular Biology Reviews, 1995, 59(2): 201-222.
[11] LIN Yun-long, BLASCHEK H P. Butanol Production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth[J]. Applied and Environment Microbiology, 1983, 45(3): 966-973.
[12] HONICKE D. JANSSEN H,GRIMLER C, et al. Global transcriptional changes of Clostridium acetobutylicum cultures with increased butanol∶acetone ratios[J]. New Biotechnology, 2012, 29(4): 485-493.
[13] 李汉广.高产丁醇菌株选育及其耐受丁醇机制研究[D].无锡:江南大学, 2014.
[14] GUO Ting, HE Ai-yong, DU Teng-fei, et al. Butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor-tolerance [J]. Bioresource Technology, 2013, 135(3):379-385.
[15] MILLER G L. Use of dinitrosalicylic acid reagent for determ ination of reducing sugar [J]. Analytical Biochemistry, 1959, 31(3): 426-428.
[16] 刘志明,唐彦君,吴海舟,等. 苯酚-硫酸法测定葡萄酒中总糖含量的样品处理[J].中国酿造,2011(2):158-161.
[17] 周灿灿. 丙酮丁醇梭菌的选育及高强度丁醇发酵的研究[D]. 无锡:江南大学, 2012.
[18] GIOVANNONI S J, RAPPE M. Evolution diversity and molecular ecology of marine prokaryotes [J]. Microbial Ecology of the Oceans, 2000, 5: 47-84.
[19] 田毅红,朱志豪,高媛,等. 红薯发酵产丁醇的工艺优化[J].化学与生物工程, 2016, 33(2): 64-66.
[20] 宋钢,郑璞,倪晔,等. 木薯发酵产丁醇的研究[J]. 生物加工过程,2012, 10(2): 6-10.
[21] 谭秀花. 丁醇高产菌的选育及小麦淀粉废水与木薯发酵生产丁醇的研究[D].无锡:江南大学, 2010.
[22] GU Yang, HU Shi-yuan, CHEN Jun, et al. Ammonium acetate enhances solvent production by Clostridium acetobutylicum EA2018 using cassava as a fermentation medium [J]. Journal of Industrial Microbiology & Biotechnology, 2009, 36(9): 1 225-1 232.
[23] 冯志彬,王东阳,徐庆阳,等. 氮源对L-苏氨酸发酵的影响[J]. 中国生物工程杂志, 2006(11): 54-58.
[24] 董军,赵勇胜,刘莹莹. 含水层介质对垃圾渗滤液污染缓冲性研究[J]. 环境科学, 2009, 30(2): 484-487.
[25] 诸葛健,王正祥.工业微生物实验技术手册[M]. 北京:中国轻工业出版社, 1994.
