摘要
随着石化资源日益耗竭,各国开始重视可再生资源的利用。丁醇是重要的有机溶剂和化工原料,作为生物燃料,性能优于乙醇。丁醇发酵中,原料成本占总成本的比例高达60%～70%,并且由于丁醇对菌体的毒害作用,一般总溶剂产量只有18～20g/L,溶剂得率为0.34g/g。溶剂产量低、生产成本高,使发酵法无法与石油化工竞争。本文通过丁醇浸泡及N+束注入诱变筛选丁醇耐受性强、溶剂产量高的菌种,并用小麦淀粉废水与木薯等廉价原料代替玉米,降低生产成本,提高生物丁醇的竞争力。
通过丁醇浸泡处理,筛得高耐受性菌株BR30-2,丁醇产量达11.77g/L,比出发菌提高16.65%。再经N+束注入诱变,筛得高产菌BH-9,丁醇与总溶剂产量分别达14.51g/L23.14g/L,较出发菌提高43.81%、38.28%,BH-9稳定性良好。
研究了电子转移介体、有机酸盐、pH缓冲剂、还原剂以及震荡和通气对丁醇发酵的影响。刃天青低于0.50%o时可用作筛选高还原力菌株的指示剂。电子转移介体中,中性红显著提高丁醇比例；亚甲基蓝调控作用不明显；甲苯胺蓝抑制作用较强。有机酸盐均可提高溶剂产量,添加0.30%乙酸钠或0.10%丁酸钠时,丁醇产量分别达16.20g/L、16.59g/L。pH缓冲剂中,K2HPO4可以促进溶剂生成,浓度为0.40%时丁醇产量达16g/L；CaCO3、两种缓冲盐(Na2HPO4:KH2PO4=1:1)不利于溶剂生产。还原剂Na2S、L-半胱氨酸盐酸盐、Vc均不利于丁醇发酵。间歇震荡对溶剂产量影响不大；不同发酵时期通气对发酵影响效果不同；相同发酵时期,通气时间越长,氧气对菌体抑制作用越明显。
小麦淀粉废水中添加木薯进行丁醇发酵试验,通过单因子试验和正交试验确定的优化培养基配方为：木薯粉5%,蛋白A 3%,丁酸钠0.10%, K2HPO40.10%。最适培养条件为：温度37℃,初始pH7,种龄24h,接种量3%,装液量110mL/250mL,发酵周期80h。丁醇与总溶剂产量达19.32g/L、32.34g/L,溶剂产量与溶剂得率分别比玉米丁醇提高61.70%、21.91%,生产成本大大降低。
With the depletion of fossil resources, people paid attention to using renewable resources. Butanol is an important organic solvent and chemical material. As a promising new biofuel, butanol is better than ethanol. However, raw materials price almost accounts for 60%～70% of the total production costs. As butanol is toxic to the bacteria, solvent and productivity are only 18～20g/L and 0.34g/g respectively. When competing with chemical synthesis, fermentation had no advantage because of low solvent yield and high production cost. In this paper, high butanol tolerance and butanol-yielding strains were bred after high concentration of butanol soaking and N+ beam implantation. Using wheat starch wastewater and cassava instead of corn as raw materials for butanol fermentation, production costs were reduced, competitivity was improved accordingly.
A high butanol tolerance strain BR30-2 was bred after high concentration butanol soaking, and the output of butanol reached 11.77g/L, increasing by 16.65% compared with the original strain. Using BR30-2 as original strain for N+ beam implantation, a higher butanol-yielding strain BH-9 was bred, of which butanol and total solvent were up to 14.51g/L and 23.14g/L, increasing by 43.81% and 38.28% respectively compared with BR30-2. And BH-9 had a good stability.
Effects of electron transfer mediator, acetate, butyrate, pH buffers, reducing agents, agitation and ventilation on butanol fermentation were studied. Resazurin (<0.50%o) could be used as a indicator for breeding high reducing strains. Neutral red increased the ratio of butanol significantly; methylene blue had little effect on solvent production while toluidine blue inhibited growth strongly. Acetate and butyrate increased solvents. When added 0.30% sodium acetate or 0.10% sodium butyrate, butanol reached 16.20g/L and 16.59g/L respectively. K2HPO4 stimulated the formation of solvent, and butanol reached 16g/L when its concentration was 0.40%; CaCO3, two buffer salts (Na2HPO4:KH2PO4=1:1) were not conducive to butanol fermentation. Na2S, Vc and L-cysteine hydrochloride as reducing agents were not good for solvent production either. Intermittent agitation had little effect on solvent; Ventilation had different effects in different fermentation periods. During the same period, the inhibition effect from oxygen was increased while extending ventilation time.
Using wheat starch wastewater and cassava as raw materials for butanol fermentation, the optimal fermentation medium and conditions were gotten through uni-factor test and orthogonal design. The optimal medium compositions were:cassava power 5%, protein A 3%, butyric acid sodium 0.10% and K2HPO4 0.10%. The optimal culture conditions were: temperature 37℃, initial pH7, seed age 24h, inoculum 3%, medium volume 110mL/250mL, fermentation time 80h. Under the optimal conditions, butanol and total solvent reached 19.32g/L and 32.34g/L respectively, solvent and productivity increased 61.70% and 21.91% compared with corn, and production costs were decreased remarkably.
引文
1.沈兆兵,杜风光,史吉平,等.丙酮丁醇生产技术进展[J].广州化工,2007,35(5)：8-19
2.陈驹声,陆祖祺.发酵法丙酮和丁醇生产技术[M].北京：化学工业出版社,1991.1-238
3.何景昌.丙酮丁醇高产菌株的选育及其发酵新工艺的研究[D]：[硕士学位论文].杭州：浙江工业大学生物与环境工程学院,2009
4. Schwarz WH, Gapes JR. Butanol-rediscovering a renewable fuel [J]. Bio World Europe, 2006(1):16-19
5.王鑫昕.发酵分离耦合系统高产丁醇的工艺优化研究[D]：[硕士学位论文].城都：四川师范大学生命科学学院,2009
6.王一雷.发酵法制备丙酮、丁醇的研究[D]：[硕士学位论文].天津：天津大学化工学院,2008
7.董晓玲,袁自强.正丁醇市场动态及生产工艺探讨[J].甘肃科技,2009(20)：42-44
8. Jones D T, Woods D R. Acetone-butanol fermentation revisited [J]. Microbiol Rev,1986, 50(4):484-524
9.姜岷,韦萍,卢定强,等.后化石经济时代工业生物技术发展的若干思考[J].化工进展,2006(10)：1119-1123
10. Stefanie Keis, Ranad Shaheen, David T Jones. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov[J]. Int J Syst Evol Microbiol,2001,51 (6):2095-2103
11.靳孝庆,王桂兰,何冰芳.丙酮丁醇发酵的研究进展及其高产策略[J].化工进展,2007(12)：1727-1732
12.廖东栋.一株丙酮丁醇梭菌高产丁醇培养基条件优化[D]:[硕士学位论文].武汉：华中农业大学生命科学与技术学院,2009
13. Sang Yup Lee, Jin Hwan Park, Seh Hee Jang, et al. Fermentative butanol production by Clostridia [J]. Biotechnol Bioeng,2008,101(2):209-228
14.唐波.丙酮丁醇梭菌的改良及发酵工艺的研究[D]:[硕士学位论文].无锡：江南大学生物工程学院,2008
15.孙志浩,王舒,吴燕.用固定化细胞连续生产丙酮丁醇的研究[J].1988,18(3)：12-16
16.张益棻,陈军,杨蕴刘,等.高丁醇比丙酮丁醇梭菌的选育与应用[J].工业微生物,1996(4)：1-6
17.县永平.丙酮丁醇梭菌固定化技术用于丙酮丁醇发酵的研究[J].西北师范大学学报(自然科学版),2001(3)：70-73
18.李冬敏,陈洪章.汽爆秸秆膜循环酶解耦合丙酮丁醇发酵[J].过程工程学报,2007(6)：1212-1216
19.靳孝庆,周华,吴薛明,等.丙酮-丁醇发酵生产菌的快速筛选方法[J].过程工程学报,2008(6)：1185-1189
20.王鑫昕.原位萃取发酵耦合工艺高产丁醇的初步研究[J].河北农业大学学报,2008(6)：62-64
21. Benoit Faucon BP, ABF and DuPont to Build Biofuel Plant.Wall Street Journal (Eastern edition) [N]. New York,2007-06-28
22. Kristin Brekke. Butanol-an energy alternative? [J]. Ethanol Today,2007:36-39
23. Gevo receives funding from virgin fuels and khosla ventures to make biobutanol [EB/OL]. http://biopact.com/2007/07/gevo-receives-funding-from-virgin-fuels.html,2007-07-19
24. Green biologies awarded €855,000 to boost biobutanol fuel development [EB/OL]. http://biopact.com/2007/01/green-biologics-awarded-855000-to_22.html,2007-12-07
25.韩加大新能源的研发投入[EB/OL]. http://www.bioon.com/biology/news/316518. shtml,2007-11-21
26.周京波.英计划用甜菜生产生物丁醇,用作车辆驱动燃料[J].功能材料信息,2006(5)：47
27. Annous, B.A.,and H.P. Blaschek. Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity [J]. Appl Environ Microbiol,1991, 57(9):2544-2548
28. Nolling J, Breton G, Omelchenko M V, et al. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum [J]. J Bacteriol,2001, 183(16):4823-4838
29. Copeland A, Lucas S,Lapidus A,et al.Clostridium beijerinckii NCIMB 8052,complete genome[M/OL]. http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=nucleotide&val= CP000721
30. Mermelstein L D, Papoutsakis E T, Petersen D J, et al. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon [J]. Biotechnol Bioeng, 1993,42(9):1053-1060
31. Nair RV, Green EM, Watson DE. Regulation of the sollocus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor [J]. J Bacteriol,1999,181(1):319-330
32. Harris LM, Blank L, Desai RP, et al. Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene [J]. J Ind Microbiol Biotechnol,2001,27(5):322-328
33. Sillers R, Al-Hinai MA, Papout sakis ET. Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations [J]. Biotechnol Bioeng, 2009,102(1):38-49
34. Inui M, Suda M, Kimura S, et al. Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli [J]. Appl Microbiol Biotechnol,2008,77(6):1305-1316
35. At sumi S, Cann AF, Connor MR, et al. Metabolic engineering of Escherichia coli for 1-butanol production [J]. Metab Eng,2007:[Epub ahead of print]
36. Zhu Y, Yang ST. Adaptation of Clostridium tyrobutyricum for enhanced tolerance to butyric acid in a fibrous-bed bioreactor [J]. Biotechnol Prog,2003,19(2):365-372
37. David Edward Ramey, Reynoldsburg Ohio. Continuous two stage, dual path anaerobic fermentation of butanol and other organic solvents using two different strains of bacteria [P]. USA, US5753474,1998-5-19
38. The next generation in biofuels:A new approach to the production of biofuels [EB/OL]. http://www.energeneticsusa.com
39. Qureshi N, Blaschek H P. Recovery of butanol from fermentation broth by gas stripping [J]. Renew. Energ,2001,22(4):557-564
40. Qureshi N, Blaschek H P. Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation [J]. Appl Biochem Biotechnol,2000,84-86:225-235
41. Huang W C, Ramey D E, Yang S T. Continuous production of butanol by Clostridium acetobutylicum immobilized in a fibrous bed bioreactor [J]. Appl Biochem Biotechnol,2004. 113-116:887-898
42. Ishizaki A, Michiwaki S, Crabbe E, et al. Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in bach culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) [J]. J.Biosci.Bioeng,1999,87(3):352-356
43. Ezeji T C, Qureshi N, Blaschek H P. Production of acetone butanol (AB) from liquefied corn starch, a commercial substrate, using Clostridium beijerinckii coupled with product recovery by gas stripping [J]. J Ind Microbiol Biotechnol,2007,34(12):771-777
44. Ezeji T C, Qureshi N, Blaschek H P. Acetone butanol ethanol (ABE) production from concentrated substrate:reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping [J]. Appl Microbiol Biotechnol,2004,63(6):653-658
45. Isken S, Heipieper HJ. Encyclopedia of Environmental Microbiology:Toxicity of Organic Solvents to Microorganisms [J]. New York:Wiley,2002:3147-3155
46. Sardessai Y, Bhosle S. Tolerance of bacteria to organic solvents [J]. Res Microbiol,2002, 153(5):263-268
47.江欢,舒正玉,吴继光,等.耐有机溶剂极端微生物的耐受机制及应用[J].微生物学通报,2009(11)：1744-1749
48.王鑫昕,王少华,李维,等.细菌的有机溶剂耐受机制[J].生物工程学报,2009(5)：641-649
49. Bowles L K, W L Ellefson. Effects of butanol on Clostridium acetobutylicum [J]. Appl Environ Microbiol,1985,50(5):1165-1170
50. Gottwald M, G Gottschalk. The internal pH of Clostridium acetobutylicum and its effect on the shift from acid to solvent formation. Arch[J]. Microbiol,1985(143):42-46
51. Moreira A R, D C Ulmer, J C Linden. Butanol toxicity in the butylic fermentation. Biotechnol. Bioeng [J]. Symp,1981(11):567-579
52. Shirley H Bare, HANS P Blaschek, Terrance L Smith. Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824 [J]. Appl. Environ. Microbiol,1984(47):193-194
53. Brosseau J D, J Yan, K V Lo. The relationship between hydrogen gas and butanol production by Clostridium saccharoperbutylacetonicum [J]. Biotechnol Bioeng,1986,28(3): 305-310
54. Rogers P. Genetics and biochemistry of Clostridium relevant to development of fermentation processes [J]. Appl. Microbiol,1984(31):1-60
55. Costa J M, and A R Moreira. Growth inhibition for kinetics of the acetone-butanol fermentation,1983:501-512. In H. W. Blanch, E. T. Papoutsakis, and G. Stephanopoulos(ed.), Foundations of biochemical engineering, kinetics and thermodynamics in biological systems. ACS Symp. Ser. no.207. American Chemical Society, Washington, D.C
56. Yun-Long LIN, Hans P Blaschek. Butanol production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth [J]. Appl. Environ. Microbiol,1983, 45(3):966-973
57. Shirley H Bare, Hans P Blaschek, Terrance L Smith. Effect of butanol and temperature on lipid composition and memberane fluidity of butanol-tolerant Clostridium acetobutylicum [J]. 1987,53(12):2854-2861
58.左方梅,翟衡.低能离子束在生物学领域的应用[J].山东农业大学学报(自然科学版),2003(3)：451-454
59.陈义光,李铭刚,徐丽华,等.新型物理诱变方法及其在微生物诱变育种中的应用进展 [J].长江大学学报(自科版),2005(5)：46-48
60.陈宇,林梓鑫,张峰,等.离子注入微生物的生物效应研究[J].中国抗生素杂志,1998,23(6)：415-419
61.虞龙,许安,王纪,等.低能离子注入在Vc高产菌株选育中的应用[J].激光生物学报,1999(3)：217-220
62.姚建铭,朱皖宜,王纪,等.离子注入利福霉素产生菌诱变选育研究[J].激光生物学报,1999(3)：214-216
63.姚建铭,王纪,王相勤.离子注入花生四烯酸-产生菌诱变育种[J].生物工程学报,2000,4(16)：478-482
64.张艳玲,黄俊生.淀粉废水处理与资源化利用的研究现状与发展前景[J].江苏农业科学,2009(3)：388-391
65.袁超,刘亚伟,杨宝,等.小麦淀粉与谷朊粉生产[J].西部粮油科技,2003(1)：34-36
66.杨劲峰,赵继红.小麦淀粉生产废水资源化及处理技术研究[J].粮食与油脂,2009(2)：6-8
67.何国庆,朱辉,陈忠明,等.小麦淀粉工业废水的水质特征及酶法预处理条件研究[J].浙江农业学报,1997(5)
68.许泳清,汤浩,蔡南通,等.木薯生产利用现状及福建省发展木薯产业可行性分析[J].中国农学通报,2008(5)：414-417
69.谢铭,李肖.广西木薯生物燃料乙醇产业发展分析[J].江苏农业科学,2010(3)：471-474
70.韦家少.近十年世界木薯生产与贸易概况[J].中国热带农业,2006(1)：18-20
71.姜卫红,阳顾,杨蕴刘,等.一种木薯发酵制备丙酮、丁醇、乙醇的方法[P].中国专利,CN101311272A.2008-11-26
72.沈兆兵,杜风光,史吉平.薯类发酵生产丙酮丁醇及其工艺优化[J].化工进展,2008(27)：134-136
73.菲利普·索凯勒.以高产率生物产生正丁醇的方法[P].中国专利,CN101528935A.2009-09-09
74.何景昌,张正波,裘娟萍.生物丁醇合成途径中关键酶及其基因的研究进展[J].食品与发酵工业,2009,35(2)：116-120
75. Lee J, Yun H, Feist AM, et al. Genome-scale reconstruction and in silico analysis of the Clostridium acetobutylicum ATCC 824 metabolic network [J]. Applied Microbiology and Biotechnology,2008,80(5):849-862
76.杨明,刘力强,牛昆,等.丙酮丁醇发酵菌的分子遗传改造[J].中国生物工程杂志,2009 (10)：109-114
77.牛晓乐,史仲平,张龙云.添加电子载体耦联原位液液萃取的高效丁醇发酵[J].安徽农业科学,2008(26)：11188-11189
78. Shinsaku Hayashida, Sadazo Yoshino. Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells [J]. J Ind Microbiol Biotechnol,2001,27(5):314-321
79. Laurence Girbal, Isabel Vasconcelos, Silvie Saint-Amans, et al. How neutral red modified carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH [J]. FEMS Microbiology Reviews,1995(16):151-162
80.杨影.以生物柴油为萃取剂的丙酮丁醇发酵研究[D].江南大学,2008
81.宋道军,李红,余增亮.N+离子注入对不同辐射敏感性微生物超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的影响[J].生物物理学报,1998,14(2)：325-330
82.全国食品工业标准化技术委员会.GB/T 5514-2008.粮油检验粮食、油料中淀粉含量测定[S].北京：中华人民共和国国家质量监督检验检疫总局,2008
83.中国国家标准化管理委员会.GB/T 5009.10-2003.植物类食品中粗纤维的测定[S].北京：中华人民共和国卫生部,2003
84.中国国家标准化管理委员会.GB/T 5009.10-2003.植物类食品中粗纤维的测定[S].北京：中华人民共和国卫生部,2003
85.史建国,杨俊慧,马耀宏,等.斐林氏法测定还原糖的误差分析[J].发酵科技通讯,2002(4)：1-2
86.徐芳.丙酮丁醇梭菌生产丁醇及代谢调控的初步研究[D]:[硕士学位论文].无锡：江南大学生物工程学院,2009
87.宋道军,姚建铭,邵春林,等.离子注入微生物产生“马鞍型”存活曲线的可能作用机制[J].核技术,1999(3)
88.沈娟,别小妹,陆兆新,等.N+注入诱变芽孢杆菌选育高产抗菌物质菌株[J].核农学报,2006(4)：296-298
89.宋道军,吴丽芳,陈若雷.N+束和γ射线对两种微生物生物膜辐射损伤效应的比较研究[J].激光生物学报,2000,9(2)：89-94
90.蔡强,苑惠娟,周真.色差法在乳品菌群浓度检测中的应用[J].计量与测试技术,2005(12)：26-27
91.高新义,李绍中,潘祥美.以有机染料为媒介体的酶生物传感器研究进展[J].食品工程,2006(3)：24-26
92. Martin J R, Petitdemange H, Ballongue J, et al. Effects of acetic and butyric acid on solvents production by Clostridium acetobutylicum [J]. Biotechnol Letters,1985(2):89-94
93. Husemann M H, Papoutsakis, et al. Effects of propionate and acetate additions on solvent production in batch cultures of Clostridium acetobutylicum [J]. Appl Environ Microbiol,1990, 56(5):1497-1500
94. Chen C K, Blaschek H P. Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101 [J]. Applied Microbiology and Biotechnology, 1999(52):170-173
95.王少华,张延平,寅李.一株丙酮丁醇梭菌及其应用[P].中国发明专利,CN101250496A.2008-08-27
96. Madihah Md Salleh, Liew Shiau Tsuey, Arbakariya Bin Ariff. The proile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 utilizing different pH control strategies [J]. Biotechnology and Bioprocess Engineering,2008,13:33-39
97.刘立明,李寅,堵国成,等.碳酸钙促进丙酮酸发酵过程中α-酮戊二酸的形成[J].生物工程学报,2003(6)：745-749
98.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,1994.682-688
99.于长青,张日俊.瘤胃真菌的营养特性及应用前景[J].粮食与饲料工业,2004(2)：40-41
100.刘宏娟,张建安,刘娅,等.一种微生物间歇供氧补料发酵生产丁醇的工艺[P].中国发明专利,CNl01429527.2009-05-13
101.刘淑玲.固体发酵对木薯皮有毒成分的作用[J].广西轻工业,1994(3)：48-50