摘要
化石燃料的大量使用给人类带来巨大的资源和环境问题。如:化石燃料燃烧产生的CO_2是造成全球气候变暖的主要原因。据有关文献报道,如果要将全球的平均气温控制在2℃的上升幅度内,在2020年之前,非碳能源的消耗要上升到占总能的30%。为此各国开始寻找替代化石燃料的可持续发展的能源。氢作为最清洁的可再生能源,被认为是最具应用前景的可再生能源之一。利用生物本身的生理代谢特性制取氢气是未来一种很有潜力的能源生产技术。特别是生物质发酵法制氢,由于其具有发酵产氢潜力巨大,原料来源广泛且成本低廉,在获得氢能的同时,还可以处理有机污染物,变废为宝等优点,被能源界称为是可再生替代能源研究应用领域中最具发展前景、经济、无污染的重要技术之一,备受国内外能源界的关注。
产氢的稳定性和持久性是发酵产氢中一直存在的问题,如何在稳定持续产氢的基础上提高产氢的速率和转化率是生物质发酵产氢研究中的重要内容,也是能否实现产业化应用的关键性因素。鉴于上述种种问题,本研究首次创造性地在微生物发酵产氢中引入脱氢酶理论,将其作为指标并证实了“脱氢酶与产氢微生物密切相关”的假设,获得以下结果:
1、建立了一种简便、快捷且适用于不同发酵产氢体系的单菌株筛选方法;
2、以脱氢酶活性为关键指标,筛选出产氢微生物,作为本实验的研究对象;
3、以产氢微生物为接种物,通过生物质发酵产氢的动态过程表征:脱氢酶活性与产氢菌株产气随时间的变化呈现明显的规律性趋势,体现出脱氢酶在微生物产氢过程中起到的先导作用。这是对深入研究脱氢酶对其揭示生物质发酵产氢过程中自然规律研究的良好开端;
4、依据脱氢酶理论与产氢的关系规律,采用正交实验L_(27)(3~(13))设计研究,极大地提高原料利用率和产氢得率。在优化的产氢菌种产氢的工艺条件下,发酵产气的启动时间比原始条件工艺提前了33h,发酵系统产氢能力达到45.3mmol/mol葡萄糖,比菌株在原始条件下的对照组提高了273%;
5、针对试验研究所得的规律,对后续研究与发展提出了合理化的几点建议。
It brings mankind the huge problem of resources and environment,that fossil fuel drives a great deal of depletion,such as:the CO_2 coming from the fossil fuel is a main reason to make the global weather warmer.The research indicates recently,if the average temperature in the world is controlled in 2℃ascension ranges,the amount of the non-carbon depletion energy would have 30%in total depletion energy by 2020.For this,researchers in all countries start to look for a renewable energy which can replace fossil fuel.As an most detergent renewable energy,hydrogen energy is one of attractive energy.The method that microorganism producing hydrogen from biomass has great future,It is widely anticipated that bio-hydrogen production which has the advantages of high hydrogen production rates and of the capability to convert widely organic wastes in the environment into more valuable energy could be one of the most promising,economical,clean biotechnology in the applied domain of renewable energy.
The Stability and persistence are lasting problems in fermentative hydrogen processes. Moreover,how to improve hydrogen production rate and substrate conversion efficiency is also important in the research of fermentative hydrogen production,which is also the key factor to realize industrialization.In view of these questions,this study firstly puts forward and validates 'There was a good correlation between hydrogen-producing bacteria and dehydrogenase activity(DHA)' with innovation.The conclusions are as follow:
1、A convenient,quick technical method was developed for screen pure bacterium in fermentation system;
2、It is used in this research that pure hydrogen-producing bacteria with the dehydrogen activity as key index is screened;
3、With hydrogen-producing bacteria for inoculums,The research indicates that dehydrogenase activity changes in obvious regulation trend with production of hydrogen in dynamic hydrogen fermentation,which is a meaningful start for the further research of dehydrogenase to reveal the law and effect of dehydrogenase in fermentation hydrogen production from biomass;
4、The hydrogen production rate and substrate conversion efficiency were dramatically improved by optimizing hydrogen-producing bacteria with adding promoting component which obtained by L_(27)(3~(13)) orthogonal test since it could activate the DHA of hydrogen-producing bacteria.By using the batch fermentation bioreactor with optimizing microorganism,the hydrogen yield from PDB was was 45.3mmol/mol glucose,which was raised 273%compared with contrast test without optimizing component of culture medium respectively.Moreover,the start-up time was about 33 hours ahead of contrast test;
5、Put forward rationalization to the follow-up research and the development according to the research regulation gained by examination of what time suggestion.
引文
[1]世界能源供需现状与发展趋势[EB╱OL],华经纵横,http://www.enpinfo.com/Boiler/Tech/Expert/200701/8181.html
[2]周永刚,2006世界能源统计:全球石油储量可供生产40年[N/OL],中华工商时报,http://news2.eastmoney.com/060703,436995.html
[3]李会霞,南堡油田:我国石油储备的天赐良机[N/OL],上海证券报,http://www.stock2000.com.cn/refresh/arch/2007/05/09/1061748.htm
[4]毛宗强,氢能—21世纪的绿色能源[M],北京:化学工业出版社,2005:4
[5]刘刚,吴长春,中国石油储备体系探讨及对策[J].能源工程,2005,(1):1-6
[6]马欢,脱氢酶在生物质发酵产氢过程中的规律与作用研究[D],昆明:云南师范大学,2006:1
[7]贺江华,乘用车油耗强制性国标出台[N/OL],新快报,http://www.ycwb.com/gb/content/2004-11/03/content_787993.htm
[8]王晴,调查表明我国石油进口依存度2010年达50%[N/OL],第一财经日报,http://auto.tom.com/1440/1441/2006718-97548.html
[9]陶梓,我国成品油供需形势及石油安全对策[EB/OL],http://gdpe.cn/second_detail.jsp?N_id=287580157445
[10]刘广志,新能源近期的发展态势[J],中国工程科学,2003,5(7):29-32
[11]柏杰,发展生物柴油大有可为[J],中国科技产业,2002,(9):63-64
[12]廖艳芬,王树荣,谭洪等,生物质热裂解制取液体燃料技术的发展[J],新能源及工艺,2002(2):1
[13]张正敏,朱俊生,燃料电池21世纪电力行业的主力军[J],中国能源,1999,(9):99-11
[14]赵玉山,张鹏,细菌产氢的研究[D],北京:北京化工大学,2005:2
[15]Nanqi R,Sengupta S.Microbial production of hydrogen:An overview[J].Critical Review in Microbiology,1998,24(1):61-84
[16]王艳辉,吴迪镛,迟建,氢能及利用的应用技术现状及发展趋势[J],化工进展,2001,(1):6-8
[17]李建政,任南琪,生物制氢技术的研究与发展[J],能源工程,2001,2:18-20
[18]樊耀亭,廖新成,卢会杰等,有机废弃物氢发酵制备生物氢气的研究[J],环境科学,2003. 24(3):132
[19]周谨,王树东,世界氢能和燃料电池发展规划[C],第五届全国氢能学术会议,441-445
[20]吴承康,徐建中,金红光,世界科技研究与进展[J],2000,22(4):1-6
[21]Casper M S.Hydrogen manufacture by electrolysis,thermal decomposition and unusual techniques [M].New JerseY:Nayes Data Corp,1978:181-193
[22]Lodhi M A K.Hydrogen production from renewable sources of energy[J].Int J Hydrogen Energy,1987,(12):461-469
[23]SastroM V C.India's hydrogen energy program—A status report[J].Int J Hydrogen Energy,1989,(14):507-520
[24]Cox K E,William son K D.Hydrogen:Its technology and implications[M].Boca Raton,FL:CRC Press,1979:154-165
[25]Rosen M A,Scott D S.Comparative efficiency assessments for arrange of hydrogen production processes[J].Int J Hydrogen Energy,1998,(23):653-662
[26]季亚英,陈艳馨,李文钊等,催化干气选择氧化制氢[J],石油与天然气化工,1999,28(3):190-192
[27]冯玉全,杨颖,燃料电池21世纪电力行业的主力军—从国家安全及战略高度出发大力发展我国的燃料电池技术[J],中国能源,1999(11):21-24
[28]陈哲良,太刚能光电氢能系统[J],能源工程,2001,(1):12-14
[29]朱起名,魏俊梅,徐柏庆,中国氢气的生产和应用—现状和I前景[J/OL],中国能源网
[30]Gest H,Kamen M D.Science[J],1949,109:558-559
[31]袁传敏,颜涌捷,营建勤,生物质制氢气的研究[J],煤炭转化,2002,25(1):18-19
[32]陈丹之,氢能[M],西安:西安交通大学出版社,1990
[33]Kosourov S,Tsygankov A,Seibert M,et al.Sustained hydrogenphoto production by chlamy-domonas reinhardtii[J].Effects of culture parameters Biotechnology and Bioengineering,2002,78(7):731-740
[34]Koku H,Eroglu I,et al.Kinetics of biological hydrogen production by Rhodobacter sphae-roides O.U.001[J].Int J Hydrogen Energy,2003,(28):381-388
[35]Yetis M,Gunduz U,et al.Photoproduction of hydrogen from sugar refinery wastewater by Rhodobac -ter sphaeroides O.U.001[J].Int J Hydrogen Energy,2000,(25):1035-1041
[36]Tsygankov A.Hydrogen production by purple bacteria:immobilizedversus suspension cultures[C].In: Miyake J,Matsunaga T,San PietroA.Biohydrogen II an approach to enviro-nmentally acceptable technology,UK:Elsevier Science Ltd,2001
[37]Melis A,Zhang L P,Forestier M,et al.Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomanas reinhardtii[J].Plant Physiol,2000,(122):127-136
[38]Kondo T,Arakawa Met al.Enhancement of hydrogen production by aphotosynthetic bacterium mutant with reduced pigment[J].J Biosci Bioeng,2002,93(2):145-150
[39]Patrick C H,Benemann J R.Biological hydrogen production:fundamentals and limiting process[J].International J of Hydrogen Energy,2000,(27):1185-1193
[40]李永峰,任南琪,史英,有机废水生物制氢的连续流发酵工艺[J],新能源及工艺,2004,(6):24
[41]张希衡,废水厌氧生物处理工程[M],北京:中国环境科学出版社,1996:10-15
[42]Ren Nanqi,Wang Baozhen,Huang Juchang.Biotechnol Bioeng[J],1997,54(5):428-433
[43]Donanyos,M,et al.Water Sci Technol,1985,17:191
[44]Breure,A.M,etal.Appl Microbiol Biotech,1984,20:40
[45]Cohen A,et al.Influence of Phase Separation on the Anaerobic Digestion of Glc0se[J].Water Res,1982,(16):445-449
[46]Kisaalita,W.S,et al.Acidogenic Fermentation of Lactose[J].Biotech & Bioeng,1987,(30):88-95
[47]Ren N Q,Wang B Z,Huang J C.Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor[J].Biotechnol Bioeng,1997,(54):428-433
[48]Thauer R.K,Jungermann K,Decher K.Bacteriol Rev,1997,41:100-180
[49]Thanisho S.Kamiya N Wakao N.Biochem Biophys Acta,1989,973:1-6
[50]樊耀亭,李晨林,侯红卫等,天然厌氧微生物氢发酵生产生物制氢气的研究[J]。中国环境科学,2002,22(4):370-374
[51]Ren Nanqi(任南琪).Bio-producing H_2 Priciples and Controlling Strategy for Organic Wasterwater Treatment[D].Harbin:Harbin university for Architecture and Engineering(哈尔滨建筑大学),1993(in Chinese)
[52]Li Jianzheng(李建政).The Bio-producing hydrogen technology of Organic Wasterwater Fermentation[D].Harbin:Harbin University of Architecture and Engineering(哈尔滨建筑大学), 1999(in chinese)
[53]Li Baikun(李白昆).The Priciples of Biohydrogen Production by Heavy Organic Wastewater-mechanism and Ability of Hydrogen-producing Bacterum[D].Harbin:Harbin University of Architecture and Engineering(哈尔滨建筑大学),1995(in chinese)
[54]Lay J J.Modelling and optimization of anaerobic digested sludge conberting starch to hydrogen[J]Biotechnology and Bioengineering,2000,68(3):269-278
[55]Yokoi H,Maki R,Hirose J,et al.Microbioal production of hydrogen from starch-manufacturing wastes[J].Biomass & Bioenergy,2002,(22):389-395
[56]李永峰,任南琪,丁杰等,生物制氢:工程应用问题[J],地球科学进展,2004,19(增刊):544
[57]Hawker F R,Dinsdale R,Hawkesb D L,et al.Int J Hydrogen Energy,2002,27:1339-1347
[58]Lee Y J,Miyahara T,Noike T.Chem Technol Biotechnol,2002,77:694-698
[59]TANISHO S,LSHIWATA Y.Continuous hydrogen production from molasses by bacterium Enterobaceter aerogenes[J].Int J Hydrogen Energy,1994,19(10):807-812
[60]TAGUCHI F,MIZUKAMI N,HASEGAWA K,et al.Microbial conbersion of arabinose and xylose to hydrogen by a newly isolated Clostridium sp.No.2[J].Can J Microbiol,1994,(40):228-233
[61]TAGUCHI F,CHANGJ D,MIZUKAMI N,et al.Isolation of a hydrogen-producing bacterium,Clostridium beijerinckii strain AM21B,from termites[J].Can J Microbiol,1993,(39):726-730
[62]JAMES D,BROSSEAU,ZAJIC J E.Hydrogen-ga production with Citrobacter intermedius and Clostridium pasteurianum[J].J Chem Tech Biotechnol,1982,(32):496-502
[63]KUMAR N,DAS D.Enhancement of hydrogen production by Enterobacter cloacae IIT-BT08[J].Proc Biochem,2000,(35):589-593
[64]OOSIMA H,TAKAKUWA S,KATSUDA T,et.al.Production of hydrogen by a hydrogenase-deficient mutant of Rhodobacter capsulatus[J].J Fermernt Bioeng,1998,85(5):470-475
[65]SATZ H.Critical behavior in finite temperature QCD[J].Phys Rep,1982,88(5):349-364
[66]BJORKEN J D.Highly relativistic nucleus-nucleus collisions:the central rapidity region[J].Phys Rev D,1983,27(1):140-151
[67]WEI NER R M.Boson interferometry in high energy physics[J].Phys Rep,2000,327(5):249-346
[68]WIEDE MANN U A,HEI NZ U.Particle interferometry for relativistic heavy ion collisions[J].Phys PEP,1999,319(4):145-230
[69]朱核光,史家樑,生物产氢技术研究进展[J],应用与环境生物学报,2002,8(1):98-104
[70]任南琪,李建政,林明等,产酸发酵细菌产氢机理探讨[J],太阳能学报,2002,23(1):124-128
[71]朱建良,何世颖,活性污泥降解有机物制氢技术[J],化工纵横,2003,17(4):5-8
[72]管英富,邓麦村,金美芳等,微藻光生物水解制氢技术[J],中国生物工程杂志,2003,23(4):8-13
[73]刘士清,张无敌,固定化酵母的污染处理[J],天津微生物,1995,(4):16-18
[74]Momirlan M,Veziroglu T.Recent directions of world hydrogen production[J].Renewable and Sustainable Energy Reviews,1999,(3):219-231
[75]任南琪,王宝贞,有机废水处理生物制氢技术[J],中国环境科学,1994,14(6):411-415
[76]Suzuki Y.On hydrogen as fuel gas[J].In J Hydrogen Energy,1982,(7):227-230
[77]Bockris J M.The econom ics of hydrogen as a fuel[J]..Int J Hydrogen Energy,1981,(6):223-264
[78]宫曼丽,任南琪,邢德峰,丁酸型发酵生物制氢反应器的运行特性研究[J],环境科学学报,2005,25(2):275
[79]Xing X H,Zhang CH.Chin J Bioprocess Eng,2005,3(1):1-8
[80]卢文玉,刘铭辉,陈宇等,厌氧发酵法生物制氢的研究现状和发展前景[J],中国生物工程杂志,2006,26(7):99-103
[81]Tanisho S,Ishiwata Y.Continuous'hydrogen production from molasses by the bacterium Enterobacter aerogenes[J].Int J Hydrogen Energy,1994,19(10):807-812
[82]Taguchi F,Mizukami N,Hasegawa K,et al.Microbial conversion of arabinose and xylose to hydrogen by a newly isolated Clostridium sp.No.2[J].Can J Microbial,1994,(40):228-233
[83]Taguchi F,Chang J D,Mizukami N,et al.Isolation of a hydrogen-produing bacterium,Clostridium beijerinckii strain AM21B,from termites[J].Can J Microbio,1993,(39):726-730
[84]James D,Brosseau,Zajic J E.Hydrogen gas production with Citrobacter intermedius and Clostridium pasteurianum[J].J Chert Tech Biotechnol,1982,(32):496-502
[85]刘波,王海岩,赵静玫等,几株产氢微生物的产氢能力及协同作用[J],食品与发酵工业,2002,29(8):23-26
[86]Taguchi F,Chang J D,Takiguchi S,et al.Efficient hydrogen production from starch by a bacterium isolated from termites[J].J Ferment Bioeng,1992,73(3):244-245
[87]Taguchi F,Chang J D,Mizukami N,et al.Isolation of a hydrogenproducing bacterium,Clostridium beijerickii strain AM21B,from terimites[J].Can J Microbiol,1993,(39):726-730
[88]Osamu M,Hawkes D,Hawkes F R,et al.Enhancement of hydrogen production from glucose by nitrogen gas sparging[J].Bioresoure Technology,2000,(73):59-65
[89]Brosseau J D,Zajic J E.Hydrogen gas production with CitrobaCter intermedius and Clotridium pasteurianum[J].J Chem Technol Biotechnol,1982,(32):496-502
[90]陈双雄,东秀珠,一个新的产氢细菌的鉴定及产氢特性的研究[J],微生物学报,2004,44(4):411-416
[91]李建昌,张无敌,宋洪川等,pH值凋控对发酵产氢的影响[J],能源工程,2003,(2):26-28
[92]MITSU GI C,HARUMI A,KENZO F.We-net:Japanese hydrogen program[J].Int J Hydrogen Energy,1998,23(3):159-165
[93]CATHERINE E,GREGOIRE P.Hydrogen,the onceand future fuel[J].Energy & Fuels,1998,12(1):1-2
[94]PEKHOTA F N,RUSANOV V D,MAL YSHENKO S P.Russian federal hydrogen energy program [J].Int J Hydrogen Energy,1998,23(10):967-970
[95]ARASON B,SIGFUSSON T I.Iceland—a future hy2drogen economy[J].Int J Hydrogen Energy,2000,(25):389-394
[96]YOKOI H,OHKAWARA T,HIROSE J,et al.Char2 acteristics of hydrogen production by aciduric Enterobacter aerogenes strain HO-39[J].J Ferment Bioeng,1995,80(6):571-574
[97]林明,任南琪,王爱杰等,几种金属离子对高效产氢细菌产氢能力的促进作用[J],哈尔滨工业大学学报,2003,35(2):147-151
[98]洪天求,郝小龙,俞汉青,Na~+离子浓度对厌氧发酵产氢气影响的实验研究[J],水处理技术,2004,(30):270-275
[99]REN Nan-qi,DING Jie,DING Lan.Effect of Cu~(2+) concentration on hydrogen fermentation by mixed culture[J].Journal of Harbin Institute of Technology(New Series),2004,11(1):11-16
[100]杨素萍,赵春贵,曲音波等,铁和镍对光和细菌生长和产氢的影响[J],微生物学报,2003,43(2):257-263
[101]丁杰,任南琪,刘敏等,Fe和Fe~(2-)对混合细菌产氢发酵的影响[J],环境科学,2004,25(4):48.53
[102]王勇,任南琪,孙寓姣,Fe对产氢发酵细菌发酵途径及产氢能力影响[J],太阳能学报,2003,24(2):222-225
[103]Tessier A,Campbell P C,Bission M.Sequential extraction procedure for the Speciation of particulate trace metals[J].Analytical Chemistry,1975,(51):844-851
[104]Junelles AM,Janati-Idrissi R,petitdemange H,Gay R.Iron effect on acetone-butanol fermentation[J].Curr.Microbiol,1988,(17):299-303
[105]GRAY C T,GEST H.Biological formation of molecu2 lar hydrogen[J].Sci,1964,(148):186-192
[106]闵 肮,陈美慈,赵宁华等,厌氧微生物学[M],杭州:浙江大学出版社,1993
[107]陈天寿,微生物培养基的制造与使用[M],北京:中国农业出版社,1995
[108]Chung K T.Inhibitory effects of H_2 on growth of clostridium cellobioparum[J].Applied and Enviromental Microbiology,1976,(31):342-348
[109]Bahl H,Gottschalk G.Parameters affecting solvent production by clostridium acetobutyli-cum in continuous culture[J].Biotechnology and Bioengineering Symposium,1984,(14):215-223
[110]Yerushalmi L,Volesky B.Importance of agitation in acetone-butanol fermentation[J].Biotechnol Bioeng,1985,(27):1297-1305
[111]Yerus halmi L,Volesky B,Szczesny T.Effect of increased hydrogen partial pressure on the acetone-butanol fermentation by clostridium acetobutylicum[J].Applied Microbiology and Biotechnology,1985,(22):103-107
[112]Tanisho S,Kuromoto M,Kadokura N.Effect of CO_2 removal on hydrogen production by fermentation[J].Int.J.Hydrogen Energy,1988,(23):593-598
[113]Lee Y J,Miyahara T,Noike T.Effect of pH on microbialhydrogen fermentation[J].J.Chem.Technol Biotechnol,2002,(77):694-698
[114]Van Ginkel S,Sung T,Lay J J.Bio-hydrogen production as a function of pH and substrate concentration[J].Environ.Sci.Technol,2001,(35):4726-4730
[115]Yu H Q,Fang H P.Inhibition on acido-genesis of dairy waste water by zinc and copper[J].Environmental Technology,2001,(22):1459-1465