沼泽红假单胞菌phbC-hupL缺失突变株构建及产氢特性研究
|
摘要
化石燃料的大量使用造成环境污染和温室效应日益严重,加上能源短缺的威胁,使得清洁能源的开发与应用成为大势所趋。氢能不仅燃烧后的惟一产物为水,而且具有比较高的热值和较容易转化为其它能源形式等特点,因此被认为是最洁净、最具有开发潜力的能源之一。生物制氢技术反应条件温和、能耗低、能妥善解决能源与环境的矛盾,其中能将太阳能利用、氢能开发和有机废水净化处理有机结合的光合细菌制氢技术是一种的低成本、低耗能的氢能生产绿色技术。光合细菌中的沼泽红假单胞菌(Rhodopseudomonas palustris)是一种古老的能够在光合异养条件下产生氢气的紫色非硫细菌,是目前国内外比较常用的光合产氢模式菌株。
在光合细菌的产氢代谢途径中,固氮酶催化产生氢气,吸氢酶催化氢生成质子和电子重新供给固氮酶,起到回收能量的作用。聚羟基丁酸酯合成酶,其主要作用是把电子还原力以能源物质PHB的形式储存起来。一般说来,H2的分解可以直接减少细胞的产氢量,而聚羟基丁酸酯(PHB)的积累,会消耗细胞内的还原力,从而可以间接减少固氮酶产生的氢气量.本研究利用湖底淤泥分离的沼泽红假单胞菌(Rhodopseudomonas palustris)CQU01与该菌株吸氢酶基因hupL缺失菌株(Rhodopseudomonas palustris)CQU012作为出发菌株,分别构建聚羟基丁酸酯合成酶基因phbC单突变株及聚羟基丁酸酯合成酶基因phbC与吸氢酶基因hupL双突变株,以提高其在光照培养条件下的产氢量。
首先PCR扩增phbC两侧的基因片段5′侧翼与3′侧翼作为双交换时的同源交换臂,连入pMD18-T载体进行测序验证,pSUP202载体上含有BamHⅠ和HindⅢ酶切位点,通过BamHⅠ和HindⅢ双酶切并回收大片段载体,将两目的基因扩增产物与大片段载体连接形成前自杀载体‘pSUP202-phbC’。最后将Em红霉素抗性筛选标记片段连入前自杀载体,构建出pYMT自杀载体。把载体转化具有接合转移作用的E.coli S17-1中,然后再与沼泽红假单胞菌混合,利用接合转移作用完成对目的基因的敲除。最后,经PCR筛选验证,成功获得了沼泽红假单胞菌phbC单突变株R. palustris CQU013及phbC-hupL双突变株R. palustris CQU014。
在相同条件下,以野生菌株为对照,测定了单突变和双突变菌株的生长特性和产氢特性。结果显示出,突变菌株生长曲线与野生菌株有明显差异。两个突变菌株的菌量增加主要发生在120h以内,其后的菌量增加不再明显,但野生菌株的菌量增加一直持续到168h,然后开始下降;突变菌株对数生长期较野生菌株要长,但是最高生物量却明显低于野生菌株。这可能是由于突变工程菌株大量产氢消耗了能量,因此菌株生物量较出发菌株有一定降低。产氢量测定的结果,也显示出两株突变菌株的产氢量较野生菌株产氢量有显著提高。相同发酵条件下,野生菌株R. palustris CQU01的产氢量为344mL/L,而单突变株R. palustris CQU013的产氢量为454mL/L,双突变株R. palustris CQU014产氢量为604mL/L,其产氢量分别是野生菌株的1.31和1.76倍。特别是双突变菌株,可望为微生物光合产氢和工业废水的生物治理提供高效工程菌株。
研究显示出双突变菌株产氢能力较phbC基因和hupL基因单突变菌株的产氢能力有明显提高,进一步证明两个基因都参与了光合产氢代谢过程。双突变菌株比单突变菌株可以显著提高菌株产氢量,但是由于细菌代谢途径的可变性和多样性,可能有其他代谢途径对两个基因敲除后的代谢起补偿作用,因此使得产氢量的提高有限。对沼泽红假单胞菌光合产氢能力的提高还有待于对菌株代谢途径和产氢机理的更深入的了解。
Fossil fuel's massive uses cause the environmental pollution and the greenhouse day by day serious, in addition the shortage of energy the threat. They make the clean energy the development and applly into ultimately. Hydrogen is not only the only product of combustion is water, but also has relatively high heat value and more easily converted into other energy forms and so on. So it is considered the cleanest, most development potential of energy sources. Biological hydrogen has mild reaction conditions, low energy consumption, use of solar energy, which can properly resolve the contradiction between energy and environment. Hydrogen energy development and treatment of organic waste water purification of organic combination of hydrogen production by photosynthetic bacteria is a low-cost, low power hydrogen production of green technology. Rhodopseudomonas palustris is an ancient the purple non-sulfur bacteria, which can produce hydrogen under photosynthetic heterotrophic conditions. Recently, it becomes a mode of photosynthetic hydrogen production strain at home and abroad.
Hydrogen production metabolic pathway in photosynthetic bacteria, the nitrogenase enzyme catalyzes the hydrogen gas. Uptake hydrogenase catalyzed hydrogen proton and electron and re-supply of nitrogen-fixing enzyme, playing a role in energy recovery. Polyhydroxybutyrate synthase is to restore power in the form of material PHB. In general, H2 decomposition can reduce the amount of hydrogen. Poly-hydroxybutyrate (PHB) can reduce power consumption, reducing the amount of hydrogen produced by nitrogenase.Our aim is to construct a di-mutant bioengineering strain of Rhodopseudomonas palustris CQU01 with phbC (PHB synthase) and hupL) gene knocked-out, and improve the strain’s hydrogen produce ability. We applied R. palustris CQU01 isolated from silt as initial strain, for phbC gene knocked out, and R. palustris CQU012 with hupL knocked out which be constructed before in our lab strain as the initial strains.
First of all, PCR amplified phbC both sides of the 5 'flank and 3' flank as a pair of homologous exchange during the exchange of arms. These two genes were jointed in the pMD18-T vector and sequenced to verify. The pSUP202 vector contains BamHⅠand HindⅢrestriction sites. And correct-sequenced genes 5 'flank and 3' flank were cloned into the BamHⅠand HindⅢ-digested pSUP202, generating the previous vector‘pSUP202-phbC’. The Emr cassette was cloned into the‘pSUP202-phbC’, generating the suicide gene vector pYMT. The pYMT was transformed into E. coli S17-1, obtaining the donor bacteria. The donor was mixed with the original strain (receptor bacteria) R. palustris CQU01. The correct integration of the mutants was confirmed by PCR test and subsequent sequence analysis. The single mutant strain R. palustris CQU013 with phbC knocked out and double mutant strain R. palustris CQU014 with phbC-hupL knocked out were obtained.
Under the same conditions, the bacterial growth curves and hydrogen production characteristics of the mutant strains showed that the biological characters of the mutant strains had some difference with the wild strain R. palustris CQU01. Two mutant strains of bacteria increased mainly in less than 120h, and the bacteria was not significantly increased. But the wild strain of the bacteria increased until 168h, and then began to decline; Mutant strain has logarithmic growth phase longer than the wild strain. But the maximum biomass was significantly lower than the wild strain. The mutant strains may consume a large number of energy, so strain biomass is lower than the wild strain. Hydrogen production results also show that the two mutant strains have the remarkable enhancement of hydrogen. The same fermentation conditions, The total H2 yield of R. palustris CQU013 and R. palustris CQU014 increased by 31% and 76% respectively comparing to wild strain, which up to 454mL and 604mL per liter fermentation broth in 216 hours. Both the phbC gene and hupL gene affected the yield of hydrogen produce. In particular, the double mutant strain is expected as an effective engineering strain for photosynthetic hydrogen production and microbial biological treatment of industrial wastewater.
The studies have shown that hydrogen production capacity of double mutant strain is more than the phbC gene and the single hupL gene mutant. Two genes are involved in the metabolism of photosynthetic hydrogen production. Double mutant can improve the hydrogen production significantly. But the result of bacterial metabolic pathways variability and diversity, there may be other metabolic pathways. So the increase is limited. On R. palustris hydrogen production capabilities, we have to understand the hydrogen metabolic pathway and the mechanism.
在光合细菌的产氢代谢途径中,固氮酶催化产生氢气,吸氢酶催化氢生成质子和电子重新供给固氮酶,起到回收能量的作用。聚羟基丁酸酯合成酶,其主要作用是把电子还原力以能源物质PHB的形式储存起来。一般说来,H2的分解可以直接减少细胞的产氢量,而聚羟基丁酸酯(PHB)的积累,会消耗细胞内的还原力,从而可以间接减少固氮酶产生的氢气量.本研究利用湖底淤泥分离的沼泽红假单胞菌(Rhodopseudomonas palustris)CQU01与该菌株吸氢酶基因hupL缺失菌株(Rhodopseudomonas palustris)CQU012作为出发菌株,分别构建聚羟基丁酸酯合成酶基因phbC单突变株及聚羟基丁酸酯合成酶基因phbC与吸氢酶基因hupL双突变株,以提高其在光照培养条件下的产氢量。
首先PCR扩增phbC两侧的基因片段5′侧翼与3′侧翼作为双交换时的同源交换臂,连入pMD18-T载体进行测序验证,pSUP202载体上含有BamHⅠ和HindⅢ酶切位点,通过BamHⅠ和HindⅢ双酶切并回收大片段载体,将两目的基因扩增产物与大片段载体连接形成前自杀载体‘pSUP202-phbC’。最后将Em红霉素抗性筛选标记片段连入前自杀载体,构建出pYMT自杀载体。把载体转化具有接合转移作用的E.coli S17-1中,然后再与沼泽红假单胞菌混合,利用接合转移作用完成对目的基因的敲除。最后,经PCR筛选验证,成功获得了沼泽红假单胞菌phbC单突变株R. palustris CQU013及phbC-hupL双突变株R. palustris CQU014。
在相同条件下,以野生菌株为对照,测定了单突变和双突变菌株的生长特性和产氢特性。结果显示出,突变菌株生长曲线与野生菌株有明显差异。两个突变菌株的菌量增加主要发生在120h以内,其后的菌量增加不再明显,但野生菌株的菌量增加一直持续到168h,然后开始下降;突变菌株对数生长期较野生菌株要长,但是最高生物量却明显低于野生菌株。这可能是由于突变工程菌株大量产氢消耗了能量,因此菌株生物量较出发菌株有一定降低。产氢量测定的结果,也显示出两株突变菌株的产氢量较野生菌株产氢量有显著提高。相同发酵条件下,野生菌株R. palustris CQU01的产氢量为344mL/L,而单突变株R. palustris CQU013的产氢量为454mL/L,双突变株R. palustris CQU014产氢量为604mL/L,其产氢量分别是野生菌株的1.31和1.76倍。特别是双突变菌株,可望为微生物光合产氢和工业废水的生物治理提供高效工程菌株。
研究显示出双突变菌株产氢能力较phbC基因和hupL基因单突变菌株的产氢能力有明显提高,进一步证明两个基因都参与了光合产氢代谢过程。双突变菌株比单突变菌株可以显著提高菌株产氢量,但是由于细菌代谢途径的可变性和多样性,可能有其他代谢途径对两个基因敲除后的代谢起补偿作用,因此使得产氢量的提高有限。对沼泽红假单胞菌光合产氢能力的提高还有待于对菌株代谢途径和产氢机理的更深入的了解。
Fossil fuel's massive uses cause the environmental pollution and the greenhouse day by day serious, in addition the shortage of energy the threat. They make the clean energy the development and applly into ultimately. Hydrogen is not only the only product of combustion is water, but also has relatively high heat value and more easily converted into other energy forms and so on. So it is considered the cleanest, most development potential of energy sources. Biological hydrogen has mild reaction conditions, low energy consumption, use of solar energy, which can properly resolve the contradiction between energy and environment. Hydrogen energy development and treatment of organic waste water purification of organic combination of hydrogen production by photosynthetic bacteria is a low-cost, low power hydrogen production of green technology. Rhodopseudomonas palustris is an ancient the purple non-sulfur bacteria, which can produce hydrogen under photosynthetic heterotrophic conditions. Recently, it becomes a mode of photosynthetic hydrogen production strain at home and abroad.
Hydrogen production metabolic pathway in photosynthetic bacteria, the nitrogenase enzyme catalyzes the hydrogen gas. Uptake hydrogenase catalyzed hydrogen proton and electron and re-supply of nitrogen-fixing enzyme, playing a role in energy recovery. Polyhydroxybutyrate synthase is to restore power in the form of material PHB. In general, H2 decomposition can reduce the amount of hydrogen. Poly-hydroxybutyrate (PHB) can reduce power consumption, reducing the amount of hydrogen produced by nitrogenase.Our aim is to construct a di-mutant bioengineering strain of Rhodopseudomonas palustris CQU01 with phbC (PHB synthase) and hupL) gene knocked-out, and improve the strain’s hydrogen produce ability. We applied R. palustris CQU01 isolated from silt as initial strain, for phbC gene knocked out, and R. palustris CQU012 with hupL knocked out which be constructed before in our lab strain as the initial strains.
First of all, PCR amplified phbC both sides of the 5 'flank and 3' flank as a pair of homologous exchange during the exchange of arms. These two genes were jointed in the pMD18-T vector and sequenced to verify. The pSUP202 vector contains BamHⅠand HindⅢrestriction sites. And correct-sequenced genes 5 'flank and 3' flank were cloned into the BamHⅠand HindⅢ-digested pSUP202, generating the previous vector‘pSUP202-phbC’. The Emr cassette was cloned into the‘pSUP202-phbC’, generating the suicide gene vector pYMT. The pYMT was transformed into E. coli S17-1, obtaining the donor bacteria. The donor was mixed with the original strain (receptor bacteria) R. palustris CQU01. The correct integration of the mutants was confirmed by PCR test and subsequent sequence analysis. The single mutant strain R. palustris CQU013 with phbC knocked out and double mutant strain R. palustris CQU014 with phbC-hupL knocked out were obtained.
Under the same conditions, the bacterial growth curves and hydrogen production characteristics of the mutant strains showed that the biological characters of the mutant strains had some difference with the wild strain R. palustris CQU01. Two mutant strains of bacteria increased mainly in less than 120h, and the bacteria was not significantly increased. But the wild strain of the bacteria increased until 168h, and then began to decline; Mutant strain has logarithmic growth phase longer than the wild strain. But the maximum biomass was significantly lower than the wild strain. The mutant strains may consume a large number of energy, so strain biomass is lower than the wild strain. Hydrogen production results also show that the two mutant strains have the remarkable enhancement of hydrogen. The same fermentation conditions, The total H2 yield of R. palustris CQU013 and R. palustris CQU014 increased by 31% and 76% respectively comparing to wild strain, which up to 454mL and 604mL per liter fermentation broth in 216 hours. Both the phbC gene and hupL gene affected the yield of hydrogen produce. In particular, the double mutant strain is expected as an effective engineering strain for photosynthetic hydrogen production and microbial biological treatment of industrial wastewater.
The studies have shown that hydrogen production capacity of double mutant strain is more than the phbC gene and the single hupL gene mutant. Two genes are involved in the metabolism of photosynthetic hydrogen production. Double mutant can improve the hydrogen production significantly. But the result of bacterial metabolic pathways variability and diversity, there may be other metabolic pathways. So the increase is limited. On R. palustris hydrogen production capabilities, we have to understand the hydrogen metabolic pathway and the mechanism.
引文
[1]王革华.能源与可持续发展[M].北京:化学工业出版社, 2005.
[2]张全国,李刚.生物制氢技术现状及其发展潜力[J].新能源产业, 2007, 4 (1): 17–21.
[3] Zhou H. Discussion on development strategy and policy measure for renewable energy during the tenth five-year-plan [Z]. In Proceeding Clean Energy Technology Seminar, Beijing, China. 2001.
[4]赵玉山,腾晓峰,张鹏,谭天伟.混合厌氧微生物产氢研究[J].北京化工大学学报, 2005, 32 (4): 1-4.
[5] Ebru O zgur, Astrid E. Mars, Begum Peksel, et al. Biohydrogen production from beet molasses by sequential dark and photofermentation [J]. International Journal of Hydrogen Energy, 2010, 35: 511-517.
[6]曾晓波,王海英.微藻生物制氢技术[J].北京化工大学学报, 2005, 12 (5): 60-63.
[7]刘颖,王爱杰,邓娴,刘冰峰,丁杰.联合生物制氢方法及发展趋势[J].生物加工过程, 2007, 5 (3): 6-9.
[8]李永峰,任南琪,杨传平,胡立杰,吴忆宁,张文启.生物制氢系统产氢菌的富集培养与分离技术[J].南京理工大学学报, 2006, 30 (3): 366-370.
[9]梁建光,吴永强.生物产氢研究进展J].微生物学通报, 2002, 29 (6): 81-85.
[10]高广义,陈素华.谈谈光合细菌的生态和应用[J].生物学通报, 1992 , 5 (1): 14–15.
[11]顾钢.国外氢能技术路线图及对我国的启示[J].国际技术经济研究, 2004.7(4): 34-37.
[12] National Research Council. The Hydrogen Economy: Opportunities, Costs, Barriers, and R&D Needs [J]. National Academy Press (Washington DC), 2004.
[13] Pietro Carlozzi, Maurizio Lambardi. Fed-batch operation for bio-H2 production by Rhodopseudomonas palustris (strain 42OL) [J]. Renewable Energy, 2009, 34: 2577-2584.
[14] Miriam Rosenbaum, Zhen He, Largus T Angenent. Light energy to bioelectricity: photosynthetic microbial fuel cells [J]. Current opinion in Biotechnology, 2010, 21: 1-6.
[15]蒋志城.生物发酵制氢技术的研究及进展[J].国际技术经济研究, 2008.39(2): 14-17.
[16]中国科学院可持续发展战略研究组.中国可持续发展战略报告-建设资源节约型和环境友好型社会[M].北京:科学出版社, 2006.
[17]毛宗强.氢能——21世纪的绿色能源[M].北京:化学工业出版, 2005.
[18]朱核光,史家木粱.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.
[19] Hyung-Sool Lee1, Wim F.J. Vermaas, Bruce E. Rittmann. Biological hydrogen production: prospects and challenges [J]. Cell.
[20]韩静淑,赵振英,周礼恺,刘增柱编.生物细胞的固定化技术及其应用[M].北京:科学出版社, 1993.
[21] Chen YD, Li L B. 11th the international symposiumon photosynthesis[J].Acta Biophysica Sinica,1999,15(1):232-235.
[22]王建龙.聚乙烯醇包埋固定化微生物的研究及进展[M].化学反应工程与工艺, 1998, 8 (12): 35–39.
[23] Mayhew, S. G., M. E. O, connor. Trends Biochim [J]. Socie January, 1982, (3): 18–21.
[24] Gest H, Ormerod, JG, Ormerod, KS. Photometabolism of R.rubrum. Light-dependent dissimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic citric acid cycle [J]. Arch Biochem Biophys, 1962 , 97: 21.
[25] Stefano F. Simoni 1, Anke Schafer, Hauke Harms, Alexander J. B. Zehnder. Factors affecting mass transfer limited biodegradation in saturated porous media [J]. Journal of Contaminant Hydrology, 2001, (50): 99–120.
[26] Arzu Y., Dursun, Zumriye Aksu. Biodegradation kinetics of ferrous (II) cyanide complex ions by immobilized Pseudomonas fluorescens in a packed bed column reactor [J]. Process Biochemistry, 2000, (35): 615–622.
[27] Klein J., Vorlop K D., Wagner, F., Ann.N.y. Acad.Evidence for a nitrogenase system in the photosynthetic bacterium Rhodospirillum rubrum [J]. Sci., 1984, (3): 434–437.
[28] Daniel Graham, Rui Pereira, Dominic Barfield, Don Cowan. Yeast as a Cell factor [J]. Enzyme and Microbial Technology, 2000, (26): 368–373.
[29] Tsuey-Ping Chung, Hsiu-Ya Tseng, Ruey-Shin Juang Mass transfer effect and intermediate detection for phenol degradation in immobilized Pseudomonas putida systems [J]. Process Biochemistry, 2003, (38): 1497–1507.
[30] Bahatyrova S, Frese R N, Siebert C A, et al. The native architecture of a photosynthetic membrane [J]. Nature, 2004, 430: 1058―1062.
[31] Michael A. van der Horst, Jason Key, Klaas J. Hellingwerf. Photosensing in chemotrophic, non-phototrophic bacteria: let there be light sensing too [J]. Mol Biol, 2003, 326: 1523―1538.
[32] Papiz M Z, Prince S M, Howard T, et al. The structure and thermal motion of the B800-850 LH2 complex from Rps. acidophila at 2.0 ? resolution and 100K: New structural features and functionally relevant motions [J]. TRENDS in Microbiology, 2007, 15(12): 554-562.
[33] Arluison V, Seguin J, Robert B. The reaction order of the dissociation reaction of the B820 subunit of Rhodospirillum rubrum light-harvesting I complex [J]. FEBS Lett, 2002, 516: 40―42.
[34] Martin F. Richter, Jurgen Baier, June Southal, et al. Refinement of the x-ray structure of theRC–LH1core complex from Rhodopseudomonas palustris by single-molecule spectroscopy [J]. PNAS, 2007, 104(51): 20280-20284.
[35] Feng Juan, Li Xuefeng, Liu Yan. Effects of pH on the peripheral light-harvesting antenna complex for Rhodopseudomonas palustris [J]. Science in China Series C: Life Sciences, 2008, 51(8): 760-766.
[36] Gregory J. S. Fowler, C. Neil Hunter. The synthesis and assembly of functional high and low light LH2 antenna complexes from Rhodopseudomonas palustris in Rhodobacter sphaeroides [J]. PNAS, 1996, 271(23): 13356-13361.
[37] Gokhan Kars, Ufuk Gunduz, Gabor Rakhely, et al. Improved hydrogen production by uptake hydrogenase de?cient mutant strain of Rhodobacter sphaeroides O. U. 001 [J]. International Journal of Hydrogen Energy, 2008, 37(1): 1-5.
[38] R. Dembczynski, T. Jankowski,Growth characteristics and acidifying activity of Lactobacillus rhamnosus in alginate/starch liquid-core capsules [J]. Enzyme and Microbial Technology, 2002, (31): 111–115.
[39] Voselsang C, Gollembiewski K, Ostgaard K. Effect of preservation techniques on the regeneration of gel entrapped nitrifying sludge [J]. Water Res 1999, 33 (1): 164–168.
[40]宗文明,于瑞嵩,樊美珍,周志华.一株利用生物柴油废水产氢的光合菌的筛选、鉴定[J].中国环境科学, 2009,27(4):413-418.
[41]张明,史家梁.光合细菌光合产氢机理研究进展[J].应用与环境生物学报, 1999, 5 (增): 25–29.
[42]吴永强,陈秉俭,仇哲.浑球红假单胞菌在暗处发醉生长时的固氮酶、吸氢酶以及放氢机制研究.微生物学通报, 1991, 18(2): 71~74.
[43]李清彪,陈洪芳.固定化细胞及其载体中组分扩散性质研究[J].离子交换与吸附, 1992, 8 (4): 355–361.
[44] Nelson P.Guerra, Lorenzo Pastrana.Modelling the influence of pH on the kinetics of both nisin and pediocin production and characterization of their functional properties[J]. Process Biochemistry, 2002, 37: 1005–1015.
[45]刘如林,梁凤来,刁虎欣等.光合细菌及其应用[M].北京:中国农业科技出版社, 1991.
[46] Weaver P F, Lien S, Senbert M. Photobiological production of hydrogen [J]. Solar Energy. 1980, (24): 3–45.
[47] Reza Yegani, Satoshi Yoshimura, Kazunori Moriya. Improvement of growth stability of photosynthetic bacterium Rhodobacter capsulatus [J]. Journal of Bioscience and Bioengineering, 2005,100(6): 672-677.
[48] Yong-Zhong Wang, Qiang Liao, Xun Zhu, et al. Characteristics of hydrogen production andsubstrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor [J]. Bioresource Technology, 2010, 101: 4034-4041.
[49] Rostron WM, Stuckey DC, Young AA. Nitrification of high strength ammonia wastewaters: comparative study of immobilization media [J]. Water Res, 2001, 35(5): 1169–1178.
[50]朱瑞艳,王迪, Yaoping Zhang,李季伦.深红红螺菌draTGB hupL双突变株在不同光照条件下的放氢[J].科学通报,2006 ,5 1(17): 2045–2051.
[51] Toshihiko kondo, Masayasu arakawa. Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment [J]. Arch Biochem Biophys, 2002, 93(2): 145-150.
[52] I. -H. Lee, J. Y. Park, D. H. Kho, et al. Reductive effect of H2 uptake and poly-β- hydroxybutyrate formation on nitrogenase-mediated H2 accumulation of Rhodobacter sphaeroides according to light intensity [J]. Appl Microbiol Biotechnol, 2002,60:147-153.
[53]殷幼平,张宝鹏,廖强,等.沼泽红假单胞菌高效产氢hupL缺失突变株的构建[J].中国环境科学, 2009,27(4):413-418.
[54] Larimer F W, Chain P, Hauser L, et al. Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris [J]. Nature Biotechnology, 2004, 22(1): 55-61.
[55] Laurence Casalot, Marc Rousset. Maturation of the [NiFe] hydrogenases [J]. Microbiology, 2001,9(5) :228-237.
[56] Paulette M. Vignais, Jean-Pierre Magnin, John C. Willison. Increasing biohydrogen production bymetabolic engineering [J]. International Journal of Hydrogen Energy, 2006, 31(7): 1478-1483.
[57] Federico E. Rey, Yasuhiro Oda, and Caroline S. Harwood. Regulation of Uptake Hydrogenase and Effects of Hydrogen Utilization on Gene Expression in Rhodopseudomonas palustris [J]. Journal of Bacteriology. 2006, 188(17): 6143-6152.
[58] Eilert Hustede, Alexander Steinbiichel, Hans G. Schlege. Relationship between the photoproduction of hydrogen and the accumulation of PHB in non-sulphur purple bacteria [J]. Appl Microbiol Biotechnol, 1993,39:87-93.
[59] Deniz Ozgur Yigit, Ufuk Gunduz, Lemi Turker, et al. Identi cation of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O. U. 001 grown in the waste water of a sugar re nery [J]. Journal of Biotechnology, 1999,4:25–131.
[60] Roberto De Philippis, Alba Ena, Monica Guastini, et al. Factors affecting poly-β- hydroxybutyrate accumulation in cyanobacteria and in purple non-sulfur bacteria [J]. Microbiol Rev, 1992,103:187-194.
[61]岳文洁,夏元生,张小凡.红色非硫磺菌生长及聚β-羟基丁酸积累的研究[J].环境与可持续发展, 2008,3(2):18-20.
[62] Kim JH, Lee JK. Cloning, nucleotide sequence and expression of gene coding for polyhydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 [J]. Microbiol Biotechnol, 1997,7:229–236.
[63] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxy alkanoates [J]. Microbiol Rev, 54:450–472.
[64] Yasuhiro Oda, Sudip K. Samanta, Federico E. Rey, et al. Functional Genomic Analysis of Three Nitrogenase Isozymes in the Photosynthetic Bacterium Rhodopseudomonas palustris[J]. Journal of Bacteriology. 2005, 187(22): 7784-7794.
[65] Mi-Sun Kim, Jin-Sook Baek, Jeong K. Lee. Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase de?cient mutant [J]. International Journal of Hydrogen Energy, 2006,31:121-127.
[66]王永忠,廖强,朱恂,等.底物初始浓度对光合细菌产氢动力学特性的影响[J].环境工程学报, 2007,7(1):125-129.
[67]王中康,胡小容,廖强等.几株紫色非硫细菌的分离鉴定与产氢特性分析[J].应用与环境生物学报, 2009, 15(1):120-124.
[68] Simon. R., U. Priefer, and A. Puhler. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gramnegative bacteria[J]. Bio/Technology 1983, 1: 784-791.
[69]吴海珍,李莉,张惠展,刘和.荚膜红细菌接合转移系统的建立及优化[J]华东理工大学学报2000-02 Vol.26 No.1.
[70] Davis J , Donohue I J , Kaplan S. Construction characterization, and complementation of a duf mutant of Rhodobacter Sphaeroides [J]. J Bacterio l, 1988, 170: 320-329.
[71] Klipp W., M asepoh B, Puhler A. Identification and mapping of nitrogen fixation of Rhodobacter capsulatus: duplication of nif A-nif B region [J]. J Bacteriol,1998.170: 693-699.
[2]张全国,李刚.生物制氢技术现状及其发展潜力[J].新能源产业, 2007, 4 (1): 17–21.
[3] Zhou H. Discussion on development strategy and policy measure for renewable energy during the tenth five-year-plan [Z]. In Proceeding Clean Energy Technology Seminar, Beijing, China. 2001.
[4]赵玉山,腾晓峰,张鹏,谭天伟.混合厌氧微生物产氢研究[J].北京化工大学学报, 2005, 32 (4): 1-4.
[5] Ebru O zgur, Astrid E. Mars, Begum Peksel, et al. Biohydrogen production from beet molasses by sequential dark and photofermentation [J]. International Journal of Hydrogen Energy, 2010, 35: 511-517.
[6]曾晓波,王海英.微藻生物制氢技术[J].北京化工大学学报, 2005, 12 (5): 60-63.
[7]刘颖,王爱杰,邓娴,刘冰峰,丁杰.联合生物制氢方法及发展趋势[J].生物加工过程, 2007, 5 (3): 6-9.
[8]李永峰,任南琪,杨传平,胡立杰,吴忆宁,张文启.生物制氢系统产氢菌的富集培养与分离技术[J].南京理工大学学报, 2006, 30 (3): 366-370.
[9]梁建光,吴永强.生物产氢研究进展J].微生物学通报, 2002, 29 (6): 81-85.
[10]高广义,陈素华.谈谈光合细菌的生态和应用[J].生物学通报, 1992 , 5 (1): 14–15.
[11]顾钢.国外氢能技术路线图及对我国的启示[J].国际技术经济研究, 2004.7(4): 34-37.
[12] National Research Council. The Hydrogen Economy: Opportunities, Costs, Barriers, and R&D Needs [J]. National Academy Press (Washington DC), 2004.
[13] Pietro Carlozzi, Maurizio Lambardi. Fed-batch operation for bio-H2 production by Rhodopseudomonas palustris (strain 42OL) [J]. Renewable Energy, 2009, 34: 2577-2584.
[14] Miriam Rosenbaum, Zhen He, Largus T Angenent. Light energy to bioelectricity: photosynthetic microbial fuel cells [J]. Current opinion in Biotechnology, 2010, 21: 1-6.
[15]蒋志城.生物发酵制氢技术的研究及进展[J].国际技术经济研究, 2008.39(2): 14-17.
[16]中国科学院可持续发展战略研究组.中国可持续发展战略报告-建设资源节约型和环境友好型社会[M].北京:科学出版社, 2006.
[17]毛宗强.氢能——21世纪的绿色能源[M].北京:化学工业出版, 2005.
[18]朱核光,史家木粱.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.
[19] Hyung-Sool Lee1, Wim F.J. Vermaas, Bruce E. Rittmann. Biological hydrogen production: prospects and challenges [J]. Cell.
[20]韩静淑,赵振英,周礼恺,刘增柱编.生物细胞的固定化技术及其应用[M].北京:科学出版社, 1993.
[21] Chen YD, Li L B. 11th the international symposiumon photosynthesis[J].Acta Biophysica Sinica,1999,15(1):232-235.
[22]王建龙.聚乙烯醇包埋固定化微生物的研究及进展[M].化学反应工程与工艺, 1998, 8 (12): 35–39.
[23] Mayhew, S. G., M. E. O, connor. Trends Biochim [J]. Socie January, 1982, (3): 18–21.
[24] Gest H, Ormerod, JG, Ormerod, KS. Photometabolism of R.rubrum. Light-dependent dissimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic citric acid cycle [J]. Arch Biochem Biophys, 1962 , 97: 21.
[25] Stefano F. Simoni 1, Anke Schafer, Hauke Harms, Alexander J. B. Zehnder. Factors affecting mass transfer limited biodegradation in saturated porous media [J]. Journal of Contaminant Hydrology, 2001, (50): 99–120.
[26] Arzu Y., Dursun, Zumriye Aksu. Biodegradation kinetics of ferrous (II) cyanide complex ions by immobilized Pseudomonas fluorescens in a packed bed column reactor [J]. Process Biochemistry, 2000, (35): 615–622.
[27] Klein J., Vorlop K D., Wagner, F., Ann.N.y. Acad.Evidence for a nitrogenase system in the photosynthetic bacterium Rhodospirillum rubrum [J]. Sci., 1984, (3): 434–437.
[28] Daniel Graham, Rui Pereira, Dominic Barfield, Don Cowan. Yeast as a Cell factor [J]. Enzyme and Microbial Technology, 2000, (26): 368–373.
[29] Tsuey-Ping Chung, Hsiu-Ya Tseng, Ruey-Shin Juang Mass transfer effect and intermediate detection for phenol degradation in immobilized Pseudomonas putida systems [J]. Process Biochemistry, 2003, (38): 1497–1507.
[30] Bahatyrova S, Frese R N, Siebert C A, et al. The native architecture of a photosynthetic membrane [J]. Nature, 2004, 430: 1058―1062.
[31] Michael A. van der Horst, Jason Key, Klaas J. Hellingwerf. Photosensing in chemotrophic, non-phototrophic bacteria: let there be light sensing too [J]. Mol Biol, 2003, 326: 1523―1538.
[32] Papiz M Z, Prince S M, Howard T, et al. The structure and thermal motion of the B800-850 LH2 complex from Rps. acidophila at 2.0 ? resolution and 100K: New structural features and functionally relevant motions [J]. TRENDS in Microbiology, 2007, 15(12): 554-562.
[33] Arluison V, Seguin J, Robert B. The reaction order of the dissociation reaction of the B820 subunit of Rhodospirillum rubrum light-harvesting I complex [J]. FEBS Lett, 2002, 516: 40―42.
[34] Martin F. Richter, Jurgen Baier, June Southal, et al. Refinement of the x-ray structure of theRC–LH1core complex from Rhodopseudomonas palustris by single-molecule spectroscopy [J]. PNAS, 2007, 104(51): 20280-20284.
[35] Feng Juan, Li Xuefeng, Liu Yan. Effects of pH on the peripheral light-harvesting antenna complex for Rhodopseudomonas palustris [J]. Science in China Series C: Life Sciences, 2008, 51(8): 760-766.
[36] Gregory J. S. Fowler, C. Neil Hunter. The synthesis and assembly of functional high and low light LH2 antenna complexes from Rhodopseudomonas palustris in Rhodobacter sphaeroides [J]. PNAS, 1996, 271(23): 13356-13361.
[37] Gokhan Kars, Ufuk Gunduz, Gabor Rakhely, et al. Improved hydrogen production by uptake hydrogenase de?cient mutant strain of Rhodobacter sphaeroides O. U. 001 [J]. International Journal of Hydrogen Energy, 2008, 37(1): 1-5.
[38] R. Dembczynski, T. Jankowski,Growth characteristics and acidifying activity of Lactobacillus rhamnosus in alginate/starch liquid-core capsules [J]. Enzyme and Microbial Technology, 2002, (31): 111–115.
[39] Voselsang C, Gollembiewski K, Ostgaard K. Effect of preservation techniques on the regeneration of gel entrapped nitrifying sludge [J]. Water Res 1999, 33 (1): 164–168.
[40]宗文明,于瑞嵩,樊美珍,周志华.一株利用生物柴油废水产氢的光合菌的筛选、鉴定[J].中国环境科学, 2009,27(4):413-418.
[41]张明,史家梁.光合细菌光合产氢机理研究进展[J].应用与环境生物学报, 1999, 5 (增): 25–29.
[42]吴永强,陈秉俭,仇哲.浑球红假单胞菌在暗处发醉生长时的固氮酶、吸氢酶以及放氢机制研究.微生物学通报, 1991, 18(2): 71~74.
[43]李清彪,陈洪芳.固定化细胞及其载体中组分扩散性质研究[J].离子交换与吸附, 1992, 8 (4): 355–361.
[44] Nelson P.Guerra, Lorenzo Pastrana.Modelling the influence of pH on the kinetics of both nisin and pediocin production and characterization of their functional properties[J]. Process Biochemistry, 2002, 37: 1005–1015.
[45]刘如林,梁凤来,刁虎欣等.光合细菌及其应用[M].北京:中国农业科技出版社, 1991.
[46] Weaver P F, Lien S, Senbert M. Photobiological production of hydrogen [J]. Solar Energy. 1980, (24): 3–45.
[47] Reza Yegani, Satoshi Yoshimura, Kazunori Moriya. Improvement of growth stability of photosynthetic bacterium Rhodobacter capsulatus [J]. Journal of Bioscience and Bioengineering, 2005,100(6): 672-677.
[48] Yong-Zhong Wang, Qiang Liao, Xun Zhu, et al. Characteristics of hydrogen production andsubstrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor [J]. Bioresource Technology, 2010, 101: 4034-4041.
[49] Rostron WM, Stuckey DC, Young AA. Nitrification of high strength ammonia wastewaters: comparative study of immobilization media [J]. Water Res, 2001, 35(5): 1169–1178.
[50]朱瑞艳,王迪, Yaoping Zhang,李季伦.深红红螺菌draTGB hupL双突变株在不同光照条件下的放氢[J].科学通报,2006 ,5 1(17): 2045–2051.
[51] Toshihiko kondo, Masayasu arakawa. Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment [J]. Arch Biochem Biophys, 2002, 93(2): 145-150.
[52] I. -H. Lee, J. Y. Park, D. H. Kho, et al. Reductive effect of H2 uptake and poly-β- hydroxybutyrate formation on nitrogenase-mediated H2 accumulation of Rhodobacter sphaeroides according to light intensity [J]. Appl Microbiol Biotechnol, 2002,60:147-153.
[53]殷幼平,张宝鹏,廖强,等.沼泽红假单胞菌高效产氢hupL缺失突变株的构建[J].中国环境科学, 2009,27(4):413-418.
[54] Larimer F W, Chain P, Hauser L, et al. Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris [J]. Nature Biotechnology, 2004, 22(1): 55-61.
[55] Laurence Casalot, Marc Rousset. Maturation of the [NiFe] hydrogenases [J]. Microbiology, 2001,9(5) :228-237.
[56] Paulette M. Vignais, Jean-Pierre Magnin, John C. Willison. Increasing biohydrogen production bymetabolic engineering [J]. International Journal of Hydrogen Energy, 2006, 31(7): 1478-1483.
[57] Federico E. Rey, Yasuhiro Oda, and Caroline S. Harwood. Regulation of Uptake Hydrogenase and Effects of Hydrogen Utilization on Gene Expression in Rhodopseudomonas palustris [J]. Journal of Bacteriology. 2006, 188(17): 6143-6152.
[58] Eilert Hustede, Alexander Steinbiichel, Hans G. Schlege. Relationship between the photoproduction of hydrogen and the accumulation of PHB in non-sulphur purple bacteria [J]. Appl Microbiol Biotechnol, 1993,39:87-93.
[59] Deniz Ozgur Yigit, Ufuk Gunduz, Lemi Turker, et al. Identi cation of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O. U. 001 grown in the waste water of a sugar re nery [J]. Journal of Biotechnology, 1999,4:25–131.
[60] Roberto De Philippis, Alba Ena, Monica Guastini, et al. Factors affecting poly-β- hydroxybutyrate accumulation in cyanobacteria and in purple non-sulfur bacteria [J]. Microbiol Rev, 1992,103:187-194.
[61]岳文洁,夏元生,张小凡.红色非硫磺菌生长及聚β-羟基丁酸积累的研究[J].环境与可持续发展, 2008,3(2):18-20.
[62] Kim JH, Lee JK. Cloning, nucleotide sequence and expression of gene coding for polyhydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 [J]. Microbiol Biotechnol, 1997,7:229–236.
[63] Anderson AJ, Dawes EA. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxy alkanoates [J]. Microbiol Rev, 54:450–472.
[64] Yasuhiro Oda, Sudip K. Samanta, Federico E. Rey, et al. Functional Genomic Analysis of Three Nitrogenase Isozymes in the Photosynthetic Bacterium Rhodopseudomonas palustris[J]. Journal of Bacteriology. 2005, 187(22): 7784-7794.
[65] Mi-Sun Kim, Jin-Sook Baek, Jeong K. Lee. Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase de?cient mutant [J]. International Journal of Hydrogen Energy, 2006,31:121-127.
[66]王永忠,廖强,朱恂,等.底物初始浓度对光合细菌产氢动力学特性的影响[J].环境工程学报, 2007,7(1):125-129.
[67]王中康,胡小容,廖强等.几株紫色非硫细菌的分离鉴定与产氢特性分析[J].应用与环境生物学报, 2009, 15(1):120-124.
[68] Simon. R., U. Priefer, and A. Puhler. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gramnegative bacteria[J]. Bio/Technology 1983, 1: 784-791.
[69]吴海珍,李莉,张惠展,刘和.荚膜红细菌接合转移系统的建立及优化[J]华东理工大学学报2000-02 Vol.26 No.1.
[70] Davis J , Donohue I J , Kaplan S. Construction characterization, and complementation of a duf mutant of Rhodobacter Sphaeroides [J]. J Bacterio l, 1988, 170: 320-329.
[71] Klipp W., M asepoh B, Puhler A. Identification and mapping of nitrogen fixation of Rhodobacter capsulatus: duplication of nif A-nif B region [J]. J Bacteriol,1998.170: 693-699.