【作者】 田远明；

【导师】 王中康；

【作者基本信息】 重庆大学 ， 生物学， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原