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首页> 外文学位 >Improvements in Fermentative Hydrogen Production through Physiological Manipulation and Metabolic Engineering.
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Improvements in Fermentative Hydrogen Production through Physiological Manipulation and Metabolic Engineering.

机译：通过生理操纵和代谢工程改善发酵产氢。

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摘要

Biological hydrogen (H2) production represents a possible technology for the large scale sustainable production of H 2 needed for a future hydrogen economy. However, the major obstacle to developing a practical process has been the low yields that are obtained, typically around 25%, well below those achievable for the production of other biofuels from the same feedstock. The goal of this thesis was to improve H 2 production through physiological manipulation and metabolic engineering.;One investigated hypothesis was that H2 production could be improved and made more economical by using a microaerobic dark fermentation process since this could provide the extra reducing power required for driving substrate conversion to completion and hence more H2 production might be obtained without using light energy. The optimal O2 concentrations for microaerobic H2 production were examined as well as the impact of carbon and nitrogen sources on the process. The research reported here proved the capability of Rhodobacter capsulatus JP91 hup- (an uptake-hydrogenase deficient mutant) to produce H2 under microaerobic dark conditions with limiting amounts of O2 and fixed nitrogen. Further work should be undertaken to increase H2 yields using this technology.;In addition, a photofermentation process was established to improve H 2 yield from glucose using R. capsulatus JP91 hup- strain either in batch and/or continuous culture conditions. Some technical challenges in establishing the proper operational conditions for increased H2 yield were overcome. A maximum yield of 3.3 mols of H2/ mol of glucose was found for batch cultures whereas in continous cultures it was 10.3 mols H2/ mol glucose, much higher than previously reported and close to the theoretical maximum value of 12 mols H2/ mol glucose. In batch cultures the maximum light conversion efficiency was 0.7% whereas it was 1.34% in continuous cultures with a maximum conversion efficiency of the heating value of glucose of 91.14%. Various approaches to further increasing yields in photofermentation processes are proposed. The overall results suggest that an efficient single stage photofermentative H2 production process from glucose using continuous cultures in photobioreactors could be developed which would be a much more promising alternative process to the previously studied two stage photofermentation or co-culture approaches.;Furthermore, the heterologous expression of hydrogenases was used as a metabolic engineering strategy to improve fermentative H2 production. The capability of expressing a hydrogenase from one species with the maturation genes from another was examined. One strategy demonstrated that the orphan hydA of R. rubrum is functional and active when co-expressed in E. coli with hydE, hydF and hydG from different organisms. Co-expression of the [FeFe]-hydrogenase structural and maturation genes in microorganisms that lack a native [FeFe]-hydrogenase can successfully result in the assembly and biosynthesis of active hydrogenases. However, other factors may be required for significantly increased protein yields and hence the specific activity of the recombinant hydrogenases.;Another strategy was to overexpress one of the highly active [FeFe]- hydrogenases in a suitable E. coli host strain. Expression of a hydrogenase that can directly interact with NADPH is desirable as this, rather than reduced ferredoxin, is naturally produced by its metabolism. However, the successful maturation of this type of hydrogenase in E. coli had not been previously reported. The Desulfovibrio fructosovorans hnd operon (hndA, B, C, and D genes), encoding a NADP-dependent [FeFe]-hydrogenase, was expressed in various E. coli strains with the maturation genes hydE, hydF and hydG of Clostridium acetobutylicum. Hydrogenase activities were detected in vitro, thus a multi-subunit NADP-dependent [FeFe]-active hydrogenase was successfully expressed and matured in E. coli for the first time. Future research could lead to the expression of this hydrogenase in E. coli host strains that overproduce NADPH, setting the stage for increased hydrogen yields via the pentose phosphate pathway.;Keywords: Hydrogen production, Photofermentation, Microaerobic dark fermentation, Heterologous expression of hydrogenases.
机译：生物氢气（H2）的生产代表了未来氢气经济所需的大规模可持续生产H 2的可能技术。但是，开发实用方法的主要障碍是所获得的低产率，通常约为25％，远低于从相同原料生产其他生物燃料所能达到的产率。本论文的目的是通过生理操纵和代谢工程来提高H 2的产生。；一个研究的假设是通过使用微需氧的暗发酵工艺可以提高H 2的产生并使其更经济，因为这可以提供H2产生所需的额外还原能力。驱动底物转化为完成反应，因此无需使用光能即可获得更多的氢气产量。检查了用于产生好氧的最佳氧气浓度，以及碳和氮源对工艺的影响。此处报道的研究证明了荚膜红细菌JP91 hup-（一种摄取氢酶的缺陷型突变体）在微需氧的黑暗条件下（具有少量的O2和固定的氮）产生H2的能力。使用该技术应进一步开展工作以提高H2的产量。此外，采用光发酵工艺，通过在分批和/或连续培养条件下使用荚膜罗氏杆菌JP91 hup-train提高葡萄糖的H 2产量。克服了在建立适当的操作条件以提高氢气产量方面的一些技术难题。对于分批培养，发现最大产率为3.3 mol H2 / mol葡萄糖，而在连续培养中，最高产率为10.3 mol H2 / mol葡萄糖，比先前报道的要高得多，并且接近理论最高值12 mol H2 / mol葡萄糖。在分批培养中，最大的光转换效率为0.7％，而在连续培养中为1.34％，其中葡萄糖热值的最大转换效率为91.14％。提出了在光发酵过程中进一步增加产量的各种方法。总体结果表明，可以开发在光生物反应器中使用连续培养物从葡萄糖产生有效的单阶段H2发酵过程的方法，这将是以前研究的两阶段光发酵或共培养方法的一个更有希望的替代方法。氢化酶的表达被用作代谢工程策略以提高发酵H2的产生。检验了从一种物种表达具有成熟基因的氢化酶的能力。一种策略证明，当与来自不同生物体的hydE，hydF和hydG在大肠杆菌中共表达时，红球红球菌的孤品hydA具有功能性和活性。在缺乏天然[FeFe]-加氢酶的微生物中，[FeFe]-加氢酶的结构和成熟基因的共表达可成功导致活性加氢酶的组装和生物合成。但是，可能还需要其他因素来显着提高蛋白质产量，从而提高重组氢化酶的比活性。另一策略是在合适的大肠杆菌宿主菌株中过表达一种高活性[FeFe]-氢化酶。可以直接与NADPH相互作用的加氢酶的表达是理想的，因为它不是通过其代谢而天然产生的还原铁氧还蛋白。但是，以前没有报道过这种类型的氢化酶在大肠杆菌中的成功成熟。编码NADP依赖性[FeFe]氢化酶的Desulfovibrio fructosovorans hnd operon（hndA，B，C和D基因）在各种大肠杆菌菌株中表达，并带有丙酮丁醇梭菌的成熟基因hydE，hydF和hydG。体外检测到氢化酶活性，因此首次成功在大肠杆菌中成功表达了多亚基NADP依赖性[FeFe]活性氢化酶并使其成熟。未来的研究可能会导致这种氢化酶在过量生产NADPH的大肠杆菌宿主菌株中表达，从而为通过戊糖磷酸途径增加氢气产量奠定了基础。关键词：氢的产生，光发酵，微需氧的黑暗发酵，异源表达的氢酶。

著录项

  • 作者

    Abo-Hashesh, Mona Talaat Aly.;

  • 作者单位

    Universite de Montreal (Canada).;

  • 授予单位 Universite de Montreal (Canada).;
  • 学科 Biology Microbiology.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 245 p.
  • 总页数 245
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 肿瘤学;
  • 关键词

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