Nitrogen fixation and hydrogen production by photosynthetic bacteria

光合细菌固氮产氢

• 批准号: RGPIN-2014-04515
• 负责人: Hallenbeck, Patrick
• 金额: $ 1.89万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566571
• 关键词: Nitrogen; fixation; hydrogen; production; photosynthetic

The non-sulfur purple photosynthetic bacteria serve as excellent model organism for research on a series of fundamental and applied basic biological areas of interest; photosynthesis, nitrogen fixation, and biotechnological conversions. The photosynthetic bacteria are thus excellent organisms for studying both general and unique aspects of nitrogen fixation and its regulation. Besides the regulation at the transcriptional level with respect to oxygen and fixed nitrogen shared by most nitrogen fixing organisms, the photosynthetic bacteria are capable of regulating the activity of preformed enzyme through the covalent and metabolic regulation of nitrogenase. We have shown that the regulation of nitrogenase with respect to the sudden external addition of ammonium requires the membrane bound ammonia sensor/transporter AmtB. Amt/Rh family members are found in almost every sequenced organism and are present in archaea, bacteria, protists, plants, fungi and invertebrates. We will continue our investigations into the structure/function of AmtB and its role in controlling the the nitrogenase regulatory system. In particular, we will be applying site directed mutagenesis to understanding the role that specific amino acids play in ammonia sensing and/or transport. The nitrogenase enzymatic machinery is also responsible for hydrogen production by photosynthetic bacteria. These organisms, which have the potential capacity to use a variety of feedstocks, are well known for their light driven conversion of organic acids to hydrogen and carbon dioxide. Thus, they are ideal candidates for two stage or co-culture systems which derive additional hydrogen from the effluents of dark fermentations. In addition, various industrial and agricultural waste streams rich in organic acids can potentially serve as substrates for photofermentation. We have recently demonstrated a number of improvements in hydrogen production including the demonstration of the stoichiometric conversion to hydrogen of glycerol produced as a waste product of biodiesel manufacture and the generation of appreciable amounts of hydrogen from various molasses fractions. In a series of investigations we have also recently shown that significant amounts of hydrogen, up to 9 moles of H2 per mole of glucose, can be obtained in a one step, single stage fermentation of glucose. Of some interest, we have also demonstrated for the first time the production of hydrogen thought microaerobic fermentation. This process, which promises to enable the release of additional hydrogen from substrates by coupling further degradation with the generation of energy from limited respiration, will be further investigated under the present grant. This technology is of interest in the broader context of industrial biotechnology as it would allow the development of “unbalanced’ fermentations. Two approaches will be used; development of bioreactor technology using a redox stat to control the oxygen levels for achieving maximum substrate conversion, and metabolic engineering to increase metabolic flux in the desired direction. Metabolic engineering will also be used, in conjunction with metabolic modeling to further increase photofermentative hydrogen production. Thus, the current research is of both fundamental and applied interest. Further research on the molecular details of the involvement of AmtB in nitrogenase regulation should shed more light on this unique form of metabolic regulation. In addition, increasing hydrogen production through both physiological manipulation and metabolic engineering will pave the way for the production of other chemicals by these organisms as well as possibly providing a practical means of sustainable hydrogen generation from various waste materials.

无硫紫色光合细菌是一系列基础和应用基础生物学领域研究的优良模式生物；光合作用、固氮和生物技术转化。因此，光合细菌是研究固氮及其调节的一般和独特方面的优秀生物体。除了大多数固氮生物所共有的关于氧和固定氮的转录水平调节之外，光合细菌还能够通过固氮酶的共价和代谢调节来调节预先形成的酶的活性。我们已经表明，固氮酶对突然外部添加铵的调节需要膜结合氨传感器/转运蛋白 AmtB。 Amt/Rh 家族成员几乎存在于所有已测序的生物体中，并且存在于古细菌、细菌、原生生物、植物、真菌和无脊椎动物中。我们将继续研究 AmtB 的结构/功能及其在控制固氮酶调节系统中的作用。特别是，我们将应用定点诱变来了解特定氨基酸在氨传感和/或运输中发挥的作用。 固氮酶酶机械还负责光合细菌产生氢气。这些生物体具有使用各种原料的潜在能力，以其光驱动的有机酸转化为氢气和二氧化碳而闻名。因此，它们是两阶段或共培养系统的理想候选者，该系统从暗发酵的流出物中获取额外的氢气。此外，富含有机酸的各种工业和农业废物流可以作为光发酵的底物。我们最近展示了氢气生产方面的许多改进，包括展示了作为生物柴油生产废品生产的甘油化学计量转化为氢气，以及从各种糖蜜馏分中产生大量氢气。在一系列研究中，我们最近还表明，在葡萄糖的一步、单阶段发酵中可以获得大量的氢气，每摩尔葡萄糖最多可产生 9 摩尔的氢气。有趣的是，我们还首次展示了通过微氧发酵产生氢气的方法。该过程有望通过进一步降解与有限呼吸产生的能量相结合，从而从底物中释放出额外的氢，该过程将在目前的资助下进行进一步研究。这项技术在更广泛的工业生物技术背景下很受关注，因为它将允许发展“不平衡”发酵。将使用两种方法；开发生物反应器技术，使用氧化还原状态来控制氧气水平以实现最大底物转化，以及代谢工程以增加所需方向的代谢通量。还将使用代谢工程，与代谢模型结合以进一步增加 光发酵产氢。因此，当前的研究既具有基础意义又具有应用意义。对 AmtB 参与固氮酶调节的分子细节的进一步研究应该可以更好地阐明这种独特的代谢调节形式。此外，通过生理操作和代谢工程增加氢气产量将为这些生物体生产其他化学物质铺平道路，并可能提供一种实用的方法 从各种废料中可持续地产生氢气。