Resilience of a marine, methylotrophic denitrifying biofilm to environmental changes in a lab-scale denitrification system.

在封闭的循环水生态系统中，如水族馆或水产养殖池，硝酸盐的快速增加是一个常见的问题，往往达到对生态系统的居民有毒的水平。废水生物过滤单元封闭的生态系统需要增加一个反硝化步骤。反硝化作用发生在细菌细胞中，其中硝酸盐在N2中被还原，并在O2耗尽期间作为末端电子受体而不是氧气（O2）用于能量产生。虽然反硝化系统的工程组成部分是很好地掌握，我们的理解如何在这些系统中的微生物群落的运作是不完整的，特别是在海洋/盐条件下。本实验室一直在研究海洋甲基营养型反硝化生物膜的微生物群落特征。结果表明，该群落中60%~ 80%的甲基营养细菌属于嗜甲基菌属和丝微菌属。我们从该生物膜中分离出两种菌株，Methylophaga nitratireducenticrescens JAM 1和Hyphomicrobium nitrativorans NL 23，它们负责分解活性。菌株JAM 1具有部分反硝化途径。它能够将硝酸盐还原为亚硝酸盐，但不能将亚硝酸盐还原为一氧化氮。菌株NL 23可以完成完整的反硝化途径，但不能在纯培养物中在盐条件下生长，这是特殊的，因为它起源于海洋系统。然而，共培养这两种菌株允许在海洋条件下发生完全反硝化，这表明两种菌株之间的合作。我们还表明，生物膜是非常有弹性的变化，因为它可以维持在不同的pH值和温度，并在不同浓度的甲醇，硝酸盐和NaCl的生物活性。我们还证明了在生物膜中存在异养的非甲基营养型硝化细菌，这表明生物膜具有适应异养的非甲基营养型环境的潜力。总之，这些结果表明，我们的海洋甲基营养型生物膜的可塑性，以适应不断变化的环境。我的研究计划的总体目标是确定反硝化系统的操作条件的变化如何影响菌株JAM 1和NL 23，并评估社区其他成员在支持系统恢复力方面的重要性。关于海洋条件下反硝化作用的深入研究仍然相对较少，尤其是在这些独特条件下关注甲基营养菌的研究，这使得我的研究计划具有很强的原创性。更好地了解生物膜中的微生物群落如何适应变化，将有助于开发更稳定、更有效的脱氮系统，如闭路系统或海水养殖系统。

In closed circuit water ecosystems, such as aquarium or aquaculture tanks, rapid increases in nitrate is a common problem, often reaching toxic levels for the ecosystem's inhabitants. Wastewater biofiltration units to closed ecosystems requires addition of a denitrification step. Denitrification takes place in bacterial cells where nitrate is reduced in N2 and serve as terminal electron acceptors instead of oxygen (O2) for energy production during O2 depletion. Although the engineering component of denitrification systems is well mastered, our understanding of how the microbial community operates in these systems is incomplete, especially under marine/salty conditions. My laboratory has been characterizing the microbial community of marine methylotrophic denitrifying biofilm obtained from a denitrification system. We found that the community is composed of 60% to 80% of methylotrophic bacteria affiliated to Methylophaga and Hyphomicrobium. We isolated two strains, Methylophaga nitratireducenticrescens JAM1 and Hyphomicrobium nitrativorans NL23, from this biofilm that are responsible for the denitrifying activities. Strain JAM1 has a partial denitrification pathway. It is capable of reducing nitrate to nitrite, but not reducing nitrite to nitric oxide. Strain NL23 can accomplish the full denitrification pathway, but cannot grow in pure cultures under saline conditions, which is peculiar because it originates from a marine system. However, co-culturing these two strains allows full denitrification to occur under marine conditions, suggesting cooperation between the two strains. We also showed that the denitrifying biofilm was very resilient to changes as it could sustain denitrifying activities at different pH and temperatures, and at different concentrations of methanol, nitrate and NaCl. We also demonstrated the presence of heterotrophic non-methylotrophic denitrifying bacteria in the biofilm suggesting that the biofilm has the potential to adapt to heterotrophic, non-methylotrophic environments. Taken together, these results demonstrate the plasticity of our marine methylotrophic denitrifying biofilm in adapting to changing environments. The general aim of my research program is to determine how changes in the operating conditions of a denitrification system can affect strains JAM1 and NL23, and to assess the importance of other members of the community in supporting the resilience of the system. In-depth studies on denitrification under marine conditions remain relatively scarce, even more so those focusing on methylotrophic bacteria under these unique conditions, which makes my research program highly original. Better understanding of how the microbial community in a biofilm adapts to changes will lead to the development of more stable and efficient denitrification systems, such as those found in closed-circuit systems or in marinocultures.