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

在闭路水生态系统中，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，对生态系统的居民来说往往达到有毒的水平。封闭生态系统的废水生物过滤单元需要增加一个反硝化步骤。反硝化作用发生在细菌细胞中，硝酸盐在氮气中被还原，并在氧气耗尽过程中作为终端电子受体而不是氧气(O2)来产生能量。虽然我们已经很好地掌握了反硝化系统的工程组成部分，但我们对微生物群落如何在这些系统中运行的了解是不完整的，特别是在海洋/盐分条件下。我的实验室一直在研究从反硝化系统中获得的海洋甲基营养反硝化生物膜的微生物群落。我们发现，该群落由60%至80%的甲基噬菌属和下位微生物属的甲基营养细菌组成。我们从这个生物膜中分离到了两株负责反硝化活性的菌株，甲基食腐杆菌NAM1和亚硝酸盐次生微生物NL23。菌株JAM1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NL23可以完成完整的反硝化途径，但不能在盐碱条件下的纯培养中生长，这一点很特殊，因为它来自海洋系统。然而，共同培养这两个菌株允许在海洋条件下发生充分的反硝化作用，这表明这两个菌株之间的合作。反硝化生物膜在不同的pH、温度以及不同浓度的甲醇、硝酸盐和氯化钠条件下都能保持反硝化活性，对环境变化具有很强的适应性。我们还证明了异养非甲基营养反硝化细菌在生物膜中的存在，这表明生物膜具有适应异养、非甲基营养环境的潜力。综上所述，这些结果表明我们的海洋甲基营养反硝化生物膜在适应变化的环境中具有可塑性。我的研究计划的总体目标是确定反硝化系统运行条件的变化如何影响菌株JAM1和NL23，并评估社区其他成员在支持该系统的弹性方面的重要性。对海洋条件下反硝化作用的深入研究还相对较少，尤其是针对这些独特条件下的甲基营养细菌的研究更是如此，这使得我的研究项目具有很高的原创性。更好地了解生物膜中的微生物群落如何适应变化将导致开发更稳定和高效的反硝化系统，如闭路系统或海水养殖中的系统。

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.