Distribution,Enrichment And Influence Factors Of Denitrifying Anaerobic Methane Oxidizing Microbes In Taihu Lake

Denitrification Anaerobic Methane Oxidation（DAMO）is a biochemical process with methane as the electron donor and nitrate/nitrite as the electron acceptor,which plays an important role in the reduction of methane emission in the natural environment.Compared with the traditional sewage denitrification process（which requires an additional organic carbon source）,the DAMO process can utilize the methane generated by the anaerobic digestion of sludge in situ,thereby realizing the recycling of energy.On one hand,the ecological distribution and ecological functions of DAMO processes in the natural environment are not fully understood.On the other hand,the DAMO microbial enrichment process is lengthy,and easily affected by external conditions.Therefore,its application in biological denitrification of sewage is limited.Taihu Lake,one of the five major freshwater lakes in China,was the research object of this article.The distribution characteristics of DAMO microorganisms in the sediments of Taihu Lake was explored.And the survival preference of DAMO microbes in different eutrophic regions was analyzed.After that,some reactors were built in the laboratory to compare the effects of carbon source,electroactive medium addition and different influence factors on the DAMO microbial enrichment process.The main findings of this paper were as follows:（1）The survival characteristics of DAMO microbes in Taihu Lake were evaluated.The results of qPCR and high-throughput sequencing showed that The abundance of DAMO bacteria was 1.82×10⁴-6.11×10⁶copies g^-1sediment,which was the highest in D1 sample of the mildly eutrophic region.The relative abundance of DAMO archaea was relatively high in D7 and D8 samples of the moderately eutrophic region.When constructing a microbial network and analyzing the interaction between DAMO bacteria/archaea and other methane/nitrogen cycling microorganisms in different eutrophic lakes,it was found that DAMO bacteria and DAMO archaea showed different survival preference.DAMO bacteria showed an advantage in competition with other microorganisms and became key species in the mildly eutrophic region.Comparatively,DAMO archaea was more suitable to survive in moderately eutrophic lake region where they show stronger cooperation with other methane/nitrogen-cycling microorganisms.Meanwhile,DAMO microbes were affected by antagonism of Rhodocyclaceae and Anammox bacteria,and DAMO bacteria were more affected than DAMO archaea by Anammox bacteria.Additionally,structural equation model analysis showed that p H,DO,geospatial,nitrite and sulfate had positive effects on DAMO bacterial abundance,while microbial community diversity and Anammox bacteria had negative effects on DAMO bacterial abundance influence within the actual detection range.Geographic space and nitrate had significant positive effects on DAMO archaea,and community diversity had negative effects on DAMO archaea.（2）The enrichment effects of DAMO microbes under different conditions were compared.In the biochar-added R4 reactor,the enrichment of bacterial population contained the highest proportion of DAMO microbes,and the r CH₄/r NO₂in the system is closest to the stoichiometric ratio of the DAMO reaction.In the reactors with sodium acetate added（R2 and R3）,the proportion of DAMO microbes was also significantly higher than that in the control reactor（R1）,indicating that both biochar and sodium acetate additions in this study could promote the enrichment of DAMO microbes.In addition,high-throughput sequencing showed that the DAMO bacteria in R4 increased from 0.13%to 7.2%in only 105 days after the stimulation and recovery phase,which meant that biochar made a potential contribution in helping DAMO bacteria adapt to changes in the system’s environment.The results of isotope activity verification showed that the addition of sodium acetate and biochar could stimulate the activity of DAMO.Moreover,the stimulation effect of biochar on DAMO microbial activity was the most significant,and the stimulation effect of 0.1 mmol/L sodium acetate was better than that of 0.5 mmol/L sodium acetate.Cyclic voltammetry and extracellular polymer analysis found that the addition of sodium acetate and biochar could improve the electrochemical activity of the system,promote the secretion of polysaccharides and proteins,thereby accelerating the enrichment of DAMO microbes.In addition,combining scanning electron microscopy and Fourier transform infrared spectroscopy analysis,it was found that the high specific surface area and pore volume of biochar could provide attachment sites for microorganisms,which helped slow-growing microorganisms to form biofilms.The oxygen-containing unsaturated functional groups in biochar,especially the carbon-oxygen double bond,played an important role in the electron transfer/storage of biochar.（3）The activity changes of DAMO microbes under different influence factors were explored.With the increase of temperature（25℃-35℃）,the activity of n-DAMO and the proportion of DAMO bacteria increased significantly,indicating that the optimal temperature of DAMO bacteria was 35℃under the experimental conditions.The trace element Ni²⁺had no significant effect on the n-DAMO activity,while Fe²⁺could promote the n-DAMO activity.The denitrification activity of DAMO bacteria was the highest under the condition of 20μmol/L Fe²⁺,and the mean consumption rate of nitrite was increased by 22.6%compared to the control group.In addition,within a certain range,the n-DAMO activity increased with the improvement of methane partial pressure,and the optimal methane partial pressure under this experimental condition was 98 k Pa.In conclusion,this study elucidated the distribution characteristics and survival strategies of DAMO microbes in Taihu Lake sediments.Thus,biotic and abiotic factors affecting DAMO microbial abundance were identified.And through long-term enrichment experiments and short-term isotope labeling experiments,it was verified that the addition of an appropriate amount of biochar and sodium acetate had a certain stimulating effect on the DAMO process.In addition,the optimal environmental conditions of DAMO microbes were preliminarily explored through influencing factor experiments.The results were helpful to systematically understand the ecological functions of DAMO microbes and provide a theoretical basis for the potential application of DAMO in biological denitrification of wastewater.