| Bacteria in nature can be roughly divided into Gram-positive bacteria and Gramnegative bacteria according to different cell wall compositions.In microbial electrochemistry,Gram-negative bacteria are often the main functional microorganisms(e.g.,Geotrichum spp.,Shiva spp.),so most researches focus on negative bacteria.However,there are also a large number of Gram-positive bacteria in microbial electrochemistry system.In some natural environments,it has also been reported that Gram-positive bacteria have the ability of extracellular electron transfer,but the knowledge of their ecological function and mechanism in microbial electrochemistry system is very limited.Therefore,in this paper,the microbial electrolytic cell was taken as the research object,and the cell walls of Gram-positive bacteria were specifically dissolved by lysozyme,so as to investigate the effects of Gram-positive bacteria in anode biofilm at different stages(start-up and operation)on the current,coulombic efficiency,electron transfer efficiency,gas production and composition,COD removal efficiency,etc.The changes of biofilm structure at start-up and operation stages were studied by using three-dimensional fluorescence,in-situ fluorescence hybridization and laser confocal techniques.With the help of amplicon sequencing technology of q PCR and16 Sr RNA genes,the changes of microbial community composition and relative abundance in biofilm at different stages,and the main metabolic pathways and the ecological network relationship between Gram-positive bacteria and Gram-negative bacteria were established.The main conclusions are as follows:(1)At the start-up stage,the time for the current of the experimental group to reach stability is 2.12 times that of the control group,and the coulombic efficiency and cathode electron transfer efficiency of the experimental group and the control group reach stability in the ninth cycle and the fourth cycle respectively;The results of in situ hybridization showed that the distribution of total bacteria in anode of the experimental group was lower than that of the control group,and the proportion of Gram-positive bacteria(7.75±0.75%)was slightly lower than that of the control group(8.57±0.10%),with no significant difference(P = 0.26),but actinomycetes showed significant difference between the experimental group and the control group(P = 0.046),indicating that adding lysozyme mainly affected Gram-positive bacteria.(2)The current in the stable stage was kept at 3.5-3.7 m A,and there was no significant difference in electron transfer efficiency between coulombic efficiency and cathode.The results of laser confocal scanning and Gram staining showed that adding lysozyme had no significant effect on anode and solution,but mainly affected the proportion of Gram-positive bacteria in cathode,and the relative abundance changed from8.10±0.40% to 2.41±0.43%,with significant difference(P < 0.05),It shows that Grampositive bacteria can affect the formation of cathode biofilm,but have no obvious effect on anode biofilm in stable operation stage.(3)The results of community ecological network analysis show that the ratio of extremely strong negative correlation(-1 ≤r ≤-0.9)in the experimental group at the start-up stage is 36.17%,which is higher than that in the control group(27.92%),which indicates that adding lysozyme at the start-up stage will significantly enhance the negative correlation between gram-positive bacteria and gram-negative bacteria.In the stable stage,lysozyme had little effect on anode,mainly affecting the correlation between Grampositive bacteria and Gram-negative bacteria at cathode,but had little effect on the extremely negative correlation(-1 ≤ r ≤-0.9),which only increased from 0.00% to 2.31%without enzyme,and mainly affected the correlation between Firmicutes and Desulfobacterota.To sum up,this paper studies the role of Gram-positive bacteria in biological film formation from the aspects of the operation effect of the reactor,the structure of biofilm,the change of microbial community structure and relative abundance,and the relative relationship between film-forming microorganisms and Gram-positive bacteria in ecology.It lays a theoretical foundation and provides technical support for rapid film formation in subsequent practical projects. |
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