Relationship Between Community Assembly And Hydrogen Producing Performance Of Anodic Biofilms In Microbial Electrolysis Cells

Microbial electrolysis cell(MEC)has attracted widespread attention as an emerging technique for bio-hydrogen generation.This technology can apply electrode microbes to treat extensive organic substrates under a small external voltage,and eventually produce clean energy such as hydrogen,indicating that it has great potential in the field of organic wastewater treatment.At present,related research has made breakthroughs in reactor configuration and electrode materials.However,there is relatively little research on the structure and assembly mechanisms of microbial communities in this system.Therefore,revealing the relationship between the assembly mechanism of anode biofilm community and hydrogen production performance is of great significance for the industrial application of microbial electrochemical technology.However,due to the complex structure of MEC anodic biofilm communities and the diverse interactions of functional groups,it is impossible to fully analyze the inherent rules using traditional molecular analysis methods.To this end,taking the single-chamber MEC reactor as the research object in this dissertation,the quantitative methodology of microbial community analysis was established by coupling the high-throughput sequencing technology and quantitative results of total environmental microbes;On this basis,the relationship between the community assembly processes involving in biodiversity,functional structure and interaction of anode biofilms and macroscopic performance were further investigated.It aims to elucidate the microbial ecology regularity in functional microorganisms,and provide theoretical guidance for its performance optimization.Main research contents are as follows:Five different microbial quantitative methods were used to compare the total amount of bacteria in soil microbial communities from Beijing and Tibet meadows.The results of four measurements,including adenosine tri-phosphate,flow cytometry,quantitative real-time PCR,and phospholipid fatty acids,showed a good repeatability,where the absolute amount of bacteria in Beijing was significantly higher than that in Tibet,while the microbial biomass carbon quantification showed obvious inconsistency with others.According to the composition analysis of the two sites by Illumina high-throughput sequencing,the Proteobacteria and Actinobacteria were the most abundant bacterial phyla in both Beijing and Tibet grasslands,respectively.Then,the absolute abundance of different populations estimated by coupling the relative abundance with the quantitative results(adenosine tri-phosphate,flow cytometry,quantitative real-time PCR,and phospholipid fatty acids measurements)of total bacteria,indicated distinct trends with the corresponding relative abundance.The results showed that quantitative analysis of microbial communities could reflect the abundance changes of bacterial population more truly by coupling high-throughput sequencing analysis with absolute quantification,providing a theoretical basis for further experiments.The relationship between the community assembly processes of MEC anodic biofilms and reactor performance was investigated.Four groups of MEC reactors were inoculated with gradient diluted activated sludge,which was collected from wastewater treatment plant,and showed obvious different performance under identical operational conditions.With the decrease of the inoculation concentration,the reactor performance showed a trend of increasing first and then decreasing,where the optimal performance of hydrogen production(1.24 ± 0.09 mL H2/mL reactor cycle)was obtained by 102-time dilution.Two different measurements,including quantitative PCR and volatile suspended solids,were used to quantify the biofilm biomass on the anode.The results of both two methods showed that there was no obvious difference in the microbial biomass of biofilms operated with diluted inoculations.However,the biodiversity of the anodic biofilm communities reduced with the decrease of inoculated concentrations,and the significant difference in community structure was also observed.Null model analysis indicated that both deterministic and stochastic processes played important roles in controlling the assembly of the anodic biofilm communities in MECs,and the most important ecological process(relative ratio>50%)in shaping the microbial diversity changed from stochasticity to determinism with the increasing dilution ratio of inoculations.When deterministic assembly dominates this formation,the higher diversity of biofilm community would generally show better reactor performance.However,when the stochasticity dominates the assembly process,the bioreactor performance would decline.Different environmental samples,including the activated sludge from reclaimed water plant,effluent of bioelectrochemical treatment device,soil,artificial wetland sediment and the marine sediment,were collected and inoculated MEC reactor with identical microbial biomass.The reactor inoculated from the artificial wetland sediment sample had the longest start-up time(18 days)but had the best hydrogen production performance(1.10 ± 0.22 mL H2/mL reactor cycle),while the hydrogen production rate was the slowest after inoculation by marine sediment(0.88 ± 0.17 mL H2/mL reactor cycle).The influence of the inherent differences on structure and functions of biofilm communities was investigated using the high-throughput sequencing with 16S rRNA amplicon.The great difference of those inoculated sources led to the significant difference in the structure and composition on the anodic biofilm communities,besides,the differences of the biofilm communities decreased with the identical operational conditions.Under the selective pressure of reactor operation,the biofilm communities of each group presented significantly changes,where the Geobacter as the main electrogenerating were enriched to be the dominant bacteria genes.The whole genome sequencing results of biofilm samples in each reactor showed that the community differences of different habitat sources ultimately led to the functional differences in the construction of anode biofilm communities.The resilience of MEC reactor to environmental disturbance,the response mechanism of the community structure and microbial interactions in anode biofilms were investigated.The reactor was subjected to a short-term impact by adding a high concentration of the antibiotic florfenicol(FLO),which caused the reactor to lose its stable hydrogen production capacity.After the addition of FLO was stopped,the performance of the reactor could be restored to a stable performance for a period of time,and it was improved(19.5%)compared with that before the impact,showing a strong recovery ability.The FLO perturbation showed a significant influence on the electroactive biofilms with a distinct reformation of the community structure.The Geobacter was the most dominant genus with the relative abundance more than 60%,and showed no significant changes during the experiment period.The molecular ecological network analysis indicated that the microbial interactions in the biofilm communities become much tighter after recovery than those before disturbance,in which the 91.6%increase of positive correlations with the predominant genus Geobacter,was more than that of negative links increased by 32.73%.In addition,some non-electrogenic microorganisms become keystone species in response to the FLO disturbance,of which the synergistic interactions with other microorganisms could facilitate the restoration of the community structure and function.