Microbial Remediation And Mechanism Study Of Nitrate/Fe(?) Combined Contaminated Groundwater

With the rapid development of social economy and industry,groundwater nitrate/Fe???combined contamination has become a serious environmental problem that threatens the safety of drinking water.However,the interaction mechanism between the two contaminants in the anaerobic treatment process is unclear.In this study,heterotrophic denitrifying bacteria were used for simultaneous denitrification and Fe???oxidation.The interaction and mechanism of the two contaminants in the simultaneous removal process were discussed.This study provides a new approach for the remediation of nitrate/Fe???combined contaminated groundwater.First,the ability of simultaneous denitrification and oxidation of Fe???by heterotrophic denitrifying bacteria was confirmed at different concentrations of nitrate and Fe???with synthetic groundwater.Only when Fe???concentration reaching 200mg/L,the activity of heterotrophic denitrifying bacteria was inhibited.Under other Fe???concentration conditions??100 mg/L?,nitrate removal efficiency all reached around 100%.The oxidation efficiency of Fe????12.5%-100%?is positively correlated with the initial concentration and reduction of nitrate.Then,the optimal temperature of24.93^oC,pH 7.23 and C/N ratio of 1.43 for the denitrification coupling with Fe???oxidation were obtained by Box-Behnken design and response surface methodology?RSM?.Besides,real groundwater was used to verify the capacity for heterotrophic denitrifiers coupling denitrification with Fe???oxidation,and the reaction rate was higher than that of synthetic groundwater.It was identified in the above studies that the metabolic activity of microorganisms,the diversity,evenness and richness of microbial community could be promoted when the concentration of Fe???was low.Comparably,under high Fe???concentration conditions,the denitrification process exhibited a significant lag phase and the nitrite accumulation increased during the reaction.The high concentration of Fe???in the system is of toxicity to microorganisms.Through the analysis of microbial electron transport system activity?ETSA?,it was found that the inhibitory concentration of Fe???on heterotrophic denitrifying bacteria was 13.84 mg/L and the median inhibitory concentration(IC₅₀)was 45.19mg/L.The wheat-rice stone was added into the reaction system to produce a variety of metal elements necessary for microorganisms such as Ca,Mn,Mo etc.,which could significantly improve the microbial denitrification rate and the resistance to stress.By the addition of phosphate rock into the system providing phosphorus source,the denitrification capacity of microorganisms was also improved.At the same time,it was found that dosage of natural minerals?wheat-rice stone,phosphate rock?can effectively promote the diversity and richness of microbial communities.The relative quantification of the genes of denitrification-related enzymes?narG,napA,nirS and nirK?at different reaction times was carried out.The inhibitory effect of Fe???on gene expression of denitrification-related enzyme occurred at a high concentration of 100 mg/L;when Fe???concentration was 50 mg/L,gene expression delayed but was not significantly inhibited.The decrease in denitrification rate at 50mg-Fe???/L attribute to the influence of Fe???on the translation process or inhibition on the activity of denitrifying enzymes.Through the high throughput sequencing analysis,microbial community structure?phylum level?was more similar in composition than percentage of each phylum.The phylum structure was similar under different conditions,while the proportion of each phylum was different.The most predominant phylum was Betaproteobacteria?relative abundance 39.9%-84.1%?,followed by Bacteroidetes?relative abundance 4.7%-16.5%?.For genus level,the most dominant one was Methyloversatilis,a denitrifier able to use C-1 compounds as carbon source and energy source.Other denitrifiers included Denitratisoma,Azonexus,Dechloromonas etc.A comprehensive analysis of microbial communities under different experimental conditions showed that the influence of Fe???and natural minerals on microorganisms was reflected on the level of species composition rather than its functional composition;the experimental conditions had an effect on both genus level structure of microbial community and the proportion of each genus.Distinct microorganisms were both cooperative and competitive,and microbial cooperation and competition constitute the dynamic equilibrium on macroscopic level.In this study,the reaction mechanism and metabolic pathway model of simultaneous denitrification and Fe???oxidation by heterotrophic denitrifying bacteria with methanol as carbon source was obtained.Nitrate reduction and Fe???oxidation reactions were carried out by nitrate reductase in cytoplasm,and the rate of Fe???entrying into cells determines its rate of oxidation.This study revealed the process and reaction mechanism of simultanenous denitrification coupling with Fe???oxidation by heterotrophic denitrifiers,which laid a theoretical foundation for the remediation of nitrate/Fe???combined contaminated groundwater and provided technical support for the application of this process.