Bioremediation of nitrate and Fe(ii) combined contamination in groundwater by heterotrophic denitrifying bacteria and microbial community analysis

Abstract

Species of denitrifying bacteria are capable of nitrate reducing and Fe(II) oxidizing. The optimal temperature, pH and C/N ratio for denitrification coupled with Fe(II) oxidation in circumneutral anaerobic groundwater was investigated by a Box–Behnken design and response surface methodology. The microbial diversities were analyzed by high-throughput sequencing. The results showed that the optimal condition was achieved at a temperature of 24.93 °C, pH of 7.23 and C/N ratio of 1.43, at which almost 100% of nitrate-N and Fe(II) was removed with little byproduct accumulation. Within the temperature range 15–25 °C, pH 7–8.25 and C/N ratio 1.23–1.47, both nitrate and Fe(II) concentration could be removed effectively. The distributions of the microbial taxonomy composition (e.g., phylum, genus) differed under different temperatures, pH values and C/N ratios. Betaproteobacteria and Bacteroidetes dominated the bacterial phyla, and Methyloversatilis was the predominant bacterial genus in the enrichment cultures. Complex species of bacteria were attributed to the sophisticated reaction system. Though heterotrophic denitrification and Fe(II) oxidation dominated the system, autotrophic denitrification, H₂ and acetate production may, somehow, have contributed to the entire reaction system. Denitrifiers (e.g., Azonexus), bacteria capable of nitrate dependent Fe(II) oxidation (NDFO) (e.g., Dechloromonas) and bacteria which could produce acetate as co-substrates for NDFO (e.g., Acetobacterium) were unevenly distributed. These findings provide support for nitrate and Fe(II) contaminated groundwater bioremediation, and a better understanding of the microbial ecosystem of the enriched heterotrophic denitrifiers.