Metagenomic analysis reveals abundance of mixotrophic, heterotrophic, and homoacetogenic bacteria in a hydrogen-based membrane biofilm reactor.

Heterotrophic microorganisms are frequently observed in hydrogenotrophic denitrification systems and are presumed to contribute to their improved performance. However, their roles and metabolic pathways in the hydrogen-based membrane biofilm reactor (H2-MBfR) system remain unclear. The objective of this study was to elucidate the underlying mechanisms driving heterotrophic denitrification. For this purpose, metagenomic analysis was conducted on an H2-MBfR showing higher denitrification performance, focusing on the metabolic function of the microbial community. Functional genes related to H2 oxidation, organic carbon metabolism, and denitrification were the major targets of interest. This analysis revealed a substantial number of genes associated with the oxidation of organic carbon compounds in the biofilm, suggesting its potential for heterotrophic denitrification. Investigation of the genes of interest in metagenome-assembled genomes (MAGs) has demonstrated a predominance of mixotrophs or heterotrophs rather than obligate autotrophs. Notably, MAGs exhibiting the highest abundance of genes of interest were affiliated with Hydrogenophaga and Thauera, implying their significant role in denitrifying the H2-MBfR as mixotrophs utilizing both H2 and organic substrates. The identification of 11 MAGs, presumed to originate from homoacetogens suggested that acetate might contribute to the proliferation of heterotrophs. Based on these metagenomic findings, possible metabolic pathways were identified to explain heterotrophic denitrification within the H2-MBfR biofilms.

Optimization of nitrogen removal and microbial mechanism of a hydrogen-based membrane biofilm reactor.

The hydrogen-based membrane biofilm reactor (H2-MBfR) is an emerging biological nitrogen removal technology characterized by high efficiency, energy-saving capability, and environmental friendliness. The technology achieves denitrification and denitrogenation of microorganisms by passing hydrogen as an electron donor from inside to outside through the hollow fibre membrane module, and eventually the hydrogen reachs the biofilm attached to the surface of the fibre membrane. H2-MBfR has obtained favourable outcomes in the treatment of secondary biochemical effluent and low concentration nitrogen polluted water source. The experiment was optimized by s single-factor testing and response surface methodology-based optimization (RSM), and the optimal operational conditions were obtained as follows: an influent flow rate of 2 mL/min, hydrogen pressure of 0.04 MPa, and influent nitrate concentration of 24.29 mg/L. Under these conditions, a high nitrate removal rate of 98.25% was achieved. In addition, Proteobacteria and Bacteroidetes were the dominant bacteria in all stages, and the genus Hydrogenophaga was sufficiently enriched, occurring at 13.0%-49.0% throughout the reactor operation. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for nitrate reduction and inorganic carbon utilization by microorganisms in the H2-MBfR was explored through comparison with the KEGG database. The results provided a mechanistic explanation for the denitrification and carbon sequestration capacity of the H2-MBfR.

Biodegradation of nitrate and p-bromophenol using hydrogen-based membrane biofilm reactors in parallel.

ABSTRACTP-bromophenol (4-BP) is a toxic halogenated phenolic organic compound. The conventional treatment processes for 4-BP elimination are costly and inefficient, with complete mineralization remaining a challenge for water treatment. To overcome these limitations, we investigated the treatment of 4-BP in a membrane biofilm reactor (MBfR) using hydrogen as an electron donor. The pathway of 4-BP degradation within the H2-MBfR was investigated through long-term operational experiments by considering the effect of nitrate and 4-BP concentrations, hydrogen partial pressure, static experiments, and microbial community diversity, which was studied using 16S rRNA. The results showed that H2-MBfR could quickly remove approximately 100% of 4-BP (up to 20 mg/L), with minimal intermediate product accumulation and 10 mg/L of nitrate continuously reduced. The microbial community structure showed that the presence of H2 created an anaerobic environment, and Thauera was the dominant functional genus involved in the degradation of 4-BP. The genes encoding related enzymes were further enhanced. This study provides an economically viable and environmentally friendly bioremediation technique for water bodies that contain 4-BP and nitrates.

Liquid-gas phase transition enables microbial electrolysis and H2-based membrane biofilm hybrid system to degrade organic pollution and achieve effective hydrogenotrophic denitrification of groundwater.

Electron-donor Lacking was the limiting factor for the denitrification of oligotrophic groundwater and hydrogenotrophic denitrification provided an efficient approach without secondary pollution. In this study, a hybrid system with microbial electrolysis cell (MEC) assisted hydrogen-based membrane biofilm reactor (MBfR) was established for advanced groundwater denitrification. The liquid-gas phase transition prevented the potential pollution from organic wastes in MEC to groundwater, while the bubble-free diffusion of MBfR promoted hydrogen utilization efficiency. The negative-pressure extraction from MEC and the positive pressure for gas supply into MBfR increased the hydrogen proportion and current density of MEC, and improved the kinetic constant K of the denitrification reaction in MBfR. With actual groundwater, the MEC-MBfR hybrid system achieved a nitrate reduction of 97.8% with an effluent NO3--N of 2.2 ± 1.0 mg L-1. The hydrogenotrophic denitrifiers of Thauera, Pannonibacter, and Azonexus, dominated the denitrification biofilm on the membrane and elastic filler in MBfR.

Insights into selenate removal mechanism of hydrogen-based membrane biofilm reactor for nitrate-polluted groundwater treatment based on anaerobic biofilm analysis.

The selenate removal mechanism of hydrogen-based membrane biofilm reactor (MBfR) for nitrate-polluted groundwater treatment was studied based on anaerobic biofilm analysis. A laboratory-scale MBfR was operated for over 60 days with electron balance, structural analysis, and bacterial community identification. Results showed that anaerobic biofilm had an excellent removal of both selenate (95%) and nitrate (100%). Reduction of Selenate → Selenite → Se0 with hydrogen was the main pathway of anaerobic biofilm for selenate removal with amorphous Se0 precipitate accumulating in the biofilm. The element selenium was observed to be evenly distributed along the cross-sectional thin biofilm. A part of selenate (3%) was also reduced into methyl-selenide by heterotrophic bacteria. Additionally, Hydrogenophaga bacteria of ß-Proteobacteria, capable of both nitrate and selenate removal, worked as the dominant species (over 85%) in the biofilm and contributed to the stable removal of both nitrate and selenate. With the selenate input, bacteria with a capacity for both selenate and nitrate removal were also developed in the anaerobic biofilm community.

Effects of sulfate on simultaneous nitrate and selenate removal in a hydrogen-based membrane biofilm reactor for groundwater treatment: Performance and biofilm microbial ecology.

Effects of sulfate on simultaneous nitrate and selenate removal in a hydrogen-based membrane biofilm reactor (MBfR) for groundwater treatment was identified with performance and biofilm microbial ecology. In whole operation, MBfR had almost 100% removal of nitration even with 50 mg mL-1 sulfate. Moreover, selenate degradation increased from 95% to approximate 100% with sulfate addition, indicating that sulfate had no obvious effects on nitrate degradation, and even partly promoted selenate removal. Short-term sulfate effect experiment further showed that Gibbs free energy of reduction (majority) and abiotic sulfide oxidation (especially between sulfate and selenate) contributed to degradable performance with sulfate. Microbial ecology showed that high percentage of Hydrogenophaga (≥75%) was one of the contributors for the stable and efficient nitrate degradation. Chemoheterotrophy (ratio>0.3) and dark hydrogen oxidation (ratio>0.3) were the majority of functional profile for biofilm in MBfR, and sulfate led to profiles of sulfate respiration and respiration of sulfur compounds in biofilm. Additionally, no special bacteria for selenate degradation was identified in biofilm microbial ecology, and selenate degradation was relied on Hydrogenophaga (75% of ecology percentage with sulfate addition) and Desulfovibrionaceae (4% of ecology percentage with sulfate addition). But with overloading sulfate, Desulfovibrionaceae was prior to sulfate degradation for energy supply and thus inhibited selenate removal.