Molecular characterization of a microbial consortium involved in methane oxidation coupled to denitrification under micro‐aerobic conditions

Abstract

Methane can be used as an alternative carbon source in biological denitrification because it is nontoxic, widely available and relatively inexpensive. A microbial consortium involved in methane oxidation coupled to denitrification (MOD) was enriched with nitrite and nitrate as electron acceptors under micro-aerobic conditions. The 16S rRNA gene combined with pmoA phylogeny of methanotrophs and nirK phylogeny of denitrifiers were analysed to reveal the dominant microbial populations and functional microorganisms. Real-time quantitative polymerase chain reaction results showed high numbers of methanotrophs and denitrifiers in the enriched consortium. The 16S rRNA gene clone library revealed that Methylococcaceae and Methylophilaceae were the dominant populations in the MOD ecosystem. Phylogenetic analyses of pmoA gene clone libraries indicated that all methanotrophs belonged to Methylococcaceae, a type I methanotroph employing the ribulose monophosphate pathway for methane oxidation. Methylotrophic denitrifiers of the Methylophilaceae that can utilize organic intermediates (i.e. formaldehyde, citrate and acetate) released from the methanotrophs played a vital role in aerobic denitrification. This study is the first report to confirm micro-aerobic denitrification and to make phylogenetic and functional assignments for some members of the microbial assemblages involved in MOD.

Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1

“…Previous studies showed that CH 4 is an adequate electron donor for denitrification in mixed communities at micro‐aerobic conditions in both engineered systems and natural environments (Costa et al ., ; Knowles, ; Liu et al ., ). These studies concluded that the observed denitrification process was driven by collaboration of at least two different cohorts in the microbial consortium: methane‐oxidizing bacteria that export organic compounds (acetate, formaldehyde, methanol, citrate) under O 2 limitation and heterotrophic bacteria that use the reduced organic compounds to denitrify.…”

“…Nitrite-dependent anaerobic methane oxidation facilitated by intra-oxygenic methane-oxidizing bacteria in the NC10 phylum has been recently identified as a major process in oxygen-depleted habitats (Ettwig et al, 2010). While denitrification coupled to aerobic methane oxidation has also been studied previously, these studies focused on microbial consortia in which chemoorganoheterotrophic denitrifiers utilize organic compounds released by methanotrophs as sources of energy, reductant and carbon (Costa et al, 2000;Knowles, 2005;Liu et al, 2014). In contrast, the study of methanedependent denitrification in pure cultures of aerobic methanotrophs has been largely controversial in that previous studies of aerobic methanotrophy in hypoxic culture concluded that nitrous oxide (N 2O) production from NO3 − or NO2 − was the result of abiotic processes (Yoshinari, 1985;Ren et al, 2000;Knowles, 2005).…”

Biogas‐based denitrification in a biotrickling filter: Influence of nitrate concentration and hydrogen sulfide

“…In our particular study, NO 2 − accumulation was not observed, which suggests that NO 3 − reduction was limited by the activity of DAMO archaea (Cai et al, ; Hu et al, ). Recent studies also demonstrated the capability of aerobic methanotrophs to reduce nitrate under hypoxia (e.g., gas phase concentrations <2% O 2 ) (Kits et al, ; Liu et al, ). In this regard, the detection of aerobic methanotrophs in our consortium, based on PCR‐amplification of the pmoA gene, also indicates the presence of micro‐aerobic niches, which likely supported an increase in the CH 4 EC to the levels attained by this bacterial group (Fig.…”

Optimum O2:CH4 Ratio Promotes the Synergy between Aerobic Methanotrophs and Denitrifiers to Enhance Nitrogen Removal

“…The basal medium contained the following components in 1 liter (L) distilled water: 1,250 mg KHCO 3 , 50 mg KH 2 PO 4 , 300 mg CaCl 2 ·2H 2 O, 200 mg MgSO 4 ·7H 2 O, 345 mg NaNO 2 , 1.0 mL acidic trace element solution and 1.0 mL alkaline trace element solution. Constituents of the acidic and alkaline trace element solution were described by Liu et al (2014). Inorganic nitrogenous compounds were prepared from NaNO 2 to result in a concentration of ${\text{NO}}_2^{-}$-N in the basal medium of 70 mg/L.…”