Responses of mixed methanotrophic consortia to variable Cu²⁺/Fe²⁺ ratios

Methane mitigation in landfill top cover soils is mediated by methanotrophs whose optimal methane (CH₄) oxidation capacity is governed by environmental and complex microbial community interactions. Optimization of CH₄ remediating bio-filters need to take microbial responses into account. Divalent copper (Cu²⁺) and iron (Fe²⁺) are present in landfills at variable ratios and play a vital role in methane oxidation capacity and growth of methanotrophs. This study, as a first of its kind, therefore quantified effects of variable Cu²⁺ and Fe²⁺ (5:5, 5:25 and 5:50 μM) ratios on mixed methanotrophic communities enriched from landfill top cover (LB) and compost soils (CB). CH₄ oxidation capacity, CH₄ removal efficiencies, fatty acids content/profiles and polyhydroxybutyrate (PHB; a biopolymer) contents were also analysed to quantify performance and potential co-product development. Mixed methanotroph cultures were raised in 10 L continuous stirred tank reactors (CSTRs, Bioflo^® & Celligen^® 310 Fermentor/Bioreactor; John Morris Scientific, Chatswood, NSW, Australia). Community structure was determined by amplifying the V3-V4 region of 16s rRNA gene. Community structure and, consequently, fatty acid-profiles changed significantly with increasing Cu²⁺/Fe²⁺ ratios, and responses were different for LB and CB. Effects on methane oxidation capacities and PHB content were similar in the LB- and CB-CSTR, decreasing with increasing Cu²⁺/Fe²⁺ ratios, while biomass growth was unaffected. In general, high Fe²⁺ concentration favored growth of the type -II methanotroph Methylosinus in the CB-CSTR, but methanotroph abundances decreased in the LB-CSTR. Increase in Cu²⁺/Fe²⁺ ratio increased the growth of Sphingopyxis in both systems, while Azospirllum was co-dominant in the LB- but absent in the CB-CSTR. After 13 days, methane oxidation capacities and PHB content decreased by ∼50% and more in response to increasing Fe²⁺ concentrations. Although methanotroph abundance was ∼2% in the LB- (compared to >50% in CB-CSTR), methane oxidation capacities were comparable in the two systems, suggesting that methane oxidation capacity was maintained by the dominant Azospirllum and Sphingopyxis in the LB-CSTR. Despite similar methanotroph inoculum community composition and controlled environmental variables, increasing Cu²⁺/Fe²⁺ ratios resulted in significantly different microbial community structures in the LB- and CB-CSTR, indicative of complex microbial interactions. In summary, our results suggest that a detailed understanding of allelopathic interactions in mixed methanotrophic consortia is vital for constructing robust bio-filters for CH₄ emission abatement.