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.

Culture scale-up and immobilisation of a mixed methanotrophic consortium for methane remediation in pilot-scale bio-filters.

Robust methanotrophic consortia for methane (CH4) remediation and by-product development are presently not readily available for industrial use. In this study, a mixed methanotrophic consortium (MMC), sequentially enriched from a marine sediment, was assessed for CH4 removal efficiency and potential biomass-generated by-product development. Suitable packing material for bio-filters to support MMC biofilm establishment and growth was also evaluated. The enriched MMC removed ∼7-13% CH4 under a very high gas flow rate (2.5 L min-1; 20-25% CH4) in continuous-stirred tank reactors (∼10 L working volume) and the biomass contained long-chain fatty acids (i.e. C16 and C18). Cultivation of the MMC on plastic bio-balls abated ∼95-97% CH4 in pilot-scale non-sterile outdoor-operated bio-filters (0.1 L min-1; 1% CH4). Contamination by cyanobacteria had beneficial effects on treating low-level CH4, by providing additional oxygen for methane oxidation by MMC, suggesting that the co-cultivation of MMC with cyanobacterial mats does not interfere with and may actually be beneficial for remediation of CH4 and CO2 at industrial scale.

Influence of nutrients on oxidation of low level methane by mixed methanotrophic consortia.

Low-level methane emissions from coal mine ventilation air (CMV-CH4; i.e., 1 % CH4) can significantly contribute to global climate change, and therefore, treatment is important to reduce impacts. To investigate CMV-CH4 abatement potential, five different mixed methanotrohic consortia (MMCs) were established from soil/sediment sources, i.e., landfill top cover soil, bio-solid compost, vegetated humus soil, estuarine and marine sediments. Enrichment conditions for MMCs were as follows: nitrate mineral salt (NMS) medium, pH ~ 6.8; 25 °C; 20-25 % CH4; agitation 200 rpm; and culture period 20 days, in mini-bench-top bioreactors. The enriched cultures were supplemented with extra carbon (methanol 0.5-1.5 %, formate 5-15 mM, and acetate 5-15 mM), nitrogen (nitrate 0.5-1.5 g L(-1), ammonium 0.1-0.5 g L(-1), or urea: 0.1-0.5 g L(-1)), and trace elements (copper 1-5 µM, iron 1-5 µM, and zinc 1-5 µM) in different batch experiments to improve low-level CH4 abatement. Average CH4 oxidation capacities (MOCs) of MMCs varied between 1.712 ± 0.032 and 1.963 ± 0.057 mg g(-1)DWbiomass h(-1). Addition of formate improved the MOCs of MMCs, but the dose-response varied for different MMCs. Acetate, nitrate and copper had no significant effect on MOCs, while addition of methanol, ammonium, urea, iron and zinc impacted negatively. Overall, MMCs enriched from marine sediments and landfill top cover soil showed high MOCs which were largely resilient to nutrient supplementation, suggesting a strong potential for biofilter development for industrial low-level CH4 abatement, such as those present in CMV.

Effect of CH4/O2 ratio on fatty acid profile and polyhydroxybutyrate content in a heterotrophic-methanotrophic consortium.

Understanding the role of heterotrophic-methanotrophic (H-Meth) communities is important for improvement of methane (CH4) oxidation capacities (MOC) particularly in conjunction with bio-product development in industrial bio-filters. Initially, a H-Meth consortium was established and enriched from marine sediments and characterized by next generation sequencing of the 16s rDNA gene. The enriched consortium was subjected to 10-50% CH4 (i.e., 0.20-1.6 CH4/O2 ratios) to study the effects on MOCs, biomass growth, fatty acid profiles and biopolymer (e.g. polyhydroxybutyrate; PHB) content. Methylocystis, Methylophaga and Pseudoxanthomonas dominated the H-Meth consortium. Culture enrichment of the H-Meth consortium resulted in 15-20-folds higher MOC compared to seed sediments. Increasing CH4 concentration (and decreased O2 levels) yielded higher MOCs, but did not improve total fatty acid contents. PHB contents varied between 2.5% and 8.5% independently of CH4/O2 ratios. The results suggest that H-Meth consortia could potentially be used in industrial bio-filters for production of biopolymer/biofuel precursors from CH4.