Characterisation of 'Candidatus Methylobacter titanis' sp. nov., a putative novel species of Methylobacter clade 2 and their distribution in sediments of freshwater lakes in maritime Antarctica.

Global warming has a strong impact on the polar regions, in particular, the Antarctic Peninsula and nearby islands. Methane (CH4) is a major factor in climate change and mitigation of CH4 emissions can be accomplished through microbial oxidation by methanotrophic bacteria. Understanding this biological process is crucial given the shortage of research carried out in this geographical area. The aim of this study was to characterise psychrophilic enrichment cultures of aerobic methanotrophs obtained from lake sediments of the Fildes Peninsula (King George Island, South Shetland Islands) and revealing the distribution of the genus Methylobacter in different lake sediments of the peninsula. Four stable methanotrophic enrichment cultures were obtained and analysed by metagenome-assembled genomes (MAGs). The phylogeny of methanotroph MAGs recovered from these enrichment cultures based on the 16S rRNA gene showed that K-2018 MAG008 and D1-2020 MAG004Ts clustered within the Methylobacter clade 2, with high similarity to Methylobacter tundripaludum SV96T (97.88 and 98.56% respectively). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with M. tundripaludum were < 95% (84.8 and 85.0%, respectively) and < 70% (30.2 and 30.3%, respectively), suggesting that they represent a putative novel species for which the name 'Ca. Methylobacter titanis' is proposed. This is the first species of clade 2 of the genus Methylobacter obtained from Antarctica. The bacterial diversity assessed by 16S rRNA gene sequencing of 21 samples of different lakes (water column and sediments) revealed 54 ASVs associated with methanotrophs and the genus Methylobacter as the most abundant. These results suggest that aerobic methanotrophs belonging to the Methylobacter clade 2 would be the main responsible for CH4 oxidation in these sediments.

Oxidation of methane in biotrickling filters inoculated with methanotrophic bacteria.

The oxidation of methane (CH4) using biofilters has been proposed as an alternative to mitigate anthropogenic greenhouse gas emissions with a low concentration of CH4 that cannot be used as a source of energy. However, conventional biofilters utilize organic packing materials that have a short lifespan, clogging problems, and are commonly inoculated with non-specific microorganisms leading to unpredictable CH4 elimination capacities (EC) and removal efficiencies (RE). The main objective of this work was to characterize the oxidation of CH4 in two biotrickling filters (BTFs) packed with polyethylene rings and inoculated with two methanotrophic bacteria, Methylomicrobium album and Methylocystis sp., in order to determine EC and CO2 production (pCO2) when using a specific inoculum. The repeatability of the results in both BTFs was determined when they operated at the same inlet load of CH4. A dynamic mathematical model that describes the CH4 abatement in the BTFs was developed and validated using mass transfer and kinetic parameters estimated independently. The results showed that EC and pCO2 of the BTFs are not identical but very similar for all the conditions tested. The use of specific inoculum has shown a faster startup and higher EC per unit area (0.019 gCH4 m-2 h-1) in comparison to most of the previous studies at the same CH4 load rate (23.2 gCH4 m-3 h-1). Global mass balance showed that the maximum reduction of CO2 equivalents was 98.5 gCO2eq m-3 h-1. The developed model satisfactorily described CH4 abatement in BTFs for a wide range of conditions.

Methanogenic activity inhibition by increasing the linear alkylbenzene sulfonate (LAS) concentration.

The effect of the initial concentration of linear alkylbenzene sulfonate (LAS) on specific methanogenic activity (SMA) was investigated in this work. Six anaerobic flasks reactors with 1 L of total volume were inoculated with anaerobic sludge (2 g VSS L(-1)). The reactors were assayed for 42 days, and fed with volatile fatty acids, nutrients, and LAS. The initial LAS concentrations were 0, 10, 30, 50, 75, and 100 mg L(-1) for the treatment flasks T1 (control), T2, T3, T4, T5, and T6, respectively. When compared with T1, T2 exhibited a 30% reduction in maximum SMA and total methane production (TMP). In treatment T3 through T6, the reductions were 44-97% (T3-T6) for SMA, and 30-90% (T3-T6) for TMP. Total LAS removal increased following the increase in the initial LAS concentration (from 36% at T1 to 76% at T6), primarily due to the high degree of sludge adsorption. LAS biodegradation also occurred (32% in all treatments), although this was most likely associated with the formation of non-methane intermediates. Greater removal by adsorption was observed in long-chain homologues, when compared to short-chain homologues (C13 > C10), whereas the opposite occurred for biodegradation (C10 > C13). The C13 homologue was adsorbed to a great extent (in mass) in T4, T5 and T6, and may also have inhibited methane formation in these treatments.

Oxidation of methane by Methylomicrobium album and Methylocystis sp. in the presence of H2S and NH 3.

Oxidation of methane by methanotrophs, Methylomicrobium album and Methylocystis sp., was measured at several initial concentrations of H2S and NH3 in the headspace of stoppered flasks, at the same initial concentration of methane as sole carbon and energy source: 15 % (v/v). No effect was observed at 0.01 % (v/v) H2S and 0.025 % (v/v) NH3 in gas phase but over 0.05 and 0.025 % (v/v), respectively, they inhibited the oxidation of methane. The effect of H2S was stronger in Methylocystis sp. and both microorganisms were similarly affected by NH3. Depending on their concentrations in gas phase, H2S and NH3 can thus affect the rate of oxidation of methane and biomass growth of both methanotrophs.

Abundance and distribution of diverse membrane-bound monooxygenase (Cu-MMO) genes within the Costa Rica oxygen minimum zone.

Diverse copper-containing membrane-bound monooxygenase-encoding sequences (Cu-MMOs) have recently been described from the marine environment, suggesting widespread potential for oxidation of reduced substrates. Here, we used the well-defined oxygen and methane gradients associated with the Costa Rican oxygen minimum zone (OMZ) to gain insight into the physico-chemical parameters influencing the distribution and abundance of Cu-MMO-encoding marine microorganisms. Two Methylococcales-related Cu-MMO-encoding lineages, termed groups OPU1 and OPU3, demonstrated differences in their relative abundance, with both pmoA and candidate 16S rRNA genes correlating significantly with reduced environmental oxygen concentrations and depth. In contrast, a newly identified Cu-MMO-encoding lineage, Group C, was primarily associated with the oxygenated euphotic zone. An updated phylogenetic analysis including these sequences, a marine pxmABC gene cluster, ethylene-utilizing Cu-MMO-encoding lineages and previously reported planktonic Cu-MMOs (Groups W, X, Z and O) demonstrates the breadth of diversity of Cu-MMO-encoding marine microorganisms. Groups C and X affiliated phylogenetically with ethane- and ethylene-oxidizing Cu-MMOs, Groups W and O affiliated phylogenetically with the recently described Cu-MMO 'pXMO', and Group Z clustered with Cu-MMOs recovered from soils. Collectively, these data demonstrate widespread genetic potential in ocean waters for the oxidation of small, reduced molecules and advance our understanding of the microorganisms involved in methane cycling in the OMZ environment.

Methane carbon supports aquatic food webs to the fish level.

Large amounts of the greenhouse gas methane (CH(4)) are produced by anaerobic mineralization of organic matter in lakes. In spite of extensive freshwater CH(4) emissions, most of the CH(4) is typically oxidized by methane oxidizing bacteria (MOB) before it can reach the lake surface and be emitted to the atmosphere. In turn, it has been shown that the CH(4)-derived biomass of MOB can provide the energy and carbon for zooplankton and macroinvertebrates. In this study, we demonstrate the presence of specific fatty acids synthesized by MOB in fish tissues having low carbon stable isotope ratios. Fish species, zooplankton, macroinvertebrates and the water hyacinth Eichhornia crassipes were collected from a shallow lake in Brazil and analyzed for fatty acids (FA) and carbon stable isotope ratios (δ(13)C). The fatty acids 16:1ω8c, 16:1ω8t, 16:1ω6c, 16:1ω5t, 18:1ω8c and 18:1ω8t were used as signature for MOB. The δ(13)C ratios varied from -27.7‰ to -42.0‰ and the contribution of MOB FA ranged from 0.05% to 0.84% of total FA. Organisms with higher total content of MOB FAs presented lower δ(13)C values (i.e. they were more depleted in (13)C), while organisms with lower content of MOB signature FAs showed higher δ(13)C values. An UPGMA cluster analysis was carried out to distinguish grouping of organisms in relation to their MOB FA contents. This combination of stable isotope and fatty acid tracers provides new evidence that assimilation of methane-derived carbon can be an important carbon source for the whole aquatic food web, up to the fish level.

Evaluating most probable number method to count and isolate viable methylotrophs

Nine different receptacles were tested with the MPN method to determine which receptacle was most reliable and economical for MPN counts. Results showed that 96 well PCR plate were the best vessels for this type of analysis and facilitated the isolation of viable Methylotrophs.

Methylogaea oryzae gen. nov., sp. nov., a mesophilic methanotroph isolated from a rice paddy field.

A novel methanotroph, designated strain E10(T), was isolated from a rice paddy field in Uruguay. Strain E10(T) grew on methane and methanol as sole carbon and energy sources. Cells were Gram-negative, non-motile, non-pigmented, slightly curved rods showing type I intracytoplasmic membranes arranged in stacks. The strain was neutrophilic and mesophilic; optimum growth occurred at 30-35 °C with no growth above 37 °C. The strain possessed only a particulate methane monooxygenase (pmoA). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was most closely related to the moderately thermophilic strains Methylocaldum szegediense OR2(T) (91.6 % sequence similarity) and Methylococcus capsulatus Bath (91.5 %). Comparative sequence analysis of pmoA genes also confirmed that strain E10(T) formed a new lineage among the genera Methylocaldum and Methylococcus with 89 and 84 % derived amino acid sequence identity to Methylococcus capsulatus Bath and Methylocaldum gracile VKM-14L(T), respectively. The DNA G+C content was 63.1 mol% and the major cellular fatty acid was C(16 :0) (62.05 %). Thus, strain E10(T) (=JCM 16910(T) = DSM 23452(T)) represents the type strain of a novel species within a new genus, for which the name Methylogaea oryzae gen. nov., sp. nov. is proposed.

Activity and diversity of methanotrophs in the soil-water interface and rhizospheric soil from a flooded temperate rice field.

AIMS: To combine molecular and cultivation techniques to characterize the methanotrophic community in the soil-water interface (SWI) and rhizospheric soil from flooded rice fields in Uruguay, a temperate region in South America. METHODS AND RESULTS: A novel type I, related to the genus Methylococcus, and three type II methanotrophs were isolated from the highest positive dilution steps from the most probable number (MPN) counts. Potential methane oxidation activities measured in slurried samples were higher in the rhizospheric soil compared to the SWI and were stimulated by N-fertilization. PmoA (particulate methane monooxygenase) clone libraries were constructed for both rice microsites. SWI clones clustered in six groups related to cultivated and uncultivated members from different ecosystems of the genera Methylobacter, Methylomonas, Methylococcus and a novel type I sublineage while cultivation and T-RFLP (terminal restriction fragment length polymorphism) analysis confirmed the presence of type II methanotrophs. CONCLUSIONS: Cultivation techniques, cloning analysis and T-RFLP fingerprinting of the pmoA gene revealed a diverse methanotrophic community in the rice rhizospheric soil and SWI. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports, for the first time, the analysis of the methanotrophic diversity in rice SWI and this diversity may be exploited in reducing methane emissions.

Biodegradability potential of two experimental landfills in Brazil

Durante um ano foi realizado o monitoramento da biodegradabilidade anaeróbia de resíduos sólidos, do potencial de geração de metano e da composição microbiológica de dois aterros sanitários experimentais. Observou-se que, apesar da grande heterogeneidade dos resíduos sólidos, os resultados em termos de estabilização de matéria orgânica durante a fase de produção de metano foram similares para os dois aterros. Ambos os sistemas apresentaram as mesmas faixas de produção de metano (91.5 L CH4 / kg STV - sólidos totais voláteis) e de ácidos orgânicos, principalmente ácidos acético e butírico. Isolou-se ainda, culturas bacterianas dos gêneros Megasphaera, Selenomonas, Methanobacterium, Methanobrevibacter and Methanosarcina.

Comparison of EPR-visible Cu(2+) sites in pMMO from Methylococcus capsulatus (Bath) and Methylomicrobium album BG8.

X-band (9.1 GHz) and S-band (3.4 GHz) electron paramagnetic resonance (EPR) spectra for particulate methane monooxygenase (pMMO) in whole cells from Methylococcus capsulatus (Bath) grown on (63)Cu and (15)N were obtained and compared with previously reported spectra for pMMO from Methylomicrobium album BG8. For both M. capsulatus (Bath) and M. album BG8, two nearly identical Cu(2+) EPR signals with resolved hyperfine coupling to four nitrogens are observed. The EPR parameters for pMMO from M. capsulatus (Bath) (g( parallel) = 2.244, A( parallel) = 185 G, and A(N) = 19 G for signal one; g( parallel) = 2.246, A( parallel) = 180 G, and A(N) = 19 G for signal two) and for pMMO from M. album BG8 (g( parallel) = 2.243, A( parallel) = 180 G, and A(N) = 18 G for signal one; g( parallel) = 2. 251, A( parallel) = 180 G, and A(N) = 18 G for signal two) are very similar and are characteristic of type 2 Cu(2+) in a square planar or square pyramidal geometry. In three-pulse electron spin echo envelope modulation (ESEEM) data for natural-abundance samples, nitrogen quadrupolar frequencies due to the distant nitrogens of coordinated histidine imidazoles were observed. The intensities of the quadrupolar combination bands indicate that there are three or four coordinated imidazoles, which implies that most, if not all, of the coordinated nitrogens detected in the continuous wave spectra are from histidine imidazoles.