Peter F. Dunfield

Methane production and consumption in temperate and subarctic peat soils: Response to temperature and pH

Abstract Rates of methane (CH4) production under anaerobic conditions and CH4 consumption under aerobic conditions were studied in slurries of peat samples kept at different temperatures (0–35°C) and pH values (buffered at pH 3.5–8). Apparent Xn, CH4 for consumption was 1 μm. Optimum temperatures for both processes were about 25°C but CH4 production showed much more temperature-dependence (activation energies 123–271 kJ mol−1, Q10 values 5.3–16) than did CH4 consumption (activation energies 202−80 kJ mol−1 values 1.4–2.1). In the 0–10°C range, CH4 production was negligible but CH4 consumption was 13–38% of maximum. Both processes showed optimum pH values which were about 2 pH units higher than the native peat pH in acidic peats and only 0–1 pH unit higher in the more alkaline peats. We conclude that the microflora involved in CH4 metabolism is not well adapted to either low temperatures or low pH values.

Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia

Aerobic methanotrophic bacteria consume methane as it diffuses away from methanogenic zones of soil and sediment. They act as a biofilter to reduce methane emissions to the atmosphere, and they are therefore targets in strategies to combat global climate change. No cultured methanotroph grows optimally below pH 5, but some environments with active methane cycles are very acidic. Here we describe an extremely acidophilic methanotroph that grows optimally at pH 2.0–2.5. Unlike the known methanotrophs, it does not belong to the phylum Proteobacteria but rather to the Verrucomicrobia, a widespread and diverse bacterial phylum that primarily comprises uncultivated species with unknown genotypes. Analysis of its draft genome detected genes encoding particulate methane monooxygenase that were homologous to genes found in methanotrophic proteobacteria. However, known genetic modules for methanol and formaldehyde oxidation were incomplete or missing, suggesting that the bacterium uses some novel methylotrophic pathways. Phylogenetic analysis of its three pmoA genes (encoding a subunit of particulate methane monooxygenase) placed them into a distinct cluster from proteobacterial homologues. This indicates an ancient divergence of Verrucomicrobia and Proteobacteria methanotrophs rather than a recent horizontal gene transfer of methanotrophic ability. The findings show that methanotrophy in the Bacteria is more taxonomically, ecologically and genetically diverse than previously thought, and that previous studies have failed to assess the full diversity of methanotrophs in acidic environments.

Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia

Aerobic methanotrophic bacteria are capable of utilizing methane as their sole energy source. They are commonly found at the oxic/anoxic interfaces of environments such as wetlands, aquatic sediments, and landfills, where they feed on methane produced in anoxic zones of these environments. Until recently, all known species of aerobic methanotrophs belonged to the phylum Proteobacteria, in the classes Gammaproteobacteria and Alphaproteobacteria. However, in 2007-2008 three research groups independently described the isolation of thermoacidophilic methanotrophs that represented a distinct lineage within the bacterial phylum Verrucomicrobia. Isolates were obtained from geothermal areas in Italy, New Zealand and Russia. They are by far the most acidophilic methanotrophs known, with a lower growth limit below pH 1. Here we summarize the properties of these novel methanotrophic Verrucomicrobia, compare them with the proteobacterial methanotrophs, propose a unified taxonomic framework for them and speculate on their potential environmental significance. New genomic and physiological data are combined with existing information to allow detailed comparison of the three strains. We propose the new genus Methylacidiphilum to encompass all three newly discovered bacteria.

Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

ABSTRACT Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with 13CH4 at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of 13C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.

Methylocella Species Are Facultatively Methanotrophic

All aerobic methanotrophic bacteria described to date are unable to grow on substrates containing carbon-carbon bonds. Here we demonstrate that members of the recently discovered genus Methylocella are an exception to this. These bacteria are able to use as their sole energy source the one-carbon compounds methane and methanol, as well as the multicarbon compounds acetate, pyruvate, succinate, malate, and ethanol. To conclusively verify facultative growth, acetate and methane were used as model substrates in growth experiments with the type strain Methylocella silvestris BL2. Quantitative real-time PCR targeting the mmoX gene, which encodes a subunit of soluble methane monooxygenase, showed that copies of this gene increased in parallel with cell counts during growth on either acetate or methane as the sole substrate. This verified that cells possessing the genetic basis of methane oxidation grew on acetate as well as methane. Cloning of 16S rRNA genes and fluorescence in situ hybridization with strain-specific and genus-specific oligonucleotide probes detected no contaminants in cultures. The growth rate and carbon conversion efficiency were higher on acetate than on methane, and when both substrates were provided in excess, acetate was preferably used and methane oxidation was shut down. Our data demonstrate that not all methanotrophic bacteria are limited to growing on one-carbon compounds. This could have major implications for understanding the factors controlling methane fluxes in the environment.

Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase.

Two strains of aerobic methanotrophic bacteria, AR4(T) and SOP9, were isolated from acidic (pH 3.8-4.0) Sphagnum peat bogs in Russia. Another phenotypically similar isolate, strain LAY, was obtained from an acidic (pH 4.0) forest soil in Germany. Cells of these strains were Gram-negative, non-pigmented, non-motile, thin rods that multiplied by irregular cell division and formed rosettes or amorphous cell conglomerates. Similar to Methylocella species, strains AR4(T), SOP9 and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4(T), SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 °C (optimum 20-23 °C). The major cellular fatty acid was 18 : 1ω7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4(T), SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4(T), SOP9 and LAY. Strain AR4(T) ( = DSM 22108(T)  = LMG 25277(T)  = VKM B-2543(T)) is the type strain of Methyloferula stellata.

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria, Acidobacteria, Chloroflexi, Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria, Firmicutes, Thermus/Deinococcus, Actinobacteria and Bacteroidetes, as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria, Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.

Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2

The molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small‐scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC, were adjacent to two genes, mmoR and mmoG, encoding a σ54 transcriptional activator and a putative GroEL‐like chaperone, located downstream (3′) of mmoC. Transcriptional analysis revealed that the genes were all cotranscribed from a σ54‐dependent promoter located upstream (5′) of mmo X. The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane‐bound methane monooxygenase enzyme in M. silvestris BL2 and the copper‐mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

Effect of nitrogen fertilizers and moisture content on CH4 and N2O fluxes in a humisol: Measurements in the field and intact soil cores

Field and laboratory studies were conducted to determine effects of nitrogen fertilizers and soil water content on N2O and CH4 fluxes in a humisol located on the Central Experimental Farm of Agriculture Canada, Ottawa. Addition of 100 kg N ha−1 as either urea or NaNO3 had no significant effect on soil CH4 flux measured using chambers. Fertilization with NaNO3 resulted in a significant but transitory stimulation of N2O production. Inorganic soil N profiles and the potential nitrification rate suggested that much of the NH4+ from urea hydrolysis was rapidly nitrified. CH4 fluxes measured using capped soil cores agreed well with fluxes measured using field chambers, and with fluxes calculated from soil gas concentration gradients using Ficks diffusion law. This humisol presents an ideal, unstructured, vertically homogeneous system in which to study gas diffusion, and the influence of gas-filled porosity on CH4 uptake. In soil cores gradually saturated with H2O, the relationship of CH4 flux to gas-filled porosity was an exponential rise to a maximum. Steepening CH4 concentration gradients partially compensated for the decreasing diffusion coefficient of CH4 in soil matrix air as water content increased, and diffusion limitation of CH4 oxidation occurred only at water contents > 130% (dry weight), or gas-filled porosities < 0.2.

Edaphobacter modestus gen. nov., sp. nov., and Edaphobacter aggregans sp. nov., acidobacteria isolated from alpine and forest soils.

The phylum Acidobacteria is currently represented mostly by environmental 16S rRNA gene sequences, and the phylum so far contains only four species with validly published names, Holophaga foetida, Geothrix fermentans, Acidobacterium capsulatum and Terriglobus roseus. In the present study, two novel strains of acidobacteria were isolated. High-throughput enrichments were set up with the MicroDrop technique using an alpine calcareous soil sample and a mixture of polymeric carbon compounds supplemented with signal compounds. This approach yielded a novel, previously unknown acidobacterium, strain Jbg-1T. The second strain, Wbg-1T, was recovered from a co-culture with a methanotrophic bacterium established from calcareous forest soil. Both strains represent members of subdivision 1 of the phylum Acidobacteria and are closely related to each other (98.0 % 16S rRNA gene sequence similarity). At a sequence similarity of 93.8-94.7 %, strains Jbg-1T and Wbg-1T are only distantly related to the closest described relative, Terriglobus roseus KBS 63T, and accordingly are described as members of the novel genus Edaphobacter gen. nov. Based on the DNA-DNA relatedness between strains Jbg-1T and Wbg-1T of 11.5-13.6 % and their chemotaxonomic and phenotypic characteristics, the two strains are assigned to two separate species, Edaphobacter modestus sp. nov. (the type species), with strain Jbg-1T (=ATCC BAA-1329T =DSM 18101T) as the type strain, and Edaphobacter aggregans sp. nov., with strain Wbg-1T (=ATCC BAA-1497T =DSM 19364T) as the type strain. The two novel species are adapted to low carbon concentrations and to neutral to slightly acidic conditions.