Selective stimulation of type I methanotrophs in a rice paddy soil by urea fertilization revealed by RNA-based stable isotope probing

Methane-oxidizing bacteria (MOB) in soil are not only controlled by their main substrates, methane and oxygen, but also by nitrogen availability. We compared an unfertilized control with a urea-fertilized treatment and applied RNA-stable-isotope-probing to follow activity changes upon fertilization as closely as possible. Nitrogen fertilization of an Italian rice field soil increased the CH4 oxidation rates sevenfold. In the fertilized treatment, isopycnic separation of 13C-enriched RNA became possible after 7 days when 300 micromol 13CH4 g(dry soil)(-1) had been consumed. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprints and clone libraries documented that the type I methanotrophic genera Methylomicrobium and Methylocaldum assimilated 13CH4 nearly exclusively. Although previous studies had shown that the same soil contains a much larger diversity of MOB, including both type I and type II, nitrogen fertilization apparently activated only a small subset of the overall diversity of MOB, type I MOB in particular.     

Interactions between Thaumarchaea,Nitrospira and methanotrophs modulate autotrophic nitrification in volcanic grassland soil

Ammonium/ammonia is the sole energy substrate of ammonia oxidizers, and is also an essential nitrogen source for other microorganisms. Ammonia oxidizers therefore must compete with other soil microorganisms such as methane-oxidizing bacteria (MOB) in terrestrial ecosystems when ammonium concentrations are limiting. Here we report on the interactions between nitrifying communities dominated by ammonia-oxidizing archaea (AOA) and Nitrospira-like nitrite-oxidizing bacteria (NOB), and communities of MOB in controlled microcosm experiments with two levels of ammonium and methane availability. We observed strong stimulatory effects of elevated ammonium concentration on the processes of nitrification and methane oxidation as well as on the abundances of autotrophically growing nitrifiers. However, the key players in nitrification and methane oxidation, identified by stable-isotope labeling using ¹³CO₂ and ¹³CH₄, were the same under both ammonium levels, namely type 1.1a AOA, sublineage I and II Nitrospira-like NOB and Methylomicrobium-/Methylosarcina-like MOB, respectively. Ammonia-oxidizing bacteria were nearly absent, and ammonia oxidation could almost exclusively be attributed to AOA. Interestingly, although AOA functional gene abundance increased 10-fold during incubation, there was very limited evidence of autotrophic growth, suggesting a partly mixotrophic lifestyle. Furthermore, autotrophic growth of AOA and NOB was inhibited by active MOB at both ammonium levels. Our results suggest the existence of a previously overlooked competition for nitrogen between nitrifiers and methane oxidizers in soil, thus linking two of the most important biogeochemical cycles in nature.     

Selective grazing of methanotrophs by protozoa in a rice field soil

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10⁴ MPN (g soil dry weight)⁻¹], and three out of four Methylocystis spp. yielded significantly fewer numbers [10²–10³ MPN (g soil dry weight)⁻¹]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.     

Diversity and activity of methanotrophs in landfill cover soils with and without landfill gas recovery systems

Aerobic CH₄ oxidation plays an important role in mitigating CH₄ release from landfills to the atmosphere. Therefore, in this study, oxidation activity and community of methanotrophs were investigated in a subtropical landfill. Among the three sites investigated, the highest CH₄ concentration was detected in the landfill cover soil of the site (A) without a landfill gas (LFG) recovery system, although the refuse in the site had been deposited for a longer time (∼14–15 years) compared to the other two sites (∼6–11 years) where a LFG recovery system was applied. In April and September, the higher CH₄ flux was detected in site A with 72.4 and 51.7 g m⁻² d⁻¹, respectively, compared to the other sites. The abundance of methanotrophs assessed by quantification of pmoA varied with location and season. A linear relationship was observed between the abundance of methanotrophs and CH₄ concentrations in the landfill cover soils (R = 0.827, P < 0.001). The key factors influencing the methanotrophic diversity in the landfill cover soils were pH, the water content and the CH₄ concentration in the soil, of which pH was the most important factor. Type I methanotrophs, including Methylococcus, Methylosarcina, Methylomicrobium and Methylobacter, and type II methanotrophs (Methylocystis) were all detected in the landfill cover soils, with Methylocystis and Methylosarcina being the dominant genera. Methylocystis was abundant in the slightly acidic landfill cover soil, especially in September, and represented more than 89% of the total terminal-restriction fragment abundance. These findings indicated that the LFG recovery system, as well as physical and chemical parameters, affected the diversity and activity of methanotrophs in landfill cover soils.     

Identification of glucose-fermenting bacteria present in an in vitro model of the human intestine by RNA-stable isotope probing

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.     

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by ¹³CO₂ after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the ¹³C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the ¹³C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that ¹³CO₂ assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.     

Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing

Arctic lakes are a significant source of the greenhouse gas methane (CH₄), but the role that methane oxidizing bacteria (methanotrophs) play in limiting the overall CH₄ flux is poorly understood. Here, we used stable isotope probing (SIP) techniques to identify the metabolically active aerobic methanotrophs in upper sediments (0–1 cm) from an arctic lake in northern Alaska sampled during ice‐free summer conditions. The highest CH₄ oxidation potential was observed in the upper sediment (0–1 cm depth) with 1.59 µmol g wet weight^?1 day^?1 compared with the deeper sediment samples (1–3 cm, 3–5 cm and 5–10 cm), which exhibited CH₄ oxidation potentials below 0.4 µmol g wet weight^?1 day^?1. Both type I and type II methanotrophs were directly detected in the upper sediment total communities using targeted primer sets based on 16S rRNA genes. Sequencing of 16S rRNA genes and functional genes (pmoA and mxaF) in the ¹³C‐DNA from the upper sediment indicated that type I methanotrophs, mainly Methylobacter, Methylosoma, Methylomonas and Methylovulum miyakonense, dominated the assimilation of CH₄. Methylotrophs, including the genera Methylophilus and/or Methylotenera, were also abundant in the ¹³C‐DNA. Our results show that a diverse microbial consortium acquired carbon from CH₄ in the sediments of this arctic lake.     

Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing

Soda lakes are an environment with an unusually high pH and often high salinity. To identify the active methanotrophs in the Soda lake sediments, sediment slurries were incubated with a 10% (v/v) (13)CH(4) headspace and the (13)C-labelled DNA was subsequently extracted from these sediments following CsCl density gradient centrifugation. This DNA was then used as a template for PCR amplification of 16S rRNA genes and genes encoding PmoA and MmoX of methane monooxygenase, key enzymes in the methane oxidation pathway. Phylogenetic analysis of 16S rRNA genes, PmoA and MmoX identified that strains of Methylomicrobium, Methylobacter, Methylomonas and 'Methylothermus' had assimilated the (13)CH(4). Phylogenetic analysis of PmoA sequences amplified from DNA extracted from Soda lake sediments before Stable Isotope Probing (SIP) treatment showed that a much wider diversity of both type I and type II methanotroph sequences are present in this alkaline environment. The majority of methanotroph sequences detected in the (13)C-DNA studies were from type I methanotrophs, with 50% of 16S rRNA clones and 100% of pmoA clones from both Lake Suduntuiskii Torom and Lake Gorbunka suggesting that the type I methanotrophs are probably responsible for the majority of methane oxidation in this environment.     

Use of DNA-stable isotope probing and functional gene probes to investigate the diversity of methyl chloride-utilizing bacteria in soil

Enrichment and isolation of methyl chloride-utilizing bacteria from various terrestrial environments, including woodland and forest soils, resulted in the identification of seven methyl chloride-utilizing strains belonging to the genus Hyphomicrobium, an Aminobacter strain TW23 and strain WG1, which grouped closely with the genus Mesorhizobium. Methyl chloride enrichment cultures were dominated by Hyphomicrobium species, indicating that these bacteria were most suited to growth under the enrichment and isolation conditions used. However, the application of culture-independent techniques such as DNA-stable isotope probing and the use of a functional gene probe targeting cmuA, which encodes the methyltransferase catalysing the first step in bacterial methyl chloride metabolism, indicated a greater diversity of methyl chloride-utilizing bacteria in the terrestrial environment, compared with the diversity of soil isolates obtained via the enrichment and isolation procedure. It also revealed the presence of as yet uncultured and potentially novel methyl chloride-degrading bacteria in soil.     

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [¹³C]-α-toluene or [¹³C₇]-toluene as growth substrates. Cells incubated with [¹³C]-α-toluene or [¹³C₇]-toluene incorporated 8–15 at.%¹³C and 51–57 at.%¹³C into total lipid fatty acids, respectively, indicating a lower specific incorporation of ¹³C from [¹³C₇]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [¹³C]-α-toluene and [¹³C₇]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.     

Identification of anthracene degraders in leachate-contaminated aquifer using stable isotope probing

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in landfill leachate-contaminated aquifer. It is necessary to identify the microorganisms truly responsible for PAH degradation if bioremediation can be applied as an effective technology. DNA-based stable isotope probing (SIP) in combination with terminal restriction fragment length polymorphism (TRFLP) was used to identify the active anthracene degraders in the contaminated aquifer sediment. One kind of degrader was classified as Variovorax species within class ??-proteobacteria, but another belonged to unclassified bacteria. These findings also suggest novel microorganisms involved in PAH-degrading processes.     

Enterobacteriaceae facilitate the anaerobic degradation of glucose by a forest soil

Anoxic micro zones that occur in soil aggregates of oxic soils may be temporarily extended after rainfall and thus facilitate the anaerobic degradation of organic compounds in soils. The microbial degradation of glucose by anoxic slurries of a forest soil yielded acetate, CO₂, H₂, succinate, and ethanol, products indicative of mixed acid fermentation. Prokaryotes involved in this process were identified by time-resolved 16S rRNA gene-targeted stable isotope probing with [¹³C-U]-glucose. All labeled phylotypes from the ¹³C-enriched 16S rRNA gene were most closely related to Rahnella and Ewingella , enterobacterial genera known to catalyze mixed acid fermentation. These results indicate that facultative aerobes, in particular Enterobacteriaceae , (1) can outcompete obligate anaerobes when conditions become anoxic in forest soils and (2) may be involved in the initial decomposition of monosaccharides in anoxic micro zones of aerated forest soils.     

Links between methanotroph community composition and CH4 oxidation in a pine forest soil

The main gap in our knowledge about what determines the rate of CH₄ oxidation in forest soils is the biology of the microorganisms involved, the identity of which remains unclear. In this study, we used stable-isotope probing (SIP) following ¹³CH₄ incorporation into phospholipid fatty acids (PLFAs) and DNA/RNA, and sequencing of methane mono-oxygenase ( pmoA ) genes, to identify the influence of variation in community composition on CH₄ oxidation rates. The rates of ¹³C incorporation into PLFAs differed between horizons, with low ¹³C incorporation in the organic soil and relatively high ¹³C incorporation into the two mineral horizons. The microbial community composition of the methanotrophs incorporating the ¹³C label also differed between horizons, and statistical analyses suggested that the methanotroph community composition was a major cause of variation in CH₄ oxidation rates. Both PLFA and pmoA -based data indicated that CH₄ oxidizers in this soil belong to the uncultivated 'upland soil cluster α'. CH₄ oxidation potential exhibited the opposite pattern to ¹³C incorporation, suggesting that CH₄ oxidation potential assays may correlate poorly with in situ oxidation rates. The DNA/RNA-SIP assay was not successful, most likely due to insufficient ¹³C-incorporation into DNA/RNA. The limitations of the technique are briefly discussed.     

Identification of cellulolytic bacteria in soil by stable isotope probing

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. ¹³C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the ¹³C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.