Unexpected stimulation of soil methane uptake as emergent property of agricultural soils following bio-based residue application.

Intensification of agriculture to meet the global food, feed, and bioenergy demand entail increasing re-investment of carbon compounds (residues) into agro-systems to prevent decline of soil quality and fertility. However, agricultural intensification decreases soil methane uptake, reducing, and even causing the loss of the methane sink function. In contrast to wetland agricultural soils (rice paddies), the methanotrophic potential in well-aerated agricultural soils have received little attention, presumably due to the anticipated low or negligible methane uptake capacity in these soils. Consequently, a detailed study verifying or refuting this assumption is still lacking. Exemplifying a typical agricultural practice, we determined the impact of bio-based residue application on soil methane flux, and determined the methanotrophic potential, including a qualitative (diagnostic microarray) and quantitative (group-specific qPCR assays) analysis of the methanotrophic community after residue amendments over 2 months. Unexpectedly, after amendments with specific residues, we detected a significant transient stimulation of methane uptake confirmed by both the methane flux measurements and methane oxidation assay. This stimulation was apparently a result of induced cell-specific activity, rather than growth of the methanotroph population. Although transient, the heightened methane uptake offsets up to 16% of total gaseous CO2 emitted during the incubation. The methanotrophic community, predominantly comprised of Methylosinus may facilitate methane oxidation in the agricultural soils. While agricultural soils are generally regarded as a net methane source or a relatively weak methane sink, our results show that methane oxidation rate can be stimulated, leading to higher soil methane uptake. Hence, even if agriculture exerts an adverse impact on soil methane uptake, implementing carefully designed management strategies (e.g. repeated application of specific residues) may compensate for the loss of the methane sink function following land-use change.

Remarkable recovery and colonization behaviour of methane oxidizing bacteria in soil after disturbance is controlled by methane source only.

Little is understood about the relationship between microbial assemblage history, the composition and function of specific functional guilds and the ecosystem functions they provide. To learn more about this relationship we used methane oxidizing bacteria (MOB) as model organisms and performed soil microcosm experiments comprised of identical soil substrates, hosting distinct overall microbial diversities(i.e., full, reduced and zero total microbial and MOB diversities). After inoculation with undisturbed soil, the recovery of MOB activity, MOB diversity and total bacterial diversity were followed over 3 months by methane oxidation potential measurements and analyses targeting pmoA and 16S rRNA genes. Measurement of methane oxidation potential demonstrated different recovery rates across the different treatments. Despite different starting microbial diversities, the recovery and succession of the MOB communities followed a similar pattern across the different treatment microcosms. In this study we found that edaphic parameters were the dominant factor shaping microbial communities over time and that the starting microbial community played only a minor role in shaping MOB microbial community.

The intrinsic methane mitigation potential and associated microbes add product value to compost.

Conventional agricultural activity reduces the uptake of the potent greenhouse gas methane by agricultural soils. However, the recently observed improved methane uptake capacity of agricultural soils after compost application is promising but needs mechanistic understanding. In this study, the methane uptake potential and microbiomes involved in methane cycling were assessed in green compost and household-compost with and without pre-digestion. In bottle incubations of different composts with both high and near-atmospheric methane concentrations (∼10.000 & ∼10 ppmv, respectively), green compost showed the highest potential methane uptake rates (up to 305.19 ± 94.43 nmol h-1 g dw compost-1 and 25.19 ± 6.75 pmol h-1 g dw compost-1, respectively). 16S, pmoA and mcrA amplicon sequencing revealed that its methanotrophic and methanogenic communities were dominated by type Ib methanotrophs, and more specifically by Methylocaldum szegediense and other Methylocaldum species, and Methanosarcina species, respectively. Ordination analyses showed that the abundance of type Ib methanotrophic bacteria was the main steering factor of the intrinsic methane uptake rates of composts, whilst the ammonium content was the main limiting factor, being most apparent in household composts. These results emphasize the potential of compost to contribute to methane mitigation, providing added value to compost as a product for industrial, commercial, governmental and public interests relevant to waste management. Compost could serve as a vector for the introduction of active methanotrophic bacteria in agricultural soils, potentially improving the methane uptake potential of agricultural soils and contributing to global methane mitigation, which should be the focus of future research.

Interactions between Cyanobacteria and Methane Processing Microbes Mitigate Methane Emissions from Rice Soils.

Cyanobacteria play a relevant role in rice soils due to their contribution to soil fertility through nitrogen (N2) fixation and as a promising strategy to mitigate methane (CH4) emissions from these systems. However, information is still limited regarding the mechanisms of cyanobacterial modulation of CH4 cycling in rice soils. Here, we focused on the response of methane cycling microbial communities to inoculation with cyanobacteria in rice soils. We performed a microcosm study comprising rice soil inoculated with either of two cyanobacterial isolates (Calothrix sp. and Nostoc sp.) obtained from a rice paddy. Our results demonstrate that cyanobacterial inoculation reduced CH4 emissions by 20 times. Yet, the effect on CH4 cycling microbes differed for the cyanobacterial strains. Type Ia methanotrophs were stimulated by Calothrix sp. in the surface layer, while Nostoc sp. had the opposite effect. The overall pmoA transcripts of Type Ib methanotrophs were stimulated by Nostoc. Methanogens were not affected in the surface layer, while their abundance was reduced in the sub surface layer by the presence of Nostoc sp. Our results indicate that mitigation of methane emission from rice soils based on cyanobacterial inoculants depends on the proper pairing of cyanobacteria-methanotrophs and their respective traits.

Methane-Derived Carbon as a Driver for Cyanobacterial Growth.

Methane, a potent greenhouse gas produced in freshwater ecosystems, can be used by methane-oxidizing bacteria (MOB) and can therefore subsidize the pelagic food web with energy and carbon. Consortia of MOB and photoautotrophs have been described in aquatic ecosystems and MOB can benefit from photoautotrophs which produce oxygen, thereby enhancing CH4 oxidation. Methane oxidation can account for accumulation of inorganic carbon (i.e., CO2) and the release of exometabolites that may both be important factors influencing the structure of phytoplankton communities. The consortium of MOB and phototroph has been mainly studied for methane-removing biotechnologies, but there is still little information on the role of these interactions in freshwater ecosystems especially in the context of cyanobacterial growth and bloom development. We hypothesized that MOB could be an alternative C source to support cyanobacterial growth in freshwater systems. We detected low δ13C values in cyanobacterial blooms (the lowest detected value -59.97‰ for Planktothrix rubescens) what could be the result of the use of methane-derived carbon by cyanobacteria and/or MOB attached to their cells. We further proved the presence of metabolically active MOB on cyanobacterial filaments using the fluorescein isothiocyanate (FITC) based activity assay. The PCR results also proved the presence of the pmoA gene in several non-axenic cultures of cyanobacteria. Finally, experiments comprising the co-culture of the cyanobacterium Aphanizomenon gracile with the methanotroph Methylosinus sporium proved that cyanobacterial growth was significantly improved in the presence of MOB, presumably through utilizing CO2 released by MOB. On the other hand, 13C-CH4 labeled incubations showed the uptake and assimilation of MOB-derived metabolites by the cyanobacterium. We also observed a higher growth of MOB in the presence of cyanobacteria under a higher irradiance regime, then when grown alone, underpinning the bidirectional influence with as of yet unknown environmental consequences.

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Distribution and diversity of Gallionella-like neutrophilic iron oxidizers in a tidal freshwater marsh.

Microbial iron oxidation is an integral part of the iron redox cycle in wetlands. Nonetheless, relatively little is known about the composition and ecology of iron-oxidizing communities in the soils and sediments of wetlands. In this study, sediment cores were collected across a freshwater tidal marsh in order to characterize the iron-oxidizing bacteria (FeOB) and to link their distributions to the geochemical properties of the sediments. We applied recently designed 16S rRNA primers targeting Gallionella-related FeOB by using a nested PCR-denaturing gradient gel electrophoresis (DGGE) approach combined with a novel quantitative PCR (qPCR) assay. Gallionella-related FeOB were detected in most of the samples. The diversity and abundance of the putative FeOB were generally higher in the upper 5 to 12 cm of sediment than in deeper sediment and higher in samples collected in April than in those collected in July and October. Oxygen supply by macrofauna appears to be a major force in controlling the spatial and temporal variations in FeOB communities. The higher abundance of Gallionella-related FeOB in April coincided with elevated concentrations of extractable Fe(III) in the sediments. Despite this coincidence, the distributions of FeOB did not exhibit a simple relationship to the redox zonation inferred from the geochemical depth profiles.

Complete and Draft Genome Sequences of Aerobic Methanotrophs Isolated from a Riparian Wetland.

Wetlands are important sources of methane emissions, and the impacts of these emissions can be mitigated by methanotrophic bacteria. The genomes of methanotrophs Methylomonas sp. strain LL1 and Methylosinus sp. strain H3A, as well as Methylocystis sp. strains H4A, H15, H62, and L43, were sequenced and are reported here.

Impacts of inter- and intralaboratory variations on the reproducibility of microbial community analyses.

With the advent of molecular biological techniques, especially next-generation sequencing and metagenomics, the number of microbial biogeography studies is rapidly increasing. However, these studies involve the synthesis of data generated by different laboratories using different protocols, chemicals, etc., all with inherent biases. The aim of this study was to assess inter- as well as intralaboratory variations in microbial community composition when standardized protocols are applied to a single soil sample. Aliquots from a homogenized soil sample from a rice field in Italy were sent to five participating laboratories. DNA was extracted by two investigators per laboratory using an identical protocol. All DNA samples were sent to one laboratory to perform DNA quantification, quantitative PCR (QPCR), and microarray and denaturing gradient gel electrophoresis (DGGE) analyses of methanotrophic communities. Yields, as well as purity of DNA, were significantly different between laboratories but in some cases also between investigators within the same laboratory. The differences in yield and quality of the extracted DNA were reflected in QPCR, microarray, and DGGE analysis results. Diversity indices (Shannon-Wiener, evenness, and richness) differed significantly between laboratories. The observed differences have implications for every project in which microbial communities are compared in different habitats, even if assessed within the same laboratory. To be able to make sensible comparisons leading to valid conclusions, intralaboratory variation should be assessed. Standardization of DNA extraction protocols and possible use of internal standards in interlaboratory comparisons may help in rendering a "quantifiable" bias.

Niche Differentiation of Host-Associated Pelagic Microbes and Their Potential Contribution to Biogeochemical Cycling in Artificially Warmed Lakes.

It has been proposed that zooplankton-associated microbes provide numerous beneficial services to their "host". However, there is still a lack of understanding concerning the effect of temperature on the zooplankton microbiome. Furthermore, it is unclear to what extent the zooplankton microbiome differs from free-living and particle-associated (PA) microbes. Here, we explicitly addressed these issues by investigating (1) the differences in free-living, PA, and zooplankton associated microbes and (2) the impact of temperature on these microbes in the water column of a series of lakes artificially warmed by two power plants. High-throughput amplicon sequencing of the 16S rRNA gene showed that diversity and composition of the bacterial community associated to zooplankton, PA, and bacterioplankton varied significantly from one another, grouping in different clusters indicating niche differentiation of pelagic microbes. From the abiotic parameters measured, temperature significantly affected the diversity and composition of all analyzed microbiomes. Two phyla (e.g., Proteobacteria and Bacteroidetes) dominated in zooplankton microbiomes whereas Actinobacteria was the dominant phylum in the bacterioplankton. The microbial species richness and diversity was lower in zooplankton compared to bacterioplankton and PA. Surprisingly, genera of methane-oxidizing bacteria, methylotrophs and nitrifiers (e.g., Nitrobacter) significantly associated with the microbiome of zooplankton and PA. Our study clearly demonstrates niche differentiation of pelagic microbes and their potential link to biogeochemical cycling in freshwater systems.

Ethyl tert-butyl ether (EtBE) degradation by an algal-bacterial culture obtained from contaminated groundwater.

EtBE is a fuel oxygenate that is synthesized from (bio)ethanol and fossil-based isobutylene, and replaces the fossil-based MtBE. Biodegradation of EtBE to harmless metabolites or end products can reduce the environmental and human health risks after accidental release. In this study, an algal-bacterial culture enriched from contaminated groundwater was used to (i) assess the potential for EtBE degradation, (ii) resolve the EtBE degradation pathway and (iii) characterize the phylogenetic composition of the bacterial community involved in EtBE degradation in contaminated groundwater. In an unamended microcosm, algal growth was observed after eight weeks when exposed to a day-night light cycle. In the fed-batch reactor, oxygen produced by the algae Scenedesmus and Chlorella was used by bacteria to degrade 50 µM EtBE replenishments with a cumulative total of 1250 µM in a day/night cycle (650 lux), over a period of 913 days. The microbial community in the fed-batch reactor degraded EtBE, using a P450 monooxygenase and 2-hydroxyisobutyryl-CoA mutase, to tert-butyl alcohol (TBA), ethanol and CO2 as determined using 13C nuclear magnetic resonance spectroscopy (NMR) and gas chromatography. Stable isotope probing (SIP) with 13C6 labeled EtBE in a fed-batch vessel showed no significant difference in community profiles of the 13C and 12C enriched DNA fractions, with representatives of the families Halomonadaceae, Shewanellaceae, Rhodocyclaceae, Oxalobacteraceae, Comamonadaceae, Sphingomonadaceae, Hyphomicrobiaceae, Candidatus Moranbacteria, Omnitrophica, Anaerolineaceae, Nocardiaceae, and Blastocatellaceae. This is the first study describing micro-oxic degradation of EtBE by an algal-bacterial culture. This algal-bacterial culture has advantages compared with conventional aerobic treatments: (i) a lower risk of EtBE evaporation and (ii) no need for external oxygen supply in the presence of light. This study provides novel leads towards future possibilities to implement algal-bacterial consortia in field-scale groundwater or wastewater treatment.

Resistance and Recovery of Methane-Oxidizing Communities Depends on Stress Regime and History; A Microcosm Study.

Although soil microbes are responsible for important ecosystem functions, and soils are under increasing environmental pressure, little is known about their resistance and resilience to multiple stressors. Here, we test resistance and recovery of soil methane-oxidizing communities to two different, repeated, perturbations: soil drying, ammonium addition and their combination. In replicated soil microcosms we measured methane oxidation before and after perturbations, while monitoring microbial abundance and community composition using quantitative PCR assays for the bacterial 16S rRNA and pmoA gene, and sequencing of the bacterial 16S rRNA gene. Although microbial community composition changed after soil drying, methane oxidation rates recovered, even after four desiccation events. Moreover, microcosms subjected to soil drying recovered significantly better from ammonium addition compared to microcosms not subjected to soil drying. Our results show the flexibility of microbial communities, even if abundances of dominant populations drop, ecosystem functions can recover. In addition, a history of stress may induce changes in community composition and functioning, which may in turn affect its future tolerance to different stressors.

Environmental legacy contributes to the resilience of methane consumption in a laboratory microcosm system.

The increase of extreme drought and precipitation events due to climate change will alter microbial processes. Perturbation experiments demonstrated that microbes are sensitive to environmental alterations. However, only little is known on the legacy effects in microbial systems. Here, we designed a laboratory microcosm experiment using aerobic methane-consuming communities as a model system to test basic principles of microbial resilience and the role of changes in biomass and the presence of non-methanotrophic microbes in this process. We focused on enrichments from soil, sediment, and water reflecting communities with different legacy with respect to exposure to drought. Recovery rates, a recently proposed early warning indicator of a critical transition, were utilized as a measure to detect resilience loss of methane consumption during a series of dry/wet cycle perturbations. We observed a slowed recovery of enrichments originating from water samples, which suggests that the community's legacy with a perturbation is a contributing factor for the resilience of microbial functioning.

New DGGE strategies for the analyses of methanotrophic microbial communities using different combinations of existing 16S rRNA-based primers.

Methane-oxidising microbial communities are studied intensively because of their importance for global methane cycling. A suite of molecular microbial techniques has been applied to the study of these communities. Denaturing gradient gel electrophoresis (DGGE) is a diversity screening tool combining high sample throughput with phylogenetic information of high resolution. The existing 16S rRNA-based DGGE assays available for methane-oxidising bacteria suffer from low-specificity, low phylogentic information due to the length of the amplified fragments and/or from lack of resolving power. In the present study we developed new combinations of existing primers and applied these on methane-oxidising microbial communities in a freshwater wetland marsh. The designed strategies comprised nested as well as direct amplification of environmental DNA. Successful application of direct amplification using combinations of universal and specific primers circumvents the nested designs currently used. All developed assays resulted in identical community profiles in wetland soil cores with Methylobacter sp. and Methylocystis sp.-related sequences. Changes in the occurrence of Methylobacter-related sequences with depth in the soil profile may be related to the decrease in methane-oxidizing activity.

Compositional and functional stability of aerobic methane consuming communities in drained and rewetted peat meadows.

The restoration of peatlands is an important strategy to counteract subsidence and loss of biodiversity. However, responses of important microbial soil processes are poorly understood. We assessed functioning, diversity and spatial organization of methanotrophic communities in drained and rewetted peat meadows with different water table management and agricultural practice. Results show that the methanotrophic diversity was similar between drained and rewetted sites with a remarkable dominance of the genus Methylocystis. Enzyme kinetics depicted no major differences, indicating flexibility in the methane (CH4) concentrations that can be used by the methanotrophic community. Short-term flooding led to temporary elevated CH4 emission but to neither major changes in abundances of methane-oxidizing bacteria (MOB) nor major changes in CH4 consumption kinetics in drained agriculturally used peat meadows. Radiolabeling and autoradiographic imaging of intact soil cores revealed a markedly different spatial arrangement of the CH4 consuming zone in cores exposed to near-atmospheric and elevated CH4. The observed spatial patterns of CH4 consumption in drained peat meadows with and without short-term flooding highlighted the spatial complexity and responsiveness of the CH4 consuming zone upon environmental change. The methanotrophic microbial community is not generally altered and harbors MOB that can cover a large range of CH4 concentrations offered due to water-table fluctuations, effectively mitigating CH4 emissions.

Spatial patterns of methanotrophic communities along a hydrological gradient in a riparian wetland.

Microbial communities display a variety of biogeographical patterns mainly driven by large-scale environmental gradients. Here, we analysed the spatial distribution of methane-oxidizing bacteria (MOB) and methane oxidation in a strongly fluctuating environment. We investigated whether the spatial variability of the MOB community can be explained by an environmental gradient and whether this changes with different plot sizes. We applied a pmoA-specific microarray to detect MOB, measured methane oxidation, methane emissions and soil properties. All variables were measured in a 10 × 10 m, 1 × 1 m and 20 × 20 cm plot and interpreted using a geostatistical approach. Methane oxidation as well as MOB displayed spatial patterns reflected in the underlying flooding gradient. Overlapping and contrasting spatial patterns for type I and type II MOB suggested different ecological life strategies. With smaller plot size, the environmental gradient could not explain the variability in the data and local factors became more important. In conclusion, environmental gradients can generally explain variability in microbial spatial patterns; however, we think that this does not contribute to a mechanistic explanation for microbial diversity because the relevant scales for microorganisms are much smaller than those normally measured.

Microbial minorities modulate methane consumption through niche partitioning.

Microbes catalyze all major geochemical cycles on earth. However, the role of microbial traits and community composition in biogeochemical cycles is still poorly understood mainly due to the inability to assess the community members that are actually performing biogeochemical conversions in complex environmental samples. Here we applied a polyphasic approach to assess the role of microbial community composition in modulating methane emission from a riparian floodplain. We show that the dynamics and intensity of methane consumption in riparian wetlands coincide with relative abundance and activity of specific subgroups of methane-oxidizing bacteria (MOB), which can be considered as a minor component of the microbial community in this ecosystem. Microarray-based community composition analyses demonstrated linear relationships of MOB diversity parameters and in vitro methane consumption. Incubations using intact cores in combination with stable isotope labeling of lipids and proteins corroborated the correlative evidence from in vitro incubations demonstrating γ-proteobacterial MOB subgroups to be responsible for methane oxidation. The results obtained within the riparian flooding gradient collectively demonstrate that niche partitioning of MOB within a community comprised of a very limited amount of active species modulates methane consumption and emission from this wetland. The implications of the results obtained for biodiversity-ecosystem functioning are discussed with special reference to the role of spatial and temporal heterogeneity and functional redundancy.

Structural and functional response of methane-consuming microbial communities to different flooding regimes in riparian soils.

Climate change will lead to more extreme precipitation and associated increase of flooding events of soils. This can turn these soils from a sink into a source of atmospheric methane. The latter will depend on the balance of microbial methane production and oxidation. In the present study, the structural and functional response of methane oxidizing microbial communities was investigated in a riparian flooding gradient. Four sites differing in flooding frequency were sampled and soil-physico-chemistry as well as methane oxidizing activities, numbers and community composition were assessed. Next to this, the active community members were determined by stable isotope probing of lipids. Methane consumption as well as population size distinctly increased with flooding frequency. All methane consumption parameters (activity, numbers, lipids) correlated with soil moisture, organic matter content, and conductivity. Methane oxidizing bacteria were present and activated quickly even in seldom flooded soils. However, the active species comprised only a few representatives belonging to the genera Methylobacter, Methylosarcina, and Methylocystis, the latter being active only in permanently or regularly flooded soils.This study demonstrates that soils exposed to irregular flooding harbor a very responsive methane oxidizing community that has the potential to mitigate methane produced in these soils. The number of active species is limited and dominated by one methane oxidizing lineage. Knowledge on the characteristics of these microbes is necessary to assess the effects of flooding of soils and subsequent methane cycling therein.

Spatial patterns of iron- and methane-oxidizing bacterial communities in an irregularly flooded, riparian wetland.

Iron- and methane-cycling are important processes in wetlands with one connected to plant growth and the other to greenhouse gas emission, respectively. In contrast to acidic habitats, there is scarce information on the ecology of microbes oxidizing ferrous iron at circumneutral pH. The latter is mainly due to the lack of isolated representatives and molecular detection techniques. Recently, we developed PCR-DGGE and qPCR assays to detect and enumerate Gallionella-related neutrophilic iron-oxidizers (Ga-FeOB) enabling the assessment of controlling physical as well as biological factors in various ecosystems. In this study, we investigated the spatial distribution of Ga-FeOB in co-occurrence with methane-oxidizing bacteria (MOB) in a riparian wetland. Soil samples were collected at different spatial scales (ranging from meters to centimeters) representing a hydrological gradient. The diversity of Ga-FeOB was assessed using PCR-DGGE and the abundance of both Ga-FeOB and MOB by qPCR. Geostatistical methods were applied to visualize the spatial distribution of both groups. Spatial distribution as well as abundance of Ga-FeOB and MOB was clearly correlated to the hydrological gradient as expressed in moisture content of the soil. Ga-FeOB outnumbered the MOB subgroups suggesting their competitiveness or the prevalence of Fe(2+) over CH(4) oxidation in this floodplain.

Whole-community genome amplification (WCGA) leads to compositional bias in methane-oxidizing communities as assessed by pmoA-based microarray analyses and QPCR.

Whole-genome amplification (WGA) using multiple displacement amplification (MDA) has recently been introduced to the field of environmental microbiology. The amplification of single-cell genomes or whole-community metagenomes decreases the minimum amount of DNA needed for subsequent molecular community analyses. The resolution of profiling methods of environmental microbial communities will increase substantially by the use of the whole-community genome amplification (WCGA) procedure, assuming that the original community composition is not affected qualitatively as well as quantitatively. The present study aims to test if WCGA introduces a bias when applied to aerobic proteobacterial methanotrophic communities. For this, first, we subjected samples from freshwater lake sediment to WCGA, and amplified using primers targeting the pmoA gene coding for the α-subunit of the methane monooxygenase enzyme. Second, we analysed community composition using a diagnostic microarray and quantitative PCR (QPCR) assays. These methods clearly demonstrated that the WCGA amplification introduced a bias. Thus, numbers of γ-proteobacterial methanotrophs ('type Ia') increased significantly while the α-proteobacterial methanotrophs ('type II') were not amplified by the WCGA procedure. It is hypothesized that this bias is caused by the differences in GC content, which may compromise the efficiency of the MDA reaction.