Claudia Lüke

Conceptualizing functional traits and ecological characteristics of methane-oxidizing bacteria as life strategies

Methane-oxidizing bacteria (MOB) possess the ability to use methane for energy generation and growth, thereby, providing a key ecosystem service that is highly relevant to the regulation of the global climate. MOB subgroups have different responses to key environmental controls, reflecting on their functional traits. Their unique features (C1-metabolism, unique lipids and congruence between the 16S rRNA and pmoA gene phylogeny) have facilitated numerous environmental studies, which in combination with the availability of cultured representatives, yield the most comprehensive ecological picture of any known microbial functional guild. Here, we focus on the broad MOB subgroups (type I and type II MOB), and aim to conceptualize MOB functional traits and observational characteristics derived primarily from these environmental studies to be interpreted as microbial life strategies. We focus on the functional traits, and the conditions under which these traits will render different MOB subgroups a selective advantage. We hypothesize that type I and type II MOB generally have distinct life strategies, enabling them to predominate under different conditions and maintain functionality. The ecological characteristics implicated in their adopted life strategies are discussed, and incorporated into the Competitor-Stress tolerator-Ruderal functional classification framework as put forward for plant communities. In this context, type I MOB can broadly be classified as competitor-ruderal while type II MOB fit more within the stress tolerator categories. Finally, we provide an outlook on MOB applications by exemplifying two approaches where their inferred life strategies could be exploited thereby, putting MOB into the context of microbial resource management.

Potential of pmoA Amplicon Pyrosequencing for Methanotroph Diversity Studies

ABSTRACT We analyzed the potential of pmoA amplicon pyrosequencing compared to that of Sanger sequencing with paddy soils as a model environment. We defined operational taxonomic unit (OTU) cutoff values of 7% and 18%, reflecting methanotrophic species and major phylogenetic pmoA lineages, respectively. Major lineages were already well covered by clone libraries; nevertheless, pyrosequencing provided a higher level of diversity at the species level.

Biogeography of wetland rice methanotrophs.

We focused on the functional guild of methane oxidizing bacteria (MOB) as model organisms to get deeper insights into microbial biogeography. The pmoA gene was used as a functional and phylogenetic marker for MOB in two approaches: (i) a pmoA database (> 4000 sequences) was evaluated to obtain insights into MOB diversity in Italian rice paddies, and paddy fields worldwide. The results show a wide geographical distribution of pmoA genotypes that seem to be specifically adapted to paddy fields (e.g. Rice Paddy Cluster 1 and Rice Paddy Cluster 2). (ii) On the smaller geographical scale, we designed a factorial experiment including three different locations, two rice varieties and two habitats (soil and roots) within each of three rice fields. Multivariate analysis of terminal restriction fragment analysis profiles revealed different community patterns at the three field sites, located 10-20 km apart. Root samples were characterized by high abundance of type I MOB whereas the rice variety had no effect. With the agronomical practice being nearly identical, historical contingencies might be responsible for the field site differences. Considering a large reservoir of viable yet inactive MOB cells acting as a microbial seed bank, environmental conditions might have selected and activated a different subset at a time thereby shaping the community.

One millimetre makes the difference: high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic-anoxic interface in a flooded paddy soil

Aerobic methane-oxidizing bacteria (MOB) use a restricted substrate range, yet >30 species-equivalent operational taxonomical units (OTUs) are found in one paddy soil. How these OTUs physically share their microhabitat is unknown. Here we highly resolved the vertical distribution of MOB and their activity. Using microcosms and cryosectioning, we sub-sampled the top 3-mm of a water-saturated soil at near in situ conditions in 100-μm steps. We assessed the community structure and activity using the particulate methane monooxygenase gene pmoA as a functional and phylogenetic marker by terminal restriction fragment length polymorphism (t-RFLP), a pmoA-specific diagnostic microarray, and cloning and sequencing. pmoA genes and transcripts were quantified using competitive reverse transcriptase PCR combined with t-RFLP. Only a subset of the methanotroph community was active. Oxygen microprofiles showed that 89% of total respiration was confined to a 0.67-mm-thick zone immediately above the oxic–anoxic interface, most probably driven by methane oxidation. In this zone, a Methylobacter-affiliated OTU was highly active with up to 18 pmoA transcripts per cell and seemed to be adapted to oxygen and methane concentrations in the micromolar range. Analysis of transcripts with a pmoA-specific microarray found a Methylosarcina-affiliated OTU associated with the surface zone. High oxygen but only nanomolar methane concentrations at the surface suggested an adaptation of this OTU to oligotrophic conditions. No transcripts of type II methanotrophs (Methylosinus, Methylocystis) were found, which indicated that this group was represented by resting stages only. Hence, different OTUs within a single guild shared the same microenvironment and exploited different niches.

Recovery of methanotrophs from disturbance: population dynamics, evenness and functioning

Biodiversity is claimed to be essential for ecosystem functioning, but is threatened by anthropogenic disturbances. Prokaryotes have been assumed to be functionally redundant and virtually inextinguishable. However, recent work indicates that microbes may well be sensitive to environmental disturbance. Focusing on methane-oxidizing bacteria as model organisms, we simulated disturbance-induced mortality by mixing native with sterilized paddy soil in two ratios, 1:4 and 1:40, representing moderate and severe die-offs. Disturbed microcosms were compared with an untreated control. Recovery of activity and populations was followed over 4 months by methane uptake measurements, pmoA-qPCR, pmoA-based terminal restriction fragment length polymorphism and a pmoA-based diagnostic microarray. Diversity and evenness of methanotrophs decreased in disturbed microcosms, but functioning was not compromised. We consistently observed distinctive temporal shifts between type I and type II methanotrophs, and a rapid population growth leading to even higher cell numbers comparing disturbed microcosms with the control. Overcompensating mortality suggested that population size in the control was limited by competition with other bacteria. Overall, methanotrophs showed a remarkable ability to compensate for die-offs.

Ageing well: methane oxidation and methane oxidizing bacteria along a chronosequence of 2000 years

Rice is the staple food for more than half of the worlds growing population. While the area planted to wetland rice is expected to increase further, virtually nothing is known about the long-term development of the respective microbial communities, and how these might influence biogeochemistry. Focusing on methane oxidizing bacteria, we studied a chronosequence of paddy fields in China aged 50-2000 years. Potential methanotrophic activity increased substantially with age of soil. Community composition was relatively similar in all fields. However, growth and activity of one particular subgroup of methanotrophs correlated to soil age suggesting an intricate abiotic control on methanotrophs evolving with time. Our results demonstrate that continuous rice agriculture does not only shape the microbial community, but also modifies the micro-environment in a way enabling faster growth and higher activity of selected populations.

Succession of methanotrophs in oxygen–methane counter-gradients of flooded rice paddies

Little is known about population dynamics and contribution of specific taxa to methane oxidation in flooded rice paddies. In this article we investigate the succession of methanotrophs in oxygen–methane counter-gradients. We used a gradient microcosm system that simulates oxic–anoxic interfaces of a water-saturated paddy soils, and measured pmoA-based (gene encoding particulate methane monooxygenase) terminal restriction fragment length polymorphism (T-RFLP) profiles at both the transcription (mRNA) and the population (DNA) levels. The DNA T-RFLP profiles indicated that the methanotrophic community present clearly differed from the active methanotrophic community. We observed a succession of the methanotrophic community over time without any direct effect of pore water chemistry on the community structure. Both the total population and the active subpopulation changed with time, whereas methane oxidation rates remained nearly constant. Hence, we suggest that a diverse microbial seed bank of methanotrophs is important in maintaining the function in a dynamic ecosystem.

Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN

The classification of high-throughput sequencing data of protein-encoding genes is not as well established as for 16S rRNA. The objective of this work was to develop a simple and accurate method of classifying large datasets of pmoA sequences, a common marker for methanotrophic bacteria. A taxonomic system for pmoA was developed based on a phylogenetic analysis of available sequences. The taxonomy incorporates the known diversity of pmoA present in public databases, including both sequences from cultivated and uncultivated organisms. Representative sequences from closely related genes, such as those encoding the bacterial ammonia monooxygenase, were also included in the pmoA taxonomy. In total, 53 low-level taxa (genus-level) are included. Using previously published datasets of high-throughput pmoA amplicon sequence data, we tested two approaches for classifying pmoA: a naïve Bayesian classifier and BLAST. Classification of pmoA sequences based on BLAST analyses was performed using the lowest common ancestor (LCA) algorithm in MEGAN, a software program commonly used for the analysis of metagenomic data. Both the naïve Bayesian and BLAST methods were able to classify pmoA sequences and provided similar classifications; however, the naïve Bayesian classifier was prone to misclassifying contaminant sequences present in the datasets. Another advantage of the BLAST/LCA method was that it provided a user-interpretable output and enabled novelty detection at various levels, from highly divergent pmoA sequences to genus-level novelty.

Structure and function of methanotrophic communities in a landfill-cover soil.

In landfill-cover soils, aerobic methane-oxidizing bacteria (MOB) convert CH(4) to CO(2), mitigating emissions of the greenhouse gas CH(4) to the atmosphere. We investigated overall MOB community structure and assessed spatial differences in MOB diversity, abundance and activity in a Swiss landfill-cover soil. Molecular cloning, terminal restriction-fragment length polymorphism (T-RFLP) and quantitative PCR of pmoA genes were applied to soil collected from 16 locations at three different depths to study MOB community structure, diversity and abundance; MOB activity was measured in the field using gas push-pull tests. The MOB community was highly diverse but dominated by Type Ia MOB, with novel pmoA sequences present. Type II MOB were detected mainly in deeper soil with lower nutrient and higher CH(4) concentrations. Substantial differences in MOB community structure were observed between one high- and one low-activity location. MOB abundance was highly variable across the site [4.0 × 10(4) to 1.1 × 10(7) (g soil dry weight)(-1)]. Potential CH(4) oxidation rates were high [1.8-58.2 mmol CH(4) (L soil air)(-1) day(-1) ] but showed significant lateral variation and were positively correlated with mean CH(4) concentrations (P < 0.01), MOB abundance (P < 0.05) and MOB diversity (weak correlation, P < 0.17). Our findings indicate that Methylosarcina and closely related MOB are key players and that MOB abundance and community structure are driving factors in CH(4) oxidation at this landfill.

Aerobic methanotroph diversity in Riganqiao peatlands on the Qinghai–Tibetan Plateau

The Zoige Plateau is characterized by its high altitude, low latitude and low annual mean temperature of approximately 1°C and is a major source of atmospheric methane in the Qinghai-Tibetan Plateau. Methanotrophs play an important role in the global cycling of CH4, but the diversity, identity and activity of methanotrophs in this region are poorly characterized. Soils were collected from hummocks and hollows in the Riganqiao peatland and the methanotroph community was analysed by qPCR and sequencing methane monooxygenase (pmoA and mmoX) genes. The pmoA genes ranged between 10(7) and 10(8) copies g(-1) fresh soil, with a somewhat greater abundance in hummocks than hollows. The pmoA genes were analysed by amplicon pyrosequencing and the mmoX genes by cloning and sequencing. Methylocystis species were found to be the most abundant methanotrophs, but numerous clades were present including three novel pmoA and three novel mmoX clusters. There were differences between the methanotroph communities in the hummocks and hollows, with the most significant being an increased abundance of uncultivated type Ib methanotrophs in the hollows. The results indicate that aerobic methanotrophs are abundant in Riganqiao peatland and include previously undetected clades in this geographically isolated and distinctive environment.