Sascha Krause

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.

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.

Hydrology is reflected in the functioning and community composition of methanotrophs in the littoral wetland of a boreal lake

In lake ecosystems a major proportion of methane (CH(4) ) emissions originate from the littoral zone, which can have a great spatial variability in hydrology, soil quality and vegetation. Hitherto, spatial heterogeneity and the effects it has on functioning and diversity of methanotrophs in littoral wetlands have been poorly understood. A diagnostic microarray based on the particulate methane monooxygenase gene coupled with geostatistics was used to analyse spatial patterns of methanotrophs in the littoral wetland of a eutrophic boreal lake (Lake Kevätön, Eastern Finland). The wetland had a hydrology gradient with a mean water table varying from -8 to -25 cm. The wettest area, comprising the highest CH(4) oxidation, had the highest abundance and species richness of methanotrophs. A high water table favoured the occurrence of type Ib methanotrophs, whereas types Ia and II were found under all moisture conditions. Thus the spatial heterogeneity in functioning and diversity of methanotrophs in littoral wetlands is highly dependent on the water table, which in turn varies spatially in relation to the geomorphology of the wetland. We suggest that changes in water levels resulting from regulation of lakes and/or global change will affect the abundance, activity and diversity of methanotrophs, and consequently CH(4) emissions from such systems.

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.

Habitat preferences of an endangered insect species, Cepero’s ground-hopper (Tetrix ceperoi)

Preventing the global decline in biodiversity is a major task for conservation biologists. Although habitat loss has been identified as a key factor driving extinction processes, our knowledge on the habitat requirements of many endangered species, particularly invertebrates, is still sparse. We present a feasible method to study the microhabitat preferences of insect species. In Central Europe, the endangered Cepero’s ground-hopper, Tetrix ceperoi, is believed to have its only remaining natural habitats in dune slacks of the Wadden Sea Islands. Our results suggest that this species performs an active habitat choice of damp, bare patches with high temperatures. While ponds and fens in dune slacks provide large areas of damp bare ground and algal mats, grasslands, degraded dune slacks and the transitional zone between salt marsh and dunes are less suited as habitats. The major threat for T. ceperoi is found in the succession of its pioneer habitats due to the reduced natural dynamics. In industrialized countries, pioneer habitats and species are threatened substantially by coastal protection and floodplain regulation. This is only compensated in part by anthropogenic creation of secondary habitats, such as different kinds of pits or coal heaps. Nevertheless, there is a strong need for restoration of dynamic habitats by floodplain revitalization and dune slack restoration.

Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses

ABSTRACT 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.

Intersexual niche segregation in Cepero's Ground-hopper, Tetrix ceperoi

Sexual differences in habitat preferences have been reported from a variety of animal taxa. However, the ultimate causes for this intersexual niche segregation remain poorly understood. It has been suggested that sexual dimorphism is a consequence of dimorphic niches based upon different reproductive costs and activities of the sexes. Here we provide evidence from field data to examine this hypothesis by studying the behavioral background of niche segregation in Tetrix ceperoi. Our data revealed distinct sexual differences in the substrates on which the insects perched and in the solar radiation of these locations. Males were found at brighter locations and more often on bare ground than females. Incorporation of behavioral data in our analysis showed that patches of bare ground were mainly utilized during mating behavior, in which males invested more time than females. In contrast, females spent more time resting and feeding in the vegetation. Intersexual differences in the proportion of autotomized individuals indicate that males might suffer higher predation risks. These patterns support the dimorphic niches hypothesis, which suggests that differential habitat utilization is caused by differences in the life history strategies of males and females, since males should accept a higher predation risk due to the benefits of multiple matings. Females should invest more time in gaining nutrients and energy for egg production and survival, whereas males should spend more time with searching for mates. We suggest that behavioral covariates should more often be implemented in ecological analyses, since these might have a strong explanatory power.

Methane source strength and energy flow shape methanotrophic communities in oxygen–methane counter‐gradients

The role of microbial diversity for ecosystem functioning has become an important subject in microbial ecology. Recent work indicates that microbial communities and microbial processes can be very sensitive to anthropogenic disturbances. However, to what extent microbial communities may change upon, resist to, or overcome disturbances might differ depending on substrate availability. We used soil from an Italian rice field in gradient microcosms, and analysed the response of methanotrophic communities to an NH4 (+) pulse as a potential disturbance under two different CH4 source strengths. We found a significant influence of source strength, i.e. the energy flow through the methanotrophic community, while NH4 (+) had no effect. Our data suggest that historical contingencies, i.e. nitrogen fertilization, led to an ammonium-tolerant MOB community. Methanotrophs were able to oxidize virtually all CH4 diffusing into the oxic-anoxic boundary layer regardless of NH4 (+) addition. Total and active methanotrophic communities were assessed by a pmoA-specific microarray. From the reservoir of dormant methanotrophs, different species became active with Methylobacter and an environmental cluster affiliated with paddy soils being indicative for high CH4 source strength. Thus, a microbial seed bank is an important prerequisite to maintain functioning in a fluctuating environment.

Spatial heterogeneity of methanotrophs: a geostatistical analysis of pmoA‐based T‐RFLP patterns in a paddy soil

Despite numerous studies on methanotrophs, virtually nothing is known about their spatial heterogeneity in nature. These patterns, however, have strong influences on the interpretations made from analysing microbial processes and community structure. Here we report the first use of geostatistics to analyse the spatial heterogeneity of methanotrophs in a rice field soil (Vercelli, Italy). We used the gene encoding the particulate methane monooxygenase, pmoA, for terminal restriction fragment length polymorphism (T-RFLP) analysis. The profiles obtained were compared using a pseudo-variogram analysis to study autocorrelation as a function of distance. We demonstrated that there was no large-scale spatial structure at this study site, but a micro-scale spatial structure could not be excluded. A species accumulation curve with all terminal restriction fragments revealed that even 75 samples were insufficient to cover the diversity of methanotrophs in a rice field. However, a species accumulation curve of methanotrophs defined as operational taxonomic units validated from a clone library with 90% coverage demonstrated saturation after approximately 15 samples. The results of this study have consequences for studying the diversity and function of methanotrophs. In this agroecosystem population structure showed no spatial pattern implying that both a systematic and random sampling design would be adequate.