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Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000 p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales (Gammaproteobacteria) and methylotrophic Methylophilaceae (Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed.

Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000?p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales(Gammaproteobacteria) and methylotrophic Methylophilaceae(Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed.

The biodegradation of organic pollutants in aquifers is often restricted to the fringes of contaminant plumes where steep countergradients of electron donors and acceptors are separated by limited dispersive mixing. However, long-distance electron transfer (LDET) by filamentous ‘cable bacteria’ has recently been discovered in marine sediments to couple spatially separated redox half reactions over centimeter scales. Here we provide primary evidence that such sulfur-oxidizing cable bacteria can also be found at oxic–anoxic interfaces in aquifer sediments, where they provide a means for the direct recycling of sulfate by electron transfer over 1–2-cm distance. Sediments were taken from a hydrocarbon-contaminated aquifer, amended with iron sulfide and saturated with water, leaving the sediment surface exposed to air. Steep geochemical gradients developed in the upper 3 cm, showing a spatial separation of oxygen and sulfide by 9 mm together with a pH profile characteristic for sulfur oxidation by LDET. Bacterial filaments, which were highly abundant in the suboxic zone, were identified by sequencing of 16S rRNA genes and fluorescence in situ hybridization (FISH) as cable bacteria belonging to the Desulfobulbaceae. The detection of similar Desulfobulbaceae at the oxic–anoxic interface of fresh sediment cores taken at a contaminated aquifer suggests that LDET may indeed be active at the capillary fringe in situ.

The biodegradation of organic pollutants in aquifers is often restricted to the fringes of contaminant plumes where steep countergradients of electron donors and acceptors are separated by limited dispersive mixing. However, long-distance electron transfer (LDET) by filamentous ‘cable bacteria’ has recently been discovered in marine sediments to couple spatially separated redox half reactions over centimeter scales. Here we provide primary evidence that such sulfur-oxidizing cable bacteria can also be found at oxic–anoxic interfaces in aquifer sediments, where they provide a means for the direct recycling of sulfate by electron transfer over 1–2-cm distance. Sediments were taken from a hydrocarbon-contaminated aquifer, amended with iron sulfide and saturated with water, leaving the sediment surface exposed to air. Steep geochemical gradients developed in the upper 3?cm, showing a spatial separation of oxygen and sulfide by 9?mm together with a pH profile characteristic for sulfur oxidation by LDET. Bacterial filaments, which were highly abundant in the suboxic zone, were identified by sequencing of 16S rRNA genes and fluorescence in situhybridization (FISH) as cable bacteria belonging to the Desulfobulbaceae. The detection of similar Desulfobulbaceaeat the oxic–anoxic interface of fresh sediment cores taken at a contaminated aquifer suggests that LDET may indeed be active at the capillary fringe in situ.

At the heart of microbial ecology lies a true scientific dichotomy. On the one hand, we know microbes are responsible for processes on which all other life on Earth is dependent; their removal would mean the cessation of all known life. However, in opposition, the majority of extant microbial species in natural environments have never been cultured or studied in a laboratory as living organisms. Owing to these factors, the question of “who does what?” has been a major barrier to understanding how microbially mediated ecosystem level events occur. Recently, the use of stable isotopes (13C) to trace carbon from specific substrates into microbes that assimilate carbon from that substrate has significantly advanced our understanding of the relationship between environmental processes and microbial phylogeny.

Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4–5 days, with much of the time being committed to the ultracentrifugation step.

Addition of straw to anoxic rice field soil stimulates production of CH4, an important greenhouse gas. The archaeal community colonizing rice straw was investigated by molecular methods targeting the small subunit ribosomal RNA gene. Cloning and sequencing of 60 clones detected predominantly relatives of Methanobacteriumspp. (38 clones) and Methanosarcinaspp. (16 clones). Terminal restriction fragment length polymorphism (T‐RFLP) analysis confirmed the dominance of Methanobacteriaceae and Methanosarcinaceae, and in addition showed restriction fragments characteristic for Rice cluster I (RC‐I) methanogens. A new oligonucleotide probe specific for RC‐I was designed. Quantitative slot blot hybridization of extracted rRNA with this probe indicated the presence of an active population of RC‐I methanogens. Other methanogenic groups (e.g. Methanomicrobiaceae, Methanosaetaceae), although present and active in soil, could not be conclusively detected on rice straw. The methanogenic community pattern on straw, as revealed by T‐RFLP and quantitative rRNA probing, was fairly constant with incubation time (8–57 days), but the total activity of methanogenic Archaea almost doubled. Our results indicate that the methanogens colonizing rice straw are less diverse than those inhabiting the soil.

Addition of straw to anoxic rice field soil stimulates production of CH4, an important greenhouse gas. The archaeal community colonizing rice straw was investigated by molecular methods targeting the small subunit ribosomal RNA gene. Cloning and sequencing of 60 clones detected predominantly relatives of Methanobacteriumspp. (38 clones) and Methanosarcinaspp. (16 clones). Terminal restriction fragment length polymorphism (T-RFLP) analysis confirmed the dominance of Methanobacteriaceae and Methanosarcinaceae, and in addition showed restriction fragments characteristic for Rice cluster I (RC-I) methanogens. A new oligonucleotide probe specific for RC-I was designed. Quantitative slot blot hybridization of extracted rRNA with this probe indicated the presence of an active population of RC-I methanogens. Other methanogenic groups (e.g. Methanomicrobiaceae, Methanosaetaceae), although present and active in soil, could not be conclusively detected on rice straw. The methanogenic community pattern on straw, as revealed by T-RFLP and quantitative rRNA probing, was fairly constant with incubation time (8–57 days), but the total activity of methanogenic Archaea almost doubled. Our results indicate that the methanogens colonizing rice straw are less diverse than those inhabiting the soil.

The methane production potential of rice soils, which are situated in different geographical regions, shows inherent variations and is catalyzed by archaeal methanogens. We therefore investigated the archaeal community structure in 11 rice field soils which represent a range of climatic conditions (temperate to subtropical zones) and soil properties. Retrieval of environmental partial SSU rDNA sequences from the rice soils of Shenyang (China) and Gapan (The Philippines) showed that the communities were different from each other. However, despite the differences in soil properties and geographical region the sequences clustered in similar phylogenetic groups to those obtained earlier from rice fields of Vercelli (Italy). The archaeal community structure in the other rice field soils was compared using terminal restriction fragment length polymorphism (T‐RFLP) analysis targeting the SSU rRNA gene and the methyl‐coenzyme M reductase α‐subunit gene (mcrA). The relative abundance of each terminal restriction fragment (T‐RF) was determined by fluorescence peak area integration. The 182‐bp SSU rDNA T‐RF (representing members of Methanosarcinaceaeand rice cluster (RC) VI) was dominant (40–80% contribution) in Chinese soils (Zhenjiang, Changchun, Jurong, Beiyuan, Shenyang) and the Philippine soil of Gapan. The other Philippine soils (Luisiana, Guangzhou, Pila) and the Italian soils (Vercelli, Pavia) showed a dominant 389‐bp T‐RF (35–40% contribution), representing mainly the novel methanogenic RC‐I. All the other T‐RF (80, 88, 280, 375 and >800 bp) contributed <20%. Prolonged anoxic incubation (30–200 days) of the air‐dried soils resulted in the production of CH4, which was in some soils preceded by a characteristic halt phase. T‐RFLP analysis revealed that the soils with a methanogenic halt phase also showed dramatic archaeal population dynamics which were related to the length of the halt phase. Our results show that the archaeal communities in rice field soils of different geographical origin are highly related, but nevertheless exhibit individual patterns and dynamics, thus providing evidence for the active participation of the community members in energy and carbon flow.

The methane production potential of rice soils, which are situated in different geographical regions, shows inherent variations and is catalyzed by archaeal methanogens. We therefore investigated the archaeal community structure in 11 rice field soils which represent a range of climatic conditions (temperate to subtropical zones) and soil properties. Retrieval of environmental partial SSU rDNA sequences from the rice soils of Shenyang (China) and Gapan (The Philippines) showed that the communities were different from each other. However, despite the differences in soil properties and geographical region the sequences clustered in similar phylogenetic groups to those obtained earlier from rice fields of Vercelli (Italy). The archaeal community structure in the other rice field soils was compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the SSU rRNA gene and the methyl-coenzyme M reductase α-subunit gene (mcrA). The relative abundance of each terminal restriction fragment (T-RF) was determined by fluorescence peak area integration. The 182-bp SSU rDNA T-RF (representing members of Methanosarcinaceaeand rice cluster (RC) VI) was dominant (40–80% contribution) in Chinese soils (Zhenjiang, Changchun, Jurong, Beiyuan, Shenyang) and the Philippine soil of Gapan. The other Philippine soils (Luisiana, Guangzhou, Pila) and the Italian soils (Vercelli, Pavia) showed a dominant 389-bp T-RF (35–40% contribution), representing mainly the novel methanogenic RC-I. All the other T-RF (80, 88, 280, 375 and >800 bp) contributed <20%. Prolonged anoxic incubation (30–200 days) of the air-dried soils resulted in the production of CH4, which was in some soils preceded by a characteristic halt phase. T-RFLP analysis revealed that the soils with a methanogenic halt phase also showed dramatic archaeal population dynamics which were related to the length of the halt phase. Our results show that the archaeal communities in rice field soils of different geographical origin are highly related, but nevertheless exhibit individual patterns and dynamics, thus providing evidence for the active participation of the community members in energy and carbon flow.