Niels Iversen

The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

Methane distribution in European tidal estuaries

Methane concentrations have been measured along salinity profilesin nine tidal estuaries in Europe (Elbe, Ems, Thames, Rhine,Scheldt, Loire, Gironde, Douro and Sado). The Rhine, Scheldt andGironde estuaries have been studied seasonally. A number ofdifferent methodologies have been used and they yieldedconsistent results. Surface water concentrations ranged from0.002 to 3.6 μM, corresponding to saturation ratios of 0.7 to1580 with a median of 25. Methane concentrations in thefresh-water end-members varied from 0.01 to 1.4 μM. Methaneconcentrations in the marine end-members were close to saturationoffshore and on the order of 0.1 μM in estuarine plumes. Methaneversus salinity profiles in river-dominated, stratified estuaries(Rhine and Douro) appeared rather erratic whereas those in thewell mixed, long-residence time estuaries (Elbe, Ems, Thames,Scheldt, Loire, Gironde and Sado) revealed consistent trends. Inthese systems dissolved methane initially decreases withincreasing salinity, then increases to a maximum at intermediateto high salinities before decreasing again going offshore. Tidalflats and creeks were identified as a methane source to estuarinewaters. The global estuarine flux of methane to the atmospherehas been calculated by combining the median water-air methanegradient (68.2 nmol dm−3) with a global area weighted transfercoefficient and the global area of estuaries. Estuaries emit 1.1to 3.0 Tg CH4 yr−1, which is less than 9% of the global marinemethane emission.

Microbial diversity and activity in a Danish Fjord with anoxic deep water

Abstract Microbial diversity and activity were studied in a stratified basin of Mariager Fjord, Denmark in August 1994. The basin is about 30 m deep and the lower half of the water column is anoxic and sulphidic. The hydrographical and biological features of the system are described. Based on chemical gradient profiles and measurements of process rates, we found that the relative importance of sulphate reduction, denitrification and methanogenesis in terms of anaerobic terminal mineralisation was about 5:1:0.4. It is possible, however, that methanogenesis is underestimated because an unknown fraction of the methane production escaped by ebullition. It was estimated that 10–15 % of the net primary production is mineralised anaerobically. The mean residence time of methane, sulphide and ammonia beneath the chemocline is within the range 1.6–2.3 yrs. Chemolithotrophic production in the chemocline (sulphide oxidation and nitrification) accounted for about 3% of the net primary production of the system. Methan...

Isotope array analysis of Rhodocyclales uncovers functional redundancy and versatility in an activated sludge

Extensive physiological analyses of different microbial community members in many samples are difficult because of the restricted number of target populations that can be investigated in reasonable time by standard substrate-mediated isotope-labeling techniques. The diversity and ecophysiology of Rhodocyclales in activated sludge from a full-scale wastewater treatment plant were analyzed following a holistic strategy based on the isotope array approach, which allows for a parallel functional probing of different phylogenetic groups. Initial diagnostic microarray, comparative 16S rRNA gene sequence, and quantitative fluorescence in situ hybridization surveys indicated the presence of a diverse community, consisting of an estimated number of 27 operational taxonomic units that grouped in at least seven main Rhodocyclales lineages. Substrate utilization profiles of probe-defined populations were determined by radioactive isotope array analysis and microautoradiography-fluorescence in situ hybridization of activated sludge samples that were briefly exposed to different substrates under oxic and anoxic, nitrate-reducing conditions. Most detected Rhodocyclales groups were actively involved in nitrogen transformation, but varied in their consumption of propionate, butyrate, or toluene, and thus in their ability to use different carbon sources in activated sludge. This indicates that the functional redundancy of nitrate reduction and the functional versatility of substrate usage are important factors governing niche overlap and differentiation of diverse Rhodocyclales members in this activated sludge.

Modeling Diffusion and Reaction in Soils: II. Atmospheric Methane Diffusion and Consumption in a Forest Soil

A mixed hardwood forest with a maximum potential atmospheric methane consumption at 4 to 6 cm depth was investigated. Vertical variation of soil-water content, gas diffusivity and atmospheric methane uptake was measured with high spatial resolution in intact soil cores (2-5 cm depth intervals). Gas diffusivity increased rapidly with decreasing soil-water potential and a linear relationship between gas diffusivity, and the logarithm to the volumetric soil-water content was found (R 2 ≥ 0.98). Using this relationship in a simple, dynamic diffusion-reaction model, the vertical methane concentration profiles in intact soil cores were simulated. Only diffusion of methane in the soil air and variable methane consumption with depth was considered in the model. An excellent agreement between simulated and measured methane profiles indicated that a main control of methane consumption in non-waterlogged soils is methane diffusion in the soil air. Simulated methane uptake rates, calculated by summing up the methane oxidation at each 1-cm-depth interval, agreed well with measured methane fluxes into the soil cores. Model sensitivity analyses showed an accurate estimation of the effective gas diffusion coefficient at and above the zone of maximum methane consumption to be the most critical parameter for a realistic simulation of methane concentration profiles and total uptake rates.

Direct fingerprinting of metabolically active bacteria in environmental samples by substrate specific radiolabelling and lipid analysis

Substrate specific radio assays were used for enumeration and fingerprinting of microorganisms in environmental samples. Direct fingerprinting was based on incorporation of 14C-labelled substrates into microbial lipids. A radioactive fingerprint was obtained by subsequent radio analysis of whole sample phospholipid ester-linked fatty acids (14C-PLFA fingerprint). This approach provided a 14C-PLFA fingerprint of the organism actively metabolizing the added 14C-labelled substrate. Labelled and unlabelled PLFAs were analysed as methyl ester derivatives by gas–liquid chromatography with flame ionization detection. The presence of 14C-PLFAs were determined by collection of 14CO2 produced after combustion of the fatty acids. Additional analysis of the microbial community was carried out by analysis of the radioactivity assimilated into poly-β-hydroxyalkanoates relative to that assimilated into total phospholipids (14C-PHA/14C-PL ratio). The number of organisms involved in the degradation of a 14C-labelled substrate was estimated using a 14C-most-probable-number technique. These different 14C-based methods were evaluated by studying [14C]methane oxidation in agricultural soil, and [14C]phenanthrene degradation in activated sludge and marine sediment. The radio assays resulted in distinct fingerprints of the bacterial populations capable of degrading the different radiolabelled substrates. Manipulation of the incubation conditions (e.g., oxygen status) resulted in significant changes in population specific metabolic activity and labelling pattern. Phenotypically related microorganisms appeared to dominate [14C]phenanthrene degradation in activated sludge and marine sediment under oxic conditions. Anaerobic [14C]phenanthrene degraders in activated sludge produced a very different 14C-PLFA fingerprint. In methane enriched agricultural soil, aerobic methane oxidation was dominated by organisms most similar to the Type I methanotrophic bacteria. Several of the findings obtained by the 14C-PLFA analysis could not have been established on the basis of conventional PLFAs analysis alone. The results suggest that variations of this simple 14C-fingerprinting method may be applicable to studies of substrate metabolism in mixed microbial communities. Direct fingerprinting based on substrate specific radiolabelling may also aid in phenotypic characterization of heterotrophic microorganisms without the need for enrichment or cultivation.

Degradation of organic pollutants by methane grown microbial consortia

AbstractMicrobial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge (“AAE-A2”), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 °C was 3.1 ± 1.2 mg l−1 and 8.7 ± 1.1 mg DBP (g protein × h)−1, respectively. AAE-A2 also showed fast degradation of BTX (230 ± 30 nmol benzene (mg protein × h)−1 at 20 °C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinustrichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83–92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas,Flavobacterium,Janthinobacterium and Rubivivax).

Aspects of the Biogeochemistry of Methane in Mono Lake and the Mono Basin of California

Above-ambient levels of methane and higher hydrocarbons were detected in the atmosphere of the Mono Basin. These gases emanated from several different sources, including natural gas seeps (thermogenic and biogenic), and methanogenic activity in sediments. Seeps were distributed over nearly 33% of the lake bottom and were also present in the exposed former lakebed. They originated from one or more natural gas deposits that underlie the basin. Seeps associated with hot springs had a thermogenic character, whereas the others had the characteristics of bacterially formed gases. The radiocarbon content of methane in all seep gases was low (5-11% modern carbon), indicating an age of greater than about 20,000 years.

Uptake and persistence of human associated Enterococcus in the mussel Mytilus edulis: relevance for faecal pollution source tracking

Aims:  Micro‐organisms and molecular markers for microbial source tracking (MST) in coastal waters are often present at low numbers, and often exhibit significant variability in time and space. In this study, we investigated the uptake, accumulation, and persistence of human associated Enterococcus in the mussel Mytilus edulis.

Methane Oxidation in Coastal Marine Environments

Methane oxidation has been observed in both the water column and sediments of marine environments. The number of publications is relatively limited but does indicate that methane oxidation is an important process regulating methane emission from the marine areas.