Microbial communities along biogeochemical gradients in a hydrocarbon-contaminated aquifer.

Micro-organisms are known to degrade a wide range of toxic substances. How the environment shapes microbial communities in polluted ecosystems and thus influences degradation capabilities is not yet fully understood. In this study, we investigated microbial communities in a highly complex environment: the capillary fringe and subjacent sediments in a hydrocarbon-contaminated aquifer. Sixty sediment sections were analysed using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting, cloning and sequencing of bacterial and archaeal 16S rRNA genes, complemented by chemical analyses of petroleum hydrocarbons, methane, oxygen and alternative terminal electron acceptors. Multivariate statistics revealed concentrations of contaminants and the position of the water table as significant factors shaping the microbial community composition. Micro-organisms with highest T-RFLP abundances were related to sulphate reducers belonging to the genus Desulfosporosinus, fermenting bacteria of the genera Sedimentibacter and Smithella, and aerobic hydrocarbon degraders of the genus Acidovorax. Furthermore, the acetoclastic methanogens Methanosaeta, and hydrogenotrophic methanogens Methanocella and Methanoregula were detected. Whereas sulphate and sulphate reducers prevail at the contamination source, the detection of methane, fermenting bacteria and methanogenic archaea further downstream points towards syntrophic hydrocarbon degradation.

High mitochondrial haplotype diversity of Coleps sp. (Ciliophora: Prostomatida).

To date the awareness of the temporal population structure in eukaryotic microbes is very limited. This is exemplified in the scarce knowledge about the intraspecific genetic variation in ciliates. To elucidate the genetic variation of Coleps (Ciliophora: Prostomatida), we employed the analysis of the mitochondrial apocytochrome b gene of the Coleps community in a young lake in Germany. The analysis of 111 isolates, sampled from April 2005 to September 2006, revealed a high genetic variation for the two dominant Coleps species (11 mitochondrial haplotypes in Coleps spetai, nine in Coleps hirtus hirtus). The study represents one of the largest datasets of intraspecific diversity in a microbial eukaryote and demonstrates for the first time the suitability of a mitochondrial gene for the detection of genetic variation within populations of eukaryotic microbes. However, the results of our study warrant caution in the application of such an approach, as we amplified some non-orthologous cob-like sequences, whose uncritical acceptance would have led to the erroneous discovery of cryptic species.

Fluorescence in situ hybridization (CARD-FISH) of microorganisms in hydrocarbon contaminated aquifer sediment samples.

Groundwater ecosystems are the most important sources of drinking water worldwide but they are threatened by contamination and overexploitation. Petroleum spills account for the most common source of contamination and the high carbon load results in anoxia and steep geochemical gradients. Microbes play a major role in the transformation of petroleum hydrocarbons into less toxic substances. To investigate microbial populations at the single cell level, fluorescence in situ hybridization (FISH) is now a well-established technique. Recently, however, catalyzed reporter deposition (CARD)-FISH has been introduced for the detection of microbes from oligotrophic environments. Nevertheless, petroleum contaminated aquifers present a worst case scenario for FISH techniques due to the combination of high background fluorescence of hydrocarbons and the presence of small microbial cells caused by the low turnover rates characteristic of groundwater ecosystems. It is therefore not surprising that studies of microorganisms from such sites are mostly based on cultivation techniques, fingerprinting, and amplicon sequencing. However, to reveal the population dynamics and interspecies relationships of the key participants of contaminant degradation, FISH is an indispensable tool. In this study, a protocol for FISH was developed in combination with cell quantification using an automated counting microscope. The protocol includes the separation and purification of microbial cells from sediment particles, cell permeabilization and, finally, CARD-FISH in a microwave oven. As a proof of principle, the distribution of Archaea and Bacteria was shown in 60 sediment samples taken across the contaminant plume of an aquifer (Leuna, Germany), which has been heavily contaminated with several ten-thousand tonnes of petroleum hydrocarbons since World War II.