Metabolic flexibility of a prospective bioremediator: Desulfitobacterium hafniense Y51 challenged in chemostats.

Desulfitobacterium hafniense Y51 has been widely used in investigations of perchloroethylene (PCE) biodegradation, but limited information exists on its other physiological capabilities. We investigated how D. hafniense Y51 confronts the debilitating limitations of not having enough electron donor (lactate), or electron acceptor (fumarate) during cultivation in chemostats. The residual concentrations of the substrates supplied in excess were much lower than expected. Transcriptomics, proteomics and fluxomics were integrated to investigate how this phenomenon was regulated. Through diverse regulation at both transcriptional and translational levels, strain Y51 turned to fermenting the excess lactate and disproportionating the excess fumarate under fumarate- and lactate-limiting conditions respectively. Genes and proteins related to the utilization of a variety of alternative electron donors and acceptors absent from the medium were induced, apparently involving the Wood-Ljungdahl pathway. Through this metabolic flexibility, D. hafniense Y51 may be able to switch between different metabolic capabilities under limiting conditions.

Formulation and stabilization of an Arthrobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation.

Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities.

Physiological and Transcriptome Response of the Polycyclic Aromatic Hydrocarbon Degrading Novosphingobium sp. LH128 after Inoculation in Soil.

Soil bioaugmentation involves the inoculation of pollutant-degrading bacteria to accelerate pollutant degradation. Often the inoculum shows a dramatic decrease in Colony Forming Units (CFU) upon soil inoculation but this behavior is not well-understood. In this study, the physiology and transcriptomic response of a GFP tagged variant of Novosphingobium sp. LH128 was examined after inoculation into phenanthrene spiked soil. Four hours after inoculation, strain LH128-GFP showed about 99% reduction in CFU while microscopic counts of GFP-expressing cells were identical to the expected initial cell density, indicating that the reduction in CFU number is explained by cells entering into a Viable But Non-Culturable (VBNC)-like state and not by cell death. Transcriptome analysis showed a remarkably higher expression of phenanthrene degradation genes 4 h after inoculation, compared to the inoculum suspension concomitant with an increased expression of genes involved in stress response. This indicates that the cells were active in phenanthrene degradation while experiencing stress. Between 4 h and 10 days, CFU numbers increased to numbers comparable to the inoculated cell density. Our results suggest that strain LH128-GFP enters a VBNC-like state upon inoculation into soil but is metabolically active and that VBNC cells should be taken into account in evaluating bioaugmentation approaches.

Resilience of Soil Microbial Communities to Metals and Additional Stressors: DNA-Based Approaches for Assessing "Stress-on-Stress" Responses.

Many microbial ecology studies have demonstrated profound changes in community composition caused by environmental pollution, as well as adaptation processes allowing survival of microbes in polluted ecosystems. Soil microbial communities in polluted areas with a long-term history of contamination have been shown to maintain their function by developing metal-tolerance mechanisms. In the present work, we review recent experiments, with specific emphasis on studies that have been conducted in polluted areas with a long-term history of contamination that also applied DNA-based approaches. We evaluate how the "costs" of adaptation to metals affect the responses of metal-tolerant communities to other stress factors ("stress-on-stress"). We discuss recent studies on the stability of microbial communities, in terms of resistance and resilience to additional stressors, focusing on metal pollution as the initial stress, and discuss possible factors influencing the functional and structural stability of microbial communities towards secondary stressors. There is increasing evidence that the history of environmental conditions and disturbance regimes play central roles in responses of microbial communities towards secondary stressors.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars.

Interactions between accumulated copper, bacterial community structure and histamine levels in crayfish meat during storage.

BACKGROUND: Pollution in aquaculture areas may negatively impact edible species and threaten seafood quality and safety. The aim of this study was to determine the interaction between copper and bacteria in the aquatic habitat and their impact upon crustaceans. Marbled crayfish was chosen as a model of aquatic crustaceans and the influence of metal contamination on bacterial community structure in water used to culture crayfish and in crayfish themselves was investigated. Histamine, an allergen commonly formed by certain groups of bacteria in crustacean edible tissue during storage, was also determined. RESULTS: Copper exposure increased its concentration in crayfish meat by 17.4%, but the copper concentration remained within acceptable food safety limits. Elevated copper levels affected the bacterial community both in the water used to cultivate crayfish and in the marbled crayfish themselves. Cluster analysis of 16S rRNA-gene based microbial community fingerprints revealed that copper impacted the bacterial community in the water and in the crayfish meat. However, copper exposure reduced the formation of histamine in crayfish meat during storage by 66.3%. CONCLUSION: Copper from the habitat appears to reduce histamine accumulation in crayfish meat during storage by affecting the bacterial community structure of the cultivation water and most likely also in the intestine of the crayfish. From a food safety point of view, copper treatment during the aqua culturing of crustaceans has a positive impact on the postharvest stage.

Impacts of shallow geothermal energy production on redox processes and microbial communities.

Shallow geothermal systems are increasingly being used to store or harvest thermal energy for heating or cooling purposes. This technology causes temperature perturbations exceeding the natural variations in aquifers, which may impact groundwater quality. Here, we report the results of laboratory experiments on the effect of temperature variations (5-80 °C) on redox processes and associated microbial communities in anoxic unconsolidated subsurface sediments. Both hydrochemical and microbiological data showed that a temperature increase from 11 °C (in situ) to 25 °C caused a shift from iron-reducing to sulfate-reducing and methanogenic conditions. Bioenergetic calculations could explain this shift. A further temperature increase (>45 °C) resulted in the emergence of a thermophilic microbial community specialized in fermentation and sulfate reduction. Two distinct maxima in sulfate reduction rates, of similar orders of magnitude (5 × 10(-10) M s(-1)), were observed at 40 and 70 °C. Thermophilic sulfate reduction, however, had a higher activation energy (100-160 kJ mol(-1)) than mesophilic sulfate reduction (30-60 kJ mol(-1)), which might be due to a trade-off between enzyme stability and activity with thermostable enzymes being less efficient catalysts that require higher activation energies. These results reveal that while sulfate-reducing functionality can withstand a substantial temperature rise, other key biochemical processes appear more temperature sensitive.

Testing potential effects of maize expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) on mycorrhizal fungal communities via DNA- and RNA-based pyrosequencing and molecular fingerprinting.

The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF.

Community assembly, species richness and nestedness of arbuscular mycorrhizal fungi in agricultural soils.

Understanding how communities assemble is a central goal of ecology. This is particularly relevant for communities of arbuscular mycorrhizal fungi (AMF), because the community composition of these beneficial plant symbionts influences important ecosystem processes. Moreover, AMF may be used as sensitive indicators of ecological soil quality if they respond to environmental variation in a predictable way. Here, we use a molecular profiling technique (T-RFLP of 25S rRNA gene fragments) to test which factors determine AM fungal community composition in 40 agricultural soils in the Netherlands. In particular, we test whether species richness, dominance structure and community nestedness are influenced by management type (in pairs of organically and conventionally farmed fields), and we examine the contribution of crop species (maize vs. potato), soil type (sand vs. clay-textured soils) and habitat (plant root vs. bulk soil) on AMF community characteristics. AMF richness varied from 1 to 11 taxa per field. Communities from species-poor fields were found to be subsets of those in richer fields, indicating nestedness and a progressive 'loss' from the species pool. AMF taxa richness and occurrence in soil and plant roots were highly correlated, and richness was related to management intensity (phosphate availability and grass-cropping history together explained 32% and 50% of richness in roots and soils). Soil type together with soil chemical parameters explained only 17% of variance in AMF community structure. We synthesize these results by discussing the potential contribution of a 'bottleneck effect' on AMF communities through increased stochastic effects under environmental stress.

The influence of long-term copper contaminated agricultural soil at different pH levels on microbial communities and springtail transcriptional regulation.

Copper has long been applied for agricultural practises. Like other metals, copper is highly persistent in the environment and biologically active long after its use has ceased. Here we present a unique study on the long-term effects (27 years) of copper and pH on soil microbial communities and on the springtail Folsomia candida an important representative of the soil macrofauna, in an experiment with a full factorial, random block design. Bacterial communities were mostly affected by pH. These effects were prominent in Acidobacteria, while Actinobacteria and Gammaroteobacteria communities were affected by original and bioavailable copper. Reproduction and survival of the collembolan F. candida was not affected by the studied copper concentrations. However, the transcriptomic responses to copper reflected a mechanism of copper transport and detoxification, while pH exerted effects on nucleotide and protein metabolism and (acute) inflammatory response. We conclude that microbial community structure reflected the history of copper contamination, while gene expression analysis of F. candida is associated with the current level of bioavailable copper. The study is a first step in the development of a molecular strategy aiming at a more comprehensive assessment of various aspects of soil quality and ecotoxicology.

Correlating biodegradation kinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the dynamics of microbial communities originating from soil in Vietnam contaminated with herbicides and dioxins.

We studied the succession of bacterial communities during the biodegradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The communities originated from a mesocosm with soil from Bien Hoa airbase in Vietnam heavily contaminated with herbicides and dioxins. They were grown in defined media with different carbon and Gibbs energy sources and 2,3,7,8-TCDD. Cultures with dimethyl sulfoxide (DMSO) as the sole carbon and energy source degraded about 95% of 2,3,7,8-TCDD within 60 days of cultivation. Those with an additional 1 mM of vanillin did that in roughly 90 days. Further 16S rRNA gene amplicon sequencing showed that the increase in relative abundance of members belonging to the genera Bordetella, Sphingomonas, Proteiniphilum, and Rhizobium correlated to increased biodegradation of 2,3,7,8-TCDD in these cultures. A higher concentration of vanillin slowed down the biodegradation rate. Addition of alternative carbon and Gibbs energy sources, such as amino acids, sodium lactate and sodium acetate, even stopped the degradation of 2,3,7,8-TCDD completely. Bacteria from the genera Bordetella, Achromobacter, Sphingomonas and Pseudomonas dominated most of the cultures, but the microbial profiles also significantly differed between cultures as judged by non-metric multidimensional scaling (NMDS) analyses. Our study indicates that 2,3,7,8-TCDD degradation may be stimulated by bacterial communities preadapted to a certain degree of starvation with respect to the carbon and energy source. It also reveals the succession and abundance of defined bacterial genera in the degradation process.

Molecular diversity and distribution of aromatic hydrocarbon-degrading anaerobes across a landfill leachate plume.

Natural attenuation of the mono-aromates benzene, toluene, ethylbenzene and xylene occurs under iron-reducing conditions in a leachate-contaminated aquifer near the Banisveld landfill, the Netherlands. The diversity of mono-aromate-degrading microorganisms was studied by targeting functional genes encoding benzylsuccinate synthase α-subunit (bssA) and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase (bamA). Sixty-four bssA and 188 bamA variants were sequenced from groundwater sampled along the pollution plume in 1999 and 2004. Species containing bssA sequences closest affiliated (> 91%) with the betaprotebacterium Georgfuchsia toluolica were the dominant alkylbenzene degraders (89% of bssA sequences). bssA genes were found at more than 10-fold lower copy numbers than bamA genes, of which only a small fraction (< 2%) was closely related to the genes of Georgfuchsia. bamA gene diversity was high and bamA-based community composition was primarily affected by dissolved organic carbon (DOC) and ferrous iron concentrations. bamA sequences closest related to Geobacteraceae were dominantly (43.2%) observed and the presence of Geobacteraceae-related bamA sequences was associated with DOC. Our results indicate a key role for specialized Georgfuchsia spp. in the degradation of alkylbenzenes, whereas Geobacteraceae are involved in degradation of aromatics other than toluene and xylene.

High biodiversity in a benzene-degrading nitrate-reducing culture is sustained by a few primary consumers.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.

Positive effects of organic farming on below-ground mutualists: large-scale comparison of mycorrhizal fungal communities in agricultural soils.

*The impact of various agricultural practices on soil biodiversity and, in particular, on arbuscular mycorrhizal fungi (AMF), is still poorly understood, although AMF can provide benefit to plants and ecosystems. Here, we tested whether organic farming enhances AMF diversity and whether AMF communities from organically managed fields are more similar to those of species-rich grasslands or conventionally managed fields. *To address this issue, the AMF community composition was assessed in 26 arable fields (13 pairs of organically and conventionally managed fields) and five semi-natural grasslands, all on sandy soil. Terminal restriction fragment length polymorphism community fingerprinting was used to characterize AMF community composition. *The average number of AMF taxa was highest in grasslands (8.8), intermediate in organically managed fields (6.4) and significantly lower in conventionally managed fields (3.9). Moreover, AMF richness increased significantly with the time since conversion to organic agriculture. AMF communities of organically managed fields were also more similar to those of natural grasslands when compared with those under conventional management, and were less uniform than their conventional counterparts, as expressed by higher beta-diversity (between-site diversity). *We suggest that organic management in agro-ecosystems contributes to the restoration and maintenance of these important below-ground mutualists.

How Geobacteraceae may dominate subsurface biodegradation: physiology of Geobacter metallireducens in slow-growth habitat-simulating retentostats.

Geobacteraceae dominate many iron-reducing subsurface environments and are associated with biodegradation of organic pollutants. In order to enhance the understanding of the environmental role played by Geobacteraceae, the physiology of Geobacter metallireducens was investigated at the low growth rates found in its subsurface habitat. Cultivation in retentostats (a continuous culturing device with biomass retention) under electron acceptor and electron donor limitation enabled growth rates as low as 0.0008 h(-1). The maximum growth yield was between 0.05 and 0.09 C-mol biomass per C-mol acetate and comparable to that observed in batch experiments. Maintenance energy demand is among the lowest reported for heterotrophic bacteria, under both acetate and AQDS limitation. The cells were able to use alternative electron acceptors directly, without requiring de novo protein synthesis. We discuss how the extremely low maintenance energy demand and the ability to readily use alternative electron acceptors may help Geobacter species to become ubiquitous and dominant microorganisms in many iron-reducing subsurface settings.

Marine microorganisms make a meal of oil.

Hundreds of millions of litres of petroleum enter the environment from both natural and anthropogenic sources every year. The input from natural marine oil seeps alone would be enough to cover all of the world's oceans in a layer of oil 20 molecules thick. That the globe is not swamped with oil is testament to the efficiency and versatility of the networks of microorganisms that degrade hydrocarbons, some of which have recently begun to reveal the secrets of when and how they exploit hydrocarbons as a source of carbon and energy.

Seasonal dynamics in leachate hydrochemistry and natural attenuation in surface run-off water from a tropical landfill.

Open waste dump systems are still widely used in Indonesia. The Jatibarang landfill receives 650-700 tons of municipal waste per day from the city of Semarang, Central Java. Some of the leachate from the landfill flows via several natural and collection ponds to a nearby river. The objectives of the study were to identify seasonal landfill leachate characteristics in this surface water and to determine the occurrence of natural attenuation, in particular the potential for biodegradation, along the flow path. Monthly measurements of general landfill leachate parameters, organic matter-related factors and redox-related components revealed that leachate composition was influenced by seasonal precipitation. In the dry season, electrical conductivity and concentrations of BOD, COD, N-organic matter, ammonia, sulphate and calcium were significantly higher (1.1-2.3 fold) than during the wet season. Dilution was the major natural attenuation process acting on leachate. Heavy metals had the highest impact on river water quality. Between the landfill and the river, a fivefold dilution occurred during the dry season due to active springwater infiltration, while rainwater led to a twofold dilution in the wet season. Residence time of leachate in the surface leachate collection system was less than 70 days. Field measurements and laboratory experiments showed that during this period hardly any biodegradation of organic matter and ammonia occurred (less than 25%). However, the potential for biodegradation of organic matter and ammonia was clearly revealed during 700 days of incubation of leachate in the laboratory (over 65%). If the residence time of leachate discharge can be increased to allow for biodegradation processes and precipitation reactions, the polluting effects of leachate on the river can be diminished.

Ample Arsenite Bio-Oxidation Activity in Bangladesh Drinking Water Wells: A Bonanza for Bioremediation?

Millions of people worldwide are at risk of arsenic poisoning from their drinking water. In Bangladesh the problem extends to rural drinking water wells, where non-biological solutions are not feasible. In serial enrichment cultures of water from various Bangladesh drinking water wells, we found transfer-persistent arsenite oxidation activity under four conditions (aerobic/anaerobic; heterotrophic/autotrophic). This suggests that biological decontamination may help ameliorate the problem. The enriched microbial communities were phylogenetically at least as diverse as the unenriched communities: they contained a bonanza of 16S rRNA gene sequences. These related to Hydrogenophaga, Acinetobacter, Dechloromonas, Comamonas, and Rhizobium/Agrobacterium species. In addition, the enriched microbiomes contained genes highly similar to the arsenite oxidase (aioA) gene of chemolithoautotrophic (e.g., Paracoccus sp. SY) and heterotrophic arsenite-oxidizing strains. The enriched cultures also contained aioA phylotypes not detected in the previous survey of uncultivated samples from the same wells. Anaerobic enrichments disclosed a wider diversity of arsenite oxidizing aioA phylotypes than did aerobic enrichments. The cultivatable chemolithoautotrophic and heterotrophic arsenite oxidizers are of great interest for future in or ex-situ arsenic bioremediation technologies for the detoxification of drinking water by oxidizing arsenite to arsenate that should then precipitates with iron oxides. The microbial activities required for such a technology seem present, amplifiable, diverse and hence robust.

Eukaryotic diversity in an anaerobic aquifer polluted with landfill leachate.

Eukaryotes may influence pollutant degradation processes in groundwater ecosystems by activities such as predation on bacteria and recycling of nutrients. Culture-independent community profiling and phylogenetic analysis of 18S rRNA gene fragments, as well as culturing, were employed to obtain insight into the sediment-associated eukaryotic community composition in an anaerobic sandy aquifer polluted with landfill leachate (Banisveld, The Netherlands). The microeukaryotic community at a depth of 1 to 5 m below the surface along a transect downgradient (21 to 68 m) from the landfill and at a clean reference location was diverse. Fungal sequences dominated most clone libraries. The fungal diversity was high, and most sequences were sequences of yeasts of the Basidiomycota. Sequences of green algae (Chlorophyta) were detected in parts of the aquifer close (<30 m) to the landfill. The bacterium-predating nanoflagellate Heteromita globosa (Cercozoa) was retrieved in enrichments, and its sequences dominated the clone library derived from the polluted aquifer at a depth of 5 m at a location 21 m downgradient from the landfill. The number of culturable eukaryotes ranged from 10(2) to 10(3) cells/g sediment. Culture-independent quantification revealed slightly higher numbers. Groundwater mesofauna was not detected. We concluded that the food chain in this polluted aquifer is short and consists of prokaryotes and fungi as decomposers of organic matter and protists as primary consumers of the prokaryotes.