Christian Griebler

Present state and future prospects for groundwater ecosystems.

SUMMARY Ecological and socioeconomic aspects of subterranean hydrosystems have changed during the past 40‐50 years. The major environmental pressures (mainly anthropogenic ones) impact the quantity and quality of groundwater resources and the state of subsurface ecosystems, and it is expected that the environmental pressures on groundwater will continue, at least until 2025, unless new environmental policies change this state of affairs. The world demographic increase and the general rise of water demand constitute one of the major environmental pressures on groundwater

Depth-Resolved Quantification of Anaerobic Toluene Degraders and Aquifer Microbial Community Patterns in Distinct Redox Zones of a Tar Oil Contaminant Plume

ABSTRACT Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (Geobacter and Desulfocapsa spp.), as well as clostridial fermenters (Sedimentibacter spp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate—an important electron acceptor—meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-cluster bssA genes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota.

Anaerobic degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are among the most important contaminants of groundwater. The 2- and 3-ring PAHs are of particular concern because they are water soluble in the 1-200 mug/l range and are transported with the groundwater over significant distances. Anaerobic degradation of PAH has been demonstrated in several microcosm studies with nitrate, ferric iron, or sulfate as electron acceptors and under methanogenic conditions. The biochemical degradation pathways were studied with naphthalene-degrading pure and enrichment cultures and revealed that 2-naphthoic acid is a central metabolite. Naphthalene is activated by addition of a C(1)-unit to generate 2-naphthoic acid, whereas methylnaphthalene is activated by addition of fumarate to the methyl group and further degraded to 2-naphthoic acid. In the central 2-naphthoic acid degradation pathway the ring system is reduced prior to ring cleavage generating e.g. 5,6,7,8-tetrahydro-2-naphthoic acid. The ring cleavage produces metabolites such as 2-carboxycyclohexylacetic acid indicating that further degradation goes via cyclohexane derivatives and not via aromatic compounds. Anaerobic degradation of PAH has also been demonstrated in situ in contaminated aquifers by identification of compound specific metabolites and using stable isotope fraction studies. Detection of specific metabolites of anaerobic PAH degradation such as naphthyl-2-methylsuccinate indicated anaerobic degradation of 2-methylnaphthalene in situ whereas 2-naphthoic acid was indicative of naphthalene and 2-methylnaphthalene degradation. Other carboxylic acids that were detected in groundwater indicated anaerobic degradation of a wide range of PAH and heterocyclic compounds. Degradation of naphthalenes in contaminated aquifers could also be confirmed by carbon stable isotope shifts in the residual substrate fraction.

Effects of thermal energy discharge on shallow groundwater ecosystems

The use of groundwater as a carrier of thermal energy is an important source of sustainable heating and cooling. However, the effects of thermal use on geochemical and biological aquifer characteristics are poorly understood. Here, we have assessed the impacts of heat discharge on an uncontaminated, shallow aquifer by monitoring the hydrogeochemical, bacterial and faunal parameters at an active thermal discharge facility. The observed variability between wells was considerable. Yet, no significant temperature impacts on bacterial or faunal abundance and on bacterial productivity were observed. Also, we did not observe an improved survival or growth of coliforms with temperature. In contrast, the diversity of bacterial terminal restriction fragment (T-RF) length polymorphism fingerprints and faunal populations was either positively or negatively affected by temperature, respectively, and the abundance of selected T-RFs was clearly temperature dependent. Canonical correspondence analysis indicated that both the impact of temperature and of surface water from a nearby river, were important drivers of aquifer biotic variability. These results demonstrate that aquifer thermal energy discharge can affect aquifer bacteria and fauna, while at the same time controlling only a minor part of the total seasonal and spatial variability and therefore posing no likely threat to ecosystem functioning and drinking water protection in uncontaminated, shallow aquifers.

Biodegradation: Updating the Concepts of Control for Microbial Cleanup in Contaminated Aquifers

Biodegradation is one of the most favored and sustainable means of removing organic pollutants from contaminated aquifers but the major steering factors are still surprisingly poorly understood. Growing evidence questions some of the established concepts for control of biodegradation. Here, we critically discuss classical concepts such as the thermodynamic redox zonation, or the use of steady state transport scenarios for assessing biodegradation rates. Furthermore, we discuss if the absence of specific degrader populations can explain poor biodegradation. We propose updated perspectives on the controls of biodegradation in contaminant plumes. These include the plume fringe concept, transport limitations, and transient conditions as currently underestimated processes affecting biodegradation.

Enhanced biodegradation by hydraulic heterogeneities in petroleum hydrocarbon plumes

In case of dissolved electron donors and acceptors, natural attenuation of organic contaminant plumes in aquifers is governed by hydrodynamic mixing and microbial activity. Main objectives of this work were (i) to determine whether aerobic and anaerobic biodegradation in porous sediments is controlled by transverse dispersion, (ii) to elucidate the effect of sediment heterogeneity on mixing and biodegradation, and (iii) to search for degradation-limiting factors. Comparative experiments were conducted in two-dimensional sediment microcosms. Aerobic toluene and later ethylbenzene degradation by Pseudomonas putida strain F1 was initially followed in a plume developing from oxic to anoxic conditions and later under steady-state mixing-controlled conditions. Competitive anaerobic degradation was then initiated by introduction of the denitrifying strain Aromatoleum aromaticum EbN1. In homogeneous sand, aerobic toluene degradation was clearly controlled by dispersive mixing. Similarly, under denitrifying conditions, microbial activity was located at the plumes fringes. Sediment heterogeneity caused flow focusing and improved the mixing of reactants. Independent from the electron accepting process, net biodegradation was always higher in the heterogeneous setting with a calculated efficiency plus of 23-100% as compared to the homogeneous setup. Flow and reactive transport model simulations were performed in order to interpret and evaluate the experimental results.

Biogeochemical and isotopic gradients in a BTEX/PAH contaminant plume: model-based interpretation of a high-resolution field data set.

A high spatial resolution data set documenting carbon and sulfur isotope fractionation at a tar oil-contaminated, sulfate-reducing field site was analyzed with a reactive transport model. Within a comprehensive numerical model, the study links the distinctive observed isotope depth profiles with the degradation of various monoaromatic and polycyclic aromatic hydrocarbon compounds (BTEX/PAHs) under sulfate-reducing conditions. In the numerical model, microbial dynamics were simulated explicitly and isotope fractionation was directly linked to the differential microbial uptake of lighter and heavier carbon isotopes during microbial growth. Measured depth profiles from a multilevel sampling well with high spatial resolution served as key constraints for the parametrization of the model simulations. The results of the numerical simulations illustrate particularly well the evolution of the isotope signature of toluene, which is the most rapidly degrading compound and the most important reductant at the site. The resulting depth profiles at the observation well show distinct differences between the small isotopic enrichment in the contaminant plume core and the much stronger enrichment of up to 3.3 per thousand at the plume fringes.

Isotopic fractionation by transverse dispersion: flow-through microcosms and reactive transport modeling study.

Flow-through experiments were carried out to investigate the role of transverse dispersion on the isotopic behavior of an organic compound during conservative and bioreactive transport in a homogeneous porous medium. Ethylbenzene was selected as model contaminant and a mixture of labeled (perdeuterated) and light isotopologues was continuously injected in a quasi two-dimensional flow-through system. We observed a significant fractionation of ethylbenzene isotopologues during conservative transport at steady state. This effect was particularly pronounced at the plume fringe and contrasted with the common assumption that physical processes only provide a negligible contribution to isotope fractionation. Under the experimental steady state conditions, transverse hydrodynamic dispersion was the only process that could have caused the observed fractionation. Therefore, the measured isotope ratios at the outlet ports were interpreted with different parameterizations of the transverse dispersion coefficient. A nonlinear compound-specific parameterization showed the best agreement with the experimental data. Successively, bioreactive experiments were performed in two subsequent stages: a first oxic phase, involving a single strain of ethylbenzene degraders and a second phase with aerobic and anaerobic (i.e., ethylbenzene oxidation coupled to nitrate reduction) degradation. Significant fractionation through biodegradation occurred exclusively due to the metabolic activity of the anaerobic degraders. We performed analytical and numerical reactive transport simulations of the different experimental phases which confirmed that both the effects of physical processes (diffusion and dispersion) and microbially mediated reactions have to be considered to match the observed isotopic fractionation behavior.

High Resolution Analysis of Contaminated Aquifer Sediments and Groundwater—What Can be Learned in Terms of Natural Attenuation?

High-resolution depth-resolved monitoring was applied to groundwater and sediments samples in a tar oil contaminated aquifer. Today, it is not fully clear, whether groundwater-based lines of evidence are always sufficient to adequately assess natural attenuation (NA) potentials and processes going on in situ. Our data unveiled small-scale heterogeneities, steep physical-chemical and microbial gradients, as well as hot spots of contaminants and biodegradation in the supposedly homogeneous sandy aquifer. The comparison of basic geochemical data revealed a fairly good agreement between sediment and groundwater samples. Nevertheless, a comprehensive understanding of both BTEX and PAH distribution, as well as redox processes involving insoluble electron acceptors, i.e., iron reduction, clearly asks for consideration of both, sediment and groundwater analysis. A thin BTEX plume right below the groundwater table was visible only in groundwater, while significant amounts of PAHs were present in sediments from deeper zones of the aquifer. Indications for sulfate reduction as a dominant process involved in BTEX degradation were largely obtained from groundwater, while the role of iron reduction in degradation and possible sulfide oxidation at the capillary fringe and the upper BTEX plume fringe, as well as in deeper PAH-contaminated zones was evident from sediments. Moreover, sediment analyses were essential to meaningfully recover cell abundances, distribution, activity and composition of the bacterial community. Sediments harbored > 97.7% of bacterial cells and displayed enzyme activities 5 to 6 orders of magnitude higher than groundwater samples. Bacterial community T-RFLP fingerprints revealed important distinctions, but also similarities in depth-resolved microbial community distribution in sediments and water. An apparently highly specialized degrader population was found to dominate the lower BTEX plume fringe. However, even though sediment data seemed to comprise most community information found also in groundwater, this relation did not apply vice versa. In summary, our results show that groundwater sampled at an appropriate scale may contain sufficient information to identify and localize dominant redox reactions, but clearly fails to unravel natural attenuation potentials. This clearly emphasizes the importance of both groundwater and sediment samples for truly assessing natural attenuation potentials and activities at organically contaminated aquifers.

Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture

Anaerobic benzene degradation is an important process in contaminated aquifers but is poorly understood due to the scarcity of microbial cultures for study. We have enriched a ferric iron-reducing culture that completely mineralizes benzene to CO(2). With (13)C(6)-labelled benzene as the growth substrate, ring-labelled benzoate was identified as a major intermediate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of culture supernatants. With increasing incubation time, (13)C(7)-labelled benzoate appeared, indicating that the carboxyl group of benzoate derived from CO(2) that was produced from mineralization of labelled benzene. This was confirmed by growing the culture in (13)C-bicarbonate-buffered medium with unlabelled benzene as the substrate, as the label appeared in the carboxyl group of benzoate produced. Phenol was also identified as an intermediate at high concentration. However, it was clearly shown that phenol was formed abiotically by autoxidation of benzene during the sampling and analysis procedure as a result of exposure to air. The results suggest that, in our culture, anaerobic benzene degradation proceeds via carboxylation and that caution should be exercised in interpreting hydroxylated benzene derivatives as metabolic intermediates of anaerobic benzene degradation.