Mini sediment columns and two-dimensional sediment flow-through microcosms: Versatile experimental systems for studying biodegradation of organic contaminants in groundwater ecosystems.

Groundwater ecosystems are our most important source for drinking water supply. The increasing pressure to our groundwater reservoirs from anthropogenic contamination is a major threat not only to the ecosystem but also to human health. Microbial transformation of quantitatively important organic contaminants, such as petroleum hydrocarbons, in aquifers is an ecosystem service of ecological as well as economic importance. However, key controls and limitations of biodegradation in situ are still poorly understood. Facing the limited accessibility of the subsurface, the complex structural heterogeneity, and the hidden temporal physical–chemical and biotic dynamics, bench-top experimental systems are necessary tools for a systematic and controlled investigation of key variables in contaminant removal processes at appropriate micro- and meso-scales. Here, we introduce mini sediment columns and two-dimensional sediment flow-through microcosms as complementary versatile experimental systems that offer a high degree of simplification, experimental control, and replication

Contaminant concentration versus flow velocity: Drivers of biodegradation and microbial growth in groundwater model systems.

Aromatic hydrocarbons belong to the most abundant contaminants in groundwater systems. They can serve as carbon and energy source for a multitude of indigenous microorganisms. Predictions of contaminant biodegradation and microbial growth in contaminated aquifers are often vague because the parameters of microbial activity in the mathematical models used for predictions are typically derived from batch experiments, which don't represent conditions in the field. In order to improve our understanding of key drivers of natural attenuation and the accuracy of predictive models, we conducted comparative experiments in batch and sediment flow-through systems with varying concentrations of contaminant in the inflow and flow velocities applying the aerobic Pseudomonas putida strain F1 and the denitrifying Aromatoleum aromaticum strain EbN1. We followed toluene degradation and bacterial growth by measuring toluene and oxygen concentrations and by direct cell counts. In the sediment columns, the total amount of toluene degraded by P. putida F1 increased with increasing source concentration and flow velocity, while toluene removal efficiency gradually decreased. Results point at mass transfer limitation being an important process controlling toluene biodegradation that cannot be assessed with batch experiments. We also observed a decrease in the maximum specific growth rate with increasing source concentration and flow velocity. At low toluene concentrations, the efficiencies in carbon assimilation within the flow-through systems exceeded those in the batch systems. In all column experiments the number of attached cells plateaued after an initial growth phase indicating a specific "carrying capacity" depending on contaminant concentration and flow velocity. Moreover, in all cases, cells attached to the sediment dominated over those in suspension, and toluene degradation was performed practically by attached cells only. The observed effects of varying contaminant inflow concentration and flow velocity on biodegradation could be captured by a reactive-transport model. By monitoring both attached and suspended cells we could quantify the release of new-grown cells from the sediments to the mobile aqueous phase. Studying flow velocity and contaminant concentrations as key drivers of contaminant transformation in sediment flow-through microcosms improves our system understanding and eventually the prediction of microbial biodegradation at contaminated sites

Dynamics of suspended and attached aerobic toluene degraders in small-scale flow-through sediment systems under growth and starvation conditions.

The microbially mediated reactions, that are responsible for field-scale natural attenuation of organic pollutants, are governed by the concurrent presence of a degrading microbial community, suitable energy and carbon sources, electron acceptors, as well as nutrients. The temporal lack of one of these essential components for microbial activity, arising from transient environmental conditions, might potentially impair in-situ biodegradation. This study presents results of small scale flow-through experiments aimed at ascertaining the effects of substrate-starvation periods on the aerobic degradation of toluene by Pseudomonas putida F1. During the course of the experiments, concentrations of attached and mobile bacteria, as well as toluene and oxygen were monitored. Results from a fitted reactive-transport model, along with the observed profiles, show the ability of attached cells to survive substrate-starvation periods of up to four months and suggest a highly dynamic exchange between attached and mobile cells under growth conditions and negligible cell detachment under substrate-starvation conditions. Upon reinstatement of toluene, it was readily degraded without a significant lag period, even after a starvation period of 130 days. Our experimental and modeling results strongly suggest that aerobic biodegradation of BTEX-hydrocarbons at contaminated field sites is not hampered by intermittent starvation periods of up to four months

Organic contamination versus mineral properties: Competing selective forces shaping bacterial community assembly in aquifer sediments.

Multiple factors have been shown to influence the assembly of sediment microbial communities. We hypo thesized that in an organically polluted aquifer, the degree of contamination controls bacterial distribution patterns, superimposing other selective forces such as sediment and mineral properties. Groundwater and sediment samples were analyzed from distinct zones of a petroleum hydrocarbon contaminated sandy aquifer that correspond to different degrees of contamination: Zone 1, with a high concentration of dissolved contaminants (benzene, toluene, ethylbenzene, and xylenes); Zone 2, with high concentrations of sediment-bound polycyclic aromatic hydrocarbons (PAHs); and Zone 3, with only minor PAH contamination. Sediment analysis concentrated on 2 mineral fractions differing in many sediment properties, i.e. translucent quartz (TQ) and mica. Sediment bacterial communities were analyzed by DNA fingerprinting (terminal restriction fragment length polymorphism) and total cell counts. While Zone 1 exhibited highly similar communities on TQ and mica, the selective sorption of PAHs to mica revealed sediment bacterial communities with hardly any taxonomic units shared in Zone 2. Typical selective forces active in sediments of oligotrophic habitats, such as sediment mineral content and surface roughness, only gained influence in Zone 3. Similarly, the least contamination revealed the most pronounced differences in Shannon diversity, evenness, and total cell counts between the mineral fractions tested, with mica characterized by highest biomass and bacterial diversity. The role of contamination as a selective force is also underlined by the zone-specific dominance of key microbes involved in petroleum hydrocarbon degradation. Our results demonstrate that typical selective forces shaping aquifer sediment microbial communities are outcompeted by organic contamination

Spherical particles of halophilic archaea correlate with exposure to low water activity - implications for microbial survival in fluid inclusions of ancient halite.

Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (aw). Gradual formation of spheres of about 0.4 mu m diameter occurred in 4 m NaCl buffer of aw = 0.75, but exposure to buffered 4 m LiCl (aw = 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering aw should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times