Biodegradation during contaminant transport in porous media: 4. Impact of microbial lag and bacterial cell growth

Miscible-displacement experiments were conducted to examine the impact of microbial lag and bacterial cell growth on the transport of salicylate, a model hydrocarbon compound. The impacts of these processes were examined separately, as well as jointly, to determine their relative effects on biodegradation dynamics. For each experiment, a column was packed with porous medium that was first inoculated with bacteria that contained the NAH plasmid encoding genes for the degradation of naphthalene and salicylate, and then subjected to a step input of salicylate solution. The transport behavior of salicylate was non-steady for all cases examined, and was clearly influenced by a delay (lag) in the onset of biodegradation. This microbial lag, which was consistent with the results of batch experiments, is attributed to the induction and synthesis of the enzymes required for biodegradation of salicylate. The effect of microbial lag on salicylate transport was eliminated by exposing the column to two successive pulses of salicylate, thereby allowing the cells to acclimate to the carbon source during the first pulse. Elimination of microbial lag effects allowed the impact of bacterial growth on salicylate transport to be quantified, which was accomplished by determining a cell mass balance. Conversely, the impact of microbial lag was further investigated by performing a similar double-pulse experiment under no-growth conditions. Significant cell elution was observed and quantified for all conditions/systems. The results of these experiments allowed us to differentiate the effects associated with microbial lag and growth, two coupled processes whose impacts on the biodegradation and transport of contaminants can be difficult to distinguish.     

The monitoring of biofilm formation in a mulch biowall barrier and its effect on performance

Lab scale mulch biofilm biowall barriers were constructed and tested to monitor the effect of biofilm formation on the performance of the biobarrier. Naphthalene, a two-ring polycyclic aromatic hydrocarbon (PAH), was used as the model compound. With column reactors, the amounts of viable naphthalene degraders and biofilm formation were monitored, as was the performance of the biobarrier. The sorption capacity of the mulch, the increase in biomass and the extracellular polymeric substance (EPS) content of the biofilm created a strong affinity for naphthalene and induced an increase in the number of slowly growing hydrocarbon degraders, resulting in a higher degradation rate and more stable PAH removal. Concentration profiles of pore water naphthalene and electron acceptors indicated that dissolved oxygen (DO) was preferentially used as the electron acceptor, and the greatest removal occurred at the inlet to the column reactor where DO was highest. However, when using nitrate as an alternative electron acceptor, both biofilm formation and continual degradation of naphthalene also occurred. Microprofiles of DO in the biofilm revealed that oxygen transport in the biofilm was limited, and there might be sequential utilization of nitrate for naphthalene removal in the anoxic zones of the biofilm. These results provide insight into the distribution of viable biomass and biofilm EPS production in engineered permeable reactive mulch biobarriers.     

新型人工浮岛流场数值模拟与结构优化分析

近期提出的新型人工浮岛是通过两种不同透水能力基质材料的填充在系统内部形成2个不同水流与溶解氧浓度区域,两区域的过流能力及两者之间水流交换量对系统脱氮除磷效果具有较大的影响。为实现这一新型人工浮岛基质结构设计参数的优化,进一步提高净化能力,应用数值模拟方法对不同结构条件下流场进行了模拟。模拟结果发现,影响两区域水流交换量及其过流能力的主要因素包括：基质材料的填充形状、填充深度及透水能力,其中“钟乳状”形态较“层状”填充形态人工浮岛具有更好的两区域间水流交换能力及过流能力,理论上具有更好的净化效果。根据模拟结果确定的设计参数构建小试试验系统进行对比试验研究,试验结果与数值模拟结论吻合。可见,采用数值模拟方法对人工浮岛内部水流状态进行模拟分析,能够为人工浮岛结构优化设计提供依据。     

In situ modification of an aquifer material by a cationic surfactant to enhance retardation of organic contaminants

Sorption of hexadecyltrimethylammonium chloride (HDTMA), a cationic surfactant, on aquifer material from Columbus AFB, Mississippi, U.S.A., was examined. Transport studies using flow-through columns and a box model aquifer showed that an almost stationary zone of HDTMA-modified aquifer material could be produced in situ without a significant decrease in hydraulic conductivity.Perchloroethylene (PCE) and naphthalene sorption isotherms on the HDTMA-modified aquifer material were linear, and sorption coefficients were increased by over two orders of magnitude relative to the unmodified material. The retardation of PCE by insitu emplaced HDTMA zones within a column was examined. Agreement between batch- and column-derived sorption coefficients and breakthrough curve symmetry indicates that local equilibrium was attained. Significant retardation of a naphthalene plume by an in situ emplaced surfactant zone was demonstrated in the box model aquifer system.The experimental results indicate that it is feasible to create in situ a sorbent zone within an aquifer using cationic surfactants. In most situations, the sorbent zone concept needs to be coupled with contaminant degradation processes for sorbent emplacement to be a practical tool in the remediation of groundwater contamination sites. Sorbent zones may be of benefit in the engineering of suitable environments for microbial or abiotic degradation reactions and by providing time slow reactions to occur.     

The influence of system complexity on bacterial transport in saturated porous media

A series of miscible-displacement column experiments were conducted under saturated flow conditions to systematically investigate the influence of physical and biological complexity on bacterial activity and fate in the presence and absence of a non-sorbing growth substrate, salicylate. Bacterial elution was monitored for three different systems; System I--a sterilized, inoculated, well-sorted sand, System II--a sterilized, inoculated, heterogeneous loamy sand (Hayhook), and System III--two different unsterilized loamy sands (Hayhook and Vinton) each with their associated indigenous microbial community. Results show that System I behaved ideally with respect to both cell and substrate transport, wherein: (1) growth occurred in response to substrate addition, (2) cell elution increased in response to the substrate pulse, and (3) breakthrough curves were reproducible for both substrate and cell elution. In contrast, System II showed ideal behavior with respect to substrate transport but showed variable behavior for cell transport. Further, there was no measurable growth in response to substrate addition and no increase in cell elution during the salicylate pulse. System III exhibited non-ideal behavior for both substrate and cell transport. Of particular interest is the fact that the indigenous communities of the two soils behaved differently. Specifically, for the Hayhook soil, an increased elution response was observed for the heterotrophic population while the salicylate-degrading community was preferentially retained in the column. In contrast for the Vinton soil, the substrate pulse did not elicit an elution response from either the heterotrophic or salicylate-degrading community from the culturable, indigenous Vinton microorganisms. For Systems II and III, the observed variability appears to be associated with the biological component of the system, since sterile controls were reproducible. This type of systematic study is critical for understanding cell and substrate transport behavior in complex, heterogeneous systems, and illustrates the potential uncertainty associated with measurements in such systems.     

Influence of substrate exposure history on biodegradation in a porous medium

This study investigates the influence of fluctuating toluene concentrations on aerobic toluene degradation in a sandy porous medium colonized with Ralstonia pickettii PKO1. Column effluent toluene concentrations were found to increase after a temporary decrease in influent toluene concentration. Subsequent examination of the spatial gradient of toluene degradative activity in the column suggested that the observed increase in effluent toluene concentrations was attributable to an adverse effect of toluene limitation on the biodegradative activity of attached cells. The traditional Michaelis-Menten-type biodegradation equation associated with batch-measured Vmax (2.26 mg toluene/mg living cell/day) and KS (1.20 mg toluene/1) of nonstarved cells was unable to predict the observed toluene breakthrough behavior when the column had been previously exposed to no-toluene conditions. An alternative modeling approach was developed based upon the assumptions that (i) degradative activity was completely deactivated within the no-toluene exposure period (53.5 h) and (ii) a lag-phase was present prior to the subsequent reactivation of degradative activity in previously toluene-starved cells. These assumptions were independently verified by batch microbial investigations, and the modified model provided a good fit to the same observed toluene breakthrough curve. Application of single lag-time and threshold concentration values, however, failed to predict observed toluene breakthrough under different toluene exposure conditions. Results of this experimental and modeling investigation suggested that substrate exposure history, including the length of the starvation period and the level of substrate concentration, affected the induction of biodegradation in the porous medium.     

Methanotrophic activity in a diffusive methane/oxygen counter-gradient in an unsaturated porous medium

Microbial methane (CH4) oxidation is a main control on emissions of this important greenhouse gas from ecosystems such as contaminated aquifers or wetlands under aerobic onditions. Due to a lack of suitable model systems, we designed a laboratory column to study this process in diffusional CH4/O2 counter-gradients in unsaturated porous media. Analysis and simulations of the steady-state CH4, CO2 and O2 gas profiles showed that in a 15-cm-deep active zone, CH4 oxidation followed first-order kinetics with respect to CH4 with a high apparent first-order rate constant of approximately 30 h(-1). Total cell counts obtained using DAPI-staining suggested growth of methanotrophic bacteria, resulting in a high capacity for CH4 oxidation. This together with apparent tolerance to anoxic conditions enabled a rapid response of the methanotrophic community to changing substrate availability, which was induced by changes in O2 concentrations at the top of the column. Microbial oxidation was confirmed by a approximately 7 per thousand enrichment in CH4 stable carbon isotope ratios along profiles. Using a fractionation factor of 1.025+/-0.0005 for microbial oxidation estimated from this shift and the fractionation factor for diffusion, simulations of isotope profiles agreed well with measured data confirming large fractionation associated with microbial oxidation. The designed column should be valuable for investigating response of methanotrophic bacteria to environmental parameters in future studies.     

Two-dimensional distribution of microbial activity and flow patterns within naturally fractured chalk

The two-dimensional distribution of flow patterns and their dynamic change due to microbial activity were investigated in naturally fractured chalk cores. Long-term biodegradation experiments were conducted in two cores ( approximately 20 cm diameter, 31 and 44 cm long), intersected by a natural fracture. 2,4,6-tribromophenol (TBP) was used as a model contaminant and as the sole carbon source for aerobic microbial activity. The transmissivity of the fractures was continuously reduced due to biomass accumulation in the fracture concurrent with TBP biodegradation. From multi-tracer experiments conducted prior to and following the microbial activity, it was found that biomass accumulation causes redistribution of the preferential flow channels. Zones of slow flow near the fracture inlet were clogged, thus further diverting the flow through zones of fast flow, which were also partially clogged. Quantitative evaluation of biodegradation and bacterial counts supported the results of the multi-tracer tests, indicating that most of the bacterial activity occurs close to the inlet. The changing flow patterns, which control the nutrient supply, resulted in variations in the concentrations of the chemical constituents (TBP, bromide and oxygen), used as indicators of biodegradation.     

Illuminating reactive microbial transport in saturated porous media: demonstration of a visualization method and conceptual transport model

We demonstrate a method to study reactive microbial transport in saturated translucent porous media using the bacteria Pseudomonas fluorescens 5RL genetically engineered to carry a plasmid with bioluminescence genes inducible by salicylate. Induced bacteria were injected into a cryolite grain filled chamber saturated with a sterile non-growth-promoting (phosphorus limited) chemical mixture containing salicylate as an aromatic hydrocarbon analogue. The amount of light produced by the bacteria serves as an estimator of the relative efficiency of aerobic biodegradation since bioluminescence is dependent on both salicylate and oxygen but only consumes oxygen. Bioluminescence was captured with a digital camera and analyzed to study the evolving spatial pattern of the bulk oxygen consuming reactions. As fluid flow transported the bacteria through the chamber, bioluminescence was observed to initially increase until an oxygen depletion zone developed behind the advective front. Bacterial transport was modeled with the advection dispersion equation and oxygen concentration was modeled assuming bacterial consumption via Monod kinetics with consideration of additional effects of rate-limited mass transfer from residual gas bubbles. Consistent with previous measurements, bioluminescence was considered proportional to oxygen consumed. Using the observed bioluminescence, model parameters were fit that were consistent with literature values and produced results in good agreement with the experimental data. These findings demonstrate potential for using this method to investigate the complex spatial and temporal dynamics of reactive microbial transport in saturated porous media.     

Impact of microbial activity on the hydraulic properties of fractured chalk

The impact of microbial activity on fractured chalk transmissivity was investigated on a laboratory scale. Long-term experiments were conducted on six fractured chalk cores (20 cm diameter, 23-44 cm long) containing a single natural fracture embedded in a porous matrix. Biodegradation experiments were conducted under various conditions, including several substrate and oxygen concentrations and flow rates. 2,4,6-Tribromophenol (TBP) was used as a model contaminant (substrate). TBP biodegradation efficiency depended mainly on the amount of oxygen. However, under constant oxygen concentration at the core inlet, elevating the flow rates increased the removal rate of TBP. Transmissivity reduction was clearly related to TBP removal rate, following an initial slow decline and a further sharp decrease with time. The fracture's transmissivity was reduced by as much as 97% relative to the initial value, with no leveling off of the clogging process. For the most extreme cases, reductions of 262 and 157 microm in the equivalent hydraulic apertures were recorded for fractures with initial apertures of 495 and 207 microm, respectively. The reductions in fracture transmissivity occurred primarily because of clogging by bacterial cells and extracellular polymeric substances (EPS) produced by the bacteria. Most of the biodegradation activity was concentrated near the fracture inlet, where the most suitable biodegradation conditions (nutrients and oxygen) prevailed, suggesting that the clogging had occurred in that vicinity. The clogging must have changed the structure of the fracture void, thereby reducing the active volume participating in flow and transport processes. This phenomenon caused accelerated transport of non-reactive tracers and doubled the fracture's dispersivity under constant flow rates.     

Laboratory studies to characterize the efficacy of sand capping a coal tar-contaminated sediment

Placement of a microbial active sand cap on a coal tar-contaminated river sediment has been suggested as a cost effective remediation strategy. This approach assumes that the flux of contaminants from the sediment is sufficiently balanced by oxygen and nutrient fluxes into the sand layer such that microbial activity will reduce contaminant concentrations within the new benthic zone and reduce the contaminant flux to the water column. The dynamics of such a system were evaluated using batch and column studies with microbial communities from tar-contaminated sediment under different aeration and nutrient inputs. In a 30-d batch degradation study on aqueous extracts of coal tar sediment, oxygen and nutrient concentrations were found to be key parameters controlling the degradation rates of polycyclic aromatic hydrocarbons (PAHs). For the five PAHs monitored (naphthalene, fluorene, phenanthrene, anthracene, and pyrene), degradation rates were inversely proportional to molecular size. For the column studies, where three columns were packed with a 20-cm sand layer on the top of a 5 cm of sediment layer, flow was established to sand layers with (1) aerated water, (2) N(2) sparged water, or (3) HgCl(2)-sterilized N(2) sparged water. After steady-state conditions, PAH concentrations in effluents were the lowest in the aerated column, except for pyrene, whose concentration was invariant with all effluents. These laboratory scale studies support that if sufficient aeration can be achieved in the field through either active and passive means, the resulting microbially active sand layer can improve the water quality of the benthic zone and reduce the flux of many, but not all, PAHs to the water column.     

Biodegradation of hydrocarbons vapors: Comparison of laboratory studies and field investigations in the vadose zone at the emplaced fuel source experiment, Airbase Vaerløse, Denmark

The natural attenuation of volatile organic compounds (VOCs) in the unsaturated zone can only be predicted when information about microbial biodegradation rates and kinetics are known. This study aimed at determining first-order rate coefficients for the aerobic biodegradation of 13 volatile petroleum hydrocarbons which were artificially emplaced as a liquid mixture during a field experiment in an unsaturated sandy soil. Apparent first-order biodegradation rate coefficients were estimated by comparing the spatial evolution of the resulting vapor plumes to an analytical reactive transport model. Two independent reactive numerical model approaches have been used to simulate the diffusive migration of VOC vapors and to estimate degradation rate coefficients. Supplementary laboratory column and microcosm experiments were performed with the sandy soil at room temperature under aerobic conditions. First-order kinetics adequately matched the lab column profiles for most of the compounds. Consistent compound-specific apparent first-order rate coefficients were obtained by the three models and the lab column experiment, except for benzene. Laboratory microcosm experiments lacked of sensitivity for slowly degrading compounds and underestimated degradation rates by up to a factor of 5. Addition of NH3 vapor was shown to increase the degradation rates for some VOCs in the laboratory microcosms. All field models suggested a significantly higher degradation rate for benzene than the rates measured in the lab, suggesting that the field microbial community was superior in developing benzene degrading activity.     

Microbial growth and transport in porous media under denitrification conditions: experiments and simulations

Soil column experiments were conducted to study bacterial growth and transport in porous media under denitrifying conditions. The study used a denitrifying microbial consortium isolated from aquifer sediments sampled at the U.S. Department of Energy's Hanford site. One-dimensional, packed-column transport studies were conducted under two substrate loading conditions. A detailed numerical model was developed to predict the measured effluent cell and substrate concentration profiles. First-order attachment and detachment models described the interphase exchange processes between suspended and attached biomass. Insignificantly different detachment coefficient values of 0.32 and 0.43 day⁻¹, respectively, were estimated for the high and low nitrate loading conditions (48 and 5 mg l⁻¹ NO₃, respectively). Comparison of these values with those calculated from published data for aerobically growing organisms shows that the denitrifying consortium had lower detachment rate coefficients. This suggests that, similar to detachment rates in reactor-grown biofilms, detachment in porous media may increase with microbial growth rate. However, available literature data are not sufficient to confirm a specific analytical model for predicting this growth dependence.     

The reactive transport of trichloroethene is influenced by residence time and microbial numbers

The dechlorination rate in a flow-through porous matrix can be described by the species specific dechlorination rate observed in a liquid batch unless mass transport limitations prevail. This hypothesis was examined by comparing dechlorination rates in liquid batch with that in column experiments at various flow rates (3-9-12 cm day(-1)). Columns were loaded with an inoculated sand and eluted with a medium containing 1mM trichloroethene (TCE) for 247 days. Dechlorination in the column treatments increased with decreasing flow rate, illustrating the effect of the longer residence time. Zeroth order TCE or cis-DCE degradation rates were 4-7 folds larger in columns than in corresponding batch systems which could be explained by the higher measured Geobacter and Dehalococcoides numbers per unit pore volume in the columns. The microbial numbers also explained the variability in dechlorination rate among flow rate treatments marked by a large elution of the dechlorinating species' yield as flow increased. Stop flow events did not reveal mass transport limitations for dechlorination. We conclude that flow rate effects on reactive transport of TCE in this coarse sand are explained by residence time and by microbial transport and that mass transport limitations in this porous matrix are limited.     

The spatial distribution of eubacteria and archaea in sand-clay columns degrading carbon tetrachloride and methanol

The spatial distribution of microbial communities was investigated in anaerobic sand-clay columns fed methanol and carbon tetrachloride (CT). Microbial communities were characterized through analysis of soil samples with denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR) for archaea and eubacteria. Increasing CT inlet concentrations to 29 microM lead to complete inhibition of methanol consumption in both columns. Although low levels of eubacteria and archaea were initially present in the clay soils in both columns, there was no significant microbial growth over 400 days in the clays beyond the interface with the sand zone. Thus, the potential for increased contaminant attenuation in heterogeneous sand-clay systems through biodegradation in the clay matrix zones may be limited in many systems.     

Iron-rich dune grasslands: Relations between soil organic matter and sorption of Fe and P

Effects of high atmospheric nitrogen-deposition partly depend on availability of phosphate. Lime-poor, but iron-rich dune grasslands are supposedly protected from grass-encroachment, due to P-fixation in iron phosphate. However, in iron-rich Dutch hinterdunes, dunes have low, but dry former beach plains high grass-encroachment. To test whether these zones differ in nutrient availability, and whether this changed with duration of grass-encroachment, we measured net N-mineralization, microbial characteristics and different fractions of P and Fe from pioneer and shortgrass to tallgrass stages approximately 10, 20 and >25 years old. N-mineralization did not differ between zones, but increased in older tallgrass stages in the organic layer. P-availability was significantly lower in the low grass-encroachment zone, with SOM values below 3% and mineral Fe above 40% allowing for P-fixation in iron phosphates. In the high grass-encroachment zone, however, P-availability increased, because SOM increased and Fe became incorporated in organic matter complexes, with more reversible P-sorption.     

Biodegradation of petroleum hydrocarbon vapors: laboratory studies on rates and kinetics in unsaturated alluvial sand

Predictions of natural attenuation of volatile organic compounds (VOCs) in the unsaturated zone rely critically on information about microbial biodegradation kinetics. This study aims at determining kinetic rate laws for the aerobic biodegradation of a mixture of 12 volatile petroleum hydrocarbons and methyl tert-butyl ether (MTBE) in unsaturated alluvial sand. Laboratory column and batch experiments were performed at room temperature under aerobic conditions, and a reactive transport model for VOC vapors in soil gas coupled to Monod-type degradation kinetics was used for data interpretation. In the column experiment, an acclimatization of 23 days took place before steady-state diffusive vapor transport through the horizontal column was achieved. Monod kinetic parameters Ks and vmax could be derived from the concentration profiles of toluene, m-xylene, n-octane, and n-hexane, because substrate saturation was approached with these compounds under the experimental conditions. The removal of cyclic alkanes, isooctane, and 1,2,4-trimethylbenzene followed first-order kinetics over the whole concentration range applied. MTBE, n-pentane, and chlorofluorocarbons (CFCs) were not visibly degraded. Batch experiments suggested first-order disappearance rate laws for all VOCs except n-octane, which decreased following zero-order kinetics in live batch experiments. For many compounds including MTBE, disappearance rates in abiotic batch experiments were as high as in live batches indicating sorption. It was concluded that the column approach is preferable for determining biodegradation rate parameters to be used in risk assessment models.     

Consolidation of degraded ornamental porous limestone stone by calcium carbonate precipitation induced by the microbiota inhabiting the stone

Although it has already been shown that calcareous stone can be consolidated by using a bacterially inoculated culture medium, a more user-friendly method is the in situ application of a sterile culture medium that is able to activate, among the microbial community of the stone, those bacteria with a potential for calcium carbonate precipitation. In order to test this new method for stone consolidation, non-sterilized decayed porous limestone was immersed in sterile nutritional media. Results were compared to those of the runs in which stone sterilized prior to the treatment was used. The effects of the microbial community on stone consolidation were determined by recording the evolution of the culture media chemistry. The treated stone was tested for mechanical resistance and porosity. Results demonstrate that the tested media were able to activate bacteria from the microbial community of the stone. As a consequence of the growth of these bacteria, an alkalinization occurred that resulted in calcium carbonate precipitation. The new precipitate was compatible with the substrate and consolidated the stone without pore plugging. Therefore, a good candidate to in situ consolidate decayed porous limestone is the application of a sterile culture medium with the characteristics specified in the present study.     

Column experiments to assess the effects of electron donors on the efficiency of in situ precipitation of Zn,Cd, Co and Ni in contaminated groundwater applying the biological sulfate removal technology

BACKGROUND, AIMS AND SCOPE: In a previous study, we explored the use of acetate, lactate, molasses, Hydrogen Release Compound (HRC, which is based on a biodegradable poly-lactate ester), methanol and ethanol as carbon source and electron donor to promote bacterial sulfate reduction in batch experiments, this with regards to applying an in situ metal precipitation (ISMP) process as a remediation tool to treat heavy metal contaminated groundwater at the site of a nonferrous metal work company. Based on the results of these batch tests, column experiments were conducted with lactate, molasses and HRCI as the next step in our preliminary study for a go-no go decision for dimensioning an on site application of the ISMP process that applies the activity of the endogenous population of sulfate-reducing bacteria (SRB). Special attention was given to the sustainability of the metal precipitation process under circumstances of changing chemical oxygen demand (COD) to [SO4(2-)] ratios or disrupted substrate supply. METHODS: To optimize the ISMP process, an insight is needed in the composition and activity of the indigenous SRB community, as well as information on the way its composition and activity are affected by process conditions such as the added type of C-source/ electron donor, or the presence of other prokaryotes (e.g. fermenting bacteria, methane producing Archaea, acetogens). Therefore, the biological sulfate reduction process in the column experiments was evaluated by combining classical analytical methods [measuring heavy metal concentration, SO4(2-)-concentration, pH, dissolved organic carbon (DOC)] with molecular methods [denaturing gradient gel electrophoresis (DGGE) fingerprinting and phylogenetic sequence analysis] based on either the 16S rRNA-gene or the dsr (dissimilatory sulfite reductase) gene, the latter being a specific biomarker for SRB. RESULTS AND DISCUSSION: All carbon sources tested promoted SRB activity, which resulted within 8 weeks in a drastic reduction of the sulfate and heavy metal contents in the column effluents. However, unexpected temporal decreases in the efficiency of the ISMP process, accompanied by the release of precipitated metals, were observed for most conditions tested. The most dramatic observation of the failing ISMP process was observed within 12 weeks for the molasses amended column. Subsequent lowering the COD/ SO4(2-) ratio from 1.9 to 0.4 did not alter the outcome of sulfate reduction and metal precipitation efficiency in this set-up. Remarkably, after 6 months of inactivity, bacterial sulfate reduction was recovered in the molasses set up when the original COD/ SO4(2-) ratio of 1.9 was applied again. Intentional disruption of the lactate and HRC supplies resulted in an immediate stagnation of the ISMP processes and in a rapid release of precipitated metals into the column effluents. However, the ISMP process could be restored after substrate amendment. 16S rDNA-based DGGE analysis revealed that the SRB population, in accordance with the results of the previously performed batch experiments, consisted exclusively of members of the genus Desulfosporosinus. The community of Archaea was characterized by sequencing amplicons of archaeal and methanogen-specific PCR reactions. This approach only revealed the presence of non-thermophilic Crenarchaeota, a novel group of organisms which is only distantly related to methane producing Euryarchaeota. DGGE on the dsrB genes was successfully used to link the results of the ISMP process to the community composition of the sulfate reducing bacteria. CONCLUSIONS: In the case of an intentional disruption of substrate supply, the ISMP process failed most likely because the growth and activity of the indigenous SRB community stopped due to a lack of a carbon and electron donor. On the other hand, the cause of the sudden temporal shortcomings of the ISMP process in the presence of different substrates was not immediately clear. It was first thought to be the result of competition between methanogenic prokaryotes (MP) and sulfate reducers, since the formation of small amounts of CH4 (0.01-0.03 ppm ml(-1) was detected. However, the results of molecular analyzes indicate that methanogens do not constitute a major fraction of the microbial communities that were enriched in the column experiments. Therefore, we postulate that the SRB population becomes inhibited by the formed metal sulfides. RECOMMENDATION AND PERSPECTIVE: Our results indicate that the ISMP process is highly dependent on SRB-stimulation by substrate amendments and suggest that this remedial approach might not be viable for long-term application unless substrate amendments are continued and environmental conditions are strictly controlled. This will include the removal of affected aquifer material from the metal precipitation zone at the end of the remediation process, or removal of metal precipitates when the microbial activity decreases. Additional tests are necessary to investigate what will happen when clear groundwater passes through the reactive zone while no more C-sources are amended and all indigenous carbon is consumed. Also, the effects of dramatic increases in sulfate- or HM-concentrations on the SRB-community and the concomitant ISMP process need to be studied in more detail.