On-site biostimulation evaluation of a remediated perchloroethene-contaminated acquifer located in Jablunkov, Czech Republic

Chlorinated ethenes solvents (CEs) are one of the most common contaminants identified in groundwater ecosystems. Long used as a degreaser in industry, perchloroethene (PCE) and trichloroethene (TCE) are particularly persistent in the environment as recalcitrant to oxic degradation, and classified as unhealthy and dangerous for the environment. Under anaerobic conditions, CEs can be degraded naturally in the environment by organohalide respiring bacteria (OHRB) through dehalogenase enzymes reducing them into ethene, a non-hazardous end product. However, toxicity of the intermediate daughter compounds of the degradation (TCE, DCE and VC) is higher than PCE. As this natural attenuation is a slow process, intermediate compounds accumulate in the environment and biostimulation is often used to enhance the complete degradation of CEs. Substrates are added to the contaminated aquifer and used by fermentative bacteria releasing by-product such as H2 that is essential for the dehalorespiration of CEs. Biostimulation has been studied in a PCE contaminated-aquifer located in Jablunkov (Czech Republic). Cheese whey (CW) has been injected into the groundwater in November 2012 and June 2013. The objective of the current project is to evaluate the efficiency of the CW applications by analysing microbiologic and physico-chemical parameters describing the aquifer at six sampling dates from September 2012 to December 2013 within six wells allocated over the aquifer. Global bacterial community structures were analysed in laboratory using Polymerase Chain Reaction (PCR) and T-RFLP (Terminal Restriction Fragment Length Polymorphism). OHRB were analysed as well by specific PCR targeting 16S rRNA and dehalogenase genes of bacteria known to degrade completely the CEs. Then, contaminants and ethene concentrations as well as others Terminal Electron Acceptors implicated in the respiratory processes (Terminal Electron Accepting Processes – TEAPs) were measured to give information about the redox conditions within the aquifer. Finally, others hydrogeological parameters such as pH and temperature were measured. All data were analysed using Multiple Factor Analysis (MFA) in the R software. Interpretations provided a global model indicating the characteristics and functioning of the aquifer. The successive CW applied on-site have a gradual impact on the environmental conditions and on the structures of bacterial communities. After the two CW applications, the aquifer has partly changed to conditions favourable for complete dechlorination. It was the case for three wells over six. Two CW injections have been necessary to allow the production of ethene for two wells whereas only one was needed for the other one. Biostimulation were efficient but not sufficient to completely degrade the CEs on the whole aquifer. In one well, the accumulation of DCE were more important than the production of ethene. In two other wells, ethene was not produced but the statistical analysis showed that they tend toward complete dechlorination conditions. New CW applications would be necessary to completely remediate this aquifer.

An ecological approach for understanding the reductive dechlorination process of chlorinated ethenes in contaminated aquifers

Christof Holliger, Pierre Rossi, Sonia Estelle Tarnawski

Chlorinated ethenes, frequently encountered contaminants in groundwater, are often found to be incompletely dechlorinated to dichloroethene and vinyl chloride, more toxic products that accumulate in the aquifer. Some of the concerns in designing remediation approaches based on biological dechlorination are i) the presence of bacteria capable of completely reducing chlorinated ethenes, and ii) the competition events for available electron donors between classical biogeochemical processes and the reductive dechlorination process. For this purpose, an assessment methodology was developed to explore the existing relations between autochthonous bacterial communities, degradation state of chlorinated ethenes and redox processes, including reductive dechlorination. Three steps are involved with first the acquisition of abiotic environmental data, followed by assessing the biochemical potential for complete dechlorination and an analysis of bacterial community structures, and finally a statistical Multiple Factor Analysis combining datasets from geochemistry and microbiology. This approach was applied for diagnosing a Swiss aquifer for its in situ potential of complete reductive dechlorination, and for monitoring a Czech aquifer under bioremediation by successive cheese whey injections. The Swiss site contained the microbiological potential for complete dechlorination but the global redox conditions were not enough reducing and dichloroethene reduction was in competition locally with manganese reduction. The follow-up of the Czech site has shown that during the remediation process, complete reductive dechlorination was favored under sulfate-reducing conditions, but was in competition with other anaerobic bacterial processes leading to a transient accumulation of dichloroethene and vinyl chloride. In both cases, antagonist biogeochemical processes were identified as the reasons restricting the reductive dechlorination at the level of toxic intermediates. This approach is now formalized in a user-oriented manual, and could be integrated as tool for eligibility investigation of aquifers for a bioremediation strategy through natural attenuation.

2015

Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Noam Shani

Groundwater is essential for human activities and is sometimes referred to as a non-renewable resource in the same way as oil and gas. During the past century, precious groundwater reserves worldwide have been threatened by anthropogenic release of chemical compounds. Among these, chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) belong to the most common class of groundwater contaminants. In oxic conditions, PCE and TCE are recalcitrant to any form of degradation and constitute a long-term source of groundwater contamination. It has been reported that CEs can be degraded biologically in anoxic conditions, serving as electron acceptors of an anaerobic respiratory process called organohalide respiration (OHR). In this process, PCE is sequentially reduced to TCE, cis-dichloroethene (cDCE), vinyl chloride (VC), and finally to harmless ethene (Eth). Engineering strategies based on reductive dechlorination, such as monitored natural attenuation (MNA), have been designed for the bioremediation of aquifers contaminated with CEs. However, stalling of the sequence of dechlorination has often been observed, resulting from incomplete or impeded biodegradation of the highly toxic daughter molecules (cDCE and VC) and leading to their accumulation in situ. It was shown in laboratory experiments that OHR of CEs is more efficient in mixed cultures and that organohalide-respiring bacteria (OHRB) live in association with other microorganisms in microbial consortia. In the aquifer ecosystem, OHRB rely on complex interactions between members of bacterial communities for their electron donors and carbon supplies. On the other hand, they are in competition for these resources with other terminal electron-accepting processes (TEAPs). These complex interactions together with the intrinsic structural heterogeneity of aquifers make it difficult to understand the reasons for lower CEs accumulation, to predict the fate of OHR, and to design bioremediation strategies. This thesis aimed at elucidating the reasons for lower CEs accumulation in situ. An ecological approach considering the aquifer ecosystem in its whole complexity was developed and applied to the specific cases of two contaminated sites showing accumulation of lower CEs. The proposed methodology relies on the precise depiction of both aquifer habitat and microbial communities interacting therein. However, the potential impact of the first technical step, namely the impact of the pumping parameters on groundwater samples used for the description of the bacterial communities, was not known at the start of this thesis work. Results of investigations addressing this topic showed that parameters related to the tubing characteristics did not impact the apparent bacterial community structures (BCS) in a laboratory experiment. However, the study revealed a significant impact of the pumping flow rate on apparent BCS extracted from the aquifer. Terminal-restriction fragment length polymorphism (T-RFLP), coupled with an appropriate groundwater sampling strategy, enabled an accurate profiling of the BCS. However no indication was provided concerning the identities of the community members. A bioinformatics tool called PyroTRF-ID was developed to overcome this obstacle. The software enabled affiliating terminal-restriction fragments (T-RFs) to precise phylotypes by coupling T-RFLP and pyrosequencing data. An additional function enables in silico assessment of restriction enzymes for designing appropriate T-RFLP procedures. The developed methodology was applied all along the thesis work. Optimized tools and procedures were used to investigate the reasons for the accumulation of VC, and to a lesser extent cDCE, in the first aquifer showing relatively homogeneous lithological composition and slow groundwater fluxes. Statistical findings indicated that VC reduction was outcompeted by Fe(III) reduction in some sections of the aquifer. T-RFs showing significant correlations with VC reduction variables were identified by sequencing and with PyroTRF-ID. Results showed sequences closely affiliated to uncultured bacteria of the "Lahn Cluster" (LC) within the class Dehalococcoidetes, previously reported as PCE-to-cDCE dechlorinating microorganisms, and to the genus "Dehalococcoides". According to present knowledge, only members of this genus are able to reduce cDCE and VC. A major T-RF negatively correlated with the LC T-RF affiliated to the genus Rhodoferax, containing iron-reducing bacteria (IRB). Furthermore, indications were obtained of a mutualistic interaction between IRB and sulfate-reducing bacteria, potentially playing an important role by reducing the Fe(III) contents locally. The importance of the aquifer hydrogeological structure and functioning was exemplified in the study of the second aquifer ecosystem, where discrepancies in the fate of OHR were observed. In the immediate vicinity of the source zone, complete OHR was occurring, with production of Eth, whereas cDCE was accumulating further downstream, in apparent similar redox conditions. The profiling and metagenomic techniques revealed drastically different bacterial communities in the zones displaying different OHR fate. The source zone displayed bacterial populations typically found in highly reduced environments and involved in OHR, such as Dehalococcoides sp. In contrast, the downstream zone showing an accumulation of cDCE displayed bacterial populations typically found in more oxidized environments, such as aerobic bacteria, nitrifiers, and denitrifiers. In this zone, the conditions were probably slightly oxidized periodically by the combination of aquifer recharge, a specific lithological configuration, and lower organic matter content. Finally, and based on these considerations, a methodology for the investigation of the reasons for lower CEs accumulation in contaminated aquifers is proposed and discussed. This methodology follows on a multidisciplinary ecological approach for the description of the functioning of the aquifer ecosystem, and enables formulating scenarii of the reasons for lower CEs accumulation.

EPFL2012

Assessment of bioremediation potential and monitoring of biological reductive dechlorination in sites contaminated with chlorinated ethenes

Christof Holliger, Pierre Rossi, Sonia Estelle Tarnawski

Chlorinated ethenes (CEs), such as perchloroethene (PCE) and trichloroethene (TCE), are one of the most common class of groundwater contaminants. Aquifers are considered nowadays as dynamic ecosystems, showing multiple interactions between the physical, chemical and biotic components. The assessment of the intrinsic contaminant biodegradation capacities of these complex ecosystems was carried out using an integrative methodology, combining both molecular microbial ecology and water geochemical data. The bioremediation potential of CEs was evaluated in two contaminated sites, presenting both dichloroethene (DCE) and vinyl chloride (VC) accumulation. These aquifers were investigated using multivariate statistics applied to data matrix combining environmental variables, bacterial community structures determined by terminal-restriction fragment length polymorphism and detection of dechlorinating bacteria and genes by specific PCR. The multifactorial analysis of data obtained from 25 groundwater samples from a PCE-contaminated site in Switzerland showed that manganese reduction (MR) was a key terminal electron accepting process, suggesting a potential competition between MR and DCE degradation to VC. Ethene was detected in low concentrations as well as Dehalococcoides sp. and VC reductive dehalogenase genes. Therefore, the potential for the complete biodegradation of PCE was present in this aquifer. However, the reduction of DCE will be strongly inhibited under the local conditions as long as oxidized manganese resources are present. The second site (Velamos, Czech Republic) sampled at 7 different dates making a total of 35 groundwater samples, was under active biostimulation process. Statistical analysis of the environmental parameters showed that successive cheese whey injections modified the aquifer conditions, becoming favourable for the complete dechlorination, and also for sulfate reduction (SR) and methanogenesis. However, DCE and VC accumulated along with the production of ethene, methane and hydrogen sulphide. This indicated a competition between CEs dechlorination and SR and methanogenesis, and possibly explained a transitional slower reaction of CEs dechlorination during the remediation process. Bacterial populations correlated with ethene production were related to Geobacter sp. and Dehalococcoides sp. In conclusion, statistical analysis combining the whole environmental and bacterial data allows evaluation of the bioremediation potential present in contaminated aquifers and elucidating mechanisms responsible for the incomplete dechlorination of CEs.

2013

Assessment of bioremediation potential and monitoring of biological reductive dechlorination in sites contaminated with chlorinated ethenes

Christof Holliger, Pierre Rossi, Sonia Estelle Tarnawski

2013

Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Noam Shani

EPFL2012

An ecological approach for understanding the reductive dechlorination process of chlorinated ethenes in contaminated aquifers

Christof Holliger, Pierre Rossi, Sonia Estelle Tarnawski

2015