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Microbial degradation of chlorinated ethenes in hyporheic zones: processes and development of mitigation strategies
Chlorinated aliphatic compounds (CAHs) like tetrachloroethene (PCE), trichloroethene (TCE) and their daughter products cis</>-dichloroethene (cDCE) and vinyl chloride (VC) are major groundwater contaminants threatening water quality and human health. At many industrial sites, CAH contaminated groundwater plumes discharge into surface water bodies like rivers and lakes. </>Research and technology development aiming at mitigationof contamination of surface water by discharging CAH groundwater plumesis largely directed to source confinement and plume remediation in the aquifer compartment. However, riverbed </>sediments- known as the hyporheic zone- </>can support</> a broad spectrum of natural CAH attenuation processes such as sorption, dilution and biodegradation. B</>iodegradation is the only mechanism that results into destruction of CAH compounds in the environment and involves bacteria mediated metabolic processes like organohalide respiration (OHR) under anoxic conditions and aerobic degradation under oxic conditions</>. </>However, despite the </>critical role of biodegradation processes in determining contaminant fate and fluxes toward receiving ecosystems, and in estimating the need for additional remedial actions, information on CAH biodegradation processes in hyporheic zones is scarce. Particularly, the potential for a combination of aerobic/anaerobic CAH biodegradation in hyporheic sediments has receivedlimited attention. Therefore, the general objective of this work </>wasto study the underlying processes of as well anaerobic as aerobic microbial degradation of CAHs in hyporheic zones and the development of mitigation strategies to reduce or prevent CAH discharge into receiving surface water bodies. The study focused on the hyporheic zone of a particularindustrial site of the Zenne River in Belgium where a VC/cDCE polluted groundwater plume discharges into the river. A previous study showed that the hyporheic zone at that site displays cDCE and VC OHR activity. </>
Since hyporheic surficial river-bed sediments are often characterized by sharp redox gradients between the oxic benthic sediment and underlying anoxic sediment, it can be hypothesized that it represents an ideal niche for both aerobic and anaerobic VC degraders. To test this hypothesis, in the first part of the study, the fate of VC and the dynamics of bacterial guilds involved in aerobic and anaerobic degradation of VC was studied in microcosms containing surficial sediments of the hyporheic zone of the study location under anoxic and oxygen-exposed static conditions. After degradation of 3 </>consecutive </>VC spikes, quantitative PCR(qPCR) showed that Dehalococcoides</> </>mccartyi</></> </>16S rRNA andVC reductive dehalogenase-encoding genes (vcrA</>, bvcA</>) were enriched more than four orders of magnitude in anoxic microcosms, concomitant with stoichiometric conversion of VC to ethene. In oxygen-exposed microcosms, etnC</> and etnE</> involved in aerobic ethene/VC-oxidation, were enriched more than one order of magnitude with concomitant low or no accumulation of ethene. However, D.</> </>mccartyi</></> </>16S rRNA gene, vcrA</> and bvcA</> copy numbers were also enriched in oxygen-exposed microcosms containing sediment characterized by a high organic carbon content, a small grain size and limited oxygen penetration, whereas they were reduced more than two order of magnitude in oxygen-exposed sediment characterized by low organic carbon content, a larger grain size and extensive oxygen penetration. These results suggest the co-existence and co-activity of anaerobic and aerobic VC degraders in the same small volume of surficial sediment of the Zenne River, and that oxygen distribution, as determined by sediment grain size and organic matter content, affects the local VC degrading bacterial community and VC biodegradation pathway.</>
In the hyporheic zone, </>fluctuations in redox conditions are regularly occurring.</> </>Penetration of oxygenated surface water into the riverbed sediment in hyporheic zones and resulting redox fluctuationsmight affect site-specific aerobic and anaerobic microbial guilds and their contribution to degradation of contaminants such as </>cDCE and VC in discharging groundwater. To test this hypothesis, in the second part of the study, the resistance and resilience of aerobic and anaerobic VC/cDCE degraders to fluctuating redox conditions was studied in microcosmsprepared from surficial Zenne sediment. The microcosms were incubated under anoxic static condition and each time when the degradation </>of 3 </>consecutive </>VC/cDCE spikes was completed,</> the redox and incubation conditions were changed with respect to oxygen exposure and/or shaking. The results show that</> oxygen exposure under static condition resulted in an 1.5-3.4 folds increased VC degradation rate as compared with anoxic static incubations and concomitant enrichment of the catabolic genes etnC</> </>and etnE</>. However, </>under oxygen-exposed shaking condition, real oxidative VC assimilation was only noticed in sediments with low organic carbon content leading to 5 folds higher degradationrate as compared with anoxic incubations. In these sediments, VC/cDCE respiration activity was impaired under oxygen-exposed shaking condition,resulting in a more than two orders of magnitude decay of D. mccartyi</>. The impacted resilience of </>organohalide respiring bacteria (</>OHRB) led to irreversible hindrance of OHR under subsequent anoxic static conditions. Furthermore, oxidative VC assimilators did not show the ability to assimilate cDCE. On the other hand, in the sediments with high organic carbon content where OHRB were protected from oxygen under oxygen-exposed static condition, VC/cDCE removal appeared to be non-assimilative, since in these microcosms ethenotrophic assimilation of the ethene that was produced by VC/cDCE respiration, led to mischaracterized aerobic degradation. These results suggest the role of oxygen penetration and organic carbon distribution in hyporheic zones not only in instant pathway selection but also in long-term adaptation toward assimilative aerobic VC degradation. </>
In situ</></> bioreactive capping is a promising technology for mitigation of surface water contamination by enhancing biodegradation of contaminants that discharge into the river from either polluted sediments or discharging groundwater. In bioreactive caps, contaminants are transformed into harmless products through microbially mediated reactions. Stimulation of CAHs degradation in bioreactive caps can beachieved through incorporation of solid polymeric organic materials (SPOMs) in the cap in order to provide a sustainable electron source for reductive dechlorination by </>OHRB.</> In the third part of the study, the possibility of application of natural SPOMs to stimulate CAH dechlorination in a bioreactive cap approach, was examined. Five different SPOMs,i.e., wood chips, hay, straw, tree bark and shrimp waste, were assessedfor their long term applicability as an electron donor for reductive dechlorination of cDCE and VC in sediments of the Zenne River. The partitioning of reducing equivalents between OHR and methanogenesis as well as the dynamics of associated microbial guilds were studied with the aim offinding an electron source that preferentially stimulated OHR over methanogenesis. The initial fast release of fermentation products such as acetate, propionate and butyrate led to 171, 152, and 112 times higher methane production </>in the microcosms amended with shrimp waste, straw and hay as compared with natural attenuation while no considerable stimulation of VC/cDCE</> respiration</> was obtained in any of the SPOM stimulated microcosms. However, in the longer term, accumulation of short chain </>fatty acids decreased as well as methanogenesis while sustained dechlorination rates of both VC and cDCE were established with concomitant increase in the number of D.</> </>mccartyi</></> </>and corresponding catabolic genes vcrA</> and bvcA</> both in the sediment and on the surface of the SPOM materials. </>The </>rapid and persistent colonization oftree bark by D.</> </>mccartyi</></> </>combined with 4-12 folds lower stimulation of methanogenesis compared with other stimulated conditions selected tree bark as a SPOM of interest for use in bioreactive caps forlong term stimulation of dehalorespiration of CAHs. </>
In the fourth part of the study, the bacterial community composition was examined inthe hyporheic sediment of the Zenne River before and after the installation of a wastewater treatment plant (WWTP) which was expected to decrease the organic load of the river. It was hypothesized that the OHRB thatwas fueled by the organic-rich untreated wastewater input of the river,might be impacted after the installation of the WWTP due to reduced organic carbon input. Bacterial 16S rRNA gene sequences amplified from DNA extracts of horizontal sediment layers, collected two years before (in 2005) and after WWTP construction (2010 and 2011) were obtained by pyrosequencing. Major differences in bacterial community composition were observed in the sediments of 2010 and 2011 compared with those of 2005 whichcould be associated with a reduction in organic content. Proteobacteria </></>was the most dominant phylum in the sediments of 2005 followedby the Chloroflexi</> phylum that includes OHRB like D.</> </></>mccartyi.</></> </>The relative abundance of Chloroflexi</> declined dramatically from the average of 14.8% of the qualified bacterial reads in sediments of 2005 to 4.21 and 0.7 % in sediments of 2010 and 2011, respectively. </>The more oxic and oligotrophic sediments of 2011 were associated with a strong decrease of copiotrophic and anaerobic microbial groups </></></>and </>enrichment of oligotrophic members of Alpha</>- and Betaproteobacteria. </>Moreover, the results indicated </></>lower species richness and diversity in the sediments of 2011. </>The results of qPCR indicated a more than two orders of magnitude decrease in the number of bacteria and D.</> </>mccartyi</></> 16S rRNA gene </>in the sediments with depth and time </>concomitant with a substantial decrease ofanaerobic VC respiration potential in sediments microcosms over time. Although the installation of the WWTP and subsequent reduced organic carbon load led to </></>reduced bacterial biomass and diversity, it also decreased</>OHR potential and natural attenuation capacity of the hyporheic sediments in the Zenne River. </></>
Overall, the study shows the role of aerobic assimilative VC degradation in hyporheic sediments and that in addition to OHR, it is important to consider that activity to fully evaluate the importance of biodegradation in mitigation of CAH discharge into surface water. Moreover, it shows the potential of SPOM to be incorporated into in situ</> bioactive caps as slowly degrading natural solid organic material to stimulate OHR for improved protection of rivers against discharging CAH contaminated groundwater. The results from the last part of the study accentuate the role of organic matter in OHR in hyporheic zones and the impact of river habilitation on that activity. Thelocal implementation of in situ</> bioactive caps or the activity of aerobic CAH degraders can result in river protection from CAH contaminateddischarging groundwater.</>
Since hyporheic surficial river-bed sediments are often characterized by sharp redox gradients between the oxic benthic sediment and underlying anoxic sediment, it can be hypothesized that it represents an ideal niche for both aerobic and anaerobic VC degraders. To test this hypothesis, in the first part of the study, the fate of VC and the dynamics of bacterial guilds involved in aerobic and anaerobic degradation of VC was studied in microcosms containing surficial sediments of the hyporheic zone of the study location under anoxic and oxygen-exposed static conditions. After degradation of 3 </>consecutive </>VC spikes, quantitative PCR(qPCR) showed that Dehalococcoides</> </>mccartyi</></> </>16S rRNA andVC reductive dehalogenase-encoding genes (vcrA</>, bvcA</>) were enriched more than four orders of magnitude in anoxic microcosms, concomitant with stoichiometric conversion of VC to ethene. In oxygen-exposed microcosms, etnC</> and etnE</> involved in aerobic ethene/VC-oxidation, were enriched more than one order of magnitude with concomitant low or no accumulation of ethene. However, D.</> </>mccartyi</></> </>16S rRNA gene, vcrA</> and bvcA</> copy numbers were also enriched in oxygen-exposed microcosms containing sediment characterized by a high organic carbon content, a small grain size and limited oxygen penetration, whereas they were reduced more than two order of magnitude in oxygen-exposed sediment characterized by low organic carbon content, a larger grain size and extensive oxygen penetration. These results suggest the co-existence and co-activity of anaerobic and aerobic VC degraders in the same small volume of surficial sediment of the Zenne River, and that oxygen distribution, as determined by sediment grain size and organic matter content, affects the local VC degrading bacterial community and VC biodegradation pathway.</>
In the hyporheic zone, </>fluctuations in redox conditions are regularly occurring.</> </>Penetration of oxygenated surface water into the riverbed sediment in hyporheic zones and resulting redox fluctuationsmight affect site-specific aerobic and anaerobic microbial guilds and their contribution to degradation of contaminants such as </>cDCE and VC in discharging groundwater. To test this hypothesis, in the second part of the study, the resistance and resilience of aerobic and anaerobic VC/cDCE degraders to fluctuating redox conditions was studied in microcosmsprepared from surficial Zenne sediment. The microcosms were incubated under anoxic static condition and each time when the degradation </>of 3 </>consecutive </>VC/cDCE spikes was completed,</> the redox and incubation conditions were changed with respect to oxygen exposure and/or shaking. The results show that</> oxygen exposure under static condition resulted in an 1.5-3.4 folds increased VC degradation rate as compared with anoxic static incubations and concomitant enrichment of the catabolic genes etnC</> </>and etnE</>. However, </>under oxygen-exposed shaking condition, real oxidative VC assimilation was only noticed in sediments with low organic carbon content leading to 5 folds higher degradationrate as compared with anoxic incubations. In these sediments, VC/cDCE respiration activity was impaired under oxygen-exposed shaking condition,resulting in a more than two orders of magnitude decay of D. mccartyi</>. The impacted resilience of </>organohalide respiring bacteria (</>OHRB) led to irreversible hindrance of OHR under subsequent anoxic static conditions. Furthermore, oxidative VC assimilators did not show the ability to assimilate cDCE. On the other hand, in the sediments with high organic carbon content where OHRB were protected from oxygen under oxygen-exposed static condition, VC/cDCE removal appeared to be non-assimilative, since in these microcosms ethenotrophic assimilation of the ethene that was produced by VC/cDCE respiration, led to mischaracterized aerobic degradation. These results suggest the role of oxygen penetration and organic carbon distribution in hyporheic zones not only in instant pathway selection but also in long-term adaptation toward assimilative aerobic VC degradation. </>
In situ</></> bioreactive capping is a promising technology for mitigation of surface water contamination by enhancing biodegradation of contaminants that discharge into the river from either polluted sediments or discharging groundwater. In bioreactive caps, contaminants are transformed into harmless products through microbially mediated reactions. Stimulation of CAHs degradation in bioreactive caps can beachieved through incorporation of solid polymeric organic materials (SPOMs) in the cap in order to provide a sustainable electron source for reductive dechlorination by </>OHRB.</> In the third part of the study, the possibility of application of natural SPOMs to stimulate CAH dechlorination in a bioreactive cap approach, was examined. Five different SPOMs,i.e., wood chips, hay, straw, tree bark and shrimp waste, were assessedfor their long term applicability as an electron donor for reductive dechlorination of cDCE and VC in sediments of the Zenne River. The partitioning of reducing equivalents between OHR and methanogenesis as well as the dynamics of associated microbial guilds were studied with the aim offinding an electron source that preferentially stimulated OHR over methanogenesis. The initial fast release of fermentation products such as acetate, propionate and butyrate led to 171, 152, and 112 times higher methane production </>in the microcosms amended with shrimp waste, straw and hay as compared with natural attenuation while no considerable stimulation of VC/cDCE</> respiration</> was obtained in any of the SPOM stimulated microcosms. However, in the longer term, accumulation of short chain </>fatty acids decreased as well as methanogenesis while sustained dechlorination rates of both VC and cDCE were established with concomitant increase in the number of D.</> </>mccartyi</></> </>and corresponding catabolic genes vcrA</> and bvcA</> both in the sediment and on the surface of the SPOM materials. </>The </>rapid and persistent colonization oftree bark by D.</> </>mccartyi</></> </>combined with 4-12 folds lower stimulation of methanogenesis compared with other stimulated conditions selected tree bark as a SPOM of interest for use in bioreactive caps forlong term stimulation of dehalorespiration of CAHs. </>
In the fourth part of the study, the bacterial community composition was examined inthe hyporheic sediment of the Zenne River before and after the installation of a wastewater treatment plant (WWTP) which was expected to decrease the organic load of the river. It was hypothesized that the OHRB thatwas fueled by the organic-rich untreated wastewater input of the river,might be impacted after the installation of the WWTP due to reduced organic carbon input. Bacterial 16S rRNA gene sequences amplified from DNA extracts of horizontal sediment layers, collected two years before (in 2005) and after WWTP construction (2010 and 2011) were obtained by pyrosequencing. Major differences in bacterial community composition were observed in the sediments of 2010 and 2011 compared with those of 2005 whichcould be associated with a reduction in organic content. Proteobacteria </></>was the most dominant phylum in the sediments of 2005 followedby the Chloroflexi</> phylum that includes OHRB like D.</> </></>mccartyi.</></> </>The relative abundance of Chloroflexi</> declined dramatically from the average of 14.8% of the qualified bacterial reads in sediments of 2005 to 4.21 and 0.7 % in sediments of 2010 and 2011, respectively. </>The more oxic and oligotrophic sediments of 2011 were associated with a strong decrease of copiotrophic and anaerobic microbial groups </></></>and </>enrichment of oligotrophic members of Alpha</>- and Betaproteobacteria. </>Moreover, the results indicated </></>lower species richness and diversity in the sediments of 2011. </>The results of qPCR indicated a more than two orders of magnitude decrease in the number of bacteria and D.</> </>mccartyi</></> 16S rRNA gene </>in the sediments with depth and time </>concomitant with a substantial decrease ofanaerobic VC respiration potential in sediments microcosms over time. Although the installation of the WWTP and subsequent reduced organic carbon load led to </></>reduced bacterial biomass and diversity, it also decreased</>OHR potential and natural attenuation capacity of the hyporheic sediments in the Zenne River. </></>
Overall, the study shows the role of aerobic assimilative VC degradation in hyporheic sediments and that in addition to OHR, it is important to consider that activity to fully evaluate the importance of biodegradation in mitigation of CAH discharge into surface water. Moreover, it shows the potential of SPOM to be incorporated into in situ</> bioactive caps as slowly degrading natural solid organic material to stimulate OHR for improved protection of rivers against discharging CAH contaminated groundwater. The results from the last part of the study accentuate the role of organic matter in OHR in hyporheic zones and the impact of river habilitation on that activity. Thelocal implementation of in situ</> bioactive caps or the activity of aerobic CAH degraders can result in river protection from CAH contaminateddischarging groundwater.</>
Date:1 Oct 2009 → 18 Jun 2013
Keywords:River sediment, VOCL, River basin, Biodegradation
Disciplines:Microbiology, Systems biology, Laboratory medicine, Biochemistry and metabolism, Medical biochemistry and metabolism, Sustainable and environmental engineering, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering
Project type:PhD project