Bacterial communities play a pivotal role in biogeochemical cycle, however there is still no consensus on the effect of pesticide contamination on bacterial community function, especially on their ability to reduce nitrate, which is an issue in several pesticides impacted sites. Atrazine is an herbicide which have been widely used for weed control in corn, soja and sorgho cultures, until 2003 when it has been withdrawn in France. Desethylated atrazine (DEA) is among its metabolite the one most observed in soil and groundwater and it has been reported with higher effect to aquatic life, than ATZ. Ten years after its withdrawn, ATZ and DEA concentrations exceeding the legal EU thresholds for groundwater and drinking waters (0.1 µg/L) are still reported.
Our objective was to assess the effect of pesticide mixtures on groundwater microbial abundance, community structure and their function in the nitrate reduction at the catchment level. This is, to our best knowledge, the first study on pesticide impact on groundwater microbial community diversity structure and function; it has the potential to provide sound-based arguments to be considered when improving the current strategy to manage water quality, as well as when proposing end points to monitor the microbial community in the biodiversity objective under the European water directive framework.
Two-year monitoring. The Ariège alluvial plain (France) is contaminated with a large panel of pesticides with concentations up to the ppm level. Water (1 L) was sampled from 17 selected springs on a monthly basis during 2 years (March 2012- March 2014, n = 50).
Microcosm. Water was sampled in July 2014 in two wells having different contamination profils. Water (700 mL) was placed in 1 L microcosm and ATZ, DEA or ATZ+DEA was spiked at 0, 1 and 10 µg/L. Units were sacrificed at the start and following 15-day and 30-day incubation (n = 58).
Bacterial analyses. Water was filtered through 0.22 µm filters and microbial DNA was extracted. Abundance of the universal marker (16S rRNA gene) and of nitrate-reducing bacteria (narG and napA genes) were assessed by quantitative PCR (qPCR). Diversity was assessed using fingerprinting technic, CE-SSCP (Capillary Electrophoresis-Single Strand Conformational Polymorphism).
Chemical analyses. Water samples for pesticides were analyzed by LC-MS/MS following an on line-solid phase extraction, and samples for anions and cations were analyzed by ion chromatography.
Statistical analyses. Divergence between diversity profiles were analyzed with StatFingerprints software. Diversity indexes were also calculated (richness, Shannon-weaver index, eveness). Statistical differences were analyzed using two-way ANOVA with treatments, chemical or incubation time as factors (p < 0.05). Principal component analyses (PCA) were performed using XLSTAT Version 2011.2.02.
Biodiversity was higher thorough the experiment in the water C+, historically contaminated with various pesticides than in the water C- where none of the 51 pesticides monitored was observed during 2 years (Figure 1). Pesticide concentrations in the water historically contaminated often exceeded the legal EU threshold for groundwater and drinking waters (e.g. 2-year mean: 0.09 ± 0.01 µg ATZ /L, 0.43 ± 0.06 µg DEA /L (n = 23)). On the other side, during microcosm incubation, biodiversity decreased when spiked-chemical concentration increased from 1 to 10 µg/L. In the water C+, no effect of the incubation duration was noticed. In the water C-, biodiversity decreased with the chemical concentration and increased with the incubation duration when exposed to 1 µg/L, while no increase was observed when the community was exposed to 10 µg/L, suggesting that adaptation to pesticides might occur and this process depends on the chemical concentration. In both waters, there was no difference in the ATZ, DEA or ATZ+DEA effect to the microbial diversity. Abundance of bacteria reducing nitrate among the total community drastically decreased during the experiment, but this decrease was also observed in the control unit (p < 0.05). Total biomass was similar in both waters and during the whole experiment (p > 0.05). No biodegradation of ATZ and DEA was observed during the 1-month exposure.
Analyses of the two-year monitoring at an agricultural catchment level are undergoing; preliminary results suggests that biomass is similar in all samples while biodiversity and nitrate-reducing bacteria show important differences between samples.
The undergoing analyses on natural waters monitored during two years at a catchment level will hopefully show boundaries within the complex relationship between biodiversity and chemical concentrations observed in the microcosm, which is positive at the environmental level (between water C- and C+) and negative at the spiked-concentratoin level (between 1 and 10 µg/L).
In the microcosms, ATZ, DEA or ATZ+DEA exhibited similar effect on the microbial community. Comparison at the catchment level of the effect of the 51 monitored pesticides taken individually or summed, on the microbial biodiversity will enable to assess the mixture effect on the microbial community.
Biomass was similar in all conditions (p > 0.05), suggesting that this is not a sensitive endpoint to assess water quality.
Relationship between pesticide contamination and bacterial nitrate-reducing community will be assessed further to consider the risk of nitrate acumulation or of inhibition effect of nitrate on pesticide biodegradation.
Acknowledgement - The authors thank the Water Agency Adour-Garonne (France), the FEDER grants (Europe) and the BRGM for their financial support.
Successful bioremediation of subsurface environments, such as contaminated soil or groundwater, can depend on a good understanding of microbial degradation processes. Taking into account the complexity of interactions that occur between the solid matrix, indigenous microorganisms and pollutants, initial on-site characterization and in situ monitoring of microbial communities over time are essential. One of the major challenges with subsurface systems has been the development of sampling techniques for microbiological investigations. Reliable sampling is highly critical since detection of microbes and/or their expression, and the quantification of genes involved in degradation processes are often used to design and monitor remediation processes. Conventional (active) sampling usually relies on the collection of individual spot samples and may often lead to an underestimation of the abundance and diversity of the community as well as an important variability.
The objective of our work was to develop and optimize tools for reliable on-site passive sampling of microbial communities in non-destructive manner. Different matrices ranging from activated carbon to coarse sand were tested for enrichment of bacterial growth in monitoring wells. Samplers were maintained over 30 days and the microbial communities enriched on the different matrices compared to the communities of the surrounding soil and interstitial water using molecular tools (e.g., Next Generation Sequencing, RISA, Phylogenetic microarrays). Results obtained showed that the amount of biomass and structure of the communities are different on the different matrices tested. Some solid supports seem less adapted than others in order to sample in a reliable manner the microbial communities present in the surrounding water and soil. Therefore, the choice of the matrix selected to passively sample subsurface microbial communities is highly critical and some material are to be avoided. Data obtained with the different matrices in different environments will be presented and the advantages and limitations of such approach for different applications will be discussed.
Title: Microbial responses to biostimulation and bioaugmentation – a 2-year long pilot trial to evaluate molecular sampling techniques
Helena Branzén*, Lennart Larsson, Märta Ländell, Anja Enell
Swedish geotechnical institute, 581 93 Linköping
*speaker, helena.branzen@swedgeo.se +46-709-73 01 13
To evaluate different approaches to sample microbes, a two year-long field study was performed at a site contaminated by chlorinated ethenes. In the study, the outcome of using common groundwater samples was compared to the outcome from two different molecular sampling tools; a sampling tool with an artificial carrier for the collection of microbes over time and a sampling tool containing soil from the site. The pilot test was initiated in 2012, and performed in parallel to the assessment of reductive dechlorination as a potential remediation technique at a dry cleaning site in Alingsås, Sweden.
Background
For a bioremediation project to be successful, reliable methods to predict the degradation potential is crucial. It is important that sampling methods, as well as the molecular analysis carried out, reflect the true degradation potential of the subsurface system. Active microbial populations develop and thrive in environments offering nutrients and substrates necessary for respiration and cell growth. The tendency for microorganisms to attach to sediment particles is well-known, and bacterial density in communities attached to sediment may well be a factor 103 – 104 higher, compared to the density of free-living communities in groundwater. Of practical reasons, molecular analyses to a large extent focus on bacteria collected in the pumped groundwater, i.e. rendering a snapshot of the bacterial density. However, due to the organisms preferences for particles, the absence (or very low densities) of singled out specimens in groundwater may not be conclusive with corresponding absence in soil. To assess the potential for reductive dechlorination, or to subsequently monitor performance during remediation, techniques focusing on attached communities is supposed to offer a more reliable decision basis. Passive sampling tools that collects microbes over time has thus been developed. The Standard BioTrap® consists of a carrier material that mimics soil particles and stimulates colonization of active bacteria. With a larger bacterial density, the registration of background levels and evaluation of steps taken to enhance reductive dechlorination is considered to be more reliable. However, the results generated by this sampling tool does not reflect the soil ”history”, like competing microorganisms or predatory microorganisms (for example those feeding on the dechlorination bacteria). Neither chemical composition, nor structure of the carrier material is identical to that of the soil. Over the years, attempts have been made to use soil mesocosms to even better simulate natural conditions. However, for economical and practical reasons, in-situ mesocosms have not become a common practice in the field. Mesocosms may be described as small perforated containers filled with anaerobic handled soil from the contaminated site. The mesocosms are deployed in the groundwater wells from where they originally were collected and harvested at different stages of the trial.
In this study, we have evaluated the consequences of applying these different sampling techniques (i.e. a) conventional ground water sampling, b) Standard Biotrap® and c) in-situ mesocosms), in relation to the assessment of dechlorination potential, such as choosing remediation technique or assessing the need for additional biostimulation or bioaugmentation. The detection levels and sensitivity for variations in the amount of selected microorganisms and their gene-potential for dechlorination, triggered by biostimulation and and bioaugmentation have been compared. The hypothesis was that analysis of mesocosms, compared to analysis of groundwater and artificial Bio-Trap®, would give a more reliable result for presence of specific microorganisms and better register changes in the microbial composition after biostimulation and bioaugmentation, due to the larger amount of colonizing bacteria.
Aim
The aim of this presentation is to share experiences and conclusions from the pilot trial, performed during 2012-2014. More specific we wish to present /show apparent responses from the different sampling techniques/tools to biostimulation and bioaugmentation by evaluating detection sensitivity and sensitivity to variations in the microbe gene sequences over time.
Results
During the trial period, microbes were collected from three closely situated groundwater wells. All three wells were subjected to biostimulation (molasse and Newman Zone®), while two of the sampling wells were bioaugmented with KB-1® culture and smaller amount of lactate.
• Preliminary results from the trial show higher bacterial density (a factor 102 – 103) in soil mesocosms compared to both groundwater and Bio-Trap®.
• Bacteria dechlorinating PCE and TCE (Dehalobacter restrictus, and Desulfuromonas spp.) were continuously identified by the soil mesocosms, while groundwater and the artificial samplers did not always identify Desulfuromonas spp.
• After bioaugmentation, the groundwater (snapshot) and Bio-Trap® (passive sampler) showed quick responses, measured as Dehalococcoides spp. and gene copies involved in VC and ethene/ethane formation in comparison with the development in the mesocosms. The responses from the soil mesocosms were delayed, and while VC and ethane production declined, the number of gene copies, involved in the formation of VC and ethane, were increasing.
Integrated characterization of the development in natural attenuation of a PCE plume over 7 years after thermal remediation of the source zone with use of dual stable isotope and microbial techniques
Mette M. Broholm1, Alice Badin2, Carsten S. Jacobsen3, Philip Dennis4, Niels Just5, and Daniel Hunkeler2
1Technical University of Denmark, 2University of Neuchatel, 3GEUS, 4SiREM, 5Region of Southern Denmark.
PCE DNAPL contamination at the former central dry cleaning facility in Rødekro, Denmark, was subject to thermal (steam) source zone remediation in late 2006. A > 2 km long plume of chlorinated ethenes (PCE and chlorinated degradation products) which has migrated downgradient from the source zone has not undergone active remediation. A study of the natural degradation within the plume prior to source treatment including stable isotope monitoring was conducted in 2006(-2007) by Hunkeler et al. (2010). This investigation documented complete degradation of PCE via TCE to DCE by reductive dechlorination 1-1.5 km downstream the source area, where the plume descends into more reduced groundwater. It further proved that cDCE was further degraded by reductive dechlorination to VC, and that VC was not accumulated but further degraded, potentially by another pathway (not reductive dechlorination). Detection (< quantification limit) of specific degraders (Dehalococcoides) enforced that cDCE degradation was biotic reductive dechlorination. The understanding of the degradation within the plume, not least the documentation of VC degradation, was essential in the risk evaluation of the plume.
The scope of the new (2014) study is to evaluate how the source remediation has impacted the plume and in particular the natural attenuation within the plume.
The evolution in plume composition and attenuation has been monitored by the Region of Southern Denmark on an annual basis since the remediation, and in 2014 a large monitoring campaign including redox, chlorinated ethenes, non-chlorinated degradation products, carbon and chlorine stable isotope composition, specific degraders and their activity and next generation sequencing (454 pyrotag) for bacterial composition was conducted.
The source remediation has, in addition to direct reduction of the concentration level in and flux from the source area, resulted in the release of dissolved organic matter and some geochemical changes. This has had an effect on redox conditions and biodegradation by reductive dechlorination particularly in the near source area. However, also in the further downstream area of the plume redox and contaminant levels have changed, suggesting an evolution in natural attenuation at significant distance (>1 km downgradient) from the treated source area. Dual isotope analysis are currently being conducted. Dual isotope and microbial data will be processed for interpretation of the changes in redox and degradation processes within the plume.
The understanding of the degradation processes within chlorinated solvent plumes and the effects of source remediation on these is essential for the risk evaluation of the plumes, and it has significant influence on decisions regarding costly plume remediation efforts. This project is unique in the integrated characterization approach for line of evidence evaluation of the natural attenuation of cDCE and VC in the DCE dominated plume and the monitoring of the effects of source remediation on plume natural attenuation.
Reference:
Hunkeler, D., Abe, Y., Jeannottat, S., Westergaard, C., Jacobsen, C.S., Aravena, R., and Bjerg, P.L., (2011). Assessing chlorinated ethene degradation in a large scale contaminant plume by dual carbon-chlorine isotope analysis and quantitative PCR, J. Contam. Hydrol., 119, 69-79.
Polycyclic aromatic hydrocarbons (PAHs) are among the most abundant contaminants in the environment which mostly originate from anthropogenic sources like mineral oil spills or former gas plants. Due to their toxic, mutagenic, and carcinogenic effects to humans and animals, PAHs are pollutants of particular concern, thus the effort to reduce their environmental impact is of paramount importance. Biodegradation of PAHs has been demonstrated in laboratory studies under both oxic and anoxic conditions. However, the role of biodegradation for in situ reduction of PAHs at polluted field sites is only partially understood, in particular due to the limited number of approaches to evaluate the biodegradation of PAHs within contaminated aquifers.
In the present study, the biodegradation of four PAHs (naphthalene, fluorene, phenanthrene, and acenaphthene) was investigated in an oxic aquifer at the site of a former gas plant using a novel integrated approach comprising in situ and laboratory microcosms amended with 13C-labelled PAHs as tracer compounds. In situ microcosms with 13C-labelled substrates (BACTRAP®s) aim to enrich indigenous groundwater microorganisms on site and subsequent analysis of their community structure and carbon assimilation patterns (for review see [1]). BACTRAP®s were amended with either 13C-labelled naphthalene or fluorene and were incubated for a period of over two months in two groundwater wells located at the contaminant source and plume fringe, respectively. Subsequently, the assimilation of 13C-carbon derived from the 13C-labelled PAHs into amino acids extracted from BACTRAP®-grown cells was analysed. Amino acids showed significant 13C-enrichments with 13C-fractions of up to 30.4% for naphthalene and 3.8% for fluorene, thus providing clear evidence for the in situ biodegradation and assimilation of those PAHs at the field site. In contrast to several laboratory microcosm studies, showing potential inhibitory effects of high PAH concentrations or complex contaminant mixtures on PAH biodegradation, microbial degradation of naphthalene and fluorene was observed in situ in both the contaminant source and the plume fringe. Recently, we could identify members of the orders Burkholderiales, Actinomycetales and Rhizobiales as the most active microorganisms in the naphthalene degrading microbial community by analysing 13C-labelled proteins extracted from the BACTRAP®s [2].
In order to provide quantitative information on the PAH biodegradation, a laboratory microcosm study was additionally conducted. Groundwater and BACTRAP®-grown cells were used as inoculum. All laboratory microcosms were incubated under in situ-like conditions, using 13C-labelled naphthalene, fluorene, phenanthrene, and acenaphthene as tracers. Mineralisation of 13C-labelled PAHs was detected with high sensitivity and quantified by analysing the formation of 13C-CO2 [3, 4]. Observed PAH mineralisation rates ranged between 17 mg L-1 d-1 and 1639 mg L-1 d-1. On the basis of our results, we consider Monitored Natural Attenuation (MNA) as a potential management strategy for this field site.
References:
1. Bombach, P., Richnow, H.H., Kästner, M., Fischer, A., 2010. Current approaches for the assessment of in situ biodegradation. Appl. Microbiol. Biot. 86 (3), 839-852.
2. Herbst, F.A., Bahr, A., Duarte, M., Pieper, D.H., Richnow, H.H., von Bergen, M., Seifert, J., Bombach, P., 2013. Elucidation of in situ polycyclic aromatic hydrocarbon degradation by functional metaproteomics (protein-SIP). Proteomics 13 (18-19), 2910-2920.
3. Morasch, B., Höhener, P., Hunkeler, D., 2007. Evidence for in situ degradation of mono-and polyaromatic hydrocarbons in alluvial sediments based on microcosm experiments with C-13-labeled contaminants. Environ. Pollut. 148 (3), 739-748.
4. Nijenhuis, I., Stelzer, N., Kästner, M., Richnow, H.H., 2007. Sensitive detection of anaerobic monochlorobenzene degradation using stable isotope tracers. Environ. Sci. Technol. 41 (11), 3836-3842.