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Trichloroethene (TCE) has extensively been used as a solvent in metal processing and dry cleaning industries. Improper storage, however, has resulted in widespread groundwater contaminations. In the subsoil, its low solubility (8.4 mM) and high density cause the formation of dense non-aqueous phase liquid (DNAPL). Such a DNAPL only dissolves slowly and, therefore, forms a source of long-term groundwater contamination. Remediation of TCE groundwater contaminations can rely on the biodegradation of TCE, since several anaerobic bacteria reductively dechlorinate TCE to cis-dichloroethene (cis-DCE). Reductive dechlorination in the vicinity of a DNAPL can even enhance the DNAPL dissolution rate and, as such, reduce the time required for remediation. Bio-enhanced DNAPL dissolution is defined as the biological enhancement of DNAPL dissolution in comparison to abiotic dissolution. The different factors controlling bio-enhanced DNAPL dissolution, however, are not yet well understood. The general objective of this work was to assess the biological and chemical factors that control bio-enhanced TCE DNAPL dissolution. The general hypothesis is that the characteristics of the dechlorinating bacteria determine the DNAPL dissolution enhancement, unless chemical factors, such as electron donor limitations and acidification, limit the dechlorination rate.In a first part, TCE self-inhibition, i.e. the inhibition of the dechlorination reaction due to the toxicity of elevated TCE concentrations, was examined. An experiment with liquid batch systems combined four different initial TCE concentrations (1.4 - 3.0 mM) and three different inoculation densities (4.0×105 - 4.0×107 Geobacter cells∙mL-1). A KB-1TM subculture with Geobacter as dechlorinator was used as inoculum. Dechlorination was observed in all treatments with an initial TCE concentration of 2.5 mM and lower. The time required for complete conversion of TCE to cis-DCE increased with an increasing initial TCE concentration and a decreasing inoculation density. At an initial TCE concentration of 3.0 mM, dechlorination only proceeded at the highest inoculation density. A Monod model, incorporating TCE self-inhibition, successfully fitted the observed dechlorination. This model explained that high Geobacter cell densities can overcome the TCE self-inhibition above 2.6 mM TCE only in batch conditions.In a second part, a 1-D diffusion-cell was developed to enable the quantification of bio-enhanced DNAPL dissolution and the measurement of the conditions near the DNAPL-water interface. The 12 cm long diffusion-cell setup consists of a 5.5 cm central sand layer, a lower 3.5 cm DNAPL layer and a top 3 cm aqueous layer. The DNAPL layer maintains the saturated TCE concentration at the lower boundary of the sand layer, while the top layer is frequently refreshed to remove the chloroethenes and to provide electron donor at the upper boundary. Abiotic and biotic diffusion-cells were tested, the latter with a homogeneous inoculation of the sand layer. In the abiotic diffusion-cells, a linearsteady-state TCE concentration profile between the DNAPL and the top layer developed. In the biotic diffusion-cells, TCE was completely converted to cis-DCE at 2.5 cm distance from the DNAPL. Dechlorination was likely inhibited till a distance of 1.5 cm from the DNAPL, as at this distance the TCE concentration exceeded 2.6 mM. The DNAPL dissolution flux was calculated from the measured concentration gradients and was in the biotic diffusion-cells a factor 2.4 ± 0.2 larger than in the abiotic diffusion-cells.In a third part, the distribution of the dechlorinating community in diffusion-cells was examined. A 16S rRNA gene clone library analysis showed that the used KB-1TM subculture consisted of dechlorinating bacteria similar to Geobacter lovleyi SZ and fermentative micro-organisms related to Clostridium. qPCR and RFLP analysis showed a stratified microbial community composition in the diffusion-cells. Geobacter dominated where TCE was dechlorinated, i.e. in the lower 2.5 cm of the sand layer. Even at 0.5 cm distance from the DNAPL layer, where toxic TCE concentrations were expected, Geobacter cell densities were two orders of magnitude higher than at inoculation. In the upper 2.5 cm of the sand layer, where TCE was depleted, apparently fermenting populations prevailed, which corresponded to Clostridium in some diffusion-cells.A fourth part examined if microbial migrationtowards a TCE DNAPL could initiate bio-enhanced DNAPL dissolution. A first diffusion-cell experiment, which used top layer inoculation, showed that Geobacter migrated slower in the presence than in absence of TCE. Random motility coefficients were fitted on the observed migration. A second diffusion-cell experiment measured dechlorination after the inoculation of either the sand or the top layer. In the latter treatment, migrating Geobacter cells started to dechlorinate in the sand layer, thereby enhancing the DNAPL dissolution flux. The DNAPL dissolution enhancement was after 19 days, surprisingly, only 1.3 times lower than with sand layer inoculation. Different inoculation densities did not affect the dechlorination rate. A diffusion-reaction model, incorporating random motility, well described the observed dechlorination. This model suggested that the combined effect of random motility and growth on TCE explained the fast colonization of the sand layer by Geobacter.In a fifth part, the relation between the DNAPL dissolution enhancement and the electron donor supply rate was examined. Top layers of diffusion-cells were amended with different concentrations of formate (0 – 16 mM) as electron donor. The TCE DNAPL dissolution rate increased from no enhancement compared to abiotic dissolution without formate, to a 2.4 times enhancement with 16 mM formate. With 2, 4 and 8 mM formate, the TCE diffusion flux out of the DNAPL layer equaled the formate diffusion flux out of the top layer, illustrating their stoichiometric interdependence under electron donor limiting conditions. In contrast, with 16 mM formate, the TCE diffusion flux was lower than the formate diffusion flux, demonstrating that the dechlorination kinetics likely limited the DNAPL dissolution enhancement. The DNAPL dissolution rate under electron donor limiting conditions could readily be predicted from the sand layer length and the electron donor concentration in the top layer.In a final part, the effect of acidification on bio-enhanced TCE DNAPL dissolution was examined. In batch systems, dechlorination was optimal at pH 7.1 – 7.5, but was completely inhibited below pH 6.2. In addition, a diffusion-cell experiment was performed with three different pH buffer concentrations (0.0 mM – 30.0 mM MOPS) and with lactate or formate as electron donor. Measurement of the pore water pH showed that acidification was less with an increased pH buffer concentration or with formate instead of lactate as electron donor. In the lactate fed diffusion-cells, the DNAPL dissolution enhancement factor increased from 1.5 to 2.2 with an increasing pH buffer concentration. In contrast, in the formate fed diffusion-cells, the dissolution enhancement factor (2.4 ± 0.3) was unaffected by the pH buffer concentration. These results suggest that the significant impact of acidification on bio-enhanced DNAPL dissolution could be overcome by the amendment of a pH buffer or by using formate as electron donor.In conclusion, this work demonstrated the feasibility of bio-enhanced TCE DNAPL dissolution, even though TCE toxicity inhibited dechlorination in the vicinity of the TCE DNAPL. High Geobacter cell densities were present adjacent to the TCE DNAPL, but these cells were probably inactive and likely resulted from random motility towards all parts of the sand layer. In addition, it was shown that the migration of the dechlorinators initiated bio-enhanced TCE DNAPL dissolution. The inoculation density did not affect bio-enhanced DNAPL dissolution, although this could determine the time before the onset of the dechlorination. Moreover, it was confirmed that electron donor limitations and acidification significantly reduce the DNAPL dissolution enhancement in comparison to that expected from the dechlorination kinetics. The motility mechanisms and the potential adaptation of the dechlorinators to elevated TCE concentrations warrant future research. In addition, the factors affecting bio-enhanced DNAPL dissolution should be examined under more field-like conditions to validate the current concepts.

Eau souterraine --- groundwater --- Bioremédiation --- Bioremediation --- Dissolution --- Dissolving --- Academic collection --- Theses --- Bioremediation. --- Dissolving. --- Déchlorination --- Geobacter