Microbial monitoring and degradation of lindane in soil

Lindane (γ-hexachlorocyclohexane, γ-HCH), once the most applied insecticide worldwide, is still considered a serious threat to the environment, due to its persistence in soil and water and to its potential to bioaccumulate in the food chain. Other ubiquitous HCH-isomers, formed as waste products during the production of lindane, also pose a major environmental threat. Because of the widespread pollution by lindane (and other HCH-isomers), there is a need for risk assessment tools to monitor its environmental impact. Monitoring of polluted sites is as yet often restricted to the quantification of the contaminant by means of chemical analyses. In soil the use of microorganisms as indicator tools in monitoring procedures offers many opportunities, because of their ability to respond and rapidly adapt to changes in environmental conditions. As yet, removal of lindane from the environmental compartments has attained the highest priority. A possible approach is bioremediation, the process by which living organisms degrade the contaminant. A technology often used in bioremediation is bioaugmentation, in which specialized microorganisms are introduced into the environment in order to metabolize the targeted compound. Despite its long-term use in bioremediation, bioaugmentation of contaminated sites with microbial cells continues to be a source of controversy within environmental microbiology. This largely results from its notoriously unreliable performance record. In a first part of this research, the applicability of methane oxidation, an important soil function performed by methanotrophic bacteria, was evaluated as a microbial indicator for the monitoring of HCH pollution. The effect of HCH on both the activity and the structure of the methanotrophic community was assessed, using methane oxidation assays and PCR-DGGE (polymerase chain reaction – denaturing gradient gel electrophoresis) respectively. Methane oxidation assays with historically polluted soils revealed that in the long-term methane oxidation was inhibited by HCH pollution. PCR-DGGE and diversity analysis based on Lorenz curves showed that the type I methanotrophic community was less evenly distributed in historically HCH polluted soils compared to less polluted reference soils. Short-term experiments with methane enriched consortia further demonstrated that only γ and δ isomers inhibited methane oxidation. Type I methanotrophs of methane enriched microbial consortia that received γ- or δ-HCH evolved towards higher species richness. Apparently, for historically HCH polluted soils, a narrow community remained after long-term exposure, while in case of short-term exposures, methane enriched consortia were converted into less active, but richer communities when they were stressed by the presence of γ- or δ-HCH. These results demonstrated that methane oxidation activity and structural analysis of type I methanotrophic communities, can be valuable tools in risk assessment studies and the monitoring of HCH pollution. The second part of this doctoral research aimed to develop a promising bioaugmentation technology for the aerobic biodegradation of lindane by surveying microbiological inoculants and by designing a suitable inoculation procedure for their introduction into the environment. The occurrence of HCH degraders was investigated in several contaminated soils. Thirty five enrichment cultures grown on different HCH-isomers yielded 16 cultures in which HCH was removed. HCH-degrading populations were clearly associated with HCH-contaminated soils. Nine HCH-degrading isolates could subsequently be isolated, which were all Sphingomonas spp. All isolates degraded α- and γ-HCH, while degradation of β- and δ-HCH was highly strain dependent. However, none of the isolates grew on HCH as a sole organic substrate in pure culture. DGGE analyses evaluated whether enrichment of HCH degraders occurred in situ at contaminated sites. Most soils were characterized by diverse bacterial communities, although one soil contained a predominant ribotype, possibly selected by the HCH contamination. However, the latter population was not selected in the enrichment culture from that soil. Furthermore, DGGE analyses indicated that the isolates represented predominant populations in the enrichment cultures, but additional predominant populations, including some Pseudomonas spp. , could not be isolated. Isolated strains were subsequently used to assess the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane. Slow-release inoculation of Sphingomonas sp. γ1-7 was established in both liquid and soil slurry microcosms, using open ended silicone tubes in which the bacteria are encapsulated in a protective nutrient rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free living cells. Encapsulation of cells in silicone tubes caused removal of lindane by adsorption to the silicone tubes, but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer, for liquid and soil slurry microcosms respectively, compared to inoculation with free cells. While inoculation of free living cells led to a loss in lindane degrading activity in limited time intervals, encapsulation in these tubes allowed for a more stable actively degrading population. The loss in degrading activity was linked to the loss of the linA gene, encoding γ-HCH dehydrochlorinase (LinA) which is involved in the initial steps of the lindane degradation pathway. Further, the sustained degradation of lindane by Sphingomonas sp. γ1-7 in open ended tubes was evaluated over an extended period of time. It was shown that the slow-release approach by inoculation of encapsulated cells in open ended tubes provided stable lindane degradation for over 4 months, whereas freely inoculated cells could not maintain degradation for longer than one month. Several factors that could possibly regulate this sustained lindane degradation by encapsulated Sphingomonas spp. were systematically assessed in liquid medium. The obtained results indicated that in a relatively nutrient rich medium, nutrient conditions inside the tubes did not influence the rate of lindane degradation. Also, initial cell density inside the tubes only exerted a temporary positive effect on lindane degradation. Quorum sensing regulated gene expression was also assessed, but most likely did not occur. Physical protection from high lindane concentrations or potentially toxic metabolites is likely the prevailing advantage provided by the encapsulation in tubes, although preliminary results also indicate the potential role of increased plasmid stability. In a third and final experimental part of this work, an alternative lindane removal technique was proposed, making use of the catalytic reduction of HCH over a metal catalyst, namely palladium (Pd[0]). Since specific surface area plays an important role in reactivity of catalysts, this study investigated the use of bioPd(0), i.e. nano-scale Pd(0) particles precipitated on the biomass of Shewanella oneidensis, for the removal of lindane. It was demonstrated that bioPd(0) has catalytic activity towards dechlorination of lindane, with the addition of formate as electron donor, and that dechlorination with bioPd(0) was more efficient than with commercial powdered Pd(0). The biodegradable compound benzene was formed as a reaction product and other HCH-isomers could also be dechlorinated. Subsequently, bioPd(0) was implemented in a membrane reactor technology for the treatment of lindane polluted water. In a fed-batch process configuration with formate as electron donor, a removal percentage of 98% of γ-HCH saturated water (10 mg l-1) was achieved within 24 hours. The measured chloride mass balance approached the theoretical value. These results demonstrate, a complete, efficient and fast removal of lindane using catalytic reduction with bioPd(0). In conclusion, this work has shown that in risk assessment and ecotoxicological studies of lindane, the soil methanotrophs represent a sensitive bacterial group, with great potential to be used as indicators for the monitoring of HCH soil pollution. Further research is recommended for the development and validation of methane oxidation bioassays, based on the findings of this research. Slow-release bioaugmentation with encapsulated lindane degrading Sphingomonas spp. in open ended tubes was demonstrated to be a promising tool for soil bioremediation. Although several insights into the mechanisms of the ‘hot spot’ tubes were provided by the present research, more work needs to be done to unravel their true identity. Future research should certainly focus on the further upscaling of this technique for (in situ) soil applications. Finally, the application of an alternative lindane degradation technique - biocatalytic reduction with bioPd(0) - resulted in a complete, efficient and fast removal of lindane, and was demonstrated to be applicable as a membrane based reactor technology.