Past operational practices at chemical manufacturing facilities and widespread use of synthetic chemicals in agriculture, industry, and military operations have introduced many anthropogenic compounds to the biosphere. Some of them are readily biodegradable as a likely consequence of bacterial evolution of efficient degradation pathways, whereas others are partially degraded or persistent in the environment. Insight about biodegradation mechanisms and distribution of bacteria responsible provide the basis to predict the fate of synthetic chemicals in the environment and to enable bioremediation. The main focus of the research described here encompasses basic science to discover pathways and evolutionary implications of aerobic biodegradation of two specific synthetic chemicals, cis-dichloroethene (cDCE) and diphenylamine (DPA). cDCE is a suspected carcinogen that frequently accumulates due to transformation of perchloroethene and trichloroethene at many contaminated sites. Polaromonas sp. strain JS666 is the only isolate able to use cDCE as the growth substrate, but the degradation mechanism was unknown. In this study, the degradation pathway of cDCE by strain JS666 and the genes involved were determined by using heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. The requirement of oxygen for cDCE degradation and inhibition of cDCE degradation by cytochrome P450 specific inhibitors suggested that cytochrome P450 monooxygenase catalyzes the initial steps of cDCE degradation. The finding was supported by the observation that an E. coli recombinant expressing cytochrome P450 monooxygenase catalyzes the transformation of cDCE to dichloroacetaldehyde and small amounts of the epoxide. Both the transient accumulation of dichloroacetaldehyde in cDCE degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 further support a pathway involving the degradation of cDCE through dichloroacetaldehyde. Molecular phylogeny of the cytochrome P450 gene and organization of neighboring genes suggest that the cDCE degradation pathway evolved in a progenitor capable of degrading dichloroacetaldehyde by the recruitment of the cytochrome P450 monooxygenase gene from alkane assimilating bacteria. The discovery provides insight about the evolution of the aerobic cDCE biodegradation pathway and sets the stage for field applications. DPA has been widely used as a precursor of dyes, pesticides, pharmaceuticals, and photographic chemicals and as a stabilizer for explosives, but little was known about the biodegradation of the compound. Therefore, bacteria able to use DPA as the growth substrate were isolated by selective enrichment from DPA-contaminated sediment and the degradation pathway and the genes that encode the enzymes were elucidated. Transposon mutagenesis, the sequence similarity of putative open reading frames to those of well characterized dioxygenases, and 18O2 experiments support the conclusion that the initial reaction in DPA degradation is catalyzed by a multi-component ring-hydroxylating dioxygenase. Aniline and catechol produced from the initial reaction of DPA degradation are then completely degraded via the common aniline degradation pathway. Molecular phylogeny and organization of the genes involved were investigated to provide insight about the evolution of DPA biodegradation. The fate and transport of toxic chemicals are of a great concern at several historically contaminated sites where anoxic contaminant plumes emerge into water bodies. The release of toxic chemicals to overlying water poses a potential source of environmental exposure. Bench scale studies were conducted to evaluate the impact of biodegradation on the transport of toxic chemicals across the sediment/water interface. These studies demonstrated that substantial populations of bacteria associated with organic detritus at the interface rapidly biodegrade toxic chemicals as they migrate from contaminated sediment to overlying water, suggesting that the natural attenuation processes serve as a remedial strategy for contaminated sediments and protect the overlying water.

<p>Soil bottles and soil slurry experiments were conducted to investigate the effect of some additives on the aerobic and anaerobic biodegradation of chlorinated aliphatic hydrocarbons; tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) in a contaminated soil from Startvätten AB Linköping Sweden. For the aerobic degradation study the soil sample was divided into two groups, one was fertilised. The two groups of soil in the experimental bottles were treated to varying amount of methane in pairs. DCE and TCE were added to all samples while PCE was found in the contaminated soil. Both aerobic and anaerobic experiments were conducted. For aerobic study air was added to all bottles to serve as electron acceptor (oxygen). It was observed that all the samples showed a very small amount of methane consumption while the fertilised soil samples showed more oxygen consumption. For the chlorinated compounds the expected degradation could not be ascertained since the control and experimental set up were more or less the same.</p><p>For the anaerobic biodegradation study soil slurry was made with different media i.e. basic mineral medium (BM), BM and an organic compound (lactate), water and sulphide, phosphate buffer and sulphide and phosphate buffer, sulphide and ammonia. To assure anaerobic conditions, the headspace in the experimental bottles was changed to N2/CO2. As for the aerobic study all the samples were added DCE and TCE while PCE was found in the contaminated soil. The sample without the soil i.e. the control was also given PCE. It was observed that there was no clear decrease in the GC peak area of the pollutants in the different media. The decrease in GC peak area of the pollutants could not be seen, this may be so because more susceptible microorganisms are required, stringent addition of nutrients and to lower the risk of the high concentration of PCE and petroleum products in the soil from Startvätten AB.</p>

Soil bottles and soil slurry experiments were conducted to investigate the effect of some additives on the aerobic and anaerobic biodegradation of chlorinated aliphatic hydrocarbons; tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) in a contaminated soil from Startvätten AB Linköping Sweden. For the aerobic degradation study the soil sample was divided into two groups, one was fertilised. The two groups of soil in the experimental bottles were treated to varying amount of methane in pairs. DCE and TCE were added to all samples while PCE was found in the contaminated soil. Both aerobic and anaerobic experiments were conducted. For aerobic study air was added to all bottles to serve as electron acceptor (oxygen). It was observed that all the samples showed a very small amount of methane consumption while the fertilised soil samples showed more oxygen consumption. For the chlorinated compounds the expected degradation could not be ascertained since the control and experimental set up were more or less the same. For the anaerobic biodegradation study soil slurry was made with different media i.e. basic mineral medium (BM), BM and an organic compound (lactate), water and sulphide, phosphate buffer and sulphide and phosphate buffer, sulphide and ammonia. To assure anaerobic conditions, the headspace in the experimental bottles was changed to N2/CO2. As for the aerobic study all the samples were added DCE and TCE while PCE was found in the contaminated soil. The sample without the soil i.e. the control was also given PCE. It was observed that there was no clear decrease in the GC peak area of the pollutants in the different media. The decrease in GC peak area of the pollutants could not be seen, this may be so because more susceptible microorganisms are required, stringent addition of nutrients and to lower the risk of the high concentration of PCE and petroleum products in the soil from Startvätten AB.

No description available.

Microtransplantation of mammalian neurolemma into Xenopus laevis oocytes has been used to study ion channels in terms of their structure and function in the central nervous system. Use of microtransplanted neurolemma is advantageous in that tissue can be obtained from various sources, ion channels and receptors are present in their native configuration and they can be used to evaluate numerous channelpathies caused by environmental toxicants. Here we show that Xenopus oocytes injected with fragments of rat brain neurolemma successfully express functional native ion channels that are assembled in their own plasma membrane. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin (TTX), omega-conotoxin MVIIC, and tetraethylammonium (TEA) were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium, calcium and potassium channels, respectively). In this current research, a “proof-of-principle” experiment was conducted where TTX-sensitive voltage-sensitive sodium channel (VSSC) currents were measured. VSSCs are a well-established site of action for 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) but not for its non-toxic metabolite 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE). A differential sensitivity of DDT versus DDE on TTX-sensitive sodium current in neurolemma-injected oocytes was determined. DDT elicited an increase in depolarization-dependent, TTX-sensitive sodium current while DDE had no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE has no similar effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs by injecting cRNA into Xenopus oocytes. By demonstrating the classic structural activity relationship of DDT and DDE on mammalian voltage-gated sodium channels isolated in rat brain neurolemma, this study supports the use of automated high-throughput electrophysiology to study the effects of various environmental toxicants on multiple mammalian cellular targets. More importantly, using rat brain neurolemma ensures that the proteins of interest have been transcribed and have undergone all the necessary post-translational modifications before they were injected and expressed in the Xenopus oocytes which is not the case for traditional heterologous expression.