Contamination of perchlorate and chlorate in nature is primarily the result of various industrial processes. The microbial respiration of these oxyanions of chlorine plays a major role in reducing the society’s impact on the environment. The focus with this thesis is to investigate the oxygen-dependent regulation of key components involved in the chlorate respiration in the gram‑negative bacterium Ideonella dechloratans. Chlorate metabolism is based on the action of the enzymes chlorate reductase and chlorite dismutase and results in the end products molecular oxygen and chloride ion. Up‑regulation of chlorite dismutase activity in the absence of oxygen is demonstrated to occur at the transcriptional level, with the participation of the transcriptional fumarate and nitrate reduction regulator (FNR). Also, the chlorate reductase enzyme was shown to be regulated at the transcriptional level with the possible involvement of additional regulating mechanisms as well. Interestingly, the corresponding chlorate reductase operon was found to be part of a polycistronic mRNA which also comprises the gene for a cytochrome c and a putative transcriptional regulator protein.

Chlorate and perchlorate pollutions have become a problem in the environment in the last decades. Studies have shown that some bacteria can degrade these substances into unharmful substances such as chloride and molecular oxygen. One of these chlorate degrading bacteria is Ideonella dechloratans that uses chlorate reductase and chlorite dismutase to process chlorate. In the promoter gene sequence of chlorite dismutase there might be regulator sequences such as fumarate and nitrate reductase regulator (FNR) and aerobic respiration control protein (ArcA) that might control the transcription of this enzyme. This promoter sequence was placed in a pBBR1MCS-4-LacZ reporter vector and the possible regulatory sequences were changed through site-directed mutagenesis and tested on activity through beta-galactosidase assays. The changes in the FNR binding sequence gave beta-galactosidase activity that was close to a negative control which might give conclusions that either FNR has an important role or an important part of the promoter was hit. The changes in the ArcA regulator binding sequence did not give such big differences and no certainty can be given if this made important changes to the promoter.

Perchlorate is a known inhibitor of the human thyroid gland. Perchlorate is destroyed by ubiquitous perchlorate-reducing bacteria. The bacteria often lack sufficient electron donor. Research was undertaken to evaluate the relationship between plants and perchlorate-reducing bacteria. To what degree can plant-produced electron donors stimulate perchlorate reduction in low organic carbon (LOC) and high organic carbon (HOC) soil? A complication is that plants have been shown to influence redox conditions which may inhibit perchlorate reduction. The removal of perchlorate in a flow-through reactor was monitored with variables of soil organic carbon, hybrid poplar trees, and bioaugmentation. The biodiversity was monitored using denaturing gradient gel electrophoresis. Low oxidation-reduction potential (ORP) was shown to indicate the capacity for greater perchlorate removal in soil. However, in planted LOC soil systems, evidence suggests that perchlorate reduction may also be possible at higher bulk redox conditions than previously observed. Increased hydraulic retention time was shown to both lower bulk ORP and increase perchlorate removal. Radiolabeled perchlorate was used to find that in planted systems as much as 11.7% of the influent perchlorate mass was taken up into the tree and 82% of the perchlorate taken up was accumulated in the leaves. The plant contribution to total perchlorate removal in nonbioaugmented LOC soil was 39%, with the balance of the removal being attributed to microbial reduction. In bioaugmented soil the microbial contribution to perchlorate removal was increased. Just planting poplar trees decreased the diversity of perchlorate reducers in the soil. However, when LOC soil was both planted and bioaugmented, the diversity of perchlorate reducers was not decreased. In HOC soil, the presence of an indigenous population of microorganisms competed with perchlorate reducers. At the increased ORP observed in planted HOC soil, the non-perchlorate-reducing bacteria appear to outcompete the perchlorate reducers and perchlorate removal is decreased. Engineering implications of this research are that perchlorate remediation in HOC soil does not benefit from planting hybrid poplar trees but that remediation in LOC soil is stimulated by planting and bioaugmentation.

Microbial chlorate respiration plays an important role in the turnover of oxochlorates in nature and industrial waste management. This thesis deals with the characterization of the molecular components of chlorate respiration in Ideonella dechloratans. Chlorate respiration utilizes two soluble periplasmic enzymes, chlorate reductase and chlorite dismutase, to convert chlorate to chloride and oxygen. The genes encoding the enzymes participating in the chlorate degradation have been sequenced, and are found in close proximity, forming a gene cluster for chlorate metabolism. This work also includes the successful recombinant expression of three genes from Ideonella dechloratans. Two of the gene products, chlorite dismutase and the C subunit of chlorate reductase, participate in the chlorate respiration. The third gene, which is found close to the gene cluster for chlorate metabolism, encodes a soluble c-type cytochrome. The localization of the gene suggests the corresponding protein as a candidate for a role as electron donor to chlorate reductase. Also, the role of soluble periplasmic c cytochromes of Ideonella dechloratans in chlorate respiration was studied. At least one of the soluble c cytochromes was found capable of serving as electron donor for chlorate reduction. This c cytochrome, and several others, can also donate electrons to a terminal oxidase for subsequent reduction of oxygen, as required for the branched electron flow during chlorate respiration.