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Bioaugmentation with Dehalococcoides-containing cultures is a successful technology for the remediation of chlorinated ethene-contaminated roundwater. The overall goal of this research was to identify and characterize genes that are used by a Dehalococcoides-containing culture, KB-1, during degradation of Trichloroethene (TCE) to ethene, via cis-Dichloroethene (cDCE) and vinyl chloride. Firstly, the diversity and dynamics of the microbial populations within KB1 was assessed using 16S rRNA clone libraries and quantitative PCR analyses. Secondly, reductive-dehalogenase-homologous- (RDH) genes in KB1 were identified, sequenced and their transcription compared through RNA-generated RDH cDNA clone libraries. Finally, to elucidate functionally important genes within the community, Shotgun metagenome microarrays were constructed and used to investigate transcription during dechlorination. Results of the phylogenetic analyses indicated that KB1 is a diverse community of microorganisms whose stability is enhanced by functional redundancy within the culture. To fully understand this diverse community of uncultivated microorganisms a metatranscriptome approach was used. Experiments with shotgun metagenome microarrays identified spots which were statistically significantly differentially expressed during dechlorination. These spots were then sequenced, revealing Dehalococcoides and non-Dehalococcoides-genes which are important during dechlorination. These results demonstrated that shotgun microarrays can be constructed without prior sequence knowledge and used to effectively examine differential transcription within an uncultivated community. Subsequently, all of the spots of the array were sequenced, and additional array experiments were conducted. Sequencing identified 24 reductive dehalogenase genes in the culture, and analysis of the microarray results indicated that many of these RDH genes were differentially expressed in response to certain chlorinated compounds. Interspecies interactions were also highlighted as results suggested that non- Dehalococcoides microorganisms provide partial corrinoids which Dehalococcoides salvages to synthesize cobalamin which is essential for reductive dehalogenation. Transcription of CRISPR-associated genes also indicated interaction between phage and other microorganism in the KB1 community. Overall these results provided sequence and transcription information about possible biomarkers for reductive dechlorination by KB1 and can be used for more effective design and monitoring of bioremediation technologies.

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Hydrocarbons in soil may assert acute or chronic impacts to plants, animals and microbial processes if contacted. These have raised political and scientific concerns. Consequential research efforts corroborated that constitutive microorganisms contact the compounds for their metabolic activities. This may result in mineralisation, transformation and/or detoxification (biodegradation) of the compounds. Hydrocarbon biodegradation is relatively cost-effective and ecological, but often marred with limited availability to plant or animal cells (bioavailability) for metabolism. Several authors reported that growth of some plants or administration of requisite rootexudates into soil with hydrocarbons often increases hydrocarbon bioavailability for enhanced biodegradation. However, development of knowledge about this respite from plants is often founded on impacts of plants on single dose or selected mixture of hydrocarbons in soils or culture solutions. These do not; and cannot represent the heterogeneous complex mixture of numerous organic and inorganic compounds in soils where plants grow naturally. In this study, synthetic root-exudates, seedlings of lupin and ryegrass were applied separately into respective soils that were contaminated with aged and/or fresh petroleum hydrocarbons. Individual impacts of the treatments on bulk hydrocarbon concentrations, rate of microbial respiration and total numbers of culturable bacterial colonies in the soils were investigated. Results suggested that application of lupin, ryegrass or synthetic root-exudates into the soils significantly (p ≤ 0.05) induced reduction or upsurge of hydrocarbon biodegradation end-points relative to the type and concentration of hydrocarbons in soil. Thus, it is inferred that growth of plants or administration of root-exudates into hydrocarbon contaminated soils could result in enhanced biodegradation of hydrocarbons in soil.

New economically viable methods for the detection and removal of organic pollutants from water samples are needed to meet modern safety requirements. Development of techniques to produce antibody fragments in Escherichia coli (E. coli) have provided the opportunity to exploit antibodies as specialised chemicals for affinity detection and removal technologies. The variable heavy and light antibody domains of the anti-paraquat monoclonal antibody PQXB1/2 have been cloned into the single-chain antibody (scAb) expression vector, pBG1. Anti-paraquat scAb (0.1-0.4 mg/L bacterial culture) was expressed in E. coli. Characterisation of scAb by antigen binding profile and competition ELISA showed it to have a sensitivity one order of magnitude below that of the parent monoclonal. ScAb was purified as a monomer or dimer and analysed by HPLC size exclusion chromatography. When immobilised on polystyrene beads the scAb could remove 85% of paraquat-bovine serum albumin conjugate from solution in a single step.