Molecular investigation of chloroethene reductive dehalogenation by the mixed microbial community KB1

Bioaugmentation with Dehalococcoides-containing cultures is a successful technology for the remediation of chlorinated ethene-contaminated groundwater. 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.