Tetrachloroethene (PCE) and trichloroethene (TCE) can be effectively biodegraded in anaerobic environments by reductive dechlorination processes, and recent progress has been made towards exploiting these processes for bioremediation applications. There remains a need, however, for appropriate and cost-effective biomarkers to assess, monitor, and optimize performance. Commonly, biomarker development has focused on identifying nucleic acid sequences, peptides, proteins, or lipids of organisms that catalyze biodegradation reactions of interest. Although promising, such approaches are limited in reductive dechlorination processes as they do not address the roles of other organisms required to support and/or enhance the activity of the dechlorinating organisms. Novel biomarkers that quantify the presence, abundance, and activity of supporting organisms are therefore needed to more effectively assess and optimize dechlorination potentials.
The objective of this project was to identify 16S-rRNA-based phylogenetic and mRNA-based functional biomarkers diagnostic of microbial communities that support the robust growth and activity of chlorinated ethene-degrading organisms. In particular, researchers focused on biomarkers indicative of organisms that support the growth and activity of Dehalococcoides (Dhc) species.
In this project, researchers applied state-of-the-art microarray-based tools to identify relevant biomarkers. Dechlorination activities, as well as community structures were characterized in dechlorinating microbial communities, including laboratory-scale enrichment cultures, semi-batch microcosm, continuous-flow chemostat, and environmental samples from contaminated field sites.
Firmicutes (mostly Clostridium spp.), Bacteroidetes (mostly Bacteroides spp.), as well as Proteobacteria (mostly sulfate-reducer, i.e. Desulfovibrio spp.) were the most commonly found supportive microorganisms in the characterized communities. Since most of them are capable of fermenting organic compounds such as lactate and whey to acetate and hydrogen, these supportive microorganisms might play a role in providing hydrogen to Dhc. Moreover, since Clostridium spp. and Desulfovibrio spp. were reported to have an up-stream corrinoid biosynthesis pathway, these species might also play an important role in providing corrinoids to Dhc.
Defined consortia were further constructed by growing pure Dhc species (strain 195) with some of the detected supportive microorganisms. Results indicated that Desulfovibriovulgaris Hildenborough exhibited the most successful syntrophic growth with strain 195. Whole-genome expression microarray results exhibited that in the co-culture of strain 195 and D. vulgaris, hydrogenase-encoding genes, cobalamin-associated genes as well as some genes involved in amino-acid biosynthesis were down-regulated compared to the pure isolate, which indicated a supportive relationship in the aspects of hydrogen transfer, corrinoid and amino-acid availabilities. Furthermore, a genus-wide microarray targeting four known Dehalococcoides genomes was designed, validated and applied to pure cultures and communities. The application of mRNA-based whole-genome and genus-wide microarrays combined with qPCR tools indicated that the 16S rRNA gene of Dhc had a good correlation with the sum of well-known functional RDase genes (tceA, bvcA and vcrA). However, it is the expression of functional RDase genes rather than the 16S rRNA gene that determines the dechlorination activity in a community, and the functional RDase genes were not restricted to a specific genome.
The method of fluorescence-activated cell sorting (FACS) in combination with whole genome amplification and microarray techniques was successfully established and applied, which would allow examination of communities with dilute quantities of Dhc without time-consuming and laborious culturing steps. Expression microarray results of strain 195 grown under different corrinoid availabilities indicated corrinoid riboswitches in strain 195 played important roles in corrinoid salvaging mechanisms of strain 195. Furthermore, diverse corrinoid and lower ligand forms were identified and quantified by LC/MS/MS. Results exhibited that cobalamin was the dominant species in the ANAS culture, while p-cresol cobamide was the most dominant one produced by supportive microorganisms in groundwater enrichments, and Dhc in these enrichments modified this unfavorable form to the favorable cobalamin in the presence of the associated lower ligand 5, 6-dimenthylbenzimidazole (DMB). The corrinoid species and DMB also could serve as diagnostic “biomarkers” in bioremediation of chlorinated solvents.
The broadest significance of this research is that it will lead to improved strategies for optimizing in situ bioremediation technologies. The biomarkers developed could shorten the bioremediation process feedback cycle by replacing traditional diagnostics, such as microcosm responses that are monitored over weeks, with appropriate 16S-rRNA-based and gene expression-based diagnostics that can be monitored within hours. Furthermore, insights gained on ecological interactions improve the ability to design, construct, and optimize bioaugmentation and biostimulation systems.