Enhancing selection for perchlorate-reducing bacteria in presence of competing electron acceptors.

Perchlorate is a ubiquitous water contaminant that inhibits thyroid function. Standards for perchlorate in drinking water range from 2 to 18 μg L-1 in United States and Europe. A major natural source of perchlorate contamination is Chile saltpeter, found in the Atacama Desert in Chile. This dissertation starts by providing a literature review on the unique situation of perchlorate contamination in Chile. The review discusses perchlorate sources, presence in environmental media and in the human population, possible steps to mitigate its health impacts, and opportunities for bioprospecting. Microbial degradation is a promising strategy to remediate perchlorate, as it is reduced to innocuous chloride and oxygen. However, perchlorate is typically found in the μg/L range, and exerts a weak selective pressure for perchlorate-reducing bacteria (PRB). Also, nitrate can inhibit perchlorate reduction, so low nitrate levels are needed. Low nitrate levels can favor sulfatereducing bacteria (SRB). Sulfate reduction has also been related to inhibition of perchlorate reduction. Thus, the overarching goal of this research was to devise strategies to enrich PRB when perchlorate is at low concentrations, together with inhibition of sulfate reduction. The proposed strategy was the addition of the perchlorate analogs chlorate or chlorite. Both are intermediates in the perchlorate reduction pathway, and could have a stronger selective pressure for PRB and also inhibit SRB. The addition of chlorate and chlorite was tested in a hydrogen-based membrane biofilm reactor (H2-MBfR). In this type of reactor, H2 is supplied as electron donor through a hollow fiber membrane and bacteria grow on the surface using nitrate, perchlorate and others as electron acceptors. Chlorate was added for 30 days to a H2-MBfR reducing oxygen, nitrate, perchlorate and sulfate. Before chlorate addition, nitrate and perchlorate were reduced to low levels, but after 17 days sulfate reduction took place, leading to a decrease in perchlorate reduction. When chlorate was added, it increased perchlorate reduction and decreased sulfate reduction. Interestingly, analysis of the microbial community with 16S rRNA high-throughput sequencing suggested that the SRB (Desulfovibrionaceae) relative abundance increased. This was probably due to their role in sulfur cycling, although it cannot be ruled out that they played a role in chlorate reduction. To further understand the effect of chlorate on the microbial community, we tested chlorate addition in a H2-MBfR reducing nitrate, perchlorate and sulfate with similar bulk concentrations as before, but the bulk chlorate concentration was 10 times higher. Although the effect on perchlorate could not be evaluated, chlorate exerted a strong selective pressure for PRB, doubling the abundance of Dechloromonas, a common genus of PRB. As before, our results suggest that chlorate addition inhibited sulfate reduction. To understand the effect of chlorite, we initially determined the potential of chlorite to serve as an indirect electron acceptor and support bacterial growth, as it is dismutated to O2 during perchlorate metabolism. This was successfully proven by first determining O2 production and consumption rates after chlorite additions to PRB and CRB enrichments. Subsequently, the bacterial growth on chlorite was also demonstrated by measurements of the optical density of PRB and CRB cultures. Finally, we evaluated the selective pressure of chlorite in a H2-MBfR reducing nitrate, perchlorate and sulfate. This showed that chlorite had a minor selective pressure. This research provided evidence that adding chlorate and chlorite to a perchlorate H2-MBfR improved perchlorate reduction. Although it should be further studied, the results suggest this strategy could be helpful, particularly in the case of chlorate.