Rapid Enrichment of Dehalococcoides-Like Bacteria by Partial Hydrophobic Separation

Abstract

Organohalide-respiring bacteria can be difficult to enrich and isolate, which can limit research on these important organisms. The goal of this research was to develop a method to rapidly (minutes to days) enrich these organisms from a mixed community. The method presented is based on the hypothesis that organohalide-respiring bacteria would be more hydrophobic than other bacteria as they dehalogenate hydrophobic compounds. The method developed tests this hypothesis by separating a portion of putative organohalide-respiring bacteria, those phylogenetically related to Dehalococcoides mccartyi, at the interface between a hydrophobic organic solvent and an aqueous medium. This novel partial separation technique was tested with a polychlorinated biphenyl-enriched sediment-free culture, a tetrachloroethene-enriched digester sludge culture, and uncontaminated lake sediment. Significantly higher fractions, up to 20.4 times higher, of putative organohalide-respiring bacteria were enriched at the interface between the medium and either hexadecane or trichloroethene. The selective partial separation of these putative organohalide-respiring bacteria occurred after 20 min, strongly suggesting that the separation was a result of physical-chemical interactions between the cell surface and hydrophobic solvent. Dechlorination activity postseparation was verified by the production of cis-dichloroethene when amended with tetrachloroethene. A longer incubation time of 6 days prior to separation with trichloroethene increased the total number of putative organohalide-respiring bacteria. This method provides a way to quickly separate some of the putative organohalide-respiring bacteria from other bacteria, thereby improving our ability to study multiple and different bacteria of potential interest and improving knowledge of these bacteria.IMPORTANCE Organohalide-respiring bacteria, bacteria capable of respiring chlorinated contaminants, can be difficult to enrich, which can limit their predictable use for the bioremediation of contaminated sites. This paper describes a method to quickly separate Dehalococcoides-like bacteria, a group of organisms containing organohalide-respiring bacteria, from other bacteria in a mixed community. From this work, Dehalococcoides-like bacteria appear to have a hydrophobic cell surface, facilitating a rapid (20 min) partial separation from a mixed culture at the surface of a hydrophobic liquid. This method was verified in a polychlorinated biphenylenriched sediment-free culture, an anaerobic digester sludge, and uncontaminated sediment. The method described can drastically reduce the amount of time required to partially separate Dehalococcoides-like bacteria from a complex mixed culture, improving researchers' ability to study these important bacteria.KEYWORDS dechlorination, Dehalococcoides, enrichment, organohalide-respiring bacteria, separation

Assessing the Biodegradation of BTEX and Stress Response in a Bio-Permeable Reactive Barrier Using Compound-Specific Isotope Analysis

“…On the contrary, norank_p_Aminicenantes , which appeared with a high abundance, played an important role in the bio-PRB under TCE stress. Although Acidovorax has been described as capable of degrading BTEX [ 24 , 67 , 68 ], its poor tolerance to TCE stress meant that it hardly contributed to BTEX biodegradation in our experiment.…”

“…Besides the small percentage of unclassified bacteria, Proteobacteria were the most abundant (relative abundance of 35.9–79.7%) phylum, followed by Bacteroidetes (1.51–20.9%) and Ignavibacteriae (1.15–9.82%) ( Figure 9 A). Since Ignavibacteriae , Bacteroidetes , Aminicenantes , and Proteobacteria are frequently found in BTEX-impacted environments [ 24 , 66 , 67 , 68 ], the present work focused on these four dominant phyla. The relative abundance of Proteobacteria increased with the influent concentration of benzene and toluene, which indicated that Proteobacteria had a strong tolerance to a high concentration of benzene and toluene.…”

“…The dominant bacterial community ( Ignavibacteriae and Bacteroidetes at the phylum level, Ignavibacteria and Bacteroidia at the class level) varied significantly. The newly appeared Aminicenantes , responsible for the biodegradation of TCE, became the most dominant phylum (relative abundance = 44.6%) [ 67 ], while the relative abundance of Proteobacteria (35.9%), Bacteroidetes (1.53%), and Ignavibacteriae (2.91%) drastically decreased. Aminicenantes are anaerobic bacteria that can be found in hydrocarbon-polluted environments.…”