Enrichment and Isolation of Metal Respiring Hydrocarbon Oxidizers

Abstract

Converging lines of research point to chloroxyanion-respiring hydrocarbon oxidizers as an important group of microorganisms that merit expanded isolation and characterization. Respiration of the chloroxyanions perchlorate (ClO4?) and chlorate (ClO3?), collectively termed (per)chlorate, is the only non-phototrophic process shown to produce molecular oxygen (O2). Accordingly, strains that respire (per)chlorate while oxidizing hydrocarbons have been of longstanding interest for bioremediation of hydrocarbons in anaero- bic environments (Coates et al., Nature 396(6713):730, 1998). Perchlorate also shows promise as a corrective to corrosive sulfidogenesis in oil reservoirs. The perchlorate ion is a putative competitive inhibitor of ATP sulfurylase, a key enzyme in the sulfate reduction pathway (Carlson et al., ISME J 9:1295–1305, 2014); (per)chlorate reduction can be coupled to the oxidation of sulfide into sulfur and sulfate (Gregoire et al., Environ Microbiol Rep 6(6):558–564, 2014); and in reservoirs amended with perchlorate, sulfate- respiring microorganisms could be competitively excluded by perchlorate-respiring hydrocarbon oxidizers (Engelbrektson et al., Frontiers Microbiol 5, 2014; Liebensteiner et al., Frontiers Microbiol 5(428), 2014). The discovery of mesophilic and hyperthermophilic (per)chlorate-reducing microorganisms that can consume hydrocarbons (Carlstro ¨m et al., Appl Environ Microbiol 81(8):2717–2726, 2015; Coates et al., Nature 411(6841):1039–1043, 2001; Liebensteiner et al., Science 340(6128):85–87, 2013) emphasizes the potential for bioremediation and oil reservoir souring control and provides exciting motivation to characterize additional (per)chlorate-reducing microorganisms from similar environments. This protocol provides an overview of anaerobic culturing and handling hydrocarbons (reviewed exten- sively in Davidova and Sulfita, Methods Enzymol 397(05):17–34, 2005) and specific techniques for the enrichment and isolation of chloroxyanion-respiring hydrocarbon oxidizers from several environments of interest.