Detection and monitoring of arsenate respiring bacteria in soil

The capacity to couple growth to arsenate [As(V)] reduction may be widespread in soil bacteria. Microrganisms involved in arsenate reduction can be classified as either arsenic resistant bacteria (ARB) or dissimilatory arsenate respiring bacteria (DARB). In ARBs the cytoplasmic arsenate reductase is encoded by an arsC gene that is part of an arsenic resistance system. Differently, DARBs use As(V) as electron acceptor in respiration and the reduction is catalyzed by a periplasmic arsenate respiratory reductase (encoded by arrAB gene). DARBs are able to reduce both sorbed and dissolved As(V) (Oremland and Stolz, 2006) in environment with redox potential below +135mV. ArrA genes have been identified in several Proteobacteria and Gram-positive bacteria (Malasarn et al., 2004; Pérez-Jiménez et al., 2005). We investigated the occurrence of DARBs in an As polluted soil when it was spiked with glucose under flooding condition by real-time PCR quantification (qPCR) of the functional gene arrA. As(V) reduction was also monitored during the incubation time. The presence of DARBs was confirmed by using enrichment cultures established by mixing 0.5 g of soil in 45 ml of a modified Niggemyer enrichment medium containing sodium lactate (10 mM) and sodium arsenate (10 mM) (Niggemyer et al, 2001). Anaerobic conditions were achieved by flushing the vials, sealed with rubber stoppers, with N2. Four transplants were performed, in which As(V) reduction and arrA quantification were determined. Cloning and sequencing of arrA amplified in soil microcosms and in enrichment cultures were also performed. In glucose amended soil As(V) reduction occurred in the first 15 days of incubation, when reducing conditions occurred; copy numbers of arrA gene were detected only at 15 days of incubation at levels of 10 copy number g-1 soil, close to the detection limit. After the fourth transplant of the enrichment culture, As(V) reduction was 85% within 3 days and the qPCR analysis revealed an increase of copy numbers of arrA gene in concomitance of As(V) reduction, thus demonstrating the presence of DARBs in this polluted soil and a their potential activity in As solubilization.