Phage of Sulfate Reducing Bacteria Isolated from High Saline Environment

Sulfate reducing bacteria cause significant problems in the petroleum industry through corrosion of metal infrastructures and by the degradation of petroleum in reservoirs (reservoir souring) and storage tanks.  Current control measures include mechanical removal through pipeline pigging often in conjunction with expensive and environmentally undesirable chemical biocides.  The significant damage and costs associated with microbially influenced corrosion and reservoir souring warrants investigations into new technologies and methodologies for the control of petroleum industry associated bacteria.  We are actively investigating the potential use of bacteriophage as control agents of sulfate reducing bacteria.  Bacteriophage, aka phage, are the ubiquitous, natural predators of bacteria.  A characteristic of phage that has particular implications for their use in limiting bacterial populations is that phage are typically very host-specific.  Most phage only infect a single bacterial species or only limited number of strains of a single bacterial species.  This extreme host limitation has positive ramifications in that treatments that are very targeted can be developed to control only the problem bacteria, leaving all other bacterial species as well as all non-bacterial life forms unaffected.  However, this extreme host limitation also means that there needs to be some understanding of the bacteria present in the target populations of bacteria associated with any corrosion or reservoir souring event.  In order to develop a working phage preparation, the bacteria present in a target site need to be identified and at least some need to be cultured for use as bait in phage hunts.  Here, we report on the identification of bacteria present in a saturated brine pond associated with a saltern cavern oil storage system.  Total bacterial population was analyzed in the raw sample, as well as in samples cultured in 10% salinity media, by the use of temperature gradient gel electrophoresis of the product of a PCR reaction using 16s universal primers and DNA isolated from the various samples.  Sequence analysis indicated that bacteria found from this site included members of the Class Clostridia and sulfate reducing members of the delta-Proteobacteria.  These were found to be most closely related to strains isolated from other anaerobic, high salinity environments.  Sulfate reducing bacteria from this site could not be cultured to homogeneity but appear to require co-culturing with bacteria most closely related to Halanaerobium (Firmicute, Clostridia).  Phage specific for some of the bacteria cultured from this site were isolated and examined by electron microscopy.  These phages were used in assays designed to determine if the phages are capable of reducing the levels of bacterial cells relative to untreated samples.  These results will be discussed in the context of their prospective use as control agents.