Proposal of a phage cocktail for sulfate-reducing bacteria biofilms control in environments related to the oil and gas industry

Abstract

In the oil industry, biofilms formed by Sulfate-Reducing Bacteria (SRB) cause great damage related to the corrosion of tanks and pipes, and produce toxic gases such as H2S. The application of biocides is currently the most used form of treatment to control these microorganisms. However, these biocides are often ineffective against biofilms. Thus, the use of bacteriophages (or phages) has been studied as an alternative treatment. Phages are viruses that infect bacteria and may have the ability to degrade polymeric substances in the biofilm matrix, exposing the bacteria to infection by new phage particles or physical and chemical stresses. In this context, the present work aimed to formulate phage cocktails and evaluate their potential to control the biofilm formed by an SRB mixed culture of unknown composition. For this, 10 phages were isolated from sewage samples and analyzed by transmission electron microscopy (TEM). Fast-growing bacteria were then used to screen these isolated phages for their potential to reduce biofilm formation and also to assess their host spectrum. The host spectrum was investigated by analyzing the growth curve of each bacterium in the presence and absence of the phage, while the biofilm control potential was investigated by quantifying the biofilm biomass, using the crystal violet staining method. As all phages showed promise in controlling these biofilms, all were tested separately against a mixed SRB culture. From the results obtained with the phages tested separately, 6 cocktails were formulated by combining the isolates, which were then tested in the control of biofilm formation by the SRB culture. The members of the cocktails were also analyzed for stability after incubation under different conditions of temperature and pH, as well as in seawater. Microscopy analysis revealed the presence of 4 myoviruses, 3 siphoviruses and 3 podoviruses. Three of these isolates were able to infect more than one bacterial genus, being, therefore, polyvalent phages. All phages were able to, separately, reduce the biofilm formation of at least one non-host bacterial species, and 5 were able to reduce the biofilm formation by the SRB culture. Three of the six cocktails tested, Coq3, Coq5 and Coq6, reduced biofilm formation by the SRB culture by 15%, 44% and 43%, respectively. Although Coq5 showed the highest percentage of reduction (albeit with little difference from Coq6), Coq6 was the most promising since these phages had better stability under the different conditions studied compared to Coq5 phages. All Coq6 phages remained completely stable when incubated in seawater and showed viability above 50% when incubated at pH 4, interesting characteristics for a field application in the oil industry where low pH values and high salinity can be reached. In contrast, only one Coq6 phage maintained 100% viability when tested at higher temperatures (55 °C). Future analysis of the phages' genomes and identification of the component species of the mixed SRB culture should be carried out to obtain a better under...