Aims: Forty Bacillus strains isolated from a Brazilian oil reservoir were tested against each other to select strains producing antimicrobial substances (AMS). Three strains, Bacillus subtilis (LFE-1), Bacillus firmus (H 2 O-1) and Bacillus licheniformis (T6-5), were selected due to their ability to inhibit more than 65% of the Bacillus strains tested. These three strains were also investigated for their capability to inhibit sulphate-reducing bacteria (SRB). Furthermore, physiological and biochemical characteristics of the antimicrobial compounds produced by the selected strains were determined. Methods and Results: Among the forty strains tested, 36 (90%) strains were able to inhibit at least one Bacillus strain used as indicator in plate assays and three of them (LFE-1, T6-5 and H 2 O-1) were able to inhibit 65, 70 and 97AE5% of the 40 strains studied here respectively. Clear zones of inhibition were observed when H 2 O-1 was tested against SRB-containing consortium T6-lab and Desulfovibrio alaskensis strain NCIMB 13491, while strain T6-5 was able to inhibit only the D. alaskensis strain. The three substances showed to be insensitive to different enzymes and chemicals, were heat stable and the substances produced by strains T6-5 and H 2 O-1 were active over a wide pH range. Conclusions: Three different AMS produced by Bacillus strains from an oil reservoir, two of them with activity against SRB, are presented here. Significance and Impact of the Study: The preliminary characterization of these AMS points to their potential use as biocides in the petroleum industry for controlling problems associated with SRB.

Microbial colonization of petroleum industry systems takes place through the formation of biofilms, and can result in biodeterioration of the metal surfaces. In a previous study, two oil reservoir Bacillus strains (Bacillus licheniformis T6-5 and Bacillus firmus H(2)O-1) were shown to produce antimicrobial substances (AMS) active against different Bacillus strains and a consortium of sulfate-reducing bacteria (SRB) on solid medium. However, neither their ability to form biofilms nor the effect of the AMS on biofilm formation was adequately addressed. Therefore, here, we report that three Bacillus strains (Bacillus pumilus LF4 -- used as an indicator strain, B. licheniformis T6-5, and B. firmus H(2)O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H(2)O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H(2)O-1 reduced the viability and attachment of the SRB consortium biofilm by an order of magnitude. Our results suggest that the AMS produced by Bacillus strains T6-5 and H(2)O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.

Microorganisms, especially sulphate-reducing bacteria (SRB), have long been implicated in metal corrosion in the petroleum industry. SRB can appear in planktonic or adherent forms, interacting with surfaces to produce thick, consortial biofilms. We carried out an ultrastructural investigation of biofilms and planktonic bacteria obtained from samplers on offshore platforms operated in southeast Brazil. A great variety of Gram-negative bacteria were observed. The surface coat and extracellular matrix of these cells stained strongly with ruthenium red, indicating their anionic character. Sessile bacteria were found to be enmeshed in an extensive extracellular matrix. The inner ultrastructure of sessile bacteria was characterized by vacuoles, inclusions and internal membranes. ESI analysis revealed the presence of corrosion products associated with the surface coat of these bacteria.

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Anaerobic sulphate-reducing bacteria (SRB) are among the most important microorganisms involved in industrial biocorrosion (7,11). Like other microorganisms, SRB attach to metal surfaces and develop firmly adherent biofilms, in which microcolonies of different bacterial species can grow, immersed in an extensive extracellular matrix (EM) of anionic polysaccharide nature (6, 7). This complex tissue-like structure influences many physiological parameters of microbial colonization, including rate of growth, nutrient concentration and resistance to biocides. The development of an adherent biofilm defines two phases in which SRB can appear: planktonic/swimming and sessile/adherent (7). Direct observations of bacteria growing in natural or industrial environments have shown that these cells are always surrounded by some form of glycocalyx or coat (6,15,16), both in the planktonic and in the sessile phase. As described for the petroleum industry of the North Sea (11), in Brazil, biocorrosion caused by SRB is also a serious problem, affecting marine offshore platforms operated by PETROBRAS. Mixed populations of anaerobic bacteria, rich in SRB, can be obtained from samplers installed in the water injection system of the platforms, at points with a suitable environment for