Elisa Korenblum

Production of antimicrobial substances by Bacillus subtilis LFE‐1, B. firmus H2O‐1 and B. licheniformis T6‐5 isolated from an oil reservoir in Brazil

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 (H2O‐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.

Bacterial diversity in water injection systems of Brazilian offshore oil platforms

Biogenic souring and microbial-influenced corrosion is a common scenario in water-flooded petroleum reservoirs. Water injection systems are continuously treated to control bacterial contamination, but some bacteria that cause souring and corrosion can persist even after different treatments have been applied. Our aim was to increase our knowledge of the bacterial communities that persist in the water injection systems of three offshore oil platforms in Brazil. To achieve this goal, we used a culture-independent molecular approach (16S ribosomal RNA gene clone libraries) to analyze seawater samples that had been subjected to different treatments. Phylogenetic analyses revealed that the bacterial communities from the different platforms were taxonomically different. A predominance of bacterial clones affiliated with Gammaproteobacteria, mostly belonging to the genus Marinobacter (60.7%), were observed in the platform A samples. Clones from platform B were mainly related to the genera Colwellia (37.9%) and Achromobacter (24.6%), whereas clones obtained from platform C were all related to unclassified bacteria. Canonical correspondence analyses showed that different treatments such as chlorination, deoxygenation, and biocide addition did not significantly influence the bacterial diversity in the platforms studied. Our results demonstrated that the injection water used in secondary oil recovery procedures contained potentially hazardous bacteria, which may ultimately cause souring and corrosion.

Antimicrobial action and anti-corrosion effect against sulfate reducing bacteria by lemongrass (Cymbopogon citratus) essential oil and its major component, the citral

The anti-corrosion effect and the antimicrobial activity of lemongrass essential oil (LEO) against the planktonic and sessile growth of a sulfate reducing bacterium (SRB) were evaluated. Minimum inhibitory concentration (MIC) of LEO and its major component, the citral, was 0.17 mg ml-1. In addition, both LEO and citral showed an immediate killing effect against SRB in liquid medium, suggesting that citral is responsible for the antimicrobial activity of LEO against SRB. Transmission electron microscopy revealed that the MIC of LEO caused discernible cell membrane alterations and formed electron-dense inclusions. Neither biofilm formation nor corrosion was observed on carbon steel coupons after LEO treatment. LEO was effective for the control of the planktonic and sessile SRB growth and for the protection of carbon steel coupons against biocorrosion. The application of LEO as a potential biocide for SRB growth control in petroleum reservoirs and, consequently, for souring prevention, and/or as a coating protection against biocorrosion is of great interest for the petroleum industries.

Molecular analysis of the bacterial communities in crude oil samples from two brazilian offshore petroleum platforms.

Crude oil samples with high- and low-water content from two offshore platforms (PA and PB) in Campos Basin, Brazil, were assessed for bacterial communities by 16S rRNA gene-based clone libraries. RDP Classifier was used to analyze a total of 156 clones within four libraries obtained from two platforms. The clone sequences were mainly affiliated with Gammaproteobacteria (78.2% of the total clones); however, clones associated with Betaproteobacteria (10.9%), Alphaproteobacteria (9%), and Firmicutes (1.9%) were also identified. Pseudomonadaceae was the most common family affiliated with these clone sequences. The sequences were further analyzed by MOTHUR, yielding 81 operational taxonomic units (OTUs) grouped at 97% stringency. Richness estimators also calculated by MOTHUR indicated that oil samples with high-water content were the most diverse. Comparison of bacterial communities present in these four samples using LIBSHUFF and Principal Component Analysis (PCA) indicated that the water content significantly influenced the community structure only of crude oil obtained from PA. Differences between PA and PB libraries were observed, suggesting the importance of the oil field as a driver of community composition in this habitat.

Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation

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 H2O-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 H2O-1), and an oil reservoir SRB consortium (T6lab) were grown as biofilms on glass surfaces. The AMS produced by strains T6-5 and H2O-1 prevented the formation of B. pumilus LF4 biofilm and also eliminated pre-established LF4 biofilm. In addition, the presence of AMS produced by H2O-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 H2O-1 may have a potential for pipeline-cleaning technologies to inhibit biofilm formation and consequently reduce biocorrosion.

Comparative Bioremediation of Crude Oil-Amended Tropical Soil Microcosms by Natural Attenuation, Bioaugmentation, or Bioenrichment

Bioremediation is an efficient strategy for cleaning up sites contaminated with organic pollutants. In this study, we evaluated the effectiveness of monitored natural attenuation, bioenrichment, and bioaugmentation using a consortium of three actinomycetes strains in remediating two distinct typical Brazilian soils from the Atlantic Forest and Cerrado biomes that were contaminated with crude oil, with or without the addition of NaCl. Microcosms were used to simulate bioremediation treatments over a 120-day period. During this period, we monitored total petroleum hydrocarbons (TPHs) and n-alkanes degradation and changes in bacterial communities. Over time, we found the degradation rate of n-alkanes was higher than TPH in both soils, independent of the treatment used. In fact, our data show that the total bacterial community in the soils was mainly affected by the experimental period of time, while the type of bioremediation treatment used was the main factor influencing the actinomycetes populations in both soils. Based on these data, we conclude that monitored natural attenuation is the best strategy for remediation of the two tropical soils studied, with or without salt addition.

Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria

BackgroundBacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells.ResultsA lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched β-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu.Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 μg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces.ConclusionAMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Streptomyces lunalinharesii Strain 235 Shows the Potential to Inhibit Bacteria Involved in Biocorrosion Processes

Four actinomycete strains previously isolated from Brazilian soils were tested for their antimicrobial activity against Bacillus pumilus LF-4 and Desulfovibrio alaskensis NCIMB 13491, bacteria that are well known to be involved in biofilm formation and biocorrosion. Strain 235, belonging to the species Streptomyces lunalinharesii, inhibited the growth of both bacteria. The antimicrobial activity was seen over a wide range of pH, and after treatment with several chemicals and heat but not with proteinase K and trypsin. The antimicrobial substances present in the concentrated supernatant from growth media were partially characterized by SDS-PAGE and extracellular polypeptides were seen. Bands in the size range of 12 to 14.4 kDa caused antimicrobial activity. Transmission electron microscopy of D. alaskensis cells treated with the concentrated supernatant containing the antimicrobial substances revealed the formation of prominent bubbles, the spherical double-layered structures on the cell membrane, and the periplasmic space completely filled with electron-dense material. This is the first report on the production of antimicrobial substances by actinomycetes against bacteria involved in biocorrosion processes, and these findings may be of great relevance as an alternative source of biocides to those currently employed in the petroleum industry.