- Research article
- Open Access
Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria
© Korenblum et al.; licensee BioMed Central Ltd. 2012
Received: 12 July 2012
Accepted: 1 November 2012
Published: 7 November 2012
Bacillus 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.
A 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.
AMS 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.
Sulfide accumulation in petroleum reservoirs is generally described as souring. Biogenic souring is usually due to the hydrogen sulfide that is produced by sulfate reducing bacteria (SRB), a diverse group of anaerobes that use sulfate as a final electron acceptor . The souring process can be intensified when the petroleum reservoir is subjected to water flooding for secondary oil recovery . Because seawater is often used in water flooding in offshore oil fields, sulfate amounts raise downhole and further stimulate SRB growth, resulting in increased risk of souring. The hydrogen sulfide can reach concentrations in the reservoir that may be toxic and/or explosive. Hence, a sulfate reducing bacteria control strategy is mandatory in the oil and gas industries. Biocorrosion is also a common process in reservoirs that are subjected to secondary oil recovery . In order to avoid the risks associated with the injection of sea water, the water is pretreated before being injected. The treatment usually consists of deaeration and the addition of biocides. Although different strategies of sulfide production control have been developed, the most commonly used strategy is biocide dosing with inorganic substances (chlorine; ozone) or organic compounds (quaternary ammonium salts; glutaraldehyde; tetrakis hydroxymethyl phosphonium sulphate) . Quaternary ammonium salts are widely used in the Brazilian petroleum industry as a continuous biocide treatment . Glutaraldehyde has been extensively applied as both batch and continuous treatment to prevent sulfate reducing bacteria growth [4, 5]. However, the cost and the environmental impact of using these compounds should always be considered. A cost estimation of billions of dollars per year is predicted in oil and gas production industries due to lost material and the resources required to monitor and to prevent sulfide production, including biocide treatment . For these reasons, alternative sources for avoiding or limiting the production of biogenic sulfide are needed, and the identification of new antimicrobial substances that are active against sulfate reducing bacteria is an important area of research.
Many members of the genus Bacillus are able to produce different types of biologically active compounds . Many Bacillus strains are well-known for their ability to produce antimicrobial substances, including bacteriocins, exoenzymes, RNA-degrading enzymes, cell wall lytic enzymes and peptide and lipopeptide antibiotics [8–13]. Some of these substances are active only against the same species or a closely related species , while others have a broad spectrum of activity [15, 16]. A well-known lipopeptide that is produced by Bacillus subtilis is surfactin, a compound named for its strong interfacial activity . The structure of surfactin consists of a peptide loop of seven amino acids (L-asparagine, L-leucine, glutamic acid, L-leucine, L-valine and two D-leucines) and a hydrophobic fatty acid chain with thirteen to fifteen carbons that allows surfactin to penetrate cellular membranes. Other surfactin analogues that have been described include pumilacidin , bacircine  and lichenysin . Those molecules are classified as biosurfactants because of their abilities to decrease surface tension and act as emulsifying agents . Biosurfactants are amphiphilic compounds  that can be applied in many fields that require their capacities as detergents, emulsifying agents, lubricants, foams, wetting agents or their solubilizing and phase dispersion abilities [22–24]. Most of them also exhibit antimicrobial, anti-adhesive and anti-corrosion properties . These properties are desirable for control corrosion, colonization with sulfate reducing bacteria and biofilm formation in oil facilities.
In our laboratory, an antimicrobial substance produced by a petroleum reservoir bacterium, the Bacillus sp. H2O-1, has been previously shown to inhibit the sessile and planktonic growth of the SRB strain Desulfovibrio alaskensis NCIMB 13491 . This antimicrobial substance was stable at a wide pH range and at a variety of temperatures. However, further detailed biochemical studies of the antimicrobial substance produced by the Bacillus sp. H2O-1 strain (AMS H2O-1) were necessary to evaluate its potential use in the petroleum industry.
Therefore, this study presents the taxonomic affiliation of Bacillus sp. H2O-1, the structure of AMS H2O-1 and its effects on sulfate reducing bacteria cells. Furthermore, the surface free energy and the hydrophilic or hydrophobic characteristics of different surfaces conditioned with the antimicrobial substance produced by strain H2O-1 were determined and compared to surfaces treated with a surfactin produced by B. subtilis ATCC 21332.
The antimicrobial substance producer strain Bacillus sp. H2O-1 was originally isolated from an oil reservoir in Brazil and previously described by Korenblum et al. . This strain was grown in Luria-Bertani broth (LB), pH 7.0-7.2, containing 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl per liter of distilled water. The strain Desulfovibrio alaskensis NCIMB 13491 was used as a sulfate reducing bacteria indicator (AMS H2O-1 sensitive) and was grown at 30°C in Postgate E medium  purged with a N2 flux to achieve anaerobiosis. Bacillus subtilis ATCC 21332 was used to produce surfactin as described by Nitschke .
The bacterial strain H2O-1 was characterized by using the kit API 50CH (Apparéils et Prócédes d′Identification – bioMérieux sa, Lyon, France) as described by the manufacturer.
In addition, the 16S rRNA gene was amplified by PCR from H2O-1 genomic DNA using the universal primers 27f (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492r (5’-GGTTACCTTGTTACGACTT-3’). DNA was extracted from Bacillus sp. H2O-1 grown overnight at 30°C in LB broth using the ZR Fungal/Bacterial DNA MiniPrepTM kit (ZYMO Research, Irvine, CA, USA) according to the manufacturer’s instructions. The full 16S rRNA gene sequencing (GenBank accession number JX575798) was carried out by the Macrogen Genomic Division, South Korea, using ABI PRISM Big Dye Terminator Cycle Sequencing technology (Applied BioSystems, Foster city, CA, USA). The sequence obtained was compared with 16S rRNA gene sequences of closely related type strains using RDP database (http://rdp.cme.msu.edu/). Alignment and phylogenetic tree construction were performed using the Tree Builder tool from RDP website.
Isolation and purification of the lipopeptide
The Bacillus sp. H2O-1 was cultured in LB broth at 30°C for three days and then harvested by centrifugation at 12,500 x g for 30 min. The supernatant was adjusted to pH 2.0 with concentrated HCl and allowed to stand overnight at 4°C. The precipitate was then dissolved in 0.4 M HCl and extracted with chloroform-methanol (2:1 v/v) . The mixture was shaken vigorously and then left static for phase separation. The organic phase was concentrated at reduced pressure at 40°C, yielding the crude extract containing the lipopeptide. The AMS H2O-1 lipopeptide extract was applied to a silica gel 60 column chromatography (particle size 0.063-2 mm) and eluted with chloroform-methanol 9:1 v/v and methanol. The collected fractions were analyzed by thin layer chromatography (TLC) that was developed with CHCl3/CH3OH/ 2M NH4OH (40:10:1 v/v), and the spots were visualized with iodine and by spraying them with orcinol/H2SO4. The methanol fraction containing the partially purified lipopeptide was then analyzed by ESI-MS in the positive and negative ionization modes.
Gas chromatography–mass spectrometry (GC-MS) of fatty acids
The lipids (1 mg) were methanolyzed in 0.5 ml of 1 M HCl-MeOH for 4 h at 100°C. The product containing the fatty acid methyl esters (FAMEs) was partitioned by adding H2O (0.5 ml) and extracting with 1 ml of n-hexane . The MeOH/H2O phase was dried under N2 stream and was acetylated in pyridine-MeOH-Ac2O (1:1:4, v/v) with heating at 100°C for 60 min . The samples were then analyzed using a GC-MS-ion trap detector (Varian, Saturn-2000R) with a capillary column DB-1-MS (J&W) that was 30 m x 0.25 mm x 0.25 μm in size. The chromatograph temperature was programmed to increase from 50 to 280°C at 20°C/min and was then held constant for 30 min. FAMEs were identified on the basis of their relative retention time in comparison with the standard of 3-hydroxy-hexadecanoate methyl ester (Sigma-Aldrich, SP, Brazil) and by their MS-fragmentation profile at electron ionization (EI – 70 eV).
Electrospray ionization-mass spectrometry (ESI-MS)
The approximately 300 μg/ml suspension of lipids in MeOH–H2O (3:1, v/v) containing HCl at 1 mmol/l was submitted to positive and negative mass spectrometry at atmospheric pressure ionization and recorded on a triple quadrupole, Quattro LC (Waters) with N2 as the nebulization and desolvation gas. Offline analyses were performed with an infusion pump at a flow rate of 10 μl/min. The energies were set at 3.5 kV on the capillary and 100 V on the cone (negative mode) or at 3.5 kV and 90 V (positive mode). Tandem-MS was obtained by collision-induced dissociation-mass spectrometry (CID-MS) using argon as collision gas and a collision energy of 40 eV.
In order to confirm the antimicrobial activity of the partial purified lipopeptide fraction, approximately 100 μl of the extract were applied to two thin layer chromatography (TLC) plates (10 cm × 20 cm) and developed with CHCl3/CH3OH/ 2M NH4OH (40:10:1 v/v). One plate was used as the reference chromatogram, and the spots were visualized with iodine and by spraying them with orcinol/H2SO4. The other one was used for bioautography in a Petri dish. A suspension (15 ml) containing 105 cells/ml of D. alaskensis NCIMB 13491 was poured over the TLC plate. After solidification of the medium, the TLC plate was incubated for 7 days at 30°C in an anaerobic chamber. Clear growth inhibition zones were observed against a blackish background.
Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
To determine the minimum concentration that the lipopeptide inhibits D. alaskensis NCIMB 13491 growth, the AMS H2O-1 lipopeptide extract obtained was used in microdilution susceptibility tests, which were carried out according to Das et al. . A working solution of the AMS H2O-1 lipopeptide extract was prepared in distilled water (80 μg/ml) and sterilized by passing it through a 0.45 μm filter. This working solution was serially diluted to a lowest concentration of 1.2 μg/ml in sterile Postgate E medium in 96-well microtiter plates to determine the minimum inhibitory and the minimum bactericidal concentrations. The indicator strain D. alaskensis was grown for 7 days at 32°C in Postgate E medium; this culture was diluted to yield a final SRB inoculum of 105 cells/ml. All of the controls and test concentrations were prepared as five replicates. The microtiter plates were incubated for 7 days at 32°C. The D. alaskensis growth was detected by observing the blackish color of the medium caused by iron sulfide precipitation in Postgate E medium. The minimum inhibitory concentration (MIC) was determined as the least amount of antimicrobial substance added that did not result in blackish color of the medium. To perform the minimum bactericidal concentration test, an aliquot of 10 μl of the treated and untreated cell suspensions from the MIC plate were used to inoculate fresh Postgate E medium (90 μl) and incubated for 7 days at 32°C. The minimum bactericidal concentration (MBC) was determined as the lowest concentration of antimicrobial substance that resulted in no growth of D. alaskensis indicator strain. All of the inoculation procedures and incubations were performed in an anaerobic chamber (PlasLabs Inc., USA).
Preparation of cells for transmission electron microscopy (TEM)
Electron microscopy examination was used to study the biocidal effect of the AMS H2O-1 lipopeptide extract on D. alaskensis cells. After incubating 105 bacterial cells/ml with AMS H2O-1 (at MIC, 0.5x MIC and 2x MIC) at 30°C for 24 hours, the cells were fixed overnight at 4°C in 2.5% glutaraldehyde in sodium cacodylate buffer 0.1M prepared in artificial sea water, washed in the same buffer, post-fixed in osmium tetroxide 1% in sodium cacodylate buffer 0.1M, washed again in the same buffer, dehydrated in an acetone series and embedded in Polybed 812. All of the ultra-thin sections were obtained using a Leica ultramicrotome, contrastained with uranyl acetate and lead citrate and observed with a FEIMorgagni TEM at 80 kV. The samples of the AMS H2O-1 treated cells and the untreated control samples were prepared in duplicate. The transmission electron microscopy preparation was also performed twice at different times.
The following parameters were analyzed in order to compare the tensoactive properties of Bacillus sp. H2O-1 lipopeptide extract with the one produced by B. subtilis ATCC 21332, respectively: surface tension, interfacial tension and critical micellar concentration. These parameters were determined using the pendant drop technique (DSA 100S Goniometer, Model: OF 3210), according to Song and Springer . The results were expressed as the mean value of at least ten pendant drops at 23°C and 55% relative humidity. Biosurfactant serial dilutions in water were performed and analyzed using the pendant drop technique described above to determine the critical micellar concentration . The measurements were taken until the surface tension was close to the one of water.
Analysis of conditioned surfaces
The surfaces samples were 2 cm2 coupons of stainless steel AISI 304, stainless steel AISI 430, carbon steel, galvanized steel and polystyrene. All of them were cleaned by immersing them in 99% ethanol (v/v), placing them in an ultrasonic bath for 10 min, rinsing them with distilled water, immersing them in a 2% aqueous solution of commercial detergent and ultrasonic cleaning them for 10 more minutes. The coupons were washed with distilled water and then sterilized at 121°C for 15 min. The cleaned coupons were then conditioned with aqueous solutions 5% (w/v) of the dried powder obtained after neutralization of AMS H2O-1 lipopeptide extract, surfactin or water (control) by immersing them in the solutions for 24 h at room temperature. The samples were then washed with water and left to dry at room temperature until further analysis.
The water, formamide and ethylene glycol drop angles were measured to determine the surface free energy and hydrophilic and hydrophobic characteristics of the metal and non-metal surfaces after they were conditioned with the AMS H2O-1 lipopeptide extract, surfactin, or water (control). The assays were performed using a Krüss DSA 100S goniometer (model: OF 3210) to measure the contact angles between the liquids and the different surfaces (stainless steel AISI 304, stainless steel AISI 430, carbon steel, galvanized steel and polystyrene). The results are expressed as the mean value of at least ten drops (10 μl) at 23°C and 55% relative humidity.
where: θ is the contact angle between the liquid and the surface; γTOT is the total surface free energy; γLW is the Lifshitz-van der Waals component; γAB is the Lewis acid–base property; γ+ and γ- are the electron acceptor and donor components, respectively; .
Taxonomic affiliation of the Bacillussp. H2O-1
Some biochemical characteristics that differentiate strain H2O-1 from reference strains of phylogenetically related Bacillus species
Acid production from:
After being released from the lipopeptides by methanolysis, the fatty acid compositions were determined by GC-MS of the FAMEs. Five main peaks on the chromatogram were consistent with fatty acids ranging from C13 to C16. They had MS-fragmentation profile similar to that of β-hydroxy-palmitic acid methyl ester (3-OH-C16:0-O-Me), with a main fragment ion at m/z 103. This is derived from the cleavage of an alpha hydroxylated carbon (C3) that results in a diagnostic fragmentation pattern for this group that differs from the α-hydroxyl fatty acid methyl esters from a McLafferty rearrangement that result in a diagnostic fragment at m/z 90 . Curiously, the chromatogram showed two main peaks that appeared close together and had retention times somewhat lower than the 3-OH-C16:0-O-Me. This result might be attributed to the presence of equivalent amounts of iso- and anteiso-β-OH-C15, as observed for surfactins from Bacillus subtilis.
No monosaccharides were observed in the MeOH/H2O phase after acetylation, indicating the absence of glycolipids. Instead, the compounds that were observed were identified as amino acids by comparison with our previous data bank . The amino acids present were leucine (or isoleucine), glutamate, aspartate and valine (data not shown) and indicated a surfactin-like lipopeptide.
Minimum inhibitory and bactericidal concentrations of AMS H2O-1 against D. alaskensisNCIMB 13491
Transmission electron microscopy analysis
Physico-chemical properties (surface tension –ST, Interfacial tension – IT and critical micellar concentration – CMC) of AMS H2O-1 and surfactin
26.8 ± 0.1
21.8 ± 2.8
83.7 ± 0.8
27.1 ± 1.6
15.6 ± 1.4
27.6 ± 0.1
Surface conditioning analysis
Energy properties of conditioned surfaces including the total surface free energy, the Lifshitz-van der Waals component, the Lewis acid–base properties, the electron acceptor component, the electron donor component and the surface hydrophobicity
The stainless steel AISI 304 and 430 and the galvanized steel donated more electrons after both treatments, while the carbon steel remained less likely to donate electrons than the control. The AMS H2O-1 treatment of the polystyrene increased its ability to donate electrons, while surfactin decreased this property.
The Lifshitz van der Waals component increased with both treatments on stainless steel 304 and 430. This component was maintained on carbon steel, galvanized steel and polystyrene with surfactin but decreased on galvanized steel and increased on polystyrene when treated with the AMS H2O-1.
The surface free energy increased on stainless steel 304 and 430 and polystyrene, was maintained on carbon steel and decreased on galvanized steel for both molecules.
Although synthetic surfactants are able to control corrosion and the growth of sulfate reducing bacteria, these substances may cause human and environmental health risks . An alternative is the use of biosurfactants to replace the chemically synthesized surfactant compounds. Biosurfactants are biodegradable and have low toxicity . The AMS H2O-1 produced by Bacillus sp. H2O-1 has already been shown to inhibit the growth of sulfate reducing bacteria (SRB) [11, 26]. In this study, the AMS H2O-1 was characterized and was shown to have a surfactin-like lipopeptide structure. Surfactin is a biosurfactant, or an amphipathic molecule, that is a well-known product from the secondary metabolism of B. subtilis.
A comparative 16S rRNA gene sequence-based phylogenetic analysis placed strain H2O-1 in a clade with the species Bacillus subtilis, B. amyloliquefaciens and B. methylotrophicus and revealed pairwise similarities higher than 99.5%. API 50CH tests were further used to help the assignment of H2O-1 in one of these species but the fermentation of 49 sugar substances or derivatives was not sufficient for that. Therefore, the essential features for description of new taxa of the aerobic endospore-forming bacteria  should be used to achieve a reliable identification of strain H2O-1. In this study, this strain was considered only as a member of the genus Bacillus since the purification and characterization of AMS H2O-1 were the main purposes.
Different surfactin-like compounds are non-ribosomally synthesized in Bacillus spp., and the enzymes that are involved in those syntheses are closely related . AMS H2O-1, like every surfactin-like analogue, consists of a cyclic peptide containing seven amino acid residues (mostly hydrophobic amino acids) linked to a lipidic chain. The lipophilic portion may vary in length and ramification or in the amino acid content . The original surfactin molecule contains the heptapeptide sequence Glu-Leu-Leu-Val-Asp-Leu-Leu, the same found in AMS H2O-1, and a varying lipid portion of C13-C15 β-hydroxy-fatty acids that was also observed in AMS H2O-1. However, an additional lipid portion, a C16 β-hydroxy-fatty acid, was also produced by the Bacillus sp. H2O-1 strain under the selected conditions (shaken in a flask of LB broth at 30°C for three days). LB broth has been used in most cases for biosurfactant production from Bacillus strains . Previous studies have shown that the length and composition of the fatty acid depends on the growth medium and may result in higher specific surfactant activity [19, 49]. Regardless of the similarities between the structures of surfactin and AMS H2O-1, one of the genes required for surfactin biosynthesis, sfp, could not be detected in Bacillus sp. H2O-1 by PCR (data not shown) using primers previously described by Hsieh et al. . These authors were able to amplify the sfp gene from different strains of Bacillus subtilis and from other surfactin-producing Bacillus spp. Bacillus sp. H2O-1 either has a mutant allele of sfp that could not be detected by this pair of primers or has a slightly different homologue. The expression of different homologues or different ratios of the same homologues will confer different surface tension characteristics .
The AMS H2O-1 lipopeptide extract was further compared with the crude extract of surfactin produced by B. subtilis for its ability to decrease interfacial tension and surface tension, and their critical micellar concentration (CMC) were determined. The results showed that the properties of both molecules were similar, although the CMC of the AMS H2O-1 lipopeptide extract was much lower (3 times), probably because of differences between the mixture of homologues produced by each species. Previous studies showed that the surfactin produced by B. subtilis LB5a using cassava waste water as substrate presented different CMC values [24, 28, 52].
Biosurfactants are now being widely studied because of their ability to adsorb to surfaces and delay microbial attachment. Banat et al. , Araujo et al.  and many other authors have been able to decrease microbial adhesion and biofilm development on many surfaces through the pre-treatment of the surfaces with a variety of biosurfactants. The anti-adhesive effects of a biosurfactant is due to its capacity to adsorb to a solid surface and change the hydrophobicity; the apolar portion interacts with the hydrophobic surface, while the polar portion is exposed to the aqueous environment, resulting in a decrease in the hydrophobicity of the surface. This change interferes with the microbial adhesion on this surface and therefore alters biofilm development . The inhibitory activity of AMS H2O-1 on the formation of SRB biofilms on glass has been previously demonstrated .
Biofilm formation is a complex phenomenon that is usually divided into five steps: reversible adhesion, irreversible adhesion, EPS production, maturation and dispersion. The first and second steps involve microbial adhesion to surfaces are the most important to the initiation of biofilm formation. These steps involve physico-chemical interactions that can be mediated by non-specific interactions with long-range forces, including Lifshitz–van der Waals interactions, electrostatics, acid–base interactions, Brownian motion forces  and the presence or absence of cellular appendages . In addition to cellular appendages, the hydrophobic interactions between the abiotic surface and the microorganism have a major role in the initial microbial adhesion and, therefore, biofilm development in biological systems .
Because of the ability of biosurfactants to change surface characteristics and potentially inhibit microbial adhesion and delay the corrosion of metallic surfaces , surfaces were conditioned with each of the biosurfactants in order to analyze their potential as a tool to control sulfate reducing bacteria and the formation of destructive biofilms in oil production facilities. The results indicated that the studied surfaces became less hydrophobic when conditioned by AMS H2O-1, with the exception of carbon steel, which became hydrophobic. Our surface hydrophobicity results agree with those of previous studies, such as the studies of Guillemot  and Meylheuc et al. , which analyzed the hydrophobic character of stainless steel conditioned with biosurfactants compared to unconditioned stainless steel (control). These authors also found that polystyrene maintained the same degree of hydrophobicity. Similar results were obtained by Araujo et al. , who analyzed the hydrophobic character of treated and untreated polystyrene. The anti-adhesive property of biosurfactants is due to their ability to adsorb to a surface and change its hydrophobicity according to the orientation of the molecules adsorbed; usually the apolar portion interacts with hydrophobic surfaces, and the polar portion is exposed to the aqueous environment, resulting in a decrease in the hydrophobicity of the surface . When the surfaces are hydrophilic, the inverse may occur.
Stainless steel AISI 304 and 430 and galvanized steel became more electron-donating with both treatments, while carbon steel remained less electron-donating than the control. The electron-donating ability of polystyrene increased after treatment with AMS H2O-1 extract, but decreased after treatment with surfactin. Nitschke et al.  reported that stainless steel AISI 304 that had been conditioned with surfactin for 24 hours showed a great increase as an electron-donor and a decrease as an electron-acceptor. They concluded that surfactin modifies the surface and generates a more basic (electron-donor) surface that reduces the hydrophobicity. Our results are closely related to those found on that work, and therefore, we can state that the mixture of homologues produced by Bacillus sp. H2O-1 also presents these characteristics for polystyrene, stainless steel AISI 430 and galvanized steel.
Hydrophilic repulsions and hydrophobic attractions are principally due to Lewis acid–base interactions; the apolar or Lifshitz-van der Waals interactions usually only play a minor role . The Lifshitz van der Waals component increased with both treatments on stainless steel 304 and 430; this component was maintained on carbon steel, galvanized steel and polystyrene with surfactin but decreased on galvanized steel and increased on polystyrene treated with the AMS H2O-1 extract. The surface free energy increased on stainless steel 304 and 430 and polystyrene, was maintained on carbon steel and decreased on galvanized steel for both molecules. These surface characteristics are strictly related to microbial adhesion and biofilm formation, and if these properties are altered by AMS H2O-1 lipopeptide extract, as demonstrated in our results, it is likely to interfere with microbial adhesion .
When D. alaskensis NCIMB 13491 was treated with AMS H2O-1 lipopeptide extract at the MIC (5 μg/ml), many cells with extracted cytoplasm were observed in transmission electron micrographs, and the cytoplasms of some cells were full of electron dense granules and condensed nucleoids. Although we observed cells in the micrographs after treatment, the MBC assay showed that these cells were no longer viable. The AMS H2O-1 lipopeptide extract had a bactericidal effect against the sulfate reducing bacteria tested. The surfactin-like lipopeptide critical micellar concentration (CMC) value (27.6 μg/ml) was approximately 5 times greater than the MIC (5 μg/ml), and cell shape modifications and cytoplasm electron density alterations were observed at 0.5x MIC concentration. Then, the antimicrobial effect of AMS H2O-1 is observed at concentrations lower than the CMC.
Biosurfactants in aqueous solutions form aggregates and then exhibit a lytic activity against an extensive range of microbes, possibly by forming pores and disintegrating membranes [61, 62]. Sotirova and coworkers  observed, by scanning electron microscopy, that a biosurfactant (rhamnolipid) affects cell shape at concentrations greater than the CMC. However, Bharali and coworkers  observed that the rhamnolipid produced by Pseudomonas aeruginosa OBP1 had a CMC value of 45 μg/ml and an MIC value of 8 μg/ml against different bacteria.
Other antimicrobial compounds produced by Bacillus species have been tested against sulfate reducing bacteria. For example, Jayaraman et al.  described a peptide antibiotic produced by the gramicidin-S-overproducing Bacillus brevis Nagano strain that prevents sulfate reducing bacteria growth in biofilms and significantly reduced the biocorrosion of mild steel and stainless steel. The same strain has been shown to inhibit Desulfosporosinus orientis biofilms in situ. The Bacillus strain B21, which was isolated from injection water obtained from an Algerian Sahara oilfield, was recently shown to inhibit a SRB consortium in co-culture  better than the biocide tetrakis hydroxymethyl phosphonium sulphate - THPS. However, the mode of action of strain B21 against sulfate reducing bacteria growth was not elucidated. Nevertheless, this growth inhibition might be due to biosurfactant production, as strain B21 was reported to produce this compound previously .
The use of antimicrobial substances isolated from Bacillus species has been of interest for SRB control in oilfields, and patents have being submitted in this field to use antimicrobials produced by Bacillus strains [69, 70]. In order to be applied in the petroleum industry, the production of the described herein surfactin-like lipopeptide has to be optimized and scaled up, even though only a low inhibitory concentration is necessary. Because the antimicrobial lipopeptides produced by Bacillus generally are active against a wide range of bacteria, these molecules are also useful in the agricultural, chemical, food, and pharmaceutical industries [7, 32, 71]. Furthermore, in the petroleum industry, biosurfactants are important tools to assist in the biodegradation of oil spills in contaminated environments  and in EOR (enhanced oil recovery) or MEOR (Microbial EOR), which is a tertiary oil recovery strategy that increases petroleum yields by decreasing the surface and interfacial tensions of the oil to enable oil flow . Moreover, the surfactin-like lipopeptide is produced by a bacterium that was isolated from a petroleum reservoir and could be reintroduced to the oilfield or other industrial systems in order to produce the AMS H2O-1 in situ.
The methanol fraction of the AMS H2O-1 lipopeptide extract was analyzed by GC-MS and ESI-MS and was identified as a mixture of four surfactin-like homologues. This mixture showed excellent tensoactive properties and a lower critical micellar concentration than the surfactin produced by B. subtilis. These characteristics are of great importance for industrial applications because a lesser amount of the product is required to achieve the aim of application. The antimicrobial activity of this fraction was detected by bioautography and was observed by transmission electron microscopy. The micrographs suggested that these molecules are able to disrupt the cell walls of the strain D. alaskensis NCIMB 13491 at concentrations as low as 5 μg/ml. In addition, AMS H2O-1 surfactin-like lipopeptide has physico-chemical characteristics that are similar to those of the biosurfactant produced by B. subtilis ATCC 21332 (surfactin). Both biosurfactants adsorbed to the surface samples and changed their energy characteristics; the changes that occurred may be of great value for their ability to inhibit/decrease the initial adhesion of sulfate reducing bacteria to the surfaces. Thus, the lipopeptide biosurfactant that is produced by Bacillus sp. H2O-1 in this study was shown to be a potential antimicrobial biosurfactant that may be used in the petroleum industry to replace synthetic surfactants for sulfate reducing bacteria control.
This study was financial supported in part by PETROBRAS project grant, CAPES, CNPq and FAPERJ.
- Muyzer G, Stams AJ: The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol. 2008, 6: 441-454.PubMedGoogle Scholar
- Nemati M, Jenneman GE, Voordouw G: Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs. Biotechnol Bioeng. 2001, 74: 424-434. 10.1002/bit.1133.PubMedView ArticleGoogle Scholar
- Videla HA, Herrera LK: Microbiologically influenced corrosion: looking to the future. Int Microbiol. 2005, 8: 169-180.PubMedGoogle Scholar
- Korenblum E, Valoni E, Penna M, Seldin L: Bacterial diversity in water injection systems of Brazilian offshore oil platforms. Appl Microbiol Biotechnol. 2010, 85: 791-800. 10.1007/s00253-009-2281-4.PubMedView ArticleGoogle Scholar
- Videla HA: Prevention and control of biocorrosion. Inter Biodeterd Biodegrad. 2001, 4: 259-270.Google Scholar
- NACE International - the corrosion society: http://www.nace.org/,
- Ongena M, Jacques P: Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 2008, 16: 115-125. 10.1016/j.tim.2007.12.009.PubMedView ArticleGoogle Scholar
- Abriouel H, Franz CM, Ben Omar N, Gálvez A: Diversity and applications of Bacillus bacteriocins. FEMS Microbiol Rev. 2011, 35: 201-232. 10.1111/j.1574-6976.2010.00244.x.PubMedView ArticleGoogle Scholar
- Cherif A, Chehimi S, Limem F, Hansen BM, Hendriksen NB, Daffonchio D, Boudabous A: Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9. J Appl Microbiol. 2003, 95: 990-1000. 10.1046/j.1365-2672.2003.02089.x.PubMedView ArticleGoogle Scholar
- Hyronimus B, Le Marrec C, Urdaci MC: Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4. J Appl Microbiol. 1998, 85: 42-50. 10.1046/j.1365-2672.1998.00466.x.PubMedView ArticleGoogle Scholar
- Korenblum E, der Weid I, Santos AL, Rosado AS, Sebastián GV, Coutinho CM, Magalhães FC, Paiva MM, Seldin L: Production of antimicrobial substances by Bacillus subtilis LFE-1, B. firmus HO-1 and B. licheniformis T6-5 isolated from an oil reservoir in Brazil. J Appl Microbiol. 2005, 98: 667-675. 10.1111/j.1365-2672.2004.02518.x.PubMedView ArticleGoogle Scholar
- Naruse N, Tenmyo O, Kobaru S, Kamei H, Miyaki T, Konishi M, Oki T: Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. J Antibiot (Tokyo). 1990, 43: 267-280. 10.7164/antibiotics.43.267.View ArticleGoogle Scholar
- Stein T: Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol. 2005, 56: 845-857. 10.1111/j.1365-2958.2005.04587.x.PubMedView ArticleGoogle Scholar
- Tagg JR, Dajani AS, Wannamaker LW: Bacteriocins of gram-positive bacteria. Bacteriol Rev. 1976, 40: 722-756.PubMedPubMed CentralGoogle Scholar
- Azuma T, Harrison GI, Demain AL: Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system. Appl Microbiol Biotechnol. 1992, 38: 173-178.PubMedView ArticleGoogle Scholar
- Fujita-Ichikawa Y, Tochikubo K: Quantitative analysis of polymyxin B released from polymyxin B-treated dormant spores of Bacillus subtilis and relationship between its permeability and inhibitory effect on outgrowth. Microbiol Immunol. 1993, 37: 935-941.PubMedView ArticleGoogle Scholar
- Arima K, Kakinuma A, Tamura G: Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commu. 1968, 31: 488-494. 10.1016/0006-291X(68)90503-2.View ArticleGoogle Scholar
- Prokof'eva NG, Kalinovskaya NI, Luk'yanov PA, Shentsova EB, Kuznetsova TA: The membranotropic activity of cyclic acyldepsipeptides from bacterium Bacillus pumilus, associated with the marine sponge Ircinia sp. Toxicon. 1999, 37: 801-813. 10.1016/S0041-0101(98)00219-0.PubMedView ArticleGoogle Scholar
- Yakimov MM, Timmis KN, Wray V, Fredrickson HL: Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl Environ Microbiol. 1995, 61: 1706-1713.PubMedPubMed CentralGoogle Scholar
- Banat IM, Makkar RS, Cameotra SS: Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol. 2000, 53: 495-508. 10.1007/s002530051648.PubMedView ArticleGoogle Scholar
- Cooper DG: Biosurfactants. Microbiol Sci. 1986, 3: 145-149.PubMedGoogle Scholar
- Desai JD, Banat IM: Microbial production of surfactants and their commercial potential. Microbiol Mol Biol Rev. 1997, 61: 47-64.PubMedPubMed CentralGoogle Scholar
- Mulligan CN: Environmental applications for biosurfactants. Environ Pollut. 2005, 133: 183-198. 10.1016/j.envpol.2004.06.009.PubMedView ArticleGoogle Scholar
- Nitschke M, Pastore GM: Biossurfactantes: Propriedades e Aplicações. Química Nova. 2002, 25 (5): 772-776.View ArticleGoogle Scholar
- Freire DMG, Araujo LV, Kronemberger FA, Nitschke M: Biosurfactants as emerging additives in food processing. Innovation in Food Engineering: New Techniques and Products. Edited by: Passos ML, Ribeiro CP. 2009, CRC Press, EUA, 685-705.View ArticleGoogle Scholar
- Korenblum E, Sebastián GV, Paiva MM, Coutinho CM, Magalhães FC, Peyton BM, Seldin L: Action of antimicrobial substances produced by different oil reservoir Bacillus strains against biofilm formation. Appl Microbiol Biotechnol. 2008, 79: 97-103. 10.1007/s00253-008-1401-x.PubMedView ArticleGoogle Scholar
- Postgate JR: The sulfate reducing bacteria. 1984, Cambridge University Press, CambridgeGoogle Scholar
- Nitschke M: Produção e caracterização de biossurfactante de Bacillus subtilis utilizando manipueira como substrato. 2004, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, Brasil, 88-Google Scholar
- Monteiro SA, Sassaki GL, de Souza LM, Meira JA, de Araújo JM, Mitchell DA, Ramos LP, Krieger N: Molecular and structural characterization of the biosurfactant produced by Pseudomonas aeruginosa DAUPE 614. Chem Phys Lipids. 2007, 147: 1-13. 10.1016/j.chemphyslip.2007.02.001.PubMedView ArticleGoogle Scholar
- Souza LM, Iacomini M, Gorin PA, Sari RS, Haddad MA, Sassaki GL: Glyco- and sphingophosphonolipids from the medusa Phyllorhiza punctata: NMR and ESI-MS/MS fingerprints. Chem Phys Lipids. 2007, 145: 85-96. 10.1016/j.chemphyslip.2006.11.001.PubMedView ArticleGoogle Scholar
- Sassaki GL, Souza LM, Serrato RV, Cipriani TR, Gorin PA, Iacomini M: Application of acetate derivatives for gas chromatography–mass spectrometry: novel approaches on carbohydrates, lipids and amino acids analysis. J Chromatogr A. 2008, 24: 215-222.View ArticleGoogle Scholar
- Das P, Mukherjee S, Sen R: Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J Appl Microbiol. 2008, 104: 1675-1684. 10.1111/j.1365-2672.2007.03701.x.PubMedView ArticleGoogle Scholar
- Song B, Springer J: Determination of interfacial tension from the profile of a pendant drop using computer-aided image processing: 2 Experimental. J Colloid Interface Sci. 1996, 184: 77-91.PubMedGoogle Scholar
- Sheppard JD, Mulligan CN: The production of surfactin by Bacillus subtilis grown on peat hydrolysate. App Microbiol Biotecnol. 1987, 27: 110-116.Google Scholar
- Van Oss CJ: Hydrophobicity of biosurfaces – Origin, quantitative determination and interaction energies. Colloids Surf B Biointerfaces. 1995, 5: 91-110. 10.1016/0927-7765(95)01217-7.View ArticleGoogle Scholar
- Van Oss CJ, Chaudhury MK, Good RJ: Interfacial Lifshitz-van der Waals and Polar Interactions in Macroscopic Systems. Chem Rev. 1988, 88: 927-941. 10.1021/cr00088a006.View ArticleGoogle Scholar
- Madhaiyan M, Poonguzhali S, Kwon SW, Sa TM: Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil. Int J Syst Evol Microbiol. 2010, 60: 2490-2495. 10.1099/ijs.0.015487-0.PubMedView ArticleGoogle Scholar
- Ryhage R, Stenhagen E: Mass spectrometry in lipid research. J Lipid Res. 1960, 1: 361-390.PubMedGoogle Scholar
- Liu XY, Yang SZ, Mu BZ: Production and characterization of a C15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121. Process Biochem. 2009, 44: 1144-1151. 10.1016/j.procbio.2009.06.014.View ArticleGoogle Scholar
- Souza LM, Cipriani TR, Serrato RV, da Costa DE, Iacomini M, Gorin PA, Sassaki GL: Analysis of flavonol glycoside isomers from leaves of Maytenus ilicifolia by offline and online high performance liquid chromatography-electrospray mass spectrometry. J Chromatogr A. 2008, 1207 (1–2): 101-109.PubMedView ArticleGoogle Scholar
- Souza LM, Müller-Santos M, Iacomini M, Gorin PA, Sassaki GL: Positive and negative tandem mass spectrometric fingerprints of lipids from the halophilic Archaea Haloarcula marismortui. J Lipid Res. 2009, 50: 1363-1373. 10.1194/jlr.M800478-JLR200.PubMedPubMed CentralView ArticleGoogle Scholar
- Hue N, Serani L, Laprévote O: Structural investigation of cyclic peptidolipids from Bacillus subtillis by high-energy tandem mass spectrometry. Rapid Commun Mass Spectrom. 2001, 15: 203-209. 10.1002/1097-0231(20010215)15:3<203::AID-RCM212>3.0.CO;2-6.PubMedView ArticleGoogle Scholar
- Savadogo A, Tapi A, Chollet M, Wathelet B, Traoré AS, Jacques P: Identification of surfactin producing strains in Soumbala and Bikalga fermented condiments using polymerase chain reaction and matrix assisted laser desorption/ionization-mass spectrometry methods. Int J Food Microbiol. 2011, 151: 299-306. 10.1016/j.ijfoodmicro.2011.09.022.PubMedView ArticleGoogle Scholar
- Negm NA, Kandile NG, Aiad IA, Mohammad MA: New eco-friendly cationic surfactants: Synthesis, characterization and applicability as corrosion inhibitors for carbon steel in 1N HCl. Colloid Surface A. 2011, 391: 224-233. 10.1016/j.colsurfa.2011.09.032.View ArticleGoogle Scholar
- Kosaric N: Biosurfactants in industry. Pure Appl Chern. 1992, 64: 1731-1737. 10.1351/pac199264111731.View ArticleGoogle Scholar
- Logan NA, Berge O, Bishop AH, Busse H-J, De Vos P, Fritze D, Heyndrickx M, Kämpfer P, Rabinovitch L, Salkinoja-Salonen MS, Seldin L, Ventosa A: Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol. 2009, 59: 2114-2121. 10.1099/ijs.0.013649-0.PubMedView ArticleGoogle Scholar
- Kalinovskaya NI, Kuznetsova TA, Ivanova EP, Romanenko LA, Voinov VG, Huth F, Laatsch H: Characterization of surfactin-like cyclic depsipeptides synthesized by Bacillus pumilus from ascidian Halocynthia aurantium. Mar Biotechnol (NY). 2002, 4: 179-188. 10.1007/s10126-001-0084-4.View ArticleGoogle Scholar
- Thaniyavarn J, Roongsawang N, Kameyama T, Haruki M, Imanaka T, Morikawa M, Kanaya S: Production and characterization of biosurfactants from Bacillus licheniformis F2.2. Biosci Biotechnol Biochem. 2003, 67: 1239-1244. 10.1271/bbb.67.1239.PubMedView ArticleGoogle Scholar
- Youssef NH, Duncan KE, McInerney MJ: Importance of 3-hydroxy fatty acid composition of lipopeptides for biosurfactant activity. Appl Environ Microbiol. 2005, 71: 7690-7695. 10.1128/AEM.71.12.7690-7695.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Hsieh FC, Li MC, Lin TC, Kao SS: Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR. Curr Microbiol. 2004, 49: 186-191.PubMedView ArticleGoogle Scholar
- Bodour AA, Drees KP, Maier RM: Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid Southwestern soils. Appl Environ Microbiol. 2003, 69: 3280-3287. 10.1128/AEM.69.6.3280-3287.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Cooper DG, Macdonald CR, Duff SJ, Kosaric N: Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Appl Environ Microbiol. 1981, 42: 408-412.PubMedPubMed CentralGoogle Scholar
- Araujo LV, Abreu F, Lins U, Santa Anna LMM, Nitschke M, Freire DMG: Rhamnolipid and surfactin inhibit Listeria monocytogenes adhesion. Food Res Int. 2011, 44: 481-488. 10.1016/j.foodres.2010.09.002.View ArticleGoogle Scholar
- Gomes MZV, NITSCHKE M: Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria. Food control. 2012, 25: 441-447. 10.1016/j.foodcont.2011.11.025.View ArticleGoogle Scholar
- Cerca N, Pier GB, Vilanova M, Oliveira R, Azeredo J: Quantitative analysis of adhesion and biofilm formation on hydrophilic and hydrophobic surfaces of clinical isolates of Staphylococcus epidermidis. Res Microbiol. 2005, 156: 506-514. 10.1016/j.resmic.2005.01.007.PubMedPubMed CentralView ArticleGoogle Scholar
- Chae MS, Schraft H, Truelstrup Hansen L, Mackereth R: Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass. Food Microbiol. 2006, 23: 250-259. 10.1016/j.fm.2005.04.004.PubMedView ArticleGoogle Scholar
- Guillemot G, Vaca-Medina G, Martin-Yken H, Vernhet A, Schmit P, Mercier-Bonin M: Shear-flow induced detachment of Saccharomyces cerevisiae from stainless steel: Influence of yeast and solid surface properties. Colloids Surf B Biointerfaces. 2006, 49: 126-135. 10.1016/j.colsurfb.2006.03.001.PubMedView ArticleGoogle Scholar
- Meylheuc T, Renault M, Bellon-Fontaine MN: Adsorption of a biosurfactant on surfaces to enhance the disinfection of surfaces contaminated with Listeria monocytogenes. Int J Food Microbiol. 2006, 109: 71-78. 10.1016/j.ijfoodmicro.2006.01.013.PubMedView ArticleGoogle Scholar
- Nitschke M, Araújo LV, Costa SG, Pires RC, Zeraik AE, Fernandes AC, Freire DM, Contiero J: Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces. Lett Appl Microbiol. 2009, 49: 241-247. 10.1111/j.1472-765X.2009.02646.x.PubMedView ArticleGoogle Scholar
- Bellon-Fontaine MN, Rault J, Van Oss CJ: Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid–base properties of microbial cells. Colloids Surf B Biointerfaces. 1996, 7: 47-53. 10.1016/0927-7765(96)01272-6.View ArticleGoogle Scholar
- Ostroumova OS, Malev VV, Ilin MG, Schagina LV: Surfactin activity depends on the membrane dipole potential. Langmuir. 2010, 26: 15092-15097. 10.1021/la102691y.PubMedView ArticleGoogle Scholar
- Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M: Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev. 2010, 34: 1037-1062.PubMedView ArticleGoogle Scholar
- Sotirova AV, Spasova DI, Galabova DN, Karpenko E, Shulga A: Rhamnolipid-biosurfactant permeabilizing effects on gram-positive and gram-negative bacterial strains. Curr Microbiol. 2008, 56: 639-644. 10.1007/s00284-008-9139-3.PubMedView ArticleGoogle Scholar
- Bharali P, Konwar BK: Production and physico-chemical characterization of a biosurfactant produced by Pseudomonas aeruginosa OBP1 isolated from petroleum sludge. Appl Biochem Biotechnol. 2011, 164: 1444-1460. 10.1007/s12010-011-9225-z.PubMedView ArticleGoogle Scholar
- Jayaraman A, Hallock PJ, Carson RM, Lee CC, Mansfeld FB, Wood TK: Inhibiting sulfate-reducing bacteria in biofilms on steel with antimicrobial peptides generated in situ. Appl Microbiol Biotechnol. 1999, 52: 267-275. 10.1007/s002530051520.PubMedView ArticleGoogle Scholar
- Zuo R, Wood TK: Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms. Appl Microbiol Biotechnol. 2004, 65: 747-753. 10.1007/s00253-004-1651-1.PubMedView ArticleGoogle Scholar
- Gana ML, Kebbouche-Gana S, Touzi A, Zorgani MA, Pauss A, Lounici H, Mameri N: Antagonistic activity of Bacillus sp. obtained from an Algerian oilfield and chemical biocide THPS against sulfate-reducing bacteria consortium inducing corrosion in the oil industry. J Ind Microbiol Biotechnol. 2011, 38: 391-404. 10.1007/s10295-010-0887-2.PubMedView ArticleGoogle Scholar
- Kebbouche-Gana S, Gana ML, Khemili S, Fazouane-Naimi F, Bouanane NA, Penninckx M, Hacene H: Isolation and characterization of halophilic Archaea able to produce biosurfactants. J Ind Microbiol Biotechnol. 2009, 36: 727-738. 10.1007/s10295-009-0545-8.PubMedView ArticleGoogle Scholar
- Wood TK, Jayaraman A, Earthman JC: Patent US6630197. Inhibition of sulfate-reducing-bacteria-mediated degradation using bacteria which secrete antimicrobials. 2003Google Scholar
- Wood TK, Jayaraman A, Earthman JC: Patent US7060486. Inhibition of sulfate-reducing-bacteria-mediated degradation using bacteria which secrete antimicrobials. 2006Google Scholar
- Roongsawang N, Washio K, Morikawa M: Diversity of nonribosomal Peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants. Int J Mol Sci. 2010, 12: 141-172. 10.3390/ijms12010141.PubMedPubMed CentralView ArticleGoogle Scholar
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