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Biosurfactant-producing bacteria were isolated from the production water of an oil field. Isolates were screened for biosurfactant production using surface tension test. The highest reduction of surface tension was achieved with a bacterial strain which was identified by 16S rRNA gene sequencing as Brevibacilis brevis HOB1. It has been investigated using different carbon and nitrogen sources. It showed that the strain was able to grow and reduce the surface tension of the broth to 29 mN/m on commercial sugar and maltose, and to 32 mN/m on glucose after 72 h of growth. The maximum amount of biosurfactant was obtained when nitrate ions were supplied as nitrogen source. Biosurfactant produced by Brevibacilis brevis HOB1 was confirmed as a lipopeptide class of biosurfactant using TLC test and mass spectra. Lipopeptide isoforms were isolated from cell-free supernatants by acid-precipitation followed by one step of chromatographic separation on solid-phase ODS C18 column. The separation was confirmed by HPLC and ESI Q-TOF MS spectroscopy. Comparing the mass data obtained and the mass numbers reported for the lipopeptide complexes from other strains, it can be concluded that the major lipopeptide product of Brevibacilis brevis HOB1 is the surfactin isoform. This lipopeptide showed strong antibacterial and antifungal activity. It is a candidate for the biocontrol of pathogens in agriculture and other industries.

A biosurfactant-producing strain was isolated from the production water of an oil-field and was identified as Bacillus subtilis HOB2 by 16S rRNA gene sequencing. The production of biosurfactant by Bacillus subtilis HOB2 has been investigated using different carbon and nitrogen sources, under thermophilic and mesophilic conditions. The strain was able to grow and to produce surfactant, reducing the surface tension of medium to 27 mN/m on sucrose, and 28 mN/m on glucose after 24 h of cultivation. The strain was able to produce the maximum amount of biosurfactant when ammonium ions were used as nitrogen source. The surface-active compound was stable during exposure to elevate temperature (100degreesC), high salinity (25% NaCl) and a wide range of pH values (5.0-11.0). The biosurfactant was capable of forming a promising emulsification index (E(24)= 68%) with kerosene. The kinetic studies revealed that biosurfactant production is a cell growth-associated process. Preliminary chemical characterization revealed that the surfactant has a lipopeptide composition similar to surfactin as confirmed by TLC and IR analysis. Properties and characteristics of the biosurfactant produced by Bacillus subtilis HOB2 suggesting potential commercial applications, such as enhanced oil recovery, bioremediation of soil and marine environments, and food industries.

Brevibacillus brevis HOB1 was isolated from the formation water of an oil field and found to produce lipopeptides. The separation of lipopeptides was successfully achieved by reversed-phase high-performance liquid chromatography (HPLC) leading to nine separated peaks. The chemical structures of these lipopeptides were studied by means of electrospray ionization mass spectrometry (ESI-MS), gas chromatography-mass spectrometry (GC/MS), HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS). As the results, all the lipopeptides had peptide parts with the same amino acid composition of Asp, Glu, Val, and Leu in the molar ratio 1:1:1:4, while the lipid part was composed of C(13)-C(15) beta-hydroxy fatty acids. As the sequence of fraction 1 was determined to be N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C, the same as surfactin, they were proposed to be surfactin isoforms. Fraction 4 (C(15) surfactin) exhibited a good surface activity of 26.8 mN/m with CMC of 9 x 10(-6 )M. Surfactin is a powerful biosurfactant possessing biological activities. As far as we know, Br. brevis is a new surfactin-producing species.

Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a beta-hydroxy fatty acid. A biosurfactant-producing strain, Bacillus subtilis HSO121, was isolated from the production water of an oil field. The strain was able to produce eight surfactin isoforms which have been isolated by acid-precipitation followed by extraction into methanol. A novel procedure for the purification of surfactin was achieved. It consists of a solid-phase extraction on C(18) gel followed by reversed-phase high performance liquid chromatography using Prep. HiQ sil C18W, column. The surfactin isoforms were eluted by linear acetonitrile gradient from 80-100%. The peaks were analyzed by TLC on silica gel, and after acid hydrolysis their amino acid compositions were determined by HPLC analysis. Eight isoforms of surfactin had nearly the same amino acid composition and appeared a single spot in TLC. According to the R(f) values with the amino acid composition, these peaks belong to the surfactin group of lipopeptides. Infrared analysis of the purified samples also revealed a pattern similar to that of surfactin. This is a very effective method for isolating and fractionating lipopeptides, of the same or different nature.