Biosurfactant synthesis by Pseudomonas aeruginosa LBI isolated from a hydrocarbon-contaminated site

Aims: Pseudomonas aeruginosa LBI (Industrial Biotechnology Laboratory) was isolated from hydrocarbon-contaminated soil as a potential producer of biosurfactant and evaluated for hydrocarbon biodegradation. The emulsifying power and stability of the product was assessed in the laboratory, simulating water contamination with benzene, toluene, kerosene, diesel oil and crude oil at various concentrations. Methods and Results: Bacteria were grown at 30°C and shaken at 200 rpm for 168 h, with three repetitions. Surface tension, pH and biosurfactant stability were observed in the cell-free broth after 168 h of incubation. The strain was able to produce biosurfactant and grow in all the carbon sources under study, except benzene and toluene. When cultivated in 30% (w/v) diesel oil, the strain produced the highest quantities (9·9 g l⁻¹) of biosurfactant. The biosurfactant was capable of emulsifying all the hydrocarbons tested. Conclusion: The results from the present study demonstrate that Ps. aeruginosa LBI can grow in diesel oil, kerosene, crude oil and oil sludge and the biosurfactant produced has potential applications in the bioremediation of hydrocarbon-contaminated sites. Significance and Impact of the Study: Pseudomonas aeruginosa LBI or the biosurfactant it produces can be used in the bioremediation of environmental pollution induced by industrial discharge or accidental hydrocarbon spills.     

一株石油烃降解菌的细胞疏水性及其乳化性质

【目的】从新疆油田石油污染土壤中分离到一株在25 °C条件下利用烃类产生生物表面活性剂的菌株红球菌(Rhodococcus sp.) HL-6, 对其菌体细胞疏水性及所产表面活性剂进行研究。【方法】通过细胞粘附性、表面张力及乳化活性测定对菌株所产表面活性剂进行性质研究。【结果】菌株HL-6在亲水性和疏水性基质中均能产生生物表面活性剂, 在疏水性基质中可以将培养液表面张力由初始的62.487 mN/m降到30.667 mN/m, 培养液在pH 6?9及NaCl浓度1%?5%范围内乳化效果良好, 在4 °C到55 °C范围内乳化效果均为100%, 菌株对柴油的耐受能力很高, 在30%柴油浓度下依然生长良好并且有44%的乳化活性。【结论】HL-6菌株的细胞表面具有很强的疏水性, 这有助于菌体细胞对烃类的摄取。该菌株能够利用烃类基质生产生物表面活性剂, 可以明显降低培养液表面张力并且对石油烃具有良好的乳化作用。说明菌株HL-6能够适应海洋滩涂石油污染的环境, 并可用于严重石油污染区域的生物修复。     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SNP0614 and its Effect on Biodegradation of Petroleum

An efficient biosurfactant-producing strain was isolated and cultured from Dagang oil field (China) using crude oil as sole source of carbon. Based on partial sequenced 16S rDNA analysis, the isolated strain was identified as Pseudomonas aeruginosa SNP0614. The bacterium P. aeruginosa SNP0614 produced a type of biosurfactant with excessive foam-forming properties. After microbial cultivation at 37°C and 150 rpm for 12 h, the produced biosurfactant was found to reduce the surface tension to 25.4 mN/m with critical micelle concentration (CMC) of 45.0 mg/L. After 20 days of incubation, the biosurfactant exhibited 90% emulsification activity (E₂₄) on crude oil. FTIR spectroscopy of extracted biosurfactant indicated the biosurfactant as lipopeptide. The significant synergistic effect between P. aeruginosa SNP0614 and the mixed oildegrading bacteria resulted in increasing n-alkanes degradation rate by 30%. The strain P. aeruginosa SNP0614 represented as a promising biosurfactant producer and could be applied in a variety of biotechnological and industrial processes, particularly in microbial enhanced oil recovery and the bioremediation of oil pollution.     

Biosurfactant production by Mucor circinelloides: Environmental applications and surface‐active properties

Biosurfactants are structurally a diverse group of surface‐active molecules widely used for various purposes in industry. In this study, among 120 fungal isolates, M‐06 was selected as a superior biosurfactant producer, based on different standard methods, and was identified as Mucor circinelloides on the basis of its nucleotide sequence of the internal transcribed spacer (ITS) gene. M. circinelloides reduced the surface tension to 26 mN/m and its EI₂₄ index was determined to be 66.6%. The produced biosurfactant exhibited a high degree of stability at a high temperature (121°C), salinity (40 g/L), and acidic pH (2–8). The fermentation broth's ability to recover oil from contaminated sand was 2 and 1.8 times higher than those of water and Tween 80, respectively. The ability of biosurfactant to emulsify crude oil in the sea and fresh water was 64.9 and 48% respectively. This strain could remove 87.6% of crude oil in the Minimal Salt Medium (MSM) crude oil as the sole carbon source. The results from a primary chemical characterization of crude biosurfactant suggest that it is of a glycolipid nature. The strain and its biosurfactant could be used as a potent candidate in bioremediation of oil‐contaminated water, soil, and for oil recovery processes.     

Crude biosurfactant from thermophilic Alcaligenes faecalis: Feasibility in petro-spill bioremediation

The thermophilic bacterium Alcaligenes faecalis isolated from the crude oil contaminated soil of Upper Assam, India. The isolated bacterium was first screened for the ability to produce biosurfactant. The strain growing at 42 °C could produce higher amount of biosurfactant in medium supplemented with 2% (v/v) diesel as sole source of carbon and energy. Biochemical characterizations including FT-IR and MS studies suggested the biosurfactant to be glycolipid. Tensiometric studies revealed that the biosurfactant produced by the bacterial strain could decrease the surface tension (??) at air-water interface from 71.6 to 32.3 mNm⁻¹ after 96 h of growth on hydrocarbon and possessed a low critical micelle concentration (CMC) value of approximately 38 mgl⁻¹, indicating high surface activity. The culture supernatant containing the biosurfactant was found to be functionally stable at varying pH (2-12), temperature (100 and 121 °C) and salinity (1-6% NaCl, w/v) conditions. Both the culture broth and the cell free supernatant exhibited high emulsifying activity against the different hydrocarbons and the crude oil components. The increase in cell surface hydrophobicity and glycolipid production by the strain suggested the existence of biosurfactant enhanced interfacial uptake of the hydrocarbons. Moreover, the partially purified biosurfactant exhibited antimicrobial activity by inhibiting the growth of several bacterial and fungal species. The strain represented a new class of biosurfactant producers and could be a potential candidate for the production of glycolipid biosurfactant which could be useful in a variety of biotechnological and industrial processes, particularly in the oil industry.     

Characterization and properties of biosurfactants produced by a newly isolated strain <Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> DCS1 and their applications in enhancing solubility of hydrocarbon

Six biosurfactant-producing bacteria were isolated from hydrocarbon contaminated soils in Sfax, Tunisia. Isolates were screened for biosurfactant production by different conventional methods including hemolytic activity, surface tension reduction, drop-collapsing and oil displacement tests. All these screening tests show that all the isolates behave differently. Among the isolated bacteria, DCS1 strain was selected for further studies based on its highest activities and it was identified as Bacillus methylotrophicus DCS1. This strain was found to be a potent producer of biosurfactant when cultivated in mineral-salts medium supplemented with diesel oil (2 %, v/v) as a sole carbon source. Physicochemical properties and stability of biosurfactants synthesized by B. methylotrophicus DCS1 were investigated. The produced biosurfactants DCS1, from Landy medium, possess high surface activity that could lower the surface tension of water to a value of 31 from 72 mN m^?1 and have a critical micelle concentration (CMC) of 100 mg L^?1. Compared with SDS and Tween 80, biosurfactants showed excellent emulsification activities against different hydrocarbon substrates and high solubilization efficiency towards diesel oil. Biosurfactants DCS1 showed good stability in a wide range of temperature, pH and salinity. These results suggested that biosurfactants produced by B. methylotrophicus DCS1 could be an alternative to chemically synthesized surfactants for use in bioremediation processes to enhance the solubility of hydrophobic compounds.     

Inoculation of 1-aminocyclopropane-1-carboxylate deaminase–producing bacteria along with biosurfactant application enhances the phytoremediation efficiency of Medicago sativa in hydrocarbon-contaminated soils

Phytoremediation efficiency of Alfa alfa (Medicago sativa) was evaluated in hydrocarbon-contaminated soil with the combined application of 1-aminocyclopropane-1-carboxylate (ACC) deaminase–producing Bacillus sp. PVMX4 and an isolated biosurfactant from this strain. Results on the plant growth–promoting (PGP) traits of Bacillus sp. PVMX4 revealed that phosphate (P) solubilization, indole-3-acetic acid (IAA) production, and ACC deaminase activity were not affected by low-concentration hydrocarbon amendment in the form of crude oil. Bacillus sp. PVMX4 was able to utilize crude oil as a sole carbon source in mineral salt medium (MSM), and this strain synthesized significant quantities of biosurfactant in growth medium quantified by an emulsification index of 69.2 EI_24% and surface tension reduction of 26.2 mN/m at the end of the experimental period. Biosurfactant, when partially purified and characterized by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR), revealed it to be a lipopeptide-type biosurfactant. Pilot-scale phytoremediation studies conducted under growth chamber conditions in hydrocarbon-contaminated soil using Medicago sativa along with combined application of ACC deaminase–containing bacteria and biosurfactant recorded 76.4% hydrocarbon degradation.     

Isolation and characterization of a biosurfactant-producing heavy metal resistant <Emphasis Type="Italic">Rahnella</Emphasis> sp. RM isolated from chromium-contaminated soil

Objective of the study was to isolate heavy metal resistant bacteria from chromium-contaminated subsurface soil and investigate biosurfactant production and heavy metal bioremediation. Based on 16S rRNA gene sequence and phylogenetic analysis, the isolate was identified as Rahnella sp. RM. The biosurfactant production by heavy metal resistant Rahnella sp. RM was optimized using Box- Behnken design (BBD). The maximum emulsification activity was obtained 66% at 6% soybean meal in pH 7.0 and 33.5°C. The biosurfactant was characterized using Field emission scanning electron microscopy (FE-SEM), Fourier transform infrared spectroscopy (FT-IR) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). The highest metal removal rates using the biosurfactant were found 74.3, 72.5, and 70.1%, respectively, at the 100 mg/L amended flasks at 48 h. This study indicated the biosurfactant from heavy metal resistant Rahnella sp. RM could be used as a potential tool to remediate the metals in contaminated environments.     

Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa

A gamma ray-induced mutant of Pseudomonas aeruginosa strain S8, capable of hyperproduction of biosurfactant from hydrocarbons, was isolated and named as EBN-8. The mutant showed 3–4 times more hydrocarbon emulsification/conversion as compared to the parent when grown on Khaskheli crude oil in minimal medium. Enhanced biosurfactant production and hydrocarbon utilization by the mutant was also observed during growth on heptadecane in minimal medium as indicated by emulsion index and surface tension of cell-free culture broth. Using heptadecane as carbon and energy source, time course for the growth (cfu ml^-1) and biosurfactant production were compared for both parent and mutant. These studies were carried out for 24 d at 30 ± 2°C and for 20 d at 37°C. Growth of EBN-8 was much faster compared to the parent as well as being 2–3 times more hyperproductive.     

Optimization,production and characterization of glycolipid biosurfactant from the marine actinobacterium,Streptomyces sp. MAB36

A potential glycolipid biosurfactant producer Streptomyces sp. MAB36 was isolated from marine sediment samples. Medium composition and culture conditions for the glycolipid biosurfactant production by Streptomyces sp. MAB36 were optimized, using two statistical methods: Plackett–Burman design was applied to find out the key ingredients and conditions for the best yield of glycolipid biosurfactant production and central composite design was used to optimize the concentration of the four significant variables, starch, casein, crude oil and incubation time. Fructose and yeast extract were the best carbon and nitrogen sources for the production of the glycolipid biosurfactant. Biochemical characterizations including FTIR and MS studies suggested the glycolipid nature of the biosurfactant. The isolated glycolipid biosurfactant reduced the surface tension of water from 73.2 to 32.4 mN/m. The purified glycolipid biosurfactant showed critical micelle concentrations of 36 mg/l. The glycolipid biosurfactant was effective at very low concentrations over a wide range of temperature, pH, and NaCl concentration. The purified glycolipid biosurfactant showed strong antimicrobial activity. Thus, the strain Streptomyces sp. MAB36 has proved to be a potential source of glycolipid biosurfactant that could be used for the bioremediation processes in the marine environment.     

Surfactant activity of a naphthalene degrading Bacillus pumilus strain isolated from oil sludge

We studied the growth, biosurfactant activities and petroleum hydrocarbon compounds utilisation of strain 28-11 isolated from a solid waste oil. The isolate was identified as Bacillus pumilus. It grew well in the presence of 0.1% (w/v) of crude oil and naphthalene under aerobic conditions and utilised these substances as carbon and energy source. The capacity of strain 28-11 to emulsify crude oil and its ability to remove hydrocarbons looks promising for its application in environmental technologies.     

Utilization of palm oil decanter cake as a novel substrate for biosurfactant production from a new and promising strain of Ochrobactrum anthropi 2/3

A biosurfactant-producing bacterium, isolate 2/3, was isolated from mangrove sediment in the south of Thailand. It was evaluated as a potential biosurfactant producer. The highest biosurfactant production (4.52 g/l) was obtained when the cells were grown on a minimal salt medium containing 25 % (v/v) palm oil decanter cake and 1 % (w/v) commercial monosodium glutamate as carbon and nitrogen sources, respectively. After microbial cultivation at 30 °C in an optimized medium for 96 h, the biosurfactant produced was found to reduce the surface tension of pure water to 25.0 mN/m with critical micelle concentrations of 8.0 mg/l. The stability of the biosurfactant at different salinities, pH and temperature and also its emulsifying activity was investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pH and salt concentrations. The biosurfactant obtained was confirmed as a glycolipid type biosurfactant by using a biochemical test, fourier-transform infrared spectroscopy, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance polyaromatic hydrocarbons solubility.     

Bioremediation of multi-metal contaminated soil using biosurfactant — a novel approach

An unconventional nutrient medium, distillery spent wash (1:3) diluted) was used to produce di-rhamnolipid biosurfactant by Pseudomonas aeruginosa strain BS2. This research further assessed the potential of the biosurfactant as a washing agent for metal removal from multimetal contaminated soil (Cr-940 ppm; Pb-900 ppm; Cd-430 ppm; Ni-880 ppm; Cu-480 ppm). Out of the treatments of contaminated soil with tap water and rhamnolipid biosurfactant, the latter was found to be potent in mobilization of metal and decontamination of contaminated soil. Within 36 hours of leaching study, di-rhamnolipid as compared to tap water facilitated 13 folds higher removal of Cr from the heavy metal spiked soil whereas removal of Pb and Cu was 9–10 and 14 folds higher respectively. Leaching of Cd and Ni was 25 folds higher from the spiked soil. This shows that leaching behavior of biosurfactant was different for different metals. The use of wastewater for production of biosurfactant and its efficient use in metal removal make it a strong applicant for bioremediation.     

Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant

The present investigation dealt with the use of previously isolated and studied gamma-ray mutant strain Pseudomonas aeruginosa EBN-8 for the production of biosurfactant by using different hydrocarbon substrates viz. n-hexadecane, paraffin oil and kerosene oil, provided in minimal medium, as the sole carbon and energy sources. The batch experiments were conducted in 250 mL Erlenmeyer flasks, containing 50 mL minimal salt media supplemented with 1% (w/v) hydrocarbon substrate, inoculated by EBN-8 and incubated at 37 degrees C and 100 rpm in an orbital shaker. The sampling was done on 24 h basis for 10 d. The surface tension of cell-free culture broth decreased from 53 to 29 mN/m after 3 and 4 d of incubation when the carbon sources were paraffin oil and n-hexadecane, respectively. The largest reduction in interfacial tension from 26 to 0.4 mN/m was observed with n-hexadecane, while critical micelle dilution was obtained as 50 x CMC for paraffin oil as carbon source. When grown on n-hexadecane and paraffin oil, the EBN-8 mutant strain gave 4.1 and 6.3 g of the rhamnolipids/L, respectively. These surface-active substances subsequently allowed the hydrocarbon substrates to disperse readily as emulsion in aqueous phase.     

Serratia sp. SVGG16: a promising biosurfactant producer isolated from tropical soil during growth with ethanol-blended gasoline

The strain SVGG16 isolated from tropical soil in Brazil and identified as Serratia sp. was selected as a promising biosurfactant producer using atypic growth conditions including ethanol-blended gasoline as the sole carbon source. Important parameters for biosurfactant production were selected through experimental design. Results demonstrated that this strain was able to reduce surface tension of the medium to 34 mN m⁻¹, showing potential to be used in bioremediation processes.     

Role of Sophorolipid Biosurfactant in Degradation of Diesel Oil by Candida tropicalis

The yeast Candida tropicalis, isolated from petroleum-contaminated soil in India, was found to be the potent producer of biosurfactant in mineral salt media containing diesel oil as the carbon source and found to be an efficient degrader of diesel oil (98%) over a period of 10 days. The crude biosurfactant decreased the surface tension of cell-free broth, 78 to 30 mN/m, with a large oil displacement area and highly positive drop collapse test. The crude biosurfactant was purified using silica gel column chromatography followed by dialysis. With the use of Fourier transform infrared (FT-IR) spectroscopy, in combination with gas chromatography–mass spectrometry (GC-MS) analysis, chemical structures of the purified biosurfactant was identified as sophorolipid species. Involvement of biosurfactant in physiological mechanism of diesel adsorption on yeast cell surface was characterized based on zeta potential. When diesel oil was emulsified with biosurfactant, the surface charge of the diesel was modified, resulting in more adsorption of diesel on yeast cell surface. Biosurfactant production by yeast species was monitored using scanning electron microscopy (SEM) analysis and found that yeast species could form thick mat of mucilaginous biosurfactant that could interconnect the individual cells. Uptake of diesel oil by C. tropicalis was elucidated through transmission electron microscopy (TEM) analysis. Interestingly, it was observed that internalization of diesel oil droplet was taking place, suggesting a mechanism similar in appearance to active pinocytosis.     

Hydrocarbon Degradation and Biosurfactant Production by an Acenaphthene-degrading Pseudomonas Species

An acenaphthene-degrading bacterium putatively identified as Pseudomonas sp. strain KR3 and isolated from diesel-contaminated soil in Lagos, Nigeria was investigated for its degradative and biosurfactant production potentials on crude oil. Physicochemical analysis of the sampling site indicates gross pollution of the soil with high hydrocarbon content (2100 mg/kg) and detection of various heavy metals. The isolate grew luxuriantly on crude oil, engine oil and acenaphthene. It was resistant to septrin, amoxicillin and augmentin but was susceptible to pefloxacin, streptomycin and gentamycin. It was also resistant to elevated concentration of heavy metals such as 1–15 mM lead, nickel and molybdenum. On acenaphthene, the isolate exhibited specific growth rate and doubling time of 0.098 day^?1 and 3.06 days, respectively. It degraded 62.44% (31.2 mg/l) and 91.78% (45.89 mg/l) of 50 mg/l acenaphthene within 12 and 21 days. On crude oil, the specific growth rate and doubling time were 0.375 day^?1 and 1.85 days with corresponding percentage degradation of 33.01% (903.99 mg/l) and 87.79% (2403.71 mg/l) of crude oil (2738.16 mg/l) within 9 and 18 days. Gas chromatographic analysis of residual crude oil at the end of 18 days incubation showed significant reductions in the aliphatic, alicyclic and aromatic fractions with complete disappearance of benzene, propylbenzene, pristane, phytane, and nC₁₈-octadecane fractions of the crude oil. The isolate produced growth-associated biosurfactant on crude oil with the highest emulsification index (E₂₄) value of 72% ± 0.23 on Day 10 of incubation. The partially purified biosurfactant showed zero tolerance for salinity and had its optimal activity at 27°C and pH 2.0.     

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

Aims: To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria. Methods and Results: Eight Tunisian hydrocarbonoclastic oilfields bacteria have been isolated and selected for further characterization studies. Phylogenetic analysis revealed that three thermophilic strains belonged to the genera Geobacillus, Bacillus and Brevibacillus, and that five mesophilic strains belonged to the genera Pseudomonas, Lysinibacillus, Achromobacter and Halomonas. The bacterial strains were cultivated on crude oil as sole carbon and energy sources, in the presence of different NaCl concentrations (1, 5 and 10%, w/v), and at 37 or 55°C. The hydrocarbon biodegradation potential of each strain was quantified by GC–MS. Strain C450R, phylogenetically related to the species Pseudomonas aeruginosa, showed the maximum crude oil degradation potentiality. During the growth of strain C450R on crude oil (2%, v/v), the emulsifying activity (E24) and glycoside content increased and reached values of 77 and 1·33 g l^?1, respectively. In addition, the surface tension (ST) decreased from 68 to 35·1 mN m^?1, suggesting the production of a rhamnolipid biosurfactant. Crude biosurfactant had been partially purified and characterized. It showed interest stability against temperature and salinity increasing and important emulsifying activity against oils and hydrocarbons. Conclusions: The results of this study showed the presence of diverse aerobic bacteria in Tunisian oilfields including mesophilic, thermophilic and halotolerant strains with interesting aliphatic hydrocarbon degradation potentiality, mainly for the most biosurfactant produced strains. Significance and Impact of the Study: It may be suggested that the bacterial isolates are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon‐contaminated sites.     

Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions

Aims:  To isolate and characterize an efficient hydrocarbon-degrading bacterium under hypersaline conditions, from a Tunisian off-shore oil field.
Methods and Results:  Production water collected from 'Sercina' petroleum reservoir, located near the Kerkennah island, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated after enrichment on crude oil, in the presence of 100 g l⁻¹ NaCl and at 37°C. This strain was aerobic, Gram-negative, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that it was related to members of the Halomonas genus. The degradation of several compounds present in crude oil was confirmed by GC–MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. Strain C2SS100 was able to degrade hexadecane (C₁₆). During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant by strain C2SS100.
Conclusions:  A halotolerant bacterial strain Halomonas sp. C2SS100 was isolated from production water of an oil field, after enrichment on crude oil. This strain is able to degrade hydrocarbons efficiently. The mode of hydrocarbon uptake is realized by the production of a biosurfactant which enhances the solubility of hydrocarbons and renders them more accessible for biodegradation.
Significance and Impact of the Study:  The biodegradation potential of the Halomonas sp. strain C2SS100 gives it an advantage for possibly application on bioremediation of water, hydrocarbon-contaminated sites under high-salinity level.     

A halotolerant and thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. degrades hydrocarbons and produces tensio-active emulsifying agent

A Bacillus sp. strain DHT, isolated from oil-contaminated soil, grew and produced biosurfactant when cultured in variety of substrate at salinities of up to 100 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, various pure alkanes and PAHs as a sole carbon and energy source across a wide range of temperature and salinity. Over the range evaluated, the degradation of hydrocarbon and biosurfactant production was not influenced by salinity (0–10% wv⁻¹) and temperature (30–45°C). The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as the best substrate and toluene as the poorest. From 16S rDNA analysis, strain DHT was related to Bacillus licheniformis.