Isolation and functional characterization of a biosurfactant produced by a new and promising strain of Oleomonas sagaranensis AT18

Biosurfactant-producing bacteria were isolated from mangrove sediment in southern Thailand. Isolates were screened for biosurfactant production by using the surface tension test. The highest reduction of surface tension was achieved with a bacterial strain which was identified by 16S rRNA gene sequencing as Oleomonas sagaranensis AT18. It has also been investigated using different carbon and nitrogen sources. It showed that the strain was able to grow and reduce the surface tension of the culture supernatant to 25?mN/m. In all 5.30?g of biosurfactant yield was obtained after 54?h of cultivation by using molasses and NaNO(3) as carbon and nitrogen sources, respectively. The biosurfactant recovery by chloroform:methanol extraction showed a small critical micelle concentration value (8?mg/l), thermal and pH stability with respect to surface tension reduction. It also showed emulsification activity and a high level of salt concentration. The biosurfactant obtained was confirmed as a glycolipid by using a biochemical test, FT-IR and mass spectra. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.     

Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites

Biosurfactant-producing bacteria were isolated from terrestrial and marine samples collected in areas contaminated with crude oil or its byproducts. Isolates were screened for biosurfactant/bioemulsifier production in different carbon sources (glucose, fructose, sucrose and kerosene) using the qualitative drop-collapse test. Glucose produced the highest number of positive results (17 of 185 isolates). All 17 isolates produced emulsions with kerosene and 12 exhibited high emulsion-stabilizing capacity, maintaining 50% of the original emulsion volume for 48 h. Eight of the 17 isolates reduced the growth medium surface tension below 40 mN m(-1) with 5 exhibiting this capacity in cell-free filtrates. Onset of biosurfactant production differed among the isolates, with some initiating synthesis during the exponential growth phase and others after the stationary phase was reached. Increasing temperature from 25 to 35 degrees C accelerated onset of biosurfactant production in only two isolates while pH (6.5-7.6) had no effect in any isolate tested. Isolation from petroleum contaminated sites using the screening protocol presented proved to be a rapid and effective manner to identify bacterial isolates with potential industrial applications.     

Production and characterization of a glycolipid biosurfactant from Bacillus megaterium using economically cheaper sources

Criteria selected for screening of biosurfactant production by Bacillus megaterium were hemolytic assay, bacterial cell hydrophobicity and the drop-collapse test. The data on hemolytic activity, bacterial cell adherence with crude oil and the drop-collapse test confirmed the biosurfactant-producing ability of the strain. Accordingly, the strain was cultured at different temperatures, pH values, salinity and substrate (crude oil) concentration in mineral salt medium to establish the optimum culture conditions, and it was shown that 38°C, 2.0% of substrate concentration, pH 8.0 and 30‰ of salt concentration were optimal for maximum growth and biosurfactant production. Laboratory scale biosurfactant production in a fermentor was done with crude oil and cheaper carbon sources like waste motor lubricant oil and peanut oil cake, and the highest biosurfactant production was found with peanut oil cake. Characterization of partially purified biosurfactant inferred that it was a glycolipid with emulsification potential of waste motor lubricant oil, crude oil, peanut oil, diesel, kerosene, naphthalene, anthracene and xylene.     

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.     

Characterization and antimicrobial properties of lipopeptide biosurfactants produced by Bacillus subtilis SJ301 and Bacillus vallismortis JB201

Biosurfactant producing bacteria, terrestrial Bacillus subtilis SJ301 and marine Bacillus vallismortis JB201 were isolated from sites contaminated with crude oil and its by-products. Cellular growth and biosurfactant production of the isolates were studied with different carbon sources (glucose, fructose, glycerol and petrol). Both bacterial isolates synthesized biosurfactants in the presence of glucose at late log phase and in the presence of petrol at stationary phase at 35°C. Biosurfactants obtained from both bacteria reduced the surface tension of the growth medium below 33 mN/m and exhibited this capacity in cell-free filtrates also. Raising the temperature from 25 to 35°C, accelerated onset of biosurfactant production in both the isolates, however, change in pH values from 6.5 to 7.5 had no effect. Functional and structural characterization of the crude biosurfactants was carried out by FTIR and ¹H and ¹³C NMR spectroscopy and the compounds were identified as surfactin lipopeptides. Biosurfactant produced by the terrestrial B. subtilis SJ301 showed antimicrobial activity against Escherichia coli and Shigella dysenteriae whereas the marine B. vallismortis JB201 revealed antimicrobial activity against Klebsiella pneumoniae, Salmonella typhi and Streptococcus pneumoniae.     

Isolation and characterization of a biosurfactant‐producing Fusarium sp. BS‐8 from oil contaminated soil

This study reports characterization of a biosurfactant‐producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS‐8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant‐producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum biosurfactant production using 9% (v/v) inoculum at 30°C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m^?1) with a critical micelle concentration of ≥ 1.2 mg mL^?1. During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L^?1 pure biosurfactant having significant emulsifying index (E₂₄, 70%) and oil‐displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS‐8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1065–1075, 2014     

Co-Utilization of Canola Oil and Glucose on the Production of a Surfactant by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

Candida lipolytica synthesized a surfactant in a cultivation medium supplemented with canola oil and glucose as carbon sources. Measurements of biosurfactant production and surface tension indicated that the biosurfactant was produced at 48 h of fermentation. The surface-active species is constituted by the protein–lipid–polysaccharide complex in nature. The cell-free broth was particularly influenced by the addition of salt, the pH and temperature depending on the emulsified substrate (hexadecane or a vegetable oil). After comparison between ethyl acetate and mixtures of chloroform and methanol as solvent systems for surfactant recovery, it was found that ethyl acetate was able to extract crude surfactant material with high product recovery (8.0 g/L). The isolated biosurfactant decreased the surface tension to values of 30 mN/m at the critical micelle concentration. Emulsification properties of the biosurfactant produced were compared to those of commercial emulsifiers and other microbial surfactants.     

产表面活性剂石油降解菌BS-5的特性研究

目的确定培养条件对产表面活性剂菌株BS-5的生长及降解特性的影响。方法利用紫外分光光度法、表面张力测定法和气相色谱-质谱联用仪(GC-MS),分别以菌体浓度、培养液表面张力及原油降解率为评价指标。结果菌体生长与产表面活性物质的能力及降解能力呈正相关,且确定最优碳源为0.5%的可溶性淀粉,氮源为1.0%的玉米浆,降解时间为6d,在此条件下原油降解率最高达42.3%。结论菌株培养条件的优化提高了菌株自身生长、产表面活性剂及降解原油的能力,为石油污染修复提供理论依据。     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

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.     

Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by <Emphasis Type="Italic">Pseudomonas nitroreducens</Emphasis> isolated from soil

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from petroleum-contaminated soil was investigated. The effects of carbon, nitrogen and carbon to nitrogen ratio on biosurfactant production were examined using mineral salts medium as the growth medium. The tenso-active properties (surface activity and critical micelle concentrations of the produced biosurfactant were also evaluated. The best carbon source, nitrogen source were glucose and sodium nitrate giving rhamnolipid yields of 5.28 and 4.38 g l⁻¹, respectively. The maximum rhamnolipid production of 5.46 g l⁻¹ was at C/N (glucose/sodium nitrate) of 22. The rhamnolipid biosurfactant reduced the surface tension of water from 72 to ~37 mN/m. It also has critical micelle concentration of ~28 mg l⁻¹. Thus, the results presented in our reports show that the produced rhamnolipid can find wide applications in various bioremediation activities such as enhanced oil recovery and petroleum degradation.     

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.     

A Comparative Study on Biosurfactant Activity of Crude Oil–Degrading Bacteria and Its Correlation to Total Petroleum Hydrocarbon Degradation

In this study, 11 bacteria isolated from Tapis crude oil–contaminated sites were identified by using biochemical tests and 16S rDNA gene sequencing. Their abilities to biodegrade Tapis crude oil was determined by gas chromatography before they were further screened for biosurfactant activity by employing qualitative (blood agar hemolysis, microplate assay, drop-collapse test), semiquantitative (emulsification formation), and quantitative (surface tension measurement) methods. Four isolates, namely, Acinetobacter baumanii UKMP-12T, Pseudomonas aeruginosa UKMP-14T, Rhodococcus sp. UKMP-5T, and Rhodococcus sp. UKMP-7T, exhibited high percentages in total petroleum hydrocarbon (TPH) degradation. A strong correlation between the emulsification index (E ₂₄) and surface tension measurement (r _s = +.866) as shown by Spearman rank correlation analysis suggested that these two methods were more reliable to predict biosurfactant activity. The TPH removal was also positively correlated to the ability of bacterial isolates to reduce the surface tension of growth medium, as revealed by Pearson correlation test (r_p = +.886). In conclusion, not all the biosurfactant detection protocols employed were effective. Nevertheless, the measurement of surface tension and E ₂₄ determination provided a rather rapid, easy, reproducible, and accurate result in identifying bacteria with biosurfactant-producing ability.     

Production and partial characterization of biosurfactant produced by crude oil degrading bacteria

Production of biosurfactant by crude oil degrading bacteria for use in microbial enhanced oil recovery was investigated. Crude oil utilizing bacteria were isolated from soil by enrichment method on oil agar at 30 °C for 5 days. The isolates were identified and screened for biosurfactant production using blood haemolysis and emulsification tests. IR and GC–MS analyses were carried out to detect the type of biosurfactant. The biosurfactant was purified and its stability at various pH, temperature and salinity levels was studied. The organisms were identified as: Achromobacter xylosoxidans subspecies xylosoxidans, Bacillus licheniformis, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Sphingomonas paucimobilis and Micrococcus kristinae. Emulsification test (E₂₄) revealed that Serratia marcescens had the highest emulsification index of 87%. GC–MS indicated the biosurfactants as lipopeptides. The biosurfactant can be used in EOR under various environmental conditions.     

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.     

Production and characterization of lipopeptide biosurfactant by a sponge-associated marine actinomycetes <Emphasis Type="Italic">Nocardiopsis alba</Emphasis> MSA10

A sponge-associated marine actinomycetes Nocardiopsis alba MSA10 was screened and evaluated for the production of biosurfactant. Biosurfactant production was confirmed by conventional screening methods including hemolytic activity, drop collapsing test, oil displacement method, lipase production and emulsification index. The active compound was extracted with three solvents including ethyl acetate, diethyl ether and dichloromethane. The diethyl ether extract was fractionated by TLC and semi-preparative HPLC to isolate the pure compound. In TLC, a single discrete spot was obtained with the R _f 0.60 and it was extrapolated as valine. Based on the chemical characterization, the active compound was partially confirmed as lipopeptide. The optimum production was attained at pH 7, temperature 30°C, and 1% salinity with glucose and peptone supplementation as carbon and nitrogen sources, respectively. Considering the biosurfactant production potential of N. alba, the strain could be developed for large-scale production of lipopeptide biosurfactant.     

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.