Production of water-soluble surface-active exolipids by Torulopsis apicola

Summary Several Torulopsis yeasts were screened for production of extracellular surface-active compounds. One strain, Torulopsis apicola IMET 43747, was studied in greater detail. Both on nalkanes and on carbohydrates it produced a mixture of water-soluble biosurfactants with remarkable interfacial activities and surface-tension values around 30 mN m^-1 and interfacial tension below 1 mN m^-1. Most of the biosurfactants are produced in the late exponential and in the early stationary growth phase. Production was increased by using hydrophobic compounds as the carbon source. The yields on n-alkanes were influenced by the concentrations of both the carbon source and the yeast extract. The effects of one purified biosurfactant on microbial growth on nalkanes and its antibacterial and antiphagal activities reveal new physiological aspects of biosurfactant generation by T. apicola.     

In Situ Biosurfactant Production by Bacillus Strains Injected into a Limestone Petroleum Reservoir

Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO₂ and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 ± 0.01 h⁻¹), carbon balances (107% ± 34%), biosurfactant production rates (0.02 ± 0.001 h⁻¹), and biosurfactant yields (0.015 ± 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica

Candida antarctica (sp. SY16) required avegetable oil as the carbon source to produce a biosurfactant, mannosylerythritol lipid (MEL-SY16). Biosurfactant production was 31 g l^–1 after 7 days in a batch culture and was not growth associated. In a two-stage culture, glycerol and oleic acid were used as an initial and a feeding carbon source, respectively, and 41 g l^–1 biosurfactant was produced after 8 days.     

Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant Activity

Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r² = 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r² = 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.     

A Biosurfactant-Producing Pseudomonas aeruginosa Strain

A Pseudomonas aeruginosa strain producing an extracellular surfactant (biosurfactant) was isolated. The growth of this strain, referred to as 50.3, on a mineral glycerol-containing medium produces an emulsifying activity (60%) and decreases the surface tension of the culture liquid by a factor of 2.8 (to 25 mN/m). The optimum conditions for its growth and production of biosurfactants are intense aeration, pH 7.0–8.0, and the presence of Mg²⁺. The optimum biosurfactant properties were achieved when glucose was used as the only source of carbon and energy and NH₄Cl was used as a source of nitrogen. The biosurfactant was isolated from the culture liquid by extraction and precipitation.     

Initial Stages in the Biodegradation of the Surfactant Sodium Dodecyltriethoxy Sulfate by Pseudomonas sp. Strain DES1

The biodegradation of the surfactant sodium dodecyltriethoxy sulfate by Pseudomonas sp., strain DES1 (isolated from activated sludge plant effluent) has been studied. Growth of the organism when the ³⁵S-labeled surfactant was present as the sole source of carbon and energy led to the appearance in the culture fluid of five ³⁵S-labeled organic metabolites. These have been identified as mono-, di-, and triethylene glycol monosulfates (major metabolites) and acetic acid 2-(ethoxy sulfate) and acetic acid 2-(diethoxy sulfate), authentic samples of which have been prepared and characterized. Evidence is presented that the major metabolites were produced by rupture of one or another of the three ether linkages present in the surfactant molecule, probably via the agency of a single etherase enzyme. Acetic acid 2-(ethoxy sulfate) and acetic acid 2-(diethoxy sulfate) were formed by the oxidation of the free alcohol groups of di- and triethylene glycol monosulfates, respectively, and increased in amount during the stationary phase of growth. Inorganic ³⁵S-sulfate also appeared in significant quantities in culture fluids and arose from the parent surfactant (presumably via the action of an alkylsulfatase) and not from any of the five metabolites. The appearance of sulfated organic metabolites during the exponential phase of growth and their quantitative relationship remained remarkably constant, even when additional carbon and energy sources (succinate or yeast extract) were also present in the growth media.     

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.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.     

Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry

The production of biosurfactant by Bacillus subtilis LSFM-05 was carried out using raw glycerol, obtained from a vegetable oil biodiesel plant in Brazil, as the sole carbon source. Production of the biosurfactant was carried out in a 15-L bench-top fermentor and the surfactant was obtained from the foam produced. The crude surfactant was purified by silica gel column chromatography with a yield of 230 mg of the purified biosurfactant per liter of foam. TLC, IR spectroscopy, ¹H and ¹³C NMR and Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization (ESI-FTMS) were used to characterize the purified surfactant. The isolated surfactant was identified as a surfactin lipopeptide. MS/MS data identified the amino acid sequence as GluOMe-Leu-Leu-Asp-Val-Leu-Leu and showed that the fatty acid moiety contained 14 carbons in iso, anteiso or normal configurations. The critical micelle concentration of the C₁₄/Leu₇ surfactin was 70 μM, with emulsification efficiency after 24 h (E₂₄) of 67.6% against crude oil. Raw glycerol represents an abundant and renewable carbon source and provides an opportunity for reducing the cost of biosurfactant production and may add value to biodiesel production by creating new commercial applications for this by-product.     

Uptake modes of octadecane by Pseudomonas sp. DG17 and synthesis of biosurfactant

Aims: In order to gain more insight into the uptake modes of octadecane by bacteria. Methods and Results: A strain that could utilize octadecane well was isolated from crude oil contaminated soil, and named as Pseudomonas sp. DG17 by 16S rDNA analysis. Culture growth result showed that Pseudomonas sp. DG17 grew well in the addition of 200 and 400 mg l^?1 of octadecane, which showed that physical contact between substrate and bacteria was important in the substrate biodegradation. Meanwhile, Pseudomonas sp. DG17 produced rhamnolipids biosurfactant that contains 10 congeners, thus causing the surface tension of the culture medium decline and facilitating the contact between hydrocarbon and bacteria. Scanning‐electron‐microscopy results showed that a disruption of the surface membranes in certain zones was observed in some of the cells grown in 400 mg l^?1 octadecane at 176 h compared with the cells in exponential phase at 72 h due to the production of biosurfactant‐rhamnolipid. Conclusions: These results indicated the possibility that the direct contact with insoluble octadecane droplets occurred before the contact with pseudosolubilization smaller oil droplets. Significance: This report throws more light on the uptake mechanisms of octadecane by bacteria, and proposes the possibility that role of biosurfactant is to increase the contact between hydrocarbon and bacteria by changing the cell membrane structure which needs studied in depth. Impact of Study: Results of this study are useful in the bioremediation of petroleum polluted soil.     

Isolation and characterization of hydrocarbon-degrading and biosurfactant-producing yeast strains obtained from a polluted lagoon water

Microbial surfactants are environmentally friendly products with amazing properties and spectrum of applications. It is therefore, not surprising that research has increased in recent time with the objectives of sourcing for novel surface-active compounds with dual functions in oil and pharmaceutical industries. Evaluation of hydrocarbon degrading potentials and emulsifying activities indicated that biosurfactants were produced by two newly isolated and promising yeast strains, Saccharomyces cerevisiae and Candida albicans, obtained from a polluted lagoon water. Both strains were able to grow effectively on crude oil and diesel as sole sources of carbon and energy. Growth curves on diesel were obtained to establish the relation between cell growth and biosurfactant production. The growth peak was on the 8th day while the specific growth rate ranged insignificantly (P < 0.05) between 0.46 and 0.48 day⁻¹. Interestingly, biosurfactant was detected on the 2nd day when growth was almost inexistent, with maximal production obtained at stationary/death phase of growth. The partially-purified biosurfactants exhibited antimicrobial activities by completely inhibiting the growth of clinical strains of Escherichia coli and Staphylococcus aureus at all concentrations tested. Although C. albicans appeared to be a better diesel-utilizer and biosurfactant-producer (E₂₄ = 64.2%), the potency of its surfactant was smaller than that of S. cerevisiae. These strains represent a new class of biosurfactant producers that have potential for use in a variety of biotechnological and industrial processes particularly in the pharmaceutical industry.     

Microbial Degradation of Natural Rubber Vulcanizates

An actinomycete, Nocardia sp. strain 835A, grows well on unvulcanized natural rubber and synthetic isoprene rubber, but not on other types of synthetic rubber. Not only unvulcanized but also various kinds of vulcanized natural rubber products were more or less utilized by the organism as the sole source of carbon and energy. The thin film from a latex glove was rapidly degraded, and the weight loss reached 75% after a 2-week cultivation period. Oligomers with molecular weights from 10⁴ to 10³ were accumulated during microbial growth on the latex glove. The partially purified oligomers were examined by infrared and ¹H nuclear magnetic resonance and ¹³C nuclear magnetic resonance spectroscopy, and the spectra were those expected of cis-1, 4-polyisoprene with the structure, OHC—CH₂—[—CH₂—C(—CH₃)=CH —CH₂—]_n—CH₂—C(=O)— CH₃, with average values of n of about 114 and 19 for the two oligomers.     

Nutritional requirements and growth characteristics of a biosurfactant-producingRhodococcus bacterium

The nutritional requirements and growth characteristics of a biosurfactant-producingRhodococcus bacterium isolated from Kuwaiti soil were determined. Maximum cell yields (6.6 g/l) and biosurfactant production were achieved with a medium containing 2% (v/v)n-paraffin as a carbon and energy source, 0.2% lactose broth, optimal concentrations of nitrogen (nitrate), phosphorus, iron, magnesium and sodium sources, and minimal concentrations of potassium and trace element sources. The optimal pH was 6.8 for surfactant production and optimal temperature was 37°C. The biosurfactant produced after 16 to 33 h growth in a 7 I fermenter decreased both surface tension and interfacial tension of culture broth to below 27 and 1.8 mN/m, respectively, and was effective at critical micelle dilutions of 10^–3. Data on biosurfactant biosynthesis suggest that the product is produced as a primary metabolite and, therefore, could be produced effectively under continuous fermentation conditions.A.S. Abu-Ruwaida, S. Haditirto and A. Khamis are with the Kuwait Institute for Scientific Research, Biotechnology Department, P.O. Box 24885, 13109, Safat, Kuwait. I.M. Banat is now in Londonderry, Northern Ireland but was at the Kuwait Institute for Scientific Research at the time this paper was written. A.S. Abu-Ruwaida is the corresponding author.In view of the annexation of Kuwait by Iraq in August 1990, this paper has been accepted without return to the author for attention to minor details. The Editor-in-Chief therefore assumes full responsibility for any errors or omissions.     

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.     

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.     

Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions

The lipopeptide, surfactin, is produced by Bacillus subtilis. A study has been made on large-scale production of this surfactant. A good yield was obtained from a glucose substrate fermentation by continuously removing the product by foam fractionation. The surfactin could be easily recovered from the collapsed foam by acid precipitation. The yield was also improved by the addition of either iron or manganese salts. Hydrocarbon addition to the medium, which normally increases biosurfactant production, completely inhibited surfactin production by B. subtilis.     

Continuous production of the lipopeptide biosurfactant of Bacillus licheniformis JF-2

Bacillus licheniformis JF-2 synthesizes a surfactin-like lipopeptide that is the most effective biosurfactant known. In shake-flask cultures the biosurfactant is produced by actively growing cells (mid-linear phase), but subsequently it becomes rapidly internalized by the cells as soon as the culture enters the stationary phase. This deactivation phenomenon is a major hurdle in the efficient production of the biosurfactant. We have shown that the synthesis of the JF-2 lipopeptide is strongly dependent on O₂ concentration with substantial production observed only in cultures grown under O₂-limiting conditions. In continuous cultures the biosurfactant was produced only within a narrow window of low dilution rates. At a dilution rate of 0.12 h^–1 and low dissolved O₂, the biosurfactant concentration was maintained at 33 mg/l, which is virtually the same as the maximum concentration obtained in optimized batch fermentations.     

Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater

A biosurfactant-producing strain S6 was isolated from oil-containing wastewater and identified as Pseudomonas aeruginosa based on physiological and biochemical tests together with 16S rDNA sequence analysis. Thin layer chromatography (TLC) and high-performance liquid chromatography electrospray ionization mass spectra (HPLC-ESI-MS) worked together to reveal that the strain S6 produced rhamnolipid biosurfactant. Mass spectrometry confirmed the presence of some major components in the rhamnolipid surfactant showing m/z of 675.8, 529.6, 503.3 and 475.4, which corresponded to RhaRhaC₁₀C_12:1, RhaC_12:1C₁₀, RhaC₁₀C₁₀ and RhaC₈C₁₀, respectively. The biosurfactant produced by strain S6 had the ability to decrease the surface tension of water from 72 to 33.9 mN m^?1, with the critical micelle concentration (CMC) of 50 mg L^?1. Emulsification experiment indicated that this biosurfactant effectively emulsified the crude petroleum and the measurements of surface tension demonstrated that the biosurfactant possessed stable surface activity at variable ranges of pH and salinity. The biosurfactant also exhibited good performance of phenanthrene solubilization with about 23 times higher solubility of phenanthrene in water than the control. Thus, this biosurfactant may have a potential for application in bioremediation of crude oil contamination.     

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.     

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.