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.     

Biosurfactant production by free and alginate entrapped cells of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Production of biosurfactant by free and alginate-entrapped cells of Pseudomonas fluorescens Migula 1895-DSMZ was investigated using olive oil as the sole carbon and energy source. Biosurfactant synthesis was followed by measuring surface tension and emulsifying index E24 over 5 days at ambient temperature and at neutral pH. Diffusional limitations in alginate beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. Nevertheless, the emulsion stability was improved and fewer by-products interfered with the biosurfactant activity. A decrease in pH down to 5 in the case of immobilized cells was observed during the first 3 days, after which it returned to its initial value. The minimum values of surface tension were 30 and 35 dynes cm⁻¹ achieved after 40 and 72 h with free and immobilized cells, respectively, while the corresponding maximum E24 values were 67 and 62%, respectively. After separation by acetone precipitation, the biosurfactant showed a rhamnolipid-type in nature, and had a good foaming and emulsifying activities. The critical micellar concentration was found to be 290 mg l⁻¹. The biosurfactant also showed good stability during exposure to high temperatures (up to 120 °C for 15 min), to high salinity (10% NaCl) and to a wide range of pH (4–9).     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

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.     

Pyrene-degradation potentials of Pseudomonas species isolated from polluted tropical soils

Three Pseudomonas species isolated from oil polluted soils in Lagos, Nigeria were studied for their pyrene degradation potentials. These isolates exhibited broad substrate specificities for hydrocarbon substrates including polycyclic aromatic hydrocarbons, petroleum fractions and chlorobenzoates. All three isolates tolerated salt concentrations of more than 3%. They resisted ampicillin, cenfuroxime, but susceptible to ofloxacin and ciprofloxacin. Pseudomonas sp. strain LP1 exhibited growth rates and pyrene degradation rates of 0.018 h⁻¹ and 0.111 mg l⁻¹ h⁻¹ respectively, while P. aeruginosa strains LP5 and LP6 had corresponding values of 0.024, 0.082 and 0.017, 0.067 respectively. The overall respective percentage removal of pyrene obtained for strains LP1, LP5 and LP6 after a 30-day incubation period were 67.79, 66.61 and 47.09. Resting cell assay revealed that strain LP1 had the highest uptake rate. Strains LP1, LP5, and LP6 also used the ortho-cleavage pathway. Enzyme study confirmed activity of catechol 1,2-dioxygenase in all with values 0.6823, 0.9199, and 0.8344 μmol min⁻¹ mg⁻¹ respectively for LP1, LP3 and LP6. To the best of our knowledge, ours is the first report of pyrene-degraders from the sub-Saharan African environment.     

Production and properties of a biosurfactant synthesized byArthrobacter protophormiae — an antarctic strain

This study reports the production of biosurfactant by a psychrophilic strain ofArthrobacter protophormiae during growth on an immiscible carbon source, w-hexadecane. The biosurfactant reduces the surface tension of the medium from 68.0 mN/m to 30.60 mN/m and exhibits good emulsification activity. The strain could grow and produce biosurfactant in the presence of high NaCl concentrations (10.0 to 100.0 g/1). Although the biosurfactant was isolated by growing the organism under psychrophilic conditions (10‡C) it exhibited stable activity over a wide range of temperature (30‡C to 100‡C). It retained its surface-active properties at p^H2 to 12. The biosurfactant was effective in recovering up to 90% of residual oil from an oil saturated sandpack column, indicating its potential value in enhanced oil recovery.     

Biosurfactant production and hydrocarbon-degradation by halotolerant and thermotolerant Pseudomonas sp.

A hydrocarbon degrading and biosurfactant producing, strain DHT2, was isolated from oil-contaminated soil. The organism grew and produced biosurfactant when cultured in variety of substrates at salinities up to 6 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, alkanes and PAHs as carbon source across the wide range of temperature (30–45°C) and salinity (0–6%). Over the range evaluated, the salinity and temperature did not influence the degradation of hydrocarbon and biosurfactant productions. Isolate DHT2 was identified as Pseudomonas aeruginosa by analysis of 16S rRNA sequences (100% homology) and biochemical analysis. PCR and DNA hybridization studies revealed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by DHT2 during growth on both, water miscible and immiscible substrates, including PAH. The biosurfactants lowered the surface tension of medium from 54.9 to 30.2 dN/cm and formed a stable emulsion. The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as best substrate and toluene was the poorest. These findings further indicate that the isolate could be useful for bioremediation and bio-refining application in petroleum industry.     

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.     

Biosurfactant Production by Azotobacter chroococcum Isolated from the Marine Environment

Preliminary characterization of a biosurfactant-producing Azotobacter chroococcum isolated from marine environment showed maximum biomass and biosurfactant production at 120 and 132 h, respectively, at pH 8.0, 38°C, and 30‰ salinity utilizing a 2% carbon substrate. It grew and produced biosurfactant on crude oil, waste motor lubricant oil, and peanut oil cake. Peanut oil cake gave the highest biosurfactant production (4.6 mg/mL) under fermentation conditions. The biosurfactant product emulsified waste motor lubricant oil, crude oil, diesel, kerosene, naphthalene, anthracene, and xylene. Preliminary characterization of the biosurfactant using biochemical, Fourier transform infrared spectroscopy, and mass spectral analysis indicated that the biosurfactant was a lipopeptide with percentage lipid and protein proportion of 31.3:68.7.     

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.     

Marine Bacterium Derived Lipopeptides: Characterization and Cytotoxic Activity Against Cancer Cell Lines

A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension up to 27 mN m⁻¹. The microorganism produced 1.64 ± 0.1 g l⁻¹ of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products were found to be 90 and 40 mg l⁻¹ respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human colon cancer cell lines HCT-15 (IC₅₀ 80 μg ml⁻¹) and HT-29 (IC₅₀ 120 μg ml⁻¹).     

Laboratory production and characterization of a new biosurfactant from <Emphasis Type="Italic">Candida glabrata</Emphasis> UCP1002 cultivated in vegetable fat waste applied to the removal of hydrophobic contaminant

Biosurfactant production by Candida glabrata was studied using vegetable fat waste as substrate. A factorial design was initially carried out to investigate the effects and interactions of waste, yeast extract and glucose on the surface tension after 144 h cultivation. Maximum surface tension reduction was achieved with vegetable fat waste at 5% and yeast extract at 0.2%. The biosurfactant containing cell-free broth retained its surface-active properties after incubation at high temperatures, at a wide range of pH values and salt concentrations. Comparison between three solvent systems for surfactant recovery showed that ethyl acetate extracted both crude extracellular and intracellular biosurfactant with high product recovery. The isolated extracellular biosurfactant showed a CMC of 1% and the surface tension at that point was 24 mN m⁻¹. Preliminary chemical composition revealed the presence of carbohydrates, proteins and lipids. The application of the crude biosurfactant to a soil–water-hydrophobic contaminant system was investigated and the apparent critical micelle concentration was determined at 7% of the broth, although the best oil removal (92.6%) had been obtained with 10% of the cell-free broth. The cost of application of the biosurfactant in soils was estimated based on the cost of a commercial biosurfactant.     

Biodegradation of diesel oil and production of fatty acid esters by a newly isolated Pseudomonas citronellolis KHA

The ability of a newly isolated Pseudomonas citronellolis KHA to degrade diesel oil and to synthesize fatty acid esters has been screened in aerobic batch cultures. The microorganism was able to grow with diesel oil at initial concentrations up to 126 g/l, with optimal growth at 25 g/l. Strain KHA has produced compounds showing strong emulsifying properties (E₂₄ = 75% at the end of the exponential growth phase). The crude extract reduces the surface tension of water from 72 mN m⁻¹ down to 35 mN m⁻¹ with a corresponding minimal concentration value of 60 mg/l. GC and GC–MS analysis of crude product show that the major components are those of hexadecanoic acid propyl ester and octadecanoic acid propyl ester, which have potential for applications in cosmetics, pharmaceutical and foods industries. In addition, strain KHA represents a valuable source of compounds with surface-active properties and potential for the application in clean up of the sites contaminated with hydrocarbons.     

Annual changes of microalgae biomass in Antarctic sea ice contaminated by crude oil and diesel fuel

The influence of diesel fuel and “Arabian light” crude oil contamination were investigated on the land fast ice located in the continental shelf of Terre Adélie, Antarctica, during the austral winter 1993. Autotrophic biomass exhibited a clear seasonal pattern. In uncontaminated sea ice, chlorophyll a concentration showed two maxima, one in April (50 mg m⁻³) during sea-ice formation, and the second one in spring just before the ice thaw (20 mg m⁻³). The crude oil and diesel fuel contamination induced a negative effect on ice-microalgae biomass, which remained at a weak level throughout the ice-covered period. However, the inhibitory effect of diesel contamination was immediate while the crude oil effect occurred after the autumn phytoplankton bloom. Addition of fertilizer (Inipol EAP-22) to diesel and crude oil had a clear favourable effect on ice-microalgae. Chlorophyll a biomass exhibited the same seasonal pattern in fertilized and uncontaminated areas. Accepted: 10 November 1998     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from Renewable Resources

This study deals with production and characterization of biosurfactant from renewable resources by Pseudomonas aeruginosa. Biosurfactant production was carried out in 3L fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (11.6 mg/ml) and biosurfactant production (8.6 mg/ml) occurred with peanut oil cake at 120 and 132 h respectively. Characterization of the biosurfactant revealed that, it is a lipopeptide with chemical composition of protein (50.2%) and lipid (49.8%). The biosurfactant (1 mg/ml) was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene, comparatively the emulsification activity was higher than the activity found with Triton X-100 (1 mg/ml). Results obtained in the present study showed the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources. Emulsification activity found with the biosurfactant against different hydrocarbons showed its possible application in bioremediation of environments polluted with various hydrocarbons.     

Evaluation Antimicrobial and Antiadhesive Properties of the Biosurfactant Lunasan Produced by <Emphasis Type="Italic">Candida sphaerica</Emphasis> UCP 0995

Different groups of biosurfactants exhibit diverse properties and display a variety of physiological functions in producer microorganisms; these include enhancing the solubility of hydrophobic/water-insoluble compound, heave metal binding, bacterial pathogenesis, cell adhesion and aggregation, quorum sensing and biofilm formation. Candida sphaerica was grown in a low cost medium, consisting of distilled water supplemented with 9% refinery residue of soybean oil and 9% corn steep liquor, for 144 h at 28°C and 150 rpm. The cell-free supernatant obtained at the end of the experiments was submitted to extraction, and afterward the biosurfactant was isolated using methanol with a yield of 9 g l⁻¹. The critical micelle concentration of the biosurfactant was found to be 0.25 mg ml⁻¹ with a surface tension of 25 mN m⁻¹. Several concentrations of the biosurfactant (0.625–10 mg ml⁻¹) were used to evaluate its antimicrobial and antiadhesive activities against a variety of microorganisms. The biosurfactant showed antimicrobial activity against Streptococcus oralis (68%), Candida albicans (57%), and Staphylococcus epidermidis(57.6%) for the highest concentration tested. Furthermore, the biosurfactant at a concentration of 10 mg ml⁻¹ inhibited the adhesion between 80 and 92% of Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus sanguis12. Inhibition of adhesion with percentages near 100% occurred for the higher concentrations of biosurfactant used. Results gathered in this study point to a potential use of the biosurfactant in biomedical applications.     

Characterisation of a biosurfactant produced by a Bacillus cereus strain tolerant to cadmium and isolated from green coffee grain

In this work, a Gram-positive bacterium with bacillus-type morphology was isolated from low-quality coffee beans in a nutritive medium supplemented with 178 μM of Cd [Cd(NO₃)₂ 4H₂O]. PCR showed 99% similarity of the isolated bacteria with Bacillus cereus QD232. This bacterium produced a biosurfactant after 120 h of growth with an average production of 480 mg l⁻¹ and was able to emulsify various hydrocarbons such as diesel (60%), cyclohexane (48%), benzene (48%), isooctane (47%) and toluene (40%). The molecular weight of the biosurfactant, as determined by high-performance liquid chromatography, was 34,194 Da. The cell-free media had a surface tension of 45 mN m⁻¹ and a critical micellar concentration in the range of 0.2–2.5% (v/v), as evaluated by surface tension and conductance, respectively. The emulsifying agent maintained its properties over a pH range from 6 to 10. The composition of the biosurfactant was 53% proteins, 44.4% lipids and 2.6% carbohydrates. Only a few reports have described the production of biosurfactant from Bacillus cereus strains, and the results from this study show that the biosurfactant properties of Bacillus cereus may have potential environmental applications.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

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.     

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.