Biosurfactant production and use in oil tank clean-up

A proprietary bacterial strain (Pet 1006) produced biosurfactants when grown on both glucose and an immiscible hydrocarbon as carbon sources. Pilot-plant-scale (1500 I) production gave, on repeated batch runs, 2 tonnes of culture broth containing active biosurfactant. The product was used as a substitute for chemical surfactants in a clean-up demonstration test carried out by Cargo Fleet Chemical Company Ltd. (UK) on an oil storage tank belonging to Kuwait Oil Company, Kuwait. The clean-up was successful in removing the sludge from the tank bottom, and it also allowed the recovery of more than 90% of the hydrocarbon trapped in the sludge. The recovered hydrocarbon had excellent properties and could be sold after being blended with fresh crude.I.M. Banat is at 5, Upper Galliagh Road, Londonderry, Northern Ireland BT48 8LW, UK but was at the Kuwait Institute for Scientific Research at the time this paper was written. The remaining authors are with the Kuwait Institute for Scientific Research, Biotechnology Department, P.O. Box 24885, 13109, Safat, Kuwait. I.M. Banat 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 and for approval of certain editorial changes that have been made. The Editor-in-Chief therefore assumes full responsibility for any errors or omissions.     

Isolation of biosurfactant-producing bacteria,product characterization,and evaluation

A gram-positive, nonfermentative, rod-shaped bacterium designated ST-5, identified as Rhodococcus, was isolated from Kuwait soil. Grown on hydrocarbon, such as kerosene and n-paraffin, the bacterium produced surface-active compounds (biosurfactants). Measurements of surface tension, critical micelle dilution and emulsifying activity indicated that the biosurfactant is produced as a primary metabolite. The ST-5 culture surface-active component is mainly glycolipid in nature. Whole-culture broth dropped surface tension to values below 27 mN/m and was stable during exposure to high salinity (10% NaCl), elevated temperatures (120°C for 15 min) and a wide range of pH values. The culture broth was effective in recovering up to 86% of the residual oil from oil-saturated sand packs, indicating potential value in enhanced oil-recovery processes.     

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.     

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.     

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     

Isolation and characterization of a biosurfactant producing strain, <Emphasis Type="Italic">Brevibacilis brevis</Emphasis> HOB1

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

Enhancement of stability of biosurfactant produced by <Emphasis Type="Italic">Candida lipolytica</Emphasis> using industrial residue as substrate

This work describes experimental results carried out on the fermentation of Candida lipolytica, which produced a new biosurfactant when grown on a vegetable oil refinery residue as substrate. The cell-free culture broth containing the biosurfactant formed stable emulsions with hydrophobic natural compounds. Emulsification properties of the biosurfactant were not affected by salinity; however, treatment at a higher temperature decreased the emulsification activity, indicating applications in oil recovery. The isolated biosurfactant corresponds to a yield of 4.5 g/l, and the surface tension of water was reduced from 71 to 32 mN/m. Preliminary chemical characterizations showed that the biosurfactant consisted of protein (50%), lipid (20%), and carbohydrate (8%).     

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.     

Isolation of auxotrophic mutants and development of conjugation system in KISR methylotrophs

Isolation of auxotrophic mutants and conjugal transfer of plasmids are successfully demonstrated in four obligate methylotrophic strains isolated at Kuwait Institute for Scientific Research (KISR)Out of 11 auxotrophic, mutants, three required methionine and two leucine. Complete transfer of plasmid R68.45 from Pseudomonasto KISR methylotrophs and vice versa have been achieved with a low transfer rate.     

Experimental design for the production of tensio-active agent by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

The strategy of optimization using sequential factorial design was employed to enhance the tensio-active emulsifying agent produced by Candida lipolytica using soybean oil refinery residue as substrate. A full factorial design was used to evaluate the impact of three fermentation factors—amounts of refinery residue, glutamic acid and yeast extract. This allowed exclusion of the yeast extract. Full factorials designs were then sequentially used to optimize the levels of the residue and glutamic acid. The surface tension value was finally reduced to 25.29 mN/m. The maximum emulsifier activity using different substrates was within 40 h of cultivation. The surface tension of the cell-free broth containing the biosurfactant remained very stable during exposure to a wide range of pH (2–12), temperatures (0–120°C) and salinity (2–10% NaCl). The combination of an industrial waste and a cheap substrate therefore seems to be very promising for the low-cost production of potent biosurfactant.     

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.     

An efficient biosurfactant-producing and crude-oil emulsifying bacterium Bacillus methylotrophicus USTBa isolated from petroleum reservoir

An efficient biosurfactant-producing bacterium was isolated and cultured from petroleum reservoir in northeast China. Isolate was screened for biosurfactant production using haemolytic assay, Cetyl Trimethyl Ammonium Bromide agar plate assay (CTAB) and the qualitative oil-displacement test. Based on partial sequenced 16S rDNA analysis of isolate, USTBa, identified as Bacillus methylotrophicus with 100% identity. This bacterium was able to produce a type of biosurfactant with excessive foam-forming properties. The maximum biosurfactant production was obtained when the cells were grown on minimal salt medium containing 2% (v/v) crude-oil as the sole source of carbon at 35 °C and 180 rpm after 192 h. This strain had a high emulsification activity and biosurfactant production of 78% and 1.8 g/L respectively. The cell free broth containing biosurfactant could reduce the surface tension to 28 mN/m. Fourier transform infrared (FT-IR) spectrum of extracted biosurfactant indicates the presence of carboxyl, hydroxyl and methoxyl functional groups. Elemental analysis of the biosurfactant by Energy dispersive X-ray spectroscopy (EDS) reveals that the biosurfactant was anionic in nature. The strain USTBa represented as a potent biosurfactant-producer and could be useful in variety of biotechnological and industrial processes, particularly oil industry.     

Distillery and curd whey wastes as viable alternative sources for biosurfactant production

Biosurfactant production from synthetic medium and industrial waste, viz. distillery and whey wastes was investigated by using an oily sludge isolate Pseudomonas aeruginosa strain BS2. In synthetic medium separately supplemented with glucose and hexadecane as water-soluble and -insoluble carbon sources, respectively, strain BS2 reduced the surface tension of the fermentation broth from 57 to 27 mN/m. The culture produced biosurfactant during the stationary growth phase and its yield was 0.97 g/l. The culture utilized distillery and whey wastes for its growth, as maximum cell counts reached to 54 × 10⁸ and 64 × 10⁹ c.f.u./ml from an initial inoculum size of 1 × 0⁵ c.f.u./ml, respectively, within 48 h of incubation and in these wastes the yields of biosurfactant obtained were 0.91 and 0.92 g/l, respectively. In synthetic medium, distillery and whey wastes, strain BS2 produced a crystalline biosurfactant which belonged to the category of secondary metabolites and its maximum production occurred after the onset of nitrogen-limiting conditions. After recovering biosurfactant from the fermented waste, the chemical oxygen demand (COD) of distillery and whey wastes was significantly reduced by 81 and 87%, respectively. Total acids, nitrogen and phosphate levels in distillery waste were reduced by 90, 92 and 92%, respectively, while in case of whey waste the concentration of these nutrients was reduced by 88, 95 and 93%, respectively. The isolated biosurfactant possessed potent surface active properties, as it effectively reduced the surface tension of water from 72 to 27 mN/m and formed 100% stable emulsions of a variety of water-insoluble compounds such as hydrocarbons, viz. hexadecane, crude oil, kerosene and oily sludge and pesticides, viz. dichlorodiphenyltrichloroethane (DDT) and benzene hexachloride (BHC). The effectiveness of biosurfactant was also evident from its low critical micellar concentration (CMC) which was 0.028 mg/ml.     

Bacterial biosurfactant increases ex situ biodiesel bioremediation in clayey soil

The contamination of soils by oily compounds has several environmental impacts, which can be reversed through bioremediation, using biosurfactants as auxiliaries in the biodegradation process. In this study, we aimed to perform ex situ bioremediation of biodiesel-contaminated soil using biosurfactants produced by Bacillus methylotrophicus. A crude biosurfactant was produced in a whey-based culture medium supplemented with nutrients and was later added to biodiesel-contaminated clayey soil. The produced lipopeptide biosurfactant could reduce the surface tension of the fermentation broth to 30.2 mN/m. An increase in the microbial population was observed in the contaminated soil; this finding can be corroborated by the finding of increased CO₂ release over days of bioremediation. Compared with natural attenuation, the addition of a lower concentration of the biosurfactant (0.5% w/w in relation to the mass of diesel oil) to the soil increased biodiesel removal by about 16% after 90 days. The added biosurfactant did not affect the retention of the contaminant in the soil, which is an important factor to be considered when applying in situ bioremediation technologies.

     

Screening of biosurfactant-producing Bacillus strains using glycerol from the biodiesel synthesis as main carbon source

Glycerol, a co-product of biodiesel production, was evaluated as carbon source for biosurfactant production. For this reason, seven non-pathogenic biosurfactant-producing Bacillus strains, isolated from the tank of chlorination at the Wastewater Treatment Plant at Federal University of Ceara, were screened. The production of biosurfactant was verified by determining the surface tension value, as well as the emulsifying capacity of the free-cell broth against soy oil, kerosene and N-hexadecane. Best results were achieved when using LAMI005 and LAMI009 strains, whose biosurfactant reduced the surface tension of the broth to 28.8?±?0.0 and 27.1?±?0.1?mN?m(-1), respectively. Additionally, at 72?h of cultivation, 441.06 and 267.56?mg?L(-1) of surfactin were produced by LAMI005 and LAMI009, respectively. The biosurfactants were capable of forming stable emulsions with various hydrocarbons, such as soy oil and kerosene. Analyses carried out with high performance liquid chromatography (HPLC) showed that the biosurfactant produced by Bacillus subtilis LAMI009 and LAMI005 was compatible with the commercially available surfactin standard. The values of minimum surface tension and the CMC of the produced biosurfactant indicated that it is feasible to produce biosurfactants from a residual and renewable and low-cost carbon source, such as glycerol.     

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.     

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.     

1株耐高温生物表面活性剂产生菌的特性研究

研究了耐高温生物表面活性剂产生菌ZY-3的生理生化特性,并通过测定发酵液的菌体密度、表面张力和乳化活性等指标,研究不同碳源和初始pH对菌株ZY-3生长和产生物表面活性剂的影响,同时对其所产生物表面活性剂进行了初步分离和性质分析。菌株ZY-3被初步鉴定为芽胞杆菌属(Bacillus),具有产酸、不产H_2S、还原硝酸盐等特性。在以淀粉为碳源、初始pH 6.0的培养基中发酵,产生物表面活性剂多且稳定;在种子培养基和发酵培养基中都有淀粉的条件下,菌体生长较多,降低表面张力和乳化的作用均较强,所产生物表面活性剂可以使发酵液的表面张力从72.1 mN/m降到53.1 mN/m,乳化活性从0升高到24%。初步判断产物为糖脂类阴离子表面活性剂。     

Indoor cultivation of a marine algae species in a thin-layer unit

Summary Chlorella capsulata UTEX LB 2074 was cultivated in a thin-layer intensive algae cultivation unit located indoors. The specific growth rate of the species under the applied conditions was found to be about 0.11 d^–1. A steady maximum cell number of about 15×10⁶ml^–1 was observed. Nutritional analysis revealed 33.12% amino-acids and 21.17%w 3 highly unsaturated fatty acidsKuwait Institute for Scientific Research, Publication No.KISR 1931 Kuwait.     

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.