Characteristics of bioemulsifiers synthesised in crude oil media by <Emphasis Type="Italic">Halomonas eurihalina</Emphasis> and their effectiveness in the isolation of bacteria able to grow in the presence of hydrocarbons

Halomonas eurihalina strains F2-7, H28, H96, H212 and H214 were capable of producing large amounts of exopolysaccharides (EPS) in MY medium with added crude oil. The biopolymers showed lower carbohydrate and protein content than those synthesised in control medium without oil. Nevertheless, the percentages of uronic acids, acetyls and sulphates were higher. The emulsifying activity of biopolymers was measured; crude oil was the substrate most efficiently emulsified. Furthermore, all the EPS tested emulsified higher volumes of crude oil than the commercial surfactants used as controls. We have also proved the effectiveness of both Halomonas eurihalina strains and their EPS to select indigenous bacteria able to grow in the presence of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene and pyrene) from waste crude oil. The majority of isolated strains belonged to the genus Bacillus.     

Characteristics of bioemulsifier V2-7 synthesized in culture media added of hydrocarbons: chemical composition, emulsifying activity and rheological properties

The bioemulsifier V2-7 is an exopolysaccharide (EPS) synthesized by strain F2-7 of Halomonas eurihalina and it has the property of emulsifying a wide range of hydrocarbons i.e. n-tetradecane, n-hexadecane, n-octane, xylene mineral light and heavy oils, petrol and crude oil. Characteristics of exopolysaccharide V2-7 produced in media supplemented with various hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol or crude oil) were studied. Yield production varied from 0.54 to 1.45 g L(-1) according to the hydrocarbon added, in the same way chemical composition, viscosity and emulsifying activity of EPS varied with the culture conditions. Respect to chemical composition, percentage of uronic acids found in exopolymers produced in hydrocarbon media was always higher than that described for V2-7 EPS (1.32%) obtained with glucose. This large amount of uronic acid present could be useful in biodetoxification and waste water treatment. On the other hand, the highest amount of biopolymer was synthesized with mineral light oil, while the most active emulsifiers were those obtained from media added with petrol and n-octane. Furthermore, all EPS were capable of emulsifying crude oil more efficiently than the three chemical surfactants tested as control (Tween 20, Tween 80 and Triton X-100). The capacity of strain F2-7 to grow and produce bioemulsifier in presence of oil hydrocarbons together with the high emulsifying activity and low viscosity power of the biopolymers synthesized in hydrocarbons media could be considered highly beneficial for application of both bioemulsifier and producing strain in bioremediation of oil pollutants.     

Studies on surfactant-biopolymer interaction. I. Microcalorimetric investigation on the interaction of cetyltrimethylammonium bromide (CTAB) and sodium dodecylsulfate (SDS) with gelatin (Gn), lysozyme (Lz) and deoxyribonucleic acid (DNA)

The interaction of the surfactants cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) with the biopolymers gelatin (Gn), lysozyme (Lz) and deoxyribonucleic acid (DNA) was studied by isothermal titration microcalorimetry at varied biopolymer concentration, pH and temperature. The nature of interaction of the surfactants with the biopolymers was assessed from the observed enthalpy-[surfactant] profiles. The biopolymer-induced aggregation of the surfactants was observed. The enthalpies of aggregation of amphiphiles, binding of aggregates with macromolecules, organisational change of bound aggregates, and threshold concentrations for micelle formation of surfactants in the presence of biopolymers were estimated. The results collected on the three biopolymers were analysed and compared.     

Microbial biosurfactant mediated removal and/or solubilization of crude oil contamination from soil and aqueous phase: An approach with Bacillus licheniformis MTCC 5514

This study highlights the role of marine microbial biosurfactants on solubilization/removal of crude-oil contamination from four different soils in an aqueous phase. Soil of four different types, viz., sandy, fine sand soil, clay, and clay loam, were collected and saturated with crude oil. Marine isolate MTCC 5514 (Bacillus licheniformis) was chosen for the study and comparisons were made with synthetic surfactants and commercially available biosurfactant. In-situ studies were carried out with different percentages of crude oil to assess the growth and the percentage removal of oil. For ex-situ studies, soils were pre-saturated with crude oil and then treated with the chosen biosurfactant at a 10% concentration level using flask and column methods. After time intervals of 30–120 min, samples were collected and then subjected to extraction with hexane and the percentage removal was calculated. Results revealed, at 2% concentration of crude oil, that complete solubilization was achieved. With regard to ex-situ studies, clay soil absorbed the maximum percentage of oil compared to other soil types, and with regard to the removal, all the synthetic surfactants showed <60% removal irrespective of soil type. In the case of biosurfactants even at 10% concentration, >85% removal was achieved.     

Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture.

The effects of surfactant physicochemical properties, such as the hydrophile-lipophile balance (HLB) and molecular structure, on the biodegradation of 2% w/v Bow River crude oil by a mixed-bacterial culture were examined. Viable counts increased 4.6-fold and total petroleum hydrocarbon (TPH) biodegradation increased 57% in the presence of Igepal CO-630, a nonylphenol ethoxylate (HLB 13, 0.625 g/L). Only the nonylphenol ethoxylate with an HLB value of 13 substantially enhanced biodegradation. The surfactants from other chemical classes with HLB values of 13 (0.625 g/L) had no effect or were inhibitory. TPH biodegradation enhancement by Igepal CO-630 occurred at concentrations above the critical micelle concentration. When the effect of surfactant on individual oil fractions was examined, the biodegradation enhancement for the saturate and aromatic fractions was the same. In all cases, biodegradation resulted in increased resin and asphaltene concentrations. Optimal surfactant concentrations for TPH biodegradation reduced resin and asphaltene formation. Chemical surfactants have the potential to improve crude oil biodegradation in complex microbial systems, and surfactant selection should consider factors such as molecular structure, HLB, and surfactant concentration.     

Optimization of Nutrient Requirements and Culture Conditions for the Production of Rhamnolipid from Pseudomonas aeruginosa (MTCC 7815) using Mesua ferrea Seed Oil

Environmental awareness has led to a serious consideration for biological surfactants and hence non-edible vegetable oils may serve as a substitute carbon source for bio-surfactant production (rhamnolipid) which might be an alternative to complex synthetic surfactants. There are reports of rhamnolipid production from plant based oil giving higher production than that of glucose because of their hydrophobicity and high carbon content. Therefore the contribution of non-edible oil such as Mesua ferrea seed oil could serve as a good carbon source for rhamnolipid production. Moreover the use of rhamnolipid production from non-edible plant based seed oil has not been reported elsewhere. The present work focus on the optimal production of rhamnolipid by considering both micro and macro nutrients and culture conditions using response surface methodology. The study observes that micronutrients play a significant role in rhamnolipid production from Pseudomonas aeruginosa (MTCC 7815). The investigation results with the statistically optimize parameters able to produce a higher rhamnolipid production and this methodology could be used to optimize the nutrients requirements and culture conditions. The present findings would assist in bioremediation of crude oil contaminated ecosystems.     

Characterization of bacterial strains able to grow on high molecular mass residues from crude oil processing

Oil residues containing high molecular mass hydrocarbons, rich in polyaromatic compounds, are frequent end-products of crude oil processing and are poorly biodegradable. Their disposal poses an environmental problem. Through batch-enrichments from contaminated soils we have isolated and characterized seven bacterial strains that can use a residue from crude oil processing as a source of carbon and energy. The residue was a complex mixture of high molecular mass compounds, including saturated, aromatic and polycyclic aromatic hydrocarbons (PAHs). Analysis of the metabolic profiles of the strains isolated showed that they could all metabolize long-chain-length alkanes efficiently, but not PAHs. Strains degrading naphthalene, a simple PAH, did exist in the soil inocula used, but could be isolated only when enrichments were performed using pure naphthalene as the sole carbon source. All strains tested emulsified the oil residue and their ability to produce surfactants was studied.     

Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture

Three bacterial strains isolated from waste crude oil were selected due to their capacity of growing in the presence of hydrocarbons and production of bioemulsifier. The genetic identification (PCR of the 16S rDNA gene using fD1 and rD1 primers) of these strains showed their affiliation to Bacillus subtilis, Alcaligenes faecalis and Enterobacter sp. These strains were able to emulsify n-octane, toluene, xylene, mineral oils and crude oil, look promising for bioremediation application. Finally, chemical composition, emulsifying activity and surfactant activity of the biopolymers produced by the selected strains were studies under different culture conditions. Our results showed that chemical and functional properties of the bioemulsifiers were affected by the carbon source added to the growth media.     

Effects of surfactant mixtures, including Corexit 9527, on bacterial oxidation of acetate and alkanes in crude oil

Mixtures of nonionic and anionic surfactants, including Corexit 9527, were tested to determine their effects on bacterial oxidation of acetate and alkanes in crude oil by cells pregrown on these substrates. Corexit 9527 inhibited oxidation of the alkanes in crude oil by Acinetobacter calcoaceticus ATCC 31012, while Span 80, a Corexit 9527 constituent, markedly increased the oil oxidation rate. Another Corexit 9527 constituent, the negatively charged dioctyl sulfosuccinate (AOT), strongly reduced the oxidation rate. The combination of Span 80 and AOT increased the rate, but not as much as Span 80 alone increased it, which tentatively explained the negative effect of Corexit 9527. The results of acetate uptake and oxidation experiments indicated that the nonionic surfactants interacted with the acetate uptake system while the anionic surfactant interacted with the oxidation system of the bacteria. The overall effect of Corexit 9527 on alkane oxidation by A. calcoaceticus ATCC 31012 thus seems to be the sum of the independent effects of the individual surfactants in the surfactant mixture. When Rhodococcus sp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the presence of a mixture of Tergitol 15-S-7 and AOT even though the Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT did not affect it. This indicated that there was synergism between the two surfactants rather than an additive effect like that observed for A. calcoaceticus ATCC 31012.     

Binding of cationic surfactants to DNA, protein and DNA-protein mixtures

Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale.     

Potential for industrial products from the halophilic <Emphasis Type="Italic">Archaea</Emphasis>

The halophilic Archaea are a group of microorganisms that have not been extensively considered for biotechnological applications. This review describes some of the enzymes and products and the potential applications of this unique group of microorganisms to various industrial processes. Specifically, the characteristics of the glycosyl hydrolases, lipases and esterases, proteases, biopolymers and surfactants, as well as some miscellaneous other activities will be described.     

Oil desorption from mineral and organic materials using biosurfactant complexes produced by Rhodococcus species

Rhodococcus strains from the culture collection at the Institute of Ecology and Genetics of Microorganisms, Perm, Russia were examined for biosurfactant production during growth on n-alkanes and the ability to remove oil associated with contaminated sands and oil shale cuttings. Members of the genus, particularly R. ruber, were shown to produce low toxicity surfactants effective in removing oil from surfaces. The extent of desorption was inversely related to the concentration of high molecular weight hydrocarbons, namely asphaltenes and resins. In addition, crude surfactant complexes enhanced the degradation of crude oil, in the short term, when added to contaminated agricultural soil during bioremediation studies utilizing biopiling technology.     

The prospects of using bacteria of the genus Rhodococcus and microbial surfactants for the degradation of oil pollutants

The possibility of accelerating oil degradation by an enrichment culture of oil-oxidizing microorganisms in the presence of bacteria of the genus Rhodococcus and microbial surfactants was studied. It was shown that the degree of consumption of crude oil (2% v/v) after 192 h of enrichment culture growth reached 84%. Inoculation of the active hydrocarbon-oxidizing strain Rhodococcus erythropolis EK-1 and exogenous surfactants produced by Pseudomonas sp. PS-27 increased this degree to 90% and 93-94%, respectively. On the grounds of these results, efficient methods of purification of the environment from oil pollutants can be developed.     

Effects of Surfactant Mixtures, Including Corexit 9527, on Bacterial Oxidation of Acetate and Alkanes in Crude Oil

Mixtures of nonionic and anionic surfactants, including Corexit 9527, were tested to determine their effects on bacterial oxidation of acetate and alkanes in crude oil by cells pregrown on these substrates. Corexit 9527 inhibited oxidation of the alkanes in crude oil by Acinetobacter calcoaceticus ATCC 31012, while Span 80, a Corexit 9527 constituent, markedly increased the oil oxidation rate. Another Corexit 9527 constituent, the negatively charged dioctyl sulfosuccinate (AOT), strongly reduced the oxidation rate. The combination of Span 80 and AOT increased the rate, but not as much as Span 80 alone increased it, which tentatively explained the negative effect of Corexit 9527. The results of acetate uptake and oxidation experiments indicated that the nonionic surfactants interacted with the acetate uptake system while the anionic surfactant interacted with the oxidation system of the bacteria. The overall effect of Corexit 9527 on alkane oxidation by A. calcoaceticus ATCC 31012 thus seems to be the sum of the independent effects of the individual surfactants in the surfactant mixture. When Rhodococcus sp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the presence of a mixture of Tergitol 15-S-7 and AOT even though the Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT did not affect it. This indicated that there was synergism between the two surfactants rather than an additive effect like that observed for A. calcoaceticus ATCC 31012.     

Yield production,chemical composition,and functional properties of emulsifier H28 synthesized by <Emphasis Type="Italic">Halomonas eurihalina </Emphasis>strain H-28 in media containing various hydrocarbons

Halomonas eurihalina strain H-28 is a moderately halophilic bacterium that produces an extracellular polysaccharide not only in media with glucose but also in media supplemented with hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol, or crude oil). In this study we investigated yield production, chemical composition, viscosity, and emulsifying activity of exopolysaccharides (EPS) extracted from the different media used. The largest amounts of biopolymer were synthesized in media with glucose and n-hexadecane. Chemical composition varied with culture conditions; thus EPS from cultures grown in the presence of hydrocarbons had lower contents of carbohydrates and proteins than EPS from media with glucose. However, the percentages of uronic acids, acetyls, and sulfates were always higher than glucose EPS. Crude oil was the substrate most effectively emulsified. All EPS were capable of emulsifying crude oil more efficiently than the three control surfactants tested (Tween 20, Tween 80, and Triton X-100). All polymers gave low viscosity solutions. EPS H28 could be attractive for application in the oil industry and/or in bioremediation processes, bearing in mind not only its functional properties, but also the capacity of producer strain H-28 to grow in the presence of high salt concentrations and oil substrates.     

Changes in mutagenicity during crude oil degradation by fungi

Two fungal strains, Cunninghamella elegans and Penicillium zonatum, that grow with crude oil as a sole carbon source were exposed to three crude oils that exhibit a range of mutagenic activity. At regular time intervals following fungal incubation with the various crude oils, extracts were tested for the presence of mutagenic activity using the spiral Salmonella assay. When the most mutagenic of the oils, Pennsylvania crude oil, was degraded by C. elegans or by P. zonatum, its mutagenicity was significantly reduced; corresponding uninoculated (weathered) controls of Pennsylvania crude remained mutagenic. West Texas Sour crude oil, a moderately mutagenic oil, exhibited little change in mutagenicity when incubated with either C. elegans or P. zonatum. Swanson River Field crude oil from Cook Inlet, Alaska is a slightly mutagenic oil that became more mutagenic when incubated with C. elegans; weathered controls of this oil showed little change in mutagenicity. Mycelial mat weights measured during growth on crude oils increased corresponding to the biodegradation of about 25% of the crude oil.     

The prospects of using bacteria of the genus Rhodococcus and microbial surfactants for the degradation of oil pollutants

The possibility of accelerating oil degradation by an enrichment culture of oil-oxidizing microorganisms in the presence of bacteria of the genus Rhodococcus and microbial surfactants was studied. It was shown that the degree of consumption of crude oil (2vol %) after 192 h of enrichment culture growth reached 84%. Inoculation of the active hydrocarbon-oxidizing strain Rhodococcus erythropolis EK-1 and exogenous surfactants produced by Pseudomonas sp. PS-27 increased this degree to 90 and 93–94%, respectively. On the grounds of these results, efficient methods of purification of the environment from oil pollutants can be developed.     

Engineering bacterial biopolymers for the biosorption of heavy metals; new products and novel formulations

Bioremediation of heavy metal pollution remains a major challenge in environmental biotechnology. One of the approaches considered for application involves biosorption either to biomass or to isolated biopolymers. Many bacterial polysaccharides have been shown to bind heavy metals with varying degrees of specificity and affinity. While various approaches have been adopted to generate polysaccharide variants altered in both structure and activity, metal biosorption has not been examined. Polymer engineering has included structural modification through the introduction of heterologous genes of the biosynthetic pathway into specific mutants, leading either to alterations in polysaccharide backbone or side chains, or to sugar modification. In addition, novel formulations can be designed which enlarge the family of available bacterial biopolymers for metal-binding and subsequent recovery. An example discussed here is the use of amphipathic bioemulsifiers such as emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1, that forms stable, concentrated (70%), oil-in-water emulsions (emulsanosols). In this system metal ions bind primarily at the oil/water interface, enabling their recovery and concentration from relatively dilute solutions. In addition to the genetic modifications described above, a new approach to the generation of amphipathic bioemulsifying formulations is based on the interaction of native or recombinant esterase and its derivatives with emulsan and other water-soluble biopolymers. Cation-binding emulsions are generated from a variety of hydrophobic substrates. The features of these and other systems will be discussed, together with a brief consideration of possible applications. Received: 2 February 2000 / Received revision: 2 June 2000 / Accepted: 3 June 2000     

Treatment of petroleum industry oil sludge in soil

Petroleum refining unavoidably generates large volumes of oil sludge. The environmentally acceptable disposal of oil sludge is a current challenge to the petroleum industry. Many soil microorganisms possess a remarkable capacity to degrade various components of crude oil. The land treatment of oil sludge — land farming — is an environmentally acceptable and economically feasible disposal method. The development of efficient hydrocarbon-degrading microorganisms and their use for cleaning oil sludge in soil are discussed.     

Biological souring of crude oil under anaerobic conditions

Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil–water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring.