Cell Surface Measurements in Hydrocarbon and Carbohydrate Fermentations

Acinetobacter calcoaceticus was grown in 11-liter batch fermentations with hexadecane or sodium citrate as the sole source of carbon. Surface and interfacial tension measurements of the microbial broth indicated that surface-active compounds were being produced only during growth on the hydrocarbon substrate. Contact angle measurements of an aqueous drop on a smooth lawn of cells in a hexadecane bath indicated a highly hydrophobic surface of the cells in the initial stages of the hydrocarbon fermentation (120° contact angle). At this stage, the entire cell population was bound to the hydrocarbon-aqueous interface. The contact angle dropped rapidly to approximately 45° after 14 h into the fermentation. This coincided with a shift of the cell population to the aqueous phase. Thus, the cells demonstrated more hydrophilic characteristics in the later stages of the fermentation. Contact angles on cells grown on sodium citrate ranged from 18 to 24° throughout the fermentation. The cells appear to be highly hydrophilic during growth on a soluble substrate. From the contact angle and aqueous-hydrocarbon interfacial tension, the surface free energy of the cells was calculated along with the cell-aqueous and cell-hydrocarbon interfacial tension. The results of these measurements were useful in quantitatively evaluating the hydrophobic nature of the cell surface during growth on hydrocarbons and comparing it with the hydrophilic nature of the cell surface during growth on a soluble substrate.     

生物表面活性剂发酵液的组成及表面活性

假单胞菌（Pseudomonas sp.)生长在正烷烃或植物油中,能生产出表面活性物质,分析其发酵液,类脂物和多糖是主要代谢产物,发酵液中表面活性物质主要是糖脂化合物及甘油单脂,发酵液稀释到5%,能将表面张力降到27mN/m,表面性能在广泛pH(2-12),高矿化度溶液中和高温下都非常稳定,发酵液的良好表面性能显示了它在三次采油,土壤处理等领域中应用潜力。     

Properties of hydrocarbon-in-water emulsions stabilized by Acinetobacter RAG-1 emulsan

Emulsan is a polymeric extracellular emulsifying agent produced by Acinetobacter RAG-1. Hydrocarbon-in-water emulsions (V(f) of hydrocarbon of 0.01-0.10) were stabilized by small quantities of emulsan (0.02-0.2 mg/mL). Although both aliphatic and aromatic hydrocarbon emulsions were stabilized by emulsan, mixtures containing both aliphatics and aromatics were better substrates for emulsan than the individual hydrocarbon by themselves. The emulsan remained tightly bound to the hydrocarbon even after centrifugation as determined by (a) residual emulsan in the aqueous phase and (b) the fact that the resulting "cream" readily dispersed in water to reform stable emulsions. With hexadecane-to-emulsan weight ratio of 39 and 155, the noncoalescing oil droplets had average droplet diameters of 2.0 and 4.0 mum, respectively. Dialysis studies showed that the water-soluble dye Rhodamine B adsorbed tightly to the interface of hexadecane-emulsan droplets although the dye did not bind to either hexadecane or emulsan alone. At saturating concentrations of dye, 2.2 mumol of dye were bound per mg emulsan.     

Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater

The production and properties of a biosurfactant, synthesized by Bacillus subtilis LB5a strain, using cassava wastewater as substrate were investigated. The microorganism was able to grow and to produce surfactant on cassava waste, reducing the surface tension of medium to 26.6 mN/m and giving a crude surfactant concentration of 3.0 g/L after 48 h. The surface-active compound retained its properties during exposure to elevate temperatures (100 degrees C), high salinity (20% NaCl) and a wide range of pH values. The surfactant was capable of forming stable emulsions with various hydrocarbons. Preliminary chemical characterization revealed that the surfactant has a lipopeptide composition with a CMC value of about 33 mg/L. Cassava wastewater proved to be a suitable substrate for biosurfactant biosynthesis, providing not only bacterial growth and product accumulation but also a surfactant that has interesting and useful properties with potential for many industrial applications.     

Hydrocarbon assimilation by Candida lipolytica: Formation of a biosurfactant; Effects on respiratory activity and growth

Summary Candida lipolytica is shown to produce an extracellular polymer with emulsifying properties when grown on n-tetradecane or a mixture of linear hydrocarbons. A device for biosurfactant isolation is presented. The polymers recovered from the fermentation broth were found to be complex molecules with a protein, a lipid and a carbohydrate moiety. Their surface active properties suggest a possible role in hydrocarbon uptake by cells. Effectively, the addition of crude polymer resulted in an enhancement of respiration rate which was dependent on n-alkane concentration with glucose grown cells. Likewise in batch culture, maximum growth rate, cell productivity and yield were increased by the presence of the biosurfactant.     

The production and release of an extracellular polysaccharide during starvation of a marine Pseudomonas sp. and the effect thereof on adhesion

A marine Pseudomonas sp. S9 produced and released an extracellular polysaccharide during complete energy and nutrient starvation in static conditions. The presence of the polysaccharide on the cell surface, demonstrable by immune transmission electron microscopy, correlated with changes in the degree of adhesion to hydrophobic surfaces. Polysaccharide coated cells showed a lower degree of adhesion than did cells devoid of the polymer. After 10 h of starvation, no ruthenium red stained antibody stabilized polysaccharides could be observed on the cell surface. The polysaccharide was not produced during growth since lysates of mid-log phase cells did not precipitate the antiserum. The relative proportions of sugars in the polysaccharide were 28% glucose, 35% N-acetylglucosamine and 37% N-acetylgalactosamine. The released polysaccharide did not significantly alter the physical parameters of surface tension and viscosity of the starvation regime. Cells starved in agitated conditions did not produce any extracellular polysaccharides and exhibited a different adhesion pattern to hydrophobic surfaces.Non-standard abbreviations FSS Four salt solution - GLC gas liquid chromatography - MS Mass spectrometry - NSS nine salt solution     

Biodegradability of bacterial surfactants

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO₂ evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO₂ emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.     

Mineralization of phenanthrene by a Mycobacterium sp.

A Mycobacterium sp., designated strain BG1, able to utilize the polycyclic aromatic hydrocarbon phenanthrene as the sole carbon and energy source was isolated from estuarine sediment following enrichment with the hydrocarbon. Unlike other phenanthrene degraders, this bacterium degraded phenanthrene via 1-hydroxy-2-naphthoic acid without accumulating this or other aromatic intermediates, as shown by high-performance liquid chromatography. Degradation proceeded via meta cleavage of protocatechuic acid. Different nonionic surfactants (Tween compounds) solubilized the phenanthrene to different degrees and enhanced phenanthrene utilization. The order of enhancement, however, did not correlate perfectly with increased solubility, suggesting physiological as well as physicochemical effects of the surfactants. Plasmids of approximately 21, 58, and 77 megadaltons were detected in cells grown with phenanthrene but not in those which, after growth on nutrient media, lost the phenanthrene-degrading phenotype. Given that plasmid-mediated degradations of aromatic hydrocarbons generally occur via meta cleavages, it is of interest that the addition of pyruvate, a product of meta cleavage, supported rapid mineralization of phenanthrene in broth culture; succinate, a product of ortho cleavage, supported growth but completely repressed the utilization of phenanthrene. The involvement of plasmids may have given rise to the unusual degradation pattern that was observed.     

Microbial assimilation of hydrocarbons. I. The fine-structure of a hydrocarbon oxidizing Acinetobacter sp.

1. The fine-structure analysis of the hydrocarbon oxidizing microorganism, Acinetobacter sp., demonstrated a cytoplasmic modification resulting from growth on paraffinic and olefinic hydrocarbons. 2. Intracytoplasmic hydrocarbon inclusions were documented by electron microscopy with chemical identifications obtained by gas chromatography and X-ray diffraction. 3. These results demonstrate the ability of a microorganism to accumulate hydrocarbon substrates intracellularly which, in turn, indicates the transport across the cell membrane.     

Evidence for surfactant production by the haloarchaeon Haloferax sp. MSNC14 in hydrocarbon-containing media

The potential for surfactant production by the extreme halophilic archaeon Haloferax sp. MSNC14 in the presence of individual hydrocarbon substrates was studied. This strain was selected for its ability to grow on different types of hydrocarbons at high NaCl concentrations. Linear (n-heptadecane or C17) and isoprenoid (pristane) alkanes, a polyaromatic hydrocarbon (phenanthrene) and ammonium acetate (highly water-soluble control compound) were used as growth substrates. The adherence potential was demonstrated by the ability of the cells to adhere to liquid or solid hydrocarbons. The biosurfactant production was indicated by the reduction of the surface tension (ST) and by the emulsification activity (EA) of cell-free supernatants. Growth on acetate was accompanied by a low EA (lower than 0.1) and a high ST (~70 mN/m), whereas an important EA (up to 0.68 ± 0.08) and a reduction of ST (down to 32 ± 2.3 mN/m) were observed during growth on the different hydrocarbons. Both ST and EA varied with the growth phase. The adhesion to hydrocarbons was higher when cells were grown on C17 (by 60–70 %) and pristane (by 30–50 %) than on phenanthrene (~25 %). The results demonstrated that strain MNSC14 was able to increase the bioavailability of insoluble hydrocarbons, thus facilitating their uptake and their biodegradation even at high salt concentration.     

厌氧产脂肽工程菌的构建及其代谢活性评价

利用微生物产生生物表面活性剂驱油,是微生物采油的重要技术之一.但油藏的缺氧环境使得大多数生物表面活性剂产生菌的代谢活性受到限制.本研究以筛选自油田采出水中的好氧产脂肽类表面活性剂的解淀粉芽孢杆菌BQ-2和兼性厌氧的施氏假单胞菌DQ-1为亲本菌,通过原生质体融合的方法构建了一株能在厌氧条件下迅速生长并能产脂肽类表面活性剂的融合子JD-3.融合子JD-3的菌落形态与亲本菌株BQ-2相似,其16S rDNA序列与BQ-2的相似度达99%;在厌氧条件下培养36 h,融合子JD-3发酵液的表面张力由初始的63.0 mN·m^-1降至32.5 mN·m^-1;薄层层析及红外光谱分析结果显示,JD-3在厌氧条件下的产物为脂肽类表面活性剂;该表面活性剂的临界胶束浓度(CMC)为90 mg·L^-1,且对原油、液体石蜡、煤油等有较好的乳化活性.JD 3在厌氧条件下可以蔗糖、葡萄糖或甘油为碳源,以蛋白胨等为氮源合成脂肽类表面活性剂,且经多次厌氧传代培养仍保持稳定的产生物表面活性剂功能,显示该菌株在油藏厌氧条件下对提高原油采收率具有较好的应用潜力.     

Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀ were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.     

A novel lipopeptide, an inhibitor of bacterial adhesion, from the thermophilic and halotolerant subsurface Bacillus licheniformis strain 603

A new Bacillus licheniformis strain, 603, isolated from a mixture of drilling fluid and subsurface thermal water, has been found to produce a cyclic lipopeptide which is released into cultural medium as well as present in cells as the major lipid constituent (57% of the total cell lipids extractable with 2:1 chloroform-methanol). The quantitative ratio of the extracellular and intracellular lipopeptide has been estimated as 23:10. The metabolite represents a heptapeptide, L-Asp-->L-Leu-->L-Leu-->L-Val-->L-Val-->L-Glu-->L-Leu, N-acylated to the N-terminal amino acid, L-Asp, by a 3-hydroxy fatty acid (from 13:0 to 17:0 with n-, iso-, and anteiso-chains), the 3-OH group of which is esterified by the C-terminal amino acid, L-Leu. The chemical structure of the lipopeptide has been established by means of infrared (IR), 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionisation (ESI) mass spectrometry (MS), including secondary ion mass spectrometry, along with chemical and enzymatic degradation. Although a diversity of similar metabolites synthesised by various B. licheniformis strains are presently known, such a structure has not been reported thus far. Added to the growth medium of strain 603 at the concentration of 1.6 microg/ml, the lipopeptide prevents adhesion of cells to a glass surface. Also, it exhibits a considerable growth-inhibiting activity against Corynebacterium variabilis and a much lower activity against Acinetobacter sp.     

Stabilization of Oil-Water Emulsions by Hydrophobic Bacteria

Formation of oil-water emulsions during bacterial growth on hydrocarbons is often attributed to biosurfactants. Here we report the ability of certain intact bacterial cells to stabilize oil-in-water and water-in-oil emulsions without changing the interfacial tension, by inhibition of droplet coalescence as observed in emulsion stabilization by solid particles like silica.     

Stabilization of oil-water emulsions by hydrophobic bacteria

Formation of oil-water emulsions during bacterial growth on hydrocarbons is often attributed to biosurfactants. Here we report the ability of certain intact bacterial cells to stabilize oil-in-water and water-in-oil emulsions without changing the interfacial tension, by inhibition of droplet coalescence as observed in emulsion stabilization by solid particles like silica.     

Physical and metabolic interactions of Pseudomonas sp. strain JA5-B45 and Rhodococcus sp. strain F9-D79 during growth on crude oil and effect of a chemical surfactant on them

Methods to enhance crude oil biodegradation by mixed bacterial cultures, for example, (bio)surfactant addition, are complicated by the diversity of microbial populations within a given culture. The physical and metabolic interactions between Rhodococcus sp. strain F9-D79 and Pseudomonas sp. strain JA5-B45 were examined during growth on Bow River crude oil. The effects of a nonionic chemical surfactant, Igepal CO-630 (nonylphenol ethoxylate), also were evaluated. Strain F9-D79 grew attached to the oil-water interface and produced a mycolic acid-containing capsule. Crude oil emulsification and surface activity were associated with the cellular fraction. Strain JA5-B45 grew in the aqueous phase and was unable to emulsify oil, but cell-free supernatants mediated kerosene-water emulsion formation. In coculture, stable emulsions were formed and strain JA5-B45 had an affinity for the capsule produced by strain F9-D79. Igepal CO-630 inhibited F9-D79 cells from adhering to the interface, and cells grew dispersed in the aqueous phase as 0.5-microm cocci rather than 2.5-microm rods. The surfactant increased total petroleum hydrocarbon removal by strain JA5-B45 from 4 to 22% and included both saturated compounds and aromatics. In coculture, TPH removal increased from 13 to 40% following surfactant addition. The culture pH normally increased from 7.0 to between 7.5 and 8.5, although addition of Igepal CO-630 to F9-D79 cultures resulted in a drop to pH 5.5. We suggest a dual role for the nonylphenol ethoxylate surfactant in the coculture: (i) to improve hydrocarbon uptake by strain JA5-B45 through emulsification and (ii) to prevent strain F9-D79 from adhering to the oil-water interface, indirectly increasing hydrocarbon availability. These varied effects on hydrocarbon biodegradation could explain some of the known diversity of surfactant effects.     

Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1.

An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG-1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10-fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X-100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8-9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 degrees C, with maximal activity observed at 55 degrees C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG-1 lipase offers potential for use as a biocatalyst.     

Production of biosurfactants by Cladosporium resinae

Summary Cladosporium resinae produces extracellular biosurfactants when growing in a hydrocarbon source such as the jet fuel JP8. This production of biosurfactants was observed by the reduction of the surface tension of the aqueous phase of growing medium, and by the increase in emulsion and foaming properties. A partial purification by collapsed foam gave better physical properties by decreasing surface tension and increasing foaming power and stabilization of emulsions. Surface active substances were purified by reversed phase chromatography. Six compounds representing over 75% of fraction containing surface activity were present. This fraction gave an improvement of all surface properties.     

Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa

A gamma ray-induced mutant of Pseudomonas aeruginosa strain S8, capable of hyperproduction of biosurfactant from hydrocarbons, was isolated and named as EBN-8. The mutant showed 3–4 times more hydrocarbon emulsification/conversion as compared to the parent when grown on Khaskheli crude oil in minimal medium. Enhanced biosurfactant production and hydrocarbon utilization by the mutant was also observed during growth on heptadecane in minimal medium as indicated by emulsion index and surface tension of cell-free culture broth. Using heptadecane as carbon and energy source, time course for the growth (cfu ml^-1) and biosurfactant production were compared for both parent and mutant. These studies were carried out for 24 d at 30 ± 2°C and for 20 d at 37°C. Growth of EBN-8 was much faster compared to the parent as well as being 2–3 times more hyperproductive.     

Surface-Active Agents from Two Bacillus Species

Two Bacillus species were studied which produced bioemulsifiers; however, they were distinctly different compounds. Bacillus sp. strain IAF 343 produced unusually high yields of extracellular biosurfactant when grown on a medium containing only water-soluble substrates. The yield of 1 g/liter was appreciably better than those of most of the biosurfactants reported previously. This neutral lipid product, unlike most lipid biosurfactants, had significant emulsifying properties. It did not appreciably lower the surface tension of water. On the same medium, Bacillus cereus IAF 346 produced a more conventional polysaccharide bioemulsifier, but it also produced a monoglyceride biosurfactant. The bioemulsifier contained substantial amounts of glucosamine and originated as part of the capsule layer. The monoglyceride lowered the surface tension of water to 28 mN/m. It formed a strong association with the polysaccharide, and it was necessary to use ultrafiltration to effect complete separation. The removal of the monoglyceride caused the polysaccharide to precipitate. It is suggested that earlier reports of biopolymers which both stabilized emulsions and lowered surface tension were actually similar aggregates of lipid and bioemulsifier.