Isolation and characterization of biosurfactant production under extreme environmental conditions by alkali-halo-thermophilic bacteria from Saudi Arabia

Twenty three morphologically distinct microbial colonies were isolated from soil and sea water samples, which were collected from Jeddah region, Saudi Arabia for screening of the most potent biosurfactant strains. The isolated bacteria were selected by using different methods as drop collapse test, oil displacement test, blue agar test, blood hemolysis test, emulsification activity and surface tension. The results showed that the ability of Virgibacillus salarius to grow and reduce surface tension under a wide range of pH, salinities and temperatures gives bacteria isolate an advantage in many applications such as pharmaceutical, cosmetics, food industries and bioremediation in marine environment. The biosurfactant production by V. salarius decreased surface tension and emulsifying activity (30 mN/m and 80%, respectively). In addition to reducing the production cost of biosurfactants by tested several plant-derived oils such as jatropha oil, castor oils, jojoba oil, canola oil and cottonseed oil. In this respect the feasibility to reusing old frying oil of sunflower for production rhamnolipids and sophorolipids, their use that lead to solve many ecological and industrial problems.     

Screening and Evaluation of Biosurfactant-Producing Strains Isolated from Oilfield Wastewater

The six biosurfactant-producing strains, isolated from oilfield wastewater in Daqing oilfield, were screened. The production of biosurfactant was verified by measuring the diameter of the oil spreading, measuring the surface tension value and emulsifying capacity against xylene, n-pentane, kerosene and crude oil. The experimental result showed three strains (S2, S3, S6) had the better surface activity. Among the three strains, the best results were achieved when using S2 strain. The diameter of the oil spreading of the biosurfactant produced by S2 strain was 14 cm, its critical micelle concentration (CMC) was 21.8 mg/l and the interfacial tension between crude oil and biosurfactant solution produced by S2 strain reduced to 25.7 mN/m. The biosurfactant produced by S2 strain was capable of forming stable emulsions with various hydrocarbons, such as xylene, n-pentane, kerosene and crude oil. After S2 strain treatment, the reduction rate of oil viscosity was 51 % and oil freezing point reduced by 4 °C.     

泡菜水中产生物表面活性剂酵母菌株的筛选

[背景]由微生物产生的生物表面活性剂(biosurfactant,BS)具有低毒性、高效性、生物可降解性等多种特性,能在一定程度上缓解化学表面活性剂所造成的环境问题,因此筛选高产、安全的BS生产菌株备受研究者的关注.[目的]从泡菜水中筛选能代谢合成药食两用型BS的微生物菌株.[方法]运用滴崩法和排油圈法从传统发酵食品泡...     

Role of Sophorolipid Biosurfactant in Degradation of Diesel Oil by Candida tropicalis

The yeast Candida tropicalis, isolated from petroleum-contaminated soil in India, was found to be the potent producer of biosurfactant in mineral salt media containing diesel oil as the carbon source and found to be an efficient degrader of diesel oil (98%) over a period of 10 days. The crude biosurfactant decreased the surface tension of cell-free broth, 78 to 30 mN/m, with a large oil displacement area and highly positive drop collapse test. The crude biosurfactant was purified using silica gel column chromatography followed by dialysis. With the use of Fourier transform infrared (FT-IR) spectroscopy, in combination with gas chromatography–mass spectrometry (GC-MS) analysis, chemical structures of the purified biosurfactant was identified as sophorolipid species. Involvement of biosurfactant in physiological mechanism of diesel adsorption on yeast cell surface was characterized based on zeta potential. When diesel oil was emulsified with biosurfactant, the surface charge of the diesel was modified, resulting in more adsorption of diesel on yeast cell surface. Biosurfactant production by yeast species was monitored using scanning electron microscopy (SEM) analysis and found that yeast species could form thick mat of mucilaginous biosurfactant that could interconnect the individual cells. Uptake of diesel oil by C. tropicalis was elucidated through transmission electron microscopy (TEM) analysis. Interestingly, it was observed that internalization of diesel oil droplet was taking place, suggesting a mechanism similar in appearance to active pinocytosis.     

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.     

Identification and ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry characterization of biosurfactants,including a new surfactin,isolated from oil‐contaminated environments

Biosurfactant‐producing bacteria were isolated from samples collected in areas contaminated with crude oil. The isolates were screened for biosurfactant production using qualitative drop‐collapse test, oil‐spreading and emulsification assays, and measurement of their tensoactive properties. Five isolates tested positive for in the screening experiments and displayed decrease in the surface tension below 30 mN m^?1. The biosurfactants produced by these isolates were further investigated and their molecular identification revealed that they are bacteria related to the Bacillus genus. Additionally, the biosurfactants produced were chemically characterized via UHPLC‐HRMS experiments, indicating the production of surfactin homologues, including a new class of these molecules.     

Bench-Scale Production of Biosurfactants and their Potential in Ex-Situ MEOR Application

The fermentative production of biosurfactants by five Bacillus strains in a bench-scale bioreactor and evaluation of biosurfactant-based enhanced oil recovery using sand pack columns were investigated. Adjusting the initial dissolved oxygen to 100% saturation, without any further control and with collection of foam and recycling of biomass, gave higher biosurfactant production. The microorganisms were able to produce biosurfactants, thus reducing the surface tension and interfacial tension to 28 mN/m and 5.8–0.5 mN/m, respectively, in less than 10 hours. The crude surfactant concentration of 0.08–1.1 g/L, and critical micelle concentration (CMC) values of 19.4–39 mg/L, corresponding to the biosurfactants produced by the different Bacillus strains, were observed. The efficiency of crude biosurfactant preparation obtained from Bacillus strains for enhanced oil recovery, by sand pack column studies, revealed it to vary from 30.22–34.19% of the water flood residual oil saturation. The results are indicative of the potential of the strains for the development of ex-situ, microbial-enhanced, oil recovery processes.     

Physicochemical and structural characterization of biosurfactant produced by <Emphasis Type="Italic">Pleurotus djamor</Emphasis> in solid-state fermentation

Biosurfactants are amphiphilic compounds produced by several microorganisms that reduce the surface tension. Low toxicity, optimal activity in extreme conditions, biodegradability and production from several wastes are main advantages of biosurfactants as compared to synthetic surfactants. Production of biosurfactant by a white rot fungus Pleurotus djamor on sunflower seed shell in solid-state fermentation was determined by emulsification indexes, oil spreading activity and surface tension (28.82 ± 0.3mN/m) measurement. The critical micelle concentration was detected as 0.964 ± 0.09 mg/mL. Also, the chemical and physicochemical properties of the biosurfactant produced were investigated. Considering the results of the chemical contents analysis, HPLC, FT-IR and ¹H-NMR, it can be concluded that the produced biosurfactant has a complex structure. Besides, resistance of its activity to environmental factors such as temperature, pH and salt concentration, as well as its thermal stability, were investigated. Additionally, the produced biosurfactant formed stabile emulsions with different hydrocarbons. Lastly, the performance of removing waste frying oil from contaminated sand of produced biosurfactant was detected as 76.57 ± 6%. Owing to its high emulsification capacity, low surface tension and critical micelle concentration, the biosurfactant, shows great potential for use in hydrocarbon removal applications.     

Optimization of low-cost biosurfactant production from agricultural residues through response surface methodology

Biosurfactants are surface-active compounds capable of reducing surface tension and interfacial tension. Biosurfactants are produced by various microorganisms. They are promising replacements for chemical surfactants because of biodegradability, nontoxicity, and their ability to be produced from renewable sources. However, a major obstacle in producing biosurfactants at the industrial level is the lack of cost-effectiveness. In the present study, by using corn steep liquor (CSL) as a low-cost agricultural waste, not only is the production cost reduced but a higher production yield is also achieved. Moreover, a response surface methodology (RSM) approach through the Box–Behnken method was applied to optimize the biosurfactant production level. The results found that biosurfactant production was improved around 2.3 times at optimum condition when the CSL was at a concentration of 1.88 mL/L and yeast extract was reduced to 25 times less than what was used in a basic soybean oil medium (SOM). The predicted and experimental values of responses were in reasonable agreement with each other (Pred-R² = 0.86 and adj-R² = 0.94). Optimization led to a drop in raw material price per unit of biosurfactant from $47 to $12/kg. Moreover, the biosurfactant product at a concentration of 84 mg/L could lower the surface tension of twice-distilled water from 72 mN/m to less than 28 mN/m and emulsify an equal volume of kerosene by an emulsification index of (E24) 68% in a two-phase mixture. These capabilities made these biosurfactants applicable in microbial enhanced oil recovery (MEOR), hydrocarbon remediation, and all other petroleum industry surfactant applications.     

一株糖脂表面活性剂产生菌的筛选及干酪根降解

【目的】从油页岩环境中筛选可降解油页岩干酪根的产生物表面活性剂菌株。【方法】从抚顺油页岩矿废水样品中用血平板法初筛,排油圈法、乳化法和表面张力法复筛,获得产生物表面活性剂菌株。对目标菌株进行生理生化鉴定、16S r RNA基因序列和系统发育分析,用薄层色谱鉴定其发酵液表面活性成分,优化产表面活性剂的培养条件,初步考察其对油页岩干酪根的降解能力。【结果】筛选到一株产糖脂表面活性剂菌株B-1,初步鉴定为Pseudomonas sp.,该菌株有良好的排油和乳化能力以及较低的表面张力,可利用烷烃、不饱和脂肪酸和糖类作为碳源。在30-34°C范围内添加0.3%Na Cl的葡萄糖培养基(p H 7.0)中该菌生长旺盛,发酵液表面张力最低为27 m N/m。菌株B-1在添加一定量葡萄糖的无机盐培养基中作用30 d后对干酪根的降解率为2.85%,高于不添加葡萄糖无机盐培养基对照组的降解率(1.04%)。【结论】菌株B-1是一株性能良好的产糖脂表面活性剂细菌,有降解干酪根的潜力。     

Biosurfactant-Mediated Biodegradation of Polycyclic Aromatic Hydrocarbons—Naphthalene

The present study is aimed at the naphthalene degradation with and without biosurfactant produced from Pseudomonas aeruginosa isolated from oil-contaminated soil. The present study was carried out to isolate the bacterial strains for the naphthalene degradation and also for biosurfactant production. The isolated strains were screened for their ability to degrade the naphthalene by the methods of optimum growth rate test and for the production of biosurfactants by cetyltrimethylammonium bromide, blood agar medium, and thin-layer chromatography. The present study also focused on the effect of biosurfactant for the degradation of naphthalene by isolate-1. Two bacterial strains were isolated and screened, one for biodegradation and another for biosurfactant production. The second organism was identified as Pseudomonas aeruginosa by 16S rRNA analysis. The purified biosurfactant reduces the surface tension of water and also forms stable emulsification with hexadecane and kerosene. The end product of naphthalene degradation was estimated as salicylic acid equivalent by spectrophotometric method. The results demonstrated that Pseudomonas aeruginosa has the potential to produce biosurfactant, which enhances the biodegradation of naphthalene. The study reflects the potential use of biosurfactants for an effective bioremediation in the management of contaminated soils.     

一株石油烃降解菌的细胞疏水性及其乳化性质

【目的】从新疆油田石油污染土壤中分离到一株在25 °C条件下利用烃类产生生物表面活性剂的菌株红球菌(Rhodococcus sp.) HL-6, 对其菌体细胞疏水性及所产表面活性剂进行研究。【方法】通过细胞粘附性、表面张力及乳化活性测定对菌株所产表面活性剂进行性质研究。【结果】菌株HL-6在亲水性和疏水性基质中均能产生生物表面活性剂, 在疏水性基质中可以将培养液表面张力由初始的62.487 mN/m降到30.667 mN/m, 培养液在pH 6?9及NaCl浓度1%?5%范围内乳化效果良好, 在4 °C到55 °C范围内乳化效果均为100%, 菌株对柴油的耐受能力很高, 在30%柴油浓度下依然生长良好并且有44%的乳化活性。【结论】HL-6菌株的细胞表面具有很强的疏水性, 这有助于菌体细胞对烃类的摄取。该菌株能够利用烃类基质生产生物表面活性剂, 可以明显降低培养液表面张力并且对石油烃具有良好的乳化作用。说明菌株HL-6能够适应海洋滩涂石油污染的环境, 并可用于严重石油污染区域的生物修复。     

Core flooding tests to investigate the effects of IFT reduction and wettability alteration on oil recovery during MEOR process in an Iranian oil reservoir

Microbial enhanced oil recovery (MEOR) refers to the process of using bacterial activities for more oil recovery from oil reservoirs mainly by interfacial tension reduction and wettability alteration mechanisms. Investigating the impact of these two mechanisms on enhanced oil recovery during MEOR process is the main objective of this work. Different analytical methods such as oil spreading and surface activity measurements were utilized to screen the biosurfactant-producing bacteria isolated from the brine of a specific oil reservoir located in the southwest of Iran. The isolates identified by 16S rDNA and biochemical analysis as Enterobacter cloacae (Persian Type Culture Collection (PTCC) 1798) and Enterobacter hormaechei (PTCC 1799) produce 1.53 g/l of biosurfactant. The produced biosurfactant caused substantial surface tension reduction of the growth medium and interfacial tension reduction between oil and brine to 31 and 3.2 mN/m from the original value of 72 and 29 mN/m, respectively. A novel set of core flooding tests, including in situ and ex situ scenarios, was designed to explore the potential of the isolated consortium as an agent for MEOR process. Besides, the individual effects of wettability alteration and IFT reduction on oil recovery efficiency by this process were investigated. The results show that the wettability alteration of the reservoir rock toward neutrally wet condition in the course of the adsorption of bacteria cells and biofilm formation are the dominant mechanisms on the improvement of oil recovery efficiency.     

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.     

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.     

Production of biosurfactant and antifungal compound by fermented food isolate Bacillus subtilis 20B

A biosurfactant producing strain, Bacillus subtilis 20B, was isolated from fermented food in India. The strain also showed inhibition of various fungi in in-vitro experiments on Potato Dextrose Agar medium. It was capable of growth at temperature 55 degrees C and salts up to 7%. It utilized different sugars, alcohols, hydrocarbons and oil as a carbon source, with preference for sugars. In glucose based minimal medium it produced biosurfactant which reduced surface tension to 29.5 mN/m, interfacial tension to 4.5 mN/m and gave stable emulsion with crude oil and n-hexadecane. The biosurfactant activity was stable at high temperature, a wide range of pH and salt concentrations for five days. Oil displacement experiments using biosurfactant containing broth in sand pack columns with crude oil showed 30.22% recovery. The possible application of organism as biocontrol agent and use of biosurfactant in microbial enhanced oil recovery (MEOR) is discussed.     

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.     

Characterization and optimization of biosurfactants produced by Acinetobacter baylyi ZJ2 isolated from crude oil-contaminated soil sample toward microbial enhanced oil recovery applications

The present work aims to investigate the surface activity of the biosurfactant produced by Acinetobacter baylyi ZJ2 isolated from crude oil-contaminated soil sample in China and evaluate its potential application in microbial enhanced oil recovery. The biosurfactant produced by A. baylyi ZJ2 was identified as lipopeptide based on thin-layer chromatography, Fourier transform infrared spectroscopy and nuclear magnetic resonance techniques. This biosurfactant could reduce the surface tension of water from 65 mN/m to 35 mN/m, and interfacial tension against oil from 45 mN/m to 15 mN/m. Moreover, surface activity stability results showed that this biosurfactant was effective when the salinity was lower than 8% and the pH value was 4–9, and it was especially stable when the salinity was lower than 4% and pH was 6–7. Based on the result of gas chromatography, there was a decrease in heavy components and an increase in light components, which indicated that A. baylyi ZJ2 exhibited the biodegradability on the heavy components of crude oil. Furthermore, the ability of recovering oil from oil-saturated core showed that nearly 28% additional residual oil was displaced after water flooding. The lipopeptide biosurfactant produced by A. baylyi ZJ2 presented a great potential application in microbial enhanced oil recovery process, owing its good surface activity and satisfying degradation ability to crude oil.     

A Comparative Study on Biosurfactant Activity of Crude Oil–Degrading Bacteria and Its Correlation to Total Petroleum Hydrocarbon Degradation

In this study, 11 bacteria isolated from Tapis crude oil–contaminated sites were identified by using biochemical tests and 16S rDNA gene sequencing. Their abilities to biodegrade Tapis crude oil was determined by gas chromatography before they were further screened for biosurfactant activity by employing qualitative (blood agar hemolysis, microplate assay, drop-collapse test), semiquantitative (emulsification formation), and quantitative (surface tension measurement) methods. Four isolates, namely, Acinetobacter baumanii UKMP-12T, Pseudomonas aeruginosa UKMP-14T, Rhodococcus sp. UKMP-5T, and Rhodococcus sp. UKMP-7T, exhibited high percentages in total petroleum hydrocarbon (TPH) degradation. A strong correlation between the emulsification index (E ₂₄) and surface tension measurement (r _s = +.866) as shown by Spearman rank correlation analysis suggested that these two methods were more reliable to predict biosurfactant activity. The TPH removal was also positively correlated to the ability of bacterial isolates to reduce the surface tension of growth medium, as revealed by Pearson correlation test (r_p = +.886). In conclusion, not all the biosurfactant detection protocols employed were effective. Nevertheless, the measurement of surface tension and E ₂₄ determination provided a rather rapid, easy, reproducible, and accurate result in identifying bacteria with biosurfactant-producing ability.