pH对兼性嗜碱菌Alcaligenes sp.XJ-T-1产生物破乳剂性能的影响

从受石油污染土壤中筛选出一株生物破乳剂产生菌Alcaligenes sp.XJ-T-1,生长的最适初始pH范围为9.0～11.0,为兼性嗜碱菌,能在初始pH 6.0～pH 11.0生长并产生生物破乳剂,但只有在碱性环境下才产生胞外产物。XJ-T-1菌悬液和胞外产物溶液可将蒸馏水表面张力分别降低到30 mN/m左右和35 mN/m左右。经过TLC分析,初步推测XJ-T-1产生的胞外产物为脂肽类和糖脂类的混合物。XJ-T-1菌悬液主要针对O/W型乳状液破乳,胞外产物主要针对W/O型乳状液破乳,初始pH 9.0培养下的菌悬液和胞外产物破乳效果最好。投加210 mg/L菌悬液可使O/W型乳状液在24 h的破乳率达77.5%,投加40 mg/L胞外粗产物溶液可使W/O型乳状液在24 h的破乳率达90.0%。通过透射电镜照片推测,随着培养pH提高到9.0以上,胞外产物的产生使XJ-T-1利用石蜡的过程发生变化。     

生物破乳剂产生菌的筛选及其方法研究

针对生物破乳剂产生菌筛选难的问题, 采用显色法、溶血细胞测试法、表面张力测定法和排油圈法从6种不同菌源对生物破乳菌产生菌进行了筛选。通过试验筛选得到了17株生物破乳剂产生菌, 其中24h内破乳率高于70%的破乳菌有5株; 油田含油污泥、采油废水生物处理污泥和污水处理厂剩余污泥是筛选破乳菌的较好的菌源; 显色法、溶血圈法存在检测范围的局限性; 表面张力测定法和排油圈法是最为简易和准确的生物表面活性剂产生菌的筛选方法, 采用模型乳状液对生物破乳剂产生菌进行筛选最为直接和准确, 但工作量大、所需时间长, 因此在筛选高效破乳菌时, 建议采用表面张力、排油圈法进行初筛, 而后通过模型乳状液破乳进行验证。     

生物破乳剂产生菌的筛选及其方法研究

针对生物破乳剂产生菌筛选难的问题,采用显色法、溶血细胞测试法、表面张力测定法和排油圈法从6种不同菌源对生物破乳菌产生茵进行了筛选.通过试验筛选得到了17株生物破乳剂产生茵,其中24h内破乳率高于70%的破乳菌有5株;油田含油污泥、采油废水生物处理污泥和污水处理厂剩余污泥是筛选破乳菌的较好的菌源:显色法、溶血圈法存在检测范围的局限性;表面张力测定法和排油圈法是最为简易和准确的生物表面活性剂产生茵的筛选方法,采用模型乳状液对生物破乳剂产生菌进行筛选最为直接和准确,但工作量大、所需时间长,因此在筛选高效破乳菌时,建议采用表面张力、排油圈法进行初筛,而后通过模型乳状液破乳进行验证.     

Dietzia sp.S-JS-1利用煎炸废油生产生物破乳剂及其性能研究

以前期研究中筛选得到的破乳剂产生菌Dietzia sp.S-JS-1为研究对象,采用煎炸废油为培养碳源,考察菌株的生物量和表面张力,研究处理方式、温度、乳状液pH对破乳剂在两种模型乳状液W/O型(water in oil)和O/W型(oilin water)中破乳性能的影响,并初步分析生物破乳剂成分。结果表明:菌株最大生物量为2.6 g/L,其产生的破乳剂能够将纯水表面张力从72.0 mN/m降低到32.5 mN/m。冻融对破乳剂效果的影响小于高温灭菌;破乳剂经冷冻干燥处理后的破乳效果明显好于烘干处理;破乳剂在35℃～75℃时具有较好的破乳效果,脱水率均在75%以上;破乳剂在W/O型乳状液中的效果随着pH变大而逐渐增加,pH=10时的脱水率高达99.8%,而在O/W型乳状液中,pH=7时的脱水率最高,为90%左右。薄层色谱结果表明S-JS-1利用煎炸油生产的生物破乳剂可能含有5种脂肽类物质。     

pH对破乳菌Alcaligenes sp. S-XJ-1破乳性能的影响

从受石油污染的土壤中筛选出一株破乳菌Alcaligenes sp. S-XJ-1,该菌在初始pH6.0-11.0均可生长,其最适初始pH为10.0。在初始pH10.0条件下培养,破乳菌产量最高可达4.8g/L,菌悬液投加量为1000mg/L时,24h破乳率在85%以上。同时,该培养条件下得到的生物破乳菌细胞表面疏水性最大,对碳氢化合物的吸附能力达72.7%,接触角达115°。采用稳定性分析仪分别对pH7.0和pH10.0条件下培养得到的菌体的破乳效果进行分析,结果发现与初始pH7.0相比,初始pH10.0培养得到的破乳菌可以加速分散相粒径的增大,最终使乳状液破乳速率大幅提高。     

Dietzia sp. S-JS-1利用废弃油脂生产生物破乳剂的研究

生物破乳剂是近期开发出来的用于油水分离的新型破乳剂。本研究利用从受石油污染的土壤中筛选得到、并采用16S rRNA鉴定为Dietzia sp.的一株破乳剂产生菌, 在以废弃油脂MWFO、SWFO为碳源培养下, 得到的生物破乳剂的粗重为4 g/L、3.5 g/L; 对于W/O、O/W模型乳状液的破乳效果均可超过以液体石蜡产生的破乳剂, 且以SWFO废弃油脂培养得到的生物破乳剂可以同时应用于两种模型乳状液的使用。对于碳源利用方面Dietzia sp.在利用两种废弃油脂脂肪酸的过程中, 都是优先利用C16和C18的脂肪酸, 但对于两种废弃油脂的利用率上存在一定差异。采用TLC和FTIR分析发现, 3种碳源培养得到的生物破乳剂均为脂肽类生物破乳剂, 其破乳剂的化学结构还有待进一步研究。     

Effective biodemulsifier components secreted by Bacillus mojavensis XH-1 and analysis of the demulsification process

The purpose of the present study was to investigate the effective components of the demulsifying bacterial strain Bacillus mojavensis XH-1 and its demulsification process. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the shotgun LC–MS/MS method were used to separate and identify proteins with efficient demulsification activity. The zeta potential changes of the emulsion before and after addition of the biodemulsifier were tested, and the relationships between oil-in-water interfacial tension, the demulsification efficiency and the biodemulsifier structure were examined. The study results indicate that the effective biodemulsifier components were extracellular proteins attached to the cells or secreted into the culture solution that presented as a 50–80 kDa band observed by SDS-PAGE. Six of the proteins were unknown or unnamed, and the demulsifying functions of another 14 proteins had not been previously reported. The main demulsification mechanisms were determined to be solubilization and replacement. When the concentration of the biodemulsifier was low, the replacement mechanism dominated, and the demulsification ratio increased with the biodemulsifier concentration. Solubilization dominated when a high concentration of biodemulsifier was provided, and the demulsification ratio decreased as the biodemulsifier concentration increased.     

生物破乳剂产生菌混合培养及其发酵条件的优化

【目的】对菌株L1和XH1的混合发酵条件进行优化,为混合菌发酵生物破乳剂的实际生产和应用提供理论依据。【方法】利用响应面实验(RSM)的中心组合旋转设计方法(CCRD)针对混合菌的发酵条件进行优化,通过对模型乳状液进行破乳实验,以排油率作为发酵液破乳效能的评价标准。【结果】经模型的分析与验证,确定最佳发酵条件为：种子液比例(L1:XH1)为3:2,葡萄糖投加时间为第4天,投加葡萄糖后再培养21 h,液体石蜡含量3.6% (体积比)。【结论】与破乳菌XH1和L1单独培养相比,经混合培养后获得复合生物破乳剂具有投加量少、破乳接触时间短的优势。同时双株破乳菌复配培养有效地提高了培养基中主要营养物质的利用率,减少了对底物的浪费。     

Chemically modified surface functional groups of Alcaligenes sp. S-XJ-1 to enhance its demulsifying capability

Cell-surface functional groups (amino, carboxyl, hydroxyl, as well as phosphate) were chemically modified in various ways to enhance the demulsification capability of the demulsifying bacteria Alcaligenes sp. S-XJ-1. Results demonstrated that the demulsifying activity was significantly inhibited by amino enrichment with cetyl trimethyl ammonium bromide, amino methylation, hydroxyl acetylation, and phosphate esterification, but was gradually promoted by carboxyl blocking with increasing the extents of esterification. Compared with the raw biomass, an optimal esterification of carboxyl moieties enhanced the demulsification ratio by 26.5% and shortened the emulsion half-life from 24 to 8.8 h. The demulsification boost was found to be dominated by strengthened hydrophobicity (from 53° to 74°) and weakened electronegativity (from −34.6 to −4.3 mV at pH 7.0) of the cell surface, allowing the rapid dispersion and adsorption of cells onto the oil-water interface. The chemical modification of the functional groups on the biomass surface is a promising tool for the creation of a high-performance bacterial demulsifier.

     

Characterization of antarctic hydrocarbon-degrading bacteria capable of producing bioemulsifiers.

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, two heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from Terra Nova Bay (Ross sea, Antarctica) by using aliphatic and artomatic hydrocarbons as the principal carbon source. These gram-positive, aerobic, cocci-shaped bacteria use a various number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and biphenyl. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both an extracellular and cell-bound surface-active mixture of trehalose lipids which reduced the surface tension of water from 72 mN/m to 32mN/m. This class of glycolipids was found to be produced only by marine rhodococci. The 16S-rRNA gene sequence analysis showed that both strains are members of the G + C rich gram-positive group of the phylum Proteobacteria and was found to be almost identical to that of Rhodococcus fascians DSM 20669. The potential of these strains for in situ bioremediation of contaminated cold marine environment is discussed in the present study.     

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.     

生物破乳菌形态、表面性质和物质特征研究方法与进展

生物破乳菌在石油开采与加工行业的研究已经引起各界的广泛关注,然而由于生物破乳菌菌体形态、表面性质和表面物质的复杂性,使菌体的破乳活性特征尚未被揭示。本文介绍了生物破乳剂的来源、合成及破乳机制;归纳了影响生物破乳菌破乳活性的菌体形态、表面性质和表面物质三方面因素的研究进展,特别是总结了相关研究的方法;最后在此基础上对今后研究方向提出展望。     

Investigation of the relationship between cell surface hydrophobicity and demulsifying capability of biodemulsifier-producing bacteria

Background

Cell surface hydrophobicity (CSH) is one of the key physicochemical features of biodemulsifier-producing bacteria that influence their demulsification capability maintenance in petroleum contaminated environments.

Methods

In present study, biodemulsifier-producing bacteria were isolated from petroleum contaminated environments using different isolation media and the correlation between their CSH and demulsifying ability was investigated. The demulsifying ability of isolates was measured through demulsification tests on water in kerosene emulsions. The microbial adhesion to the hydrocarbon (MATH) assay was used to denote their CSH.

Results

The evaluation of CSH showed that majority of biodemulsifier producing bacteria have high CSH which indicating a positive correlation between CSH and demulsifying capability.

Conclusions

According to these results it can be concluded that CSH can be used as an indicator for assessment of biodemulsifier-producing bacteria and screening of new isolates for their biodemulsifier production.

     

Investigation of potential biosurfactant production among phytopathogenic corynebacteria and related soil microbes

PhytopathogenicCorynebacterium species and related soil microbes were examined for their ability to produce surfactants from hydrocarbon substrate. Surfactant production was detected by the lowering of both surface and interfacial tensions, by the formation of high reciprocal critical micelle concentrations (CMCs), and by the possession of properties of emulsification and demulsification. Whole broth cultures of severalC. fascians strains,C. insidiosum ICPB CI13A,Arthrobacter paraffineus NRRL B-3453, andNocardia amarae NRRL B-8176 lowered surface tensions from 58.5 mN/m to 27–33 mN/m, lowered interfacial tensions from 33.5 mN/m to 0.55–4.0 mN/m, and had high reciprocal CMCs. Whole-broth cultures ofN. amarae NRRL B-8176 had excellent demulsification ability; those of someC. fascians strains andC. insidiosum ICPB CI13A produced unusual gel-like emulsions. These properties are believed to aid in the attachment of phytopathogenic corynebacteria to the surfaces of host plants.     

Marine biosurfactants. IV. Production,characterization and biosynthesis of an anionic glucose lipid from the marine bacterial strain MM1

Summary During screening for biosurfactants among marine, n-alkane-utilizing bacteria, low- and high-molecular surface-active substances were detected. The marine bacterial strain MM1 was found to synthesize a novel glycolipid that has not so far been cited in the literature. Both ¹H, ¹³C-nuclear magnetic resonance spectroscopic and positive ion fast atom bombardment mass spectrometer studies led to the identification of a glucose lipid consisting of four 3-OH-decanoic acids, which are linked together by ester bonds. The lipophilic moiety is coupled glycosidically with C-1 of glucose. The glucolipid reduced the surface tension from 72 mN/m to 30 mN/m while the minimum interfacial tension towards n-hexadecane was lowered to values smaller than 5 mN/m. Correspondence to: S. Lang     

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.     

Comparison of methods to detect biosurfactant production by diverse microorganisms

Three methods to detect biosurfactant production, drop collapse, oil spreading, and blood agar lysis, were compared for their ease of use and reliability in relation to the ability of the cultures to reduce surface tension. The three methods were used to test for biosurfactant production in 205 environmental strains with different phylogenetic affiliations. Surface tension of select strains that gave conflicting results with the above three methods was also measured. Sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mN/m. There was a very strong, negative, linear correlation between the diameter of clear zone obtained with the oil spreading technique and surface tension (rs = -0.959) and a weaker negative correlation between drop collapse method and surface tension (rs = -0.82), suggesting that the oil spreading technique better predicted biosurfactant production than the drop collapse method. The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production. The large number of false negatives and positives obtained with the blood agar lysis method and its poor correlation to surface tension (rs = -0.15) demonstrated that it is not a reliable method to detect biosurfactant production.     

Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites

Biosurfactant-producing bacteria were isolated from terrestrial and marine samples collected in areas contaminated with crude oil or its byproducts. Isolates were screened for biosurfactant/bioemulsifier production in different carbon sources (glucose, fructose, sucrose and kerosene) using the qualitative drop-collapse test. Glucose produced the highest number of positive results (17 of 185 isolates). All 17 isolates produced emulsions with kerosene and 12 exhibited high emulsion-stabilizing capacity, maintaining 50% of the original emulsion volume for 48 h. Eight of the 17 isolates reduced the growth medium surface tension below 40 mN m(-1) with 5 exhibiting this capacity in cell-free filtrates. Onset of biosurfactant production differed among the isolates, with some initiating synthesis during the exponential growth phase and others after the stationary phase was reached. Increasing temperature from 25 to 35 degrees C accelerated onset of biosurfactant production in only two isolates while pH (6.5-7.6) had no effect in any isolate tested. Isolation from petroleum contaminated sites using the screening protocol presented proved to be a rapid and effective manner to identify bacterial isolates with potential industrial applications.     

Palmitoylation of a pulmonary surfactant protein C analogue affects the surface associated lipid reservoir and film stability

Surfactant protein C (SP-C) is a lipopeptide that contains two thioester-linked palmitoyl groups and is considered to be important for formation of the alveolar surface active lipid film. Here, a non- or dipalmitoylated SP-C analogue (SP-C(Leu)), in which all helical Val residues were replaced with Leu and Cys-5 and Cys-6 were replaced with Ser, was tested for surface activity in a captive bubble system (CBS). SP-C(Leu), either palmitoylated at Ser-5 and Ser-6 or non-palmitoylated, was added to mixtures of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidyl glycerol (PG)/palmitic acid (PA), 68:22:9, (by mass) at a concentration of 2 and 5%. With 2% peptide, surface film formation was rapid, reaching a surface tension below 25 mN/m within 5 s, but the samples with 5% SP-C(Leu) required more than 20 s to reach values below 25 mN/m. Minimum surface tension for the samples with dipalmitoylated SP-C(Leu) was below 1.5 mN/m and very stable, as the surface tension increased by less than 0.5 mN/m within 10 min at constant bubble volume. Minimum surface tension for the non-palmitoylated SP-C(Leu) was approximately 2 and 5 mN/m for 2 and 5% peptide, respectively, but the films were less stable as seen by frequent bubble clicking at low surface tensions. Films with dipalmitoylated SP-C(Leu) that were dynamically cycled at 20-30 cycles/min were substantially less compressible at a surface tension of 20 mN/m (0.007 m/mN) than those that contained the non-palmitoylated peptide (0.02 m/mN). After subphase depletion, the incorporation of lipids into the surface active film during initial bubble expansion occurred at a relatively low surface tension (about 35 mN/m) for the samples with dipalmitoylated SP-C(Leu) compared to approximately 45 mN/m for those containing the non-palmitoylated peptide. Furthermore, for samples that contained non-palmitoylated SP-C(Leu), the ability to reach near zero stable surface tension was lost after a few adsorption steps, whereas with the dipalmitoylated peptide the film quality did not deteriorate even after more than 10 expansion steps and the incorporation of reservoir material equivalent to more than two monolayers. It appears that the covalently linked palmitoyl groups of the SP-C analogue studied are important for the mechanical stability of the lipid film, for the capacity to incorporate material from the reservoir into the surface active film upon area expansion, and for the low film compressibility of dynamically cycled films.     

Screening of bioemulsifier-producing micro-organisms isolated from oil-contaminated sites

Eighty-eight micro-organisms were isolated from oil-contaminated soils and checked for their extracellular bioemulsifier producing potential. The micro-organisms were screened on the basis of oil spread, drop collapse and emulsification index. Most efficient strains were characterized as Lysinibacillus sp. SP1025 and Bacillus cereus SP1035. In Lysinibacillus sp. SP1025, the E-24 index, surface tension and production of crude bioemulsifier were found to be 83.3 % with diesel, 34.20?±?0.03 mN/m and 3.07?±?0.62 g/L, respectively, whereas in the case of Bacillus cereus SP1035, the E-24 index, surface tension and production of crude bioemulsifier recorded were 76.5 % with diesel, 43.42?±?0.03 mN/m and 3.90?±?0.3 g/L. Crude biomemulsifier produced by selected micro-organisms was stable, withstanding a wide temperature and pH range with an E-24 Index value greater than 50 %. All emulsions formed were oil-in-water type. Emulsions formed with tested aliphatic and aromatic hydrocarbons, except those formed with ester based oils, were 100 % stable with the entire organic layer converted into emulsion. To the best of our knowledge this is the first report for bioemulsifier production from the genus Lysinibacillus.