烟碱降解菌以烷烃为碳源产生物表面活性剂的研究

通过测定发酵过程中菌体浓度和发酵上清液的表面张力,研究了烷烃碳源和发酵条件对烟碱降解菌(Ochrobactrum sp.)产生物表面活性剂的影响。结果表明,菌株Ochrobactrum sp.以十三烷和十六烷为碳源生长较好,而利用液体石蜡可产生较多生物表面活性剂。以2%液体石蜡为碳源,装液量为40%(250 m L三角瓶),于30℃,120 r/min培养4 d时,发酵液表面张力能降低至42.1m N/m。     

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。     

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。     

一株高效生物表面活性剂产生菌的筛选鉴定及其性能研究

从新疆克拉玛依油田受石油污染的土壤中筛选到一株高效生物表面活性荆产生菌,编号为XJ-T-1,经16S rDNA同源性分析和生理生化试验鉴定为产碱杆菌.该菌株具有很强的产表面活性剂能力,全培养液的排油图直径能达到13.0cm,表面张力可降至30.0mN/m.投加10%(V/V)培养7d的XJ-T-1全培养液可使原油乳状液150min脱水率达到90%以上.XJ-T-1对原油具有高效降解作用,投加量为2%～5%(V/V),pH为中性或碱性,降解时间为7d时,XJ-T-1对7500mg/L高浓度含油废水的降解率可达80%以上.     

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。     

一株可乳化降解原油的Compostibacillus的分离及特性

【背景】通过实施多轮次微生物采油,华北油藏产出液菌浓达到了106个/mL以上,油藏内部已经形成了较稳定的微生物发酵场,从其中筛选出能够乳化降解原油的微生物,并在地面对其进行扩大培养,然后再应用到微驱油藏,以进一步提高微生物采油实施效果。【目的】筛选乳化降解原油性能良好的菌株,对其进行多相分类学鉴定和性能评价。【方法】利用原油为底物筛选乳化降解性能良好的菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等确定菌株的分类地位。通过乳化能力、降解率等方法确定菌株的原油乳化降解特性。【结果】从华北油田采集的地层水样品中分离得到一株乳化原油的菌株BLG74,经多相分类鉴定表明其是土壤堆肥芽孢杆菌(Compostibacillus humi)的新菌株,亲源性99.6%。该菌株的生长温度为30-60℃ (最适温度45℃),pH6.5-9.5(最适pH7.0),NaCl浓度0%-7%(质量体积比)。菌株BLG74在玉米浆培养基中培养,其发酵液的表面张力为56.3 mN/m,乳化力约95%,在初始原油质量浓度0.5%、温度45℃的条件下培养20d,对原油的降解率可达40.8%。【结论】菌株BLG74是可乳化降解原油的新成员,其在热盐条件下乳化降解原油的特性在石油开采中有一定的潜力。     

海洋石油降解菌AH07的分离鉴定及其石油降解性能分析

以原油为唯一碳源,从大连新港石油污染区域海底沉积物中分离获得1株石油高效降解菌AH07。通过形态学观察、生理生化特征检验及16S rDNA序列分析,确定菌株AH07为人苍白杆菌(Ochrobactrum anthropi),GenBank序列登录号为KT831449。通过单因素试验确定了菌株AH07的最优生长条件,即培养温度为30℃,培养基pH 7.0。为了进一步了解并提高菌株AH07的降解性能,选用5种氮源考察不同氮源对菌株石油降解性能的影响。结果表明,玉米粉为最佳有机氮源,NH_4NO_3为最佳无机氮源;28℃、150 r/min振荡培养10 d,菌株AH07对原油的降解率分别为58.25%和31.98%。     

营养要素对原油降解菌生长的影响

目的:研究营养要素对原油降解菌生长的影响.方法:通过紫外分光光度计于600nm处测量菌体OD值检测6种碳源、5种氮源、无机离子含量和氯化钠含量对原油降解菌K5生长的影响.通过正交实验确定了原油降解菌K5生长的最佳营养条件.结果:当蔗糖含量0.6%、硫酸铵含量0.2%、氯化钠含量0.6%、微量元素含量0.1%时,原油降解菌生长得最好.结论:确定了原油降解菌K5生长的最佳营养条件,并且发现了培养基中的氮源和碳源对菌体生长量的影响最大以及不同微量元素对菌体生长量有不同程度的促进或抑制作用.     

一株产脂肽类表面活性剂的碱性Dietzia菌及特性研究

【目的】筛选降解性能良好的产生物表面活性剂的菌株,对其进行分类学鉴定,确定所产表面活性剂物质并对各影响因素进行评价。【方法】利用液体石蜡为底物筛选降解性能良好的产生物表面活性剂菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等实验确定菌株的分类地位。通过排油圈活性、表面张力值、薄层层析等方法确定生物表面活性剂的性质,分析碳、氮源和温度、pH、盐浓度各因素对菌株产生物表面活性剂的影响。【结果】从大连新港采集的样品中分离得到一株产表面活性剂的嗜碱菌株3372,经分类鉴定表明其是Dietzia cercidiphylli的新菌株。嗜碱菌3372发酵液粗提物的排油直径为6.1 cm,表面张力可从67.62 mN/m降到32.95 mN/m,经薄层层析分析,初步鉴定为脂肽类表面活性剂。综合各因素对发酵液表面活性的影响,菌株3372在pH为9.0、适盐浓度为3%的培养基中,经30°C培养可将发酵液表面张力值降到最低。【结论】嗜碱菌3372是脂肽类生物表面活性剂产生菌的新成员,其在高盐碱条件下产生表面活性剂的特性在工业应用上有一定的潜力。     

低能离子诱变烃降解菌所产表面活性剂的研究

菌株产表面活性剂的能力直接影响其对石油烃的降解和利用,大量的研究表明,生物表面活性剂可以通过胶束来渗透、润湿、乳化、增溶、发泡、消泡等作用促进石油的利用,有效提高石油烃的降解,加快油污土壤的生物修复过程。对菌株23产表面活性剂和菌株生长的关系,发酵液中表面活性剂的提取鉴定,以及生物表面活性剂的临界胶束浓度,对温度、pH、盐度的稳定性,对石蜡的乳化活性等理化性质进行了初步分析研究,为该菌株进一步的研究以及今后实际应用提供较多的资料和信息,为其应用领域提供理论依据,以便更好的发挥其在实际生产中的功能。     

Characterization of new biosurfactant produced by Klebsiella sp. Y6-1 isolated from waste soybean oil

To obtain predominant bacteria degrading crude oil, we isolated some bacteria from waste soybean oil. Isolated bacterial strain had a marked tributyrin (C4:0) degrading activity as developed clear zone around the colony after incubation for 24h at 37 degrees C. It was identified as Klebsiella sp. Y6-1 by analysis of 16S rRNA gene. Crude biosurfactant was extracted from the culture supernatant of Klebsiella sp. Y6-1 by organic solvent (methanol:chloroform:1-butanol) after vacuum freeze drying and the extracted biosurfactant was purified by silica gel column chromatography. When the purified biosurfactant dropped, it formed degrading zone on crude oil plate. When a constituent element of the purified biosurfactant was analyzed by TLC and SDS-PAGE, it was composed of peptides and lipid. The emulsification activity and stability of biosurfactant was measured by using hydrocarbons and crude oil. The emulsification activity and stability of the biosurfactant showed better than the chemically synthesized surfactant. It reduced the surface tension of water from 72 to 32 mN/m at a concentration of 40 mg/l.     

Isolation and Characterization of Biosurfactant-and Bioemulsifier-Producing Bacteria from Petroleum Contaminated Sites in Western Canada

Biosurfactant-producing bacteria were isolated from two petroleum contaminated sites in western Canada. Seven potential biosurfactant/bioemulsifier-producing isolates were screened and characterized. All of the seven isolates were able to form emulsions. Emulsion-stabilizing capacity was also measured up to 48 hrs. Strain C-111-2 and C-203-2 would lead to highly reduced surface tension. For strain C-203-2, the optimum conditions that supported bacteria growth and production were investigated. The influences of carbon sources, medium pH values, and temperature were taken into account. The experimental results indicated that the crude oil and glucose were promising carbon sources for biosurfactants production; the isolated strains produced a maximum concentration of biosurfactant in a neutral pH environment and showed a higher surface activity under the temperature level of 35°C than that under 10°C. To further optimize the carbon and nitrogen source for biosurfactant production, response surface methodology (RSM) was applied to explore the favorable concentration of two carbon sources: glucose, crude oil, and one nitrogen source, NaNO₃. The optimal concentration of 8.1g/L, 4% and 3.9 g/L for glucose, crude oil, and NaNO₃, respectively, which can be obtained through RSM analysis.     

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.     

The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

一种脂肽类生物表面活性剂产生菌的筛选

从油田地层水中筛选分离得到1株能够产生表面活性剂的细菌,经鉴定为枯草芽孢杆菌。分析了该菌株的生理形态和生长特性,以及该菌株代谢产生的生物表面活性剂的性质。薄层色谱与原位水解显色和红外光谱分析表明,培养后菌株代谢产生的生物表面活性为脂肽。它能使水的表面张力降低到26mN/m,其临界胶束浓度为0.025mg/mL。     

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.     

Biosurfactant production by Pseudomonas aeruginosa SP4 using sequencing batch reactors: effects of oil loading rate and cycle time

In this present study, sequencing batch reactors (SBRs) were used for biosurfactant production from Pseudomonasaeruginosa SP4, which was isolated from petroleum-contaminated soil in Thailand. Two identical lab-scale aerobic SBR units were operated at a constant temperature of 37 degrees C, and a mineral medium (MM) with palm oil was used as the culture medium. The effects of oil loading rate (OLR) and cycle time on the biosurfactant production were studied. The results indicated that the optimum conditions for the biosurfactant production were at an OLR of 2 kg/m(3)days and a cycle time of 2 days/cycle, which provided a surface tension reduction of 59%, a chemical oxygen demand (COD) removal of 90%, and an oil removal of 97%. Under the optimum conditions, it was found that the biosurfactant production was maximized at an aeration time of 40 h. These preliminary results suggest that the SBR can potentially be adapted for biosurfactant production, and perhaps further developed, potentially for large-scale biosurfactant production.     

Inoculation of 1-aminocyclopropane-1-carboxylate deaminase–producing bacteria along with biosurfactant application enhances the phytoremediation efficiency of Medicago sativa in hydrocarbon-contaminated soils

Phytoremediation efficiency of Alfa alfa (Medicago sativa) was evaluated in hydrocarbon-contaminated soil with the combined application of 1-aminocyclopropane-1-carboxylate (ACC) deaminase–producing Bacillus sp. PVMX4 and an isolated biosurfactant from this strain. Results on the plant growth–promoting (PGP) traits of Bacillus sp. PVMX4 revealed that phosphate (P) solubilization, indole-3-acetic acid (IAA) production, and ACC deaminase activity were not affected by low-concentration hydrocarbon amendment in the form of crude oil. Bacillus sp. PVMX4 was able to utilize crude oil as a sole carbon source in mineral salt medium (MSM), and this strain synthesized significant quantities of biosurfactant in growth medium quantified by an emulsification index of 69.2 EI_24% and surface tension reduction of 26.2 mN/m at the end of the experimental period. Biosurfactant, when partially purified and characterized by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR), revealed it to be a lipopeptide-type biosurfactant. Pilot-scale phytoremediation studies conducted under growth chamber conditions in hydrocarbon-contaminated soil using Medicago sativa along with combined application of ACC deaminase–containing bacteria and biosurfactant recorded 76.4% hydrocarbon degradation.     

Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997