生物破乳菌的筛选及发酵条件的优化

生物破乳剂的开发可以降低油田乳状液对石油工业和生态环境的负面影响并减少化学破乳剂的使用量。本研究建立了一套高效、便捷的破乳菌筛选方法, 并对破乳菌的特性进行研究。利用大庆油田受石油污染土壤作为菌种来源, 将Tween 60-water(0.072%, V/V)和Span 60-oil (0.028%, V/V)以6.5:3.5体积比配置出可以稳定200 h以上的O/W型乳状液, 用于破乳菌破乳效能的评价。经过分离纯化、血平板试验、排油试验和破乳试验最终筛选出2株24 h平均排油率在80%以上的优势破乳菌, 初步鉴定为芽孢杆菌属(Bascillus)。通过该破乳菌发酵条的件优化得到, 当温度为25°C, 摇床转数为160 r/min, pH值为9, 接菌量为20%时对该破乳菌生长速率最快, 积累发酵产物的量最多; 当温度为35°C, 摇床转数为120 r/min, pH值为9, 接菌量为2%时该破乳菌代谢产物的破乳活性最高。     

枯草芽孢杆菌发酵条件优化及其破乳效能

本文对枯草芽孢杆菌在不同碳源、氮源培养基的生长及破乳效能进行了研究, 并通过正交试验对枯草芽孢杆菌的发酵条件进行优化结果表明, 单一碳源葡萄糖和混合碳源葡萄糖 + 液体石蜡的培养基可提高枯草芽孢杆菌的发酵产量; 单一碳源葡萄糖、混合碳源葡萄糖 + 汽油和以硝酸铵 + 酵母膏为氮源的菌液有较高的破乳效能; 在正交试验中, 培养温度对枯草芽孢杆菌的发酵产量影响最大, 其最优组合为: 培养温度25oC, 摇床转数140 r/min, 培养pH值7.0, 接菌量6 mL, 培养时间24 h。摇床转数对枯草芽孢杆菌的发酵产物的破乳效能影响最大, 优化结果为: 培养温度25oC, 摇床转数140 r/min, pH值7.0, 培养时间20 h。     

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。     

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。     

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。     

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。     

植物乳杆菌对重金属Pb~(2+)、Cr~(6+)和Cu~(2+)的耐受性与吸附作用相关性比较

从四川矿区泡菜样品中分离得到1株对重金属铅(Pb)、铬(Cr)和铜(Cu)具有较高耐受性的菌株,经16S rDNA初步鉴定为1株植物乳杆菌。研究重金属铅、铬和铜对该植物乳杆菌的最小抑制浓度(MIC)。比较不同初始pH、初始离子浓度、吸附时间和菌体加入量对植物乳杆菌吸附3种重金属的影响,探讨MIC与吸附作用相关性。使用MIC的方法测定重金属对该菌的最小抑制浓度,原子吸收法测定对重金属的吸附效果。研究表明,该菌对Pb~(2+)、Cr~(6+)和Cu~(2+)的耐受性分别为6.67、0.67和2.17 mmol/L;其吸附性最适初始pH分别为4、6和6;最优初始离子浓度分别为100、100和50 mg/L;最优加菌量分别为3、6和5 g/L;最佳吸附时间分别为12、2和8 h。在100 mg/L的初始离子浓度下对Pb~(2+)、Cr~(6+)和Cu~(2+)的吸附率最高分别可达96%、61%和49%。MIC与吸附作用没有明显相关性。结果表明该菌具有优良的吸附性能,为今后含有乳酸菌的食品或饲料制剂的开发提供了新的乳酸菌种。     

高糖和氮源对鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸发酵的影响

鼠李糖乳杆菌经实验室耐高糖高酸选育,能够在高糖浓度下高效高产L-乳酸。以酵母粉为氮源和生长因子,葡萄糖初始浓度分别为120 g/L和146 g/L,摇瓶培养120h,L-乳酸产量分别为104g/L和117.5g/L,L-乳酸得率分别为86.7%和80.5%。高葡萄糖浓度对菌的生长和乳酸发酵有一定的抑制。增加接种量,在高糖浓度发酵条件下,可以缩短发酵时间,但对增加乳酸产量效果不明显。乳酸浓度对鼠李糖乳杆菌生长和产酸有显著的影响。初始乳酸浓度到达70g/L以上时,鼠李糖乳杆菌基本不生长和产酸,葡萄糖消耗也被抑制。酵母粉是鼠李糖乳杆菌的优良氮源,使用其它被测试的氮源菌体生长和产酸都有一定程度的下降。用廉价的黄豆粉并补充微量维生素液,替代培养基中的酵母粉,可以使产酸浓度和碳源得率得以基本维持。     

固定化植物乳杆菌的制备及其发酵条件优化

采用海藻酸钠包埋植物乳杆菌并通过测定固定化细胞发酵清液的抑菌效果,优化得到的固定化最佳工艺条件为:海藻酸钠浓度为3%,CaCl2浓度为1.5%,菌悬液体积为3.5 mL(4.0×108 cfu/mL).固定化细胞重复发酵多批次效果良好.固定化细胞发酵条件优化结果表明:最适pH为7.0,最适温度为36℃,培养基中添加0....     

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。     

Evaluation of screening methods for demulsifying bacteria and characterization of lipopeptide bio-demulsifier produced by Alcaligenes sp

In this paper, surface tension measurement, oil-spreading test and blood-plate hemolysis test were attempted in the screening of demulsifying bacteria. After the comparison to the screening results obtained in demulsification test, 50 mN/m of surface tension of culture was proposed as a preliminary screening standard for potential demulsifying bacteria. For the identification of efficient demulsifying strains, surface tension level was set at 40 mN/m. The detected strains were further verified in demulsification test. Compared to using demulsification test alone as screening method, the proposed screening protocol would be more efficient. From the screening, a highly efficient demulsifying stain, S-XJ-1, was isolated from petroleum-contaminated soil and identified as Alcaligenes sp. by 16S rRNA gene and physiological test. It achieved 96.5% and 49.8% of emulsion breaking ratio in W/O and O/W kerosene emulsion within 24h, respectively, and also showed 95% of water separation ratio in oilfield petroleum emulsion within 2h. The bio-demulsifier was found to be cell-wall combined. After soxhlet extraction and purification through silicon-gel column, the bio-demulsifier was then identified as lipopeptide biosurfactant by TLC and FT-IR.     

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.     

Ecofriendly demulsification of water in oil emulsions by an efficient biodemulsifier producing bacterium isolated from oil contaminated environment

Objective

Water in oil emulsions increase oil processing costs and cause damage to refinery equipment which necessitates demulsification. Since chemical demulsifiers cause environmental pollution, biodemulsifiers have been paid more attention. This study aims to identify biodemulsifier-producing bacteria from petroleum contaminated environments.

Results

As a result, several biodemulsifier producing strains were found that Stenotrophomonas sp. strain HS7 (accession number: MF445088) which produced a cell associated biodemulsifier showed the highest demulsifying ratio, 98.57% for water in kerosene and 66.28% for water in crude oil emulsion after 48 h. 35 °C, pH 7, 48 h incubation and ammonium nitrate as nitrogen source were optimum conditions for biodemulsifier production. Furthermore, it was found that hydrophobic carbon sources like as liquid paraffin is not preferred as the sole carbon source while a combination of various carbon sources including liquid paraffin will increase demulsification efficiency of the biodemulsifier.

Conclusions

The appropriate potential of this biodemulsifier strengthens the possibility of its application in industries especially petroleum industry.

     

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 the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

Comparison between waste frying oil and paraffin as carbon source in the production of biodemulsifier by Dietzia sp. S-JS-1

In order to lower the production cost, waste frying oils were used in the biosynthesis of demulsifier by Dietzia sp. S-JS-1, which was isolated from petroleum contaminated soil. After 7 days of cultivation, the biomass concentration of the most suitable waste frying oil (WFO II) culture reached 3.78 g/L, which was 2.4 times the concentration of paraffin culture. The biodemulsifier produced with WFO II culture broke the emulsions more efficiently than that produced with paraffin culture, given the same volume ratio of carbon source in the culture medium and the same cultivation conditions. It achieved 88.3% of oil separation ratio in W/O emulsion and 76.4% of water separation ratio in O/W emulsion within 5 h. With the aid of thin layer chromatography (TLC) and Fourier transform infrared (FTIR) spectrometry, biodemulsifiers produced from both paraffin and WFO II were identified as a mixture of lipopeptide homologues. The subtle variation in the distribution of these homologues and high biomass concentration of WFO II cultures may account for the afore-mentioned good demulsification performance.