一株破乳菌破乳有效成分分析及其强化培养条件优化
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摘要
生物破乳剂是多种有效成分共存的复杂体系。目前对破乳有效成分缺乏足够的认识,难于人工调控活性物质的产量导致破乳能力不稳定、效率不高是其无法大规模生产应用的根本原因。系统分析生物破乳剂成分可使代谢调控、改善其破乳性能成为可能;同时寻找新的手段和方法提高破乳效率也可为生物破乳剂的发展注入新的活力。以破乳菌的分离、筛选为基础,摸索破乳菌培养条件,明确环境因素对其破乳效能的影响;深入研究生物破乳剂的破乳有效成分,优化提取、纯化手段,分离出破乳活性物质并利用现代化表征技术加以解析;初步探讨破乳菌株混合强化培养对破乳性能的提升。
结合生物表面活性剂的筛选方法及破乳性能测试,从大庆原油污染土壤中分离出一株高效破乳菌,经16SrDNA鉴定,明确该菌为莫海威芽孢杆菌(Bacillus mojavensis),编号XH-1。通过破乳实验及生物量的测定,对影响破乳菌生长和菌体发酵液破乳能力的培养条件进行考察,使用MMSM培养基,得出最适于培养液破乳的发酵条件为:培养基初始pH6.5,培养温度30℃,摇床转数140rpm,培养时间24h。该菌株培养液表现出优良的破乳性能:室温下,接触时间48h,40%投加量可实现O/W模型乳状液完全破乳;46℃下,接触时间10h,排油率在80%以上;55℃下,接触时间10h,排油率为100%。
通过对破乳活性成分分布及有效成分耐热性、耐酸碱性的考察,借助红外光谱、紫外可见光谱等分析手段确定蛋白类物质为该生物破乳剂的破乳有效成分之一,同时蛋白在溶液中的构象对其破乳能力至关重要。破乳活性物质存在于细胞外,粘连在菌体周围或游离于发酵液中,菌体本身不具有破乳能力。采用反复冻融手段及蛋白变性剂处理后,除菌上清液仍保持至少30%破乳活性,证明非蛋白类组分在破乳过程中的作用。
提取破乳有效成分,通过对粗提产物破乳能力、产量的比较,选择最佳粗提方法。结果发现:XH-1上清液中提取出的多糖、脂类、脂肽类物质基本不具有破乳能力;25%~45%硫酸铵分离及有机溶剂沉淀法均可有效地将破乳活性物质提取出来。使用乙醇沉淀法进行粗提,提取产率高,产物破乳效果好,方法可重复性强。改进提取工艺,粗提产率可达91%,室温下48h粗提产物排油率接近100%。通过对粗提产物成分及分子量分布分析发现:粗提物是多种蛋白、多糖等大分子物质和分子量低于1000D的小分子产物共存的复杂体系。
选用HiTrap Butyl-S FF疏水相互作用层析,结合Superdex75凝胶排阻层析的分离方法,纯化粗提产物,得到破乳活性物质。纯化产物溶液经液相色谱分离得到单峰,通过质谱鉴定确定为具有PEG结构单元的小分子。这些物质分子量分布范围从200~1000D,在溶液中聚结为胶团,在破乳过程承担重要作用。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)比较破乳能力不同组分间差异。酶切差异条带后进行质谱分析,并在数据库中比对,得到四种蛋白,其中一种为疏水蛋白。确认疏水蛋白为破乳有效成分之一,鉴定为Phosphate-binding protein。
将破乳菌XH-1与枯草芽胞杆菌(Bacillus subtilis)在改进的无机盐液体培养基中强化培养可以有效地提高生物破乳剂的破乳效率。强化培养液在室温下,接触时间48h可使O/W型模型乳状液完全破乳。与单一菌株发酵全培养液相比,缩短了发酵时间(提高6h以上),降低了破乳剂投加量,提升了破乳能力和稳定性。乳状液pH 3~7的条件下,强化培养液可维持较高破乳活性。具有良好耐温性,乳状液温度在20~120℃,破乳剂排油率变化率在15%以内。强化培养后,生物破乳剂仍主要依靠发酵过程中产生代谢产物破乳,上清液排油效果为全培养液的87%。强化培养是提高破乳活性的更为简便、有效的手段。
Microbial de-emulsifier is a complex system containing many kinds of effective ingredients. At the present time, the limit understanding of de-emulsification substances and uncontrolled productivity result in unstable de-emulsifying effectiveness and low de-emulsification activity. For this reason, the use of microbial de-emulsifier is limited. Profound investigation on effective de-emulsification substances would make it possible to adjust and control the performance of the microbial de-emulsifier. At the same time, seeking for new ways and methods to develop de-emulsification performance would also help to make big progress in bio-demulsifier research. Experiments were carried out to optimized fermentation factors for more de-emulsifer production based on superior demulsifying bacteria isolation. Environmental factors effected de-emulsification activity was clarified. More researches focused on analysis of effective de-emulsification substances. These effective ingredients were extracted, purified then characterized by modern analysis techniques. Basic studies using intensified culture were carried out to explore a simple and available way improving de-emulsification activity.
Referring to methods of isolation bio-surfactant producing bacteria combining with de-emulsifying test, a superior strain was isolated. The strain was identified by 16SrDNA as Bacillus mojavensis, named as XH-1. The fermentation conditions which affected its growth characteristics and de-emulsification ability of cell culture were investigated by testing biomass and oil drain rate of the culture. The experimental results indicated that the optimal cultivation conditions using MMSM medium were: medium pH 6.5, cultivation temperature 30℃, rotary speed 140 rpm/min, and cultivation time 24h. Under this fermentation condition, the cell culture exhibited best de-emulsifying ability, with 40 %( v/v) input amount, the oil drain rate on O/W model emulsions was: 100% under room temperature in 48h; 80% at 46℃in 10h and 100% at 55℃in 10h.
Distribution of de-emulsification activity in the culture and the stability of the active substances against high temperature and pH were studied with the help of UV, IR spectrum. The results revealed that protein was one of the active ingredients of the de-emulsifier. The active substances which were attached around the bacterial surface or suspended in the culture existed outside of bacterial cells. The bacterial cells itself did not have de-emulsification capability. Repeated freezing and thawing method and denaturant treating were used to evaluate the de-emulsification activity of the supernatant. At least 30% of its activity remained which indicated the existence of non-protein ingredients.
De-emulsification substances were extracted from the supernatant. Optimized method was chosen by comparing the de-emulsification activity and productivity of the extracted products. The results showed that polysaccharides, lipoid, lipopeptide extracted from XH-1 supernatant could not break any O/W model emulsion. Effective substances could be precipitated by 25%~45% (NH4)2SO4 and organic solvents. Extracted products settled by ethanol showed high productivity and high de-emulsification activity. The extracted method using ethanol also showed good repeatability. With the optimizing extraction techniques, extracted productivity would reach the maximum point of 91%. The oil drain rate of the crude products solution to model emulsions was 100% under room temperature in 48h. The results of ingredients measuring and molecular weight distribution analysis showed that the extracted product was a complex system containing macromolecule substances as protein, polysaccharides and micromolecule substances which molecular weight was under 1000D.
HIC and SEC purifying methods were conducted to separate and purify the activity ingredients. Only one elution peak was detected when eluted the purifying product solution through HLPC. Mass spectrograpHic analysis showed micromolecule substances with PEG structure played important role in de-emulsifying process. The molecular weight of these substances which existed as micelle in solution varied from 200-1000D. SDS-PAGE revealed the differences between fractions with varied de-emulsification activity. The differential band was digested by enzyme and characterized by mass spectrogram. Comparing the results with data base, four reliable proteins were identified. Only one of them was hydropHobic protein which was confirmed as activity ingredient, identified as pHospHate-binding protein.
To intensify the de-emulsification activity of the microbial de-emulsifier, two superior strains (Bacillus mojavensis, Bacillus subtilis) were cultured in a mineral salt medium to obtain a microbial compound de-emulsifier with high capability of de-emulsification. The cell culture could demulsify the O/W model emulsion completely in 48 hours under room temperature. Comparing with that of the single strain culture, the fermentation process of the mixed culture was shortened more than 6 hours. With less addition volume, the mixed culture showed higher de-emulsification activity and stability. The mixed culture kept high de-emulsification activity in the emulsion with different pH value ranged from 3~7. Thermo stability was detected. When the emulsion temperature varied from 20~120℃, changing rate of the oil drain rate was under 15%. After the strains were mixed cultured, the metabolites produced during the fermentation process still took the main function of de-emulsification. The supernatant remained 87% de-emulsification ability of the cell culture while the bacterial cells could hardly break the emulsion. Intensified and mixed culturing is an effective way to enhance their de-emulsification abilities.
结合生物表面活性剂的筛选方法及破乳性能测试,从大庆原油污染土壤中分离出一株高效破乳菌,经16SrDNA鉴定,明确该菌为莫海威芽孢杆菌(Bacillus mojavensis),编号XH-1。通过破乳实验及生物量的测定,对影响破乳菌生长和菌体发酵液破乳能力的培养条件进行考察,使用MMSM培养基,得出最适于培养液破乳的发酵条件为:培养基初始pH6.5,培养温度30℃,摇床转数140rpm,培养时间24h。该菌株培养液表现出优良的破乳性能:室温下,接触时间48h,40%投加量可实现O/W模型乳状液完全破乳;46℃下,接触时间10h,排油率在80%以上;55℃下,接触时间10h,排油率为100%。
通过对破乳活性成分分布及有效成分耐热性、耐酸碱性的考察,借助红外光谱、紫外可见光谱等分析手段确定蛋白类物质为该生物破乳剂的破乳有效成分之一,同时蛋白在溶液中的构象对其破乳能力至关重要。破乳活性物质存在于细胞外,粘连在菌体周围或游离于发酵液中,菌体本身不具有破乳能力。采用反复冻融手段及蛋白变性剂处理后,除菌上清液仍保持至少30%破乳活性,证明非蛋白类组分在破乳过程中的作用。
提取破乳有效成分,通过对粗提产物破乳能力、产量的比较,选择最佳粗提方法。结果发现:XH-1上清液中提取出的多糖、脂类、脂肽类物质基本不具有破乳能力;25%~45%硫酸铵分离及有机溶剂沉淀法均可有效地将破乳活性物质提取出来。使用乙醇沉淀法进行粗提,提取产率高,产物破乳效果好,方法可重复性强。改进提取工艺,粗提产率可达91%,室温下48h粗提产物排油率接近100%。通过对粗提产物成分及分子量分布分析发现:粗提物是多种蛋白、多糖等大分子物质和分子量低于1000D的小分子产物共存的复杂体系。
选用HiTrap Butyl-S FF疏水相互作用层析,结合Superdex75凝胶排阻层析的分离方法,纯化粗提产物,得到破乳活性物质。纯化产物溶液经液相色谱分离得到单峰,通过质谱鉴定确定为具有PEG结构单元的小分子。这些物质分子量分布范围从200~1000D,在溶液中聚结为胶团,在破乳过程承担重要作用。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)比较破乳能力不同组分间差异。酶切差异条带后进行质谱分析,并在数据库中比对,得到四种蛋白,其中一种为疏水蛋白。确认疏水蛋白为破乳有效成分之一,鉴定为Phosphate-binding protein。
将破乳菌XH-1与枯草芽胞杆菌(Bacillus subtilis)在改进的无机盐液体培养基中强化培养可以有效地提高生物破乳剂的破乳效率。强化培养液在室温下,接触时间48h可使O/W型模型乳状液完全破乳。与单一菌株发酵全培养液相比,缩短了发酵时间(提高6h以上),降低了破乳剂投加量,提升了破乳能力和稳定性。乳状液pH 3~7的条件下,强化培养液可维持较高破乳活性。具有良好耐温性,乳状液温度在20~120℃,破乳剂排油率变化率在15%以内。强化培养后,生物破乳剂仍主要依靠发酵过程中产生代谢产物破乳,上清液排油效果为全培养液的87%。强化培养是提高破乳活性的更为简便、有效的手段。
Microbial de-emulsifier is a complex system containing many kinds of effective ingredients. At the present time, the limit understanding of de-emulsification substances and uncontrolled productivity result in unstable de-emulsifying effectiveness and low de-emulsification activity. For this reason, the use of microbial de-emulsifier is limited. Profound investigation on effective de-emulsification substances would make it possible to adjust and control the performance of the microbial de-emulsifier. At the same time, seeking for new ways and methods to develop de-emulsification performance would also help to make big progress in bio-demulsifier research. Experiments were carried out to optimized fermentation factors for more de-emulsifer production based on superior demulsifying bacteria isolation. Environmental factors effected de-emulsification activity was clarified. More researches focused on analysis of effective de-emulsification substances. These effective ingredients were extracted, purified then characterized by modern analysis techniques. Basic studies using intensified culture were carried out to explore a simple and available way improving de-emulsification activity.
Referring to methods of isolation bio-surfactant producing bacteria combining with de-emulsifying test, a superior strain was isolated. The strain was identified by 16SrDNA as Bacillus mojavensis, named as XH-1. The fermentation conditions which affected its growth characteristics and de-emulsification ability of cell culture were investigated by testing biomass and oil drain rate of the culture. The experimental results indicated that the optimal cultivation conditions using MMSM medium were: medium pH 6.5, cultivation temperature 30℃, rotary speed 140 rpm/min, and cultivation time 24h. Under this fermentation condition, the cell culture exhibited best de-emulsifying ability, with 40 %( v/v) input amount, the oil drain rate on O/W model emulsions was: 100% under room temperature in 48h; 80% at 46℃in 10h and 100% at 55℃in 10h.
Distribution of de-emulsification activity in the culture and the stability of the active substances against high temperature and pH were studied with the help of UV, IR spectrum. The results revealed that protein was one of the active ingredients of the de-emulsifier. The active substances which were attached around the bacterial surface or suspended in the culture existed outside of bacterial cells. The bacterial cells itself did not have de-emulsification capability. Repeated freezing and thawing method and denaturant treating were used to evaluate the de-emulsification activity of the supernatant. At least 30% of its activity remained which indicated the existence of non-protein ingredients.
De-emulsification substances were extracted from the supernatant. Optimized method was chosen by comparing the de-emulsification activity and productivity of the extracted products. The results showed that polysaccharides, lipoid, lipopeptide extracted from XH-1 supernatant could not break any O/W model emulsion. Effective substances could be precipitated by 25%~45% (NH4)2SO4 and organic solvents. Extracted products settled by ethanol showed high productivity and high de-emulsification activity. The extracted method using ethanol also showed good repeatability. With the optimizing extraction techniques, extracted productivity would reach the maximum point of 91%. The oil drain rate of the crude products solution to model emulsions was 100% under room temperature in 48h. The results of ingredients measuring and molecular weight distribution analysis showed that the extracted product was a complex system containing macromolecule substances as protein, polysaccharides and micromolecule substances which molecular weight was under 1000D.
HIC and SEC purifying methods were conducted to separate and purify the activity ingredients. Only one elution peak was detected when eluted the purifying product solution through HLPC. Mass spectrograpHic analysis showed micromolecule substances with PEG structure played important role in de-emulsifying process. The molecular weight of these substances which existed as micelle in solution varied from 200-1000D. SDS-PAGE revealed the differences between fractions with varied de-emulsification activity. The differential band was digested by enzyme and characterized by mass spectrogram. Comparing the results with data base, four reliable proteins were identified. Only one of them was hydropHobic protein which was confirmed as activity ingredient, identified as pHospHate-binding protein.
To intensify the de-emulsification activity of the microbial de-emulsifier, two superior strains (Bacillus mojavensis, Bacillus subtilis) were cultured in a mineral salt medium to obtain a microbial compound de-emulsifier with high capability of de-emulsification. The cell culture could demulsify the O/W model emulsion completely in 48 hours under room temperature. Comparing with that of the single strain culture, the fermentation process of the mixed culture was shortened more than 6 hours. With less addition volume, the mixed culture showed higher de-emulsification activity and stability. The mixed culture kept high de-emulsification activity in the emulsion with different pH value ranged from 3~7. Thermo stability was detected. When the emulsion temperature varied from 20~120℃, changing rate of the oil drain rate was under 15%. After the strains were mixed cultured, the metabolites produced during the fermentation process still took the main function of de-emulsification. The supernatant remained 87% de-emulsification ability of the cell culture while the bacterial cells could hardly break the emulsion. Intensified and mixed culturing is an effective way to enhance their de-emulsification abilities.
引文
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