CN114457135A - 一种糖蛋白类型生物乳化剂及其应用 - Google Patents
一种糖蛋白类型生物乳化剂及其应用 Download PDFInfo
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Abstract
一种糖蛋白类型生物乳化剂及其应用,所述乳化剂是从保藏号为CGMCC No.1228的嗜热脱氮地芽孢杆菌NG80‑2菌株发酵液中制备的,其主要组分为糖蛋白。本发明获得的生物乳化剂可用于稠油降黏及提高原油采收率。所述乳化剂具有良好的乳化活性(乳化柴油达80%以上),且在高温、高盐及极端酸碱性条件下具有良好的乳化稳定性。本发明获得的生物乳化剂对稠油有明显的降黏效果,可将辽河稠油和鲁克沁特稠油的乳化粘度分别降低81.03%和55.27%,并在模拟高温储层条件下有效提高了原油采收率至15.6%。本发明获得乳化剂的发酵过程短,发酵碳源廉价易得,极大降低了生产成本,产量可达3‑5g/L,展现出了重大经济效益和规模化应用的潜力。
Description
技术领域
本发明属于生物工程技术领域,涉及一种耐高温、耐盐且耐酸碱的生物乳化剂的生产及其在原油乳化降黏及提高采收率中的应用。
背景技术
与化学采油技术相比,微生物采油技术(MEOR)具有操作简单、成本效益高、环境友好等优点,近年来发展迅速。MEOR利用微生物及其代谢产物(如生物表面活性剂、聚合物和气体)回收地下残余原油。在这些代谢产物中,生物表面活性剂可以通过改变岩石润湿性和乳化原油来显著改善油在多孔介质中的流动性,引起了世界范围内的广泛关注。因此,越来越多的研究致力于分离表面活性剂生产菌株,并试图利用这些分离的微生物提高高含水量油藏的采收率。同时,石油碳氢化合物成分复杂,包括芳香烃、正构烷烃、树脂和沥青质组分,以及含有氮、氧和硫的不同有机化合物,它们流动性差,生物毒性强,其造成的污染会对生态系统构成严重威胁。
生物表面活性剂是由疏水性和亲水性基团组成的两亲性物质。根据分子量大小,生物表面活性剂分为两类:(i)降低表面张力和界面张力的低分子量生物表面活性剂和(ii)高分子量生物表面活性剂,即生物乳化剂,其在稳定油水乳状液方面更为有效,而不会显著降低表面张力。生物乳化剂,也被称为胞外多糖(EPSs)或高分子量生物聚合物,由脂多糖、杂多糖或多糖蛋白等复杂化合物组成。生物乳化剂的化学组成和乳化性能受到外部因素的强烈影响,如营养物质的有效性(碳水化合物、氮和磷)、pH值、盐度和温度等。
极端微生物所处环境明显比嗜中温或中性微生物分泌胞外乳化剂的环境压力更不宜生存,这就意味着其更能适应油藏或污染场地复杂的集高温、高压、低氧及高矿化度的极端环境。极端微生物必须通过独特的机制适应恶劣的环境,胞外聚合物的生物合成是其重要的生存机制之一。目前为止,人们普遍认为极端微生物将为生物技术的新工艺开发提供宝贵的资源,包括合成独特的生物乳化剂。栖息在不同极端环境中的极端微生物被认为是生物乳化剂最有前途的产生菌,并且对极端微生物(嗜热菌、嗜盐菌、嗜碱菌、嗜冷菌和嗜酸菌)产生的乳化剂研究显示了它们丰富的新特性,在工业应用中具有巨大的潜力。
在极端微生物中,耐热生物乳化剂产生菌由于其胞外多糖独特的化学组成和性质,在各个工业领域都有着广泛的应用。耐热生产菌株的主要优点包括发酵过程短,较好的质量传递和发酵液较低黏度。嗜热芽孢杆菌可以利用廉价碳源和氮源在相对简单的培养基中迅速生长,因而受到特别关注。嗜热芽孢杆菌包括芽孢杆菌属,土壤杆菌属,厌氧芽孢杆菌属,苍白芽孢杆菌属和短芽孢杆菌属,其中仅有少数被报道过可生产生物乳化剂。因此,嗜热芽孢杆菌产生的新型生物乳化剂化学组成和功能的表征将为其在生物技术的新应用开辟前景。
发明内容
本发明的目的是解决现有生物乳化剂的化学组成和乳化性能受到外部因素强烈影响的问题,提供一种糖蛋白类型生物乳化剂,其对温度、pH及盐度有着优越的耐受能力,能够大幅度降低原油的乳化黏度,并能够显著提高原油采收率。
本发明的技术方案
一种糖蛋白类型生物乳化剂,其是从嗜热脱氮地芽孢杆菌NG80-2(CGMCCNo.1228)的发酵液中制备得到的。具体发酵条件为:将存于菌种保藏管中的NG80-2在LB平板划线活化,挑取单菌落接于LB液体培养基中45-70℃过夜并转接于新的LB培养基中培养至对数后期OD 1.3-1.6随即按5%-10%的比例接种到乳化剂生产的培养基中进行发酵。
所述的生物乳化剂主要由糖类和蛋白质组成,其中糖含量0.81mg/mg,蛋白含量0.084mg/mg。
所述的生物乳化剂对原油、葵烷、十四烷、十六烷、苯、二甲苯和柴油具有良好的乳化活性,及良好的温度、盐度和pH耐受性。
本发明还提供了一种生物乳化剂的制备方法,该方法包括:
菌株NG80-2在以多种有机物为碳源的培养基中进行发酵,之后至少经过离心除去菌体,三倍冰乙醇沉淀出生物乳化剂粗品,粗品透析冻干,离子交换层析进一步分离得到纯化后的生物乳化剂。
在上述的生物乳化剂制备方法中,所述的发酵培养基配方按照g/L或mL/L计为,碳源2g-20g,磷酸氢二钾4.8g,磷酸二氢钾1.5g,硫酸铵1g,柠檬酸三钠0.5g,硫酸镁0.2g,酵母提取物0.1g,微量元素1mL,pH 7。
所述微量元素配方按照g/L为:氯化钙2g,氯化锰0.4g,氯化镍0.4g,硫酸锌0.4g,氯化铁0.2g,钼酸钠0.2g。
本发明提供的发酵培养基中的碳源可以为但不限于单糖(葡萄糖、木糖、阿拉伯糖),二糖(蔗糖,纤维二糖),聚合物(木质素,木聚糖)、石油,甘油,豆油及单烷烃。优选为0.2g/L的葡萄糖。
本发明还提供了上述生物乳化剂在乳化油品中的应用。根据本发明的具体实施方式,优选地,所述油品为原油、葵烷、十四烷、十六烷、苯、二甲苯和柴油中的一种或几种组合。
本发明提供的生物乳化剂具有较高的乳化活性和抗逆性。当浓度为1.0g/100mL时,该生物乳化剂溶液对柴油的乳化指数可达80%以上。且该生物乳化剂在不同的盐度条件和pH条件下及广泛的温度范围内均对柴油具有较高的乳化活性。
本发明还提供了上述乳化剂在原油乳化降黏中的应用。根据本发明的具体实施方式,本发明的乳化剂可有效降低辽河稠油和鲁克沁特稠油的乳化粘度至原始样品的21%和42%。
本发明还提供了上述生物乳化剂在提高原油采收率中的应用。根据本发明的具体实施方式,本发明的乳化剂在模拟高温储层条件下,能够提高的原油采收率为15.6%。
本发明的优点和有益效果在于:
1)本发明所公开的生物乳化剂为嗜热菌发酵产生,其发酵过程短,发酵碳源廉价易得,极大降低了生产成本,产量可达3-5g/L。
2)本发明提供的生物乳化剂具有较好的乳化性能,且在高温、高盐及极端酸碱性条件下都具有良好的乳化稳定性,这使得其在高温、高矿化度的油藏及复杂的石油污染修复环境如地下蓄水层或石油化工带来的高温石油废水的处理中具有广阔的应用前景。
3)本发明提供的生物乳化剂可有效降低稠油的乳化黏度,将辽河稠油和鲁克沁特稠油的乳化粘度分别降低了81.03%和55.27%,并在模拟高温储层条件下有效提高了原油采收率至15.6%。一方面,作为微生物采油的重要作用方式之一,该乳化剂的乳化作用可改善油藏中极难开采的稠油的流动性,从而有利于原油的开采并提高石油采收率。另一方面,原油的乳化作用是微生物摄取碳氢化合物底物的重要手段,该乳化剂可有效提高微生物利用碳氢化合物的效率,从而促进石油污染的修复进程。
附图说明
图1生物乳化剂的分离纯化。
图2生物乳化剂的温度、盐度和酸碱耐受性。
图3生物乳化剂降黏率。
图4生物乳化剂提高原油采收率。
具体实施方式
下面通过具体实施例并结合附图对本发明作进一步的详细说明。以下各实施例仅仅是用于说明而不是限制本发明。需要特别说明的是实施例中所用到的试剂均由市售,嗜热脱氮地芽胞杆菌NG80-2(CGMCC No.1228)已保藏在国家菌种保藏中心。
实施例1
本实施例用于说明生物乳化剂的制备及组分分析
1、菌株和培养条件
NG80-2适宜生长温度范围为45-75℃。取出冻存于-80℃的NG80-2菌株,在LB固体平板上划线,60℃静置培养约12h直至长出单菌落。挑取单菌落至5mL LB液体培养基中于60℃振荡培养至OD为1.3-1.6,按照5:100的比例转接于含有300mL基本发酵培养基的500mL锥形瓶中,60℃,200rpm振荡培养以获取发酵产物。基本发酵培养基按照g/L或mL/L计为,碳源2g-20g,磷酸氢二钾4.8g,磷酸二氢钾1.5g,硫酸铵1g,柠檬酸三钠0.5g,硫酸镁0.2g,酵母提取物0.1g,微量元素1mL,pH 7。所述微量元素配方按照g/L为:氯化钙2g,氯化锰0.4g,氯化镍0.4g,硫酸锌0.4g,氯化铁0.2g,钼酸钠0.2g。
2、生物乳化剂提取
首先将发酵后的培养基通过离心(8000×g,20min,4℃)及通过孔径为0.47μm的HVLP滤膜除去培养物中的菌体,向上清液中加入两倍体积的预冷无水乙醇并不断搅拌,4℃过夜,离心(12000×g,20min,4℃)回收沉淀,ddH2O透析48h后沉淀并冻干。
3、生物乳化剂的纯化
将粗品(80mg)溶解于蒸馏水中(8mL),以10000rpm离心20min以去除不溶物。将获得的上清液用DEAE-Sepharose CL-6B柱层析分离。用梯度浓度的NaCl溶液(0,10%,20%)以1.6mL/min的流速逐步洗脱。使用自动分馏收集器在试管中收集洗脱液(8mL/管),并测定每管糖和蛋白的含量,绘制洗脱曲线。将各主要部分的洗脱液汇集、浓缩,并用蒸馏水透析3天,以去除NaCl(分子量截止值10kDa),然后冷冻干燥,得到一种酸性糖蛋白。图1为生物乳化剂即糖蛋白的分离纯化洗脱曲线图。
4、生物乳化剂的化学组成测定
1)生物乳化剂糖含量测定
采用硫酸-苯酚法对生物乳化剂中多糖含量进行测定,
表1多糖含量测定步骤及体系
2)生物乳化剂中糖醛酸含量测定
采用咔唑-硫酸法测定生物乳化剂中的糖醛酸含量
表2糖醛酸含量测定步骤及体系
3)生物乳化剂中蛋白含量测定
Bradford法测定生物乳化剂中蛋白含量
表3考马斯亮蓝G-250标准液配置
按照上表顺序依次加入试剂,用ddH2O定容至1L,过滤后绘制标准曲线。
表4蛋白含量测定标准曲线绘制
牛血清白蛋白(BSA)母液浓度为1mg/mL,NaCl母液浓度为0.15M。测定标准曲线时,按照上表在0-10号10mL离心管中加入各组分,混匀后置于37℃孵育30min,测定各离心管中溶液在595nm处的吸光值,并以此为纵坐标,蛋白浓度为横坐标,绘制标准曲线。
测定纯化的蛋白浓度时,取待测样品2μL,ddH2O 18μL及1mL已知标准曲线的考马斯亮蓝G-250标准液,混匀后37℃孵育10min,测定其595nm处的吸光值,带入标准曲线计算得出样品中蛋白含量(mg/mL)。
实验结果表明所述的生物乳化剂主要由糖类和蛋白质组成,其中糖含量0.81mg/mg,蛋白含量0.084mg/mg。
实施例2
本实施例用于说明生物乳化剂的乳化活性及酸碱、温度和盐度的耐受性
为了测定生物乳化剂的乳化活性,将5mg冻干的乳化剂溶解在5mL蒸馏水中,置于5mL的刻度试管中,同时分别加入相同体积的碳氢化合物(即己烷、癸烷、十四烷、十六烷、苯、二甲苯、柴油和原油),漩涡振荡2min,再于60℃放置24h测定乳化指数(Emusificationindex,EI-24)。乳化指数评估为乳化层高度与总混合液体高度的比率。我们还评估了不同的环境参数对乳化剂乳化稳定性的影响,以确定乳化剂的应用范围。将乳化剂溶液在4,20,30,40,50,60,70,80,90和100℃水浴1h,冷却至室温后分别测定其乳化热稳定性。为了研究pH稳定性,使用1.0M NaOH或1.0M HCl将乳化剂溶液pH调节至不同值(3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0),再于60℃放置24h分别测定其乳化活性。为了确定盐度(NaCl)对乳化活性的影响,向乳化剂溶液中添加不同浓度的NaCl(0,10,20,30,40,50,60,70,80,90,100,g/L)混匀直到完全溶解,再于60℃放置24h并测定其乳化活性。以上实验均以柴油作为测试的碳氢化合物。实验结果表明,乳化剂可以成功乳化多种纯烷烃(己烷、癸烷、十四烷和十六烷)和芳香烃(苯和二甲苯),以及作为芳香烃和烷烃混合物的柴油和原油,并形成水包油的乳状液,显示出了广泛的底物特异性。30-70℃的温度范围对乳化剂的乳化活性没有显著影响,但当温度升高到70℃以上时,乳化剂的乳化活性略有降低,这可能是由于多糖或蛋白质的功能结构受损所致。当盐(NaCl)浓度为0-30g/L时,乳化剂的乳化指数普遍较高,这可能是由于少量盐离子可以稳定糖蛋白的结构。对于pH来说,尽管当pH≤4或pH≥9时,乳化剂乳化活性略有降低,但E24普遍高于60%。因此,乳化剂表现出较好的耐酸性和耐盐性,以及对中链烷烃较高的乳化活性(见图2)。
实施例3
本实施例用于说明生物乳化剂在稠油降黏中的应用
在发酵培养基中添加2%的稠油,并将活化好的NG80-2按照5:100的比例接入含有稠油的发酵培养基,培养7天,14天和28天。将培养基中的油水混合样在120rpm/min振荡培养14h,为防止轻质成分挥发,震荡培养过程中加盖密闭性好的硅胶塞,然后利用Brookfield CAP2000+锥板粘度计1#转子,在60℃、900rpm/min条件下测定稠油采出样及培养7d、14d、28d的油水混合样的乳化粘度。图3表明乳化剂具有优异的乳化降黏功能,可将辽河稠油和鲁克沁特稠油的乳化粘度分别降低81.03%和55.27%。
实施例4
本实施例用于说明生物乳化剂在提高原油采收率中的应用
乳化剂在非原位MEOR中的应用是通过填砂管岩心驱油实验进行的。具体而言,填好的岩心真空饱和地层水24h,然后通过干燥岩心到饱和岩心的重量计算孔隙体积(PV)。将原油注入岩心(0.5mL/min),直到没有水流出,饱和24h。原油置换的水体积为OOIP。用于乳化剂驱油实验的岩心参数为:岩心长度30mm;岩心直径20mm;PV,40mL;OOIP,37.5mL。原油饱和度93.75%。老化5天后,将地层水注入岩心(0.5mL/min)直到驱出液体的含水量达到98%以上,并计算残油饱和度。在乳化剂驱油期间,将2.5PV菌株发酵液以0.5mL/min的恒定流速注入岩心,随后以0.5mL/min的恒定流速继续注入地层水直至观察到没有原油的采出,以模拟二次注水。所有岩心驱替实验均在60℃下进行。原油的采出量与岩芯中初始原油量之比,乘以100%,即为原油的采收率。图4表明,整个驱油过程约有61.2%的原油被采出,其中乳化剂提高原油采收率为15.6%。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (10)
1.一种糖蛋白类型生物乳化剂,其特征在于:所述的生物乳化剂是从保藏号为CGMCCNo.1228的嗜热脱氮地芽孢杆菌NG80-2菌株的发酵液中制备得到的;具体发酵条件为:将存于菌种保藏管中的NG80-2在LB平板划线活化,挑取单菌落接于LB液体培养基中45-70℃过夜并转接于新的LB培养基中培养至对数后期OD 1.3-1.6随即按5%-10%的比例接种到乳化剂生产的培养基中进行发酵。
2.根据权利要求1所述的生物乳化剂,其特征在于所述的生物乳化剂的组成主要为多糖和蛋白,其中糖含量0.81mg/mg,蛋白含量0.084mg/mg。
3.根据权利要求1所述的生物乳化剂,其特征在于所述的生物乳化剂对原油、葵烷、十四烷、十六烷、苯、二甲苯和柴油具有良好的乳化活性,及良好的温度、盐度和pH耐受性。
4.权利要求1至3任一项所述的生物乳化剂的制备方法,其特征在于制备步骤如下:将所述的菌株NG80-2在发酵培养基中进行发酵,之后至少经过离心除去菌体,三倍冰乙醇沉淀出生物乳化剂粗品,粗品透析冻干,离子交换层析进一步分离得到纯化后的生物乳化剂;
所述的发酵培养基配方按照g/L或mL/L计为,碳源2g-20g,磷酸氢二钾4.8g,磷酸二氢钾1.5g,硫酸铵1g,柠檬酸三钠0.5g,硫酸镁0.2g,酵母提取物0.1g,微量元素1mL,pH 7。
5.根据权利要求4所述的生物乳化剂的制备方法,其特征在于所述微量元素配方按照g/L为:氯化钙2g,氯化锰0.4g,氯化镍0.4g,硫酸锌0.4g,氯化铁0.2g,钼酸钠0.2g。
6.根据权利要求4所述的生物乳化剂的制备方法,其特征在于所述碳源为但不限于单糖,二糖,聚合物,石油,甘油,豆油或单烷烃。
7.根据权利要求6所述的生物乳化剂的制备方法,其特征在于所述碳源中的单糖为葡萄糖、木糖或阿拉伯糖;所述二糖为蔗糖或纤维二糖;所述聚合物为木质素或木聚糖;优选为0.2g/L的葡萄糖。
8.权利要求1至3任一项所述的生物乳化剂在乳化油品中的应用。
9.权利要求1至3任一项所述的生物乳化剂在稠油降黏中的应用。
10.权利要求1至3任一项所述的生物乳化剂在提高原油采收率中的应用。
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