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Ambiente

使用生物表面活性剂组合提高石油采收率

Published: June 03, 2022
doi:
10.3791/63207
, , ,
1Department of Biochemical Engineering and Biotechnology,Indian Institute of Technology Delhi, 2Indian Oil Corporation, Research and Development Faridabad

Summary

我们说明了筛选和鉴定产生生物表面活性剂的微生物的方法。还介绍了生物表面活性剂的色谱表征和化学鉴定方法，确定了生物表面活性剂在提高残油采收率方面的工业适用性。

Abstract

生物表面活性剂是表面活性化合物，能够降低两个不同极性的相之间的表面张力。生物表面活性剂已成为化学表面活性剂的有前途的替代品，因为它具有较低的毒性，高生物降解性，环境相容性和对极端环境条件的耐受性。在这里，我们说明了用于筛选能够产生生物表面活性剂的微生物的方法。使用滴落塌陷，油扩散和乳液指数测定来鉴定产生生物表面活性剂的微生物。通过确定由于微生物构件的生长而导致的介质表面张力的降低，验证了生物表面活性剂的生产。我们还描述了生物表面活性剂表征和鉴定所涉及的方法。对提取的生物表面活性剂进行薄层色谱，然后对板进行差异染色以确定生物表面活性剂的性质。LCMS， 1H NMR和FT-IR用于化学鉴定生物表面活性剂。我们进一步说明了评估所生产生物表面活性剂组合在模拟砂堆积柱中提高残余油采收率的方法。

Introduction

生物表面活性剂是由微生物产生的两亲表面活性分子，具有降低表面和两相1之间的界面张力的能力。典型的生物表面活性剂含有亲水部分，其通常由糖部分或肽链或亲水氨基酸组成，疏水部分由饱和或不饱和脂肪酸链2组成。由于其两亲性质，生物表面活性剂在两相之间的界面处组装并降低边界处的界面张力，这有助于一相分散到另一相1，3中。迄今为止报道的各种类型的生物表面活性剂包括糖脂，其中碳水化合物 通过 酯键（例如，鼠李脂，三卤脂和桔梗脂）与长链脂肪族或羟基脂肪族酸相连，脂质附着在多肽链上的脂肽（例如，表面活性素和地衣官能素），以及通常由多糖 – 蛋白质复合物组成的聚合物生物表面活性剂（例如， 乳聚糖，脂质，阿拉桑和脂甘露聚糖）4。由微生物产生的其他类型的生物表面活性剂包括脂肪酸、磷脂、中性脂质和颗粒生物表面活性剂5.研究最多的一类生物表面活性剂是糖脂类，其中大多数研究都报告了鼠李糖脂6。鼠李糖含有一个或两个鼠李糖分子（形成亲水部分），与一个或两个长链脂肪酸分子（通常是羟基癸酸）相连。鼠李糖脂是首先从 铜绿假单胞菌7 中报告的原代糖脂。

与化学表面活性剂相比，生物表面活性剂因其提供的各种独特和独特的特性而受到越来越多的关注8.这些包括更高的特异性，更低的毒性，更大的多样性，易于制备，更高的生物降解性，更好的发泡性，环境相容性和极端条件下的活性9。生物表面活性剂的结构多样性（图S1）是另一个优点，使它们比化学对应物10更具优势。它们在较低浓度下通常更有效和高效，因为它们的临界胶束浓度（CMC）通常比化学表面活性剂11低几倍。据报道，它们具有高度耐热性（高达100°C），并且可以耐受更高的pH值（高达9）和高盐浓度（高达50 g / L）12 因此在需要暴露在极端条件下的工业过程中具有多种优势13。生物降解性和较低的毒性使它们适用于环境应用，如生物修复。由于它们提供的优势，它们在食品，农业，洗涤剂，化妆品和石油工业等各个行业中得到了越来越多的关注11.生物表面活性剂在石油修复中也引起了很多关注，以去除石油污染物和有毒污染物14。

在这里，我们报告了由 红球菌 IITD102， Lysinibacillus sp. IITD104和 Paenibacillus sp. IITD108生产的生物表面活性剂的生产，表征和应用。 图1概述了筛选，表征和应用生物表面活性剂组合以提高石油采收率所涉及的步骤。

Figure 1
图1：使用生物表面活性剂组合提高石油采收率的方法。 图中显示了逐步工作流程。这项工作分四个步骤进行。首先，通过各种测定对微生物菌株进行培养和筛选，以生产生物表面活性剂，包括滴坍试验，油扩散测定，乳化指数测定和表面张力测量。然后，从无细胞肉汤中提取生物表面活性剂，并使用薄层色谱法鉴定其性质，并使用LCMS，NMR和FT-IR进一步鉴定它们。在下一步中，将提取的生物表面活性剂混合在一起，并使用砂包柱技术确定所得混合物提高石油采收率的潜力。 请点击此处查看此图的大图。

通过滴坍，油铺，乳液指数测定和测定由于微生物生长引起的无细胞培养基表面张力的降低，对这些微生物菌株进行筛选以产生生物表面活性剂。通过LCMS， 1H NMR和FT-IR提取，表征和化学鉴定生物表面活性剂。最后，制备了这些微生物产生的生物表面活性剂混合物，并用于回收模拟砂包柱中的残余油。

本研究仅说明了生物表面活性剂组合在提高残油采收率方面的筛选、鉴定、结构表征和应用方法。它没有提供由微生物菌株15，16产生的生物表面活性剂的详细功能表征。执行各种实验，例如关键胶束测定，热重分析，表面润湿性和生物降解性，以对任何生物表面活性剂进行详细的功能表征。但由于本文是方法论文，因此重点在于生物表面活性剂组合在提高残油采收率方面的筛选、鉴定、结构表征和应用;这些实验尚未纳入本研究。

Protocol

1. 微生物菌株的生长 称取 2 克 Luria 肉汤粉，并在 250 mL 锥形瓶中加入 50 mL 蒸馏水。混合内容物，直到粉末完全溶解，并使用蒸馏水将体积制成100 mL。 同样，再准备两个100 mL的Luria肉汤烧瓶，并将棉塞放在烧瓶的颈部。 用铝箔覆盖棉塞，并在121°C和15 psi下高压灭菌烧瓶15分钟以灭菌培养基。 高压灭菌后，让培养基冷却至室温。 对于菌株的原代培?…

Representative Results

通过各种测定筛选了三种细菌菌株（红球菌 属IITD102， 利西尼巴西勒 属IITD104和 帕尼巴西鲁斯 属IITD108）用于生产生物表面活性剂，包括滴塌法，油置换测定，乳化指数测定和表面张力降低。所有三种细菌菌株的无细胞上清液和化学表面活性剂溶液导致滴剂塌陷，因此生物表面活性剂的存在评分为阳性（图4a）。另一方面，水滴没有塌陷，因此生物表面活…

Discussion

生物表面活性剂是最通用的生物活性成分之一，正在成为化学表面活性剂的有吸引力的替代品。它们具有更好的润湿性，较低的CMC，多样化的结构和环保性，因此在洗涤剂，油漆，化妆品，食品，制药，农业，石油和水处理等众多行业中具有广泛的应用18.这导致人们越来越有兴趣发现更多能够生产生物表面活性剂的微生物菌株。在这里，我们说明了筛选，鉴定和应用由红球菌…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者要感谢印度政府生物技术部的财政支持。

Materials

1 ml pipette Eppendorf, Germany G54412G
1H NMR Bruker Avance AV-III type spectrometer,USA
20 ul pipette Thermo scientific, USA H69820
Autoclave JAISBO, India Ser no 5923 Jain Scientific
Blue flame burner Rocker scientific, Taiwan dragon 200
Butanol GLR inovations, India GLR09.022930
C18 column Agilent Technologies, USA 770995-902
Centrifuge Eppendorf, Germany 5810R
Chloroform Merck, India 1.94506.2521
Chloroform-d SRL, India 57034
Falcon tubes Tarsons, India 546041 Radiation sterilized polypropylene
FT-IR Thermo Fisher Scientific, USA  Nicolet iS50
Fume hood Khera, India 47408 Customied
glacial acetic acid Merck, India 1.93002
Glass beads Merck, India 104014
Glass slides Polar industrial Corporation, USA Blue Star 75 mm * 25 mm
Glass wool Merk, India 104086
Hydrochloric acid Merck, India 1003170510
Incubator Thermo Scientific, USA MaxQ600 Shaking incubator
Incubator Khera, India Sunbim
Iodine resublimed Merck, India 231-442-4  resublimed Granules
K12 –Kruss tensiometer Kruss Scientific, Germany K100
Laminar air flow cabnet Thermo Scientific, China 1300 Series A2
LCMS Agilent Technologies, USA 1260 Infinity II
Luria Broth HIMEDIA, India M575-500G Powder
Methanol Merck, India 107018
Ninhydrin Titan Biotech Limited, India 1608
p- anisaldehyde Sigma, USA 204-602-6
Petri plate Tarsons, India 460090-90 MM Radiation sterilized polypropylene
Saponin Merck, India 232-462-6
Sodium chloride Merck, India 231-598-3
Test tubes Borosil, India 9800U06 Glass tubes
TLC plates Merck, India 1055540007
Vortex GeNei, India 2006114318
Water Bath Julabo, India SW21C
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Tags

Keywords: BiosurfactantsEnhanced Oil RecoveryScreeningCharacterizationMicrobial StrainsDrop Collapse AssayOil Spreading AssaySand Pack Column Technique
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Nissar Zargar, A., Patil, N., Kumar, M., Srivastava, P. Enhanced Oil Recovery using a Combination of Biosurfactants. J. Vis. Exp. (184), e63207, doi:10.3791/63207 (2022).
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