1. 石油石化污染物控制与处理国家重点实验室，北京 102206;
2. 中国石油集团安全环保技术研究院有限公司，北京 102206;
3. 上海交通大学 生命科学技术学院 微生物代谢国家重点实验室，上海 200240
收稿日期：2021-02-10；接收日期：2021-05-21；网络出版时间：2021-09-08
基金项目：国家自然科学基金(No. 31570099)，上海市自然科学基金(No. 19ZR1475500)，中国石油集团安全环保技术研究院项目(No. 2018D-4616) 资助
作者简介：梁如冰??上海交通大学生命科学技术学院(微生物代谢国家重点实验室) 副研究员、系副主任。主要进行微生物降解石油烃、多环芳烃、激素等污染物的分子机制、调控网络及应用模拟等研究。已在J Hazard Mater、Front Microbiol、Appl Microbiol Biot、Appl Environ Microb、J Bacteriol、Mol Microbiol、Sci Total Environ等期刊发表论文30多篇，申请发明专利12项。主持国家自然科学基金、上海市自然科学基金、浦江人才计划等。为美国微生物学会、中国微生物学学会、中国遗传学会、上海微生物学会等会员，多个国际期刊审稿人.
摘要：微生物修复被认为是去除石油污染物和修复石油污染土壤的一种经济、高效且无二次污染的绿色清洁技术。受土壤环境条件和石油污染物性质等因素制约，土壤中土著石油降解微生物常存在数量不足、活性偏低、生长缓慢等问题，导致修复效果不佳、修复周期偏长。微生物强化修复技术可有效提高微生物降解效能，通过投加具有降解效能的功能菌株或菌剂、营养物质、表面活性剂、生长基质及固定化微生物等手段，可改善提升土著微生物对石油污染土壤的修复效果。文中梳理了已报道的石油降解微生物的种类，总结了微生物修复石油污染土壤的主要影响因素，阐述了微生物强化修复石油土壤的多种有效策略，提出了微生物强化修复石油污染的未来发展方向。
关键词：石油污染生物修复生物强化生物刺激降解微生物
Advances in the bioaugmentation-assisted remediation of petroleum contaminated soil
Jin Zheng1,2*, Yali Fu3*, Quanwei Song1,2, Jiacai Xie1,2, Shuangjun Lin3, Rubing Liang3
1. State Key Laboratory of Petroleum Pollution Control, Beijing 102206, China;
2. CNPC Research Institute of Safety & Environmental Technology, Beijing 102206, China;
3. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Received: February 10, 2021; Accepted: May 21, 2021; Published: September 8, 2021
Supported by: National Natural Science Foundation of China (No. 31570099), Natural Science Foundation of Shanghai, China (No. 19ZR1475500), Grant of Safety and Environmental Technology Research Institute of CNPC, China (No. 2018D-4616)
Corresponding author: Rubing Liang. Tel: +86-21-34204192; E-mail: icelike@sjtu.edu.cn.
*These authors contributed equally to this study.
Abstract: Bioremediation is considered as a cost-effective, efficient and free-of-secondary-pollution technology for petroleum pollution remediation. Due to the limitation of soil environmental conditions and the nature of petroleum pollutants, the insufficient number and the low growth rate of indigenous petroleum-degrading microorganisms in soil lead to long remediation cycle and poor remediation efficiency. Bioaugmentation can effectively improve the biodegradation efficiency. By supplying functional microbes or microbial consortia, immobilized microbes, surfactants and growth substrates, the remediation effect of indigenous microorganisms on petroleum pollutants in soil can be boosted. This article summarizes the reported petroleum-degrading microbes and the main factors influencing microbial remediation of petroleum contaminated soil. Moreover, this article discusses a variety of effective strategies to enhance the bioremediation efficiency, as well as future directions of bioaugmentation strategies.
Keywords: petroleum pollutionbioremediationbioaugmentationbiostimulationbiodegrading microorganisms
随着社会经济的发展，人们对石油及其产品的需求快速增加。石油开采、炼制、运输、存储和利用而导致的石油泄漏，严重破坏了生态环境，污染了水体、大气和土壤。石油中不仅含有可致癌、致畸、致突变的石蜡、烯烃和芳香烃等有机化合物，还含有钒、镉、铅、镍、汞等重金属[1]。石油污染物进入水体后，会对水体生态系统造成持久性伤害，污染地下水资源，进而会影响生产生活用水，危害人类健康。进入土壤的石油污染物则会破坏土壤结构、影响土壤通透性、改变土壤理化性质，破坏土壤中的微生物群落结构、导致农作物的产量和品质显著下降等[2]。因此，石油污染修复是关系着人类社会可持续发展的重大环境与社会问题。
目前，石油污染土壤的治理方法有物理修复法、化学修复法和生物修复法[1]。物理修复法本质上是污染物的转移，污染物本质并未改变，成本高，操作困难。化学修复法则是通过添加外源化合物去除污染物的处理技术，易破坏土壤结构，导致二次污染。生物修复法是指利用植物或微生物对石油的吸收、转化和降解来实现土壤修复的一项技术。微生物修复技术因操作简单、成本低、不破坏土壤环境、无二次污染且处理效果好等优点，逐渐成为石油污染治理方面的理想技术[3]。
石油污染物进入土壤后，会抑制土壤中土著微生物的生长，微生物种类下降，可利用石油的土著微生物数量不足且活性降低，故其自然衰减速度十分缓慢，修复周期很长。微生物强化修复技术，是指通过人工投加具降解效能的功能菌株或菌剂及固定化微生物等方法(生物投加法)，或通过添加可刺激土著微生物生长的营养物质、表面活性剂、生长基质等手段(生物刺激法)，有效改善提升微生物对土壤中石油污染物的降解效能与环境修复效果。近年来，微生物强化修复技术已成为石油修复研究的热点。
本文针对石油污染土壤的微生物修复技术，梳理了微生物修复石油污染土壤的研究工作进展与主要影响因素，着重阐述了多种微生物强化修复石油土壤的有效策略，并提出了相关新技术和未来的发展方向。
1 石油降解微生物微生物修复石油污染土壤是利用可降解石油污染物的微生物对污染土壤进行修复的方法。在长期受到石油污染的土壤中，微生物生态系统已发生改变；通过竞争筛选或者遗传进化等，石油降解微生物的比例会上升，在总微生物群落中的占比可从原有的低于0.1%增加1%–10%[4]。因此，长期受石油污染土壤中的土著微生物往往具备一定的石油降解潜力，可通过进一步的人为处理，达到土壤修复的目的。
目前，已报道的石油降解微生物大多是分离自不同的石油污染环境，种类超过200种，包括细菌、真菌和藻类；细菌是最主要的石油降解微生物[5]。表 1列出了已报道的石油烃降解菌株。常见的可降解石油烃化合物的细菌包括假单胞菌Pseudomonas sp.[6-8]、芽孢杆菌Bacillus sp.[9]、分枝杆菌Mycobacterium sp.[10-11]、红球菌Rhodococcus sp.[12-13]、鞘氨醇杆菌Sphingobacterium sp.[14-15]、食烷菌Alcanivorax sp.[16-17]、诺卡氏菌Nocardia sp.[18]、无色杆菌Achromobacter sp.[19]、不动杆菌Acinetobacter sp.[20-21]、固氮弧菌Azoarcus sp.[22-23]、苍白杆菌Ochrobactrum sp.[24]，其中，假单胞菌属的菌株最多，降解效率也较高。真菌中可降解石油烃的菌株主要来源于曲霉属Aspergillus sp.[25]、青霉菌属Penicillium sp.[26]、假丝酵母属Candida sp.[27]、毕赤酵母属Pichia sp.[28]、白腐菌属Phlebia sp.[29]等。石油烃降解藻类主要有斜生栅藻Scenedesmus obliquus[30]、小球藻Chlorella sp.[31]、月形藻Closterium lunula[32]等。由于不同种属微生物的代谢特点不同，其石油烃的代谢机制也有所差异，因此，其底物选择性与环境适应性等方面具有各自特点[33]。如从淤泥沉积物中分离的新鞘氨醇单胞菌Novosphingobium pentaromativorans US6-1可降解菲、芘和苯并(a)芘等多环芳烃，24 h后对菲的降解率可达86.62%[34]。无色杆菌LH-1则可产生表面活性剂，提高多环芳烃的生物利用度；当菲浓度为100 mg/L时，LH-1的降解率为94%，矿化率为40%[4]。脱硫菌TMJ1T和UKTLT可利用单芳香烃如甲苯、苯酚和对甲酚等多种芳香烃[35]。
表 1 石油降解微生物的基本特性Table 1 Characteristics of the petroleum-degrading microorganisms
| Types | Species | Strains | Sources | Substrates | References |
| Bacteria | Achromobacter sp. | Achromobacter sp. HZ01 | Ocean | C12–C27 n-alkanes, PAH | [19] |
| Acinetobacter sp. | Acinetobacter sp. EB104 | Soil | n-alkanes | [20] | |
| Acinetobacter sp. DSM17874 | Soil | C32–C36 n-alkanes | [21] | ||
| Azoarcus sp. | Azoarcus sp. HxN1 | Soil | C6–C8 n-alkanes, C2–C16 fatty acid | [22] | |
| Azoarcus sp. EB1 | Soil | Ethylbenzene | [23] | ||
| Sphingobacterium sp. | Sphingomonas sp. | Soil | PAH | [14] | |
| Sphingobium yanoikuyae SJTF8 | Soil | PAH | [15] | ||
| Alcanivorax sp. | Alcanivorax borkumensis AP1 | Ocean | C6–C20 n-alkanes | [16] | |
| Alcanivorax borkumensis SK2 | Ocean | C6–C32 n-alkanes | [17] | ||
| Nocardia sp. | Nocardiopsis VITSISB | Ocean | n-alkanes, PAH | [18] | |
| Ochrobactrum sp. | Ochrobactrum sp. VA1 | Soil | PAH | [24] | |
| Pseudomonas sp. | Pseudomonas putida GPo1 | Soil | Propane, butane and C5–C12 n-alkanes | [6] | |
| Pseudomonas aeruginosa SJTD-1 | Soil | C14–C32 n-alkanes | [7] | ||
| Pseudomonas aeruginosa NCIM 5514 | Soil | C8–C36 n-alkanes | [8] | ||
| Bacillus sp. | Bacillus licheniformis | Soil | C9–C30 n-alkanes | [9] | |
| Mycobacterium sp. | Mycobacterium vanbaalenii PYR-1 | Soil | n-alkanes, PAH | [10] | |
| Mycobacterium sp. TY-6 | Soil | n-alkanes, PAH | [11] | ||
| Rhodococcus sp. | Rhodococcus erythropolis 20S-E1-c | Soil | Aromatic hydrocarbon | [12] | |
| Rhodococcus erythropolis M-25 | Soil | C14–C20 n-alkanes | [13] | ||
| Fungi | Aspergillus sp. | Aspergillus niger | Aerobiotic | PAH | [25] |
| Candida sp. | Candida tropicalis ATCC 20336 | Facultative anaerobic | n-alkanes, fatty acid | [27] | |
| Penicillium sp. | Penicillium sp. | Aerobiotic | n-alkanes, PAH | [26] | |
| Pichia sp. | Pichia | Aerobiotic | n-alkanes | [6] | |
| Phlebia sp. | White-rot fungi | Aerobiotic | PAH | [29] | |
| Algae | Chlorella sp. | Chlorella | Aerobiotic | n-alkanes, PAH | [31] |
| Closterium lunula sp. | Closterium lunula | Aerobiotic | Dimethyl phthalate | [32] | |
| Scenedesmus obliquus sp. | Scenedesmus obliquus | Aerobiotic | n-alkanes, PAH | [30] |
表选项
2 影响微生物降解石油效能的主要因素石油一般指直接从地下开采且未经提炼的原油，主要是由各种烷烃、环烷烃和芳香烃构成的混合烃类物质，其余为少量卟啉、沥青烯、痕量金属和含硫、氮、氧等非烃类化合物，组分与理化性质十分复杂。因此，生物降解石油污染物的效能不仅受到石油污染物本身性质的影响，也会受到多种环境因素的影响[36]。
2.1 石油性质对微生物降解效能的影响生物修复过程中微生物的转化率取决于其对石油的有效利用效能，包括微生物与污染物的接触面积、吸收效果和代谢速率等。石油污染物的生物降解会受到石油物质的组成、种类、浓度和理化性质等因素的影响[37-38]。一般来说，短链烷烃比长链烷烃(C10–C25) 对生物的毒性更大，长链烷烃因疏水性强，水溶性差，很难被降解；支链烷烃和环烷烃的降解速度比普通烷烃的降解速度慢，一些高分子化合物如多环芳烃、树脂和沥青等极难被降解。微生物降解不同石油烃类的难易程度依次为正烷烃 < 分支烷烃 < 低分子量芳香烃 < 多环芳烃 < 高分子量芳香烃 < 胶质和沥青质[36]。同时，石油烃浓度也会影响生物降解活性。一定范围内，石油烃的去除效率会随其浓度增加而升高；但石油烃浓度过高则会抑制微生物生长，降低微生物的降解效率[38]。
2.2 不同环境因素对微生物降解效能的影响微生物降解石油物质，不仅与自身代谢机制有关，还会受多种环境因素的影响，如温度、土壤pH、营养成分、盐度、含水量、氧气和土壤中的电子受体等[39]。
2.2.1 温度温度会改变石油的物理性质、化学组成及降解微生物的生长速率和代谢水平，进而影响微生物降解石油的效能。低温环境会使石油黏度增加，挥发性减弱，对微生物的毒性随之增大，影响烃类物质的生物降解；低温也会抑制石油降解酶的活力，降低石油的生物降解速率。在一定范围内，温度升高会促进石油烃的降解；但当温度高于40 ℃时，石油烃的膜毒性增大，有些微生物分泌的石油降解酶会变性，微生物降解被抑制。因此，不同石油烃降解微生物均在一定的最佳温度范围内能保持较好的降解效能，如嗜冷菌是低于20 ℃，中温菌是在15–45 ℃，而嗜热菌则是在50 ℃以上。如嗜冷菌Rhodococcus sp. Q15能在0–5 ℃降解多种短链烷烃及十六烷、二十八烷、三十二烷等长链烷烃[40]。嗜冷多环芳烃降解菌在7 ℃时对多环芳烃的去除率为39%，而高于20 ℃其去除率为31%[41]。嗜热芽孢杆菌NG80-2能在45–73 ℃温度下生长，可利用长链烷烃(C15–C36) 为碳源[42]。除嗜冷、嗜热菌外，一般情况下，石油降解微生物大多在20–35 ℃最活跃，温度过低或过高都会影响其对石油的降解效能。
2.2.2 土壤pH环境酸碱条件也会影响微生物的降解效果；大部分微生物的最适生长pH为7.0–7.8，过酸或过碱条件均不利于微生物生长和其石油降解效能[39]。环境酸碱条件会影响微生物对营养物的吸收速率，也会影响微生物胞外酶的产量与活性，还会因改变石油物质的溶解度影响微生物对石油的可利用度。多数菌株在碱性条件下对石油的降解率较高，这是由于在中性偏碱的pH条件下石油的溶解度增大，且碱性条件下有利于微生物对氮磷的吸收，微生物的生长降解效能提高所致。如铜绿假单胞菌在pH 8.0时，其对石油的生物降解率最高。但有一些菌株则适合在中性偏酸的环境中生存，偏酸性环境利于其降解石油。如链霉菌在pH 7.0时，其对萘的7 d降解率可达99.14%；而当pH为8.0时，其对萘的降解率显著下降[43]。由于微生物降解过程中产生的酸性物质可在土壤中积累，导致土壤pH降低；因此，可向土壤中添加适量酸碱缓冲液，调控土壤酸碱条件，从而达到微生物高效降解石油的效果。
2.2.3 土壤营养成分碳、氢、氧、氮、磷、硫、镁等均是微生物生长所必需的元素。石油污染土壤中，大量石油烃化合物能满足微生物生长所需的碳、氢元素，而氮、磷、硫等微量元素的供给是否充分，成为了微生物生长代谢的限制因素。当土壤中营养物质不足时，微生物种群数量会减少[44]；在土壤中添加适当浓度和比例的氮磷元素，可显著促进石油降解微生物的生长，有助于污染物的降解[45-46]。研究表明，环境中碳氮磷的最适比例为100︰10︰1时，其石油污染物的生物降解效果较好[47]。埃克森瓦尔迪兹号油轮泄漏后，在石油污染的海岸线上施用化肥，大大提高了石油烃生物降解的速度[48]。另一方面，营养物浓度过量也会抑制生物降解的活性。研究表明，添加过量氮磷等营养元素对石油烃的降解会产生消极影响[49]。
2.2.4 土壤盐度盐度通过改变土壤微生物的生长速率和多样性，直接影响土壤中石油污染物的生物修复效果[50]。在土壤中添加一定量的盐类，有助于微生物的生长；一定范围内，石油烃降解与土壤盐度成正相关[51]。但过高的盐度则会干扰土壤中微生物的酶促反应进程、膜平衡和渗透压水平，抑制微生物的生长代谢。可耐受较高盐度的石油污染物降解菌株具有更大的实际应用潜能。如钠白菌Natrialba sp. C21能在25% (W/V) 的氯化钠上生长，以苯酚、萘和芘作为唯一碳源[52]。从渤海湾石油污染沉积物中分离的迪茨氏菌Dietzia sp. CN-3，即使氯化钠浓度达到85 g/L，其对长链烷烃和芳香烃的降解速率几乎不受影响。
2.2.5 土壤含水量石油降解微生物主要在油水界面发生其代谢作用，因此，土壤中含有适量水分可促进微生物的生长代谢。土壤中水分含量不足时，营养物质和石油污染物不能有效传递给微生物，抑制了微生物生长和石油污染物代谢，影响生物修复效果。当土壤含水量过高时，土壤中毛细孔隙被水充满，则会影响土壤透气性，妨碍氧的供应，干扰微生物的生长和石油代谢。研究表明，微生物生长的最佳土壤水分含量取决于土壤类型，含水量从30%–90%不等[38]；真菌对石油污染的修复，土壤含水量一般需保持在60%以上[53]。因此，干旱地区的油污土壤处理，可采取间断性的喷淋等手段增强土壤湿度来提高微生物降解效率。
2.2.6 土壤含氧量土壤含氧量是微生物降解深层石油污染物的主要限制因素。氧气是微生物好氧呼吸的最终电子受体，还参与生物修复过程中部分物质的合成过程；因此，含氧量会直接影响降解菌株的生长过程与石油烃降解效能[38]。部分微生物能在厌氧条件下降解石油，但降解速率相对较低。表层受石油污染的土壤，可采用翻耕法提升氧的供应量；深层污染土壤的修复，则可采用通气法[54]。研究表明，当空气注入量为81.504 m3/d (Run 1) 和407.52 m3/d (Run 2) 时，石油污染物的挥发降解和生物降解比率分别为0.57︰1和0.89︰1，表明较低的喷气率可满足微生物对氧气的需求，大大提高了生物降解率，降低运行成本[55]。
2.2.7 土壤中的电子受体微生物厌氧降解石油时，外源电子的供给是影响石油烃生物降解的主要因素之一。研究表明，NO33-、Fe3+、SO42-、Mn4+均可代替氧分子来用作微生物代谢过程中的电子受体[56-58]；羧酸盐可为土壤微生物群落提供直接且易降解的能源，增加磷的供应，提高污染物的生物利用度[38]。如硝酸盐和亚硝酸盐存在时，微杆菌对苯并芘的降解效率增加[59]。NaHCO3添加剂除了使石油降解过程中CO2含量增加，对石油烃生物降解和其他参数并无影响，这可能是由于NaHCO3作为电子受体，更易被厌氧菌利用，产生更多的CO2[60]。
3 石油污染的微生物修复技术按照修复方式分类，微生物修复石油污染方法包括原位微生物修复法和异位微生物修复法(图 1)。
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| 图 1 生物修复石油污染土壤主要技术 Fig. 1 The main bioremediation techniques for petroleum-contaminated soil. |
| 图选项 |
原位微生物修复技术指在原污染地利用微生物直接对石油污染土壤进行生物修复，主要包括生物通气法、生物强化法(投菌法)、生物刺激法(生物培养法) 等[61-63]。生物通气法通常在污染地至少打两口井，安装鼓风机和抽真空机，向石油污染地供给空气或氧气促进生物生长，加快污染物降解，同时排出土壤中的二氧化碳。生物强化法是向污染地投入高效降解菌的同时提供营养物质，促进土壤中石油污染物的生物降解。生物刺激法则是向石油污染土壤中添加氮磷等营养物质或H2O2、O2等电子受体，刺激土著石油降解菌的生长，加快污染物降解。原位生物修复法的工艺简单、费用低，但相对处理速度慢，适用于渗透性好的土壤的治理。
异位微生物修复法是指将污染物转移到专门的修复场地进行处理，主要包括堆肥法、生物预制床法及生物泥浆法等[5, 64-65]。堆肥法是将受污染土壤与适当的材料混合堆放，依靠堆肥过程中微生物的生长代谢来降解石油烃类；在此过程中，可加入土壤调理剂，以促进微生物的生长和石油的降解。生物预制床法是将受污染土壤转移到底部铺满高密度乙烯或粘土的预制床上进行处理，通过调节pH、施肥和灌溉，或添加石油降解微生物和表面活性剂，来促进污染物的微生物降解。预制床的设计可使污染物的迁移量减至最小，被认为是最为有效的石油污染土壤修复方法之一。生物泥浆法是将挖出的石油污染土壤与水混合成泥浆，接种石油降解微生物后，再转入反应器内进行处理，期间可通过控制条件促进石油的微生物降解。与原位微生物修复技术相比，异位微生物修复法操作复杂、费用昂贵，一般只有在土壤污染严重时才采用该技术。
此外，利用植物根际微生物来修复石油污染也是一种有效的生物修复方法。与植物共生的微生物向植物提供其生长必需的氨基酸和细胞分裂素，促进植物生长，植物则可向根际释放氧气、氨基酸、碳水化合物和有机酸等。有机酸的羧酸根为微生物提供了易于降解的能源，可提高微生物对石油烃的利用度，促进根际微生物群落对石油烃的降解[66]。如黑麦可降低油泥中52%的石油成分，而羊草根际微生物也可有效降解土壤中的多环芳烃[34, 67]。
4 微生物强化修复石油污染土壤的方法微生物强化修复是指在生物处理污染物过程中，通过接种高效降解菌、增加营养物质或活性因子等方式，提高微生物代谢活动从而达到去除污染物的方法。微生物强化修复是石油污染生物修复的主要与有效方式，除了接种高效降解菌株/群/剂和添加营养物质或活性因子外，近年来出现的固定化和纳米颗粒等方法也为微生物强化修复提供了新技术。
4.1 接种高效降解菌株/菌群或复合菌剂石油是一种含有许多复杂成分的混合物质，对石油污染环境的有效修复需要高效稳定的降解菌株，也要避免单一菌株降解石油性质的局限性。一方面，分离获得环境适应性好的高效降解菌株是提升生物修复石油污染的效率根本。如嗜热脂肪土芽孢杆菌A-2，能在40–75 ℃条件下生长；表面疏水性较强，可产生生物乳化剂来降低油脂粘度，优先利用原油中的重组分和多环芳烃[68]。从渤海湾石油污染沉积物中分离的迪茨氏菌属菌株CN-3，能以C14–C31烷烃、芳香化合物和原油等生长，对高盐度耐受性高，在85 g/L的氯化钠浓度时，降解效率几乎不变；在pH值为6.0–9.0的范围内，该菌对原油和饱和烃的降解率高达80%，可适用于多种胁迫环境条件下的石油污染修复[69]。另一方面，筛选和组合不同的石油降解复合菌群，利用复合菌群的共生协同作用可大大提高石油的降解率[70]。由枯草芽孢杆菌DM-04、铜绿假单胞菌M和NM组成的复合菌群，可有效降解苯、甲苯和二甲苯，均比单一菌株降解效率高；外源加入0.01% H2O2、硝酸盐和磷酸盐作为微生物的额外氧源、电子受体和营养素，可进一步提高无氧或限氧条件下复合菌群对石油的降解效率[71]。研究发现，将土著菌群与枯草芽孢杆菌按2︰1的接种量接种到石油污染环境中，7 d后复合菌群对石油的降解率为85.01%，明显高于原土著菌群的石油降解率(71.32%)[72]。因此，复合菌群在石油降解中表现出巨大潜力，是微生物强化石油修复的新思路。
4.2 添加生物表面活性剂生物表面活性剂是指由动植物和微生物产生的天然表面活性物质，是一种具有乳化、增溶、消泡、润湿或破乳、反润湿等作用的两亲性分子。与化学合成的表面活性剂相比，生物表面活性剂具有适应范围广、无毒、无二次污染、耐酸碱能力强且稳定性好等优点[73]。通过改变油水接触面的表面张力，生物表面活性剂可增加微生物与石油烃的接触面积，提高石油烃的降解效率[74-75]。研究发现，在土壤中添加鼠李糖脂和营养物，可提高微生物的生存和代谢活性，加快土壤的生物修复[76]。鼠李糖脂能促进铜绿假单胞菌对异构烷烃(C12–C19) 的降解，降解率可达23%–100%[77]。可产生表面活性剂的微生物较其他石油烃降解菌降解效果更好，20 d内对石油烃的降解率超过80%，对难降解组分的降解率达10%–81%[78]。
4.3 固定化技术固定化技术是指使用物理或化学方法将微生物或酶控制于限定的空间、使其保持生物活性并可反复使用的技术[46]。固定化载体不仅具有缓冲作用，为微生物提供有利的环境条件，同时还具有吸附作用，可有效提高微环境中的微生物浓度，使微生物和污染物更好地接触[79]。高盐度培养条件下，固定化可显著提高细菌对原油的降解率[80]。利用多孔活性炭载体固定的微生物，其石油降解率显著提升；用藻酸盐珠粒固定的细菌则可重复使用，石油降解活性稳定[81-82]。生物炭是固定化修复石油污染技术中应用最广泛的固定化基质。生物炭是一种用天然材料制成的新型土壤吸附剂，可吸附土壤中的多种污染物，增强微生物与污染物的接触，孔隙率高，比表面积大，可改善土壤的结构性质，为微生物提供良好的生存环境，提升微生物对土壤的修复效率[46]。利用生物炭固定的菌株修复石油污染土壤60 d后，固定化菌株对正构烷烃的降解效果好，半衰期短[83]。以鸡粪、麦秸和木屑作为生物炭添加到微生物原位强化修复石油污染土壤中，经223 d修复后，添加了营养物质磷的鸡粪组对烷烃去除效率最高，添加了营养物质氮的麦秸组则对芳香烃去除效率最高[84]。因此，固定化技术可有效提高微生物修复石油污染的有效性和可行性，具有广泛的应用前景。
4.4 其他方法除上述技术外，纳米颗粒和吸附剂等也可用于石油污染的微生物修复。以聚乙烯吡咯烷酮(polyvinyl pyrrolidone，PVP) 包覆的纳米颗粒(Nanoparticles，NPs) 可与石油降解菌协同作用，纳米颗粒对石油的吸附和微生物对石油的降解相结合，可显著提高菌株的石油降解效率[85]。利用细菌吸附剂(沸石) 和包裹剂(γ-PGA或壳聚糖) 可对石油污染的海洋沉积物进行原位生物修复，70 d内石油的生物降解率超过50%。该法成本低，降解石油效率高，在海洋石油行业应用前景广[86]。利用低电压电解海水产氧气，可加快烃类的生物降解速率[87]。此外，构建高效的石油降解工程菌有助于解决单一菌株应用的局限性和混合菌竞争抑制问题，提高石油污染的微生物修复效率[88]。
5 总结与展望社会进步与工业发展使全球对石油的需求量逐年上升，石油污染日益严重，已经成为严重的全球性公害。石油污染物具有毒性强、稳定性高和难降解等特点，需对其进行高效稳定的修复。相对于物理与化学修复技术，微生物修复技术因成本低、无污染和效果好等优点被用于石油污染的修复中。但是，目前微生物强化修复石油污染中仍存在较多问题[89]。如土壤中高浓度石油对微生物有毒害作用，降低了微生物修复效果，导致微生物强化修复次数增加，成本提高[90]；微生物生长缓慢，修复周期长，易受环境因素影响，难以实现石油污染物的彻底降解[91]；石油类污染物疏水性强，微生物与其接触面较小，亲和性较低，不利于微生物的利用[38]。此外，微生物对石油烃的降解机理并不十分明确，生物降解中涉及的功能基因、降解酶和调控机理等仍有待深入研究[92]。
针对上述问题，可从以下几方面来深化微生物强化修复石油污染的研究：(1) 加强挖掘石油降解菌株/群，设计稳定高效的复合菌剂：高效降解菌株/菌群是微生物强化修复石油污染的核心与根本。通过挖掘与组合环境适应性好且降解效能稳定的降解微生物，探索不同微生物菌株之间和微生物与环境生物之间的协同互作效应；优化各组分功效，如优化营养物质的比例，寻找更高效的表面活性剂，优化固定化载体和生物炭材料等，提升生物刺激和生物强化效能，使之更快速、更经济、更有效[93]；依据实际污染环境，有针对性地组合与设计环境适应性高且降解效果强大的微生物菌群/修复菌剂[94-95]。(2) 多学科交叉，多种手段联合运用，解决复杂难处理问题：处理石油污染严重的土壤时，可采用物理方法、化学方法与生物修复方法结合运用的策略，取长补短，互为补充[96]。交叉运用多个学科的技术，开发新的污染物去除与环境修复方法，如将磁性纳米颗粒与生物电化学技术结合，可有效提升厌氧环境下微生物对石油污染物的降解效能，提升生物修复的效果[97]。(3) 加快微生物降解石油机理解析，开发高效工程菌株/群/剂。运用多种组学(宏基因组、宏转录组、蛋白组、代谢组等) 技术与分子生物学技术，明确微生物降解石油污染物的关键功能基因与降解酶，解析微生物降解石油污染物的代谢途径，明晰不同的微生物之间及微生物与环境生物之间的代谢互作网络，开发高效降解石油污染物的工程菌株/群/剂[98-100]。
综上所述，微生物修复石油污染的技术不仅与微生物自身代谢能力相关，还会受环境条件、营养物质、固有微生物群落等多种因素的影响。研究人员需要结合实际待修复场地的环境条件与生物条件，依据降解微生物自身性质，创造有利于其生存、繁殖、代谢和降解的最适环境，运用多种策略和多种方法，提升微生物降解石油污染物的效能，实现石油污染土壤的高效安全修复。
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