Bacteria in Crude Oil Survived Autoclaving and Stimulated Differentially by Exogenous Bacteria

Autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. This may be potentially useful for bioaugmentation and microbial enhanced oil recovery (MEOR). However, it is not entirely clear if “endogenous” bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the “exogenous” bacterial strains. To test this, we inoculated autoclaved crude oil medium with six exogenous bacterial strains (three Dietzia strains, two Acinetobacter strains, and one Pseudomonas strain). The survival of the spore-forming Bacillus and Paenibacillus and the non-spore-forming mesophilic Pseudomonas, Dietzia, Alcaligenes, and Microbacterium was detected using a 16S rRNA gene clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis. However, neither bacteria nor bacterial activity was detected in three controls consisting of non-inoculated autoclaved crude oil medium. These results suggest that detection of endogenous bacteria was stimulated by the six inoculated strains. In addition, inoculation with Acinetobacter spp. stimulated detection of Bacillus, while inoculation with Dietzia spp. and Pseudomonas sp. stimulated the detection of more Pseudomonas. In contrast, similar exogenous bacteria stimulated similar endogenous bacteria at the genus level. Based on these results, special emphasis should be applied to evaluate the influence of bacteria capable of surviving autoclaving on the hydrocarbon-degrading abilities of exogenous bacteria, in particular, with regard to bioaugmentation and MEOR. Bioaugmentation and MEOR technologies could then be developed to more accurately direct the growth of specific endogenous bacteria that may then improve the efficiency of treatment or recovery of crude oil.

采用高压蒸汽灭菌（autoclaving）处理原油的方法，常被用于评估细菌的烃类降解能力，该方法在生物强化（bioaugmentation）与微生物强化采油（microbial enhanced oil recovery, MEOR）领域具备潜在应用价值。然而，原油内及表面的"内源"细菌（如芽孢）能否在高压蒸汽灭菌过程中存活，以及其是否会干扰后续对"外源"菌株烃类降解能力的评估，目前尚未完全明晰。为验证上述问题，我们将6株外源细菌接种至经高压蒸汽灭菌的原油培养基中，其中包含3株迪茨氏菌属（Dietzia）菌株、2株不动杆菌属（Acinetobacter）菌株与1株假单胞菌属（Pseudomonas）菌株。本研究采用16S rRNA基因克隆文库（16S rRNA gene clone library）与末端限制性片段长度多态性（terminal restriction fragment length polymorphism, T-RFLP）分析技术，检测了产芽孢的芽孢杆菌属（Bacillus）、类芽孢杆菌属（Paenibacillus），以及非产芽孢的嗜温假单胞菌属、迪茨氏菌属、产碱杆菌属（Alcaligenes）和微杆菌属（Microbacterium）的存活情况。但在3组未接种外源细菌的高压蒸汽灭菌原油培养基对照中，均未检测到细菌或细菌活性。上述结果表明，6株接种的外源菌株可促进内源细菌的检出。此外，接种不动杆菌属菌株可促进芽孢杆菌属的检出，而接种迪茨氏菌属菌株与假单胞菌属菌株则会促进更多假单胞菌属的检出。与之相对，在属分类水平上，同属的外源细菌可刺激对应的相似内源细菌增殖。基于上述结果，在评估外源细菌的烃类降解能力时，应特别关注可耐受高压蒸汽灭菌的细菌所带来的影响，尤其针对生物强化与微生物强化采油相关研究。后续可通过优化生物强化与微生物强化采油技术，更精准地调控特定内源细菌的生长，进而提升原油处理或采油效率。