French Katherine E, Terry Norman
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, United States.
Front Microbiol. 2019 Jun 19;10:1318. doi: 10.3389/fmicb.2019.01318. eCollection 2019.
Over the past 100 years, oil spills and long-term waste deposition from oil refineries have significantly polluted the environment. These contaminants have widespread negative effects on human health and ecosystem functioning. Natural attenuation of long chain and polyaromatic hydrocarbons is slow and often incomplete. Bioaugmentation of polluted soils with indigenous bacteria that naturally consume petroleum hydrocarbons could speed up this process. However, the characterization of bacterial crude oil degradation efficiency - which often relies upon expensive, highly specialized gas-chromatography mass spectrometry analyses - can present a substantial bottleneck in developing and implementing these bioremediation strategies. Here, we develop a low-cost, rapid, high-throughput fluorescence-based assay for identifying wild-type bacteria that degrade crude oil using the dye Nile Red. We show that Nile Red fluoresces when in contact with crude oil and developed a robust linear model to calculate crude oil content in liquid cell cultures based on fluorescence intensity (FI). To test whether this assay could identify bacteria with enhanced metabolic capacities to break down crude oil, we screened bacteria isolated from a former Shell Oil refinery in Bay Point, CA, and identified one strain ( sp. OPK) with superior crude oil depletion efficiencies (up to 83%) in only 3 days. We further illustrate that this assay can be combined with fluorescence microscopy to study how bacteria interact with crude oil and the strategies they use to degrade this complex substance. We show for the first time that bacteria use three key strategies for degrading crude oil: biofilm formation, direct adherence to oil droplets, and vesicle encapsulation of oil. We propose that the quantitative and qualitative data from this assay can be used to develop new bioremediation strategies based on bioaugmentation and/or biomimetic materials that imitate the natural ability of bacteria to degrade crude oil.
在过去的100年里，炼油厂的石油泄漏和长期废物沉积对环境造成了严重污染。这些污染物对人类健康和生态系统功能产生了广泛的负面影响。长链和多环芳烃的自然衰减缓慢且往往不完全。用天然消耗石油碳氢化合物的本地细菌对污染土壤进行生物强化可以加速这一过程。然而，细菌原油降解效率的表征——通常依赖于昂贵、高度专业化的气相色谱-质谱分析——可能会成为开发和实施这些生物修复策略的一个重大瓶颈。在这里，我们开发了一种低成本、快速、高通量的基于荧光的检测方法，用于识别使用染料尼罗红降解原油的野生型细菌。我们发现尼罗红与原油接触时会发出荧光，并建立了一个强大的线性模型，根据荧光强度（FI）计算液体细胞培养物中的原油含量。为了测试这种检测方法是否能够识别具有增强代谢能力以分解原油的细菌，我们筛选了从加利福尼亚州贝波特的前壳牌炼油厂分离出的细菌，并鉴定出一种菌株（sp. OPK），该菌株在仅3天内就具有高达83%的优异原油消耗效率。我们进一步说明，这种检测方法可以与荧光显微镜结合使用，以研究细菌如何与原油相互作用以及它们用于降解这种复杂物质的策略。我们首次表明，细菌使用三种关键策略来降解原油：生物膜形成、直接附着于油滴以及油的囊泡包裹。我们建议，该检测方法的定量和定性数据可用于开发基于生物强化和/或模仿细菌降解原油天然能力的仿生材料的新生物修复策略。
