Deep sea microbial community: potential to remediate petroleum hydrocarbons. Deep sea microbial community: potential to remediate petroleum hydrocarbons

The present work aims to contribute for the study of deep sea microbial resources and for their exploitation for the development of biotechnological tools that can be used for bioremediation of petroleum hydrocarbon polluted environments.Deep sea sediment samples (~1000 m depth) were collected from four sample sites in North Atlantic Ocean. Autochthonous seafloor microbial assemblages were initially described by next-generation sequencing of the V4–V5 hypervariable region of the 16S ribosomal RNA (rDNA) gene.Then, the remediation potential of microorganisms previously isolated from the sentiment sampled and enriched in petroleum for 15 days was tested.A total of 26 isolates recovered from the enrichment process were identified. Among them, 8 strains from the first location (L1) were then assembled, and their degradation potential was tested in bioremediation experiments. The abundance of hydrocarbon degrading bacteria (HD), in the beginning and the end of the experiment, was estimated based on most probable number method (MPN). Prior to bioremediation experiments, petroleum and acetate enrichments were performed, with the assembled microbial consortia, in Bushnell-Haas medium (supplemented with 2% of NaCl) for 4 days. Then, in a microcosm experiment, 4 different treatments were tested: natural attenuation (N), biostimulation (BS), bioaugmentation with petroleum enrichment (BAp) and bioaugmentation with acetate enrichement (BAa) for 15 days. Additionally, the microbial composition of cultures from the treatments BAp and BAa were analysed. At end of the experiment, 14 morphological different isolates were recovered from the two treatments, including most of those that were initially used to establish the microbial consortium. Furthermore, total petroleum hydrocarbons (TPHs) were determined by Fourier transform infrared spectroscopy analysis (FTIR) showing rates of crude oil degradation between 18% and 30%. Despite these degradation rates being relatively low, when compared with other studies, this is one of the first studies to describe the use of deep-sea microorganism for bioremediation experiments.

本研究旨在为深海微生物资源的研究与开发利用提供支撑，以研发可用于石油烃污染环境生物修复的生物技术工具。研究从北大西洋4个采样点位采集了深度约1000米的深海沉积物样品。首先通过对16S核糖体RNA（16S ribosomal RNA，rDNA）基因的V4-V5高变区进行下一代测序（next-generation sequencing），解析了原位海底微生物群落的组成。随后，本研究对前期从沉积物样品中分离、经石油富集培养15天得到的微生物的修复潜力进行了测试。本次富集培养过程中共获得26株分离菌株并完成鉴定。其中，来自第一个采样点位（L1）的8株菌株被用于构建复合微生物菌剂，随后在生物修复实验中测试了其降解性能。实验初始与结束阶段，基于最大似然数法（most probable number method，MPN）对烃降解菌（hydrocarbon degrading bacteria，HD）的丰度进行了估算。在正式开展生物修复实验前，本研究以添加2%氯化钠的巴斯德-哈斯培养基（Bushnell-Haas medium）为底物，对构建的复合微生物菌剂开展了为期4天的石油与乙酸盐富集培养。随后在微宇宙实验（microcosm experiment）中，设置4组不同处理并开展为期15天的实验：自然衰减组（natural attenuation，N）、生物刺激组（biostimulation，BS）、石油富集型生物强化组（bioaugmentation with petroleum enrichment，BAp）以及乙酸盐富集型生物强化组（bioaugmentation with acetate enrichment，BAa）。此外，本研究对BAp与BAa两组处理的培养物的微生物群落组成进行了分析。实验结束后，从两组处理中分离得到14株形态各异的菌株，其中包含大部分用于构建初始复合菌剂的菌株。此外，本研究通过傅里叶变换红外光谱（Fourier transform infrared spectroscopy，FTIR）分析测定了总石油烃（total petroleum hydrocarbons，TPHs）的含量，结果显示原油降解率介于18%至30%之间。尽管与其他相关研究相比，本次实验的降解率相对偏低，但本研究仍是首批将深海微生物应用于生物修复实验的研究之一。