EpicPCR-based identification of diversity and community structure of environmental alkBs-carrying bacteria

Oil spills during transport have resulted in severe hydrocarbon contamination in soil and water. As endogenous microorganisms, hydrocarbon oxidizing bacteria (HOB) play a vital role in degrading hydrocarbons in oil-contaminated environments. However, accurately identifying and tracking HOB distribution remain difficult due to the low conservative and high diversity of their key alkane monooxygenase genes (alkBs). Here, we proposed a workflow with multiple-primers-based epicPCR processes to identify HOBs in the environment. Epic-alkB database, which contained 14 times more genera and 6 times more phyla than the known database (AlkBase, 23 genera and 4 phyla) was built successfully with 5 pairs of major primers, and some of the genera coexist in both databases or specific to the epic-alkB database were functionally validated by newly isolated HOBs. The addition of multiple primer pairs enabled us to obtain accurate HOBs with higher coverage and diversity than AlkBase. Besides, the alkB genes varied among microorganisms. unclassified_Ignavibacteriales, Pseudomonas, and Thalassolituus were mainly amplified by alkB_4/5/6 respectively, with alkB_6 being the most prevalent and abundant. In conclusion, an epicPCR-based workflow was provided to facilitate the accurate identification of HOBs and the exploration of the distribution of alkB genes in the biosphere, which greatly expands the bacterial species for aerobic hydrocarbon degradation and offers a universal approach for the identification and quantification of isozyme genes in the environment.