Hydrocarbons are a class of compounds that consist entirely of carbon and hydrogen. Typical hydrocarbons and their derivatives, including aromatic hydrocarbons and alkanes, are widespread in the environments, leading to serious problems worldwide. Understanding the microbial processes responsible for degradation of hydrocarbons can assist in developing efficient strategies for bioremediation of organic pollutants and mitigating global climate changes. Both aerobic and anaerobic microorganisms play an important role for removal of hydrocarbons under natural settings. Microbes and the underlying reactions catalyzing biodegradation of organic pollutants under aerobic conditions have been intensively studied since several decades. However, the in situ degradation processes occurred in natural complex environments like soil and sediments, as well as the microbiology of anaerobic hydrocarbon degradation remain elusive.
In this study, we applied real-time PCR (RT-PCR), DNA-stable isotope probing (DNA-SIP) and nanoscale secondary ion mass spectrometry (NanoSIMS) to identify and visualize the active microorganisms responsible for in situ biodegradation of aromatic hydrocarbons in soil environments. Furthermore, fluorescence in situ
hybridization (FISH), metagenomics, metaproteomics and metabolomics were utilized to reveal the microorganisms and underlying reactions catalyzing biological consumption of alkanes under anoxic conditions. The main content and results are as followings:
1) qPCR and clone library were used to monitor the dynamic response of functional microbes and genes to pyrene contamination in an agricultural soil and an industrial soil. Over 80% of added pyrene was dispersed in both soils after 35-day incubation. Concurrently, gene abundances targeting pyrene-degrading bacteria,
including Gram-positive bacteria harboring PAH-ring hydroxylation dioxygenase (PAH-RHDα GP) and Mycobacterium, were dramatically enriched. In addition, the abundances of pyrene dioxygenase encoded by nidA and nidA3, increased significantly during pyrene incubation, suggesting that two underlying catabolic pathways are responsible for pyrene degradation in both soils. The different behaviors of two soil microbial communities in response to pyrene stress indicates that soil conditions may affect in situ processes of pyrene biodegradation.
2) To identify active pyrene-degrading bacteria, we applied 13C-pyrene incubations, DNA stable isotope probing (DNA-SIP) and high-throughput sequencing in an agricultural soil and an industrial soil. After 35-day incubation, pyrene in both soil was mineralized, with industrial soil showed higher rates than agricultural soil. As revealed by DNA-SIP, the uncultivated members of Pseudonocardia were the dominant pyrene degraders in agricultural soil; their relative abundances increased by more than 3 orders of magnitude. In contrast, Arthrobacter sp. was found as the major pyrene-degrading populations in industrial soil, whereas Pseudonocardia was not detected. In both soils, Mycobacterium was found to be actively involved in pyrene
3) By applying 13C-biphenyl incubation followed by NanoSIMS analysis, we visualized the biphenyl-degrading populations in four PCB-contaminated sediments and soils. 13CO2 measurement in headspace of microcosms indicated that biphenyl was significantly mineralized during 4-day incubation. NanoSIMS analysis on soil
microorganisms showed that biphenyl-degrading microbial populations comprised 5%-10% of the whole communities. These results emphasize that the numerous inconspicuous microbes play a key role in biphenyl/PCB degradation in the environments. Using 13C/12C ratio, the carbon assimilation rate was estimated for each metabolically active cell. The relative abundances and catabolic activities of the biphenyl-degraders differ significantly among four sediments/soils.
4) We reveal hitherto unknown ethane-utilizing archaea via specific enrichment, fluorescence in situ hybridization, metagenomic and metaproteomic and metabolic analyses. The enrichment culture, which coupled ethane oxidation to sulfate reduction,was predominated by an archaeon named Candidatus Argoarchaeum ethanivorans;
other members include sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum encodes all genes for a methyl-coenzyme M reductase with all subunits detected in protein extracts. Consistently, ethyl-coenzyme M was identified as an intermediate by liquid chromatography/mass spectrometry. This indicated that Ca. Argoarchaeum activates ethane by forming ethyl-CoM, which is analogous to the anaerobic butane-oxidizing archaea Ca. Syntrophoarchaeum. Further metagenomic and proteomic analysis suggest oxidation of acetyl-CoA to CO2 through the oxidative Wood-Ljungdahl pathway.
This study expanded our knowledge of microbial processes and mechanisms involved in hydrocarbon degradation. The identification and visualization of environmentally-relevant microorganisms responsible for degradation of aromatic hydrocarbons will have a potential impact in defining future bioremediation strategies. Furthermore, we revealed the microorganisms and reactions that catalyze anaerobic oxidation of ethane. These results fill a gap in our knowledge of microorganisms that specifically oxidize members of homologous alkanes series without oxygen.