A novel method for in situ identification and sorting of cycloalkane-degrading microbes at single-cell level in marine water samples

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Microbes RACS-Seq

Highlights

• Cycloalkane-degrading cells in seawater can be readily identified by “redshift” in single-cell Raman spectra (SCRS) of cells collected from seawater samples incubated with stable isotope 13C-labeled cyclohexane
• Using RACS-Seq®, Raman-activated Optical Tweezers based Cell Sorter, cells that undergo Raman redshift were identified and isolated. They have been confirmed to be  cycloalkane-degrading bacteria by 16S rRNA sequencing. RACS-Seq® can directly link the phenotype of microbes with their genotype at the single-cell level
• Using RACS-Seq®, a new uncultured marine bacterium genus C1-B045 capable of degrading cycloalkanes was discovered, and the cycloalkane degradation pathway was proposed through metagenomic sequencing data

Abstract

Cycloalkanes are hydrocarbon compounds commonly present in crude oil and condensate, and they are degraded in the marine environment mostly by low-temperature microorganisms. Most of these microorganisms are difficult to culture in the laboratory, and therefore the majority of the active species involved in marine cycloalkane degradation have not been identified yet. This study used a combination of stable isotope labeling, single-cell Raman sorting technology, and 16S rRNA gene sequencing to discover a new genus of uncultured marine bacteria, C1-B045, that can effectively consume methylcyclohexane (MCH) in the marine environment. We used RACS-Seq® to link metabolic phenotype and genotype at single-cell resolution, and proposed an ε-caprolactone MCH degradation pathway. This pathway may involve multiple key enzymes, including alkyl monoxygenase, cyclohexanone monoxygenase, and 6-caprolactone hydrolase.

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