Variances in cellular sedimentation behavior as an effective enrichment method of hydrocarbon-overproducing Micrococcus luteus strains

Angel Angelov,Maria Übelacker,Wolfgang Liebl

doi:10.1186/s13068-018-1286-6

Abstract

BackgroundAliphatic hydrocarbons of microbial origin are highly interesting candidate biofuels because these molecules are identical or very similar to the main components of petroleum-based gasoline and diesel fuels. The high-GC Gram-positive bacterium Micrococcus luteus is capable of naturally synthesizing long-chain, iso- and anteiso-branched alkenes which are formed via the head-to-head condensation of fatty acid thioesters by a dedicated enzyme system. The present study describes the relation we observed between olefin production and cell buoyancy in Micrococcus luteus and the use of this phenotype to simply and efficiently separate cells from a mixture based on their hydrocarbon content.MethodsWe generated M. luteus mutants producing different amounts of olefins and used them in mixing and sedimentation experiments, olefin content analysis by GC-MS and in equilibrium centrifugation in Percoll gradients.ResultsWe found well-detectable differences in the buoyant densities of the examined strains, which correlated with the amounts of hydrocarbons produced by the cells. We also demonstrate how our observations can be used to simply and efficiently fractionate cells based on their hydrocarbon content.ConclusionsIn summary, we show that cultures of M. luteus cells sediment at distinct rates depending on the amounts of alkenes produced. Our results indicate that buoyant cell density is the primary cause for the observed differences in sedimentation behaviour. The simple separation strategy described here can be a valuable tool in various mutagenesis and enrichment protocols, aimed at generating and isolating strains with increased olefin productivity.

Highlights

Aliphatic hydrocarbons of microbial origin are highly interesting candidate biofuels because these molecules are identical or very similar to the main components of petroleum-based gasoline and diesel fuels
The present study describes the relation we observed between cell buoyancy and olefin production in Micrococcus luteus and the use of this phenotype to and efficiently separate cells from a mixture based on their hydrocarbon content
During our work on engineering the olefin biosynthetic pathway in Micrococcus luteus, we repeatedly observed that the cells of liquid cultures expressing different amounts of olefins show distinct sedimentation behavior

Summary

Introduction

Aliphatic hydrocarbons of microbial origin are highly interesting candidate biofuels because these molecules are identical or very similar to the main components of petroleum-based gasoline and diesel fuels. Angelov et al Biotechnol Biofuels (2018) 11:288 spectrometry (GC–MS) This is the most accurate and reliable method to measure such compounds, it is not well suited for high-throughput settings, for example, in a screening of a random mutagenesis library. For high-throughput assays, the fluorescence emitted by the hydrophobic dye Nile Red has been established as a convenient proxy for the amounts of hydrocarbons and ketones in bacteria [8] and Nile Red-based screenings have been employed in the isolation of free fatty acid overproducing mutants of Escherichia coli [9]. With the population heterogeneity in hydrocarbon-producing M. luteus cells described by us, it may be feasible to apply similar fermentation strategies to increase productivity

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