Abstract

Vinasse is generated as the main by product of the ethanol industry and is composed mainly of water, minerals, organic acids, glycerol and sucrose. Polyhydroxyalkanoates (PHAs) are natural bioplastics produced by many bacteria from different substrates, and can replace petrochemical plastics besides being biodegradable and biocompatible. Ralstonia eutropha is a model organism for the study of biosynthesis of PHA, and can accumulate over 90 % of its cell dry weight as PHA. R. eutropha is able to grow on glycerol but on previous experiments in the laboratory it was observed that when growing on vinasse it was unable to utilize all the carbon available, especially glycerol. The aim of this work was to develop a R. eutropha strain optimized for the utilization of vinasse as a carbon source for the production of PHA. To improve glycerol utilization and PHA production, putative proteins of the phosphoenolpyruvate-carbohydrate photransferase system (PEP-PTS), the fruAptsHI endogenous operon, was overexpressed. These genes were previously described as responsible for limited cell growth and reduced PHA accumulation in media containing glycerol as sole carbon source. The operon fruAptsHIwas cloned on pBBR1MCS-2 generating the plasmid pTRANS which was inserted in R. eutropha. The resulting strain was tested on vinasse and on glycerol as sole carbon source to study growth and PHA accumulation. During tests on pure vinasse, complemented with nitrogen to avoid nutrient limitation, the engineered strain (H16/pTRANS) presented 4.6 g/L of final biomass while the control strain (H16/pBBR1MCS-2) reached 2.17 g/L, before growth stopped. Final PHA content of 0.1 g/L was similar for both strains. The overexpression of the genes allowed R. eutropha to consume more carbon available in the vinasse than the control strain. On test using glycerol as the sole carbon source and nitrogen limitation it was observed a final production of 26.3 % of PHB accumulated by the engineered strain, which is 1.6 times higher than the control strain in the conditions tested, while residual biomass was the same. Engineering R. eutropha to improve the consumption of the carbon sources available in the vinasse is one of the challenges of using this feedstock as substrate for a biopolymer production process. The overexpression of a fruAptsHI operon in R. eutropha enhanced PHA production when growing on glycerol and enhanced biomass production when growing on vinasse.