Produção de 2,3-Butanodiol a partir de glicerol por klebsiella oxytoca ATCC 8724

Abstract

Glycerol is a by-product of biodiesel production, which accounts for approximately 10% of the biofuel. Since a large volume of it is formed in industry, glycerol could be used as a substrate to obtain other compounds, such as 2,3-butanediol, through fermentative routes. This substance, which has potential for application in industry and as fuel, can be obtained by glycerol fermentation with the facultative anaerobe bacterium Klebsiella oxytoca. In this work, parameters and operational conditions for the cultivation of K. oxytoca ATCC 8724, envisaging 2,3-butanediol production from glycerol, were assessed. Due to the equilibrium between 2,3-butanediol and acetoin, in a significantly higher proportion for the first product, in the fermentative metabolism of K. oxytoca, the discussion of this work takes in account the sum of concentrations of these compounds. The results have shown that the microorganism is able to efficiently metabolize glycerol once cell growth rate on this substrate, after 8 hours of cultivation in shake flasks, was 0.44 g/L/h, higher than that obtained with glucose (0.38 g/L/h) under the same conditions. As previously described for saccharides, it was observed that the microbial metabolism can be divided into two phases: in the first phase, without limitation of oxygen, an intense cell growth occurs due to the prevalence of respiration, that results in a substantial gain of energy for the microorganism, whereas in the second phase, under oxygen limitation, most of carbon flux is driven to the formation of fermentation products. Thus, in batch runs carried out in a bench bioreactor, the use of an intermediate characteristic oxygen transfer rate (OTR) of about 17 mmol/L/h, led to superior results in terms of 2,3-butanediol production, with conversion of 60 g/L of glycerol to 17.3 g/L of product. In batch mode, the highest conversion of glycerol to product was achieved with 129 g/L of initial substrate concentration, with the production of 53.6 g/L 2,3-butanediol, in 48 hours, which means a yield of 83% in relation to the theoretical maximum. With initial glycerol concentrations up to 60 g/L, no inhibitory effect on cell growth was observed. On the other hand, the use of 160 g/L of glycerol led to a drastic decrease in the process yield to 56.3% and to a residual concentration of substrate of 98 g/L after 72 h of cultivation. By carrying out the process in fed-batch mode, as an alternative to overcome the substrate inhibitory effects, remarkable results have been attained with the mass of glycerol that would be needed to have an initial concentration of 202 g/L in batch mode. Under these conditions, a final concentration of 92.1 g/L of 2,3-butanediol, corresponding to a yield of 92.6%, was achieved after 84 hours of fermentation. The results show clearly the high potential of the use of glycerol as a substrate for 2,3- butanediol production in industrial scale.