Produção de 2,3-butanodiol por Klebsiella oxytoca e Enterobacter aerogenes a partir de diferentes fontes de carbono e regime de operação

Abstract

Different carbon sources have been used in fermentation processes for 2,3-butanediol (2,3- BDO) and acetoin (acetyl methyl carbinol; AMC) production. These substrates can be obtained from agro-industrial by-products, improving the economy of the respective processes. Considering the sugar and alcohol industry, lignocellulosic hydrolysates from residual sugarcane bagasse could be used as carbon source for this purpose. However, lignocellulosic hydrolysates normally have low concentration of sugars, which makes impossible the attainment of the desired levels of products. As such, sugarcane molasses, a relatively low-cost raw material that is rich in sucrose, could be added to the hydrolysate, resulting in higher concentration of carbohydrates. The aim of this work was to evaluate the microbial growth and the production of 2,3-BDO and AMC by Klebsiella oxytoca and Enterobacter aerogenes in mineral medium, using glucose, xylose, fructose, and sucrose as carbon sources in order to establish the basic knowledge that could be applied to the production process from mixtures of the pentose-rich fraction of lignocellulosic hydrolysates and sugarcane molasses. Preliminary experiments were carried out in Erlenmeyer flasks under reciprocal agitation, using mineral medium supplemented with the carbon sources, either pure or mixed, at different concentrations, at initial pH 5.5 and 37ºC. Furthermore, cultivations were also performed in batch and fed-batch modes, in a bench-scale bioreactor. Among the experiments carried out, a central composite design was used for optimizing pH and temperature values of cultivation, aiming to increase 2,3-BDO+AMC production. The studies also allowed the definition of the most suitable micr oorganism to metabolise the set of carbohydrates evaluated. When sugars were used one at a time, the cell growth of both cultures was favoured by sucrose. Xylose metabolism was slower probably due to a longer microbial adaptation phase. In the shaken flasks tests, in medium with 60 g/L of xylose, K. oxytoca presented productivity 22% superior to that obtained with E. aerogenes. In batch or fed-batch cultivations, using dif consumption of glucose, rather than the other sugars. K. oxytoca was more able to metabolise the carbohydrate mixture tested in fed-batch runs, in which final concentrations around 75 g/L of 2,3-BDO+AMC were attained, with yield and productivity higher than those obtained with E. aerogenes. Thus, K. oxytoca was defined for further studies. In the pH and temperature optimization experiments, 5.7 and 34.5ºC were defined as the best values for the process. In fed-batch runs under such conditions, approximately 80 g/L of 2,3-BDO+AMC were achieved, resulting in a productivity of 2.32 g/L/h, which means an average increase of 27% when compared with data previously reported in the literature. From the results of this study, a set of relevant information related to the application of by-products of the sugar and alcohol industry for 2,3-BDO and AMC production was obtained. In particular, the data from fed-batch experiments conducted under optimised pH and temperature indicate the feasibility of carrying out larger-scale fermentation studies envisaging the industrial production of 2,3-butanediol and acetoin.