Biotransformação do glicerol - subproduto da produção de biodiesel

Abstract

The increasing goal for escalating the national production of biodiesel will result in the generation of an increasing amount of glycerol, a byproduct of this process, with the consequent devaluation of its price in the domestic market and increased need for new routes of application of this material. Thus, we proposed in this thesis, the study of the microbial metabolism of glycerol and the main variables affecting it, and additionally, the isolation of a bacterium which can potentially transform this raw material into products of higher commercial value. A strain was isolated from soil samples, inoculating it with media whose carbon source was glycerol standard PA. The formulation of the isolation media and fermentation conditions was well-founded on a theoretical analysis of several studies on the degradation of glycerol by fermentation, available in the literature. The isolated bacterium was identified by biochemical tests as Klebsiella oxytoca. The data indicated that bacterial growth and glycerol consumption were favored at pH 7.4, with urea as nitrogen source and low initial substrate concentration (12.6 g L-1). Under these conditions, for a reaction time of 48 h, the glycerol consumption reached 99.77%. When high concentrations of substrate (75.6 g L-1) were used, the best result in terms of consumption of glycerol (69.83%) was obtained for tests with the absence of tryptone, using thioglycollate as an oxygen reducing agent and high urea concentration (4.9 g L-1). The tests also showed that the addition of tryptone (2.0 g L-1) had a positive effect only when the urea concentration in the medium was low (1.5 g L-1). The main products formed were 1,3-propanediol, formate, 2,3-butanediol, lactate and acetate being produced very small amounts of ethanol. The highest rate of production of 1,3-propanediol occurred during the logarithmic phase of growth for both PA glycerol (5.67 mmol L-1 h-1) and for crude glycerin (5.28 mmol L-1 h-1). The product profile analysis showed that as the gases formed by fermentation were removed there was an increase in the ethanol production. However, when CO2 and H2 were not taken from the fermentative broth, production of ethanol was low (0.15 g L-1). The degradation of formate is linked to the process of regeneration of NADH. However, the mechanism of consumption of hydrogen formed from formate has not been completely explained. This mechanism serves as an alternative to maintaining the redox balance of the system and was more active in the final stage of the logarithmic growth phase or stationary phase. The yield of 1,3-propanediol derived from 75,6 g L-1 of glycerol and the isolated strain was equal to 42.90% and 39.60% for standard PA glycerol and crude glycerin, respectively. These results are very close to those determined for other strains of K. oxytoca and K. pneumoniae found in the literature, which values the selected strain. During fermentation of glycerol the imbalance between the reactions of the oxidative and reductive branches of metabolism leads to accumulation of 3-hydroxypropionaldehyde and cessation of bacterial growth. This phenomenon is probably due to a high in vivo activity of the enzyme glycerol-dehydratase, when compared to other enzymes of the anaerobic metabolism of glycerol under our experimental conditions. The data also indicated that this imbalance can be reduced by maintaining microaerobic conditions in the fermentation media. The mechanism of regeneration of NADH present in the bacterium Klebsiella oxytoca seems not to be restricted to a single direction of this conversion (formation of reduced or oxidized forms of NAD), because the enzyme reactions catalyzed by the enzyme glycerol dehydrogenase (oxidative branch) and an the enzyme 1,3-propanediol dehydrogenase (reductive branch) indicated that the microorganism consumed H2 partially to regenerate NADH. This seems to be an alternative metabolic compensation to stabilize or reduce the concentration of 3-hydroxypropionaldehyde.