Biotechnological valorisation of crude glycerol from biodiesel manufacturing processproduction of 1,3-propanediol by clostridium butyricum

Resumen

1,3-Propanediol can be biologically produced from glycerol by several microorganisms, namely clostridia. Different strategies have been tried to optimize this conversion and set up a practicable process. In this work, the production of 1,3- propanediol from crude glycerol for the biodiesel industry was investigated, testing different process strategies using Clostridium butyricum DSM 10703. In order to optimize the operation conditions that maximize the production of 1,3- propanediol, the influence of operation variables (temperature, pH and initial glycerol concentration) in 1,3-propanediol biosynthesis was studied. Furthermore, different culture techniques (batch, fed-batch and chemostat cultures) were tested with pure glycerol (Sigma 99 % (wt/wt)) as substrate for further comparison with crude glycerol experiments. A first set of experimental runs, based on a factorial design of experiments, were conducted in a batch system with pure glycerol to obtain the operating conditions that maximize both 1,3-PD concentration and yield factor. The inhibitory effect of the initial substrate concentration on fermentation to 1,3-PD was also studied for pure glycerol. Assays with biodiesel crude glycerol were carried out under the operation conditions that maximize 1,3-propanediol concentration and yield. The concentration of 1,3-propanediol reached a maximum of 35.9 g/L, slightly lower than the concentration reached using pure glycerol. The yield factor was 0.51 g of 1,3-PD/g glycerol, also below the value reached in the fermentation of pure glycerol. The main by-product was butyric acid (7.9 g/L) followed by acetic acid (2.7 g/L). In this case, butyric acid production was higher than when pure glycerol was used, while the acetic acid concentration was quite similar for both substrates. The maximum initial crude glycerol concentration tolerated by C. butyricum, while maintaining a high production of 1,3-PD, was 70 g/L, lower than the 100 g/L for pure glycerol experiments. Batch fermentation of glycerol by Clostridium butyricum DSM 10702 was also successfully simulated by the kinetic Contois type-model. In fed-batch, using crude glycerol as substrate, the maximum 1,3-propanediol concentration was 36.1 g/L, close to the 37.3 g/L reached in fed-batch pure glycerol experiments. Acetic and butyric acid concentrations reached, respectively, 2.5 g/L and 9.0 g/L. Continuous cultures provided 27.5 g/L of 1,3-propanediol at a dilution rate of 0.05 h-1 and 70 g/L of glycerol in the fed stream. Nevertheless, at D=0.15 h-1, a high 1,3-PD concentration was achieved (¿ 22 g/L), with a lower production of butyrate and acetate but higher productivity. A pretreatment process for biodiesel-based crude glycerol was evaluated to eliminate a number of impurities generated during the transesterification process and reduce the concentrations of growth inhibitors, such as salts and pigment. Two different pretreatments were tested based on ion exchange resins. Serum bottle batch fermentations by C. butyricum DSM 10703 were run, at different initial substrate concentrations, to select the most effective pretreatment in terms of 1,3-propanediol concentration, yield, productivity and high substrate tolerance. The production of 1,3-propanediol by C. butyricum DSM 10702, using pretreated glycerol, was feasible in the two pretreatments tested. The serum bottle results showed that the presence of salts between 4 and 5 wt% in crude glycerol do not originate C. butyricum DSM 10702 inhibition. For the two different fermentation modes tested (batch and fed-batch systems), the proposed pretreatment improves the results reached in fermentations carried out with untreated crude glycerol. An immobilization technique was evaluated, and the use of sodium alginate gel as the immobilization material on C. butyricum cell growth and 1,3-propanediol production from crude glycerol was studied. The concentration and yield of 1,3-propanediol under batch and continuous cultures was also studied and compared with the results of free cell culture. The production of 1,3-propanediol by immobilized C. butyricum in alginate biobeads demonstrated desirable characteristics, including repeated cultivations with a high biomass density. The batch fermentation results with immobilized C. butyricum provided a higher concentration of 1,3-propanediol (13.77 g/L, an increase of 2.21 g/L compared to the free cells) and substantially higher biomass concentrations (2.3-fold higher than that of the free cell culture). Furthermore, the acetic acid route was affected, and this acid was not detected in the effluent. In continuous fermentations, the results were similar to those obtained with free cell continuous fermentations of crude glycerol with the exception of acetic acid concentration, which was considerably below.