## 3 Results and Discussion
### Impact of Temperature  
To investigate the effect of temperature on the fermentation, fed-batch fermentations were performed at temperatures ranging from 27 to 36 \({}^{\circ}\)C. The changes in dry cell weight (DCW) and metabolite levels (1,3-PD, lactic acid, acetic acid, succinic acid, ethanol) produced as the result of glycerol bioconversion were depicted in Figure 1. The conducted experiments revealed that the temperature of the process affects the dynamics of _C. freundii_ AD119 growth and the accumulation of metabolites. Table 1 presents the final concentrations of the produced metabolites and DCW and the maximum concentration of 3-HPA observed in the process. Figure 1: Time course of dry cell weight (DCW) and metabolite production (1,3-propanediol (1,3-PD), lactic acid, acetic acid, succinic acid, ethanol) with _C. freundii_ AD119 in a fed-batch culture with a fixed pH value of 7.0 and stirring rate of 80 rpm at (**a**) 27 \({}^{\circ}\)C, (**b**) 30 \({}^{\circ}\)C, (**c**) 33 \({}^{\circ}\)C, and (**d**) 36 \({}^{\circ}\)C.  
The best results were obtained at 30 \({}^{\circ}\)C, in which case 43.30 g/L of 1,3-PD were produced. The highest concentrations of the accompanying metabolites were also observed at this temperature. Lactic (10.82 g/L) and acetic (9.65 g/L) acids were the dominating by-products. Succinic acid and ethanol were also present in the fermentation broth at the concentrations of 4.85 g/L and 1.86 g/L, respectively. Throughout the process, glycerol concentration was maintained in the range of 30-45 g/L. Interestingly, in the experiments performed at temperatures surpassing 30 \({}^{\circ}\)C, cessation of product formation and cell growth was observed. At these temperatures, the final concentrations of 1,3-PD were around four times lower than in the process conducted at 30 \({}^{\circ}\)C. The titers of the accompanying fermentation products were lower when compared to those observed in the process performed at 30 \({}^{\circ}\)C, and the fed-batch mode of fermentation prevented depletion of glycerol. Thus, the inhibition could neither be attributed to an excessive concentration of fermentation by-products nor to starvation. Available data indicate that the accumulation of 3-HPA, an intermediate of the conversion pathway of glycerol to 1,3-PD, leads to such a result [2,17]. Moreover, in an experiment in which exogenous 3-HPA was added to a culture of actively growing bacteria, immediate growth cessation was observed [17]. In a report by [PERSON] et al. [30], a genetically modified strain of _C. freundii_, which overexpressed 1,3-PD oxidoreductase, showed superior capability for 1,3-PD production (35.6 g/L, vs. 25.5 g/L of the wild strain) accompanied by a decreased accumulation of 3-HPA. The overexpressed enzyme is responsible for catalyzing the final step of the pathway--transformation of 3-HPA to 1,3-PD. The concentration of 3-HPA was thus determined (Figure 2). In all the cultures in which cessation of fermentation was observed, the maximal concentration of this intermediate was found to be at least two-fold increased, compared to the process performed at 30 \({}^{\circ}\)C.  
In the experiments performed at temperatures higher than 30 \({}^{\circ}\)C, the concentration of 3-HPA reached its maximum between 8 and 13 h of fermentation, at which time also the premature cessation of the processes was observed. Although the accumulation of 3-HPA in the medium was transient, the fermentation has not been observed to resume after the titer of the intermediate had decreased. It is noteworthy that when the strain was cultured in a medium with the glycerol having been replaced with glucose, no inhibition of the fermentation was observed even at a temperature of 37 \({}^{\circ}\)C (Figure 3). As expected, no 1,3-PD production was observed in this case as glycerol is the sole substrate that can be converted to the diol in microorganisms not subjected to genetic manipulation [4].