| 1,3-Propanediol (1,3-PD) is a bulk chemical, which is used in the fields of food industry, medicines and cosmetics, in particular as a monomer to produce a new type of polysester, polytrimethylene terephthalate (PTT). However, it is expensive to synthesize 1,3-PD chemically for the predicted shortage of fossil fuel and pollution of environment. Considerable attention is being paid to the production of 1,3-PD for its advantages, such as use of renewable resources and sustainable development. The bioconversion of glycerol to 1,3-PD is usually carried out by Klebsiellia pneumonia under anaerobic conditions. But low biomass, concentration and productivity of 1,3-PD are unsatisfactory. In this work, the effects of aeration in microaerobic conditions on metabolism of 1,3-PD were investigated, and these experiments were compared with anaerobic conditions in batch, fed-batch and continuous fermentations. Based on the distribution of carbon flux under different conditions, the mechanism of glycerol metabolism was analyzed. Additionally, considering the advantage of microaerobic and anaerobic fermentation of K.pneumoniae, a novel multi-stage fed continuous culture for 1,3-PD production was investigated by combining microaerobic with anaerobic aeration.To begin with, a high performance liquid chromatographic method was set up by means of a column of Shodex RSpak KC-811 for rapid measuring the concentrations of a-ketoglutaric acid, pyruvic acid, citric acid, malic acid, fumaric acid, succinic acid, lactic acid, formic acid and acetic acid. The mobile phase was water and perchloric acid solution (4 mmol/L, pH 2.4) at a flow rate of 1 mL/min. The ratio of perchloric acid to water was controlled by a gradient elution program. The assay was carried out at a column temperature of 60℃and detection wavelength of 210 nm. Eight organic acids in 1,3-PD fermentation broths could be effectively separated and detected by this method in 15 min. The recovery and RSD were 96.8～102.5% and 0.97-2.72%, respectively. All the correlation coefficients (R) were not less than 0.9992. This precise and accurate method could be used to analyze the organic acids in 1,3-PD fermentation broths.In addition,1,3-PD production by K. pneumoniea and the distribution of carbon flow in exponential phase were studied in batch and fed-batch cultures under microaerobic and anaerobic conditions. The experimental results showed that the microaerobic condition with 0.04 vvm air sparged was preferable, which resulted in high concentration of 17.30 g/L and 57.49 g/L, high molar yield of 0.52 and 0.55 mol/mol, and high productivity of 2.47 and 3.03 g/(L-h) in batch and fed-batch fermentation. Although the analysis of carbon flow distribution confirmed that the flux into the reductive pathway decreased with the increasing of air flow, the flux of 1,3-PD (46.15%) with 0.04 vvm air sparged was still higher than that in anaerobic condition, mainly because of the lower releasing ratio of 3-hydroxypropionaldehyde in the reductive pathway. The same ratio (1:3) of 0.04 vvm air and with nitrogen demonstrated that qEtOH/qHAc could indicate the balance between generation of NADH and ATP. Analysis of carbon flow distribution of organic acids in tricarboxylic acid (TCA) cycle showed that TCA cycle is not completed under microaerobic condition. Otherwise, the analysis of distribution of fed-batch fermentation showed the flux of 1,3-PD was highest with 0.04 vvm air sparged under microaerobic conditions which was 93% of the value under anaerobic condition.What’s more, the effects of glycerol concentration and oxygen supply on glycerol dissimilation in K. pneumonia were investigated in chemostat cultures at steady states. Cell growth, formation of 1,3-PD and by-products, and specific activities of three key enzymes were investigated at three glycerol concentrations under four aeration conditions at a dilution rate of 0.2 h-1. According to these results, the airflow rate of 0.04 vvm was preferable, with the highest 1,3-PD concentration of 19.7 g/L and a yield of 0.41 mol/mol under high initial glycerol condition. However, the highest concentration and yield of 1,3-PD were achieved for low initial glycerol and medium initial glycerol concentrations under anaerobic conditions. Under microaerobic conditions, the availability of NADH could be used for producing 1,3-PD theoretically. But experimental results showed that more carbon flux was shifted to the oxidative pathway instead of the reductive pathway, and used to produce lactic acid and 2,3-BD under microaerobic conditions. Changing the vEtOH/vHAc ratio is one of the ways to regulate the balance between NAD+ and NADH. In this case, the bottleneck to enhance the concentration of 1,3-PD is not the pool of NADH, but the regulation of the distribution of carbon flow at glycerol dissimilation point. Enzyme activity assays showed that the dha operon was not repressed but promoted under microaerobic conditions. Furthermore, on the basis of the specific activities of GDH, PDOR and GDHt measured in the cell extracts, the differences in the regulation mechanism for 1,3-PD production exist not only at the level of metabolism, but may also be at the level of gene expression.Finally, a new process for the fermentation of 1,3-PD by K. pneumoniae combining microaerobic with anaerobic aeration in a multi-stage fed-continuous culture was performed at different initial glycerol concentration in the first stage and residual glycerol concentration in the second stage. In the first stage, high cell biomass (OD650=5.1-7.0) was obtained under microaerobic condition (0.04 vvm air). Furthermore, in order to prevent limit or inhibit effect of substrate on cell growth, glycerol feed concentration was chosen to be low (40 g/L) at a high dilution rate (D=0.2 h-1) or high (70～110 g/L) at a low dilution rate (0.1 h-1). In the second stage, glycerol was fed continuously into the culture to maintain the residual concentrations of glycerol about 15～45 g/L, so that the concentration and yield of 1,3-PD was expected to be improved by anaerobic aeration (0.04 vvm N2). In the last stage, the residual glycerol was consumed to low level (1～13 g/L) and further enhance of the concentration and yield of the 1,3-PD was achieved under anaerobic condition (0.04 vvm N2). Under these conditions, a final 1,3-PD concentration of 36.7～46.2 g/L, a volumetric productivity of 4.0～8.1 g/(L·h) can be obtained. The highest concentration of 1,3-PD (46.2 g/L) was achieved at initial glycerol concentration of 110 g/L and dilute rate of 0.1 h-1, however, the low productivity and high residual glycerol in final broth was not benefit for the industrial production. The initial glycerol concentration of 40 g/L at dilute rate of 0.2 h-1 when residual glycerol was controlled at 30.9 g/L was preferable, corresponding to the high 1,3-PD concentration of 36.7 g/L, productivity of 7.3 g/(L·h) and low residual glycerol concentration of 1.7 g/L. This can be concluded that this novel fermentation process could increase the biomass, concentration and productivity of 1,3-PD, meanwhile, decrease the final concentration of residual glycerol, which is benefit for the separation of 1,3-PD. |
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