Downstream processing of biotechnological produced succinic acid.

Succinic acid is a promising chemical which has a wide range of applications and can be biologically produced. The separation of succinic acid from fermentation broth makes more than 50 % of the total costs in their microbial production. This review summarizes the present state of methods studied for the recovery and purification of biologically produced succinate. Previous studies on the separation of succinic acid primarily include direct crystallization, precipitation, membrane separation, extraction, chromatography, and in situ separation. No single method has proved to be simple and efficient, and improvements are especially needed with regard to yield, purity, and energy consumption. It is argued that separation technologies coupled with upstream technology, in situ product removal, and biorefining strategy deserve more attentions in the future.

Ammonium and phosphate limitation in 1,3-propanediol production by Klebsiella pneumoniae.

Excretion of 1,3-propanediol (1,3-PD) by K. pneumoniae was compared in ammonium- and phosphate-limited chemostat cultures running with an excess of glycerol. 59 and 43% catabolic flux were directed to 1,3-PD in ammonia-limited cultures and phosphate-limited cultures at dilution rate of 0.1 h(-1), respectively. Ammonia-limited fed-batch cultures produced 61 g 1,3-PD l(-1) and a total of 15 g l(-1) organic acid in 36 h. However, phosphate-limited fed-batch cultures excreted 61 g lactate l(-1) and 44 g 1,3-PD l(-1).

Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae HR526, a new isolated 1,3-propanediol (1,3-PD) producer, exhibited great productivity. However, the accumulation of lactate in the late-exponential phase remained an obstacle of 1,3-PD industrial scale production. Hereby, mutants lacking D-lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D-lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed-batch fermentation. In experiments using pure glycerol as feedstock, the 1,3-PD concentrations, conversion, and productivity increased from 95.39 g L(-1), 0.48 and 1.98 g L(-1) h(-1) to 102. 06 g L(-1), 0.52 mol mol(-1) and 2.13 g L(-1) h(-1), respectively. The diol (1,3-PD and 2,3-butanediol) conversion increased from 0.55 mol mol(-1) to a maximum of 0.65 mol mol(-1). Lactate would not accumulate until 1,3-PD exceeded 84 g L(-1), and the final lactate concentration decreased dramatically from more than 40 g L(-1) to <3 g L(-1). Enzymic measurements showed LDH activity decreased by 89-98% during fed-batch fermentation, and other related enzyme activities were not affected. NADH/NAD(+) enhanced more than 50% in the late-exponential phase as the D-lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3-PD industrial production.

Physiologic mechanisms of sequential products synthesis in 1,3-propanediol fed-batch fermentation by Klebsiella pneumoniae.

The glycerol fed-batch fermentation by Klebsiella pneumoniae CGMCC 1.6366 exhibited the sequential synthesis of products, including acetate, 1,3-propanediol (1,3-PD), 2,3-butanediol, ethanol, succinate, and lactate. The dominant flux distribution was shifted from acetate formation to 1,3-PD formation in early- exponential growth phase and then to lactate synthesis in late-exponential growth phase. The underlying physiological mechanism of the above observations has been investigated via the related enzymes, nucleotide, and intermediary metabolites analysis. The carbon flow shift is dictated by the intrinsic physiological state and enzymatic activity regulation. Especially, the internal redox state could serve as a rate-controlling factor for 1,3-PD production. The q(1,3-PD) formation was the combined outcomes of regulations of glycerol dehydratase activity and internal redox balancing. The q(ethanol)/q(acetate) ratios demonstrated the flexible adaptation mechanism of K. pneumoniae preferring ATP generation in early-exponential growth phase. A low PEP to pyruvate ratio corresponded LDH activity increase, leading to lactate accumulation in stationary phase.

Influence of dhaT mutation of K. pneumoniae on 1,3-propanediol fermentation.

Metabolic role of 1,3-propanediol oxidoreductase (PDOR) in the production of 1,3-propanediol (1,3-PDO) with K. pneumonia was investigated by knocking out the coded gene dhaT. Fermentation with both the wide-type and mutant were studied in 5 l fermentor. A PDOR-deficient mutant K. pneumonia T1.9131 with 19% PDOR activity of the wild type was constructed. The cultures of the mutant indicated that PDOR inactivation had great effect on the other dha regulon enzymes: activity of glycerol dehydratase decreased by 70% while activity of glycerol dehydrogenase increased by 68%. Fed-batch fermentation showed that more metabolic flux of glycerol was directed to lactate and ethanol in the mutant. Lactate was identified as major metabolite and received an increase in the final concentration from 45 to 91 g l(-1), while the concentration of 1,3-PDO production dropped from 94 to 36 g l(-1). The results demonstrated PDOR was not indispensable in glycerol metabolism but was crucial in high 1,3-PDO productivity. It is postulated that a hypothetical oxidoreductase was expressed and replaced the function of PDOR. Blocking the pathway towards lactate and ethanol could be a plausible scheme to enhance 1,3-PDO productivity.