Two-step fermentation

There are a total of three ascorbic acid processes either in place or in late stages of development with firm expectations of commercialization (i) the traditional chemical Reichstein-Griissner synthesis (ii) the two-step fermentation process to 2-ketogulonic acid with subsequent chemical esterification/lactonization to ascorbic acid and (iii) the one-step fermentation to 2-ketogulonic acid with the same last chemical step. Figure 20.8 and Table 20.3 provide an overview of the three processes. 

Two-Step Fermentation Process to 2-Ketogulonic Acid with Chemical Step to Ascorbic Add... 

Ethyl alcohol can be bacterially oxidized to acetic acid in the following two-step fermentation sequence ... 

An additional two-step fermentation, catalyzed, however, by pure cultures of methane organisms is the decomposition of CO (21). 

In general, two-step fermentation processes are realized. In the first step, the breeding of the biological material takes place, and in the second step the accumulation of the PHAs takes place by limiting essential nutrients like N2, O2, P, S, or Mg. In order to isolate and purify the produced polymers out of the bacterial cells, a process of centrifugation and freeze drying can be executed after the harvest. 

Natural and kosher tuberose lactone is one of the lactones that was produced using a two-step fermentation and the a schematic process of tuberose lactone is described in Figure 1. Tuberose lactone, a mixture of saturated and unsaturated C12 lactones, was produced from hydrolyzed flaxseed oil. Flaxseed oil contains high concentration of C-18 unsaturated fatty acids (>90%),... 

As mentioned above for high cell density fed-batch cultures, the two-step fermentation strategy has often been applied, where the cell division is separated from the PHA accumulation phase. With use of octanoic acid as a substrate, PHA contents of up to 75% (w/w) at a cell concentration of 55 gL" and a volumetric productivity of 0.63 gL h were obtained with P. putida GPol under nitrogen limitation (Kim et al. 2002). With Pseudomonas IPT 046, cell concentrations of up to 50 gL with a PHA content of 63% (w/w) and a volumetric productivity of 0.8 gL" h were reached under phosphate limitation using glucose and fructose as a mixed carbon source (Diniz et al. 2004). When oleic acid was used as a substrate for the cultivation of P. putida KT2442, a cell concentration of 141 gL" with a PHA content of 51% (w/w) and a volumetric productivity of 1.91 gL" h were obtained under phosphate limitation (Lee et al. 2000). 

Serizawa N (1997) Development of two-step fermentation-based production of pravastatin, a HMG-CoAreductase. J Synth Org Chem Jpn 55 334-338... 

This chapter focuses on the production of pravastatin. Pravastatin is produced by a two-step fermentation the first step is production of ML-236B, and the second is hydrox-ylation of ML-Z36B. The mechanism of microbial hydroxylation o( ML-Z36B and industrialization of the production of pravastatin are described here in detail. 

II. raODUdlON BY TWO-STEP FERMENTATION A. ML-234B Production by PenieiUiuiR eftruimn... 

To produce microbial products in an industrial Scale fermentaCton, several kinds of tech niques are required. Strain and medium improvements are very effective in increasing the yield of fermentation products. However, as the yield increases, problems often arise, such as product inhibition or limitation of dissolved oxygen (DO). To overcome these problents, it is imponant to study the characteristics of the strains and detennine which biochemical engineerii approaches are effective. In this section, we describe mainly a mycelial morpholep control and computer application (or both twO Step fermentations. 

Pravastatin is produced by a two-step fermentation first, ML-236B is produced by P. cim num, followed by the hydroxylation of ML-236B by S. oirbophilus to fonn pravastatin- In an effort to increase the productivity of these fermentation ocesses, new technologies have been developed, and the mechanism of hydroxylation has been extensively investigated. 

Abstract Vitamin C, an important organic add, is widely used in the industries of pharmaceuticals, cosmetics, food, beverage and feed additives. Compared with the Reichstein method, biotechnological production of vitamin C is an attractive approach due to the low cost and high product quality. In this chapter, biosynthesis of vitamin C, including one-step fermentation processes and two-step fermentation processes are discussed and compared. Furthermore, the prospects of the biotechnological production of vitamin C are also presented. 

According to the production process, vitamin C production by biotechnology can now be divided into two-step fermentation and one-step fermentation. The two-step fermentation including tandem fermentation process that uses glucose as substrate and the fermentation process that uses D-sorbitol as substrate (Fig. 12.2). For the... 

Fig. 12.2 Classical two-step fermentation process. The D-glucose was hydrogenated to form D-sorbitol. The D-sorbitol was converted into L-sorbose by acetic acid bacteria. The L-sorbose was further oxidized with a mixture culture system with B. megaterium and K. vulgare to form 2-KLG. The 2-KLG was then esterified and lactonized to form vitamin C. The only difference between the classical two-step process and the Reichstein process is the replacement of low efficient protective oxidation with a fermentation process. The G. oxydans here was further identified to be K. vulgare...

Previous Research on the Classical Two-Step Fermentation Process... 

The miCTobial transformation of D-sorbitol to L-sorbose is the most important industrial fermentation process of vitamin C in Reichstein process and two-step fermentation. Microorganisms are inhibited severely by high concentration of... 

Three bacteria were involved in the classical two-step fermentation process, for example G. oxydans, B. megaterium and K vulgare. The denomination of the bacteria for G. oxydans and K vuglare is highly speculative because of the lack of efficient strain identification methods. Furthermore, there are also some updates on the two strains. In early times, G. oxydans were named as G. melanogenus, Acetobacter suboxydans, or A melanogenus. Because most of these stains could not... 

The classical two-step fermentation process is the most successful route for vitamin C production for its high yield of 2-KLG on D-sorbitol. Though there are two fermentation process, the yield of L-sorbose on D-sorbitol and the yield of 2-KLG on L-sorbose could achieve to more than 99.5 and 97%, respectively. Few of the industrial process could achieve this level. Therefore, the metabolic engineering on the classical two-step fermentation process is always undergoing. 

Previously, researchers from both industry and academia have invariably noted the inherent disadvantages of the two-step fermentation vitamin C production process, such as long period fermentation, additional sterilizing, control of the mix-culture... 

However, after the report of the innovative two-step fermentation process, the research on the classical two-step based one-step fermentation process seems to be suspended. Few literatures about metabolic engineering of G. oxydans for one-step vitamin C production could be found after then. 

Though the classical two-step fermentation process has got highly promoted results, it still needs D-sorbitol as substrate, which need additional hydrogenation step by chemical process. Therefore, the direct fermentation of D-glucose to 2-KLG is still presumed. There have been some attempts to add an additional step that could convert the D-glucose to D-sorbitol, in order to supplement the classical two-step fermentation process. However, the lack of efficient enzyme makes this impossible. 

Therefore, an innovative two-step fermentation process were discovered to resolve this problem. In the new innovative two-step fermentation process, the D-glucose was converted into 2,5-diketo-gluonic acid (2,5-DKG) by Erwinia herhicola ATCC 21988 or some similar strains with glucose dehydrogenase, gluconate dehydrogenase... 

Based on this innovative two-step fermentation process, Anderson et al. (1985) has expressed a 2,5-DKG reductase from Corynebacterium ATCC 31090 into the Erwinia herbicola ATCC 21988 with the expression vector ptrpl-35. The final recombinant E. herbicola strain could produce 1 g/L of 2-KLG from saturated D-glucose solution. By the protoplast fusion, Lin et al. (1999) fused an Erwinia herbicola and a Corynebacterium strain. The resultant strain could produce 2.07 g/L of 2-KLG (Fig. 12.5). 

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