Integrated Project

Vitamin C is an essential nutrient that can be found in various foods and is also produced as dietary supplements by chemical companies. It plays a crucial role in treating scurvy and is widely utilized in the cosmetics industry due to its antioxidant properties. However, many individuals, including medical professionals, are unaware of the dual nature of vitamin C. It consists of two distinct sides: the (-) side called “L”-Ascorbic Acid and the (+) side known as D-Ascorbic Acid. The L side is the active component of Vitamin C and offers significant benefits for humanity.

The body usually eliminates the D-side of Vitamin C as it is deemed ineffective according to research. Hence, people only derive benefits from the “L” side of Vitamin C, mainly present in foods such as oranges [1]. Consequently, the objective during Vitamin C production is to generate L-ascorbic acid. The industrial manufacturing of Vitamin C has been ongoing for around 70 years and has led to the development of several efficient methods including the Reichstein Process, Two-Step Fermentation Process, and Single Step Fermentation Process.

The two most commonly used methods for producing vitamin C are the Reichstein Process and the Two-Step Fermentation Process. The classical Reichstein process, which was introduced in the 1930s by Tadeus Reichstein and his colleagues, involves a single pre-fermentation process followed by a purely chemical route that consists of five steps. This process typically utilizes glucose or sugar as the raw material, with D-glucose being specifically used for vitamin C production [1][2].

The first step of the process involves converting D-glucose into D-sorbitol using hydrogenation with nickel as the catalyst. This conversion occurs in a large boiler under high temperature (140-150°C) and pressure (80-125 atm). Afterwards, sorbitol is oxidized by microorganisms to produce L-sorbose. This oxidation process takes place at a pH range of 4 to 6 and a temperature of 30°C, facilitated by Acetobacter suboxydans bacteria. These bacteria consume specific hydrogen atoms, allowing for the formation of double bonds with oxygen. In the protection stage, acetone acts as a catalyst to preserve the four remaining hydroxyl groups in sorbose by creating acetal linkages.

The result of this stage is Diacetone-L-sorbose. The next stage involves organic oxidation using potassium permanganate, followed by heating with water at temperatures exceeding 100°C[3]. The hydroxyl group without protection undergoes chemical oxidation and becomes carboxylic acid. The most challenging aspect of this stage is ensuring that the correct part is oxidized. Ultimately, 2-keto-L-glucanic acid is generated [4]. The remaining task is to combine the acid with alcohol and eliminate the water, known as Gamma Lactonisation. Hydrochloric acid and ethanol are introduced.

Hydrolysis with acid eliminates the acetal groups, causing the acid to bond with the alcohol in a cyclic formation. This leads to the production of crystallized Vitamin C [4]. The Reichstein process involves a series of chemical reactions: D-glucose to D-sorbitol to L-sorbose to L-ascorbic acid (Vitamin C) to 2-keto-L-glucanic acid. See Diagram 1 [5]. However, the Reichstein process has numerous drawbacks. It requires six separate procedures and is challenging to perform continuously. As a result, several researchers are keen on discovering alternative methods to enhance the traditional Reichstein process.

Using the “two-step fermentation” process is a successful route in the biotechnology industry. This process has been utilized on an industrial scale for more than 40 years [6]. It has undergone advancements and now incorporates bacteria like Corynebacterium sp., which rely on enzymatic-based approaches [7]. Corynebacterium, a type of rod-shaped bacteria found in the Actinobacteria group within the Corynebacteriaceae family, are small, non-motile, non-sporulating gram-positive organisms that form grayish colonies with a granular appearance [8].

Chinese letters, or groups of Corynebacteria, have a slow growth rate even in nutrient-rich media. All Corynebacteria require biotin (also known as vitamin H) for growth [9]. The method described here uses a two-step fermentation process that involves only a few chemical steps compared to the Reichstein Process. This process utilizes fermentation techniques with bacteria such as Corynebacterium sp. and Erwinia herbicola. The steps are as follows:

1. Sugar (D-glucose) fermentation by Erwinia herbicola
2. 2,5-diketo-D-gluconic acid fermentation by Corynebacterium sp.
3. Gamma lactonization of keto-L-gulonate (a precursor of vitamin C)
4. L-ascorbic Acid (vitamin C)

Figure 2: The flow diagram of Vitamin C production [10]. The two-step fermentation system is a practical approach to produce 2-keto-L-gulonate (a precursor in the synthesis of L-ascorbic acid) from D-glucose using biotechnological methods. The final product can only be obtained after several fermentation processes of glucose using specific microbacteria or enzymes [11]. In the first step, D-glucose is oxidized to 2,5-diketo-D-gluconic acid by Erwinia strains via the intermediates D-gluconic acid and 2-keto-D-gluconic acid.

In the experiment, a mutant strain of Erwinia sp. was employed to convert D-glucose into 2,5-diketo-D-gluconic acid. The medium used contained D-glucose, corn steep liquor, ammonium hydrogen phosphate (NH4)2HPO4, and CaCO3. After 26 hours of cultivation, a yield of 94.5% for 2,5-diketo-D-gluconate was attained from D-glucose. Instead of eliminating the cells with sodium dodecyl sulfate, the broth was directly utilized for the subsequent conversion.

The next step involved using a different mutant strain called Corynebacterium sp., along with an enzyme known as 2,5-diketo-D-gluconate reductase. This combination allowed for the stereospecific reduction of 2,5-diketo-D-gluconate to form 2-keto-L-gulonate.

When the cell growth reached its peak after around 16 hours, a medium containing various components was used. These components include D-glucose, corn steep liquor, NaNO3, potassium dihydrogen phosphate KH2PO4, and trace elements. At this point in time, NaNO3 was added to the culture. Following that, a 2,5-diketo-D-gluconate broth was provided for approximately 50 hours. In order to fulfill the hydrogen donor requirement for reduction of the mutant strain, the calcium 2,5-diketo-D-gluconate broth was combined with D-glucose before being introduced. The concentration of 2-keto-L-gulonate in the broth remained constant throughout the process. Importantly, no intermediates such as 2-keto-Dgluconic acid or 5-keto-D-gluconic acid were detected in the final broth [10].

The Gamma Lactonisation process, which involves adding hydrochloric acid and ethanol, is used to extract L-ascorbic acid from 2-keto-L-gulonate. This process causes hydrolysis of the acid, removing two acetal groups and resulting in the formation of a circular structure when combined with alcohol. After water is removed, Vitamin C crystals are formed. This step is similar to the final stage in the Reichstein process, ultimately producing L-ascorbic acid or Vitamin C[13]. The single-step fermentation process is considered the most innovative method for producing L-ascorbic acid or Vitamin C from d-glucose[12].

Further development and research are required before applying this method to vitamin C production in the industry [14]. The one-step fermentation process involves enhancing the pathway of Erwina herbicola through gene cloning techniques. This process utilizes microbes to convert d-glucose to 2-keto-L-gluconate in a single step. The gene responsible for encoding the enzyme 2,5-diketo-D-gluconate reductase was cloned from Corynebacterium sp. and expressed in E. herbicola. By optimizing culture conditions, these genetically modified strains of E. herbicola can produce approximately 60% L-ascorbic acid as the final product.

The production of Vitamin C using this new process had a lower yield compared to the two-step fermentation process. However, it has the potential to pave the way for future economical methods of Vitamin C production[15]. In conclusion, the Reichstein process and the two-step fermentation process are two different methods used for Vitamin C production. The newer Chinese-developed two-step fermentation process utilizes biological oxidation instead of chemical oxidation as seen in the Reichstein process [16]. Each method has its own advantages and disadvantages. The Reichstein process involves the use of heavy machinery and incurs costs for maintenance, waste disposal, and labor. It also requires various organic and inorganic solvents and reagents such as acetone, hydrochloric acid, and oxidizers. Many of these reagents cannot be recycled and have high disposal costs [17]. Additionally, a significant amount of toxic “three-waste” gases are produced during production, posing a high risk of fire and explosion [18]. Overall, this process demands high energy consumption, a large amount of organic solvent, and can lead to severe environmental pollution.

Compared to the classical Reichstein process, the two-step fermentation process offers several benefits. These include cost reductions in raw materials and grain requirements, as well as a decrease in the presence of explosive, flammable, and toxic gases. Additionally, the simplicity of this process means less equipment is necessary and production time is shortened [19]. Overall, out of the three methods discussed, the two-step fermentation system proves to be the most productive approach.

The use of microbiological techniques in the production of vitamin C is a safer and more cost-effective option compared to Tadareus Reichstein’s classical method [20]. However, this modern approach requires extensive knowledge of biochemistry for optimal production. References: [1] [3] [4] [7].

F., Payton, M.A., van de Pol, H., Hardy, K.G., 1988. Conversion of glucose to 2keto-l-gulonate, an intermediate in l-ascorbate synthesis, by a recombinant strain of Erwinia citreus. Appl. Environ. Microbiol. 54, 1770–1775. [9]

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[11] Source: BASF.

Skatrud,T.J.andHuss,R.J.
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[13] Inventors.about.com (1905) History of Vitamins.
[14] Rosenberg, H.R., 1942. Chemistry and Physiology of the Vitamins.

Hofmann, H., Bill, W., Chemie-Ing.