http://fhs.gcal.ac.uk/bio/micro/drjrattray/amb/ascor.htm

L-Ascorbic Acid - vitamin C
Used in food industry as a preservative, health industry as a supplement, antioxidant, animal feeds


Bioconversion/Biotransformation process.

1. Glucose reduced to Sorbitol - chemical electrolysis using bleach (caustic soda) and HCl, nickel
catalyst and lots of electricity. Hydrogenation.

2. D-Sorbitol oxidised to L-Sorbose by Gluconobacter. Selective oxidations - fermentation. Sorbose
crystallised for step 3.

3. L-Sorbose converted to Diacetone sorbose. Acetone block put on to sorbose in the presence of
H2SO4 releasing NaSO4. The acetone block protects hydroxyl groups on the sorbose allowing
easier oxidation in subsequent steps.

4. Diacetone Sorbose oxidised to Diacetone ketogulonic acid in the presence of bleach (NaOCl) and a
nickel catalyst.

5. Diacetone ketogulonic acid converted to ascorbic acid by the removal of the acetone block. This
molecular rearrangement gives activity and functionality to the molecule.

6. Recovery, purification (charcoal column to decolourize) and crystallisation of product.

Fermentation - Reichstein - Grussner synthesis

An aerobic and exogenic process - needs constant cooling.

Culture - Gluconobacter (Acetobacter) suboxydans.
stored at -70°C on slopes. Plates and flasks are subculture for inoculums. The 750ml
flasks have large baffles allowing maximum aeration. Culture must be 99.5% pure -
potential problems from yeasts, therefore daily checking.

Produces enzyme - Sorbitol Dehydrogenase for oxidation.

Medium - Submerged process
98 % glucose (90% liquid and 10% solid) - converted chemically to Sorbitol
Sorbitol is added at around 20% initially.
Vigorous stirring and aeration needed
2% other sugars
0.5% Corn steep liquor for N and C
Water - quality check using GLC.
NH4 PO4.
Acetic acid to maintain/reduce pH (kept around pH4) which may give better cell activity.
(higher concentration gives higher cell activity).
Oleic acid and silicone used as antifoam - reduced O2 tension - added on demand

Fermentation - 600l fermenter , media added following heat exchange sterilisation (135°C for 10 mins then
cooled to 37°C in batches or continuous)
and filter sterilisation.
air filtered through 0.8µm and 0.2µm filters.
Air sparged through paddles in fermenter.
Continual cooling to maintain temperature at 35°C.
Run under +ve pressure to stop back flow through ports.

Fermenter train - 1st fermenter 30% conversion after 24hours at 36°C. Contents split into
two vessels, more sorbitol added which boosts yield and conversion. Fermenters then
pooled and eventually 95% conversion and 50% yield (1kg sorbitol from initial 2kg
glucose) achieved (after a few more fermenter stages - Cascade- semi continuous).

Reaction stopped by cooling, can be held for up to 14 days for next processing stage.

Chemical byproducts used in glass industry and recycled for reuse


CURRENT DEVELOPMENTS IN L-ASCORBIC ACID PRODUCTION.

A two stage fermentation process in currently being investigated to simplify the production of Ascorbic Acid.

1. Glucose ----------------- 2,5 diketo D gluconic acid

A 26 hour fermentation (oxidation reaction) by Erwinia sp. via gluconic acid and 2 keto D gluconic acid which is 94% efficient.

2. 2,5 diketo D gluconic acid --------------- 2 keto L gulonic acid

Corynebacterium sp. is grown for 16 hours and then the sterilised broth from stage 1 is added. Incubation is for 66 hours during which time their is a reduction reaction by the enzyme 2,5 DKG reductase. This stage is 92% efficient.

The 2 keto gulonic acid is then chemically converted to ascorbic acid.

Overall 86% efficiency using this method. Yield .60%

Scientists now trying to clone the gene for 2.5 DKG reductase from the Corynebacterium sp. into the Erwinia sp. to enable a one step fermentation reaction to occur.

However a) the hybrid cell has a low tolerance to high glucose concentrations.
b) the hybrid cell is giving low yields
c) conversion of 2,5 L-KG -----> L. Idonic acid (by cytoplasmic enzyme --> delete)

This needs further work before commercial application.

Work is also being done on producing a defined media which can be used in the fermentation stage to minimise batch variations. It is hoped eventually to exclude corn steep liquor and to use defined media instead. Small scale fermentations are currently being done on different defined media to optimise production whilst excluding corn steep liquor, and also in general optimisation of the process.

Immobilised enzymes - entrapment in polyacrylamide.

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