Industrial Production of Ascorbic Acid

[Guest guest]

The Commercial Production of Ascorbic Acid

[this document followed below by a technique that someone is developing

to minimize the use of energy and harmful chemicals in the manufacture

of vitamin C.]

 

http://www.roccomanzi.it/IMP-VITAMINERALI/SCIENZIATI/scienziati-docu/cathcart/as\

cor_file/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.

 

=====================================================

 

Development of a Yeast-Based Single-Step Process for the Manufacture of

L-Ascorbic Acid (vitamin C)

Funding:

 

http://www.scri.sari.ac.uk/SCRI/Web/Site/home/ResearchAreas/Theme2~GenestoProduc\

ts/QHN/External/vitaminC.asp

 

 

Scottish Enterprise Proof of Concept Fund

 

Project Duration:

1 August 2002 – 31 July 2004

 

Staff:

Roberto Viola; ; Rob Hancock; Jane Shaw

 

Contact person:

Roberto Viola

 

Project Aim:

 

To develop an environmentally friendly and cost effective alternative

for the industrial manufacture of vitamin C

Rationale

 

Vitamin C is used in a wide range of industrial applications including

the food and beverage industry, the pharmaceutical industry and in

livestock rearing and aquaculture. Global production is currently

estimated at approximately 110,000 tonnes annually. The vast majority of

this is synthesised by the 60 year old Reichstein process (figure 1)

which is highly energy consuming and relies on the use of a number of

environmentally hazardous chemicals. We aim to develop yeast strains

with the capacity to synthesise vitamin C from inexpensive sugar

precursors providing an alternative and less damaging method for vitamin

C manufacture.

 

D-Glucose is converted to vitamin C via a series of chemical steps and

one bacterial fermentation. The figure shows steps using environmentally

hazardous chemicals (orange boxes) or steps requiring high energy

consumption (purple boxes). The present project aims to reduce

environmental harm in vitamin C manufacturing by replacing this chemical

synthesis with a yeast fermentation.

 

[Note -- nice graphic is provided on the web site]

 

Current Status of Research

A number of fermentation methods currently exist for the synthesis of

intermediates in the Reichstein process (figure 2), however, no

microbial fermentations have been developed to date that result in the

economical synthesis of vitamin C in a single step.

 

Purpose of the Research

Through our knowledge of the comparative biochemistry of plant pathways

for vitamin C synthesis and yeast pathways for D-erythroascorbic acid

synthesis (a five carbon analogue of vitamin C) we intend to produce

improved yeast strains with the capacity for vitamin C biosynthesis from

cheap starting materials. Once the yeast strains are produced, we will

optimise culture conditions for vitamin C biosynthesis and harvesting at

the benchtop scale.