Acetone-Butanol Fermentation

An example of anaerobic fermentation as well as a mixed fermentation where different products are obtained by using different species of Clostridium. The production of butanol by butyric acid bacteria was first observed by Louis Pasteur in the 19^th century. Before World War-I processes involving microorganisms were developed for the production of butadiene which is required for the production of synthetic rubber. Cham Weizmann reported that Clostridium acetobutylicum is capable of producing acetone, butanol and ethanol in an economically feasible quantity.

During World War-I, acetone was in great demand to manufacture the explosive trinitrotoluene (TNT). Hence, the acetone-butanol fermentation rapidly expanded. But after war, the demand for acetone decreased and butanol increased, as it was required as a solvent for the rapid drying of nitrocellulose paints in automobile industry. Thus, the commercial process of acetone-butanol survived even after a lack of demand of acetone after World War-I.

But after World War II petroleum based processes replaced biological fermentation processes of acetone-butanol production, which lead to the closure of many industries. However, the fermentative production of acetone-butanol is still being carried out in certain countries where the carbon source material, specially, starchy material are available at cheaper rate. Riboflavin/Vitamin B₂ is produced as a byproduct in this fermentation process.

Now, produced predominantly from petroleum based raw materials.

Uses of Acetone-Butanol:

1. Butanol is extensively used in brake fluid, antibiotic recovery procedures, urea, formaldehyde resins, amines for gasoline additives and as ester in the protective coating industry.

2. Butanol is also used for the synthesis of butadiene which is used in the preparation of synthetic rubber.

3. Acetone is used as a universal organic solvent and also in the preparation of explosives like trinitrotoluene.

 Microorganism: Clostridium spp.

Raw material: Starch, molasses, sucrose, wood hydrolysates

Products: Butyric acid, Butanol, Acetone, Isopropanol and some acetic acid, H₂, CO_2.

The relative proportion of the products depend on bacterial strain used and fermentation conditions.

Biosynthesis of acetone-butanol

 

Fermentation Process of Acetone-Butanol:

Acetone-butanol fermentation process has the following phases:

(i) Production of inoculum

(ii) Fermentation process

(iII) Harvest and recovery

     Two species of Clostridium, Clostridium acetobutylicum and Cl. saccharoacetobutylicum are generally employed for acetone-butanol fermentation. They differ slightly in their nutritional requirements and fermentation factors. For Clostridium acetobutylicum, potato/corn medium is used and for Cl. saccharoacetobutylicum, molasses medium. Inoculum growth and fermentative production of the solvents are carried out at 31° to 32°C for Cl. saccharoacetobutylicum and at approximately 37°C for Cl. acetobutylicum.

(a) Production of Inoculum

Inoculum of Cl. saccharoacetobutylicum is developed in media with molasses, calcium carbonate, ammonium sulphate or phosphate and sometimes corn-steep liquor.

Clostridia are spore formers and are easily maintained as soil stocks in contrast to the vegetative cells. The spores are not very sensitive to oxygen. However, prolonged storage of these spores leads to decrease in the acetone butanol production.

Spores from soil stocks are initially added to deep tubes of semisolid potato-glucose medium for molasses cultures. As the spores are added to the bottom of these tubes they along with soil particles sink to the bottom of the tubes. The submerged location of the spores can protect the vegetative cells from oxygen after germination of spores.

Inoculated tubes are heat shocked and rapidly cooled to incubation temperature to select heat resistant spores. The tubes are then incubated at 31° to 32°C for 20 hours. The growth that occurs in the tubes are used as inoculum for larger batch of molasses medium present in inoculum tanks. The inoculum is grown for 24 to 26 hours before addition to production tank.

Large volumes of inoculum are produced by successive transfers of inoculum by volume to larger media with incubation period of 20 to 24 hours for each batch.

With Cl. acetobutylicum, Stages of inoculum preparation are generally similar to that using Cl. saccharoacetobutylicum except:

1. Spores of soil stock are added to the deep tubes containing corn medium

2. The tubes are incubated at 37°C for 20 hours.

Corn medium is more prone to contamination so sterile conditions should be ensured, periodically.

    Various inoculum stages, particularly the last stage, before the inoculum is transferred to production tank, are checked for contamination by testing :

1. pH

2. Density of the inoculum

3. Rate of gas evolution

4. Presence of facultative anaerobe tested by aerobic plating on agar medium

5. Microscopic observation for contamination by hanging drop method

6. Determination of titratable acidity is done with corn medium in addition to the above tests.

 Building up of inoculum over different stages ensure:

ü  Greater resistance to contamination

ü  Shorter fermentation times

ü  Greater yields

ü  More substrate utilization

 At each of these stages of inoculum transfer, anaerobic conditions are produced/ ensured by:

1. The reducing condition of the medium

2. Immediate use of freshly sterilized and cooled medium before air becomes incorporated

3. Evolution of fermentation gasses

4. By filling the head space of the inoculum tank with sterile inert gas