Acetone-Butanol Fermentation - Steps

Acetone-butanol fermentation process can be described under the following phases:

(i) Production of inoculum

(ii) Fermentation process

(iii) Harvest and recovery 

Medium:

(a) Molasses Medium:Molasses (black strap) which is a by-product in the sugar industry, is used a 5-7% concentration. Nitrogen source is added in the form of ammonium sulphate. In addition, calcium carbonate and sometimes corn-steep liquor are also added. pH of the medium is 5.5 to 6.5 for molasses medium.

Calcium carbonate is added to prevent development of gross acidity in the medium. Excess addition of this salt lowers the production of the solvents. Ammonium sulphate is added at 18 to 24 hours of fermentation.

(b) Corn Medium: Corn meal obtained after corn oil extraction is added at 8% to 10% to water with or without stillage (residue from the preceding fermentation). It is then heated for 20 minutes at 65°C to gelatinize the starch before sterilization of the medium. The pH of the medium is 5.0 to 6.5 for corn medium.

The media are then sterilized and employed in fermentation depending upon the type of Clostridium species used in the fermentation. 

Stillage, that is, residue formed in the previous fermentation is added to the medium approximately at 30% to 40%. It results in the addition of certain nutrients like proteins, carbohydrates and minerals to the freshly prepared medium. It may contain some toxic compounds since it is the result of a previous fermentation, so excess amounts should not be added.                         

(ii) Fermentation Process:

Fermentation is carried out under anaerobic conditions. Production tanks of the capacity of 50,000 to 2.5 lakhs gallons are used in the fermentation. The incubation period is 36 h (2 to 2 ½ days). If the freshly steamed molasses medium is employed, approximately 2 to 4% of inoculum is needed, while for freshly steamed corn medium slightly less inoculum is employed. The inoculum is added first to the production fermenter followed by the addition of medium. This ensures thorough mixing of inoculum with the medium and maintaining anaerobic condition. Before and after inoculation, fermenters gassed with CO₂

 Fermentation generally passes through three phases:

(a) First Phase (0-18 h):

In this phase rapid growth of the bacterium and formation of acetic acid and butyric acid in large amounts along with the production of large quantities of carbon dioxide and hydrogen gases. The pH of the medium which was initially 5.0 to 6.5 for corn medium and 5.5 to 6.5 for molasses medium, decreases to 5.2. This phase, lasts for approximately 13 to 17 hours of incubation. The titratable acidity increases to a maximum and adaptive enzymes are produced which convert acids to neutral solvents.

(b) Second Phase (Next 18 h):

A sharp decrease in the titratable acidity due to conversion of more acids into acetone and butanol. As acids are metabolized to neutral solvents, acetone and butanol, there is an increase in pH, this process is called as acid break. It gets delayed if there is contamination. The rate of gas formation reaches maximum after acid break. It gradually slows as the fermentation process proceeds further.

(c) Third Phase:

The rate of solvent production decreases along with decreased rate of gas formation.  A final pH of 4.2 to 4.4 in the corn medium and 5.2 to 6.2 in the molasses medium. Many cells undergo autolysis at this point resulting in the release of riboflavin into the medium. Products should be recovered before this. 

Anaerobic conditions ensured by:

1. Immediate use of freshly sterilized and cooled medium before air becomes incorporated
2. Active evolution of fermentation gasses 
3. Addition of sterile inert gas/fermentation gasses derived from parallel fermentationsto the headspace of the fermenter
4.  Gassing with carbon dioxide before and after inoculation

Yield:

The ratio of yield of acetone, butanol and ethanol differ slightly depending on the fermentation medium. But, generally the yield is approximately equal to 30% conversion of carbohydrate to solvents.

In a corn medium the ratio of butanol, acetone and ethanol are 6:3:1 respectively, but in molasses medium the ratios of butanol, acetone and ethanol are 6:5:3.

Apart from these solvents, carbon dioxide and hydrogen are also produced as byproducts of the fermentation.

(Gases produced form approximately half of the sugar medium. Total weight of the gases will be one and half times more than the solvents

The acetone-butanol fermentation yields several by products in addition to the gases - include isopropanol, formic acid, acetic acid, butyric acid, acetyl methyl carbinol and yellow oil, which is a mixture of higher alcohols and acids, which are industrially very important.   

 (iv) Harvest and Recovery:

The individual solvents present in the solvent mixture are separated by fractional distillation. Acetone and butanol are collected in separate fractions. Ethanol and isopropanol are collected as a single fraction and sold as a general solvent.

Carbon dioxide converted to solid CO2/dry ice. The residue contains riboflavin and other B vitamins as well as considerable quantity of bacterial cells. The residue is concentrated and dried and used as vitamin feed supplement. 

A beer still is used for the recovery of the products from the fermentation broth. 

Drawbacks/To be taken care of

1)   Contamination due to bacteriophages and Lactobacillus is a common problem which can be prevented by undertaking absolute sterilization.

2)      Maintenance of absolute sterility

3)      Product inhibition

4)      High costs of distillation

5)      Mixture of fermentation products- add to the cost of recovery

 (1 gallon=3-4 L)

References

1.     Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications)

2.     A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese Crueger)

3.     Principles of Fermentation Technology- Peter Stanbury, Allan Whitaker, Stephen Hall