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 19th 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. But after World War II petroleum based processes replaced biological fermentation processes of acetone-butanol production, which lead to the closure of many industries. Now, Acetone-Butanol is produced predominantly from petroleum based raw materials.
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.
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, CO2.
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
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:
- Immediate use of freshly sterilized and cooled medium before air becomes incorporated
- Active evolution of fermentation gasses
- Addition of sterile inert gas/fermentation gasses derived from parallel fermentationsto the headspace of the fermenter
- 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)
No comments:
Post a Comment