The ABE Fermentation

The ABE fermentation is an acronym for acetone-butanol-ethanol fermentation. This particular fermentation is extremely well studied and was one of the first to be examined in detail. Louis Pasteur in 1862 first discovered the production of butanol and thin 1911, Fernbach conducted further research into a number of bacteria that could conduct ABE fermentation. At the turn of the 20th Century, it was realised that the industrial production of both acetone and n-butanol (butan-1-ol) was extremely commercially important. Acetone was used as a solvent and in the manufacture of explosives whilst n-butanol was used to make synthetic rubbers. Butanol can also be dehydrated to 1-butene and then to longer-chain aviation fuels.

In 1916 Chaim Weizmann developed the acetone-butanol fermentation further by fermenting one of its most important bacteria – Clostridium acetobutylicum. It is a fermentation also known as the Weizmann process. Later on Clostridium beijerinckii was used on an industrial basis but C. acetobutylicum has become firmly associated with the process. It is one of the classic industrial processes of the modern age.

The bacteria of the Clostridia genus are often used in fermentation of glucose, the main substrate to produce acetone, butanol and ethanol. It is strongly associated with bioethanol production. The fermentation process is anaerobic and all the strains used are strict anaerobes too. The ABE fermentation produces the products acetone, butanol and ethanol in the ratio of parts of 3:6:1 respectively.

Biobutanol has a 30% higher energy content with lower water miscibility, flammability, volatility and corrosiveness than ethanol. It is in many ways a more attractive type of biofuel than bioethanol because it can slot relatively easily into fuel production and can be used in automobile engines without too much modification if any.

Both Clostridia bacteria were used on commercial fermentations in the USA and Europe right up to the 1970s but now only China exploits them on an industrial scale. These bacteria are rods and are both obligate anaerobes, Gram-positive and endospore-forming. They are also non-pathogenic to animals and plants which is an important safety consideration.

The metabolic pathway has two characteristic phases. Glucose as the principal substrate follows a form of glycolysis which leads to the production of acetyl-CoA from pyruvate. This intermediate can either follow a pathway called the acid fermentation phase other wise called acidogenesis which produces acetate or butyrate or it can be diverted to the solvent fermentation phase (called solventogenesis) which produces ethanol, or acetone or butanol. It is quite a complex pathway.

Preparing the Inoculum

An inoculum of Cl. acetobuytlicum is grown on molasses, calcium carbonate, ammonium phosphate or sulphate. Corn steep liquor (CSL) is also popular. because Clostridia are mostly spore-former excepts for a few outliers, they are usually grown as soil stocks which is where they originally hail from. The spores are not sensitive to oxygen hence they are often kept in the spore form.

Fermentation Medium 

The ABE fermentation is usually conducted in batch mode.  The glucose is usually supplied in the form of hydrolysates of corn and molasses. Molasses is a by-product of the sugar industry and this usually in plentiful supply. The sugar content in the form of sucrose is usually maintained at 6% w/w. The nitrogen source is usually an ammonium salt of sulphate. The ammonium salt is added about 18 to 24 hours into the fermentation.

Calcium carbonate is used to prevent the fermentation becoming too acidic. 

The corn meal which is also exploited is prepared by passing it through a magnetic field to remove metallic contamination and dust. Any corn oil is removed from the sprouted germ. This degermed corn is then pulverised to a fine powder using a hammer or roller mill. Corn meal is added to water with or without stillage which is the residue from the previous fermentation. The amount in solution is between 8 and 10% by weight. The suspension is treated by heating at 65ºC for 20 minutes so the starch is gelatinized and then sterilized. 

The stillage is the residue from the previous fermentation and can form 30 to 40% of the next fermentation. It contains plenty of nutrient including protein, carbohydrates and minerals.

Both media, molasses and corn meal are sterilized. One or other is used depending on the Clostridium bacteria to be used. 

The Process of Fermentation

A purely anaerobic fermentation. It is also one of great large-scale type taking place for between 2 and 2.5 days. When molasses is used, only between 2 and 4% inoculum volume is needed but even less is required when freshly steamed corn liquor is employed.

There are three phases to this fermentation. In the initial phase, the bacteria produces large amounts of acetic and butyric acid as well as plenty of hydrogen gas and carbon dioxide. The pH of the medium usually starts at between 5 and 6.5 if corn medium is used and 54.5 to 6.54 if molasses used. This decreases becoming more acidic and remains constant for the rest of the fermentation. The whole initial phases lasts between 13 and 17 hours. The titratable acidity rises to a maximum value which triggers the generation of enzymes that convert these acids to neutral solvents.

In the next phase known as the acid break, the titratable acidity starts dropping quickly because more acid is converted to acetone and butanol. The fermentation is susceptible to contamination and can lose its potency as a result. The gas formed reaches its peak rate immediately after the acid break.

The rate of solvent production begins to drop as does the rate of gas formation. The titratable acidity begins to slowly increase, rising to between pH 4.2 and pH 4.4 if corn medium is used or pH 5.2 to 6.2 if molasses is used. the fermentation begins to come to a halt with many bacteria dying through autolysis and with it the result of riboflavin into the medium. 

The product of fermentation containing butanol etc. is distilled and the three chemicals separated industrially using this process.

A number of Clostridia bacteria have been tested and explored for ABE fermentation

As a process, every commercial concern involved in the fermentation is interested in a particular product. Biobutanol production is an attractive proposition but does not compete with butanol produced chemically from other conventional sources such as oil. The yields of fermented biobutanol are still too poor compared to the chemical process. Commercial application is also stymied by high production costs. There is considerable effort to develop better strains and improved processing strategies but it still suffers from extremely poor productivity and cannot compete with the petrochemical industry.

Industrial concerns would like to conduct a continuous fermentation so that the butanol is produced on a constant basis. Unfortunately this type of fermentation suffers from typical issues of cell washout, culture degeneration and increased contamination by other bacteria. It also appears to be a difficult process to control because of the complexity of the different phases – acidogenesis, solventogenesis, and sporulation. At the genetic level, there appears to be a large number of gene regulators involved and it these are all highly regulated. They involve various kinases, transcription factors, and interlocking signal transduction pathways.

One of the main issues with the ABE fermentation is the role of product inhibition. Here, the product concentration cannot be exceeded and as a consequence there is a threshold level which cannot be effectively exceeded. It means that a great deal of effort has been expended in overcoming this issue.

Immobilizing bacteria is a popular approach to fermentation generally and none more so than with the ABE fermentation.

An early example of an immobilized reactor was one using a trickle bed reactor. In this example, two serial columns were packed with C. acetobutylicum trapped on a natural sponge. The cell loadings were between 2 and 5.5 g dry cells/g sponge (Park et al., 1989)..

In one example a non-spore forming (asporogenous) C. acetobutylicum was immobilized in a fibrous-bed bioreactor (FBB) which was a single pass (Chang et al., 2022). In this example, butyrate and glucose were the feedstocks supplied at different dilution rates. The butyric acid in the feed medium ensured the cells were in the solventogenic phase so that butanol could be produced without too much issue. The fermentation conditions were a dilution rate of 1.88 h⁻¹, with butanol produced at 9.55 g/L,  a yield of 0.24 g/g and productivity of 16.8 g/L/h. The researchers claimed this was the highest productivity level they had witnessed and was 80x better than a standard ABE fermentation. They reasoned that the high productivity level was down to the capability of supporting a high viable cell density within the fibrous matrix. That level was about 100g/L with over 70% viability of the cells. They also suggested that these cells could tolerate butanol as the product and butyric acid much more effectively. It is the case that whilst immobilization of cells alters the kinetics of production and makes it slower, the cells are better protected and preserved in such a system. This particular fermentation could run without too much trouble for over a month.

Other fermentations have been built around the construction of biofilms which have then been operated continuously. Qureshi et al., (2004) have tried this using acetate, butyrate and corn steep liquor as substrates.

To overcome product inhibition during fermentation, a variety of in situ product recovery systems have been examined. The most commonly encountered is distillation but pervaporation and gas stripping have shown promise. It is possible to find examples using membrane distillation and separation a long with liquid-liquid extraction. 

References

Chang, W. L., Hou, W., Xu, M., & Yang, S. T. (2022). High‐rate continuous n‐butanol production by Clostridium acetobutylicum from glucose and butyric acid in a single‐pass fibrous‐bed bioreactor. Biotechnology and Bioengineering, 119(12), 3474-3486 (Article).

Ezeji, T., Milne, C., Price, N. D., & Blaschek, H. P. (2010). Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms. Applied microbiology and Biotechnology, 85, pp. 1697-1712.

Park, C. H., Okos, M. R., & Wankat, P. C. (1989). Acetone–butanol–ethanol (ABE) fermentation in an immobilized cell trickle bed reactor. Biotechnology and Bioengineering, 34(1), pp. 18-29 (Article).

Qureshi, N., Karcher, P., Cotta, M., Blaschek, HP. (2004). High-productivity continuous biofilm reactor for butanol production: Effect of acetate, butyrate, and corn steep liquor on bioreactor performance. Appl. Biochem. Biotechnol 113–116 pp. 713–721