Clostridium acetobutylicum

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A Microbial Biorealm page on the genus Clostridium acetobutylicum

Image of Clostridium acetobutylicum courtesy of NCBI.

Classification

Higher order taxa

Bacteria (Domain); Firmicutes (Phylum); Clostridia (Class); Clostridiales (Order); Clostridiaceae (Family); Clostridium (Genus)

Species

Clostridium acetobutylicum

Clostridium acetobutylicum ATCC 824 is considered the type strain.

NCBI: Taxonomy

Description and significance

Clostridium acetobutylicum is a Gram-positive bacillus (1). C. acetobutylicum is most often soil dwelling, although it has been found in a number of different environments. It is mesophilic with optimal temperatures of 10-65°C. In addition, the organism is saccharolytic (can break down sugar) (1) and capable of producing a number of different commercially useful products; most notably acetone, ethanol and butanol (2).

C. acetobutylicum requires anaerobic conditions in order to grow in its vegetative state. In its vegetative states, it is motile via flagella across is entire surface. It can only survive up to several hours in aerobic conditions, in which it will form endospores that can last for years even in aerobic conditions. Only when these spores are in favorable anaerobic conditions will vegetative growth continue (1).

It was first isolated between 1912 and 1914 (2). Chaim Weizmann cultured the bacteria to produce produce acetone, ethanol and butanol in a process called the ABE method. Thus, it is fitting that C. acetobutylicum is often called the "Weizmann organism." The products were then used in the production of TNT and gunpowder in the first World War (3). Following WWI, the ABE process was widely used until the 1950's when petrochemical processes became more cost-effective due to the cost and availability of petroleum fuel sources. The recent fossil fuel crisis has spurred more research into C. acetobutylicum and the utilization of the ABE process (2).

In addition to being an important bacteria for industrial use, C. acetobutylicum is studied as model for endospore formation in bacteria. It has been compared to the most frequently studied endospore bacteria, Bacillus subtilis (2). Understanding the pathways of endospore formation is important because many endospore forming bacteria are human pathogens, in both the Bacillus and Clostridium genera.

The most commonly studied strain is the type-strain, ATCC 824. This strain was discovered and isolated in soil from a Connecticut garden in 1924. Research has indicated that the widely studied ATCC 824 is closely related to the Weizmann strain used in the early industrial production of acetone (2).

Genome structure

The genome of Clostridium acetobutylicum ATCC 824 has been sequenced using the shotgun approach. This is the model strain for solvent-producing bacteria. The genome consists of one circular chromosome and a circular plasmid. The chromosome contains 3,940,880 base pairs. There is little strand bias with approximately 51.5% of genes being transcribed from forward strand and 49.5% from the complementary strand (2).

Noted genes common to bacteria include the 11 operons which code for ribosomes. It is interesting that each of these operons is near the oriC (origin of replication) and oriented in the direction of the leading strand of the replication fork. (2). This is a characteristic commonly observed known as gene dosage, in which highly transcribed genes are placed near the oriC. Due to the orientation of these genes, they will be transcribed in greater number while DNA is in the process of being replicated and there are additional copies of the gene present within the cell.

In addition, the genome contains of one large plasmid (called a megaplasmid). This plasmid seems to be contain nearly all genes involved with solvent production and is aptly named pSOL1. pSOL1 contains 192,000 base pairs and codes for 178 polypeptides. Examination of the plasmid indicates no bias in which strand is the coding strand (2).

When Clostridium acetobutylicum is cultured in continuous culture or undergoes many transfers, the strain slowly degenerates in that it loses its ability for solvent production. Experiments to determine what causes degeneration have shown that pSOL1 contains four genes which are vital for alcohol and acetone production. Over the course of many transfers or continued vegetative growth, this plasmid is lost. Further evidence for the loss of this plasmid leading to strain degeneration is that mutants lacking these genes and unable to produce solvent resume acetone and alcohol production upon complementation of the genes via plasmids (4).

Other, less studied strains of C. acetobutylicum such as ATCC 4259 have shown similar degeneration. The plasmid in this strain is named pWEIZ. Again, degeneration due to serial culturing of this strain is thought to occur because of eventual loss pWEIZ. This strain is worth noting because, interestingly, these degenerate strains also do not sporulate. This has spurred the idea that genes involved in sporulation also exist on the plasmid in both ATCC 4259 as well as the type strain, ATCC 824 (4, 2).

Energy metabolism and byproducts

Clostridium acetobutylicum is a chemoorganotroph. It obtains energy via substrate phosphorylation by fermentation. As with all fermentation, the substrate are organic molecules which act as the electron donor and acceptor. It follows that it is heterotrophic with its source of carbon coming from organic molecules. In particular, C. acetobutylicum requires a carbohydrate source capable of undergoing fermentation to survive (1).

In addition, C. acetobutylicum is an obligate anaerobe. It can only survive hours in an aerobic environment before undergoing sporulation as a means to survive for much longer periods of time in the aerobic environment. It displays no activity of catalase, an enzyme important for aerobic organisms in order to convert a toxic byproduct of oxygen metabolism, hydrogen peroxide, to water and oxygen (5). However, it contains many enzymes that allow it survive in microoxic environments, such as superoxide dismutase. These enzymes are upregulated in the presence of oxygen and contribute to short term cell survival in microoxic environments (6).

C. acetobutylicum is able to use a number of different fermentable carbohydrates as an energy, as well as carbon, source. The genome codes for proteins that aid in the breakdown of xylan, levan, pectin, starch, and other polysaccharides (2). Interestingly, while genes which commonly code for cellusomes, protein complexes which breakdown crystalline cellulose, are present the organism is unable to grow solely on cellulose substrates (7).

Considerable research has been invested into metabolic pathways of Clostridium acetobutylicum in order to improve industrial fermentation operations. The metabolic pathways which produce industrial useful solvents are most notable in C. acetobutylicum. The solvents acetone, acetate, butanol, butyrate, and ethanol are all derived from the common precursor, acetyl-CoA (2). In addition to these products, CO₂ and H₂ are produced (1).

Another notable metabolic pathway is that some Clostridia (including C. acetobutylicum) are capable of "fixing" atmospheric nitrogen. The process of nitrogen fixation reduces atmospheric N₂ into ammonia which is then incorporated into molecules via biosynthesis. This was determined using a labeled form of nitrogen, ¹⁵N₂. After sequencing, C. acetobutylicum ATCC 824, a series of genes very similar to the nitrogen fixing genes in C. pasteurianum were found, further confirming the bacterium's ability to utilize atmospheric nitrogen (8).

Cell structure and development

During early cell development, C. acetobutylicum stains Gram-positive, however, it can stain Gram-negative as the culture ages. During vegetative growth, the cell has peritrichous flagella (flagella which cover the entire surface of the cell) (1). Increased motility of the bacteria have been implicated in increased solvent production due to chemotaxis. Attractants include butyric acid and sugar. Notable repellents include acetone, butanol, and ethanol. This mechanism is logical in allowing the cell to find nutrients and move away from byproducts produced by its own metabolism (9).

In addition, different byproducts are produced at different phases of growth in C. acetobutylicum. During exponential growth phase, primary products are acetate and butyrate. During this time, nitrogen fixation is also taking place (8). Some time after the cell enters stationary phase (18 hours), the production of butanol and acetone peak (1). This temporal separation of nitrogen fixation and production of solvent is advantageous in order to avoid competition for reductants by the two process (8).

The major stage of cell development is characterized by the formation of an endospore. An endospore is the most resistant cell type known. Upon certain environmental cues, the vegetative cell produces a subterminal septum( 1), an event which can be viewed with electron microscopy . This septum eventually become another cell, called the forespore, engulfed by the original cell, termed the mother cell. The forespore is composed of a layer of cortex (primarily peptidoglycan) and coat proteins. These two highly resistant layers surround the core, which is a highly dehydrated cytoplasm. The core is defined by absolutely no metabolism occurring within the cell. The mother cell lyses releasing the mature spore. This mature spore is resistant to high temperature, chemicals and many types of radiation allowing it to survive for extraordinary number of years. Upon other environmental cues, such as an anoxic environment, the cell germinates and begins the vegetative cycle again (10).

Spore formation begins when the cell is exposed to unfavorable conditions. Aerobic conditions, formation of organic byproducts, and dissipation of the proton gradient outside the cytoplasmic membrane all lead to sporulation. This is in contrast to model organism of endospore formation, Bacillus subtilis, which forms endospores primarily due to limitation of nutrients (10).

Ecology

While the type strain of C. acetobutylicum was isolated from soil, C. acetobutylicum is ubiquitous. It has been found in "lake sediment, well water, and clam gut" (1). In addition, it has been recorded in a number of different feces specimens, including human, bovine, and canine feces (1). A search of the literature reveals that pathogenic or symbiotic relationships are not documented.

Pathology

C. acetobutylicum is completely benign to both plants and animals, however, many other species in the Clostridium genus are known pathogens, including: Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringen. In particular, C. botulinum and C. tetani, produce some of the most deadly neurotoxins known (11).

C. acetobutylicum has been found in the human colon, however, it is not known to be a part of normal human flora (3). In addition, because the organism does not appear to be toxic to mammals through the production of intracellular or extracellular substances, the organism would have to be present in enormous quantities to produces any threat (12).

The only issue of pathology with C. acetobutylicum is acquiring genes from pathogenic Clostridium such as C. tetani or C. botulinum. While there are no reported cases of C. acetobutylicum acquiring these genes, there have been incidents in the literature in which other Clostridium species have caused infant botulism with toxins very similar to those present in C. botulinum. The similarity of the toxins suggest that the normally non-toxigenic Clostridium strain acquired toxin-coding genes from C. botulinum, which are likely present on a plasmid (13).

Application to Biotechnology

Clostridium acetobutylicum has played an important role in biotechnology throughout the 20th century. Initially, acetone was needed in the production of synthetic rubber. Chaim Weizmann was hired to work on the problem at Manchester University and fermentation became an attractive route in which to acquire the acetone necessary for the process. Between 1912 and 1914, Weizmann isolated a number of strains. The best producing would later come to be known as Clostridium acetobutylicum. The ABE method devised by Weizmann offered the advantage of increased efficiency over other fermentation processes. In addition, it could use maize starch as a substrate, whereas other processes required the use of potatoes (3).

The outbreak of World War I in 1914 resulted in a huge increase in the need for acetone. It would prove a pivotal point in the development of the ABE process utilizing Weizmann's organism. The acetone was to be used in the production of smokeless gunpowder, known as cordite. Over the course of the next few years, Weizmann's process would be utilized in a number of large industrial factories through Great Britain. When Great Britain was cutoff from access to grain during the war, the process was moved to factories in Canada. When the United States entered the war in 1917, it also opened a number of factories using the Weizmann method. After the war ended, the need for acetone abruptly dropped. However, factories were still used to produce butanol, a useful solvent in the production of lacquers for the expanding automobile industry. Previously, butanol had a been a waste product of the process when the focus was on the production of acetone. Throughout the late 1920's, the demand for butanol continued to escalate due to the growing automobile industry and a number of new plants opened with enormous output capacity. Two such plants put out 100 tons of acetone every day. In addition to butanol, industrial ethanol was being produced for a variety of purposes. The hydrogen gas given off by the process was used to hydrogenate oils used for food. At about this time, molasses became the leading substrate for ABE fermentation. It was cheaper and more efficient than maize starch. When the patent on the Weizmann strain expired in 1937,more new plants were opened throughout the country as well as internationally (3).

However, in the late 1950's and 1960's, the petroleum industry began climbing at an unbelievable rate. In addition, the price of molasses used in fermentation began to climb steeply. While more efficient fermentation methods were developed, they ultimately could not compete with petrochemical production of the industrial solvents and most plants were shut down by 1957(3). However, with the continued rise of petroleum prices, there have been since studies in order to reconsider fermentation as a source of industrial solvents. Some of these processes have attempted to increase the efficiency of the process using genetic manipulation (14). Others have examined using waste products such as whey or wood shavings as a substrate (15).

Current Research

C. acetobutylicum has been the focus of research as a specific mechanism of delivery of therapeutic drugs to cancerous regions of the body. C. acetobutylicum is necessarily anaerobic and therefore intravenous injection of spores will result in germination only in hypoxic regions of solid tumors in the body. Genetic manipulation of C. acetobutylicum in order to produce enzymes which will activate pro drugs within the tumorous region provides an extremely specific delivery mechanism to these tumor sites (16).

Some of the newest research has investigated alternative methods to produce the industrial solvents which C. acetobutylicum has been used for the last century to produce. In particular, butanol has received particular attention as a possible alternative fuel source for automobiles. Butanol and ethanol, both products of fermentation by C. acetobutylicum, have been studied intensely. Of the two, butanol has advantages over ethanol as a fuel source, as well as many possible benefits over current fuel sources, in that it may offer lower emissions and increased efficiency. The most important factor in the cost of butanol production is associated with the cost and availability of the substrate. Studies therefore have been geared toward novel methods of utilizing cheap substrates. In a 2006 study, butanol fermentation via a new patented process in replacement to the ABE process was proposed. It involves use of corn fiber (specifically xylem), as a substrate for C. acetobutylicum, to produce cheap butanol. The major advantage of this technique is that corn fiber is a byproduct in many agriculture processes and provides an abundant source of substrate (17).

Another intense source of study for C. acetobutylicum is hydrogen gas production as an alternative energy source. Hydrogen gas contains a large amount of energy, which could be an extremely beneficial alternative gasoline. In particular, use of hydrogen gas produces no carbon dioxide or greenhouse gases. Most hydrogen gas is currently produced using nonrenewable sources; an alternative means of production via fermentation would be extremely valuable if yields could be increased tremendously. Thus, a number of different fermentation methods that could be used to improve yields are being explored in the most recent research involving C. acetobutylicum. In particular, a trickle bed reactor that uses glucose as a substrate has been presented as a possibility, though yields are too low to be used industrially. However, some sort of application of a trickle bed is seen as a possible means of productions in the future (18).

References

Taxonomy:NCBI

(1) Cato, E.P., W.L. George, and S.M. Finegold. 1986. Genus Clostridium, pp. 1141-1200. In: P. H. A. Sneath et al. (eds.), Bergey's Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins, Baltimore, MD.

(2) Nolling J et al., "Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum.", J Bacteriol, 2001 Aug;183(16):4823-38.

(3) Jones, D. T., and D. R. Woods. 1986. Acetone-butanol fermentation revisited. Microbiol. Rev. 50:484-524.

(4) Cornillot, E., R. V. Nair, E. T. Papoutsakis, and P. Soucaille. 1997. The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain. J. Bacteriol. 179:5442-5447.

(5) Zhang H, Bruns MA, Logan BE.(5) Keis, S., Shaheen, R., and Jones, D.T. "Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov." Int. J. Syst. Evol. Microbiol. (2001) 51:2095-2103.

(6) Kawasaki, S., Y. Watamura, M. Ono, T. Watanabe, K. Takeda, and Y. Niimura. 2005. Adaptive responses to oxygen stress in obligatory anaerobes Clostridium acetobutylicum and Clostridium aminovalericum. Appl. Environ. Microbiol. 71:8442-8450.

(7) Fabrice Sabathe, Anne Belaıch, Philippe Soucaille (2002) Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum FEMS Microbiology Letters 217 (1), 15–22.

(8) Chen, J.S., Toth, J., and Kasap, M. (2001) Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii. J Ind Microbiol Biotechnol 27: 281–286.

(9) Gutierrez, Noemi A., Maddox, Ian S. Role of Chemotaxis in Solvent Production by Clostridium acetobutylicum Appl. Environ. Microbiol. 1987 53: 1924-1927.

(10) P. Durre and C. Hollergschwandner, Initiation of endospore formation in Clostridium acetobutylicum, Anaerobe 10 (2004), pp. 69–74.

(11) Hill, E. O. 1981. The genus Clostridium (Medical aspects), pp. 1756-1766. In: M. P. Starr et al. (eds.), The Prokaryotes, Volume II. Springer-Verlag, New York.

(12) Gill, D.M. 1982. Bacterial toxins: A table of lethal amounts. Microbiol. Rev. 46:86-94.

(13) Gimenez, J.A. and H. Sugiyama. 1988. Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E. Infection and Immunity 56:926-929.

(14) Harris, L. M., R. P. Desai, N. E. Welker, and E. T. Papoutsakis. 2000. Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol. Bioeng. 67:1-11.

(15) McNeil, B. and B. Kristiansen. 1986. The acetone butanol fermentation. Adv. Appl. Microbiol. 31:61-92.

(16) Nuyts S, Van Mellaert L, Theys J, Landuyt W, Lambin P, and Anne J. Clostridium spores for tumor-specific drug delivery. Anticancer Drugs. 2002 Feb;13(2):115-25.

(17) Nasib Qureshi, Xin-Liang Li, Stephen Hughes, Badal C. Saha, and Michael A. Cotta Butanol Production from Corn Fiber Xylan Using Clostridium acetobutylicum Biotechnol. Prog.; 2006; 22(3) pp 673 - 680.

(18) Zhang H, Bruns MA, Logan BE. Biological hydrogen production by Clostridium acetobutylicum in an unsaturated flow reactor. Water Res. 2006 Feb;40(4):728-34.

Edited by Mark Hower, student of Rachel Larsen and Kit Pogliano