General Microbiology - Schlegel, H. 1987

Types of fermentation
Butyric acid and acetone-butanol fermentation; Clostridia

Butyric acid (butyrate), n-butanol, acetone, 2-propanol, and several other organic acids and alcohols are typical products of carbohydrate Fermentation by anaerobic spore-forming Bacteria (clostridia). Therefore, clostridia, as well as certain specialized species that ferment exclusively ethanol, Amino Acids, or other substances, are discussed here in relation to the Butyric acid fermentation they induce.

Characteristics. The genus Clostridium belongs to the family Bacillaceae. Like other members of this family (Bacillus, Sporolactobacillus, Desulfotomaculum, and Sporosarcina), clostridia are Gram-positive. Thanks to their peritrichous flagella, they are highly motile. Vegetative Cells are rod-shaped, though their Morphology can vary depending on environmental conditions. Oval or spherical endospores alter the shape of the rod-shaped mother Cell, as their diameter is generally greater than the thickness of The Cell. The spores are heat-resistant.

Physiologically, clostridia are characterized by a pronounced fermentative type of METABOLISM and high sensitivity to oxygen, growing strictly under anaerobic conditions. However, seemingly all transitional forms exist—ranging from strictly anaerobic species (Clostridium pasteurianum, C. kluyveri) to nearly aerotolerant ones (e.g., C. histolyticum, C. acetobutylicum). As a rule, clostridia lack Hemoproteins (Cytochromes, catalase). Some species are capable of synthesizing cytochromes, however, if their precursors are present in the growth medium. Starch-like Polysaccharides are widespread as reserve Materials.

The optimal growth Temperature for most known Clostridium species lies between 30 and 40°C. Along with these mesophilic representatives, There are many thermophilic species with an optimum of 60–75°C, such as C. thermoaceticum and C. thermohydrosulfuricum. Like other members of Bacillaceae, clostridia can grow only at a neutral (or alkaline) environmental pH. Consequently, their growth, which is often undesirable, can be completely suppressed by acidification (e.g., in sauerkraut, silage, canned fruits, and raw sausages).

Substrates. Clostridia vary widely in the range of substrates they can utilize and ferment. Some species are undemanding and utilize a broad spectrum of compounds, whereas others are highly specialized and capable of fermenting only one or a few substrates. Overall, clostridia can access a multitude of different natural compounds. They are capable of degrading polysaccharides (starch, Glycogen, Cellulose, hemicelluloses, Pectins), Nucleic Acids, Proteins, amino acids, Purines, and Pyrimidines. While some clostridia require complex nutrient media or growth factors, others do not. Some can utilize molecular gas nitrogen as their sole source of this element, fixing nitrogen at a high rate (Clostridium pasteurianum)¹.

¹ In recent years, it has been established that certain Clostridium species are capable of autotrophic growth utilizing molecular hydrogen. These include C. thermoautotrophicum, C. aceticum, and C. thermoaceticum (see Section 9.5). — Ed. note.

Based on their ability to utilize various substrates, clostridia can be divided into several groups. Saccharolytic clostridia predominantly break down polysaccharides or sugars. Peptolytic clostridia degrade proteins, peptones, and amino acids. These groups, in turn, are categorized according to their fermentation types and products (Tables 8.7 and 8.8).

Enrichment cultures of Clostridium species can be obtained by taking advantage of certain unique traits. Because their spores are heat-resistant, the inoculum is pasteurized beforehand. Establishing strictly anaerobic conditions rules out the growth of all aerobic bacteria in advance. Since some polysaccharide-degrading bacteria are attached to particle surfaces—such as starch grains or cellulose particles in the rumen of ruminants or in certain sediments—these particles can first be washed to free them from other bacteria and then used as inoculation material. To maintain high fermentative activity during subsequent work with isolated strains, pasteurized inocula must always be used.

Biochemistry of fermentation and its products. Fermentation yields various proportions of acids (butyric, acetic, lactic), alcohols (butanol, ethanol, 2-propanol), as well as acetone and gaseous products (H₂ and CO₂). Clostridia break down glucose via the fructose bisphosphate pathway. The hydrogen released during the dehydrogenation of glyceraldehyde 3-phosphate is typically transferred to organic acids or ketones formed from Pyruvate or acetyl-CoA. The fermentation prototypically carried out by clostridia can be exemplified by the glucose fermentation of Clostridium butyricum and C. acetobutylicum, which yields butyrate, acetate, butanol, ethanol, acetone, 2-propanol, CO₂, and H₂. Product yields vary depending on conditions.

Class="center">Table 8.7. Clostridia classified by fermentation type

Butyric acid (butyrate) is the Condensation product of two acetyl-CoA molecules mediated by thiolase, yielding acetoacetyl-CoA, which is subsequently reduced (Fig. 8.4). Acetoacetyl-CoA is reduced to β-hydroxybutyryl-CoA by NADH₂ in the presence of β-hydroxybutyryl-CoA dehydrogenase. Water is then split off from the latter by crotonase. Crotonyl-CoA is reduced to butyryl-CoA by the flavin enzyme butyryl-CoA dehydrogenase. Coenzyme A can be transferred from butyryl-CoA to acetate via CoA transferase, releasing butyric acid, which passes into the medium. Free acetate can be generated from acetyl-CoA via phosphotransacetylase and acetate kinase, a process coupled with the synthesis of ATP from ADP (see also p. 264).

Table 8.8. Special Types of Fermentation

In pure butyric acid fermentation, the hydrogen released during pyruvate oxidation is evolved as a gas. When glucose is fermented According to the equation

Glucose → Butyrate + 2CO₂ + 2H₂

the hydrogen balance closes; three moles of ATP are formed per mole of glucose.

Butanol, butyrate, acetone, and 2-propanol are produced during Glucose fermentation by Clostridium acetobutylicum cells. Initially, butyric acid is also released; however, as the medium becomes acidic (Fig. 8.5), Enzymes begin to be synthesized (including acetoacetate decarboxylase), the action of which leads to the accumulation of acetone and butanol. The pathways leading to these substances are closely interconnected. The decarboxylation of a portion of the acetoacetate eliminates a potential hydrogen acceptor that could otherwise have added 2[H] twice during reduction to butyrate. This hydrogen must somehow be transferred to other acceptors, including the newly formed butyrate. For reduction to butanol, butyrate must first be activated by conversion into butyryl-CoA. The reactions involved in The formation of butyrate, acetone, and butanol are illustrated in Fig. 8.4. During fermentation in an alkaline environment (e.g., in the presence of CaCO₃; Table 8.9), C. acetobutylicum behaves like C. butyricum. Some strains reduce acetone and release 2-propanol.

Fig. 8.4. Formation of acetate, ethanol, n-butanol, butyrate, acetone, and 2-propanol during fermentations carried out by clostridia. The initial breakdown of glucose proceeds via the fructose bisphosphate pathway; pyruvate is dehydrogenated by pyruvate:ferredoxin oxidoreductase. The scheme shows only the reaction sequences starting from acetyl-CoA. DH - dehydrogenase.

Fig. 8.5. Kinetics of glucose fermentation by Clostridium acetobutylicum. (Davis R., Stephenson M., Biochem. J., 35 [1941], 1320.)

Table 8.9. Acetone-butanol fermentation by Clostridium acetobutylicum in the presence and absence of CaCO₃ (Bernhauer et al., Biochem. Zeitschrift, 287 [1936], 61)

Ethanol is formed As a result of acetyl-CoA reduction.

Molecular hydrogen can originate from NADH₂, which is produced both during pyruvate breakdown and the dehydrogenation of glyceraldehyde 3-phosphate (see p. 265). The more hydrogen that can be generated in this process, the fewer hydrogen acceptors (acetoacetyl-CoA) need to be synthesized. Thus, the bond energy of acetyl-CoA can be conserved in the form of ATP. Therefore, if the fermentation of glucose by C. butyricum yields more than two moles of H₂ per mole of glucose—and consequently less butyrate and correspondingly more acetate—the ATP yield may exceed 3 moles (see below regarding Ruminococcus).

Since acetone, 2-propanol, and butanol serve as important organic Solvents, clostridial fermentation is of great technical significance. It was precisely in connection with The production of these substances that industry first faced The Challenge of carrying out microbiological syntheses under conditions precluding any possibility of contamination.

Ethanol and acetate fermentation. From enrichment cultures of Methanobacterium omelianskii containing ethanol as a substrate, an anaerobic spore-forming bacterium was isolated that requires both ethanol and acetate. This bacterium—Clostridium kluyveri—converts a mixture of acetic acid and ethanol into butyric and caproic acids along with molecular hydrogen (Fig. 8.6). Acetate serves as an additional hydrogen acceptor and is formed during the course of fermentation. ATP is synthesized solely via the acetate kinase reaction.

Lactate and acetate fermentation. From enrichment cultures containing lactate as a substrate, the bacterium Clostridium tyrobutyricum was isolated, which also requires acetate as an additional hydrogen acceptor when lactate or glycerol serves as the primary substrate:

C. tyrobutyricum ferments glucose following the pathway known for C. butyricum, without requiring an exogenous hydrogen acceptor.

Fig. 8.6. Fermentation of Ethanol and acetate with the formation of butyrate and molecular hydrogen by Clostridium kluyveri. Fd, ferredoxin.

Glutamic acid fermentation. Among the numerous fermentation processes in which Amino acids are converted under anaerobic conditions into Fatty acids, CO₂, and ammonia, we will examine only the degradation of glutamate by the bacterium Clostridium tetanomorphum. This fermentation has attracted attention because it served as the model for elucidating The biochemical function of vitamin B₁₂. C. tetanomorphum is usually isolated from an enrichment culture with histidine as the substrate. Histidine is degraded via glutamate. The fermentation of glutamate yields butyric and acetic acids, ammonia, CO₂, and molecular hydrogen. The glutamate catabolic pathway involves a series of unusual reactions (Fig. 8.7). First, the bonds between carbon atoms 2 and 3 are cleaved, and new bonds are formed between atoms 2 and 4, resulting in a branched-chain amino acid, methylaspartic acid. A coenzyme—a derivative of vitamin B₁₂—participates in this process. Deamination occurs only at this stage. Water is added to the unsaturated mesaconic (methylfumaric) acid, and the resulting citramalic (2-methylmalic) acid is subsequently cleaved into acetic and pyruvic acids. Acetic acid is released, while pyruvic acid is converted into Butyric Acid and CO₂ via the pathway discussed above.

Coupled fermentation of Two amino acids (Stickland reaction).

Peptolytic clostridia (Table 8.7, V) hydrolyze proteins and subsequently utilize amino acids. Many amino acids are fermented only in conjunction with certain others. As Stickland established (1934), Clostridium sporogenes rapidly ferments a mixture of alanine and glycine, but cannot utilize either of these amino acids individually. Judging by the overall equation

Alanine + 2 Glycine + 2H₂O → 3 Acetate + 3NH₃ + CO₂

alanine serves as the hydrogen donor and glycine as the acceptor:

Fig. 8.7. Glutamate fermentation by Clostridium tetanomorphum via the mesaconate pathway.

The energy source is evidently a coupled oxidation-reduction reaction. Hydrogen Donors can include, for example, alanine, leucine, isoleucine, valine, Serine, Methionine, etc. Hydrogen acceptors may be glycine, Proline, Arginine, Tryptophan, etc. The donor amino acid is deaminated to an oxoacid, which is then converted into a fatty acid via oxidative decarboxylation. This step is coupled with phosphorylation and thus represents an energy-yielding reaction. The hydrogen transferred in the process to ferredoxin is bound again during the reductive deamination of the acceptor amino acid. However, not all amino acids are utilized by all peptolytic clostridia.

Fig. 8.8. Glucose fermentation by Ruminococcus albus and utilization of the generated hydrogen in a mixed culture with Vibrio succinogenes. (An example of Symbiosis involving interspecies hydrogen transfer.) Fd, ferredoxin.

Butyric and acetic acid fermentation conducted by non-spore-forming bacteria. Some genera of anaerobic bacteria are closely related to clostridia in terms of their fermentation products; however, they do not form spores, and many of them are Gram-negative. Most of these butyric- and acetic-acid-producing bacteria have been isolated from the rumen, where they participate in the degradation of cellulose, starch, and other CARBOHYDRATES. They liberate large amounts of molecular hydrogen and CO₂, thereby creating favorable conditions for methanogenic bacteria to produce methane. Among the ruminal bacteria that produce butyric acid, Butyrivibrio fibrosolvens deserves special mention, while Ruminococcus albus is a notable ruminal producer of acetic acid. The latter species is a strict anaerobe that utilizes cellulose, Xylan, and numerous sugars. It is capable of converting 1 mole of glucose into 2 moles of acetate, 2 moles of CO₂, and 4 moles of H₂—provided, however, that the concentration of dissolved hydrogen is kept at a low level. This is achieved in a mixed culture with a hydrogen-consuming bacterium such as Vibrio succinogenes (Fig. 8.8). As the scheme shows, up to 4 moles of ATP can be generated per mole of glucose during pure acetate fermentation. Such high efficiency of ATP regeneration is possible, however, only if all reducing equivalents released during glucose Cleavage are liberated in the form of molecular hydrogen (see above).

Clostridia as disease agents and toxin producers. Certain peptolytic clostridia can cause diseases in wound infections (gas gangrene and tetanus) as well as food poisoning. Their spores are widespread in the soil. When spores of Clostridium histolyticum or C. septicum enter an open wound lacking oxygen access—or where aerobic bacteria consume oxygen and create anaerobic conditions—these clostridia begin to proliferate, break down Collagen and other proteins using proteinases, and cause the formation of foul-smelling Fermentation products and gas. In former times, such gas gangrene ("hospital gangrene") could be combated only by amputating the affected limb. Tetanus, which is still encountered fairly often today, is likewise associated with wound infection. It is caused by Clostridium tetani. During its growth, this bacterium secretes a potent neurotoxin that triggers tonic Muscle spasms.

¹ Acetic acid fermentation should not be confused with the aerobic process carried out by acetic acid bacteria. — Tr.

The most severe form of food poisoning, botulism, is caused by Clostridium botulinum. This is a widespread soil bacterium capable of growing in insufficiently sterilized meat products and canned legumes. It derives its name from its association with sausage (from Latin botulus, sausage). If contaminated food is consumed, the toxin it produces can cause death resulting from nerve paralysis, specifically respiratory paralysis. Fortunately, this toxin is thermolabile and is rapidly inactivated (within 15 min) by boiling.