Model organism for the solventogenic clostridia is C. acetobutylicum. The metabolism of this organism is well understood, and the genes and enzymes needed for butanol production are already identified and characterized (see below and Fig. 10.2). C. acetobutylicum typically performs a biphasic fermentation, often referred to as ABE (for acetone/butanol/ethanol) fermentation (Durre, 2005a; Jones and Woods, 1986).

During exponential growth (in the so-called acidogenic phase or acidogenesis),

C. acetobutylicum follows the standard butyric acid pathway producing acetate, butyrate, CO2 and H2 (see Fig. 10.2). In addition, small amounts of ethanol and

Table 10.1 Butanol-producing microorganisms

Genus Species Reference

Clostridium septicum Clostridium chavoei — Clostridium aurantibutyricum Clostridium tetanomorphum

Clostridium sporogenes Clostridium pasteurianum Clostridium carboxidivorans — Butyribacterium methyotrophicum — T. thermosaccharolyticum Hyperthermus butylicus

10.1 Relationship of butanol-producing microorganisms. Tree was created with Ribosomal Database Project (Cole et al., 2007) on the basis of 16s rRNA gene sequences.

Acetaldehyde —

Thiolase (Th1A)

Acetoacetyl-CoA

I 3-Hydroxybutyryl-CoA dehydrogenase (Hbd) 3-Hydroxybutyryl-CoA

I Crotonase (Crt)

Crotonyl-CoA

I Butyryl-CoA dehydrogenase (Bed)

► Butyraldehyde —

Butyraldehyde dehydrogenase Butanol dehydrogenase (AdhE) (AdhE, BdhA/B)

10.2 Catabolic pathways of acid and solvent formation in Clostridium acetobutylicum. The single reactions shown do not represent stoichiometric fermentation balances.

acetoin are formed successively, and under certain conditions, lactate is produced as well. Typically, about twice as much butyrate is formed compared to acetate. Accumulation of the excreted acids causes a rapid decrease in pH of the surrounding medium. This poses a serious threat to C. acetobutylicum, since anaerobic bacteria are unable to maintain a constant internal pH, which is generally 1 unit higher than the external pH (Durre et al., 1988; Gottwald and Gottschalk, 1985; Huang et al., 1985). When the external pH drops to the critical point of 4.5, considerable levels of undissociated acetic and butyric acid are present (pKa of acetic acid = 4.75 and pKa of butyric acid = 4.82), which can then pass the cytoplasmatic membrane via diffusion. Due to the higher internal pH, these acids dissociate into salts and protons again and thus destroy the essential proton gradient across the membrane needed for energy conservation and several transport mechanisms.

To avoid this deleterious effect, a major metabolic shift takes place in C. acetobutylicum at the end of exponential growth. The organism takes up acetate and butyrate and converts these organic acids into the solvents acetone and butanol, respectively (solventogenic phase or solventogenesis; see Fig 10.2). A butanol/acetone ratio of 2:1 is typical for C. acetobutylicum, whereas some strains of C. beijerinckii form isopropanol instead of acetone. While the reassimilation of
the excreted acids leads to an increased pH, the solvents acetone, isopropanol and, especially, butanol are also toxic for the cell (see Section 10.5). However, the cell gains enough time to initiate the formation of endospores and thus secures long-time survival.