C. acetobutylicum is well known for its biphasic ABE fermentation metabolism and consequently rose as a model organism for nonpathogenic clostridia in the past 30 years (L¨utke-Eversloh and Bahl, 2011). Thus, the clostridial ABE fer- mentation is well characterized, whereas the detailed regulation between acid and solvent formation still remains unknown. However, the typical ABE fermentation of clostridial strains can be divided into three major growth phases (Figure 9.1).
The first growth phase is highlighted by exponentially growing vegetative cells converting carbohydrates by substrate-level phosphorylation to the acids acetate and butyrate. Thus, the exponential growth phase is also named as “acidogene- sis”. Consequently, the accumulation of acetate and butyrate in the medium leads to a significantly decrease of the pH (<5). In reaction to a lowering pH, sol- ventogenic clostridia switch their metabolism and cellular morphology. This sec- ond growth phase, well known as stationary phase, is characterized by markedly swollen cigar-shaped cells which accumulate a carbohydrate reserve material known as granulose. These cigar-shaped cells are named “clostridial stages” (Jones et al., 1982). Interestingly, for several clostridial strains, for example,Clostridium pasteuri- anum, the intracellular storage compound granulose was determined asα-1,4-linked polyglucan with less than 2% of branchedα-1,6-linked chains (Hobson and Nasr, 1951; Whyte and Strasdine, 1972; Darvill et al., 1977), whereas for other strains, for example, Clostridium saccharobutylicum(formerly known, C. acetobutylicum P262) (Shaheen et al., 2000; Keis et al., 2001), onlyα-1,4-linked polyglucan units
FIGURE 9.1 The cell cycle ofClostridium acetobutylicumwith its different growth phases, respective cell forms, and major fermentation products.
were determined (Reysenbach et al., 1986). Thus, granulose shows similar charac- teristics like amylopectin or glycogen. However, during stationary growth phase, the characteristic “clostridial stage” cells also re-assimilate prior produced acids, acetate, and butyrate, and convert them to the neutral solvents, acetone, butanol, and ethanol. This growth phase is well known as “solventogenesis.” The char- acteristic change in product formation is also named “metabolic switch” and is dependent on several environmental factors, that is, change of the pH, the concen- tration of the carbon source, a threshold of produced acids, acetate, and butyrate, and/or the intracellular NADH/NAD+ratio (Jones and Woods, 1986). Concomitant with the “metabolic switch” the microorganism initiates the sporulation process and uses the stored carbohydrate granulose to finalize the energy-consuming steps of spore formation in the third growth phase, at the end of late stationary phase. Further- more, spores are able to survive bad environmental conditions caused by different stress factors, for example, UV light, heat, drought, or frost. However, the germina- tion of spores under better growth conditions, for example, improved availability of specific nutrition, finalizes the clostridial cell cycle (Figure 9.1).
9.4.2 Physiology and Enzymes of the Central Metabolic Pathway Solventogenic clostridia, for example, C. acetobutylicumand Clostridium beijer- inckii, are able to consume a variety of sugars. At the beginning of the fermentation process exponential growing cells using the Embden–Meyerhof–Parnas Pathway convert a carbohydrate, for example, glucose, to pyruvate by generation of two molecules ATP and two molecules NADH (Figure 9.2). In general, the enzyme
Glucose Glycolysis Pyruvate Ldh Pta Ptb ButyrylphosphateButyryl-CoAButyraldehydeBuk
Pdc Acetyl-CoAAdhE AdhEAdhE, Bdh
Thl CtfA/B
AdhE AcetaldehydeAcetylphosphateAck Adc Hbd Crt Bcd, EtfA/B
Acetate Butyrate
AcetoneAcetoacetateAcetoacetyl-CoA β-Hydroxybutyryl-CoA Crotonyl-CoA
Ethanol Butanol
H2Fdred+ Pfor
Lactate
NaD+ NaD+ NAD+ ATPADPCoAPi
NaD+ + CoA NaD+ + CoA
CoA CoA CoA
ATPADPPi Acetyl-CoA
CO2 CO2 H2O
CO2
NADH + H+2 ATP; 2 NADH + H+ NADH + H+ NADH + H+ NADH + H+ NADH + H+
NaD(P)+NAD(P)H + H+ NAD(P)+NAD(P)H + H+ FIGURE9.2PhysiologyofABEfermentationmetabolismofClostridiumacetobutylicumwiththerespectiveenzymesandproducts.CoA, coenzymeA;Ldh,lactatedehydrogenase;Pdc,pyruvatedecarboxylase;Pfor,pyruvate::ferredoxinoxidoreductase;Fdred,ferredoxinreduced;Th thiolase;Hbd,β-hydroxybutyryl-CoAdehydrogenase;Crt,crotonase;Bcd,butyryl-CoAdehydrogenase;Etf,electrontransferflavoprotein;Pta, phosphotransacetylase;Ack,acetatekinase;Ptb,phosphotransbutyrylase;Buk,butyratekinase;CtfA/B,acetoacetyl-CoA:acyl-CoAtransferase;Adc, acetoacetatedecarboxylase;AdhE,aldehyde/alcoholdehydrogenase;Bdh,butanoldehydrogenase.
234
pyruvate ferredoxin::oxidoreductase (Pfor) utilizes the formed pyruvate using a molecule CoA to release acetyl-CoA and CO2, whereas the lactate dehydrogenase (Ldh) only shows remarkable influences under certain growth conditions, that is, iron limitation (Bahl and Gottschalk, 1984; Bahl et al., 1986). At this time the organ- ism also produces significant amounts of hydrogen by interplay of Pfor, reduced and oxidized ferredoxins and hydrogenases. Furthermore, during acidogenic growth the central molecule acetyl-CoA is converted to the acids, acetate, and butyrate.
Here, the enzymes phosphotransacetylase (Pta) and acetate kinase (Ack) are respon- sible for the synthesis of acetate (Hartmanis and Gatenbeck, 1984; Boynton et al., 1996). First, Pta converts acetyl-CoA to acetylphosphate, and finally Ack metabo- lizes acetylphosphate to acetate. On the other hand, for the butyrate production the enzyme thiolase (Thl) combines two molecules of acetyl-CoA to acetoacetyl-CoA.
Furthermore, acetoacetyl-CoA is converted in sequential steps to butyryl-CoA by the enzymesβ-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and butyryl- CoA dehydrogenase (Bcd). Here, the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) convert butyryl-CoA in a two-step process to butyrate (Hart- manis, 1987; Walter et al., 1993). Ptb produces butyrylphosphate which is used by Buk to finalize butyrate production. Notably, each acid-forming pathway generates one molecule of the important energy molecule ATP.
As mentioned above, the significant production of acids, acetate, and butyrate leads to a drastically decrease of the external pH. Interestingly, solventogenic clostridia are unable to maintain a constant intracellular pH, as described for other bacteria (Booth, 1985; Krulwich et al., 2011). Thus, the internal pH follows the external pH with a difference ofΔpH∼1 (Gottwald and Gottschalk, 1985; Terracciano and Kashket, 1986). Consequently, in response to the changed pH the organism switches its metabolism from acidogenesis to solventogenesis. Therefore, the prior produced acids, acetate, and butyrate are re-assimilated by the enzyme acetoacetyl-CoA:acyl- CoA transferase (CtfA/B). The CtfA/B enzyme converts acetate or butyrate, based on a molecule acetoacetyl-CoA, to their respective CoA-derivate, acetyl- or butyryl- CoA, and to a molecule acetoacetate (Wiesenborn et al., 1989). In a further step, the acetoacetate decarboxylase (Adc) metabolizes acetoacetate to acetone (Petersen and Bennett, 1990). The synthesized CoA molecules, acetyl- and butyryl-CoA, are the precursors for the neutral solvents ethanol and butanol, respectively. In a NAD(P)H- dependent two-step process the bifunctional aldehyde/alcohol dehydrogenase (AdhE) forms acetaldehyde and/or butyraldehyde, and furthermore the respective alcohols ethanol and butanol. Interestingly, solventogenic clostridia possess more than one AdhE (e.g., AdhE1 and AdhE2 inC. acetobutylicum) and further additional single alcohol dehydrogenases or butanol dehydrogenases (BdhA and BdhB) (Walter et al., 1992). The activity of different Bdh’s and AdhE’s, involved in butanol or ethanol pro- duction, is dependent on NADH or NADPH as well as on influenced redox potentials (Chen, 1995). Recently, a NAD(P)H pool-influenced mutant ofC. acetobutylicum (C. acetobutylicum rex::int; redox-sensing protein) demonstrated earlier solvent pro- duction and in consequence higher final ABE concentrations (Wietzke and Bahl, 2012). In general, the butanol:acetone ratio is defined as 2:1. However, cultures with blocked hydrogen production and consequently with a reduced redox potential, for
example, by gassing with carbon-monoxide or addition of methyl-viologen, showed significant increased butanol:acetone ratios by different expressed AdhE’s (Fontaine et al., 2002; H¨onicke et al., 2012). Nevertheless, further investigations are required to unravel detailed regulation of the different aldehyde/alcohol dehydrogenases in solventogenic clostridia strains under certain growth conditions.