SCIENCE, ENGINEERING AND ECONOMICS
2.2 Biochemistry of the ABE Fermentation
BIOBUTANOL:SCIENCE,ENGINEERING AND ECONOMICS
Urbana-Champaign [45-51] that specifically address issues such as substrates and reactor systems for fermentation, genetic strain improvement, immobilization techniques and solvent recovery techniques. Other reviews published in past 5 years are from Wu et al. [52] who have assessed life cycle energy and greenhouse gas emission effect using corn-based biobutanol, Karakashev et al. [53] who have given an overview of production of liquid alcoholic fuels such as ethanol and butanol, and Lee et al. [54] who have critically evaluated various strategies for strain improvement, fermentation processes and downstream processing.
This chapter will touch upon various aspects of ABE fermentation, including: (1) microbial cultures for fermentation and metabolic pathway; (2) substrates for fermentation;
(3) fermentation protocols and reactor design (including immobilized systems); (4) solvent recovery techniques; (5) mathematical modeling; and (6) economics of the process. Genetic and metabolic engineering aspects of clostridial species is outside the scope of this review and reader is referred to the reviews by Blaschek and White [32], Woods [31], Ezeji et al.
[51] and Lee et al. [54]. Moreover, this review mainly focuses on literature published since 1985; for earlier literature we refer the reader to Jones and Woods [12].
Substrate
Glucose Cellulose
Hemicellulose Xylose
D-Xylose
Glucose D-Xylulose
Xylose isomerase
Xylulose kinase
D- Xylulose 5 phosphate
Glyceraldehyde -3 phosohate
transaldolase & transketolase
Glucose-6-phosphate
Fructose-6-phosphate
Glyceraldehyde-3-phosphate
Pyruvate Acetyl Co-A Acetoacetyl CoA
Butyryl-CoA Butanol
Butyrate
Ethanol Acetone Acetate
Fruits / vegetables
Fructose
Fructose
Fructose1-6 bis phosphate
Fructose 1-phosphate
cellulose hydrolysis
Fibrous biomass
(wheat straw, rice straw)
Starch (corn)
starch hydrolysis hemicellulose hydrolysis
E1
E2
E5, E6
E7, E8
E9, E10, E11
E12, E13 E14, E15
PPP Pathway
Phosphotransferase System
& EMP Pathway
E3, E4 Hydrolysis
Figure 2.2: The metabolic pathway of ABE fermentation by Clostridia. Abbreviations: E1 – pyruvate ferrodoxin oxidoreductase; E2 – thiolase;
E3 – phosphate acetyltransferase; E4 – acetate kinase; E5 – acetaldehyde dehydrogenase; E6 – ethanol dehydrogenase; E7 – CoA transferase; E8 – acetoacetate decarboxylase; E9 – 3-hydroxybutyryl-CoA dehydrogenase; E10 – crotonase; E11 – butyryl CoA dehydrogenase; E12 – phosphate butyryl transferase; E13 – butyrate kinase; E14 – butyraldehyde dehydrogenase; E15 – butanol dehydrogenase
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growth have normally been semi-defined and defined media in which the main carbohydrate source is supported by various vitamins and minerals depending upon the microbial strains.
The optimum temperature for the ABE fermentation is between 30–40oC. Two principal phases in the fermentation are characterized by: (1) an acid production phase or acidogenesis and (2) a solvent production phase or solventogenesis. The pH of the fermentation broth, initially at 6.8 to 7, drops to 4.5–5 during acidogenic phase. This phase is associated with the rapid growth of cells and the secretion of the carboxylic acids, acetate and butyrate. The switchover from acidogenesis to solventogenesis occurs at reduced pH. Bahl et al. [55] have suggested that the switchover is an adaptive response of the cells to the low pH of the medium, while Ballongue et al. [56] have suggested that acids produced in the acidogenesis phase act as inducers for the biosynthesis of solventogenic enzymes. We briefly describe below the metabolic pathway for the ABE fermentation employing the clostridia. Gheshlagi et al. [57] have recently reviewed in detail the metabolic pathway of the clostridia. For greater details on the enzymes involved in various steps of the metabolic pathway, we refer the reader to this review.
Fig.2.2 shows the metabolic pathway for clostridial cultures employing various substrates. Among the various nutrient transport mechanisms, anaerobes (obligate/facultative) accumulate sugars via the Phosphoenol Pyruvate (PEP) dependent Phosphotransferase system (PTS) [58, 59]. On the whole, hexose sugars are metabolized by the Embden Meyerhoff Pathway (EMP), while pentose sugars go through the Pentose Phosphate pathway (PPP) to produce pyruvate. Glycolysis utilizes Glucose (1mol) and produces pyruvate (2 mol), while PPP results in production of CO2 (6 mol). During Glycolysis energy is stored as ATP and NADH (2 mol), while during PPP energy is stored as NADPH. The most common substrate for the ABE fermentation is starch, which is converted to glucose following acid/enzyme hydrolysis. Glucose (6C) is first phosphorylated to glucose-6-phosphate, which is
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subsequently converted to pyruvate (3C) via EMP. Other fermentation substrates containing hemicellulose or cellulose (e.g. fibrous biomass such as rice straw or wheat straw) can be converted to xylose and glucose respectively, following hydrolysis [60]. Glucose enters the metabolic pathway in the same manner as stated earlier, while xylose (existing naturally in the form of D-xylose, an aldose) undergoes the ―Isomerase pathway‖ in which, the enzyme xylose isomerase convert D-xylose to D-xylulose. Furthermore, D-xylulose undergoes phosphorylation to form D-xylulose-5-phosphate. D-xylulose-5-phosphate dissimilates by means of transketolase and transaldolase through non-oxidative pentose phosphate resulting in the production of glyceraldehydes 3-Phosphate and fructose-6-phosphate, which finally enters the EMP pathway for further conversion [12, 57, 61]. Fermentation of hexose sugar (1 mol) results in generation of 2 mol of ATP and 2 mole of reduced NADH, while fermentation of pentose sugar yields 5 mol of ATP and 5 mol of NADH [12]. ATP produced during the consumption of glucose results in the exponential growth of cells.
The pyruvate formed during glycolysis (or EMP pathway) is cleaved by pyruvate ferredoxin oxidoreductase in presence of co-enzyme A (CoA) producing CO2, acetyl-CoA and reduced ferredoxin. Conversion of acetyl-CoA to acetate is achieved by the enzymes phosphate acetyltransferase and acetate kinase, while conversion of butyryl-CoA (formed through sequential conversion of acetyl-CoA, as explained later) to butyrate is catalyzed by the enzymes phosphate butyltransferase and butyl kinase. The pH of medium decreases as these conversions proceeds with accumulation of butyric and acetic acids in the medium.
These acids can permeate the cell membrane and are involved in triggering the of solventogenic phase.
During the solventogenic phase, the products of the preceding acidogenic phase are reassimilated and converted to acetone and butanol. The enzyme catalyzing this conversion is Co-A transferase, which converts CoA from acetoacetyl-CoA (formed from acetyl-CoA by
TH-1146_08615103
BIOBUTANOL:SCIENCE,ENGINEERING AND ECONOMICS
action of thiolase) either to acetate forming acetyl-CoA or to butyrate resulting in butyryl- CoA. Out of these, acetyl-CoA can be converted to acetone, butanol and ethanol, while butyryl-CoA can only be converted to butanol, as explained below.
1. Acetyl-CoA can be converted to acetaldehyde (catalyzed by acetaldehyde dehydrogenase) and further to ethanol (catalyzed by ethanol dehydrogenase).
2. Alternatively, two moles of acetyl-CoA are converted to one mole of acetoacetyl-CoA by thiolase (as noted earlier). Removal of CoA from acetoacetyl-CoA by enzyme CoA- transferase yields acetoacetate, which is further converted to acetone by enzyme acetoacetate decarboxylase with release of CO2.
3. Acetoacetyl-CoA can simultaneously undergo reduction to 3-hydroxybutyryl-CoA catalyzed by hydroxybutyryl-CoA dehydrogenase. Dehydration of 3-hydroxybutyryl-CoA catalyzed by crotonase results in crotonyl-CoA, which is reduced to butyryl-CoA by action of NADH and butyryl-CoA dehydrogenase. Butyryl-CoA can be further converted to butyraldehyde (via the action of NADH and butyraldehyde dehydrogenase), and further to butanol (via the action of NADH and butanol dehydrogenase).
4. The fate of butyryl-CoA is unique, as there is no metabolic pathway to regenerate acetyl-CoA from butyryl-CoA. It initially undergoes conversion to butyraldehyde by the action of NADH and butyraldehyde dehydrogenase, and further to butanol by the action of NADH and butanol dehydrogenase.
2.2.1 Inhibition of fermentation
The products of both phases of the ABE fermentation, i.e. acidogenesis and solventogenesis, cause inhibition of fermentation after reaching a certain concentration level in the medium. The presence of butanol in the cell membrane increases membrane fluidity causing destabilization of membrane [12]. For most clostridial cultures, the maximum amount of solvents (i.e. acetone, butanol and ethanol) that cells can tolerate is 20 g/L. Due to
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this limitation, the maximum amount of sugar utilized in a batch fermentation is 60 g/L. An obvious solution to the problem of inhibition is continuous removal of solvents from the fermentation broth or development of new strains of clostridia that are more resistant and tolerant towards butanol. In the subsequent sections of this review, we deal with these issues in greater detail.