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Chapter 22 : Carbohydrate Fermentations

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Abstract:

This chapter reviews some of the more common carbohydrate fermentation pathways, schematics of the flow of carbon in these pathways, characteristic products produced, typical energy yields, and assay procedures for the key enzymes. Fermentations are classified according to the key fermentation end products of each as exemplified by bacterial ethanolic, homolactic, heterolactic, propionic, mixed-acid, butyrate-butanol, homoacetogenic, and other fermentations. Much of one's understanding of early fermentations came from alcoholic fermentation of sugar by the yeast . The enterobacteria carry out mixed acid and butanediol fermentation, and this is the basis for the methyl red/Voges Proskauer test that is used to distinguish the genera. Propionate is an end product of many fermentative bacteria. In contrast, in the propionate-succinate pathway, the formation of propionate involves pyruvate and succinate as intermediates and appears to be much more widespread. The phosphorylation of glucose is carried out by either glucokinase or hexokinase. It appears that among the archaea so far examined, the organisms that contain glucokinase appear to use ADP as the phosphoryl donor for the phosphorylation of glucose. However, in the chapter, only the acetate fermentative pathway of hexoses is considered.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22

Key Concept Ranking

Lactic Acid Fermentation
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Acetyl Coenzyme A
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High-Performance Liquid Chromatography
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Image of FIGURE 1
FIGURE 1

Fermentation products formed from pyruvate by various organisms.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 2
FIGURE 2

Ethanolic fermentation of glucose by the EMP pathway. 1, hexokinase; 2, glucose-6-phosphate isomerase; 3, PFK; 4, fructose bisphosphate aldolase; 5, triose phosphate isomerase; 6, GAPDH; 7, 3-phosphoglycerate kinase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase; 11, pyruvate decarboxylase; 12, ADH.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 3
FIGURE 3

Ethanolic fermentation of glucose by the ED pathway. 1, glucokinase; 2, glucose-6-phosphate dehydrogenase; 3, 6-phosphogluconate dehydratase; 4, KDPG aldolase; 5, GAPDH; 6, 3-phosphoglycerate kinase; 7, phosphoglycerate mutase; 8, enolase; 9, pyruvate kinase; 10, pyruvate decarboxylase; 11, ADH.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 4
FIGURE 4

Flow of carbon when [C]glucose is fermented to ethanol and CO via the EMP and ED pathways.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 5
FIGURE 5

Homolactate fermentation. 1, PEP-PTS; 2 to 6, conversion of glucose-6-phosphate to pyruvate, which is accomplished by the same enzymes as in Fig. 2 ; 7 , lactate dehydrogenase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 6
FIGURE 6

Heterolactate fermentation. 1, PEP-PTS; 2, glucose-6-phosphate dehydrogenase; 3, 6-phosphogluconate dehydrogenase; 4, ribulose 5-phosphate 3-epimerase; 5, phosphoketolase; 6, conversion of GAP to pyruvate, which is accomplished by the same enzymes as in Fig. 2 and 3 ; 7 , lactate dehydrogenase; 8, phosphotransacetylase; 9, acetaldehyde dehydrogenase; 10, ADH.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 7
FIGURE 7

Formation of acetate and lactate from glucose by the heterofermentative bifidum pathway. 1, PEP-PTS; 2, fructose-6-phosphate phosphoketolase; 3, transaldolase; 4, transketolase; 5, ribose-5-phosphate isomerase; 6, ribulose 5-phosphate 3-epimerase; 7, xylulose 5-phosphate phosphoketolase; 8, acetate kinase; 9, conversion of GAP to pyruvate, which is accomplished by the same enzymes as in Fig. 2 and 3 ; 10 , lactate dehydrogenase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 8
FIGURE 8

Butyrate-butanol-acetone-isopropanol fermentation in . 1, PEP-PTS and EMP pathway enzymes; 2, pyruvate-ferredoxin oxidoreductase; 3, acetyl-CoA acetyltransferase (thiolase); 4, l-(+)-β-hydroxybutyryl-CoA dehydrogenase; 5, crotonase; 6, butyryl-CoA dehydrogenase; 7, butyraldehyde dehydrogenase; 8, butanol dehydrogenase; 9, NADH-ferredoxin oxidoreductase and hydrogenase; 10, hydrogenase; 11, acetaldehyde dehydrogenase; 12, ethanol dehydrogenase; 13, lactate dehydrogenase; 14, phosphotransacetylase; 15, acetate kinase; 16, acetoacetyl-CoA:acetate/butyrate:CoA transferase; 17, acetoacetate decarboxylase; 18, isopropanol dehydrogenase; 19, phosphotransbutyrylase; 20, butyrate kinase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 9
FIGURE 9

Ethanol-acetate fermentation by . 1, ADH; 2, acetaldehyde dehydrogenase; 3, H-evolving enzyme system; 4, thiolase; 5, l-(+)-β-hydroxybutyryl-CoA dehydrogenase; 6, crotonase; 7, butyryl-CoA dehydrogenase; 8, CoA transferase; 9, phosphotransacetylase; 10, acetate kinase. The substrates, ethanol and acetate, are enclosed in shaded rectangles, while the major product, butyrate, and the minor product, acetate, are enclosed in rectangles. Formation of acetate primarily yields the energy required for growth of the organism.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 10
FIGURE 10

Mixed acid (top) and butanediol (bottom) fermentation. 1, PEP-PTS; 2, pyruvate kinase; 3, lactate dehydrogenase; 4, PFL; 5, FHL; 6, acetaldehyde dehydrogenase; 7, ADH; 8, phosphotransacetylase; 9, acetate kinase; 10, PEP carboxylase; 11, malate dehydrogenase; 12, fumarase; 13, fumarate reductase; 14, α-acetolactate synthase; 15, α-acetolacate decarboxylase; 16, acetoin reductase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 11
FIGURE 11

Fermentation of lactate to propionate by the acrylate pathway. 1, lactate racemase; 2, propionyl-CoA transferase; 3, lactyl-CoA dehydratase; 4, acrylyl-CoA reductase; 5, d-lactate dehydrogenase; 6, pyruvate-ferredoxin oxidoreductase; 7, phosphotransacetylase; 8, acetate kinase. ETFP, electron-transferring flavoprotein.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 12
FIGURE 12

Fermentation of lactate by the succinate-propionate pathway. 1, lactate dehydrogenase (H acceptor is flavoprotein); 2, ()-methylmalonyl-CoA-pyruvate transcarboxylase; 3, malate dehydrogenase; 4, fumarase; 5, fumarate reductase; 6, CoA transferase; 7, ()-methylmalonyl-CoA mutase; 8, methylmalonyl-CoA racemase; 9, pyruvate-ferredoxin oxidoreductase; 10, phosphotransacetylase; 11, acetate kinase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 13
FIGURE 13

Acetate fermentation of glucose by bacteria. 1, enzymes of the EMP pathway; 2, pyruvate-ferredoxin oxidoreductase; 3, phosphotransacetylase; 4, acetate kinase; 5, formate dehydrogenase; 6, formyl-THF synthetase; 7, methenyl-THF cyclohydrolase; 8, 5,10-methylene-THF dehydrogenase; 9, 5,10-methylene-THF reductase; 10, THF:B methyltransferase; 11, CO dehydrogenase/acetyl-CoA synthase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Image of FIGURE 14
FIGURE 14

Acetate fermentation in archaea. 1, kinase (glucokinase/hexokinase); 2, glucose-6-phosphate isomerase; 3, PFK; 4, fructose bisphosphate aldolase; 5, triose phosphate isomerase; 6, GAPDH; 7, GAP ferredoxin oxidoreductase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase; 11, pyruvate-ferredoxin oxidoreductase; 12, acetyl-CoA synthetase (ADP forming).

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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Tables

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TABLE 1

Glucose fermentation by : calculation of carbon recovery, O/R balance, and available hydrogen balance

Data in this column are taken from reference .

This value is the product of the number of carbons × the number in column [2]. Carbon recovered = 578/600 × 100 = 96.3%; O/R balance = 408/384 = 1.06; balance of available H = 2,400/2,264 = 1.06.

This value is the product of column [2] × column [4].

Calculation of available [H] is based on the following equations: CHO + 6HO → 24H + 6HO; CHO + 6HO → 20H + 4CO; CHO + 2HO → 8H+ 2CO; CHO + 7HO → 24H + 4CO; CHO + 5HO → 18H + 3CO.

This value is the product of column [2] × column [6].

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 2

Assay procedures for enzymes of the EMP pathway

Most reagents and enzymes described are readily available from various commercial sources, such as Sigma-Aldrich.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 3

Assay procedures for the enzymes of the ED pathway

BSA, bovine serum albumin; MES, morpholineethanesulfonic acid.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 4

Assay procedures for enzymes of the homolactate fermentation pathway

Bacterial culture is washed twice with ice-cold 50 mM KPO buffer (pH 6.5) containing 2 mM MgSO (KPM buffer), resuspended in 1/100 of culture volume of KPM buffer containing 20% (wt/vol) glycerol, and rapidly frozen in liquid nitrogen and kept at –80°C until use. After thawing,cells are washed once with KPM buffer; resuspended in 50 mM KPO buffer (pH 6.5) containing 12.5 mM NaF, 5 mM MgCl, and 2.5 mM dithiothreitol (DTT); and permeabilized. First, 2.5 μl of toluene/acetone (1:9, vol/vol) is added per 250 μl of cell suspension and vortexed for 5 min at 4°C. The cells are then centrifuged (150× at 4°C for 2 min) and the cell pellet is resuspended in the same buffer (optical density at 600 nm of 50) and again treated with toluene/acetone as described above ( ). After 5 min of vortexing, permeabilized cells are kept on ice.

The product, [C]hexose-phosphate, is separated from excess C-substrate by ion-exchange chromatography. The incubation mixtures are diluted with 0.5 ml of water and transferred to columns, 0.8 by 9 cm, of analytical-grade, chloride form, ion-exchange resin (AG l-X2, 50 to 100 mesh; Bio-Rad); the excess C-substrate is washed from each column with 15 ml of water; and the labeled product is eluted from the column with 6 ml of 1.0 M LiCl. Each elute is collected in a liquid scintillation spectrometer vial, and the C is counted after the addition of 15 ml of a mixture containing 333 ml of Triton X-100 (Packard Instrument Company), 666 ml of toluene, 5.5 g of 2,5-diphenyloxazole, and 125 mg of dimethyl-1,4-bis[2-(5-phenyloxazolyl)] benzene (dimethyl POPOP). The efficiency of the counting system is determined with C-standards (see chapter 17).

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 5

Assay procedures for enzymes of the heterolactate fermentation pathway

CHES, 2(-cyclohexylamino) ethanesulfonate; DTT, dithiothreitol.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 6

Assay procedures for enzymes of the heterofermentative bifidum pathway

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 7

Assay procedures for the enzymes of the butyrate-butanol-acetone-isopropanol fermentation in

DTT, dithiothreitol; KF, potassium fluoride; GSH, glutathione (reduced); FAD, flavin adenine dinucleotide; MOPS, morpholinepropanesulfonic acid.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 8

Assay procedures for the enzymes involved in ethanol and acetate fermentation by

FAD, flavin adenine dinucleotide; RS, regenerating system.

H is quantified by gas chromatography. Sample column: Length, 4 m; inner diameter, 2 mm; material, steel; temperatures, 50°C for both injection port and column;carrier gas, argon; detection, thermal conductivity detector; gas samples, 2 ml of 15-ml gas phase is injected with a gastight syringe.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 9a

Assay procedures for the enzymes of the mixed acid and butanediol fermentation

DTT, dithiothreitol; MOPS, morpholinepropanesulfonic acid; BSA, bovine serum albumin.

The sugar phosphates used are glucose-6-phosphate with [C]glucose, fructose-1-phosphate with [C]fructose, and mannitol-1-phosphate with [C]mannitol. The resin used for separating C-sugar from C-sugar-phosphate is AG1-X2, 50- to 100-mesh size, chloride form.

The tube is evacuated and filled with O-free argon, and 10 μl of 20 mM Fe(NH)(SO) is then added to the first arm. The tube is preincubated under argon for 30 min. The contents of the two arms are mixed and the tube is illuminated by a daylight lamp. The enzyme is fully activated usually within 30 min. The larger chloroplast fragments are centrifuged and the mixture is stored at 0°C. The low-molecular-weight components of the activated enzyme solution are removed by gel filtration under argon with a Sephadex G-25 column (1.6 by 20 cm) with an anaerobic buffer containing 50 mM MOPS (pH 7.6), 9 mM DTT, and 0.2 mM Fe(NH)(SO). Temperature, 4°C; flow rate, 2 ml/min. The protein fraction is collected into argon-flushed tubes and stored at 0°C.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 9b

Assay procedures for the enzymes of the mixed acid and butanediol fermentation

DTT, dithiothreitol; MOPS, morpholinepropanesulfonic acid; BSA, bovine serum albumin.

The sugar phosphates used are glucose-6-phosphate with [C]glucose, fructose-1-phosphate with [C]fructose, and mannitol-1-phosphate with [C]mannitol. The resin used for separating C-sugar from C-sugar-phosphate is AG1-X2, 50- to 100-mesh size, chloride form.

The tube is evacuated and filled with O-free argon, and 10 μl of 20 mM Fe(NH)(SO) is then added to the first arm. The tube is preincubated under argon for 30 min. The contents of the two arms are mixed and the tube is illuminated by a daylight lamp. The enzyme is fully activated usually within 30 min. The larger chloroplast fragments are centrifuged and the mixture is stored at 0°C. The low-molecular-weight components of the activated enzyme solution are removed by gel filtration under argon with a Sephadex G-25 column (1.6 by 20 cm) with an anaerobic buffer containing 50 mM MOPS (pH 7.6), 9 mM DTT, and 0.2 mM Fe(NH)(SO). Temperature, 4°C; flow rate, 2 ml/min. The protein fraction is collected into argon-flushed tubes and stored at 0°C.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 10

Assay procedures for the enzymes of the acrylate pathway for propionate production

BSA, bovine serum albumin.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 11

Assay procedures for the enzymes of the succinate pathway for propionate production

DBC [(5,6-dimethyl benzimidazolyl) Co-5′-deoxyadenosine cobamide] should be stored in a light-proof container. Benzimidazolylcobamide or adenylcobamide can also be used as coenzymes.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 12

Assay procedures for the enzymes of the bacterial acetate fermentation pathway

DTT, dithiothreitol.

All reagents are prepared anaerobically by boiling the water containing reagents and storing them under nitrogen. The assay is performed in nitrogen-filled, serum-stoppered anaerobic cuvettes and the enzyme is added via a syringe. NAD reduction is monitored by measuring the absorbance at 340 nm, and methyl viologen reduction is measured at 600 nm.

The reaction is initiated with the addition of acetyl-CoA and is allowed to proceed for 15 min at 55°C. The reaction is stopped by adding 0.04 ml of 4 M acetic acid, which drops the pH to 3.5. The solution is then made alkaline with the addition of 0.2 ml of 2 N NaOH to hydrolyze acetyl-CoA over a period of 6 h at room temperature. Carrier acetic acid (0.0032 mM) is added and the acetate is then isolated using chromatography on Celite before assaying for C.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 13

Phosphoryl donors for kinases of archaea

ADP-GK, ADP-glucokinase; ATP-HK, ATP-hexokinase.

In , ADP-GK and ADP-PFK activities are carried out by a single bifunctional enzyme.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22
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TABLE 14

Assay procedures for the enzymes of the acetate fermentation pathway in archaea

DHAP, dihydroxyacetone phosphate; EPPS, -(2-hydroxyethyl) piperazine-9-3-propanesulfonic acid; ACS, acetyl-CoA synthetase.

Citation: Meganathan R, Ranganathan Y, Reddy C. 2007. Carbohydrate Fermentations, p 558-585. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch22

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