Elucidation of Enzymes in Fermentation Pathways

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for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. ... Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2000, p. 246–251 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 66, No. 1

Elucidation of Enzymes in Fermentation Pathways Used by Clostridium thermosuccinogenes Growing on Inulin JAYANTH SRIDHAR,1 MARK A. EITEMAN,1*

AND

JUERGEN W. WIEGEL2

Center for Molecular BioEngineering, Department of Biological and Agricultural Engineering,1 and Department of Microbiology,2 University of Georgia, Athens, Georgia 30602 Received 16 August 1999/Accepted 20 October 1999

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.

Clostridium thermosuccinogenes is a strictly anaerobic sporeforming gram-positive bacterium that can ferment inulin to succinate and acetate as major products and to lactate, ethanol, formate, hydrogen, and carbon dioxide as minor products (7). Inulin is a carbohydrate found in the roots or tubers of some plants, consisting of 20 to 30 fructose molecules connected to a terminal glucose residue by a ␤ (231) linkage. High concentrations of inulin are found in the roots of Jerusalem artichoke (80% [weight/dry weight]), chicory (75 % [wt/ wt]), and dahlia (72% [wt/wt]) (16). Succinic acid is a four-carbon aliphatic dicarboxylic acid having a pKa1 of 4.2 and a pKa2 of 5.6 which has applications in the manufacture of specialty chemicals and in agriculture, food, medicine, textiles, plating, and waste gas scrubbing (41). Industrially, succinic acid is currently produced by hydrogenation of maleic anhydride to succinic anhydride followed by hydration to succinic acid (5, 41). Succinic acid can be produced by many anaerobic microorganisms, usually near neutral pH (5). For example, one method to produce succinic acid microbially employs the strict anaerobe Anaerobiospirillum succiniciproducens (5, 10, 11, 24). Under optimal conditions, these bacteria produce succinic acid from glucose with a yield of 87% and a final concentration of 35 g/liter (11, 24). Recently, Guettler et al. (15) isolated the facultatively anaerobic gram-negative bacterium Actinobacillus sp. strain 130Z, which produced a final succinate concentration of 50 g/liter while growing on a complex medium. While several succinate-forming mesophilic bacteria have been isolated and their biochemical pathways have been elucidated (4, 12, 43), C. thermosuccinogenes is the only known

thermophilic succinate-forming anaerobic bacterium. The advantages of using thermophilic processes are that there is generally less risk of contamination and the processes are more rapid (1, 40, 43). A thermophilic process involving the fermentation of renewable inulin (which is water soluble at 58°C) to form succinic acid might be an attractive alternative to the existing chemical process for succinic acid production. Drent et al. (7) isolated four strains of C. thermosuccinogenes (DSM 5806 through DSM 5809) from fresh cow manure, beet pulp from the extraction column of a sugar refinery, soil immediately around Jerusalem artichoke tubers, and pond sediment. Two of the strains (DSM 5807 and DSM 5809) grow optimally at 58°C on inulin but at 70°C on fructose, while they differ in product formation and growth rate on either substrate. Interestingly, strain DSM 5807 produces the same fermentation products regardless of whether bacteria are fermenting fructose, glucose, or inulin. The presence of cell-bound inulinase activity was demonstrated in DSM 5807. Maximum inulinase activity was observed at 58°C and at pH 6.8 (7). In order to understand the regulation of metabolic carbon flux towards the different end products in C. thermosuccinogenes, the fermentative pathway (conversion of phosphoenolpyruvate [PEP] to the different end products) needs to be elucidated. Since all the strains produced identical products whether growing on inulin or glucose, the fermentative pathways used by C. thermosuccinogenes from these substrates would likely be identical. Metabolic flux analysis conducted on this pathway would provide insight into product formation and provide a basis for controlling fermentation process variables, such as temperature, pH, and redox potential, to alter the distribution of desired end products. Preliminary studies in our laboratory indicated that strain DSM 5809 has the highest final succinate concentration and cell growth among the four strains. Our objective in this study is to elucidate the fermen-

* Corresponding author. Mailing address: 408 Driftmier Engineering Center, University of Georgia, Athens, GA 30602. Phone: (706) 542-0833. Fax: (706) 542-8806. E-mail: [email protected] 246

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TABLE 1. Enzyme assay conditions Enzyme (reference)

Assay conditionsa

Glycolytic enzymes 1-Phosphofructokinase (37) ..................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 2 mM ATP, 5 mM MgSO4, 0.3 mM NADH, 50 mM KCl, 2 U of aldolase, 2 U of triose phosphate isomerase, 10 U of glycerophosphate dehydrogenase, 2 mM fructose 1-phosphate, cell extract 6-Phosphofructokinase (37) ..................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 2 mM ATP, 5 mM MgSO4, 0.3 mM NADH, 50 mM KCl, 2 U of aldolase, 2 U of triose phosphate isomerase, 10 U of glycerophosphate dehydrogenase, 2 mM fructose 6-phosphate, cell extract Lactate branch Lactate dehydrogenase (35)..................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 0.25 mM NADH, 10 mM pyruvate, 1 mM FDP, cell extract Succinate branch PEPC (26, 28).........................................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 0.15 mM NADH, 10 mM MgCl2, 25 mM NaHCO3, 1 U of MDH, 5 mM PEP, cell extract PEPCK (26) ............................................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 10 mM MgCl2, 10 mM ADP, 25 mM NaHCO3, 10 mM PEP, 2 U of MDH, 0.15 mM NADH, cell extract PEPCTrP (26).........................................................0.1 M K2HPO4 (pH 7.0), 2 mM PEP, 10 mM MgCl2, 30 mM NaHCO3, 0.1 mM CoCl2, 2 U of MDH, 0.15 mM NADH, cell extract Malate dehydrogenase (42)...................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 2 mM OAA, 0.3 mM NADH, cell extract Fumarase (6)...........................................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 15 mM L-malate, cell extract Fumarate reductase (26) .......................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 0.15 mM NADH, 5 mM fumarate, cell extract Acetate branch Phosphotransacetylase (26)...................................0.1 M Tris-HCl (pH 6.5), 1 mM CoA, 30 mM NH4Cl, 10 mM DTT, 2 mM acetyl phosphate, cell extract Acetate kinase (21) ................................................0.1 M Tris-HCl (pH 6.5), 3 mM MgCl2, 2 mM glucose, 0.5 mM NADP, 1 U of hexokinase, 1 U of glucose-6-phosphate dehydrogenase, 1 mM ADP, 4 mM acetyl phosphate, cell extract Ethanol branch Acetaldehyde dehydrogenase (21) .......................0.1 M Tris-HCl (pH 6.5), 1 mM NAD(P), 1 mM DTT, 0.1 mM CoA, 7 mM sodium arsenate, 10 ␮M acetaldehyde, 0.5 U of phosphotransacetylase, cell extract Alcohol dehydrogenase (31) .................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 0.3 mM NAD(P)H, 10 mM acetaldehyde, cell extract Formate branch Formate dehydrogenase (37) ................................0.1 M K2HPO4 (pH 7.0), 2 mM benzyl viologen, 5 mM formate, cell extract Pyruvate formate lyase (37) ..................................0.1 M Tris-HCl (pH 6.5), 0.2 mM CoA, 10 mM DTT, 1 mM NAD, 5 mM L-malate, 4 U of citrate synthase, 20 U of MDH, 50 mM pyruvate, cell extract Other enzymes Pyruvate kinase (26) ..............................................50 mM Tris-HCl (pH 6.5), 10 mM DTT, 10 mM MgCl2, 5 mM PEP, 5 mM ADP, 1 mM FDP, 10 U of LDH, 0.2 mM NADH, cell extract Pyruvate ferredoxin oxidoreductase (21) ............50 mM K2HPO4 (pH 7.0), 0.1 mM CoA, 20 mM DTT, 2 mM benzyl viologen, 5 mM pyruvate, cell extract Pyruvate decarboxylase (18) .................................0.1 M Tris-HCl (pH 6.5), 10 mM pyruvate, 0.4 mM NADH, 30 mM acetaldehyde, 1.8 U of alcohol dehydrogenase, cell extract Pyruvate carboxylase (30)......................................0.1 M Tris-HCl (pH 6.5), 10 mM DTT, 5 mM ATP, 10 mM MgCl2, 10 mM NaHCO3, 0.2 mM acetyl CoA, 0.1 mM DTNB, 1 U of citrate synthase, 10 mM pyruvate, cell extract Hydrogenase (21) ...................................................0.1 M Tris-HCl (pH 6.5), 2 mM benzyl viologen, 2 mM DTT, 1 atm of H2 gas, cell extract NAD(P)-dependent malic enzyme (23) ..............0.1 M Tris-HCl (pH 6.5), 2 mM malate, 0.5 mM NAD(P)H, 5 mM MnCl2, 20 mM NH4Cl, cell extract a

DTNB, 5,5-dithiobis(2-nitrobenzoic acid); LDH, lactate dehydrogenase; MDH, malate dehydrogenase.

tative enzymes in C. thermosuccinogenes DSM 5809. Since DSM 5809 was isolated from mesobiotic environments and grows optimally on inulin at higher temperatures, the effects of four temperatures (37, 47, 58, and 70°C) on the activities of different enzymes of the fermentative pathways in strain DSM 5809 were investigated. The observed enzyme activities were compared with results from batch fermentations of C. thermosuccinogenes growing on inulin at the respective temperatures. MATERIALS AND METHODS

C. thermosuccinogenes DSM 5809, obtained from the German Culture Collection, was routinely cultivated at 58°C with 5 g of chicory inulin (Fructafit IQ; Imperial Suiker Unie, Sugarland, Tex.)/liter in a modified basal medium prepared under

an atmosphere of 85% N2–15% CO2 and with the following composition (pH 7.2) (7): NaCl, 1.2 g/liter; MgCl2 䡠 6H2O, 0.056 g/liter; KCl, 0.3 g/liter; CaCl2 䡠 2H2O, 0.056 g/liter; NH4Cl, 0.27 g/liter; KH2PO4, 0.21 g/liter; Na2SO4, 0.1 g/liter; Na2HPO4, 0.2 g/liter; yeast extract, 1 g/liter; Casamino acids, 0.03 g/liter; FeCl2 䡠 4H2O, 1.5 mg/liter; ZnCl2, 0.07 mg/liter; MnCl2 䡠 4H2O, 0.1 mg/liter; H3BO3, 0.006 mg/liter; CoCl2 䡠 6H2O, 0.19 mg/liter; CuCl2 䡠 2H2O, 0.002 mg/liter; NiCl2 䡠 6H2O, 0.024 mg/liter; Na2MoO4 䡠 2H2O, 0.036 mg/liter; biotin, 0.02 mg/liter; folic acid, 0.02 mg/liter; pyridoxine-HCl, 0.1 mg/ liter; thiamine-HCl, 0.05 mg/liter; nicotinic acid, 0.05 mg/liter; calcium pantothenate, 0.05 mg/liter; vitamin B12, 0.001 mg/ liter; p-aminobenzoic acid, 0.05 mg/liter; lipoic acid, 0.05 mg/ liter; resazurin, 1 mg/liter; NaHCO3, 2.5 g/liter, and Na2S 䡠

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9H2O, 0.35 g/liter. For preparation of cell extracts, 100 ml of C. thermosuccinogenes DSM 5809 grown anaerobically was harvested when the cells were in late exponential phase (i.e., an optical density at 620 nm of 0.35 to 0.45). The dry cell matter correlation for DSM 5809 was 0.44 g of cells for an optical density at 620 nm of 1.0. After centrifugation (8,000 ⫻ g for 10 min), the pellet was washed with 0.1 M Tris HCl (pH 6.5) containing 10 mM dithiothreitol (DTT) and recentrifuged twice (8,000 ⫻ g for 10 min). The pellet was finally resuspended in 4 ml of 0.1 M Tris-HCl with 10 mM DTT and passed through a mini-French Press (SLM Aminco, Urbana, Ill.) at 20,000 lb/in2. The cell debris was centrifuged (40,000 ⫻ g for 60 min) to separate the membrane fraction (pellet) from the soluble cytosolic fraction (supernatant). Table 1 summarizes the assay conditions and original references. Most assays were based on the oxidation of NADH to NAD (ε ⫽ 6.2 mM⫺1 cm⫺1) under aerobic conditions in a cuvette with a path length of 1 cm. The activity of fumarase was measured by the formation of fumarate at 250 nm (ε ⫽ 1.450 mM⫺1 cm⫺1), phosphotransacetylase activity was detected by acetyl coenzyme A (CoA) formation at 233 nm (ε ⫽ 4.44 mM⫺1 cm⫺1), and pyruvate carboxylase activity was based on reduction of (5,5dithiobis (2-nitrobenzoic acid) at 412 nm (ε ⫽ 13.6 mM⫺1 cm⫺1) by the CoA liberated during the reaction. Anaerobic enzyme assays were carried out with an 80% N2–20% CO2 atmosphere according to the procedure outlined by Lamed and Zeikus (21). Enzyme activities were obtained from three replicates of at least two separate cell extract preparations at each temperature. The cell protein content was determined by a modified Lowry assay (kit P5656; Sigma Chemical Co., St. Louis, Mo.). One unit of enzyme activity was defined as the amount of enzyme that could convert a micromole of substrate into product per minute for each milligram of total cell protein. Fermentations from growing cultures were carried out in numerous 100-ml serum bottles with 5 g of inulin/liter with an initial atmosphere of 85% N2–15% CO2 at a pH of 7.2 The fermentations were terminated when the pH reached 6.5. Soluble fermentation products were analyzed by high-pressure liquid chromatography (8). The column was eluted with 4 mN H2SO4 at 60°C. Succinate, lactate, acetate, formate and ethanol were simultaneously detected by a differential refractive index detector (model 2410; Waters, Milford, Mass.). Hydrogen was analyzed by gas chromatography with a thermal conductivity detector (HP 5870; Hewlett-Packard, Palo Alto; Calif.). The carrier gas used was N2 at a flow rate of 100 ml/min, and the oven, injector, and detector temperatures were 120, 115, and 170°C, respectively. The quantity of inulin present was determined by hydrolyzing a 2.5-ml sample with 100 ␮l of 37% HCl for 30 min and assaying it for hexose units with dinitrosalicylic acid (27). The molecular weight of the cells was based on 26.0 g/mol, a value used previously for anaerobic bacteria (9). RESULTS AND DISCUSSION Determination of fermentation enzymes. C. thermosuccinogenes DSM 5809 breaks down inulin to fructose and glucose with an inulinase characterized by Drent et al. (7). Both DSM 5807 (7) and DSM 5809 (33) are known to utilize either fructose or glucose as a carbon source. Activities for the two enzymes 1-phosphofructokinase (catalyzing the conversion of fructose 1-phosphate to fructose 1,6-diphosphate [FDP]) and 6-phosphofructokinase (catalyzing the conversion of fructose 6-phosphate to FDP) were detected in cell extracts. Since FDP is a characteristic intermediate of the Embden-Meyerhof-Parnas (EMP) pathway (13), the presence of these two enzymes suggested that fructose and glucose are metabolized via the

APPL. ENVIRON. MICROBIOL.

FIG. 1. Fermentative pathways of C. thermosuccinogenes DSM 5809 with the following enzymes: (1) inulinase, (2) 6-phosphofructokinase, (3) 1-phosphofructokinase, (4) PEPC, (5) malate dehydrogenase, (6) fumarase, (7) fumarate reductase, (8) pyruvate kinase, (9) lactate dehydrogenase, (10) pyruvate formate lyase, (11) pyruvate ferredoxin oxidoreductase, (12) hydrogenase, (13) phosphotransacetylase, (14) acetaldehyde dehydrogenase, (15) alcohol dehydrogenase, and (16) acetate kinase.

EMP pathway for the conversion of inulin to pyruvate. For 13 of 19 possible enzymes (Table 1) in the assumed pathway for regeneration of NAD(P), substantial activities were obtained. Based on the measured activities, the pathway shown in Fig. 1 is proposed. For discussion, the proposed pathway is divided into five branches according to the final fermentation products: the lactate, succinate, acetate, ethanol, and formate branches. (i) Lactate branch. Lactate is usually formed by the NADHdependent reduction of pyruvate. Like other clostridial species that produce lactate (3, 35), C. thermosuccinogenes showed lactate dehydrogenase activity. The lactate dehydrogenase in C. thermosuccinogenes had an absolute requirement for FDP, unlike the enzymes in Clostridium acetobutylicum (3) and Clostridium thermohydrosulfuricum (35), where activity was observed in the absence of FDP. (ii) Succinate branch. Four steps for the conversion of PEP into succinate are proposed (Fig. 1). The first step involves the carboxylation of PEP to form oxaloacetate (OAA). Three enzymes are known that catalyze carboxylation of PEP: PEP carboxytransphosphorylase (PEPCTrP), PEP carboxylase (PEPC), and PEP carboxykinase (PEPCK) (36). No PEPCTrP activity was detected in C. thermosuccinogenes. This result agrees with the observation that PEPCTrP has been found only in propionic acid bacteria and some protozoans (36). Significant PEPC activity, however, was detected. Previously, PEPC has been detected in Escherichia coli, which produces succinate

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TABLE 2. Effect of temperature on enzyme activities Enzyme

6-Phosphofructokinase 1-Phosphofructokinase PEPC Malate dehydrogenase Fumarase Fumarate reductase Pyruvate kinase Lactate dehydrogenase Pyruvate formate lysate Pyruvate ferredoxin oxidoreductase Hydrogenase Phosphotransacetylase Acetaldehyde dehydrogenase Alcohol dehydrogenase Acetate kinase a

Activity ata: 37°C

47°C

58°C

70°C

0.11 (0.01) 0.07 (0.01) 0.16 (0.01) 0.14 (0.06) 0.50 (0.04) 0.06 (0.01) 0.17 (0.02) 0.30 (0.02) 0.21 (0.06) 0.21 (0.02)

— — 0.41 (0.03) 0.28 (0.06) 0.69 (0.04) 0.11 (0.01) 0.67 (0.02) 0.49 (0.03) — —

— — 0.77 (0.05) 0.43 (0.08) 0.95 (0.11) 0.15 (0.01) 1.20 (0.05) 1.05 (0.06) — —

— — 1.04 (0.13) 0.65 (0.04) 1.56 (0.12) 0.27 (0.02) 2.02 (0.29) 2.16 (0.13) — —

0.06 (0.01) 0.97 (0.05) 0.07 (0.00)

— 1.28 (0.04) —

— 1.61 (0.10) —

— 2.54 (0.29) —

0.13 (0.01) 0.29 (0.01)

0.26 (0.02) 0.48 (0.02)

0.42 (0.03) 0.59 (0.04)

0.43 (0.03) ND

In micromoles per minute per milligram of protein. ND, not detected; —, not determined. The standard errors of the measurements are in parentheses.

as a minor product (2). Like that in E. coli (28), PEPC in C. thermosuccinogenes was found to be activated by 10 mM FDP by a factor of 2.5. In contrast, PEPCK has been detected in succinate-producing A. succiniciproducens (31). The conversion of PEP to OAA by PEPCK is reversible (36), and therefore enzyme activity was examined in both directions. Unlike PEPC, PEPCK has an absolute requirement for nucleotide diphosphates, and this difference was used to distinguish the two enzymes. Since addition of ADP and appropriate metal ions did not increase conversion, PEPCK appears not to be present in C. thermosuccinogenes. Malate dehydrogenase catalyzes the conversion of OAA to malate in other succinate-forming anaerobes (31), and this enzyme was detected in the cell extracts. Fumarase catalyzes the conversion of malate to fumarate in other anaerobes (6, 31), and fumarase activity was also detected in C. thermosuccinogenes cell extracts. The fourth enzyme in the sequence to succinate, fumarate reductase, was detected in the cell extracts. Dorn et al. (6) also observed fumarate reductase activity in the cytosolic fraction of Clostridium formicoaceticum. As in the case of C. formicoaceticum, fumarate reductase in C. thermosuccinogenes may be linked to energy-deriving electron transport phosphorylation (13). (iii) Acetate branch. The acetate-forming branch is usually energy conserving and linked to the formation of one ATP molecule. The first enzyme in this branch, phosphotransacetylase, was detected by the method outlined by Klotzsch (20), who demonstrated the presence of the enzyme in Clostridium kluyveri. Acetate kinase, which catalyzes the conversion of acetyl phosphate to acetate with the formation of one ATP, was also detected. (iv) Ethanol branch. NADH-dependent alcohol dehydrogenase was not detected under either aerobic or anaerobic conditions. Lamed and Zeikus (22) showed that the NADH-dependent alcohol dehydrogenase was more oxygen sensitive than NADPH-dependent alcohol dehydrogenase. By using the assay of Lamed and Zeikus (22), NADPH-dependent alcohol dehydrogenase activity was detected in the cell extracts under aerobic conditions. NADH-dependent CoA-acetylating acetaldehyde dehydrogenase was detected only under anaerobic conditions.

(v) Formate branch. By the assay of Van der Werf et al. (37), formate dehydrogenase was not observed in DSM 5809. However, pyruvate formate lyase activity was detected in the cell extract only under anaerobic conditions. (vi) Other enzymes forming or utilizing pyruvate. The key enzyme involved in pyruvate formation from PEP, pyruvate kinase, was detected in the cell extract. Ammonium ions stimulated pyruvate kinase activity. The presence of several pyruvate-catabolizing enzymes was investigated. While pyruvate ferredoxin oxidoreductase was detected under anaerobic conditions, neither pyruvate decarboxylase, an enzyme not found so far in clostridia, nor pyruvate carboxylase was detected. Interestingly, the presence of both pyruvate formate lyase and pyruvate ferredoxin oxidoreductase results in redundancy in the formation of acetyl CoA from pyruvate, an observation previously made for C. kluyveri (13). Hydrogenase activity, which is proposed to be linked to oxidation of the clostridial ferredoxin, was also detected in the cell extract. Neither NAD-dependent malic enzyme nor NADP-dependent malic enzyme was detected in the cell extracts. Effect of temperature on enzyme activities. Drent et al. (7) demonstrated that C. thermosuccinogenes DSM 5807 grew opTABLE 3. Eas of enzymes in C. thermosuccinogenes and other organismsa Enzyme

Temp range (°C)

Ea (kcal/mol)

nb

PEPC Malate dehydrogenase Fumarase Fumarate reductase Pyruvate kinase Lactate dehydrogenase Phosphotransacetylase Alcohol dehydrogenase Acetate kinase

37–58 37–70 37–70 37–70 37–58 37–70 37–58 37–58 37–58

21.5 (1.3) 15.0 (1.3) 10.1 (1.2) 13.2 (1.1) 25.6 (2.2) 18.5 (1.0) 7.0 (1.0) 16.0 (1.5) 9.8 (1.1)

27 30 27 27 18 30 21 30 18

Reported Ea (reference)

13–15 (25) 8.7 (17) 14–20 (29) 16.0 (39) 8.2 (19) 5.0 (38)

a Eas in C. thermosuccinogenes were calculated from activities shown in Table 2. Eas for other organisms were obtained from the literature. The standard errors of calculated Eas are indicated in parentheses. b Number of measurements used in each Arrhenius plot.

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TABLE 4. Molar yields of products during fermentation of inulin at 37, 47, 58, and 70°C Parameter

Product yield (mol product/mol hexose unit utilized) Succinate Acetate Lactate Formate Ethanol Hydrogen Carbon dioxideb Biomass yield (g/mol hexose unit) Carbon recovery O/R ratio a b

Valuea 37°C

47°C

58°C

70°C

0.16 (0.01) 0.48 (0.02) 0.01 (0.00) 0.45 (0.01) 1.03 (0.08) 0.10 (0.00) 0.90 21.7 (0.5) 0.98 1.12

0.16 (0.01) 0.60 (0.03) 0.01 (0.00) 0.60 (0.03) 0.72 (0.03) 0.14 (0.00) 0.56 27.2 (0.2) 0.92 1.19

0.23 (0.01) 0.79 (0.02) 0.10 (0.01) 0.65 (0.05) 0.45 (0.04) 0.15 (0.01) 0.36 29.5 (1.0) 0.97 1.52

0.20 (0.01) 0.03 (0.00) 0.19 (0.01) 0.50 (0.02) 1.16 (0.05) 0.25 (0.01) 0.49 19.2 (0.2) 0.91 0.65

Standard errors of measurements are in parentheses. Molar carbon dioxide yield calculated by the following equation: carbon dioxide yield ⫽ acetate yield ⫹ ethanol yield ⫺ succinate yield ⫺ formate yield.

timally on inulin at 58°C and on fructose at 70°C. Since DSM 5809 has a different product and growth profile than DSM 5807, the effects of four temperatures (37, 47, 58, and 70°C) on the enzyme activities of the nine oxygen-insensitive enzymes from DSM 5809 were investigated (Table 2). Lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase activities consistently increased between 37 and 70°C, indicating for each a higher temperature for optimal activity than the optimal growth temperature on inulin (58°C). PEPC, pyruvate kinase, and phosphotransacetylase activities showed exponential increases from 37 to 58°C but increased at a lower rate between 58 and 70°C. Alcohol dehydrogenase activity remained unchanged between 58 and 70°C. Acetate kinase activity was not detected at 70°C, indicating inactivation at this high temperature, an observation which has also been made with Clostridium thermocellum, Thermoanaerobacterium brockii, and Clostridium thermoaceticum (22, 32). Due to the enzyme’s involvement in energy metabolism, inactivation of acetate kinase might be responsible for the decrease in cell growth of DSM 5809 at 70°C. The Arrhenius plot profiles (42) were linear between 37 and 58°C or 37 and 70°C and were used to calculate the Arrhenius energies (Eas) of these enzymes in crude cell extracts (Table 3). Values for activation energies for clostridial fermentative enzymes are scarce in the literature. Tolman et al. (34) reported that activation energies of early glycolytic enzymes in C. thermocellum were around 25 kcal/mol. A few activation energies for these enzymes in other microorganisms have also been reported. These published values for Ea are in the same range as those obtained for C. thermosuccinogenes (Table 3). Effect of temperature on product formation. Serum bottle fermentations were carried out on 5 g of inulin/liter at 37, 47, 58, and 70°C, and the product yields were calculated (Table 4). The observed product yields support the enzyme activity measurements. Acetate kinase inactivation at 70°C corresponds with the low acetate yield at this temperature compared to those at the three lower temperatures. The yields of acetate and succinate were highest at 58°C (0.79 and 0.23 mol of product/mol hexose unit, respectively), which corresponds to the optimum growth temperature of the organism and also to the maximum biomass yield (29.5 g/mol). The yield of lactate increased with increasing temperature, an observation which is in agreement with the particularly high Ea for lactate dehydrogenase (and hence proportionally greater activity at higher temperatures). The yield of hydrogen also increased with increasing temperature. The yield of ethanol decreased from 37 to 58°C but increased between 58 and 70°C. This increase

might be associated with a cell’s inability to produce significant acetate at 70°C, and ethanol is another product that can be formed from generated acetyl CoA. No trend was apparent with the yield of formate at the various temperatures. Of course, final product distributions are related not only to in vitro enzyme activities but also to the balance of oxidized and reduced cofactors. Carbon balances and ratios of oxidized products to reduced products (O/R ratios) were calculated by the technique of Gottschalk (13). The carbon recoveries were between 0.91 and 0.98. The quantity of CO2 was estimated by the following equation: CO2 ⫽ acetate ⫹ ethanol ⫺ formate ⫺ succinate. This takes into account CO2 produced by the pyruvate ferredoxin oxidoreductase reaction and CO2 utilized in the CO2 fixation step while succinate is formed (14, 37). The O/R ratios at 37, 47, and 58°C were 1.12, 1.19, and 1.52, respectively. It is not clear why these ratios are greater than 1.0, particularly in light of the carbon balances, which were nearly 1.0. However, the O/R ratio decreased sharply to only 0.65 at 70°C. This decreased O/R ratio reflects the increased formation of lactate, ethanol, and hydrogen and the decreased formation of acetate in comparison to those at the other temperatures. One possible explanation for this observation is that some other medium components (e.g., yeast extract and Casamino acids) might increasingly serve as additional electron donors at the more elevated temperature of 70°C. The increased hydrogen production relative to acetate production from pyruvate at this highest temperature is in agreement with the assumption of additional, unrecognized electron donors. A similar decrease in the O/R ratio from 0.84 to 0.50 was observed with Actinobacillus sp. when that organism was grown in a hydrogen atmosphere, an observation which similarly reflected increased succinate formation and decreased formate production (37). ACKNOWLEDGMENTS We thank the Georgia Experiment Stations for financial support to M.A.E. We thank Imperial Suiker Unie for providing the chicory inulin used in the study. REFERENCES 1. Canganella, F., and J. Wiegel. 1993. The potential of thermophilic clostridia in biotechnology, p. 391–429. In D. R. Woods, (ed.), The clostridia and biotechnology. Butterworths Publications, Stoneham, Mass. 2. Clark, D. P. 1989. The fermentation pathways of Escherichia coli FEMS Microbiol. Rev. 63:223–234. 3. Contag, P. R., M. G. Williams, and P. Rogers. 1990. Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in

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