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Applied and Environmental Microbiology, January 2000, p. 246-251, Vol. 66, No. 1
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 spore-forming
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 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 fermentative 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.
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 · 6H2O, 0.056 g/liter; KCl, 0.3 g/liter; CaCl2 · 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 · 4H2O, 1.5 mg/liter; ZnCl2, 0.07 mg/liter;
MnCl2 · 4H2O, 0.1 mg/liter; H3BO3, 0.006 mg/liter; CoCl2
· 6H2O, 0.19 mg/liter; CuCl2 · 2H2O, 0.002 mg/liter; NiCl2 · 6H2O, 0.024 mg/liter; Na2MoO4 · 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 · 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 (
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Elucidation of Enzymes in Fermentation Pathways
Used by Clostridium thermosuccinogenes Growing on
Inulin
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
(2
1) 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).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
= 6.2 mM
1 cm1) 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 ( = 1.450 mM1 cm1),
phosphotransacetylase activity was detected by acetyl coenzyme A (CoA)
formation at 233 nm ( = 4.44 mM1 cm1),
and pyruvate carboxylase activity was based on reduction of (5,5-dithiobis (2-nitrobenzoic acid) at 412 nm ( = 13.6 mM1 cm1) 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.
TABLE 1.
Enzyme assay conditions
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 µl 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).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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 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.
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(i) Lactate branch. Lactate is usually formed by the NADH-dependent 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 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 optimally 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).
|
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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).
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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).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* Corresponding author. Mailing address: 408 Driftmier Engineering Center, University of Georgia, Athens, GA 30602. Phone: (706) 542-0833. Fax: (706) 542-8806. E-mail: eiteman{at}bae.uga.edu.
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Abstract
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Materials and Methods
Results and Discussion
References
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