JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 212-217
Vol. 172, No. 1
0021-9193/90/010212-06$02.00/0 Copyright C) 1990, American Society for Microbiology
Biotransformations of Carboxylated Aromatic Compounds by the Acetogen Clostridium thermoaceticum: Generation of GrowthSupportive CO2 Equivalents under C02-Limited Conditions TSUNGDA HSU, STEVEN L. DANIEL, MARY F. LUX, AND HAROLD L. DRAKE* Microbial Physiology Laboratories, Department of Biology, The University of Mississippi, University, Mississippi 38677 Received 31 July 1989/Accepted 2 October 1989
Clostridium thermoaceticum ATCC 39073 converted vanillate to catechol. Although carboxylated aromatic compounds which did not contain methoxyl groups were not by themselves growth supportive, protocatechuate and p-hydroxybenzoate (nonmethoxylated aromatic compounds) were converted to catechol and phenol, respectively, during carbon monoxide-dependent growth. Syringate is not subject to decarboxylation by C. thermoaceticum (Z. Wu, S. L. Daniel, and H. L. Drake, J. Bacteriol. 170:5705-5708, 1988), and sustained growth at the expense of syringate-derived methoxyl groups was dependent on supplemental CO2. In contrast, vanillate was growth supportive in the absence of supplemental C02, and 14CO2 was the major 14C-labeled product during [carboxyl-"4C]vanillate-dependent growth. Furthermore, the decarboxylation of protocatechuate and p-hydroxybenzoate supported methanol- and 1,2,3-trimethoxybenzene-dependent growth (CO2 is required for growth at the expense of these substrates) when supplemental CO2 was depleted from the growth medium, and the decarboxylation of protocatechuate was concomitant with improved cell yields of methanol cultures. These findings demonstrate that (i) C. thermoaceticum is competent in the decarboxylation of certain aromatic compounds and (ii) under certain conditions, decarboxylation may be integrated to the flow of carbon and energy during acetogenesis.
Acetogenic bacteria utilize a unique form of autotrophic metabolism termed acetogenesis. Some of the key features of this autotrophic process have been elucidated, and the pathway utilized for the autotrophic synthesis of acetate and biomass has been termed the acetyl coenzyme A (acetylCoA) (Wood) pathway (21, 37, 56). Recent studies have shown that various aromatic compounds are growth-supportive substrates for acetogenic bacteria (3, 8, 10, 12, 13, 18-20, 33, 51, 57) and may thus be important to other microorganisms which can serve as syntrophic partners of acetogens (15, 28, 32, 39, 41, 47, 52a, 54, 58). The natural habitats of acetogens range from anaerobic sediments to the gastrointestinal tracts of humans and other animals, and lignin-derived aromatic compounds probably constitute important naturally occurring substrates in these environments (7, 8, 16, 22, 26, 28, 34, 36, 38, 43, 53, 55). Despite this recent evidence implicating acetogens as an important biological group in the anaerobic breakdown of aromatic compounds, most attention to date has centered on the use of aromatic methoxyl and acrylate groups during acetogenesis, and metabolic potentials for the use of other aromatic substituents remain poorly defined. Clostridium thermoaceticum (17) has been the classic acetogen used to elucidate many of the biochemical features central to acetogenesis (37, 56). For methoxy-based acetogenesis, both vanillate and syringate are growth-supportive substrates for this acetogen (10). Syringate is sequentially 0-demethylated to 5-hydroxyvanillate and gallate, and an inducible carbon monoxide-dependent 0-demethylating enzyme system functions in concert with acetyl-CoA synthetase (CO dehydrogenase) during methoxyl-coupled acetogenesis (57). Since gallate is the only aromatic product detected from syringate with C. thermoaceticum (57), aromatic carboxyl groups appeared to be nonreactive with this *
acetogen. However, as outlined in the following study, the carboxyl groups of other aromatic compounds are subject to decarboxylation by C. thermoaceticum and can serve as growth-supportive CO2 equivalents under C02-limited conditions. (Portions of this work have been presented at the 89th Annual Meeting of the American Society for Microbiology, New Orleans, La., 14 to 18 May 1989.) MATERIALS AND METHODS Cultivation of C. thermoaceticum. C. thermoaceticum ATCC 39073 (17) was cultivated in either undefined or defined medium at 55°C in crimp-sealed culture tubes (18 by 150 mm; Bellco series 2048; Bellco Glass Inc., Vineland, N.J.). Undefined medium contained (in milligrams per liter): NaHCO3, 5,000; KH2PO4, 500; NaCl, 400; NH4Cl, 400; MgCl 6H20, 330; CaCl2. 2H20, 50; resazurin, 1; yeast extract, 1,000; nicotinic acid, 0.25; cyanocobalamin, 0.25; p-aminobenzoic acid, 0.25; calcium D-pantothenate, 0.25; thiamine hydrochloride, 0.25; riboflavin, 0.25; lipoic acid, 0.15; folic acid, 0.1; biotin, 0.1; pyridoxal hydrochloride, 0.05; sodium nitrilotriacetate, 7.5; MnSO4. H20, 2.5; FeSO4- 7H20, 0.5; Co(NO3)2 6H20, 0.5; ZnCl2, 0.5; NiCl2 *6H20, 0.25; CuS04 5H20, 0.05; AIK(S04)2 12H20 0.05; H3BO3, 0.05; and Na2MoO4 2H20, 0.05. Defined medium was undefined medium without yeast extract. Sodium sulfide-cysteine reducer (46) was added after the media were boiled and cooled under C02; media were subsequently dispensed (7 ml per tube) under C02, and the tubes were crimp sealed and autoclaved. Glucose and methanol were added at concentrations of 10 and 60 mM, respectively, before autoclaving. For cultivation at the expense of CO, culture tubes were pressurized at room temperature to 70 kPa (10 lb/in2) over atmospheric pressure (171.3 kPa final total pressure) with 100% CO prior to incubation. Growth was initiated by injecting 0.5 ml of inoculum per culture -
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Corresponding author. 212
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tube. The total volume of the culture tubes approximated 28 ml, and the ratio of gas phase to liquid phase approximated 3:1. Stock solutions of vanillate and syringate (383.5 mM) were prepared in distilled and deionized water titrated with 5 N NaOH until dissolved. Unless otherwise indicated, stock solutions of all other aromatic compounds (383.5 mM) were prepared in 80% ethanol (the resulting ethanol concentration in culture media approximated 1% and did not significantly influence growth). Stock solutions of aromatic compounds were filter sterilized and added via syringe to sterile tubes of medium to the concentrations indicated. The aromatic compounds studied were stable under the conditions used; transformations were not observed in the absence of cells. In this study, no distinction is made between the acid and dissociated ionic forms of the aromatic carboxylic acids. Carbon dioxide-free medium was medium in which the NaHCO3 and KH2PO4 were replaced with Na2HPO4 (5.68 g/liter) and NaH2PO4 (7.2 g/liter). The initial gas phase of the C02-free medium was 100% N2 unless otherwise indicated. The initial pH of all media at the time of inoculation approximated 6.4 to 6.5. Cultivation of C. formicoaceticum and C. aceticum. C. formicoaceticum ATCC 27076 (2) and C. aceticum DSM 1496 (1, 6, 52) were cultivated at 37°C in undefined medium in which the NaHCO3 concentration was modified to 6 g/liter and Na2CO3 was added to a final concentration of 12 g/liter. Fructose and methanol, when added, were used at initial concentrations of 10 and 60 mM, respectively. For C02-free undefined medium, carbonates were not included, and the following phosphates were added (in milligrams per liter): Na2HPO4, 680; Na3PO4. 12H20, 60. The initial gas phase of C02-free media was 100% N2. The initial pH of these media at the time of inoculation approximated 7.8.
Measurement of '4C-labeled products from [carboxyl-'4C] vanillate. Cells were cultivated in undefined medium containing 5 mM [carboxyl-14C]vanillate (approximately 2.7 x 104 dpm/Lmol). Following growth, a sample of culture medium was removed for analysis of 14C in acetate and biomass. For analysis of 14CO2, the culture tube was connected to duplicate 1 N KOH CO2 traps and acidified with 0.75 ml of 2 N H2SO4. The gas phase of the culture tube was flushed through the traps for 5 min with a steady stream of N2, and the trapped 14C02 was quantitated by liquid scintillation in Safety Solve (Research Products International Corp., Mount Prospect, Ill.) with a Beckmann Instruments, Inc. (Fullerton, Calif.) LS 6800 liquid scintillation counter. 14C-labeled cells were harvested by microfiltration, washed four times, and then analyzed for 14C by liquid scintillation as described previously for the analysis of 63Ni-labeled biomass (58). Analytical methods. Aromatic compounds were obtained from Sigma Chemical Co. (St. Louis, Mo.) and Aldrich Chemical Co., Inc. (Milwaukee, Wis.). [Carboxyl-14C]vanillate was from Research Products International Corporation. Growth was monitored at 660 nm with a Spectronic 501 spectrophotometer (Bausch & Lomb, Inc., Rochester, N.Y.), and cell dry weights were determined as described previously (45). None of the compounds studied influenced the turbidity (A660) of culture media, and a linear relationship was shown to exist between biomass and absorbance. Aromatic compounds were analyzed by high-pressure liquid chromatography with a Hewlett-Packard Co. (Palo Alto, Calif.) 1090L high-performance liquid chromatograph equipped with a Bio-Rad Laboratories (Richmond, Calif.) fermentation monitoring column and a Spectra-Physics (Bedford, Mass.) 4290 integrator; the column temperature
Cultivation Time (h) FIG. 1. Conversion of vanillate (V) to catechol
(C) by C. thermoaceticum. Undefined culture medium contained 5 mM vanillate; inoculum was from an undefined vanillate medium culture. P, Protocatechuate.
600C, the mobile phase was 0.01 N H2SO4 at 0.8 ml/min, the sample size was 10 ,ul, and the detector was at 210 nm. Cells were removed by microcentrifugation and microfiltration prior to chromatographic analysis of fermentation broths. The authenticity of aromatic compounds was verified by UV absorption spectral analysis with a Gilford Systems (Oberlin, Ohio) 2600 recording spectrophotometer.
was
RESULTS
Growth-dependent transformations of carboxylated aromatic compounds. During vanillate-dependent growth, vanillate was converted to catechol (Fig. 1). Protocatechuate was a transient product, and catechol was the sole aromatic end product detected upon completion of growth under these
conditions. Given the transient appearance of protocatechuate during vanillate-dependent growth, the capacity to convert protocatechuate to catechol was investigated by using CO as the energy source for growth. Although protocatechuate did not support the growth of C. thermoaceticum in the absence of additional energy sources (unpublished data), protocatechuate was rapidly converted to catechol during CO-dependent
Ec 0 0 -
E 1-
in
la
C
0l.E33
0
0 C
n 4)
0
U
LI.
.5
U
E0
o
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Cultivation Time (h) FIG. 2. Conversion of protocatechuate (P) to catechol (C) during of C. thermoaceticum. Undefined culture CO-dependent growth medium contained 5 mM protocatechuate and a CO-CO2 (40:60 at 140 kPa over atmospheric pressure) gas phase. Inoculum was from an undefined CO medium culture.
214
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HSU ET AL.
TABLE 1. Distribution of '4C from [carboxyl-'4C]vanillate after vanillate-dependent growth by C. thermoaceticum Distribution of 14C (dpm) C 0.18 0* (0 (0
0.1 2 .0 L_ 0
Q06
0 0
125 250 0 125 250 Cultivation Time (h) FIG. 3. Effects of CO2 on syringate-dependent (A) and vanillatedependent (B) growth of C. thermoaceticum. The C02-free undefined culture medium was supplemented as indicated: 0, aromatic compound plus 70 kPa of CO2 over pressure; 0, aromatic compound without supplemental C02; O, 70 kPa of CO2 over pressure without aromatic compound; *, no supplements. (A) Inoculum was from a syringate-CO2 undefined medium culture. (B) Inoculum was from a C02-free undefined vanillate medium culture (maintained for four
Expt
Initial 14C (dpm)
1st CO2 trap
2nd CO2 Biomass trap
Supernatanta
% of
initial dpm
recovered
982,388 502,136 107,456 3,115 152,179 78 978,938 279,4% 63,664 3,126 52 161,667 100 3b 1,058,115 16,560 13,800 NAC 1,027,686 a 14C remaining in the supernatant fluid after cells were removed from
1 2
culture medium by microcentrifugation. b Uninoculated culture medium (control). NA, Not applicable.
sparging and to incomplete trapping of CO2 in the two KOH traps. Based on these findings, we reasoned that the carboxyl group of certain aromatic compounds might serve as a CO2 equivalent during acetogenesis. Methanol and 1,2,3-trimethoxybenzene cultures, both of which are dependent upon supplemental CO2 for sustained growth (unpublished data), were used to test this hypothesis. As depicted in Fig. 4, cell + 40 ,mol
CO2 + 40 umol P
transfers).
growth (Fig. 2). The overall specific activity for the transformation of protocatechuate to catechol approximated 7.0 nmol of protocatechuate converted per min per mg (dry weight) of cells. Protocatechuate was also converted to catechol during growth at the expense of methanol or glucose (data not shown). In addition, p-hydroxybenzoate was converted in near stoichiometry to phenol in similar growth studies; as with protocatechuate, p-hydroxybenzoate did not support the growth of C. thermoaceticum in the absence of additional energy sources (data not shown). Benzoate, o-hydroxybenzoate, and m-hydroxybenzoate were not transformed (data not shown). Capacity of other acetogens to decarboxylate aromatic compounds. In contrast to C. thermoaceticum, both C. formicoaceticum and C. aceticum converted vanillate to protocatechuate rather than catechol (data not shown). Furthermore, neither C. formicoaceticum nor C. aceticum was capable of transforming protocatechuate or p-hydroxybenzoate when cultivated at the expense of fructose. Function of aromatic carboxyl groups: origin of growthsupportive CO2 equivalents. As noted above, C. thermoaceticum does not decarboxylate syringate (57). Significantly, only marginal growth was obtained with syringate in the absence of supplemental CO2 (Fig. 3A). In contrast, vanillate supported substantial growth without supplemental CO2 (Fig. 3B). In CO2-free medium, vanillate was also converted in near stoichiometry to catechol (data not shown; product profile similar to that illustrated in Fig. 1). Collectively, these results suggested that vanillate was subject to decarboxylation. This possibility was confirmed by growth studies with [carboxyl-'4C]vanillate (Table 1). Under standard growth conditions in undefined vanillate medium, "4CO2 was the predominant 14C-labeled product detected after completion of growth. The low recovery of 14C may have been due to leakage of gas during growth and
c '-
0
Q3 B
0 L-
p
Cultivation Time (h) FIG. 4. Effects of CO2 and protocatechuate (P) on methanolsupported growth of C. thermoaceticum. (A) C02-free undefined medium with 60 mM methanol (inoculum was from undefined methanol-protocatechuate medium culture). (B) C02-free defined medium with 60 mM methanol (inoculum was from defined methanol-protocatechuate medium culture). Amounts of protocatechuate and CO2 are per culture tube; the protocatechuate stock solution was the salt form.
VOL. 172, 1990 -
Ec
USE OF CARBOXYLATED AROMATICS BY C. THERMOACETICUM
U.2u A
215
TABLE 2. Protocatechuate-dependent stimulation of C. thermoaceticum during growth at the expense of methanol
0
(0
Protocatechuate concn' (mM)
Acetate
0
Cu
0 1 10 10C
34.1 35.3 34.6 16.2
0.15)
0~
u 0.10 O
10
20
Cultivation Time (h) FIG. 5. Effects of C02, protocatechuate, and p-hydroxybenzoate on trimethoxybenzene-supported growth of C. thermoaceticum. The C02-free undefined culture medium was supplemented as indicated: O, 1,2,3-trimethoxybenzene (5 mM) pIus C02 (70 kPa over pressure); A, 1,2,3-trimethoxybenzene plus protocatechuate (5 mM); O, 1,2,3-trimethoxybenzene plus p-hydroxybenzoate (5 mM); *, 1 ,2,3-trimethoxybenzene; *, protocatechuate or p-hydroxybenzoate. Inoculum was from an undefined 1,2,3-trimethoxybenzene medium culture.
yields of methanol-protocatechuate-grown cultures were nearly identical to those of methanol-CO2-grown cultures when protocatechuate was added at concentrations equivalent to those of C02. The capacity for protocatechuate to replace C02 was observed with both undefined and defined cultures, demonstrating that components in yeast extract were not responsible for the protocatechuate-dependent effects. Catechol was the only aromatic product detected from the methanol-protocatechuate cultures. Furthermore, both protocatechuate and p-hydroxybenzoate served as C02 replacements during 1,2,3-trimethoxybenzene-dependent growth of C. thermoaceticum (Fig. 5). In contrast, vanillate-dependent growth of C. formicoaceticum did not occur in the absence of supplemental CO2 (data not shown). In addition, supplemental C02 was required for the methanol-dependent growth of C. formicoaceticum (6) and could not be replaced by p-hydroxybenzoate (data not shown). Effect of decarboxylation of protocatechuate on methanol dependent cell yields. Since C. thermoaceticum is homoacetogenic, the synthesis of biomass is obligately coupled to acetogenesis, i.e., the capacity to synthesize biomass is strictly dependent upon the cell's ability to conserve energy during the formation of acetate. Therefore, the ratio of acetate formed per unit of biomass synthesized can be taken as a direct reflection of cell energetics (29, 45, 51). In this regard, the decarboxylation of protocatechuate to catechol
(MM)
Acetate/
1.18 1.32 1.51
28.9 26.8 22.9 24.0
0.67
ratiob
a Undefined methanol (60 mM) culture medium was supplemented with protocatechuate as indicated. Inoculum was from a methanol-protocatechuate medium culture. Protocatechuate was converted stoichiometrically to catechol. b Acetate formed (millimolar) per gram (dry weight) of biomass per liter of medium. 'C02-free undefined methanol (60 mM) culture medium.
DISCUSSION In the present study, C. thermoaceticum was shown to be competent in decarboxylating certain aromatic compounds, and this metabolic potential was found to replace CO2 during methanol- and 1,2,3-trimethoxybenzene-dependent growth. This is the first report correlating the growth of an acetogen with the utilization of an aromatic carboxyl group. That vanillate is growth supportive in the absence of supplemental CO2 corroborates such an essential role for aromatic carboxyl groups under C02-limited conditions. C. formicoaceticum and C. aceticum did not display this metabolic potential, indicating that the use of aromatic carboxyl groups is not characteristic of all acetogenic bacteria. The present findings suggest that decarboxylation of aromatic compounds by C. thermoaceticum yields CO2 equivalents which can subsequently be utilized in the acetyl-CoA pathway (Fig. 6). CO2 plays essential roles in the bioenergetics and carbon flow of acetogenic bacteria (21, 37, 56), and how tightly coupled decarboxylation is to acetogenesis is not clear. Although the aromatic carboxyl group gave rise to CO2 under CO2-enriched conditions, decarboxylation may be more tightly coupled to the acetyl-CoA pathway when CO2 is limited (i.e., utilized directly as a one-carbon unit without being released from the cell as free CO2). Carbon monoxide exerts a strong influence on the flow of CO2-derived carbon during acetogenesis by C. thermoaceticum (40), and the relative concentration of exogenous CO2 is critical to the growth efficiency of the closely related acetogen Clostridium thermoautotrophicum (46). Thus, growth conditions may dictate whether aromatic compound-derived CO2H
C0C°2 CO
OH
H20
2H
(P)
/
was concomitant with a decrease in the acetate to biomass
ratio during methanol-coupled growth (Table 2). The amount of acetate formed remained relatively constant in the presence or absence of protocatechuate with CO2-enriched methanol medium, but the capacity to synthesize biomass increased coincident with decarboxylation. The acetateto-biomass ratio of CO2-free protocatechuate-methanol cultures was also less than that of CO2-enriched methanol cultures (Table 2). Protocatechuate was stoichiometrically converted to catechol, and the synthesis of biomass carbon from protocatechuate would therefore not account for the observed increase in biomass formation.
Biomass formed (g [dry wt]Aiter)
C
I1OH OH (C)
J
HC02H
THF2 CH3-THF THF
HSCoA
CH3-CO-SCoA (acety l-CoA)
FIG. 6. Hypothetical pathway for the integration of aromatic carboxyl groups into the acetyl-CoA pathway. Abbreviations: P, protocatechuate; C, catechol; THF, tetrahydrofolate; HSCoA, coenzyme A.
216
HSU ET AL.
"CO2" equivalents enter the carbonyl or methyl carbon of acetyl-CoA. Likewise, the enzyme(s) responsible for decarboxylation and integration of these CO2 equivalents into the acetyl-CoA pathway may be subject to regulation by available substrates. The 0-demethylating enzyme system is induced by utilizable methoxylated aromatic compounds but repressed by glucose (57). Certain decarboxylation reactions yield energy gains under anaerobic conditions (4, 9, 11, 14, 25, 27, 48), and a recent homoacetogenic isolate, Sporomusa malonica, grows at the expense of decarboxylation of malonate or succinate (12). Although C. thermoaceticum did not grow at the sole expense of decarboxylation of aromatic compounds, decarboxylation was concomitant with improved methanol-dependent growth efficiencies, as determined by the cell's capacity to synthesize biomass during acetogenesis. Thus, decarboxylation of aromatic compounds may enhance the cell's potential to conserve energy from other energy-yielding acetogenic substrates. Under certain conditions, aromatic carboxyl groups may constitute a more effective origin of CO2 equivalents for acetogenesis, or the decarboxylation reaction may be coupled to energy flow by a mechanism independent of acetogenesis. Based on standard thermodynamic tables, the decarboxylation of p-hydroxybenzoate is exergonic (50). Some decarboxylation reactions are coupled to the energy state of the cell via ion transport or cycling (14). A growing body of evidence indicates that carboxylation and decarboxylation reactions play important initiating roles in the anaerobic breakdown of aromatic compounds (5, 15, 23, 24, 30, 31, 35, 44, 49, 59). However, the specific microorganisms responsible for these reactions have not been well defined. The present study demonstrates that acetogenic bacteria may play important roles at this level in the anaerobic biodegradation of aromatic compounds. Rhodopseudomonas palustris degrades benzoates anaerobically via benzoyl-CoA derivatization and subsequent reduction of the aromatic ring (42). In this case, decarboxylation is apparently not involved in the initial catabolism of the aromatic compound. C. thermoaceticum was first isolated from horse manure as a strict heterotroph (17) and has served as the model acetogen for resolution of the acetyl-CoA (Wood) pathway (37, 56). Recent studies have further resolved the diverse metabolic potentials of this acetogen to include CO- and H2-dependent chemolithotrophic growth, methoxyl-based acetogenesis, and the oxidation and reduction of aromatic aldehydes during CO-dependent growth (10, 57; M. F. Lux, E. Keith, T. Hsu, and H. L. Drake, FEMS Microbiol. Lett., in press; S. L. Daniel Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, K99, p. 261 [manuscript in preparation]). The present study demonstrates that the metabolic potentials of this acetogen have not been fully elucidated and furthermore illustrates that aromatic compounds devoid of methoxyl and acrylate groups can play essential roles in the growth of acetogenic bacteria. From the present study, it is tempting to speculate that the capacity to utilize aromatic carboxyl groups may confer a competitive advantage to acetogens in certain C02-limited environments. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant A121852 and Research Career Development Award AI00722 (H.L.D.) from the National Institute of Allergy and Infectious Diseases, and by a National Institutes of Health Biomedical Research Support grant to the University of Mississippi.
J. BACTEIOL.
LITERATURE CITED 1. Adamse, A. D. 1980. New isolation of Clostridium aceticum (Wieringa). Antonie van Leeuwenhoek J. Microbiol. Serol. 46:523-531. 2. Andreesen, J. R., G. Gottschalk, and H. G. Schlegel. 1970. Clostridium formicoaceticum nov. spec. isolation, description and distinction from C. aceticum and C. thermoaceticum. Arch. Mikrobiol. 72:154-174. 3. Bache, R., and N. Pfennig. 1981. Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields. Arch. Microbiol. 130:255-261. 4. Barik, S., W. J. Brulla, and M. P. Bryant. 1985. PA-1, a versatile anaerobe obtained in pure culture, catabolizes benzenoids and other compounds in syntrophy with hydrogenotrophs, and P-2 plus Wolinella sp. degrades benzenoids. Appl. Environ. Microbiol. 50:304-310. 5. Berry, D. F., A. J. Francis, and J.-M. Bollag. 1987. Microbial metabolism of homocyclic and heterocyclic aromatic compounds under anaerobic conditions. Microbiol. Rev. 51:43-59. 6. Braun, M., F. Mayer, and G. Gottschalk. 1981. Clostridium aceticum (Wieringa), a microorganism producing acetic acid from molecular hydrogen and carbon dioxide. Arch. Microbiol. 128:288-293. 7. Breznak, J. A., and J. M. Switzer. 1986. Acetate synthesis from H2 plus CO2 by termite microbes. Appl. Environ. Microbiol. 52:623-630. 8. Breznak, J. A., J. M. Switzer, and H.-J. Seitz. 1988. Sporomusa termitida sp. nov., an H2/C02-utilizing acetogen isolated from termites. Arch. Microbiol. 150:282-288. 9. Brulla, W. J., and M. P. Bryant. 1989. Growth of the syntrophic anaerobic acetogen, strain PA-1, with glucose or succinate as energy source. Appl. Environ. Microbiol. 55:1289-1290. 10. Daniel, S. L., Z. Wu, and H. L. Drake. 1988. Growth of thermophilic acetogenic bacteria on methoxylated aromatic acids. FEMS Microbiol. Lett. 52:25-28. 11. Dehning, I., and B. Schink. 1989. Malonomonas rubra gen. nov. sp. nov., a microaerotolerant anaerobic bacterium growing by decarboxylation of malonate. Arch. Microbiol. 151:427-433. 12. Dehning, I., M. Stieb, and B. Schink. 1989. Sporomusa malonica sp. nov., a homoacetogenic bacterium growing by decarboxylation of malonate or succinate. Arch. Microbiol. 151:421-426. 13. DeWeerd, K. A., A. Saxena, D. P. Nagle, Jr., and J. M. Suflita. 1988. Metabolism of the '80-methoxy substituent of 3-methoxyl-benzoic acid and other unlabeled methoxybenzoic acids by anaerobic bacteria. Appl. Environ. Microbiol. 54:1237-1242. 14. Dimroth, P. 1987. Sodium ion transport decarboxylases and other aspects of sodium ion-cycling bacteria. Microbiol. Rev. 51:320-340. 15. Evans, W. C., and G. Fuchs. 1988. Anaerobic degradation of aromatic compounds. Annu. Rev. Microbiol. 42:289-317. 16. Finegold, S. M., V. L. Sutter, and G. E. Mathison. 1983. Normal indigenous intestinal flora, p. 3-31. In D. A. Hentges (ed.), Human intestinal flora in health and disease. Academic Press, Inc., New York. 17. Fontaine, F. E., W. H. Peterson, E. McCoy, M. J. Johnson, and G. J. Ritter. 1942. A new type of glucose fermentation by Clostridium thermoaceticum n. sp. J. Bacteriol. 43:701-715. 18. Frazer, A. C., and L. Y. Young. 1985. A gram-negative anaerobic bacterium that utilizes 0-methyl substituents of aromatic acids. Appl. Environ. Microbiol. 49:1345-1347. 19. Frazer, A. C., and L. Y. Young. 1986. Anaerobic C1 metabolism of the O-methyl-'4C-labeled substituents of aromatic acids. Appl. Environ. Microbiol. 51:84-87. 20. Frazer, A. C., I. Bossert, and L. Y. Young. 1986. Enzymatic aryl-O-methyl-14C labeling of model lignin monomers. Appl. Environ. Microbiol. 51:80-83. 21. Fuchs, G. 1986. CO2 fixation in acetogenic bacteria: variations on a theme. FEMS Microbiol. Rev. 39:181-213. 22. Genthner, B. R. S., C. L. Davis, and M. P. Bryant. 1981. Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species. Appl. Environ. Microbiol. 42:12-19. 23. Genthner, B. R. S., W. A. Price II, and P. H. Pritchard. 1989.
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Anaerobic degradation of chloroaromatic compounds in aquatic sediments under a variety of enrichment conditions. Appl. Environ. Microbiol. 55:1466-1471. 24. Genthner, B. R. S., W. A. Price H, and P. H. Pritchard. 1989. Characterization of anaerobic dechlorinating consortia derived from aquatic sediments. Appl. Environ. Microbiol. 55:14721476. 25. Gottschalk, G. 1986. Bacterial metabolism, 2nd ed. SpringerVerlag, New York Inc., New York. 26. Hansen, B., M. Bokranz, P. Schonheit, and A. Kroger. 1988. ATP formation coupled to caffeate reduction by H2 in Acetobacterium woodii NZval6. Arch. Microbiol. 150:447-451. 27. Hilpert, W., B. Schink, and P. Dimroth. 1984. Life by a new decarboxylation-dependent energy conservation mechanism with Na+ as coupling ion. EMBO J. 3:1665-1670. 28. Jones, J. G., and B. M. Simon. 1985. Interaction of acetogens and methanogens in anaerobic freshwater sediments. Appl. Environ. Microbiol. 49:944-948. 29. Kellum, R., and H. L. Drake. 1986. Effects of carbon monoxide on one-carbon enzymes and energetics of Clostridium thermoaceticum. FEMS Microbiol. Lett. 34:41-45. 30. Knoll, G., and J. Winter. 1989. Degradation of phenol via carboxylation to benzoate by a defined, obligate syntrophic consortium of anaerobic bacteria. AppI. Microbiol. Biotechnol. 30:318-324. 31. Knoll, G., and J. Winter. 1987. Anaerobic degradation of phenol in sewage sludge: benzoate formation from phenol and carbon dioxide in the presence of hydrogen. Appl. Microbiol. Biotechnol. 25:384-391. 32. Kreikenbohm, R., and N. Pfennig. 1985. Anaerobic degradation of 3,4,5-trimethoxybenzoate by a defined mixed culture of Acetobacterium woodii, Pelobacter acidigallici, and Desulfobacter postgatei. FEMS Microbiol. Ecol. 31:29-38. 33. Krumholz, L. R., and M. P. Bryant. 1985. Clostridium pfennigii sp. nov. uses methoxyl groups of monobenzenoids and produces butyrate. Int. J. Syst. Bacteriol. 35:454-456. 34. Krumholz, L. R., and M. P. Bryant. 1986. Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate, methoxybenzenoids or Methanobrevibacter as electron acceptor systems. Arch. Microbiol. 143:313-318. 35. Krumholz, L. R., and M. P. Bryant. 1986. Eubacterium oxidoreducens sp. nov. requiring H2 or formate to degrade gallate, pyrogallol, phloroglucinol and quercetin. Arch. Microbiol. 144: 8-14. 36. Lajoie, S. F., S. Bank, T. L. Miller, and M. J. Wolin. 1988. Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces. Appl. Environ. Microbiol. 54: 2723-2727. 37. Ljungdahl, L. G. 1986. The autotrophic pathway of acetate synthesis in acetogenic bacteria. Annu. Rev. Microbiol. 40: 415-450. 38. Lorowitz, W. H., and M. P. Bryant. 1984. Peptostreptococcus productus strain that grows rapidly with CO as an energy source. Appl. Environ. Microbiol. 47:961-964. 39. Mah, R. A. 1982. Methanogenesis and methanogenic partnerships. Phil. Trans. R. Soc. London B 297:599-616. 40. Martin, D. R., A. Misra, and H. L. Drake. 1985. Dissimilation of carbon monoxide to acetic acid by glucose-limited cultures of Clostridium thermoaceticum. Appl. Environ. Microbiol. 49: 1412-1417. 41. Mclnerney, M. J., and M. P. Bryant. 1980. Metabolic stages and energetics of microbial anaerobic digestion, p. 91-98. In D. A.
217
Stafford, B. I. Wheatley, and D. E. Huges (ed.), Anaerobic digestion. Applied Science Publishers, Ltd., London. 42. Merkel, S. M., A. E. Eberhard, J. Gibson, and C. S. Harwood. 1989. Involvement of coenzyme A thioesters in anaerobic metabolism of 4-hydroxybenzoate by Rhodopseudomonas palustris. J. Bacteriol. 171:1-7. 43. Prins, R. A., and A. Lankhorst. 1977. Synthesis of acetate from CO2 in the cecum of some rodents. FEMS Microbiol. Lett. 1:255-258. 44. Samain, E., G. Albagnac, and H.-C. Dubourguier. 1986. Initial steps in the catabolism of trihydroxybenzenes in Pelobacter acidigallici. Arch. Microbiol. 144:242-244. 45. Savage, M. D., and H. L. Drake. 1986. Adaptation of the acetogen Clostridium thermoautotrophicum to minimal medium. J. Bacteriol. 165:315-318. 46. Savage, M. D., Z. Wu, S. L. Daniel, L. L. Lundie, Jr., and H. L. Drake. 1987. Carbon monoxide-dependent chemolithotrophic growth of Clostridium thermoautotrophicum. Appl. Environ. Microbiol. 53:1902-1906. 47. Schink, B., and N. Pfennig. 1982. Fermentation of trihydroxybenzenes by Pelobacter acidigallici gen. nov. sp. nov., a new strictly anaerobic, non-sporeforming bacterium. Arch. Microbiol. 133:195-201. 48. Schink, B., and N. Pfennig. 1982. Propionigenium modestum gen. nov. sp. nov., a new strictly anaerobic nonsporing bacterium growing on succinate. Arch. Microbiol. 133:209-216. 49. Sleat, R., and J. P. Robinson. 1984. The bacteriology of anaerobic degradation of aromatic compounds. J. Appi. Bacteriol. 57:381-394. 50. Stull, D. R., E. F. Westrum, Jr., and G. C. Sinke. 1969. The chemical thermodynamics of organic compounds. John Wiley & Sons, Inc., New York. 51. Tschech, A., and N. Pfennig. 1984. Growth yield increase linked to caffeate reduction in Acetobacterium woodii. Arch. Microbiol. 137:163-167. 52. Wieringa, K. T. 1936. Over het verdwijnen van waterstof en koolzuur onder anaerobe voorwaarden. Antonie van Leeuwenhoek J. Microbiol. Serol. 3:263-273. 52a.Winter, J. U., and R. S. Wolfe. 1980. Methane formation from fructose by syntrophic associations of Acetobacterium woodii and different strains of methanogens. Arch. Microbiol. 124: 73-79. 53. Wolin, M. J. 1981. Fermentation in the rumen and human large intestine. Science 213:1463-1468. 54. Wolin, M. J., and T. L. Miller. 1982. Interspecies hydrogen transfer: 15 years later. ASM News 48:561-565. 55. Wolin, M. J., and T. L. Miller. 1983. Carbohydrate fermentation, p. 147-165. In D. A. Hentges (ed.), Human intestinal flora in health and disease. Academic Press, Inc., New York. 56. Wood, H. G., S. W. Ragsdale, and E. Pezacka. 1986. The acetyl-CoA pathway of autotrophic growth. FEMS Microbiol. Rev. 43:345-362. 57. Wu, Z., S. L. Daniel, and H. L. Drake. 1988. Characterization of a CO-dependent 0-demethylating enzyme system from the acetogen Clostridium thermoaceticum. J. Bacteriol. 170:57055708. 58. Yang, H., S. L. Daniel, T. Hsu, and H. L. Drake. 1989. Nickel transport by the thermophilic acetogen Acetogenium kivui. Appl. Environ. Microbiol. 55:1078-1081. 59. Young, L. Y., and A. C. Frazer. 1987. The fate of lignin and lignin-derived compounds in anaerobic environments. Geomicrobiol. J. 5:261-293.
Vol. 172, No. 1
0021-9193/90/010212-06$02.00/0 Copyright C) 1990, American Society for Microbiology
Biotransformations of Carboxylated Aromatic Compounds by the Acetogen Clostridium thermoaceticum: Generation of GrowthSupportive CO2 Equivalents under C02-Limited Conditions TSUNGDA HSU, STEVEN L. DANIEL, MARY F. LUX, AND HAROLD L. DRAKE* Microbial Physiology Laboratories, Department of Biology, The University of Mississippi, University, Mississippi 38677 Received 31 July 1989/Accepted 2 October 1989
Clostridium thermoaceticum ATCC 39073 converted vanillate to catechol. Although carboxylated aromatic compounds which did not contain methoxyl groups were not by themselves growth supportive, protocatechuate and p-hydroxybenzoate (nonmethoxylated aromatic compounds) were converted to catechol and phenol, respectively, during carbon monoxide-dependent growth. Syringate is not subject to decarboxylation by C. thermoaceticum (Z. Wu, S. L. Daniel, and H. L. Drake, J. Bacteriol. 170:5705-5708, 1988), and sustained growth at the expense of syringate-derived methoxyl groups was dependent on supplemental CO2. In contrast, vanillate was growth supportive in the absence of supplemental C02, and 14CO2 was the major 14C-labeled product during [carboxyl-"4C]vanillate-dependent growth. Furthermore, the decarboxylation of protocatechuate and p-hydroxybenzoate supported methanol- and 1,2,3-trimethoxybenzene-dependent growth (CO2 is required for growth at the expense of these substrates) when supplemental CO2 was depleted from the growth medium, and the decarboxylation of protocatechuate was concomitant with improved cell yields of methanol cultures. These findings demonstrate that (i) C. thermoaceticum is competent in the decarboxylation of certain aromatic compounds and (ii) under certain conditions, decarboxylation may be integrated to the flow of carbon and energy during acetogenesis.
Acetogenic bacteria utilize a unique form of autotrophic metabolism termed acetogenesis. Some of the key features of this autotrophic process have been elucidated, and the pathway utilized for the autotrophic synthesis of acetate and biomass has been termed the acetyl coenzyme A (acetylCoA) (Wood) pathway (21, 37, 56). Recent studies have shown that various aromatic compounds are growth-supportive substrates for acetogenic bacteria (3, 8, 10, 12, 13, 18-20, 33, 51, 57) and may thus be important to other microorganisms which can serve as syntrophic partners of acetogens (15, 28, 32, 39, 41, 47, 52a, 54, 58). The natural habitats of acetogens range from anaerobic sediments to the gastrointestinal tracts of humans and other animals, and lignin-derived aromatic compounds probably constitute important naturally occurring substrates in these environments (7, 8, 16, 22, 26, 28, 34, 36, 38, 43, 53, 55). Despite this recent evidence implicating acetogens as an important biological group in the anaerobic breakdown of aromatic compounds, most attention to date has centered on the use of aromatic methoxyl and acrylate groups during acetogenesis, and metabolic potentials for the use of other aromatic substituents remain poorly defined. Clostridium thermoaceticum (17) has been the classic acetogen used to elucidate many of the biochemical features central to acetogenesis (37, 56). For methoxy-based acetogenesis, both vanillate and syringate are growth-supportive substrates for this acetogen (10). Syringate is sequentially 0-demethylated to 5-hydroxyvanillate and gallate, and an inducible carbon monoxide-dependent 0-demethylating enzyme system functions in concert with acetyl-CoA synthetase (CO dehydrogenase) during methoxyl-coupled acetogenesis (57). Since gallate is the only aromatic product detected from syringate with C. thermoaceticum (57), aromatic carboxyl groups appeared to be nonreactive with this *
acetogen. However, as outlined in the following study, the carboxyl groups of other aromatic compounds are subject to decarboxylation by C. thermoaceticum and can serve as growth-supportive CO2 equivalents under C02-limited conditions. (Portions of this work have been presented at the 89th Annual Meeting of the American Society for Microbiology, New Orleans, La., 14 to 18 May 1989.) MATERIALS AND METHODS Cultivation of C. thermoaceticum. C. thermoaceticum ATCC 39073 (17) was cultivated in either undefined or defined medium at 55°C in crimp-sealed culture tubes (18 by 150 mm; Bellco series 2048; Bellco Glass Inc., Vineland, N.J.). Undefined medium contained (in milligrams per liter): NaHCO3, 5,000; KH2PO4, 500; NaCl, 400; NH4Cl, 400; MgCl 6H20, 330; CaCl2. 2H20, 50; resazurin, 1; yeast extract, 1,000; nicotinic acid, 0.25; cyanocobalamin, 0.25; p-aminobenzoic acid, 0.25; calcium D-pantothenate, 0.25; thiamine hydrochloride, 0.25; riboflavin, 0.25; lipoic acid, 0.15; folic acid, 0.1; biotin, 0.1; pyridoxal hydrochloride, 0.05; sodium nitrilotriacetate, 7.5; MnSO4. H20, 2.5; FeSO4- 7H20, 0.5; Co(NO3)2 6H20, 0.5; ZnCl2, 0.5; NiCl2 *6H20, 0.25; CuS04 5H20, 0.05; AIK(S04)2 12H20 0.05; H3BO3, 0.05; and Na2MoO4 2H20, 0.05. Defined medium was undefined medium without yeast extract. Sodium sulfide-cysteine reducer (46) was added after the media were boiled and cooled under C02; media were subsequently dispensed (7 ml per tube) under C02, and the tubes were crimp sealed and autoclaved. Glucose and methanol were added at concentrations of 10 and 60 mM, respectively, before autoclaving. For cultivation at the expense of CO, culture tubes were pressurized at room temperature to 70 kPa (10 lb/in2) over atmospheric pressure (171.3 kPa final total pressure) with 100% CO prior to incubation. Growth was initiated by injecting 0.5 ml of inoculum per culture -
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Corresponding author. 212
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tube. The total volume of the culture tubes approximated 28 ml, and the ratio of gas phase to liquid phase approximated 3:1. Stock solutions of vanillate and syringate (383.5 mM) were prepared in distilled and deionized water titrated with 5 N NaOH until dissolved. Unless otherwise indicated, stock solutions of all other aromatic compounds (383.5 mM) were prepared in 80% ethanol (the resulting ethanol concentration in culture media approximated 1% and did not significantly influence growth). Stock solutions of aromatic compounds were filter sterilized and added via syringe to sterile tubes of medium to the concentrations indicated. The aromatic compounds studied were stable under the conditions used; transformations were not observed in the absence of cells. In this study, no distinction is made between the acid and dissociated ionic forms of the aromatic carboxylic acids. Carbon dioxide-free medium was medium in which the NaHCO3 and KH2PO4 were replaced with Na2HPO4 (5.68 g/liter) and NaH2PO4 (7.2 g/liter). The initial gas phase of the C02-free medium was 100% N2 unless otherwise indicated. The initial pH of all media at the time of inoculation approximated 6.4 to 6.5. Cultivation of C. formicoaceticum and C. aceticum. C. formicoaceticum ATCC 27076 (2) and C. aceticum DSM 1496 (1, 6, 52) were cultivated at 37°C in undefined medium in which the NaHCO3 concentration was modified to 6 g/liter and Na2CO3 was added to a final concentration of 12 g/liter. Fructose and methanol, when added, were used at initial concentrations of 10 and 60 mM, respectively. For C02-free undefined medium, carbonates were not included, and the following phosphates were added (in milligrams per liter): Na2HPO4, 680; Na3PO4. 12H20, 60. The initial gas phase of C02-free media was 100% N2. The initial pH of these media at the time of inoculation approximated 7.8.
Measurement of '4C-labeled products from [carboxyl-'4C] vanillate. Cells were cultivated in undefined medium containing 5 mM [carboxyl-14C]vanillate (approximately 2.7 x 104 dpm/Lmol). Following growth, a sample of culture medium was removed for analysis of 14C in acetate and biomass. For analysis of 14CO2, the culture tube was connected to duplicate 1 N KOH CO2 traps and acidified with 0.75 ml of 2 N H2SO4. The gas phase of the culture tube was flushed through the traps for 5 min with a steady stream of N2, and the trapped 14C02 was quantitated by liquid scintillation in Safety Solve (Research Products International Corp., Mount Prospect, Ill.) with a Beckmann Instruments, Inc. (Fullerton, Calif.) LS 6800 liquid scintillation counter. 14C-labeled cells were harvested by microfiltration, washed four times, and then analyzed for 14C by liquid scintillation as described previously for the analysis of 63Ni-labeled biomass (58). Analytical methods. Aromatic compounds were obtained from Sigma Chemical Co. (St. Louis, Mo.) and Aldrich Chemical Co., Inc. (Milwaukee, Wis.). [Carboxyl-14C]vanillate was from Research Products International Corporation. Growth was monitored at 660 nm with a Spectronic 501 spectrophotometer (Bausch & Lomb, Inc., Rochester, N.Y.), and cell dry weights were determined as described previously (45). None of the compounds studied influenced the turbidity (A660) of culture media, and a linear relationship was shown to exist between biomass and absorbance. Aromatic compounds were analyzed by high-pressure liquid chromatography with a Hewlett-Packard Co. (Palo Alto, Calif.) 1090L high-performance liquid chromatograph equipped with a Bio-Rad Laboratories (Richmond, Calif.) fermentation monitoring column and a Spectra-Physics (Bedford, Mass.) 4290 integrator; the column temperature
Cultivation Time (h) FIG. 1. Conversion of vanillate (V) to catechol
(C) by C. thermoaceticum. Undefined culture medium contained 5 mM vanillate; inoculum was from an undefined vanillate medium culture. P, Protocatechuate.
600C, the mobile phase was 0.01 N H2SO4 at 0.8 ml/min, the sample size was 10 ,ul, and the detector was at 210 nm. Cells were removed by microcentrifugation and microfiltration prior to chromatographic analysis of fermentation broths. The authenticity of aromatic compounds was verified by UV absorption spectral analysis with a Gilford Systems (Oberlin, Ohio) 2600 recording spectrophotometer.
was
RESULTS
Growth-dependent transformations of carboxylated aromatic compounds. During vanillate-dependent growth, vanillate was converted to catechol (Fig. 1). Protocatechuate was a transient product, and catechol was the sole aromatic end product detected upon completion of growth under these
conditions. Given the transient appearance of protocatechuate during vanillate-dependent growth, the capacity to convert protocatechuate to catechol was investigated by using CO as the energy source for growth. Although protocatechuate did not support the growth of C. thermoaceticum in the absence of additional energy sources (unpublished data), protocatechuate was rapidly converted to catechol during CO-dependent
Ec 0 0 -
E 1-
in
la
C
0l.E33
0
0 C
n 4)
0
U
LI.
.5
U
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o
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Cultivation Time (h) FIG. 2. Conversion of protocatechuate (P) to catechol (C) during of C. thermoaceticum. Undefined culture CO-dependent growth medium contained 5 mM protocatechuate and a CO-CO2 (40:60 at 140 kPa over atmospheric pressure) gas phase. Inoculum was from an undefined CO medium culture.
214
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HSU ET AL.
TABLE 1. Distribution of '4C from [carboxyl-'4C]vanillate after vanillate-dependent growth by C. thermoaceticum Distribution of 14C (dpm) C 0.18 0* (0 (0
0.1 2 .0 L_ 0
Q06
0 0
125 250 0 125 250 Cultivation Time (h) FIG. 3. Effects of CO2 on syringate-dependent (A) and vanillatedependent (B) growth of C. thermoaceticum. The C02-free undefined culture medium was supplemented as indicated: 0, aromatic compound plus 70 kPa of CO2 over pressure; 0, aromatic compound without supplemental C02; O, 70 kPa of CO2 over pressure without aromatic compound; *, no supplements. (A) Inoculum was from a syringate-CO2 undefined medium culture. (B) Inoculum was from a C02-free undefined vanillate medium culture (maintained for four
Expt
Initial 14C (dpm)
1st CO2 trap
2nd CO2 Biomass trap
Supernatanta
% of
initial dpm
recovered
982,388 502,136 107,456 3,115 152,179 78 978,938 279,4% 63,664 3,126 52 161,667 100 3b 1,058,115 16,560 13,800 NAC 1,027,686 a 14C remaining in the supernatant fluid after cells were removed from
1 2
culture medium by microcentrifugation. b Uninoculated culture medium (control). NA, Not applicable.
sparging and to incomplete trapping of CO2 in the two KOH traps. Based on these findings, we reasoned that the carboxyl group of certain aromatic compounds might serve as a CO2 equivalent during acetogenesis. Methanol and 1,2,3-trimethoxybenzene cultures, both of which are dependent upon supplemental CO2 for sustained growth (unpublished data), were used to test this hypothesis. As depicted in Fig. 4, cell + 40 ,mol
CO2 + 40 umol P
transfers).
growth (Fig. 2). The overall specific activity for the transformation of protocatechuate to catechol approximated 7.0 nmol of protocatechuate converted per min per mg (dry weight) of cells. Protocatechuate was also converted to catechol during growth at the expense of methanol or glucose (data not shown). In addition, p-hydroxybenzoate was converted in near stoichiometry to phenol in similar growth studies; as with protocatechuate, p-hydroxybenzoate did not support the growth of C. thermoaceticum in the absence of additional energy sources (data not shown). Benzoate, o-hydroxybenzoate, and m-hydroxybenzoate were not transformed (data not shown). Capacity of other acetogens to decarboxylate aromatic compounds. In contrast to C. thermoaceticum, both C. formicoaceticum and C. aceticum converted vanillate to protocatechuate rather than catechol (data not shown). Furthermore, neither C. formicoaceticum nor C. aceticum was capable of transforming protocatechuate or p-hydroxybenzoate when cultivated at the expense of fructose. Function of aromatic carboxyl groups: origin of growthsupportive CO2 equivalents. As noted above, C. thermoaceticum does not decarboxylate syringate (57). Significantly, only marginal growth was obtained with syringate in the absence of supplemental CO2 (Fig. 3A). In contrast, vanillate supported substantial growth without supplemental CO2 (Fig. 3B). In CO2-free medium, vanillate was also converted in near stoichiometry to catechol (data not shown; product profile similar to that illustrated in Fig. 1). Collectively, these results suggested that vanillate was subject to decarboxylation. This possibility was confirmed by growth studies with [carboxyl-'4C]vanillate (Table 1). Under standard growth conditions in undefined vanillate medium, "4CO2 was the predominant 14C-labeled product detected after completion of growth. The low recovery of 14C may have been due to leakage of gas during growth and
c '-
0
Q3 B
0 L-
p
Cultivation Time (h) FIG. 4. Effects of CO2 and protocatechuate (P) on methanolsupported growth of C. thermoaceticum. (A) C02-free undefined medium with 60 mM methanol (inoculum was from undefined methanol-protocatechuate medium culture). (B) C02-free defined medium with 60 mM methanol (inoculum was from defined methanol-protocatechuate medium culture). Amounts of protocatechuate and CO2 are per culture tube; the protocatechuate stock solution was the salt form.
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Ec
USE OF CARBOXYLATED AROMATICS BY C. THERMOACETICUM
U.2u A
215
TABLE 2. Protocatechuate-dependent stimulation of C. thermoaceticum during growth at the expense of methanol
0
(0
Protocatechuate concn' (mM)
Acetate
0
Cu
0 1 10 10C
34.1 35.3 34.6 16.2
0.15)
0~
u 0.10 O
10
20
Cultivation Time (h) FIG. 5. Effects of C02, protocatechuate, and p-hydroxybenzoate on trimethoxybenzene-supported growth of C. thermoaceticum. The C02-free undefined culture medium was supplemented as indicated: O, 1,2,3-trimethoxybenzene (5 mM) pIus C02 (70 kPa over pressure); A, 1,2,3-trimethoxybenzene plus protocatechuate (5 mM); O, 1,2,3-trimethoxybenzene plus p-hydroxybenzoate (5 mM); *, 1 ,2,3-trimethoxybenzene; *, protocatechuate or p-hydroxybenzoate. Inoculum was from an undefined 1,2,3-trimethoxybenzene medium culture.
yields of methanol-protocatechuate-grown cultures were nearly identical to those of methanol-CO2-grown cultures when protocatechuate was added at concentrations equivalent to those of C02. The capacity for protocatechuate to replace C02 was observed with both undefined and defined cultures, demonstrating that components in yeast extract were not responsible for the protocatechuate-dependent effects. Catechol was the only aromatic product detected from the methanol-protocatechuate cultures. Furthermore, both protocatechuate and p-hydroxybenzoate served as C02 replacements during 1,2,3-trimethoxybenzene-dependent growth of C. thermoaceticum (Fig. 5). In contrast, vanillate-dependent growth of C. formicoaceticum did not occur in the absence of supplemental CO2 (data not shown). In addition, supplemental C02 was required for the methanol-dependent growth of C. formicoaceticum (6) and could not be replaced by p-hydroxybenzoate (data not shown). Effect of decarboxylation of protocatechuate on methanol dependent cell yields. Since C. thermoaceticum is homoacetogenic, the synthesis of biomass is obligately coupled to acetogenesis, i.e., the capacity to synthesize biomass is strictly dependent upon the cell's ability to conserve energy during the formation of acetate. Therefore, the ratio of acetate formed per unit of biomass synthesized can be taken as a direct reflection of cell energetics (29, 45, 51). In this regard, the decarboxylation of protocatechuate to catechol
(MM)
Acetate/
1.18 1.32 1.51
28.9 26.8 22.9 24.0
0.67
ratiob
a Undefined methanol (60 mM) culture medium was supplemented with protocatechuate as indicated. Inoculum was from a methanol-protocatechuate medium culture. Protocatechuate was converted stoichiometrically to catechol. b Acetate formed (millimolar) per gram (dry weight) of biomass per liter of medium. 'C02-free undefined methanol (60 mM) culture medium.
DISCUSSION In the present study, C. thermoaceticum was shown to be competent in decarboxylating certain aromatic compounds, and this metabolic potential was found to replace CO2 during methanol- and 1,2,3-trimethoxybenzene-dependent growth. This is the first report correlating the growth of an acetogen with the utilization of an aromatic carboxyl group. That vanillate is growth supportive in the absence of supplemental CO2 corroborates such an essential role for aromatic carboxyl groups under C02-limited conditions. C. formicoaceticum and C. aceticum did not display this metabolic potential, indicating that the use of aromatic carboxyl groups is not characteristic of all acetogenic bacteria. The present findings suggest that decarboxylation of aromatic compounds by C. thermoaceticum yields CO2 equivalents which can subsequently be utilized in the acetyl-CoA pathway (Fig. 6). CO2 plays essential roles in the bioenergetics and carbon flow of acetogenic bacteria (21, 37, 56), and how tightly coupled decarboxylation is to acetogenesis is not clear. Although the aromatic carboxyl group gave rise to CO2 under CO2-enriched conditions, decarboxylation may be more tightly coupled to the acetyl-CoA pathway when CO2 is limited (i.e., utilized directly as a one-carbon unit without being released from the cell as free CO2). Carbon monoxide exerts a strong influence on the flow of CO2-derived carbon during acetogenesis by C. thermoaceticum (40), and the relative concentration of exogenous CO2 is critical to the growth efficiency of the closely related acetogen Clostridium thermoautotrophicum (46). Thus, growth conditions may dictate whether aromatic compound-derived CO2H
C0C°2 CO
OH
H20
2H
(P)
/
was concomitant with a decrease in the acetate to biomass
ratio during methanol-coupled growth (Table 2). The amount of acetate formed remained relatively constant in the presence or absence of protocatechuate with CO2-enriched methanol medium, but the capacity to synthesize biomass increased coincident with decarboxylation. The acetateto-biomass ratio of CO2-free protocatechuate-methanol cultures was also less than that of CO2-enriched methanol cultures (Table 2). Protocatechuate was stoichiometrically converted to catechol, and the synthesis of biomass carbon from protocatechuate would therefore not account for the observed increase in biomass formation.
Biomass formed (g [dry wt]Aiter)
C
I1OH OH (C)
J
HC02H
THF2 CH3-THF THF
HSCoA
CH3-CO-SCoA (acety l-CoA)
FIG. 6. Hypothetical pathway for the integration of aromatic carboxyl groups into the acetyl-CoA pathway. Abbreviations: P, protocatechuate; C, catechol; THF, tetrahydrofolate; HSCoA, coenzyme A.
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"CO2" equivalents enter the carbonyl or methyl carbon of acetyl-CoA. Likewise, the enzyme(s) responsible for decarboxylation and integration of these CO2 equivalents into the acetyl-CoA pathway may be subject to regulation by available substrates. The 0-demethylating enzyme system is induced by utilizable methoxylated aromatic compounds but repressed by glucose (57). Certain decarboxylation reactions yield energy gains under anaerobic conditions (4, 9, 11, 14, 25, 27, 48), and a recent homoacetogenic isolate, Sporomusa malonica, grows at the expense of decarboxylation of malonate or succinate (12). Although C. thermoaceticum did not grow at the sole expense of decarboxylation of aromatic compounds, decarboxylation was concomitant with improved methanol-dependent growth efficiencies, as determined by the cell's capacity to synthesize biomass during acetogenesis. Thus, decarboxylation of aromatic compounds may enhance the cell's potential to conserve energy from other energy-yielding acetogenic substrates. Under certain conditions, aromatic carboxyl groups may constitute a more effective origin of CO2 equivalents for acetogenesis, or the decarboxylation reaction may be coupled to energy flow by a mechanism independent of acetogenesis. Based on standard thermodynamic tables, the decarboxylation of p-hydroxybenzoate is exergonic (50). Some decarboxylation reactions are coupled to the energy state of the cell via ion transport or cycling (14). A growing body of evidence indicates that carboxylation and decarboxylation reactions play important initiating roles in the anaerobic breakdown of aromatic compounds (5, 15, 23, 24, 30, 31, 35, 44, 49, 59). However, the specific microorganisms responsible for these reactions have not been well defined. The present study demonstrates that acetogenic bacteria may play important roles at this level in the anaerobic biodegradation of aromatic compounds. Rhodopseudomonas palustris degrades benzoates anaerobically via benzoyl-CoA derivatization and subsequent reduction of the aromatic ring (42). In this case, decarboxylation is apparently not involved in the initial catabolism of the aromatic compound. C. thermoaceticum was first isolated from horse manure as a strict heterotroph (17) and has served as the model acetogen for resolution of the acetyl-CoA (Wood) pathway (37, 56). Recent studies have further resolved the diverse metabolic potentials of this acetogen to include CO- and H2-dependent chemolithotrophic growth, methoxyl-based acetogenesis, and the oxidation and reduction of aromatic aldehydes during CO-dependent growth (10, 57; M. F. Lux, E. Keith, T. Hsu, and H. L. Drake, FEMS Microbiol. Lett., in press; S. L. Daniel Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, K99, p. 261 [manuscript in preparation]). The present study demonstrates that the metabolic potentials of this acetogen have not been fully elucidated and furthermore illustrates that aromatic compounds devoid of methoxyl and acrylate groups can play essential roles in the growth of acetogenic bacteria. From the present study, it is tempting to speculate that the capacity to utilize aromatic carboxyl groups may confer a competitive advantage to acetogens in certain C02-limited environments. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant A121852 and Research Career Development Award AI00722 (H.L.D.) from the National Institute of Allergy and Infectious Diseases, and by a National Institutes of Health Biomedical Research Support grant to the University of Mississippi.
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