Scholarly article on topic 'Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.'

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp. Academic research paper on "Industrial Biotechnology"

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Academic research paper on topic "Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp."

Arch Microbiol (1990) 153:200-204

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Microbiology

© Springer-Verlag 1990

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Beate Schuppert* and Bernhard Schink

Lehrstuhl Mikrobiologie I der Eberhard-Karls-Universitat, Auf der Morgenstelle 28, D-7400 Tübingen, Federal Republic of Germany

Abstract. Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffcred medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6—10 g dry matter per mol methyl group. H2/C02, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4 — 2.2 Uxmg protein-1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carhinolicum. One freshwater isolate, strain KoMacl, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.

Keywords: Anaerobic ether cleavage — Methoxyacetate — 2-Methoxyethanol — Demethylation — Homoacetogenic bacteria — Acetobacterium sp.

Ether linkages are cleaved aerobically usually by oxygenase reactions which hydroxylate one of the vicinal ether carbon atoms and thus transform the ether compound into a comparably unstable hemiacetal structure (Heydeman 1974). Accordingly, phenylmethyl ethers such as lignin monomers are aerobically oxidized to the corresponding phenols and formaldehyde (Axclrod 1956; Cartwright and Smith 1967; Bernhardt et al. 1970; Taylor 1983; Sutherland 1986).

Acetobacterium woodii was the first bacterium reported to be able to cleave phenylmethyl ether linkages in the ab-

* Present address: Fraunhofer-Tnstitut für Grcn/flächen- und Bio-verfahrcnstcchnik, Nobclstr. 12, D-7000 Stuttgart RO, FRG

Offprint requests to: B. Schink

sence of molecular oxygen, and to ferment the methyl residues, analogous lo methanol, with C02 to acetate (Bache and Pfennig 1981). Similar findings were made later on with other homoacetogenic bacteria (Krumholtz and Bryant 1985; Frazer and Young 1985, 1986; Mountfort and Asher 1986) and even with an Enterobacter strain (Grbic-Galic and LaPat-Polasko 1985). All these bacteria could remove the methyl groups only from aromatic methyl ethers; cleavage of methyl alkyl ethers was never observed.

Another case of anaerobic ether cleavage was observed with polyethylene glycol degradation by Pelobacter ve-netianus (Schink and Stieb 1983). The first degradation intermediate detected was acetaldehyde, suggesting that the terminal hydroxyl group of the polymer is shifted to the subterminal carbon atom, analogous to a diol dehydratase reaction, which in this case transforms the ether linkage again into a hemiacetal bond (Straß and Schink 1986). It was shown by feeding experiments that anaerobic polyethylene glycol degradation depended on a free terminal hydroxyl function, and that terminally methylated polyethylene glycols were not degraded. A different pathway of anaerobic polyethylene glycol degradation was assumed to occur in other fermenting and sulfate-reducing bacteria, however, the ether cleavage reaction was not studied (Dwyer and Tiedje 1986).

In the present study, we chose methoxyacetate as a model substrate of a methylalkylether which cannot be attacked by a diol dehydratase because it does not contain a free alcohol function. New strains of homoacetogcnic bacteria were isolated which degraded methoxyacetate, and very slow anaerobic degradation was also found in enrichment cultures with dimethoxyethane.

Materials and methods

Sources of organisms

Strains KoMacl and GoMacl were enriched and isolated from anoxic digestor sludges of municipal sewage plants at Gottingen and Konstanz, FRG, respectively. Strain RMMacl was isolated from a saltwater sediment sample taken from Rio Marin in the city of Venice, Italy. An enrichment culture degrading dimethoxyethane (culture WoDME) was obtained with an inoculum from a polluted freshwater creek near Konstanz, FRG.

Media and growth conditions

All procedures for cultivation and isolation as well as all methods for analysis of metabolic products were essentially as described in earlier papers (Widdel and Pfennig 1981; Schink and Pfennig 1982). The mineral medium for enrichment, isolation and cultivation contained 30 mM sodium bicarbonate as buffer, 1 mM sodium sulfide as reducing agent, the trace element solution SL 10 (Widdel et al. 1983) and a vitamin solution (Pfennig 1978). Freshwater medium contained 0.5 g NaCl and 0.4 g MgCl2 x 6 H20, saltwater medium 20.0 g and 3.0 g, respectively, per liter. The pH was 7.1 — 7.3. Growth experiments were carried out at 30°C. For isolation of pure cultures, the agar shake dilution method was applied (Pfennig 1978). Gram staining was carried out after Magee et al. (1975); the Gram type was also checked by the KOH method (Gregersen 1978).

Chemical determinations and chemicals

Sulfide was determined after Cline (1969). nitrite after Pro-chazkova (1959), protein after Zamcnhoff (1957), carbon monoxide dehydrogenase after Diekert and Thauer (1978). Fatty acids and methoxyacetate were determined by direct gas chromatography on a packed column (Schink and Pfennig 1982), glycolic acid on a glas capillary column after methylation (Dehning and Schink 1989).

All chemicals used were of analytical or reagent grade quality and were obtained from Merck, Darmstadt, Sigma, Miinchen, and Fluka, Neu-Ulm, FRG.

Results

Enrichment and isolation

Enrichment cultures with freshwater or saltwater medium containing 10 mM methoxyacetate were inoculated with about 5 ml sediment and sludge samples from various sources, including marine and freshwater sediments, as well as sewage sludge from two municipal sewage plants. Gas production started m all cases after 3 — 5 weeks, and subcultures were subsequently inoculated with 5 ml culture fluid, usually after 2-3 weeks of incubation. Pure cultures were isolated from these cultures by two subsequent agar shake dilution series with methoxyacetate as substrate.

Freshwater cultures formed yellowish lens-shaped colonies, marine cultures small white colonies in the agar. Pure

cultures were finally obtained with the enrichment cultures from two sewage plants (strains KoMacl and GoMacl) and from Rio Marin, Venice (strain RMMacl). Purity was chccked by microscopical examination after growth in minimal medium and in complex medium (AC- medium, Difco, Ann Arbor, Michigan, USA).

Similar enrichment cultures with dimethoxyethane (ethylene glycol dimethyl ether) exhibited gas production only in one case (sediment from a polluted freshwater creek) after 5 months of incubation. The dimethoxyethane-degrading bacteria could be transferred over 16 subcultures with cultivation times of 4 —6 weeks every time. Pure cultures could not be isolated. In the enrichment cultures, complete conversion to CH4 and CO2 with intermediate formation of acetate could be demonstrated (data not shown).

Cytological properties of pure cultures

The two freshwater strains KoMacl and GoMacl were motile rods, 0.9 —1,0 x 2.0—4.0 nm in size, with slightly pointed ends (Fig. 1 a, b). In older cultures, they tended to form chains of several cells which were sometimes swollen in the middle. Both strains stained Gram-positive; results of the KOH test indicated a Gram-positive cell wall architecture as well. Cells of strain RMMacl were immotile, rather small rods, 0,5—0.7x 1 — 1,5 |rm in size, with rounded ends (Fig. 1 c). They stained Gram-negative, but both the KOH test as well as electron microscopic examination indicated a Gram-positive cell wall architecture (Fig. 2). In Indian ink preparations, thin slime capsules became visible. No strain formed spores, neither in defined medium nor in a special sporulation medium (Hollaus and Sleytr 1972). Pasteurized cultures did not show growth after more than 6 weeks of incubation. The guanine-plus-cytosine content of the DNA was 42.7 ± 1.0 mol% with strain KoMacl, 44.4 ± 1.0 mol% with strain GoMacl, and 47.8 ± 1.0 mol% with strain RMMacl. No cytochromes could be detected by redox difference spectroscopy of crude cell extracts of either strain.

Physiology

The strains KoMacl and RMMacl grew well in mineral medium containing NaCl from 0.05 up to 2,0% and MgCl2 x 6 H20 from 0.04 to 0.3 % (w/v), strain GoMacl grew in medium with 0.05-1.5% NaCl and 0.04-0.4% MgCl2 x 6 H20. Phosphate inhibited growth of strains

> » ^ J s ^ r / r

fc \ * ** *

Fig. la — c

Phase-contrast photomicrographs of cells of the new isolates, a Strain KoMacl, b GoMacl, c strain RMMaci. Bar equals 10 urn for all three pictures

Fig. 2. Electron micrograph of an ultrathin section of cells of strain RMMacl. Bar equals 0.2 urn

TIME Id)

Fig. 3. Fermentation time course of strain KoMacl growing with methoxyacetate. Experiments were performed at 30 ' Cin half-filled 100 ml culture bottles with rubber septa. Samples were taken at times indicated and the headspaces were flushed with N3/C02 gas mixture. (•) cell density (oD540); (■) methoxyacetate; (♦) acetate

Tabic 1. Substrates tested for growth with the new strains KoMacl. GöMacl, and RMMacl. All tests were carricd out in mineral medium with 0.05% (w/v) yeast extract

Substrate degraded KoMacl GöMacl RMMacl

h2/co2 + 4- +

Formate + + —

Hexamethylene tetramine + - —

Methanol + + +

Methoxyacetate + + +

Vanillate + + +

Syringale + + _

Sinapinate + + _

Trimethoxybcnzoate + + —

Betaine + + +

Ethanol ± - —

Lactate + + —

Pyruvate + 1 +

D,L-Malate + +

Fumarate — — +

Fructose + + —

Glycerol + + —

Triacetin + —

F.thylene glycol + + —

Di ethylene glycol + + —

Tri ethylene glycol + — —

Ethoxyethanol + + —

KoMacl and RMMacl at > 30 mM, of strain GoMacl at >10 mM concentration. Yeast extract was not required for growth, but all strains grew faster and to higher cell densities in the presence of yeast extract. Optimal growth with methoxyacetate was found at 30CC with the following growth rates and doubling times. Strains GoMacl and KoMacl: ft = 0.049 h~l, fd = 14 h; strain RMMacl: n = 0.037 h \ — 19 h. The temperature limits of growth were 15 and 34°C, the pH optima 6.8-7.0, and the pH ranges from 6.0 — 8.0 with the freshwater isolates and 6.6 — 7.4 with strain RMMacl.

The substrates utilized by the various strains are listed in Table 1. Besides those substrates used by many other homoacetogenic bacteria, methoxyacetate and betaine were found to be used as growth substrates. The methoxylated aromatic compounds were fermented to the corresponding phenols. UV spectrophotometry examination indicated that the acrylic acid side chain of sinapic acid was reduced to trihydroxyphenylpropionic acid. Hexamethylcne tetramine was used as a formaldehyde-yielding substrate; many homoacetogenic bacteria ferment formaldehyde to acetate (Schink 1987). Neither nitrate, sulfate, sulfite, thiosulfate, nor sulfur was reduced during growth with methoxyacetate. Strain KoMacl appeared to be the metabolically most versatile isolate. In Fig. 3, a growth curve of this strain growing with methoxyacetate is shown. Similar results were obtained with the other isolates.

Acetate was the main or sole fermentation product in all cases. Fermentation balances and growth yields were measured with all isolates. In Table 2, the data obtained with strain KoMacl are presented. Similar results were obtained with the other strains as well The amounts of products formed indicated that the substrates were fermented completely. The methyl groups of all methoxyaromatics, methoxyacetate, and betaine were fermented with C02 to acetate, in the same manner as methanol, but the cell yields obtained were by nearly 50% higher than with methanol.

Carbon monoxide dehydrogenase activity was detected in crude extracts of cells of all three isolates grown with methoxyacetate. The specific activities [ umol CO oxidized per min and mg protein) were 1.1 with strain KoMacl, 2.2 with strain GoMacl, and 0.37 with strain RMMacl.

Discussion

Physiology and taxonomy of the new isolates

In the present study, anaerobic degradation of melhylalkyl ether compounds is documented for the first time. Three strains of homoacetogenic bacteria are described which ferment methoxyacetate with carbon dioxide to glycolate and acetatc, according to the following equation (all calculations of free energy changes after Thaucr ct al.; 1977; Dimroth 1983):

2 C02 + 2 H20

3 CH3COO + 3H4

No growth was found with the following substrates: Di-methoxyethane, diethoxyethanc, 1-propanol, 1-butanol, glucose, ar-abinose, xylose, glycolate. glycine, ethanolamine, trimethylamine, citrate. D-erythritol

4 CH3OCH2COO" 4HOCH2COO"

zlG'o - -85.2 kJ per mol methoxyacetate .

The free energy change of this reaction is considerably higher than that of the analogous fermentation of methanol and COj to acetate and water (/1Gb ~ - 55.5 kJ per mol methanol).

Table 2. Stoichiometry of substrate conversion and growth yields of strain KoMacl with various substrates

Substrate degraded Amount of Cell dry matter Acetate Acetate formed Growth yield Electron

substrate supplied formed a assimilated11 (|jmol) (g/mol) recovery

(|iiii(ilj (mg) (|imol) (%)

Formate 440 0.52 10.6 103.4 1.2 104

Hexamethylene tetramine 44 1.5 30.8 94.4 5.7 95

Methanol 110 0.72 14.7 65.3 6.5 97

Methoxyacetate 220 1.9 39.2 1237.6 8.6 101

Vamllate 110 1.05 19.8 61.6 9.5 99

Syringate 110 2.3 47.1 132 20.8 109

Sinapinate 110 2.1 42.9 114.4 19 95

Trimethoxybenzoate 110 3.1 63.8 206 27.9 109

Betaine 220 2.16 44 144.5 9.8 114

Lactatc 220 2.16 44 320.5 9.8 110

Pyruvate 220 2.35 48.4 226 10.7 100

D,L-Malate 220 1.44 28.6 142.3 13.0 104

Fructose 44 1.18 24.2 108.5 26.8 101

Glycerol 220 2.18 28.6 151.1 6.5

Triacetin 110 2.06 41.8 464.2 18.7 97

Ethylene glycol 220 0.98 19.8 261.1 4.5 102

Diethylene glycol 220 1.83 37.4 530.2 8.3 103

Triethylene glycol 220 2.18 52.8 675.4 9.9

Ethoxyethanol 220 0.85 17.4 259.6 3.9 101

All growth experiments were carried out in scrcw cap tubes containing 22 ml mineral medium with 0.05% yeast extract 3 Cell dry matter values were calculated via cell density using an experimentally determined conversion factor (0.1 0D540 = 29.7 mg dry matter/1)

11 Acetate assimilated was calculated according to the formula: 17 CH3COO - 17 II1 8 <QII-.0-) 4- 2 CO, + 6 H20

The product of betaine fermentation was not identified. From the stoichiometry of acctatc formation, it appears that only one methyl group per substrate molecule was fermented and therefore dimethylglycine was formed as dcmethylatcd product:

4 (CH3)3N+CH2COO + 2 C02 + 2HaO 4 (CH3)2N+HCH2COO + 3CH3C00 + 3H+ .

A similar methyl group fermentation pattern was found with Eubacterium limosum (Mtiller et al. 1981) and Aceto-bacterium carbinolicum (Eichler and Schink 1984).

The newly isolated homoacetogens differ from those of the species described so far by their ability to demethylate methoxyacetate to glycolate. Our freshwater isolates resemble A. carbinolicum most among the described types of homoacetogens, also with respect to triacctin utilization (Emde and Schink 1987), and we suggest that they should be grouped with this species as new biotypes. The taxonomic status of the morphologically atypical marine isolate RMMac! would require further studies. Strain KoMacl was deposited with the Deutsche Sammlung fur Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.

Biochemistry of anaerobic methylether cleavage

The chemistry of anaerobic phenylmethyl ether cleavage by homoacetogenic bacteria is not yet understood. Ether linkages are too stable to be split by a simple hydrolysis reaction. Studies on kinetics and yields of growth of A. woodii on phenylmethyl ether compounds and on methanol indicated that free methanol is not an intermediate (Tschech and Pfennig 1984). Degradation experiments with Eubacterium limosum on lsO-labelled methoxybenzoates demonstrated recently that the ether oxygen stays with the aromatic ring and is not released into acetatc or water (deWeerd

et al. 1988). This supports the concept that the ether linkage is not cleaved by a hydrolysis reaction but that the methyl residue is transferred directly to a methyl carrier such as coenzyme B12, possibly as a methyl radical. A similar chemistry of ether linkage cleavage must be involved in methoxyacetate degradation by our new isolates.

It is open at present whether ail the demethylation reactions carried out by our new isolates are catalyzed by the same enzyme system. From the fermentation stoichiometries and growth yields it is obvious that methyl groups bound to nitrogen or ether oxygen give about 50% higher growth yields than methanol, in close agreement with the free energy changes of these reactions (see above). This rules out again a formation of free methanol as intermediate in demethylation and indicates that methanol, unlike the linked methyl groups, has to be transferred to a methyl carrier by an energy-consuming system. Cross-feeding experiments with cell suspensions grown with either methyl substrate indicated that separate demethylating activities had to be induced for methanol, vanillic acid, methoxyacetate, and betaine, respectively (data not shown). However, since these specificities may as well be due to different substrate uptake systems, these results cannot rule out that a common enzyme system may be responsible for the respective demethylation and methyl group transfer reactions.

Anaerobic demethylation of methoxyacetate by our new isolates opens a further pathway for methanogenic degradation of methoxy ethanol, an important industrial solvent. In anoxic sludge degrading this substrate, methoxyacetate was found and was assumed to be an intermediate product in a degradation pathway leading from methoxyethanol via 2-methoxyacetate and glycolate to methane plus carbon dioxide, however, bacteria catalyzing such a reaction sequence could not be cultivated (Tanaka et al. 1986). Instead, 2-methoxyethanol-degrading homoacetogenic and other fer-

meriting bacteria isolated from this culture were found to use a different pathway similar to that of anaerobic polyethylene glycol degradation: they formed acetate and free methanol, probably via 1-methoxyethanol as intermediate (Tanaka and Pfennig 1988). Our new isolates prove that the pathway first suggested is possible as well, and perhaps is used in the same culture to a minor extent. It is worth emphasizing that in both hypothetical pathways coenzyme B12 plays a key role in ether destabilization or ether cleavage.

The freshwater strain KoMacl was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.

Acknowledgement. The authors are indebted to Prof. Dr. F. Mayer and his coworkers for electron microscopic examination of strain RMMacl.

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Received August 22, 1989/Acccpted October 10, 1989