Isolation from a Shea Cake Digester of a Tannin ... - Springer Link

CURRENT MICROBIOLOGY Vol. 44 (2002), pp. 341–349 DOI: 10.1007/s00284-001-0020-x

Current Microbiology An International Journal © Springer-Verlag New York Inc. 2002

Isolation from a Shea Cake Digester of a Tannin-Tolerant Escherichia coli Strain Decarboxylating p-Hydroxybenzoic and Vanillic Acids Mohamed Chamkha,1 Eric Record,2 Jean-Louis Garcia,1 Marcel Asther,2 Marc Labat1 1

Laboratoire de Microbiologie IRD,IFR-BAIM, Universite´s de Provence et de la Me´diterrane´e, ESIL case 925, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France. 2 Laboratoire de Biotechnologie des Champignons Filamenteux INRA, IFR-BAIM, Universite´s de Provence et de la Me´diterrane´e, ESIL case 925, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France Received: 30 July 2001 / Accepted: 17 August 2001

Abstract. A facultatively anaerobic, mesophilic, Gram-negative, non-motile, non-sporulated bacterium, designated strain C2, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds and previously inoculated with anaerobic sludge from the pit of a slaughterhouse, after enrichment on tannic acid. The straight rods occurred singly or in pairs. Strain C2 fermented numerous carbohydrates (fructose, galactose, glucose, lactose, mannose, maltose, melibiose, raffinose, rhamnose, ribose, saccharose, sorbitol, trehalose, and xylose) and peptides (Biotrypcase, Casamino acids, and yeast extract), producing acid and gas, and had a G ⫹ C content of 51.6 ⫾ 0.1 mol %. Strain C2 was very closely related to Escherichia coli (⫽ DSM 30083T) phylogenetically (similarity of 99%), genotypically (DNA homology of 79%), and phenotypically. The isolate tolerated tannic acid (hydrolyzable tannin) and decarboxylated by non-oxidative decarboxylation only p-hydroxybenzoic and vanillic acids to their corresponding phenol and guaicol, under anaerobic and aerobic conditions without further degradation. Adding glucose increased growth and the rate of conversion. High concentrations of p-hydroxybenzoic acid or vanillic acid inhibited growth, and decarboxylation could not occur completely, suggesting phenol toxicity. In contrast, the type strain of E. coli cannot metabolize p-hydroxybenzoic and vanillic acids, anaerobically or aerobically, with or without glucose added.

Tannins are water-soluble polyphenols that differ from other natural phenolic compounds by their ability to precipitate proteins from solution. They are common in a large array of herbaceous and woody plants. Their molecular weight ranges from 500 to 3000 g mole⫺1 [40]. Two groups are distinguished according to their structure: hydrolyzable and condensed tannins [49]. Hydrolyzable tannins are mainly polyesters of 3,4,5-trihydroxybenzoic acid (gallic acid) or gallotannins, also containing low amounts of gallic acid dimer, ellagic acid (ellagitannins). They are readily hydrolyzed chemically by acidification or biologically by an enzyme known as tannase (Tannin acyl hydrolase EC 3.1.1.20) [3]. The biodegradability of hydrolyzable tannins depends to some extent on the tannin type [18]. Condensed tannins are polymers of flavonoid-type monomers that are linked together by covalent bonds between the C4 and C8. They Correspondence to: M. Labat; email: [email protected]

are not readily hydrolyzed. Tannic acid is a gallotannin consisting of esters of gallic acid and glucose, containing up to five galloyl groups esterified directly to the glucose molecule, with additional galloyl groups esterified to the core galloyl groups [42]. Tannins present in industrial effluents are environmental pollutants because of their high toxicity [40] and their resistance to biological degradation. They have negative impacts in aquatic environment. They are also toxic to microorganisms involved in biological methods of wastewater treatment. Concentrations of tannins ranging from 325 to 3000 mg L⫺1 inhibit methanogenic bacteria, which are the key microorganisms involved in anaerobic treatment systems and nitrifying bacteria, which are important for the wastewater post-treatment. The presence of high concentrations of tannin (1–2%) reduces the overall decomposition of organic materials applied to the soil [18]. The growth of Clostridium

342 cellulosolvens, Cellvibrio fulvus, Bacillus subtilis, and Sporocytophaga myxococcoides is inhibited by tannic acid at 10, 12, 30, and 45 mg L⫺1, respectively [29]. Tannins inhibit Azotobacter spp. and Escherichia coli respiration [4]. Various filamentous fungi including Aspergillus niger, Penicillium sp., Trichoderma viride, Botrytis cinerea, and yeasts including Saccharomyces cerevisiae have also been reported to be susceptible to the toxic effects of tannins [40]. On the other hand, microorganisms degrading or tolerating tannins have been isolated. Soil fungi belonging to the genera Aspergillus and Penicillium, yeasts of the genus Pichia, and bacteria of the genera Klebsiella, Bacillus, Corynebacterium, and Achromobacter are the most frequently observed degraders of hydrolyzable tannins [18]. Aerobic degradation of tannins is largely reported; interest in the anaerobic degradation of hydrolyzable tannins has resulted from the increasing application of anaerobic systems for the treatment of tannin-containing wastewaters. Several species of bacteria degrading trihydroxyphenols in pure anaerobic culture have been isolated. They include Pelobacter acidigallici [41], Coprococcus sp. [44, 45], and Eubacterium oxidoreducens [24]. Streptococcus gallolyticus, a facultatively anaerobic bacterium, hydrolyzes tannins and releases gallic acid, which is then decarboxylated to pyrogallol by gallate decarboxylase without further degradation [35]. During wastewater treatment, performed in continuous anaerobic reactor fed with shea cake, high removal rates of tannins and production of organic acids and methane were consistently observed [37]. Previously we isolated from this anaerobic digester a strain of Streptococcus gallolyticus capable of hydrolyzing tannic acid (hydrolyzable tannin) and decarboxylating gallic acid to pyrogallol (data not published). Here we report on the isolation and the characterization of a strain of Escherichia coli that tolerated tannic acid and decarboxylated, by non-oxidative decarboxylation, only p-hydroxybenzoic and vanillic acids to their corresponding phenol and guaiacol, under anaerobic and aerobic conditions without further degradation. Materials and Methods Source of strains. Strain C2 (⫽ DSMZ 14166) was isolated from an anaerobic digester fed with shea cake situated in Burkina Faso. The digester has previously been inoculated with anaerobic sludge from the pit of a slaughterhouse and contained tannins and various aromatic compounds as carbon and energy sources. The type strain of Escherichia coli (⫽ ATCC 11775T ⫽ DSM 30083T) used as reference culture was obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). Culture media. The anaerobic techniques of Hungate [19, 25, 28] were used throughout this work. The basal medium contained (L⫺1): 0.4 g NH4Cl, 0.5 g KH2PO4, 0.4 g NaCl, 0.33 g MgCl2 䡠 6H2O, 0.05 g

CURRENT MICROBIOLOGY Vol. 44 (2002) CaCl2 䡠 2H2O, 0.25 g cysteine-HCl, 2 g yeast extract (Difco), 1 ml trace element mineral solution [47], and 1 mg resazurin. The pH was adjusted to 7 with 10 M KOH solution; the medium was then boiled under a stream of O2-free N2 gas and cooled to room temperature. Five-ml aliquots were dispensed into Hungate tubes, degassed under N2-CO2 (80:20%, vol/vol) and subsequently sterilized by autoclaving at 110°C for 45 min. Prior to inoculation, 0.05 ml of 10% (wt/vol) NaHCO3 and 0.05 ml of 5% (wt/vol) Na2S 䡠 9H2O were injected from sterile stock solutions. Substrates were injected from concentrated anaerobic sterile stock solutions to obtain the desired final concentration. Escherichia coli (⫽ DSM 30083T) was grown aerobically at 37°C using LB medium containing (L⫺1): 10 g tryptone, 5 g yeast extract, and 10 g NaCl. The pH was adjusted to 7.0 with 10 M KOH solution. Enrichment and isolation procedure. The liquid samples (0.5 ml) taken from the digester were inoculated into 5 ml basal medium containing 1 g L⫺1 tannic acid (Aldrich) and then incubated at 37°C. The enrichment culture that developed was subcultured several times under the same conditions prior to isolation. For isolation purposes, the culture was serially diluted tenfold, and the few single, well-isolated colonies that developed in roll tubes (basal medium containing 1 g L⫺1 tannic acid and 1.6% agar) were picked and serially diluted in fresh media. This procedure was repeated until only one type of colony was observed. Purity was checked by microscopy on cultures grown in basal medium amended with 10 mM glucose and 0.2% Biotrypcase. The isolate was maintained in basal medium containing 1 g L⫺1 tannic acid. Morphology. Light and electron microscopy were performed as previously described [15]. Growth parameters. For all experiments, basal medium containing 0.2% yeast extract and 10 mM glucose was used. The pH of the pre-reduced anaerobic medium was adjusted with 5% NaHCO3, 5% Na2CO3, or 0.1 M HCl to obtain a range of initial pH between 5.0 and 10.0. Different amounts of NaCl were weighed directly in Hungate tubes prior to dispensing 5 ml medium to obtain the desired NaCl concentration (range 0 –90 g L⫺1). The temperature range for growth was determined between 15° and 50°C. Electron acceptors. Sulfate, thiosulfate, sulfite (20 mM), and elemental sulfur (2% wt/vol) were tested as electron acceptors in basal medium containing 10 mM glucose. Substrate utilization. Experiments were performed in duplicate with an inoculum subcultured at least once under the same test conditions. The substrates tested for utilization were injected into Hungate tubes that contained 5 ml presterilized basal medium, from presterilized and concentrated stock solutions. The following substrates were used: 20 mM carbohydrates (arabitol, cellobiose, fructose, galactose, glucose, lactose, mannose, maltose, melibiose, melezitose, myo-inositol, raffinose, rhamnose, ribose, saccharose, sorbitol, sorbose, trehalose, and xylose); 20 mM organic acids (acetate, adipate, butyrate, citrate, crotonate, formate, isobutyrate, lactate, propionate, succinate, and valerate); 20 mM alcohols (butanol, ethanol, isobutanol, isopropanol, methanol, and propanol); 10 g L⫺1 proteins/peptides (Biotrypcase, Casamino acids, gelatin, and yeast extract), 20 mM phenylalanine, and 5 mM of the following aromatic compounds: benzoate; hydroxylated benzoic acids [m-hydroxybenzoate, p-hydroxybenzoate, 3,4-dihydroxybenzoate (protocatechuic acid), 3,5-, 2,4,- 2,6-dihydroxybenzoates (␣-, ␤-, ␥-, resorcylic acids), 2,4,6-trihydroxybenzoate (phloroglucinolcarboxylic acid), and 3,4,5-trihydroxybenzoate (gallic acid)]; methoxylated benzoic acids [2,4-, 2,6-, 3,5-dimethoxybenzoates, 3,4dimethoxybenzoate (veratric acid), and 3,4,5-trimethoxybenzoate]; mixed hydroxylated/methoxylated benzoic acids [4-hydroxy-3-me-

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M. Chamkha et al.: Decarboxylation of Aromatics by E. coli thoxybenzoate (vanillic acid), 3-hydroxy-4-methoxybenzoate (isovanillic acid), and 4-hydroxy-3,5-dimethoxybenzoate (syringic acid)]; 3,4-dimethylbenzoate; 3,4,5-trihydroxybenzene (pyrogallol), 2,4,6-trihydroxybenzene (phloroglucinol); cinnamic acids [cinnamate, o-, m-, p-hydroxycinnamate (coumaric acid), 3,4-dihydroxycinnamate (caffeic acid), and 4-hydroxy-3-methoxycinnamate (ferulic acid)]; and phenylpropionic acids [3-phenylpropionate (hydrocinnamic acid) and 3,4dihydroxyphenylpropionate (hydrocaffeic acid)]. Concentrated stock solutions were prepared, neutralized if necessary, rendered anaerobic by gassing with O2-free N2, and sterilized by filtration (pore size 0.2 ␮m; Millipore). Aromatic compounds were tested with or without 5 mM glucose. Autotrophic growth was tested with H2/CO2 (20:80%, vol/vol) at a final pressure of 2 bars. An increase in OD580 in tubes containing added substrates, compared with control tubes lacking a substrate, was considered to be positive growth. The type strain of E. coli (⫽ DSM 30083T) was tested for its ability to metabolize p-hydroxybenzoic, vanillic, m-hydroxybenzoic, 3,4-dihydroxybenzoic, and isovanillic acids at 5 mM, anaerobically in basal medium and aerobically in LB medium, with or without 5 mM glucose added. Analytical techniques. Bacterial growth was monitored by measuring OD580 directly from anaerobic Hungate tubes inserted into the cuvette holder of a spectrophotometer (Shimadzu UV 160A). Aromatic compounds were measured by HPLC by using a chromatograph (Consta Metric 200; LDC-Analytical) equipped with a C18 symmetry 5 ␮mparticle-size column 250 mm long, 4.6 mm inside diameter (Waters Chromatography). The column temperature was maintained at 35°C. An isocratic mobile phase of 30:69.5:0.5 (by vol) acetonitrile/distilled water/acetic acid was used at a flow rate of 0.6 ml min⫺1. The volume of the injection loop was 25 ␮l. Aromatic compounds were quantified at 240 nm with a Shimadzu SPD-6A UV detector connected to a CR-6A Shimadzu integrator. Carbohydrates, volatile fatty acids, and alcohols were measured by HPLC (Spectra Series 100 model; Thermo Separation Products) equipped with an Aminex HPX-87X 300 mm long, 7.8 mm i.d. column (Biorad) connected to a differential refractometer (RID-6A, Shimadzu). Analysis was performed with a CR-6A Shimadzu integrator. The mobile phase was 2.5 mM H2SO4 at a flow rate of 0.5 ml min⫺1, and the column temperature was 35°C. The volume of the injection loop was 20 ␮l. H2 and CO2 were measured as previously described [14]. Determination of G ⴙ C content. The G ⫹ C content of DNA was determined by the DSMZ. The DNA was isolated and purified by chromatography on hydroxyapatite, and the G ⫹ C content was determined by using HPLC [27]. Non-methylated lambda DNA (Sigma) was used as the standard. DNA-DNA hybridization. DNA was isolated by chromatography on hydroxyapatite by the procedure of Cashion et al. [8]. DNA-DNA hybridization was performed at the DSMZ as described by De Ley et al. [12], with the modification described by Escara and Hutton [13] and Huss et al. [20] by using a Gilford System model 2600 equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program by Jahnke and Bahnweg [22] and Jahnke [21]. DNA extraction and 16S rRNA gene amplification. DNA was extracted from the isolate as described by Redburn and Patel [39] and Andrews and Patel [2]. The universal primers Fd1 and Rd1 were used to obtain a PCR product of approximately 1.5 kb corresponding to base position 8 to 1542 based on Escherichia coli numbering of the 16S rDNA [48]. A 50-␮l reaction contained 1–20 ng of genomic DNA, 1 ␮M of each primer, 5 ␮l of 10⫻ buffer, 200 ␮M of dNTP, 3.5 mM of MgCl2, and 2.5 U of Taq polymerase (Promega). PCR was carried out

by an initial denaturation at 94°C for 7 min, then 29 cycles of annealing at 55°C for 2 min, extension at 72°C for 4 min, denaturation at 94°C for 1 min, and finally an extension cycle of 55°C for 2 min and 72°C for 20 min. Direct PCR products sequencing. PCR products were purified with a QIAquick Kit (Qiagen). The DNA concentration of purified PCR product was estimated by comparison with the Low Mass Ladder (Gibco-BRL) on an agarose gel containing ethidium bromide. Sequencing was performed on an ABI 373A sequencer using the ABI PRISM Dye Terminator Cycle Sequencing Kit containing AmpliTaq FS DNA polymerase under the following conditions. A 10-␮l reaction contained 35 ng of PCR product, 4 ␮l of cycle sequencing reaction mix, 3.2 pmol of primer [2], and 2.5 ␮g of BSA. Thermal cycling was performed with a Rapid Cycler (Idaho Technology) at a temperature transition slope of 2, an initial denaturation of 94°C for 15 s, then 25 cycles of denaturation at 94°C for 0 s, annealing at 50°C for 10 s, extension at 60°C for 3 min. Sequence alignments and phylogenetic inferences. The new sequence data that were generated were aligned to an almost full-length consensus 16S rRNA gene sequence assembled and checked for accuracy manually by using the alignment editor, ae2 [26]. These were compared with other sequences in the GenBank database [5] using BLAST [1], and in the Ribosomal Database Project, version 7.0 using SIMILARITY_RANK and SUGGEST_TREE [26]. Pairwise evolutionary distances based on 1235 unambiguous nucleotides were computed using DNADIST [23] and NEIGHBOR-JOINING programs that form part of the PHYLIP suite of programs [16]. TREECON was used extensively for bootstrap analysis [46]. Nucleotide sequence accession number. The GenBank accession number for the 16S rRNA gene sequence of strain C2 is AF403733.

Results Enrichment and isolation. Enrichment cultures developed in medium containing 1 g L⫺1 tannic acid within 3 weeks of incubation at 37°C, as shown by growth and organic acid production. After several transfers in the liquid medium, a stable microbial population tolerating tannic acid developed. Several isolates were obtained by using the roll-tube method [19], and one of these, designated strain C2, was studied further. Morphology. Cells of strain C2 were Gram-negative, straight rods (1.0 –1.3 ⫻ 2.1– 6.0 ␮m), non-motile, and occurred singly or in pairs. Spores were not observed. Physiology. Strain C2 is a mesophilic and facultative anaerobe. The temperature range for growth was 20 – 45°C, with optimal growth occurring at 37°C. No growth occurred at 15 and 50°C. The pH range for growth was between pH 6.0 and 9.6 with an optimum at 8.0 – 8.8. NaCl concentration range for growth was between 0 and 80 g L⫺1. Yeast extract stimulated but was not required for growth. Strain C2 fermented a wide range of carbohydrates including fructose, galactose, glucose, lactose, mannose, maltose, melibiose, raffinose, rhamnose, ribose, saccharose, sorbitol, trehalose, and xylose, produc-

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ing formate, acetate, lactate, succinate, ethanol, H2, and CO2 as end products. Peptides including Biotrypcase, Casamino acids, and yeast extract were fermented into formate, acetate, succinate, and CO2. The following compounds were not used by isolate C2: arabitol, cellobiose, melezitose, myo-inositol, sorbose, acetate, adipate, butyrate, citrate, crotonate, formate, isobutyrate, lactate, propionate, succinate, valerate, butanol, ethanol, isobutanol, isopropanol, methanol, propanol, phenylalanine, and H2/CO2. Gelatin was not hydrolyzed. Sulfate, thiosulfate, sulfite, or elemental sulfur could not be used as electron acceptors. G ⴙ C content, 16S rRNA sequence analysis, and DNA-DNA relatedness. The G ⫹ C content of strain C2 was 51.6 ⫾ 0.1 mol% as determined by the HPLC method. Phylogenetic analysis revealed that strain C2 was a member of the Gram-negative Enterobacteriaceae family in the ␥ subdivision of the class Proteobacteria in the domain Bacteria. Strain C2 was found to be closely related to E. coli (99% of similarity between the two 16S rRNA sequences). The level of DNA-DNA relatedness between strain C2 and E. coli (⫽ DSM 30083T) was 78.9%. Metabolism of aromatic compounds. Strain C2 did not hydrolyze tannic acid (hydrolyzable tannin) at 1 g L⫺1, with or without 5 mM glucose added. But its growth was not inhibited by 1 and 8.5 g L⫺1 tannic acid. Tannin concentrations greater than 20 g L⫺1 inhibited completely the growth of strain C2. Gallic acid, a monomer of hydrolyzable tannic acid, was not metabolized, with or without glucose added. The anaerobic metabolism of other benzoic acid derivatives was then studied with strain C2, with or without supplementation of glucose. Only p-hydroxybenzoic acid (4-hydroxybenzoate) and vanillic acid (4hydroxy-3-methoxybenzoate) were decarboxylated to phenol and to guaiacol, respectively, producing CO2 (Fig. 1). The corresponding phenol and CO2 were the end products, and the aromatic ring was not degraded after 1 month of incubation. Decarboxylation of p-hydroxybenzoic acid occurred faster than that of vanillic acid (Fig. 2). Sixteen hours were sufficient for strain C2 to decarboxylate half of the p-hydroxybenzoic acid added in the medium (Fig. 2a). In contrast, 35 h were needed to decarboxylate half of the added vanillic acid. Addition of 5 mM glucose accelerated by about two-times these conversions (Fig. 2b). By contrast, benzoate; hydroxylated benzoic acids (m-hydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-, 2,4-, 2,6-dihydroxybenzoates, and 2,4,6-trihydroxybenzoate); methoxylated benzoic acids (2,4-, 2,6-, 3,4-, 3,5-dimethoxybenzoates, and 3,4,5-trimethoxybenzoate); mixed hydroxylated/methoxylated benzoic acids

Fig. 1. Metabolism of p-hydroxybenzoic and vanillic acids by strain C2.

(3-hydroxy-4-methoxybenzoate and 4-hydroxy-3,5-dimethoxybenzoate); and 3,4-dimethylbenzoate were not metabolized by strain C2, with or without glucose added. In addition, the ability of strain C2 to decarboxylate cinnamic and phenylpropionic acids, with or without glucose added, was studied. Cinnamic compounds including cinnamic, o-, m-, p- coumaric, caffeic, and ferulic acids, and phenylpropionic acids including hydrocinnamic and hydrocaffeic acids were not metabolized. Other phenols tested, including pyrogallol and phloroglucinol, were not used by strain C2, with or without glucose added. Metabolism of p-hydroxybenzoic and vanillic acids, under aerobic conditions, was similar but faster than that observed under anaerobic conditions (Fig. 3). The metabolism implied non-oxidative decarboxylation of phydroxybenzoic and vanillic acids to their corresponding phenols, i. e., phenol and guaiacol (Fig. 1). Our experiments revealed that 7 h and 9 h were enough to decarboxylate half of the added p-hydroxybenzoic acid and vanillic acid respectively under aerobic conditions (Fig. 3a). Adding glucose accelerated this decarboxylation, i. e., less than 6 h was needed to perform both decarboxylations of half of the added hydroxybenzoic acids (Fig. 3b). The amount of phenol and guaiacol produced by strain C2 equalled that of p-hydroxybenzoic acid and vanillic acid decarboxylated, in anaerobiosis (Fig. 2) and aerobiosis (Fig. 3), respectively. The ratio of phenols to their corresponding benzoic acid derivatives in the medium increased during the course of the experiment until substrate utilization was complete. With all aromatic compounds tested and even after 1 month of incubation, no ring cleavage was observed.

M. Chamkha et al.: Decarboxylation of Aromatics by E. coli

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Fig. 2. Anaerobic growth of strain C2 in basal medium containing 5 mM p-hydroxybenzoate and 5 mM vanillate, without (a) and with (b) 5 mM glucose added. Symbols: 䊐, concentration of p-hydroxybenzoate; ■, concentration of phenol; E, concentration of vanillate; F, concentration of guaiacol; Œ, optical density.

Fig. 3. Aerobic growth of strain C2 in LB medium containing 5 mM p-hydroxybenzoate and 5 mM vanillate, without (a) and with (b) 5 mM glucose added. Symbols: 䊐, concentration of p-hydroxybenzoate; ■, concentration of phenol; E, concentration of vanillate; F, concentration of guaiacol; Œ, optical density.

The type strain of E. coli (⫽ DSM 30083T) did not metabolize p-hydroxybenzoic and vanillic acids, tested at 5 mM, anaerobically or aerobically, with or without 5 mM glucose added. m-Hydroxybenzoic, 3,4-dihydroxybenzoic, and isovanillic acids were not metabolized by E. coli (⫽ DSM 30083T). Metabolism and growth at increasing concentrations of p-hydroxybenzoic and vanillic acids, tolerance of phenol and guaiacol. Addition of p-hydroxybenzoic acid in LB medium inhibited growth of strain C2. Decarboxylation was not complete at concentrations greater than 20 mM of p-hydroxybenzoic acid, and phenol produced did not

exceed 26 mM (Fig. 4a). With 90 mM of p-hydroxybenzoic acid, decarboxylation decreased markedly, and at 120 mM, decarboxylation and growth were completely inhibited. Phenol was not metabolized and inhibited markedly growth of strain C2 at concentrations higher than 22.5 mM (Fig. 5). When strain C2 was grown in LB medium containing vanillic acid, cell yield was very low at concentrations greater than 20 mM, and guaiacol produced did not exceed 23 mM, suggesting guaiacol toxicity. Neither growth nor decarboxylation occurred with 130 mM of vanillic acid (Fig. 4b). Strain C2 did not grow at guaiacol concentrations greater than 26 mM (Fig. 5).

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Fig. 4. Growth of strain C2 at varying concentration of p-hydroxybenzoate (a) and vanillate (b) in LB medium. Symbols: ■, concentration of phenol produced; F, concentration of guaiacol produced; Œ, optical density after 2 days of aerobic incubation.

Fig. 5. Maximum growth of strain C2 with different levels of phenol (■) and guaiacol (F) in LB medium.

Discussion 16S rRNA sequence analysis indicated that strain C2 is closely related to Escherichia coli (99% of similarity). DNA-DNA hybridization showed a homology of 78.9% between both strains. The DNA G ⫹ C content of strain C2 is similar to that observed with strains of E. coli (48 –52 mol%) [6]. Phenotypically, both strains are Gram-negative, facultatively anaerobic, straight rods, non-sporulating, occurring singly or in pairs. Isolate C2 is non-motile; E. coli strains are motile by peritrichous flagella or non-motile [6]. They grow at mesophilic temperatures and ferment a wide range of carbohydrates and peptides, including Biotrypcase, Casamino acids, and yeast extract. Acid and often gas

are produced during fermentation of glucose and other carbohydrates. The phylogenetic, genotypic, and phenotypic characteristics of isolate C2 indicate that it is a member of the genus Escherichia in the Gramnegative Enterobacteriaceae family and a strain of Escherichia coli. Escherichia coli’s natural habitat is in the lower part of the intestine of most warm-blooded animals [6]. Isolation of strain C2 from the anaerobic digester fed with shea cake, after enrichment on tannic acid, is not surprising, because the digester was rich in tannins and aromatic compounds and has previously been inoculated with anaerobic sludge from the pit of a slaughterhouse. We already isolated, from the same digester, a new strain of the facultative anaerobe, Streptococcus gallolyticus, after enrichment on tannic acid, able to hydrolyze tannic acid into gallic acid and to decarboxylate gallic, protocatechuic, and some hydroxycinnamic acids without further degradation (data not published). A strictly anaerobic bacterium, Papillibacter cinnamivorans [10], was isolated in our laboratory from the same digester after enrichment on cinnamic acid. It transforms cinnamic acid into acetic and benzoic acids. Strain C2 tolerated 8.5 g L⫺1 tannic acid. Bacteria degrading or tolerating tannins have been isolated from sewage sludge [17], the alimentary tracts of koalas [32], goats [7, 29], and horses [38]. Six bacteria that can tolerate high levels of hydrolyzable and condensed tannins were isolated from ruminal contents in Europe, North America, and South America [30]. The ability of ruminal bacteria to tolerate and/or degrade tannins is widespread. Among six isolates, four were most closely related to ruminal strains of Streptococcus bovis and S.

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gallolyticus, one clustered with the clostridia, and one was an Enterobacteriaceae tolerating up to 2 g L⫺1, i.e., one-fourth that of strain C2. Tannin-protein complexdegrading enterobacteria, Gram-negative and facultatively anaerobic, were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas [32]. They constitute a phylogenetically and phenotypically novel group within the family Pasteurellaceae [36]. Osawa et al. [36] proposed the name Lonepinella koalarum gen. nov. sp. nov., which produces gallic acid from tannic acid and decarboxylates gallic acid to pyrogallol. Nemoto et al. [31] suggested that Lonepinella koalarum plays a significant role in digestion of a tannin-rich diet in the alimentary tract of host animal. Two type strains, Haemophilus actinomycetemcomitans and Haemophilus segnis, taxonomically classified into the family Pasteurellaceae, were isolated from the human oral cavity and were detected positive for tannase [31]. Many Streptococcus bovis biotype I strains hydrolyze tannins to release gallic acid, which is then decarboxylated to pyrogallol by gallate decarboxylase [33, 34]. All strains of S. bovis that exhibited gallate decarboxylase activity belonged to a single DNA homology group described as Streptococcus gallolyticus [35, 43]. From the different aromatic substrates tested, with or without glucose added, including 18 benzoic acids, 6 cinnamic acids, 2 phenylpropionic acids, and 4 phenols, only p-hydroxybenzoic and vanillic acids were metabolized stoichiometrically by strain C2 to their corresponding phenol derivatives, phenol and guaiacol, and to CO2 without further degradation. The mechanism involves a non-oxidative decarboxylation. It appears that an unsubstituted para-hydroxyl group on the benzene ring was required for the decarboxylation. Other benzoic acids without a hydroxyl group in the para position and with another substituent than -H or -OCH3 in the meta position to the carboxyl group were not metabolized, since various benzoic acids including benzoate, m-hydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-, 2,4-, 2,6-dihydroxybenzoates, 2,4,6-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 2,4-, 2,6-, 3,4-, 3,5-dimethoxybenzoates, 3,4,5-trimethoxybenzoate, 3-hydroxy-4-methoxybenzoate, 4-hydroxy-3, 5-dimethoxybenzoate, and 3,4-dimethylbenzoate were not decarboxylated, indicating a specific selection. All two decarboxylations proceeded without or with O2 without further degradation. Generally it has been observed that microorganisms under aerobic conditions convert vanillic or p-hydroxybenzoic acid to protocatechuic acid prior to dearomatization of the ring with a protocatechuate dioxygenase. Some observations indicated that the classic separations of catabolic pathways leading to specific ring-fission substrates such as proto-

catechuate and catechol are often interconnectable by single enzymatic transformations, usually a decarboxylation [11]. Supplementation of the medium with glucose accelerated the rate of growth and increased these conversions of p-hydroxybenzoic and vanillic acids. Growth and decarboxylation were accelerated under aerobic conditions. p-Hydroxybenzoic acid was decarboxylated more rapidly than vanillic acid. We could suggest that the hydroxyl group in the para position of the benzene ring only seems to be preferential for the binding of the substrate to the decarboxylase activity. Strain C2 tolerated high concentrations of p-hydroxybenzoic and vanillic acids, but growth was markedly inhibited, decarboxylations were not complete at concentrations greater than 20 mM, and production of phenol and guaiacol did not exceed 26 and 23 mM, respectively, suggesting phenol toxicity. It was shown that accumulation of guaiacol, which is highly bactericidal [9], generally inhibited further microbial activities. Similarly, phenol resulting from the degradation of p-hydroxybenzoic acid was reported to inhibit Klebsiella sp. [38]. Each of the following hypotheses might explain the formation of phenol and the decarboxylation of p-hydroxybenzoic acid: the decarboxylation is catalyzed by an enzyme which has another function; the phenol produced inhibits competing organisms more than the one producing it; the reaction was useful in a previous stage of evolution; the reaction is now an imperfection of metabolism. The type strain of E. coli cannot metabolize p-hydroxybenzoic and vanillic acids, anaerobically or aerobically, with or without glucose added. It is possible that strain C2, after adaptation to the digester conditions, acquired this function of decarboxylation to excrete in the medium compounds (phenol and guaiacol) more toxic than initial substrates (phydroxybenzoate and vanillate) for inhibition of competitive microorganisms that cannot tolerate these high phenol concentrations. ACKNOWLEDGMENTS We thank Pierre Roger (IRD) for his comments on the manuscript, Frederic Verhe´ (IRD) for his technical assistance, and Pr Alfred Traore (Burkina Faso University) for providing the microbial material used in this study.

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