The âinorganic fermentationâ of sulfur oxyanions by strictly anaerobic sulfate-reducing bacteria was first reported for two isolates obtained in pure culture (Bak ...
Arch Microbiol (1996) 166 : 184–192
© Springer-Verlag 1996
O R I G I N A L PA P E R
Peter H. Janssen · Alexandra Schuhmann · Friedhelm Bak · Werner Liesack
Disproportionation of inorganic sulfur compounds by the sulfate-reducing bacterium Desulfocapsa thiozymogenes gen. nov., sp. nov. Received: 29 January 1996 / Accepted: 31 May 1996
Abstract A new strictly anaerobic, gram-negative bacterium was isolated from the sediment of a freshwater lake after enrichment with thiosulfate as the energy source. The strain, named Bra2 (DSM 7269), is able to grow by disproportionation of thiosulfate or sulfite to sulfate plus sulfide. Elemental sulfur is also disproportionated to sulfate and sulfide, but this only supports growth if free sulfide is chemically removed from the culture, e.g., by precipitation with amorphous ferric hydroxide. Growth is also possible by coupling the reduction of sulfate to sulfide with the oxidation of ethanol, propanol, or butanol to the corresponding fatty acid. The cells are rod-shaped, motile, and have genomic DNA with a mol% G+C content of 50.7. Cytochromes are present, but desulfoviridin is not. The new strain was shown to be related to, but distinct from members of the genus Desulfobulbus on the basis of physiological characteristics and by comparative sequence analysis of its 16S rDNA. Strain Bra2 is described as the type strain of a new taxon, Desulfocapsa thiozymogenes gen. nov., sp. nov. Key words Sulfur · Thiosulfate · Sulfite · Disproportionation (thiosulfate, sulfite) · Sulfate reduction · Ferric iron · Desulfocapsa thiozymogenes · Desulfobulbus
Introduction The “inorganic fermentation” of sulfur oxyanions by strictly anaerobic sulfate-reducing bacteria was first reported for
P. H. Janssen (Y)1 · A. Schuhmann · F. Bak2 · W. Liesack Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany Tel. +49-6421-178830; Fax +49-6421-178809 Present address: of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia 2 Friedhelm Bak passed away on 27 December 1992 1 Department
two isolates obtained in pure culture (Bak and Cypionka 1987; Bak and Pfennig 1987), and later studies revealed that this capability is also found in other sulfate-reducing bacteria (Krämer and Cypionka 1989; Schnell et al. 1989; Mohn and Tiedje 1990). Recently, it has also been shown that anaerobic bacteria are able to catalyze the disproportionation of inorganic sulfur (Bak 1993; Thamdrup et al. 1993; Lovley and Phillips 1994; Fuseler and Cypionka 1995). The disproportionation of sulfur is endergonic under standard conditions, but the reaction is greatly enhanced by the presence of sulfide-scavenging compounds such as ferric or manganic oxides (Thamdrup et al. 1993; Lovley and Phillips 1994). Sulfate is formed in this reaction along with reduced iron or manganese sulfides. The addition of manganic oxide (Aller and Rude 1988; King 1990) and ferric oxide (Elsgaard and Jørgensen 1992) to anoxic sediments also resulted in the formation of sulfate, but from sulfide. In this latter reaction, the metal oxides apparently act as electron acceptors, perhaps in analogy to sulfide oxidation to sulfur coupled to the reduction of oxygen, nitrate, and fumarate or malate (Wolfe and Pfennig 1977; Macy et al. 1986; Schumacher et al. 1992). During the disproportionation of elemental sulfur, in contrast, metal oxides fulfill another function, namely to remove one of the products (sulfide) chemically. Using sensitive analytical techniques, Fuseler and Cypionka (1995) have measured sulfur disproportionation to sulfate and sulfide by cell suspensions of Desulfobulbus propionicus in the absence of a chemical sulfide sink. However, for growth to be possible, the reaction must be exergonic. The removal of sulfide by precipitation by metal oxides thus apparently renders the overall reaction thermodynamically favorable. Here we report on the characteristics of a sulfate-reducing bacterium with the ability to grow by the disproportionation of inorganic sulfur compounds, including sulfur. Strain Bra2, briefly reported earlier (Bak 1993), is described as the type strain of a new genus and species, Desulfocapsa thiozymogenes gen. nov., sp. nov.
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Materials and methods Source of bacterial strains Sediment from Lake Brabrand, in the western part of the city of Århus, Denmark (Elsgaard and Jørgensen 1992), was used to inoculate the enrichment culture from which strain Bra2 was isolated. Desulfobulbus propionicus strain 1pr3 (DSM 2032) was from our culture collection. Desulfobulbus elongatus strain FP (DSM 2908), Desulfobulbus sp. strain 2pr4 (DSM 2033), and Desulfobulbus “marinus” strain 3pr10 (DSM 2058) were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Media and cultivation techniques Strain Bra2 was isolated and cultivated in a bicarbonate-buffered, sulfide-reduced mineral medium containing (per liter): 1.0 g NaCl, 0.4 g MgCl2 · 6H2O, 0.1 g CaCl2 · 2H2O, 0.1 g NH4Cl, 0.2 g KH2PO4, 0.5 g KCl, 2.52 g NaHCO3, 0.33 g Na2S · 8H2O, and 2.0 ml trace element solution (Widdel 1986). The pH was adjusted to between 7.0 and 7.2. The medium was prepared under an O2-free, N2 plus CO2 atmosphere [N2 : CO2, 4 :1 (v/v)] as described by Widdel and Pfennig (1981). Sulfur compounds, electron acceptors, and carbon sources were added from sterile stock solutions before inoculation. To adjust the pH of the medium, sterile 1 M HCl or 0.5 M Na2CO3 was added as required. H2 was used in a mixture with CO2 [H2:CO2, 4 :1 (v/v)] in the gas phase of bottles sealed with black rubber stoppers. The Hungate technique was used for gassing of culture vessels (Widdel and Bak 1992). Stock solutions of Na2S2O3 and Na2SO3 were filter-sterilized and stored in the dark under N2. Flowers of sulfur was prepared as an aqueous slurry and autoclaved at 110° C for 30 min. Amorphous ferric hydroxide was prepared as described by Lovley and Phillips (1986) and sterilized by autoclaving. Pure cultures were obtained by the repeated application of agardeep dilutions as described by Widdel and Bak (1992). Cultures were checked for purity with microscopic controls and with growth tests in the medium described above with 0.25% (w/v) yeast extract, 5 mM pyruvate, 5 mM glucose, and 5 mM fumarate. The pure culture obtained was routinely grown in the mineral medium described above with 10 mM Na2S2O3 as energy source. Unless noted otherwise, 2 mM acetate was added as a supplementary carbon source when inorganic growth substrates were employed. Completely filled 20-ml screw-capped tubes or 50-ml screwcapped bottles were normally used; the lids were lined with rubber seals. Incubations were at 30° C in the dark, unless noted otherwise. Stock cultures were stored at 4° C. Growth kinetics and substrate transformations were followed in 200 ml medium in 250-ml bottles sealed with black rubber stoppers and under a headspace of N2 plus CO2 [N2 : CO2, 4 :1 (v/v)], from which samples were taken by syringe. Strains of Desulfobulbus spp. were cultivated in the sulfide-reduced, bicarbonate-buffered defined multipurpose medium of Widdel and Bak (1992) with 10 mM propionate as carbon and energy source and 20 mM Na2SO4 as electron acceptor. For Desulfobulbus “marinus” strain 3pr10, the NaCl and MgCl2 · 6H2O concentrations were increased to 20 g/l and 3 g/l, respectively. Propionate and sulfate were omitted when other substrates were tested (details in results). Measurement of growth Culture density was routinely followed by measuring the turbidity at 440 nm in a 10-mm cuvette. Dry mass growth yields were calculated from culture densities using a gravimetrically determined conversion factor obtained from washed (25 mM ammonium acetate) cell pellets harvested from two 1-l cultures by centrifugation and dried to a constant mass at 105° C. Direct cell counts at 400 ×
magnification were made on 10-µl samples evenly spread under 10-mm × 10-mm coverslips on agar-coated microscope slides (Pfennig and Wagener 1986), and about 100 fields were counted using a calibrated grid in the microscope eyepiece. An optical density of 1.00 at 440 nm corresponded to 355 mg cell dry mass/l and 8 × 1011 cells/l. Analysis of inorganic and organic compounds Sulfate, thiosulfate, sulfite, nitrate, and nitrite were analyzed by ion chromatography (Bak et al. 1991). Ammonia was analyzed colorimetrically (Chaney and Marbach 1962). Samples for sulfide determinations were collected without contact with air and were added to 2% (w/v) zinc acetate; the amount of sulfide trapped as ZnS was determined by the spectrophotometric methylene blue method (Cline 1969). Organic acids and alcohols were analyzed by high-pressure liquid chromatography (Krumböck and Conrad 1991). Analysis of iron species Fe2+ was quantitatively determined by photometric measurement of the red-violet complex formed with ferrozine [3-(2-pyridyl)-5, 6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid, monosodium salt, monohydrate]. Samples of 0.5 ml were taken from the cultures by syringe and added to 4.5 ml of 0.5 M HCl. The samples were frozen at –18° C for up to 4 weeks before the determinations were made. Samples were thawed before use, mixed well, and diluted as necessary in 0.5 M HCl. Subsamples of 100 µl were added to 1 ml of 50 mM N-2-hydroxyl-ethylpiperazine-N′-2-ethanesulfonic acid at pH 7 (adjusted with NaOH) containing 0.1% (w/v) ferrozine (Fluka, Neu-Ulm, Germany). This was mixed and centrifuged for 4 min at 5000 × g; the absorbance of the supernatant was measured at 562 nm. Standard curves were prepared from FeCl2 · 4H2O. Total iron was determined by first reducing Fe3+ to Fe2+ using hydroxylamine as the reducing agent. Suitably diluted subsamples of 100 µl from the samples taken for the Fe2+ analysis were added to 2 ml of 250 mM hydroxylamine hydrochloride in 250 mM HCl, and incubated at 60° C for 2 h. Aliquots of 100 µl were then analyzed for Fe2+ as described above. All determinations were carried out in duplicate. Due to the difficulties in taking uniformly mixed samples of the cultures containing iron, the proportion of the total iron present as Fe2+ was determined in each sample, and the amounts of Fe3+ and Fe2+ were then calculated knowing that iron, as amorphous ferric hydroxide, had been added at 30 mmol/l at the beginning of the experiment. No significant iron encrustations were formed on the culture vessel walls. Characterization Oxygen tolerance was determined in agar-deep cultures (Widdel and Bak 1992) incubated under N2:CO2:O2 [3 :1:1 (by vol)]. Gelatin hydrolysis was determined with gelatin [12% (w/v) final concentration] deep cultures under N2:CO2 [4:1 (v/v)] using the agar-deep culture method with gelatin instead of agar. Methods for testing catalase activity, indole production, aesculin hydrolysis, and urea hydrolysis have been described previously (Janssen and Harfoot 1990). Spore formation was checked microscopically under various growth conditions, and in cultures grown with thiosulfate supplemented with 10% (v/v) soil extract. Soil extract was prepared as described by Cote and Gherna (1994). Crude cell-free extracts of thiosulfate-grown cells were prepared by French press treatment as described by Janssen and Schink (1993), and spectrophotometric scans were made with a Hitachi U-2000 spectrophotometer. Desulfoviridin was analyzed in crude cell-free extracts both spectrophotometrically (Postgate 1956) and by a fluorescence test (Postgate 1959). Genomic DNA was isolated and its mol% G+C content determined by high-pressure liquid chromatography as described elsewhere (Janssen et al. 1996).
186 Gram staining was carried out by the method of Magee et al. (1975). Phase-contrast micrographs were made using agar-coated microscope slides (Pfennig and Wagener 1986) and a Zeiss Axiophot photomicroscope. Negatively-stained [1% (w/v) uranyl acetate] preparations on Formvar-plus-carbon-coated copper grids were examined with a Philips EM 301G electron microscope. Comparative 16S rDNA analysis DNA isolation, PCR-mediated amplification of the almost complete 16S rDNA, and sequence analysis were performed as described by Liesack and Finster (1994). The 16S rDNA sequences of strain Bra2 and of Desulfobulbus elongatus strain FP were manually aligned and compared with reference sequences selected from publicly available databases [EMBL, Ribosomal Database Project (Maidak et al. 1994)]. The phylogenetic position of these strains was deduced by comparing their 16S rDNA sequences with various representatives of the main branches of the empire Eubacteria (Trüper 1994). Sequence similarities were calculated by comparing 790 nucleotide positions. Stretches that either were not sequenced in one or more reference organisms or were of uncertain alignment were not used for the derivation of the phylogenetic position, i. e., positions 28–106, 182–218, 451–479, 833–853, 998– 1040, 1132–1142, and 1359–1491 (International Union of Biochemistry of Escherichia coli 16S rRNA numbering). Evolutionary distances between pairs of microorganisms were determined using the Jukes-Cantor equation (Jukes and Cantor 1969) implemented in the DNADIST program of the PHYLIP v.3.5 package (Felsenstein 1993). A dendrogram estimating the phylogenetic relationships was derived using the FITCH program of the same package with a random-order input of sequences and the global rearrangement option. Nucleotide sequence accession numbers The 16S rDNA sequences of strain Bra2 and of Desulfobulbus elongatus strain FP have been deposited in the EMBL, GenBank, and DDBJ nucleotide sequence databases under the accession numbers X95181 and X95180 respectively. The EMBL and GenBank data base accession numbers for sequences used as references for phylogenetic analysis are as follows: “Desulfoarculus baarsii”, M34403; Desulfobacter postgatei, M26633; Desulfobacterium “vacuolatum”, M34408; Desulfobulbus “marinus”, M34411; Desulfobulbus propionicus, M34410; Desulfomicrobium baculatus, M37311; Desulfomonile tiedjei, M26635; Desulforhabdus amnigenus, X83274; Desulfosarcina variabilis, M26632; Desulfovibrio desulfuricans, M34113; Desulfuromonas acetoxidans, M26634; Escherichia coli, J01695; Geobacter metallireducens, L07834; Pelobacter propionicus, X70954; Syntrophobacter wolinii, X70905; Syntrophus buswellii, X85131; Syntrophus gentianae, X85132.
Results Isolation and morphology Strain Bra2 was enriched from the sediment of a freshwater lake using 15 mM acetate plus 10 mM thiosulfate. After repeated transfer, the enrichment culture was transferred to medium containing 15 mM acetate plus 20 mM sulfate, but no growth occurred. The strain was isolated in pure culture by agar-shake dilution series using acetate and thiosulfate as the growth substrates. Cells of strain Bra2 were round-ended rods (Fig. 1), 0.8–0.9 µm wide and 2–3.5 µm long. Division was by binary fission. Cells showed a propellerlike motility and possessed a single, subpolarly inserted flagellum. Cells stained gram-nega-
Fig. 1 Phase contrast photomicrograph of thiosulfate-grown cells of strain Bra2 (Bar 10 µm)
tively. No spores were ever detected, not even when 10% soil extract was added to the growth medium. The mol% G+C content of genomic DNA from strain Bra2, determined by liquid chromatography, was 50.7. Absorption spectra (dithionite-reduced minus air-oxidized) of cell extracts revealed peaks at 420 nm, 524 nm, and 552.5 nm, indicating the presence of a c-type cytochrome. Absorption spectra and fluorescence tests did not indicate the presence of desulfoviridin. Electron donors with sulfate Strain Bra2 was able to grow with ethanol, propanol, and butanol (each tested at 10 mM), with sulfate as electron acceptor. Ethanol (7.35 mmol l–1) was oxidized to acetate (6.14 mmol l–1) coupled to the reduction of sulfate (3.51 mmol l–1) to sulfide (3.77 mmol l–1), and with a specific growth yield of 5.3 g cell dry mass/mol ethanol. On propanol and butanol, propionate and butyrate were produced, respectively. There was no growth with the following electron donors and carbon sources (concentration tested is given in mM in brackets; all tested with 20 mM sulfate as electron acceptor): acetate (10), propionate (10), butyrate (10), isobutyrate (5), valerate (5), 2-methylbutyrate (5), 3-methylbutyrate (5), hexanoate (5), octanoate (2), decanoate (2), dodecanoate (2), myristate (1), palmitate (1), stearate (1), isopropanol (10) ± acetate (2), methanol (10) ± acetate (2), pentanol (5), hexanol (5), DL-3-hydroxybutyrate (10), crotonate (10), L-malate (10), fumarate (10), succinate (10), L-lactate (10), pyruvate (10), glycerol (10) ± acetate (2), citrate (5), glutarate (5), pimelate (5), D-ribose (2), D-xylose (2), D-glucose (2), D-fructose (2), D-galactose (2), benzoate (2), nicotinate (2), gallate (2), pyrogallol (2), 4-hydroxybenzoate (2), syringate (2), 3,4,5-trimethoxybenzoate (2), glycine (5), L-glutamate (5), alanine (5), L-aspartate (5), L-threonine (5), and L-valine (5).
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Strain Bra2 did not grow with 20 mM formate (± 2 mM acetate) or with H2 plus CO2 [H2:CO2, 4 :1 (v/v), ± 2 mM acetate] in the presence or absence of sulfate. There was no fermentative growth on ethanol, propanol, and butanol in the absence of sulfate. Other potential electron acceptors Amorphous ferric hydroxide (30 mM), nitrate (20 mM), or fumarate (20 mM) did not support growth and were not reduced with 10 mM acetate, propionate, L-lactate, or ethanol as potential electron donors. When sulfite (10 mM), thiosulfate (20 mM), or sulfur (excess) was added as electron acceptor with acetate, propionate, or L-lactate, sulfide was produced, but the organic compounds were not degraded. In these cases, the sulfur compounds were disproportionated. With 10 mM ethanol as electron donor, sulfate was used as electron acceptor. Ethanol was oxidized to acetate with concomitant growth and sulfide production from sulfate. When strain Bra2 was grown on ethanol plus thiosulfate or sulfite, the sulfur oxyanion was apparently first disproportionated, and the resulting sulfate was then utilized as electron acceptor for ethanol oxidation when the inorganic energy-yielding substrate was exhausted. Sulfide was also produced from ethanol plus sulfur, but it was not determined if sulfur itself, or sulfate produced by disproportionation (see below) was the actual electron acceptor.
Fig. 2 Time courses of growth of strain Bra2 with a thiosulfate (x), and b sulfite (G), showing the increase in cell density (P) concomitant with the disproportionation of the sulfur oxyanions to sulfate (g) and sulfide (p)
Disproportionation of elemental sulfur and inorganic sulfur compounds Strain Bra2 was able to disproportionate thiosulfate, sulfite, and sulfur. In the cases of thiosulfate (Fig. 2a) and sulfite (Fig. 2b), disproportionation of the inorganic sulfur compound was linked to an increase in the culture density due to an increase in the cell numbers. Thiosulfate (10.54 mmol l–1) was disproportionated to sulfate (10.51 mmol l–1) and sulfide (9.36 mmol l–1) with a specific growth yield of 2.2 g cell dry mass/mol thiosulfate. Sulfite (8.40 mmol l–1) was disproportionated to sulfate (5.86 mmol l–1) and sulfide (2.50 mmol l–1) with a specific growth yield of 3.6 g cell dry mass/mol sulfite. There was no increase in cell numbers in cultures disproportionating sulfur alone (Figs. 3, 4a). Addition of amorphous ferric hydroxide to cultures disproportionating sulfur increased the rate of sulfate production and resulted in the formation of Fe2+ (Fig. 4b). Direct cell counts showed that bacterial numbers increased from 8 × 105 cells˙ml–1 to 7 × 106 cells˙ml–1 during the disproportionation of sulfur in the presence of amorphous Fe3+ (Fig. 3). The specific growth yield, calculated from the increase in cell numbers, was about 0.1 g cell dry mass/mol sulfur, and 7.81 mmol sulfate and 14.22 mol Fe2+ were produced per liter. The fine-grained, black Fe2+ salts produced during growth were not attracted by a magnet. No free sulfide was detectable. Strain Bra2 grew more rapidly by sulfite disproportionation [td (doubling
Fig. 3 Increase in cell numbers (P) in a culture of strain Bra2 growing on S0 plus amorphous ferric hydroxide. There was no increase in numbers in cultures with only S0 (p) or only amorphous ferric hydroxide (k)
Fig. 4 a Production of sulfide (p) and sulfate (g) from S0 by strain Bra2 in the absence of amorphous ferric hydroxide. b Production of Fe2+ (X) and sulfate (g) from S0 and amorphous ferric hydroxide by strain Bra2
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Fig. 5 Dendrogram of evolutionary distances based on the 16S rDNA sequences derived for strain Bra2 and 17 reference organisms of the delta subgroup of proteobacteria. The 16S rDNA of Escherichia coli was used as an outgroup sequence. The scale bar represents 5% difference in nucleotide sequences as determined by measuring the horizontal branch lengths connecting two species
time) = 9.1 h] and thiosulfate disproportionation (td = 13.6 h) than by sulfate reduction with ethanol as electron donor (td = 25.7 h). A doubling time of approximately 32–33 h was calculated from the rates of product formation when grown with sulfur plus amorphous ferric hydroxide. Miscellaneous characteristics Strain Bra2 did not hydrolyze gelatin, urea, or aesculin, and indole was not formed from L-tryptophan, not even when 10 mM thiosulfate (+ 2 mM acetate) was added as the energy source. Strain Bra2 displayed no catalase activity.
Phylogenetic position The dendrogram of evolutionary distances based on comparative 16S rDNA sequencing placed strain Bra2 in the delta subgroup of the proteobacteria. Within this radiation, the phylogenetically most closely related, but distinctly separated group of microorganisms is that of the sulfate-reducing bacteria of the genus Desulfobulbus, i.e., D. propionicus, D. “marinus”, and D. elongatus (Fig. 5).
Sulfur disproportionation by related bacteria Desulfobulbus sp. strain 2pr4 produced 2 mM sulfate from sulfur in the presence of amorphous ferric hydroxide after 21 days. Strain Bra2 produced about 8 mM sulfate within this time. No sulfate formation by strain 2pr4 was observed in the absence of amorphous ferric hydroxide. D. propionicus strain 1pr3, D. elongatus strain FP, and D. “marinus” strain 3pr10 produced less than 0.5 mM sulfate from sulfur, either in the presence or absence of amorphous ferric hydroxide.
Culture conditions Strain Bra2 could grow after repeated transfers [5% (v:v) inocula] in mineral medium with thiosulfate as energy source without added vitamins or acetate. Strain Bra2 grew optimally at pH values of 7.3–7.5, and within a range of pH 6.8–8.0. Thiosulfate disproportionation still occurred at pH 6.6, but without growth. The optimal temperature for growth was 30° C. Growth was possible at 20° C, but not at 15 or 35° C. Growth was slowed by the addition of NaCl to the medium and was still possible at 10 g l–1, but not at 15 g l–1. No growth occurred under oxic or micro-oxic conditions, and a reductant such as sulfide was necessary for the initiation of growth.
Discussion Disproportionation of elemental sulfur and inorganic sulfur compounds Strain Bra2 is able to grow by the disproportionation of inorganic sulfur oxyanions and by the disproportionation of inorganic sulfur. Thiosulfate and sulfite are disproportionated to sulfate and sulfide in agreement with the following equations [free energy changes calculated using data from Thauer et al. (1977) and from Sigg and Stumm (1989)]:
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S2O32– + H2O → SO42– + HS– + H+ ∆G°′ = –21.9 kJ/mol S2O32–
(1)
4 SO32– + H+ → 3 SO42– + HS– ∆G°′ = –58.9 kJ/mol SO32–
(2)
Strain Bra2 is also able to grow by disproportionating elemental sulfur with amorphous ferric hydroxide as sulfide sink, as shown by an increase in cell numbers. Desulfobulbus propionicus strain 1pr3 has been reported to form sulfate and to grow by sulfur disproportionation in the presence of ferric iron as sulfide sink (Lovley and Phillips 1994). We found no or very little sulfate production from elemental sulfur by three strains of Desulfobulbus spp. Recent sensitive measurements by Fuseler and Cypionka (1995) have detected sulfur disproportionation by cell suspensions of D. propionicus in the absence of a chemical sulfide sink. The disproportionation of inorganic sulfur alone does not support growth of Desulfobulbus spp. or strain Bra2. This reaction is thermodynamically unfavorable: 4 S0 + 4 H2O → SO42– + 3 HS– + 5 H+ ∆G°′ = +10.2 kJ/mol S0
(3)
The addition of amorphous ferric hydroxide to cultures disproportionating elemental sulfur, however, did result in an increase in cell numbers. This was also found by Lovley and Phillips (1994) with D. propionicus. In our experiments, the sulfide formed reacted chemically with Fe3+, resulting in the precipitation of reduced iron sulfides. Other potential sulfide scavengers, such as phototrophic bacteria, Fe2+, or manganese species, remain to be tested. Approximately 2 mol Fe2+ were formed per mole SO42–, suggesting the reaction of sulfide with Fe3+ as follows [(Gf° values for FeS and amorphous FeOOH from Sigg and Stumm (1989)]: 3 HS– + 2 FeOOH + 3 H+ → S0 + 2 FeS + 4 H2O ∆G°′ = –143.9 kJ/mol S0
(4)
The overall reaction stoichiometry is thus (Eqs. 3 plus 4): 3 S0 + 2 FeOOH → SO42– + 2 FeS + 2 H+ ∆G°′ = –34.4 kJ/mol S0
(5)
The chemical removal of sulfide by ferric iron is, thus, the sum of two reactions: the oxidation of sulfide to sulfur, and the precipitation of iron sulfides. These chemical reactions apparently result in favorable thermodynamics of the biological sulfur disproportionation reaction by removing one of the metabolic end products (sulfide). There was a clear increase in cell numbers during growth on sulfur and amorphous ferric hydroxide that corresponds to a dry mass yield of 0.1 g/mol sulfur. Cultures supplemented with only sulfur or with only amorphous ferric hydroxide showed no increase in cell numbers. Thus, the disproportionation reaction is able to be coupled to the conservation of energy. The growth yield is extremely small. This may be only partially due to the slow growth rate with an associated high maintenance energy component. The cultures disproportionating sulfur in the absence of amorphous ferric hydroxide produced only low amounts of sulfide and
sulfate. The free energy change (∆G′) once the reaction ceased, calculated using the final concentrations of products, was about –4 kJ/mol S0. Growth obviously requires a more negative free energy change. Calculations of the free energy change under conditions allowing growth in the presence of amorphous ferric hydroxide give values of about –90 kJ/mol S0 (Bak 1993; Thamdrup et al. 1993). Enrichment cultures able to grow by disproportionating elemental sulfur, also coupled to the chemical removal of sulfide by oxidation and by precipitation with oxidized metal species, have been described by Thamdrup et al. (1993). A culture obtained with ferric hydroxide and sulfur was dominated by rod-shaped cells similar to those of strain Bra2. This culture also produced iron sulfides. Magnetite was not formed in the enrichment culture (Thamdrup et al. 1993) or by strain Bra2. This suggests the chemical nature of the reaction between sulfide and ferric iron. Many anaerobic bacteria are able to reduce ferric iron directly to form magnetite (Bell et al. 1987; Lovley and Phillips 1988; Roden and Lovley 1993; Caccavo et al. 1994). However, a direct participation of ferric iron in the energy metabolism of sulfur-disproportionating bacteria cannot be ruled out at present although the disproportionation reaction, without growth, is carried out in the absence of ferric iron (Fig. 4; Fuseler and Cypionka 1995). The reaction stoichiometries obtained with strain Bra2 agree with those obtained by Lovley and Phillips (1994) for D. propionicus, and with those of Thamdrup et al. (1993) for their enrichment culture. The growth yields of strain Bra2 on thiosulfate and sulfite were comparable to those obtained with Desulfovibrio sulfodismutans (Bak and Pfennig 1987). The mechanism of energy conservation during thiosulfate and sulfite disproportionation by strain Bra2 is likely to be similar to that of D. sulfodismutans and D. desulfuricans strain CSN (Krämer and Cypionka 1989). In the case of sulfur disproportionation, the mechanism of energy conservation remains to be investigated. Recently, Fuseler and Cypionka (1995) have proposed a pathway for D. propionicus that could also operate in strain Bra2. Strain Bra2 is apparently able to grow autotrophically since the strain could be repeatedly subcultured on thiosulfate in the absence of acetate. No measurements of CO2 fixation were carried out. The enrichment culture obtained on ferric hydroxide and sulfur by Thamdrup et al. (1993) has also been reported to grow autotrophically. Ecological aspects Sulfur can be a product of the anoxic sulfide oxidation observed in sediments (Elsgaard and Jørgensen 1992), and it can be produced from sulfide by chemical oxidation as well as biologically by some phototrophic bacteria and by some aerobic and microaerophilic sulfide-oxidizing bacteria. The presence of large amounts of elemental sulfur in the sediments of freshwater lakes has been reported (Dévai 1990). Thus, elemental sulfur may become available for disproportionating bacteria that ferment sulfur to
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sulfide and sulfate. Amorphous ferric oxides and mixed Fe3+/Fe2+ compounds appear to be the major iron species in aquatic sediments (Coey et al. 1974; Phillips et al. 1993), and thus are available to chemically remove the sulfide produced. In addition to chemical removal of sulfide to form metal sulfides, the biological use of these products may also play a role in ensuring favorable thermodynamics for the microorganisms carrying out this transformation. Sulfides in sediments are commonly depleted in 34S by 45–70%, while sulfate-reducing bacteria produce sulfide depleted in 34S by 4–46% as compared to the sulfate they reduce [see Canfield and Thamdrup (1994)]. Enrichment cultures of sulfur-disproportionating bacteria produce sulfide depleted in 34S by about 8% (Canfield and Thamdrup 1994). It has, therefore, been suggested that a repeated cycle of sulfide oxidation and subsequent disproportionation leads to the observed 34S depletion in sedimentary sulfides (Canfield and Thamdrup 1994). Thiosulfate has been identified as an important intermediate in the anoxic parts of the sulfur cycle in Braband Lake and in other anoxic environments (Jørgensen 1990a,b; Jørgensen and Bak 1991). In the sediment of Braband Lake, from which strain Bra2 was isolated, 44% of added thiosulfate was disproportionated (Jørgensen 1990a), suggesting a significant role for such bacteria in this habitat. Apparently, bacteria such as strain Bra2 participate in the anoxic parts of the sulfur cycle by disproportionating thiosulfate and elemental sulfur and by reducing sulfate. The contribution of the disproportionation of elemental sulfur to the total rate of sulfur transformation in such systems still remains to be investigated. Phylogenetic position Strain Bra2, in addition to being able to couple the disproportionation of inorganic sulfur compounds to growth, is also able to grow by the oxidation of a limited range of organic compounds with associated sulfate reduction. This is a mode of metabolism found in many of the species shown to group phylogenetically with strain Bra2, namely
Table 1 Comparison of characteristics of strain Bra2 and its nearest known relativesa
a Data from: Fuseler and Cypionka (1995), Lovley and Phillips (1994), Samain et al. (1984), Stackebrandt et al. (1995), Widdel and Pfennig (1982) b Data from this study c Janssen, unpublished data
the sulfate-reducing members of the delta subgroup of the proteobacteria. Comparative analysis of the 16S rDNA sequence showed the nearest known relatives of strain Bra2 to be Desulfobulbus spp. However, in contrast to these organisms, strain Bra2 cannot grow with hydrogen, or with propionate or lactate as electron donor (Table 1). Given the phylogenetic distance between strain Bra2 and Desulfobulbus spp. of about 10% based on comparative analysis of the 16S rDNA sequences, and the differences in other characteristics of these organisms (Table 1), we propose the placement of strain Bra2 in a new genus and species as Desulfocapsa thiozymogenes gen. nov., sp. nov. The following descriptions of the new genus and species are based on the characteristics of strain Bra2 only. Description of Desulfocapsa gen. nov. De.sul.fo.cap′sa. L. pref. de from; L. n. sulfur sulfur; L. n. capsa box; ML. fem. n. Desulfocapsa, sulfate-reducing box. The cells are gram-negative, round-ended rods that can be motile. Multiplication is by binary fission. No spores are formed. Cytochromes are present, and desulfoviridin is absent. Metabolism requires strictly anoxic conditions. Simple carbon compounds are incompletely oxidized and coupled to the dissimilatory reduction of sulfate to sulfide. Inorganic sulfur compounds are disproportionated to sulfate and sulfide and support growth under thermodynamically favorable conditions. The genus Desulfocapsa belongs to the delta subgroup of the proteobacteria, as revealed by comparative sequence analysis of the 16S rDNA, and is phylogenetically affiliated with, but distinct from, Desulfobulbus species. Description of Desulfocapsa thiozymogenes sp. nov. thi.o.zy.mo´ge.nes. Gr. n. thios sulfur; Gr. n. zyme leaven, ferment; Gr. v. gennaio to produce; ML. adj. thiozymogenes causing a fermentation of sulfur.
Character
Strain Bra2b
Desulfobulbus propionicus
Desulfobulbus elongatus
Desulfobulbus “marinus”
Cell shape Flagellum mol% G+C
rod sub-polar 50.7
oval no 59.9
rod polar 59.0
oval polar 47.3c
Sulfate reduction with: H2 + CO2 Propionate Lactate Pyruvate Ethanol 2-Methylbutyrate
– – – – + –
+ + + + + +c
+ + + + + +c
+ + + + + –c
Autotrophic growth Sulfur disproportionation
+ +
– +
– –b
– –b
191
Cells are round-ended rods, 0.8–0.9 µm wide and 2–3.5 µm long. The cells are motile by means of a single, subpolarly inserted flagellum. The mol% G+C content of the genomic DNA is 50.7. Cytochromes are present. Ethanol, propanol, and butanol are used as electron donors with sulfate as electron acceptor and are oxidized to their respective fatty acids. Volatile fatty acids, organic and amino acids, and sugars are not utilized. Amorphous ferric hydroxide, fumarate, and nitrate are not used as electron acceptors. Thiosulfate and sulfite are disproportionated to sulfate and sulfide, concomitant with growth. Sulfur is disproportionated to sulfate and sulfide, and in the presence of ferric iron this reaction can support growth. Fermentative growth with organic compounds is not possible. Growth occurs within a pH range of 6.8–8.0, with an optimum of 7.3–7.5. Growth is possible at 20–30° C, but not at 15 or 35° C. NaCl concentrations of 15 g l–1 and higher inhibit growth. Vitamins are not required. When the cells grow by disproportionation of inorganic sulfur compounds, carbon dioxide can act as the sole carbon source. The type strain, Bra2, was isolated from a freshwater lake in Århus, Denmark, and has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) under accession number DSM 7269. Acknowledgements The authors thank H. Hippe (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) for helpful discussion, Sonja Fleissner for technical assistance, and the reviewers for making very useful suggestions.
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