Three Strains of Sulfate-Reducing Bacteria Utilizing H2 As the Electron

J. Gen. Appl. MicrobioI., 42, 109-120 (1996)

PHYSIOLOGICAL PROPERTIES OF A SULFATE-REDUCING BACTERIUM ISOLATED FROM MUNICIPAL SEWAGE SLUDGE AND ITS POSSIBLE ROLE AS A SYNTROPHIC ACIDOGEN IN THE ECOSYSTEM

MANABU NAKAMOTO, ATSUKO UEKI, AND KATSUJI UEKI*

Department of Bioproduction, Faculty of Agriculture, Yamagata University, Tsuruoka 997, Japan

(Received August 10, 1995; Accepted December 22, 1995)

Three strains of sulfate-reducing bacteria utilizing H2 as the electron donor of sulfate reduction were isolated from anaerobic digester slurry of municipal sewage sludge. The isolates were Gram-negative, non-spore- forming, motile, curved rods with a single polar flagellum. Cytochrome c3 and desulfoviridin were present. The DNA base compositions (mol% G + C) were 59.6±0.7. The isolates used H2, formate, lactate, pyruvate, fumarate, malate, alcohols and amino acids as the electron donor for sulfate reduction. Alcohols were oxidized to corresponding mono- carboxylates, and other organic compounds were to acetate. Sulfate, sulfite and thiosulfate, but not nitrate, were used as the electron acceptor and reduced to sulfide. The isolates grew with pyruvate, fumarate, or malate as the energy source in the absence of oxidized sulfur compounds. Pyruvate was oxidized to acetate with the production of formate and H2. Fumarate and malate were oxidized to acetate, and the oxidation was coupled with the reduction of fumarate or malate to succinate. From the morphological and physiological properties, the isolates were considered to belong to a Desulfovibrio sp. In association with Methanobacterium formicicum as a hydrogen-consuming partner, one of the isolates could also grow in the absence of oxidized sulfur compounds by utilizing organic substrates other than pyruvate, malate and fumarate. In the coculture of the isolate and the methanogen, all the organic compounds other than malate and fumarate, which could serve as the electron donor for sulfate reduction in the pure culture of isolates, were syntrophically oxidized with the production of methane. These results indicate that the isolates of sulfate-reducing bacteria can play a role as a syntrophic

* Address reprint requests to: Dr . Katsuji Ueki, Department of Bioproduction, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997, Japan.

109 110 NAKAMOTO, UEKI, and UEKI VOL. 42

degrader of a wide range of organic compounds in the methanogenic sewage sludge devoid of sulfate.

Sulfate-reducing bacteria, as well as methanogens, can play a role as the terminal hydrogen-scavenger in the anaerobic degradation process of organic matter. There are many reports on the competition between sulfate reduction and methanogenesis for the utilization of electron donors in various anaerobic environments, and sulfate reduction is generally thought to dominate methanogenesis due to the differences in kinetic and thermodynamic properties (1, 6, 7, 10,11). As reported previously (15,17-20), however, sulfate reduction does not appar- ently compete with methanogenesis in nutritionally rich environments such as the anaerobic digester slurry of municipal sewage sludge or cattle waste, even when sulfate reduction was markedly enhanced by adding sulfate to the slurry. Sulfate- reducing bacteria can be generally enumerated at relatively high densities (>i0 5 cells/ml) in such environments even practically devoid of sulfate (18,20). Besides, it is known that some strains of sulfate-reducing bacteria can grow as the syntrophic degrader of lactate and ethanol (2, 4, 5), or propanediols and glycerol (9) in coculture with the hydrogenotrophic methanogen in the absence of oxidized sulfur compounds. These facts may provide insight into the ecological role of sulfate- reducing bacteria as the syntrophic acidogen in various methanogenic environments devoid of sulfate. In the present study, three strains of sulfate-reducing bacteria utilizing H2 as the electron donor for sulfate reduction were isolated from anaerobic digester slurry of municipal sewage sludge and characterized. The ability of the sulfate- reducing bacteria to grow as the syntroph in a binary coculture with a hydrogen- utilizing methanogen, Met hanobacterium formicicum, was examined, and a possible role of the sulfate-reducing bacteria as the syntrophic acidogen in the methanogenic digester slurry was discussed.

MATERIALS AND METHODS

Organisms. Three strains (HS-l, HS-2, HS-3) of sulfate-reducing bacteria were isolated from anaerobic digester slurry of municipal sewage sludge sampled from an anaerobic digester in the Wastewater Treatment Center of Tsuruoka City in Japan. Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans NCIMB 8307 were obtained from the National Collection of Industrial and Marine Bacteria (Scotland, U.K.), and Methanobacterium formicicum DSM 1535 was from the Deutsche Sammlung von Mikroorganismen GmbH (Braunschweig, Germany). Media and culture conditions. All procedures for cultivation of bacteria were carried out under strictly anoxic conditions. Cultures (ca. 10 ml) were grown at 30°C under 02-free atmosphere in test tubes (18>< 180 mm) sealed with butylrubber double stoppers. 1996 Syntrophic Role of Sulfate-reducing Bacteria 111

For the cultivation of sulfate-reducing bacteria, the basal medium (16) containing (per liter) 0.5 g KH2PO4, 1.0 g NH4C1, 1.0 g Na2SO4, 2.0 g MgSO4.7H2O, 0.1 g CaC12.2H2O, 0.5 g yeast extract (Difco Lab., Detroit, MI, U.S.A.), 10 ml trace element solution, 1 ml vitamin solution, 0.5 g cysteine-HCI, and 1 mg resazurin-Na was amended with various organic compounds or H2 as the electron donor. The pH was adjusted to 7.2-7.4. Sulfate-reducing bacteria were usually main- tained on the medium with Na-lactate (20 mM) or H2 as the electron donor under 02-free N2 or H2/N2 (40/60, v/v) atmosphere, respectively. In experiments for substrate utilization, cultures (0.5 ml) grown with H2 as the electron donor for sulfate reduction were inoculated to fresh media (10 ml) amended with organic compounds as the electron donor and incubated at 30°C on a reciprocal shaker, unless otherwise stated. Sulfate-reducing bacteria were isolated from the anaerobic digester slurry of municipal sewage sludge by the anaerobic roll tube method. The medium for sulfate-reducing bacteria was used with H2 as the electron donor, except that cysteine-HC1 was omitted and 0.1 g Na-thioglycollate, 0.1 g ascorbic acid, 2.5 g Na2S204, and 2.5g FeSO4(NH4)2SOe6H2O (per liter) were added (16). Black colonies formed on roll tubes inoculated with 0.3 ml of 105 dilutions of the digester slurry were picked up and purified by repeating the colony isolation by the roll tube method. For the cultivation of M formicicum DSM 1535, the basal medium (DSM 119, modified) containing (per liter) 0.5 g KH2PO4, 0.6 g NH4C1, 0.4 g NaCI, 0.2 g MgC12.6H2O, 0.05 g CaCl2.2H2O, 5 mg FeCl2.4H2O, 1.0 g yeast extract (Difco Lab.), 1.0g Trypticase (BBL, Cockeysville, MD, U.S.A.), 10 ml trace element solution, 1 ml vitamin solution, 0.3 g cysteine-HCI, 0.3 g Na2S, and 1 mg resazurin- Na was used. The medium with H2 or Na-formate (40 mM) as the energy source was used under H2/C02 (80/20, v/v) or N2/C02 (80/20, v/v) atmosphere, respectively. The pH was adjusted to 7.0. For the coculture of strain HS-1 plus M formicicum, the media (chlorides were substituted for sulfates) amended with various organic compounds (20 mM) as the electron donor were used under N2/C02 (80/20, v/v) atmosphere. Strain HS-1 and M formicicum were separately grown with H2, and then the cultures (each 0.1 ml) were inoculated to a fresh coculture medium (10 ml). The growth was followed by measuring the optical density at 660 nm with a spectrophotometer (Hitachi U-100) . Analytical methods. DNA base composition and pigments were determined in cells grown to the early stationary phase with lactate as the electron donor for sulfate reduction. DNA was extracted as described by Marmur (8) and hyrolyzed with nuclease P, using the DNA-GC Kit (Yamasa Shoyu Co., Ltd., Choshi, Japan). The mol% G+C was determined by HPLC (Shimadzu LC-6A) equipped with YMC-Pack ODS-AQ column (YMC Inc., Kyoto, Japan). Cytochrome c3 was determined in whole cells by measuring redox difference spectra (Na2S204-reduced minus air-oxidized) with a spectrophotometer (Hitachi 112 NAKAMOTO, UEKI, and UEKI VOL. 42

150-20). Desulfoviridin was partially purified and determined according to Seki et al. (12). Gases, alcohols and fatty acids were determined by gas chromatography as described previously (19). Gas samples were taken from the headspace of culture tubes through the stopper with a pressure lock syringe (Precision Sampling Co., Baton Rouge, U.S.A.) and injected into a gas chromatograph (Hitachi 163). Culture fluids sampled for the analysis of fatty acids and alcohols were deprotein- ized and injected to a gas chromatograph (Hitachi 263). Sulfate was measured with an ion chromatograph (Dionex 2000i). A mobile phase composed of 1.8 mM Na2C03 and 1.7 mM NaHC03 was used at a flow rate of 1 ml/min.

RESULTS

Properties of isolates of H2-utilizing sulfate-reducing bacteria Morphological and physiological properties of three isolates designated as strains HS-1, HS-2 and HS-3 are presented in Table 1. For comparison, properties of type strains of D. vulgaris (NCIMB 8303) and D. desulfuricans (NCIMB 8307) are also presented. Morphology: Three isolates were Gram-negative, non-spore-forming, curved rods motile with a single polar flagellum. The cells were usually single, and 0.5-0.6 um in diameter and 2.2-2.8 gymin length. DNA base compositions and pigments: The G + C contents (mol%) of the isolates were 59.6+0.7%. The redox difference spectra of isolates exhibited a pattern characteristic of cytochrome c3 with maxima at 420, 523 and 553 nm, and the partially purified preparation of desulfoviridin showed the characteristic ab- sorption band at about 630 nm. Substrate for growth: Three isolates used H2, formate, pyruvate, lactate, malate, fumarate, ethanol, propanol, butanol, isobutanol, serine, and alanine as the electron donor for sulfate reduction. Methanol, isopropanol, pentanol, 3-methyl- butanol, hexanol, 1,2-propanediol, 1,3-propanediol, glycerol, amino acids other than serine and alanine, acetate, propionate, butyrate, glucose, fructose, phenol, benzoate and choline were not used as the electron donor. Since no significant difference existed among isolates in kinetics of substrate utilization during the culture, data from experiments with strain HS-1 are presented in the following figures. Pyruvate, lactate, malate, fumarate, serine, and alanine were incompletely oxidized to acetate and C02 with the reduction of sulfate. During the growth with formate or pyruvate (Fig. 1A), H2 transiently accumulated at the early stage of the logarithmic phase. Succinate was also produced during the growth with malate or fumarate (Fig. 1B), indicating that these compounds could also serve as the electron acceptor together with sulfate. Ethanol (Fig. 1C), propanol (Fig. 1D), 1996 Syntrophic Role of Sulfate-reducing Bacteria 113

Table 1. Morphological and physiological properties of isolates.° 114 NAKAMOTO, UEKI, and UEKI VOL. 42

Fig. 1. Utilization of pyruvate (A), fumarate (B), ethanol (C) or propanol (D) by strain HS-1 in pure culture in the presence of sulfate. Changes in O.D. (0) and concentrations of pyruvate (0), fumarate (o), ethanol (0), propanol (ELI),acetate (•), propionate (A), succinate (•), sulfate (o), CO2 (•) and HZ (Y) are presented. The value of gases represents the amount evolved in the headspace of culture tubes. Values are averages of duplicate cultures. butanol, and isobutanol were oxidized to acetate, propionate, butyrate, and isobutyrate, respectively, with the reduction of sulfate. The stoichiometry of substrate degradation by isolates during the culture in the presence of sulfate can be proposed as follows. The oxidation of each 2 mol of lactate, ethanol, propanol, butanol, isobutanol, serine and alanine or 4 mol of pyruvate is coupled with the reduction of 1 mol of sulfate. During the culture with malate or fumarate, the reduction of malate or fumarate to succinate proceeded simultaneously with sulfate reduction. The oxidation of 2 mol of malate or fumarate is theoretically coupled with the reduction of 1 mol of sulfate or with the reduction of 4 mol of malate or fumarate. Thus, in all strains, about 67 or 62% of electrons generated by the oxidation of malate or fumarate was estimated to be used for sulfate reduction, respectively, and about 33 or 38% for the reduction of malate or fumarate, respectively. Pyruvate, malate and fumarate were used as the energy source even in the absence of oxidized sulfur compounds. Pyruvate was oxidized to acetate, C02 and H2 (Fig. 2A). Formate transiently accumulated in the culture with pyruvate. Malate and fumarate (Fig. 2B) were oxidized to acetate and C02, coupled with the reduction to succinate. In all strains, sulfate, sulfite and thiosulfate, but not nitrate, could serve as the electron acceptor and were reduced to sulfide (Table 1). 1996 Syntrophic Role of Su Ifate-reducing Bacteria 115

Fig. 2. Utilization of pyruvate (A) or fumarate (B) by strain HS-1 in pure culture in the absence of sulfate. Changes in O.D. (0) and concentrations of pyruvate (0), fumarate (o), acetate (•), succinate (•), formate (7), CO2 (•) and Hz (V) are presented. The value of gases represents the amount evolved in the headspace of culture tubes. Values are averages of duplicate cultures.

Nutritional requirements and optima of pH and temperature: Yeast extract at more than 0.05% (w/v) was required for good growth of isolates. Without the addition of yeast extract, strains HS-1 and HS-2 finally oxidized lactate only by 2- 3 mmol/l and reduced sulfate only by 1-1.5 mmol/l, and strain HS-3 finally oxidized lactate only by less than 1 mmol/l. The addition of NaCI at 25-100 mM slightly enhanced the growth of strains, but that at more than 200 mM markedly inhibited growth. Temperature optima of growth were at about 30°C in all strains, and no growth was observed at 7 or 50°C. The pH optima of growth were at pH 6.0-7.0, and no growth was observed at pH 5.3. The maximum 40.D. was reduced at pH 7.8 to 40-50% of the optimal growth in strains HS-1 and HS-2, but to about 70% in strain HS-3.

Syntrophic growth of the isolate in a coculture with M, formicicum in the absence of oxidized sulfur compounds Since the three isolates showed essentially identical properties in morphology and physiology, strain HS-1 was chosen for further examinations. Strain HS-1 did not grow in pure culture on the coculture medium amended with the organic compounds other than pyruvate, malate and fumarate independently of the atmosphere of culture tubes, since sulfate was absent in the medium. The methanogen also did not grow in pure culture on the medium, unless formate 116 NAKAMOTO, UEKI, and UEKI VOL. 42 or H2 was amended as the energy source. In coculture of strain HS-1/MMformici- cum, however, the two species of bacteria grew well even on the medium amended with the compounds which could support only the sulfidogenic growth of strain HS- 1 in pure culture, as shown in Table 2. Lactate (Fig. 3A), serine (Fig. 3B) and alanine were degraded to acetate with the production of methane. Ethanol (Fig. 3C), propanol (Fig. 3D), butanol and isobutanol were oxidized to acetate, pro-

Table 2. Substrate utilization and methanogenesis in the coculture of strain HS- 1/Methanobacterium formicicum in the absence of sulfate.

Fig. 3. Utilization of electron donors and methanogenesis in the coculture of strain HS-1/Methanobacterium formicicum with lactate (A), serine (B), ethanol (C) or propanol (D) in the absence of sulfate. Changes in O.D. (0) and concentrations of lactate (LII), ethanol (0), propanol (ELI),acetate (•), propionate (A), C02 (•) and CH4 (V) are presented. Serine was not determined. The value of gases represents the amount evolved in the headspace of culture tubes. Values are averages of duplicate cultures. 1996 Syntrophic Role of Sulfate-reducing Bacteria 117

Fig. 4. Utilization of electron donors and methanogenesis in the coculture of strain HS-1/Methanobacterium formicicum with pyruvate (A) or fumarate (B) in the absence of sulfate. Changes in O.D. (0) and concentrations of pyruvate (LI), fumarate (0), acetate (•), succinate (U), CO2 (•) and CH4 (Y) are presented. The value of gases represents the amount evolved in the headspace of culture tubes. Values are averages of duplicate cultures. pionate, butyrate and isobutyrate, respectively, with the production of methane. With pyruvate as the energy source, the coculture also grew well and pyruvate was degraded to acetate with the production of methane (Fig. 4A). Neither H2 nor formate accumulated to detectable levels during the growth of coculture with lactate, pyruvate, amino acids, or alcohols as the energy source. With malate or fumarate (Fig. 4B) as the energy source, acetate, C02 and succinate were produced with the growth of strain HS-1, but the methanogen did not grow and no methane was formed. The coculture did not grow with propionate or butyrate.

DISCUSSION

Morphological, biochemical and physiological properties of three isolates (strains HS-1, HS-2 and HS-3) of sulfate-reducing bacteria were essentially identical except for the slight difference in the requirement of yeast extract and the growth at high pH. In particular, the presence of desulfoviridin and cytochrome c3 indicated that these isolates belonged to the genus Desulfovibrio. The isolates resembled D. desulfuricans and D. vulgaris in terms of substrate utilization, and were slightly different from D. vulgaris in G + C content. As presented in Table 1, however, the isolates differed from D. vulgaris in the ability to utilize alanine, glycerol and malate as the electron donor for sulfate reduction, and the ability to 118 NAKAMOTO, UEKI, and UEKI VOL. 42 utilize pyruvate, malate and fumarate in the absence of sulfate. The isolates also differed from D. desulfuricans in the ability to utilize methanol, glycerol, choline and alanine for the sulfate reduction, and malate and choline in the absence of sulfate, and nitrate as the electron acceptor. Thus, these isolates seem to represent a new species of the genus Desulfovibrio, but further study such as 16S rRNA sequence analysis is required for classification. As reported previously (20), sulfate-reducing bacteria in the sewage sludge were generally enumerated at the levels of 105colony forming units (CFU)/ml with H2 or lactate as the electron donor, and methanogenic bacteria at the levels of 106 CFU/ml with acetate as the substrate or 104CFU/ml with H2 as the substrate. Bacteria morphologically resembling to the isolates were found in almost all black colonies formed on roll tubes of the enumeration medium with H2 or lactate as the electron donor, when 104 or 105 dilutions of the sewage sludge were inoculated to the roll tubes (data not shown). Thus, the isolates may represent dominant hydrogen-utilizing sulfate-reducing bacteria inhabiting the sewage sludge and may act as a dominant H2-scavenger under the condition that sulfate is present at available levels. The isolates grew with pyruvate, malate or fumarate as the energy source independently of the presence of sulfate. With pyruvate as the energy source, formate transiently accumulated at the early logarithmic phase and H2 accumulated during fermentative growth, but only H2 transiently accumulated at the early logarithmic phase of the sulfidogenic growth. Pyruvate catabolism to acetate in sulfate-reducing bacteria has been generally thought to proceed via the pyruvate dehydrogenase reaction (3), and, in fact, we can detect relatively high activity of pyruvate dehydrogenase in a cellular-soluble fraction of isolates (data not shown). The formate formation during the fermentative growth, however, presented evi- dence for the involvement of pyruvate: formate lyase reaction in the pyruvate catabolism of isolates. When the isolates are grown with malate or fumarate in the presence of sulfate, the oxidation of malate or fumarate is allowed to proceed by coupling with sulfate reduction or reduction of the organic compounds to succinate. Free energy changes (dG°', kJ/mol substrate oxidized) of the redox couples are -191.2 kJ/mol in the malate oxidation/malate reduction couple, - 202.4 kJ/mol in the fumarate oxidation/fumarate reduction couple, -102.4 kJ/mol in the malate oxidation/sulfate reduction couple, and -106.1 kJ/mol in the fumarate oxidation/sulfate reduction couple (13). Thus, the reduction of malate or fumarate seems to be energetically more advantageous than sulfate reduction. The present results, however, showed that sulfate reduction proceeded simultaneously with the reduction of malate or fumarate to succinate, and that more than 60% of reducing equivalents generated by the oxidation of malate or fumarate was distributed to sulfate reduction. The partition of electrons may reflect the control of electron flow depending on the biochemical parameters of electron transport machinery of isolates. The present results also indicated that the isolate could grow as a syntrophic 1996 Syntrophic Role of Sulfate-reducing Bacteria 119 acidogen in the binary coculture with MMformicicum, utilizing all the organic compounds tested except for malate and fumarate, which could serve as the energy source for growth with sulfate reduction in pure culture. In the syntrophic association, reducing equivalents generated in the oxidation of substrate by syntrophic bacteria are generally thought to be transferred to the partner bacteria by the interspecies-hydrogen transfer. The interspecies-formate transfer is, however, also known to be involved in the syntrophic association (14). In the present study, formate may have been produced together with H2 in the coculture at least when pyruvate was added as the substrate. The fact that neither H2 nor formate accumulated at detectable levels during our methanogenic coculture with any of the substrates tested indicated that reducing equivalents could be efficiently transferred between members of the coculture. The involvement of interspecies-formate transfer, however, remains unknown, since formate as well as H2 could be con- sumed by M. formicicum and rapidly degraded to H2 and CO2 by the isolate. The ability to grow syntrophically in association with hydrogenotrophic methanogens has already been demonstrated in some strains of Desulfovibrio spp. Only few compounds, however, are known to serve as the energy source for syntrophic growth. Syntrophic utilization of lactate and ethanol in coculture with a methanogen has been demonstrated for D. vulgaris (2), D. desulfuricans (2), Desulfovibrio gigas (S), and Desulfotomaculum nigrificans (4), and that of 1,2-propanediol, 1,3- propanediol and glycerol has been demonstrated for Desulfovibrio alcoholovorans (9). Therefore, the ability of our isolate to utilize all of the compounds which serve as the electron donor for sulfate reduction as the energy source for syntrophic growth is surprising. The results in the present study indicate that the isolate of sulfate-reducing bacteria can play a role as a syntrophic degrader of a wide range of organic compounds in the methanogenic sewage sludge devoid of sulfate. The ability of isolate may well explain for the reason why sulfate-reducing bacteria can inhabit the digester slurry at rather high densities despite of the depletion of sulfate. It is of interest to examine whether other sulfate-reducing bacteria can also grow as the syntroph under conditions devoid of sulfate, utilizing all the organic compounds which serve as the energy source in growth with sulfate reduction.

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