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Degrading Syntrophs Smithella Propionica Gen. Nov., Sp. Nov. and Syntrophobacter Wolinii

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Degrading Syntrophs Smithella Propionica Gen. Nov., Sp. Nov. and Syntrophobacter Wolinii International Journal of Systematic Bacteriology (1 999),49, 545-556 Printed in Great Britain Characterization of the anaerobic propionate- degrading syntrophs Smithella propionica gen. nov., sp. nov. and Syntrophobacter wolinii Yitai Liu,' David L. Balkwill,2 Henry C. Aldri~h,~Gwendolyn R. Drake2 and David R. Boone' Author for correspondence: David R. Boone. Tel: + 1 503 725 3865. Fax: + 1 503 725 3888. e-mail: booned@,pdx.edu 1 Department of A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT Environmental Biology, (= OCM 661T; T = type strain), was isolated and proposed as representative of a Portland State University, PO Box 751, Portland, OR new genus and new species, Smithella propionica gen. nov., sp. nov. The strain 97207-0751, USA was enriched from an anaerobic digestor and isolated. Initial isolation was as a * Department of Biological monoxeni c propi on at e-d eg ra d i ng co-cuIt u re conta i n i ng Methanospirillum Science, Florida State hungateii JF-lT as an H,- and formate-using partner. Later, an axenic culture University, Ta Ila hassee, FL, was obtained by using crotonate as the catabolic substrate. The previously USA described propionate-degrading syntrophs of the genus Syntrophobacter also 3 Department of grow in co-culture with methanogens such as Methanospirillum hungateii, Microbiology and Cell Science, University of forming acetate, CO, and methane from propionate. However, Smithella Florida, Gainesville, FL, propionica differs by producing less methane and more acetate; in addition, it USA forms small amounts of butyrate. Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products. Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly. Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate. Syntrophobacter wolinii and Syntrophobacterpfennigii grow on propionate plus sulfate, whereas Smithella propionica did not. Comparisons of 165 rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter. Keywords : Swithella propionica, propionate-oxidizing bacteria, anaerobic digestor INTRODUCTION electron carriers for this transfer. Propionate oxidation with H, and formate production as the electron sink is Propionate is an important intermediate in methano- exergonic only when H, and formate concentrations genic fermentation: it is the precursor of a large are very low (Boone & Bryant, 1980; McInerney & fraction of methane from anaerobic digestors (Boone, Bryant, 198 1; Schink, 1997) (Table 1, equation A). 1982, 1984; Kaspar & Wuhrmann, 1978a), and its The propionate-degrading bacterium Syntrophobacter & accumulation inhibits anaerobic digestion (Kaspar wolinii was first isolated in co-cultures in which one or Wuhrmann, 1978b). Propionate is oxidized to acetate & both of these catabolic products were continuously and CO, (Boone, 1984; Stadtman Barker, 1951), removed by methanogens or sulfate-reducing bacteria with the electrons generated from this oxidation (Boone & Bryant, 1980). More recently, Syntropho- ultimately transferred to methanogens that form meth- bacter wolinii was shown to be phylogenetically related ane by CO, reduction. H, (Boone & Bryant, 1980; to sulfate-reducing bacteria (Harmsen et al., 1993). Wolin, 1982) and formate (Boone et al., 1989; Stams & Syntrophobacter et al., This led to the demonstration that Dong, 1995; Thiele 1988) are the interspecies wolinii can grow slowly on propionate with sulfate as electron acceptor, and allowed the first isolation of this The GenBank accession number for the 165 rRNA sequence of strain LYPT bacterium in axenic culture (Wallrabenstein et al., is AFI 26282. 1994). Stams et al. (1993) showed that propionate 00911 0 1999 IUMS 545 Y. Liu and others Table 1. Possible catabolic reactions during the anaerobic degradation of propionate Reaction Equation AQ H, at equilibrium* (kJ/reaction)t Pa nM I. Hydrogen-producing reactions A CH,CH,COO- + 2H,O -, CH,COO- + CO, + 3H, 71.7 12.1 89.2 B CH,CH,CH,COO- + 2H,O + 2CH,COO- + H++ 2H, 48.3 275 2024 11. Hydrogen-consuming reactions C CO, + 4H, + CH, + 2H,O - 130.7 0.3 17 2.33 D CH,CH,COO- + CO, + 3H, -+ CH,CH,CH,COO- + 2H,O -71.5 12-3 90.6 E(D + B) CH,CH,COO- + CO, + H, -, 2CH,COO- + H+ - 23.3 0.025 0.18 111. Combined hydrogen-producing and hydrogen-consuming reactions F(B + 1/2C) CH,CH,CH,COO- + 1/2CO, + H,O + - 17.1 2CH,COO- + H++ 1/2CH, G(4A + 3C) 4CH,CH,COO- + 2H,O --+ 4CH,COO- + CO, + 3CH, - 105.6 H(A + D) 2CH,CH,COO- -, CH,COO- + CH,CH,CH,COO- 0.12 I(3F +A) 4CH,CH,COO- + 2C0, + 2H,O + 7CH,COO- + 3H+ 1.91 * H, activity for AG = 0 with the following activities: CH,, 0.5 atm; CO,, 0-5 atm; acetate, propionate and butyrate at 1 mM; pH = 7. j. H,, CO,, and CH, in the gaseous state; H' at lo-' mol activity per kg; all other substances in aqueous solution at 1 mol of activity per kg; 25 "C (Thauer et al., 1977). enrichment cultures could also grow in the absence of generated internally, such as by propionate oxidation syntrophic interactions by fermenting fumarate to (Table 1, equation H) or, in part, by butyrate oxidation succinate and CO,, and demonstrated the enzymes of (Table 1, equation D). a fermentative pathway that includes acetyl-CoA oxidation to CO, (Plugge et al., 1993). They were In this paper, we report the isolation of strain LYPT, a unable to grow Syntrophobacter wolinii axenically on bacterium that syntrophically degrades propionate fumarate because fumarate was metabolized by the with butyrate as a small but significant product, and sulfate-reducing bacterium present in that syntrophic that also slowly degrades butyrate. We propose it as Smithellapropionica culture (Stams et al., 1993). In this paper, we report the the type strain of gen. nov., sp. isolation of the type strain of Syntrophobacter wolinii nov. This strain's 16s rDNA sequence, whose de- from its co-culture with Desulfovibrio strain GI1 in termination is reported herein, has been used to monoxenic co-culture with Methanospirillurn hungateii develop primers that detect phylogenetically similar and in axenic culture with fumarate as a catabolic organisms in anaerobic digestors (Sauer et al., 1997). substrate. Portions of this material were reported previously (Liu et al., 1998). Propionate degradation by Syntrophobacter wolinii (Houwen et al., 199 l), anaerobic sediments (Schink, 1985), anaerobic digestors (Robbins, 1987, 1988 ; METHODS Tholozan et al., 1988) and other methanogenic en- Source of inoculum and cultures. The source from which vironments (Boone, 1984; Houwen et al., 1987, 1990; strain LYPT was enriched was an up-flow anaerobic filter Koch et al., 1983) occurs mainly by the methyl-malonyl inoculated with digested domestic sewage sludge and op- pathway, with succinate as a symmetrical intermediate. erated with propionate as the major feedstock for 6 months. This pathway leads to the production of 1 mol acetate, Cultures were obtained from the Oregon Collection of 1 mol CO, and three pairs of electrons (i.e. a total of Methanogens (OCM) : Syntrophobacter wolinii DBT in co- culture with Desulfovibrio G11 (= OCM 467), Syntro- 3 mol H, and formate) per mol propionate degraded phomonas wolfeii LYB (= OCM 65), which is a butyrate- (Table 1, equation A). However, some propionate in oxidizing syntroph (Boone et al., 1989). Methanosarcina anaerobic digestors is reductively carboxylated to barkeri NIH-1 (= OCM 266) was purified from Methano- butyrate (Samain et al., 1984; Tholozan et al., 1988), sarcina barkeri NIH (= OCM 265) (Blaylock & Stadtman, and this reduction can occur in the absence of 1966) by roll-tube isolation in MS medium with 50 mM methanogenesis (Samain et al., 1984). This six-electron methanol. Methanospirillurn hungateii JF- lT( = OCM 16T) reduction (e.g. Table 1, equation D) probably does not is a H,- and formate-utilizing methanogen. use H, as electron donor if it is a catabolic reaction, Media and culture technique. We used the anaerobic because the reaction is not exergonic at the low H, techniques of Hungate (1969) with some modifications concentrations found in anaerobic digestors. However, (Miller & Wolin, 1974; Sowers & Noll, 1995). Except where the electrons to accomplish this reduction could be noted, we used MS medium (Boone et al., 1989), an 546 international Journal of Systematic Bacteriology 49 Smithella propionica gen. nov., sp. nov. anaerobic bicarbonate-buffered medium with 2 g 1-1 each of cycle sequencing method according to the manufacturer’s Trypticase peptone and yeast extract; MES (0.5 g 1-l) and procedures (McBride et al., 1989). The following primers Na,S. 9H,O (0.25 g 1-I) were the reducing agents. MS en- were used for sequencing : C (5’ ACGGGCGGTGTGTAC) richment medium was the same as MS medium except that (Lane et al., 1985), corresponding to positions 1406-1392 in yeast extract and Trypticase peptone were reduced to 0.5 g the 16s rDNA nucleotide sequence of Escherichia coii of each per litre. We also used MS low-nutrient medium in (Brosius et al., 1978) ; C-complement (5’ GTACACACCG- which the amount of yeast extract was reduced to CCCGT; E. coli positions 1392-1406); H (5’ ACACGA- 0.01 g 1-land Trypticase peptone was omitted. Agar media GCTGACGACAGCCA; positions 1075-1056); G (5’ CC- contained 18 g purified agar 1-l. For isolations, we placed AGGGTATCTAATCCTGTT ; positions 800-78 1) ; G- cultures into anaerobic jars with an anoxic gas to eliminate complement (5’ AACAGGATTAGATACCCTGG ; posi- oxygen diffusion through the stoppers, thus allowing longer tions 78 1-800) ; A (5’ GTATTACCGCGG[C/G]TGCTG ; anoxic incubations. positions 536519) ; P (5’ CTGCTTCCTCCCGTAGGAG; positions 357-339) ; P-complement (5’ CTACGGGAGGC- Analytical techniques. Methane was quantified by GC with & AGCAG; positions 342-357); and F,C (5’ AGAGTT- flame-ionization detection (Maestrojuan Boone, 199 1) TGATC[A/C]TGGCTC ; positions 8-25).
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