Sulfate Reduction and Anaerobic Glycerol Degradation by a Mixed Microbial Culture

Transcription

1 CURRENT MCRBLGY Vol. 22 (1991), pp Current Mirobiology 7' h Springer-Verlag New York n Sulfate Redution and Anaerobi Glyerol Degradation by a Mixed Mirobial Culture L Abdel-llah Qatibi,1,2 André Bories, and Jean-Louis Garia2 'Laboratory of Biotehnology of A.A.. Environment, Narbonne; 2Laboratory of Mirobiology RSTM, University of Provene, Marseilles, Frane Abstrat. Anaerobi glyerol degradation by a mixed mirobial ulture from a fermenter fed with industrial alohol distillation waste water, was investigated in the absene or presene of sulfate, at 37 C and at a onstant ph of 7.2. n the absene of sulfate, glyerol utilization was found to be haraterized by the transient formation of 173-propanediol prior to propionate and aetate aumulation. n the presene of sulfate, 173-propanediol prodution was minor, and the arbon balane refleted a onsiderable aumulation of intermediate(s). A study of the role of sulfate redution and methanogenesis on anaerobi 1,3-propanediol degradation showed that onsumption of this substrate by the mixed mirobial ulture required a terminal eletron aeptor. The number of fermentative and sulfate-reduing bateria with glyerol or 173-propanediol as arbon and energy soure revealed that sulfate-reduing bateria outompete fermentative bateria for these substrates. The possible eologial role of sulfate-reduing bateria in the metabolism of these redued substrates is disussed. The lassial Desulfouibrio speies utilize a rather limited range of eletron donors for sulfate redution. Latate, pyruvate, ethanol, malate, formate, and hydrogen are the ommon energy substrates for these bateria [27]. Reently, new types ofdesulfouibrio speies were isolated that utilize frutose [6, 261, surose [16], and amino aids [35]. t has also been reently reported that glyerol an be used as energy and arbon soure by some Desulfouibrio speies in pure ulture [lo, 17,24-26, 28, 30, 311. Little is known, however, about the utilization of glyerol by sulfate-reduing bateria [31]. Glyerol is a major omponent of waste water from bioethanol prodution plants (distillery stillage), whih also ontain sulfate [2, 31; and anaerobi digestion is one of the main purifiation proesses used on these wastes [2, 31. The purpose of the present study was to examine whether sulfate-reduing bateria play a role in anaerobi glyerol and 173-propanediol digestion, and to quantify the populations involved in this proess. Materials and Methods Soures of miroorganisms. The mirobial mixed ulture ame from a ontinuous fermenter (20-liter Biolafitte reator; agitation rate, 50 rpm, temperature, 37 C; sequential feeding, 1 liter every 3 days with a mean organi load of 0.4 g CD L-' d-' and hydroli residene time of 60 days) fed with waste water from an industrial distillery (NRA-Narbonne, Frane). The main substrates present in the waste were glyerol and lati aid. Conentrations of sulfate varied between 6 and 12 mm. The inoulum was from an anaerobi lagoon of distillery waste waters [2, 31. Media. The basal sulfate-free salt solution mineral-medium used for bath ulture experiments had the following omposition (g/ L): K2HP04, 0.4; KH2P0,, 0.4; NH,Cl, 0.9; NaCl, 0.9; MgCl,. 6H20, 0.1; CaCl,. 2H20, 0.1; ysteine-hc1. H20 (as a reduing agent), 0.4; resazurin, A basal biarbonate buffered and sulfide-redued medium with vitamins and trae elements was omposed and prepared as desribed by Widdel and Pfennig [39] and modified by Widdel [37]; it was supplemented with 0.01% yeast extrat (Difo) for fermentative and sulfate-reduing baterial ounts. Growth onditions. Bath ulture experiments were arried out at 37 C either in 1-L magnetially stirred reators with ph ontrol or in 60-ml serum bottles losed with butyl-rubber stoppers, inubated in a gyratory shaker, without ph ontrol. Prior to the bath ulture experiments, the inoulum was anaerobially diluted to 50% (vol/vol) with the sterilized sulfate-free salt solution under 02-free N,, with a Hungate tehnique [15] as modified for the use of syringes [20]. The diluted samples were inubated under 100% N,, at 37"C, for 3 or 4 days to stimulate baterial growth and to ensure that the sulfate derived from the samples was ompletely onsumed. Samples were then distributed among reators and Address reprint requests to: Dr. J.L. Garia, RSTM Laboratoire de Mirobiologie, Université de Provene, 3, plae V-Hugo, Marseille édex 3, Frane. Q. R- S. Ta. M, FQMJ~ ~~~~~~~n~~~~~ N g , QX

2 4s CURRENT MCRBLGY Vol. 22 (1991) bottles under anaerobi onditions. rgani substrates, sulfate, and hloroform (an inhibitor of methanogenesis) were added from separately sterilized stok solutions. Baterial ounts. Fermentative and sulfate-reduing bateria were ounted by the most probable number (MPN) method with the following substrates: glyerol (20 mm) and 1,3-propanediol (20 mm). Sulfate-reduing bateria were ounted in the presene of sulfate (20 mm). Total flora was estimated by the aridine orange epifluoresene method [14]. The MPN tubes were inubated at 37 C for 4 weeks; growth of fermentative and sulfate-reduing bateria was determined from opti density measurements at 580 nm in Baush and Lomb Spetroni 70 spetrophotometer and sulfide prodution respetively. Metabolites produed from the various substrates were also determined. Analytial methods. Sulfide prodution ould not be demonstrated under these onditions by spetrophotometri methods beause of the strong olor of the waste used in the experiments. The sulfate redution ativity was, therefore, estimated from sulfate depletion in the ulture medium. Sulfate was analyzed by the HPLC tehnique under the following onditions: pump, Shimadzu LC 6A; eluant, phthali aid (3 mm) neutralized with Na-tetraborate at ph 4.9; flow rate, 2 ml min-'; injetion loop, 100 pl; olumn, Vyda anion exhange phase 302 C (25 m long); room temperature; ondutivity detetion was performed with a Shimadzu model CDD 6A detetor; reorder integrator, Shimadzu hromatopak CR 3A; external standard. Sulfide was determined spetrophotometrially in the form of olloidal CuS, as desribed by Cord-Ruwish 151. Glyerol was determined by the enzymati method developed by Batlle and Collon [l]. 1,3-propanediol was measured by means of FD gas hromatography (THEED 10% olumn on arbopak [SUPELC, n.] 1 m long, 2 mm inner diameter; olumn temperature, 125 C; arrier gas, nitrogen; internal standard, ethyleneglyol). Volatile fatty aids were analyzed by means of FD gas hromatography (Glass olumn, 2 m long; 4 mm inner diameter; filled with Chromosorb (SUPELC, n.), oated with 10% SP-1200 and 1% H3P4; arrier gas, nitrogen; olumn temperature, 105 C; internal standard, ethyl 2-butyri aid. Alohol and nonvolatile fatty aids were analyzed by HPLC measurements (olumn, Aminex HPX- 87H (BRAD, Rihmond); detetor, differential refratometer (Knauer); external standard). Methane and hydrogen were measured with TCD gas hromatography as desribed by Qatibi and Bories [29]. Total organi arbon (TC) was measured on a Dohrmann DC 80 analyzer [ 181. Results Anaerobi glyerol degradation. n the absene of sulfate, with ph ontrol at 7.2, glyerol was degraded with transient l,3-propanediol formation before propionate and aetate prodution ourred; the 173-propanediol aumulated was slowly degraded into propionate and aetate (Fig. la). The kinetis of the total organi arbon measured and alulated revealed the presene of unknown intermediate(s) (Fig. lb). n the presene of sulfate (Fig. 2a), with ph maintained at 7.2, glyerol was degraded and a parallel sulfate depletion ourred, o- 'F W L e 4 o e u qn. "" Fig. 1. Time ourse of anaerobi glyerol degradation by (a) the mixed mirobial ulture and (b) total organi arbon (TC) alulated and measured. The experiment was performed in the absene of sulfate at onstant temperature and ph (37 C and 7.2 respetively). Symbols: W, glyerol;, 1,3-propanediol; A, propionate; A, aetate; x, TC measured; +, TC alulated. iniding with a transient formation of 173-propanedio1 and other intermediate(s), before propionate (then onverted into aetate by sulfate redution) and aetate prodution took plae. A little 1,3-propanediol was produed, and the arbon balane (Fig. 2b) showed that onsiderable aumulation of the intermediate(s) had ourred. Effets of sulfate redution and methanogenesis on anaerobi 1,3-propanediol degradation. To determine the relationship between sulfate redution and methanogenesis on anaerobi 1,3-propanedio degradation, experiments were arried out in whih hloroform was added to inhibit methanogenesis (final onentration, 0.003% vol/vol) in the absene or presene of sulfate. As shown in Table 1, when b

3 A.-. Qatibi et al.: Sulfate Redution and Anaerobi Glyerol Degradation 49 n r Y E 'C W L u o s n r E u + ;ells were ounted per ml by the epifluoresene h tehnique with aridine orange. n low-sulfate glya erol medium, 2.5 x lo9 fermentative bateria were 40 estimated per ml by MPN ounts. n the last positive tubes, propionate, aetate, methane, and presum- / ably arbon dioxide were produed. Propionobaterium sp., Desulfovibrio sp., and Metlzanibaterium sp. were observed by mirosopi examination. n high-sulfate glyerol medium, 1.4 x lo9 sulfate-reduing bateria were ounted per ml; aetate, sulfide, and presumably arbon dioxide aumulated. Speies of Desulfovibrio were identified as the main glyerol degraders, from their morphology and fermentation produts; 4.0 X lo7 fermentative bateria were ounted per ml with 1,3-propanediol as sole substrate in the absene of sulfate. Putative speies 2oo of Pelobater and Desulfovibrio were observed in 50 u r- b the last positive tubes; propionate, aetate, methane, and persumably arbon dioxide were produed, whereas in the presene of sulfate, 2.5 x lo7 sulfatereduing bateria were estimated per ml. Speies of Desulfovibrio were identified as the main 1,3- propanediol degraders in the last positive tubes with aumulation of aetate, sulfide, and presumably arbon dioxide. r "1, +-i' Fig. 2. Time ourse of anaerobi glyerol degradation by (a) the mixed mirobial ulture and (b) total organi arbon (TC) alulated and measured. The experiment was performed in the presene of sulfate at onstant temperature and ph (37 C and 7.2 respetively). Symbols: B, glyerol; U, 1,3-propanediol; A, propionate; A, aetate; &, sulfate; x, TC measured; +, TC alulated. hloroform was added to the waste with or without sulfate, methane prodution from 1,3-propanediol as arbon and energy soure was entirely inhibited. n the absene of sulfate, and in the presene of hloroform, hydrogen aumulated; it was onsumed when sulfate was added, and the amount of 1,3-propanedio1 degraded was higher in the presene than in the absene of sulfate. This suggests that 1,3-propanedio1 degradation required either a terminal eletron aeptor suh as sulfate or syntrophi ooperation with methanogeni hydrogenotrophi bateria. Baterial ounts. Table 2 shows results from baterial ounts. n total, approximately 1' baterial Disussion Anaerobi degradation of glyerol requires the redution of eletron aeptors suh as fumarate, as ours in Esherihia oli [32] or Streptoous faealis 1131 or C2 in the ase of homoaetogeni bateria [S, 91. The prodution of 1,3-propanediol as a redued end produt from glyerol has been observed in Clostridiurn pasteurianurn [23], Clostridium butylium [ 121, Klebsiella pneurnoriiae [ 1 11, Latobaillus brevis, and Latobaillus bulzneri [33], Citrobater freundii [22], and Klebsiella aerogenes [36]. n this study, it was demonstrated that in the absene of sulfate, anaerobi glyerol degradation by the mixed mirobial ulture studied was haraterized by the transient formation of 1,3-propanediol prior to propionate and aetate aumulation. No latate [19], ethanol and butyrate [12], arolein [34], suinate, or pyruvate were produed. The presene of sulfate as a terminal eletron aeptor seems to diminish the prodution of 1,3-propanediol by the mixed mirobial ulture. Thus, in the presene of sulfate, the problem of exess eletron release during glyerol degradation is solved. These results suggest that sulfate-reduing bateria may be involved in glyerol degradation with sulfate as terminal eletron aeptor. The results of our enu- Y

4 50 CURRENT MCRBLGY Vol. 22 (1991) Table 1. Effets of sulfate redution and methanogenesis on anaerobi 1,3-propanediol degradation by the mixed mirobial ulture. nubation at 3TC, for 4 weeks nitial onentration (mn/r) Final onentration (mm) Substrates 1,3-H' C3 2 sul 1,3-H 3 2 sul CH4 H2 1,3-H 17.7 l ,3-H t Sul ,3-H + hl ,3-H + Sul + hl ' Symbols: 1,3-H, 1,3-propanediol; C3, propionate; C2, aetate; sul, sulfate; hl, hloroform. Table 2. MPN baterial ounts of fermentative and sulfate-reduing bateria involved in anaerobi glyerol and 1,3-propanediol degradation by a mixed mirobial ulture. Baterial ounts Substrates (viable ells/ml) Produts of fermentation - sulfate Glyerol 1,3-propanediol t sulfate Glyerol 1,3-propanediol Total florab 2.5 x x x x '0 propionate t aetate + methane propionate + aetate + methane sulfide t aetate' sulfide t aetate' nd ' Traes of propionate were also deteted. Estimated by the aridine epifluoresene method [15]. merations are onsistent with this hypothesis. Sulfate-dependent glyerol oxidation was only reently unequivoallyproved [lo, 17,24-26,28,30,31,35]. Furthermore, the values obtained in the absene of sulfate (53% arbon reovery when glyerol has disappeared, versus 91% at the end) and in the presene of sulfate (12.5% arbon reovery when glyerol has disappeared, versus 100% approximately at the end) are quite similar to those reported on pure ulture of Desulfovibrio earbitzolius growing on glyerol with or without sulfate [24]; they strongly suggest the possibility that the intermediate produed during glyerol degradation by mixed mirobial ulture was 3-hydroxypropionate, although it is diffiult to ompare the results obtained beause the media used were different, and the behavior of bateria in pure ulture differs greatly from that in mixed ultures. Anaerobi oxidation of 1,3-propanediol has been reported with D. arbinolius in the presene of sulfate [25] and with a strain of Pelobater arbinolius that oxidizes 1,3-propanediol into aetate only in the presene of a methanogeni hydrogen- otrophi baterium [7]. ur finding that 1,3-propanediol degradation did not our when methanogenesis was inhibited in the absene of sulfate (Table 1) suggests that 1,3-propanediol degradation requires a terminal eletron aeptor suh as sulfate or a methanogeni hydrogenotrophi baterium. t is interesting to note that in low-sulfate medium tubes from enumerations with glyerol and l,3- propanediol, Desulfovibrio speies were observed by mirosopi examination. t has, in fat, been demonstrated that Desulfovibrio speies utilize glyerol and diols suh as 1,2- and 1,3-propanediol by interspeies hydrogen transfer [28, 30, 311. ur results on glyerol and 1,3-propanediol degradation by the mixed mirobial ulture agree with the onept that anaerobi degradation of redued produts of lassial fermentations suh as alohols or fatty aids usually requires the redution of eletron aeptors suh as sulfate [38], or a syntrophi assoiation of fermenting and methanogeni bateria, for example [4, 211. ur results also show that glyerol and 1,3-propanediol (two substrates reently desribed as potential donors of reduing equivalents

5 A.-. Qatibi et al.: Sulfate Redution and Anaerobi Glyerol Degradation 51 4 * P ' *i for sulfate redution) may be used by sulfate-reduing bateria in the methanogeni eosystem studied, possibly thanks to the presene of glyerol and sulfate in the original fermenter. The isolation and haraterization of the main sulfate-reduing bateria using glyerol and 1 &propanediol as energy and arbon soures from the positive tubes of enumerations will be desribed elsewhere [28]. ACKNWLEDGMENTS The authors wish to thank J.L. Batlle for glyerol assays, NRA experimental station (Pesh-Rouge-Narbonne, Frane), and J. Blan for revising the manusript. A.. Qatibi's work was supported by RSTM and 1.N.R.A grants. Literature Cited 1. Batlle JL, Collon Y (1979) Dosage enzymatique en flux ontinu du glyérol dans les vins. Conn Vigne Vin 1: Bories A (1981) Méthanisation des eaux résiduaires de distilleries. Trib Cebedeau, 456: Bories A, Raynal J, Jover JP (1982) Fixed film reator with plasti media for methane fermentation of distilleries. n: Strub A, Chartier P, Shleser G (eds) Energy from biomass. London: Applied Siene Publisher, pp Bryant MP, Wolin EA, Wolin MJ, Wolfe RS (1967) Metkanobaillus oinelianskii, a symbioti assoiation of two speies of bateria. Ark Mikrobiol'-59: Cord-Ruwish R (1985) A quik method for the determination of dissolved and preipitated sulfides in ultures of sulfatereduing bateria. J Mirobiol Methods 4: Cord-RuwishR, llivier B, Garia JL (1986) Frutose degradation by Desulfouibrio sp. in pure ulture and in oulture with Metliatzospirillum hungatei. CUT Mirobiol. 13: Dubourguier HC, Samain E, Premier G, Albagna G (1986) Charaterisation of two strains of Pelobater arbinolius isolated from anaerobi digesters. Arh Mirobiol 145: Eihler B, Shink B (1984) xidation of primary aliphati alohols by Aetobaterium arbinolium sp. nov., a homoaetogeni anaerobe. Arh Mirobiol 140: Emde R, Shink B (1987) Fermentation of triaetin and glyerol by Aetobaterium sp. No energy is onserved by aetate exretion. Arh Mirobiol 149: Esnault G, Caumette P, Garia JL (1988) Charaterization of Desulfouibrio giganteus sp. nov., a sulfate-reduing baterium isolated from a brakish oastal lagoon. System Appl Mirobiol 10: Forage RG, Foster MA (1982) Glyerol fermentationinklebsiellapneumoniae: funtions of the oenzyme B 12-dependent glyerol and diol dehydratases. J Bateriol 149: Forsberg CW (1987) Prodution of 1,3-propanediofromglyero1 by Clostridium aetoburyliutn and other Clostridium speies. Appl Environ. Mirobiol 53: Gunsalus C (1947) Produts of anaerobi glyerol fermentation by Streptoous faealis. J Bateriol54: Hobbie JE, Daley RJ, Jasper S (1977) Use of nuleopore filters for ounting bateria by fluoresene mirosopy. Appl Environ Mirobiol 33: Hungate RE (1960) Mirobial eology of rumen. Bateriol Rev 24: Joubert WA, BritzTJ (1987) solation of saharolyti dissimilatory sulfate-reduing bateria. FEMS Mirobiol Lett 48: Kremer DR, Hansen TA (1987) Glyerol and dihydroxyaetone dissimilation in Desulfouibrio strains. Arh Mirobiol 147: Landre J (1983) Détermination du arbone organique total dans les eaux potables par oxydation U.V. Eau ndustries Nuisanes 71: Larohe M (1983) Métabolisme intermédiaire des aides gras volatils en fermentation méthanique. Thése dotorat, nstitut National Des Sienes Appliquées, Toulouse, Frane 20. May JM, Snellen JE, Hungate RE (1972) Use of syringe methods for anaerobiosis. Am J Clin Nutr 25: Mnerney MJ, Bryant MP, Pfennig N (1979) Anaerobi baterium that degrades fatty aids in syntrophi assoiation with methanogens. Arh Mirobiol 122: Mikelson MN, Werkman CH (1940) The dissimilation of glyerol by Coli-aerogenes intermediates. J Bateriol 39: Nakas JP, Chaedle MS, Parkinson CM, Coonley CE, Tannen-, baum SW (1983) System development of linked fermentation prodution of solvents from algal biomass. Appl Environ Mirobiol 46: Nanninga HJ, Gottshal JC (1986) solation of a sulfate-reduing baterium growing with methanol. FEMS Mirobiol EC 38: NanningaHJ, Gottshal JC (1987) Properties ofdesulfouibrio arbinolius sp. nov. and other sulfate-reduing bateria isolated from an anaerobi-purifiation plant. Appl Environ Mirobiol51: llivier B, Cord-RuwishR, HathikianEC, GariaJL (1988) Charaterization of Desulfouibrio fiutosouoratis sp. nov. Arh Mirobiol Postgate JR (1984) The sulfate reduing bateria, 2nd ed London: Cambridge University Press 28. Qatibi A (1990) Fermentation du latate, du glyérol et des diols par les batéries sulfato-rédutries du genre Desulfouibrio. Thèse dotorat, Univer. Aix-Marseille, Frane 29. Qatibi A, Bories A (1988) Glyerol fermentation and sulfate utilization during the anaerobi digestion proess. n: Tilhe A, Rozzi A (eds) Fifth ntern. Symp. Anaerobi Digestion, Monduzzi Editore, Bologna, pp Qatibi A, Garia JL (1989) Glyerol degradation by Desulfouibrio sp., in pure ulture and in oulture with Metlianospiriluin hutigatei. n: FEMS Symp. mirobiology and biohemistry of strit anaerobes involved in interspeies hydrogen transfer. New York Plenum Publ.Corp., in press 31. Qatibi A, Cayo1 JL, Garia JL (1989) 1,2- and 1,3-propanedio1 degradation by Desulfouibrio aloholouorans sp. nov., in pure ulture or through interspeies transfer. n: FEMS Symp. mirobiology and biohemistry of strit anaerobes involved in interspeies hydrogen transfer. New-York: Plenum Publ. Corp., in press 32. Quastel JH, Stephenson M, Whetham MD (1925) Some reations of resting bateria in relation to anaerobi growth. Biohem J 19: Shutz H, Radler F (1984) Anaerobi redution of glyerol to propanediol-1,3 by Latobaillus brevis and Latobaillus buhneri. Syst Appl Mirobiol. 5: il 1: Ø h

6 ì Serjak WC, Day WH, Van Lanen JM, Boruff CS (1954) Arolein prodution by bateria found in distillery grain mashes. Appl Mirobio1 2: Stams A, HansenTA, SkyringGW (1985) Utilisationofamino aids as energy substrates by two marine Desir[fouibrio strains. FEMS Mirobio1 Lett 31:ll Thiamann KV (1955) The life of bateria. New York: MaMillan Publishing Co 37. Widde F (1980) Anaerober Abbau von Fettsaüren und Benzoesaiire durh neu isolierte Arten Sulfat-reduzierender CURRENT MCRBLGY Vol. 22 (1991) Bakterien. Dotoral thesis, Univ Göttingen, FRG 38. Widdel F (1988) Mirobiology and eology of sulfate- and sulfur-reduing bateria. n: Zehnder AJB (ed) Biology of anaerobi miroorganisms. New York: John Wiley, pp Widdel F, Pfennig N (1977) A new anaerobi sporing aetate-oxidizing, sulfate-reduing baterium, Desulfotomnulum (emend) aetoxidans. Arh Mirobiol 112: F i 't