Jan 18, 1996 - of Microbiology,3 Wageningen Agricultural University, Wageningen, The Netherlands. Received 18 .... analyzed immediately or stored at -20°C until analysis. Nuclear ..... way, i.e. at the level of propionyl-CoA, which cor-.
JOURNALOF FERMENTATION AND Vol. 82, No. 4, 387-391. 1996
BIOENGINEERING
Propionate Degradation by Mesophilic Anaerobic Sludge: Degradation Pathways and Effects of Other Volatile Fatty Acids PIET N. L. LENS,’ VINCENT
O’FLAHERTY,’ COR DIJKEMA,2 ALFONS J. M. STAMS3*
EMER COLLERAN,’
AND
Department of Microbiology, University College, Galway, Ireland, I Wageningen NMR Centre,= and Department of Microbiology,3 Wageningen Agricultural University, Wageningen, The Netherlands Received 18 January 1996/Accepted 22 July 1996
The degradation of volatile fatty acids by flocculant mesophilic (35+2”C) syntrophic sludge grown in an anaerobic hybrid reactor fed with a mixture of propionate, n-butyrate and ethanol (1: 1: 2 on COD basis) at a volumetric loading rate of 3.7 kg COD per m3 per day was examined. The propionate degradation rate amounted to 1.1 mmol per g volatile suspended solids per day. The same propionate degradation rate was measured in the presence of 10 mM acetate, but it decreased by 30% when 10 mM n-butyrate was added. 13CNuclear magnetic resonance spectroscopy demonstrated interconversion of 1% to 10% of both the propionate and butyrate pool, during their simultaneous degradation. Propionate formation from [2-13C]butyrate was not via a direct decarboxylation. ‘SC-Propionate was converted via at least three different pathways. The first pathway was syntrophic conversion of propionate via the randomising pathway, evidenced by scrambling of [3J3C]propionate into [2J3C]propionate. Secondly, reductive carboxylation occurred, i.e. [3J3Clpropionate and [2,3J3C]propionate were partly (2 to 10%) converted into [4-13C]butyrate and [3,4J%]butyrate, respectively. Reductive carboxylation probably involved a transcarboxylase, as 13C-bicarbonate was not incorporated in the caboxyl group of butyrate. Thirdly, propionate was converted into higher fatty acids: [2,3J3Clpropionate was converted into [4,5-*3C]valerate and 2-methyl[2,3-*3C]butyrate. [Key words: propionate,
reductive carboxylation,
butyrate,
acetate]
(combining a sludge blanket and a biofilm) reactor (11) operating at a volumetric loading rate of 3.7 kg COD per m3 per day and a hydraulic retention time of 3.4 d. The reactor treated a synthetic wastewater (pH 7.6+0.2), consisting of a mineral NaHC03 buffered (10 g/l) medium (11) and a total chemical oxygen demand (COD) of 12g/l. It contained propionate, butyrate and ethanol as the carbon sources in a ratio of 1 : 1 : 2 on COD basis. Sludge samples were taken during steady-state operation to characterize its metabolic properties. Reactor experiment To study propionate metabolism in the reactor, steady-state conditions were perturbated by increasing the influent propionate concentration from 3.0 g COD/I (27.0mM) to 4.5 g COD/f (40.5 mM) from day 250 till day 270. To maintain the volumetric loading rate at 3.7 kg COD per m3 per day, butyrate and ethanol influent concentrations were decreased from 3 g COD/I (18.7 mM) to 2.5 g COD/I (15.6 mM) and from 6 g COD/I (62.3 mM) to 5 g COD/I (51.9 mM), respectively. Activity tests The pressure bottle technique was used to determine maximum specific conversion rates (5), substrate affinity constants (4) and 50% inhibitory concentrations (8). The effect of VFAs on propionate degradation was investigated in batch experiments. Serum vials (120 ml) containing about 50 mg of volatile suspended solids (VSS) in 25 ml of anaerobic buffer (38mM NaHC03; pH 7.2kO.2) were incubated under a 80%N2-20%C02 atmosphere at 35 (-+2) “C. Prior to the anaerobic addition of the substrates (10 mM final concentration unless specified otherwise), the vials were incubated overnight to allow complete degradation of residual substrate in
The thermodynamics (24), biochemistry (2, 13, 14, 2022), kinetics (3, 6) and inhibition (7) of anaerobic propionate degradation have been well documented in defined cocultures and enrichment cultures. Propionate degradation has been studied in more complex microbial ecosystems as well, e.g. in sludges growing in different bioreactor types (12, 19, 23, 25). These studies, mainly carried out to improve bioreactor performance, showed that besides the syntrophic oxidation of propionate to the methanogenic substrates Hz, formate and acetate, reduced end products such as alcohols (23) and higher (C&Z,) volatile fatty acids (VFAs) (18, 25, 27, 29) can be formed from propionate as well. In general, these side reactions are associated with suboptimal reactor performance (23), but little is known about the biochemistry involved and their in situ role in organic matter removal in bioreactors. The aim of the present study was to characterize possible alternative propionate conversion routes present in a flocculant sludge grown on a mixture of propionate, butyrate and ethanol. The propionate degradation capacity of the sludge is described, and the effects of other VFAs on propionate degradation were investigated with an emphasis on the interrelationship of the propionate and butyrate degrading pathways. MATERIALS
isomerisation,
AND METHODS
Source of biomass Biomass was sampled anaerobically from the sludge blanket of a laboratory scale (17 r) mesophilic (35 k2”C) quarter packed anaerobic hybrid * Corresponding author. 387
388
J. FERMENT. BIOENG.,
LENS ET AL.
the sludge. At regular time intervals, the supernatant was sampled, centrifuged for 1Omin at 13,000 x g and analyzed immediately or stored at -20°C until analysis. The fate Nuclear magnetic resonance spectroscopy of 13C-labeled compounds was studied in batch experiments as described above, except that 13C-enriched substrates were added at time zero. The r3C proton-decoupled NMR spectra of the supernatants (2 ml) were recorded in a 10mm (o.d.) NMR tube on a Bruker spectrometer (AMX-300) tuned at 75.47 MHz as described previously (15), except that spectra were acquired at 294 K during 120min (14400 scans). The 13C chemical shifts were referenced to the carbon-2 (C,) of propionate (31.6 ppm). Labeled compounds were quantified after correction for differences in relaxation and NOE behavior by referring to preadded r3C-enriched glucose (5 mM). Presented spectra are representative of the results of replicate experiments. Analytical methods Methane and hydrogen were analyzed by gas chromatography (14). VFAs were measured both by high-performance liquid chromatography (6) and gas chromatography (11). Chemicals r3C-labeled compounds (> 99 atom % r3C) were obtained from Isotec Inc. (Pixie Corporatie B.V., Tjuchem, The Netherlands). All other chemicals were reagent grade. Gasses were supplied by Hoek-Loos (Schiedam, The Netherlands). RESULTS The Metabolic properties of the anaerobic sludge sludge present in the reactor under steady-state conditions reached a maximum specific conversion rate of 1 mmol CH4/g VSS.d with propionate (Table 1). Compared to the other feed constituents, a comparable maximum specific conversion rate was measured for n-butyrate, whereas ethanol was converted at a 4 times higher rate. Operating the reactor for a period of 226 d doubled the maximum specific conversion rate with propionate, while that of butyrate increased with 60% (Table 1). In contrast, the maximum specific conversion rate with ethanol remained the same. Also the maximum specific conversion rates with the direct methanogenic substrates acetate and HZ/CO2 (about 4 mmol CHJg VSS .d) did not vary much during reactor operation (Table 1). At concentrations higher Propionate degradation than 5 mM, the methane formation rate from propionate was independent of the initial propionate concentration. From the specific conversion rates measured for different initial propionate concentrations, the apparent K, of the sludge for propionate was 2.8 mM. During the degradation of propionate, only minor quantities of acetate (CZ) or higher VFAs (> C,) could be detected in the effluent. Since perturbating steady-state reactor operation did not reveal how the various degradation routes are interrelated, batch experiments using r3C-labeled substrates were performed, especially to differentiate butyrate formed from
INTERACTIVE DEGRADATION
VOL. 82, 1996
OF PROPIONATE
AND BUTYRATE
389
P3
B
A
P2
A2 BES2 \
40
I
35
30
I
,
I
25
20
II
15
10
5
7,
nna
40
35
30
25
20
15
10
5
PPB
FIG. 1. High resolution ‘H-decoupled 13CNMR spectra of medium sample from [3J3C]propionate degradation by the syntrophic sludge recorded after 77 h incubation. Partially inhibited incubation by 5 mM BES (A). Incubation in the presence of butyrate (B). A, Acetate; B, butyrate; BES, bromoethanesulfonate; P, propionate; U, unidentified peak. The numbers following the one-letter abbreviations give the positions of the carbon in the molecule.
propionate
and butyrate
added as the substrate. When sludge was incubated with 10 mM [3J3C]propionate and 10 mM butyrate, little scrambling occurred (Table 2), which is in agreement with the inhibition of propionate degradation by butyrate. More importantly, butyrate with unequally labeled carbon atoms was observed in the NMR spectra (Fig. lB, Table 2) as indicated by an i3C-enrichment at the Cq position of 70% above its nature abundant level. The labeling pattern, formation of [4-13C]butyrate out of [3J3C]propionate, is in agreement with the reductive carboxylation mechanism (25). After 77 h of incubation, about 100,nm [4J3C]butyrate was formed, which accounted for 1% of the total butyrate pool.
Batch incubations
TABLE 2.
Incubating sludge with equimolar quantities (initially 1OmM) of [2,3J3C]propionate and butyrate resulted in [3,4-13C]butyrate formation. The latter accounted for up to 10% of the total butyrate pool (1.24 mM after 134 h incubation). To further investigate the reductive carboxylation mechanism, experiments with [‘%]HC03were performed (Fig. 2). In case bicarbonate present in the medium is incorporated into the propionate molecule, [2,3-i3C]propionate would yield [1,3,4-13C]butyrate. However, no butyrate with a [13C]labeled carboxyl group could be detected upon concomitant incubation of [2,3J3C]propionate (10 mM) and [13C]HC03- (50 mM), with or without butyrate (10mM). However, [2,3-13C] propionate (with typical doublet structures for the C2
Labeling positions of propionate and butyrate during batch degradation of i3C-labeled sodium propionate Fractional distribution of W-atoms
Incubation” 3-Propionate 3_Propionate+acetate 3-Propionate + butyrate 3-Propionate + BES
Time of sampling (h)
Propionate degraded (%)
12 77 12 77 12 77 12 77
24 76 25 75 9 30 N.D. 45
propionate
in
butyrate
2-‘3C
3-“C
0.01 0.27 0.01 0.26 0.01 0.10 N.D. 0.28
0.99 0.73 0.99 0.74 0.99 0.90 N.D. 0.72
2-13~
/ / / 0.12 0.33 0.28 N.D. /
?-‘3C
I-‘3C
/ /
/ /
0.50 0.29 0.33 0.22 N.D. /
0.50 0.59 0.33 0.50 N.D. /
a Incubations were performed with 10 mM VFA. Propionate degradation was partially inhibited by the addition of 5 mM BES. /, Below detection limit. N.D., Not determined.
390
LENS ET AL.
J. FERMENT. BIOENG.,
P2
84 + M2
B2
FIG. 2. High resolution rH-decoupled r3C NMR spectrum of medium samples from [2,3-“Clpropionate degradation by the syntrophic sludge in the presence of butyrate and [t3C]HCOI-, recorded after 134 h incubation. A, Acetate; B, butyrate; M, methyl-butyrate; P, propionate; V, valerate. The numbers following the one-letter abbreviations give the positions of the carbon in the molecule.
and C3 resonances, separated by 34 Hz due to spin-spin coupling between both labeled carbon atoms), appeared to be incorporated in [4,5-13C]valerate and 2 methyl-[2,3r3C]-butyrate (Fig. 2). The formation of propionate from butyrate was studied by incubating sludge with [2-Wlbutyrate (10 mM) in the presence of propionate (10 mM). After 77.5 h of incubation, [4-13C]butyrate (92 /IM, 1.4% of total butyrate pool) as well as [3-r3C]butyrate (64 PM) were formed from [2-13C]butyrate (6.3 mM). Over that time interval, 300 ,uM [2-13C]propionate and 100 /*M [3-13C] propionate had been formed. The labeled propionate fraction was 6% of the total propionate pool present. DISCUSSION Interactive degradation of propionate and butyrate 13C-NMR spectroscopy showed that interconversion of the propionate and butyrate degradation routes occurs during their simultaneous degradation, involving about 1 to 10% of both the propionate and butyrate pools. One of those interconversion routes is, as evidenced by 13CNMR spectroscopy, reductive carboxylation (Fig. 1B). So far, this mechanism has only been described in sludge growing in a mesophilic anaerobic contact digester treating vegetable cannery wastewater, in which a somewhat higher fraction (up to 20%) of the 13C-propionate pool was involved (25). It should, nevertheless, be noted that these quantifications were obtained in batch experiments with high initial substrate concentrations and may not reflect the in situ role of these pathways in anaerobic bioreactors. Organisms capable of propionate oxidation to acetate via the succinate pathway were probably present in the sludge, as suggested by the exchange between the CZ and C3 atoms of propionate (Table 2). In contrast to the succinate pathway (13, 21, 22), reductive carboxylation preserves the carbon skeleton of the propionate molecule, i.e. [3-13C]propionate is converted into [4-13C]
butyrate (Fig. 1B). Further degradation of butyrate ($oxidation) yields [Z13C]acetate, as observed in the BESinhibited incubation (Fig. 1A). This labelling pattern of acetate, also observed by Tholozan et al. (27) in BESinhibited enrichment cultures degrading [3-13C]propionate, disagrees with acetogenesis via the succinate pathway, where degradation of [3-r3C]propionate would yield acetate equally labeled at the carboxyl and the methyl group (16). This indicates that a non-randomizing pathway, i.e. reductive carboxylation, is more important in acetogenesis from propionate in methanogenic syntrophic sludges. Alternatively, the succinate pathway is involved but the label at the C-l position of acetate is rapidly exchanged with unlabeled CO2 by methanogens or homoacetogens. Our data suggest that the reductive carboxylation mechanism is performed by a transcarboxylase, as [13C]HC03- present in the medium was not involved in the reaction (Fig. 2). Involvement of a carboxylase using intracellular HC03- can nevertheless not be excluded if cells have a low permeability for [13C]HC03-. The lack of accumulation of intermediates other than acetate in the degradation experiments hampers the outlining of the detailed biochemical route of the reductive carboxylation reaction. The observed labeling patterns of 13Cbutyrate, however, allow to eliminate some theoretically possible butyrate formation routes. Condensation of two acetyl-CoA molecules would yield randomly labeled butyrate, which disagrees with the conservation of the propionate skeleton (Fig. 1B). The observed labeling pattern also disagrees with butyrate formation from succinyl-CoA either directly via 4_hydroxybutyrate, as observed in Clostridium kluyveri (28), or after carboxylation of succinyl-CoA to 2-oxoglutarate (25). These pathways involving succinyl-CoA as an intermediate would also (partly) randomize the label in the butyrate molecule, e.g. [3-Wlpropionate would be converted into a mixture of [3-13C]butyrate and [4-13C]butyrate. Consequently, the carboxylation of the propionate molecule should occur in an earlier step in the degradation pathway, i.e. at the level of propionyl-CoA, which corroborates with the conclusions of Tholozan et al. (25). However, none of the intermediates of the pathway proposed by the latter authors could be detected in our NMR-spectra. The formation of r3C-propionate out of r3C-butyrate is not in accordance with the f-oxidation pathway used by syntrophic butyrate degraders (30). Direct decarboxylation of butyrate into propionate has been proposed as a possible mechanism for n-butyrate removal in sequentially fed anaerobic filters (10). In that case, [213C]butyrate would be converted into [l-13C]propionate, which was not observed in our experiments. The labeling pattern of the propionate molecule observed in the spectra recorded in the present study indicates that propionate is formed from labeled acetate (during ;3-oxidation of [2J3C]butyrate) in a pattern which is the reverse of the propionate degradation reaction. The same mechanism can explain the partial randomization of label in the butyrate molecule. The presence of isobutyrate in the reactor effluents during the perturbation experiment indicates that n-butyrate was involved in isomerization reactions, which has also been observed in other syntrophic sludges (1, 17). As isobutyrate was not detected in the batch degradation tests performed with much lower substrate concentra-
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tions (Figs. 1 and 2), NMR work showing that isomerization of n-butyrate is due to the migration of its carboxyl group (26) could not be confirmed.
15.
ACKNOWLEDGMENTS This research was supported by the EC Human Capital and Mobility network “Improved Application of Anaerobic Digestion Technology” (ERBCHRXCT930262) and the Wageningen NMR Centre (ERBCHGECT940061).
16.
REFERENCES
17.
1. Aguilar, A., Casas, C., Lafuente, J., and Lema, J. M.: Kinetic modelling of isomerization and anaerobic degradation of nand i-butyrate. J. Ferment. Bioeng., 69, 261-264 (1990). 2. Boone, D. R.: Propionate exchange reactions in methanogenic ecosystems. Appl. Environ. Microbial.. 48. 863-864 (1984). 3. Boone, D. R.-and Xun, L.: Effect of pH, temperature and nutrients on propionate degradation by a methanogenic enrichment culture. Appl. Environ. Microbial., 53, 1589-1592 (1987). 4. Coates, J. and Collerao, E.: Effect of initial agitation on the start-up and operational performance of anaerobic hybrid reactors treating a synthetic feed. Process Biochem., 10, 162-171 (1990). 5. Colleran, E. and Pistilli, A.: Activity test system for determining the toxicity of xenobiotic chemicals to the methanogenic process. Ann. Microbial. Enzymol., 44, l-20 (1994). 6. Dong, X., Plugge, C. M., and Stams, A. J. M.: Anaerobic degradation of propionate by a mesophilic acetogenic bacterium in coculture and triculture with different methanogens. Appl. Environ. Microbial., 60, 2834-2838 (1994). I. Fukuzaki, S., Nisbio, N., Sbobayashi, M., and Nagai, S.: Inhibition of the fermentation of propionate to methane by hydrogen, acetate, and propionate. Appl. Environ. Microbial., 56, 719-723 (1990). 8. Golden, T., Molyneaux, E., and Colleran, E.: Toxicity of aromatic chemicals to the bacterial populations involved in anaerobic digestion, p. 19-22. In Preprints seventh international symposium on anaerobic digestion, Cape Town, South Africa (1994). 9. Gottschalk, G.: Bacterial metabolism, 2nd ed. Springer-Verlag, New York (1985). 10. Gourdon, R., Comel, C., Vermande, P., and Veron, J.: NButyrate catabolism in the treatment of a semi-synthetic landfill leachate on anaerobic filter under sequential feeding conditions. Biomass, 15, 11-24 (1988). 11. Henry, M. H., Doolon, B.A., Lens, P. N., and Colleran, E. M.: Use of anaerobic hybrid reactors for treatment of synthetic pharmaceutical wastewaters containing organic solvents. J. Chem. Technol. Biotechnol., 66, 251-264 (1996). 12. Heppner, B., Zellner, G., and Diekmann, H.: Start-up and operation of a propionate-degrading fluidized-bed reactor. Appl. Microbial. Biotechnol., 36, 810-816 (1992). 13. Houwen, F. P., Dijkema, C., Schoenmakers, C. H. H., Stams, A. J. M., and Zehnder, A. J. B.: “C NMR studv of urooionate degradation by a merhanogcnic coculture. FEMS ‘Midrobiol. Lett., 41, 269-274 (1987). 14. Houwen, F. P., Dijkema, C., Stams, A. J. M., and Zehnder,
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
DEGRADATION
OF PROPIONATE
AND BUTYRATE
391
A. J. B.: Propionate metabolism in anaerobic bacteria; determination of carboxylation reactions with i3C-NMR spectroscopy. Biochim. Biophys. Acta, 1056, 126132 (1991). Kengen, S. W. M., de Bok, F. A. M., van Loo, N.-D., Dijkema, C., Stams, A. J. M., and de Vos, W. M.: Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus. J. Biol. Chem., 269, 17537-17541 (1994). Koch, M. E., Dolfing, J., Wuhrmann, K., and Zehnder, A. J.B.: Pathways of propionate degradation by enriched methanogenic cultures. Appl. Environ. Microbial., 45, 14111414 (1983). Lin, C. Y. and Hu, Y. Y.: Mesophilic degradation of butyric acid in anaerobic digestion. J. Chem. Tech. Biotechnol., 56, 191-194 (1993). Lin, C. Y., Sato, K., Noike, T., and Matsumoto, J.: Methanogenie digestion using mixed substrate of acetic, propionic and butyric acids. Water Res., 20, 385-394 (1986). Mawson, A. J., Earle, R. L., and Larsen, V. F.: Degradation of acetic and propionic acids in the methane fermentation. Water Res., 25, 1549-1554 (1991). Plugge, C. M., Dijkema, C., and Stams, A. J. M.: Acetyl-CoA cleavage pathway in a syntrophic propionate oxidizing bacterium growing on fumarate in the absence of methanogens. FEMS Microbial. Lett., 110, 71-76 (1993). Robbins, J. E.: 13C Nuclear magnetic resonance studies of propionate catabolism in methanogenic cocultures. Appl. Environ. Microbial., 53, 2260-2261 (1987). Robbins, J. E.: A proposed pathway for catabolism of propionate in methanogenic cocultures. Appl. Environ. Microbiol., 54, 1300-1301 (1988). Smith, D. P. and McCarty, P. L.: Reduced product formation following perturbation of ethanol- and propionate-fed methanogenie CSTRs. Biotechnol. Bioeng., 34, 885-895 (1989). Thauer, R. K., Decker, K., and Jungermann, K.: Energy conservation in chemotrophic anaerobic bacteria. Bacterial. Rev., 41, 100-180 (1977). Tholozan, J. L., Samain, E., Grivet, J. P., Moletta, R., Dubourguier, H. C., and Albagnac, G.: Reductive carboxylation of propionate to butyrate in methanogenic ecosystems. Appl. Environ. Microbial., 54, 441-445 (1988). Tholozan, J. L., Samain, E., and Grivet, J. P.: Isomerization between n-butyrate and isobutyrate in enrichment cultures. FEMS Microbial. Ecol., 53, 187-191 (1988). Tholozan, J. L., Samain, E., Grivet, J. P., and Albagnac, G.: Propionate metabolism in a methanogenic enrichment culture. Direct reductive carboxylation and acetogenesis pathways. FEMS Microbial. Ecol., 73, 291-298 (1990). Wolff, R. A., Urben, G. W., O’Herrin, S. M., and Kenealy, W. R.: Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri. Appl. Environ. Microbial., 59, 1876-1882 (1993). Wu, W.-M., Jain, M. K., and Zeikus, J. G.: Anaerobic degradation of normal- and branched-chain fatty acids with four or more carbon atoms to methane by a syntrophic methanogenic triculture. Appl. Enrivon. Microbial., 60, 2220-2226 (1994). Zhao, H., Yang, D., Woese, C. R., Bryant, M. P.: Assignment of fatty acid-$-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16s rRNA sequences. Int. J. Syst. Bacterial., 43, 278-286 (1993).
