Glycolytic pathway and hydrogen yield studies of the extreme thermophile Caldicellulosiruptor saccharolyticus.

NMR analysis of (13)C-labelling patterns showed that the Embden-Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H(2) per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g(-1) h(-1) and 20 mmol l(-1) h(-1). An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.

Pathways of methanol conversion in a thermophilic anaerobic (55 degrees C) sludge consortium.

The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H(2)/CO(2) and acetate. The deprivation of inorganic carbon species [Sigma(HCO(3)(-)+CO(2))] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H(2)) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-(13)C]acetate, [1-(13)C]acetate and [2-(13)C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO(3)(-) was the preferred source of the carboxyl group. A small amount of the added H(13)CO(3)(-) was reduced to form the methyl group of acetate and a small amount of the added (13)CH(3)OH was oxidised and found in the carboxyl group of acetate when (13)CH(3)OH was converted. The recovery of [(13)C]carboxyl groups in acetate from (13)CH(3)OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when (13)CH(3)OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO(2 )prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO(2), with CO(2) being incorporated into the carboxyl group of acetate.

Metabolic response of roots to osmotic stress in sensitive and tolerant cereals--qualitative in vivo [31P] nuclear magnetic resonance study.

High resolution [31P] nuclear magnetic resonance (NMR) spectroscopy was used to investigate the changes in phosphate metabolism and intracellular pH in intact root segments of relatively osmotic stress sensitive species maize (Zea mays L) and insensitive species pearl millet (Pennisetron americanum (L) Leeke) exposed to hyper osmotic shock. The results were used to understand the adaptive mechanism of the two species. The hyper osmotic shock resulted in large build-up of phosphocholine and decrease in glucose 6-phosphate (G-6P) and UDPG levels in both the crops. The osmotic shock produced a large vacuolar alkalinization and decrease in pH across tonoplast membrane in maize roots. However, the roots of pearl millet were able to adapt to the stress and maintained pH gradient across tonoplast with marginal vacuolar alkalinization. This may be attributed to the sustained activity of primary tonoplast pumps and increased activity of H+-ATPase that normally maintain pH gradient across tonoplast.

Intracellular PHB conversion in a Type II methanotroph studied by 13C NMR.

Poly-p-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [13C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by 13C NMR. During PHB synthesis in the presence of labelled acetate, low levels of beta-hydroxybutyrate, butyrate, acetone, isopropanol, 2,3-butanediol and succinate were observed. Subsequent anaerobic PHB breakdown showed enhanced levels of these products at the expense of PHB. Fermentative metabolism occurring during anaerobic PHB degradation was confirmed in experiments with fully 13C-enriched cells, which were grown on 13C-labelled methane. beta-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were detected as multiple 13C-labelled compounds in the culture medium. Our results suggest that intracellular PHB degradation can be used as a reserve energy source by methanotrophs under anoxic conditions.

The effect of oxygen on methanol oxidation by an obligate methanotrophic bacterium studied by in vivo 13C nuclear magnetic resonance spectroscopy.

13C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35-25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of 13C-labelled methanol to carbon dioxide was observed at low (1-5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically.

Energy yield of respiration on chloroaromatic compounds in Desulfitobacterium dehalogenans.

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO2. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.

The effect of oxygen on methanol oxidation by an obligate methanotrophic bacterium studied by in vivo (13)C nuclear magnetic resonance spectroscopy.

13C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35-25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of (13)C-labelled methanol to carbon dioxide was observed at low (1-5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically.

Intracellular PHB conversion in a Type II methanotroph studied by (13)C NMR.

Poly-beta-hydroxybutyrate (PHB) formation under aerobic conditions via incorporation of [(13)C-2]acetate as a cosubstrate and its intracellular degradation under anaerobic conditions in a Type II methanotroph was studied by (13)C NMR. During PHB synthesis in the presence of labelled acetate, low levels of beta-hydroxybutyrate, butyrate, acetone, isopropanol, 2,3-butanediol and succinate were observed. Subsequent anaerobic PHB breakdown showed enhanced levels of these products at the expense of PHB. Fermentative metabolism occurring during anaerobic PHB degradation was confirmed in experiments with fully (13)C-enriched cells, which were grown on (13)C-labelled methane. beta-hydroxybutyrate, butyrate, acetate, acetone, isopropanol, 2,3-butanediol and succinate were detected as multiple (13)C-labelled compounds in the culture medium. Our results suggest that intracellular PHB degradation can be used as a reserve energy source by methanotrophs under anoxic conditions.

Pathway of propionate oxidation by a syntrophic culture of Smithella propionica and Methanospirillum hungatei.

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by (13)C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-(13)C]propionate was converted to [2-(13)C]acetate, with no [1-(13)C]acetate formed. Butyrate from [3-(13)C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-(13)C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-(13)C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-(13)C-labeled propionate yielded both [1-(13)C]acetate and [2-(13)C]acetate. When (13)C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, (13)C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B891.

Lactococcus lactis subsp. cremoris B891 grown on whey permeate produced an exopolysaccharide containing D-Gal and D-Glc in a molar ratio of 2:3. The polysaccharide was partially O-acetylated. By means of HF solvolysis, O-deacetylation, enzymic modification, sugar linkage analysis and ID/2D NMR studies the exopolysaccharide was shown to be composed of repeating units with the following structure: [structure: see text].

Denitrification with methane as electron donor in oxygen-limited bioreactors.

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10(7) cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10(7) cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10(4) cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. "In vivo" 13C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification.

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B39.

Lactococcus lactis subsp. cremoris B39 grown on whey permeate produced an exopolysaccharide containing L-Rha, D-Gal and D-Glc in a molar ratio of 2:3:2. The polysaccharide was modified using an enzyme preparation from Aspergillus aculeatus, resulting in the release of Gal and a polymer with approximately the same hydrodynamic volume as the native polysaccharide. Linkage analysis and 1H NMR studies of both the native and modified exopolysaccharides elucidated that terminally linked Gal was released during modification and that the chemical structure of the branches within the repeating units is: beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. 2D NMR experiments (both 1H-1H and 1H-13C) revealed that exopolysaccharide B39 consists of a branched heptasaccharide repeating unit with the following structure: [structure: see text].

Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger.

31P nuclear magnetic resonance (31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5-10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca(2+)-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min-1 compared with 4 ml min-1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (delta pH = 1.6 and 1.4, respectively).

Analysis of sugar metabolism in an EPS producing Lactococcus lactis by 31P NMR.

Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.

Introduction of polyphosphate as a novel phosphate pool in the chloroplast of transgenic potato plants modifies carbohydrate partitioning.

Potato plants (Solanum tuberosum L., cv. Désirée) were transformed with the polyphosphate kinase gene from Escherichia coli fused to the leader sequence of the ferredoxin oxidoreductase gene (FNR) from Spinacea oleracea under the control of the leaf specific St-LS1 promoter to introduce a novel phosphate pool in the chloroplasts of green tissues. Transgenic plants (cpPPK) in tissue culture developed necrotic lesions in older leaves and showed earlier leaf senescence while greenhouse plants showed no noticeable phenotype. Leaves of cpPPK plants contained less starch but higher concentrations of soluble sugars. The presence of polyphosphate in cpPPK leaves was demonstrated by toluidine blue staining and unambiguously verified and quantified by in vitro 31P-NMR of extracts. Polyphosphate accumulated during leaf development from 0.06 in juvenile leaves to 0.83 mg P g-1 DW in old leaves and had an average chain length of 18 residues in mature leaves. In situ 31P-NMR on small leaf pieces perfused with well-oxygenated medium showed only 0.036 mg P g-1 DW polyphosphate that was, however, greatly increased upon treatment with 50 mM ammonium sulfate at pH 7.3. This phenomenon along with a yield of 0.47 mg P g-1 DW polyphosphate from an extract of the same leaf material suggests that 93% of the polyphosphate pool is immobile. This conclusion is substantiated by the observation that no differences in polyphosphate pool sizes could be discerned between darkened and illuminated leaves, leaves treated with methylviologen or anaerobis and control leaves, treatments causing a change in the pool of ATP available for polyPi synthesis. Results are discussed in the context of the chelating properties of polyphosphates for cations and its consequences for the partitioning of photoassimilate between starch and soluble sugars.

In vivo qualitative changes of 31P NMR in stressed maize roots vis-à-vis carbon substrate determining the degree of stress.

High resolution 31P nuclear magnetic resonance spectroscopy was used to investigate the changes in phosphate metabolism and intracellular pH in intact maize (Zea mays L) root segments to hyper osmotic shock. The results were compared with the happenings under field conditions, when the stress was given gradually. Effect of sugar substrate on adaptation of tissue to both kinds of situations was also studied. The hyper osmotic shock resulted in large vacuolar alkalinization and a decrease in pH across tonoplast membrane. There was gradual build up of phosphocholine and decrease in glucose 6P and UPDG levels. In gradual stress, the root segments were able to adapt to the stress and maintained pH gradient across tonoplast, with marginal alkalinization of vacuoles. The presence of sugar substrate reduced the impact of stress significantly, commensurate with the increased activity of plasmalemma H(+)-ATPase. The latter providing the driving force for uptake of organic molecules and ions required for osmoregulation.

Slow desorption of PCBs and chlorobenzenes from soils and sediments: relations with sorbent and sorbate characteristics.

The kinetics of slow desorption were studied for four soils and four sediments with widely varying characteristics [organic carbon (OC) content 0.5-50%, organic matter (OM) aromatic content (7-37%)] for three chlorobenzenes and five polychlorinated biphenyls (PCBs). Slowly and very slowly desorbing fractions ranged from 1 to 50% (slow) and 3 to 40% (very slow) of the total amount sorbed, and were observed for all compounds and all soils and sediments. In spite of the wide variations in sorbate K(OW) (factor 1000) and sorbent characteristics, the rate constants of slow (k(slow), around 10(-3) h(-1)) and very slow (k(very slow), 10(-5)-10(-4) h(-1)) desorption appeared to be rather constant among the sorbates and sorbents (both within a factor of 5). There was a good correlation (r(2) above 0.9) between the distribution over the slow, very slow and rapid sediment fractions and log K(OC), indicating that sorbate hydrophobicity may be important for this distribution. No correlation could be found between sorbent characteristics [OC, N, and O in the organic matter, polarity index C/(N+O), OC aromaticity as determined by CP-MAS (13)C-NMR] and slow desorption parameters (slowly/very slowly desorbing fractions+corresponding rate constants). The absence of (1) a correlation between k(slow) and k(very slow), respectively, and OC content, and (2) the narrow range of k(slow) and k(very slow) values, indicates that intra-OM diffusion is not the mechanism of slow or very slow desorption, because on the basis of this mechanism it would be expected that increasing OC content would lead to longer diffusion pathlengths and, consequently, to smaller rate constants. In addition, it was tested whether differential scanning calorimetry would reveal a glass transition in the soils/sediments. In spite of the sensitivity of the equipment used (changes in heat flow in the micro-Watt range were measurable), a glass transition was not observed. This means that activation enthalpies of slow desorption can be calculated from desorption measurements at various temperatures. In the present study these values ranged from 60 to 100 kJ/mol among the various soils and sediments studied.

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 alpha-galactosidase.

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an alpha-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083T. It was found to act with retention of configuration (alpha-->alpha), releasing alpha-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As alpha-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of alpha-galactosides using the transgalactosylation activity of Bi. adolescentis alpha-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 degrees C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and tetrasaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp- (1-->6)-alpha-D-Galp-(1-->6)-D-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride alpha-D-Galp-(1-->6)-alpha- D-Galp-(1-->6)-D-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.

Endoglucanase V and a phosphatase from Trichoderma viride are able to act on modified exopolysaccharide from Lactococcus lactis subsp. cremoris B40.

EPS B40 from Lactococcus lactis subsp. cremoris consists of a repeating unit of-->4)-beta-D-Glcp-(1-->4)-[alpha-L-Rhap-(1 -->2)][alpha-D-Galp-1-PO4-3]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. A phosphatase from Trichoderma viride was able to release phosphate, but only after removal of rhamnosyl and galactosyl residues by mild CF3CO2H treatment. Purified endoV from T. viride was able to act on the backbone of the polymer, but only if rhamnosyl substituents and phosphate had been removed. After complete removal of phosphate and partial removal of rhamnosyl residues by HF treatment, incubation with endoV resulted in a homologous series of oligomers. Purification of these oligomers and subsequent characterisation by NMR demonstrated that endoV was able to cleave the beta-(1-->4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-treated EPS B40 is discussed on the basis of the subsite model described for endoV [J.-P. Vincken, G. Beldman, A.G.J. Voragen, Carbohydr. Res., 298 (1997) 299-310].

13C-NMR study of propionate metabolism by sludges from bioreactors treating sulfate and sulfide rich wastewater.

Applications of nuclear magnetic resonance (NMR) to study a variety of physiological and biochemical aspects of bacteria with a role in the sulfur cycle are reviewed. Then, a case-study of high resolution 13C-NMR spectroscopy on sludges from bioreactors used for treating sulfate and sulfide rich wastewaters is presented. 13C-NMR was used to study the effect of sulfate and butyrate on propionate conversion by mesophilic anaerobic (methanogenic and sulfate reducing) granular sludge and microaerobic (sulfide oxidizing) flocculant sludge. In the presence of sulfate, propionate was degraded via the randomising pathway in all sludge types investigated. This was evidenced by scrambling of [3-13C]propionate into [2-13C]propionate and the formation of acetate equally labeled in the C1 and C2 position. In the absence of sulfate, [3-13C]propionate scrambled to a lesser extend without being degraded further. Anaerobic sludges converted [2,3-13C]propionate partly into the higher fatty acid 2-methyl[2,3-13C]butyrate during the simultaneous degradation of [2,3-13C]propionate and butyrate. [4,5-13C]valerate was also formed in the methanogenic sludges. Up to 10% of the propionate present was converted via these alternative degradation routes. Labeled butyrate was not detected in the incubations, suggesting that reductive carboxylation of propionate does not occur in the sludges.