Glutamate racemization and catabolism in Fusobacterium varium

The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.     

Sodium ion pumps and hydrogen production in glutamate fermenting anaerobic bacteria

Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B12-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na+ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na+ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na+ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP 'richer' than that of NADH.     

Analysis of the fermentation pathways of clostridia using double labelled glutamade

l-(4-¹⁴C, 3-³H)Glutamate was used as a tool to elucidate the pathway of its fermentation. The methylaspartate pathway was found in strains of Clostridium tetanomorphum, C. malenominatum, C. limosum, C. lentoputrescens and C. tetani, whereas the hydroxyglutarate pathway was detected in C. sporosphaeroides. These data were supported by measurement of enzyme activities present in cell-free extracts.     

Unusual enzymes involved in five pathways of glutamate fermentation

Anaerobic bacteria from the orders Clostridiales and Fusobacteriales are able to ferment glutamate by at least five different pathways, most of which contain enzymes with radicals in their catalytic pathways. The first two pathways proceed to ammonia, acetate and pyruvate via the coenzyme B12-dependent glutamate mutase, which catalyses the re-arrangement of the linear carbon skeleton to that of the branched-chain amino acid (2S,3S)-3-methylaspartate. Pyruvate then disproportionates either to CO2 and butyrate or to CO2, acetate and propionate. In the third pathway, glutamate again is converted to ammonia, CO2, acetate and butyrate. The key intermediate is (R)-2-hydroxyglutaryl-CoA, which is dehydrated to glutaconyl-CoA, followed by decarboxylation to crotonyl-CoA. The unusual dehydratase, containing an iron-sulfur cluster, is activated by an ATP-dependent one-electron reduction. The remaining two pathways require more then one organism for the complete catabolism of glutamate to short chain fatty acids. Decarboxylation of glutamate leads to 4-aminobutyrate, which is fermented by a second organism via the fourth pathway to acetate and butyrate, again mediated by an unusual dehydratase which catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The fifth pathway is the only one without decarboxylation, since the gamma-carboxylate of glutamate is reduced to the amino group of delta-aminovalerate, which then is fermented to acetate, propionate and valerate. The pathway involves the oxidative dehydration of 5-hydroxyvaleryl-CoA to 2,4-pentadienoyl-CoA followed by reduction to 3-pentenoyl-CoA and isomerisation to 2-pentenoyl-CoA.     

Pathways of Propionate Degradation by Enriched Methanogenic Cultures

A mixed methanogenic culture was highly enriched in a growth medium containing propionate as the sole organic carbon and energy source. With this culture, the pathways of propionate degradation were studied by use of ¹⁴C-radiotracers. Propionate was first metabolized to acetate, carbon dioxide, and hydrogen by nonmethanogenic organisms. Formate was not excreted. The carbon dioxide originated exclusively from the carboxyl group of propionate, whereas both [2-¹⁴C]- and [3-¹⁴C]propionate lead to the production of radioactive acetate. The methyl and carboxyl groups of the acetate produced were equally labeled, regardless of whether [2-¹⁴C]- or [3-¹⁴C]propionate was used. These observations suggest that in the culture, propionate was degraded through a randomizing pathway.     

Dissimilatory amino Acid metabolism in human colonic bacteria

The abilities of slurries of human faecal bacteria to ferment 20 different amino acids were investigated in batch culture incubations. Ammonia, short chain fatty acids, and in some cases, amines, were the principal products of dissimilatory metabolism. The types of SCFA produced were dependent on the chemical compositions of the test substrates. Thus, acetate and butyrate were formed from the acidic amino acid glutamate, while acetate and propionate predominated in aspartate fermentations. Breakdown of the basic amino acids lysine and arginine was rapid, and yielded butyrate and acetate, and ornithine and citrulline, respectively. The major products of histidine deamination were also acetate and butyrate. However, fermentation of sulphur-containing amino acids was slow and incomplete. Acetate, propionate and butyrate were formed from cysteine, whereas the main products of methionine metabolism were propionate and butyrate. The simple aliphatic amino acids alanine and glycine were fermented to acetate, propionate and butyrate, and acetate and methylamine, respectively. Branched-chain amino acids were slowly fermented by colonic bacteria, with the main acidic products being branched-chain fatty acids one carbon atom shorter than the parent amino acid. Low concentrations of amines were also detected in these fermentations. Aliphatic-hydroxy amino acids were rapidly deaminated by large intestinal microorganisms. Serine was primarily fermented to acetate and butyrate, while threonine was mainly metabolised to propionate. Proline was poorly utilized by intestinal bacteria, but hydroxyproline was efficiently fermented to acetate and propionate. The aromatic amino acids tyrosine, phenylalanine and tryptophan were broken down to a range of phenolic and indolic compounds.     

Genomes of rumen bacteria encode atypical pathways for fermenting hexoses to short‐chain fatty acids

Bacteria have been thought to follow only a few well‐recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here, we show that well‐recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% of bacteria had an atypical pathway for propionate or succinate formation; 6% of bacteria had an atypical pathway for butyrate formation and 33% of bacteria had an atypical or incomplete Embden–Meyerhof–Parnas pathway. This study shows that reconstruction of metabolic pathways – a common goal of omics studies – could be incorrect if well‐recognized pathways are used for reference. Furthermore, it calls for renewed efforts to delineate fermentation pathways biochemically.     

Anaerobic fermentation: Microbes from ruminants

Fed-batch fermentation of biomass could provide a route for direct conversion of renewable resources to commercially significant chemicals. The ecosystem in the forestomach (rumen) of ruminants provides a highly reduced environment (oxidation-reduction potential of ?250 to ?450 mV) in which anaerobic bacteria directly utilize cellulose, hemicellulose, and other fermentable biomass constituents to produce acetate, butyrate, propionate, methane and carbon dioxide at pH 5.7 to 7.3. The cellulose fermentation in the rumen is impacted by the physically and chemically heterogeneous character of the insoluble substrate, as well as the properties of the mixed culture responsible for fibre hydrolysis and carbohydrate utilization. The rumen system provides an interesting case study in the context of possible process concepts for direct fermentation of biomass to commercially important chemicals such as acetate, propionate, succinate, lactate and ethanol. The role of the chemical and physical characteristics of the substrate, the microbes in the rumen system and the metabolic pathways of soluble carbohydrates are discussed in the context of cellulose and hemicellulose fermentation.     

Changes of fermentation pathways of fecal microbial communities associated with a drug treatment that increases dietary starch in the human colon.

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-13C]glucose and 12CO2 or with unlabeled glucose and 13CO2, the distribution of 13C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO2. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO2 reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.     

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70°C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65°C site, with naturally high hydrogen levels, and a 55°C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65°C mat.     

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.     

Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.     

Formation of n-Butanol from d-Glucose by Strains of the "Clostridium tetanomorphum" Group

A clostridial strain has been isolated that produced n-butanol, ethanol, butyrate, and acetate as major fermentation products from glucose but no acetone. At a pH of 6.6, n-butanol was formed by this microorganism only during growth. On the basis of its physiological characteristics and DNA-DNA homology data, the strain was assigned to the "Clostridium tetanomorphum" group (S. Nakamura, I. Okado, T. Abe, and S. Nishida, J. Gen. Microbiol. 113:29-35, 1979). All members of this group were shown to produce n-butanol from glucose as the major fermentation product, whereas C. cochlearium produced it in only minor amounts.     

Contribution of 13C-NMR spectroscopy to the elucidation of pathways of propionate formation and degradation in methanogenic environments

Propionate is an important intermediate in the anaerobic degradation of complex organic matter to methane and carbon dioxide. The metabolism of propionate-forming and propionate-degrading bacteria is reviewed here. Propionate is formed during fermentation of polysaccharides, proteins and fats. The study of the fate of 13C-labelled compounds by nuclear magnetic resonance (NMR) spectroscopy has contributed together with other techniques to the present knowledge of the metabolic routes which lead to propionate formation from these substrates. Since propionate oxidation under methanogenic conditions is thermodynamically difficult, propionate often accumulates when the rates of its formation and degradation are unbalanced. Bacteria which are able to degrade propionate to the methanogenic substrates acetate and hydrogen can only perform this reaction when the methanogens consume acetate and hydrogen efficiently. As a consequence, propionate can only be degraded by obligatory syntrophic consortia of microorganisms. NMR techniques were used to study the degradation of propionate by defined and less defined cultures of these syntrophic consortia. Different types of side-reactions were reported, like the reductive carboxylation to butyrate and the reductive acetylation to higher fatty acids.     

Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates

Abstract From chemostat enrichments conducted at dilution rates of 0.025, 0.12 and 0.25 h⁻¹ glutamate- and aspartate-fermenting bacteria were isolated. The dominant aspartate-fermenting strains in all these enrichments belonged to the genus Campylobacter , whereas 3 dissimilar types of glutamate-fermenting bacteria predominated at the different dilution rates. One of these strains was identified as Clostridium cochlearium . The remaining two were designated as strain DKglu16 (glutamate → acetate + propionate + ammonium + carbon dioxide) and DKglu21 (glutamate → acetate + formate + ammonium + carbon dioxide). Grown in continuous culture under glutamate limitation, strain DKglu16 (μ_max= 0.13 h⁻¹; K _s= 1.9 μM) outcompeted C. cochlearium (μ_max= 0.36 h⁻¹; K _s= 7 μM) at low dilution rates, but was outgrown at higher rates of dilution (0.044 h⁻¹). In glutamate-limited continuous culture the competitiveness of strain DKglu16 increased considerably when lactate was added to the feed in addition to glutamate.     

Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is an anaerobic, rod-shaped, gram-positive bacterium that produces butyrate, acetate, hydrogen, and carbon dioxide from various saccharides, including glucose and xylose. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. In this work, effects of ptb knockout by homologous recombination on metabolic flux and product distribution were investigated. When compared with the wild type, the activities of PTB and butyrate kinase in ptb knockout mutant decreased 76 and 42%, respectively; meanwhile, phosphotransacetylase and acetate kinase increased 7 and 29%, respectively. However, ptb knockout did not significantly reduce butyric acid production from glucose or xylose in batch fermentations. Instead, it increased acetic acid and hydrogen production 33.3-53.8% and ≈ 11%, respectively. Thus, the ptb knockout did increase the carbon flux toward acetate synthesis, resulting in a significant decrease (28-35% reduction) in the butyrate/acetate ratio in ptb mutant fermentations. In addition, the mutant displayed a higher specific growth rate (0.20 h(-1) vs. 0.15 h(-1) on glucose and 0.14 h(-1) vs. 0.10 h(-1) on xylose) and tolerance to butyric acid. Consequently, batch fermentation with the mutant gave higher fermentation rate and productivities (26-48% increase for butyrate, 81-100% increase for acetate, and 38-46% increase for hydrogen). This mutant thus can be used more efficiently than the parental strain in fermentations to produce butyrate, acetate, and hydrogen from glucose and xylose.     

Changes of Fermentation Pathways of Fecal Microbial Communities Associated with a Drug Treatment That Increases Dietary Starch in the Human Colon

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-¹³C]glucose and ¹²CO₂ or with unlabeled glucose and ¹³CO₂, the distribution of ¹³C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO₂. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO₂ reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.     

Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor

A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities. Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C. The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors. The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique. In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide. Cell numbers of glutamate-degrading organisms increased 400-fold over the first year. In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation. After 2 years of reactor operation the predominant organisms were isolated and characterized. Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively. The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively. The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii. It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide. Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.