Formation of bacterial aggregates in a continuous-flow reactor: Influence of carrier availability

Summary A dilution-rate shift-up was employed to induce bacterial hold-up in a continuous-flow gas-lift reactor. A minimum carrier concentration (sand, 2–5 g/l) was found a prerequisite for formation of bacterial aggregates, which fermented glucose either to propionate/acetate or to butyrate/acetate. Higher levels of sand did not affect the onset of propionate/acetate-forming aggregates, but decreased the rate at which they subsequently grew. Reversely, butyrate/acetate-producing aggregates grew at a constant rate but the onset of their formation was progressively retarded by increasing sand concentrations. In both cases, completion of start-up was most rapid at low sand concentrations.     

Production of flavour esters by Rhizopus oryzae

Microbial production of different alipathic esters with flavour characteristic has been studied. Lyophilized whole cells of Rhizopus oryzae CBS 112-07 were found to be particularly suitable to catalyse the synthesis of different flavour esters (hexyl acetate, propionate, butyrate, caprylate; geranyl acetate, propionate, butyrate and 2- and 3-methylbutyl acetate, butyrate) in n-heptane. This strain was therefore utilized for the semipreparative production of geranyl butyrate by semicontinous and continous addition of the substrates with satisfactory yields (144 g l^–1 in 264 h and 142 g l^–1 in 48 h respectively).     

DNA Microarray Analyses of the Long-Term Adaptive Response of Escherichia coli to Acetate and Propionate

In its natural environment, Escherichia coli is exposed to short-chain fatty acids, such as acetic acid or propionic acid, which can be utilized as carbon sources but which inhibit growth at higher concentrations. DNA microarray experiments revealed expression changes during exponential growth on complex medium due to the presence of sodium acetate or sodium propionate at a neutral external pH. The adaptive responses to acetate and propionate were similar and involved genes in three categories. First, the RNA levels for chemotaxis and flagellum genes increased. Accordingly, the expression of chromosomal fliC′-′lacZ and flhDC′-′lacZ fusions and swimming motility increased after adaptation to acetate or propionate. Second, the expression of many genes that are involved in the uptake and utilization of carbon sources decreased, indicating some kind of catabolite repression by acetate and propionate. Third, the expression of some genes of the general stress response increased, but the increases were more pronounced after short-term exposure for this response than for the adaptive response. Adaptation to propionate but not to acetate involved increased expression of threonine and isoleucine biosynthetic genes. The gene expression changes after adaptation to acetate or propionate were not caused solely by uncoupling or osmotic effects but represented specific characteristics of the long-term response of E. coli to either compound.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

A method for the analysis of acetate turnover in a coastal marine sediment

The concentrations of volatile fatty acids were measured in the pore water of sediment from the Limfjorden, Denmark. The pore water was freeze-dried and the acids, which were redissolved in formic acid, were analyzed by gas chromatography on a Carbopack column. The limit of detection was 0.1 mol l^–1 pore water. The concentration ranges (mol l^–1 pore water) were as follows: 0.1 to 6.0 for acetate; <0.1 to 0.6 for propionate, and <0.1 to 0.5 for butyrate. The rate constants for the disappearance of injected tracer concentrations of U-¹⁴C-acetate were measured at 2 cm depth intervals in sediment strata (0 to 10 cm). The rate constant for acetate turnover at 4 to 6 cm depth did not vary greatly with season, 2.1 h^–1, SD 0.6 for 7 values. In spring, the rate constants were highest in the 0 to 2 cm stratum and decreased with sediment depth. The calculated rates for acetate turnover of 7.2 mmol m^–2 day^–1 for early spring (2°C) and of 19.6 mmol m^–2 day^–1 for late autumn (7°C) were higher than would be expected from published values for carbon oxidation by sulfate in these sediments.     

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 ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]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. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C 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.     

New motile anaerobic bacteria growing by succinate decarboxylation to propionate

Three strains of new anaerobic, gram-negative bacteria which grew with succinate as sole source of carbon and energy were isolated from anoxic marine and freshwater mud samples. Cells of the three strains were small, non-spore-forming, motile rods or spirilla. The guanine-plus-cytosine content of the DNA of strain US2 was 52.6±1.0 mol%, of strain Ft2 63.5±1.4 mol%, and of strain Ft1 62.6±1.0 mol%. Succinate was fermented stoichiometrically to propionate and carbon dioxide. The growth yields were 1.2–2.6 g dry cell mass per mol succinate degraded. Strains US2 and Ft2 required 0.05% w/v yeast extract in addition to succinate for reproducible growth. Optimal growth occurred at 30°–37°C and pH 6.8–8.0. Addition of acetate as cosubstrate did not stimulate growth with any strain. Strain Ft2 grew only under strictly anaerobic conditions, whereas strains US2 and Ft1 tolerated oxygen up to 20% in the headspace. Strains US2 and Ft2 grew only with succinate. Strain Ft1 also converted fumarate, aspartate, and sugars to propionate and acetate. This strain also oxidized propionate with nitrate to acetate. Very low amounts of a c-type cytochrome were detected in propionate plus nitrate- or glucose-grown cells of this strain (0.4 g x g protein^-1). Moderate activities of avidin-sensitive methylmalonyl-CoA decarboxylase were found in cell-free extracts of all strains.     

In vitro gas production and ruminal fermentation of glycerol, propylene glycol and molasses

Ruminal fermentation pattern and in vitro gas production was determined for three energy sources for ruminants, glycerol, propylene glycol and molasses with ruminal fluid from sheep. Substrates incubated were alfalfa, corn silage, glycerol (320 and 640 μl), propylene glycol (320 and 640 μl) and molasses (320 μl). The greater volume of gas produced was observed at the highest dose of glycerol which also showed the slowest rate of gas production and the longest lag time (P<0.05). Propylene glycol presented the minor volume of gas and was rapidly metabolized with short lag time. Molasses presented typical characteristics of a rapidly available substrate, with the fastest rate of gas production (P<0.05). Glycerol fermentation resulted in a reduction of acetate, a slight increase in propionate and an increment in percentage of butyrate. Incubations with propylene glycol also reduced acetate and butyrate, but increased propionate (P<0.05). Molasses fermentation reduced acetate and increased propionate and butyrate. Increasing dose of energy sources resulting in a greater volume of gas produced. In conclusion, glycerol fermentation reduced acetate and increased the molar proportion of butyrate and propionate was the main product of fermentation of propylene glycol.     

Oxidation of glycerol,lactate, and propionate by Propionibacterium freudenreichii in a poised-potential amperometric culture system

Growth of Propionibacterium freudenreichii was studied with glycerol, lactate, and propionate as energy sources and a three-electrode poised-potential amperometric electrode system with hexacyanoferrate (III) as mediator. In batch culture experiments with glycerol and lactate as substrates, hexacyanoferrate (III) was completely reduced. Growth yields increased and the fermentation patterns were shifted towards higher acetate formation with increasing hexacyanoferrate (III) concentrations (0.25–8.0 mM). In experiments with regulated electrodes, glycerol, lactate, and propionate were oxidized to acetate and CO₂, and the electrons were quantitatively transferred to the working electrode. Growth yields of 29.0, 13.4 and 14.2 g cell material per mol were calculated, respectively. The high cell yield obtained during propionate oxidation cannot be explained solely by substrate level phosphorylation indicating that additional energy was conserved via electron transport phosphorylation. Furthermore, this result indicated complete reversibility of the methyl-malonyl-CoA pathway in propionic acid bacteria.     

Production of volatile compounds by the edible fungus Rhizopus oryzae during solid state cultivation on tropical agro-industrial substrates

When Rhizopus oryzae was grown on medium containing cassava bagasse plus soybean meal (5:5 w/w), CO2 production was at its highest (200 ml.l–1) while highest volatile metabolite production was with amaranth grain as substrate (282.8 ml.l–1). In the headspace, ethanol was the most abundant compound (more than 80%). Acetaldehyde, 1-propanol, ethyl acetate, ethyl propionate and 3-methyl butanol were also present. CO2 and volatile metabolite productions reached their maxima around 20 h and 36 h, respectively. © Rapid Science Ltd. 1998     

End product inhibition in methane fermentations: Effects of carbon dioxide and methane on methanogenic bacteria utilizing acetate

Summary The effects of pCO₂ and pCH₄ in the interval 0–1 bar on rates of acetate degradation and methane formation by methanogens as well as methane yields were studied in enrichment cultures in batch and continuous fermentations.In batch fermentations the rate of acetate utilization by methanogens was 1,000–1,500 mg/l · d at low levels of pCO₂. CO₂ was inhibitory and degradation rates were around 350 mg/l · d in 1 bar CO₂. The degradation of acetate was almost linear. In continuous culture maximum rates of acetate utilization around 2,500 mg/l · d were obtained and the acetate concentration in the substrate was reduced by 98–99%.The yields of methane on acetate substrates were close to the theoretical value (1 mole CH₄ per mole HAc) in the interval pCO₂-0–0.5 bar. In 1 bar CO₂ yields decreased by 20–30%.CH₄ was found to be only slightly inhibitory; the inhibiting effects of 1 bar CH₄ on acetate degradation rates were comparable to the effects of 0.3 bar CO₂. Also gas sparging and rapid mixing had small effects compared with a non-sparged, slowly mixed culture.The redox potential was usually around –200 mV during fermentations and no connection was found between acetate degradation rate, E_h and pCO₂.Acetate and propionate degradation were the reactions most sensitive to pCO₂ and to obtain maximum rates as well as maximum methane yields pCO₂-levels around 0.2 bar were found to be optimal.     

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.     

Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min⁻¹ mg of protein⁻¹) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO₂, with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.     

Uptake of Acetate and Propionate by Isolated Nerve Endings from the Electric Organ of Torpedo marmorata and Their Incorporation into Choline Esters

Abstract: The uptake and incorporation into choline esters of acetate and propionate by electric organ synaptosomes were compared, with the aim of better understanding the basis for the selectivity of choline ester synthesis shown by this tissue for acetate. It was found that propionate uptake, like acetate uptake, was a temperature-dependent, saturable process. Both uptake mechanisms had similar affinities for their substrates, but the maximal velocity of propionate uptake was considerably lower than that of acetate uptake; and less of the accumulated propionate was used for choline ester synthesis than of the accumulated acetate. While acetate was a good inhibitor of propionate uptake, propionate was a very poor inhibitor of acetate uptake. This finding, in addition to the observation that the two uptakes were not affected in the same way by changes in pH, led to the suggestion that acetate uptake and propionate uptake reflect different processes. In both cases, however, the pH dependence of uptake indicated that these substrates cross the membrane as the charged species. Acetate uptake and acetylcholine synthesis remained closely associated under various experimental conditions, while propionate uptake could be dissociated from the synthesis of propionylcholine. Hence, it appears that acetate is taken up by a specific, high-velocity mechanism linked to acetylcholine synthesis, whereas propionate uptake may represent a less specific mechanism.     

Influence of exogenous enzymes on nutrient digestibility, extent of ruminal fermentation as well as milk production and composition in dairy cows

This experiment studied effects of a mixture of exogenous enzymes (ZADO^®) from anaerobic bacteria on ruminal fermentation, feed intake, digestibility, as well as milk production and composition in cows fed total mixed rations (TMRs; 0.7 corn silage and 0.3 of a concentrate mixture). Twenty lactating multiparous Brown Swiss cows (500 ± 12.4 kg live weight) were randomly assigned into two experimental groups of 10 immediately after calving and fed a TMR with or without (CTRL) addition of 40 g/cow/d of enzymes for 12 weeks. Addition of enzymes increased (P<0.05) rumen microbial N synthesis. Intake of dry matter (DM) and organic matter (OM) was positively influenced (P<0.05) by supplementation, and digestibility of all nutrients was higher (P<0.05) in the total tract of supplemented cows, although the magnitude of the improvement varied among nutrients, with the highest improvement in aNDFom and ADFom (418–584 and 401–532 g/kg respectively; P<0.05) than the other nutrients. Supplementation of enzymes also increased (P<0.05) rumen ammonia N and total short chain fatty acid (SCFA) concentrations, and individual SCFA proportions were also altered with an increase in acetate (61.0–64.8 mol/100 mol; P=0.05) before feeding, and acetate and propionate increased 3 h post-feeding (60.0–64.0 and 18.3–20.8 mol/100 mol respectively; P<0.05). Milk and milk protein production was higher (12.8–15.7 and 0.45–0.57 kg/d respectively; P<0.05) for cows fed the ZADO^® supplemented diet. This exogenous enzyme product, supplemented daily to the TMR of cows in early lactation, increased milk production due to positive effects on nutrient intake and digestibility, extent of ruminal fermentation and microbial protein synthesis.     

Digesta passage and functional anatomy of the digestive tract in the desert tortoise (Xerobates agassizii)

The herbivorous tortoise Xerobates agassizii contents with large fluctuations in the quality and abundance of desert pastures. Responses to grass (Schismus barbatus), herbage (Sphaeralcea ambigua) and pelleted diets were studied in captive animals. Digestive anatomy was investigated in wild tortoises. Cornified esophageal epithelia and numerous mucus glands along the digestive tract indicated a resistance to abrasive diets. Gastric contents were acidic whereas hindgut digesta were near neutral pH. The colon was the primary site of fermentation with short-chain fatty acids mainly comprised of acetate (69–84%), propionate (10–15%) and n-butyrate (1–12%). Fibre digestion was extensive and equivalent to 22–64% of digestible energy intakes. Large particles of grass (25 mm Crmordants) were excreted as a pulse but retained longer than either fluids (Co-EDTA) or fine particles (2 mm; Yb). Patterns of marker excretion suggested irregular mixing of only the fluid and fine particulate digesta in the stomach and the colon. Mean retention times of Crmordants were 14.2–14.8 days on the grass and highfibre pellets. Intakes of grass were low and accompanied by smaller estimates of digesta fill than for the high-fibre pellets. Digestive capacity was large and estimated at 11–21% of body mass on these diets. The capacious but simple digestive anatomy of the tortoise may provide the greatest flexibility in utilizing a variety of forages in its unreliable habitat.Abbreviations bm body mass - DM drymatter - EDTA ethylene-diamine tetra-acetic acid - MRT mean retention time - NDF neutral detergent fibre - SCFA short-chain fatty acid(s) - T _max time to maximum marker concentration     

The kinetics of glucose fermentation bySelenomonas ruminantium HD4 grown in continuous culture

Summary The production of organic acids (acetate, lactate, and propionate) by the anaerobic, ruminal bacteriumSelenomonas ruminantium HD4 was investigated in both glucose-limited and glucose-sufficient (phosphate-limited) continuous cultures. The fermentation pattern of products exhibited a shift upon release of glucose limitation from acetate and propionate to lactate at a dilution rate of 0.2 h^–1. Glucose sufficiency brought about two-to fourfold increase in specific glucose utilization rate, lactate productivity, and lactate yield relative to glucose-limited growth conditions. The increased lactate production under glucose-sufficient growth conditions was attributed to the overutilization of excess glucose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Mixed-Culture Fermentor for Simulating Methanogenic Digestors

Propionate degradation in an anaerobic digestor degrading animal waste (10-day retention time, 5.75 g liter⁻¹ day⁻¹ volatile solids loading rate, 40°C) was 0.304 mM h⁻¹, measured with [2-¹⁴C]propionate; this value indicated that CH₄ produced from propionate accounted for 14.8% of the CH₄ produced in the digestor (34.5%, including acetate produced from propionate). The mean propionate concentration was 0.67 mM, giving a propionate turnover rate of 0.46 h⁻¹. A continuous-, mixed-culture fermentor was developed to mimic the digestor. When degradation rates of methanogenic precursors (H₂, CO₂, and acetate) equalled those measured in the digestor, propionate degradation was inhibited. When the H₂ turnover rate was lowered by decreasing addition of H₂-generating substrates or by allowing a portion of the H₂ degradation to occur in an isolated compartment, propionate degradation in the fermentor resumed. The possibility is discussed that in digestors, much of the H₂ is produced and degraded within microenvironments associated with particles. Thus, the gross turnover rate of H₂ measured in digestors is an average, and specific microenvironments within the digestor may have different rates of turnover.     

Fermentation of cellulose and fatty acids with enrichments from sewage sludge

Summary A mixed culture enriched from sewage sludge and anaerobic digestor effluent was able to degrade cellulose and acetate rapidly and quantitatively to methane and carbon dioxide. The maximum specific rate of gas production was 87 ml/gm cell-h, corresponding to a rate of cellulose utilization of 0.1 g/g cells-h. Acetate, an intermediate in cellulose degradation, was fermented much more rapidly than butyrate or propionate; its maximum utilization rate was first order with a rate constant of 0.34 h^–1. Addition of 2-¹⁴C-acetate to a digestor fed cellulose showed tht 2% of the methyl groups were oxidized to carbon dioxide. When 1-¹⁴C-acetate was added to a similar digestor, 51% of the carboxyl groups were reduced to methane, suggesting that not all the carbon dioxide during simultaneous cellulose and acetate utilization is treated equally. The pulse addition of large amounts of acetate, propionate and butyrate to a cellulose fed digestor was also examined.