In vitro study of the effect of different ionophore antibiotics and of certain derivatives on rumen fermentation and on protein nitrogen degradation

An in vitro study was carried out to evaluate the effect of different ionophore antibiotics and some of their derivatives on rumen fermentation and on the degradation of peanut meal nitrogen. The increase in the production of propionic acid at the expense of acetic acid, observed with lonomycin, nigericin, cationomycin and lysocellin, was identical to that noted with monensin. The decrease in methanogenesis observed in the presence of monensin was also found with cationomycin and lysocellin. With the exception of lysocellin, which greatly reduced protein degradation of peanut meal, and of nigericin, which had no effect on this parameter, the 2 other molecules presented the same action as monensin. The negative effect of monensin on microbial ammonia uptake was demonstrated with the same intensity in the presence of cationomycin; it was slightly higher with nigericin and particularly accentuated with lonomycin and lysocellin. Three ester derivatives of monensin (monensin acetate, monensin propionate and monensin butyrate) had a similar action to that of monensin on the orientation of rumen fermentations. The monensin isobutyrate derivative appeared to be more active than monensin and only weakly altered microbial ammonia uptake. The oxolonomycin and hydroxolonomycin derivatives behaved identically to lonomycin with respect to microbial metabolism and protein nitrogen degradation. Unlike the molecules from which they derive, the deacylated cationomycin and nigericic acid had no effect on the orientation of rumen fermentations. Of the compounds tested and presenting a potential 'growth-promoting action' at least comparable to that of monensin, and which demonstrated lower toxicity on mice, three molecules (oxolonomycin, lysocellin and cationomycin) appeared to present a zootechnical interest as feed additives for growing cattle.     

Monensin does not increase yields of propionic acid fermentations

Summary Monensin, a polyether antibiotic, inhibited growth and acid production by five species ofPropionibacterium. The ratio of propionate to acetate produced was not affected. Neither subinhibitory levels of monensin nor adaptation of two propionibacteria to growth in the presence of monensin resulted in increased yields of propionic acid. Monensin cannot be used to increase yields in propionic acid fermentations.     

Phenylacetic acid in an anaerobic swine manure digester

Summary The presence of phenylacetic acid (PAA) in an anaerobic swine manure digester was determined by gas chromatography of the butyl ester and confirmed by mass spectroscopy. PAA concentration increased during start-up of a digester and with low carbon, high nitrogen loading. Unlike acetate, propionate and butyrate, the concentration of PAA varied little through the day in a stable digester loaded once per day. The laboratory scale digester was loaded at 4 g of swine manure solids/liter digester volume per day. The retention time and temperature were 15 days and 37°C. PAA is a microbial intermediate which is produced by one group of anaerobic bacteria and converted to methane by other members of the bacterial community in the digester. As such, it may be a useful indicator of the relative metabolic activity of the bacterial groups and thus of the overall stability of the anaerobic process.     

Effect of precursors on biosynthesis of monensins A and B

Precursors of monensins (acetate, propionate, butyrate, isobutyrate) affect the total production and the relative proportion of monensins A and B. Addition of propionate into the fermentation medium causes a prevalence of monensin B whereas butyrate and isobutyrate stimulate the production of monensin A and suppress the production of monensin B.     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

Performance of anaerobic granules for degradation of pentachlorophenol.

Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28 degrees C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99% was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14C]PCP to 14CH4 and 14CO2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP.     

Methanogenic Decomposition of Ferulic Acid, a Model Lignin Derivative

Ferulic acid, a model lignin derivative, was observed to be biodegradable to methane and carbon dioxide under strict anaerobic conditions. This conversion appears to be carried out by a consortium of bacteria similar to that previously described for the methanogenic degradation of benzoic acid. A temporary buildup of acetate in these cultures indicates that it is a likely intermediate and precursor for methane formation. An analog of coenzyme M, 2-bromoethanesulfonic acid (BESA), inhibited gas production and enhanced the buildup of propionate, butyrate, isobutyrate, and isovalerate. Phenylacetate, cinnamate, 3-phenylpropionate, benzoate, cyclohexane carboxylate, adipate, and pimelate were also detected in BESA-inhibited cultures. A pathway is proposed which includes these various acids as possible intermediates in the methanogenic degradation of ferulic acid. This model overlaps previously described benzoic acid degradation pathways, suggesting that this type of anaerobic degradation may be common for aromatic compounds.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures.

The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied. The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days. Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor. The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection. Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium. Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent. However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture. In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM. However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated. All experiments were conducted at pH 7.0 to 7.7. The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature. Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of acrylic acid formation in Megasphaera elsdenii in the presence of 3-butynoic acid

Summary The formation of acrylic acid from lactic acid in the anaerobic rumen bacterium Megasphaera elsdenii was detected in the presence of 3-butynoic acid. While the major end products of lactic acid fermentation in the absence of the inhibitor were propionate, acetate, valerate, and butyrate, the presence of 3-butynoic acid led to the production of propionate, acetate, acrylate, and butyrate. An improvement in the chemical synthesis and purification of 3-butynoic acid was developed.     

Methanogenic bacterial consortia degrading 2-methylbutyric acid

Two co-cultures, each of a coccobacillus plus a Gram-negative rod, were isolated from a cattle waste digester. The cultures were distinguished by production of methane from acetate only, or from either acetate or butyrate. 2-Methylbutyrate, added initially as a growth factor, was degraded to propionate but only during growth on acetate or butyrate. Valerate and 2- and 3-methyl valerate were not degraded.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Effect of Dietary Monensin or Chlortetracycline on Methane Production from Cattle Waste

Wastes from feedlot cattle fed finishing diets containing either monensin, chlortetracycline, or no antibiotic were investigated as substrates for methane production. We used continuously mixed anaerobic fermentors with 3-liter working volumes at 35 and 55°C; these fermentors were fed once per day. Within a few days after waste from animals fed monensin was added, the volume of methane produced began to decrease in the 55°C fermentors. After 9 days of daily feeding, methane production was severely inhibited, the pH dropped from 7.6 to 5.9, and the concentration of volatile acids increased from 543 to 6,300 mg/liter (as acetate). Although additions of waste from cattle fed monensin were discontinued after 9 days, the fermentors did not resume gas production within 8 weeks. The addition of waste from cattle which had been fed chlortetracycline reduced the methane production rate approximately 20%; however, pH and volatile acid values were comparable to control fermentor values after 40 days. Similar effects were observed with the 35°C fermentors. In a batch fermentation experiment in which 50-g portions of volatile solids from waste of animals fed monensin, chlortetracycline, or no antibiotics were added to fermentors, monensin delayed the onset of methane production for about 40 days, but then these fermentors began to produce methane at a rate comparable to the control rate. The ultimate methane yields from the three types of waste after 180 days were not significantly different. These studies indicate that monensin has a detrimental effect on the conversion of feedlot wastes to methane, unless microorganisms can be adapted to the levels that are present in these wastes.     

Effects of 2-Bromoethanesulfonic Acid and 2- Chloroethanesulfonic Acid on Acetate Utilization in a Continuous-Flow Methanogenic Fixed-Film Column

2-Bromoethanesulfonic acid (BESA) and 2-chloroethanesulfonic acid (CESA) have been reported to be potent inhibitors of methane formation during methanogenic decomposition in batch cultures. However, in a laboratory-scale continuous-flow methanogenic fixed-film column containing a predominance of acetate-decarboxylating methanogens, BESA at 6 × 10⁻⁴ M produced only a 41% inhibition of acetate utilization, and CESA at 5.4 × 10⁻⁴ M produced a 37% inhibition of acetate utilization. BESA and CESA concentrations were not monitored in the effluent, so their fate is unknown. The organisms in the column were capable of degrading trace halogenated aliphatic compounds (~30 μg/liter) with acetate (100 mg/liter) as the primary substrate. Previous exposure of the cells to halogenated organic compounds may have conferred resistance to BESA and CESA. Degradation of the inhibitor compounds is another possible explanation for the observed effects.     

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.     

Effects of Organic Acid Anions on the Growth and Metabolism of Syntrophomonas wolfei in Pure Culture and in Defined Consortia

The effects of organic acid anions on the growth of Syntrophomonas wolfei was determined by varying the initial concentration of the acid anion in the medium. The addition of 15 mM acetate decreased the growth rate of a butyrate-catabolizing coculture containing Methanospirillum hungatei from 0.0085 to 0.0029 per hour. Higher initial acetate concentrations decreased the butyrate degradation rate and the yield of cells of S. wolfei per butyrate degraded. Inhibition was not due to the counter ion or the effect of acetate on the methanogen. Initial acetate concentrations above 25 mM inhibited crotonate-using pure cultures and cocultures of S. wolfei. Benzoate and lactate inhibited the growth of S. wolfei on crotonate in pure culture and coculture. Lactate was an effective inhibitor of S. wolfei cultures at concentrations greater than 10 mM. High concentrations of acetate and lactate altered the electron flow in crotonate-catabolizing cocultures, resulting in the formation of less methane and more butyrate and caproate. The inclusion of the acetate-using methanogen, Methanosarcina barkeri, in a methanogenic butyrate-catabolizing coculture increased both the yield of S. wolfei cells per butyrate degraded and the efficacy of butyrate degradation. Butyrate degradation by acetate-inhibited cocultures occurred only after the addition of Methanosarcina barkeri. These results showed that the metabolism of S. wolfei was inhibited by high levels of organic acid anions. The activity of acetate-using methanogens is important for the syntrophic degradation of fatty acids when high levels of acetate are present.     

Effect of Monensin and Lasalocid-Sodium on the Growth of Methanogenic and Rumen Saccharolytic Bacteria

It is thought that monensin increases the efficiency of feed utilization by cattle by altering the rumen fermentation. We studied the effect of monensin and the related ionophore antibiotic lasalocid-sodium (Hoffman-LaRoche) on the growth of methanogenic and rumen saccharolytic bacteria in a complex medium containing rumen fluid. Ruminococcus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens were inhibited by 2.5 μg of monensin or lasalocid per ml. Growth of Bacteroides succinogenes and Bacteroides ruminicola was delayed by 2.5 μg of monensin or lasalocid per ml. Populations of B. succinogenes and B. ruminicola that were resistant to 20 μg of either drug per ml were rapidly selected by growth in the presence of each drug at 5.0 μg/ml. Selenomonas ruminantium was insensitive to 40 μg of monensin or lasalocid per ml. Either antibiotic (10 μg/ml) inhibited Methanobacterium MOH, Methanobacterium formicicum, and Methanosarcina barkeri MS. Methanobacterium ruminantium PS was insensitive to 40 μg of monensin or 20 μg of lasalocid per ml. The methanogenic strain 442 was insensitive to 40 μg of monensin but sensitive to 10 μg of lasalocid per ml. The results suggest that monensin or lasalocid acts in the rumen by selecting for succinate-forming Bacteroides and for S. ruminantium, a propionate producer that decarboxylates succinate to propionate. The selection could lead to an increase in rumen propionate formation. Selection against H₂ and formate producers, e.g. R. albus, R. flavefaciens, and B. fibrisolvens, could lead to a depression of methane production in the rumen.     

Kinetic modelling of isomerization and anaerobic degradation of n- and i-butyrate

Very clear experimental evidence of isomerization between n- and i-butyrate during their anaerobic degradation was presented. A first experiment in the presence of bromoethane sulfonic acid (BESA), an inhibitor of methanogenesis, allowed the equilibrium distribution of n-, i-butyrate and acetate to be determined. To elucidate the mechanism of the isomerization process, a kinetic analysis was employed. The results suggested that i-butyrate was isomerized into n-butyrate prior to being oxidized to acetate. A mechanism for butyrate degradation, based on the values of the kinetic parameters obtained, was proposed.     

Inhibition of propionate degradation by acetate in methanogenic fluidized bed reactors

Summary The degradation of acetate, propionate and butyrate was monitored during start-up of five lab-scale methanogenic fluidized bed reactors on an artificially prepared waste water. The acetate concentration in the reactor content was found to influence the degradation of propionate but not of butyrate. In general, at acetate levels over 200 mg/l the degradation of propionate was below 60%, whereas the degradation was complete at acetate levels under 100 mg/l. The rationale of the inhibition of propionate degradation by acetate is discussed.