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.     

Interconversions and production of volatile fatty acids in the sheep rumen

1. Sheep fed at a constant rate were infused intraruminally with [1-(14)C]-acetate, -propionate or -butyrate during 5hr. periods. 2. Volatile fatty acids were estimated in the rumen contents and steady-state conditions were obtained. 3. Of the butyric acid carbon 60% was in equilibrium with 20% of the acetic acid carbon, and 2-3g.atoms of carbon were interconverted/day. 4. Little interconversion took place between propionic acid, acetic acid or butyric acid. 5. The net production rates for acetic acid, propionic acid and butyric acid were 3.7, 1.0 and 0.7moles/day respectively. 6. The production of volatile fatty acids accounted for 80% of the animal's energy expenditure.     

Maintenance of the Rumen Microbial Population in Continuous Culture

The design and use of an apparatus for continuous in vitro cultivation of the mixed bovine rumen microbial population are described. Data relative to the concentrations and proportions of volatile fatty acids produced, methanogenesis, carbon dioxide production, and survival of bacteria and protozoa indicated that an essentially normal in vivo fermentation pattern was maintained in vitro for experimental periods of from 3 to 10 days. The continuous cultures were responsive to major changes in type of feed intake. A change from grain to hay resulted in increased acetate and decreased propionate production, whereas decreased acetate and increased propionate resulted when feed intake was changed from hay to grain. Methanogenesis, volatile fatty acid production, and the numbers of microorganisms in the cultures were proportional to the amount of feed materials added up to levels calculated to approximate the in vivo maintenance requirement of the adult bovine.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

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.     

THE PRODUCTION OF VOLATILE FATTY ACIDS BY BACTERIA OF THE DYSENTERY GROUP

These studies show: 1. A close agreement exists among all the organisms studied in the total quantity of volatile fatty acids produced and in the ratio of formic to acetic, under aerobic conditions, and in the presence of 1 per cent of glucose. 2. When grown upon peptone alone, with free access of air to the cultures, volatile fatty acids are produced in appreciable quantities, although the reaction of the solution has gone more alkaline as shown by colorimetric pH tests. Formic acid is not found, but in its place we obtain propionic acid. 3. Upon exhaustion of air from the non-sugar medium the bacteria again produce formic acid, and in addition some butyric. This is true for both Shiga and non-Shiga cultures. The reaction is distinctly more acid. 4. The presence of glucose in the medium from which the air has been pumped furnishes a condition which provokes about the same type and degree of fermentation that operates in the glucose medium bathed in air at atmospheric pressure. 5. The enormous quantity of formic acid produced by these bacteria may play a significant part in the digestive disturbances and toxic symptoms accompanying their infection of the human intestinal tract.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Combined removal of nitrate and carbon in granular sludge: Substrate competition and activities

Granular sludge from an upflow anaerobic sludge blanket reactor treating synthetic waste water containing a mixture of volatile fatty acids and nitrate showed a removal efficiency of nearly 100% for both nitrogen and carbon. This activity was achieved by a combined process of denitrification and methanogenesis under conditions of surplus carbon. Under batch conditions the two processes proceeded clearly separated in time with first denitrification dominating and excluding methanogenesis. However, as soon as nitrate was depleted, methane production was initiated, showing that the inhibition of methanogenesis by nitrate was reversible. Of the volatile fatty acids supplied to the reactor, i.e. acetate, propionate, and butyrate, the denitrifying population clearly preferred butyrate and propionate even though acetate could also be metabolized. Consequently, growth of syntrophic volatile fatty acid degraders was suppressed by the denitrifiers in cases of low C:N ratios in the medium, leaving acetate as the major substrate for methanogenesis.Abbreviations UASB upflow anaerobic sludge blanket - COD chemical oxygen demand - VFA volatile fatty     

Efect of the volatile fatty acids on phosphate uptake parameters by pure cultures of Acinetobacter

E.RUSTRIAN, J.P. DELGENES AND R. MOLETTA. 1996. Experiments were performed to examine the effect of volatile fatty acids(VFA) as carbon source, on the phoshate uptake parameters in four Acinetobacter strains. Acetic and butyric acids were equally good carbon sources for phosphate removal, while propionic acid was the least efficient substrate. The best ratios of assimilated phosphate vs VFA consumed were 0-178 wit acetic acid by Ac.calcoaceticus NRRL 4270, 0.21 with propionic acid by Ac.calcoaceticus NRRL 4270 AND 0.187 with butyric acid by Acinetobacter sp.SUCT 5.     

Culture and Metabolism of the Rumen Ciliate Epidinium ecaudatum Crawley

The rumen ciliate Epidinium ecaudatum was cultured for 6 months in the presence of two strains of bacteria, starch, alfalfa, linseed oil meal, and buffered saline. The cultures required daily transfer and addition of fresh substrate. The protozoan degraded starch, soybean oil meal, linseed oil meal, and cottonseed oil meal, and the fermentation end products from the breakdown of starch were acetic and butyric acids with traces of formic, propionic, and lactic acids, carbon dioxide, and hydrogen. The relationship of E. ecaudatum to other species of rumen oligotrichs and bacteria is discussed.     

The effect of propionic to acetic acid ratio on anaerobic-aerobic (low dissolved oxygen) biological phosphorus and nitrogen removal

In this paper, three lab-scale sequencing batch reactors (SBR-A, B, and C) operated with anaerobic/aerobic (low dissolved oxygen, 0.15-0.45 mg L(-1)) configuration were long-term cultured, respectively with single acetic acid and propionic/acetic acid of 1/1 and 2/1 (carbon molar ratio), and the comparisons of anaerobic and aerobic transformations of phosphorus and nitrogen among them were made. With the increase of propionic/acetic acid, lower anaerobic phosphorus release and higher phosphorus release to short-chain fatty acids uptake ratio were observed, and less anaerobic and aerobic transformations of glycogen and poly-3-hydroxybutyrate as well as total polyhydroxyalkanoates occurred, but the transformations of poly-3-hydroxyvalerate and poly-3-hydroxy-2-methyvalerate increased. The phosphorus removal efficiency was respectively 81, 94 and 97% in SBR-A, B and C. Almost all ammonium was removed and no significant nitrite was accumulated at different propionic/acetic acid ratios. However, the nitrate accumulation and total nitrogen removal were observed to be affected by propionic/acetic acid ratio. The total nitrogen removal efficiency was 61, 68 and 82%, and the aerobic end nitrate concentration was 8.05, 6.40 and 3.54 mg L(-1) in three SBRs, respectively. All the above studies indicated that the sole acetic acid caused more nitrate accumulation than propionic and acetic acids mixture, and a pertinent increase of wastewater propionic/acetic acid ratio was of benefit to both nitrogen and phosphorus removal in an anaerobic/aerobic (low dissolved oxygen) biological wastewater treatment process.     

Identification of putative pheromones in bovine (Bos taurus) faeces in relation to estrus detection

Ten different volatile compounds were detected in bovine faeces using three chromatograms. The chemical profiles of estrus faeces were distinguished significantly from other phases by the presence of three specific substances, viz. acetic acid and propionic acid and 1-iodo undecane. The estrus specific synthetic compounds were rubbed onto the genital region of nonestrus animals (dummy cows), and the bulls were allowed to sniff the genital region and observed sexual behaviours. The statistical significance was higher (P<0.001) in bulls exhibiting repeated flehmen and mounting behaviours towards the mixture of acetic acid, propionic acid and 1-iodo undecane. The bioassay revealed that the fatty acids viz., acetic acid; propionic acid and 1-iodo undecane produced in faeces during estrus appear to be estrus specific. The results suggest that these compounds may be used as estrus indicator in cow, probably involved in bovine biocommunication.     

Inhibition of Rumen Methanogenesis by Methane Analogues

Extremely low concentrations of chloroform and carbon tetrachloride and somewhat larger concentrations of methylene chloride inhibited the formation of methane by the rumen microbiota in the presence or absence of added substrate. The accumulation of hydrogen at these low concentrations indicates a selective inhibition of methanogenesis. Presumably, these inhibitors affect one or more of the reactions by which methane is formed from hydrogen and carbon dioxide.     

The effect of pectin, corn and wheat starch, inulin and pH on in vitro production of methane, short chain fatty acids and on the microbial community composition in rumen fluid

Methane emission from livestock, ruminants in particular, contributes to the build up of greenhouse gases in the atmosphere. Therefore the focus on methane emission from ruminants has increased. The objective of this study was to investigate mechanisms for methanogenesis in a rumen fluid-based in vitro fermentation system as a consequence of carbohydrate source (pectin, wheat and corn starch and inulin) and pH (ranging from 5.5 to 7.0). Effects were evaluated with respect to methane and short chain fatty acid (SCFA) production, and changes in the microbial community in the ruminal fluid as assessed by terminal-restriction fragment length polymorphism (T-RFLP) analysis. Fermentation of pectin resulted in significantly lower methane production rates during the first 10 h of fermentation compared to the other substrates (P = 0.001), although total methane production was unaffected by carbohydrate source (P = 0.531). Total acetic acid production was highest for pectin and lowest for inulin (P < 0.001) and vice versa for butyric acid production from pectin and inulin (P < 0.001). Total propionic acid production was unaffected by the carbohydrate source (P = 0.791). Methane production rates were significantly lower for fermentations at pH 5.5 and 7.0 (P = 0.005), sustained as a trend after 48 h (P = 0.059), indicating that there was a general optimum for methanogenic activity in the pH range from 6.0 to 6.5. Decreasing pH from 7.0 to 5.5 significantly favored total butyric acid production (P < 0.001). Principle component analysis of T-RFLP patterns revealed that both pectin and pH 5.5 resulted in pronounced changes in the microbial community composition. This study demonstrates that both carbohydrate source and pH affect methane and SCFA production patterns, and the microbial community composition in rumen fluid.     

Catabolism of Lysine by Mixed Rumen Bacteria

Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ ml of lysine was decomposed to give ether-soluble substances and CO₂ by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. δ-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did.     

Production and recovery of propionic and acetic acids in electrodialysis culture of Propionibacterium shermanii

The simultaneous production and recovery of propionic and acetic acids in cultures of Propionibacterium shermanii were examined while maintaining the concentrations of these acids in the fermentation broth at low values with electrodialysis to alleviate end product inhibition. By this method, maximum cell concentration increased 1.36 fold and the average productivities of propionic and acetic acids increased 1.4 fold and 1.31 fold, respectively, compared to cultures without electrodialysis. The mass ratio of propionic and acetic acids recovered was the same as that for the acids produced by fermentation. The inhibitory effect of propionic acid was more significant on the growth of cells than on the production of propionic acid. Some kinetic equations are presented for simulating the dry cell concentration in the fermentor and the concentrations of propionic and acetic acids in the permeate solution of an electrodialysis culture.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.