No abstract

Rumen contents were obtained from a cow fed 2.5 kg concentrates and 2.5 kg timothy hay twice daily, and the mixed rumen bacterial inoculum (pH 6.3) was separated from large feed particles and protozoa by centrifugation. Bacteria were incubated in artificial media at pH 6.7 and 6.0. When starch, sucrose, cellobiose, xylan, pectin, or a mix of carbohydrates were provided to pH 6.7 incubations at 1 mM/h for 10 h, fermentation was carbohydrate-limited, less than 20% of the carbohydrate was converted to lactate, molar ratio of methane to hexose fermented was as great as 49%, and pH remained constant. When initial pH was lowered to 6.0, methane production was nearly eliminated, volatile fatty acid production was reduced, and there was a small decrease (less than .5) in final pH. At pH 6.0, lactate production was increased in all incubations except xylan and pectin, and pH had a greater effect on acid production than the type of carbohydrate provided. Bacterial protein synthesis was reduced 34 to 69% when initial pH was 6.0, but these reductions were greater than decreases in carbohydrate utilization, increases in lactate, and associated decreases in adenosine 5'-triphosphate production. Because less adenosine 5'-triphosphate was used for protein synthesis, it appeared that low pH diverted energy to nongrowth functions.

When mixed ruminal bacteria were inoculated into semicontinous cultures (25% transfer every other day) containing lactate, dulcitol, pectin, or xylose and Trypticase (1 g/liter) as the sole nitrogen source, the specific activity of ammonia production increased. The greatest enrichment was observed with lactate and xylose, and in these cases the specific rate of ammonia production was eightfold higher than that of the ruminal fluid control (approximately 35 nmol of ammonia per mg of protein per min). Isolates with different morphologies were obtained from each of the enrichments, but in no case did the specific activity of any isolate exceed that of the mixed ruminal bacteria. If Trypticase (15 g/liter) was used as the only energy and nitrogen source, there was an even greater increase in ammonia production, and two monensin-sensitive bacteria, a Peptostreptococcus species and a Clostridium species, were obtained. The Peptostreptococcus species was unable to grow on any of 25 carbohydrate or carbohydrate derivatives tested; but the Clostridium species was able to use glucose, maltose, fructose, cellobiose, trehalose, sorbitol, and salicin as energy sources. Neither organism was able to grow in the absence of an amino acid source, but growth rates on Trypticase were >0.35/h. The specific activities of ammonia production were 346 and 427 nmol/mg of protein per min for strains of Peptostreptococcus and Clostridium, respectively. Megasphaera elsdenii and Bacteroides ruminicola, previously isolated ruminal ammonia producers, had specific activities of only 11 and 19 nmol of ammonia per mg of protein per min, respectively. The most probable number of Clostridium species in ruminal fluid was <103/ml, but the Peptostreptococcus species was present at 108/ml. Since the Peptostreptococcus species was present in significant numbers and was a very active ammonia producer, it could play an important role in the fermentation of dietary amino acids.

When PrevoteUa ruminicola 23 was grown in a defined medium containing a vitamin mixture, significant amounts of propionate were formed. Succinate and acetate were the major fermentation acids produced when vitamins were omitted, and further experiments demonstrated that propionate formation was dependent on vitamin B12. When the organism was grown in continuous culture at dilution rates of less than 0.20 h-', propionate and acetate were the predominant fermentation products and little succinate was formed when vitamin B12 was present. However, at higher dilution rates, propionate formation declined and succinate accumulated. Since cell protein yields were reduced 15 to 25% in the absence of vitamin B12, the pathway for propionate formation may contain an energy-conserving step.

Mixed ruminal bacteria (n = 4) were incubated in anaerobic media for 24 h in vitro with either hay, corn meal, protein hydrolyzate or hydrogen gas as the substrate. The ionophore monensin and the polypeptide antibiotic bacitracin were added to the incubation flasks at concentrations ranging from 0 to 10 or 40 mg/liter. As was expected, monensin decreased methane production, increased the ratio of propionate to acetate and decreased the deamination of amino acids. Monensin had little effect on methane production, however, if hydrogen gas was the fermentation substrate. Bacitracin, another gram-positive antibiotic with a distinctly different cellular target, was somewhat less potent than monensin, but it produced strikingly similar responses. This similarity of fermentation patterns suggested that monensin action in the rumen is probably due to its activity as a gram-positive antibiotic, and that any gram-positive antibiotic not suppressed by resistance may produce fermentation effects similar to those of monensin. The cellular action of monensin as an ionophore in membranes is probably little more than a means of inhibiting sensitive species. Many gram-positive antibiotics have little affect on ciliate protozoa or coccidia.