This chapter will deal mainly with the characteristics of bacteria from the rumen that have been successfully cultivated in the laboratory. For some ecosystems, particularly those dominated by slow-growing or specialized microorganisms, it has become clear that only a very small fraction (often <1%) of the total microbial diversity has been recovered by cultural methods (Amann et al., 1995) and that descriptions of the ecosystem based on the available isolated strains can be highly misleading. These discrepancies are apparent both from comparison of direct microscopic and culturable counts, and from direct analyses of ribosomal RNA sequence diversity. In the rumen, organisms surviving in significant numbers must have growth rates sufficient to counteract the constant dilution due to turnover of rumen contents, and there are indications that the discrepancies may be less extreme. Leedle et al (1982) found that the culturable count fluctuated with time after feeding between 14% and 74% of the direct microscopic count in the rumens of animals fed on two different diets. Since the viability of several rumen species is known to change upon starvation, the lower figure could partly reflect changes in the viability of known, culturable species. Thus it is to be hoped that the major obstacles to cultivation of the most numerous rumen bacteria have been overcome by the development of sufficiently rigorous anaerobic methods and of suitable isolation media. It remains likely, however, that some functionally important groups (e.g. obligate syntrophs) may not have been recovered; Mclnerney et al (1981) used co-culture with Desulfovibrio in the presence of sulphate to isolate a fatty acid-oxidizing bacterium similar to Syntrophomonas wolfei from bovine rumen contents.

The rumen bacteria

The use of chemically defined media is gaining popularity in some commercial fermentations, particularly for the preparation of biological products. Although these media are still not frequently developed for industrial processes, they do exhibit favorable characteristics at large scale that are not observed with traditional complex media. This review focuses on the application, development, and practical considerations, especially process economics, of fermentations in chemically defined media in an industrial environment.

Chemically defined media for commercial fermentations

Streptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7% (w/v) tannic acid and 4% (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1% (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.

Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S-caprinus) and Streptococcus bovis

Svalbard reindeer (Rangifer tarandus platyrhynchus) live under austere nutritional conditions on the high-arctic archipelago of Svalbard, while semi-domesticated Norwegian reindeer (R. tarandus tarandus) migrate between lush coastal summer pastures and inland winter pastures with lichens on mainland Norway. Svalbard reindeer are known to have high rumen concentrations of cellulolytic bacteria, ranging from 15% of the viable population in summer to 35% in winter, compared to only 2.5% in Norwegian reindeer. Their rumen bacterial diversity was investigated through comparative analyses of 16S rRNA gene sequences (∼1.5 kb in length) generated from clone libraries (n = 121) and bacterial isolates (n = 51). LIBSHUFF comparisons of the composition of the two 16S rRNA libraries from Norwegian reindeer showed a significant effect of artificial feeding compared to natural pasture, but failed to yield significant differences between libraries from Norwegian reindeer and Svalbard reindeer. The combined sequences from reindeer were not significantly different from those reported in wild Thompson’s gazelle in Kenya but did differ from those reported in domestic cattle in Japan. A total of 90 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 97% similarity, while the Chao1 index estimated the reindeer bacterial rumen population richness at 698 OTUs. The majority of the clone library sequences (92.5%) represented novel strains with <97% identity to any known sequence in the public database, most of them affiliated with the bacterial phylum Firmicutes (low G+C Gram-positives) related to the order Clostridiales (76.7%), while Gram-negative bacteria in the Bacteriodales (Prevotella–Bacteroides group) contributed to 22.5%. Also, six of the isolates were putatively novel strains, possibly representing new species in the Clostridium subphylum (cluster XIVa), Actinomyces and Butyrivibrio.

Novel Rumen Bacterial Diversity in Two Geographically Separated Sub-Species of Reindeer

Selenomonas ruminantium is one of the more prominent and functionally diverse bacteria present in the rumen and can survive under a wide range of nutritional fluctuations. Selenomonas is not a degrader of complex polysaccharides associated with dietary plant cell wall components, but is important in die utilization of soluble carbohydrates released from initial hydrolysis of these polymers by other ruminal bacteria. Selenomonads have multiple carbon flow routes for carbohydrate catabolism and ATP generation, and subspecies differ in dieir ability to use lactate. Some soluble carbohydrates (glucose, sucrose) appear to be transported via the phosphoenolpyruvate phosphotransferase system, while arabinose and xylose are transported by proton symport. High cell yields and the presence of electron transport components in Selenomonas strains has been documented repeatedly and this may partially account for the energy partitioning observed between energy consumed for growth and maintenance functions. Most...

Ecology, metabolism, and genetics of ruminal Selenomonads

A completely defined growth medium has been developed to determine the nitrogen requirements for several species of ruminal bacteria, and has revealed two strains which are impaired in de novo biosynthesis of certain amino acids. Using NH4Cl as a sole nitrogen source, the medium supported growth of Butyrivibrio, Selenomonas, Prevotella and Streptococcus species. One strain of B. fibrisolvens (E14) and one strain of P. ruminicola (GA33) did not grow in the presence of NH4Cl until the medium was supplemented with amino acids or peptides. For B. fibrisolvens strain E14, methionine was identified as the specific growth-limiting amino acid although methionine alone did not support growth in the absence of NH4Cl. For P. ruminicola strain GA33, any individual amino acid other than methionine or cysteine could supplement the medium and support growth. Enzyme assays confirmed a lack of NADH and NADPH-dependent glutamate dehydrogenase (GDH) activities in this strain.

A defined medium for rumen bacteria and identification of strains impaired in de novo biosynthesis of certain amino acids

Butyrovibrio fibrisolvens strain E14 has an absolute requirement for methionine. Metabolism of L-[β- 14 C]-serine to methionine occurred in the methionine-independent B, fibrisolvens strain H17c but not in strain E14. The absolute requirement for methionine in strain E14 could be met by addition of S-adenosylmethionine to the medium, but incorporation was not due to the presence of free methionine in the S-adenosylmethionine preparation. The results show that B. fibrisolvens strain E14 is unable to synthesize methionine de novo, probably due to a lack of methionine synthase. Butyrivibrio fibrisolvens may also possess an alternative pathway of methionine biosynthesis from S-adenosylmethionine.

N. Nili

Papers

A defined medium for rumen bacteria and identification of strains impaired in de novo biosynthesis of certain amino acids

Lack of methionine biosynthesis de novo in Butyrivibrio fibrisolvens strain E14