Early cultural studies by Bryant and Robinson (1962) established that most of the species of rumen bacteria they tested required amino acids for growth. Exactly which amino acid(s) are required has not received systematic analysis. Some rumen Prevotella spp. require methionine (Pittman and Bryant, 1964), whereas Fibrobacter succinogenes requires phenylalanine (Bryant et al., 1959; Atasoglu et al., 2001), which can be supplied in the form of phenylacetic acid (Allison, 1965). Stimulation of growth of Ruminococcus spp. by phenylalanine or its precursors, phenylacetic acid and phenyl-propionic acid, is also well established (Allison, 1965; Morrison et al., 1990; Stack et ah, 1983). The cellulolytic species are stimulated by short-chain fatty acid precursors of aliphatic amino acids (Allison et al., 1958). Otherwise, absolute auxotrophic amino acid requirements are not known, to our knowledge. The question then moves on to which amino acids stimulate growth rate/yield, similar to the mixed population above.
Bacteria appear to fall into two categories with respect to their amino acid requirements for optimum growth, namely cellulolytic bacteria and non-structural carbohydrate (NSQ fermenters (Russell et al., 1992). Generally speaking, the cellulolytic bacteria use ammonia for growth, whereas NSC fermenters use many more amino acids when they are available. Why amino acid requirements should be linked to cellulolytic activity has never been explained. Perhaps the slow growth that is inevitable on cellulose because of its resistant structure has meant, as the cellulolytic bacteria evolved, that the use of amino acids did not provide a selective advantage over ammonia use. The short-chain fatty acid precursors of various amino acids which stimulate cellulolytic species occur in rumen fluid as a result of amino acid catabolism by other species, so the energetic advantage of preformed C skeletons is not necessarily lost by not taking up the intact amino acid.
It is sometimes assumed that rumen cellulolytic bacteria, because they do not require amino acids, do not benefit from or incorporate amino acids. Early studies by Bryant and Robinson (1961, 1962, 1963) with pure cultures of cellulolytic bacteria indicated that Ruminococcus albus and R. flavefaciens incorporated large amounts of ammonia and very small amounts of amino acids into cell N. Several recently published results are not consistent with the conclusion that cellulolytic bacteria do not use amino acids, however. The amino acid transport experiments of Ling and Armstead (1995) indicated that F. succinogenes accumulated radioactivity from 14C-labelled peptides and amino acids. Furthermore, there is experimental evidence that preformed amino acids stimulate fibre digestion in the mixed population in vivo and in vitro (Merry et al., 1990; Chikunya et al., 1996; Griswold et al., 1996; Carro and Miller, 1999), and pure cellulolytic species grow faster on cellobiose when peptides are added to the medium (Cruz Soto et a I., 1994). In addition, bacteria most closely associated with solids derived a substantial proportion of their cell N from sources other than ammonia (Komisarczuk et a I., 1987; Carro and Miller, 1999; Dixon and Chanchai, 2001). The explanation may lie in the concentration of amino acids available to the bacteria under different conditions. The proportion of cell N formed from amino acids and ammonia has been shown to vary according to the concentrations of both. In the mixed population, increasing the concentration of ammonia increased the proportion of microbial protein derived from ammonia; conversely, as the peptide concentration increased, the proportion of cell-N derived from ammonia declined (Fig. 15.3; Atasoglu et al., 1999). At amino acid concentrations typical of the liquid phase of rumen fluid, ammonia would account for 80% or more of amino acid-N in the cellulolytic bacteria (Atasoglu et al., 2001). If the concentration in a microenvironment increased tenfold above the liquid phase, less than half the amino acid-N would be derived from ammonia (Atasoglu et al., 2001). The high peptide concentration in bacterial culture media may therefore provide misleading information about N metabolism of rumen microbes in vivo.
The influence of growth conditions on de novo amino acid synthesis was determined in pure cultures of cellulolytic (Atasoglu et ah, 2001) and non-cellulolytic bacteria (Atasoglu et al., 1998) by following the incorporation of 15N from 15NH3 into individual amino acids. Different patterns emerged with the two
Concentration of ammonia/trypticase (mg N h1)
Fig.15. 3. Influence of peptide concentration on ammonia uptake by mixed rumen microbes. Based on the data of Atasoglu etal. (1999).
categories of bacteria. Phenylalanine synthesis was insignificant in F. succinogenes, and was generally lower than that of the other amino acids in the ruminococci. In contrast, proline synthesis was most responsive to preformed amino acids in non-cellulolytic bacteria. The most enriched amino acids in both types of bacteria were glutamate, aspartate and alanine, reflecting the predominant mechanisms of ammonia assimilation and transamination in these bacteria.
The Cornell model (Russell etal., 1992) assumes that bacteria which ferment NSC derive 66% of their N from preformed amino acids and the remaining 34% from ammonia when both are available. The situation may be more variable because of the recently discovered concentration-dependence, described above. The results of other studies (Hristov and Broderick, 1994; Atasoglu et al. 1999) demonstrate the dependence of bacterial N assimilation on the type of protein and concentration of amino acids present, also as described above, which in turn may be a concentration-dependence effect.
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