Variations in the Uptake and Metabolism of Peptides and Amino Acids by Mixed Ruminal Bacteria In Vitro

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1993, p /93/1336-7$2./ Copyright 1993, American Society for Microbiology Vol. 59, No. 1 Variations in the Uptake and Metabolism of Peptides and Amino Acids by Mixed Ruminal Bacteria In Vitro IAN P. ARMSTEAD AND JOHN R. LING* Department of Biochemistry, School of Life Sciences, The University of Wales, Aberystwyth SY23 3DD, Wales, United Kingdom Received 18 May 1993/Accepted 31 July 1993 Mixed ruminal bacteria, isolated from sheep (Q and W) fed a concentrate and hay diet, were anaerobically incubated with either 14C-peptides or "4C-amino acids. Experiment 1 showed that uptake of both "4C-labeled substrates was rapid, but the rate for amino acids was twofold greater than for peptides (molecular weight, 1, to 2) initially but was similar after 1 min. Experiment 2 demonstrated that metabolism was also rapid; at least 9%o of either "4C-labeled substrate was metabolized by 3 min. Of the radioactivity remaining in bacteria, approximately 3% was in the form of "4C-amino acids, but only in leucine, tyrosine, and phenylalanine. Supernatant radioactivity was contained only in tyrosine, phenylalanine, and mostly proline for incubations with "4C-amino acids but in up to 1 amino acids when 14C-peptides were the substrates. Short-term incubations (<5 min; experiment 3) confirmed previous uptake patterns and showed that the experimental system was responsive to substrate competition. Experiment 4 demonstrated that bacteria from sheep Q possessed initial and maximum rates of "4C-amino acid uptake approximately fourfold greater (P <.1) than those of "4C-peptides, but with no significant differences (P >.1) between four "4C-peptide substrate groups with molecular weights of 2, to <2. By contrast, bacteria from sheep W showed no such distinctions (P >.1) between rates for "4C-peptides and "4C-amino acids. Calculations suggested that peptides could supply from 11 to 35% and amino acids could supply from 36 to 68% of the N requirements of mixed ruminal bacteria. The variations of these findings and their implications for future research are discussed. The degradation of dietary proteins by ruminal microorganisms inevitably produces peptides and amino acids (7). Furthermore, there is substantial indirect evidence that these intermediates of degradation are also important substrates for biosynthesis by ruminal microorganisms. For instance, it has been demonstrated that in some circumstances only 4% of bacterial N synthesized in the rumen was derived from ammonia; the remaining 6% was presumably supplied by peptide and amino acid substrates (2). Our understanding of these catabolic and anabolic processes within the rumen is apparently sufficient to allow their incorporation into schemes for feeding ruminant animals (1, 2, 26). Yet several important aspects remain unclear; for example, many would still agree with the statement that, once they are produced, the role of peptides and amino acids within the rumen is "uncertain" (1). The fate of peptides and amino acids in the ruminal ecosystem has been studied from a number of perspectives, including measuring their effects on microbial growth (3, 15, 22), their fermentation and production of ammonia (5, 9, 1), their persistence in rumen fluid (8, 35), the activities of microbial peptidase enzymes (3, 31), the utilization of specific peptides and amino acids (6, 27, 37), and the utilization of mixtures of either of these substrates (14, 25, 38). Despite these and other studies, progress in this research area has been hampered by considerable controversy. For example, disagreements about the extent of peptide accumulation in ruminal fluid appear to have been due to differences in analytical techniques used (11, 12, 33). Variations in the utilization of peptides can be attributed in part to differences in their molecular composition; hydrophilic peptides seem to be metabolized more rapidly than * Corresponding author. 336 hydrophobic peptides (13). Molecular size also seems to be an important, but disputed, metabolic parameter; some (14, 25) have unexpectedly found that uptake of large peptides can be more rapid than that of small peptides or amino acids. Furthermore, the use of imprecise nomenclature has added to the confusion; for example, the same experimental data have been presented as both "peptide metabolism" (33) and "peptide uptake" (34). Reported variations concerning peptide and amino acid utilization by ruminal bacteria thus appear to have been caused largely by variations in the substrates, experimental techniques, and microorganisms used. The work reported here examined some of these variations by simultaneously comparing the uptake and metabolism of peptides and amino acids by mixed ruminal bacteria. The substrates used were 14C labeled and derived from plant biomass; the peptides were of mixed residue composition and grouped according to molecular size. MATERIALS AND METHODS When appropriate, all media and solutions were autoclaved at 13 kpa for 15 min. Anaerobic conditions were maintained by gassing (approximately 1 ml/min) with CO2 that had passed through a column of copper turnings heated at 35 C. Rumen microbial preparations. Samples of mixed ruminal microorganisms were obtained for experiments 1 to 4 from a mature (approximately 5-year-old) wether sheep (sheep Q) and also from a similar animal (sheep W) for experiment 4; each was fitted with a permanent cannula in the rumen. The animals' daily dietary allowance was 6 g of grain-based concentrate and 5 g (fresh weight) of chopped hay, and this was given in two equal portions (at 9 and 16 h). Water was available at all times. Downloaded from on May 4, 218 by guest

2 VOL. 59, 1993 PEPTIDE AND AMINO ACID UTILIZATION BY RUMINAL BACTERIA 3361 Ruminal digesta were removed approximately 2 h after the morning feed and strained through several layers of cheesecloth, and the strained ruminal fluid was centrifuged (1,3 x g, 5 min). The resulting supernatant, which was examined (magnification, x 1) to ensure the absence of protozoa, was used as the source of mixed ruminal bacteria. Preparation of "'C-labeled substrates. Samples of "'Cproteins were prepared by growing barley plants (Hordeum vulgare) in an atmosphere of 1'CO2, harvesting the leaves, and extracting the water-soluble "'C-proteins as previously described (18). The "'C-peptides were prepared by sequentially digesting these "'C-proteins with proteolytic enzymes (18) and separating the products from contaminating amino acids by elution from columns of copper-chelex resin (4). The 14C-peptides were then fractionated by elution from a column of Sephadex G-25 fine (23 mm by 25 mm), which had been calibrated with standards of known molecular weights (18). Eluted fractions were pooled to produce four 14C-peptide substrate groups (A to D), each with a different molecular weight range. Substrate group E, composed of mixed 14C-amino acids, was prepared by acid hydrolysis of the 14C-proteins (17). In vitro incubations and sample preparations. Samples (.95 ml each) of mixed ruminal bacteria were anaerobically transferred and rapidly mixed with samples of the various "4C-labeled substrates (5,ul each, containing 25 Bq and from 5 to 9 nmol of total amino acids). These reaction mixtures were layered onto 4-,ul volumes of silicone fluid (Dow Corning 55 and 556, mixed 3:2 [vol/vol]; Hopkins & Williams, Chadwell Heath, United Kingdom) contained in 1.5-ml Eppendorf tubes. The tubes were anaerobically sealed and maintained at 39 C for up to 3 min (experiments 1 and 2) or 5 min (experiments 3 and 4). In experiment 3, nonradiolabeled peptides (prepared by removing free amino acids by method 1 of Armstead and Ling [4] from tryptone [Oxoid Ltd., London, United Kingdom]) and nonradiolabeled amino acids (obtained from an acid hydrolysate of casein [Casamino Acids; Oxoid Ltd.]) were also added at five times the concentrations of the "C-labeled substrates. Incubations were terminated by rapid centrifugation (14, x g, 6 s). The supernatants were removed and stored, with the tubes containing the bacterial pellets, at -2 C. Methods of analysis. The bottom of each frozen Eppendorf tube, containing the bacterial pellet, was cut off and thoroughly mixed with 4 ml of liquid scintillant (Ecoscint; National Diagnostics, Manville, N.J.). Duplicate samples of bacterial pellets were lysed by adding 1 ml of.2 M NaOH and heating at 1 C for 5 min. These lysates were centrifuged (15, x g, 1 min), and the protein contents of the supernatants were estimated by the method of Lowry et al. (19). Substrate and incubation samples were acid hydrolyzed by refluxing in 6 M HCI and subsequently prepared by the methods of Ling and Buttery (17). Amino acids were separated with an amino acid analyzer (model 5; Locarte Co., London, United Kingdom), detected with ninhydrin, and quantified with a data management system (Roseate; Drew Scientific, London, United Kingdom). Radioactivity contents of samples were detected by the addition of a scintillant (Ecoscint) and measured in a scintillation counter (model SL 3; Intertechnique SA, Plaisir, France). When appropriate, quenching was corrected by the external-standard channel ratio facility. Experimental design and statistical analysis. Results from experiments 1 and 3 were obtained from triplicate incubations of bacteria prepared from sheep Q on the same day. TABLE 1. Amino acid composition of the substrate groups of 14C-peptides (A to D) and 14C-amino acids (E) incubated with mixed ruminal bacteria Amino Amt" in substrate group: acid A (91.23) B (81.25) C (5.4) D (58.65) E (89.3) Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe His Lys 1.35 NDb ND ND 6.2 Arg a In nanomoles of amino acid per 5 1J (this volume also had a radioactivity content of 25 Bq). Totals are given in parentheses. Molecular weights: A, 2, to 1,; B, 1, to 5; C, 5 to 2; D, <2. b ND, not determined. Results from experiment 2 are single values for sheep Q. Incubations in experiment 4 were performed in triplicate with bacteria from sheep Q and W obtained on three consecutive days. Analysis of variance and Tukey's modified honestly significant difference test (16) were used to compare differences between the uptake rates of the 14Clabeled substrates. RESULTS Characteristics of the "4C-peptide and "4C-amino acid substrates. The approximate molecular weight ranges of the substrate groups were 2, to 1, (A), 1, to 5 (B), 5 to 2 (C), and <2 (D); group E consisted of free amino acids. The amino acid contents of the substrate groups are listed in Table 1. Analysis showed each of these substrate groups to be of high chemical purity (>98%), 14C labeled in the general form, of mixed amino acid composition, and with all amino acids presumably in the L configuration. Uptake and metabolism of 14C-peptides and "4C-amino acids by ruminal bacteria for 3 min (experiments 1 and 2). Experiment 1 compared the uptake of 14C-peptides, composed of a combination of substrate groups B and C (specific radioactivity, 416 Bq/,umol; molecular weight, 1, to 2), with that of 14C-amino acids (group E; specific radioactivity, 28 Bq/,umol) in 3-min incubations by mixed ruminal bacteria isolated from sheep Q. The uptake of radiolabel from 14C-peptides resulted in a plateau value of approximately 1.2 Bq/mg of bacterial protein after 1 min, as shown in Fig. 1. The uptake pattern for the 14C-amino acids was quite different; the peak value (1.7 Bq/mg of bacterial protein) was approximately 5% greater than that of the 14C-peptides and was attained after only 2.5 min. This subsequently declined, so that by 1 min the uptake values were similar for both "4C-labeled substrates. Experiment 2 examined the uptake of the same 14C_ labeled substrates and their subsequent distributions between the bacterial pellets and supernatants. Table 2 shows that after 3 min of incubation only 5.7% (2.4 and 3.3% from Downloaded from on May 4, 218 by guest

3 3362 ARMSTEAD AND LING APPL. ENvIRON. MICROBIOL C) C13 S.. Cw to Incubation time (min) FIG. 1. Patterns of radioactivity in pellets of mixed ruminal bacteria, isolated from sheep Q, after incubation with either '4Cpeptides (molecular weight, 1, to 2) (@) or 14C-amino acids () for up to 3 min (experiment 1). Values are means of triplicate observations with their standard deviations (bars). the pellet and supematant, respectively) of the radioactivity added as 14C-peptides was detectable in the form of radiolabeled amino acids in the acid-hydrolyzed samples. When the substrates were '4C-amino acids, the equivalent data were only slightly higher: a total of 11.%, 6.% from the pellet and 5.% from the supernatant. These values changed little and exhibited no discernible trend during the total 3-min incubations; these pellet values represented about a third (mean + standard deviation, 31.8% + 5.2%) of the total radioactivity remaining in each pellet, regardless of which 14C-labeled substrate had been added. Table 3 shows that this radioactivity was attributable to only three '4C-amino acids (Leu, Tyr, and Phe). Table 3 also shows that the corresponding results for the acid-hydrolyzed supernatants were quite different. When '4C-peptides were the substrates as many as 1 amino acids were radiolabeled, whereas when 14C-amino acids were the substrates only 3 amino acids (Tyr, Phe, and Pro) were radiolabeled, with an average of 74% of the radiolabel present in the last throughout the 3-min incubation period. Uptake and metabolism of '4C-peptides and "4C-amino acids by ruminal bacteria for 5 min (experiments 3 and 4). It TABLE 2. Radioactivity present as 14C-amino acids (experiment 2) % Radioactivity' after incubation with: Incubation l4c-peptides "4C-amino acids Pellet Supernatant Pellet Supernatant aof the 25 Bq added to each incubation. b Molecular weight, 1, to 2. was evident from the preceding 3-min experiments that important uptake and metabolic processes were occurring rapidly. Experiments 3 and 4 were therefore undertaken to examine these events during the first 5 min of incubation. Figure 2 shows the results from experiment 3 with the same "'C-labeled substrates previously used. It confirmed the general pattern seen in experiment 1 (Fig. 1); namely, the uptake of ' C-peptides plateaued after about 3 min, it was considerably less than that from "'C-amino acids, and the uptake of the latter peaked at 2 min and then declined towards the 14C-peptide uptake values. Figure 2 also demonstrates that when nonradiolabeled peptides or amino acids were added after 2 min to the incubations, the uptake of both "'C-peptides and 14C-amino acids declined markedly (by approximately 25%) within the next 6 s when new, but quantitatively lower, plateau values were established. Experiment 4 measured the uptake of each of the five "'C-labeled substrate groups (A to E) by bacteria isolated from sheep Q and W. Figure 3 shows that bacteria from sheep Q exhibited uptake patterns and quantitative values similar to those previously measured for this animal (experiment 3; Fig. 2). The uptake rates of the various "'C-peptides (A to D) were not significantly different (P >.1) within the bacterial populations of sheep Q or W, but there were significant differences between these bacterial populations. Though the initial rates (up to 3 s) were similar (P >.1), after 5 min of incubation bacteria from sheep W were taking up 14C-peptides faster (P <.5) than those from sheep Q. In addition, while the rates of uptake of "'C-amino acids and 14C-peptides by bacteria from sheep W were not significantly different, sheep Q exhibited "'C-amino acid rates much greater (P <.1), both initially and after 5 min, than the rates for "'C-peptides. Nevertheless, while the initial "'Camino acid uptakes were quite different (1.6 versus.8 Bq/mg of bacterial protein; P <.1), after 5 min they were more similar (1.9 versus 1.6 Bq/mg of bacterial protein) for bacteria from sheep Q and W, respectively. DISCUSSION General approach of the present study. In contrast to our understanding of some aspects of protein degradation and synthesis by ruminal microorganisms, surprisingly little is known about the processes of uptake and metabolism of those inevitable intermediates, amino acids, and in particular, peptides. This lack of knowledge has in part been caused by confusion in the reported literature, due largely to differences in substrates, experimental methods, and ruminal microorganisms used. The choice of substrates is of paramount importance in peptide research. Because of the vast array of different peptides, the selection of a typical peptide is almost meaningless. This problem is not easily overcome. Substrates used in ruminal studies have included derivatized peptides (6, 29), single amino acid residue peptides (33, 37), defined mixed residue peptides (6, 37), and physiologically high concentrations of commercially available peptides (22). None of these is entirely satisfactory, because ruminal bacteria will never encounter such substrates in vivo. Furthermore, their use can be misleading. For example, studies with single amino acid residue peptides have underestimated the persistence of peptides in extracellular ruminal fluid (34). Commercially available enzymatic digests of proteins (13, 22) are also unsuitable sources of peptides, because they typically contain high proportions of free amino acids (4a) and those that have been sufficiently purified are usually Downloaded from on May 4, 218 by guest

4 VOL. 59, 1993 PEPTIDE AND AMINO ACID UTILIZATION BY RUMINAL BACTERIA 3363 TABLE 3. Distribution of radioactivity associated with "'C-amino acids (experiment 2) % Radioactivity' in: Amino Pellet Supematant acid l4c_peptidesb "'C-amino acids "'C-peptides 14C-amino acids 3 min 15 min 3 min 3 min 15 min 3 min 3 min 15 min 3 min 3 min 15 min 3 min Asp Thr Ser Glu Pro Gly 6 4 Ala 1 Val Ile Leu Tyr Phe His 5 Lys Arg 8 6 aof the total radioactivity present in the pellet or supernatant after the indicated incubation period. b Molecular weight, 1, to 2. bioactive, expensive, and unavailable as radiolabeled mixtures. Since the rumen contains relatively low concentrations of underivatized, mixed amino acid residue peptides plus mixed amino acids derived mainly from plant biomass and since the most physiological and sensitive methods for studying them involve the use of radiolabeled plant material (21), we devised procedures (4, 18) to produce "'C-peptide and "'C-amino acid substrates that were pure, cheap, of. c; co. E C; m u- 8 cc la cc Incubation time (min) FIG. 2. Patterns of radioactivity in pellets of mixed ruminal bacteria, isolated from sheep Q, after incubation with either 14Cpeptides (molecular weight, 1, to 2) (closed symbols) or "4C-amino acids (open symbols) for up to 5 min with (triangles) or without (circles) the addition of nonradiolabeled peptides (A) or amino acids (A) after 2 min (arrowheads) (experiment 3). Values are means of triplicate observations with their standard deviations (bars). 3 mixed molecular composition, and derived from plant biomass. These are the "'C-labeled substrates whose characteristics are listed in Table 1. The protocols used for terminating incubations and assaying the uptake and metabolism of substrates will affect the interpretation of any study. In the present study, incubations were terminated by physically separating bacteria from the "'C-labeled substrate by centrifuging them through silicone fluid. The pellets produced were composed of bacteria plus whatever "C-label had been taken up. This uptake would include some radiolabel bound to cell surfaces (14), but mainly it would consist of 14C-substrates transported into the bacterial cells, where they may have remained intact or been metabolized; subsequently, they may have been incorporated into cellular material or been excreted. The radioactivity in the pellets was therefore the net result of the processes of uptake and metabolism of any 14C-labeled substrate; that in the supernatants was the net result of any uptake, metabolism, and excretion. All methods of assessing peptide utilization have limitations. None of several methods for measuring peptide concentrations in ruminal fluid was considered reliable (11, 32). For instance, the direct ionexchange chromatography method (35) cannot determine whether released amino acids are derived from peptide substrates or from microbial sources, and because of this, Wallace et al. (33) have had "to revise some of their earlier conclusions" (6). One of the advantages of the present study's protocols is that any radioactivity detected in the bacterial pellets and supernatants must unequivocally have originated from whichever 14C-labeled substrate was added. The final key decision to be made in this area of research is whether to use single or mixed species of ruminal bacteria; for the present study the latter, incubated in extracellular rumen fluid and at cell densities typical of those found in the rumen, were chosen to mimic the ruminal environment more closely. The uptake of peptides and amino acids by mixed ruminal bacteria. Figure 1 illustrates the basic patterns of uptake of 14C-peptides and 14C-amino acids by mixed bacteria from the rumen of sheep Q; these were confirmed and defined more Downloaded from on May 4, 218 by guest

5 3364 ARMSTEAD AND LING APPL. ENvIRON. MICROBIOL cc. u co U m1-._._ a 8 Ca %.5 Sheep W Incubation time (min) Incubation time (min) FIG. 3. Patterns of radioactivity in pellets of mixed ruminal bacteria, isolated from sheep Q and W, after incubation with either "4C-peptides grouped by molecular weight (*, 2, to 1, [A]; A, 1, to 5 [B]; *, 5 to 2 [C]; *, <2 [D]) or 14C-amino acids () (E) for up to 5 min (experiment 4). Values are means of three daily observations, but for clarity their standard deviations are omitted. precisely by the results obtained in the short-term studies (experiments 3 and 4; Fig. 2 and 3). Maximum rates for both substrates generally occurred prior to 3 min; after that time metabolic equilibria, with uptake similar to efflux, were attained. In addition, experiment 3 (Fig. 2) showed that the protocols used to measure uptake were, as expected, responsive to perturbation, for when nonradiolabeled peptides or amino acids were added 14C label uptakes declined significantly as a result of substrate competition. In experiments 1, 2, and 3 with bacteria from sheep Q, "4C-amino acids were taken up, at least initiall Y, at a much greater (approximately threefold) rate than any 4C-peptides. This suggests the existence of different uptake sites and mechanisms for the two substrate types. Experiment 4 confirmed this general pattern for sheep Q, but the results for sheep W were quite different. Yet despite these differences, when the data (Fig. 3) are expressed as initial uptake rates of amino acid residues (Table 4), the trend for sheep Q and W is E > A > D > B > C. These observations conflict with several reports that ruminal bacteria prefer to take up peptides rather than amino acids (23, 25, 38). They also disagree with the findings of Cooper and Ling (14), who, using similar techniques in the same laboratory as that used for the present study, demonstrated a positive correlation between molecular size and uptake rate. These differences are difficult to explain; they are perhaps partly related to the different substrates used. Those of Cooper and Ling (14) were probably contaminated with free amino acids, because in the present study substrate groups A to D contained 1, 7, 14, and 39% amino acids, respectively, prior to their removal by the methods of Armstead and Ling (4). However, probably the major cause of variation among these studies is the different composition of the bacterial populations used. The present study provides evidence to support this hypothesis, because in experiment 4 the uptake patterns of bacteria from sheep W were most unlike those of bacteria derived from sheep Q (Fig. 3), even though these sheep were similar in breed, age, and weight, had been fed the same type and amount of diet for several months, and were housed together. Possible differences in the microbial populations of these animals were not assessed. The metabolism of peptides and amino acids by mixed ruminal bacteria. Once taken up, the metabolism of the 14C-labeled substrates was rapid (experiment 2); within 3 min, less than 6% of the added 14C-peptides or 11% of the 14C-amino acids was detectable as 4C-amino acids in the acid-hydrolyzed pellets plus supernatants (Table 2). This means that about 9% of the radiolabel was unaccounted for; it was certainly not present in intact amino acids (that is, as proteins, peptides, or amino acids). It had most probably been metabolized to volatile compounds, such as 1 CO2 and 14C-volatile fatty acids, and lost during the rotary evaporation step of sample preparation. Such metabolism is apparently faster than that indicated by Yang and Russell (39), who considered Lys-Ala to be rapidly metabolized, yet after 6 h only 8% of that dipeptide had been degraded, though its initial concentration was as much as 4-fold greater than that used in the present study. Nevertheless, individual substrates did vary considerably Substrate group TABLE 4. Initial rates of uptake of substrate groups of 14C-peptides (A to D) and 14C-amino acids (E) (experiment 4) Mol wt Rate of uptakea for sheep: A 2,-1, 3.21 (.1) 5.62 (.22) B 1, (.12) 3.44 (.14) C (.39) 2.34 (.11) D < (.14) 3.75 (.23) E (.44) 5.86 (.34) a In nanomoles of amino acid per minute per milligram of bacterial protein. The data are means, with standard deviations in parentheses, of three daily observations. Downloaded from on May 4, 218 by guest

6 VOL. 59, 1993 PEPTIDE AND AMINO ACID UTILIZATION BY RUMINAL BACTERIA 3365 in their rates of metabolism. Of the total radioactivity remaining within the bacterial pellets, about a third was consistently detectable as '4C-amino acids, even after 3 min, and surprisingly, this was contained in just three amino acids, regardless of whether the substrates added were 14C-peptides or 14C-amino acids. These hydrophobic amino acids (Leu, Tyr, and Phe), in either the free or the peptidyl form, are apparently resistant to bacterial metabolism. In contrast, the radioactivity in the acid-hydrolyzed incubation supernatants was present in as many as 1 14C-amino acids when 14C-peptides were added but in only 3 (Tyr, Phe, and Pro) when ' C-amino acids were the substrates; Pro dominated, especially with the latter substrates. This reinforces the interesting observation (39) that proline-containing peptides appear to be especially resistant to degradation, and possibly uptake, by ruminal bacteria. General considerations of peptide and amino acid uptake and metabolism. The importance of peptides and amino acids to the N economy of the rumen bacterial population may be judged by their abilities to supply intracellular amino acids. Using substrate group C data (molecular weight, 5 to 2), the initial uptake rates for these 14C-peptides by bacteria from sheep Q and W are 1.85 and 2.34 nmol of amino acid per min per mg of bacterial protein, respectively (Table 4) or, on the basis that mixed ruminal bacteria contain 48% protein (17),.89 and 1.12 nmol of amino acid per min per mg of bacterial dry matter. These are minimum rates, because rapid effluxes of amino acids and their products are not included. Even so, these values are within the range of.85 to 2.59 nmol of amino acid per min per mg of dry matter reported (6) for mixed ruminal organisms (which presumably, unlike those in the present study, included some protozoa) incubated with individual mixed residue di- and tripeptides at concentrations up to fivefold greater than that used in the present study. However, on the basis that the average molecular weight of an amino acid is 137 (11), these values are only about 1% of the rate (.7 g of peptide per h per g of dry cellular matter) reported by Russell et al. (26), though they did use concentrations as much as 4-fold higher. The comparable rates for amino acid uptake by bacteria from sheep Q and W (11.14 and 5.86 nmol/min/mg of bacterial protein; Table 4) are as much as 1-fold higher than those reported for Bacteroides ruminicola (28), though the substrates for the latter were approximately 1-fold lower. By using the minimum and maximum rates of peptide uptake (1.85 and 5.62 nmol of amino acid per min per mg of bacterial protein; Table 4) and the assumptions that the average amino acid molecule contains 1.3 nitrogen atoms (11), that the average generation time of mixed ruminal bacteria is 6 h (24), and that their growth rate is linear and ignoring any turnover of N, the mean contributions made by peptides during the total generation time can be calculated to vary from 8.7 to 26.6,ug of amino acid N, values which are equivalent to 11.3 and 34.5% of the N requirements of such ruminal bacteria. Similar calculations show that the uptake of amino acids could account for 35.5 to 68.2% of the N required. Of course, these are tentative estimates of potential N supply, but the amino acid/peptide ratio of approximately 2.5:1 and the mean proportion of bacterial N originating from peptides plus amino acids of 75% (range, 47 to 13%) appear to be realistic and sensible, and the latter value is similar to the 6% estimate of Nolan et al. (2). Nevertheless, the rapid metabolism and lack of incorporation into cellular material of many of the '4C-labeled substrates seen in the present study suggest that they may have been used mainly as energy-yielding, rather than N-supplying, substrates. The present in vitro study has demonstrated variations in the patterns and rates of peptide and amino acid uptake and metabolism by mixed ruminal bacteria and that these patterns and rates can vary between similar sheep fed the same diets; presumably, these variations have their basis in differences between individual bacterial species. Additional research to study these differences and the effects of physical binding of substrates, extracellular hydrolysis, intracellular metabolism, and subsequent excretion with both mixed and single ruminal species is now needed. When these processes are quantitatively assessed, peptide and amino acid utilization by ruminal bacteria will be better understood and more likely to be successfully manipulated to the benefit of the host animal (36). ACKNOWLEDGMENT We gratefully acknowledge the financial support of the Agricultural and Food Research Council, Swindon, United Kingdom. REFERENCES 1. Agricultural Research Council The nutrient requirements of ruminant livestock. Commonwealth Agricultural Bureaux, Slough, England. 2. Agricultural Research Council The nutrient requirements of ruminant livestock, supplement 1. Commonwealth Agricultural Bureaux, Slough, England. 3. Argyle, J. L., and R. L. Baldwin Effects of amino acids and peptides on rumen microbial growth yields. J. Dairy Sci. 72: Armstead, I. P., and J. R. Ling Chromatographic separation of mixed peptides from amino acids in biological digests with volatile buffers. J. Chromatogr. 586: a.Armstead, I. P., and J. R. Ling. Unpublished observations. 5. Bladen, H. A., M. P. Bryant, and R. N. Doetsch A study of bacterial species from the rumen which produce ammonia from protein hydrolyzate. Appl. Microbiol. 9: Broderick, G. A., R. J. Wallace, and N. McKain Uptake of small neutral peptides by mixed rumen microorganisms in vitro. J. Sci. Food Agric. 42: Broderick, G. A., R. J. Wallace, and E. R. Orskov Control of rate and extent of protein degradation, p In T. Tsuda, Y. Sasaki, and R. Kawashima (ed.), Physiological aspects of digestion and metabolism in ruminants. Academic Press, San Diego, Calif. 8. Chalupa, W Degradation of amino acids by the mixed rumen microbial population. J. Anim. Sci. 43: Chen, G., and J. B. Russell Fermentation of peptides and amino acids by a monensin-sensitive ruminal peptostreptococcus. Appl. Environ. Microbiol. 54: Chen, G., and J. B. Russell More monensin-sensitive, ammonia-producing bacteria from the rumen. Appl. Environ. Microbiol. 55: Chen, G., J. B. Russell, and C. J. Sniffen A procedure for measuring peptides in rumen fluid and evidence that peptide uptake can be a rate-limiting step in ruminal protein degradation. J. Dairy Sci. 7: Chen, G., C. J. Sniffen, and J. B. Russell Concentration and estimated flow of peptides from the rumen of dairy cattle: effects of protein quantity, protein solubility, and feeding frequency. J. Dairy Sci. 7: Chen, G., H. J. Strobel, J. B. Russell, and C. J. Sniffen Effect of hydrophobicity on utilization of peptides by ruminal bacteria in vitro. Appl. Environ. Microbiol. 53: Cooper, P. B., and J. R. Ling The uptake of peptides and amino acids by rumen bacteria. Proc. Nutr. Soc. 44: Cotta, M. A., and J. B. Russell Effect of peptides and amino acids on efficiency of rumen bacterial protein synthesis in continuous culture. J. Dairy Sci. 65: Downloaded from on May 4, 218 by guest

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