flavefaciens Isolated from the Rumen of Cattle

Transcription

1 APPLIED MICROBIOLOGY, May 1969, p Copyright 1969 American Society for Microbiology Vol. 17, No. 5 Printed in U.S.A. Growth Factor Requirements of Ruminococcus flavefaciens Isolated from the Rumen of Cattle Fed Purified Diets1 L. L. SLYTER AND J. M. WEAVER Agricultural Research Service, Aninal Husbandry Research Division, U.S. Department of Agriculture, Beltsville, Maryland Received for publication 8 January 1969 Eight strains of cellulolytic cocci were isolated from a 10- dilution of rumen ingesta and were presumptively identified as Ruminococcusflavefaciens. Four strains were isolated from a steer fed a purified diet which contained isolated soy protein, and four strains were isolated from a steer fed a purified diet which contained urea. Certain growth factor requirements of these bacteria were determined. All strains grew with clarified rumen fluid added to the medium. However, fatty acids could substitute for rumen fluid in four strains. Two strains isolated from each steer either required or their growth was stimulated by isobutyric and/or isovaleric and/or 2-methyl-butyric acid. These results indicate that, even when a diet was fed which contained no branched-chain amino acids, the carbon skeleton precursors of branched-chain fatty acids, the cattle were still able to maintain a large population of cellulolytic bacteria that require fatty acids for growth. Therefore, the fatty acids appear to be provided by other bacteria, by protozoa, or by the host animal. Previous results by Oltjen and Putnam (19) indicated that, when cattle were fed a basal purified diet in which urea was replaced by isolated soy protein, the concentrations in blood plasma of valine, isoleucine, leucine, and phenylalanine were significantly reduced. Reports from the same laboratory (R. R. Oltjen et al., J. Animal Sci., in press) also indicated that there was less ruminal isobutyric, isovaleric, or 2- methyl-butyric acid, or less of all three, present in cattle fed purified diets which contained urea than in cattle fed purified diets which contained soy protein. Low levels of ruminal branchedchain acids have also been found in sheep fed purified diets which contained urea as the primary dietary nitrogen source (11, 18). Isobutyric, 2-methyl-butyric, and isovaleric acid have been shown to be required for growth by many rumen cellulolytic bacteria (1, 6, 7, 13). It has been suggested that these acids are formed mainly through dietary protein degradation and amino acid deamination (12, 14). This experiment was conducted to determine whether the predominant cellulolytic bacteria isolated from cattle fed highfiber purified diets in which the dietary nitrogen I Presented in part at the meeting of the North Atlantic Section of the American Society of Animal Science, June 28, 1967, Ithaca, N.Y. was supplied by urea have growth factor requirements, particularly in regard to the branchedchain fatty acids, similar to those of bacteria isolated from cattle fed purified diets in which the dietary nitrogen was supplied by isolated soy protein. MATERIALS AND METHODS Bacterial strains. Bacteria were obtained from the rumen ingesta of ruminal fistulated identical twin steers. The steers and the high wood pulp supplemented purified diets used in this study were those used in experiment 3 by 0rskov and Oltjen (20). One steer was fed the diet which contained 4.7% urea, and the other was fed the diet which contained 14.9% isolated soy protein. The diets were isonitrogenous. The urea- and isolated soy-supplemented diets contained 74 and 63.8% wood pulp, respectively, to compensate for urea and isolated soy weight differences. Each diet contained 12.8% corn starch. The steers were fed equal portions of the diet twice daily in amounts at each feeding equivalent to 0.65% of the steer's body weight. The diets were fed to the steers for 3 weeks before sampling the rumen ingesta. The eight strains of cellulolytic cocci used in this study were chosen from the 63 cellulolytic bacteria isolated because they were among the most active in causing the disappearance of cellulose in broth medium. The strains were isolated from 10-8 dilutions of rumen ingesta into nonselective medium 98-5 of Bryant and 737

2 738 SLYTER AND WEAVER APPL. MICROBIOL. Robinson (8). The anaerobic roll tube technique of Hungate (17) was used to isolate the bacteria. The strains were presumptively identified as Ruminococcus flavefaciens by the procedure of Bryant et al. (4, 10), except that a modified ph glucose medium was used (22). The starch cellulose broth medium, used to detect cellulolytic activity among bacteria isolated in pure culture, contained the same ingredients as the rumen fluid-glucose-cellobiose-agar (RGCA) medium of Bryant and Burkey (5), except that agar, glucose, and cellobiose were excluded and 0.1% ball-milled cellulose, 0.05% starch, and 0.1% Trypticase were included. Strains 1607, 1615, 1625, and 1734 were isolated from the steer fed the purified diet in which urea supplied the dietary nitrogen, and strains 1578, 1593, 1664, and 1708 were isolated from the steer fed the isolated soy protein-supplemented diet. Nutritional studies. All media were prepared and inoculated under CO2 by use of the anaerobic technique of Hungate (17). Cells in the log phase of growth, in the basal medium plus 10% rumen fluid, were centrifuged and resuspended twice in the dilution fluid of Bryant and Burkey (5) which had been modified to exclude resazurin. Samples (0.1 ml) of a cell suspension, with an optical density (OD) of 0.1 at 600 nm in tubes (13 by 100 mm), were used to inoculate duplicate 4-ml volumes for growth factor determinations. Growth was assayed as the turbidity measured at 600 nm in tubes (13 by 100 mm) during 1 week of incubation at 38 C. The basal medium contained (in grams/100 ml of medium): cellobiose, 0.3; vitamin-free Casitone (Difco), 0.4; (NH4)2S04, 0.066; Na2CO3, 0.4; KH2PO4, 0.09; NaCl, 0.09; CaCl2, 0.002; MgCl2* 6H20, 0.002; MnCl2-4H20, 0.001; CoCl2-6H20, 0.001; hemin, ; resazurin, ; cysteine hydrochloride, 0.05; Na2S 9H20, 0.05; thiamine hydrochloride, ; calcium D-pantothenate, ; nicotinamide, ; riboflavine, ; pyridoxine hydrochloride, ; p-amino benzoic acid, ; biotin, ; folic acid, ; and vitamin B12, The basal medium solution, minus cysteine hydrochloride, Na2S 9H20, vitamins, and Na2CO3, was prepared and adjusted to ph 6.5, boiled under C02, and autoclaved for 15 min at 120 C and 15 psi. Cysteine hydrochloride, Na2S 9H20, vitamins, and Na2CO3 were prepared and added separately to the autoclaved medium by the procedure of Bryant and Robinson (7), except that the reducing solution was added before the medium was tubed. In experiments 1 to 3, the test media were prepared by adding the test compounds before the ph of the media was adjusted to 6.5. The same was true in experiment 4, except for medium which contained the additional vitamin mixtures. In this medium, the vitamins were added separately after the basal medium was autoclaved. All strains were inoculated into basal medium in experiment 1. The strains were also inoculated into media into which the following test ingredients had been added to the basal medium: (i) the residue from the clarified rumen fluid which was steam-distilled at ph 3 for 48 hr and neutralized to ph 6.5 with NaOH before it was added to the basal medium at a final concentration of 10%; (ii) the above residue plus a complete fatty acid mixture which contained (in millimoles/100 ml of medium) acetic, 4.0; propionic, 1.3; butyric, 0.65; caproic, 0.64; isobutyric, 0.13; 2-methylbutyric, 0.13; isovaleric, 0.13; n-valeric, 0.13; and phenylacetic, 0.13; (iii) 10% clarified rumen fluid prepared as the clarified rumen fluid 1 (CRF1) of Bryant and Robinson (8); and (iv) the complete fatty acid mixture. In experiment 2, in addition to clarified rumen fluid and complete fatty acid mixture media, the following test media were prepared by adding one of three acid mixtures (millimoles/100 ml of medium) to the basal medium: straight chain mixture, acetic, 4.0; propionic, 1.3; butyric, 0.65; caproic, 0.64; branched-chain mixture, acetic, 4.0; isobutyric, 0.13; 2-methyl-butyric, 0.13; isovaleric, 0.13 ; and branched-chain mixture with n-valeric and phenylacetic, acetic, 4.0; isobutyric, 0.13; 2-methyl-butyric, 0.13; isovaleric, 0.13; n-valeric, 0.13; and phenylacetic, Although each of the test media contained acetic acid, they also contained other combinations of fatty acids and were named, for convenience of discussion, on the basis of these differences. In experiment 3, culture fluid was obtained for media from a fermentor which had been inoculated with ruminal contents and incubated as a continuous culture for more than 3 weeks. During the period of continuous culture, the culture received a nitrogenfree purified diet and was sparged with nitrogen gas (L. L. Slyter et al., J. Animal Sci. 27: 1510). The culture contents were prepared as clarified culture fluid by the same procedure used to prepare CRF 1 (8). The clarified culture fluid was added to give final concentrations of 5, 10, 20, and 40% in the media. In experiment 4, the basal and complete volatile fatty acid (VFA) media were prepared as negative controls. Media were also prepared which contained the following test compounds: (i) residue from clarified rumen fluid which was steam-distilled at ph 3 for 4 hr and added to give a final residue concentration of 10%; (ii) distillate from the acidified clarified rumen fluid which was distilled for 4 hr and which was added to the basal medium to give a final concentration of 10%; (iii) the residue and the distillate, each of which was added to give a final concentration of 10% in the medium; (iv) 10% CRF; (v) 40% clarified culture fluid; (vi) 0.5% yeast extract; (vii) 0.002% adenine and guanine; and (viii) solutions to give a final concentration of 0.002% each of vitamin A, vitamin D, DL-a-tOcopherol, lipoic acid, inositol, and ascorbic acid. The first four ingredients were prepared as an ethanolic mixture and were added in 0.1-ml amounts to empty, sterile culture tubes (13 by 100 mm). The ethyl alcohol was evaporated away by flushing the tube with CO2 before the rest of the medium was added. The latter two ingredients were added as an aqueous filter-sterilized solution with the basal medium. Chemical determinations. The completeness of removal of the branched-chain fatty acids from the clarified rumen fluid residue by steam distillation was determined by gas chromatography (22). Although the branched-chain acids were removed from the residue much earlier, the steam distillation was continued

3 VOL. 17, 1969 GROWTH FACTOR REQUIREMENTS OF CELLULOLYTIC BACTERIA 739 for 48 hr to remove most of the steam VFA. Even after 48 hr of distillation, small quantities of acetic acid remained with the residue. Deoxyribonucleic acid (DNA) determinations were conducted as previously described (21), except that the contents were extracted three times for DNA rather than once. RESULTS AND DISCUSSION The substitution of branched-chain fatty acids, the complete mixture of fatty acids, the residue from clarified rumen fluid which had been acidified and steam-distilled, or a combination of the fatty acids and the residue did not replace the clarified rumen fluid requirement for strains 1607 and 1625, isolated from the steer fed the urea-supplemented purified diet, or for strains 1593 and 1664, isolated from the steer fed the isolated soy protein-supplemented purified diet (Table 1). Nor did the distillate and/or residue from the acidified steam-distilled clarified rumen fluid satisfy the nutritional requirements of three of these strains (Table 2). Other compounds which failed to support good growth were adenine and guanine, lipoic acid, inositol, menadione, DL-a-tocopherol, vitamins A and D, ascorbic acid, and yeast extract (Table 2). Clarified culture fluid, in final concentrations of 20 and 40%, allowed relatively good growth of these strains (Table 2). The culture which provided this fluid was maintained in vitro and contained no protozoa. This suggests that the unidentified growth factor(s) in the animal may have been provided for these bacteria by other ruminal bacteria. The possibility also exists that the ruminant animal or the ruminal protozoa, or both, were major contributors of the growth factor(s). We have little evidence to indicate whether strains 1593, 1607, or 1625 require the nonvolatile acidic material of rumen fluid found essential for growth of a strain of R. albus by D. W. Fletcher (Ph.D. Thesis, State College of Washington, Pullman, 1956). The growth factor(s) may be non-volatile since the distillate, in final concentrations as high as 40% in the basal medium, did not support growth of these strains. Presumably the unidentified factor was destroyed by the acidsteam distillation. Neither guanine nor adenine, which Ayers (3) found essential for growth of a strain thought to be R. flavefaciens, satisfied the nutritional requirements of our strains (Table 2). The growth rates of strains 1578 and 1708, from the steer fed soy protein, were stimulated by a branched-chain fatty acid mixture (Table 1), but these strains did not require it for growth. In TABLE 1. Growth response of ruminal cellulolytic cocci isolated from cattle fed urea- or isolated soy protein-supplemented purified diets Growth of strains (OD X 100) isolated6 from steers fed Addition to basal medium5 Urea Soy Experiment 1 No addition... 2(168) 1(168) 0(168) 2 (168) 41 (168) 4 (168) 1 (168) 3 (68) Complete fatty acid mixture 2(41) 58 (34) 0(168) 22(49) 73(34) 4(168) 1 (168) 48 (41) Clarified rumen fluid... 50(52) 50(63) 47(50) 11(70) 76 (26) 56 (67) 30(160) 61 (41) Residue from acid-steam distillate... 0(168) 1(168) 0(168) 1(168) 31(168) 2(137) 1 (168) 1(45) Residue + complete fatty acid mix (168) 58 (34) 0(168) 20 (53) 77 (30) 3 (168) 1 (168) 40(53) Experiment 2 Normal straight-chain fatty acids. 1 (168) 8 (168) 35 (82) 54(120) Acetic + branched-chain fatty acids.68(28) 38(36) 62(24) 42(52) Straight + branched + phenylacetic.65 (26) 35 (36) 61 (24) 44(48) Complete fatty acid mixture. 68(28) 23 (33) 68 (24) 44(52) Clarified rumen fluid 68(26) 27(62) 63(26) 55(26) a The levels added were 10% clarified rumen fluid or an amount equivalent to that present in that quantity of rumen fluid, with the exception of fatty acids which were added in the quantities indicated in Materials and Methods. The parentheses enclosed the number of hours of incubation required for a strain to reach maximum growth.

4 740 SLYTER AND WEAVER APPL. MICROBIOL. TABLE 2. Growth response of ruminal cellulolytic cocci isolated from cattle fed urea or isolated soy protein supplemented diets Addition to basal medium Growth of strains (OD X 100)a isolated from steers fed Urea Soy (1593) Experiment 3 Clarified culture fluid 5% 0 4(47) 2(143) 10% 6(96) 11(110) 5(72) 20% 32(89) 37(49) 16(96) 40% 37 (49) 35 (63) 19 (96) Experiment 4b Adenine + guanine 0 2(71) 4(65) Vitamin mix Acid-steam distillate (ASD) ASD + residue from ASD Residue from ASD % Yeast extract 8 (97) 9 (116)1 1(116) a The parentheses enclose the number of hours of incubation required for a strain to reach maximum growth. b No growth was obtained in medium which contained branched-chain fatty acids in this experiment. The maximum growth of strains 1607, 1625, and 1593 in 10% clarified rumen fluid plus basal medium, presented as OD X 100, was 40, 32, and 35, respectively. These same strains grew to a maximum OD X 100 of 16, 21, and 10, respectively, in 40% clarified culture fluid. The final concentration of the different ingredients added to the basal medium are indicated in the methods section. comparing the growth of strain 1708 in experiments 1 and 2, a straight-chain fatty acid growth factor is indicated. It could be argued that, since rather large amounts of acids were added, one or more of the straight-chain acids were contaminated with a branched-chain acid. However, there was no indication of contamination with a branched-chain acid when the individual straightchain acids were assayed by gas chromatography. Presumably, the amino acids in Casitone may have spared any branched-chain acid requirement for growth of strain Since chemically defined medium minus Casitone was not included, it is not possible to rule out that this strain had limited ability to synthesize the branched-chain carbon skeletons essential for synthesis of the required amino acids, aldehydes, or long branched-chain acids. However, there was no growth by strain 1578 when it was inoculated into medium in which Casitone was replaced by a 2% final concentration of the urea purified diet. The failure to note DNA increases in this medium suggests that this strain does not have the ability to synthesize the required branchedcarbon skeletons. DNA content was used to measure growth instead of OD, because the cellulose of the diet was not soluble in the basal medium. Therefore turbidity could not be used as a growth measurement. The addition of branched-chain acids to the medium allowed growth of the strain, as indicated by an increase in DNA content. These results are interpreted to suggest that strain 1578 could not synthesize the required branched acids and that there was no appreciable amount of the required branchedchain fatty acid(s) contaminating the urea purified diet. That the concentration of contaminating amino acids in the diet is low is indicated by the report of Oltjen et al. (R. R. Oltjen, J. Animal Sci., in press), who found that amino acid nitrogen comprised only 0.4% of the total nitrogen of a urea-supplemented purified diet which contained the same ingredients, although in somewhat different proportions, as the urea-supplemented purified diet used in this study. The two remaining strains from the steer fed urea, 1615 and 1734, required the branched-chain fatty acids for growth (Table 1). At the time the ruminal samples were obtained (4 hr after the steers were fed), the combined concentration of isobutyric, isovaleric, and 2-methyl butyric was 0.6 and 3.2 pmoles per ml of ruminal ingesta for the steers fed the purified diets which contained urea and isolated soy protein, respectively. In spite of the reduced levels of ruminal branchedchain acids in cattle fed urea purified diets, the acids were present in quantities above the minimum required for good growth of ruminal cellulolytic bacteria in batch culture. The minimal concentration of fatty acids which allowed good growth of ruminal cellulolytic bacteria in pure culture has been reported to be between 0.1 to 0.3 (2, 6) and 0.01 and 0.1 jimoles per ml (13). Ruminal bacteria which are noncellulolytic have also been shown to have a nutritional requirement for branched-chain fatty acids (9, 23), although the minimal concentration of branched-chain acids which allows good growth has not been established. However, the minimal concentration of branched-chain acids which allows good growth of cellulolytic bacteria in batch culture may not reflect the minimal requirements for optimal growth of bacteria in continuous culture in the ruminant. Although the data from the present study do not answer this question, they clearly indicate that the amino acids, known to be precursors of the branchedchain fatty acids, do not have to be provided by

5 VOL. 17, 1969 GROWTH FACIOR REQUIREMENTS OF CELLULOLYTIC BACTERIA 741 the diet, per se, in order to maintain large numbers of the cellulolytic bacteria which require the branched-chain acids for growth. There are reports indicating that cellulose digestion in ruminants is stimulated by the addition of branched-chain acids (11, 15). However, increased cellulose digestion by the ruminant has not always been obtained after the addition of the branched-chain acids to the ruminant's diet (11, 16). Growth factors for the cellulolytic bacteria isolated in the present study were produced by organisms grown in mixed culture. This is evidence for a symbiotic relationship between the two groups of bacteria. The exact nature of the relationship, however, needs to be established. Work is also required to determine whether changes in the composition of the diet fed to the ruminant affect the production of bacterial growth factors within the rumen. This type of information would be useful if specific dietary entities could be shown to increase the availability of particular growth factors. Presumably, the application of dietary changes could then be used to increase the availability of growth factors which would otherwise be present in rate-limiting concentrations for optimal growth of bacteria in the rumen. ACKNOWLEDGMENTS We thank R. R. Oltjen for making available the diets and animals from which the bacteria in this study were obtained. LITERATURE CITED 1. Allison, J. M., M. P. Bryant, and R. N. Doetsch Volatile fatty acid growth factor for cellulolytic cocci of bovine rumen. Science 128: Allison, M. J., M. P. Bryant, and R. N. Doetsch Studies on the metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. L. Incorporation of isovalerate into leucine. J. Bacteriol. 83: Ayers, W. A Nutrition and physiology of Runinococcus flavefaciens. J. Bacteriol. 76: Bryant, M. P., B. F. Barrentine, J. F. Sykes, I. M. Robinson, C. V. Shawver, and L. W. Williams Predominant bacteria in the rumen of cattle on bloat-provoking ladino clover pasture. J. Dairy Sci. 43: Bryant, M. P., and L. A. Burkey Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36: Bryant, M. P., and R. N. Doetsch Factors necessary for the growth of Bacteroides succinogenes in the volatile acid fraction of rumen fluid. J. Dairy Sci. 38: Bryant, M. P., and I. M. Robinson Some nutritional requirements of the genus Ruminococcus. Appl. Microbiol. 9: Bryant, M. P., and L M. Robinson An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44: Bryant, M. P., and L. M. Robinson Some nutritional characteristics of predominate culturable ruminal bacteria. J. Bacteriol. 84: Bryant, M. P., I. M. Robinson, and I. L. Lindahl A note on the flora and fauna in the rumen of steers fed a feedlot bloat-provoking ration and the effect of penicillin. Appl. Microbiol. 9: Cline, T. R., U. S. Garrigus, and E. E. Hatfield Addition of branched- and straight-chain volatile fatty acids to purified lamb diets and effects on utilization of certain dietary components. J. Animal Sci. 25: Dehority, B. A., R. R. Johnson, 0. G. Bentley, and A. L. Moxon Metabolism of valine, proline, leucine, isoleucine by rumen microorganisms in vitro. Arch. Biochem. Biophys. 78: Dehority, B. A., H. W. Scott, and P. Kowaluk Volatile fatty acid requirements of cellulolytic rumen bacteria. J. Bacteriol. 94: El-Shazly, K Degradation of protein in the rumen of the sheep. I. The action of rumen micro-organisms on amino acids. Biochem. J. 51: Hemsley, J. A., and R. J. Moir The influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep. Aust. J. Agr. Res. 14: Hungate, R. E., and I. A. Dyer Effect of valeric and isovaleric acids on straw utilization by steers. J. Animal Sci. 15: Hungate, R. E The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14: Matrone, G., C. R. Bunn, and J. J. McNeill Study of purified diets for growth and reproduction of the ruminant. J. Nutr. 86: Oltjen, R. R., and P. A. Putnam Plasma amino acids and nitrogen retention by steers fed purified diets containing urea or isolated soy protein. J. Nutr. 89: Orskov, E. R., and R. R. Oltien Influence of carbohydrate and nitrogen sources on the rumen volatile fatty acids and ethanol of cattle fed purified diets. J. Nutr. 93: Slyter, L. L., W. 0. Nelson, and M. J. Wolin Modifications of a device for maintenance of the rumen microbial population in continuous culture. Appl. Microbiol. 12: Slyter, L. L., and P. A. Putnam In vivo vs. in vitro continuous culture of ruminal microbial populations. J. Animal Sci. 26: Wegner, G. H., and E. M. Foster Fatty acid requirements of certain rumen bacteria. J. Dairy Sci. 43: