[57] FERMENTATION IN THE RUMEN OF THE SHEEP

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1 [57] FERMENTATION IN THE RUMEN OF THE SHEEP IV. THE NATURE AND ORIGIN OF THE VOLATILE FATTY ACIDS IN THE RUMEN OF THE SHEEP BY F. V. GRAY, A. F. PILGRIM, H. J. RODDA AND R. A. WELLER From the Division of Biochemistry and General Nutrition of the Commonwealth Scientific and Industrial Research Organization, University of Adelaide, South Australia {Received 3 May 1951) Early workers in the field of ruminant physiology were aware of the presence of formic, acetic, propionic and butyric acids (Tappeiner, 1884), and of smaller amounts of higher members of the saturated fatty acid series (Mangold, 1934), in the rumen fluid. Accurate data were not reported, however, until recently, when partition chromatography became available as an analytical procedure for the analysis of complex mixtures of these acids (Elsden, 1946). By this means acetic, propionic and butyric acids were established as the main components of the mixture of fatty acids in the rumen fluid of sheep and of other ruminants. The butyric acid fraction, however, was thought to contain some higher acid or acids, and it was claimed that formic acid was not a component of the mixture (Elsden, 1945). Using modifications of the older partition and distillation methods Gray (1946) found similar proportions of the three main acids, but also stated that small amounts of formic acid were normally present. Earlier communications in this series (Gray, Pilgrim & Weller, 1951; Gray & Pilgrim, 1951) dealt with the production of acetic, propionic and butyric acids during the rumen fermentation in vitro and in vivo. A more detailed analysis of the fatty acids of the rumen fluid is reported in the present work, and an attempt has been made to trace the origins of some of these acids by incorporating 14 C-labelled acetic and propionic acids into the inoculum used to initiate the fermentation of wheaten hay and lucerne hay in vitro. The investigation was prompted by the observation that when such labelled acids were introduced into the normal rumen, some of the labelled carbon appeared later in the ' butyric' fraction of the fatty acid mixture. EXPERIMENTAL (1) The procedures used in the collection of rumen fluid and of fermentation liquor, and the method employed for separation of the fatty acids from other substances have been described in an earlier publication (Gray et al. 1951). (2) Methods for the separation, identification and determination of the saturated fatty acids Q to C,. The acids were separated from one another by partition chromatography, using three different columns: Column 1. For separation into (a) formic, (b) acetic, (c) propionic and (d) the

2 58 F. V. GRAY AND OTHERS remaining higher acids. Preparation and use of the column have already bee"ff described (Gray et al. 1951). Column 2. For separation of the group of higher acids from the first column into (a) butyric, (b) valeric and (c) higher acids. The column was prepared in two parts, separation being effected in the upper, buffered portion (cf. Moyle, Baldwin & Scarisbrick, 1948). The lower portion of the column was similar to column 1, but the indicator used was Brom-cresol purple. Immediately before use, dilute ammonia was added to a solution of 0-2 mg./ml. of this indicator, in just sufficient quantity to give a purple colour; 1-5 ml. of the resulting solution was added to 2-5 g. of ' Celite' in a mortar and mixed thoroughly and rapidly. Developing solvent (BB7-5, as used in column 1) was added to form a slurry before sufficient atmospheric CO 2 had been absorbed to change the colour from purple to yellow. The mixture was packed in the column (12 mm. diameter) to a height of 5 cm. The upper portion of the column was made by mixing 2 ml. of 2M-K2HPO 4 and 1 ml. of 2M-KH 2 PO 4 with 5 g. celite. A slurry was prepared with BBy-5 and the column packed to a height of 10 cm. above the indicator portion. The column was made ready for use by passing through it a mixture of acids of approximately the same composition as the mixture to be examined. Butyric, valeric and the group of higher acids became visible as yellow bands passing through the lower portion of the column. The eluates were titrated with N/100-barium or sodium hydroxide in a CO g -free atmosphere, using phenol red as indicator. Column 3. For separation of the higher acids eluted from column 2, into (a) caproic and (b) heptoic and higher acids. This column differed from the previous one only in the reaction of the buffered portion, which contained am-k^elpc^ alone. Preparation of acids for columns 2 and 3. The group of acids eluted from column 1 was neutralized (phenol red) with sodium or barium hydroxide and taken to dryness on a steam-bath in a 10 ml. centrifuge tube. Ten drops of wet BB75 were added, and then sufficient dry powdered potassium bisulphate was stirred in to produce a pink colour in the mixture. After centrifuging for a short time, a small bulbpipette was used to transfer the liquid to the column. The extraction was repeated about ten times, using sufficient BB 7-5 to give a total volume of extract of about 1 ml. Behaviour of pure adds on columns 2 and 3. Approximate determinations of R values were made by measuring the total movement of the surface of the developer required for the leading edges of the bands to reach the centre of the indicator portion of the column. As these R values varied considerably with acid concentration, the relative amounts of pure acids used were of the same order as those found in rumen fluid. Column 2. R determinations on pure acids and mixtures showed that the first rapidly eluted band contained caproic and higher acids. The n-valeric band appeared next, followed by the other valeric isomers. These isomers could not be clearly separated unless concentration relationships were ideal. The butyric acids appeared well behind the valeric band, but as the R values of iso- and n-butyric acids were almost identical, no separation could be obtained. Propionic acid was firmly held in the buffered portion of the column.

3 Fermentation in the rumen of the sheep 59 Column 3. Amounts greater than o-6 mg. of heptoic acid were necessary to produce a visible band on the column. This band was eluted well ahead of the caproic band and excellent separations were possible. Valeric acid moved very slowly through the buffered column and was not easily eluted. Recovery of acids. Some acid is not recovered in the first use of these celite columns, and they should therefore be prepared for use by passing through them a mixture of acids similar in composition to the mixture to be separated. Even with this precaution recoveries varied from 86 to 96 % on column 2 and from 67 to 100% on column 3. They were, however, very nearly complete on column 1. Identification and determination of iso-butyric acid. The presence of iso-butyric acid in the mixture of the two butyric acid isomers was demonstrated by applying the test described by Ramsey & Patterson (1945) in which iso-butyric acid is oxidized to acetone by acid permanganate in the presence of silver sulphate. The acetone, after distillation, gives a red colour with salicylaldehyde. This procedure, when carried out under strictly standardized conditions, provided a quantitative colorimetric determination of iso-butyric acid. Pure /z-butyric and iso-butyric acids were used to prepare a series of standards in which the varying quantity of iso-butyric acid amounted to 5 % of the total butyric acid in each case. The standards, together with a blank and the salt under test were oxidized and distilled, and the distillates used simultaneously to develop the colours with salicylaldehyde. After cooling, the colour densities were measured in a spectrophotometer (wave-length 530 m/i) and the amount of iso-butyric acid in the salt determined graphically. The presence of barium did not affect the acetone yield. Formic acid in the lowest boiling fraction of the mixture of acids present in the steam distillate from rumen fluid was identified by its conversion to formaldehyde and the colour reaction with chromotropic acid (Feigl, 1939). Estimation by this procedure (Grant, 1948) and by a gravimetric method involving reduction of mercuric chloride (Scott, 1925) gave similar results. (3) Incorporation of li C-labelled acetic and propionic acids in the rumen fermentation in vitro. The procedure for reproducing the rumen fermentation of wheaten hay and lucerne hay, in vitro, has already been described (Gray et al. 1951). In the present experiments, 25 g. of the fodder were used and approximately 750 ml. of rumen fluid were employed as inoculum. To this inoculum was added a small amount of either acetic or propionic acid labelled with 14 C in the carboxyl carbon. The active acids were prepared by carbonation of the Grignard reagent in the usual manner. In the majority of the experiments enough of the labelled acid was added to give an activity of approximately 200 counts/min./ml. of inoculum, after plating out in the manner described below; in two of the experiments a much larger amount of the active acid was added. Six of the fermentations were made with wheaten hay as substrate and one with lucerne hay. (4) Measurement of the activities of the acids. The neutral solutions obtained by titration of the acid fractions from the chromatographic columns were brought to dryness on a water-bath. The residues were taken up in exactly 2 ml. of water and aliquots (0-5 ml.) of these solutions were spread uniformly on plates of oxidized

4 Table I. Fermentations in the presence of CH3WOOH or CH3CH,1'COOH. The fatty acids and their actiwities in the nunen fluid used as i h, and in the fmtation liquor remaining at the end of the fermentation in vitro The same type of fodder, but not the same sample, wan fed to the sheep from which the i nda were taken. t Given in black figures. Fodder fermented' Fluid analyaed Acetic acid Acids present (rnl. N/IOO volatile acidlml. of fluid) Activitica of acida (wuntslmin.lml. of fluid)t Propionic acid Butyric acid Valeric acid Caproic and higher acids, aarnple I Wheatem hay, sample I, sample I, aample 2 Lucerne hay Whwten hay, aample I, aample 2 Inoculum Inmlum Inoculum Inoculum Inoculurn Inoculurn Inoculum 3' Nil f " yr I Nil Nil

5 Fermentation in the rumen of the sheep 61 Bopper 2-3 cm. in diameter with a central depression of 1-9 cm. diameter. The contents of the plates were evaporated to dryness on a hot plate ( C.) and finally heated in an air oven at C. for 15 min., and the prepared plates were kept in a desiccator over CaCl 2 until counted. Preliminary trials indicated that reproducible films could be obtained in this way. Counting was carried out using a shielded end-window G-M tube (background 8 counts/min.) which was standardi2ed during each series of counts against a standard of uranium glass. Corrections for thickness of sample were made in the usual manner from an absorption curve previously prepared. The corrected activities reported are the means of triplicate samples. (5) Analyses. In Exp. 1 the acids were separated into three groups only acetic, propionic, and higher acids. In Exps. 2, 3, 5 and 6 the butyric and valeric acids were separated but the caproic and higher acids were measured as a group, while in Exps. 4 and 7 the final separation of caproic from higher acids was also carried out. In Exps. 3, 5 and 6, the butyric fraction was recovered after its activity had been measured and the proportions of wo-butyric and n-buryric acids were determined. In Exp. 5 two isomers of valeric acid were separated, one of which was n-valeric acid. Formic acid was not determined in any of the in vitro experiments. (6) Results. The amounts and activities of the fatty acids in the inocula and in the liquors remaining at the end of the fermentation period are summarized in Table 1. The molecular proportions of the individual acids in the rumen fluid used as inocula in these experiments are given in Table 2. This table also includes the further analyses of the butyric, valeric and caproic fractions. Table 2. Molecular proportions of fatty acids in the rumen fluid 1-2 hr. after feeding Exp. no. Fodder Acetic Propionic Butyric Valeric Caproic and higher acids Further separations i Lucerne hay ' i-o ' i-6 2-2,. 0-4 i-o i-o n-butyric, 8-6%; tiobutyric, 0-3 % 'Heptoic', 0-04%* n-butyric, 6-i %; itobutyric, o-6%; n- valeric, 2-1 % n-butyric, 8-i %; isobutyric, 0-4% 'Heptoic', 0-05%* Approximate values. DISCUSSION (1) The nature of the fatty acids. The experiments have established the presence of formic, acetic, propionic, n-butyric, wo-butyric, valeric, caproic and probably heptoic acid in the rumen fluid of sheep fed on wheaten hay or on lucerne hay.

6 62 F. V. GRAY AND OTHERS In the case of lucerne hay the valeric acid was present as n-valeric and another isome the identity of which was not finally established. (2) The proportion of each acid in the rumen fluid. The proportions in which the various acids are present in the rumen fluid are shown in Table 2. It must be stressed that these proportions were found in fluid drawn soon after feeding, for it has already been reported (Gray & Pilgrim, 1951) that the composition of the mixture changes subsequent to feeding, the proportion of propionic acid increases and that of acetic or butyric acid decreases for some hours, after which these changes are reversed and the mixture gradually returns to its original composition. The presence of formic acid was again established, but it was not determined quantitatively in the in vitro experiments. However, numerous analyses of rumen fluid (Gray & Pilgrim, ) have shown that it may constitute 0-5% of the total fatty acid present. In three of the experiments, the wo-butyric acid was determined and found to vary from 3 to 8 % of the total butyric acid production. In the experiment in which lucerne hay was fermented, the valeric acid contained a fraction identified by its R value to be n-valeric, the quantity of which accounted for over 60 % of the total valeric acid present. In Exps. 4 and 7 the caproic acid was separated from a faster moving acid which may have been heptoic acid. The amount of this last acid was quite small, sufficient to titrate but showing as a definite band only in Exp. 7. It was present to the extent of about io % of the caproic acid. The composition of the volatile acids of the rumen fluid* may be summarized as follows: Formic acid Acetic acid Propionic acid n-butyric acid o- 5 62^ ito-butyric acid Valeric acid Caproic acid 'Heptoic' acid 0-3 -o O'04-o-os (3) The origin of the fatty acids. The intermediate steps leading to the formation of volatile fatty acids in the rumen have as yet received little consideration. Many organisms have been subcultured from rumen material and their attack on cellulose in vitro has been examined, with varying results, but in view of our more recent knowledge of the organisms responsible for cellulose digestion in the rumen most of this work may be disregarded (cf. Sypesteyn, 1948). However, the production of acetic and propionic acids from pure cellulose by rumen organisms in vitro has been demonstrated (Elsden, 1945; Marston, 1948), and two organisms isolated from the rumen, which have strong claims to be active cellulose fermenters there, have It is interesting that the feeding of two very different fodders such as lucerne hay and wheaten hay should lead to much the same mixture of acids in the rumen, for there is strong evidence that the proportions of various acids actually produced are by no means the same with these two substrates (Gray et al. 1051). The same phenomenon has been noticed by other workers (Elsden, Hitchcock, Marshall & Phillipson, 1946; McCIymont, 1051) for fodders of even wider difference; moreover, it has been shown that the same general composition applies for different animals and for different parts of the alimentary tract where plant structures are digested. It is a matter for speculation whether this miiture represents an equilibrium brought about by the factors which govern the relative rates of absorption of the acids through the wall of the gut. There is a clear but maybe quite fortuitous parallel between the proportions of the acids in the mixture and their distribution coefficients between oil and water.

7 Fermentation in the rumen of the sheep 63 n studied in detail (Sypesteyn, 1948). One of these organisms was shown to produce from cellulose a mixture of propionic and acetic acids, and the other a mixture of succinic and acetic acids. Later it was demonstrated (Johns, 1949; Elsden & Sypesteyn, 1950) that succinic acid is readily decarboxylated to propionic acid either by suspensions of rumen organisms, or when introduced into the rumen itself. Table 3. Relative activities of the acids in the fermentation Exp. no.* Fodder Lucerne hay Acetic Propionic Butyric Valeric Nil o no Nil Nil The mean molar activities of the originally labelled acids have been given the value of ioo and the molar activities of the other acids formed in the fermentation have been calculated accordingly. It is notable that there is no evidence for the production of significant amounts of fatty acids higher than propionic, from cellulose. The present experiments suggest that a very considerable part of the higher fatty acids in the rumen fluid may be produced by secondary reactions from both acetic and propionic acids. It will be seen from the data in Table 1 that at the end of the fermentation some of the carbon originally present in the carboxyl group of acetic acid is distributed among all of the higher fatty acids that were examined, while part of the carboxyl carbon of propionic acid appears in valeric but not in butyric acid. The extent to which this occurs is best shown by comparing the specific (i.e. molar) activities of the acids formed during the fermentation with the mean molar activity of the acid originally labelled with 14 C. The comparison is made in Table 3 where the mean molar activity of the acetic acid in Exps. 2-5 and of the propionic acid in Exps. 6 and 7 is given the value of 100 in each case. It will be seen from this table that when labelled acetic acid was added to the inoculum the molar activity of the butyric acid produced in the fermentation averaged 90 %, and the valeric acid 49 %, of the mean activity of the acetic acid. When labelled propionic acid was included in the inoculum, however, the butyric acid formed contained no active carbon at all, while the valeric acid was active to the extent of 40 % of the mean molar activity of the propionic acid. These findings suggest very strongly that a large part of the butyric and valeric acids may be formed from the lower acids by the mechanism which has recently been studied in considerable detail in the synthesis of higher volatile fatty acids by Clostridium kluyveri (Bornstein & Barker, 1948; Stadtmann, Stadtmann & Barker, 1949); further work would be necessary, however, to establish this as a fact. It should be noted, incidentally, that some of the carboxyl carbon of acetic acid regularly appeared in the propionic acid formed, and that practically all (90-100%) of the labelled carbon in acetic acid was recovered in the various fatty acids at the

8 64 F. V. GRAY AND OTHERS end of the fermentation. Rather less (64-83 %) of the 14 C in propionic acid recovered. The presence of small amounts of isomers of n-butyric and n-valeric acid is interesting for it is known that they occur as products of the amino-acid metabolism of a number of anaerobes of the Clostridium genus (Cohen-Bazire, Cohen & PreVot, 1948). They might therefore arise from proteins in the rumen and no doubt some of the normal acids could have a similar origin. Finally, it is to be expected that the dissimilation of polysaccharides other than cellulose will account for a considerable part of the fatty acids produced in the fermentation of plant structures in the rumen. SUMMARY 1. The mixture of volatile fatty acids in the rumen of the sheep has been shown to include formic acid, acetic acid, propionic acid, n-butyric acid, wo-butyric acid, n-valeric acid, another valeric acid isomer, caproic acid and an acid which is probably heptoic acid. The proportions in which they are present have been determined. 2. When acetic acid labelled with 14 C in the carboxyl group was incorporated in the rumen fermentation in vitro, active carbon appeared later in all the higher acids. When labelled propionic acid was included in the fermentation, active carbon appeared in the valeric but not in the butyric acid. The results suggest a synthesis of the higher acids by condensation of the lower ones with 2-C compound in equilibrium with acetic acid. The extent of such syntheses and other possible modes of origin of the fatty acids are discussed. The authors are indebted to the Chief of the Division, Mr H. R. Marston, F.R.S., for his interest in this work and for his criticism and help in the preparation of the manuscript. REFERENCES BORNSTEIN, B. T. & BARKER, H. A. (1948). The energy metabolism of Clostridktm hhtyveri and the synthesis of fatty acids. J. Biol. Chan. 172, COHEN-BAZIRE, G., COHEN, G. N. & PREVOT, A. R. (1948). Nature et mode de formation des acides volatils dans lea cultures de quelques bactiries ana^robies proteoh/tiques du groupe de Clostridium tporogenes. Formation par reaction de Stickland des acides isobutyrique, isovalerianique et valerianique optiquement actif. Ann. Inst. Pasteur, 75, ELSDEN, S. R. (1945). The fermentation of carbohydrates in the rumen of the sheep. J. Exp. Biol. 23., ELSDEN, S. R. (1946). The application of the silica gel partition chromatogram to the estimation of volatile fatty acids. Biochem. J. 40, ELSDEN, S. R. & SYPESTHYN, A. K. (1950). The decarboxylation of succinic acid by washed suspensions of rumen bacteria. J. Gen. Microbiol. 4, xi. ELSDEN, S. R., HITCHCOCK, M. W. S., MARSHALL, R. A. & PHILLIPSON, A. T. (1946). Volatile acid in the digests of ruminants and other animals. J. Exp. Biol. 33, FEIGL, F. (1939). Spot Tests. Second English ed. New York: Nordman Publishing Co. Inc. GRANT, W. M. (1948). Colorimetric microdetermination of formic acid based on reduction to formaldehyde. Anal. Chem. ao, GRAY, F. V. (1946). Thesis. University of Adelaide. GRAY, F. V. & PILGRIM, A. F. (1947-8). Unpublished data. GRAY, F. V. & PILGRIM, A. F. (1951). Fermentation in the rumen of the sheep. II. The production and absorption of volatile fatty acids during the fermentation of wheaten hay and lucerne hay in the rumen. J. Exp. Biol. 38,

9 Fermentation in the rumen of the sheep 65,Y, F. V., PILGRIM, A. F. & WBLLKR, R. A. (1951). Fermentation in the rumen of the sheep. I. The production of volatile fatty adds and methane during the fermentation of wheaten hay and lucerne hay in vitro by micro-organisms from the rumen. J. Exp. Biol. 28, JOHNS, A. T. (1949). Mechanism of propionic acid formation in bacteria. Nature, Land., 164, MCCLYMONT, G. L. (195 I). Identification of the volatile fatty add in the peripheral blood and rumen of cattle and the blood of other spedes. Aust. J. Agric. Res. a, MANGOLD, E. (1934). The digestion and utilization of crude fibre. Nutr. Abttr. Rev. 3, MARSTON, H. R. (1948). The fermentation of cellulose in vitro by organisms from the rumen of sheep. Biochem. J. 4a, MOYLE, V., BALDWIN, E. & SCARISBRICK, R. (1948). Separation and estimation of saturated C,-C«fatty acids by buffered partition columns. Biochem. J. 43, RAMSEY, L. L. & PATTERSON, W. I. (1945). Separation and identification of the volatile saturated fatty adds Cj-C,. J. Aisoc. Off. Agric. Chem., Wash., a8, SCOTT, W. W. (1925). Standard Methods of Chemical Analysis, 4th ed. New York: D. Van Nostrand Co. Inc. SYPESTEYN, A. K. (1948). Thesis. University of Leyden. STADTMAN, E. R., STADTMAN, T. C. & BARKER, H. A. (1949). Tracer experiments on the mechanism of synthesis of valeric and caproic adds by Clostridium kluyveri. J. Biol. Chem. 178, TAPPEINER, H. (1884). Untersuchungen Uber die Gfirung der Cellulose insbesondere Uber deren Losung im Dannkanale. Z. Biol. ao, JIB. 29, 1