Factors Affecting the Cellulolytic Activity of Rumen Contents

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mr. 1977, p Copyright 1977 Americn Society for Microbiology Vol. 33, No. 3 Printed in U.S.A. Fctors Affecting the Cellulolytic Activity of Rumen Contents COLIN S. STEWART Rowett Reserch Institute, Bucksburn, Aberdeen AB2 9SB, United Kingdom Received for publiction 3 August 1976 The cellulolytic ctivity ofrumen contents ws ssyed by mesuring losses in weight nd tensile strength of cotton yrn incubted in rumen contents in the presence of dietry dditives (brley, tllow) nd t different ph vlues. The ddition of brley depressed cellulolysis nd the titer of filter pper-degrding bcteri only if the ph ws llowed to fll. Lowering the ph from 7. to 6. by ddition of HCl lmost completely inhibited ttck of cotton nd gretly reduced the titer of filter pper-degrding bcteri. The lyering of tllow on cotton inhibited ttck of cotton but did not decrese the titer of filter pper-degrding bcteri. The results re discussed with specil reference to the importnce of the study of cellulosic substrtes, which require known enzyme or mixture of enzymes for ttck. The criteri normlly used to define the rumen cellulolytic bcteri re the ttck of filter-pper strips (16) or the clering of filterpper powder (23). Truly cellulolytic microorgnisms, however, re distinguished by the production of n enzyme (C,) tht fcilittes ttck of highly ordered forms of cellulose, such s cotton, by the cellulse complex. Experiments with the cellulse of the fungus Trichoderm hve indicted tht the ttck of filter pper does not require C, (18). It seems likely, therefore, tht filter pper cn be degrded by pseudocellulolytic (s defined by Wood [24]) s well s truly cellulolytic microorgnisms. There hve been number of studies on the ttck of cotton by rumen contents. However, mny of these studies used cotton sewing thred (1, 7, 21), nd it is doubtful if such bleched nd lkli-treted cotton is suitble substrte for the ssy oftrue cellulolytic ctivity (25). It hs been demonstrted, though, tht dewxed ntive cotton fibers cn be degrded by rumen contents (9) nd some, but not ll, strins of cellulolytic rumen bcteri (1). A strin of Bcteroides succinogenes tht hd been shown to degrde cotton fibers (1) ws prticulrly ctive in the degrdtion of forge cellulose when tested in pure culture (4). It seems likely, therefore, tht bcteri tht produce C1 enzymes ply n importnt prt in the degrdtion of cellulose by rumen bcteri, nd the responses of the true cellulolytic ctivity of rumen contents to chnges in environmentl conditions re therefore of specil ecologicl interest. Cotton yrn tht hd hd the minimum chemicl tretment ws incubted in vitro in rumen contents from sheep fed roughge. Dietry dditives (brley, tllow) known to ffect the digestibility of forge cellulose were dded, nd their effect on the bility of rumen contents to cuse losses in weight of cotton ws deternined. The true cellulolytic ctivity of rumen contents deternined in this wy ws compred with the "titer" of bcteri ble to degrde filter pper, to find out whether chnges in true cellulolytic ctivity were correlted with chnges in the totl numbers of"cellulolytic" bcteri. Finlly, weight losses of cotton were compred with losses in tensile strength of the sme smples, since if close reltionship exists between these two prmeters, s some previous studies suggest (19), estimtion of strength losses would be simple lterntive to the determintion of weight losses. 497 MATERIALS AND METHODS Rumen smples. Unless otherwise stted, rumen contents were from cnnulted sheep (no. 283) fed 35 g of hy nd 175 g of dried grss twice dily. The sme niml ws lso used for estimtions in vivo. The contents were tken 2 h fter the morning feed nd were strined through one thickness of surgicl guze before use. Incubtion of rumen contents. Rumen contents (1 ml), or mixture of rumen contents nd rtificil sliv (15) s specified in the text, were incubted for 24 h t 39 C in vessels similr to the culture vessel of Hobson (11), with provision for gssing with 2-free CO2 (1 ml/min), bunsenvlve gs outlet, nd in some cses provision for the mintennce of constnt ph by the utomted ddition of 1 M NOH (11). When required, rumen contents were diluted by ddition to rtificil sliv, which hd been thoroughly gssed with CO2 to ph 6.8 to 6.9. Different ph vlues were chieved by dding the required mount of 1 M HCl to the C2- gssed rtificil sliv before dding the rumen con-

2 498 STEWART APPL. ENVIRON. MICROBIOL. tents. Diluted rumen contents incubted in this wy remined within.1 ph unit of the strting ph. Chnges in weight nd strength of cotton. The cotton yrn (Egyptin Menoufi cotton) ws gift from J. & P. Cots Ltd., Pisley, Renfrewshire, U.K., nd hd been treted by the mnufcturer with cold.2% Lisspol ND (to remove geltin nd oxidizble oils),.2 M HCl, nd.125% N2HPO4. The yrn ws dewxed by successive soks in chloroform nd methnol (7 g of yrn; 7 ml of solvent), wshed in hot wter nd distilled wter, dried t 37 C, nd stored in desicctor. Weighed mounts (c. 3 mg) of cotton yrn, wound into coils nd ttched to glss weights, were incubted in rumen contents in the vessels described bove. For in vivo estimtions, similr coils of yrn were suspended in the rumen for 24 h, using methods similr to those of Blch nd Johnson (1). After incubtion, the yrn ws wshed in the solvents described by Hlliwell (9), dried, nd reweighed. The residul tensile strength of the yrn ws then determined using lbortory-built, tensile-testing mchine. The loss of tensile strength ws clculted by comprison with the strength of controls incubted in sterile rtificil sliv nd wshed nd dried s bove. Tretment of cotton with tllow or ftty cids. Weighed coils of cotton yrn (c. 3 mg) were soked for 3 min in solutions of tllow or ftty cids in 15 ml of chloroform. The treted yrn ws dried with hot ir blower nd reweighed. The required mounts of tllow or ftty cids in the solution were determined by tril nd error; for ech 1 mg of ft in solution, 1 mg ws normlly found to be bsorbed onto the cotton. It ws not possible to obtin ccurcy of greter thn +2% in bsorbing ft onto yrn, so the results in Tble 2 were obtined by interpoltion. Fermenttion products nd vible counts of bcteri. The nlyticl nd culturl methods used were the sme s in previous study (13). Counts of filter pper-degrding bcteri were done by the method of Mnn (16). Chemicls. Chemicls were of Anlr grde (BDH Ltd., Poole, Dorest, U.K.) except K2HPO4, which ws lbortory grde. Higher ftty cids were Puriss (Koch-Light Lbortories Ltd., Colnbrook, Bucks, U.K.), nd tllow ws gift from Isc Spencers Ltd., Aberdeen, U.K. RESULTS Effect of brley. In n ttempt to deternine how much of the effect of brley on the cottonsolubilizing ctivity or rumen contents is result of the cid conditions tht develop on fermenttion of the brley, rumen contents from roughge-fed sheep were incubted with brley in seprte vessels. The contents of one vessel were mintined t ph 6.6; the contents of the second vessel were llowed to fll in ph during the incubtion. A third vessel, contining rumen contents held t ph 6.6 but without dded brley, ws set up s control. The results (Tble 1) of this study show tht mrked. C C.) *S.... C.) C.) Co c) c;4 4.5 _O o.-5) r.l 4g C.s ri X4 X UX o co ttd o > c ~C. co ico4 4 6 cli '-- d; r- V- V- cq; o co ti-l:- Co r-4cq cq. co ) f- f. CD *. Coo _ co. C. ri t- C-. Lo Co.. oo _- ox_qr-s "s c t- C Co IC cos U IC) o ox- N r- eq4-4 oo t6 Do Q XO Lo Co bco- Co Co I CoC CA Co DC~C X _' CoO ) I"4 C t-.. CUV CoO SD X) C) co C- -! to t cwz3 z '4 IC;._ el U: '3 4 4C.. b.ce^ Q 4),t ~H = Im IC ~. -Z4 C 11 4) '. V U) v > 4 -.4) 5 c; 4 e 4 ~ U) V > '4D. C.)1 O 5 : 5)& C4en : 3~. o or 5). n

3 VOL. 33, 1977 depression in cotton-solubilizing ctivity of rumen contents, nd mrked depression in the titer of filter pper-degrding bcteri, ws obtined only if the ph of the brley-supplemented rumen fluid ws llowed to fll. Selective counts of some groups of bcteri tht would be expected to thrive on the ddition of brley nd nlyses of fermenttion products were mde, s n indiction of the extent to which the system used recreted events tht would be expected to occur in vivo on ddition of concentrtes to roughge diet. The ddition of brley incresed the concentrtions of voltile ftty cids (VFA) nd chnged the cette-propionte rtio from bout 4:1 to 2.4:1 independently of ph chnges. Smll nd pproximtely equl mounts of isobutyrte, isovlerte, nd vlerte were found in ll of the smples. The CO2 flow probbly removed some VFA from the brley-supplemented rumen fluid incubted t low ph, nd this my ccount for the VFA concentrtion being lower in this cse thn tht in the brleysupplemented rumen contents t ph 6.6. Although lctte-producing bcteri (prticulrly lctobcilli) incresed in numbers when the ph of brley-supplemented rumen contents ws llowed to fll, lctte ccumulted to only smll extent (Tble 1). There ws n increse in the numbers of the lctte utilizer Veillonell under these conditions. The smple of rumen contents nd brley incubted t low ph lso showed on Veillonell gr lrge number of colonies (corresponding to 9 x 17/ml of rumen contents) of grm-negtive, curved rod similr in morphology to Selenomons. None of the other smples showed these colonies. Microscopic observtions ofthe protozo present in the rumen contents under these conditions showed tht n A-type (5) popultion ws initilly present. A lrge proportion of this popultion (holotrichs, entodini, nd Polyplstron together with flgelltes) survived in the rumen contents incubted t ph 6.6. When the ph ws llowed to fll to 5.2, however, ll of the protozo ppered to die. The increse in bcteril numbers tht occurred when brley-supplemented rumen fluid ws incubted t low ph might hve been consequence of the deth of protozo. However, (qulittively) similr increse in bcteril numbers occurred when rumen contents from cilite-free sheep were incubted under similr conditions. Rumen contents from this cilitefree sheep lso cused weight losses of cotton. Effect of ph. The limited number of vessels vilble for mintennce of ph by utomted ddition of lkli restricted the number of incubtions tht could be studied simultneously. CELLULOLYSIS IN THE RUMEN 499 This led to tests on mixtures of rumen contents nd buffer (rtificil sliv) to find mixture with high ctivity ginst cotton nd miniml fll in ph during incubtion. The results ofthis study re shown in Fig. 1. The ddition of 1 ml of rumen contents to 1 ml of rtificil sliv resulted in the greter weight loss of cotton per milliliter of rumen contents, nd the ph remined with +.1 unit of the strting ph. Mixtures of 1 ml of rumen contents nd 1 ml of rtificil sliv were then used in study of the effect of ph on the cellulolytic ctivity of rumen contents, the results of which re shown in Fig. 2. The decrese in ctivity on lowering the ph ws found to be ccompnied by decrese in the numbers of filter pper-degrding bcteri. Thus, fter incubtion t ph 6.9, the titer of filter pper-degrding bcteri ws 16/.S 15 z I.- o 12 IL 5 I - IF o L I I I I tl zo O4-4 LZO I1 CA U) z U.LW L) 6 wc 5 C, E ml RUMEN CONTENTS ADDED FIG. 1. Cellulolytic ctivity of mixtures of different mounts of rumen contents dded to 1 ml of rtificil sliv nd incubted in vitro. Symbols:, totl cellulolytic ctivity;, cellulolytic ctivity per milliliter of rumen contents. Brs = stndrd errors. o B L _ z 2 - -J I I I OF ph FIG. 2. Effect of ph on the cellulolytic ctivity of rumen contents incubted in vitro. Brs = stndrd errors.

4 5 STEWART ml; fter incubtion t ph 6. the count decresed to 13/ml. The dilution of rumen contents by ddition to rtificil sliv produced lower concentrtion ofvfa thn tht in the originl rumen smple. Incresing the VFA concentrtion in the diluted rumen contents to the level in the originl smp'e (57 mm cette, 17.4 mm propionte, 8.7 nmm butyrte) without lowering the ph decresed the weight losses of cotton by bout 25%. The ddition of 2 mm mixture of brnched-chin ftty cids without lowering the ph (isobutyrte, isovlerte, dl-2-methylbutyrte) lso decresed cellulolytic ctivity, by bout 18%. Effeut of niml tllow nd ftty cids. On incub-.tion in diluted rumen contents of cotton tretea with tllow, it ws found tht the ddition of tllow depressed the ttck on cotton by the rumen microorgnisms (Tble 2). Anlysis of the tllow by gs-liquid chromtogrphy (8) showed tht it consisted principlly of the following cids (percentge, by weight): oleic, 41.2; plmitic, 25.9; steric, 17; linoleic, 4.1; plmitoleic, 2.7; myristic cid, 3. The effect of some of these cids, when dded to cotton t 15% (by weight), re lso listed in Tble 2. At this concentrtion, oleic cid hd the most mrked inhibitory effect. The presence of tllow did not significntly ffect the totl number of rumen bcteri present (17/ml), nd there ws no evidence of n effect on the titer of filter pper-degrding bcteri (16/ml). Tensile-strength losses. The weighed smples of cotton yrn, which hd been incubted in the bove experiments, were tested for their residul tensile strength. Sttisticl nlysis showed tht correltion could be obtined by the formul Y = X -.17X2 between losses in tensile strength (Y) nd weight (X) of cotton yrn. The correltion coefficient ws.87; residul stndrd devition ws 7.7. TABLE 2. Effect of niml tllow nd ftty cids on weight losses of cotton fter incubtion in rumen contents t 39 C for 24 h Tretment (%, by wt) None (control) Tllow, 5 Tllow, 1 Tllow, 15 Steric cid, 15 Plmitic cid, 15 Myristic cid, 15 Oleic cid, 15 Wt loss of cotton (mg) b 51.3c 38.6d 36.9d 36.2d 32. le 6.5' Mens followed by different superscripts re significntly different t P <.1. APPL. ENVIRON. MICROBIOL. Although this is sttisticlly highly significnt reltionship, there ws mrked sctter of the tensile-strength loss vlues corresponding to prticulr weight loss, nd there would not seem to be ny obvious biologicl reson or reltionship of this form. DISCUSSION The system used for incubtion of rumen contents in vitro seemed to ccurtely reproduce conditions in vivo in tht the gross chnges in concentrtion of fermenttion products nd bcteril numbers were consistent with in vivo effects noted previously (12, 17). Direct comprison of the results of the present study with those from previous work is difficult, becuse there re often differences in the form of cellulose or the experimentl conditions used. Even so, the weight loss of cotton in vitro in the present study (up to 1 mg/ml of rumen contents in 24 h) ws considerbly greter thn tht previously reported in comprble experiments (9). It seems tht the correct incubtion conditions re very importnt for demonstrtion of the mximum cellulolytic ctivity of rumen contents in vitro. The most effective cotton-solubilizing rumen bcterium so fr described, strin S85 ofb. succinogenes, lmost completely digested 2-mg smples of ntive cotton in 7 dys (1). In view of the present results, it would be of interest to mesure the ctivity of strin S85 when tested with lrger mounts of cotton t constnt ph to see wht proportion of the cotton-solubilizing ctivity of the rumen could be contributed by this pure culture. The present studies on cotton led to generlly similr conclusions bout fctors ffecting cellulolytic ctivity in the rumen, s do erlier experiments on the digestion of forge cellulose. In comprble experiments on the effects of concentrtes nd ph on the digestibility of forge cellulose in vitro (14, 22), cellulose digestion ws found to be mrkedly inhibited by fll in ph, s in the present study. The effect of tllow on cotton solubiliztion ws comprble to effects of the sme tllow on grss cellulose digestion in vivo in experiments tht hve been reported seprtely (J. Kovlczyk, E. R. Orskov, J. J. Robinson, nd C. S. Stewrt, Br. J. Nutr., in press). These results re comprble to those obtined in rnge of studies on the effects of dietry fts on cellulose digestion (3). On the other hnd, the conclusions from studies on chemiclly purified or mechniclly modified cellulose re more diverse. Much of this diversity undoubtedly results from differences in experimentl conditions nd the criteri used to estimte cellulolysis, but there must

5 VOL. 33, 1977 be doubt bout which enzyme ctivity is being mesured when different, reltively degrded forns of cellulose re studied. In the present work, for exmple, the titer of filter pperdegrding bcteri showed shrp fll on lowering the ph of the rumen contents. This result is consistent with the finding (9) tht Whtmn cellulose powder is not ttcked by rumen contents t ph 5.9 nd lso with generl effect of ph, in tht experiments suggest tht the optimum ph for mny of the importnt hydrolytic rumen bcteri in the forge-fed ruminnt is bove ph 6. A study of the digestion of filter pper in vivo nd in vitro (6), however, suggested tht ph chnges were not minly responsible for the inhibitory effect of strchy concentrtes on the digestion of cellulose, view tht differs from the conclusions drwn from the present results nd the studies on forge cellulose digestion previously mentioned. The ddition of tllow, lthough suppressing digestion of grss cellulose nd cotton, did not depress the titer of filter pper-degrding bcteri. This suggests more specilized effect of the tllow only on the group of true cellulolytic bcteri ssyed by cotton degrdtion. Supposing the previously mentioned B. succinogenes to be representtive ofthis group, possible mechnism is given by Brynt et l., who suggested tht fts could specificlly depress growth ofb. succinogenes by depriving the bcterium of essentil C2+ ions (2). If the bcteri with the C1 enzyme were only smll frction of the totl ble to degrde filter pper, then decline in the number of the former might not significntly lter the numbers of the ltter s counted here. In ny cse, it is cler tht the numbers of filter pper-degrding bcteri do not give guide to the cellulolytic ctivity of rumen contents under ll conditions. The effect of VFA on the cotton-solubilizing ctivity of rumen contents is comprble to inhibitory effects ofvfa on some non-cellulolytic rumen bcteri previously reported (2). Tensile-strength losses pprently do not give n ccurte guide to weight losses of cotton yrn. Presumbly sitution exists in the rumen comprble to tht described for Trichoderm cellulse (26) in which strength nd weight losses re primrily cused by different mixtures of enzymes of the cellulse complex. The results emphsize tht, s hs previously been implied (1), detiled understnding of cellulose hydrolysis in the rumen cn only be ssembled by experiments with cellulosic mterils, which require known enzymes or mixtures of enzymes for ttck. Cotton fibers, which require the whole of the cellulse CELLULOLYSIS IN THE RUMEN 51 complex, re prticulrly suitble in this respect. ACKNOWLEDGMENTS It is plesure to thnk J. M. Edie for ccess to the cilite-free sheep, P. N. Hobson nd Dr. Edie for helpful discussions, W. R. H. Duncn for the gs-liquid chromtogrphy nlysis, nd Elizbeth Prsons for technicl ssistnce. LITERATURE CITED 1. Blch, C. C., nd V. W. Johnson Fctors influencing the rte of brekdown of cellulose (cotton thred) in the rumen of the cow. Br. J. Nutr. 4: Brynt, M. P., I. M. Robinson, nd H. Chu Observtions on the nutrition ofbcteroides succinogenes - ruminl cellulolytic bcterium. J. Diry Sci. 42: Bull, L. S Fts in ruminnt rtions, p In Proceedings of the 2th Mrylnd Nutrition Conference. University of Mrylnd, College Prk. 4. Dehority, B. A., nd H. W. Scott Extent of cellulose nd hemicellulose digestion in vrious forges by pure cultures of rumen bcteri. J. Diry Sci. 5: Edie, J. M The development of rumen microbil popultions in lmbs nd clves under vrious conditions of mngement. J. Gen. Microbiol. 29: el-shzly, K., B. A. Dehority, nd R. R. Johnson Effect of strch on the digestion of cellulose in vitro nd in vivo by rumen microorgnisms. J. Anim. Sci. 2: Evns, E. W., J. Burnett, nd J. A. Bines A study of the effect ofexposure in the reticulo-rumen of the cow on the strength of cotton, grss, hy nd strw. Br. J. Nutr. 31: Grton, G. A., F. D. DeB. Hovell, nd W. R. H. Duncn Influence of dietry voltile ftty cids on the ftty cid composition of lmb triglycerides, with specil reference to the effect of propionte on the presence of brnched-chin components. Br. J. Nutr. 28: Hlliwell, G Cellulolysis by rumen micro-orgnisms. J. Gen. Microbiol. 17: Hlliwell, G., nd M. P. Brynt The cellulolytic ctivity of pure strins of bcteri from the rumen of cttle. J. Gen. Microbiol. 32: Hobson, P. N Continuous culture of rumen bcteri: pprtus. J. Gen. Microbiol. 38: Lthm, M. J., M. E. Shrpe, nd J. D. Sutton The microbil flor of the rumen of cows fed hy nd high cerel rtions nd its reltionship to the rumen fermenttion. J. Appl. Bcteriol. 34: Lysons, R. J., T. J. L. Alexnder, P. D. Wellsted, P. N. Hobson, S.. Mnn, nd C. S. Stewrt Defined bcteril popultions in the rumen of gnotobiotic lmbs. J. Gen. Microbiol. 94: McCullough, M. E Influence of hy or silge nd rtio of flked corn or beet pulp to forge or cellulose disppernce in vitro. J. Anim. Sci. 27: McDougll, E. I Studies on ruminnt sliv. 1. The composition nd output of sheep sliv. Biochem. J. 43: Mnn, S An improved method of determining cellulolytic ctivity in nerobic bcteri. J. Appl. Bcteriol. 31: Mnn, S Some effects on the rumen microorgnisms of over feeding high brley rtion. J. Appl. Bcteriol. 33: Selby, K The purifiction nd properties of the C, component of the cellulse complex, p In G. J.

6 52 STEWART APPL. ENVIRON. MICROBIOL. Hjny nd E. T. Reese (ed.), Cellulses nd their pplictions. Advnces in Chemistry series 95. Americn Chemicl Society, Wshington, D.C. 19. Siu, R. G. H Microbil decomposition of cellulose, p Reinhold Publishing Corp. New York. 2. Stewrt, C. S Some effects of phosphte nd voltile ftty cid slts on the growth of rumen bcteri. J. Gen. Microbiol. 89: Storry, J. E., nd J. D. Sutton The effect of chnge from low-roughge to high-roughge diets on rumen fermenttion, blood composition nd milk ft secretion in the cow. Br. J. Nutr. 23: Terry, R. A., J. M. A. Tilley, nd G. E. Outen Effect of ph on cellulose digestion under in vivo conditions. J. Sci. Food Agric. 2: vn Gylswyk, N A comprison of two techniques for counting cellulolytic bcteri. J. Gen. Microbiol. 6: Wood, T. M The reltionship between cellulolytic nd pseudo-cellulolytic microorgnisms. Biochim. Biophys. Act 192: Wood, T. M Cellulose nd cellulolysis, p In G. H. Bourne (ed.), World review of nutrition nd dietetics, vol. 12. S. Krger, New York. 26. Wood, T. M., nd S. I. McCre The cellulse complex of Trichoderm koningii, p In M. Biley, T.-M. Enri, nd M. Lindo (ed.), Symposium on enzymtic hydrolysis of cellulose. The Finnish Ntionl Fund for Reserch nd Development (Sitr), Helsinki. Downloded from on April 6, 219 by guest