Relation of Lignin and Cellulose

8/13/2019 Relation of Lignin and Cellulose

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THE REL TION OF CELLULOSE ND LIGNINCONTENT TO THE NUTRITIVE V LUEOF NIM L FEEDS

E . W . C BAMPTO ND ep ar tm en t o f A nim al H usb and ry M cG ul U niv ersity M ac don ald C olleg e P Q

ANDL. A. M AYN ABD

L ab or ato ry o f A ni ma l N utr it io n C or ne ll U ni ve rs ity I th ac a(E eceived for publication O ctober 20, 1937)

For the past 40 years, anim al feeding stuffs have, for generaldescription, been divided by proximate analysis into sixfractions: moisture, ether extract, protein, ash, crude fiberand nitrogen-free extract. This analysis has been the commonbasis of the nutritional classification of feeds, particularly thethree fractions (protein, fat and fiber) which have been usedin feed control measures. Through an extension of thisanalysis to the feces voided by animals on controlled feedingtests, the usual digestibility coefficients for feeds are calculated. The significance of the specific values obtained, however, has frequently been over-estimated. This has beentrue especially of the crude fiber fraction.In terms of feeding value, crude fiber is intended as am easure of the quantity of the fibrous, poorly digested m aterialin the feed. On the other hand, one has but to recall thechem ical procedure by w hich this fraction is isolated to understand that any relation crude fiber may have to the digestibility of a feed may be in part fortuitous. (For a full discussion of this subject, the reader is referred to the work ofNorm an, 35).1 The experim ental w ork of this study w as carried out largely in the L aboratory

of A nim al N utrition, C ornell U niversity, and excepting w here otherw ise noted,the data are taken from a thesis presented by E. W . Crampton in partial fulfillm ent of the requirem ents for the degree of doctor of philosophy.38 3T HE J OU RN AL O F N UT RI TI ON V OI 1 5 N O 4


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8 E W CH AM P IO N AN D L A M A YN AR D

T hat the digestibility of the dietary carbohy drate does notfollow its partition into crude f iber and nitrogen-f ree ex tractw ith any m ark ed certainty , especially in the case of roughages,is ev ident f rom table 1 calculated f rom digestion coef f icientsreported by M orrison ( 37).T he published literature also contains am ple ev idence thatthe crude f iber of forages m ay be as w ell digested as theprotein (N ew lander and Jones, 32; M itchell and Ham ilton,33 ; M o rrison, 37). It w ould, therefore, seem that the justif i

cation for partition of the carbohy drate f raction of f eeds intocrude f iber as the poorly , and nitrogen-free ex tract as thehighly digestible parts, m ay be open to som e question.

T ABL Ee la tive di ge stibi li ty o f We ende c rude f ibe r a nd n itro ge n f re e e xtra ct

K IND OFEEDDry

roughagesGreenroughagesSilagesCo ncentratesAll

feedsNUMBER

AVERAGED110612588284AVKRAI;KCOEFFICIENTrDIGESTIB ILITY Crude

fiber52.463.558.253.355.6N-free

extract59.576.364.678.569.5PEB

CEN T OF OA SESITHrulliiK l 1 :1 :1 ;.S HOW ING A gCOMPLETEIGESTIBILITYA SN-FBEBEXT RA CT 3920281025

Furtherm ore, it is logical to believ e that if a div ision of thecarbohydrate fraction could be m ade into parts w hich w ereeither biological or chem ical units, the usefulness of thefeeding stuf f s analy sis in predicting probable feeding v aluew ould be enhanced. T he problem of such a partition resolv esitself largely into a consideration of the chief constituents ofthe cell w all carbohy dratescellulose, hem icellulose andlignin.

Chemical and biological nature of lignin cellulose andhemicelluloseLignin occurs in plants chiefly as lignocellulose There is

support for the belief that substances of the glucosanx ylanseries are the forerunners of lignin, but neither its ex act


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C ELLU LO SE A N D LIG N IN IN A N IM A L FEED S 385

chem ical structure nor the m anner in w hich it is com binedw ith cellulose are fully understood. Its behavior in nutritionis likewise unsettled, different feeding tests yielding conflicting results (D ietrich and K nig,1871; K nigand Becker, '18;Paloheim o, '25; Eogozinski and Starzew ska, '27; Rubner,'28; Phillips, '29, '34; Fuchs, '36; W oodm an and Stew art,'32; Prjanischnikow and Tom m e, '36). Proof for or againstits utilization by the anim al is difficult to establish, for untilits m olecular structure is know n, no criterion of the accuracyof a quantitative test for lignin is possible.In prelim inary tests of this study, and using chem icalprocedures to be later described, 97.8 and 99.3 of the dietarylignin w ere recovered in the feces of rabbits and a steer,respectively. Subsequent tests of the digestibility of certaindietary constituents of m ixed pasture herbage clippings substantiated this finding w ith rabbits as seen in the follow ingfigures :

D a te of c lippin g P er cen t dieta r y lign inof gr a tt fed r ecover ed in fecetM ay 12 97.56June 3 95.18June 20 93.39July 9 100.32July 9 103.67July 24 96.87A ugust 20 102.73A verage 98.53

These data support the rather m ore general opinion thatdietary lignin is not appreciably m etabolized by anim als.C ellulose N either the chem ical nature of cellulose northe steps by w hich it eventually yields products nutritionallyuseful to an anim al, can be discussed here (see Pochon, '35;W oodm an and Stew art, '28). U ndoubtedly the extent to w hichsuch end products are produced m ay be influenced by a num berof factors (M angold, '34). A m ong them , the possible role'D igestibility data from nutrition laboratory, M acdonald C ollege and obtained

for each clipping by pooling feed and feces figures for three rabbits fed individually on identical diets. The rabbit feeding w as carried out by M r. Robert P.Forshaw and the analyses of the feces by M r. A . J. Sutherland, graduate assistantsat M acdonald C ollege.T heir assistance is m uch appreciated and gladly acknowledged.


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386 E. W . C H A M PIO N A N D L. A . M A Y N A K D

of lignin is of special interest in this study. Lignified planttissues apparently are not attacked by alim entary bacteria(W oodm an and Stew art, '32), perhaps because of a certaindegree of antiseptic action of the lignin resulting from itsphenolic nucleus (H ibbert).3

This obviously suggests that if lignification differs in degreebetw een sam ples of forage either because of species or environm ental conditions, the feeding value of such m aterialm ay be changed as a result. This m ight, in part, explain theobserved decrease in nutritive value of pasture grasses duringseasons or parts of seasons w hen conditions are not favorableto rapid growth, but instead favor lignification of the planttissues. O n the other hand, it m ay not follow that, otherfactors constant, a decrease in digestibility of the feed w illreflect an increase in total lignin, inasm uch as the m anner ofits deposition in the plant m ay be a factor of im portance inthis respect (W oodm an and Stew art, '32).Hemicellulose a ccording to Armstrong a nd Armstrong('34), is perhaps m ost usefully visualized as consisting of a

m ixture of the com ponents of incom pletely form ed w oodysubstances containing, in association, true cellulose chains,xylan and polyglucuronates (see also N orm an, '36). Theusually accepted m ethod of quantitative estim ation (fur-furaldehyde) is open to the criticism that hem icellulose, ofanim al forage at least, contains appreciable quantities ofhexosans as w ell as pentosans. B iologically, this group ofcarbohydrates follows m uch the sam e paths of degradation ascellulose. H ow ever, there is som e evidence (Schm idt-O tt, '36;Iw ata, '35; W illiam s and O lm sted, '35) that they m ay besom ew hat m ore com pletely utilized by the anim al system , thusstanding interm ediate betw een cellulose and the easily solublestarches and sugars.The large num ber of substances w hich appear logically tobe classed as hem icellulose, together w ith the confusion w hichat present exists in the exact definition of this group, m akes1H ibbert, H . Pulp and Paper Besearch Institute, M cG ill U niversity, personal

com m unication.


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the possibility of a satisfactory chem ical estim ation ratherrem ote. There appears to be no advantage for purposes of afeeding stuffs analysis in determ ining a part of the group,as, for exam ple, pentosans, and still leaving an undeterm inedfraction. A s an alternate plan, estim ation by difference ofthe total hem icelluloses plus any other carbohydrates notcellulose, seem s to be a m ore logical procedure and w asadopted in this study.Chemica l estima tion of lignin a nd cellulose

Chemica l determina tion of lignin. Spa ce does not permita full discussion of the problem s of lignin determ ination, andfor such m aterial the reader is referred especially to papersby N orm an and Jenkins ('34), G oss and Phillips ('36), C ohen('36), C ohen and H arris ('37), W aksm an and Stevens ('30),Boss and Potter ('30), and Ross and H ill ('29). Consideration of these and other studies led us to the conclusion thatat the present tim e som e form of the '72 H 2S04' m ethodis the m ost satisfactory for the quantitative estim ation ofthe lignin content of anim al forage. It consists in solutionin concentrated H 2S04 of the sam ple w hich has been pre-treated to rem ove fats, sugars and proteins w hich otherw isew ould interfere w ith the lignin values obtained.

The special problem , in the case of forage and anim al feces,lies in the rem oval of the protein w ithout a sim ultaneousrem oval of a part of the lignin. A ccording to present inform ation, lignin is soluble in varying degrees in dilute alkali(hot or cold), boiling w ater, and dilute m ineral acid (1.25H 2S04) at boiling tem perature. Pretreatm ent by enzym edigestion, how ever, w ould seem to be a suitable possibility.W illiam s and O lm sted ('35), w orking w ith hum an diets andfeces, proposed the use of pancreatin in a solution bufferedat pH 8. In their proposal, cellulose and hem icellulose w eredeterm ined on the filtrate rem aining on rem oval of the lignin ;but because som e hem icellulose is rem oved by the alkalinepancreatin solution, a correction is m ade by running an additional sam ple of the diet w ith the pancreatin om itted.


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388 E. W . C H A M PIO N A N D L. A . M A Y N A K D

Lignin values are taken from the sam ple digested w ith thepancreatiti. But if lignin w ere isolated from both the enzym eand no-enzym e residues, any effect of the pretreatm ent onlignin w ould be seen. (Fats, starches and soluble sugars arerem oved by ether extraction and autoclaving previous to theenzym e treatm ent.)W hen this procedure w as tried w ith sheep and steer dietsof grain and hay, the results in every case show ed a sm allerlignin value in the pancreatin treated sam ple, as indicated intable 2. TA BLE 2Effect on lignin va lues of pretr ea tment of sheep a nd steer diets by pa ncrea tin

digestionD ESC RIPTIO N Sheep

dietrain + alfalfa haySheep dietrain + tim othy haySteer dietrain + alfalfa hayM O N I

If (A S PIK C EN T O r A IK D R Y SA M PL EW ithpancreatiti

4.764.212.81W ithout

pancreatin5.405.783.40

It seem s probable that the long exposure of the sam ples(72 hours at 45 C.)in a solution buffered at pH 8 using N aO Has the alkali, m ay have dissolved som e of the lignin and thusresulted in the low ered lignin values.A nother difficulty w as also encountered in the use of theW illiam s and O lm sted procedure. It w as found im possiblein the case of the anim al diets to effect com plete solution inthe concentrated H 2S04 of the enzym e digest residue. Subsequently, w hen a lignin balance w as struck from a steerdigestion trial, 25 m ore lignin w as recovered in the fecesthan w as consum ed.Pepsin, on the other hand, is active in acid m edium ; andithas not yet been shown that lignin is soluble in dilute m ineralacid at tem peratures at w hich this enzym e is active. Theeffectiveness of this enzym e in rem oving proteins w as uncertain in view of studies by H orw itt et al. ('36) in w hich not

m ore than 89 of the nitrogen w as rem ovable from spinachleaf by pepsin digestion. There w as the possibility, how ever,


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C EL L U LO SE A ND L IG N IN IN A NIM A L FEE D S 389

that the protein of the materials would be reduced by pepsindigestion to a level no longer seriously interfering withlignin determ ination. If the hypothesis is accepted that ligninis not utilized by the animal, the usefulness of the pepsinpretreatment and subsequent analytical steps would be indicated by a lignin balance trial. Such a test yielded the datagiven in table 3. The practically quantitative recovery of thedietary lignin obtained argues well for the procedure used.T A B L E 3

ig es tib ility o f lig n in b y r b bits n d s te erITDrymatterPer

centigninWeightofigninBalance

Per c en t r ec ov er yConsumed

InecesInfeedInecesCo nsum edVoidedBABBITS228

grams79rams9.03 25.48 20.58

grams20.15rams0.43grains97.8 STET.K53.00

pounds10.75ounds5.5627.12 2.95pounds2.92ounds0.03

pounds99.3The problem of completely and rapidly dissolving the undigested residue in the strong acid was solved by Boss andHill ( 29) who found that lignified tissues would dissolve

promptly (10 to 15 minutes) in 72 H2S04 if first moistenedwith formalin. W ith the sample in contact with the acid forso short a tim e, the difficulty w ith the form ation of substancesfrom the carbohydrates (pentoses or hexoses) which mightadd to the lignin value is presumably largely avoided (Eitteret al., 32). The use of a granulating reagent (chloroform-acetic acid) in the precipitation of the lignin to hasten thetim e necessary for filtration w as proposed by Boss and Potter( 30), and has been recommended by Baillie ( 36), in a micro-technic. It appeared, therefore, that pepsin digestion of theether extracted sample, followed by solution of the residuein 72 H2S04 and subsequent precipitation of the ligninaccording to the Boss and Hill, and Boss and Potter procedures could be successfully used for lignin determ inationin anim al feeds and feces.


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39 E W C R A M P TO N A N D L A M A Y N A E D

Specifically, the procedure eventually adopted in this studyis as follows : Place the oven-dry, ether extracted residue froma 1 gm. sample of feed (or feces) in a 50 cc. glass stopperedErlenmeyer and add 40 cc. of a 2.0 solution of pepsin inN /10 H C1. Digest for 12 hours at 40 C.,shaking frequentlyespecially during the first 4 or 5 hours. Recover the non-digested residue by filtration through bolting silk and washsuccessively with hot water, hot alcohol, hot benzene, hotalcohol, and ether. Transfer the washed residue to a 100 cc.beaker, and remove the last traces of ether with mild heat.Moisten the residue with 4 cc. of 40 formaldehyde. Thenadd 4 cc. of 72 H2SO4, and allow it to penetrate the sample(2 minutes). Add 6 cc. of concentrated H2S04 and stirvigorously with a glass rod to aid in solution of the samplewhich should be complete in 10 to 15 m inutes. Partially immerse the beaker in a cold water bath, if necessary, to preventthe temperature from rising above about 70C. When dissolved, stir in 35 cc. of a granulating reagent consisting of a1:6 mixture (by volume) of chloroform and acetic acid,and then pour the whole into 500 cc. distilled water in an800 cc. beaker. Boil gently until the chloroform has beendriven off (15 minutes), after which the solution should clearand the lignin settle in granular form . Filter on a Goochwith suction. W ash in not less than 200 cc. of 5 HC1.Dry at 110C.and determ ine lignin by loss on ignition. Thisprocedure has given reproducible results in the hands of twoanalysts.D etermination of cellulose For a sum m ary of the manym ethods w hich have been used for the quantitative estim ationof cellulose, the reader is referred to papers by Krschnerand Hoff e r ( 31), and Krschner a nd Hanak ( 30). According to the former procedure, the sample was freed of non-cellulose, organic constituents by digestion w ith an alcohol-nitric acid reagent. The treatment involved boiling thesample with the reagent for two and sometimes three successive hour periods. In the latter procedure, acetic acid issubstituted for the alcohol in the digestion reagent and thetime of treatment reduced to 20 minutes.


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Com parative tests of these tw o procedures resulted inpractically the sam e values for cellulose from feces m aterial,but low er values from certain feeds w ith the acetic acid-H N Oreagent, as is show n in table 4.

TA B LE 4C ellulose a s per cent of moistur e fr ee sa mple

SteerecesB abbitfecesSteerfeed (grain + alfalfaay)B abbitfeed (grass clippings)A LC O H O IrH N O j2L730.414.722.4A C ETIC

A CID -H N O j2l l31.011.422.3

A possible explanation of this m ight lie in a difference inthe resistance of the cellulosan fractions of m ature hay andgrain as com pared to those in im m ature grasses. Certainly,the CH 3CO O H -H N 03 m ixture is the m ore pow erful reagent.Incidentally, the results w ith the feces m ight be interpretedthat the anim al w as able to rem ove m ost of the dietarycellulosans.These results, together w ith its greater sim plicity, led tothe choice of the acetic acid reagent for the cellulose determ ination. It w as found, how ever, that by using alcoholinstead of w ater for the first w ashings to free the cellulosefrom the digesting reagent, this process could be carried outby centrifuging, w hich considerably facilitated the w ashingoperations.The details of the procedure used are as follow s: Place a1 gm .,air dry sam ple in a 150 cc.round-bottom ed, w ide-neckedflask fitted w ith a reflux condenser. A dd 15 cc. of 80 aceticacid and 1.5 con.H N 03. Boil gently for 20 m inutes. Transferthe sam ple and liquid to a 50 cc. centrifuge tube ; add about20 cc.of alcohol and centrifuge 10 m inutes. D ecant the liquid.W ash (in centrifuge tube) w ith alcohol. Transfer the residue(w ith aid of a stream of alcohol from a w ash bottle) into analundum crucible and w ash successively w ith hot benzene,hot alcohol, and etherusing suction. D ry. Calculate cellulose


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392 E. W . C H A M PIO N A N D L. A . M A Y N A EDas loss on ignition. O ur experience has show n this m ethod togive reproducible results.

A test of a modified procedure for feeding stuffs a na lysisThis study w as originally undertaken in connection w ith theproblem of the nutritive value of pasture herbage w here the

TA BLE 5Ana lysis o f feed s a nd feces a nd a pp ar en t d ig estibility of d iets

A N IM A I.Y earling

A ngusteerweight850oundsD iet:

equal partslfalfahayandrainD rym atteraten:53poundsD ryfeces: 10.75oundsM ature

rabbitD iet:

pasturerassclippingsD rym atteraten:228gm .D ryfeces 79 gm .PR O C EIH 7 SStandardM odifiedStandardM odifiedEther

extractProtein(N X 6.25)A shCrudefiberN -freeextractEtherextractProteinA shLigninC elluloseO ther

carbohydrateEtherextractProteinA shC rude

fiberN -freeextractEtherextractProteinA shLigninC elluloseO ther

carbohydrateAN A LY SISPer

centdiet6.5912.193.6412.1165.476.5912.193.645.5611.4260.605.1entieces7.6118.739.9125.6638.087.6118.739.9127.21CE2TD IGE STIBILITY 76.668.857.088.276.668.80.762.594.861

51.766.761.980.0.

.2.252.986.4

lack of correlation betw een the 'standard' feeding stuffsanalysis and feeding value as m easured by the grow th ofrabbits had been especially noted. To test the value of thedata obtained by m odifying the standard m ethod of analysisby the inclusion of the determ inations for cellulose and lignin,analyses w ere m ade of the feed and feces from a digestiontrial w ith rabbits and a steer. The feeds and feces w ere


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analyzed for w ater, protein, ash, fat and fiber by the usualprocedures and for cellulose and lignin by the m ethods previously outlined. The term 'other carbohydrates' w as used todenote the residue obtained by difference. These data arepresented in table 5.These data suggest that the m odified procedure m akes asharper distinction am ong the carbohydrates as regards theirdigestibility. The carbohydrates are partitioned into threefractions: a practically non-digested portion, lignin; ahighly digestible fraction, other carbohydrates; and intocellulose w hich is both biologically and chem ically a recognizable unit. The digestibility of the latter fraction by agiven anim al m ay be expected to vary inversely w ith thedegree (or nature) of its lignification.A s a m eans of estim ating their feeding value, the m odifiedschem e of analysis here proposed w ould seem to offer distinctadvantages over the standard procedure for the analysis ofrations, especially of H erb vora. Further study is neededof the application of the m ethods proposed to various kindsof feeds, and of the correlation of the analytical data w ith theresults of feeding trials. Such studies are now in progress atM acdonald College.

SU K M A B YD ata are presented w hich show the lim itations, as m easuresof digestibility and nutritive value, of the crude fiber andnitrogen-free extract partition of the carbohydrates in anim al

feeds. Based on studies of various procedures, m ethods areproposed for the determ ination of cellulose and lignin infeeds and feces. The usefulness of the inclusion of thesedeterm inations in feed analyses is show n by data obtained indigestion trials w ith rabbits and steers. The data indicatethat, at least for H erb vora,a partition of the carbohydrateportion of a feed into lignin, cellulose and other carbohydratesm ay have m ore biological significance, and hence be of greaterusefulness in predicting feeding values than the presentdivision into crude fiber and nitrogen-free extract.


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394 E. W . C R A M PTO N A N D L. A . M A Y N A B D

LITER A TU R E C ITEDA R M ST RO N G , . F., A N D K . P. A R M STR O N O 1934 The C arbohydrates, M onographs on Biochem istry. Longm ans, G reen and Co., N . Y .I'.A ILM F..A . J. 1936 M ikro determ ination of ligniti. M ikrochem ie, vol. 19,

p. 98.C O H E N ,W . E . 1936 T he chem istry of A ustralian tim bers. V . Study of thelignin determ ination. C oun. Sei. Ind. R es. A ustralia Tech. Paper no.20,p. 791.COH EN,W . E., AN D E. E. HA RRIS 1937 Pretreatm ent of wood with hot diluteacid. Its effect on lignin values. Ind. Eng. Chem . A nal. Ed. 9,p. 235.D IETRICH ,T., A N D J. K N IG 1871 Zusam m ensetzung und V erdaulichkeit derneben der cellulose in der Rohfaser des W iesenheus vorkom m endenSubstanz. Landw . V ers. Sta., Bd. 13, 8. 122. (Cited by Phillips,C hem . R ev., vol. 14, p 158, 1934.)FUCH S,W . M . 1936 The chem ical nature of lignin. Paper Trade J., vol. 102,TA PPI sec.p. 181.G O SS,M . J., A N D M . PHILLIPS 1936 Studies on the quantitative estim ation oflignin. Factors affecting the determ ination by the fum ing H C 1 m ethod.J. A ssoc. O fficial A gr. C hem ., vol. 19, p. 341.Hoew iTT, M .K ., G. R. CO W GILLAN D L. B. M END EL 1936 Availability of carbohydrate and fats of green leaf together w ith som e observations oncrude fiber. J. N utrition, vol. 12,p. 237.IW A TA ,H . 1935 Biochem ical studies on xylan. Im p. Coll. A gr. and Forestry,M orioka, N ippon. B ui. 21.K NIG ,., A N DE . BE CK ER 1918 D ie Bestandteile des H olzes und ihre w irtschaftliche V erw ertung. V er ffentlichungen d. Landw.-K am m er f. d. Prov.W estfalen, B d.26. (C ited by Phillips, C hem .R ev. 14,p. 158,1934. Seealso Joseph K nig (M unster i. W .), Sein Leben und seine A rbeit.V erlagsbuchhandlung Paul Parey, B erlin, p. 141-150, 1928.)

K RSCH N ER,., A N DA . K A N A K 1930 D eterm ination of cellulose. Z. U ntersuch.Lebensm ., vol. 59, p. 484.K RSCH N ER,., A N D A . H O PPER 1931 A new quantitative cellulose determ ination. C hem . Zeit., vol. 55, p. 161, 181.M AN GO LD,. 1934 Digestion and utilization of crude fibre. Nutr. Abs. andRev., vol. 3,p. 647.M IT C H E LL ,H . H ., A N D T . 8. H A M IL TO N 1933 D igestibility of oat hulls bysw ine. J. A gr. R es., vol. 47, p. 427.M O RR ISON ,. B. 1937 Feeds and Feeding, A ppendix Table 1,M orrison Publishing C o.,Ithaca, N . Y ., p. 953.N EW LA N D ER ,. A ., A N D C. H . JO N ES 1932 The digestibility of artificially driedgrass. V t. A gr. Exp. Sta. Bul. 348.NO RM AN ,A. G. 1935 The com position of crude fibre. J. Agr. Res., vol 25,p. 529.1936 The association of xylan w ith cellulose in certain structuralcelluloses. Biochem . J., vol. 30, p. 2054.N O RM A N , .G .,AN DS.H .JEN K IN S 1934 The determ ination of lignin. Biochem .J., vol. 28,p. 2147:2160.


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C ELLU LO SE A N D LIG N IN IN A N IM A L FEED S 395PALO H EIM O ,L. 1925 D ie V erw endung der Saurehydrolyse zur Ligninbestim -

m nng. Biochem . Z., vol. 165, p. 463. (Cited by Phillips, Chem . Eev.14, p. 158, 1934.PHILLIPS,M . 1934 The chem istry of lignin. Chem . Rev., vol. 14, p. 103.PHILLIPS,M ., ET A L. 1929 The dem ethoxylation of lignin in the anim al. Proe.Soc. Exp. Biol. and M ed., vol. 26, p. 320.PO C H O N ,J. 1935 B ole of a cellulolytic bacterium in the rum en in the digestion ofcellulose. A nn. Inst. Pasteur, vol. 55, p. 676. A bstracted in N utr.A bs. and Eev., vol. 6,p. 95.PRJAN ISCHN IKO W ,J. E., AN D M . F. TOM M E 1936 Effect of lignin on digestibility of rye straw . B iederm ann's Zentr. B . Tierernahr., vol. 8,p. 104.A bstracted in N utr. A bs. and Eev., vol. 6,p. 231.EITTER,G -.J., E. M . SEBOR OA ND R. L. M ITCHELL 1932 Factors affectingquantitative determ ination of lignin by the 72 per cent H 38O 4 m ethod.Ind. Eng. Chem . A nal. Ed. 4, p. 202.RO O O ZIN SKI,., A N DM .STA RZEw sK A 1927 The digestion of lignin by rum inants.Intern. R ev. A gr., vol. 18,p. 413.Eoss, J. H ., A N DA . C.H ILL 1929 D eterm ination of lignin by form aldehyde andsulphuric acid. Pulp and Paper M ag. Can., vol. 27, p. 451.Ross, J. H., AN D J. C. POTTER 1930 The determ ination of lignin by form aldehyde and sulphuric acid, II. Pulp and Paper M ag. Can., vol. 29,p. 567.BU BN ER,M . 1928 The physiological significance of certain im portant constituents of vegetables w ith particular reference to lignin. Sitzb.Preuss. A kad. W iss., vol. 12, p. 127. (Cited by Phillips, C hem . R ev.,vol. 14, 1934.SCH M ID T-O TT,A . 1936 D igestion of cellulose. II. The utilization of com plexcarbohydrates. A rch. V erdauungs-K rankh., vol. 59,p. 143.

W A K S M A N , 8. A ., A N D K . R . ST EV E N S 1930 System of proxim ate chem icalanalysis of plant m aterials. Ind. Eng. C hem . A nal. Ed., vol. 2,p. 167.W ILLIAM S,E. D., AN D W . H. OLM STED 1935 Indigestible residue in feces.J. Biol. C hem ., vol. 108, p. 653.W OO DM AN ,. E., AN D J. STEW AR T 1928 The m echanism of cellulose digestionin the rum inant organism . The transform ation of cellulose intoglucose by the agency of cellulose-splitting bacteria. J. A gr. Sci.,vol. 18,p. 713.1932 The m echanism of cellulose digestion in the rum inant organism .The action of cellulose-splitting bacteria on the fiber of certain typicalfeeding stuffs. J. A gr. Sei., vol. 22, p. 527.

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Relation of Lignin and Cellulose