Vol. 54, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1988, p. 1163-11690099-2240/88/051163-07$02.00/0Copyright © 1988, American Society for Microbiology

Effects of Alkaline Hydrogen Peroxide Treatment on In VitroDegradation of Cellulosic Substrates by Mixed Ruminal

Microorganisms and Bacteroides succinogenes S85SHERRY M. LEWIS,t LARRY MONTGOMERY, KEITH A. GARLEB, LARRY L. BERGER, AND

GEORGE C. FAHEY, JR.*

Department ofAnimal Sciences, 126 Animal Sciences Laboratory, University of Illinois, Urbana, Illinois 61801

Received 9 November 1987/Accepted 9 February 1988

The effects of sodium hydroxide (NaOH) and alkaline hydrogen peroxide (AHP) treatments on wheat straw(WS) and various cellulosic substrates were determined by measuring susceptibility to degradation by mixedruminal organisms or Bacteroides succinogenes S85. In vitro incubations were used to measure differences infermentation resulting from each successive step in the AHP treatment process. In vitro incubations through48 or 108 h were conducted to measure these differences. The AHP treatment of WS increased (P < 0.05) drymatter, neutral detergent fiber, and acid detergent fiber degradation over control WS when these substrateswere incubated with mixed ruminal microorganisms or B. succinogenes S85. Fermentations containingAHP-treated WS had greater (P < 0.05) microbial purine (RNA) and volatile fatty acid concentrations by 12h compared with those containing untreated or NaOH-treated WS. Xylose in AHP-treated WS was utilizedmore extensively (P < 0.05) by 12 h compared with the xylose of untreated or NaOH-treated WS. Treatmentwith AHP removed 23% of the alkali-labile phenolic compounds from WS. When substrates with high levelsof crystalline cellulose (raw cotton fiber, Solka floc, and Sigmacell-50) were treated with NaOH or AHP andincubated for 108 h with B. succinogenes S85, extent of acid detergent fiber degradation of cotton fiber andSigmacell-50 was similar to that of their respective controls. Sodium hydroxide and AHP treatments were

effective in increasing acid detergent fiber degradation of the Solka floc which contained, on average, 3.3 and4.8 percentage units more acid detergent lignin and hemicellulose, respectively, than cotton fiber andSigmacell-50. The present studies provide evidence that cellulose substrates which have a greater degree ofcrystallinity or lower amounts of lignin and hemicellulose or both are not rendered more degradable by AHPtreatment. Microbial degradation of substrates containing greater amounts of lignin and hemicellulose isenhanced by AHP treatment.

The digestibility and nutritive value for ruminants ofagricultural residues are greatly influenced by certain com-ponents of the plant cell wall. The cell wall, and its diversechemical structure, is susceptible to various treatments thatenhance microbial degradation of complex carbohydrates.

Physical treatments, such as grinding and ball-milling,have been applied to increase available surface area (9, 10)and to disrupt the crystalline structure of cellulose microfi-brils (11). Chemical treatment with alkali removes part of thelignin, increasing accessibility of structural carbohydrates(19). Physical and chemical treatments generally increasecell wall digestibility, but those tested to date have beenneither economical nor practical for treatment of largequantities of lignocellulosics. Oxidative agents have re-ceived some attention as lignocellulosic pretreatments.Ozone, sulfur dioxide, and sodium chlorite improved digest-ibility of cereal straws (4, 5, 12). Treatments which combinealkaline hydrolysis and oxidation with hydrogen peroxide(H202) appear to offer the greatest potential for improvingfiber degradation by ruminal microorganisms (25, 27) and toprovide sufficient energy from wheat straw (WS) to supportgrowth of ruminants (28).The objectives of this study were to determine the (i)

effects of successive steps of the alkaline hydrogen peroxide(AHP) treatment process on in vitro WS degradation by

* Corresponding author.t Present address: The Bionetics Corporation, National Center

for Toxicological Research, Jefferson, AR 72079.

ruminal microorganisms, (ii) changes in microbial RNA andvolatile fatty acid (VFA) concentrations as well as in thexylose/glucose (X/G) ratio for substrates during fermenta-tion, (iii) effects of NaOH and AHP treatments on aciddetergent fiber (ADF) degradation of different types ofcellulose with varying degrees of crystallinity, and (iv)degradation of cellulosic substrates by mixed ruminal micro-organisms or pure cultures of Bacteroides succinogenes S85.

MATERIALS AND METHODS

Substrate preparations. (i) Experiment 1. Coarsely ground(10 mm) WS was washed to remove soil contaminants, mostnotably Fe3", which catalyzes the breakdown of H202 (14).The washed WS was air dried prior to subsequent treatment.WS was used untreated (control WS) or treated in one of thefollowing manners: to prepare hydrated WS, 160 g of WS(10%, wt/vol) was soaked in H2O for 24 h; NaOH-treatedWS was prepared by soaking 160 g of WS (10%, wt/vol) inH2O and NaOH (0.72%, wt/vol) for 24 h; AHP-treated WSwas prepared by soaking 160 g of WS for 12 h in 1,550 ml ofH20 adjusted to pH 12.0 with NaOH, after which 51 ml of30% H202 was added and the treatment was continued for 12h. The pH was maintained at 11.5, the PKa for H202dissociation (14), by NaOH addition (0.68%, wt/vol, finalconcentration). Substrates were not washed following treat-ment; solids were removed from the reaction solutions byfiltration, dried at 55°C, and ground through a 425-,um screenprior to medium preparation.

(ii) Experiment 2. Cellulose degradation in vitro was

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TABLE 1. Composition of complex medium supplemented withvarious cellulosic substrates (experiment 2)

Component % in medium

Cellulosic substrate ................... 0.4Soln A.................... 33.0Soln Bb.................... 33.0Trace mineral soln SL4 . .................. 1.0Vitamin mixd ................... 2.0Hemin solne................... 0.25Resazurin................... 0.10Yeast extract (wt/vol) ................... 0.05Trypticase (wt/vol)................... 0.05Na2CO3 (wt/vol) ................... 1.67Cysteine-HCI-H20 (wt/vol)................... 0.05VFAf................... 0.31

a Concentrations (grams per liter): NaCl, 5.4; KH2PO4, 2.7; CaC12 * H20,0.159; MgCl * 6H20, 0.12; MnCl2 - 4H20, 0.06; CoC12 * 6H20, 0.06;(NH4)2SO4, 5.4.

b Concentration (grams per liter): K2HPO4, 2.7.c Components: EDTA Triplex III, 500 mg; FeSO4 - 7H20, 200 mg; H20,

900 ml; SL6, 100 ml. Mineral concentration of SL6: ZnSO4 * 7H20, 40 mg;MnCI2 * 4H20, 12 mg; H3PO4, 120 mg; CoC12 - 6H20, 80 mg; CuC12 - 2H20, 4mg; NiCl2 - 6H20, 8 mg; Na2MoO4 - 2H20, 12 mg; H20, 400 ml.

d Prepared by the method of Scott and Dehority (32).e Prepared by the method of Holdeman et al. (18).f Prepared by the method of Caldwell and Bryant (6).

compared for five lignocellulosic materials: WS; WS cellu-lose prepared by the method of Crampton and Maynard (8),designated C/M cellulose; raw cotton fiber (CF); Solka floc;and Sigmacell (type 50, microcrystalline cellulose; averageparticle size, 50 p.m; Sigma Chemical Co., St. Louis, Mo.).Each untreated, ground (10 mm) cellulosic material served

as a control for its NaOH- and AHP-treated counterpart.Solka floc and Sigmacell were used in powdered form. WhenNaOH treatment was used, approximately 50 g of eachsubstrate was added to 450 ml of H20 and sufficient 6 NNaOH (0.46%, wt/vol) to increase the pH to 12.5 to 12.8during a 24-h treatment period. When AHP treatment wasused, approximately 50 g of each substrate was added to 450ml of H2O and sufficient 6 N NaOH to allow the pH toincrease to 12.5 to 12.8 during a 12-h alkaline presoak, afterwhich 14 ml of 30% H202 was added to the substrate mixtureand treatment was continued for an additional 12 h. The finalconcentration of NaOH in the AHP treatment mixture was,on average, 0.63% (wt/vol) for the CF, Solka floc, andSigmacell, whereas WS and C/M cellulose required, onaverage, 0.88% (wt/vol) NaOH. Treated substrates werewashed thoroughly to remove residual chemicals and solu-bilized products. Solids were prepared as described forexperiment 1. Untreated substrates were also groundthrough a 425-p.m screen.

In vitro protocols. (i) Experiment 1. Substrates were fer-mented in vitro by a one-stage modification of the Tilley andTerry method (36) with 1.25% (wt/vol) substrate and 10%(vol/vol) inoculum of ruminal contents which had beenstrained through four layers of cheesecloth. Urea was addedto provide the equivalent of 10% crude protein.

(ii) Experiment 2. The composition of the complex mediumcontaining various cellulosic sources (0.4%, wt/vol) is pre-sented in Table 1. The medium was prepared under CO2 gasphase, adjusted to pH 6.8, and tubed in 15-ml aliquots. TheB. succinogenes S85 inoculum was grown for 12 h in asimilar medium containing cellobiose in place of cellulose.The culture was diluted to 0.3 optical density units (600 nm)in anaerobic dilution solution, and 0.2 ml was inoculated intoeach tube. When ruminal microorganisms served as inocu-

lum, contents were collected from a Holstein donor cowmaintained on alfalfa hay. Fluid contents (500 ml) werecentrifuged under CO2 at 160 x g for 10 min to remove largeparticulate debris. The supernatant was decanted anaerobi-cally and centrifuged at 4,200 x g for 10 min to concentratemicrobial cells. The microbial pellet was suspended to 30 mlin anaerobic dilution solution, and 0.2 ml of this preparationserved as inoculum. All fermentations were incubated at370C.Chemical analyses. Subsamples of all treatments were

dried at 550C and ground through a 850-p.m screen prior toanalysis for cellulose by the procedure of Crampton andMaynard (8). Neutral detergent fiber (NDF), ADF, and aciddetergent lignin (ADL) were determined by the methods ofGoering and Van Soest (13). The extent of lignocellulosefermentation (experiment 2) was determined by ADF analy-sis (17). Nitrogen was determined by the Kjeldahl method(2). Ash was determined by loss of organic elements uponcombustion. Total microbial purine concentration (milli-grams of total purine per gram of original substrate) wasdetermined (39) on supernatant and pellets.Samples were prepared for VFA analysis by centrifuga-

tion and acidification of the supematant with 25% (wt/vol)metaphosphoric acid (34). Internal standard used was 2-ethylbutyrate. VFA were analyzed with a 5890 gas chromato-graph (Hewlett-Packard Co., Palo Alto, Calif.) equippedwith a flame ionization detector and a column of 15%SP-1220-1% H3PO4 on 100/120-mesh Chromosorb WAW(Supelco, Inc., Bellefonte, Pa.).

Substrates and fermentation residues were acid hydro-lyzed (31) to release neutral sugars. One milliliter of 72%(wt/wt) H2S04 was added to 250 mg of residue and vigor-ously agitated at 370C for 1 h. A 28-ml amount of deionizedH20 was added prior to autoclaving for 1 h at 121°C.Erythritol was added as an internal standard. The hydroly-sate was neutralized with BaCO3 at 600C, centrifuged, andfiltered (30). The filtrate was lyopholized and then solubi-lized in 6 ml of acetonitrile-H20 (2:1) prior to high-perfor-mance liquid chromatographic analysis. Solubilized sample(50 p.l) was analyzed with a 1084B Hewlett-Packard high-performance liquid chromatograph fitted with a APS-Hy-persil NH2 column (200 by 4.6 mm; 5-p.m particle size). Themobile phase consisted of acetonitrile-H20 (87:13) pumpedat 1.5 ml/min. Column and solvent were maintained at 35°C.The Hewlett-Packard 79877A refractive index detector wasused to analyze samples.

Alkali-labile phenolic acids were extracted from 500-mgsubsamples by the procedure of Hartley and Buchan (16) asmodified by Jung et al. (24), with the exception that 2 NNaOH was used instead of 1 N NaOH. Alkali-extractablephenolic acids were dried under N2 and reconstituted in 5 mlof methanol for quantification by high-performance liquidchromatography. Sample (40 p.1) was injected into a 1084BHewlett-Packard high-performance liquid chromatographfitted with a column (250 by 4.6 mm) packed with Spheri-sorb-C18 (5-p.m particle size; Supelco, Inc.). The solventconsisted of H20-glacial acetic acid-butanol (350:1:7, byvolume) pumped at 2.5 ml/min. The column temperature was350C. The UV detector was programmed at 272 nm for thefirst 11.2 min of each determination and at 308 nm thereafter.

Statistical analyses. Experiment 1 was repeated twice. Sixfermentation tubes per time interval (12, 18, 24, 36, and 48 h)were inoculated for each substrate so that 12 observationswere used to estimate dry matter (DM) degradation. Foreach time interval, two of the six tubes were used fordetermination of NDF, ADF, and total microbial purine

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TABLE 2. Chemical composition of washedWS substrates (experiment 1)

% (DM basis)Treatmenta

NDF ADF ADL N Celluloseb Ash

WS 84.0 59.8 8.9 0.30 48.1 4.4WS + H20 82.9 52.9 9.3 0.34 48.5 5.0WS + NaOH 81.7 61.2 7.5 0.24 57.5 5.4WS + AHP 75.5 64.4 3.6 0.18 61.8 7.3

" See text for explanation of treatments.b Cellulose was assayed by the method of Crampton and Maynard (8).

concentration, so that each mean consisted of four observa-tions. Neutral sugar determinations were made on one tubeper time interval. Phenolic acids were determined on dupli-cate samples of each substrate.

Incubation with B. succinogenes S85 was repeated twice.Three tubes were inoculated for a total of six tubes pertreatment per time interval. Due to limited availability ofsubstrate, the 36- and 60-h incubations with mixed ruminalinoculum were not repeated; however, triplicate tubes wereinoculated per time interval. Time and treatment main ef-fects were the factors considered in a completely random-ized design. Data were blocked by replication when appro-priate. Statistical analyses were performed by using analysisof variance obtained from the General Linear Models pro-cedure of Statistical Analysis Systems (33), using least-squares calculation of treatment means and F-protectedcomparisons.

RESULTS

Experiment 1. Experiment 1 was designed to test effects ofsequential steps of the AHP treatment process (i.e., notreatment versus the additive effects of hydration, NaOH,and NaOH plus H202) on composition of the substrates,DM, NDF and ADF degradation, and microbial biomasssynthesis during fermentation. Due to the potential for DMloss during the treatment sequence, substrate DM contentwas measured prior to treatment and after drying of thetreated material at 55°C. During the washing process, WSlost 22% of total DM, whereas NaOH and AHP treatmentsremoved 30.1 and 34.1% of the total DM, respectively.Losses due to washing included soil contaminants, ash,soluble components, and fine straw particles. With NaOH,alkali-labile cell wall substituents, cell wall nitrogenouscompounds, and other alkali extractables (e.g., waxes andcutin) were likely removed. Addition of H202 resulted infurther DM losses due to its strongly oxidative nature (14).Losses of NDF, ADL, and N, measured in the residuefollowing treatment (Table 2), resulted in increases in ADFand cellulose concentrations. Increases in ash with NaOHand AHP treatments were partially due to the NaOH addedto maintain an alkaline pH.DM degradation of AHP-treated WS was greater (P <

0.05) between 18 and 48 h than that of other WS treatments(Table 3). At 48 h, the DM degradation of AHP-treated WSwas 1.3 and 2.9 times that of NaOH-treated and untreatedWS substrates, respectively.

Hydrating WS had no apparent effect on DM degradationas the extent of degradation throughout the incubation wassimilar to that of the untreated control; however, all WSsubstrate was washed prior to experimental treatment. Thebeneficial effects ofAHP treatment were evident in NDF andADF degradation (Table 3). At 36 and 48 h, degradation of

TABLE 3. Degradation in vitro of DM, NDF, and ADF of WSsubstrates by mixed ruminal microorganisms (experiment 1)

% Degradation'Item Treatment"

12 h 18 h 24 h 36 h 48 h

DM WS 1.7a 3.9a 9.4a 15.2a 25. laWS + H20 1.9a 3.0a 8.9a 16.3a 25.3aWS + NaOH 4.4a.b 10.2a 24 ob 35.6b 55.8bWS + AHP 9.7b 21.2b 38.3c 52.3c 73.2c

SEM 1.66 2.38 2.94 2.29 3.17

NDF WS 0.0 3.3 4.5a 8.0a 11.7aWS + H20 2.3 4.1 7.1a.b 6.2a 12.3aWS + NaOH 3.7 3.8 7.4a.b 18 oa 31.5bWS + AHP 1.4 4.2 14.3b 52.8b 63.2c

SEM 2.13 2.97 2.17 5.89 2.61

ADF WS 0.1 1.6 7.1 11.0a 14.4aWS + H20 2.8 2.6 7.3 12.la 14.2aWS + NaOH 1.6 2.2 9.7 28.la 35.8aWS + AHP 4.7 6.2 21.9 72.6b 72 lb

SEM 2.35 4.80 5.32 8.21 7.45

"See text for explanation of treatments.b Means for each component within a column without common superscripts

differ (P < 0.05).

NDF and ADF was greatest (P < 0.05) for the AHP-treatedWS substrate.At 12 h, total microbial purine concentration of incubated

residue plus supernatant was greater (P < 0.05) for AHP-treated WS substrate than for control and hydrated WS.Total microbial purines of NaOH- or AHP-treated WSsubstrates were greater (P < 0.10) at 18 h.At 24 h, AHP-treated WS residues had a maximal total

purine concentration of 11.2 mg/g (adjusted for 0 h), greater(P < 0.05) than total purines of NaOH-treated, hydrated, orcontrol WS, 9.1, 6.4, or 6.1 mg/g, respectively. By 48 h,purine concentration of the AHP-treated residue had de-creased to 6.2 mg/g, the lowest concentration for thatsubstrate and similar to total purine concentrations for theremaining substrates.VFA concentration (millimolar) reflected the increased

degradability of AHP- and NaOH-treated WS. Total VFAconcentration (corrected for 0 h) was consistently greatestfor the AHP-treated WS and was greater (P < 0.05) than thatof NaOH-treated, hydrated, or control WS at 12 h (9.0 mMversus 4.6, 3.4, or 4.3 mM VFA, respectively). At 24 h,NaOH-treated WS fermentations were similar to that ofAHP-treated WS fermentations, 24.2 and 33.3 mM VFA,respectively, and were greater (P < 0.05) than VFA concen-trations of either hydrated or control WS. By 48 h, thepattern of VFA concentrations was similar to that at 12 h,when AHP-treated WS was again greater (P < 0.05) thanother substrates: 66.8 mM versus 55.6, 30.2, and 32.4 mMVFA, for AHP-treated, NaOH-treated, hydrated, and con-trol WS, respectively. Acetate was the primary acid in allsubstrate fermentations. The acetate/propionate ratio, 2.3,of the AHP-treated WS fermentation did not differ from thatof the WS control and suggests a fiber-type fermentation.

Concentration of glucose (as percentage of total neutralsugars) was similar for all treatments, 70.0, 68.8, 63.7, and66.1%, for AHP-treated, NaOH-treated, hydrated, and con-trol WS, respectively. Concentration of xylose was alsosimilar for all substrates, 30.1, 28.0, 33.3, and 29.7% for

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1.0 -

0.9 -

0-0 Ws

0-* WS + H20A-A WCZ J KInNluWLI Li vv.a Fr IN UV

a 0.8-- A-A WS + AHP, 0.7-.0 0.6

.o 0.40.3

0.2A0.1

0.0

0 12 24 36 48Time (h)

FIG. 1. X/G ratios for substrates and residues following in vitrofermentation.

AHP-treated, NaOH-treated, hydrated, and control WS,respectively. Arabinose concentration was similar forNaOH-treated, hydrated, and control WS, 3.2, 3.0, and4.2%, respectively. Arabinose was not detected in the AHP-treated WS.X/G ratios were similar for all substrates (Fig. 1, 0 h). At

12 h, the proportion of xylose had decreased in all fermen-tation residues, indicating a rapid utilization of a readilyfermentable pool of xylose. The most rapid proportional rateof xylose disappearance occurred in the AHP-treated WSresidue, resulting in a decrease (P < 0.05) in the X/G ratiofrom 0.44 to 0.11. Between 12 and 24 h, there was no changein the X/G ratio for untreated WS, whereas the X/G ratioincreased gradually for the remaining treatments. Ratios forthe untreated and hydrated WS at 48 h, 0.62 and 0.59,respectively, exceeded initial values. The X/G ratio for theNaOH-treated WS residue at 48 h was 0.48, similar to that at0 h; however, the X/G ratio for the AHP-treated WS was

59% lower than the 0-h value, 0.18 versus 0.44, respectively.Concentrations of the hydroxycinnamic acids, para-cou-

maric and ferulic acids, were greatest among the alkali-labilephenolic monomers measured in all substrates (Table 4). Thecontent of para-coumaric acid was lowered (P < 0.10) byAHP treatment of WS, while ferulic acid concentration was

decreased (P < 0.10) by both NaOH and AHP treatments.Experiment 2. Experiment 2, using mixed ruminal orga-

nisms or B. succinogenes S85, was designed to examine

TABLE 4. Concentrations of alkali-labile phenolic compounds incontrol and treated WS cell walls (experiment 1)'

Concn (,ug/g of NDF)after given treatmentb

Phenolic compound SEMWS WS WS WS

+ H20 + NaOH + AHP

Protocatechuic acid 9.1 6.6 4.7 0 4.6para-Hydroxyben- 8.2 5.6 4.4 0 4.8

zoic acid

para-Hydroxybenzal- 15.2a 12.5a 6.5b 10.1a.b 2.4dehyde

Vanillic acid 14.1 18.4 13.8 10.4 5.5Syringic acid 11.3 5.1 10.2 9.8 5.3Vanillin 26.0 10.5 14.7 37.1 15.3para-Coumaric acid 2,077.7a 1,934.4ab 1,979.4a 1,679.8b 144.0Ferulic acid 905.9a 928.6a 511.4b 626.6b 86.1

aCell walls were prepared as NDF (13).b See text for explanation of treatments. Means in a row without common

superscripts differ (P < 0.10).

TABLE 5. Chemical composition of various cellulosic sources,treated with sodium hydroxide or AHP (experiment 2)

Substrate and % (DM basis)treatment" NDF ADF ADL Ash

WS 80.2 54.4 7.5 7.6WS + NaOH 93.0 64.0 9.5 4.0WS + AHP 89.4 66.9 5.6 4.6

C/M cellulose 90.1 87.9 1.2 10.5C/M cellulose + NaOH 94.4 90.1 0.7 3.5C/M cellulose + AHP 96.6 90.4 0.3 2.1

CF 96.0 93.0 0.7 0.9CF + NaOH 96.2 95.5 0.5 0.4CF + AHP 97.0 95.5 0.4 0.6

Solka floc 98.9 91.3 4.1 0.2Solka floc + NaOH 98.7 92.3 4.9 0.2Solka floc + AHP 99.4 95.3 2.2 0.2

Sigmacell 98.3 97.7 0.3 0.04Sigmacell + NaOH 98.1 97.2 0.4 0.08Sigmacell + AHP 99.1 98.2 0.4 0.13

" Substrates are defined in the text. Treatments: untreated substrates andeach substrate treated with NaOH or AHP.

whether NaOH or AHP treatment of various cellulosicsubstrates altered the average rate of microbial cellulosedegradation by partially removing lignin or altering thestructure of the substrate or both. Concentrations of NDFand ADF increased with NaOH or AHP treatment of allcellulosic substrates after they were washed to removetreatment chemicals (Table 5). Solubilized components, par-ticularly hemicellulose and lignin, were also washed from thesubstrate. Percentages of ADL and ash in WS decreasedwith AHP treatment, but ADL concentration increased by27% when WS was treated with NaOH. Due to the oxidativenature of the AHP treatment, a part of the lignin moiety wasremoved from the residue, whereas solubilization of poly-saccharides increased the ADL concentration in the NaOH-treated residue. ADL and ash each comprised <1% of CFand Sigmacell and changed little with treatment. Both ADLand ash concentrations decreased when C/M cellulose wastreated with NaOH or AHP. Solka floc contained 4.1%ADL, which was decreased 46% by AHP treatment; ashconcentration of all Solka floc substrates was 0.2%.When B. succinogenes S85 was the inoculum, ADF deg-

radation of WS was most improved (P < 0.05) by AHPtreatment at 24 h (Table 6). By 108 h, however, NaOHtreatment was similar to AHP in improving ADF degradationof WS. The least degradable substrate was C/M cellulose.Treatment with NaOH or AHP did not improve utilization ofthis substrate. Untreated CF was more extensively (P <0.05) utilized than treated CF at 24 h. By 108 h, ADFdegradation of CF was similar for all treatments. Sodiumhydroxide treatment of Solka floc was equal to AHP treat-ment in increasing ADF degradation by 60 and 180 h.Utilization of Sigmacell ADF was most improved (P < 0.05)by NaOH treatment at 24 h. Extent of ADF degradation at108 h was the same for all Sigmacell treatments.When substrates were incubated for 60 h with mixed

ruminal microorganisms, WS was the only one for whichAHP treatment improved degradation. Only NaOH treat-ment increased ADF degradation of C/M cellulose (Table 7).At 36 h, sodium hydroxide treatment was effective (P <

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TABLE 6. ADF degradation of various cellulosic substratesinoculated with B. succinogenes S85 (experiment 2)

Substrate and % Degradation'treatmenta 24 h 36 h 60 h 108 h

Ws 19.6a,b 34.4a 38.4a 48.9a-bWS + NaOH 18.5a,b 29.6a,b 44.4ab.c 568 b,cWS + AHP 28.5' 37.9a 59.8d 66.8c.d

C/M cellulose 9.2c de 10.3e.f 12.6e 14.1eC/M cellulose + NaOH 8.ld.e 11.2e.f 11.8e 14.9eC/M cellulose + AHP 5.3d,e 9.5f 13.2e 13.4e

CF 16.2a.b 24 lb.c 53.6c,d 69.3dCF + NaOH 4.4d,e 21.6b,c.d 51.4b.c,d 70 ldCF + AHP 79d.e 17.0c.d.e.f 43.4a,b 58.6b.c,d

Solka floc 11.2b,c.d 12.5d.e.f 20 oe 19.2eSolka floc + NaOH 8.1d.e 18.7c.d.e.f 39.6a 52.0a.bSolka floc + AHP 15.7a,b 20.4b,c.d.e 395a 51.1a,b

Sigmacell 5.1d,e 20.3b.c.d.e 40.2a.b 490a.bSigmacell + NaOH 15.4a,b,c 24 lb,c 40.2ab 488 a.bSigmacell + AHP 4.0e 16.7c,d.e.f 36.6a 44.5a

SEM 2.23 3.29 3.86 3.93

a See footnote a, Table 5.b Means within a column without common superscripts differ (P < 0.05).

0.05) in increasing ADF degradation of C/M cellulose, CF,Solka floc, and Sigmacell, but not WS.

DISCUSSION

Degradation in vitro of DM and fiber components of WStreated sequentially with the various chemicals of the AHPprocess demonstrated the improvement in digestibility withAHP treatment compared with NaOH treatment (Table 3).

TABLE 7. ADF degradation of cellulosic substrates incubatedwith mixed ruminal microorganisms (experiment 2)

Substrate and % Degradationtreatmenta 36 h 60 h

WS 45.0a 54.5aWS + NaOH 37.6b.c 61 a,b.cWS + AHP 47.7a 83.sd

C/M cellulose 11.4e 34.6eC/M cellulose + NaOH 32.8c 74.4c.dC/M cellulose + AHP 23.5d 28.8e

CF 33.6c 76.7c.dCF + NaOH 41.3a.b 78.0c.dCF + AHP 33.1c 73 b,c.d

Solka floc 12.7e 80.3dSolka floc + NaOH 42.8a.b 83.7dSolka floc + AHP 33.9c 73 b.c.d

Sigmacell 32.3c 591a.b.cSigmacell + NaOH 46.6a 56.9a.bSigmacell + AHP 23.6d 57.4a.b

SEM 2.36 5.46a See footnote a, Table 5.b Means within a column without common superscripts differ (P < 0.05).

From 18 to 48 h, DM degradation of AHP-treated WSsubstrate was greater (P < 0.05) than that of the othersubstrates, clearly indicating the potential of AHP treatmentto increase structural carbohydrate degradation and shortenthe lag phase of fermentation.NDF and ADF degradation of all substrates followed the

same general trends as DM degradation. By 36 h, theimprovement in NDF and ADF degradation due to AHPtreatment was greater than the improvement in DM degra-dation. A comparison of 36- and 48-h NDF and ADFdegradation indicates that ADF degradation exceeded that ofNDF, particularly for the NaOH- or AHP-treated WS. Asimilar response was noted by Lesoing et al. (26), whotreated WS with variable levels of NaOH and Ca(OH)2 andmeasured in vitro hemicellulose and cellulose digestibility.With chemical treatment, the extent of cellulose digestibilityexceeded that of hemicellulose at 48 h. It is possible that thesolubilization of hemicellulose by NaOH and AHP treat-ments effectively removed the hemicellulosic fraction mostavailable for microbial utilization, leaving a more refractoryhemicellulosic fraction.

Total microbial purine concentration, an indicator of mi-crobial biosynthesis, indicated rapid cell growth during earlyfermentation of WS treated with NaOH or AHP. Maximaltotal microbial purine concentrations occurred at 18 and 24 hfor NaOH- or AHP-treated WS, 10.0 and 11.2 mg/g, respec-tively. Microbial purine concentrations declined consider-ably, even before DM degradation reached its maximum.John (20) found that total RNA concentration in rumina ofsheep fed once daily can be considered in three phases: (i) arapid increase to maximal values 4 h postfeeding, (ii) a 10-hphase during which RNA concentrations decrease to nearprefeeding values, and (iii) a 10-h phase during which RNAconcentrations decline at a slower rate than in the secondphase. In contrast to the situation in the rumen, where RNAconcentration decreased 23% during the second phase, totalpurines of control and hydrated WS fermentations decreased12.5 and 6.3%, respectively, from their maxima. However,total microbial purines decreased more extensively for theNaOH- and AHP-treated WS, 28 and 45%, respectively,suggesting a decreased average growth rate presumably dueto substrate limitation for nonfibrolytic microbes or end-product inhibition or both.VFA concentrations in fermentation vessels containing

AHP-treated WS were twofold greater than that for un-treated WS at all sampling times. The final VFA concentra-tion of the AHP-treated WS fermentation at 48 h was 20%greater than for the NaOH-treated WS and 106% above thatof the untreated control.

Neutral sugars were measured to examine potential shiftsin the X/G ratio throughout fermentation, as affected bychemical treatment. Despite the hydrolytic and oxidativenature of the AHP treatment, the relative concentrations ofdetectable neutral sugars in AHP-treated WS were similar tothose of the other WS substrates except for arabinose, whichwas presumably removed by chemical treatment.The decline in the X/G ratio of all WS substrates at 12 h

demonstrates the rapid utilization of xylose (Fig. 1). Whileglucose was always available and presumably being utilized,two distinct xylose pools may exist, one readily availableand another more refractory to microbial degradation. Be-tween 24 and 48 h, rapid glucose utilization contributed tothe increase in X/G ratios for all but the AHP-treated WSfermentations, suggesting that the refractory xylose wasmade more available by AHP treatment.The phenolic acids and aldehydes present in cell walls are

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bound to various constituents. Some are esterified to arabi-noxylan of the cell wall and can be released by treatmentwith aqueous alkali (35). The AHP treatment may be effec-tive in disrupting the ester linkage between phenolic acidsand polysaccharides as demonstrated by a 23% reduction inconcentrations of para-coumaric and ferulic acids (Table 4).Attempts to relate the presence of para-coumaric or ferulicacids to a depression in DM or cellulose degradation haveproduced conflicting results. DM (35) and cellulose (1, 22)degradation in vitro are depressed more by para-coumaricacid; however, ferulic acid was found to be more inhibitoryto cellulose digestibility in vivo (23). These findings suggestthat profound differences may occur when phenolic acids are

metabolically transformed (7, 21, 35) in the more dynamicruminal system as compared with in vitro systems. Ferulicacid was shown to be more inhibitory than para-coumaricacid to cellulose digestion when B. succinogenes was thepredominant cellulolytic species in mixed ruminal cultures(21) and in pure cultures of B. succinogenes or Rumino-coccus flavefaciens (7); para-coumaric acid was more inhib-itory to Ruminococcus albus (7).The concentration of a particular phenolic compound and

the form in which it exists in the cell wall (unbound or boundto other phenolics or sugars or both) is important in theevaluation of its inhibitory nature. In the current research,para-coumaric and ferulic acids comnprised 0.30% of the dryweight of untreated WS, far below the levels (3.0 to 9.0%,DM) found to be inhibitory (21) when unbound. Chesson etal. (7) reported these phenolics to comprise 0.48% of driedgrass and 1.19% of barley straw. Cell walls of barley strawcontained 1.02% total para-coumaric and ferulic acids (35).The concentration of total phenolic compounds in un-

treated WS was decreased 15 and 23% by NaOH and AHP,respectively. Reduced concentrations of phenolic acids as

well as decreased lignin content and increased celluloseavailability may have been a contributing factor in increasingthe extent of DM, NDF, and ADF degradation (Table 3). Inlight of these results and others, the effect ofAHP treatmenton phenolic acid content and ester bonding of phenolics tohemicellulose warrants further research.WS crystallinity is difficult to assess by standard methods,

but WS appears to be less crystalline than cotton cellulosewhen measured by X-ray diffraction (3). Extracted WScellulose (C/M cellulose) may be more crystalline thannatural WS cellulose due to the acid treatment used in itspreparation; it should be relatively free of hemicellulose andlignin (8) compared with control WS (Table 5). Cotton is 95%cellulose (38); it has the highest crystallinity of any naturalcellulosic material, estimated at 60 to 100% depending on themethod of determination. Sigmacell-50 is a microcrystallineform of cellulose from which noncrystalline cellulose hasbeen removed by mineral acid and is reported to be between85 and 100% crystalline (11). Solka floc, a hammer-milledsulfite pulp, is less crystalline than Sigmacell, but is stillhighly crystalline (11).The lack of response of CF, Solka floc, and Sigmacell to

treatment may be directly related to the degree of crystal-linity and the low levels of lignin (Table 5). B. succinogenes

S85, used to test the degradation of the various cellulosicsubstrates because of its ability to utilize crystalline forms of

cellulose (15), degraded the ADF of AHP-treated WS more

effectively than other substrates through 60 h. By 108 h,control and NaOH-treated CF were utilized to an equal

extent (Table 6).Untreated CF was degraded to a greater extent at 24 h

than NaOH- or AHP-treated CF. This was not expected, as

chemical treatment would theoretically "de-wax" the CFand allow for greatest utilization. CF contained approxi-mately 3% hemicellulose (NDF minus ADF, Table 5) and<1.0% lignin and thus has low levels of components topotentially react with NaOH or AHP during treatment. Infact, AHP treatment tended to impair ADF degradation ofthe CF at all incubation times tested. Prepared celluloses andcotton often show a longer lag time for digestion than doescellulose in intact forages (37).When untreated substrates and their NaOH- or AHP-

treated counterparts were incubated with mixed ruminalmicroorganisms (Table 7), ADF degradation of all untreatedand treated substrates was higher than with B. succinogenesS85, due principally to the much higher microbial concen-tration in the mixed inoculum. The B. succinogenes S85inoculum was grown on cellobiose as the energy source,which may have contributed to a prolonged lag period beforefiber degradation reached maximal rates. Using mixed cul-tures, ADF degradation of the three substrates containinghighly crystalline cellulose (CF, Sigmacell, and Solka floc)was less affected by either NaOH or AHP treatment thanwas WS or C/M cellulose (Table 7).

In summary, AHP treatment of WS allowed greater DM,NDF, and ADF degradation in vitro, with greater apparentmicrobial RNA and VFA production. The neutral sugars,glucose and xylose, were more rapidly and extensivelyfermented during incubation following AHP treatment of WS(data not shown). To some degree, the efficacy of AHPtreatment depends on the nature of the cellulose sourcebeing treated. Overall, fiber sources having greater crystal-linity or lower levels of lignin and hemicellulose (CF, Sig-macell, or Solka floc) or both were less responsive tochemical treatment. Sodium hydroxide treatment of themore highly crystalline cellulose sources had a greater effecton ADF degradation than did AHP treatment when mixedruminal microorganisms were used. These cellulose sourcescontain less hemicellulose and lignin, the cell wall compo-nents most affected by AHP treatment. Many agriculturalresidues should benefit from AHP treatment as a result ofpartial lignin removal by hydrolysis and oxidation. The AHPtreatment of agricultural residues to increase in vitro and invivo cellulose utilization is superior to previously usedchemical treatments and provides a useful tool for directpractical and basic research applications.

LITERATURE CITED1. Akin, D. E. 1982. Forage cell wall degradation and p-coumaric,

ferulic and sinapic acids. Agron.l. 74:424-428.2. Association of Official Analytical Chemists. 1984. Nitrogen anal-

ysis, p. 153-154. In S. Williams (ed.), Official methods ofanalysis, 14th ed. The William Byrd Press, Inc., Richmond, Va.

3. Baker, T. I., G. V. Quicke, 0. G. Bentley, R. R. Johnson, andA. L. Moxon. 1959. The influence of certain physical propertiesof purified celluloses and forage celluloses on their digestibilityby rumen microorganisms in vitro. J. Anim. Sci. 18:655-662.

4. Ben-Ghedalia, D., and J. Miron. 1981. Effect of sodium hydrox-ide, ozone and sulphur dioxide on the composition and in vitrodigestibility of wheat straw. J. Sci. Food Agric. 32:224-228.

5. Ben-Ghedalia, D., and J. Miron. 1984. The response of wheatstraw varieties to mild sulphur dioxide treatment. Anim. FeedSci. Technol. 10:269-276.

6. Caldwell, D. R., and M. P. Bryant. 1966. Medium without rumenfluid for nonselective enumeration and isolation of rumen bac-teria. Appl. Microbiol. 14:794-801.

7. Chesson, A., C. S. Stewart, and R. J. Wallace. 1982. Influence ofplant phenolic acids on growth and cellulolytic activity of rumenbacteria. Appl. Environ. Microbiol. 44:597-603.

8. Crampton, E. W., and L. A. Maynard. 1938. The relation of

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cellulose and lignin content to the nutritive value of animalfeeds. J. Nutr. 15:383-388.

9. Dehority, B. A. 1961. Effect of particle size on the digestion rateof purified cellulose by rumen cellulolytic bacteria in vitro. J.Dairy Sci. 44:687-692.

10. Dehority, B. A., and R. R. Johnson. 1961. Effect of particle sizeupon the in vitro cellulose digestibility of forages by rumenbacteria. J. Dairy Sci. 44:2242-2249.

11. Fan, L. T., Y. H. Lee, and D. H. Beardmore. 1980. Mechanismof the enzymatic hydrolysis of cellulose: effects of major struc-tural features of cellulose or enzymatic hydrolysis. Biotechnol.Bioeng. 22:177-199.

12. Goering, H. K., L. W. Smith, P. J. Van Soest, and C. H. Gordon.1973. Digestibility of roughage materials ensiled with sodiumchlorite. J. Dairy Sci. 56:233-240.

13. Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses(apparatus, reagents, procedures, and some applications). Ag-riculture Handbook 379. U.S. Government Printing Office,Washington, D.C.

14. Gould, J. M. 1984. Alkaline peroxide delignification of agricul-tural residues to enhance enzymatic saccarification. Biotechnol.Bioeng. 26:46-52.

15. Halliwell, G., and M. P. Bryant. 1963. The cellulolytic activity ofpure strains of bacteria from the rumen of cattle. J. Gen.Microbiol. 32:441-448.

16. Hartley, R. D., and H. Buchan. 1979. High performance liquidchromatography of phenolic acids and aldehydes derived fromplants or from the decomposition of organic matter in soil. J.Chromatogr. 180:139-143.

17. Hiltner, P. A., and B. A. Dehority. 1983. Effect of solublecarbohydrates on digestion of cellulose by pure cultures ofrumen bacteria. Appl. Environ. Microbiol. 46:642-648.

18. Holdeman, L. V., E. P. Cato, and W. E. C. Moore. 1977.Anaerobic laboratory manual, 4th ed. Virginia PolytechnicInstitute Anaerobe Laboratory, Blacksburg.

19. Jackson, M. G. 1977. Review article: The alkali treatment ofstraws. Anim. Feed Sci. Tech. 2:105-131.

20. John, A. 1984. Effects of feeding frequency and level of feedintake on chemical composition of rumen bacteria. J. Agric. Sci.102:45-57.

21. Jung, H. G. 1985. Inhibition of structural carbohydrate fermen-tation by forage phenolics. J. Sci. Food Agric. 36:74-80.

22. Jung, H. G., and G. C. Fahey, Jr. 1983. Interactions amongphenolic monomers and in vitro fermentation. J. Dairy Sci.66:1255-1263.

23. Jung, H. G., and G. C. Fahey, Jr. 1984. Influence of phenolicacids on forage structural carbohydrate digestion. Can. J. Anim.Sci. 64(Suppl.):50-51.

24. Jung, H. G., G. C. Fahey, Jr., and N. R. Merchen. 1983. Effects

of ruminant digestion and metabolism on phenolic monomers offorages. Br. J. Nutr. 50:637-651.

25. Kerley, M. S., G. C. Fahey, Jr., L. L. Berger, J. M. Gould, andF. L. Baker. 1985. Alkaline hydrogen peroxide treatment un-locks energy in agricultural by-products. Science 230:820-822.

26. Lesoing, G., T. Klopfenstein, I. Rush, and J. Ward. 1981.Chemical treatment of wheat straw. J. Anim. Sci. 51:263-269.

27. Lewis, S. M., D. P. Holzgraefe, L. L. Berger, G. C. Fahey, Jr.,J. M. Gould, and G. F. Fanta. 1987. Alkaline hydrogen peroxidetreatments of crop residues to increase ruminal dry matterdisappearance in sacco. Anim. Feed Sci. Technol. 17:179-199.

28. Lewis, S. M., M. S. Kerley, G. C. Fahey, Jr., L. L. Berger, andJ. M. Gould. 1987. Use of alkaline hydrogen peroxide-treatedwheat straw as an energy source for the growing ruminant.Nutr. Rep. Int. 35:1093-1104.

29. Miron, J., and D. Ben-Ghedalia. 1982. Effect of hydrolyzing andoxidizing agents on the composition and degradation of wheatstraw monosaccharides. Eur. J. Appl. Microbiol. Biotechnol.15:83-87.

30. Neilson, M. J., and J. A. Marlett. 1983. A comparison betweendetergent and nondetergent analyses of dietary fiber in humanfoodstuffs, using high-performance liquid chromatography tomeasure neutral sugar composition. J. Agric. Food Chem.31:1342-1347.

31. Saeman, J. F., W. E. Moore, and M. A. Millett. 1963. Sugarunits present: Hydrolysis and quantitative paper chromatogra-phy, p. 54-69. In R. L. Whistler (ed.), Methods in CarbohydrateChemistry. Vol. 3. Academic Press, New York.

32. Scott, H. W., and B. A. Dehority. 1965. Vitamin requirements ofseveral cellulolytic rumen bacteria. J. Bacteriol. 89:1169-1175.

33. Statistical Analysis Systems. 1982. SAS user's guide: statistics.Statistical Analysis Systems Institute, Inc., Cary, N.C.

34. Supelco, Inc. 1975. GC separation of VFA C2-C5. Bulletin 749C.Supelco, Inc., Bellefonte, Pa.

35. Theodorou, M. K., D. J. Gascoyne, D. E. Akin, and R. D.Hartley. 1987. Effect of phenolic acids and phenolics from plantcell walls on rumen-like fermentation in consecutive batchculture. Appl. Environ. Microbiol. 53:1046-1050.

36. Tilley, J. M., and R. A. Terry. 1963. A two-stage technique forthe in vitro digestion of forage crops. J. Br. Grassland Soc.18:104-111.

37. Van Soest, P. J. 1973. The uniformity and nutritive availabilityof cellulose. Fed. Proc. 32:1804-1808.

38. Young, R. A. 1986. Structure, swelling and bonding of cellulosefibers, p. 91-128. In R. A. Young and R. M. Rowell (ed.),Cellulose structure, modification and hydrolysis. John Wiley &Sons, Inc., New York.

39. Zinn, R. A., and F. N. Owens. 1986. A rapid procedure forpurine measurement and its use for estimating net ruminalprotein synthesis. Can. J. Anim. Sci. 66:157-166.

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