ruminal microorganisms, (ii) changes in microbial RNA and volatile fatty acid (VFA) concentrations as well as in the

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1988, p /88/ $02.00/0 Copyright 1988, American Society for Microbiology Vol. 54, No. 5 Effects of Alkaline Hydrogen Peroxide Treatment on In Vitro Degradation of Cellulosic Substrates by Mixed Ruminal Microorganisms and Bacteroides succinogenes S85 SHERRY M. LEWIS,t LARRY MONTGOMERY, KEITH A. GARLEB, LARRY L. BERGER, AND GEORGE C. FAHEY, JR.* Department of Animal Sciences, 126 Animal Sciences Laboratory, University of Illinois, Urbana, Illinois 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 mixed ruminal organisms or Bacteroides succinogenes S85. In vitro incubations were used to measure differences in fermentation resulting from each successive step in the AHP treatment process. In vitro incubations through 48 or 108 h were conducted to measure these differences. The AHP treatment of WS increased (P < 0.05) dry matter, neutral detergent fiber, and acid detergent fiber degradation over control WS when these substrates were incubated with mixed ruminal microorganisms or B. succinogenes S85. Fermentations containing AHP-treated WS had greater (P < 0.05) microbial purine (RNA) and volatile fatty acid concentrations by 12 h compared with those containing untreated or NaOH-treated WS. Xylose in AHP-treated WS was utilized more extensively (P < 0.05) by 12 h compared with the xylose of untreated or NaOH-treated WS. Treatment with AHP removed 23% of the alkali-labile phenolic compounds from WS. When substrates with high levels of crystalline cellulose (raw cotton fiber, Solka floc, and Sigmacell-50) were treated with NaOH or AHP and incubated for 108 h with B. succinogenes S85, extent of acid detergent fiber degradation of cotton fiber and Sigmacell-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 and 4.8 percentage units more acid detergent lignin and hemicellulose, respectively, than cotton fiber and Sigmacell-50. The present studies provide evidence that cellulose substrates which have a greater degree of crystallinity or lower amounts of lignin and hemicellulose or both are not rendered more degradable by AHP treatment. Microbial degradation of substrates containing greater amounts of lignin and hemicellulose is enhanced by AHP treatment. The digestibility and nutritive value for ruminants of agricultural residues are greatly influenced by certain components of the plant cell wall. The cell wall, and its diverse chemical structure, is susceptible to various treatments that enhance 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 microfibrils (11). Chemical treatment with alkali removes part of the lignin, increasing accessibility of structural carbohydrates (19). Physical and chemical treatments generally increase cell wall digestibility, but those tested to date have been neither economical nor practical for treatment of large quantities of lignocellulosics. Oxidative agents have received some attention as lignocellulosic pretreatments. Ozone, sulfur dioxide, and sodium chlorite improved digestibility of cereal straws (4, 5, 12). Treatments which combine alkaline hydrolysis and oxidation with hydrogen peroxide (H202) appear to offer the greatest potential for improving fiber degradation by ruminal microorganisms (25, 27) and to provide sufficient energy from wheat straw (WS) to support growth 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 ruminal microorganisms, (ii) changes in microbial RNA and volatile fatty acid (VFA) concentrations as well as in the xylose/glucose (X/G) ratio for substrates during fermentation, (iii) effects of NaOH and AHP treatments on acid detergent fiber (ADF) degradation of different types of cellulose with varying degrees of crystallinity, and (iv) degradation of cellulosic substrates by mixed ruminal microorganisms 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, most notably 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 the following manners: to prepare hydrated WS, 160 g of WS (10%, wt/vol) was soaked in H2O for 24 h; NaOH-treated WS was prepared by soaking 160 g of WS (10%, wt/vol) in H2O and NaOH (0.72%, wt/vol) for 24 h; AHP-treated WS was prepared by soaking 160 g of WS for 12 h in 1,550 ml of H20 adjusted to ph 12.0 with NaOH, after which 51 ml of 30% H202 was added and the treatment was continued for 12 h. The ph was maintained at 11.5, the PKa for H202 dissociation (14), by NaOH addition (0.68%, wt/vol, final concentration). Substrates were not washed following treatment; solids were removed from the reaction solutions by filtration, dried at 55 C, and ground through a 425-,um screen prior to medium preparation. (ii) Experiment 2. Cellulose degradation in vitro was

2 1164 LEWIS ET AL. TABLE 1. Composition of complex medium supplemented with various cellulosic substrates (experiment 2) Component % in medium Cellulosic substrate Soln A Soln Bb Trace mineral soln SL Vitamin mixd Hemin solne Resazurin Yeast extract (wt/vol) Trypticase (wt/vol) Na2CO3 (wt/vol) Cysteine-HCI-H20 (wt/vol) VFAf a Concentrations (grams per liter): NaCl, 5.4; KH2PO4, 2.7; CaC12 * H20, ; MgCl * 6H20, 0.12; MnCl2 4H20, 0.06; CoC12 * 6H20, 0.06; (NH4)2SO4, 5.4. b Concentration (grams per liter): K2HPO4, 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, mg; 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 cellulose 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; average particle 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. When NaOH treatment was used, approximately 50 g of each substrate was added to 450 ml of H20 and sufficient 6 N NaOH (0.46%, wt/vol) to increase the ph to 12.5 to 12.8 during a 24-h treatment period. When AHP treatment was used, approximately 50 g of each substrate was added to 450 ml of H2O and sufficient 6 N NaOH to allow the ph to increase to 12.5 to 12.8 during a 12-h alkaline presoak, after which 14 ml of 30% H202 was added to the substrate mixture and treatment was continued for an additional 12 h. The final concentration of NaOH in the AHP treatment mixture was, on average, 0.63% (wt/vol) for the CF, Solka floc, and Sigmacell, whereas WS and C/M cellulose required, on average, 0.88% (wt/vol) NaOH. Treated substrates were washed thoroughly to remove residual chemicals and solubilized products. Solids were prepared as described for experiment 1. Untreated substrates were also ground through a 425-p.m screen. In vitro protocols. (i) Experiment 1. Substrates were fermented in vitro by a one-stage modification of the Tilley and Terry method (36) with 1.25% (wt/vol) substrate and 10% (vol/vol) inoculum of ruminal contents which had been strained through four layers of cheesecloth. Urea was added to provide the equivalent of 10% crude protein. (ii) Experiment 2. The composition of the complex medium containing various cellulosic sources (0.4%, wt/vol) is presented in Table 1. The medium was prepared under CO2 gas phase, adjusted to ph 6.8, and tubed in 15-ml aliquots. The B. succinogenes S85 inoculum was grown for 12 h in a similar 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 into each tube. When ruminal microorganisms served as inocu- APPL. ENVIRON. MICROBIOL. lum, contents were collected from a Holstein donor cow maintained on alfalfa hay. Fluid contents (500 ml) were centrifuged under CO2 at 160 x g for 10 min to remove large particulate debris. The supernatant was decanted anaerobically and centrifuged at 4,200 x g for 10 min to concentrate microbial cells. The microbial pellet was suspended to 30 ml in anaerobic dilution solution, and 0.2 ml of this preparation served as inoculum. All fermentations were incubated at 370C. Chemical analyses. Subsamples of all treatments were dried at 550C and ground through a 850-p.m screen prior to analysis for cellulose by the procedure of Crampton and Maynard (8). Neutral detergent fiber (NDF), ADF, and acid detergent lignin (ADL) were determined by the methods of Goering and Van Soest (13). The extent of lignocellulose fermentation (experiment 2) was determined by ADF analysis (17). Nitrogen was determined by the Kjeldahl method (2). Ash was determined by loss of organic elements upon combustion. Total microbial purine concentration (milligrams of total purine per gram of original substrate) was determined (39) on supernatant and pellets. Samples were prepared for VFA analysis by centrifugation and acidification of the supematant with 25% (wt/vol) metaphosphoric acid (34). Internal standard used was 2-ethyl butyrate. VFA were analyzed with a 5890 gas chromatograph (Hewlett-Packard Co., Palo Alto, Calif.) equipped with a flame ionization detector and a column of 15% SP % H3PO4 on 100/120-mesh Chromosorb WAW (Supelco, Inc., Bellefonte, Pa.). Substrates and fermentation residues were acid hydrolyzed (31) to release neutral sugars. One milliliter of 72% (wt/wt) H2S04 was added to 250 mg of residue and vigorously agitated at 370C for 1 h. A 28-ml amount of deionized H20 was added prior to autoclaving for 1 h at 121 C. Erythritol was added as an internal standard. The hydrolysate was neutralized with BaCO3 at 600C, centrifuged, and filtered (30). The filtrate was lyopholized and then solubilized in 6 ml of acetonitrile-h20 (2:1) prior to high-performance liquid chromatographic analysis. Solubilized sample (50 p.l) was analyzed with a 1084B Hewlett-Packard highperformance liquid chromatograph fitted with a APS-Hypersil NH2 column (200 by 4.6 mm; 5-p.m particle size). The mobile phase consisted of acetonitrile-h20 (87:13) pumped at 1.5 ml/min. Column and solvent were maintained at 35 C. The Hewlett-Packard 79877A refractive index detector was used to analyze samples. Alkali-labile phenolic acids were extracted from 500-mg subsamples by the procedure of Hartley and Buchan (16) as modified by Jung et al. (24), with the exception that 2 N NaOH was used instead of 1 N NaOH. Alkali-extractable phenolic acids were dried under N2 and reconstituted in 5 ml of methanol for quantification by high-performance liquid chromatography. Sample (40 p.1) was injected into a 1084B Hewlett-Packard high-performance liquid chromatograph fitted with a column (250 by 4.6 mm) packed with Spherisorb-C18 (5-p.m particle size; Supelco, Inc.). The solvent consisted of H20-glacial acetic acid-butanol (350:1:7, by volume) pumped at 2.5 ml/min. The column temperature was 350C. The UV detector was programmed at 272 nm for the first 11.2 min of each determination and at 308 nm thereafter. Statistical analyses. Experiment 1 was repeated twice. Six fermentation tubes per time interval (12, 18, 24, 36, and 48 h) were inoculated for each substrate so that 12 observations were used to estimate dry matter (DM) degradation. For each time interval, two of the six tubes were used for determination of NDF, ADF, and total microbial purine

3 VOL. 54, 1988 MICROBIAL UTILIZATION OF AHP-TREATED CELLULOSE SOURCES 1165 Treatmenta TABLE 2. Chemical composition of washed WS substrates (experiment 1) % (DM basis) NDF ADF ADL N Celluloseb Ash WS WS + H WS + NaOH WS + AHP " 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 observations. Neutral sugar determinations were made on one tube per time interval. Phenolic acids were determined on duplicate samples of each substrate. Incubation with B. succinogenes S85 was repeated twice. Three tubes were inoculated for a total of six tubes per treatment per time interval. Due to limited availability of substrate, the 36- and 60-h incubations with mixed ruminal inoculum were not repeated; however, triplicate tubes were inoculated per time interval. Time and treatment main effects were the factors considered in a completely randomized design. Data were blocked by replication when appropriate. Statistical analyses were performed by using analysis of variance obtained from the General Linear Models procedure of Statistical Analysis Systems (33), using leastsquares calculation of treatment means and F-protected comparisons. RESULTS Experiment 1. Experiment 1 was designed to test effects of sequential steps of the AHP treatment process (i.e., no treatment versus the additive effects of hydration, NaOH, and NaOH plus H202) on composition of the substrates, DM, NDF and ADF degradation, and microbial biomass synthesis during fermentation. Due to the potential for DM loss during the treatment sequence, substrate DM content was measured prior to treatment and after drying of the treated material at 55 C. During the washing process, WS lost 22% of total DM, whereas NaOH and AHP treatments removed 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 nitrogenous compounds, and other alkali extractables (e.g., waxes and cutin) were likely removed. Addition of H202 resulted in further DM losses due to its strongly oxidative nature (14). Losses of NDF, ADL, and N, measured in the residue following treatment (Table 2), resulted in increases in ADF and cellulose concentrations. Increases in ash with NaOH and AHP treatments were partially due to the NaOH added to 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 WS was 1.3 and 2.9 times that of NaOH-treated and untreated WS substrates, respectively. Hydrating WS had no apparent effect on DM degradation as the extent of degradation throughout the incubation was similar to that of the untreated control; however, all WS substrate was washed prior to experimental treatment. The beneficial effects of AHP treatment were evident in NDF and ADF degradation (Table 3). At 36 and 48 h, degradation of TABLE 3. Degradation in vitro of DM, NDF, and ADF of WS substrates by mixed ruminal microorganisms (experiment 1) Item Treatment" % Degradation' 12 h 18 h 24 h 36 h 48 h DM WS 1.7a 3.9a 9.4a 15.2a 25. la WS + H20 1.9a 3.0a 8.9a 16.3a 25.3a WS + NaOH 4.4a.b 10.2a 24 ob 35.6b 55.8b WS + AHP 9.7b 21.2b 38.3c 52.3c 73.2c SEM NDF WS a 8.0a 11.7a WS + H a.b 6.2a 12.3a WS + NaOH a.b 18 oa 31.5b WS + AHP b 52.8b 63.2c SEM ADF WS a 14.4a WS + H la 14.2a WS + NaOH la 35.8a WS + AHP b 72 lb SEM "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-treated WS substrate. At 12 h, total microbial purine concentration of incubated residue plus supernatant was greater (P < 0.05) for AHPtreated WS substrate than for control and hydrated WS. Total microbial purines of NaOH- or AHP-treated WS substrates 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, or control WS, 9.1, 6.4, or 6.1 mg/g, respectively. By 48 h, purine concentration of the AHP-treated residue had decreased to 6.2 mg/g, the lowest concentration for that substrate and similar to total purine concentrations for the remaining substrates. VFA concentration (millimolar) reflected the increased degradability of AHP- and NaOH-treated WS. Total VFA concentration (corrected for 0 h) was consistently greatest for the AHP-treated WS and was greater (P < 0.05) than that of NaOH-treated, hydrated, or control WS at 12 h (9.0 mm versus 4.6, 3.4, or 4.3 mm VFA, respectively). At 24 h, NaOH-treated WS fermentations were similar to that of AHP-treated WS fermentations, 24.2 and 33.3 mm VFA, respectively, and were greater (P < 0.05) than VFA concentrations of either hydrated or control WS. By 48 h, the pattern of VFA concentrations was similar to that at 12 h, when AHP-treated WS was again greater (P < 0.05) than other substrates: 66.8 mm versus 55.6, 30.2, and 32.4 mm VFA, for AHP-treated, NaOH-treated, hydrated, and control WS, respectively. Acetate was the primary acid in all substrate fermentations. The acetate/propionate ratio, 2.3, of the AHP-treated WS fermentation did not differ from that of the WS control and suggests a fiber-type fermentation. Concentration of glucose (as percentage of total neutral sugars) was similar for all treatments, 70.0, 68.8, 63.7, and 66.1%, for AHP-treated, NaOH-treated, hydrated, and control WS, respectively. Concentration of xylose was also similar for all substrates, 30.1, 28.0, 33.3, and 29.7% for

4 1166 LEWIS ET AL. APPL. ENVIRON. MICROBIOL Ws 0-* WS + H A-A WCZ J KInNluW LI Li vv.a Fr IN UV a A-A WS + AHP, o A Time (h) FIG. 1. X/G ratios for substrates and residues following in vitro fermentation. AHP-treated, NaOH-treated, hydrated, and control WS, respectively. Arabinose concentration was similar for NaOH-treated, hydrated, and control WS, 3.2, 3.0, and 4.2%, respectively. Arabinose was not detected in the AHPtreated WS. X/G ratios were similar for all substrates (Fig. 1, 0 h). At 12 h, the proportion of xylose had decreased in all fermentation residues, indicating a rapid utilization of a readily fermentable pool of xylose. The most rapid proportional rate of xylose disappearance occurred in the AHP-treated WS residue, resulting in a decrease (P < 0.05) in the X/G ratio from 0.44 to Between 12 and 24 h, there was no change in the X/G ratio for untreated WS, whereas the X/G ratio increased gradually for the remaining treatments. Ratios for the untreated and hydrated WS at 48 h, 0.62 and 0.59, respectively, exceeded initial values. The X/G ratio for the NaOH-treated WS residue at 48 h was 0.48, similar to that at 0 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-coumaric and ferulic acids, were greatest among the alkali-labile phenolic monomers measured in all substrates (Table 4). The content of para-coumaric acid was lowered (P < 0.10) by AHP 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 organisms or B. succinogenes S85, was designed to examine TABLE 4. Concentrations of alkali-labile phenolic compounds in control and treated WS cell walls (experiment 1)' Phenolic compound Concn (,ug/g of NDF) after given treatmentb WS WS WS WS + H20 + NaOH + AHP SEM Protocatechuic acid para-hydroxyben zoic acid 10.1a.b 2.4 Vanillic acid Syringic acid Vanillin para-coumaric acid 2,077.7a 1,934.4ab 1,979.4a 1,679.8b Ferulic acid 905.9a 928.6a 511.4b 626.6b 86.1 para-hydroxybenzaldehyde 15.2a 12.5a 6.5b 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 WS + NaOH WS + AHP C/M cellulose C/M cellulose + NaOH C/M cellulose + AHP CF CF + NaOH CF + AHP Solka floc Solka floc + NaOH Solka floc + AHP Sigmacell Sigmacell + NaOH Sigmacell + AHP " Substrates are defined in the text. Treatments: untreated substrates and each substrate treated with NaOH or AHP. whether NaOH or AHP treatment of various cellulosic substrates altered the average rate of microbial cellulose degradation by partially removing lignin or altering the structure of the substrate or both. Concentrations of NDF and ADF increased with NaOH or AHP treatment of all cellulosic substrates after they were washed to remove treatment chemicals (Table 5). Solubilized components, particularly hemicellulose and lignin, were also washed from the substrate. Percentages of ADL and ash in WS decreased with AHP treatment, but ADL concentration increased by 27% when WS was treated with NaOH. Due to the oxidative nature of the AHP treatment, a part of the lignin moiety was removed from the residue, whereas solubilization of polysaccharides increased the ADL concentration in the NaOHtreated residue. ADL and ash each comprised <1% of CF and Sigmacell and changed little with treatment. Both ADL and ash concentrations decreased when C/M cellulose was treated with NaOH or AHP. Solka floc contained 4.1% ADL, which was decreased 46% by AHP treatment; ash concentration of all Solka floc substrates was 0.2%. When B. succinogenes S85 was the inoculum, ADF degradation of WS was most improved (P < 0.05) by AHP treatment at 24 h (Table 6). By 108 h, however, NaOH treatment was similar to AHP in improving ADF degradation of WS. The least degradable substrate was C/M cellulose. Treatment with NaOH or AHP did not improve utilization of this substrate. Untreated CF was more extensively (P < 0.05) utilized than treated CF at 24 h. By 108 h, ADF degradation of CF was similar for all treatments. Sodium hydroxide treatment of Solka floc was equal to AHP treatment 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 at 108 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 which AHP treatment improved degradation. Only NaOH treatment increased ADF degradation of C/M cellulose (Table 7). At 36 h, sodium hydroxide treatment was effective (P <

5 VOL. 54, 1988 MICROBIAL UTILIZATION OF AHP-TREATED CELLULOSE SOURCES 1167 TABLE 6. ADF degradation of various cellulosic substrates inoculated with B. succinogenes S85 (experiment 2) % Degradation' Substrate and treatmenta 24 h 36 h 60 h 108 h Ws 19.6a,b 34.4a 38.4a 48.9a-b WS + NaOH 18.5a,b 29.6a,b 44.4ab.c 568 b,c WS + AHP 28.5' 37.9a 59.8d 66.8c.d C/M cellulose 9.2c de 10.3e.f 12.6e 14.1e C/M cellulose + NaOH 8.ld.e 11.2e.f 11.8e 14.9e C/M cellulose + AHP 5.3d,e 9.5f 13.2e 13.4e CF 16.2a.b 24 lb.c 53.6c,d 69.3d CF + NaOH 4.4d,e 21.6b,c.d 51.4b.c,d 70 ld CF + 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 19.2e Solka floc + NaOH 8.1d.e 18.7c.d.e.f 39.6a 52.0a.b Solka 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 490 a.b Sigmacell + NaOH 15.4a,b,c 24 lb,c 40.2ab 488 a.b Sigmacell + AHP 4.0e 16.7c,d.e.f 36.6a 44.5a SEM 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 WS treated sequentially with the various chemicals of the AHP process demonstrated the improvement in digestibility with AHP treatment compared with NaOH treatment (Table 3). TABLE 7. ADF degradation of cellulosic substrates incubated with mixed ruminal microorganisms (experiment 2) Substrate and % Degradation treatmenta 36 h 60 h WS 45.0a 54.5a WS + NaOH 37.6b.c 61 a,b.c WS + AHP 47.7a 83.sd C/M cellulose 11.4e 34.6e C/M cellulose + NaOH 32.8c 74.4c.d C/M cellulose + AHP 23.5d 28.8e CF 33.6c 76.7c.d CF + NaOH 41.3a.b 78.0c.d CF + AHP 33.1c 73 b,c.d Solka floc 12.7e 80.3d Solka floc + NaOH 42.8a.b 83.7d Solka floc + AHP 33.9c 73 b.c.d Sigmacell 32.3c 591a.b.c Sigmacell + NaOH 46.6a 56.9a.b Sigmacell + AHP 23.6d 57.4a.b SEM a 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 WS substrate was greater (P < 0.05) than that of the other substrates, clearly indicating the potential of AHP treatment to increase structural carbohydrate degradation and shorten the lag phase of fermentation. NDF and ADF degradation of all substrates followed the same general trends as DM degradation. By 36 h, the improvement in NDF and ADF degradation due to AHP treatment was greater than the improvement in DM degradation. A comparison of 36- and 48-h NDF and ADF degradation indicates that ADF degradation exceeded that of NDF, particularly for the NaOH- or AHP-treated WS. A similar response was noted by Lesoing et al. (26), who treated WS with variable levels of NaOH and Ca(OH)2 and measured in vitro hemicellulose and cellulose digestibility. With chemical treatment, the extent of cellulose digestibility exceeded that of hemicellulose at 48 h. It is possible that the solubilization of hemicellulose by NaOH and AHP treatments effectively removed the hemicellulosic fraction most available for microbial utilization, leaving a more refractory hemicellulosic fraction. Total microbial purine concentration, an indicator of microbial biosynthesis, indicated rapid cell growth during early fermentation of WS treated with NaOH or AHP. Maximal total microbial purine concentrations occurred at 18 and 24 h for NaOH- or AHP-treated WS, 10.0 and 11.2 mg/g, respectively. Microbial purine concentrations declined considerably, even before DM degradation reached its maximum. John (20) found that total RNA concentration in rumina of sheep fed once daily can be considered in three phases: (i) a rapid increase to maximal values 4 h postfeeding, (ii) a 10-h phase during which RNA concentrations decrease to near prefeeding values, and (iii) a 10-h phase during which RNA concentrations decline at a slower rate than in the second phase. In contrast to the situation in the rumen, where RNA concentration decreased 23% during the second phase, total purines of control and hydrated WS fermentations decreased 12.5 and 6.3%, respectively, from their maxima. However, total microbial purines decreased more extensively for the NaOH- and AHP-treated WS, 28 and 45%, respectively, suggesting a decreased average growth rate presumably due to substrate limitation for nonfibrolytic microbes or endproduct inhibition or both. VFA concentrations in fermentation vessels containing AHP-treated WS were twofold greater than that for untreated WS at all sampling times. The final VFA concentration of the AHP-treated WS fermentation at 48 h was 20% greater than for the NaOH-treated WS and 106% above that of the untreated control. Neutral sugars were measured to examine potential shifts in the X/G ratio throughout fermentation, as affected by chemical treatment. Despite the hydrolytic and oxidative nature of the AHP treatment, the relative concentrations of detectable neutral sugars in AHP-treated WS were similar to those of the other WS substrates except for arabinose, which was 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). While glucose was always available and presumably being utilized, two distinct xylose pools may exist, one readily available and another more refractory to microbial degradation. Between 24 and 48 h, rapid glucose utilization contributed to the increase in X/G ratios for all but the AHP-treated WS fermentations, suggesting that the refractory xylose was made more available by AHP treatment. The phenolic acids and aldehydes present in cell walls are

6 1168 LEWIS ET AL. bound to various constituents. Some are esterified to arabinoxylan of the cell wall and can be released by treatment with aqueous alkali (35). The AHP treatment may be effective in disrupting the ester linkage between phenolic acids and polysaccharides as demonstrated by a 23% reduction in concentrations of para-coumaric and ferulic acids (Table 4). Attempts to relate the presence of para-coumaric or ferulic acids to a depression in DM or cellulose degradation have produced conflicting results. DM (35) and cellulose (1, 22) degradation in vitro are depressed more by para-coumaric acid; however, ferulic acid was found to be more inhibitory to cellulose digestibility in vivo (23). These findings suggest that profound differences may occur when phenolic acids are metabolically transformed (7, 21, 35) in the more dynamic ruminal system as compared with in vitro systems. Ferulic acid was shown to be more inhibitory than para-coumaric acid to cellulose digestion when B. succinogenes was the predominant cellulolytic species in mixed ruminal cultures (21) and in pure cultures of B. succinogenes or Ruminococcus flavefaciens (7); para-coumaric acid was more inhibitory to Ruminococcus albus (7). The concentration of a particular phenolic compound and the form in which it exists in the cell wall (unbound or bound to other phenolics or sugars or both) is important in the evaluation of its inhibitory nature. In the current research, para-coumaric and ferulic acids comnprised 0.30% of the dry weight of untreated WS, far below the levels (3.0 to 9.0%, DM) found to be inhibitory (21) when unbound. Chesson et al. (7) reported these phenolics to comprise 0.48% of dried grass and 1.19% of barley straw. Cell walls of barley straw contained 1.02% total para-coumaric and ferulic acids (35). The concentration of total phenolic compounds in untreated WS was decreased 15 and 23% by NaOH and AHP, respectively. Reduced concentrations of phenolic acids as well as decreased lignin content and increased cellulose availability may have been a contributing factor in increasing the extent of DM, NDF, and ADF degradation (Table 3). In light of these results and others, the effect of AHP treatment on phenolic acid content and ester bonding of phenolics to hemicellulose warrants further research. WS crystallinity is difficult to assess by standard methods, but WS appears to be less crystalline than cotton cellulose when measured by X-ray diffraction (3). Extracted WS cellulose (C/M cellulose) may be more crystalline than natural WS cellulose due to the acid treatment used in its preparation; it should be relatively free of hemicellulose and lignin (8) compared with control WS (Table 5). Cotton is 95% cellulose (38); it has the highest crystallinity of any natural cellulosic material, estimated at 60 to 100% depending on the method of determination. Sigmacell-50 is a microcrystalline form of cellulose from which noncrystalline cellulose has been removed by mineral acid and is reported to be between 85 and 100% crystalline (11). Solka floc, a hammer-milled sulfite pulp, is less crystalline than Sigmacell, but is still highly crystalline (11). The lack of response of CF, Solka floc, and Sigmacell to treatment may be directly related to the degree of crystallinity and the low levels of lignin (Table 5). B. succinogenes S85, used to test the degradation of the various cellulosic substrates 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 APPL. ENVIRON. MICROBIOL. chemical treatment would theoretically "de-wax" the CF and allow for greatest utilization. CF contained approximately 3% hemicellulose (NDF minus ADF, Table 5) and <1.0% lignin and thus has low levels of components to potentially react with NaOH or AHP during treatment. In fact, AHP treatment tended to impair ADF degradation of the CF at all incubation times tested. Prepared celluloses and cotton often show a longer lag time for digestion than does cellulose in intact forages (37). When untreated substrates and their NaOH- or AHPtreated counterparts were incubated with mixed ruminal microorganisms (Table 7), ADF degradation of all untreated and treated substrates was higher than with B. succinogenes S85, due principally to the much higher microbial concentration in the mixed inoculum. The B. succinogenes S85 inoculum was grown on cellobiose as the energy source, which may have contributed to a prolonged lag period before fiber degradation reached maximal rates. Using mixed cultures, ADF degradation of the three substrates containing highly crystalline cellulose (CF, Sigmacell, and Solka floc) was less affected by either NaOH or AHP treatment than was WS or C/M cellulose (Table 7). In summary, AHP treatment of WS allowed greater DM, NDF, and ADF degradation in vitro, with greater apparent microbial RNA and VFA production. The neutral sugars, glucose and xylose, were more rapidly and extensively fermented during incubation following AHP treatment of WS (data not shown). To some degree, the efficacy of AHP treatment depends on the nature of the cellulose source being treated. Overall, fiber sources having greater crystallinity or lower levels of lignin and hemicellulose (CF, Sigmacell, or Solka floc) or both were less responsive to chemical treatment. Sodium hydroxide treatment of the more highly crystalline cellulose sources had a greater effect on ADF degradation than did AHP treatment when mixed ruminal microorganisms were used. These cellulose sources contain less hemicellulose and lignin, the cell wall components most affected by AHP treatment. Many agricultural residues should benefit from AHP treatment as a result of partial lignin removal by hydrolysis and oxidation. The AHP treatment of agricultural residues to increase in vitro and in vivo cellulose utilization is superior to previously used chemical treatments and provides a useful tool for direct practical and basic research applications. LITERATURE CITED 1. Akin, D. E Forage cell wall degradation and p-coumaric, ferulic and sinapic acids. Agron.l. 74: Association of Official Analytical Chemists Nitrogen analysis, p In S. Williams (ed.), Official methods of analysis, 14th ed. The William Byrd Press, Inc., Richmond, Va. 3. Baker, T. I., G. V. Quicke, 0. G. Bentley, R. R. Johnson, and A. L. Moxon The influence of certain physical properties of purified celluloses and forage celluloses on their digestibility by rumen microorganisms in vitro. J. Anim. Sci. 18: Ben-Ghedalia, D., and J. Miron Effect of sodium hydroxide, ozone and sulphur dioxide on the composition and in vitro digestibility of wheat straw. J. Sci. Food Agric. 32: Ben-Ghedalia, D., and J. Miron The response of wheat straw varieties to mild sulphur dioxide treatment. Anim. Feed Sci. Technol. 10: Caldwell, D. R., and M. P. Bryant Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14: Chesson, A., C. S. Stewart, and R. J. Wallace Influence of plant phenolic acids on growth and cellulolytic activity of rumen bacteria. Appl. Environ. Microbiol. 44: Crampton, E. W., and L. A. Maynard The relation of

7 VOL. 54, 1988 MICROBIAL UTILIZATION OF AHP-TREATED CELLULOSE SOURCES 1169 cellulose and lignin content to the nutritive value of animal feeds. J. Nutr. 15: Dehority, B. A Effect of particle size on the digestion rate of purified cellulose by rumen cellulolytic bacteria in vitro. J. Dairy Sci. 44: Dehority, B. A., and R. R. Johnson Effect of particle size upon the in vitro cellulose digestibility of forages by rumen bacteria. J. Dairy Sci. 44: Fan, L. T., Y. H. Lee, and D. H. Beardmore Mechanism of the enzymatic hydrolysis of cellulose: effects of major structural features of cellulose or enzymatic hydrolysis. Biotechnol. Bioeng. 22: Goering, H. K., L. W. Smith, P. J. Van Soest, and C. H. Gordon Digestibility of roughage materials ensiled with sodium chlorite. J. Dairy Sci. 56: Goering, H. K., and P. J. Van Soest Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agriculture Handbook 379. U.S. Government Printing Office, Washington, D.C. 14. Gould, J. M Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccarification. Biotechnol. Bioeng. 26: Halliwell, G., and M. P. Bryant The cellulolytic activity of pure strains of bacteria from the rumen of cattle. J. Gen. Microbiol. 32: Hartley, R. D., and H. Buchan High performance liquid chromatography of phenolic acids and aldehydes derived from plants or from the decomposition of organic matter in soil. J. Chromatogr. 180: Hiltner, P. A., and B. A. Dehority Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria. Appl. Environ. Microbiol. 46: Holdeman, L. V., E. P. Cato, and W. E. C. Moore Anaerobic laboratory manual, 4th ed. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg. 19. Jackson, M. G Review article: The alkali treatment of straws. Anim. Feed Sci. Tech. 2: John, A Effects of feeding frequency and level of feed intake on chemical composition of rumen bacteria. J. Agric. Sci. 102: Jung, H. G Inhibition of structural carbohydrate fermentation by forage phenolics. J. Sci. Food Agric. 36: Jung, H. G., and G. C. Fahey, Jr Interactions among phenolic monomers and in vitro fermentation. J. Dairy Sci. 66: Jung, H. G., and G. C. Fahey, Jr Influence of phenolic acids on forage structural carbohydrate digestion. Can. J. Anim. Sci. 64(Suppl.): Jung, H. G., G. C. Fahey, Jr., and N. R. Merchen Effects of ruminant digestion and metabolism on phenolic monomers of forages. Br. J. Nutr. 50: Kerley, M. S., G. C. Fahey, Jr., L. L. Berger, J. M. Gould, and F. L. Baker Alkaline hydrogen peroxide treatment unlocks energy in agricultural by-products. Science 230: Lesoing, G., T. Klopfenstein, I. Rush, and J. Ward Chemical treatment of wheat straw. J. Anim. Sci. 51: Lewis, S. M., D. P. Holzgraefe, L. L. Berger, G. C. Fahey, Jr., J. M. Gould, and G. F. Fanta Alkaline hydrogen peroxide treatments of crop residues to increase ruminal dry matter disappearance in sacco. Anim. Feed Sci. Technol. 17: Lewis, S. M., M. S. Kerley, G. C. Fahey, Jr., L. L. Berger, and J. M. Gould Use of alkaline hydrogen peroxide-treated wheat straw as an energy source for the growing ruminant. Nutr. Rep. Int. 35: Miron, J., and D. Ben-Ghedalia Effect of hydrolyzing and oxidizing agents on the composition and degradation of wheat straw monosaccharides. Eur. J. Appl. Microbiol. Biotechnol. 15: Neilson, M. J., and J. A. Marlett A comparison between detergent and nondetergent analyses of dietary fiber in human foodstuffs, using high-performance liquid chromatography to measure neutral sugar composition. J. Agric. Food Chem. 31: Saeman, J. F., W. E. Moore, and M. A. Millett Sugar units present: Hydrolysis and quantitative paper chromatography, p In R. L. Whistler (ed.), Methods in Carbohydrate Chemistry. Vol. 3. Academic Press, New York. 32. Scott, H. W., and B. A. Dehority Vitamin requirements of several cellulolytic rumen bacteria. J. Bacteriol. 89: Statistical Analysis Systems SAS user's guide: statistics. Statistical Analysis Systems Institute, Inc., Cary, N.C. 34. Supelco, Inc 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 Effect of phenolic acids and phenolics from plant cell walls on rumen-like fermentation in consecutive batch culture. Appl. Environ. Microbiol. 53: Tilley, J. M., and R. A. Terry A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassland Soc. 18: Van Soest, P. J The uniformity and nutritive availability of cellulose. Fed. Proc. 32: Young, R. A Structure, swelling and bonding of cellulose fibers, p 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 A rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66: