Effects of Alkaline Hydrogen Peroxide Treatment on In Vitro

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1988, p. 1163-1169 0099-2240/88/051163-07$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 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 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

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

* Corresponding author. t Present address: The Bionetics Corporation, National Center for Toxicological Research, Jefferson, AR 72079.

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LEWIS ET AL.

APPL. ENVIRON. MICROBIOL.

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 SL4 . .................. Vitamin mixd ................... Hemin solne................... Resazurin ...................

0.4 33.0 33.0 1.0 2.0 0.25 0.10 Yeast extract (wt/vol) ................... 0.05 0.05 Trypticase (wt/vol) ................... Na2CO3 (wt/vol) ................... 1.67 0.05 Cysteine-HCI-H20 (wt/vol) ................... VFAf................... 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, 4 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-

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-1220-1% 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

VOL. 54, 1988

MICROBIAL UTILIZATION OF AHP-TREATED CELLULOSE SOURCES

TABLE 2. Chemical composition of washed WS substrates (experiment 1) NDF

WS WS + H20 WS + NaOH WS + AHP

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

% (DM basis)

Treatmenta

84.0 82.9 81.7 75.5

ADF

59.8 52.9 61.2 64.4

ADL

8.9 9.3 7.5 3.6

N

0.30 0.34 0.24 0.18

1165

Celluloseb

Ash

48.1 48.5 57.5 61.8

4.4 5.0 5.4 7.3

Item

12 h

DM

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

b

NDF

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

% Degradation'

Treatment"

ADF

18 h

24 h

36 h

48 h

3.9a

9.4a

15.2a 16.3a

25. la

3.0a

35.6b

55.8b


1.7a 1.9a 4.4a.b

10.2a

8.9a 24 ob

9.7b

21.2b

38.3c

52.3c

73.2c

SEM

1.66

2.38

2.94

2.29

3.17


0.0 2.3 3.7 1.4

3.3 4.1 3.8 4.2

4.5a

7.1a.b 7.4a.b 14.3b

8.0a 6.2a 18 oa

11.7a 12.3a

52.8b

63.2c

SEM

2.13

2.97

2.17

5.89

2.61


0.1 2.8 1.6 4.7

1.6 2.6 2.2 6.2

11.0a 12.la 28.la

72.6b

14.4a 14.2a 35.8a 72 lb

SEM

2.35

4.80

8.21

7.45

7.1 7.3 9.7 21.9 5.32

25.3a

31.5b

"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

APPL. ENVIRON. MICROBIOL.

LEWIS ET AL.

1166

1.0 -

0.9

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

0-0 Ws

0-* WS + H20

-

A-A WCZ J KInNluW Li vv.a

LI

a 0.8-, 0.7-. 0 0.6

A-A

Fr

IN UV

WS + AHP

Substrate and treatment"

.o 0.4 0.3 0.2A 0. 1 0.0

24 36 48 Time (h) FIG. 1. X/G ratios for substrates and residues following in vitro fermentation.

12

0

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

SEM

Phenolic compound WS

Protocatechuic acid

para-Hydroxyben-

+

WS H20

+

WS NaOH

9.1 8.2

6.6 5.6

4.7 4.4

15.2a

12.5a

6.5b

+

WS AHP

0 0

4.6 4.8

zoic acid

para-Hydroxybenzaldehyde

para-Coumaric acid Ferulic acid aCell walls

were

2.4

5.5 10.4 13.8 5.3 10.2 9.8 14.7 37.1 15.3 2,077.7a 1,934.4ab 1,979.4a 1,679.8b 144.0 905.9a 928.6a 511.4b 626.6b 86.1 14.1 11.3 26.0

Vanillic acid Syringic acid Vanillin

10.1a.b

prepared

as

18.4 5.1 10.5

NDF (13).

See text for explanation of treatments. Means in a row without common superscripts differ (P < 0.10). b

NDF

% (DM basis) ADL ADF

Ash

54.4

7.5

7.6

64.0 66.9

9.5 5.6

4.0 4.6

87.9

1.2

10.5

90.1

0.7

3.5

0.3

2.1

WS WS + NaOH WS + AHP

80.2 93.0 89.4

C/M cellulose C/M cellulose + NaOH C/M cellulose + AHP

90.1 94.4

96.6

90.4

CF CF + NaOH CF + AHP

96.0

93.0

0.7

0.9

96.2 97.0

95.5

0.5

0.4

95.5

0.4

0.6

Solka floc Solka floc + NaOH Solka floc + AHP

98.9 98.7 99.4

91.3 92.3 95.3

4.1 4.9 2.2

0.2 0.2 0.2

Sigmacell Sigmacell + NaOH Sigmacell + AHP

98.3 98.1 99.1

97.7 97.2 98.2

0.3 0.4

0.04 0.08

0.4

0.13

" 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