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In vitro digestibility of barley and wheat straws treated with hydrogen peroxide, sodium hydroxide and sodium peroxide under various conditions
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ANIMAL FEED SCIENCE AND TECHNOLOGY
~
E LS EV 1ER
Animal Feed Science and Technology 50 (1994) 1- 15
In vitro digestibility of barley and wheat straws treated with hydrogen peroxide, sodium hydroxide and sodium peroxide under various conditions A.S. C h a u d h r y * , E . L . M i l l e r Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0JQ, UK Received 14 October 1993; accepted 25 May 1994
Abstract Five factorial experiments were conducted to evaluate the use of different amounts (0600 g kg- t ) of hydrogen peroxide ( H 2 0 2 ) in improving the in vitro digestibility of barley and wheat straws under different pH (5.6, 11.5 ), storage times (2, 7, 14, 2 l, 28 days) and straw/liquid ratios (8:1, 4:1, 2:1, l:l, 1:6). The use of NaOH (50 g k g - ' straw at 8:1, 4:1 and l:l ratios) and Na202 ( 15 and 45 g kg -~ straw at 1:1 ratio) was also tested. In vitro digestibility of untreated (UBS) and ammonia-treated barley straw increased linearly (Experiment 1 ) with increase in H202 at pH 11.5 (alkaline H202, AHP). In Experiment 2, however, the digestibility of UBS increased curvilinearly (P < 0.001 ) with increase in AHP. In contrast digestibility decreased curvilinearly (P < 0.001 ) when H202 was applied at pH 5.6 (Experiment 2). In vitro digestibility increased with AHP in a dose related manner at different storage times (Experiments 3 and 4). In vitro digestibility of wheat straw was also increased (P < 0.001 ) with AHP treatments. However, the effect was not as great as with Na202 (45 g kg-~ ) or NaOH (50 g kg-J ) when compared at same ratios of l:l (Experiment 5), NaOH being the most effective (P < 0.001 ) in improving digestibility. The full potential of AHP was perhaps masked by inhibitory soluble products released from straws which may have hampered the in vitro microbial digestion. Further studies are suggested to exploit the full potential of AHP.
1. Introduction H y d r o g e n p e r o x i d e ( H 2 0 2 ) significantly increased in vitro dry m a t t e r ( D M D ) a n d cell wall digestibility ( C W D ) o f cereal straw ( C h a u d h r y a n d Miller, 1994). * Corresponding author: Division of Animal Health and Husbandry, University of Bristol, Langford, BSl8 7DU, UK. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10377-8401 ( 94 )00685-3
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A.S. Chaudhry, E.L. Miller/Animal FeedScience and Technology50 (1994) 1-15
However, the degree of improvement was not very great. H202 is known to play an important role in degrading lignin (Crawford and Crawford, 1984; Gould, 1984; Mackie and Kistner, 1985; Hartley et al., 1987) under certain conditions. High alkaline pH is one of these conditions at which H202 is split to give the hydroxyl free radical which then reacts with various resistant bonds present in the lignin molecule (Chaudry, 1990). Gould (1985) found pH 11.5 most effective for increasing straw degradability in a short (24 h ) in vitro incubation. However, his use of very large amounts of H202 (500 g kg-~ straw) and liquid (50 1 kg-~ straw) would limit practical application. Therefore, a series of in vitro experiments was conducted to examine if H202 can effectively be applied, at pH 11.5, to cereal straws under conditions easy to scale up. Sodium hydroxide (NaOH) and sodium peroxide (Na202) were also employed to compare their effects with those of H202 treatments.
2. Materials and methods 2.1. Substrate used
The medium (ground through a 1.4 m m sieve) fraction of the two types, untreated- (UBS) and ammonia-treated (ABS) barley straw, was used in Experiment 1 whereas only UBS (medium) was used in Experiments 2-4. In Experiment 5 however the Avalon variety of wheat straw (WS) was used as wheat straw has been (Reeves III, 1985 ) the most responsive substrate to all the oxidizing treatments. Rectangular bales of the wheat straw were procured from Cambridge University Farm in Autumn 1986. The straw bales were chopped by a SKIOLD chopper (SAEBY, Denmark) through a 16 m m sieve, a practical size for 'onfarm' chemical treatments. In Experiments 1-3, duplicate samples of only 0.5 g straw were used for each chemical treatment. In contrast the samples were increased to 10 g and 1 kg for each chemical treatment in Experiments 4 and 5, respectively. 2.2. Preparation o f chemical solutions 2.2.1. H202 Solutions of 0.078, 0.294, 0.588, 1.176, 1.764, 2.353 M were freshly prepared by diluting the 30% solution (Everrard Ltd. Cheshire, UK) in distilled water or in 0.1 M citrate-phosphate (CP) buffer (pH 5.6). Alkaline hydrogen peroxide (AHP) solutions were prepared by adding 10 M NaOH (Everrard Ltd. Cheshire, UK), drop by drop, into the H202 solutions to achieve pH 11.5; 8.82 M AHP was prepared by adjusting the pH of 30% H202 with 10 M NaOH. 2.2.2. Alkali blank (0 M AHP) Distilled water was adjusted to pH 1 1.5 by adding 10 M NaOH.
A.S. Chaudhry, E.L. Miller/Animal FeedScience and Technology50 (1994) 1-15 2.2.3. CP buffer (pH 5.6) CP buffer was prepared as described by Dawson et al. ( 1986 ). 2.2.4. Na202 The powder form of Na202 was used. 2.3. Chemical treatments 2.3.1. General description The test straw (ABS or UBS or WS) was placed in a centrifuge tube or in a beaker or on a plastic sheet, test solutions were added, mixed by hand and were kept open in tubes or were kept closed in beakers and plastic bags at room temperature for the required reaction times. After the completion of the reactions the samples were either filtered and washed (Experiments 1-3) as described by Chaudhry and Miller (1994) or the whole material (Experiments 4 and 5 ) was freeze dried, ground by hammer mill followed by a laboratory mill (Cyclotech, Tecator Ltd.) through a 1 m m sieve and then 0.5 g of this ground straw was weighed into centrifuge tubes in duplicate for the determination of in vitro DMD and CWD. 2.3.2. Experiment 1 This experiment was planned as a 2 × 4 factorial design, in duplicate, incorporating two types of straw (ABS and UBS) with four levels of AHP (0, 15, 60, and 600 g kg -~ straw) being provided by adding 3 ml of 0, 0.078, 0.294 and 2.94 M solutions, respectively, to each 0.5 g straw samples. The ratio of straw/liquid was 1:6. After mixing the contents by swirling, the centrifuge tubes were stood for 40 h at room temperature, with occasional shaking by hand. UBS samples were reacted with the same levels of AHP for 40 h at room temperature followed by filtration and washing and then analysed for acid detergent lignin content as described by Goering and Van Soest (1970). 2.3.3. Experiment 2 The experiment was planned to see the effect o f p H ( 5.6, 11.5 ) and strength of H202 solutions. Intermediate strengths of H202 (0.588, 1.176, 1.764, and 2.353 M) in addition to those used in Experiment 1, were tested using UBS only. Previous work with H202 alone did not show any significant interaction of the improvement in digestibility with straw type (Chaudhry and Miller, 1994). The experiment was arranged as a 2 × 8 factorial design, in duplicate, incorporating two pH levels and eight H202 solutions. H202 solutions were prepared as described earlier; 3 ml of solution were added to each of the tubes containing 0.5 g of straw to provide 0, 15, 60, 120, 240, 360, 480 and 600 g H202 kg- 1 straw. The remaining procedure was as described in Experiment 1.
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A.S. Chaudhry, E.L. Miller/Animal FeedScience and Technology50 (1994) 1-15
2.3.4. Experiment 3
The amount of AHP used in Experiments 1 and 2 was very high (up to 600 g kg- ~ straw) whereas small quantities may equally be successful if the treatment conditions are changed. Therefore, an experiment was planned to assess the efficacy of low levels of AHP at the same straw/liquid ratio (1:6) for longer durations. A 3 X 3 factorial design, in duplicate, was used in this experiment, incorporating three solutions (0, 0.294 M, 0.588 M) of AHP and three storage times (2, 7 and 14 days); 2 days were used instead of 40 h on the assumption that there was no significant difference between 40 and 48 h (2 days). Three millilitres of each solution were added into the centrifuge tubes containing 0.5 g of straw. The estimated H202 being provided by these solutions was 0, 60, 120 g kg-1 straw. The contents were thoroughly mixed in the tubes which were then kept at room temperature for the designated times. After each storage time the test materials were subjected to in vitro digestibility followed by chemical analysis as described in the previous experiments. 2.3.5. Experiment 4
The amounts of liquid used in Experiments 1, 2 and 3 were very high (6 ml g- 1 straw) which may be a limiting factor in scaling-up the technique. An experiment was therefore planned to see the effect of applying nearly similar amounts (per unit of substrate) of AHP using smaller volumes of concentrated solutions for various durations, on the DMD and CWD of straw. The effect of different straw/ liquid ratios with same amount of AHP was also tested. A 4 × 2 × 5 factorial design, in duplicate, was arranged by applying four sol-ations (0, 0.588, 2.353, 8.82 M) of AHP on each 10 g sample of straw, each with two levels of liquid (5 ml, 10 ml) and then storing the treated samples for five different durations (2, 7, 14, 21 and 28 days). Ten grams of straw sample were weighed into each of 80 400-ml beakers; 5 ml of various AHP solutions were added to all the beakers. For the series at a total volume of 10 ml, 5 ml alkaline solution (0 M at pH 11.5) were added. The calculated amounts of H202 provided through these solutions were 0, 10, 40 and 150 g of H202 for each kg of straw. The straw/liquid ratios were 2:1 and 1:1 for added amounts of 5 ml and 10 ml liquid, respectively. After thorough mixing of the solution and straw samples, the beakers were covered with cling film and kept at room temperature for the required times. 2.3.6. Experiment 5
In this experiment, effectiveness of AHP treatment in improving the digestibility of wheat straws was evaluated at different levels of H202, in combination with various levels of NaOH and at different straw/liquid ratios. A much larger sample size was used. Na202 is known to generate H202 and NaOH when it is mixed with water, so this chemical was also applied to evaluate its effects compared with those treated with AHP. The amount of NaOH used to achieve pH 11.5 was substantial. As NaOH alone, used at 50 g kg- ~ straw, is known to improve digestibility, the effect of NaOH alone was also compared with that of AHP.
A.S. Chaudhry, E.L. Miller/AnimalFeedScience and Technology50 (1994) 1-15
5
A completely randomized design was followed in treating straws with 12 different solutions in duplicate. The test straws were prepared by applying the chemicals using hand sprayer to each 1 kg straw in the following combinations. ( 1 ) Control (A): untreated chopped wheat straw served as a control. (2) NaOH treatment (50 g NaOH kg- ~straw): three straws were prepared to test the effect of increasing moisture levels on the action of NaOH in improving the digestibility. The required amount of NaOH was provided by adding 10 M NaOH solution to each of the following combinations: (B) straw+ 125 ml NaOH (straw/liquid, 8:1); (C) straw+ 125 ml NaOH+ 125 ml water (4:1); (D) straw+ 125 ml NaOH+875 ml water (1:1). (3) Na202 treatment: Na202 was sprinkled onto the wet straw as follows: (E) straw+ 1 I water+ 15 g Na202 ( l:l ); (F) straw+ 1 I water+ 45 g Na202 ( l:l ). (4) AHP treatments: AHP treated straws were prepared as described below. The straw/liquid ratios in parentheses were not exactly as shown but were the nearest approximates: (G) straw + 20 ml NaOH + 250 ml 2.352 M H202 (4:1); (H) straw+20 ml NaOH+ 1000 ml 0.588 M H202 ( l:l ); (I) straw+20 ml NaOH+267 ml 8.820 M H202 (4:1); (J) straw+20 ml NaOH+ 1000 ml 2.352 M H202 ( 1:1 ); (K) straw+ 80 ml NaOH+ 267 ml 8.820 M H202 (4:1); (L) straw+ 80 ml NaOH+ 1000 ml 2.352 M H202 ( l:l ). The above test straws were thoroughly mixed by hand, filled into plastic bags, tied up and stored in an open shed for 21 days for maximum reaction. 2.4. Chemical analysis
Neutral detergent fibre (NDF) representing total cell wall of the test straws was determined as described by Goering and Van Soest (1970). 2.5. D M D and C W D
In vitro digestibility was determined by the method of Van Soest et al. (1966) as described by Chaudhry and Miller (1994). 2.6. Statistical analysis
The results were subjected to analysis of variance (ANOVA) using Genstat (Lawes Agricultural Trust, 1984). Individual comparisons between treatments were made using non-orthogonal and orthogonal polynomials.
3. Results
3.1. D M and N D F composition o f A B S and UBS
The straws contained 878 g DM kg- ~and 868 g DM kg- ~and 832 g NDF kgand 809 g NDF kg- ~DM for UBS and ABS, respectively.
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A.S. Chaudhry, E.L. M i l l e r / A n i m a l Feed Science and Technology 50 (1994) 1-15
Table 1 Experiment 1. Effect of various levels of alkaline hydrogen peroxide (AHP) at a fixed straw/liquid (1:6) on lignin composition and in vitro DMD and CWD (g kg -~ DM) of ammonia-treated (ABS) and untreated (UBS) barley straws (means with standard error of difference, SED) Treatments
DMD
Straw + distilled water at pH 11.5 AHP (g kg - l straw) Straw+ 15 (0.078 M) Straw+60 (0.29 M) Straw+600 (2.94 M)
CWD
Lignin
ABS
UBS
ABS
UBS
UBS
573
422
504
325
103
562 610 881
451 594 905
483 544 858
351 519 888
98 89 66
25
ND
SED
20
Linear effect of AHP
***
***
***
ND
***
ND, not determined; M, molar concentrations of AHP solutions. ***Significant at P< 0.001.
1000 IVDMD
(SED=
18.7)
~
-
-
-_Q
900"
800"
o 700"
Y
6oo-
©
©
(~
pH
II.5
•
pH
5.6 i J
8 500-
Q.
400 !
a
300-
ZOO
1
IO0 0
® i SO
i 100
i 150
i 200
i 250
i 300
i 350
i 400
i 450
i 500
, 550
j 600
HZ Oz (g/kg straw) Fig. 1. Effect of treating barley straw with H202 at a straw/liquid ratio of 1:6 for 40 h at different pH on its in vitro dry matter digestibility (IVDMD). Solid lines are values fitted according to y = a + bx + cx 2. SED, standard error of difference.
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) 1-15
7
iooo
(SED= 20.5)
IVCWD
0
904)-
800-
700 -
6oo-
(~
v
pH 11.5 /
500-
pH
5.6
]
400e',
20tl-
lO0-
0 S0
i
i
100
150
t
Z00
i
250
l
300
H 2 0 Z (g/kg
i
350
|
i
400
450
t 500
550
i 600
straw)
Fig. 2. Effect of treating barley straw with H202 at a straw/liquid ratio of 1:6 for 40 h at different pH on its in vitro cell wall digestibility (IVCWD). Solid lines are values fitted according to y = a + bx + cx 2. SED, standard error of difference.
3.2. Experiment 1 The mean D M D and CWD are presented in Table 1. Although both straw type and AHP treatment effects were significant (P < 0.001 ) the interaction between straw type and AHP was also significant (P < 0.001 ). Therefore, the means for each treatment combination are presented. D M D and CWD of ABS were slightly reduced (P > 0.05) when it was treated with 0.078 M AHP ( 15 g kg-~ straw), whereas a slightly increased response (P > 0.05) was observed when UBS was treated with the same amount of AHP. The D M D and CWD of both ABS and UBS were increased linearly (P < 0.001 ) when AHP was increased from 0 to 2.94 M (600 g kg -1 straw). The response in D M D and CWD per unit AHP declined from 0.294 M (60 g kg-x straw) to 2.94 M (600 g kg- 1 straw) compared with that of 0.078 M (15 g kg -~ straw) to 0.292 M (60 g kg -1 straw) for both ABS and UBS. The overall response per g of AHP was greater ( D M D 0.804 g; CWD 0.937 g) for UBS compared with ( D M D 0.513 g; CWD 0.589 g) for ABS. Both types of straw showed almost the same digestibility at the 2.94 M (600 g kg-~ straw) concentration of AHP; indeed a slightly better digestibility was observed when untreated straw was used as a substrate (Table 1 ). The mean g kg- 1 DM values of acid detergent lignin are presented in Table 1. Lignin decreased in treated straw as the concentration of AHP increased. Using
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994} 1-15 800
I I IVDMD
(S~D=16,7)
[]
I)t. k}T~'
[]
r,~!g \lip
•
12(~g\ill'
700-
600-
500
40O
g c3
300-
,~00.
100
O. ?
7
Storage
time
14
(days)
Fig. 3. Effect of storage time and AHP (g kg- ' straw) on in vitro dry matter digestibility (IVDMD) of barley straw. SED, standard error of difference.
2.94 M AHP, lignin decreased by 36% but even using 0.294 M AHP the reduction was 14%. 3.3. Experiment 2 The DMD and CWD increased curvilinearly (P < 0.001 ) with the increase in concentration of H202 at pH 11.5 (Figs. 1 and 2). In contrast these values decreased curvilinearly (P < 0.001 ) when the same concentrations were applied at pH 5.6 (Figs. 1 and 2). The response in DMD and CWD per unit H202 at pH 11.5 was greater when 0 (0 g kg-i straw) to 0.588 M ( 120 g kg-1 straw) was compared with 0.588 ( 120 g kg-~ straw) to 2.94 M (600 g kg-~ straw). Maximum percent increase ( 117.5 DMD; 184.8 CWD) was found in samples treated with 2.352 M H202 (480 g kg -~ straw) solution at pH 11.5. No further response was observed when concentration was increased to 2.94 M (600 g kgstraw). In contrast at pH 5.6, maximum decrease ( - 6 1 . 3 % DMD; -88.4% CWD) was observed in samples treated with 2.352 M H202 with no further decrease, rather a slight increase, at 2.94 M H202.
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) 1-15
Y
a
2
Z Storage
time
14 (days)
Fig. 4. Effect of storage time and AHP on in vitro cell wall digestibility (IVCWD) of barley straw. SED, standard error of difference.
3.4. Experiment 3 The storage t i m e × A H P interaction was significant (P < 0.001 ) and so the DMD and CWD for each treatment combination together with their SED are given in Figs. 3 and 4, respectively. AHP increased (P < 0.001 ) DMD and CWD at each storage time. The increase was greater with 120 g than with 60 g AHP kg-1 straw. With 60 g AHP kg-t straw DMD and CWD increased with storage time. However, with 120 g AHP kg-~ straw DMD and CWD were greatly increased at 2 days, lower at 7 days than at 2 days (P < 0.001 ) and then increased again (P < 0.001 ) at Day 14 compared with Day 7. A small increase in DMD and CWD was apparent after 7 days with the control treatment of 0 AHP (water at pH 11.5) but not at 14 days. In summary, the storage time enhanced the effect of 60 g but not 120 g AHP kg- l straw.
3.5. Experiment 4 The mean DMD and CWD for all treatment combinations are presented in Table 2. Although the main effects o f A H P (P < 0.001 ), storage time (P < 0.001 )
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A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) 1-15
Table 2 Experiment 4. Effect of various levels of alkaline hydrogen peroxide (AHP) when applied at different straw/liquid ratios for different days on the in vitro dry matter (DMD) and cell wall (CWD) digestibility (g kg - ~DM ) of barley straw (means with SED ) Parameters
DMD
CWD
AHP (g kg-I straw) 0 10 40 Straw/liquid ratio 2:1 1:1 2:1 1:1 2:1 Storagetime (days) 2 7 14 21 28 SED
346 432 421 466 338 49.7
Main effects AHP Storage time Straw/liquid
(SED 16) (SED18) (SED 11)
319 461 409 446 420
435 492 364 481 452
463 452 437 475 393
485 458 433 535 303
AHP (g kg-~ straw) 0 10 40
150
150
1:1 2:1
Straw/liquid ratio 1:1 2:1 1:1 2:1 1:1 2:1
1:1 2:1
1:1
486 472 462 526 491
539 465 544 521 387
410 386 374 444 408
469 380 478 454 294
462 439 486 551 384
P<0.001 P<0.001 P<0.10
238 334 324 391 229 56.6
206 367 309 358 322
(SED 18) (SED20) (SED 13)
350 357 266 392 380
384 361 353 383 302
392 368 336 455 199
378 347 405 473 289
P<0.001 P<0.001 P<0.05
Table 3 Experiment 4. Effect of storage time × AHP (alkaline hydrogen peroxide) interaction on the in vitro dry matter (DMD) and cell wall (CWD) digestibility (g kg-1 DM) of barley straw (means with SED) Storage time (days)
DMD
CWD
AHP (g kg-lstraw) 0 10 40
150
AHP (g kg-lstraw) 0 10 40
150
2 7 14 21 28
333 447 415 456 379
501 452 515 536 385
222 350 316 375 276
423 363 441 463 291
SED
35*
449 472 400 478 423
486 465 448 531 397
367 359 310 388 341
401 377 355 450 304
40*
* Significant at P< 0.05.
and straw/liquid ratios ( D M D P < 0.1; CWD P < 0.05) were significant the interaction between storage time and AHP was also significant ( D M D P < 0.030; CWD P < 0.025). Therefore, the results of this interaction, presented in Table 3, are examined more carefully. None of the other two or three way interactions were significant (P > 0.05 ).
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) I-15
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Table 4 Experiment 5. Effect of various levels of alkaline hydrogen peroxide (AHP), NaOH and NazOz on the in vitro dry matter ( D M D ) and cell wall digestibility (CWD) of wheat straw (means with SED) Parameters
Treatments A
Chemicals used (g kg- 1 straw) NaOH H2Oz NazO/ Straw/liquid Digestibility (g kg- J DM ) DMD CWD
B
. . -
C
SED D
E
F
G
H
I
J
K
50 50 50 8 8 8 8 32 . . . . . 20 20 80 80 80 . . . 15 45 . . . . . . 8:1 4:1 l:l 1:1 1:1 4:1 1:1 4:1 1:1 4:1
L
32 80 1:1
551 739 789 811 637 783 628 543 645 626 735 694 415 660 748 768 546 729 499 402 537 516 661 601
11"** 12"**
***Significantat P < 0.001.
DMD and CWD increased with AHP in a dose related manner at 2 and 14 days of storage (P < 0.001 ) but changes at other times were not significant. The control solution (0 AHP at pH 11.5) increased DMD and CWD at 7 and 21 days over that observed at 2 days (P < 0.001 ) Maximum percent improvements (DMD 61.23; CWD 108.7) were observed when samples, treated with 150 g of AHP, were stored for 21 days. A significant depression (P < 0.001 ) was observed in almost all the samples treated with any level of AHP, except in those treated with 10 g of AHP, where it was non-significant (P > 0.05 ), when storage time was increased from 21 to 28 days. Increasing the volume of liquid at a constant amount of AHP increased DMD on average by 20 g kg- t units (P=0.078 SED 11.1 ) and CWD by 27 g kg- l (p < 0.05 SED 12.7).
3.6. Experiment 5 The mean DMD and CWD are given in Table 4. The treatments significantly (P < 0.001 ) affected both DMD and CWD of wheat straw. The DMD and CWD of wheat straw showed positive responses to almost all the treatments except those of treatment H where a slight negative, though non-significant (P > 0.05 ), response was obtained. Maximum percent improvement (47.34, DMD; 85.06, CWD ) was observed in Samples (D) treated with 50 g NaOH alone at low ( 1:1 ) straw/liquid ratio. The response decreased quadratically (P < 0.001, DMD and CWD) with the increase in the straw/liquid ratios (Treatments B and C). In contrast significantly greater (P < 0.001 ) responses were observed when any level of H202 in combination with 8 g NaOH at the 4:1 straw/liquid ratio (Treatments G and I) was compared with that of 1:1 (H and J ). The effect of 80 g H202 (I + J ) was significantly greater (P < 0.001 ) than that of 20 g ( G + H ) when both
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A.S. Chaudhry, E.L. Miller / Animal Feed Science and Technology 5 0 (1994) 1-15
were applied in combination with 8 g NaOH. The efficiency of 80 g H202 was significantly improved (P < 0.001 ) when the added amount of NaOH was increased from 8 ( I + J ) to 32 g ( K + L ) . However, H202 appeared to be most efficient when 80 g of H202 and 32 g of NaOH were mixed with each kg of straw at a straw/liquid ratio of 4:1 (K). The effect of Na202 was significantly improved (P < 0.001 ) when the amount was increased from 15 to 45 g kg- 1 of straw. However, the improvement in samples treated with 50 g NaOH kg- 1 straw at a straw/liquid ratio of l:l was significantly greater (P < 0.001 ) than those of treated with Na202 at the same ratio. In contrast to the results of Experiment 4, the DMD and CWD were greater when small volumes of liquid were applied in Treatments G, I and K in comparison with those of H, J and L.
4. Discussion AHP treatment was effective in improving the in vitro digestibility of straw in these experiments. Similar effectiveness was reported by Kerley et al. (1986) who examined the effect of AHP-treated wheat straw on the site and extent of digestion in sheep. They noticed significantly increased DMD and NDF digestibility (NDFD) when AHP treated straw was fed to wethers in different straw/ concentrate proportions, pH-regulated AHP treatment showed greater percent increase in the in vivo NDFD and cellulose digestibility than that of DMD in experiments conducted by Kerley et al. (1987). Similar responses are reflected in the current findings (Figs. 1 and 2). In contrast, treatment with H202 at pH 5.6 reduced digestibility possibly due to residual H202 or the formation of some substances which inhibited microbial digestion in the in vitro system. Almost equal (Table 1 ) responses of ABS and UBS to high AHP (60 and 600 g kg- l straw), in terms of digestibility, are in agreement with those of Bhargava et al. (1989) who studied the effect of suspending wheat straw in 1% AHP solution on its in sacco degradability. They observed increased potential degradability in both UBS and ABS treated with AHP and supported the potential use of AHP in improving the straw digestibility. Improvement in the digestibility of crop residues by AHP treatment is mainly mediated through cell wall modifications, i.e. lignin degradation. Gould (1984) demonstrated that soaking wheat straw in AHP at p H I 1.5 released about one half of the lignin as water soluble products. Similar effects of AHP on the lignin composition of straw are shown in Experiment 1 (Table l ). However, the magnitude of reduction (14%) was lower than that ( > 50%) reported by Gould (1985) which can perhaps be attributed to the low AHP (60 g kg-1 vs. 500 g kg- i straw). Even at 600 g AHP kg- 1 straw, an AHP level close to that used by Gould ( 1985 ), lignin reduced to only 36%. The lower effect of AHP in reducing lignin was possibly due to the higher ( 1:6 vs. 1:50) straw/liquid ratio and differences in the preparation of the treated residues. However, increasing AHP at a constant straw/liquid ratio of 1:6 increased DMD and CWD up to 900 g kg-1
A.S. Chaudhry, E.L. Miller / Animal Feed Science and Technology 5 0 (1994) 1-15
13
(Table 1 and Figs. 1 and 2) similar to values achieved by Gould ( 1985 ). AHP was least effective at low levels (15 g kg-1 straw) which is in agreement with those reported by Flachowsky and Sundstol ( 1988 ) and Adebowale et al. ( 1989 ) who did not find any significant improvements in the in vitro DMD (IVDMD) and rumen DMD when straws treated with AHP were compared with their NaOH controls. However, the present work clearly shows that increasing H202 concentration at pH 11.5 greatly increases digestibility compared with the control maintained at pH 11.5. Furthermore, the DMD and CWD of UBS treated with AHP under the same conditions are shown to be repeatable (Table 1, Figs. 1 and 2 ). While AHP was effective at very high levels (Experiments 1 and 2) the response was less dramatic at lower levels ( < 200 g AHP kg -1 straw). This can partly be compensated by extending the time of storage/reaction (Experiments 3 and 4). A positive effect of increasing storage time is clearly presented in Experiment 3 (Figs. 3 and 4). Shahiduzzaman and Owen (1987) also demonstrated similar effects on digestibility when Ca (OH)2-treated straws were stored for longer durations. However, in Experiment 4 (Tables 2 and 3) the proportional increase in the digestibility was not maintained with the increase in storage time, rather poor digestibilities were noted compared with those of Experiment 3. The low responses of DMD and CWD towards AHP may be attributed to the release of certain inhibitory substances like phenolic acids which remained in the unwashed samples and probably affected the ultimate digestibility. This was also supported by Lewis et al. ( 1987 ) who observed lower NDFD and ADFD values in the unwashed samples of AHP treated wheat straw retaining the solubles. The IVDMD values of NaOH in Experiment 5 are closer to those reported by Wanapat et al. ( 1985, 1986) and Adebowale et al. (1989). Various straw/liquid ratios did affect the IVDMD and in vitro CWD (IVCWD) which is in agreement with those stated by Wanapat et al. ( 1986 ) where they found NaOH as the most effective, amongst various treatments, in improving the digestibility of straw when applied together with plenty of liquid. Increased digestibility obtained from samples treated with either amounts of Na202 supports the earlier findings of Jones and Klopfenstein (1967) who observed significantly increased IVDMD and in vivo DMD values in corn cobs treated with 4% solution of Na202. Improvement in the digestibility values of straws treated with AHP is in agreement with the previous findings (Chaudhry and Miller, 1990a,b). Chopped wheat straw was equally effective as a substrate (Reeves III, 1985 ). The response with AHP in Experiment 5 was not as great as with NaOH alone. Since all samples were freeze dried before being subjected to in vitro tests, a possibility that the full potential of AHP was masked by production of inhibitory soluble substances cannot be ruled out. If inhibitory soluble phenolics are released and must be washed away, as in Experiments 1, 2 and 3 according to the Gould (1985) method, the loss of soluble carbohydrates, the considerable extra cost of washing and drying and the problem of affluent disposal are the limitations which needs to be addressed before suggesting AHP as an alternative to
14
A.S. Chaudhry, E.L. Miller / Animal Feed Science and Technology 5 0 (1994) 1-15
other alkali treatments for the nutritional improvement of cereal straws (Chaudhry, 1990).
Acknowledgments ASC is grateful to Cambridge Commonwealth Trust and Government of Pakistan for their financial support.
References Adebowale, E.A., Orskov, E.R. and Horton, P.M., 1989. Rumen degradation of straw. 8. Effect of alkaline hydrogen peroxide treatment on degradation of straw using NaOH and ammonia as source of alkali. Anim. Prod., 48: 553-560. Bhargava, P.K., Orskov, E.R. and Walli, T.K., 1989. Effect of soaking, ensilingand hydrogen peroxide treatment of barley straw on rumen degradability. Anim. Feed Sci. Technol., 22: 295-303. Chaudhry, A.S., 1990. Nutritional improvement of cereal straws. PhD Thesis. University of Cambridge, Cambridge, UK. Chaudhry, A.S. and Miller, E.L., 1990a. Effect of treating wheat straw with sodium hydroxide and calcium oxide alone or in combination with hydrogen peroxide on the in vitro dry matter digestibility. Anim. Prod., 50:118 (abstract). Chaudhry, A.S. and Miller, E.L., 1990b. Effect of calcium oxide, sodium hydroxide and alkaline hydrogen peroxide treatments on the nutrient digestion of wheat straw by sheep. Anim Prod., 50: 119 (abstract). Chaudhry, A.S. and Miller, E.L., 1994. Effect of H202-dependent enzyme systems on the in vitro digestibility of untreated and NH3 treated barley straws. J. Sci. Food Agric., 64:371-378. Crawford, R.L. and Crawford, D.L., 1984. Recent advances in studies of the mechanisms of microbial degradation of lignin. A Review. Enzymol. Microbiol. Technol., 6:433-442. Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M., 1986. Data for Biochemical Research. Oxford University Press, Oxford. Flachowsky, G. and Sundstol, F., 1988. Effect of NaOH and H202 on the degradability of straw in ruminants. Arch. Anim. Nutr. Berlin., 38: 955-964. Goering, H.K. and van Soest, P.J., 1970. Forage Fibre Analysis. US Dep. Agric. Handbook No. 379. US Dep. Agric., Washington, DC, USA. Gould, J.M., 1984. Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification. Biotech. Bioeng., 26: 46-52. Gould, J.M., 1985. Studies on the mechanism of alkaline hydrogen peroxide delignification of agricultural residues. Biotech. Bioeng., 27:225-23 I. Hartley, B.S., Broda, P.M.A. and Senior, P.J., 1987. Technology in the 1990s: utilization of lignocellulosic wastes. Proc. R. Soc. London. Jones, M.J. and Klopfenstein, T.J., 1967. Chemical treatment of poor quality roughages. J. Anim. Sci., 26:1492 (abstract). Kerley, M.S., Fahey, Jr., G.C., Berger, L.L, Merchen, N.R. and Gould, J.M., 1986. Effect of alkaline hydrogen peroxide treatment on site and extent of digestion of sheep. J. Anim. Sci., 53: 868-878. Kerley, M.S., Fahey, Jr., G.C., Berger, L.L. and Merchen, N.R., 1987. Effects of treating wheat straw with pH-regulated solutions of alkaline hydrogen peroxide on nutrient digestion by sheep. J. Dairy Sci., 70: 2078-2084. Lawes Agricultural Trust, 1984. Genstat IV. Rothamsted Experiment Station, Rothamsted, UK. Lewis, S.M., Holzgraefe, P.D., Berger, G.L., Fahey, Jr., G.C., Gould, J.M. and Fanta, G.C., 1987.
A.S. Chaudhry, E.L. Miller / Animal Feed Science and Technology 5 0 (1994) 1-15
15
Alkaline hydrogen peroxide treatment of crop residues to increase ruminal dry matter disappearance in sacco. Anim. Feed. Sci. Technol., 17: 179-199. Mackie, R.I. and Kistner, A. 1985. Some frontiers of research in basic ruminant nutrition. S. Aft. J. Anim. Sci., 15: 72-85. Reeves III, J.B., 1985. Lignin composition of chemically treated feeds as determined by nitrobenzene oxidation and its relationship to digestibility. J. Dairy Sci., 68:1976-1983. Shahiduzzaman, Md. and Owen, E., 1987. Treatment of barley straw with mixtures of calcium hydroxide and urea. Effect on the intake and digestibility in sheep. Anim. Prod., 44:68 (abstract). Van Soest, P.J., Wine, R.H. and Moor, L.A., 1966. Estimation of true digestibility of forages by in vitro digestion of cell walls. Proc. 10th Int. Grassld. Congr., 7-16 July 1966, Helsinki. Finnish Grassland Association, Helsinki, pp. 438-44 I. Wanapat, M., Sundstol, F. and Garmo, T.H., 1985. A comparison of alkali treatment methods to improve the nutritional value of straw. I. Digestibilityand metabolizability. Anim. Feed Sci. Technol., 12: 29-309. Wanapat, M., Sundstol, F. and Hall, J.M.R., 1986. A comparison of alkali treatment methods used to improve the nutritive value of straw. II. In sacco and in vitro degradation relative to in vivo digestibility. Anim. Feed Sci. Technol., 14:215-220.
ANIMAL FEED SCIENCE AND TECHNOLOGY
~
E LS EV 1ER
Animal Feed Science and Technology 50 (1994) 1- 15
In vitro digestibility of barley and wheat straws treated with hydrogen peroxide, sodium hydroxide and sodium peroxide under various conditions A.S. C h a u d h r y * , E . L . M i l l e r Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0JQ, UK Received 14 October 1993; accepted 25 May 1994
Abstract Five factorial experiments were conducted to evaluate the use of different amounts (0600 g kg- t ) of hydrogen peroxide ( H 2 0 2 ) in improving the in vitro digestibility of barley and wheat straws under different pH (5.6, 11.5 ), storage times (2, 7, 14, 2 l, 28 days) and straw/liquid ratios (8:1, 4:1, 2:1, l:l, 1:6). The use of NaOH (50 g k g - ' straw at 8:1, 4:1 and l:l ratios) and Na202 ( 15 and 45 g kg -~ straw at 1:1 ratio) was also tested. In vitro digestibility of untreated (UBS) and ammonia-treated barley straw increased linearly (Experiment 1 ) with increase in H202 at pH 11.5 (alkaline H202, AHP). In Experiment 2, however, the digestibility of UBS increased curvilinearly (P < 0.001 ) with increase in AHP. In contrast digestibility decreased curvilinearly (P < 0.001 ) when H202 was applied at pH 5.6 (Experiment 2). In vitro digestibility increased with AHP in a dose related manner at different storage times (Experiments 3 and 4). In vitro digestibility of wheat straw was also increased (P < 0.001 ) with AHP treatments. However, the effect was not as great as with Na202 (45 g kg-~ ) or NaOH (50 g kg-J ) when compared at same ratios of l:l (Experiment 5), NaOH being the most effective (P < 0.001 ) in improving digestibility. The full potential of AHP was perhaps masked by inhibitory soluble products released from straws which may have hampered the in vitro microbial digestion. Further studies are suggested to exploit the full potential of AHP.
1. Introduction H y d r o g e n p e r o x i d e ( H 2 0 2 ) significantly increased in vitro dry m a t t e r ( D M D ) a n d cell wall digestibility ( C W D ) o f cereal straw ( C h a u d h r y a n d Miller, 1994). * Corresponding author: Division of Animal Health and Husbandry, University of Bristol, Langford, BSl8 7DU, UK. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10377-8401 ( 94 )00685-3
2
A.S. Chaudhry, E.L. Miller/Animal FeedScience and Technology50 (1994) 1-15
However, the degree of improvement was not very great. H202 is known to play an important role in degrading lignin (Crawford and Crawford, 1984; Gould, 1984; Mackie and Kistner, 1985; Hartley et al., 1987) under certain conditions. High alkaline pH is one of these conditions at which H202 is split to give the hydroxyl free radical which then reacts with various resistant bonds present in the lignin molecule (Chaudry, 1990). Gould (1985) found pH 11.5 most effective for increasing straw degradability in a short (24 h ) in vitro incubation. However, his use of very large amounts of H202 (500 g kg-~ straw) and liquid (50 1 kg-~ straw) would limit practical application. Therefore, a series of in vitro experiments was conducted to examine if H202 can effectively be applied, at pH 11.5, to cereal straws under conditions easy to scale up. Sodium hydroxide (NaOH) and sodium peroxide (Na202) were also employed to compare their effects with those of H202 treatments.
2. Materials and methods 2.1. Substrate used
The medium (ground through a 1.4 m m sieve) fraction of the two types, untreated- (UBS) and ammonia-treated (ABS) barley straw, was used in Experiment 1 whereas only UBS (medium) was used in Experiments 2-4. In Experiment 5 however the Avalon variety of wheat straw (WS) was used as wheat straw has been (Reeves III, 1985 ) the most responsive substrate to all the oxidizing treatments. Rectangular bales of the wheat straw were procured from Cambridge University Farm in Autumn 1986. The straw bales were chopped by a SKIOLD chopper (SAEBY, Denmark) through a 16 m m sieve, a practical size for 'onfarm' chemical treatments. In Experiments 1-3, duplicate samples of only 0.5 g straw were used for each chemical treatment. In contrast the samples were increased to 10 g and 1 kg for each chemical treatment in Experiments 4 and 5, respectively. 2.2. Preparation o f chemical solutions 2.2.1. H202 Solutions of 0.078, 0.294, 0.588, 1.176, 1.764, 2.353 M were freshly prepared by diluting the 30% solution (Everrard Ltd. Cheshire, UK) in distilled water or in 0.1 M citrate-phosphate (CP) buffer (pH 5.6). Alkaline hydrogen peroxide (AHP) solutions were prepared by adding 10 M NaOH (Everrard Ltd. Cheshire, UK), drop by drop, into the H202 solutions to achieve pH 11.5; 8.82 M AHP was prepared by adjusting the pH of 30% H202 with 10 M NaOH. 2.2.2. Alkali blank (0 M AHP) Distilled water was adjusted to pH 1 1.5 by adding 10 M NaOH.
A.S. Chaudhry, E.L. Miller/Animal FeedScience and Technology50 (1994) 1-15 2.2.3. CP buffer (pH 5.6) CP buffer was prepared as described by Dawson et al. ( 1986 ). 2.2.4. Na202 The powder form of Na202 was used. 2.3. Chemical treatments 2.3.1. General description The test straw (ABS or UBS or WS) was placed in a centrifuge tube or in a beaker or on a plastic sheet, test solutions were added, mixed by hand and were kept open in tubes or were kept closed in beakers and plastic bags at room temperature for the required reaction times. After the completion of the reactions the samples were either filtered and washed (Experiments 1-3) as described by Chaudhry and Miller (1994) or the whole material (Experiments 4 and 5 ) was freeze dried, ground by hammer mill followed by a laboratory mill (Cyclotech, Tecator Ltd.) through a 1 m m sieve and then 0.5 g of this ground straw was weighed into centrifuge tubes in duplicate for the determination of in vitro DMD and CWD. 2.3.2. Experiment 1 This experiment was planned as a 2 × 4 factorial design, in duplicate, incorporating two types of straw (ABS and UBS) with four levels of AHP (0, 15, 60, and 600 g kg -~ straw) being provided by adding 3 ml of 0, 0.078, 0.294 and 2.94 M solutions, respectively, to each 0.5 g straw samples. The ratio of straw/liquid was 1:6. After mixing the contents by swirling, the centrifuge tubes were stood for 40 h at room temperature, with occasional shaking by hand. UBS samples were reacted with the same levels of AHP for 40 h at room temperature followed by filtration and washing and then analysed for acid detergent lignin content as described by Goering and Van Soest (1970). 2.3.3. Experiment 2 The experiment was planned to see the effect o f p H ( 5.6, 11.5 ) and strength of H202 solutions. Intermediate strengths of H202 (0.588, 1.176, 1.764, and 2.353 M) in addition to those used in Experiment 1, were tested using UBS only. Previous work with H202 alone did not show any significant interaction of the improvement in digestibility with straw type (Chaudhry and Miller, 1994). The experiment was arranged as a 2 × 8 factorial design, in duplicate, incorporating two pH levels and eight H202 solutions. H202 solutions were prepared as described earlier; 3 ml of solution were added to each of the tubes containing 0.5 g of straw to provide 0, 15, 60, 120, 240, 360, 480 and 600 g H202 kg- 1 straw. The remaining procedure was as described in Experiment 1.
4
A.S. Chaudhry, E.L. Miller/Animal FeedScience and Technology50 (1994) 1-15
2.3.4. Experiment 3
The amount of AHP used in Experiments 1 and 2 was very high (up to 600 g kg- ~ straw) whereas small quantities may equally be successful if the treatment conditions are changed. Therefore, an experiment was planned to assess the efficacy of low levels of AHP at the same straw/liquid ratio (1:6) for longer durations. A 3 X 3 factorial design, in duplicate, was used in this experiment, incorporating three solutions (0, 0.294 M, 0.588 M) of AHP and three storage times (2, 7 and 14 days); 2 days were used instead of 40 h on the assumption that there was no significant difference between 40 and 48 h (2 days). Three millilitres of each solution were added into the centrifuge tubes containing 0.5 g of straw. The estimated H202 being provided by these solutions was 0, 60, 120 g kg-1 straw. The contents were thoroughly mixed in the tubes which were then kept at room temperature for the designated times. After each storage time the test materials were subjected to in vitro digestibility followed by chemical analysis as described in the previous experiments. 2.3.5. Experiment 4
The amounts of liquid used in Experiments 1, 2 and 3 were very high (6 ml g- 1 straw) which may be a limiting factor in scaling-up the technique. An experiment was therefore planned to see the effect of applying nearly similar amounts (per unit of substrate) of AHP using smaller volumes of concentrated solutions for various durations, on the DMD and CWD of straw. The effect of different straw/ liquid ratios with same amount of AHP was also tested. A 4 × 2 × 5 factorial design, in duplicate, was arranged by applying four sol-ations (0, 0.588, 2.353, 8.82 M) of AHP on each 10 g sample of straw, each with two levels of liquid (5 ml, 10 ml) and then storing the treated samples for five different durations (2, 7, 14, 21 and 28 days). Ten grams of straw sample were weighed into each of 80 400-ml beakers; 5 ml of various AHP solutions were added to all the beakers. For the series at a total volume of 10 ml, 5 ml alkaline solution (0 M at pH 11.5) were added. The calculated amounts of H202 provided through these solutions were 0, 10, 40 and 150 g of H202 for each kg of straw. The straw/liquid ratios were 2:1 and 1:1 for added amounts of 5 ml and 10 ml liquid, respectively. After thorough mixing of the solution and straw samples, the beakers were covered with cling film and kept at room temperature for the required times. 2.3.6. Experiment 5
In this experiment, effectiveness of AHP treatment in improving the digestibility of wheat straws was evaluated at different levels of H202, in combination with various levels of NaOH and at different straw/liquid ratios. A much larger sample size was used. Na202 is known to generate H202 and NaOH when it is mixed with water, so this chemical was also applied to evaluate its effects compared with those treated with AHP. The amount of NaOH used to achieve pH 11.5 was substantial. As NaOH alone, used at 50 g kg- ~ straw, is known to improve digestibility, the effect of NaOH alone was also compared with that of AHP.
A.S. Chaudhry, E.L. Miller/AnimalFeedScience and Technology50 (1994) 1-15
5
A completely randomized design was followed in treating straws with 12 different solutions in duplicate. The test straws were prepared by applying the chemicals using hand sprayer to each 1 kg straw in the following combinations. ( 1 ) Control (A): untreated chopped wheat straw served as a control. (2) NaOH treatment (50 g NaOH kg- ~straw): three straws were prepared to test the effect of increasing moisture levels on the action of NaOH in improving the digestibility. The required amount of NaOH was provided by adding 10 M NaOH solution to each of the following combinations: (B) straw+ 125 ml NaOH (straw/liquid, 8:1); (C) straw+ 125 ml NaOH+ 125 ml water (4:1); (D) straw+ 125 ml NaOH+875 ml water (1:1). (3) Na202 treatment: Na202 was sprinkled onto the wet straw as follows: (E) straw+ 1 I water+ 15 g Na202 ( l:l ); (F) straw+ 1 I water+ 45 g Na202 ( l:l ). (4) AHP treatments: AHP treated straws were prepared as described below. The straw/liquid ratios in parentheses were not exactly as shown but were the nearest approximates: (G) straw + 20 ml NaOH + 250 ml 2.352 M H202 (4:1); (H) straw+20 ml NaOH+ 1000 ml 0.588 M H202 ( l:l ); (I) straw+20 ml NaOH+267 ml 8.820 M H202 (4:1); (J) straw+20 ml NaOH+ 1000 ml 2.352 M H202 ( 1:1 ); (K) straw+ 80 ml NaOH+ 267 ml 8.820 M H202 (4:1); (L) straw+ 80 ml NaOH+ 1000 ml 2.352 M H202 ( l:l ). The above test straws were thoroughly mixed by hand, filled into plastic bags, tied up and stored in an open shed for 21 days for maximum reaction. 2.4. Chemical analysis
Neutral detergent fibre (NDF) representing total cell wall of the test straws was determined as described by Goering and Van Soest (1970). 2.5. D M D and C W D
In vitro digestibility was determined by the method of Van Soest et al. (1966) as described by Chaudhry and Miller (1994). 2.6. Statistical analysis
The results were subjected to analysis of variance (ANOVA) using Genstat (Lawes Agricultural Trust, 1984). Individual comparisons between treatments were made using non-orthogonal and orthogonal polynomials.
3. Results
3.1. D M and N D F composition o f A B S and UBS
The straws contained 878 g DM kg- ~and 868 g DM kg- ~and 832 g NDF kgand 809 g NDF kg- ~DM for UBS and ABS, respectively.
6
A.S. Chaudhry, E.L. M i l l e r / A n i m a l Feed Science and Technology 50 (1994) 1-15
Table 1 Experiment 1. Effect of various levels of alkaline hydrogen peroxide (AHP) at a fixed straw/liquid (1:6) on lignin composition and in vitro DMD and CWD (g kg -~ DM) of ammonia-treated (ABS) and untreated (UBS) barley straws (means with standard error of difference, SED) Treatments
DMD
Straw + distilled water at pH 11.5 AHP (g kg - l straw) Straw+ 15 (0.078 M) Straw+60 (0.29 M) Straw+600 (2.94 M)
CWD
Lignin
ABS
UBS
ABS
UBS
UBS
573
422
504
325
103
562 610 881
451 594 905
483 544 858
351 519 888
98 89 66
25
ND
SED
20
Linear effect of AHP
***
***
***
ND
***
ND, not determined; M, molar concentrations of AHP solutions. ***Significant at P< 0.001.
1000 IVDMD
(SED=
18.7)
~
-
-
-_Q
900"
800"
o 700"
Y
6oo-
©
©
(~
pH
II.5
•
pH
5.6 i J
8 500-
Q.
400 !
a
300-
ZOO
1
IO0 0
® i SO
i 100
i 150
i 200
i 250
i 300
i 350
i 400
i 450
i 500
, 550
j 600
HZ Oz (g/kg straw) Fig. 1. Effect of treating barley straw with H202 at a straw/liquid ratio of 1:6 for 40 h at different pH on its in vitro dry matter digestibility (IVDMD). Solid lines are values fitted according to y = a + bx + cx 2. SED, standard error of difference.
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) 1-15
7
iooo
(SED= 20.5)
IVCWD
0
904)-
800-
700 -
6oo-
(~
v
pH 11.5 /
500-
pH
5.6
]
400e',
20tl-
lO0-
0 S0
i
i
100
150
t
Z00
i
250
l
300
H 2 0 Z (g/kg
i
350
|
i
400
450
t 500
550
i 600
straw)
Fig. 2. Effect of treating barley straw with H202 at a straw/liquid ratio of 1:6 for 40 h at different pH on its in vitro cell wall digestibility (IVCWD). Solid lines are values fitted according to y = a + bx + cx 2. SED, standard error of difference.
3.2. Experiment 1 The mean D M D and CWD are presented in Table 1. Although both straw type and AHP treatment effects were significant (P < 0.001 ) the interaction between straw type and AHP was also significant (P < 0.001 ). Therefore, the means for each treatment combination are presented. D M D and CWD of ABS were slightly reduced (P > 0.05) when it was treated with 0.078 M AHP ( 15 g kg-~ straw), whereas a slightly increased response (P > 0.05) was observed when UBS was treated with the same amount of AHP. The D M D and CWD of both ABS and UBS were increased linearly (P < 0.001 ) when AHP was increased from 0 to 2.94 M (600 g kg -1 straw). The response in D M D and CWD per unit AHP declined from 0.294 M (60 g kg-x straw) to 2.94 M (600 g kg- 1 straw) compared with that of 0.078 M (15 g kg -~ straw) to 0.292 M (60 g kg -1 straw) for both ABS and UBS. The overall response per g of AHP was greater ( D M D 0.804 g; CWD 0.937 g) for UBS compared with ( D M D 0.513 g; CWD 0.589 g) for ABS. Both types of straw showed almost the same digestibility at the 2.94 M (600 g kg-~ straw) concentration of AHP; indeed a slightly better digestibility was observed when untreated straw was used as a substrate (Table 1 ). The mean g kg- 1 DM values of acid detergent lignin are presented in Table 1. Lignin decreased in treated straw as the concentration of AHP increased. Using
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994} 1-15 800
I I IVDMD
(S~D=16,7)
[]
I)t. k}T~'
[]
r,~!g \lip
•
12(~g\ill'
700-
600-
500
40O
g c3
300-
,~00.
100
O. ?
7
Storage
time
14
(days)
Fig. 3. Effect of storage time and AHP (g kg- ' straw) on in vitro dry matter digestibility (IVDMD) of barley straw. SED, standard error of difference.
2.94 M AHP, lignin decreased by 36% but even using 0.294 M AHP the reduction was 14%. 3.3. Experiment 2 The DMD and CWD increased curvilinearly (P < 0.001 ) with the increase in concentration of H202 at pH 11.5 (Figs. 1 and 2). In contrast these values decreased curvilinearly (P < 0.001 ) when the same concentrations were applied at pH 5.6 (Figs. 1 and 2). The response in DMD and CWD per unit H202 at pH 11.5 was greater when 0 (0 g kg-i straw) to 0.588 M ( 120 g kg-1 straw) was compared with 0.588 ( 120 g kg-~ straw) to 2.94 M (600 g kg-~ straw). Maximum percent increase ( 117.5 DMD; 184.8 CWD) was found in samples treated with 2.352 M H202 (480 g kg -~ straw) solution at pH 11.5. No further response was observed when concentration was increased to 2.94 M (600 g kgstraw). In contrast at pH 5.6, maximum decrease ( - 6 1 . 3 % DMD; -88.4% CWD) was observed in samples treated with 2.352 M H202 with no further decrease, rather a slight increase, at 2.94 M H202.
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) 1-15
Y
a
2
Z Storage
time
14 (days)
Fig. 4. Effect of storage time and AHP on in vitro cell wall digestibility (IVCWD) of barley straw. SED, standard error of difference.
3.4. Experiment 3 The storage t i m e × A H P interaction was significant (P < 0.001 ) and so the DMD and CWD for each treatment combination together with their SED are given in Figs. 3 and 4, respectively. AHP increased (P < 0.001 ) DMD and CWD at each storage time. The increase was greater with 120 g than with 60 g AHP kg-1 straw. With 60 g AHP kg-t straw DMD and CWD increased with storage time. However, with 120 g AHP kg-~ straw DMD and CWD were greatly increased at 2 days, lower at 7 days than at 2 days (P < 0.001 ) and then increased again (P < 0.001 ) at Day 14 compared with Day 7. A small increase in DMD and CWD was apparent after 7 days with the control treatment of 0 AHP (water at pH 11.5) but not at 14 days. In summary, the storage time enhanced the effect of 60 g but not 120 g AHP kg- l straw.
3.5. Experiment 4 The mean DMD and CWD for all treatment combinations are presented in Table 2. Although the main effects o f A H P (P < 0.001 ), storage time (P < 0.001 )
10
A.S. Chaudhry, E.L. Miller/Animal Feed Science and Technology 50 (1994) 1-15
Table 2 Experiment 4. Effect of various levels of alkaline hydrogen peroxide (AHP) when applied at different straw/liquid ratios for different days on the in vitro dry matter (DMD) and cell wall (CWD) digestibility (g kg - ~DM ) of barley straw (means with SED ) Parameters
DMD
CWD
AHP (g kg-I straw) 0 10 40 Straw/liquid ratio 2:1 1:1 2:1 1:1 2:1 Storagetime (days) 2 7 14 21 28 SED
346 432 421 466 338 49.7
Main effects AHP Storage time Straw/liquid
(SED 16) (SED18) (SED 11)
319 461 409 446 420
435 492 364 481 452
463 452 437 475 393
485 458 433 535 303
AHP (g kg-~ straw) 0 10 40
150
150
1:1 2:1
Straw/liquid ratio 1:1 2:1 1:1 2:1 1:1 2:1
1:1 2:1
1:1
486 472 462 526 491
539 465 544 521 387
410 386 374 444 408
469 380 478 454 294
462 439 486 551 384
P<0.001 P<0.001 P<0.10
238 334 324 391 229 56.6
206 367 309 358 322
(SED 18) (SED20) (SED 13)
350 357 266 392 380
384 361 353 383 302
392 368 336 455 199
378 347 405 473 289
P<0.001 P<0.001 P<0.05
Table 3 Experiment 4. Effect of storage time × AHP (alkaline hydrogen peroxide) interaction on the in vitro dry matter (DMD) and cell wall (CWD) digestibility (g kg-1 DM) of barley straw (means with SED) Storage time (days)
DMD
CWD
AHP (g kg-lstraw) 0 10 40
150
AHP (g kg-lstraw) 0 10 40
150
2 7 14 21 28
333 447 415 456 379
501 452 515 536 385
222 350 316 375 276
423 363 441 463 291
SED
35*
449 472 400 478 423
486 465 448 531 397
367 359 310 388 341
401 377 355 450 304
40*
* Significant at P< 0.05.
and straw/liquid ratios ( D M D P < 0.1; CWD P < 0.05) were significant the interaction between storage time and AHP was also significant ( D M D P < 0.030; CWD P < 0.025). Therefore, the results of this interaction, presented in Table 3, are examined more carefully. None of the other two or three way interactions were significant (P > 0.05 ).
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Table 4 Experiment 5. Effect of various levels of alkaline hydrogen peroxide (AHP), NaOH and NazOz on the in vitro dry matter ( D M D ) and cell wall digestibility (CWD) of wheat straw (means with SED) Parameters
Treatments A
Chemicals used (g kg- 1 straw) NaOH H2Oz NazO/ Straw/liquid Digestibility (g kg- J DM ) DMD CWD
B
. . -
C
SED D
E
F
G
H
I
J
K
50 50 50 8 8 8 8 32 . . . . . 20 20 80 80 80 . . . 15 45 . . . . . . 8:1 4:1 l:l 1:1 1:1 4:1 1:1 4:1 1:1 4:1
L
32 80 1:1
551 739 789 811 637 783 628 543 645 626 735 694 415 660 748 768 546 729 499 402 537 516 661 601
11"** 12"**
***Significantat P < 0.001.
DMD and CWD increased with AHP in a dose related manner at 2 and 14 days of storage (P < 0.001 ) but changes at other times were not significant. The control solution (0 AHP at pH 11.5) increased DMD and CWD at 7 and 21 days over that observed at 2 days (P < 0.001 ) Maximum percent improvements (DMD 61.23; CWD 108.7) were observed when samples, treated with 150 g of AHP, were stored for 21 days. A significant depression (P < 0.001 ) was observed in almost all the samples treated with any level of AHP, except in those treated with 10 g of AHP, where it was non-significant (P > 0.05 ), when storage time was increased from 21 to 28 days. Increasing the volume of liquid at a constant amount of AHP increased DMD on average by 20 g kg- t units (P=0.078 SED 11.1 ) and CWD by 27 g kg- l (p < 0.05 SED 12.7).
3.6. Experiment 5 The mean DMD and CWD are given in Table 4. The treatments significantly (P < 0.001 ) affected both DMD and CWD of wheat straw. The DMD and CWD of wheat straw showed positive responses to almost all the treatments except those of treatment H where a slight negative, though non-significant (P > 0.05 ), response was obtained. Maximum percent improvement (47.34, DMD; 85.06, CWD ) was observed in Samples (D) treated with 50 g NaOH alone at low ( 1:1 ) straw/liquid ratio. The response decreased quadratically (P < 0.001, DMD and CWD) with the increase in the straw/liquid ratios (Treatments B and C). In contrast significantly greater (P < 0.001 ) responses were observed when any level of H202 in combination with 8 g NaOH at the 4:1 straw/liquid ratio (Treatments G and I) was compared with that of 1:1 (H and J ). The effect of 80 g H202 (I + J ) was significantly greater (P < 0.001 ) than that of 20 g ( G + H ) when both
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A.S. Chaudhry, E.L. Miller / Animal Feed Science and Technology 5 0 (1994) 1-15
were applied in combination with 8 g NaOH. The efficiency of 80 g H202 was significantly improved (P < 0.001 ) when the added amount of NaOH was increased from 8 ( I + J ) to 32 g ( K + L ) . However, H202 appeared to be most efficient when 80 g of H202 and 32 g of NaOH were mixed with each kg of straw at a straw/liquid ratio of 4:1 (K). The effect of Na202 was significantly improved (P < 0.001 ) when the amount was increased from 15 to 45 g kg- 1 of straw. However, the improvement in samples treated with 50 g NaOH kg- 1 straw at a straw/liquid ratio of l:l was significantly greater (P < 0.001 ) than those of treated with Na202 at the same ratio. In contrast to the results of Experiment 4, the DMD and CWD were greater when small volumes of liquid were applied in Treatments G, I and K in comparison with those of H, J and L.
4. Discussion AHP treatment was effective in improving the in vitro digestibility of straw in these experiments. Similar effectiveness was reported by Kerley et al. (1986) who examined the effect of AHP-treated wheat straw on the site and extent of digestion in sheep. They noticed significantly increased DMD and NDF digestibility (NDFD) when AHP treated straw was fed to wethers in different straw/ concentrate proportions, pH-regulated AHP treatment showed greater percent increase in the in vivo NDFD and cellulose digestibility than that of DMD in experiments conducted by Kerley et al. (1987). Similar responses are reflected in the current findings (Figs. 1 and 2). In contrast, treatment with H202 at pH 5.6 reduced digestibility possibly due to residual H202 or the formation of some substances which inhibited microbial digestion in the in vitro system. Almost equal (Table 1 ) responses of ABS and UBS to high AHP (60 and 600 g kg- l straw), in terms of digestibility, are in agreement with those of Bhargava et al. (1989) who studied the effect of suspending wheat straw in 1% AHP solution on its in sacco degradability. They observed increased potential degradability in both UBS and ABS treated with AHP and supported the potential use of AHP in improving the straw digestibility. Improvement in the digestibility of crop residues by AHP treatment is mainly mediated through cell wall modifications, i.e. lignin degradation. Gould (1984) demonstrated that soaking wheat straw in AHP at p H I 1.5 released about one half of the lignin as water soluble products. Similar effects of AHP on the lignin composition of straw are shown in Experiment 1 (Table l ). However, the magnitude of reduction (14%) was lower than that ( > 50%) reported by Gould (1985) which can perhaps be attributed to the low AHP (60 g kg-1 vs. 500 g kg- i straw). Even at 600 g AHP kg- 1 straw, an AHP level close to that used by Gould ( 1985 ), lignin reduced to only 36%. The lower effect of AHP in reducing lignin was possibly due to the higher ( 1:6 vs. 1:50) straw/liquid ratio and differences in the preparation of the treated residues. However, increasing AHP at a constant straw/liquid ratio of 1:6 increased DMD and CWD up to 900 g kg-1
A.S. Chaudhry, E.L. Miller / Animal Feed Science and Technology 5 0 (1994) 1-15
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(Table 1 and Figs. 1 and 2) similar to values achieved by Gould ( 1985 ). AHP was least effective at low levels (15 g kg-1 straw) which is in agreement with those reported by Flachowsky and Sundstol ( 1988 ) and Adebowale et al. ( 1989 ) who did not find any significant improvements in the in vitro DMD (IVDMD) and rumen DMD when straws treated with AHP were compared with their NaOH controls. However, the present work clearly shows that increasing H202 concentration at pH 11.5 greatly increases digestibility compared with the control maintained at pH 11.5. Furthermore, the DMD and CWD of UBS treated with AHP under the same conditions are shown to be repeatable (Table 1, Figs. 1 and 2 ). While AHP was effective at very high levels (Experiments 1 and 2) the response was less dramatic at lower levels ( < 200 g AHP kg -1 straw). This can partly be compensated by extending the time of storage/reaction (Experiments 3 and 4). A positive effect of increasing storage time is clearly presented in Experiment 3 (Figs. 3 and 4). Shahiduzzaman and Owen (1987) also demonstrated similar effects on digestibility when Ca (OH)2-treated straws were stored for longer durations. However, in Experiment 4 (Tables 2 and 3) the proportional increase in the digestibility was not maintained with the increase in storage time, rather poor digestibilities were noted compared with those of Experiment 3. The low responses of DMD and CWD towards AHP may be attributed to the release of certain inhibitory substances like phenolic acids which remained in the unwashed samples and probably affected the ultimate digestibility. This was also supported by Lewis et al. ( 1987 ) who observed lower NDFD and ADFD values in the unwashed samples of AHP treated wheat straw retaining the solubles. The IVDMD values of NaOH in Experiment 5 are closer to those reported by Wanapat et al. ( 1985, 1986) and Adebowale et al. (1989). Various straw/liquid ratios did affect the IVDMD and in vitro CWD (IVCWD) which is in agreement with those stated by Wanapat et al. ( 1986 ) where they found NaOH as the most effective, amongst various treatments, in improving the digestibility of straw when applied together with plenty of liquid. Increased digestibility obtained from samples treated with either amounts of Na202 supports the earlier findings of Jones and Klopfenstein (1967) who observed significantly increased IVDMD and in vivo DMD values in corn cobs treated with 4% solution of Na202. Improvement in the digestibility values of straws treated with AHP is in agreement with the previous findings (Chaudhry and Miller, 1990a,b). Chopped wheat straw was equally effective as a substrate (Reeves III, 1985 ). The response with AHP in Experiment 5 was not as great as with NaOH alone. Since all samples were freeze dried before being subjected to in vitro tests, a possibility that the full potential of AHP was masked by production of inhibitory soluble substances cannot be ruled out. If inhibitory soluble phenolics are released and must be washed away, as in Experiments 1, 2 and 3 according to the Gould (1985) method, the loss of soluble carbohydrates, the considerable extra cost of washing and drying and the problem of affluent disposal are the limitations which needs to be addressed before suggesting AHP as an alternative to
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other alkali treatments for the nutritional improvement of cereal straws (Chaudhry, 1990).
Acknowledgments ASC is grateful to Cambridge Commonwealth Trust and Government of Pakistan for their financial support.
References Adebowale, E.A., Orskov, E.R. and Horton, P.M., 1989. Rumen degradation of straw. 8. Effect of alkaline hydrogen peroxide treatment on degradation of straw using NaOH and ammonia as source of alkali. Anim. Prod., 48: 553-560. Bhargava, P.K., Orskov, E.R. and Walli, T.K., 1989. Effect of soaking, ensilingand hydrogen peroxide treatment of barley straw on rumen degradability. Anim. Feed Sci. Technol., 22: 295-303. Chaudhry, A.S., 1990. Nutritional improvement of cereal straws. PhD Thesis. University of Cambridge, Cambridge, UK. Chaudhry, A.S. and Miller, E.L., 1990a. Effect of treating wheat straw with sodium hydroxide and calcium oxide alone or in combination with hydrogen peroxide on the in vitro dry matter digestibility. Anim. Prod., 50:118 (abstract). Chaudhry, A.S. and Miller, E.L., 1990b. Effect of calcium oxide, sodium hydroxide and alkaline hydrogen peroxide treatments on the nutrient digestion of wheat straw by sheep. Anim Prod., 50: 119 (abstract). Chaudhry, A.S. and Miller, E.L., 1994. Effect of H202-dependent enzyme systems on the in vitro digestibility of untreated and NH3 treated barley straws. J. Sci. Food Agric., 64:371-378. Crawford, R.L. and Crawford, D.L., 1984. Recent advances in studies of the mechanisms of microbial degradation of lignin. A Review. Enzymol. Microbiol. Technol., 6:433-442. Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M., 1986. Data for Biochemical Research. Oxford University Press, Oxford. Flachowsky, G. and Sundstol, F., 1988. Effect of NaOH and H202 on the degradability of straw in ruminants. Arch. Anim. Nutr. Berlin., 38: 955-964. Goering, H.K. and van Soest, P.J., 1970. Forage Fibre Analysis. US Dep. Agric. Handbook No. 379. US Dep. Agric., Washington, DC, USA. Gould, J.M., 1984. Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification. Biotech. Bioeng., 26: 46-52. Gould, J.M., 1985. Studies on the mechanism of alkaline hydrogen peroxide delignification of agricultural residues. Biotech. Bioeng., 27:225-23 I. Hartley, B.S., Broda, P.M.A. and Senior, P.J., 1987. Technology in the 1990s: utilization of lignocellulosic wastes. Proc. R. Soc. London. Jones, M.J. and Klopfenstein, T.J., 1967. Chemical treatment of poor quality roughages. J. Anim. Sci., 26:1492 (abstract). Kerley, M.S., Fahey, Jr., G.C., Berger, L.L, Merchen, N.R. and Gould, J.M., 1986. Effect of alkaline hydrogen peroxide treatment on site and extent of digestion of sheep. J. Anim. Sci., 53: 868-878. Kerley, M.S., Fahey, Jr., G.C., Berger, L.L. and Merchen, N.R., 1987. Effects of treating wheat straw with pH-regulated solutions of alkaline hydrogen peroxide on nutrient digestion by sheep. J. Dairy Sci., 70: 2078-2084. Lawes Agricultural Trust, 1984. Genstat IV. Rothamsted Experiment Station, Rothamsted, UK. Lewis, S.M., Holzgraefe, P.D., Berger, G.L., Fahey, Jr., G.C., Gould, J.M. and Fanta, G.C., 1987.
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15
Alkaline hydrogen peroxide treatment of crop residues to increase ruminal dry matter disappearance in sacco. Anim. Feed. Sci. Technol., 17: 179-199. Mackie, R.I. and Kistner, A. 1985. Some frontiers of research in basic ruminant nutrition. S. Aft. J. Anim. Sci., 15: 72-85. Reeves III, J.B., 1985. Lignin composition of chemically treated feeds as determined by nitrobenzene oxidation and its relationship to digestibility. J. Dairy Sci., 68:1976-1983. Shahiduzzaman, Md. and Owen, E., 1987. Treatment of barley straw with mixtures of calcium hydroxide and urea. Effect on the intake and digestibility in sheep. Anim. Prod., 44:68 (abstract). Van Soest, P.J., Wine, R.H. and Moor, L.A., 1966. Estimation of true digestibility of forages by in vitro digestion of cell walls. Proc. 10th Int. Grassld. Congr., 7-16 July 1966, Helsinki. Finnish Grassland Association, Helsinki, pp. 438-44 I. Wanapat, M., Sundstol, F. and Garmo, T.H., 1985. A comparison of alkali treatment methods to improve the nutritional value of straw. I. Digestibilityand metabolizability. Anim. Feed Sci. Technol., 12: 29-309. Wanapat, M., Sundstol, F. and Hall, J.M.R., 1986. A comparison of alkali treatment methods used to improve the nutritive value of straw. II. In sacco and in vitro degradation relative to in vivo digestibility. Anim. Feed Sci. Technol., 14:215-220.