Effect of hydrogen peroxide treatment on the utilization of lignocellulosic residues by rumen micro-organisms

Biological Wastes 28 (1989) 133-141

Effect of Hydrogen Peroxide Treatment on the Utilization of Lignocellulosic Residues by Rumen Micro-organisms D. V. R e d d y , U s h a R. M e h r a & U. B. Singh

Animal Nutrition Division, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India (Received 26 March 1988; revised version received 15 May 1988; accepted 26 June 1988)

ABSTRACT Oat straw, wheat straw and sugar-cane bagasse were treated by incubation for 21 days with various combinations of hydrogen peroxide, ammonia and urea. The polyester bag (in sacco) technique was employed to measure tureen microbial degradation of treatedfeeds. Analyses of treated material indicated that urea, and peroxide plus urea, were significant in solubilizing lignin and thus enhancing the cell contents. I-I202 treatment enhanced (P< 0.01) the extent of digestion of DM, NDF, ADF and cellulose over 48 h for wheat straw and sugar-cane bagasse. Ammoniation increased (P < 0.01) the tureen degradability of DM and CWC of the crop residues studied, though the response with sugar-cane bagasse was poor. Hydrogen peroxide in combination wih aqueous ammonia treatments gave significantly ( P < 0"01) higher in sacco degradability values than other alkaline I-I202 treatments. However, H202 treatment of sugar-cane bagasse increased (P < 0.01) the fermentability of structural carbohydrates more than alkaline hydrogen peroxide treatments.

INTRODUCTION Lignocellulosic crop residues contain relatively large amounts o f lignin and silica, the presence o f which adversely affects the extent o f digestion o f fibrous feeds in the rumen (Mertens, 1977). This m a y in turn result in a low 133 Biological Wastes 0269-7483/89/$03.50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain

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D. V. Reddy, Usha R. Mehra, U. B. Singh

yield of microbial cells, possibly due to the relatively large energy requirements for maintenance of slow-growing bacteria, and in a slow flow of digesta and microbial protein down the ruminant gastro-intestinal tract (Hungate, 1975). To utilize the energy of cellulosic material, whose gross energy equals that of starch in cereal grains, delignification and simultaneous reduction of cellulose crystallinity is needed. For delignifying or disrupting the lignin-carbohydrate complex several chemical treatments using NaOH (Jackson, 1977; Klopfenstein, 1978), calcium oxide (Prasad et al., 1985), aqueous or anhydrous ammonia (Sundstol et al., 1978; Kumar et al., 1982) or ammoniation through hydrolysis of urea (Jayasuriya & Perera, 1982; Dass et al., 1987) have been tested. Of all chemicals, ammonia treatment is by far the most appropriate, since the ammonia, having served as alkali to improve the potential rate and extent of digestion, subsequently serves as an essential nutrient for microbes to ensure efficient rumen fermentation. It was reported that the hydroxyl radical ('OH) derived from H202 plays an integral role in natural delignification by fungi and bacteria (Forney et al., 1982; Kutsuki and Gold, 1982). Gould (1984) developed a procedure to delignify lignocellulosic material by using an alkahne H202 solution (1% H202 solution with added NaOH to obtain a pH of 11.5) which decreased lignin content and apparently disrupted the ordered structure of the cellulose polymers (Gould, 1985). The powerful oxidizing agent, H202, in combination with urea solution and NH4OH to provide alkalinity to H202, was tested on lignocellulosic crop residues (oat and wheat straw) and an agro-industrial by-product (sugar-cane bagasse). The present paper reports the effect of these treatments on crude protein (CP), cell wall constituents (CWC) and in sacco degradability of dry matter (DM) and CWC of the residues.

METHODS Treatments

Samples of chaffed oat straw and wheat straw and sugar-cane bagasse were mixed thoroughly with the respective chemical solutions (Table 1) and sealed in airtight polythene bags. Addition of NH4OH caused a change ofcolour to yellowish. Bleaching ofcolour was noticed with H202 treatment. The H202 and NH4OH solution combination generated heat instantaneously. After a 21-day incubation period the bags were opened and the contents were dried in air under shade.

Peroxide treatment of lignocellulosic wastes

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TABLE 1

Details of Treatments

Treatment

Chemicals applied

Quantity applied/60 g

Description

T1

Water

37 ml

Sprinkled with water

T2

Water and hydrogen peroxide (H202)

Sprinkled with water and H202 mixture

T3

Water and urea

7 and 30 ml of 30% (w/v) solution respectively 34 ml and 3 g respectively

T4

Ammonium hydroxide

8".5ml of 25% solution

Ammonium hydroxide solution sprinkled

T5

H202 and ammonium hydroxide

30ml of 30% solution and 8-5 ml 25% solution respectively

Sprinkled with H202 and ammonium hydroxide solution

T6

H202 and ammonium hydroxide

Same

Sprinkled with H202 and kept for 1 day followed by 20 day incubation with ammonium hydroxide solution

T7

H202 and ammonium hydroxide

Same

Sprinkled with ammonium hydroxide and kept for 20 clays followed by 1 day incubation with H202 solution

Ts

Water, H20 2 and urea

7ml, 30ml of 30% solution and 3"0g respectively

Urea dissolved in water and sprinkled accompanied by sprinkling with H202 solution

T9

Water, H202 and urea

Same

Sprinkled with H202 and kept for l day followed by 20 day incubation with urea dissolved in water

Tlo

Water, H202 and urea

Same

Sprinkled with urea solution and kept for 20 days followed by 1 day incubation with H20 2 solution

Urea dissolved in water and sprinkled

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D.V. Reddy, Usha R. Mehra, U. B. Singh

Polyester bag technique The in sacco degradability of D M and C W C was determined by the polyester bag (size o f bag: 8 x 12 cm; pore size 44 pm) technique with 3 g o f dry sample (Mehrez & Orskov, 1977). Three fistulated buffalo calves o f 300 kg body weight fed on a m m o n i a t e d wheat bhoosa ad lib with 500 g wheat bran were used for this purpose and two bags for each sample were kept for 48 h in the rumen o f each animal (i.e. six bags for each sample).

Analytical methods The dried samples were milled to pass through a 1 m m sieve. The CP (N x 6.25) content o f samples was determined by the auto-kjelteck m e t h o d (AOAC, 1975). The samples were analysed for cell wall fractions (Goering & Van Soest, 1970). D a t a was processed statistically.

RESULTS AND DISCUSSION

Effect of treatments on chemical composition Samples o f treatments I and 2 o f oat straw were discarded because o f fungus growth. H 2 0 2 treatment with either N H 4 O H (T 5, T6, T7) or urea solution (Ts, T9, Tlo ) increased crude protein (CP) content, probably due to binding o f a m m o n i a (Table 2). F o r T 2, T 3 and T 4 the decreases in N D F , A D F and TABLE 2 Crude Protein Content (% DM Basis) of Treated LignocellulosicResidues° Treatment

Oat straw

Wheat straw

Sugar-cane bagasse

T1 T2 T3 T4 T5 T6 T7 T8 T9 Tlo

--7.96 10.14 14'8 13.9 10"8 22.81 21"52 23.31

3.15 3-88 5-31 5.78 7"36 9-35 7"57 18.90 18.29 17.53

2.46 2"54 8-60 4.96 8"56 9"63 6.23 16"0 17"56 17-25

° Each value is the average of three replications.

-- Not tested, see text.

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137

ADL and increases in NDS values were significant for H 2 0 2 treatment (T2). Comparisons among treatments 5-7 over treatment 4 revealed the beneficial effect of combined H 2 0 2 and NH4OH treatments. Treatments 2, 6, 8 and 9 appeared to be better in solubilizing lignin and increasing NDS (Table 3). Buettner et al. (1982) also reported that ammoniation decreases NDF, TABLE 3 Cell W a l l C o n s t i t u e n t s o f T r e a t e d LignocellulosicResidues(% DM Basis) a Treatments

NDF

T1

. 83-59 90-00 . 70"22 66-09 65"87 78"32 81"58 62.00 77"86 82.49 56'90 74"70 76"33 55"60 70"37 69"37 59"11 77"28 78.54 61"59 68"46 65-74 63"38 70"03 65"37 64"41 76-20 72"84

T2

T3

"1"4

T5

T6

T7

Ts

T9

Tlo

OS ~ WS SB OS WS SB OS WS SB OS WS SB OS WS SB OS WS SB OS WS SB OS WS SB OS WS SB OS WS SB

ADF

.

ADL

. 57-65 62-53

. . 48"82 45.60 49'87 55'83 54.41 43"19 55.00 53"73 43-01 54.07 51.03 42-39 53"57 45"75 41"03 55.21 52"79 40"22 46"44 42"86 41"63 45-68 41-20 41"13 50"19 43"27

. 8"05 13"38 . 2'64 2"48 6"77 7"80 9"88 4"30 6.60 9.24 4.20 4"11 8"57 2"70 3"99 2"86 3"41 5-49 8-22 3"30 2"88 2"92 3-27 3"30 3"00 3"38 4"33 6"62

NDS

Hemicellulose

. 16.41 10,00 . 29"78 33.91 34-13 21"68 18.42 38.00 22.14 17.51 43.10 25.30 23-67 44.40 29.63 30"63 40"89 22.72 21"46 38"41 31"54 34"26 36"62 29"97 34"63 35"59 23.80 27"16

. 25.94 27"47 . 21"40 20.49 16.00 22.49 27.17 18'81 22"86 28"76 13.88 20.63 25.30 13.21 16.80 23"62 18.08 22.07 25.75 21"37 22"02 22"88 21"75 24"35 24.17 23"28 26.01 29"57

° Each value is the average of three replications. OS, oat straw; WS, wheat straw; SB, sugar-cane bagasse. For other abbreviations see text. - - Not tested, see text.

Cellulose

AcM mso~b& ash

42.78 46.64

6"82 2-51

41.16 41.92 34.61 42.86 43-55 34.51 40-69 43.30 34.62 43.71 41.26 35.25 44-38 41.94 33-68 44"16 43.24 32"62 39-27 39"06 33"31 39"28 37"69 31"99 38"02 35"74

5"02 1-20 8"49 5"17 0"98 4"38 7"71 1"19 4'19 6'25 1"20 4"44 5"20 0"95 3'94 5"56 1"33 4"30 4"29 0"88 5"05 3"10 0"51 5"76 5.74 0"91

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D. V. Reddy, Usha R. Mehra, U. B. Singh

hemicellulose and lignin, and infra-red spectroscopy showed that ammonia ruptured ester bonds, the major type of linkage between lignin and polysaccharides. Treatment of wheat straw with alkaline (NaOH) H202 released 50% of the lignin as water-soluble degradation products (Gould, 1984, 1985). The better solubilization oflignin obtained in the present study might be due to the higher concentration of HzO 2 used (0.55 g vs 0.26 g H202/g substrate). Effect of treatments on rumen degradability Hydrogen peroxide treatment

H 2 0 2 treatment enhanced (P < 0.01) the extent of in sacco degradability of DM, NDF, A D F and cellulose of wheat straw and sugar-cane bagasse (Table 4). As reported by Owen (1978), the increase in digestibility was greater with residues of low than with high initial digestibility. Chandra and Jackson (1971) reported an increase from 40% (untreated) to 55% in rumen degradability of DM over 72 h, by treating with 60 g H202/kg maize cobs. Treatment with ammonia

Ammoniation (T a and T4) increased the in sacco degradability of DM and CWC in all the lignocellulosics studied (assuming oat straw to be similar to wheat straw) though sugar-cane bagasse gave a poor response which is in agreement with our earlier findings (Kumar et al., 1982). Oat straw showed a better response to aqueous ammonia treatment, whereas T 3 increased (P < 0.01) in sacco degradability of DM and CWC in wheat straw. In a comparative study of cereal straws treated with anhydrous ammonia, Horton (1981) reported that digestibility response was greater with wheat straw (17%) than with oat and barley straws (8%). Alkaline hydrogen peroxide treatment In sacco degradability data for oat straw showed significant (P < 0.01)

beneficial effect from HzO 2 along with either NH4OH (Ts-T7) or urea solution (Ts-Tlo) over T4 and T 3 respectively. In the case of wheat straw, the alkaline hydrogen peroxide treatments 6 and 9 were effective in enhancing the in sacco degradability of DM and CWC, of which the former was superior (P < 0.01) to the latter. H20 2 treatment followed by aqueous ammonia (Te) gave higher ruminal degradability of wheat straw. Kerley et al. (1985) also reported that treatment of wheat straw with alkaline (NaOH) hydrogen peroxide allowed more bacterial colonization and more rapid degradation of the cell wall. Among the alkaline hydrogen peroxide treatments T6, T 8 and T 9 improved (P < 0.01) the ruminal degradability of sugar-cane bagasse (these were not

TABLE

4

-39"92 4.14

-39-41 12-45

-41.91 13.36

-48-40 23.94

Oat straw Wheat straw Sugar-cane bagasse

Oat straw Wheat straw Sugar-cane bagasse

Oat straw Wheat straw Sugar-cane bagasse

Oat straw Wheat straw Sugar-cane bagasse

CD, Critical difference. ° Significant (P < 0.01). - - Not tested, see text.

7"1

Substrate

-73-20 83.41

-68.35 78-06

-68"73 79.19

-70"20 81.32

T2

63.87 65-89 38.22

57"81 60.95 22.51

52-91 56.00 23.60

56"27 56"86 22.20

T3

75"57 64.32 28.65 83.45 70.92 42.10

N D F disappearance ° 61.89 79"51 69-52 48-52 56.27 72.15 19-85 31.34 63.18 Cellulose disappearance ° 74.07 86-49 79.82 56.26 64-40 8(~33 35.81 45-82 71.97

76-96 63.63 64.62

66.11 56-73 55-25

66.01 55"14 58.48

Ts

73"65 61"95 33-26

T7

A D F disappearance ° 63-65 77-69 69"15 48"10 56-37 71"65 20.12 33.12 65.46

T6

70"95 59"56 63.03

1"5

Dry matter disappearance ° 68"49 82.81 77.06 79-38 49"24 59"77 73"73 66.23 20.33 35-36 68-40 31.67

7"4

83.73 67.99 64.60

69.62 58.52 56.21

72"28 61-00 55.84

74"27 64-03 62.62

T9

72.85 58.21 34.09

63"29 53.66 26.23

62"71 52"39 16.47

68-10 54.42 26-59

Tl 0

1.86 1.79 1-82

2"72 2.13 2.35

2"70 1"97 2.18

1"97 l'81 2.08

SE Means

The in sacco Degradability (% DM Basis) of Dry Matter and Cell Wall Constituents of Treated Lignocellulosic Residues

7.10 6.77 6"88

10-39 8.07 8-91

10"32 7"47 8-26

7"54 6"87 7"88

CD

Fo

t~

¢

140

D. II. Reddy, Usha R. Mehra, U. B. Singh

significantly different among themselves) over T a or T 4. The in sacco degradability values obtained from T6, T s and T 9 were significantly less than those obtained from hydrogen peroxide-treated (T2) sugar-cane bagasse. The in sacco degradability and other results indicated that alkaline H 2 0 2 treatments with aqueous a m m o n i a appeared to be better than urea solution for oat and wheat straws, while sugar-cane bagasse gave better response with hydrogen peroxide treatment.

ACKNOWLEDGEMENTS The authors thank D r P. N. Bhat, Director, Indian Veterinary Research Institute, Izatnagar, for facilities and encouragement. Shri U.S. Srivastava rendered competent technical assistance.

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Jayasuriya, M. C. N. & Perera, H. G. D. (1982). Urea ammonia treatment of rice straw to improve its nutritive value for ruminants. Agric. Wastes, 4, 143-50. Kerley, M. S., Fahey, G. C. Jr, Berger, L. L., Gould, J. M. & Baker, F. L. (1985). Alkaline hydrogen peroxide treatment unlocks energy in agricultural byproducts. Science, 230, 820-2. Klopfenstein, T. (1978). Chemical treatment of crop residues. J. Anita. Sci., 46, 841-8. Kumar, N., Verma, D. N., Dass, R. S. & Singh, U. B. (1982). Effect of concentration and period of ammonia treatment on the chemical composition and nutritive value of sugar-cane bagasse. Indian J. Nutr. Diet., 19, 381-8. Kutsuki, H. & Gold, M. H. (1982). Generation of hydroxyl radical and its involvement in lignin degradation by Phanerochaete chrysosporium. Biochem. biophys. Res. Commun., 109, 320-7. Mehrez, A. Z. & Orskov, E. R. (1977). A study of the artificial fibre bag technique for determining the digestibility of feeds in the rumen. J. Agric. Sci. Camb., 88, 645-50. Mertens, D. R. (1977). Dietary fiber components: relationship to the rate and extent of ruminal digestion. Fed. Proc., 36, 187-92. Owen, E. (1978). Processing of roughages. Proceedings of the 12th Nutrition Conference for Feed Manufacturers, Nottingham, pp. 127--48. Prasad, R., Agrawal, I. S. & Verma, M. L. (1985). The effect of lime treatment of wheat straw and its supplementation with diammonium phosphate on growth in heifers, lndian J. Anita. Nutr., 2, 114-19. Sundstoll F., Coxworth, E. and Mowat, D. N. (1978). Improving the nutritive value of straw and other low quality roughages by treatment with ammonia. Wld Anim. Rev., 26, 13-21.