or SO2 on the compositional and histological characteristics of sorghum stover

ANIMAL FEED SCIENCE AND TECHNOLOGY

ELS EV 1ER

Animal Feed Science and Technology 47 (1994) 141-150

The effect of NH3 and/or S O 2 o n the compositional and histological characteristics of sorghum stover Myriam Lea1*'a, A r m a n d o

S h i m a d a b, E l i s e o H e r n ~ m d e z a

aCoordinaci6n de Estudios de Posgrado, Facultad de Estudios Superiores-Cuautitl&n, Universidad Nacional Aut6noma de Mbxico, Cuautitldm-lzcalli, Mbx., Mexico bCentro Nacional de Investigaci6n en Fisiologia y Mejoramiento Animal, Divisi6n Pecuaria, Instituto Nacional de Investigaciones Forestales y Agropecuarias, Ajuchitldn, Qro., Mexico

(Received 26 January 1993; accepted 26 October 1993)

Abstract Two experiments were conducted to determine the effects of chemical treatments on the compositional and histological characteristics of sorghum stover. In Experiment 1, chopped sorghum stover was placed inside plastic bags, where 5% of either NH3 applied as ammonium hydroxide or SO2 applied as gas, was injected. After 21 days in contact with the respective gas, bags were opened, aerated for 7 days and prepared for chemical analysis and microscopical observations. Compared with an untreated control, treatment with NH3 increased total nitrogen, non-protein nitrogen, protein nitrogen, acid detergent fiber nitrogen, and in vitro dry matter disappearance and decreased the acid detergent fiber (ADF) contents of stover; SO2-treated stover had a lower ADF content. In Experiment 2, each chemical was applied to stover previously treated with the alternate gas. In general, the compositional effects followed the same trend as in the previous experiment, although they seemed to be more severe. Scanning electron microscopy showed the extent of the stover tissue damage inflicted by the chemical additives applied.

1. Introduction T h e chemical t r e a t m e n t o f straws and other crop residues has the objective o f increasing the digestibility o f the material for r u m i n a n t s (Klopfenstein, 1978), and can be accomplished through the use o f either alkaline c o m p o u n d s (Waiss *Corresponding author: CeNIFMA. Apartado postal 29-A, Quer6taro, Qro. 76020, Mexico. Elsevier Science B.V. SSD10377-8401 (93)00569-H

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and Guggolz, 1972; Rounds and Klopfenstein, 1974; Sundstol, 1984; Mason et al., 1990a; Goto et al., 1993 ) or oxidizing compounds (Ben-Ghedalia et al., 1980; Ben-Ghedalia and Miron, 1981; 1984a; Bunting et al., 1984; Kerley et al., 1987; Amjed et al., 1992; Sultan et al., 1992). The mode of action of alkaline compounds consists of a partial hydrolysis of the cell wall, with the rupturing of ester bonds between hemicellulose and lignin, without removing the latter (Klopfenstein, 1978). Oxidizing agents seem to cause a complete solubilization of hemicellulose and a reduction of the lignin content of the treated material, possibly creating hollow spaces within the cellulose matrix that make the cell walls more accessible to ruminal microbial action (Shefet and Ben-Ghedalia, 1982 ). The purpose of the experiments reported here was to assess the effect of treating sorghum stover with NH3 and/or SO2, on its chemical and histological characteristics.

2. Materials and methods Two experiments were conducted. Chopped sorghum stover was commercially purchased, and batches of 2 kg were placed inside each of three 0.2-mm-thick plastic bags. This material was then treated with 5% (w/w) of either NH3 (applied as NH4OH ) or SO2 (applied as gas), the bags were sealed and the contents allowed to react for 21 days at ambient temperature (average of 25°C). At the end of this period, the bags were opened, the contents aerated for 7 days, and sampled. In Experiment 1, the treated material (NH3 or SO2 ) was compared with an untreated control. In Experiment 2, the same procedure was followed, but the treated material was subsequently treated with the alternate chemical: i.e. the NH3 treatment was followed by SO2 treatment and vice versa. At the end of the second treatment the material was compared with an untreated control. Stover samples were ground through a Wiley mill using a 1 mm screen and analyzed to determine dry matter (DM) and ash (Association of Official Analytical Chemists (AOAC), 1990 ), total nitrogen by the Kjeldahl method (AOAC, 1990), non-protein nitrogen (npN) (Jacobs, 1965 ), acid detergent fiber nitrogen (ADF-N), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin and cellulose (Goering and Van Soest, 1975 ), in vitro dry matter disappearance (IVDMD), following the method of Minson and McLeod (1972), modified by Barnes and Lynch (1969) (after a 48 h incubation, 1 ml of 6 N HCI and 0.2 g pepsin 1:10 000 NFX1 were added to each tube and then incubated for 48 h at 39 ° C; samples were then filtered, dried and weighed). In addition, stems were observed in a JMS-25-SII scanning electron microscope. Samples were cut either transversally or longitudinally and attached to brass sample holders with adhesive silver paint and coated with a 150 ~ layer of gold in a fine coat 'ion sputter' JFC-1100. All observations and photographs were made at accelerating voltage between 12.5 and 15.0 kV and at both low and high magnifications.

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143

In each experiment, a one-way classification analysis of variance was used, along with an SNK comparison of means (Anderson and McLean, 1974).

3. Results and discussion

3.1. Experiment I Treatment of sorghum stover with NH3 (Table 1 ) increased the total nitrogen content by 12.9 g kg-1 compared with the untreated control, the results being partially explained by both the nitrogen added and the ammonolysis reaction between the carbohydrates and the alkali (Saenger, 1982) as has been previously reported (Dryden and Kempton, 1983; Alibez et al., 1984; Mason et al., 1990b; Kondo et al., 1992 ). The non-protein nitrogen increase (Table 1 ) was simply the result of the ammonia nitrogen added. The latter probably also increased the ADFN content (Table 1 ) of the stover owing to its union with the lignin, forming an aromatic amine (Muller and Bergner, 1975; Dryden and Kempton, 1983 ); the resulting nitrogen fraction probably being completely undigestible. The increase in the total nitrogen content of SO2-treated stover may be due to increases in ammonia-nitrogen, presumably as a result of the liberation of some nitrogen compounds (amines or amides) from the lignin molecules owing to SO2 treatment related attacks on the inner covalent bonds of the lignin molecule. However, the improvement in the protein-N content of the stover following its treatment with NH3 (Table 1 ) could be attributed to the action of the alkali on the primary wall of the residue, which resulted in the hydrolysis of the ester bonds of the lignoceUulosic complex and was reflected as 77.7 g kg-1 solubilization of the NDF (Table 2). The reduction in the ADF content of the NH3-treated stover (Table 2 ) is also associated with the solubilization of the lignin and the cellulose, presumably as a result of the decreased crystallinity of the latter, and also its swelling and the Table 1 Experiment 1: effect o f treatment o f sorghum stover with NH3 DM)

Control NH3 SO2 SEM

or

802 on its nitrogen fractions (g k g -

Total nitrogen

Ammonia nitrogen

Acid detergent fiber nitrogen

Non-protein nitrogen

Protein nitrogen

6.8 c 19.7 a 8.0 b 1.93E-03

0.7 b 14.0 a 3.7 c 1.64E-04

3.4 c 4.8 a 3.8 b 8.90E-05

2.2" 13-9 a 2"3b 1.32E-04

4.6 b 5.8a 5'9~ 2.29E-03

For values within columns, different superscripts indicate statistically significant differences ( P < 0.01 ). Values are based on duplicate determinations.

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Table 2 Experiment 1: effect of treatment of sorghum stover with NH3 or SO2 on its fiber fractions (g kg-1 DM)

Control NH3 SO2 SEM

NDF

ADF

Lignin

Cellulose

IVDMD

712.8 b 635.1a 704.4 b 1.0

585.4 b 506.1 a 504.3 ~ 0.2

76.3 c 64.6 a 71.2 b 0.2

422.0 b 358.5 a 350.6 a 0.6

365.9 b 455.2 a 367.6 b 21.0

For values within columns, different superscripts indicate statistically significant differences ( P < 0.01 ). Values are based on duplicate determinations.

Fig. 1. Transversal section of an untreated sorghum stover stem. Magnification, × 150; bar, 100/~m. P, parenchyma; Ph, phloem; X, xylem.

saponification of the ester bonds between lignin and hemicellulose (Klopfenstein et al., 1972; Jackson, 1977; Capper et al., 1977; Saenger, 1982 ). The significant increase ( P < 0.01 ) in the IVDMD of the NH3-treated stover was probably a result of the increments in the nitrogen fractions and the removal of the lignin and crystalline cellulose microfibers that limit the digestion of the structural carbohydrates by the rumen microbes. Using SO2 under ambient conditions (25 ° C) with a 21 day reaction time probably solubilized part of the lignocellulosic complex of the cell wall (Table 2);

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145

Fig. 2. Transversal section of an NH3-treated sorghum stover stem. Magnification, × 100; bar. 100 gm. P, parenchyma; Ph, phloem; X, xylem.

Fig. 3. Transversal section of SO2-treated sorghum stover stem. Magnification, × 200; bar, 100/~m. P, parenchyma; Ph, phloem; X, xylem.

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Table 3

Experiment 2: effect of treatment of sorghum stover with NH3 and SO2 on its nitrogen fractions (g k g - l dry matter)

Control NH3-SO 2 SO2-NH 3 SEM

Total nitrogen

Ammonia nitrogen

Acid detergent fiber nitrogen

Non-protein nitrogen

Protein nitrogen

6.8 b 21.5 a 21.6 a 3.66E-03

0.7 c 9.0 b l 0.0 a 1.25E-03

3.4 c 4.2 b 4.5 a 1.54E-04

2.2 c 13.5 b 17.0 a 4.19E-03

4.6 b 8.3 ~ 4.6 b 6.66E-03

For values within columns, different superscripts indicate statistically significant differences ( P < 0.01 ). Values are based on duplicate determinations.

Table 4 Experiment 2: effect of treatment of sorghum stover with NH3 and SO2 on its fiber fractions (g kg-1 dry matter) NDF

ADF

Lignin

Cellulose

In vitro DMD

Control NH3-SO2 SO2-NH3 SEM

712.8 b 621.2 a 634.2 a 9.40E-01

585.4 ¢ 490.7 a 503.9 b 2.26E-01

76.3 c 57.9 a 64.8 b 4.67E-02

422.0 b 359.5 a 356.5 a 2.27E-01

365.9 b 469.2 a 472.8 ~ 16.6

For values within columns, different superscripts indicate statistically significant differences ( P < 0.01 ). Values are based on duplicate determinations.

however, that was not enough to improve its IVDMD. In this respect, Ben-Ghedalia and Miron (1984b) suggest that in addition to the solubilization of the polysaccharides of the cell wall, SO2 also releases some phenolic oligosaccharides that can inhibit the digestibility of the soluble fraction of the cell wall. Figure 1 shows the arrangement of the cells in vascular bundles and also of the cells of the parenchyma of an untreated stem. This may be compared with the deformation of the tissue and expansion of the cell walls in an NH3-treated stem, depicted in Fig. 2. The vascular tissue of this stem was also deformed, especially at the xylem which appears swelled. This supports the findings of Klopfenstein (1978), who reported changes in the lignin-polysaccharide complex bonds and swelling of the cell wall material as a result of alkaline treatment. This is confirmed by the analysis of the fiber fractions in this study (Table 2), which demonstrated the partial solubilization of the cell wall components of sorghum stover. Figure 3 shows the effect of SO2 treatment to be the partial removal of xylem and phloem (cellulose and lignin) from the cell wall as a result of their solubilization, and of cellulose-containing parenchymatous tissue, causing a loss of struc-

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147

Fig. 4. Transversal section of NH3-SO2 treated sorghum stover stem. Magnification, × 100; bar, 100 #m. P, parenchyma; VB, vascular bundles.

Fig. 5. Transversal section of an SO2-NHa treated sorghum stover stem. Magnification, × 100; bar, 100/zm. P, parenchyma; VB, vascular bundles.

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tural rigidity. Although SO2 also has an effect on cellulose, it can be observed that in this case the characteristic swelling of the tissues due to NH3 is not present; instead, the cells were apparently collapsed.

3.2. Experiment 2 When both treatments were used, the increase in the total nitrogen content (Table 3) was higher than that resulting from the single NH3 treatment (Table 1 ); it is possible that NH3 and SO2 react to form sulfite or ammonium bisulfite, considered to be a water-soluble source of npN that can be used by the rumen microbiota as efficiently as urea nitrogen (Dryden and Leng, 1988 ). In the case of the SO2-NH3 treatment, the initial SO2 treatment apparently allowed a more efficient distribution of nitrogen from NH3, as can be observed from the npN content. In the case of the combined NH3-SO2 treatment, the supply ofnpN was similar to that recorded for NH3 alone, which reinforces the conclusion that previous action by SO2 improves NH3 fixation. The ADF-N content was increased as a result of both treatments (Table 3). However, this increase was larger in the SO2-NH3 treatment, possibly owing to a better fixation of the N derived from NH3, this being a result of the previous attack of SO2 on the cell wall, resulting in the lignin-N-NH3 complex. The reduction in the NDF content in both combined treatments is attributed to the presence of ammonia (Table 4) because SO2 alone did not show any effect on this fraction (Table 1 ). The reductions in the lignin and cellulose (and consequently the ADF) contents of both combined treatments (Table 4), added to the increases in the nitrogen fractions, probably resulted in the higher IVDMD. However, differences between the combined treatments and the single application of NH3 (Table 2) were very small. The latter was probably due to the fact that the cellulose content for all four treatments (NH3, SO2, NH3-SO2, SO2-NH3) decreased in a similar fashion and thus the differences in ADF between them were very small and influenced mostly by changes in lignin content. In the combined treatments it can be observed (Figs. 4 and 5 ) that the structural damage corresponds to the effect of the last chemical agent that was applied. The lignified tissues (xilema and sclerenchyma) are characterized by being difficult to remove, so a larger improvement in the digestibility of the combined treatment residue could be expected; however, from the nutritional point of view (Table 4), the quantitative removal of lignin was probably not as important as the qualitative one, and the type of end-products that are obtained by its solubilization.

Acknowledgments This study formed part of the requirements of a Master's degree (senior author) at the National University of Mexico. The authors wish to acknowledge the technical review of this paper by Irma Tejada-Hern~tndez and Dr. Guadalupe Suarez-Ramos in the chemical and botanical aspects, respectively.

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