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Ammoniation of barley straw. Effect on cellulose crystallinity and water-holding capacity
ANIMAL FEED SCIENCE AND TECHNOLOGY
ELSEVIER
Animal Feed Science Technology 58 (1996) 239-247
Ammoniation of barley straw. Effect on cellulose crystallinity and water-holding capacity Madam
Goto *, Yasuhiko Yokoe
Faculty of Bioresources, Mie University, 1515 Kamihama-cho, Tsu 514, Japan
Received 5 April 1995;accepted9 August 1995
Abstract The X-ray diffraction patterns of untreated and ammonia-treated barley (Hordeum v&are L. cv. Klaxon) straws showed spectra typical of cellulosic materials, having the major peak at a diffraction angle (20) of 23” and a broader, secondary peak at 16”. Two measures of cellulose crystallinity calculated from the X-ray diffraction pattern indicated that treatment of barley straw with 30 g NH, kg-’ straw produced a reduction in crystallinity index values of 14-24%. The water retention of ammonia-treated barley straw was approximately 25% greater than that of the untreated straw following centrifugation at 300 g and 3000 g. A slightly higher swollen volume was also observed for water-saturated ammonia-treated barley straw, although the difference was not significant. It is suggested that two major consequences of treatment, the effect of ammonia as a weak base on ester bonding within the cell wall and the ability of ammonia in an undissociated form to affect cellulose crystallinity, combine to increase the degradability of the treated straw. Keywords: Ammonia treatment; Barley straw; Cellulose crystallinity; X-ray diffraction; Water retention
1. Introduction The improvement in cell-wall degradability of forage plants following treatment with ammonia appears to be associated with a number of small changes in cell-wall composition. These are usually seen in cereal straws as an increase in the nitrogen content of the cell-wall fraction and as a partial loss of ester-linked phenolic acids and acetyl groups. Otherwise, there appears to be little detectable difference in the gross
* Corresponding
author. E-mail: [email protected].
0377-8401/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0377-8401(95)00903-5
240
M. Goto, Y. Yokoe/Animal
Feed Science Technology 58 (1996) 239-247
composition of treated and untreated materials (Ito et al., 1981a; Barton et al., 1986; Goto et al., 1991, 1993). However, the overall effects of ammonia treatment clearly produce changes to the physical properties of the cell-wall fraction and its digestion characteristics. These have been demonstrated in feeding experiments with ruminants by the lower resistance to mastication, the higher outflow rate, and the shorter retention time in the digestive tract of the treated straw (Zorilla-Rios et al., 1985; Ramanzin et al., 1986; Okamoto and Miyazaki, 1990; Nakashima, 1994). Structural changes associated with ammonia treatment can also be seen in straw tissues examined by electron microscopy. A collapse of vascular bundle sheath cells in treated rice straw (Ito et al., 1981b) and the substantial rupture of inner cuticular surfaces and separation from adjacent ground parenchyma in treated wheat straw (Harbers et al., 1982), may have contributed to the observed increases of rate and extent of cell-wall degradation. Goto et al. (1993) observed that ammonia treatment promoted access by mmen micro-organisms to the luminal surface of barley parenchyma and sclerenchyma cells apparently due to an alteration in the fragility of a thinner, rigid layer covering the inner surface of cell walls. The increased friability of straw tissues following ammonia treatment argues for a change having occurred in the physical properties of individual cell walls. This would, in turn, require a modification to the way in which cell-wall polymers are organised. The ability of ammonia to cleave ester bonds, demonstrated by the release of acetic and phenolic acids, could lead to reduction in the number of cross-linkages involving ester groups formed between wall polymers (Lam et al., 1992). The increase in the fiber saturation point of rice hulls with ammonia treatment, defined as the amount of water held by a water-saturated cell wall, suggests that such a loosening of wall structure, with its concomitant increase in water-holding capacity, does occur (Terashima et al., 1984). Another effect of ammonia is suggested by retention of added nitrogen in the cell wall. The ability of liquid ammonia to form an ammonia-cellulose complex and to decrease the crystallinity of cellulose is well known (Isogai and Usuda, 1992). Any reduction in the crystallinity of the cellulose forming the microfibrils of the cell wall might be expected to contribute to an increased fragility of the wall and, possibly, to an increased susceptibility to attack by cellulolytic micro-organisms. However, there is no direct evidence that ammonia, at the concentrations applied to cereal straw, has any direct effect on cellulose. The objective of this study was to confirm the changes in water retention of the cell walls of barley straw following ammonia treatment and to determine whether X-ray diffraction patterns of the cell wall provided any evidence of reduced crystallinity which could be related to the improved degradability characteristics of the treated straw.
2. Materials and methods Spring barley (Hordeum vulgare L.) cv. Klaxon, was grown in 1986 during variety testing at the North of Scotland College of Agriculture, UK, and harvested at the grain-matured stage 24 weeks after sowing. Samples of untreated and ammonia-treated barley straw were those investigated previously (Goto et al., 1993; Goto and Takabe,
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Feed Science Technology 58 (1996) 239-247
241
1995). Treatment with gaseous ammonia (30 g kg- ’ straw) was in an ammonia oven at 80°C for 20 h after the moisture content of the straws had been adjusted to 15% by addition of water. The stem fractions (including chaff) of the treated and untreated samples were ground to pass a 1 mm screen for measurements of chemical composition and to pass a 80 pm screen for measurements of X-ray diffraction patterns, water retention and swollen volume. Acid and neutral detergent fibers were measured according to the standardised methods (Van Soest et al., 19911, and cellulose was by the calorimetric method of Updegraff (1969). Lignin content was determined by the acetyl bromide method (Morrison, 1972). Neutral sugars in hemi-celluloses were determined as their alditol acetates recovered from 2 M trifluoroacetic acid hydrolysates (Albersheim et al., 1967) and separated by gas chromatography (30 m X 0.25 mm i.d. SP-2380 fused silica capillary column; Supelco, USA). Nitrogen content was measured by a micro-Kjeldahl method (Association of Official Analytical Chemists, 1970). Total soluble sugars were determined by a phenol-sulfuric acid method (Dubois et al., 1956). X-ray diffraction patterns were obtained with a diffractometer with a Geiger tube connected to a scaler and a continuous synchronised recorder (Rikagaku Co, Tokyo, Japan). Powdered samples of the straws were held in an aluminum cell and exposed to an X-ray beam (30 kV, 15 mA) from the CuK, radiation which was passed through a Ni filter. The width of both divergence and scatter slits was 1” and that of receiving slit was 0.4 mm. The samples were scanned with a diffraction angle (28) ranging from 5” to 40”. The ratios of height:width at half peak height of the main peak (Baker et al., 1959) and crystalline:amorphous areas (Komiya et al., 1987) were calculated to provide crystallinity index values. The upper area (AC in Fig. 1) which was separated with the smooth curve connecting each point of the minimum intensity corresponded to the crystalline portion and the lower area was background containing the amorphous portion (Aa in Fig. 1). A a was assumed to be the upper area separated with the straight line joining the two points of intensity at 35” and 5” in the background. Water retention (water-holding capacity, g H,O g-’ dry matter) of the powdered samples was determined after soaking of the samples overnight in excess water under vacuum. The amount of water held in the samples against centrifugal forces of 300 g and 3000 g maintained for 50 min was measured gravimetrically.The swollen volume (cm3 g- ’dry matter) was determined by applying the same hydrated samples to the top of glass columns of known diameter with a glasswool plug at the bottom and leaving to stand for 3 h. The height of the column of solid sample above the plug was then measured and the packed volume calculated. Results in Tables 2 and 3 were analyzed using analysis of variance and means were separated by a Student’s t-test when the F-test was significant at a less than 0.05 probability level (Steel and Torrie, 1980).
3. Results and discussion Dry matter (DM) degradability of ammonia-treated barley straw was higher than that of untreated straw, while the gross compositions of both barley straws, except for total
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Diffraction
angle(28)
Fig. 1. X-ray diffraction patterns of untreated and ammonia-treated barley straw. AC and Aa indicate crystalline and amorphous portions in the X-ray diffractogram, respectively. W is measured at half peak of the height (H) of the 23” peak.
nitrogen content, were similar (Table 1). A slightly higher nitrogen content in acid detergent fiber (NIADF) detected for ammonia-treated barley straw was consistent with a previous result of Ito et al. (1981a,b) who showed that 16% of the nitrogen applied to rice (Oryza sarim L.) straw as ammonia was insoluble in hot water and could be detected in the neutral and acid detergent fiber fractions. The X-ray diffraction patterns of untreated and ammonia-treated barley straw similarly showed spectra typical of cellulosic materials (Fig. l>, having the main and secondary peaks at 28 of 23” and 16”, respectively (Atalla, 1983). The main peak is
M. Goto, Y. Yokoe/Animal Table 1 Gross composition Degradability/
and rumen degradability
composition
Dry matter degradability
(g kg-
’) a
Feed Science Technology 58 (1996) 239-247
of untreated
and ammonia-treated
243
barley straw
Untreated
Ammonia-treated
268+z3
376+2
854&3 409+ 12 303zt 12 7.3 69.8 12.1 10.8 126k2 2.5 29.8 4.6 * 0.1
882+2 416*6 303* 15 7.3 70.9 9.6 12.2 123+3 6.4 32.8 4.8kO.l
Dry matter composition (g kg- ’1 Neutral detergent fiber Cellulose Neutral sugars b Arabinose ’ Xylose Glucose Others Lignin Total nitrogen Soluble sugars NlADF d
a The dry matter degradabilities were determined by a nylon-bag method and presented previously 1993). b Neutral sugars contributing hemi-celluloses. ’ Percentages of arabinose, xyloses, glucose and others of the amount of neutral sugars. d Nitrogen content in the acid detergent fiber.
(Goto et al.,
taken as indicative of the presence of highly organised ‘crystalline’ cellulose, while the second, broader peak is a measure of a less organised polysaccharide structure. Xylans and other non-cellulosic polysaccharides contribute to this ‘amorphous’ peak. Various measures relating the two peaks have been devised to provide a value indicative of the extent of crystallinity known as the crystallinity index (CI). Ammonia-treated barley straw had a 24% lower ratio of height to width at half peak height of the main peak (P < 0.05) and a 14% lower ratio of crystalline:amorphous regions than did the untreated straw (Table 2). These observations are consistent with the result of 13C-NMR analysis of highly crystalline fecal fiber from alfalfa-grass hay observed following treatment with liquid ammonia. This showed that the order of two crystalline forms of cellulose in the fecal material was drastically decreased by treatment (Cyr et al., 1990).
Table 2 Ratios of height:width treated barley straw Untreated/treated
Untreated Ammonia-treated
of the main X-ray peak and crystalline-amorphous
Height:width
7.0 a 5.3 b
Crystalline-amorphous
regions in untreated
regions
AC (cm’)
Aa (cm’ )
Ac/(Ac
8.0 f 0.3 6.5 + 0.2
10.4*0.4 11.1*0.3
0.43 b 0.37 b
AC and Aa were indicated in Fig. 1. Means with different superscripts in the same column were significantly t-test.
and ammonia-
different
+ Aa)
(P < 0.05) by a Student’s
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Isogai and Usuda (1992) similarly reported on the X-ray diffraction pattern and solid-state i3C-NMR spectra of cellulose I which converted to cellulose III with very low crystallinity or to an almost wholly disordered structure when treated with liquid ammonia. The formation of a cellulose-ammonia complex was also reported, since ammonia molecules, which could not be removed from the lattice by heating at 60°C for 1 day under vacuum, were readily removed by extraction with dry methanol. This was the result of the low dissociation constant of ammonia and its capacity to diffuse readily into the cellulose microfibril. Therefore, the two measures of CI used here similarly indicated that ammonia treatment produced a significant loss of crystallinity. Any base of sufficient strength is capable of disrupting hydrogen bonding in cellulose microfibrils leading, in aqueous solutions, to water uptake and swelling of cellulose (Immergut, 1963). However, the response of barley straw cellulose to ammonia cannot simply be due to its action as an alkali. Sodium hydroxide, a far stronger alkali, was reported to have no effect on cellulose crystallinity in wheat and barley straw as measured by X-ray diffraction and infrared spectrometry, even when applied at 200 g NaOH kg-’ straw; five to seven times the concentration normally applied to cereal straws (Chesson, 1981). Richter et al. (1992) similarly reported that 7.5% NaOH had no effect on cellulose crystallinity. Given the concentration of hydroxyl ions supplied as gaseous ammonia in this study and their low pK, the observed reduction in crystallinity of the treated barley straw samples could only have been a consequence of the formation of a cellulose-ammonia complex, as shown by some increase of NIADF in ammoniatreated straw. It is of interest, therefore, to examine whether this sort of complex is formed in response to the conditions (temperature, time, moisture, ammonia level, etc.) and whether it is related to changes in cellulose crystallinity and rumen degradability. The effects of varying these conditions of treatment on water retention and swollen volume of barley straw presented later in this study, also require further investigation. Ammonia-treated barley straw had an approximately 25% higher water retention than did the untreated straw (Table 3, P < 0.05). A slightly higher swollen volume was also observed for ammonia-treated barley straw, although this difference was not significant. These results are consistent with observed alteration in fiber saturation point of ammonia-treated rice hulls (Terashima et al., 1984). The extent to which any change in cellulose crystallinity might affect water retention is unclear. However, since the cellulose contents of the untreated and treated straws were virtually identical while water retention varied, it is likely that the two measures are not closely related. Water retention
Table 3 Water retention and swollen volume of untreated Untreated/
treated
Untreated Ammonia-treated Means with different superscripts r-test. a Centrifugation force.
and ammonia-treated
g H,O g-’ dry matter 300aa
3000,1za
1.73 b 2.36 ’
0.79 b 1.02 c
in the same column
were significantly
barley straw Swollen volume (cm3 g- ’dry matter) 5.55 b 5.87 b different
(P < 0.05) by a Student’s
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Feed Science Technology 58 (1996) 239-247
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would appear more likely to reflect the degree to which ester cross-linking between wall polymers was disrupted by treatment allowing the wall as a whole, rather than just the cellulose microfibrils, to swell (Lau and Van Soest, 1981; Hargreaves et al., 1984; Nelson et al., 1984; Manson et al., 1988). The higher water retention of treated barley straw obtained was not wholly consistent with previous results from this laboratory which showed a less extensive effect of ammonia on the water retention of straw samples from the five barley cultivars (Goto et al., 1991). This inconsistency probably relates to the lower particle size used in this study and the exclusion of leaf blade and leaf sheath materials present in the straw samples previously examined. While there is little doubt that the crystallinity of an isolated cellulose has a marked effect on its enzymatic digestion (Soltes, 1983; Hoshino et al., 1992, 1993; Romas et al., 1993), there is still considerable confusion in the literature as to whether cellulose crystallinity has any effect on forage digestion in vivo. Cyr et al. (1990>, for example, found that the fecal fiber from alfalfa-grass hay had a much higher crystallinity index than the original hay, implying that amorphous cellulose was preferentially digested in the animal. It was also concluded from 13C-NMR spectra of grass and clover that, at all incubation periods, highly crystalline cellulose was significantly less degraded than moderately crystalline cellulose (McBride, 1991). However, neither of these reports comments on the distribution of the more crystalline cellulose. It is possible that the results simply reflect the preferential loss of leaf tissue rich in amorphous cellulose, compared with stem with a higher crystalline cellulose content. Others working with forage recovered from the rumen (Beveridge and Richards, 1975) or digestion made with rumen microbes in vitro (Weimer et al., 1990; Hatfield, 1993) have concluded that crystallinity has, at best, only a minor effect on fiber digestion. The results of this and previous work with the same samples suggest that ammonia has two distinct effects which combine to increase the degradability of treated straw. The first, related to ammonia as an alkali, is the limited cleavage of interpolymer bridging structures containing ester bonds. This results in a loosening of wall structure and the observed increase in wall hydration. The second relates to the ability of ammonia to form complexes with cellulose and so to reduce its crystallinity. While this reduction in crystallinity may act to promote the rate of enzymatic digestion, a more important effect would be seen on the fragility of wall structure. A more rapid fragmentation of ingested plant material would increase the surface area available for attack by rumen micro-organisms and the rate of breakdown would increase the rate of passage of treated straw through the digestive tract.
Acknowledgements
The authors thank Dr. Andrew Chesson, Rowett Research Institute, for valuable discussion and critical reading of this paper. Dr. Chesson’s Mie University was supported by International Programs of the Japan Promotion of Science. This work was supported in part by a Scientific Ministry of Education, Science and Culture of Japan (No. 05660309).
Aberdeen, UK, official visit to Society for me Grant from the
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References Albersheim, P., Nevins, D.J., English, P.D. and Karr, A., 1967. A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res., 5: 340-345. Association of Official Analytical Chemists, 1970. Official Methods of Analysis, 1lth edn. Association of Official Analytical Chemists, Washington DC, pp. 122- 131. Atalla, R.H., 1983. The structure of cellulose: Recent developments. In: E.J. Soltes (Editor), Wood and Agricultural Residues-Research on Use for Feed, Fuels, and Chemicals. Academic, New York, pp. 59-77. Baker, T.I., Quicke, G.V., Bentley, O.G., Johnson, R.R. and Moxon, A.L., 1959. The influence of certain physical properties of purified celluloses and forage celluloses on their digestibility by rumen microorganisms in vitro. J. Anim. Sci., 18: 655-662. Barton, F.E., Wolsink, J.H. and Vedder, H.M., 1986. Near infrared reflectance spectroscopy of untreated and ammoniated barley straw. Anim. Feed Sci. Technol., 15: 189-196. Beveridge, R.J. and Richards, G.N., 1975. Digestion of polysaccharide constituents of tropical pasture herbage in the bovine rumen. VI. Investigation of the digestion of cell wall polysaccharides of spear grass and of cotton cellulose by viscometry and by X-ray diffraction. Carbohydr. Res., 43: 163- 172. Chesson, A., 1981. Effects of sodium hydroxide on cereal straws in relation to the enhanced degradation of structural polysacchatides by rumen microorganisms. J. Sci. Food Agric., 32: 745-758. Cyr, N., Elofson, R.M. and Mathison. G.W., 1990. Determination of crystallinity of carbohydrates by 13C cross polarization/magic angle spinning NMR with applications to the nutritive value of forages. Can. J. Anim. Sci., 70: 695-701. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Goto, M. and Takabe, K., 1995. Histochemistry and UV-microspectrometty of cell walls of untreated and ammonia-treated barley straw and their degradation by rumen microorganisms. Food Struct., in press. Goto, M., Gordon, A.H. and Chesson, A., 1991. Effect of gaseous ammonia on barley straws showing different rumen degradabilities. J. Sci. Food Agric., 56: 141-153. Goto, M., Yokoe, Y., Takabe, K., Nisikawa, S. and Morita. 0.. 1993. Effects of gaseous ammonia on chemical and structural features of cell walls in spring barley straw. Anim. Feed Sci. Technol., 40: 207-221. Harbers, L.H., Kretner, G.L. and Davis, G.V.. 1982. Ruminal digestion of ammonium hydroxide-treated wheat straw observed by scanning electron microscopy. J. Anim. Sci., 54: 1309- 1319. Hargreaves, A., Huber, J.T., Arroyoluna, J. and Kung, L., 1984. Influence of adding ammonia to corn stalkage on feeding value for dairy cows and on fermentation changes. J. Anim. Sci., 59: 567-575. Hatfield, R.D., 1993. Cell wall polysaccharide interactions and degradability. In: H.G. Jung, D.R. Buxton, R.D. Hatfield and J. Ralph (Editors), Forage Cell Wall Structure and Digestibility. ASA-CSSA-SSSA Inc., Madison, pp. 285-314. Hoshino, E., Kanda, T., Sasaki, Y. and Nisizawa, K., 1992. Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus rulipiferae) on cellulose with different crystallinities. J. B&hem. (Tokyo). 111: 600605. Hoshino, E., Sasaki, Y., Okazaki, M., Nisizawa K. and Kanda, T., 1993. Mode of action of exo-and endo-type cellulases from Irpex lacreus in the hydrolysis of cellulose with different crystallinities. J. Biochem. (Tokyo), 114: 230-235. Immergut. E.H., 1963. Cellulose. In: B.L. Browning (Editor), The Chemistry of Wood. Interscience. New York, pp. 103-190. Isogai, A. and Usuda, M., 1992. X-ray diffraction and solid-state I3C-NMR analyses of celluloses treated with ammonia. Mokuzai Gakkaishi, 38: 562-569. Ito, H., Terashima, Y., Ueda, K. and Takahashi, Y., 198la. Nitrogen distribution in rice straw and rice hulls treated with sodium chlorite and ammonia. Jpn. J. Zootech. Sci., 52: 519-524. Ito, H., Terashima, Y. and Hayashizaki, A., 198lb. Ammoniated rice straw and rice hulls and rumen microbial degradation investigated by scanning electron microscopy. Jpn. J. Zootech. Sci., 52: 671-679. Komiya, T.. Yamada, T. and Nara, S., 1987. Crystallinity of acid treated corn starch. Staerke, 39: 308-311. Lam, T.B.T., Iiyama, K. and Stone B.A., 1992. Cimtamic acid bridges between cell wall polymers in wheat and phalaris internodes. Phytochem., 31: 1179-l 183.
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Lau, M.M. and van Soest, P.J., 1981. Titratable digestibility of chemically treated roughages.
groups and soluble phenolic compounds as indicators Anim. Feed Sci. Technol., 6: 123- 13 1.
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Manson, V.C., Hartley, R.D., Keene, A.S. and Cobby, J.M., 1988. The effect of ammoniation on the nutritive value of wheat, barley and oat straws. I. Changes in chemical composition in relation to digestibility in vitro and cell wall degradability. Anim. Feed Sci. Technol., 19: 159- 171. McBride, B.W., 1991. ‘3C-CP-MAS NMR spectrometry of cellulose and forages. N.Z. J. Agric. Res., 34: 257-262. Morrison, I.M., 1972. A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage crops. J. Sci. Food Agric., 23: 455-463. Nakashima, Y., 1994. Effects of ammonia treatment on the rumen degradation characteristics and retention time of rice straw in sheep. J. Jpn. Grassl. Sci., 40: 106-109. Nelson, M.L., Klopfenstein, T.J. and Britton, R.A., 1984. Protein supplementation of ammoniated roughages. I. Corncobs supplemented with a blood-meal-corn gluten meal mixture-lamb studies. J. Anim. Sci., 59: 1601-1609. Okamoto, M. and Miyazaki, H., 1990. Particle size reduction of reticula-ruminal contents in sheep fed rice straw and ammoniated rice straw. Jpn. J. Zootech. Sci., 61: 424-427. Ramanzin, M., 0rskov. E.R. and Tuah, A.K., 1986. Rumen degradation of straw. 2. Botanical fractions of straw from two barley cultivars. Anim. Prod., 43: 27 1-278. Richter, U., Krause, T. and Schempp, W., 1992. Strukur%nderungen von cellulose durch alkalibehandlung: physikalische und chemische beurteilungsmethoden. Papier (Darmstadt), 46: 318-323. Romas, L.P., Nazhad, M.M. and Saddler, J.N., 1993. Effect of enzymic hydrolysis on the morphology and fine structure of pretreated cellulosic residues. Enzyme Microb. Technol., 15: 82 1-83 1. Soltes, E.J., 1983. Cellulose: Elusive component of the plant cell wall. In: W.A. Cot (Editor), Biomass Utilization. Plenum, New York, pp. 271-298. Steel, R.G.D. and Torrie, J.H., 1980. Principles and Procedures of Statistics. McGraw-Hill, New York. Terashima, Y., Tohrai, N. and Ito, H., 1984. Effect of ammonia treatment on free carboxyl group content and fiber saturation point of rice straw and rice hulls. Jpn. J. Zootech. Sci., 55: 569-575. Updegraff, D.M., 1969. Semi-micro determinations of cellulose in biological materials. Anal. Biochem., 32: 420-424. Van Soest, P.J., Robertson, J.B. and Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci., 74: 3583-3597. Weimer, P.J., Lopez-Guisa, J.M. and French, A.D., 1990. Effect of cellulose fine structure on kinetics of its digestion by mixed ruminal microorganisms in vitro. Appl. Environ. Microbial., 56: 2421-2429. Zorilla-Rios, J., Owens, F.N., Horn, G.W. and McNew, R.W., 1985. Effect of ammoniation of wheat straw on performance and digestion kinetics in cattle. J. Anim. Sci., 60: 814-821.
ELSEVIER
Animal Feed Science Technology 58 (1996) 239-247
Ammoniation of barley straw. Effect on cellulose crystallinity and water-holding capacity Madam
Goto *, Yasuhiko Yokoe
Faculty of Bioresources, Mie University, 1515 Kamihama-cho, Tsu 514, Japan
Received 5 April 1995;accepted9 August 1995
Abstract The X-ray diffraction patterns of untreated and ammonia-treated barley (Hordeum v&are L. cv. Klaxon) straws showed spectra typical of cellulosic materials, having the major peak at a diffraction angle (20) of 23” and a broader, secondary peak at 16”. Two measures of cellulose crystallinity calculated from the X-ray diffraction pattern indicated that treatment of barley straw with 30 g NH, kg-’ straw produced a reduction in crystallinity index values of 14-24%. The water retention of ammonia-treated barley straw was approximately 25% greater than that of the untreated straw following centrifugation at 300 g and 3000 g. A slightly higher swollen volume was also observed for water-saturated ammonia-treated barley straw, although the difference was not significant. It is suggested that two major consequences of treatment, the effect of ammonia as a weak base on ester bonding within the cell wall and the ability of ammonia in an undissociated form to affect cellulose crystallinity, combine to increase the degradability of the treated straw. Keywords: Ammonia treatment; Barley straw; Cellulose crystallinity; X-ray diffraction; Water retention
1. Introduction The improvement in cell-wall degradability of forage plants following treatment with ammonia appears to be associated with a number of small changes in cell-wall composition. These are usually seen in cereal straws as an increase in the nitrogen content of the cell-wall fraction and as a partial loss of ester-linked phenolic acids and acetyl groups. Otherwise, there appears to be little detectable difference in the gross
* Corresponding
author. E-mail: [email protected].
0377-8401/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0377-8401(95)00903-5
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Feed Science Technology 58 (1996) 239-247
composition of treated and untreated materials (Ito et al., 1981a; Barton et al., 1986; Goto et al., 1991, 1993). However, the overall effects of ammonia treatment clearly produce changes to the physical properties of the cell-wall fraction and its digestion characteristics. These have been demonstrated in feeding experiments with ruminants by the lower resistance to mastication, the higher outflow rate, and the shorter retention time in the digestive tract of the treated straw (Zorilla-Rios et al., 1985; Ramanzin et al., 1986; Okamoto and Miyazaki, 1990; Nakashima, 1994). Structural changes associated with ammonia treatment can also be seen in straw tissues examined by electron microscopy. A collapse of vascular bundle sheath cells in treated rice straw (Ito et al., 1981b) and the substantial rupture of inner cuticular surfaces and separation from adjacent ground parenchyma in treated wheat straw (Harbers et al., 1982), may have contributed to the observed increases of rate and extent of cell-wall degradation. Goto et al. (1993) observed that ammonia treatment promoted access by mmen micro-organisms to the luminal surface of barley parenchyma and sclerenchyma cells apparently due to an alteration in the fragility of a thinner, rigid layer covering the inner surface of cell walls. The increased friability of straw tissues following ammonia treatment argues for a change having occurred in the physical properties of individual cell walls. This would, in turn, require a modification to the way in which cell-wall polymers are organised. The ability of ammonia to cleave ester bonds, demonstrated by the release of acetic and phenolic acids, could lead to reduction in the number of cross-linkages involving ester groups formed between wall polymers (Lam et al., 1992). The increase in the fiber saturation point of rice hulls with ammonia treatment, defined as the amount of water held by a water-saturated cell wall, suggests that such a loosening of wall structure, with its concomitant increase in water-holding capacity, does occur (Terashima et al., 1984). Another effect of ammonia is suggested by retention of added nitrogen in the cell wall. The ability of liquid ammonia to form an ammonia-cellulose complex and to decrease the crystallinity of cellulose is well known (Isogai and Usuda, 1992). Any reduction in the crystallinity of the cellulose forming the microfibrils of the cell wall might be expected to contribute to an increased fragility of the wall and, possibly, to an increased susceptibility to attack by cellulolytic micro-organisms. However, there is no direct evidence that ammonia, at the concentrations applied to cereal straw, has any direct effect on cellulose. The objective of this study was to confirm the changes in water retention of the cell walls of barley straw following ammonia treatment and to determine whether X-ray diffraction patterns of the cell wall provided any evidence of reduced crystallinity which could be related to the improved degradability characteristics of the treated straw.
2. Materials and methods Spring barley (Hordeum vulgare L.) cv. Klaxon, was grown in 1986 during variety testing at the North of Scotland College of Agriculture, UK, and harvested at the grain-matured stage 24 weeks after sowing. Samples of untreated and ammonia-treated barley straw were those investigated previously (Goto et al., 1993; Goto and Takabe,
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Feed Science Technology 58 (1996) 239-247
241
1995). Treatment with gaseous ammonia (30 g kg- ’ straw) was in an ammonia oven at 80°C for 20 h after the moisture content of the straws had been adjusted to 15% by addition of water. The stem fractions (including chaff) of the treated and untreated samples were ground to pass a 1 mm screen for measurements of chemical composition and to pass a 80 pm screen for measurements of X-ray diffraction patterns, water retention and swollen volume. Acid and neutral detergent fibers were measured according to the standardised methods (Van Soest et al., 19911, and cellulose was by the calorimetric method of Updegraff (1969). Lignin content was determined by the acetyl bromide method (Morrison, 1972). Neutral sugars in hemi-celluloses were determined as their alditol acetates recovered from 2 M trifluoroacetic acid hydrolysates (Albersheim et al., 1967) and separated by gas chromatography (30 m X 0.25 mm i.d. SP-2380 fused silica capillary column; Supelco, USA). Nitrogen content was measured by a micro-Kjeldahl method (Association of Official Analytical Chemists, 1970). Total soluble sugars were determined by a phenol-sulfuric acid method (Dubois et al., 1956). X-ray diffraction patterns were obtained with a diffractometer with a Geiger tube connected to a scaler and a continuous synchronised recorder (Rikagaku Co, Tokyo, Japan). Powdered samples of the straws were held in an aluminum cell and exposed to an X-ray beam (30 kV, 15 mA) from the CuK, radiation which was passed through a Ni filter. The width of both divergence and scatter slits was 1” and that of receiving slit was 0.4 mm. The samples were scanned with a diffraction angle (28) ranging from 5” to 40”. The ratios of height:width at half peak height of the main peak (Baker et al., 1959) and crystalline:amorphous areas (Komiya et al., 1987) were calculated to provide crystallinity index values. The upper area (AC in Fig. 1) which was separated with the smooth curve connecting each point of the minimum intensity corresponded to the crystalline portion and the lower area was background containing the amorphous portion (Aa in Fig. 1). A a was assumed to be the upper area separated with the straight line joining the two points of intensity at 35” and 5” in the background. Water retention (water-holding capacity, g H,O g-’ dry matter) of the powdered samples was determined after soaking of the samples overnight in excess water under vacuum. The amount of water held in the samples against centrifugal forces of 300 g and 3000 g maintained for 50 min was measured gravimetrically.The swollen volume (cm3 g- ’dry matter) was determined by applying the same hydrated samples to the top of glass columns of known diameter with a glasswool plug at the bottom and leaving to stand for 3 h. The height of the column of solid sample above the plug was then measured and the packed volume calculated. Results in Tables 2 and 3 were analyzed using analysis of variance and means were separated by a Student’s t-test when the F-test was significant at a less than 0.05 probability level (Steel and Torrie, 1980).
3. Results and discussion Dry matter (DM) degradability of ammonia-treated barley straw was higher than that of untreated straw, while the gross compositions of both barley straws, except for total
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Diffraction
angle(28)
Fig. 1. X-ray diffraction patterns of untreated and ammonia-treated barley straw. AC and Aa indicate crystalline and amorphous portions in the X-ray diffractogram, respectively. W is measured at half peak of the height (H) of the 23” peak.
nitrogen content, were similar (Table 1). A slightly higher nitrogen content in acid detergent fiber (NIADF) detected for ammonia-treated barley straw was consistent with a previous result of Ito et al. (1981a,b) who showed that 16% of the nitrogen applied to rice (Oryza sarim L.) straw as ammonia was insoluble in hot water and could be detected in the neutral and acid detergent fiber fractions. The X-ray diffraction patterns of untreated and ammonia-treated barley straw similarly showed spectra typical of cellulosic materials (Fig. l>, having the main and secondary peaks at 28 of 23” and 16”, respectively (Atalla, 1983). The main peak is
M. Goto, Y. Yokoe/Animal Table 1 Gross composition Degradability/
and rumen degradability
composition
Dry matter degradability
(g kg-
’) a
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of untreated
and ammonia-treated
243
barley straw
Untreated
Ammonia-treated
268+z3
376+2
854&3 409+ 12 303zt 12 7.3 69.8 12.1 10.8 126k2 2.5 29.8 4.6 * 0.1
882+2 416*6 303* 15 7.3 70.9 9.6 12.2 123+3 6.4 32.8 4.8kO.l
Dry matter composition (g kg- ’1 Neutral detergent fiber Cellulose Neutral sugars b Arabinose ’ Xylose Glucose Others Lignin Total nitrogen Soluble sugars NlADF d
a The dry matter degradabilities were determined by a nylon-bag method and presented previously 1993). b Neutral sugars contributing hemi-celluloses. ’ Percentages of arabinose, xyloses, glucose and others of the amount of neutral sugars. d Nitrogen content in the acid detergent fiber.
(Goto et al.,
taken as indicative of the presence of highly organised ‘crystalline’ cellulose, while the second, broader peak is a measure of a less organised polysaccharide structure. Xylans and other non-cellulosic polysaccharides contribute to this ‘amorphous’ peak. Various measures relating the two peaks have been devised to provide a value indicative of the extent of crystallinity known as the crystallinity index (CI). Ammonia-treated barley straw had a 24% lower ratio of height to width at half peak height of the main peak (P < 0.05) and a 14% lower ratio of crystalline:amorphous regions than did the untreated straw (Table 2). These observations are consistent with the result of 13C-NMR analysis of highly crystalline fecal fiber from alfalfa-grass hay observed following treatment with liquid ammonia. This showed that the order of two crystalline forms of cellulose in the fecal material was drastically decreased by treatment (Cyr et al., 1990).
Table 2 Ratios of height:width treated barley straw Untreated/treated
Untreated Ammonia-treated
of the main X-ray peak and crystalline-amorphous
Height:width
7.0 a 5.3 b
Crystalline-amorphous
regions in untreated
regions
AC (cm’)
Aa (cm’ )
Ac/(Ac
8.0 f 0.3 6.5 + 0.2
10.4*0.4 11.1*0.3
0.43 b 0.37 b
AC and Aa were indicated in Fig. 1. Means with different superscripts in the same column were significantly t-test.
and ammonia-
different
+ Aa)
(P < 0.05) by a Student’s
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Isogai and Usuda (1992) similarly reported on the X-ray diffraction pattern and solid-state i3C-NMR spectra of cellulose I which converted to cellulose III with very low crystallinity or to an almost wholly disordered structure when treated with liquid ammonia. The formation of a cellulose-ammonia complex was also reported, since ammonia molecules, which could not be removed from the lattice by heating at 60°C for 1 day under vacuum, were readily removed by extraction with dry methanol. This was the result of the low dissociation constant of ammonia and its capacity to diffuse readily into the cellulose microfibril. Therefore, the two measures of CI used here similarly indicated that ammonia treatment produced a significant loss of crystallinity. Any base of sufficient strength is capable of disrupting hydrogen bonding in cellulose microfibrils leading, in aqueous solutions, to water uptake and swelling of cellulose (Immergut, 1963). However, the response of barley straw cellulose to ammonia cannot simply be due to its action as an alkali. Sodium hydroxide, a far stronger alkali, was reported to have no effect on cellulose crystallinity in wheat and barley straw as measured by X-ray diffraction and infrared spectrometry, even when applied at 200 g NaOH kg-’ straw; five to seven times the concentration normally applied to cereal straws (Chesson, 1981). Richter et al. (1992) similarly reported that 7.5% NaOH had no effect on cellulose crystallinity. Given the concentration of hydroxyl ions supplied as gaseous ammonia in this study and their low pK, the observed reduction in crystallinity of the treated barley straw samples could only have been a consequence of the formation of a cellulose-ammonia complex, as shown by some increase of NIADF in ammoniatreated straw. It is of interest, therefore, to examine whether this sort of complex is formed in response to the conditions (temperature, time, moisture, ammonia level, etc.) and whether it is related to changes in cellulose crystallinity and rumen degradability. The effects of varying these conditions of treatment on water retention and swollen volume of barley straw presented later in this study, also require further investigation. Ammonia-treated barley straw had an approximately 25% higher water retention than did the untreated straw (Table 3, P < 0.05). A slightly higher swollen volume was also observed for ammonia-treated barley straw, although this difference was not significant. These results are consistent with observed alteration in fiber saturation point of ammonia-treated rice hulls (Terashima et al., 1984). The extent to which any change in cellulose crystallinity might affect water retention is unclear. However, since the cellulose contents of the untreated and treated straws were virtually identical while water retention varied, it is likely that the two measures are not closely related. Water retention
Table 3 Water retention and swollen volume of untreated Untreated/
treated
Untreated Ammonia-treated Means with different superscripts r-test. a Centrifugation force.
and ammonia-treated
g H,O g-’ dry matter 300aa
3000,1za
1.73 b 2.36 ’
0.79 b 1.02 c
in the same column
were significantly
barley straw Swollen volume (cm3 g- ’dry matter) 5.55 b 5.87 b different
(P < 0.05) by a Student’s
M. Goto. Y. Yokoe/Animal
Feed Science Technology 58 (1996) 239-247
245
would appear more likely to reflect the degree to which ester cross-linking between wall polymers was disrupted by treatment allowing the wall as a whole, rather than just the cellulose microfibrils, to swell (Lau and Van Soest, 1981; Hargreaves et al., 1984; Nelson et al., 1984; Manson et al., 1988). The higher water retention of treated barley straw obtained was not wholly consistent with previous results from this laboratory which showed a less extensive effect of ammonia on the water retention of straw samples from the five barley cultivars (Goto et al., 1991). This inconsistency probably relates to the lower particle size used in this study and the exclusion of leaf blade and leaf sheath materials present in the straw samples previously examined. While there is little doubt that the crystallinity of an isolated cellulose has a marked effect on its enzymatic digestion (Soltes, 1983; Hoshino et al., 1992, 1993; Romas et al., 1993), there is still considerable confusion in the literature as to whether cellulose crystallinity has any effect on forage digestion in vivo. Cyr et al. (1990>, for example, found that the fecal fiber from alfalfa-grass hay had a much higher crystallinity index than the original hay, implying that amorphous cellulose was preferentially digested in the animal. It was also concluded from 13C-NMR spectra of grass and clover that, at all incubation periods, highly crystalline cellulose was significantly less degraded than moderately crystalline cellulose (McBride, 1991). However, neither of these reports comments on the distribution of the more crystalline cellulose. It is possible that the results simply reflect the preferential loss of leaf tissue rich in amorphous cellulose, compared with stem with a higher crystalline cellulose content. Others working with forage recovered from the rumen (Beveridge and Richards, 1975) or digestion made with rumen microbes in vitro (Weimer et al., 1990; Hatfield, 1993) have concluded that crystallinity has, at best, only a minor effect on fiber digestion. The results of this and previous work with the same samples suggest that ammonia has two distinct effects which combine to increase the degradability of treated straw. The first, related to ammonia as an alkali, is the limited cleavage of interpolymer bridging structures containing ester bonds. This results in a loosening of wall structure and the observed increase in wall hydration. The second relates to the ability of ammonia to form complexes with cellulose and so to reduce its crystallinity. While this reduction in crystallinity may act to promote the rate of enzymatic digestion, a more important effect would be seen on the fragility of wall structure. A more rapid fragmentation of ingested plant material would increase the surface area available for attack by rumen micro-organisms and the rate of breakdown would increase the rate of passage of treated straw through the digestive tract.
Acknowledgements
The authors thank Dr. Andrew Chesson, Rowett Research Institute, for valuable discussion and critical reading of this paper. Dr. Chesson’s Mie University was supported by International Programs of the Japan Promotion of Science. This work was supported in part by a Scientific Ministry of Education, Science and Culture of Japan (No. 05660309).
Aberdeen, UK, official visit to Society for me Grant from the
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