Ensiling of whole crop wheat with
cellulase±hemicellulase based enzymes
3. Comparing effects of urea or enzyme treatment
on forage composition and stability
T. Adogla-Bessa
a,1, E. Owen
a, A.T. Adesogan
b,* aDepartment of Agriculture, The University of Reading, Earley Gate,P.O. Box 236, Reading RG6 6AT, UK
bWelsh Institute of Rural Studies, University of Wales, Aberystwyth, Llanbadarn Campus,
Aberystwyth SY23 4AL, Wales, UK
Received 21 October 1998; received in revised form 25 June 1999; accepted 20 July 1999
Abstract
This work compared the effect of ensiling technique and application of urea or a cellulase± hemicellulase enzyme mixture (FSO2) on the conservation of whole crop wheat. Awheat crop harvested
at 600 g dry matter (DM) kgÿ1was conserved with or without the application of urea (40 g kgÿ1DM)
and four levels of FSO2 (0, 1750, 3500 and 15 503 ml tÿ1DM). Each forage (5 kg DM) was conserved
in polythene-bag silos that were evacuated and sealed immediately, sealed immediately but not evacuated or evacuated and sealed after 24 h. After at least 42 days of conservation, the silos were opened and analysed for chemical composition, rumen fluid-pepsin in vitro digestibility and aerobic stability. In non-urea-treated forages, increasing enzyme application rate did not affect in vitro digestibility, but increased water soluble carbohydrate and lactic acid contents, and reduced pH, neutral detergent fibre, acid detergent fibre and cellulose contents. Compared to the FSO2 only treatments, urea treatment increased pH and N content and reduced ensiling DM loss, neutral detergent fibre, acid
detergent fibre, acid detergent lignin and cellulose contents. Application of FSO2 and urea (FSO2U)
produced forages with higher in vitro digestibility and lower cell wall contents than in FSO2 only
forages. NDF and ADF contents were also 5±10% lower in FSO2U forages than in those conserved
with only urea. Immediate evacuation of silos did not enhance fermentation quality. Delaying silo sealing by 24 h increased lactic acid content and aerobic stability relative to either of the immediate seal treatments. Urea treatment alone and the high enzyme level alone also enhanced aerobic stability.
However, increasing the enzyme application rate in the FSO2U treatments did not enhance
82 (1999) 51±61
*Corresponding author. Tel.:44-1970-624471; fax:44-1970-611264
E-mail address: ata@aber.ac.uk (A.T. Adesogan)
1Present address: University of Botswana, Botswana College of Agriculture, Bag 0027, Gaborone, Botswana.
stability. These findings indicate that in whole crop wheat, the medium (3500 ml tÿ1DM) and high
(15 503 ml tÿ1DM) levels of FSO2 application are as effective as urea in degrading cell walls and
maintaining aerobic stability. However, nutritive value is optimised when whole crop wheat is
conserved using a mixture of urea and the low level (1750 ml tÿ1DM) of FSO2.#1999 Elsevier
Science B.V. All rights reserved.
Keywords: Whole crop wheat; Chemical composition; In vitro digestibility; Aerobic stability
1. Introduction
Whole crop wheat (WCW) can be harvested and successfully conserved over a wide range of crop maturities (350±700 g DM kgÿ1; Adogla-Bessa and Owen, 1995) but the optimum dry matter (DM) yield is attained at the medium-dough growth stage (550±600 g DM kgÿ1) (Corral et al., 1977). However, at such advanced growth stages, water soluble carbohydrate content (WSC) is low and therefore fermentation capacity is reduced. Urea application has been successfully used to overcome this problem as the resulting alkaline preservation obviates the need for a high WSC content in the crop. However, several studies have shown disappointing production results when dairy cows were fed urea-treated WCW harvested at optimal DM yield (Leaver and Hill, 1992). This problem is thought to relate largely to the lignified pericarp in mature WCW, which prevents digestion of the grain. Although cellulolytic enzymes have been successfully used to hydrolyse forage polysaccharides in grass silage (Huhtanen et al., 1985) and whole crop sorghum silage (Laytimi et al., 1988), little is known about their potential for conserving and enhancing the nutritive value of WCW. Therefore, this work aimed to determine how cellulolytic enzymes compare with urea for conserving mature WCW. To this end, the study evaluated the effect of urea or enzyme treatment on the chemical composition, in vitro digestibility and aerobic stability of WCW harvested at optimum DM yield. Since WCW is particularly prone to aerobic deterioration, this work also investigated the effect of different ensiling techniques on the conservation of WCW. The study is particularly relevant to the current UK situation where farmers are looking for alternatives to urea treatment for conserving WCW harvested at optimal DM yield. This is because of the inefficient utilisation of such forages due to losses of undigested cereal grains in the faeces. This study follows on from previous work (Adogla-Bessa and Owen, 1995) which showed that specific cellulase±hemicellulase enzymes enhanced the fermentation of immature (< 500 g kgÿ1 DM) WCW but did not enhance digestibility or reduce fibre content. A different enzyme mixture with a higher hemicellulase activity (FSO2) was investigated in this study.
2. Materials and methods
2.1. Forage preparation and additive treatments
Whole crop winter wheat (cv. `Brock') was cut and harvested with a New Holland 717 metered-chop, forage harvester, at the medium-dough stage (600 g DM kgÿ1). The crop
was precision chopped and conserved with an enzyme additive (FSO2, a cellulase± hemicellulase experimental enzyme mixture from Finnish Sugar, Helsinki, Finland) applied at rates of 0, 1750 ml tÿ1 DM (low), 3500 ml tÿ1 DM (medium) and 15 503 ml tÿ1DM (high). Additional treatments included the application of urea (40 g kgÿ1DM) alone and the application of a mixture of urea and each of the enzyme levels (FSO2U). The enzyme was obtained in liquid form, diluted to a standard volume and applied using a high velocity pressure sprayer (Kremlin pneumatic sprayer, model J4, Kremlin Spray Painting Equipment Ltd. Slough, UK). Enzyme activities (IU mlÿ1) for FSO2 were as follows: cellulase 2.08; carboxymethyl cellulase 54.00; cellobiase 3.24; xylanase 496.9; (Jacobs, 1989).
Each forage (5 kg) was conserved in a polythene bag silo using the procedure outlined by Adogla-Bessa and Owen (1995). The silos were evacuated as completely as possible with a vacuum pump (Edwards Hi-Vac vacuum pump, Crawley, UK) and sealed immediately, not evacuated and sealed immediately, or evacuated and sealed after 24 h.
2.2. Forage stability measurements
The forages were ensiled for a minimum of 42 days and ensiling DM loss determined as the weight difference between silo DM content at sealing and opening. Silos were exposed for 55 days in a controlled environment room (15±178C). Exposure DM loss was determined as the weight difference between silo DM content at opening and at the end of the exposure period. Silo temperatures were monitored daily during the exposure period, as an index of bacterial activity and aerobic deterioration. Thermocouple wires (Ni±Al) were inserted into the geometric centre of each silo and left in situ. The wires were connected to a central junction and recording box (Wheatstone Bridge, Comark 1621, Comark Electronics, Littlehampton, UK) which enabled temperature to be read without disturbing the silo.
2.3. Chemical analyses
2.4. Experimental design and statistical analysis
The design was a 243 factorial arrangement with 2 replicates per treatment. Data were analysed using the Genstat Statistical Package (Lawes Agricultural Trust, 1988) and differences between means were tested using the studentst-test.Fratios were inspected and where the main effects were positive and much larger than the interaction effect, they were also discussed (Mead and Curnow, 1983).
3. Results
3.1. Effect of additive type
Table 1 shows the effect of additive type on forage chemical composition and DM loss. Silo DM losses during ensiling and exposure were lower (P< 0.01) in forages treated FSO2U than in FSO2-treated forages (Table 1). The FSO2U treatment also gave higher (P< 0.01) in vitro DOMD and total N contents, and lower (P< 0.05) ADF, cellulose, ADL and NDF (P< 0.05) contents than the FSO2 treatment. FSO2U application also resulted in higher (P< 0.01) pH and acetic acid levels and lower (P< 0.01) lactic and butyric acid levels. Aerobic spoilage was inhibited for up to 30 days
Table 1
Main effect of enzyme (FSO2) and enzymeurea (FSO2U) treatment on silo dry matter loss, chemical composition and fermentation products in WCW forages
Additive SEDb
FSO2a FSO2U
pH 4.7 8.9 0.02
In vitro organic matter digestibility (g kgÿ1DM)
530 620 11.0
Silo dry matter loss (g kgÿ1DM)
Ensiling 11 3 0.2
Exposure 160 126 4.1
Composition of dry matter (g kgÿ1DM)
Total nitrogen 14.8 21.2 0.19
Neutral detergent fibre 492 441 13.5 Acid detergent fibre 301 274 10.9
Cellulose 240 220 9
Acid detergent lignin 52 49 1.8
Fermentation products
Lactic acid (g kgÿ1DM) 7.6 2.4 0.44
Acetic acid (g kgÿ1DM) 5.4 10.5 0.34
Butyric acid (g kgÿ1DM) 1.9 0.1 0.07
Ammonia nitrogen (g kgÿ1N) 67 379 10 aAn experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland. bNumber of observations per mean was 24.
by FSO2U application except at the low enzyme level (Fig. 1). Whereas deterioration was apparent within 10 days in the FSO2-treated forages.
Compared to the FSO2-treated forages, urea treatment increased pH and N content and reduced ensiling DM loss, NDF, ADF, ADL and cellulose contents (Table 2).
3.2. Effect of enzyme application rate and interaction with additive type
Table 2 shows the effect of enzyme concentration on chemical composition. Increasing the rate of enzyme application did not affect in vitro DOMD or exposure DM losses. In FSO2-treated forages, increasing enzyme concentration increased WSC content whilst
Table 2
Effect of enzyme (FSO2a) application rate on chemical composition, in vitro digestibility and fermentation products in WCW forages conserved with enzymes or with an enzyme±urea mixture (FSO2U)
Enzyme application rate (ml tÿ1DM) SEDb
0
Lactic acid 4.7 4.4 4.3 6.6 0.63
Acetic acid 8.0 8.1 7.6 8.2 0.42
Digestible organic matter
Total nitrogen FSO2 14.5 14.3 14.7 15.6 0.39 (g kgÿ1DM) FSO2U 21.2 21.4 21.3 20.7
Neutral detergent fibre FSO2 526 526 485 432 27.1 (g kgÿ1DM) FSO2U 448 461 418 469
Acid detergent fibre FSO2 333 320 288 263 21.8 (g kgÿ1DM) FSO2U 276 262 256 301
Cellulose FSO2 250 250 240 200 17.0 (g kgÿ1DM) FSO2U 22 21 20 23
Water soluble carbohydrates FSO2 64 69 89 118 5.3 (g kgÿ1DM) FSO2U 77 82 82 76
Butyric acid FSO2 2.5 2.0 1.7 1.9 0.14 (g kgÿ1DM) FSO2U 0.1 0.1 0.1 0.1
Ammonia-nitrogen (g kgÿ1N) FSO2 74 79 57 57 19.2
FSO2U 87 446 487 498
aAn experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland. bNumber of observations per mean was 6.
Fig. 1. Temperature changes as an indication of aerobic deterioration in untreated, enzyme-treated (FS2), urea treated or ureaenzyme treated (FS2U) WCW harvested and ensiled immediately (IMM) or after 24 h (DEL), with or without evacuation (NOVAC) of air. (&ambient;untreated; * low-enzyme;&medium enzyme; x high-enzyme;}urea).
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decreasing (P< 0.05) pH, ensiling DM losses, and contents of butyric acid, NDF, ADF and cellulose. Such trends were absent in the FSO2U treated forages. In contrast, ammonia nitrogen content increased with increasing enzyme application rate in FSO2U treated forages but was unaffected by FSO2 treatment.
3.3. Effect of silo sealing treatment
Table 3 shows the effect of the silo sealing treatment on chemical composition and aerobic stability. Delaying sealing by 24 h significantly reduced (P< 0.01) DM losses during exposure. Ensiling DM loss was unaffected by sealing treatment in FSO2 treatments, but was lowest (P< 0.05) in silos which were not evacuated after FSO2U application.
Table 3
Effect of silo sealing treatment on chemical composition, aerobic stability and fermentation products in WCW forages conserved with enzyme (FSO2a) and an enzyme±urea mixture (FSO2U)
Silo sealing treatmentb SEDc
Immediate digestibility (g kgÿ1DM)
570 590 570 14.0
Total nitrogen 17.9 18.1 17.8 0.24 Neutral detergent fibre 477 451 484 16.6 Acid detergent fibre 288 273 301 13.3
Cellulose 23 22 23 1.0
Ensiling DM loss FSO2 11.0 11.0 10.9 0.26 (g kgÿ1DM) FSO2U 3.9 2.2 3.9
Butyric acid FSO2 2.1 2.0 1.6 0.12 (g kgÿ1DM) FSO2U 0.1 0.1 0.1
aAn experimental cellulase±hemicellulase enzyme mixture from Finnish Sugar Company, Helsinki, Finland. bMean of FSO2 and FSO2U treatments.
Seal treatment did not affect N content, in vitro DOMD or structural carbohydrate contents. An exception was the ADF content which was highest (P< 0.05) when sealing was delayed for 24 h and lowest (P< 0.05) when silos were not evacuated. WSC content was higher (P< 0.01) in non-evacuated silos. Delaying silo sealing by 24 h increased pH (P< 0.01) reduced lactic and butyric acid levels (P< 0.01) compared to the immediately sealed silos (Table 3). Other fermentation products were unaffected by sealing treatment. Irrespective of silo seal treatment, untreated and FSO2-treated forages tended to deteriorate within a week of exposure (Fig. 1) but FSO2U forages remained stable for about 30 days. Also enzyme level had no major effect on aerobic stability.
4. Discussion
4.1. Effect of additive type and level
The FSO2U treatment was better than the FSO2 treatment at degrading cell wall carbohydrates, enhancing digestibility, minimising DM losses and maintaining aerobic stability. The presence of urea which as found previously (Deschard et al., 1988; Tetlow, 1992; Adesogan et al., 1998), enhances digestibility and N content and decreases structural carbohydrate content in WCW, may have contributed to the effectiveness of the FSO2U treatment. The urea present would have also promoted alkaline conditions that inhibit the proliferation of fungi and saccharolytic clostridia that cause aerobic spoilage and DM losses, respectively (McDonald et al., 1991). Hill and Leaver (1991) and Tetlow (1992) also reported urea treatment inhibited aerobic deterioration in WCW. In the rumen, the alkalinity resulting from urea treatment would have buffered rumen acidity and provided a conducive environment for cellulolysis and growth of rumen bacteria (Mgheni et al., 1994) and protozoa (Mould and Orskov, 1984). A synchronous supply of the additional volatile N from the urea and the fermentable energy in the whole crop forages could have also contributed to the enhancement of microbial growth.
The fact that forages to which FSO2U was applied had 5±10% lower NDF and ADF contents than urea-treated forages suggests that the effects of the enzyme and urea were complimentary. Other authors have also shown that combining urea and cellulase enzyme treatments resulted in higher digestibility and lower cell wall contents in grass, maize and sorghum silages than either of the treatments alone (Baintner et al., 1989; Jakhmola et al., 1990). However, Jacobs (1989) reported reductions in FSO2 activity with increasing pH and other workers have shown that cellulase±hemicellulase enzymes are most effective at pH 4.2 (Pitt, 1990). These findings suggest that the alkaline pH resulting from urea application would inhibit cellulase±hemicellulase enzyme activity. Therefore, the benefits of combining urea and enzyme treatments in this and previous studies may reflect differences in the time taken for the treatments to act. While cellulase enzymes can commence cell wall degradation shortly after they are applied, it can take up to two weeks for urea application to take effect and substantially increase the pH. The benefits of the combination of urea and enzyme may therefore reflect an initial degradation of xylans (hemicellulose) and cellulose by the enzymes followed by further degradation of lignocellulose bonds in the cell walls (Chesson and érskov, 1984) by the ammonia
produced by the urea in the presence of microbial urease. However, increasing enzyme concentration did not affect the chemical composition of FSO2U-treated forages in this study. This suggests that the benefits of the FSO2U treatment are optimised at the low level of enzyme application.
In line with the findings of Selmerolsen et al. (1993), increasing the concentration of the enzyme (when applied alone), increased the WSC content and decreased the ensiling DM losses, structural carbohydrate and ammonia N contents. These effects which are probably related to the improved fermentation that often accompanies increasing enzyme concentration (Woolford, 1984), suggest that the high rate of enzyme application is most effective for conserving WCW. Indeed the medium and high rates of enzyme application were comparable or superior to urea treatment at degrading structural carbohydrates and increasing WSC content, but urea application was consistently superior to the low enzyme application level.
4.2. Effect of silo sealing treatment
As found previously (Adogla-Bessa and Owen, 1995), exposure DM losses were lower (P< 0.01) when sealing was delayed for 24 h. This probably reflects the fact that less volatile constituents were available, and therefore lost from the delayed seal silos due to the poorer fermentation that occurred. Such poor fermentation in delayed seal silos is due to the utilisation of WSC by the intrinsic plant enzymes before anaerobiosis prevails (McDonald et al., 1991). Although this suggests that benefits could arise from wilting whole crop clamps under practical conditions, the loss in WSC content and attendant decrease in nutritive value would probably outweigh the benefits of the reduced DM losses. It is interesting to note that immediate evacuation of silos did not improve the fermentation or aerobic stability. This suggests that the volume of air trapped in the silo was not large enough to adversely influence the fermentation process. Thus, the trapped oxygen was rapidly used up by the post-sealing respiratory process and did not hinder the establishment of anaerobic conditions (Honig, 1975). It therefore appears that WCW can be adequately conserved without vacuum evacuation of oxygen in small scale, laboratory silos. However, this does not minimise the need for adequate compaction and air exclusion from WCW clamps in practice.
The improvement in aerobic stability resulting from urea application agrees with the findings of Hill and Leaver (1991) and Adesogan et al. (1996). Tetlow (1983) also demonstrated that urea treatment of perennial ryegrass decreased the silo temperature relative to ambient for one month after opening and concluded that urea treatment was effective against fungi, actinomycetes and yeasts.
Acknowledgements
The Finnish Sugar Co. Ltd., Helsinki, Finland is gratefully acknowledged for financing the study at the institute of Grassland and Environmental Research, Hurley, UK (IGER), and providing a postgraduate scholarship to Tsatsu Adogla-Bessa. The authors wish to acknowledge help from personnel of the IGER Hurley in particular R.M. Tetlow and R.D. Baker who assisted during the initiation and conduct of the experiments. The assistance of the Department of Applied Statistics and Faculty of Agriculture and Food Analytical Service of Reading University is also acknowledged.
References
Adesogan, A.T., Givens, D.I., Owen, E., 1996. The aerobic stability of fermented and urea-treated whole crop wheat harvested at three stages of maturity. Proceedings of the British Grassland Society Winter Meeting titled 'Grass and forage for cattle of high genetic merit', Great Malvern.
Adesogan, A.T., Givens, D.I., Owen, E., 1998. The chemical composition, digestibility and energy value of fermented and urea-treated whole crop wheat harvested at three stages of maturity. Grass and Forage Sci. 53, 66±75.
Adogla-Bessa, T., Owen, E., 1995. Ensiling of whole crop wheat with cellulase-hemicellulase based enzymes 1. Effect of crop growth stage and enzyme on silage composition and stability. Anim. Feed Sci. Technol. 55, 335±347.
Baintner, F., Kissno, B., Kelemen, G., Harangozo, F., 1989. Effect of cellulase and microbial inoculant on the quality and digestibility of maize and mixed maize-sorghum silages. Proceedings of the 16th International Grassland Congress, Nice, pp. 973±974.
Chesson, A., érskov, E.R., 1984. Microbial degradation in the digestive tract. In: Sundstél, F., Owen, E. (Eds.), Straw and Other Fibrous By-products as Feed, Elsevier, Amsterdam, Ch. 11.
Corral, A.J., Heard, A.J., Fenlon, J.S., Terry, Cora, P., Lewis, G.C., 1977. Whole crop silage. Grassland Research Institute, Hurley, Tech. Rep. No. 22, 35pp.
Deschard, G., Mason, V.C., Tetlow, R.M., 1988. Treatment of whole crop cereals with alkali 4. Voluntary intake and growth in steers given wheat ensiled with sodium hydroxide, urea or ammonia. Anim. Feed Sci. Technol. 19, 55±56.
Hill, J., Leaver, J.D., 1991. Effect of stage of growth and urea addition on the nutritive value of whole crop wheat. Animal Production 52, 606.
Honig, H., 1975. Changes and losses by aerobic deterioration. Das W. Futter. 21, 25±32.
Huhtanen, P., Hissa, K., Jaakkola, S., Poutiainen, E., 1985. Enzymes as silage additives. Effect on fermentation quality, digestibility in sheep, degradability in sacco and performance in growing cattle. J. Agric. Sci. Finland 57, 284±292.
Jacobs, J.L., 1989. Enzyme Additives for Grass Silage. PhD Thesis, University of Reading, 186 p.
Jakhmola, R.C., Weddell, J.R., Greenhalgh, J.F.D., 1990. Ensiling grass with straw 1. Effect of straw, cellulase enzyme and urea on chemical composition of grass and grass/legume silages. Anim. Feed Sci. Technol. 28, 39±50.
Lawes Agricultural Trust, 1988. Genstat, a general statistical programme. Rothamstead Experimental Station, Herts.
Laytimi, A., Bolsen, K., Schurhamner, J., Kirch, B., 1988. Effect of enzyme and inoculant additives on preservation and feeding value of wheat and sorghum silages. 75th Cattlemans Day Progress Report 539, Kansas State University, pp. 199±207.
Leaver, J.D., Hill, J., 1992. Feeding cattle on whole crop cereals. In: Stark, B.A., Wilkinson, J.M. (Eds.), Whole Crop Cereals, 2nd ed., Chalcombe Publications, Kent, pp. 59±72.
McDonald, P., Henderson, N., Heron, S., 1991. The Biochemistry of Silage, 2nd ed., Chalcombe Publications, Marlow, 340 p.
McDonald, P., Dewar, W.A., 1960. Determination of dry and volatiles in silage. J. Sci. Food Agric. 11, 566±570. Mead, R., Curnow, R.M., 1983. Statistical Methods in Agriculture and Experimental Biology, Chapman & Hall,
London, 335 p.
Mgheni, D.M., Kimambo, A.E., Sundstol, F., Madsen, J., 1994. The influence of urea supplementation or treatment of rice straw and fish meal supplementation on rumen environment and activity in sheep. Anim. Feed Sci. Technol. 49, 223±235.
Mould, F.L., Orskov, E.R., 1984. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry matter degradation and the rumen microflora of sheep offered either hay or concentrate. Anim. Feed Sci. Technol. 16, 1±14.
Pitt, R.E., 1990. Silage and hay preservation, NRAES, New York, 53 p.
Selmerolsen, I., Henderson, A., Robertson, S., McGinn, R., 1993. Cell-wall degrading enzymes for silage 1. The fermentation of enzyme-treated ryegrass in laboratory silos. Grass and Forage Sci. 48, 45±54.
Smith, D., Groteluschen, R.D., 1966. Carbohydrates in grasses I. Crop Sci. 6, 263±270.
Tetlow, R.M., 1983. The effect of urea on the preservation and digestibility in vitro of perennial ryegrass. Anim. Feed Sci. Technol. 10, 49±63.
Tetlow, R.M., 1992. A decade of research into whole crop cereals. In: Stark, B.A., Wilkinson, J.M. (Eds.), Whole Crop Cereals, 2nd ed., Chalcombe Publications, Kent, pp. 1±20.
Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of silage crops. J. Br. Grassl. Soc. 18, 104±111.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3568±3597.
