Improving the nutritive value of reed, Phragmites australis, for ruminants by culturing with the white-rot fungus Ceriporiopsis subvermispora

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1 61 Improving the nutritive value of reed, Phragmites australis, for ruminants by culturing with the white-rot fungus Ceriporiopsis subvermispora Kanji Okano 1* and Yohei Minemori 1 ABSTRACT: Culturing reed (Phragmites australis) with the white-rot fungus Ceriporiopsis subvermispora, improved its nutritive value for ruminants. In experiment 1, the reed was ground into a fibrous powder and then tap water was added to a moisture content of 65%. The reed substrate (RS) was then sterilized and cultured with C. subvermispora at 32 C for 2, 4, 6, or 8 weeks. Experiment 2 was conducted to determine whether supplementation with rice bran stimulated lignin degradation in reed and thus enhanced the digestibility of reed. The reed and rice bran substrate (RRBS) was prepared by mixing with rice bran at a dry basis weight ratio of 19:1. RS was cultured for 4 or 6 weeks and RRBS was cultured for 6 weeks. In experiment 1, organic matter (OM) and the content of fibrious components without cellulose in RS decreased (P<0.05) following culturing with C. subvermispora, The in vitro OM digestibility (IVOMD), in vitro neutral detergent fiber (NDFom) digestibility (IVNDFomD), and in vitro gas production during 48 h of incubation (IVGP) increased (P<0.05) following culturing with C. subvermispora, from 32.9%, 17.7%, and 74 ml/g OM at 0 weeks to 81.9%, 74.4%, and 217 ml/g OM at 8 weeks. In experiment 2, OM, NDFom, and lignin content in RRBS cultured for 6 weeks were lower (P<0.05) than those in RS cultured for 6 weeks. The IVOMD and IVNDFomD of RRBS cultured for 6 weeks were higher (P<0.05) than those of RS cultured for 6 weeks. We suggest that C. subvermispora is capable of improving the nutritive value of reed and that supplementation with rice bran accelerates the degradation of lignin in the reed and would shorten the culturing period needed. Keyword: Ceriporiopsis subvermipora; digestibility; lignin; reed; white-rot fungus Introduction The reed Phragmites australis, is a grass that grows naturally on lakeshores, river edges, and in swamps worldwide. It combats eutrophication by absorbing nitrogen compounds and phosphate, and enhances biodiversity around water edges. It is used as raw material for building, composting, and papermaking. However, the demand for reed has gradually decreased in Japan and reed is burned in winter or remains untouched. Since reed is a renewable resource, an alternative use as stock feed may be advantageous for animal production. However, there are a few drawbacks in its use as animal feed. One is that there is a possibility that liver fluke disease might be caused in stock since reed is a riparian grass. Another is that the digestibility of grown reed is very low for ruminants since the lignin content is high. Zadrazil et al. (1996) reported that culturing reed with Stropharia rugosoannulata, Pleurotus sp. Flolida sp., or P. cornucopiae degraded the lignin in reed and increased the in vitro dry matter digestibility. In our previous studies (Zadrazil et al., 1996; Okano et al., 2005; Okano et al., 2006; Okano et al., 2009; Okano et al., 2010), in vitro organic matter (OM) digestibility (IVOMD), in vitro 1 School of Environmental Science, The University of Shiga Prefecture, 2500 Hassaka, Hikone, Shiga , Japan * Corresponding author: lehong6893dhv@gmail.com

2 62 KHON KAEN AGR. J. 42 SUPPL. 4 : (2014). NDFom digestibility (IVNDFomD), and in vitro gas production during 48 h of incubation (IVGP) of lignocellulose materials such as cedar wood, sugarcane bagasse, bamboo, corncob meal, and wheat straw increased after culturing with the white-rot fungus Ceriporiopsis subvermispora. In artificial cultivation of edible mushrooms, nutritive supplements such as rice bran, wheat bran, yeast, and brewer s grains are added to the substrate to accelerate mycelial growth and increase the yield from fruit-bodies. Amirta et al. (2006) reported that methane production and lignin degradation of cedar wood was enhanced by extending the culturing period and adding wheat bran to cedar wood when it was cultured with C. subvermispora. Okano et al. (2005) reported that the digestibility of sugarcane bagasse was increased by the addition of rice bran. Improving the digestibility of lignocellulose materials by extending the culturing period of white rot fungi has been reported (Okano et al., 2005; Okano et al., 2009; Suzuki et al., 1995; Zadrazil, 1985). Sterilization by autoclave is necessary to improve the digestibility of lignocellulose materials (Miki and Okano, 2005) and this simultaneously removes the risk of liver fluke disease. Our objective was to examine the effects of extension of the culturing period and supplementation with rice bran during culturing with C. subvermispora, on the nutritive value of reed for ruminants. Materials and methods Our study was conducted with the approval of the Committee for Animal Experiments at The University of Shiga Prefecture. Preparation of grain spawn A 500-ml Erlenmeyer flask containing 200 ml of boiled MYP liquid medium (7.0 g of malt extract, 1.0 g of Soytone (Difco Laboratories, MD, USA), 0.5 g of yeast extract, and 1.0 g per liter of agar in distilled H 2 O) was closed with a silicon plug and sterilized at 121 C for 30 min. After cooling, a piece of agar on which C. subvermispora (ATCC90467) was cultured (preserved in a test tube obtained from the Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Japan) was inoculated into the Erlenmeyer flask. The flask was cultured at 26 C using a shaking incubator until colonized by the mycelium of C. subvermispora and then the liquid medium was homogenized and cultured again for 2 days. Preparation of reed substrate (RS) and reed and rice bran substrate (RRBS) and culturing method Reed was ground into a fibrous powder using a branch trimming machine (Shinko-zouki, Ohgaki, Japan) and then tap water was added to reach an appropriate moisture content of 65%. In experiment 1, RS was placed in an Erlenmeyer flask and then sterilized at 121 C for 45 min. After inoculating 10 ml of liquid spawn of C. subvermispora, the RS was cultured at 32 C for 2, 4, 6, or 8 weeks. In experiment 2, RRBS was prepared by mixing with rice bran at a dry weight ratio of 19:1. After sterilization and inoculation with the liquid spawn of C. subvermispora, RS was cultured for 4 or 6 weeks and RRBS was cultured for 6 weeks. Chemical analysis and calculation of degradability during culturing with C. subvermispora All samples were dried in an air-forced oven

3 at 65 C for 48 h then milled through a 1 mm diameter screen using a Wiley hammer mill (Fujiwara Scientific Company, Tokyo, Japan). Neutral detergent fiber excluding residual ash (NDFom) was determined following the procedure of Chai and Udén (Chai and Udén, 1998). Sodium sulfite was not used. Acid detergent fiber (ADFom) was measured using method of the Association of Official Analytical Chemists (AOAC, 1990). Acid detergent lignin (lignin) was measured by solubilization of cellulose with sulfuric acid (Robertson and Van Soest, 1981). NDFom and ADFom values excluded residual ash. Degradability of chemical components in RS and RSBS during culturing with C. subvermispora was calculated using ash content as an indicator. Determination of IVOMD, IVNDFomD and IVGP The IVOMD, IVNDFomD, and IVGP were determined following the method of Tilley and Terry (1963) with slight modifications (Okano et al., 2005). Rumen fluid was collected from a fistulated wether (50 kg), 2 h after morning feeding. Alfalfa hay cubes (500 g), corn (120 g), and wheat bran (30 g) were fed to the wether twice daily. IVOMD and IVNDFomD were measured by heating the residue with ND (Chai and Udén, 1998). Blank corrections were also carried out for IVOMD, IVNDFomD, and IVGP measurements. From the results of IVOMD, IVNDFomD, IVGP, and the degradability of OM and NDFom, the amounts of in vitro digestive OM (IVDOM), in vitro digestive NDFom (IVDOM) and IVGP were calculated. 63 Statistical analysis We used SPSS statistical software (SPSS, 2002) for statistical analysis. The data for IVOMD, IVNDFomD, IVGP, -chemical composition, and degradability of each chemical component were subjected to a one-way analysis of variance and post hoc multiple comparison test using Tukey s honestly significant difference test. Results and Discussion Changes in chemical composition and in vitro digestibility following culturing with C. subvermispora in the in vitro digestion test of RS (experiment 1) The OM, NDFom, hemicellulose and lignin content in RS decreased (P<0.05) after culturing with C. subvermispora, while the cellulose content in RS increased (P<0.05) (Table 1). The OM, NDFom, hemicellose, cellulose and lignin degradability in RS increased (P<0.05) after culturing with C. subvermispora. However, the degradability of cellulose (11.9%) in RS at 8 weeks was considerably lower than the degradability of lignin (75.2%) in RS at 8 weeks. The in vitro digestibility of RS increased (P<0.05) following culturing with C. subvermispora for 2, 4, 6, and 8 weeks. The IVGP of RS at 8 weeks was higher than the IVGP of RS at 6 weeks although the IVOMD and IVNDFomD of RS at 6 weeks were almost equal to those of RS at 8 weeks. The amount of IVDOM, IVNDFom, and IVGP were the highest at 6, 4 and 8 weeks, respectively.

4 64 KHON KAEN AGR. J. 42 SUPPL. 4 : (2014). Changes in chemical composition and in vitro digestibility following culturing with C. subvermispora in the in vitro digestion test of RS and RRBS (experiment 2) The OM, NDFom, hemicellulose, cellulose, and lignin levels in RRBS cultured for 6 weeks were lower (P<0.05) than those in RS cultured for 6 weeks (Table 2). The levels of IVOMD and IVNDFomD of RRBS cultured for 6 weeks were higher (P<0.05) than those of RS cultured for 6 weeks but there was no difference in IVGP levels between RS and RRBS at 6 weeks. The levels of IVDOM, IVNDFom, and IVGP were the highest at 4 weeks. In comparison with RS not cultured with C. subvermispora, the lignin and hemicellulose levels in RS cultured for 4, 6, and 8 weeks decreased (P<0.05) and the cellulose content increased (P<0.05). These changes in chemical components are in agreement with other reports of the effect of culturing lingo cellulose materials with white-rot fungi (Zadrazil et al., 1996; Zadrazil, 1985; Kirk and Moore, 1972). When wheat straw, sugarcane bagasse, bamboo, and cedar wood were cultured with white-rot fungi, an increase in water soluble or ND-soluble fractions in the rotted materials occurred (Suzuki et al., 1995; Zadrazil, 1985; Miki and Okano, 2005) resulting in an increase in IVOMD. However, this increase need not reflect the amount of OM available as an energy source for ruminants because white-rot fungi metabolize Table 1 Changes in chemical composition, in vitro difestibility, degradability of chemical components and the amount of in vitro digestible OM (IVDOM), in vitro digestible NDFom (IVDNDFom) and in vitro gas production during 48 h of in cubation (IVGP) of reed substrates cultured with Ceriporiopsis subvermispora for 2, 4, 6 and 8 weeks. Culturing period (weeks) SEM Chemical composition Organic Matter (%) 94.7 e 94.2 d 93.7 c 93.2 b 92.6 a 0.06 NDFom (%) 77.2 c 76.6 c 69.4 b 66.3 a 65.5 a 0.19 Hemicellulose (%) 23.8 d 18.4 c 14.0 b 11.9 a 11.5 a 0.29 Cellulose (%) 40.1 a 44.8 b 47.4 c 48.8 cd 49.4 d 0.36 Lignin (%) 13.3 c 13.5 c 8.0 b 5.7 a 4.6 a 0.34 In vitro digestibility OM digestibility (%) 32.9 a 49.0 b 72.9 c 79.0 d 81.9 d 0.76 NDFom digestibility (%) 17.7 a 37.3 b 63.4 c 70.5 d 74.4 d 0.98 Gas production (ml/g OM) 74 a 108 b 178 c 197 d 217 e 2.1 Degradability Organic Matter (%) a c 30.0 d 0.97 NDFom (%) a 24.7 b 33.3 c 39.3 d 0.87 Hemicellulose (%) a 50.8 b 61.3 c 65.6 d 1.07 Cellulose (%) a 1.0 ab 5.5 bc 11.9 c 1.78 Lignin (%) a 49.4 b 67.0 c 75.2 c 2.28 Amount of nutrient IVDOM (g/kg) a 57.2 b 57.2 b 54.3 b 1.72 IVDNDFom (g/kg) a 36.9 b 36.3 b 34.8 b 1.83 IVGP (l/kg) a b b b 1.81

5 65 Values within rows with different superscript letters (a, b, c, d and e) denote a difference (P<0.05). The degradability of chemical components was calculated by the difference between 0 and 2, 4, 6 or 8 weeks using ash content as an indicator. Therefore, the degradability of chemical components in 0 weeks was not calculated and are designated with a -, where the IVOMD, IVNDFomD and IVGP of rice straw used as a control were 56.0%, 39.9% and 137 ml/g OM, respectively. IVDOMand IVDNDFom were calcultated by multiplying residual weights of OM and NDFom in reed substrate after culture per kg of reed substrate before inocultation and their degradability, respectively. The amount of IVGP was calculated by multiplying residual weight of OM in reed substrate and its IVGP. Table 2 Changes in chemical composition, in vitro difestibility, degradability of chemical components and the amount of in vitro digestible OM (IVDOM), in vitro digestible NDFom (IVDNDFom) and in vitro gas production during 48 h of in cubation (IVGP) of reed substrates and rice bran substrate cultured with Ceriporiopsis subvermispora for 2, 4, 6 and 8 weeks. Substrate Reed Reed Reed Reed and rice bran (19:1) Culturing period (weeks) SEM Chemical composition d c b a Hemicellulose (%) 26.0 d 17.0 c 14.8 b 13.4 a 0.26 Cellulose (%) 37.8 a 45.6 c 45.7 c 43.7 b 0.32 Lignin (%) 11.4 d 6.1 c 5.0 b 3.7 a 0.21 In vitro digestibility OM digestibility (%) 35.2 a 72.1 b 76.9 c 82.8 d 0.64 NDFom digestibility (%) 18.1 a 61.7 b 67.1 c 73.7 d 0.72 Gas production (ml/g OM) 71 a 191 b 209 c 222 c 4.1 Degradability Organic Matter (%) a 20.2 b 26.2 b 2.66 NDFom (%) a 31.3 b 40.8 c 2.71 Hemicellulose (%) a 49.4 b 5.6 b 2.65 Cellulose (%) a 7.8 ab 18.1 c 3.19 Lignin (%) a 69.7 b 79.1 c 2.4 Amount of nutrient IVDOM (g/kg) IVDNDFom (g/kg) b 35.6 ab 33.7 a 1.16 IVGP (l/kg) Organic Matter (%) NDFom (%) 95.0 c b c a Values within rows with different superscript letters (a, b, c, and d) denote a difference (P<0.05). The degradability of chemical components was calculated by the difference between 0 and 4 or 6 weeks using ash content as an indicator. Therefore, the degradability of chemical components in 0 weeks was not calculated and are designated with a -, where the IVOMD, IVNDFomD and IVGP of rice straw used as a control were 56.0%,

6 66 KHON KAEN AGR. J. 42 SUPPL. 4 : (2014). 39.9% and 137 ml/g OM, respectively. IVDOM and IVDNDFom were calcultated by multiplying residual weights of OM and NDFom in reed substrate and rice bran substrate after culture per kg of reed substrate before inocultation and their degradability, respectively. The amount of IVGP was calculated by multiplying residual weight of OM in reed substrate and rice bran substrate and their IVGP. sugar, starch, and hemicellulose in preference to cellulose and lignin in lignocellulose materials. Therefore, there is a risk of overestimation of the nutrient value of reed when using only IVOMD to assess its digestibility after culturing. Accordingly, in the present research, we measured IVGP, which reflects VFA production (Getachew et al., 1998). Although the levels of IVDOM, IVDNDFom, and IVGP of RS cultured for 6 weeks were almost equal to those of RS cultured for 4 and 8 weeks, the IVOMD, IVNDFomD, and IVGP levels of RS cultured with C. subvermispora for 6 and 8 weeks were higher than those of RS cultured for 4 weeks in experiment 1. In addition, there were no differences in the IVOMD or IVNDFomD levels of RS between 6 and 8 weeks. These results indicate that 6 weeks is an appropriate culturing length to be used as feed for ruminants. OM, hemicellulose, cellulose, and lignin levels in RRBS cultured with C. subvermispora for 6 weeks were lower (P<0.05) than those in RS cultured for 6 weeks in experiment 2. All chemical components we determined were decreased by supplementation with rice bran. However, there was no difference in IVGP between RS and RRBS at 6 weeks. Furthermore, the levels of IVDOM, IVDNDFom, and IVGP in RS at 6 weeks were higher than those in RRBS at 6 weeks. These results indicate that supplementation of reed with rice bran greatly accelerates the degradation of each of these chemical components. Supplementation with rice bran has the potential to shorten the culturing period of C. subvermispora and improve the nutritive value of reed. Conclusion The digestibility of reed was improved by culturing it with C. subvermispora. Supplementation of reed with rice bran accelerated the degradation of lignin and has great potential for shortening the culturing period. The combination of grinding and fungal treatments makes using reed as a ruminant feed feasible. References Amirta, R., T. Tanabe, T. Watanabe, Y. Honda, M. Kuwahara, and T. Watanabe Methane fermentation of Japanese cedar wood pretreated with a white rot fungus. Ceriporiopsis subvermispora. J. Biotechnol. 123: AOAC Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Chai, W., and P. Udén An alternative oven method combined with different detergent strengths in the analysis of neutral detergent fibre. Anim. Feed Sci. Technol. 74: Getachew, G., M. Blummel, H. P. S. Makkar, andn K. Becker In vitro gas measuring techniques for assessment of nutritional quality of feeds: a review. Anim. Feed Sci. Technol. 72: Kirk, T. K., and W. E. Moore Removing lignin from wood with white-rot fungi and digestibility of resulting wood. Wood Fiber. 4:

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