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The effect of particle size and amount of inoculum on fungal treatment of wheat straw and wood chips

The effect of particle size and amount of inoculum on fungal treatment of wheat straw and wood chips Background: The aim of this study was to optimize the fungal treatment of lignocellulosic biomass by stimulating the colonization. Wheat straw and wood chips were treated with Ceriporiopsis subvermispora and Lentinula edodes with various amounts of colonized millet grains (0.5, 1.5 or 3.0 % per g of wet weight of substrate) added to the substrates. Also, wheat straw and wood chips were chopped to either 0.5 or 2 cm. Effectiveness of the fungal treatment after 0, 2, 4, 6, or 8 wk of incubation was determined by changes in chemical composition, in vitro gas production (IVGP) as a measure for rumen degradability, and ergosterol content as a measure of fungal biomass. Results: Incomplete colonization was observed for C. subvermispora treated wheat straw and L. edodes treated wood chips. The different particle sizes and amounts of inoculum tested, had no significant effects on the chemical composition and the IVGP of C. subvermispora treated wood chips. Particle size did influence L. edodes treatment of wheat straw. The L. edodes treatment of 2 cm wheat straw resulted in a more selective delignification and a higher IVGP than the smaller particles. Addition of 1.5 % or 3 % L. edodes inoculum to wheat straw resulted in more selective delignification and a higher IVGP than addition of 0.5 % inoculum. Conclusion: Particle size and amount of inoculum did not have an effect on C. subvermispora treatment of wood chips. At least 1.5 % L. edodes colonized millet grains should be added to 2 cm wheat straw to result in an increased IVGP and acid detergent lignin (ADL) degradation. Keywords: Amount of inoculum, Fungal treatment, In vitro rumen degradability, Lignin degradation, Lignocellulosic biomass, Particle size Background thus rumen degradability, lignin should be removed Cellulose is one of the most abundant carbohydrates in before feeding lignocellulosic biomass to ruminants. Lig- the world and is, next to starch, an important carbon nin removal can be achieved using several pre-treatment source in a ruminants diet. In plant cell walls, cellulose methods [3, 4], for example specific lignin degrading fun- is bound to hemicellulose and lignin in the lignocellu- gal or bacterial species [5] These biological pre-treatments losic complex. Lignin is a polymer that is difficult to are a relatively cheap and environmental friendly alterna- degrade, and is only degradable under aerobic conditions tives for chemical and physical pre-treatments [6]. [1]. In the anaerobic rumen, lignin is hardly affected and Although some bacterial species show lignin degrading as a result, the lignocellulosic complex has a limited activities, fungal systems are more oxidatively powerful degradability [2]. As a result, cellulose and hemicellulose compared to bacterial systems [7]. Furthermore lignin is a in this complex have a limited availability to the rumen complex substrate that is known to require maximum microbes. To increase this availability of cellulose, and oxidative enzymes to its degradation. That is best done in a solid state fermentation for which fungi are more suited. * Correspondence: vankuijk.sandra@gmail.com Bacteria flourish better in a watery environment [7]. In the Animal Nutrition Group, Wageningen University, De Elst 1, 6708WD current study, white rot fungi were selected based on their Wageningen, The Netherlands selectivity for lignin degradation. Pre-treatments using Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 2 of 9 selective lignin degrading fungi, such as Ceriporiopsis sub- geographic availability and the fact that they are widely vermispora and Lentinula edodes, were proven to increase studied. Two different particle sizes, 0.5 and 2 cm length, the in vitro rumen degradability as a consequence of of both substrates were used with different amounts of increased cellulose availability of wheat straw and sugar- inoculum (colonized millet) added. cane bagasse [8, 9]. Also, in vivo studies reported in the scientific literature show an increased digestibility of fun- Methods gal treated biomass; e.g. treatment with C. subvermispora Fungal strains increased digestibility of bamboo in sheep [10], treatment Ceriporiopsis subvermispora (strain MES 13094) and with Ganoderma spp. increased the digestibility of wheat Lentinula edodes (strain MES 11910) were initially cul- straw in goats [11] and a treatment with Pleurotus spp. tured on malt extract (10 g malt extract and 17.5 g agar increased the digestibility, however only a maximum of per L, Oxoid LP0039, Thermo Scientific, Hampshire, 17 % dietary inclusion rate of fungal treated material UK) agar until it was almost fully colonized. Agar pieces was accepted by cows [12]. (approximately 1 cm ) were added to sterilized millet The time needed to obtain maximum cellulose avail- grains. Inoculated millet grains were incubated at 24 °C ability to increase in vitro rumen degradability was 6 to until full colonization, which occurred 5 wk after the 12 wk [8, 9, 13]. Although the substrates after the fungal start of incubation. Fully colonized grains were used as pre-treatment were enriched in cellulose, hemicellulose spawn to inoculate the substrates. was partially used by the fungi as lignin was degraded. This relatively long treatment time and consumption of Substrates part of the carbohydrate fraction are major drawbacks Wheat straw and wood chips (oak) were used as and optimization of both is needed to make this method substrates. Both substrates were chopped to particles competitive with current chemical and physical pre- with average sizes of 0.5 cm or 2 cm. The substrates treatments. were submerged in water for 3 d after which the excess Fungal pre-treatment starts with the inoculation of water was removed by draining through nets and the the substrate. In scientific literature, inoculation for C. substrates divided over 1.2 L autoclavable polypropylene subvermispora or L. edodes treatments has been done containers and covered with a lid containing a filter using agar plugs [14–16] or using liquid medium [17–19]. (model TP1200 + TPD1200 XXL Combiness, Nazareth, However, in the commercial mushroom production Belgium) through which air can pass. To each container process spawn is produced from grains [20]. Grain based 200 g of wet substrate was added and, since the water spawn can be produced on a large scale and is easy holding capacity of both substrates was different, this to mix through the substrate. Previous studies have represented approximately 100 and 50 g dry matter of used spawn made from wheat, millet or sorghum grains wood chips and wheat straw, respectively. The containers [8–10, 13, 21]. Compared to other grains, sorghum grains with substrate were sterilized for 1 h at 121 °C. The steril- are relatively large, meaning less inoculation points per g ized substrates were kept at room temperature until of inoculum added. The use of a smaller grain, like millet, further use. Autoclaved, uninoculated samples were taken would increase the amount of inoculation points per g to serve as control. of inoculum added. The latter can initiate a faster colonization of the lignocellulose biomass [22]. Inoculation Rapid and complete colonization of the lignocellulosic Colonized millet grains were added to each container in material is key for a competitive fungal pre-treatment. a concentration of either 0.5, 1.5 or 3 % of the weight of During colonization, the fungus starts degrading the the wet substrate. The content of the containers were outer layer of the material before reaching the inside mixed under aseptic conditions to divide the inoculum [23]. By decreasing the particle size of the substrate, the equally over the substrate. All conditions were tested in surface to volume ratio is increased. The latter will result triplicate, in which each sample started with 100 g dry in more contact points on the surface and reach the matter (in case of wheat straw, divided over two inner part of the material faster [22]. containers). Samples were incubated for 2, 4, 6 and 8 wk The aim of this study was to optimize the colonization in a climate controlled chamber at 24 °C and 70 % conditions of the fungal pre-treatment to obtain the most relative humidity. After incubation, the two containers of selective delignification, with minimal carbohydrate deg- wheat straw were pooled to represent one sample of 100 g radation and a high in vitro rumen degradability. Based on dry. Part of each sample (approximately 10 %) was freeze- the work of Tuyen et al. [8], C. subvermispora and L. dried for ergosterol measurements and the remaining part edodes were chosen as selective lignin degraders. Wheat of the sample was air-dried at 70 °C for 1 wk to be used straw and wood chips were selected for the fungal treat- for chemical analyses and in vitro gas production mea- ment, because of their high content of lignocellulose, surements. The dried wheat straw was ground to 1 mm van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 3 of 9 using a Peppink 100 AN cross beater mill (Peppink, De- reversed phase C18 column (250 × 4.6 mm, Phenomex venter, The Netherlands). The dried wood chips were aqua 5 μm). The liquid phase was 90 % methanol and 10 % finely ground over a 1 mm sieve using a Retch ZM100 (1:1) 2-propanol/hexane. Areas under the peak were cor- centrifugal mill (Retsch, Haan, Germany) to obtain a rected for the extraction efficiency based on the internal homogenous sample. standard cholecalciferol (vitamin D )(9.6 μg added) (Sigma The remaining colonized and uninoculated millet Aldrich, St. Louis, Missouri, USA), using Empower 2 soft- grains that were not used for inoculation were used for ware (Waters Corporation, Milford, Massachusetts, USA). further analysis. Approximately 10 % of each sample Mycelium of C. subvermispora and L. edodes grown on was freeze-dried, and the remaining part was air-dried at malt extract plates covered with cellophane was freeze 70 °C for 1 wk. The dried millet was ground to 1 mm dried and subjected to ergosterol extraction. The ergosterol using a Peppink 100 AN cross beater mill (Peppink, content of the pure mycelium was used to calculate the Deventer, The Netherlands). amount of fungal biomass formed. In vitro gas production technique Statistical analysis In vitro gas production (IVGP) was measured for all sam- The effect of amount of inoculum, particle size and in- ples by the IVGP technique, according to Cone et al. [24]. cubation on ergosterol content, detergent fiber compos- In summary, 60 mL of buffered rumen fluid, collected ition and IVGP was tested using the generalized linear from non-lactating cows, was added to 0.5 g air-dried model (GLM) analysis in SAS software version 9.3 (SAS material. Incubations were done in shaking water baths at Institute Inc., Cary, North Carolina, USA). The following 39 °C. Gas production was measured continuously for 72 model was used: h. Results were expressed as mL gas produced after 72 h Y ¼ μ þ α þ β þ γ þ ω ijk i ijk j k per g organic matter (OM). in which Y is the observation at incubation time i; μ is ijk Chemical analysis the overall mean; α is the fixed effect of amount of in- Fiber analysis was performed according to Van Soest oculum i; β is the fixed effect of particle size j; γ is the j k et al. [25], using an Ankom fiber analyser 2000 fixed effect of incubation time k; ω is the random ijk (ANKOM Technology, Macedon, New York, USA). error. The hemicellulose content was calculated as the differ- The results of ergosterol measurements, chemical ence between neutral detergent fiber (NDF) and acid analysis and IVGP at different incubation times of the detergent fiber (ADF) contents. The cellulose content fungal treatment of each substrate, for each amount of was calculated as the difference between ADF and acid inoculum and particle size combination, were compared detergent lignin (ADL) contents. using the generalized linear model (GLM) analysis in For dry matter (DM) determination air-dried material SAS software version 9.3 (SAS Institute Inc., Cary, North was dried for 4 h at 103 °C. Ash content was determined Carolina, USA). Post-hoc multiple comparison with by combustion for 3 h at 550 °C in a muffle furnace. Tukey’s significant test at a level of α = 0.05 was per- Starch content of the millet grains was determined enzy- formed to determine the significance of differences be- matically according to ISO15914. tween the treatments. The following model was used: Fungicide analysis was performed on autoclaved, un- treated, air-dried wheat straw using gas chromatography/ Y ¼ μ þ α þ ω ij i ij mass spectrometry according to NEN EN 12393. in which Y is the observation j at incubation time i; μ is ij Ergosterol the overall mean; α is the fixed effect of incubation time Ergosterol determination of freeze-dried material was i; ω is the random error. ij based on Niemenmaa et al. [26]. In summary, 200 mg of the material was saponified in 10 % KOH in methanol Results for 1 h at 80 °C. After cooling, hexane and distilled water Only results of C. subvermispora treated wood chips and were added for extraction. The samples were shaken for L. edodes treated wheat straw are presented. Visually no 10 min and centrifuged for 15 min at 4,000 rpm. The or limited growth was observed in L. edodes treated wood hexane phase was collected and the hexane-water ex- chips and C. subvermispora treated wheat straw. This low traction was repeated once. The hexane phases of the 2 growth was caused by unknown external factors. extractions were pooled and evaporated under vacuum. The extracted ergosterol was dissolved in 1 mL methanol Inoculum before ergosterol content was determined by a high per- The chemical composition and the IVGP of millet used formance liquid chromatography (HPLC) fitted with a as spawn at the moment of inoculation (5 wk of van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 4 of 9 colonization) are presented in Table 1. Ergosterol con- the first 4 wk of incubation as the ergosterol content in- tents showed that the millet was colonized by both C. creased during the first 4 wk, after which the growth rate subvermispora and L. edodes.Toestimatethe amount reduced (Fig. 1). Only after 4 wk of incubation differences of mycelial biomass we have measured the amount of were seen between treatments. Addition of 3 % inoculum ergosterol per unit dried mycelium for each fungus after to 0.5 cm wood chips resulted in a higher (P < 0.05) ergos- growth until full colonization (approximately 10 d) on malt terol content compared to the other treatments. Only the extract plates. C. subvermispora contained 9.6 mg addition of 3 % inoculum to 0.5 cm wood chips caused a ergosterol/g mycelium and L. edodes contained 6 mg significant decrease (P < 0.05) in cellulose content (g/kg ergosterol/g mycelium. Each g of colonized millet contains DM) and absolute amount of cellulose (g). thus approximately 14.3 mg C. subvermispora mycelium or Generally, in all tested conditions, the ADL and hemi- 32.1 mg L. edodes mycelium at the moment of inoculation. cellulose content and their absolute amounts were also However, it should be noted that the ergosterol contents of decreasedduringthe first4wk with C. subvermispora in- pure mycelium grown on malt extract agar may be differ- cubation. After 4 wk, the lowest values (P < 0.05) of hemi- ent for the ergosterol contents of mycelium grown on mil- cellulose and cellulose (contents and absolute amounts) let grains. Colonized millet had a lower (P < 0.05) ADL and were reached in the treatments with 3 % inoculum added cellulose content than the uninoculated grains (control). to 2 cm wood chips compared to the other treatments. Although L. edodes numerically decreased the hemicellu- The lower carbohydrate and unchanged ADL degradation lose content, this was not significant, whereas C. subver- suggest a more selective delignification process when 3 % mispora significantly decreased the hemicellulose content. inoculum was added to 2 cm wood chips. The least select- Interestingly, the starch content of the C. subvermispora ive delignification was observed when 0.5 % inoculum was spawn was not different from the control, whereas L. added to 0.5 cm wood chips, since most hemicellulose and edodes significantly degraded the starch in the millet a similar amount of ADL was degraded by C. subvermis- grains. The spawn showed a high IVGP of approximately pora. The higher hemicellulose degradation did not result 300 mL/g OM, which suggests that the addition of spawn in a higher ergosterol content or a lower IVGP. The IVGP to the substrates may have contributed to the increase in increased (P < 0.05) during the first 4 wk of incubation. IVGP at the start of fungal treatment. Most treatments reached a plateau level, whereas addition of 1.5 % inoculum to 0.5 cm wood chips and addition of 3 C. subvermispora treatment of wood chips % inoculum to 2 cm wood chips caused a continuous in- The probability values of the effects of the amount of crease in IVGP. As a result the addition of 1.5 % inoculum inoculum added, particle size and incubation time are to 0.5 cm wood chips resulted in the highest IVGP after 8 shown in Table 2. The amount of inoculum had only a wk of C. subvermispora incubation. significant effect on the cellulose degradation during the C. subvermispora treatment of wood chips. Chan- L. edodes treatment of wheat straw ging theparticlesizeofwoodchips hadsignificant The significance of the effect of the amount of inoculum, effects on the hemicellulose and cellulose degradation particle size and incubation time are shown in Table 2. by C. subvermispora. No significant effects were ob- The main differences observed in L. edodes treatment of served for the amount of inoculum (content P = 0.1435, wheat straw were seen between particle sizes and incu- absolute amounts P = 0.4626) and particle size (con- bation times. The particle size of wheat straw had a sig- tent P = 0.5688, absolute amount P = 0.9148) on the nificant effect on all variables measured, except the ADL degradation. The time of incubation showed effects absolute amounts of cellulose. The time of incubation (P < 0.05) on all variables measured. Tables 3 and 4 show had an effect (P < 0.05) on all variables measured. The that most changes occurred during the first 4 wk of incu- amount of inoculum had a significant effect on the bation. C. subvermispora colonized the wood chips within Table 1 Chemical composition (g/kg DM) of spawn at the moment of inoculation Treatment ADL Hemicellulose Cellulose Starch IVGP Ergosterol a a a a c Control 14.4 54.0 76.3 699.0 319.7 0.0 b b b a b C. subvermispora 2.3 30.3 50.4 680.2 285.8 138.1 b ab b b a L. edodes 3.5 40.9 43.2 408.2 302.5 192.7 SEM 1.58 3.31 2.65 5.56 16 10.61 P-value 0.003 0.0068 0.0003 <0.0001 0.3855 <0.0001 Values with different superscripts within column are significantly (P < 0.05) different ADL acid detergent lignin, IVGP in vitro gas production in rumen fluid van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 5 of 9 Table 2 Probability values for effects of the amount of inoculum added (0.5, 1.5 and 3.0 %), particle size (0.5 and 2.0 cm) and incubation time (2, 4, 6 and 8 wk) on chemical composition, ergosterol content and in vitro gas production of two fungal treated substrates (wheat straw and wood chips) C. subvermispora treated wood chips L. edodes treated wheat straw Item Amount of inoculum Particle size Incubation time Amount of inoculum Particle size Incubation time ADL, g/kg DM 0.1435 0.5688 <0.0001 0.0346 <0.0001 <0.0001 ADL, g 0.4626 0.9148 <0.0001 0.1640 <0.0001 <0.0001 HC, g/kg DM 0.3704 <0.0001 <0.0001 0.0003 <0.0001 <0.0001 HC, g 0.2533 <0.0001 <0.0001 0.0054 <0.0001 <0.0001 Cell, g/kg DM 0.0002 <0.0001 <0.0001 0.0026 0.0003 <0.0001 Cell, g 0.0197 <0.0001 0.0012 0.4314 0.1179 0.0120 Ergosterol, mg/g 0.5085 0.3220 <0.0001 <0.0001 <0.0001 <0.0001 IVGP, mL/g OM 0.4508 0.6735 <0.0001 0.1910 <0.0001 <0.0001 ADL acid detergent lignin, HC hemicellulose, Cell cellulose, IVGP in vitro gas production after 72h in rumen fluid ergosterol content, absolute amounts of hemicellulose material, because of its small grain size. Addition of the and ADL and hemicellulose and cellulose contents. same weight of spawn will result in more inoculation Addition of 1.5 or 3 % L. edodes inoculum to 0.5 cm points when using a smaller grain. The IVGP was not wheat straw particles resulted in a continuous growth different between the different amounts of inoculum during 8 wk, whereas addition to 2 cm wheat straw added. If the spawn would contribute to IVGP, this particles resulted in growth which stopped after 4 wk would be mainly because millet contains starch, espe- of incubation. No changes in ergosterol content were cially in C. subvermispora treated biomass since this fun- observed throughout the entire incubation period when gus did not degrade starch upon colonization. The 0.5 % inoculum was added to 0.5 cm wheat straw observation that C. subvermispora did not utilize starch particles. is interesting as the publically available genome data of Regardless how much inoculum was added, the ADL C. subvermispora shows the presence of genes encoding content and absolute amounts of 0.5 cm wheat straw for amylase. The ergosterol content data show that C. did not change after 4 to 6 wk of L. edodes incubation subvermispora grew on both millet and wood chips, (Tables 5 and 6). In contrast, the ADL in 2 cm wheat suggesting millet can be used as spawn for C. subvermis- straw decreased continuously during the 8 wk of incu- pora. The data presented here, indicate that hemicellu- bation (Tables 5 and 6). After 8 wk of incubation, the lose and to some extent cellulose are the substrates used ADL content of 2 cm wheat straw was lower (P <0.05) by C. subvermispora to grow on millet grains. The esti- than that of the 0.5 cm particles (Tables 5 and 6). The mation of the amount of fungal biomass on grains has hemicellulose content decreased (P <0.05) during the been done previously for the button mushroom Agaricus first 4 to 6 wk (Table 5). The final hemicellulose con- bisporus using three independent methods including the tent and the absolute amounts after 6 and 8 wk of incu- ergosterol method [27]. These authors calculated that bation were lower for the 2 cm particles than for the spawn contains circa 10 mg dry mycelial biomass per 0.5 cm particles (Tables 5 and 6). The cellulose content gram of grain which is in the same range as we have increased more (P < 0.05) for the 0.5 cm particles than measured here (14 to 32 mg for C. subvermispora and L. for the 2 cm particles (Table 5). This effect was more edodes respectively). visible by the absolute amounts of cellulose, where only C. subvermispora, in contrast to L. edodes, showed a a significant decrease in time was seen when 1.5 % in- low growth on wheat straw. In some treatments, such as oculum was added to 0.5 cm wheat straw particles addition of 0.5 % inoculum to 0.5 cm wheat straw parti- (Table 6). cles, also a low growth of L. edodes was found compared Six to eight wk of L. edodes treatment increased the to other studies [8, 13]. A possible explanation for the IVGP of the 2 cm wheat straw particles more (P < 0.05) low efficiency of the fungal treatment of wheat straw in than that of the 0.5 cm wheat straw particles (Fig. 2b). the present study is the presence of fungicides. Analysis of the wheat straw showed that it contained 0.767 mg Discussion tebuconazole/kg dry substrate. Tebuconazole is a fungi- The aim of this study was to optimize the colonization cide used in wheat production to control plant pathogenic conditions of fungal treatments. To do so, the amount fungi. The minimal inhibitory concentration of tebucona- of inoculum added and the particle size of the substrate zole has been shown to be different for different basidio- was varied. Here, millet was chosen as inoculation mycete species [28]. Possibly C. subvermispora is more van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 6 of 9 Table 3 Changes in detergent fiber content (g/kg DM) over an Table 4 Changes in absolute amounts of detergent fiber (g) eight wk period of 0.5 and 2 cm wood chips incubated with over an eight wk period of 0.5 and 2 cm wood chips incubated three different amounts of C. subvermispora inoculum with three different amounts of C. subvermispora inoculum Particle size 0.5 cm 2 cm Particle size 0.5 cm 2 cm Inoculum, % 0.5 1.5 3.0 0.5 1.5 3.0 Inoculum, % 0.5 1.5 3.0 0.5 1.5 3.0 Wk Wk a a a a a a a a a a a a ADL 0 214.8 214.8 214.8 214.8 214.8 214.8 ADL 0 31.2 31.2 31.2 31.2 31.2 31.2 a b b b b b a b b b b b 2 197.8 162.4 162.6 178.3 163.7 156.5 2 27.5 22.3 22.4 24.3 23.0 21.3 b c c c c c b c c c c c 4 127.0 130.0 126.3 133.2 126.8 134.3 4 16.2 16.9 16.3 17.3 16.6 18.1 b d c d c c b d c d c cd 6 128.3 118.4 123.4 111.8 126.4 126.4 6 16.4 14.9 15.8 14.0 16.1 16.5 b cd c c c c b d c d c d 8 112.0 123.6 129.4 126.0 121.2 120.7 8 13.8 15.1 16.4 15.5 15.5 15.2 SEM 9.95 2.07 2.18 2.31 5.41 2.95 SEM 1.60 0.30 0.31 0.35 0.80 0.52 P-value <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 P-value <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 a a a a a ab a a a a a a HC 0 151.5 151.5 151.5 151.5 151.5 151.5 HC 0 22.0 22.0 22.0 22.0 22.0 22.0 a a a a a a a a a a a ab 2 154.1 160.7 170.7 151.7 156.2 158.3 2 21.4 22.1 23.5 20.7 21.9 21.6 b b b bc b abc b b b bc b bc 4 90.0 98.5 92.8 109.4 114.1 129.9 4 11.5 12.8 12.0 14.2 14.9 17.5 b b b ab b bc b b b b b c 6 107.4 85.6 87.0 126.8 106.2 122.6 6 13.7 10.8 11.1 15.9 13.6 16.0 b b b c b c b b b c b c 8 74.5 73.1 74.8 94.9 107.3 112.1 8 9.2 9.0 9.5 11.7 13.7 14.1 SEM 8.21 10.80 9.82 5.50 4.32 6.55 SEM 1.19 1.47 1.30 0.83 0.59 0.94 P-value 0.0001 0.0005 0.0001 <.0001 <.0001 0.0025 P-value <.0001 0.0001 <.0001 <.0001 <.0001 0.0005 a b ab b b b a Cell 0 381.3 381.3 381.3 381.3 381.3 381.3 Cell 0 55.5 55.5 55.5 55.5 55.5 55.5 ab ab b b b b ab 2 390.3 393.1 375.3 408.2 396.8 387.4 2 54.1 54.0 51.7 55.7 55.7 52.7 ab ab ab ab a a b 4 417.0 403.3 384.5 414.7 430.3 422.2 4 53.2 52.3 49.8 53.9 56.3 57.0 ab ab a a a a ab 6 417.9 415.4 405.4 455.1 446.0 419.3 6 53.2 52.3 51.9 57.0 57.0 54.6 a a ab ab a a b 8 430.3 419.1 397.1 424.2 438.4 419.2 8 52.9 51.3 50.5 52.1 55.9 52.9 SEM 10.34 7.90 5.98 9.97 6.82 6.42 SEM 1.59 1.05 0.81 1.25 0.91 0.91 P-value 0.0349 0.0362 0.0301 0.0054 0.0002 0.0019 P-value 0.7836 0.1072 0.0048 0.133 0.7735 0.0402 Values with different superscripts within column are significantly (P < 0.05) Values with different superscripts within column are significantly (P < 0.05) different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM standard error of the mean standard error of the mean sensitive to this fungicide than L. edodes. Tebuconazole Possibly L. edodes is more sensitive to water activity than inhibits the formation of ergosterol in fungi [28]. C. C. subvermispora. subvermispora contains more ergosterol per g of myce- The particle size of the substrate had only an effect on lium (9.6 mg ergosterol/g mycelium) than L. edodes (6 mg the L. edodes treatment of wheat straw. Sachs et al. [23] ergosterol/g mycelium), which may be the reason why C. showed that P. chrysosporium formed more mycelium subvermispora is more sensitive to this fungicide. Since on the surface of aspen wood chips compared to the in- the use of fungicides is general practice in wheat produc- side of the wood chips. Therefore, it is expected that tion, it is advised for future fungal treatments to use with a decreasing particle size, and thus a larger surface organically grown wheat straw. However, L. edodes to volume ratio, the substrate is more accessible and showed a reduced growth on wood chips. A possible more mycelium can be formed. Despite the different sur- explanation may be the different moisture content be- face to volume ratio, particle size did not have a major tween both wood chips and wheat straw. The moisture influence on the P. chrysosporium growth patterns [23]. content of wheat straw was around 60 %, while that of In the present study, particle size had a significant effect wood chips was around 45 %. This difference is inherent on the ergosterol content, although only addition of to the manner of moisturizing the substrates. After full 0.5 % inoculum to 0.5 cm wheat straw particles had a penetration of the water, the ‘free’ water is removed by remarkable low ergosterol content. Probably the contrast draining. The water holding capacity of wood chips is between the surface to volume ratios between particle lower than that of wheat straw, causing a difference in sizes was not large enough to see effects on colonization. moisture content. However there may be a different water Interestingly, 2 cm particles resulted in a lower ADL and a activity for both substrates, which was not measured. higher IVGP upon the fungal treatment. Gómez [29] van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 7 of 9 Fig. 1 Results of ergosterol measurements. (a) C. subvermispora on wood chips, (b) L. edodes on wheat straw. ♦ 0.5% inoculum per g wet substrate (0.5 cm) ■ 1.5 % inoculum per g wet substrate (0.5 cm) ▲ 3 % inoculum per g wet substrate (0.5 cm) ◊ 0.5 % inoculum per g wet substrate (2 cm) □ 1.5 % inoculum per g wet substrate (2 cm) Δ 3 % inoculum per g wet substrate (2 cm) demonstrated an increased expression of cellulases and carbohydrates in large particles, the fungi first have to de- xylanases by Trametes sp., when grown on smaller parti- grade more lignin. On the other hand, not more carbohy- cles of corn straw, compared to larger particles. Carbohy- drate degradation was observed in the smaller particles. drates in smaller particles are possibly more exposed at It has to be noted that the detergent fiber analysis the surface compared to large particles. To reach the method was used in this study. The detergent fiber Table 5 Changes in detergent fiber content (g/kg DM) over an Table 6 Changes in absolute amounts of detergent fiber (g) eight wk period of 0.5 and 2 cm wheat straw incubated with over an eight wk period of 0.5 and 2 cm wheat straw incubated three different amounts of L. edodes inoculum with three different amounts of L. edodes inoculum Particle size 0.5 cm 2 cm Particle size 0.5 cm 2 cm Inoculum, % 0.5 1.5 3.0 0.5 1.5 3.0 Inoculum, % 0.5 1.5 3 0.5 1.5 3 Wk Wk abc ab a b a a a ab a b a a ADL 0 90.8 90.8 90.8 90.8 90.8 90.8 ADL 0 17.7 17.7 17.7 17.7 17.7 17.7 a a a a a a a a a a a a 2 95.7 100.2 97.9 107.3 97.3 92.6 2 18.4 19.5 19.2 20.6 18.8 18.0 ab ab ab b b b a ab ab b b b 4 92.7 95.4 82.6 89.1 79.5 79.3 4 17.5 18.3 16.0 16.8 15.2 15.2 bc ab a c c c ab b a c c c 6 81.9 80.5 95.6 68.2 64.1 64.5 6 15.7 15.2 18.3 12.7 12.0 12.2 c b b d d d b b b d d d 8 78.6 79.0 69.9 56.5 54.6 52.2 8 14.6 14.7 13.2 10.4 10.1 9.7 SEM 2.74 4.40 3.82 1.80 1.63 2.36 SEM 0.60 0.85 0.77 0.33 0.32 0.43 P-value 0.0056 0.0261 0.0025 <.0001 <.0001 <.0001 P-value 0.0062 0.0113 0.0019 <.0001 <.0001 <.0001 a a a a a a a a a a a a HC 0 287.1 287.1 287.1 287.1 287.1 287.1 HC 0 56.0 56.0 56.0 56.0 56.0 56.0 a a ab a a b a a ab a a b 2 288.8 277.8 262.8 286.9 273.6 262.1 2 55.6 54.0 51.6 55.2 52.8 51.1 b ab bc a b c b ab c a b c 4 253.4 260.7 235.4 263.7 242.5 233.1 4 47.9 50.1 45.6 49.8 46.3 44.7 c bc bc b c d c bc bc b c d 6 224.7 228.9 247.2 211.4 204.8 190.7 6 43.1 43.2 47.3 39.3 38.2 36.1 bc c c b c d bc c c b c d 8 240.8 223.6 221.0 199.9 188.2 196.2 8 44.8 41.7 41.8 36.8 34.8 36.4 SEM 5.22 7.96 6.04 8.39 4.80 3.44 SEM 0.96 1.49 1.29 1.59 0.92 0.64 P-value <.0001 0.0006 0.0002 <.0001 <.0001 <.0001 P-value <.0001 0.0001 0.0001 <.0001 <.0001 <.0001 b b ab b bc ab Cell 0 474.1 474.1 474.1 474.1 474.1 474.1 Cell 0 92.5 92.5 92.5 92.5 92.5 92.5 ab b b b c b 2 480.4 470.5 461.1 493.5 475.6 467.1 2 92.5 91.4 90.6 94.9 91.7 91.1 ab a a a abc a 4 489.5 503.1 485.5 481.3 500.3 486.9 4 92.6 96.6 94.0 90.9 95.5 93.5 a ab ab a a ab 6 493.8 491.2 478.7 500.7 498.3 503.0 6 94.7 92.8 91.6 93.2 92.9 95.1 ab ab ab a ab b 8 489.8 479.6 478.1 508.3 507.4 494.1 8 91.2 89.5 90.3 93.6 93.9 91.7 SEM 4.04 6.20 4.63 8.32 4.17 4.62 SEM 1.38 1.03 1.08 1.59 0.83 1.06 P-value 0.035 0.0221 0.0403 0.0835 0.0005 0.0016 P-value 0.5141 0.0076 0.1953 0.5183 0.0667 0.1257 Values with different superscripts within column are significantly (P < 0.05) Values with different superscripts within column are significantly (P < 0.05) different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM standard error of the mean standard error of the mean van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 8 of 9 Fig. 2 Results of in vitro gas production (IVGP) measurements. (a) C. subvermispora on wood chips, (b) L. edodes on wheat straw. ♦ 0.5 % inoculum per g wet substrate (0.5 cm) ■ 1.5 % inoculum per g wet substrate (0.5 cm) ▲ 3 % inoculum per g wet substrate (0.5 cm) ◊ 0.5% inoculum per g wet substrate (2 cm) □ 1.5 % inoculum per g wet substrate (2 cm) Δ 3% inoculum per g wet substrate (2 cm) method is developed to estimate feeding value of forage does not seem to be the limiting factor for C. subvermis- for ruminants. It is not developed to precisely determine pora. The L. edodes treatment of wheat straw was influ- the chemically defined fractions, and as a result it under- enced by the particle size of the substrate. Larger (2 cm) estimates the lignin fraction and overestimates the carbo- particles resulted in a more selective delignification and hydrate fractions [30, 31]. Nevertheless, the acid detergent a higher IVGP than smaller (0.5 cm) particles. The lignin is highly correlated to rumen degradability for both amount of inoculum to some extent affects L. edodes wheat straw and wood chips [13], and changes in the treatment of wheat straw. At least 1.5% spawn should be lignin and carbohydrate fractions upon fungal treatment added to obtain a more selective lignin degradation and are a good indication for carbohydrate accessibility. For a an increased IVGP. more detailed chemical analysis of plant cell walls it is advised to measure lignin and carbohydrates in more de- Abbreviations tail using pyrolysis coupled to gas chromatography and ADF, acid detergent fiber; ADL, acid detergent lignin; Cell, cellulose; DM, dry matter; GLM, generalized linear model; HC, hemicellulose; HPLC, high mass spectrometry and sugar analysis. performance liquid chromatography; IVGP, in vitro gas production; NDF, neutral Particle size did not have an effect on the C. subver- detergent fiber; OM, organic matter; SEM, standard error of the mean mispora treatment of wood chips. If a larger contrast between the particle sizes was used, maybe an effect Acknowledgements This research was supported by the Dutch Technology Foundation (STW), would have been seen. In the scientific literature how- which is part of the Netherlands Organization for Scientific Research (NWO), ever, 15 mm size corn stover yielded less glucose upon which is partly funded by the Dutch Ministry of Economic Affairs. This enzymatic saccharification than 10 and 5 mm corn sto- project (11611) was co-sponsored by Agrifirm, Purac, DSM, Den Ouden, Hofmans, the Dutch commodity boards for dairy and horticulture, and ver after a C. subvermispora treatment [32]. The same Wageningen University. We thank Agrifirm for practical assistance with authors concluded that moisture content, time and the analysis of dry matter and ash contents. We thank DSM for practical temperature were also important factors influencing assistance with the analysis of fungicides. the effectiveness of the fungal treatment. Different sub- Authors’ contributions strates show different water holding capacities, which SvK carried out the experiments and drafted the manuscript. AS, JB, WH and also depends on the particle size, explaining why the JC were involved in the study design, data interpretation and manuscript effect of particle size may be different for different preparation. All authors read and approved the final manuscript. substrates. Competing interests The results of this study show that the particle sizes The authors declare that they have no competing interests. (0.5 or 2 cm) tested do not have a major influence on the C. subvermispora treatment of wood chips. Using Author details Animal Nutrition Group, Wageningen University, De Elst 1, 6708WD wheat straw particles smaller than 2 cm does not have Wageningen, The Netherlands. Plant Breeding, Wageningen University, beneficial effects on the L. edodes treatment. Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands. Received: 18 January 2016 Accepted: 21 June 2016 Conclusions The major changes in chemical composition, IVGP and ergosterol content occur during the first 4 wk of C. sub- References vermispora treatment of wood chips. The particle size 1. Isroi, Millati R, Syamsiah S, Niklasson C, Nur Cahyanto M, Lundquist K, Taherzadeh MJ. Biological pretreatment of lignocelluloses with white-rot (0.5 or 2 cm) of the wood chips and the amount of in- fungi and its applications: a review. Bioresour. 2011;6:5224–59. oculum (0.5, 1.5 or 3 %) added do not have significant 2. Jung HG, Allen MS. Characteristics of plant cell walls affecting intake and effects on the C. subvermispora treatment. Colonization digestibility of forages by ruminants. J Anim Sci. 1995;73:2774–90. van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 9 of 9 3. Sarnklong C, Cone JW, Pellikaan W, Hendriks WH. Utilization of rice straw 26. Niemenmaa O, Galkin S, Hatakka A. 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Bioresour Technol. 2012;111:336–42. J Nutr. 1997;127:S810–3. 9. Tuyen DV, Phuong HN, Cone JW, Baars JJP, Sonnenberg ASM, Hendriks WH. 32. Wan C, Li Y. Microbial pretreatment of corn stover with Ceriporiopsis Effect of fungal treatments of fibrous agricultural by-products on chemical subvermispora for enzymatic hydrolysis and ethanol production. Bioresour composition and in vitro rumen fermentation and methane production. Technol. 2010;101:6398–403. Bioresour Technol. 2013;129:256–63. 10. Okano K, Ohkoshi N, Nishiyama A, Usagawa T, Kitagawa M. Improving the nutritive value of madake bamboo, Phyllostachys bambusoides, for ruminants by culturing with the white-rot fungus Ceriporiopsis subvermispora. Anim Feed Sci Technol. 2009;152:278–85. 11. Shrivastava B, Nandal P, Sharma A, Jain KK, Khasa YP, Das TK, Mani V, Kewalramani NJ, Kundu SS, Kuhad RC. Solid state bioconversion of wheat straw into digestible and nutritive ruminant feed by Ganoderma sp. rckk02. 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Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal Submit your manuscript at nutrition. J Dairy Sci. 1991;74:3583–97. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science and Biotechnology Springer Journals

The effect of particle size and amount of inoculum on fungal treatment of wheat straw and wood chips

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Copyright © 2016 by The Author(s).
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Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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2049-1891
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10.1186/s40104-016-0098-4
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27418962
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Abstract

Background: The aim of this study was to optimize the fungal treatment of lignocellulosic biomass by stimulating the colonization. Wheat straw and wood chips were treated with Ceriporiopsis subvermispora and Lentinula edodes with various amounts of colonized millet grains (0.5, 1.5 or 3.0 % per g of wet weight of substrate) added to the substrates. Also, wheat straw and wood chips were chopped to either 0.5 or 2 cm. Effectiveness of the fungal treatment after 0, 2, 4, 6, or 8 wk of incubation was determined by changes in chemical composition, in vitro gas production (IVGP) as a measure for rumen degradability, and ergosterol content as a measure of fungal biomass. Results: Incomplete colonization was observed for C. subvermispora treated wheat straw and L. edodes treated wood chips. The different particle sizes and amounts of inoculum tested, had no significant effects on the chemical composition and the IVGP of C. subvermispora treated wood chips. Particle size did influence L. edodes treatment of wheat straw. The L. edodes treatment of 2 cm wheat straw resulted in a more selective delignification and a higher IVGP than the smaller particles. Addition of 1.5 % or 3 % L. edodes inoculum to wheat straw resulted in more selective delignification and a higher IVGP than addition of 0.5 % inoculum. Conclusion: Particle size and amount of inoculum did not have an effect on C. subvermispora treatment of wood chips. At least 1.5 % L. edodes colonized millet grains should be added to 2 cm wheat straw to result in an increased IVGP and acid detergent lignin (ADL) degradation. Keywords: Amount of inoculum, Fungal treatment, In vitro rumen degradability, Lignin degradation, Lignocellulosic biomass, Particle size Background thus rumen degradability, lignin should be removed Cellulose is one of the most abundant carbohydrates in before feeding lignocellulosic biomass to ruminants. Lig- the world and is, next to starch, an important carbon nin removal can be achieved using several pre-treatment source in a ruminants diet. In plant cell walls, cellulose methods [3, 4], for example specific lignin degrading fun- is bound to hemicellulose and lignin in the lignocellu- gal or bacterial species [5] These biological pre-treatments losic complex. Lignin is a polymer that is difficult to are a relatively cheap and environmental friendly alterna- degrade, and is only degradable under aerobic conditions tives for chemical and physical pre-treatments [6]. [1]. In the anaerobic rumen, lignin is hardly affected and Although some bacterial species show lignin degrading as a result, the lignocellulosic complex has a limited activities, fungal systems are more oxidatively powerful degradability [2]. As a result, cellulose and hemicellulose compared to bacterial systems [7]. Furthermore lignin is a in this complex have a limited availability to the rumen complex substrate that is known to require maximum microbes. To increase this availability of cellulose, and oxidative enzymes to its degradation. That is best done in a solid state fermentation for which fungi are more suited. * Correspondence: vankuijk.sandra@gmail.com Bacteria flourish better in a watery environment [7]. In the Animal Nutrition Group, Wageningen University, De Elst 1, 6708WD current study, white rot fungi were selected based on their Wageningen, The Netherlands selectivity for lignin degradation. Pre-treatments using Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 2 of 9 selective lignin degrading fungi, such as Ceriporiopsis sub- geographic availability and the fact that they are widely vermispora and Lentinula edodes, were proven to increase studied. Two different particle sizes, 0.5 and 2 cm length, the in vitro rumen degradability as a consequence of of both substrates were used with different amounts of increased cellulose availability of wheat straw and sugar- inoculum (colonized millet) added. cane bagasse [8, 9]. Also, in vivo studies reported in the scientific literature show an increased digestibility of fun- Methods gal treated biomass; e.g. treatment with C. subvermispora Fungal strains increased digestibility of bamboo in sheep [10], treatment Ceriporiopsis subvermispora (strain MES 13094) and with Ganoderma spp. increased the digestibility of wheat Lentinula edodes (strain MES 11910) were initially cul- straw in goats [11] and a treatment with Pleurotus spp. tured on malt extract (10 g malt extract and 17.5 g agar increased the digestibility, however only a maximum of per L, Oxoid LP0039, Thermo Scientific, Hampshire, 17 % dietary inclusion rate of fungal treated material UK) agar until it was almost fully colonized. Agar pieces was accepted by cows [12]. (approximately 1 cm ) were added to sterilized millet The time needed to obtain maximum cellulose avail- grains. Inoculated millet grains were incubated at 24 °C ability to increase in vitro rumen degradability was 6 to until full colonization, which occurred 5 wk after the 12 wk [8, 9, 13]. Although the substrates after the fungal start of incubation. Fully colonized grains were used as pre-treatment were enriched in cellulose, hemicellulose spawn to inoculate the substrates. was partially used by the fungi as lignin was degraded. This relatively long treatment time and consumption of Substrates part of the carbohydrate fraction are major drawbacks Wheat straw and wood chips (oak) were used as and optimization of both is needed to make this method substrates. Both substrates were chopped to particles competitive with current chemical and physical pre- with average sizes of 0.5 cm or 2 cm. The substrates treatments. were submerged in water for 3 d after which the excess Fungal pre-treatment starts with the inoculation of water was removed by draining through nets and the the substrate. In scientific literature, inoculation for C. substrates divided over 1.2 L autoclavable polypropylene subvermispora or L. edodes treatments has been done containers and covered with a lid containing a filter using agar plugs [14–16] or using liquid medium [17–19]. (model TP1200 + TPD1200 XXL Combiness, Nazareth, However, in the commercial mushroom production Belgium) through which air can pass. To each container process spawn is produced from grains [20]. Grain based 200 g of wet substrate was added and, since the water spawn can be produced on a large scale and is easy holding capacity of both substrates was different, this to mix through the substrate. Previous studies have represented approximately 100 and 50 g dry matter of used spawn made from wheat, millet or sorghum grains wood chips and wheat straw, respectively. The containers [8–10, 13, 21]. Compared to other grains, sorghum grains with substrate were sterilized for 1 h at 121 °C. The steril- are relatively large, meaning less inoculation points per g ized substrates were kept at room temperature until of inoculum added. The use of a smaller grain, like millet, further use. Autoclaved, uninoculated samples were taken would increase the amount of inoculation points per g to serve as control. of inoculum added. The latter can initiate a faster colonization of the lignocellulose biomass [22]. Inoculation Rapid and complete colonization of the lignocellulosic Colonized millet grains were added to each container in material is key for a competitive fungal pre-treatment. a concentration of either 0.5, 1.5 or 3 % of the weight of During colonization, the fungus starts degrading the the wet substrate. The content of the containers were outer layer of the material before reaching the inside mixed under aseptic conditions to divide the inoculum [23]. By decreasing the particle size of the substrate, the equally over the substrate. All conditions were tested in surface to volume ratio is increased. The latter will result triplicate, in which each sample started with 100 g dry in more contact points on the surface and reach the matter (in case of wheat straw, divided over two inner part of the material faster [22]. containers). Samples were incubated for 2, 4, 6 and 8 wk The aim of this study was to optimize the colonization in a climate controlled chamber at 24 °C and 70 % conditions of the fungal pre-treatment to obtain the most relative humidity. After incubation, the two containers of selective delignification, with minimal carbohydrate deg- wheat straw were pooled to represent one sample of 100 g radation and a high in vitro rumen degradability. Based on dry. Part of each sample (approximately 10 %) was freeze- the work of Tuyen et al. [8], C. subvermispora and L. dried for ergosterol measurements and the remaining part edodes were chosen as selective lignin degraders. Wheat of the sample was air-dried at 70 °C for 1 wk to be used straw and wood chips were selected for the fungal treat- for chemical analyses and in vitro gas production mea- ment, because of their high content of lignocellulose, surements. The dried wheat straw was ground to 1 mm van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 3 of 9 using a Peppink 100 AN cross beater mill (Peppink, De- reversed phase C18 column (250 × 4.6 mm, Phenomex venter, The Netherlands). The dried wood chips were aqua 5 μm). The liquid phase was 90 % methanol and 10 % finely ground over a 1 mm sieve using a Retch ZM100 (1:1) 2-propanol/hexane. Areas under the peak were cor- centrifugal mill (Retsch, Haan, Germany) to obtain a rected for the extraction efficiency based on the internal homogenous sample. standard cholecalciferol (vitamin D )(9.6 μg added) (Sigma The remaining colonized and uninoculated millet Aldrich, St. Louis, Missouri, USA), using Empower 2 soft- grains that were not used for inoculation were used for ware (Waters Corporation, Milford, Massachusetts, USA). further analysis. Approximately 10 % of each sample Mycelium of C. subvermispora and L. edodes grown on was freeze-dried, and the remaining part was air-dried at malt extract plates covered with cellophane was freeze 70 °C for 1 wk. The dried millet was ground to 1 mm dried and subjected to ergosterol extraction. The ergosterol using a Peppink 100 AN cross beater mill (Peppink, content of the pure mycelium was used to calculate the Deventer, The Netherlands). amount of fungal biomass formed. In vitro gas production technique Statistical analysis In vitro gas production (IVGP) was measured for all sam- The effect of amount of inoculum, particle size and in- ples by the IVGP technique, according to Cone et al. [24]. cubation on ergosterol content, detergent fiber compos- In summary, 60 mL of buffered rumen fluid, collected ition and IVGP was tested using the generalized linear from non-lactating cows, was added to 0.5 g air-dried model (GLM) analysis in SAS software version 9.3 (SAS material. Incubations were done in shaking water baths at Institute Inc., Cary, North Carolina, USA). The following 39 °C. Gas production was measured continuously for 72 model was used: h. Results were expressed as mL gas produced after 72 h Y ¼ μ þ α þ β þ γ þ ω ijk i ijk j k per g organic matter (OM). in which Y is the observation at incubation time i; μ is ijk Chemical analysis the overall mean; α is the fixed effect of amount of in- Fiber analysis was performed according to Van Soest oculum i; β is the fixed effect of particle size j; γ is the j k et al. [25], using an Ankom fiber analyser 2000 fixed effect of incubation time k; ω is the random ijk (ANKOM Technology, Macedon, New York, USA). error. The hemicellulose content was calculated as the differ- The results of ergosterol measurements, chemical ence between neutral detergent fiber (NDF) and acid analysis and IVGP at different incubation times of the detergent fiber (ADF) contents. The cellulose content fungal treatment of each substrate, for each amount of was calculated as the difference between ADF and acid inoculum and particle size combination, were compared detergent lignin (ADL) contents. using the generalized linear model (GLM) analysis in For dry matter (DM) determination air-dried material SAS software version 9.3 (SAS Institute Inc., Cary, North was dried for 4 h at 103 °C. Ash content was determined Carolina, USA). Post-hoc multiple comparison with by combustion for 3 h at 550 °C in a muffle furnace. Tukey’s significant test at a level of α = 0.05 was per- Starch content of the millet grains was determined enzy- formed to determine the significance of differences be- matically according to ISO15914. tween the treatments. The following model was used: Fungicide analysis was performed on autoclaved, un- treated, air-dried wheat straw using gas chromatography/ Y ¼ μ þ α þ ω ij i ij mass spectrometry according to NEN EN 12393. in which Y is the observation j at incubation time i; μ is ij Ergosterol the overall mean; α is the fixed effect of incubation time Ergosterol determination of freeze-dried material was i; ω is the random error. ij based on Niemenmaa et al. [26]. In summary, 200 mg of the material was saponified in 10 % KOH in methanol Results for 1 h at 80 °C. After cooling, hexane and distilled water Only results of C. subvermispora treated wood chips and were added for extraction. The samples were shaken for L. edodes treated wheat straw are presented. Visually no 10 min and centrifuged for 15 min at 4,000 rpm. The or limited growth was observed in L. edodes treated wood hexane phase was collected and the hexane-water ex- chips and C. subvermispora treated wheat straw. This low traction was repeated once. The hexane phases of the 2 growth was caused by unknown external factors. extractions were pooled and evaporated under vacuum. The extracted ergosterol was dissolved in 1 mL methanol Inoculum before ergosterol content was determined by a high per- The chemical composition and the IVGP of millet used formance liquid chromatography (HPLC) fitted with a as spawn at the moment of inoculation (5 wk of van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 4 of 9 colonization) are presented in Table 1. Ergosterol con- the first 4 wk of incubation as the ergosterol content in- tents showed that the millet was colonized by both C. creased during the first 4 wk, after which the growth rate subvermispora and L. edodes.Toestimatethe amount reduced (Fig. 1). Only after 4 wk of incubation differences of mycelial biomass we have measured the amount of were seen between treatments. Addition of 3 % inoculum ergosterol per unit dried mycelium for each fungus after to 0.5 cm wood chips resulted in a higher (P < 0.05) ergos- growth until full colonization (approximately 10 d) on malt terol content compared to the other treatments. Only the extract plates. C. subvermispora contained 9.6 mg addition of 3 % inoculum to 0.5 cm wood chips caused a ergosterol/g mycelium and L. edodes contained 6 mg significant decrease (P < 0.05) in cellulose content (g/kg ergosterol/g mycelium. Each g of colonized millet contains DM) and absolute amount of cellulose (g). thus approximately 14.3 mg C. subvermispora mycelium or Generally, in all tested conditions, the ADL and hemi- 32.1 mg L. edodes mycelium at the moment of inoculation. cellulose content and their absolute amounts were also However, it should be noted that the ergosterol contents of decreasedduringthe first4wk with C. subvermispora in- pure mycelium grown on malt extract agar may be differ- cubation. After 4 wk, the lowest values (P < 0.05) of hemi- ent for the ergosterol contents of mycelium grown on mil- cellulose and cellulose (contents and absolute amounts) let grains. Colonized millet had a lower (P < 0.05) ADL and were reached in the treatments with 3 % inoculum added cellulose content than the uninoculated grains (control). to 2 cm wood chips compared to the other treatments. Although L. edodes numerically decreased the hemicellu- The lower carbohydrate and unchanged ADL degradation lose content, this was not significant, whereas C. subver- suggest a more selective delignification process when 3 % mispora significantly decreased the hemicellulose content. inoculum was added to 2 cm wood chips. The least select- Interestingly, the starch content of the C. subvermispora ive delignification was observed when 0.5 % inoculum was spawn was not different from the control, whereas L. added to 0.5 cm wood chips, since most hemicellulose and edodes significantly degraded the starch in the millet a similar amount of ADL was degraded by C. subvermis- grains. The spawn showed a high IVGP of approximately pora. The higher hemicellulose degradation did not result 300 mL/g OM, which suggests that the addition of spawn in a higher ergosterol content or a lower IVGP. The IVGP to the substrates may have contributed to the increase in increased (P < 0.05) during the first 4 wk of incubation. IVGP at the start of fungal treatment. Most treatments reached a plateau level, whereas addition of 1.5 % inoculum to 0.5 cm wood chips and addition of 3 C. subvermispora treatment of wood chips % inoculum to 2 cm wood chips caused a continuous in- The probability values of the effects of the amount of crease in IVGP. As a result the addition of 1.5 % inoculum inoculum added, particle size and incubation time are to 0.5 cm wood chips resulted in the highest IVGP after 8 shown in Table 2. The amount of inoculum had only a wk of C. subvermispora incubation. significant effect on the cellulose degradation during the C. subvermispora treatment of wood chips. Chan- L. edodes treatment of wheat straw ging theparticlesizeofwoodchips hadsignificant The significance of the effect of the amount of inoculum, effects on the hemicellulose and cellulose degradation particle size and incubation time are shown in Table 2. by C. subvermispora. No significant effects were ob- The main differences observed in L. edodes treatment of served for the amount of inoculum (content P = 0.1435, wheat straw were seen between particle sizes and incu- absolute amounts P = 0.4626) and particle size (con- bation times. The particle size of wheat straw had a sig- tent P = 0.5688, absolute amount P = 0.9148) on the nificant effect on all variables measured, except the ADL degradation. The time of incubation showed effects absolute amounts of cellulose. The time of incubation (P < 0.05) on all variables measured. Tables 3 and 4 show had an effect (P < 0.05) on all variables measured. The that most changes occurred during the first 4 wk of incu- amount of inoculum had a significant effect on the bation. C. subvermispora colonized the wood chips within Table 1 Chemical composition (g/kg DM) of spawn at the moment of inoculation Treatment ADL Hemicellulose Cellulose Starch IVGP Ergosterol a a a a c Control 14.4 54.0 76.3 699.0 319.7 0.0 b b b a b C. subvermispora 2.3 30.3 50.4 680.2 285.8 138.1 b ab b b a L. edodes 3.5 40.9 43.2 408.2 302.5 192.7 SEM 1.58 3.31 2.65 5.56 16 10.61 P-value 0.003 0.0068 0.0003 <0.0001 0.3855 <0.0001 Values with different superscripts within column are significantly (P < 0.05) different ADL acid detergent lignin, IVGP in vitro gas production in rumen fluid van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 5 of 9 Table 2 Probability values for effects of the amount of inoculum added (0.5, 1.5 and 3.0 %), particle size (0.5 and 2.0 cm) and incubation time (2, 4, 6 and 8 wk) on chemical composition, ergosterol content and in vitro gas production of two fungal treated substrates (wheat straw and wood chips) C. subvermispora treated wood chips L. edodes treated wheat straw Item Amount of inoculum Particle size Incubation time Amount of inoculum Particle size Incubation time ADL, g/kg DM 0.1435 0.5688 <0.0001 0.0346 <0.0001 <0.0001 ADL, g 0.4626 0.9148 <0.0001 0.1640 <0.0001 <0.0001 HC, g/kg DM 0.3704 <0.0001 <0.0001 0.0003 <0.0001 <0.0001 HC, g 0.2533 <0.0001 <0.0001 0.0054 <0.0001 <0.0001 Cell, g/kg DM 0.0002 <0.0001 <0.0001 0.0026 0.0003 <0.0001 Cell, g 0.0197 <0.0001 0.0012 0.4314 0.1179 0.0120 Ergosterol, mg/g 0.5085 0.3220 <0.0001 <0.0001 <0.0001 <0.0001 IVGP, mL/g OM 0.4508 0.6735 <0.0001 0.1910 <0.0001 <0.0001 ADL acid detergent lignin, HC hemicellulose, Cell cellulose, IVGP in vitro gas production after 72h in rumen fluid ergosterol content, absolute amounts of hemicellulose material, because of its small grain size. Addition of the and ADL and hemicellulose and cellulose contents. same weight of spawn will result in more inoculation Addition of 1.5 or 3 % L. edodes inoculum to 0.5 cm points when using a smaller grain. The IVGP was not wheat straw particles resulted in a continuous growth different between the different amounts of inoculum during 8 wk, whereas addition to 2 cm wheat straw added. If the spawn would contribute to IVGP, this particles resulted in growth which stopped after 4 wk would be mainly because millet contains starch, espe- of incubation. No changes in ergosterol content were cially in C. subvermispora treated biomass since this fun- observed throughout the entire incubation period when gus did not degrade starch upon colonization. The 0.5 % inoculum was added to 0.5 cm wheat straw observation that C. subvermispora did not utilize starch particles. is interesting as the publically available genome data of Regardless how much inoculum was added, the ADL C. subvermispora shows the presence of genes encoding content and absolute amounts of 0.5 cm wheat straw for amylase. The ergosterol content data show that C. did not change after 4 to 6 wk of L. edodes incubation subvermispora grew on both millet and wood chips, (Tables 5 and 6). In contrast, the ADL in 2 cm wheat suggesting millet can be used as spawn for C. subvermis- straw decreased continuously during the 8 wk of incu- pora. The data presented here, indicate that hemicellu- bation (Tables 5 and 6). After 8 wk of incubation, the lose and to some extent cellulose are the substrates used ADL content of 2 cm wheat straw was lower (P <0.05) by C. subvermispora to grow on millet grains. The esti- than that of the 0.5 cm particles (Tables 5 and 6). The mation of the amount of fungal biomass on grains has hemicellulose content decreased (P <0.05) during the been done previously for the button mushroom Agaricus first 4 to 6 wk (Table 5). The final hemicellulose con- bisporus using three independent methods including the tent and the absolute amounts after 6 and 8 wk of incu- ergosterol method [27]. These authors calculated that bation were lower for the 2 cm particles than for the spawn contains circa 10 mg dry mycelial biomass per 0.5 cm particles (Tables 5 and 6). The cellulose content gram of grain which is in the same range as we have increased more (P < 0.05) for the 0.5 cm particles than measured here (14 to 32 mg for C. subvermispora and L. for the 2 cm particles (Table 5). This effect was more edodes respectively). visible by the absolute amounts of cellulose, where only C. subvermispora, in contrast to L. edodes, showed a a significant decrease in time was seen when 1.5 % in- low growth on wheat straw. In some treatments, such as oculum was added to 0.5 cm wheat straw particles addition of 0.5 % inoculum to 0.5 cm wheat straw parti- (Table 6). cles, also a low growth of L. edodes was found compared Six to eight wk of L. edodes treatment increased the to other studies [8, 13]. A possible explanation for the IVGP of the 2 cm wheat straw particles more (P < 0.05) low efficiency of the fungal treatment of wheat straw in than that of the 0.5 cm wheat straw particles (Fig. 2b). the present study is the presence of fungicides. Analysis of the wheat straw showed that it contained 0.767 mg Discussion tebuconazole/kg dry substrate. Tebuconazole is a fungi- The aim of this study was to optimize the colonization cide used in wheat production to control plant pathogenic conditions of fungal treatments. To do so, the amount fungi. The minimal inhibitory concentration of tebucona- of inoculum added and the particle size of the substrate zole has been shown to be different for different basidio- was varied. Here, millet was chosen as inoculation mycete species [28]. Possibly C. subvermispora is more van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 6 of 9 Table 3 Changes in detergent fiber content (g/kg DM) over an Table 4 Changes in absolute amounts of detergent fiber (g) eight wk period of 0.5 and 2 cm wood chips incubated with over an eight wk period of 0.5 and 2 cm wood chips incubated three different amounts of C. subvermispora inoculum with three different amounts of C. subvermispora inoculum Particle size 0.5 cm 2 cm Particle size 0.5 cm 2 cm Inoculum, % 0.5 1.5 3.0 0.5 1.5 3.0 Inoculum, % 0.5 1.5 3.0 0.5 1.5 3.0 Wk Wk a a a a a a a a a a a a ADL 0 214.8 214.8 214.8 214.8 214.8 214.8 ADL 0 31.2 31.2 31.2 31.2 31.2 31.2 a b b b b b a b b b b b 2 197.8 162.4 162.6 178.3 163.7 156.5 2 27.5 22.3 22.4 24.3 23.0 21.3 b c c c c c b c c c c c 4 127.0 130.0 126.3 133.2 126.8 134.3 4 16.2 16.9 16.3 17.3 16.6 18.1 b d c d c c b d c d c cd 6 128.3 118.4 123.4 111.8 126.4 126.4 6 16.4 14.9 15.8 14.0 16.1 16.5 b cd c c c c b d c d c d 8 112.0 123.6 129.4 126.0 121.2 120.7 8 13.8 15.1 16.4 15.5 15.5 15.2 SEM 9.95 2.07 2.18 2.31 5.41 2.95 SEM 1.60 0.30 0.31 0.35 0.80 0.52 P-value <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 P-value <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 a a a a a ab a a a a a a HC 0 151.5 151.5 151.5 151.5 151.5 151.5 HC 0 22.0 22.0 22.0 22.0 22.0 22.0 a a a a a a a a a a a ab 2 154.1 160.7 170.7 151.7 156.2 158.3 2 21.4 22.1 23.5 20.7 21.9 21.6 b b b bc b abc b b b bc b bc 4 90.0 98.5 92.8 109.4 114.1 129.9 4 11.5 12.8 12.0 14.2 14.9 17.5 b b b ab b bc b b b b b c 6 107.4 85.6 87.0 126.8 106.2 122.6 6 13.7 10.8 11.1 15.9 13.6 16.0 b b b c b c b b b c b c 8 74.5 73.1 74.8 94.9 107.3 112.1 8 9.2 9.0 9.5 11.7 13.7 14.1 SEM 8.21 10.80 9.82 5.50 4.32 6.55 SEM 1.19 1.47 1.30 0.83 0.59 0.94 P-value 0.0001 0.0005 0.0001 <.0001 <.0001 0.0025 P-value <.0001 0.0001 <.0001 <.0001 <.0001 0.0005 a b ab b b b a Cell 0 381.3 381.3 381.3 381.3 381.3 381.3 Cell 0 55.5 55.5 55.5 55.5 55.5 55.5 ab ab b b b b ab 2 390.3 393.1 375.3 408.2 396.8 387.4 2 54.1 54.0 51.7 55.7 55.7 52.7 ab ab ab ab a a b 4 417.0 403.3 384.5 414.7 430.3 422.2 4 53.2 52.3 49.8 53.9 56.3 57.0 ab ab a a a a ab 6 417.9 415.4 405.4 455.1 446.0 419.3 6 53.2 52.3 51.9 57.0 57.0 54.6 a a ab ab a a b 8 430.3 419.1 397.1 424.2 438.4 419.2 8 52.9 51.3 50.5 52.1 55.9 52.9 SEM 10.34 7.90 5.98 9.97 6.82 6.42 SEM 1.59 1.05 0.81 1.25 0.91 0.91 P-value 0.0349 0.0362 0.0301 0.0054 0.0002 0.0019 P-value 0.7836 0.1072 0.0048 0.133 0.7735 0.0402 Values with different superscripts within column are significantly (P < 0.05) Values with different superscripts within column are significantly (P < 0.05) different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM standard error of the mean standard error of the mean sensitive to this fungicide than L. edodes. Tebuconazole Possibly L. edodes is more sensitive to water activity than inhibits the formation of ergosterol in fungi [28]. C. C. subvermispora. subvermispora contains more ergosterol per g of myce- The particle size of the substrate had only an effect on lium (9.6 mg ergosterol/g mycelium) than L. edodes (6 mg the L. edodes treatment of wheat straw. Sachs et al. [23] ergosterol/g mycelium), which may be the reason why C. showed that P. chrysosporium formed more mycelium subvermispora is more sensitive to this fungicide. Since on the surface of aspen wood chips compared to the in- the use of fungicides is general practice in wheat produc- side of the wood chips. Therefore, it is expected that tion, it is advised for future fungal treatments to use with a decreasing particle size, and thus a larger surface organically grown wheat straw. However, L. edodes to volume ratio, the substrate is more accessible and showed a reduced growth on wood chips. A possible more mycelium can be formed. Despite the different sur- explanation may be the different moisture content be- face to volume ratio, particle size did not have a major tween both wood chips and wheat straw. The moisture influence on the P. chrysosporium growth patterns [23]. content of wheat straw was around 60 %, while that of In the present study, particle size had a significant effect wood chips was around 45 %. This difference is inherent on the ergosterol content, although only addition of to the manner of moisturizing the substrates. After full 0.5 % inoculum to 0.5 cm wheat straw particles had a penetration of the water, the ‘free’ water is removed by remarkable low ergosterol content. Probably the contrast draining. The water holding capacity of wood chips is between the surface to volume ratios between particle lower than that of wheat straw, causing a difference in sizes was not large enough to see effects on colonization. moisture content. However there may be a different water Interestingly, 2 cm particles resulted in a lower ADL and a activity for both substrates, which was not measured. higher IVGP upon the fungal treatment. Gómez [29] van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 7 of 9 Fig. 1 Results of ergosterol measurements. (a) C. subvermispora on wood chips, (b) L. edodes on wheat straw. ♦ 0.5% inoculum per g wet substrate (0.5 cm) ■ 1.5 % inoculum per g wet substrate (0.5 cm) ▲ 3 % inoculum per g wet substrate (0.5 cm) ◊ 0.5 % inoculum per g wet substrate (2 cm) □ 1.5 % inoculum per g wet substrate (2 cm) Δ 3 % inoculum per g wet substrate (2 cm) demonstrated an increased expression of cellulases and carbohydrates in large particles, the fungi first have to de- xylanases by Trametes sp., when grown on smaller parti- grade more lignin. On the other hand, not more carbohy- cles of corn straw, compared to larger particles. Carbohy- drate degradation was observed in the smaller particles. drates in smaller particles are possibly more exposed at It has to be noted that the detergent fiber analysis the surface compared to large particles. To reach the method was used in this study. The detergent fiber Table 5 Changes in detergent fiber content (g/kg DM) over an Table 6 Changes in absolute amounts of detergent fiber (g) eight wk period of 0.5 and 2 cm wheat straw incubated with over an eight wk period of 0.5 and 2 cm wheat straw incubated three different amounts of L. edodes inoculum with three different amounts of L. edodes inoculum Particle size 0.5 cm 2 cm Particle size 0.5 cm 2 cm Inoculum, % 0.5 1.5 3.0 0.5 1.5 3.0 Inoculum, % 0.5 1.5 3 0.5 1.5 3 Wk Wk abc ab a b a a a ab a b a a ADL 0 90.8 90.8 90.8 90.8 90.8 90.8 ADL 0 17.7 17.7 17.7 17.7 17.7 17.7 a a a a a a a a a a a a 2 95.7 100.2 97.9 107.3 97.3 92.6 2 18.4 19.5 19.2 20.6 18.8 18.0 ab ab ab b b b a ab ab b b b 4 92.7 95.4 82.6 89.1 79.5 79.3 4 17.5 18.3 16.0 16.8 15.2 15.2 bc ab a c c c ab b a c c c 6 81.9 80.5 95.6 68.2 64.1 64.5 6 15.7 15.2 18.3 12.7 12.0 12.2 c b b d d d b b b d d d 8 78.6 79.0 69.9 56.5 54.6 52.2 8 14.6 14.7 13.2 10.4 10.1 9.7 SEM 2.74 4.40 3.82 1.80 1.63 2.36 SEM 0.60 0.85 0.77 0.33 0.32 0.43 P-value 0.0056 0.0261 0.0025 <.0001 <.0001 <.0001 P-value 0.0062 0.0113 0.0019 <.0001 <.0001 <.0001 a a a a a a a a a a a a HC 0 287.1 287.1 287.1 287.1 287.1 287.1 HC 0 56.0 56.0 56.0 56.0 56.0 56.0 a a ab a a b a a ab a a b 2 288.8 277.8 262.8 286.9 273.6 262.1 2 55.6 54.0 51.6 55.2 52.8 51.1 b ab bc a b c b ab c a b c 4 253.4 260.7 235.4 263.7 242.5 233.1 4 47.9 50.1 45.6 49.8 46.3 44.7 c bc bc b c d c bc bc b c d 6 224.7 228.9 247.2 211.4 204.8 190.7 6 43.1 43.2 47.3 39.3 38.2 36.1 bc c c b c d bc c c b c d 8 240.8 223.6 221.0 199.9 188.2 196.2 8 44.8 41.7 41.8 36.8 34.8 36.4 SEM 5.22 7.96 6.04 8.39 4.80 3.44 SEM 0.96 1.49 1.29 1.59 0.92 0.64 P-value <.0001 0.0006 0.0002 <.0001 <.0001 <.0001 P-value <.0001 0.0001 0.0001 <.0001 <.0001 <.0001 b b ab b bc ab Cell 0 474.1 474.1 474.1 474.1 474.1 474.1 Cell 0 92.5 92.5 92.5 92.5 92.5 92.5 ab b b b c b 2 480.4 470.5 461.1 493.5 475.6 467.1 2 92.5 91.4 90.6 94.9 91.7 91.1 ab a a a abc a 4 489.5 503.1 485.5 481.3 500.3 486.9 4 92.6 96.6 94.0 90.9 95.5 93.5 a ab ab a a ab 6 493.8 491.2 478.7 500.7 498.3 503.0 6 94.7 92.8 91.6 93.2 92.9 95.1 ab ab ab a ab b 8 489.8 479.6 478.1 508.3 507.4 494.1 8 91.2 89.5 90.3 93.6 93.9 91.7 SEM 4.04 6.20 4.63 8.32 4.17 4.62 SEM 1.38 1.03 1.08 1.59 0.83 1.06 P-value 0.035 0.0221 0.0403 0.0835 0.0005 0.0016 P-value 0.5141 0.0076 0.1953 0.5183 0.0667 0.1257 Values with different superscripts within column are significantly (P < 0.05) Values with different superscripts within column are significantly (P < 0.05) different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM different. ADL acid detergent lignin, HC hemicellulose, Cell cellulose, SEM standard error of the mean standard error of the mean van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:39 Page 8 of 9 Fig. 2 Results of in vitro gas production (IVGP) measurements. (a) C. subvermispora on wood chips, (b) L. edodes on wheat straw. ♦ 0.5 % inoculum per g wet substrate (0.5 cm) ■ 1.5 % inoculum per g wet substrate (0.5 cm) ▲ 3 % inoculum per g wet substrate (0.5 cm) ◊ 0.5% inoculum per g wet substrate (2 cm) □ 1.5 % inoculum per g wet substrate (2 cm) Δ 3% inoculum per g wet substrate (2 cm) method is developed to estimate feeding value of forage does not seem to be the limiting factor for C. subvermis- for ruminants. It is not developed to precisely determine pora. The L. edodes treatment of wheat straw was influ- the chemically defined fractions, and as a result it under- enced by the particle size of the substrate. Larger (2 cm) estimates the lignin fraction and overestimates the carbo- particles resulted in a more selective delignification and hydrate fractions [30, 31]. Nevertheless, the acid detergent a higher IVGP than smaller (0.5 cm) particles. The lignin is highly correlated to rumen degradability for both amount of inoculum to some extent affects L. edodes wheat straw and wood chips [13], and changes in the treatment of wheat straw. At least 1.5% spawn should be lignin and carbohydrate fractions upon fungal treatment added to obtain a more selective lignin degradation and are a good indication for carbohydrate accessibility. For a an increased IVGP. more detailed chemical analysis of plant cell walls it is advised to measure lignin and carbohydrates in more de- Abbreviations tail using pyrolysis coupled to gas chromatography and ADF, acid detergent fiber; ADL, acid detergent lignin; Cell, cellulose; DM, dry matter; GLM, generalized linear model; HC, hemicellulose; HPLC, high mass spectrometry and sugar analysis. performance liquid chromatography; IVGP, in vitro gas production; NDF, neutral Particle size did not have an effect on the C. subver- detergent fiber; OM, organic matter; SEM, standard error of the mean mispora treatment of wood chips. If a larger contrast between the particle sizes was used, maybe an effect Acknowledgements This research was supported by the Dutch Technology Foundation (STW), would have been seen. In the scientific literature how- which is part of the Netherlands Organization for Scientific Research (NWO), ever, 15 mm size corn stover yielded less glucose upon which is partly funded by the Dutch Ministry of Economic Affairs. This enzymatic saccharification than 10 and 5 mm corn sto- project (11611) was co-sponsored by Agrifirm, Purac, DSM, Den Ouden, Hofmans, the Dutch commodity boards for dairy and horticulture, and ver after a C. subvermispora treatment [32]. The same Wageningen University. We thank Agrifirm for practical assistance with authors concluded that moisture content, time and the analysis of dry matter and ash contents. We thank DSM for practical temperature were also important factors influencing assistance with the analysis of fungicides. the effectiveness of the fungal treatment. Different sub- Authors’ contributions strates show different water holding capacities, which SvK carried out the experiments and drafted the manuscript. AS, JB, WH and also depends on the particle size, explaining why the JC were involved in the study design, data interpretation and manuscript effect of particle size may be different for different preparation. All authors read and approved the final manuscript. substrates. Competing interests The results of this study show that the particle sizes The authors declare that they have no competing interests. (0.5 or 2 cm) tested do not have a major influence on the C. subvermispora treatment of wood chips. Using Author details Animal Nutrition Group, Wageningen University, De Elst 1, 6708WD wheat straw particles smaller than 2 cm does not have Wageningen, The Netherlands. Plant Breeding, Wageningen University, beneficial effects on the L. edodes treatment. Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands. 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Journal of Animal Science and Biotechnology – Springer Journals

Published: Jul 13, 2016

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