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Background: The present work investigated the influence of lignin content and composition in the fungal treatment of lignocellulosic biomass in order to improve rumen degradability. Wheat straw and wood chips, differing in lignin composition, were treated with Lentinula edodes for 0, 2, 4, 8 and 12 wk and the changes occurring during fungal degradation were analyzed using pyrolysis-gas chromatography-mass spectrometry and detergent fiber analysis. Results: L. edodes preferentially degraded lignin, with only limited cellulose degradation, in wheat straw and wood chips, leaving a substrate enriched in cellulose. Syringyl (S)-lignin units were preferentially degraded than guaiacyl (G)-lignin units, resulting in a decreased S/G ratio. A decreasing S/G ratio (wheat straw: r = −0.72, wood chips: r = −0.75) and selective lignin degradation (wheat straw: r = −0.69, wood chips: r = −0.88) were correlated with in vitro gas production (IVGP), a good indicator for rumen degradability. Conclusions: L. edodes treatment increased the IVGP of wheat straw and wood chips. Effects on IVGP were similar for wheat straw and wood chips indicating that lignin content and 3D-structure of cell walls influence in vitro rumen degradability more than lignin composition. Keywords: Fungal treatment, In vitro rumen degradability, Lignocellulosic biomass, Py-GC/MS Background more cost effective and less harmful for animals and the Carbohydrates in plant cell walls can be an important environment compared to current pre-treatment methods. source of nutrients for ruminants. However, these carbo- Studies reported in the scientific literature describe treat- hydrates are bound to lignin, which can be degraded ments by different fungi to pre-treat various substrates only under aerobic conditions by fungi and some bac- suitable as ruminant feed ingredients . Among them, teria , and as such cannot be broken down in the low the white rot fungus Lentinula edodes was found to be oxygen environment of the rumen. Currently, several highly promising due to its selective lignin degradation chemical and physical pre-treatments are used to make pattern [4–10]. In addition, this fungus has a ‘generally the carbohydrates in lignocellulosic substrates more regarded as safe’ (GRAS) status and is, therefore, a poten- available for degradation in the rumen [2, 3]. Biological tially suitable fungus for fungal pre-treatment of feed in- treatments using fungi that selectively degrade lignin, gredients . However, due to the GRAS status of edible without or with limited cellulose degradation, may be mushrooms of this fungal genus, hitherto the majority of research has focused on mushroom production [6–8]. Scientific studies describing L. edodes treatment of lig- * Correspondence: email@example.com 1 nocellulosic biomass to increase rumen degradability Animal Nutrition Group, Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands often report changes in lignin content as measured by 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) 7:55 Page 2 of 14 the Van Soest acid detergent lignin (ADL) methodology plates at 24 °C until most of the plate surface was cov- [3–5, 11]. However, ADL does not represent the total ered with mycelium. Pieces of colonized agar culture lignin content, since acid soluble lignin is not included (~1 cm )wereaddedtosterilizedsorghum grains (Sor- in the ADL fraction leading to an underestimation ghum bicolor var. bicolor, type red dari) and incubated [12, 13]. Lignin is a complex aromatic polymer produced at 24 °C until all grains were colonized by mycelium. by the oxidative coupling of three main monolignols, sina- This spawn was kept at 4 °C until the substrates were pyl, coniferyl and p-coumaryl alcohols, differing in their ready to use (~1 wk). degree of methoxylation . When incorporated into the lignin polymer, these monolignols give rise to the syringyl Substrate preparation (S), guaiacyl (G) and p-hydroxyphenyl (H) units, respect- The substrates used were wheat straw and municipal ively, generating a variety of structures and linkages trimmings consisting of a mixture of chips from differ- within the polymer, including β–O–4′ alkyl-aryl ent wood species. Both substrates were chopped into ethers, phenylcoumarans and resinols, amongst others. pieces of approximately 3 cm length and submerged in Lignin composition is different for each plant species, tap water (pH was not adjusted) for 3 d at room for example, lignin in grasses and herbaceous plants temperature to allow moisture to fully penetrate. After consists of S-, G-, and H-units, whereas softwoods removal of excess water, substrates were autoclaved present mainly G-lignin units and hardwoods present twice with the first sterilization performed in autoclava- S- and G-units in different proportions . Lignin ble bags at 121 °C for 1 h. After cooling, the material composition can be rapidly assessed using pyrolysis was weighed into 1.2 L polypropylene containers fitted coupled to gas chromatography-mass spectrometry with a cover containing a filter allowing gas exchange, (Py-GC/MS) . During pyrolysis the plant material but preventing contamination (model TP1200 + TPD1200 is heated at high temperatures (usually around 500– XXL Combiness, Nazareth, Belgium). Each container 700 °C) in an oxygen-free environment to break down was filled with approximately 80–90 g dry matter of the macromolecular components of plant cell walls to wheat straw or wood chips before being autoclaved a smaller compounds, which are subsequently analyzed second time at 121 °C for 1 h to kill the remaining in a GC/MS system. As such, Py-GC/MS is a useful germinated spores in the substrates. After cooling, the tool to monitor the extent of fungal degradation of sterile substrates were kept in the container at room lignocellulosic constituents, which cannot be straight- temperature until further use. Three of these auto- forwardly detected with the standard gravimetric claved containers were used as an uninoculated con- methods, such as the detergent fiber method [16–18]. trol (0 wk treatment). In this study it is hypothesized that in addition to the total lignin content, the lignin composition (in terms of Substrate inoculation relative abundances of the S-, G-, and H-lignin units) To each remaining container, approximately 8–10 g of and the 3D-structure formed by lignin and carbohy- spawn (sorghum grains colonized with a pure culture of drates in plant cell walls also determine the efficiency of L. edodes) was added and mixed to distribute the spawn the fungal treatment to improve rumen degradability. In equally over the substrate. Both handlings were per- this paper, two substrates (wheat straw and wood chips) formed aseptically. Each container was then incubated differing in lignin content and composition were treated for 2, 4, 8 or 12 wk at 24 °C and 70 % relative humidity with L. edodes and analyzed for cell wall components in a climate controlled chamber. All conditions were using the detergent fiber method, and lignin compos- tested in triplicate. ition using Py-GC/MS. Rumen degradability of treated After incubation, the substrate was air-dried at 70 °C substrates was measured by the in vitro gas production until constant weight. The dried wheat straw was technique. ground with a Peppink 100 AN cross beater mill (Peppink, Deventer, The Netherlands) over a 1 mm Methods sieve. The dried wood chips were first coarsely ground Fungal strains and spawn preparation over a 1 mm sieve using a Retch SM2000 cutting mill Lentinula edodes (strain MES 11910) was used in this (Retch, Haan, Germany) before being ground over a study. The strain MES11910 originates from the CCBAS 1 mm sieve using a Retch ZM 100 centrifugal mill culture collection of basidiomycetes (Institute of micro- (Retch, Haan, Germany). Samples were stored at 4 °C biology, academy of sciences of the Czech Republic until chemical analyses. (http://www.biomed.cas.cz/ccbas/fungi.htm). The strain has been isolated in 1961 in Japan and found on Passania Fiber analysis wood. The species identity was confirmed with ITS se- Samples were analyzed according to the Van Soest quencing. L. edodes was cultured on malt extract agar method . The hemicellulose content was calculated van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 3 of 14 as the difference between the neutral detergent fiber autoclaved, uninoculated control of each substrate. The (NDF) and the acid detergent fiber (ADF). The lignin following model was used: content was determined as the ‘acid detergent lignin’ (ADL) content, that was defined as the part of the cell Y ¼ μþα þ ω ij i ij wall that is not soluble in acid detergent reagent and 72 % sulphuric acid. The cellulose content was calcu- in which Y is the observation j in treatment i; μ is ij lated as the difference between ADF and ADL. For dry the overall mean; α is the fixed effect of treatment i; matter (DM) determination, air-dried material was dried ω is the random error. Post-hoc multiple comparison ij at 103 °C for 4 h. Ash content was determined by com- with Tukey’s significant test at a level of α =0.05 was bustion for 3 h at 550 °C in a muffle furnace. The data performed to determine the significance between the for three replicate samples were averaged and expressed treatments. as g/kg DM. Regression analysis between IVGP and fiber compos- ition of fungal treated substrates was analyzed in SAS 9.3. The correlation between IVGP and Py-GC/MS data Pyrolysis-GC/MS was analyzed in SAS 9.3. The correlations are provided Pyrolysis-GC/MS (approximately 1 mg) was performed as the Pearson correlation coefficient (r). with a 3030 μ-furnace pyrolyzer (Frontier Laboratories Ltd.) connected to an Agilent 7820A GC using a DB- Results 1701 fused-silica capillary column (60 m × 0.25 mm, Composition of wheat straw and wood chips during 0.25 μm film thickness) and an Agilent 5975 mass select- fungal treatment ive detector (EI at 70 eV). The pyrolysis was performed The wood chips used in the current study originated at 500 °C. The oven temperature of the gas chromato- from municipal trimmings consisting of a mixture of dif- graph was programmed from 100 °C (4 min) to 280 °C ferent wood species, which all contain a different lignin at a rate of 3 °C/min and held at the maximum composition. The composition presented in this study is temperature for 2 min. The transfer line was set to therefore representative for this mixture, since both 290 °C. Helium was the carrier gas with a constant technical (each sample was measured in duplicate) and flow of 1 mL/min. The compounds were identified by biological (each treatment was measured in triplicate) comparing their mass spectra with those of the Wiley replicates are presented here. (John Wiley and Sons, Hoboken, NJ, USA) and NIST/ The ADL, hemicellulose and cellulose content of wheat EPA/NIH 2011 (National Institute of Standards and straw and wood chips before and after L. edodes treatment Technology, Gaithersburg, MD, USA) mass spectral li- for 2, 4, 8 and 12 wk is shown in Table 1. Untreated wheat braries and those reported in literature [19, 20]. Peak straw had a lower ADL, higher hemicelluloses and similar areas corrected for molecular weight were calculated cellulose content compared to untreated wood chips. for the carbohydrate and lignin-degradation products, the Upon L. edodes treatment, the content of ADL and hemi- summed areas were normalized, and the data for three rep- celluloses of wheat straw decreased (P <0.05), while the licate samples were averaged and expressed as percentages. cellulose content increased (P < 0.05) compared to the un- treated control. Expressed in absolute amounts, ADL de- In vitro gas production technique creased (P < 0.05) up to 87 %, hemicelluloses (P <0.05) up The in vitro gas production (IVGP) technique was per- to 77 % and cellulose was degraded up to 20 %, but this formed according to the procedure previously described was not significant (P = 0.07) (Table 1). The amounts of . In short, rumen fluid of 2 fistulated non-lactating dry matter significantly decreased by 30 % after 12 wk. cows fed a grass silage based diet was mixed with a Fungal treatment of wood chips also resulted in a decrease buffer solution under anaerobic conditions. Air dried (P < 0.05) in the ADL content during the 12 wk of incuba- samples (500 mg) were incubated in 60 mL buffered tion. No significant decrease in hemicelluloses and cellu- rumen fluid (final dilution 3 times) for 72 h at 39 °C. lose content was observed after 12 wk incubation The gas production was automatically recorded as previ- compared to the autoclaved control. The same applied to ously described , and the data for three replicate the absolute amounts of each compound, only ADL de- samples were averaged and expressed as mL gas/g creased significantly (P < 0.05) over time during L. edodes organic matter (OM). treatment of wood chips compared to the uninoculated control. Statistical analysis A generalized linear model (GLM) analysis in SAS 9.3 In vitro gas production (IVGP) of fungal treated samples was used to compare fiber composition and IVGP of IVGP of wheat straw increased (P = 0.058) from the fungal treatment at each incubation time to the 252.8 mL/g OM in the untreated control sample (0 wk van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 4 of 14 Table 1 Chemical composition of autoclaved wheat straw and wood chips before and after treatment with L. edodes for 2, 4, 8 and 12 wk Substrate Treatment Contents, g/kg DM Amounts, g DM IVGP, mL/g time, wk loss, % OM ADL HC Cell ADL HC Cell a a b,c a a c a,b Wheat straw 0 81.1 260.1 479.6 7.5 24.2 44.6 0 252.8 b b c b b c b 2 74.6 202.7 461.2 6.4 17.4 39.5 8 247.8 c c b c c b,c a,b 4 57.0 148.1 496.3 4.7 12.2 41.0 11 277.0 d d a d d b a,b 8 33.2 94.6 537.3 2.4 6.9 39.1 22 287.3 e d a e d a a 12 15.5 87.2 544.2 1.0 5.7 35.6 30 311.2 RMSE 2.22 11.48 11.80 0.29 1.22 3.21 0.05 22.40 P-value <0.01 <0.01 <0.01 <0.01 <0.01 0.07 <0.01 0.03 1 a c a a c Wood chips 0 198.2 140.8 445.5 15.4 11.0 35.3 0 54.0 a,b a,b b a a,b c 2 187.3 96.3 486.2 15.1 7.8 39.2 0 55.4 b a,b a,b b a,b b 4 163.8 103.6 497.4 10.1 6.8 31.1 20 120.7 c b a b b a 8 126.6 79.9 520.6 8.8 5.6 36.1 11 169.6 c a,b c b a,b a 12 107.0 105.6 436.5 6.9 6.8 28.5 17 177.4 RMSE 9.49 16.97 11.59 1.44 1.81 4.53 1.2 16.12 P-value <0.01 0.02 <0.01 <0.01 0.04 0.10 0.16 <0.01 Values with different superscripts within column are significantly (P < 0.05) different ADL acid detergent lignin, HC hemicellulose, Cell cellulose, DM loss dry matter loss, IVGP in vitro gas production, RMSE root-mean-square error treatment) to 311.2 mL/g OM after 12 wk of L. edodes The relative abundances of lignin-derived phenols treatment, a 23 % increase (Table 1). For fungal treated decreased with incubation time of wheat straw and wood chips, the IVGP of the uninoculated control (0 wk wood chips with L. edodes. In the case of wheat straw de- treatment) was 54.0 mL/g OM and a significant increase graded by L. edodes (Fig.1, Table 2),the percentage of com- (P < 0.05) was already seen after 4 wk treatment, and pounds released that were derived from carbohydrates continued to increase during the 12 wk treatment up to upon Py-GC/MS varied from 59.6 % in the control sample a value of 177.4 mL/g OM, representing a nearly 230 % (0 wk incubation time) to 92.9 % after 12 wk of incubation, increase compared to the untreated sample (Table 1). while the lignin-derived phenols (H + G + S) varied from Regression analysis between IVGP (mL/g OM) and cell 40.4 % in the control sample to only 7.1 % after 12 wk of in- wall composition (g/kg) yielded the following equation: cubation. In the case of wood chips incubated with L. edodes (Fig. 2, Table 3), the percentage of carbohydrate- derived compounds released upon Py-GC/MS varied from IVGP ¼ 0:26 hemicelluloses þ 0:33 47.9 % in the control sample (0 wk incubation time) to cellulose−1:34 ADL þ 135:86 73.1 % after 12 wk of incubation, while the lignin-derived phenols varied from 52.1 % in the control sample to 26.9 % This equation indicates that changes in ADL have after 12 wk of incubation. The lignin/carbohydrate (L/C) ra- more effect on IVGP than hemicelluloses and cellulose. tio estimated upon Py-GC/MS decreased from 0.7 in the The large influence of ADL on IVGP is the reason to wheat straw control sample (0 wk incubation time) to 0.1 in study lignin in more detail. the wheat straw degraded for 12 wk (Table 2), and from 1.1 in the wood chips control sample (0 wk incubation time) to Py-GC/MS analyses of uninoculated and fungal treated 0.4 for the wood chips degraded for 12 wk (Table 3). substrates Among the lignin-derived phenols, the pyrograms The pyrograms of wheat straw and wood chips during of wheat straw show compounds derived from p- fungal treatment with L. edodes are shown in Figs. 1 and hydroxyphenyl (H), guaiacyl (G) and syringyl (S) 2. The identities and relative abundances (mean average lignin units, whereas lignin in wood chips contained of three replicates) of the compounds released are pro- mainly G- and S-units (Tables 2 and 3). For wheat vided in Table 2 (wheat straw) and Table 3 (wood chips). straw, S/G ratio gradually decreased with incubation time The pyrolysis of untreated wheat straw (Fig. 1a) and from 0.7 in the control sample (0wkincubation time) to a wood chips (Fig. 2a) released a similar set of compounds final value of 0.4 after 12 wk (Table 2). However, in wheat derived from the carbohydrate and lignin moieties, straw, 4-vinyl-guaiacol (compound 17) may also arise from although in different proportions (Tables 2 and 3). van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 5 of 14 100% 24 30 4 36 3 7 23 31 5 12 26 1 14 32 37 39 15 29 40 10 19 27 8 13 21 41 100% 17 b 16 35/36 7 9 31 1 5 23 26 15 29 38 32 37 27 40 10 41 13 18 100% 35/36 16/17 4 14 1 5 20 2 9 26 7 30 31 15 29 32 12 23 34 10 40 18 21 100% 14 16/17 2 31 26 30 10 15 29 34 39 23 40 13 18 21 100% 2 7 16/17 10 20 29 34 39 15 26 37 8 23 10 15 20 25 30 35 40 45 Retention time , min Fig. 1 Py-GC/MS chromatograms of wheat straw degraded with the fungus Lentinula edodes. a untreated wheat straw control (0-wk incubation); b wheat straw degraded for 2 wk; c 4wk; d 8wk; e 12 wk. The identities and relative abundances of the compounds represented by the numbered peaks are listed in Table 2 Relative abundance Relative abundance Relative abundance Relative abundance Relative abundance van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 6 of 14 100% 6 35 9 26 14 18 29 34 39 4 21 31 7 19 27 1 5 8 10 11 100% 3 20 32 39 31 34 4 38 18 27 1 7 2 8 13 10 11 15 41 100% 39 40 4 19 1 28 34 5 21 38 2 8 11 41 10 13 15 100% 3 6 20 30 12 16 26 4 25 7 37 28 39 10 32 2 38 18 27 11 15 33 41 8 13 21 6 35 100% 23 31 1 4 12 16 26 29 7 19 37 39 5 32 40 2 10 34 38 11 13 15 21 41 10 15 20 25 30 35 40 45 Retention time , min Fig. 2 Py-GC/MS chromatograms of wood chips degraded with the fungus Lentinula edodes. a untreated wood chips (0-wk incubation); b wood chips degraded for 2 wk; c 4wk; d 8wk; e 12 wk. The identities and relative abundances of the compounds represented by the numbered peaks are listed in Table 3 Relative abundance Relative abundance Relative abundance Relative abundance Relative abundance van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 7 of 14 Table 2 Identities and relative abundance (mean average of three replicates) of the compounds released upon pyrolysis GC/MS of autoclaved wheat straw before and after treatment with L. edodes for 2, 4, 8 and 12 wk Label Compound Origin Time, wk 0 248 12 1(2H)-furan-3-one C 3.4 4.1 4.4 4.9 5.2 2 Propanal C 6.1 5.4 4.4 4.7 6.7 3 Furfural C 6.0 10.1 10.4 11.2 9.7 4 2,3-dihydro-5-methylfuran-2-one C 6.0 5.4 5.1 5.4 7.0 5(5H)-furan-2-one C 4.6 3.8 3.9 3.8 4.9 6 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one C 7.9 10.9 10.8 9.1 6.5 7 2-hydroxy-3-methyl-2-cyclopenten-1-one C 3.4 3.0 3.8 5.4 5.9 8 Phenol LH 1.1 0.9 1.0 1.1 1.0 9 Guaiacol LG 3.4 2.5 2.4 2.2 1.6 10 3-hydroxy-2-methyl-(4H)-pyran-4-one C 1.0 1.1 1.7 2.5 2.7 11 4-hydroxymethyl-1,4-butyrolactone C 2.9 3.1 2.7 2.8 3.9 12 4-methylguaiacol LG 1.3 1.0 0.7 0.5 0.3 13 4-ethylguaiacol LG 0.4 0.3 0.2 0.1 0.1 14 5-hydroxymethyl-2-tetrahydrofuraldehyde-3-one C 1.7 1.5 2.4 2.8 3.0 15 1,4-anhydroarabinofuranose C 1.7 1.7 1.9 1.6 1.3 16 4-vinylphenol LH/PCA 9.3 6.3 3.1 2.2 0.8 17 4-vinylguaiacol LG/FA 8.3 5.3 2.9 2.1 0.8 18 Eugenol LG 0.4 0.2 0.2 0.1 0.0 19 5-hydroxymethyl-2-furfuraldehyde C 1.7 3.3 3.6 3.2 4.7 20 Syringol LS 2.9 2.1 1.7 0.8 0.5 21 cis-isoeugenol LG 0.2 0.1 0.1 0.1 0.0 22 1,4-dideoxy-D-glycerohex-1-enopyranos-3-ulose C 0.8 1.0 1.4 2.0 1.7 23 trans-isoeugenol LG 1.5 0.9 0.6 0.4 0.2 24 1,4-anhydroxylofuranose C 2.4 2.1 3.5 2.8 2.4 25 4-methylsyringol LS 1.4 1.2 0.7 0.2 0.1 26 Vanillin LG 1.8 1.0 1.0 0.6 0.3 27 4-ethylsyringol LS 0.2 0.1 0.1 0.0 0.0 28 vanillic acid methyl ester LG 0.1 0.3 0.2 0.1 0.1 29 Acetovanillone LG 0.4 0.5 0.5 0.5 0.3 30 4-vinylsyringol LS 2.2 1.2 0.7 0.3 0.2 31 Guaiacylacetone LS 0.4 0.3 0.2 0.1 0.1 32 4-allyl-syringol LS 0.6 0.3 0.1 0.1 0.0 33 Propiovanillone LG 0.1 0.1 0.1 0.1 0.0 34 cis-4-propenylsyringol LS 0.4 0.3 0.1 0.1 0.0 35 trans-4-propenylsyringol LS 2.0 1.5 0.4 0.3 0.1 36 Levoglucosane C 10.0 15.6 21.6 25.1 27.3 37 syringaldehyde LS 0.9 0.5 0.4 0.2 0.1 38 syringic acid methyl ester LS 0.1 0.2 0.2 0.1 0.1 39 acetosyringone LS 0.6 0.6 0.4 0.2 0.1 40 syringylacetone LS 0.3 0.3 0.2 0.1 0.0 41 propiosyringone LS 0.1 0.1 0.0 0.0 0.0 % Lignin 40.4 27.9 18.1 12.6 7.1 van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 8 of 14 Table 2 Identities and relative abundance (mean average of three replicates) of the compounds released upon pyrolysis GC/MS of autoclaved wheat straw before and after treatment with L. edodes for 2, 4, 8 and 12 wk (Continued) % Carbohydrates 59.6 72.1 81.9 87.4 92.9 Lignin/Carbohydrate ratio 0.7 0.4 0.2 0.1 0.1 H 10 743 2 G1812974 S 12 853 1 Syringyl/Guaiacyl ratio 0.7 0.7 0.5 0.4 0.4 (Syringyl/Guaiacyl) ratio 1.0 1.0 0.7 0.5 0.4 except vinyl Ph-C0-2/Ph-C3 5.7 6.0 8.2 9.5 10.9 %Cα-oxidized lignin 10.2 11.5 15.2 15.2 16.4 %Cα-oxidized G-units 5.9 6.5 9.4 10.4 11.3 %Cα-oxidized S-units 4.4 4.9 5.9 4.8 5.1 C, carbohydrate-derived compounds; LH, p-hydroxycinnamyl lignin-derived compounds; LG, guaiacyl-lignin derived compounds; S, syringyl-lignin derived compounds; PCA, p-coumarates; FA: ferulates. All G- and S-derived peaks were used for the estimation of the S/G ratio, except 4-vinylguaiacol (which also arises from ferulates), and the analogous 4-vinylsyringol. Ratio of lignin-derived phenols with none, 1 and 2 carbons in the side-chain to lignin-derived phenols with 3 carbons in the side-chain ferulates on arabinoxylans and, therefore, the lignin S/G In addition, some Cα-oxidized phenolic compounds ratio may be underestimated . Hence, a more accurate such as aromatic aldehydes, acids and ketones were S/G ratio of the lignin in wheat straw could be obtained by found during pyrolysis of wheat straw and wood chip ignoring 4-vinylguaiacol (compound 17) (and the analogous samples treated with L. edodes (Tables 2 and 3). 4-vinylsyringol, compound 30). This S/G ratio estimated a Among them, the relative abundance of lignin-derived value of 1.0 in the untreated wheat straw and shows a compounds oxidized at the α-carbon, clearly increase continuous decrease until a value of 0.4 after 12 wk of after fungal treatment of wheat straw and wood chips incubation (Table 2). Likewise, in the case of wood chips, with L. edodes (Tables 2 and 3). In the case of wheat a decrease of the lignin S/G ratio was also observed dur- straw treated with L. edodes, the percentage of the ing fungal incubation time. The S/G ratio estimated by Cα-oxidized compounds increases continuously during Py–GC/MS for the untreated wood chips sample was fungal incubation from 10.2 % in the control sample 0.9, and decreased from 4 wk on steadily during incuba- (0 wk incubation) up to 16.4 % in the wheat straw tion down to a value of 0.6 in the wood chips incubated sample after 12 wk of fungal incubation. Similarly, in for 12 wk (Table 3). The S/G ratio in wood chips was the case of the wood chips treated with L. edodes,the not corrected for 4-vinylguaiacol and 4-vinylsyringol, percentage of Cα-oxidized compounds also increases since ferulates are abundant in grasses, but not import- during fungal incubation from 12.3 % in the control ant in wood . sample (0 wk incubation) up to 19.2 % after 12 wk of The Ph-C0-2/Ph-C3 ratio represents the ratio between fungal degradation. Interestingly, more Cα-oxidized lignin units with short (C0-C2) side chains (Ph-C0-2) lignin compounds were found originating from G- and intact C3 side-chains (Ph-C3). The Ph-C0-2/Ph-C3 lignin units (compounds 26, 28, 29 and 33) than from ratio in wheat straw increased from 5.7 in the untreated S-lignin units (compounds 37, 38, 39 and 41) in both sample up to 10.9 in the 12-wk treated sample. In wood wheat straw and wood chips, as reflected in Fig. 4. chips the Ph-C0-2/Ph-C3 ratio increased from 2.3 in the untreated sample up to 3.6 in the 12-wk treated sample. Correlations between IVGP and composition of the As seen in Fig. 3, Ph-C3 compounds originating from both substrates S- and G-units decreased at a similar rate while Ph-C0-2 IVGP showed a linear relationship to changes in cell wall compounds originating from S-units (compounds 20, 25, composition (ADL to carbohydrate (hemicellulose + cel- 27, 30, 37, 38 and 39) are degraded faster than Ph-C0-2 lulose) ratio) as determined by the detergent fiber ana- compounds originating from G-units (compounds 9, 12, lysis (Fig. 5). This relation was similar for both wheat 13, 16, 26, 28 and 29). As a result, more Ph-C0-2 com- straw and wood chips. pounds originating from G-units than from S-units were The increase in IVGP of fungal treated wheat found in both the fungal degraded wheat straw and wood straw was negatively correlated with the L/C ratio chips, in accordance with the fact that L. edodes degraded (r = −0.69, P < 0.01) determined by Py-GC/MS (Fig. 6). more S-units than G-units. S/G ratio was also negatively correlated with IVGP van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 9 of 14 Table 3 Identities and relative abundance (mean average of three replicates) of the compounds released upon pyrolysis GC/MS of autoclaved wood chips before and after treatment with L. edodes for 2, 4, 8 and 12 wk Label Compound Origin Time, wk 0 24812 1(2H)-furan-3-one C 2.7 3.2 3.4 3.9 4.0 2 Propanal C 2.5 2.1 2.2 2.2 2.2 3 Furfural C 6.1 8.3 8.6 8.8 9.2 4 2,3-dihydro-5-methylfuran-2-one C 4.0 3.4 3.5 4.1 3.8 5(5H)-furan-2-one C 2.6 2.7 2.5 2.7 2.4 6 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one C 5.5 7.8 7.3 7.1 7.4 7 2-hydroxy-3-methyl-2-cyclopenten-1-one C 4.3 3.3 3.7 4.3 3.9 8 Phenol LH 1.8 1.8 1.8 1.6 1.5 9 Guaiacol LG 4.2 3.9 4.3 4.5 4.9 10 3-hydroxy-2-methyl-(4H)-pyran-4-one C 1.3 1.0 1.1 1.4 1.4 11 4-hydroxymethyl-1,4-butyrolactone C 1.1 1.0 1.0 0.7 0.8 12 4-methylguaiacol LG 4.9 3.6 3.3 2.3 2.3 13 4-ethylguaiacol LG 0.8 0.4 0.4 0.4 0.2 14 5-hydroxymethyl-2-tetrahydrofuraldehyde-3-one C 1.7 1.8 2.1 2.6 2.9 15 1,4-anhydroarabinofuranose C 0.9 0.8 0.7 0.8 0.7 16 4-vinylphenol LH 1.0 1.0 0.7 0.7 0.6 17 4-vinylguaiacol LG 5.8 4.9 4.4 3.2 2.5 18 Eugenol LG 1.3 0.9 0.8 0.6 0.5 19 5-hydroxymethyl-2-furfuraldehyde C 1.6 2.5 2.4 2.7 3.0 20 Syringol LS 3.8 3.3 2.8 2.4 1.9 21 cis-isoeugenol LG 0.9 0.6 0.5 0.3 0.2 22 1,4-dideoxy-D-glycerohex-1-enopyranos-3-ulose C 0.4 1.3 1.3 1.3 1.6 23 trans-isoeugenol LG 4.4 2.7 2.3 1.6 1.3 24 1,4-anhydroxylofuranose C 0.8 1.2 1.1 1.3 1.4 25 4-methylsyringol LS 3.2 2.6 1.9 1.4 1.0 26 Vanillin LG 1.9 2.0 1.7 1.4 1.5 27 4-ethylsyringol LS 0.6 0.4 0.3 0.2 0.1 28 vanillic acid methyl ester LG 0.3 0.5 0.6 0.7 0.8 29 acetovanillone LG 1.0 1.0 1.0 0.9 0.9 30 4-vinylsyringol LS 4.4 3.5 2.4 1.8 1.3 31 guaiacylacetone LS 0.7 0.6 0.6 0.4 0.4 32 4-allyl-syringol LS 1.3 0.9 0.6 0.5 0.3 33 propiovanillone LG 0.2 0.2 0.6 0.5 0.3 34 cis-4-propenylsyringol LS 0.8 0.7 0.5 0.3 0.6 35 trans-4-propenylsyringol LS 5.2 3.6 2.5 1.9 1.6 36 Levoglucosane C 12.6 17.0 22.2 26.3 28.5 37 syringaldehyde LS 1.8 1.7 1.1 0.8 0.7 38 syringic acid methyl ester LS 0.1 0.2 0.2 0.3 0.3 39 acetosyringone LS 0.9 0.9 0.7 0.6 0.5 40 syringylacetone LS 0.6 0.7 0.6 0.5 0.4 41 propiosyringone LS 0.3 0.2 0.2 0.1 0.1 % Lignin 52.1 42.7 36.6 29.9 26.9 van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 10 of 14 Table 3 Identities and relative abundance (mean average of three replicates) of the compounds released upon pyrolysis GC/MS of autoclaved wood chips before and after treatment with L. edodes for 2, 4, 8 and 12 wk (Continued) % Carbohydrates 47.9 57.3 63.4 70.1 73.1 Lignin/Carbohydrate ratio 1.1 0.7 0.6 0.4 0.4 H 3 3322 G 26 21 20 17 16 S 23 19 14 11 9 Syringyl/Guaiacyl ratio 0.9 0.9 0.7 0.7 0.6 Ph-C0-2/Ph-C3 2.3 2.8 3.1 3.5 3.6 %Cα-oxidized lignin 12.3 15.7 16.5 17.4 19.2 %Cα-oxidized G-units 6.4 8.5 10.7 11.3 13.0 %Cα-oxidized S-units 5.9 7.2 5.8 6.1 6.2 C, carbohydrate-derived compounds; LH, p-hydroxycinnamyl lignin-derived compounds; LG, guaiacyl-lignin derived compounds; S, syringyl-lignin derived compounds. Ratio of lignin-derived phenols with none, 1 and 2 carbons in the side-chain to lignin-derived phenols with 3 carbons in the side-chain (r = −0.72, P < 0.01), while a positive correlation was temperature for L. edodes may be higher, meaning that a found between IVGP and the percentage of Cα- faster colonization and delignification could be achieved. oxidized lignin compounds (r = 0.77, P < 0.01) and the To make sure only L. edodes was present in the culture, Ph-C0-2/Ph-C3 ratio (r =0.51, P = 0.05) determined the substrates were sterilized before inoculation. In this by Py-GC/MS (Fig. 6). Similar to wheat straw, IVGP study, a 2 h cycle was used to eliminate the spores. of fungal treated wood chips was also negatively cor- However, autoclaving for 2 h may compare to a severe related to the L/C ratio (r = −0.88, P <0.01) and S/G thermal treatment, which may result in changes in the ratio (r = −0.75, P < 0.01) (Fig. 6), while a positive cell wall. For this reason, a less severe sterilization step correlation between IVGP of wood chips and Ph-C0- before fungal inoculation should be used in future stud- 2/Ph-C3 ratio (r =0.77, P < 0.01) and %Cα-oxidized ies. Also, sterilization causes a loss in moisture. The lignin (r = 0.62, P = 0.01) was found (Fig. 6). moisture content in this study was controlled by incuba- tion in a room with 70 % relative humidity. Because of Discussion the small size of the containers (1.2 L), which are cov- Fungal incubations using L. edodes were used as a pre- ered with a lid, it is expected that moisture loss upon in- treatment for wheat straw and wood chips for their sub- cubation is minimal. Regression analysis showed that sequent use as ruminant feed. The fungal incubations changes in ADL content influence changes in IVGP were done at 24 °C to make it comparable to earlier most. This is in line with a previous paper  that found studies. However, it should be noted that the optimal that ADL content and IVGP were negatively correlated. Fig. 3 Relative amounts of Ph-C3 and Ph-C0-2 compounds originating from S- and G-units present in wheat straw and wood chips during Lentinula edodes treatment. □ Ph-C0-2 compounds originating from S-units ■ Ph-C3 compounds originating from S-units Δ Ph-C0-2 compounds originating from G-units ▲ Ph-C3 compounds originating from G-units van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 11 of 14 Fig. 4 Percentage Cα-oxidized lignin units originating from S- and G-units present in wheat straw and wood chips during Lentinula edodes treatment. ▲total Cα-oxidized phenols ♦ Cα-oxidized products originating from S-units ■ Cα-oxidized products originating from G-units ADL represents the non-degradable fraction that is de- results confirm the preferential lignin degradation fined as lignin . However, it does not include acid found by L. edodes in beech and wheat straw [4, 17]. soluble lignin, and due to a filtration step, it might not The higher lignin degradation found in wheat straw include smaller fragments of lignin (such as degradation compared to wood chips is likely due to the higher products) [12, 13]. The detergent fiber analysis method lignin content in wood (L/C ratio of 0.3) compared to used in the current study used filter bags with pore size wheat straw (L/C ratio of 0.1) and possibly to phys- of 25 μm, meaning that smaller, unbound compounds ical differences in the two types of substrate (such as will be lost during analysis. Also, fungal biomass might density of tissues and the surface to content ratio). It be analyzed as ADL, since Jurak et al.  also observed must be noted, however, that the L/C estimated by a Klason lignin fraction in button mushrooms. To deter- Py-GC/MS ratios do not reflect the real content of mine the effect of lignin composition, without con- each moiety since pyrolysis is known to be more sen- tamination of the lignin fraction with fungal biomass, sitive for lignin as the cellulose is significantly under- lignin was studied in more detail using Py-GC/MS. A estimated due to intense charring and extensive preferential lignin degradation occurred in both sub- degradation to non-chromatographed products . stratesproducing a residue enriched in cellulose, However, the L/C ratios observed upon pyrolysis can which was more pronounced in wheat straw. These still be used for comparison of the relative amounts of individual moieties of lignocellulose in the analyzed samples and visualize the direction in which com- pound concentrations changes. For future studies it is advised to include a carbohydrate analysis to study the fate of these compounds more in detail. The lig- nin degradation is confirmed by the increasing occur- rence of lignin compounds with shorter side chains (Ph-C0-2) and the occurrence of Cα-oxidized lignin compounds. Interestingly, Ph-C0-2 compounds were also detected in the untreated, autoclaved control. These compounds are generated during the pyrolysis of condensed lignin structures. Since G-units form more condensed structures, more Ph-C0-2 com- pounds originating from G-lignin were found (Fig. 3). Fig. 5 Relation between in vitro gas production (IVGP) and the The Py-GC/MS data showed a change in lignin com- lignin to carbohydrates ratio (lignin/hemicellulose + cellulose) as position by L. edodes. Two different phases in lignin determined by the detergent fiber analysis. □ wheat straw ■ wood composition changes by L. edodes can be defined. The chips, dashed line: potential maximum IVGP first phase is characterized by radical attack of lignin, van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 12 of 14 Fig. 6 Correlations between in vitro gas production (IVGP) and lignin/carbohydrate ratio, S/G ratio, Ph-C0-2/Ph-C3 ratio and %Cα-oxidized lignin. □ wheat straw ■ wood chips since lignin degrading enzymes cannot enter the intact they have a higher predominance of β-O-4 ether linkages cell walls during the initial phase of fungal delignifica- compared to G-units that are more recalcitrant to fungal tion . These radicals are likely less specific resulting attack due to the formation of condensed linkages . in a simultaneous degradation of S- and G-units during However, it has recently been indicated that S-rich the first 2 to 4 wk of L. edodes treatment. On the other transgenic poplar woods exhibited improved resistance hand, lignin side-chain oxidation does occur at Cα under to fungal degradation , which suggests that besides the influence of both the radicals and direct enzymatic composition of lignin, other features such as the 3D- degradation , resulting in an increased percentage of structure of cell walls is important in degradation. To Cα-oxidized lignin units released upon Py-GC/MS confirm this, future studies should include additional within the first 2 wk of L. edodes treatment as was also 2D-NMR analysis to obtain information about linkanges shown for other fungal treated lignocellulosic samples within the cell wall. [16, 18]. During the second phase, the final stages of the Here, the S/G ratio is correlated to IVGP (Fig. 6), sug- fungal treatment, lignin degradation shifts from radical gesting that lignin composition has an influence on degradation toward enzymatic degradation . With rumen degradability. Changes in S/G ratio of wood chips enzymatic degradation preferential S- over G-unit have a larger influence on rumen degradability than degradation starts after 4 wk of L. edodes treatment. changes in S/G ratio of wheat straw. A similar trend for In wood chips, this preferential S-unit degradation is Ph-C0-2/Ph-C3 ratio was found in wood chips and accompanied by a significant increase in IVGP. In the wheat straw (Fig. 6). In the current study, with the deg- calculation of the S/G ratio for wheat straw, 4- radation of lignin, also its structure, composition and vinylguaiacol (compound 17) and 4-vinylsyringol (com- content are changing. The relatively large effect of pound 30) were excluded. The presence of these com- changes in S/G ratio and Ph-C0-2/Ph-C3 ratio are re- pounds is mostly due to the presence of p-coumarates and lated to the high lignin content of wood chips. Small ferulates, which decarboxylate efficiently under pyrolytic changes in lignin will have a relatively large effect on ac- conditions producing these vinyl compounds, as cessibility of rumen microbes. However, there is a high previously shown . The decrease of 4-vinylguaiacol linear correlation between the L/Cratio and IVGP (compound 17) in wheat straw suggests a degradation of (Fig. 5), where both substrates with different lignin com- ferulates or the arabinoxylans to which ferulates are position show an identical trend. This indicates that lig- bound. This result is in accordance with the hemicel- nin content, which was different between wheat straw lulose degradation found by the detergent fiber and wood chips, is more important than lignin compos- method. The increase in the relative amounts of ition, which does not have any effect on IVGP. The lat- partly degraded G-units at a later stage of fungal deg- ter is in line with previous works that reported no effect radation might indicate that S-units are degraded fur- of lignin composition on the degradation of polysaccha- ther and are not detected anymore, which is in rides in maize cell walls by enzymes of Trichoderma ree- accordance with the preferential S-unit degradation sei and Aspergillus niger or rumen microbes . It is by L. edodes. important to note that lignin degradation started during The higher biodegradability of the S-lignin compared the first 2 to 4 wk, while both S/G ratio and IVGP did to the G-lignin has already been shown in several other not change. This suggests that lignin structure, i.e., the studies showing the decrease of the S/G ratio during binding to carbohydrates, rather than lignin composition degradation of other lignocellulosic substrates by differ- is important for rumen degradability. Access of rumen ent white-rot fungi [9, 16–18, 26, 27]. S-lignin units are microbes to the fermentable carbohydrates seems to be more prone to enzymatic fungal degradation because physically blocked by the presence of bound lignin in van Kuijk et al. Journal of Animal Science and Biotechnology (2016) 7:55 Page 13 of 14 the cell wall matrix. This is demonstrated by the theoret- was co-sponsored by Agrifirm, Purac, DSM, Den Ouden, Hofmans, the Dutch commodity boards for dairy and horticulture, and Wageningen University. We ical potential maximum IVGP if all ADL would be re- would like to thank Agrifirm for practical assistance on analysis for dry matter, moved (dashed line in Fig. 5). The maximum IVGP will ash, crude protein, NDF and ADF. This study has also been partially funded by be approximately 350 ml/g OM, since removal of total the Spanish projects AGL2011-25379, AGL2014-53730-R and CTQ2014-60764-JIN (co-financed by FEDER funds), the CSIC project 2014-40E-097 and the ADL will result in pure carbohydrates. The maximum EU-project INDOX (KBBE-2013-7-613549). IVGP decreases slightly, since ADL is diluting the carbo- hydrates, i.e., at a higher ADL/(hemicelluloses + cellu- Funding lose) ratio a lower amount of carbohydrates are present This study has been funded by Dutch Technology Foundation (STW), which is part of the Netherlands Organization for Scientific Research (NWO). STW (Fig. 6). This suggests that both the content of lignin did approve the experimental set up and final manuscript made by the and composition of lignin (S/G ratio) are not influencing authors. This study has also been partially funded by the Spanish projects IVGP during the first 2 to 4 wk. Probably the linkages AGL2011-25379, AGL2014-53730-R and CTQ2014-60764-JIN (co-financed by FEDER funds), the CSIC project 2014-40E-097 and the EU-project INDOX between lignin and carbohydrates  and the 3D- (KBBE-2013-7-613549). These parties have funded the CSIC institute and thus structure of lignin block the rumen microbes. If this is the analysis using Py-GC/MS. true, removal of the linkages between lignin and carbo- hydrates would result in a theoretical IVGP of approxi- Availability of data and materials mately 350 ml/g OM (dashed line in Fig. 5). This value The dataset supporting the conclusions of this article (are) available upon request at the secretariat of the Animal Nutrition Group of Wageningen would decrease with an increasing lignin/carbohydrate University or via the authors of this manuscript. ratio, because of the diluting effect of lignin. This theor- etical IVGP is only true under the assumption that lignin Authors’ contributions is not toxic for rumen microbes. SvK carried out the fungal treatment and fiber analysis and drafted the manuscript. JCdR, JR, AG performed the Py-GC/MS analysis and were involved Diverse and complex products are generated during in data interpretation and manuscript preparation. AS, JB, WH and JC were degradation of lignin by L. edodes. Phenolic compounds involved in the study design, data interpretation and manuscript preparation. originating from lignin such as cinnamic acid and vanil- All authors read and approved the final manuscript. lin are described to inhibit cellulose degradation by rumen microbes , but it should be noted that in sci- Competing interests The authors declare that they have no competing interests. entific literature these compounds were added in pure form. In contrast, when these compounds are present in Author details the matrix of fungal treated biomass, they do not inhibit Animal Nutrition Group, Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands. Instituto de Recursos Naturales y IVGP as observed in the current study. Also, other stud- Agrobiologica de Sevilla (IRNAS-CSIC), Avenida Reina Mercedes, 10, 42012 ies describing fungal treatment of lignocellulose found Seville, Spain. 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