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957
Research Article
Received: 25 June 2018 Revised: 8 August 2018 Accepted article published: 20 August 2018 Published online in Wiley Online Library: 16 October 2018
(wileyonlinelibrary.com) DOI 10.1002/jsfa.9320
Improving ruminal digestibility of various
wheat straw types by white-rot fungi
Nazri Nayan,a* Gijs van Erven,bMirjam A Kabel,bAnton SM Sonnenberg,c
Wouter H Hendriksaand John W Conea
Abstract
BACKGROUND: This study investigated the ruminal degradability of various wheat straw types by the white-rot fungi Ceripori-
opsis subvermispora (CS) and Lentinula edodes (LE). Different cultivars (CV) of wheat straw at different maturity stages (MS) were
treated with the fungi for 7 weeks and assessed for chemical composition and in vitro gas production (IVGP).
RESULTS: Both fungi showed a more pronounced degradation of lignin on a more mature straw (MS3; 89.0%) in comparison
with the straw harvested at an earlier stage (MS1; 70.7%). Quantitative pyrolysis coupled to gas chromatography and mass
spectrometry, using 13C lignin as an internal standard 13C-IS Py-GC/MS revealed that lignin in more mature straw was degraded
and modified to a greater extent. In contrast, cellulose was less degraded in MS3, as compared to MS1 (8.3% versus 14.6%).
There was no effect of different MS on the IVGP of the fungus-treated straws. Among the different straw cultivars, the extent
of lignin degradation varied greatly (47% to 93.5%). This may explain the significant (P<0.001) effect of cultivar on the IVGP
of the fungal-treated straws. Regardless of the factors tested, both fungi were very capable of improving the IVGP of all straw
types by 15.3% to 47.6%, (as compared to untreated straw), with CS performing better than LE – on different MS (33.6% versus
20.4%) and CVs (43.2% versus 29.1%).
CONCLUSION: The extent of lignin degradation caused by fungal treatment was more pronounced on the more mature
and lignified straw, while variable results were obtained with different cultivars. Both fungi were capable of improving the IVGP
of various straw types.
© 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of
Chemical Industry.
Supporting information may be found in the online version of this article.
Keywords: white-rot fungi; wheat straw cultivar; wheat straw maturity; in vitro gas production; ruminant feed; lignocellulosic biomass
INTRODUCTION
The valorization of lignocellulosic biomass, such as wheat straw,
as an alternative feed ingredient is important for sustainable
animal production. To unlock the energy potential of lignocel-
lulose, various physicochemical methods have been used, such
as hydrothermal treatment, ammonia fiber expansion, acids,
and alkaline media.1–3 These methods are used to increase the
enzymatic accessibility of the structural carbohydrates by break-
ing the lignin barrier and thus improving the degradability of
the feedstock by ruminants. Although they are attractive for
industrial-scale applications due to time efficiency, their potential
adverse impact on the environment is a major concern.4The
use of biological pretreatments, particular fungi, has become a
preferred approach.5–7 Some strains of white-rot fungi possess
unique capabilities for degrading highly recalcitrant lignocel-
lulosic biomass, whereby lignin is being selectively degraded,
increasing the available carbohydrate contents of the biomass.
The effectiveness of a fungal pretreatment can be influenced
by many factors, such as fungal strain, substrate, and culture
conditions.6,8,9 Variability in substrate and culture conditions leads
to difficulties in comparing the effectiveness of fungi across dif-
ferent studies.9The quality of the straw, i.e. the ratio of lignin to
total carbohydrates, is not only an important factor influencing its
digestibility in the rumen10 but may also affect the effectiveness of
a fungal pretreatment.9Unfortunately, standardizing the straw for
an optimal result is difficult and impractical, considering dispari-
ties among the straw types and conditions, as well as the differ-
ences in post-harvest residue management.11 The present study
∗Correspondence to: N Nayan, Animal Nutrition Group, Wagenin-
gen University, P.O. Box 338, 6700 AH, Wageningen, the Netherlands.
E-mail: nazri.nayan@live.com
aAnimal Nutrition Group, Wageningen University & Research, Wageningen,The
Netherlands
bLaboratory of Food Chemistry, Wageningen University & Research, Wagenin-
gen, The Netherlands
cPlant Breeding, Wageningen University & Research, Wageningen, The Nether-
lands
© 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
958
www.soci.org N Nayan et al.
was conducted to assess the effect of straw quality on lignocellu-
lose degradation by fungi and the subsequent effect on the rumi-
nal degradability of the treated straw.
Straw quality is affected by many factors including maturity
stage, cultivar, and other factors related to management and the
environment.12,13 The maturity of the plant, in particular, correlates
with the extent of lignin deposition in the plant tissues.13 The total
fiber content increases as the plant matures (lower leaf-to-stem
ratio14) and the lignin, cellulose, and hemicellulose composition
also changes. As straw consists mainly of fiber (∼85%), changes in
the cell-wall composition with maturity may thus influence the lig-
nocellulose degradation by fungi. Another important factor is the
wheat straw genotype. In wheat breeding, nutrient-use efficiency
and disease resistance are among the selection parameters, in con-
trast to straw quality.12,15
Recently, high-potential strains of the white-rot fungi species
Ceriporiopsis subvermispora and Lentinula edodes have been
selected for the bioprocessing of wheat straw into ruminant
feed.16 To determine whether these high-potential strains could
perform well across different straw types, two independent trials
were carried out in the present study. These trials investigated the
effect of two main sources of straw variation, (i) different maturity
of the straw around harvest, and (ii) different straw cultivars, on
fungal growth and their persistency in improving the in vitro
degradability of the straw. The lignin degradation characteristics
of wheat straw with different maturities were assessed using
pyrolysis coupled to gas chromatography with mass spectrometry
(Py-GC/MS).17
MATERIALS AND METHODS
Effect of different maturity stages
Harvesting wheat straw with different maturities
Wheat straw (Triticum aestivum L.) of the same variety (Quintus)was
harvested during the summer of 2015 from the experimental field
UNIFARM of Wageningen University, Netherlands. Each harvest
covered a plot size of 8.5 ×1.5 m. Zadoks et al.18 codes were used
to characterize the maturity stages of the wheat plant. The first
harvest was carried out on July 14 (MS1, code 83 – soft dough
development, 37.3% dry weight), the second harvest on July 28
(MS2, code 87 – hard dough development, 41.2% dry weight) and
the third harvest on August 11 (MS3, code 91 – ripening,64.6% dr y
weight). The wheat plants were cut at 10 cm above the ground and
the fresh plants were dried on a drying panel for a week (∼60 ∘C).
Prior to chopping, the spike containing the wheat grains was
removed, leaving the straw (leaves and stalk) intact. The trimmed
straw was chopped into approximately 3 cm pieces.
Fungal pretreatment of the wheat straw
Cultures of the white-rot fungi, (from the collection of Plant Breed-
ing, Wageningen University, the Netherlands) C. subvermispora (CS;
CBS 347.63; origin: USA) and L. edodes (LE; sh 03/08; origin: Japan),
were maintained on malt agar extract containing 20.0 g L−1of
malt extract, 0.5 g L−1of KH2PO4,0.5gL
−1of MgSO4.7H2O, and
0.5 g L−1of Ca(NO3)2·4H2O, (all chemicals were purchased from
Sigma-Aldrich, St. Louis, MO) with pH5.4 at 24 ∘C. Once the fungi
fully colonized the agar surface, spawn was prepared by placing a
piece of agar culture (1.5 ×2.0 cm) into sterilized (121 ∘C, 20 min)
sorghum grains (Wageningen, the Netherlands). The spawn was
incubated at 24 ∘C for 4 or 5 weeks. The chopped straw was soaked
in water for 3 days at room temperature and excess water was
drained off for 5 h. Adjustments were made based on the final
moisture content of the straw – which ranged from 77 (MS1) to
80% (MS3) for each container (172 ×110 ×70 mm; Combiness,
Nevele, Belgium) to contain 41.7 ±0.3g of dry matter. After auto-
claving at 121 ∘C for 1 h, the straw was aseptically inoculated with
the previously prepared spawn at 10% of the dry weight. Con-
trol (untreated) and fungal-treated wheat straw were incubated in
triplicate under solid-state fermentation at 24 ∘C for 7 weeks in a
climate-controlled chamber (Wageningen University, the Nether-
lands). All weekly samples were freeze dried and ground over a
1 mm sieve using a cross beater mill (100AN: Peppink, Olst, Nether-
lands).
Chemical analysis
Each sample was analyzed for dry matter (DM; ISO 6496, 1999)
and ash (ISO 5984, 2002) content. Crude protein was calculated
by multiplying the nitrogen content (ISO 5983, 2005) by 6.25. The
Van Soest et al.19 method was used to determine the cell-wall com-
position. Neutral detergent fiber (NDF) was determined using a
heat-stable amylase (thermamyl) and alcalase; acid detergent fiber
(ADF) and acid detergent lignin (ADL) were determined by boiling
the sample in an acid detergent solution, and the latter was further
treated with 72% v/v H2SO4. All fiber contents were corrected for
ash. Hemicellulose was calculated as the difference between NDF
and ADF, while cellulose was calculated as the difference between
ADF and ADL. Reducing sugars in the ethanol-extract of the straw
were determined by measuring the oxidation reaction between
the hydrolyzed monosaccharides with copper (II) and neocuproine
at 460 nm. Absolute amounts (g) of each component were calcu-
lated from the remaining amount (g) of the freeze-dried samples.
Ergosterol estimation
Fungal biomass was estimated by determining the content
of ergosterol. Details of the procedure have previously been
described.5,20 Samples (∼200 mg) were saponified with 10% (1:9)
KOH/methanol solution at 80 ∘C for 60 min. After cooling, the
ergosterol was extracted from the samples through a series of
mixing with 1 mL of water and 2mL of hexane. The hexane layers
from the same sample were collected by centrifuging and pooled
into a tube. The pooled hexane layer was dried in a vacuum evap-
oration system (Rapidvap, Kansas, MO, USA) before re-dissolving
in methanol. The solution was filtered into a high-performance
liquid chromatography (HPLC) vial for Waters HPLC-PDA analysis
(Alliance HPLC system, Milford, USA). Cholecalciferol (vitamin
D3) was used as an internal standard. The ergosterol peak was
detected at 280 nm.
In vitro gas production (IVGP)
In vitro gas production (IVGP) was used to assess the ruminal
degradability of the wheat straw. The gas production experiment
was performed according to the procedures described by Cone
et al.21 In brief, rumen fluid was collected from two non-lactating
cows that were fed 1kg concentrate and grass silage ad libitum.
The rumen fluid was filtered through a cheesecloth and mixed
(1:2 v/v) with phosphate-bicarbonate buffer solution, which also
contained trace elements, hydrolyzed casein, redox indicator and
reducing agent.21 The samples (0.5 g) were incubated in 60 mL
of buffered rumen fluid for 72h and the gas production was
automatically registered. The gas-production data were fitted to
a biphasic model22 to determine the kinetic parameters (An,Bn,
Cn,tRmn,Rmn), where nis the phase number (1 or 2). Anis the
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asymptotic gas production (mL g−1OM) of phase n;Bnis the half
time of the maximum gas production (h); Cnis a parameter to
determine the steepness of the curve; tRmnis the time of the
maximum fractional rate of substrate degradation (h), and Rmnis
the maximum fractional rate of substrate degradation (h−1).
Quantitative pyrolysis GC/MS with 13C lignin as internal standard
Prior to Py-GC/MS, ground wheat straw (1 mm) was ball-milled in a
MM200 mixer mill (Retsch, Haan, Germany) and biological tripli-
cates were mixed to one replicate. Pyrolysis of the ball-milled sam-
ple was carried out as previously described in detail by Van Erven
et al.17 Briefly, 10 μLofa13 C lignin internal standard (IS) solution
(1 mg mL−1)wasmixedwith∼80 μg of sample and dried before
analysis. Lignin-derived pyrolysis products were monitored in full
mass spectrometry mode on the two most abundant fragments
per compound (both 12Cand13C). The area for each compound
was normalized by dividing by the respective relative response fac-
tor (RRF). Relative response factor values were updated to system
performance by recalculation to obtain an identical relative abun-
dance of lignin-derived pyrolysis products of the 13CISaddedtoa
wheat straw reference sample. Lignin content (% w/w) was deter-
mined as the sum of lignin-derived pyrolysis products, where RRF
corrected areas for each compound were multiplied by the molec-
ular weight of the respective compound and summed instead of
the application of the published correction factor of 1.057.17 Rel-
ative abundances of lignin-derived pyrolysis products were based
on areas normalized for the 13C analogues from the IS present in
the same sample to distinguish matrix and treatment effects. Areas
were not corrected for molecular weight before relative abun-
dance determination as previously described by Del Río et al.23 as
RRF values are mole based. Compounds were classified according
to their structural features (Table S1, File S1 in the supplementary
material) and summed. All samples were prepared and analyzed in
triplicate.
Effect of different wheat cultivars
An independent experiment assessing the effects of different
straw cultivars on the fungal pretreatment was carried out. Wheat
straw from five winter wheat cultivars – Britannia (CV1), Cellule
(CV2), Henrik (CV3), Residence (CV4) and Tabasco (CV5), were pur-
chased from Limagrain (Rilland, Netherlands). Any remaining spike
from the straw was removed prior to chopping. The wheat straw
was chopped at 3 cm length. The chopped straw underwent
the same processing and treatment procedures as previously
described with the same fungal strains used as mentioned above.
Solid state fermentation of the inoculated wheat straw was carried
out at 24 ∘C for 7 weeks in a climate controlled room with mini-
mum exposure to light. The weekly samples were weighed, freeze
dried, and ground over a 1mm sieve for further analysis. All sam-
ples were subjected to the same chemical and ergosterol analyses
as well as to the IVGP procedure described above. No Py-GC/MS
was conducted on these samples.
Statistics
Data from both experiments were independently analyzed
by analysis of variance using the generalized linear model (GLM)
in SAS 9.3, followed by post-hoc multiple group comparisons
using least significance differences. The statistical model for the
ergosterol and IVGP data included the effect of levels in each
factor (maturity stage 1 to 3; or cultivar 1 to 5), treatment (control,
C. subvermispora and L. edodes), week, and the interaction effects
of all three terms. For chemical analyses, the end point data were
analyzed by excluding week and its interaction terms from the pre-
vious model. Analysis of pyrolysis data of different maturity stages
reflected analytical rather than biological variances as three bio-
logical replicates of the same treatment (with an equal amount)
were mixed prior to the analysis. The minimum significance
threshold level was set at P<0.05. Pearson’s product – moment
correlation (r) coefficients were also determined among the
measured variables.
RESULTS AND DISCUSSION
Effect of different maturity stages
Mass balances for the wheat straw at different maturities
The dry matter, ash content, and the mass balances of different
wheat straw maturities treated with CS and LE for 7 weeks are
reported in Table 1. The water-holding capacity of the straw was
increased with straw maturity, as is evident by a 7% lower DM
content of MS3 straw as compared to MS1 and MS2 (Table 1).
However, the total amounts of OM were not different for the
untreated straw at different maturities. To allow comparison across
different maturity stages, the amounts of the quantified nutrients
were expressed per 100 g of starting OM for the respective stages.
For the untreated straws, higher amounts of ADL (12.2%) and
cellulose (6%) were seen with increasing maturity. The untreated
MS3 straw contained a significantly (P<0.01) higher amount
of ADL compared to the rest. Hemicellulose was significantly
(P<0.001) lower in the MS3 straw compared to its counterparts,
whereas high (P<0.01) amounts of free sugars and crude protein
were observed for the MS1 straw.
There were significant (P<0.001) losses of OM after 7 weeks of
fungal pretreatment (17.5% to 25.3%), with high losses observed
for fungal-treated MS1 straw. Lentinula edodes resulted in a signif-
icantly (P<0.01) higher loss of OM than CS at any maturity stage.
Overall, both fungi significantly (P<0.001) degraded all cell-wall
components and increased the amount of soluble sugars. A lesser
amount of cellulose was degraded when both fungi grew on MS3
as compared to MS1 (∼8.3% versus 14.6%, respectively). Interest-
ingly, lignin degradation by both fungi was more pronounced on
mature straw. The delignification by CS, for instance, was simi-
lar when grown on MS1 and MS2 straw (∼82%), whereas on MS3
straw, CS degraded 98.2% of the total amount of ADL. The figures
for lignin degradation presented here were higher than those in
previous reports (33% to 60%), which were carried out under sim-
ilar treatment conditions,5–7 but at an unknown stage of maturity.
Meanwhile, a similar trend was also observed for the hemicellulose
with a higher percentage of degradation (66.2% to 79.3%) for both
fungi. Ceriporiopsis subvermispora consistently resulted in higher
level of degradation of ADL and hemicellulose than LE for all straw
maturities. In addition, both fungi also increased (P<0.001) the
amount of free sugars in the treated straw, which may have arisen
from the breakdown of the cell-wall polysaccharides. The appar-
ent susceptibility of a more mature straw to fungal delignification
is of particular interest here. Toi nvestigate the delignification char-
acteristics of both fungi, the samples from the control (week 0)
and after 7 weeks of fungal pretreatment were subjected to quan-
titative pyrolysis coupled to gas chromatography and mass spec-
trometry, using 13C lignin as an internal standard 13C-IS Py-GC/MS
analysis.Therewasanaveragevariationof8%intheADLmeasure-
ment of biological replicates of the same treatment. Hence, prior
to Py-GC/MS, the biological replicates were thoroughly mixed to
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960
www.soci.org N Nayan et al.
Table 1. Dry matter, ash content and mass balances for wheat straw of different maturity stages, treated with C. subvermispora (CS) and L. edodes
(LE) for 7 weeks
Amount (g per 100 g of starting OM)b
Maturity stageaTreatment DM(g kg−1)Ash(gkg
−1DM) OM Cell Hcell ADL Sugar CP NI
MS1 Control 184.3a18.9d100.0a47.7b34.4a7.4c0.7d3.3b6.6d
CS 158.4c24.7ab 78.7c40.9de 8.3e1.4f1.8bc 3.5a22.7a
LE 156.9c23.6b74.7d40.5e11.6c2.9d1.8bc 3.2bc 14.5c
MS2 Control 183.3a20.7c100.0a49.6a34.1a7.8b0.5e2.5e5.5d
CS 154.5cd 25.5ab 82.5b42.3d8.7e1.4f2.1a3.1c24.8a
LE 150.7d24.3ab 79.0c44.4c10.8cd 2.2e1.9b2.8d16.9b
MS3 Control 171.3b19.9cd 100.0a50.7a30.8b8.4a0.5e2.6e7.0d
CS 150.9d25.8a82.2b46.2b6.4f0.2g2.1a2.9d24.5a
LE 145.3e25.3a78.5c46.8b9.9d1.7f1.7c2.8d15.7bc
RMSE 2.36 0.96 0.74 0.89 0.67 0.23 0.06 0.09 1.27
aMaturity stages around harvest (MS1: Zadoks code 83; MS2: code 87; MS3: code 91).
bCalculated from the remaining materials for each treatment (g), using respective starting OM (week 0) for each maturity stage.
Values with different superscripts within a column are significantly (P<0.05) different.
DM, dry matter; OM, organic matter; Cell, cellulose; Hcell, hemicellulose; ADL, acid detergent lignin; Sugar, free reducing sugar from ethanol-extract
of wheat straw; CP, crude protein (N ×6.25); NI, unaccounted OM; RMSE, root mean square error.
Table 2. Characterization of lignin and its structural moieties in different straw maturities (MS), treated with C. subvermispora (CS) and L. edodes (LE)
for 7 weeks, using quantitative 13 C-IS Py-GC/MS
MS1aMS2 MS3
Parameter Control CS LE Control CS LE Control CS LE
Lignin content (% w/w DM) 23.74.78.122.64.86.923.53.55.9
Amount of lignin (g per 100 g OM) 24.23.86.223.14.15.624.03.04.8
Lignin aromatic unit (%)b
H8.913.311.19.313.311.98.813.911.4
G62.661.363.362.559.362.961.259.362.4
S28.525.525.628.127.525.230.026.826.2
S/G 0.46 0.42 0.40 0.45 0.46 0.40 0.49 0.45 0.42
Structural moieties (%)
Unsubstituted 4.811.47.74.712.29.35.213.710.0
Methyl 2.03.72
.92.13.83.02.34.53.5
Ethyl 0.10.20.20.10.20.20.20.20.2
Vinyl 29.924.828.430.523.927.330.420.326.9
C𝛼-oxidized 3.220.18.43.421.310.23.226.110.6
C𝛽-oxidized 1.63.72.31.53.72.61.63.92.7
C𝛾-oxidized 57.046.852.056.246.851.155.747.849.8
Miscellaneous 1.91.92.11.91.81.82.01.72.0
aMS: maturity stages around harvest (MS1: Zadoks code 83; MS2: code 87; MS3: code 91).
bG: guaiacyl lignin subunit, H: p-hydroxyphenyl unit, S: syringyl unit.
Values are averages of three technical replicates. No statistics were carried out on the data.
allow analytical triplicates for detailed and more accurate lignin
analysis.
Quantitative 13C-ISPy-GC/MSofthewheatstraw
A total of 34 lignin-derived compounds were released on pyrolysis
and monitored (see supporting information, Table S1, File S1).
The lignin content, as quantified using 13C-IS Py-GC/MS, and
its structural features are summarized in Table 2. Overall, the
untreated straws showed comparable amounts of lignin (23.8g
per 100 g OM), in contrast with the ADL method. It is inferred that
mature straw may contain a high amount of recalcitrant resid-
ual lignin that was retained in the ADL fraction, which explains
a higher ADL in MS3 straw than its counterparts. Acid deter-
gent lignin is known to underestimate the total lignin content,
as it does not take into account the acid-soluble lignin.24 Both
methods, however, seem in agreement on the extent of delig-
nification by both fungi. Ceriporiopsis subvermispora showed a
higher delignification capability on MS3 straw (87.6%) than MS1
(84.4%) and MS2 (82.4%). Similar observations were also recorded
for LE-treated MS3 straw (80.1%), as compared to MS1 and
MS2 (∼75%).
To explain this observation, we assessed the lignin structural
features of all straws. The untreated MS3 straw contained 6%
more syringyl (S) unit compounds than the other straws. This also
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0
40
80
120
160
200
(A) (B)
01234567
Ergosterol (µg g-1 substrate)
Colonization week
0
100
200
300
400
500
600
01234567
Ergosterol (µg g-1 substrate)
Colonization week
Figure 1. Growth (based on ergosterol data) of C. subvermispora (A) and L. edodes (B) on the wheat straw har vested at different maturity stages – MS1 ( ),
MS2 ( )andMS3( ), for 7 weeks. Error bars indicate standard deviations.
resulted in a higher S to guaiacyl (G) ratio in MS3 (0.49) than MS1
and MS2 straws (∼0.45). Fungal pretreatment resulted in minor
changes in lignin subunit composition, with slight preference of
fungi towards attacking the S units, which is in agreement with
previous reports.25,26 The results also indicate that both fungi can
degrade all lignin subunits at the magnitudes of lignin removal.
The S-units result in more linear structures and have a lower redox
potential than the G- units,23,27 which made them more suscep-
tible to the fungal attack.25 Hence, the relatively high S/G ratio
may partly explain the higher fungal delignification of MS3 straw.
Nevertheless, the S/G ratio was poorly correlated with the total
amount of lignin (Pearson’s r=−0.17), due to a smaller change in
the S/G ratio. Fungal pretreatment resulted in reduced amounts
of vinyl products, mainly 4-vinylguaicol (supplementary material,
Table S1 in File S1). These compounds are also derived from fer-
ulic acids that link lignin to its associated carbohydrates.28 The
reduction of vinyl products was more pronounced on the more
mature straw, particularly for the CS treatment. Other notable
changes include the increase in unsubstituted and C𝛼-oxidized
compounds with increasing straw maturity, particularly with the
CS treatment, despite no difference being observed in the abun-
dances of these compounds in the untreated straws. These obser-
vations clearly indicate more extensive degradation of inter-unit
linkages within the lignin macromolecule of the more mature
straw, which resulted in residual lignin with lowintac t linkages.25,29
The ADL and Py-GC/MS assessments confirm the preference of
both fungi for degrading the lignin of more mature straw – both
by decreasing the amount of residual lignin and structural modifi-
cations.
Fungal growth
Successful colonization of the substrate is an important prereq-
uisite for effective fungal pretreatment.9We therefore assessed
the growth of both fungi during the 7 week colonization period
using an ergosterol assay (Fig. 1).20,30 The baseline ergosterol con-
tent at week 0 was significantly (P<0.001) lower in the untreated
MS1 straw and increased with increasing maturity. The higher
ergosterol content in MS3 straw may be due to its high water
potential (high osmotic gradient), which leads to high activity
and the growth of field fungi.31 Another possible explanation is
post-harvest fungal growth, which is a common occurrence.32
Although all processed straw was autoclaved prior to the fungal
pretreatment, the persistence of ergosterol in the sterilized straw
has been reported in several other studies.5,30 The differences in
the baseline ergosterol contents among the straw types produced
unique colonization characteristics of both fungi, especially dur-
ing the early growth stage. The growth of both fungi on MS3 at
the beginning of the colonization weeks appeared slower, com-
pared to their growth on MS1 and MS2. It is possible that existing
ergosterol (or other components) belonging to the endogenous
field fungi might have ‘masked’ the initial growth of CS and LE.
On MS3 straw, both fungi might have recycled these endogenous
compounds and incorporated them in their own biomass, erro-
neously indicating a slower growth or the inability of these fungi to
colonize the straw during the early weeks. The variation in the fun-
gal growth rate could be seen throughout the colonization period,
especially for CS, which showed a low persistence in growth. This
observation indicates a weaker colonization trait for this fungus on
different straw types, as compared to LE.
Assessing in vitro rumen degradability of the straw
The effectiveness of both fungi in improving the rumen degrad-
ability of the straw was assessed by the IVGP technique, which
was used as a decisive parameter in selecting the strains used
in the present study.16 Table 3 summarizes the IVGP and its kinetic
parameters for straw of different maturities, treated with the two
fungal species. The changes in IVGP have been explained well
by the changes in the cell-wall composition of the substrate.7,33
Although the control straws were noticeably different in their
cell-wall compositions, the IVGPs of all straw maturities were not
different. There was no effect of maturity stage on the IVGP of
the fungal-treated wheat straws. Ceriporiopsis subvermispora and
LE significantly (P<0.05) improved the IVGP of all straw types
by 33.6% and 20.4%, respectively, which was within the range of
previous reports.5,16 Ceriporiopsis subvermispora performed signif-
icantly (P<0.05) better than LE in improving the degradability of
the straw at all stages. The highest increase in the IVGP by both
fungal pretreatments was observed on MS1 straw with 38.4% and
23.3% increases for CS and LE, respectively.
The kinetic parameters were determined using a biphasic model
approach22 to differentiate the IVGP profile of all straws. This
approach has been used to differentiate the fermentability of
two fungal-treated wheat straws with similar total IVGP.5All
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Table 3. In vitro gas production and its kinetic parameters for wheat straw of different maturity stages, treated with C. subvermispora (CS) and L.
edodes (LE) for 7 weeks
Kinetic parameters
Maturity stageaTreatment IVGP A1A2B2C2tRm2 Rm2
MS1 Control 241.4a32.0a193.6a13.93c2.39a15.99bc 0.087a
CS 334.2de 61.4b262.8de 10.52a2.88cde 13.09a0.143c
LE 297.7bc 52.3b231.5b12.72b2.74b15.57b0.112b
MS2 Control 250.6a34.5a198.1a14.47c2.50a17.01d0.088a
CS 323.6cde 61.5b253.2cde 10.70a2.94de 13.39a0.145c
LE 307.0bcd 50.3b243.8bc 13.15b2.79bc 16.20bcd 0.111b
MS3 Control 254.5a34.5a202.3a14.36c2.48a16.84cd 0.088a
CS 339.5e60.6b269.8e10.48a3.00e13.20a0.152c
LE 293.4b37.3a245.2bcd 13.06b2.79bc 16.09bc 0.112b
RMSE 17.00 7.87 10.98 0.39 0.07 0.52 0.005
aMaturity stages around harvest (MS1: Zadoks code 83; MS2: code 87; MS3: code 91).
Values with different superscripts within a column are significantly (P<0.05) different.
RMSE, Root mean square error; IVGP, cumulative in vitro gas production af ter 72 h (mL g−1OM); A1,A2, asymptotic gas production (mL g−1OM) phase
1 and 2, respectively; B2, half time to the maximum gas production of phase 2 (h); C2, parameters determine the curvature of the graph; tRm2,timeof
the maximum fractional rate of substrate degradation (h); Rm2, maximum fractional rate of substrate degradation (h−1).
fungus-treated straws showed a better kinetic profile than the con-
trol – among others, a shorter half time to the asymptotic gas pro-
duction (B2) and a steeper curve (greater C2). There was no effect
of straw maturity on the kinetic parameters of the fungal-treated
straws, although a numerical increase of fractional fermentation
rate at phase 2 (Rm2) could be seen for CS-treated straws with
increasing maturity. This observation indicates that both fungi
were very capable of improving the degradability of these straws
to a similar extent.
Effect of different cultivars
Mass balances for wheat straw of different cultivars
Table 4 summarizes the mass balances of the different wheat straw
cultivars, treated and not treated with fungi. There was consid-
erable variation in the amount of nutrients among the differ-
ent straw cultivars, particularly in the DM and ash contents. The
absolute amounts of OM in the starting materials were com-
parable among the different untreated straw cultivars (ranging
from 36.3 to 37.6 g; Table 4). As in the previous experiment, the
amount of nutrients was expressed per 100 g of starting OM. The
starting amounts of all cell-wall components were comparable
among the different untreated straw cultivars, with CV5 high in
lignin. Besides showing a high ash content, the untreated CV2 and
CV3 straws contained lower amounts of CP than the other straw
cultivars.
There were significant (P<0.05) losses of OM in all
fungal-treated straws, with a relatively higher loss observed
for CV1 straw (∼12.2%). The fungal degradation of lignin and
hemicellulose for most straw cultivars was in the range of previous
reports.5–7 The fungal-treated CV1 straw, however, showed high
losses of lignin (∼86.5%) and hemicellulose (∼67.0%). Previously,
CS had been shown to be superior over LE in its delignification
capability.7,16 In the present study, the delignification capability
of CS was surprisingly comparable to LE for most straw culti-
vars (56.2% versus 53.3%). Ceriporiopsis subvermispora degraded
a significantly (P<0.01) higher amount of lignin in CV1 than
LE (93.5% versus 79.6%). Significant losses of cellulose were
observed for both fungi for the CV1 straw. Other fungal-treated
straws showed slight changes in their cellulose contents but
a significant increase in cellulose was observed for LE-treated
CV2 (4%) and CV4 straws (6.5%). Similar observations were also
reported in several other studies.6,7 The difficulties in the accurate
quantification of cellulose with regards to the interference of
fungal biomass have been described previously.16 Nonetheless,
the variable observation in cellulose content among studies
(based on the gravimetric method) triggers an intriguing ques-
tion regarding how these different types and batches of straw
affect the fungal growth and the extent of utilization of the
polysaccharides.
Fungal growth
The growth of both fungi on wheat straw of different cultivars is
illustrated in Fig. 2. The baseline ergosterol content varied con-
siderably among all five cultivars, ranging from 28.4 μgg
−1(CV3)
to 71.4 μgg
−1(CV5). Lentinula edodes showed a more consistent
growth on all straw cultivars, as compared to CS, although its over-
all growth was noticeably lower on CV2 and CV3 straw. Similar
to the growth on MS3 in an earlier experiment (with high base-
line ergosterol content), the growth of both fungi were more chal-
lenged on the CV5 straw. Due to a characteristically smaller for-
mation of the mycelium, CS was more affected by the variable
baseline ergosterol content, as compared to LE. On CV2 and CV3
straws, the CS growth appeared ‘stunted’ after a rapid increase in
ergosterol at week 1, before its growth continued at a slower rate
towards the end of the colonization weeks. There were also signifi-
cant (P<0.05) decreases in the ergosterol content of CS grown on
CV 1 and CV4 after week 5. These observations further indicate a
weaker colonization trait of CS as compared to LE on various types
of straw used.
Assessing in vitro rumen degradability of the straw
The IVGP and its kinetic parameters for straw of different cul-
tivars treated with CS and LE are summarized in Table 5. There
were no differences in the IVGP between the different untreated
straws, although a significantly (P<0.05) lower Rm2 was observed
for CV3 straw as compared to CV1 and CV5. Although both fungi
significantly (P<0.001) increased the IVGP of the straws (as com-
pared to controls), the magnitude of the effects was significantly
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963
Ruminal degradability of wheat straw by fungi www.soci.org
Table 4. Mass balances for wheat straw of different cultivars, treated with C. subvermispora (CS) and L. edodes (LE) for 7 weeks
Amount (g per 100 g of starting OM)b
CultivaraTreatment DM(g kg−1)Ash(gkg
−1DM) OM Cell Hcell ADL Sugar CP NI
CV1 Control 173.9ef 26.9k100.0a51.1abc 32.9a6.5b0.5h1.8fg 7.2d
CS 156.9gh 35.0hi 87.5e46.8f9.6g0.4e2.4a2.9ab 25.5a
LE 154.2h33.0i88.0e49.2de 12.2f1.3d2.3ab 2.8ab 20.3bc
CV2 Control 193.5ab 75.1e100.0a50.4cde 33.8a6.3b0.5h1.5gh 7.5d
CS 177.0cdef 85.1c97.1b49.7cde 19.9b3.0c1.8e2.2cdef 20.6bc
LE 186.5bcde 78.9d96.2b52.5ab 17.9bcd 3.3c2.1bcd 2.1def 18.2c
CV3 Control 203.1a84.7c100.0a50.9bcd 33.4a6.1b0.4h1.2h7.9d
CS 189.0abcd 95.2a96.6b50.5bcde 19.3bc 2.8c1.5f2.2cdef 20.4bc
LE 190.4abc 90.7b95.7bc 52.5ab 17.4cd 3.3c2.1cd 2.3cde 18.3c
CV4 Control 191.2abc 30.7j100.0a49.6cde 33.6a6.6ab 0.7g1.9ef 7.6d
CS 163.2fgh 38.5f96.4b50.1cde 16.6de 2.9c1.9de 2.6bc 22.2b
LE 175.3def 35.8gh 94.2cd 52.8a14.7e2.8c2.4a2.4cd 19.1c
CV5 Control 192.2ab 29.9j100.0a49.2de 33.0a7.2a0.6gh 2.2cde 7.9d
CS 170.1fg 37.3fg 95.4bc 48.3ef 16.0de 2.7c2.0de 3.2a23.1ab
LE 171.0fg 35.2ghi 92.9d50.7bcd 15.3e2.9c2.2abc 2.8ab 19.0c
RMSE 9.02 1.36 1.11 1.11 1.08 0.34 0.11 0.24 1.54
aCV1: Britannia;CV2:Cellule;CV3:Henrik;CV4:Residence;CV5:Tab a s co .
bCalculated from the remaining materials for each treatment (g), using respective starting OM for each maturity stage.
Values with different superscripts within a column are significantly (P<0.05) different.
DM, dry matter; OM, organic matter; Cell, cellulose; Hcell, hemicellulose; ADL, acid detergent lignin; Sugar, free reducing sugar from ethanol-extract
of wheat straw; CP, crude protein (N ×6.25); N.I, unaccounted OM. RMSE, root mean square error.
(A) (B)
0
40
80
120
160
200
240
01234567
Ergosterol (µg g-1 substrate)
Colonization week
0
100
200
300
400
500
01234567
Ergosterol (µg g-1substrate)
Colonization week
Figure 2. Growth (based on ergosterol data) of C. subvermispora (A) and L. edodes (B) on the wheat straw of different cultivars – CV1 ( ), CV2 ( ), CV3 ( ),
CV4 ( )andCV5( ) for 7 weeks. Error bars indicate standard deviations.
(P<0.001) affected by the different straw cultivars. The IVGP of the
fungus-treated CV2 and CV3 was noticeably lower than the other
treated straw cultivars with the difference between CS-treated
CV3 and CV4 being significant (P<0.05). The Rm2 of CV2 and
CV3 treated with both fungi were also significantly (P<0.05)
lower than the other treated straw cultivars. Overall, CS treatment
resulted in a significantly (P<0.05) higher increase in the IVGP
than LE (∼43 versus 29%).
Correlating the IVGP with the fungal growth (ergosterol) is rather
complex, although its relationship was statistically significant
(r=0.56; P<0.001). Ceriporiopsis subvermispora, which possessed
a weaker colonization trait on any straw type, resulted in a more
degradable straw than LE. Hence, the growth ‘inconsistency’ seen
in CS more likely suggests a higher dynamic nutrient utiliza-
tion and recycling of the field fungi biomass, contributing to an
apparently slow growth rate. It does not indicate the inability of
this particular fungus to colonize the different straw types success-
fully. When scaling-up the bioprocess in practice, the slow growth
of CS may lead to a problem with undesirable microbial growth
on the substrate. Nevertheless, an improved and optimized inoc-
ulation method can be used to ensure a quick colonization of
the substrate. In both trials above, these high-potential strains
showed a high persistency in improving the degradability of the
straw, although different straw cultivars affected the IVGP of the CS
and LE treatments. Significant (P<0.05) correlations were found
between the IVGP and the cell-wall compositions in both trials,
with stronger correlations to lignin (r∼−0.91) and hemicellulose
(r∼−0.92) than to cellulose (r∼0.51). Nonetheless, the current
results indicate that a careful assessment has to be made when
describing the chemical changes (using the unspecific gravimetric
J Sci Food Agric 2019; 99: 957–965 © 2018 The Authors. wileyonlinelibrary.com/jsfa
Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
964
www.soci.org N Nayan et al.
Table 5. In vitro gas production and its kinetic parameters for wheat straw of different cultivars, treated with C. subvermispora (CS) and L. edodes (LE)
for 7 weeks
Kinetic parameters
CultivaraTreatment IVGP A1A2B2C2tRm2 Rm2
CV1 Control 223.8a16.9b199.9a14.45de 2.41a16.68bc 0.085bcd
CS 308.4fg 49.4g255.6ef 11.20a2.86d13.91a0.135j
LE 282.7bcde 32.1de 243.7de 12.57b2.67bcd 15.22ab 0.110h
CV2 Control 205.4a6.0a191.3a15.38ef 2.41a17.76cde 0.080abc
CS 294.6defg 41.9efg 245.0de 13.31bcd 2.55abc 15.79bc 0.099efg
LE 269.4bc 33.5de 224.6bc 14.39de 2.53abc 17.01bc 0.090de
CV3 Control 203.9a9.2ab 183.5a16.96g2.44a19.68e0.073a
CS 289.2cdef 36.7def 240.7cde 14.14cde 2.51abc 16.63bc 0.091def
LE 260.7b27.5cd 219.6b14.81e2.49abc 17.37cd 0.086cd
CV4 Control 213.1a9.4ab 192.8a16.28fg 2.47ab 19.04de 0.077ab
CS 314.4g37.9def 266.9f12.86bc 2.73cd 15.69abc 0.111h
LE 281.0bcde 30.4d241.1cde 13.40bcd 2.59abc 16.04bc 0.099fg
CV5 Control 209.9a18.2bc 183.1a15.12ef 2.44ab 17.56cd 0.082bcd
CS 305.2efg 47.4fg 252.9ef 12.23ab 2.89d15.24ab 0.124i
LE 269.6bcd 34.9de 225.8bc 12.58b2.62abc 15.12ab 0.107gh
RMSE 13.38 6.00 10.28 0.71 0.13 1.07 0.005
aCV1: Britannia;CV2:Cellule;CV3:Henrik;CV4:Residence;CV5:Taba s co.
Values with different superscripts within a column are significantly (P<0.05) different.
RMSE, Root mean square error; IVGP, cumulative in vitro gas production at 72 h (mL g−1OM); A1,A2, asymptotic gas production (mL g−1OM) phase 1
and 2, respectively; B2, half time of the maximum gas production of phase 2 (h); C2, parameters determine the curvature of the graph; tRm2,timeof
the maximum fractional rate of substrate degradation (h); Rm2, maximum fractional rate of substrate degradation (h−1).
method) and relate them to the subsequent changes in IVGP. As
mentioned above, the interference of fungal biomass has to be
taken into account. Based on this consideration, the IVGP is, there-
fore, the most potent method for assessing the success and effec-
tiveness of a particular fungus in improving the ruminal degrad-
ability of different straw types.
CONCLUSION
Different straw types influenced the characteristics of C. subver-
mispora and L. edodes in degrading lignin. A more pronounced
degradation of lignin was observed on mature straw, which was
further confirmed by quantitative 13C-IS Py-GC/MS of the wheat
straw. Variable results were observed for lignin degradation with
different straw cultivars. Both high-potential strains of C. subver-
mispora and L. edodes were able to improve the ruminal degrad-
ability of wheat straw, regardless of the various straw types (matu-
rity and cultivar) investigated. The magnitude of the effect, how-
ever, was only affected by different straw cultivars but not by dif-
ferent maturity stages when harvested. Under all circumstances, C.
subvermispora was more capable than L. edodes of improving the
degradability of wheat straw. Lentinula edodes was more adapted
for colonizing different straw types than C. subvermispora.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge financial support from
the Wageningen UR Fund (WUF) as part of the project ‘More Meat
and Milk from Straw’, which is sponsored by DEKA, Forfarmers,
and the Victam Foundation. The authors would like to acknowl-
edge the scholarship provided by the Ministry of Higher Education
Malaysia and Universiti Putra Malaysia. Limagrain is acknowledged
for providing the different straw cultivars.
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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