Embed Size (px)
Citation preview
ORIGINAL PAPER
Bioprocessing of wheat and paddy straw for their nutritionalup-gradation
Rakesh Kumar Sharma • Daljit Singh Arora
Received: 10 May 2013 / Accepted: 15 December 2013
� Springer-Verlag Berlin Heidelberg 2014
Abstract Solid-state bioprocessing of agricultural resi-
dues seems to be an emerging and effective method for the
production of high quality animal feed. Seven strains of
white-rot fungi were selected to degrade wheat and paddy
straw (PS) under solid-state conditions. Degradation of
different components, i.e., hemicellulose, cellulose and
lignin was evaluated along with nutritional parameters
including; in vitro digestibility, crude protein, amino acids,
total phenolic contents (TPC) etc. Effect of nitrogen-rich
supplements on degradation of lignocellulosics was eval-
uated using two best selected fungal strains (Phlebia
brevispora and Phlebia floridensis). The best selected
conditions were used to upscale the process up to 200 g
batches of wheat and PS. Lignin was selectively degraded
up to 30 % with a limited loss of 11–12 % in total organic
matter. Finally, the degraded agro-residues demonstrated
50–62 % enhancement in their digestibility. Two–threefold
enhancement in other nutritional quality (amino acids,
TPCs and antioxidant activity) fortifies the process. Thus
the method is quite helpful to design an effective solid-state
fermentation system to improve the nutritive quality of
agricultural residues by simultaneous production of ligno-
cellulolytic enzyme production and antioxidants.
Keywords Bioprocessing � Lignocellulosic residues �Ligninolysis � Nutritive value � White-rot fungi
Introduction
Wheat (Triticum aestivum) and paddy (Oryza sativa) are
important cereal crops and produce a large quantity of
agricultural waste in the form of residual straw. This
residual biomass is invariably used in various industries as
raw material, but a large amount of the residues left are
incinerated or disposed off. Apart from waste disposal and
environmental pollution, the crisis of animal feed also exists
because of the scarcity of green forage. Different ligno-
cellulosic residues are generally fed to animals along with
green fodder. As compared to green fodder, cereal straws
have higher lignin content, which results in the lower
digestibility and nutritive value of these straws, thus limit-
ing their use as animal feed. Removal of lignin from the
lignocellulose and reduction of the crystallinity of cellulose
to loosen the cellulose structure increase the effective
contact area of the cellulose with beneficial microorganisms
[1]. Degradation of lignin by means of biological treatments
has got the potential to upgrade the quality of straw [2].
Unlike fermentation of agro-residues in a landfill and
composting, biodegradation using white-rot fungi results in
the production of nutritionally rich animal feed by simul-
taneous production of industrially important enzymes and
other phenolic bioactive compounds [3].
These white-rot fungi are well-known lignin degraders,
but simultaneous degradation of other energy-rich poly-
saccharide fibers like hemicellulose and cellulose limits the
efficiency of treatment because lesser biomass left behind
for ruminants. Thus, it necessitates looking for some more
selective lignin degrading organisms and the answer lies
R. K. Sharma � D. S. Arora (&)
Microbial Technology Laboratory, Department of Microbiology,
Guru Nanak Dev University, Amritsar 143005, Punjab, India
e-mail: [email protected]
R. K. Sharma
e-mail: [email protected]
Present Address:
R. K. Sharma
Department of Microbiology and Biotechnology Centre,
The Maharaja Sayajirao University of Baroda,
Vadodara 390002, India
123
Bioprocess Biosyst Eng
DOI 10.1007/s00449-013-1116-y
with Phlebia species [4, 5] which may thus possess the
potential capability for improvement of digestibility of
agro-residues [6, 7].
A part of the work has already been published using
either one substrate or one organism [3, 6, 8–10], while the
present article represents the overall picture including two
substrates and seven fungal treatment finally sorting down
to the best two fungi to make the study more comprehen-
sive and clear.
Materials and methods
Substrate procurement and its preparation
Two agro-residues wheat straw (WS) and paddy straw (PS)
used in the present study were obtained from the fields of
Guru Nanak Dev University, Amritsar (31�380 N 74�520 E),
India. Both the straw samples (WS and PS) were milled
(particle size 2 mm ± 0.5), washed and dried at 90 �C.
Organisms
Seven white-rot fungi including Ceriporiopsis subvermis-
pora (FP-90031), Daedalea flavida (MTCC-145), Phan-
erochaete chrysosporium (BKM-F-1767), Phlebia
brevispora (HHB-7030), Phlebia fascicularia (FP-70880),
Phlebia floridensis (HHB-5325) and Phlebia radiata
(MJL-1198) were selected for the study. All the fungi were
received from Forest Product Laboratories, Madison, USA,
except, D. flavida, which was procured from Microbial
Type Culture Collection, IMTECH, Chandigarh, India. The
cultures were maintained by regular subculturing on yeast
extract glucose agar (YGA) slants and stored at 4 �C.
Experimental setup for lignocellulosic degradation
Biodegradation of agro-residues by white-rot fungi
All the organisms were tested for their ability to degrade
both the agro-residues under solid-state fermentation (SSF)
conditions using 5 g straw (WS or PS) moistened with
25 ml of 0.5 % (w/v) malt extract, sterilized (at 15 lbs for
15 min), inoculated and incubated at 25 �C as described
earlier [6]. The processing of the substrate was carried out
after 30 days of incubation to monitor the biochemical
changes in straw constituents. The contents of each flask
were filtered through a tared filter paper and dried at 90 �C
till constant weight to obtain total organic matter (TOM),
which comprises of degraded WS and fungal biomass. Loss
in TOM was calculated from the difference between the
uninoculated control and inoculated flasks.
Optimization of degradation process using statistical
method
For optimizing the degradation of agro-residues and their
in vitro digestibility (IVD), three independent variables,
i.e., moisture content, one organic and one inorganic sup-
plement on the basis of the results obtained from the pre-
vious experiments [3, 11] were selected. All the three
variables significantly affect the degradation process as
moisture content regulates the production of extracellular
enzymes and its concentration; organic nitrogen source
provides carbon and nitrogen required for initial growth of
fungi, while inorganic nitrogen source is a capital nitrogen
source which further regulates the enzyme production. The
standard concentrations of these variables were obtained
with response surface methodology (RSM) using a Box–
Behnken design. Each variable was studied at three dif-
ferent levels (1, 0, -1). The experimental design included
17 flasks with five central points. Each 250 ml conical flask
contained 5 g of WS, 0–100 mg of supplement and
moistened with 1–12 ml of distilled water per gram of
straw.
The flasks containing straw were sterilized, inoculated
with three mycelial discs and incubated at 27 �C. The
processing was done after 20 days of incubation and the
dried biomass obtained was used to analyze changes in
water solubles, hemicellulose, cellulose, lignin and IVD.
The mathematical relationship of response G (for each
parameter) and independent variables X1, X2, and X3 was
calculated by the quadratic model Eq. 1.
G ¼ b0 þ b1X1 þ b2X2 þ b3X3 þ b11X21 þ b22X2
2
þ b33X23 þ b12X1X2 þ b13X1X3 þ b23X2X3. . . ð1Þ
where, G is the predicted response; b0, intercept; b1, b2,
and b3, linear coefficients; b11, b22 and b33, squared coef-
ficients and b12, b13 and b23 interaction coefficients. MI-
NITAB and statistical software package Design Expert
version 8.0 (Stat ease, Inc, Minneapolis, USA) were used to
obtain optimal working conditions and generate response
surface graphs.
Experimental setup for scaling up
The SSF was scaled up from 5 to 200 g of straw under the
selected conditions. Two hundred grams of straw were
taken in an autoclavable polyethylene bag with selected
concentrations of distilled water and supplements on the
basis of results obtained from above experiments. The
bag was autoclaved, placed inside a surface sterilized
plastic container, inoculated and incubated as described
earlier [3].
Bioprocess Biosyst Eng
123
Analytical methods for estimation of biochemical
composition of agro-residue
The sequential fractionation of lignocellulosics for esti-
mation of water solubles, hemicellulose, cellulose and
lignin was carried out according to Datta [12] with slight
modifications as described earlier [6]. Ash content,
moisture content, swelling capacity and pH of the sound
straw were determined as described by Dill and Kraepelin
[13].
Analysis of nutritive quality
IVD of sound and degraded straw was estimated according
to Akhter et al. [14], with slight modifications using fecal
inoculum and acidified pepsin. The weight loss in dry
matter during the incubation has been expressed as IVD
[6]. Total nitrogen was estimated by micro-Kjeldahl
method and crude protein of degraded and sound straw was
determined by multiplying total nitrogen content by the
factor 6.25. Total amino acid assay was carried out using
ninhydrin assay and the results were compared with
asparagine standard curve [3]. Total polyphenolic contents
were determined colorimetrically by Folin–Ciocalteu (FC)
method using gallic acid as standard. Antioxidant activity
was assayed using DPPH [3, 15].
Chitin estimation
Chitin content of the fungus and fermented straw was
measured according to Chen and Johnson [16] using Ehr-
lich reagent.
Statistical analysis
The data were represented as mean and analyzed by one-
way or two-way analysis of variance (ANOVA). Pear-
son’s correlation was used to correlate different
parameters.
Results and discussion
White-rot fungi have got the necessary potential to degrade
lignin, which makes them suitable candidates for deligni-
fication of agro-residues. Fungal delignification of such
lignocellulosics not only enhances the digestibility of the
feed, but also improves their nutritional value [17]. The
loss of cellulose and hemicellulose along with lignin from
the feed is not economically desirable because less biomass
then remains available to the animal as feed for energy. P.
chrysosporium a widely studied fungus degrades lignin
efficiently, but also cause a high loss in TOM [18]. To
overcome this problem, selective ligninolysis is of great
importance in feed as well as pulp & paper industry. Cell
wall constituents of straw play an important role in deter-
mining its quality as animal feed. In several studies, lignin
loss enhanced the IVD and selective lignin degradation
minimized the TOM loss [19].
Biochemical analysis of sound (undegraded)
lignocellulosic residues
Both the substrates, i.e., sound WS and PS were analyzed
for their biochemical composition before subjecting them
to degradation studies. In comparison to PS, WS contained
slightly higher hemicellulose and lignin while cellulose
was higher in the former. Crude protein and IVD were
higher for PS as compared to WS (Table 1).
Biodegradation of wheat straw
SSF of WS was carried out using seven white-rot fungi to
find out their potential to degrade different plant cell wall
components. After fungal treatment of WS, loss in TOM,
hemicellulose, cellulose and lignin was analyzed
(Table 2).
During 30 days of incubation, P. chrysosporium grew
vigorously on WS and caused maximum loss in TOM, i.e.,
54 %, followed by D. flavida (22 %). Around 16 % loss in
TOM was caused by P. fascicularia, C. subvermispora, P.
brevispora and P. floridensis, while P. radiata caused a
maximum loss of 9.8 % only (Table 2). Next to P. chry-
sosporium, maximum ligninolysis was caused by P. brev-
ispora (30.6 %), followed by P. radiata (27.9 %) and P.
floridensis (27.5 %). C. subvermispora degraded 25.2 % of
lignin followed by P. fascicularia (23.1 %) and D. flavida
(18.7 %). Other cell wall constituents, i.e., hemicellulose
and cellulose were degraded up to variable extents as
presented in Table 2.
Table 1 Biochemical composition (%) of sound wheat and paddy
straw
Properties Wheat straw Paddy straw
Moisture content 12 ± 0.3 13 ± 0.4
Swelling capacity 12.8 ± 0.5 17.4 ± 0.6
pH 6.2 ± 0.2 5.6 ± 0.2
Water soluble part 10 ± 0.2 12 ± 0.15
Hemicellulose 33 ± 0.4 29 ± 0.2
Cellulose 37 ± 0.5 40.5 ± 0.4
Lignin 20.5 ± 0.2 18.5 ± 0.1
Ash 7.2 ± 0.3 10.2 ± 0.4
Crude protein 1.4 ± 0.04 2.1 ± 0.05
In vitro digestibility 17.2 ± 0.15 18.5 ± 0.2
Bioprocess Biosyst Eng
123
Nutritional value of degraded wheat straw
Undegraded WS had an IVD of 17.2 %, which increased
during SSF. Maximum IVD was observed in the WS
degraded by P. brevispora (28.7 %) and P. fascicularia
(28.4 %), followed by P. floridensis (26.9 %), C. subver-
mispora (25.4 %) and P. radiata (24.5 %) while in P.
chrysosporium it was minimum (21 %). No enhancement
in IVD was observed in WS degraded by D. flavida
(Table 2). Crude protein content of WS significantly
increased during its fungal degradation as compared to
control (1.4 %). Maximum crude protein content (3.7 %)
was observed in WS degraded by P. chrysosporium, while
the crude protein content of WS degraded by all other fungi
ranged between 1.8 and 2 %.
Amino acid content of degraded WS was also maximum
in the case of P. chrysosporium (0.36 %) followed by P.
floridensis and P. brevispora, while in other fungi it ranged
from 0.16 to 0.19 %. As compared to sound WS, total
phenolic content (TPC) was also higher in degraded WS. P.
chrysosporium and P. floridensis produced maximum TPC,
which was almost similar in both the cases (statistically
insignificant P [ 0.05). DPPH assay and TPC showed a
strong positive correlation as the DPPH free radical scav-
enging activity increased along with TPC (Table 2).
The present study is in consonance with earlier obser-
vations on corn stover degradation by P. brevispora which
enhanced the digestibility of the substrate while P. chry-
sosporium degraded maximum lignin along with a large
amount of cellulose [20]. The same study has reported that
P. chrysosporium reduced the digestibility of substrate,
which is in contrast to the current findings, where an
increase from 17.2 to 21 % was observed. Overall, P.
brevispora was more effective for the enhancement of fiber
digestibility as also observed in the present studies. During
the cell wall degradation study on maize, Chen et al. [21]
concluded that P. brevispora also exhibited stronger ability
to degrade cell wall-bound phenolic acids, which might be
a reason for its better degradation ability.
Biodegradation of paddy straw
SSF of PS was carried out with seven white-rot fungi up to
60 days because of the slower growth of fungi on the
substrate. All the tested fungi were able to grow on PS
under the experimental conditions and similar parameters
were used to assay the PS quality as used for WS analysis
(Table 3).
Of the different fungi, P. chrysosporium caused maxi-
mum loss in TOM of 46.4 % during 60 days of incubation.
The organism was fast growing and degraded all the
components up to its maximum extent. D. flavida followed
P. chrysosporium and degraded 17.6 % of TOM, followed
by C. subvermispora (16.8 %) and P. radiata (13.9 %). P.
floridensis and P. fascicularia caused a similar loss of
10.8 %, while P. brevispora caused the lowest loss (9.8 %)
in TOM.
Next to P. chrysosporium, P. radiata degraded a max-
imum of 22.8 % of lignin followed by P. floridensis
(21.8 %), P. fascicularia (21 %), P. brevispora (20 %), D.
flavida (19.4 %) and C. subvermispora (18.8 %), respec-
tively. Variable amount of other cell wall components was
degraded by these fungi as presented in Table 3.
White-rot fungi are known to attack initially the hemi-
cellulose lignin matrix [22], which was also clearly
observed during the present study. The experiments were
designed to study the profile of WS and PS degradation vis-
a-vis their IVD. All the Phlebia spp. degraded higher
amount of lignin selectively during the degradation of WS,
though in PS hemicellulose and lignin both were degraded
simultaneously during initial period, while cellulose was
not degraded during same period and it remained low on
further incubation also. However, hemicellulose and lignin
degradation continued up to the end of the experiment to a
Table 2 Changes in biochemical constituents (%) and nutritive quality of wheat straw during its 30 days solid-state fermentation by white-rot
fungi
Organisms TOM loss Cellulose loss HMCL loss Lignin loss Crude protein Amino acid TPC Antioxidant
potential
IVD
Control WS – – – – 1.40 0.07 0.10 32.4 17.2
C. subvermispora 16.4 25.2 28.5 25.2 1.92 0.19 1.46 65.4 25.4
D. flavida 22.0 20.9 40.7 18.7 1.94 0.16 0.96 66.7 15.8
P. chrysosporium 54.0 59.2 67.2 47.6 3.70 0.36 2.04 78.6 21.0
P. brevispora 16.3 29.1 16.9 30.6 1.83 0.25 1.68 66.1 28.7
P. fascicularia 16.7 29.4 22.2 23.1 1.87 0.17 1.06 57.2 28.4
P. floridensis 16.3 17.1 22.6 27.5 2.05 0.28 1.86 70.8 26.9
P. radiata 9.8 19.2 26.3 27.9 1.99 0.17 1.38 65.2 24.5
TOM total organic matter, HMCL hemicellulose, TPC total phenolic contents, IVD in vitro digestibility, WS wheat straw
Bioprocess Biosyst Eng
123
reasonable extent [9]. During 60 days of PS degradation,
both the fungi P. chrysosporium and P. brevispora
enhanced the maximum IVD from 18.5 (control) to 25 %
with a respective loss of 39.4 and 20 % lignin (Table 3).
Nevertheless, P. chrysosporium was non-selective in lig-
ninolysis and degraded all the cell wall constituents
simultaneously which resulted in more holocellulose loss
and thus degraded a large amount (46.4 %) of TOM. On
the other hand, P. brevispora degraded only 9.8 % TOM
during the fermentation process thus accounting for its
more selective ligninolytic ability and leaving behind a
sufficient amount of TOM. Thus, a reasonable amount of
easily digestible degraded biomass is available to the ani-
mal as feed. For practical purposes, higher TOM loss
severely limits the use of P. chrysosporium, which is in
consonance with earlier observations [18].
Nutritional value of degraded paddy straw
P. chrysosporium and P. brevispora maximally enhanced
the IVD of degraded PS from 18.5 to 25 %, while the IVD
ranged from 23 to 25 % for remaining fungi, during
60 days of incubation period. Crude protein and amino
acids content increased at least 1.4-fold in PS degraded by
different fungi. TPCs and antioxidant activity also
increased significantly ranging from three to ninefold in
degraded PS. Except TPC and antioxidant activity, P.
chrysosporium favored the other nutritional factors, but a
huge biomass loss (TOM) was the limiting factor for its use
(Table 3).
During the present study, D. flavida degraded lignin
efficiently and enhanced protein content, but was unable to
enhance the IVD. Similarly, earlier study on solid-state
cultivation of the white-rot fungus Lentinula edodes on
wheat bran demonstrated that total and insoluble dietary
fiber and crude protein content increased with fungal
growth while the in vitro dry matter enzyme digestibility
decreased [23]. In another report, about 50 % of the white-
rot fungal strains decreased the in vitro substrate digest-
ibility during screening experiments carried out using WS
as substrate [24]. Barahona et al. [25] reported that despite
high nitrogen content in most of the tropical legumes, IVD
estimates were low, which was also found true in the case
of straw degraded by D. flavida during present study.
Optimization of degradation of lignocellulosics
and IVD using response surface methodology
The data obtained from the design were analyzed by
applying multiple regression analysis method based on
Eq. 1 and found to be significant. It is verified by F value
and the analysis of variance (ANOVA) by fitting the data
of all independent observations in response surface qua-
dratic model. Lack of fit was insignificant in all the cases
and R2 value for all the responses was [85 %, which
showed suitable fitting of the model in the designed
experiments. The obtained concentration of each variable
was validated by repeating the experiment in duplicate
flasks.
The selected variables on the basis of results obtained
from above experiments were studied at three different
levels (1, 0 and -1). Each 250 ml conical flask contained
5 g of straw, inorganic and/or organic nitrogen-rich sup-
plement (0–100 mg) and 1–10 ml of distilled water/g of
substrate [3]. The flasks were sterilized, inoculated, incu-
bated and processed as described in ‘‘Materials and meth-
ods’’. Whole experiment was repeated and validated using
optimized concentration of supplements as predicted by the
RSM.
Effect of different variables on WS degradation
by P. brevispora and P. floridensis
Maximum TOM loss (18.8 %), ligninolysis (29.5 %) and
IVD (28 %) occurred at lowest concentration of NH4Cl and
highest concentration of malt extract and at a moisture
Table 3 Changes in biochemical constituents (%) and nutritive quality of paddy straw during its 60 days solid-state fermentation by white-rot
fungi
Organisms TOM loss Cellulose loss HMCL loss Lignin loss Crude protein Amino acid TPC Antioxidant
potential
IVD
Control PS – – – – 2.10 0.13 0.14 21.2 18.5
C. subvermispora 16.8 28.7 30.8 18.8 2.99 0.26 0.62 46.8 24.0
D. flavida 17.6 12.5 14.2 19.4 3.02 0.25 0.80 51.4 18.8
P. chrysosporium 46.4 50.9 52.0 39.4 3.55 0.32 0.44 35.7 25.4
P. brevispora 9.8 12.8 8.7 20.0 4.08 0.38 0.98 54.7 25.2
P. fascicularia 10.7 19.6 10.3 21.0 2.84 0.25 0.92 52.9 23.7
P. floridensis 10.8 18.8 11.4 21.8 3.25 0.33 1.34 60.8 23.2
P. radiata 13.9 21.6 13.4 22.8 3.64 0.32 0.86 50.7 24.8
TOM total organic matter, HMCL hemicellulose, TPC total phenolic contents, IVD in vitro digestibility, PS paddy straw
Bioprocess Biosyst Eng
123
level of 5.5 ml/g of WS degraded by P. brevispora [3]
(Fig. 1a).
As predicted by the RSM plot, during WS degradation
by P. floridensis, maximum TOM loss (14 %) was sup-
ported by a moisture content of 7 ml, malt extract 90 and
10 mg NH4Cl/g of WS. Lignin degradation was well
optimized under the experimental conditions, i.e., 8 ml of
moisture content, 55 mg of malt extract and 50 mg of
NH4Cl/g of WS (Fig. 1b). A maximum of 29 % lignin was
degraded over 20 days of incubation period. The optimized
conditions for the maximum enhancement in IVD required
7 ml of moisture, a minimum concentration of NH4Cl and
a maximum concentration of malt extract [11].
Effect of different variables on PS degradation
by P. brevispora and P. floridensis
As predicted by the RSM plot, maximum TOM loss (8 %)
was supported by moisture content 8 ml, maximum amount
of peptone (100 mg) and lowest NH4Cl (0–10 mg), during
PS degradation by P. brevispora. Lignin degradation was
well optimized under the experimental conditions, i.e.,
6.5 ml of moisture content, 60 mg of peptone and 75 mg of
NH4Cl/g of PS (Fig. 2a). IVD was also well optimized by
the model. The optimized conditions for the maximum
increase in IVD from 18.5 (sound PS) to 26 % required
mid level of moisture content (6–7 ml), 70 mg of NH4Cl
and 20 mg of peptone (Fig. 2b).
During PS degradation by P. floridensis, Maximum
TOM loss (8 %) occurred at 6 ml of moisture, minimum
concentration of NH4Cl and a maximum concentration of
soya bean meal (100 mg/g of PS). Lignin was degraded
optimally at a moisture level of 6.5 ml/g of PS, 50 mg of
NH4Cl and 60 mg of soya bean meal. Maximum
enhancement in IVD (from 18.5 to 26 %) required 6.5 ml
of moisture, a minimum concentration of NH4Cl (10 mg)
and a maximum concentration of soya bean meal
(90–100 mg) per gram of substrate [26].
The experiment was scaled up from 5 to 200 g of straw
under optimized conditions for 20 days as described in
‘‘Materials and methods’’. Each substrate was subjected to
degradation by the fungus under optimized conditions as
above giving maximum enhancement in IVD and
NH4Cl (mg)
NH4Cl (mg)
0.00 25.00 50.00 75.00 100.000.00
25.00
50.00
75.00
100.00
22
23
23
24
24
25
25
26
27
Mal
t ext
ract
(m
g)
0.00 25.00 50.00 75.00 100.000.00
25.00
50.00
75.00
100.00
24
26
26
28
30
Mal
t ext
ract
(m
g)
(a)
(b)
Fig. 1 Contour plots showing a maximum IVD, hold value moisture
6 ml, b optimum lignin degradation, hold value moisture 7.5 ml,
during 20 days of SSF of WS by P. brevispora
2.00 4.00 6.00 8.00 10.000.00
25.00
50.00
75.00
100.00
3
4
45
6
Moisture (ml)
Moisture (ml)
NH
4C
l (m
g)
2.00 4.00 6.00 8.00 10.000.00
25.00
50.00
75.00
100.00
20
22
22
24
26
NH
4C
l (m
g)
(a)
(b)
Fig. 2 Contour plots showing a optimum lignin degradation, hold
value peptone 60 mg, b optimum IVD, hold value peptone 20 mg,
during 20 days of SSF of PS by P. brevispora
Bioprocess Biosyst Eng
123
minimum loss in TOM. Different biochemical analyses
along with nutritive quality were done before and after the
degradation of straw (Table 4).
Scaling up of wheat straw biodegradation
To scale up the experiment, 200 g of straw was taken in an
autoclavable plastic bag along with the optimized con-
centrations of supplements, i.e., 5 ml distilled water, 20 mg
NH4Cl and 75 mg malt extract/g of WS for P. brevispora
and 5.5 ml distilled water, 25 mg NH4Cl and 60 mg ME/g
of WS for P. floridensis, keeping in mind minimum loss in
TOM and enhancement in IVD.
Both the organisms caused about 10.5 % loss in TOM.
P. brevispora caused 29 % loss in lignin with a concomi-
tant rise in the IVD from 17.2 to 28 %, thus resulting in
62 % enhancement in IVD. Total amino acid content
increased almost four times as compared to control. About
fivefold enhancement in TPC was observed accompanied
by twofold enhancement in antioxidant activity [3].
P. floridensis caused a lignin loss of 24 % and increased
the IVD from 17.2 to 24.6 % (43 % enhancement).
Enhancement in crude protein (1.5-folds), total amino acid
(3.4-folds) and TPC (threefold) accompanied by propor-
tionate enhancement in antioxidant activity was recorded
(Table 4).
The up scaling of SSF was conducted successfully. SSF
is an advantageous method to degrade lignin and improve
the digestibility of lignocellulosics. Fungi grown under
these conditions perform better ligninolysis and the addi-
tion of fungal mycelium contributes to the total protein
content of the feed [27]. The results also demonstrated the
increase in amino acid content, TPC and antioxidant
property of the fungal degraded straw, thus reflecting an
enhancement in nutritional qualities. Enhancement in
antioxidant activity has been earlier reported during the
SSF of some agricultural residues by Rhizopus stolonifer
[28]. Thus, the strategy may be used for upgrading low
quality agro-wastes to develop healthy animal feed sup-
plements [15, 29]. The use of lignocellulosic residues for
the production and extraction of bioactive phenolic com-
pounds has been well studied and reviewed recently [30].
Scaling up of paddy straw biodegradation
During PS degradation by P. brevispora, 5 ml distilled
water, 70 mg NH4Cl and 25 mg peptone/g of PS were
used. The fungus degraded 8 % TOM accompanied by 6 %
lignin loss and increased the IVD from 18.2 to 26 %, thus
resulting in 45 % enhancement in IVD. Total amino acid
contents increased up to twofold and TPC up to threefold,
while antioxidant property increased by twofold.
During up scaling of PS degradation by P. floridensis,
5.5 ml distilled water, 80 mg NH4Cl and 25 mg soya bean
meal/g of PS were used. It influenced a loss in TOM of 6 %
accompanied by lignin loss of 6.2 % with a concomitant
enhancement in the IVD from 18.2 to 25.8 %, thus
resulting in 42 % enhancement in IVD. Enhancement in
TPC (sixfold), total amino acid (threefold) and antioxidant
activity (threefold) was observed [26] (Table 4).
The fermented straw is a mixture of straw and fungal
biomass. Estimation of the fungal biomass in fermented
straw is very difficult as the experiment was performed
under solid-state, which did not allow separating the fungus
Table 4 Biochemical and nutritional properties (%) of wheat and paddy straw after 20 days of SSF under optimized conditions
Properties Wheat straw Paddy straw
Controla P. brevispora P. floridensis Controla P. brevispora P. floridensis
TOM loss – 10.5 ± 0.3 10 ± 0.5 – 8 ± 0.6 6 ± 0.4
Water solubles 10 ± 0.5 15 ± 0.4 9 ± 0.4 12 ± 0.4 11.5 ± 0.5 11 ± 0.3
Hemicellulose loss – 13.6 ± 0.4 9.5 ± 0.5 – 6.5 ± 0.3 5.8 ± 0.4
Cellulose loss – 12 ± 0.2 8.7 ± 0.4 – 3.2 ± 0.4 4.7 ± 3
Lignin loss – 29 ± 0.4 24 ± 0.3 – 6 ± 0.3 6.2 ± 0.3
Ash content 7 ± 0.3 7.6 ± 0.2 10 ± 0.2 10.2 ± 0.4 10.8 ± 0.2 11.6 ± 0.2
IVD 17.2 ± 0.4 28 ± 0.5 24.6 ± 0.5 18.2 ± 0.3 26 ± 0.4 25.8 ± 0.4
Protein content 1.4 ± 0.05 1.88 ± 0.08 2.01 ± 0.08 2.1 ± 0.07 2.5 ± 0.08 3.05 ± 0.08
Total Amino acid 0.07 ± 0.003 0.27 ± 0.01 0.24 ± 0.01 0.08 ± 0.003 0.15 ± 0.005 0.22 ± 0.005
TPC 0.1 ± 0.04 0.55 ± 0.02 0.3 ± 0.015 0.12 ± 0.01 0.37 ± 0.015 0.73 ± 0.015
Antioxidant activity 32.4 ± 0.1 68 ± 0.61 65.4 ± 0.5 21.2 ± 0.5 58 ± 0.6 61.4 ± 0.8
Fungal biomass ND 6.6 ± 0.15 6.8 ± 1 ND 6.4 ± 0.1 5.8 ± 1
TOM total organic matter, IVD in vitro digestibility, TPC total phenolic content, ND not detecteda Undegraded straw
Bioprocess Biosyst Eng
123
from the straw. Roche et al. [31] have proposed a method
to measure the fugal biomass by estimating the chitin
content. The idea was adopted to calculate the fungal
biomass present in the degraded straw and found to be
helpful, which revealed that finally the total degraded
biomass comprised 6–7 % of fungal biomass (Table 4).
During microbial processes for conversion of lignocel-
lulosic wastes into feed, at least one of the three objectives
must be achieved: an increase in the digestibility of the
lignocellulosic material, an increase in the protein level and
an improvement in the dry product palatability, although
the last factor can be easily improved by ensiling or mixing
the substrate with other more palatable foods [32]. As
evident from the present study, first two important objec-
tives were achieved successfully. P. brevispora and P.
floridensis were the best organisms to provide a practically
promising approach in selective ligninolysis and enhance-
ment of IVD of WS and PS (Fig. 3).
Conclusions
It can be concluded that the selected strains, i.e., P. brev-
ispora and P. floridensis will be useful in value-addition of
the agro-wastes, towards their utilization as healthy feed
supplements in animal husbandry. Furthermore, the
enrichment of the substrates, particularly in protein content
and antioxidant activities may reduce the level of fortifi-
cation in the preparation of animal feeds as it is done at
present, thereby reducing the cost of producing the feeds.
Thus, the study is an effort to provide a strategy and use of
eco-friendly system to convert a large amount of agricul-
tural residues, like wheat and PS into nutritive animal feed.
The major benefits can be summarized as pollution-free
environment, waste management and finally to provide a
natural, effective, economical and safe feed for the
ruminants.
Acknowledgments Rakesh Kumar Sharma is thankful to CSIR,
India for the award of Senior Research Fellowship, File No. 09/254
(0226)/2110-EMR-I.
References
1. Kejun S, Juntao Z, Ying C, Zongwen L, Lin R, Cong L (2011)
Accelerating the degradation of green plant waste with chemical
decomposition agents. J Environ Manag 92:2708–2713
2. Bisaria R, Madan M, Vasudevan P (1997) Utilisation of agro-
residues as animal feed through bioconversion. Bioresour Tech-
nol 59:5–8
3. Arora DS, Sharma RK (2011) Effect of different supplements on
bioprocessing of wheat straw by Phlebia brevispora: changes in
its chemical composition, in vitro digestibility and nutritional
properties. Bioresour Technol 102:8085–8091
4. Dong YC, Wang W, Hu ZC, Fu ML, Chen QH (2012) The
synergistic effect on production of lignin-modifying enzymes
through submerged co-cultivation of Phlebia radiata, Dichomitus
squalens and Ceriporiopsis subvermispora using agricultural
residues. Bioprocess Biosyst Eng 35:751–760
5. Arora DS, Gill PK (2000) Laccase production by some white rot
fungi under different nutritional conditions. Bioresour Technol
73:283–285
6. Arora DS, Sharma RK (2009) Comparative ligninolytic potential
of Phlebia species and their role in improvement of in vitro
digestibility of wheat straw. J Animal Feed Sci 18:151–161
7. Sharma RK, Arora DS (2013) Fungal degradation of lignocellu-
losic residues: an aspect of improved nutritive quality. Crit Rev
Microbiol. doi:10.3109/1040841X.2013.791247
8. Arora DS, Sharma RK (2009) Enhancement in in vitro digest-
ibility of wheat straw obtained from different geographical
regions during solid state fermentation by white rot fungi. Bio-
resources 4:909–920
9. Sharma RK, Arora DS (2010) Changes in biochemical constitu-
ents of paddy straw during degradation by white rot fungi and its
impact on in vitro digestibility. J Appl Microbiol 109:679–686
10. Sharma RK, Arora DS (2011) Biodegradation of paddy straw
obtained from different geographic locations by means of Phlebia
spp. for animal feed. Biodegradation 22:143–152
11. Sharma RK, Arora DS (2010) Production of lignocellulolytic
enzymes and enhancement of in vitro digestibility during solid
state fermentation of wheat straw by Phlebia floridensis. Biore-
sour Technol 101:9248–9253
12. Datta R (1981) Acidogenic fermentation of lignocelluloses—acid
yield and conversion of components. Biotechnol Bioeng
23:2167–2170
13. Dill I, Kraepelin G (1986) Palo Podrido: model for extensive
delignification of wood by Ganoderma applanatum. Appl Envi-
ron Microbiol 52:1305–1312
14. Akhter S, Owen E, Theodorou MK, Butler EA, Minson DJ (1999)
Bovine faeces as a source of micro-organisms for the in vitro
digestibility assay of forages. Grass Forage Sci 54:219–226
15. Sharma RK, Chandra P, Arora DS (2010) Antioxidant properties
and nutritional value of bioprocessed wheat straw by Phanero-
chaete chrysosporium and Daedalea flavida. J Gen Appl Micro-
biol 56:519–523
16. Chen GC, Johnson BR (1983) Improved colorimetric determi-
nation of cell wall chitin in wood decay fungi. Appl Environ
Microbiol 46:13–16
17. Okano K, Iida Y, Samurai M, Prasetya B, Usagawa T, Watanabe
T (2006) Comparison of in vitro digestibility and chemical
composition among sugarcane bagasses treated by four white rot
fungi. Animal Sci J 77:308–313
0
10
20
30
40
50
60
WS PS WS PS
P. brevispora P. floridensis
(% e
nhan
cem
ent i
n IV
D)
Undegraded straw Before optimization After optimization
Fig. 3 Percentage enhancements in IVD during 20 days of degrada-
tion of agro-residues before and after optimization with respect to
control
Bioprocess Biosyst Eng
123
18. Jung HG, Valdez FRJ, Abad AR, Blanchette RA, Hatfield RD
(1992) Effect of white rot basidiomycetes on chemical compo-
sition and in vitro digestibility of oat straw and alfalfa stems.
J Animal Sci 70:1928–1935
19. Cohen R, Persky L, Hadar Y (2002) Biotechnological applica-
tions and potential of wood degrading mushrooms of the genus
Pleurotus. Appl Microbiol Biotechnol 58:582–594
20. Chen J, Fales SL, Varga GA, Royse DJ (1995) Biodegradation of
cell wall components of maize stover colonized by white-rot
fungi and resulting impact on in vitro digestibility. J Sci Food
Agric 68:91–98
21. Chen J, Fales SL, Varga GA, Royse DJ (1996) Biodegradability
of free monomeric and cell-wall-bound phenolic acids in maize
stover by two strains of white-rot fungi. J Sci Food Agric
71:145–150
22. Martinz AT, Speranza M, Duenas FJR, Ferreira P, Camarero S,
Guillen F, Martınez MJ, Gutierrez A, Rıo JC (2005) Biodegra-
dation of lignocellulosics: microbial, chemical, and enzymatic
aspects of the fungal attack of lignin. Int Microbiol 8:195–204
23. Lena GD, Patroni E, Quaglia GB (1997) Improving the nutri-
tional value of wheat bran by a white-rot fungus. Int J Food Sci
Technol 32:513–519
24. Capelari M, Zadrazil F (1997) Lignin degradation and in vitro
digestibility of wheat straw treated with Brazilian tropical species
of white rot fungi. Folia Microbiol 42:481–487
25. Barahona R, Lascano CE, Narvaez N, Owen E, Morris P, The-
odorou MK (2003) In vitro degradability of mature and immature
leaves of tropical forage legumes differing in condensed tannin
and non-starch polysaccharide content and composition. J Sci
Food Agric 83:1256–1266
26. Sharma RK, Arora DS (2011) Solid state degradation of paddy
straw by Phlebia floridensis in the presence of different supple-
ments for improving its nutritive status. Int Biodeterior Biodegrad
65:990–996
27. Fazaeli H (2007) Nutritive value index of treated wheat straw
with Pleurotus fungi. Biotechnol Animal Husb 23:169–180
28. Lateef A, Oloke JK, Kana GEB, Oyeniyi SO, Onifade OR,
Oyeleye AO, Oladosu OC, Oyelami AO (2008) Improving the
quality of agro-wastes by solid state fermentation: enhanced
antioxidant activities and nutritional qualities. World J Microbiol
Biotechnol 24:2369–2374
29. Arora DS, Sharma RK, Chandra P (2011) Biodelignification of
wheat straw and its effect on in vitro digestibility and antioxidant
properties. Int Biodeterior Biodegrad 65:352–358
30. Martins S, Mussatto SI, Martınez-Avila G, Montanez-Saenz J,
Aguilar CN, Teixeira JA (2011) Bioactive phenolic compounds:
production and extraction by solid-state fermentation: a review.
Biotechnol Adv 29:365–373
31. Roche N, Venague A, Desgranges C, Durand A (1993) Use of
chitin measurement to estimate fungal biomass in solid state
fermentation. Biotechnol Adv 11:677–683
32. Kamra DN, Zadrazil F (1988) Microbiological improvement of
lignocellulosics in animal feed production: a review. In: Zadrazil
F, Reiniger P (eds) Treatment of lignocellulosics with white-rot
fungi. Elsevier, Essex, pp 56–63
Bioprocess Biosyst Eng
123
