Screening of lignin-degrading fungi for their ability to decay cassava residue
To screen applicable fungi for their ability to decay cassava residue, nineteen lignin-degrading fungi were isolated by guaiacol and azure B plates. Cassava residue decayed characteristics of the isolates were evaluated systematically by principal component analysis (PCA) of weight and components lo...
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| Цитувати: | Screening of lignin-degrading fungi for their ability to decay cassava residue / Bin Xu, Huixing Li, Chaojun Du, Ying Wang, Bin Li // Functional Materials. — 2016. — Т. 23, № 4. — С. 668-675. — Бібліогр.: 26 назв. — англ. |
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Bin Xu Huixing Li Chaojun Du Ying Wang Bin Li 2017-06-14T14:14:42Z 2017-06-14T14:14:42Z 2016 Screening of lignin-degrading fungi for their ability to decay cassava residue / Bin Xu, Huixing Li, Chaojun Du, Ying Wang, Bin Li // Functional Materials. — 2016. — Т. 23, № 4. — С. 668-675. — Бібліогр.: 26 назв. — англ. 1027-5495 DOI: doi.org/10.15407/fm23.04.668 https://nasplib.isofts.kiev.ua/handle/123456789/121496 To screen applicable fungi for their ability to decay cassava residue, nineteen lignin-degrading fungi were isolated by guaiacol and azure B plates. Cassava residue decayed characteristics of the isolates were evaluated systematically by principal component analysis (PCA) of weight and components loss data, and compared with those of Phanerochaete chrysosporium and Trametes sp. SYBC-L4. Four groups of decayed cassava residue were identified. Fungus N1 and N3 grouped together and showed high lignin-degrading selectivity. Fungus N5 and N8 grouped with P. chrysosporium and showed high degradation ability. During the four weeks incubation, the lignin-degrading selectivity value of fungus N3 ranged from 1.14 to 1.38 and was the best, the weight loss of fungus N5 and N8 achieved 30.93% and 33.34%, respectively. Fungus N3, N5, and N8 were identified as Pleurotus sp., Trametes sp. and Coriolopsis sp. based on 18S rDNA gene sequences, respectively. PCA is an effective method in recognizing cassava residue decayed characteristics of fungi and is helpful to screen fungi for their potential application. The three screened out fungi could be used to decay cassava residue for enhancement of its bioconversion efficiency. en НТК «Інститут монокристалів» НАН України Functional Materials Green Chemical Screening of lignin-degrading fungi for their ability to decay cassava residue Article published earlier |
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Screening of lignin-degrading fungi for their ability to decay cassava residue |
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Screening of lignin-degrading fungi for their ability to decay cassava residue Bin Xu Huixing Li Chaojun Du Ying Wang Bin Li Green Chemical |
| title_short |
Screening of lignin-degrading fungi for their ability to decay cassava residue |
| title_full |
Screening of lignin-degrading fungi for their ability to decay cassava residue |
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Screening of lignin-degrading fungi for their ability to decay cassava residue |
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Screening of lignin-degrading fungi for their ability to decay cassava residue |
| title_sort |
screening of lignin-degrading fungi for their ability to decay cassava residue |
| author |
Bin Xu Huixing Li Chaojun Du Ying Wang Bin Li |
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Bin Xu Huixing Li Chaojun Du Ying Wang Bin Li |
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Green Chemical |
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Green Chemical |
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2016 |
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English |
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Functional Materials |
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НТК «Інститут монокристалів» НАН України |
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Article |
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To screen applicable fungi for their ability to decay cassava residue, nineteen lignin-degrading fungi were isolated by guaiacol and azure B plates. Cassava residue decayed characteristics of the isolates were evaluated systematically by principal component analysis (PCA) of weight and components loss data, and compared with those of Phanerochaete chrysosporium and Trametes sp. SYBC-L4. Four groups of decayed cassava residue were identified. Fungus N1 and N3 grouped together and showed high lignin-degrading selectivity. Fungus N5 and N8 grouped with P. chrysosporium and showed high degradation ability. During the four weeks incubation, the lignin-degrading selectivity value of fungus N3 ranged from 1.14 to 1.38 and was the best, the weight loss of fungus N5 and N8 achieved 30.93% and 33.34%, respectively. Fungus N3, N5, and N8 were identified as Pleurotus sp., Trametes sp. and Coriolopsis sp. based on 18S rDNA gene sequences, respectively. PCA is an effective method in recognizing cassava residue decayed characteristics of fungi and is helpful to screen fungi for their potential application. The three screened out fungi could be used to decay cassava residue for enhancement of its bioconversion efficiency.
|
| issn |
1027-5495 |
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https://nasplib.isofts.kiev.ua/handle/123456789/121496 |
| citation_txt |
Screening of lignin-degrading fungi for their ability to decay cassava residue / Bin Xu, Huixing Li, Chaojun Du, Ying Wang, Bin Li // Functional Materials. — 2016. — Т. 23, № 4. — С. 668-675. — Бібліогр.: 26 назв. — англ. |
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668 Functional materials, 23, 4, 2016
ISSN 1027-5495. Functional Materials, 23, No.4 (2016), p. 668-675
doi:https://doi.org/10.15407/fm23.04.668 © 2016 — STC “Institute for Single Crystals”
Screening of lignin-degrading fungi for their
ability to decay cassava residue
Bin Xu, Huixing Li, Chaojun Du, Ying Wang, Bin Li
School of Biological and Chemical Engineering,
Nanyang Institute of Technology, Nanyang 473004, P.R. China
Received July 22, 2016
To screen applicable fungi for their ability to decay cassava residue, nineteen lignin-degrad-
ing fungi were isolated by guaiacol and azure B plates. Cassava residue decayed characteristics
of the isolates were evaluated systematically by principal component analysis (PCA) of weight
and components loss data, and compared with those of Phanerochaete chrysosporium and Tram-
etes sp. SYBC-L4. Four groups of decayed cassava residue were identified. Fungus N1 and N3
grouped together and showed high lignin-degrading selectivity. Fungus N5 and N8 grouped with
P. chrysosporium and showed high degradation ability. During the four weeks incubation, the
lignin-degrading selectivity value of fungus N3 ranged from 1.14 to 1.38 and was the best, the
weight loss of fungus N5 and N8 achieved 30.93% and 33.34%, respectively. Fungus N3, N5, and
N8 were identified as Pleurotus sp., Trametes sp. and Coriolopsis sp. based on 18S rDNA gene se-
quences, respectively. PCA is an effective method in recognizing cassava residue decayed charac-
teristics of fungi and is helpful to screen fungi for their potential application. The three screened
out fungi could be used to decay cassava residue for enhancement of its bioconversion efficiency.
Keywords: Lignin-degrading fungi, cassava residue, decay, principal component analysis,
screening.
Исследованы девятнадцать грибов, разрушающих лигнин с целью разрушения отходов
маниоки. Характеристики разрушенных отходов маниоки были оценены методом главных
компонент (PCA�� по данным потерь в весе и сравнивались с такими �е характеристикамиPCA�� по данным потерь в весе и сравнивались с такими �е характеристиками�� по данным потерь в весе и сравнивались с такими �е характеристиками
для Phanerochaete chr��o��or��� chr��o��or���chr��o��or��� andand Tra�ete� sp. SYBC-L4. �ыли определены �етыре груп-sp. SYBC-L4. �ыли определены �етыре груп-. SYBC-L4. �ыли определены �етыре груп-SYBC-L4. �ыли определены �етыре груп--L4. �ыли определены �етыре груп-L4. �ыли определены �етыре груп-4. �ыли определены �етыре груп-
пы грибов, разлагавших отходы маниоки. Грибы N1 и N3 были выделены в одну группу,N1 и N3 были выделены в одну группу,1 и N3 были выделены в одну группу,N3 были выделены в одну группу,3 были выделены в одну группу,
и они показали высокую избирательность при разрушении лигнина. Грибы N5 и N8 былиN5 и N8 были5 и N8 былиN8 были8 были
помещены в одну группу с P�� chr��o��or����� chr��o��or���chr��o��or��� и показали высокую способность к деградации
лигнина. В те�ение �етырех недель инкубации гриба N3 зна�ение его лигнин разрушаю-N3 зна�ение его лигнин разрушаю-3 зна�ение его лигнин разрушаю-
щей селективности было наилу�шим и колебалось от 1,14 до 1,38, потеря в весе для грибов
N5 и N8 достигла 30,93% и 33,34%, соответственно. Грибы N3, N5 и N8 были иденти�и-5 и N8 достигла 30,93% и 33,34%, соответственно. Грибы N3, N5 и N8 были иденти�и-N8 достигла 30,93% и 33,34%, соответственно. Грибы N3, N5 и N8 были иденти�и-8 достигла 30,93% и 33,34%, соответственно. Грибы N3, N5 и N8 были иденти�и-N3, N5 и N8 были иденти�и-3, N5 и N8 были иденти�и-N5 и N8 были иденти�и-5 и N8 были иденти�и-N8 были иденти�и-8 были иденти�и-
цированы как Ple�rot�� sp.,., Tra�ete�Tra�ete� sp. and. andand �or�olo�����or�olo���� sp., основанные на генных пос-., основанные на генных пос-
ледовательностях 18S рекомбинантной ���, соответственно. PCA является ���ективнымS рекомбинантной ���, соответственно. PCA является ���ективным рекомбинантной ���, соответственно. PCA является ���ективнымPCA является ���ективным является ���ективным
методом в выявлении способности грибов к разрушению отходов маниоки. Установлено, �то
из подвергшихся скринингу образцов три гриба мо�но использовать для повышения ��-
�ективности биоконверсии отходов маниоки.
Скринінг грибів, що руйнують лігнін, на предмет їх здатності розкладати
відходи маніоки. Бінь Сюй, Хуей Сін Лі, Чао Цзюнь Ду, Ін Ван, Бінь Лі
�ослід�ено дев’ятнадцять грибів, що руйнують лігнін з метою руйнування відходів
маніоки. Характеристики зруйнованих відходів маніоки були оцінені системати�н о за
допомогою методу головних компонент (PCA�� за даними втрат у вазі і в компонентах, і
порівнювалися з такими � характеристиками для Phanerochaete chr��o��or��� and Tra�ete�
��. SYBC-L4. �ули визна�ені �отири групи грибів, що розкладають відходи маніоки. Гриби
N1 і N3 були виділені в одну групу, і вони показали високу вибірковість при руйнуванні
Functional materials, 23, 4, 2016 669
Bin Xu et al. / Screening of lignin-degrading fungi for ...
1. Introduction
Cassava residue is a lignocellulosic waste,
which is generated from cassava-based ethanol
production [1]. Approximate 0.3 million tons
(dry matter) of cassava residue are generated
annually in China [2]. Handle of the waste is a
problem and this problem will become increas-
ing severe with increased industrial produc-
tion of cassava-based ethanol. Direct discharge
of cassava residue will cause serious environ-
mental pollution and also be a huge waste of
carbohydrate resources [3]. Currently, cassava
residue is mainly used as raw material for pro-
duction of methane or protein feed. However,
the bioconversion efficiency of cassava resi-
due is low due to its recalcitrant lignocellulose
structure [1,4]�� Hence, to improve the biocon-
version efficiency of cassava residue, a promis-
ing method must be explored to decompose its
lignocellulosic components, in particular the
lignin component [5]. From the economic and
environmental perspectives, fungal decay with
lignin-degrading microorganisms has received
great interest as an alternative to physical and
chemical methods since it is a simple process
and low energy requirement [6].
The application of fungal decay in various
lignocellulosic biomass, e��g�� wheat straw, rice
straw, cotton stalks, and woody biomass have
been reported [7-10]. The low lignin-degrading
selectivity and long decay time are core prob-
lems associated with fungal decay. Therefore,
fungi with high lignin-degrading selectivity
and high degradation ability are preferred to be
applied in lignocellulosic biomass decayed [11].
However, a fungal species does not exhibit the
same characteristic to decay different lignocel-
lulosic biomass and the fungal decay specific-
ity has been reported [12]. As a consequence of
the specificity, researchers cannot screen out a
“universal” strain, and instead need to screen
appropriate fungi to decay specific lignocellu-
losic biomass [13,14].
To the best of our knowledge, there are no
reports of fungal decay in cassava residue.
Hence, the purpose of this study was to isolate
lignin-degrading fungi that decay cassava resi-
due with high lignin-degrading selectivity or
high degradation ability. Moreover, to evaluate
systematically cassava residue decayed charac-
teristics of the isolates, principle components
analysis (PCA) was applied since it has been
proved to be a useful tool to extract impor-
tant information from a large group of dataset
[12,15-17].
2. Materials and methods
2.1 Raw Material
Cassava residue was obtained from the
Yong Xiang Ethanol Co. Ltd., Wujiang, Chi-
na. The raw material was dried at 105 °C and
milled to pass through 40-mesh screen. Cas-
sava residue components were as follows: total
organic carbon (TOC�� 33.85±0.17%; hemicellu-
lose 10.49±0.70%; lignin 20.19±0.22%; cellulose
31.46±0.63%.
2.2 Fungal sources and sampling
Eighty-two samples were collected from vari-
ous places in China, including Wuxi, Hefei, Nan-
jing, Nanyang and Hangzhou. The isolates were
obtained from various fruit bodies. Phanerochaete
chr��o��or��� (CICC 40719�� and Tra�ete� sp.
SYBC-L4 (NCBI accession number: HQ891288��
were cultured for comparison with the isolates. P��
chr��o��or��� was obtained from Beina Chuan-
glian Biological Research Institute, Tra�ete� sp.
SYBC-L4 was donated by Professor Liao, Jiang-
nan University, Wuxi.
2.3 Isolation of lignin-degrading fungi
Samples were cleaned using sterile water
and then inoculated onto potato dextrose agar
(PDA�� plates containing 400 mg/L of guaiacol or
50 mg/L of azure B dye. Lignin-degrading fungi
would make the guaiacol become red or azure
B dye decolorization [18,19]. After ten days of
incubation, isolates exhibiting diffusible guaia-
col-reddening or azure B decolorization zone
were lignin-degrading fungi.
2.4 Genetic Analyses
Fungal DNA was extracted from 100 mg of
mycelia grown on potato dextrose broth (PDB��.
Total DNA was extracted using a UNIQ-10
Fungal Genomic DNA Prep Kit (Sangon Bio-
tech Co., Ltd., China) and following the manu-
facturer’s instructions.
лігніну. Гриби N5 і N8 були поміщені в одну групу з P�� chr��o��or��� і показали високу
здатність до деградації лігніну. Протягом �отирьох ти�нів інкубації гриба N3 зна�ення його
лігнін руйнує селективності було найкращим і коливалося від 1,14 до 1,38, втрата у вазі для
грибів N5 і N8 досягла 30,93% і 33,34%, відповідно. Гриби N3, N5 і N8 були іденти�іковані
як Ple�rot�� ��., Tra�ete� ��. and �or�olo���� ��., засновані на генних послідовностей 18S
рекомбінантної ���, відповідно. PCA є е�ективним методом у виявленні здатності грибів
до руйнування відходів маніоки, він виявився корисним при проведенні скринінгу грибів з
метою їх мо�ливого застосування. Встановлено, що з зазнали скринінгу зразків три гриба
мо�на використовувати для підвищення е�ективності біоконверсії відходів маніоки.
670 Functional materials, 23, 4, 2016
Bin Xu et al. / Screening of lignin-degrading fungi for ...
Polymerase chain reaction (PCR) was per-
formed in a 25 �L reaction volume containing�L reaction volume containingL reaction volume containing
1 �L of template, 0.5 �L dNTP mix (10 �m�L of template, 0.5 �L dNTP mix (10 �m of template, 0.5 �L dNTP mix (10 �m�L dNTP mix (10 �m dNTP mix (10 �m
each��, 0.2 uL Taq (5 �/�L��, 2.5�L 10�Taq reac-�/�L��, 2.5�L 10�Taq reac-/�L��, 2.5�L 10�Taq reac-�L��, 2.5�L 10�Taq reac-��, 2.5�L 10�Taq reac-�L 10�Taq reac- 10�Taq reac-
tion buffer, and 0.5 �L (10 ���� of each primer�L (10 ���� of each primer (10 ���� of each primer���� of each primerM) of each primer
(NS1: 5’GTAGTCATATGCTTGTCTC 3’, NS6:
5’ GCATCACAGACCTGTTATTGCCTC 3’��. The
amplification program was consisted of an ini-
tial preheating for 5 min at 94 °C, followed by
35 cycles of denaturation at 94 °C for 30 s, an-
nealing at 55 °C for 35 s, and extension at 72 °C
for 1 min, with a final extension of 72 °C for 8
min. The PCR products were separated in aga-
rose (1.5%, w/v�� gels using Tris-borate (TBE��
buffer, and were directly sequenced by Sangon
Biotech Co., Ltd (Shanghai, China). Sequences
were subjected to BLAST searching using the
National Center for Biotechnology Information
(NCBI�� database, and phylogenetic position was
inferred [8,19]
2.5 Fungal decay cassava residue
Fungi were maintained on PDA plates at 4
°C and periodically subcultured. The fungal colo-
nies were harvested from seven days old cultures
that were grown on PDA plates. Cultivations
were in 250-mL Erlenmeyer flasks containing
10 g cassava residue, 25 mL distilled water, and
four 10 mm agar plugs removed from the PDA
plates. The flasks were incubated at 30 °C and
humidity of 75% and shaken every three days.
After harvesting, solid fractions were dried at
105 °C until the constant weight was obtained.
Dry solids were then weighed and their content
tested [10,20].
2.6 Determination of lignin, cellulose
and hemicellulose
Lignin, cellulose and hemicellulose were de-
termined by Van Soest method [21]. The cellu-
lose was considered the difference between the
acid detergent fiber (ADF�� and acid detergent
lignin (ADL). Lignin was considered the differ-
ence between the ADL and ash content. The
hemicellulose was considered the difference
between neutral detergent fiber (NDF�� and the
ADF.
The loss ratio (%�� of weight, lignin, cellulose
and hemicellulose were calculated using the fol-
lowing equation (1��:
Loss (%�� = (�0 – ��� / �0 × 100% (1��
Where �0(g) is the initial weight of cassava resi-
due or lignocellulosic components (lignin, cellu-
lose and hemicellulose), and �(g) is the weight
of cassava residue or lignocellulosic components
after incubation.
Cassava residue is a waste byproduct gener-
ated during cassava-based ethanol production,
and it would be used as raw material for the pro-
duction of methane or protein feed [1-4]. There-
fore, lignin-degrading selectivity (LDS) was de-
fined as the following equation (2��. When LDS
value of fungi is greater than 1.0, indicating that
the fungi possess high lignin-degrading selectiv-
ity since they are prefer to degrade lignin.
LDS = lignin loss (%�� / weight loss (%�� (2��
3. Results
3.1 Primary screening of lignin-degrad-
ing fungi
Using of guaiacol or azure B are rapid and re-
liable methods to screen lignin-degrading fungi
because the structures of guaiacol and azure B
resemble to lignin. Positive fungi showed red
in guaiacol-containing plates or/and decoloriza-
tion zones in azure B-containing plates [18,19].
In the present study, nineteen lignin-degrad-
ing fungi were screened out, two fungi had a
positive reaction on both guaiacol and azure B
plates, while six fungi had a positive reaction
on guaiacol plate and eleven fungi had a posi-
tive reaction on azure B plate. Positively react-
ing fungus N1, N3, N5 and N8 were shown in
Fig. (1) since the lignin-degrading selectivity of
fungus N1 and N3 and degradation ability of
fungus N5 and N8 was better than those of oth-
ers which will be shown in following results.
3.2 Characteristics of Fungal Decay
Cassava Residue
Table 1 showed weight losses, lignocellulos-
ic components (cellulose, hemicellulose and lig-
nin) losses, and LDS values of cassava residue
which were decayed by nineteen screened fungi,
P�� chr��o��or��� and Tra�ete� sp. SYBC-L4 in
two weeks incubation. All tested fungi degrad-
ed amount of hemicellulose, and its losses were
between 17.70% and 69.88%. Lignin losses
were between 0.97% and 25.10%. Weight losses
and cellulose losses were ranged from 7.45% to
29.09%, and 7.38% to 64.32%, respectively. The
best degradation ability of tested fungi was P��
chr��o��or���, and the weight loss achieved by
29.09%. �eanwhile, the best lignin-degrading
selectivity of tested fungi was fungus N3, and
the LDS value was 1.38.
For a large number of samples, it is difficult
to achieve comprehensive and concise conclu-
sions through qualitative analysis above. Prin-
cipal component analysis (PCA) is suitable for
recognition and comparison of large numbers
of samples, and visually displays the differ-
ences in ordination maps [12,16]. Therefore,
the data in Table 1 were standardized and then
analyzed by PCA, the results were shown in
Table 2. The polynomial of the first principal
component (PC1�� and the five variables includ-
Functional materials, 23, 4, 2016 671
Bin Xu et al. / Screening of lignin-degrading fungi for ...
ing LDS value, weight loss, cellulose loss, hemi-
cellulose loss, and lignin loss was calculated by
the following equation (3��. PC1 with a variance
contribution of 58.76% showed a positive rela-
tionship with five variables, reflecting the deg-
radation ability of fungi.
PC1 = 0.55×weight loss +
+ 0.50×lignin loss+0.51×cellulose loss +
+ 0.35×hemicellulose loss+0.24×LDS (3��
The polynomial of the second principal com-
ponent (PC2�� and the five variables were calcu-
lated by the following equation (4). PC2 with a
variance contribution of 29.21% was negative
in relation to weight loss, cellulose loss, and
hemicellulose loss, but showed a positive rela-
tionship with lignin loss and LDS value, thus re-
flecting the fungal lignin-degrading selectivity.
PC2 = -0.20×weight loss +
+ 0.42×lignin loss-0.24×cellulose loss –
– 0.44×hemicellulose loss+0.73×LDS (4��
The variance contribution of two principal
components (PC1 variance 58.76% and PC2
variance 29.21%�� was 87.97%, which meet the
requirement of more than 85% [22]�� The PCA
biplot of two principal components showed the
decay characteristics of fungi (Fig. 2). Four
main groups can be discerned, set apart along
the two component axes. Group A was located
in the upper left quadrant, included fungus
N1 and N3, which were high in lignin-degrad-
ing selectivity but poor in degradation ability.
Group B, in the lower left quadrant, included
fifteen fungi that were poor in both degradation
ability and lignin-degrading selectivity. Group
C included fungus N5 and N8, which were high
in degradation ability but poor in lignin-de-
grading selectivity, and grouped with P�� chr��o-
��or����� Tra�ete� sp. SYBC-L4 was in group
D and exhibited a certain level of degradation
ability and lignin-degrading selectivity.
3.3 Cassava Residue Decayed by Iso-
lated Fungi in Four Weeks Incubation
Based on the PCA analysis above, fungus
N1 and N3 showed lignin-degrading selectivity,
while fungi N5 and N8 showed high degrada-
tion ability. To further investigate the degrada-
tion ability and lignin-degrading selectivity of
these four fungi, cassava residue was decayed
by these fungi for four weeks. Tra�ete� sp.
SYBC-L4 was selected to be compared with the
four fungi because it simultaneously exhibited
a certain level of degradation ability and lig-
nin-degrading selectivity. The results of weight
losses, components losses, and LDS value were
shown in Table 3. In terms of selectivity, fungus
N3 was better than other fungi, the LDS value
of N3 ranged from 1.14 to 1.38 and was greater
than that of other fungi. In terms of degrada-
tion ability, fungus N5 and N8 were better than
others. �oreover, fungus N5 and N8 mainly de-
cayed cassava residue in the first two weeks. To
summarize, fungus N3, N5 and N8 were finally
screened out on the basis of their characteris-
tics of cassava residue decayed.
3.4 Identification of the Isolated Fungi
The 18S rDNA of the fungus N3, N5, and
N8 were amplified by PCR and sequenced. As
shown in Table 4, comparison of the 18S rDNA
sequences with data in the NCBI database in-
dicated that the sequence of fungus N3 showed
99% identity with Ple�rot�� sp., the sequence
Fig. 1. Positively Reacting Fungus N1, N3, N5,
N8 Cultured in Different Plates. ((a�� PDA, (b��
PDA Contained 400 mg/L Guaiacol, (c�� PDA
Contained 50 mg/L Azure B dye��.
Fig. 2. PCA Plot from the Data of Weight loss,
Components losses, and LDS value of Cassava
Residue Decayed by Fungi.
672 Functional materials, 23, 4, 2016
Bin Xu et al. / Screening of lignin-degrading fungi for ...
of fungus N5 showed 99% identity with Tra�-
ete� sp., and the sequence of fungus N8 showed
99% identity with �or�olo���� sp. The identified
species are both white rot fungi, which produce
ligninolytic enzymes including laccase and
manganese peroxidase (�nP��, but no lignin
peroxidase (LiP�� [23-25].
4. Discussions
At present, cassava residue is mainly used
as raw material to produce methane or protein
feed [1-4]�� Methane is produced by anaerobic
digestion including three steps: hydrolysis,
acidogenesis and methanogenesis [1]. Protein
feed is produced by co-culturing of fungi and
yeast, supplying a certain amount of inorganic
nitrogen under solid state fermentation [4].
Bioconversion efficiency of cassava residue by
anaerobic digestion or co-culturing of fungi and
yeast is low due to the recalcitrant lignocellu-
lose structure of cassava residue, in particular
the lignin since it is a cross-linked hydrophobic
polymer that is fairly resistant to degradation
[5]. Therefore, isolation and characterization of
lignin-degrading fungi to decay cassava residue
is favorable for bioconversion of cassava resi-
due. Moreover, fungi with high lignin-degrad-
Table 1. Weight loss, components losses, and LDS of cassava residue decayed by lignin-degrading fungi
after two weeks incubation
Strain Weight loss (%�� Lignin Loss (%�� Cellulose Loss (%�� Hemicellulose
Loss (%�� LDS Value
N1 7.45 ± 0.64 6.99 ± 0.80 9.21 ± 0.51 22.69 ± 0.89 0.94
N2 12.17 ± 0.86 7.63 ± 0.40 33.47 ± 1.22 19.48 ± 0.25 0.63
N3 9.65 ± 0.48 13.32 ± 0.26 7.38 ± 0.47 45.98 ± 0.82 1.38
Tra�� �� 20.18 ± 0.42 20.31 ± 0.22 39.61 ± 0.99 38.40 ± 0.47 1.01
N5 24.74 ± 0.53 17.18 ± 0.27 44.09 ± 0.68 62.88 ± 0.40 0.69
N6 16.45 ± 0.74 7.36 ± 0.81 35.50 ± 0.43 17.70 ± 0.66 0.45
N7 15.79 ± 0.90 1.18 ± 0.28 24.55 ± 1.09 47.64 ± 1.62 0.07
N8 28.10 ± 0.82 22.54 ± 0.97 52.35 ± 0.54 63.33 ± 1.07 0.80
N9 12.80 ± 0.55 3.27 ± 0.89 21.29 ± 0.35 30.72 ± 0.40 0.26
N10 16.70 ± 0.94 3.07 ± 0.80 31.26 ± 1.04 60.16 ± 0.48 0.18
N11 15.85 ± 0.85 1.72 ± 0.59 32.74 ± 1.20 50.98 ± 0.48 0.11
N12 15.75 ± 0.56 2.64 ± 0.77 35.44 ± 0.24 38.01± 0.50 0.17
N13 17.40 ± 0.99 2.89 ± 0.52 34.64 ± 0.31 45.82± 1.08 0.17
N14 15.86 ± 0.64 3.62 ± 0.45 29.29 ± 0.10 61.20 ± 1.24 0.23
N15 14.78 ± 0.77 4.65 ± 0.64 29.45 ± 0.87 41.63 ± 0.26 0.31
N16 17.24 ± 0.75 3.51 ± 0.42 32.11 ± 1.03 40.19 ± 0.20 0.20
N17 14.89 ± 0.38 1.19 ± 0.67 28.94 ± 0.27 56.09 ± 0.20 0.08
N18 14.72 ± 0.64 1.11 ± 0.24 27.77 ± 0.17 53.38 ± 0.95 0.07
N19 16.46 ± 0.75 3.24 ± 0.60 30.49 ± 0.92 61.62 ± 0.64 0.20
N20 12.11 ±0.66 0.97 ± 0.50 23.51 ± 0.21 42.67 ± 0.39 0.08
Ph�� c 29.09 ± 0.85 25.10 ± 0.72 64.32 ± 1.10 69.88 ± 0.40 0.86
Table 2. Eigenvalues of the Variables for Principal Components and Variance Explained by PCA
Variables Principal Compo-
nent 1 (PC1�� PC2 PC3 PC4 PC5
Weight loss 0.55 –0.20 0.18 -0.69 –0.39
Lignin loss 0.50 0.42 –0.02 -0.14 0.74
Cellulose loss 0.51 –0.24 0.50 0.66 –0.08
Hemicellulose loss 0.35 –0.44 –0.81 0.19 0.03
LDS value 0.24 0.73 –0.26 0.21 –0.54
Variance explained (%�� 58.76 29.21 11.11 0.74 0.18
Functional materials, 23, 4, 2016 673
Bin Xu et al. / Screening of lignin-degrading fungi for ...
ing selectivity and/or degradation ability are
preferred. Fungi with high lignin-degrading se-
lectivity have been proved to enhance methane
production from lignocellulosic biomass [5,7,9].
Fungi with high degradation ability decay lig-
nocellulosic biomass rapidly and throughly to
enhance yeast production for improvement of
protein content and palatability of feed [2]. In
the present investigation of cassava residue de-
cayed, fungus N3 showed high lignin-degrading
selectivity and fungus N5 and N8 showed high
degradation ability. These fungi would facili-
tate the decomposing of cassava residue to en-
hance production of methane or protein feed.
The three fungus N3, N5 and N8 screened
out were identified as Ple�rot�� sp., Tra�ete�
sp. and �or�olo���� sp. respectively. Ple�rot��
sp. has shown high lignin-degrading selectivity
in different types of lignocellulosic biomass de-
cayed and has been used to enhance methane
production [9,24]. �üller and Trösch [9] con-
cluded that Pleurotus florida showed the fast-
est delignification among twenty-two tested
fungi. Wheat straw pretreated by this fungus
was fermented anaerobically to biogas, and
the gas yield was twice the amount of the un-
treated straw. The screened fungus N3, a Ple�-
rot�� sp. in the present study, also showed high
lignin-degrading selectivity in cassava residue
decayed with a maximum LDS value of 1.38,
suggesting that fungus N3 is useful for enhanc-
ing methane production from cassava residue.
Tra�ete� sp. generally shows no lignin-degrad-
ing selectivity but good degradation ability in
lignocellulosic biomass decayed [9,12] and has
been found to produce protein feed from ligno-
cellulosic biomass. JalČ et al�� [26] reported that
Tra�ete� g�bbo�a incubated on wheat straw
could significantly improve the crude protein
content and �n v�tro dry matter digestibility.
Zhu et al. [25] concluded that Tra�ete� ver��-
color has potential for laccase production us-
ing solid state fermentation, as well as the si-
multaneous improvement of the crude protein
content in corn stover. �or�olo���� sp. secrete
extracellular oxidase laccase, and have been
used to eliminate toxic waste, e.g. nonylphenol,
bisphenol A, triclosan, and azo dyes, but have
rarely been used to decay lignocellulosic bio-
mass[23]. The screened fungus N5 and N8 were
Tra�ete� sp. and �or�olo���� sp., respectively.
They both showed high degradation ability in
cassava residue decayed, and grouped with P��
chr��o��or���, a typical white rot fungus with
high degradation ability [11]. Decayed charac-
teristics of fungus N5 and N8 suggesting that
they are favorable of protein feed production
from cassava residue.
PCA was an effective method of identifying
lignocellulosic biomass decay characteristics
Table 3. Weight loss, Components losses, and LDS of Cassava Residue Decayed by the Isolates during
Four Weeks Incubation
Strain Weeks Weight Loss (%�� Lignin Loss (%�� Cellulose Loss
(%��
Hemicellulose
Loss (%��
LDS
Value
N1
1 3.37 ± 0.52 3.18 ± 0.23 3.44 ± 0.60 13.39 ± 0.17 0.94
2 7.45 ± 0.64 6.99 ± 0.80 9.21 ± 0.51 22.69 ± 0.89 0.94
3 11.22 ± 0.31 8.56 ± 0.27 18.05 ± 0.49 28.04 ± 0.14 0.76
4 15.06±0.80 11.89 ± 0.81 27.8 ± 0.61 32.17 ± 0.92 0.79
N3
1 8.61 ± 0.64 10.57 ± 0.70 7.14 ± 0.51 31.31 ± 0.65 1.23
2 9.65 ± 0.48 13.32 ± 0.26 7.38 ± 0.47 45.98 ± 0.82 1.38
3 12.54 ± 0.59 14.23 ± 0.44 10.25 ± 0.87 62.77 ± 0.72 1.14
4 16.23 ± 0.84 18.89 ± 0.20 16.12 ± 0.98 63.48 ±1.03 1.16
Tra�ete� ��.
SYBC-L4
1 16.50 ± 0.80 11.23 ±0.99 31.22 ± 0.41 30.4 ± 1.01 0.68
2 20.18 ± 0.42 20.31 ± 0.22 39.61 ± 0.99 38.4 ± 0.47 1.01
3 23.02 ± 0.88 23.96 ± 0.76 45.18 ± 1.33 40.31± 0.63 1.04
4 25.28 ± 0.97 25.82 ± 0.96 48.28 ± 1.27 45.96 ± 0.51 1.02
N5
1 19.96 ± 0.83 13.06 ± 0.50 38.03 ± 1.10 58.8 ± 0.80 0.65
2 24.74 ± 0.53 17.18 ± 0.27 44.09 ± 0.68 62.88 ± 0.40 0.69
3 27.12 ± 0.68 26.12 ± 1.00 50.32 ± 0.40 68.19 ± 0.37 0.96
4 30.93 ± 0.97 30.54 ± 0.84 54.47 ± 1.05 70.64 ± 1.14 0.98
N8
1 22.78 ± 0.50 16.56 ± 0.67 40.3 ± 0.48 60.29 ± 0.56 0.73
2 28.10 ± 0.82 22.54 ± 0.97 52.35 ± 0.54 63.33 ± 1.07 0.80
3 31.70± 0.77 25.49 ± 0.62 55.77 ± 1.05 67.16 ± 0.70 0.80
4 33.34 ± 0.98 27.66 ± 0.90 56.95 ± 1.04 70.99 ± 1.16 0.83
674 Functional materials, 23, 4, 2016
Bin Xu et al. / Screening of lignin-degrading fungi for ...
instead of electron microscopy, cytochemistry
and conventional chemical analysis. Ferraz et
al. [12] used PCA to identify and group eight
basidiomycetes and two ascomycetes cultured
on Eucalyptus grandis wood. The results
showed that PCA was very efficient in recog-
nizing wood decay characteristics. Yu [14] used
PCA to recognize the characteristics of forty-
nine fungi in Moso bamboo decayed, the results
showed that PCA was a useful tool for analyz-
ing large weight and components losses data-
set. In this study, PCA was used to favor fungal
screening for cassava residue decayed. Based
on the results of PCA, twenty-one fungi were
placed in four groups, and their decay charac-
teristics displayed visually in ordination maps
(Fig. 2��.As a consequence, fungus N3 with high
lignin-degrading selectivity and fungus N5 and
N8 with high degradation ability were screened
out conveniently.
5. Conclusions
Principal component analysis was applied
successfully to recognize the characteristics of
fungal decay in cassava residue, and is help-
ful of screening fungi for their potential ap-
plications. Three lignin-degrading fungi were
screened out for cassava residue decayed. Fun-
gus N3, a Ple�rot�� sp. exhibited high lignin-
degrading selectivity. Fungus N5 and N8 were
Tra�ete� sp.and �or�olo���� sp., respectively,
which exhibited high degradation ability. The
three screened fungi possess potential applica-
tion in the bioconversion of cassava residue to
added value products.
Acknowledgements
This work is financially supported by Key
Technology Program of Henan Province (No.
152102110156��
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Fungi Accession Number Sequence Identity
N3 a KC422248 —
Pleurotus cystidiosus strain P-24 b FJ379283.1 99%
Ple�rot�� corn�co��ae strain P-38 b FJ869181.1 99%
Ple�rot�� o�treat�� strain Po-13 b FJ379284.1 99%
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N5 a KC422247 —
Tra�ete� sp. CPCC 480671b FJ515315.1 99%
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Tra�ete� ver��color strain BCRC 36387 b AY309019.1 99%
Tra�ete� ver��color strain ATCC 11235 b AY309018.1 99%
N8 a KC422249 —
�or�olo���� gall�ca strain RLG 7630-SP b AY336772.1 99%
�or�olo���� b�r��na strain CRM-46 b AY336773.1 99%
�or�olo���� �ol�zona strain OH-184-SP b AY336771.1 99%
�or�olo���� b�r��na b JN546141.1 99%
a The isolates submitted to the National Center for Biotechnology Information (NCBI�� GenBank .
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