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CELLULOSE CHEMISTRY AND TECHNOLOGY
Cellulose Chem. Technol., 54 (9-10), 977-982(2020)
HYPSIZYGUS MARMOREUS –
A NOVEL POTENT DEGRADER OF LIGNOCELLULOSE RESIDUES
MILICA GALIĆ, MIRJANA STAJIĆ and JASMINA ĆILERDŽIĆ
University of Belgrade, Faculty of Biology, Takovska 43, 11000 Belgrade, Serbia
Corresponding author: Jasmina Ćilerdžić, simonicj@bio.bg.ac.rs
Received June 4, 2020
Hypsizygus marmoreus is an edible and medicinal commercially cultivated mushroom, whose ligninolytic potential is
still insufficiently known. Therefore, the goals of this study were to profile its Mn-oxidizing peroxidases and laccases
activity and its potential to depolymerize common agro-forestry residues. Raspberry and blackberry sawdust induced
the production of highly active laccases (26006.83 U L
-1
and 17576.79 U L
-1
, respectively), while the activity peaks of
Mn-dependent peroxidase (134.47 U L
-1
) and Mn-independent peroxidase (184.40 U L
-1
) were recorded on plum
sawdust and wheat straw, respectively. The pretreatment of corn stalks resulted in the highest lignin removal (57.15%),
while the highest lignin/cellulose removal ratio (selectivity) was noted on oak sawdust (17.69). The maximal (20%) and
the minimal (8.30%) dry matter loss were obtained for corn stalks and oak sawdust, respectively. The presented results
showed the exceptional potential of the ligninolytic system of H. marmoreus HAI 605 and the possibility of its
application in numerous biotechnological processes.
Keywords: agro-forestry residues, Hypsizygus marmoreus, ligninolytic enzymes, lignocelluloses depolymerization
INTRODUCTION
One of the main trends of modern society is to
provide sufficient amounts of high-value food and
energy due to the geometric progression of the
human population and intensive industrial and
technological development. However, it leads to
great pressure on natural resources and
ecosystems in general, and therefore there is a
need for the creation of environmentally friendly
procedures for food and energy production,
aiming to preserve the integrity of ecosystems.
1
Nowadays, numerous studies are focused on the
definition of conditions for the most efficient
usage of lignocellulosic wastes, which are
produced annually in huge quantities, but are not
used, despite their great potential, and mostly
improperly deposited or combusted, releasing
CO
2
and other pollutants, and adversely affecting
the environment.
2-4
These wastes are mainly
generated in agriculture and forestry, in the food
industry as well as municipal wastes (yard
trimmings and wood), and could be excellent
renewable sources for the production of not only
feed and high nutritional value food (such as
mushroom fruiting bodies), but also biofuels,
chemicals, and industry-related products.
5
According to the estimation of the
International Renewable Energy Agency
(IRENA),
6
the consumption of renewable energy
resources in the European Union will increase
from 17% in 2015 to 34% in 2030, and biomass
will remain a key energy source. Nowadays,
bioethanol, one of the most widely used
alternative energy sources, is mainly produced by
fermentation of dominant crops waste, i.e. rice,
corn, or wheat residues.
7
However, numerous
challenges lie ahead for producers, such as what
plant residues should be used, how to
economically and primarily environmentally
friendly degrade lignin, the most recalcitrant
molecule in the plant cell wall, etc. Preference
should be given to biomass that could not be used
as food/feed and has a more favorable energy
input/output ratio, as well as to biological
delignification.
1,8
The most efficient participants
in pretreatment, the first phase in the process of
bioethanol production, are white-rot fungi, due to
their remarkable capacity to produce ligninolytic
enzymes, which oxidize lignin and facilitate
access to holocellulose.
9
However, the efficiency
of these enzymes varies from species to species,
MILICA GALIĆ et al.
978
even from strain to strain, and depends on
cultivation conditions.
10-12
Therefore, the most
effective and selective lignin remover and
optimized conditions for the production of its
most active enzyme isoforms should be found.
Hypsizygus marmoreus (Peck) Bigelow is an
edible and medicinal mushroom, which is
cultivated commercially, especially in East Asia.
13
This species was the object of numerous studies
focused on its nutritional value, bioactive
compounds, and healthcare functions, while its
potential to degrade lignin has only been the topic
of several researches.
14-17
Therefore, the
objectives of this study were to determine the Mn-
oxidizing peroxidases and laccases activity profile
in H. marmoreus cultivated on eight common
European agro-forestry residues, as well as its
capacity to delignify selected plant materials.
EXPERIMENTAL
Organism and growth conditions
The culture of the studied Hipsizygus marmoreus
HAI 605 was obtained from the Institute of Evolution,
University of Haifa, Israel (HAI), and maintained on
Malt agar medium in the culture collection of the
Institute of Botany, Faculty of Biology, University of
Belgrade.
The inoculum was obtained by inoculation of
synthetic glucose/NH
4
NO
3
/yeast extract medium with
mycelium of 7-day old culture, incubation at room
temperature on a rotary shaker for 7 days, washing the
harvested biomass with sterile distilled water (dH
2
O),
and its homogenization with dH
2
O in a laboratory
blender (Waring, USA).
11
The inoculum, in the amount
of 3.0 mL, was used for inoculation of 6.0 g of tested
agro-forestry residue (apple-, blackberry-, grapevine-,
oak -, plum-, and raspberry sawdust, corn stalks, and
wheat straw) soaked with 30.0 mL of the modified
synthetic medium (without glucose). Solid-state
cultivation was carried out in 250 mL flasks at 25 °C
for 21 days.
Assays of enzyme activity and total protein
production
The ligninolytic enzymes were extracted by the
method of Ćilerdžić et al.,
11
i.e. by sample stirring with
50.0 mL of dH
2
O at 4 °C for 10 min. The supernatant
obtained after extract centrifugation (4 °C, 3000 rpm,
15 min) was used for measurement of the activity of
MnP-oxidizing peroxidases [Mn-dependent peroxidase
(MnP, EC 1.11.1.13) and Mn-independent peroxidase
(MnIP, EC 1.11.1.16)] and laccase (EC 1.10.3.2), as
well as total protein content, by a spectrophotometer
(CECIL CE2501 (BioQuest), UK).
The activities of Mn-oxidizing peroxidases and
laccases were determined by using 3 mM phenol red
(ε
610
= 22000 M
-1
cm
-1
) and 2,2
'
-azino-bis-[3-
ethyltiazoline-6-sulfonate] (ABTS) (ε
436
= 29300 M
-1
cm
-1
), respectively.
11
Enzymatic activity was expressed
in U L
-1
, and the activity of 1U presents the amount of
enzyme that transforms 1.0 µmol of substrate per min.
The total protein content was determined by the
method of Silva et al.,
18
utilizing Bradford’s reagent
and bovine serum albumin as standard, and expressed
in mg mL
-1
. The value was used for the definition of
the specific enzyme activity (U mg
-1
), an indicator of
enzyme purity.
Determination of lignin, cellulose, and hemicellulose
contents
The loss of substrate dry matter (%) was
determined by the equation (Mi – Mf)/Mi x 100, where
Mi represents the initial lignocellulosic mass and Mf –
the mass after fermentation.
The contents of lignin, cellulose, and
hemicelluloses were determined by modified methods
of Kirk and Obst,
19
and Van Soest et al.
20
A dried
ground sample (1.0 g) was treated with a neutral
detergent/Na
2
SO
3
mixture under refluxing conditions
to remove soluble sugars, proteins, lipids, and vitamins.
The obtained biomass presented neutral detergent
fibers (NDF). Acidic detergent fibers (ADF) were
obtained by the treatment of the samples with acidic
detergent solution, and the difference between NDF
and ADF represented the hemicelluloses amount. The
lignin content (LC), expressed as the percentage of
quantity present in the initial sample, was defined after
sample incubation with 72% H
2
SO
4
at 30 °C and its
hydrolysis at 120 °C. The cellulose content was
calculated as the difference between ADF and LC.
The selectivity index, i.e. the ratio between the
amounts of removed lignin and cellulose, was used as
an indicator of selectivity in lignin degradation.
Statistical analyses
All the experiments were done in three replicates
and the results were expressed as mean ± standard
error. Assaying any significant differences among
means was performed by one-way analysis of variance
(ANOVA) and Tukey’s test, using STATISTICA,
version 6.0 (StatSoft, Inc., Tulsa, USA). Statistical
significance was declared at p<0.05.
RESULTS AND DISCUSSION
The majority of the tested agro-forestry
residues stimulated the production of laccase and
Mn-oxidizing peroxidases by Hypsizygus
marmoreus HAI 605 (Fig. 1). The synthesized
laccase isoforms were characterized by extremely
high activity, especially those produced during the
fermentation of raspberry sawdust (26006.83 U L
-
1
). Blackberry-, grapevine- and oak sawdusts were
also good inducers of laccase activity (17576.79
U L
-1
, 13139.93 U L
-1
, and 9914.67 U L
-1
,
Lignocellulose degradation
979
respectively), while apple- and plum sawdusts and
wheat straw inhibited the synthesis of this enzyme.
Contrary to laccase, the activities of the
synthesized Mn-oxidizing peroxidases were
remarkably lower (Fig. 1). H. marmoreus
produced more active isoforms of MnIP than of
MnP. The most favorable substrate for MnIP
activity was wheat straw (184.40 U L
-1
);
grapevine-, plum- and blackberry sawdusts were
also good inducers of the activity (126.89 U L
-1
,
123.11 U L
-1
, and 109.85 U L
-1
, respectively),
while apple sawdust and corn stalks inhibited the
synthesis of the enzyme. In the case of MnP, plum
sawdust was the best stimulator of the activity
(134.47 U L
-1
), and further, the activity gradually
decreased in the following order: 62.50 U L
-1
(corn stalks) > 56.82 U L
-1
(blackberry sawdust) >
35.98 U L
-1
(raspberry sawdust) > 28.41 U L
-1
(grapevine sawdust) > 15.10 U L
-1
(wheat straw).
Apple sawdust also inhibited the synthesis of
MnP.
The agro-forestry residues used in the study
also affected the protein production and,
consequently, the specific activities of the
enzymes. The highest protein content was noted
after corn stalk fermentation (3.8 mg mL
-1
) and
the lowest – after apple sawdust fermentation (0.6
mg mL
-1
). A significant value of specific enzyme
activities was noted for laccase produced during
H. marmoreus cultivation on plum sawdust (22.34
U mg
-1
), while specific activities of Mn-oxidizing
peroxidases were insignificant, less than 1.0 U
mg
-1
.
Owing to the ability to synthesize the
mentioned enzymes, H. marmoreus HAI 605 was
a good degrader of lignocellulosic residues.
Namely, it caused the loss of corn stalk dry matter
of even 20.00%, the reduction was 5-7% lower
after the fermentation of raspberry-, apple-,
grapevine- blackberry- and plum sawdust, and
wheat straw, while this percentage was the
smallest in oak sawdust (8.30%) (Table 1).
The type of plant residues also affected the
enzymes’ efficiency to cause degradation of
lignocellulosic polymers. After 21 days of
fermentation, the maximal reduction of lignin
content was noted in corn stalks (57.15%),
slightly lower in apple sawdust (49.12%), and the
lowest in plum sawdust (5.72%) (Table 1).
However, H. marmoreus HAI 605 was not only a
highly effective delignifier of corn stalks and
apple sawdust, but also a depolymerizer of
hemicelluloses (24.89% and 28.74%, respectively)
and cellulose (33.36% and 24.94%, respectively),
which was reflected in low selectivity indices
(1.71 and 1.97, respectively).
Figure 1: Effect of agro-forestry residues on activity of Mn-dependent peroxidase, Mn-independent peroxidase and
laccase produced by Hypsizygus marmoreus HAI 605 during solid-state cultivation
MILICA GALIĆ et al.
980
Table 1
Extent of dry mass loss and depolymerisation of selected lignocellulose residues by Hypsizygus marmoreus HAI 605
Fiber composition of samples
(mg)
Extent of polymer degradation
(%)
Agro-
forestry
residues
Studied
species
Sample
weight
(g) Lignin Cellulose Hemicelluloses
Dry
matter
loss (%) Lignin Cellulose Hemicelluloses
Selectivity
index
Apple
wood
sawdust
Control
HAI 605
6.00
5.08
1158.00
589.16
2808.00
2107.79
1176.00
838.04
/
15.33
/
49.12
/
24.94
/
28.74
/
1.97
Blackberry
sawdust
Control
HAI 605
6.00
5.21
1218.00
770.78
2712.00
1963.42
1038.00
843.70
/
13.17
/
36.72
/
27.60
/
18.72
/
1.33
Corn
stalks
Control
HAI 605
6.00
4.80
594.00
254.51
2796.00
1863.18
1860.00
1397.38
/
20.00
/
57.15
/
33.36
/
24.89
/
1.71
Grapevine
sawdust
Control
HAI 605
6.00
5.15
1421.41
963.24
2652.00
2420.97
887.08
710.84
/
14.15
/
32.23
/
8.17
/
19.87
/
3.94
Oak
sawdust
Control
HAI 605
6.00
5.50
1530.00
1034.75
2808.00
2757.50
1159.00
891.65
/
8.30
/
32.37
/
1.83
/
23.13
/
17.69
Plum
sawdust
Control
HAI 605
6.00
5.23
1837.49
1732.45
2544.00
1648.71
1368.00
743.23
/
12.62
/
5.72
/
35.19
/
45.67
/
0.16
Raspberry
sawdust
Control
HAI 605
6.00
5.09
1200.00
951.46
2160.00
1897.82
1308.00
803.90
/
15.20
/
20.71
/
12.14
/
38.54
/
1.71
Wheat
straw
Control
HAI 605
6.00
5.13
666.00
554.26
2418.00
2217.02
1692.00
1339.45
/
14.50
/
16.78
/
8.31
/
20.84
/
2.02
Lignocellulose degradation
981
The highest selectivity index (17.69) was
noted after oak sawdust fermentation, indicating a
significant reduction of lignin content (32.37%)
and cellulose loss of only 1.83%. Likewise, this
species showed a significantly high
delignification potential during cultivation on
corn stalks (57.15%), apple sawdust (49.12%),
blackberry sawdust (36.72%), and grapevine
sawdust (32.23%). However, these substrates also
induced the synthesis of highly active forms of
holocellulases, causing cellulose
depolymerization and low selectivity indices
(1.33–3.94). The lowest selectivity index, of only
0.16, was obtained for plum sawdust. Namely,
during cultivation on this residue, the enzymatic
system of
H. marmoreus
was more efficient in
cellulose and, especially, hemicellulose removal
(35.19% and 45.67%, respectively), in
comparison with lignin, of which only 5.72% was
degraded (Table 1).
The noted positive correlation between
primarily laccase activity and the extent of lignin
removal from some tested plant residues during
solid-state fermentation indicated that laccase was
a key enzyme in lignin mineralization. On the
contrary, Songulashvili
et al
.
15
noted that
submerged cultivation of this species on mandarin
peels and residues of ethanol production from
wheat grain was not favorable for the synthesis of
laccase and especially MnP, whose activity was
not even detected. On the other hand, Xu
et al
.
16
reported the production of MnP with significant
activity (~250 U L
-1
) by this species after only 7-
day long submerged fermentation of malt–yeast
extract–peptone–glucose–bran extract, and thus
emphasized that these two groups of enzymes
(peroxidases and oxidases) have a synergistic
effect on lignin degradation, namely at the
beginning of the process, peroxidases possess a
key role in delignification, while over the time
this role is taken by oxidases,
i.e.
laccases.
However, in some cases, solid cultivation also
caused the absence of some ligninolytic enzymes,
for example,
Hypsizygus tessulatus
did not
produce laccase in the solid fermentation of wheat
straw.
11
Based on sequencing and characterization
of the gene responsible for laccase synthesis in
H.
marmoreus
(
lcc1
), Zhang
et al
.
17
proposed that
lignocellulosic materials regulate the synthesis of
the enzyme on the transcriptional level. Generally,
H. marmoreus
,
together with
Pleurotus
spp.,
makes a group of highly potential laccase
producers. Namely, Dong
et al
.
21
and
Ć
ilerdž
ić et
al
.
22
detected remarkably high laccase activity
(35000 U L
-1
) synthesized after a few weeks of
sugarcane bagasse fermentation by
P. ostreatus
and oak sawdust fermentation by
P. pulmonarius
.
In comparison with other white-rot species,
H.
marmoreus
had multi-fold stronger potential for
the production of highly active laccase isoforms.
Thus,
Auricularia auricula
synthetized a low
active laccase isoform only in blackberry sawdust
based medium,
12
while this enzyme was not
produced by
Ganoderma lucidum
during wheat
straw solid-state fermentation.
11
According to the high capacity of laccase
production,
H. marmoreus
was a significantly
stronger delignifier than numerous fungal species.
Thus,
H. marmoreus
was a better and more
selective degrader of corn residues than
Irpex
lacteus
and
Phanerochaete chrysosporium
.
22,23
Namely,
I. lacteus
caused the loss of 37.6% corn
stalk lignin, but after as many as 42 days of
pretreatment, while ~50% of corn stover lignin
was degraded by
Ph. chrysosporium
after 30 days.
Although
H. marmoreus
showed a similar
potential of oak sawdust delignification to that of
Ganoderma applanatum
strain studied by
Ć
ilerdž
ić et al
.,
24
its selectivity was remarkably
higher,
i.e.
in contrast with
H. marmoreus
,
G.
applanatum
simultaneously mineralized a high
percentage of both lignin and holocellulose.
However,
Hypsizygus tessulatus
was a better
delignifier than
H. marmoreus
HAI 605, but the
degradation selectivity was similar.
CONCLUSION
Despite the studies already perfomed on
Hypsizygus marmoreus
, these results demonstrate
for the first time high diversity in the profile of
ligninolytic enzymes produced on until now
untested agro-forestry residues.
H. marmoreus
HAI 605 is proven as a highly potent producer of
enzymes and consequently a remarkable
delignifier of the selected residues, which makes
it a promising participant for various
biotechnological processes. The demonstrated
high selectivity for lignin over holocellulose
degradation by
H. marmoreus
, in comparison to
other wood rot fungi, is one more contribution of
the present research. According to data from the
Food and Agricultural Organization, Serbia is a
significant global producer of raspberry, plum,
blackberry, apple, and grape, and their residues
could be efficiently biotransformed into various
value-added products. This offers special
economical and ecological importance to our
results. Finally,
H. marmoreus
HAI 605 deserves
MILICA GALIĆ et al.
982
further investigations, aiming to introduce new
biotechnological applications.
ACKNOWLEDGEMENTS
: This study was
carried out under project No. 173032, financially
supported by the Ministry of Education, Science
and Technological Development of the Republic
of Serbia.
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