J. Microbiol. Biotechnol. (2015), 25(6), 803–813
http://dx.doi.org/10.4014/jmb.1410.10054
Research Article
Review
jmb
Fungal Growth and Manganese Peroxidase Production in a Deep
Tray Solid-State Bioreactor, and In Vitro Decolorization of Poly R-478
by MnP
Xinshan Zhao, Xianjun Huang, JuntaoYao, Yue Zhou, and Rong Jia*
College of Life Science, Anhui University, Hefei 230601, P.R. China
Received: October 23, 2014
Revised: December 11, 2014
Accepted: December 13, 2014
First published online
December 24, 2014
*Corresponding author
Phone: +86-551-6386-1005;
Fax: +86-551-6386-1281;
E-mail: [email protected]
The growth of Irpex lacteus F17 and manganese peroxidase (MnP) production in a selfdesigned tray bioreactor, operating in solid-state conditions at a laboratory scale, were
studied. The bioreactor was divided into three layers by three perforated trays. Agroindustrial residues were used both as the carrier of bound mycelia and as a nutrient medium
for the growth of I. lacteus F17. The maximum biomass production in the bioreactor was
detected at 60 h of fermentation, which was consistent with the CO2 releasing rate by the
fungus. During the stationary phase of fungal growth, the maximum MnP activity was
observed, reaching 950 U/l at 84 h. Scanning electron microscopy images clearly showed the
growth situation of mycelia on the support matrix. Furthermore, the MnP produced by I.
lacteus F17 in the bioreactor was isolated and purified, and the internal peptide sequences
were also identified with mass spectrometry. The optimal activity of the enzyme was detected
at pH 7 and 25°C, with a long half-life time of 9 days. In addition, the MnP exhibited
significant stability within a broad pH range of 4-7 and at temperature up to 55°C. Besides
this, the MnP showed the ability to decolorize the polymeric model dye Poly R-478 in vitro.
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2015 by
The Korean Society for Microbiology
and Biotechnology
Keywords: Irpex lacteus F17, tray bioreactor, solid-state fermentation, manganese peroxidase,
polymeric dye Poly R-478, decolorization
Introduction
White-rot fungi produce extracellular ligninolytic enzymes,
such as manganese peroxidase (MnP, E.C. 1.11.1.13), lignin
peroxidase (LiP, E.C. 1.11.1.14), and laccase (Lac, E.C.
1.10.3.2) that can efficiently degrade lignin as well as many
recalcitrant organic pollutants. Among these enzymes, MnP
is one of the most frequently used for the transformation of
environmental pollutants, such as synthetic dye decolorization
as well as polycyclic aromatic hydrocarbons and tetracycline
degradation [7, 11, 33]. MnP is a glycosylated hemecontaining enzyme and can catalyze the oxidation of Mn2+
to Mn3+ in a H2O2-dependent reaction. Mn3+ is stabilized by
organic acid, and the Mn3+-acid complex acting as the
active redox mediator can attack phenolic lignin structures,
resulting in the formation of unstable free radicals that
tend to disintegrate spontaneously. A large number of
white-rot fungi, including Phanerochaete chrysosporium, Trametes
versicolor, Pleurotus ostreatus, Bjerkandera sp. BOS55, and
others, have been reported to produce MnP [10].
Owing to the potential applications of MnP in treating
environmental pollutants, there is general interest in
producing the enzyme biotechnologically. However, largescale enzyme production from white-rot fungi is limited
mainly owing to the lack of suitable bioreactor configurations
for fungal growth and their metabolism [27]. Although
several studies on the development of innovative bioreactor
systems have already been carried out [7, 25, 32], an ideal
bioreactor for ligninolytic enzyme production from white-rot
fungi has not yet been shown [6, 8]. Furthermore, the high
production cost is another barrier to the commercialization
of these enzymes.
Solid-state fermentation (SSF), which is carried out without
the use of free-flowing water, is the most economic process
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for ligninolytic enzyme production owing to the use of
agro-industrial residues as the nutrient medium for whiterot fungi. This solid matrix can also be employed as a
natural support for the development of filamentous fungi
in SSF. In addition, these agro-industrial residues are rich
in lignin or cellulose, which are inducers for ligninolytic
enzyme production, thus stimulating much higher enzyme
production in SSF processes. In fact, SSF has become an
alternative large-scale production system for enzymes and
secondary metabolites [9, 30].
I. lacteus F17 (CCTCC AF 2014020) — a local white-rot
basidiomycete isolated by our laboratory — has been
found to be a producer of MnP. The fungus I. lacteus has
been reported to be very efficient in degrading a wide
variety of recalcitrant organic pollutants, including various
types of dyes and polycyclic aromatic hydrocarbons (PAH)
[2, 13, 19, 26, 28]. MnP proved to be the main enzyme
during the decolorization and PAH degradation by I. lacteus
[2, 13, 29]. These features suggest this organism to be a
good candidate for further applications at a larger scale
and make it an interesting objective to scale up ligninolytic
enzymes production.
In the present study, a laboratory-scale bioreactor was
designed to examine the feasibility of utilizing agro-industrial
residues as the nutrient and support for the growth of the
white-rot fungus I. lacteus F17, as well as for scale-up of
MnP production. Furthermore, the MnP produced by
I. lacteus F17 in the bioreactor was purified and partially
characterized to gain more information on its stability for
application. Finally, the ability of the MnP to degrade
aromatic pollutants was also assessed by monitoring the
decolorization of the polymeric model dye Poly R-478.
121°C for 20 min. The grown mycelial mat collected from several
slants was washed with sterile water and homogenized by using a
Waring blender. Then, 500 ml flasks containing 150 ml of liquid
medium (potato extract 200 g/l, glucose 20 g/l, KH2PO4 3.0 g/l,
and MgSO4·7H2O 1.5 g/l) were inoculated with 20 ml of the
mycelial suspension and incubated at 28°C on a rotary shaker at
120 rpm for 3 days for the formation of mycelial pellets.
The homogenized mycelia obtained by crushing the mycelial
pellets in a Waring blender were inoculated into a 7.23 L bioreactor
containing 300 g of solid substrate and 300 ml of water (the water
holding capacity was 1 ml/g (1 ml water per gram dry medium)).
Fermentations were performed at 28°C for 7 days.
Bioreactor Configuration and Operating Conditions
The designed tray bioreactor used in this study contains a
cylindrical glass vessel with 16.0 cm diameter, 36.0 cm height, and
7 L working volume (Fig. 1). The outer frame of the bioreactor and
the air-intake casing were made of glass. The bioreactor was
vertically divided into three layers: top (10 cm height), middle (10 cm
Materials and Methods
Microorganism and Growth Medium
I. lacteus F17 was isolated from a decayed wood chip pile in the
vicinity of Hefei, China, and was stored in the China Center for
Type Culture Collection (CCTCC AF 2014020). The fungus was
cultured on potato dextrose agar (PDA) slants for 7 days at 28°C
and then preserved at 4°C. The PDA medium contains 200 g/l of
potato extract, 20 g/l of glucose, and 20 g/l of agar.
Solid-State Fermentation
SSF substrate preparation was based on results of our previous
research. Dried, ground, and sifted pine sawdust, rice straw, and
soybean powder mixed at the ratio of 0.52:0.15:0.33 in weight were
used both as an attachment place and to supply nutrients to the
I. lacteus F17.
Prior to use, these solid materials were mixed and autoclaved at
J. Microbiol. Biotechnol.
Fig. 1. The solid-state deep tray bioreactor.
(A) Schematic diagram of external circulation of air. 1, power; 2, air
pump; 3, air filter; 4, NaOH solution; 5, bioreactor. (B) Schematic
diagram of the right and front views of the solid-state reactor. 6, gas
exhaust; 7, cover; 8, air inlet; 9, sampling port.
Fungal Growth and MnP Production in a Tray Bioreactor
height), and bottom (10 cm height). Each layer was composed of a
removable perforated tray, where 100 g initial dry weight of the
mixed solid matrix was placed, forming a layer of about 3.5 cm
thickness, respectively. Each perforated tray, made of stainless
steel, had 34 circular holes with a diameter of 0.5 mm. The 10 cm
height between each tray in the deep-bed bioreactor was designed
for better temperature control and metabolic heat removal. Three
sampling ports (inner diameter 2.4 cm) were arranged above each
tray. The bioreactor had two air inlets and a gas exhaust from the
outside to ensure effective circulation of air. The air pumped into
the bioreactor was separated through five vents at each layer,
which kept the atmosphere near saturation and uniform distribution.
A ground glass covered the top of the bioreactor, and three rubber
plugs were used to seal the sampling ports. A constant flow
ventilation was achieved in the bioreactor through an air pump
that moved the air through a solution of NaOH, which absorbed
CO2. During the process of SSF, the entire bioreactor was kept
inside a constant-temperature incubator maintained at 28°C,
and humidified air was supplied continuously at airflow rate of
0.4 l/min. The samples were removed from three sampling ports
at regular intervals of 12 or 24 h and were analyzed for enzyme
activity and biomass. The experiments were conducted in triplicate
and the results described are the mean of three replicates.
Biomass Analysis
In the present study, the biomass produced during SSF was
quantitatively analyzed each day by measuring the nucleic acids
present in the fermentation medium [20]. Samples of the medium
(0.4 g) from each layer were collected every 24 h, except that the
first sample was taken at 12 h after the start of SSF. After
pulverizing the sample in a white porcelain mortar, 25 ml of 5%
trichloroacetic acid was added and the mixture was constantly
stirred for 25 min at 80°C. Subsequently, the mixture was cooled
in an external ice bath. After centrifugation of the mixture (for
15 min at 5,653 ×g and 4°C), the absorbance of the supernatant at
260 nm was measured, and the medium without inoculation
through the same treating process was used as the blank control.
Different amounts of mycelia were weighted precisely and dealt
with the method as mentioned above. The linear relationship
between the weight of mycelia and the absorbance of the
supernatant at 260 nm was calculated. The biomass was expressed
as grams per gram matrix. Furthermore, CO2 emission during SSF
was also tested by carrying out acid–base titration at every 12 h to
analyze the fungal growth condition. All the experiments were
performed in triplicate, and the results were expressed as the
mean values. Deviation values are standard deviations based on
triplicate determinations.
Scanning Electron Microscopy
The status of I. lacteus F17 mycelium growth in the tray
bioreactor was observed by scanning electron microscopy (SEM).
The mycelium samples collected from the middle layer of the
bioreactor at different fermentation periods were fixed in 0.1 M
805
phosphate buffer (pH 7.4) with 5% (v/v) glutaraldehyde for 2 h at
4°C. Then, the samples were dehydrated sequentially using 20%,
40%, 60%, 80%, and 100% ethanol [10]. The samples were coated
with gold before examination under a scanning electron microscope
(Hitachi S-4800 SEM, Japan). The substrate without fungus was
considered as the blank. All the images were taken at a magnification
of 500×.
Enzyme Activity Assays
To obtain enzyme extracts, 5 ml/g dry matrix of 10 mM sodium
acetate buffer (pH 4.5) was added to the sample in an Erlenmeyer
flask, and the flask was incubated on a rotary shaker at 120 rpm
for 30 min. Then, the supernatants were used for enzyme activity
assays.
The MnP activity in the supernatant was measured with Mn2+ in
0.11 M sodium lactate buffer. Oxidation of Mn2+ to Mn3+ was
determined by the increase in absorbance at 240 nm [14]. One unit
(U) of MnP activity was defined as the oxidization of 1 µmol of
Mn2+ per minute. The enzyme activities were expressed in U/l.
The lignin peroxidase (LiP) assay was based on the oxidation of
veratryl alcohol to veratraldehyde, determined by the increase in
absorbance at 310 nm [12]. One unit (U) of LiP activity was
defined as the oxidation of 1 µmol of veratryl alcohol per minute.
The laccase (Lac) assay was based on the oxidation of ABTS,
determined by the increase in absorbance at 420 nm [35]. One unit
(U) of Lac activity was defined as the oxidation of 1 µmol of ABTS
per minute. All the experiments were performed in triplicate, and
all the enzyme activity assays were carried out at 25°C.
Enzyme Purification
In order to investigate the enzyme production by I. lacteus F17
during SSF in the bioreactor, enzyme activities were analyzed
daily. The enzyme extracts containing the highest MnP activity
were concentrated by ammonium sulfate (70%) and dialyzed
against 10 mM of sodium acetate buffer (pH 4.5). After this, the
crude enzyme was loaded onto a DEAE-cellulose column (Whatman
DE52, England) (2.6 × 30 cm), which had been equilibrated with
10 mM of sodium acetate buffer (pH 4.5) at 4°C. Elution of the
proteins was achieved with a flow rate of 100 ml/h by establishing
a linear gradient first with 10 mM of sodium acetate buffer
(pH 4.5) and then with 0–0.5 M of NaCl at 4°C. Fractions with
MnP activity were pooled, concentrated by a Stirred Ultra
filtration Cell Model 8010 system (10 kDa cut-off polyethersulfone
membranes; Millipore Corporation, USA) to approx. 1 ml, and
then loaded to a Sephadex G-75 gel filtration column (Fluka, USA)
(1.7 × 100 cm). Proteins from this column were eluted with 10 mM
of sodium acetate buffer (pH 4.5) at a flow rate of 20 ml/h and
temperature of 4°C. The peak containing MnP was collected,
concentrated, and then applied to FPLC (FPLC with Superdex 75
10/300 column (Pharmacia)), which was previously equilibrated
with 10 mM of sodium acetate buffer (pH 4.5). Elution of proteins
from this column was detected by continuous recordings of
absorbance at 280 and 406 nm. The enzyme solution was stored at
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4°C for the characterization and decolorization experiments.
Enzyme Characterization
Enzyme electrophoresis and mass spectrometry. Electrophoresis
on polyacrylamide gel in denatured conditions (SDS-PAGE) was
carried out using a 10% Tris/HCl separation gel and 3% Tris/HCl
stacking gel. After SDS-PAGE, the gels were stained with Coomassie
Brilliant Blue R-250.
The protein was excised from the gel and digested with trypsin.
Then the resulting peptide mixture was analyzed by LC/MS (liquid
chromatography-mass spectrometry) with a mass spectrometer,
ProteomeX-LTQ (ThermoFisher Scientific, USA). All complete or
partial sequences of this protein were searched against those of the
proteins from BLAST similarity search to obtain the identifications
with higher confidence.
Determination of Kinetic Parameters of MnP
The Km and kcat values of MnP were calculated by the
hyperbolic non-linear least-square method, using MnSO4 as the
substrate at varying concentrations (0.025-0.2 mM). The reactions
were carried out in 0.11 M of sodium lactate buffer (pH 4.5) with
0.1 mM of H2O2.
Effects of Temperature and pH on MnP Activity and Stability
Determination of the optimum temperature for MnP activity
was carried out in 0.11 M of sodium lactate buffer over temperatures
from 15°C to 65°C at pH 4.5. To evaluate the thermal stability of
the purified MnP, the enzyme was incubated at different
temperatures ranging from 25°C to 65°C for 1-3 days and aliquots
of samples were removed at every 24 h for analysis.
The effect of pH on MnP was determined at 25°C using citrate–
phosphate buffer with a pH range of 2.2–8. The pH stability was
assayed by incubating the enzyme at different pH values (pH 3–7,
citrate–phosphate buffer) at 25°C for 0-35 h. The remaining activities
were measured using the enzyme activity assays mentioned above.
In Vitro Decolorization of Poly R-478 by MnP
Dye decolorization was measured spectrophotometrically at
520 nm (WFV UV-2100 Spectrophotometer; Shanghai, China),
which is the maximum visible absorbance of the dye Poly R-478.
The reaction mixture contained citrate–phosphate buffer (62.8 mM),
MnSO4 (1 mM), H2O2 (0.2 mM), Poly R-478 dye (0.001%), and
200 µl of the purified MnP or 100 µl of crude MnP in a total
volume of 1 ml. The MnP activity employed was 900 U/l. The
reaction was initiated by the addition of H2O2 and the mixture was
incubated at different temperatures and pH conditions for 1 h.
The decolorization of Poly R-478 represents the percentage decrease
in the peak of absorbance at 520 nm. Control samples, without
enzyme or H2O2, were done in parallel under identical conditions.
The optimal temperature and pH conditions for dye decolorization
were carried out in vitro with purified MnP and crude enzyme
preparations.
J. Microbiol. Biotechnol.
The decolorization efficiency was calculated according to the
following Eq. (1):
A0 – At
Decolorization ( % ) = ---------------- × 100%
A0
(1)
where A 0 is the initial absorbance at 520 nm and At refers to the
absorbance at 520 nm at reaction time t. The data were the mean
values of triplicates.
Results
Production of Biomass in the Deep Tray Bioreactor
In SSF, the mycelium cannot be directly quantified
because of its strong adherence to the solid matrix; hence,
indirect methods such as analyses of ATP, enzymatic
activity, respiration rate, nutrient consumption, and cell
constituents are employed to evaluate the biomass. Nucleic
acids evolution could be used as an indirect measurement
of the growth of the white-rot fungus. The biomass
production profile of I. lacteus F17 in the tray bioreactor is
depicted in Fig. 2A. In SSF using agricultural waste as the
substrate, the biomass of the fungus increased rapidly in
the bioreactor at the beginning of fermentation, peaked at
60 h, and then reached a plateau until 156 h. The maximum
biomass production at the top, middle, and bottom layers
of the bioreactor was 0.25, 0.23, and 0.24 g per gram matrix,
respectively. The rate of CO2 released by I. lacteus F17
under SSF is shown in Fig. 2A, which indicated the metabolic
activity of the fungus in the tray bioreactor. The release rate
rapidly increased for 36 h, reached to the highest value at
60 h, and then reduced significantly. When compared with
biomass production, the fungus grew rapidly by consuming
carbon sources at the beginning of the 60 h fermentation,
leading to high rate of CO2 release. After 60 h, the biomass
production remained almost constant and the CO2 release
rate decreased, indicating that the respiratory metabolism
of fungus was becoming increasingly stressed.
Production of MnP in the Deep Tray Bioreactor
Fig. 2B presents the MnP production by I. lacteus F17
during SSF in tray bioreactor. MnP appeared after 36 h of
fermentation, and then started to increase rapidly to a high
level. The activity of MnP was approx 950 U/l, with maximum
activity observed at the bottom layer of the bed at 84 h,
along with LiP and Lac activities of about 31 U/l and 17 U/l,
respectively. After that, the MnP activity decreased drastically
until the end of cultivation. When compared with the
biomass, the MnP activity started to increase and gradually
reached the maximum value while the biomass remained
Fungal Growth and MnP Production in a Tray Bioreactor
807
Fig. 3. SEM of the carriers in the bioreactor at different times.
(A) Substrate without fungus at a magnification of 500×. (B) Samples
after 24 h of inoculation at a magnification of 500×, exhibiting the
beginning of mycelium production. (C) Micrograph showing lush
growth of mycelium after 48 h of inoculation at a magnification of
500×. (D) Micrograph showing mycelium closely entangled in the
support matrix after 60 h of inoculation.
environment for the fungus.
Fig. 2. Tray bioreactor data determined at different times.
(A) The total CO2 release rate and biomass at the three levels of the
tray bioreactor measured from 12 to 156 h of SSF. (B) The MnP
activity at the three levels of the bioreactor measured every 12 h of
SSF. Deviation values are standard deviations based on triplicate
determinations.
stable, indicating the production of a secondary metabolite
by the fungus when its growth rate was zero.
SEM Observation
The morphology of I. lacteus F17 grown on support
matrix was examined by SEM. Fig. 3 shows the SEM
images of the mycelium growth status after 0, 24, 48, and
60 h of SSF. The mycelia could be easily observed on the
surface of the medium mixed with pine sawdust, rice
straw, and soybean powder. They were densely entangled
in the support matrix after 60 h of SSF (Fig. 3D). These
results were consistent with the data regarding I. lacteus
F17 biomass production in the bioreactor, indicating that
the local tray bioreactor provided an appropriate growth
Fermentation Kinetics Model Fitting
A logistic model can be used as the mathematical model
to reveal the fungal growth condition [7, 36], which can be
defined as follows:
Nm
N t = --------------------------------------------------------1 + ( N m ⁄ N 0 – 1 )exp –µt
(2)
The Luedeking–Piret equation has also been used for the
kinetic analysis of extracellular enzyme production. According
to this model, the product formation rate depends on both
the instantaneous biomass concentration N and the growth
rate dN/dt:
dP
------- = α dN
--------- + βN
dt
dt
(3)
By substituting Eq. (2) into Eq. (3) and integrating it, the
following equation could be obtained:
µt
exp
α
α - + --β- ln ⎛ 1 + -----------------------Pt = Nm --------------------------------------------------- – ----------------⎞
–µt
( Nm ⁄ N0 – 1)exp + 1 Nm ⁄ N0 µ ⎝ Nm ⁄ N0 – 1⎠
(4)
where Nt is the biomass synthesized during the time
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Zhao et al.
interval considered (g per gram matrix), N0 is the initial
biomass of the inoculum (g per gram matrix), Nm is the
maximum biomass during fermentation (g per gram matrix),
µ is the maximum specific growth rate (h−1), t is the
differential time interval (h), α is the growth-associated
product formation, and β is the non-growth-associated
product formation coefficient and other variables.
The average biomass and MnP production evaluated at
the three layers can be used as a parameter to measure the
ability of the entire bioreactor to enhance fungal growth
and MnP production. Eqs. (2) and (4) were solved by a data
processing software, Origion 8.0. Through the nonlinear
least-squares fitting technique, the program provided the
result as the output. The fitted equation can be given as
follows:
0.23575
N t = ----------------------------------------------------1 + 3.37869exp –0.06681t
(5)
– 4569.29145
P t = 0.23575 ----------------------------------------------------- + 1043.52993
3.37869exp –0.06681t + 1
0.06681t
exp
+ 1737.20581ln ⎛⎝ 1 + ----------------------⎞⎠
3.37869
12h ≤ t ≤ 84h
(6)
The Adj. R2 of the nonlinear regression of the biomass
and MnP production was 0.95 and 0.96, respectively,
indicating that Eq. (5) can be used to determine the
relationship between production of biomass and time of
fermentation. Eq. (6) represents the relationship between
MnP production and time of fermentation.
Purification and Identification of MnP
The MnP was isolated and purified for further examination
of its enzyme properties. Crude enzyme was extracted
from SSF culture of I. lacteus F17 in the tray bioreactor at
84 h, corresponding to the maximum MnP activity, and
concentrated by ammonium sulfate precipitation. Fractionation
of MnP was performed by DEAE-cellulose anion-exchange
chromatography, followed by Sephadex G-75 gel filtration
chromatography. SDS-PAGE (Fig. 4) revealed that the
molecular mass of the purified MnP was about 43.2 kDa,
which agreed well with the molecular mass range of the
MnP family.
Four peptides with m/z of 689.33, 979.52, 1,054.53, and 1,625.74
corresponding to the amino acid sequences VACPDGK,
PNPVAAAPDK, LQSDHDLAR, and TACEWQSFVNNQAK,
were obtained by LC-MS. Then, they were searched against
a nonredundant protein database using BLASTp in NCBI.
Table 1 shows the homology of the internal sequences of
I. lacteus F17 MnP with those of representative MnPs. The
J. Microbiol. Biotechnol.
Fig. 4. SDS-PAGE of samples containing MnP from various
purification steps.
Molecular mass markers are shown on the left of the gel; lane 1:
purified MnP; lane 2: samples after Sephadex G-75 gel filtration; lane
3: samples after DEAE-Cellulose DE52 column; lane 4: crude enzyme.
amino acid sequences of four peptides showed low levels
of identity with MnP from P. chrysosporium (57%, 30%, 67%,
and 71%, respectively). It also showed relatively high levels
of identity with MnPs from P. ostreatus and T. versicolor,
ranging from 42% to 100%. Besides this, the amino acid
sequences of these four peptides were in full accordance
with those of the MnP of I. lacteus F17 (AGO86670.2), which
was predicted by the full-length cDNA sequence obtained
by our laboratory. Namely, the purified MnP in this work
may be the deduced MnP (AGO86670.2) or its isozymes
from I. lacteus F17.
Kinetic Parameters of the Purified MnP
The kinetic constants of the purified MnP were determined
based on typical Michaelis–Menten behavior. The Km and
kcat values of MnP for Mn2+ were found to be 80 µmol/l and
273.9 s-1, respectively. Furthermore, the catalytic efficiency
(specificity constant, kcat/Km) of the purified enzyme was
3.4 × 106 mol-1 l s-1.
Effects of Temperature and pH on the Activity and
Stability of Purified MnP
As illustrated in Fig. 5A, the purified MnP showed its
maximal activity at 25°C, and had a relatively high activity
at 25–55°C. Furthermore, the enzyme was stable at low and
moderate temperatures, maintaining more than 90% of its
maximal activity after incubation at temperature ranging
from 25°C to 45°C for 24 h. However, a rapid loss of
stability occurred at temperatures higher than 55°C. The
half-lives of MnP at pH 4.5 were 19, 18, 18, and 5 days, and
Fungal Growth and MnP Production in a Tray Bioreactor
809
Table 1. Sequence homology for internal peptides of I. lacteus F17 MnP by NCBI BLAST short sequence search.
[M+H]+
Amino acid sequences
689.33
VACPDGK
Similar sequences in the database of the NCBI (Accession No.)
100
26
42
26
86
VACPDGK(AGO86670.2)
ATCADGR(BAA33009.2)
VACPDGV(CAA83148.1)
358
IAHCPDG(AAA33744.1)
979.52
PNPVAAAPDK
LQSDHDLAR
100
154
40
160
PDATQPAPDL(CAA83148.1)
44
18
AAPTAVCPD (AAA33744.1)
30
PNPVAAAPDK(AGO86670.2)
255
LQSDHDLAR(AGO86670.2)
89
262
78
259
67
LQSDSELAR(CAA83148.1)
LQSDFALAH (AAA33744.1)
TACEWQSFVNNQAK
100
255
IQSDHDLAR(BAA33009.2)
1625.74
57
153
PPATAASPNG(BAA33009.2)
1054.53
Identity (%)
27
267
100
267
100
274
100
271
71
TACEWQSFVNNQAK(AGO86670.2)
TACEWQSFVNNQAK(BAA33009.2)
TACEWQSFVNNQAK(CAA83148.1)
TACIWQGFVNEQAF(AAA33744.1)
AGO86670.2: MnP of I. lacteus; BAA33009.2: MnP of Pleurotus ostreatus; CAA83148.1: MnP of Trametes versicolor; AAA33744.1: MnP of Phanerochaete chrysosporium.
Fig. 5. Effects of temperature and pH on the activity and stability of purified MnP from I. lacteus F17.
(A) The optimum temperature and thermostability of purified MnP. For thermal stability, the enzyme was incubated for 24 h at various
temperatures. The residual MnP activity was assayed under standard assay conditions (maximum activity = 100%). (B) The optimum pH and pH
stability of purified MnP. For pH stability, the purified MnP was incubated for 24 h at 25°C in various pHs in citrate–phosphate buffer. The
residual MnP activity was assayed under standard assay conditions (maximum activity=100%). Deviation values are standard deviations based on
triplicate determinations.
2.1 h at 25°C, 35°C, 45°C, 55°C, and 65°C, respectively.
In addition, the effect of varying the pH on the activity of
the purified MnP was also examined. Fig. 5B shows the
influence of pH in the range of 2.2–8 on MnP activity. The
enzyme was stable in the pH range of 4-7, exhibited an
optimum pH of about 7, and retained more than 90% of the
maximal activity after 24 h incubation at 25°C in citrate–
phosphate buffer with different pH values. In addition, the
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Zhao et al.
half-lives of MnP at 25°C were 3, 14, 28, 28, and 9 days at
pH 3, 4, 5, 6, and 7, respectively.
Therefore, the purified MnP exhibited great stability in
these conditions of the temperature ranging from 25°C to
55°C and the pH range of 3-7.
In Vitro Decolorization of Poly R-478
Poly R-478 is a charged, sulfonated, anthraquinone
polymeric dye, which has been shown to be useful for the
screening of fungi to degrade lignin. This dye was found to
be one of the recalcitrant organic compounds and could
remain colored for a long time [18]. Thus, it was chosen to
examine the degrading capability of both crude and purified
MnP.
The optimal temperature and pH for crude MnP to
decolorize Poly R-478 was first evaluated (Fig. 6A). Efficient
decolorization was only observed at pH 4.0 and decreased
rapidly at other values. Thus, for practical application, an
acidic pH system is required for the enzymatic decolorization.
The optimal temperature for decolorization was around
45°C. However, the effective range of temperature was
broader compared with pH. Furthermore, the effects of
temperature and pH on decolorization efficiency by the
purified MnP were evaluated, and optimum values of 35°C
and 4.0 were detected (Fig. 6B). The corresponding maximum
decolorization percentages were 21% for crude enzyme and
14% for purified enzyme after 1 h of reaction (Fig. 6). Then,
the decolorization of Poly R-487 was enhanced slowly
along with the reaction time extension. The decolorization
for prolonged times is in accordance with the thermostability
of this enzyme.
Discussion
For the cultivation of I. lacteus F 17 on a solid substrate, a
cylindrical static bed bioreactor, aerated from the two sides
with moist air, was set up in this study for batch SSF at a
laboratory scale (see Fig. 1). The design of this deep tray
bioreactor combined ideas of both the solid-state tray
reactor and packed bed reactor to overcome some problems,
such as the requirement of large areas and the occurrence
of a dry bed, as well as mycelium damage. Three perforated
trays in the bioreactor formed a thin layer, allowing good
attachment of the fungus to the support composed of agroindustrial residues, as well as facilitating appropriate
oxygen and nutrient diffusion. The support material for the
attachment and growth of I. lacteus F 17 was a mixture of
pine sawdust, rice straw, and soybean powder. After 60 h
of incubation, the support was completely penetrated and
J. Microbiol. Biotechnol.
Fig. 6. Poly R-478 decolorization by crude MnP and purified
MnP at different pH values and temperatures.
(A) The effect of pH (black triangle) and temperature (black square)
on Poly R-478 decolorization by crude MnP. The reaction mixture
contained citrate–phosphate buffer (62.8 mM), MnSO4 (1 mM), H2O2
(0.2 mM), Poly R-478 dye (0.001%), and 100 µl of crude MnP (900 U/l)
in a total volume of 1 ml. (B) The effect of pH (black triangle) and
temperature (black square) on Poly R-478 decolorization by purified
MnP. A 200 µl aliquot of purified MnP (900 U/l) was used to
decolorize the Poly R-478 dye (0.001%) in a total volume of 1 ml, and
other conditions are the same as in (A). Deviation values are standard
deviations based on triplicate determinations.
its surface was covered with a layer of fungal hyphae (see
Fig. 3D). The major components of rice straw are cellulose
and hemicelluloses, which are macromolecules composed
of different sugar monomers. Pine sawdust is not only a
carbon source but also an important inducer for ligninolytic
enzymes production by white-rot fungi. Thus, the support
Fungal Growth and MnP Production in a Tray Bioreactor
material used in this study is not only a good immobilization
matrix with very low cost but also provides all the nutrients
needed for fungal growth and enzyme production. Taken
together, the main advantage of this bioreactor is the
reduction of MnP production costs as well as the ability to
mimic the natural environment for rapid mycelium growth.
In addition, this bioreactor is simple and has less stringent
control of operative conditions.
Moilanen et al. [16] reported that the maximum MnP
activity (340 nkat/g DM) occurred after 14 days of fermentation
in a solid-state reactor with an internal conveyor, but their
MnP activity stayed at almost the same level at 100 g-scale
in plastic jars and 4 kg-scale in bioreactor cultivation. A
similar study was carried out by Vivekanand et al. [32]
using a bioreactor that contained four circular aluminum
trays, and the maximum MnP activity (1,339.0 ± 131.23 U/l)
was obtained after 6 days of SSF by Aspergillus fumigatus
VkJ2.4.5. Phanerochaete chrysosporium BKM-F-1767 had also
produced 1,293 U/l of MnP activity, in a fixed-bed bioreactor
on the 7th day [17]. Compared with these reports, 950 U/l
of MnP activity was achieved after 3.5 days of SSF in our
bioreactor, indicating a higher MnP activity obtained from
our bioreactor in a relatively short time. In addition, the
MnP activity was 1.68-fold higher than that observed in
flask cultures under the same conditions. In fact, the tray
bioreactors have been proposed as an appropriate type of
bioreactor for enzyme production through solid-state
fermentation [31, 24]. Couto et al. [4, 22] demonstrated that the
tray bioreactor is an ideal configuration for the production
of laccase by Trametes versicolor and Trametes hirsuta under
solid-state conditions when compared with other types of
configurations. Rosales et al. [23] focused on obtaining high
laccase activity levels by T. hirsuta in a 1.8 L static tray
bioreactor under solid-state conditions, and they exhibited
a very high laccase activity of around 12,000 U/l. From
these research results, it can be indicated that the tray
bioreactor is a successful scale-up for the production of
enzymes from filamentous fungi under SSF conditions and
it is economical and accessible.
Nucleic acid, ergosterol, and glucosamine have been
used to evaluate the biomass of fungal Aspergillus terreus in
SSF [34]. Nucleic acid proved to be a good biomass
indicator because its content changed only with mycelial
growth during the cultivation period, whereas the amount
of ergosterol and glucosamine could not accurately represent
the biomass changes. In the present study, the nucleic acid
content of the mycelium was chosen as the indicator to
estimate the biomass of I. lacteus F17 during the cultivation
811
period in the deep tray bioreactor. Furthermore, CO2
measurements also provided valuable information about
the progress of mycelium growth and metabolism on the
solid medium tested in the bioreactor. The profile of CO2
emission is shown in Fig. 2A, which was noted to be in
agreement with the amount of I. lacteus F17 biomass
produced. During early growth, the nucleic acid content was
higher and there was a strong increase in CO2 production
before 60 h of fermentation, indicating aerobic metabolism in
the tray bioreactor. During the late growth phase, the
nucleic acid content remained almost constant, and the
MnP activity of I. lacteus F17 grown on the solid medium
was high, while the CO2 production decreased consistently
through the end of fermentation, indicating that the
digestion of carbon sources present in the growth medium
gradually became more difficult and that secondary
metabolism was initiated. The causes of deceleration of
growth may be the accumulation of inhibitory metabolites
in the medium, exhaustion of readily utilizable nutrients, or
the onset of oxygen limitation. To analyze the bioreactor
performance, logistic and Luedeking–Piret equations were
applied to determine the different kinetic parameters related
to fungal biomass production and MnP synthesis. The
results calculated using the models were comparable with
the experimental data, and the model equations could
actually reflect the enzymatic fermentation process and
kinetic mechanism.
The purified MnP showed an optimum activity at 25°C,
which is slightly lower than that of MnP obtained from
other organisms such as Pleurotus ostreatus (30°C) [1],
I. lacteus (35°C) [26], and Lentinula edodes (40°C) [3]. Moreover,
the enzyme was quite stable up to 55°C, and did not lose
activity for at least 24 h. Furthermore, it is worth noting
that this MnP had stronger half-lives within a broad
temperature range of 25-55°C among MnPs reported so far
[3, 15, 28]. In fact, good thermostability is a desirable
feature of an enzyme for enzymatic industrial applications.
The purified MnP obtained in the present study was stable
in the pH range of 3-7, with an optimum pH of 7 at 25°C,
whereas most of the MnPs reported previously exhibited
maximum activity at an acidic pH ranging from 4.5 to 5.0
[3, 21, 28].
In the present study, Poly R-478 was selected as the
lignin model compound, whose strong structure is especially
difficult to degrade through most of the available technologies
[5]. The effects of temperature and pH on the decolorization
of Poly R-478 by both crude and purified MnP produced
from I. lacteus F17 were analyzed. It was noticed that the
June 2015 ⎪ Vol. 25 ⎪ No. 6
Zhao et al.
optimal temperature for crude and purified MnP to
decolorize was 45°C and 35°C, respectively, greatly different
from the optimum temperature of 25°C for the MnP
activity. It is probably attributed to the strong thermal
stability of the MnP at a broad temperature range of 2545°C, and the enzyme retained more than 90% of its
original activity for at least 3 days, and exhibited a half-life
of 18 days at 45°C. However, the crude enzyme lost its
ability of decolorization with further increases of the reaction
temperature to 55°C. The purified MnP was effective for
decolorization of the polymeric dye, but the decolorization
rate was a little low when compared with the crude MnP
(Fig. 6), even though the concentration of the purified MnP
was 2-fold higher than that of the crude enzyme. From a
practical point of view, crude enzyme is preferred owing to
its low cost. Therefore, the use of crude enzyme for in vitro
decolorization may be a viable technology for the degradation
of some recalcitrant organic compounds. Furthermore,
the decolorization of Poly R-478 was found to be strictly
dependent on the pH of the reaction system, and the
optimum pH for the decolorization by both types of MnP
from I. lacteus F17 was detected at pH 4. The acidic
optimum pH in dye oxidation by MnP has already been
reported and was suggested to be due to the increased
redox potential of the oxidized heme at low pH [15].
A systematic investigation on the growth of I. lacteus F17
and MnP production in the designed bioreactor was
presented, and MnP purification as well as its kinetic
properties and application in decolorization of the polymeric
model dye were reported in this study. The results indicated
that the proposed bioreactor is an effective system for MnP
production by I. lacteus F17 at laboratory scale. The
experiments of in vitro decolorization of Poly R-478 by MnP
indicate this enzymatic degrading ability. Thus, if the
conditions permit the scale-up of enzyme production
through an efficient production system without operational
problems, the enzymatic biodegradation can be selected as
a viable option for treatment of dye wastewater at an
industrial scale. Therefore, further studies are needed to
optimize the bioreactor configuration and environmental
factors, as well as to further enhance the enzyme product
yield.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 31070109) and by the Nature
Science Research Programme of the Education Office of
Anhui Province (No. KJ2010A027).
J. Microbiol. Biotechnol.
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