ORIGINAL PAPER Biomass, laccase and endoglucanase production by Lentinula edodes during solid state...

ORIGINAL PAPER

Biomass, laccase and endoglucanase production by Lentinulaedodes during solid state fermentation of reed grass, bean stalksand wheat straw residues

A. Philippoussis • P. Diamantopoulou •

K. Papadopoulou • H. Lakhtar • S. Roussos •

G. Parissopoulos • S. Papanikolaou

Received: 11 November 2009 / Accepted: 19 May 2010 / Published online: 3 June 2010

� Springer Science+Business Media B.V. 2010

Abstract Mycelium growth rates, biomass concentration

(estimated as glucosamine content) and laccase and endo-

glucanase secretion were monitored during solid state fer-

mentation (SSF) of wheat straw (WS), reed grass (RG) and

bean stalk (BS) residues by Lentinula edodes strains 119,

121, and 122. In a first experiment, these strains were

subjected to screening regarding their growth rates and

biomass yield, where strain 121 proved to be the fastest

colonizer. However, the greater biomass yield at the end of

colonization was demonstrated by strain 122 on BS

(465.93 mg g-1 d.w.). In a second experiment, growth

characters, as well as endoglucanase and laccase produc-

tion patterns of the selected strains 121 and 122 were

monitored at three intervals i.e., at 33, 66, and 100% of

substrate colonization. BS furnished the highest endoglu-

canase production for strain 121, while RG for strain 122.

A strain and substrate-dependent behaviour of the enzyme

secretion was detected, with strain 122 presenting maximal

endoglucanase activity in all substrates at the initial (33%)

and final (100%) stages of colonization (0.64–0.90 and

0.79–0.97 U g-1, respectively). However, in strain 121 the

peak of endoglucanase production was detected in the early

stages of colonization (at 33% on WS and at 66% on RG

and BS). Laccase activity showed increased values (max-

ima on WS, 353.68 and 548.67 U g-1 by strains 121 and

122, respectively) at 66% of colonization. Correlation

analysis of growth data demonstrated negative relations

between growth rate and biomass yield and between lac-

case and endoglucanase activities on WS and RG sub-

strates fermented by strain 122. Finally, possible relations

of growth parameters with nutritional constituents of the

substrates were investigated.

Keywords Lentinula edodes � Solid state fermentation �Lignocellulosic residues � Chemical constituents �Correlations � Growth rate � Glucosamine �Mycelial biomass � Laccase � Endoglucanase �Enzyme activities

Introduction

Among applications of solid state fermentation (SSF),

mushroom cultivation has proved its economic and eco-

logical importance for efficient utilization and biotrans-

formation of agro-residues into value-added products

(Chang 2006). Lentinula edodes (Berk.) Pegler, the second

most popular edible mushroom in the world because of its

flavour, taste, nutritional and medicinal properties (Smith

et al. 2002; Wasser 2002; Nikitina et al. 2007; Silva et al.

2007; Israilides et al. 2008), is an efficient biodegrader of

hardwood (preferably oak-wood). However, logs and wood

A. Philippoussis (&) � P. Diamantopoulou � G. Parissopoulos

Laboratory of Edible and Medicinal Fungi, National Agricultural

Research Foundation, IAMC, 61 Democratias St., Ag. Anargyri

Attikis, 13561 Athens, Greece

e-mail: [email protected]

K. Papadopoulou

School of Chemical Engineering, National Technical University

of Athens, 15700 Athens, Greece

H. Lakhtar � S. Roussos

IRD, Unite 185 Biotrans, IMEP Case 441, FST Saint Jerome,

Universite Paul Cezanne, Av. Escadrille Normandie-Niemen,

13397 Marseille cedex 20, France

S. Papanikolaou

Department of Food Science and Technology, Laboratory

of Food Microbiology and Biotechnology, Agricultural

University of Athens, Iera Odos 75, 11855 Athens, Greece

123

World J Microbiol Biotechnol (2011) 27:285–297

DOI 10.1007/s11274-010-0458-8

residues have been over-exploited in many areas, so the

need for diversification is necessary (Chiu et al. 2000).

The last decades, the artificial log (or bag-log) method

of cultivation on residue-based substrates has been devel-

oped, with shorter time and greater efficiency being the

major advantages (Royse 2004; Philippoussis et al. 2007a).

Wheat straw and other agricultural by-products resulting

after processing of maize, cotton, sugarcane, sunflower,

grape, coffee etc. have been examined as alternative sub-

strates for its cultivation (Philippoussis et al. 2002, 2003;

Rossi et al. 2003; Fan and Soccol 2005; Mata and Savoie

2005; Gaitan-Hernandez et al. 2006; Ozcelik and Peksen

2007, Royse and Sanchez 2007; Philippoussis 2009).

Substrate formulation, strain genotype and length of the

incubation period have been identified as important vari-

ables in L. edodes efficient production on residue-based

substrates used in bag-logs (Royse and Bahler 1986; Kal-

berer 1995; Philippoussis et al. 2001).

Lentinula edodes has a two-phase life cycle, mycelium

growth and fruiting bodies production, both of which are

affected by cellulose, hemicellulose and lignin proportions

along with nitrogen content of the cultivation substrate

(Philippoussis et al. 2003; Mata and Savoie 2005). Sup-

plements containing easily available carbohydrates and

nitrogen (e.g., cereal brans) are usually added to residues to

speed-up growth and increase mushroom yield (Ohga and

Royse 2001; Silva et al. 2007). The extent of colonization

of the substrate, determined by mycelium extension rates,

biomass yield and enzyme activities, is of great importance

for the success of the cultivation (Leatham 1985; Di Lena

et al. 1997; Philippoussis et al. 2003; Silva et al. 2005a).

The ‘glass-tube’ method (Philippoussis et al. 2001) has

been effectively used for L. edodes growth evaluation and

fructification assay to provide a quick estimate of the

bioconversion potential of various agro-residues to myce-

lial biomass and mushroom basidiomata (Philippoussis

et al. 2002, 2003, 2007a).

For lignocellulose degradation, this white-rot fungus

secretes a plethora of hydrolytic (cellulases and hemicel-

lulases) and oxidative (ligninolytic) extracellular enzymes

(Makkar et al. 2001; Kachlishvili et al. 2005). Among

them, endoglucanase and laccase are produced by L. ed-

odes strains, their activities being related to and dependent

on substrate composition and environmental factors (Bu-

swell et al. 1996; Silva et al. 2005b; Lakhtar et al. 2007,

Elisashvili et al. 2008). These enzymes have remarkable

biotechnological importance in industrial field. Cellulases

and related enzymes, accounting for approximately 20% of

the world enzyme market, are employed in different food

processing industries, in refinement of fodder quality, in

textile and laundry, in pulp and paper industries (Bhat

2000, Ghorai et al. 2009). Moreover, laccase, being able to

catalyse the oxidation of phenolic and non-phenolic

compounds and to degrade dyes, is very suitable for

application to several bioprocesses such as biopulping,

biobleaching and decolourisation of industrial effluents

(Bollag et al. 1988; Bourbonnais and Paice 1990, Lakhtar

et al. 2010).

The last decade, various grass and reed species have

been researched and proposed as suitable substrates for

mushroom producing basidiomycetes (Srijumpa 2003; Lin

2005). In our previous work we have studied the suitability

of reed grass residues for the cultivation of Ganoderma

species (Philippoussis et al. 2007b). The objective of the

present work was to evaluate WS, RG and BS, cheap and

mass-produced residues, as growth media of L. edodes. To

provide additional information on bioconversion process,

in this study the rate of residues colonization was evaluated

through mycelium growth rate, biomass yield and endo-

glucanase and laccase activities. Moreover, analysis of the

substrates’ constituents was performed in order to investi-

gate the impact of these substrates to L. edodes growth

characters, which is a prerequisite for their valorization

through mushroom biotechnology.

Materials and methods

Fungal strains and culture medium

Three strains of Lentinula edodes (Berk.) Pegler deposited

at the fungal culture collection of the National Agricultural

Research Foundation/Laboratory of Edible and Medicinal

Fungi with the accession numbers AMRL 119, 121, and

122 were used in this study. The culture medium used for

routine culture and storage purposes was Potato Dextrose

Agar (PDA; Merck, Germany).

Substrate preparation and analysis of their constituents

Residue-substrates, i.e., WS (Triticum aestivum), RG

(mixture of Typha angustifolia, Carex pseudocyperus and

Phragmites australis) and BS (Phaseolus coccineus) were

prepared (all ingredients based on oven dry substrate

weight) in a ratio of 80% residue to 20% supplements i.e.,

12% wheat bran, 7% soybean flour and 1% CaCO3 (Phil-

ippoussis et al. 2007b). The residues were soaked in water

for 12–24 h and after drainage, supplements were added

and mixed.

For substrate analysis, samples were dried to constant

weight in a 60�C oven and milled to size \0.3 mm in

Cyclotec TM 1093 sample mill (Foss, France). Carbon and

nitrogen concentrations were determined using a combus-

tion-gas chromatography technique (Flash EA 1212 Ele-

mental Analyzer, France). The instrument was calibrated

using aspartic acid standard.

286 World J Microbiol Biotechnol (2011) 27:285–297

123

For cellulose, hemicellulose and lignin determinations,13C cross-polarization CP magic angle spinning MAS NMR

spectroscopy was used. Dried samples were used directly in

NMR experiments without special preparation. Solid-state13C CPMAS NMR spectra of samples were obtained on a

Bruker Avance-400 MHz spectrometer (Bruker, Bremen,

Germany) operating at a 13C resonance frequency of

100.7 MHz. Samples were placed in a 7-mm zirconium

rotor and spun at the magic-angle at 6 kHz. All measure-

ments were made at room temperature. The 13C chemical

shifts were referenced to tetramethylsilane and calibrated

with the glycine carbonyl signal, set at 172.5 ppm (Albrecht

et al. 2008).

Deconvolution of the NMR spectra was performed using

the DmFit software (Massiot et al. 2002). This software

adjusts the spectra to obtain the linewidth and the peak

positions (in ppm.) and to integrate each peak so as to

obtain the percentage of each contribution. The lignin

content was calculated according to the model of Haw et al.

(1984), which takes into account the total lignin carbon.

These authors calibrated lignin content from aromatic

carbons plus aliphatic carbons of lignin. This model was

applied to each spectrum to determine lignin content. The

cellulose and hemicellulose were calculated according to

the model of Gilardi et al. (1995).

SSF conditions and extension rate measurement

Solid state fermentation was performed as previously

described by Philippoussis et al. (2001) in glass-tubes

(200 9 30 mm) uniformly filled with the substrates to a

80 ml volume and sterilized twice for 1 h at 121�C. The

moisture content of the sterilized substrates was 62–65%,

while the initial pH was 5.48, 5.34, and 5.53 for WS, RG

and BS, respectively. In a completely randomized design,

fifteen replicate tubes per strain and substrate were inocu-

lated with two agar plugs (6 mm diam.) cut from the

periphery of actively growing mycelium on PDA, trans-

ferred onto the top of the substrate and incubated at 26�C in

the dark.

The growth rate of mycelium (mm day-1), was recorded

daily in a set of three test tubes by measuring the visible

penetration of mycelia into the substrate in two perpen-

dicular directions and the extension rate Kr (mm day-1)

was calculated after the mycelium front has reached more

than 30 mm.

Substrate sampling and biomass determination

Twelve replicate tubes per substrate and strain were used

for biomass estimation and enzymes assays. At predeter-

mined for each experiment percentages of substrate volume

colonization (i.e., intervals of 4–7 days, depending on the

strain and substrate used), two replicate tubes per substrate

and strain were withdrawn for biomass and enzymes

activity determinations. Samples, comprising the entire

solid fermented medium (substrate and mycelium), were

frozen (-20�C, 48 h) and dried by a Heto LyoLab 3000

freeze-dryer (Heto-Holten Als, Denmark), milled and

sieved.

The glucosamine content of the fungal cell wall was

used to monitor L. edodes biomass. At the beginning,

glucosamine standard curve (glucosamine vs. absorbance)

was obtained. Moreover, glucosamine content of mycelia

(curves biomass vs. glucosamine) for each individual strain

were determined through liquid state fermentation (LSF)

for 35 days, in 100 ml Erlenmeyer flasks with 50 ml Malt

Extract Broth (MEB; Merck, Germany), at 26�C under

static conditions.

The biomass was determined by the method of the

fungal chitin hydrolysis into N-acetylglucosamine, as

described by Scotti et al. (2001). For each replicate of solid

state culture, 2 g of dry sample were treated at 25�C with

5 ml of 72% H2SO4 (Merck, Germany) on a rotary shaker

(MPM M301-OR, Italy) at 130 rpm for 30 min. After

dilution with 54 ml deionized water, the hydrolysis was

carried out by autoclaving the mixture at 121�C for 2 h.

The hydrolyzate was neutralized to pH 7.0, initially with a

10 M and then 0.5 M NaOH (Merck, Germany) solution,

as described by Rigas et al. (2009).

Glucosamine was quantified by the colorimetric method

of Ride and Drysdale (1972). In a sample of 3 ml from the

previous step, an equal volume (3 ml) of 5% (w/v) NaNO2

(Merck, Germany), and 5% KHSO4 (Merck, Germany)

was added, and the solution was shaken for 15 min and

then centrifuged (Hettich Micro22R, Germany) at 1,500 g,

for 2 min at 2�C. Then, two samples, 3 ml each, of the

supernatant were removed with a pipette. To each of them

1 ml of 12.5% NH4SO3NH2 (Merck, Germany) was

added, shaken for 5 min and then 1 ml solution (prepared

daily) of 0.5% 3-methyl-2-benzothiazolone hydrazone

hydrochloride (MBTH; Sigma, Germany) was added. The

mixture was heated in a boiling water bath for 3 min (at

this step the color changed), cooled and 1 ml of 0.5%

FeCl3 (Alfa Aesar, Germany), stored at 4�C and discarded

after 3 days, was added. After standing for 30 min and

centrifugation, the absorbance at 650 nm was read by a

Jasco V-530 UV/VIS spectrophotometer (Jasco, Japan).

The blank was the medium without inoculum. Results

were expressed as mg fungal biomass per g of dry

substrate.

Enzymes extraction and activities determination

For enzymes extraction, frozen colonized substrate of each

replicate (equivalent to 2 g of dry sample) and 20 ml

World J Microbiol Biotechnol (2011) 27:285–297 287

123

0.05 M sodium acetate buffer (pH = 5.0) were transferred

to 100 ml Erlenmeyer flasks and extracellular enzymes

were extracted after agitation at 100 rpm for 1 h at room

temperature in an orbital shaker (MPM M301-OR, Italy).

The crude extracts were recovered by double filtration

through filter paper (Whatman No2, England) and the fil-

trates were centrifuged (10,500 rpm for 15 min at 4�C) to

remove the fine particles using a Hettich Micro22R (Het-

tich, Germany) centrifuge. The pH was measured and clear

supernatants were frozen (-20�C) and used as the enzyme

source. Each activity value of both enzymes tested repre-

sents the mean of 6 replicates (3 sample replicates 9 2

assay replicates). Absorbance measurements were con-

ducted by the Jasco V-530 UV–VIS spectophotometer and

enzyme activities were expressed as U g-1 (units g-1 of

dry substrate).

Endoglucanase (cellulase, EC 3.2.1.4) activity was

determined using as substrate 0.5 ml of 1% (w/v) car-

boxymethylcellulose sodium salt (CMC; Sigma, Germany),

in 0.05 M sodium citrate buffer (Merck, Germany), pH 4.8,

incubated with 0.5 ml crude enzyme extract at 50�C for

30 min (Mandels et al. 1976). The reducing sugars formed

were measured by the DNS (3,5-dinitrosalicylic acid;

Merck, Germany) method (Miller 1959), monitored

through the change in absorbance per minute at 540 nm.

One unit of endoglucanase is defined as the amount of

enzyme producing 1 lmol of reducing sugar (glucose

equivalent) in 1 min, under the conditions used. Standard

curve was obtained with glucose for CMC.

Laccase (EC 1.10.3.2) activity was measured spectro-

photometrically, using syringaldazine (4-hydroxy-3,5-

dimethoxybenzaldehydeazine, Sigma, Germany) as substrate

at 525 nm (extinction coefficientTM525 = 65,000 M-1 cm-1)

for about 10 min. Reaction was carried out in 3-ml cuvettes

containing 0.2 ml of crude enzyme extract, 1.7 ml of 0.1 M

sodium phosphate buffer at pH 6.8 and 0.1 ml of 1 mM sy-

ringaldazine dissolved in absolute ethanol (Riedel-de-Haen,

Germany). Crude enzyme extracts were first incubated at

30�C for 10 min in sodium phosphate buffer at pH 6.8. Then

the reaction was initiated by addition of the syringaldazine

(Harkin and Obst 1973; Ride 1980). One unit of laccase is

defined as the amount of enzyme required to produce a

change in absorbance of 0.001 per minute, under the assay

conditions used.

Experimental design and statistical analysis

In all experiments, a completely randomized design was

applied, using fifteen replicates per strain and substrate.

Variance analysis was performed by Statgraphics Plus

version 5.1 statistical package, using the Least Significant

Difference (LSD) test at 5% level of probability to compare

mean values of growth rate, biomass production, and en-

doglucanase and laccase activities. Correlations were

elaborated between the achieved values of these parameters

and the constituents of the substrates.

Results

Mycelium growth rates and biomass accumulation

during SSF of agro-residues

Initially, the growth characteristics (mycelium growth rate,

full colonization period) of the three L. edodes strains were

comparatively evaluated in glass tubes filled with WS, RG,

and BS residue-substrates. As shown in Fig. 1, the periods

required to L. edodes strains for complete colonization of

the substrates varied between 28 and 40 days. The shortest

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

L. edodes119

L. edodes121

L. edodes122

L. edodes119

L. edodes121

L. edodes122

L. edodes119

L. edodes121

L. edodes122

SBGRSW

Strains/Growth substrates

Gro

wth

rat

e K

r (m

m d

ay-1

)

0

3

69

12

15

18

2124

27

30

33

3639

42

45Fu

ll co

loni

zatio

n tim

e (d

ays)

Kr (mm day-1) Full colonization time (days)Fig. 1 Growth rate Kr

(mm day-1) and colonization

period (days) of L. edodesstrains AMRL 119, 121, 122 on

WS, RG, and BS lignocellulosic

residues. Bar plots represent

growth rates, while dotsrepresent colonization periods.

Error bars indicate standard

deviations


123

colonization periods were recorded on RG and WS by

strain 121 (28 and 30 days, respectively), while the longest

by 119 on RG and BS (36 and 40 days, respectively).

Growth rate assay (Fig. 1) demonstrated significant

differences among strains. Strain 121 proved to be the

fastest colonizer, presenting growth rates Kr 6.11, 5.24, and

5.23 mm day-1 on RG, WS, and BS, respectively. Strain

122 followed by 119 presented inferior growth results. The

former exhibited growth rates 4.69–5.15 mm day-1, while

for the latter the respective extension rates were signifi-

cantly lower, especially on BS (3.53 mm day-1). More-

over, correlation analysis, taking into account all strains

and substrates studied, revealed a significant negative

relation (R2 = 0.85) between time needed for full substrate

colonization and L. edodes mycelium growth rate.

The suitability of the residue-substrates to support

growth of the tested strains was further evaluated by

recording over a 5-week period the mycelial quantity

during SSF, at 25, 50, 75, and 100% of substrate coloni-

zation. At first, in order to determine the relationship

between glucosamine content and dry biomass, L. edodes

strains were grown for 35 days in liquid state culture, in

flasks containing Malt Extract Broth. Correlations between

glucosamine (mg) and mycelial biomass (g) for the tested

strains are shown in Table 1. The glucosamine content of

the mycelial biomass varied among strains: 1.94–2.48% of

fungal dry matter (average 2.08% ± 0.39) for strain

AMRL 119, 0.84–0.90% (average 0.87% ± 0.03) for

AMRL 121 and 1.74–1.95% (average 1.91% ± 0.08) for

AMRL 122. The linear regression equations of glucosa-

mine versus biomass were used to convert glucosamine

content (expressed in mg of glucosamine per g of biomass

dry weight) to mycelial biomass in solid culture conditions.

The courses of biomass production (mg g-1 dry sub-

strate) as well as biomass production rate (mg g-1 dry

substrate day-1) during SSF of WS, RG and BS substrates

are presented in Table 2. In general, the strain as well as

the nature of the substrate used had a pronounced effect on

biomass quantity. From the beginning to the end of the

fermentation process, BS furnished significantly higher

biomass accumulation in all tested strains. Among them,

strain 122 presented significantly (P \ 0.05) the highest

overall biomass production efficiency, followed by strain

121. The former strain, exhibiting high biomass production

rate that averaged 13.47, 9.46, and 9.72 mg biomass

g-1 substrate day-1 on BS, RG, and WS, respectively,

furnished 465.93, 372.10, and 357.48 mg dry biomass g-1

BS, RG, and WS substrates by the end of cultivation

(35 days). The respective dry biomass yields obtained by

strain 121 were 510.28 mg g-1 BS in 33 days (the highest

biomass yield and production rate), but only 210.28 and

187.96 mg g-1 RG and WS, respectively. Strain 119 was

the least efficient biomass producer, as colonization of all

substrates was the slowest and least extensive. Finally, a

negative correlation was found between growth rate and

biomass production rate, this relationship being more sig-

nificant for strain 121 (R2 = 0.82).

Patterns of biomass, endoglucanase and laccase

production during SSF

In a following experiment, growth characters (extension

rate, biomass concentration) as well as endoglucanase and

laccase production of strains AMRL 121 and 122, selected

from the previous experiment, were monitored at three

intervals of the vegetative growth (33%, 66% and 100% of

colonization corresponding respectively to 2nd, 3.5th, and

5th weeks after inoculation). Both strains exhibited sig-

nificantly better growth rates on RG and the inferior growth

results on WS. Also, strain 121 presented better growth

results than strain 122. In general, growth rate Kr presented

a similar pattern throughout colonization of all substrates.

The lowest Kr values were recorded in the initial coloni-

zation stage, increased significantly until mycelium colo-

nized 66% of the substrate volume and then they slightly

declined until the end of incubation. The respective

extension rates at the three colonization intervals of RG

were 5.03, 6.56, and 5.74 mm day-1 for strain 121 and

4.07, 5.20, and 4.90 mm day-1 for strain 122.

The courses of biomass production (mg g-1 dry sub-

strate) during colonization of WS, RG, and BS substrates

are presented in Table 3. Although a steady increase of

biomass accumulation was recorded in all substrates, the

biomass profiles obtained from the RG and BS substrates

were remarkably different from the Kr ones. From the

beginning to the end of the fermentation process, BS

Table 1 Linear regression equations of glucosamine (mg) and mycelial biomass (g) of L. edodes strains AMRL 119, 121, 122 grown on MEB in

flasks under static conditions

L. edodesstrains

Linear regression equation

biomass (x) vs. glucosamine (y)

Linear regression equation

glucosamine (x) vs. biomass (y)

Correlation

coefficient R2

AMRL 119 y = 36.537x - 6.775 y = 0.0272x ? 0.186 0.996

AMRL 121 y = 9.384x - 1.097 y = 0.105x ? 0.141 0.986

AMRL 122 y = 19.757x - 0.554 y = 0.051x ? 0.304 0.998


123

furnished significantly the highest biomass accumulation in

both tested strains, with strain 122 presenting significantly

higher biomass production efficiency than strain 121,

especially in the first two stages of substrate colonization

(33 and 66%).

Regarding the production of the hydrolytic enzyme en-

doglucanase (Table 3), BS furnished the higher overall

endoglucanase production for strain 121, while for strain

122 endoglucanase production was more pronounced on

RG. On all substrates, strain 122 presented similar pattern

of endoglucanase production, showing increased activities

at the early stage of the vegetative growth and at the end of

incubation and considerably lower values at 66% of sub-

strate volume colonization (3.5th week), especially on WS

and BS. However, a differentiated pattern of endoglucan-

ase production was detected in strain 121, as it demon-

strated higher endoglucanase production in the early two

stages rather than the final stage of vegetative growth,

presenting maxima the 2nd week on WS and the 3.5th

week on RG and BS, while minima were recorded at

complete colonization.

Regarding the production of the oxidative enzyme lac-

case (Table 3), obtained results of both strains showed an

increased laccase activity occurred at 66% of substrate

colonization, whereas the corresponding activity was sig-

nificantly lower at the early stage of the vegetative growth

and at complete colonization. This pattern, contrary to

endoglucanase production, was more pronounced in strain

122. Regarding the maxima obtained at 66% of coloniza-

tion, the significantly (P \ 0.05) highest activity was

recorded on WS by both strains (548.67 and 353.68 U g-1

for strains 122 and 121, respectively). However, for strain

122 BS supported the highest cumulative laccase produc-

tion, but with completely different time course than WS, as

laccase activity on BS was high (282.06 U g-1) at the

initial stage of the vegetative growth, then declined and

peaked (390.31 U g-1) at the end of incubation.

Effects of substrates on colonization characters

and their correlations

The time courses of mycelium growth rate, biomass pro-

duction rate as well as biomass, endoglucanase, and laccase

production during SSF of WS, RG and BS substrates by

L. edodes strain 122 are presented in Fig. 2. The type of

substrate had a notable effect on biomass and enzymes

Table 2 Mycelial biomass production (mg g-1 substrate d.w.) and biomass production rate (mg g-1 substrate day-1) during solid state

fermentation of WS, RG and BS substrates by L. edodes strains AMRL 119, 121 and 122

Colonization (%) 25 (%) 50 (%) 75 (%) 100 (%)

Period (weeks/days) (2/11–14) (3/18–22) (4/24–30) (5/32–40)

Strains Substrates

Biomass production (mg g-1 substrate d.w.)

119 WS 84.03 a1 99.24 a,b 110.75 a 137.95 a

RG 69.58 a 89.36 a 102.71 a 152.36 a,b

BS 108.03 b,c 131.77 c 176.66 c 215.22 c

121 WS 76.19 a 94.31 a 143.00 b 187.96 b,c

RG 86.14 a,b 122.86 b,c 155.32 b 210.28 c

BS 117.24 c 207.15 e 324.18 e 510.28 f

122 WS 144.12 d 174.69 d 256.30 d 357.48 d

RG 149.19 d 179.50 d,e 267.61 d 372.10 d

BS 224.17 e 261.12 f 363.44 f 465.93 e

Biomass production rate (mg g-1 substrate day-1)

119 WS 7.64 a,b 5.51 a,b,c 4.61 b 4.31 a,b

RG 6.33 a 4.26 a 3.54 a 4.23 a

BS 9.00 b,c 5.99 b,c 5.52 c 5.38 b,c

121 WS 7.62 a,b 5.24 a,b 5.96 c 6.27 c,d

RG 7.83 a,b 6.83 c,d 6.75 d 7.25 d

BS 9.77 c,d 10.36 f 12.01 g 15.46 g

122 WS 12.01 e 8.32 e 8.84 e 10.21 e

RG 10.66 d,e 8.16 d,e 9.56 f 10.63 e

BS 16.01 f 11.87 g 12.53 g 13.31 f

1 Mean values of each parameter within the same column not sharing common letters are significantly different (P \ 0.05)


123

levels, and on the timing of their peaks production. In all

substrates, growth rates and biomass accumulation rates

(found to be negatively but not significantly related)

presented opposite curves, as biomass production acceler-

ated by the end of colonization process, when growth rate

declined.

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

2.0 3.5 5.0 2.0 3.5 5.0 2.0 3.5 5.0

SBGRSW

Cultivation time (weeks)/ Substrates

Lac

case

pro

duct

ion

(U g

-1 )

0

60

120

180

240

300

360

420

480

540

600

End

oglu

cana

se p

rodu

ctio

n (U

g-1

)

Endoglucanase Growth rate Biomass productionLaccase Biomass production rate

Bio

mas

s p

rodu

ctio

n (g

g-1)-

prod

ucti

on ra

te (g

-1 g

-1da

y)

Gro

wth

rate

(cm

day

-1)

480

0

300

60

120

180

240

360

420

600

540

1.20

0.00

0.75

0.15

0.30

0.45

0.60

0.90

1.05

1.50

1.35

L. edodes strain 122Fig. 2 Time courses of growth

rate, biomass production,

biomass production rate and

endoglucanase and laccase

activities of L. edodes strain 122

grown on WS, RG, and BS

residues for 5 weeks. Error barsindicate standard deviations

Table 3 Patterns of biomass, endoglucanase and laccase production, monitored at 33, 66, and 100% of WS, RG and BS colonization, during

their solid state fermentation by L. edodes strains AMRL 121, 122

L. edodes strains Substrates Substrate colonization (%)/period (weeks) from inoculation

33%/(2 Weeks) 66%/(3.5 Weeks) 100%/(5 Weeks)

Biomass production (mg g-1 dry substrate)

121 WS 85.25 a1 137.08 a 187.96 a

RG 104.50 a 139.09 a 210.28 a

BS 162.19 b 265.66 c 510.28 c

122 WS 159.41 b 215.49 b 357.48 b

RG 164.35 b 223.56 b 372.10 b

BS 242.64 c 312.28 d 465.93 c

Endoglucanase production (U g-1 dry substrate)

121 WS 0.78 a,b 0.59 b 0.59 b

RG 0.66 a 0.85 c 0.33 a

BS 0.62 a 1.39 d 0.43 a

122 WS 0.67 a 0.30 a 0.79 c

RG 0.90 b 0.80 b,c 0.97 d

BS 0.64 a 0.17 a 0.93 d

Laccase production (U g-1 dry substrate)

121 WS 51.16 a,b 353.68 b 101.82 a

RG 207.12 c 236.44 a 31.57 a

BS 68.06 a,b 159.45 a 101.11 a

122 WS 86.48 b 548.67 c 54.91 a

RG 41.21 a 257.69 a,b 45.49 a

BS 282.06 d 175.06 a 390.31 b

1 Mean values of each parameter within the same column not sharing common letters are significantly different (P \ 0.05)


123

Furthermore, WS and RG substrates presented similar

patterns for the change of endoglucanase and laccase

activities with time (Fig. 2). In the beginning of coloniza-

tion process laccase activity was low, while biomass pro-

duction rate and endoglucanase levels were comparatively

higher. Thereafter, laccase reached its peak production

(fivefold increase) while biomass accumulation and endo-

glucanase presented their lower values which peaked again

at the final stage of colonization, simultaneously with

laccase activity diminution. In these substrates, laccase and

endoglucanase activities were found to be negatively cor-

related (R2 values 0.97 and 0.85 for WS and RG, respec-

tively). Moreover, Kr and biomass production rate presented

high positive correlation with endoglucanase production

(R2 values 0.85 and 0.96 for WS and RG, respectively) and

high negative relation with laccase activity (respective R2

values 0.96 and 0.97 for WS and RG).

The respective BS curves were very different, as high

levels of both enzymes and biomass production were

recorded from the initial stage of colonization (Fig. 2),

which decreased by 66% colonization of the substrate

volume and again increased by the end of incubation,

resulting in massive biomass production. In this substrate,

laccase and endoglucanase activities demonstrated a sig-

nificant positive correlation (R2 = 0.90).

Finally, possible relations of the tested growth parame-

ters with chemical constituents of substrates were investi-

gated. Initial composition of substrates regarding cellulose,

hemicellulose and lignin percentages, as well as carbon and

nitrogen contents, are given in Table 4. Correlation studies

revealed that biomass production rate is significantly

(P \ 0.05) positively correlated with nitrogen content of

the substrates (R2 = 0.99), with their hemicellulose content

(R2 = 0.78) and not significantly with their cellulose to

hemicellulose ratio (R2 = 0.55). Moreover, a significant

negative relation of biomass yield was found with cellu-

lose ? hemicellulose per N ratio (R2 = 0.97). However,

substrates as RG, with higher lignin and lower cellulose

content appeared to support higher levels of endoglucanase

and lower values of laccase. Correlation analyses demon-

strated significant positive relation of endoglucanase pro-

duction with initial hemicellulose content of the substrates

(R2 = 0.76) and negative relation with cellulose per hemi-

cellulose ratio (R2 = 0.90) and of laccase activity with

lignin per hemicellulose ratio (R2 = 0.86).

Discussion

Although hardwood sawdust is the most used substrate for

the cultivation of L. edodes (Royse and Sanchez-Vazquez

2001), its limited availability in many regions brought up

the necessity to find cheap alternatives (Salmones et al.

1999; Pire et al. 2001; Philippoussis 2009). For the success

of L. edodes cultivation, vigorous mycelia growth and

reduction of colonization period is of prime importance

(Philippoussis et al. 2002). While L. edodes mycelium

growth kinetics on LSF and on agar media are widely

reported in the literature (Lomberh et al. 2002, Montini

et al. 2006), fewer data are available on the fungus growth

and especially on mycelial biomass yield during SSF of

agro-residues (Di Lena et al. 1997; Silva et al. 2005b). The

present work provide important information on the rate of

lignocellulosic residues colonization, through monitoring

and comparing mycelium growth, biomass yield and

enzyme activities in SSF of WS, RG, and BS substrates.

Moreover, the work aimed at detecting growth differences

among substrates and stains tested, as well as to investigate

possible relations of the mentioned growth parameters with

the nutritional elements of these substrates.

It has been reported that genotype and substrate com-

position influence L. edodes mycelial growth, which can

affect mushroom yield (Royse and Bahler 1986; Diehle and

Royse 1986; Oei 2003). The initial nitrogen content and C

Table 4 Analysis of the main constituents of WS, RG and BS residue-substrates at the beginning of SSF

Carbon

(%)

Nitrogen

(%)

C

N-1Cellulose

(%)

Hemicellulose

(%)

Lignin

(%)

Cellulose

lignin-1Cellulose hemi-

cellulose-1(Cellulose ?

hemicellulose)N-1

Residuesa

WS 37.70 0.64 58.81 80.03 6.32 8.51 9.40 12.66 134.92

RG 45.30 0.63 72.02 74.68 6.78 13.13 5.69 11.01 129.30

BS 45.60 0.80 57.07 80.39 5.73 8.41 9.56 14.03 107.65

Substratesb

WS 40.0 1.26 31.82 68.93 11.16 7.58 9.10 6.18 63.56

RG 46.1 1.25 36.94 64.65 11.53 11.27 5.73 5.61 60.94

BS 46.34 1.38 33.48 69.22 10.69 7.50 9.23 6.48 57.91

a Raw residues (before mixing with supplements)b Cultivation substrates (after mixing with 12% wheat bran, 7% soybean flour and 1% CaCO3)


123

per N ratio varied among substrates. Among nutritional

constituents, nitrogen has been recognized as a key growth

factor for L. edodes cultivation (Boyle 1998; Kalberer

2000), while our previous work showed the influence of

low C per N ratio values of substrates to the earliness of

fructification and crop cycle duration (Philippoussis et al.

2007a).

The tested raw residues were supplemented with wheat

bran and soybean flour to obtain a final C per N ratio in the

range of 30–40 1-1, which is favourable for L. edodes

growth (Song and Chow 1987). Regarding the rate of

substrate colonization, our previous observations, relying

exclusively on extension rate measurements, associated

fast mycelial growth to the low C per N ratio values of the

substrates (Philippoussis et al. 2002, 2003). However, the

present investigation, comprising both growth measure-

ment and biomass yield estimation, demonstrated a stron-

ger correlation of initial nitrogen concentration of the

substrates with biomass production rate rather than with

mycelium growth rate. In fact, BS that contained higher

amounts of available nitrogen furnished significantly

increased biomass accumulation, while the lower N content

of WS and RG substrates resulted in equally low biomass

quantity. Moreover, data obtained through monitoring the

entire SSF process indicated that there is a negative relation

between biomass production and mycelium growth rates.

Analysis of substrates showed also variations in the

concentrations of hemicellulose, cellulose and lignin. Our

finding that biomass yield is significantly positively cor-

related with their hemicellulose content and negatively to

cellulose ? hemicellulose per N ratio, can be explained by

the preference of white-rot fungi to metabolize water-sol-

uble sugars, starch and hemicellulose rather than cellulose

and lignin (Okano et al. 2006). Moyson and Verachtert

(1991) indicated that substrate decomposition by L. edodes

is initially associated to its hemicellulose content. Also,

other studies demonstrated that hemicellulose exercises a

positive effect during the initial active growth phase, prior

to break down of lignin (Leatham 1985; Buswell et al.

1996; Silva et al. 2005a). Moreover, Silva et al. (2005a)

obtained the best biomass yields on eucalypt residue with

high nitrogen level obtained by 20% supplementation with

soya bran. It seems reasonable to assume that cellu-

lose ? hemicellulose per N ratio reflects better the com-

bined effects of these growth factors and can be indicative

for evaluation of substrates regarding their bioconversion

potential to L. edodes biomass.

Apart from substrates, the genotype of strains exercised

a considerable influence on mycelium growth. Their rates

of substrate colonization, determined simultaneously by the

mycelium extension, the biomass yield and by the levels of

enzymes were found significantly different. Our results

regarding glucosamine content of L. edodes mycelia, used

for quantification of fungal growth, are comparable to those

recently reported on MEB (Di Mario et al. 2008). However,

mycelial glucosamine content varied among strains and

remarkable differences were observed regarding their

growth characters. It was also noticeable that our best

growth results were furnished by strains 121 and 122 with

lower glucosamine content in their mycelial biomass.

Strain 121 proved to be the fastest colonizer, while strain

122 presented the highest biomass production efficiency.

This observation requires further investigation as the

selection of substrate-adapted genotypes is a useful tool to

improve L. edodes production characters (Silva et al.

2005a).

The utilization of lignocellulosic substrates by mush-

rooms depends on the production of a pool of hydrolytic

and oxidative enzymes able to convert lignocellulosic

media, of high molecular weight, into low molecular

compounds that can be assimilated by the mycelium (Bu-

swell and Chang 1993). Regarding endoglucanase activity

(which was weak relatively to laccase), Leatham (1985),

Buswell et al. (1996) and Pereira Junior et al. (2003)

reported L. edodes as a moderate cellulolytic enzyme pro-

ducer, as it hydrolyzes hemicellulose more efficiently than

cellulose (Moyson and Verachtert 1991; Silva et al. 2005a).

The positive relation of endoglucanase activity with the

hemicellulose content of the substrates reported here sup-

port these previously drawn conclusions, as the weak en-

doglucanase production of L. edodes mycelia is accelerated

in substrates as RG with high hemicellulose content. This is

further supported by the positive correlation of biomass

and endoglucanase production found in the present study,

as the higher endoglucanase level could be due to higher

biomass production. Arai et al. (2005) demonstrated that

exocarbohydrase and particularly xylanase production

correlate with growth promotion of L. edodes mycelium.

Our results of weak endoglucanase activity of L. edodes

on tested substrates are in accordance with those previously

reported by Silva et al. (2005a, b) on supplemented euca-

lyptus residues, but are quite lower than the enzyme levels

obtained by Kachlishvili et al. (2005) on supplemented

beech tree leaves and wheat straw. It is well known that the

type and the composition of the lignocellulosic substrate

appear to determine the amount of enzyme produced by the

wood-rotting basidiomycetes (Elisashvili et al. 2008).

Our results also revealed two different patterns regard-

ing cellulose activity peaks (strain 121 one peak, strain 122

two peaks), under the same culture conditions, which

could be attributed to inherited characteristics of the

strains. Ohga et al. (2000), studying laccase and cellulose

activity changes of L. edodes during colonization and

fruiting stages on sawdust based substrate, demonstrated

that secretion of the enzymes is controlled at the level of

gene transcription. Cavallazzi et al. (2004) demonstrated


123

different behavior of L. edodes strains regarding lignocel-

lulolytic enzymes profiles in SSF of eucalyptus bark-based

medium, particularly during the colonization phase.

The observed two peaks of endoglucanase activity by

strain 122, with the first (2nd week) related to the early

mycelial growth and the second, greater peak (5th week),

related to the complete substrate colonization, just before

primordation, are in accordance with the two peaks of this

enzyme activity observed during cultivation on wheat

straw by Mata and Savoie (1998). They related the first

peak to the initial colonization stage and the second to the

fructification stage. Moreover, endoglucanase alteration

has been observed by Pereira Junior et al. (2003) during a

10 day growth of the fungus in liquid media containing

carboximethylcellulose or microcrystalline cellulose, while

two activity peaks of the enzyme, followed by activity

decrease, where shown by Cavallazzi et al. (2004) in the

first 40 days of strain UFV 77 growth on eucalyptus bark

substrate. However, in the same work, strain UFV 53

presented only one endoglucanase peak, as was presented

in our results by strain 121, rather early on WS or later on

RG and BS.

In the literature, contradictory evidence exists for the

effects of the nature and concentration of the nitrogen

source on lignolytic enzyme production. Previous inference

by Dill and Kraepelin (1988) that lignin degradation by

many white-rot fungi is stimulated by low nitrogen content

in the culture medium is in contrast with results indicating

higher laccase activity of L. edodes in media supplemented

with nitrogen sources (Kaal et al. 1995, Kachlishvili et al.

2005). Our results indicate that the expression of L. edodes

lignolytic potential is strain-dependent as well as substrate-

dependent. Strain 121 furnished high laccase levels on WS,

with lower N content, while strain 122 produced high

laccase activities on BS, a substrate with relatively higher

N content. Previous reports mention the occurrence of

quantitative variations of enzyme activity on different

L. edodes strains and culture media (Mata and Savoie 1998;

Elisashvili et al. 2008). In general, all tested strains pro-

duced high levels of laccase activities. Regarding their

production patterns, the sole maximum pattern of laccase

activity obtained by all strains at 66% of colonization on

both substrates, is in accordance with the single peak pat-

tern observed by Panichajakul et al. (1991) in liquid culture

between 30 and 40 days of growth and by Mata and Savoie

(1998) on wheat straw during the first weeks of cultivation.

Furthermore, Moyson and Verachtert (1991), culturing

L. edodes on wheat straw, pointed out that lignin was

degraded only after several weeks.

The controversial patterns of endoglucanase and laccase

production observed on WS and RG substrates by strain

122, could be explained as follows. The supplementation of

the tested substrates with wheat bran and soybean flour

increased N pool and readily available carbohydrates level,

stimulating so initial endoglucanase and biomass produc-

tion (Silva et al. 2005a, Arai et al. 2005). The deceleration

of growth rate after the 3rd week of colonization, being in

agreement with previous results (Leatham 1985), indicates

the limitation of utilizable nutrients and soluble carbon

sources (Mata and Savoie 1998). As a result, endoglucan-

ase activity of this moderate cellulolytic fungus decreased.

At the same time, laccase production rapidly increased,

after an initial lag in activity at the start of colonization

(Leatham 1985, Elisashvili et al. 2008). The breakdown of

the lignin polymers by L. edodes, known to be an effective

lignin degrader, permits the fungus to gain access to car-

bohydrates that are embedded in the lignin matrix. As a

result, biomass production peaked and nutrients and N

became limited. These are prerequisites, along with envi-

ronmental stimulus (cold shock) to induce fruiting. At the

end of colonization, just before fruiting, a second cycle of

endoglucanase activity was observed. Decrease of laccase

activity and a complementary increase in the cellulose

activity has been associated with primordium formation

(Ohga 1992; Ohga et al. 2000). Through this pattern, at the

sporophore production phase the polysaccharides are easily

available as carbon source to support fructification process.

In the case of BS substrate, it can be speculated that its

relatively higher N content along with the nature of this

ligninocellulosic substrate promoted a simultaneous deg-

radation of cellulose and lignin, as indicated by initially

high endoglucanase and laccase activities. This is sup-

ported by previously published relevant results indicating

stimulation of ligninolytic activity of L. edodes (Leatham

and Kirk 1983) as well as cellulolytic activity and biomass

production (Silva et al. 2005b) by increased nitrogen in the

substrate. This positive correlation of laccase and endo-

glucanase activities indicates that a high biological poten-

tial of the strain along with high metabolic activity in the

first days of substrate colonization are prerequisites to

achieve vigorous mycelium growth and high biomass yield.

Conclusions

As the length of the incubation period of L. edodes relates

to both the duration cycle and the reduction of potential

contamination risks, it is of prime importance to select fast

growing substrate-adapted strains. Moreover, due to the

differences among substrates it is necessary to investigate

the influence of the nature of the lignocellulosic substrates

to mycelium growth and enzymes secretion during their

bioconversion. This work, investigating the rate of sub-

strate colonization in SSF of L. edodes, shows the impor-

tance of simultaneous evaluation of mycelium growth rate,

biomass yield and activities of hydrolytic and oxidative


123

enzymes, along with analysis of constituents of the sub-

strates. The results obtained showed that high mycelium

growth rate and biomass yield, as well as high endoglu-

canase and laccase production during vegetative growth of

L. edodes are usually negatively related variables. Their

desirable coincidence depends on the fungal strain and is

strongly influenced by the nature and composition of the

lignocellulosic substrate. Moreover, data indicated the

patterns of secretion of these enzymes in substrate matrix

during the entire colonization period. BS substrate proved

to support good growth and mycelial biomass production,

supporting high levels of hydrolytic and oxidative enzymes.

The obtained results support the potential effective and

profitable utilization of bean stalk (BS), alone or in mixtures

with reed grass (RG) and wheat straw (WS) residues as of

L. edodes cultivation substrate. However, farther experi-

ments investigating the impact of these substrates on

L. edodes sporophore yield and quality have to be conducted.

Acknowledgments The authors acknowledge financial support

given partially by the General Secretariat of Research and Technol-

ogy through the 05 PAV 105 project and partially by the Institute of

Agricultural Engineering of the National Agricultural Research

Foundation (NAGREF). They also thank Dr. Y. Kazoglou, (Society

for the Protection of Prespa) for providing the raw substrate material

and Dr. C. Mountzouris (Agricultural University of Athens) for

helping in raw material processing.

References

Albrecht R, Ziarelli F, Alarcon-Gutierrez E, Petit JL, Terrom G,

Perissol C (2008) 13C solid-state NMR assessment of decompo-

sition pattern during co-composting of sewage sludge and green

wastes. Eur J Soil Sci 59(3):445–452

Arai Y, Shirasaka N, Yoshikawa K, Kitamoto Y, Suzuki A, Sakamoto

R, Sata H, Terashita T (2005) The stimulation of extracellular

carbohydrases of edible mushrooms by the hot water-soluble

fraction from corn fiber. Mycoscience 46:235–240

Bhat MK (2000) Cellulases and related enzymes in biotechnology.

Biotechnol Adv 18:355–383

Bollag JM, Shuttleworth LK, Anderson HD (1988) Laccase mediated

detoxification of phenolic compounds. Appl Environ Microbiol

54:3086–3091

Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic

substrates: an expanded role of laccase in lignin biodegradation.

FEBS Lett 267:99–102

Boyle CD (1998) Nutritional factors limiting the growth of Lentinulaedodes and other white-rot fungi in wood. Soil Biol Biochem

30(6):817–823

Buswell JA, Chang ST (1993) Edible mushrooms: attributes and

applications. In: Chang ST, Bushwell JA, Miles PG (eds)

Genetics and breeding of edible mushrooms. Gordon and Bearch

Scientific Publishers, Philadelphia, pp 297–324

Buswell JA, Cai YJ, Chang ST (1996) Ligninolytic enzyme

production and secretion in edible mushroom fungi. In: Royse

DJ (ed) Mushroom biology and mushroom products. Penn State

University, Pennsylvania, pp 113–122

Cavallazzi JRP, Brito MS, Oliveira MGA, Villas-Boas SG, Kasuya

MCM (2004) Lignocellulolytic enzymes profile of three

Lentinula edodes (Berk.) Pegler strains during cultivation on

eucalyptus bark-based medium. Food Agr Env 2(1):291–297

Chang ST (2006) The world mushroom industry: trends and

technological development. Int J Med Mushrooms 8(4):297–314

Chiu SW, Law SC, Ching ML (2000) Themes for mushroom

exploitation in the 21st century: sustainability, waste manage-

ment, and conservation. J Gen Appl Microbiol 46:269–282

Di Lena G, Vivanti V, Quaglia GB (1997) Amino acid composition of

wheat milling by-products after bioconversion by edible myce-

lia. Nahrung 41(5):285–288

Di Mario F, Rapana P, Tomati U, Galli E (2008) Chitin and chitosan

from basidiomycetes. Int J Biol Macromol 43:8–12

Diehle DA, Royse DJ (1986) Shiitake cultivation on sawdust:

evaluation of selected genotypes for biological efficiency and

mushroom size. Mycologia 78(6):929–933

Dill I, Kraepelin G (1988) Der Abbau von lignin/cellulose durch

weissfaule-pilze: Einfluss spezifischer okologischer faktoren.

Forum Mikrobiol 11(88):484–489

Elisashvili V, Penninckx M, Kachlishvili E, Tsiklauri N, Metreveli E,

Kharziani T, Kvesitadze G (2008) Lentinus edodes and Pleuro-tus species lignocellulolytic enzymes activity in submerged and

solid-state fermentation of lignocellulosic wastes of different

composition. Biores Technol 99:457–462

Fan L, Soccol CR (2005) Coffee residues. In: Gush R (ed) Mushroom

growers’ handbk 2. Mush World, Seoul, pp 92–95

Gaitan-Hernandez RG, Esqueda M, Gutierrez A, Sanchez A, Beltran-

Garcia M, Mata G (2006) Bioconversion of agrowastes by

Lentinula edodes: the high potential of viticulture residues. Appl

Microbiol Biotechnol 71:432–439

Ghorai S, Banik SP, Verma D, Chowdhury S, Mukherjee S, Khowala

S (2009) Fungal biotechnology in food and feed processing.

Food Res Int 42:577–587

Gilardi G, Abis L, Cass AEG (1995) Carbon-13 CP/MAS solid-state

NMR and FT-IR spectroscopy of wood cell wall biodegradation.

Enzyme Microb Technol 17(3):268–275

Harkin JM, Obst JR (1973) Syringaldazine, an effective reagent for

detecting laccase and peroxidase in fungi. Experientia 29(4):

381–508

Haw JF, Maciel GE, Schroeder HA (1984) C-13 nuclear magneticre-

sonance spectrometric study of wood and wood pulping with cross

polarization and magic-angle spinning. Anal Chem 56:1323–1329

Israilides C, Kletsas D, Arapoglou D, Philippoussis A, Pratsinis H,

Ebrgerova A (2008) Cytostatic and immunomodulatory proper-

ties of the medicinal mushroom Lentinula edodes. Phytomedi-

cine 15:512–519

Kaal EE, Field JA, Joyce TW (1995) Increasing lignolytic enzyme

activities in several white-rot basidiomycetes by nitrogen-

sufficient media. Bioresour Technol 53:133–139

Kachlishvili E, Penninckx MJ, Tsiklauri N, Elisashvili V (2005)

Effect of nitrogen source on lignocellulolytic enzyme production

by white-rot basidiomycetes under solid-state cultivation. World

J Microbiol Biotechnol 22(4):391–397

Kalberer PP (1995) An investigation of the incubation phase of a

shiitake (Lentinus edodes) culture. Mushroom Sci 14:375–383

Kalberer PP (2000) Influence of urea and ammonium chloride on crop

yield and fruit body size of shiitake (Lentinula edodes).

Mushroom Sci 15:361–366

Lakhtar H, Ismaili-Alaoui M, Philippoussis A, Macarie H, Roussos S

(2007) Preliminary studies on Lentinula edodes strains able to

degrade polyphenols in olive wastewater. In: XVI Nat bioproc-

ess symposium—SINAFERM 2007, Curitiba

Lakhtar H, Ismaili-Alaoui M, Philippoussis A, Perraud-Gaime I,

Roussos S (2010) Screening of strains of Lentinula edodesgrown on model olive mill wastewater in solid and liquid state

culture for polyphenol biodegradation. Int Biodet Biodeg

64(3):167–172


123

Leatham GF (1985) Extracellular enzymes produced by the cultivated

mushroom Lentinus edodes during degradation of a lignocellu-

losic medium. Appl Environ Microbiol 50:859–867

Leatham GF, Kirk TK (1983) Regulation of ligninolytic activity by

nutrient nitrogen in white-rot basidiomycetes. FEMS Microbiol

Lett 16:65–67

Lin Z (2005) Grass. In: Gush R (ed) Mushroom growers’ handbk 2.

Mush World, Seoul, pp 96–99

Lomberh ML, Solomko EF, Buchalo AS, Kirchhoff B (2002) Studies

of medicinal mushrooms in submerged cultures. In: Sanchez JE,

Huerta G, Montiel E (eds) Mushroom biology and mushroom

products. UAEM, Cuernavaca, pp 367–377

Makkar RS, Tsuneda A, Tokuyasu K, Mori Y (2001) Lentinulaedodes produces a multi-component protein complex containing

manganese (II)-dependent peroxidase, laccase and beta-glucosi-

dase. FEMS Lett 200:175–179

Mandels M, Andreotti R, Roche C (1976) Measurement of saccha-

rifying cellulase. Biotechnol Bioeng Symp 6:21–33

Massiot D, Fayon F, Capron M, King I, Le Calve S, Alonso B,

Durand J-O, Bujoli B, Gan Z, Hoatson G (2002) Modeling one-

and two-dimensional solid state NMR spectra. Magn Reson

Chem 40:70–76

Mata G, Savoie JM (1998) Extracellular enzyme activities in six

Lentinula edodes strains during cultivation in wheat straw.

World J Microbiol Biotechnol 14:513–519

Mata G, Savoie JM (2005) Whet straw. In: Gush R (ed) Mushroom

growers’ handbk 2. Mush World, Seoul, pp 105–109

Miller GL (1959) Use of dinitrosalicylic acid reagent for determina-

tion of reducing sugars. Anal Chem 31:426–428

Montini RMC, Passos JRS, Eira AF (2006) Digital monitoring of

mycelium growth kinetics and vigor of shiitake (Lentinulaedodes (Bergk.) Pegler) on agar medium). Braz J Microbiol 37:

90–95

Moyson E, Verachtert H (1991) Growth of high fungi on wheat straw

and their impact on the digestibility of the substrate. Appl

Microbiol Biotechnol 36:421–424

Nikitina VE, Tsivileva OM, Pankratov AN, Bychkov A (2007)

Lentinula edodes biotechnology—from lentinan to lectins. Food

Technol Biotechnol 45(3):230–237

Oei P (2003) Mushroom cultivation. In: Oei P (ed) Appropriate

technology for mushroom growers, CTA, 3rd edn. Backhuys

Publishers, Leiden, pp 1–7

Ohga S (1992) Comparison of extracellular enzyme activities among

different strains of Lentinus edodes grown on sawdust-based

cultures in relationship to their fruiting abilities. J Jpn Wood Res

Soc 38:310–316

Ohga S, Royse DJ (2001) Transcriptional regulation of laccase and

cellulose genes during growth and fruiting of Lentinula edodeson supplemented sawdust. FEMS Microbiol Lett 201:111–115

Ohga S, Cho NS, Thurston CF, Wood DA (2000) Transcriptional

regulation of laccase and cellulase in relation to fruit body

formation in the mycelium of Lentinula edodes on a sawdust-

based substrate. Mycoscience 41:149–153

Okano K, Iida Y, Samsuri M, Prasetya B, Usagawa T, Watanabe T

(2006) Comparison of in vitro digestibility and chemical

composition among sugarcane bagasses treated by four white-

rot fungi. Anim Sci J 77:308–313

Ozcelik E, Peksen A (2007) Hazelnut husk as a substrate for the

cultivation of shiitake mushroom (Lentinula edodes). Biores

Technol 98:2652–2658

Panichajakul S, Yindeeyoungyeon W, Triratana S (1991) Alteration

of laccases and acid phosphatase in mono-and dikaryotic

mycelia of Lentinus edodes. In: Maher MJ (ed) Science and

cultivation of edible fungi. Balkema, Rotterdam, pp 241–249

Pereira Junior JA, Correia MJ, Oliveira NT (2003) Cellulase activity

of a Lentinula edodes (Berk.) Pegl. strain grown in media

containing carboximetilcellulose or microcrystalline cellulose.

Braz Arch Biol Technol 46(3):333–337

Philippoussis A (2009) Production of mushrooms using agro-indus-

trial residues as substrates. In: Sing Nigam P, Pandey A (eds)

Biotechnology for agro-industrial residues processing. Springer,

Berlin, pp 163–196

Philippoussis A, Zervakis G, Diamantopoulou P (2001) Bioconver-

sion of lignocellulosic wastes through the cultivation of the

edible mushrooms Agrocybe aegerita, Volvariella volvacea and

Pleurotus spp. World J Microbiol Biotechnol 17(2):191–200

Philippoussis A, Diamantopoulou P, Zervakis G (2002) Monitoring of

mycelium growth and fructification of Lentinula edodes on

several agricultural residues. In: Sanchez JE, Huerta G, Montiel

E (eds) Mushroom biology and mushroom products. UAEM,

Cuernavaca, pp 279–287

Philippoussis A, Diamantopoulou P, Zervakis G (2003) Correlation of

the properties of several lignocellulosic substrates to the crop

performance of the shiitake mushroom Lentinula edodes. World

J Microbiol Biotechnol 19(6):551–557

Philippoussis A, Diamantopoulou P, Israilides C (2007a) Productivity

of agricultural residues used for the cultivation of the medicinal

fungus Lentinula edodes. Int Biodeterior Biodegrad 59(3):216–

219

Philippoussis A, Diamantopoulou P, Kokkinis G, Kazoglou Y,

Parisopoulos G (2007b) Potential use of littoral vegetation

residues from Lake Mikri Prespa, as substrate for mushrooms

cultivation. In: Proceedings of international conference on

environment management, engineering, planning and economics

(CEMEPE 2007), Skiathos, pp 1663–1669

Pire DG, Wright JE, Alberto E (2001) Cultivation of shiitake using

sawdust from widely available local woods in Argentina. Micol

Apl Int 13(2):87–91

Ride JP (1980) The effect of induced lignification on the resistance of

wheat cell walls to fungal degradation. Physiol Plant Pathol

16:187–196

Ride JP, Drysdale RB (1972) A rapid method for the chemical

estimation of filamentous fungi in plant tissue. Physiol Plant

Pathol 2:7–15

Rigas F, Papadopoulou K, Philippoussis A, Papadopoulou M,

Chatzipavlidis J (2009) Bioremediation of lindane contaminated

soil by landfarming technology. Water Air Soil Pollut 197:

121–129

Rossi IH, Monteiro AC, Machado JO (2003) Shiitake Lentinulaedodes production on a sterilized bagasse substrate enriched with

rice bran and sugarcane molasses. Braz J Microbiol 34:66–71

Royse DJ (2004) Specialty mushrooms, mushroom fact sheet. Mush-

room Spawn Laboratory, Penn State University, Pennsylvania

Royse DJ, Bahler CC (1986) Effects of genotype, spawn run time and

substrate formulation on biological efficiency of shiitake. Appl

Environ Microbiol 52(6):1425–1427

Royse DJ, Sanchez JE (2007) Ground wheat straw as a substitute for

portions of oak wood chips used in Shiitake (Lentinula edodes)

substrate formulae. Biors Technol 98:2137–2141

Royse DJ, Sanchez-Vazquez JE (2001) Influence of substrate wood-

chip particle size on shiitake (Lentinula edodes) yield. Biores

Technol 76:229–233

Salmones D, Mata G, Ramos LM, Waliszewski KN (1999) Cultivation

of shiitake mushroom, Lentinula edodes in several lignocellulosic

materials originating from the subtropics. Agron 19(1):13–19

Scotti CT, Vergoignan C, Feron G, Durand A (2001) Glucosamine

measurement as indirect method for biomass estimation of

Cunninghamella elegans grown in solid state cultivation condi-

tions. Biochem Eng J 7:1–5

Silva EM, Machuca A, Milagres AMF (2005a) Effect of cereal brans

on Lentinula edodes growth and enzyme activities during

cultivation on forestry waste. Lett Appl Microbiol 40:283–288


123

Silva EM, Machuca A, Milagres AMF (2005b) Evaluating the growth

and enzyme production from Lentinula edodes strains. Process

Biochem 40:161–164

Silva ES, Cavallazzi JRP, Muller G (2007) Biotechnological appli-

cations of Lentinus edodes. J Food Agric Environ 5(3&4):403–

407

Smith JE, Rowan NJ, Sullivan R (2002) Medicinal mushrooms: a

rapidly developing area of biotechnology for cancer therapy and

other bioactivities. Biotechnol Lett 24:1839–1845

Song CH, Chow KY (1987) A synthetic medium from the production

of submerged cultures of Lentinus edodes. Mycol 79:866–876

Srijumpa N (2003) Use of grasses as a substrate for the cultivation of

oyster mushroom. Food and Fertilizer Technology Center

(FFTC), Taipei

Wasser S (2002) Medicinal mushroom as a source of antitumor and

immunomodulating polysaccharides. Appl Microbiol Biotechnol

60:258–274


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ORIGINAL PAPER Biomass, laccase and endoglucanase production by Lentinula edodes during solid state...

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