nyl:guaiacyhsyringyl) and Ac/(A1 + Ke) (acid to aide- g/lOOg g/lOOg. 3'° l. A. 3.o- ..... 185) and the ECLAIR Program of the European Community (EC contract no.
Appl Microbiol Biotechnot(1991) 35:817-823 017575989100228T
Applied Microbiology Biotechnology © Springer-Verlag 1991
Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus lignin and polysaccharide alteration and production of related enzymatic activities Manuel Valmaseda, Maria Jesfis Martinez, and Angel T. Martinez Centro de InvestigacionesBiol6gicas,Consejo Superior de InvestigacionesCientlficas,Velfizquez 144, E-28006 Madrid, Spain Received 15 March 1991/Accepted 24 May 1991
Summary. The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two successive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of sidechains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Laccase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of HzOz-producing aryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus.
Straw treatment with Pleurotus ostreatus was first studied by Zadrazil (1977) and Lindenfeiser et al. (1979), and preferential removal of lignin, increasing in-vitro digestibility, has been obtained with Pleurotus species (Kamra and Zadrazil 1986; Valmaseda et al. 1990). Trametes versicolor produces simultaneous degradation of lignin and polysaccharides, but under certain growth conditions it can be used for straw delignification (Zafar et al. 1989). Although most of the above studies are connected with improving straw for animal feeding, the use of T. versicolor and P. ostreatus in paper pulp production has also been suggested (Kirpatrick et al. 1989; Schiesser et al. 1989). Important progress in the knowledge of the enzymatic aspects of lignin degradation has been produced since lignin peroxidase was described in Phanerochaete chrysosporium. Similar ligninases have been found in other white-rot fungi (Dodson et al. 1987; Muheim et al. 1990; Niku-Paavola et al. 1988), and the mechanisms of lignin depolymerization and degradation are now partially established (Higuchi 1990). However, little is known about the production and activity of the enzymes responsible for lignin degradation during lignocellulose transformation under natural or SSF conditions (Mishra and Leatham 1990).
Introduction Materials and methods Solid-state fermentation (SSF) has traditionally been used in composting agricultural wastes for mushroom cultivation and the production of organic fertilizers. Straw SSF with ligninolytic fungi increases the enzymatic hydrolysis of polysaccharides (feed and fuel production), but may also be used for reducing pulping costs. Different aspects of straw SSF have been investigated, such as strain screening and process control for optimum lignin degradation (Kamra and Zadrazil 1988; Zadrazil 1985). However, structural changes in straw lignin during fungal delignification are still little known (Agosin et al. 1985). Offprint requests to: A. T. Martinez
Fungal strains and SSF conditions. T. versicolor IJFM A137 and P. ostreatus IJFM A163 were selected from 45 fungal strains (Valma-
seda et al. 1990). Wheat straw SSF was performed in a horizontal multi-vessel rotary fermentor using the conditions described by Valmaseda (1989). The process was monitored by COz release, and the straw from a fermentationvessel (initiallycontaining25 g of straw and 75 ml of 0.25% NH4NO3) was freeze-dried and analysed after 0, 4, 7, 10, 20, 30, 40, 50 and 60 days. Substrate characterization and lionin analysis. Weight loss, pH, ash content, extractives,solubilityin water and alkali, Klason and acid-soluble lignin, fungal biomass (from chitin content) and digestibility were estimated. Sugars in water-soluble fractions and polysaccharide hydrolysateswere analysed. Alkali lignins were obtained by acid precipitation after alkaline extraction, and the
818 distribution of apparent molecular size was evaluated using Sephadex G-100 chromatography. The conditions for these analyses were as reported by Valmaseda et al. (1990). Protein was estimated after trichloroacetic acid precipitation (Ulmer et al. 1981). Lignin alteration was analysed by CuO alkaline degradation (Hedges and Ertel 1982). One hundred milligrams of extracted straw was degraded at 170 ° C (3 h, N2) in teflon bombs with 2 g CuO, 200rag Fe(NHa)2(SO4)~.6H20 and 14ml of 2M NaOH. The degradation products were recovered with ether, suspended in 100 ~1 pyridine (with ethylvanilline as internal standard), derivatized with bis(trimethylsilyl)-trifluoroacetamide, analysed by gas chromatography (GC) (30mx0.25mm SP-2100 column, 100°C to 270°C at 4°C/min) and identified using an ion trap mass-detector. Response factors were determined from mixtures of standards.
Statistical analyses. Principal component analysis was performed using the programmes of Orlrci and Kenkel (1985). Samples of straw treated with the two fungi studied were compared on the basis of average data obtained from substrate analyses, lignin characteristics and production of enzymatic activities. The correlation matrix between variables was calculated, and a set of new axes (eigenvectors) was obtained as linear combinations between the original variables (the loading factor of each variable on these axes was calculated). The two series of samples corresponding to the straw SSF with the fungi studied were represented on the first two axes, explaining the highest percentage of the total variance.
Estimation of enzymatic activities. The enzymatic activities were estimated in a crude enzyme extract from freeze-dried straw (25 mg/ml), and fresh material was used for peroxidase and oxidase estimation. One activity unit (U) was defined as the amount of enzyme producing 1 ~tmol/min of reaction product (endocellulase and laccase units are defined below). The cellulase activities (Wood and Bhatt 1988) were estimated in 50 mM acetate buffer (pH 4.8). Exocellulase (EC 3.2.1.91) was tested using 0.2 ml crude extract and 0.8 ml of 20 mg/ml Avicel (4 h, 50 ° C), and reducing sugars were estimated by the SomogyiNelson method. Endocellulase (EC 3.2.1.4) was tested using a 300 Cannon-Fenske viscometer, after incubation of 0.5 ml crude extract and 4.5 ml of 20 mg/ml Na carboxymethylcellulose for 5 min at 50 ° C. One unit was defined as the enzyme producing 1% viscosity reduction/min, fl-Glucosidase (EC 3.2.1.21) was determined using 0.1 ml crude extract and 0.9 ml of 1 mg/ml p-nitrophenyl-fl-D-glucopyranoside (4h, 60°C). Xylanase (EC 3.2.1.8) was assayed using 0.1 ml crude extract and 0.9 ml of a 70 ixg/ml larchwood xylan suspension in the same buffer, and reducing sugars were estimated after 4 h at 37 ° C. Laccase (EC 1.10.3.2) was assayed using 50 ~tl crude extract, 0.9 ml water and 50 ixl of 1% syringaldazine in ethanol (Harkin and Obst 1973). One unit was defined as the enzyme producing one absorption unit/min at 527 nm. Lignin peroxidase activity was estimated as the oxidation of veratryl alcohol (Tien and Kirk 1988) using 50 ~tl crude extract, 0.75 ml of 4 mM veratryl alcohol in 50 mM sodium tartrate (pH 3.0), and 0.2 ml of 3 mM H202. Aryl-alcohol oxidase was assayed as lignin peroxidase without H202 addition (Guillrn et al. 1990b). Mn(II)-dependent peroxidase was investigated using 0.25 ml crude extract, 0.25 ml of 0.1 mM H202 and 0.5 ml of phenol red solution (Kuwahara et al. 1984).
Substrate transformation during fungal treatment
g/lOOg
Results
T h e e v o l u t i o n o f t h e CO2 r e l e a s e d f r o m s t r a w d u r i n g S S F w i t h b o t h f u n g i is s h o w n in Fig. 1. W h e a t s t r a w c o m p o s i t i o n , w e i g h t loss, d i g e s t i b i l i t y a n d a l k a l i solub i l i t y d u r i n g S S F a r e p r e s e n t e d in T a b l e 1. T h e f u n g a l b i o m a s s was c o r r e l a t e d with t h e p r o t e i n c o n t e n t s h o w n in T a b l e 1, r e a c h i n g 9.5% with T. versicolor a n d 7.3% w i t h P. ostreatus. The s t r a w p H o n l y c h a n g e d d u r i n g the first S S F w e e k , d e c r e a s i n g f r o m 5 to 4.5. F r e e s u g a r s d e c r e a s e d s i m u l t a n e o u s l y , b u t i n c r e a s e d later. T h e c o m p o s i t i o n o f s t r a w p o l y s a c c h a r i d e s is s h o w n in T a b l e 1. T h e g l u c a n / p e n t o s a n ratio i n c r e a s e d a f t e r f u n g a l treatment, but simultaneous degradation of both types o f p o l y s a c c h a r i d e s was i n i t i a l l y p r o d u c e d b y T. versicolor (Fig. 2a). T h e a l k a l i - s o l u b l e m a t e r i a l was i n c r e a s e d b y t h e fungi ( T a b l e 1). A n i n c r e a s e in t h e a l k a l i - s o l u b l e l i g n i n y i e l d was p r o d u c e d d u r i n g t h e first S S F d a y s a n d d e c r e a s e d later. T h e m o l e c u l a r size d i s t r i b u t i o n o f s o m e o f t h e lignins is s h o w n in Fig. 3.
Changes in lignin composition G a s c h r o m a t o g r a p h y after C u O a l k a l i n e d e g r a d a t i o n o f s t r a w a f t e r 60 d a y s o f f u n g a l S S F a n d c o n t r o l a r e s h o w n in Fig. 4. M o l a r H : G : S (p-hydroxyphen y l : g u a i a c y h s y r i n g y l ) a n d A c / ( A 1 + K e ) ( a c i d to a i d e -
Fig. 1. Carbon dioxide release (g/100 g initial straw) during solid-state fermentation (SSF) with Trametes versicolor ( A ) and Pleurotus ostreatus (B)
g/lOOg
3'°l
A
2.6-
2.0
2.0
1.6
1,6
1.0
1.0
0.6-
0,6
10
20
30
Days
40
50
B
3.o-
2.6
60
0
10
20
30
Days
40
60
60
819
T.versicolor 60d
75
7o
P.0s _ t r e a t u ~
6.06'06'05.0
50d
65
4,0-
/~
50 i c o n t r o l /
I~.0-
[]
[]
A
~o~
/
45 ~
20d
~olX / ~"
a.~ ~
2.0
0
,
,
10
20
,
,
!
,
30
40
50
60
.~>
~60d
~o
,.o-
Fig. 2. G l u c a n / p e n t o s a n ( G l u / P e n ) ratio (A) and straw digestibility vs weight loss (B) during straw solid-state fermentation (samples from 0 to 60 days are given in B)
% Digestibility
Glu/Pen
a5 -~
~ .
0
10
Days
B .
.
.
20
.
30
40
60
% Weight loss
O.D. 0.6-
O.D.
0.3-
0.4"
Fig. 3. Gel chromatography (Sephadex G-100) of alkali-soluble lignins from control straw (A, D) and from straw after 30 days (B, E) and 60 days (C, F) SSF: kay, elution vol - void voi total vol - void vol ' O.D., optical density
rotusostreatus D
Trametesversicolor 0,2
0,3
0,20.1
0,1
0
!
0
0,5
1
o.
1,6
~
~
0
~
O.~i
Kay
1
1.6
Kay
Table 1. Changes in straw composition with solid-state fermentation time (g/100 g straw) Parameters
Control
Trametes versicolor
4 Weight loss Digestibility Alkali solubility Ash Protein Extractives Water-soluble Klason lignin Soluble lignin Glucose a Xylose a Arabinose a
0.0 46.8 53.5 6.6 1.6 9.3 11.6 15.2 1.5 37.6 10.5 4.6
a From polysaccharide hydrolysis
7
10
2.0 7.0 8.6 40.6 39.6 45.9 49.6 44.2 45.5 6.7 7.8 8.1 1.8 1.9 1.9 6.6 4.5 5.9 8.7 10.8 13.2 15.2 15.0 13.9 1.2 1.1 1.2 43.1 43.2 39.4 10.6 10.3 10.3 4.5 4.2 4.2
20
20.4 55.5 51.3 10.1 2.5 6.5 17.8 11.8 1.5 .34.7 10.0 4.1
Pleurotus ostreatus
30 40 (days) 31.9 65.9 55.7 11.7 3.3 8.5 19.7 10.7 1.7 29.9 9.4 3.6
41.8 66.9 61.7 13.6 3.7 9.2 21.5 10.0 1.8 28.4 5.7 3.0
50
60
4
45.4 68.3 65.4 14.0 4.2 8.2 24.9 9.4 1.8 28.3 4.3 2.0
54.3 70.5 64.3 15.8 4.8 12.1 27.7 8.4 2.1 22.5 2.8 1.5
0.7 1.2 9.7 16.0 46.3 44.4 43.3 46.3 46.1 45.7 43.9 46.6 6.6 7.1 7.6 8.0 1.4 1.9 1.8 2.1 7.1 5.8 4.8 3.7 10.7 9.0 8.0 11.7 14.9 15.4 14.5 13.5 1.3 1.3 1.4 1.5 42.8 47.2 47.1 47.1 10.6 8.0 8.1 7.4 3.4 3.3 4.6 3.7
7
10
20 30 (days)
40
50
21.9 55.1 48.0 8.6 2.4 3.9 13.8 13.0
26.8 58.8 50.8 9.4 2.5 5.6 15.2 12.1
27.7 28.3 59.5 61.5 53.7 56.5 9.8 10.0 3.0 3.8 6.0 6.3 17.7 17.8 11.0 10.2
1.6
1.7
1.7
60
1.8
44.0 41.4 39.8 40.1 7.2 5.7 4.6 4.4 3.2 2.7 2.7 2.2
820 Control H:G~S. 9:41:50 Cin/(H*Q*S) -O.41 Ac/(AI+Ke)- O.24
(peak d) were obtained (Fig. 4). Small amounts of 3,4dihydroxybenzoic acid were also detected (peak c). Some aliphatic compounds (dicarboxylic and fatty acids) were identified after CuO degradation, and the azelaic (nonanedioc) acid (peak b), a product from the oxidative cleavage of oleic and other C9:Clo unsaturated fatty acids, was the most abundant.
I1 12 10
tot ion. b
Lionocellulose-deoradin O enzymatic activities 9
I
~
I
T. v e r s i c o l o r H:G:S" 15:50:35
Cin/(H+G*$).O.22 Ac/(AI*Ke) • 0.56
10
I0t ion.
12
4 56
10~,
I
I
i
~
I
~
I
~
t
The evolution of enzymatic activities responsible for cellulose degradation is shown in Fig. 5a, b. The decrease in the initial straw glucan content is presented simultaneously. Very low exocellulase activity was found in straw treated with P. ostreatus. Xylanase and xylan degradation are presented in Fig. 5c, d. Lignin peroxidase of T. versicolor was found for a short period, showing the highest lignin degradation rate (Fig. 5e), and Mn(II)-dependent peroxidase was not detected. Laccase was found in straw treated with both fungi, and high aryl-aleohol oxidase levels were produced by P. ostreatus (Fig. 5e, f).
P. o s t r e a t u s H:G:S- 2 1 ; 4 2 : 3 7 Cin/(H÷G*S)-0.35 A c / ( A I * K e ) - 1.43
Principal component analysis
10
T01
b
I011, 8
~
I
i
~
I
J
[,,
12
II
?
i
I
i
I
'
I
8~ 12~ l~l~ 2~ ~.c~ 13:21 2~:81 2~:~ 33:21 ,~n Fig. 4. Gas chromatography and molar ratios of CuO degradation products from control straw and from straw after 60 days SSF with T. versicolor and P. ostreatus. Peaks: 1, p-OH-benzaldehyde; 2, p-OH-acetophenone; 3, vanillin; 4, ethy]vanillin (standard); 5, acetovanillone; 6, p-OH-benzoic acid; 7, syringaldehyde; 8, acetosyringone; 9, vani]lic acid; 10, syringic acid; 11, p-coumafic acid; 12, fe~]ic acid; a, m-OH-benzoic acid; b, azelaic acid; c, 3,4diOH-benzoic acid; d, sinapic acid
hyde+ketone) ratios are also shown. Cinnamic acids (Cin) were not included in the above molar contents, and the Cin/(S + G + H) ratio is also given. During SSF a linear decrease in the relative S content and an increase in the H content were observed with both fungi (p > 0.95), and the degradation of cinnamic acids by P. ostreatus also showed a linear decrease. The Ac/ (AI+ Ke) ratio increased during SSF, but did not increase further after the removal of 30-40% of straw lignin. Although the recovery of CuO oxidation products was not exhaustive, the CuO-yield/Klason-lignin ratio was constant during the treatment with T. versicolor, and slightly decreased by P. ostreatus (both variables showed a very significant correlation). m-Hydroxybenzoic acid (peak a) was less abundant than p-hydroxybenzoic acid, and traces of sinapic acid
The results of principal component analysis are shown in Fig. 6. The disposition of the samples on the two first axes (Fig. 6a) showed the temporal evolution of the straw SSF with the two fungi studied. The variables with the highest loading factors on these two axes, explaining 62% of the total variance, are presented as vectors (Fig. 6b). The whole SSF process, the different phases observed, and the transformation pattern produced by each fungus are discussed on the basis of the tendencies evidenced by these variables.
Discussion
Two phases could be defined during fungal SSF of wheat straw. The "colonization phase" was characterized by a strong increase in respiratory activity and decrease in free sugars, extractives and in vitro-digestibility. Its duration was estimated to be 7 days for T. versicolor and 10 days for P. ostreatus. The "degradation phase" represented fungal attack on the lignin and polysaccharides and an increase in straw digestibility and protein content. The SSF phases and the fungal degradation patterns showed up better after principal component analysis. The colonization phase was shown by the displacement on the second axis, and the first axis shows the straw degradation tendency (Fig. 6a). The above-mentioned variables present the highest loading factors (Fig. 6b) on both axes. When the increases in straw digestibility were considered together with the weight losses produced by the fungi (Fig. 2b), it became clear that no substrate improvement was obtained after 30-40 days of treatment. Straw digestibility and Klason lignin were very signifi-
821 Trametes
versicolor
Pleurotus
ostreatus
0.3
100 ~
~ ~
Endocellulase
~
-~-
B-Qlucosldase
--
Qlucan
0.2
~
Exocellulase 8O i
°°°
g ~/;~-
40
0.1
~
~^,,.
~ ~
0 C ~
~
~
T
~-, .~
,o
o
I
I
"w /
Xylanase
~-
8o
~,0 ~
0.2
~
";
60
~X
g
o.~
~
~e
/
~
~
~
~
, ~
~
~
I
~
c I/ ~
~
~
~
~
I ~00
~4 12
80
lO 60 ~
r-
~" ~,
4
20 ~
~ 10
20
~
~
,
30
40
50
-
;~/_. 60
10
,
,
~
20
30
40
F
~ 50
60
Days
Fig. 5. Changes in enzymatic activities (U/g initial straw) involved in cellulose (A, B), hemicellulose (C, D) and lignin (E, F) degradation during SSF with T. versicolor (A, C, E) and P. ostreatus (B, D, F), AAO, aryl-alcohol oxidase
cantly correlated during SSF. The same was observed by Agosin et al. (1985), who suggested that soluble lignin may be involved in increasing digestibility. However, a small increase in soluble lignin was produced during straw lignin degradation, and in-vitro digestibility was mainly related to the changes in the water-soluble fraction. Protein enrichment is obtained by straw SSF with cellulolytic fungi (Ulmer et al. 1981), but the ligni-
nolytic species studied can increase both digestibility and protein content. The low-molecular-size lignin fractions disappeared after fungal treatment, especially in the case of T. versicolor (Fig. 3). This may correspond to the preferential degradation of these fractions, since repolymerization of the degradation products, as reported during lignin enzymatic treatment (Haemmerli et al. 1986), could be discarded under SSF conditions.
822
20d
-- 30~1
10~\ I
A
P. ostreatus
,,o ,i K
B
d 60d
7d ~, ' ~//-~20d~" ]7d'~10d
1(46%)
$
,
~~AI!
WLO
.......
60d
T. versicolor
() control
II (16%)
The straw lignin composition from CuO degradation (Fig. 4) was similar to that reported by Lapierre and Monties (1989) using thioacidolysis (H:G: S = 5:43:53). Degradation of cinnamic acids, reduction in S and increase in H content in lignin were observed during fungal treatment. Cinnamic acids are involved in ligninpolysaccharide linkages (Iiyama et al. 1990) and their degradation could increase lignin extractability, as observed during the first SSF phase. Since degradative methods predominantly depolymerize uncondensed lignin, S/G and S/H decreases cannot be attributed to preferential biodegradation of ether-linked units and must be considered the possibility of fungal demethoxylation and differences in S and G-S lignin distribution. The CuO-yield/Klason-lignin ratio was stable while treating straw with T. ~ersicolor, suggesting simultaneous degradation of condensed and uncondensed lignin. Fungal breakdown of fl-O-4 and 8-1 dimers has been extensively investigated, and the degradation of biphenyl structures by T. versicolor has been also demonstrated (Katayama et al. 1989). Fungal degradation of lignin side-chains could be responsible for the increase in aromatic acid yield from the transformed straw. High carboxyl content was found in lignin from wood decayed by Ganoderma australe (Martinez et al. 1990), and an increased vanillic acid yield was reported after treating wood with Phlebia tremellosus (Hedges et al. 1988). Strong increases in the Ac/(AI+Ke) ratio were observed while straw was treated with P. ostreatus. The highest increase corresponded to the G compounds, and the abundance of altered G-units in residual lignin may be connected with their participation in aryl-aryl linkages. The aromatic acid amounts did not increase while treating straw with T. versicolor, and a similar result was reported by Hedges et al. (1988) after wood decay by this fungus. Side-chain alteration in residual lignin seems to be characteristic of fungi such as P. tremellosus and P. ostreatus, producing preferential degradation of lignin.
2x,
Fig. 6. Principal component analysis of straw SSF, showing the position of samples from 0 to 60 days (A), and the variables with the highest loading factors on the two first axes (B). Variables: AAK, Ac/ (A1+ Ke) ratio; AAO, aryl alcohol oxidase; ARA, arabinan; DIG, digestibility; ENX, xylanase; EXT, extractives; FSU, free sugars; KLI, Klason lignin; pH; PRO, protein; S, syringyl content; SLI, soluble lignin; WSO, watersoluble; WLO, weight loss; XYL, xylan
Polysaccharide-degrading enzymes have been studied during straw SSF with ascomycetes, and less frequently with basidiomycetes (Milstein et al. 1986). T. versicolor showed higher cellulase levels than P. ostreatus (Fig. 5a, b) and the latter produced the highest xylanase level, although the most intense removal of both polysaccharides was obtained with T. versicolor (Fig. 5c, d). Lignin peroxidase of T. versicolor has been described by Dodson et al. (1987), and its detection in transformed straw (Fig. 5e) constitutes the first report of its production under SSF conditions. Immobilization of the enzyme in lignocellulose may be responsible for lowering the free enzyme level, and the interference of water-soluble aromatic compounds in the lignin peroxidase test must be investigated. A Mn(II)-dependent peroxidase has been purified from lignocellulosic material decayed by Lentinula edodes (Forrester et al. 1987) and from liquid cultures of other white-rot fungi, but this enzyme was not detected in straw SSF. Enzymatic sources for extracellular H202 were investigated in ligninolytic fungi, and an aryl-alcohol oxidase has been described in Pleurotus sajor-caju (Bourbonnais and Paice 1988), P. eryngii (Guillrn et al. 1990b) and Bjerkandera adusta (Muheim et al. 1990). The enzyme oxidizes different polyunsaturated primary alcohols (Guillrn et al. 1990a) and its role in fungal metabolism is being investigated. To our knowledge, the present results (Fig. 5f) constitute the first report of aryl-alcohol oxidase in P. ostreatus and a demonstration of the enzyme production under SSF conditions. Relatively high levels of laccase were found during straw SSF with the two fungi studied. Degradation of phenolic lignin models by T. versicolor laccase has been demonstrated by Kawai et al. (1988). Limited degradation of wood lignin can be caused by laccase, but it could play a role in the degradation of lignins with a high content of free phenols, such as wheat lignin (Lapierre and Monties 1989). The information on the kinetics of straw SSF with T. oersicolor and P. ostreatus will contribute to optimising the straw treatments with these ligninolytic fungi. The transformations of the carbohydrate fraction could be
823 r e l a t e d to c h a n g e s in e n z y m a t i c activities, b u t m o r e studies on the nature of lignin-degrading enzymes are still n e c e s s a r y to e s t a b l i s h t h e i r p a r t i c i p a t i o n in l i g n i n degradation under natural and SSF conditions. Acknowledgements. The authors are indebted to A. Pdeto for her help in gas chromatography-mass spectrometry. This research has been supported by the Spanish Biotechnoiogy Program (BIO88185) and the ECLAIR Program of the European Community (EC contract no. AGRE-CT90-0047-(SMA)).
References Agosin E, Monties B, Odier E (1985) Structural changes in wheat straw components during decay by lignin-degrading white-rot fungi in relation to improvement of digestibility for ruminants. J Sci Food Agric 36:925-935 Bourbonnais R, Paice MG (1988) Veratryl-alcohol oxidase from the lignin-degrading basidiomycete Pleurotus sajor-caju. Biochem J 255:445-450 Dodson PJ, Evans CS, Harvey PJ, Palmer JM (1987) Production and properties of an extracellular peroxidase from Coriolus versicolor which catalyses C~-Ca cleavage in a lignin model compound. FEMS Microbiol Lett 42:17-22 Forrester IT, Grabski AC, Burgess RR, Leatham GF (1987) Manganese, Mn-dependent peroxidases and the biodegradation of lignin. Biochem Biophys Res Commun 157:992-999 Guill~n F, Martinez AT, Martinez MJ (1990a) Characterization and purification of an aryl-alcohol oxidase from Pleurotus eryngii. In: Reisinger A, Bresinsky A (eds) Abstracts of the 4 tr~ International Mycological Congress, Regensburg, August 1990, p 232 Guillrn F, Martinez AT, Martinez MJ (1990b) Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Appl Microbiol Biotechnol 32:465469 Haemmerli SD, Leisola MSA, Fiechter A (1986) Polymerization of lignins from Phanerochaete chrysosporium. FEMS Microbiol Lett 35:33-36 Harkin JM, Obst JR (1973) Syringaldazine, an effective reagent for detecting laccase and peroxidase in fungi. Experientia 29:381-387 Hedges JI, Blanchette RA, Weliky K, Devol AH (1988) Effects of fungal degradation on the CuO oxidation products of lignin: a controlled laboratory study. Geochim Cosmochim Acta 52:2717-2726 Hedges JL, Ertel JR (1982) Characterization of lignin by gas capillary chromatography of cupric oxide oxidation products. Anal Chem 54:174-178 Higuchi T (1990) Lignin biochemistry: biosynthesis and biodegradation. Wood Sci Technol 24:23-63 Iiyama K, Lam TBT, Stone BA (1990) Phenolic acid bridges between polysaccharides and lignin in wheat internodes. Phytochemistry 29:733-737 Kamra DN, Zadrazil F (1986) Influence of gaseous phase, light and substrate pretreatment on fruit-body formation, lignin degradation and in vitro digestibility of wheat straw fermented by Pleurotus spp. Agric Wastes 18:1-17 Kamra DN, Zadrazil F (1988) Microbiological improvement of lignoceilulosics in animal feed production - a review. In: Zadrazil F, Reiniger P (eds) Treatment of lignocellulosics with white-rot fungi. Elsevier Applied Science, London, pp 56-63 Katayama Y, Nishida T, Morohoshi N, Kuroda K (1989) The metabolism of biphenyl structures in lignin by the wood-rotting fungus Coriolus vesicolor. FEMS Microbiol Lett 61:307-314 Note added in proof." The AAO of Postreatus has been recently purified and characterized by Sannia et al. (Biochim Biophys Acta Gen Sub 1991, 1073: 114)
Kawai S, Umezawa T, Higuchi T (1988) Degradation mechanisms of phenolic fl-1 lignin substructure model compounds by laccase of Coriolus versicolor. Arch Biochem Biophys 262:99110 Kirpatrick N, Reid ID, Ziomek E, Ho C, Paice MG (1989) Relationship between fungal biomass production and the brightening of hardwood kraft pulp by Coriolus versicolor. Appl Microbiol Biotechnol 55:1147-1152 Kuwahara M, Glenn JK, Morgan MA, Gold MH (1984) Separation and characterization of two extracellular HzOz-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett 169:247-250 Lapierre C, Monties B (1989) Structural information gained from the thioacidolysis of grass lignins and their relation with alkali solubility. In: TAPPI (eds) Proceedings of the International Symposium on Wood Pulp Chemistry, Raleigh, NC, May 1989, pp 615-619 Lindenfelser LA, Detroy RW, Ramstack JM, Worden KA (1979) Biological modification of the lignin and cellulose components of wheat straw by Pleurotus ostreatus. Dev Ind Microbiol 20:41-551 Martinez AT, Barrasa JM, Almendros G, Gonzfilez AE (1990) Fungal transformation of lignocellulosics as revealed by chemical and ultrastructural analyses. In: Coughlan MP, AmaralCollaqo MT (eds) Recent advances on fungal treatment of lignocellulosic materials. Elsevier Applied Science, London, pp 129-147 Milstein O, Vered Y, Gressel J, Flowers HM (1986) Heat and microbial treatments for nutritional upgrading of wheat straw. Biotechnol Bioeng 28:381-386 Mishra C, Leatham GF (1990) Recovery and fractionation of the extracellular degradative enzymes from Lentinula edodes cultures cultivated on a solid lignocellulose substrate. J Ferment Bioeng 69:8-15 Muheim A, Leisola MSA, Schoemaker HE (1990) Aryl-alcohol oxidase and lignin peroxidase from the white-rot fungus Bjerkandera adusta. J Biotechnol 13 : 159-167 Niku-Paavola M-L, Karhunen E, Salola P, Raunio V (1988) Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 254:877-884 Orlrci L, Kenkel NC (1985) Introduction to data analysis. International Co-operative Publishing House, Fairland, Md. Schiesser A, Filippi C, Totani G, Lepidi AA (1989) Fine structure and mechanical properties of straw filaments invaded by Pleurotus ostreatus. Biol Wastes 27: 87-100 Tien M, Kirk TK (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol 160B:238-249 Ulmer DC, Tengerdy RP, Murphy VG (1981) Solid-state fermentation of steam-treated feedlot waste fibers with Chaetomium cellulolyticum. Biotechnol Bioeng Symp 11:449-461 Valmaseda M (1989) Fermentacirn en estado s61ido por hongos ligninoliticos. Thesis, Universidad Complutense, Madrid Valmaseda M, Almendros G, Martinez AT (1990) Substrate-dependent degradation patterns in the decay of wheat straw and beech wood by ligninolytic fungi. Appl Microbiol Biotechnol 33: 481-484 Wood TM, Bhat KM (1988) Methods for measuring cellulase activity. Methods Enzymol 160A:87-112 Zadrazil F (1977) The conversion of straw into feed by basidiomycetes. Eur J Appl Microbiol Biotechnol 4:273-281 Zadrazil F (1985) Screening of fungi for lignin decomposition and conversion of straw into feed. Angew Bot 59:433-452 Zafar SI, Sheeraz Q, Abdullah N (1989) Degradation of the lignocellulosic component on wheat straw - Coriolus versicolor solid state fermentation under nitrogen-starved conditions. Biol Wastes 27:67-70
