Bioconversion of organosoluble lignins by different types of fungi

Resources and Conservation, 13 (1986) 37-51 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

BIOCONVERSION OF ORGANOSOLUBLE TYPES OF FUNGI

LIGNINS

37

BY DIFFERENT

A. HAARS, D. TAUTZ and A. HUTTERMANN Forstbotanisches

Znstitut der Universitiit Giittingen (F.R.G.)

(Received August 9, 1985; accepted in revised form March 24, 1986)

ABSTRACT Technical organosoluble lignins could serve as growth substrate for several fungi of different ecotypes. Certain species of fungi increased the water solubility of the lignin, depending on the glucose concentration in the medium. The solubilization rate was increased also by successive growth of fungi on the lignin. All types of organosoluble lignin induced high activities of extracellular phenoloxidase (E.C.1.14.18.1) in cultures of white-rot fungi. The enzyme activities obtained on organosoluble lignins were higher than on lignosulfonates. Due to the action of phenoloxidases two water-soluble, acidprecipitable lignin polymerizates with different physical properties were formed. The nitrogen and glucose content in the medium plays an important role in the polymerization. INTRODUCTION

Separating lignin from cellulose by the “organosolv” process [ 11, which now is being performed in a pilot plant, causes fewer environmental problems than the sulfite and sulfate pulping method. On the other hand, the energy costs of this process are rather high, so that this pulping method can only become technically and economically feasible if, besides the cellulose, the other main components such as lignin and hemicellulose are converted to useful chemicals too. Organosoluble lignin derived from ethanol-water delignification, proved to be mostly unchanged, resembling to some extent analytical Bjorkman lignins [2--51. In contrast to lignosulfonate and kraft lignin, no studies have yet been carried out on the biotransformation of technical organosoluble lignin. It is to be expected that this lignin is more accessible to microorganisms than the ones mentioned above, since it is less condensed, purer than the other technical by-product lignins and has a rather low molecular weight. Extracellular phenoloxidase of white-rot fungi was recently found to polymerize lignosulfonates by a radical mechanism so that these polyphenolic substances are converted to an active binder for wood materials [6]. As the molecular weight plays an important role for the curing behaviour of ligninbased adhesives [7], the purpose of this work was to find out if technical

0166-3097/86/$03.50

0 1986 Elsevier Science Publishers B.V.

38

organosoluble lignin can serve as growth substrate for fungi and, if so, whether different fungi are able to transform this lignin in order to obtain lignin derivatives with new properties, e.g. enhanced water solubility or increased molecular weight of the water-soluble fraction. Therefore, the effect of lignin on the production of extracellular phenoloxidase activity was studied. MATERIALS

AND METHODS

Materials Beech and spruce lignins from different organosolvent pulping stages (kindly supplied by Dr. Feckl, MD Nicolaus GmbH & Co. KG, Munich) are listed in Table 1. The content of residual non-dialysable polysaccharides in the organosoluble lignin was below l%, according to information from Dr. Feckl and former investigations [4]. The organosolvent pulping process is described by Edel [8]. Calcium lignosulfonate (CaLs) was obtained from Roth, Karlsruhe, F.R.G., and Sephadex LH gels from Pharmacia, Freiburg, F.R.G. TABLE

1

Organosoluble

lignins

No.

Abbreviation

Wood

Solvent

eZBOW’ cm“)

1

BI SI B II s II BI+II s1+11

beech spruce beech spruce beech spruce

CH,OH/H,O CH,OH/H,O CH,OH/NaOH CH,OH/NaOH mixed lignins from both pulping stages

20 20 24 24 24 24

2 3 4 5 6

Organisms and culture conditions The following fungi were studied in the presence of 1% organosoluble lignin (OL): White-rot fungi: Heterobasidion annosum, strain 215 (H.a.), Sporotrichum pulverulentum (S.p.), Schizophyllum commune (S.C.), strain 440, Pleurotus ostreatus subsp. florida (P.f.), Polyporus versicolor (P.v.), DSM 1977, Ischnoderma benzoinum (1.b.). Brown-rot fungus: Gloephyllum trabeum (G.f.), DSM 1398. Mycorrhizal fungi: Tricholoma aumntium (T.a.), Schff. ex Fr./Ricken No. 36247, Cenococcum geophilum (e.g.), CBS 14751 (Baarn). Soil fungus: Botrytis cinerea Pers. ex Nocca & Balb., own isolate (B.C.). After autoclaving, the lignin in the media was homogenized 1 min with an Ultraturrax homogenizer.

39

Two media with different nitrogen content were used: F-medium [9], which has a high N-content (2500 mg/L asparagin) and B-medium [lo], containing 650 n-&L asparagin. Unless otherwise indicated, 1% glucose was used as additional carbon source. The fungi were grown in 500 mL erlenmeyer flasks containing 50 mL medium, pH 6.0-6.5, and incubated for 14-28 days at 24°C in the dark as standing cultures, until they reached the stationary growth phase. Transfer of mycelia was performed using the method described by Zweck et al. [ 111. The cultures (4 replicates) were harvested using the scheme described in Fig. 1 in order to separate water-soluble, acid-precipitable lignin (WSAPL), aromatic substances (AS), water-insoluble lignin (WIL) and myceliumbound lignin (ML). Uninoculated lignin-containing medium (C,) as well as 4 X 50 ml - Cultures 1 flltratlon S&S 1506

1% Llqnln

I 2.

i flltercake(= mplllml+WIL+ML)

1. filtrate

1 washry with 1W ml H20,pH 2.U

1 nk?aSurlng pl,1acca5.?

1 solveWIL in 50 ml dioxane, stirringuntil lignlnis dissolv& f 15mln)

I +Ha pi 2.8 standingovem~ght at roan terpera+ure t centrifugation 3Ormn 26W rp

i filtration S&S 1506

t 2. filtercake (=mycelimn lwwdliqninj

1 t

, t

2. filtrate .___

l.sedimnt 1 washingwith loo ml 0.1 N Hcl, centrifug.

add XD ml Xk~'cknight I suspxl. in 20 ml 0.2aNaal

I filtration S&S 6/1X+, ,

,

I I 1. suprnatant A280 I extractulq3x wth Mm1 ether

I 2.tiinent

i evaprationof

:"y

z/Y~~=

1 filtration S&S 1506 1 4. filtrate + HCL #I 2.8

3. filtercake 3.filtrate p-

1 f1ltratlon S&S lxx

I dryingat 40°C 2Oh

WSAPL

AS =

1 5. filtercake

1

drying2Oh at 40°c 1 &&

9 WIL

Fig.1. Scheme for the separation and isolation of bioconversedlorganosoluble

lignin.

40

inoculated cultures without lignin (C,) served as controls. The yield of lignin recovered gravimetrically in C1 cultures by this procedure was ca. 90%. Methods Molecular weight distribution Changes in the molecular weight distribution after fungal attack were determined using the gelchromatographic system developed by Concin et al. [12]: 1 mL samples containing 1.5 mg lignin or 10 O.D./mL at 280 nm, respectively, were applied to Sephadex LH 20 and LH 60 columns using dioxane:water 7:3 as solvent. In this solvent no adsorption and association effects occurred as was tested with acetylated samples. The columns were packed according to the method of Determann [13], and calibrated with polystyrols according to Concin et al. [12]. Low molecular weight aromatics Qualitative changes of ether-extractable low molecular weight aromatics were determined by thin-layer chromatography (TLC) on precoated Silicagel F-254 plates (Merck, Darmstadt, F.R.G.) using benzene:dioxane:acetic acid 90:25:4 (v/v)‘as a solvent system. Aromatic substances with and without phenolic hydroxyl groups were detected by UV radiation and 0.1% p-nitrobenzenediazonium fluoroborate (NBDF) [ 141. Phenoloxidase (PO) activity Extracellular PO activity in the 1. filtrate (Fig. 1) was determined using 2,6-dimethoxyphenol in McIlvaine buffer, pH 4.5, as substrate [15]. One unit of enzymatic activity was expressed as 1 U = AE X 1 cm/l min. RESULTS

Growth of fungi on the lignins Organosoluble lignins are soluble in organic solvents and alkali. However, at pH 4-5 (the pH-region which is known to favour lignin biodegradation by many fungi, especially Sporotrichum pulverulentum), the technical organosoluble lignins were nearly insoluble. Therefore, pH 6.5, which still allowed good growth of the fungi, was chosen for the cultivation. At this pH, 6% of the total lignin was dissolved. In order to saturate the lignolytic system, the lignin was applied in excess (1%). All fungi were grown as stationary cultures, because of the high amount of insoluble material, which may be converted only when the contact with the membrane is not interrupted by shaking. All types of organosoluble lignin could serve as growth substrates for the fungi listed above, as was determined by mycelial wet-weight measurements after separation of the water-insoluble part of the lignin (WIL). Compared to

41

CZ controls a slight increase of biomass in lignin-containing cultures was observed for the following fungi: 5’. pulverulentum, P. versicolor, G. trabeum and T. aumntium. Phenoloxidase activity All types of lignin induced extracellular phenoloxidase activity (E.C. 1.14.18.1) in I. benzoinum, P. versicolor, P. florida and H. annosum cultures; S. pulverulentum, S. commune, C. geophilum and B. cinerea produced only negligible amounts. The values given in Table 2 refer to the activity in the stationary growth phase. In many cases mixed lignins (I + II) had a stronger effect on the phenoloxidase activity than lignins from the first pulping stage (I). In P. versicolor cultures the enzyme activity could be increased up to 170 U/mL after a cultivation time of 19 days. The same yield of extracellular phenoloxidase activity was obtained when the fungi were cultivated only on the water-soluble fraction of the lignin (WSAPL). Compared with the phenoloxidase production in other technical lignins (CaLS) this yield was nearly six times higher. The enzyme solution could be concentrated to 3000 U/mL and stored at -20°C without loss of activity. TABLE 2 Extracellular lactase production lignins

of different white-rot fungi as induced by organosoluble

Fungus

Type of lignin

Medium

Lactase activity W/mL)

B.C. c.g. S.C. s.p. H.a. H.a. H.a. H.a. P.f. P.f. P.f. 1.b. 1.b. P.V. P.V. P.V.

all types all types s1+11 s1+11 BI BI+II SI SI+II BI BI+II,SI SI+II B I + II, S I + II BI SI+II SI+II CaLS

B B B B B B B B B B B F F F F F

0.3-0.4 0.2 0.2 0.2 9 17 11 30 3 4 8 4-5 3 59 170 29

aIncrease of activity as compared to the control (without lignin).

Induction*

4 fold 2 fold 4 fold 2.5 fold 5 fold 10 fold 15 fold 30 fold 80 fold 60 fold 7 fold 19 fold 3 fold

42

Bioconversion of organosolv lignin fractions

Eignins: Proportions

of water-soluble

At the end of the cultivation period the lignin was fractionated according to the scheme given in Fig. 1, which is based on the procedure of Janshekar et al. [16,17]. The relative amount of each lignin fraction was determined gravimetrically. Figure 2 shows the results for the WSAPL and AS fractions. The low molecular weight aromatic substances decreased in all cases. As was found by TLC (data not shown), certain phenols were totally metabolized and new aromatic substances, which were probably degradation products of

L&l rwL-!i

10

5

0

Kr

Cl

P.f.

Lb

rp

ac

GL

1.0

c*

Fig.2. Relative amounts of water-soluble lignin fractions (AS and WSAPL) in fungal cultures.

OS-

,I”\

0.4

‘;I/+ I ‘, 0

‘1

\ I

0.1 02

I

a3

1

1

1

0.4

0.5

0.6

1

0.7

I

0.8

1

0.9

’ I.O'b

1

Fig.3. Sepbadex LH 20 elution profiles of low molecular weight aromatics and acid soluble lignin (AS) before (-) and after (----) attack by Pleurotus florida.

43

the lignins, were released. After fungal growth the concentration of AS in the medium was very low (50 mg/L). The molecular weight distribution of this fraction bef re and after attack of a white-rot fungus is shown in Fig. 31 Due to phenoloxidase activity the molecular weight of a part of the aromatic substances was increased. The content of water-soluble, acid-precipitable lignin (WSAPL) was also decreased in many cases except in H. annosum and I. benzoinum cultures. G. trubeum and C. geophilum did not change the amount of WSAPL. In the cases where the WSAPL content decreased drastically, the medium was acidified by the fungi during the cultivation period. Two fungi -H. annosum and I. benzoinum - solubilized remarkable amounts of lignin (up to 17%). The solubilization rate obtained in H. annosum cultures was dependent on the glucose concentration in the medium. At higher glucose concentrations the amount of solubilized lignin was lower as is shown in Table 3: in the presence of 2% glucose only 5.4% WSAPL could be recovered, whereas in absence of glucose 16.7% lignin was solubilized. TABLE 3 WSAPL content in H.a. cultures (S I + II) containing different glucose concentrations % WSAPL

Culture S I + II

without glucose + 0.75% glucose + 1 .O% glucose + 2.0% glucose

16.7 13.4 10.3 5.4

Spruce

0

02

OffI

0.6

08

1

l.O%V

Fig.4. Elution profiles of organosoluble llgnins in the system Sephadex LH 60 - dioxane: water 7 :3. (- -) lignin from the first stage (CH,OH) I; (--) lignin from the second stage (CH,OH/NaOH) II (compare Table 1).

44

Molecular

weight distribution

of lignin fractions

Native lignin The amount of high molecular weight material was higher in the lignins from the second pulping stage II (Fig. 4). This was already found using the GPC system Sephadex LH6O/dimethylformamide [18]. Mixed lignins from both pulping stages exhibited the same pattern as stage-11 lignins. Autoclaving of the lignins for 15 min as 1% suspensions in media pH 6.5 had only minor effects on the molecular weight of WSAPL. That was to be expected [ 181, because the pulping was performed at 180°C. The amount of acidprecipitable, water-soluble lignin (WSAPL) increased after autoclaving. This was found for kraft lignin as well [ 191. Because this lignin fraction is expected to be attacked more easily by microorganisms, autoclaving was used throughout all experiments. Lignin fractions after fungal attack Figure 5 shows that WSAPL contained a high part of low molecular weight material. The WIL (elution pattern not shown), however, contained a large amount of high molecular weight material so that the elution pattern

b

d2

dl

d6

d6

10

0

02

0‘

06

08

"0"

Fig.5. Sephadex LH 60 elution profiles of the WSAPL spruce and beech mixed lignins after attack by Heterobasidion annosum (H.a.), Pleurotus florida (P.f.), Sporotrichum pulverulentum (S.p.), Gloephyllum trabeum (G.t.), Botrytis cinerea (B.C.), Cenococcum geophilum (C.g.) and Tricholoma aurantium (T.a.). (-) C, WSAPL; (- -) fungal WSAPL from spruce (S I + II); (. . . .) fungal WSAPL from beech (B I + II).

45

was similar to WSAPL after fungal polymerization (= WSAPL-P). The WIL was not attacked by the fungi. Remarkable changes in the molecular weight distribution were obtained for WSAPL after fungal attack (Fig. 5). Each type of lignin was tested with each fungus, but because the fungal effect on lignins of different pulping stages was nearly identical, only I + II-elution patterns are shown. The white-rot fungi, B. cinerea and C. geophilum, polymerized parts of the lignin. This polymerizate is called WSAPL-P. The rate of polymerization was dependent on the wood type and the carbon and nitrogen content in the medium. Spruce lignin was polymerized to a higher extent than beech lignin. In contrast to WIL the high molecular weight part of WSAPL-P (= lignin polymerized by fungi) was water soluble. In all fungal cultures the low molecular weight fraction of WSAPL-P or metabolization. (K,, = 0.4) decreased considerably due to polymerization It is an interesting fact that phenoloxidase activities of 0.2 U/mL (C. geophilum) are sufficient to cause a polymerization of WSAPL. After drying at 40°C for 20 h the C1-WSAPL could be redissolved in dioxane:water (7:3), whereas the WSAPL-P from fungal cultures could only partly be redissolved. A certain amount of the fungal polymerizate (Table 4) formed a brown “lacquer” which was insoluble in several organic solvents and only partly soluble after 3 days in alkali. It can be concluded from Table 4 that the formation of the “lacquer” corresponds with extracellular lactase activity (compare Table 2), because fungi without or only with traces of extracellular lactase (G. trabeum, T. aurantium and C. geophilum) did not produce this “lacquer”. TABLE 4 Dioxane:water-soluble Lignin

material (%) of WSAPL and WSAPL-P after drying

Fungus C,

G.t.

T.a.

c.g.

S.p.

B.C.

H.a.

P.f.

SI s1+11 BI BI+II

100 100 100 100

100

100

100

100

100

100

41 41 69 66

91 75 95 90

28 33 64 46

17 19 32 60

Lactase activity

no

no

no

traces

low

low

high

high

The type of wood is important too: the amount of insoluble material was higher in spruce lignins. This corresponds very well to the higher degree of polymerization of spruce lignins. The formation of WSAPL-polymerizate in I. benzoinum cultures was inversely correlated to the nitrogen content in the medium: only in Bmedium (low N-content) a polymerizate (WSAPL-P) was formed, whereas

46

in F-medium (high N-content) no WSAPL-P was formed (Fig. 6). The amount of ML (= mycelium-bound lignin) was 3 times higher (12%) on N-poor medium than on N-rich medium. The solubilization rate (= relative amount of water-soluble fraction) was also higher on N-poor medium. For H. anlzosum also the effect of the glucose concentration on the rate of polymerization was investigated. Similar to the effect of N-content, observed for I. benzoinum, the polymerization was higher at low glucose concentration.

OL- t

I\

0.3-

1 ‘\

02-

j

‘+, \

0.1-

I ‘.. -_ I p....................y:’ I I I, 0 01 02 0.3

.* I 0.4

*...... ?? ... . ..’ ‘. ‘. 0. ,c. *. ‘u ‘\ ‘a.* /’ -._ * I I I I I I a5 0.6 07 0.9 09 10

.** ..** . ..* ,/

Fig.6. Sephadex LH 60 elution profiles poor (. . . .) I. benzoinum cultures.

I, bv

of I-WSAPL-P

in nitrogen-rich

(--)

and nitrogen-

The same effect was observed with regard to the solubilization rate (relative amount of WSAPL) which also was higher with low glucose concentration in the media. In contrast to white-rot fungi, mycorrhizal fungi (e.g., T. auruntium) had only minor effects on the lignin, though adsorption of lignin to the mycelial surface was observed as well. The mycelium-bound lignin in all cultures exhibited a similar elution pattern as WIL. Dependent on the type of the fungus, from 2.3% (G. trabeum) up to 4.6% (T. aumntium) of the total lignin was bound to the mycelium and released after NaOH treatment. Successivegrowth of different fungi on organosoluble lignins The insoluble, remaining lignin (WIL) from B. cinereu, S. pulverulentum, P. florida, and H. annosum cultures was isolated (WILF) and then was inoculated with a second fungus (Table 5) in order to see if parts of the lignin could be solubilized or converted after successive growth of two different fungi. On purpose, successive and not mixed cultivation was chosen because the data obtained by Janshekar et al. [16,17] showed generally poorer results with mixed than with the pure cultures. The concentration of lignin in the medium was 0.8%. Two fungi (H. annosum and P. florida) collected the lignin at the bottom side of their mycelium; correspondingly, the amount of solubilized lignin was higher than in cultures of B. cinerea and S. pulverulentum (Table 5). The last two fungi produce only low lactase activities (Table 4). The rate of lactase induction by WILF was

47 TABLE

5

WSAPL proportions (W) after successive growth remaining after growth of the first fungus)

of two different

fungi on WILF (= WIL

Sp.

s.p. --t P.f.

Lignin

C,

H.a. --* B.C.

B.C. + H.a.

P.f.-

BI BI+II SI SI+II

0 0 0 0

0 0 0 0

1.15 1.16 2.28 1.32

0 0.48 0.58 0.62

Binding of lignin to mycelial surface

-

+

Lactase activity of the first fungus

high

low

medium

low

Lactase activity of the second fungus

low

high

low

medium

2.48 4.04 2.10 1.30 +

the same as by OL. A good solubilization rate was obtained when the lactase activity of the second fungus was medium or high. Growth induction compared to CZ cultures was observed for B. cinerea and H. annosum. DISCUSSION

The production of biomass on organosoluble lignins was in most cases not significantly increased. The mycelial yields of H. annosum, which produced 2-5 times more biomass on lignosulfonate than on sugars [ 201, could not be increased by addition of any organosoluble lignin. This is probably due to the low water solubility of organosoluble lignins. For production of lactase, however, the lignin gave much better results than lignosulfonates. The cause for this effect may be the high content of phenolic hydroxyl groups in organosoluble lignins, which is 4.8% thus 2.5 times higher than the phenolic hydroxyl content in lignosulfonate (1.9% according to Glasser [21]). This affect would be worth further investigation in order to obtain high yields of enzyme for the binding system for wood materials on the basis of lignin/ phenoloxidase [6]. The phenolic hydroxyl content seems to be important for lactase induction, because lignins from the second pulping stage and mixed lignins (I + II) have a higher phenolic hydroxyl content [22] and induced higher levels of lactase, e.g., in H. annosum and I. benzoinum. The decrease of aromatic substances and acid-soluble oligomeric lignin can be attributed to metabolization or polymerization in phenoloxidasecontaining cultures. It is a known fact that phenols can serve as sole and predominant carbon source for fungi. Polymerization forms products of higher molecular weight which are no longer soluble in acid and will be found in the WSAPL fraction. In the cultures the overall concentration of

38

acid-soluble lignin (measured as A zxo-absorbing material) decreased. However, a release of new molecular weight substances as a response to the presence of lignin was observed. Other authors [23,24] also reported on the production of acid-soluble lignin depending on the age of the culture. Eriksson et al. [23] assume, therefore, that this acid-soluble material is an intermediate product in lignin metabolism. Summarizing, it has to be stated that the yield of aromatics in the culture tluid was too low to be of industrial interest. In accordance with the results reported here are the findings of Meier and Schweers [3], who degraded ethanol-water lignins by catalytic hydrolysis and compared the results with other technical lignins. They also stated that economical utilization of their method is not possible, because of small yields and heterogeneous composition. Two fungi, H. alznosum and I. benzoinum, increased the amount of WSAPL in the culture fluid. The decrease of the WSAPL portion in other cultures may be attributed not only to metabolization but also to the low solubility of the organosoluble lignins at pH values below 6.5. Especially 5’. puluerulentum acidified the medium by secretion of organic acids [ 251. Because a higher water solubility of the organosoluble lignins is desirable for many applications further investigations of the “solubilizing system” and optimization of cultural conditions for I. benzoinum should follow. The formation of WSAPL-P was not only observed in cultures of whiterot fungi but also in cultures containing very low phenoloxidase activities, The increase of molecular weight of a still water-soluble e.g., C. geophilum. product may be interesting for application of the lignins as adhesives. It is a known fact that the curing behaviour is dependent on the molecular weight [ 71. The formation of an intermediate acid-precipitable polymeric lignin (APPL) was also reported for Streptomyces viridosporus growing on corn stover lignocellulose [26]. Using the radiorespirometric method, Chua et al. [27] also found an increase of high molecular weight material after incubation of Phanerochaete chrysosporium with synthetic C-labeled lignin. The glucose concentration had a negative effect on the polymerization rate of spruce lignin by H. annosum. This effect of co-substrate has already been reported for Pleuro tus ostrea tus. As reported for other fungi as well [28,29], polymerization of lignin was predominant in the absence of glucose, whereas in the presence of glucose or other carbohydrates (cellulose), depolymerization was favoured. This effect is caused by the enzyme cellobiose-quinone-oxidoreductase which reduces the quinones, thus preventing polymerization. This enzyme was also found in the culture fluid of Heterobasidion annosum [30]. The nitrogen content is a critical cultural parameter too, e.g., for the formation of WSAPL and binding of lignin to the mycelium as was shown by conversion experiments with Ischnoderma benzoinum in two different media of high and low asparagine concentration. This was found for Sporotrichum pulverulentum [31] as well. Other fungi, e.g., Pleurotus ostreatus and Chaetomium cellulo-

49

Zyticum, however, gave better lignin degradation results under conditions of sufficient nitrogen. Therefore, the increase of lignolytic activity by nitrogen starvation cannot be regarded as a general principle [ 16,17,32]. It is supposed that lactase can increase the water solubility of milled-wood lignin [33]. This would explain the fungus with high extracellular lactase why Heterobasidion annosum, activity, increased the amount of WSAPL, whereas the fungi with low lactase activity decreased the WSAPL amount. The findings that lactase is involved in lignin solubilization is in contrast with results of Kirk and Fenn [31] reporting that certain exceptionally good lignin-degrading white-rot fungi (e.g., Sporotrichum p.) produce barely detectable levels of phenoloxidases. The LH-60 chromatograms show that besides the polymerization a degradation had occurred preferentially for the low molecular size components, which was reported for other lignin types as well [34]. Similar results were obtained by Ferm and Nilsson [35] : during the fungal degradation of commercial lignin sulfonate, the microorganisms preferred the low molecular portion. Besides white-rot fungi, which are known to have a good lignolytic activity, a brown-rot fungus was also included in these studies because the latter group of fungi lacks the efficient system of ring-fission enzymes so that phenolic hydroxyl groups are newly introduced. Unfortunately G. trabeum had no significant effect on the lignins, a finding which is in accordance with the results of Trojanowski et al. [ 361. Successive growth of different white-rot fungi with high and low lactase activity on the remaining water-insoluble part of lignin after growth of another white-rotter resulted in an increase of WSAPL provided that the lactase activity of the second fungus is high enough. This finding again supports the hypothesis of Konishi and Inoue [33] that lactase may be involved in the solubilization of lignin. ACKNOWLEDGEMENTS

This work was supported munity.

by a grant BOS 007-D of the European

Com-

REFERENCES 1 2

3

Kleinert, T.N., 1974. Organosolv pulping with aqueous alcohol. Tappi, 57 : 99-102. Schweers, W., 1979. Utilization of lignins isolated under mild conditions from wood or wood waste for the production of useful chemicals and other chemical products. Mitt. Bundesforschungsanst. Forst-Holzwirtsch., Hamburg, 124 : 179-189. Meier, D. and Schweers, W., 1981. Dber Eigenschaften und Abbaubarkeit von mit Alkohol-Wasser-Gemischen isolierten Ligninen. 4. Mitteilung: Katalytische Hydrogenolyse zur Erzeugung monomerer Phenole. Holzforschung, 35: 81-85.

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