World Journal
of Microbiology
& Biotechnology
12. 16-21
Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five Iignocellulose-degrading fungi by ferulic acid F.O. Asiegbu,” A. Paterson & J.E. Smith At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurob sajor-caju. At 0.5 g/l, however, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-cajtr) also secreted laccases. Laccasesecreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested. Key words: Biomass protein, cellulases, ferulic acid, laccases, spruce wood, wood-decay
Lignocejluloses in the form of agricultural residues, including straw and forestry wastes, constitute a major renewable source of organic matter (Smith et al. 1987). Such materials consist of three principal classes of polymers-celluloses, hemicelluloses and lignins-with minor quantities of lowmolecular-weight compounds such as phenolics, lignans and tannins (Wayman & Parekh 1990). To date, biological conversion of lignocellulosic plant materials into addedvalue products has been limited, largely due to the economics of the process but partly because of our limited understanding of the biochemistry and physiology of microbial breakdown of glycolignin (Paterson 1989). This material forms the lignin hemicellulose matrix separating cellulose protofibrils in the interrupted lamella model of the lignified plant cell wall (Young 1986). Lignified tissues resist microbial attack partly because of the limited ability of cellulolytic enzymes to penetrate cell
F.O. Asiegbu was and A. Paterson and J.E. Smith are with the of Bioscience and Biotechnology, University of Strathclyde, Street, Glasgow Gl IXQ. UK. F.O. Asiegbu is now with the of Forest Mycology and Pathology, Swedish University of Sciences, P.O. Box 7026. S-750 07 Uppsala, Sweden; fax: 46 ‘Corresponding author. @ 1996 Rapid Science
Publishers
Department 204 George Department Agricultural 16 30 92 45.
fungi.
walls (Cowling & Kirk 1976). However, lignified tissues may also contain substantial quantities of phenolic compounds, such as ferulic or hydroxycinnamic acid, that contribute to the bonding between carbohydrates or between hemicelluloses and lignin (Hartley & Ford 1989). Such compounds may also be present in locally high concentrations in sap wood. Two processes that have gained economic significance in lignocellulose fermentation are mushroom cultivation (Smith et al. 1987, 1989) and, more recently, the conversion of wood into animal feed (palo podrido) in Chile (Gonzalez et al. 1989). It is possible that, with an increased understanding of the physiology of lignin-degrading fungi, further solid-substrate processes for enhancing digestibility of lowvalue animal lignocellulosic feed can be developed (Reid 1989). In vitro studies using purified lignin peroxidases have indicated that the value of such extracellular enzymes per se will be limited (Jurasek 1990). Lignin peroxidases and laccases are regularly associated with fungal degradation of the lignin component of lignocellulose materials (Evans 1985). The activities of these enzymes in two white-rot fungi, Trametes versicolor and Phanerochaefe chrysosporium, have been extensively studied (Leatham & Kirk 1983;
Ferulic
Evans et al. 1984; Janshekar & Fiechter 1988). However, data on the physiological regulation and role of lactase production iniigninolytic fungi are limited. Many have studied the effect of addition of alternate carbohydrate sources on lignocellulose fermentations (Reid 1989). Detailed studies on the effects of plant cell-wall phenolic constituents on the biodegradability of spruce wood by other wood-inhabiting fungi has not received as much attention, except for studies with Phanaerochaete Chrisosporium (Ander & Eriksson 1975; Kirk ef al. 1976). It is likely that the behaviour of other saprophytes on this substrate may differ. Furthermore, the stimulatory effects of the key, cell-wall phenolic, ferulic acid, on fungal saccharification and on rumen cellulolytic activity are well documented (Akin et al.. 1988; Bomeman et al. 1990). The effects of such exogenous addition on enzymes important in lignocellulose breakdown has not been correlated with breakdown of natural substrates. The present study is on the influence of differing concentrations of ferulic acid on enzymes important in lignocellulose degradation and aims to clarify how such plant cellwall constituents might influence saccharification of natural substrates.
Materials
and Methods
Orgunisms Trichodermu harziunum was isolated from decayed palm wood and identified at the International Mycological Institute, UK. Phanerochuefe chrysosporium IMI 747691, Trumefes versicolor IMI 210864, Chuefomium cel/uioiyficum IMI 188965 and P/ear&s sujo-cuju IMI 19976 were also used. Substrates and Chemicals Spruce (Piceu sitchensls) sawdust was from the Economic try Commission, Dunoon, UK. Fen&c acid (4-hydroxy, methoxy cinnamic acid) and syringaldazine were Aldrich.
Fores3from
Media The minimal medium (pH 5.5) employed contained (g/l): NH,H,PO,, 2.0; KH,PO,, 0.6; K,HPO,, 0.4; MgS0,.7H,O, 0.5; CaCI,, 0.2; FeSO,, 0.01; and thiamine, 0.001; and 1 ml micronutrient solution (per 1) containing (g/l): Z&O,, 6.6; MnS0,.4H,O, 5.0; CoC1,.6H,O, 1.0; and CuS0,.5H,O, 1.0. Phosphates, sulphates and chlorides were dissolved separately, mixed and made up to the required volume with distilled water (Asiegbu et al. 1994). Starter Cttltures Starter cultures for biomass and enzyme determinations were prepared by growing mycelia for 4 to 10 days in minimal medium supplemented with 17 g malt extract, 11 g peptone and 1 g cellulose/l. Flasks were incubated statically and mycelia were harvested, washed, and fragmented with a sterile laboratory blender. Hyphal homogenates (2.5 ml) were used to inoculate 50. ml batches of minimal medium containing 1% (w/v) crystalline cellulose powder. Experimental flasks contained ferulic acid at 0.5,
acid and glyculignin-attacking
enzymes
1.0 or 5.0 g/l. Sets of static flasks were set up so that duplicate cultures were harvested every 48 h for 14 days. Solid-stlbsfrufe Fermentation: Exogenotls Addition of Ferulic Acid Spruce sawdust was moistened with ferulic acid (0.5, I.0 or 5.0 g/l) in minimal medium to a moisture content of 70%. Moistened substrates were equilibrated at 4°C for 48 h and fermented as described by Cuero et al. (1985). Briefly, aliquots (20 g) of moistened substrate were dispensed into micro-porous bags, autoclaved at 121°C for 30 min, cooled and inoculated with a suspension of fragmented mycelia (1 ml/bag). Substrates inoculated with T. harzianum, P. sajor-caju or T. uersicolor were incubated at 28”C, those with C. cellulolyficMm at 37.5”C and those with P. chrysospo-
rictm at 40"C, all in environmental cabinets (Fisons Ltd, Loughborough, UK) at 98% relative humidity for 14 days. Substrates moistened treated.
with
unsupplemented
minimal
medium
were
similarly
Preparation of Extracellular Enzymes and Biomass Cultures were harvested and centrifuged at 5000 x g at 4°C for 30 min. Residual biomass was dried at 75°C and quantified as total protein-nitrogen by Kjeldahl determination. Enzyme Assays Saccharification of crystalline cellulose was determined as glucose produced from hydrolysis of absorbent cotton wool (200 mg) by 3 ml culture supematant with 2 ml 0.1 M sodium citrate buffer, pH 4.8, at 50°C for 24 h (Mandels et al. 1974), using the glucose oxidase method (Karkalas 1985). Carboxymethyl cellulase and filter paper cellulase were determined as described by Mandels et crl. (1976). p-Glucosidase was estimated using the procedure of Yamanobe et ui. (1987). Lactase (phenol oxidase) activity was estimated using a modification of methods described by Eriksson et al. (1983) and Nigam et al. (1987) in which syringaldazine is oxidized to a quinone. An assay mixture containing 0.5 ml 0.1% (w/v) syringaldazine in 99% ethanol and 1.5 ml sodium acetate (0.2 M, pH 5.6) was made up to 3 ml with culture supernatant and assays were incubated at 30°C. Soluble Squrs, Total Polysucchuride and Cellulose Total soluble sugars were estimated as reducing sugar using the Somogyi method. Total polysaccharide and cellulose were determined as reducing sugars and glucose, respectively, in acid hydrolysates, using previously described methods (Englyst and Cummings 1988; Asiegbu 1992). Sfufisficul Analyses The experiments were repeated at ieast two times. Results analysed using basic statistical method (Student’s t-tests).
were
Results Saccharificafion
of Cellulose
The growth and saccharification of crystalline cellulose by three fungi (7. harzianum, C. cellulolyticum and P. chrysosporittm) was suppressed by ferulic acid (Table 1). The other two strains had normal saccharification except in the presence of 5.0 g ferulic acid/l (Table 1). A similar observation was made for /I-glucosidase (Table 2) and CM-cellulase activity (data not shown). Values for initial cellulose depolymerizing activity, assayed as reducing sugar released
World Journal of Microbiology 6 Biotechnology. Vol 12, 1996
17
F.O. Asiegbu, A. Pakrson and J E. Smith Table 1. Effect on cellulose-saccharlfylng cultures of five fungi growlng on crystalline Fungus
Activity
enzyme (exo-glucanase) cellulose.
(pg glucose
released
from
actlvitles
of addition
200 mg cotton/ml)
of ferulic
with ferulic
acid
0
0.5
1.0
5.0
C. cellulolyticum T. harzianum
71 61
P. chrysosporium P. sajor-caju T. versicolor
45 28 113
3ot 2% 1st 53 133
17t 11t 23 86 117
ND W w 40 75
Samples analysed t Significantly lower ND-Not determined.
l
Table 2. Effect cellulose.
10 days post than control
on /?-glucosldase
Fungus
cellulolyticum harzianum chrysosporium sajor-caju versicolor
* Samples analysed t Significantly lower
at (g/l):*
inoculation.
of addltfon
(pg glucose
of ferullc
released
0 C. T. P. P. T.
to
value (p < 0.05).
activities
Activity
acid
25 32 26 32 100
from
acid
to cultures
cellobiose/ml)
of live
with ferullc
fungi
growing
on
acid at (g/l):’
0.5
1.0
5.0
22 22 gt 36 787
gt W 1st 50 143
1st st gt 1st sot
10 days post inoculation. than control value (p < 0.05).
from filter paper cellulose over a 60-min incubation, appeared to be stimulated for all strains with increasing concentrations of the phenolic (data not shown). Lactase Acfiuities Lactase activities, assayed as the oxidation of syringaldazine by culture supernatants, were determined every second day for 14 days. Of the five strains of fungi studied, only i? sajor-caju and T. versicolor produced detectable lactase activity and then only under some of the experimental conditions (Table 3). Under control conditions, in the absence of ferulic acid, Tra. versicolor produced appreciable lactase activity whereas P. sajor-caju had no demonstrable activity. With l? sajor caju in the presence of 0.5 or 1.0 g fen&c acid/l, lactase activity increased with time, reaching a maximum at 12 (0.5 g/l) or 14 days (I g/l). Growth in the presence of 5.0 g ferulic acid/l yielded lactase activities approximately two orders of magnitude below those with I g ferulic acid/l. The pattern of lactase activity observed with T. versicolor in the presence of 0.5 or 1.0 g ferulic acid/l was more complex, with maximum activities at 6 and 14 days, respectively. No lactase activity was detected in the presence of 5.0 g ferulic acid/l.
Total Soluble Sugars Significantly increased concentrations of soluble sugars accumulated in cultures growing in the presence of 5.0 g ferulic acid/l (Table 4). Only low amounts of reducing sugar were detected in supematants of cultures grown in the absence of ferulic acid. Biomass Prod&on Biomass production (as total protein) was estimated in 12day-old cultures growing on crystalline cellulose in the presence of varying concentrations of ferulic acid. The results obtained show that growth was severely repressed by 5.0 g ferulic acid/l in all five species tested (Figure I). Ferulic acid at 0.5 g/l appeared to stimulate growth in P. sajor-caju and T. versicolor. A different pattern was observed for T. harzianum, P. chysosporium and C. cellulolyficum, where increasing ferulic acid concentration resulted in decreased biomass production (Figure 1). Effect of Fertllic Acid on Solid-substrate Fermentation (SSF) Spruce sawdust was supplemented with ferulic acid in solid-substrate fermentation to determine if exogenous addition of monomeric forms of phenolic influenced cellulose saccharification. More polysaccharide was lost at 0.5 g to
Fen&c acid and ~lycolignin-attacking Table
3. Effect
Ferulic (“/I
acid
of ferulic
acid
T. versicofor P. sajor-caju
0.05
T. P. T. P. T.
0.5
production
by cultures
of T. versicolor Activity
Fungus
0
0.1
(FA) on lactase
versicolor sajor-caju versicolor sajor-caju versicolor
Table fungal
lncubatlon
on 1% (w/v)
cellulose.
for (days):’
2
4
6
8
10
12
14
17 (1.0) -
20 (3.0) -
25 (4.0) -
35 (5.0) -
32 (5.0) -
27 (3.0) -
(6.0) (0.0) (3.0) (0.0)
67 7 a3 12
(4.8) (1.0) (5.0) (2.0) -
109 37 157 21
-
* One unit of lactase enzyme activity and (standard deviations) for duplicate - - No detectable lactase activity.
(u) after
growing
9 (2.0) 55 1 37 2
P. sajor-caju
and P. sajor-caju
enzymes
is defined as the amount determinations.
(13.0) (2.0) (9.0) (1.0)
C.cellulolyticum T. harzianum P. chrysosporium P. saior-caju T. versicolor
Reducing
(9.0) (5.0) (2.0) (3.0)
0.4
0.5
that will produce
a change
4. Effect of ferulic acid on accumulation saccharfflcatlon of crystalline cellulose.
Fungus
96 68 a7 42
sugars
(ma/ml)
43 124 45 92
(1.0) (1.0) (3.0) (7.0)
97 165 44 82
0.6
of reducing
in A,,,
sugars
wlth ferulic
(4.0) (4.0) (2.0) (4.0) -
127 167 73 110
0.5
of 0.001
per min. Values
(5.0) (8.0) (6.0) (5.0) 1.0
are
means
durlng
acid at (g/l):*
0
0.5
1.0
5.0
0.03 0.04 0.01 0.03 0.06
0.02 0.08 0.02 0.08 0.13
0.03 0.08 0.10t 0.10 0.13
0.3t 0.16t 0.20t 0.207 0.3t
* Samples were analysed 10 days post inoculation. t Significantly different from control value (p < 0.05).
1.0 g fen&c acid/l (Table 5) than at 5.0 g/l (data not shown) when wood was fermented with l? chrysosporium. Cellulose breakdown was enhanced, in proportion to the ferulic acid added, in wood fermented with T. uersicolur or T. harzianum (Table 5). Also, at 0.5 g ferulic acid/l, polysaccharide loss was slightly higher than in controls without the acid in fermentations with P. sajor-caju (Table 5). In contrast, exogenous addition of fen&c acid repressed polysaccharide breakdown by C. cellulolyticum.
Discussion Fungi have different enzymatic responses to ferulic acid in lignocellulosic plant residues. Under favourable conditions, the extracellular glycolignin-degrading enzymatic activities of three fungi studied (I? chrysosporium, P. sajor-caju, T. versicolor) increased linearly with growth (unpublished work). An exception was lactase production in submerged culture, which showed a cyclical pattern in the two organisms (P. sajor-caju and T. vet&o/or) shown to secrete the enzymes with this activity. Such patterns were not observed for ligninase production, in surface culture after 7 days
(unpublished work), although cyclical production of ligninases in liquid culture has been observed elsewhere (Janshekar & Fiechter 1988). Smith ef al. (1989) reported cyclical production of lactase during growth of Agaricus bisporus in solid-substrate culture. The patterns of lactase production by T. versicolor and P. sajor-caju were different. Although T. versicolor secretes both constitutive and inducible laccases (Fahraeus ef al. 1958; Evans ef al. 1984), to our knowledge, inductive lactase production in P. sajor-caju has not been previously reported. In the present study, lactase activity was enhanced by adding ferulic acid to the liquid culture. However, ferulic acid at the highest concentration tested inhibited biomass production by T. uersicolor and consequently no lactase could be detected. In contrast, lactase activity in P. sajor-caju was inducible by ferulic acid at all concentrations in liquid culture and no basal level of constitutive enzyme activity was detected in the absence of the phenolic compound. The present results also revealed that P. sajor-caju and T. versicolor, which secreted phenol oxidizing laccases, showed stimulation of both depolymerizing cellulase and /3-glucosidase activities. In contrast, strains that did not
World
Joumf of Micmbdogy
6 Biotechnology, Vol 12, 1996
19
F.O. Asiegbu. A. Paterson and J.E. Smith Table 5. Effect of ferulic acid breakdown In fungal-fermented Fungus
on total polysaccharide spruce wood.
Loss
(% of control
values)
(NSP)
with
and cellulose
lerulic
acid
0
1.0
15.7 (2.2)
14.7 (4.5)
0.0 (0.0)
10.3 (1.5)
12.3 (1.2)
19.2 (3.1) 11.5 (2.0)
(CP)
at (g/l):
T. versicolof NSP CP P. sajor-caju NSP CP
8.1 (1.0)
P. chrysosporium NSP
15.7 (1.0)
CP T. harzianum NSP CP
7.1 (1.0)
18.4 (4.0) 13.9 (5.0)
8.5 (1.4) 2.0 (0.2)
12.3 (3.5) 10.7 (2.6)
21.0 (1.6) 11.2 (2.6)
11.4 (2.0) 4.3 (0.7)
C. cellulolyticum NSP CP ‘Values fermentation
are
means in solid-state
and
(standard
deviations)
recorded
after
14
days’
cultures.
I
Figure 1. Effect of adding ferulic acid, at 0.5 (m), 1.0 (@) or 5 ) g/l, on biomass production by P. chrysosporium (l), P. sajor-ca/u (2), T. versicolor (3), C. cellulolyticum (4) and T. harzianum (5) after 12 days’ growth on cellulose. Vertical lines represent standard deviations. W-Control with no ferulic acid.
secrete this class of enzyme showed repression of j?-glucosidase and endoglucanase although initial cellulose depolymerization was stimulated (unpublished work). A similar observation was reported for T. versicolor by Muller et al. (1988), who concluded that cellobiono-lactone from oxidative attack on carbohydrate was responsible for the stimulation. Analyses of accumulated sugars in the growth media indicated that, in the five organisms studied, soluble sugars accumulated in the media in parallel with increasing ferulic acid concentrations. In all five fungi, high concentrations of ferulic acid inhibited growth. Whereas the amount of accumulated sugars with 0.5 g ferulic acid/l was similar to control values, it was much higher when 5 g ferulic acid/l was used. The varying rates of polysaccharide loss obtained in solid-substrate cultures were similar to those reported earlier (Reid 1985; Agosin et al. 1986). The increased cellulose loss recorded with lignolytic fungal species in the presence of ferulic acid may be explained either as induction of phenol-detoxifying enzymes by the acid (GiovannozziSermanni eI al. 1982; Reihammer 1984) or stimulation of cellulolytic activity in stressed cells. Finally, it is suggested that the presence of certain phenolic compounds in feeds will suppress mycelial biomass production while inducing high activities of cellulolytic and ligninolytic enzymes leading to accumulation of degradation products. Such catabolic products, which might not be readily utilized by aerobic fungi, may be of value in ruminant nutrition. Obviously, fungi will differ in physiological response to substrate composition and so this should be optimized for each organism.
Fe&c
Acknowledgement This
study
was
Association
of
supported
partly
Commonwealth
by
a scholarship Universities
from (ACU)
the to
FOA.
References Agosin, E., Tollier, M.T., Brillouet, J.M., Thivend, P. & Odier, E. 1986 Fungal pretreatment of wheat straw: effects on the biodegradability of cell wall structural polysaccharide, lignin and phenolic acids by rumen micro-organisms. ]oarnul of the Science of Food and AgriczdtMre 37, 97-106. Akin, D.E., Rigsby, CC., Theodorou, M.K. & Hartley, R.D. 1988 Population changes of fibrolytic rumen bacteria in the presence of phenolic acids and plant extracts. Animul Feed Science and Technology 19, 261-275. Ander, P. & Eriksson, K.E. 1975 Influence of carbohydrates in lignin degradation by white rot fungus Sporolrichum pulveralenhum. Svensk Pupperstidn 78, 643-652. Asiegbu, F.O. 1992 Fungal delignification of lignocellulosic materials; Physiological aspects and enhancement of rumen fermentation. PhD Thesis. University of Strathclyde, Glasgow, UK. Asiegbu, F.O., Morrison, I.M., Paterson, A. & Smith, J.E. 1994 Analyses of fungal fermentation of lignocellulosic substrates using continuous culture rumen simulation. Journal of General and Applied Microbiology 40, 305-318. Bomeman, W.S., Hartley, R.D., Morisson, W.H., Akin., D.E. & Ljundahl, L.G. 1990 Feruoyl and p-coumaryl esterase from anaerobic fungi in relation to plant cell wall degradation. Applied Microbiology and Biotechnology 109, l-8. Cowling, E.B. & Kirk, T.K. 1976 Properties of cellulose and lignocellulosic materials as substrates for enzymic conversion processes. Biotechnology and Bioengineering Symposium 6, 9&
123. Cuero, R.G., Smith, J.E. & Lacey, J. 1985 A novel containment system for laboratory scale solid particulate fermentation. Biotechnology Letters 7, 463-466. Englyst, H.N. & Cummings, J.H. 1988. Improved method for measurement of dietary fiber as non starch polysaccharides in plant foods. ]otlrnul of the Associution of Official Analytical Chemists 71, 808-814. Eriksson, K.E., Johhnsrud, SC. & Vallander, L. 1983 Degrading of lignin and lignin model compounds by various mutants of the white rot fungus Sporotrichtrm pu/veru/entum. Archives of Microbiorogy 35, 161-168. Evans, SC. 1985 Lactase activity in lignin degradation by Coriolus versicolor in vivo and in vitro studies. FEMS Microbiology Letters
27,339-343. Evans, SC., Farmer, J.Y. & Palmer, J.M. 1984 An haem protein from Coriolus versico/or. Phytochemistry
extracellular
23, 1247-
1250. Fahraeus, G., Tullander, high lactase yields
V. & Ljunggren, H. 1958 Production of in cultures of fungi. Physiologiu PIantnnrm
ll,631-639. Giovannozzi-Sermanni, G. Badiani, M. & Luna, M. 1982 Protein production and lactase activity in Aguricus bisporas. Biotechnology Letters 4, 507-512. Gonzalez, A.E., Martinez, A.T., Almendros, G. & Grribergs, J. 1989 A study of yeasts during the delignification and fungal transformation of wood into cattle feed in Chilean rain forest. Antonie vun Leewenhoek 55, 221-236.
acid and glycolignin-attacking
enzymes
Hartley, R.D. & Ford, C.W. 1989 Phenolic constituents of plant cell walls and wall biodegradability. In Plant Cell Wall Polymers: Biogenesis and Biodegradation eds Lewis, N.G. & Paice, W.G. pp. 138-145. Washington DC: American Chemical Society. Janshekar, H. & Fiechter, A. 1988 Cultivation of Phanmochuete chrysosporiMm and production of lignin peroxidases in submerged stirred tank reactors. @trnul of Biotechnology 8, 97-112. Jurasek, L. 1990 Biological pulping. In Proceedings of the 4th International Mycological Congress, Regensbttrg, Germany. Eds. Reisinger A. & Bresinsky A., p. 237. Karkalas, J, 1985 An improved enzymic method for the determination of native and modified starch. loamal of Ihe Science of Food undAgric&re 36, 1019-1027. Kirk, T.K., Conors, WJ. & Zeikus, J.G. 1976 Requirement for growth substrate during lignin decomposition by wood rotting fungi. Applied and Environmental Microbiology 32, 192-194. Leatham, G.F. & Kirk, T.K. 1983 Regulation of lignolytic activity by nutrient nitrogen in white rot basidiomycetes. FEMS Microbiology Letters 16, 65-71. Mandels, M., Andreotti, R. & Roche, C. 1976 Measurement of saccharifying cellulase. Biotechnology and Bioengineering Symposium 6, 21-33. Mandels, M., Honzt, I. & Nystrom, J. 1974 Enzymatic hydrolysis of waste cellulose. Biotechnology and Bioengineering 26, 1471-
1493. Muller, H.W., Trosch, W. & Kulbe, C. 1988 Effect of phenolic compounds on cellulose degradation by some white rot basidiomyces. FEMS Microbiology Letters 49, 87-93. Nigam, P., Pandey, A. & Prabhu, K.A. 1987 Lignolytic activity of two basidiomycetes cultures in the decomposition of bagasses. Biological Wastes 21, l-10. Paterson, A. 1989 Biodegradation of lignin and cellulolysic materials. In Biotechnology for Livestock Production, Olds, RJ. pp. 245261. New York: Plenum Press. Reid, I.D. 1985 Biological delignification of aspen wood by solid state fermentation with white rot fungus Met&w trernellosus. Applied and Environmentul Microbiology 50, 133-139. Reid, I.D. 1989 Optimization of solid state fermentation for selective delignification of aspen wood with Phlebia tremellosa. Enzyme and Microbial Technology 11, 804-809. Reinhammer, B. 1984 Lactase. In Copper Proteins and Copper Enzymes, Vol. 3, ed Lonte, R. pp. l-36. Boca Raton: CRC Press. Smith, J.E., Anderson, J.G., Senior, E. & Aiddo, K. 1987 Bioprocessing of lignocelluloses. Philosophical Transactions of the Royal Society of London, Series A 321, 507-521. Smith, J.F., Claydon, N., Love, M.N., Alban, M. & Wood, D.A. 1989 Effect of substrate dept on extracellular endocellulase lactase production of Aguricw bispolw. Mycological Research 93,
292-296. Wayman, M. & Parekh, S.R. 1990 Biotechnology for Biomass Conversion. Milton Keynes, UK: Open University Press, Yamanobe, T., Mitsuichi, Y. & Takasaki, Y. 1987 Isolation of cellulolytic enzyme producing micro-organism: culture conditions and properties of the enzymes. Agrical~ttrul and Biological Chemistry 5, 65-74. Young, R.A. 1986 Structure, swelling and bonding of cellulose fibres. In Celltllose Structttre, Modificufion and Hydrolysis, eds Young, R.A. & Rowell, R.M. pp. 91-128. New York: Interscience.
(Received in revised form
28 July 1995; accepted 2 August 7 995)
World looumal ofMicrobiology 6 Bmtechnology, Voi 12, 1996
21
of Microbiology
& Biotechnology
12. 16-21
Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five Iignocellulose-degrading fungi by ferulic acid F.O. Asiegbu,” A. Paterson & J.E. Smith At 5 g/l, ferulic acid, a plant cell-wall phenolic, severely repressed growth of the lignocellulose-degrading fungi Trichoderma harzianum, Chaetomium cellulolyticum, Phanaerochaete chrysosporium, Trametes versicolor and Pleurob sajor-caju. At 0.5 g/l, however, it slightly stimulated growth of the latter two organisms. Two classes of extracellular enzymes involved in cellulose and glycolignin breakdown were assayed: cellulases; and phenol oxidases as laccases. All of the strains depolymerized cellulose but two (T. versicolor and P. sajor-cajtr) also secreted laccases. Laccasesecreting fungal species had normal levels of cellulose saccharification except in the presence of 5 g ferulic acid/l, whereas saccharification by the other strains was suppressed at all concentrations of the phenolic tested. Key words: Biomass protein, cellulases, ferulic acid, laccases, spruce wood, wood-decay
Lignocejluloses in the form of agricultural residues, including straw and forestry wastes, constitute a major renewable source of organic matter (Smith et al. 1987). Such materials consist of three principal classes of polymers-celluloses, hemicelluloses and lignins-with minor quantities of lowmolecular-weight compounds such as phenolics, lignans and tannins (Wayman & Parekh 1990). To date, biological conversion of lignocellulosic plant materials into addedvalue products has been limited, largely due to the economics of the process but partly because of our limited understanding of the biochemistry and physiology of microbial breakdown of glycolignin (Paterson 1989). This material forms the lignin hemicellulose matrix separating cellulose protofibrils in the interrupted lamella model of the lignified plant cell wall (Young 1986). Lignified tissues resist microbial attack partly because of the limited ability of cellulolytic enzymes to penetrate cell
F.O. Asiegbu was and A. Paterson and J.E. Smith are with the of Bioscience and Biotechnology, University of Strathclyde, Street, Glasgow Gl IXQ. UK. F.O. Asiegbu is now with the of Forest Mycology and Pathology, Swedish University of Sciences, P.O. Box 7026. S-750 07 Uppsala, Sweden; fax: 46 ‘Corresponding author. @ 1996 Rapid Science
Publishers
Department 204 George Department Agricultural 16 30 92 45.
fungi.
walls (Cowling & Kirk 1976). However, lignified tissues may also contain substantial quantities of phenolic compounds, such as ferulic or hydroxycinnamic acid, that contribute to the bonding between carbohydrates or between hemicelluloses and lignin (Hartley & Ford 1989). Such compounds may also be present in locally high concentrations in sap wood. Two processes that have gained economic significance in lignocellulose fermentation are mushroom cultivation (Smith et al. 1987, 1989) and, more recently, the conversion of wood into animal feed (palo podrido) in Chile (Gonzalez et al. 1989). It is possible that, with an increased understanding of the physiology of lignin-degrading fungi, further solid-substrate processes for enhancing digestibility of lowvalue animal lignocellulosic feed can be developed (Reid 1989). In vitro studies using purified lignin peroxidases have indicated that the value of such extracellular enzymes per se will be limited (Jurasek 1990). Lignin peroxidases and laccases are regularly associated with fungal degradation of the lignin component of lignocellulose materials (Evans 1985). The activities of these enzymes in two white-rot fungi, Trametes versicolor and Phanerochaefe chrysosporium, have been extensively studied (Leatham & Kirk 1983;
Ferulic
Evans et al. 1984; Janshekar & Fiechter 1988). However, data on the physiological regulation and role of lactase production iniigninolytic fungi are limited. Many have studied the effect of addition of alternate carbohydrate sources on lignocellulose fermentations (Reid 1989). Detailed studies on the effects of plant cell-wall phenolic constituents on the biodegradability of spruce wood by other wood-inhabiting fungi has not received as much attention, except for studies with Phanaerochaete Chrisosporium (Ander & Eriksson 1975; Kirk ef al. 1976). It is likely that the behaviour of other saprophytes on this substrate may differ. Furthermore, the stimulatory effects of the key, cell-wall phenolic, ferulic acid, on fungal saccharification and on rumen cellulolytic activity are well documented (Akin et al.. 1988; Bomeman et al. 1990). The effects of such exogenous addition on enzymes important in lignocellulose breakdown has not been correlated with breakdown of natural substrates. The present study is on the influence of differing concentrations of ferulic acid on enzymes important in lignocellulose degradation and aims to clarify how such plant cellwall constituents might influence saccharification of natural substrates.
Materials
and Methods
Orgunisms Trichodermu harziunum was isolated from decayed palm wood and identified at the International Mycological Institute, UK. Phanerochuefe chrysosporium IMI 747691, Trumefes versicolor IMI 210864, Chuefomium cel/uioiyficum IMI 188965 and P/ear&s sujo-cuju IMI 19976 were also used. Substrates and Chemicals Spruce (Piceu sitchensls) sawdust was from the Economic try Commission, Dunoon, UK. Fen&c acid (4-hydroxy, methoxy cinnamic acid) and syringaldazine were Aldrich.
Fores3from
Media The minimal medium (pH 5.5) employed contained (g/l): NH,H,PO,, 2.0; KH,PO,, 0.6; K,HPO,, 0.4; MgS0,.7H,O, 0.5; CaCI,, 0.2; FeSO,, 0.01; and thiamine, 0.001; and 1 ml micronutrient solution (per 1) containing (g/l): Z&O,, 6.6; MnS0,.4H,O, 5.0; CoC1,.6H,O, 1.0; and CuS0,.5H,O, 1.0. Phosphates, sulphates and chlorides were dissolved separately, mixed and made up to the required volume with distilled water (Asiegbu et al. 1994). Starter Cttltures Starter cultures for biomass and enzyme determinations were prepared by growing mycelia for 4 to 10 days in minimal medium supplemented with 17 g malt extract, 11 g peptone and 1 g cellulose/l. Flasks were incubated statically and mycelia were harvested, washed, and fragmented with a sterile laboratory blender. Hyphal homogenates (2.5 ml) were used to inoculate 50. ml batches of minimal medium containing 1% (w/v) crystalline cellulose powder. Experimental flasks contained ferulic acid at 0.5,
acid and glyculignin-attacking
enzymes
1.0 or 5.0 g/l. Sets of static flasks were set up so that duplicate cultures were harvested every 48 h for 14 days. Solid-stlbsfrufe Fermentation: Exogenotls Addition of Ferulic Acid Spruce sawdust was moistened with ferulic acid (0.5, I.0 or 5.0 g/l) in minimal medium to a moisture content of 70%. Moistened substrates were equilibrated at 4°C for 48 h and fermented as described by Cuero et al. (1985). Briefly, aliquots (20 g) of moistened substrate were dispensed into micro-porous bags, autoclaved at 121°C for 30 min, cooled and inoculated with a suspension of fragmented mycelia (1 ml/bag). Substrates inoculated with T. harzianum, P. sajor-caju or T. uersicolor were incubated at 28”C, those with C. cellulolyficMm at 37.5”C and those with P. chrysospo-
rictm at 40"C, all in environmental cabinets (Fisons Ltd, Loughborough, UK) at 98% relative humidity for 14 days. Substrates moistened treated.
with
unsupplemented
minimal
medium
were
similarly
Preparation of Extracellular Enzymes and Biomass Cultures were harvested and centrifuged at 5000 x g at 4°C for 30 min. Residual biomass was dried at 75°C and quantified as total protein-nitrogen by Kjeldahl determination. Enzyme Assays Saccharification of crystalline cellulose was determined as glucose produced from hydrolysis of absorbent cotton wool (200 mg) by 3 ml culture supematant with 2 ml 0.1 M sodium citrate buffer, pH 4.8, at 50°C for 24 h (Mandels et al. 1974), using the glucose oxidase method (Karkalas 1985). Carboxymethyl cellulase and filter paper cellulase were determined as described by Mandels et crl. (1976). p-Glucosidase was estimated using the procedure of Yamanobe et ui. (1987). Lactase (phenol oxidase) activity was estimated using a modification of methods described by Eriksson et al. (1983) and Nigam et al. (1987) in which syringaldazine is oxidized to a quinone. An assay mixture containing 0.5 ml 0.1% (w/v) syringaldazine in 99% ethanol and 1.5 ml sodium acetate (0.2 M, pH 5.6) was made up to 3 ml with culture supernatant and assays were incubated at 30°C. Soluble Squrs, Total Polysucchuride and Cellulose Total soluble sugars were estimated as reducing sugar using the Somogyi method. Total polysaccharide and cellulose were determined as reducing sugars and glucose, respectively, in acid hydrolysates, using previously described methods (Englyst and Cummings 1988; Asiegbu 1992). Sfufisficul Analyses The experiments were repeated at ieast two times. Results analysed using basic statistical method (Student’s t-tests).
were
Results Saccharificafion
of Cellulose
The growth and saccharification of crystalline cellulose by three fungi (7. harzianum, C. cellulolyticum and P. chrysosporittm) was suppressed by ferulic acid (Table 1). The other two strains had normal saccharification except in the presence of 5.0 g ferulic acid/l (Table 1). A similar observation was made for /I-glucosidase (Table 2) and CM-cellulase activity (data not shown). Values for initial cellulose depolymerizing activity, assayed as reducing sugar released
World Journal of Microbiology 6 Biotechnology. Vol 12, 1996
17
F.O. Asiegbu, A. Pakrson and J E. Smith Table 1. Effect on cellulose-saccharlfylng cultures of five fungi growlng on crystalline Fungus
Activity
enzyme (exo-glucanase) cellulose.
(pg glucose
released
from
actlvitles
of addition
200 mg cotton/ml)
of ferulic
with ferulic
acid
0
0.5
1.0
5.0
C. cellulolyticum T. harzianum
71 61
P. chrysosporium P. sajor-caju T. versicolor
45 28 113
3ot 2% 1st 53 133
17t 11t 23 86 117
ND W w 40 75
Samples analysed t Significantly lower ND-Not determined.
l
Table 2. Effect cellulose.
10 days post than control
on /?-glucosldase
Fungus
cellulolyticum harzianum chrysosporium sajor-caju versicolor
* Samples analysed t Significantly lower
at (g/l):*
inoculation.
of addltfon
(pg glucose
of ferullc
released
0 C. T. P. P. T.
to
value (p < 0.05).
activities
Activity
acid
25 32 26 32 100
from
acid
to cultures
cellobiose/ml)
of live
with ferullc
fungi
growing
on
acid at (g/l):’
0.5
1.0
5.0
22 22 gt 36 787
gt W 1st 50 143
1st st gt 1st sot
10 days post inoculation. than control value (p < 0.05).
from filter paper cellulose over a 60-min incubation, appeared to be stimulated for all strains with increasing concentrations of the phenolic (data not shown). Lactase Acfiuities Lactase activities, assayed as the oxidation of syringaldazine by culture supernatants, were determined every second day for 14 days. Of the five strains of fungi studied, only i? sajor-caju and T. versicolor produced detectable lactase activity and then only under some of the experimental conditions (Table 3). Under control conditions, in the absence of ferulic acid, Tra. versicolor produced appreciable lactase activity whereas P. sajor-caju had no demonstrable activity. With l? sajor caju in the presence of 0.5 or 1.0 g fen&c acid/l, lactase activity increased with time, reaching a maximum at 12 (0.5 g/l) or 14 days (I g/l). Growth in the presence of 5.0 g ferulic acid/l yielded lactase activities approximately two orders of magnitude below those with I g ferulic acid/l. The pattern of lactase activity observed with T. versicolor in the presence of 0.5 or 1.0 g ferulic acid/l was more complex, with maximum activities at 6 and 14 days, respectively. No lactase activity was detected in the presence of 5.0 g ferulic acid/l.
Total Soluble Sugars Significantly increased concentrations of soluble sugars accumulated in cultures growing in the presence of 5.0 g ferulic acid/l (Table 4). Only low amounts of reducing sugar were detected in supematants of cultures grown in the absence of ferulic acid. Biomass Prod&on Biomass production (as total protein) was estimated in 12day-old cultures growing on crystalline cellulose in the presence of varying concentrations of ferulic acid. The results obtained show that growth was severely repressed by 5.0 g ferulic acid/l in all five species tested (Figure I). Ferulic acid at 0.5 g/l appeared to stimulate growth in P. sajor-caju and T. versicolor. A different pattern was observed for T. harzianum, P. chysosporium and C. cellulolyficum, where increasing ferulic acid concentration resulted in decreased biomass production (Figure 1). Effect of Fertllic Acid on Solid-substrate Fermentation (SSF) Spruce sawdust was supplemented with ferulic acid in solid-substrate fermentation to determine if exogenous addition of monomeric forms of phenolic influenced cellulose saccharification. More polysaccharide was lost at 0.5 g to
Fen&c acid and ~lycolignin-attacking Table
3. Effect
Ferulic (“/I
acid
of ferulic
acid
T. versicofor P. sajor-caju
0.05
T. P. T. P. T.
0.5
production
by cultures
of T. versicolor Activity
Fungus
0
0.1
(FA) on lactase
versicolor sajor-caju versicolor sajor-caju versicolor
Table fungal
lncubatlon
on 1% (w/v)
cellulose.
for (days):’
2
4
6
8
10
12
14
17 (1.0) -
20 (3.0) -
25 (4.0) -
35 (5.0) -
32 (5.0) -
27 (3.0) -
(6.0) (0.0) (3.0) (0.0)
67 7 a3 12
(4.8) (1.0) (5.0) (2.0) -
109 37 157 21
-
* One unit of lactase enzyme activity and (standard deviations) for duplicate - - No detectable lactase activity.
(u) after
growing
9 (2.0) 55 1 37 2
P. sajor-caju
and P. sajor-caju
enzymes
is defined as the amount determinations.
(13.0) (2.0) (9.0) (1.0)
C.cellulolyticum T. harzianum P. chrysosporium P. saior-caju T. versicolor
Reducing
(9.0) (5.0) (2.0) (3.0)
0.4
0.5
that will produce
a change
4. Effect of ferulic acid on accumulation saccharfflcatlon of crystalline cellulose.
Fungus
96 68 a7 42
sugars
(ma/ml)
43 124 45 92
(1.0) (1.0) (3.0) (7.0)
97 165 44 82
0.6
of reducing
in A,,,
sugars
wlth ferulic
(4.0) (4.0) (2.0) (4.0) -
127 167 73 110
0.5
of 0.001
per min. Values
(5.0) (8.0) (6.0) (5.0) 1.0
are
means
durlng
acid at (g/l):*
0
0.5
1.0
5.0
0.03 0.04 0.01 0.03 0.06
0.02 0.08 0.02 0.08 0.13
0.03 0.08 0.10t 0.10 0.13
0.3t 0.16t 0.20t 0.207 0.3t
* Samples were analysed 10 days post inoculation. t Significantly different from control value (p < 0.05).
1.0 g fen&c acid/l (Table 5) than at 5.0 g/l (data not shown) when wood was fermented with l? chrysosporium. Cellulose breakdown was enhanced, in proportion to the ferulic acid added, in wood fermented with T. uersicolur or T. harzianum (Table 5). Also, at 0.5 g ferulic acid/l, polysaccharide loss was slightly higher than in controls without the acid in fermentations with P. sajor-caju (Table 5). In contrast, exogenous addition of fen&c acid repressed polysaccharide breakdown by C. cellulolyticum.
Discussion Fungi have different enzymatic responses to ferulic acid in lignocellulosic plant residues. Under favourable conditions, the extracellular glycolignin-degrading enzymatic activities of three fungi studied (I? chrysosporium, P. sajor-caju, T. versicolor) increased linearly with growth (unpublished work). An exception was lactase production in submerged culture, which showed a cyclical pattern in the two organisms (P. sajor-caju and T. vet&o/or) shown to secrete the enzymes with this activity. Such patterns were not observed for ligninase production, in surface culture after 7 days
(unpublished work), although cyclical production of ligninases in liquid culture has been observed elsewhere (Janshekar & Fiechter 1988). Smith ef al. (1989) reported cyclical production of lactase during growth of Agaricus bisporus in solid-substrate culture. The patterns of lactase production by T. versicolor and P. sajor-caju were different. Although T. versicolor secretes both constitutive and inducible laccases (Fahraeus ef al. 1958; Evans ef al. 1984), to our knowledge, inductive lactase production in P. sajor-caju has not been previously reported. In the present study, lactase activity was enhanced by adding ferulic acid to the liquid culture. However, ferulic acid at the highest concentration tested inhibited biomass production by T. uersicolor and consequently no lactase could be detected. In contrast, lactase activity in P. sajor-caju was inducible by ferulic acid at all concentrations in liquid culture and no basal level of constitutive enzyme activity was detected in the absence of the phenolic compound. The present results also revealed that P. sajor-caju and T. versicolor, which secreted phenol oxidizing laccases, showed stimulation of both depolymerizing cellulase and /3-glucosidase activities. In contrast, strains that did not
World
Joumf of Micmbdogy
6 Biotechnology, Vol 12, 1996
19
F.O. Asiegbu. A. Paterson and J.E. Smith Table 5. Effect of ferulic acid breakdown In fungal-fermented Fungus
on total polysaccharide spruce wood.
Loss
(% of control
values)
(NSP)
with
and cellulose
lerulic
acid
0
1.0
15.7 (2.2)
14.7 (4.5)
0.0 (0.0)
10.3 (1.5)
12.3 (1.2)
19.2 (3.1) 11.5 (2.0)
(CP)
at (g/l):
T. versicolof NSP CP P. sajor-caju NSP CP
8.1 (1.0)
P. chrysosporium NSP
15.7 (1.0)
CP T. harzianum NSP CP
7.1 (1.0)
18.4 (4.0) 13.9 (5.0)
8.5 (1.4) 2.0 (0.2)
12.3 (3.5) 10.7 (2.6)
21.0 (1.6) 11.2 (2.6)
11.4 (2.0) 4.3 (0.7)
C. cellulolyticum NSP CP ‘Values fermentation
are
means in solid-state
and
(standard
deviations)
recorded
after
14
days’
cultures.
I
Figure 1. Effect of adding ferulic acid, at 0.5 (m), 1.0 (@) or 5 ) g/l, on biomass production by P. chrysosporium (l), P. sajor-ca/u (2), T. versicolor (3), C. cellulolyticum (4) and T. harzianum (5) after 12 days’ growth on cellulose. Vertical lines represent standard deviations. W-Control with no ferulic acid.
secrete this class of enzyme showed repression of j?-glucosidase and endoglucanase although initial cellulose depolymerization was stimulated (unpublished work). A similar observation was reported for T. versicolor by Muller et al. (1988), who concluded that cellobiono-lactone from oxidative attack on carbohydrate was responsible for the stimulation. Analyses of accumulated sugars in the growth media indicated that, in the five organisms studied, soluble sugars accumulated in the media in parallel with increasing ferulic acid concentrations. In all five fungi, high concentrations of ferulic acid inhibited growth. Whereas the amount of accumulated sugars with 0.5 g ferulic acid/l was similar to control values, it was much higher when 5 g ferulic acid/l was used. The varying rates of polysaccharide loss obtained in solid-substrate cultures were similar to those reported earlier (Reid 1985; Agosin et al. 1986). The increased cellulose loss recorded with lignolytic fungal species in the presence of ferulic acid may be explained either as induction of phenol-detoxifying enzymes by the acid (GiovannozziSermanni eI al. 1982; Reihammer 1984) or stimulation of cellulolytic activity in stressed cells. Finally, it is suggested that the presence of certain phenolic compounds in feeds will suppress mycelial biomass production while inducing high activities of cellulolytic and ligninolytic enzymes leading to accumulation of degradation products. Such catabolic products, which might not be readily utilized by aerobic fungi, may be of value in ruminant nutrition. Obviously, fungi will differ in physiological response to substrate composition and so this should be optimized for each organism.
Fe&c
Acknowledgement This
study
was
Association
of
supported
partly
Commonwealth
by
a scholarship Universities
from (ACU)
the to
FOA.
References Agosin, E., Tollier, M.T., Brillouet, J.M., Thivend, P. & Odier, E. 1986 Fungal pretreatment of wheat straw: effects on the biodegradability of cell wall structural polysaccharide, lignin and phenolic acids by rumen micro-organisms. ]oarnul of the Science of Food and AgriczdtMre 37, 97-106. Akin, D.E., Rigsby, CC., Theodorou, M.K. & Hartley, R.D. 1988 Population changes of fibrolytic rumen bacteria in the presence of phenolic acids and plant extracts. Animul Feed Science and Technology 19, 261-275. Ander, P. & Eriksson, K.E. 1975 Influence of carbohydrates in lignin degradation by white rot fungus Sporolrichum pulveralenhum. Svensk Pupperstidn 78, 643-652. Asiegbu, F.O. 1992 Fungal delignification of lignocellulosic materials; Physiological aspects and enhancement of rumen fermentation. PhD Thesis. University of Strathclyde, Glasgow, UK. Asiegbu, F.O., Morrison, I.M., Paterson, A. & Smith, J.E. 1994 Analyses of fungal fermentation of lignocellulosic substrates using continuous culture rumen simulation. Journal of General and Applied Microbiology 40, 305-318. Bomeman, W.S., Hartley, R.D., Morisson, W.H., Akin., D.E. & Ljundahl, L.G. 1990 Feruoyl and p-coumaryl esterase from anaerobic fungi in relation to plant cell wall degradation. Applied Microbiology and Biotechnology 109, l-8. Cowling, E.B. & Kirk, T.K. 1976 Properties of cellulose and lignocellulosic materials as substrates for enzymic conversion processes. Biotechnology and Bioengineering Symposium 6, 9&
123. Cuero, R.G., Smith, J.E. & Lacey, J. 1985 A novel containment system for laboratory scale solid particulate fermentation. Biotechnology Letters 7, 463-466. Englyst, H.N. & Cummings, J.H. 1988. Improved method for measurement of dietary fiber as non starch polysaccharides in plant foods. ]otlrnul of the Associution of Official Analytical Chemists 71, 808-814. Eriksson, K.E., Johhnsrud, SC. & Vallander, L. 1983 Degrading of lignin and lignin model compounds by various mutants of the white rot fungus Sporotrichtrm pu/veru/entum. Archives of Microbiorogy 35, 161-168. Evans, SC. 1985 Lactase activity in lignin degradation by Coriolus versicolor in vivo and in vitro studies. FEMS Microbiology Letters
27,339-343. Evans, SC., Farmer, J.Y. & Palmer, J.M. 1984 An haem protein from Coriolus versico/or. Phytochemistry
extracellular
23, 1247-
1250. Fahraeus, G., Tullander, high lactase yields
V. & Ljunggren, H. 1958 Production of in cultures of fungi. Physiologiu PIantnnrm
ll,631-639. Giovannozzi-Sermanni, G. Badiani, M. & Luna, M. 1982 Protein production and lactase activity in Aguricus bisporas. Biotechnology Letters 4, 507-512. Gonzalez, A.E., Martinez, A.T., Almendros, G. & Grribergs, J. 1989 A study of yeasts during the delignification and fungal transformation of wood into cattle feed in Chilean rain forest. Antonie vun Leewenhoek 55, 221-236.
acid and glycolignin-attacking
enzymes
Hartley, R.D. & Ford, C.W. 1989 Phenolic constituents of plant cell walls and wall biodegradability. In Plant Cell Wall Polymers: Biogenesis and Biodegradation eds Lewis, N.G. & Paice, W.G. pp. 138-145. Washington DC: American Chemical Society. Janshekar, H. & Fiechter, A. 1988 Cultivation of Phanmochuete chrysosporiMm and production of lignin peroxidases in submerged stirred tank reactors. @trnul of Biotechnology 8, 97-112. Jurasek, L. 1990 Biological pulping. In Proceedings of the 4th International Mycological Congress, Regensbttrg, Germany. Eds. Reisinger A. & Bresinsky A., p. 237. Karkalas, J, 1985 An improved enzymic method for the determination of native and modified starch. loamal of Ihe Science of Food undAgric&re 36, 1019-1027. Kirk, T.K., Conors, WJ. & Zeikus, J.G. 1976 Requirement for growth substrate during lignin decomposition by wood rotting fungi. Applied and Environmental Microbiology 32, 192-194. Leatham, G.F. & Kirk, T.K. 1983 Regulation of lignolytic activity by nutrient nitrogen in white rot basidiomycetes. FEMS Microbiology Letters 16, 65-71. Mandels, M., Andreotti, R. & Roche, C. 1976 Measurement of saccharifying cellulase. Biotechnology and Bioengineering Symposium 6, 21-33. Mandels, M., Honzt, I. & Nystrom, J. 1974 Enzymatic hydrolysis of waste cellulose. Biotechnology and Bioengineering 26, 1471-
1493. Muller, H.W., Trosch, W. & Kulbe, C. 1988 Effect of phenolic compounds on cellulose degradation by some white rot basidiomyces. FEMS Microbiology Letters 49, 87-93. Nigam, P., Pandey, A. & Prabhu, K.A. 1987 Lignolytic activity of two basidiomycetes cultures in the decomposition of bagasses. Biological Wastes 21, l-10. Paterson, A. 1989 Biodegradation of lignin and cellulolysic materials. In Biotechnology for Livestock Production, Olds, RJ. pp. 245261. New York: Plenum Press. Reid, I.D. 1985 Biological delignification of aspen wood by solid state fermentation with white rot fungus Met&w trernellosus. Applied and Environmentul Microbiology 50, 133-139. Reid, I.D. 1989 Optimization of solid state fermentation for selective delignification of aspen wood with Phlebia tremellosa. Enzyme and Microbial Technology 11, 804-809. Reinhammer, B. 1984 Lactase. In Copper Proteins and Copper Enzymes, Vol. 3, ed Lonte, R. pp. l-36. Boca Raton: CRC Press. Smith, J.E., Anderson, J.G., Senior, E. & Aiddo, K. 1987 Bioprocessing of lignocelluloses. Philosophical Transactions of the Royal Society of London, Series A 321, 507-521. Smith, J.F., Claydon, N., Love, M.N., Alban, M. & Wood, D.A. 1989 Effect of substrate dept on extracellular endocellulase lactase production of Aguricw bispolw. Mycological Research 93,
292-296. Wayman, M. & Parekh, S.R. 1990 Biotechnology for Biomass Conversion. Milton Keynes, UK: Open University Press, Yamanobe, T., Mitsuichi, Y. & Takasaki, Y. 1987 Isolation of cellulolytic enzyme producing micro-organism: culture conditions and properties of the enzymes. Agrical~ttrul and Biological Chemistry 5, 65-74. Young, R.A. 1986 Structure, swelling and bonding of cellulose fibres. In Celltllose Structttre, Modificufion and Hydrolysis, eds Young, R.A. & Rowell, R.M. pp. 91-128. New York: Interscience.
(Received in revised form
28 July 1995; accepted 2 August 7 995)
World looumal ofMicrobiology 6 Bmtechnology, Voi 12, 1996
21
