Cellulase and ligninase production by basidiomycete culture in solid-state fermentation

Biological Wastes 20 (1987) 1-9

Cellulase and Ligninase Production by Basidiomycete Culture in Solid-State Fermentation P o o n a m N i g a m , * A s h o k P a n d e y & K. A. P r a b h u Biochemistry Division, National Sugar Institute, Kanpur-208017, India (Received 20 January 1986; revised version received 1 June 1986; accepted 10 June 19861

A BS TRA C T Two Basidiomycete isolates, Polyporus BH 1 and P. BW x, were studied for cellulases and ligninase activities in the degradation of bagasse in solid-state .fermentation (85% moisture). In the solid-fermentation system the cultures were grown on whole bagasse (untreated) alone and in admixture with cellulose pulp. Production of enzymes by cultures was studied at different periods of growth and with different particle sizes of bagasse. Cellulase activities reached maximum in 4-5 days and ligninase in 2 to 3 weeks time of growth. Air drying of fermented bagasse showed a loss in cellulase and ligninase enzyme activities. Acetone treatment of the fermented residue before drying helped in retention o[ both the enzyme activities.

INTRODUCTION Most of the research on the production of cellulases and single cell protein from cellulosic materials has been performed with slurry-state systems with 1 % - 5 % of substrate. The fermentation systems with about 20% w/v solid are referred to as 'solid-state fermentations' (Cannal & Moo-Young, 1980) (SSF). SSF systems are ideal for use of fungi on insoluble substrates and have m a n y potential advantages ( H a n & Anderson, 1975; Han et al. 1976; Harseltine 1977). * Present address: Siiddeutsche Zucker-AG, Zentral-Laboratorium, Postfach 1127, D-6718 Griinstadt 1, West Germany. 1

Biological Wastes 0269-4607/87/$03.50 (@ Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain

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Poonam Nigam, ,4shok Pandey, K. A. Prabhu

Production of cellulases from cellulosic substrates by SSF has been studied in detail by Toyama & Ogawa (1972, 1978). Most of the experiments on cellulase production in SSF are with treated cellulose as substrate. There are no reports so far on ligninase production in SSF and with untreated substrates. The present work deals with the cultivation of two Basidiomycete spp. on whole bagasse in a solid-state fermentation and estimation of extracellular cellulases and ligninase in the extract at different periods of fermentation, using different particle sizes of bagasse. Effect of drying and acetone treatment of the fermented sample, before enzyme extraction, on enzyme activity was also studied.

METHODS

Microorganisms Two isolates Polyporus BH 1 and P. BW 1 (Nigam & Prabhu, 1985a) were used.

Substrate Whole bagasse powder (untreated) was washed with hot water, dried and further powdered to 35-65 mesh size. Filter paper (ordinary, used for routine filtrations) was soaked in water and homogenized in a blender to make pulp. Two parts (dry weight) were mixed with eight parts bagasse powder.

lnoculum preparation and B W 1 cultures were grown on malt agar plates for 3 4 days at 30°C for inoculum preparation. Erlenmeyer flasks (100ml) containing 40ml malt extract medium (3%, w/v) and six pieces of mycelium (10mm diameter) were incubated for 72 h at room temperature (27°C-30°C) on a rotary shaker. The biomass formed was asceptically washed with sterile water and used as inoculum for each 4 g of substrate. BH 1

Preparation of SSF system In the studies of solid-state cultivation, bagasse powder (16 g) (without and with filter paper, two paper:eight bagasse) was suspended in Norkran's medium (Eriksson & Larson, 1975) (pH 5.5) at 85% w/v moisture (dry

Enzyme production by Basidiomycetes

3

basis) in 1-1itre Roux bottles and autoclaved for 15min at 120°C. The bottles were cooled and the substrate was spread at 1-2 cm thickness and inoculated, and the bottles were incubated at 3 0 _ I°C in a humid atmosphere. At regular intervals, the contents of the bottles were removed for extraction of enzymes. Separate sets of bottles were used in each study.

Enzyme extraction The enzyme extracts were prepared by extracting the fermented residue with three volumes of 0"IM acetate buffer (pH 4.8 for cellulases and pH 5.6 for ligninase) at room temperature for 1 h (Toyama & Ogawa, 1978). The extracts were filtered through a sintered-glass crucible (G-I) under vacuum. The residual substrate was washed with the respective buffer and reextracted for 1 h at room temperature. Both extracts, together with washings, were collected and centrifuged at 4°C and 5000 rpm for 5 min. The supernants were used for cellulases and ligninase assays.

Effect of mesh size Bagasse powder, of mesh sizes 35, 48, 65 and 80, was taken in 4-g quantities with 25-ml Norkran's medium in 250-ml Erlenmayer flasks. After inoculation the flasks were incubated for 5 to 6 days at 3 0 _ I°C.

Air drying A weighed portion of fermented bagasse was spread on a plate at room temperature (27°C-30°C) for about 10-12h to dry it completely, then enzymes in the dried sample were extracted as before.

Acetone treatment prior to air drying A weighed portion of fermented residue was treated with cold acetone

(Methods in enzymology, 1955), filtered and then left at room temperature for drying for 10-15 min. The acetone-dried sample was then extracted for enzymes after storing for 4-5 days at 5°C.

Assay of enzymes

Cellulase Filter paper decomposing (FPD), carboxymethyl cellulase (CM-Cellulase) and fl-glucosidase (Salicinase) activities were estimated in enzyme extracts by the procedure of Mandels & Weber (1969). Reducing sugars produced in

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Poonam Nigam, Ashok Pandey, K. A. Prabhu

the enzyme reaction mixtures were estimated by dinitrosalicyclic acid (Miller, 1959). Cellulase units are expressed as milligrams of reducing sugars released in one hour by the total amount of enzyme extracted from 1 g of substrate and specific activity expressed as cellulase units per milligram of enzyme protein. Ligninase Ligninase activity in extracts was measured by the procedure of Kirk & Kelman (1965) using guaiacol as substrate with 0"IM acetate buffer (pH 5.6). The increase in optical density was measured at 465 nm in a Spectronic 20, after allowing the reaction mixture to stand for 30min at room temperature (30°C). A ligninase unit is defined per gram of substrate and the specific activity was expressed as ligninase units per milligram of protein as for the cellulase. Enzyme protein Protein in enzyme extracts was precipitated by cold trichloroacetic acid (10%) and estimated by the method of Lowry et al. (1951). Lignin Free lignin in the extracts was precipitated (Toyama & Ogawa, 1977) by adjusting the pH to 2.0 in the cold and estimated by Denis reagent (Berk & Schroeder, 1942).

RESULTS AND DISCUSSION Polyporus BH1 and P. BW t mould isolates showed cellulolytic and ligninolytic activities in submerged fermentation (Nigam & Prabhu, 1985a,b). The cultures showed good growth in solid-state cultivation and the extracts obtained at regular intervals from solid cultures contained cellulases and also ligninase activities. In BH1 culture FPD, CM-Cellulase and salicinase activities reached maximum in 4-5 days and remained constant up to 18 days of fermentation and then decreased at about 30-45 days (Fig. l). In BW~ culture all the cellulase activities decreased appreciably after 5 days of growth (Fig. 2). Addition of cellulose pulp decreased cellulolytic enzyme activities in both the cultures. Ligninase activity was maximum at 13-18 days and decreased after 30-45 days of growth in both the cultures. Ligninase activity in BW 1 culture was about four times that in BH 1 (Figs 1 and 2). Addition of cellulose also inhibited ligninase activity. Similar results were obtained in shake-flask cultures

Enzyme production by Basidiomycetes

5

12

1-

4"J

/

I 5

0

Fig. 1.

9

Days

13

18

,:

45

0

Enzyme activities at different periods of SSF (BH1). ©, FPD. I-1, CM-Cellulase. 0 , Salicinase. A, Ligninase. - - - - , Bagasse. -, Bagasse + cellulose.

where cellulase activities decreased after I week, and ligninase after 15 days, of growth (Nigam & Prabhu, 1985a,b). The presence of free cellulose inhibited ligninase enzyme production (Figs 1 and 2). In shake cultures also, ligninase production and lignin degradation were decreased in the presence of cellulose (Nigam & Prabhu, 1985b). Free lignin (degraded) and protein contents in extracts increased

E9

~ ~1//," l Ill ,/ ..'- - ' ~~ - _ ~ ~ -. ~ -_-

._

~~ I " . - ' "

Ig~=c-'-"~'--__

0

5

9

13

~ ' ~\\\'o

°

N

18

45

Days

Fig. 2, Enzyme activities

at different periods

O, Salicinase. /X, L i g n i n a s e . -

of SSF (BW~). Q, FPD. D, CM-Case.

Bagasse.

Bagasse + cellulose.

Poonam Nigam, Ashok Pandey, K. A. Prabhu

6

with the period of growth and reached a maximum at about 3 weeks (Table 1). These, also, were decreased by free cellulose. Specific activities of cellulase increased up to 13 days of growth in BH 1, but in BW 1 maximum specific activities were found on the fifth day (Table 1). In the case of ligninase, maximum specific activity in BH1 culture was on day 13, whereas, in BW1, activity increased up to 19 days. Specific activities of all the enzymes were lower in the presence of cellulose in both the cultures. Similar suppressions of enzyme activities have been reported by different workers (Kirk & Chang, 1975). The presence of free lignin in culture filtrates indicated the decomposition of lignocellulosic material. Air drying of the fermented residue before extraction of enzyme gave loss in cellulase and ligninase activities. Salicinase activity in BW~ culture was affected most by drying and addition of cellulose pulp (Table 2). However, Deschamps & Huet (1984), using beet pulp as substrate in SSF, reported that drying of fermentation residues was of great use in preserving the salicinase activity in concentrated form. TABLE 1 Specific Activities of Cultures in Solid-State Fermentation Details

FPD a b

B H 1 days

B W I days

5

13

19

5

13

19

4.13 2.88

6.74 3.93

2"67 1.57

4'13 4.02

3"75 3.1

2-01 1.34

5.05 3.17

8-70 1-07

2.53 0'57

4.90 2-14

2.14 1.03

1.74 0.14

4.74 2.31

3.35 1.07

2.12 0.57

3.52 1.34

1.71 0-26

1.76 0-14

3.87 8-85

6.01 5.26

3-23 1'98

4.28 3'57

15-00 5'52

17.12 5.88

2-45 1.30

1.73 1.21

4.02 2'64

2.45 1"40

1.80 1'45

3.87 3.51

19.72 18.01

25-92 24"53

14"50 11"89

17-98 15.08

24.66 20.55

CM-Cellulase a b

Salicinase a b

Ligninase a b

Protein (rag g 1) a b

Free lignin (mgg- 1) a b

12.76 11.02

a--Bagasse, b---Bagasse + cellulose.

Enzyme production by Basidiomycetes

7

TABLE 2 Effect of Drying and Acetone Treatment on Enzyme Activity Details

Activity units per

gram a

BHI

BW 1

Wet

Dry

AT

Loss

Wet

Dr)'

AT

Loss

FPD a

10"13

6.71

9"98

10-12

6-37

9"70

b

3-75

2.71

3"55

33"77 (1'47) 27'62 (5.33)

5.63

2-81

5'33

37.13 (4.24) 50-11 (5.24)

CM-Cellulase a 12.37

6'79

45.12 (2.22) 40.70 (2.78)

12.00

5-94

11-95

3.94

1.71

3.84

63'41 (0.26) 90-24 (0.67)

8"63

1"41

8'50

1.87

Nil

1.83

21.16 (3.16) 28-60 (5"56)

10.50

9"55

10-36

5.00

3"90

4.85

b

Salicinase a b

Ligninase a b

12.1

4-13

2.45

4'01

11.63

4.25

11"60

3.00

0.29

2.98

9"50

7-49

9"20

5.00

3-57

4.72

50"52 (0.42) 56'55 (2.54) 83"63 (1.45) 100.00 (2.4) 9"05 (1"33) 22.00 (3"0)

a Per gram wet fermented bagasse used in each preparation. Wet-untreated, Dry-air dried and AT-acetone treated bagasse. a--Bagasse, ~ - B a g a s s e + cellulose. (Values in parentheses are per cent loss in acetone treatment).

Acetone treatment of the fermented residue before drying stabilized the cellulase and ligninase activities in the residue without much loss and the dried material could be preserved for a long time even at room temperature. It can be concluded that, to avoid any loss in activity of the samples, they should be extracted immediately or treated with cold acetone if the sample is to be stored unextracted for many days. The use of fine and coarse bagasse increased specific cellulase and ligninase production, respectively (Table3). Maximum cellulase specific activities were found at 65 and 48 mesh, whereas maximum ligninase specific activities were produced in 48 and 35 mesh size bagasse particles in

8

Poonarn Nigam, Ashok Pandey, K. A. Prabhu TABLE 3

Effect of Mesh Size of Bagasse on Enzyme Production (Specific Activities) Details

Mesh BH 1 35

FPD CM-Cellulase Salicinase Ligninase Proteina Lignina (in extract)

48

B WI 65

80

1'74 2'55 9"51 8'36 1.36 1.45 9-33 7-82 1.86 1.27 8.97 8.73 2'64 2'91 1-53 1-2 3.63 2.475 2.557 2.475 20.88 21.93 27-84 28.71

35

48

65

80

1-54 2-21 2-1 2.06 0-75 1.29 1.6 1.19 0.37 0.91 0.87 0.84 4.33 4.1 3-55 2-56 3.19 2.31 2.42 2.42 17-1 35.96 37.12 38.05

a Milligrams per gram of substrate. BH 1 and BW 1 cultures, respectively. The total enzyme activities were similarly affected. Lignin in the extracts increased with the increase in mesh size (finesses of bagasse) in both cultures. After extraction o f enzyme from fermented samples the spent bagasse could be used as animal feed because of its good nutritional value and the soft and loose texture of the bagasse particles produced by the enzyme action (Nigam & Prabhu, 1985b). So solid-state fermentation serves two p u r p o s e s - - p r o d u c t i o n o f concentrated cellulolytic and ligninolytic enzymes at less cost than shaken submerged fermentation and the production of good animal feed. The fermented bagasse can be kept as a source of enzymes after acetone treatment without appreciable losses of enzyme activities.

ACKNOWLEDGEMENT Thanks are due the Director, National Sugar Institute, for providing necessary facilities. One of the authors (Poonam Nigam) is thankful to the Ministry o f F o o d and Civil Supplies (Department of Food), Government of India, for the award o f a Junior Research Fellowship.

REFERENCES Berk, A. A. & Schroeder, W. C. (1942). Ind. Eng. Chem. A n a l (14th edn), 456. Cannal, E. & Moo-Young, M. (1980). Process Biochem. 15, 2.

Enzyme production by Basidiomycetes

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Deschamps, F. & Huet, M. C. (1984). Biotechnology Letters, 6(1), 55-60. Eriksson, K. E. & Larson, K. (1975). Biotech. Bioeng., 17, 327. Han, Y. W. & Anderson, A. W. (1975). Appl. Microbiol., 30, 930. Han, Y. W., Grant, C. A., Anderson, A. W. & Yu, P. L. (1976). FoodStuffs, 48(17), 17. Harseltine, L. W. (1977). Process Biochem., 12(6), 24. Kirk, T. K. & Keiman, A. (1965). Phytopathology, 55, 739. Kirk, T. K. & Chang, H. M. (1975). Holzforschung, 29, 56. Lowry, O. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. L. (1951). J. Biol. Chem., 193, 265. Mandels, M. & Weber, J. (1969). Adv. Chem. Set., 95, 391. Methods in enzymology (1955). Vol. II. Academic Press Inc., New York, 704. Miller, G. L. (1959). Anal, Chem., 31,426. Nigam, P. & Prabhu, K. A. (1985a). Agricultural Wastes, 12(4), 273. Nigam, P. & Prabhu, K. A. (1985b). Folia Microbiol.--Communicated. Nigam, P. & Prabhu, K. A. (1985c). International Sugar Journal, 87(1033), 17. Toyama, N. & Ogawa, K. (1972). Proc. 4th Int. Ferment. Symp. (Tervi, G. (Ed)), 743. Toyama, N. & Ogawa, K. (1978). In: Proc. Int. Symp. on Bioconversion of Cellulosic Substances into Energy. Chemicals and Microbial Proteins (Ghose, T. K. Ghose (Ed), Vol. III. Delhi, India, 305. Toyama, N. & Ogawa, K. (1977). In: Proc. Bioconversion Symp., liT, Delhi, 375.