Applied

Appl Microbiol Biotechnol (1986) 24:454--458

Microbiology Biotechnology © Springer-Verlag 1986

Production of cellulases from alkali-treated bagasse in Trichoderma reesei* Mikio Kawamori ~, Yasushi Morikawa 2, Yutaka Ado 3, and Seigo Takasawa 1 1 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Asahimachi, Machida-shi, Tokyo 194, Japan 2 Research and Development Division, Kyowa Hakko Kogyo Co. Ltd., Otemachi, Chiyoda-ku, Tokyo 100, Japan 3 Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Kyowamachi, Hofu-shi, Yamaguchi 747, Japan

Summary. Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X31, in which strong cellulase activity is inducible by L-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.

Introduction There is an increasing interest in the bioconversion of cellulosic biomass to produce chemical and liquid fuels such as ethanol, thereby conserving petroleum. The main technical problem of this process is the cost of enzyme production. Many investigators, including our own group, have tried to improve productivity of enzyme by screening for improved mutant strains of T. reesei, the most potent cellulase producer amongst microorganisms, and by optimizing fermentation conditions (Montenecourt and Eveleigh 1978; Gallo et al. 1978; Labudovfi and Farkfis 1983; Morikawa et al. 1985 ; Kawamori et al. 1985). From the point of view of economical cellulase production, it is de*

Production of Ethanol from Biomasses Part IV. This work was supported by the funds of the Research Association for Petroleum Alternative Development

Offprint requests to: M. Kawamori

sirable to use inexpensive materials in cellulase fermentation by T. reesei. Cellulosic biomasses such as bagasse, rice straw, wheat straw and sawdust are promising for this purpose. There has been some research on cellulase production from cellulosic biomasses (Peitersen 1975). A problem is presented by the lignified structure of cellulosic biomasses making them unsuitable for cellulase production as raw materials. Pretreatment of cellulosic biomasses to achieve effective saccharification by cellulase has been investigated (Millett et al. 1976; Toyama 1976; Ghedalia and Miron 1981); of all the methods tested, treatment with alkali has been found to be one of the most convenient and economical methods for saccharification. Many mutant strains of T. reesei were examined for cellulase production on these alkalitreated cellulosic biomasses. The mutant strains PC-3-7 and X-31, which had effective cellulase activity inducible by L-sorbose (Kawamori et al. 1986), were found to be capable of this. In this report, growth and cellulase production of T. reesei mutants, mainly on alkali-treated bagasse, are described.

Materials and methods Microorganisms used. The experiments were carried out with the mutants of T. reesei. The genealogy of these mutants and the screening procedure were described previously (Kawarnori et al. 1986). Treatment of cellulosic biomasses with alkali. Sugarcane bagasse was mechanically milled to 30--80 mesh with Willey mill and treated with various concentrations of NaOH for 15 min at 120°C for delignification. Culture conditions. The production of cellulase occurred in 300-ml Erlenmeyer flask culture containing 50 ml of the medium; culture time was 7--8 days at 28°C on a rotary shaker

455

M. Kawamori et al.: Cellulases from alkali-treated bagasse Table 1. Assay conditions of cellulase activities Termination

Product

45 ° C 60 min

Boiling

Reducing sugar

Enzyme soln. 0.05 ml 0.1 M AB (pH 5.0) 0.45 ml 1% C M C - N a 0.5 ml

45°C 30 min

Boiling

Reducing sugar

Enzyme soln. 0.02 ml 0.05 M AB (pH 5.0) 1 ml p - N P G 2 ~tmoles

45°C 10 min

Addition of 2 ml of 1 M Na2CO3

p-NP

Enzyme

Reaction mixture

Temperature and time

Avicelase

Enzyme soln. 1 ml 0.1 M AB (pH 5.0) 4 ml Avicel SF 150 mg

CMCase

fl-Glucosidase

AB: Acetate buffer, p - N P G : p-Nitrophenyl-fl-D-glucopyranoside, p-NP: p-Nitrophenol

at 220 rpm. The media were composed of various concentrations of carbon sources such as Avicel and alkali-treated bagasse and others, described previously (Morikawa et al. 1985; Kawamori et al. 1985). The culture broth was centrifuged and the supernatants were analysed for cellulolytic activities,

Analytical method. Enzyme activities were determined by the modified methods of Mandels et al. (1976) for CMCase, Bergham et al. (1973) for Avicelase, and Hagerdal et al. (1979) for fl-glucosidase. Assay conditions of the enzymes are summarized in Table 1. Reducing sugars were determined by the dinitrosalicylic acid method (Miller 1969). The enzyme unit is expressed in each case as the amount of enzyme required to produce 1 ~tmole of reducing sugar (glucose or glucose equivalent) or p-nitrophenol per min under the given conditions. Protein was determined by the Lowry's method (1951). Mycelial weight was determined by the method described previously (Morikawa et al. 1985).

trol strain and PC-3-7 as a representative effective cellulase producer from alkali-treated bagasse.

Effect of conditions for alkali treatment of bagasse on CMCase production The CMCase productivities of T. reesei KY 746 and PC-3-7 were examined on the bagasses treated with various concentrations of alkali (Table 3). The CMCase activity in KY 746 remained more or less constant, regardless of the conditions of alkali treatment of bagasse. The CMCase activTable 2. CMCase production from Avicel and alkali-treated

bagasse and CMCase induction by L-sorbose in Trichoderma reesei mutants

Results and discussion

Strain

Selection of strain for CMCase production from alkali-treated bagasse The CMCase productivities from alkali-treated (0.3 N NaOH) bagasse by various mutants of T. reesei were examined and compared with those from Avicel and CMCase induction by L-sorbose (Table 2). Most of the strains showed high activities of CMCase in Avicel medium, but their CMCase productivity in alkali-treated bagasse medium was low. PC-l-4, PC-3-7, X-30 and X-31, which were selected as mutant strains with high CMCase inducibility by L-sorbose (Kawamori et al. 1986), showed almost the same CMCase activities from alkali-treated bagasse as those from Avicel. There was some correlation between CMCase inducibility by L-sorbose and CMCase productivity from alkali-treated bagasse. In the following experiments, we used KY 746 as a con-

CMCase production" (U/ml)

CMCase induction b ( U / r a g of myc.)

Alkali-treated bagasse

Avicel

KY 746 MCG-77 KDR-11 KDD-10 KDR-27 KDG-12

10 13 15 18 15 9

30 40 44 38 44 38

0.03 0.03 0.03 0.15 trace 0.02

X-30 X-31 PC-l-4 PC-3-7

32 36 31 32

33 38 36 38

0.50 0.60 0.55 0.65

a

b

CMCase production was carried out for 7 days at 28°C by the flask culture. Alkali-treated bagasse and Avicel were used as carbon sources at 1% concentration. The data were obtained from the broth of the 7th day of the culture and usually consistent with the maximum activities Data of CMCase induction were derived from a previous paper (Kawamori et al. 1986)

456

M. Kawamori et al.: Cellulases from alkali-treated bagasse

Table 3. Effect of concentration of alkali for pretreatment o f bagasse on CMCase production by T. reesei mutants a Treatment with alkali

None 0.1N 0.3 N 1.0N 3.0 N

NaOH NaOH NaOH NaOH

Saccharification b (%)

6.6 40 56 50 36

CMCase (U/ml) KY 746

PC-3-7

4.0 9.0 10 10 10

11 28 32 33 30

CMCase production was carried out for 7 days at 28 ° C by the flask culture. The bagasses treated with various concentrations of NaOH for 15 min at 120°C were used as carbon sources at 1% concentration The sacchariflcation was carried out for 4 h at 45 ° C. Each substrate and the cellulases of T. reesei PC-3-7 were used at 5% and 50 U / m l (CMCase), respectively in 0.1 M acetate buffer (pH 5.0). The quantity of reducing sugars was determined with Nihon Bunko Trirotor III HPLC apparatus equipped with refractive index detector, and Shodex Ionpak C-811 column (Showa Denko Co. Ltd.) and 0.1% H3PO4 as an eluent were used

ity in PC-3-7, however, increased after treatment of bagasse with alkali; treatment with 0.3--3.0 N NaOH brought about the highest CMCase activity. Treatment with 0.3 N NaOH brought about the most effective saccharification of bagasse, while a higher concentration did not improve saccharification. The mutants which had enhanced CMCase inducibility by L-sorbose showed higher CMCase productivity from alkali-treated bagasse than other mutants, regardless of the condition of alkali treatment. Because alkali-treated bagasse was an easily saccharified substrate in comparison with raw bagasse and Avicel (saccharification was 16% in the conditions of the experiments shown in Table 3), CMCase productivity from easily saccharified substrate is much higher in PC-3-7 than in KY 746. Table 4. CMCase production from easily saccharified substrates by T. reesei mutants a Carbon source

CMCase production from other easily saccharified substra tes CMCase production from easily saccharified substrates other than alkali-treated bagasse was examined in T. reesei KY 746 and PC-3-7 (Table 4). CMCase productivity by KY 746 from other easily saccharified substrates was as low as from alkali-treated bagasse; CMCase productivity of PC3-7 was higher than that of KY 746. The results obtained for PC-l-4, X-30 and X-31 were the same as for PC-3-7. Consequently, the mutants with strong inducibility of CMCase by L-sorbose were advantageous to the CMCase production from easily saccharified substrates. In a previous paper (Kawamori et al. 1986), it was suggested that the cell wall of PC-3-7 might be different from that of KY 746. This alteration of cell wall might not only be advantageous for the induction of CMCase by L-sorbose but also for the production of CMCase from easily saccharified substrates.

Effect of the concentration of alkali-treated bagasse on CMCase production The time course of CMCase production and mycelial weight of T. reesei KY 746 and PC-3-7 in various concentrations of alkali-treated (0.3 N NaOH) bagasse is shown in Fig. 1. Although the mycelial weight in KY 746 increased with increase in NaOH concentration (1--3%), CMCase

80 6O

~' 20

a

b

I

0

PC-3-7

10 16 8 1

32 35 16 4

CMCase production was carried out for 7 days at 28° C by the flask culture. Carbon sources were used at 1% concentration Treatment with alkali was carried out by 0.3 N NaOH for 15 min at 120°C

..z~~ ' ' ~ ' ~ ,.C>--C>--0

CMCase (U/ml) KY 746

PC-3-7

40

-~

Alkali-treated bagasse b Alkali-treated rice straw b Walseth's cellulose CMC- Na

KY 746

l

I

t

!

l

4

'.

E

2 % u

F //'q.. -.'o I

0

2

4

t

v

i "~'~

6 8 2 Incubation time (day)

I'~

I 4

I 6

I

I 8

Fig. 1. Effect of the concentration of alkali-treated bagasse on the CMCase production in Trichoderma reesei K Y 746 and PC-3-7. CMCase production was carried out for 8 days at 28 o C by the flask culture. The alkali-treated bagasse obtained by 0.3N NaOH for 15rain at 120°C was used as a carbon source at 1% (O), 2% (O), 3% (9

M. Kawamori et al.: Cellulases from alkali-treated bagasse

457

Table 5. Cellulase production from Avicel and alkali-treated bagasse by 5-1 jar fermentor culture in T. reesei KY 746, PC-3-7 and X-31 a Strain

Carbon source

CMCase (U/ml)

Avicelase (U/ml)

fl-Glueosidase (U/ml)

Protein (mg/ml)

KY 746

Avicel 6% Alkali-treated bagasse 4%

120 20

10 4.4

3.5 0.7

14 4.5

PC-3-7

Avicel 6% Alkali-treated bagasse 4%

160 92

18 16

5.7 2.1

23 24

X-31

Avicel 6% Alkali-treated bagasse 4%

165 100

20 17

3.4 1.3

23 12

a

A 5-1jar fermentor (working volume: 2.5 1) was inoculated with 500 ml of a 4-day flask culture of the fungus, and cultured for 7 days at 28 ° C. The pH was maintained at 4.0 by addition of 1 N NH4OH, and the air flow and agitation frequency were 1 VVM and 450 rpm, respectively

activity did not show much increase. In the case of PC-3-7, however, both CMCase and mycelial weight increased with increasing the concentration of alkali-treated bagasse.. The growth rate of PC-3-7 was lower than that of KY 746 in the alkali-treated bagasse medium. PC-3-7 thus seems to utilize alkali-treated bagasse effectively as an inducer for the CMCase production by lowering the growth rate.

Cellulase production from alkali-treated bagasse in 5-1jar fermentor culture In Table 5, cellulase production by T. reesei KY 746, PC-3-7 and X-31 from 4% alkali-treated (0.3 N NaOH) bagasse in 5-1 jar fermentor culture is compared with that from 6% Avicel. CMCase productivity from alkali-treated bagasse reached about two thirds that from Avicel in PC-3-7 and X-31. When alkali-treated bagasse of 4% or more was used, CMCase activity was the same in PC3-7 and X-31. Cellulases consist of endo-fl-l,4glucanase (EC 3.2.1.4), exo-fl-l,4-glucanase (EC 3.2.1.91) and fl-glucosidase (EC 3.2.1.21). In our experiments, CMCase was used as an enzyme showing endo-fl-l,4-glucanase activity, and Avicelase as an enzyme showing exo-fl-l,4-glucanase activity. For the effective saccharification of cellulose to glucose, these enzyme activities are required in an appropriate ratio. The activity of flglucosidase obtained by culture with alkalitreated bagasse was not sufficiently high, even in PC-3-7 and X-31 compared with those of CMCase and Avicelase. Thus although PC-3-7 and X-31 appear to be promising strains for cellulase production with alkali-treated bagasse, even in large-scale culture, some problems remain with

regard to culture conditions required in order to improve the ratio of cellulase components. Acknowledgement. We gratefully acknowledge the skillful technical assistance of Miss Hiroko Nodake.

References Berghem LER and Pettersson LG (1973) The mechanism of enzymatic cellulose degradation: Purification of a cellulolytic enzyme from Trichoderma viride active on highly ordered cellulose. Eur J Biochem 37:21--30 Gallo BJ, Andreotti R, Rhoche C, Ryu D, Mandels M (1978) Cellulase production by a new mutant strain of Trichoderma reesei MCG-77. Biotechnol Bioeng Symp No 8:89--101 Ghedalia BD, Miron J (1981) The effect of combined chemical and enzyme treatments on the saccharification and in vitro digestion rate of wheat straw. Biotechnol Bioeng 23:823--831 Hagerdal B, Harris H, Pye EK (1979) Association offl-glucosidase with intact cells of Thermoactinomyces. Biotechnol Bioeng 21:345--355 Kawamori M, Morikawa Y, Shinsha Y, Takayama K, Takasawa S (1985) Preparation of mutants resistant to catabolite repression of Trichoderma reesei. Agric Biol Chem 49:2875--2879 Kawamori M, Morikawa Y, Takasawa S (1986) Induction and production of cellulases by L-sorbose in Trichoderma reesei. Appl Microbiol Biotechnol (in press) Labudovfi I, Fark~s V (1983) Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase. FEMS Microbiology Letters 20:211--215 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:265--275 Mandels M, Andreotti R, Roche C (1976) Measurement of saccharifing cellulase. Biotechnol Bioeng Symp No. 6: 21 --33 Miller GL (1969) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426--428 Millett MA, Baker AJ, Satter LD (1976) Physical and chemical pretreatments for enhancing cellulose saccharification. Biotechnol Bioeng Symp No. 6:125--153

458 Montenecourt BS, Eveleigh DE (1978) Hypercellulolytic mutants and their role in saccharification. Proc 2nd Ann Symp on fuels from biomass. 613--625 Morikawa Y, Kawamori M, Ado Y, Shinsha Y, Oda F, Takasawa S (1985) Improvement of cellulase production in Trichoderma reesei. Agric Biol Chem 49:1869--1871 Peitersen N (1975) Production of cellulase and protein from barley straw by Trichoderma viride. Biotechnol Bioeng 27:361--374

M. Kawamori et al.: Cellulases from alkali-treated bagasse Toyama N (1976) Feasibility of sugar production from agricultural and urban cellulosic wastes with Trichoderma viride cellulase. Biotechnol Bioeng Symp No 6:207--219

Received September 12, 1985/Revised January 18, 1986