Isolation of new anaerobic, thermophilic and cellulolytic bacteria-JT strains and their cellulase production

[ j . Ferment. Technol., Vol. 66, No. 4, 389-395. 1988]

Isolation of New Anaerobic, Thermophilic and Cellulolytic Bacteria-JT Strains and Their Cellulase Production FENGXIE JIN9 a n d KIYOSHI TODA*

Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Six microbial strains (JT) of endospore-forming, anaerobic, thermophilic and cellulolytic bacteria were isolated from camel feces, compost, soil and hot spring water in Japan. These strains are gram negative and classified as the genus Clostridium. Strains JT3-1, JT3-2 and JT3-3 can digest starch. All of the strains produce a high activity of extracellular cellulases in cellobiose and cellulose media. StrainJT1 produced 1.36 units/ml ofCMCase (endo-fl-l,4-D-glucanase, EC 3.2.1.4), 66.2 units/ml of fl-glucosidase (EC 3.2.1.21) and 39.9 units/ml of fl-xylosidase (EC 3.2.1.37) in 1% cellobiose medium. Strain JT3-3 produced 1.87 units/ml of CMCase, 166.3 units/ml offl-glucosidase and 23.6 units/ml offl-xylosidase in 1% cellulose medium.

Cellulose is the most abundant natural biopolymer and a potentially important resource for the production of useful materials such as fuels and chemicals. T h e utilization of cellulose as a chemical and energy, source has been extensively reviewed.s) However, studies on the microbial degradation of cellulosic material have been mainly confined to mesophilic microorganisms. Evidence suggests that cellulolysis is very active in thermophiles.2-4) In this study, anaerobic, thermophilic and cellulolytic bacteria were isolated, and some characteristics of the isolated bacteria as well as cellulase production were investigated.

taxononic features of the bacterial strains. Isolation method The strains of J T were isolated from camel feces, compost, soil and hot spring water by enrichment culture. Enrichment cultures were initiated by inoculating 0.2-0.6 g of source materials into a test tube (1.8× 18 cm) containing about 15 ml of cellulose medium. After purging with oxygen-free N2 gas, the test tubes were tightly plugged with butyl rubber stoppers and then incubated at 55-60°C for 6-10d. 0.1-0.3 ml of the culture was then transferred to 10-15 ml of cellulose medium in test tubes, and again anaerobically cultured at 55-60°C for 4-8 d. This operation was carried out several times. 0.1-0.2 ml of the culture was inoculated at 60°C into 10 ml of cellulose medium solution containing 2% agar in a test tube. After purging the oxygen in the test tube using N, gas, the tube was horizontally Materials and Methods rolled 8) with cooling to form a thin agar layer on the Media CM3 medium 5) was used for the isolation inner wall. The rolled tubes were incubated at 55of the bacterial strains and cellulase production with 60°C in a horizontal position. After 4--7 d, the colonies some modifications. It contained (per liter) : with clear zones of cellulose digestion were transferred K~HPO4, 4.4g; urea, 2 g; MgCI2.6H~O, 0.5g; individually into the cellulose medium under a flow CaCI~.2H20, 0.05 g; cysteine monohydrochloride of N2 gas. monohydrate, 1.0g; 3-(n-morpholino)propanesulfonic Characteristics of the organisms The gram acid (MOPS), 20 g; yeast extract, 6.0 g; and cellulose reaction, utilization of carbohydrates, fermentation or cellobiose, 10 g. products from carbohydrates and other taxononic PY medium7) was used for determination of the features of the test microorganisms were determined by the methods described in Bergey's Manual ot * Corresponding author Systematic Bacteriology.7) § Present address: Dalian College of Light Industry, G + C c o n t e n t o f DNA The G + C mol % content of DNA in the bacterial cells was determined Dalian, China

390

JIN and TODA

by the method of Saito and Miura.S) Analysis of enzynae activity CM-cellulase (CMCase, endo-fl-l,4-D-glucanase, EC 3.2.1.4) was determined according to the method of Creuzet and Frixon9~ with some modifications: 10 ml of 1% (w/v) CMC (Daiichi Kogyo Seiyaku, WS-C) in 0.05 M citrate buffer (pH 6.5) and 9 ml of distilled water were added to the BL Viscometer (Tokyo Keiki in Japan) and incubated at 60°C for 10 rain. Then 0.I-1 ml of the culture sample was added (the final volume was 20ml, the CMC was 0.5%), and the reduction in viscosity was measured. One unit of enzyme activity was defined as the amount of enzyme decreasing one cp of viscosity per minute in 20 ml of 0.5% CMC solution. fl-Glucosidase (EC 3.2.1.21) was determined by the method of Herr et al. lo) using p-nitrophenyl-/~-Dglucose (PNPG) (Nakarai Chemicals) as a suhstrate with some modifications. The assay mixture contained 0.9 ml of 2 mM PNPG in 0.05 M citrate buffer (pH6.5) and 0.1 ml of the diluted sample. After incubation at 60°C for 10-40 rain, the reaction was stopped by the addition of 2.0 ml of I M Na~CO~ solution. Based on the absorbancy at 410nm of p-nitrophenol produced from the reaction, the activity was calculated. One unit offl-glueosidase was defined as the amount of enzyme liberating 1 nmol of p-nitrophenol per minute under the assay conditions. ~-Xylosidase was determined using p-nitrophenylfl-D-xylose as the substrate. The method was the same as that for/~-glucosidase. Glucose concentration The concentration of glucose was determined with the Glucostat Reagent (Sigma Chemicals).

Results and Discussion Taxononic

features of the JT strains

Six strains o f endospore-forming, a n a e r o b i c , t h e r m o p h i l i c a n d cellulolytic b a c t e r i a were

[j. Ferment. Technol.,

isolated. T h e characteristics of these strains, n a m e d J T , a r e shown in T a b l e 1. S t r a i n J T 1 was isolated from c a m e l feces at the T o b u Z o o l o g i c a l G a r d e n ; J T 2 , from soil at T o k y o U n i v e r s i t y ; J T 3 - 1 , J T 3 - 2 a n d J ~ 3-3 from cattle feces a n d grass compost at the Tanashi Farm of Tokyo University and JT5 from hot spring at K i n u g a w a , J a p a n . A l l o f the strains were g r a m negative, r o d cells (0.4-0.7 b y 2.0-6.5 # m ) . T h e velocities o f f o r m i n g spores for J T 2 , J T 3 - 1 , J T 3 - 2 a n d J T 3 - 3 were faster t h a n those o f J T 1 a n d J T 5 . Strains J T 2 , J T 3 - 1 , J T 3 - 2 a n d J T 3 - 3 p r o d u c e d spores after c u l t u r i n g for 15-20 h. T h e s e spores ( a b o u t 1.2 b y 1 . 6 # m ) were formed in the t e r m i n a l s o f the cells. T h e p H g r o w t h r a n g e was 5.5-8.0 ( T a b l e 1). T h e o p t i m u m p H value for strains J T 1 a n d J T 2 was 7.3, while for strains J T 3 - 1 , J T 3 - 2 , J T 3 - 3 a n d J T 5 it was 6.5. T h e p H profile for the g r o w t h rate o f s t r a i n J T 1 is shown in Fig. 1. T h e r a n g e o f g r o w t h t e m p e r a t u r e was 47-74°C ( T a b l e 1). T h e o p t i m u m tempera t u r e for strains J T 3 - 1 , J T 3 - 2 a n d J T 5 was 60°C; for strain J T 2 , 55°C; a n d 60-65°C for J T 3 - 3 . The optimum growth tempera t u r e for s t r a i n J T 1 was 55-60°C as shown in Fig. 2. T h e G + C tool % contents o f D N A for the strains were; J T 1 , 3 7 . 7 % ; J T 2 , 3 7 . 8 % ; JT3-1, 36.9%; JT3-2, 36.7%; JT3-3, 3 7 . 2 % ; a n d J T 5 , 37.0o/o. All o f the strains could utilize glucose, fructose, cellobiose, cellulose a n d most other

Table 1. Characteristics of the JT strains.

Source of isolation pH range for growth Temperature range for growth (°C) Gram reaction H2 and CO2 production HaS production Ethanol production Acetic acid production Butyric acid production

JT 1

JT2

camel feces

soil

6.0-8. 0 50-68 . + ÷ ÷ ÷ +

5. 5-8. 0 47-63 . . + + ÷ + ÷

JT3-1

JT3-2

JT3-3

compost of cattle feces and grass 5. 5-8. 0 50-70 . + + ÷ + ÷

5. 5-8. 0 50-70 . . + + ÷ ÷ +

5. 5-8. 0 50-74 ÷ ÷ ÷ ÷ ÷

JT5 hotspring 5. 5-8. 0 50-70 + + ÷ + +

V o l . 66, 1988]

New Thermophilic, Cellulolytic Anaerobes

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£

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-

80 ..c

60

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S w

20

0

20 X I 5,0

L

,

I

6,0

7,0 pH

8,0

F i g . 1. E f f e c t o f p H o n g r o w t h r a t e . at 60°C in 1% cellobiose medium) Table

2.

9.0

I

30

(strain JT1,

40

50 60 Temperoture (%)

JT 2

L-Arabinose

-

D-Glucose

w

D-Fructose

w

D-Galactose

w

L-Rhamnose

-

D-Ribose

.

Cellobiose

w

J T 3-1

JT 3-2

w

w

w

w

w

w

+

w

+

w

+

w

+

+

w

+

--

w

w

w

-

-

w

-

w

+

w

w

Lactose

w

w

-

w

w

w

w

w

.

.

JT 3-3

.

JT 5

w

Maltose

w

+

+

w

w

w

Mannose

w

w

w

w

+

w

Sucrose

-

-

w

-

w

-

Raffinose

.

Trehalose

--

w

Melezitose

w

.

Sorbitol

-

-

-

Salicin

w

-

w

Inulin

w

w

Soluble starch

-

Glycogen

.

80

F i g . 2. E f f e c t o f t e m p e r a t u r e on growth rate. ( s t r a i n J T 1 , a t p H 7.0 i n 1 % c e l l o b i o s e m e d i u m )

Acid production from carbohydrate fermentation. JT 1

70

.

.

w w

w

w

w

w

-

-

w

-

w

-

-

-

-

,

,

,

-

w

w

-

w

w

-

Amygdalin

--

w

w

w

+

--

Cellulose

w

w

w

w

w

w

.

w .

.

.

Symbols: +, reaction positive; --, reaction negative; w, weak reaction; ., weak reaction in culture medium. The media were PY+ 1% carbohydrate and fermentat i o n f o r 4 d. ( P o s i t i v e r e a c t i o n l i s t e d f o r c a r b o h y d r a t e f e r m e n t a t i o n r e p r e s e n t a p H b e l o w 5.5 a n d a d e c r e a s e i n p H o f a t l e a s t 0 . 5 p H u n i t s b e l o w t h e c o n t r o l laY b a s a l m e d i u m c u l t u r e . W e a k a c i d p r o d u c t i o n r e p r e s e n t s a p i t o f 5 . 5 - 5 . 9 a n d a t l e a s t 0.3 p H u n i t s b e l o w t h e P Y c u l t u r e c o n t r o l . A p H o f 5.9 o r a b o v e , o r w i t h i n 0.3 p H u n i t s o f t h e P Y c u l t u r e c o n t r o l p H , is c o n s i d e r e d n e g a t i v e . )

JIN and TODA

392

carbohydrates, although there were some differences with respect to producing acids (Table 2). Strains J T 3 - 1 , J T 3 - 2 and J T 3 - 3 could hydrolyze soluble starch. All strains produced carbon dioxide, hydrogen, ethanol, lactic acid, acetic acid and butyric acid in cellulose and ceUobiose fermentation (Table 1). For example, these strains produced 0.02-0.14% (v/v) acetic acid, 0.07-0.36% (v/v) n-butyric acid and a trace amount of lactic acid in P Y + I % cellobiose medium (Table 3). All strains produced hydrogen sulfide. It is presumed from the above observation that these bacteria are members of the genus

[J. Ferment. Technol., /3-×yiosidase

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B-Glucosidose

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CMCose

Clostridium. Cellulase medium

production

in

ceHobiose

All six strains produced extracellular cellulase in cellobiose medium, although to different degrees (Fig. 3). Strains JT1, J T 3 - 1 and J T 3 - 3 exhibited somewhat higher extracellular cellulase activity among the six strains. Strain JT1 produced 1.36 units/ml of CMCase, 66.2 units/ml of /~glucosidase and 39.9 units/ml of t3-xylosidase in 1% cellobiose medium. High production of ~-xylosidase was a special characteristic of strain JT1. Strain J T 3 - 3 demonstrated high CMCase and /3-glucosidase activities, but the production of/3-xylosidase was low. Strain J T 3 - 1 had high activity for the three cellulases. High cellulase production was observed after the log growth phase for all strains, i.e., after cultivation for about 40-60h. For instance, the results of a batch culture of

o

JTI

JT2 JT3-1 01"3-2 JT3-3 JTS

Fig. 3. Cellulase production in cellobiose medium. (initial pH, 7.06; temperature, 55-60°C; batch culture in 1% cellobiosemedium for 2-3 d) strain JT1 are presented in Fig. 4. The time period of the log growth phase of strain JT1 was about 14-40 h, with the activity of CMCase reaching a maximum at about 40 h. The activities of ~-glucosidase and /~-xylosidase reached maximums at about 60 h. The concentration of glucose in the cuhure was generally 0.1-0.5 g]l with the doubling time being about 1.8-2.3 h for all strains in cellobiose medium. Cellulase production in cellulose medium All six strains produced extracellular

cellulase in the cellulose medium, although

Table 3. Acidsproducts from cellobiosefermentation (batch culture in 1% cellobiosemedium). Acetic acid JT 1 JT 2 JT3-1 JT 3-2 JT 3-3 JT5

0. 09 0. 03 0.04 0. 03 0. 14 0.02

Propionic acid -------

/so-Butyric acid 0. 05 r 0. 10 --r

Symbole: --, not produced; r, weakly recognized.

n-Butyric acid 0. 07 0. 36 0.36 0. 30 0. 15 0. 19

V a l e r i c Lactic acid acid -----

r r r r

--

0. 08

--

--

Vol. 66, 1988]

New Thermophilic, CellulolyticAnaerobes

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0 I0 20 5040 50 6 0 7 0 8 0 9 0

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Fermentation time (h)

Fig. 4. Batch culture of strain JT1 in cellobiose medium. (initial pH, 7.06; temperature, 5560°(]; 1% cellobiose medium I , (]M(]ase; ~, fl-glucosidase; &, /~-xylosidase; El, dry cell; ~, pH; ©, cellobiose)

with differing abilities. A comparison of cellulase production in cellulose medium is shown in Fig. 5. CMCase production for all strains was similar (about 1.80-1.97 units/ml). However, the production of flglucosidase was the highest in strain JT3-3 among the six strains. The activity of fl-glucosidase of strain .]T3-3 was 166.3 40

8-Xylosidase

T: B-Glu~sidase

150

-~1oo 50

C M ~

r:

JT1

JT2

JT3-1 J T 3 - 2 JT3-3 JT5

Fig. 5. Cellulase production in cellulose medium. (batch culture in 1% cellulose medium for 3-5 d; initial pH, 7.06; temperature, 55--60°C)

/

50 -~ 40 "~

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-~~ / oA /

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~

. . . . . ~, , o 0 20 4 0 6 0 80 100120140160180200 Fermentation time (h)

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Fig. 6. Batch culture of J T I in cellulose medium. (initial pH; 7.05; temperature, 55-60°C; in 1% cellulose medium. I , CMcase; 0, fl-glucosidase; &, fl-xylosidase; A, pH; ©, reducing sugar)

units/ml, a value about 4-6 times greater than that for the others. Comparing the productions of cellulases for all strains in cellulose medium with those in cellobiose medium, the production of CMCase was increased, but those of fl-glucosidase and fl-xylosidase were decreased, except strain JT3-3. StrainJT3-3 produced 1.87 units/ml of CMCase, 166.3 units/ml of fl-glucosidase and 23.6 units/ml of fl-xylosidase. The production of cellulase by strain JT3-3 in cellulose medium was much higher than that in cellobiose medium. The characteristics of batch culture cellulase production for the six strains in cellulose medium were similar; the example of strain JT1 is shown in Fig. 6. A high concentration of cellulases was observed after culturing for 80-120 h, i.e., after digestion of more than 800/0 of the cellulose (in 1% cellulose medium). The concentration of reducing sugar (as glucose) produced in the cellulose medium during fermentation was below 0.6-1.0g/l for all strains, with more than 50-80% of the total reducing sugar being glucose. Strains JT1, JT3-1, JT3-3 and JT5 showed higher rates of cellulose digestion. When cultured in a 1% cellulose medium, 80-900//0 of the cellulose was digested after 3-5 d. Comparison of JT strains with other cellulolytic and thermophilic strains of Clostridium genus When the cellulase

394

JIN and TODA

[J. Ferment. Technol.,

Table 4. Comparison of taxonomic features among the Glostridium genus. Strains JT

C thermocellum

C. stercorarium

G. thermolacticum

+ _j

+ _*

+ +

weak +

+ ±

-

_ _

+ +

Acid production Lactic acid Butyric acid

+ +

+ -

+

~

H~S production

+

-

G+C content of DNA

36, 7

38. 0

39.0

40.9

(%)

--37.8

--39.0

Fermentation of Cellulose Glucose Fructose Starch

* + in our-experiment.

-42.3

-- . . . . . . . . . . . . . . . . . . .

production of strain J T 3 - 3 was compared with that of Clostridium thermocellum A T C C 27405 in a batch culture using 1% cellulose medium, CMCase production was similar, however, the fl-glucosidase and fl-xylosidase productions were about 50% more than that of C. thermocellum. T h e ability to hydrolyze cellulose was estimated by determining the reduction of the cellulose weight in a mixture consisting of 15 g of cellulose powder and 500 ml of cellfree cultured medium, after incubating at 60°C for 3 5 h with shaking. Cellulose hydrolysis by the strain J T 3 - 3 culture fikrate was 10% higher than that by C. thermocellum A T C C 27405 culture filtrate. J T strains are different from other anaerobic, thermophilic and cellulolytic clostridia presently known. A comparison of the taxonomic features T) of the thermophilic cellulolytic clostridia is shown in T a b l e 4. Clostridium thermocellum A T C C 27405~,~,11) cannot utilize fructose, digest soluble starch or xylan, and does not produce butyric acid or hydrogen sulfate. T h e G + C mol% content of D N A for most species of C. thermocellum is 38--39%; the J T strains can utilize fructose, hydrolyze xylan, and produce butyric acid and hydrogen sulfate, and the G + C content is 36.7-37.8%. In addition

to strain J T 3 - 1 , J T 3 - 2 and J T 3 - 3 can also digest soluble starch. It is clear that the J T strains are different from C. thermocellurn. C. stercorarium12) cannot utilize fructose or soluble starch, and does not produee butyric acid or hydrogen sulfate. The G + C content is 39 %. These properties are clearly different from those of the J T strains. C. thermolacticumla) only slightly digested cellulose, and produced a lot of lactic acid, but did not produce butyric acid or hydrogen sulfate. T h e G + C mol % content of the D N A is 40.9-42.3%, a value which is about 4 % greater than for J T strains. T a y a et alA 4) reported that strain T x was an anaerobic, thermophilic and cellulolytic bacterium classified in the Clostridium genus. Strain T x is g r a m positive, and cannot produce butyric acid or hydrogen sulfate. For the above reasons, it is suggested that the J T strains are a new species in the Clostridium genus having the ability to produce ceUulase and digest cellulose. References

1) Wilke, C.R." Biotechnol. Bioeng. Symposium No. 5, p. 134 (1975). 2) Ng, T.K., Weimer, P.J., Zeikus, J.G." Arch. Mierobiol., 114, 1 (1977). 3) Saikl, T.: Biosdence & Industry in Japan, 44 (7),

Vol. 66, 1988]

New Thermophilic, Cellulolytic Anaerobes

55 (1986). 4) Jurgen, W., Michael, D.: Appl. Microbiol. Bioterhnol., 20, 59 (1984). 5) Garcia-Martinez, D.V., Shinmyo, A., Madia, A., Demain, A.L.: Appl. Microbiol. Biotechnol., 9, 189 (1980). 6) Hungate, R.E.: Methods in Microbiology, (Norris, J. R., Ribbons, D. W.), Vol. 3b, pp. 117-132, Academic Press, London, New York (1969). 7) Murray, R. G.E.: Bergey's Manual of Systematic Bacteriology, Vol. 2, p. 1142, Williams & Wilkim, Baltimore (1986). 8) Saito, H., Miura, K.: Bioehem. Biophys. Acta, 72, 619 (1963).

395

9) Creuzet, N., Frixon, C.: Biochime, 65, 149 (1983). 10) Herr, D., Baumer, F., Dellweg, H.: Eur. J. Appl. Mierobiol. Bioteehnol., 5, 29 (1978). 11) Mcbee, R.H.: J. Bact., 67, 505 (1954). 12) Madden, R.H.: Int. J. Syst. Bacteriol., 33, 837

(1983). 13) Ruyet, P.L., Duborguier, H.C., Albagnac, G., Premier, G.: System. Appl. Microbiol., 26, 196 (1985). 14) Taya, M., Suzuki, Y., Kobayashl, T.: jr. Ferment. Technol., 62, 229 (1984). (Received December 24, 1987)