Applied Mwrobiology Biotechnology
Appl Microbiol Biotechnol (1988) 29:474--479
© Springer-Verlag 1988
Isolation and characterization of an extremely thermophilic, cellulolytic, anaerobic bacterium Masahito Taya, Haruyuki Hinoki, Toshiyuki Yagi, and Takeshi Kobayashi Department of Chemical Engineering, Faculty of Engineering, Nagoya University, Chikusa-ku, Nagoya 464, Japan
Summary. A cellulolytic, obligately anaerobic, extreme thermophile (strain NA10) was isolated from an alkaline hot spring in Nagano Prefecture, Japan. The microorganism was a non-spore-forming, flagellated rod which had a negative reaction to Gram stain, and occurred singly or in pairs. The growth temperature was between 50°C and 85 °C with the optimum at 75 ° C, and the growth pH was between 6.0 and 9.5 with the optimum at 8.1. The anaerobe characteristically fermented cellulose, and produced acetic acid, H2, CO2 (main products) and lactic acid (minor product). The DNA had a base composition of 37.7 mol% guanine + cytosine content.
Introduction Extremely thermophilic anaerobes which grow above 70°C have been isolated from geothermal environments (Zeikus 1979; Ljungdahl 1979). The isolation and characterization of these bacteria have extended our knowledge of microbial species diversity and ecology in nature and have encouraged technological applications of thermostable enzymes. To date, however, few have been known which decompose cellulose under extremely thermophilic and obligately anaerobic conditions except for the reports of Reynolds et al. (1986) and Sissons et al. (1987). We have examined samples collected from hot springs in Japan, in order to isolate thermophilic, cellulolytic anaerobes. As reported briefly in our previous paper (Taya et al. 1985), we isolated an extremely thermophilic, cellulolytic anaerobe (strain NA10), which required tungsten as an esOffprint requests to: T. Kobayashi
sential growth factor. The present paper reports on the physiological characterization of this isolate.
Materials and methods Samples for enrichment and isolation. Water samples containing mud and sediments were collected from various hot springs including Nozawa Hot Spring (Nagano Prefecture, Japan). The temperature and pH of Nozawa Hot Spring water were 75°--85°C and 9.0, respectively. Microorganisms and culture conditions. Strain NA10 is a representative of cellulolytic thermophiles isolated from one of the samples from Nozawa Hot Spring by the enrichment and isolation procedures described below. Clostridium thermocellum ATCC 27405 (a thermophilic, cellulolytic anaerobe) and Thermoanaerobacter ethanolicus ATCC 31550 (an extremely thermophilic anaerobe) were used as type strains, obtained from the American Type Culture Collection, USA. Ruminococcus albus F-40 (a mesophilic cellulolytic anaerobe) was also used (Taya et al. 1980). Basal medium from screening was the same as reported previously (Taya et al. 1985). Unless otherwise noted, the concentration of carbon sources was 6 g/1. Cellulose, KC-floc W300 (Sanyo Kokusaku Pulp Co., Iwakuni, Japan) was used after ball-milling in a 3% suspension for 3 days. The pH of the medium after autoclaving at 121°C for 10 min was about 8.1 and the culture temperature was 75°C, unless otherwise stated. Physiological tests for the isolate were carried out using PRAS media (Scott Laboratories, New York, USA) with 0.4 rag/1 Na2WO4.2H20, based on PY medium described in the Anaerobe Laboratory Manual (Holdeman et al. 1977). C. thermocellum (culture temperature: 60 ° C) and T. ethanolicus (culture temperature: 65 ° C) were cultivated using the media of Weimer et al. (1977) and of Wiegel and Ljungdahl (1981), respectively. Ruminoeoccus albus was cultivated at 37 °C as described previously (Taya et al. 1980). Medium preparation and microbe transfers were carried out under strictly anaerobic condition using O2-free N2 gas following Hungate (1969). Analytical methods. Gaseous products were determined by a gas chromatograph (Model GC-7AG, Shimadzu Co., Kyoto, Japan) equipped with a thermal conductivity detector and a glass column (3ram by lm) packed with 30/60-mesh activated
M. Taya et al.: Extremely thermophilic, cellulolytic anaerobe
475
charcoal (Gasukuro Kogyo Co., Tokyo, Japan). Helium was used as the carrier gas. According to the Anaerobe Laboratory Manual (Holdeman et al. 1977), volatile and non-volatile acids (except for formic acid), and alcohols were analysed with a gas chromatography (Model GC-6A, Shimadzu Co.) equipped with a flame ionization detector and a glass column (3mm by 2m) containing 80/100-mesh Chromosorb W-AW-DMCS coated with 10% Reoplex 400 (Shimadzu Co.). Nitrogen was used as the carrier gas. Formic acid was colorimetrically analysed (Lang and Lang 1972). Dry cell weight and concentrations of total reducing sugar and cellulose were measured by the methods reported elsewhere (Taya et al. 1979; 1980). DNA base composition and DNA hybridization. The D N A was extracted and purified from the cells by the method of Marmur (1961). The D N A base composition was calculated from the thermal denaturation temperature using calf thymus D N A (42 mol% guanine plus cytosine ( G + C) content) as a standard. For the D N A homology test, Southern blot hybridization (Southern 1975) was carried out among chromosomal DNAs of strain NA10, C. thermocellum, T. ethanolicus and R. albus. Five micrograms of chromosomal D N A obtained from the four anaerobes were cut with Hind III, fractionated on a 0.8% agarose gel, transferred to a nitrocellulose filter, and hybridized with the Hind III-digested, 32P-labelled chromosomal D N A of strain NA 10.
Fig. 1. Electron micrograph of strain NA10. The bar represents 1 ~m. Cells from a young culture growing on glucose medium were negatively stained with 1% phosphotungstic acid (pH 6.2). Electron micrographs were taken with a Model HS-9 electron microscope (Hitachi Co., Tokyo, Japan)
Results
Isolation of thermophilic cellulolytic anaerobe, strain NAIO Samples from various hot spring waters were transferred into the cellulose medium under strict anaerobiosis. After several subcultures, many positive enrichment cultures were obtained at 80°C and an initial p H of 8.5, and strain NA10 was selected for further study of cellulose degradation. Through repeated roll-tube cultures (Hungate 1969) at 65°C, strain NA10 was purified from a well-isolated single colony. Its homogeneity was ensured by microscopic observations.
4.0 Ixm in length) with rounded ends, and had a few flagella arranged peritrichously. Microscopic observation showed that the cells occurred singly or in pairs. Spore formation of strain NA10 was not observed in all spore detection tests (Holdeman et al. 1977). Moreover, no survivals were found after heat treatment (95°C for 3--10 min) or alcohol treatment (50% ethanol for 1 h at room temperature). C. thermocellum, a spore-forming thermophile, survived under similar conditions. Thus we concluded that strain NA10 was not a spore former. Cells from both young and old cultures had a negative reaction to Gram stain.
Colony and cell morphology Growth properties and fermentation pattern Deep agar colonies of strain NA10 were 0.5--0.8 mm in diameter, and lenticular. The surface colonies were uniformly round and grew to a diameter of 1--2 mm after 4 - - 6 days at 65 ° C. All colonies developed with clear zones around them on the cellulose medium, which appeared as the result of cellulose hydrolysis by the anaerobe. No pigmentation was observed in any growth phase. Figure l shows an electron micrograph of a strain NA10 cell. The micrograph revealed that the ceils were rods (0.6--0.9 ~tm in width by 1.8--
Figure 2 shows the relationship between growth of strain NA10 and temperature. The optimum temperature was 75 °C. No growth was observed below 50°C and above 85°C even after 2 days. The effect of initial p H on growth of the strain is shown in Fig. 3. In this experiment, basal medium was modified for p H adjustment at different values. The optimum p H for strain NA10 was 8.0-8.3, and it grew between p H 6.0 and 9.5. The fermentation test of carbohydrates for
476
M. T a y a et al. : E x t r e m e l y t h e r m o p h i l i c , cellulolytic a n a e r o b e 1.o
Table 1. Utilization o f c a r b o h y d r a t e s by strain N A 1 0
0.8 "6
O.6
"~
0.4
~-
0.2
E
0
o
50
40
50
60 7 0
8 0 90 100
Temperature ('C)
Fig. 2. Effect o f t e m p e r a t u r e on g r o w t h o f strain NA10. T h e strain was g r o w n in b a s a l m e d i u m c o n t a i n i n g glucose at t h e t e m p e r a t u r e indicated. G r o w t h was d e t e r m i n e d by m e a s u r i n g optical d e n s i t y at 570 n m after 8.5 h cultivation
strain NA10 was investigated as shown in Table 1. The carbohydrate utilization was checked both by product formation and by pH drop. The strain characteristically fermented cellulose besides several other saccharides. The main products of this strain from cellulose or glucose were acetic acid, H2 and CO2. Lactic acid was also detectable as a minor fermentation product; ethanol and formic acid were not detectable. Figure 4 shows the results obtained using cellulose as a carbon source. The amount of cellulose decomposed during 30 h was 7.3 g/l; the amount of acetic acid produced was 2.8 g/1 during cultivation.
DNA base composition and other biological properties The D N A isolated from strain NA10 had a base composition of 37.7 mo1% G + C content. The indole production test, and nitrate and sulphate re-
0.5 >"
o-Galactose D-Glucose D-Fructose D-Mannose L-Rhamnose L-Arabinose D-Ribose D-Xylose
+ + + + +
Cellobiose Lactose Melibiose Maltose Sucrose
+ + + +
Trehalose
-
Melezitose Raffinose
+
Cellulose Dextrin G u m Arabic Glycogen Inulin Pectic acid Pectin Starch Xylan
+ + +
Adonitol Dulcitol Erythritol Glycerol Inositol D-Mannitol D-Sorbitol
--
Amygdalin Esculin Salicin -
duction tests were negative for this strain. Catalase activity also was not detected. Hydrogen sulphide production and milk coagulation tests were positive. The main characteristics of strain NA10 mentioned above are summarized in Table 2.
Discussion
As already reported in our previous paper (Taya et al. 1985), strain NA10 required tungsten as an essential growth factor. The characteristics of the strain described above show that the strain was significantly different from asporogeneous, noncellulolytic, anaerobic, extreme thermophiles reported previously such as Thermobacteroides acetoethylicus (Ben-Bassat and Zeikus 1981), Thermoanaerobium brocldi (Zeikus et al. 1979) and Dictyoglomus thermophilum (Saiki et al. 1985). The main characteristics of the strain were compared with those of T. ethanolicus ATCC 31550 and C. thermocellum ATCC 27405 (Table 2) which are representatives of non-cellulolytic and
0.4 0.3
N .t-
~
E~,
0.2 0.1
8
bg:
0 5
6
7 8 pH (-)
9
Zx-~x10 11
Fig. 3. Effect o f p H o n g r o w t h o f strain NA10. T h e strain w a s g r o w n in b a s a l m e d i u m c o n t a i n i n g g l u c o s e at t h e initial p H v a l u e s indicated. T h e b a s a l m e d i u m was m o d i f i e d to reinforce the b u f f e r capacity b y u s i n g t h e following b u f f e r s y s t e m s : O , K H 2 P O n - - N a z H P O 4 s y s t e m (60 m M as p h o s p h a t e ) ; O , N a 2 C O 3 - - N a H C O 3 s y s t e m (60 m M as c a r b o n a t e ) ; x, N a 2 H P O 4 - - N a O H (60 m M as p h o s p h a t e ) . G r o w t h w a s determ i n e d b y m e a s u r i n g the optical d e n s i t y at 570 n m after 5 h cultivation
~'6~
4
~ g 2 o 0
10
20
50
CuLtivation time (hr)
Fig. 4. T i m e c o u r s e o f cellulose d e c o m p o s i t i o n by strain NA10. T h e strain w a s g r o w n in b a s a l m e d i u m c o n t a i n i n g cellulose at 75 ° C. I , cellulose; A , dry cell m a s s ; O , acetic acid; x, reducing sugars
M. Taya et al. : Extremely thermophilic, cellulolytic anaerobe
477
Table 2. Comparison of morphological and physiological characteristics of strain NA10 with those of representative strains
Shape and cell size Flagellum Spore Gram stain G + C content (mol%) Optimum temperature (° C) Optimum pH Cellulose utilization Main product
Relation to oxygen
Strain NAIO
T. ethanolicus ATCC 31550
C. thermocellum ATCC 27405
C. thermohydrosulfuricum
Strain TP8.T
Rods, 0.6-0.9 × 1.8-4.0 I-tm Peritrichous Not observed Negative 37.7
Rods, 0.5-0.8 x 3.0-5.0 ~tm Peritrichous Not observed Negative 32.0 (37-39)
Rods, 0.4-0.8 × 2.0-5.0 I,tm Peritrichous Observed Negative 38.9
Rods, 0.4-0.6 x 1.8-13.0 ixm Peritrichous Observed Variable 35-37
Rods, ca. 0 . 4 x 4 I,tm -Not observed Negative --
75 8.1
70 (5.8-8.5)
60 6.7
67-69 6.9-7.5
Growth at 75 °C --
Yes Acetic acid, H2, CO2
No Ethanol, acetic acid, H2, CO2
Yes Ethanol, acetic acid, H2, CO2
Yes Ethanol, acetic acid,
Strictly anaerobic
Strictly anaerobic
Strictly anaerobic
No Ethanol, acetic and lactic acids, H2, CO2 Strictly anaerobic
Strictly anaerobic
The data in parenthesis for T. ethanolicus ATCC 31550 are cited from the literature (Wiegel and Ljungdahl 1981). The descriptions for C. thermohydrosulfuricum and strain TP8.T are cited from the literature (Hollaus and Sleytr 1972; Sneath et al. 1986) and (Sissons et al. 1987), respectively
cellulolytic thermophiles, respectively. C. thermocellum was similar to our isolate in its ability to digest cellulose, but was definitely different in sporulation and optimum growth temperature. We cloned a cellulase gene of strain NA10 in Escherichia coli and showed that the gene did not hybridize with the chromosomal D N A of C. thermocellum (Honda et al. 1987). This fact suggests that the cellulase produced by strain NA10 is dif-
Fig. 5 A, B. Southern blot analysis of chromosomal DNAs of strain NA10, Clostridium thermocellum ATCC 27405, Thermoanaerobacter ethanolicus ATCC 31550 and Rurninococcus albus F-40. A. Agarose gel electrophoresis. B. Autoradiograph of 3ep-labelled chromosomal D N A (strain NA10) hybridized to HindlII-digested DNAs. Lane 1, R. albus; lane 2, strain NA10; lane 3, LDNA (molecular weight marker); lane 4, C. thermocellure; lane 5, T. ethanolicus. The period of exposure was 40 h
ferent from that produced by C. thermocellum. As shown in Fig. 5, the D N A homology test revealed that the probe (Hind Ill-digested chromosomal D N A of strain NA10) did not hybridize to the chromosomal DNAs of C. thermocellum, T. ethanolicus or R. albus. Even during long-term exposure (11 days), hybridization was not observed among these DNAs and the probe (data not shown). This suggests that strain NA10 is different from the other cellulolytic anaerobes or thermophiles tested. Among members of the genus Clostridium, C. thermohydrosulfuricum is known as an extreme thermophile, for which the upper temperature limit permitting growth is 74°--76°C (Hollaus and Sleytr 1972), as shown in Table 2. Clostridium thermohydrosulfuricum differs from strain NA10 in cellulose digestion and spore formation. In Bergey's Manual of Systematic Bacteriology, vol 2 (Sneath et al. 1986), however, some similarities are suggested between C. thermohydrosulfuricum and non-spore-forming genera (Thermoanaerobacter and Thermoanerobium). Strain NA10 resembled Thermoanaerobacter ethanolicus in its growth at high temperature and lack of spore formation, although there were differences in carbohydrate utilization and product formation. The main product was ethanol for T. ethanolicus, whereas it was acetic acid for strain NA10. Leigh et al. (1981) isolated Acetogenium kivui, a thermophilic, non-spore-forming bacterium
478
M. Taya et al.: Extremely thermophilic, cellulolytic anaerobe
w h i c h p r o d u c e d principally acetic acid f r o m glucose. A c e t o g e n i u m kivui is a p p a r e n t l y different f r o m strain N A 1 0 in cellulose digestion a n d g r o w t h temperature. Recently, Sissons et al. (1987) isolated some extremely thermophilic, cellulolytic anaerobes (a representative strain, TP8.T) f r o m thermal p o o l s in N e w Zealand. T h e y r e p o r t e d that the isolate was a n o n - s p o r e - f o r m i n g rod, w h i c h stained gram negative a n d grew at 75 ° C (Table 2). T h e y p r o p o s e d Caldocellum saccharolyticum for strain TP8.T. A l t h o u g h detailed physiological data on the t h e r m o p h i l e is not available, strain N A 1 0 seems to be similar to strain TP8.T. We c o u l d n o t find an a n a e r o b e identical to o u r strain in Bergey's M a n u a l o f Determinative Bacteriology ( B u c h a n a n et al. 1974), Bergey's M a n u a l o f Systematic Bacteriology, vol 1 (Krieg et al. 1984) a n d vol 2 (Sneath et al. 1986), or App r o v e d Lists o f Bacterial N a m e s ( S k e r m a n et al. 1980). Thus, strain N A 1 0 seems to be distinct f r o m all previously established genera. I n o r d e r to identify strain NA10, further studies will be necessary, including t a x o n o m i c c o m p a r i s o n b e t w e e n the strain a n d closely related anaerobes. There are potential a d v a n t a g e s for the use o f cellulases f r o m extremely t h e r m o p h i l i c bacteria b e c a u s e they are very thermostable. To date, however, little has been k n o w n a b o u t the cellulase c o m p l e x f r o m extremely t h e r m o p h i l i c anaerobes. We have c l o n e d [~-glucanase genes o f strain N A 1 0 in E. coli a n d in Saccharomyces cerevisiae ( H o n d a et al. 1987, 1988a a n d b) but further studies will be n e e d e d to clarify the e n z y m o l o g i c a l a n d genetic properties o f the cellulases o f strain N A 1 0 for e x t e n d e d application.
anaerobic, thermophilic bacterium. Arch Microbiol 128:365--370 Buchanan RE (ed) (!974) Bergey's manual of determinative bacteriology, 8th edn. Williams & Wilkins, Baltimore Holdeman LV, Cato EP, Moore WEC (1977) Anaerobe laboratory manual, 4th edn. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va Hollaus F, Sleytr U (1972) On the taxonomy and fine structure of some hyperthermophilic saccharolytic clostridia. Arch Microbiol 86:129--146 Honda H, Naito H, Taya M, Iijima S, Kobayashi T (1987) Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable [3glucanase. Appl Microbiol Biotechnol 25:480--483 Honda H, Iijima S, Kobayashi T (1988a) Cloning and expression in Saccharomyces cerevisiae of an endo-[~-glucanase gene from a thermophilic cellulolytic anaerobe. Appl Microbiol Biotechnol 28:57--58 Honda H, Saito T, Iijima S, Kobayashi T (1988b) Isolation of a new cellulase gene from a thermophilic anaerobe and its expression in Escherichia coli. Appl Microbiol Biotechnol 29: 264--268 Hungate RE (1969) A roll tube method for cultivation of strict anerobes. In: Norris JR, Ribbons DW (eds) Methods in microbiology, vol 36. Academic Press, London pp 117-132 Krieg NR (ed) (1984) Bergey's manual of systematic bacteriology, vol 1. Williams & Wilkins, Baltimore Lang E, Lang H (1972) Spezifische Farbreaktion zum direkten Nachweis der Ameisens/iure. Z Anal Chem 260:8--10 Leigh JA, Mayer F, Wolfe RS (1981) Acetogenium kivui, a new thermophilic hydrogen-oxidizing, acetogenic bacterium. Arch Microbiol 129:275--280 Ljungdahl LG (1979) Physiology of thermophilic bacteria. In" Rose AH, Morris JG (eds) Advances in microbial physiology, vol 19. Academic Press, London, pp 149--243 Marmur J (1961) A procedure for isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3"208--218 Reynolds PHS, Sissons CH, Daniel RM, Morgan HW (1986) Comparison of cellulolytic activities in Clostridium thermocellum and three thermophilic, cellulolytic anaerobes. Appl Environ Microbiol 51:12--17 Saiki T, Kobayashi Y, Kawagoe K, Beppu T (1985) Dictyoglomus thermophilum gen. nov., sp. nov., a chemoorganotrophic, anerobic, thermophilic bacterium. Int J Syst Bacteriol 35:253--259 Sissons CH, Sharrock KR, Daniel RM, Morgan HW (1987) Isolation of cellulolytic anaerobic extreme thermophiles from New Zealand thermal sites. Appl Environ Microbiol 53:832--838 Skerman VBD, McGowan V, Sneath PHA (eds) (1980) Approved lists of bacterial names. Int J Syst Bacteriol 30:225--420 Sneath PHA (ed) (1986) Bergey's manual of systematic bacteriology, vol 2. Williams & Wilkins, Baltimore Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503--517 Taya M, Ito Y, Ohmiya K, Kobayashi T, Shimizu S (1979) Anaerobic digestion of cellulose and utilization of digestion products in a consecutive cultivation system. J Ferment Technol 57:178-- 185 Taya M, Kobayashi T, Shimizu S (1980) Synthetic medium for the cellulolytic anaerobe, Ruminococcus albus. Agric Biol Chem 44: 2225-- 2227
Acknowledgements. The authors thank Drs. S. Mizushima and T. Nogami (Faculty of Agriculture, Nagoya Univ.), and Drs. T. Mitsuoka and Y. Benno (Institute of Physical and Chemical Research) for the electron microscopy, and for determining the DNA base composition, respectively. We are also grateful to Drs. K. Ueno and K. Watanabe (Faculty of Medicine, Gifu Univ.), and S. Iijima and H. Honda (Faculty of Engineering, Nagoya Univ.) for useful suggestions. This work was supported in part by the Biomass Conversion Project of the Ministry of Agriculture, Forestry, and Fisheries, Japan (BCP 88V-I), a Grant-in-Aid (No. 63603525) for Scientific Research from the Ministry of Education, Science and Culture of Japan and the Asahi Glass Foundation for Industrial Technology.
References Ben-Bassat A, Zeikus JG (1981) Thermobacteroides acetoethylicus gen. nov. and sp. nov., a new chemoorganotrophic,
M. Taya et al.: Extremely thermophilic, cellulolytic anaerobe Taya M, Hinoki H, Kobayashi T (1985) Tungsten requirement of an extremely thermophilic, cellulolytic anaerobe (strain NA10). Agric Biol Chem 49:2513--2515 Weimer PJ, Zeikus JG (1977) Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence and presence of Methanobacterium thermoautotrophicum. Appl Environ Microbiol 33:289--297 Wiegel J, Ljungdahl LG (1981) Thermoanaerobacter ethanolicus gen. nov., sp. nov., a new, extreme thermophilic, anaerobic bacterium. Arch Microbiol 128:343 348 Zeikus JG (1979) Thermophilic bacteria: ecology, physiology and technology. Enzyme Microb Technol 1:243--252
479 Zeikus JG, Hegge PW, Anderson MA (1979) Thermoanaerobium brockii gen. nov. and sp. nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium. Arch Microbiol 122:41--48
Received August 17, 1987/Accepted May 26, 1988