System. Appl. Microbiol. 15, 52-58 (1992) O Gustav Fischer Verlag, Stuttgartmew York

Clostridium termitidis sp. nov., a Cellulolytic Bacterium from the Gut of the Wood-feeding Termite, Nasutitermes lujae

PIERRE HETHENER, ALAIN BRAUMAN, and JEAN-LOUIS GARCIA"

Laboratoire de Microbiologie ORSTOM, Université de Provence, 13331 Marseille Cédex 3, France

Received July 31, 1991

Summary

An anaerobic, mesophilic, spore-forming, cellulolytic bacterium was isolated from the gut of a wood- feeding termite, Nasutitermes lujae. The cells were Gram-positive rods, motile by peritrichous flagella, and formed oval terminal spores which swelled the cells. The deep colonies were circular, punctiform and slightly yellow; the surface colonies spread rapidly and were diffuse. Cellulose, cellobiose, glucose, fruc- tose, galactose, lactose, mannose, ribose, sorbose, xylose, maltose, melibiose, mannitol and sorbitol served as carbon source; glycerol was poorly used. The fermentation products were acetate, ethanol, Ha, COZ. Optimal growth occurred at 37°C and at p H 7.5. The deoxyribonucleic acid composition was 39.2 mol% guanine plus cytosine. The name Clostridium termitidis sp.nov. is proposed. The type strain is strain CT1112 (= DSM 5398).

~

Key words: Clostridium termitidis sp. nov. - Cellulose - Mesophilic - Wood-feeding termites - Nasutiter- mes h jae - Intestinal tract

Introduction

During the last decade, the potential for converting cel- lulosic wastes into industrial substrates has stimulated cur- rent interest in cellulose fermentation. Many cellulolytic microorganisms have been isolated from various biotopes, particularly those belonging to the genus Clostridizrm from the rumen (Hzazgate, 1944; Van Gylszuyh et al., 1980; Kelly et al., 1987; Varel, 1989), anaerobic digestors (Ng et al., 1977; Slent et al., 1984; Sleat and Mah, 1985; Palop et al., 1989; Yang et al., 1990), compost (Madden, 1983; Petitdemange et al., 1984; Suhhumavnsi et al., 1988; Yanling et al., 1991) estuarine sediments (Madden et al., 1982) and riverside mud (Mendez et al., 1991). Little is known about the anaerobic cellulolytic bacteria from termite gut, although these insects are very efficient lignocellulose-decomposers. Recent studies (Brauman et al., submitted for publication) have indicated that their gut microbiota may provide a suitable model system to under- stand the electron flow involved during biopolymer degra- dation by anaerobic microbial communities.

In this paper, we report on a new cellulolytic Clostri- dizwz species, strain CT1112, isolated from the gut of a :' Corresponding author

wood-feeding termite, Nasutitermes lztjae, from the May- ombe tropical rainforest, Congo, Central Africa.

Materials and Methods

Culture methods and media. Hungate's anaerobic techniques (Httngate, 1950; Hungate, 1969; Macy et al., 1972) were used throughout these experiments. Strain CT1112 was cultured on the mineral medium described by Balch et al. (1979) containing per liter: K2HP04, 0.3 g; KH2P04, 0.3 g; (NH&SO+ 0.3 g; NaCl 0.6 g; MgS04, 7H20, 0.13 g; CaCIZ, 2H20, 8 mg; cystein-HC1, 0.5 g; resazurin 0.2%, 1 ml; trace element solution (hd70ff- Stzrckle and Pfenttig, 1983), 1 ml; NH&l, 1 g. After autoclaving at 110'C for 40 min, the medium was cooled under a stream of 02-free NZ-COZ (80-20%). NaHC03, 4.5 g/l, Na$, 0.4 g/1 and carbon source were added from separately sterilized anoxic solu- tions; filter-sterilized growth factor solutions (yeast extract, Bio- trypcase, vitamins) were added when required. The media were finally adjusted to pH 7.0 and distributed into Hungate tubes, serum bottles or roll tubes containing 1.8 YO agar. The cellulose used was cellulose MN 300, 20 ym (SERVA, Heidelberg, FRG). Growth was measured in Hungate tubes at 580 nm. All charac- terization tests were carried out in at least duplicate cultures.

Isolatioiz procedure. Anaerobically dissected and ground guts from living wood-feeding termites were incubated on enriched liquid medium containing cellulose or directly on solid medium in roll tubes. Colonies in roll tubes showing clear zones of digested cellulose were serially diluted onto cellulose-agar in roll tubes .until the pure culture was obtained. Purity was confirmed by subculturing the isolate on enriched liquid medium supplemented with 1 g/l yeast extract and Biotrypcase (Biomérieux, Crappone, France). Strain CT1112 isolated from Nusutiteriizes lujae was then routinely cultured both on 5g/l cellulose MN 300 and 2 g/l cellobiose.

Analytical techniques. All biochemical and physiological tests were carried out from subcultures on cellobiose minimum medium, without any growth factor. The degradation of cellulose and reducing sugars was measured using the phenol-sulfuric acid method (Dubois et al., 1956) and the ferrocyanide method (Park and Jobnso?~, 1949), respectively. Volatile fatty acids and al- cohols were analysed as previously described (Cord-Ruwiscb et al., 1986). Hydrogen was measured using a gas chromatographic method with a thermal conductivity detector, with a 1.80 m Car- bosphere (60-80 mesh) column operated at 85 OC; gas sampling was performed with a gastight pressure lock syringe.

DNA buse composition. The mol percent guanine plus cy- tosine of the DNA was determined at the DSM, Braunschweig, FRG. After disruption with a French pressure cell, the DNA was isolated by means of HAP chromatography (Cusbioiz et al., 1977) and determined by HPLC using the method described by Mesh- bah et al. (1989).

Electroiz microscopy. Negative staining was performed with uranyl acetate (4% w/v in distilled water). Cells were fixed for 1 h in sodium cacodylate buffer (0.07 M, p H 7.3) containing glutaraldehyde (1.2% w/v) and ruthenium red (0.05% w/v). Af-

Clost?idiunz ternzitidis sp. nov. 53

ter washing in cacodylate buffer with ruthenium red, the samples were postfixed in Oso4 (1% w/v in cacodylate buffer 0.07 M). Embedding was performed in Epon and ultrathin sections were stained with uranyl acetate (2% w/v in ethanol 50% w/v) and then with lead citrate. Micrographs were talcen on a JEOL 1200 CX electron microscope.

Results Isolation

T h e enrichment and isolation procedures yielded three anaerobic cellulolytic isolates froin which strain CT1112 was selected for identification.

Colony morphology

Colonies developed to about 1 inm in diameter in 7 days, when grown on cellulose-agar at 37 O C . The colonies inside the agar were circular and punctiform in shape and slightly yellow. Clearing cellulolysis zones reached up to 5-6 mm in diameter with prolonged incubation. The sur- face colonies spread rapidly and were diffuse.

Cellular morphology

The cells of strain CT1112 were straight to slightly curved rods about 0.5 pm wide by 4-6 pm long (Fig. la) . The oval spores were terminal. The mature spores were 0.7 pm in width and 1.2 pm in length and caused a marked swelling of the cells (Fig. l a ) . Endospores were still viable

Fig. 1. Morphology of strain CT 11 12. a, phase contrast micrograph; b, transmission electron micrograph, phosphotungstate negative staining; c, transmission electron micrograph, thin section.

54 P. Hethener, A. Brauman, and J.-L. Garcia

after heating at 100°C for 10 min. Strain CT1112 was motile by peritrichous flagella (Fig. l b ) and stained Gram positive; the transmission electron micrograph of vegeta- tive cells and showed a Gram positive cell wall profile with a thick single layer and an S-layer (Fig. IC). Chromato- graphic separation of the amino acids showed that the cell well contains m-DAI? (Kandler, personal communication).

Nutrition and growth conditions

No yeast extract, Biotrypcase, or added vitamins were required for growth, but their presence enhanced the growth. The optimum temperature for growth was 37 OC, and growth occurred in a temperature range of 20 to 48 "C (Fig. 2). The optimum pH for growth was 7.5, and the pH range was 5.0 to 8.2 (Fig. 3).

Metabolic characteristics

Strain CT1112 is an obligate anaerobe that ferments cellulose, cellobiose, glucose, fructose, galactose, lactose, mannose, ribose, sorbose, xylose, maltose, melibiose, mannitol, sorbitol and poorly glycerol. It does not ferment arabinose, rhamnose, salicine, sucrose, trehalose, melezit- ose, raffinose, adonitol, dulcitol, pectin, starch, glycogen or gelatin. H2, COZ, acetate and ethanol are fermentation products. A doubling time of 6 h was obtained on 6 g/l cellobiose (Fig. 4).

Mol% G+C. The mol% G+C of strain CT 1112 was 39.2 k 0.1.

0.0)

=: 0.Of k a, c F c

2 L

cn o o .- .t. .-

% 0.04

0.02 20 30 40 50 60

Temperature (OC)

Fig. 2. Effect of temperature on growth of strain CT 1112.

Clostridium termitidis sp. nov. 55

t

PH

Fig. 3. Effect of pH on growth of strain CT 1112.

0.11

0.10

F 0.09 E.

F

g

al .I-

c 2 0.08

u)

o o a,

.- c .-

$- 0.07

0.06

, . , . , . I . 0.05

Cellobiose (g/l)

Fig. 4. Effect of cellobiose concentration on growth of strain CT 1112.

Table 1. Differentiation of strain C T l l l 2 from other mesophilic cellulolytic Clostridia

Charac- teristic

adonitol arabinose dulcitol esculin fructose galactose gelatin glucose göycerol lactose maltose mannitol mannose melezitose melibiose pectin raffinose rhamnose ribose salicin sorbitol sorbose sucrose trehalose xylose

Gram motility spore

optimal temp. GC%

C. aldricbii

ND

ND + ND

-

- - - ND - - - - - - - - - - - - ND - - -

+ + polar oblong subterm. 35 40

C. celere- crescens

+ + + + + + + W + + + W W ND + + + + W W + + + + peritr. spherical terminal 30 to 37 38

-

-

-

C. cellobio- C. cellirlo- C. cellulo- C. cellulo- C. chartata- C. iosui C. lento- C. lonai- C. PaDyro- C. poly- C. poptileti Strain

-

panini feniientaizs lyticitm vorans bidiim - . . _ . . . .

cellitin sporunz solvens saccharo- a 1112 lyticzrni

+ W W + + + ND

- -

-

+ + + + + + ND +

-

-

+ + +

+ peritr. + peritr. + peritr. spher./oval spher./oval spherical terminal terminal terminal 30 to 37 37 to 40 32 to 35 28 34 41

ND

ND ND + + + + + ND + ND + ND

ND + ND +

-

-

-

-

-

-

- - - - central subterm. 37 27/27

ND ND ND ND + + + ND V

-

- - - ND

W ND ND ND + ND ND + + + + peritr. long subterm. 39 31

-

-

- ND + ND - ND + + V + ND

ND + + ND

- - -

- -

ND ND ND ND ND ND ND

ND ND ND

-

-

ND + ND ND + + 3. + - - + ND - - ND + ND

ND ND

ND +

-

-

- - + fimbriae + peritr. spher./oval long terminal subterm. 40 35 to 42 36 33

- (wall +) - (wall +) - x peritr. x peritr. + spherical ovalkpher. oval terminal terminal terminal 2 5 t o 3 0 3 0 t o 3 8 35 30 42 28

- - - + + + + + W + + + + + -

- - - - - + + - - + + + peritr. oval terminal 37 39.2

- +, positive; -, negative (?), ztncertaiii; W, weak; ND, itot determined; V, variable; peitr., peritrichous flagella; spher., sphe?icd; subterm., subterminal.

Clostridium ternzitidis sp. nov. S i

Clostridium cellulofermentans (Yanling , et al., 1991) and Clostridium lentocellunz (Murray et al., 1986) use raf- finose, salicin, sucrose and trehalose but not sorbitol; they have a lower G+C% content of DNA (34-36) than that of strain CT1112 (39).

According to our results, strain CT1112 is a new mesophilic cellulolytic bacterium using sorbitol, mannitol and lactose but not arabinose and pectin, and producing mainly acetate, ethanol, H2 and COZ. We propose that strain CT1112 be placed in the genus Clostridium as a new species, Clostridium termitidis sp.nov.

Description of Clostridium termitidis sp. nov.: ter.- mi'ti.dis. L.n. tarnzes, tarmit- (L.L.var. termes, termit-) worm that eats wood; M.L. adj. termitidis pertaining to the termite.

Discussion

Cellulolysis occurs in the lower termites through sym- biotic associations between protozoa and endo- or ec- tosymbionts (Breznak, 1982; O'Brien and Slaytor, 1982). In some higher termites, cellulolysis is known to be linked to the activity of gut microflora such as the actinomyceta of the genera Streptomyces and Micromonospora [Pasti and Belli, 1985), which need aerobic conditions to de- velop. Cellulolysis can be also linked to enzymatic ac- tivities in the insects' salivary glands (Veivers et al., 1982) or in posterior parts of their gut (Rouland et al., 1988).

The cellulolytic strain CT1112 has been isolated from the gut of a wood-feeding higher termite able to assimilate the main part of wood constituants such as cellulose, hemicellulose and lignin (Seifert and Becker, 1965; Noirot and Noirot-Timothée, 1969). As cellulolysis is an impor- tant source of nutriment for wood-feeding termites, selec- tive enrichments of anaerobic celluloytic bacteria from the gut of such termites may have been successful.

Several strictly anaerobic bacterial strains have been iso- lated recently from the gut of termites, such as the homo- acetogens Sporomusa termitida (Breznack et al., 1988) and Clostridium nzayombii (Kane et al., 1991) or sulfate reducers (Brauman e t al., 1990; Trinker1 et al., 1990).

Seventeen species of the cellulolytic genus Clostridium have been described up to now, four of which are ther- mophilic, namely Clostridium thermocellz~nz (Ng. et al., 1977), Clostridiunz stercorarium (Madden, 1983), Clos- tridium tl~ernzopapyrolyticum (Mendez et al., 1991) and Clostridium cellulosi (Yangling et al., 1991). Strain CT1112 can therefore be compared with the thirteen mesophilic Clostridium species described in the literature. Table 1 gives the main characteristics of these species as compared to strain CT1112.

Strain CT1112 is a Gram-positve slightly curved rod, motile by peritrichous flagella and forms swollen oval ter- minal spores. Strain CT1112 differs greatly from Clos- tridium celerecrescens (Palop et al., 1989), Clostridium populeti (Sleat and Muh, 1985), Clostridium longisporunt (Varel et al., 1989) and Clostridium chartatabidunz (Kelly et al., 1987) in the shape of spores and the utilisation of substrates, especially gelatin. Clostridium josui (Sukhu- nzavasi et al., 1988) has an optimal temperature of 45 OC, is not motile and uses a limited range of substrates for growth.

Clostridium cellobioparum (Hungate, 1944), Clostri- dium papyrosolvens (Madden et al., 1982) and Clostri- dium cellulolyticum (Petitdemange et al., 1984) differ in substrate utilisation, and their spores are spherical. Clos- tridium aldrichii (Yang et al., 1990) has a polar tuft of flagella and an oblong subterminal spore. Clostridium polysaccharolyticunz (Van Gylswyk et al., 1980) uses slightly cellobiose, a common substrate of strain CT1112, and does not use fructose, xylose, maltose or lactose. Clos- tridium cellulovorans (Sleat et al., 1984) utilises lactose as is observed for strain CT1112, but forms central to subter- minal spores, and is not motile; furthermore the G+C % content of its DNA (26-27) is lower than that of strain CT1112 (39).

Cells are straight to slightly curved Gram-positive rods, 0.5 pm by 4-6 pm, peritrichously flagellated. Oval termi- nal spores cause a marked swelling of the cells.

The deep colonies which develop in cellulose-agar roll tubes are small, circular and slightly yellow. The surface colonies are widespread on agar.

Obligate anaerobe. Ferments cellulose, cellobiose, glu- cose, fructose, galactose, lactose, mannose, ribose, sor- bose, xylose, maltose, melibiose, mannitol, sorbitol and poorly glycerol. Acetate, ethanol, H2 and CO2 are fermen- tation products.

The temperature optimum for growth is 37°C. The range is 20 to 48 OC. The pH optimum for growth is 7.5. The range is 5 to 8.2. Growth factors are not required, but yeast extract, Biotrypcase or vitamins enhanced growth.

The mol% G+C is 39.2-t.0.1. Isolated from the guts of the wood-feeding termite

Nasutitermes lujae, from the Mayombe tropical rainfor- est, Congo, Central Africa.

The type strain is strain CT1112 (DSM 5398).

Acknowledgements. We are indebted to K. D. Jahnke (DSM) for determining the DNA-base ratio, O. Kaizdler for determining the cell wall content of m-DAP, C. Allasia for preparing the electron micrographs, B. Ollivier for helpful discussion, and J . Blanc for revising the English.

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Dr. Jean-Louis Garcia, ORSTOM case 87, Université de Provence, 3 Place Victor-Hugo, F-13331 Marseille Cédex 3, France

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