Cultural, morphological, and physiological characteristics of Thermomonospora fusca (strain 190Th)l DONL. CRAWFORD

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Deparrr~le~lf of Biology, George Mason University, Fairfax, Virginia 22030 Accepted July 28, 1975 CRAWFORD, D. L. 1975. Cultural, morphological, and physiological characteristics of Tl~ern~o~no~losporci firscn (strain 190Th). Can. J . Microbiol. 21: 1842-1848. The cultural, morphological, and physiological properties of Thermo~nonospornfirsca(strain 190Th) are described. Its physiological properties show that this species is primarily a carbohydrate-degrading actinomycete which can use a wide range ofplant sugars and polymeric carbohydrates as sources of carbon and energy. The culture does not use proteins or amino acids for carbon and energy, or as a nitrogen source. A few organic acids are utilized. Ammonia is the preferred nitrogen source. The culture has trace nutrient requirements which include biotin and an undetermined number of amino acids. These and other physiological characteristics are discussed in relation to the roles that T.firsccr carries out as a saprophytic bacterium in nature. Its cultural and morphological properties are discussed in relation to the taxonomic status of this species in the literature. CRAWFORD, D. L. 1975. Cultural, morphological, and physiological characteristics of Ther~nornonosporafirscn(strain 190Th). Can. J. Microbiol. 21: 1842-1848. Nous y decrivons les proprietes culturales, morphologiques et physiologiques de Thermolnonospora Jirscrr (lignie 190Th). s e s propriites physiologiques montrent que cette espece est premierement un actinomycete degradant les hydrates de carbone et qui peut utiliser une grande variete de sucres des plantes et d'hydrates de carbone polymeriques comme sources de carbone et d'energie. Cette lignee n'utilise pas les proteines ou les acides amines comme sources de carbone et d'inergie, ou comme source d'azote. Quelques acides organiques sont utilises. L'ammonia est la source d'azote prifiree. Cette espice a des exigences pour des traces d'elements qui incluent la biotine et unnombre indetermine d'acidesamines. Cescaracteristiques et d'autres caracteristiques physiologiques sont discutees en relation des rBles que T. f i ~ s c ajoue comme bacterie saprophyte dans la nature. Ses propriites morphologiques et culturales sont discuties en relation du statut taxonomique de cette espece dans la litterature. [Traduit par le journal]

Introduction It is generally assumed that in nature the obligately thermophilic actinomycetes play an important role in the decomposition of organic compounds in composting plant materials (12). However, the exact nature of their activities remains incompletely understood because few of the physiological and enzymatic studies needed to define them more clearly have been done. Certain of the thermophilic actinomycetes are known to produce extracellular hydrolytic enzymes. Thermoactinomyces vulgaris has been shown to produce very active extracellular amylases (9) as well as several extracellular proteinases (6). Thermomonospora fusca and T. curvata produce highly active extracellular cellulases (4, 15). Thermomonospora fusca will also utilize lignocellulose for carbon and energy (2,3). These are the only definitive physiological studies 'Received june 5, 1975.

that have been done with obligately thermophilic actinomycete species, and all have concentrated on single physiological properties of specific isolates. No study of the general physiological characteristics of any of these species has been carried out. In this paper we report on the physiological properties of T . fusca. In addition, we report the cultural and morphological characteristics of this strain which identify it as T.fusca. It is important that the identifying characteristics of T. fusca be reported in light of the uncertain status of this species in the taxonomic literature (1). Materials and Methods Culture Used Thermomonospora fusca (strain I90Th) = (ATCC2 27730) = (NCIB3 11185) was used throughout this work. This culture was originally isolated, but not identified (Forbes (7)). Stock cultures were kept as slants either o n 2ATCC, American Type Culture Collection. 3NCIB, National Collection of Industrial Bacteria.

CRAWFORD: THERMOMONOSPORA FUSCA (STRAIN 190Th)

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a tryptone - yeast extract -glucose (TYG) agar previously described (3), or on brain heart infusion (BHI) agar (Difco). Stocks were inoculated and then incubated at 55 "C for 2 4 days, by which time good sporulation had occurred. Sporulated stocks were stored at 4 "C, and transfers were made bimonthly. Observations of Cultural and Morphological Cl~aracteristics Cultures were grown on agar plates of nutrient agar (Difco), TYG agar, BHI agar, Czapek's agar (16), and glucose asparagine agar (16). Spore-inoculated plates were incubated at 55 "C in a humid incubator for 2-4 days. Colonies were observed for color and amount of aerial mycelium, color of substrate mycelium, and total amount of colony growth. Sporulation was observed at 400 x , or under oil immersion lens (1000 x ) directly on the surface of agar plates, as recommended by Lechevalier (13). Determination of P/~ysiologicalProperties Temperature Ranqe for Growth Spores from TYG slants were inoculated to fresh slants and incubations carried out at 5 "C intervals between 20 "C and 70 "C. Maximal, minimal, and optimal growth temperatures were determined visually after 2-7 days of incubation at a given temperature. Hydrolysis of Casein, Gelatin, and Starch Hydrolysis of casein, gelatin, and starch was determined on TYG agar plates containing 1 . 0 z of the substrate to be tested. Spore-inoculated plates were incubated at 55 "C for 7 days. Plates were wrapped in plastic wrap to prevent excessive drying. Casein and gelatin hydrolysis were determined after 7 days by flooding plates with saturated (NH,)*S04 solution, and starch hydrolysis. by flooding plates with Lugol's iodine solution. Clear zones around the colonies were indicative of hydrolysis. Other Media Media used for the further physiological characterization of T . fit.~cuwere as follows: nitrate broth (0.1z KNO,, 0 . 2 z tryptone, 0.05z yeast extract, pH 7.2-7.4); litmus milk (Difco); potato slants (14); carbohydrate utilization broth (a mineral salts solution (3) plus 0 . 0 5 z yeast extract with or without specific carbohydrate at 0.3z); organic acid utilization broth (same as the carbohydrate utilization broth except that specific organic acids at 0.3Z were substituted for carbohydrate in the medium); nitrogen utilization broth (nitrogen-free mineral salts solution plus 0.05z yeast extract with or without added nitrogen source, plus 1 . 0 z glucose as the carbon and energy source). All of these media were autoclaved or filter-sterilized as required, and the pH was adjusted to 7.2-7.4 before sterilization.

Results Spore formation by T. fusca is shown in Figs. 1 and 2. The culture produces single spores borne only in the aerial mycelium. This is characteristic of the genus (8). Spores are formed in clusters, each singly on the tip of a short sporophore, with spore clusters originating from primary laterally

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branched aerial hyphae (Fig. 1). Cross and Goodfellow (5) described this structure as a repeatedly branched sporophore producing dense spore heads. A single spore head is shown in Fig. 2. Spores are oval, about 0.6-0.7 by 0.70.9 pm. On TYG or BHI agar plates, colonies take 3-4 days to develop at 55 "C. Sporulation occurs after about 3 days, as evidenced by the appearance of an abundant white aerial mycelium. The substrate mycelium appears translucent to slightly yellowish. On Czapek's or glucose asparagine agar plates, growth is very poor. These are not suitable media for T. fusca. On nutrient agar growth is fair. Colonies are thin and transparent and sporulation occurs only a t the edges of the colonies. This culture does not generally produce a diffusible pigment on the agar media used. In liquid media containing yeast extract, however, the broth becomes slightly yellowish to golden brown as a result of growth by T. fusca. This may be the result of soluble pigment production. Table 1 gives the general taxonomically identifying physiological properties of T. fusca. Temperature range for growth is about 35 "C to 65 "C. No growth was observed after 7 days of incubation at 30°C or 70°C. Optimal growth temperature was 50-55 "C. Litmus milk remained unchanged through 7 days of incubation at 55 "C. Casein hydrolysis on TYG plates was negative; however, gelatin hydrolysis appeared slightly positive after 7 days of incubation. In broth cultures neither casein nor gelatin will support the growth of T. fusca when they are supplied as the primary source of carbon and energy. Starch is actively hydrolyzed by T. fusca. Cellulose is an excellent carbon source. Nitrate reduction to nitrite in nitrate broth is weakly positive after 3-4 days of growth. On potatb slants growth is good. A white aerial mycelium is produced, and the slant turns brown. The physiological characteristics listed in Table 2 can be used to further characterize this strain. They show that T. fusca has a rather distinctive physiology. This organism is quite versatile in its ability to use carbohydrates as sources of carbon and energy. Of the sugars tested, only lactose did not support growth well, and of the carbohydrate polymers tested, only chitin and pectin were not utilized. Organic acids do not support growth as well as carbohydrates; however, acetate and pyruvate do make good carbon

CAN. I. MICROBIOL. VOL. 21, 1975

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FIG.1. Aerial sporophores of T. fusca showing multibranched sporophore structure, after 48 h of growth on brain heart infusion agar; taken at 1000 x under oil immersion lens.

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CRAWFORD: THERMOMONOSPORA FUSCA (STRAIN 190Th)

FIG.2. Spore cluster showing dense spore head, after 48 h of growth of T. fusca on brain heart infusion agar: taken at 1000 x under oil immersion lens.

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C A N . J. MICROBIOL. VOL. 21, 1975

TABLE 1 Taxonomically identifying physiological properties of T. fusca (strain 190Th) Physiological test

Result

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Growth temperatures Casein hydrolysis Gelatin hydrolysis Starch hydrolysis Growth on cellulose Growth on litmus milk Nitrate reduction Growth on potato slants

Minimal, 35 "C Maximal, 65 OC Optimal, 50-55 "C Negative Weakly positive after 7 days Strongly positive Excellent Negative; no change in 7 days Weakly positive after 4 days Good; with production of a n aerial mycelium

and energy sources. Citrate, malate, and succinate support limited growth. In glucose broth, of those tested, ammonia is the best nitrogen source for growth. Nitrate and urea support only limited cell growth. When these two nitrogen sources were used, large numbers of spores germinated, but the individual colonies which normally develop from each spore remained very small. With ammonia as the nitrogen source, these spores germinated and then developed into large mycelial balls.

When a mixture of equal amounts of the 20 common amino acids was supplied to T. fusca under various cultural conditions, it was observed that this culture could not utilize them for either carbon and energy or as a nitrogen source. The data in Table 3 show that a 0.3% level of the amino acids will not support growth, either with or without added yeast extract. Nor can T.fusca substitute the amino acids for ammonia as a nitrogen source. The amino acids alone cannot replace yeast extract as a source of trace nutrients for T. fusca. However, a mixture of the amino acids plus biotin can fulfill the culture's trace nutrient requirements, although growth still does not resemble that obtained with yeast extract. Since biotin alone does not fulfill these requirements, we conclude that a t least some of the amino acids are required by T. fusca. Intact proteins (casein and gelatin), as pointed out earlier, are not used for carbon and energy. It seems that T. fusca is not adapted for growth at the expense of proteinaceous substrates, either intact or hydrolyzed.

Discussion The morphological and physiological properties mentioned identify this culture as T. fusca. These properties correspond well with previous descriptions of the species (5, 8, 16). The nomina

TABLE 2 Utilization of carbon and nitrogen sources by T. frtsco (strain 190Th) Compound tested Sugars Arabinose Cellobiose Galactose Glucose Fructose Lactose Maltose Mannose Mannitol Sucrose Xylose

Amount of growth "

Compound tested

Amount of growth"

Compound tested

Amount of growtha

Carbohydrate polymers Filter paper Absorbent cottonc Carboxymethyl cellulose (CMC) Chitin Pectin Starch (soluble) Xylan Proteins Gelatin Casein

+2 +2 +2

Organic acids Acetate Citrate Fumarate Lactate Malate Pyruvate Succinate Tartrate Nitrogen sources Ammonia Nitrate Urea Peptone

+3 +1

+,

t ?

+3 +2 t t

?

-

+1 +3 +1 k

+3 t ?

t

*Amount of growth was measured as follows: -, no growth; same as on yeast extract alone (10-20 mg dry weight cells/100 ml medium); + 1, fair growth (50-60 mg dry weight ceIIsI100 ml medium; + 2, good growth (90-100 mg dry weight cells/100 ml medium); +3. excellent growth (150-175mg dry weight cells1100 ml med~um). bWhatman N o . 1 filter oaoer, blended In a Warine Blendor.

Cultural, morphological, and physiological characteristics of Thermomonospora fusca (strain 190Th).

The cultural, morphological, and physiological properties of Thermomonospora fusca (strain 190Th) are described. Its physiological properties show tha...
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