Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 9, Num. 3, 1998

S. DEY and S. R. CHAPHALKAR

Division of Microbial Sciences, Agharkar Research Institute, G.G.Agarkar Road, Pune - 411004, India

The thermophilic flora from a meteoritic crater was investigated. Five actinomycetes were selected on the basis of their bioactivity and antibiosis. The isolates belong to the genus Streptomyces and were identified by numerical taxonomy as members of the species S. thermovulgaris and S. thermoviolaceus. All isolates produce proteases, amylases and lipases at 55°C. Following induction protease and amylase (but not lipase) production increases by 46.2% and 49.8% respectively. The crude preparation is thermostable up to 85°C with a half life of 90 min at 85°C and activity (Ca⁺⁺ independent) at a pH range of 7.5 to 12. Culture broths from all isolates show antimicrobial activity against B. subtilis, E. coli, M. luteus and Aspergillus niger.

Located on the floor of a large circular crater (Fig. 1), the inland saline, highly alkaline, Lonar lake is unique in the world (Clarke, 1924; Subrahmanyam, 1985). It appears to have been formed towards the end of the Cretaceous period by an explosion (Friedriksson et al., 1973; Jingran & Rao, 1954) or by a giant meteor (La Touche & Christie, 1911).

Lonar lake is a closed system without outlets and regular influents are responsible for its existence (Nandy & Deo, 1961). However, there is no evidence of fossils from the lake sediments nor information about biota from the lake environment, except for algae (Round, 1973; Singh, 1961) and a passing reference on the activity of lower organisms responsible for precipitation of sodium salts and H₂S production (Badve et al., 1993).

It was believed that, due to alkalinity, there could be no life in the lake, however later on it was observed that the environment could represent one of the oldest biological habitats (Anteibi & Fishlock, 1985). The recent interest in biotechnology, coupled with the discovery of novel thermophiles, has prompted the search for new phenotypes in untouched areas. Interest in thermophiles, especially streptomycetes, centres on their widespread distribution in soil (Zenova et al., 1984), their role in the production of antibiotics, enzymes, growth promoters (Williams & Wellington, 1982) as well as the inherent stability of their proteins and hypobiosis (Bierly & Bierly, 1986; Kutzner, 1981). The majority of novel metabolites with the greatest chemical diversity is obtained from streptomycetes (Yarbrough et al., 1993; Sanglier et al., 1993), therefore isolation of thermophilic actinomycetes from this unique ecosystem is justified.

In the present note the results of a screening program, carried out on a number of silt and water samples from Lonar lake, in a search for thermophilic actinomycetes producing multiple enzymes, with broad pH and temperature ranges, and antibiotic metabolites are reported.

Several silt and water samples were collected over a two-year period from five locations. Silt samples were heated to 100°C for 10 min (Athalye et al., 1981) and used as inoculum. Water samples were filtered through a 0.22 µm membrane (Al Diwany et al., 1978) and the membranes were suspended in 2 ml of sterile water. All samples were plated onto a nutrient agar (neutral and alkaline pH), with a salt composition similar to that of the lake water.

Generic placement was carried out by spore chain morphology, using scanning electron microscopy (Chaphalkar et al., 1993), cell wall composition (Lechevalier & Lechevalier, 1970), G+C mol % (Mandel & Marmur, 1968) and fatty acid profiles (Ballio et al., 1965). Species were identified by chemotaxonomy (Locci, 1989), using MICRO-IS (Potyrata & Krichvesky, 1993) and TAXAN (Information Resources Group, 1993) programs.

For enzyme and antibiotic production, isolates were grown for 18 hrs as shaken cultures on GYP medium containing (% w/v) glucose (1), yeast extract (0.5), peptone (0.5), NaCl (0.5) and CaCl₂.

Hydrolytic activity of cell free broths was assessed on substrates containing casein, starch and tributyrin respectively. Isolates able to hydrolyse the substrates and active against at least two test organisms were selected for further studies. Strains were grown in 100 ml of different synthetic media, at pH 7.2 and 9, containing glucose, starch and tributyrin respectively as carbon sources (Collee & Marr, 1989) and incubated at 55°C for 24 h, using a 100 µl spore suspension as inoculum.

The cell free broth was used as a crude enzyme preparation and the activity was determined by the azocaseinase (Ginther, 1979), DNS (Bernfield, 1955) and phospholipid method (Fisk-Subba, 1959). Xylanase, cellulase and pectinase were determined by recording the clearing of xylan, cellulose and pectin containing media. Protein content of the broth was determined according to Lowry et al. (1954). The stain removing property of the broths was tested by the Fabric Test (Chaplin & Bucke, 1972) using different stains in presence and absence of detergents.

Antimicrobial activity was determined by the standard cup method evaluating the MIC values of broths concentrated under vacuum in comparison with those of standard antibiotics against Bacillus subtilis, Escherichia coli, Micrococcus luteus and Aspergillus niger. Each experiment was carried out in triplicate and the mean value of ± SD determined from 3 different analyses.

Out of a total of 51 colonies recovered on neutral media incubated at 55°C a first set of 13 strains was selected on the basis of enzyme and antibiotic potential and, of these, three showed enzyme activity also at pH 10. In parallel 23 colonies were obtained by incubation at the same temperature on GYP at pH 10. Twelve strains were selected and, of these, two were also active when grown at pH 7.2.

These five isolates were subjected to further investigation (Table 1). All strains were aerobic, salt tolerant (10%) Streptomyces species and were identified as S. thermoviolaceus (SD1, SD3 and SD9) and S. thermovulgaris (SD6 and SD7).

Sporulation takes place after 18 hrs at 55°C. Cell mass increases substantially by switching incubation temperatures from 45° to 65°C and the lag phase becomes shorter. Growth and sporulation time are the same at 55° and 65°C after 18 hrs.

Table 1. Identification, origin and bioactivit of the strains isolated from Lonar lake (A: amylase, C: chitinase, L: lipase, P: proteinase, Pc: pectinase, X: xylanase) * identification score according to MICRO-IS).

Ability to hydrolyse casein, starch and egg yolk (Nitch & Kutzner, 1969) was investigated on GYP (pH 8) containing the above components separately and all strains gave positive results.

Enzyme activity was quantified at both neutral and alkaline pH (Table 2). Induction with casein, starch and oil induces a 45 to 50% increase in protease and amylase activity, but has slight effect in the case of lipase.

Table 2. Enzyme activity of cell-free broths of the five strains at neutral and basic pH before (BI) and after (AI) induction (A: amylase, L: lipase, P: protease).

As proteases account for most enzyme sales, further purification was carried out. PAGE profiles and post electrophoretic reactivity of the purified enzymes reveal the presence of multiple proteases. Comparative studies with commercially available proteases point out the novelty of these enzymes (Table 3). The proteins are highly stable in alkali up to pH 12 and active up to 85°C with a half life of 20-90 min. They retain their activity in the presence of different chaotrophic agents even in the absence of Ca⁺⁺ thus making them interesting for industrial applications.

The Fabric Test shows that these enzymes could be very useful for detergent industries even in a crude form.

Antimicrobial activity of the isolates, compared with that of standard antibiotics, is summarised in Table 4.

Table 4. MIC of the strain broths compared with that of known antibiotics

One of the interesting features of the isolates is the early sporulation time (18hrs) which in mesophiles vary from 2 to 7 days. As the sporulation time is associated with the production of secondary metabolites, the biotechnological potential of the strains is undeniable.

Thermophiles represent unique and important genetic resources as their macromolecules are stable at higher temperatures (Brock, 1985). In the present study antibiotic and enzyme production were assessed with regard to their biotechnological potential. Activity at a broad pH range and salt and alkali tolerance of thermophilic streptomycetes are quite important under the harsh conditions of bioreactors in the production of pharmaceuticals and enzymes.

Present and potential applications of industrially important enzymes, e.g., proteases, amylases and lipases are quite diverse. They are used as digestive aids and debridement of wounds (Inada et al., 1986), disc herniation (Wood et al., 1984), membrane cleaning (Schwimmer, 1981), in breweries (Gerhertz, 1990) and in detergent (Godfrey & Reichelt, 1983) as well as leather industries (Gerhertz, 1990). Industrial processes are run under specific conditions which cannot always be adjusted to the optimal values required for the enzyme activity. This is especially true for pH and temperature.

A final comment on the suggested promotion of Lonar lake as a tourist attraction. The site is important not only geologically but microbiologically and biotechnologically too. Making the lake accesible to tourists may destroy this important ecosystem.

Al Diwany, L. J., B. A. Unsworth & T. Cross (1978). Enumeration of microorganisms in water. J.Appl. Bacteriol., 45: 249 - 258

Athalye, M., J. Lacey & M. Goodfellow (1981). Isolation of thermophiles from soil. J. Appl. Bacteriol ., 51: 289 - 298

Anteibi, E. & D. Fishlock (1985). Biotechnology - Strategies for Life. Academic Press, New York

Badve, R. M., K. P. N. Kumaran & G. Rajshekhar (1993). Eutrophication of Lonar Lake. Curr. Sci., 65: 347-354

Ballio, A., S. Barcellona & L. Boniferti (1965). Detection of fatty acid profiles of microorganisms. Biochem. J., 94: 11c-13c

Bernfield, M, (1955). Estimation of amylases. In: Colowick & Kaplan (eds.) Methods in Enzymology. Vol. 1. Academic Press, pp. 149-150

Brierly, J. A. & G. L. Brierly (1986). Thermostability of the macromolecules in thermophiles. In: T. D. Brock (ed.) Thermophiles. pp. 279-306

Brock, T. D. (1985). Life at high temperature. Science, 238: 132 - 138

Chaphalkar, S., R. Dongre, D. Joshi & S. Dey (1993). Rapid replica method of microbial sample preparation for SEM. Biotechn. Histochem., 68: 166-168

Chaplin, M. F. & R. Buck (1972). Enzymes as digestive aid. In: M. F. Chaplin & R. Buck (eds.) Enzyme Technology. Cambridge University Press, Cambridge, pp. 220-222

Clarke, F.W. (1924). The Data of Geochemistry. Williams & Wilkins, New York

Collee, J. G. & W. Marr (1989). In: J. G. Collee, J. P. Duguid, A. G. Fraser & B. P. Marmion (eds.) Practical Medical Microbiology. Churchill Livingstone, New York, p. 109

Fredriksson, K., A. Dube, D. J. Milton & M. S. Balasundaram (1973). Chemical analysis of Lonar Lake water. Science, 180: 862-864

Fisk-Subba, R. (1959). Estimation of phospholipids. J. Biol. Chem., 234: 466-468.

Gerhertz, W. (1990). Industrial uses of enzymes. Proteolytic enzymes. In: W. Gerhertz (ed.) Enzymes in Industry - Production and Application. VCH Verlagsgesellschaft, Weinheim, pp. 127-130

Ginther, C. L. (1979). Sporulation and the production of serine protease and cephamycin by S. lactamadureus. Antimicrob. Ag. Chemther., 15: 522-526

Godfrey, T. & J. R. Reichelt (1983). Industrial Enzymology. The Nature Press, New York

Inada, Y., T. Yoshimoto, A. Matshisu & Y. Saito (1986). Proteases in debridement of wounds. Trends. Biotechnol., 4: 68-73

Information Resources Group (1993). TAXAN a Numerical Taxonomy Program, Release 2.0. Maryland Biotechnology Institute, USA

Jingran, A. G. & K. V. Rao (1954). Geological aspects of Lonar lake. Geol. Survey India Publ., 85: 313 - 334

Kutzner, H. J. (1981). The family Streptomycetaceae. In: In: M. P. Starr, H. Stolp, H. G. Trüper, A. Balows, H. G. Schlegel (eds.) The Prokaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria. Vol. 2. Springer Verlag, Berlin, 1981, pp. 2028- 2090

La Touche , T. H. D. & W. A. K. Christie (1911). Origin of Lonar lake. Geol. Survey India Publ., 14: 266 - 285

Lechevalier, M. P. & H. A. Lechevalier (1970). A critical evaluation of the genera of aerobic actinomycetes. In: H. Prauser (ed.) The Actinomycetales. VEB G. Fisher Verlag, Jena, pp. 311-316.

Locci, R. (1989). Streptomycetes and related genera. In : S. T. Williams, M. E. Sharpe & J. G. Holt (eds.). Bergey's Manual of Systematic Bacteriology. Williams & Willkins, Baltimore, pp. 2452-2508

Lowry, O. H., N. J. Rosenbrough, A. L. Fair & R. J. Randell (1954). Protein measurement by Folin Phenol reagent. J. Biol. Chem., 193: 265

Mandel, M. & J. Marmur (1968). Use of CsCl density gradient analysis for determining the G+C content of DNA. In: L. Grossman & K. Noldave (eds.) Methods in Enzymology , Vol. XII. Academic press, New York, pp. 195-206

Nandy, N. C. & V. B. Deo (1961). Origin of Lonar lake and its alkalinity. J. Tata Iron & Steel Co. , 8: 1- 12

Nitch, B. & H. G. Kutzner (1969). Egg yolk agar as a diagnostic medium for streptomycetes. Experientia,. 25: 220-221

Potyrata, D.A. & M. K. Krichvesky (1993). Microbial database management and analysis system. Binary, 4: 31-37

Round, F. E. (1973). The Biology of Algae. Edward Arnold, London

Sanglier, J. J., H. Haag, T. A. Huck & T. Fehr (1993). Novel bioactive compounds from actinomycetes. Res. Microbiol., 144: 633 - 642

Schwimmer, S. (1981). Source Book of Food Enzymology. The Ann. Publ.Comp., Westpost, Conn

Singh, R. N. (1961). Role of blue green Algae. ICAR, pp 135 - 139.

Subramanyan, B. 1985). Origin of Lonar crater. J. Geol. Soc. India, 26: 326-335

Williams, S. T. & E. M. H. Wellington (1982). In: J. D. Bulock (ed.) Bioactive Microbiological Products. Academic Press, London, pp. 9-21

Wood, W. I., D. J. Capen, C. C. Simonseu & D. L. Eaton (1984). Proteases in disk herniation. Nature, 312: 330-346

Yarbrough, G. G., D. P. Taylor, R. T. Rowlands, M. S. Crawford & L. L. Lacuce (1993). Screening of microbial metabolites for new drugs. J. Antib., 46: 535 - 544

Zenova, G. M., T. A. Gracheva & A. A. Likhachera (1994). Actinomycetes in terrestrial ecosystems. Microbiologya, 63: 313-317 (in Russian).

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