1) TITLE OF THE RESEARCH
Microbial Biodiversity of Khumbu Region : Study of Antibiotic
Activity of Actinomycetes
2) PROJECT LEADER
Prof. Dr. Vishwanath P. Agrawal, Academician, Royal Nepal Academy of Science
and Technology (RONAST), Khumaltar, Lalitpur, P.O.Box 3323, Kathmandu ,
NEPAL; Tel. 977-1-547720; Fax 977-1-547713; email ronast@mos.com.np
3) COLLABORAT ING INSTITUTIONS
(a) Faculty of Science, RONAST
(b) Research Laboratory for Agricultural Biotechnology and Biochemistry
(RLABB), Sanepa – Indrayani, Ringroad, Lalitpur, P.O.Box 2128,
Kathmandu, Nepal (a private research organization)
Tel : 528313; Fax : 977-1-526318; email : rlabb@wlink.com.np
4) AIMS OF THE SCIENTIFIC PROGRAM
The over all goal of this project is to survey and inventory the microbial diversity of
the Khumbu region. The specific aim of the project is to map the actinomycetes
population of this region.
5) DETAILED SCIENTIFIC PROGRAM
Introduction
The Himalaya has always been a source of fascination and inspiration for the naturalists
and scientists since time immemorial. It has such an unusual rich fauna and flora that
enticed the biologist all over the world. It provides the multiplicity of varied
microclimates and ecological niches not only for higher plants but for microorganisms as
well. Since, the Himalaya has been recognized as a hot spot for biodiversity, its microbial
diversity also deserves the attention of scientist community.
Microbial biodiversity is vast frontier and potential goldmine for the biotechnology
industry because it offers countless new genes and biochemical pathways to probe for
enzymes, antibiotics and other useful molecules. Worldwide, the economic value of
microorganisms is estimated to be “at least many tens of billions of US dollars”.
Pharmaceutical of microbial origin account for sales of approximately $35-50 billion per
annum in the North. It is the invisible world of microbes that has given us more than
3,000 antibiotics, for example, many derived from soil samples. In 1993, five of the
pharmaceutical industry’s top-selling drugs were derived from microbes; accounting for
more than $4,500 million in annual sales. The commercial value of microbials extends
beyond pharmaceuticals. The total world market or industrial enzymes, all produced by
microorganisms, is “1,300 million.
With the use of modern biotechnology, the potential applications of potential applications
of microorganisms is vast. The starting point for biotechnological development is the
search for and discovery of exploitable biological phenomenon. Scientists are
experimenting with genetically engineered bacteria that are capable of producing
products such as biodegradable plastics, artificial skin, and fibres that are as strong as
spider silk. Maize, rice, potato and cotton are among the crops that have been genetically
engineered with insecticidal genes from a common soil bacterium, Bacillus thuringiensis
(Bt).. Today, transnational microbe hunters are especially interested in exotic and hostile
environments – including boiling hot springs, undersea hydrothermal vents, alkali lakes
and the frozen tundra of Antarctica – as a source of unexplored microbial diversity.
Bioprospecting for microbes goes, quite literally, to the ends of the hearth.
A short description of actinomycetes
Bergey’s manual divides actniomycetes in eight diverse groups (J.G.Holt, 1989). They
are aerobic gram- positive bacteria that from branching , usually nonfragmenting hyphea
and asexual spores .They comprise 63 genera and are relatively well documented
because of their value in drug discovery (L.J.Nisbet and F.M.Fox 1991 ). The
morphology and arrangement of spores, cell wall chemistry, physiology and the types of
sugars present in the cell extracts are particularly important in actinomycetes taxonomy.
Actinomycetes have considerable practical impact because they play major role in the
mineralization of organic matter in the soil and are the primary source of most naturally
synthesized antibiotics. Streptomyces represents the largest genus in actinomycetes.
Another important genus is Frankia which grows in symbiotic association with the roots
of at least eight families of higher nonleguminous plants and fixes atmospheric nitrogen.
Technical Work Plan
The plan contains the following major steps : (a) Collection of soil samples from different
places in the Khumbu region and (b) Isolation and Characterization of Actinomycetes to
the genus and species level, if possible.
Collection of soil samples:
Following factors will be considered in soil collection (Bull A. T. et al 1992)
(a) thickly and sparsely populated areas, (b) areas with thick and thin vegetation, (c) areas
having tourist activities, (d) pollutted and unpollutted areas, (e) multiple samples from a
given site, (f) sampling of a given site during different seasons and (g) soil type: Dry –
richest in actinomycetes; Sandy soil favors: Streptomyces; Organic rich soil favors:
Microminospora.
The Khumbu region will be divided in 100 sampling sites and at least three samples will
be collected from each site. The soil samples will be collected in sterile plastic bags from
6 cm – 10 cm depth. Soil samples can be pretreated by air drying for 48 hours to
maximize actinomycetes population.
Isolation and characterization of Actinomycetes (Bergey’s manual, 1974; Khala and
Chandrika, 1993; M.P. Starr et al, 1981; You and Yong, 1996; D. Singh, 1999)
Methods for bacterial isolation and characterization given below are just guidelines. They
will be modified as the situation arises. Soil will be suspended in water and will be
progressively diluted in water. Actinomycetes will be selectively grown using starchcasein or glycerol-arginine media. Further purification will be carried out in the same
media. Characterization will be done using biochemical tests, morphology and
pigmentation. At least 300 soil samples will be analysed.
A representative example of Isolation of Actinomycetes from Soil
1) Create a suspension by mixing 1 g of soil with 99 mls of sterile ddH20. Take 1 ml of
this and dilute it into 9 ml sterile ddH20. 2) Heat the suspension for 16 hours at 45 C. 3)
Plate 0.1 ml and 0.2 ml of the aqueous extract onto sodium caseinate plates. (Sodium
Caseinate 0.20 g/l; K2HPO4 0.50 g/l; MgSO4 0.20 g/l; FeCl3 0.01 g/l; Agar 15.0-16.0 g/l;
pH 6.5-6.7), 4) Incubate for 5-7 days at room temperature.
Examination of the plates
The actinomycetes on the plates are simply identified as the colored, dried, rough, with
reegular7regular margin; generally convex colony as described by Williams and Cross
(1971). Such colonies are distinctly different from the other bacterial and fungal colonies.
Purification of actinomycetes :
Streak plate method is used to purify cultures of actinomycetes contaminated by bacteria
and fungi. The observed actinomycetes colonies are picked up either by straight wire or
by loop according to the condition. The picked up specks of the colonies are streaked
over the dried agar medium divided into sectors radially. After streaking, the plates were
incubated for 5 days at 27°C. After purification the actinomycetes from the bacteria and
fungi, they may become contaminated with a propagule of another actinomycetes from an
external source, because there are several ways in which internal contamination may arise
(e.g. dry nature of the colony, handling error, etc.). Hence, to purify the actinomycete
individual sp. From other actonomices, they are streaked over the next plate. After
isolation of the pure colonies based on their colonial morphology, colour of hyphae, color
of aerial mycelium, they are individually plated on another but the same agar medium as
described by Williams and Cross, 1971.
Examination of morphology : the morphological examination of the actinomycetes is
done by using cellophane tape method (Williams and Cross, 1971) and cover slip-buried
method (Williams and Cross 1971).
Physiological and biochemical tests: Different physiological and biochemical tests are
performed to identify the species of actinomycetes. The tests generally used are gelatin
hydrolysis, starch hydrolysis, urea- hydrolysis, acid production, resistance to NaCl,
temperature tolerance test, hydrogen sulphide production test, motility test, triple sugar
iron (TSI) agar test, citrate utilization test, indole test, methyl red test, voges-proskauer
(Acetoin Production) test, catalase test, oxidase test (Singh, D., 1999).
References:
Bergey’s manual of Determinative Bacteriology (1974) Part 17 Actinomcetales pp. 657-881.
Bull, A.T., Goodfellow, M. and Slater, J.H. (1992) Biodiversity as a source of innovation in biotechnology,
Ann. Rev. Microbiol., 46: 219-252.
Holt, J.G., (1989) Bergey’s manual of systematic bacteriology, vol. 4, ed. S.T.Williams and M.E.Sharpe,
Baltimore, Md : Williams and Williams
Kala, R.R. and Chandrika, V (1993) Effect of different media for isolation, growth and maintenance of
actinomycetes from mangrove sediments. Indian Journal of Marine Sciences. Vol. 22(4): 297-299
Nisbet, L.J. and F.M.Fox (1991) The importance of microbial biodiversity to biotechnology, In., The
biodiversity of microorganisms and invertebrates : its role in sustainable Agriculture, ed. D.L.Hawksworth,
pp. 229 – 244, CAB International
Singh, D. (1999) Antibacterial activity of actinomycetes isolated from various geographical regions of
Nepal and characterization of their antibacterial agents. A M.Sc. thesis submitted to the Central Dept. of
Microbiology, Tribhuvan University
Starr, M.P.; Stolp, H.; Truper, H.G.; Balows, A.; and Schlegel, H.G. (1981) The prokaryotes: a handbook
on habitats, isolation and identification of bacteria. Vol. 2 Springer - Verlag, New York
Williams, S.T. and Cross, T. (1971) Actinomycetes In J.R.Norris and D.W.Robbins (eds) Methods in
Microbiology, vol.4, pp.295-334, London, New York : Academic Press
You, Kyung Man and Yong, Keun Park (1996) A new method for selective isolation of actinomycetes from
soil. Biotechnology Techniques 10(7): 541-546.
6) LIST OF RESEARCHERS:
Prof. V. P. Agrawal (Ph.D. in Biochemistry )…. Supervisor and coordinator of the
project
Deepak Singh ( M.Sc. in Microbiol.) Research Scientist in RLABB
Vijay Dhakal (M.Sc. in Microbiol.) Research Scientist in RLABB
Research scientist form RONAST to be identified