Actinomycetes are Grams-positive bacteria frequently filamentous and
sporulating with DNA rich in G+C from 55-75% (Ho et al.,
2002). The name Actinomycetes derived from Greek aktis (a ray) and
mykes (fungus) was given to these organisms from initial observation of their
morphology. Streptomycetes is the dominant among Actinomycetes.
The non-streptomycetes are called rare Actinomycetes, comparing
approximately 100 genera numbers of the Actinomycetes, which live in
marine environment, are poorly understood and only few reports are available
pertaining to Actinomycetes from mangroves (Kumar,
2001). Actinomycetes are the most fruitful source for production
of bioactive secondary metabolites. Actinomycetes a total 7899 (100%)
compounds has been identified up to 1988; 67% from Actinomycetes, 12%
from bacteria and 20% fungi. Japanese and American scientists (Tanaka
and Omura, 1990) contributed greatly to the discovery of Actinomycetes
products, 286 compounds are produced by Streptomyces hygroscopicus, 189
by S. griseus, 129 by S. lavendulae, 383 by Micromonospora
app., 278 by Nocardia sp. The isolation and characterization of Actinomycetes
were performed in different biochemical methods (Dhanasekaran
et al., 2009). Morphological examination of the Actinomycetes
was done by using cellophane tape and cover slip-buried methods (Williams
and Cross 1971). The mycelium structure, color and arrangement of conidiophores
and arthrospore on the mycelium were examined under oil immersion (1000X). The
observed structure compared with Bergays manual of Determinative Bacteriology,
Ninth edition (2000) for identification Streptomyces sp., under the group
of Actinomycetes. Different biochemical tests were performed to characterize
the Actinomycetes. The tests generally used are starch hydrolysis, Triple
Sugar Iron (TSI) agar test, citrate utilization test, indole test, methyl red
test, vogus-proskauer (Acetone Production) test, Catalase test (Holt,
1989). The Actinomycetes were originally considered an intermediate
group between bacteria and fungi then were recognized as prokaryotic organisms.
Actinomycetes species synthesize a numerous natural metabolites with
diverse biological activity such as antibiotics. Antibiotics of Actinomycetes
origin evidence a wide variety of chemical structure, including amino glycosides,
anthracyclines, β-lactams, nucleosides, peptides, polyenes, actinomycins
and tetracycline (Barrios-Gonzalez et al., 2005).
Generally, antibiotics are non-growing associated secondary metabolites. It
is therefore very difficult to produce them by continuous cultivation of microorganisms.
Application of immobilized growing cells may overcome these problems by controlling
cultivation conditions the growth phase of cells will be maintained in preference
to antibiotic production and also culture fluids will not contain heavy cell
mass. In recent years, much interest has been focused on the use of immobilized
microbial cells for producing useful bioactive compounds (Manjula
et al., 2009). The antibiotics are widely produced by fermentation
using free cell cultures to enhance the productivity. Among another strategies
adopted, the whole cell immobilization appears to be more effective for antibiotic
production. Immobilized cells have been used in a wide spectrum of application
such as production of ethanol, Biosensors and production of oxytetracycline.
The method of cell immobilization includes nonspecific adsorption, covalent
attachment and entrapment. Entrapment of living cells with natural polymers
such as agar, agarose, alginate etc., is principally carried out by ionotropic
or thermal gelatin used to increase the yield of antibiotics. Some of the methods
demonstrated the advantages of use of immobilized growing cells for the continuous
production of antibiotics. For example, the immobilization of Streptomyces
rimoses leads to increase in the production of oxytetracycline (Yang
and Yueh, 2001), the enhanced production of neomycin by immobilization of
Streptomyces marinensis on calcium alginate matrix (Srinivasulu
and Ellaiah, 2005). The present study was to compare the efficacy of immobilized
and free Actinomycetes for their antibiotic production and its antimicrobial
property against pathogens. Proteins are polypeptides, which are made up of
many amino acids linked together as a linear chain. The structure of an amino
acid contained amino group, a carboxyl group and an R group, which is, usually
carbon based gives the amino acid its specific properties. These properties
determine the interactions between atoms and molecules, which are vander waals
force between temporary dipoles, ionic interactions between charged groups and
attractions between polar groups. The Actinomycetes strains were prepared
in crude protein. Then, the crude protein was determined by using SDS PAGE method.
MATERIALS AND METHODS
Isolation of Actinomycetes from soil sample: The soil samples
were collected from near my institute (2009), Muthayammal College of Arts and
Science (MCAS) in corn and Soya fields and air dried and used. A total soil
sample was collected from two areas, collected in sterile plastic bags and brought
into sterile condition. Actinomycetes were isolated by soil dilution
plate technique using Arginine-Glycerol-Salt (AGS) medium, (El-Nakeeb
and Lechevalier, 1962). The organism was screened by purification method,
streaking on AGS medium and incubated at 30°C for 4 days.
Biochemical characterization of Actinomycetes: The potent Actinomycetes were characterized by morphological methods consist of macroscopic and microscopic methods. The mycelium structure, color and arrangement of conidiophores and arthrospore on the mycelium were observed through the oil immersion (100X). The observed structure was compared with Berbeys manual of Determinative Bacteriology and the organism was identified. Various biochemical tests performed for the identification of potent isolates are as follows: Starch hydrolysis, fermentation of citrate, nitrate reduction and IMVIC tests.
Fermentation process: The Actinomycetes were cultured at 30°C
for 120 h in a jar fermentor containing 1 L of a medium containing of maltose
4%, sodium glutamate 1.2%, K2HPO4 0.01%, MgSO4
0.05%, CaCl2 0.01% and FeSO4 0.005% with or without sodium
alginate beads by the ionotropic method. At the end of fermentation cycle, the
sodium alginate beads were aseptically separated from the fermentation broth
by filtration using a sterile Bunchner funnel (Pandey et
Isolation of antibacterial metabolites: Antibacterial compound was purified
from the filtrate by solvent extraction method; Ethyl acetate was added to the
filtrate in ratio of 1:1 (v/v) and shaken vigorously for 1 h complete extraction.
The ethyl acetate phase contains antibiotic substances separated from the aqueous
phase. It was evaporated to dryness in water bath and the residue obtained was
weighed (Westley et al., 1979). The obtained
compound thus used to determine the antibacterial activity.
Determination of antibacterial activity: The actinomycete isolates often encounted show antibiotic activity on agar but not in liquid culture. The results of screening method were that most of the active isolates were active against Gram-positive and Gram-negative pathogen. Antibacterial activity was tested in vitro against pathogenic bacteria: Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 441. Antibacterial activities were performed by disc-diffusion assay and effectiveness was measured by zone of inhibition on bacterial culture plates.
Determination of protein profile from Actinomycetes
Protein profile: For the determination of protein profile from Actinomycetes
using SDS-PAGE method as described by Laemmli (1970).
Preparation of bacterial sample: The actinomycete was grown in their respective broth at 30°C for 54 h. The cells were harvested by centrifugation at 8000 rpm for 1 min. The pellet was washed with 1 M Tris HCl buffer (pH = 6.8) and resuspended in 10 mL of the same buffer and vortexed. Then 80 mL of the sample buffer (1 M Tris HCl, pH = 6.8, 20% SDS, 20% glycerol, 10% b-mercaptoethanol and 0.005% Bromophenol blue) and 6.5 mL of b-mercaptoethanol were added to the preparations and boiled immediately in water bath at 100°C for 5 min. After boiling, the samples were placed on ice for 5 min and centrifuged at 8000 rpm for 1 min.
Preparation of SDS-PAGE: For SDS-PAGE 12.5%, separating gel and 6.5%
resolving gel were prepared. A volume of 30 μL of each sample was loaded
on gel and was run on mini gel electrophoresis at 100 V for 2 h and stained
in a solution containing 0.1% (by mass per volume) Coomassie blue, 10% (by volume)
acetic acid and 40% (by volume) methanol. De staining was performed in a solution
containing 10% (by volume) acetic acid and 45% (by volume) methanol. The protein
molecular mass marker (100 kDa) was used as standard.
Out of three Actinomycetes subjected for primary screening process and
subjected for purification method by streak plate method (Fig.
1a-c). The identification of the potent antibiotic producing
strains reveal strain belongs to the genus streptomyces. The biochemical
tests were performed in positive isolates of Actinomycetes. The isolated
microorganism is Gram-positive, branching and filamentous bacteria, it has also
shown positive results in methyl red, indole test, starch hydrolysis, vogus-proskauer
test, catalase test and triple sugar iron test and shown negative result in
fermentation of citrate, the results were represented at Table
Out of three isolates, the one isolate was showed in positive result. The selected
isolate was performed in whole cell immobilization and free cells. The isolates
were selected for fermentation based on their broad spectrum of activity and
largest zone of inhibition.
|| (a-c) Isolation and screening of microorganism
||Bio chemical characterization of Actinomycetes strains
from soil sample
||Antibacterial activity for free and immobilized cell extract
||Isolation of crude protein from Actinomycetes. L1:
Marker (100 kDa); L2 to L7: Crude protein from Actinomycetes
The isolates were cultivated in specific fermentation liquid medium for 120
h. After fermentation process, the Antibacterial compound was purified from
the filtrate by solvent extraction method. The isolate showed the activity against
tested organisms. The determination of antibiotic activity of selected strain
was determined using Escherichia coli MTCC 50, Pseudomonas aeruginosa
MTCC 424 and Bacillus subtilis MTCC 441. The comparative studies on the
total antibiotic production with free and immobilized cells are shown in Fig.
2. It was found that, the immobilized cells in sodium alginate were more
efficient for the production of antibiotics, which was conformed by zone of
inhibition on bacterial culture plates.
The positive Actinomycetes strains were prepared in crude protein and the protein profile were determined by SDS-PAGE method. The results were identical to that of the same genous from various species. The crude protein separated has different molecular weight from lane 2 to 7 (Fig. 3).
The composition of an arginine-glycerol-salt medium (AGS), Suitable for the
selective isolation of aerobic Actinomycetes, was given. When soil samples
were treated with calcium carbonate and plated on the AGS medium, higher total
and relative plate counts of Actinomycetes were obtained than when other
media and methods were used (El-Nakeeb and Lechevalier et
al., 1962). In this present study, we are using AGS medium for isolation
of Actinomycetes from soil sample. The results were observed as filamentous,
branching bacteria with a fungal type of morphology. Various biochemical tests
were performed, to identify but it was unable to identify the Actinomycetes
up to species level due to the lack of other tests. Apart from proper identification
of genera and species of Actinomycetes, besides morphological and physiological
properties (Kuster, 1972). In this study, we use various
biochemical tests such as Starch hydrolysis, Triple sugar iron test, Fermentation
of citrate were performed for the identification of the potent isolates (Abbas,
2006). Some of the Actinomycetes are characterized by the production
of various pigments on natural or synthetic media. Then, the isolates were cultivated
in fermentation liquid medium.
To demonstrate use of immobilized cells of S. rimosus for the continuous
production of oxytetracycline it would be improved with calcium alginate immobilization
in submerged fermentation compared with free cells. The results showed that
in 1 mL culture broth, free cells produced 121 to 124 μg of oxytetracycline;
where as immobilized cells produced 153 to 252 μg (Yang
and Yueh, 2001). In this study, we are using the whole cell immobilization
technique in an efficient way to overcome the difficulty in producing antibiotics
by continuous fermentations with free-cells, since the growth and metabolite
yields, have been immobilized various microbial species on different support
matrices for antibiotic production. The Actinomycetes isolates, which
are often encounted, show antibiotic activity on agar but not in liquid culture.
In this present study, six Actinomycetes strains were isolated from
soil available in the local area and screened with regard to their potential
against Gram-positive and negative bacteria. Among the six isolated strains
(A1, A2, A3, A4, A5 and A6) three isolates A2, A3 and A5 were found to be effective
against tested organisms. These three strains were further cultivated in fermentation
liquid medium for 120 h and immobilized using sodium alginate (Manjula
et al., 2009). A comparative profile on the total antibiotic sensitivity
of the free cells and immobilized cells showed that the immobilized strains
were found to be effective against the tested microorganism then the free cells.
Further, the most potent of the producer strain was selected and identified,
based on the cultural and physiological characteristics. From the results, it
was concluded that the three isolates showed very promising activities against
tested multi-drug resistant bacteria.
The immobilized cells of Actinomycetes were found to be more efficient
for the production of antibiotics with batch fermentation. The possible routes
of antibiotic synthesis and its biological efficiency are currently under investigation
(Pandey et al., 2001). In the present study also
we are using the same methods, the results of screening method was that most
of the active isolates were active against Gram-positive and Gram- negative
pathogen (Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC
424 and Bacillus subtilis MTCC 441). The morphological differences between
these microorganisms show that different sensitivity occurred. The reason is
that Gram-negative bacteria have an outer polysaccharide membrane carrying the
structural lipo polysaccharide compounds. This makes the cell wall impermeable
to lipophilic solutes.
The Gram-positive should be more susceptible and only an outer peptide glycon layer which is not an effective permeability barrier. The Actinomycetes isolates are often encounted and show antibiotic activity on agar but not in liquid culture. The results of screening method was that most of the active isolates were active against Gram-positive and Gram-negative pathogen (Escherichia coli MTCC 50, Pseudomonas aeruginosa MTCC 424 and Bacillus subtilis MTCC 441).
Due to the morphological differences between these microorganismss different
sensitivity occurred. The reason was that a Gram-negative bacterium has an outer
polysaccharide membrane carrying the structural lipopolysaccharide compounds.
This makes the cell wall impermeable to lipophilic solutes. The Gram-positive
should be more susceptible having only an outer peptide glycon layer which is
not an effective permeability barrier. From the results, it is concluded that
the immobilized cells of Actinomycetes are more efficient for the production
of antibiotics. The possible routes of antibiotic synthesis and its biological
efficiency are currently under investigation. The crude protein samples were
determined by using SDS-PAGE method (Laemmli 1970).
I express my grateful and my heartfelt gratitude to Dr. N.Selvan, M.Sc., Ph.D., Head, Centre for Biotechnology, Muthayammal College of Arts and Science for his immense help for the completion of my project.