ISOLATION, CHARACTERIZATION, TAXONOMIC STUDIES AND ...

www.wjpr.net Vol 8, Issue 9, 2019.

Kundu et al. World Journal of Pharmaceutical Research

723

ISOLATION, CHARACTERIZATION, TAXONOMIC STUDIES AND

BIOLOGICAL ACTIVITIES OF AN ANTIBIOTIC PRODUCING SOIL

BACTERIUM FROM MANGROVE REGION OF SUNDARBANS,

WEST BENGAL, INDIA

Bikas Kumar Kundu1* and Prasanta Kumar Sircar

2

1Microbiology Department, Maulana Azad College, Kolkata, India.

2Department of Botany, Ballygunge Science College, University of Calcutta, India.

ABSTRACT

The aim of this study was to characterize, taxonomic studies and

biological activities of antibiotic producing soil bacterium isolated

from Mangrove region of Sundarban, West Bengal, India, designated

as IIIM-A. For the taxonomic placement of the organism,

morphological studies of the isolated organism were done primarily

under light microscope growing the isolated organism on the solid

media and in liquid media. The nature of growth, colony morphology,

colour and nature of mycelia and diffusible pigments were studied in

different bacteriological media. Physiological characters of the

organism were determined by the studies of melanin pigment formation, proteolysis, urease

activity, starch hydrolysis; H2S gas production, utilization of different carbon sources and

antagonistic activity in addition to the study of cell wall composition. Tube dilution technique

and agar cup assay were employed to determine the antimicrobial activities against gram

positive bacteria, gram negative bacteria and few species of fungi as test organisms.

KEYWORDS: Soil, Bacteria, Mangrove, Sundarban, West Bengal, India, Characterization,

Taxonomy, Antagonism.

INTRODUCTION

The Actinomycetes is a large group of microorganisms consisting of a variable number of

genera and species. The group was considered as a transition group between fungi and

bacteria but recent evidences prove that the group is more closely related to bacteria.

World Journal of Pharmaceutical Research SJIF Impact Factor 8.074

Volume 8, Issue 9, 723-738. Research Article ISSN 2277– 7105

Article Received on

27 May 2019,

Revised on 17 June 2019,

Accepted on 07 July 2019,

DOI: 10.20959/wjpr20199-15320

*Corresponding Author

Dr. Bikas Kumar Kundu

Microbiology Department,

Maulana Azad College,

Kolkata, India.



724

Morphological features of Actinomycetes undoubtedly play an important role for the

classification of this group but generic identification based on morphology alone may be

controversial in some cases. Waksman and Henrici (1943), suggested a comprehensive

system of classification in which the Actinomycetes were subdivided into four genera and

formation of a new genus Streptomyces where division was solely based on morphological

characters like production of aerial mycelium and catenulate spores. The color of aerial and

substrate mycelium was used for the separation of species. Waksman (1961) proposed a

system of classification for the genus Streptomyces with series based on color and growth of

substrate and aerial mycelium.[15][16]

Biochemical characters drew more attention for the classification of Actinomycetes. Pridham

and Gottlieb (1948) emphasized on the utilization of acetate, malate, pyruvate, propionate

and succinate as species characters.[12][13]

Krasilinikov (1960) did not agree to accept the

characters such as proteolysis, starch hydrolysis, sucrose hydrolysis, cellulose utilization and

nitrate reduction as taxonomic criteria because the characters are found in almost all

Actinomycetes, but utilization of urea, creatine and certain amino acids, casein, tyrosine and

xanthine could have more significance in classification. Quantitative production of antibiotics

(Krasilinikov, 1960),[4][5][6][7]

production of sulphide on peptone agar (Tresner and Danga,

1958)[13]

were useful criteria for Actinomycetes classification. Cell wall composition and

whole cell hydrolysate proved to be the practical use for the classification of

Actinomycetes.[8]

This group was subdivided into nine broad groups based on the distribution

of amino acids and sugars in the cell wall (Cumming, 1962). Presence of DL-dap

(Diaminopimelic acid) and LL-DAP in peptidoglycan and sugar compositions of

polysaccharides in the cell wall were used as an important marker for classification of

Actinomycetes (Kroppenstedt and Krtzner, 1976). Lipid pattern was considered to separate

Streptomyces from Nocardioform group.[9]

Present study deals with the characterization of a bacterial strain isolated from Mangrove

region of Sunderbans, India considering morphological, cultural, physiological and

biochemical characteristics and generic assignment with taxonomic placement of the strain

on the basis of determined characters.

The potency of antibiotic can be determined by chemical, physical and biological means. An

assay is made to determine the ability of an antibiotic to kill or inhibit the growth of living

microorganisms. Species and strains of microorganisms have varying degrees of



725

susceptibility to different antibiotics. The susceptibility of an organism to a given antibiotic

may change, especially during treatment. Therefore, microbiologists should make an accurate

microbiological diagnosis to determine the susceptibility of an organism to antibiotic. The

susceptibility of an organism to antibiotic can be determined by the tube -dilution technique

or the paper -disc-plate technique.[11]

1. Tube dilution technique

By the tube dilution technique, one can determine the smallest amount of antibiotic required

to inhibit the growth of the organisms in vitro. This amount is referred to as the minimal

inhibitory concentration (MIC). In this technique increasing amounts of antibiotic under

examination are placed in a series of culture tubes containing a suitable amount broth

medium and incubated with the test organism keeping a control group of tubes having no

antibiotic. After incubation, the concentration of antibiotic required to inhibit the growth of

the organism is determined by the absence of growth.

2. Paper-disc-plate technique

The paper-disc-plate technique is the most commonly used technique for determining

susceptibility of microorganisms to antibiotic. Small paper discs of the same size

impregnated with known amounts of antibiotic are placed upon the surface of an inoculated

plate. After incubation the plates were observed for any zones of inhibition surrounding the

discs. A zone of inhibition around the disc indicates that the organism is inhibited by the

antibiotic which diffused into the agar from the disc.

All incubations were made at 37°C for bacterial species, 30°C for fungal species, sterilized at

15lb for 20 minutes, inoculated aseptically where necessary, observations were made with

five replica unless stated otherwise. Readings were taken 3 days of incubations.

MATERIALS AND METHODS

In course of our screening programs a strain of soil bacteria isolated from Mangrove region

of Sundarbans, India, showed antimicrobial activity against gram positive and gram negative

bacteria but not against fungi. So we were intrigued to characterize and identify the strain in

our laboratory for taxonomic placement in order to assign proper systematic position of the

organism. The identification was based on the criteria suggested by International

Streptomyces Project (ISP), other characters were also studied.[9]



726

All incubations were made at 32°-35°C unless stated otherwise. Observations were made

with 5 replicas.

Morphological Characteristics

Morphological characteristics of the organism were studied on the solid and liquid culture

media. Nutrient broth and agar medium (modified after Waksman, 1961) and Glucose Yeast

extract broth and agar medium were used for morphological studies.

Plates were prepared with media and sterile square cover slips were dipped at an angle of 45°

into the solidified medium. The organism was spread along the cover slip-medium interface.

A number of replicas were prepared and after incubation one set was observed daily.

Morphological characteristics of the organism were also studied growing the organism in

submerged condition.

Cultural Characteristics

Plates were made with respective agar media (Table – 1). The organism was streaked on the

solidified agar medium and incubated at 32°-35°C for 14 days. The nature of growth,

morphology of colony, colour and nature of mycelium and formation of diffusible pigments

were noted. All compositions of the media were in g/L. The agar media contained 20.0g of

agar, sterilized at 15lb pressure for 20 minutes.

Physiological characteristics

1) Melanin formation

Melanin formation (Melanoid pigments) was studied on agar slants.

Melanin formation medium (Waksman, 1961)

Components of the medium Amount in g/L

Yeast extract 1.0

L-Tyrosine 1.0

NaCl 8.5

Bacteriopeptone-Yeast extract-Iron agar medium (Shirling and Gottlieb, 1966)


Bacteriopeptone (Difco) 15.0

Protease peptone (Difco) 5.0

Ferric ammonium citrate 0.5

Dipotassium phosphate 1.0

pH of the medium was adjusted at 7.0-7.2



727

Agar slants of these media were inoculated and incubated for 8 days. Formation of diffusible

pigments were noted.

2) Proteolytic activity (Gordonand Mihm, 1959)

Gelatin slabs containing 20g gelatin in 100mL distilled water were inoculated and incubated

for 7 days. The cultures were kept at 4°C for one hour and observed for

solidification/liquefaction.

3) Urease activity (Nitsch and Kutzner, 1969).[10]


Peptone 1.0

NaCl 5.0

Glucose 1.0

Phenol red 0.012

Urea 20.0

Agar 3.0

pH of the N/45 Phosphate buffer was adjusted to 6.8.

(Urea was dissolved in a small amount of buffer and sterilized by Seitz filtration and

incorporated into the medium).

The semisolid slabs were inoculated and the colour change from phenol red to darker red due

to liberation of NH3 from urea was noted for five consecutive days and compared to the

uninoculated control set without urea.

4) Hydrolysis of starch

Starch agar (Waksman, 1961)


Soluble starch 10.0

NaNO3 0.3

K2HPO4 0.3

NaCl 0.5

MgCO3 1.0

The starch agar plates were spot inoculated and after 14 days of inoculation the plates were

flooded with 2.0mL of Lugol’s iodine solution (3.0g KI and 2.0g Iodine dissolved in 300mL

distilled water). Hydrolysis of starch was noted by the formation of clear zones around the

colonies.



728

5) Production of H2S

Lead acetate agar (Kuster and Williams, 1964)


Bactopeptone 15.0

Protease peptone 5.0

Dextrose 1.0

Sodium thiosulphate 0.08

Lead acetate 0.2

The slants were inoculated with the organism and after 7 days of growth the formation of

dark brown pigment in the medium was noted.

6) Antagonistic property (Waksman, 1945)

Antimicrobial activity was tested against bacteria and fungi by cup assay method. Organism

was grown in nutrient broth medium (Waksman, 1961, modified) for 6 days. Bacillus subtilis

was placed in the nutrient agar medium and Saccharomyces cerevisiaewas placed in PDA

medium. Cups were cut in the middle of the plate, 0.1mL of the spores free culture filtrate

was put into the cup and incubated for 7 days. Inhibition of bacterial and fungal growth

around the agar cups indicated the antagonistic activity.

7) Utilization of carbon sources (Shirling and Gottlieb, 1966)

The carbon sources were filtered through a 0.45μ millipore filter before added to the

sterilized basal mineral salt agar medium.

Basal mineral salt agar


(NH4)2SO4 2.64

KH2PO4 2.38

K2HPO4 5.65

MgSO4.7H2O 1.0

The medium containing different carbon sources were plated and inoculated with the

organism. Plates containing D-glucose was considered as positive control.

Analysis of cell wall

Cell wall composition of the organism was analyzed according to the method of Boone and

Pine (1968).[1]

Culture was grown for 6 days in 50ml of modified medium of Waksmam,

1961 containing 0.5% peptone and 0.25% beef extract in 250ml conical flask. Mycelia were

collected by centrifugation and thrice with sterile water. About 0.5gm of mycelia was



729

extracted with 5ml of 0.1 (N) NaOH in tightly sealed screw capped tubes for one hour in

water bath. The mixture was cooled, centrifuged and washed with sterile water. The alkali

extract was discarded and the cell wall was suspended in 1.0ml of water and 0.7ml was

utilized for the detection of cell wall sugars and 0.3ml was utilized for the detection of cell

wall amino acids.

Sugars

A sample of 0.7ml was made to 1.0ml with 2 (N) HCl and taken in a tightly sealed screw-

capped and placed in a boiling water bath for 2 hours. The hydrolyzed materials were

transferred to a small beaker and dried on the water bath adding sterile water time to time for

evaporation of acid-water. Finally the material was suspended in 0.5ml sterile water and used

for chromatography.

Amino acids

The rest sample of 0.3ml was hydrolyzed with 6 (N) HCl and dried in the same way as

mentioned in sugars.

Detection

Both samples each of 0.1ml were spotted on Whatman number one paper separately and two

dimensional descending chromatography was performed. Primary migration of sugars and

amino acids was done with phenol: water (80ml 90% phenol: 20ml water) at 30°C for 6

hours. After complete evaporation of phenol and water the chromatography papers were

subjected to second dimensional run at the same temperature in n Butanol : Acetic acid :

Water (25 :6 : 25 v/v). For detecting sugars, the chromatograms were dipped in 25ml acetone

containing 0.1ml 50% AgNO3 and dried at room temperature and again dipped in 0.5 (N)

KOH in 90% Ethanol. On drying for 2 to 3 minutes dark brown spots appeared and the

chromatograms were dipped in 1.0% Sodium thiosulphate to remove dark background.

Whereas amino acids were detected by spraying the chromatograms with 0.2% ninhydrin

solution in nButanol and finally dried at 80°C for 5 minutes.

Antimicrobial action

Preparation of inocula

Bacterial inocula were prepared from 24 hours broth culture of each test organism in their

respective media. Bacteria were inoculated in 250ml Erlenmayer flask containing 50ml broth

medium consisting of ingredients as required for growth. The inoculated flasks were



730

incubated at 37°C for 24 hours at 120 r.p.m. for agitation. The prepared bacterial broth

cultures were employed in antimicrobial activity tests. The fungal inocula were prepared

from the spore suspensions or cell suspensions. The fungal spores/cell from the solid culture

were suspended in sterile distilled water to make the inocula for antimicrobial susceptibility

tests.

Preparation of serially diluted antibiotic fractions

Stock solution of antibiotic compound was prepared with weighed amount of lyophilized

antibiotic in sterile distilled water (1000μg/ml). From the stock solution, diluted fractions of

antibiotic were prepared. The concentrations of antibiotic in the diluted fractions were

500μg/ml, 200μg/ml, 100μg/ml, 50μg/ml, 20μg/ml and 10μg/ml. After the determination

inhibitory concentration range, the fractions were again serially diluted within the range and

assay was performed to obtain the approximate MIC against test organisms.

Microorganisms employed for the antimicrobial activity

Gram -ve and gram +ve bacterial species including the species of Mycobacterium and some

species of fungi were employed in this test (the list of microbial species is given in Table –

5).

Methods employed for the determination of MIC

Tube dilation technique was employed for the determination of antimicrobial activities and

MIC. Increasing amounts of antibiotics, 10μg/ml, 20μg/ml, 50μg/ml, 100μg/ml, 200μg/ml

and 500μg/ml, were placed in a series of culture tubes containing 5ml suitable broth medium

inoculated with 0.1ml of the test organism. In each case, control set of culture tubes was kept

where only antibiotic was absent. The inoculated culture tubes were incubated on the rotary

shaker (120 r.p.m.) at 37°C temperature for bacterial species and 30°C for fungal species.

After 72 hours of incubation, the concentration of antibiotic required to inhibit the growth of

the microorganisms was determined by colorimetric studies.



731

Fig. 1: Submerged 2 days culture of organism IIIMA showing branched mycelia with

well developed hyphae (×1000). Grown in nutrient broth medium composing 0.5%

peptone and 0.25% beef extract, incubated at 35°C.

Fig. 2: Submerged 7 days culture of organism IIIMA showing branched aerial mycelia

which are sporulated to form coccoid spores (×1000). Grown in nutrient broth medium

composing 0.5% peptone and 0.25% beef extract, incubated at 35°C.

Fig. 3: Substrate mycelia of organism IIIMA showing fragmentation. The

microorganisms grown on cover slip – solid medium interface (×1000). Grown in

nutrient broth medium composing 0.5% peptone and 0.25% beef extract, incubated at

35°C for 7 days.



732

Fig. 4: Aerial mycelia of the organism IIIMA showing chains of spores. The

microorganisms grown on cover slip – solid medium interface (×1000). Grown in

nutrient broth medium composing 0.5% peptone and 0.25% beef extract, incubated at

35°C for 14 days.

RESULTS AND DISCUSSIONS

The microorganism

The microorganism produced both substrate and aerial mycelium. The substrate mycelium

were found penetrated into agar medium and ramified in it. The substrate mycelium was

branched and yellowish in colour, fragmented at maturity, fragments are of unequal in length

and bears chains of nonmotile coccoid spores. The microorganism formed whitish yellow to

brown aerial mycelia which were sporulated. Spores were brownish in colour, coccoid in

shape, grew in chains, chains were not curved, number of spores in chain varied from 5 to 30,

aerial spores were smaller than spores of substrate mycelia. There were no distinguishable

sporophore, and absence of sporangia, sclerotia, synnemata or other special structures. The

microorganism was aerobic, heterotroph, could grow on different media with varying colony

morphology, yellow or brown diffusible pigment was secreted on some media. The

microorganism was gram positive, non-acid fast, grew well from 15°C to 40°C with optimum

range 30°C to 35°C, melanoid pigments were not produced, could not decompose cellulose

and showed no urease activity but the microorganism could liquefy gelatin, could hydrolyze

starch, produced H2S and showed antagonistic activity against gram positive and gram

negative bacteria but not against fungi. The microorganism could utilize arabinose, glucose,

maltose, moderately utilize galactose and fructose but could not utilize urea, lactose, fucose,

sorbitol, mannitol and inositol. The cell wall contained meso-diaminopimelic acid, arabinose

as detectable sugar and the cell wall belongs to type IV.



733

Taxonomic placement

The taxonomic placement of the strain was based on Bergey’s Manual of Determinative

Bacteriology, Group -22,1994 and Bergy’s Manual of Systematic Bacteriology,Vol-

4,1989,[3][18]

The strain can be excluded from the following groups of Actinomycetes by the characteristics

mentioned in each group.

1) Exclusion of Group II (Actinomycetes with multilocular sporangia)

i) Multilocular sporangia absent

ii) Aerial mycelia formed

iii) Filamentous phase present

2) Exclusion of Group III (Actinoplanetes)

i) Aerial mycelia formed

ii) Absence of single spore or sporangia on the hypha

3) Exclusion of Group IV (Streptomyces and related genera)

i) Substrate mycelia fragmented

ii) The strain contains meso-diaminopimelicacid and wall type IV

iii) Arabinose found as detectable sugar in the cell wall

iv) Spore chains on aerial mycelia not curles or spiral

4) Exclusion of Group V (Maduromycetes)

i) Substrate mycelia fragmented

ii) Arabinose detected in the cell wall

5) Exclusion of Group VI (Thermomonospora and related genera)

i) Single spore on the hypha not formed

6) Exclusion of Group VII (Thermoactinomycetes)

i) Absence of single or two spores on dichotomized hypha

7) Exclusion of Group VIII (Other genera)

i) Cell wall type IV

ii) Cell wall diagnostic sugar arabinose

Both substrate and aerial mycelia sporulated, mycelia fragmented at maturity, fragmented

hyphae of different length bearing short to long chains of spores, chain of spores on aerial

mycelia not curled or spiral, sporophore not distinguishable, no special structures like



734

sporangia, sclerotia, synnemata etc., cell wall type Iv with arabinose as detectable sugar in the

cell wall.

………………….Nocardioform Actinomycetes

Extensively branched substrate mycelia growing on the surface and penetrating agar media,

substrate mycelia of older culture fragmented into coccoid elements, aerial mycelia visible

only microscopically, short to long chains of spores formed on aerial mycelia, no endospore,

sporangia, sclerotia or synnemata formed, gram positive, non-acid fast, colonies white in

young culture, brown in older culture, colonies velvety in appearance, raised and heaped,

yellow or brown diffusible pigment produced, cell wall contained meso -diaminopimelic acid

and arabinose belonging to type IV.

…………………..Nocardia

Among the known species of Nocardia the isolated strain might be close to Nocardia carnea

as per the characteristics of the strain IIIMA determined.

Antagonistic properties

The antimicrobial activities of the antibiotic substance produced by the strain were

determined and approximate MIC was determined (Table – 5). The strain showed strong

inhibitory activity against gram positive and gram negative bacteria but there was no

antifungal activity.

Table – 1: Cultural characteristics of the organisms.

Media Colony morphology Soluble pigment

Nutrient agar (Waksman, 1961)

Round to convex, center

brownish, outer ring whitish,

outermost pale yellow and thin,

center colony raised, powdery,

heaped and velvety

Deep yellow

Glycerol Asparagines agar

(Pridham & Lyons, 1961)

Scanty growth, radiating, colony

not well organized Light yellow

Czapek’s medium agar

Around the small brown center a

thin whitish ring, outer ring

radiating and faint

Yellowish

Emerson’s agar (Waksman,

1961)

Round yellowish colony turned

white then brown, dotted brown

center surrounded by broad white

zone, outermost ring yellowish

Brown

Glucose asparagine agar

(Waksman, 1961)

Round yellowish colony turned

white then brown, dotted brown Yellowish brown



735

center surrounded by broad white

zone, outermost ring yellowish

Egg albumin agar (Waksman,

1961) Very scanty growth None

Glucose yeast extract agar

(Waksman, 1961)

Raised, powdery, center brown,

whitish ring outside, margin

radiating, lensoid

Yellow

Cellulose agar (Waksman, 1959) None None

Inorganic salt starch agar

(Waksman, 1959)

Greyish brown center, outer ring

chocolate brown, radiating,

slightly raised

Light brown

Potato glucose agar (Waksman,

1961)

Uneven circles, raised, purplish

white ring, radiating None

Potato plug (Waksman, 1961) Growth throughout the surface of

the plug, no distinguished colony Deep yellow

Inorganic salt agar (Waksman,

1961) None None

Observations were made after incubation at 32°-35°C for 21 days.

Table – 2: Utilization of carbon sources by the organism.

Carbon source Utilization

Glucose +

Galactose +

Fructose +

Maltose +

Mannose +

Sucrose +

Arabinose +

Glycerol +

Lactose -

Carboxymethyl cellulose -

Fucose -

Mannitol -

Sorbitol -

Inositol -

Urea -

Utilization of carbon sources was investigated by using the procedure of Shirling and

Gottlieb (1966)

+: utilized

-: not utilized



736

Table – 3: Physiological properties of the strain.

Physiological properties Response

Temperature range for growth (°C) 15-40

Optimum temperature (°C) 30-35

Gram nature Gram +ve.; non-acid fast

Formation of melanoid pigment (Shirling &

Gottlieb, 1966) -

Liquefaction of gelatin at 28°C (Gordon and

Mihm, 1962) +

Hydrolysis of starch (Lyons and Pridham, 1962) +

Decomposition of cellulose (Waksman, 1961) -

Production of H2S (Kuster and Williams, 1964) +

Urease activity (Nitsch and Kutzner, 1968) -

Antagonistic property (Waksman, 1965) + (Positive against species of bacteria but not

against species of fungi)

+: test positive; - : test negative

Results of cell wall analysis

Analysis of cell wall indicated L-Arabinose as diagnostic sugar and meso-DAP as diagnostic

amino acid present in the cell wall.

Table – 4: Comparison of isolated Nocardia sp. With related species.

Species of Nocardia Decomposition

of tyrosine

Production of

urease

Growth on sole carbon source (%,

w/v)

Arabinose

(1.0)

Inositol

(1.0)

Mannitol

(1.0)

N. amarae - + - + +

N. asteroids - + - - -

N. brasiliensis + + - + +

N. brevicatena - - - + +

N. carnea - - + - -

N. farcinia - + - - -

N. nova ND + - - -

N. otidiscaviarum - + - + +

N. pinensis - + ND ND ND

N. sterilae - - ND ND -

N. transvalensis - + - - -

N. vaccinii - + D + +

Isolated Nocardia sp. - - + - -

+ : test positive; - : test negative; ND : not determined; D : 11-89% strains positive



737

Table – 5: Antimicrobial activities of antibiotic.

Test organisms MIC (μg/ml)

Gram positive bacteria

Bacillus subtilis >20

Bacillus licheniformis >20

Bacillus cereus >20

Staphylococcus aureus >20

Staphylococcus albus >20

Sarcinalutea >20

Micrococcus flavus >20

Mycobacterium phlei >50

Gram negative bacteria

Escherichia coli >10

Proteus sp. >20

Salmonella gallinarum >10

Pseudomonas sp. >20

Serratiamarcescens >20

Alkaligenesviscolactis >20

Klebsiella sp. >20

Providensia sp. >20

Fungi

Saccharomyces cerevisiae -

Penicilliumnotatum -

Aspergillus sp. -

Rhizopus sp. -

ACKNOWLEDGEMENTS

The study was supported by the Department of Botany, Calcutta University and

Microbiology Department, Maulana Azad College, Kolkata.

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738

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