Sethi et al., IJPSR, 2013; Vol. 4(8): 2967-2973.

E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2013), Vol. 4, Issue 8 (Research Article)

Received on 21 March, 2013; received in revised form, 10 May, 2013; accepted, 27 July, 2013; published, 01 August, 2013

ANTIBIOTIC PRODUCTION BY MICROBES ISOLATED FROM SOIL

Sonia Sethi*, Ravi Kumar and Saksham Gupta
Dr. B. Lal Institute of Biotechnology, Malviya Nagar, Jaipur-302017, Rajasthan, India
Keywords: ABSTRACT: Antibiotic is one of the most important commercially exploited
secondary metabolites produced by bacteria, fungi and Streptomyces and
Antibiotic, Bacitracin, Bacillus, employed in a wide range. Most of the antibiotics used today are from the
Penicillium, Streptomyces,
microbes. Bacteria are easy to isolate, culture, maintain and to improve their
Antimicrobial activity
strain. Bacillus species being the predominant soil bacteria because of their
Correspondence to Author: resistant endospore formation and production of vital antibiotic like
polymyxin, bacitracin etc. are always found inhibiting the growth of the other
Dr. Sonia Sethi organisms. In the present research study, screening of bacteria, fungi and
Streptomyces with potential antibiotic activity was carried out. Among the
Associate Professor, Department of microbes isolated and identified, Bacillus subtilis, Penicillium chrysogenum
Biotechnology, Dr. B. Lal Institute of and Streptomyces sp were selected on the basis of their anti-bacterial activity.
Biotechnology, Jaipur, Rajasthan,
The inhibitory activities of the isolated microorganisms were checked against
India
Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC
E-mail: [email protected] 27853), Escherichia coli (ATCC 25922) and Klebsiella pneumonia (ATCC
4352). It was observed that Penicillium chrysogenum metabolites showed
maximum antimicrobial activity against Staphylococcus aureus,
Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia with a
zone of inhibition of 17mm, 11mm, 19.8mm and 8.2mm respectively.
Bacillus subtilis metabolites showed activity against Staphylococcus aureus
and Pseudomonas aeruginosa measured as zone of inhibition of 13.4mm and
13.8mm respectively whereas Streptomyces sp showed minimum activity
against all the four tested organisms.

INTRODUCTION: Antibiotics, in one form or With advances in organic chemistry many

another, have been in use for centuries. The vast antibiotics are now also obtained by chemical
majority of novel antibiotics have been detected by synthesis, such as the sulfa drugs. Drugs used in the
screening of “wild isolates” obtained from soil and chemotherapy of infectious diseases are classified
other natural habitats. Although a wide taxonomic into two groups. Drugs that have been synthesized
range of microbes have the ability to produce by chemical procedures in the laboratory are called
antibiotics. Thus, over 55% of the antibiotics synthetic drugs while those produced by bacteria
detected between 1945 and 1978 originated from the and fungi are called antibiotics 2.
genus Streptomyces, representing a total of more
than 5,000 compounds 1. The antibiotics are widely distributed in the nature,
where they play an important role in regulating the
QUICK RESPONSE CODE microbial population of soil, water, sewage, and
DOI:
10.13040/IJPSR.0975-8232.4(8).2967-73
compost.
Of the several hundred naturally produced
Article can be accessed online on: antibiotics that have been purified, only a few have
www.ijpsr.com
been sufficiently non-toxic to be of use in medical
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.4(8).2967-73 practice.

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Those that are currently of greatest use have derived Penicillin was discovered accidentally in 1928 by
from a relatively small group of microorganisms Fleming, who showed its efficacy in laboratory
belonging to the genera Penicillium, Streptomyces, cultures against many disease producing bacteria.
Cephalosporium, Micomonospora and Bacillus 3. This discovery marked the beginning of the
development of antibacterial compounds produced
Antibiotics are low molecular-weight (non-protein) by living organisms.
molecules produced as secondary metabolites,
mainly by microorganisms that live in the soil. Another antibiotic, streptomycin was isolated in
While many antibiotics are known to exist, 1944 by Waksman, a Microbiologist, from a species
efforts to discover new antibiotics still continue. of soil bacteria, called Streptomyces griseus,
Therefore, many species such as Streptomyces, particularly tubercle bacilli, and has proved to be
Bacillus and Penicillium have been studied very valuable against tuberculosis. A vigorous
continuously for their ability to produce antibiotics search for more antibiotics was on at this time and
4
. in 1947, another antibiotic, chloromycetin was
discovered by Burkholder 14, 15. It was isolated from
In addition, due to the fact that Bacillus species have S. venezuelae. It has a powerful action on a wide
produced antibiotics in the soluble protein range of infectious bacteria both Gram positive and
structure and that these antibiotics have been Gram negative.
found to be cheaper and more effective in
studies conducted to date, these microorganisms Most of the peptide antibiotics produced by Bacillus
are preferable for commercial production. are active against Gram positive bacteria 16.
However, compounds such as polymyxin, colistin,
Currently, the target is to produce antibiotics such as and circulin exhibit activity almost exclusively upon
polymyxin and bacitracin from Bacillus 5, 6. It was Gram-negative forms, whereas bacillomycin,
reported that members of the species Bacillus mycobacillin, and fungistatin are effective agents
generally produced polypeptide type bacteriocines, against molds and yeasts 17. As more antibiotics
and that these antibiotics generally affect Gram were discovered, designed and studied, scientists
positive bacteria 7, 8. The apparent increase of the found that they had different properties. Some of
occurrence of antibiotic resistance among bacteria these properties include their source, range of
during the past years and its possible implication in activity and their kinds. These were used to classify
public health has led to an intensified surveillance of those 14.
bacterial resistance in many countries.
The objective of the present study is production,
Treatment of infectious diseases caused by extraction and assay of antimicrobial metabolites
pathogenic bacterial and fungal strains was one of from bacterial, fungal and Streptomyces isolates
the most traditional problems in the clinical field 9, using soil as source.
10
. This necessity encouraged the investigators to
synthesize novel and more potent inhibitory MATERIAL AND METHODS:
compounds (like azoles and quinolones derivatives)
11, 12 Isolation and screening of microbial isolates: In
to fight them. However, the adverse effects and
also appearance of bacterial or fungal resistances the present study, soil sprinkle technique was used
persuaded the investigators to study on natural to isolate antibiotic producing bacilli. For this
products from microorganisms or herbal extracts to purpose about 20-30 particles of soil were sprinkled
discover novel and safe lead compounds 9, 10. on the surface of nutrient agar plates seeded with the
test organism one Gram positive Staphylococcus
It was not until 1940 with the discovery of aureus (ATCC 29213) and three Gram-negative
penicillin, the first, best-known and most widely Escherichia coli (ATCC 25922), Pseudomonas
used antibiotic 13, 14 in 1928 by an English aeruginosa (ATCC 27853), Klebsiella pneumonia
Bacteriologist, late Sir Alexander Fleming that the (ATCC 4352). The plates were incubated at 30°C
first clinical trials of penicillin were tried on for 24 hours. Antibiotic activity was checked by
humans. This antibiotic was obtained from a blue zone of inhibition, surrounding a colony.
green mould of the soil called Penicillium notatum.

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Different colonies having zones of inhibition were Identification of antibiotic producing soil
picked and streaked on separate nutrient agar plates microbes: Isolated bacterial strain was identified
to get pure cultures. These isolates were used as the morphologically (shape, gram staining, spore
source of antibiotic producing microbes. All strains staining, spore shape, sporangium dilatation and
were stored at 4°C and subcultured periodically. motility) and biochemically (sugar utilization,
starch utilization, casein hydrolysis, indole
The soil fungi were isolated by both the Direct Soil production, citrate utilization, methyl red-voges
Inoculation and the Soil Dilution Techniques using proskauer (MR-VP), oxidase production, catalase
the pour plate method. The Media used for the production, nitrate reduction, gas production from
isolation were Potato Dextrose Agar (PDA). Plates glucose according to the Bergey’s Manual of
were incubated for 5 days at 28˚C. Pure cultures of Determinative Bacteriology 19.
fungal isolates were identified using both
macroscopic (cultural) and microscopic (morpho- Pure cultures of fungal isolates were identified using
logical) features with reference to Barnett and both macroscopic (cultural) and microscopic
Hunter 18. (morphological) features with reference to Barnett
and Hunter 18.
Each fungal isolate was streaked on Nutrient Agar
as a straight line and incubated at 30˚C. After two For Streptomyces, a medium containing 10 g of
days of incubation, the test organisms, one gram yeast extract per liter and 10 g of glucose per liter
positive Staphylococcus aureus (ATCC 29213) and and pH 7.2 (yeast extract-glucose medium) was
three gram-negative Escherichia coli (ATCC used for general cultivation of actinomycetes
25922), Pseudomonas aeruginosa (ATCC 27853), strains. The media used for morphological
Klebsiella pneumonia (ATCC 4352) were streaked characterization of strain were those described by
perpendicular to the streaked line of the growing Shirling and Gottlieb 20. Cultural characteristics of
fungus. cells in various media were recorded after
incubation for 14 days at 30°C and morphological
This was then incubated at 37˚C for 24 hours, after observations were made with a light microscope by
which the zone of inhibition of each test organism using the method of Shirling and Gottlieb 20.
from the streaked line of the growing fungus was
measured. Inoculum preparation: The inoculum of Bacillus
species, identified as Bacillus subtilis, selected on
For Streptomyces, 1g of the soil were suspended the basis of maximum activity against the test
in 100 ml of physiological water (NaCl 8.5 g/1) organisms, was prepared in Tryptic Soy broth
then incubated in an orbital shaker incubator at (pH7.3) at concentration of 10% (v/v) by incubating
28 0 C with shaking at 200 rpm for 30 min. Mixtures at 30°C for 72 hours.
were allowed to settle, and serial dilutions up to
10-5 were prepared using sterile physiological The fungus was sub-cultured on PDA plates and
water and agitated with the vortex at maximum incubated at 30 0C for 3-5 days to obtain the spores
speed. An aliquot of 0.1 ml of each dilution from used for antibiotics production. Spores were washed
10-2 to 10-5 was taken and spread evenly over the into a sterile beaker using 0.1% Tween 80 in 0.1M
surface of starch casein agar medium. potassium phosphate buffer at pH 7.0. The spore
suspension was standardized such that 1 in 10
The media are added to antibiotic to inhibit dilutions has an absorbance of 0.48 at 530nm.
bacterial and fungal contamination, respectively.
Plates were incubated at 28o and 37oC and The inoculum of Streptomyces specie, selected on
monitored after 48, 72, and 96 h. Repeated the basis of maximum activity against the test
streaking on starch casein agar plates led to organisms, was prepared in Streptomyces growth
purify bacterial colonies that showed an medium was used at concentration of 10% (v/v) by
actinomycetes like appearance. The isolated strains incubating at 28°C for 4 days.
are preserved at 4oC during two months and
maintained for longer period by serial subculture.

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Production medium for antimicrobial versatile method for removing the insoluble from
compounds: the broth.

1. For Bacteria: About 50 ml of the Synthetic Penicillin rich aqueous broth was treated with
medium (g/L); L-glutamic acid 5.0; KH2PO4 activated charcoal to remove pigments and
0.5; K2HPO4 0.5; MgSO4.7H2O 0.2; MnSO4. impurities. After filtration and carbon treatment,
H2O 0.01; NaCl 0.01; FeSO4.7H2O 0.01; penicillin recovery was done by liquid-liquid
CuSO4.7H2O 0.01; CaCl2.2H2O 0.015; Glucose extraction (solvent extraction). Penicillin was
10; and pH 7), was taken in 250 ml flask as extracted from an aqueous phase into the solvent
production medium. After sterilization of butyl acetate. Potassium ions were added to
synthetic media concentrated glucose solution precipitate the salt of penicillin and this was further
previously sterilized by 0.2 μm pore size filter used for antimicrobial activity.
paper, was added to give a final concentration
of 1% in the medium. The purification of antibiotic from the production
media of Streptomyces began with filtration of the
2. For Penicillium: About 50 ml of the Synthetic broth. In the first stage, large solids and microbial
medium (g/L); 6.0g Ammonium acetate, 0.5g cells were separated by filtration, as filtration is the
NaSO3, 0.02g ZnSO4.7H2O, 0.25g MgSO4. most versatile method for removing the in soluble
7H2O, 6.0g KH2PO4, 0.02g FeSO4.7H2O, 0.5g from the broth and cell free filtrate was further used
Phenylalanine and 10.0g carbon source (lactose) for antimicrobial activity.
(pH-6.0) was taken in 250 ml flask used as
production medium. The flasks were incubated Antimicrobial activity by Agar diffusion assay:
with intermittent shaking for 21 days after Agar well diffusion method was used to check the
which the contents were sieved through cotton cultures for the production of antimicrobial
wool and filter to remove cells. metabolites. Twenty-four hours fresh cultures of one
Gram positive Staphylococcus aureus (ATCC
3. For Streptomyces: About 50 ml of the 29213) and three Gram-negative Escherichia coli
Synthetic medium (g/L); 40g Sucrose, 12.8g (ATCC 25922), Pseudomonas aeruginosa (ATCC
H3C6H5O7.H2O, 6.0g (NH4)2SO4, 0.25g MgSO4. 27853), Klebsiella pneumonia (ATCC 4352) were
7H2O, 0.15g K2HPO4, 11g CaCO3,0.25g diluted with pre-sterilized normal saline. A sterilized
MnSO4.4H2O, 0.04g ZnSO4.7H2O, 0.016 g cotton swab was dipped in the diluted cultures and
K2Cr2O7 was taken in 250 ml flask used as lawns were prepared over the agar surface.
production medium. After sterilization of
synthetic media then add 5% mycelium Wells were made in the inoculated plates using
inoculums. And incubate at 28oC with sterile corn borer. About 80 μl cell free supernatants
intermittent shaking (200 rpm) for 6-7 days. were added in the wells and the plates were
incubated at 37ºC for 24 hours. After 24 hours, the
Antibiotic production condition and Purification: zones of inhibition were observed. The diameter of
Shake flask fermentation method was used for the zone of inhibition was measured in mm with
bacterial antibiotic production. Seventy two hours well size of 6mm.
old inoculum was prepared in Tryptic Soy Broth
(pH 7.3) at concentration of 10% (v/v). Inoculum RESULTS AND DISCUSSION:
was added to the production medium and incubated
for 24 hours in orbital shaker at 120 rpm. After Isolation, Screening and Identification of
incubation, culture was centrifuged to get cell free microbial isolates: By using soil sprinkle
supernatant and was further used for antimicrobial technique, five different bacterial colonies having
activity. zones of inhibition were picked and streaked on
separate nutrient agar plates to get pure cultures. Out
The purification of penicillin from the production of 5 bacterial isolates only one bacterial isolate
media began with filtration of the broth. In the first showing maximum inhibition zone was selected and
stage, large solids and microbial cells were identified as Bacillus subtilis (Table 1).
separated by filtration, as filtration is the most

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TABLE 1: MORPHOLOGICAL AND BIOCHEMICAL By using soil dilution technique three different
TESTS FOR THE IDENTIFICATION OF BACILLUS fungal cultures having zones of inhibition were
SUBTILIS
Morphological and
picked and streaked on separate potato dextrose agar
Test plates to get pure cultures. Out of three fungal
Biochemical tests
Grams staining + strains, one isolate showing maximum inhibition
Shape Cocco bacilli zone was selected and characterized as Penicillium
Spore formation + chrysogenum by its shiny blue green mold
Motility + surrounded by white mycelium on potato dextrose
Gelatin + agar. Such colonies were visualized under light
Indole production -
microscope using phenol blue to confirm the
Citrate utilization +
Mannitol + mycelium and conidia of the fungi.
Methyl red (MR) -
Voges-Proskauer (VP) + By using soil sprinkle technique seventeen different
Oxidase + Streptomyces colonies were obtained. But five
Catalase + species having zones of inhibition were picked and
Starch hydrolysis + streaked on separate starch casein agar plates to get
Nitrate reduction + pure cultures. Out of five species only one
Casein hydrolysis + Streptomyces species (Isolate 3) showing maximum
Gas production from glucose - inhibition zone was selected (Table 2).
TABLE 2: MORPHOLOGICAL IDENTIFICATION OF STREPTOMYCES BASED ON PIGMENTATION
CHARACTERISTICS
Characteristic Observed Isolate Spore-mass colour Substrate mycelium colour Diffusible pigment production
Isolate 3 White to pink Pink +

Antimicrobial activity by Agar diffusion assay: and was found inhibiting some gram positive and
Samples drawn during batch fermentations were some gram negative organism both Bacillus lentus
subjected to agar well diffusion assay, using and Bacillus alvei also shows antibacterial activity
Staphylococcus aureus as test organisms. against Staphylococcus aureus. Bacillus pumillus
Antimicrobial activity was measured in terms of only show slight zone of inhibition on Proteus spp
zone of inhibition. The incubated samples were while it is inactive against others.
evaluated and optimum antimicrobial activity of
inoculum of Bacilius species was ensured at The culture extracts of P. chrysogenum exhibited
48hours. significant antibacterial effects against isolates. The
results show that the organism produces antibiotics
It was observed that Penicillium chrysogenum that are effective against the bacterial isolates. This
metabolites showed maximum antimicrobial activity is consistent with the fact that most strains of
against Staphylococcus aureus, Pseudomonas Penicillium chrysogenum produce β-lactam
aeruginosa, Escherichia coli and Klebsiella antibiotics, mainly penicillins 22.
pneumonia with a zone of inhibition of 17mm,
11mm, 19.8mm and 8.2mm while Bacillus Out of 22,500 microbial bioactive metabolites,
metabolites showed activity against Staphylococcus 10,100 metabolites are produced by Actinomycetes
23
aureus and Pseudomonas florescens measured as . Streptomyces are especially prolific, producing
zone of inhibition of 13.4mm and 13.8mm and around 80% of total antibiotic products 24.
Streptomyces sp showed minimum activity against
all the four tested organisms with inhibition zone of The present study was focused on antimicrobial
7.2mm, 7.5mm, 10.6mm and no zone was observed metabolites production from soil microbial isolates.
in case of Klebsiella pneumonia (Table 3). The strains showed good inhibition against all
bacteria in cross streak method were selected.
Schallmey 21, isolated soil bacteria which shows
antibiotic activity under normal growth condition

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TABLE 3: ANTIMICROBIAL ACTIVITY OF MICROBIAL METABOLITES AGAINST TEST ORGANISMS

Test Organism Control Bacillus metabolite Penicillium metabolite Streptomyces metabolite
Staphylococcus aureus
26 mm 13.4 mm 17mm 7.2mm
(ATCC 29213)
Pseudomonas aeruginosa
27 mm 13.8 mm 11.0mm 7.5mm
ATCC 27853)
Escherichia coli
25 mm 0 mm 19.8mm 10.6mm
(ATCC 25922)
Klebsiella pneumonia
25 mm 0 mm 8.2mm 0mm
(ATCC 4352)

CONCLUSION: In search of new antibiotics, Zinsser Microbiology, Prentice Hall International, UK

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How to cite this article:

Sethi S, Kumar R and Gupta S: Antibiotic production by Microbes isolated from Soil. Int J Pharm Sci Res 2013: 4(8); 2967-
2973. doi: 10.13040/IJPSR. 0975-8232.4(8).2967-73
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