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Advances in Environmental Biology, 7(1): 7-11, 2013

ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLE

Soil Screening For Antibiotic - Producing Microrgansms

1

Ahmed, Risikat Nike, 2Sani, Al Hassan,3Ajijolakewu, 4Alamu, folake bosede

1,2,3,4

Department of Microbiology, University of Ilorin, Ilorin, Nigeria.

Ahmed, Risikat Nike, Sani, Al Hassan, Ajijolakewu, Alamu, folake bosede; Soil Screening For
Antibiotic - Producing Microrgansms.

ABSTRACT
Naturally soil is rich in microorganisms capable of antibiotic synthesis but the frequency with which
synthesis occurs at ecologically significant levels in has been much less clear. Over the past decade, however,
genetic and molecular techniques, have been applied to demonstrate conclusively that microorganisms
synthesize a variety of antibiotics, even under field conditions, in the rhizosphere. Soil sample from the Post
Graduate Hostel of the Permanent Site campus,University of Ilorin, Nigeria was screened for antibioticproducing microorganisms by agar sensitivity assay. Seven bacterial species and one fungus were isolated. The
bacterial species were identified by their cellular characteristics, colonial morphology and biochemical tests.
The bacterial isolates include; Staphylococcus aureus, Proteus vulgaris, Bacillus spp., Pseudomonas
aeruginosa, Micrococcus luteus, Escherichia coli and Micrococcus varians. The fungus was identified to be
Rhizopus stolonifer using lactophenol in- cotton blue staining technique and possessed inhibitory action
against test isolates. Only Bacillus spp exhibited antibacterial activity of all the bacteria isolated.
Key words: Soil screening, Antibiotic production, Sensitivity.
Introduction
Technically defined, antibiotics include a
chemically heterogeneous group of small organic
molecules of microbial origin that, at low
concentrations, are deleterious to the growth or
metabolic activities of other microorganisms. Over
the past decade, however, genetic and molecular
techniques, have been applied to demonstrate
conclusively that microorganisms synthesize a
variety of antibiotics, even under field conditions, in
the rhizosphere i.e. that portion of the soil enriched in
carbon and energy resources released by plant roots.
That Soil is rich in microorganisms capable of
antibiotic synthesis is well accepted, but the
frequency with which synthesis occurs at
ecologically significant levels in nature has been
much less clear [1].
Actinomycetes are best known for their ability to
produce antibiotics and are gram positive bacteria
which comprise a group of branching unicellular
microorganisms.
Among actinomycetes, the Streptomycetes are th
e dominant. Important species of bacteria known to
produce antibiotics is the Bacillus spp. Over 5000
antibiotics have been identified from the culture of
gram positive, gram negative organisms and
filamentous fungi, but only a few have been
commercially used to treat human, animal and plant

diseases. The genus Streptomycete is responsible for

the formation of more than 60% of known antibiotics
[7]. The trend of search for antibiotics in the past and
in recent times as a result of drug resistance by
microbial species has required combing the earth for
various sources of antibiotics including the soil.
Materials and Methods
Sample Collection:
The debris from the soil surface of the sampling
sites was first removed before commencement of
sampling. A hand trowel sterilized with 70% ethanol
was used for the collection of soil samples from the
front of Block C at the Post Graduate Hostel,
University of Ilorin (Main Campus). The sample
was collected in a sterile polythene bag and taken to
the laboratory for analysis.
Isolation of Microorganisms from Soil Sample:
One gram of the soil sample was weighed and
diluted serially up to 10-5 with distilled water.
Dilutions of 10-2 and 10-4 were taken and introduced
into differenly labelled sterile Petri-dishes. Unto the
petri dishes, molten agar was added and swirled
evenly to ensure homogeneity of the mixture and to
discrete colonies. The plates were left to set for some

Corresponding Author
Ahmed, Risikat Nike, Department of Microbiology, University of Ilorin, Ilorin, Nigeria.
E-mail : anrisikat @gmail.com, TEL: +2348063109301.

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Adv. Environ. Biol., 7(1): 7-11, 2013

minutes and incubated at 370C for 24 hours for

bacteria while in the case of fungi incubation was
done at room for about 48-72 hours.Distinct colonies
were selected from the cultured plates after
incubation onto sterile culture media to obtain a
subculture of each colony, using a sterile inoculating
loop and inoculating needle for the bacterial species
and fungal species respectively. Slants were prepared
as stock cultures and kept in the refrigerator for
further use.
Characterization and Identification Of Bacterial/
Fungal Isolates:
The
characterization
and
subsequent
identification of isolates were carried out based on
colonial and microscopic observation according to
Fawole and Oso [4].
Screening Of Soil Isolates For Antibiotic Activity:
A sensitivity assay was carried out to determine
the antibiotic producing ability of some of the
isolates (Bacteria and fungi) against the other test
organisms isolated. This technique was aimed at
demonstrating the Alexander Flemings observation
of the discovery of penicillin. For the bacterial
sensitivity test, the isolate being screened for
antibiotic production was streaked horizontally at a
particular portion on sterile nutrient agar while the
other organisms on which the activity was tested
against were streaked vertically very close to the
screened one. Plates were incubated for 24 hours at
370C ,during which any substance produced by the
screened isolate was expected to lyse the tested
organisms growing perpendicularly and produce
zones of inhibition.
For the fungal isolate, the Sabouraud dextrose
agar plates were seeded with the test organisms to be
inhibited on one end of the plate and the isolate to be
screened was smeared nearby the other. The plates
were sincubated at room temperature for 42-72
hours.

Results:
In the screening of the soil sample taken from
Post Graduate Hostel, University of Ilorin,
Permanent Site, a total of Seven Bacterial species
and a fungus specie were isolated employing the soil
dilution plate technique. The bacterial isolates
include; Staphylococcus aureus, Proteus vulgaris,
Bacillus spp., Pseudomonas aeruginosa Micrococcus
luteus, Escherichia coli and Micrococcus varians..
The result of the bacterial identification is presented
in Table 1. The fungus Rhizopus stolonifer was
identified based on cellular and structural
morphology as shown in the list below.
Characterization and Identification of Rhizopus
stolonifer:
Surface colour White
Undersurface colour Yellow
Filament colour White
Colour of colour Black
Shape of spore Circular
The colonies on SDA were fluffy to cotton white
at first, becoming grayish brown, spreading widely
by means of aseptate stolons (completely filling the
Petri-dishes within 36-48 hours). The mycelia
appeared
multinucleated
and
non-septate,
sporangiophore erect, sporangia globose, shiny white
at first then later turning black as spores matured.
The sensitivities of the tested organisms to the
screened bacterial isolates are presented in Table 2.
Of all the screened isolates Bacillus spp. Was the
only bacterial isolate that demonstrated antibiotic
producing ability against the tested organisms,
showing zones of inhibition around the colonies of
two other tested bacteria. Table 3 shows the
sensitivity test of the fungi to the screened fungus
isolate. The sensitivity assays for both bacteria and
fungi are illustrated in plates 1 and 2.

Plate 1: Clear zone around the colony of E. coli due to sensitivity to the surrounding Bacillus species.
KEY:

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Adv. Environ. Biol., 7(1): 7-11, 2013

A = Bacillus species (Screened isolate)

B = Zone of inhibition around E. coli
C = Pseudomonas aeroginosa
D = Escherichia coli
Table 1: Identification of Bacterial Isolates.
SH
COLONIAL MORPHOLOGY
AP
E

COAGULASE

INDOLE

CITRATE

STARCHHYDROLY
SIS
SPORE STAINNING

OXIDASE

GLUCOSE

SUCROSE

LACTOSE

But
yro
us

Clu
ster
s

Irre
gul
ar

Un
dul
ate

Tran
spare
nt

Pal
e

Conv
ex

But
yro
us

A
G

A
G

Cir
cul
ar
Ro
und

Ent
ire

Tran
spare
nt
Tran
spare
nt

Yel
low
ish
Blu
ish
Gre
en

Raise
d

Gra
nula
r
But
yro
us

Sin
gle
rod
s
Ro
ds

S
m
oo
th
Ro
ug
h
Ro
ug
h

Ro
ds

A
G

Ent
ire

ELEVATION
Flat

CELLULAR
ARRANGEMNT

Flat

CONSISTENCY

Gol
den
Yel
low

PIGMENTATION

Opaq
ue

OPTICAL
CHARACTEISTICS

Ent
ire

SURFACE
TEXTURE
EDGE
S
m
oo
th

SHAPE
Cir
cul
ar

S/N

CATALASE

SUGAR
FERMENT
ATION

Cir
cul
ar

S
m
oo
th

Ent
ire

Opaq
ue

Yel
low
ish

Conv
ex

Vis
cid

Clu
ster
s

A
G

A
G

Ro
und

Gl
os
sy

Ent
ire

Tran
spa=r
ent

Wh
itis
h

Raise
d

But
yro
us

Ro
ds

A
G

A
G

A
G

Irre
gul
ar

Ro
ug
h

Lo
bat
e

Tran
spare
nt

Yel
low
ish

Raise
d

But
yro
us

Clu
ster

A
G

A
G

KEY:
+ =Indicate Positive
-=Indicate Negative

IDE
NTI
FIE
D
ORG
ANI
SM
Stap
hylo
cocc
us
aure
us
Prot
eus
vulg
aris
Bacil
lus
spp.
Pseu
dom
onas
aeru
gino
sa
Micr
ococ
cus
luteu
s
Esch
erich
ia
coli
Micr
ococ
cus
varia
ns

A=Acid Production
AG=Acid and Gas Production

Table 2: Antibiotic activity of bacterial isolates from soil.

Screened
Sampled organisms to be inhibited
Isolates
Micrococcu
Bacillus
Escherichia
s luteus
spp.
coli
Staphylococcus
_
_
aureus
Proteus vulgaris
_
_
Bacillus spp.
_
_
Pseudomonas
_
_
aeruginosa
Micrococcus
_
_
luteus
Escherichia
_
_
coli
Micrococcus
_
_
varians
KEY: + = inhibition, - = No inhibition.

BIOCHEMICA L TESTS

MOTILITY

GRAMS REACTION

CELLULAR
CHARACTERI
STICS

Pseudomona
s
aeruginosa
_

Proteus
vulgaris

Micrococcus
varians

Staphylococcu
s
aureus
_

_
+
_

_
+
_

_
_
_

_
_
_

_
_
_

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Adv. Environ. Biol., 7(1): 7-11, 2013

Table 3: Antibiotics activity of fungal isolate from the soil by seeding technique.
Screened
Sampled organisms to be inhibited
Isolate
Trichophyton rubrum
Aspergillus niger
Epidermophyton
floccosum
Rhizopus stolonifer
_
_
+
KEYS:
+ = indicates Inhibition
_ = indicates No inhibition

Trichophyton mentagrophytes
_

Plate 2: Inhibition of the growth of E. floccosum by R. stolonifer

KEY
A = Epidermophyton floccosum
B = Zone of inhibition
C = Rhizopus stolonifer
Discussion:
The antibiotic - producing microorganism
isolated from the soil sample taken from the Post
Graduate Hostel of the University of Ilorin,
Permanent Site was Bacillus sp. of all the bacterial
species originally isolated. The results conform to
many reviews of literature that Bacillus spp. are
known to produce bioactive secondary metabolites
(antibiotic). According to Prescott et al. [6] spore
forming bacteria and other members of the Bacillus
genus possess genes for the catabolism of diverse
carbon source and antibiotic synthesis. Muaz and
Shahida, [5] in a research discovered the production
of bacitracin and subtilin by Bacillus sp. Prescott et
al., [6] reported that Bacitracin produced by Bacillus
sp. inhibits Escherichia coli and Staphylococcus
aureus which corroborated the result in the present
study. The other Bacterial isolates including
Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli, Proteus vulgaris, Micrococcus
luteus, and Micrococcus varians were found to be
incapable of exhibiting antibiotic activity against the
various test organisms employed for antibiotic
sensitivity testing. This may be due to the fact that
these strains of bacterial species do not naturally
have the tendency to produce antibiotic substances.
No
actinomycetes
antibioticproducing
organism was isolated. This may have been due to
reasons such as the textures of soil and other
prevailing environmental activities at the location

where the sample was collect. Also, Actinomycetes

will take a longer time to grow than the other
bacteria as it has been reported by some literatures.
This could possibly be the reason why
Actinomycetes did not grow on the media used.
The results of antibiotic sensitivity testing of the
fungal isolate by seeding method (from table 2)
revealed that Rhizopus stolonifer did not inhibit
majority of the test organisms which include;
Trichophyton rubrum, Aspergillus niger, and
Trichophyton mentagrophytes which may be because
these organisms are not susceptible to the
antimicrobial substance produced by Rhizopus
stolonifer . However, it exhibited inhibitory effect
against Epidermophyton floccosum. This is in
agreement with some previous literatures where
Rhizopus spp were reported to exhibit amylo-lytotic
and antibiotic activity including Rhizopus stolonifer.
Prescott et al., [6] reported that Rhizopus secretes a
toxin. It was discovered that the toxin was produced
by Burkholderia which was found growing within
the fungus although this association is yet to be
clarified. Hence its inhibition of E. floccosum may be
as result of its production of antibiotics or toxin to
which E. floccosum is susceptible.
In conclusion, the extracellular substance
produced by Bacillus spp. or Rhizopus stolonifer may
equally become potent and exhibit marked effect as
antibiotics if harnessed and purified as those of the
commercially available antibiotics

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Adv. Environ. Biol., 7(1): 7-11, 2013

The discovery of antibiotics is one of the most

useful discoveries in the field of science and
particularly in the field of microbiology. This intense
search for antibiotics has paid off over the past few
decades because more of them have been
discovered,though surprisingly only a relatively
small number have been applicable to chemotherapy
, because finding a new antimicrobial substance is
only a first step in drug development.

3.

4.

5.

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