Scientific articles

Rhizosphere actinomycetes ISOLATION ON GRASS

Teki ( Cyperus rotundus) AND TEST POTENTIAL AS
PRODUCING ANTIBIOTICS

BY

Zulfikri S POU NIM:

431 411 163

GORONTALO STATE UNIVERSITY FACULTY OF

MATHEMATICS AND IPA
DEPARTMENT OF BIOLOGY
2015
 APPROVAL SHEET GUIDE

The article entitled Isolation of actinomycetes in the rhizosphere Grass Puzzle

( Cyperus rotundus and)Test
Cyperusrotundus) and Potential as a producer
Test Potential of Antibiotics
as a producer of Antibiotics

By

Zulfikri S POU

Department of Biology
 Isolation of actinomycetes in the rhizosphere Grass Puzzle ( Cyperus rotundus) and

Potential Test as Producing Antibiotics

Zulfikri S Pou 1, Wirnangsi D. Uno 2 Y. Novri Kandowangko 3

1) Students of the Department of Biology, 2) Lecturer Department of Biology, 3) Lecturer Department of Biology

Study Program of Biology, Faculty of Science, University of Gorontalo

E-mail: zulfikripou93@gmail.com

Abstract: This study aims to determine the presence of actinomycetes in the rhizosphere isolates
sedges ( Cyperus rotundus) and recognizing the actinomycetes isolates that have potential as
producers of antibiotics. The method used in this research is descriptive method. Data collection
techniques are secondary metabolites produced by these two isolates tested against bacterial
inhibition tests are Escherichia coli and Staphyloccocus aureus

to determine their potential as producers of antibiotics. Data analysis technique used is to

measure the inhibition zone is formed. Based on the test results of inhibition, one isolate which
isolates art1 able to inhibit the growth of bacteria Staphyloccocus aureus with inhibition zone
diameter of 6 mm and including a weak category while isolates ART2 not able to inhibit both
bacteria test well Escherichia coli nor

Staphyloccocus aureus. Based on the results of this study concluded that the rhizosphere sedges
( Cyperus rotundus) Actinomycetes include 2 isolates and one isolate (art1) could inhibit the
growth of bacteria
Staphylococcus aureus with a diameter of 6 mm zone of inhibition that has the potential of
producing antibiotics with weak category.

Keywords : Isolation, actinomycetes, rhizosphere, Grass Puzzle (Cyperus rotundus),

Antibiotics.

1 Yulandari Nusi Students of the Department of Biology

2 Wirnangsi D. Uno, S. Pd, M. Kes Biology Lecturer As Supervisor 1

3 Dr. Y. Novri Kandowangko, MP Lecturer of Biology As Supervisor 2
 PRELIMINARY
Appearance various kind
infectious diseases requiring antibiotics and the
nature of some pathogens that are resistant to
existing antibiotics, to encourage continued
research to find new antibiotics. Antibiotics are
metabolic products generated in a specific
organism, which is a very small amount is
detrimental or inhibit other microorganisms
(Pelczar and Chan,
1988).
Actinomycetes are a group of bacteria
producing antibiotics are most at around 70%
of antibiotics that have been found mainly
produced by actinomycetes of the genus
Streptomyces, so that targeted screening of
bacteria producing antibiotics aimed at
group actinomycetes
(Alcamo in Rofiq, 2011). More than 90% of
antibiotics produced by various species of
Streptomyces is used for the treatment of
infectious diseases caused by bacteria
(Rahayu,
2006). These bacteria are commonly found in a
variety of soil types and has the greatest
abundance which play an important role in the
decomposition process (Nurkanto, 2007).
In general, the population of
microorganisms in the rhizosphere is much
higher than other populations in the soil. The
number of microorganisms including
actinomycetes in the rhizosphere is due to the
roots of plants have the ability to remove
exudate containing organic material which is
useful as an energy source for microorganisms
that live around the roots (Ambarwati, 2007).
Rhizosphere is a portion of land
directly affected by roots
plant. Rhizosphere limit starts from the root
surface to the extent that the roots are no
longer directly affect the lives of
microorganisms (can reach 5 mm) (Saraswati et
al., 2007). Studies that have been done with
regards to actinomycetes in the rhizosphere is
Ambarwati study (2007), who studied the plant
rhizosphere microorganisms shy daughter and
cat and mouse. Based on these results
obtained 7-cetes Actinomy isolates from the
rhizosphere shy daughter ( Mimosa pudica
L.) and 1 isolates from rhizosphere
kucingkucingan ( Acalypha indica L.). Five
isolates were recovered from the rhizosphere
shy daughter and one strain of rhizosphere cat
and mouse can inhibit the growth of Escherichia
coli and
Staphylococcus aureus. Meanwhile Ambarwati et
al., ( 2013) also managed to find eight isolates
of actinomycetes from rice rhizosphere ( Oryza
sativa) potential
as producers of anti-
bacterium which could hamper
Salmonella typhosa and S. aureus
with weak and moderate categories.
Actinomycetes are saprophytic bacteria
which grow decomposing materials
organic so that
population increases when there are a lot of
organic material. According Rahayu (2006),
grassroots has
the ability to remove exudate (fluid
that comes out of cells around the root), as well
as on other plants. The results of root
exudation then spread to the grass rhizosphere
soil. As a result around the grass roots can be
found in many microorganisms. Studies that
have been done with regards to
rhizosphere grass is
Rahayu research (2006) that done research for
grass rhizosphere bacterial isolate pangola find new antibiotics. Substance
( Digitaria decumbens), antibiotics produced by microorganisms more
obtained seven isolates with different profitable than the antibiotic substance
fermentation times capable of inhibiting E. coli multiresistant.
produced by plants, this was due to the
Four isolates potentially very strong antibiotics, regeneration of microorganisms that time much
the bacteria is suspected as actinomycetes. shorter than the time to grow a crop. Bacteria
The presence of exudate released by the grass can grow and multiply within a few hours,
roots is also possible the actinomycetes can be actinomycetes within approximately one month,
obtained in the rhizosphere while the plant to produce the active ingredient
takes many years (Ambrawati, 2007).

grass Credits ( Cyperus

rotundus).
Sedges is a chronic herb that grows wild
and could care less shortly. Grass
Credits
is a plant that can easily be found in the open RESEARCH METHODS
and are often considered Place and time of research
as weeds. Research this implemented in
rhizome grass Credits ( Cyperus Microbiology laboratory department
rotundus) contains alkaloids, cineol, pinene, Biology Faculty UNG. The timing of that in
siperon, rotunol, siperenon, April to June 2015.
tannins, siperol, as well as flavonoids (Murnah,
2012). Koen research results (2012), shows Object of research
that the nut-grass rhizome extract made of The object of this research is Isolates from
sweets can be used as an alternative medicine the rhizosphere actinomycetes sedges ( Cyperus
pain of canker sores, this is due to the high rotundus) which has potential as a producer of
content of antibiotics in the nut-grass rhizome antibiotics.
extract. In addition, research Roekistiningsih et
al. ( 2012) mem-prove that the nut-grass Research methods
rhizome extract has antimicrobial activity The method used in this research is
against Escherichia coli. Their ability to extract descriptive method.
Tools and materials
Tools used: Laminar airflow, oven,
incubator, autoclave,
grass Credits Erlenmeyer, micropipette, test tubes, petri dishes, glass
as antimicrobial actinomycetes may objects ,, centrifuges, shakers
affect the ability to produce incubator, colony counter,
metabolite water bath, ose, microscope and camera.
secondary, one of which is an antibiotic. Materials used: rhizosphere soil
grass Credits ( Cyperus
The importance of this research because of rotundus), medium Starch Casein Agar, Nystatin,
their nature of some pathogens that Sterptomycin, distilled water, Ringer solution of
resistant to alcohol, carbol gentian violet, safranin, microbes
existing antibiotics, encouraging continued form
Escherichia coli and Staphylococcus aureus, Nutreint 14 days (Sembiring et al., in
order, Muller Hilton To and the fermentation Ambarwati, 2013).
medium After incubation, each colony has a
Data collection technique different appearance isolated on media SCA
sample Collection
samples for isolation to obtainable pure isolates.
actinomycetes obtained from soil Furthermore incubated at 25 0 C. for 4-14 days.
rhizosphere grass Credits ( Cyperus
rotundus). Soil sampling as many as three morphological observation
areas sedges rhizosphere of different Growing actinomycetes observed
individuals. Rhizosphere limit starts from the morphologic characters include forms colony, the
root surface to the extent that the roots are no colony surface, the edge of the colony and the
longer directly influence the microbial life (it can colony color. Cell morphological observation is
reach 5 based on the Gram stain method.

mm) (Saraswati et al., 2007). Samples are Earnings Test Antibiotics

placed in a container sterile Test income antibiotics
then taken to the laboratory for further conducted through stages
treatment. following:
Isolation a) Cultivation in liquid medium isolates Isolates of
samples rhizosphere soil in actinomycetes that
weigh as much as 1 gram then placed on were then used to test all income inability
test tube. antibiotic compounds. Actinomycetes isolates
Once it is added 9 ml of Ringer's solution and were grown on agar slant at 28 0 C for 2 weeks,
shaken for 5 minutes (this suspension is then the mature spores inoculated in the
dilution 10- 1). Taken four test tubes, each filled medium-kan International
with 9 ml of Ringer solution with a sterile
pipette. Inserted 1 ml Streptomyces Project
2 (ISP 2) as much as 100 ml (0.4 g yeast extract, 1
suspension of g of malt extract, 0.4 g of glucose, 100 ml of
dilution 10- 1 mistake one tube containing 9 ml of distilled water) (Eka cider,
Ringer solution, and shaken evenly, this 2011) and incubated at 30 0 C rotary shaker ( 160rpm)
suspension has a dilution rate of 10- 2. for 12 days (Baskaran et al.,
in Joseph,
In the same way, be made 2012).
suspension with a dilution rate of 10- 3 10- 4 and b) Isolation of Antibiotics To obtain antibiotic
10- 5. From each dilution, 1 ml samples were liquid phase, liquid medium that has been
taken and inoculated by surface plate on a terfermen-tation
medium Casein strach order centrifuged on
10,000 rpm with a temperature of 4 o C for 20
(SCA), which has been supplemented with minutes. The resulting supernatant was
25μg.ml- 1 nystatin to prevent the growth of fungi collected as a sample antibiotic (Baskaran et al., in
(Waluyo, Joseph, 2012).
2010). Media were inoculated were incubated c) Antibiotic Activity Test
at 28 0 C for 4 - Activities antibiotics could
determined by looking at the ability of
metabolite Secondary produced by bacteria using chloroform: methanol (4: 1) as a solvent
isolates against per- system. Spot formed
plant microorganisms test by disc diffusion on chromatogram
method (method KirbyBauer). The medium vapaour visualized in iodine chamber and the
used for the determination of power resistor is UV chamber.
medium TLC can be used for identification test
Muller Hilton Agar. A total of 100 mL of each raw compound. For
test microorganism suspension ( S. aureus and E.TLC analyzed data, the parameters used are
coli) the value Resolution Funjungtion
inoculated-kan on a petri dish and coupled with (Rf). Two compound
MHA medium as much as ± 15 ml and allowed is said to be identical if it has the same Rf value
to solidify. The supernatant as much as 20 mL when measured on the same TLC conditions
is dripped on paper discs and dried, (Gandjar and Rohman,
2008). Rf value this is defined
then placed on a medium as the ratio between the distance of the
had conceived compound with a developer solvent distance.
test microorganisms (Naid, 2013).
Then incubated for 2 x 24 hours at a The distance traveled compound Rf =
temperature of 36-37 0 C (Safinah,
2008). The distance traveled solvent
Observations inhibition zone formed developers (Arista, 2010)
can be seen from the area of ​the zone is
formed. According to Lee and Hwang (in
Ambarwati et al. Data analysis technique
2009), when the barrier region diameter of 5-9 To analyze the data in this research
mm the categorized inhibitory activity data analysis techniques
weak, in descriptive. isolates
10.00 to 19.00 mm is average and more than Actinomycetes found in the rhizosphere
or equal to 20 mm is categorized strong. If the grass Credits ( Cyperus
area of ​the inhibition zone is formed in the rotundus) inhibition test for the presence of
strong category, isolates that have the potential as a producer of
will next analysis antibiotics.
Thin Layer Chromatography (TLC) for phase
identification of antibiotics produced.

identification of antibiotic
RESULTS AND DISCUSSION
Determining the type of antibiotics
result
produced by actinomycetes isolates using thin
Based on the results of this research is that
layer chromatography (TLC). Prepared Silica gel
the grass rhizosphere region
plates size 10 x 20 cm and a thickness of 1 mm
Credits earned two isolates of
and activated at a temperature of 150 0 C for 30
actinomycetes which each have different
minutes. Ethyl acetate fraction as much 10μl
characteristics.

and antibiotics marker

( streptomycin) placed on the plate and the
chromatogram developed

(A) (B)
Figure 1. Isolate actinomycetes rhizos-
fer Teki grass ( Cyperus
rotundus) ( a) rhizosphere actinomycetes
Puzzle (Isolate 1); (B) the rhizosphere
actinomycetes Puzzle (Isolates 2)
In addition to observing the colony
morphology isolates obtained, also made
observations on the morphology of the cell,
namely through the Gram staining method.
(A) (B)
Figure 2. Gram stain on Isolates
(A) ART 1 and (b) ART 2
Based on the results observation
cell morphology Actinomycetes isolates
obtained using the Gram stain method, showed
morphology
Actinomycetes cell
has the shape of basil on 1 isolates and
isolates 2 and is a gram-positive for binding the
color purple. Actinomycetes isolates that were
isolated from the rhizosphere sedges ( Cyperus
rotundus) income test is then performed
secondary metabolites (antibiotics) using
methods Diffusion test ( Kirby-
Bauer). These secondary metabolites income
test uses two test bacteria that S. aureus and E.
coli.
Based on the test results metabolites income
sekuder from isolates
Actinomycetes obtained, that one
isolates could inhibit the growth of test
bacteria that isolates ART 1 that can inhibit the
growth of
bacterium test
Staphylococcus aureus that is equal to 6
mm. Inhibition zone is formed is included in the
weak category. In the test bacteria Escherichia
coli no
inhibition zone formed by isolates ART
1. Based on observations, the isolates ART 2 is
not formed inhibitory zone on the test bacteria S.
aureus
or bacteria test E. coli.
Discussion
actinomycetes constitute
filamentous Gram-positive bacteria, and can act
as a producer of a variety of bioactive
compounds that can serve among other things
as antibiotics, enzyme inhibitors, and other
bioactive compounds.
actinomycetes could
found in the area because the root rhizosphere
plants have
the ability to remove exudate containing
organic material which is useful as an energy
source for microorganisms that live around the
roots.
In this study took the three regions
rhizosphere of sedges ( Cyperus rotundus) at a
different location. Based on the results
isolation
two isolates obtained from the rhizosphere
actinomycetes sedges ( Cyperus rotundus) taken
from a location that has
to-ground
lembabannya low, while on the rhizosphere
grass Credits ( Cyperus
rotundus) taken at the location where the land is
more humid
found their actinomycetes isolates. acquired included in Gram-positive bacteria.
Rhizosphere area sedges more moist than the
surrounding soil, so it is possible the cause of Two isolates of actinomycetes
at least actinomycetes isolates were obtained. obtained further testing secondary metabolites
As which aims to determine the ability of
secondary metabolites as antibiotics. These
presented by Ambarwati (2007), secondary metabolites derived from the results
that unlike most bacteria love moist, of centrifugation medium supernatant
actinomycetes tend to live in conditions of low actinomycetes isolates were previously
humidity and the dry ground. Besides other incubated for 12 days. According to Cross, (in
causes Rofiq 2011) secondary metabolites are often
produced in large quantities and mostly
secreted into the culture medium. In the normal
is their activity life cycle, microbes will grow in the appropriate
Antimicrobial tuber sedges ( Cyperus rotundus) may
medium and produce maximum cell number.
affect After the growth stops and enters
the existence of micro-
organisms in the rhizosphere The.
As noted Rahayu (2006), that a plant capable
of producing secondary metabolites, some of phase
which have antimicrobial activity or chemicals stationary, and then entered the death phase of
toxic to microorganisms in the soil, including vegetative cell death occurs
actinomycetes. (Lysis) or sporulation. In the
stationary phase cells stop dividing and begin
production of secondary metabolites.

based on result isolation, The results showed that the metabolite

two isolates were obtained each having a
different colony morphology. ART isolates
colony morphology 1 color gray-green colonies
with wrinkled surfaces while ART 2 isolates
colony color is white with a smooth surface.
This isolates the color is affected by pigmented
hyphae. In addition to observing colony
morphology, actinomycetes isolates obtained
are also carried out observations of cell
morphology by Gram staining method.
Based on the results of Gram stain
showed actinomycetes isolates have shaped
cell morphology binding basil and purple
indicates that both isolates
secondary that
produced by ART 1 is able to inhibit bacteria S.aureus
whereas bacteria E. coli not able to be
inhibited. The diameter of inhibition zone
formed on the test bacteria S.aureus of 6 mm
that are included in the weak category.
Because potency samples
secondary metabolites actinomycetes isolates
obtained has a weak category, so in this study
did not proceed on Thin Layer Chromatography
test for the identification of antibiotics
produced. According Ambarwati, et al. ( 2012)
test purpose Thin Layer Chromatography
(TLC) is knowing
type chemical compound
as producer substanceantibiotics.
Pratama (2008) states that the
Thin layer chromatography (TLC)
detected presence compound
Antimicrobial because the location can be
determined although patches are in a complex
mixture making it possible to isolate the active
compound.
Menurtu Pelczar and Chan (1988), the
difference in the structure of cell walls causing
clogs-pok both these bacteria respond
differently to various per-lakuan and materials,
such as Gram staining and certain antibiotics.
The result of the inhibition of secondary
metabolites produced by ART isolates 2 does
not show the ability to inhibit bacterial good test
bacteria E. coli nor S. aureus. Differences
inhibition of both
isolates that
obtained is also influenced by the pigments of
the two isolates. As proposed by Schlegel (in
Rahayu, 2006) that some pigments have
antibiotic properties, the correlation between
pigmentation and the formation of secondary
metabolites diaggap as forming pigment
forming antibiotic.
based on research
Rahayu (2006), which isolate bacteria
rhizosphere pangola grass,
that isolates that have a green pigment capable metabolism that occurs in all cells of almost all
of inhibiting the growth of bacteria E. coli multiresisten
with strong compared to other isolates.
Differences in the ability of inhibitory
by metabolites secondary
of the test bacteria was also influenced by the
different structure of cell walls between Gram
positive and Gram negative bacteria. According 2011).
to Waluyo (2007), the structure of the cell wall
of Gram-negative bacteria is a layered
structure, whereas Gram-positive bacteria have
a thick layer 1 (Figure
4.2). The cell walls of Gram-negative bacteria
are more complex than Gram-positive bacteria.
The main difference is their outer membrane
layer, which includes peptidoglycan. The
presence of this membrane causes the cell wall
of Gram-negative bacteria are rich in lipids
(11-22%). Coating the outer membrane of
Gram-negative bacteria have a structure as
membrane unit. The difference is that this layer
does not only consist of just a case of the
plasma membrane phospholipids, but also
contains other lipids, polysaccharides and
proteins. Lipids and polysaccharides are closely
linked to form a distinctive structure called
lipopolysaccharide.
Most antibotik a metabolite
secondary, will
but there is antibiotics are
The primary metabolite, which is an antibiotic
formed during the exponential growth phase,
for example antibiotics
nisin polypeptide. Broadly speaking
metabolites produced by microbes are divided
into two groups, namely primary metabolites
and secondary metabolites. Primary
metabolites are generated in the biochemical
processes of anabolic and catabolic processes
that produce assimilation, respiration,
transportation, and differentiation. Primary
had either the process or the product similarity
occurring or biological function. Secondary
metabolites are chemical compounds produced
by microbes,
plants, or
animals that are not directly involved in the
growth, development, and reproduction (Rofiq,
CONCLUSION
Based on the results of research and
discussion can be concluded that:
1. Has found two isolates
Actinomycetes (art1 and ART2) from the
rhizosphere sedges ( Cyperus rotundus).
2. There is one strain that is art1
which is able to inhibit the growth of
bacteria Staphylococcus aureus diameter
zone
inhibition of 6 mm so that it has the potential
of producing antibiotics with weak category.
SUGGESTION
Suggestions in this research is
necessary to measure the factors
environment that is level
humidity to take the rhizosphere area will be
isolated actinomycetes and see
per-
comparison of the number of isolates of
actinomycetes found in the rhizosphere region
sedges ( Cyperus rotundus) who live in humid
areas and dry. In addition, it is also necessary
isolation of endophytic actinomycetes tuber
sedges ( Cyperus rotundus)
and test potential as
producing antibiotics.
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