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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 directly affected by roots
plant. Rhizosphere limit starts from the root
Appearance various kind surface to the extent that the roots are no
infectious diseases requiring antibiotics and the longer directly affect the lives of
nature of some pathogens that are resistant to microorganisms (can reach 5 mm) (Saraswati et
existing antibiotics, to encourage continued al., 2007). Studies that have been done with
research to find new antibiotics. Antibiotics are regards to actinomycetes in the rhizosphere is
metabolic products generated in a specific Ambarwati study (2007), who studied the plant
organism, which is a very small amount is rhizosphere microorganisms shy daughter and
detrimental or inhibit other microorganisms cat and mouse. Based on these results
(Pelczar and Chan, obtained 7-cetes Actinomy isolates from the
rhizosphere shy daughter ( Mimosa pudica

1988).
Actinomycetes are a group of bacteria
producing antibiotics are most at around 70% L.) and 1 isolates from rhizosphere
of antibiotics that have been found mainly kucingkucingan ( Acalypha indica L.). Five
produced by actinomycetes of the genus isolates were recovered from the rhizosphere
Streptomyces, so that targeted screening of shy daughter and one strain of rhizosphere cat
bacteria producing antibiotics aimed at and mouse can inhibit the growth of Escherichia
coli and
group actinomycetes Staphylococcus aureus. Meanwhile Ambarwati et
(Alcamo in Rofiq, 2011). More than 90% of al., ( 2013) also managed to find eight isolates
antibiotics produced by various species of of actinomycetes from rice rhizosphere ( Oryza
Streptomyces is used for the treatment of sativa) potential
infectious diseases caused by bacteria as producers of anti-
(Rahayu, bacterium which could hamper
2006). These bacteria are commonly found in a Salmonella typhosa and S. aureus
variety of soil types and has the greatest with weak and moderate categories.
abundance which play an important role in the Actinomycetes are saprophytic bacteria
decomposition process (Nurkanto, 2007). which grow decomposing materials
organic so that
In general, the population of population increases when there are a lot of
microorganisms in the rhizosphere is much organic material. According Rahayu (2006),
higher than other populations in the soil. The grassroots has
number of microorganisms including the ability to remove exudate (fluid
actinomycetes in the rhizosphere is due to the that comes out of cells around the root), as well
roots of plants have the ability to remove as on other plants. The results of root
exudate containing organic material which is exudation then spread to the grass rhizosphere
useful as an energy source for microorganisms soil. As a result around the grass roots can be
that live around the roots (Ambarwati, 2007). found in many microorganisms. Studies that
Rhizosphere is a portion of land 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
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
(A) (B) growth of
Figure 1. Isolate actinomycetes rhizos- bacterium test
fer Teki grass ( Cyperus Staphylococcus aureus that is equal to 6
rotundus) ( a) rhizosphere actinomycetes mm. Inhibition zone is formed is included in the
Puzzle (Isolate 1); (B) the rhizosphere weak category. In the test bacteria Escherichia
actinomycetes Puzzle (Isolates 2) coli no
inhibition zone formed by isolates ART
1. Based on observations, the isolates ART 2 is
In addition to observing the colony not formed inhibitory zone on the test bacteria S.
morphology isolates obtained, also made aureus
observations on the morphology of the cell, or bacteria test E. coli.
namely through the Gram staining method. 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
(A) (B)
the ability to remove exudate containing
Figure 2. Gram stain on Isolates organic material which is useful as an energy
(A) ART 1 and (b) ART 2 source for microorganisms that live around the
roots.
Based on the results observation
cell morphology Actinomycetes isolates
In this study took the three regions
obtained using the Gram stain method, showed
rhizosphere of sedges ( Cyperus rotundus) at a
morphology
different location. Based on the results
Actinomycetes cell
isolation
has the shape of basil on 1 isolates and
two isolates obtained from the rhizosphere
isolates 2 and is a gram-positive for binding the
actinomycetes sedges ( Cyperus rotundus) taken
color purple. Actinomycetes isolates that were
from a location that has
isolated from the rhizosphere sedges ( Cyperus
to-ground
rotundus) income test is then performed
lembabannya low, while on the rhizosphere
secondary metabolites (antibiotics) using
grass Credits ( Cyperus
methods Diffusion test ( Kirby-
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 secondary that
different colony morphology. ART isolates produced by ART 1 is able to inhibit bacteria S.aureus
colony morphology 1 color gray-green colonies whereas bacteria E. coli not able to be
with wrinkled surfaces while ART 2 isolates inhibited. The diameter of inhibition zone
colony color is white with a smooth surface. formed on the test bacteria S.aureus of 6 mm
This isolates the color is affected by pigmented that are included in the weak category.
hyphae. In addition to observing colony
morphology, actinomycetes isolates obtained Because potency samples
are also carried out observations of cell secondary metabolites actinomycetes isolates
morphology by Gram staining method. 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
Based on the results of Gram stain (TLC) is knowing
showed actinomycetes isolates have shaped
cell morphology binding basil and purple type chemical compound
indicates that both isolates as producer substanceantibiotics.
Pratama (2008) states that the
Thin layer chromatography (TLC) 4.2). The cell walls of Gram-negative bacteria
detected presence compound are more complex than Gram-positive bacteria.
Antimicrobial because the location can be The main difference is their outer membrane
determined although patches are in a complex layer, which includes peptidoglycan. The
mixture making it possible to isolate the active presence of this membrane causes the cell wall
compound. of Gram-negative bacteria are rich in lipids
(11-22%). Coating the outer membrane of
Menurtu Pelczar and Chan (1988), the Gram-negative bacteria have a structure as
difference in the structure of cell walls causing membrane unit. The difference is that this layer
clogs-pok both these bacteria respond does not only consist of just a case of the
differently to various per-lakuan and materials, plasma membrane phospholipids, but also
such as Gram staining and certain antibiotics. contains other lipids, polysaccharides and
proteins. Lipids and polysaccharides are closely
linked to form a distinctive structure called
The result of the inhibition of secondary lipopolysaccharide.
metabolites produced by ART isolates 2 does
not show the ability to inhibit bacterial good test
bacteria E. coli nor S. aureus. Differences Most antibotik a metabolite
inhibition of both secondary, will
isolates that but there is antibiotics are
obtained is also influenced by the pigments of The primary metabolite, which is an antibiotic
the two isolates. As proposed by Schlegel (in formed during the exponential growth phase,
Rahayu, 2006) that some pigments have for example antibiotics
antibiotic properties, the correlation between nisin polypeptide. Broadly speaking
pigmentation and the formation of secondary metabolites produced by microbes are divided
metabolites diaggap as forming pigment into two groups, namely primary metabolites
forming antibiotic. and secondary metabolites. Primary
metabolites are generated in the biochemical
based on research processes of anabolic and catabolic processes
Rahayu (2006), which isolate bacteria that produce assimilation, respiration,
rhizosphere pangola grass, transportation, and differentiation. Primary
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
had either the process or the product similarity
with strong compared to other isolates. occurring or biological function. Secondary
metabolites are chemical compounds produced
by microbes,
Differences in the ability of inhibitory
by metabolites secondary plants, or
of the test bacteria was also influenced by the animals that are not directly involved in the
different structure of cell walls between Gram growth, development, and reproduction (Rofiq,
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
CONCLUSION BIBLIOGRAPHY
Based on the results of research and
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