Hitit ZY et al. JOTCSB. 2022; 5(2): 157-166.
RESEARCH ARTICLE
The Isolation of Streptomyces species in Different Soil Sources from
Middle Anatolian Regions of Turkey
Zeynep Yilmazer Hitit1* , Sila Eldemir2 , Harun Buyukegen2 ,
Kemal Kesenci 2
, Suna Ertunc 1
and Bulent Akay 1
1
Ankara University, Department of Chemical Engineering, 06100, Ankara, Turkey.
2
Safa Tarim AS, Konya, Turkey.
Abstract: Streptomyces is the largest species of the actinomycetes group, with more than 500 defined
species, aerobic, gram-positive, and phylogenetic class filamentous (thin protruding in thread form). It is a
large group that is mostly found in the soil and as a secondary metabolite of its fermentation, it enables
the production of various and important components (antibiotics, chemotherapeutics, fungicides,
herbicides, and immunosuppressants) in the field of industry and medicine. In this study, six bacterial
isolates were isolated from soil samples in different regions of Turkey. Morphological characteristics, gram
staining, and PCR test were applied for identification. Six isolates, Streptomyces mutabilis, S. collinus, S.
peucetius, S. cyaneofuscatus, S. albogriseolus and, S. griseoflavus, were compared with the general
characteristics of the Streptomyces species in International Streptomyces Project. Air and reverse side
mycelium color were determined, and all were confirmed by the gram-positive test. Studies have shown
that the regions of Ankara and Konya are rich in Streptomyces species.
Keywords: Actinomycetes; Streptomyces cp.; Isolation; Antibiotic; Secondary metabolite.
Submitted: June 24, 2022. Accepted: October 30, 2022.
Cite this: Hitit ZY, Eldemir S, Buyukegen H, Kesenci K, Ertunc S, Akay B. The Isolation of Streptomyces
species in Different Soil Sources from Middle Anatolian Regions of Turkey. JOTCSB. 2022;5(2):157–66.
INTRODUCTION sporulation to occur. Gray spores form on light
brown agar and the colony reverses from dark
Actinomycetes are bacteria belonging to the brown to tan. Culture grows best between 27-37 °C.
Actinomycetales group known as actinobacteria. This group has a strong tendency to produce
They are spore-forming gram-positive bacteria and antibiotics with different chemical structures and
are characterized by the formation of mycelium, biological activities (3).
which protrudes on the medium and produces
asexual spores (1). Compared to yeast and other The presence and distribution of Streptomyces
microorganisms, they contain high amounts of species belonging to actinomycetes, the most
guanine and cytosine in their DNA structure (2). abundant group in soil, are highly affected by the
Within 24 hours, colonies that can only be seen physical and chemical conditions of the soil such as
under a microscope are formed. Colony formation is temperature, pH, types of organic materials, and
very slow. It takes 3-4 days to be seen with the moisture content. Acidic-resistant groups are the
naked eye. It takes 7-14 days of incubation to see most abundant actinomycetes in acidic soil, while
mature mycelium. Streptomyces avermitilis is a they are less abundant in soils with alkaline pH (4).
spore-forming, aerobic gram-positive bacteria They are widely recognized for their ability to
belonging to the mesophilic actinomycete group. produce industrially important enzymes, and
Spores are spherical or oval and usually occur in secondary metabolites during fermentation, and, in
chains. Albumin, glycerol-asparagine, inorganic salt, addition, to cover about 80% of antibiotic products
starch, and oatmeal agar media are required for (5). The identification and isolation of
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microorganisms producing secondary metabolites Standard microbiological methods, biochemical
have been the focus of research for several years analysis, and DNA sequencing are used to
(6). selectively identify the genus and species of isolated
microorganisms. Streptomyces forms stable
Chemical, molecular and taxonomic properties of filaments and can produce long-chain spores that
the genus Streptomyces have been revealed by grow in aerobic conditions. Direct and indirect
several studies (7–12). According to this, non- screening methods to determine antibiotic producers
acidic, rarely fragmented submerged hyphae (0.5-2 have often been used to isolate a particular
µm) are organisms in which reproduction occurs by microorganism. While direct screening of strains
dormant spores at the ends of the aerial hyphae includes bioassay or chemical methods, indirect
(13). A few exceptional species form short spore screening includes the correlation of antibiotic
chains in submerged mycelium; sclerotium, production with strain characteristics (11).
pycnidium, sporangium, and synnemata-like Streptomyces members are very important due to
structures can be formed (14). Colonies at the their ability to produce various types of secondary
beginning of growth are smooth and soft, then metabolites such as Valinomycin, Neomycin B,
hard-tight, cottony, granular, powdery, or velvety. Avermectin, Neomycin C, Bicosamycin, Colabomycin
Most strains can produce species-specific antibiotics A, Colabomycin C, Germakradionel, Hormaomycin,
(3). A large number of pigments responsible for the etc. (5).
color of the submerged micelle can be self-
produced, as well as secreted pigments. Isolation and identification of Streptomyces cp. were
carried out by three basic methods in the literature
The cell wall contains large amounts of L- (22–24). Siddique et al (2014) performed isolation
Diaminopimelic acid (L-DAP) in its peptidoglycan by using different types of media such as
structure. It contains no mycolic acid and saturated Actinomiycete isolation medium (AC) and Kuster's
iso and anteiso fatty acids, hexa and octa isolation medium (KU) for the single colony, Yeast
dehydrogenated menaquinone with nine isoprene Extract Malt Extract Glucose Medium (YMG) and
units as the predominant isoprenoid. It also contains Streptomyces isolation medium (SC) for preculture
complex polar oil containing phosphatidylinositol, and culture growths (22). Ariffuzzaman et al (2010)
phosphatidylglycerol, phosphatidylinositol, and tested Glycerolarginine isolation medium (GAM) for
phosphatidylinositol with these oil characteristics, it the single colony, Modified starch casein agar
belongs to the phospholipid type 2 group. The medium (MSCAM), Tryptone-soybean agar medium
Guanine+Cytosine ratio of its DNA varies between (TSA) and Yeast Extract Malt Extract Glucose
69-78%. It is mostly rotten; a few species are Medium (YMG) for the preculture and culture
rarely pathogenic in humans and animals, some in growths (23). Kumar et al (2010) determined the
plants. The number of species in the genus isolation of Streptomyces cp. by starch casein agar
Streptomyces is increasing (15). (SCAM) for a single colony and Yeast Extract Malt
Extract Glucose Medium (YMG) for the preculture
More than 650 species were reported in the German and culture growths (24).
Collection of Microorganisms and Cell Cultures
(DSMZ). Thus, this genus became a member of the In this study, Streptomyces antibiotic-producing
genus Actinobacteria with the highest number of bacteria were isolated from soil samples from
species in the order Actinomycetales (16). Based on Ankara and Konya regions. Different media were
all these classifications, Streptomyces has been used to screen pure Streptomyces strains. The
divided into 20 major, 41 minor, and 22 single- antibacterial activities of various isolates were
member groups. Large classes are considered to be evaluated, and their morphological structures were
the groups of species consisting of six or more types determined.
of strains, while small classes are considered to be a
single species consisting of 2-5 types of strains (16– MATERIALS AND METHODS
20).
Isolation and Identification of Streptomyces
Contents of the medium affect Streptomyces from Soil
isolation (5). It has been observed that the best The soil suspended in sterile water is diluted and
isolation occurs in media containing glycerol or spread on selective agar medium and then
starch as a carbon source, and arginine, casein, or subjected to aerobic incubation at 25-28 °C.
nitrate as a nitrogen source. Different antifungal
agents called nystatin, cycloheximide, and pimaric Collection and Preparation of Soil Samples
are generally used during isolation to obtain pure Soil samples were taken from Ankara and Konya
bacterial isolates. Streptomyces are identified by regions. The methods used in sampling and
their spore size, morphology, chains, pigmentation, preparation are given below.
physiological and biochemical properties, and
resistance to antibiotic resistance (21). Method 1 (22)
Soil samples are taken with a sterile spatula by
digging 3 cm from the soil surface and stored in
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clean, dry and sterilized polyethylene bags at 40 °C 5 g/L peptone from soybean meal, 5 g/L NaCl, and
until pre-treatment. Each 1-g soil sample is 15 g/L Agar.
suspended in 100 mL of sterile 0.9% NaCl solution
and incubated at 28°C for 30 min at 180 rpm in an Cultures showing growth in TSA medium are
orbital shaker. Samples are spread on petri dishes transferred to YMG medium, and the strains are
containing Actinomiycete isolation medium (AC), maintained at 4 °C for 2-month periods and serially
(pH=7, 5 g/L glycerol, 2 g/L KH 2PO4, 0.1 g/L maintained for a longer period. It is considered a
asparagine, 0.1 g/L MgSO4.7H2O, 1 mg/L Streptomyces culture if it grows in a YMG medium.
FeSO4.7H2O, and 15 g/L agar) and Kuster's isolation
medium (KU), (pH=7.1, 10 g/L soluble starch, 2 g/L Method 3 (24)
KNO3, 2 g/L K2HPO4, 2 g/L NaCl, 0.05 g/L Soil samples are taken from a depth of 10-15 cm.
MgSO4.7H2O, 0.02 g/L CaCO3, 0.01 g/L FeSO4.7H2O, The samples are air-dried for 1 week, crushed, and
and 18 g/L agar) and incubated for 7-10 days at 28 sieved. Each 1-g soil sample is suspended in 100 mL
°C. of sterile 0.9% NaCl and incubated at 28 °C in an
orbital shaker for 30 minutes at 180 rpm. Samples
Samples grown on Actinomycete isolation (AC) and are spread by taking 0.1 mL into petri dishes
Kuster’s agar media (KU) were incubated while the containing starch casein agar (SCAM) isolation
ones that could be Streptomyces were labeled, and medium and incubated at 28-30 °C and monitored
single colony screenings were made on Yeast for 48, 72, and 96 hours. SCAM (pH 7.2) consists of
Extract Malt Extract Glucose Medium (YMG) 10 g/L soluble starch, 1 g/L casein powder 50%
(pH=7.3, 4 g/L yeast extract, 10 g/L malt extract, 4 seawater, and 15 g/L agar. While the soil samples
g/L glucose, 20 g/L agar) containing 0.05 mg/mL grown in the SCAM medium are in the incubation
nystatin and incubated at 28 °C for 7-10 days for stage, bacteria that may be Streptomyces species
colony formation. are marked and a single colony is cultivated in the
SCAM medium and colony formation is observed at
Single colonies are transferred to Streptomyces 28 °C for 7-10 days. Repetitive transfers of cultures
isolation medium (SC) (pH= 7.0) consists of 5 g/L from SCAM isolation media are performed on YMG
glucose, 4 g/L L-glutamic, 19 g/L K 2HPO4, 0.7 g/L media. Maintained at 4 °C after repeated transfers.
MgSO4.7H2O, 1 g/L NaCl, 3 mg/L FeSO 4.7H2O, 25 Selected samples are cultivated in a single colony in
g/L agar, and 0.05 mg/L nystatin. YMG medium.
Method 2 (23) Morphological Characterization
Soil samples are taken with a sterile spatula by The morphology of Streptomyces colonies after
digging 4 cm from the soil surface and stored in colony proliferation was characterized based on air
clean, dry and sterilized polyethylene bags at 40 °C. mycelial morphology, and reverse side color
Each 1-g soil sample is diluted 1:10 and 1:100 with principles. Microscopic slides were prepared for all
sterile water or 0.9% NaCl solution, and the soil isolates and examined under the microscope.
suspensions are heated in a water bath at 50 °C for
10 minutes. Samples are spread on petri dishes Gram Stain
containing Glycerolarginine (GAM) isolation medium Gram-positive responses were investigated for
(pH 7.4) consists of 1 g/L L-Asparagine, 1 g/L Streptomyces isolates. A small amount of distilled
Dipotassium phosphate, 0.01 g/L FeSO 4.7H2O, water is dripped onto the coverslip in the sterile
0.001 g/L MgCl₂.4H₂O, 0.001 g/L ZnSO4. 7H2O, 20 cabinet, and a single colony is taken from the petri
g/L Agar, and 0.05 mg/L Nystatin and incubated at dish with the help of a loop and spread
25 °C for 7-10 days. homogeneously on the coverslip. The water in the
lamella is evaporated on the burner flame. Crystal
While the soil samples growing in GAM medium are violet solution is dripped onto the coverslip and left
in the incubation stage, colonies that could be for 1 minute. After a 1-minute period, the excess
Streptomyces are marked. Single colony was crystal violet solution is disposed of. Lugol solution
cultivated on modified starch casein agar medium is dripped and left for 1 minute. The preparation is
and incubated for colony formation at 28 °C for 7- washed with distilled water. Excess water on the
10 days. Modified starch casein agar medium lamella is disposed of. It is washed with a
(MSCAM) (pH=7.2) consists of 10 g/L soluble decolorizing solution and then washed with distilled
starch, 15 g/L agar, 50% sea water, and 0.05 water. It is dyed with saffron, left for 1 minute. The
mg/mL nystatin. Cultures taken from this isolation preparation is washed with plenty of distilled water
medium are plated on Modified starch-casein agar and dried. Some immersion oil is poured onto the
medium to which cyclohexamide and nystatin (0.05 dried preparation. It is examined under a
mg/mL) have been added. The formed colonies are microscope.
transferred back to Tryptone-soybean agar medium.
Colonies are observed on this medium by incubating Microorganisms seen in purple are marked as gram
at 25 °C for 2-7 days. Tryptone-soybean agar (+), and those seen in pink-red color are marked as
medium (TSA) (pH=7): 15 g/L peptone from casein, grams (-).
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DNA Isolation, Polymerase Chain Reaction for PCR reactions. Electrophoresis of DNA samples is
(PCR), and Sequence Analysis performed on gels containing 1% agarose. After
YMG liquid nutrient medium is prepared; the PCR, 5 μL of the obtained products are taken, mixed
selected samples are mixed with the aid of the loop with 1 μL of 6X loading paint and run under 100-
and microorganism proliferation is observed. At the Volt electric current. The 1 kb DNA Marker
end of the process, 2 mL of the sample taken from (Fermentas, Finland) is used to determine the size
the YMG liquid medium was used for DNA isolation. of the approximately 920 bp long amplicon. After
For isolation GeneJET Genomic DNA Purification Kit electrophoresis, the gels are stained in 0.2 μg/mL
(Thermo Cat No: K0721) was used and the following ethidium bromide (Biotium, Cat: 40042) solution for
steps are applied in order. 2 mL of liquid YMG 20 minutes and visualized and photographed under
medium with microorganisms is taken into UV light at 366 nm wavelength using a
Eppendorf tubes with the help of a micropipette transilluminator.
then sample is centrifuged at 5000 g for 10 min.
The supernatant is disposed of, and the precipitate Antimicrobial Activity with Well Management
is centrifuged once again under the same An antimicrobial activity study was planned on the
conditions. The supernatant is disposed of without determined species, and according to this study,
disturbing the pellet. In order to lyse the cells, 200 Escherichia coli (E .coli) and Staphylococcus (Staph)
µL of Lysis Buffer (20 mM Tris-HCl, pH:8, 2 mM pathogenic microorganisms were selected and the
EDTA, 1.2% TritonX-100, and 20 mg/mL lysozyme) effectiveness of antibiotic production of
is added and incubated for 45 min at 37 °C. 10 µL microorganisms on these pathogens was examined.
of Lysozyme enzyme is added and waited for a Lysogeny broth (LB) solid, LB liquid and LB soft
minute. At the end of the incubation, 200 µL of lysis media were prepared and sterilized. 5 µL of E. coli
solution and 100 µL of Proteinase K are added and and Staph were separately inoculated in 5 mL of LB
mixed thoroughly and incubated for 30 min at 56 liquid medium. Pathogens were activated by
0
C. 20 μL of RNase A is added to the solution and incubation at 37 °C at 180 rpm for 18 hours. LB
incubated for 10 min at room temperature. 400 µL solid medium was prepared in glass tubes as 5 mL
of 50% ethanol is added to the solution and the and LB solid medium was prepared in petri dishes as
suspension is transferred to the column with mixing. 10 mL. At the end of 18 hours, 1 µL of activated
After spinning down at 6000 g for a minute, the pathogens was taken and inoculated into 5-mL LB
bottom part is discarded, and the column is soft tubes after the media was melted at 95 °C and
transferred to new tubes. 500 µL of Washing mixed well, then poured onto LB solid petri dishes.
Solution I is added and centrifuged at 8000 g for a After media solidifies, wells were drilled in the
minute. The bottom liquid is discarded, and 500 µL divided areas of the petri dish with the well drilling
of Washing Solution II is added to the column and apparatus. Two of the wells were filled directly by
centrifuged at maximum speed for 3 minutes. The taking samples from the liquid fermentation media
bottom liquid is discarded, and the column is of isolated microorganisms, and two of them were
centrifuged again for 1 min at maximum speed. filled with centrifugated sample. One well was filled
After centrifugation, the column is transferred to a with physiological saline for negative control
new 1.5 mL microcentrifuge tube and 50 µL of purposes. After 10-15 minutes of waiting period,
elution buffer is added. DNA samples are stored at - petri dish was left to incubate for 18 hours, and at
20 °C after centrifugation at 8000 g for a minute. the end of the incubation antimicrobial activity was
The primers, which came in lyophilized form, were observed.
first diluted with ultrapure water to 100 μmol per
microliter. The stock is diluted to 10 μmol per RESULTS AND DISCUSSION
microliter for later use in PCR. PCR is performed
using DreamTaq DNA Polymerase (Cat: EP0703). The isolation of Streptomyces type antibiotic-
Standard 3-step PCR Cycling is as follows, initial producing bacteria was carried out from six soil
denaturation for a cycle for 5 min at 95 °C, samples in Ankara and Konya regions by using 3
denaturation for 35 cycles for 30 sec at 95 °C, 30 different nutrient media. After 5-7 days of
sec at 58 °C and a min at 72 °C, final extension for incubation in aerobic conditions, characteristic
a cycle for 10 min at 72 °C. colonies of Streptomyces are observed, and pure
culture is obtained by plating a single colony on
~920 base pair PCR amplicons are submitted for selective media.
Forward-Reverse DNA sequencing specific to the
16S rDNA gene. The nucleotide sequences resulting First, morphological characterization was performed
from the sequencing reaction are aligned to the based on the morphology of Streptomyces colonies,
NCBI database with the “Basic Local Alignment air mycelium morphology, reverse side color
Search Tool (BLAST)” algorithm. principles after colony proliferation. Their isolation
methods and the back and front mycelial images are
The obtained genomic DNA is tested in RAPD given in Figure 1.
reactions to show the suitability of the isolated DNA
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Figure 1: The isolated microorganism species and the isolation pathway.
S. mutabilis, S. albogriseolus and S. griseflavous are peucetius which is unlike the others in terms of
isolated with KU and AC agar medium with Method aerial and reverse side mycelia color and soluble
1. Although they are isolated with the same pigment color. S. mutabilis, S. collinus and S.
method, they have different aerial and reverse side cyaneofuscatus are the Streptomyces species that
mycelia color. The only Streptomyces species that is are isolated with Method 3.
formed in the GAM medium with Method 2 is S.
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Figure 2: Antimicrobial activity of isolated microorganism species on two different pathogenic
microorganisms.
In Figure 2, the activities of Streptomyces species of Konya soil, it was concluded that none of the
isolated from soils in Ankara and Konya regions on microorganisms were effective on E. coli and Staph.
Staph and E. coli pathogens by well diffusion
method are given. It was concluded that none of the DNA sequence analysis results and DNA sequences
microorganisms were effective on E. coli for Ankara are given in Figure 3 and PCR gel image of
soil. However, S. mutabilis shows the most microorganisms isolated from the regions Ankara
prominent activity for the Staph pathogen, as well and Konya are given in Figures 4 and 5.
as S. collinius. As can be seen from the petri images
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Figure 3: DNA sequence analysis results of isolated microorganism species.
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Figure 4. PCR gel image of microorganisms isolated from Ankara soil. M: 1 kb Marker. NC: Negative
control, 1- S. mutabilis, 4- S. collinus, 5- S. peucetius.
Figure 5. PCR gel image of microorganisms isolated from Konya soil. M: 1kb Marker. NC: Negative control,
1-S. cyaneofuscatus, 2- S. albogriseolus, 6- S. griseoflavus
Chemical, molecular, and taxonomic properties of Air, reverse side mycelium colors, and antimicrobial
the genus Streptomyces have been revealed by activities are determined.
several studies. Siddique et al. studied S. avermitilis
isolation from different soil samples of of Pakistan. CONCLUSION
Different media compositions were applied for the
screening of pure Streptomyces species and In this study, six Streptomyces species are isolated
antibacterial and antifungal activities of various from soil samples collected randomly from Ankara
isolates were studied (22). Arifuzzaman et al. and Konya regions. Studies showed that the regions
investigated screening Actionmycetes from of Ankara and Konya are rich in Streptomyces
Sundarbans soil for antibacterial compounds against species and Methods 1-3 can be used for the
some gram-negative pathogenic bacteria. For this isolation of Streptomyces species. If the specific
purpose, a GAM isolation medium was used and it Streptomyces species are to be isolated from soil,
was concluded that Karanjal region of Sundarbans is then the isolation medium in Method 1-3 must be
rich in Actinomyces (23). Kumar et al. studied the chosen carefully to reach that specific Streptomyces
isolation of Actinomycetes from the soil samples of species. Further study is in progress to see and
the wasteland and garden of Ghaziabad and improve antibiotics production of isolated
assessed their anti-bacterial properties. The SCAM Streptomyces species with fermentation using
medium was used according to that purpose and optimization methods.
fifteen isolates of Actinomycetes showed activity
against bacteria (24). In the present study six CONFLICT OF INTEREST
isolates, S. mutabilis, S. collinus, S. peucetius, S.
cyaneofuscatus, S. albogriseolus and S. There is no conflict of interest.
griseoflavus, are compared with the general
characteristics of the Streptomyces species with 3
different methods specific to Streptomyces species.
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