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Characterization and Identification of A

ctinomycetes Isolated From Contaminated

Soil in Riyadh

Al-Kadeeb, Siham, A . Al-Rokban ,Ahlam ,H. and Awad, F.

AbstractIn this study, twenty soil samples were collected to isolate heavy metals resistant actinom ycetes. The collection sites were chosen toward the four geographical directions, east, west, south and north of the gold factory in the second industrial city in Riyadh region at Saudi Arabia. Results has been shown that the best growth medium for actinom ycetes isolation was the Agar-Albumin medium. From soil samples collected six isolates were able to grew in media containing 1 mM/100 ml of either aluminum, silver or cobalt ions .All isolates were found to be belonging to the genus Streptomyces. The isolates were classified on the basis of color into four strains. The first isolate was characterized by white color chain and was identified as S. albus, two isolates has gray color chain and were scored as S. diastaticus and S. atroolivaceous, those with red chain and comprised two isolates ,they were identified as S. violaceus and S. exfoliate and one isolate with yellow color chain identified as S. niveus. The recorded results showed that S. diastaticus and S. albus were the most tolerant actinomycetes to the concentration 1 mM/25 ml medium of aluminum, silver and cobalt.

Index TermsAluminum, silver and cobalt resistant actinomycetes, Characterization and Identification of actinomycetes , Riyadh, Saudi Arabia.

1 INTRODUCTION

—————————— ——————————
OST of the point sources of heavy metal pollutants are industrial wastewater from mining and metal processing. The heavy metals are transported by runoff
water and contaminate water sources downstream from the
industrial site[1]. As a consequence, the environment becomes
acid and rich in heavy metals. Selecting microorganisms able
to survive in this condition , which are of great interest as bioremediation agent [2].Heavy metals resistance actinomycetes have shown in some previous studies, such as , Albarracı´n et al.(2005) isolated fifty actinomycetes from copper contaminated and non-contaminated area. Primary qualitative screening assays showed that 100% of the isolated microorganisms of the contaminated area were resistant up to
80mg L_1 of CuSO4. Polti(2006) used forty-one isolated actinomycetes to study qualitative and semi-quantitative
screening of chromium(VI) resistance. Eleven Cr(VI) resistant strains were characterized and identified as species of the genera Streptomyces (10) and Amycolatopsis (1). Schmidt, A. et al.,(2007) showed that strain, Streptomyces acidiscabies E13 toleratant high concentrations of heavy metals (Ni, Cu, Cd, Cr, Mn, Zn, Fe). Al-Kadeeb et al. (2009) isolated S. albus ,S. griseoflavus S. omiyaensis, S. lydicus, S. griseoflavus ,S. pactum, S. griseoviridis, S. cellulosae and S. sulphureus from Riyadh soil. They reported that S. griseoflavus and S. pactum were the most tolerate lead, mercury and copper at (1 mM/ 25 ml) followed by S. albus and S. griseoflavus. Zhao et al (2009) isolated a novel actinomycete, strain S187(T), from a marine sediment sample collected from Xinghai Bay, Dalian, China. On the basis of phenotypic and genotypic analyses, it is proposed that strain S187(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces xinghaiensis sp. Hence this
study aim to characterization and identification aluminum, silver and cobalt resistant actinomycete strains isolated from contaminated soils of the gold factory in the second industrial city in Riyadh region at Saudi Arabia.

2 MATERIAL AND METHODS

2.1. Soil samples

Four soil samples of a total weight of 4000g were collected from Eastern, Western, Northern and Southern directions of gold factory in second industrial city, Riyadh, Saudi Arabia mixed thoroughly. Approximately 2000 g of the mixed samples were sieved through screens with a 0.5 and 0.1 mm diameter opening to remove stones and other debris and used for isolation of actinomycetes which can tolerate the level of 1 mM /100ml concentration of aluminum, silver and cobalt ions. Samples were diluted with sterile water prior inoculation onto agar plates in three replicate.

2.2. Preparation of metals solutions

Aluminum, silver and cobalt solutions were prepared from Al(NO3)3.9H2O (Laboratory, Rasayan), AgNO3(Laba, Chemie) and CoCl2.6H2O(Shangal Chemical Reagent Works). The pH of the working solution was adjusting to 7.0. Fresh dilutions were used for each study.

2.3. Isolation and purification of actinomycetes

Isolation of aluminum, silver and cobalt resistant actinomycetes strains from the soil samples were carried out in Glycerol-asparagines agar[8]; Albumin medium[9]; Tryptone- Yeast extract broth (ISP 1) [10]Yeast extract- malt

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extract agar (ISP 2) [11]and Geodermatophilus[6], Actinomycete colonies were maintained by transfer to Agar albumin slant tubes every 3 months ,incubated at 28 oC and stored at 4 oC.

2.3.1. Antagonistic activity testing

Each streptomyces isolate was cultured on different media, namely starch-nitrate agar, glycerol-asparagine agar, starch casein agar and starch-amm. sulphate agar for 7 days at 28oC. Agar discs (6 mm diameter) were prepared (cut) by using a sterile cork bores and placed on the surface of agar plates freshly seeded with bacterial, yeast or fungal test-organisms. The plates were incubated for 24 h for bacteria and yeast and for 48 h in case of fungi [12].

2.3.2. Description and identification of the actinomycetes

Actinomycetes were carring out in Yeast extract broth (ISP
1);Yeast extract- malt extract agar[11];Glycerol- asparagine
agar [8]Starch-nitrate agar[13]; Inorganic salts-starch agar
(starch-ammonium sulphate agar)[14]; Tyrosine agar[15]; Synthetic medium for melanin formation[16]; Peptone-yeast extract iron agar[17]; Skimmed milk medium; Carbon

utilization medium[9]; Gelatin medium; Hutchinson medium;Nutrient agar, Oatmeal agar and Czapek’s solution agar[6].Method describe by (Harborne,1973) used for description of cell wall and the modification method of that described by Becker et al. (1964) used to describe whole cell sugar composition and determination of isomers 2,6- diaminopimelic acid (DAP) of the cell wall. The micromorphology of spore chains of the isolates were determined by direct microscopic examination as described by Shirling and Gottlieb (1966). The spore print technique described by Tresner et al. (1961) was applied to prepare materials for electron microscopy. Cultural characteristics, the colorful appearance of mature sporulating aerial mycelium (color of colony or spore color en masse) and color of substrate mycelium as view from the reverse side of the colony and its sensitivity to pH changes, as well as the diffusible pigment other than melanoides were determined on starch-nitrate agar, starch-amm. sulphate, yeast-malt agar, glycerol-asparagine agar, oatmeal agar and Czapek’s solution agar after 7, 14 and
21 day of incubation at 28oC.The production of melanin (or
melanoid) pigments was tested on peptone-yeast iron agar,
tyrosine agar and tryptone-yeast broth.The ability of isolates to assimilate the following carbon sources: D-glucose, D- fructose, L-arabinose, L-rhamnose, D-mannitol, D-xylose, i- inositol, raffinose and sucrose was tested(all of these sugars are chemically pure). As a negative control, no carbon source was added to the basal medium[9]. Biochemical characteristic
including Starch hydrolysis, Cellulose decomposition, Casein hydrolysis ,Gelatin liquefaction, Nitrate reductase activity and Coagulation and peptonization of milk were performed as recommended by Mansour, 1979 , Mansour & Shady, 1984and Mansour,1985.The ability of Streptomyces isolates to grow at different concentrations of NaCl was tested using starch- nitrate agar amended with 1-12% NaCl [24]and[25].

2.4. Screening for high aluminum silver and cobalt tolerance Actinomycetes


Solid albumin medium plate's with1 mM /25ml concentra- tion of aluminum, silver and cobalt were employed to isolated high aluminum silver and cobalt tolerance actinomycetes. There were three replicates per isolate. All plates were incu- bating at 28 °C for bacteria and yeast and for 48 h in case of fun- gi [12]

5 RESULTS AND DISCUSSION

5.1.Taxonomic identification of the Actinomycete isolates

5.1.1.Cell wall type determination

The results of the chromatographic analysis of the 6 actinomycete isolates revealed that all of these isolates contained LL-DAP, glycine and no characteristic sugars. Therefore, these isolates were classified as belonging the actinomycetes with cell wall type 1; notably Streptomycetes[19]and[26].

5.1.2.Classification and identification of the actinomycete isolate

Identification of strains has been carried out according the Identification Key of Nonomura (1974), Shirling and Gottlieb (1968a, 1968b, 1969 & 1972) and Bergey’s Manual (1989). According to their morphological characteristics (micromorphology of spore chains) all isolates were found to be belonging to the genus Streptomyces as observed by others. Siddiq (1995); Alkahtani(2005)and Al-Kadeeb et al.( 2009) reported that the actinomycetes isolated from Saudi Arabia soil belonging to the genus Streptomyces . Polti (2007) isolated Forty-one actinomycetes and identified as species of the genera Streptomyces (10) and Amycolatopsis (1).

5.1.3.Antagonistic characteristics and identification of isolates

Isolates No. 1 was characteristics by its aerial mass color in the yellow series (whitish-yellow to pale greenish yellow or pale olive yellow) on all media used(Table 1). Its spore chain was spirals (Fig. 1A) with smooth surface (Fig.1 B). Reverse side of colony: Pale yellow to greyish-yellow substrate mycelium was observed on all media used. The substrate mycelium pigment was not sensitive to change in pH (Table
1). Melanoid pigments were not formed on peptone. Yeast
iron agar, tyrosine agar or tyrptone yeast broth medium(Table
7). No pigment was found on any of the media used (Table 1).
D-glucose, D-fructose, and D-xylose were utilized for growth.
L-arabinose, L-rhamnose and D-mannitol favored slight or weak growth, whereas i-inositol, raffinose or sucrose were not utilized by this strain (Table 8). It was able to hydrolyze starch, liquefy gelatin, weakly coagulate milk and slightly reduce nitrate to nitrites, but was not able to decompose

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cellulose, peptones milk hydrolyze casein or produce H2S (Table 9). It appeared to exhibit good antimicrobial activity against Gram-negative and Gram-positive bacteria, but did affect the growth of yeast or fungal-test organisms(Table 10). It grow in presence of NaCl at a concentration of 6% (Table
11). Based on the above mentioned results, isolate No. 1 was classified as a strain belonging to Streptomyces niveus.

TABLE 1

CULTURE PROPERTIES OF THE ACTINOMYCETE ISOLATE NO. 1.

Medium

Color of colony

Color of substrate mycelium

Diffusible pigment

Starch-nitrate agar

Whitish yellow to pale greenish yellow

Greyish yellow, not pH sensitive

No

Starch-amm.

sulphate agar

Light greenish

yellow

Greyish yellow, not

pH sensitive

No

Yeast-malt

agar

Light greenish

yellow

Greyish yellow, not

pH sensitive

No

Glycerol

asparagine agar

Light yellow to

greenish yellow

Pale yellow, not pH-

sensitive

No

Oatmeal agar

Whitish-yellow to

greenish yellow

Pale yellow, not pH

sensitive

No

Czapek’s

solution agar

Pale olive to yellow

Pale greyish olive, not

pH sensitive

No

* In this table and the next tables, the following should be regarded: Colour of colony = color of aerial mycelium.

Reverse side of colony = color of substrate mycelium. Diffusible pigment =

pigment (s) other than melanoid.

1A 1B

Fig. 1 Streptomyces niveus morphological characteristics, Light micrograph showing spore chain morphology(A,x100) and Electron image (Spore surface: Smooth (B, x4.000)

Isolate No. 2.was characteristics by its aerial mass color in the white series on all the media used(Table 2). Its spore chain was spirals (Fig. 2A) with smooth surface(Fig. 2B). Reverse side of colony, not distinctive (colorless to pale yellow substrate mycelium was produced on all of the above mentioned media (Table 2). Melanin pigments were not produced on any of peptone-yeast iron agar, tyrosine agar or tryptone-yeast broth medium (Table 7). No diffusible pigment
was found in the medium on any of the media used (Table 2). D-glucose, D-fructose, D-xylose and D-mannitol were utilized for growth of this strain. L-arabinose and i-inositol favored slight or weak growth, whereas no growth was observed with L-rhamnose, raffinose or sucrose (Table 8). It was able to coagulate and peptones milk, liquefy gelatin, hydrolyze casein and reduce nitrate to nitrite but was unable to hydrolyze starch or decompose cellulose and did not produce H2S (Table
9). It was found to exhibit pronounced antimicrobial activity against Gram-negative and Gram-positive bacteria, but did not affect the growth of yeast or fungi(Table 10 ). It appeared
to tolerate relatively high concentration of NaCl up to
6%(Table 11) .Based on the above mentioned diagnostic features, isolate No. 2 was classified as a strain belonging to Streptomyces albus. S. albus,,was isolated before from Riyadh region by (Alkadeeb et al.,2009). This indicated that this
species is dominant in Riyadh region.

2A 2B

Fig. 2 Streptomyces albus morphological characteristics, Light micrograph showing spore chain morphology(A,x100) and Electron image (Spore surface: Smooth (B, x4.000)

TABLE 2

CULTURE PROPERTIES OF THE ACTINOMYCETE ISOLATE NO. 2.

Medium

Color of colony

Color of substrate mycelium

Diffusible pigment

Starch-nitrate agar

White

Not distinctive

No

Starch-amm.

sulphate agar

White

Not distinctive

No

Yeast-malt agar

White

Not distinctive

No

Glycerol asparagine agar

White

Pale yellow

No

Oatmeal agar

White

Colourless

No

Czapek’s solution

agar

White

Not distinctive

No


Isolate No. 3 was characteristic by aerial mass color in the red series on all of the media used(Table 3). Its spore chain was rectiflexibiles (Fig. 3A) with smooth surface((Fig. 3B). Reverse side of colony not distinctive; greyed yellow to yellowish brown on starch-nitrate agar and glycerol-asparagines agar and Czapek’s solution agar (Table 3). Melanoid pigments were not formed on all of the media used (Table 7). No diffusible pigment was found on any of the media used (Table 3).
D-glucose, D-fructose, L-arabinose, L-rhamnose, D-xylose, raffinose and sucrose were utilized for growth, whereas D-

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mannitol and i-inositol were not utilized by this strain (Table
8). It was able to hydrolyze starch, decompose cellulose,
liquefy gelatin, coagulate and peptones milk, hydrolyze casein, reduce nitrate to nitrites and produce H2S(Table 9).
It exhibited antimicrobial activity towards Gram-positive bacteria, but did not affect the growth of Gram-negative bacteria, yeast or fungi (Table 10). It appeared to tolerate NaCl concentration up to 8% (Table 11). On the basis of the above mentioned results, isolate No. 3 was identified as a strain belonging to Streptomyces exfoliates.
with L-rhamnose, i-inositol or raffinose (Table 8). It is able to hydrolyze starch, coagulate and peptones milk, hydrolyze casein, liquefy gelatin and reduce nitrate to nitrite, but did not produce H2S(Table 9). It exhibits a pronounced antimicrobial activity against yeast and fungi, but did not affect the growth of Gram-negative or Gram-positive bacteria(Table 10) . It could grow at NaCl concentration of 4%, but failed to grow at or above 6% (Table 11). According to the above mentioned results, isolate No. 4 was identified as a strain belonging to Streptomyces diastaticus.

TABLE 3

CULTURE PROPERTIES OF THE ACTINOMYCETE ISOLATE NO. 3.

TABLE 4

CULTURE PROPERTIES OF THE ACTINOMYCETE ISOLATE NO. 4.

* The substrate mycelium pigment was not pH sensitive when tested with 0.5N HCl or 0.5N NaOH.

3A 3B

Fig (3)Streptomyces exfoliates morphological characteristics, Light micrograph showing spore chain morphology(A,x100) and Electron image (Spore surface: Smooth (B, x4.000).

Isolate No. 4 was characteristic by aerial mass color in the grey series on all of the media used(Table 4), its spore chains spirals, meanwhile some spore chains appeared to be straight to flexuous, i.e. rectiflexibiles (Fig. 4A)with smooth surface (Fig. 4B). Reverse side of colony, not distinctive, greyed yellow to yellowish olive or yellowish brown substrate mycelium was produced on the media used (Table 4). The substrate mycelium pigment was not pH sensitive when tested with
0.5N HCl or 0.5N NaOH. Melanin pigments were not formed
all of the media used (Table 7). No pigment was found in the medium on any of the media used (Table 4). D-Glucose, D-
fructose, L-arabinose, D-mannitol, D-xylose and sucrose were utilized for growth of isolate No. 4. No growth was observed

* The substrate mycelium pigment was not pH sensitive when tested with 0.5N HCl or 0.5N NaOH.

4A 4B

Fig. 4. Streptomyces diastaticus morphological characteristics, Light micrograph showing spore chain morphology(A,x100) and Electron image (Spore surface: Smooth (B, x4.000).

Isolates No. 5 was characteristic by aerial mass color in the red series on all of the media used(Table 5). Its spore chain was spirals (Fig. 5A), with smooth surface(Fig. 5B). Reverse side of colony was yellowish pink or yellowish orange to red substrate mycelium was produced on starch-nitrate agar, starch-amm. sulphate agar, yeast-malt agar, oatmeal agar and Czapek’s solution agar, and violetish purple on glycerol- asparagine agar. This pigment appeared to be pH sensitive, changing from red in acidic pH to violet in alkaline pH (Table
5).Melanin pigments were formed on peptone-yeast iron agar
and tryptone yeast broth, but not formed on tyrosine agar
medium (Table 7). Meanwhile reddish orange to pink or

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violetish purple diffusible pigment was produced on starch- nitrate agar, starch-amm. sulphate agar, yeast malt agar, glycerol asparagine agar, oat meal agar and Czapek’s solution agar. This pigment was pH sensitive; exhibited the changes reported for the substrate mycelium pigment (Table 5).D- Glucose, D-fructose, L-arabinose, L-rhamnose, D-mannitol, D- xylose, i-inositol, raffinase and sucrose were all utilized for growth of isolate No. 5 (Table 8). It was able to hydrolyze starch, decompose cellulose, liquefy gelatin and reduce nitrate to nitrite, and weakly coagulate milk, but was unable to peptonize milk, hydrolyze casein and produce H2S (Table 9). It exhibited pronounced antibiotic activity against Gram- negative bacteria, Gram-positive bacteria, yeast and fungi (Table 10 ). It could tolerate relatively high salinity up to NaCl concentration of 6%(Table 11). On the basis of the above mentioned diagnostic features, isolate No. 5 was identified as a strain belonging to Streptomyces violaceus.

TABLE 5

CULTURE PROPERTIES OF THE ACTINOMYCETE ISOLATE NO. 5.

* The substrate mycelium pigment and the diffusible pigment appeared to be pH

sensitive, changing from red in acid pH to violet or purple in alkaline pH.

5A 5B

Fig. 5 Streptomyces violaceus morphological characteristics, Light micrograph showing spore chain morphology(A,x100) and Electron image (Spore surface: Smooth (B, x4.000).

isolates No. 6 characteristic by aerial mass color in the Grey series, (light grey to cinereous on starch-nitrate agar, starch- amm. sulphate agar and oatmeal agar, whitish grey on yeast- malt agar and Czapek’s solution agar, and light grey to medium grey on glycerol-asparagines agar (Table 6). Its spore chain rectiflexibles (Fig. 6A)with smooth surface(Fig.
6B).Reverse side of colony, not distinctive (grayed-yellow, grayed brown or yellowish brown) on the media used (Table
8).Melanin pigments were not formed on peptone-yeast iron agar, tyrosine agar or tryptone-yeast broth media (Table 7). No pigment was found on any of the media used (Table 6).D- Glucose, D-fructose, L-arabinose, L-rhamnose and D-mannitol were utilized for growth of isolate 6. The utilization of D- xylose, i-inositol and raffinose favored slight a weak growth, but no growth was detected with sucrose (Table 8). It could actively liquefy gelatin, coagulate milk and reduce nitrates to nitrites; weakly hydrolyze starch, decompose cellulose, peptones milk and hydrolyze casein, but did not produce H2S (Table 9). It exhibited antagonistic activity against Gram- negative and Gram-positive bacteria, but did not affect the growth of yeast or fungal test-organisms (Table 10). It could tolerate relatively high concentration of NaCl up to 8%( Table
11). On the basis of the above mentioned description (diagnostic characteristics), isolate No. 6 was classified as a strain belonging Streptomyces atroolivaceous.

TABLE 6

CULTURE PROPERTIES OF THE ACTINOMYCETE ISOLATE NO. 6.

Medium

Color of colony

Color of substrate mycelium

Diffusible pigment

Starch-nitrate agar

Light grey to

cinereous

Greyed yellow, not

pH sensitive

No

Starch-amm.

sulphate agar

Light grey to

cinereous

Greyed yellow

No

Yeast-malt agar

Whitish grey

Greyed brown

No

Glycerol

asparagine agar

Light grey to

medium grey

Yellowish brown

No

Oatmeal agar

Light grey to

cinereous

Greyed yellow

No

Czapek’s solution

agar

Whitish grey

Greyed yellow

No


6A 6B

Fig.6. Streptomyces atroolivaceous morphological characteristics, Light micrograph showing spore chain morphology(A,x100) and Electron image (Spore surface: Smooth (B, x4.000)

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TABLE 7

THE ABILITY OF ACTINOMYCETES ISOLATES TO PRODUCE MELANIN

PIGMENTS ON:

Isolate No.

Peptone-yeast iron agar

Tyrosine agar

Tryptone-yeast broth

1

-

-

-

2

-

-

-

3

-

-

-

4

-

-

-

5

+

-

+

6

-

-

-

TABLE 8

UTILIZATION OF DIFFERENT CARBON SOURCES BY THE

ACTINOMYCETES ISOLATES.

About actinomycetes tolerance to high concentration of aluminum,silver and cobalt, the recorded results(Table12) showed that S. diastaticus and S. albus were the most tolerant Actinomycetes to the concentration 1 mM/25 ml medium of Aluminum Silver and Cobalt.

TABLE 10

ANTAGONISTIC ACTIVITY OF THE ACTINOMYCETE ISOLATES.



TABLE 9.





TABLE 11

NACL TOLERANCE OF THE STREPTOMYCES ISOLATES OBTAINED.

BIOCHEMICAL PROPERTIES OF ACTINOMYCETES ISOLATES

Isolate

Concentration of NaCl% (w/v



+++ = Good growth + = Weak growth - = No growth ++ = Moderate growth


+ = Good reaction or good activity - = Weak reaction or slight activity

5.2.Effect of Media using for isolation and purification on the growth of Actinomycetes

About the effect of media using for isolation and purification on the growth of actinomycetes result in table (12) observed that the best medium for isolation of actinomycetes was albumin agar among the media screened.
This result agree with these reported by Al-Kadeeb et al.(2009),
while Alkahtani(2005)reported that the best medium for isolation of actinomycetes was starch and nitrate agar.

5.3. High aluminium silver and cobalt concentration tolerance Actinomycetes




TABLE 12

EFFECT OF MEDIA USING FOR ISOLATION AND PURIFICATION ON THE GROW TH OF ACTINOMYCETES

+ = Growth - = No growth

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TABLE 13

EFFECT OF HEAVY METAL ION ON THE GROW TH OF

ACTINOMYCETES AT 1 MM/25ML CONCENTRATIONS OF ALUMINUM, SILVER AND COBALT IONS ON ALBUMINE AGAR AFTER 7 D INCUBATION AT 28°C

Sig = 0.00

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