Egypt. J. Exp. Biol. (Bot.), 8(2): 205 – 217 (2012) © The Egyptian Society of Experimental Biology

RESEARCH ARTICLE

Nayera A.M. Abdelwahed 1 Nagwa A. Abdallah 2 Dina E. El-Ghawas 1 Said M. Badr EL-Din 3 Ahmed I. El-Diwa ny 1

ISOLATION, IDENTIFICATION AND OPTIMIZATION OF ANTIMICROBIAL METABOLITES PRODUCED BY SOIL DERIVED ACTINOMYCETES

ABSTRACT: Some actinomycete strains isolated from CORRESPONDENCE: Egyptian soil were screened for their Nayera A.M. Abdelwahed antimicrobial activities using starch casein Chemistry of Natural and Microbial Products medium. Two of the actinomycete isolates Department, National Research Centre, 12311 designated as DN.37 and DN.7 were found to Dokki, Cairo, Egypt produce a broad spectrum antimicrobial E-mail: [email protected] activity. The cultural, physiological, biochemical characteristics and the nucleotide 2 sequence of the 16S rRNA gene of the two Nagwa A. Abdallah 1 isolates strain evidenced a 98 and 99% Dina E. El-Ghawas similarity with Streptomyces cyaneus and Said M. Badr EL-Din 3 Streptomyces lavendulae respectively. The Ahmed I. El-Diwany 1 optimum antimicrobial activity occurred upon 1 using medium No. 4 for both isolates. The Chemistry of Natural and Microbial Products optimized conditions for antimicrobial Department, National Research Centre, 12311 productivity were achieved at pH (7.0-7.5); Dokki, Cairo, Egypt temperature (30 oC), agitation speed (200 2Department of Microbiology, Faculty of rpm), working volume (50 ml medium/ 250 ml Science, Ain Shams University, Cairo, Egypt conical flask) and the best incubation period 3Agricultural Microbiology Dept., National was 4 and 6 days for both St. cyaneus DN. 37 Research Centre, 12311 Dokki, Cairo, Egypt and St. lavendulae DN. 7 respectively. The optimum inoculums size was 3ml/50ml vegetative cells of 3 days old slant and spore suspension for both St. cyaneus DN. 37 and St. lavendulae DN.7. Both sugar cane molasses and corn steep liquor found to be the best carbon and nitrogen by-product sources for Streptomyces cyaneus DN. 37 while, sugar cane molasses and soybean meal for St. lavendulae DN.7. The suggest optimized medium for the highest antimicrobial productivity in case of St. cyaneus DN. 37 was sugar cane molasses, 70.0; corn steep liquor, 40.0 ml and CaCO 3, 2.0, while, for St. lavendulae DN.7 the optimized medium was sugar cane molasses, 60.0; soybean meal, 48.0, and CaCO 3, 2.0.

KEY WORDS: Streptomyces cyaneus, Streptomyces lavendulae, fermentation, optimization, secondary metabolites. ARTICLE CODE: 25.02.12

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for inhibition zone after 1-2 days for bacteria INTRODUCTION: and yeast strains and after 3 days for fungi The ability to produce a large number of strains. Strain which manifest significant chemically different secondary metabolites is inhibitory activity against all previously listed associated mostly with the filamentous pathogens was selected for further evaluation. actinomycetes. They gain special importance, Identification of the actinomycete isolate: as they are the most potent source for Colours were assessed through ISCC- production of antibiotics and other bioactive NBS Colour-Name Charts II illustrated with secondary metabolites. Each actinomycete centroid detection of the aerial, substrate strain has probably genetic potential for mycelia and soluble pigments (Kenneth and producing 10-20 secondary metabolites (Sosio Deane, 1955). Cultural characteristics were et al.; 2000; Bentley et al.; 2002). The conducted by growing the organism on yeast actinomycetes are noteworthy as antibiotic extract-malt extract agar (ISP-2), oatmeal producers and making three quarters of all agar (ISP-3), inorganic salt starch agar (ISP- known products; the Streptomyces are 4), and glycerol asparagine agar (ISP-5), especially prolific (Locci, 1989 and Nolan; peptone yeast extract-malt extract iron agar Cross, 1998). Streptomyces species are (ISP-6), tyrosine agar (ISP-7), starch casein widely recognized as industrially important agar and Czapek-Dox agar media after 14 microorganisms because of their ability to days of culturing at 30°C. Morphological produce many kinds of novel secondary characteristics of aerial hyphae, spore chain, metabolites including antibiot ics. The spore mass, spore surface, colour of aerial problems of drug resistance, patient's and substrate mycelia and diffusible pigments sensitivity and inability to control certain production were conducted by growing the infectious diseases have given an impetus for organism on ISP-4 medium for 7-days and continuous search of new antibiotics all over assessed via light and transmission electron the world. To combat the multidrug resistant microscopy (TEM). Determination of Di- organisms, introduction of new antimicrobial aminopimelic acid (DAP) and sugar pattern compounds or antibiotics from new source is were carried out according to (Becker et al.; essential (Berdy, 2005). This report describes 1964; Lechevalier and Lechevalier, 1971). the isolation of actinomycete strains DN. 37 Melanin pigment was detected according to and DN. 7 from Egyptian soil. The the method of Shirling and Gottlieb (1966), identification of these strains and its protease and lipase (Nitsh and Kutzner, extracellular activity has been assessed from 1969), pectinase (Bertheau et al., 1984) the culture broth of the submerged culture Hydrogen sulphide production was carried out fermentation. according to Kuester and Williames (1964). Nitrate reduction was performed according the MATERIAL AND METHODS: method of Cowan (1974). Xanthine and Preliminary screening: esculin degradation have been conducted according to Gordon et al. (1974). The Streptomyces spp. isolated from utilization of different carbon and nitrogen different soil in Egypt were screened for sources was carried out according to Pridham antimicrobial activities against Escherichia and Gottlieb (1948). The susceptibility of the coli ATCC 25922, Staphylococcus aureus isolates to various antibiotics was studied on ATCC 29213, Pseudomonas aeuroginosa nutrient agar plates by placing the various ATCC 27953 and the unicellular fungi antibiotic discs on the surface and incubating Candida albicans ATCC 10231 obtained from at 30°C for 7 days. (the American Type Culture Collection (ATCC), Manassas, VA, USA), as well as Molecular identification: Alcaligenes faecalis B-170, Bacillus subtilis Genomic DNA was extracted in NRRL-B-4219, Micrococcus luteus B-287 and accordance with the methods described by the filamentous fungi Aspergillus niger Pospiech and Neumann (1995) and Song et NRRL-363 obtained from the ARS culture al., (1999). Chromosomal DNAs were isolated collection (NRRL), Northern Regional by a versatile quic-prip method for of genomic Research Laboratory, Peoria, Illinois, USA, by DNA from G-positive bacteria (Streptomyces). agar well diffusion method (Kavanagh, 1972). The polymerase chain reaction (PCR) was Selected isolates were grown in starch casein performed using primers designed to amplify fermentation broth medium (Shirling and about 1000pb fragment of the DNA region of Gottlieb, 1966) at 30.0°C in a rotary shaker at Streptomyces. Phylogenetic analysis was (200 rpm) for 6 days. The culture broth was applied using Blast program filtrated and placed in the wells made on (www.ncbi.nlm.gov/blast) to assess the DNA Czapek-Dox agar plates (pH 5.6) seeded with similarities. Multiple sequence alignment and the test yeast cultures, Sabouraud dextrose molecular phylogeny were performed using agar plates (pH 5.6) seeded with the test BioEdit software (Hall, 1999). The fungal cultures and on nutrient agar plates phylogenetic tree was displayed using the seeded with the test bacterial cultures. The Tree-view program (Page, 1996). plates were incubated at 30°C and observed

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Parameters controlling antimicrobial agent Table 1. Antimicrobial activity of some locally isolated biosynthesis: actinomycetes strains under shaking cultivation conditions Seven fermentation media were Diameter of inhibition zone in (mm) inoculated with the potent strain for the Test production of antimicrobial secondary organisms Fungal Yeast Bacterial species containing the following compositions (g/l):- species species

Medium No.1, Starch casein medium

(Shirlling and Gottlieb, 1966): soluble starch,

4219

- 10.0; casein, 1.0; CaCO .2H O and K HPO , 363 3 2 2 4 B - 170

- - 287 -

B

0.5. B

B. S. E. A. A. C. M. ATCC 10231 NRRL ATCC ATCC 29213 27953 Medium No.2, Bennett’s medium (Seong NRRL ATCC 25922 Isolate No. luteus faecalis et al., 2001): D-glucose, 10.0; beef extract, Actinomycetes niger niger coli coli aureus subtilis 1.0; yeast extract, 1.0 and N-Z amine types A, P. aeuroginosa ATCC albicans 2.0. 1 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Medium No.3, Soybean medium 2 10.0 15.0 0.0 20.0 0.0 15 0.0 0.0 (Mohamed, 1969): dextrin, 15.0; soybean, 3 15.0 0.0 0.0 15.0 0.0 15.0 0.0 0.0 4 10.0 20.0 0.0 25.0 0.0 20.0 0.0 0.0 30.0; CaCO3.2H2O, 10.0 and MgSO 4.7H2O, 1.0. 5 0.0 0.0 0.0 10.0 0.0 14.0 0.0 0.0 Medium No.4, C medium (Gastaldo and 6 0.0 20.0 0.0 0.0 0.0 0.0 0.0 0.0 Marinelli, 2003): D-glucose, 10.0; soluble 7 15.0 18.0 17.0 15.0 16.0 16.0 16.0 16.0 starch, 35.0; casein hydrolysate, 5.0; yeast 8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15.0 extract, 8.0; meat extract, 3.5; soybean meal, 9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.5 and CaCO3.2H2O, 2.0. 10 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Medium No.5, ISP-2 medium (Pridham et 11 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 12 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 al.; 1957): glucose, 4.0; malt extract, 10.0 and 13 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 yeast extract, 4.0. 14 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Medium No. 6, Modified C medium: - 15 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 soluble starch, 45.0; soybean meal, 3.5 and 16 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 17 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 CaCO3, 2.0. 18 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Medium N0. 7, Modified C medium: 19 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 glucose, 30.0; yeast extract, 16.0 and CaCO 3, 20 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0. 21 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Medium N0. 8, Corn steep liquor- sugar 22 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 cane molasses medium: sugar cane molasses, 23 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 24 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 70.0; corn steep liquor, 40.0 ml and CaCO 3, 25 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0. 26 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Medium N0. 9, Soybean meal- sugar 27 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 cane molasses medium: sugar cane molasses, 28 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 29 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 60.0; soybean meal, 48.0 and CaCO 3, 2.0. 30 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 The fermentation media were incubated 31 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 º for 7 days, at about 30 C for 6 days under 32 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 shaking conditions. After incubation, 33 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 evaluation for antimicrobial effects by well- 34 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 diffusion method against previously test 35 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 pathogens was determined. 36 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 37 30.0 37.0 20.0 30.0 19.0 20.0 30.0 30.0 Optimization conditions: 38 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Optimal conditions for antimicrobial 39 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 compound(s) production include media 40 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 composition, incubation period, temperature, 41 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 initial pH, agitation speed, inoculum type, size 42 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 and age, different volume and carbon and 43 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 44 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 nitrogen sources. 45 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 46 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 RESULTS: 47 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 48 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Preliminary screening: Identification of the actinomycete isolates: The active metabolites produced by the actinomycete isolate DN.37 and DN. 7 was The identification was performed characterized by its broader antibacterial basically according to many different activity, because it affected the growth of all international keys such as Bergey’s Manual of th the tested bacteria, showing a stronger Determinative Bacteriology 8 edition. On the activity against Gram positive and Gram basis of the examination of the macro- negative bacterial, unicellular and filamentous morphological characteristics of isolates fungi under study as shown in table 1. number DN.37 and DN. 7 on starch casein

I ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org 208 Egypt. J. Exp. Biol. (Bot.), 8(2): 205 – 217 (2012) agar medium (Table 2 & Fig. 1) indicate that reference strains, they found to be belonged both isolates in relation to the closest to genus Streptomyces. Table 2. Identification of the actinomycete isolates Cultural characteristic on starch Test organism Microscopic examination Cell wall DAP Total sugar casein agar medium

Growth is good A.m. is white Spore chain is straight Not LL-DAP DN. 37 S. m. is yellow Spore surface is smooth detected D. p. is yellow

Growth is good Spore chain is rectflexibilies A. m. is whitish grey Not Spore surface is spiny LL-DAP DN. 7 S. m. is creamy detected

No diffusible pigment

Note: A.m. = aerial mycelium, S. m. =substrate mycelium and D.p. =diffusible pigment. Molecular Identification of the Streptomyces isolates: The resulted sequence from PCR amplification process was aligned with the corresponding sequences of representive Streptomyces species, figure 2. The sequence data of the both isolates was then analyzed by Blast program (www.ncbi.nlm.gov/blast) to assess the DNA similarities. Multiple sequence alignment and molecular phylogeny were performed using BioEdit software and the phylogenetic tree, figure 3 was displayed using the TREEVIEW program. The obtained results revealed that the sequence of the isolate No. 37 showed highest similarity (98%) with Streptomyces coerulescens (Streptomyces cyaneus) and the sequence of the isolate No.7 showed highest similarity (99%) with Streptomyces flavotricini (Streptomyces lavendulae).

Fig. 2. PCR product gel analysis of the isolated Streptomyces species, where (M) is the 100bp Fig. 1. TME of the actinomycetes isolates DN. 37 DNA ladder (marker), (1) is the isolated strain and No. 7 growing on starch casein medium, No. 37 and (2) is the isolated strain No. 7. (A) spore chain is straight and spore surface is smooth X 25.000; (B) spore chain is rectiflexibiles and spore surface is spiny X 16.000.

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AB045859 Streptomyces panayensis

HQ611067 Streptomyces sp. zx-10-20

EU124566 Streptomyces heteromorphus strain OSS 34

EU570714 Streptomyces coeruleofuscus strain 174514

FJ406114 Streptomyces coerulescens strain AS 4.1597

AJ399473 Streptomyces coeruleofuscus strain ISP 5144

AB184840 Streptomyces coeruleofuscus strain NBRC 12757

DQ026668 Streptomyces coeruleofuscus strain NRRL B-5417

AB045865 Streptomyces heteromorphus

AY999765 Streptomyces cinereospinus strain JCM 6917

AB184648 Streptomyces cinereospinus strain NBRC 15397

Isolate 71

FJ883739 Streptomyces fulvissimus strain cfcc3058

FJ883750 Streptomyces niveoruber strain cfcc3145

HQ143637 Streptomyces champavatii strain TZQ46

HQ143615 Streptomyces sp. strain RHH34

HQ268537 Streptomyces odorifer strain P3

HQ268538 Streptomyces limosus strain P5

AB184498 Streptomyces rosa subsp. notoensis strain NBRC 13807

AY999780 Streptomyces calvus strain ISP 5010

GU433227 Streptomyces calvus strain ABRIINW 673 0.01 A

EU196532 Streptomyces globosus clone B15 JF330117 Streptomyces sp. neau-Q2 EU841670 Streptomyces flavotricini strain HBUM174933 FJ486464 Streptomyces flavotricini strain HBUM175041 FJ532404 Streptomyces flavotricini strain HBUM175089 HQ143623 Streptomyces flavotricini strain TZH3 GQ357943 Streptomyces sp. HNDD1 GQ262795 Streptomyces toxytricini strain vh17 FJ772053 Streptomyces flavotricini strain IMER-B3-32 GQ357983 Streptomyces sp. NXHG3 EU841607 Streptomyces flavotricini strain HBUM174888 FJ772054 Streptomyces flavotricini strain IMER-B3-28 GQ357979 Streptomyces sp. NXLJ11 GU350503 Streptomyces polychromogenes strain MJM11317 DQ459019 Streptomyces lavendulae strain ZGO429 Isolate 7 EU841608 Streptomyces olivochromogenes strain HBUM174717 AB184287 Streptomyces noboritoensis strain NBRC 13065 FJ486378 Streptomyces phaeochromogenes strain HBUM174705 EU594474 Streptomyces phaeochromogenes strain NRRL B-1517 EU594477 Streptomyces phaeochromogenes strain NRRL B-2123 AB184235 Streptomyces racemochromogenes strain NBRC 12906 DQ026656 Streptomyces racemochromogenes strain NRRL B-5430 DQ442505 Streptomyces herbaricolor strain NRRL B-3299T EU699488 Actinomycetales bacterium TLI012 EU699674 Actinomycetales bacterium TLI216 D85106 STM16RRNAA Streptomyces lavendulae strain IFO 3125 EF654092 Streptomyces katrae strain NRRL B-3093 AB184409 Streptomyces katrae strain NBRC 13447 EF017710 Streptomyces roseus EU841669 Streptomyces polychromogenes strain HBUM174749 DQ645958 Streptomyces lavendulae strain xjy AB184746 Streptomyces erythrochromogenes strain NBRC 3304 GQ328750 Streptomyces polychromogenes strain DC-19 HQ538727 Streptomyces polychromogenes strain 002 FJ486455 Streptomyces polychromogenes strain HBUM173123 FJ547116 Streptomyces polychromogenes strain HBUM175164 0.01 B Fig. 3. Phylogenetic tree based on about 1000bp fragment of the DNA region sequencing for (A) DN. 37 and (B) DN. 7

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Production optimization: Different pH: Different fermentation media: The initial pH value of the present data showed a significant influence on the Among the five different media 1, 2, 3, maximum productivity of the antimicrobial 4, and 5 used in this study, both secondary metabolites, as well as on the Streptomyces spp. give the highest activity growth of the producer microorganisms. The against the test organism under study when two isolates under investigation showed a cultivated in medium No. 4 for 6 days under lower growth and production when the initial shaking cultivation conditions with an reaction of the medium was adjusted at pH 4, inhibition zone diameter of (32 and 23 mm) while higher pH values yielded better growth while static cultivation showed lower and antimicrobial secondary metabolite production under the same other cultivation production that showed the maximum conditions as shown in figure 4. antibiotic activity at an initial pH of 7.0-7.5 (Fig. 5).

34 32 Static conditions 30 30 Shaking conditions 28 26 24 22 25 20 18 16 14 20 12 10 8 6 15 4 Streptomyces cyaneus DN.37 Inhibiton zone diameter (mm) diameter zone Inhibiton 2 Streptomyces lavendulae DN.7 0 4 5 6 7 8 9 10 Intial pH

Inhibiton zone (mm) diameter Fig. 5. Effect of different initial pH on the production 5 of antimicrobial by both Streptomyces spp. isolates

0 Incubation period: 1 2 3 4 5 6 The optimum incubation period for Medium No. Streptomyces cyaneus DN.37 was found to be Streptomyces cyaneus DN. 37 4 days while, that of Streptomyces lavendulae DN.7 was 6 days, increasing incubation time to 5 and 8 days led to noticed depletion of 24 antimicrobial activity of the both Streptomyces Static conditions species. After that, a steadiness of 22 Shaking conditions antimicrobial agent(s) productivity was 20 observed as shown in figure 6. 40 18 16 35 14 30

12 25

10 20

8 15 6 10

Inhibitonzone diameter (mm) 4 5 Streptomyces cyaneus DN.37 Inhibition Zone diameter (mm) diameter Zone Inhibition 2 Streptomyces lavendulae DN.7 0 0 0 2 4 6 8 10 12 14 16 18 1 2 3 4 5 Incubation period/ day Medium No. Fig. 6. Effect of different incubation period on the Streptomyces lavendulae DN.7 production of antimicrobial compounds by both Streptomyces spp. isolates Fig. 4. Effect of different fermentation media on the production of antimicrobial compounds produced Incubation temperature: by both Streptomyces spp. isolates under Results illustrated in figure 7 show that different cultivation conditions. the maximum antimicrobial secondary

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org Abdelwahed et al., Isolation, Identification and Optimization of Antimicrobial Metabolites Produced by Soil Actinomycetes 211 metabolites production was obtained at afterward up to 45°C at which the lowest incubation temperature 30°C for the two antimicrobial productivity was obtained. selected isolates, then obvious decline 40

35

30

25

20

15

10

Inhibition zone diameter(mm) zone Inhibition 5 Streptomyces cyaneus DN.37 Streptomyces lavendulae DN.7 0 0 5 10 15 20 25 30 35 40 45 Incubation temp. (°C) Fig. 7. Effect of different temperature on the production of compounds by both Streptomyces spp. isolates Different agitation speed: antimicrobial compound increased reaching Upon studying the effect of different its maximum value of 180 rpm for both agitation speed, it was found that at low rpm Streptomyces cyaneus DN.37 and (70 rpm) there was no antimicrobial compound Streptomyces lavendulae DN.7 (37-23 mm). production for Streptomyces lavendulae DN.7, On the other hand, as the agitation speed while, for Streptomyces cyaneus DN.37 it increased to 300 rpm, the amount of gave low antimicrobial compound of 15 mm antimicrobial compound decreased for both inhibition zone diameter. Furthermore, as the Streptomyces spp (Fig. 8). agitation speed increased, the amount of 45

40

35

30

25

20

15

10

Inhibition Zone diameter (mm) diameter Zone Inhibition 5 Streptomyces cyaneus DN.37 Streptomyces lavendulae DN.7 0 0 50 100 150 200 250 300 350 Different agitation speed (rpm) Fig. 8. Effect of different agitation speed on the production of antimicrobial compounds by both Streptomyces spp. Isolates Different inoculums size, type and age: the use of vegetative cells of 1and 2 days Upon studying the effect of inoculum growth for both isolates. type, size and age on antimicrobial Carbon sources: secondary metabolites production by We explored the effect of carbon Streptomyces cyaneus DN.37 it was found sources on antimicrobial secondary that as the inoculum size of vegetative cells metabolites production by inoculating the two increased, the production increased linearly selected isolates on the basal medium through the incubation time reaching its having different carbon source. In this maximum yield after 4 days (37 mm) when 3 experiment, the previously selected C and 4 ml vegetative cells /250ml flask were medium (medium No. 4) for both isolates used. While, in case of Streptomyces were prepared and sterilized, Then the lavendulae DN.7, upon using spores as amount of carbon content were calculated inoculum, production was noticed and and replaced by equivalent amounts of increased during the incubation period till different carbon sources under test. The reached maximum production upon using 3 latter were sterilized separately and then and 4 ml spore suspension /250ml flask added individually to the sterile fermentation after 6 days of incubation time (23 mm). medium under aseptic conditions. The Concerning inoculum age, the use of maximum production was in the following vegetative cells of 3 days growth revealed manner (starch > raffinose and lactose > higher yield through the incubation time than sucrose and mannose > fructose > glucose).

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These results indicated that starch was the the following manner (glucose> lactose > best carbon source for antibiotic production sucrose > mannose > fructose > raffinose). by Streptomyces cyaneus DN.37 isolate (37 These results indicated that glucose was the mm). On the other hand, in case of best carbon source in case of Streptomyces Streptomyces lavendulae DN.7 results lavendulae DN.7 for antimicrobial secondary showed that the maximum production was in metabolites (25 mm) (Fig. 9).

Polysaccharides C o n tr o l Trisaccharides Organic acids (Streptomyces cyaneus DN.37) Streptomyces lavendulae DN.7

Monosaccharides Disaccharides Alcohol sugars 3 5

3 0

2 5

2 0

1 5

1 0 Inhibition Zone diameter (mm) diameter Zone Inhibition 5

0

Starch Ethanol Glucose Fructose Suc rose Lactose Chitosan Mannitol Glycerol mannose Raffinose Citric acid Succinic acid Carbon sources g% No. carbon source

Fig. 9. Effect of different carbon sources on the production of compounds produced by two Streptomyces spp. Nitrogen sources: and replaced by equivalent amounts of different nitrogen sources under test. The Also, the effect of different organic results in figure 10 illustrated that, soybean nitrogen sources rich in amino acids, as well meal and yeast extract was found to be the as some inorganic ones, on the production of most favorable nitrogen source for antimicrobial compounds by two Streptomyces antimicrobial compound production, reached spp. isolate. In this experiment, the previously 37 and 25 (mm) of inhibition zone diameter by selected C medium (medium No. 4) for both both Streptomyces cyaneus DN.37 and isolates were prepared and sterilized, Then Streptomyces lavendulae DN.7, respectively. the amount of carbon content were calculated C o n tro l Streptomyces cyaneus DN.37 Streptomyces lavendulae DN.7

Organic nitrogen Inorganic nitrogen 3 5

3 0

2 5

2 0

1 5

1 0

5 Inhibition zoneInhibition diameter(mm)

0

Peptone Control

Meat extract Yeast extract Soybean meal Sodium nitrate casein hydrolsate nitrogen sources g % Ammforium chloride Ammforium sulphate Ammforium phosphate Fig. 10. Effect of different nitrogen sources on the production of antimicrobial compounds produced by two Streptomyces spp. isolates

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By-product carbon and nitrogen sources: soybean meal (1.6 to 7 % (w/v)) as a by-product of carbon and nitrogen sources respectively. The It is known that the production media were results represented in figures 11 and 12 showed usually selected on the basis of their values in that, the maximum amount of antimicrobial permitting the elaboration of the antibiotics as compounds production by Streptomyces cyaneus well as their economic costs, therefore DN.37 reached 37 mm after 4 days of incubation formulating low cost medium suitable for when supplemented with 7.0% (w/v) of sugar maximum production of antimicrobial compound cane molasses and 4.0% (w/v) Corn steep liquor. produced by two Streptomyces spp. isolates. In On the other hand, results represented in figures the following experiment we substituting the 11 and 13 revealed that the maximum amount of starch and soybean meal content of medium No. antimicrobial compounds production by 6 and glucose and yeast extract content of Streptomyces lavendulae DN.7 reached 25 (mm) medium No. 7 (as a conventional carbon and after 6 days of incubation when supplemented nitrogen source, respectively) with different with 6.0% (w/v) of sugar cane molasses and 4.8 concentrations of sugar cane molasses (2.0 to % (w/v) soybean meal. 12.0% (w/v)), CSL (0.35 to 6.0% v/v) and

4 0

3 5

3 0

2 5

2 0

1 5

1 0

Inhibition zone diameter(mm) zone Inhibition 5 Streptom yces cyaneus DN.37 Streptom yces lavendulae DN.7 0 0 2 4 6 8 1 0 1 2 sugar cane m olasses conc. g % Fig. 11. Effect of different concentrations of sugar cane molasses on the production of antimicrobial compounds by two Streptomyces spp. isolates

4 5

4 0

3 5

3 0

2 5

2 0

1 5 Inhibition zone diamater (mm) diamater zone Inhibition 1 0

5

0 0 1 2 3 4 5 6 7 Different conc. of C .S.L (w /v) g% Fig. 12. Effect of diff erent concentrations of corn steep liquor on the of antimicrobial compound by Streptomyces cyaneus DN.37

4 0

3 5

3 0

2 5

2 0

1 5

Inhibition zone diameter (mm) diameter zone Inhibition 1 0

5

0 0 1 2 3 4 5 6 7 8 9 soybean m eal conc.g % Fig. 13. Effect of different concentrations of soybean meal on the production of antimicrobial compound by Streptomyces DN. 7

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cell growth and antibiotic production after 4 DISCUSSION: days. Furthermore, Sousa et al. (2002) found In our screening program of isolation that the production of actinomycin-D by St. and characterization of the most antagonistic parvulus reached their maximum values at soil actinomycetes, isolates were selected around 6 days. according to their antimicrobial activity and The maximum antimicrobial secondary broad spectra antagonistic activities. metabolites production was obtained at Identification process had been carried out incubation temperature 30°C for the two according to the Key's given in Bergey’s selected isolates, (St. cyaneus DN. 37 and St. th Manual of Determinative Bacteriology 8 lavendulae DN. 7), then obvious decline edition (Buchanan and Gibbsons, 1974), afterward up to 45°C at which low Bergey’s Manual of Determinative antimicrobial compound production was Bacteriology, Vol. 4 (Locci, 1989) and obtained. These results are in agreement with International Journal of Systematic Hassan et al. (2001) who showed that, the Bacteriology (Shirling and Gottlieb, 1968a&b). maximum antibiotic production was obtained From the taxonomic features, the most potent by S. violatus at 30°C. Also, Adinarayana et 2 isolates were found to match Streptomyces al. (2003) found that, maximum neomycin species in the morphological, physiological production by Streptomyces marinensis was and biochemical characters. Further obtained at 30°C. Upon studying the effect of identification based on 16S-rRNA indicate different agitation speed, it was found that at that, the actinomycete isolate No. 37 was low rpm (70 rpm) there was no antimicrobial found to match the character of Streptomyces compound production for St. lavendulae DN. cyaneus and the other actinomycete isolate 7, while, for St. cyaneus DN. 37 it give low No. 7 was found to match the characters of antimicrobial compound. This may attribute to Streptomyces lavendulae. The effect of the decrease of agitation rate that might different environmental and nutritional factors reduce the dissolved oxygen level in on the antimicrobial activity was studied for fermentation broth that could affect the optimizing the cultural conditions to obtain the antimicrobial metabolite production (Elattal et highest quantities of antimicrobial compounds al., 2011). Furthermore, as the agitation produced by the two Streptomyces spp. speed increased the amount of antimicrobial isolates. In concerning the effect of media compound increased reaching its maximum composition, the maximum biosynthesis of the value of 180 rpm for both Streptomyces spp. antimicrobial compounds was obtained upon isolates. These results are in agreement with using medium C (No. 4) for both isolates; those discussed by Beg et al. (2003) who these results are in harmony with that of concluded that, at this speed, aeration of the Gastaldo and Marinelli (2003) who previously culture medium was increased, and this could reported this media for optimum antimicrobial lead to sufficient supply of dissolved oxygen compound production. in the media and nutrient uptake by bacteria The maximum antimicrobial secondary also will be increased. On the other hand, as metabolite production by the two the agitation speed increased to 300 rpm, the Streptomyces spp. isolates was at an initial amount of antimicrobial compound decreased pH of 7.0-7.5. These results are in for both Streptomyces spp. isolates. This is accordance with Ghosh and Prasad (2010) due to the fact that increasing of the agitation who proved that the growth of S. speed tends to increase the amount of kanamyeticus MTCC 324 was optimum at pH dissolved oxygen available to the culture that 7 and Song et al. (2012) who demonstrated makes depletion for the cell growth and that, the optimum antimicrobial compound antimicrobial compound production (Sousa et production was obtained at pH 7. On the other al., 2002). hand, The optimum incubation period for St. On the other hand, it was found that as cyaneus DN. 37 was found to be 4 days while, the inoculum size of vegetative cells that of St. lavendulae DN.7 was 6 days, increased, the production increased linearly increasing incubation time to 5 and 8 days led through the incubation time reaching its to noticed depletion of antimicrobial activity of maximum yield after 4 days when 3 and 4 ml the both Streptomyces species, this is due to vegetative cells / 250 ml flask were used for the fact that the extent of antibiotic production St. cyaneus DN. 37. While, in case of St. equals the tolerance of the cells to the lavendulae DN. 7, upon using spores as antibiotic. Martin and Demain (1980) inoculum, production was noticed and suggested that the cessation of antibiotic increased during the incubation period till biosynthesis may be attributed to one of the reached maximum production upon using 3 following factors: a shortage of intermediate and 4 ml spore suspension / 250ml flask after precursors, the feedback effect of the 6 days of incubation time. Concerning accumulated antibiotic or the irreversible inoculum age, the use of vegetative cells of 3 decay of one or more enzymes. In addition, days growth revealed higher yield through the the present results agreed with that of incubation time than the use of vegetative Narayana and Vijayalakshmi (2008) where St. cells of 1and 2 days growth for both isolates. albidoflavus reached to maximum levels of These results were in accordance with that of

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Himabindu and Jetty (2006) who proved that, obtained when the culture medium was the inoculums age of 3 days gives maximum supplemented with yeast extract as a sole yield of gentamicin production compared to 1, nitrogen source. These results may be 2 and 4 days old slant. Mukhtar et al. (2012) attributed to the stimulatory effect of yeast found that, 2 days old inoculum at a size of extract due to the presence of 3’-(1-butyl 8% (v/v) gave best antibiotic production. phosphoryl) adenosine (the B-factor) which Where, the quantity and quality of inoculation acts as hormonal regulator for secondary material play a crucial role in the bioprocess metabolism or morphogenesis or both in results and a large inoculum was found to Streptomycetes as reported by El-Tayeb et al. suppress the production. (2004). Exploring, the effect of carbon sources Upon formulating low cost medium on antimicrobial secondary metabolites suitable for maximum production of production by inoculating the selected isolates antimicrobial compound produced by two on the basal medium having different carbon Streptomyces spp. isolates. The sugar cane sources. The maximum production was in the molasses was the best by-product carbon following manner (starch > raffinose and sources as a sole carbon source for both lactose > sucrose and mannose > fructose > isolates St. cyaneus DN. 37 and St. glucose). These results indicated that starch lavendulae DN. 7. These results were in was the best carbon source for antibiotic agreement with El-Enshasy et al. (2008) who production by St. cyaneus DN.37 isolate. found that, the addition of sugar cane Mellouli et al. (2003) reported that, antibiotic molasses as a sole carbon source at a production was only observed when starch concentration of 60 g/l gave the maximal was used as carbon source. On the other antibiotic production. This stimulatory effect hand, in case of St. lavendulae DN.7 results of sugar cane molasses may be duo to showed that the maximum production was in vitamins, minerals and growth factors. Also, the following manner (glucose> lactose > corn steep liquor and soybean meal were the sucrose > mannose > fructose > raffinose). best natural by-product nitrogen sources f or Where, glucose was the best carbon source. isolates St. cyaneus DN. 37 and St. Sultan et al. (2002) proved that, glucose as a lavendulae DN. 7, respectively. This in carbon source was the most suitable for large accordance with Mukhtar et al. (2012) who scale production of antibiotic by Streptomyces discovered that, corn steep liquor (CSL) species. Also, Ramadan (2000) Showed that greatly stimulates the production of antibiotic glucose was the most adequate carbon source and this is because it is rich source of followed by starch for antibiotic production. nutrients; proteins, vitamins, and minerals In a similar way to carbon, the nitrogen (Gupte and Kulkarni, 2002; El-Mehalawy et source was used to regulate secondary al., 2005; Narayana and Vijayalakshmi, 2008) metabolism. The best nitrogen source was who showed that the used of soybean meal as soybean meal and yeast extract at which the a nitrogen source was proved to be the best maximum biosynthesis of secondary for the production of bioactive metabolites by metabolites was attained for both St.cyaneus Streptomyces spp. DN. 37 and St. lavendulae DN. 7, respectively. These results were in agreement CONCLUSION: with Gupte and Kulkarni (2002) and Narayana Streptomyces cyaneus DN.71 and and Vijayalakshmi (2008) who showed that, Streptomyces lavendulae DN. 7 represents using of soybean meal as a nitrogen source promising actinomycetes from soil habitat and was proved to be the best for the production encourages the search for two new members of bioactive metabolites by Streptomyces spp. of Streptomycetes that may produce useful This is may be attributed to that Soybean secondary metabolites. The use of raw meal is a complex nitrogen source and material such as sugar cane molasses, corn contains a number of amino acids which led to steep liquor and soybean meal as cheap enhancement of antibiotic production (Singh carbon and nitrogen source respectively et al., 1982). On the other hand, Ripa et al. represent an applicable and economic media (2009) illustrated that; the maximum in industrial production of bioactive secondary production of antimicrobial metabolites was metabolites.

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ﻋﺰل وﺗﻌﺮﻳﻒ وﺗﺤﺪﻳﺪ اﻟﻈﺮوف اﻟﻤﺜﻠﻰ ﻟﻠﻤﻮاد اﻟﻀﺪﻣﯿﻜﺮوﺑﯿﺔ اﻟﻤﻨﺘﺠﺔ ﺑﻮاﺳﻄﺔ اﻷﻛﺘﯿﻨﻮﻣﯿﺴﯿﺘﺎت اﻟﻤﻌﺰوﻟﺔ ﻣﻦ اﻟﺘﺮﺑﺔ ﻧﯿﺮه أﺣﻤﺪ ﻣﺤﻤﺪ ﻋﺒﺪ اﻟﻮاﺣﺪ*، ﻧﺠﻮى أﺣﻤﺪ ﻋﺒﺪ اﷲ**، أﺣﻤﺪ إﺑﺮاھﯿﻢ اﻟﺪﻳﻮاﻧﻰ*، ﺳﻌﯿﺪ ﻣﺤﻤﻮد ﺑﺪر اﻟﺪﻳﻦ*، دﻳﻨﺎ ﻋﺰت اﻟﺨﻮاص* *اﻟﻤﺮﻛﺰ اﻟﻘﻮﻣﻰ ﻟﻠﺒﺤﻮث **ﻗﺴﻢ اﻟﻤﯿﻜﺮوﺑﯿﻮﻟﻮﺟﻲ، ﻛﻠﯿﺔ اﻟﻌﻠﻮم، ﺟﺎﻣﻌﺔ ﻋﯿﻦ ﺷﻤﺲ. ﺗﻢ ﻣﻦ ﺧﻼل ھﺬا اﻟﺪراﺳﺔ اﺧﺘﺒﺎر اﻟﻘﺪرة اﻟﺘﻀﺎدﻳﺔ وﺟﺪ أﻳﻀﺎ أن أﻓﻀﻞ ﻛﻤﯿﺔ ﻟﻘﺎح ھﻲ (3 ﻣﻠﻲ / 50 ﻣﻠﻲ ﻟﺒﻌﺾ اﻷﻛﺘﯿﻨﻮﻣﯿﺴﯿﺘﺎت اﻟﻤﻌﺰوﻟﻪ ﻣﻦ اﻟﺘﺮﺑﺔ اﻟﻤﺼﺮﻳﺔ وذﻟﻚ ﻣﻨﺒﺖ ﻏﺬاﺋﻲ) ﻋﻠﻲ ﺻﻮرة ﺧﻼﻳﺎ ﺧﻀﺮﻳﺔ ﻋﻤﺮ 3 أﻳﺎم ﻟﻜﻞ ﻣﻦ ﺑﺎﺳﺘﺨﺪام ﻛﺎزﻳﻦ اﻟﻨﺸﺎ ﻛﻮﺳﻂ ﻏﺬاﺋﻲ. وﻗﺪ ﺗﻢ إﺧﺘﯿﺎر اﻟﺴﻼﻟﺘﯿﻦ. وﻗﺪ أوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ أن اﻟﻤﻮﻻس ھﻮ أﻓﻀﻞ ﺳﻼﻟﺘﺎن ﻣﻦ اﻷﻛﺘﯿﻨﻮﻣﯿﺴﯿﺘﺎت (DN.37&DN.7) ذو ﻧﺸﺎط اﻟﻤﺨﻠﻔﺎت اﻟﺼﻨﺎﻋﯿﺔ ﻛﻤﺼﺪر ﻛﺮﺑﻮﻧﻲ ﻓﻲ ﺣﯿﻦ ﻛﻦ ﻣﻨﻘﻮع أﺣﯿﺎﺋﻲ واﺳﻊ اﻟﻤﺪي ﺿﺪ اﻟﻜﺎﺋﻨﺎت اﻟﺤﯿﺔ اﻟﺪﻗﯿﻘﺔ اﻟﺬرة وﻓﻮل اﻟﺼﻮﻳﺎ ھﻤﺎ أﻓﻀﻞ ﻣﺨﻠﻒ ﺻﻨﺎﻋﻲ ﻛﻤﺼﺪر اﻟﻤﺴﺘﺨﺪﻣﺔ ﻓﻲ اﻟﺪراﺳﺔ. ﺛﻢ ﺗﻢ ﺗﻌﺮﻳﻒ اﻟﺴﻼﻟﺘﺎن ﻧﯿﺘﺮوﺟﯿﻨﻲ. وﺗﻮﺻﻠﺖ اﻟﺪراﺳﺔ إﻟﻲ أن اﻟﺒﯿﺌﺔ اﻟﻤﺜﻠﻲ ﺑﺎﺳﺘﺨﺪام اﻟﺼﻔﺎت اﻟﻤﺰرﻋﯿﺔ واﻟﻔﺴﯿﻮﻟﻮﺟﯿﺔ واﻟﺒﯿﻮﻛﯿﻤﯿﺎﺋﯿﺔ اﻟﻤﻘﺘﺮﺣﺔ ﻷﻋﻠﻲ إﻧﺘﺎﺟﯿﺔ ﻣﻦ اﻟﻤﻀﺎدات اﻟﺤﯿﻮﻳﺔ ﺑﺎﻟﻨﺴﺒﺔ ل ﻛﻤﺎ ﺗﻢ ﺗﻌﺮﻳﻔﮫﻤﺎ اﻳﻀﺎ ﺑﻮاﺳﻄﺔ ﻃﺮق اﻟﺒﯿﻮﻟﻮﺟﯿﺎ اﻟﺠﺰﻳﺌﯿﺔ وﻗﺪ Streptomyces cyaneus ھﻲ:- (ﺟﻢ/ﻟﺘﺮ) ﻣﻮﻻس -70.0 اﻓﺎدت اﻟﻨﺘﺎﺋﺞ ﺗﻄﺎﺑﻘﮫﻤﺎ ﺑﻨﺴﺒﺔ 98% و 99% ﻣﻊ ﻧﻮﻋﻲ ﻣﻨﻘﻮع اﻟﺬره 40 ﻣﻠﻲ- ﻛﺮﺑﻮﻧﺎت اﻟﻜﺎﻟﺴﯿﻮم 2.0. ﺑﯿﻨﻤﺎ ﺟﻨﺲ ﺳﺘﺮﺑﺘﻮﻣﯿﺴﯿﺲ ھﻤﺎ Streptomyces lavendulae ﺑﺎﻟﻨﺴﺒﺔ ﻟﻨﻮع Streptomyces lavendulae ھﻲ:- (ﺟﻢ/ﻟﺘﺮ) & Streptomyces cyaneu ﻋﻠﻲ اﻟﺘﻮاﻟﻲ. ﻛﻤﺎ ﺗﻢ دراﺳﺔ 60.0- ﻓﻮل ﺻﻮﻳﺎ 48.0- ﻛﺮﺑﻮﻧﺎت ﻛﺎﻟﺴﯿﻮم 2.0. اﻧﺴﺐ اﻟﻈﺮوف اﻟﻔﺴﯿﻮﻟﻮﺟﯿﺔ اﻟﻤﻼﺋﻤﺔ ﻷﻋﻠﻲ إﻧﺘﺎﺟﯿﺔ ﻣﻦ اﻟﻤﺮﻛﺒﺎت اﻟﻤﻀﺎدة ووﺟﺪ أن أس ھﯿﺪروﺟﯿﻨﻲ (7.5-7)، درﺟﺔ ﺣﺮارة (م 30º )، ﺳﺮﻋﺔ دوران(200ﻟﻔﺔ/دﻗﯿﻘﺔ) ، ﺣﺠﻢ اﻟﻤﺤﻜﻤﻮن: اﻟﻮﺳﻂ اﻟﻐﺬاﺋﻲ ( 50 ﻣﻠﻲ ﻣﻨﺒﺖ ﻏﺬاﺋﻲ/250 ﻣﻠﻲ دورق) و زﻣﻦ ﻓﺘﺮة اﻟﺘﺤﻀﯿﻦ 4 و 6 اﻳﺎم ﻟﻜﻞ ﻣﻦ Streptomyces & أ.د. ﻣﺤﻤﺪ إﺑﺮاھﯿﻢ أﺣﻤﺪ ﻋﻠﻲ ﻗﺴﻢ اﻟﻨﺒﺎت، ﻋﻠﻮم اﻟﻘﺎھﺮة Streptomyces cyaneu & lavendulae ﻋﻠﻲ اﻟﺘﻮاﻟﻲ. ﻛﻤﺎ أ.د. زﻳﻨﺎت ﻛﺎﻣﻞ ﻣﺤﻤﺪ ﻗﺴﻢ اﻟﻨﺒﺎت، ﻋﻠﻮم اﻟﻘﺎھﺮة

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