Isolation, identification and extraction of antimicrobial compounds produced by Streptomyces sps from terrestrial soil

Author’s Accepted Manuscript Isolation, Identification and Extraction antimicrobial compounds produced Streptomyces sps from terrestrial soil

of by

S. Usha Nandhini, S. Sudha, Anusha Jeslin V, Manisha S www.elsevier.com/locate/bab

PII: DOI: Reference:

S1878-8181(18)30155-5 https://doi.org/10.1016/j.bcab.2018.06.024 BCAB797

To appear in: Biocatalysis and Agricultural Biotechnology Received date: 13 February 2018 Revised date: 9 May 2018 Accepted date: 30 June 2018 Cite this article as: S. Usha Nandhini, S. Sudha, Anusha Jeslin V and Manisha S, Isolation, Identification and Extraction of antimicrobial compounds produced by Streptomyces sps from terrestrial soil, Biocatalysis and Agricultural Biotechnology, https://doi.org/10.1016/j.bcab.2018.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Isolation , Identification and Extraction of antimicrobial compounds produced by Streptomyces sps from terrestrial soil S. UshaNandhini1,S.Sudha1,Anusha Jeslin.V2 and Manisha. S2 1

Assistant Professor , Department of Biotechnology, Sathyabama Institute of Science and Technology, Jeppiaar Nagar, , OldMamallapuram Road, Chennai - 600 119, Tamilnadu. 2 M.Tech Biotechnology, Department of Biotechnology, Sathyabama Institute of Science and Technology, JeppiaarNagar, OldMamallapuram Road, Chennai - 600 119, Tamilnadu. E-mail :[email protected] ABSTRACT The main objective of the present study was to find the antimicrobial compounds from terrestrial Strptomyces sps. Streptomycetes strains were isolated from terrestrial soil and subjected to primary screened by cross streak method against some pathogenic Gram-positive and Gram-negative test bacteria and fungi. Streptomycetes of which one isolate showing broad spectrum of antimicrobial was characterized on the basis of their morphological and physiological properties. The antimicrobial substances were extracted from active isolates and inoculated in ISP2 broth were fermented for 7 days at 28°C by ethyl acetate extraction method. The active crude compounds were subjected to secondary screening by agar well diffusion method to confirm activity against same pathogenic bacteria and fungi. The crude compounds were purified by Thin layer chromatography (TLC). The bioactive region was detected on the TLC plate and calculated Rf value. Two bands were detected and each band tested for agar well diffusion method for antimicrobial activity. The characterization of the bioactive compounds was further determined by using UV-visible spectrophotometer, FT-IR and GC-MS techniques. These potent isolates from terrestrial soil could be an interesting source for pharmaceutical industries to explore antibacterial and antifungal compounds. Key words : Streptomyces, antimicrobial compunds, terrestrial soil … * Corresponding author. E-mail address: [email protected] 1. Introduction

Streptomyces are Gram positive, filamentous forms of soil bacteria. They are known to produce varieties of antimicrobial compounds (Bull and Stach, 2007). The G + C content are more in their Deoxyribo Nucleic Acids and they are thermophiles. Soil microorganisms like streptomyces provide an excellent resource for the isolation and identification of therapeutically important products. This phylum alone accounting for the production of an approximately 75% of the total bioactive compounds including antibiotics with more than 70% produced by member of the genus Streptomyces (Subramani and Aalbersberg, 2012). Genus Streptomyces are prolific producers of novel compounds with a variety of biological activities including antimicrobial compounds, anti-cancer agents and other pharmaceutically useful compounds (Ser et al., 2015). Streptomycetes forms a large and important group of the microflora of most natural environments. Soil , freshwater lake and river bottom , compost and manures contain an abundance of these Strptomyces..A large number of streptomycetes have been isolated and screened from soil in the past several decades, accounting to 70-80% of relevant secondary metabolites available commercially (Baltz, 2008). Streptomycetes are potential source of many bioactive compounds,which have diverse clinical effects and important applications in human medicine (Watve et al., 2001). It has been estimated that approimately one-third of the thousands of naturally occurring antibiotics have been obtained from Strptomycetes (Takizawa et al., 1993). Strains of the genus streptomyces are superior to other actinomycete strains in their ability to produce large number and varieties of bioactive secondary metabolites. (UshaNandhini et al., 2015) The drug resistance problem demands to discover new antimicrobial compounds effective against resistant pathogenic bacteria and fungi. So, we need to screen more and more Streptomycetes from different habitats for antimicrobial activity in the hope of getting some new streptomycetes strains that produce antibiotics, which have not been discovered yet and are active against drug-resistant pathogens. 2. Materials and Methods 2.1 Soil sample collection The soil samples were collected from Nagercoil, Kanyakumari District in Tamil Nadu. The depth of 5 cm after removing the 1cm of upper soil surface by scrapping with the help of spatula in sterile vials, transfer to the laboratory and stored at the 4°C until use for the isolation process. Soil analysis was processed for the parameters such as pH, soil texture and total nitrogen contents in the soil (Bremner, 1960). 2.2 Isolation of Streptomyces The sediment samples were collected and air-dried for one week and kept at 45ºCfor 1hr to minimize the bacterial contaminants. About 1gm of sample was transferred to an Erlenmeyer’s flask containing 99ml of sterile distilled water. The sediment suspension was further diluted up to 10-5 level. 0.1ml of the diluted suspension was spread over the surface of Actinomyceteisolation-agar medium prepared in 100% distilled water to enhance the isolation of terrestrial Streptomycetes by serial dilution and spread plate method (Shirling and Gottileb, 1966) . The pH

of the selected media was adjusted to 7.2 t0 7.4. To prevent the fungal and bacterial contaminates, cycloheximide (100mg/L) and nalidixic acid (20mg/L) were added to the isolation medium. The petriplates were then incubated at 370C and the colonies were observed from fifth day onwards for one month. Based on colony morphology the streptomyces strains were selected and sub -cultured on ISP-2 medium. (Predham,1965). 2.3 Primary screening of Streptomyces The isolates were screened for antibacterial and antifungal activity by using cross streak method on modified nutrient agar against three bacterial pathogens such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis and two fungal strains such as Aspergillus niger and Candida albicans purchased from private lab (Enviro care pvt ltd, Chennai ). (Balagurunathan and Subramanian, 2001). The presence and absence of growth were noted for the primary screening. 2.4 Identification of active strain The active strains were subjected to identified based on cultural characteristics, microscopic morphology by scanning electron microscopy, biochemical and physiological characterization.(Kawato and Shinobu, 1959, Shirling and Gottileb,1966 ). 2.5 Extraction of crude compounds The extraction of crude compounds from active strain was processed by using ISP-2 medium by ethyl – acetate extraction method (Ismail et al., 2009). The active strain was inoculated in ISP-2 broth and incubated for 7 days in shaker incubator at 24°C. After incubation culture broth was centrifuged at 8,000 rpm for 15 mins and then the supernatant was collected and mixed with an equal volume of ethyl acetate. The mixture was added in the separating funnel and shake vigorously for 20mins. The mixture was kept at resting position and collected the supernatant. The extracted crude compounds were dried at 40°C. The crude compounds were processed for secondary screening by agar well diffusion method (Jennifer Emelda et al., 2012) to confirm the active antimicrobial metabolites. 2.6 Purification of crude compounds Purification of crude compounds were processed by pre coated Thin Layer chromatography method (Usha Nandhini and Masilmani selvam, 2013). The crude compound was spotted and applied a line 1.5 cm above from the bottom of TLC plates by using the capillary tube. The spots were left to dry for a minute. The TLC plate was placed vertically in a trough containing the mixture of solvent (hexane :ethyl acetate (1: 9) ratio. When the solvent moved up to 80% of TLC plate, the plate was taken out and dried. The Rf values were calculated. (Dharumaduari Dhanasekaran et al., 2008).

2.7 Secondary screening of compounds separated using TLC

Each bands were scrapped out separately and collected in different vials containing ethyl acetate. Then vials were kept for centrifugation and separated the compounds present in the bands. Then the each band compounds were checked again for antimicrobial metabolites by agar well diffusion method (Mohanraj et al., 2011) for the same bacterial strains and fungal strains. 2.8 Identification of purified compounds The active band compounds were identified by using UV-Visible spectrophotometer, FT-IR and (Gas chromatography and mass spectrometry) GC-MS techniques. The mass spectrum was recorded by using SHIMADZU QP2010. Mass spectrometer under the current (MA) 100 and the temperature at 70°C. 3 Results and Discussion 3.1 Isolation and Screening of Streptomyces Ten soil sediments were collected in sterile vials. The samples were processed for soil analysis and showed in Table 1. The 16 isolates were isolated from the terrestrial soil samples based on their different colony morphology and color variations using starch casein agar medium. These strains were purified and subcultured on ISP-2 medium (Sudha and Masilamani Selvam, 2013) These strains were selected and tested for antimicrobial activity by primary screening. Only one strain were selected from primary screening based on strong antibacterial and antifungal activity. That active strain showed activity against all the bacterial and fungal pathogens Fig 1. Total of 68 Actinomycetes were isolated from sea shore marine environment located in Bigeum Island, South West coast of South Korea. The majority of these isolates were assigned to the genus Streptomyces of which one isolate was showed broad spectrum of antimicrobial on the basis of their morphological, physiological and biochemical properties (Parthasarathi et al., 2012). Table 1: Soil analysis Parameters Result pH

7.28

Texture

Sandy loam (0.80.2mm)

Total nitrogen

1.81%

Fig 1 : Antifungal activity of active strain – cross streak method

3.2 Identification of active strain The cultural characteristics of the active strain on actinomycetes isolation agar was showed in Table 2 and scanning electron microscopy was showed as spiral spore structure and smooth surface showed in Fig 2. In the carbon utilization test most of the sugars utilized by active strain except fructose. The optimum growth temperature was confirmed as 30°C – 40°C. The optimum pH was confirmed as pH 7 Table 3. From these analysis this active strain confirmed as strptomyces sps from terrestrial soil sample. Table 2 : cultural characteristics and SEM report of active strain Growth medium

Aerial mass colour

Melanoid pigment

Reverse side pigment

Soluble pigment

Spore chain morphology (SEM Photo)

Actinomycete isolation agar

White

_

Brown

_

Spiral and smooth surface

Fig 2 SEM photo of active strain

Table 3 : Biochemical and physiological characteristics of active strain Carbon source

Active strain

Glucose Mannose Galactose Inositol Lactose Maltose Fructose Sucrose Mannitol Starch Citrate pH: 5 6 7 8 9 Temperature 10°C 20°C 30°C 40°C 50°C

+++ ++ +++ ++ ++ +++ +++ ++ +++ +++ ++ ++ +++ ++ +++ -

Note : Good growth - +++ , moderate growth - ++ , Nil growth - -3.3 Extraction of bioactive compounds The antimicrobial compounds were extracted from active strain by ethyl acetate extraction method. The antimicrobial activity was processed by agar well diffusion method for the active crude compounds. The 20 μl of active crude compounds were showed most active against Bacillus subtilis and Candida albicans. Zone of inhibition of active strain showed in Table 4. The actinomycetes isolated from Manipur terrestrial soil showed good activity against Bacillus subtilis, Pseudomonas aeroginosa, Staphylococcus aureus and Candida albicans. (Debananda, 2009)

Table 4 : Antimicrobial activity by agar well diffusion method Test Organisms

Bacillus subtilis Escherichia coli Staphylococcus aureus Aspergillus niger Candida albicans

Zone of inhibition in mm (20µl) 24 16 13 10 12

3.4 Purification of crude compounds The crude compounds were extracted from ethyl acetate extraction method and then purified and separated by Thin layer chromatography method. Two bands were observed, and then active compounds were confirmed by agar well diffusion method for the same test organisms. The Rf value of the first band was measured as 0.55 (Fig 3) and the second band was measured as 0.24 in TLC and both the band compounds were scrapped from TLC plates and checked for antimicrobial activity. Only first band compounds were showed both antibacterial and antifungal activity. Streptomyces isolated from marine sponges produced antimicrobial compounds that showed Rf values ranging from 0.40 to 0.78 in TLC analysis, which confirmed the production of polyene nature of compounds ( Selvakumar et al., 2010)

Fig 3 : TLC plate showed first band under short UV 3.5 Identification of bioactive metabolites The active band compounds were characterized by UV Visible spectrophotometer in the range of 200nm to 700nm. The active compounds were showed peaks in the range of 208nm and 406nm (Fig 4). Then the bands were analyzed for FT-IR spectrum showed the peaks in the range of

2983, 2628, 2358, 1736, 1713, 1373, 1236, 1043, 1006 indicated the presence of carboxylic acid, aldehydes, esters, hydroxyl functional group of compounds (Fig 5). More or less similar trend was observed by (Augustine et al., 2005) , when they tested the FT- IR spectrum of ethyl acetate extract of Streptomyces albidoflavus PU23 that exhibited absorption bands at 3296 and 1031.8 cm-1, which indicated hydroxyl groups and absorption at 1639 cm-1 indicating double bonding. These bands were further analyzed by GC-MS analysis. The GC-MS analysis showed the presence of 21 compounds showed in Table 5 (Fig 6). The identification of the compounds were based on the Real time, peak area percentage and peak height percentage of the compounds. The two compounds were suggested for high area percentage namely 2,6-Lutidine 3,5-dichloro-4-dodecylthio and 2,5-Piperazinedione,3,6-bis(2-methylpropyl. This may be the active compounds responsible for antimicrobial activity from active strain.

60 40 20

3500

E:\USERS

3000

DATA\INTERNAL\INT 2013\APEX\MANISHA.0

2500 2000 Wavenumber cm-1

MANISHA

1500

1000

Instrument type and / or accessory

Page 1/1

Fig 5 : FT-IR analysis for active band

Fig 6 : GC-MS analysis of active band

632.50 607.59

1043.70 1006.33 937.71 878.99 848.86 787.13

1235.61

1373.84

1736.82 1713.01

2358.26

2628.46

2983.88

0

Transmittance [%]

80

100

Fig 4 : UV – Visible spectrophotometer analysis of active band

23-03-2017

Table 5 : GC-MS report of active band Peak R.Time Name 1 6.089 Isotridecanol-SS isotridecyl alcohol SS 2 6.653 d-Glucitol, 4-O-decyl-SS 4-ODecyl-d-glucitol 3 6.740 d-Glucitol, 4-O-decyl-SS 4-ODecyl-d-glucitol 4 7.176 N-hydroxymethyl-2phenylacetamide SS N-H 5 8.628 Ethyl 4-t-butylbenzoate SSEthyl 4-tert-butylbenzoate

Area 14128208

Area% 7.47

Height 2882211

Height% 10.52

1266388

0.67

377405

1.38

7646316

4.04

1403295

5.12

10941879

5.79

1262844

4.61

18237660

9.65

5599838

20.45

6

9.617

3-(2-Phenylethyl)pyridazine

306159

0.16

42535

0.16

7 8

9.694 10.636

224896 486164

0.12 0.26

69335 97263

0.25 0.36

9 10

13.850 14.713

218665 557159

0.12 0.29

22269 139590

0.08 0.51

11 12

16.448 17.290

5446048 4801878

2.88 2.54

664286 830518

2.43 3.03

13

17.585

34470717

18.23

5202647

19.00

14

21.289

cis-vaccenic acid [1,1’Biphenyl]-2-amine SS 2Biphenylamine Di-n-decylsulfone Naphthalene,2,6-bis(1,2-dioxo2-phenylethyl 3-phenyl-1,3-pentanediol Coumarin-6-ol,3,4-dihydro4,4,5,7-tetramethyl 2,5-Piperazinedione,3,6-bis(2methylpropyl)1-Leucyl-1-O-methylthreonine

7020427

3.71

658716

2.41

15

29.158

3-Hydroxy-6-(N,Ndimethylamino) methylpyro

366051

0.19

79652

0.29

16

33.417

2077438

1.10

346000

1.26

17 18

34.165 42.617

1169166 644581

0.62 0.34

170025 154741

0.62 0.56

19

43.083

Fumaric acid, hexadecyl propargyl ester 4,4’-Dusopropylbiphenyl Acevaltrate SS Butanoic acid, 3(acetyloxy)-3Psi,.. psi- Carotene, 1,1’,2,2’tetrahydro-1,1’-du

2801178

1.48

451818

1.65

20 21

43.627 46.047

Farnesol isomer a 2,6 Lutidine 3,5-dichloro-4dodecylthio-

4114146 72159353

2.18 38.16

1005190 5928323

3.67 21.65

4 conclusions In the present study 10 sediment samples were collected from terrestrial soil at Nagercoil, Tamilnadu.. Based on different morphological colour variations 16 isolates were selected, purified and preserved. Among the 16 isolates only one strain showed strong antibacterial and antifungal activity in primary screening. The active strain was identified based on morphological, physical, and biochemical features. The active compounds were extracted from the active strain by submerged fermentation followed by ethyl acetate solvent extraction method. Secondary screening was done by agar well diffusion method and the highest zone of inhibition was observed for Bacillus subtilis and candida albicans . Crude compounds were purified by TLC and Rf value were calculated as 0.55 and 0.24. The Rf value 0.55 showed activity against all the pathogens in secondary screening. Identification of bioactive compound was done by UV Spectrum, FT-IR and GC-MS. UV-spectrum reading was found to be highest at 208nm with a peak of 1.67 absorbance. FT-IR analysis was performed and the functional groups such as aldehydes, ketones, carboxylic acids, esters, alkanes, amides, alcohol and amines were found. GC-MS analysis was identified the specific compounds and the highest peak belongs to 2,6Lutidine 3,5-dichloro-4-dodecylthio- with a peak range of 38.16. The present study reveals that terrestrial soil sample may be potentially used for extracting novel antibiotics. Acknowledgement The work was supported by the sathyabama Institute of Science and Technology.

Conflict of interest The authors declare that there is no conflict of interest. References : Augustine,SK., Bhavsar, SP., Kapadnis, BP., 2005. A Non-Polyene Antifungal Antibiotic from Streptomyces albidoflavus PU 23. J. Biosci. 30, 201–211. Balagurunathan, R., Subramanian, A., 2001. Antagonistic streptomycetes from marine sediments. AdvBiosci. 20,71-76. Baltz,RH., 2008. Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol. 8, 557–563. Bremner, JM., 1960. Determination of nitrogen in soil by Kjeldahl method. J. Agri. Sci. 55, 1133

Bull, AT., Stach, JEM., 2007. Marine actinobacteria : new opportunities for natural product search and discovery. Trends Microbiol. 15, 491-499. Debananda, S., Ningthoujam, Suchitra Sanasam, Salam Nimaichand. 2009. Screening of Actinomycete Isolates from Niche Habitats in Manipur for Antibiotic Activity. American Journal of Biochemistry and Biotechnology. 5 (4), 221-225. DharumaduariDhanasekaran., NooruddinThajuddin., AnnamalaiPanneerselvam., 2008. An Antifungal Compound: 4' phenyl -1-napthyl –phenyl acetamide from Streptomyces Sp. DPTB16”, FactaUniversitatis Medicine and Biology, 7 – 12. Ismail, SM., Fouad, A,,Quotaiba, B., Shahidi, 2009. Comparative UV-spectra fermented cultural extract of antifungal active streptomyces isolates recovered from different ecology. Curr Trends Biotechnol Pharm. 3, 2. Jennifer Emelda, E., Vijayalakshmi, N., Santhanakrishnan,T.. 2012. Antimicrobial Activity of Antibiotic Producing Streptomyces macrospores..J Pharm BiolSci 2(3),20–23. Kawato, M., Shinobu, R., 1959. Cover slip culture of Streptomyces herbaricolournov.sp. supplement; a simple technique for the microscopic observation. Mem. Osaka, Univ. lib. Art. Educ. 8, 114-119. Mohanraj, D., Bharathi, S., Radhakrishnan, M., Balagurunathan, R.,2011. Bioprospecting of Actinobacteria from Yelagiri hills with special reference to Antibacterial Activity. J. Chem. Pharm. Res. 3, 439-446. Parthasarathi, S., Sathya, S., Bupesh, G., Samy, RD., Mohan, MR., Kumar, GS. 2012. Isolation and characterization of antimicrobial compound from marine Streptomyces hygroscopicus BDUS 49. World J Fish Mar Sci. 4(3) , 268-277. Predham.T.G., 1965.Colour of Streptomycetes: Report of an International Workshop on Determination of Colour of Streptomyces.Appl Microbial, 13, 43-64. Selvakumar D, Arun K, Suguna S, Kumar D, Dhevendaran K. 2010; Bioactive potential of Streptomyces against fish and shellfish pathogens. Iran J Microbiol 2(3):157-64. Ser, H., Mutalib, A. B., Yin, N., Chan, K., Goh, B., Lee, L., 2015. Evaluation of antioxidative and cytotoxic activities of Streptomyces pluripotens MUSC 137 isolated from mangrove soil in Malaysia. Front Microbiol. 6,1398. Shirling, E.B., Gottileb, D., 1966. Methods for Characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16, 313–340. Subramani, R., Aalbersberg, W., 2012. Marine Actinomycetes: an ongoing source of novel bioactive metabolites. MicrobiolRes. 167, 571–580.

Sudha, S., Masilamani Selvam, M., 2013. In vitro cytotoxic activity of bioactive metabolite and crude extract from a new actinomycetes Streptomyces avidinii strain SU4. Int J Pharm Pharm Sci. 5(3), 612-616. Takizawa, M., Colwell, RR., Hill, RT., 1993. Isolation and diversity of actinomycetes in the chasapeake bay. Appl Environ Microbiol. 59, 997–1002. Usha Nandhini, S., Jaya bharathy, P., Rekha, S., 2015. International Journal of Pharmacy and Pharmaceutical Sciences. 7, 207 – 209. Usha Nandhini, S., Masilamani Selvam, M.. 2013. streptomyces strain. Int J Pharm Pharm Sci. 1, 176-178.

Bioactive compounds produced by

Watve,MG.,Tickoo, R., Jog, MM., Bhole, BD., 2001. How many antibiotics are produced by genus Streptomyces? Arch Microbiol. 176, 386–390.