influence of cultural conditions for improved...

Abstract The influence of culture conditions and theeffect of environmental factors on the growth andproduction of bioactive metabolites byStreptomyces cheonanensis VUK-A was thefocus of this study. The strain exhibited broadspectrum antimicrobial activity against Gram-positive, Gram-negative, unicellular andmulticellular fungi. The optimum pH andtemperature for bioactive metabolite productionwere 7 and 30°C respectively. Production ofbioactive metabolites by the strain was high inasparagine glucose broth as compared to othermedia tested. Studies on nutritional factorsrevealed that highest antimicrobial metaboliteproduction was obtained when lactose andpeptone at 1% and 0.25% were used as carbonand nitrogen sources respectively. Ninty sixhours of incubation was found to be the optimumfor bioactive metabolite production by the strain.As the strain exhibited potent antimicrobialactivity, it may be explored for biotechnologicalpurposes.

Keywords: Optimization, Bioactive metabolites,Nutritional factors, Culture conditions,Environmental Parameters.

IntroductionThere is an immediate need to discover and

develop new antibiotics as spread and prevalenceof infectious diseases resistant to chemotherapyis on the rise (1). Special attention was focusedon the microbes that have been proved as thenatural dumps for the bioactive metabolites sincedecades for resolving the problem of theantibiotic resistance (2). These microbes arecapable of producing a wide array of the potentantimicrobial compounds that have broadapplication in the field of medicine (3). Manymicrobes that thrive in extreme conditions havethe potentiality to produce unusual bioactivemetabolites that acts as a chemical defenseagainst the pathogenic microbes (4). The mostpromising source of the future antibiotics thatthe society expects is the natural microbialproducts (5). The exploration for the newantimicrobial compounds and the new strainscontinue to be the most important researchprogramme around the world (6). The microbialnatural products remain the most potent andimportant source for the novel antibiotics,although new methodologies are needed toimprove the efficiency of the discovery ofcompounds. Numerous antibiotics have been

Bioactive metabolite production by Streptomyces cheonanensis VUK-A.

Influence of Cultural Conditions for Improved Production ofBioactive Metabolites by Streptomyces cheonanensis VUK-A

Isolated from Coringa Mangrove Ecosystem

Usha Kiranmayi Mangamuri1, Sudhakar Poda2, Krishna Naragani1 andVijayalakshmi Muvva1*

1Department of Botany and Microbiology and 2Department of Biotechnology,Acharya Nagarjuna University, Guntur-522510, Andhra Pradesh, India

*For Correspondence - [email protected]

Current Trends in Biotechnology and PharmacyVol. 6 (1) 99-111 January 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)

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obtained from various microbes isolated fromthe marine environment played a significant rolein the discovery of anti-metabolites (7).Production of the secondary metabolites by themicrobes differs in quality and quantity basedon the type of strains and also species used (8).It is also a known fact that appropriatefermentation medium is critical and crucial forthe production of the secondary bioactivemetabolites (9) and prior experience andknowledge is required in developing a suitablebasal medium since it plays an important role infurther media optimization (10). Additionally,biosynthesis of the secondary metabolites isinfluenced by numerous environmental factorsincluding nutrients (nitrogen, phosphorous andcarbon sources), growth rate, feedback control,and other physical conditions (oxygen supply,temperature and pH) (11, 12, 13, 14). Therefore,influence of the growth conditions andenvironmental conditions are important toimprove the production of the secondarymetabolites.

Streptomyces species has been widelyreported for the production of a number of anti-microbial metabolites that have therapeuticapplications (15). These species have gained aspecial prominence for their characteristic abilityto produce potent antibiotics and other secondarymetabolites including anti-tumor agents (7). Eachof the strain has a genetic set up that influencesthe production of 10-20 different kinds ofsecondary compounds. The objective of thepresent study is to design an appropriate culturemedium and also optimize the cultural conditionsof the Streptomyces cheonanensis VUK-A strainin order to reduce the cost of fermentation processand improve the formation of antimicrobialcompounds.

Materials and MethodsIsolation: The Streptomyces cheonanensis VUK-A was used in the investigation. The strain was

isolated from soils of “Coringa MangroveEcosystem” of south coastal Andhra Pradesh,India by using soil dilution plate technique onstarch-casein agar medium (16) and furthermaintained on yeast extract malt extract dextrose(ISP-2) agar medium at 4°C (17). The 16s rRNAsequence of the strain Streptomyces cheonanensisVUK-A was submitted to the Genbank (accessionnumber JN087502) (48).

Incubation Period on Bioactive MetaboliteProduction: The growth pattern and bioactivemetabolite production of the strain was studiedat regular intervals up to 7 days. One week oldculture of the strain was cultivated in seedmedium (starch casein broth) at room temperaturefor 48 h. Seed culture at a rate of 10% wasinoculated into the production medium of thesame composition. The fermentation process wascarried out for one week under agitation at 120rpm. At every 24 h interval, the flasks wereharvested and the biomass was separated fromthe culture filtrate. Biomass was determined interms of total cell dry weight. Antimicrobialmetabolite production determined in terms oftheir antimicrobial spectrum (18). The culturefiltrates were extracted with ethyl acetate andevaporated to dryness in a water-bath at 80°C.The solvent extracts were concentrated and 50μlof crude extract was tested for antimicrobialactivity by employing agar well–diffusionmethod (19) against test organisms like Bacillussubtilis (ATCC 6633), Streptococcus mutans(MTCC 497), Staphylococcus aureus (MTCC3160), Escherichia coli (ATCC 35218),Pseudomonas aeruginosa (ATCC 9027) andCandida albicans (ATCC 10231).

Culture Conditions for the Optimum Productionof Bioactive Metabolites: Antimicrobialmetabolite production of the strain was optimizedby using different parameters such as pH,

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temperature, culture media, carbon and nitrogensources and minerals.

pH and Incubation Temperature on Biomassand Bioactive Metabolite Production: Todetermine the influence of initial pH on growthand bioactive metabolite production,Streptomyces cheonanensis VUK-A was culturedin the medium with different initial pH, rangingfrom 4-10 and at different starting temperatures,from 20 - 60°C. The biomass and bioactivemetabolite production were estimated todetermine optimal pH and temperature conditionswhich were used in this study (20, 21).

Culture Media on Biomass and Production ofBioactive Metabolite: In order to determine idealconditions for the maximum production ofantimicrobial metabolite from the Streptomycescheonanensis VUK-A, the strain was cultivatedin 10 different media such as asparagine-glucosebroth, tyrosine broth (ISP-7), starch inorganicsalts broth (ISP-4), glycerol-asparagine broth(ISP-5), yeast-starch broth, malt extract broth,tryptone yeast extract broth (ISP-1), czapek-doxbroth, maltose-tryptone broth and soya-bean mealbroth. The biomass accumulation and bioactivemetabolite production in each medium wasevaluated. The medium in which the strainexhibits optimum levels of bio-active metaboliteproduction was used for subsequent study.

Carbon and Nitrogen Sources on Biomass andBioactive Metabolite Production: To determinethe effect of carbon sources on biomass andbioactive metabolite production of the strain,different carbon sources like maltose, lactose,fructose, sucrose, dextrose, starch, mannitol,arabinose, xylose, glycerol and inositol (each ata concentration of 1%) were added separately tothe optimized medium. Furthermore, the effectof varying concentrations of the best carbonsource (0.5 - 5%) on bioactive metabolite

production was also determined. Similarly,influence of various nitrogen sources on bioactivemetabolite production was evaluated bysupplementing different nitrogen sources likesodium nitrate, ammonium sulfate, ammoniumoxalate, peptone, yeast-extract, tryptone, casein,tyrosine, phenyl alanine, glycine and glutamine(each at a concentration of 0.5%) to the optimizedmedium containing an optimum amount of thesuperior carbon source as determined above (22).Furthermore, the impact of varyingconcentrations of optimized nitrogen source (0.1- 2%) was studied to standardize the maximumantimicrobial metabolite production.

Minerals on Biomass and Bioactive MetaboliteProduction: Impact of minerals on the productionof biomass and bioactive metabolites was studiedby supplementing different minerals likeK

2HPO

4, MgSO

4, FeSO

4, K

2HPO

4 and ZnSO

4

each at a concentration of 0.05% (w/v) to theoptimized medium (23).

Antimicrobial Activity Against Test Organisms:The antimicrobial metabolites of the strainproduced under optimized conditions were testedagainst various strains of bacteria viz.,Streptococcus mutans (MTCC 497),Lactobacillus casei (MTCC 1423), Lactobacillusacidophilus (MTCC 495), Enterococcus faecalis(MTCC 439), Staphylococcus aureus (MTCC3160), Bacillus subtilis (ATCC 6633),Escherichia coli (ATCC 35218), Pseudomonasaeruginosa (ATCC 9027), Proteus vulgaris(MTCC 7299), Shigella flexneri (MTCC 1457)and Xanthomonas campestris (MTCC 2286) andfungi such as Candida albicans (ATCC 10231),Aspergillus niger, Aspergillus flavus, Fusariumoxysporum (MTCC 3075) and Penicillumcitrinum by agar-diffusion assay (19).

Statistical Analysis: Data obtained on thebioactive metabolite production under different



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microbial culture conditions were statisticallyanalyzed and expressed as mean ± standard errorwith one-way analysis of variance (ANOVA).

ResultsIncubation Period on Biomass and BioactiveMetabolite Production: The growth pattern ofStreptomyces cheonanensis VUK-A was studiedon starch casein broth. The stationary phase ofStreptomyces cheonanensis VUK-A extendedfrom 72 h to 120 h of incubation (Fig.1). Thesecondary metabolites obtained from four dayold culture exhibited high antimicrobial activityagainst the test microorganisms, which is inagreement with the earlier reports (24, 25, 26,27, 28).

pH and Incubation Temperature on Biomassand Bioactive Metabolite Production: Themaximum growth and antimicrobial activity ofthe strain was obtained at pH 7 (Fig. 2) suggestingits inclusion in the neutrophilic actinomycetesgroup. Bhattacharya et al. (29) reported that 7 isthe optimum pH for antibiotic production byStreptomyces hygroscopicus D1.5. Atta et al. (30)stated that the optimum initial pH valuecapable of promoting biosynthesis of anti-

microbial agents by Streptomyces torulosus KH-4 was found to be 7. Similarly, bioactivemetabolites obtained from the isolateStreptomyces sp. VITSVK 9 at pH 7 exhibitedgood antimicrobial activity (21). There was anincrease in the growth of the cell as well as theproduction of bioactivie metabolic productionwith the increase of the incubation temperaturefrom 20°C -30°C (Fig 3). However, furtherincrease in temperature (above 30°C) resulted inthe decreased growth rate and decline in theproduction of bioactive metabolite (Fig.3). Interms of its optimum temperature for growth, theorganism appeared to be mesophilic in nature.This is in agreement with earlier reports forseveral of the Streptomyces species (31, 32, 33,34, 35).

Culture Media on Biomass and BioactiveMetabolite Production: Biomass and bioactivemetabolite production of the strain was studiedin different culture media (Fig. 4). Among themedia tested, asparagine-glucose broth producedhigher levels of bioactive metabolites followedby tryptone broth (ISP 1) and tyrosine broth (ISP7). Similarly production of biomas was higher inczapek–dox broth followed by soya-bean meal

Fig 1. Growth pattern of Streptomyces cheonanensis VUK-A. Data are statistically analyzed and found tobe significant at 5%.

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Fig. 2. Effect of pH on biomass and bioactive metabolite production by Streptomyces cheonanensisVUK –A. Data are statistically analyzed and found to be significant at 5%.

Fig. 3. Effect of temperature on biomass and bioactive metabolite production by Streptomycescheonanensis VUK-A. Data are statistically analyzed and found to be significant at 5%.

Fig. 4. Effect of different culture media on cell growth and bioactive metabolite production by Streptomyces cheonanensis VUK-10. Data are statistically analyzed and found to be significant at 5%.



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broth and asparagine-glycerol broth (ISP 5).There is a significant increase in the bioactivemetabolite production cultured in asparagine-glucose broth. Saha et al. (7) reported thatczapek- dox broth favored high rates of antibioticproduction by Streptomyces sp. MNK-7 isolatedfrom soil samples from Bangladesh. Kavitha andVijayalakshmi (28) showed that maltose-tryptonebroth favored maximum production of bioactivemetabolite production by Nocardia levis MK-VL-113.

Carbon and Nitrogen Sources on Biomass andBioactive Metabolite Production: The details ofthe effect of carbon and nitrogen sources onproduction of biomass and bioactive metabolitesby Streptomyces cheonanensis VUK-A wereshowed in figs. 5 and 6. Significant productionof bioactive metabolite was obtained in lactoseamended media followed by fructose and sucrose.Similarly, the production of biomass was highwith fructose followed by sucrose and lactose.These results are comparable with Streptomyceshygroscopicus strains AK-111-81, CH-7, whichutilized lactose as carbon source for antibioticproduction (36, 37). As lactose emerged as the

most preferred carbon source for bioactivemetabolite production by the strain, varyingconcentrations of lactose (0.5-5%) was tested todetermine its optimal concentration. As shownin Fig 7, lactose at levels of 2% and 1% showedoptimal yields of biomass and bioactivemetabolites, respectively. A few reportssuggested that maximum growth and bioactivemetabolite production occurred with glucose(1%) as sole carbon source (32, 38, 39). In orderto develop effective composition of growthmedium, the roles of different nitrogen sourceswere evaluated for their influence on growth andantimicrobial agent production by the strain. Ofall examined nitrogen sources, bacteriologicalpeptone was found to be the best nitrogen sourcefor growth as well as bioactive metaboliteproduction. It should be noted that tryptone andtyrosine, as nitrogen sources, also favored goodgrowth but the antimicrobial compound yield wasless in comparison to peptone. Inorganic nitrogensources like ammonium sulfate, ammoniumoxalate, sodium nitrate and some organic nitrogensources like yeast extract and casein did not showsignificant effect on antibiotic production by thestrain. Peptone enhanced the biomass and

Fig. 5. Effect of different carbon sources on biomass and bioactive metabolite production by Streptomyces cheonanensisVUK –A. Data are statistically analyzed and found to be significant at 5%

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Fig. 6. Effect of different nitrogen sources on biomass and bioactive metabolite production by Streptomycescheonanensis VUK –A. Data are statistically analyzed and found to be significant with 5%.

Fig. 7. Effect of different concentrations of Lactose on biomass and bioactive metabolite production byStreptomyces cheonanensis VUK –A. Data are statistically analyzed and found to be significant at 5%.



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bioactive metabolite production by Streptomycescheonanensis VUK-A which is in conformitywith earlier reports (21, 40, 41, 32). Effect ofdifferent concentrations of peptone on theproduction of bioactive metabolite production hasbeen shown in Fig.8. It should be noted thatpeptone at a concentration of 1% and 0.25%exhibited optimal production of biomass andbioactive metabolite, respectively. Hassan et al.(42) recorded that NaNO

3 followed by peptone

and alanine at a concentration of 0.25% increasedantibiotic production by Streptomyces violates.Ismet et al. (43) also reported that corn steep

powder at a concentration of 0.25% enhancedcell growth and bioactive metabolite productionby Micromonospora sp. M 39.

Minerals on Biomass and Bioactive MetaboliteProduction: Effect of minerals on biomass andbioactive metabolite production by the strain hasbeen shown in Fig 9. Among the minerals tested,K

2HPO

4 supported biomass and bioactive

metabolite production whereas lowerantimicrobial metabolite production was obtainedwith FeSO

4 and ZnSO

4. Ripa et al. (44) also

reported that K2HPO

4 showed positive influence

on antibiotic production by the strain. Narayana

Fig. 9. Effect of different minerals on biomass and bioactive metabolite production by Streptomyces cheonanensisVUK –10. Data are statistically analyzed and found to be significant at 5%.

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Fig. 8. Effect of different concentrations of peptone on biomass and bioactive metabolite production by Streptomycescheonanensis VUK –A. Data are statistically analyzed and found to be significant at 5%.

and Vijayalakshmi, (18) also noted that K2HPO

4

slightly enhanced the production of cell mass andbioactive metabolites of Streptomycesalbidoflavus.

DiscussionA broad spectrum bioactive metabolite

producing actinomycetes isolate VUK-A fromCoringa mangrove ecosystem, Andhra Pradesh,India has been identified as Streptomycescheonanensis VUK-A. Ninety six hours of

incubation at 30ºC and pH 7 was found to beoptimum for bioactive metabolite production. Ithas been reported that the environmental factorslike temperature, pH and incubation time haveprofound influence on antibiotic production (45).It was found that the bioactive metaboliteproduction by Streptomyces cheonanensis VUK-A was positively influenced by the carbohydrates,nitrogen sources and minerals. The resultssuggest that antibiotic production was higher inmedium having 1% lactose as carbon source. The

bioactive metabolite production got reduced withincrease or decrease of lactose concentration.Among the nitrogen sources tested maximumantibiotic production was obtained with 0.25%peptone. In comparison with inorganic nitrogensources, organic nitrogen sources gave relativelyhigher antimicrobial agent production byStreptomyces cheonanensis VUK-A. This is inconformity with the findings of Vahidi et al., (46)which showed that the organic nitrogen sourcesare better for the production of antifungal agents.Among minerals, K

2HPO

4 favored slight

enhancement of antibiotic production. The resultsindicated that antibiotic production is greatly

influenced by medium constituents. Vilches etal., (47) stated that the nature of carbon andnitrogen sources strongly effect antibioticproduction in different organisms. In the presentstudy, the metabolites produced by Streptomycescheonanensis VUK-A grown under optimizedconditions exhibited good antimicrobial activityagainst gram positive, gram negative bacteria andfungi (Figs. 10, 11 and 12). Hence, further studiesregarding the purification, characterization andidentification of bioactive compounds producedby Streptomyces cheonanensis VUK-A are inprogress.



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Fig. 11. Figure representing the anti-microbial metabolite produced by Streptomyces cheonanensis VUK-A (presented inzone of inhibition) under optimized conditions tested against various fungi. Data are statistically analyzed andfound to be significant at 5%.

Fig. 10. Figure representing the anti-microbial metabolite produced by Streptomyces cheonanensis VUK-A (presentedin terms of zone of inhibition) under optimized conditions tested against various bacteria. Data are statisticallyanalyzed and found to be significant at 5%.

Acknowledgements:The authors are thankful to CSIR, India

for providing the financial support.

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