• IPSITA DAS Department of Botany, Microbial Biotechnology Laboratory, North Orissa University, Baripada, Odisha, India.
  • MRUNMAYA KU PANDA Department of Botany, Microbial Biotechnology Laboratory, North Orissa University, Baripada, Odisha, India.
  • CHANDI C.RATH Department of Life Sciences, Rama Devi Women’s University, Bhubaneswar, Odisha, India.



Bacillus amyloliquefaciens, Secondary metabolites, Antimicrobial activity, 16S rDNA sequencing, Phylogenetic study


Objective: The purpose of our study was to isolate and identify the bacteriocinogenic strain exhibiting broad range antimicrobial activity and to analyze the effect of different culturing conditions on the production of an antimicrobial metabolites isolated from the soil of Simlipal Biosphere Reserve, India.

Methods: In the current study, bacterial strains were screened for antimicrobial activity from soil samples of five different regions. The effect of varying culture conditions such as pH, incubation period, and temperature along with carbon and nitrogen sources with and without certain salts was studied. The characterization of the potent strain was studied by morphological, biochemical, and 16S rRNA genetic sequencing. A phylogenetic affiliation of the strain was studied.

Results: A total of 31 out of 245 strains isolated from soil were screened on the basis of antimicrobial results against the test pathogens. On the basis of bacteriocin-like inhibition studies method, one potential isolate that exhibited the highest inhibition against all the pathogens was selected. The optimization of highest antimicrobial metabolite production by the isolate with the influence of physical parameters was found as the incubation period of 3 days with 37°C temperature at pH 8 and for the chemical parameters dextrose was showed the most effective carbon sources when implemented with salts and yeast extract as the best sources of nitrogen with salts. The crude metabolite showed an absorbance peak value of 1.234 with optimum ƛ-max at 214 nm. The potent isolate showed maximum identity with Bacillus amyloliquefaciens (99% similarity) with highest query coverage on basic local alignment search tool search analysis of the 16S rDNA sequence. Phylogenetic analysis revealed close affiliation of the isolate with B. amyloliquefaciens (KC494392.1) having antimicrobial activity.

Conclusion: The findings revealed that the incubation period, temperature, pH, and the culture medium have a direct influence on the production of metabolites. These parameters can be modified for the improvement of the fermentation process.


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Author Biography

IPSITA DAS, Department of Botany, Microbial Biotechnology Laboratory, North Orissa University, Baripada, Odisha, India.

Ipsita Das Ph.D Scholar,Dept. of Botany North Orissa University,

Odisha,India, 757003


Luzhetskyy A, Pelzer S, Bechthold A. The future of natural products as a source of new antibiotics. Curr Opin Investig Drugs 2007;8:608-13.

Bĕhal V. Bioactive products from streptomyces. Adv Appl Microbiol 2000;47:113-56.

Gallo M, Amonette R, Lauber C, Sinsabaugh RL, Zak DR. Microbial community structure and oxidative enzyme activity in nitrogen-amended north temperate forest soils. Microb Ecol 2004;48:218-29.

Thomashow LS, Bonsali RF, David M. Detection of antibiotics produced by soil and rhizosphere microbes in situ. In: Karlovsky P, editor. Secondary Metabolites in Soil Ecology. Berlin Heidelberg: Springer; 2008.

Sánchez S. Microbial diversity-the bright and promising future of microbial manufacturing. Curr Opin Microbiol 2005;8:229-33.

Fravel DR. The role of antibiosis in biocontrol of plant diseases. Annu Rev Phytopathol 1988;26:75-91.

Thomsow LS, Bonsali RF, Weller DM. Antibiotic production by soil and rhizosphere microbes’ in-situ. Man Environ Microbiol 1997:493-99.

Bibb MJ. Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol 2005;8:208-15.

Leifert C, Li H, Chidburee S, Hampson S, Workman S, Sigee D, et al. Antibiotic production and biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus CL45. J Appl Bacteriol 1995;78:97-108.

Sánchez S, Chávez A, Forero A, García-Huante Y, Romero A, Sánchez M, et al. Carbon source regulation of antibiotic production. J Antibiot (Tokyo) 2010;63:442-59.

Baruah TC, Barthakur HP. Physico-chemical methods of soil analysis. In: Baruah TC, Barthakur HP, editors. A Text Book of Soil Analysis. New Delhi: Vikash Publishing House, Pvt. Ltd.; 1988. p. 34-69.

Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.

Mayr-Harting A, Hedges AJ, Berkeley RC. Methods for studying bacteriocins. Methods Microbiol 1972;7:315-422.

Lippert H, Brinkmeyer R, Mulhaupt T, Iken K. Antimicrobial activity in sub marine invertebrates. Polar Boil 2003;26:591-600.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725-9.

Saitou N, Nei M. The neighbour-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.

Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 1985;39:783-91.

Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbour-joining method. Proc Natl Acad Sci U S A 2004;101:11030-5.

Janisiewicz W, Roitman J. Biological control of blue mold and gray mold on apple and pear with Pseudomonas cepacia. Phytopathology 1988;78:1697-700.

Rooney AP, Price NP, Ehrhardt C, Swezey JL, Bannan JD. Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. In aquosorum subsp. Nov. Int J Syst Evol Microbial 2009;59:2429-36.

Fukumoto J. Studies on the production of bacterial amylase. I. Isolation of bacteria secreting potent amylases and their distribution. J Agric Chem Soci Jpn 1943;19:487-503.

Kaur S, Kaur J, Pankaj PP. Isolation and characterization of antibiotic producing microorganisms from soil samples of certain area of Punjab region of India. Int J Pharm Clin Res 2014;6:312-5.

Arguelles-Arias A, Ongena M, Halimi B, Lara Y, Brans A, Joris B, et al. Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb Cell Fact 2009;8:63.

Naclerio G, Ricca E, Sacco M, De Felice M. Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus. Appl Environ Microbiol 1993;59:4313-6.

Tsuge K, Ano T, Shoda M. Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8. Arch Microbiol 1996;165:243-51.

Pannapa P, Pattra S. Antimicrobial and enzyme activity produced by Bacillus spp. Isolated from soil. Int J Pharm Pharm Sci 2017;9:205-10.

Milner J, Raffel S, Lethbridge B, Handelsman J. Culture conditions that influence accumulation of zwittermicin A by Bacillus cereus UW 85. Appl Microbiol Biothechnol 1995;43:685-91.

Martin JF, Demain AL. Control of antibiotic biosynthesis. Microbiol Rev 1980;44:230-51.

Meyers E, Brown W, Principe P, Rathnum M, Parker WE. A new peptide antibiotic. I. Fermentation, isolation, and preliminary characterization. J Antibiot 1973;26:444-8.

Jia Z, Zhang X, Cao X. Effects of carbon sources on fungal morphology and lovastatin biosynthesis by submerged cultivation of Aspergillus terreus. Asia Pac J Chem Eng 2009;4:672-77.

Zheng G, Slavik MF. Isolation, partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain. Lett Appl Microbiol 1999;28:363-7.

Moyne AL, Shelby R, Cleveland TE, Tuzun S. Bacillomycin D: An iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol 2001;90:622-9.

El-Banna N. Antimicrobial substances producing of the air flora. Arab Gulf J Sci Res 2003;21:134-9.

Wang YH, Li YP, Zhang Q, Zhang X. Enhanced antibiotic activity of Xenorhabdus nematophila by medium optimization. Bioresour Technol 2008;99:1708-15.

Lancini G, Parenti F. Antibiotics. New York, Heidelberg, Berlin: Springer-Verlag; 1982. p. 220-41.

El-Banna N. Effect of carbon source on the antimicrobial activity of the air flora. World J Microbiol Biotechnol 2005;21:1451-4.

Berkely CW, Logan NA, Shute LA, Capey AG. Identification of Bacillus species. Method Microbial 1984;16:291-328.

Claus D, Berkely CW. The genus Bacillus. In: Sneath PH, editor. Bergeys Manual of Systematic Bacteriology. Vol. 2. Baltimore: Wilkins; 1986. p. 1105-39.

Vahidi H, Kobarfard F, Namjoyan F. Effect of cultivation conditions on growth and antifungal activity of Mycena leptocephala. Afr J Biotechnol 2004;3:606-9.

Sharma G, Dang S, Gupta S, Gabrani R. Identification and molecular characterization of bacteria having antimicrobial and antibiofilm activity. Int J Pharm Pharm Sci 2016;8:111-4.



How to Cite

DAS, I., M. KU PANDA, and C. C.RATH. “IN VITRO ANTIMICROBIAL ACTIVITY AND MOLECULAR CHARACTERIZATION OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM SIMILIPAL BIOSPHERE RESERVE, ODISHA, INDIA”. Asian Journal of Pharmaceutical and Clinical Research, vol. 12, no. 3, Mar. 2019, pp. 164-9, doi:10.22159/ajpcr.2019.v12i3.29319.



Original Article(s)