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Calcium Alginate Bead-mediated Enhancement of the Selective Recovery of a Lead Novel Antifungal Bacillomycin Variant

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Abstract

In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF1 to AF5) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF4 was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF4 purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF4 lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO4 (2 mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8 mg/L was achieved with 44% selectivity at 30 °C compared to unoptimized conditions (186.4 ± 6.1 mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF4 variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF1 and AF2. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF4 variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.

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References

  1. Perfect, J. R. (2017). The antifungal pipeline: a reality check. Nature Reviews Drug Discovery, 16(9), 603–616.

    Article  CAS  Google Scholar 

  2. Stein, T. (2005). Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56(4), 845–857.

    Article  CAS  Google Scholar 

  3. Bonmatin, A. J. M., Laprévote, O., & Peypoux, F. (2003). Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents. Combinatorial Chemistry & High Throughput Screening, 6(6), 541–556.

    Article  CAS  Google Scholar 

  4. Romero, D., de Vicente, A., Rakotoaly, R. H., Dufour, S. E., Veening, J. W., Arrebola, E., Cazorla, F. M., Kuipers, O. P., Paquot, M., & Pérez-García, A. (2007). The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Molecular Plant-Microbe Interactions, 20(4), 430–440.

    Article  CAS  Google Scholar 

  5. Ohno, A., Ano, T., & Shoda, M. (1995). Effect of temperature on production of lipopeptides antibiotics, iturin a and surfactin by dual producer, B. subtlis RB14, in solid state fermentation. Journal of Fermentation and Bioengineering, 80(5), 517–519.

    Article  CAS  Google Scholar 

  6. Bland, J. M. (1996). The first synthesis of a member of the iturin family, the antifungal cyclic lipopeptide, iturin-A2. The Journal of Organic Chemistry, 61(16), 5663–5664.

    Article  CAS  Google Scholar 

  7. Pathak, K. V. (2011). Purification and characterization of antifungal compounds produced by banyan endophytic Bacilli Ph.D. Thesis, Sardar Patel University, Vallabh Vidynagar

  8. Rangarajan, V., & Clarke, K. G. (2015). Process development and intensification for enhanced production of Bacillus lipopeptides. Biotechnology and Genetic Engineering Reviews, 31(1-2), 46–68.

    Article  Google Scholar 

  9. Chen, H. L., & Juang, R. S. (2008). Extraction of surfactin from fermentation broth with n-hexane in microporous PVDF hollow fibers: significance of membrane adsorption. Journal of Membrane Science, 325(2), 599–604.

    Article  CAS  Google Scholar 

  10. Liu, T., Montastruc, L., Gancel, F., Zhao, L., & Nikov, I. (2007). Integrated process for production of surfactin: part 1: adsorption rate of pure surfactin onto activated carbon. Biochemical Engineering Journal, 35(3), 333–340.

    Article  CAS  Google Scholar 

  11. Palmieri, G., Cassani, G., & Fassina, G. (1995). Peptide immobilization on calcium alginate beads: applications to antibody purification and assay. Journal of Chromatography B: Biomedical Sciences and Applications, 664(1), 127–135.

    Article  CAS  Google Scholar 

  12. Ramachandran, R., Chalasani, A. G., Lal, R., Roy, U. (2014). A broad-spectrum antimicrobial activity of Bacillus subtilis RLID 12.1. Scientific World Journal, 968487.

  13. Ramchandran, R., Shrivastava, M., Namitha, N. N., Thakur, R. L., Chakrabarti, A., & Roy, U. (2018). Evaluation of antifungal efficacy of three new cyclic lipopeptides of the class bacillomycin from Bacillus subtilis RLID 12.1. Antimicrobial Agents and Chemotherapy, 62, e01457–e01417.

    Google Scholar 

  14. Tabbene, O., Kalai, L., & Imen, B. S. (2011). Anti-Candida effect of bacillomycin D-like lipopeptides from Bacillus subtilis B38. FEMS Microbiology Letters, 316(2), 108–114.

    Article  CAS  Google Scholar 

  15. Deepika, S., Santi, M. M., & Rajesh, K. M. (2014). Purification and characterization of a novel lipopeptide from Streptomyces amritsarensis sp. nov. active against methicillin-resistant Staphylococcus aureus. AMB Express, 4, 50.

    Article  Google Scholar 

  16. Chakrabarti, A., Chatterjee, S. S., Das, A., & Shivaprakash, M. R. (2011). Invasive aspergillosis in developing countries. Medical Mycology, 49(Suppl 1), 35–47. https://doi.org/10.3109/13693786.2010.505206.

    Article  Google Scholar 

  17. Colombo, A. L., Padovan, A. C. B., & Chaves, G. M. (2011). Current knowledge of Trichosporon spp. and Trichosporonosis. Clinical Microbiology Reviews, 249(4), 682–700. https://doi.org/10.1128/CMR.00003-11.

    Article  CAS  Google Scholar 

  18. Cortez, K. J., Roilides, E., Quiroz-Telles, F., Meletiadis, J., Antachopoulos, C., Knudsen, T., Buchanan, W., Milanovich, J., Sutton, D. A., Fothergill, A., Rinaldi, M. G., Shea, Y. R., Zaoutis, T., Kottilil, S., & Walsh, T. J. (2008). Infections caused by Scedosporium spp. Clinical Microbiology Reviews, 21(1), 157–197.

    Article  CAS  Google Scholar 

  19. Buzina, W., Braun, H., Schimpl, K., & Stammberger, H. (2003). Bipolaris spicifera causes fungus balls of the sinuses and triggers polypoid chronic rhinosinusitis in an immunocompetent patient. Journal of Clinical Microbiology, 41(10), 4885–4887. https://doi.org/10.1128/JCM.41.10.4885-4887.2003.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Bava, A. J., Fayad, A., Céspedes, C., & Sandoval, M. (2003). Fungal peritonitis caused by Bipolaris spicifera. Medical Mycology, 41(6), 529–531. https://doi.org/10.1080/13693780310001610065.

    Article  CAS  PubMed  Google Scholar 

  21. Clinical Laboratory Standards (1998) Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard. NCCLS document M38-P. National Committee for Clinical Laboratory Standards, Wayne, Pa.

  22. Xiaomei, B., Zhaoxin, L., Fengxia, L., & Xiaoxiong, Z. (2005). Screening the main factors affecting extraction of the antimicrobial substance from Bacillus sp. fmbJ using the Plackett–Burman method. World Journal of Microbiology and Biotechnology, 21, 925–928.

    Article  Google Scholar 

  23. Gudiña, E. J., Fernandes, E. C., Rodrigues, A. I., Teixeira, J. A., & Rodrigues, L. R. (2015). Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium. Frontiers in Microbiology, 6, 59.

    PubMed  PubMed Central  Google Scholar 

  24. Gu, X. B., Zheng, Z. M., Yu, H. Q., Wang, J., Liang, F., & Liu, R. L. (2005). Optimization of medium constituents for a novel lipopeptide production by Bacillus subtilis MO-01 by a response surface method. Process Biochemistry, 40, 3196–3201.

    Article  CAS  Google Scholar 

  25. Mhatre, E., Troszok, A., Gallegos-Monterrosa, R., Lindstädt, S., Hölscher, T., Kuipers, O. P., & Kovács, Á. T. (2016). The impact of manganese on biofilm development of Bacillus subtilis. Microbiology, 162(8), 1468–1478.

    Article  CAS  Google Scholar 

  26. Eisenstadt, E., Fisher, S., Der, C. L., & Silver, S. (1973). Manganese transport in Bacillus subtilis W23 during growth and sporulation. Journal of Bacteriology, 113(3), 1363–1372.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Serp, D., Cantana, E., Heinzen, C., Von Stockar, U., & Marison, I. W. (2000). Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization. Biotechnology and Bioengineering, 70(1), 41–53.

    Article  CAS  Google Scholar 

  28. Stephen, A. C., & John, C. V. (2016). Lipopeptides from Bacillus and Paenibacillus spp.: a gold mine of antibiotic candidates. Medicinal Research Reviews, 36, 4–31.

    Article  Google Scholar 

  29. Kumar, A., & Johri, B. N. (2012). Antimicrobial lipopeptides of Bacillus: natural weapons for biocontrol of plant pathogens. In T. Satyanarayana, A. Kumar, & B. N. Johri (Eds.), Microorganisms in sustainable agriculture and biotechnology (pp. 90–111). Dordrecht: Springer.

    Google Scholar 

  30. Latham, R. H. (2000). Bipolaris spicifera meningitis complicating a neurosurgerical procedure. Scandinavian Journal of Infectious Diseases, 32(1), 102–103. https://doi.org/10.1080/00365540050164353.

    Article  CAS  PubMed  Google Scholar 

  31. Chollet-Imbert, M., Gancel, F., Slomianny, C., & Jacques, P. H. (2009). Differentiated pellicle organization and lipopeptide production in standing culture of Bacillus subtilis strains. Archives of Microbiology, 191(1), 63–71.

    Article  CAS  Google Scholar 

  32. Davis, D. A., Lynch, H. C., & Varley, J. (1999). The production of surfactin in batch culture by Bacillus subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism. Enzyme and Microbial Technology, 25(3-5), 322–329.

    Article  CAS  Google Scholar 

  33. Pryor, S. W., Gibson, D. M., Hay, A. G., Gossett, J. M., & Walker, L. P. (2007). Optimization of spore and antifungal lipopeptide production during the solid-state fermentation of Bacillus subtilis. Applied Biochemistry and Biotechnology, 143(1), 63–79.

    Article  CAS  Google Scholar 

  34. Jacques, P., Hbid, C., Destain, J., Razafindralambo, H., Paquot, M., Pauw, E., & Thonart, P. (1999). Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design. Applied Biochemistry and Biotechnology, 77(1-3), 223–233.

    Article  Google Scholar 

  35. Heron, J. R. (1966). Some observations on commercial malt extracts. Journal of the Institute of Brewing, 72(5), 452–457.

    Article  CAS  Google Scholar 

  36. Wokes, F., & Klatzkin, C. (1994). Protein in malted preparations. Journal of Pharmacy and Pharmacology, 9(1), 903–914. https://doi.org/10.1111/j.2042-7158.1949.tb12510.x.

    Article  Google Scholar 

  37. Vandana, B. (2015). Studies on the microbial production, molecular characterization and biopreservative potential of bacteriocin. Sant Longowal Institute of Engineering and Technology.

  38. Shih, I. L., Lin, C. Y., & Wu, J. Y. (2009). Production of antifungal lipopeptide from Bacillus subtilis in submerged fermentation using shake flask and fermenter. Korean Journal of Chemical Engineering, 26(6), 1652–1661.

    Article  CAS  Google Scholar 

  39. Lin, H. Y., Rao, Y. K., Wu, W. S., & Tzeng, Y. M. (2007). Ferrous ion enhanced lipopeptide antibiotic iturin a production from Bacillus amyloliquefaciens B128. International Journal of Applied Science and Engineering, 5, 123–132.

    Google Scholar 

  40. Zhao, P., Quan, C., Jin, L., Wang, L., Wang, J., & Fan, S. (2013). Effects of critical medium components on the production of antifungal lipopeptides from Bacillus amyloliquefaciens Q-426 exhibiting excellent biosurfactant properties. World Journal of Microbiology and Biotechnology, 29(3), 401–409.

    Article  CAS  Google Scholar 

  41. Wei, H., & Chu, I. M. (1998). Enhancement of surfactin production in iron-enriched media by Bacillus subtilis ATCC 21332. Enzyme and Microbial Technology, 22(8), 724–728.

    Article  CAS  Google Scholar 

  42. Shemesh, M., & Hai, Y. A. (2013). Combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling. Journal of Bacteriology, 195(12), 2747–2754.

    Article  CAS  Google Scholar 

  43. Kavamura, V. N., & Melo, I. S. (2014). Effects of different osmolarities on bacterial biofilm formation. Brazilian Journal of Microbiology, 45(2), 627–631.

    Article  Google Scholar 

  44. Xu, Z., Shao, J., Li, B., Yan, X., Shen, Q., & Zhang, R. (2013). Contribution of bacillomycin D in Bacillus amyloliquefaciens SQR9 to antifungal activity and biofilm formation. Applied and Environmental Microbiology, 79, 808–815.

    Article  CAS  Google Scholar 

  45. Luo, C., Zhou, H., Zou, J., Wang, X., Zhang, R., Xiang, Y., & Chen, Z. (2015). Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and the biocontrol of rice sheath blight induced by Rhizoctonia solani. Applied Microbiology and Biotechnology, 99(4), 1897–1910.

    Article  CAS  Google Scholar 

  46. Ohno, A., Ano, T., & Shoda, M. (1993). Effect of temperature change and aeration on the production of the antifungal peptide antibiotic iturin by Bacillus subtilis NB22 in liquid cultivation. Journal of Fermentation and Bioengineering, 75(6), 463–465.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors remain obliged to Dr. V. Vinoth Kumar, SRM University, Chennai, India, for assisting in design software analysis. The authors sincerely acknowledge Dr. Vivek Rangarajan, Department of Chemical Engineering, BITS Pilani KK Birla Goa campus, for his valuable suggestions.

Funding

The authors sincerely acknowledge the funding agency Department of Biotechnology (DBT) [File no. BT/PR14095/NDB/39/525/2015], Government of India. Ramya R and Swetha R sincerely acknowledge the funding agencies Council of Scientific and Industrial Research (CSIR), New Delhi and DBT, Govt. of India respectively, for awarding of the Senior Research Fellowships.

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Authors and Affiliations

  1. Department of Biological Sciences, Birla Institute of Technology And Science Pilani KK Birla Goa Campus, Goa, 403726, India

    Ramya Ramachandran, Swetha Ramesh, Srinath Ramkumar & Utpal Roy

  2. Department of Medical Microbiology, Post Graduate Institute of Medical Education & Research, Chandigarh, India

    Arunaloke Chakrabarti

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  1. Ramya Ramachandran
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Correspondence to Utpal Roy.

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Ramachandran, R., Ramesh, S., Ramkumar, S. et al. Calcium Alginate Bead-mediated Enhancement of the Selective Recovery of a Lead Novel Antifungal Bacillomycin Variant. Appl Biochem Biotechnol 186, 917–936 (2018). https://doi.org/10.1007/s12010-018-2778-3

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