Title : Screening of Different Media and Heat Shock Treatment Regimens for Enhancing Sporulation in Bacillus licheniformis

Vol 6 No 4 (2023): Volume 06 Issue 04 April 2023

Article Date Published : 28 April 2023 | Page No.: 2666-2674 | Google Scholar | Crossref doi for this article

Abstract :

Bacillus licheniformis is a spore-producing bacteria. The present study aims to identify maximum sporulation in Bacillus licheniformis through induced heat shock conditions using different solidified media and broth media. Suspensions of Bacillus licheniformis spores were spread-plated on nutrient agar plates before and after heat shock treatments of 60°C, 80°C and 100°C for 30 minutes. The number of spores was determined by the dilution plate count method. The result indicates that the highest spore production for Bacillus licheniformis was observed when the spores were induced in AK (Arret and Kirshbaum) Agar for seven days under a heat shock treatment of 60℃ for 30 minutes. Whereas, in the case of Nutrient Broth media, the spore production was highest after four days at heat shock treatment of 80℃ for 30 minutes. This study will help find the optimal production of spores from Bacillus licheniformis, an industrially beneficial microorganism.

Keywords :

Bacillus licheniformis, Endospore Staining, Heat shock, Spores, Sporulation

References :

  1. Tehri N, Kumar N, Raghu HV, Shukla R, Vashishth A (2018) Microbial spores: Concepts and industrial applications. In: Singh J, Sharma D, Kumar G, Sharma N, eds. Microbial Bioprospecting for Sustainable Development. Springer Singapore, 279–289.
  2. Gould GW (2006) History of science – Spores: Lewis B Perry Memorial Lecture 2005. Journal of Applied Microbiology 101(3): 507–513. doi:10.1111/j.1365-2672.2006.02888.x.
  3. Hong HA, Le HD, Cutting SM (2005) The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews 29 (4): 813–835. doi: 10.1016/j.femsre.2004.12.001.
  4. Setlow P (2006) Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat and chemicals. Journal of Applied Microbiology 101(3): 514–525. doi: 10.1111/j.1365-2672.2005.02736.x.
  5. Tarr’ HLA (1933) The relation of the composition of the culture medium to the formation of endospores by aerobic Bacilli. Journal of Hygiene 32 (4): 535–543. doi: 10.1017/s0022172400018258.
  6. Piggot PJ, Hilbert DW (2004) Sporulation of Bacillus subtilis. Current Opinion in Microbiology 7 (6): 579–586. doi: 10.1016/j.mib.2004.10.001.
  7. Curran HR, Evans FR (1945) Heat activation inducing germination in the spores of thermotolerant and thermophilic aerobic bacteria. Journal of Bacteriology 49 (4): 335–346. doi: 10.1128/jb.49.4.335-346.1945.
  8. Levinson HS, Hyatt MT (1964) Effect of sporulation medium on heat resistance, chemical composition, and germination of Bacillus Megaterium spores. Journal of Bacteriology 87 (4): 876–886. doi: 10.1128/jb.87.4.876-886.196
  9. Chen ZM, Li Q, Liu HM, Yu N, Xie TJ, Yang MY, Shen P, Chen XD (2010) Greater enhancement of Bacillus subtilis spore yields in submerged cultures by optimization of medium composition through statistical experimental designs. Applied Microbiology and Biotechnology 85 (5): 1353–1360. doi: 10.1007/s00253-009-2162-x.
  10. Verma N, Singh NA, Kumar N (2013) Screening of different media for sporulation of Bacillus megaterium National agriculture innovation project View project Autoimmune and inflammatory diseases biomarker database View project. International Journal of Microbiology Research and Reviews 1 (4): 68–73.
  11. Turnbull PCB, Frawley DA, Bull RL (2007) Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores. Journal of Microbiological Methods 68 (2): 353–357. doi: 10.1016/j.mimet.2006.09.014.
  12. Jafari M, Alebouyeh M, Mortazavian AM, Hosseini H, Ghanati K, Amiri Z, Zali MR (2016) Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers. Nutrition and Food Sciences Research 3 (1): 35–42. doi: 10.18869/acadpub.nfsr.3.1.35.
  13. Rice EW, Rose LJ, Johnson CH, Boczek LA, Arduino MJ, Reasoner DJ (2004) Boiling and Bacillus Spores. Emerging Infectious Diseases 10 (10): 1887–1888. doi: 10.3201/eid1010.040158.
  14. Byun BY, Cho HY, Hwang HJ, Mah JH, Liu Y, Tang J, Kang DH (2011) Optimization and evaluation of heat-shock condition for spore enumeration being used in thermal-process verification: Differential responses of spores and vegetative cells of clostridium sporogenes to heat shock. Food Science and Biotechnology 20 (3): 751–757. doi: 10.1007/s10068-011-0105-7.
  15. Finley N, Fields ML (1962) Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus Spores. Applied Microbiology 10 (3): 231–236. doi: 10.1128/am.10.3.231-236.1962.
  16. Chung S, Lim HM, Kim SD (2007) Formulation of stable Bacillus subtilis AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers. Applied Microbiology and Biotechnology 76 (1): 217–224. doi: 10.1007/s00253-007-0992-y.
  17. Yang WW, Crow-Willard EN, Ponce A (2009) Production and characterization of pure Clostridium spore suspensions. Journal of Applied Microbiology 106 (1): 27–33. doi: 10.1111/j.1365-2672.2008.03931.x.
  18. Raso J, Palop A, Bayarte M, Condon S, Sala FJ (1995) Influence of sporulation temperature on the heat resistance of a strain of Bacillus licheniformis (Spanish Type Culture Collection 4523). Food Microbiology 12: 357-361. doi: 10.1016/S0740-0020(95)80116-2.
  19. Baweja RB, Zaman MS, Mattoo AR, Sharma K, Tripathi V, Aggarwal A, Dubey GP, kurupati RL, Ganguli M, Chaudhury NK, Sen S, Das TK, Gade WN, Singh Y (2008) Properties of Bacillus anthracis spores prepared under various environmental conditions. Archives of Microbiology 189 (1): 71–79. doi: 10.1007/s00203-007-0295-9.
  20. Sinnelä MT, Park YK, Lee JH, Jeong KC, Kim YW, Hwang HJ, Mah JH (2019) Effects of calcium and manganese on sporulation of bacillus species involved in food poisoning and spoilage. Foods 8 (4): 119. doi: 10.3390/foods8040119.
  21. Charney J, Fisher WP, Hegarty CP (1951) Manganese as an essential element for sporulation in the genus Bacillus. Journal of Bacteriology 62(2): 145-148. doi: 10.1128/jb.62.2.145-148.1951.
  22. Weinberg ED (1964) Manganese Requirement for Sporulation and Other Secondary Biosynthetic Processes of Bacillus. Applied Microbiology 12(5):436-441. doi: 10.1128/am.12.5.436-441.1964.
  23. Vasantha N and Freese E (1979) The Role of Manganese in Growth and Sporulation of Bacillus subtilis. Journal of General Microbiology 112(2):329-336. doi: 10.1099/00221287-112-2-329.
  24. Stöckel S, Meisel S, Böhme R, Elschner M, Rosch P, Popp J (2009) Effect of supplementary manganese on the sporulation of Bacillus endospores analysed by Raman spectroscopy. Journal of Raman Spectroscopy 40 (11): 1469–1477.

doi: 10.1002/jrs.2292.

  1. Zhang J, Lu J, Liu B, Liu Q, Jin F, Zhang M, Liu Y, Song Y, Dong C, Zhang W, Han N, Deng X, Xing F (2019) Ceramic Processing Research Rapid quantification of viable spore used in healing concrete cracks by a simple spectrophotometric method. Journal of Ceramic Processing Research 20:63-69. doi: 10.36410/jcpr.2019.20..63.
  2. Sala FJ, Ibarz P, Palop A, Raso J, Condon S (1995) Sporulation Temperature and Heat Resistance of Bacillus subtilis at Different pH Values. Journal of Food Protection 58(3): 239-243. doi: 10.4315/0362-028X-58.3.239.
  3. Raso J, Palop A, Pagá R, Condó S (1998) Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. Journal of Applied Microbiology 85 (5): 849–854.

doi: 10.1046/j.1365-2672.1998.00593.x.

  1. Dragon D, Rennie R (2001) Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis spores. Letters in Applied Microbiology 33 (2): 100–105. doi: 10.1046/j.1472-765x.2001.00966.x.
  2. Marquez VO, Mittal GS, Griffiths MW (1997) Destruction and inhibition of bacterial spores by high voltage pulsed electric field. Journal of Food Science 62 (2): 399–401. doi: 10.1111/j.1365-2621.1997.tb04010.x.
  3. Feijoo SC, Hayes WW, Watson CE, Martin JH (1997) Effects of Microfluidizer® Technology on Bacillus licheniformis Spores in Ice Cream Mix. Journal of Dairy Science 80 (9): 2184–2187. doi: 10.3168/jds.S0022-0302(97)76166-6.
  4. Zheng XL, Xiong ZQ, Wu JQ (2017) The use of a simple flow cytometry method for rapid detection of spores in probiotic Bacillus licheniformis-containing tablets. Food Science and Biotechnology 26 (1): 167–171.

doi: 10.1007/s10068-017-0022-5.

  1. Schaeffer AB, Fulton MD (1933) A Simplified method of Staining Endospores. Science 77 (1990):194.

doi: 10.1126/science.77.1990.194.

  1. Oktari A, Supriatin Y, Kamal M, Syafrullah H (2017) The Bacterial Endospore Stain on Schaeffer Fulton using Variation of Methylene Blue Solution. Journal of Physics: Conference Series 812 012066. doi: 10.1088/1742-6596/812/1/012066.
  2. Scott SA, Brooks JD, Rakonjac J et al. (2007) The formation of thermophilic spores during the manufacture of whole milk powder. International Journal of Dairy Technology 60 (2): 109–117. doi: 10.1111/j.1471-0307.2007.00309.x.
  3. Paredes-Sabja D, Torres JA, Setlow P, Sarker MR (2008) Clostridium perfringens spore germination: Characterization of germinants and their receptors. Journal of Bacteriology 190 (4): 1190–1201. doi: 10.1128/JB.01748-07.
  4. Liang XS, Liu C, Long Z, Guo XH (2018) Rapid and simple detection of endospore counts in probiotic Bacillus cultures using dipicolinic acid (DPA) as a marker. AMB Express 8(1):101. doi: 10.1186/s13568-018-0633-0.
  5. McHugh AJ, Feehily C, Hill C, Cotter PD (2017) Detection and enumeration of spore-forming bacteria in powdered dairy products. Frontiers in Microbiology 8:109. doi: 10.3389/fmicb.2017.00109.
  6. Omer AM (2010) Bioformulations of Bacillus Spores for using as Biofertilizer. Life Science Journal 7 (4): 124–131.
  7. Zhang X, Al-Dossary A, Hussain M, Setlow P, Li J (2020) Applications of bacillus subtilis spores in biotechnology and advanced materials. Applied and Environmental Microbiology 86 (17): e01096-20. doi: 10.1128/AEM.01096-20.
  8. Muras A, Romero M, Mayer C, Otero A (2021) Biotechnological applications of Bacillus licheniformis. Critical Reviews in Biotechnology 41 (4): 609–627. doi: 10.1080/07388551.2021.1873239.

How to Cite :

Screening of Different Media and Heat Shock Treatment Regimens for Enhancing Sporulation in Bacillus licheniformis. Moirangthem Bidyaswori Devi, Rhea Dhar, Atanu Bhattacharjee, Dibyendu Paul , 6(4), 2666-2674. Retrieved from https://ijcsrr.org/single-view/?id=9648&pid=9853

Most read articles by the same author(s)

Moirangthem Bidyaswori Devi, Rhea Dhar, Atanu Bhattacharjee, Dibyendu Paul,

View

157

Citation

Downloads

Article Details