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A Novel Halo-Acid-Alkali-Tolerant and Surfactant Stable Amylase Secreted from Halophile Bacillus siamensis F2 and Its Application in Waste Valorization by Bioethanol Production and Food Industry

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Abstract

The extracellular amylase production level by the moderate halophile Bacillus siamensis F2 was optimized, and the enzyme was biochemically characterized. The culture parameters for NaCl, carbon, nitrogen, pH, and temperature were optimized for high titers of amylase production. Growing B. siamensis F2 cultures in Great Salt Lake-2 medium with additions of (in g/L) NaCl (100), starch (30), yeast extract (2), KNO3 (2), and MgSO4 (1) at pH 8, 30 °C resulted in the maximum amylase production (4.2 U/ml). The amylase was active across a wide range of salinities (0 to 30% NaCl), pH (5.0–10.0), and temperatures (20–70 °C) and showed good stability with surfactants (sodium dodecyl sulfate (SDS) and Triton X-100); hence, it was identified as halo-acid-alkali-tolerant and surfactant stable. Temperature, pH, and salinity were optimal for amylase activity at 50 °C, pH 7, and 5% NaCl, respectively. It also generates amylase by utilizing agricultural wastes like sugarcane bagasse, sweet potato peel, and rice husk. Based on the performance of B. siamensis F2 using agricultural wastes and synthesizing amylase, the current study attempted to produce bioethanol by coculturing with baker’s yeast using sugarcane bagasse and sweet potato peel as a substrate, which yielded 47 and 57 g/L of bioethanol, respectively. Besides bioethanol production, amylase secreted by F2 was also employed for juice clarification for better yield and clarity and for softening dough to produce better-quality buns. This novel amylase may have many potential applications in waste valorization, biorefinery sectors, and food industries.

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Data Availability

The data supporting this study’s findings are available from the corresponding author upon reasonable request. However, data have been fully presented in the manuscript, no additional data to disclose.

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References

  1. Bhatt, B., Prajapati, V., Patel, K., & Trivedi, U. (2020). Kitchen waste for economical amylase production using Bacillus amyloliquefaciens KCP2. Biocatalysis and Agricultural Biotechnology, 26, 101654.

    Google Scholar 

  2. Pranay, K., Padmadeo, S. R., & Prasad, B. (2019). Production of amylase from Bacillus subtilis sp. strain KR1 under solid state fermentation on different agrowastes. Biocatalysis and Agricultural. Biotechnology, 21, 101300.

    Google Scholar 

  3. Kanal, H., Kobayashi, T., Aono, R., & Kudo, T. (1995). Natronococcus amylolyticus sp. nov., a haloalkaliphilic archaeon. Int J Syst Bacteriol, 45(4), 762–766.

    CAS  PubMed  Google Scholar 

  4. Pérez-Pomares, F., Bautista, V., Ferrer, J. C., Pire, C., Marhuenda-egea, F. C., & Bonete, M. J. G. (2003). Amylase activity from the halophilic archaeon Haloferax mediterranei. Extremophiles : Life under extreme conditions, 7, 299–306.

    PubMed  Google Scholar 

  5. Moshfegh, M., Shahverdi, A. R., Zarrini, G., & Faramarzi, M. A. (2013). Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles : Life under extreme conditions, 17, 677–687.

    CAS  PubMed  Google Scholar 

  6. Khire, J. M. (1994). Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan. Lett Appl Microbiol, 19, 210–212.

    CAS  Google Scholar 

  7. Patel, S., Jain, N., & Madamwar, D. (1993). Production of α-amylase from Halobacterium halobium. World J Microbiol Biotechnol, 9, 25–28.

    CAS  PubMed  Google Scholar 

  8. Amoozegar, M. A., Malekzadeh, F., & Malik, K. A. (2003). Production of amylase by newly isolated moderate halophile, Halobacillus sp. strain MA-2. J Microbiol Methods, 52(3), 353–359.

    CAS  PubMed  Google Scholar 

  9. Sharma, A., Sharma, V., Saxena, J., Chandra, R., Alam, A., & Prakash, A. (2015). Isolation and Screening of Amylolytic Bacteria from Soil. International Journal of Scientific Research in Agricultural Sciences, 2, 159–165.

    CAS  Google Scholar 

  10. Deutch, C. E. (2002). Characterization of a salt tolerant extracellular alpha-amylase from Bacillus dipsosauri. Lett Appl Microbiol, 35, 78–84.

    CAS  PubMed  Google Scholar 

  11. Kanthi Kiran, K., & Chandra, T. S. (2008). Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. Strain TSCVKK Applied Microbiology and Biotechnology, 77, 1023–1031.

    Google Scholar 

  12. Du, R., Song, Q., Zhang, Q., Zhao, F., Kim, R. C., Zhou, Z., & Han, Y. (2018). Purification and characterization of novel thermostable and Ca-independent α-amylase produced by Bacillus amyloliquefaciens BH072. Int J Biol Macromol, 115, 1151–1156.

    CAS  PubMed  Google Scholar 

  13. Pranay, K., Padmadeo, S. R., Jha, V. K., & Prasad, B. (2019). Screening and identification of amylase producing strains of Bacillus. Journal of Applied Biology & Biotechnology, 7(4), 57–62.

    CAS  Google Scholar 

  14. de Souza, P. M., & de Oliveira Magalhães, P. (2010). Application of microbial α-amylase in industry - A review. Braz J Microbiol, 41, 850–861.

    PubMed  PubMed Central  Google Scholar 

  15. Kumar, S., Grewal, J., Sadaf, A., Hemamalini, R., & Khare, S. K. (2016). Halophiles as a source of polyextremophilic alpha-amylase for industrial applications. AIMS Microbiology, 2(1), 1–26.

    CAS  Google Scholar 

  16. Qian, M., Tian, S., Li, X., Zhang, J., Pan, Y., & Yang, X. (2006). Ethanol production from dilute-acid softwood hydrolysate by co-culture. Appl Biochem Biotechnol, 134, 273–283.

    CAS  PubMed  Google Scholar 

  17. Okuda, N., Ninomiya, K., Katakura, Y., & Shioya, S. (2008). Strategies for reducing supplemental medium cost in bioethanol production from waste house wood hydrolysate by ethanologenic Escherichia coli: inoculum size increase and coculture with Saccharomyces cerevisiae. J Biosci Bioeng, 105(2), 90–96.

    CAS  PubMed  Google Scholar 

  18. Fu, N., Peiris, P. U. S., Markham, J. L., & Bavor, J. (2009). A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures. Enzym Microb Technol, 45, 210–217.

    CAS  Google Scholar 

  19. Park, E. Y., Naruse, K., & Kato, T. (2012). One-pot bioethanol production from cellulose by co-culture of Acremonium cellulolyticus and Saccharomyces cerevisiae. Biotechnology for Biofuels, 5, 64–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Poosarla, V. G., & Chandra, T. S. (2014). Purification and Characterization of Novel Halo-Acid-Alkali-Thermo-stable Xylanase from Gracilibacillus sp. TSCPVG Applied Biochemistry and Biotechnology, 173, 1375–1390.

    CAS  PubMed  Google Scholar 

  21. Khusro, A., Barathikannan, K., Aarti, C., & Agastian, P. (2017). Optimization of thermo-alkali stable amylase production and biomass yield from Bacillus sp. Under Submerged Cultivation Fermentation, 3, 7.

    Google Scholar 

  22. Giridhar, P. V., & Chandra, T. S. (2010). Production of novel halo-alkali-thermo-stable xylanase by a newly isolated moderately halophilic and alkali-tolerant Gracilibacillus sp. TSCPVG Process Biochemistry, 45, 1730–1737.

    CAS  Google Scholar 

  23. Mahato, R. K., Fatema, I. T., & Rajagopalan, G. (2020). Thermostable, solvent, surfactant, reducing agent and chelator resistant alpha-amylase from Bacillus strain IBT108: A suitable candidate enables one-step fermentation of waste potato for high butanol and hydrogen production. Waste and Biomass Valorization, 12, 223–238.

    Google Scholar 

  24. Poosarla, V. G., & Chandra, T. S. (2021). Xylanase production by halophilic bacterium Gracilibacillus sp. TSCPVG under solid state fermentation. Research. J Biotechnol, 16(7), 92–100.

    Google Scholar 

  25. Simair, A. A., Qureshi, A. S., Khushk, I., Ali, C. H., Lashari, S., Bhutto, M. A., Mangrio, G. S., & Lu, C. (2017). Production and partial characterization of α-amylase enzyme from Bacillus sp. BCC 01-50 and potential applications. Biomed Res Int, 2017, 9173040.

    PubMed  PubMed Central  Google Scholar 

  26. Nissen, L., Rollini, M., Picozzi, C., Musatti, A., Foschino, R., & Gianotti, A. (2020). Yeast-free doughs by zymomonas mobilis: Evaluation of technological and fermentation performances by using a metabolomic approach. Microorganisms, 8, 792.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Thakham, N., Thaweesak, S., Teerakulkittipong, N., Traiosot, N., Kaikaew, A., Lirio, G. A. C., & Jangiam, W. (2020). Structural characterization of functional ingredient levan synthesized by Bacillus siamensis isolated from traditional fermented food in Thailand. Int J Food Sci, 2020, 7352484.

    Google Scholar 

  28. Nasir, A., Rahman, S. S., Hossain, M. M., & Choudhury, N. (2017). Isolation of Saccharomyces Cerevisiae from pineapple and orange and study of metal’s effectiveness on ethanol production. European journal of microbiology & immunology, 7, 76–91.

    CAS  Google Scholar 

  29. Zuriarrain, A., Zuriarrain, J. M. B., Villar, M., & Berregi, I. (2015). Quantitative determination of ethanol in cider by 1H NMR spectrometry. Food Control, 50, 758–762.

    CAS  Google Scholar 

  30. Roy, K., Dey, S., Uddin, M. K., Barua, R., & Hossain, M. T. (2018). extracellular pectinase from a novel bacterium Chryseobacterium indologenes strain SD and its application in fruit juice clarification. Enzyme Research, 2018, 3859752.

    PubMed  PubMed Central  Google Scholar 

  31. Deutch, C. E. (2002). Characterization of a salt-tolerant extracellular a-amylase from Bacillus dipsosauri. Lett Appl Microbiol, 35, 78–84.

    CAS  PubMed  Google Scholar 

  32. Sánchez-Porro, C., Martín, S., Mellado, E., & Ventosa, A. (2003). Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes. J Appl Microbiol, 94(2), 295–300.

    PubMed  Google Scholar 

  33. Martins, R. F., Davids, W., Al-Soud, W. A., Levander, F., Radstrom, P., & Hatti-Kaul, R. (2001). Starch-hydrolyzing bacteria from Ethiopian soda lakes. Extremophiles : Life under extreme conditions, 5, 135–144.

    CAS  PubMed  Google Scholar 

  34. Coronado, M. J., Vargas, C., Hofemeister, J., Ventosa, A., & Nieto, J. N. J. (2000). Production and biochemical characterization of an alpha-amylase from the moderate halophile Halomonas meridiana. FEMS Microbiol Lett, 183(1), 67–71.

    CAS  PubMed  Google Scholar 

  35. Salgaonkar, B. B., Sawant, D. T., Harinarayanan, S., & Braganca, J. (2019). Alpha amylase production by extremely halophilic Archaeon Halococcus strain GUVSC8. Starch-starke, 71, 1800018.

    Google Scholar 

  36. Santorelli, M., Maurelli, L., Pocsfalvi, G., Fiume, I., Squillaci, G., La Cara, F., Del Monaco, G., & Morana, A. (2016). Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica. Int J Biol Macromol, 92, 174–184.

    CAS  PubMed  Google Scholar 

  37. Kumar, S., & Khare, S. K. (2012). Purification and characterization of maltooligosaccharide-forming alpha-amylase from moderately halophilic Marinobacter sp. EMB8. Bioresour Technol, 116, 247–251.

    CAS  PubMed  Google Scholar 

  38. Prakash, B., Vidyasagar, M., Madhukumar, M. S., Muralikrishna, G., & Sreeramulu, K. (2009). Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable alpha-amylases from Chromohalobacter sp. TVSP 101. Process Biochem, 44, 210–215.

    CAS  Google Scholar 

  39. Vijayabaskar, P., Jayalakshmi, D. S., & Shankar, T. (2012). Amylase production by moderately halophilic Bacillus cereus in solid state fermentation. Afr J Microbiol Res, 6, 4918–4926.

    CAS  Google Scholar 

  40. Suganthi, C., Mageswari, A., Karthikeyan, S., & Gothandam, K. M. (2015). Insight on biochemical characteristics of thermotolerant amylase isolated from extremophile bacteria Bacillus vallismortis TD6 (HQ992818). Microbiology, 84, 210–218.

    CAS  Google Scholar 

  41. Patel, S. H., & Madamwar, D. (1994). Photohydrogen production from a coupled system of Halobacterium Halobium and Phormidium Valderianum. Int J Hydrog Energy, 19, 733–738.

    CAS  Google Scholar 

  42. Yassin, S. N., Jiru, T. M., & Indracanti, M. (2021). Screening and characterization of thermostable amylase-producing bacteria isolated from soil samples of Afdera, Afar Region, and Molecular Detection of Amylase-Coding Gene. International Journal of Microbiology, 2021, 5592885.

    PubMed  PubMed Central  Google Scholar 

  43. Bajpai, B., Chaudhary, M., & Saxena, J. (2015). Production and characterization of α-amylase from an extremely halophilic archaeon, Haloferax sp. HA10. Food Technol Biotechnol, 53, 11–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Gomes, I., Gomes, J., & Steiner, W. W. (2003). Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: Production and partial characterization. Bioresour Technol, 90(2), 207–214.

    CAS  PubMed  Google Scholar 

  45. Singh, R., Kapoor, V., & Kumar, V. (2011). Influence of carbon and nitrogen sources on the a-amylase production by a newly isolated thermophilic Streptomyces sp. MSC702 (MTCC 10772). Asian Journal of Biotechnology, 3, 540–553.

    CAS  Google Scholar 

  46. Qin, Y., Huang, Z., & Liu, Z. (2014). A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: Molecular cloning, heterologous expression and biochemical characterization. Extremophiles : Life under extreme conditions, 18, 271–281.

    CAS  PubMed  Google Scholar 

  47. Samanta, A., Mitra, D., Roy, S. N., Sinha, C., & Pal, P. (2013). Characterization and optimization of amylase producing bacteria isolated from solid waste. J Environ Prot, 2013, 647–652.

    Google Scholar 

  48. Kalpana, B. J., & Pandian, S. (2014). Halotolerant, acid-alkali stable, chelator resistant and raw starch digesting α-amylase from a marine bacterium Bacillus subtilis S8-18. J Basic Microbiol, 54, 802–811.

    CAS  PubMed  Google Scholar 

  49. Onishi, H., & Sonoda, K. (1979). Purification and some properties of an extracellular amylase from a moderate halophile, Micrococcus halobius. Appl Environ Microbiol, 38, 616–620.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Kobayashi, T., Kanai, H., Hayashi, T., Akiba, T., Akaboshi, R., & Horikoshi, K. (1992). Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36. J Bacteriol, 174, 3439–3444.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Mesbah, N. M., & Wiegel, J. (2014). Halophilic alkali- and thermostable amylase from a novel polyextremophilic Amphibacillus sp. NM-Ra2. Int J Biol Macromol, 70, 222–229.

    CAS  PubMed  Google Scholar 

  52. Shafiei, M., Ziaee, A. A., & Amoozegar, M. A. (2010). Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting, and halophilic alpha-amylase from a moderately halophilic bacterium. Nesterenkonia sp strain F Process Biochemistry, 45, 694–699.

    CAS  Google Scholar 

  53. Kusuda, M., Nagai, M., Hur, T., Ueda, M., & Terashita, T. (2003). Purification and some properties of Α-amylase from an ectomycorrhizal fungus, Tricholoma matsutake. Mycoscience, 44, 311–317.

    CAS  Google Scholar 

  54. Vidilaseris, K., Hidayat, K., Retnoningrum, D., Nurachman, Z., Noer, A., & Natalia, D. (2009). Biochemical characterization of a raw starch degrading α-amylase from the Indonesian marine bacterium Bacillus sp. ALSHL3. Biologia, 64, 1047–1052.

    CAS  Google Scholar 

  55. Belay, E., & Teshome, M. (2021). Production of bacterial amylase and evaluation for starch hydrolysis. International Journal of Microbiology and Biotechnology, 6(2), 34–44.

    Google Scholar 

  56. Roses, R. P., & Guerra, N. P. (2009). Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material. World J Microbiol Biotechnol, 25, 1929–1939.

    CAS  Google Scholar 

  57. Riungu, A. K., Salim, A. M., Njenga, J. N., Yusuf, A. O., & Waudo, W. (2014). Bioethanol production from waste crops and crop residues. IOSR Journal of Applied Chemistry, 7, 42–47.

    Google Scholar 

  58. Gebreyohannes, G. (2015). Isolation and optimization of amylase producing bacteria and actinomycetes from soil samples of Maraki and Tewedros campus, University of Gondar, North West Ethiopia. Afr J Microbiol Res, 9, 1877–1882.

    Google Scholar 

  59. Singh, A. K., Kumar, S., & Sharma, H. K. (2012). Effect of enzymatic hydrolysis on the juice yield from bael fruit (Aegle marmelos Correa) pulp. Am J Food Technol, 7, 62–72.

    CAS  Google Scholar 

  60. Vinjamuri, S. (2015). Optimization studies on enzymatic clarification of mixed fruit juices. International Journal of Latest Trends in Engineering and Technology, 5(2), 161–165.

    Google Scholar 

  61. Rakita, M., Torbica, M. A., Dokic, P. L., Tomic, M. J., Pojic, M. M., Hadnadjev, S. M., & Hadnadjev-Dapcevic, R. T. (2015). Alpha-amylase activity in wheat flour and breadmaking properties in relation to different climatic conditions. Food and Feed Research, 42(2), 91–99.

    CAS  Google Scholar 

  62. Sanz-Penella, J. M., Laparra, J. M., & Haros, M. (2014). Impact of α-amylase during breadmaking on in vitro kinetics of starch hydrolysis and glycaemic index of enriched bread with bran. Plant Foods Hum Nutr, 69, 216–221.

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the project: Research Seed Grants (RSG) Ref No: F. No 2021/0087 from GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India.

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This work was supported by the project: Research Seed Grants (RSG) Ref No: F. No 2021/0087 from GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India.

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  1. Department of Microbiology and FST (Food Science & Technology), GITAM School of Science, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, 530045, India

    Baliram Gurunath Rathod, Srinija Pandala & Venkata Giridhar Poosarla

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BGR: he carried out > 80% of experiments, analysis, and assisted in data curation. SP: she carried out > 20% of the experiments. VGP: he designed the experiments, involved in data curation, analysis, overall supervision of the entire research, and prepared the manuscript.

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Correspondence to Venkata Giridhar Poosarla.

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Rathod, B.G., Pandala, S. & Poosarla, V.G. A Novel Halo-Acid-Alkali-Tolerant and Surfactant Stable Amylase Secreted from Halophile Bacillus siamensis F2 and Its Application in Waste Valorization by Bioethanol Production and Food Industry. Appl Biochem Biotechnol 195, 4775–4795 (2023). https://doi.org/10.1007/s12010-023-04559-x

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