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Influence of nitrogen source and pH value on undesired poly(γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Bacillus spp. are used for the production of industrial enzymes but are also known to be capable of producing biopolymers such as poly(γ-glutamic acid). Biopolymers increase the viscosity of the fermentation broth, thereby impairing mixing, gas/liquid mass and heat transfer in any bioreactor system. Undesired biopolymer formation has a significant impact on the fermentation and downstream processing performance. This study shows how undesirable poly(γ-glutamic acid) formation of an industrial protease producing Bacillus licheniformis strain was prevented by switching the nitrogen source from ammonium to nitrate. The viscosity was reduced from 32 to 2.5 mPa s. A constant or changing pH value did not influence the poly(γ-glutamic acid) production. Protease production was not affected: protease activities of 38 and 46 U mL−1 were obtained for ammonium and nitrate, respectively. With the presented results, protease production with industrial Bacillus strains is now possible without the negative impact on fermentation and downstream processing by undesired poly(γ-glutamic acid) formation.

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Abbreviations

γ-PGA:

Poly(γ-glutamic acid)

DOT:

Dissolved oxygen tension (%)

OD600 :

Optical density at 600 nm (−)

OTR:

Oxygen transfer rate (mmol L−1 h−1)

References

  1. Abe K, Ito Y, Ohmachi T, Asada Y (1997) Purification and properties of two isozymes of gamma-glutamyltranspeptidase from Bacillus subtilis TAM-4. Biosci Biotechnol Biochem 61(10):1621–1625

    Article  CAS  PubMed  Google Scholar 

  2. Anderlei T, Büchs J (2001) Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J 7(2):157–162. doi:10.1016/s1369-703x(00)00116-9

    Article  CAS  PubMed  Google Scholar 

  3. Anderlei T, Zang W, Papaspyrou M, Büchs J (2004) Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochem Eng J 17(3):187–194. doi:10.1016/s1369-703x(03)00181-5

    Article  CAS  Google Scholar 

  4. Asali EC, Mutharasan R, Humphrey AE (1992) Use of NAD(P)H-fluorescence for monitoring the response of starved cells of Catharanthus roseus in suspension to metabolic perturbations. J Biotechnol 23(1):83–94. doi:10.1016/0168-1656(92)90101-E

    Article  CAS  Google Scholar 

  5. Ashiuchi M, Nakamura H, Yamamoto T, Kamei T, Soda K, Park C, Sung MH, Yagi T, Misono H (2003) Poly-gamma-glutamate depolymerase of Bacillus subtilis: production, simple purification and substrate selectivity. J Mol Catal B Enzym 23(2–6):249–255. doi:10.1016/s1381-1177(03)00087-0

    Article  CAS  Google Scholar 

  6. Bajaj I, Singhal R (2011) Poly (glutamic acid)—An emerging biopolymer of commercial interest. Bioresour Technol 102(10):5551–5561. doi:10.1016/j.biortech.2011.02.047

    Article  CAS  PubMed  Google Scholar 

  7. Bedingfield J (2012) The Novozymes Report 2012. Novozymes. Retrieved from: http://report2012.novozymes.com/service/download-report/the-novozymes-report-2012.pdf. Accessed 10 Apr 2014

  8. Bernlohr RW, Schreier HJ, Donohue TJ (1984) Enzymes of glutamate and glutamine biosynthesis in Bacillus licheniformis. Curr Top Cell Regul 24:145–152

    Article  CAS  PubMed  Google Scholar 

  9. Büchs J, Lotter S, Milbradt C (2001) Out-of-phase operating conditions, a hitherto unknown phenomenon in shaking bioreactors. Biochem Eng J 7(2):135–141. doi:10.1016/s1369-703x(00)00113-3

    Article  PubMed  Google Scholar 

  10. Buescher JM, Margaritis A (2007) Microbial biosynthesis of polyglutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries. Crit Rev Biotechnol 27(1):1–19. doi:10.1080/07388550601166458

    Article  CAS  PubMed  Google Scholar 

  11. Bulthuis BA, Frankena J, Koningstein GM, Vanverseveld HW, Stouthamer AH (1988) Instability of protease production in a rel+/rel− pair of Bacillus licheniformis and associated morphological and physiological characteristics. Antonie Van Leeuwenhoek J Microb 54(2):95–111

    Article  CAS  Google Scholar 

  12. DelMar EG, Largman C, Brodrick JW, Geokas MC (1979) Sensitive new substrate for chymotrypsin. Anal Biochem 99(2):316–320

    Article  CAS  PubMed  Google Scholar 

  13. Giese H, Azizan A, Kümmel A, Liao AP, Peter CP, Fonseca JA, Hermann R, Duarte TM, Büchs J (2014) Liquid films on shake flask walls explain increasing maximum oxygen transfer capacities with elevating viscosity. Biotechnol Bioeng 111(2):295–308. doi:10.1002/bit.25015

    Article  CAS  PubMed  Google Scholar 

  14. Giese H, Klöckner W, Peña C, Galindo E, Lotter S, Wetzel K, Meissner L, Peter CP, Büchs J (2014) Effective shear rates in shake flasks. Chem Eng Sci 118:102–113. doi:10.1016/j.ces.2014.07.037

    Article  CAS  Google Scholar 

  15. Giesecke UE, Bierbaum G, Rudde H, Spohn U, Wandrey C (1991) Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process. Appl Microbiol Biotechnol 35(6):720–724

    CAS  Google Scholar 

  16. Harrison DE, Chance B (1970) Fluorimetric technique for monitoring changes in level of reduced nicotinamide nucleotides in continuous cultures of microorganisms. Appl Microbiol 19(3):446–450

    PubMed Central  CAS  PubMed  Google Scholar 

  17. Harwood CR (1992) Bacillus subtilis and its relatives—molecular biological and industrial workhorses. Trends Biotechnol 10(7):247–256. doi:10.1016/0167-7799(92)90233-L

    Article  CAS  PubMed  Google Scholar 

  18. Herrmann HA, Good I, Läufer A (1997) Manufacturing and downstream processing of detergent enzymes. In: Van Ee J, Misset O, Baas EJ (eds) Enzymes in detergency. Dekker, New York, pp 251–297

    Google Scholar 

  19. Kambourova M, Tangney M, Priest FG (2001) Regulation of polyglutamic acid synthesis by glutamate in Bacillus licheniformis and Bacillus subtilis. Appl Environ Microb 67(2):1004–1007

    Article  CAS  Google Scholar 

  20. Kemblowski Z, Kristiansen B (1986) Rheometry of fermentation liquids. Biotechnol Bioeng 28(10):1474–1483. doi:10.1002/bit.260281005

    Article  CAS  PubMed  Google Scholar 

  21. Klöckner W, Büchs J (2012) Advances in shaking technologies. Trends Biotechnol 30(6):307–314. doi:10.1016/j.tibtech.2012.03.001

    Article  PubMed  Google Scholar 

  22. Kumar P, Patel SKS, Lee JK, Kalia VC (2013) Extending the limits of Bacillus for novel biotechnological applications. Biotechnol Adv 31(8):1543–1561. doi:10.1016/j.biotechadv.2013.08.007

    Article  CAS  PubMed  Google Scholar 

  23. Li X, Gou XY, Long D, Ji ZX, Hu LF, Xu DH, Liu J, Chen SW (2014) Physiological and metabolic analysis of nitrate reduction on poly-gamma-glutamic acid synthesis in Bacillus licheniformis WX-02. Arch Microbiol 196(11):791–799. doi:10.1007/s00203-014-1014-y

    Article  CAS  PubMed  Google Scholar 

  24. Maier B, Dietrich C, Büchs J (2001) Correct application of the sulphite oxidation methodology of measuring the volumetric mass transfer coefficient kLa under non-pressurized and pressurized conditions. Food Bioprod Process 79(C4):107–113

    Article  CAS  Google Scholar 

  25. Maurer KH (2004) Detergent proteases. Curr Opin Biotechol 15(4):330–334. doi:10.1016/j.copbio.2004.06.005

    Article  CAS  Google Scholar 

  26. Meers JL, Pedersen LK (1972) Nitrogen assimilation by Bacillus licheniformis organisms growing in chemostat cultures. J Gen Microbiol 70:277–286

    Article  CAS  PubMed  Google Scholar 

  27. Nakano MM, Yang F, Hardin P, Zuber P (1995) Nitrogen regulation of nasA and the nasB operon, which encode genes required for nitrate assimilation in Bacillus subtilis. J Bacteriol 177(3):573–579

    PubMed Central  CAS  PubMed  Google Scholar 

  28. Ogawa KI, Akagawa E, Yamane K, Sun ZW, Lacelle M, Zuber P, Nakano MM (1995) The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis. J Bacteriol 177(5):1409–1413

    PubMed Central  CAS  PubMed  Google Scholar 

  29. Richardson DJ, Berks BC, Russell DA, Spiro S, Taylor CJ (2001) Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 58(2):165–178

    Article  CAS  PubMed  Google Scholar 

  30. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50(1):1–17. doi:10.1139/W03-076

    Article  CAS  PubMed  Google Scholar 

  31. Schreier HJ (1993) Biosynthesis of glutamine and glutamate and the assimilation of ammonia. In: Sonenshein AL (ed) Bacillus subtilis and other gram positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, DC, pp 281–298

    Google Scholar 

  32. Wilming A, Begemann J, Kuhne S, Regestein L, Bongaerts J, Evers S, Maurer KH, Büchs J (2013) Metabolic studies of gamma-polyglutamic acid production in Bacillus licheniformis by small-scale continuous cultivations. Biochem Eng J 73:29–37. doi:10.1016/j.bej.2013.01.008

    Article  CAS  Google Scholar 

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Acknowledgments

The Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) and Henkel AG & Co. KGaA (Düsseldorf, Germany) are kindly acknowledged for partly funding this work through the joint research project “Industrieinitiative GenoMik Design” (Support Code 0313917D). Henkel AG & Co. KGaA (Düsseldorf, Germany) is also kindly acknowledged for providing the used Bacillus licheniformis strain.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. AVT. Biochemical Engineering, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany

    Lena Meissner, Kira Kauffmann, Timo Wengeler & Jochen Büchs

  2. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Hitoshi Mitsunaga & Eiichiro Fukusaki

Authors
  1. Lena Meissner
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  2. Kira Kauffmann
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  3. Timo Wengeler
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  4. Hitoshi Mitsunaga
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  6. Jochen Büchs
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Correspondence to Jochen Büchs.

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Meissner, L., Kauffmann, K., Wengeler, T. et al. Influence of nitrogen source and pH value on undesired poly(γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain. J Ind Microbiol Biotechnol 42, 1203–1215 (2015). https://doi.org/10.1007/s10295-015-1640-7

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  • DOI: https://doi.org/10.1007/s10295-015-1640-7

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