Applied Microbiology and Biotechnology

, Volume 102, Issue 8, pp 3649–3661 | Cite as

Effect of dilution rate on productivity of continuous bacteriophage production in cellstat

  • Dominik Nabergoj
  • Nina Kuzmić
  • Benjamin Drakslar
  • Aleš Podgornik
Biotechnological products and process engineering


Ability to efficiently propagate high quantities of bacteriophages (phages) is of great importance considering higher phage production needs in the future. Continuous production of phages could represent an interesting option. In our study, we tried to elucidate the effect of dilution rate on productivity of continuous production of phages in cellstat. As a model system, a well-studied phage T4 and Escherichia coli K-12 as a host were used. Experiments where physiology of bacteria was changing with dilution rate of cellstat and where bacterial physiology was kept constant were performed. For both setups there exists an optimal dilution rate when maximal productivity is achieved. Experimentally obtained values of phage concentration and corresponding productivity were compared with mathematical model predictions, and good agreement was obtained for both types of experiments. Analysis of mathematical model coefficients revealed that latent period and burst size to dilution rate coefficient mostly affect optimum dilution rate and productivity. Due to high sensitivity, it is important to evaluate phage growth parameters carefully, to run cellstat under optimal productivity.


Phage T4 E. coli K-12 Cellstat Continuous production Productivity 


Funding information

This study was funded by Slovenian Research Agency—ARRS (research program no. P3-0387, project no. L4-5532, and project no. 3030-37543). The study was also supported by the European Regional Development Fund and Slovenian Ministry of Education, Science and Sport (project BioPharm.Si).

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM (2011) Phage treatment of human infections. Bacteriophage 1:66–85. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ackermann HW, Prangishvili D (2012) Prokaryote viruses studied by electron microscopy. Arch Virol 157:1843–1849. CrossRefPubMedGoogle Scholar
  3. Aita T, Husimi Y (1994) Period dependent selection in continuous culture of viruses in a periodic environment. J Theor Biol 168:281–289. CrossRefPubMedGoogle Scholar
  4. Atterbury RJ (2009) Bacteriophage biocontrol in animals and meat products. Microb Biotechnol 2:601–612. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bachmann BJ (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36:525–557PubMedPubMedCentralGoogle Scholar
  6. Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462CrossRefPubMedGoogle Scholar
  7. Brown A (1956) A study of lysis in bacteriophage-infected Escherichia coli. J Bacteriol 71:482–490PubMedPubMedCentralGoogle Scholar
  8. Brüssow H, Hendrix RW (2002) Phage genomics: small is beautiful. Cell 108:13–16CrossRefPubMedGoogle Scholar
  9. Bryan D, El-Shibiny A, Hobbs Z, Porter J, Kutter EM (2016) Bacteriophage T4 infection of stationary phase E. coli: life after log from a phage perspective. Front Microbiol 7:1391. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bull JJ, Millstein J, Orcutt J, Wichman HA, Promislow AEDEL, Whitlock EMC (2006) Evolutionary feedback mediated through population density, illustrated with viruses in chemostats. Am Nat 167:E39–E51. CrossRefPubMedGoogle Scholar
  11. Chao L, Levin BR, Stewart FM (1977) A complex community in a simple habitat: an experimental study with bacteria and phage. Ecology 58:369–378. CrossRefGoogle Scholar
  12. Chen BY, Lim HC (1996) Bioreactor studies on temperature induction of the Q- mutant of bacteriophage lambda in Escherichia coli. J Biotechnol 51:1–20CrossRefPubMedGoogle Scholar
  13. Clark DW, Meyer H-P, Leist C, Fiechter A (1986) Effects of growth medium on phage production and induction in Escherichia coli K-12 lambda lysogens. J Biotechnol 3:271–280. CrossRefGoogle Scholar
  14. Clokie MR, Millard AD, Letarov AV, Heaphy S (2011) Phages in nature. Bacteriophage 1:31–45. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre A-S, Lavigne R (2012) Learning from bacteriophages—advantages and limitations of phage and phage-encoded protein applications. Curr Protein Pept Sci 13:699–722. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Esvelt KM, Carlson JC, Liu DR (2011) A system for the continuous directed evolution of biomolecules. Nature 472:499–503. CrossRefPubMedPubMedCentralGoogle Scholar
  17. García P, Martínez B, Obeso JM, Rodríguez A (2008) Bacteriophages and their application in food safety. Lett Appl Microbiol 47:479–485. CrossRefPubMedGoogle Scholar
  18. Golec P, Karczewska-Golec J, Łoś M, Węgrzyn G (2014) Bacteriophage T4 can produce progeny virions in extremely slowly growing Escherichia coli host: comparison of a mathematical model with the experimental data. FEMS Microbiol Lett 351:156–161. CrossRefPubMedGoogle Scholar
  19. Haq IU, Chaudhry WN, Akhtar MN, Andleeb S, Qadri I (2012) Bacteriophages and their implications on future biotechnology: a review. Virol J 9:9. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hathaway H, Milo S, Sutton JM, Jenkins TA (2017) Recent advances in therapeutic delivery systems of bacteriophage and bacteriophage-encoded endolysins. Ther Deliv 8:543–556. CrossRefPubMedGoogle Scholar
  21. Hayashi K, Morooka N, Yamamoto Y, Fujita K, Isono K, Choi S, Ohtsubo E, Baba T, Wanner BL, Mori H, Horiuchi T (2006) Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol Syst Biol 2:2006.0007. doi:
  22. Horne MT (1970) Coevolution of Escherichia coli and bacteriophages in chemostat culture. Science 168:992–993CrossRefPubMedGoogle Scholar
  23. Hoskisson PA, Hobbs G (2005) Continuous culture—making a comeback? Microbiology 151:3153–3159. CrossRefPubMedGoogle Scholar
  24. Hu B, Margolin W, Molineux IJ, Liu J (2015) Structural remodeling of bacteriophage T4 and host membranes during infection initiation. Proc Natl Acad Sci U S A 112:E4919–E4928. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Husimi Y (1989) Selection and evolution of bacteriophages in cellstat. Adv Biophys 25:1–43CrossRefPubMedGoogle Scholar
  26. Husimi Y, Nishigaki K, Kinoshita Y, Tanaka T (1982) Cellstat-a continuous culture system of a bacteriophage for the study of the mutation rate and the selection process of the DNA level. Rev Sci Instrum 53:517–522CrossRefPubMedGoogle Scholar
  27. Jones JB, Vallad GE, Iriarte FB, Obradović A, Wernsing MH, Jackson LE, Balogh B, Hong JC, Momol MT (2012) Considerations for using bacteriophages for plant disease control. Bacteriophage 2:208–214. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Jungbauer A (2013) Continuous downstream processing of biopharmaceuticals. Trends Biotechnol 31:479–492. CrossRefPubMedGoogle Scholar
  29. Kick B, Behler KL, Severin TS, Weuster-Botz D (2017) Chemostat studies of bacteriophage M13 infected Escherichia coli JM109 for continuous ssDNA production. J Biotechnol 258:92–100. CrossRefPubMedGoogle Scholar
  30. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay. In: Clokie MRJ, Kropinski AM (eds) Bacteriophages. Methods in Molecular Biology™. Humana Press, New York, pp 69–76Google Scholar
  31. Kutter E, Kellenberger E, Carlson K, Eddy S, Neitzel J, Messinger L, North J, Guttman B (1994) Effects of bacterial growth conditions and physiology on T4 infection. In: Karam JD (ed) Molecular biology of bacteriophage T4. American Society for Microbiology, Washington, DC, pp 406–418Google Scholar
  32. Lee SL, O’Connor TF, Yang X, Cruz CN, Chatterjee S, Madurawe RD, Moore CMV, Yu LX, Woodcock J (2015) Modernizing pharmaceutical manufacturing: from batch to continuous production. J Pharm Innov 10:191–199. CrossRefGoogle Scholar
  33. Lin DM, Koskella B, Lin HC (2017) Phage therapy: an alternative to antibiotics in the age of multi-drug resistance. World J Gastrointest Pharmacol Ther 8:162–173. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lindemann BF, Klug C, Schwienhorst A (2002) Evolution of bacteriophage in continuous culture: a model system to test antiviral gene therapies for the emergence of phage escape mutants. J Virol 76:5784–5792. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Little JW, Michalowski CB (2010) Stability and instability in the lysogenic state of phage lambda. J Bacteriol 192:6064–6076. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Loc-Carrillo C, Abedon ST (2011) Pros and cons of phage therapy. Bacteriophage 1:111–114. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Los M, Wegrzyn G, Neubauer P (2003) A role for bacteriophage T4 rI gene function in the control of phage development during pseudolysogeny and in slowly growing host cells. Res Microbiol 154:547–552. CrossRefPubMedGoogle Scholar
  38. Malik DJ, Sokolov IJ, Vinner GK, Mancuso F, Cinquerrui S, Vladisavljevic GT, Clokie MRJ, Garton NJ, Stapley AGF, Kirpichnikova A (2017) Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Adv Colloid Interfac 249:100–133. CrossRefGoogle Scholar
  39. Merabishvili M, Pirnay JP, Verbeken G, Chanishvili N, Tediashvili M, Lashkhi N, Glonti T, Krylov V, Mast J, Van Parys L, Lavigne R, Volckaert G, Mattheus W, Verween G, De Corte P, Rose T, Jennes S, Zizi M, De Vos D, Vaneechoutte M (2009) Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. PLoS One 4:e4944. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Rüger W (2003) Bacteriophage T4 genome. Microbiol Mol Biol Rev 67:86–156. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nabergoj D, Modic P, Podgornik A (2017) Effect of bacterial growth rate on bacteriophage population growth rate. MicrobiologyOpen 2017:e558. Google Scholar
  42. Nobrega FL, Costa AR, Santos JF, Siliakus MF, van Lent JWM, Kengen SWM, Azeredo J, Kluskens LD (2016) Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine. Sci Rep 6:39235. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Northrop JH (1966) Increased mutation rate of E. coli K12 lambda cultures maintained in continuous logarithmic growth. J Gen Physiol 50:369–377CrossRefPubMedPubMedCentralGoogle Scholar
  44. O’Sullivan L, Buttimer C, McAuliffe O, Bolton D, Coffey A (2016) Bacteriophage-based tools: recent advances and novel applications. F1000Res 5:2782. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Oh JS, Cho D, Park TH (2005) Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage lambda Q- vector. Bioprocess Biosyst Eng 28:1–7. CrossRefPubMedGoogle Scholar
  46. Park SH, Park TH (2000) Analysis of two-stage continuous operation of Escherichia coli containing bacteriophage λ vector. Bioprocess Eng 23:557–563. CrossRefGoogle Scholar
  47. Park TH, Seo J-H, Lim HC (1991) Two-stage fermentation with bacteriophage λ as an expression vector in Escherachia coli. Biotechnol Bioeng 37:297–302. CrossRefPubMedGoogle Scholar
  48. Paynter MJB, Bungay HK (1970) Responses in continuous cultures of lysogenic Escherichia coli following induction. Biotechnol Bioeng 12:347–351. CrossRefPubMedGoogle Scholar
  49. Pires DP, Cleto S, Sillankorva S, Azeredo J, Lu TK (2016) Genetically engineered phages: a review of advances over the last decade. Microbiol Mol Biol Rev 80:523–543. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Pirnay JP, Blasdel BG, Bretaudeau L, Buckling A, Chanishvili N, Clark JR, Corte-Real S, Debarbieux L, Dublanchet A, De Vos D, Gabard J, Garcia M, Goderdzishvili M, Górski A, Hardcastle J, Huys I, Kutter E, Lavigne R, Merabishvili M, Olchawa E, Parikka KJ, Patey O, Pouilot F, Resch G, Rohde C, Scheres J, Skurnik M, Vaneechoutte M, Van Parys L, Verbeken G, Zizi M, Van den Eede G (2015) Quality and safety requirements for sustainable phage therapy products. Pharm Res 32:2173–2179. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Podgornik A, Janež N, Smrekar F, Peterka M (2014) Continuous production of bacteriophages. In: Subramanian G (ed) Continuous processing in pharmaceutical manufacturing. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp 297–338Google Scholar
  52. Prestinaci F, Pezzotti P, Pantosti A (2015) Antimicrobial resistance: a global multifaceted phenomenon. Pathog Glob Health 109:309–318. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Reardon S (2017) Modified viruses deliver death to antibiotic-resistant bacteria. Nat News 546:586–587. CrossRefGoogle Scholar
  54. Roca I, Akova M, Baquero F, Carlet J, Cavaleri M, Coenen S, Cohen J, Findlay D, Gyssens I, Heure OE, Kahlmeter G, Kruse H, Laxminarayan R, Liébana E, López-Cerero L, MacGowan A, Martins M, Rodríguez-Baño J, Rolain J-M, Segovia C, Sigauque B, Taconelli E, Wellington E, Vila J (2015) The global threat of antimicrobial resistance: science for intervention. New Microbes New Infect 6:22–29. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Rokney A, Kobiler O, Amir A, Court DL, Stavans J, Adhya S, Oppenheim AB (2008) Host responses influence on the induction of lambda prophage. Mol Microbiol 68:29–36. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  57. Schofield DA, Sharp NJ, Westwater C (2012) Phage-based platforms for the clinical detection of human bacterial pathogens. Bacteriophage 2:105–283. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Schwienhorst A, Lindemann BF, Eigen M (1996) Growth kinetics of a bacteriophage in continuous culture. Biotechnol Bioeng 50:217–221. CrossRefPubMedGoogle Scholar
  59. Sezonov G, Joseleau-Petit D, D’Ari R (2007) Escherichia coli physiology in Luria-Bertani broth. J Bacteriol 189:8746–8749. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Simmons LA, Foti JJ, Cohen SE, Walker GC (2008) The SOS Regulatory Network. EcoSal Plus 2008. doi:
  61. Spellberg B, Guidos R, Gilbert D, Bradley J, Boucher HW, Scheld WM, Bartlett JG, Edwards J, Infectious Diseases Society of America (2008) The epidemic of antibiotic-resistant infections: a call to action for the medical community from the Infectious Diseases Society of America. Clin Infect Dis 46:155–164. CrossRefPubMedGoogle Scholar
  62. Šuster K, Podgornik A, Cör A (2017) Quick bacteriophage-mediated bioluminescence assay for detecting Staphylococcus spp. in sonicate fluid of orthopaedic artificial joints. New Microbiol 40:190–196PubMedGoogle Scholar
  63. Wang CH, Koch AL (1978) Constancy of growth on simple and complex media. J Bacteriol 136:969–975PubMedPubMedCentralGoogle Scholar
  64. Wee S, Wilkinson BJ (1988) Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations. J Bacteriol 170:3283–3286. CrossRefPubMedPubMedCentralGoogle Scholar
  65. Yap ML, Rossmann MG (2014) Structure and function of bacteriophage T4. Future Microbiol 9:1319–1327. CrossRefPubMedPubMedCentralGoogle Scholar
  66. Ziv N, Brandt NJ, Gresham D (2013) The use of chemostats in microbial systems biology. J Vis Exp.

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Dominik Nabergoj
    • 1
  • Nina Kuzmić
    • 2
  • Benjamin Drakslar
    • 2
  • Aleš Podgornik
    • 1
    • 2
  1. 1.Center of Excellence for Biosensors, Instrumentation and Process Control (COBIK)AjdovščinaSlovenia
  2. 2.Faculty of Chemistry and Chemical TechnologyUniversity of LjubljanaLjubljanaSlovenia

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