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Pilot-scale process development and scale up for antifungal production

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Abstract

A pilot-scale fermentation was developed for an antifungal compound produced by a filamentous fungus. Replacement of galactose with lactose (20-fold cost savings) and a threefold phosphate reduction (15 to 5 g/L) improved productivity 2.5-fold. Addition of supplements—glycine, cobalt chloride, and trace elements—resulted in a further twofold productivity increase, greater process robustness, and less foaming which reduced antifoam addition tenfold (30 to <3 mL/L). Mid-cycle lactose limitations were addressed by raising initial lactose levels (40 to 120 g/L) resulting in another twofold productivity increase. Overall, peak titers increased tenfold from 45 ± 9 to 448 ± 39 mg/L, and productivities improved from 3 to 25 mg/L day. Despite its high productivity, process scale up was challenged by high broth viscosity (5,000–6,000 cP at 16.8 s−1). Gassed power requirements at the 600 L scale (4.7 kW/1,000 L) exceeded available power at the 15,000 L scale (3.0 kW/1,000 L), and broth transfer to the downstream isolation facility was hindered. Mid-cycle broth dilution with up to five 10 vol% additions of 12 wt% lactose solution or whole medium-reduced viscosity three- to fivefold (1,000–1,500 cP at 16.8 s−1), gassed power within scale-up limits (2.5 kW/1,000 L), and peak titer by up to 45%. The process was scaled up to the 15,000 L working volume based on constant aeration rate (vvm) and peak impeller tip speed, raising superficial velocities at similar shear. This strategy maximized mass transfer rates at target gassed power per unit volume levels, and along with controlled broth viscosity, precluded multiple dilution additions. A final titer of 333 mg/L with one dilution addition was achieved, somewhat lower than expected, likely owing to inhibition from some unmeasured volatile compound (not believed to be carbon dioxide) during an extended period of high back-pressure in the early production phase.

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Abbreviations

A :

constant in Eq. 3

CER:

carbon evolution rate (mmol/L h)

DI :

impeller diameter (m)

ITS:

impeller tip speed (m/s)

K :

consistency index, Eq. 4

K L a :

volumetric mass transfer coefficient (h−1)

N :

agitation speed (rpm)

n :

flow behavior index, Eq. 4

OUR:

oxygen uptake rate (mmol/L h)

Pg/VL:

power per unit volume (kW/1,000 L)

RQ:

respiratory quotient

V L :

working volume (L)

v s :

superficial velocity (cm/s)

vvm:

vessel volumes per minute (min−1)

x :

relative broth strength, Eq. 4

y :

relative broth viscosity, Eq. 4

α :

constant in Eq. 3

β :

constant in Eq. 3

τ :

shear stress (Pa)

μ a :

apparent viscosity (cP)

γ :

shear strain (s−1)

References

  1. Connors N, Prevoznak R, Brix T, Seeley A, Gbewonyo K, Greasham R, Salmon P (1995) Effects of medium sterilization on the production of zaragozic acids by the fungus Leptodontidium elatius. J Ind Microbiol 15:503–508

    Article  CAS  Google Scholar 

  2. Junker B, Mann Z, Burgess B, King J, Greasham R (2001) Carbon and complex nitrogen source selection for secondary metabolite cultivation at the pilot scale. J Biosci Bioeng 91:462–468

    Article  CAS  Google Scholar 

  3. Junker B, Zhang J, Mann Z, Reddy J, Greasham R (2001) Scale up of a defined medium process for pilot scale production of illicicolin by Gliocladium roseum. Biotechnol Prog 17:278–286

    Article  CAS  Google Scholar 

  4. Junker BH, Walker A, Connors N, Seeley A, Masurekar P, Hesse M (2006) Production of indole diterpenes by Aspergillus alliaceus. Biotechnol Bioeng 95(5):919–936

    Article  CAS  Google Scholar 

  5. Pollard D, Kirschner T, Hernandez D, Hunt G, Olewinski R, Salmon P (2002) Pilot-scale process sensitivity studies for the scale-up of a fungal fermentation for the production of pneumocandins. Biotechnol Bioeng 78:270–279

    Article  CAS  Google Scholar 

  6. Junker B, Hesse M, Burgess B, Masurekar P, Connors N, Seeley A (2004) Early phase process scale up challenges for fungal and filamentous bacterial cultures. Appl Biochem Biotechnol 119:241–277

    Article  CAS  Google Scholar 

  7. Humphrey A (1998) Shake flask to fermenter: what have we learned? Biotechnol Prog 14:3–7

    Article  CAS  Google Scholar 

  8. Jem K (1989) Scale-down techniques for fermentation. BioPharm 3:30–39

    Google Scholar 

  9. Lilly M (1983) Problems in process scale-up. In: Nisbet L, Winstanley D (eds) Bioactive microbial products. Academic Press, New York, pp 79–89

    Google Scholar 

  10. Oldshue J (1981) Let’s understand mixing. Chemtech 11:554–561

    CAS  Google Scholar 

  11. Grimm LH, Kelly S, Krull R, Hempel DC (2005) Morphology and productivity of filamentous fungi. Appl Microbiol Biotechnol 69:375–384

    Article  CAS  Google Scholar 

  12. Junker B, Seeley A, Lester M, Kovatch M, Schmitt J, Borysewicz S, Lynch J, Zhang J, Greasham R (2002) Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale. Biotechnol Bioeng 79:628–640

    Article  CAS  Google Scholar 

  13. Junker B, Mann Z, Hunt G (2000) Retrofit of CD-6 (Smith) impeller in fermentation vessels. Appl Biochem Biotechnol 89:67–83

    Article  CAS  Google Scholar 

  14. Nienow AW, Hunt GR, Buckland BC (1994) A fluid dynamic study of the retrofitting of large agitated bioreactors: turbulent flow. Biotechnol Bioeng 44:1177–1185

    Article  CAS  Google Scholar 

  15. Okumura MK, Fujinaga Y, Seike Y, Honda S (1999) A simple and rapid visual method for the determination of ammonia nitrogen in environmental waters using thymol. Frensenius J Anal Chem 365:467–469

    Article  CAS  Google Scholar 

  16. Cogan EB, Birrell GB, Griffith OH (1999) A robotics-based automated assay for inorganic and organic phosphates. Anal Biochem 271:29–35

    Article  CAS  Google Scholar 

  17. Junker BH, Stanik M, Barna C, Salmon P, Paul E, Buckland BC (1998) Influence of impeller type on power input in fermentation vessels. Bioprocess Eng 18:401–412

    Article  CAS  Google Scholar 

  18. Pollard D, Hunt G, Kirschner T, Salmon P (2002) Rheological characterization of a fungal fermentation for the production of pneumocandins. Bioprocess Biosyst Eng 24:373–383

    Article  CAS  Google Scholar 

  19. Gavrilescu M, Roman RV, Efimov V (1992) Rheological behavior of some antibiotic biosynthesis liquids. Acta Biotechnol 12(5):383–396

    Article  CAS  Google Scholar 

  20. Cabral JMS, Casale B, Cooney CL (1985) Effect of antifoam agents and efficiency of cleaning procedures on the cross-flow filtration of microbial suspensions. Biotechnol Lett 7:749–752

    Article  CAS  Google Scholar 

  21. Junker B (2007) Foam and its mitigation in fermentation systems. Biotechnol Prog 23:767–784

    CAS  Google Scholar 

  22. Kroner KH, Hummel W, Volkel J, Kula MR (1986) Effects of antifoams on cross-flow filtration of microbial suspensions. In: Drioli E, Nakagaki M (eds) Membranes and membrane processes. Plenum, Stesa, pp 223–232

    Google Scholar 

  23. McGregor WC, Weaver JF, Tansey SP (1988) Antifoam effects on ultrafiltration. Biotechnol Bioeng 31:385–389

    Article  CAS  Google Scholar 

  24. Vardar-Sukan F (1992) Foaming and its control in bioprocesses. In: Vardar-Sukan F, Sukan SS (eds) Recent advances in biotechnology. Kluwer Academic Publishers, Dordecht, pp 113–146

    Google Scholar 

  25. Yamagiwa K, Ikarashi K, Ohkawa A (1989) Effects of antifoam with inverted cloud point on permeation and solute rejection in membrane filtration processes. J Chem Eng Jpn 22:693–695

    Article  CAS  Google Scholar 

  26. Lengeler J, Drews G, Schlegel H (1999) Biology of the Prokaryotes. Blackwell Science, New York

    Google Scholar 

  27. Blanch HW, Clark DS (1996) Biochemical engineering. Marcel Dekker, New York, p 168

    Google Scholar 

  28. Weinberg E (1970) Biosynthesis of secondary metabolites: roles of trace metals. Adv Microbiol Physiol 4:1–44

    Article  CAS  Google Scholar 

  29. Demain A (1968) Regulatory mechanisms and the industrial production of microbial metabolites. Lloydia 31:395–418

    CAS  Google Scholar 

  30. Martin J, Demain A (1980) Control of antibiotic synthesis. Microbiol Rev 44:230–251

    CAS  Google Scholar 

  31. Bu’Lock J (1967) Essays in biosynthesis and microbial development. John Wiley and Sons, Inc., New York

    Google Scholar 

  32. Bu’Lock J (1975) Secondary metabolism in fungi and its relationship to growth and development. In: Smith JE, Berry DR (eds) The Filamentous Fungi. Wiley, New York, pp 33–58

    Google Scholar 

  33. Vining L, Tabor W (1963) Biochemistry of industrial microorganisms. Academic Press, London

    Google Scholar 

  34. Seiboth B, Pakdaman BS, Hartl L, Kubicek CP (2007) Fungal Biol Rev 21(1):42–48

    Article  Google Scholar 

  35. Fekete E, Karaffa L, Sandor E, Banyai I, Seiboth B, Gyemant G, Sepsi A, Szentirmai A, Kubicek CP (2004) The alternative D-galactose degrading pathway of Aspergillus nidulans proceeds via L-sorbose. Arch Microbiol 181:35–44

    Article  CAS  Google Scholar 

  36. BioRad, Guide to Aminex HPLC columns

  37. Spizek J, Tichy P (1995) Some aspects of overproduction of secondary metabolites. Folia Microbiol 40(1):43–50

    Article  CAS  Google Scholar 

  38. Vanek Z, Mikulik K (1978) Microbial growth and production of antibiotics. Folia Microbiol 23:309–328

    Article  CAS  Google Scholar 

  39. Nason A, McElroy W (1963) Plant physiology. Academic Press, New York

    Google Scholar 

  40. Williams R (1967) Heavy metals in biological systems. Endeavor 26:96–100

    CAS  Google Scholar 

  41. Weinberg D (1978) Secondary metabolism: regulation by phosphate and trace elements. Folia Microbiol 23:496–504

    Article  CAS  Google Scholar 

  42. Maret W, Yetman C, Jiang L (2001) Enzyme regulation by reversible zinc inhibition: glycerol phosphate dehydrogenase as an example. Chem Biol Interact 130–132:891–901

    Article  Google Scholar 

  43. Failla L, Niehaus W (1986) Regulation of zinc uptake and versicolorin A synthesis in a mutant strain of Aspergillus parasiticus. Exp Mycol 10:35–41

    Article  CAS  Google Scholar 

  44. Marsh P, Simpson M, Trucksess M (1975) Effects of trace metals on the production of aflatoxins by Aspergillus parasiticus. Appl Microbiol 30:52–57

    CAS  Google Scholar 

  45. Niehaus W, Failla L (1984) Effect of zinc on versicolorin production by a mutant strain of Aspergillus parasiticus. Exp Mycol 8:80–84

    Article  CAS  Google Scholar 

  46. Coupland K, Niehaus W (1987) Stimulation of alternariol biosynthesis by zinc and manganese ions. Exp Mycol 11:60–64

    Article  CAS  Google Scholar 

  47. Petersen L, Hughes D, Hughes R, DiMichele L, Salmon P, Connors N (2001) Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis; impact on titer, analogue levels, and the identification of new analogues of pneumocandin B0. J Ind Microbiol Biotechnol 26:216–221

    Article  CAS  Google Scholar 

  48. Connors N, Petersen L, Hughes R, Saini K, Olewinski R, Salmon P (2000) Residual fructose and osmolality affect the levels of pneumocandins Bo and Co produced by Glarea lozoyensis. Appl Microbiol Biotechnol 54:814–818

    Article  CAS  Google Scholar 

  49. Cooper CM, Fernstrom GA, Miller SA (1944) Performance of agitated gas-liquid contactors. Ind Eng Chem 36:504–509

    Article  CAS  Google Scholar 

  50. Bhargava S, Nandakumar MP, Roy A, Wenger KS, Marten MR (2003) Pulsed feeding during fed-batch fungal fermentation leads to reduced viscosity without detrimentally affecting protein expression. Biotechnol Bioeng 81:341–347

    Article  CAS  Google Scholar 

  51. Karsheva M, Hristov J, Penchev I, Lossev V (1997) Rheological behavior of fermentation broths in antibiotic industry. Appl Biochem Biotechnol 68:187–206

    Article  CAS  Google Scholar 

  52. Olsvik E, Kristiansen B (1994) Rheology of filamentous fermentations. Biotechnol Adv 12:1–39

    Article  CAS  Google Scholar 

  53. Petersen N, Stoeks S, Gernacey KV (2008) Multivariate models for prediction of rheological characterization of filamentous fermentation broth from the size distribution. Biotechnol Bioeng 100(1):61–71

    Article  CAS  Google Scholar 

  54. Roukas T (1999) Rheological properties of pullulan fermentation broth in a stirred tank fermentor. Food Biotechnol 13:255–266

    Article  CAS  Google Scholar 

  55. Buckland BC, Gbewonyo K, DiMasi D, Hunt G, Wasterfield G, Nienow AW (1988) Improved performance in viscous mycelial fermentations by agitator retrofitting. Biotechnol Bioeng 317:737–742

    Article  Google Scholar 

  56. Sato K (1961) Rheological studies on some fermentation broths (IV): effect of dilution rate on rheological properties of fermentation broth. J Ferment Technol 39:517–520

    Google Scholar 

  57. Taguchi H, Miyamoto S (1966) Power requirement in non-Newtonian fermentation broth. Biotechnol Bioeng 8:43–54

    Article  Google Scholar 

  58. Olsvik ES, Kristiansen B (1992) On-line rheological measurements and control in fungal fermentations. Biotechnol Bioeng 40:375–387

    Article  CAS  Google Scholar 

  59. Ahn J, Jung J, Hyung W, Haam S, Shin C (2006) Enhancement of Monascus pigment production by the culture of Monascus sp. J101 at low temperature. Biotechnol Prog 22:338–340

    Article  CAS  Google Scholar 

  60. Bhargava S, Wenger KS, Marten MR (2003) Pulsed feeding during fed-batch Aspergillus oryzae fermentation leads to improved oxygen mass transfer. Biotechnol Prog 19:1091–1094

    Article  CAS  Google Scholar 

  61. Bhargava S, Wenger KS, Rane K, Rising V, Marten MR (2005) Effect of cycle time on fungal morphology, broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon source. Biotechnol Bioeng 89:524–529

    Article  CAS  Google Scholar 

  62. Ho CS, Smith MD (1986) Effect of dissolved carbon dioxide on penicillin fermentations: mycelial growth and penicillin production. Biotechnol Bioeng 28:668–677

    Article  CAS  Google Scholar 

  63. Bylinkina ES, Nihitina TS, Birykova VV, Cherkasova ON (1973) Effect of dissolved carbon dioxide on life activity of antibiotic-producing microorganisms. Biotechnol Bioeng Symposia 4:197–207

    Google Scholar 

  64. Tikhonov VV, Vandysheva TN, Bogatov LG, Tarasova SS (1983) Study of the effect of dissolved carbon dioxide on tetracycline biosynthesis in an industrial fermenter. Antibiotiki 28:652–655

    CAS  Google Scholar 

  65. Akashi K, Shibai H, Hirose Y (1979) Inhibitory effects of carbon dioxide and oxygen in amino acid fermentation. J Ferment Technol 57:317–320

    CAS  Google Scholar 

  66. Hirose Y (1986) Biochemical effects of oxygen supply and carbon dioxide removal. In: Yamada H (ed) Biotechnology of amino acid production, vol 24. Elsevier, New York, pp 67–80

    Google Scholar 

  67. McIntyre M, McNeil B, McNeil B (1997) Dissolved carbon dioxide effects on morphology, growth, and citrate production in Aspergillus niger A60. Enzyme Microb Technol 20:135–142

    Article  CAS  Google Scholar 

  68. Makagiansar HY, Shamlou PA, Thomas CR, Lilly MD (1993) The influence of mechanical forces on the morphology and penicillin production of Penicillin chrysogenum. Bioprocess Eng 9:83–90

    Article  CAS  Google Scholar 

  69. Mudge CS (1917) The effect of sterilization upon sugars in culture media. J Bacteriol 2(4):403–415

    CAS  Google Scholar 

  70. Gbewonyo K, Hunt G, Buckland B (1992) Interactions of cell morphology and transport processes in the lovastatin fermentation. Bioprocess Eng 8:1–7

    Article  CAS  Google Scholar 

  71. Pollard DJ, Buccino R, Connors NC, Kirschner TF, Olewinski RC, Saini K, Salmon P (2001) Real-time analyte monitoring of a fungal fermentation, at pilot scale, using in situ mid-infrared spectroscopy. Bioprocess Biosyst Eng 24:13–24

    Article  CAS  Google Scholar 

  72. Cooke M, Middleton JC, Bush JR (1988) Mixing and mass transfer in filamentous fermentations. BHRA, Elsevier, pp 37–64

    Google Scholar 

  73. Nienow AW (1990) Agitators for mycelial fermentations. TIBTECH, August 8, pp 224–233

  74. Gavrilescu M, Roman RV (1994) Oxygen mass transfer and gas holdup in a bubble column bioreactor with biosynthesis liquids. Acta Biotechnol 14(1):27–36

    Article  CAS  Google Scholar 

  75. Goudar CT, Strevett KA, Shah SN (1999) Influence of microbial concentration on the rheology of non-Newtonian fermentation broths. Appl Microbiol Biotechnol 51:310–315

    Article  CAS  Google Scholar 

  76. Kim JC, Lim JS, Kim JM, Kim C, Kim SW (2005) Relationship between morphology and viscosity of the main culture broth of Cephalosporium acremonium M25. Korea Aust Rheol J 17(1):15–20

    Google Scholar 

  77. Brar SS, Giam CS, Taber WA (1968) Patterns of in vitro ergot alkaloid production by Claviceps paspali and their association with different growth rates. Mycologia 60:806–826

    Article  CAS  Google Scholar 

  78. Tkacz JS, Giacobbe RA, Monaghan RL (1993) Improvement in the titer of echinocandin-type antibiotics: a magnesium-limited medium supporting the biphasic production of pneumocandins Ao and Bo. J Ind Microbiol 11:95–103

    Article  CAS  Google Scholar 

  79. Chang LT, McGrory EL, Elander RP (1990) Penicillin production by glucose-derepressed mutants of Penicillium chrysogenum. J Ind Microbiol 6:165–169

    Article  CAS  Google Scholar 

  80. Dombrowski A, Jenkins R, Raghoobar S, Bills G, Polishook J, Pelaez F, Burgess B, Zhao A, Huang L, Zhang Y, Goetz M (1999) Production of a family of kinase-inhibiting lactones from fungal fermentations. J Antibiotics 52(12):1077–1085

    CAS  Google Scholar 

  81. Mateles RI, Adye JC (1965) Production of aflatoxins in submerged culture. Appl Microbiol 13(2):208–211

    CAS  Google Scholar 

  82. Kossen NWF, Oosterhuis NMG (1985) Modelling and scaling-up of bioreactor. In: Brauer H (ed) Biotechnology, vol 2. VCH Publisher, Weinheim, pp 571–605

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Acknowledgments

The authors would like to acknowledge the key contributions of the supervisors and operators of the fermentation pilot plant, as well as former employees Gregory Russotti, Andrew Lenz, Anna Seeley, and Misti Ushio, to these efforts.

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  1. Fermentation Development and Operations, Merck Research Laboratories, Rahway, NJ, USA

    Beth Junker, Andre Walker, Michelle Hesse, Michael Lester, Diane Vesey, Jens Christensen, Bruce Burgess & Neal Connors

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  1. Beth Junker
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  2. Andre Walker
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Correspondence to Beth Junker.

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Junker, B., Walker, A., Hesse, M. et al. Pilot-scale process development and scale up for antifungal production. Bioprocess Biosyst Eng 32, 443–458 (2009). https://doi.org/10.1007/s00449-008-0264-y

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