Applied Microbiology and Biotechnology

, Volume 104, Issue 3, pp 1077–1095 | Cite as

Biokinetics of fed-batch production of poly (3-hydroxybutyrate) using microbial co-culture

  • Anusha Mohanakrishnan Subramanian
  • Sivanesh Easwaran Nanjan
  • Hariram Prakash
  • Leelaram Santharam
  • Ankitha Ramachandran
  • Vignesh Sathyaseelan
  • Deepa Perinkulum Ravi
  • Surianarayanan MahadevanEmail author
Biotechnological products and process engineering


A novel fed-batch strategy based on carbon/nitrogen (C/N) ratio in a microbial co-culture production medium broth was carried out in a biocalorimeter for improved production of poly (3-hydroxybutyrate) (PHB). Shake flask study suggested that the C/N ratio of 10 increased the yield of PHB by 2.8 times. Online parameters monitored during the C/N ratio of 10 in biocalorimeter (BioRC1e) indicated that the heat profile was maintained in the fed-batch mode resulting in a PHB yield of 30.3 ± 1.5 g/L. The oxy-calorific heat yield coefficient during the fed-batch strategy was found to be 394.24 ± 18.71 kJ/O2 due to the oxidative metabolism of glucose. The reported heat-based model adapted for PHB concentration prediction in the present fed-batch mode. The heat-based model has a Nash-Sutcliffe efficiency of 0.9758 for PHB prediction. PHB obtained by fed-batch-mode was characterized using gas chromatography-mass spectrometry (GC-MS) for the monomer-acid analysis, Thermogravimetric analysis (TGA) for thermal stability of PHB, and Fourier transform infrared spectroscopy (FT-IR) for confirmation of functional groups. Here, we establish a favorable C/N ratio for achieving optimal PHB yield and a predictive heat-based model to monitor its production.


Poly (3-hydroxybutyrate) Fed-batch Carbon/nitrogen ratio Biocalorimeter Heat-based model 



One of the authors (Anusha SM) wishes to acknowledge the Council of Scientific & Industrial Research (CSIR), New Delhi, for the CSIR-GATE fellowship. The authors express their gratitude to Prof. NR Rajagopal for continuous support. The authors thank Mr. Saravana Raj Adimoolam for valuable and helpful scientific discussions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights and informed consent

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

Supplementary material

253_2019_10274_MOESM1_ESM.pdf (919 kb)
ESM 1 (PDF 919 kb)


  1. Ahn WS, Park SJ, Lee SY (2001) Production of poly (3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli. Biotechnol Lett 23:235–240. CrossRefGoogle Scholar
  2. Anusha SM, Leelaram S, Surianarayanan M (2016) Production of poly (3-hydroxybutyric acid) by Ralstonia eutropha in a biocalorimeter and its thermokinetic studies. Appl Biochem Biotechnol 179:1–19. CrossRefGoogle Scholar
  3. Arias DM, Uggetti E, García-Galán MJ, García J (2018) Production of polyhydroxybutyrates and carbohydrates in a mixed cyanobacterial culture: effect of nutrients limitation and photoperiods. New Biotechnol 42:1–11. CrossRefGoogle Scholar
  4. Battley EH (1998) The development of direct and indirect methods for the study of the thermodynamics of microbial growth. Thermochim Acta 309:17–37. CrossRefGoogle Scholar
  5. Beyenal H, Chen SN, Lewandowski Z (2003) The double substrate growth kinetics of Pseudomonas aeruginosa. Enzym Microb Technol 32:92–98. CrossRefGoogle Scholar
  6. Braunegg G, Sonnleitner B, Lafferty RM (1978) A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol Biotechnol 6:29–37. CrossRefGoogle Scholar
  7. Clarke KG (2013) Microbial kinetics during batch, continuous and fed-batch processes, Bioprocess engineering, an introductory engineering and life science approach, Woodhead Publishing, pp. 97–146. doi: CrossRefGoogle Scholar
  8. Colombo B, Favini F, Scaglia B, Sciarria TP, D’Imporzano G, Pognani M, Alekseeva A, Eisele G, Cosentino C, Adani F (2017) Enhanced polyhydroxyalkanoate (PHA) production from the organic fraction of municipal solid waste by using mixed microbial culture. Biotechnol Biofuels 10:1–15. CrossRefGoogle Scholar
  9. Dias JML, Serafim LS, Lemos PC, Reis MAM, Oliveira R (2005) Mathematical modelling of a mixed culture cultivation process for the production of polyhydroxybutyrate. Biotechnol Bioeng 92:209–222. CrossRefPubMedGoogle Scholar
  10. Dietrich K, Dumont M, Del LF, Orsat V (2019) Sustainable PHA production in integrated lignocellulose biorefineries. New Biotechnol 49:161–168. CrossRefGoogle Scholar
  11. Doran PM (1995) Bioprocess engineering principles. Academic Press, London, p 439Google Scholar
  12. Freches A, Lemos PC (2017) Microbial selection strategies for polyhydroxyalkanoates production from crude glycerol : effect of OLR and cycle length. New Biotechnol 39:22–28. CrossRefGoogle Scholar
  13. Gerson DF, Kole MM, Ozum B (1988) Substrate concentration control in bioreactors. Biotechnol Genet Eng Rev 6:67–105. CrossRefGoogle Scholar
  14. Hrnčiřík P, Moucha T, Mareš J, Náhlík J, Janáčová D (2019) Software sensors for biomass concentration estimation in filamentous microorganism cultivation process. Chem Biochem Eng Q 33:141–151. CrossRefGoogle Scholar
  15. Huang L, Chen Z, Wen Q, Lee D (2017) Enhanced polyhydroxyalkanoate production by mixed microbial culture with extended cultivation strategy. Bioresour Technol 241:802–811. CrossRefPubMedGoogle Scholar
  16. Kampen WH (2014) Nutritional requirements in fermentation processes. In: Vogel HC, Todaro CM (eds) Fermentation and biochemical engineering handbook (Third Edition). William Andrew Publishing, Boston, pp 37–57. CrossRefGoogle Scholar
  17. Kanzow C, Yamashita N, Fukushima M (2004) Levenberg-Marquardt methods with strong local convergence properties for solving nonlinear equations with convex constraints. J Comput Appl Math 172:375–397. CrossRefGoogle Scholar
  18. Khanna S, Srivastava AK (2005) Statistical media optimization studies for growth and PHB production by Ralstonia eutropha. Process Biochem 40:2173–2182. CrossRefGoogle Scholar
  19. Kunasundari B, Arza CR, Maurer FHJ, Murugaiyah V, Kaur G, Sudesh K (2017) Biological recovery and properties of poly(3-hydroxybutyrate) from Cupriavidus necator H16. Sep Purif Technol 172:1–6. CrossRefGoogle Scholar
  20. Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36CrossRefGoogle Scholar
  21. Luedeking R, Piret EL (1959) A kinetic study of the lactic acid fermentation. Batch process at controllet pH. J Biochem Microbiol Technol Eng 1:393–412. doi: CrossRefGoogle Scholar
  22. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. CrossRefGoogle Scholar
  23. Moriasi DN, Arnold JG, Van Liew MW, Bingner RL, Harmel RD, Veith TL (2007) Model evaluation guidelines for systematic quantification of accuracy in watershed simulations. Transactions of the ASABE 50:885–900. CrossRefGoogle Scholar
  24. Mou DG, Cooney CL (1976) Application of dynamic calorimetry for monitoring fermentation processes. Biotechnol Bioeng 18:1371–1392. CrossRefPubMedGoogle Scholar
  25. Mozumder MSI, De Wever H, Volcke EIP, Garcia-Gonzalez L (2014a) A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production. Process Biochem 49:365–373. CrossRefGoogle Scholar
  26. Mozumder MSI, Goormachtigh L, Garcia-Gonzalez L, De Wever H, Volcke EIP (2014b) Modeling pure culture heterotrophic production of polyhydroxybutyrate (PHB). Bioresour Technol 155:272–280. CrossRefPubMedGoogle Scholar
  27. Mulchandani A, Luong JHT, Groom C (1989) Applied microbiology biotechnology substrate inhibition kinetics for microbial growth and synthesis of poly-β-hydroxybutyric acid by Alcaligenes eutrophus ATCC 17697. Appl Microbiol Biotechnol 30:11–17. CrossRefGoogle Scholar
  28. Pepè Sciarria T, Batlle-Vilanova P, Colombo B, Scaglia B, Balaguer MD, Colprim J, Puig S, Adani F (2018) Bio-electrorecycling of carbon dioxide into bioplastics. Green Chem 20:4058–4066. CrossRefGoogle Scholar
  29. Pérez Rivero C, Sun C, Theodoropoulos C, Webb C (2016) Building a predictive model for PHB production from glycerol. Biochem Eng J 116:113–121. CrossRefGoogle Scholar
  30. Redl B, Tiefenbrunner F (1981) Determination of hydrolytic activities in wastewater systems by microcalorimetry. Water Res 15:87–90. CrossRefGoogle Scholar
  31. Richhardt J, Bringer S, Bott M (2013) Role of the pentose phosphate pathway and the Entner-Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H. Appl Microbiol Biotechnol 97:4315–4323. CrossRefPubMedGoogle Scholar
  32. Santharam L, Samuthirapandi AB, Easwaran SN, Mahadevan S (2017) Modeling of exo-inulinase biosynthesis by Kluyveromyces marxianus in fed-batch mode: correlating production kinetics and metabolic heat fluxes. Appl Microbiol Biotechnol 101:1877–1887. CrossRefPubMedGoogle Scholar
  33. Schuler MM, Sivaprakasam S, Freeland B, Hama A, Hughes K-M, Marison IW (2012) Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures. Appl Microbiol Biotechnol 93:575–584. CrossRefPubMedGoogle Scholar
  34. Shong J, Rafael M, Diaz J, Collins CH (2012) Towards synthetic microbial consortia for bioprocessing. Curr Opin Biotechnol 23:798–802. CrossRefPubMedGoogle Scholar
  35. Solórzano L (1969) Determination of ammonia in natural waters by the phenol hypochlorite method. Limnol Oceanogr 14:799–801. CrossRefGoogle Scholar
  36. Špoljarić IV, Lopar M, Koller M, Muhr A, Salerno A, Reiterer A, Malli K, Angerer H, Strohmeier K, Schober S, Mittelbach M, Horvat P (2013) Mathematical modeling of poly[(R)-3-hydroxyalkanoate] synthesis by Cupriavidus necator DSM 545 on substrates stemming from biodiesel production. Bioresour Technol 133:482–494. CrossRefPubMedGoogle Scholar
  37. Suzuki T, Yamane T, Shimizu S (1986) Mass production of poly-β-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding. Appl Microbiol Biotechnol 24:370–374. CrossRefGoogle Scholar
  38. Tsouko E, Papanikolaou S, Koutinas AA (2016) Production of fuels from microbial oil using oleaginous microorganisms. In: Luque R, Lin CSK, Wilson K, Clark J (eds) Handbook of Biofuels Production (Second Edition). Woodhead Publishing, pp 201–236. Google Scholar
  39. Türker M (2004) Development of biocalorimetry as a technique for process monitoring and control in technical scale fermentations. Thermochim Acta 419:73–81. CrossRefGoogle Scholar
  40. Verlinden RAJ, Hill DJ, Kenward MA, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102:1437–1449. CrossRefPubMedGoogle Scholar
  41. Voisard D, von Stockar U, Marison IW (2002) Quantitative calorimetric investigation of fed-batch cultures of Bacillus sphaericus 1593M. Thermochim Acta 394:99–111. CrossRefGoogle Scholar
  42. von Stockar U, Marison I, Janssen M, Patiño R (2011) Calorimetry and thermodynamic aspects of heterotrophic, mixotrophic, and phototrophic growth. J Therm Anal Calorim 104:45–52. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Anusha Mohanakrishnan Subramanian
    • 1
  • Sivanesh Easwaran Nanjan
    • 1
  • Hariram Prakash
    • 2
  • Leelaram Santharam
    • 3
  • Ankitha Ramachandran
    • 2
  • Vignesh Sathyaseelan
    • 2
  • Deepa Perinkulum Ravi
    • 4
  • Surianarayanan Mahadevan
    • 1
    Email author
  1. 1.Chemical Engineering DepartmentCSIR-Central Leather Research Institute (CLRI)ChennaiIndia
  2. 2.Department of Chemical EngineeringBirla Institute of Technology & Science (BITS)Zuari NagarIndia
  3. 3.Bioseparation and Bioprocessing Laboratory, Department of Chemical EngineeringIndian Institute of TechnologyDelhiIndia
  4. 4.Department of Biological SciencesBirla Institute of Technology & Science (BITS)PilaniIndia

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