Applied Microbiology and Biotechnology

, Volume 102, Issue 19, pp 8291–8305 | Cite as

Stability and oscillatory behavior of microbial consortium in continuous conversion of crude glycerol to 1,3-propanediol

  • Jin-Jie Zhou
  • Jun-Tao Shen
  • Xiao-Li Wang
  • Ya-Qin Sun
  • Zhi-Long XiuEmail author
Biotechnological products and process engineering


Microbial consortium is an alternative for bioconversion of crude glycerol to value-added products whereas concerns about the process stability in long-term operation existed. The aim of this study is to evaluate the feasibility of using an anaerobic microbial consortium as inoculum for continuous conversion of crude glycerol to 1,3-propanediol (1,3-PDO). Performances of continuous fermentations with the consortium inoculum were evaluated under different dilution rates and glycerol feed concentrations. The highest 1,3-PDO production of 57.86 g/L was achieved with a productivity of 5.55 g/(L·h). Analyses of kinetic data showed that the consortium maintained a consistent pattern for 1,3-PDO production under different operating conditions despite changes in community composition. The continuous fermentation by the consortium was able to operate for a longer period of time (31 volume changes) than that using pure culture (24 volume changes) with the average 1,3-PDO concentration of 53.52 g/L and productivity of 6.69 g/(L·h) under glycerol-excess condition, which could be contributed to the intraspecies diversity among Clostridium butyricum in the consortium. Under glycerol-limited conditions, however, a spontaneous oscillation of the consortium was observed after continuous operation for about 120 h, along with severe fluctuations of the microbial community. The oscillatory behavior could be reduced by increasing the dilution rates and was likely the metabolic feature of C. butyricum.


1,3-Propanediol Microbial consortium Continuous fermentation Clostridium butyricum Stability Oscillation 


Funding information

This work was supported by the National Natural Science Foundation of China (Grant No. 21476042) and the Fundamental Research Funds for the Central Universities (Grant No. DUT17ZD209).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_9244_MOESM1_ESM.pdf (81 kb)
ESM 1 (PDF 81 kb)


  1. Abbad-Andaloussi S, Guedon E, Spiesser E, Petitdemange H (1996) Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum DSM 5431. Lett Appl Microbiol 22(4):311–314. CrossRefGoogle Scholar
  2. Agler MT, Wrenn BA, Zinder SH, Angenent LT (2011) Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends Biotechnol 29(2):70–78. CrossRefPubMedGoogle Scholar
  3. Almeida JR, Fávaro LC, Quirino BF (2012) Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste. Biotechnol Biofuels 5(1):48. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4(1):187. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Biebl H (1991) Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat. Appl Microbiol Biotechnol 35(6):701–705. CrossRefGoogle Scholar
  6. Chatzifragkou A, Papanikolaou S, Dietz D, Doulgeraki AI, Nychas GJ, Zeng AP (2011) Production of 1,3-propanediol by Clostridium butyricum growing on biodiesel-derived crude glycerol through a non-sterilized fermentation process. Appl Microbiol Biotechnol 91(1):101–112. CrossRefPubMedGoogle Scholar
  7. Chatzifragkou A, Aggelis G, Gardeli C, Galiotou-Panayotou M, Komaitis M, Papanikolaou S (2012) Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media. Appl Microbiol Biotechnol 95(6):1541–1552. CrossRefPubMedGoogle Scholar
  8. Coyte KZ, Schluter J, Foster KR (2015) The ecology of the microbiome: networks, competition, and stability. Science 350(6261):663–666. CrossRefPubMedGoogle Scholar
  9. Dietz D, Zeng AP (2014) Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium. Bioprocess Biosyst Eng 37(2):225–233. CrossRefPubMedGoogle Scholar
  10. Gallardo R, Faria C, Rodrigues LR, Pereira MA, Alves MM (2014) Anaerobic granular sludge as a biocatalyst for 1,3-propanediol production from glycerol in continuous bioreactors. Bioresour Technol 155:28–33. CrossRefPubMedGoogle Scholar
  11. Gonzalez-Pajuelo M, Andrade JC, Vasconcelos I (2005) Production of 1,3-propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity. J Ind Microbiol Biotechnol 32(9):391–396. CrossRefPubMedGoogle Scholar
  12. Gonzalez-Pajuelo M, Meynial-Salles I, Mendes F, Soucaille P, Vasconcelos I (2006) Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5). Appl Environ Microbiol 72(1):96–101. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hoskisson PA, Hobbs G (2005) Continuous culture—making a comeback? Microbiology 151(10):3153–3159. CrossRefPubMedGoogle Scholar
  14. Jiang LL, Zhou JJ, Xiu ZL (2017a) Advances in industrial microbiome based on microbial consortium for biorefinery. Bioresour Bioprocess 4(1):11. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jiang LL, Liu HF, Mu Y, Sun YQ, Xiu ZL (2017b) High tolerance to glycerol and high production of 1,3-propanediol in batch fermentations by microbial consortium from marine sludge. Eng Life Sci 17(6):635–644. CrossRefGoogle Scholar
  16. Jiang LL, Dai JY, Sun YQ, Xiu ZL (2018) The effects of ionic liquid 1-ethyl-3-methylimidazolium trifluoromethanesulfonate on 1,3-propanediol production from crud glycerol by microbial consortium. Bioprocess Biosyst Eng. CrossRefPubMedGoogle Scholar
  17. Kanjilal B, Noshadi I, Bautista EJ, Srivastava R, Parnas RS (2015) Batch, design optimization, and DNA sequencing study for continuous 1,3-propanediol production from waste glycerol by a soil-based inoculum. Appl Microbiol Biotechnol 99(5):2105–2117. CrossRefPubMedGoogle Scholar
  18. Kaur G, Srivastava AK, Chand S (2012) Advances in biotechnological production of 1,3-propanediol. Biochem Eng J 64:106–118. CrossRefGoogle Scholar
  19. Kleerebezem R, van Loosdrecht MC (2007) Mixed culture biotechnology for bioenergy production. Curr Opin Biotechnol 18(3):207–212. CrossRefPubMedGoogle Scholar
  20. Lee CS, Aroua MK, Daud WMAW, Cognet P, Pérès-Lucchese Y, Fabre PL, Reynes O, Latapie L (2015) A review: conversion of bioglycerol into 1,3-propanediol via biological and chemical method. Renew Sust Energ Rev 42:963–972. CrossRefGoogle Scholar
  21. Li T, Chen XB, Chen JC, Wu Q, Chen GQ (2014) Open and continuous fermentation: products, conditions and bioprocess economy. Biotechnol J 9(12):1503–1511. CrossRefPubMedGoogle Scholar
  22. Liu H, Xu Y, Zheng Z, Liu D (2010) 1,3-Propanediol and its copolymers: research, development and industrialization. Biotechnol J 5(11):1137–1148. CrossRefPubMedGoogle Scholar
  23. Loureiro-Pinto M, Coca M, González-Benito G, Lucas S, García-Cubero MT (2017) Continuous bioproduction of 1,3-propanediol from biodiesel raw glycerol: operation with free and immobilized cells of Clostridium butyricum DSM 10702. Can J Chem Eng 95(5):819–826. CrossRefGoogle Scholar
  24. Luedeking R, Piret EL (1959) A kinetic study of the lactic acid fermentation. Batch process at controlled pH. Biotechnol Bioeng 1(4):393–412. CrossRefGoogle Scholar
  25. Menzel K, Zeng AP, Biebl H, Deckwer WD (1996) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: I. The phenomena and characterization of oscillation and hysteresis. Biotechnol Bioeng 52(5):549–560.<549::AID-BIT2>3.0.CO;2-J CrossRefPubMedGoogle Scholar
  26. Menzel K, Zeng AP, Deckwer WD (1997) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzym Microb Technol 20(2):82–86. CrossRefGoogle Scholar
  27. Monteiro MR, Kugelmeier CL, Pinheiro RS, Batalha MO, da Silva CA (2018) Glycerol from biodiesel production: technological paths for sustainability. Renew Sust Energ Rev 88:109–122. CrossRefGoogle Scholar
  28. Mu Y, Teng H, Zhang DJ, Wang W, Xiu ZL (2006) Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations. Biotechnol Lett 28:1755–1759. CrossRefPubMedGoogle Scholar
  29. Mu XJ, Teng H, Sun YQ, Xiu ZL (2009) Techno-economic analysis on bioconversion of glycerol into 1,3-propanediol. Chin J Process Eng 9(5):947–952Google Scholar
  30. Papanikolaou S, Ruiz-Sanchez P, Pariset B, Blanchard F, Fick M (2000) High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. J Biotechnol 77(2):191–208. CrossRefPubMedGoogle Scholar
  31. Papanikolaou S, Fick M, Aggelis G (2004) The effect of raw glycerol concentration on the production of 1,3-propanediol by Clostridium butyricum. J Chem Technol Biotechnol 79(11):1189–1196. CrossRefGoogle Scholar
  32. Papanikolaou S, Rontou M, Belka A, Athenaki M, Gardeli C, Mallouchos A, Kalantzi O, Koutinas A, Kookos I, Zeng AP, Aggelis G (2017) Conversion of biodiesel-derived glycerol into biotechnological products of industrial significance by yeast and fungal strains. Eng Life Sci 17(3):262–281. CrossRefGoogle Scholar
  33. Petitdemange E, Dürr C, Andaloussi SA, Raval G (1995) Fermentation of raw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. J Ind Microbiol 15(6):498–502. CrossRefGoogle Scholar
  34. Pirt SJ (1965) The maintenance energy of bacteria in growing cultures. Proc R Soc Lond B 163(991):224–231. CrossRefPubMedGoogle Scholar
  35. Reimann A, Abbad-Andaloussi S, Biebl H, Petitdemange H (1998) 1,3-Propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow. J Appl Microbiol 84(6):1125–1130. CrossRefGoogle Scholar
  36. Ringel AK, Wilkens E, Hortig D, Willke T, Vorlop KD (2012) An improved screening method for microorganisms able to convert crudeglycerol to 1,3-propanediol and to tolerate high product concentrations. Appl Microbiol Biotechnol 93:1049–1056. CrossRefPubMedGoogle Scholar
  37. Rywińska A, Juszczyk P, Wojtatowicz M, Robak M, Lazar Z, Tomaszewska L, Rymowicz W (2013) Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications. Biomass Bioenergy 48:148–166. CrossRefGoogle Scholar
  38. Sabra W, Dietz D, Tjahjasari D, Zeng AP (2010) Biosystems analysis and engineering of microbial consortia for industrial biotechnology. Eng Life Sci 10(5):407–421. CrossRefGoogle Scholar
  39. Samul D, Leja K, Grajek W (2014) Impurities of crude glycerol and their effect on metabolite production. Ann Microbiol 64:891–898. CrossRefPubMedGoogle Scholar
  40. Sittijunda S, Reungsang A (2017) Fermentation of hydrogen, 1,3-propanediol and ethanol from glycerol as affected by organic loading rate using up-flow anaerobic sludge blanket (UASB) reactor. Int J Hydrog Energy 42(45):27558–27569. CrossRefGoogle Scholar
  41. Sun LH, Song ZY, Sun YQ, Xiu ZL (2010) Dynamic behavior of glycerol–glucose co-fermentation for 1,3-propanediol production by Klebsiella pneumoniae DSM 2026 under micro-aerobic conditions. World J Microbiol Biotechnol 26(8):1401–1407. CrossRefGoogle Scholar
  42. Sun YQ, Ma CW, Fu HX, Mu Y, Xiu ZL (2014) 1,3-Propanediol. In: Bisaria VS, Kondo A (eds) Bioprocessing of renewable resources to commodity bioproducts, 1st edn. John Wiley & Sons, Hoboken, pp 289–326CrossRefGoogle Scholar
  43. Sun YQ, Shen JT, Yan L, Zhou JJ, Jiang LL, Chen Y, Yuan JL, Feng EM, Xiu ZL (2018) Advances in bioconversion of glycerol to 1,3-propanediol: prospects and challenges. Process Biochem. CrossRefGoogle Scholar
  44. Tomaszewska L, Rywińska A, Gładkowski W (2012) Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol. J Ind Microbiol Biotechnol 39(9):1333–1343. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Van Andel JG, Zoutberg GR, Crabbendam PM, Breure AM (1985) Glucose fermentation by Clostridium butyricum grown under a self generated gas atmosphere in chemostat culture. Appl Microbiol Biotechnol 23(1):21–26. CrossRefGoogle Scholar
  46. Varrone C, Floriotis G, Heggeset TMB, Le SB, Markussen S, Skiadas IV, Gavala HN (2017) Continuous fermentation and kinetic experiments for the conversion of crude glycerol derived from second-generation biodiesel into 1,3-propanediol and butyric acid. Biochem Eng J 128:149–161. CrossRefGoogle Scholar
  47. Wang X, Zhang H, Yang F, Wang Y, Gao M (2008) Long-term storage and subsequent reactivation of aerobic granules. Bioresour Technol 99(17):8304–8309. CrossRefPubMedGoogle Scholar
  48. Willke T, Vorlop K (2008) Biotransformation of glycerol into 1,3-propanediol. Eur J Lipid Sci Tech 110(9):831–840. CrossRefGoogle Scholar
  49. Xiu ZL, Zeng AP (1999) A study progress in oscillatory and chaotic behavior in microbial continuous cultures. Prog Biotechnol 19(6):58–63Google Scholar
  50. Xiu ZL, Zeng AP (2008) Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol. Appl Microbiol Biotechnol 78(6):917–926. CrossRefPubMedGoogle Scholar
  51. Zeng AP (1996) Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum. Bioprocess Eng 14(4):169–175. CrossRefGoogle Scholar
  52. Zeng AP, Biebl H (2002) Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends. In: Tools and applications of biochemical engineering science. Springer, Berlin, pp 239–259CrossRefGoogle Scholar
  53. Zeng AP, Ross A, Biebl H, Tag C, Günzel B, Deckwer WD (1994) Multiple product inhibition and growth modeling of Clostridium butyricum and Klebsiella pneumoniae in gIyceroI fermentation. Biotechnol Bioeng 44(8):902–911. CrossRefPubMedGoogle Scholar
  54. Zeng AP, Menzel K, Deckwer W-D (1996) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: II. Analysis of metabolic rates and pathways under oscillation and steady-state conditions. Biotechnol Bioeng 52(5):561–571.<561::AID-BIT3>3.0.CO;2-H CrossRefPubMedGoogle Scholar
  55. Zhang C, Yang H, Yang F, Ma Y (2009) Current progress on butyric acid production by fermentation. Curr Microbiol 59(6):656–663. CrossRefPubMedGoogle Scholar
  56. Zhou JJ, Shen JT, Jiang LL, Sun YQ, Mu Y, Xiu ZL (2017) Selection and characterization of an anaerobic microbial consortium with high adaptation to crude glycerol for 1,3-propanediol production. Appl Microbiol Biotechnol 101(15):5985–5996. CrossRefPubMedGoogle Scholar
  57. Zhu C, Chen B, Fang B (2013) Pretreatment of raw glycerol with activated carbon for 1,3-propanediol production by Clostridium butyricum. Eng Life Sci 13(4):376–384. CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Jin-Jie Zhou
    • 1
  • Jun-Tao Shen
    • 1
  • Xiao-Li Wang
    • 1
  • Ya-Qin Sun
    • 1
  • Zhi-Long Xiu
    • 1
    Email author
  1. 1.School of Life Science and BiotechnologyDalian University of TechnologyDalianPeople’s Republic of China

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