Prospects of Solvent Tolerance in Butanol Fermenting Bacteria

  • Shuvashish Behera
  • Nilesh Kumar Sharma
  • Sachin Kumar
Part of the Biofuel and Biorefinery Technologies book series (BBT, volume 4)


Butanol tolerance is a critical factor affecting the ability of microorganisms to produce economically viable quantities of butanol through acetone-butanol-ethanol (ABE) fermentation using renewable feedstocks. However, ABE process has certain challenges like maintaining strict anaerobic conditions, slow growth rate of microorganisms, the rapid shift of pH, sensitivity to acetic acid, low butanol titer, solvent tolerance, and product inhibition. Separation of fermentation products through distillation, gas stripping, pervaporation, and adsorption also makes the process costly. Despite their importance at a biofuel platform, a limited number of butanol-tolerant bacteria have been identified so far. This problem can be eradicated through the isolation of solvent tolerating bacteria, development of bacteria through evolutionary engineering, mutation, and genetic engineering with promising product recovery techniques. In the present chapter, an overview of the butanol tolerating microbes, their solvent survival strategies, and the techniques to overcome the problem for a high concentration of butanol have been discussed.



Authors acknowledge the grant support from Science & Engineering Research Board, New Delhi, Govt. of India (File No. YSS/2015/000295). Authors also thank to Sardar Swaran Singh National Institute of Bio-Energy, Kapurthala, India as the host Institution for providing laboratory space to complete this work.


  1. Abd-Alla MH, El-Enany AWE (2012) Production of acetone-butanol-ethanol from spoilage date palm (Phoenix dactylifera L.) fruits by mixed culture of Clostridium acetobutylicum and Bacillus subtilis. Biomass Bioenergy 42:172–178CrossRefGoogle Scholar
  2. Alsaker KV, Paredes C, Papoutsakis ET (2010) Metabolite stress and tolerance in the production of biofuels and chemicals: gene-expression-based systems analysis of butanol, butyrate and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotechnol Bioeng 105:1131–1147Google Scholar
  3. Al-Shorgani N, Kalil M, Yusoff W (2012) Fermentation of sago starch to biobutanol in a batch culture using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). Ann Microbiol 62(3):1059–1070CrossRefGoogle Scholar
  4. Atsumi S, Cann AF, Connor MR et al (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10:305–311CrossRefGoogle Scholar
  5. Behera S, Sharma NK, Arora R et al (2016) Effect of evolutionary adaption on xylosidase activity in thermotolerant yeast isolates Kluyveromyces marxianus NIRE-K1 and NIRE-K3. Appl Biochem Biotechnol 179:1143–1154CrossRefGoogle Scholar
  6. Berezina OV, Brandt A, Yarotsky S et al (2009) Isolation of a new butanol-producing Clostridium strain: High level of hemicellulosic activity and structure of solventogenesis genes of a new Clostridium saccharobutylicum isolate. Syst Appl Microbiol 32:449–459CrossRefGoogle Scholar
  7. Borden JR, Papoutsakis ET (2007) Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum. Appl Environ Microbiol 73(9):3061–3068CrossRefGoogle Scholar
  8. Brynildsen MP, Liao JC (2009) An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol Syst Biol 5:277CrossRefGoogle Scholar
  9. Cai D, Chen H, Chen C et al (2016) Gas stripping–pervaporation hybrid process for energy-saving product recovery from acetone-butanol-ethanol (ABE) fermentation broth. Chem Eng J 287:1–10CrossRefGoogle Scholar
  10. Chen T, Wang J, Yang R et al (2011) Laboratory-evolved mutants of an exogenous global regulator, IrrE from Deinococcus radiodurans, enhance stress tolerances of Escherichia coli. PLoS ONE 6(1):e16228CrossRefGoogle Scholar
  11. Dong H, Zhang Y, Zhu Y et al (2011) Biofuels and bioenergy: acetone and butanol. In: Murray M-Y (ed) Comprehensive biotechnology, 2nd edn. Academic Press, New York, 71–85Google Scholar
  12. Doukyu N, Ishikawa K, Watanabe R et al (2012) Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli. J Appl Microbiol 112:464–474CrossRefGoogle Scholar
  13. Dunlop MJ (2011) Engineering microbes for tolerance to next-generation biofuels. Biotechnol Biofuels 4:32CrossRefGoogle Scholar
  14. Dunlop MJ, Dossani ZY, Szmidt HL et al (2011) Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 7:487CrossRefGoogle Scholar
  15. Durre P (2007) Biobutanol: an attractive biofuel. Biotechnol J 2:1525–1534CrossRefGoogle Scholar
  16. Durre P (2011) Fermentative production of butanol-the academic perspective. Curr Opin Biotechnol 22:331–336CrossRefGoogle Scholar
  17. Ezeji TC, Qureshi N, Blaschek HP (2007) Bioproduction of butanol from biomass: from genes to bioreactors. Curr Opin Biotechnol 18:220–227CrossRefGoogle Scholar
  18. Fatehi P (2013) Recent advancements in various steps of ethanol, butanol, and isobutanol productions from woody materials. Biotechnol Prog 29(2):297–310CrossRefGoogle Scholar
  19. Fernandes P, Ferreira BS, Cabral JMS (2003) Solvent tolerance in bacteria: role of efflux pumps and cross resistance with antibiotics. Int J Antimicrobial Agents 22:211–216CrossRefGoogle Scholar
  20. Formanek J, Mackie R, Blaschek HP (1997) Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose. Appl Environ Microbiol 63:2306–2310Google Scholar
  21. Gao X, Sun T, Wu L et al (2017) Co-overexpression of response regulator genes slr1037 and sll0039 improves tolerance of Synechocystis sp. PCC 6803 to 1-butanol. Bioresour Technol (In Press)Google Scholar
  22. Garcia V, Pakkila J, Ojamo H et al (2011) Challenges in biobutanol production: how to improve the efficiency? Renew Sustain Energy Rev 15:964–980CrossRefGoogle Scholar
  23. George HA, Chen JS (1983) Acidic conditions are not obligatory for onset of butanol formation by Clostridium beijerinckii (synonym, C. butylicum). Appl Environ Microbiol 46:321–327Google Scholar
  24. Goodarzi H, Bennett BD, Amini S et al (2010) Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli. Mol Syst Biol 6:378CrossRefGoogle Scholar
  25. Gottwald M, Hippe H, Gottschalk G (1984) Formation of n-Butanol from D-Glucose by Strains of the” Clostridium tetanomorphum” Group. Appl Environ Microbiol 48:573–576Google Scholar
  26. Honig V, Kotek M, Marik J (2014) Use of butanol as a fuel for internal combustion engines. Agron Res 12(2):333–340Google Scholar
  27. Horinouchi T, Tamaoka K, Furusawa C et al (2010) Transcriptome analysis of parallel-evolved Escherichia coli strains under ethanol stress. BMC Genom 11:579CrossRefGoogle Scholar
  28. Hou X, From N, Angelidaki I et al (2017) Butanol fermentation of the brown seaweed Laminaria digitata by Clostridium beijerinckii DSM-6422. Bioresour Technol 238:16–21CrossRefGoogle Scholar
  29. Huang H, Liu H, Gan YR (2010) Genetic modification of critical enzymes and involved genes in butanol biosynthesis from biomass. Biotechnol Adv 28:651–657CrossRefGoogle Scholar
  30. Jang YS, Malaviya A, Cho C et al (2012) Butanol production from renewable biomass by clostridia. Bioresour Technol 123:653–663CrossRefGoogle Scholar
  31. Jeong H, Kim SH, Han SS et al (2012) Changes in membrane fatty acid composition through proton-induced fabF mutation enhancing 1-butanol tolerance in E. coli. J Korean Phys Soc 61:227–233CrossRefGoogle Scholar
  32. Jiang Y, Xu C, Dong F et al (2009) Disruption of the acetoacetate decarboxylase gene insolvent-producing Clostridium acetobutylicum increases the butanol ratio. Metab Eng 11:284–291CrossRefGoogle Scholar
  33. Jiang Y, Liu J, Jiang W et al (2014) Current status and prospects of industrial bio-production of n-butanol in China. Biotechnol Adv 33:1493–1501CrossRefGoogle Scholar
  34. Jin H1, Chen L, Wang J et al (2014) Engineering biofuel tolerance in non-native producing microorganisms. Biotechnol Adv 32(2):541–548Google Scholar
  35. Kang HJ, Heo DH, Choi SW et al (2007) Functional characterization of Hsp33 protein from Bacillus psychrosaccharolyticus; additional function of HSP33 on resistance to solvent stress. Biochem Biophys Res Commun 358(3):743–750CrossRefGoogle Scholar
  36. Kanno M, Katayama T, Tamaki H et al (2013) Isolation of butanol- and isobutanol-tolerant bacteria and physiological characterization of their butanol tolerance. Am Soc Microbiol 79:6998–7005Google Scholar
  37. Kataoka N, Tajima T, Kato J et al (2011) Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host. AMB Express 1:10CrossRefGoogle Scholar
  38. Kieboom J, Dennis JJ, de Bont JA et al (1998) Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance. J Biol Chem 273(1):85–91CrossRefGoogle Scholar
  39. Kim HJ, Turner TL, Jin YS (2013) Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals. Biotechnol Adv 31:976–985CrossRefGoogle Scholar
  40. Knoshaug EP, Zhang M (2009) Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol 153:13–20CrossRefGoogle Scholar
  41. Kumar M, Gayen K (2011) Developments in biobutanol production: new insights. Appl Energy 88:1999–2012CrossRefGoogle Scholar
  42. Lee SY, Park JH, Jang SH et al (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101:209–228CrossRefGoogle Scholar
  43. Lee SJ, Lee SJ, Lee DW (2013) Design and development of synthetic microbial platform cells for bioenergy. Front Microbiol 4:92Google Scholar
  44. Lehmann D, Lutke-Eversloh T (2011) Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway. Metabolic Eng 13:464–473CrossRefGoogle Scholar
  45. Li XZ, Zhang L, Poole K (1998) Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance. J Bacteriol 180:2987–2991Google Scholar
  46. Li J, Zhao JB, Zhao M et al (2010) Screening and characterization of butanol-tolerant micro-organisms. Lett Appl Microbiol 50:373–379CrossRefGoogle Scholar
  47. Li H, Luo W, Gu Q et al (2013) Acetone, butanol, and ethanol production from cane molasses using Clostridium beijerinckii mutant obtained by combined low-energy ion beam implantation and N-methyl-N-nitro-N-nitrosoguanidine induction. Bioresour Technol 137:254–260CrossRefGoogle Scholar
  48. Li H, Ofosu FK, Li K et al (2014) Acetone, butanol, and ethanol production from gelatinized cassava flour by a new isolates with high butanol tolerance. Bioresour Technol 172:276–282CrossRefGoogle Scholar
  49. Liao Z, Zhang Y, Luo S et al (2017) Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium. J Biotechnol 252:1–10CrossRefGoogle Scholar
  50. Lin YL, Blaschek HP (1983) Butanol production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth. Appl Environ Microbiol 45:966–973Google Scholar
  51. Liu S, Qureshi N (2009) How microbes tolerate ethanol and butanol. New Biotechnol 26:117–121CrossRefGoogle Scholar
  52. Liu S, Bischoff KM, Leathers TD et al (2012) Adaptation of lactic acid bacteria to butanol. Biocatal Agric Biotechnol 1:57–61Google Scholar
  53. Liu X, Gu Q, Liao C et al (2014) Enhancing butanol tolerance and preventing degeneration in Clostridium acetobutylicum by 1-butanol–glycerol storage during long-term preservation. Biomass Bioenergy 69:192–197CrossRefGoogle Scholar
  54. Lo TM, Suong TW, Ling H et al (2013) Microbial engineering strategies to improve cell viability for biochemical production. Biotechnol Adv 31:903–914CrossRefGoogle Scholar
  55. Lopez-Contreras AM, Kuit W, Siemerink MAJ et al (2010) Production of longer-chain alcohols from lignocellulosic biomass: butanol, isopropanol and 2,3-butanediol. In: Waldron K (ed) Bioalcohol production. Woodhead Publishing, Cambridge (UK), pp 415–460CrossRefGoogle Scholar
  56. Lutke-Eversloh T, Bahl H (2011) Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotechnol 22:1–14CrossRefGoogle Scholar
  57. Maiti S, Gallastegui G, Jyoti S et al (2016) A re-look at the biochemical strategies to enhance butanol production. Biomass Bioenergy 94:187–200CrossRefGoogle Scholar
  58. Mann MS, Dragovic Z, Schirrmacher G et al (2012) Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress. Biotechnol Lett 34:1643–1649CrossRefGoogle Scholar
  59. Mariano AP, Qureshi N, Ezeji TC (2011) Bioproduction of butanol in bioreactors: new insights from simultaneous in situ butanol recovery to eliminate product toxicity. Biotechnol Bioeng 108:1757–1765CrossRefGoogle Scholar
  60. Merlet G, Uribe F, Aravena C et al (2017) Separation of fermentation products from ABE mixtures by perstraction using hydrophobic ionic liquids as extractants. J Membr Sci 537:337–343CrossRefGoogle Scholar
  61. Nicolaou SA, Gaida SM, Papoutsakis ET (2010) A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation. Metab Eng 12(4):307–331CrossRefGoogle Scholar
  62. Okochi M, Kurimoto M, Shimizu K (2007) Increase of organic solvent tolerance by overexpression of man XYZ in Escherichia coli. Appl Microbiol Biotechnol 73:1394–1399CrossRefGoogle Scholar
  63. Papoutsakis ET (2008) Engineering solventogenic clostridia. Curr Opin Biotechnol 19:420–429CrossRefGoogle Scholar
  64. Pinkart HC, Wolfram JW, Rogers R et al (1996) Cell envelope changes in solvent-tolerant and solvent sensitive Pseudomonas putida strains following exposure to o-xylene. Appl Environ Microbiol 62:1129–1132Google Scholar
  65. Qureshi N, Blaschek HP (1999) Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation. Biotechnol Prog 15:594–602CrossRefGoogle Scholar
  66. Qureshi N, Blaschek HP (2005) Butanol production from agricultural biomass. In: Shetty K, Paliyath G, Pometto A, Levin RE (eds) Food biotechnology, 2nd edn. Taylor & Francis, New York, pp 525–549Google Scholar
  67. Qureshi N, Saha BC, Cotta MA (2007) Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioprocess Biosyst Eng 30:419–427CrossRefGoogle Scholar
  68. Qureshi N, Saha BC, Dien B et al (2010a) Production of butanol (a biofuel) from agricultural residues: part I- use of barley straw hydrolysate. Biomass Bioenergy 34:559–565CrossRefGoogle Scholar
  69. Qureshi N, Saha BC, Hector RE et al (2010b) Production of butanol (a biofuel) from agricultural residues: part II- use of corn stover and switchgrass hydrolysates. Biomass Bioenergy 34:566–571CrossRefGoogle Scholar
  70. Qureshi N, Saha BC, Cotta MA, Singh V (2013) An economic evaluation of biological conversion of wheat straw to butanol: a biofuel. Energy Convers Manag 65:456–462CrossRefGoogle Scholar
  71. Rao A, Sathiavelu A, Mythili S (2016) Genetic engineering in biobutanol production and tolerance. Braz Arch Biol Technol 59:e16150612CrossRefGoogle Scholar
  72. Reyes LH, Almario MP, Kao KC (2011) Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli. PLoS ONE 6:e17678CrossRefGoogle Scholar
  73. Rochon E, Ferrari MD, Lareo C (2017) Integrated ABE fermentation-gas stripping process for enhanced butanol production from sugarcane-sweet sorghum juices. Biomass Bioenergy 98:153–160CrossRefGoogle Scholar
  74. Ruhl J, Schmid A, Blank LM (2009) Selected Pseudomonas putida strains able to grow in the presence of high butanol concentrations. Appl Environ Microbiol 75:4653–4656CrossRefGoogle Scholar
  75. Rutherford BJ, Dahl RH, Price RE et al (2010) Functional genomic study of exogenous n-butanol stress in Escherichia coli. Appl Environ Microbiol 76:1935–1945CrossRefGoogle Scholar
  76. Shah AA, Wang C, Chung YR et al (2013) Enhancement of geraniol resistance of Escherichia coli by MarA overexpression. J Biosci Bioeng 115:253–258CrossRefGoogle Scholar
  77. Sharma NK, Behera S, Arora R et al (2016) Enhancement in xylose utilization using Kluyveromyces marxianus NIRE-K1 through evolutionary adaptation approach. Bioprocess Biosystems Eng 39:835–843CrossRefGoogle Scholar
  78. Syed Q, Nadeem M, Nelofer R (2008) Enhanced butanol production by mutant strains of Clostridium acetobutylicum in molasses medium. Turkish J Biochem 33(1):25–30Google Scholar
  79. Tanaka Y, Kasahara K, Hirose Y (2017) Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance. J Biosci Bioeng (In Press)Google Scholar
  80. Tian X, Chen L, Wang J et al (2012) Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol. J Proteomics 2012:326–345Google Scholar
  81. Tomas CA, Welker NE, Papoutsakis ET (2003) Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program. Appl Environ Microbiol 69(8):4951–4965CrossRefGoogle Scholar
  82. Tomas CA, Beamish J, Papoutsakis ET (2004) Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J Bacteriol 186(7):2006–2018CrossRefGoogle Scholar
  83. Vermue M, Sikkema J, Verheul A et al (1993) Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia sp. and the gram-negative bacteria Acinetobacter and Pseudomonas sp. Biotechnol Bioeng 42:747–758CrossRefGoogle Scholar
  84. Volkers RJM, de Jong AL, Hulst AG et al (2006) Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12. Environ Microbiol 8(9):1674–1679CrossRefGoogle Scholar
  85. Vollherbst-Schneck, K, Sands JA, Montenecourt BS (1984) Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 47:193–194Google Scholar
  86. Waber FJ, Bont JM (1996) Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim Biophys Acta 1286:225–245CrossRefGoogle Scholar
  87. Wang YF, Tiana J, Ji ZH (2016) Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation. Int J Biochem Cell Biol 78:297–306CrossRefGoogle Scholar
  88. Woods DR (1995) The genetic engineering of microbial solvent production. Trends Biotechnol 13:259–264CrossRefGoogle Scholar
  89. Wu YD, Xue C, Chen LJ et al (2013) Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum. J Biotechnol 165:18–21CrossRefGoogle Scholar
  90. Zhang J, Wang S, Wang Y (2016) Biobutanol production from renewable resources: recent advances. Adv Bioenergy 1:1–68CrossRefGoogle Scholar
  91. Zheng YN, Li LZ, Xian M et al (2009) Problems with the microbial production of butanol. J Ind Microbiol Biotechnol 36:1127–1138CrossRefGoogle Scholar
  92. Zheng J, Tashiro Y, Wang Q, Sonomoto K (2015) Recent advances to improve fermentative butanol production: genetic engineering and fermentation technology. J Biosci Bioeng 119:1–9CrossRefGoogle Scholar
  93. Zhu L, Dong H, Zhang Y, Li Y (2011) Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability. Metab Eng 13:426–434CrossRefGoogle Scholar
  94. Zhu H, Ren X, Wang J et al (2013) Integrated OMICS guided engineering of biofuel butanol-tolerance in photosynthetic Synechocystis sp. PCC 6803. Biotechnol Biofuels 6:106CrossRefGoogle Scholar
  95. Zingaro KA, Papoutsakis ET (2012) GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1, 2, 4-butanetriol and ethanol with complex and unpredictable patterns. Metab Eng 15:196–205CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Biochemical Conversion DivisionSardar Swaran Singh National Institute of Bio-EnergyKapurthalaIndia
  2. 2.Department of Chemical and Biological EngineeringSouth Dakota School of Mines and TechnologyRapid CityUSA

Personalised recommendations