Biomass Conversion and Biorefinery

, Volume 3, Issue 2, pp 143–155 | Cite as

Process optimization for butanol production from developed rice straw hydrolysate using Clostridium acetobutylicum MTCC 481 strain

  • Amrita Ranjan
  • Rahul Mayank
  • Vijayanand S. MoholkarEmail author
Original Article


In this study, an attempt is made to optimize the effect of various physical and cultural parameters on butanol production by microbial strain Clostridium acetobutylicum MTCC 481 by employing L18 orthogonal array design of experiments. A set of five parameters, viz., temperature, pH, inoculum size, inoculum age, and agitation have been studied. Utilizing a pre-optimized rice straw hydrolysate medium, the clostridial strain produced maximum amount of butanol at optimum conditions of temperature 37 °C, pH 4.0 ± 0.5, inoculum size 5 % (v/v), inoculum age 18 h, and agitation 150 rpm. Among these parameters, pH, temperature, and agitation were found to be the most significant factors affecting solvent production. The optimized physical and cultural parameters were further verified at shake flask and bioreactor scale (2 L and 5 L bioreactor). Experiments using 2 and 5 L bioreactor under the optimized process condition showed nearly complete utilization of soluble sugars with the production of 15.84 g L−1 of total solvents with 12.17 g L−1 of butanol in 2 L bioreactor and 16.91 g L−1 of total solvents with 12.22 g L−1 of butanol in a 5 L of bioreactor, respectively. The experimental data were further validated by fitting it to a kinetic model reported in literature to determine the kinetic parameters of the fermentation process.


Rice straw hydrolysate Process optimization Anaerobic processes Microbial growth Fermentation Modelling 



Acetone butanol ethanol


Analysis of variance


Cooked meat medium


Dinitrosalicylic acid


Design of experiments


Specific growth rate (per hour)


Mean of squares


Microbial Type Culture Collection


National Collection of Industrial Micro-organisms


Product concentration (grams per cubic liter)


Initial product concentration (grams per cubic liter)


Maximum product concentration (grams per cubic liter)


Kinetic constant


p-Aminobenzoic acid


Reinforced clostridial agar


Reinforced clostridial medium


Root mean square deviation


Root mean square error


Rice straw


Rice straw hydrolysate


Sum of squares


Biomass concentration (grams per cubic liter)


Maximum biomass concentration (grams per cubic liter)


Biomass concentration (grams per cubic liter)


Product yield on the utilized substrate


Biomass yield on the utilized substrate



The authors acknowledge the Ministry of New and Renewable Energy for providing NRE fellowship to Ms. Amrita Ranjan. The infrastructural and analytical facilities provided by Centre for Energy and Department of Chemical Engineering, IIT Guwahati, and Spectrophotometric analysis facility provided by CIF, IIT Guwahati, are also acknowledged.


  1. 1.
    Cascone R (2008) Biobutanol—a replacement for bioethanol. Chem Eng Prog 104:S4–S9Google Scholar
  2. 2.
    Song H, Eom MH, Lee S, Lee J, Cho JH, Seung D (2010) Modeling of batch experimental kinetics and application to fed-batch fermentation of Clostridium tyrobutyricum for enhanced butyric acid production. Biochem Eng J 53:71–76CrossRefGoogle Scholar
  3. 3.
    Tran HTM, Cheirsilpa B, Hodgsonb B, Umsakulc K (2010) Potential use of Bacillus subtilis in a co-culture with Clostridium butylicum for acetone–butanol–ethanol production from cassava starch. Biochem Eng J 48:260–267CrossRefGoogle Scholar
  4. 4.
    Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101:209–228CrossRefGoogle Scholar
  5. 5.
    Ranjan A, Moholkar VS (2012) Biobutanol: science, engineering and economics. Int J Energ Res 39:277–323CrossRefGoogle Scholar
  6. 6.
    Schmidt FR (2005) Optimization and scale up of industrial fermentation processes. Appl Microbiol Biotechnol 68:425–435CrossRefGoogle Scholar
  7. 7.
    Leal MRLV, Walter AS, Seabra JEA (2012) Sugarcane as an energy source Biomass Conv. Bioref. doi: 10.1007/s13399-012-0055-1
  8. 8.
    Machado de Castro S, Machado de Castro A (2012) Assessment of the Brazilian potential for the production of enzymes for biofuels from agroindustrial materials. Biomass Conv Bioref 2:87–107CrossRefGoogle Scholar
  9. 9.
    Joensen F, Nielsen PEH, Sørensen MDP (2011) Biomass to green gasoline and power. Biomass Conv Bioref 1:85–90CrossRefGoogle Scholar
  10. 10.
    Surisetty VR, Kozinski J, Dalai AK (2012) Biomass, availability in Canada, and gasification: an overview. Biomass Conv Bioref 2:73–85CrossRefGoogle Scholar
  11. 11.
    Lenihan P, Orozco A, O’Neill O, Ahmad MNM, Rooney DW, Walker GM (2010) Dilute acid hydrolysis of lignocellulosic biomass. Chem Eng J 15:395–403CrossRefGoogle Scholar
  12. 12.
    Orozco A, Ahmad M, Rooney D, Walker G (2007) Dilute acid hydrolysis of cellulose and cellulosic bio-waste using a microwave reactor system. Proc Safety Environ Protection 85:446–449CrossRefGoogle Scholar
  13. 13.
    Cheng L, Keener TC, Lee JY, Zhou X (2012) Dilute acid pretreatment for cellulosic alcohol production. Biomass Conv Bioref 2:169–177CrossRefGoogle Scholar
  14. 14.
    Manzanares P, Ballesteros I, Negro MJ, Oliva JM, Gonzalez A, Ballesteros M (2012) Biological conversion of forage sorghum biomass to ethanol by steam explosion pretreatment and simultaneous hydrolysis and fermentation at high solid content. Biomass Conv Bioref 2:123–132CrossRefGoogle Scholar
  15. 15.
    Chiaramonti D, Rizzo AM, Prussi M, Tedeschi S, Zimbardi F, Braccio G, Viola E, Pardelli PT (2011) 2nd generation lignocellulosic bioethanol: is torrefaction a possible approach to biomass pretreatment? Biomass Conv Bioref 1:9–15CrossRefGoogle Scholar
  16. 16.
    Thiry M, Cingolani D (2002) Optimizing scale-up fermentation processes. Trends Biotechnol 20:103–105CrossRefGoogle Scholar
  17. 17.
    Stanbury PF, Whitakar A, Hall SJ (1997) Principles of fermentation technology. Aditya Books, New DelhiGoogle Scholar
  18. 18.
    Panda BP, Ali M, Javed S (2007) Fermentation process optimization. Res J Microbiol 2:201–208CrossRefGoogle Scholar
  19. 19.
    Elizalde-González MP, García-Díaz LE (2010) Application of a Taguchi L16 orthogonal array for optimizing the removal of Acid Orange 8 using carbon with a low specific surface area. Chem Eng J 163:55–61CrossRefGoogle Scholar
  20. 20.
    García IG, Martín AM, Ruiz JMO, Pérez AC (1989) Kinetic study of the production of ethanol with Saccharomyces cerevisiae immobilized on Berl saddles. Chem Eng J 42:B1–B7CrossRefGoogle Scholar
  21. 21.
    Dasu VV, Panda T, Chidambaram M (2003) Determination of significant parameters for improved griseofulvin production in a batch bioreactor by Taguchi’s method. Proc Biochem 38:877–880CrossRefGoogle Scholar
  22. 22.
    Chang MY, Tsai GJ, Houng JY (2006) Optimization of the medium composition for the submerged culture of Ganoderma lucidum by Taguchi array design and steepest ascent method. Enz Microb Tech 38:407–414CrossRefGoogle Scholar
  23. 23.
    Oskouie SFG, Tabandeh F, Yakhchali B, Eftekhar F (2007) Enhancement of alkaline protease production by Bacillus clausii using Taguchi experimental design. Afr J Biotechnol 6:2559–2564Google Scholar
  24. 24.
    Wu X, Yang H, Guo L (2010) Effect of operation parameters on anaerobic fermentation using cow dung as a source of microorganisms. Int J Hyd Energ 35:46–51CrossRefGoogle Scholar
  25. 25.
    Sanjari M, Taheri AK, Movahedi MR (2009) An optimization method for radial forging process using ANN and Taguchi method. Int J Adv Manuf Tech 40:776–784CrossRefGoogle Scholar
  26. 26.
    Hedge JE, Hofreiter BT (1962) In Whistler RL, Miller JN (ed.) Carbohydrate chemistry. Academic Press, New York, p 17Google Scholar
  27. 27.
    Miller GL (1972) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  28. 28.
    Palaniraj RI, Nagarajan P (2012) Statistical analysis of experimental variables for the production of lactic acid using Lactobacillus casei from waste potato starch by Box-Behnken design. Int J Chem Tech Res 4:1049–1064Google Scholar
  29. 29.
    Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridium acetobutylicum. Appl Environ Microbiol 50:1165Google Scholar
  30. 30.
    Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524Google Scholar
  31. 31.
    Khamaiseh EI, Kalil MS, Dada O, El-Shawabkeh I, Yusoff WMW (1012) Date fruit as carbon source in RCM-modified medium to produce biobutanol by Clostridium acetobutylicum NCIMB 13357. J Appl Sci 12:1160–1165Google Scholar
  32. 32.
    Boon-Long S, Laguerie C, Coudere JP (1978) Mass transfer from suspended solids to a liquid in agitated vessels. Chem Eng Sci 33:813CrossRefGoogle Scholar
  33. 33.
    Venil CK, Lakshmanaperumalsamy P (2009) Taguchi experimental design for medium optimization for enhanced production by Bacillus subtilis HB04. eJST 4:1–10Google Scholar
  34. 34.
    Sirisansaneeyakul S, Luangpipat T, Vanichsriratana W, Srinophakum T, Chen HHH, Chisti Y (2007) Optimization of lactic acid production by immobilized Lactococcus lactis IO-1. J Ind Microbiol Biotechnol 34:381–391CrossRefGoogle Scholar
  35. 35.
    Marchal R, Blanchet D, Vandecasteele JP (1985) Industrial optimization of acetone-butanol fermentation: a study of the utilization of Jerusalem artichokes. Appl Microbiol Biotechnol 23:92–98CrossRefGoogle Scholar
  36. 36.
    Marchal R, Ropars M, Pourqui J, Fayolle E, Vandecasteele JE (1992) Large-scale enzymatic hydrolysis of agricultural lignocellulosic biomass. part 2: conversion into acetone-butanol. Bioresourc Technol 42:205–217CrossRefGoogle Scholar
  37. 37.
    Badr HR, Hamdy MK (1992) Optimization of acetone-butanol production using response surface methodology. Biomass Bioenerg 3:49–55CrossRefGoogle Scholar
  38. 38.
    Syed Q, Nadeem M, Nelofer R (2008) Enhanced butanol production by mutant strains of Clostridium acetobutylicum in molasses medium. Turk J Biochem 33:25–30Google Scholar
  39. 39.
    Jieun L, Sen K, Kweon D, Park K, Jin Y (2009) Fermentation of rice bran and defatted rice bran for butanol production using Clostridium beijrinckii ncimb 8052. J Microbial Biotechnol 19:482–490CrossRefGoogle Scholar
  40. 40.
    Marianoa AP, Costaa CBB, Angelisc DF, de Filhob FM, Atalab DIP, Maciela MRW, Filho RM (2010) Optimisation of a continuous flash fermentation for butanol production using the response surface methodology. Chem Eng Res Design 88:562–571CrossRefGoogle Scholar
  41. 41.
    Liu Z, Li YYF, Ma C, Xu P (2010) Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran. J Ind Microbiol Biotechnol 37:495–501CrossRefGoogle Scholar
  42. 42.
    Moon C, Lee CH, Sang B, Um Y (2011) Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum. Bioresourc Technol 102:10561–10568CrossRefGoogle Scholar
  43. 43.
    Wang Y, Blaschek HP (2011) Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052. Bioresourc Technol 102:9985–9990CrossRefGoogle Scholar
  44. 44.
    Wang L, Chen H (2011) Increased fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production by removal of fermentation inhibitors. Proc Biochem 46:604–607CrossRefGoogle Scholar
  45. 45.
    Isar J, Rangaswamy V (2012) Improved n-butanol production by solvent tolerant Clostridium beijerinckii. Biomass Bioenerg 37:9–15CrossRefGoogle Scholar
  46. 46.
    Lin Y, Wang J, Wang X, Sun X (2011) Optimization of butanol production from corn straw hydrolysate by Clostridium acetobutylicum using response surface method. Chinese Sci Bulletin 56:1422–1428CrossRefGoogle Scholar
  47. 47.
    Tran HTM, Cheirsilp B, Umsakul K, Bourtoom T (2011) Response surface optimisation for acetone-butanol-ethanol production from cassava starch by co-culture of Clostridium butylicum and Bacillus subtilis. Maejo Int J Sci Technol 5:374–389Google Scholar
  48. 48.
    Ranjan A, Moholkar VS (2011) Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel doi: 10.1016/j.fuel.2011.03.030.
  49. 49.
    Mercier P, Yerushalmi L, Rouleau D, Dochain D (1992) Kinetics of lactic acid fermentation on glucose and corn by Lactobacillus amylophilus. J Chem Tech Biotech 55:111–121CrossRefGoogle Scholar
  50. 50.
    Rodrigues L, Moldes A, Teixeira J, Oliveira R (2006) Kinetic study of fermentative biosurfactant production by Lactobacillus strains. Biochem Eng J 28:109–116CrossRefGoogle Scholar
  51. 51.
    Cheng C, Che P, Chen B, Lee W, Chien LJ, Chang JS (2012) High yield bio-butanol production by solvent-producing bacterial microflora. Bioresourc Technol 113:58–64CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Amrita Ranjan
    • 1
  • Rahul Mayank
    • 2
  • Vijayanand S. Moholkar
    • 1
    • 2
    Email author
  1. 1.Centre for EnergyIndian Institute of Technology GuwahatiGuwahatiIndia
  2. 2.Department of Chemical EngineeringIndian Institute of Technology GuwahatiGuwahatiIndia

Personalised recommendations