Abstract
Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.
Similar content being viewed by others
References
Aguilera F, Peinado RA, Millan C, Ortega JM, Mauricio JC (2006) Relationship between ethanol tolerance, H+-ATPase activity and the lipid composition of the plasma membrane in different wine yeast strains. Int J Food Microbiol 110:34–42
Alsaker KV, Spitzer TR, Papoutsakis ET (2004) Transcriptional analysis of spo0A over expression in Clostridium acetobutylicum and its effect on the cell's response to butanol stress. J Bacteriol 186:1959–1971
Annous BA, Blaschek HP (1991) Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity. Appl Environ Microbiol 57:2544–2548
Ashe MP, Slaven JW, De Long SK, Ibrahimo S, Sachs AB (2001) A novel eIF2B-dependent mechanism of translational control in yeast as a response to fuel alcohols. EMBO J 20:6464–6474
Atsumi S, Liao JC (2008a) Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Appl Environ Microbiol 74:7802–7808
Atsumi S, Liao JC (2008b) Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol 19:414–419
Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC (2008a) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10:305–311
Atsumi S, Hanai T, Liao JC (2008b) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86–89
Baer SH, Blaschek HP, Smith TL (1987) Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol-tolerant Clostridium acetobutylicum. Appl Environ Microbiol 53:2854–2861
Barber JM, Robb FT, Webster JR, Woods DR (1979) Bacteriocin production by Clostridium acetobutylicum in an industrial fermentation process. Appl Environ Microbiol 37:433–437
Bermejo LL, Welker NE, Papoutsakis ET (1998) Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification. Appl Environ Microbiol 64:1079–1085
Booth I, Morris JG (1975) Proton motive force in the obligately anaerobic bacterium Clostridium pasteurianum- a role in galactose and gluconate uptake. FEBS Letters 59:153–157
Borden JR, Papoutsakis ET (2007) Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum. Appl Environ Microbiol 73:3061–3068
Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridium acetobutylicum. Appl Environ Microbiol 50:1165–1170
Brynildsen MP, Liao JC (2009) An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol Syst Biol 5:277
Cann AF, Liao JC (2008) Production of 2-methyl-1-butanol in engineered Escherichia coli. Appl Microbiol Biotechnol 81:89–98
Chen CK, Blaschek HP (1999) Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101. Appl Microbiol Biotechnol 52:170–173
Connor MR, Liao JC (2008) Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 74:5769–5775
Demuez M, Cournac L, Guerrini O, Soucaille P, Girbal L (2007) Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners. FEMS Microbiol Lett 275:113–121
Durre P (2008) Fermentative butanol production: bulk chemical and biofuel. Ann NY Acad Sci 1125:353–362
Ennis BM, Qureshi N, Maddox IS (1987) Inline toxic product removal during solvent production by continuous fermentation using immobilized Clostridium acetobutylicum. Enzyme Microb Technol 9:672–675
Evans PJ, Wang HW (1988) Enhancement of butanol fermentation by Clostridium acetobutylicum in the presence of decanol-oleyl alcohol mixed extractants. Appl Environ Microbiol 54:1662–1667
Ezeji TC, Blaschek HP (2007) Biofuel from butanol: advances in genetic and physiological manipulation of clostridia. BioWorld Europe 2:12–15
Ezeji TC, Li Y (2009) Advanced product recovery technologies. In: Vertes A, Qureshi N, Yukawa H, Blaschek H (eds) Biomass to biofuel. Wiley, Hoboken, in press
Ezeji TC, Qureshi N, Blaschek (2003) Production of butanol by Clostridium beijerinckii BA101 and in-situ recovery by gas stripping. World J Microbiol Biotechnol 19:595–603
Ezeji TC, Qureshi N, Blaschek HP (2004a) Acetone-butanol-ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping. Appl Microbiol Biotechnol 63:653–658
Ezeji TC, Qureshi N, Blaschek HP (2004b) Butanol fermentation research: upstream and downstream manipulations. Chem Rec 4:305–314
Ezeji TC, Qureshi N, Karcher PM, Blaschek HP (2005a) Improving the performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation. Bioprocess Biosystems Eng 27:207–214
Ezeji TC, Qureshi N, Blaschek HP (2005b) Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101. J Biotechnol 115:179–187
Ezeji TC, Qureshi N, Karcher P, Blaschek HP (2006) Butanol production from corn. In: Minteer SD (ed) Chapter in “Alcoholic Fuels: Fuels for Today and Tomorrow”. Taylor & Francis (Taylor & Francis Group), New York, pp 99–122
Ezeji TC, Qureshi N, Blaschek HP (2007a) Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnol Bioeng 97:1460–1469
Ezeji TC, Qureshi N, Blaschek HP (2007b) Production of acetone butanol ethanol (ABE) in a continuous flow bioreactor using degermed corn and Clostridium beijerinckii. Process Biochem 42:34–39
Ezeji TC, Qureshi N, Blaschek HP (2007c) Bioproduction of butanol from biomass: from genes to bioreactors. Curr Opin Biotechnol 18:220–227
Fischer CR, Klein-Marcuschamer D, Stephanopoulos G (2008) Selection and optimization of microbial hosts for biofuels production. Metab Eng 10:295–304
Garcia A, Iannotti EL, Fischer JL (1986) Butanol fermentation liquor production and separation by reverse osmosis. Biotechnol Bioeng 28:785–791
Green EM, Boynton ZL, Harris LM, Ruldolph FB, Papoutsakis ET, Bennett GN (1996) Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology 142:2079–2086
Groot WJ, van den Oever CE, Kossen NWF (1984) Pervaporation for simultaneous product recovery in the butanol/isopropanol batch fermentation. Biotechnol Lett 6:709–714
Guerrini O, Burlat B, Léger C, Guigliarelli B, Soucaille P, Girbal L (2008) Characterization of Two 2[4Fe4S] Ferredoxins from Clostridium acetobutylicum. Curr Microbiol 56:261–267
Hanai T, Atsumi S, Liao JC (2007) Engineered synthetic pathway for isopropanol production in Escherichia coli. Appl Environ Microbiol 73:7814–7818
Harris LM, Desai RP, Welker NE, Papoutsakis ET (2000) Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition. Biotechnol Bioeng 67:1–11
Harris LM, Blank L, Desai RP, Welker NE, Papoutsakis ET (2001) Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene. J Ind Microbiol Biotechnol 27:322–328
Hermann M, Fayolle F, Marchal R, Podvin L, Sebald M, Vandecasteele JP (1985) Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum. Appl Environ Microbiol 50:1238–1243
Herrero AA (1983) End-product inhibition in anaerobic fermentation. Trends Biotechnol 1:49–53
Herrero AA, Gomez RF, Roberts MF (1985) 3lP NMR studies of Clostridium thermocellum. Mechanism of end product inhibition by ethanol. J Biol Chem 260:7442–7451
Hu XH, Wang MH, Tan T, Li JR, Yang H, Leach L, Zhang RM, Luo ZW (2007) Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cervisiae. Genetics 175:1479–1487
Huang H.-J, Ramaswamy S, Tschirner UW, Ramarao BV (2008) A review of separation technologies in current and future biorefineries. Separations Purifications Technol 62:1–21
Hutkins RW, Kashket ER (1986) Phosphotransferase activity in Clostridium acetobutylicum from acedogenic and solvetogenic phases of growth. Appl Environ Microbiol 51:1121–1123
Ingram LO (1976) Adaptation of membrane lipids to alcohols. J Bacteriol 125:670–678
Ingram LO (1990) Ethanol tolerance in bacteria. Crit Rev Biotechnol 9:305–319
Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524
Karcher P, Ezeji TC, Qureshi N, Blaschek HP (2005) Microbial production of butanol: product recovery by extraction. In: Satyanarayana T, Johri BN (eds) Chapter in Microbial Diversity: current perspectives and potential applications. IK International Publishing House Pvt Ltd, New Delhi, pp 865–880
Knoshaug EP, Zhang M (2009) Butanol tolerance in a selection of microorganisms. Appl Biochem Biotechnol 153(1–3):13–20
Lee J, Mitchell WJ, Blashek HP (2001) Glucose uptake in Clostridium beijerinkii NIMB 8052 and the solvent-hyperproducing mutant BA101. Appl Environ Microbiol 67:5025–5031
Lee J, Mitchell WJ, Tangney M, Blaschek HP (2005) Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101. Appl Environ Microbiol 71:3384–3387
Lepage C, Fayolle F, Hermann M, Vandecasteele JP (1987) Changes in membrane lipid composition of Clostridium acetobutylicum during acetone-butanol fermentation: effects of solvents, growth temperature and pH. J Gen Microbiol 133:103–110
Lin YL, Blaschek HP (1983) Butanol production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth. Appl Environ Microbiol 45:966–973
Lin YL, Blaschek HP (1993) butanol production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth. Appl Environ Microbiol 45:966–973
Linden JC, Moreira A (1982) Anaerobic production of chemicals. In: Hollaender A (ed) Chapter in “Basic biology of new developments in biotechnology (Basic Life Sciences 25)”. Plenum Press, New York, pp 377–403
Liyanage H, Young M, Kashket ER (2000) Butanol tolerance of Clostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA. J Mol Microbiol Biotechnol 2:87–93
Lovitt RW, Longin R, Zeikus JG (1984) Ethanol production by thermophilic bacteria: physiological comparison of solvent effects on parent and alcohol-tolerant strains of Clostridium thermohydrosulfuricum. Appl Environ Microbiol 48:171–177
Maddox IS (1989) The acetone–butanol–ethanol fermentation: recent progress in technology. Biotechnol Genetic Eng Rev 7:190–220
Mermelstein LD, Welker NE, Petersen DJ, Bennett GN, Papoutsakis ET (1994) Genetic and metabolic engineering of Clostridium acetobutylicum ATCC 824. Ann NY Acad Sci 721:54–68
Meyer CL, Roos JW, Papoutsakis ET (1986) Carbon monoxide gassing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures of Clostridium acetobutylicum. Appl Microbiol Biotechnol 24:159–167
Michel GP, Starka J (1986) Effect of ethanol and heat stresses on the protein pattern of Zymolnonas mobilis. J Bacteriol 165:1040–1042
Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by clostridia. Adv Microb Physiol 39:31–130
Mitchell WJ, Booth IR (1984) Characterization of the Clostridium pasteurianum phophotransferase system. J Gen Microbiol 130:2193–2200
Mitchell WJ, Tangney M (2005) Carbohydrate uptake by the phosphotransferase system and other mechanisms. In: Durre P (ed) Handbook on Clostridia Chapter 8. Taylor and Francis, New York, pp 155–175
Mitchell WJ, Shaw JE, Andrews L (1991) Properties of glucose phophotransferase system of Clostridium acetobutylicum NCIMB 8052. Appl Environ Microbiol 57:2354–2539
Monot F, Engasser JM, Petitdemage H (1984) Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum. Appl Microbiol Biotechnol 19:422–426
Moreira AR, Ulmer D, Linden JC (1981) Butanol toxicity in the butylic fermentation. Biotechnol Bioeng Symp 11:567–579
Nielsen DR, Yoon LE, S-H TH-C, Yuan C, Prather KLJ (2009) Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 11:262–273
Nielson L, Larsson M, Holst O, Mattiasson B (1988) Adsorbents for extractive bioconversion applied to the acetone butanol fermentation. Appl Microbiol Biotechnol 28:335–339
Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR (2001) Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183:4823–4838
Okamoto H, Sone N, Hirata H, Yoshida M, Kagawa Y (1977) Purified proton conductor in proton translocating adenosine triphosphatase of a thermophilic bacterium. J Biol Chem 252:6125–6131
Osman YA, Ingram LO (1985) Mechanism of ethanol inhibition of fermentation in Zymomonas mobilis CP4. J Bacteriol 164:173–180
Ounine K, Petitdamage H, Raval G, Gay R (1985) Regulation and butanol inhibition of D-Xylose and D-Glucose uptake in Clostridium acetobutylicum. Appl Environ Microbiol 49:874–878
Peguin S, Soucaille P (1995) Modulation of carbon and electron flow in Clostridium acetobutylicum by iron limitation and methyl viologen addition. Appl Environ Microbiol 61:403–405
Petitdemange H, Cherrier C, Raval G, Gay R (1976) Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group. Biochim Biophys Acta 421:334–347
Phillips JA, Humphrey AE (1983) An overview of process technology for the production of liquid fuels and chemical feedstocks via fermentation. In: Wise DL (ed) Organic chemicals from biomass. Benjamins/Cummings Publishing, Menlo Park, Calif, pp 249–304
Quinkal I, Davasse V, Gaillard J, Moulis JM (1994) On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin. Protein Eng 7:681–687
Qureshi N, Blaschek HP (1999) Butanol recovery from model solution/fermentation broth by pervaporation: Evaluation of membrane performance. Biomass Bioenergy 17:175–184
Qureshi N, Blaschek HP (2000) Economics of butanol fermentation using hyper-butanol producing Clostridium beijerinckii BA101. Trans IChemE 78(Part C):139–144
Qureshi N, Blaschek HP (2001) Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101. J Ind Microbiol Biotechnol 27:287–291
Qureshi N, Ezeji TC (2008) Butanol (a superior biofuel) production from agricultural residues (renewable biomass): Recent progress in technology. Biofuels Bioprod Bioref 2:319–330
Qureshi N, Maddox IS, Friedl A (1992) Application of continuous substrate feeding to the ABE fermentation: relief of product inhibition using extraction, perstraction, stripping and pervaporation. Biotechnol Prog 8:382–390
Qureshi N, Meagher MM, Hutkins RW (1999) Recovery of butanol from model solutions and fermentation broth using a silicalite/silicone membrane. J Memb Sci 158:115–125
Ramos JL, Duque E, Gallegos M-T, Godoy P, Ramos-González MI, Rojas A, Terán W, Segura A (2002) Mechanisms of solvent tolerance in gram-negative bacteria. Annu Rev Microbiol 56:743–768
Rao G, Mutharasan R (1987) Altered electron flow in continuous cultures of Clostridium acetobutylicum Induced by Viologen Dyes. Appl Environ Microbiol 53:1232–1235
Riebeling V, Jungermann K (1976) Properties and function of Clostridial membrane ATPase. Biochim Biophys Acta 430:434–444
Riebeling V, Thauer RK, Jungermann K (1975) The internal alkaline pH gradient, sensitive to uncoupler and ATP ase inhibitor, in growing Clostridium pasteurianum. Eur J Biochem 55:445–453
Roffler SR, Blanch HW, Wilke CR (1987) In-situ recovery of butanol during fermentation: Part 2. Fed-batch extractive fermentation. Bioproc Eng 2:181–190
Ruhl J, Schmid A, Blank ML (2009) Selected Pseudomonas putida strains able to grow in the presence of high butanol concentrations. Appl Environ Microbiol 75:4653–4656
Russell JB, Diez-Gonzalez F (1998) The effects of fermentation acids on bacterial growth. Adv Microbial Physiol 39:205–234
Saier MH, Stiles CD (1975) Molecular dynamics in biological membranes. Springer-Verlag, New York
Schwarz WH, Slattery M, Gapes RJ (2007) The ABC of ABE. BioWorld Europe 2:8–10
Shao P, Huang RYM (2007) Polymeric membrane pervaporation. J Membr Sci 287:162–179
Shi S, Blaschek HP (2008) Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 and the hyper-butanol-producing mutant BA101 during the shift from acidogenesis to solventogenesis. Appl Environ Microbiol 2008(74):7709–7714
Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD (2008) Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact 7:36
Tashiro Y, Shinto H, Hayashi M, Baba S, Kobayashi G, Sonomoto K (2007) Novel high-efficient butanol production from butyrate by non-growing Clostridium saccharoperbutyl acetonicum N1–4 (ATCC 13564) with methyl viologen. J Biosci Bioeng 104:238–240
Taylor M, Tuffin M, Burton S, Eley K, Cowan D (2008) Microbial responses to solvent and alcohol stress. Biotechnol J 3:1388–1397
Terracciano JS, Kashket ER (1986) Intracellular conditions required for initiation of solvent production by Clostridium acetobutylicum. Appl Environ Microbiol 52:96–91
Terracciano JS, Rapaport E, Kashket ER (1988) Stress- and growth phase-associated proteins of Clostridium acetobutylicum. Appl Environ Microbiol 54:1989–1995
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 69:4951–4965
Tomas CA, Beamish J, Papoutsakis ET (2004) Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J Bacteriol 186:2006–2018
Traxler RW, Wood EM, Mayer J, Wilson MP (1985) Extractive fermentation for the production of butanol. Dev Ind Microbiol 36:519–525
Van Der Westhuizen A, Jones DT, Woods DR (1982) Autolytic activity and butanol tolerance of Clostridium acetobutylicum. Appl Environ Microbiol 44:1277–1281
Vane A (2005) Review of pervaporation for product recovery from biomass fermentation processes. J Chem Technol Biotechnol 80:603–629
Vollherbst-Schneck K, Sands JA, Montenecourtv (1984) Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 47:193–194
Yan RT, Zhu CX, Golemboski C, Chen JS (1988) Expression of solvent-forming enzymes and onset of solvent production in batch cultures of Clostridium beijerinckii (“Clostridium butylicum”). Appl Environ Microbiol 54:642–648
Zhao Y, Hindorff LA, Chuang A, Monroe AM, Lyristis M, Harrison ML, Rudolph FB, Bennett GN (2003) Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 69:2831–2841
Acknowledgements
This work was supported by funding from Northeast Sungrant (Cornell University) Award/Contract number GRT00012344, National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2006-35504-17419, NSF CAREER award (NDP) to Nathan Price, and Seed grant from Ohio Agricultural Research and Development Center (OARDC), Wooster.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ezeji, T., Milne, C., Price, N.D. et al. Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms. Appl Microbiol Biotechnol 85, 1697–1712 (2010). https://doi.org/10.1007/s00253-009-2390-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-009-2390-0