Skip to main content
SpringerLink
Log in

Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor

  • Bioenergy/Biofuels/Biochemicals - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD620 of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h−1 OD−1 L−1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Aakvik T, Lale R, Liles M, Valla S (2011) Metagenomic libraries for functional screening. In: de Bruijn F (ed) Handbook of molecular microbial ecology I, 1st edn. Wiley-Blackwell, Hoboken, New Jersey, pp 171–181. doi:10.1002/9781118010518.ch22

    Chapter  Google Scholar 

  2. Alterthum F, Ingram LO (1989) Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli. Appl Environ Microbiol 55:1943–1948

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Archer CT, Kim JF, Jeong H, Park JH, Vickers CE, Lee SY, Nielsen LK (2011) The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genom 12:1–20. doi:10.1186/1471-2164-12-9

    Article  Google Scholar 

  4. Beloqui A (2006) Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships. J Biol Chem 281:22933–22942. doi:10.1074/jbc.M600577200

    Article  CAS  PubMed  Google Scholar 

  5. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: signalP 3.0. J Mol Biol 340:783–795. doi:10.1016/j.jmb.2004.05.028

    Article  PubMed  Google Scholar 

  6. Bondioli P, Della Bella L (2005) An alternative spectrophotometric method for the determination of free glycerol in biodiesel. Eur J Lipid Sci Technol 107:153–157

    Article  CAS  Google Scholar 

  7. Cintolesi A, Clomburg JM, Rigou V, Zygourakis K, Gonzalez R (2012) Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 109:187–198. doi:10.1002/bit.23309

    Article  CAS  PubMed  Google Scholar 

  8. Ciriminna R, Pina CD, Rossi M, Pagliaro M (2014) Understanding the glycerol market. Eur J Lipid Sci Technol 116:1432–1439

    Article  CAS  Google Scholar 

  9. Clomburg JM, Gonzalez R (2013) Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol 31:20–28. doi:10.1016/j.tibtech.2012.10.006

    Article  CAS  PubMed  Google Scholar 

  10. Cofré O, Ramírez M, Gómez JM, Cantero D (2012) Optimization of culture media for ethanol production from glycerol by Escherichia coli. Biomass Bioenergy 37:275–281. doi:10.1016/j.biombioe.2011.12.002

    Article  Google Scholar 

  11. Chaudhary N, Ngadi MO, Simpson BK, Kassama LS (2011) Biosynthesis of ethanol and hydrogen by glycerol fermentation using Escherichia coli. Adv Chem Eng Sci 1:83–89. doi:10.4236/aces.2011.13014

    Article  CAS  Google Scholar 

  12. Dharmadi Y, Murarka A, Gonzalez R (2006) Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng 94:821–829. doi:10.1002/bit.21025

    Article  CAS  PubMed  Google Scholar 

  13. Durnin G, Clomburg J, Yeates Z, Alvarez PJ, Zygourakis K, Campbell P, Gonzalez R (2009) Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 103:148–161. doi:10.1002/bit.22246

    Article  CAS  PubMed  Google Scholar 

  14. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov MM, Timmis KN, Golyshin PN (2005) Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ Microbiol 7:1996–2010. doi:10.1111/j.1462-2920.2005.00920.x

    Article  CAS  PubMed  Google Scholar 

  15. Finn RD, Clements J, Eddy SR (2011) HMMER web server: interactive sequence similarity searching. Nucl Acids Res 39(suppl 2):W29–W37. doi:10.1093/nar/gkr367

  16. Geddes R, Shanmugam KT, Ingram LO (2015) Combining treatments to improve the fermentation of sugarcane bagasse hydrolysates by ethanologenic Escherichia coli LY180. Bioresour Technol 189:15–22. doi:10.1016/j.biortech.2015.03.141

    Article  CAS  PubMed  Google Scholar 

  17. Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS (2008) A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab Eng 10:234–245. doi:10.1016/j.ymben.2008.05.001

    Article  CAS  PubMed  Google Scholar 

  18. Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68:669–685. doi:10.1128/mmbr.68.4.669-685.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hu H, Wood TK (2010) An evolved Escherichia coli strain for producing hydrogen and ethanol from glycerol. Biochem Biophys Res Commun 391:1033–1038. doi:10.1016/j.bbrc.2009.12.013

    Article  CAS  PubMed  Google Scholar 

  20. Jarboe L, Grabar T, Yomano L, Shanmugan K, Ingram L (2007) Development of ethanologenic bacteria. Adv Biochem Engin Biotechnol 108:237–261. doi:10.1007/10_2007_068

    CAS  Google Scholar 

  21. Jarboe LR, Grabar TB, Yomano LP, Shanmugan KT, Ingram LO (2007) Development of ethanologenic bacteria. In: Olsson L (ed) Biofuels. Springer, Berlin, Heidelberg, pp 237–261. doi:10.1007/10_2007_068

    Chapter  Google Scholar 

  22. Johnson DT, Taconi KA (2007) The glycerin glut: options for the value-added conversion of crude glycerol resulting from biodiesel production. Environ Prog 26:338–348. doi:10.1002/ep.10225

    Article  CAS  Google Scholar 

  23. Krogh A, Larsson B, Von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580

    Article  CAS  PubMed  Google Scholar 

  24. Lai E (2014) Biodiesel: environmental friendly alternative to petrodiesel. J Pet Environ Biotechnol 5:e122

    Google Scholar 

  25. Lee SJ, Kim SB, Kang SW, Han SO, Park C, Kim SW (2012) Effect of crude glycerol-derived inhibitors on ethanol production by Enterobacter aerogenes. Bioprocess Biosyst Eng 35:85–92. doi:10.1007/s00449-011-0607-y

    Article  CAS  PubMed  Google Scholar 

  26. Loaces I, Amarelle V, Muñoz-Gutierrez I, Fabiano E, Martinez A, Noya F (2015) Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation. Appl Microbiol Biotechnol 99:9049–9060. doi:10.1007/s00253-015-6801-0

    Article  CAS  PubMed  Google Scholar 

  27. Macrelli S, Mogensen J, Zacchi G (2012) Techno-economic evaluation of 2nd generation bioethanol production from sugar cane bagasse and leaves integrated with the sugar-based ethanol process. Biotechnol Biofuels 5:1–18. doi:10.1186/1754-6834-5-22

    Article  Google Scholar 

  28. Martínez-Gómez K, Flores N, Castañeda HM, Martínez-Batallar G, Hernández-Chávez G, Ramírez OT, Gosset G, Encarnación S, Bolivar F (2012) New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol. Microb Cell Fact 11:46

    Article  PubMed  PubMed Central  Google Scholar 

  29. Matthew S, Wong ML, Black Ryan W, Le Thao Q, Puthli Sharon, Campbell Paul, Monticello Daniel J (2014) Microaerobic conversion of glycerol to ethanol in Escherichia coli. Appl Environ Microbiol 80:3276–3282

    Article  Google Scholar 

  30. Meiswinkel TM, Rittmann D, Lindner SN, Wendisch VF (2013) Crude glycerol-based production of amino acids and putrescine by Corynebacterium glutamicum. Bioresour Technol 145:254–258. doi:10.1016/j.biortech.2013.02.053

    Article  CAS  PubMed  Google Scholar 

  31. Miller EN, Jarboe LR, Yomano LP, York SW, Shanmugam KT, Ingram LO (2009) Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli. Appl Environ Microbiol 75:4315–4323. doi:10.1128/AEM.00567-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R (2008) Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 74:1124–1135. doi:10.1128/AEM.02192-07

    Article  CAS  PubMed  Google Scholar 

  33. Nancy YY, Wagner JR, Laird MR, Melli G, Rey S, Lo R, Dao P, Sahinalp SC, Ester M, Foster LJ (2010) PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26:1608–1615

    Article  Google Scholar 

  34. Nguyen ADQ, Kim YG, Kim SB, Kim CJ (2013) Improved tolerance of recombinant Escherichia coli to the toxicity of crude glycerol by overexpressing trehalose biosynthetic genes (otsBA) for the production of β-carotene. Bioresour Technol 143:531–537. doi:10.1016/j.biortech.2013.06.034

    Article  CAS  Google Scholar 

  35. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    Article  CAS  PubMed  Google Scholar 

  36. Nikel PI, Giordano AM, de Almeida A, Godoy MS, Pettinari MJ (2010) Elimination of d-Lactate synthesis increases poly(3-Hydroxybutyrate) and ethanol synthesis from glycerol and affects cofactor distribution in recombinant Escherichia coli. Appl Environ Microbiol 76:7400–7406. doi:10.1128/aem.02067-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nikel PI, Ramirez MC, Pettinari MJ, Méndez BS, Galvagno MA (2010) Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides. J Appl Microbiol 109:492–504. doi:10.1111/j.1365-2672.2010.04668.x

    CAS  PubMed  Google Scholar 

  38. Noguchi H, Park J, Takagi T (2006) MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic Acids Res 34:5623–5630. doi:10.1093/nar/gkl723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 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 79:1189–1196. doi:10.1002/jctb.1103

    Article  CAS  Google Scholar 

  40. Patil KR, Roune L, McHardy AC (2012) The phylopythias web server for taxonomic assignment of metagenome sequences. PLoS One 7:e38581

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD (2012) The Pfam protein families database. Nucleic Acids Res 40:D290–D301. doi:10.1093/nar/gkr1065

    Article  CAS  PubMed  Google Scholar 

  42. Purushe J, Fouts DE, Morrison M, White BA, Mackie RI, Coutinho PM, Henrissat B, Nelson KE (2010) Comparative genome analysis of Prevotella ruminicola and Prevotella bryantii: insights into their environmental niche. Microb Ecol 60:721–729. doi:10.1007/s00248-010-9692-8

    Article  PubMed  Google Scholar 

  43. Rodríguez MC, Loaces I, Amarelle V, Senatore D, Iriarte A, Fabiano E, Noya F (2015) Est10: a novel alkaline esterase isolated from bovine rumen belonging to the new family XV of lipolytic enzymes. PLoS One 10:e0126651. doi:10.1371/journal.pone.0126651

    Article  PubMed  PubMed Central  Google Scholar 

  44. Santero E, Floriano B, Govantes F (2016) Harnessing the power of microbial metabolism. Curr Opin Microbiol 31:63–69. doi:10.1016/j.mib.2016.03.003

    Article  CAS  PubMed  Google Scholar 

  45. Sarma SJ, Brar SK, Sydney EB, Le Bihan Y, Buelna G, Soccol CR (2012) Microbial hydrogen production by bioconversion of crude glycerol: a review. Int J Hydrog Energy 37:6473–6490. doi:10.1016/j.ijhydene.2012.01.050

    Article  CAS  Google Scholar 

  46. Sawers R, Blokesch M, Böck A (2004) Anaerobic formate and hydrogen metabolism. EcoSal Plus 2004. doi:10.1128/ecosalplus.3.5.4

  47. Senthilkumar V, Gunasekaran P (2005) Bioethanol production from cellulosic substrates: engineered bacteria and process integration challenges. J Sci Ind Res 64:845–853

    CAS  Google Scholar 

  48. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi:10.1093/molbev/mst197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Trinh CT, Srienc F (2009) Metabolic engineering of Escherichia coli for efficient conversion of glycerol to ethanol. Appl Environ Microbiol 75:6696–6705. doi:10.1128/aem.00670-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Valle A, Cabrera G, Cantero D, Bolivar J (2015) Identification of enhanced hydrogen and ethanol Escherichia coli producer strains in a glycerol-based medium by screening in single-knock out mutant collections. Microb Cell Factories 14:93

    Article  Google Scholar 

  51. Valle A, Cabrera G, Muhamadali H, Trivedi DK, Ratray NJW, Goodacre R, Cantero D, Bolivar J (2015) A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source. Biotechnol J 10:1750–1761. doi:10.1002/biot.201500005

    Article  CAS  PubMed  Google Scholar 

  52. Wu S, Zhu Z, Fu L, Niu B, Li W (2011) WebMGA: a customizable web server for fast metagenomic sequence analysis. BMC Genom 12:444

    Article  Google Scholar 

  53. Yang F, Hanna MA, Sun R (2012) Value-added uses for crude glycerol–a byproduct of biodiesel production. Biotechnol Biofuels 5:13. doi:10.1186/1754-6834-5-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Yazdani S, Gonzalez R (2008) Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products. Metabolic Eng 10:340–351. doi:10.1016/j.ymben.2008.08.005

    Article  CAS  Google Scholar 

  55. Yazdani SS, Gonzalez R (2007) Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opini Biotechnol 18:213–219. doi:10.1016/j.copbio.2007.05.002

    Article  CAS  Google Scholar 

  56. Yomano L, York S, Ingram L (1998) Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J Ind Microbiol Biotechnol 20:132–138

    Article  CAS  PubMed  Google Scholar 

  57. Yu KO, Kim SW, Han SO (2010) Engineering of glycerol utilization pathway for ethanol production by Saccharomyces cerevisiae. Bioresour Technol 101:4157–4161. doi:10.1016/j.biortech.2010.01.066

    Article  CAS  PubMed  Google Scholar 

  58. Zhang X, Shanmugam KT, Ingram LO (2010) Fermentation of glycerol to succinate by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol 76:2397–2401. doi:10.1128/aem.02902-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. doi:10.1089/10665270050081478

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are very grateful to Dr. Lonnie Ingram (University of Florida) for providing pLOI297 plasmid and LY180 strain, as well as to Dr. Liliana Borzacconi (Departamento de Ingeniería de Reactores, Facultad de Ingeniería, Universidad de la República) for providing purge sludges. Special thanks to Nikolai Guchin (ANCAP, Uruguay) and Darío Rodríguez (ALUR, Uruguay) for providing crude glycerol. This work was supported by Grants #FSE_2011_6383 from Agencia Nacional de Investigación e Innovación (ANII, Uruguay), by the Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay) and by Sistema Nacional de Investigadores (SNI, Uruguay).

Author information

Authors and Affiliations

  1. Departamento de Bioquímica y Genómica Microbianas, Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia 3318, 11600, Montevideo, Uruguay

    Inés Loaces, Cecilia Rodríguez, Vanesa Amarelle, Elena Fabiano & Francisco Noya

Authors
  1. Inés Loaces
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cecilia Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vanesa Amarelle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elena Fabiano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Francisco Noya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francisco Noya.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Loaces, I., Rodríguez, C., Amarelle, V. et al. Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor. J Ind Microbiol Biotechnol 43, 1405–1416 (2016). https://doi.org/10.1007/s10295-016-1818-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-016-1818-7

Keywords

Search

Navigation