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Bacterial production of free fatty acids from freshwater macroalgal cellulose

  • Bioenergy and Biofuels
  • Published:
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Abstract

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl–acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 μM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 μg/mL FFA) cultures grown on rich Luria–Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.

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References

  • Akerholm M, Hinterstoisser B, Salmen L (2004) Characterization of the crystalline structure of cellulose using static and dynamic FT-IR spectroscopy. Carbohydr Res 339(3):569–578. doi:10.1016/J.Carres.2003.11.012

    Article  CAS  Google Scholar 

  • Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69(2):301–315

    Article  CAS  Google Scholar 

  • Anderson WF, Akin DE (2008) Structural and chemical properties of grass lignocelluloses related to conversion for biofuels. J Ind Microbiol Biotechnol 35(5):355–366. doi:10.1007/s10295-007-0291-8

    Article  CAS  Google Scholar 

  • American Public Health Association (2005) Standard methods for the examination of water and wastewater, 21st edn. American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC, USA

  • Atsumi S, Liao JC (2008) Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Appl Environ Microbiol 74(24):7802–7808. doi:10.1128/AEM.02046-08

    Article  CAS  Google Scholar 

  • Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451(7174):86–89. doi:10.1038/nature06450

    Article  CAS  Google Scholar 

  • Beller HR, Goh E-B, Keasling JD (2010) Genes involved in long-chain alkene biosynthesis in Micrococcus luteus. Appl Environ Microbiol 76(4):1212–1223. doi:10.1128/aem.02312-09

    Article  CAS  Google Scholar 

  • Burkholder J, Wetzel R (1989) Epiphytic microalgae on natural substrata in a hardwater lake: seasonal dynamics of community structure, biomass and ATP content. Arch Hydrobiol 83(1):1–56

    Google Scholar 

  • Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306. doi:10.1016/j.biotechadv.2007.02.001

    Article  CAS  Google Scholar 

  • Cotner J, Makino W, Biddanda B (2006) Temperature affects stoichiometry and biochemical composition of Escherichia coli. Microb Ecol 52(1):26–33. doi:10.1007/s00248-006-9040-1

    Article  CAS  Google Scholar 

  • Davis LS, Hoffmann JP, Cook PW (1990a) Production and nutrient accumulation by periphyton in a wastewater treatment facility. J Phycol 26(4):617–623. doi:10.1111/j.0022-3646.1990.00617.x

    Article  Google Scholar 

  • Davis LS, Hoffmann JP, Cook PW (1990b) Seasonal succession of algal periphyton from a wastewater treatment facility. J Phycol 26(4):611–617. doi:10.1111/j.0022-3646.1990.00611.x

    Article  Google Scholar 

  • Entwisle T (1989) Phenology of the Cladophora–Stigeoclonium community in two urban creeks of Melbourne. Mar Freshwater Res 40(5):471–489. doi:10.1071/MF9890471

    Article  CAS  Google Scholar 

  • Graham LE, Graham JM, Wilcox LW (2009) Algae, 2nd edn. Cummings, San Francisco

    Google Scholar 

  • Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100(3):965–973

    Article  CAS  Google Scholar 

  • Gustafsson C, Lennholm H, Iversen T, Nystrom C (2003) Evaluation of surface and bulk characteristics of cellulose I powders in relation to compaction behavior and tablet properties. Drug Dev Ind Pharm 29(10):1095–1107. doi:10.1081/DDC-120025867

    Article  CAS  Google Scholar 

  • Higgins SN, Malkin SY, Howell ET, Guildford SJ, Campbell L, Hiriart-Baer V, Hecky RE (2008) An ecological review of Cladophora glomerata (Chlorophyta) in the Laurentian Great Lakes. J Phycol 44(4):839–854. doi:10.1111/j.1529-8817.2008.00538.x

    Article  Google Scholar 

  • Hill J (2009) Environmental costs and benefits of transportation biofuel production from food- and lignocellulose-based energy crops: a review. In: Lichtfouse E, Navarrete M. Debaeke P, Véronique S, Alberola C (eds) Sustainable agriculture. Springer, Netherlands, pp 125–139. doi:10.1007/978-90-481-2666-8_10

  • Houghton J, Weatherwax S, Ferrell J (2006) Breaking the biological barriers to cellulosic ethanol: a joint research agenda. Department of Energy, Washington, DC

    Google Scholar 

  • Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54(4):621–639. doi:10.1111/j.1365-313X.2008.03492.x

    Article  CAS  Google Scholar 

  • Igarashi K, Wada M, Hori R, Samejima M (2006) Surface density of cellobiohydrolase on crystalline celluloses. FEBS J 273(13):2869–2878. doi:10.1111/j.1742-4658.2006.05299.x

    Article  CAS  Google Scholar 

  • Igarashi K, Wada M, Samejima M (2007) Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase. FEBS J 274(7):1785–1792. doi:10.1111/j.1742-4658.2007.05727.x

    Article  CAS  Google Scholar 

  • Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77(6):1305–1316. doi:10.1007/s00253-007-1257-5

    Article  CAS  Google Scholar 

  • Kalnes T, Marker T, Shonnard DR (2007) Int J Chem React Eng 5:10

    Google Scholar 

  • Kim S, Inglett GE (2006) Molecular weight and ionic strength dependence of fluorescence intensity of the Calcofluor/beta-glucan complex in flow-injection analysis. J Food Compos Anal 19(5):466–472. doi:10.1016/J.Jfca.2005.11.006

    Article  CAS  Google Scholar 

  • Lamai C, Kruatrachue M, Prayad Pokethitiyook E, Upatham S, Soonthornsarathool V (2005) Toxicity and accumulation of lead and cadmium in the filamentous green alga Cladophora fracta (O.F. Muller ex Vahl) Kutzing: a laboratory study. ScienceAsia 31:121–127

    Article  CAS  Google Scholar 

  • Lau MW, Dale BE (2009) Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST). Proc Natl Acad Sci USA 106(5):1368–1373. doi:10.1073/pnas.0812364106

    Article  CAS  Google Scholar 

  • Lavoie A, de la Noüe J (1987) Harvesting of Scenedesmus obliquus in wastewaters: auto- or bioflocculation? Biotechnol Bioeng 30(7):852–859. doi:10.1002/bit.260300707

    Article  CAS  Google Scholar 

  • Lennen RM, Braden DJ, West RA, Dumesic JA, Pfleger BF (2010) A process for microbial hydrocarbon synthesis: overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol Bioeng 106(2):193–202. doi:10.1002/bit.22660

    Article  CAS  Google Scholar 

  • Lu X, Vora H, Khosla C (2008) Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab Eng 10(6):333–339. doi:10.1016/j.ymben.2008.08.006

    Article  CAS  Google Scholar 

  • Mihranyan A (2011) Cellulose from Cladophorales green algae: from environmental problem to high-tech composite materials. J Appl Polym Sci 119(4):2449–2460. doi:10.1002/app.32959

    Article  CAS  Google Scholar 

  • Mihranyan A, Llagostera AP, Karmhag R, Stromme M, Ek R (2004) Moisture sorption by cellulose powders of varying crystallinity. Int J Pharm 269(2):433–442. doi:10.1016/J.Ijpharm.2003.09.030

    Article  CAS  Google Scholar 

  • Mihranyan A, Edsman K, Strømme M (2007) Rheological properties of cellulose hydrogels prepared from Cladophora cellulose powder. Food Hydrocoll 21(2):267–272

    Article  CAS  Google Scholar 

  • Moraine R, Shelef G, Meydan A, Levi A (1979) Algal single cell protein from wastewater treatment and renovation process. Biotechnol Bioeng 21(7):1191–1207. doi:10.1002/bit.260210709

    Article  CAS  Google Scholar 

  • Park JBK, Craggs RJ, Shilton AN (2011) Wastewater treatment high rate algal ponds for biofuel production. Bioresour Technol 102(1):35–42. doi:10.1016/j.biortech.2010.06.158

    Article  CAS  Google Scholar 

  • Perlack RD, Wright LL, Turhollow AF, Graham RL, Stokes BJ, Erbach DC (2005) Biomass as feedstock for a bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply. Oak Ridge National Laboratory, Oak Ridge, TN

    Book  Google Scholar 

  • Rude MA, Baron TS, Brubaker S, Alibhai M, Del Cardayre SB, Schirmer A (2011) Terminal olefin (1-alkene) biosynthesis by a novel P450 fatty acid decarboxylase from Jeotgalicoccus sp. Appl Environ Microbiol 77:1844–1853. doi:10.1128/aem.02580-10

    Article  Google Scholar 

  • Sfriso A, Marcomini A, Pavoni B (1987) Relationships between macroalgal biomass and nutrient concentrations in a hypertrophic area of the Venice Lagoon. Mar Environ Res 22(4):297–312

    Article  CAS  Google Scholar 

  • Sheehan J, Dunahay T, Benemann J, Roesler P (1998) A look back at the U.S. Department of Energy’s aquatic species program: biodiesel from algae. Department of Energy, Golden, CO

    Book  Google Scholar 

  • Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171(2):73–80

    Article  CAS  Google Scholar 

  • 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. doi:10.1186/1475-2859-7-36

    Article  Google Scholar 

  • Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, Del Cardayre SB, Keasling JD (2010) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463(7280):559–562. doi:10.1038/nature08721

    Article  CAS  Google Scholar 

  • Vispute TP, Huber GW (2008) Breaking the chemical and engineering barriers to lignocellulosic biofuels. Int Sugar J 110(1311):138

    CAS  Google Scholar 

  • Voelker TA, Davies HM (1994) Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl–acyl carrier protein thioesterase. J Bacteriol 176(23):7320–7327

    CAS  Google Scholar 

  • Walker TL, Collet C, Purton S (2005) Algal transgenics in the genomic era. J Phycol 41(6):1077–1093. doi:10.1111/j.1529-8817.2005.00133.x

    Article  Google Scholar 

  • Wetzel RG, Sondergaard M (1998) Role of submerged macrophytes for the microbial community and dynamics of dissolved organic carbon in aquatic ecosystems. In: Jeppesen E, Sondergaard M, Sondergaard M, Christofferson K (eds) The structuring role of submerged macrophytes in lakes, vol 131, Ecological studies. Springer, New York, pp 133–148

    Google Scholar 

  • Wijffels RH, Barbosa MJ (2010) An outlook on microalgal biofuels. Science 329(5993):796–799. doi:10.1126/science.1189003

    Article  CAS  Google Scholar 

  • Withers ST, Keasling JD (2007) Biosynthesis and engineering of isoprenoid small molecules. Appl Microbiol Biotechnol 73(5):980–990. doi:10.1007/s00253-006-0593-1

    Article  CAS  Google Scholar 

  • Wood PJ, Weisz J (1984) Use of Calcofluor in analysis of oat beta-d-glucan. Cereal Chem 61(1):73–75

    CAS  Google Scholar 

  • Xu Q, Singh A, Himmel ME (2009) Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose. Curr Opin Biotechnol 20(3):364–371. doi:10.1016/j.copbio.2009.05.006

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by a grant from the Wisconsin Energy Independence Fund to J.Y. and by the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). A.K.B. was supported as a recipient of a Holstrom Environmental Scholarship. R.M.L. was supported as a trainee in the Chemistry–Biology Interface Training Program (NIH).

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Authors and Affiliations

  1. Great Lakes Bioenergy Research Center, 1550 Linden Drive, Madison, WI, 53706, USA

    Spencer W. Hoover, Wesley D. Marner II, Rebecca M. Lennen, Tyler M. Wittkopp & Brian F. Pfleger

  2. Chemical and Biological Engineering, University of Wisconsin—Madison, 2034 Engineering Hall, 1415 Engineering Drive, Madison, WI, 53706, USA

    Spencer W. Hoover, Amy K. Brownson, Rebecca M. Lennen & Brian F. Pfleger

  3. Bioenergy and Environment, Inc., 29W256 Oak Lane, West Chicago, IL, 60185, USA

    Jun Yoshitani

  4. Department of Botany, University of Wisconsin—Madison, 430 Lincoln Dr., Madison, WI, 53706, USA

    Shahrizim Zulkifly & Linda E. Graham

  5. Department of Civil and Environmental Engineering, University of Wisconsin, Madison, 3204 Engineering Hall 1415 Engineering Drive, Madison, WI, 53706, USA

    Sheena D. Chaston & Katherine D. McMahon

  6. Department of Bacteriology, University of Wisconsin, Madison, 1550 Linden Dr., Madison, WI, 53706, USA

    Sheena D. Chaston & Katherine D. McMahon

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  1. Spencer W. Hoover
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  2. Wesley D. Marner II
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  11. Brian F. Pfleger
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Correspondence to Brian F. Pfleger.

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Spencer W. Hoover and Wesley D. Marner II contributed equally to this work.

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Hoover, S.W., Marner, W.D., Brownson, A.K. et al. Bacterial production of free fatty acids from freshwater macroalgal cellulose. Appl Microbiol Biotechnol 91, 435–446 (2011). https://doi.org/10.1007/s00253-011-3344-x

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  • DOI: https://doi.org/10.1007/s00253-011-3344-x

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