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Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production


Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

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National renewable energy laboratory


Triflouroacetic acid


Trilaminar structure


Transmission electron microscopy


Culture collection of algae and protozoa


Formerly CCMP, Provasoli-Guillard National Center for marine algae and microbiota


American type culture collection


Culture collection of algae at the University of Texas at Austin


NREL Aquatic species program


Modified bold’s basal medium


Artificial seawater


Phosphate buffered saline


Scanning electron microscopy


  • Afi L, Metzger P, Largeau C, Connan J, Berkaloff C, Rousseau B (1996) Bacterial degradation of green microalgae: incubation of Chlorella emersonii and Chlorella vulgaris with Pseudomonas oleovorans and Flavobacterium aquatile. Org Geochem 25:117–130

    Article  CAS  Google Scholar 

  • Atkinson AW, Gunning BESJ, John PCL (1972) Sporopollenin in the cell wall of Chlorella and other algae: ultrastructure, chemistry, and incorporation of 14C-acetate, studied in synchronous cultures. Planta 107:1–32

    Article  CAS  Google Scholar 

  • Barclay W, Johansen J, Chelf P, Nagle N, Roessler PG, Lemke P (1987) Microalgae culture collection 1986–1987. SERI/SP-232-3079

  • Blokker P, Schouten S, Van Den Ende H, De Leeuw JW, Sinninghe Damaste JS (1998) Cell wall-specific w-hydroxy fatty acids in some freshwater green microalgae. Phytochemistry 49:691–695

    Article  CAS  Google Scholar 

  • Braun E, Aach HG (1975) Enzymatic degradation of the cell wall of Chlorella. Planta 126:181–185

    Article  Google Scholar 

  • Brennan L, Owende P (2010) Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sustain Energy Rev 14:557–577

    Article  CAS  Google Scholar 

  • Brown LM (1982) Production of axenic cultures of algae by an osmotic method. Phycologia 21:408–410

    Article  Google Scholar 

  • Burczyk J, Terminska-Pabis K, Smietana B (1995) Cell wall neutral sugar composition of Chlorococcalean algae forming and not forming acetolysis resistant biopolymer. Phytochemistry 38:837–841

    Article  CAS  Google Scholar 

  • Corre G, Templier J, Largeau C, Rousseau B, Berkaloff C (1996) Influence of cell wall composition on the resistance of two Chlorella species (Chlorophyta) to detergents. J Phycol 32:584–590

    Article  CAS  Google Scholar 

  • Davis R, Aden A, Pienkos PT (2011) Techno-economic analysis of autotrophic microalgae for fuel production. Appl Energy 88:3524–3531

    Article  Google Scholar 

  • Dawson SC, Pace NR (2002) Novel kingdom-level eukaryotic diversity in anoxic environments. PNAS 99:8324–8329

    PubMed  Article  CAS  Google Scholar 

  • Derenne S, Largeau C, Berkaloff C, Rousseau B, Wilhelm C, Hatcher PG (1992) Non-hydrolysable macromolecular constituents from outer walls of Chlorella fusca and Nanochlorum eucaryotum. Phytochemistry 31:1923–1929

    Article  CAS  Google Scholar 

  • Divakaran R, Pillai VNS (2002) Flocculation of algae using chitosan. J Appl Phycol 14:419–422

    Article  CAS  Google Scholar 

  • Fawley MW, Fawley KP, Buchheim MA (2004) Molecular diversity among communities of freshwater microchlorophytes. Microb Ecol 48(4):489–499. doi:10.1007/s00248-004-0214-4

    PubMed  Article  CAS  Google Scholar 

  • Fukada K, Inoue T, Shiraishi H (2006) A posttranslationally regulated protease, VheA, is involved in the liberation of juveniles from parental spheroids in Volvox carteri. Plant Cell 18:2554–2566

    PubMed  Article  CAS  Google Scholar 

  • Gelin F, Volkman JK, Largeau C, Derenne S, Sinninghe Damaste JS, De Leeuw JW (1999) Distribution of aliphatic, nonhydrolyzable biopolymers in marine microalgae. Org Geochem 30:147–159

    Article  CAS  Google Scholar 

  • Glover H (1977) Effects of iron deficiency on Isochrysis galbana (Chrysophyceae) and Phaeodactylum tricornutum (Bacillariophyceae). J Phycol 13:208–212

    CAS  Google Scholar 

  • Graves MV, Burbank DE, Roth R, Heuser J, DeAngelis PL, VanEtten JL (1999) Hyaluronan synthesis in virus PBCV-1 infected Chlorella-like green algae. Virology 257:15–23

    PubMed  Article  CAS  Google Scholar 

  • Guarnieri MT, Nag A, Smolinski SL, Darzins A, Seibert M, Pienkos PT (2011) Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga. PLoS ONE 6:e25851

    PubMed  Article  CAS  Google Scholar 

  • Guarnieri MT, Laurens LM, Knoshaug EP, Chou YC, Donohoe BS, Pienkos PT (2012) Complex systems engineering: a case study for an unsequenced microalga. In: Patnaik R (ed) Engineering complex phenotypes in industrial strains, Wiley, New York

  • Gunnison D, Alexander M (1975) Basis for the resistance of several algae to microbial decompostion. Appl Microbiol 29:729–738

    PubMed  CAS  Google Scholar 

  • Honjoh K, Suga K, Shinohara F, Maruyama I, Miyamoto T, Hatano S, Iio M (2003) Preparation of protoplasts from Chlorella vulgaris K-73122 and cell wall regeneration of protoplasts from C. vulgaris K-73122 and C-27. J Fac Agric 47:257–266

    Google Scholar 

  • Huss VAR, Frank C, Hartmann EC, Hirmer M, Kloboucek A, Seidel BM, Wenzeler P, Kessler E (1999) Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu late (Chlorophyta). J Phycol 35:587–598

    Article  CAS  Google Scholar 

  • Jaulneau V, Lafitte C, Corio-Costet M-F, Stadnik MJ, Salamagne S, Briand X, Esquerre-Tugaye M-T, Dumas B (2011) An Ulva armoricana extract protects plants against three powdery mildew pathogens. Eur J Plant Pathol 131:393–401

    Article  Google Scholar 

  • Kapaun E, Reisser W (1995) A chitin-like glycan in the cell wall of a Chlorella sp. (Chlorococcales, Chlorophyceae). Planta 197:577–582

    Article  CAS  Google Scholar 

  • Kapaun E, Loos E, Reisser W (1992) Cell wall composition of virus-sensitive symbiotic Chlorella species. Phytochemistry 31:3101–3104

    Article  Google Scholar 

  • Kim YH, Choi YK, Park J, Lee S, Yang YH, Kim HJ, Park TJ, Kim YH, Lee SH (2012) Ionic liquid-mediated extraction of lipids from algal biomass. Bioresour Technol 109:312–315

    PubMed  Article  CAS  Google Scholar 

  • Knoshaug EP, Darzins A (2011) Algal biofuels: the process. Chem Eng Progr 107:37–47

    CAS  Google Scholar 

  • Kubo T, Kaida S, Abe J, Saito T, Fukuzawa H, Matsuda Y (2009) The Chlamydomonas hatching enzyme, sporangin, is expressed in specific phases of the cell cycle and is localized to the flagella of daughter cells within the sporangial cell wall. Plant Cell Physiol 50(3):572–583. doi:10.1093/pcp/pcp016

    PubMed  Article  CAS  Google Scholar 

  • Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM (2010) Comparison of several methods for effective lipid extraction from microalgae. Bioresour Technol 101:S75–S77

    PubMed  Article  CAS  Google Scholar 

  • Malis-Arad S, Friedlander M, Ben-Arie R, Richmond AE (1980) Alkalinity-induced aggregation in Chlorella vulgaris I. Changes in cell volume and cell-wall structure. Plant Cell Physiol 21:27–35

    Google Scholar 

  • McCandless EL, Craigie JS (1979) Sulfated polysaccharides in red and brown algae. Ann Rev Plant Physiol 30:41–53

    Article  CAS  Google Scholar 

  • Monzingo AF, Marcotte EM, Hart PJ, Robertas JD (1996) Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat Struct Mol Biol 3(2):133–140

    Article  CAS  Google Scholar 

  • Ogawa K, Yamaura M, Maruyama I (1997) Isolation and identification of 2-O-methyl-l-rhamnose and 3-O-methyl-l-rhamnose as constituents of an acidic polysaccharide of Chlorella vulgaris. Biosci Biotechnol Biochem 61:539–540

    Article  CAS  Google Scholar 

  • Ogawa K, Yamaura M, Ikeda Y, Kondo S (1998) New aldobiuronic acid, 3-O-a-D-glucopyranuronosyl-l-rhamnopyranose, from an acidic polysaccharide of Chlorella vulgaris. Biosci Biotechnol Biochem 62:2030–2031

    Article  CAS  Google Scholar 

  • Ogawa K, Ikeda Y, Kondo S (1999) A new trisaccharide, a-d-glucopyranuronosyl-(1–3)-a-l-rhamnopyranosyl-(1–2)-a-l-rhamnopyranose from Chlorella vulgaris. Carbohydr Res 321:128–131

    Article  CAS  Google Scholar 

  • Ogawa K, Arai M, Naganawa H, Ikeda Y, Kondo S (2001) A new b-d-galactan having 3-O-methyl-d-galactose from Chlorella vulgaris. J Appl Glycosci 48:325–330

    Article  CAS  Google Scholar 

  • Pienkos PT, Darzins A (2009) The promise and challenges of microalgal-derived biofuels. Biofuels Bioprod Bioref 3:431–440

    Article  CAS  Google Scholar 

  • Popper ZA, Tuohy MG (2010) Beyond the green: understanding the evolutionary puzzle of plant and algal cell walls. Plant Physiol 153:373–383

    PubMed  Article  CAS  Google Scholar 

  • Ray B, Lahaye M (1995) Cell-wall polysaccharides from the marine green alga Ulva “rigida” (Ulvales, Chlorophyta). Extraction and chemical composition. Carbohydr Res 274:251–261

    Article  CAS  Google Scholar 

  • Sato M, Murata Y, Mizusawa M, Iwahashi H, Oka S (2004) A simple and rapid dual-fluorescence viability assay for microalgae. Microbiol Cult Coll 20:53–59

    Google Scholar 

  • Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, Smith AG (2010) Biodiesel from algae: challenges and prospects. Curr Opin Biotechnol 21:277–286

    PubMed  Article  CAS  Google Scholar 

  • Sheehan JT, Dunahay J, Benemann JR, Roessler PG (1998) A look back at the US department of energy’s aquatic species program—biodiesel from algae NREL/TP-580-24190

  • Shen Y, Pei Z, Yuan W, Mao E (2009) Effect of nitrogen and extraction method on algae lipid yield. Int J Agric Biol Eng 2:51–57

    CAS  Google Scholar 

  • Siddiquee MN, Rohani S (2011) Lipid extraction and biodiesel production from municipal sewage sludges: a review. Renew Sustain Energy Rev 15:1067–1072

    Article  CAS  Google Scholar 

  • Simpson AJ, Zang X, Kramer R, Hatcher PG (2003) New insights on the structure of algaenan from Botryococcus braunii race A and its hexane insoluble botryals based on multidimensional NMR spectroscopy and electrospray-mass spectrometry techniques. Phytochemistry 62:783–796

    PubMed  Article  CAS  Google Scholar 

  • Takeda H (1991) Sugar composition of the cell wall and the taxonomy of Chlorella (Chlorophyceae). J Phycol 27:224–232

    Article  CAS  Google Scholar 

  • Van Donk E, Lurling M, Hessen DO, Lokhorst GM (1997) Altered cell wall morphology in nutrient-deficient phytoplankton and its impact on grazers. Limnol Oceanogr 42:357–364

    Article  Google Scholar 

  • Van Etten JL, Lane LC, Meints RH (1991) Viruses and virus-like particles of eukaryotic algae. Microbiol Rev 55:586–620

    PubMed  Google Scholar 

  • Veldhuis MJW, Cucci TL, Sieracki ME (1997) Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. J Phycol 33:527–541

    Article  CAS  Google Scholar 

  • Walter JK, Aach HG (1987) Isolation and characterization of the enzymes involved in disintegration of the cell wall of Chlorella fusca. Physiol Plantarum 70:485–490

    Article  CAS  Google Scholar 

  • Wijffels RH, Barbosa MJ (2010) An outlook on microalgal biofuels. Science 329:796–799

    PubMed  Article  CAS  Google Scholar 

  • Wohlkonig A, Huet J, Looze Y, Wintjens R (2010) Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs. PLoS ONE 5:e15388. doi:10.1371/journal.pone.0015388

    PubMed  Article  Google Scholar 

  • Yamada T, Sakaguchi K (1982) Comparative studies on Chlorella cell walls: induction of protoplast formation. Arch Microbiol 132:10–13

    Article  Google Scholar 

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The authors would like to thank Jonathan Meuser for help with 18S RNA gene sequencing, Ben Smith for help with quantification of settling, Todd Vinzant and the Biomass Surface Characterization Laboratory at NREL for help in SEM image acquisition, and Philip Pienkos for technical discussions and manuscript review. This project was funded by NREL’s Laboratory Directed Research and Development program.

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Correspondence to Eric P. Knoshaug.

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Gerken, H.G., Donohoe, B. & Knoshaug, E.P. Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta 237, 239–253 (2013).

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  • Growth inhibition
  • Permeability
  • Lysozyme
  • Nitrogen depletion