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

Abstract

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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Abbreviations

NREL:

National renewable energy laboratory

TFA:

Triflouroacetic acid

TLS:

Trilaminar structure

TEM:

Transmission electron microscopy

CCAP:

Culture collection of algae and protozoa

NCMA:

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

ATCC:

American type culture collection

UTEX:

Culture collection of algae at the University of Texas at Austin

ASP:

NREL Aquatic species program

mBBM:

Modified bold’s basal medium

ASW:

Artificial seawater

PBS:

Phosphate buffered saline

SEM:

Scanning electron microscopy

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Acknowledgments

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). https://doi.org/10.1007/s00425-012-1765-0

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Keywords

  • Growth inhibition
  • Permeability
  • Lysozyme
  • Nitrogen depletion