Microalgae as Biofuel: Engineering a Super Algae - COSMOS

Microalgae asfigure 1Biofuel:Engineering aSuper AlgaeYsa PitmanCluster 1 - Biotechnology

What is algae?• Eukaryotic, photosyntheticorganisms• Live near or in bodies ofwater• Can be multicellular(macroalgae) or unicellular(microalgae)• Metabolize carbon,phosphorus and nitrogen• Types of algae– Greenalgae– Diatoms– Blue-green algae(cyanobacteria)– Golden algaefigure 2

Useful components of algae• Alcohols (hydrocarbons) bioethanol– Naturally produced hydrocarbons in algaelimit reproduction and growth• Oils (lipids) biodiesel– Algae in nature are lipid-rich– Light and low temperatures are shownto increase lipid concentration in algae

Why use algae?Compare to terrestrial biofuelcrops:• Faster growth rate• Higher energy yield per unit ofbiomass– 30–100 times more energyper 10,000 square meters• Can be grown on land unfit forfarmingfigure 5• More efficiently mitigatesatmospheric carbon dioxide• Consumes the least water ofall biofuel crops

Drawbacks to using algaefigure 6• Lower biomass concentrationthan other biofuel cropsfigure 7• High initial cost due tonew equipment andfacilitiesfigure 8• More fertilizer use oceanic algal blooms dead zones

Industrial techniques forgrowing algae:Pond System– Raceway pattern withpaddlewheels providingconstant churning– Less expensive thanalternatives– Open system moresusceptible tocontamination– Takes up more surfaceareafigure 9figure 10

Industrial techniques forgrowing algae:Photo-bioreactors• More expensive due toinfrastructure costs• Higher efficiency and biomassconcentration due to highsurface area to volume ratio• Shorter harvest timefigure 11figure 7• Closed system• Can regulate O2 and CO2levels in the reactor

Chlamydomonas reinhardtii• Single-celled green aglae• Has two flagella• Can maintain foreign DNA• Is a “model organism” forresearchers– Helps answerfundamental questions,including how cellschange in response tomineral nutrition• Its genome has beensequenced and mappedFigure 13

• Aceytl-CoA is a key component in the citricacid cycle of photosynthesisLiposynthesis in algaeFigure 12• Aceytl-CoA carboxylase is the first enzymeinvolved in fatty acid synthesis and regulation

Researchable Question:Will upregulating the generesponsible for Aceytl-CoAcarboxylase synthesis in c.reinhardtii yield an algal strain witha higher lipid content than the wildtype, thereby maximizing efficiencyas a biodiesel?

Procedure (1): locate acetyl-CoAcarboxylase gene in c. reinhardtiiFigure 14• Gene name: CHLREDRAFT_133238• Amplify sequence using PCR– Will need engineered restriction enzymesthat cleave on either side of the sequence

Procedure (2): Find transcriptionfactor for ACCase gene usingAffinity Chromatography• Transcription factors are proteinsthat bind to the enhancer or promoterregions of a given gene andoverexpress the gene• Attach PCR product molecules tosolid matrix beads in a column• Pour extract of c. reinhardtii cellsover the beads• The promoter’s protein will bind tothe beads (all other proteins flowthrough column)• Wash beads with buffer that releasesproteinsFigure 15

Procedure: Measure lipidcontent in control and newstrain• Add Nile Red (a fluorescent lipophilic dye) to eachsample• Perform NIR spectroscopy on the samples– NIR = “near infra-red”– NIR spectroscopy is more effective than solventextraction protocol• Needs smaller sample• More rapid process: minutes rather than days• After a period of growth, compare lipid mass in thecontrol strain and the upregulated strain

Expected results• The upregulated strain of c.reinhardtii will have a higher lipidcontent after the growth period thanthe control strain of c. reinhardtii

Bibliography• S. Scott, M. Davey, J. Dennis, I. Horst, C. Howe, D. Lea-Smith, and A.Smith, Biodiesel from algae: challenges and prospects, Current Opinionin Biotechnology 21 (2010), pp. 277-286• A. Demirbas, Use of Algae as biofuel sources, Energy Conversion andManagement 51 (2010), pp. 2738-2749• L. Beer, E. Boyd, J. Peters, M. Posewitz, Engineering algae forbiohydrogen and biofuel production, Current Opinion in Biotechnology20 (2009), pp. 264-271• R. Radakovits, R. Jinkerson, A. Darzins, M. Posewitz, Geneticengineering of algae for increased biofuel production, Eukaryotic Cell 9(2010), pp. 486-501• J. Rosenberg, G. Oyler, L. Wilkinson, M. Betenbaugh, A green light forengineered algae: redirecting metabolism to fuel a biotechnologyrevolution, Current Opinion in Biotechnology 19 (2008), pp. 430-436• Khozin-Goldberg, Z. Cohen. Unraveling algal lipid metabolism: Recentadvances in gene identification, Biochimie 93 (2011), pp. 91-100.• M. Griffiths, S. Harrison. Lipid productivity as a key characteristic forchoosing algal species for biodiesel production, Journal of AppliedPhycology (2008).• L. Laurens, E. Wolfrum, A. Darzins, Predicting Algal LipidConcentrations using NIR Spectroscopy. In Algae Industry Magazine.