Algae: Not Just Green Kool-Aid. Jonathan Meuser Matthew C. Posewitz Lab Colorado School of Mines Golden, CO

Transcription

1 Algae: Not Just Green Kool-Aid Jonathan Meuser Matthew C. Posewitz Lab Colorado School of Mines Golden, CO

2 Biofuels: A techno-evolution U.S. DOE Classification of Biofuels 1 st Generation Biofuels 2 nd Generation Biofuels 3 rd Generation Biofuels PAST...PRESENT FUTURE

3 Plant Seeds and Fruit Grain alcohols 1 st Generation Biofuels Ethanol has a positive energy balance, DOE Veggie Oil, and Biodiesel made from it.

4 Net Energy 1 st Generation Value = Energy in Ethanol Biofuels Fossil Energy Required

5 Source: Pearl G Animal Derived Fat Potential for Bioenergy Use U.S. Dept of Agriculture Oil Crops Outlook 2003

6 Source: Peterson, C.L. Potential production of biodiesel.

7 Source: Peterson, C.L. Potential production of biodiesel.

8 1 st Generation Biofuels-Biodiesel Thus, using all surplus veggie oil, half of used oil, and all plant oil produced from high-yield crops on 37 million acres of idle crop land A 500 million g/yr U.S. biodiesel industry is very ambitious... This equals approximately 1% of total U.S. Diesel demand. Even if we used all world plant oil production (26.9 B g/yr) we wouldn t get to 50% of U.S. demand. Source:

9 2 nd Generation Biofuels Corn Stover Miscanthus Lignocellulosic feedstocks Crop residues - Corn Stover - Sugar Cane Bagasse Dedicated Cellulose Crops - Poplar - Switchgrass - Mescanthus

10 2 nd Generation Biofuels Cellulase Enzyme Cellulose Hemi-Cellulose Lignin Image: NREL

11 2 nd Generation Biofuels

12 3 rd Generation Biofuels -N 4 days Microalgae/microbial phototrophs Starch alcohol Lipid SVO/Biodiesel/Green diesel Alkanes and other carbon chains direct fuel production. Hydrogen TAG Lipid body Starch Granule Image: Libessart, 1995 Source: Libessartet al., (1995) Storage, photosynthesis, and growth: the conditional nature of mutations affecting starch synthesis and structure in Chlamydomonas. The Plant Cell 7,

13 3 rd Generation Biofuels Algae Lipids Carbohydrates Proteins, etc -N 4 days Biodiesel Ethanol Butanol Animal feed Methane Diagram source: Robert Jinkerson TAG Lipid body Starch Granule Image: Libessart, 1995 Source: Libessartet al., (1995) Storage, photosynthesis, and growth: the conditional nature of mutations affecting starch synthesis and structure in Chlamydomonas. The Plant Cell 7,

14 Source: Dismukes et al., (2008) Aquatic phototrophs: efficient alternatives to landbased crops for biofuels. Current Opinion in Biotechnology 19,

15 3 rd Generation 1 st Generation 2 nd Generation Source: Dismukeset al., (2008) Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Current Opinion in Biotechnology 19,

16 3 rd Generation Biofuels-Starch Corn, Today Traditional domestication over 12,000 yrs Teosinte, 10,000 BC Insertion of a single starch gene last year...? Image credits: Wikipedia, Robert Jinkerson

17 3 rd Generation Biofuels-Starch TAP+N TAP-N Light microscopy images of algae after 96 hours. Scale bars: 10 µm. Image credits: Wikipedia, Robert Jinkerson

18 TAP+N TAP-N TAP+N TAP-N LSCF microscopy images merged with transmitted light images (left) and DIC images (right) of CC124 (wildtype), sta7, sta7c5 and sta7-c19 at 96h. Leftmost columns: chlorophyll autofluorescence (red); lipid bodies stained with Bodipy 493/503 (green). Scale bars: 10 µm. Source: Work*, Radakovits*, Jinkerson*, et al. (2010). Eukaryot. Cell, 9:

19 3 rd Generation Biofuels-Starch µg glucose / million cells TAP + N TAP -N wild + more copies of starch gene D66 D D (chl) µg glucose / ml culture TAP + N TAP -N wild + more copies of starch gene D66 D D (chl) - Current efforts using lab strains yields about 0.6 grams/liter/day.

20 3 rd Generation Biofuels-Starch µg glucose / ml culture D66 D h 120 h 240 h Growth in 30L bioreactors. Right image after 24 hours of growth. Glucose yields in photoautotrophic growth in light/dark, low light, no CO 2. Research credit: Robert Jinkerson, Dylan Franks Also thanks to Neil Watson for T5 fluorescent light banks!

21 Wild-type +starch gene Theoretical high-production strain + starch gene Corn stover Wild lab strain Sugarcane Corn

22 Theoretical high-production strain + starch gene Major potential for future food source, also. Corn Sugarcane Corn stover Wild lab strain Wild-type +starch gene

23 3 rd Generation Biofuels-Lipid Six algal strains reported to produce high levels of lipid. Credit: Randor Radakovits

24 3 rd Generation Biofuels-Lipid Credit: Randor Radakovits

25 3 rd Generation Biofuels-Lipid Credit: Robert Jinkerson

26 Algal Lipid Extraction: Wet and Dry

27 Wet Lipid Extraction: Optimization Saponification-transesterification protocol: Work et al Current optimized method. Source: Dongxu(Tom) Li Work, V. H., R. Radakovits, R. E. Jinkerson, J. E. Meuser, L. G. Elliott, D. J. Vinyard, L. M. L. Laurens, G. C. Dismukes, and M. C. Posewitz Increased lipid accumulation in the Chlamydomonasreinhardtiista7-10 starchlessisoamylasemutant and increased carbohydrate synthesis in complemented strains. Eukaryotic Cell 9:

28 Wet Lipid Extraction: Optimization Saponification-transesterification time optimization Research credit: Dongxu Li, Robert Jinkerson

29 Lipid Engineering: Modification of chain length Expression of a thioesterasefrom Laurel (U. californica) causes C14 (Lauricacid) accumulation in triacylglycerides. RandorRadakovits a, Patrick M. Eduafo a and Matthew C. PosewitzGenetic engineering of fatty acid chain length in Phaeodactylumtricornutum(2011) Metabolic Engineering, 13(1)

30 Synthetic Biology : Direct excretion of fuel Direct excretion of FFAs in Synechocystis sp PCC 6803 Liu X., BruneD., VermassW., Curtiss R. (2011) Production and secretion of fatty acids in genetically engineered cyanobacteria.

31 Synthetic Biology : Direct excretion of fuel Direct excretion of FFAs in Synechocystis sp PCC 6803 Liu X., BruneD., VermassW., Curtiss R. (2011) Production and secretion of fatty acids in genetically engineered cyanobacteria.

32 Synthetic Biology : Direct excretion of fuel Joule Unlimited, Cambridge Mass. ( Joule's Helioculture platform combines breakthroughs in genome engineering, bioprocessingand hardware engineering to convert sunlight and waste CO 2 directly into clean, fungible diesel fuel, bypassing the limitations of biofuelproduction.

33 Algal BiofuelsProcess Source: Al Darzins, NREL (Currently: DuPont)

34 Classic Raceway Open Pond Source: Sheehan. (1998) A look back at the USDOE s Aquatic Species Program

35 Classic Raceway Open Pond A proven technology in Spirulina production: Cyanotech Co, Hawaii, USA Earthrise Farms, Imperial Valley, CA

36 Tubular Photobioreactors Image: AlgaeLink

37 Tubular Photobioreactors Image: CalPoly, Oilgae.com

38 Pond vs. Tubular Photobioreactors Important Variables Capital cost Operating cost Evaporation Temp control Invasive species Mixing Harvesting Open Circular Raceway Tubular Bioreactor Image: Adapted from Jonathan Trent, NASA Ames Research Center

39 ALG-I.com ALGI (e.g. Algae, Light & Gas Instruments) is a new technical instrument start-up currently developing a commercial Clark-electrode system primarily for applications in algal biotechnology. We are currently the sole licensee of a patent-pending ultra-sensitive, environmentally-controlled polarographicsystem developed at the Colorado School of Mines in Golden, CO. Chief Executive Officer: Jonathan E. Meuser- jon@alg-i.com Chief Technical Officer: Ed Dempsey - ed@alg-i.com Chief Information Officer: Devin Karns- devin@alg-i.com Chief Financial Officer: Stephen Moore - stephan@alg-i.com Webmaster: Alex Trujillo - alex@alg-i.com

40 ALG-I.com - Measure photosynthesis, respiration, and hydrogen-exchange from microbial samples. -A custom two-probe polarographsystem and support software for determining oxygen and/or hydrogen exchange from algae or other biological/chemicalcatalysts. -Our system and associated software is ideal for Clark-type electrode measurements in a controlled environment from small, magnetically-stirred sample volumes ( mL) using YSI 5331 probes. -On-board pressure (e.g. altitude) compensation. - Software-based salinity compensation, gas calibration (2-point) and lighting calibration. - Software-based experimental design, data acquisition, and data processing.

41 ALG-I.com

42 Most Important Variable: Cost! Standardized Estimates of the Production Cost of Algal Oil Per Gallon Source: Al Darzins, NREL & Sandia