Galdieria Sulphuraria) in Inexpensive Closed Photobioreactors

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Cultivation and Lipid Profiles of Thermo- Tolerant, Mixotrophic Red Algae (Galdieria sulphuraria) in Inexpensive Closed Photobioreactors

P. J. Lammers* 1, W. Van Voorhies 1, T. Selvaratnam 1, N. Nirmalakhandan 1, M. Seger 1, A. Unc 1, H.K. Reddy 1, S. Deng 1, R. Roth 2, U. Goodenough 2, and R. Castenholz 3 1 New Mexico State University, USA; 2 Washington University, USA; 3 University of Oregon, USA Water Availability is the Major Resource Limitation for Large-Scale Algae Cultivation in the Arid Southwest • 8-9 feet pan evaporation per year • Fresh water supplies are both highly limited and legally entangled Alternatives 1. Municipal and Agricultural Waste Water Energy positive WWT with nutrient extraction 2. Produced water from Oil and Gas Extraction – 10 to 20 barrels of water per barrel of oil – Producers pay premium for re-injection – Brackish and requires clean up but useable (El Dorado Biofuels – Jal, NM) 3. Brackish and Saline Aquifers

Evaporation Control in Closed Cultivation Systems

• Plastic bioreactors with various mixing systems • Greenhouse effect – hence thermo-tolerant algae species.

• Enable efficient CO2 enrichment (1% in gas phase) with ultra-low gas- flow rates – 2 L-gas/min/400-L-liquid = 0.005 VVM

– We observe no O2 inhibition <30% peak O2 – 0.02% gas-phase evaporative water loss/day = ~200 mL of water loss/400-L of culture volume

Temperature data for 8 inch depth, “lay flat” PBR. The area between the red lines indicates acceptable temperature range for Cyanidium/Galdieria acid thermophiles.

The area between the green lines shows temperature range in which more temperate algae like Chlorella would grow. Galdieria sulphuraria • Unicellular red alga • Extremely acidophilic (pH 0-4) – self limiting • Moderately thermophilic, maximum sustained temperature is 56oC • Photoautotrophic, mixotrophic and • Heterotrophic growth (up to 100 g/L) – Graverholt, O. S., and Eriksen, N. T. (2007) Heterotrophic high- cell-density fed-batch and continuous-flow cultures of Galdieria sulphuraria and production of phycocyanin, Applied Microbiology and Biotechnology 77, 69-75. • High Rubisco CO2/O2 discrimination – Uemura, K., Anwaruzzaman, Miyachi, S., and Yokota, A. (1997) Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic red algae with a strong specificity for CO2 fixation, Biochemical and Biophysical Research Communications 233, 568-571.

Versatile Mixotrophic Metabolism

Schonknecht, G., et al. (2013) Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote, Science 339, 1207-1210.

~1% Dissolved Inorganic Carbon Levels in Below pH 5 Relative to pH 8 in Galdieria Cultivation Systems

Can this really work? Galdieria and Cyanidium strains obtained from Culture Collection of Microorganisms from Extreme Environments

Mixotrophs

Strict Autotroph

18S phylogeny of 4 strains under study received from R. Castenholz

Toplin, J. A., T. B. Norris, et al. (2008). "Biogeographic and phylogenetic diversity of thermoacidophilic Cyanidiales in Yellowstone National Park, Japan, and New Zealand." Applied and Environmental Microbiology 74(9): 2822- 2833. Galdieria sulphuraria CCMEE 5587.1 Volumetric Growth Rate in 6 mL tube cultures, 20-L Carboys is 0.11-0.12 grams/Liter/day (40 oC, pH 2.5) 200-L raceways: 10-16 g/m2/day (0.1-0.16 g/L/day) in raceway @ Variable Temps; pH 2.5; 10 cm depth; 5% CO2; ultra-low gas flow rate @ <1 PSI to inflate bag Galdieria sulphuraria growth is independent of CO2 concentration between 1%, 5% and 10% in air Outdoor Growth of Galdieria sulphuraria in an Enclosed Raceway

10 cm Depth, 5% CO2 in Air, @ 2 L/min, O2 in gas phase is ~30% during daytime Bag Growth and Temperature

3 55 Growth y = 0.165x + 0.159 R² = 0.978 Temp. 2.5 50 )

( 0.165 grams/L/day 2 2 45 e 16.5 grams/m /day

u )

D 1.5 40

(

g 1 35 e

0.5 30

0 25 May/18/13 May/27/13 Jun/6/13 Date A B starch

Chloroplast

Dedifferentiated chloroplast

Lipid body Lipid body

Starch Lipid body

Panel A: cell organization during photosynthetic growth stage, Panel B: cell morphology after nitrogen-limitation showing abundant lipid and starch bodies.

Sinetova, M. P., Markelova, A. G., and Los, D. A. (2006) The effect of nitrogen starvation on the ultrastructure and pigment composition of chloroplasts in the acidothermophilic microalga Galdieria sulphuraria, Russian Journal of Plant Physiology 53, 153- 162. Bacterial survivability test at different pH and temperature conditions: E. coli 13-B6 (wastewater isolate highly resistant to multiple antibiotics) (Adrian Unc and Mark Seger; NMSU) Viable E. coli quantification (M.I. agar)

2.E+10

40 C 2.E+10 1.E+10 9.E+09 6.E+09

mL CFU’s/100 3.E+09 0.E+00 0 10 20 30 40 50

2.E+10

2.E+10 1.E+10 48 C 9.E+09 6.E+09

CFU’s/100 mL CFU’s/100 3.E+09 0.E+00 0 10 20 30 40 50 Time (hours) Hydrothermal Processing • Hydrothermal processing uses water, temperature and pressure to convert organic feedstock into fuel • Wet process with no additional solvents • Process conditions are wet slurry at 10-20% dry solids, 160-350⁰C • Sequential HTL allows substantial pre-extraction of saccharides, amino acids and organic phosphate prior to liquefaction step

Chakraborty, C. Miao, A. McDonald, S.L. Chen. (2012) Concomitant extraction of bio-oil and value added polysaccharides from Chlorella sorokiniana using a unique sequential hydrothermal extraction technology. Fuel 95: 63-70.

15 Hydrothermal Liquefaction Results from Wet Red Algal Biomass Galdieria sulphuraria (CCMEE 5572) Ave FAME 8.9% (SD 1.2) N=3

0.40

0.35

0.30

0.25

0.20 Galdieria

0.15 % of Total FAME Totalof %

0.10

0.05

0.00 C14:0 C16:0 C16:1c C18:0 C18:1n9c c18:2n6c C18:3n3 C20:5n3 Galdieria (5572) T=275 oC, T=300 oC, 60 bar 91 bar Oil yield (%) 15.43 25.32 Authors and Collaborators:

• Wayne Van Voorhies, Nick Csakan, Adrian Unc, Mark Seger, N. Nirmalakhandan, Thinesh Selvaratnam, Shuguang Deng, Harvind Reddy (NMSU) • Tanner Schaub, Omar Holguin, Barry Dungan (NMSU) • R. Roth and Ursula Goodenough (Washington University) Funding: • DoE - EERE (National Alliance for Advanced Biofuels & Bioproducts) • Air Force Research Laboratory (Wright Patterson) • National Science Foundation (NM-EPSCoR RII4) • Office of the Vice President for Research – NMSU PBR Consultations and Donations • Algenol Biofuels • Sapphire Biofuels