1. A better understanding of culture parameters to increase exopolysaccharide production by Botryococcus braunii CCALA 778 Rafael G. Cubero, Dorinde Kleinegris, Maria Barbosa April 7 th 2016. Olhấo (Portugal)

2. SPLASH will develop a platform for bio-chemicals and biopolymers production by making use of Botryococcus braunii → Transition to a bio-based economy 1. Optimize cultivation methods to enhance the growth and production of hydrocarbons and polysaccharides by B. braunii 2. Scale up cultivation of B. braunii to produce hydrocarbons and polysaccharides 3. Assess the economics and sustainability of the integrated process SPLASH PROJECT (Sustainable PoLymers from Algae Sugars and Hydrocarbons) (http://www.eu-splash.eu/)

3. Why Botryococcus? A perfect candidate for being a factory for biochemical and biopolymers Fresh-brackish water colonial green algae Accumulate large amounts of long-chains hydrocarbons → 4 chemical races: Race A: C 25 -C 31 (odd-numbered n- alkadienes and alkatrienes) Race B: Botryococcenes (Polymethylated unsaturated triterpenes, C n H 2n-1, n=30-37) Race L: Lycopadiene (C 48 H 78 ) Race S: C 20 saturated n-alkane Excrete high quantities of polysaccharides: Gal> Fuc> Arab> Rham> Uronic acid> 3-O-methyl rhamnose, 6-O-methyl hexose AC761 → HC CCALA778 → EPS

4. Simulating outdoors conditions from Huelva (Andalusia, Spain) using indoors systems in Wageningen Costa de la luz (>3000h Sun y -1 ) 5240 Wh m -2 d -1 Irradiation on horizontal plane

5. BATCH/LUMINOSTAT (I out 30 µmol m -2 s -1 ) Temperature 26ºC pH 7.2 Air flow 0.5 vvm Initial biomass concentration 0.2 g l -1 Modified CHU-13 Medium: (+Se/-Se) Selenium in the medium? INFORS: Flatpanel, 1.8 L No differences in: Biomass concentration Colony size µ = 0.55 d -1 Same CHT content Remove Selenium

6. Stat phase →Samples for Omics Batch Luminostat Optimal temperature? Temperature influences Growth rate (Literature 20-30C)

7. B) SUMMER Sine Curve light Max Intensity 2000 µE/m 2 s Min Intensity 0 µE/m 2 s Temperature Max 36C (at noon) Min 26C D:N (16h:8h) D= 0.25 d -1 A) WINTER Sine Curve light Max Intensity 825 µE/m 2 s Min Intensity 0 µE/m 2 s Temperature Max 23C (at noon) Min 4C D:N (12h:12h) D= 0.25 d -1 Continuous cultures (chemostat) Temperature Light

8. Scenarios Biomass productivity (g l -1 d -1 ) Colony size (µm 2 ) Carbohydrates productivity (g l -1 d -1 ) EPS productivity (g l -1 d -1 ) Ratio EPS/ CHT (%) SNI (mmol NO 3 - g -1 d -1 ) 1.4 ± 0.0290.7 ± 0.30.6 ± 0.010.1 ± 0.01170.5 0.9 ± 0.02108.8 ± 2.40.3 ± 0.010.07 ± 0.0223.30.8 0.4 ± 0.02104.8 ± 0.60.1 ± 0.010.01 ± 0.00101.9 Winter conditions Samples for Omics

9. Biomass productivity (g l -1 d -1 ) Colony size (µm 2 ) Carbohydrates productivity (g l -1 d -1 ) EPS productivity (g l -1 d -1 ) Ratio EPS/ CHT (%) SNI (mmol NO 3 - g -1 d -1 ) 0.9 ± 0.0233.9± 0.80.39 ± 0.10.12± 0.03 30 0.8 1.3 ± 0.277.8 ± 0.40.9 ± 0.10.28 ± 0.05320.6 Scenarios Summer conditions Samples for Omics

10. 1)Samples for Omics (stored -80C) -Transcriptomic -Proteomic -Metabolomic 3) Evolution of EPS/ CHT 2) Size of colonies Sine light and constant temperature (Summer)

11. 0:004:008:0012:0016:0020:000:00 A) ↓↓ COLONY SIZE: during night? Reproductive events? New small colonies? Decreasing floatability in the water column? B) RATIO EPS/CHT keeps constant (30%)

12. Sine light and constant temperature (Summer)

13. Selenium not necessary Optimal temperature 26ºC (but tolerates 16-31ºC) Colony size is affected by light intensity Day:Night Cycles → ↑ Biomass and EPS productivity EPS productivity is favoured by: SNI < 1 High light intensities (When temperature controlled → EPS/CHT ratio in Summer is twofold greater than Winter) → Gal (50%) > Fuc (31%) > GalA (9%) Findings and conclusions

14. SINE LIGHT/TEMPERATURE (Summer) Samples for 24h EPS/CHT Sugar profiles Omics Size of colonies Proof of concept MILKING: Continuous extraction of EPS (Ø= 0.2 µm; 110cm 2 micro-filtration cartridge) Wageningen summer conditions (1100 µE m -2 s -1 ; T 26C) Future work

15. 2016: Outdoor reactors in AlgaeParc http://www.wageningenur.nl/en/Expertise-Services/Facilities/AlgaePARC.htm (Fotosintetica&Microbiologica, Firenze (Italy) www.femonline.it)www.femonline.it

16. Thank you very much Rafael G.Cubero (PhD Biology) Control: pH, Dilution rate, Temperature, Oxygen probes, inclination angle (tilting) Summer season (April to September) 3 FLAT PANELS for B.braunii CCALA 778 → 40L X 3FP =120 L Continuous regime (Chemostat) and continuous extraction (Milking) 2016: Outdoor reactors in AlgaeParc http://www.wageningenur.nl/en/Expertise-Services/Facilities/AlgaePARC.htm