PRODUCTION OF HAEMATOCOCCUS ASTAXANTHIN IN SOUTHERN JAPAN:

PROOF OF CONCEPT

Miguel Olaizola (Heliae Development, LLC) Kentaro Makabe (Sincere Corporation)

Scott Kuhlman (Heliae Development, LLC) Isao Fukuda (Sincere Corporation)

1 Heliae © 2015 – Confidential

JV basics

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Heliae Development, LLC Sincere Corporation

Alvita, JV

Project summary

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1.  Purpose: (BDD) Biological Due Diligence before greenlighting production facility a.  Contamination b.  Local water sources

2.  Time line a.  Preparatory work during October-December 2014 in Gilbert b.  First inoculation on Feb 20, 2015 c.  As of July 2nd, 14 runs completed

3.  Facilities a.  6 raceway reactors: 4.4 m2 surface area b.  Greenhouse c.  Temporary laboratory

4.  Personnel a.  10 Heliae and 4 Sincere personnel involved in the set up and grow out b.  Joint engineering team working on production facility c.  Several support personnel from power plant

5.  Data collected a.  Numerous data collected {

Light issues

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Location: similar latitude (Gilbert at 33°21’ N, Saga at 33°16’ N)

BUT, 40% less light in Saga City vs. Phoenix

Light issues: outcomes

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Possible outcomes: 1.  Do nothing: expect less production in Saga City

a.  Unacceptable (economics)

2.  Strategy: augment natural sunlight with artificial illumination

a.  Understanding of photosynthetic efficiency b.  Augment number of hours of light c.  Do not augment maximum light intensity d.  Test adding sodium acetate (fixed carbon)

Light issues: experiments in Arizona

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Augment natural sunlight Extend day length for 4 hrs using metal halide lamps.

Compared Phoenix vs Saga City vs Saga City plus lamps.

Light issues: Gilbert results

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Green growth Control: Phoenix conditions 40%PAR: Phoenix-40% = Saga City conditions 40%PAR: Phoenix-40% = Saga City conditions PLUS 4 hrs light

Light issues: experimental results

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Red growth (astaxanthin induction) Control: Phoenix conditions 40%PAR: Phoenix-40% = Saga City conditions 40%PAR: Phoenix-40% = Saga City conditions PLUS 4 hrs light

Low Light Issues: Possible Solution

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Augmented Natural Sunlight 1.  Extend day length for 4 hours using

metal halide lamps •  Compared Phoenix vs Saga City

vs Saga City w/ lamps •  Simulated Saga City productivity

about 30% less than Phoenix •  Simulated Saga City w/ 4 hrs

light = Phoenix 2.  Extend day length in Saga w/ Phillips

LEDs

Tests in Saga City

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1.  Extend day length for 4 hrs using Phillips LED lamps a.  2 ponds outfitted with 6 lamps each b.  Number of runs = 10 c.  Lamps on in late afternoon for 4 hrs

2.  Use sodium acetate to enhance Haematococcus productivity

a.  2 ponds had acetate added (1 g/L, every third day) b.  Number of runs = 2 c.  Added 1 g/L every 3 days to pond cultures

3.  All inoculum produced at Gilbert facility and shipped to Saga City for reddening phase.

Saga environmental data

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Main point Strong seasonality based on 5 months of data

Saga Results: Biomass and Astaxanthin

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14 Runs 1.  Daily biomass (g/L): reach

over 0.8 g/L 2.  Daily astaxanthin (%DW):

reach >4% consistently 3.  No contamination issues to

date

Saga Results: Biomass and Astaxanthin

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Quick look: regressions

•  Start date •  Date •  Culture age •  Supplemental light (hr) •  Daily light in GH •  Daily light in GH (2) •  Average Temp time point •  Average Temp daily •  Depth (cm) •  Standing biomass (g/L) •  Astax (%) •  Biomass productivity (g/L/d) •  Biomass productivity (g/m2/d) •  Astax productivity (g/m2/d)

Saga Results: Biomass productivity

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1.  Higher temperature = more productivity

2.  More light = higher productivity (but saturates)

Saga Results: Astaxanthin productivity

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1.  Higher temperature = more productivity

2.  More light = higher productivity (but saturates)

Saga Results: Light augmentation

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10 Runs

1.  Divide runs between “early” and “late” 2.  Less difference when day length and

sun is higher 3.  Possibly indicates light saturation

Saga Results: Acetate augmentation

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2 Runs

1.  Acetate enhanced biomass productivity

2.  Acetate did not enhance astaxanthin accumulation

3.  Acetate did enhance astaxanthin productivity

Saga Results: Summary

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1.  Strong seasonality in astaxanthin productivity Correlates with temperature and with light

2.  Strong seasonality in biomass productivity Correlates with temperature and with light

3.  Effectiveness of using lights varies with season a.  Points to light saturation b.  We do not recommend installing lights

4.  Acetic acid might be useful to boost biomass and astaxanthin productivity

5.  Results in Saga support initial projections from Gilbert light testing

Acknowledgements

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!  The City of Saga !  Mayor Toshiyuki Hideshima !  Deputy Mayor Yasumori Mikuriya !  Council Chairman Toshito Kuroda !  Coordinator Junji Maeda !  Satoko Morodomi

!  Saga City Biomass Industry City Promotion Section !  Takahiro Motoyama !  Hidefumi Ejima !  Shuuji Maeda !  Hiroki Iguchi !  Shinji Koga !  Atsuko Kitamura

!  Other Sincere and Heliae personnel !  Takeshi Hironaka !  Masaaki Shiga !  Tetsuko Takahashi !  Dylan Jackson !  Lyn-Marie McKenny !  Van Nguyen !  James Riley !  Anthony Shaver !  Alex Sitek !  Alicja Sztorc