Know Your Enemy Cellanas Successful Strategy for Dealing

Know Your Enemy: Cellana's Successful Strategy for Dealing with Contaminants in Algal Mass Culture Charles J. O’Kelly Director, Research 2 October 2014 © Cellana Inc. 2014 Page 1 © Cellana 2014

Know your enemy – O’Kelly Algae Are Crop Plants Crops Need Protection From Pests / Disease ∙ Page 2 © Cellana 2014

Know your enemy – O’Kelly Investing In Crop Protection • Identification (How accurately? How quickly? ) • Behavior – Prey range – Virulence – Life cycle • Transmission – Vector(s) – Susceptibilities ∙ Page 3 © Cellana 2014

Know your enemy – O’Kelly Investing In Crop Protection • Identification (How accurately? How quickly? ) • Behavior The USDA spends ca. US$7 MM annually on the Corn Insects and Crop Genetics Research Unit in Iowa – research focused mainly on a single insect, the corn borer. – Prey range – Virulence – Life cycle • Transmission – Vector(s) – Susceptibilities European corn borer, Ostrinia nubilalis (larva) ∙ Page 4 © Cellana 2014

Know your enemy – O’Kelly The State of Algal Crop Protection • Identification difficult 10 µm – Most identifications crude (“ciliates / rotifers / amoebae / foreign algae”) – “Guidebooks” nonexistent – Carney & Lane 2014 review: ▪ 16 taxa of algivorous protozoa named ▪ only 9 of the 16 named to species ▪ only 3 of the 16 studied in algal mass culture systems ▪ 2 of the algal mass culture species new to science Paraphysoderma sedebokerense, a chytrid fungus, parasitizing Haematococcus. Described 2011. Image: Hoffman Y et al. , Mycological Research 112: 70, 2008. • Behavior and Transmission poorly understood Page 5 © Cellana 2014

Crop Protection Case Study 1. Identification Page 6 © Cellana 2014

Know your enemy – O’Kelly Who’s eating the profits - really? • Culture studies on 5 cell types with ingested algae – No growth on target alga: one amoeba, two ciliates – Slow growth on target alga: one amoeba – Rapid growth on target alga: one amoeba ▪ Consistent with observations in large-scale production Page 7 © Cellana 2014

Know your enemy – O’Kelly Who’s eating the profits - really? • DNA sequence studies assign amoeba to species Neoparamoeba branchiphila – Described in 2005 – Associated with gills of salmonid fishes affected with amoebic gill disease (not the cause of the disease) • No prior record of algivory for any N. species Page 8 © Cellana 2014

Crop Protection Case Study 2 A. Behavior – Prey Range Page 9 © Cellana 2014

Know your enemy – O’Kelly Neoparamoeba: gourmet or gourmand? • Susceptible algae – Diatoms – Some Haptophytes • Resistant algae – – – Some haptophytes Green algae Eustigmatophytes Dinoflagellates Cryptophytes Page 10 © Cellana 2014

Know your enemy – O’Kelly Neoparamoeba: gourmet or gourmand? • Grows equally well, or better, on bacteria vs. algae • Not all bacteria support growth • Variations between strains in grazing on both algae and bacteria Marinobacter sp. 1 Marinobacter sp. 2 Halomonas sp. Nitratireductor sp. 1 Labrenzia aggregata Roseivivax sp. Labrenzia sp. Marinobacter hydrocarbonoclasticus Sulfitobacter pontiacus Nitratireductor sp. 2 Page 11 © Cellana 2014 Nb O 3 a Nb R 1 Nb K 3

Crop Protection Case Study 2 B. Behavior – Virulence Page 12 © Cellana 2014

Know your enemy – O’Kelly Components of virulence • Grazing rate – 1 -3 prey cells grazer-1 h-1 • Specific growth rate • Considerably more variability in specific growth rate among grazers than in grazing rate Grazing of Neoparamoeba (03 a, R 1, KPF 3), Paramoeba (08 pp), and Thecamoeba (8 t) amoebae on the diatom Skeletonema marinoi, incubated in the dark at 23 °C. Control: no amoebae. Page 13 © Cellana 2014

Know your enemy – O’Kelly The significance of specific growth rate • Appearance of organisms in batch and semi-continuous culture a function of specific growth rate (initial inoculum a relatively small contributor) – Model assumes no loss of cells • Graph at right shows progression of organism with specific growth rate of 2 d-1 and initial inoculum of 0. 001 cells L-1 Page 14 © Cellana 2014 Forehead H, O’Kelly CJ, Bioresource Technology 129: 329, 2013

Know your enemy – O’Kelly The significance of specific growth rate • Neoparamoeba branchiphila on favored prey has specific growth rate ca. 2 d-1 • Timing of appearance of N. branchiphila in large-scale production closely followed model predictions • Dashed line shows limit of detection of contaminants via microscopic monitoring. Page 15 © Cellana 2014 Forehead H, O’Kelly CJ, Bioresource Technology 129: 329, 2013

Crop Protection Case Study 2 C. Behavior – Life Cycle Page 16 © Cellana 2014

Know your enemy – O’Kelly WYSIWYG • A “one-phase” life cycle: trophic cells (amoebae) – No cysts – No resting stages – No obvious sexual reproduction • Control the trophs, control the pest Page 17 © Cellana 2014

Crop Protection Case Study 3. Transmission – Vector and Susceptibilities Page 18 © Cellana 2014

Know your enemy – O’Kelly Airborne or Waterborne? • Airborne transmission of Neoparamoeba? – No cysts – No desiccation-resistant stages – No evidence for amoebae in air column samples • Waterborne transmission of Neoparamoeba? – Ability of amoebae to subsist on bacteria suggests possibility of ‘reservoirs’ – Must survive UV, several degrees of water filtration Page 19 © Cellana 2014

Know your enemy – O’Kelly Waterborne! • Up to 12 Neoparamoeba cells per 100 L detected in filtered seawater used in production – Only 1 cell per 1000 L needed for contamination in 14 days per model • Neoparamoeba cells survived 300 m. J UV in laboratory tests – Production UV was delivering ca. 150 m. J Page 20 © Cellana 2014

Actions Page 21 © Cellana 2014

Know your enemy – O’Kelly ALDUO™ Closed System Photobioreactors (PBRs) + • Clean water • Clean procedures • Resistant strains Open System Open Raceway Ponds • Clean water • Clean inoculum • Pond cycle management Page 22 © Cellana 2014

Know your enemy – O’Kelly ALDUO™ Closed System Photobioreactors (PBRs) + Open System Open Raceway Ponds Strategies informed by Knowing The Enemy • Clean water • Clean procedures • Resistant strains • Clean water • Clean inoculum • Pond cycle management Page 23 © Cellana 2014

Know your enemy – O’Kelly ALDUO™ Closed System Photobioreactors (PBRs) + Open System Open Raceway Ponds 12 non-extremophile algal strains grown at large scale • Clean water • Clean procedures • Resistant strains • Clean water • Clean inoculum • Pond cycle management Page 24 © Cellana 2014

Thanks to: Geneva Mottet Dan Burton Yana Eglit Page 25 © Cellana 2014