page 1 © cellana 2014 © cellana inc. 2014 know your enemy: cellana's successful strategy for...

© Cellana 2014© Cellana Inc. 2014

Know Your Enemy: Cellana's Successful Strategy for

Dealing with Contaminants in Algal Mass Culture Charles J. O’Kelly

Director, Research2 October 2014

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Know your enemy – O’Kelly

Algae Are Crop Plants

Crops Need Protection From Pests / Disease

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Investing In Crop Protection• Identification (How accurately? How quickly?)• Behavior

– Prey range– Virulence– Life cycle

• Transmission– Vector(s)– Susceptibilities

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© Cellana 2014


Investing In Crop Protection• Identification (How accurately? How quickly?)• Behavior

– Prey range– Virulence– Life cycle

• Transmission– Vector(s)– Susceptibilities

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The USDA spends ca. US$7MM annuallyon the Corn Insects and Crop Genetics Research Unit in Iowa – research focused mainly on a single insect, the corn borer.

European corn borer, Ostrinia nubilalis (larva)

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The State of Algal Crop Protection• Identification difficult

– 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

• Behavior and Transmission poorly understood

10 µm

Paraphysoderma sedebokerense, a chytrid fungus, parasitizing Haematococcus. Described 2011. Image: Hoffman Y et al., Mycological Research 112: 70, 2008.

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Crop Protection Case Study1. Identification

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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

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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


Crop Protection Case Study2A. Behavior – Prey Range

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Neoparamoeba: gourmet or gourmand?• Susceptible algae

– Diatoms– Some Haptophytes

• Resistant algae– Some haptophytes– Green algae– Eustigmatophytes– Dinoflagellates– Cryptophytes

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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.

Marinobacterhydrocarbonoclasticus

Sulfitobacter pontiacus

Nitratireductor sp. 2

Nb O3a

Nb R1

Nb K3


Crop Protection Case Study2B. Behavior – Virulence

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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 (03a, R1, KPF3), Paramoeba (08pp),and Thecamoeba (8t) amoebae on the diatom Skeletonemamarinoi, incubated in the dark at 23 °C. Control: no amoebae.

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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

Forehead H, O’Kelly CJ, Bioresource Technology 129: 329, 2013

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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. Forehead H, O’Kelly CJ, Bioresource Technology 129: 329, 2013


Crop Protection Case Study2C. Behavior – Life Cycle

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WYSIWYG• A “one-phase” life cycle: trophic cells (amoebae)

– No cysts– No resting stages– No obvious sexual reproduction

• Control the trophs, control the pest


Crop Protection Case Study3. Transmission – Vector and Susceptibilities

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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

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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 mJ UV in

laboratory tests– Production UV was delivering ca. 150 mJ


Actions

© Cellana 2014


ALDUO™ Closed System

Photobioreactors (PBRs)

+ Open SystemOpen Raceway

Ponds

• Clean water• Clean procedures• Resistant strains

• Clean water• Clean inoculum• Pond cycle management


Thanks to:

Geneva Mottet Dan Burton Yana Eglit

page 1 © cellana 2014 © cellana inc. 2014 know your enemy: cellana's successful strategy for...

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