© Cellana - PowerPoint Presentation

Slide1

© Cellana

Inc. 2014

Know Your Enemy: Cellana's Successful Strategy for Dealing with Contaminants in Algal Mass Culture

Charles J. O’KellyDirector, Research2 October 2014Slide2

Know your enemy – O’KellyAlgae Are Crop Plants

Crops Need Protection From Pests / Disease Slide3

Know your enemy – O’KellyInvesting In Crop Protection

Identification (How accurately? How quickly?)BehaviorPrey rangeVirulenceLife cycleTransmissionVector(s)Susceptibilities

Slide4

Know your enemy – O’KellyInvesting In Crop Protection

Identification (How accurately? How quickly?)BehaviorPrey rangeVirulenceLife cycleTransmissionVector(s)Susceptibilities

The USDA spends ca. US$7MM annually

on 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)Slide5

Know your enemy – O’KellyThe State of Algal Crop Protection

Identification difficult Most identifications crude (“ciliates / rotifers / amoebae / foreign algae”)“Guidebooks” nonexistentCarney & Lane 2014 review: 16 taxa of algivorous protozoa namedonly 9 of the 16 named to species

only 3 of the 16 studied in algal mass culture systems2 of the algal mass culture species new to scienceBehavior 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.Slide6

Crop Protection Case Study

1. IdentificationSlide7

Know your enemy – O’KellyWho’s eating the profits - really

?Culture studies on 5 cell types with ingested algaeNo growth on target alga: one amoeba, two ciliatesSlow growth on target alga: one amoebaRapid growth on target alga: one amoebaConsistent with observations in large-scale productionSlide8

Know your enemy – O’KellyWho’s eating the profits - really

?DNA sequence studies assign amoeba to species Neoparamoeba branchiphilaDescribed in 2005Associated with gills of salmonid fishes affected with

amoebic gill disease (not the cause of the disease)No prior record of algivory for any N. speciesSlide9

Crop Protection Case Study

2A. Behavior – Prey RangeSlide10

Know your enemy – O’KellyNeoparamoeba: gourmet or gourmand?

Susceptible algaeDiatomsSome HaptophytesResistant algaeSome haptophytesGreen algae

EustigmatophytesDinoflagellatesCryptophytesSlide11

Know your enemy – O’KellyNeoparamoeba: gourmet or gourmand?

Grows equally well, or better, on bacteria vs. algaeNot all bacteria support growthVariations between strains

in grazing on both algae and bacteria

Marinobacter

sp. 1

Marinobacter sp. 2Halomonas

sp

.

Nitratireductor

sp

. 1

Labrenzia

aggregata

Roseivivax

sp.

Labrenzia

sp.

Marinobacter

hydrocarbonoclasticus

Sulfitobacter

pontiacus

Nitratireductor

sp

. 2

Nb

O3a

Nb

R1

Nb

K3Slide12

Crop Protection Case Study

2B. Behavior – VirulenceSlide13

Know your enemy – O’KellyComponents of virulence

Grazing rate1-3 prey cells grazer-1 h-1Specific growth rateConsiderably 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.Slide14

Know your enemy – O’KellyThe 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 Slide15

Know your enemy – O’KellyThe significance of specific growth rate

Neoparamoeba branchiphila on favored prey has specific growth rate ca. 2 d-1Timing of appearance of

N. branchiphila in large-scale production closely followed

model predictionsDashed line shows limit of detection of contaminants via microscopic

monitoring.

Forehead H, O’Kelly CJ,

Bioresource

Technology

129: 329, 2013

Slide16

Crop Protection Case Study

2C. Behavior – Life CycleSlide17

Know your enemy – O’KellyWYSIWYG

A “one-phase” life cycle: trophic cells (amoebae)No cystsNo resting stagesNo obvious sexual reproductionControl the trophs, control the pestSlide18

Crop Protection Case Study

3. Transmission – Vector and SusceptibilitiesSlide19

Know your enemy – O’KellyAirborne or Waterborne?

Airborne transmission of Neoparamoeba?No cystsNo desiccation-resistant stagesNo evidence for amoebae in air column samplesWaterborne transmission of Neoparamoeba?

Ability of amoebae to subsist on bacteria suggests possibility of ‘reservoirs’ Must survive UV, several degrees of water filtrationSlide20

Know your enemy – O’KellyWaterborne!

Up to 12 Neoparamoeba cells per 100 L detected in filtered seawater used in productionOnly 1 cell per 1000 L needed for contamination in 14 days per modelNeoparamoeba cells survived 300 mJ UV in laboratory tests

Production UV was delivering ca. 150 mJSlide21

ActionsSlide22

Know your enemy – O’Kelly

ALDUO™

Closed

System

Photobioreactors

(PBRs)

+

Open

System

Open Raceway Ponds

Clean water

Clean procedures

Resistant strains

Clean water

Clean inoculum

P

ond cycle management Slide23

Know your enemy – O’Kelly

ALDUO™

Closed

System

Photobioreactors

(PBRs)

+

Open

System

Open Raceway Ponds

Clean water

Clean procedures

Resistant strains

Clean water

Clean inoculum

P

ond cycle management

Strategies informed by Knowing The EnemySlide24

Know your enemy – O’Kelly

ALDUO™

Closed

System

Photobioreactors

(PBRs)

+

Open

System

Open Raceway Ponds

Clean water

Clean procedures

Resistant strains

Clean water

Clean inoculum

P

ond cycle management

12 non-extremophile algal strains grown at large scaleSlide25

Thanks to:

Geneva

Mottet Dan Burton Yana Eglit