A journey from signature lipid profiling to marine and plant biotechnology including with career transportation to Tasmania, Australia

* OCEANS & ATMOSPHERE

Peter D Nichols

AAOCS – September 11-13, 2017

Developments & Applications with Marine Lipids

• Biochemical tracers - Signature lipids, biological markers

• Australian marine oil - Product development (wax esters, shark liver oils, omega-3)

• Fecal markers - Tracking sewage

• The ‘Good Oil’ - Long-chain Omega-3

• Aquaculture - Larval nutrition, fish nutrition, new feeds

• Long-chain Omega-3 oils – Alternate sources

Major Collaborators• Biochemical Tracers / Signature Lipids – Basil Johns, DC White, Tom McMeekin,

Carol Mancuso, Rick Phleger, Jeff Drazen, Mark Hindell, Jock Young, Michaela Guest, Harry Burton, Patti Virtue

• Marine Oils – Hamish & Guy Drummond, Ortwin Bode, Richard Saul

• Fecal Markers – Rhys Leeming, Mark Rayner, Val Latham, George Cresswell, Nick Ashbolt

• Aquaculture & Livestock – Steve Battaglene, Andrew Jeffs, Arthur Ritar, Rick Phleger, Chris Carter, Aduli Malau-Aduli

• The Good Oil (Omega-3): FRDC – Patti Virtue, Ben Mooney, Nick Elliott, Gordon Yearsley

• Long-chain Omega-3 oils: GRDC / Nuseed Aust. – Allan Green, Surinder Singh, James Petrie, Sue Blackburn, Stan Robert, Peter Mansour, Rob Defeyter

• Omega-3 Centre – Wendy Morgan, Kevin Krail, Lalen Dogan, Andy Sinclair

• Nuseed Aust. – Malcolm Devine, Jason McAllister, Joe Agnew, many others

PhD Student Collaborators

• Biochemical tracers – Patti Virtue, Graham Green, Heidi Pethybridge, Gareth Wilson, Jenny Skerratt, Kerrie Swaddling, Mary-Anne Lea, Katrina Phillips, Ben Mooney, Kathryn Wheatley, Toshi Yosida, Matt Brown, Juan Dorantes Aranda, Cathryn Wynne-Edwards, Camille White, Nicole Hellessey*, Jessica Ericson*, Ben Sellers, Lauren Myer*

• Fecal markers – Rhys Leeming, Leonard Jayasinghe

• Aquaculture & Livestock – Matt Nelson, Basseer Codabaccus, Ramez Alhazzara, Waldo Ortin Nuez, Will Bignell, Ben Holman, Arash Kashani, John Otto, Aaron Flakemore, Don Nguyen, Quang Nguyen, Hung Le Van

• Long-chain Omega-3 oils – Matt Miller, Will Bignell, Julie Kimber, Kim Lee-Chang, Tom Lewis, David Nichols, Loris Fossier Marchan

Signature Microbial Lipids (FSU to CSIRO)• Biomass - Phospholipid fatty acid & Phospholipid ether lipid content

(PLFA - Eubacteria) (PLEL - Archaea)

• Community structure – FA profile

• Metabolic status – key component ratios

PLEL - HPLC separation & quantitationMancuso et al. J Lipid Res 1986 – Archaea signatures

PLFA - GC separation & quantitation

DMDS/GC-MS confirmation/quantitationVirtue et al. JMM 1996 – Novel methanotroph signatures

Signature Lipids – Biochemical Tracers (Food web applications)

‘You are what you eat’

Marine Terrestrial

Traditional techniques problematic, e.g. gut content analysis

• Prey species - unique lipid / fatty acid (FA) compositions

• Many FA transferred from prey to predator with minimal modification

• Predator FA represents a temporal integration of diet

• Can be quantitative, allowing temporal integration (cf gut contents)

• Signature fatty acids: combinations of FA preserved up food chain

• Complements other approaches

Lipids as Dietary Tracers

FA profile (by GC)

16:0

18:0

18:19

20:19

22:111

20:53 (EPA)

22:63 (DHA)

Signature Lipids - Methodology

• Compare predator profiles to

known & potential prey species.

• Similar applications with microbial

signature lipids.

• Statistical analyses

e.g. MDS, ANOVA,

Primer, etc.

• Other treatment/modeling (future)

Myctophids:

18:19

16:0

Bathylagus:

20:19

18:19

Squid:

22:63 (DHA)

16:0

Decapods:

20:53 (EPA)

16:0

Amphipods:

20:53 (EPA)

22:63 (DHA)

SAT

MUFA

PUFA

Signature FA:

SwordfishRick Phleger et al.

Myctophid

prey

Northern sampleSwordfish

& prey

(2010, Prog Oceanog, Young et al)

Coryphaenoides armatus

4100 m

Station M

Rattails – West coast USA

MEPS 2009 Drazen et al.

-30 -20 -10 0 10 20

-20

-10

0

10

20

30

40 SpeciesA. abyssorum

O. bathybia

Travesia sp.

B. nomadus

Mundiopsis sp.

Shrimp

Amphipods

D. gigas

C. armatus

C. yaquinae

PC1

55.9%20:1ω11c

18:1ω7

22:6ω3

PC

2

15.4

%

18:1

ω9c

20:5

ω3

20:4

ω6

20:1

ω9c

16:1

ω7

16:0

P. vitrea

16:0

Rattails

Squid

Paradiopatra sp.

Laetmonice sp.

Ophiocantha sp.O.mutabilis.

Protankyra sp.Cnidaria

Holothuria

Ophiuroidea and Polychaeta

Crustacea

Rattails: main findings(Jeff Drazen, Rick Phleger et al.)

Giant Humboldt squid numbers

increasing as top predators decline

due to overfishing. Squid spawn &

die after 1-2 years & sink

Signature lipids show rattail

diet likely to be carrion,

e.g. squid. Diet not known

in past due to loss of gut

contents upon ascent

(MEPS 2009 Drazen et al)

Summary – Signature Lipids

• Signature lipid approach being continually developed & applied to key Australian & southern ocean fish, squid & higher iconic species (great white shark, whale shark, manta ray)

• Food-web, taxonomic & stock discrimination information can be gained using signature lipids –

Complements other methods

• Better understanding on transport & use of essential lipids in the marine food-web & in aquaculture

• This conference – Great white shark poster (Lauren Myer) & Krill oil oral (Jess Ericson & Nicole Hellessey)

Epi-CoprostanolH

OH

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

Cholesterol5a-Cholestanol

HOH

CH3

CH3

CH3

CH3

CH3

5b-Coprostanol

(humans)

HOH

CH3

CH3

CH3

CH3

CH3

OH

Fecal

sterols

Insert presentation title

(J. Chromatog; Chem. Aust.; Water)

80

60

40

20

100

Proportions of 5b-stanols in fecal matter

Use fecal sterol profiles + microbial indicators to determine

sources of fecal pollution in marine and aquatic environments

Leeming et al. Water Res

The Good Oil I & II - Australian Seafood

• Two “Guides” for consumers/industry

• By-product / by-catch included

• Results available for marketing seafood

• Results in “Aust. Seafood Handbook”

LC Omega-3 oils

EPA+DHA

(mg per 100g)

Fish 235

Oysters 150

Prawns 130

Lobster 105

Turkey 35

Beef 22

Chicken 19

Lamb 18

Pork 0

Veal 0

Analytical Issues - NZ Fish oil capsule study / Oil Quality

NZ paper Nature Scientific Reports (Albert et al., Jan 2015)

Two issues reported:

(i) EPA+DHA did not meet label claim, markedly so for many (69%) products

(ii) Oils highly oxidized (high PV, high pAV) – health implications raised, including

in media.

NSR – NZ Product

results

In comparison ………..

• Previous Australian analyses – Industry, CSIRO, re-testing, other analyses → products generally all met specs/claims

• TGA – 2015. All 15 products tested met PV specs and EPA+DHA claims. No action with industry undertaken. But - TGA results not published.

• GOED (+ Coun. Respon. Nutr.) 2016. 2171/2187 products met PV spec(Lipid Tech – Review paper) 2092/2117 products met pAV spec

• O3C - Updates/responses placed at O3C website, and sent to - AAOCS & AOCS, Omega List, NYT/Frontline, CMG, CMA, ABC Four Corners, others

Oil Quality. I.

New O3C Analyses – 2016

• O3C – (Analyses completed June 2016). All 10 products (5 x 18/12 oils, 5 x concentrates) were purchased in Melbourne in May 2016 and tested by standard/accepted (BP) methods by a validated laboratory (ALS) for

– PV, pAV, EPA+DHA

• All 10 products met n-3 (EPA+DHA) content claims and PV specs.

8/10 met pAV spec. 2 contained additives, well known to interfere with pAV.

GOED cautions regards pAV analyses for oils with additives.

• Aust & NZ fish oil supplements –

Do meet LC Omega-3 Claims & are NOT heavily oxidized

Nichols, Dogan, Sinclair - Nutrients 2016

Oil Quality. II.

New O3C Analyses – 2016

n-3 PUFA Content

10/10 (100%) meet

label claim

In agreement with 2015

TGA analyses

NZ NSR paper:

29/32 products

90% did NOT meet

EPA+DHA label claim.

69% markedly under.

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9 10

%La

be

l n-3

PU

FA c

on

ten

t

Fish Oil Product

18/12 Oils Enriched Oils

Nichols, Dogan, Sinclair - Nutrients 2016

Summary – Oil Quality. III.

New O3C Analyses – 2016

• Aust & NZ fish oil supplements –

- Do meet LC Omega-3 Claims & are NOT heavily oxidized

- 2017: New GOED study published in NSR (40+ NZ products)

similar findings to O3C

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10P

ero

xid

e V

alu

e (

me

q/l

)

Fish Oil Product

Peroxide Value10/10 (100%) meet PV spec

NZ NSR paper: 30/36 products (83%) exceeded PV

Nichols, Dogan, Sinclair - Nutrients 2016

FA analyses – care is needed by analysts

- Issues likely due to analytical methods / sample preparation used for

NZ LC Omega-3 profiling

- Cross checking FA, PV, pAV data is needed where ‘anomalies’ occur

- Consideration of use of standard methods, reference materials, etc

Areas not covered today

- Institutional review process, Journal review process, Journal editorial

process, Media review process

Australian & NZ fish oil supplements

- Generally DO meet Omega-3 Claims & are NOT highly oxidized

Summary – Oil Quality. IV.

Positive Australian & NZ News re LC Omega-3

General:

Joint Omega-3 Symposium O3C-AAOCS,

Newcastle, November 2013: Published in

Nutrients Special Issue (2014). Book also

published in late 2014.

“Recent Advances in Omega-3: Health Benefits, Sources,

Products and Bioavailability”.

http://www.mdpi.com/journal/nutrients/special_issues/omega-3_conference

12 papers in Special Issue. Australian / NZ emphasis.

Land Plants Containing Long-chain Omega-3 Oils

AGRICULTURE / OCEANS & ATMOSPHERE

CSIRO Omega-3 Team

LC Omega-3 Oils: Coverage today

• Health – Nutritional need for & inadequate intake of LC Omega-3 (≥C20)

• Seafood – Update on LC omega-3 profiles (farmed fish)

• Supply – Resource (sustainability) aspect; alternate sources needed; Australian perspective

• Plant Technology – Results so far; further R&D occurring to increase LC Omega-3 yields in oilseed crops

• Approval & Acceptance – CSIRO consumer research on the plant technology

• Summary

Essential Fatty Acid Families

H3C COOH

18:33

3 family

a-Linolenic (ALA)

COOH

20:53 Eicosapentaenoic

EPA

H3C

COOH

22:63 Docosahexaenoic

DHA

H3C

H3C

H3C

COOH

COOH

6 family

20:4 6

18:26 Linoleic (LA)

Arachidonic

AA

Thrombotic

Inflammatory

Corn Oil

Safflower Oil

Sunflower Oil

Anti-thrombotic

Anti-inflammatory

Meat, Eggs,

Brains

Microalgae

Seafood

LC Omega-3 Oils: ≥C20, two or more double bonds

Flaxseed Oil

Canola Oil

Soybean Oil

Increasing demand from:

• Ageing populations

• High-growth economies

• Dietary supplement markets (especially preventative health)

• Pharmaceutical pipeline

Supporting evidence:

• Now over 30,000 papers (80+% positive outcomes)

Why long-chain omega-3?

Plants: short-chain ALA, some SDA (C18 )

– Limited health benefits

– Low conversion, especially to DHA

Global Fisheries – are there enough fish anyway - ?

“We estimate that large

predatory fish biomass

today is only about 10%

of pre-industrial levels.”

Aquaculture –Has the Good Oil gone missing?

July 2002, INFORM, AOCS:

“warned some species of farm-raised fish may have little or no omega-3 fatty acids……”

(Stoll, Harvard Med. School)

Australian Farmed Fish – Good Oil (2002-15)

Global fish catches static or declining • Fish oil used in aquaculture – replaced by other oils

LC Omega-3 oils have decreased cf 2002

• Content

decreased

by 10-50+%

in 2010-15

• 3/6

ratio <1

in 2012-15

Nichols et al. Nutrients 2014 - (2002-2013 results)

Changing diets:

• % LA

increasing

• % EPA+DHA

decreasing

0

500

1000

1500

2000

2500

2002Winter

2010Aut

2010Spr

2011Aut

2011Sum

2012Aut

2012Spr

2013Aut

2013Spr

2014Aut

2014Spr

2015Aut

2015Sum

Farmed Atlantic salmon - %LA, %EPA, %DHA

Changing diets:

• % LA

increasing

• % EPA+DHA

decreasing

• 3/6 ratio

decreasing with

increased Chicken Fat

in feed

• 3/6 ratio <1 since

mid 2013

Environmental

SignatureNichols et al. Nutrients 2014 - (2002-2013 results)

0

2

4

6

8

10

12

14

16

2002Winter

2010Aut

2010Spr

2011Aut

2011Sum

2012Aut

2012Spr

2013Aut

2013Spr

2014Aut

2014Spr

2015Aut

2015Sum

% LA

0

5

10

15

20

25

30

2002Winter

2010Aut

2010Spr

2011Aut

2011Sum

2012Aut

2012Spr

2013Aut

2013Spr

2014Aut

2014Spr

2015Aut

2015Sum

% EPA + DHA

Other Sources of LC Omega-3. I.

Microalgae oil: several Australian University-Industry consortia

Phototrophs (open ponds)

Heterotrophs (fermenters)

Recent move in algal biofuels R&D towards HTP rather than forming biodiesel (FAME)

LC Omega-3 directed activities (e.g. new CRC project underway)

Krill Oil : new collaboration of Aker-IMAS / Utas (+partners) underway

(ARC-Linkage), resource monitoring & catch limits

overseen by CCAMLR (Hobart HQ); MSC certified

fishery (Aker)

- Wednesday – two krill PhD students to present

Other Sources Needed. II. CSIRO Agriculture: Oilseed LC Omega-3 Oils

• LC Omega-3 oils essential for human & marine fish health

• Global fish catches static or declining

• Microalgae biosynthesize the LC omega-3 oils that fish consume & store. Fish do not make EPA+DHA

CSIRO-wide project (1997 idea; commenced 2003)Goal: Isolate omega-3 genes from microalgae & transfer them to crop plants to sustainably produce LC omega-3 oils

www.nuseed.comwww.grdc.com.auwww.csiro.au

Partnership commenced - 2010

Genes Combinations

Crop

PlantTransformation

Field trials

DNA Constructs

Application trials

The LC Omega-3 Oilseed Journey

Detailed Oil Analyses

LC Omega-3 in Marine Microalgae

0

5

10

15

20

25

30

35

Algal Classes

Perc

en

t co

mp

osit

ion

AA

EPA

DHA

Diatoms & Golden-brown

Red

Green

Yellow-green

18:118:0 18:216:0 a-18:3

LC Omega-3 Oils - engineering in land plants

∆6-des

∆5-elo

∆4-des

∆5-des

∆6-elo

18:4

DHA

EPA

SDA

20:5

20:4

22:6

22:5Marine

Algae

Land Plants

Land Plant Achievements

First land plant with EPA + DHA in its seed oil

2.4% EPA

0%

0.5% DHA

0.0

1.0

2.0

3.0

Land Plants CSIRO Model Plant

Perc

en

t L

on

g-c

hain

Om

eg

a-3

Arabidopsis thaliana

Robert et al. FPB - 2005

DHA Biosynthesis - Isolation of an efficient synthesis pathway

Rapid assessment of

seed constructs in leaf

Timeline of DHA biosynthesis in oilseedsPetrie et al.

PLOS One 2013; Inform 2013

20152013 2018-19

CSIRO Consumer Research

Trials – Australia, USA, Europe, Asia

Take home message:

A large proportion of the population are accepting of GM

land plant LC omega-3 oil that:

• Provides a health benefit,

• Was supported by health claims from a trusted source,

• Was indirectly consumed (e.g. food for farming fish)

(Cox et al. 2007, 2008, 2010)

• LC Omega-3 health benefits - ongoing recognition; marine resource issues

• Farmed seafood in Aust & NZ generally higher LC Omega-3 content than wild harvest seafood; Aust & NZ fishing considered sustainable

• LC Omega-3 content in farmed fish has decreased, as has the previously high omega-3 / omega-6 ratio; need to revisit

• Alternate sources of LC Omega-3 required for future aquaculture - Excellent progress with new land plants (Canola-DHA, CSIRO-

Nuseed- GRDC); field & application trials completed – 2014-17

1 Ha of Canola-DHA at 12% DHA = DHA from 10,000 fish

• Aust & NZ fish oil supplements & from new land plant oils –

- Do meet LC Omega-3 Claims & are NOT oxidized

Summary

Family

Carol, Lauren, Rebecca

Thank you

Peter.Nichols@csiro.au

The CSIRO-Nuseed

LC Omega-3 Team

Surinder SinghSrinivas BelideRob DefeyterAllan GreenDawar HussainYoko KennedyGeraldine LesterQing LiuLina Ma

Anne MackenziePeter MansourPeter NicholsNathalie NiesnerJames PetriePushkar ShresthaLijun TianAdam WhiteXue-Rong Zhou