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
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
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