optimized extraction of microalgae’s metabolites: a … r., lanneluc i., thiery v., lafferriere...

Optimized extraction of Optimized extraction of microalgae’smicroalgae’s metabolites: a crucial step in metabolites: a crucial step in HighHigh--Throughput Screening programs dedicated to Throughput Screening programs dedicated to

phytoplankton phytoplankton chemodiversitychemodiversity

Track 12-1 Marine natural products and biomolecules

Serive B., Kaas R., La Barre S., Bach S., Cadoret J-P.

2- Laboratory of Physiology and Biotechnology of Algae

Bioresources exploration

Van de Peer et al, 2000

Microalgaes: a high potential of biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Microalgae biodiversity

Dunaliella salinaSource : IFREMER

- Laboratory of Physiology and Biotechnology of Algae

Rhodomonas salinaSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Tetraselmis sp.Source : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Closterium baillyanumSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Euglena proximaSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Porphyridium purpureumSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Unknown cyanobacteriaSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Arthrospira platensisSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Staurastrum vestitum

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Chaetoceros muelleriSource : gt-online.pl

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Skeletonema costatumSource : IFREMER

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Asterionella formosaSource : planktonforum.eu

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Alexandrium minutumSource : Gert Hansen

Microalgae biodiversity

- Laboratory of Physiology and Biotechnology of Algae

Microalgae biodiversity

17- Laboratory of Physiology and Biotechnology of Algae

O

CH3

O

N

O

O

CH3

O

CH3

CH3

CH3

CH3CH3 HO H

O

=

Pharmacodiversity

Chemodiversity

Biodiversity

=

12 phyla eucaryota 1 phylum procaryota

Polyketides, terpenes, alkaloides, isoprenoids, oxylipins, peptides, macrolides, microlides,

lectines, glycolipids, polysaccharides, PUFAs, mycosporine-like AA, pigments

Potential against cancers, Parkinson, Alzheimer, neurodegenerative diseases, bacterial and viral

diseases, as immunostimulant, as fluorescent probes

18- Laboratory of Physiology and Biotechnology of Algae

Lutein

Zeaxanthin

Landrum & Bone, 2001

Microalgae chemodiversity and pharmacodiversity

Anti Age-related Macular Degeneration

19- Laboratory of Physiology and Biotechnology of Algae

Violaxanthine

Pasquet V., Morisset P., Ihammouine S., Chepied A., Aumailley L., Berard J-B., Serive B., Kaas R., Lanneluc I., Thiery V., Lafferriere M., Piot J-M., Patrice T., Cadoret J-P., Picot L., 2011.

Antiproliferative activity of Violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts. Marine Drugs, 9, 819-831.

Moreau et al, 2006 FucoxanthineO. aurita

D. tertiolecta

Microalgae chemodiversity and pharmacodiversity

20- Laboratory of Physiology and Biotechnology of Algae

Mimouni et al, 2012. The Potential of Microalgae for the Production of Bioactive Molecules of Pharmaceutical Interest. Current Pharmaceutical Biotechnology 13(15): 2733-2750.

Microalgae chemodiversity and pharmacodiversity

PhotoprotectantPhotoprotectant

AntiproliferativeAntiproliferativeactivityactivity

Usijirene Palythene Asterina-330 Mycosporine-glycine

Palythine Palythenic acid Porphyra-334 Shinorine

AntioxidantAntioxidant

21- Laboratory of Physiology and Biotechnology of Algae

For high-throughput screening of such metabolites, it is essential to reach the inner content of the cell.

Aims

A part of our project was the research and development of a technique enabling a high extraction yield of any metabolite, taking into account the difficulty of extracting bound and or inaccessible molecules with a wide variety of polarities.

The challenge is therefore to identify molecules issued from this biodiversity which are often poorly known in terms of chemodiversity.

The final aim is to compare without extraction bias the potential of microalgae.

22- Laboratory of Physiology and Biotechnology of Algae

Choice of recalcitrant biological modelsFor the study of the chemodiversity extraction

Porphyridium purpureum Phaeodactylum tricornutum

Length : 5-8 µm

Phylum : Rhodophyta

Features :

- Exopolysaccharides

- Thick wall

Length : 3-4 x 15-20 µm

Phylum : Ochrophyta

Features :

- Silica wall

15 µm

23- Laboratory of Physiology and Biotechnology of Algae

Method Effectiveness Reproductibility Other considerations

Soaking - - Long processing

Cryogrinding - - Liquid nitrogen handling require

Potter homogeniser - - n.r.

Homogeniser + - Blade blunted by diatoms

Bead-beater + - Particles release

Planetary micro mill +++ +++ Particles release

Sonication ++ + ROS release, hot spots

Mixer mill – stainless steel jars + + n.r.

Mixer mill – plastic grinding tube +++ +++ n.r.

→

→→→

→→→

Chemodiversity extraction

→

Main studied parameters:

Effectiveness (1-(recalcitrant cells/total))

Reproducibility

Operating cost

Temperature stability

Easy to handling

Comparison of 9 disruption techniques

24- Laboratory of Physiology and Biotechnology of Algae

Grinding optimisation on Retsch MM-400

Time (min)

Wei

ght (

g)

0 5 10 15 20 25 30 350.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Wei

ght (

g)

Time (min)0 5 10 15 20 25 30 35

0.2

0.3

0.4

0.5

0.6

0.7

0.8

A

B

+

+Size classes:( ) Fragments; ( ) Whole cells ; ( ) Agregates.

Optimisation of 3 studied variables by image analysis: creation of an index of desirability.

P. p

urpu

reum

P. t

ricor

nutu

m Optimum

Grinding time : 27 min

Beads weight : 0.92 g

Optimum

Grinding time : 30 min

Beads weight : 0.24 g

Control Optimized grinding

50 µm

50 µm

50 µm

50 µm

P. P

urpu

reum

P. t

ricor

nutu

m

Control Optimized grinding

Isoresponses overlay

Image analysis to evaluate acurately the particle size distribution

Serive et al, 2012. Bioresource Technology 124

25- Laboratory of Physiology and Biotechnology of Algae

→ In a total extract, we have different kind of molecules:

Known by the scientific community and known by us

Known somewhere in the world but unknown by our database

Unknown by everyone

→ Isoplot absorbance metabolic snapshot because of the microalgae cellular plasticity

Total extract, chemodiversity and dereplication

SK(1,2,3) SK-R(1,2,3) L(1,2,3,4)

6

7

8

9

10

11

12

13

Sco

re R

OS

Isoplot absorbance

Time (min)

Wav

elen

ght(

nm)

Bioactivity for a samestrain and different

environmental conditions

26- Laboratory of Physiology and Biotechnology of Algae

Methodology in the work flow

Met

abol

icsn

apsh

ot

Total extract

Microalgaechemodiversity

Efficient metabolite extraction OMICs studies

■ Genomic

■ Transcriptomic

■ Proteomic

■ Metabolomic

■ Pigmentomic

Serive et al, 2012. Bioresource Technology 124

27- Laboratory of Physiology and Biotechnology of Algae

Photomer project

1 2

3 4

Identify new photosensitizers frommicroalgae for PhotoDynamic Therapy

28- Laboratory of Physiology and Biotechnology of Algae

45 microalgae strains composition

→ Spectral screening of interest in pharmacognosy.

→ High interest for oceanography applications.

Pigment analysis and spectral screening

Examples of original spectra from the following strains:

A- Alexandrium minutum B- Alexandrium tamarense

C- Chlorarachnion reptans D- Phaeodactylum tricornutum

A B

C D

29- Laboratory of Physiology and Biotechnology of Algae

OCEAN CHemodiversity Against Cell cycle Targets

Research of new marine inhibitors targeted againstdesease-relevant proteins kinases

30- Laboratory of Physiology and Biotechnology of Algae

Bruker SolariX 12.0 Tesla FTMS

C

A

B

J-B Charcot

31- Laboratory of Physiology and Biotechnology of Algae

Thank you for your attention

Contact: Benoit.Serive@live.fr