On the combination of automated optical sensors for the ... · (FRRF, PAM) data we will determine constrains of the conversion factors from electron transport rate to C-fixation at

On the combination of automated optical sensors for the observation of phytoplankton dynamics in coastal

waters: implications for the monitoring of Harmful Algal Blooms (JERICO-Next European network)

Luis Felipe ARTIGAS1 [email protected] 1 UMR 8187 LOG – CNRS-ULCO-UL1 Wimereux - FR Contributors: Blauw A.2, Bonato, S.1, Claquin P.3, Créach V.4, Deneudt K.5, Grégori G.6, Grosjean, P.7, Guiselin N.1, Hamad D.8, Hébert P.-A.8, Houliez E.9,1, Karlson B.10, Kromkamp J.11, Lefebvre A.12, Lampert L.13, Lizon F.9, Petersen W.14, Poisson-Caillault E.8, Revilla M.15, Rijkeboer M.16, Rutten T.17, Terneuzen L. 5, Thyssen M.1,6 , Seppälä J.18, Stemmann L.19, Veen A. 16, Wacquet G.1,,7, 12 , Vywerman, W.20, Puillat, I.21 Conférence du GDR Phycotox / Villefranche sur Mer / France / 15-16 Mars 2016

mailto:[email protected]�

Affiliations 1 CNRS UMR 8187, Laboratoire d’Océanologie et Géosciences (LOG – CNRS, ULCO, UL1), Université du Littoral Côte d’Opale, Wimereux, FR 2 DELTARES, Delft, NL 3 CNRS UMR 7208, Biologie des Organismes et Ecosystèmes Aquatiques (BOREA- CNRS, MNHN, UPMC, IRD 207, UCN, UA) - Université de Caen, Caen, FR 4 Center for Environment, Fisheries and Aquaculture Science (CEFAS), Lowestoft, UK 5 Vlaams Instituut voor de Zee (VLIZ), Ostende, BE 6 CNRS UMR 7294 Institut Méditerranéen d’Océanologie (MIO – CNRS, IRD, UM AMU 110), Aix-Marseille Université, Marseille, FR 7 Laboratoire d’Écologie Numérique des Milieux Aquatiques, Université de Mons, Mons, BE 8 Laboratoire d’Informatique Signal et Image de la Côte d’Opale - EA 4491, Université du Littoral Côte d’Opale, Maison de la Recherche Blaise Pascal, Calais, FR 9 CNRS UMR 8187, Laboratoire d’Océanologie et Géosciences (LOG – CNRS, ULCO, UL1), Université de Lille 1, Station Marine de Wimereux, Wimereux, FR 10 Swedish Meteorological and Hydrological Institute (SMHI), Norrköping, SE 11 Royal Netherlands Institute for Sea Research (NIOZ), Yerseke, NL 12 Laboratoire Environnement Ressources, Institut Français pour l’Exploitation de la Mer (IFREMER), Boulogne sur Mer, FR 13 DYNECO PELAGOS, Institut Français pour l’Exploitation de la Mer (IFREMER), Brest, FR 14 Institute for coastal Research, Helmholtz-Zentrum Geesthacht (HZG), Hamburg, DE 15 AZTI, Marine Research Unit, Pasaia, SP 16 Centre for Water Management, Laboratory for hydrobiological analysis, Waterdienst, RWS, Lelystad, NL 17 Thomas Rutten Projects, Middelburg, NL 18 SYKE Finnish Environmental Institute, Helsinki, FI 19 CNRS UMR7093, Laboratoire d'Océanographie de Villefranche (CNRS-UPMC), Université Pierre et Marie Curie, Villefranche sur Mer, FR 20 Protistology and Aquatic Ecology, Ghent University, Ghent, BE 21 Laboratoire d’Océanographie Côtière, IFREMER Brest, FR

…The European context…

FixO3

Lifewatch -EMBRC

NEXOS

EMBRC

JERICO-NEXT 2015-2019 - H2020

JERICO-NEXT: Quicklook

34 partners

CNRS contractor… et ses 3rd parties: • UBO (Brest) • UCBN • Univ. Bordeaux • Univ. Lille1 • Univ. Du Littoral • UPMC

Partenaires Français

UMRs: • EPOC • IUEM • LEGOS • LOCEAN • LOG • LOV • MIO • SBR

& DT-INSU

…The JERICO mind… • The JERICO-NEXT community

“ We cannot understand the complexity of the coastal ocean if we do not understand the coupling between physics, biogeochemistry and biology.”

new technological developments for continuous monitoring of a larger set of parameter

a priori definition of the optimal deployment strategy

• JERICO-NEXT focus - interactions between physics, biogeochemistry and biology - not restricted to pure technological aspects : include fundamental

scientific considerations


HF radar

Sensors

Physical data Biological

data

JERICO-NEXT

New additional partners

New high quality infrastructures

& services

New competences to better understanding interraction between

physical & biological data

Extended EU coastal observatory network

Continuous and more valuable

coastal data coupling physical & biological

information

ESFRI EMOD

net

Ocean for Tomorrow

… From JERICO to JERICO-NEXT…


… Societal challenges …

MSFD

• key environmental challenges and service and/or policy requirements on:

1) pelagic biodiversity 2) benthic biodiversity 3) invasive species 4) chemical contaminant occurrence and related biological responses 5) eutrophication 6) hydrography and transport 7) carbon fluxes and carbonate system

8) operational oceanography.


… Objectives and needs…

Delivery of an harmonized research infrastructure for coastal observations, compliant with EMODNET and Copernicus

• To ensure the sustainable provision of high-quality coastal multidisciplinary observations that can support: • Progress and breakthrough in marine science • European policies and national duties • The development of business activities (e.g. marine services)

To produce a long-term strategy for further development, integration, sustainability and relevance of coastal observatories in Europe (WP1)


LIST of WPs

• WP1 - Integrated Science Strategy and Governance from local to European Scales (COVARTEC, CNRS-EPOC)

• WP2 - Harmonization of technologies and methodologies - technical strategy (OGS, HZG)

• WP5 - Data management (HCMR, EuroGOOS) • WP8 - Outreach, communication and engagement (Blue Lobster, CEFAS)

• WP6 - Virtual Access (CEFAS)

• WP7 - Transnational Access to Coastal Observatories (CNR-ISMAR)

• WP3 – Innovations in Technology and Methodology (HCMR, Ifremer)

• WP4 - Valorisation through applied joint research (Ifremer, CNRS-EPOC)

Networking Activities Transnational Activities Joint Research Activities

• Innovative sensors can provide new insights into phytoplankton detection and characterisation in the field, allowing to better understand eutrophication processes, HABs and to provide data for better parameterisation of ecosystem models.

• There is a need to work on the operability and discrimination of existing innovative techniques addressing phytoplankton diversity, functional groups distribution and/or photosynthetic parameters, based on phytoplankton morphology and/or single cell or bulk optical characteristics.

• The objective of this task is to combine and improve the use of (semi)-automated observation techniques in several European coastal and shelf seas, at high resolution, in (near) real-time, in key monitoring platforms.

WP3: Innovation in Technology and Methodology

Task 3.1.: Automated platform for the observation of phytoplankton diversity and related ecosystem services

Methods and approaches for monitoring phytoplankton & HABs

At the laboratory/ On board

Real time Real time

Profiles Continuous

pumping/recording Synoptic/regional

Real time

Remote sensing

Microscopy Pigment analysis Total/Spectral Fluorometry Flow cytometry Image Analysis Molecular tools

In vivo Total/Spectral Fluorometry Optic probes Scanning Flow cytometry Image Analysis

CTD Total/spectral Fluorometry Scanning Flow cytometry Image Analysis Molecular tools

Ocean optics from satellites Development of new Algorithms Comparison with in situ data

Techniques

Analysis

Sampling strategy

Monitoring strategy

Discrete sampling

Research / Opportunity

Vessels

Moorings

Gliders

Critical comparison of different techniques for assessing phytoplankton abundance and/or biomass equivalents, per size/functional groups, photosynthetic activity/physiological status, in order to better define their applicability in different conditions

Definition of the range of applicability of each technique, in terms of definition of cell size, concentration, speed of measurement, background light and signal, etc.

Three main approaches will be explored and used in combination in

order to build automated platforms : - image acquisition and analysis (in flow/in situ) - single-cell optical analysis (pulse-shape recording FCM) - a combination of optical bulk techniques (fluorescence induction,

spectrophotometry and spectrofluorometry)

Task 3.1.: General strategy

• Two workshops will be organised. • During the first workshop (June 2016, Wimereux), plenary

presentations will be carried out by partners and invited experts from Europe and abroad. Targeted practical discussions on the different innovative methods for assessing phytoplankton diversity and photosynthetic parameters (linked to WP 2.4.2.).

• During the second workshop (September 2016, Gotheburg), test/inter comparisons of techniques on both cultured micro-algae of interest and natural samples will be performed

• We will take benefit from the results of the current implementation of innovative sensors in the different regions covered by both JRAP#1 and JRAP#5 (Ligurian Sea and W. Mediterranean, Bay of Biscay, English Channel, North Sea, Baltic Sea)

Task 3.1.: Activities in 2016

• CNRS (LOG-Wimereux, BOREA-Caen, OSU Villefranche s/Mer, M.I.O. Marseille)

• SMHI • VLIZ (s.c.: U. Gent) • IFREMER (LER/Boulogne sur Mer) • SYKE • HZG • AZTI • RWS (s.c. NIOZ, TRP®and Cytobuoy®) • CEFAS …and external collaborators as WHOI, DAFF, U. Mons, LISIC,

IFREMER Dyneco Brest… (in the frame of JERICO-Next and other projects).

Task 3.1.: List of participants

• Pulse-shape recording Flow Cytometer (+ Image acquisition) module (VLIZ, CNRS LOG-MIO-BOREA, RWS, CEFAS)

• Imaging Flow Cytometer (SMHI coll.WHOI , SYKE)

• Flow Cytometer (HZG) • FlowCAM (IFREMER, CNRS, SYKE,

AZTI) • FastCAM (IFREMER) • Absorption meter - PSICAM (HZG,

SYKE )

• Spectral fluorometer - AOA or Fluoroprobe (CNRS-LOG-BOREA, IFREMER, SYKE)

• PAM or Phyto-PAM (CNRS LOG-BOREA, CEFAS)

• FRRF or spectral FRRF (SYKE, CNRS BOREA-LOG, VLIZ-RWS-NIOZ, CEFAS)

• Underwater Vision Profiler - UVP5 (CNRS-OSU V)

• (Semi-)Automated data analysis (CNRS LOG-MIO, RWS-TRP, IFREMER, SYKE)

Task 3.1.: List of available techniques (consortium)

• SubTask 3.1.1 Imagery Instrumentation (led by SMHI)

Exploration of different techniques based on inflow/in situ imaging analysis and comparison of their sensitivity in discriminating phytoplankton taxa and of their accuracy in counting phytoplankton cells/colonies: Imaging FlowCytobot (collaboration with WHOI, training in spring 2016), CytoSense (work on old and new data), FlowCAM (ongoing collaboration with U.Mons) and Underwater Vision Profiler (UVP5).

The imaging in flow machines as well as the related data analysis tools will be presented and discussed in the June Workshop, and tested on cultures and discrete samples in continuous flow or in situ field work in the September Workshop. Semi-automated classifiers will be developed for phytoplankton and specially for HABs detection.


JRAP #1 WP4 meeting; Mallorca, 29 Sept. 2015

The Imaging FlowCytobot (IFCB) is an automated, submersible microscope

Sosik and Olson, 2007

Jerico-Next – Kick-Off meeting - Mallorca


Flow CAM (discrete and online)


1. The imaging system UVP5, image and data flow

and storage, and data QC will be presented and discussed

(http://rade.obs-vlfr.fr/RadeZoo/RadZoo/Accueil.html )

2. The UVP5 will be tested during in situ field exercises (colonial massive cyanobacterial blooms)

3. Classifiers for plankton will be developed in the laboratory,

The Underwater Vision Profiler 5

107 plankton images

http://rade.obs-vlfr.fr/RadeZoo/RadZoo/Accueil.html�

• SubTask 3.1.2 Single-cell optical characterization (led by CEFAS)

Discrimination between populations based on their optical properties, determination of phytoplankton functional groups, definition of biological traits (based on pigments composition, size, shapes, free-living or colonial status).

Partners working with “pulse-shape recording” Cytometers (CytoSense) will continue the implementation of their machines in continuous recording systems (cruises, ferries)

They will participate in both Workshops in 2016 and will compare the advances made in both the implementation of this technique in automated platforms, as well as in improving the automation tools for classification and analysis, and the data collection, in collaboration with SMEs and external partners (LISIC)



The Pulse-shape recording Automated Flow Cytometer

The CytoSense flow cytometer (Cytobuoy©)

Cytogramme : Multidimensional representation of all cells

analysed according to their optical features.

Optical profile (fluorescence, forward scatter and sideward scatter) of a particle

(colony of Pseudonitzchia sp.) and associated image


Web app with near real-time results for Pulse Shape recording FCM

Easyclus Live web app. Instrument check; Totals per sample (concentration, chl a); Cluster plots; Biodiversity indicators – Thomas Rutten Projects

• SubTask 3.1.3 Optical Instrumentation combination (led by SYKE)

Existing and new optical techniques will be tested to study phytoplankton biomass, taxonomy and productivity, and other optically active in-water constituents.

Algorithms for in vivo spectrophotometric (PsiCAM) and spectrofluorometric (Fluoroprobe, AOA, new multi-wavelength-Multi-exciter spectral fluorometer) determination of chlorophyll a, phycoerytrhin, algae spectral (pigmentary) groups will be compared and developed further.

To approach primary production using variable fluorescence (FRRF, PAM) data we will determine constrains of the conversion factors from electron transport rate to C-fixation at various spatio-temporal scales including different phytoplankton communities.



Spectral absorption & fluorescence

www.trios.de http://www.jfe-advantech.co.jp

Objectives: Derive taxonomic (pigmentary groups) and biomass information from spectral data Challenges: QA/QC, instrument calibration, field validation, definition of pigment groups, site specific issues, biology affecting spectra Aim: provide tools and algorithms for data analysis and in JRAP1 demonstrate the usability of selected instruments + algorithms

Profiler in situ, pumping on deck and laboratory measurements http://www.bbe-moldaenke.de

Mid-Channel Waters

Coastal Waters May 2010 (05/05/10)

Spatial distribution (at high resolution) of diatoms and Phaeocystis globosa along a transect from offshore to coastal waters (English Channel)

This method is based on the discrimination of spectral groups

of algae characterised by a specific composition of pigments and, consequently, by a specific

excitation spectrum of the chlorophyll a fluorescence, following sequential light

excitation by 5 light-emitting diodes (LEDs) emitting at 450,

525, 570, 590 and 610 nm.

Picture of the Fluoroprobe (bbe Moldaenke ©)

Spectral groups of phytoplankton discriminated by the Fluoroprobe and the Algae Online Analyser (AOA)

Multi-spectral fluorometer

Profiler in situ, pumping on deck and laboratory measurements

Flow-Through Point-source integrating cavity absorption meter (ft-PSICAM)

27

ft-PSICAM: principle and setup

Measuring pure absorption without errors caused by particle scattering with high sensitivity

Absorption measurements

28 Identification of algae groups

chlorophyll-a

676 nm

total suspended matter (TSM)

700 nm

possibility of determination of chlorophyll-a and TSM

29

Differentiation of algal groups from absorption spectra


Variable fluorescence Objectives: Derive primary production estimates with optical measurements Challenges: QA/QC, instrument calibration, field validation calibrations, conversion factor between carbon production and electron transport rate Aim: Estimate conversion factor in response to phytoplankton community structure and physiology, diel variations, nutrient availability, physical forcing (light, mixing)

Fast Repetition Fluorometer in flowthrough mode


• A set of recommendations of the most suitable and relevant combination of methods according to the environment considered, their limits and ways of implementing them as complementary sensors in combined platforms, will be written.

• We will take into account the differences in optical properties, nutrient status and phytoplankton communuity structure in European coastal seas (link with WP 4.1 and WP 4.5).

• We will provide a better description of the different types of

data that will be compiled in databases (link with WP 5.2).

Task 3.1.: Deliverables and Milestones


JRAP1 : Pelagic biodiversity Biodiversity of plankton, harmful algal blooms and eutrophication

Bengt Karlson, SMHI, [email protected]

WP4 Main objectives & organisation


JRAP#1: summary

• To get closer to resolving natural variability in the sea with regard to plankton

• To improve the understanding of the development of certain algal blooms

• To exemplify how JERICO-NEXT can help address MSFD requirements (D1-Marine biodiversity for the pelagic realm, D5 Eutrophication is addressed)

• To use JERICO-NEXT observation platforms and other infrastructure

JRAP#

lead Partners Sites

1 B. Karlson, SMHI

SMHI, CEFAS, CNRS-LOV, CNRS-Univ Litt,, CNRS-MIO, Deltares, Ifremer, NIVA, RWS, SYKE, VLIZ, and DAFF

Northern Baltic, Kattegat-Skagerrak, Eastern Channel and Southern North Sea, Bay of Biscay, Ligurian Sea, Benguela Current


• To utilize developments from WP3 innovations in technology and methodology (also from WP2 review and synthesis of existing methods)

• Task 3.1 Automated observations of phytoplankton • Task 3.2 HF-radar (advection of algal blooms) • Task 3.4 Microbial and molecular sensors • Task 3.5 Carbonate system (e.g. primary production)

• To work together with other JRAP:s in WP4 • To provide data sets and novel data types to WP5

JRAP#1: summary


JRAP#1: Preliminary Strategy Carry out short terms studies of different types of algal blooms Multi discipline approach: Biological, chemical and physical

oceanography

Multi platform approach: R/V, Buoys, FerryBox systems ( & Remote sensing )

To combine novel methods with established ones • Automated water sampling and traditional water sampling • Automated in situ sensors for bio-optical parameters such as chl.

fluorescence and spectral fluorometry for photosynthetic pigments • Automated identification and enumeration of organisms

• - Pulse-shape recording Flow Cytometry (in situ and on ship) • - Imaging Flow Cytometry (in situ and on ship) • _High Troughput sequencing of 16S and 18S rDNA

• Counting and identifying organism using the light and electron microscope


Electricity

Mussel farm Scanfjord

office and factory

Raft+ IFCB

Approx. 100 m

Plans for the Tångesund observatory

Buoy

ArvorC profiler


Baltic Sea Research vessel, Utö observatory, FerryBoxes and buoys

http://www.google.se/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCIjlx_GhksgCFQH-cgodvsUEjQ&url=http://www.shipsandharbours.com/picture/number15291.asp&psig=AFQjCNEajOm5X-7wIY992F7fe09cZaJKmQ&ust=1443273226544958�

https://www.google.se/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCPqw7p6iksgCFeazcgod7JYEEg&url=https://en.wikipedia.org/wiki/MS_Silja_Serenade&psig=AFQjCNEarUWwGr2ccs364AFTmM0K8N6kyQ&ust=1443273321517673�


Baltic Sea Research vessel, Utö observatory, FerryBoxes and buoys

Different bio-chem-phys marine observations at one site

Marine and atmospheric observations at one site Different platforms (fixed, moving, trad. sampling)

en.ilmatieteenlaitos.fi/uto

Western Med Sea projects A*MIDEX CHROME (Continuous High Resolution Observation of the Mediterranean Sea). Expected start of the ferry box : December 2015-January 2016 Ferrybox management, contact: [email protected] Coupling with a cytometer, contact: [email protected]

Institut de Mathématiques de Marseille, UMR 7373

Cytobuoy cytometer with the Image in flow device

– Temperature – Conductivity/salinity – Fluorescence/Chlorophyll-a – CDOM/FDOM – pH – pCO2 – Oxygen –Phytoplankton abundance and functional description


A new platform open for installation of new sensors

Temperature Conductivity Oxygen Turbidity Chla (Fluorescence) PAR A Fast Repetition

Rate Fluorometer – ACT2 (Chelsea) Installation on the SMILE Buoy at the end of the year

Mini – « Ferrybox » Temperature Conductivity Oxygen Turbidity BBE Only Analyser

SMILE – Buoy – Bay of Seine English Channel


41

Freshwater influence

Coastal water influence

MAREL Carnot instrumented Station: High Frequency Monitoring in the eastern English Channel

Sampling frequency: 20 minutes for physico-chemical parameters and 12 hours for nutrients Preprocessed database: 131 472 x 10 non-correlated parameters over 2005-2009 Ongoing System update.

Parameters: Temperature, Salinity, Oxygen, Turbidity, pH, Fluorescence, PAR, Relative Humidity, Wind, Water level, Nitrate, Phosphate, Silicate

Contact: [email protected]


Sampling frequency: 1 min - 10 min continuous sampling mode / Spatial resolution approx. 0.5-1.5 km AOA Database Fingerprints : Green, blue-green , brown and Mixed algae Pulse-Shape recording Flow Cytometry + Image inflow system

CytoSense (CytoBuoy©)

Pocket Ferry Box (PFB) + AlgaeOnline Analyser (AOA) + CytoSense + PhytoPAM A preliminary study towards a Ferry Box line across the Dover Strait

DYPHYMA Cruises – INTERREG “2 Seas” DYMAPHY Project

R.V. « Côtes de la Manche » (INSU-CNRS)


PhytoPAM

Contacts: [email protected] and [email protected]



Continuous recording of phytoplankton in

eastern English Channel coastal

waters SFCM (cell concentration

and fluorescence) & spectral fluorescence

(fluorescence per group)

DYPHYMA Cruise SCFM fluo

SCFM cells

Ph.D. Thesis S. Bonato2015, Bonato et al., ECSC, 2015

Observation in situ qualité des eaux et phytoplancton en

Manche orientale, des réseaux existant

(REPHY-SRN, SOMLIT, RESOMAR, IGA) aux

dispositifs projets CPER NPdcP

MARCO et JERICO-Next

- Système d’analyses

MAREL Carnot, - Plateforme analytique

- Implementation

Cytomètre en flux à scanning,

-Implementation FRRF et Fluorimètre

spectral, - Ferry Box ligne Calais-Douvres

IGA EDF R&D


« Thalassa » R.V. (IFREMER) IBTS-CAMANOC-CGFS Channel cruises

Pocket Ferry Box + AOA bbe+ CytoSense

Filtration desk

Flow Cam

CAMANOC W. Channel cruise IFREMER CTD + Bottles + Fluoroprobe


VLIZ Flow Cytometer (FCM) and Fast Repetition Rate Fluorometer (FRRF)

• On board Simon Stevin • Connected to continuou

water flow system • Flow cytometry and FRR


Facilities on line on the Cefas Endeavour

Potential cruises with the Cefas R.V. Endeavour

Cytosense (PFT) PCO2

Ferrybox


Flowcytometry in routine monitoringof Dutch offshore, coastal and estuarine waters (RWS)

Machteld Rijkeboer (RWS) Arnold Veen (RWS) Thomas Rutten (TRP) Jacco Kromkamp (NIOZ – FRRF)

FRRF

FerryBox FCM

This project has received funding from the European Union's Horizon 2020 research and innovation programme

under grant agreement No 654410.

Merci pour votre attention!

Documents

On the combination of automated optical sensors for the ... · (FRRF, PAM) data we will determine constrains of the conversion factors from electron transport rate to C-fixation at