Download Development of a whole-cell biosensor based on marine algae for

Development of a whole-cell biosensor based on
marine algae for environmental monitoring at sea
ALSENSE
Claude Durrieu and Thierry Baussant.
ENTPE, Université de Lyon, France
International Research Institut of Stavanger, Norway
Development of algal marine biosensor
for monitoring sea water near offshore activities
The project is based on previous experience and proof-of-concept with freshwater whole cell
based biosensors established at ENTPE (Lyon, France).
The expertise of IRIS (Stavanger Norway) in the development and use of monitoring tools for
field assessment of petroleum discharges, including biomarkers and other techniques based on
sensing, will complement very well the vision of this project to produce an effective tool to assist
in the sustainable management of the marine ecosystem.
Offshore discharges from petroleum activities
Examples of known toxic pollutants from offshore effluents :
Heavy metals
PAHs (polyaromatic hydrocarbons)
They can produce toxic effects on marine species and
hence impact marine ecosystems
It is necessary to assess the toxicity of these various toxicants
This could be achieved with biological sensors permanently settled or
routinely deployed in the areas under control
BIOSENSORS PRINCIPLE
électrochemical
cells
photometer
Amplification
Enzymes
Micro
électronic
…
Water sample
Bioreceptor
Transducer
BIOSENSOR
Signal
Informations
Interest and advantages of algal biosensors
Algal are very sensitive
Located at the first level of the food chain
Tests are carried out on whole cell and integrate multiple effects
Biosensors can be used on the field
Potential for routine analyses of regular offshore effluents and for
use in contingency response following accidental release
Suggested development phases of this
project
 Selection and maintenance of a marine algae strain
which is readily cultured
 Measurements of relevant end point parameters like
total photosynthetic activity by fluorescence, cellular
membrane enzymatic activities (alkaline
phosphatase and esterase)
 Sensitivity tests to several classes of pollutants
relevant to the study: PAH, phenols and heavy
metals
 Selection of sensing elements: for example optical
based- and conductometric based sensing
 Construction of the biosensors: immobilisation of
algae cells on the surface of the sensing element
 Optimisation of analytical protocols and flow-cell
tests for possible in situ deployment
 Test of the biosensor using single or complex
mixtures of chemicals, and real water samples
 Validation of the prototype with other reference
monitoring tools and more traditional techniques
based on chemistry and biological markers used in
a regulatory and water management context
Conductometric and algal Biosensor
substrate
Differential measurements
Local variations of
conductivity
Synchronous
amplifier
Dunaliella tertiolecta
Change fluorescence
level measurements
Fluorometer
Phaeodactylum tricornutum
Two strains were selected which are very simple to cultivate : Dunaliella tertiolecta, green
flagellate, and Phaeodactylum trcornutum, diatom. Cells were immobilized on
conductometric electrode or on a filter near optical bundle.
The biosensor response is based on the algal phosphatase and esterase activity
measurements
8
OrigalysElectrochemSAS sensor : a field tool
Collaboration between ENTPE and Origalys
will enable the miniaturization of conductometric biosensor as a field tool
A biosensor was previously performed with freshwater
algae to detect pesticides and heavy metals
This biosensor was based on the activity
measurements of Chlorella vulgaris membrane
enzymes. Cells were immobilized by cross linking
reaction on the conductometric electrode or on self
assembled monolayer of alkanethiolate
Chlorella vulgaris cells immobilized
on SAMs
Heavy metals detected by alkaline phosphatase inhibition
Pesticides detected by esterase inhibition
90
80
% inhibition
70
100
60
50
80
40
60
30
40
20
1,28
0,64
0,32
0,16
0,08
0,04
0,02
20
10
0
Cd
0
Atr
Gly
Diu
MPt
Hg
Pb
Zn
Cu
Ni
Cr
Mpo
Enzymatic activities are early toxicity signals
PAH composition was tested 2H00, 24H00
and 48H00 after extraction
Effects of oil extract on esterase activity
17
Effect of dispersed oil on the cell growth
Cells per mL
Phaeodactylum tricornutum
Oil concentration
Conclusions
Biosensors developed in this project present an important ecological interest because
they give informations on disturbances cause by pollution at the first level of the
ecosytem organisation
Measures are possible directly on-site for surveillance or other emergency situations
Promising technology for water managers and contingency responders as generic
Early Warning tool or decision-support tool
HOWEVER…
The reproductibility of the measures have to be improved
Need to improve robustness
Need for automation and easy use for field application
Modules for continuous real-time monitoring
Thank you for your attention