Berezhetskyy A.

Chovelon J.M.

Durrieu C.

Guedri H.

I-Revues

SESSION 7.3 
NOVATECH 2007  1507 
Whole cell algal biosensors for urban waters 
monitoring 
Biocapteurs à cellules algales entières pour la surveillance des 
eaux urbaines 
Durrieu Claude 1, Guedri Houssemeddine 1,  
Berezhetskyy Andriy 1, 2, Chovelon Jean-Marc  2 
1 ENTPE, LSE, rue Maurice Audin, 69518 Vaulx-en-velin cédex, France 
2 Université Lyon I, LACE, Bd du 11 novembre 1918,  
69100 Villeubanne, France 
RESUME 
La gestion durable des eaux urbaines nécessite des outils d’alarme précoce 
permettant de détecter la présence de composés toxiques de manière à réagir 
rapidement en cas de pollution. Les biocapteurs peuvent remplir ce rôle dans la 
détection des polluants grâce à leur sensibilité, leur faible coût et leur adaptation 
aisée à un mode de surveillance in situ et en continu. Le fonctionnement des 
biocapteurs à cellules algales présentés ici est basé sur la perturbation métabolique 
de cellules immobilisées après exposition à des polluants. Dans ce travail , des 
mesures ont été réalisées au moyen d’un biocapteur conductimétrique sur des eaux 
pluviales urbaines. Les résultats ont été comparés à ceux obtenus avec des bio-
essais sur algues libres et ont permis de montrer la sensibilité des biocapteurs et leur 
intérêt pour le monitoring in situ.    
ABSTRACT 
Sustainable urban water management leads to the requirement of early warning 
system (EWS) to detect toxic compounds in order to react quickly in case of pollution. 
Biosensors for pollutants determination can act as EWS thanks to their unique 
characteristics with include their sensitivity, they low cost and their easy adaptation 
for continuous detection and on-site monitoring. Algal cells biosensors presented 
hereafter are based on metabolic perturbations of immobilized cells in the presence of 
toxicants. In this study, measurements were carried out with conductometric 
biosensors on urban stormwater. Results were compared to those obtained with 
bioassays on free algae and showed the high sensitivity of biosensors and their 
interest for on-site monitoring. 
MOTS CLES  
Biocapteurs, algues, pollution, métaux lourds, eaux pluviales urbaines. 
SESSION 7.3 
1508 NOVATECH 2007  
1 INTRODUCTION      
The environmental impact of urban waters (waste and storm)  poses a great 
challenge to environmental protection and the pursuit  of  sustainable development of 
urban areas. Their harmfull effects on ecosystems were shown in numerous studies  
(Marsalek et al., 1999; Baun et al., 2006).The necessity of a sustainable management 
of urban waters leads to the requirement  of early warning system (EWS) to detect 
different chemicals in situ at very low concentrations in order to react quickly for 
limiting impact on natural surface and ground waters. Biosensors for pollutants 
determination can act as EWS thanks for their unique characteristics which include 
their sensitivity , their low cost and their easy adaptation for continuous detection on-
site monitoring (Rogers 1995). 
A biosensor can be considered as a combination of a bioreceptor, the biological 
component, and a transducer, the detection method. The total effect of a biosensor is 
to transform a biological event into an electric signal. The first link of a biosensor is 
the bioreceptor, which has a particularly selective site that identifies the analyte and 
ensures molecular recognition. In this work, bioreceptor is Chlorella vulgaris chosen 
among unicellular algal species because they are present ubiquitously in natural 
waters and are able to metabolise a wide range of chemical compounds (Tran-Minh, 
2001).  
These whole-cell algal biosensors are based on metabolic perturbations of 
immobilized cells in the presence of toxicants and have the ability to detect different 
group of pollutants provided they affect a particular alga metabolic pathway. This is 
the case of pesticides and heavy metals which are strong inhibitors of 
acethylcholinesterase and alkaline phosphatase, both are located on chlorella 
vulgaris membranes.  
Compared to biosensors using purified enzyme, whole cell biosensor are more 
resistant to the activity loss because their enzymes and cofactors are hosted in an 
environment optimized by nature. Therefore, these biosensors are more suitable to 
meet all the requirements for environmental  surveillance (Vedrine et al., 2003). They 
can identify in situ the presence of a toxic compound as soon as it released in waste 
water or aquatic environment. Other whole cell biosensors were constructed from 
genetically modified cells (Corbisier et al.1999). Those techniques may improve the 
biosensor sensitivity and selectivity but are no longer able to reflect the ecosystem 
operating conditions. In the present work, only native cells have been used to 
preserve the ecological aspect of the media under study. 
Previous studies showed a good correlation between measurements carried out on 
river waters, polluted by heavy metals and pesticides, and results from chemical 
analysis (Chouteau et al. 2004). 
The aim of this study is to assess the potentiality of a conductometric biosensor to 
monitor urban water toxicity. Experiments were conducted on urban stormwater 
brought to the labolatory. Results were compared with measurements by bioassays. 
    
2 METHODS  
2.1 Cells culture 
The Chlorella vulgaris strain (CCAP 211 / 12)  was purchased from the culture 
collection of Algae and Protozoa at Cumbria, United Kingdom. The axenic algal strain 
was grown in the culture medium and under conditions described by the international 
Organization for Standardization (ISO 8692). 
SESSION 7.3 
NOVATECH 2007  1509 
 
2.2 Biosensor design 
The conductometric transducers were fabricated at the Institute of Chemo-and 
Biosensors (Munster Germany) (Trebbe et al. 2001). Two airs f Pt(150 nm thick) 
interdigitated electrodes were made by the lift-off process on the Pyrex glass 
substrate. The Ti intermediate layer of 50 nm thick was used to improve adhesion of 
Pt to substrate. Central part of the sensor chip was passivated  by Si3N4 layer to 
define the electrodes working area. Both the digits width and interdigital distance 
were 10 nm and their length was about 1 mm. Thus, the sensitive part of each 
electrode was about  1 mm2. 
Measurements are based on the detection of solution conductivity variation inside 
algal cells immobilized. Alkaline phosphatase induces catalytic reaction consuming / 
producing different ionic species resulting in measurable conductivity changes.     
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1 : conductometric biosensor 
 
 
2.3 Algae immobilization 
Sodium silicate (0,4 M, 4 ml) and LUDOX (8,5 M, 4 ml) were thoroughly  mixed to 
obtain a homogeneous silica solution. An HCl 4 M  solution was then added drop by 
drop until  an appropriate pH is reached to induce the gelation process. Immediately 
an algal solution containing 1.3 x 108 cells / ml and 10 % (w/w) glycerol was 
introduced and the mixture was deposited on the sensitive area of the electrode. 
 
2.4 Measurments 
2.4.1 Enzymatic reaction measurements 
For biosensors, measurements were carried out  in daylight at room temperature in a 
5 ml glass cell filled with Tris-HCl (10 mM, pH 8.4) 
con
tact
Membr
ane
30 
mm
5 mm 
30 mm 
contacts 
Epoxy membrane 
electrodes 
SESSION 7.3 
1510 NOVATECH 2007  
Biosensors were immersed in this vigorously stirred solution. After stabilisation of the 
output signal, different aliquots of the substrate stock solution were added into the 
vessel. The differential output signal (dS) was registered  using a “home made” 
conductometric laboratory apparatus and the steady state response of the biosensor  
was plotted against the substrate concentration 
For bioassays, free algae are used and measurements in microplates are based on 
fluorescence detected by a spectrofluorometer (Fluostar BMG). The alkaline 
phosphatase enzymatic reaction using MethylUmbelliferylPhosphate (MUP, Sigma) 
as substrate gives fluorescent product MethylUmbelliferone (MUF). Essays were 
carried out in 96 wells microplates.    
                      
2.4.2 Toxicity measurements 
For biosensors, dS was measured for a definite substrate concentration. The 
biosensor was then preincubated in a test solution for 15 mn . After washing  dS 
before and after exposure to the test solution were compared and the residual activity 
rate was calculated. 
For bioassays, 48 wells microplate were filled with algal solution. After sedimentation, 
culture medium could be removed and replaced by the test solution. Exposure last 
two hours. After removing the test solution and resuspending algae in distilled water, 
enzymatic activity was measured in 96 wells microplate as above-mentioned. 
   
3 RESULTS AND DISCUSSION 
3.1.1 Enzymatic activity detection using conductometric biosensors 
In a previous work, it has been proved that phosphatase alkaline activity can be 
monitored for immobilized Chlorella vulgaris using conductometric biosensors 
(Chouteau et al., 2004).  As shown in fig 2, the enzymatic activity follows a classical 
Michaelis-Menten behaviour. The relative standard deviation of the sensor did not 
exceed 8 %. 
  
0
5 0 0 0
10 0 0 0
15 0 0 0
2 0 0 0 0
2 5 0 0 0
3 0 0 0 0
0 2 4 6 8 10
substr ate concentr ation)
   
 
figure 2 : Alkaline phosphatase activity measured with a conductometric biosensor 
 
3.1.2 Biosensor calibration for heavy metal detection 
Alkaline phosphatase measurements were carried out before and after exposure  to 
heavy metals ions. Fig 3 shows phosphatase activity (expressed as percent of control 
before exposure). 
SESSION 7.3 
NOVATECH 2007  1511 
 
Figure 3 : evolution of phosphatase activity rate after exposure to different metal ions 
 
Inhibition can reach 80 % of control for 100 mg/ L heavy metal. At low concentrations 
(0.1 - 10 µg/l) metal ions as Cd, Pb, Co, Ni  enhance phosphatase activity. The 
activation of some enzymes by low metal concentrations has already been studied  
and could be explained by cellular stress : indeed to prevent the cell from metal 
damages, stress promoters are produced inducing an increase of some enzymatic 
activities (Mazora et al. 2002).    
 
3.1.3 Biosensor responses after exposure to urban stormwaters 
The biosensor developed is supposed to be used for the detection of pollutants 
especially heavy metal ions. 
Measurements were carried out after exposures to different concentrations of urban 
stormwater in the mixture assay. Stormwater was sterilized before experiment at 
130°C, 1.5 bar for suppressing bacterial phosphatase activity. In fig 4, biosensor 
response can be compared with results of fig 3.  The lowest concentrations of 
stormwater enhance phosphates activity as higher concentrations inhibit this activity. 
Heavy metal contain of stormwater tested was determinated by chemical analysis 
(resuts showed in table 1). A mixture of metal ions with a concentration between ppb 
and ppm, can explain results obtained. 
 
SESSION 7.3 
1512 NOVATECH 2007  
  
Fig ure 4 : Evolution of algal phosphatase activity rate after exposure at different  concentrations 
of urban storwater detected with conductometric biosensor 
 
 
Zn 
(µg:l) 
Pb 
(µg/l) 
Cu 
(µg/l) 
Cr 
(µg/l) 
Ni 
 (µg/l) 
Cd 
(µg/l) 
Fe 
(mg/l) 
Al 
(mg/l) 
740 46.6 146 8.8 3.30 0.23 1.81 0.94 
 
Table 1 : heavy metal contain of urban stormwater tested 
 
For the lowest concentrations of stormwater tested an activation of phosphatase 
activity was obtained due to the low concentration of metal ions. For higher 
concentrations a good correlation was obtained between concentration of sample and 
inhibition rate.     
3.1.4 Bioassay responses after exposure to urban stormwater 
Bioassays on microplate were carried out on free algae. Fig 5 shows phosphatase 
activity rate after 2 hours exposure of algal cells to different concentrations of 
stormwater samples. 
A comparison between figures 4 and 5 shows the same behaviour between 
phosphatase activity of free cells and cells immobilized on conductometric biosensor. 
(Activation for lowest concentrations and inhibition for the highest ). 
SESSION 7.3 
NOVATECH 2007  1513 
However, inhibition (or activation for low concentrations) rates are higher using 
biosensors than bioassays. It can be explained by the different ratios ‘number of algal 
cells/toxicant elements’ in both cases. Indeed on biosensors, low amounts of algae 
were immobilized compared to bioassays using free algae : for biosensors the ratio 
algae : toxicants is lower than for bioassays. As inhibition rate are inversely 
proportional to these ratios as a result, biosensors give higher inhibition rates and 
they seem to be more sensitive to detect the enzymatic activity modification.    
 
y = -50,144x + 111,98
R2 = 0,9302
0
20
40
60
80
100
120
140
0% 20% 40% 60% 80% 100% 120%
Dillution(%)
AP
A 
re
s(
%
)
 
 
Figure 5 : evolution of  phosphatase activity rate detected  on free algae after exposure to urban 
strormwater 
 
 
Algal growth inhibition test was carried out on Pseudokirchneriella sucapitata 
according to AFNOR (NF T90-375 1998). Results showed very low toxicity of 
stormwater tested on algal growth (CE 50 was not reached with 80 % of stormwater 
in assay). 
Then, changes in phosphatase activity reflect  specific interactions between pollutants 
and algae and not a total attack of the cells. 
 
 
4 CONCLUSIONS 
 
The conductometric algal biosensor presented in this study seems to be successful 
as early warning system to identify the presence of pollutants as metal compounds. 
In aquatic environment, algae are the first trophic level : any disturbances could be 
reported to upper levels. This is one of the main interest of this work since it gave the 
opportunity to follow the response of a living organism to pollutants. Contrary to 
chemical analysis, used to detect determined pollutants, algal cell biosensors can 
detect any stresses disturbing the organism metabolism.  
SESSION 7.3 
1514 NOVATECH 2007  
Interferences of real matrices with these biosensors will have to be considered in the 
next step. After that, biosensors will be placed in the area under monitoring.  
 
5 REFERENCES 
Baun A., Eriksson E., Ledin A. and Mikkelsen P.S. (2006). A methodology of ranking and hazard 
identification of xenobiotic organic compounds in urban stormwater. Science of total 
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Chouteau C., Dzyadevych S., Chovelon J.M. and Durrieu C. (2004). Development of novel 
conductometric biosensors based on immobilized whole cell Chlorella vulgaris microalgae, 
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Corbisier P., Vander lelie D., Borremans B., Provoots A., de Lorenzo V., Brown N.L., Lloyd J.R., 
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Mazora; M.T., Rubio J.A. and Blasco J. Acid and alkaline phosphatase activities in the clam 
Scrobicularia plana : kinetics characteristics and effect of heavy metals. Comp. Biochem. 
Physiol.B 131, 241-249.  
Rogers K.R. (1995). Biosensors for environmental applications. Biosensors bioelectron., 10, 533-
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Tran-Minh C.(2001). Les biocapteurs, principe, construction et applications. Paris : Masson, 158 
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Whole cell algal biosensors for urban waters monitoring

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Whole cell algal biosensors for urban waters monitoring

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