The 4th Annual Seminar of National Science Fellowship 2004

[BIO32] The development of a biosensor for the detection of PS II herbicides using green microalgae

Maizatul Suriza Mohamed, Kamaruzaman Ampon, Ann Anton

School of Science and Technology, Universiti Malaysia Sabah, Locked Beg 2073, 88999 Kota Kinabalu, Sabah, Malaysia.

Introduction Material & Methods Increasing concern over the presence of herbicides in water body has stimulated Equipments and Chemicals research towards the development of sensitive Fluorometer used was TD700 by Turner method and technology to detect herbicides Designs with 13mm borosilicate cuvettes. residue. Biosensors are particularly of interest Excitation and emission wavelength were for the monitoring of herbicides residue in 340nm-500nm and 665nm. Lamp was water body because various classes of daylight white (185-870nm). Equipment for herbicides have a common biological activity, photographing algae was Nikon which can potentially be used for their Photomicrographic Equipment, Model HIII detection. The most important herbicides are (Eclipse 400 Microscope and 35 mm film the photosystem II herbicide group that photomicrography; prism swing type, inhibits PSII electron transfer at the quinone automatic expose and built-in shutter). binding site resulting in the increase of Chlorophyll standards for fluorometer chlorophyll fluorescence (Merz et al., 1996) calibration were purchased from Turner . Designs, USA. PS II herbicides used were diuron (3-(3,4-dicholorophenyl)-1,1

Signal dimethylurea or DCMU), and propanil (3′,4′- PS II FSU herbicide dichloropropionanilide). Non PS II herbicides used as comparison were 2,4-D (2,4- Meter dichlorophenoxy)acetic acid) and Silvex Algal Chlorophyll Transducer (2,4,5-trichlorophenoxypropionic acid)

(Aldrich Sigma). Stock solutions of both Fluorescence Fluorescence (Increase) herbicides were prepared in the range of 0.001mM to 100mM in 50% (v/v) ethanol and 50% (v/v) DMSO. Media used was modified Bristol solution (K2HPO4, KH2PO4, MgSO4, Free PS II Herbicide Inhibited- Chlorophyll Herbicides chlorophyll NaNO3, NaCl, and CaCl; prepared in separate stock solutions)

FIGURE 1 Schematic diagram of a basic biosensor Test organisms for herbicide detection. In the photosynthetic Six species of unicellular microalgae from pigments of green alga, absorption of light quantum induces the transition of pigment phylum of (class of molecules into the excited state. From the ) were used in the study (Table peripheral antenna complexes, excitation is 1). efficiently transferred to the core antenna complexes near photosynthetic reaction centers, TABLE 1 Microalgae used in the development of where it can be used in the primary photochemical the biosensor. reaction of photosynthesis (dark reaction). A small fraction of the excited energy is reemitted as Algal species Order Family fluorescence. If the transferred process is blocked dimorphus (by herbicides), more of the excited energy will be Chlorella sp. Chlorococcales Chlorellaceae reemitting as fluorescence (light). Pediastrum sp. Chlorococcales Hydrodictyaceae Kircheriella sp Chlorococcales Oocystaceae Selenestrum sp. Chlorococcales Oocystaceae sp. Chlorococcales Scenedesmaceae or Coelastraceae

169 The 4th Annual Seminar of National Science Fellowship 2004

The microalgae originated from a lake near Result and Discussion Likas Bay, Kota Kinabalu, Sabah and were obtained from The Biotechnology Research Growth of Microalgae Institute, Universiti Malaysia Sabah. They The growths of the microalgae were were cultivated in a 500 ml blue capped bottle similar to the growth of other microorganism. in the modified Bristol media (Stein, 1973). Each growth curve showed a semi sigmoidal Starter cells density for all species was curve with 3 growth phase: a log phase, a lag approximately 1000 cells/ml. The temperature phase and a stationary phase. and light intensity of the test chamber were set 120 at 25°C +/- 2°C and 4000-5000 lux (12h day: Chlorella sp. 100 Coelastrum sp. Kirchneriella sp. 12h night). The growths of all microalgae Selesnestrum sp. 80 were monitored by cells counting using Pediastrum sp. haemocytometer. Chlorophyll concentration 60 was determined by 90% (v/v) acetone 40 extraction method suggested by Strickland and Parsons, 1968. 20

0 Algae Morphology 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 The photomicrograph of all the algae were Culture period (days) taken using Nikon Microphotographic FIGURE 2 Growth curves of all microalgae used. Equipment, model H-III. All species have a semi sigmodial curve with different period of growth phases and different Assay growth rate. Scenedesmus dimorphus has the Two types of PS II herbicides (diuron and highest growth rate (4.44x104 cells/day) followed propanil) with six different concentrations by Chlorella sp. (2.57x104 cells/day), Selesnestrum (0.001mM to 100mM) were used. In order to sp. (1.51x104 cells/day ), Kirchneriella sp. (7.7x103 3 study the effect of algal age, algae at four cells/day), Coelastrum sp. (7.7x10 cells/day). Pediastrum sp. has the lowest growth rate different culture ages; 7, 14, 21 and 28 days 3 were tested. The algal suspension was dark compared to all species (4.1x10 cells/day). In addition, Pediastrum sp. and Coelastrum sp culture adapted for 10 minutes prior to assay. 10 µl suspensions were easily contaminated with other aliquot of herbicide solution was added to 8ml microalgae. of algal suspensions and mix by swirling the tube. Fluorescence was measured before and Alga Morphology immediately after herbicide was added. For Coelastrum sp. was spherical cells. They control, 10 µl of 50% (v/v) solvent was used formed colonies of a fixed number of cells (4, as replacement to herbicides stock. All 8, 16 and more) arranged in 2 or 3 experiments were carried out at room dimensional. The colonies are spherical with temperature with minimum expose of light. inner empty space; cells attached to each other Fluorescence yields of inhibition were by a protrusion of cell wall, arranged in a recorded for 200-400 seconds. Three single layer (figure 3a). Selesnestrum sp. was replicates were made for each assay. The unicells. The body is crescent in shape; fluorescence yield was than plotted as a tapered at both ends (figure 3b). Chlorella sp. function of time (seconds) was single cells with round in shape. The chloroplast was cup-shape (figure 3c). Calculations and statistical Analysis Scenedesmus dimorphus was usually arranged Inhibition curves were fitted using linearly in a colony of 4 members. The cell CurveExpert v1.36. All calculations and bodies were ellipsoidal and crescent in shape statistical analysis were computed using (outer members of the colony) (figure 3d). SigmaPlot 6 (SPSS Inc., USA) and Microsoft Colonies of Pediastrum sp were crown like Excel (Microsoft Corporation). All data were shape. Each colony consists of 4 or more fixed mean of three replicates. cells. Cell body was polygonal in shape, with horn-like projections (resemble tooth shape) (figure 3e). Kirchneriella sp. was unicells

170 The 4th Annual Seminar of National Science Fellowship 2004 with bean shape (long cylindroid, strongly a) curved) (Figure 3f). 12 day 7

10 9.50 l) day 21 / g m

t ( 8 7.28 ab c n nte o 5.61

c 6 ll a y h

p 4 3.40 o r o l 2.25 h 1.79 C 2 1.62 1.44 d e f 0.68 0.57 0 S.dimorphus Chlorella sp. Selesnestrum Kircneriella sp. Pediastrum sp. sp b) Microalgae

7.00E-02 day 7 6.22E-02 ll)

e 6.00E-02 c

/ day 21 g

FIGURE 3 Photomicrographs of (a) Coelastrum u

ll ( 5.00E-02 4.65E-02 e 4.44E-02 sp. (100x10), (b) Selenestrum sp.(100x10), (c) c

r 4.19E-02 e 4.00E-02 p

Chlorella sp. (100x10), (d) Scenedesmus t n e t dimorphus (100x10), (e) Pediastrum sp. (40x10), n 3.00E-02 o (f) Kirchneriella sp. (100x10). c ll a 2.00E-02 y h

p

o 1.06E-02 r 1.00E-02 7.68E-03 lo 3.45E-03 Chlorophyll a content and fluorescence yield h

c 2.61E-03 1.29E-03 1.00E-03 Chlorophyll a content (mg/l) of all algae 0.00E+00 S.dimorphus Chlorella sp. Selesnestrum Kircneriella sp. Pediastrum sp. shows an increase with the increase of culture sp period from age 7 days to age 21 days. This Microalgae maybe due to the increase of cell density. Kirchneriella sp. has the highest total FIGURE 4 a) Chlorophyll concentration of studied chlorophyll content 3.4mg/l and 9.5 mg/l for microalgae at two culture periods; 7 days and 21 both culture ages (7 and 21 respectively) days. Kichneriella sp. has the highest chlorophyll (figure 4a). Pediastrum sp. has the highest content for both days. All species show increased in chlorophyll content when culture age was chlorophyll content per cell compare to the longer. A chlorophyll concentration for others (Figure 4b). Because chlorophyll Coelastrum sp. was not available due to concentration represents the amount of PSII contamination during cultivation. b) Chlorophyll reaction center for the herbicide binding concentration per algal cell express as ug/cell. action (Shakinaz, 1997), theoretically we can Pediastrum sp has the highest chlorophyll content assume that this species maybe the best algae per cell maybe due to their large size. to be used. However, overall fluorescence Kirchneriella sp. also showed high chlorophyll yield after herbicide inhibition of this alga was content per cell. lower than fluorescence yield of others (figure 5a). This suggested that in vivo fluorescence 180 160 Chlorella sp. of algal maybe not only depend on the amount Kirchneriella sp. Pediastrum sp. 140 Scenedesmus dimorphus of chlorophyll but also involve more complex Selesnestrum sp. interactions. In addition, the chloroplast 120 Coelastrum sp. eld (fsu) i 100 condition in the algal cells may result in false y result of chlorophyll analysis. For example, 80 cence

Kirchneriella sp. has a chloroplast full within res 60 the cell. Scedesmus dimorphus has one or fluo 40 more chloroplast in plate-like shape. 20

Chlorophyll in Kirchneriella sp. can easily be 0 extracted than chloroplast in Scencedesmus Day 7 Day 14 Day 21 Day 28 dimorphus. Culture period (days)

FIGURE 5 a) Chlorophyll fluorescence yield after 50 seconds inhibition of 1mM diuron. The yield was express as fluorescence after 50 sec inihibition

– fluorescence before inhibition.

171 The 4th Annual Seminar of National Science Fellowship 2004

the herbicide concentrations, algal species and 1.2e-3 type of herbicides used. In some species, the Chlorella sp. Kirchneriella sp. increase in the fluorescence was not detected

ll 1.0e-3 Pediastrum sp. Scenedesmus dimorphus at herbicide concentrations lower than r ce Selesnestrum sp. e Coelastrum sp.

) p 8.0e-4 0.01mM. The detection range was between u s

(f 100mM– 0.01mM for all algal species. An d l 6.0e-4 assay using non-PSII herbicide (Silvex and ce yie n 4.0e-4 2,4-D) showed no inhibition effect. sce

re o u

l 2.0e-4 f a) 0.0 140 Day 7 Day 14 Day 21 Day 28 120 Culture period (days)

) 100 u s f d (

l 80 e FIGURE 5b) Estimated Chlorophyll fluorescence i e y 60 nc yield of each algal cell after 50 sec inhibition of 0.001mM sce 40 0.01mM re diuron. 0.1mM

Fluo 1mM 20 10mM 100mM Selection of Best Microalgae 0

The best microalgae for use as biosensor 0 20406080100 was selected based on several criteria; a) Time (second) growth rate, b) easy to cultivate and maintain c) high sensitivity to PSII herbicide. The test b) algal used in biosensor must have high growth 60 rate, ubiquitous and easily cultivated to make 50 )

sure the continuous supply of the bio-receptor. u 40 Scenedesmus dimorphus suited these criteria (fs 30 well. Although the chlorophyll concentration e yield enc c in their cell is low, but the fluorescence yield 20 0.001mM 0.01mM per cell of this algal was high. Kirchneriela Fluores 10 0.1mM 1mM sp. has high chlorophyll content but produced 10mM low of chlorophyll fluorescence yield. In 0 100mM addition their growth rate was slow. 0 1020304050 Time (second) Coelastrum sp was difficult to cultivate. Their cells suspension can easily be contaminated FIGURE 6 Time-dependent Effect of PS II with other algal. From the result obtained herbicides, (a) diuron (b) propanil to the (figure 5b), the best age of algae to be used as Fluorescence yield of Scenedesmus dimorphus, biosensor was 7 days. Fluorescence yield per culture age 7 days. Solvent used was 50% (v/v) cell of all algae was highest at this age. For DMSO. The plotted data were mean of 3 further discussion, Scenedesmus dimorphus at replicates. Each curve was obtained from non- age 7 will be used as reference. linear regression of raw data.

Standard Calibration Curve and PSII The presence of PS II herbicides can be herbicide detection detected by the increased in vivo chlorophyll After herbicide was added to the fluorescence of the algae. The rate of microalgae, the fluorescence increased with increased of the chlorophyll fluorescence was time until it reached a saturating point. It was proportional to the PSII herbicide assumed that when the fluorescence reached a concentrations used. By plotting the rate of maximum, all of the chlorophyll pigments in chlorophyll increases to PS II herbicide the cells have been inhibited by herbicides. concentrations, a standard calibration curve These curves were the same as the kinetic can be obtained (figure 8a and 8b). reaction curve. All the algae tested with both Concentration of PSII herbicide in the sample herbicides had a similar curve pattern as can be estimate from the standard calibration Scenedesmus dimorhus, (figure 6a and 6b). curve. The detection was specific to PSII However the rate of the increase varies with herbicides since no inhibition effect on Hill

172 The 4th Annual Seminar of National Science Fellowship 2004 reaction centre was observed for herbicides Mohamed and for IRPA grant awarded to with different mode of action. Thus, no Kamaruzaman Ampon. The authors were also chlorophyll fluorescence increase can be grateful to Assoc. Prof Dr. Amran Ahmed, detected. School of Science and Technology, Universiti Malaysia Sabah, for his expertise and a) guidance in statistical analysis. 100 References

10 Merz, D., Geyer, M., and Moss, D.A. (1996). ) c e

s Chlorophyll Fluorescence Biosensor for the / su

(f Detection of Herbicides. Fresenius Journal of e t 1 a r

l Analytical Chemistry 354: 299-305 a i it in g

lo r ²=0.957524 0.1 Nichols, H.W. (1973). Growth Media– y=0.11+0.7x Freshwater. In Stein, J.R. (editor) Handbook Of Phycological Methods – Culture Methods 0.01 0.001 0.01 0.1 1 10 100 1000 And Growth Measurements: 7-24. London: log diuron concentration (mM) Cambridge University Press. b) Shakinaz Desa, (1997). The Development of 100 Photosystem II-Herbicide Detection Method Using Microalgae as a Biosensor, Faculty of 10 )

c Science and Environmental Studies, e s University Putra Malaysia. su/ (f e 1 l rat

ia Strickland, J.D.H and Parsons, T.R. (1968). it in

g Pigment Analysis. In Strickland, J.D.H. and lo 0.1 Parsons, T.R. (editors) A Practical Handbook r2= 0.955047 Of Seawater Analysis: 186-192. Ottawa: y= 0.47+0.96x 0.01 Fisheries Research Board of Canada. 0.001 0.01 0.1 1 10 100 log propanil concentration (mM)

FIGURE 7 Standard calibration curves for a) diuron and b) propanil using Scencedesmus dimorphus at the age of 7 days.

The sensitivity of this biosensor was around 10 µl (0.01mM). However, the sensitivity can be further improved with some modification in the assay method. The biosensor can be used to continuously monitoring the presence of PS II herbicides in the water body. Although this biosensor is specific to PSII herbicides, the biosensor can be used as primary detection of herbicides. PS II herbicides such as propanil and diuron are important active ingredient in herbicides used in agricultural area.

Acknowledgements The authors wished to thank the Ministry of Science, Technology and Innovation (MOSTI), Malaysia for the National Science Fellowship awarded to Maizatul Suriza

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