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 Chlorophyta (class of molecules into the excited state. From the chlorophyceae) 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 Scenedesmus fluorescence. If the transferred process is blocked Chlorococcales Scenedesmaceae 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 Coelastrum 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 Scenedesmus dimorphus 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.