Abstract: In present study we tried to improve the sensitivity of whole cells of cyanobacteria, spheroplasts and thylakoidal fragments of cyanobacteria in which PSII is the main target for most of the herbicides, some heavy metals and organic pollutants. We also screened out the potential of tolerance of different cyanobacterial species for herbicides. These biologically active materials are capable of oxygen evolution by photolysis of water in the presence of light. We performed growth study, chlorophyll a analysis, bioenergetic study, oxygenevolution property using clark’s type oxy electrode for the comparison of activity both in free cell stage and immobilized condition. We recorded the changes in their spectral properties, oxygen evolution and chlorophyll a concentrations in the presence of various herbicides. We determined the I50 values for whole cells of different cyanobacterial species and also recorded peak shifting.
INTRODUCTION
Heavy metal ion pollution resulting from industrialization and excess use of fertilizers, herbicides and pesticides are reasons for heavy metal toxicity in both aquatic and terrestrial environment (Alloway, 1994). The toxic level of these pollutants in water bodies affects mostly the photosynthetic processes (Campanella et al., 2001; Rutherford and Krieger-Liszkay, 2001). Current assumptions are that the changes in global climatic pattern due to green house effect would also alter the distribution of these pollutants in the soil surface. Herbicides, heavy metals and pesticides can be highly toxic to human and animal health; their jumbled use has serious environmental insinuation. To monitor such low residual levels of the herbicides there is a need for developing sensitive and reliable detection methods. About one half of the herbicides presently used in agriculture inhibit the light reactions in photosynthesis, mostly by targeting the photosystem II (PSII) complex (Draber et al., 1991). In PSII, the D1 protein is the main target of the herbicides. It is clear that herbicides are able to inhibit the Hill reaction in isolated chloroplasts and spheroplast (Good, 1961; Walker et al., 2001). On the basis of this strategy, thaylakoidal fragments/spheroplasts have been used, conversely, to detect herbicides by testing inhibition of the Hill reaction (Loranger and Carpentier, 1994; Rouillon et al., 1999) and inhibition of DCPIP photoreduction (Brewster and Lightfield, 1993; Brewster et al., 1995). Currently, some methods which are generally used in the testing of most herbicides are HPLC, GC-MS and ELISA (Pacakova et al., 1996). But, their disadvantages are that they require expensive equipments, organic solvents and purification of samples prior to assay. The prokaryotic cyanobacteria serve as an excellent tool for studying the effect of heavy metals on photosynthetic activity owing to their close similarity with the chloroplast. The most frequently used biosensing systems for monitoring photosynthetic herbicides (biological receptors) utilize intact cells of algae, cyanobacteria and diatoms to measure either changes in photocurrent (Rawson et al., 1987; Pandard and Rawson, 1993; Pandard et al., 1993; Preuss and Hall, 1995; Rouillon et al., 1999) inhibition of electron transport with artificial mediators (Rawson et al., 1989; Haggett, 1994), or changes in chlorophyll a fluorescence (Conrad et al., 1993; El-Jay et al., 1997; Van der Heever and Grobbelaar, 1998). Cyanobacteria are very sensitive microorganism, they also showed their sensitivity towards other stresses (Sudhir et al., 2005). In this study, we have worked with whole cells, spheroplasts and thylakoidal fragments as a potential biomarker for developing a cheap and reusable biosensor.
MATERIALS AND METHODS
Pigment analysis, Spectroscopic study were done at School of Life Sciences, University of Allahabad, New Delhi, India and Immobilization and Oxygen evolution study were done at Centre for Biotechnology, University of Allahabad, India.
Preparation of culture and optimization of growth conditions of the cultures: Various strains of cyanobacteria i.e., Spirulina platensis, Synechococcus PCC 7942 and Nostoc muscorum were collected from different laboratories including National Phytron Facility Centre, IARI, New Delhi. Stock cultures were maintained in a culture room continuously illuminated with cool fluorescent light (70 μM sec-2) at 25±2°C in 16 h light and 8 h dark conditions. All the culture flasks were continuously bubbled with air using air pump. The cultures were maintained under bacteria free conditions by regularly transferring the exponentially growing cultures to fresh sterile growth medium. We provided them a varying range of pH, temperature and light and recorded the best conditions for optimal growth.
Screening of different cyanobacterial species for testing their response towards different types of herbicides: We used various concentrations of herbicides, 2, 4 D, Diuron and DCMU for testing the inhibitory potential of the 3 cyanobacterial species. Varying concentrations of 2,4 D, 25, 50, 100, 200, 500, 600 and 700 μM; DCMU, 20, 40 and 60 μM and Diuron, 200 and 400 μM were taken. Readings were taken at 0th, 12th, 24th, 36th and 48th h. All the experiments were performed in triplicate sets. The spectra, growth curves and chlorophyll a with respect to control were regularly recorded (Fig. 1A-C). Growth study was done at University of Allahabad, Allahabad, India.
Pigment analysis: Homogenized culture were taken in eppendorf tubes and centrifuged at 9000 g for 5 min. The pellet was washed twice with the reaction buffer (25 mM Hepes-NaOH buffer (pH 7.5) containing 20 mM NaCl) and suspended in the same buffer. The washed pellet was resuspended in methanol. Eppendorf tubes were then kept overnight at 4°C in dark conditions. All experiments were performed under dim green light. The cultures in the tubes were subjected to repeated freezing and thawing and then centrifuged. The supernatant was taken and its absorption was measured at 660 nm. The amount of Chlorophyll a was calculated according to MacKinney (1941) (Fig. 1D-J).
| Fig. 1: | (A-C) Effect of DCMU (20 μM) on Growth pattern of Synechococcus PCC7942, Nostoc muscorum and Spirulina platensis, (D-J) Effect of DCMU (20 μM), Diuron (200 μM) and 2,4 D (600 and 700 μM) on the chlorophyll a content of Synechococcus PCC7942, Nostoc muscorum and Spirulina platensis |
Bioenergetic studies
Absorption spectra of intact cells: Cells were harvested at 9000
g for 5 min and the pellet was suspended in 3 mL of reaction buffer. The reaction
mixture contained reaction buffer, 25 mM Hepes-NaOH (pH-7.5) with and without
herbicide. The reaction mixture in the vial was stirred continuously in light
for 5 min in the case of spheroplasts/thylakoidal fragments and for time dependent
experiments incubation was done at 12th, 24th, 36th and 48th h. This cell suspension
was taken for scanning the absorption spectra using Shimadzu UV-160 and Ultrospec
from 400 to 750 nm.
Spheroplast preparation: Cells were first harvested by centrifuging them at 9000 g for 5 min. Pellet was washed once with spheroplast preparing buffer (Tricine-KOH (pH-7.5) containing 500 mM sucrose, 10 mM KCl and 10 mM EDTA) and resuspended in the same buffer. This suspension was incubated at 37°C in a water bath for 3 h with lysozyme (1 mg mL-1). After 3 h the reaction was stopped by centrifuging the suspension. The supernatant was discarded and the pellet was resuspended in the same buffer and processed for further work (Murthy et al., 1989).
Thylakoid isolation: Cells were harvested by centrifugation at 5000 g for 20 min and the pellet was suspended in 75 mM Tricine buffer containing 10 mM NaCl (pH-7.5). Cells were disrupted by ultrasonication for 4x15 sec periods and punctured by 15 sec rest periods in an ice bath. Unbroken cells were removed by spinning at 2500 g for 30 min to get a pellet of cell wall fragments. The resulting supernatant was spun at 35,000 g for 30 min at 4°C to sediment the thylakoids (Murthy et al., 1989).
Immobilization: The photosynthetic material was immobilized in an albumin glutaraldehyde matrix (BSA-GA) (Koblizek et al., 2002). All steps were performed at 4°C under a dim green light. The procedure involved mixing of 60 mM sodium phosphate buffer at pH 7.4, 15% BSA solution and 1.5% glutaraldehyde solution. The mixture was incubated for 2 min and then the photosynthetic material was added which was followed by 5 sec of agitation. This mixture was immediately distributed into working electrode. The electrode with immobilized samples were kept at -20°C for a minimum time of 12 h.
Oxygen evolution studies of immobilized and free cells: Activity of thylakoid fragments and spheroplasts were determined by measuring the rate of DCPIP photoreduction using spectrophotometer (Shimadzu, UV-3000 and Ultrospec 4000) (Giardi et al., 1996). DCBQ was used to measure the PS II catalyzed electron transport (H2O to DCBQ). The reaction mixture contained reaction buffer (25 mM Hepes-NaOH buffer pH 7.5, containing 20 mM NaCl) and freshly prepared DCBQ (Table 1).
| Table 1: | Native and entrapped whole cell analysis. Control electron transport activity (100%) is equal to 400±20 m mol O2 evolved mg Chl-1 h-1 |
RESULTS AND DISCUSSION
The growth optimization conditions for Nostoc muscorum and Synechococcus PCC 7942, was pH 7.8, light intensity of 70-80 μM sec-2 and CO2 rich air supplement of 5% (V/V) with rotation of 160 rpm. We also observed that at certain concentration of 2, 4 D (10-4 M), growth was promoted instead of showing inhibitory effect, earlier work done supported our results (Mishra and Pandey, 1989; Leganes and Fernandez-Valiente, 1992). It was found that in case of Spirulina platensis the inhibitory effect was observed only from 8 mM concentration. The concentration of 600 μM and above was inhibitory to Nostoc muscorum and Synechococcus PCC 7942. Figure 1A-J. The I50 values for DCMU, Diuron and 2,4 D were determined. This study showed that the rigid cell wall of Synechococcus PCC 7942 did not show response during short incubations with the herbicides, our results showed the similarity with earlier work done (Murthy and Mohanty, 1993). Hence, we used spheroplasts and thylakoidal fragments to improve the sensitivity of the biomaterial towards the stress. UV-Vis spectrophotometric analysis provided us the basic spectral properties of cyanobacteria. We observed four types of major peaks, one peak of chlorophyll a in red light region (680 nm) and trace of chlorophyll a in blue light region (440 nm), one peak of carotenoid (490 nm) and one peak of phycobilisomes (650 nm in our case), our results showed close similarity with earlier work done (Keren et al., 2004). All the peaks were greatly altered and showed peak shifting and changes in optical densities during the experiments with respect to the control. When the samples were treated with 2, 4 D, Diuron and DCMU, there was clear peak shifting and changes in the intensities. We performed two types of studies; one involving long incubation of the whole cells with the herbicides and secondly, short incubation using spheroplasts and thylakoidal fragments. This method give results within minutes. We immobilized the samples directly on working electrode and recorded the amount of oxygen evolution with respect to control. There was a slight decrease in activity using immobilized sample, work done earlier proved our results right (Cocquempot et al., 1981; Touloupakis et al., 2005). This technique will be exploited to design a biosensor for commercial use.
Photosystem II particles, when isolated were not very stable. They lost their activity with- in hours (Koblizek et al., 1998). It is very time consuming. Hence, we used whole cells, spheroplasts and thylakoidal fragments from cyanobacteria. It is also reported that among all PSII particles cyanobacterial PSII showed better half life (Koblizek et al., 1998). With respect to stability, the whole cells are the best followed by, spheroplasts and then, thylakoidal fragments. In regards to photosynthetic activity the thylakoidal fragments are the best followed by spheroplasts and then, the whole cells. Whole cells, spheroplasts and thylakoidal fragments prepared under optimum conditions in this laboratory showed greater stability and activity. The oxygen evolution studies on photosystem II of whole cells showed that, there was sharp decrease in amount of oxygen evolved with respect to control, which supported our study and proved that in the presence of these toxicants, the amount of oxygen evolved gets altered.
The spheroplasts/thylakoids of the three species of cyanobacteria can be used as potential biomaterials for the preparation of a biosensor for the testing of water. We can check the purity of water samples within few minutes, it is cost effective and easy to prepare. Other markers are very specific for specific type of pollutants (Antibodies/enzyme based), time consuming and costly (Nanoparticles based as SWCNT). These biomaterials can be directly used for making Amperometric/Potentiometric Biosensor. By immobilization of the biomaterial on appropriate surface such as Clarke’s type oxygen electrode, we can increase the life span of the biosensor.
ACKNOWLEDGMENT
We would like to thank the Department of Science and Technology, Government of India, for financial support.