Early detection of mercury contamination by ...

View Online

www.rsc.org/pps | Photochemical & Photobiological Sciences

PAPER

Early detection of mercury contamination by fluorescence induction of photosynthetic bacteria Emese Asztalos,a Francesca Italiano,b Francesco Milano,b Péter Marótia and Massimo Trotta*b

Downloaded by CNR Pisa on 31 August 2010 Published on 22 July 2010 on http://pubs.rsc.org | doi:10.1039/C0PP00040J

Received 2nd March 2010, Accepted 29th June 2010 First published as an Advance Article on the web 22nd July 2010 DOI: 10.1039/c0pp00040j The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg2+ ) contamination of the culture medium. The major characteristics of the induction (dark, variable and maximum fluorescence levels together with rise time) offer an easier, faster and more informative assay of indication of the contamination than the conventional techniques. The inhibition of Hg2+ is stronger in the light than in the dark and follows complex kinetics. The fast component (in minutes) reflects the damage of the quinone acceptor pool of the RC and the slow component (in hours) is sensitive to the disintegration of the light harvesting system including the loss of the structural organization and of the pigments. By use of fluorescence induction, the dependence of the diverse pathways and kinetics of the mercury-induced effects on the age and the metabolic state of the bacteria were revealed.

Introduction

Materials and methods

Heavy metal effect on living organisms is a topic of growing importance because of the widespread presence of these substances originating from both natural and anthropogenic sources. Particularly interesting are the very toxic metals, e.g. chromium, arsenic, mercury or cadmium, whose soluble compounds can heavily affect the metabolism and may result in dangerous pathologies in human beings.1 Photosynthetic organisms have also received a great deal of attention2 because of their potential application in bioremediation of sites contaminated with metals and other pollutants.3 Prokaryotes have been often investigated to gain details on how heavy metals interact with microrganisms3,4 and in the case of photosynthetic bacteria such investigations have to closely look at their effect on the photosynthetic apparatus. It was found that, depending on which heavy metal stress the bacteria is exposed to, the biosynthetic pathway of bacteriochlorophylls and heme may or may not suffer from the exposure to such chemicals.5,6 It is hence very valuable to have an affordable, reliable, sensitive and fast technique to indicate immediately the damage of the photosynthetic apparatus in photosynthetic bacteria exposed to heavy metal pollution. We set the aim to study the kinetics of damage over a wide time range with the fastest scale of seconds. As the harmful effects are manifested by changes of the induction of (bacterio)chlorophyll fluorescence (constant and variable parts and their ratio together with the rise time), this method will be used for their early detection.

Chemicals and bacterial strain

a Department of Biophysics, University of Szeged, Rerrich Béla tér 1, H-6720, Hungary b Istituto per i Processi Chimico Fisici – Consiglio Nazionale delle Ricerche, Via Orabona, 4 70124, Bari, Italy. E-mail: [email protected]; Fax: +39 080 5442128; Tel: +39 080 5442033

1218 | Photochem. Photobiol. Sci., 2010, 9, 1218–1223

HgCl2 , sodium citrate and all chemicals (vitamins, growth factors and buffers) required for bacterial cultivation were purchased from Fluka at the highest degree of purity available and used without any further purification. Complete details are given in Italiano et al.7 Cells of purple non-sulfur photosynthetic bacterium Rhodobacter (Rba.) sphaeroides strain 2.4.18 were obtained by German Collection of Microorganisms and Cell Cultures (DSM n. ¨ medium9 under 160) and were cultivated anaerobically in Sistrom stirring in 1 litre screw top flasks. The cultures of the bacteria were deoxygenated by bubbling N2 gas for 15 min. Tungsten lamps (40 W) provided continuous illumination with an irradiance of about 13 W m-2 on the surface of the vessel. The effect of ¨ medium to mercury was checked by growing cells in Sistrom which HgCl2 was added to the desired final concentration. The possible formation of poorly soluble mercury salts was avoided using equimolar amount of citrate. The anaerobic growth curves were obtained by plotting the logarithm of the relative population size, ln (N/N 0 ), against the time elapsed from the light exposure. Growth rate (m), lagphase duration (l) and the asymptotic population size (A) were determined by fitting the modified Gompertz equation to growth curves15,16

ln

⎤ ⎫⎪ ⎡ me ⎪⎧ N = A exp ⎪⎨− exp ⎢ ( l −t ) + 1⎥ ⎪⎬ ⎥⎦ ⎪⎪⎭ ⎢⎣ A ⎪⎪⎩ N0

(1)

where N is the cell concentration at the time t, N 0 is the initial cell concentration and e (the Napier number) is the base of the natural logarithm. To demonstrate the advantages of the measurement of the fluorescence induction, the cell growth experiments under constant illumination were carried out according to the following protocol: 26 mM HgCl2 was added to the culture in different phases of the cell growth. After the mercury treatment, the samples were kept

This journal is © The Royal Society of Chemistry and Owner Societies 2010

View Online

either in darkness or exposed to constant illumination and the fluorescence induction was tested on regular intervals. By splitting the cultures, simultaneous control samples were ensured with the same citrate concentration as was in the mercury poisoned culture. The concentration of the cells (N) was determined by calibrated ¨ Burker chamber under light microscope.


Fluorescence induction The kinetics of bacteriochlorophyll fluorescence upon rectangular shape of illumination was recorded by a home-built fluorometer equipped with a CW laser diode (808 nm and 1 W) that guaranteed appropriate sample excitation.10 Because the wavelength of the laser light matched the absorption band of the peripheral antenna system of Rba. sphaeroides, the excitation was very effective as indicated by the fast (about 100 ms) closure of the reaction center (see later for the rise time of the fluorescence induction). The duration and intensity of the laser pulse could be adjusted by a home-built control unit. The rise time of the laser excitation was much less than the time resolution of the device (~1 ms). The fluorescence of the bacteria was detected through an IR cutoff filter (Schott RG-850, l > 850 nm) by a red sensitive pin photodiode (10DI, UDT Sensors, Inc.) of large surface area. For fluorescence induction measurements, small volumes (200 ml) of samples were taken from the cultures, diluted to the same cell concentration and dark adapted (relaxed) under N2 atmosphere for 2 min before excitation. The characteristics of the kinetics of bacteriochlorophyll fluorescence induction are shown in Fig. 1. Two phases are clearly distinguishable: a sharp rise, representing the constant or dark fluorescence (F 0 ), and a slowly increasing phase representing the time-dependent variable fluorescence, F v (t). The two phases sum up giving the total (maximum) fluorescence level at the end of the rise (F max = F 0 + F v ). The F 0 initial phase arises from the prompt emission of those antenna bacteriochlorophylls (or other pigment molecules) whose excitation is not converted

Fig. 1 Typical fluorescence induction kinetics of intact cells of Rba. sphaeroides 2.4.1 without (ctrl) and with treatment of mercury for 2 h (+Hg2+ ) upon stepwise dark-light transition. F 0 , F v (t) and F max are the dark, the variable and the maximum levels of fluorescence, respectively and t is the rise time of the variable fluorescence. F 0 is the cutoff level of fluorescence when the excitation is switched on but no photochemistry is taking place. It is obtained by interception of two straight lines of different slopes (see inset with 5¥ expanded time scale). Similar quantities can be introduced for the mercury treated cells. Note the decrease of all parameters of the induction in Hg2+ poisoned cells compared to those in control cells.

into a charge separated state by the reaction center (RC). These bacteriochlophyll molecules are energetically uncoupled from the light harvesting system and therefore not connected to the RC. On the other hand, the F v variable part of the fluorescence originates form connected antenna molecules, therefore the F v /F 0 ratio measures the efficiency of the photochemical trapping of the absorbed photons.11,12 The rise time of the induction kinetics from F 0 to F max relates to the absorption cross section of the antenna and to the rates of complex electron transfer reactions in and out of the RC.13,14

Results Growth of bacteria Cultures supplemented with HgCl2 and citrate (Fig. 2, upper panel) or control cultures (citrate only, not shown) were carried

Fig. 2 Growth curves and response-dose curves for Rba. sphaeroides 2.4.1 cells exposed to HgCl2 . Upper panel shows the growth curves obtained at increasing concentrations of mercury. The data of the sample treated by 100 mM mercury are not shown. Citrate is always kept equimolar with Hg2+ . The dose-response curves for relative growth rate and relative asymptotic population are shown in the middle and lower panel respectively.


Photochem. Photobiol. Sci., 2010, 9, 1218–1223 | 1219


View Online

Fig. 3 Changes of the maximum (F max ) and variable fluorescence (F v /F max ) of Rba. sphaeroides 2.4.1 cells after addition of 26 mM citrate with (䊊) and without () 26 mM Hg2+ into cell culture of early exponential growth phase. The cells were kept in the light (open symbols, left panels) or in the dark (closed symbols, right panels) after the treatment.

out simultaneously. The growth parameters from control cultures were used to estimate the relative growth rate (mrel = mHg /mcontrol ) and the relative asymptotic population (Arel = AHg /Acontrol ). These dimensionless parameters minimize any effects on bacterial growth different from mercury (i.e. fluctuations in light intensity or temperature condition, etc.). The dose-response curves of Rba. sphaeroides to mercury were obtained by plotting the relative growth rate (Fig. 2 middle panel) and the relative asymptotic population increase (Fig. 2 lower panel) obtained from cultures at different Hg2+ concentrations. From these data the mercury concentration at which each parameter is halved, the EC50 , were obtained. EC50 values of 1.98 ± 0.02 mM and 3.8 ± 0.1 mM were found for mrel and Arel respectively. Maximum and variable fluorescence are sensitive indicators of exposure of cells to Hg2+ Mercury is very detrimental for photosynthetic organisms and, at the concentration employed in this paper, it inhibits any growth of Rba. sphaeroides 2.4.1. The novel method of fluorescence induction of intact cells is introduced to study the effect of Hg2+ if it is added to the culture at different (lag, exponential and stationary) phases of the cell growth. By comparing fluorescence parameters of cells kept in the light or in the dark after the mercury treatment, information on the extent of the Hg2+ detrimental effect on the light-driven metabolism can be obtained. High mercury concentration inhibits any photosynthetic growth (Fig. 2). To check if the maximum level of fluorescence (F max ) and variable fluorescence (F v /F max ) are reliable and sensitive indicators for this detrimental effect, cells in the early exponential phase of growth were treated by Hg2+ and citrate or sole citrate as control. Sample and control were split in two parts, one left under illumination and the other kept in the dark. Fluorescence 1220 | Photochem. Photobiol. Sci., 2010, 9, 1218–1223

induction was measured at regular intervals starting within 3 min from mercury treatment (Fig. 3). In control sample allowed to grow photosynthetically, F max increases in accordance with the number of cells in the culture, while Hg2+ -treated sample shows an opposite trend, with a sharp decrease of F max (Fig. 3 top left panel). F v /F max remains constant in the control, indicating a constant photochemical utilization of the photons by the photosynthetic apparatus of the cell, while decreases in the case of the Hg-treated sample (Fig. 3 lower left panel). The cells left in the dark cannot grow photosynthetically and indeed the control shows constant F max value, indicating that no more photosynthetic pigments are synthesized, and constant F v /F max value, indicating that the photochemical yield of the photosynthetic apparatus remained unchanged in the time interval of the experiment. Moreover, the decrease of the two parameters in the Hg-treated sample kept in the dark is significantly smaller than in the light exposed sample (Fig. 3 right panels). Rise time of the variable fluorescence as probe of kinetics of detrimental effects of Hg2+ The prompt and secondary changes of the rise time of the variable fluorescence of Rba sphaeroides cells upon mercury treatment are demonstrated in Fig. 4. The cells in photosynthetically growing culture were exposed to Hg2+ of final concentration of 26 mM (indicated by an arrow) and kept subsequently in the dark. As shown in Fig. 1, the maximum fluorescence decreases and the variable fluorescence becomes gradually negligible upon mercury treatment. The decrease of F v is accompanied by a decrease of its rise time, that halves within ª20 s and reaches its plateau within 2 min (Fig. 4 left panel). Such behaviour may reflect the prompt effect of mercury on the photosynthetic apparatus of the bacteria.



View Online

Fig. 4 Changes of rise time of the variable fluorescence of Rba. sphaeroides 2.4.1 cells in the stationary phase of growth after addition of 26 mM Hg2+ into the culture (arrow) in the dark (䉱). The fast drop of approximate decay time of 20 s (left side) is followed by slow (partial) recovery in the dark (䊉) or in the light (䊊) (right side).

On a much longer (hours) time scale, however, the average rise time of F v increases again, probably due to secondary effects of the mercury (Fig. 4 right panel). This longer scale behaviour is accelerated if the Hg-treated sample is kept in the light. Exposure of cells in different phases of growth to Hg2+ The sensitivity of bacteria to mercury exposure depends on the age of the cells. Cells in the lag or exponential growth phase are very vulnerable and indeed the mercury treatment causes a severe drop of F max within 1–2 h (Fig. 5 upper and middle panel). Aged cells in the late stationary phase appears more resistant to Hg2+ and in the same time range do not markedly differ from untreated cells (Fig. 5 lower panel).

Discussion

Fig. 5 Changes of the maximum fluorescence (F max ) of Rba. sphaeroides 2.4.1 cells in different phases of growth with (at t = 0, 䊊) and without () addition of 26 mM Hg2+ into the culture in the light. The cells arrived at the lag (top), early exponential (middle) and late stationary (bottom) phases 1.5, 5 and 24 h after inoculation, respectively.

The widespread diffusion of mercury in the environment poses a health threat because this ion can easily enter the human food chain. This has spurred several studies focused on the assessment of Hg-toxicity on eukaryotes and prokaryotes. Mercury is generally rather toxic and its toxicity strongly depends on the organism, with EC50 values ranging from 60–80 mM in murine macrophages17 to 0.1 mM in the sea-urchin Paracentrotus lividus18 An even smaller value of 0.025 mM was published in the embryogenesis of the larvae of the bivalve Meretrix meretrix.19 Similarly, several authors have shown that mercury has a strong detrimental effect on photosynthesis.20–25 EC50 value of 0.7 mM is found for Sesbania drummondii seedlings, while a value of one order of magnitude smaller is found for rice seedling.26 A quite comprehensive description of mercury toxicity on microorganisms is given by Boening.27 Recently Borsetti et al.2 have reviewed the effect of metals on photosynthetic bacteria, and the effect of mercury has gained special attention by Giotta et al.28 in the case of the a deletion mutant of Rba sphaeroides, the carotenoidless strain R26. This mutant29,30 lacks caroteinoids, hence cannot survive at contemporary exposure to oxygen and light. On the other hand, it also has a reduced and sturdier photosynthetic apparatus, in good agreement with its higher resistance to mercury toxicity, EC50 of 20–30 mM, compared to that of 2–4 mM obtained here in wild type strain Rba sphaeroides 2.4.1.

The effect of Hg2+ on the photosynthetic apparatus is strongly associated with interaction of the groups C O, C–N, C–S, C– ˇ senˇ et al.32 have shown SH31,32 of the amino acids. In particular Serˇ that in mercury treated spinach chloroplasts, the fluorescence emission of chlorophyll, recorded at 685 nm, decreases to roughly 30% of the untreated samples. This indicates a strong detrimental effect on the antenna pigments. Furthermore the photochemical apparatus is also found strongly impaired by exposure to mercury as shown by the disappearance of the Z+ /D+ EPR signal arising from the tyrosins of the PSII.32 In photosynthetic bacteria of Rba. sphaeroides 2.4.1, the contemporary decrease of F 0 , F max and F v /F max (Fig. 3) agrees with a double detrimental effects on both the antenna bacteriochlorophyll pigments and the special pair of bacteriochlorophylls forming the primary electron-donor. Additionally, the changes in the rise time of the variable fluorescence F v reveal well separated effects of the mercury on the photosynthetic apparatus (Fig. 4). The prompt effect operating in the seconds time scale causes decrease of the fluorescence induction rise time. To understand it, we have to keep in mind that the rise from F 0 to F max follows complex (not monoexponential) kinetics (Fig. 1) as the lightdriven charge separation (PQA QB → P+ QA - QB ) should compete with losses of charges on the donor (P+ → P) and on the acceptor




View Online

(QA - QB → QA QB - ) sites which prevent the fast formation of P+ QA - , the highest fluorescent state of the RC. As soon as (after several turnovers) these electron transfer reactions are blocked (e.g. by exhaustion of the pools), the fluorescence reaches its maximum. The prompt effect of observed decrease of the rise time of the variable fluorescence comes from the disappearance of the slow phase of the induction that reflects the inhibition of the continuous turnover (electron transfer) through the RC. Thus, the primary effect likely involves the attack of Hg2+ to the acceptor and/or donor sites of the RC. The secondary effect of Hg2+ taking place on a longer time scale (hours) destroys the BChl pigments and weakens the connectivity within the antenna, thus decreasing the absorption cross section and hence the rate of photon capture. The relative increase of the risetime is however smaller than the relative loss of the pigments. Even at significantly reduced levels of fluorescence (e.g. after Hg2+ treatment), the rise is small but remains fast (Fig. 1). The bacteria exposed to mercury loose the light harvesting BChl pigments earlier than the coupling of the antenna. They do not disappear simultaneously. Our results indicate that the interaction among the pigments can be kept intact even at decreasing number of pigments in the antenna. Besides, mercury severely inhibits the antenna organization and electron transfer of the cell, its action is influenced by the age of the cell (Fig. 5) and facilitated by (lightdriven) metabolic processes (Fig. 4). The Hg2+ ion exerts the largest inhibitory effect in the exponential phase of growth of the cells and when they were exposed to light after mercury treatment. Much smaller (if any) effects were observed using old cells (in the late stationary phase of growth) in the dark. These results suggests that the mercury ions enter the cells and the chromatophore membrane not by passive but by facilitated or active transport mechanisms supported by metabolic processes. Hg2+ ions can pass the membranes by co-transport or use channels opened for other ions. These findings suggest that mercury may have similar effects on the photosynthetic apparatus of Rba. sphaeroides as observed in plants and algae. Additionally, understanding the ways of interaction of mercury ion with photosynthetic bacteria can help us to reveal the molecular details of harmful action of other heavy metal ions and to work out strategies to early and sensitive detection of contamination.

Conclusions The effect of mercury ions on the photosynthetic apparatus of Rba. sphaeroides 2.4.1 was investigated by recording the fluorescence induction of whole cells. The data show that exposure to Hg2+ is extremely detrimental for the bacterium, heavy compromising the photosynthetic apparatus at concentrations as low as 3 mM, a higher toxicity than previously obtained for Rba. sphaeroides carotenoid deficient strain R.26. The prompt effect of the mercury treatment is expressed by dramatic decrease of the intensity of bacteriochlorophyll fluorescence of the cells within couple of seconds after the exposure. The method of detection of fluorescence induction of photosynthetic bacteria is a sensitive, fast, efficient and easy-to-use technique to screen the cultures for heavy metal contamination. To our knowledge, this is the first report in which fluorescence induction was used for kinetic investigation of short and long 1222 | Photochem. Photobiol. Sci., 2010, 9, 1218–1223

term effects of metal ions on structure and function of the photosynthetic apparatus of bacteria. The example demonstrated here, however, represents a simplified case, in which bacteria are exposed to a single chemical, while very often heavy metal pollution is accompanied by other pollutants such as other metal ions, herbicides or pesticides. The application of the fluorescence induction of purple bacteria in detection of pollutants under more realistic conditions will require further investigations.

Acknowledgements ´ for her help in fluorescence The authors thank Ms Diana Nyuli induction experiments. The support of NKTH-OTKA (K-67850), COST Action on “Molecular machineries for ion translocation across biomembranes” (CM0902) and MTA-CNR Bilateral agreement on “Bacterial photosynthesis: artificial photosystems and bioremediation” is acknowledged.

References ¨ 1 L. Barregard, E. Fabricius-Lagging, T. Lundh, J. Molne, M. Wallin, M. Olausson, C. Modigh and G. Sallsten, Cadmium, mercury, and lead in kidney cortex of living kidney donors: Impact of different exposure sources, Environ. Res., 2010, 110, 47–54. 2 F. Borsetti, P. L. Martelli, R. Casadio and D. Zannoni, Metals and Metalloids in Photosynthetic Bacteria: Interactions, Resistance and Putative Homeostasis Revealed by Genome Analysis, The Purple Phototrophic Bacteria, Springer, Dordrecht, The Netherlands, 2009, pp. 655–689. 3 A. Malik, Metal bioremediation through growing cells, Environ. Int., 2004, 30, 261–278. 4 S. Silver, Bacterial resistances to toxic metal ions—a review, Gene, 1996, 179, 9–19. 5 F. Pisani, F. Italiano, F. de Leo, R. Gallerani, S. Rinalducci, L. Zolla, A. Agostiano, L. R. Ceci and M. Trotta, Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter sphaeroides, J. Appl. Microbiol., 2009, 106, 338–349. 6 A. Buccolieri, F. Italiano, A. Dell’Atti, G. Buccolieri, L. Giotta, A. Agostiano, F. Milano and M. Trotta, Testing the photosynthetic bacterium Rhodobacter sphaeroides as heavy metal removal tool, Ann. Chim., 2006, 96, 195–204. 7 F. Italiano, A. Buccolieri, L. Giotta, A. Agostiano, L. Valli, F. Milano and M. Trotta, Response of the carotenoidless mutant Rhodobacter sphaeroides growing cells to cobalt and nickel exposure, Int. Biodeterior. Biodegrad., 2009, 63, 948–957. 8 J. F. Imhoff, Anoxygenic Photosynthetic Bacteria, Kluwer Academic Publishers, Dordrecht, The Netherlands, 1995. 9 W. R. Sistrom, The Kinetics of the Synthesis of Photopigments in Rhodopseudomonas spheroides, J. Gen. Microbiol., 1962, 28, 607–616. ´ Kinetics and yields of bacteriochlorophyll fluorescence: 10 P. Maroti, redox and conformation changes in reaction center of Rhodobacter sphaeroides, Eur. Biophys. J., 2008, 37, 1175–1184. 11 D. Bina, R. Litvin and F. Vacha, Kinetics of in vivo bacteriochlorophyll fluorescence yield and the state of photosynthetic apparatus of purple bacteria, Photosynth. Res., 2009, 99, 115–125. 12 H.-W. Trissl, Antenna organization in purple bacteria investigated by means of fluorescence induction curves, Photosynth. Res., 1996, 47, 175–185. ´ Export or recombination of charges in 13 E. Asztalos and P. Maroti, reaction centers in intact cells of photosynthetic bacteria, Biochim. Biophys. Acta, Bioenerg., 2009, 1787, 1444–1450. 14 M. Koblizek, J. D. Shih, S. I. Breitbart, E. C. Ratcliffe, Z. S. Kolber, C. N. Hunter and R. A. Niederman, Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence, Biochim. Biophys. Acta, Bioenerg., 2005, 1706, 220–231. 15 M. H. Zwietering, I. Jongenburger, F. M. Rombouts and K. van’t Riet, Modeling of the Bacterial Growth Curve, Appl. Environ. Microbiol., 1990, 56, 1875–1881.



View Online

16 M. H. Zwietering, F. M. Rombouts and K. van’t Riet, Comparison of definitions of the lag phase and the exponential phase in bacterial growth, J. Appl. Bacteriol., 1992, 72, 139–145. 17 S. H. Kim and R. P. Sharma, Mercury-induced apoptosis and necrosis in murine macrophages: role of calcium-induced reactive oxygen species and p38 mitogen-activated protein kinase signaling, Toxicol. Appl. Pharmacol., 2004, 196, 47–57. 18 N. Fernandez and R. Beiras, Combined toxicity of dissolved mercury with copper, lead and cadmium on embryogenesis and early larval growth of the Paracentrotus lividus sea-urchin, Ecotoxicology, 2001, 10, 263–271. 19 Q. Wang, B. Liu, H. Yang, X. Wang and Z. Lin, Toxicity of lead, cadmium and mercury on embryogenesis, survival, growth and metamorphosis of Meretrix meretrix larvae, Ecotoxicology, 2009, 18, 829–837. 20 H. Clijsters and F. Assche, Inhibition of photosynthesis by heavy metals, Photosynth. Res., 1985, 7, 31–40. 21 M. Gadallah, Interactive effect of heavy metals and temperature on the growth, and chlorophyll, saccharides and soluble nitrogen contents in Phaseolus plants, Biol. Plant., 1994, 36, 373–382. 22 K.-N. Lars, The Effect of Deleterious Concentrations of Mercury on the Photosynthesis and Growth of Chlorella pyrenoidosa, Physiol. Plant., 1971, 24, 556–561. 23 C. M. Lu, C. W. Chau and J. H. Zhang, Acute toxicity of excess mercury on the photosynthetic performance of cyanobacterium, S. platensis assessment by chlorophyll fluorescence analysis, Chemosphere, 2000, 41, 191–196.

24 S. Murthy and P. Mohanty, Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacterium Synechococcus 6301, J. Biosci., 1993, 18, 355–360. 25 M. Patra and A. Sharma, Mercury toxicity in plants, Bot. Rev., 2000, 66, 379–422. 26 X. Du, Y. G. Zhu, W. J. Liu and X. S. Zhao, Uptake of mercury (Hg) by seedlings of rice (Oryza sativa L.) grown in solution culture and interactions with arsenate uptake, Environ. Exp. Bot., 2005, 54, 1–7. 27 D. W. Boening, Ecological effects, transport, and fate of mercury: a general review, Chemosphere, 2000, 40, 1335–1351. 28 L. Giotta, A. Agostiano, F. Italiano, F. Milano and M. Trotta, Heavy metal ion influence on the photosynthetic growth of Rhodobacter sphaeroides, Chemosphere, 2006, 62, 1490–1499. 29 R. J. Cogdell and J. P. Thomber, Light-harvesting pigment—protein complexes of purple photosynthetic bacteria, FEBS Lett., 1980, 122, 1–8. 30 C. Rafferty, J. Bolt, K. Sauer and R. Clayton, Photooxidation of antenna bacteriochlorophyll in chromatophores from carotenoidless mutant Rhodopseudomonas sphaeroides and the attendant loss of dimeric exciton interaction, Proc. Natl. Acad. Sci. U. S. A., 1979, 76, 4429–4432. 31 M. Bernier and R. Carpentier, The action of mercury on the binding of the extrinsic polypeptides associated with the water oxidizing complex of photosystem II, FEBS Lett., 1995, 360, 251–254. ˇ sen, ˇ K. Král’ová and A. Bumbálová, Action of Mercury on the 32 F. Serˇ Photosynthetic Apparatus of Spinach Chloroplasts, Photosynthetica, 1998, 35, 551–559.

