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Evaluation and sensitivity ofcyanobacteria, Nostoc muscorum andSynechococcus PCC 7942 for heavymetals stress – a step toward biosensorMd. A. Rahman a , K.K. Soumya a , Ashutosh Tripathi a , ShanthySundaram a , Sushil Singh b & Amita Gupta ba Centre for Biotechnology, University of Allahabad , Allahabad,Uttar Pradesh, Indiab SSPL, DRDO , Timarpur, New Delhi, IndiaAccepted author version posted online: 20 Jul 2011.Publishedonline: 23 Aug 2011.

To cite this article: Md. A. Rahman , K.K. Soumya , Ashutosh Tripathi , Shanthy Sundaram ,Sushil Singh & Amita Gupta (2011) Evaluation and sensitivity of cyanobacteria, Nostoc muscorumand Synechococcus PCC 7942 for heavy metals stress – a step toward biosensor, Toxicological &Environmental Chemistry, 93:10, 1982-1990, DOI: 10.1080/02772248.2011.606110

To link to this article: http://dx.doi.org/10.1080/02772248.2011.606110

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Toxicological & Environmental ChemistryVol. 93, No. 10, December 2011, 1982–1990

Evaluation and sensitivity of cyanobacteria, Nostoc muscorum and

Synechococcus PCC 7942 for heavy metals stress – a step toward biosensor

Md. A. Rahmana, K.K. Soumyaa, Ashutosh Tripathia, Shanthy Sundarama*,Sushil Singhb and Amita Guptab

aCentre for Biotechnology, University of Allahabad, Allahabad, Uttar Pradesh, India; bSSPL,DRDO, Timarpur, New Delhi, India

(Received 2 June 2011; final version received 14 July 2011)

This study attempted to determine the effects of heavy metals on the photosyn-thetic blue-green algae for their potential to use as a biosensor. Thebioaccumulation of metals and its effects on pigments of Nostoc muscorum andSynechococcus PCC 7942 were assessed. The culture was grown in BG 11 liquidmedium supplied with different metals like mercury (Hg), lead (Pb), and cadmium(Cd) and incubated (mM 20 concentrations) for 10 days under optimal conditions.The accumulated amounts of metals were determined by atomic absorptionspectroscopy (AAS). The stress effects on photosynthetic pigment chlorophyll a(Chl a) were monitored by laser-induced fluorescence (LIF). Bio-concentrationfactor (BCF) reached a peak in cells on the 2nd day of incubation followed by agradual reduction. The highest reduction in the pigment concentrations (Chl aand � carotene) was observed at 20 mML�1 Hg treatment. The results indicatethat, cyanobacteria may serve as both potential species to be used as a biosensorand used to clean up heavy metals from contaminated water. These changes wereanalyzed with the long-term goal of exploiting cyanobacterial cells as biosensors.

Keywords: bioaccumulation; bioconcentration factor; cyanobacteria; biosensor;laser induced chlorophyll fluorescence

Introduction

Industrialization and urbanization has led to pollution which is major threat to theenvironment (Sundaram et al. 2011; Tripathi et al. 2011). Due to this, developing strategiesfor monitoring and restoring the health of the ecosystems is a major challenge forenvironmental scientists (Mallick 2003). Bioaccumulation can be used to remove theseheavy metals from the environment. It is a process in which a metal absorption ismetabolically controlled. The accumulation of heavy metals in aquatic ecosystem is highlytoxic. Heavy metals are required as micronutrients and macronutrients for cells at lowlevels but exert adverse effects at high concentrations such as those encountered in pollutedenvironment. In response, microorganisms are able to develop tolerance (Romero, Gatti,and Bruno 1999).

Cyanobacteria are a diverse group of photosynthetic prokaryotes responsible forphotosynthetic productivity. During evolution, these organisms improved different

*Corresponding author. Email: shanthy_s@rediffmail.com

ISSN 0277–2248 print/ISSN 1029–0486 online

� 2011 Taylor & Francis

http://dx.doi.org/10.1080/02772248.2011.606110

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strategies to maintain balance with heavy metal ions in surrounding medium (Perales-Vela,Pena-Castro, and Canizares-Villanueva 2006). Heavy metal toxicity, however, may resultin diverse effects, which generally depends on the types of algae, their characteristics, metalconcentration, and environmental conditions (Heng et al. 2004; Satoha et al. 2005; Greger,Malm, and Kautsky 2007). Metal toxicity may result in decreased pigments, nutrientimbalance, increasing antioxidant enzymatic activity, and induction of oxidative stress inalgae (Cervantes et al. 2001; Panda and Choudhury 2005). Growth inhibition andchlorosis are common symptoms of metal toxicity, in which, photosynthesis is the mostaffected metabolic process (Ali et al. 2006). Changes in morphology have also beenreported (Panda and Choudhury 2005).

Taking this into account, the bioaccumulation of heavy metals like mercury (Hg), lead(Pb), and cadmium (Cd) by cyanobacteria, Nostoc muscorum and Synechococcus PCC7942 and their effects on photosynthetic pigment content, laser-induced chlorophyllfluorescence and photosystem II-based electron transport and antioxidant activities weredetermined.

Materials and methods

Test organisms and culture conditions

Filamentous cyanobacteria Nostoc muscorum and unicellular non-nitrogen fixingcyanobacteria Synechococcus PCC 7942 were obtained from the Algal Laboratory,Botany Department, University of Allahabad, Allahabad, India. Nostoc muscorumand Synechococcus PCC 7942 were grown axenically in the BG-11 medium(Stanier et al. 1971) and BG-11 medium with nitrogen source, respectively, at pH 7.5and 24þ 2�C under 72 mmol photonm�2 s�1. Cultures were continuously homogenized in arotary shaker at 100 rotations per min and cultured for 10 days under the conditionsmentioned above.

Stress application

Acclimatized cells were obtained by successive cultivation (4–5 times) at concentrations ofheavy metal, Pb in the form of lead chloride, Hg in the form of mercuric chloride, and Cdin the form or cadmium chloride 20 mM (in aqueous form), as described by Attaway andSchmidt (2001).

Estimation of chlorophyll a and � carotenoid

At the end of incubation period, the cyanobacterial cultures were subjected to pigmentcontent analysis and chlorophyll a (Chl a)and �-carotenoids were extracted (Ben-Amotzand Avron 1983).

Metal bioaccumulation studies

Accumulated amount of heavy metals (mg g�1-dry weight) was determined by atomicabsorption spectroscopy (AAS) and calculated using the simple concentration differencemethod (Arunakumara, Zhang, and Song 2008).

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Photosynthetic electron transport activities

Photosynthetic electron transport activities were measured in whole cells of cyanobacteriaand spheroplasts prepared by the method of Spiller (1980). In each assay spheroplasts andwhole cells equivalent to 5 mgChl a mL�1 were used.

Laser-induced chlorophyll fluorescence

Laser-induced fluorescence spectra are recorded in the region of 600–800 nm by using405 nm violet diode laser. The excitation source with computer-controlled Acton 0.5Mtriple grating monochromator and PMT R928 as a detector were used (Pandey andGopal 2011).

Determination of osmoprotectants

Ascorbate was extracted from the pellets of 1mL test samples with 5% w/v sulfosalicylicacid. The amount of ascorbate was determined in the supernatant obtained aftercentrifugation at 12,000 � g for 10min (Oser 1979), and expressed as uL ascorbate mg�1

protein. Proline content in the cyanobacterial homogenate was measured according toBates, Waldren, and Teare (1973).

Estimation of reactive oxygen species: total peroxide radicals and lipid peroxidation(MDA content)

Total amount of hydrogen peroxide radicals was estimated (Sagisaka 1976). The red colorobtained was measured at 480 nm. Malondialdehyde (MDA) concentration was estimatedby the method of Heath and Packer (1968) for the measurement of lipid peroxidation. Theabsorbance of the supernatant was recorded at 532 nm. 0.5% thiobarbituric acid (TBA) in20% trichloroacetic (TCA) was used as the blank, malondialdehyde content wasdetermined using the solvent coefficient.

Data processing

All the procedures were performed under aseptic conditions. Percentage reductions inactivities were calculated by comparing with control samples % reduction¼ [(con-trol� treatment)/control]� 100. Data were statistically analyzed and the results wereexpressed as averages (�SD) of three independent replicates. Comparison of treatmenteffects were conducted by standard ANOVA analysis and multiple comparison test wereperformed using Exel software (Microsoft, USA).

Results and discussions

Photosynthetic pigments under heavy metal stress

Mercury at 20 mM concentration was found to produce the highest % decrease in Chl a(57%) followed by Cd (49%) and Pb (41%) in Nostoc muscorum. In Synechococcus PCC7942, Cd showed (50%) reduction followed by Hg (45%) and Pb (34%) in Chl a. Asignificant difference in Chl a levels was found to be depending on varying metals.Decrease in �-carotenoids content of Nostoc muscorum was with Hg (34%) and Pb (34%)

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in comparison to Cd (29%). In Synechococcus PCC 7942, 36% fall in �-carotenoids wasobserved in the presence of Hg and Pb while Cd showed 9% decrease (Figure 1).

Accumulation amount of heavy metals

Results of this study showed that cyanobacteria accumulate detectable levels of heavymetals from culture medium. Slotton, Goldman, and Frank (1989) found that when grownunder contaminated conditions, these species accumulate detectable levels of Hg and Pb.Accumulated amounts of heavy metals at 20 mM concentrations (Figure 2) showedsignificant differences. Cadmium showed the highest bioconcentration factor (BCF) 3.9and 3.8mgmL�1 in N. muscorum and Synechococccus PCC 7942, respectively.Accumulation of Hg was the least. Furthermore, results shows that BCF decreased asincubation progressed regardless of the heavy metal content in the medium (data notshown).

In the case of long-term accumulation of metals in microalgae, previous investigators(Kuyucak and Volesky 1989; Knauer, Behra, and Sigg 1997) documented that theaccumulation process starts with rapid passive adsorption followed by slow active uptakeand desorption. In addition, cyanobacteria both adsorb and take up metals (Bender et al.1994), the active absorption may also contribute to the initial high accumulated amountand high BCF of the present results.

Algae and cyanobacteria are well known to detoxify heavy metals (Lombardi andVieira 2000) by adsorption and precipitation of toxic metals in extracellular and cellularcompartments, such as cell walls, phosphate-rich granules, lipid bodies, vacuoles, nucleus,and physodes (Filho et al. 1999; De Andrade, Farina, and Filho 2002). Cyanobacteria alsopossess metal binding sites for specific organic-metallic compounds, such as metallopro-teins and phytochelatins (Lombardi and Vieira 2000).

Photosystem II electron transport under heavy metal stress

Mercury showed nearly 50% decrease in PSII electron transport activity of N. muscorum.In Synechococcus, Hg displayed reduction in PS II electron transport (21%). Increase in

Figure 1. Photosynthetic pigments content in cyanobacteria under heavy metal stress after 24 h.Error bars indicate SD of three replicates. Compared to control all the stress showed significantdifference (p5 0.05).

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PS II activity was found in N. muscorum with Cd (54%) and Pb (45%) (Figure 3). After24 h treatment, all stresses suppressed the oxygen yield and PSII electron transport in somecyanobacteria.

Laser-induced chlorophyll fluorescence

Nostoc muscorum and Synechococcus 7942 showed variation in fluorescence pattern incontrol as well as treated cells at room temperature. The fluorescence pattern of treatedand control cells are shown in Figures 4 and 5. In the unicellular strain, fluorescence isreabsorbed by the far red absorption band of the Chl a. Cadmium and controlSynechococcus PCC 7942 cells showed decrease in chlorophyll fluorescence. Theproportion of the fluorescence emission also decreased as heavy metals show higherchlorophyll fluorescence comparing control- and Hg-treated cells. Thisw study showed asignificant difference with varying metals.

The fluorescence signals can be incorporated for remote sensing (Beutler et al. 2003).The increase in antioxidant compounds helps to combat metal-induced stress. The findingsof our study are supported by Atri and Rai (2003).

Figure 2. Bioconcentration factor of heavy metals (mg mL�1) in N. muscorum and SynechococcusPCC 7942 grown BG11þ or � medium after 24 h. Error bars indicate SD of three replicates.Compared to control all the stress showed significant difference (p5 0.05).

Figure 3. Effect of heavy metals on PS II activity (H2O!DCPIP) in treated whole cells (W) andspheroplasts (S) of cyanobacteria. Error bars indicate SD done in triplicates. Compared to controlall the stress showed significant difference (p5 0.05).

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Accumulation of surface protectants:

There is an increase in surface protectants like ascorbate and proline after heavy metalsincubation for 24 h in caynobacteria. In N. muscorum, Hg, Pb, and Cd showed 60%, 50%,and 25% increase in ascorbate accumulation, repectively. Synechococcus 7942 displayed arise in ascorbate by 98%, 52%, and 9%, respectively. Proline accumulation also washigher in Hg-treated cells with a significant increase (175%) in N. muscorum and 283% inSynechococcus 7942 (Figure 6).

Total peroxide radicals and lipid peroxidation (MDA content):

Nostoc muscorum showed an increase by 152%, 117%, and 130% in the accumulation oftotal peroxide radicals in Hg-, Pb-, and Cd-treated cells and in Synechococcus 7942 134%,136%, and 149%, respectively. Lipid peroxidation (estimated as MDA) and a totalperoxide radical accumulation was used as reliable markers of oxidative stress (Halliwelland Guttiredge 2007). In Synechococcus 7942 there was a significant increase in lipidperoxidation of 58%, 39%, and 15% and in N. muscorum it was 28%, 6%, and �1% forHg, Pb, and Cd, respectively. (Figure 7).

Sagisaka (1976) suggested that the enhanced total peroxide production may be due toincreased cellular metabolism rate to combat stress or may be produced as the resultant of

Figure 4. Laser-induced chlorophyll fluorescence of N. muscorum under heavy metal stress.

Figure 5. Laser-induced chlorophyll fluorescence of Synechococcus PCC 7942 under heavy metalstress.

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protection for cells against stress. Increase in ascorbate, proline, and MDA contents with

heavy metal are indicative of a correlation between free radical generation and proline

accumulation (Figures 6 and 7). This is also in agreement with the earlier reports on

Spirulina platensis and Westellopis prolifica (Choudhary et al. 2007; Fatma, Khan, and

Choudhary 2007).

Conclusions

Cyanobacteria N. muscorum and Synechococcus PCC 7942 are potential accumulators of

heavy metals and thus might be used in detoxifying metal-contaminated water bodies. All

the bioenergetic studies show that whole cells of cyanobacteria may be used for designing a

biosensor or in remote sensing. The increase in antioxidant abilities may be used in

cosmetic industry.

Figure 7. Total peroxides and lipid peroxidation under heavy metal stress. Error bars indicate SDdone in triplicates. Compared to control all the stress showed significant difference (p5 0.05).

Figure 6. Accumulation of osmoprotectants under heavy metal stress. Error bars indicate SD donein triplicates. Compared to control all the stress showed significant difference (p5 0.05).

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Acknowledgments

We would like to thank Prof. Ram Gopal and Mr. Jitendra Kumar Pandey for LICF experiments.Ms. Soumya and Mr. Akhlaqur are thankful to DRDO for the financial support.

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