Comparative assessment of Ni and As(III) mediated alterations in diazotrophic cyanobacteria, Anabaena doliolum and Anabaena sp. PCC7120

The comparative effects of nickel (Ni 2+ ) and arsenite (As(III)) on two diazotrophic cyanobacterial species were investigated in terms of photosynthetic attributes. Both metals demonstrated inhibitory effects on growth, pigments (chl a and phycocyanin) and photosystem II (PS II) photochemistry. However As(III) exerted severe effects as compared to Ni reflected by (1) reduced growth (2) significant inhibition of chl a and phycocyanin, (3) reduction in maximum photochemical efficiency of PSII and (4) depleted plastoquinone pool, thus suggesting it as more toxic. Moreover, comparative analysis of two species also demonstrated interspecies variation in terms of stress adaptive strategies reflected through higher sensitivity of Anabaena doliolum over Anabaena PCC7120. Thus the study recommends application of A. PCC7120 as biofertilizer in Ni and As(III) contaminated paddy fields.


INTRODUCTION
Anthropogenic activities have altered the global biogeochemistry due to release of metals in recent years (Bhagat et al., 2016).Not only aquatic ecosystem but soil organisms are also negatively affected by metal contamination.Effect of elevated metal input on soil organism is reflected in form of reduced species diversity, abundance and biomass and changes in microbe mediated processes (Bengtsson and Tranvik, 1989;Giller et al., 1998;Vig et al., 2003).Although few metals hold prime importance for all living organisms due to their key role in basic life processes like photosynthesis and respiration, their elevated concentration in cells causes either their inappropriate binding to metal binding sites of enzymes or undesirable redox reactions thus causing lethal effects (Waldron et al., 2009a(Waldron et al., , b, 2010)).
Nickel is one such metal that plays a vital role in the cellular physiology of living organism (Poonkothai and Vijaywathi, 2012).It is coordinated by proteins either directly or through tetrapyrrole ring of coenzyme F 430 which coordinates a nickel atom in methyl-coenzyme M reductase (Ragsdale, 2003).Statistical data revealed that nickel emission from natural and anthropogenic sources are 2.9-56.8×10, respectively (Tercier-Waeber and Taillefert, 2008).Some anthropogenic sources that causes elevated Ni level into environment are energy supplying power stations (coal burning power plants, petroleum combustion and nuclear power stations), mining and associated activities, disposal of NiCd batteries, chemical industries (planting, metal finishing, pigment production, cement manufacturing) (Poonkothai and Vijaywathi, 2012;Nnorom and Osibanjo, 2009).
Apart from them, heavy metals are the non-degradable elements that occur naturally in biosphere.In past few years, their accumulation in environment as a result of their increased utilization in industrial activities such as in mining processes has raised a global concern (Huertas et al., 2014).Arsenic is a toxic metalloid and present in two biologically active forms arsenate (As V ) and arsenite (As III ).Arsenate is analogous to phosphate thus replaces phosphate from essential biochemical reactions such as glycolysis and oxidative phosphorylation causing toxic effects (Tawfik and Viola, 2011;Nriagu and Jerome, 2000).However arsenite is reported to bind dithiols, forming dithiols thus disrupting protein functions and producing reactive oxygen species (ROS) (Liu et al., 2002;Meng et al., 2004;Wysocki et al., 2001).Use of arsenic as herbicides, insecticides, rodenticides, food preservatives and byproduct of used fosil fuel are major anthropogenic activities that are challenging the environment (Flora et al., 1995).
Present study is important in the sense that the results would provide important information regarding the cyanobacteria's ability to tolerate arsenic and nickel.

Organism and growth condition
Anabaena spp., Anabaena PCC7120 and A. doliolum were cultivated photoautotrophically under sterile condition in BG-11 medium (Supplementary Table 1) (N2-fixing condition) buffered with Tris/HCl at 25 ± 2°C under day light fluorescent tubes emitting 72 μmol photon m −2 s −1 PAR (photosynthetically active radiation) light intensity with a photoperiod of 14:10 h at pH 7.5.The cultures were shaken manually 2 to 3 times daily for aeration.

Mode and source of stress application
Nickel stress was applied as NiCl2 at concentrations 0 to 32 µM and arsenite stress was applied as sodium meta arsenite at concentrations 0 to 80 mM.Sodium meta arsenite and nickel chloride autoclaved separately and calculated amount were added directly into the sterilized medium to achieve the desired concentration and working standards were obtained by further dilutions.

Measurement of survival
Exponentially growing cells of Anabaena PCC7120 and A. doliolum treated with their respective concentrations were collected at four time points (1, 7, 10 and 15 days).Cells never exposed to nickel and arsenite were used as control.Growth was estimated by measuring the OD (optical density) of the culture at 750 nm in a UV-VIS spectrophotometer (Systronics, India) up to 16th day.

Pigments
Chlorophyll a, carotenoid and phycocyanin were measured as per the method of Bennett and Bogorad (Bennett and Bogorad, 1973), by taking the absorbance at 663, 480 and 645 nm respectively.The extinction coefficient of chl a at 665 nm in absolute methanol is 74 .5 ml/mg-cm (Mackinney, 1941).

Measurement of chlorophyll fluorescence
Chl fluorescence in dark-and light-adapted control as well as treated cultures was measured using a PAM 2500 Chl fluorometer (WALZ GmbH, Effeltrich, Germany).The fluorometer was connected to a computer by the data acquisition system (PAMWIN, Walz, Germany).Prior to each measurement, the culture was darkadapted for 30 min (Guo et al., 2006).The minimal fluorescence yield of the dark-adapted state (F0) was measured by the modulated light which was too low to induce significant physiological changes in the plant, and was recorded after dark adaptation.Subsequently, a saturating pulse was given to measure the maximal fluorescence yield of the dark-adapted state (Fm) (Qin et al., 2006).The maximal photochemical quantum efficiency of PSII (Fv/Fm) was determined after a 20-min dark acclimation period in selected cultures.Other calculated fluorescence parameters was the pastoquinone pool (Fv/2) (Bolhar-Nordenkampf et al., 1989).

Statistical analysis
Each treatment consisted of three replicates; the results presented are mean values.

Measurement of growth and survival
The present study deals with assessment of comparative toxic effects of Ni and arsenite over two strains of Anabaena viz. A. doliolum and Anabaena sp.PCC7120.Being a vital component of paddy fields diazotrophic cyanobacteria have always fascinated researchers from all over the world.Figure 1a     content was noticed at all days of treatment under both Ni and As(III) stress in both species, however among both stresses As(III) caused more pronounced inhibition in both species (Figure 4a to d).

Chlorophyll fluorescence
The test metals were found to reduce maximal quantum yield in a concentration-dependent manner, which was more pronounced in A. doliolum following As(III) treatment (Figure 5). Figure 6 presents the impact of the test metals on plastoquinone pool (Fv/2) of A. doliolum and A. sp.PCC7120 after 24 h of Ni and As (III) treatment.

DISCUSSION
Growth behavior studies suggested sensitivity of A. doliolum over A. sp.PCC7120.This finds support from the studies of Singh et al. (2015), they found that A. doliolum is more sensitive as compared to Anabaena sp.PCC7120 under cadmium stress.Similarly, Agrawal et al. (2014) found following trend of tolerant behavior A. L31> Anabaena sp.PCC7120>A.doliolum under butachlor stress among three closely related species of Anabaena.This further attested the tolerant behavior A. doliolum over Anabaena sp.PCC7120 thus suggesting the presence of separate strategies to combat stress even within species.Further requirement of high concentration of As(III) as compared to Ni may be attributed to ability of Anabaena to accumulate high concentrations of As(III).Significant reductions in the photosynthetic pigments chl a and phycocyanin whereas significant increment in carotenoid content was found in both the species.Ni is known to affect the active site of O 2 -evolving complex to which it interacts, thus causing depletion of 2 extrinsic polypeptides resulting in diminished e -transport activity (Boisvert et al., 2007).However As(III) mediated chl a content inhibition may be attributed to inhibition of δaminolevulinic acid dehydrogenase, a key enzyme of chlorophyll biosynthetic pathway (Shrivastava et al., 2009).Other metals are also known to produce similar decrease in chl a content.For example, Carfagna et al. (2013), found decrease in chl a in a green alga, Chlorella sorokiniana under Cd/Pb stress.
Carotenoid content was significantly increased in A.
doliolum at 1 and 7 days of Ni treatment and at 1 and 15 days of As(III) treatment, however in A. PCC7120 at 1st day of Ni treatment and 1 and 7 day of As(III) treatment.
Carotenoids are known to be major players of antioxidant response against ROS and found to be increased under metal stress (Yu et al., 2015).Significant decrement in phycocyanin content was noticed at all days of treatment under both Ni and As(III) stress in both species.
Phycocyanin is located on exterior side of thylakoid membrane and thus possibly toxicant exposure is prolonged causing severe inhibition as compared to chl a.This observation finds support from work of Pandey et al. (2012) they observed significant reduction in phycocyanin content under As(V) stress.Maximum photochemical efficiency of PSII (efficiency at which light absorbed by PSII is used for  photochemistry when all reaction centers are open) of both test cyanobacteria following treatment with different concentrations of Ni and As(III) after 24 h was recorded.It was found to be affected significantly.The findings of our study are supported by Rahman et al. (2011).The ratio of Fv/Fm is considered as a stress indicator and designates the potential yield of the photochemical reaction (Björkman and Demmig, 1987).Fv/Fm remains high under control condition following irradiation because Q A is in oxidized state due to transfer of electrons to NADP and finally to CO 2 via Q B , the plastoquinone pool, and PSI.However under stress condition Fv/Fm may decrease because reoxidation of Q A is restricted as a result of decrease or partial block of electron transport from PS II to PSI.A noteworthy decrease in the plastoquinone pool as represented by the Fv/2 ratio (Figure 6) could be one of the possible causes for the reduced quantum yield under metal stress.
In summary, among both test cyanobacterium A. doliolum appeared as a sensitive strain towards Ni as well as As(III) exposure at low concentrations which are toxicologically and environmen-tally relevant.Both metals significantly inhibited the population growth, pigment content (chl a, phycocyanin) and maximal photochemical efficiency of PSII, which was found to be more pronounced in A. doliolum (Figure 6).However increase in carotenoid content was found thus suggesting onset of defense mechanism.Thus present study suggests Anabaena sp.PCC7120 as more efficient candidate to be used as biofertilizer as compared to A. doliolum and needs to be further investigated.Further studies exploring effect on nitrogen fixing abilities and antioxidative defence system of both test cyanobacteria is ongoing so as to present a holistic view demonstrating integrative effect as well as help in unveiling the tolerance mechanism.

Figure 1 .
Figure 1.Population growth curves of (a) A. doliolum (b) Anabaena sp.PCC7120 exposed to different concentrations of Ni 2+ (c) A. doliolum (d) A. sp.PCC7120 exposed to different concentrations of As(III).Mean values for three bioassays with three replicates ± standard deviation bars.
and b shows the growth trends for A. doliolum and Anabaena sp.PCC7120 respectively exposed to various concentrations of Ni; as displayed in the figure the cell density was inhibited significantly by all of the tested Ni concentrations except Ni (2 µM).Similarly, Figure 1c and d represents growth

Figure 2 .
Figure 2. Effect on chlorophyll a content of (a) A. doliolum (b) Anabaena sp.PCC7120 exposed to different concentrations of Ni 2+ (c) A. doliolum (d) A. sp.PCC7120 exposed to different concentrations of As(III).Mean values for three bioassays with three replicates ± standard deviation bars.
Figure 3a and b demonstrates effect of Ni on carotenoid content in A. doliolum and A. PCC7120 respectively and Figure 3c and d displays As(III) mediated alterations on carotenoid content in A. doliolum and A. PCC7120 respectively.Significant increase was found in A. doliolum at 1, 7 days of Ni treatment and at 1 and 15 days of As(III) treatment, however in A. PCC7120 at 1 st day of Ni treatment and 1 and 7 day of As(III) treatment, carotenoid content was significantly increased.Similar to chl a, significant decrease in phycocyanin

Figure 3 .
Figure 3.Effect on carotenoid content of (a) A. doliolum (b) Anabaena sp.PCC7120 exposed to different concentrations of Ni 2+ (c) A. doliolum (d) Anabaena sp.PCC7120 exposed to different concentrations of As(III).Mean values for three bioassays with three replicates ± standard deviation bars.

Figure 4 .
Figure 4. Effect on phycocyanin content of (a) A. doliolum (b) Anabaena sp.PCC7120 exposed to different concentrations of Ni 2+ (c) A. doliolum (d) Anabaena sp.PCC7120 exposed to different concentrations of As(III).Mean values for three bioassays with three replicates ± standard deviation bars.