Modication of heavy metals toxicity by Cyanobacteria Nostoc sp. N27P72 and Nostoc sp. FB71 in Culture Conditions

Purpose: Cyanobacteria are ecologically relevant prokaryotes that can be found in environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been extensively considered in cyanobacteria. So far, most studies have focused on treatment of wastewaters using microalgae leads to remarkable reduction of an array of organic and inorganic nutrients, but what takes place in the extracellular environment when cells are exposed to external supplementation with heavy metals remains largely unknown. Methods: Here, extracellular polymeric substances (EPS) production in strains Nostoc sp. N27P72 and Nostoc sp. FB71 isolated from different habitats are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that production of EPS and cell dry weight was boosted by maltose supplementation. Result: Nostoc sp. N27P72 which was isolated from lime stones was higher, resulting in 9.1 ± 0.05 µg/ml and 1.01 ± 0.06 g/l in EPS and cell dry. The cell cultures tested for their ability to remove Cu(II), Cr(III) and Ni(II) in media culture containing the maltose and without maltose as control culture. Remarkably, we showed that although these elements can be toxic, supplementing the media culture can effectively sequester their toxic effects by increasing the production of EPSs, carbohydrates and total soluble proteins in comparison to control. The crude EPS showed metal adsorption capacity assuming the order Ni(II)> Cu(II)> Cr(III) from metal-binding experiments. Nickel was preferentially biosorbed with a maximal uptake of 188.8 ± 0.14 mg (g cell dry wt) -1 crude EPS. FT-IR spectroscopy revealed treatment with Ni made changes in the functional groups and glycoside linkages in both strains. Results of Gas Chromatography Mass Spectrometry (GC–MS) to determine the biochemical composition of Nostoc sp. N27P72 showed that strong Ni(II) removal capability is suspected to be associated with the high Cyclotrisiloxane and 1,2-Benzenedicarboxylic acid content. Conclusion: The results of these investigates specied that strains Nostoc sp. N27P72 is good candidates for the commercial production of EPS and might be utilized in bioremediation eld as an alternative to synthetic and abiotic occulants.


Introduction
Nostoc cyanobacteria species are a large and morphologically diverse group of phototrophic prokaryotes which have found in various habitats. They have has drawn more attention because of the presence of outermost polysaccharidic envelopes, often coupled with the capability to release exocellular polysaccharides (RPS) into the culture medium during cell growth .
Most of these polymers are characterized by an anionic nature, owing to the presence of uronic acids and/or of other charged groups (Pereira et al., 2013). As a result, these polysaccharides typically keep very high a nity to metallic ions and can be considered very promising as chelating agents for the removal of heavy metals from water De Philippis et al. 2001;Cepoi et al., 2019;Kumaran et al., 2011;Ni et al., 2019).
Many factors in uence cell growth and metabolite accumulation in microalgal cell cultures and extracellular polymeric substances (EPSs) including nutrients, such as phosphate and nitrogen, temperature, light intensity, aeration rate, and mixotrophic condosion (Otero and Vincenzini 2003); (Helm et al. 2000). Althought the presence of EPS is extremely preserved among cyanobacteria, not much is recognized about factors that maximize EPS biosynthesis and affecting biosorption capacity (Pereira et al. 2009;Klock et al. 2007;De Philippis et al. 2000;Yoshimura et al. 2011;Gupta et al., 2017). Some trace amounts (μgL -1 ) such as copper, cobalt and nickel are essential by cyanobacteria strains as cofactors for the enzymatic activities. Unlike organic contaminants, heavy metals such as, copper and lead are main pollutants of freshwater due to their inherent nature of being persistent, toxic, recalcitrant and non-biodegradable . On the other hand, heavy metal ion concentrations at ppm (mgL -1 ) level are known to be toxic to the organisms because of irreversible inhibition of many enzymes by the heavy metal ions. Heavy metal uptake capacity of algal biomass has proved to be the highest due to the presence of polymers such as polysaccharides, proteins or lipids on the cell wall surface containing functional groups such as amino, hydroxyl, carboxyl and sulfate, which can act as binding sites for metals . A number of articles have been published on phycoremediation investigation and severlal authors have established the fact that treatment of wastewaters using algae, microalgae particularly leads to remarkable reduction of an array of organic and inorganic nutrients, including some of the toxic chemicals (Anjana et al., 2007;Essa and Mostafa., 2012;Farooqui et al., 2017;Principe et al., 2020;Devi and Mehta., 2014). Our previous research found that cell growth and EPS production are highly dependent on the culture conditions. There is no correlation between cell growth and EPS production for cultures being grown in different sources of nitrogen. In contrast, light intensity and cell growth in mixotrophic condition at 150 or 50 µmol photon m -2 s -1 have had a highly positive effect on EPS production. In salty grown cultures, thick layers of ASN_M strain prevent the cells from NaCl stress and hence its growth is maintained without inhibition under the NaCl stress (Nowruzi et al., 2013).
This study was undertaken to assess modi cation of metal removal capability of two Nostoc strains isolated from different habitats, for their ability to remove Cr(III), Cu(II) and Ni(II) from aqueous solutions. We thought that the strain of Nostoc sp. N27P72 that is isolated from lime stones of Khuzestan province have many features which include high tolerance to different abiotic stress such as drought and high density of light that make it ideal candidate for the selective removal and concentration of heavy metals, compared to aquatic strain.
The main objective of this work is the use of the mixotrophic conditions to optimize the biosorption controllable factors for the maximum metal removal e ciency of algal biomass. Lead, Nickel and copper were selected because of their contrasting toxicity and essentiality. Furthermore, cell dry weight, carbohydrates content, total soluble proteins, analysis of functional groups by fourier transformed infrared (FT-IR) spectroscopy and Chemical composition of the lyophilized EPS by coupled gas chromatography -mass spectrometry (GC-MS) have been investigated. To the best of our knowledge, modi cation of culture media by Nostoc species has not been reported for the removal of heavy metals and hence this study would be of great importance in increasing the capability of bioremediation of metals from aqueous environment.

Materials
All materials and reagents were purchased from Sigma-Aldrich unless otherwise speci ed.
Cyanobacterial strains Nostoc sp. N27P72 and Nostoc sp. FB71 obtained from the Cyanobacteria Culture Collection (CCC) and the ALBORZ Herbarium, at the Science and Research Branch, Islamic Azad University, Tehran.
Culture conditions Nostoc sp. N27P72 was belong to lime stones of Khuzestan province and Nostoc sp. FB71 was isolated from fresh water of Golestan province. Nostoc sp. N27P72 was grown at modi ed Z8IX medium and Nostoc sp. FB71 was cultured on liquid media BG11 medium (nitrate free) (Rippka et al. 1979) and pH was adjusted to 7.1. Cultures were incubated in a culture chamber at 28° C, and were provided with continuous arti cial illumination of approximately 15 µmol m -2 s -1 for two weeks (Liu et al., 2014). the cell cultures used for optimization of metal removal capability were grown in broth (25 ml in 250 ml ba ed shake asks) containing the sugars glucose, maltose, lactose, and sucrose, respectively, as additional carbon source at the concentrations 10 g/l and were grown for 48 h. Samples were taken after 6, 12, 24, and 48 h and were analysed for total produced EPSs, cell dry weight and sugar content. A parallel control experiment was carried out using broth media cultures of Z8IX and BG11 without carbon source supplementation.

Determination of cell biomass
Cyanobacteria growth was quantitatively determined by measuring cell dry weight. First cells washed and resuspended in 2 ml of 0.05 M EDTA sodium salt solution (Sardari et al., 2013) and the mixture was shaken at 4 • C for 4 h to remove any capsular EPS. Then cells were harvested and dried in an oven set at 100 • C. The cell dry weight was measured periodically until a constant weight was reached (Nowruzi et al., 2013).

Isolation of exopolysaccharides
Strains Nostoc sp. N27P72 and Nostoc sp. FB71 were taken at different times from the cultivations were centrifuged at 4000 rpm for 30 min at 4 • C (SigmaPK). The EPSs were precipitated by adding ethanol and storing overnight at 4 •C. precipitates were harvested and put in a fume hood to evaporate the remaining ethanol. The precipitates then were dissolved in milliQ water and lyophilized (Labconco freeze dry system) to obtain the crude EPSs (Nowruzi et al., 2013).

Selectivity in the heavy metal removal
The cell cultures were grown in Z8IX and BG11 media culture containing the maltose and without maltose as control culture, were tested for their ability to remove Cu(II), Cr(III) and Ni(II). The cultures (400 ml of cell suspensions in 1000 ml Erlenmeyer asks) were grown for 10-15 days in an orbital Incubator (Gallenkamp, Loughborough,UK) at 30 ± 1 0 C under continuous illumination provided by cool white uorescent tubes giving a mean photon ux of 100 µmol photon m -2 s -1 photosynthetic active radiation at the ask surface. Before their use for the experiments, aliquots of the cultures were con ned in dialysis tubing and pretreated with 0.1 N HCl and then dialysed against water. Next, the cultures were suspended into metal solutions with continuous stirring. Working solutions of 10 mg l -1 Cr(III), Cu(II) and Ni(II) were prepared, for each metal, by dilution of 1000 mg l -1 standard solutions (pH 5.0); the experiments were performed in a thermostat at a temperature of 25 ± 1 0 C. For the determination of the kinetics of metal removal, 5 ml samples were withdrawn at known intervals, centrifuged (10 min at 10 000 g) and ltered through a 0.45 lm membrane. The metal uptake was calculated from the difference between the concentrations of the metals in solution, determined with an Atomic adsorption spectrometer (SpectrAA 10 plus, Varian, CA, USA), at the beginning and end of exposure with the cyanobacterial cell suspensions; Cu(II), Ni(II) and Cr(III) concentrations were determined at 232.0, 359.9 and 324.7 nm respectively.
The amount of metals removed in blanks carried out in parallel without the addition of cyanobacterial cells was subtracted from the experimental values gained in the experiments with the cyanobacterial cultures. All the experiments were performed at least in triplicate and the data are reported as mean values ± standard deviation. The metal uptake q, expressed as mg of metal removed per g of dry biomass, was calculated as: q = (C i -C t ) / m. The concentration of the biomass was determined as dry weight (g l -1 ) by ltering the cell suspension on 0.45 lm lters and by drying the lters at 100 0 C until constant weight (De Philippis et al., 2003)  .
In order to con rm the affection of the metals and to determine the interaction of the cations within the different functional groups of the EPS, metal solution and all incubated EPS were lyophilized after the 24 h dialysis (lyophilized EPS containing maltose and metal solution of Cu(II), Cr(III) and Ni(II)) and total produced EPSs, total soluble proteins, carbohydrate content and chemical composition were measured.
Analyzing of carbohydrates content 5 mg of the sample was mixed with 2.5 cm 3 of Antrone reagent (0.65 %) in H 2 SO 4 (65 %). The mixture was incubated for 35 min at 100 °C. The absorbance was measured at 620 nm. Carbohydrates content was computed using a calibration curve (Zinicovscaia et al., 2016).
Estimation of total soluble proteins 20 mg of EPS were ressuspended in 1 ml of deionized water and sonicated and successively diluted until a homogenous solution was obtained. Protein content was determined according to the Lowry et al. (1951), and a calibration curve was constructed using serum albumin.
Analysis of functional groups by FT-IR 2 mg of lyophilized EPS was grinded with 100 mg dry KBr and pressed into a mold in a uniaxial hydraulic press. FT-IR spectra of the puri ed EPSs fractions were recorded in the 4000-400 cm −1 region using a FT-IR system (Nicolet is5, Ther-moFisher Scienti c). The determinations were performed in two independent replicates and are reported as the mean with standard deviations (Mota et al., 2016).

Chemical composition of the lyophilized EPS
Chemical composition of extracts of Nostoc sp. N27P72 was evaluated using a coupled gas chromatography -mass spectrometry (GC-MS). The separation of compounds and their analysis was performed using agilent 7000 series Quadrupole GC-MS system with electron impact ionization. The total GC run time was 32 min and the carrier gas was helium. The initial oven temperature was held at 90 0 C for 1 mina and then reached 300 0 C in 13 min, after which it was held at this temperature for 20 min. the injector temperature was 300 0 C.
Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns and Fiehn Mass Spectra Libraries. The spectrum of the unispeci ed component was equaled with the spectrum of the identi ed components stored in the NIST library. The Name, Molecular weight and Structure of the components of the test materials were ascertained (Sampathkumar and Halith., 2020).

Statistical analysis
Results of each representative experiment were analyzed by ANOVA, using the statistical software package SPSS version 24 and differences between the groups were detected with Tukey's grouping tests. P(s) values smaller than 0.05 are considered signi cant. Means and the standard deviation were calculated from the data obtained from three replicates (Nowruzi et al., 2013).

Growth and EPS production by the two Nostoc strains
The two Nostoc strains exhibited a distinct difference in growth behavior. As initial trials with the both strains showed that use of disaccharides (maltose, lactose, and sucrose) as carbon source supplementation generally resulted in higher EPS production than the use of monosaccharides (Glucose), moreover the amounts of total produced EPSs, cell dry weight and residual sugar content was higher in Nostoc sp. N27P72 in comparison to Nostoc sp. FB71 in all treatments. Consumption of the monosaccharide glucose, and the disaccharides lactose, lactose and maltose was veri ed (Fig. 1). In all situations, production of EPS was started in the exponential growth phase and was shown to reamin in the stationary phase. The consumption rate of glucose, maltose, lactose, and sucrose was nearly uniform in both strains and after 48h all glucose was not consumed and the residual concentration was 6-6.5 ± 0.1 g/l in all treatments. The change in cell concentration was very obvious in both strains in cultures adding maltose and reaching to 1.01 ± 0.06 g/l in Nostoc sp. N27P72 and 0.75 ± 0.16 g/l in Nostoc sp. FB71 at 48 h. The increase in cell dry weight might be due to consumption of produced EPS, which was reaching a nal EPS concentration of 9.1 ± 0.05 µg/ml in Nostoc sp. N27P72 and 7.04 ± 0.1 µg/ml in Nostoc sp. FB71 (at 48 h). The experiments using cultures without added sugar, showed a slightly lower maximum cell mass (0.55 ± 0.03 g/l in Nostoc sp. N27P72 and 0.323 ± 0.05 in Nostoc sp. FB71 after 48 h) and the nal EPS concentration was 3.3 µg/ml in Nostoc sp. N27P72 and 2.5 µg/ml in Nostoc sp. FB71 after 48 h. This indicates that the 10 g/L maltose addition had a small boosting effect on both cell mass and EPS production.
Addition of glucose did not stimulate cell growth and EPS production in Nostoc sp. N27P72, however it was stimulating in EPS production in Nostoc sp. FB71. The consumption rate of glucose during the rst 6 h was 1.5 g/l in Nostoc sp. N27P72 and 2 g/l in Nostoc sp. FB71 but decreased to 0. 5 g/l h (6-15 h) and reaching a cell mass of 0.59 ± 0.21 g/l in Nostoc sp. N27P72 and 0.25 g/l in Nostoc sp. FB71 at 24 h. After 48 h the cell mass nally reached 0.79 ± 0.35 g/l in Nostoc sp. N27P72 and 0.38 g/l in Nostoc sp. FB71, while production of EPS continued (1.7 µg/ml in Nostoc sp. N27P72 and 3.9 µg/ml during the whole 48 h of cultivation. The cell mass obtained in maltose supplemented cultivations resembled that of the lactose supplementation (0.4 ± 0.28 g/l in Nostoc sp. N27P72 and 0.5± 0.1 g/l in Nostoc sp. FB71 after 24 h, maintained at 24 h as 0.94 ± 0.14 g/l in Nostoc sp. N27P72 and 0.66 ± 0.09 g/l in Nostoc sp. FB71. The EPS concentration in this case reached 6.41 ± 0.13 µg/ml in Nostoc sp. N27P72 and 5.10 ± 0.26 µg/ml in Nostoc sp. FB71 (after 48 h). Sucrose supplemented cultivations showed that the maximum cell concentration was 1.15 ± 0.21 g/l and the concentration of produced EPS was 2.01 ± 0.15 µg/ml in Nostoc sp. N27P72 and 4.32 ± 0.19 µg/ml in Nostoc sp. FB71 after 48 h. The results displayed that while the effects on cell mass were slightly small, production of EPS ampli ed upon adding of the disaccharides maltose and lactose at stationary and exponential phase.

Results of optimization of metal removal capability
The time course of speci c metal removal (q), expressed as mg of metal removed per g of biomass dry weight, by Nostoc sp. N27P72 and Nostoc sp. FB71 cultivated in media culture containing (10 g/l) maltose, with copper, chromium and nickel in single-metal solutions showed that In single-metal solutions, the kinetics of sorption by the cyanobacterial cultures was always rapid for all the metals tested; the saturation of the metal removal capacity of each strain was achieved within the rst 10 min in the metal solution. The metal a nity of the two Nostoc strains generally decreased in the order Ni > Cu > Cr. The speci c metal uptake (q) was very high, in particular for Ni (Fig. 2), which generally were removed in larger amounts than Cu and Cr.
Among the strains tested, the highest q values towards Ni was 188.8 ± 0.14 mg Ni (g cell dry wt) -1 shown by Nostoc sp. N27P72, while the highest values of uptake was 185.5 ± 0.24 by Nostoc sp. FB71. In single ion solutions, both Nostoc strains tested showed the lowest a nity for Cr namely 105.65 ± 0.34 104.5 ± 0.1 mg metal for Nostoc sp. N27P72 and Nostoc sp. FB71 (g cell dry wt) -1 respectively.

Results of optimization of metal removal capability
Results of optimization of metal removal capability showed that adding maltose in culture media in both strains as carbon source supplementation generally resulted in higher EPS production, protein and neutral sugars content in compression of control. Moreover the amounts of total produced EPSs, protein and neutral sugars content was signi cantly higher in Nostoc sp. N27P72 in comparison to Nostoc sp. FB71. Exopolysaccharide concentration in medium containing maltose and metal solution of Ni(II)) was 4.87 and 3.10 µg/L, while it was 2.72 and 1.8 µg/L in control for Nostoc sp. N27P72 and Nostoc sp. FB71 respectively. Protein concentration in medium containing maltose and metal solution of Ni(II)) was 4.87 and 3.10 mg/ml, while it was 0.07 and 0.06 mg/ml in control for Nostoc sp. N27P72 and Nostoc sp. FB71 respectively. Neutral sugars content in medium containing maltose and metal solution of Ni(II)) was 5.64 and 5.16 µg/mL, while it was 2.85 and 1.74 µg/mL in control for Nostoc sp. N27P72 and Nostoc sp. FB71 respectively. Nickel removal rate was signi cantly higher in both strains, it means this metal is more absorbed by polysaccharide envelopes. The reason for removing more nickel according to the results of the diagrams is the higher amount of EPSs, proteins and neutral sugars content compared to other elements. In fact, More EPS, proteins and neutral sugars content can effectively sequester dissolved metal ions from dilute aqueous solutions (Fig.3).

Results of analysis of functional groups
Fourier Transformed Infrared (FT-IR) spectroscopy provided useful information about active functional groups that can be used in the determination of polysaccharide composition. So, the FT-IR spectra for both Nostoc sp. N27P72 and Nostoc sp. FB71 cultivated in media culture containing (10 g/l) maltose, with copper, chromium, and nickel in single-metal solutions were examined. Strong stretching vibrations for OH and weak stretching vibrations for NH 4 + at 3800 cm −1 can be detected only in Nostoc sp. Peaks at 600 cm -1 are related to The C-N stretching band (600 cm -1 ) can see in both sample, but shifted to lower wavenumbers in both strains. Collectively, after treatment of both strains with Ni, Cu, and Cr the FT-IR patterns for both strains changed obviously (Fig 4 and 5).
The total ion chromatograms (TICs) of all samples demonstrated a strong signal, large peak capacity, and reproducible retention time, indicating the reliability of metabolomic analysis. However Cyclotrisiloxane retention time was not in the same range for the Ni(II) and Cu(II) media culture (11.63 to 23.22), while 1,2-Benzenedicarboxylic acid retention time for Ni(II) and Cu(II) media culture was in the range of 23.26 to 25.14.

Discussion
The possible use of exopolysaccharide-producing cyanobacteria for the recovery of valuable metals from industrial wash waters seems to be more promising than most of the other microorganisms ( (Pereira et al., 2009). Though, several elements that can stimulus EPS production, especilly pH, dilution rate, growth phase, presence/absence of magnesium, calcium, potassium and heavy metals, as well as the addition of glycoxylate, glucose, citrate, acetate, valerate and EDTA have been sporadically studied (Pereira et al., 2009). Moreover, the responses of cyanobacteria to changes in culture conditions appear to be frequently strain-dependent, making the optimization of EPS production even more di cult (Floutya et al., 2019). It was suggested that some additives, including amino acids, vitamins and precursors, may also play an important role in EPS production during the process of cyanobacteria (Pereira et al., 2011).
In this article, we investigated the effect of adding four different sugars in the culture medium (glucose, maltose, lactose, and sucrose) as carbohydrate source and its effect on the amount of EPS and cell dry weight in two strains of Nostoc was investigated. The results of the present investigation indicated that the optimal medium for EPS production by the isolated Nostoc sp. N27P72 strain was boosted by maltose supplementation. Previous studies on several bacterial EPS production reported that amino acids and precursor's supplements showed stimulating effect on bacterial growth and EPS synthesis, while others demonstrated that neither additional carbohydrates nor amino acids supplementation affect the EPS level (Maalej et al. 2015). Moreover, among different nature of carbon sources, carbohydrate sugars are the preferred ones for EPS production. In this study, maltose were found to be the most e cient carbon sources.
Unfortunately, large amounts of toxic elements, especially heavy metals which are harmful to human beings, are daily released in nature through human and industrial activities (Tchounwou et al. 2012). Exopolysaccharides are well suggested as surface-active mixtures for the exclusionof heavy metal pollution.
Consequently, to nd out microbial EPS with decent metal adsorption selectivity may be very valuable for building up processes aiming to pick up valuable metals from industrial wastewater. Bearing this in mind, we studied supplemented media culture as an additional sugar source exopolysaccharide to evaluation of its e ciency to adsorb various toxic heavy metals (Cu, Ni and Cr) (Mohite et al., 2017). We found that using maltose as carbon sources was shown to produce a higher amount of EPS, protein and neutral sugars content and it could be a reason for high ability of metal absorbance. This statements has been underlined by Wong and Tam (1984) who stated that algal cells cultivated in the media with very high metal contents also gathered higher metal contents. In spite of this, in few cases, the metal uptake was independent on the external metal concentration, this point was coincided with the conclusion of Wetton et al. (1976). In case of N. muscorum grown in wastewater, high concentrations of Cu and Mn did not affect the growth of the microorganism but promote its growth. This may be because of the resistance of the cyanobacterium to Cu and Mn in addiotion to the occurrence of high content of organic matter, which may detoxify Cu and Mn effects (El-Enany and Issa, 2000).
In fact, the quantity and compactness of different kind of carbohydrates can help to sort the polysaccharidic layer surrounding the cells which can also prevent direct contact between the cells and toxic heavy metals. Actually, it was recently suggested that the high viscosity of the cultures, due to the solubilization in the culture medium of large amounts of a high molecular mass RPS, delayed the free diffusion of copper ions into the media culture . the presence of negatively charged polysaccharidic layers surrounding cyanobacterial cells such as uronic acids, sulphate and ketal-linked pyruvate groups may play an important role in the sequestration of metal cations, and in forrming an environment improved in those metals that are crucial for cell growth but are existing at very low concentrations in some environments (Pereira et al., 2009).
We found that strain of Nostoc sp. N27P72 that is isolated from lime stones of Khuzestan province have many features which include high amount of EPS and high tolerance to drought and high density of light that make it ideal candidate for the selective removal and concentration of heavy metals, compared to aquatic strain. Actully, in all of the possible systems in which cyanobacteria are involved, the synthesis of EPSs provides a structurally-stable and hydrated microenvironment, as well as a putative physical defensive role against some risky factors, both chemical and physical, representing a boundary between cells and the immediate outer environment, and preserve the cells from toxic heavy metal (Rossi and Philippis., 2015).
The experiments, showed that Nostoc sp. N27P72 biomass is characterized by the best e ciency in metal removal, with a qmax (maximum amount of nickel removed per biomass unit) of 188.8 ± 0.14 mg Ni (g cell dry wt) -1 nickel removed in comparison with the value of 185.5 ± 0.24 mg Ni (g cell dry wt) -1 of Nostoc sp. FB71 biomass. Thus, the metal uptake capacity of EPS under study (especially for nickel) is of great interest regarding other biosorbent e ciencies and the lowest a nity for Cr. Our results contradicted with the result of Chan et al. (1991) who found that the removal of Ni from the mixture of 90% electroplating e uent and 10% raw sewage by two species of Chlorella was comparatively low (below 20%). While Kazy et al. (2002) reported that EPS production by a copper-resistant isolate of Pseudomonas aeruginosa was considerably higher than its copper sensitive counterpart. Moreover, Ozturk and Aslim., 2008 found Cr(VI) is an important stress factor that increases EPS concentration in cyanobacteria.
Interestingly, using optimized culture conditions increases the yield of the Nostoc sp. N27P72 EPS, thus improving the chances of its commercial scale production and making it more suitable for special applications, such as the environmental industry. However nding a general pattern for the special effects of metals on EPS synthesis is very hard. It is thought that the effects on EPS synthesis are metal-speci c. In some cases, a shortage of Mg 2+ and Ca 2+ elicited production, whereas there were no effects in other cases.
The increase in EPS synthesis appears to develop the resistance to toxic metals. A study carried out by Ozturk and Aslim [9] showed that Chroococcus and Synechocystis strains resistant to Cr(VI) created larger EPS amounts compared to the Cr(VI)-sensible isolates. It is promissing that a greater EPS concentration inspired by the metal played a role in increasing its immobilization, as suggested by Pereira et al. 2013. Though, it was lately reported that in Cyanothece sp. CCY 0110, the existence of heavy metals expressively affected its protein pro le but did not improve the amount of RPS released by the cells (Mota et al. 2016).
Due to the presence of negatively charged groups ,primarily carboxyl group they have also been shown to have good sorbent capacity towards positively charged metal ions (Mathlouthi and Koenig, 1987). mechanism against other microorganisms might be able to change the permeability of the cell membrane Through interacting with proteins and lipids of the membrane, inhibiting special enzymes or by a layer around the cells. Presence compounds of Benzene derivatives in Ni and Cu cultures may be related to absorption compounds to remove the metal, actually it has been previously been shown that some benzene inhibited bketoacyl-acyl carrier protein synthase III, a condensing enzyme that initiates fatty acid biosynthesis in most cyanobacteria, leading to absorption activity of metal. Furthermore, the cytotoxic activity of the pure compound 1, 2-benzene dicarboxylic acid, mono 2-ethylhexyl ester (DMEHE) from marine derived actinomycete Streptomyces sp. VITSJK8 was examined against mouse embryonic broblast (NIH 3T3) and human keratinocyte (HaCaT) normal cell lines, human hepatocellular liver carcinoma (HepG 2) and human breast adenocarcinoma (MCF-7) cell lines by using MTT assay (Krishnan et al., 2014). 1,2-Benzenedicarboxylic acid, bis(2-methyl propyl) ester was recorded by Alghamdi et l., 2018 as plasticizer and has light and heat stability. The antibacterial properties of olive leaves extract is suspected to be associated with the high cyclotrisiloxane hexamethyl content, which has been tested by Keskin e al., 2012. lastly, as compared to solid wastes created from old approach used for the heavy metal removal, polysaccharides are natural, nontoxic and biodegradable polymers, thus reducing their polluting effects and making them attractive for potential use as metal-absorbent safe materials. In conclusion, the strain Nostoc sp. N27P72 may be a suitable candidate for mass production of an ecologically attractive EPS with a potential for use in the bioremediation eld.

Conclusion
From the above described investigational results, it seems obvious that the EPS plays an crucial role in protecting from harmful toxic heavy metal. However, in spite of the large number of studies claiming this role, only a few of them directly investigated the modi cation of metal removal capability by cyanobacteria under mixotrophic condition. In the present study, the highest EPS production e ciency was found in cultures supplemented by maltose and biomass of Nostoc sp. N27P72 possesses a high a nity and a high speci c uptake for nickel, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use Nostoc sp. N27P72 for the bioremoval of heavy metals from polluted water bodies. The FT-IR spectra showed that treatment of Ni with both strains made obvious changes in functional groups of polysaccharids and linkages. Cyclotrisiloxane and 1,2-Benzenedicarboxylic acid, major constituent of Nostoc sp. N27P72 in this study, metal removal capability of Nostoc sp. N27P72 extract is suspected to be associated with the high Cyclotrisiloxane and 1,2-Benzenedicarboxylic acid content. Lastly, although the key elements controlling the production of the cyanobacterial EPS have been recognized, invlusive strain-speci c studies taking into account the interaction between the variables to know the system reaction to changes, are still missing. This needs a better facts of the genes and metabolic paths complicated in the production of EPS in cyanobacteria.    Table 4: Chemical composition of Nostoc sp. N27P72 (Cu(II)) extracts as revealed by gas chromatography mass spectrophotometry (GC-MS).

Figure 1
Growth pro le and EPS production of Nostoc sp. N27P72 and Nostoc sp. FB71 cultivated in media culture containing (10 g/l) glucose, sucrose, lactose and maltose, separately and media culture without additional sugars as a control. Symbols indicate (•) for cell dry weight, (□) for total EPS concentration, and (∆) for sugar concentration in the media. Results are the mean of duplicate measurement.

Figure 2
Time course of speci c metal removal (q), expressed as mg of metal removed per g of biomass dry weight, by Nostoc sp. N27P72 and Nostoc sp. FB71 cultivated in media culture containing (10 g/l) maltose, with copper, chromium and nickel in single-metal solutions. Symbols represent the mean of at least three replicates and bars represent the standard deviation, if larger than the dimensions of the symbols.

Figure 3
Comparison of total produced EPSs (a), Neutral sugar content (b) and total soluble proteins (c) of Nostoc sp.
N27P72 and Nostoc sp. FB71 in lyophilized EPS containing maltose and metal solution of Cu(II), Cr(III) and Ni(II)). All the values are mean of at least three replicates ± standard deviation.

Figure 4
Fourier transform infrared (FTIR) spectra of EPS from Nostoc sp. FB71 against control or exposure to a solution containing 400 ml of cell suspensions in 1000 ml Erlenmeyer asks of Cu(II), Cr(III) and Ni(II).

Figure 5
Fourier transform infrared (FTIR) spectra of EPS from Nostoc sp. N27P72 against control or exposure to a solution containing 400 ml of cell suspensions in 1000 ml Erlenmeyer asks of Cu(II), Cr(III) and Ni(II).

Figure 6
GC-MS chromatogram of the extract of Nostoc sp. N27P72, in media culture containing the maltose and without maltose as control culture, were tested for their ability to remove Cu(II), Cr(III) and Ni(II).