Phytoremediation of Contaminated Water by Cadmium (Cd) Using Two Cyanobacteria Species (Anabaena Variabilis and Nostoc Muscorum)


 Background: Water pollution with heavy metals is a severe dilemma that worries the whole world related to its risk to nature ecosystem and human being health. The main objective is to evaluate the removal efficiency of Cd with various concentrations from contaminated aqueous solution by two Cyanobacteria species (Nostoc muscorum and Anabaena variabilis). For this purpose, a designed laboratory pilot scale was applied using two cyanobacteria species (Nostoc muscorum and Anabaena variabilis), four different initial concentrations (0, 0.5, 1.0 and 2.0 mg L−1) for 21 days. Results: N. muscorum was more efficient than A. variabilis for removing Cd (II), because the optimum value of residual Cd achieved by N. muscorum after 21 days at (0.5 mg L-1) was (0.033 mg L-1), where the removal efficiency was 93.4%, while the residual Cd (II) by A. variabilis under the same conditions was (0.054 mg L-1), and the achieved removal efficiency was 89.13%. Algal growth parameters and Photosynthetic pigments were estimated for both Cyanobacteria species through the incubation period. Conclusions: High Cd concentration had a more toxic impact on algal growth. The outcomes of this study will help to produce treated water that could be reused in agrarian activities.


Introduction
One of the global challenges is the pollution of water bodies by heavy metals. Any metal and metalloid element having density within the range of 3.5 to 7 g cm − 3 is considered poisonous even if present in low concentrations (Gautam et al., 2014). An optimum low level of concentration of heavy metals such as iron (Fe), copper (Cu) and zinc (Zn) have biological usefulness while others, including lead (Pb) and cadmium (Cd), are not useful biologically and are toxic irrespective of the level of contamination (Halttunen, 2008). such as wheat, rice and potatoes, has negative effects on human bones. In Japan, water and soil contaminated by Cd is the major cause of the Itai-itai disease (Khan et al., 2020). Cadmium causes bone disease, kidney damage and cancer. It is reported that high levels of Cd exposure causes osteoporosis, renal dysfunction and liver damage (Ghoneim et al., 2014).
Alkaline batteries manufacturing consumes about three-quarters of Cd production. The remaining one quarter of Cd production is used by processes including coating materials and as a plastic stabilizer (Jaishankar et al., 2014). Cadmium is extremely poisonous, causes plant nutrient de ciency and oxidative stress, and also impacts on the enzymatic systems of cells (Jaishankar et al., 2014;Irfan et al., 2013). Therefore, WHO (2008) recommends that the concentration of Cd in potable water should be limited to 3 µg L − 1 (Zinicovscaia, 2016). For the short-and longterm irrigation water use, the preferred threshold of Cd concentration should be 0.05 and 0.01 mg L − 1 , respectively (Fipps, 2015;Rowe and Abdel-Magid, 1995).
Human health issues related to the pollution of water bodies and soil by heavy metals resulting from pesticides, fertilizers, sewage water and industrial activities have received global attention. There is lack of precautionary measures put in place to inspect industrial facilities that discharge contaminated wastewater into agricultural drains that supply irrigation water for production of crops in many countries, be it developing or developed. Thus, people who handle the contaminated irrigation water and the resulting agricultural products put their health at risk (Tchounwou et al., 2012;Dadar et al., 2016;Avigliano et al., 2015). Many different methods of wastewater treatment (physical, chemical treatment, biological and phytoremediation) have been applied to reduce cadmium concentration in water to the recommended international standards (Chen et al., 2020). Some of the wastewater treatments applied include physicochemical processes (ion exchange and chemical precipitation), electrochemical treatments (electrocoagulation, electrodeposition and elector-oatation), adsorption (carbon nanotubes, activated carbon and wood sawdust adsorbents), and the current methods are photocatalysis, membrane ltration, and nanotechnology (Azimi et al., 2017).
However, these techniques have many disadvantages. Phytoremediation is any process that use algae to bioremediate contaminated water and wastewater (Phang et al., 2015). The characteristics of algae involve a high ratio of surface area to volume, high heavy metals tolerance, growth possibility either autotrophically or heterotrophically, the ability for genetic manipulation, phytochelatin expression and phototaxy (Kumar and Gunasundari, 2018). Biosorption using blue-green algae (cyanobacteria) is rich in vitamins and proteins. The biomass can absorb and adsorb heavy metals from aquatic solution even when the cells are dead. Unlike conventional methods, cyanobacteria processes do not produce polluting sludges, are highly effective, easy to operate and cost effective for treating large quantities of wastewater with low contaminant

Algae and culturing conditions
Two cyanobacteria species (Nostoc muscorum and Anabaena variabilis) were cultured in a BG110 liquid medium consisting of a mixture of MgSO 4 , K 2 HPO 4 , CaCl 2 , Na 2 EDTA, Na 2 CO 3 , citric acid, and ferric ammonium citrate as presented by Ripkka et al. (1979). Erlenmeyer asks were used with daily alternation of an average of and 8 hours of darkness and 16 hours of light. The temperature was controlled during the experiment at 27 ± 2°C, while the cool white light intensity ranged from 3000-3500 lx and the pH was set to 7.2. The algae cells were harvested on the 15th day, which corresponds to the middle of the logarithmic phase, and centrifuged at 3000 rpm for 10 minutes

Preparation of metal/toxin stock solutions
The cadmium-contaminated aqueous solution was prepared by adding 0.684 g of cadmium sulfate (CdSO 4 .8H 2 O) to 100 cm 3 of distilled water and stirred well to ensure that the cadmium sulfate was completely dissolved. The prepared solution was diluted using the medium to obtain the desired concentrations of 0, 0.5, 1 and 2 mg L − 1 used in the experiments. Three replicates for each concentration treatment were set up.

Experimental Setup
The experiments were carried out using plastic containers having dimensions of 26.9 cm in length, 18.75 cm in width and 12.5 cm in depth. Each container was lled with a mixture of 2 L of the prepared aqueous solution (BG110 medium) of different Cd concentrations and 110 ml of the algae medium (OD 678 ). The samples were taken from plastic containers at a rate of 5 ml every four days to measure the optical density (OD), while samples were taken in volumes of 50 ml every 7 days (D 0 , D 7 , D 14 , D 21 ) for the determination of the photosynthetic pigments, and a sample of 50 ml was taken at end of the incubation period of 21 days to measure biomass content.

Heavy metal removal e ciency
Five ml samples were taken from the contaminated media every four days to estimate the concentration of residual Cd caused by algae absorption using atomic absorption spectrometer (Perkin Elmer Analyst 400). The removal e ciency (RE) was calculated as: In Eq. (1) C 1 and C o are, respectively, the residual and initial concentrations of Cd in mg L − 1 .

Photosynthetic pigments analysis
The samples were subjected to 10 minutes centrifugation at 4000 rpm, after which the algae medium was carefully added before the distilled water was carefully poured with the algal cell suspension into a 4 ml DMSO solution. The mixture was stirred at 1000 rpm for one minute to reach homogeneity, after which it was heated for 10 minutes in a water bath at 65 o C. Six ml of 95% acetone concentration was added to the algal cells extracted from the DMSO solution and mixed thoroughly. The photosynthesis pigments concentrations of chlorophyll a and carotenoids were estimated in µg ml − 1 according to the Ritchie (2008) and Davies (1976) methods, respectively.

Statistical Analysis
The statistical analysis involved the use of a random complete block design (RCBD) with Factorial data analysis, the three factors considered being concentration (C), algae (A), and number of days (D). Three replications were implemented in order to minimize parameter errors. The least signi cant differences (LSD) and correlation coe cient (CC) test were applied (Snedecor and Cochran, 1976). MSTAT software (Mstat-c., 1989) was for the statistical analysis.

Removal of heavy metal
There was a signi cant variation of residual Cd values among the different initial Cd concentrations considered. As shown in Fig. 1, the residual Cd tends to stabilize after day 12 for all initial concentrations. N. muscorum achieved a terminal residual Cd value of 0.033, 0.175 and 0.51 ppm for the initial concentration of 0.5, 1 and 2 ppm, respectively, translating to heavy metal removal e ciency of 93.4, 82.5 and 74.5%, respectively. Terminal residual Cd values achieved by A. variabilis were slightly higher at 0.054, 0.26 and 0.632 ppm, for the initial concentration of 0.5, 1 and 2 ppm, respectively, re ecting removal e ciency values of 89.13, 74.00 and 68.38%, respectively (Fig. 1).
Cadmium was released again into the contaminated water as a result of the algae's sorption decline related to the toxic effect of Cd, and so the residual Cd marginally increased after 16 and 12 days for the initial concentrations of 1 and 2 ppm, respectively. Additionally, Inthorn et al. (1996) reported that more than 90% of Cd removal e ciency was achieved within 10 min at 1 ppm initial concentration by Tolypothrix tenuis, after which the Cd concentration remained steady. Inthorn et al. (2003) reported Cd removal e ciency of 84% and 92%, respectively, in non-treated and NaOH-treated cells of Nostoc paludosum, and in Phormidium angustissimum as 86% and 94%, respectively. The results con rmed that 30 min contact time for signi cant Cd removal and to reach the equilibrium state. Further experiments indicated 10 mins is enough for signi cant removal capacity and to attain the equilibrium state.

Alga biomass
The highest biomass value for N. muscorum was 533.3 mg L − 1 at the end of the 21 days of experiment for the control treatment (0.0 concentration), while the lowest value of Cd was 200 mg L − 1 after 21 days for the initial concentration of 2 ppm. (Fig. 2). The decline phase approximately reached under Cd concentrations of 1 and 2 ppm after 16 days of incubation period. A biomass of 300 mg L − 1 was recorded for A. variabilis in the control treatment while the lowest biomass was 50 mg L − 1 for the initial 2 ppm Cd concentration at the end of the experiment.
The highest initial concentration of Cd reduced growth of biomass and led to cyanobacteria death (Hazarika et al., 2015). Only 5 to 6 days of incubation period was required for cadmium to delay the algae growth. Cd replaced the Mg in the chlorophyll molecule of the algae and affected photosynthesis, leading to a reduced growth of the cells, particularly the more sensitive N. muscorum cells (Kupper et al. 1996(Kupper et al. , 1998

Optical Density (OD)
The growth rate of algae cells was affected slightly at 0.5 ppm concentration of Cd, whereas concentration levels of 1 and 2 ppm completely inhibited growth at the middle of the experiment for both cyanobacteria species (Fig. 3). Rangsayatorn et al. (2002) speci ed that optical density of Spirulina platensis was affected by higher concentrations of Cd that caused the death of cyanobacterial cells, while insigni cant growth suppression was observed at lower concentrations.

Pigments
The reduction in chlorophyll a was minimal at 0.5 ppm initial Cd concentration during the incubation period (Fig. 4), but signi cant reductions were detected at concentrations of 1 and 2 ppm in the cases of the two cyanobacteria species. Likewise, carotenoids displayed a similar trend, even though N. muscorum showed nearly a constant value for the 0.5 ppm initial concentration (Fig. 5).
The results obtained for pigments were in agreement with Atri and Rai (2003) who stated that higher dosages of Cd reduced the Chlorophyll a and carotenoids of Anabaena, Microcystis, and Nostoc. Similarly, Goswami et al. (2015) showed that higher concentrations of cadmium decreased the pigments of Anabaena doliolum. Mota et al. (2015) reported a decline in chlorophyll a content for the cultures treated with Cd for Cyanothece species CCY 0110 at 24 h exposure, and the decline gradually continued thereafter. Lamaia et al. (2005) observed that high concentrations of Cd could eliminate chloroplasts in Cladophora fracta (Lamaia et al., 2005). In a similar study, Arunakumara and Zhang (2009) showed that pigments content (Chlorophyll a and carotenoids) decreased with increasing Cd concentration, and the damage to the photosynthetic pigments is related to Cd toxicity (Leborans and Novillo, 1996).

Statistical Analysis
The results of A. variabilis and N. muscorum were signi cantly different at the 0.05 level for the individual treatments (Table 1). However, the 3 factors (concentrations, days and algae) did not show any signi cant difference with respect to biomass at the 0.05 signi cance level. It is observed that N. muscorum has a higher removal e ciency of Cd from pigments and contaminated water, and thus preferred. Nevertheless A. variabilis showed superior quality in OD values.

Conclusion
The study has presented the removal e ciency of Cd by two cyanobacteria species (Anabaena variabilis and Nostoc muscorum) from contaminated water. At the end of the 21 days study period, N. muscorum achieved a maximum removal e ciency of Cd of 93.4% whereas A. variabilis recorded 89.13% for the initial metal concentration of 0.5 mg L − 1 . It is observed that N. muscorum is more e cient for Cd removal compared with A. variabilis. Higher concentrations of cadmium had a more toxic effect on the growth of algae. Our study con rms the potential of cyanobacteria for phytoremediation. The removal of Cd from the aqueous solution was attributed to biosorption of cyanobacteria.

Declarations
Ethics approval and consent to participate: All authors are approval in participation in this research.
Consent for publication: All authors are consent of publication of the research paper.
Availability of data and materials: The researchers provided the experiment requirements of raw materials, algae, and system design through their own nancial resources, while the data for previous studies and research was done through the Cairo University platform, which provides research on a regular basis.
Con icts of interest: The authors declare no con icts of interest.  Effect of Cd concentrations on Biomass.

Figure 3
Effect of Cd concentration on optical density.

Figure 4
Page 16/16 Effect of Cd concentrations on Chlorophyll a.

Figure 5
Effect of Cd concentrations on Carotenoids.