Optical density (O.D.) is an important parameter of growth of Spirulina platensis which is used indicator of risk assessment of different contaminants especially heavy metals. Results obtained of optical density after 8 days of culturing represented in figure (1) revealed that, the effective concentration (EC50) of Nickel, Copper, and Zinc was about 2.0 mg/l, according to the results obtained (theses results obtained after doing different experiments to determined EC50 of the three heavy metals). Consequently, in this work, five concentrations were chosen (two concentrations below and two higher than 2.0 mg/l) i.e. l.0, 1.5, 2.0, 2.5 and 3.0 mg/l for each element besides control. The results showed that copper had a stronger inhibitory effect on growth evaluated in optical density than nickel and zinc at all concentrations tested. The lowest concentration (1.0 mg/L) accelerated the growth but increasing concentration of copper inhibited algal growth. Our results go in agreement with those obtained by (Budi et al., 2020) who indicated that, with the treatment of various levels of Cu, the extreme growth rate of Spirulina plantesis displays that the treatment by adding 1 mg/L of heavy metal is needed for increasing growth but the higher the concentration of Cu given, the lower the density of Spirulina plantesis.
We discovered that Zn2+ ions were more toxic than Ni2+ ions, that lower quantities of zinc and nickel accelerated development, and that they are considered growth accelerators of Spirulina platensis when utilized in the lowest concentrations. These findings are in agreement with authors (Meenakshi et al., 2007) they observed that by utilizing 2 mg/l for both heavy metals, they discovered a considerable reduction in growth in Spirulina platensis culture due to copper toxicity, which was higher than zinc. Heavy metal toxicity can be observed by changes in growth circumstances and reduction in the growth of the test microorganism.
Akbarnezhad et al.,(2019) found that there was a gradual increasing on optical density of Spirulina platensis culture at low concentrations of zinc and cooper. On the contrary, another important fact obtained by Zinicovscaia et al., (2021), who observed that Spirulina platensis has a higher bioaccumulation capacity for Zn than Ni and Cu ions, and they believe it is an attractive option for environmental bioremediation because of its strong capacity in biosorption and bioaccumulation for heavy metal ions.
Infrared spectroscopy has distinct advantages over other conventional methods of biochemical analysis in that it is rapid, reliable and requires a relatively small sample size and simple sample preparation procedure (Kansiz et al. 1999). Infrared spectra of the total cell constituents showed the band assignments, which are based on the studies on whole cells, organelles and macromolecules of S. platensis at the region 4000-250 Cm-1. The data obtained revealed that there are a number of spectra regions that can account for the chemical differences in this species.
The obtained infrared peaks of the major cell constituents of S. platensis cultured for 8 days under the stress effect of different concentrations of Ni2+, Cu2+ and Zn2+ions metals were recorded in figures 2,3 and 4. It is clear – when compared to control – that some peaks disappeared, others appeared new and still others remained unchanged. The new peaks that appeared under the stress of these three elements may be either from the changes of the position of some side chains or from the degradation of some compounds having high molecular weight to others with low molecular weights. These concepts are in line with those obtained by Al-Osaimi (2010).
At 1.0 mg/l Ni2+ and Zn2+ the obtained number of peaks was higher than control (18 and 16 peaks, respectively) while in case of Cu2+ under the same concentration, the number of peaks decreased to 14 peaks as in case of control. However, at the concentrations more than 1.0 mg/l of Ni2+ (1.5, 2.0, 2.5 and 3.0 mg/l), the number of peaks decreased but still higher than control. Under this concentration (1.0 mg/l) in case of Ni2+, 9 new peaks appeared at frequencies; 4000-3500, 2500-2000 and 1500-1000 cm-1; and five peaks disappeared. The disappeared peaks were at frequencies 4500-4000 and 3500-3000 cm-1 which contained the asymmetric and symmetric C-H of methylene groups. At concentrations 1.0 and 1.5 mg/l Cu, the number of peaks are the same as at control (14 peaks); while at concentrations 2.0, 2.5 and 3.0 mg/l, the number of peaks decreased to 13, 12 and 7 peaks, respectively. Also, the total disappeared peaks were 5, 5, 6, 8 and 7 peaks at 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l respectively. The new peaks that appeared at concentrations from 1.0 to 2.0 mg/l are five peaks while at concentration 2.5 mg/l they reached six peaks. At concentration 3.0 mg/l Cu only one peak appeared. Although zinc shows evidence of some toxicity but appears to be only about one-fifth as toxic as copper (Miller, 1946).
It is clear therefore that the tested algal species S. platensis cultured at different concentrations of Ni, Cu and Zn that there are new compounds, and hence new peaks in the spectra appeared while other compounds disappeared when compared to untreated cells. These peaks may be resulted from the disappearance of some compounds, the weak rate of their synthesis, and the changes in the position of some side chains of the same compound and/or to the dissociation of complex compounds to simple one. Our findings are consistent with those obtained by El-Agawany and Kaamoush,(2022) who found that, the obtained infrared peaks of the major cell constituents of Dunaliella tertiolecta showed the appearance of new peaks and the disappearance of others which indicates changes in cell constituents due to the presence of different concentrations of zinc element.
In all of the tested elements the new peaks appeared at frequencies from 2500 – 1000 cm-1 which represents the amides associated with proteins together with the function groups of others from lipids and fatty acids (Williams and Feleming 1996). The higher number of peaks that appeared in case of control and the amended cultures with Ni2+, Zn2+ and Cu2+ under all the tested concentrations were at frequencies 1500 - 1000 cm-1. These frequencies represent the amides associated with protein and the phosphodiester back bone of nucleic acids (Noctor and Foyer, 1998). Finally, it could be concluded that Cu2+ metal ion is more toxic than Ni2+ and Zn2+ metal ions and the degree of stress depends mainly on the concentration and type of the element together with the length of the culture period. This could be proved from the results obtained by (El-Sheikh et al., 1999) which revealed that toxic effects of heavy metal depend on the type of the element and its concentration.
Environmental conditions can affect both the relative proportions of fatty acids as well as the total amounts of lipids. Also, Lipid class and fatty acids composition of microalgal cells at different growth phases can differ significantly (El-Maghrabi 2002). Fatty acids have an important role in prevention and modulation of certain diseases like coronary heart disease, (William, 2000). The fatty acids composition of dietary microalgae is linked to the growth and survival of aquaculture. It is well known fact that lipid production usually differed between genera, species and strains of microalgae. However, total lipid fractions in healthy phytoplankton vary substantially from less than 1% to more than 40% of dry weight (Dubinsky et al., 1978). Spirulina is a plentiful source of highly valuable phytocompounds with unique functional characteristics including phenolic acids, carotenoids, and both omega-3 and omega-6 polyunsaturated fatty acids (Pyne et al., 2017).
Concerning total fatty acids content of the tested alga S. platensis after 10 days of incubation in relation to the five concentrations of the chosen elements, it is quite evident that all the three groups of fatty acids are greatly affected especially at high concentrations of the elements. However, the toxic effect of these elements differed according to type of fatty acids group. So, by increasing the concentration of the element, the total content of the three groups of fatty acids decreased. This decrease was found to be very highly significant. The toxic effect of Ni2+, Zn2+ and Cu2+ was found to be concentration dependent i.e. the toxic effect increased with increasing the element concentration. El-Sheikh et al. (1999) recorded that toxicity of the element was a concentration dependent. Spirulina platensis suffered greatly from Cu2+ concentration than both Zn2+ and Ni2+. The toxic effect of all groups of fatty acids ranged from highly significant at low concentrations to very highly significant at higher ones. At higher concentrations, the synthesis of all groups of fatty acids was inhibited by the five concentrations used, but the degree of inhibition was more prominent at higher concentrations especially in the content of polyunsaturated fatty acids. The results prove that the toxic effect of the tested element on fatty acids content in S. platensis was more prominent in case of mono-unsaturated and poly-unsaturated fatty acids than saturated ones.
Under normal conditions, the organism synthesizes 27 fractions of fatty acids at the 10th day of culturing. Dempester and Sommerfeld, (1998) reported that nutrient deficiencies may lead to an increase in the cell lipid content and consequently cells of old healthy cultures are richer in fatty acids than those cells grown at the lag phase of growth. The mono unsaturated fatty acid C18:1 and the poly-unsaturated fatty acid C18:2 were the most dominant ones. Cohen,(1991) reported that microalgae are excellent sources of polyunsaturated fatty acids such as lindonic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. It was reported by many authors that linolinic acid (C18:2) fatty acid is essential for the survival and growth of many juvenile aqua culture organisms, (El-Maghrabi, 2002).
Anent the results recorded in table (1) and graphed in figures (5&6) which explained the effect of different concentrations of Ni2+ ions (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) on the content of the three groups of fatty acids fractions (saturated, mono- and poly- unsaturated fatty acids) in S. platensis, it is clear that Ni2+ ions have weak toxic effect on the content of fatty acids in S. platensis compared to control. The content of saturated fatty acids at all the tested concentrations of Ni increased by 12.36, 10.01, 10.66, 9.02 and 18.40% compared to control. Also at concentration 2.0 and 2.5 mg/l Ni2+ the saturated fatty acids (C20:0 and C21:0) disappeared completely. However, total saturated fatty acids increased under the effect of the different concentrations of Ni ions.
It is clear from data recorded in table (2) and graphed in figures (7&8) that, at concentration 1.0 mg/l Cu2+ the total content of the saturated fatty acids decreased by 8.51% compared to control. These results may confirm those results obtained for growth where the organism showed weak growth for the same concentration compared to control. The saturated fatty acid C6:0 at this concentration (1.0 mg/l) increased by 287.56% over control. The results cleared also that the mono-unsaturated fatty acids decreased under all the tested Cu2+ concentrations with different values which depends on the concentration of the element. On the contrary, although the total poly-unsaturated fatty acids increased under the effect of all the studied concentrations of Cu yet C20:5 was not detected at concentrations 2.5 and 3.0 mg/l, C20:3 at control and at concentration 3.0 mg/l, C20:2 and C22:6 was not detected at concentrations 1.5, 2.5 and 3.0 mg/l. C22:2 which was detected at concentrations 1.0 and 2.0 mg/l but was not detected at control and 1.5, 2.5 and 3.0 mg/l.However, the grand total fatty acids decreased under all the concentrations tested.
Taking into consideration the effect of the five tested concentrations of Zn2+ on the content of the three groups of fatty acids (saturated, mono- and poly- unsaturated fatty acids) in S. platensis, it is clear from table (3) and figures (9&10) that the grand total of the three groups of fatty acids increased at concentration 1.0, and 1.5 mg/l Zn2+ (4.993 and 0.006% over control) while at concentrations 2.0, 2.5 and 3.0 mg/l Zn the total content decreased by 0.009, 0.009 and 0.005% below control, respectively. The content of saturated fatty acids increased under all the tested concentrations of Zn ions and the percent of increase depended on the concentration of Zn2+. At concentration 1.0 mg/l Zn2+ the percent of increase reached 16.74% while at concentration 3.0 mg/l Zn reached 0.74% over control. Concerning the mono-unsaturated fatty acids. Our results go with harmony with those obtained by Alam et al. (2010) and Balaji, (2015) who observed that, heavy metals have the potential to alter the rate of photosynthesis by disturbing chloroplast structure leading to the changes in the fatty acid composition. Another fact which support our results obtained by (El-Agawany and Kaamoush, 2022) who observed that in Dunaliella tertiolecta culture by increasing zinc, the total content of the three fatty acid groups decreased and the toxic effect of Zn+2 was more prominent in the case of mono-unsaturated and poly-5-unsaturated fatty acids than in the case of saturated ones.
It was reported by Dempester and Sommerfeld (1998) that neutral lipid production of some diatoms was noticeably influenced by specifically altering the MgCl2 concentration in the culture medium. Roessler,(1989) reported that the activity of acetyl-CoA carboxylase, an enzyme utilized early in fatty acid synthesis, was dependent on the presence of divalent metal cations especially magnesium (Mg2+). The same author observed reduced acetyl-CoA activity when manganese (Mn2+) was the only divalent metal present and no acetyl-CoA activity when only cobalt (Co2+) was present. Increased lipid yield was observed with increasing salt concentration, which may cause physiological stress in Botryococcus braunii and Isochrysis species (Ben-Amotz et al. 1985) and in Chlorella species (Tadros 1985). El-Maghrabi, (2002) recorded that one of the major factors that enhanced lipid biosynthesis may be nutrient limitation. The same results were also obtained in our study. However, cyanobacteria do not show significant changes in their lipid content and fatty acid composition in response to nitrogen supply (Becker, 2004). It was found that nitrogen limitation is an effective method to increase lipid content, mostly at the expense of protein (Piorreck et al., 1984).
Simonopoulos, (1991) found that microlagae were a good source for Omega-3 fatty acids which are protective factor against chronic diseases, coronary-heart diseases, diabetes and cancer. Chu and Dupuy, (1980) concluded that the changes in the relative amounts of polyunsaturated fatty acids may be attributed to effects on the desaturation pathways of fatty acids. Xu et al. (1997 and 1998), reported that the reduction in polyunsaturated fatty acids fractions might be due to reduction in membrane fluidity and permeability. Dowidar, (1983), mentioned that saturated fatty acids were more dominant than unsaturated ones under stress conditions. The same conclusion was also reported in our results.
The gel plate of the total soluble protein profile for control and the treated organism with different concentrations (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) of Ni2+, Cu2+ and Zn2+showed bands distributed through gel plates were illustrated in tables (4 – 9) and plates (1- 3). The sum of the bands that appeared on the gel plate and confirmed by scanning using the band peaks were 20 bands for Zn and 17 bands for both Ni and Cu. Some of these bands were common in the control and the treated organism; others were common only in the treated organism under the effect of the five different concentrations of the three tested elements (Ni2+, Cu2+ and Zn2+). Most of them appeared in the region between 25 KDa and 212 KDa in nearly all the lanes. However, the number of these bands at all the concentrations of Ni2+, Cu2+ and Zn2+ usually increased with the increase in the concentration of the element.
It is clear also in figures (11, 12 and 13) that, the destructive effect of the heavy metal ions on protein profile was more prominent under the stress effect of Cu2+ than in case of Ni2+ and Zn2+. These results seemed to be in conformity with findings of many authors (Ahmed, 2010 ; El Taher, 2012 and El-Agawany and Kaamoush, 2022). It is clear from these results that the obtained bands of the protein profile are distributed throughout the gel plate. Some bands are cathodic, others are anodic, but most of bands are cathodic anodic symmetry. The sum of the bands that appeared on the gel plate and confirmed by scanning using the band peaks were 20 bands for Zn and 17 bands for both Ni and Cu. Some of these bands were common in the control and the treated organism; others were common only in the treated organism under the effect of the five different concentrations of the three tested elements (Ni2+, Cu2+ and Zn2+). Another important fact, The total protein content of Spirulina platensis was 82.63 % in 50 % effluent of wastewater, it was suggested that heavy metals in wastewater at low concentrations accelerate protein production in S. platensis, (Balaji et al., 2015).
However, in nearly all the lanes most of the bands appeared in the region between 25 KDa and 212 KDa. A glimpse at the number of bands obtained for control only 13 bands were observed, while this number of bands increased or decreased depending on type and concentrations of the tested element. In case of Ni, the sum of bands increased by increasing the concentration of the element. At 1.0 and 1.5 mg/l Zn the percent of increase in the sum of bands reached 15.4%, while at concentrations 2.0, 2.5 and 3.0 mg/l Ni, the percent of increased of bands reached 30.8% over control for all the three tested concentrations of Ni. The newly formed bands at concentrations 1.0 and 1.5 mg/l Ni were 3 bands while at concentrations 2.0, 2.5 and 3.0 the number of the newly formed bands increased to 4 bands. It is clear from this data that most of the newly formed bands appeared at the region having low molecular weight, while most of the disappeared bands were recorded at the region of the high molecular weight.
It is clear also that by increasing the concentration of Cu from 1.0 mg/l to 2.0 mg/l, the newly formed bands were 3 bands, while at 2.5 mg/l the newly formed ones were 2 bands while at 3.0 mg/l the newly formed bands was one band only. Also the total bands in case of Cu at 1.0, 1.5 and 2.5 mg/l increased to 15 bands while at concentration 2.0 mg/l Cu the bands increased to 16 bands. At concentration 3.0 mg/l Cu; the number of bands decreased to 14 bands. In case of Zn the number of bands increased by increasing the concentration of the element. The newly formed bands increased by increasing the concentration of the element nearly 5, 3 and 4 bands at concentrations 2.0, 2.5 and 3.0 mg/l Zn, respectively. Only one band disappeared at concentrations 2.0 and 2.5 mg/l Zn. This map could explain the fact that Spirulina platensis is more sensitive to Cu ions followed by Zn then Ni ions. El-Agawany and Kaamoush, (2022) confirmed that, in Dunaliella tertiolecta culture, the percentage of increase in the number of bands was found to depend on the toxicity of zinc element. It is clear also that at concentration 25 mg/L, the organism greatly suffered from the toxic effect of zinc element.
In addition, some heavy metals can affect protein profile in algae. Chernicova et al., (2006) observed that increasing of concentrations of manganese does not inhibit growth considerably altered cell ultrastructure and changed the protein profile in Spirulina platensis. Sinha and Hader, (1996), found that Anabaena species cultured at stress conditions did not show any changes in protein pattern. On the other hand, Fulda et al., (1999) found that the composition of periplasmic proteins obtained from cells of Synechocystis species grown under stress showed clear differences. Hoyos and Zhang, (2000) are in agreement with those the above mentioned results, they found that reversible protein phosphorylation/dephosphorylation plays an important role in signaling the plant adaptive response to stress. The results of Salah El-Din, (1994) confirmed that, most of the algal species have similar physiological functions which are related to biosynthesis or biodegradation of some macromolecules. This conclusion seems to explain the different changes of the amount of total soluble protein bands in stressed algae.). Ahmed, (2010) and El Taher, (2012) reported that, the tolerance of an organism to stress conditions could be achieved through synthesis or accumulation of new proteins. These results are nearly in harmony with our results for Spirulina platensis. Protein deficiency in human nutrition is a major concern for most of developing countries; so there is a need to develop protein sources a new unconventional ones. The high protein concentration of various microalgal species specially Spirulina platensis makes them an excellent source in the supply of this nutrient,(Anne et al., 2016).