Statement of Novelty

Determine the suitable and optimum conditions for production of chelated metals. Use the leather waste hydrolysate for production of chelated five different metals. First time for cultivation of blue-green alga in media enriched with metals chelated with LW.

Introduction

Chelation is a chemical process that involves binding an organic molecule (as amino acids) to a mineral in two or more sites to form a ring. The molecule encircles and protects the mineral from any negative interactions [1]. Micronutrients such as Zn, Cu, Fe, and Mn are essential metals that enhance growth and yield of plant and algae but these minerals in inorganic form affected by change of pH degree and converted to non-absorbable, furthermore the minerals in chelated form are coated with amino acids which precipitation and absorption complications [2] Moreover, some factors were taken in consideration such as time, ratio, temperature and pH to improve the chelation process, [1].

Arthrospira platensis is a filamentous photoautotrophic cyanobacterium that inhabits various environments [3]. Its cell wall is formed from mucopolysaccharides which consist of protein, carbohydrates, lipids, polyunsaturated fatty acids, vitamins and minerals [4].

A. platensis has attracted great attention as a source of high value products, which could be used as antioxidant agents like phenolic compounds, pigments, long-chain polyunsaturated fatty acids and sulfated polysaccharides [5,6,7]. In addition, researchers have turned their interest towards using A.platensis as one of the biodiesel sources fatty acids (methyl esters) sustainable for supplying the world's need for fuels [8,9,10]. Biodiesel is an environmentally-friendly, non-toxic, renewable and biodegradable fuel made from the reactions of either, animal fats, algal lipids or vegetable oils with short-chain alcohol (methanol or ethanol) in the presence of a catalyst [11].

The main objective of the present work is to optimize the factors controlling the formation of chelated amino acids with different minerals and to enhance antioxidant and biodiesel production from Arthrospira platensis cultivated under stress of chelated amino acids. with various metals which prepared by using leather waste hydrolysate as a source of amino acids or glycine.

Materials and Methods

Chemicals and Reagents

Pure acetone, methanol, ethanol, petroleum ether, chloroform were purchased from Merck Co. (Darmstadt, Germany). 2, 2 diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Vitamin C, Copper sulfate, Manganese sulfate, Zinc sulfate, Magnesium sulfate, Ferrous sulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cyanobacteria Culture

A cyanobacterium (Arthospira platensis) was obtained from Phycology lab in Botany Dept., Faculty of Science, Cairo University, Giza, Egypt. It was cultivated on Zarrouk media as control and modified culture by replacing some of macro and micronutrients using chelated minerals with amino acids (during spring season of 2021) as described by Jacob et al. [1].

Cultivation of Cyanobacteria

The blue green microalga Arthrospira platensis used in this study was kindly isolated in Phycology Lab. and identified by Dr. Sanaa Shanab, Professor of Phycology in the Department of Botany and Microbiology, Faculty of Science, Cairo University, according to [12].

Leather Wastes Source

Waste samples were used, chrome-containing leather waste (CCLW), as animal waste, was obtained from a commercial leather tannery, Al Basatin, Al Maadi, Cairo, Egypt. Samples were kept at 4 °C in refrigerator for further analysis to avoid any fermentation.

Preparation of Chelated Glycine

Glycine solution was prepared in distilled water or leather waste hydrolysate with CaO as described by Jacob et al. [1] mixed with each minerals salt (CuSO4⋅5H2O, ZnSO4⋅7H2O, FeSO4⋅7H2O, MnSO4⋅H2O and MgSO4) separately with molar ratio 1:2. The mixture was shaken for one minute and kept at room temperature till crystal formation. For iron chelate complex, before mixing the iron salt (FeSO4⋅7H2O), a 1% antioxidant (ascorbic acid) was added. The product obtained was concentrated by evaporation to dryness then washed with ethanol (95%). The washed products were air dried for about 12 h. this method was described by Jie et al. [13] with some modification.

Factors Affecting the Synthesis of Chelated Minerals

There are many factors affecting the formation of crystals such as ratio, temperature, time, pH that are needed to optimize the conditions of chelation rate.

$$\% {\text{ Chelation rate}}\, = \,\left( {{\text{chelated micronutrient}}/{\text{total micronutrient}}} \right)\, \times \,{1}00$$

The followings are the methods used for studying the effect of physical conditions or parameters on chelation synthesis between minerals and glycine (as amino acids)

Ratio

Different ratios (1:2 and 1:3) from glycine and minerals were used for determination the effect of these ratios on chelation formation, the preparation method was mentioned in details in the previous paragraph (no. 3: Preparation of chelated glycine) according to Jie et al. [13] with some modification.

Temperature

Using different temperature (R.T 25 °C and 60 °C) in the incubation period of mixed glycine with minerals by using the ratio (1:2) as mentioned by Jie et al. [13], compare the chelation rate between them to select the optimum temperature.

Time

Mixed glycine with minerals solution was incubated during various periods from one to 10 days with two types of ratio (1:2) and (1:3) to determine the time required for chelation formation and obtain the maximum chelation rate as mentioned by Jie et al. [13].

pH

Preparation of chelated glycine with minerals in ratio (1:2), division of the solution to equal volumes then change each one to different pH degree from 4 to 10 as mentioned by Xixi et al. [14] and Jie et al. [13]. Select the optimum pH degree or range of preparation by maintaining the chelation in its stability without sedimentation.

Lambda Max (Absorption in UV–Vis)

The maximum absorption (λ max) for chelated glycine in comparison with free glycine was determined using UV–Visible spectrophotometer T60UV PG instrument, in details: a stock solution of free glycine and chelated glycine prepared by dissolving 10 mg in 10 ml distilled water to obtain a final concentration 1000 ug/ml, the obtained solutions were scanned every 1 nm (from 200 to 800 nm) against distilled water as blank according to Prasad and Thireesha [15].

Ninhydrin Assay

The 2,2-Dihydroxyindane-1,3-dione (Ninhydrin) was prepared in acetone, glycine standard and chelated glycine were prepared in distilled water (1000 µg/ml). One ml of standard or chelated glycine was mixed with 1 ml of ninhydrin reagent, incubate in boiling water bath (98 °C) for 15 min then cooling the tubes and measure the absorbance at 570 nm against blank.

Autobiography by Ninhydrin

Standard solutions (1 mg/mL) of amino acid (glycine) and chelated glycine were prepared in distilled water and spotted on the precoated silica gel TLC plates. Plate was air-dried and subjected to TLC jar using n-butanol-acetic acid–water, (4:1:1) as mobile phase. After development, plates were dried and sprayed with 2% Ninhydrin in acetone and again dried in air for complete evaporation of solvent. The plates were then heated at 60 °C for 10 min in an oven and the colors were recorded again. Colors were always observed visually.

Antioxidant Activity and Antiradical by DPPH

The 2, 2 diphenyl-1-picrylhydrazyl (DPPH) tests were carried out as described by [16]. One mL of tested sample (algal extract or chelated minerals) was mixed with 1 mL DPPH reagent (0.03% w/v) prepared in methanol. After incubation in the dark at room temperature for 30 and 60 min, the absorbance was measured at 517 nm (using PG instrument T60 UV–Visible spectrophotometer). The same procedure repeated and measured after incubation 20, 40, 80, 160, 320 and 500 s to determine antiradical unit. This test was carried out in duplicate and the antioxidant activity was calculated as the following:

$${\text{Activity }}\left( \% \right)\, = \,{\text{Ac}} - {\text{At}}/{\text{Ac}}\, \times \,{1}00$$

Where: At was the absorbance of samples and Ac the absorbance of methanolic DPPH solution.

$${\text{Antiradical Unit }}\left( {{\text{AU}}} \right)\, = \,{\text{A}}_{0} - {\text{A}}_{{\text{t}}}$$

Where: At was the absorbance of samples in different times and A0 the absorbance of methanolic DPPH solution.

Fourier Transform Infrared (FTIR) Spectroscopy

Fourier transform infrared spectroscopy (FTIR) analysis was done for glycine, zinc glycinate, copper glycinate, ferrous glycinate, manganese glycinate, magnesium glycinate and biodiesel of treated algal with Shimadzu FTIR spectrometer at room temperature over the range of 4000 − 400 cm−1 at a resolution of 3 cm−1 in KBr pellets according to the method recorded by Jacob et al. [1].

Biodiesel Preparation

The preparation of biodiesel was described by Afify et al. [8].

Algal Oil Extract

One gram fresh weight from cultivated Arthrospira platensis was extracted by chloroform: methanol (2:1), evaporate the solvent in boiling water bath to obtain the extracted oil.

Trans Esterification

The extracted oil was esterified by mixing with alcoholic NaOH (0.25 g NaOH/24 ml methanol), stirring 20 min and shake for 3 h/3000 rpm. Keep the mixture 16 h then mixed with distilled water and petroleum ether in separating funnel to separate esterified layer in petroleum ether (biodiesel).

Separation of Pigments and Glycerol

The watery layer in separating funnel contained pigments and glycerol, mixed with active charcoal to adsorb the pigments and filtrate to obtain glycerol. Washing the charcoal by acetone to obtain the pigments.

Identification of Biodiesel Fatty Acids Using GLC

The GLC analysis was carried out with a Thermo-GC gas chromatography, with a dual flame ionization detector. The capillary column TRACE TR-FAME (100 m × 0.25 mm × 0.2 mm) was packed with 1% OV-17. Temperature of injector and detector were 240 °C. The column was hold at 200 °C for 3 min then Programmed from 60 to 200 °C (at rate of 10 °C/min). Helium was a carrier gas, hydrogen and air gases were used at flow rates of 10, 35 and 350 ml/min, respectively. The identification of fatty acids was accomplished by comparing the peaks of retention times with those of the corresponding standards. The quantity of individual compounds was determined by comparing the produced peak area by known weight of biodiesel with standard curve of the authentic substances which expressed the relation between the different concentrations and their peak area. This method was described by Afify et al. [8].

Statistical Analysis

All the data are expressed as mean ± standard deviation of three determinations. Statistical comparison was performed via a one- way analysis of variance followed by Duncan’s multiple range test (DMRT). P-Values of less than 0.05 (P˂0.05) were considered as significant.

Results and Discussion

Cultivation of Arthrospira platensis

Cultivation of Arthrospira platensis on modified Zarrouk media with substitute some of minerals in sulfate form by chelated form with glycine and different amino acids in leather waste hydrolysate such as Mg++, Mn++, Fe++, Cu++ and Zn.++ was determined by optical density in zero time and after 4 weeks. The obtained results in Fig. 1 showed that the growth rate of Arthrospira platensis with Cu-glycinate higher than M-LWH and Zarrouk media. The obtained results agree with our previous study Jacob et al. [1]

Fig. 1
figure 1

Growth rate of A. platensis as optical density treated by chelated minerals with glycine and leather waste hydrolysate at zero time and after cultivation 28 day

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Antioxidant Activity of Cultivated Alga

The antioxidant activity of different extracts of Arthrospira platensis cultivated on Zarrouk media supplemented with chelated minerals with different amino acids was determined using DPPH radical assay. The obtained results in Table 1 showed that the water extract gave high antioxidant activity than acetone extract in all treatments when compared with untreated culture (Zarrouk’s medium). In addition to LW treatments present antioxidant activity more than glycinate treatments and have a positive correlation with the incubation time in most cases. Moreover, the treated cultures with Cu-LW and Mix-glycinate in water extracts exhibited the highest antioxidant activity (59.82, 63.90 and 59.36, 60.50%) when compared with untreated culture (29.99 and 32.25%) during 30 and 60 min, respectively.

Table 1 Antioxidant activity (%) of different extracts from Arthrospira platensis cultivated under varies conditions against DPPH radical assay
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The obtained results may be due to the effect of chelated minerals on gene expression and growth rate of Arthrospira platensis which led to an increase of antioxidant compounds such as phenolic, flavonoids and natural pigments. These results are in agreement with our previous study Abou Elalla and Shalaby [17], Jacob et al. [1] they mentioned that Arthrospira platensis treated by chelated minerals contained high amount of chlorophyll and phycobiliprotein pigments when compared with untreated cells, these pigments may increase the radical scavenging activity of algal extracts. While, treated Arthrospira platensis with Mn-glycinate, Fe-glycinate, Mix-Gly, Cu-Lw, Zn-Lw and Mix-Lw have high content of chlorophyll, carotenoids and phycobiliproteins. Shalaby and Shanab [18] mentioned that water extract showed the highest antioxidant activity (95.3%), which may be due to total phycopilin pigments. In addition, Ali et al. [19] reported that the type of solvent affect the percentage of antioxidant activity and this is due to the presence of phenolic and carotenoid compounds. Also, Colla et al. [20] and Lordan et al. [21] reported that the component responsible for antioxidant activity was the phycocyanin. Furthermore, Safari et al. [22] revealed that there were positive correlations between concentration of C-phycocyanin and antioxidant activity.

Antiradical Units

The antiradical unit of different extracts of Arthrospira platensis cultivated on chelated minerals with different amino acids was determined by using DPPH. The obtained results in Figs. 2a, b and 3a, b revealed that the water extract recorded the highest antiradical effect than acetone extract and untreated culture in both extracts. As well as, the glycinate treatments have antiradical unite more than LW treatments when compared the antiradical unit at the different incubation time (from 20 to 500 Sec). Furthermore, the highest antiradical unites were recorded with Mix-glycinate and Cu-LW in water extracts (0.401, 0.524 and 0.371, 0.465 AU at 20 and 500 Sec. respectively) when compared with untreated culture (0.264, 0.312 AU at 20 and 500 Sec.) followed by Mn-glycinate treatment in acetone extract (0.472, 0.487 AU at 20 and 500 Sec. respectively) which exhibited high activity more than control culture (0.387 and 0.395 AU at 20 and 500 Sec.)

Fig. 2
figure 2

a, b Antiradical unit (AU) of acetone extract from Arthrospira platensis cultivated under varies culture conditions against DPPH radical, a algal cultivated with M-glycinate and b algal cultivated with M-LW

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Fig. 3
figure 3

a, b Antiradical unit (AU) of water extract from Arthrospira platensis cultivated under varies culture conditions against DPPH radical, a algal cultivated with M-glycinate and b algal cultivated with M-LW

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These results were synergistically related with antioxidant activity to the same treatments and may be due to an increased content of phycobiliprotein pigments especially allophycocyanin and C-phycoerythrin in water extract which have significant antiradical and antioxidant activity as mentioned by Shalaby and Shanab [18]. Also, the same authors revealed that the antiradical and antioxidant of aqueous extract may be due to the presence of phenolic compounds. These findings were in agreement with Alyasiri et al. [23] who reported that the aqueous extract of A. plantensis had highly antiradical activity at concentration1000 µg/ml of DPPH. Also, Bellahcen et al. [24] revealed that the antiradical and antioxidant activity of A. plantensis were characterized by the presence of phenolic and flavonoid compounds that presented in ethanolic extract.

Factors Affecting the Synthesis of Chelation

Ratio

The optimum ratio between amino acids as glycine and different minerals (Zn, Cu, Fe, Mg and Mn) was investigated by determining the material balance for each ratio 2:1 and 3:1. The obtained results in Table 2 presented some of the investigated minerals were gave highest yield with ratio 2:1 such as Fe, Mg and Cu (52.57, 48.98 and 47.15% respectively). While, Fe produced the largest yield with the ratio 3:1 than 2:1 (81.41 and 52.57% respectively) in both glycinate and leather waste treatments and the crystals shape with both ratios showed in Fig. 4. The obtained results may be due to the potency or capacity of each mineral to be able to form the chelation with amino acids. These results are in agreement with those of Jie et al. [13] who reported that the molar ratio affect the stability and the structure of chelating agents and the optimum ratios between amino acids and salts were three ratios (1:2, 1:2.5 and 1:3).

Table 2 Optimum ratios for formation of glycine with different minerals production rate
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Fig. 4
figure 4

Comparison between the yield production of chelated minerals with different ratios (2:1 and 3:1)

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Temperature

The impact of temperature on the production rate of chelated glycine with different minerals was measured by the quantity formed over time with uniform molar ratio (2:1). The obtained results in Fig. 5 and Fig. 6 revealed that the temperature led to acceleration the chelate formation with a decrease in the time required in most cases for chelation reaction. Moreover, the highest temperature (60 °C) increased the yield with Mg-Gly, Zn-Gly, Cu-Gly and Fe-Gly (22.60, 21.51, 17.10and 16.85 respectively). These results may be due to the fact that temperature has a catalytic role in chemical reactions. However, Jie et al. [13] revealed that the temperature degrees which investigated (20, 40, 60 and 80 °C) in the chelate preparation with different minerals had no effect on the chelation rate.

Fig. 5
figure 5

Comparison between the yields of chelated glycine with different minerals prepared at 25 °C and 60 °C

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Fig. 6
figure 6

Comparison between the yields of chelated minerals prepared at 25 °C and 60 °C

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Time

The optimization of metals amino acids ratios to achieve the maximum chelation rate in short time was investigated by using two molar ratios 2:1 and 3:1. The obtained results in Fig. 7 revealed that the ratio 3:1 reduced the time in most cases which required to crystals formation of chelated minerals such as Zn-Gly, Fe-Gly (decreased 5 days with both) and Cu-Gly (decreased 2 days).

Fig. 7
figure 7

Comparison between the required duration (Time) for formation of chelation at different ratios

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pH

The optimization of stability of chelated amino acids with different minerals was affected by the change in pH values from 4 to 10 as shown in Table 3. These results presented to the variation in pH between free metal salt and its chelated form with glycine. Furthermore, the chelation stability when the pH varies through a change in color or the precipitate appearance showed in Fig. 8. In most cases, the chelation form decrease in the pH than free form of metal salt especially in Zn-Gly and Cu-Gly. In addition, all of chelated forms were stabled at pH = 4 forming clear solutions.

Table 3 Stability of different chelated forms at different pH (from 4.0 to 10) compared to individual mineral salts
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Fig. 8
figure 8

pH degree and the stability of chelated glycine with different minerals

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Lambda Max (λ max)

The identification of the wave length which gave the maximum absorption of chelated amino acids with different minerals presented in Fig. 9a, b. The obtained results revealed that the wave length for all chelated amino acids with different minerals in UV area that ranged between 205 and 234 nm. The obtained results may be due to the presence of n–π* bond in the carbonyl group of amino acids. McConnell et al. [25] mentioned that unsaturation in the carbon–oxygen bond was produced to a transition in n–π* of the carboxylate carbon and gave the UV absorption in the range 190 to 210 nm.

Fig. 9
figure 9

a, b Maximum absorption of chelated amino acids with different minerals, a M-glycinate and b M-LW

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Ninhydrin Reagent

The amino acid concentration was determined by using ninhydrin reagent. The obtained results in Table 4 showed that the highest concentration of amino acid with all chelated minerals with glycine (Mn-Gly > Mg-Gly > Zn-Gly > Fe-Gly) except Cu-glycinate which exhibit the lowest concentration (0.670 µg/ml) when compared with glycine standard (1000 µg/ml). These results are synergistically agreed with Khan et al. [26] who mentioned that the reaction mechanism of copper-amino acids complexes with ninhydrin and revealed that the coordination bonds between copper ion and lone pair of nitrogen (amino group) and oxygen (carbonyl group) prevent the penetration of ninhydrin to react with amino acids. However, there was type of reactions called CLAM (combination of two ligands attached to the same metal ion) occasion with both amino acid and ninhydrin with the outer sphere of the same copper ion by coordination bonds in fast rate of reaction and rearrange of its coordination bonds in slow rate to bind the nitrogen with both amino acids and produced yellow color. In addition, there were some factors affecting on the reaction between complex and ninhydrin such as pH and temperature.

Table 4 Amino acid content (as µg/ml) of chelated minerals with glycine as amino acid
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TLC Chromatogram of Chelated Glycine

The Rf identification of chelated glycine with different minerals which prepared at different temperature to observe the impact of temperature on the Rf of M-glycinate. The results showed that there are only one spot with the same Rf of all chelated glycine with different minerals which matched with Rf of free glycine. Therefore, the different minerals which bind to glycine at different temperature can’t effect on the migration of amino acid on TLC plate as showed in Fig. 10. (Reference).

Fig. 10
figure 10

TLC chromatogram of chelated glycine with different metal ions prepared at 25 °C and 60 °C

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Antioxidant Activity

The antioxidant activity of chelated glycine with different minerals was determined by using DPPH radical assay. The obtained results in Fig. 11 revealed that the chelation of glycine with Fe, Mg and Mn exhibited the highest antioxidant activity when compared with glycine as control (80.04, 40.10, 34.31 and 12.63%, respectively).

Fig. 11
figure 11

Antioxidant activity (as %) of chelated minerals with glycine using DPPH radical assay

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The obtained results may be due to the traces of ascorbic acid which existed after washing Fe-glycinate crystals. These results agreed with Afanas’ ev et al. [27] who revealed that some of minerals exhibit antioxidant activity such as Zn, Cu, Mn and Mg aspartate. In several studies, they were focused on the impact of chelated amino acids applied on different plants or animals and their content from antioxidant compounds or antioxidant biomolecules such as antioxidant enzymes as mentioned by Ibrahim et al. [28], Shafeek et al. [29] and Li et al. [30].

FTIR of Chelated Amino Acids

The beneficial technique to identify the functional groups and its links with other atoms in the chemical structure is FTIR technique.

FTIR spectroscopy was used to characterize the variation between free amino acid (as glycine) and chelated-glycine with different metals such as Zn++, Cu++, Fe++, Mn++ and Mg++ as shown in Table 5. The obtained results of FTIR spectra revealed the presence of different atom groups in all treatments such as amino (–NH2), hydroxyl (–OH), methylene (–CH2) and carbonyl group (–C = O) with wavenumber (3667.73, 3200, 1412.23 and 1615 cm−1) for untreated glycine and slightly shifting with chelated glycine such as Zn-glycinate (3554.11, 3204.60, 1494.83 and 1642.82 cm−1). There are shifting and change in band shape of the stretching vibration for –OH and –NH2 groups. Moreover, the band which appeared around 1610 cm−1 indicate on the vibration of carboxylate group (–COO−) in the coordination bond of chelated glycine when compared with free carboxylate that located around 1400 cm−1 as mentioned by Al-Wasidi et al. [31].

Table 5 Wave number (cm−1) of different chelated glycine with minerals
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Biodiesel Production From Treated Algal Species

Arthrospira platensis cultivated on Zarrouk medium supplemented with chelated amino acids with metals was showed an increase in algal pigments and lipids with Mn-LW, Zn-LW and Mg-LW treatments as showed in Table 6. Which not only induced an increment in antioxidant activity but also we can benefit of the high lipid content to produce biodiesel and the related pigment and glycerol from the transesterification process. (reference).

Table 6 Biodiesel, pigments, and glycerol content of different treated Arthrospira platensis when compare with LW and untreated biomass
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Biodiesel Content

The results in Table 6 showed that the percentage of biodiesel which extracted from promising treatments of A. platensis and compared with the biodiesel which obtained from extracted lipid of leather waste. The results showed that the produced biodiesel percentage was observed with M-LW treatments, which was more than that of glycinate treatments, untreated culture (Zarrouk) and LW biodiesel. Therefore, the highest biodiesel percentages were founded with Zn, Mn and Mg-LW (5.37, 5.25 and 4.86% respectively).

FTIR of Produced Biodiesel

FTIR technique was used to check the preparation of biodiesel through the absence of hydroxyl group (–OH) band in the spectra. The obtained results in Table 7 revealed that there was a similarity in the wavenumber of –CH and –CH2 with all treatments which appeared around 2964 cm−1 and 2877 cm−1. However, there was shifting in the wavenumber bands of some treatments with C=C and C=O.

Table 7 FTIR of biodiesel produced from Arthrospira platensis cultivated in Zarrouk media rich with different chelated minerals
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Pigments Content

The results in Table 6 present the pigments concentration which prepared from the biodiesel production deposits of promising treatments. The obtained results in Table 6 revealed that the LW treatments have a positive effect on the synthesis of Chl a and T. carotenoid when compared with untreated culture. Furthermore, the highest amount of Chl a with Mn, Zn and Mg-LW treatments (0.0106, 0.0095 and 0.0087 mg/g respectively). As for the concentration of T. carotenoid increased with Mn, Zn-LW treatments and LW (1.6470, 1.6073 and 1.5408 mg/g respectively).

There are coincidences between the biodiesel percentage and pigments content resulted with the same treatments in addition to LW. This compatibility may be due to the stress effect of different chelated minerals with amino acids which have a positive effect on the biosynthesis of lipid and pigments especially T. carotenoid that increased to resist the oxidative stress as mentioned by Afify et al. [8]. Moreover, Reinbothe et al. [32],Shanab et al. [33] and El-Sheekh et al. [11] revealed that the concentration of some minerals such as iron impact on the growth of A. platensis and also the pigments production.

Glycerol Concentration

The concentration of glycerol in water fraction of biodiesel preparation showed in Table 6. The obtained results revealed that the highest concentration of glycerol in the water fraction of LW biodiesel preparation than untreated culture (0.0515 µg/ml) followed in descending order by untreated culture and Mix-glycinate (recorded 0.0438 µg/ml with both treatments).

Gas Liquid Chromatography (GLC)

The relative percentage of fatty acids of produced biodiesel from extracted lipids from different promising extracts of A. platensis treatments with different chelated minerals with amino acids and compared with biodiesel produced from the extracted lipid of leather waste was shown in Table 8. The obtained results recorded that a significantly increase in the total saturated fatty acids with all algal treatments (ranged 60.29–64.73%) when compared with untreated alga (control, 56.94%). On the other hand, there was a decrease in the total unsaturated fatty acids than untreated culture. The highest ratio of TSFAs was observed with Mn-gly followed by Zn-LW and Mn-LW when compared with untreated alga (Zarrouk medium) by (64.73, 63.80, 63.08 and 56.94 respectively). However, the untreated alga recorded the highly relative percentage in unsaturated fatty acids (USFA) by 43.06% when compared with other algal treatments as showed in Table 8

Table 8 Fatty acids composition (as relative percentage) of biodiesel produced from highly lipid content sample (as promising treatments) of Arthrospira platensis cultivated with different chelated minerals
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The obtained data in this investigation may be because of some minerals such as zinc, manganese, magnesium and ferrous on the synthesis of saturated and unsaturated fatty acids. These results were in agreement with the results obtained by El-Sheekh et al. [11] and Miazek et al. [34] who reported that there was an effect of some metals on the lipid production from A. Platensis such as ferrous ion which enhance the synthesis of palmitic, oleic, linoleic, γ-linolenic and docosahexaenoic acid through the formation of oxidant compounds such as reactive oxygen to abolish or reduce the oxidant compound injury. Also, Afify et al. [8] mentioned that the nitrogen concentration in the culture media have a negative effect on the production and the structure of unsaturated fatty acids according to the composition of growth medium. Moreover, an increase in the concentration of saturated fatty acids than unsaturated fatty acids in produced biodiesel enhances the physico-chemical quality of product for using in wagon as described by European standard. Furthermore, Murad & Al-Dawody [35] and El-Shimi et al. [9] revealed that the highest percentage of saturated fatty acid for biodiesel production was palmitic acid (C16:0) which coincided with the obtained results in this study.

From the obtained data we can conclude that the optimum conditions for production of chelated amino acid from the reaction of amino acids and different minerals were ratio (2:1 M), temperature at 60 °C, the duration between 4:5 days and the suitable or stability of chelation at pH = 4, the growth rate of A. platensis with Cu-glycinate was higher than Cu-leather waste and Zarrouk media as control media. The antioxidant activity results of different extracts of Arthrospira platensis showed that the water extract gave high antioxidant activity against DPPH radical assay than acetone extract in all treatments when compared with untreated culture (Zarrouk’s medium). Arthrospira platensis cultivated on Zarrouk medium supplemented with chelated amino acids with metals was showed an increase in algal pigments and lipids with Mn-LW, Zn-LW and Mg-LW treatments. Also, the results showed that the produced biodiesel was observed with M-LW treatments, which was more than that of glycinate treatments, untreated culture (Zarrouk) and LW biodiesel. Therefore, the highest biodiesel percentages were founded with Zn, Mn and Mg-LW. The recorded results and material balance data (Fig. 12) concluded that possibility for use the chelated minerals (glycine and leather wastes) as plant fertilizer in future because its high yield and low fees for production.

Fig. 12
figure 12

Material and energy balance for recycling of leather waste into plant fertilizer

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