2.1 Reagents
Potassium dichromate (Fisher Scientific; Pittsburgh, PA, USA) was used as a source of hexavalent chromium, diphenylcarbazide (Merck Germany), Chromium (III) chloride hexahydrate (Analar 97%, England), sulfuric acid (98%), hydrochloric acid (85%), and sodium hydroxide were all of the analytical reagent grades and obtained from Sigma (St. Louis, MO, USA). Deionized water was used for all solutions and dilutions.
2.2 Extraction of tamarind leaves
Tamarind leaves were collected by a research assistant from Pharmacognosy Research Laboratory, Ahmadu Bello University Zaria. The leaves were washed with clean water and dried before being ground into fine powder. Extraction was carried out by maceration through soaking 500 g leaves powder in 70% methanol for two days. The solution was decanted and filtered through Whatman No. 4 filter paper. The mixture was concentrated to a thick consistency, and the resulting extract was kept in a desiccator for further use.
2.3 Preparation of reagents
The extract solution was prepared by dissolving 0.1 g in deionized water and subsequently diluted to 100 ml to obtain a 1 mg/ml extract solution. Stock Cr (VI) solution (50 mg L−1) was prepared by dissolving 0.05 g of potassium dichromate (K2Cr2O7) (294.18 g mol−1) in 1 L of deionized water. The pH of Cr (VI) solution was adjusted to 7.0 using 0.1 M NaOH or 0.1 M HCl before being filtered using a 0.45-μmWhatman filter paper and sterilized. The working solution was prepared by diluting the stock solution with deionized water to give the appropriate concentration (10 mg L–1) of the solution and 0.20 g of 1,5-diphenylcarbazide was added in 100 ml of 95% acidified ethanol and store in sterilized and dried brown colored bottle.
2.4 DPPH radical scavenging activity
The method of Shahidi and Liyana-Pathiranan [40] was used for the determination of scavenging activity of DPPH free radical in the extract solution. A solution of 0.135 mM DPPH in methanol was prepared, and 1.0 ml of this solution was mixed with 1.0 ml of extract solution prepared in methanol containing 0.025–0.5 mg/ml of the plant extracts and standard separately (ascorbic acid). The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. The ability of the plant extract to scavenge DPPH radical was calculated by the equation:
$${\text{DPPH radical scavenging activity}} = \frac{{({\text{Abs control}}{-}{\text{Abs sample}})}}{{({\text{Abs control}})}} \times 100$$
where Abs control is the absorbance of DPPH radical + methanol; Abs sample is the absorbance of DPPH radical + sample extract or standard.
2.5 Hydrogen peroxide scavenging assay
The scavenging capacity for hydrogen peroxide was measured according to the method of Rahmat [35]. A solution of hydrogen peroxide (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically at 230 nm absorption using molar extinction coefficient for hydrogen peroxide of 81 M−1 cm−1. Then, 1 ml of various concentrations (25–250 μg/ml) of extract solution and ascorbic acid in triplicate was transferred into the test tubes and their volumes made up to 4 ml with 50 mM phosphate buffer (pH 7.4) or solvent (methanol). After addition of 6 ml hydrogen peroxide solution, tubes were vortexed and absorbance of the hydrogen peroxide at 230 nm was determined after 10 min, against a blank containing 50 mM phosphate buffer without hydrogen peroxide. Hydrogen peroxide scavenging ability was calculated using the formula:
$$\%{\text{Scavenging}} = \left( {1 - \frac{{A_{{\text{e}}} }}{{A_{{\text{o}}} }}} \right) \times 100$$
where Ao is the absorbance without sample, and Ae is absorbance with sample.
2.6 Determination of Cr (VI) concentration
The experiment for the reduction of Cr (VI) was conducted in batch as described by Chen et al. [6] with some modifications. The reaction mixtures were obtained by adding 100 ml of Cr (VI) solution (10 mg/L) into a 250-ml Erlenmeyer flask, adjusting the pH value, and adding 100 ml Tamarindus indica methanol leaves extract (1 mg/ml) unless otherwise specified. The initial pH of the solution was adjusted with sulfuric acid solution (0.5 M) and/or sodium hydroxide solution (1.0 M). All experiments were conducted at room temperature (25 °C) unless otherwise specified. Cr (VI) was measured spectrophotometrically at 540 nm according to the diphenylcarbazide method.
2.7 Effect of process parameters
The batch experiment explained above was conducted in 250-ml Erlenmeyer flasks with a working volume of 100 ml. The following set of factors was chosen as the standard conditions: 10 mg/L of initial Cr (VI) concentration, pH 7, room temperature (25 °C), and reaction time of 30 min, and one of the parameters was varied at a time, while others were kept constant.
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1.
Effect of initial extract concentration
To determine the effect of initial Tamarindus indica methanol leaves extract concentration on reduction of Cr(VI), 100 ml of Cr (VI) solution (10 mg/L) was reacted with 100 ml of Tamarind extract at different initial concentrations, namely 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, and 0.2 mg/ml, respectively, at pH of 2, 7, 9, and room temperature. The solution was intermittently sampled and centrifuged at 3000 rpm for 5 min, after which the Cr (VI) concentration was quantified using the 1, 5-diphenylcarbazide method.
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2.
Effect of initial Cr (VI) concentration
To determine the effect of initial Cr (VI) concentration on the reduction of Cr (VI) by Tamarindus indica methanol leaves extract, 100 ml of Cr (VI) at initial concentrations of 5, 10, 15, 20, and 25 mg/L was reacted with 100 ml of Tamarind extract at 1 mg/ml, respectively, at pH of 2, 7, 9, and room temperature. The solution was sampled and centrifuged at 3000 rpm for 5 min, after which the Cr (VI) concentration was quantified using the 1, 5-diphenylcarbazide method.
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3.
Effect of hydrogen ion concentration
The effect of pH on the reduction of Cr (VI) by Tamarindus indica methanol leaves extract was determined by varying the pH of the reaction mixture, viz. 2–9 (± 0.1), respectively. For each 100 ml solution of Cr (VI) solution (10 mg/L) in 250-ml Erlenmeyer flask, pH was adjusted (change in working volume due to the addition of NaOH or H2SO4 was negligible), and then, 100 ml Tamarind extract solution (1 mg/ml) was added. The solution was sampled and centrifuged at 3000 rpm for 5 min, after which the Cr (VI) concentration was quantified using the 1, 5-diphenylcarbazide method.
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4.
Effect of reaction time
The reaction mixture of the batch experiment at the pH of 2, 7, 9, and temperature of 25 °C was allowed to stand for the different duration (5–65 min) to determine the effect of reaction time on the reduction of Cr (VI) by Tamarindus indica methanol leaves extract. The solution was sampled and centrifuged at 3000 rpm for 5 min, after which the Cr (VI) concentration was quantified using the 1, 5-diphenylcarbazide method.
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5.
Effect of temperature
The reaction mixture of the batch experiment was incubated at different temperatures; 5–45 °C (± 10C) under pH 2, 7, and 9 to determine the effect of temperature on the reduction of Cr (VI) by Tamarindus indica methanol leaves extract. The solution was sampled and centrifuged at 3000 rpm for 5 min, after which the Cr (VI) concentration was quantified using the 1, 5-diphenylcarbazide method.
2.8 Kinetics studies of Cr (VI) bioreduction
Batch experiments were carried out as described by Chen et al. [6] under optimum conditions to ascertain Cr (VI) reduction kinetics properties.
Reaction order | Kinetic equation |
|---|
First order | \(\log q_{{\text{t}}} = \log q_{{\text{e}}} - \frac{k}{2.303}t\quad (1)\) |
Pseudo-first order | \(\log (q_{{\text{e}}} - q_{{\text{t}}} ) = \log q_{{\text{e}}} - \frac{k}{2.303}t\quad (2)\) |
Second order | \(\frac{1}{{q_{{\text{t}}} }} = \frac{1}{{kq_{{\text{e}}}^{2} }} + \frac{1}{{q_{{\text{e}}} }}t\quad (3)\) |
Pseudo-second order | \(\frac{t}{{q_{{\text{t}}} }} = \frac{1}{{kq_{{\text{e}}}^{2} }} + \frac{1}{{q_{{\text{e}}} }}t\quad (4)\) |
Half-life time | \(t_{1/2} = \frac{{{\text{In}}2}}{k}\quad (5)\) |
Other kinetic parameters such as rate constant (k) and equilibrium concentration (\(q_{{\text{e}}}\)) were derived from the equations above.
2.9 Thermodynamics studies of Cr (VI) bioreduction
Batch experiment was carried out under optimum conditions to determine the thermodynamic parameters for the reduction process such as free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) as described by Mekonnen et al. [23] with slight modification.
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1.
Gibbs free energy
Change in Gibb’s free energy (ΔG°) was determined using the equation:
$$\Delta G = - RT\;\ln \;K_{{\text{c}}}$$
(6)
where R is the gas constant (8.314 J mol−1 K−1), and T is the absolute temperature (K).
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2.
Equilibrium constant
The equilibrium constant Kc was evaluated at each temperature using the relationship:
$$K_{{\text{c}}} = \frac{{\text{Cr (III) }}}{{\text{Cr (VI)}}}$$
(7)
where Cr (VI) is the equilibrium concentration of Cr (VI) in solution in mg L−1 and Cr (III) is the amount of Cr (VI) reduced at equilibrium.
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3.
Enthalpy and entropy
The change in enthalpy (ΔH°) and change in entropy (ΔS°) were calculated, respectively, from the slope and intercept of van’t Hoff’s plot of lnKc against 1/T. The equilibrium constant Kc can be expressed in terms of the ΔH° (Kcal mol−1) and ΔS° (Kcal mol−1 K−1) as a function of temperature:
$${\text{In}}\;K_{{\text{c}}} = \frac{{\Delta S^{0} }}{R} - \frac{{\Delta H^{0} }}{RT}$$
(8)
2.10 Statistical analysis
All the experiments were carried out in triplicate and the results presented as mean ± SD.
