2.1 Materials
The raw material selected for starch extraction was green plantain (Musa paradisiaca) peel, obtained from the local food public market in Cartagena (Colombia). The original sample contained 5 kg plantain peels, which were processed immediately after peeling the fruits and separated in 150 g samples [6].
The reagents used to develop the starch chemical modification and water treatment experiments were: Sodium hypochlorite, Hydrochloric Acid, and Ascorbic Acid (AppliChem-Panreac®); Sodium Hydroxide (Merck®); Aluminum Sulfate and Acetic Anhydride (J.T. Baker®).
2.2 Native starch isolation method
The method used to extract the starch from green plantain peels with the best starch yield conditions was optimized and described in detail in a previous work of Hernández-Carmona, et al. [6], where it was evaluated how the extraction parameters, antioxidant concentration, and immersion time affect the starch production yield and purity. The method is a dry extraction process that starts with the crushing of the green plantain peel wastes in acidic solution (ascorbic acid, 5% w/v) with 5 min contact time, to promote the starch separation from the lignocellulosic fraction. The settled material is smashed to obtain a paste that is washed and filter before decanting the starch product. Finally, the starch is dehydrated at 40 °C during at least 10 h obtaining a dry starch powder.
2.3 Starch chemical modification
To chemically modify the starch, 40 g of native starch were weighed, adding 100 mL distilled water, and homogenizing the suspension with a magnetic stirrer platform in order to maintain a uniform suspension. The initial pH was adjusted to 8.0-8.5 adding a few drops of 3% NaOH, which acts as a catalyst. The acetic anhydride was slowly added (dropwise), simultaneously adjusting the pH between 8.0-8.5 until the required volume of acetic anhydride was added (volumes of 3.0 and 6.0 mL were used to obtain different degrees of substitution (DS)). Then the system was left to react for 10 minutes on the magnet stirring platform. Subsequently, the reaction was stopped by adding 0.5 N HCl solution, washing the starch 3 times with water and centrifuging the solution at 2500 rpm for 10 minutes. The last washing stage was performed with absolute alcohol and centrifuged at 2500 rpm for 10 minutes, and then dried in a tray oven at 40 °C for 12 hours [14, 15] .
2.4 Physicochemical characterization
The native starch and the modified starch were characterized to determine their main physicochemical properties including moisture and ash content, starch detection, purity degree, iodine test, Fourier Transform Infrared (FTIR) spectroscopy, gelatinization temperature, acetylation percentage (acetyl %), and degree of substitution (DS).
Moisture content in plantain peels and starch (native and acetylated) was determined with a MB 45 OHAUS moisture analyzer. Ashes content was measured by incineration at 550°C during 3.5 h (according to AOAC 2000 method). Both moisture and ash content were expressed as % of the sample.
The Lugol test was performed with Lugol iodine, also known as Lugol solution, which was used as a reagent for starch detection in routine laboratory. The Lugol solution was prepared with 5 g of I2 and 10 g of KI diluted with 100 mL distilled water, giving a brown solution with total iodine concentration of 150 mg mL-1 [5, 16].
The native starch purity was characterized to determine the purity degree using method AOAC 920.44, for starch determination in baking powders by means of acid hydrolysis [17] and method AOAC 906.03, for invert sugar determination in sugars and syrups by Munson-walker general method. The amylose-amylopectin ratio was then calculated using the colorimetric method described by Morrison and Laignelet [18–20].
Samples of native and acetylated starch were analyzed in a FTIR SHIMATZU 8400S spectrophotometer, using the KBr pellet method according to the ASTM-E168 and ASTM-E1252 standards, with the objective of obtaining information on the characteristic functional groups. Each spectrum was analyzed in the resolution range from 400 to 4000 cm-1 [21].
To determine the gelatinization temperature, 10 g of starch were dissolved in 100 mL distilled water and the suspension was heated to 85 °C. Then, 50 mL of the suspension were taken and introduced into an 85 °C water bath. The starch slurry was constantly stirred until a paste was formed and the temperature was stable for a few seconds. Finally, the gelatinization temperature was read with a thermocouple [22].
The acetyl % and the DS for both native and acetylated starch, were determined titrimetrically following the methods described in Sodhi et al., Bello-Perez et al., and Lee Phillips et al. [23, 24]. Initially, 1.0 g sample was placed in a 250 mL flask with 50 mL of distilled water, and the suspension was stirred for 60 min at 25 °C. Phenolphthalein was added as an indicator, the solution was neutralized with NaOH 0.45 N until equilibrium was reached and stirred for 30 min. Then the samples were titrated with HCl 0.2 N to the end point. A blank sample consisting of native starch was also used for comparison. The acetyl % was determined with Eq. (1).
Finally, the DS is defined as the average number of sites per glucose unit that possess a substituent group [25] and calculated with Eq. (2).
2.5 Coagulation-flocculation tests
To evaluate the coagulant behavior of the modified starch and the hybrid coagulation agent (Al2(SO4)3 + acetylated starch), we performed coagulation-flocculation tests. The raw water samples for the evaluation were taken from the Canal del Dique, a 118 km artificial water channel that connects Cartagena’s Bay to the Magdalena River (Bolivar Department, northern Colombia), since the Canal del Dique is the surface water used by the water company in Cartagena (serving a population over 1 million inhabitants), and several smaller towns and population settlements located along its course.
The turbidity value for the raw water was measured with a VELP® TB1 model turbidimeter, using ASTM method D7315-07a (Standard Test Method for Determination of Turbidity above 1 Turbidity Unit (TU) in Static Mode), with and average value of 385.9 NTU. The turbidity removal efficiency of the acetylated starch and the hybrid coagulant agent was calculated with the difference between the initial and final turbidities as a percentage respect to the initial turbidity. The pH values were also measured for each experiment.
The flocculator used the perform the coagulation-flocculation experiments was a 6-jar apparatus from VELP® Scientifica (Figure 1), following the procedure indicated in ASTM method D2035-13. Initially, 6 samples of 1 L the raw water were put into the coagulation flasks and rapidly stirred at 200 rpm. Then, the acetylated or the hybrid coagulant were added to each flask, and the samples were stirred for another 5 min at the same speed to ensure the coagulation process under rapid mixing conditions. After that, the samples were stirred at 45 rpm for the next 15 min to perform the flocculation or slow mixing step. Finally, the samples were allowed to settle for 20 min. After sedimentation, the supernatant liquid was collected and analyzed for pH and turbidity. The effect of the acetylated starch was investigated using 100 and 350 mg L-1 doses and two different DS values (Low and High), that correspond to the different acetyl % obtained. Also, the effect of the hybrid coagulant was investigated using a mixture of acetylated / Al2(SO4)3 coagulant at different ratios with two different DS. All experiments were performed at room temperature (25 ± 1 °C), and each experimental test was performed in four replicates [4, 26–28].
Experimental design was applied in order to investigate the effects of acetylated starch dose, the DS, and Al2(SO4)3 / acetylated starch relationships. Two different experimental design tests, with two levels and two factors each (2x2), were proposed and 4 independent replications were taken at each of the 2x2 treatment combinations. The first experimental design test was applied to the experiments with acetylated starch as the main coagulant, while the second one was applied to the hybrid coagulant Al2(SO4)3 / acetylated starch. The details of the first and second experimental designs are presented in Table 1 and Table 2, respectively. The design size was N=2×2×4=16 and was carried out randomly. The response variable for the two designs was the turbidity removal percentage [29].
Table 1 Experimental design test 1
Factor
|
Factor definition
|
Levels
|
Symbol
|
A
|
Acetylated starch dose
|
100 mg/L
|
Low
|
-
|
350 mg/L
|
High
|
+
|
B
|
Degree of Subs. (DS)
|
0.361
|
Low
|
-
|
0.562
|
High
|
+
|
Table 2 Experimental design test 2
Factor
|
Factor definition
|
Levels
|
Symbol
|
C
|
Wight ratio: Aluminum Sulfate/Acetylated starch
|
3:1
|
Low
|
-
|
1:1
|
High
|
+
|
D
|
Degree of Subs. (DS)
|
0.361
|
Low
|
-
|
0.562
|
High
|
+
|
2.6 Statistical Analysis
A statistical significance analysis was performed to test the hypothesis for the means difference. In order to demonstrate whether there was a significant difference in the means of the response values (turbidity) of the experiment, they were divided into two groups characterized by the proportion of acetylated starch as an adjuvant, then a test of difference of means (two-sample t-Test) was accomplished [30].
Based on the data obtained, an analysis of variance (ANOVA) was performed to determine which factors have the greatest influence on the final turbidity degrees. The results were later analyzed with PAST v3.14® and STATGRAPHICS CENTURION XVI ® software for statistical evaluation. A two-way ANOVA test was performed, to evaluate whether the chemical acetylation, the DS, and Al2(SO4)3 / acetylated starch mixtures relationships affect the turbidity in raw water, and if there are interactions between them [31].