## Abstract

The main objective of this study was to investigate the effectiveness of protein extraction from mature-dried Moringa oleifera seeds by three methods: simple pulverization, oil-extraction, and protein-fractionation. Using an electrochemically activated solution as a solvent of the protein extraction process was found to be an extremely effective method due to its significant performance, environmental qualities, chemicals reduction usage and low cost. The chemical composition of protein-fractionation (MO3) indicated that the percent of protein was high at 84% ±0.5% while the percentage of oil was low at 0.5% ±0.1%. This leads to increased efficiency in clarifying low turbidity water. In addition, the new bio-coagulant tackled the limitation of previous research, namely the loading of organic compounds into the water after the coagulation process by Moringa oleifera seeds. Three substances extracted from Moringa oleifera seeds can be used as an alternative to the commercial coagulant polyaluminum chloride (PAC) for water treatment in both lake water and municipal wastewater. The results showed that MO3 showed the highest efficiency in removing turbidity, chemical oxygen demand (COD), and Escherichia coli from lake water at 99.3%, 98.46%, and 100%, respectively, compared with results from municipal wastewater at 95.44%, 82.4%, and 99.1%, respectively.

## INTRODUCTION

Recently, coagulation and flocculation processes have been widely used for treating water. Common coagulants such as aluminum sulfate and iron salt are usually expensive and affect human health. Many studies have been reported about the relationship between Alzheimer's disease and redundant aluminum (Grabow et al. 1985; Ndabigengesere & Narasiah 1998). As recommended by the World Health Organization (Galal-Gorchev et al. 1998), the amount of aluminum in drinking water should not exceed 0.2 mg/L, whereas the level of aluminum in standard drinking water in Vietnam is 2 mg/L (QCVN 01:2009/BYT). Moreover, using chemical coagulants leads to a change of pH (alkaline in water after treatment), to the formation of non-biodegradable sludge and to the incomplete removal of toxic compounds in the water (Ndabigengesere & Narasiah 1998; Valverde et al. 2014). Another disadvantage is the high water-treatment cost for poor and developing countries making provision of a clean water supply and water treatment difficult. Therefore, natural coagulants are offering new possibilities in the quest for sustainable water treatment technologies (Jung et al. 2018).

Moringa oleifera is a tropical plant belonging to the family of Moringaceae. Its seeds have been shown to be one of the most effective main bio-coagulants for water treatment, including the turbidity of surface water, alkalis, organic contaminants through biological oxygen demand (BOD5), chemical oxygen demand (COD) in municipal wastewater, and industrial wastewater including textiles (Vilaseca et al. 2014), coffee fermentation (Garde et al. 2017), pharmaceutical wastewater (Eri et al. 2018), microalgae (Barrado-Moreno et al. 2016; Camacho et al. 2017), protozoa (Petersen et al. 2016), total coliform (Nguyen 2016) and Escherichia coli (Dasgupta et al. 2016). Many studies have described the predominant mechanisms for toxic reduction utilizing Moringa oleifera for adsorption, charge neutralization, and destabilizing particles between the cationic proteins of Moringa oleifera and the colloid of water (Jung et al. 2018; Villaseñor-Basulto et al. 2018). The bioactive agents of Moringa oleifera seeds have been determined to be cationic proteins with low molecular weight ranging from 6–16 kDa with isoelectric point above pI 10 (Ndabigengesere et al. 1995). Amino acids in Moringa oleifera seed include glutamine, arginine and proline, with a total of 60 other residues. The protein peptide assists eight positively charged amino acids (7 arginines and 1 histidine) and 15 glutamine residues. Baptista et al. (2017) extracted the Moringa oleifera proteins and determined that globulin presented the best performance, promoting high turbidity and color reduction efficiency in low turbidity waters. Also, it is able to remove microorganisms due to the consistency of the antioxidant and antimicrobial. Moringa oleifera seed extracts have been observed against S. aureus, E. coli and V. cholerae in the shrimp pond water (Viera et al. 2010). In addition, biofilms of microorganisms of clinical interest were removed by Moringa oleifera seeds, such as S. aureus and P. aeruginosa and the yeast C. albicans. The bio-compounds most likely involved in this activity are saponins, tannins, isothiocyanates and phenolic compounds, such as alkaloids, particularly flavonoids, which exist in high concentrations in the seeds (Brilhante et al. 2017).

In the past, researchers such as Bichi (2013), Petersen et al. (2016), and Nguyen (2016) only studied crushed Moringa oleifera seeds after removing the seed coat and wings, and grinding to clarify synthetic water from turbidity. However, these substances had effective results in high and medium turbidity (≤200 NTU). Thus, development in methods of derivative extraction was investigated. Distilled water, salts and solvents were used to extract the active compound of Moringa oleifera before treating the pollutants in wastewater. Sodium chloride solution was used to extract protein from the Moringa oleifera seeds (Okuda et al. 1999). This substance was found to have 7.4 times higher coagulation ability than that using distilled water due to the increase in protein dissociations, and protein solubility for coagulation. Madrona et al. (2010) described that the reduction in the color and turbidity of aqueous water by Moringa oleifera extract prepared by using potassium chloride (KCl) solution was more efficient than with distilled water. Most of the methods of extraction for Moringa oleifera seeds were the oil-extract by solvent extraction such as hexane (Muyibi et al. 2010; Garcia-Fayos et al. 2016), ethanol (Garcia-Fayos et al. 2016; Camacho et al. 2017) and acetone (Garcia-Fayos et al. 2016). On the other hand, the organic, nitrate, and phosphate concentrations in water have been illustrated to grow in the case of powder with either water or salt extracted. Therefore, to solve this limitation of Moringa oleifera extract, the purification methods, for instance dialysis, precipitation, delipidation, centrifugation, ion exchange, and lyophilization, have been described. This will improve the processing costs and difficulty for large-scale practical use. Thus, the challenge in the coagulation process is production of new coagulants with high efficiency, eco-friendliness, and biogradated sludge formation to achieve sustainable development.

In the past decade, electrochemically activated solutions (ECAS) have been applied widely in food production industry, biotechnology, healthcare settings and drinking water treatment. Aider et al. (2012) concluded that ECAS played a role as self-formation of acidic (anolyte) or base conditions (catholyte), selected protein and fiber extraction, preventing the development of microorganisms on the food or bio-material. According to Cao et al. (2017), ECAS including catholyte and anolyte were used for gelatin extraction from fish skins. Using ECAS is an eco-friendly and effective method. As mentioned in this research, the protein extraction of Moringa oleifera seeds has been improving this utilization of ECAS as extracted solvent after the oil-extraction step by n-hexane. Protein of Moringa oleifera seeds was approximately dissolved into ECAS then precipitated and dried. This extracted product is rarely biodegraded by microorganisms due to the antioxidant from ECAS.

Polyaluminium chloride (PAC) has become popular in the coagulation–flocculation process due to its many advantages over metallic salts (aluminum and iron salts). Many researchers found that PAC had better efficiency at low temperature (Yu et al. 2007), fewer aluminum residuals, less sludge formation, a wide pH range (5.5–8) of raw water for activity, and more rapid flocculation (Wei et al. 2015). Recently, most studies on the coagulation mechanisms of PAC conclude towards the combination of the adsorption–charge neutralization process, such as electrostatic patch, sweep coagulation and bridge aggregation, due to the high positive charges of Al13 [AlO4Al12(OH)24(H2O)127+] and Al30 [Al30O8(OH)56(H2O)2418+] (Popa et al. 2010; Lin et al. 2014). Gregory & Duan (2001) have investigated the effectiveness of different pre-hydrolysing coagulants for wastewater treatment and concluded that PAC products give more rapid flocculation and stronger flocs than alum at optimal dosage. Yarahmadi et al. (2009) reported the comparison of the effect of the Moringa oleifera seeds extraction by different concentrations of sodium chloride with the traditional coagulant PAC in the purification of turbid water from 10 to 1,000 NTU. These authors showed that Moringa oleifera seeds were more efficient than PAC in treating high turbidity water. In contrast, PAC had a better result for purification in low turbidity, whereas the defatted presscake from Moringa oleifera seeds was found to be more effective than the commercial coagulation agents PAC to treat black dye 19 in pH 5–8 at any temperature (Tie et al. 2015). Thus, it is necessary to investigate the use of Moringa oleifera seeds extract and the difference between Moringa oleifera seed and the commercial coagulant PAC to treat surface waters and wastewaters.

The use of a bio-coagulant based on plants such as Moringa oleifera seeds would assist sustainable, economic environmental practices by presenting great benefits such as biodegradability, low index of residual sludge formation and non-toxicity, and a non-corrosive nature, and harnessing them for water and wastewater treatment would help reduce chemical dependency (Oladoja 2014). The objectives of this study were: (1) to analyse the influence of extracted forms Moringa oleifera as a coagulant, and to characterize the Moringa oleifera extract powder by its chemical constituents; (2) to compare the effect on turbidity, COD and E. coli removal in lake water and municipal wastewater between the Moringa oleifera extract and the conventional coagulant PAC; and (3) to suggest the optimum dosage of coagulants for treatment of water.

## MATERIAL AND METHODS

### Chemicals

The following chemicals were used in, whereas this study. n-hexane, potassium dichromate (K2CrO7), silver sulfate (Ag2SO4), sulfuric acid (H2SO4), ammonium iron(II) sulfate (NH4)2Fe(SO4)2.6H2O, 1,10-phenanthroline and mTEC agar were purchased from Merck, Germany with a purity of 99%. Polyaluminium chloride (PAC), named PAC-HB or Alw(OH)x(Cl)y­(SO4)z, was a light-yellow powder with the following characteristics: 30% Al2O3, basicity at 71.3%, pH 4.14 (10 g/solution) and water insoluble at 0.2%. PAC-HB was obtained from the Viet Tri Chemical Joint-Stock Company, Vietnam.

### Extraction of Moringa oleifera seeds

Moringa oleifera seeds were harvested from Binh Thuan province, Vietnam. Seeds were chosen that were not decayed, old, or infected with seed pests. These seeds were dried in the oven (Blinder, FD 115, USA) for 24 h at 40 °C. After that, the seeds were crushed into powder using a blender (Sunhouse, SHD5580, Vietnam). This powder was known as MO1.

Continuously, the MO1 was defatted by extracting with n-hexane. The extraction process was carried out 3 times with an n-hexane/powder ratio of 3:1 (v/w) at 45 °C in 15 minutes under ultrasound by the ultrasonic cleaner (Sharpertek, SH80-2 L, USA). The solid was collected by filter papers (Whatman, 4, UK) and then dried at 50 °C (Blinder, FD 115, USA) (MO2).

The defatted flour was suspended in an electrochemically activated solution which was produced using an electroactivator (Aquapribor, AP1, Russia) with pH 9.0 in a relation 1/10 (w/v). The suspension was stirred by magnetic stirrer (IKA, RT5P, China) for 1 hour at room temperature and then centrifuged (Hettich, Eba 200, Germany) at 9,000 rpm for 20 min at 10 °C. The precipitate was freeze-dried at room temperature about 20–25 °C (MO3). All three materials were stored in a desiccator (Boeco, GLA 415262302, Germany).

### The coagulant stock

#### Moringa oleifera extract powder stock

One gram of Moringa oleifera powder extract was dissolved in 1,000 mL of distilled water to obtain the stock coagulant. The mixture was blended using a magnetic stirrer (IKA, RT5P, China) for 30 min at high speed to extract the active proteins of Moringa oleifera. The suspension was then filtered through a filter paper (Whatman, 41, UK) with a pore size 20–25 μm in a beaker to get a stock solution of 1 g/L.

#### Preparation of the PAC stock

The PAC powder (PAC-HB, Viet Tri, Vietnam) was dissolved into deionized water to prepare a concentrated stock solution (2 mol Al/L). To enhance reproducibility and avoid degradation of the solution, a fresh coagulant solution was prepared from the stock solution the day before each set of experiments.

### Water samples

The lake water samples were taken at Van Quan Lake, Ha Dong district, Ha Noi, and the municipal wastewater was collected from To Lich River which is the main artificial drain of domestic wastewater in Ha Noi. Tables 1 and 2 show the quality of lake water and river water, respectively. All samples were stored in the pharmaceutical refrigerator (Panasonic MPR-S313, Japan) at 4 °C within 24 hours.

Table 1

Characteristic of the water samples

Parameter Unit Lake water samples Municipal wastewater samples QCVN 08 − 2015/BTNMT B1
t° °C 28.5 ± 0.02 29 ± 0.02 –
pH  7.64 ± 0.06 7.1 ± 0.05 5.5–9
DO mg/L 2.2 ± 0.1 1.2 ± 0.1 ≥4
Turbidity NTU 44 ± 1 81.2 ± 2.5 –
TSS mg/L 53.24 ± 0.5 103.24 ± 0.5 100
BOD5 mg/L 60.55 ± 0.3 111.5 ± 0.2 15
COD mg/L 93.77 ± 0.25 159.3 ± 0.7 30
NH4+ mg/L 1.3 ± 0.05 19.3 ± 0.05 0.9
NO2 mg/L 0.37 ± 0.3 9.37 ± 0.2 0.05
NO3 mg/L 1.2 ± 0.1 27.2 ± 0.1 10
E. coli CFU/mL 118 ± 1.5 535 ± 0.6 50
Total coliform MPN/mL 9,700 <10,000 7,500
Parameter Unit Lake water samples Municipal wastewater samples QCVN 08 − 2015/BTNMT B1
t° °C 28.5 ± 0.02 29 ± 0.02 –
pH  7.64 ± 0.06 7.1 ± 0.05 5.5–9
DO mg/L 2.2 ± 0.1 1.2 ± 0.1 ≥4
Turbidity NTU 44 ± 1 81.2 ± 2.5 –
TSS mg/L 53.24 ± 0.5 103.24 ± 0.5 100
BOD5 mg/L 60.55 ± 0.3 111.5 ± 0.2 15
COD mg/L 93.77 ± 0.25 159.3 ± 0.7 30
NH4+ mg/L 1.3 ± 0.05 19.3 ± 0.05 0.9
NO2 mg/L 0.37 ± 0.3 9.37 ± 0.2 0.05
NO3 mg/L 1.2 ± 0.1 27.2 ± 0.1 10
E. coli CFU/mL 118 ± 1.5 535 ± 0.6 50
Total coliform MPN/mL 9,700 <10,000 7,500

Sample location and description: Lake water Van Quan Lake, Ha Dong province, Ha Noi city, 19–23 February 2018, green water and smell; Municipal water – To Lich River, Dong Da province, Ha Noi city, 19–23 February 2018, high turbidity water and foul smell.

Table 2

Chemical composition of protein Moringa oleifera seed extract

Moisture (%) Protein (%) Lipid (%) Mineral (%) Carbohydrate (%)
MO1 7.2 ± 0.3 33.4 ± 0.2 34.5 ± 0.4 6.4 ± 0.7 18.5 ± 0.3
MO2 6.8 ± 0.3 46.2 ± 0.3 1.5 ± 0.1 16.4 ± 0.4 29.1 ± 0.2
MO3 8.8 ± 0.2 84.0 ± 0.5 0.5 ± 0.1 1.5 ± 0.3 5.2 ± 0.1
Moisture (%) Protein (%) Lipid (%) Mineral (%) Carbohydrate (%)
MO1 7.2 ± 0.3 33.4 ± 0.2 34.5 ± 0.4 6.4 ± 0.7 18.5 ± 0.3
MO2 6.8 ± 0.3 46.2 ± 0.3 1.5 ± 0.1 16.4 ± 0.4 29.1 ± 0.2
MO3 8.8 ± 0.2 84.0 ± 0.5 0.5 ± 0.1 1.5 ± 0.3 5.2 ± 0.1

Notes: MO1 - simple powder of Moringa oleifera seeds; MO2 - fat-extracted Moringa oleifera seeds; MO3 - fractionation protein of Moringa oleifera seeds.

### Experimental design

The design of the Jar Test experiment was based on Nguyen (2016) with a coagulant dosage of 0–60 mg/L for the lake water. The municipal wastewater used 0–120 mg/L of coagulants. The coagulation process was carried out using the Jar Test (VELP, JLT6, Italia) and involved a rapid mixing of the mixture coagulants into the water, slow mixing, and a sedimentation stage in a batch process. In order to reproduce the coagulation and ﬂocculation stage in the water treatment, in each round of the experiment, 1 L water sample (lake water or municipal wastewater) was poured into the beaker and stirred rapidly for 4 minutes with Moringa oleifera seed extracts predetermined concentration then stirred for 20 minutes (the rotational speed of the stirring paddle for fast and slow stirring was 100 and 40 rpm, respectively). Next, the suspensions were left to stand for 30 minutes. After the coagulation process, pH, turbidity, COD, and E. coli were identified in the lake water and the municipal wastewater. These parameters represent pollution of particulates, organic matter, and microorganisms. All experiments took place at room temperature (25 °C ±0.5). In Vietnam, a tropical country, the temperature of surface waters and domestic wastewaters is relatively stable. Thus, temperature is not a significant factor in the optimal dosage of coagulants. The initial pH of the sample did not change during the coagulation process because the pH of both samples was neutral at around 7. These parameters were in the optimal range for coagulation process by Moringa oleifera extract powders and PAC.

### Analytical methods

Turbidity was measured by a portable turbidimeter (Hach 2100Q, USA), pH values of samples were tested by a pH meter (Sl Analytics, Lab 850, Germany).

E. coli were analyzed by the membrane filtration method following method 1603 of United States Environmental Protection Agency (USEPA 2014). The result provides a direct count of E. coli in water or wastewater based on the growth of colonies on the surface of a membrane filter. A sample is filtered through the membrane, so the bacteria is retained on the surface membrane. After that, the membrane is placed on a selective and differential culture, modified mTEC agar, then incubated at 44.5 ± 0.2 °C for 22 ± 2 hours (Incucell 111, MMM Medcenter Einrichtungen GmbH, Germany). The results are that the number of colonies on the modified mTEC agar is red or magenta in color after the incubation period.

COD was calculated by the open reflux method SMEWW 5220.B:2012 using a COD reactor, (Hach, DRB 200, USA). Potassium dichromate (K2Cr2O7) is used as a strong oxidant to degraded matters. After digestion, the remaining unreduced dichromate K2Cr2O7 is titrated with ferrous ammonium sulfate ((NH4)2Fe(SO4)2.6H2O) to find the amount of K2Cr2O7 consumed and the oxidizable compounds are calculated in terms of oxygen equivalent. Some samples with very low COD or with highly miscellaneous solids content may need to be analyzed in replicate to yield the most positive data. Results are further enhanced by reacting a maximum quantity of dichromate, showing that some residual dichromate remains.

The turbidity, COD, and E. coli removal efficiency (H%) were calculated using Equation (1) as follows:
(1)
where C0 and C are the initial and final concentration of turbidity (NTU), COD (mg/L) and E. coli (CFU/mL) in water, respectively.

The chemical composition of Moringa oleifera extract seed powders (moisture, ash, fat and protein contents) was determined according to standard methods 934.01, 942.05, 991.36 and 954.01, respectively (AOAC 2000).

### Statistical analysis

All the experiments were conducted in triplicate to ensure reproducibility of results. The final results were the average of the three. All statistical analyses of the data were performed using a Student's paired t-test. Statistical significance was defined as p < 0.05, analysed using Microsoft Excel 2010.

## RESULTS AND DISCUSSION

### Chemical composition of protein Moringa oleifera extract

Table 2 displays the chemical composition of three materials from Moringa oleifera extract. The results illustrated that the extraction methods of Moringa oleifera seeds was as expected: Moringa oleifera integral powder (MO1) shows a higher oil content than Moringa oleifera seed after fatty extractions (MO2) and protein fractions (MO3). They were reduced by 95.65% and 98.55% for MO2 and MO3, respectively. The oil-free component was the important step in the Moringa oleifera extraction because it increases organic pollutants by the addition of fatty acid into water. Moreover, it is a valuable product that can be collected and reused (Nordmark et al. 2016). Table 2 also demonstrates that the protein fractionation MO3 had the best protein value, around 84%. The percentage of extracted protein was much higher than that extracted by salt, or ethanol (Madrona et al. 2012; Camacho et al. 2017). The values were 32% and 42.9%, respectively. The MO3 samples were mostly protein (on dry weight) and the low lipid, ash and carbohydrate content showed the effect of extraction by electrochemical activated water (catholyte with pH 9.0). Generally, alkaline or acid solution were used in protein extraction. Proteins of Moringa oleifera seeds were observed as a natural cationic polyelectrolyte and display the main characteristics for coagulation capability. They connected with suspended particles in the water by charge neutralization to form the floc, which was removed easily from water in the sedimentation process (Baptista et al. 2017; Villaseñor-Basulto et al. 2018).

### Quality of sample water

Quality of the lake water (Van Quan) and municipal wastewater (To Lich River) were determined and are shown in Table 1. Comparing Vietnam's standard value for surface water quality class B1 for irrigation (QCVN 08: 2015/BTNMT, 2015), the pH value of both water samples standardized at neutral pH. In general, wastewater from To Lich River is neutral because a major source is wastewater from the daily activities of the city's residential area, and industrial wastewater or production wastewater only account for a small part. The pH of Van Quan lake water is slightly basic because of development of algae. DO values for the water samples were low at about 2.2 and 1 mg/L for the lake water and municipal wastewater, respectively. They did not meet the conditions of the habitat for aquatic plants and animals. Both water samples were seriously polluted with high BOD5 and COD levels. BOD5 levels were as high as 65.55 to 111.5 mg/L for the lake water and municipal wastewater, respectively. The COD value for the To Lich River exceeds 5 times that for class B1 of Vietnam's standard of surface water quality, whereas the COD of the lake was, at 93.77 mg/L, more than three times the COD value in QCVN 08:2015/BTNMT (collum B1: 30 mg/L). The main component of To Lich River and Van Quan lake is wastewater, so the concentration of nutrients in the river water is often very high. The results of analysis for NH4+, NO2, NO3 of To Lich River were approximately 19.3, 9.37, 27.2 mg/L, many times higher than the prescribed standard in QCVN 08:2015/BTNMT column B1 (0.9, 0.05, 10 mg/L, respectively). For the Van Quan lake, these results were lower than those for To Lich river, however, they were still higher than the standard of Vietnam's surface water quality. Microbial pathogens in both water samples were determined by E. coli and total coliform parameters. Both water samples had values higher than the value defined in the Standard and exceeded it by many times. Although the turbidity of water samples was not modified in Standard QCVN 08:2015/BTNMT, we can conclude that the two water samples were polluted with particles using the total suspended solids (TSS) parameter. TSS parameter in both water samples exceeded the Standard (class B1 at 50 mg/L). In summary, both water samples were polluted with regard to turbidity, organic contaminants, and microorganisms.

Coagulation is utilized as a primary process of surface water and wastewater treatment in the control of particulates, microorganisms, and natural and synthetic organic matter. Thus, in this research, we chose three parameters: turbidity, COD, and E. coli, to investigate the efficiency of the proposed coagulation process. They not only represent types of pollution for surface water and municipal wastewater but also were easily analyzed.

### Treatment of lake water with different Moringa oleifera extract powders

Figures 1 and 2 show different parameters (pH, the turbidity, COD and E. coli) analyzed from the lake water after the coagulation process by simply crushed Moringa oleifera seeds (MO1), oil-extraction Moringa oleifera seeds (MO2), fraction protein of Moringa oleifera seeds (MO3) and PAC.

Figure 1

(a) pH residual and (b) turbidity removal (%) in lake water with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

Figure 1

(a) pH residual and (b) turbidity removal (%) in lake water with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

Figure 2

(a) COD removal and (b) E. coli removal in lake water with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

Figure 2

(a) COD removal and (b) E. coli removal in lake water with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

It can be seen from Figure 1(a) that three substances did not significantly affect the pH value, which remained almost constant at around 7.64 for all dosages tested. In contrast, the pH value decreased from 7.64 to 5.51 for PAC. Coagulant PAC contains aluminum in the form of a polymer, and also forms aluminum hydroxide in contact with water, becoming cationic through the reaction with the alkalinity of water. Increased acidity may happen due to the aluminum trivalent cation that acts as a Lewis acid and accepts ion-electron pairs. Formation of the H+ ions to reduce the pH of the water followed Equation (2). This was similar to previous research on the coagulation process using PAC (Yan et al. 2008; Hendrawati et al. 2016),
(2)
In contrast, substances produced from Moringa oleifera seeds are composed of the organic molecules produced and ready for application in the cationic form. The absence of chemical reaction between these cationic proteins and the alkalinity in water is also the reason why the three substances do not decrease the pH of the water under treatment. The steady pH of Moringa oleifera extracted powder is an advantage as a replacement for the traditional coagulant PAC in water treatment, due to the reduction in the amount and cost of chemical agents required to adjust the pH for the next water treatment process (Camacho et al. 2017).

The pH and alkalinity strongly influence the coagulation ability of inorganic coagulants like aluminum sulfate, iron sulfate and PAC. Each coagulant has a different optimal pH range, for example 3–9 for iron salts coagulant, and 5.5–8 for aluminum-based coagulants (Yan et al. 2008).

#### Effect of pH on aluminum hydroxide solubility (PAC)

pH effects aluminum hydroxide solubility as it is a typical amphoteric hydroxide. The pH of the water can be too basic or too acidic to make it dissolve, resulting in excess aluminum content in the water. pH also influences aluminum hydroxide particles. The charge of the colloidal particles in the water is related to the ion composition of the water, in particular the H+ ion.

#### Influence of pH on organic matter in water (humic acid)

When the pH is neutral, the gel solution of humic acid is negatively charged so is easily degraded. PAC coagulant does not execute well for organic compound removal when pH <5.5. This can be explained by the fact that when the solution condition is acidic, the carboxyl groups of organic compounds are hardly hydrolyzed because of the high concentration of H+; furthermore, H+ could react with organic molecules which compete with the complex reaction between aluminum pieces and organic matter. At higher pH, humic acid becomes completely soluble and is difficult to remove efficiently. In addition, formation of colloids are influenced by the pH value of water with regard to potential and the isoelectric point. If the alkalinity of the water is too low, it will not be sufficient to remove the acid produced by the hydrolysis of coagulants. As a result, the pH value of the water after the coagulation process decreases rapidly. Then the alkali is used to adjust the pH of the water and thus the effect of alkalinity to the coagulation is relative to the pH value.

According to Ndabigengesere & Narasiah (1996), the effect of pH was not significant to the coagulation process using Moringa oleifera extracted seeds. This is because the main protein of Moringa oleifera has a positive charge with an isoelectric value of around 10. This means the protein remains positively charged as long as the pH is below 10. On the contrary, colloids in water are mostly negatively charged. Thus, the reactions between coagulant and colloids happen and are then removed from water in the settling process.

This study did not investigate the effect of pH on the coagulation process extensively (these results were not displayed). We agree that the optimum pH for PAC coagulant is 5.5–8 and for Moringa oleifera extracted powders pH is not a significant factor for coagulation capability. Based on the analysis, the optimum pH obtained using Moringa oleifera, a bio-coagulant, is pH 6–8. At this point, amino acids are ionized to form a carboxylate ion and proton, and directly attract electrons (colloids) to form neutral groups and produce floc so as to remove the contaminants from the water (Ndabigengesere & Narasiah 1996).

From Figure 1(b) it can be seen that coagulants extracted from Moringa oleifera significantly change the highest turbidity of the lake water at 59.89%, 87.04% and 99.32% for optimal dosages of coagulation agents of 60 mg/L, 50 mg/L and 40 mg/L, respectively. In comparison, the turbidity decreased considerably at 91.67% of the optimum PAC dose of 50 mg Al/L. Thus, the coagulation ability of Moringa oleifera seeds depends on the quality of the cationic protein and the oil in the seed. While the cationic protein has the role of crosslink to colloids of water, as a means of neutralizing their negative charge and weakening the electrostatic double layer rejection such that van der Waals forces reduce turbidity, the oil of the Moringa oleifera seed was put into the water to prevent connection between the coagulant and flocs. It is more likely that the cationic proteins adsorb the kaolin particles.

Compared with previous studies which treated surface water with low turbidity, this MO3 powder coagulated more effectively. Vo et al. (2012) illustrated removal of only 50% of river water where turbidity was 44 NTU. Or Baptista et al. (2017) reported on the fractionation protein by n-hexane 1% and 5% Moringa oleifera seeds to reduce turbidity by 89% in the low turbid water (<50–100 NTU).

The COD of the influence of Van Quan lake was 93.77 mg/L. After treatment, the following results were obtained: 59.37, 42.75, 1.44 and 49.7 mg/L for the optimum dosage of MO1, MO2, MO3, and PAC, respectively, as shown in Figure 2(a). Most results were less than 30 mg/L which was standardized at class B1 (water quality for navigation purposes) of the National Technical Regulation on Surface Water Quality (QCVN 08-MT:2015/BTNMT). Treated water can be used for water supply purpose (<10 mg/L) after utilizing MO3 coagulant.

Many authors have reported on the mechanism of removing COD by the coagulated ion process. First of all, COD removal was combined with the decrease of turbidity in water. Following on from this, adsorption was one of the main mechanisms for the coagulation process by Moringa oleifera seeds extract. Real-Olvera et al. (2015) found that the interaction between protein/amino acids and ions consisting of organic compounds from slaughter wastewater fit the second-order kinetics, while Sivakumar (2013) reported that the adsorption of COD in leachate wastewater followed the Elovich kinetic models when using crushed Morinaga oleifera seeds.

The concentrations of residual E. coli are illustrated in Figure 2(b). It was clear that using the Moringa oleifera extracted powder was better than utilizing PAC for removing E. coli. Simple crushed Moringa oleifera seed reduced E. coli to 30 CFU/mL while PAC only removed it to 42 CFU/mL after water treatment process. In addition, both Moringa oleifera extracted powder by fatty extraction and fully protein fraction removed 100% of E. coli. The reasons for this are as follows. Firstly, coagulation processes can remove microorganisms through the coagulation and precipitation process associated with turbidity and suspended solids in water. Secondly, according to Shebek et al. (2015), this Moringa oleifera cationic protein can destroy the inner and outer membranes of E. coli cells by antioxidant compound, αL-rhamnosyloxy-benzyl isothiocyanate (Eilert et al. 1981). Therefore, coagulants of Moringa oleifera play an important role in water disinfection (Bichi 2013). Madsen et al. (1987) observed 80–99.5% turbidity reduction and 90–99.99% bacterial removal during 1–2 hours of treatment with Moringa. oleifera seed extracted in treating Sudanese water. Ghebremichael et al. (2005) investigated the coagulation and antimicrobial ability of the purified protein for surface water treatment (the River Meuse and Delft Canal waters). The results were up to 97% and 86% turbidity removal for high turbid and low turbid waters, respectively, and E. coli removed completely. Poumaye et al. (2012) tested Moringa oleifera dried powder for purifying the river M'Poko surface water. Streptococci, Clostridium, and E. coli were reduced by 62%, 95%, and 47%, respectively. Dasgupta et al. (2016) illustrated detailed antimicrobial agents of the seed extract of Moringa oleifera to remove a maximum of 93.2% for E. coli and 96.2% for Bacillus subtilis. Nguyen (2016) also found that it is possible to remove the whole of the coliform bacteria from Hong River, belonging to an Escherichia coli family, by using the simple crushed Moringa oleifera seeds.

### Treatment of municipal wastewater by different Moringa oleifera extract powders

Figures 3 and 4 illustrate the efficiency in removing pH (Figure 3(a)), the turbidity (Figure 3(b)), organic pollutants through parameter COD (Figure 4(a)) and E. coli (Figure 4(b)) as a function of coagulant concentrations of 20, 40, 60, 80, 100 and 120 mg/L for four coagulants.

Figure 3

(a) pH residual and (b) turbidity removal in municipal wastewater with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

Figure 3

(a) pH residual and (b) turbidity removal in municipal wastewater with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

Figure 4

(a) COD removal and (b) E. coli removal in municipal wastewater with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

Figure 4

(a) COD removal and (b) E. coli removal in municipal wastewater with Moringa oleifera seed extract flours and PAC and optimum coagulant dosages.

As with lake water, protein fractionation of Moringa oleifera seeds had the best results in treating municipal wastewater for turbidity, COD, and E. coli. These results were 95.44%, 82.4% and 99.1%, respectively, at the optimum dosage of 80 mg/L (except for E. coli removal at 120 mg/L) of coagulant MO3. In fact, Hendrawati et al. (2016) described how when Moringa oleifera extract exceeded the optimum dosage, turbidity and COD reduced because all colloids in water had been neutralized, then formed bigger particles. Therefore, the overwhelming coagulant dosage caused the increase of turbidity and COD in water as shown in Figures 3(b) and 4(a) as they did not react with oppositely charged colloidal particles. Based on this, treated water may discharge to a water resource used for supplied water for navigation without a secondary treatment process (QCVN 14:2015/BTNMT class B2).

The lowest effect for treating domestic wastewater was the Moringa oleifera integral powder. According to Figure 3(b), the turbidity removal efficiency increased with the rise of the dosage of Moringa oleifera seed simple extracted powder by around 62.65% (turbidity decreased from 81.67 NTU to 30.48 NTU). Compared with MO3 (where turbidity in water after the treatment process was 3.72 NTU), the turbidity in wastewater after the coagulation process using MO1 was more than 26 NTU. Therefore, the purified protein in Moringa oleifera seeds extraction is affected by coagulation availability, whereas the reduction of COD in the wastewater is shown in Figure 4(a). The COD removal reached its maximum of 62.72% for a MO1 dosage of 100 mg/L. At the same with an optimal dosage of coagulant MO1 at 100 mg/L, residual E. coli were reduced by about 81.25%. Furthermore, the safety of utilizing Moringa oleifera in water treatment also received some attention. Muyibi & Evison (1995) recommended that further investigations need to be carried out to guarantee the complete safety of utilizing Moringa oleifera in water treatment and ensure there is no toxic effect on human health. In addition, it was also found to possess an antimicrobial activity and was able to clear water of several waterborne human pathogens. Compared with PAC, a conventional coagulant, the effect of treating municipal wastewater was lower than both MO2 and MO3. The results were only shown with 92% for turbidity removal, 42.16% for COD removal and 71.25% for E. coli reduction. Also, the optimum dosage of PAC was more than fractionation protein MO3 and equal to the optimal of MO2 coagulants at 100 mg Al/L. Thus, Moringa oleifera seeds from the oil extract and the fractionated protein by ECAS solution were extremely efficient for clarifying turbidity, degradation of COD, infected microorganisms and stable pH of water, whereas the decrease in pH reduced the efficiency of the coagulant PAC in the municipal wastewater treatment process (Yarahmadi et al. 2009). Kane et al. (2016) discussed the coagulating and antibacterial effects of the coagulant Moringa oleifera extract to remove pollutants from domestic wastewaters. The results showed that the combination usage between alum and Moringa oleifera protein molecules powder as coagulant within a ratio of 50:50 (w/w) provided a more than 90% decrease of turbidity and 75% COD removal. Adeniran et al. (2017) used completely randomized design (CRD) experimental design to investigate the potential of a new bio-coagulant protein purified from Moringa oleifera to treat the municipal wastewater. The turbidity value was increased drastically after treating. COD reduced from 81.6 mg/L to 72 mg/L after the coagulation process. Generally, the above results showed that the higher the quantity of three Moringa oleifera seeds extracted as applied to sewage, the better the purification of the sewage.

### Cost of protein extracted from Moringa oleifera seeds and sustainable development

Moringa oleifera is a common plant in the middle-south of Vietnam. This plant is reported as a vegetable with high nutritional value and medicinal properties. Each tree can produce around 15,000 to 25,000 seeds/year with a weight of 0.3 g/seed. The kernel-to-hull ratio is 75:257 (Ganatra et al. 2012). Thus, it helps to reduce the cost for producing bio-coagulants process. According to cost estimation, the cost of producing the bio-coagulant from Moringa oleifera seeds includes: the cost of raw materials, chemical compounds (n-hexane), the electrochemically activated solution, energy consumption, labor costs and cost for storage to keep the substance. In this case, we can save raw material and substance storage prices. The reason is that Moringa oleifera seeds are a solid waste from cultivating Moringa oleifera trees. Recently, the planted area of Moringa oleifera has been expanding rapidly throughout Vietnam. Thus, it is the greatest resource for producing the bio-coagulant. Another reason is that using ECAS promotes the bio-coagulants preservation effectively. This means that the coagulant is more resistant to attack by bacteria or moulds. In addition, we can gain income from the sale of oil extracted from Moringa oleifera seeds and the fatty decake for reuse in composting. The cost benefits of Moringa oleifera seed in Vietnam are employed to the net-zero cost of its press cake. However, in Malaysia, the growth cost of 1 kg of Moringa oleifera was evaluated to be approximately US$2. Similarly, in south Iran, the cultivation cost of every kilogram of Moringa oleifera seed was also US$2 (Yarahmadi et al. 2009), whereas the cost of 1 kg of seeds has been estimated to be more expensive at US$27, higher than the production cost of poly aluminum chloride. Thus, Vietnam has the potential to develop the bio-coagulant from Moringa oleifera seeds because of abundant and cheap raw material. Hence, the cost for protein fraction from 1 kg (5,000 seeds) of Moringa oleifera is approximately 8,000 VND (0.4 US$). This is cheaper than PAC (approximately 10.000 VND (0.5 US\$) per 1 kg). And on top of that, protein extract of Moringa oleifera seeds is of more advantage to communities in terms of health, ecology, and economy.

Using a bio-coagulant based on plants is an eco-friendly operation that often involves energy efficiency, recycling, safety and health concerns, and renewable resources. The aim is that this study informs developments in this currently growing field with a focus on sustainability, in other words meeting the needs of society in ways that can continue indefinitely into the future while protecting the environment and sustainable use of natural resources.

Coagulants from Moringa oleifera seeds have been investigated to generate not only a much smaller (five times) sludge volume (Ndabigengesere et al. 1995) but also a higher organic sludge value. As such, sludge after the coagulation process can biodegrade, has lower toxicological residue and handling costs are cheaper, making it safer for human health, aquatic or river life, and consequently having a smaller environmental impact (Choy et al. 2014; Olyaie et al. 2014). Another advantage of using renewable materials, such as reusing locally available solid waste, is not only a low-cost alternative to chemical coagulants, but also their minimal net effect on global warming. Moreover, using Moringa oleifera seed extract is observed as environmentally friendly behavior: the process does not consume alkali, so pH adjustments can be limited, thus resulting in a lower number of chemical agents entering into aquatic life, and is therefore more sustainable (Wang et al. 2014). The seed extracts are also non-corrosive, which eliminates concerns about pipe destruction (Choy et al. 2014). The Moringa oleifera seed extracts are easy to use as coagulants require less processing and can provide a sustainable means of treatment.

## CONCLUSION

The method of protein fractionation from Moringa oleifera seed discussed in this study is simple, and easy to apply on an industrial scale. Using hexane to remove oil and extracting the protein with electrochemically activated solution is a novel method of producing new bio-coagulant. In comparison with PAC coagulant, the most popular coagulation agent, fractionation protein MO3 was not only more effective for removing turbidity and COD, but also E. coli in treating lake and domestic wastewater. It has the potential to become a commercial product – bio-coagulant to replace common chemical coagulants (PAC) – with many benefits such as improved water treatment efficiency, more eco-friendliness, less sludge formation, biodegradability and lower costs.

In this study, the challenge presented by the coagulation process is the complexity of scaling-up the process on the water treatment plants. Different parameters of coagulation–flocculation technology require accurate control to attain the efficiency of the process. Thus, bench-scale experiment or trial and error testing is always required to determine the most efficient coagulant, pH range and alkalinity value for particular wastewater at a certain dosage.

## ACKNOWLEDGEMENTS

The authors thank the Environmental Engineering Laboratory at Thuyloi University and Institute of Marine Biochemistry, VAST for providing the Jar Test procedure and access to their facilities.

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