Effect of Moringa oleifera seed husk and extraction techniques on coagulation efficiency for cyanobacteria removal Open Access

The utilization of natural coagulants in water treatment as an alternative to chemical coagulants and flocculants is a promising approach. These bio-coagulants can be obtained from microorganisms like algae, bacteria, and fungi (Ahmad et al. 2021); plant materials such as leaves, seeds, vegetables, and fruit wastes (Fard et al. 2021); and animal sources such as crustaceans and chitosan (Bakshi et al. 2020). Moringa oleifera, commonly known as the Moringa tree, has gained significant attention for its coagulation properties (Sulaiman et al. 2017; Magalhães et al. 2021). Especially, Moringa seeds have been found to contain water-soluble cationic proteins having a positive charge that can effectively remove various pollutants, such as oil, heavy metals, surfactants, algae, and cyanobacteria, from water, while also significantly reducing turbidity (Magalhães et al. 2021). These proteins are nontoxic to both humans and animals, produce minimal residual sludge, and are biodegradable, making them a sustainable and cost-effective alternative to conventional chemical coagulants (Choudhary & Neogi 2017; Magalhães et al. 2021). Their non-corrosive properties help prevent pipe erosion, while their ability to reduce sludge production by up to five times lowers operational costs and supports sustainable water purification (Choy et al. 2014; Ribau Teixeira et al. 2017a). Moreover, their use in developing countries can improve economic conditions and expand access to clean water in remote rural regions (Ndabigengesere & Subba Narasiah 1998; Moulin et al. 2019).

Numerous laboratory experiments conducted thus far have demonstrated the potent coagulation capabilities of M. oleifera seeds (Ndabigengesere & Subba Narasiah 1998; Muyibi & Evison 1995; Choudhary & Neogi 2017; Magalhães et al. 2021). Various interactions can occur between Moringa seed coagulants and water contaminants, including electrostatic interactions, hydrogen bonding, and π-stacking (Gomes et al. 2022). These interactions play crucial roles in the coagulation process, wherein positively charged components of Moringa seeds attract and bind to negatively charged contaminants in water through electrostatic forces (Sulaiman et al. 2017). Additionally, hydrogen bonds facilitate the formation of stable aggregates by promoting molecular associations between the coagulant and contaminants. Furthermore, π-stacking interactions contribute to the cohesion of these aggregates by facilitating the stacking of benzene rings present in the molecules involved (Raji et al. 2023). Coagulation is facilitated by active agents found within Moringa seeds. Specifically, cationic peptides of low molecular weight (6–16 kDa) molecules (Bhuptawat et al. 2007) adhere to the surface of negatively charged particles, inducing charge neutralization. This neutralization subsequently reduces electrostatic repulsion, causing particles to agglomerate. Typically, a portion of the protein macromolecules is adequate to bind to one negatively charged particle, thereby leaving the majority free to bind to additional negatively charged particles, ultimately resulting in agglomeration and the formation of flocs via bridging mechanisms. Nevertheless, some researchers have suggested that the size of Moringa seed proteins may be insufficient to promote bridging flocculation (Ueda Yamaguchi et al. 2021). However, Moringa seed coagulant is generally not recommended for drinking water treatment as it can increase the organic matter in the aqueous phase, potentially leading to the formation of disinfection byproducts when in contact with chlorine. Therefore, it is crucial to maintain a lower dosage when using it for drinking water treatment. While M. oleifera seeds have demonstrated potent coagulation capabilities, there remains a gap in optimizing their extraction methods and comparing the coagulant properties with and without the presence of Moringa seed husk. In this study, we employed two different solvents, distilled water and 1 M NaCl, to extract Moringa seed coagulant from seeds both with and without husks, yielding four coagulant suspensions. We conducted jar tests to determine the most effective type of Moringa seed coagulant and its optimum dosage for the removal of Microcystis aeruginosa in water treatment.

Readymade BG-11 culture medium was used for the cultivation of M. aeruginosa cells. Samples were obtained from previously established M. aeruginosa monocultures to initiate new cultures. To ensure sterility, flasks, and rubber stoppers were subjected to autoclaving at 121 °C and 0.12 MPa for a duration of 20 min using an autoclave (SP300, Yamato Scientific Co., Ltd, Tokyo, Japan). Subsequently, the cultures were inoculated and maintained in an incubator (LH-55RDS, NK Systems Limited, Tokyo, Japan) at a temperature of 25 ± 1 °C, with alternating 12-h light and dark periods and a photon flux of photosynthetically active radiation measuring 60 μmol m⁻² s⁻¹. Cells were allowed to grow until they reached the exponential growth phase.

A cell suspension was prepared to establish the starting point for the experiments. The suspension was adjusted to an optical density (OD) of 0.8 at 730 nm, a turbidity level of 150 NTU, and a cell density of approximately 2 × 10⁶ cells/mL.

As per the World Health Organization, lakes were classified into three magnitude classes of cyanobacterial cell abundance to assess exposure health risk: low (≤20,000 cells/mL), moderate (20,000 ≤ cells/mL ≤ 1 × 10⁵), and high (>1 × 10⁵ cells/mL) (Mishra et al. 2019). With regard to the magnitude class ‘high,’ an initial cell count of approximately 2 × 10⁶ was chosen. The M. aeruginosa cell cultures were harvested during the exponential growth phase and subsequently diluted about five times to achieve the desired initial cell count.

The characterization of Moringa seed coagulants was carried out using physicochemical and spectroscopic analyses. The physicochemical properties, including pH, particle size distribution, and zeta potential, were measured to assess their stability and coagulation potential. Additionally, FTIR analysis was conducted to identify the key functional groups responsible for coagulation activity.

The particle size distribution and zeta potential were measured using a particle size analyzer (ELSZ-2000, Otsuka Electronics Co., Ltd, Osaka, Japan), providing insights into the colloidal characteristics of the coagulant. The pH of the samples was determined using a portable digital meter (Orion 3 Star, Thermo Fisher Scientific Co., Ltd, Massachusetts, USA) to assess their acidity or alkalinity, which can influence coagulation performance (Table 1).

Fourier-transform infrared spectroscopy (FTIR) analysis was conducted to identify the chemical composition of Moringa seed powders, both with and without husk. The analysis mainly aimed to identify the presence of functional groups that could potentially influence the coagulation properties. Moringa seed powders were prepared by grinding seeds with and without husk to a fine powder. FTIR spectra were recorded using a TENSOR II FTIR spectrometer (Bruker Optics Inc., Billerica, USA) equipped with a platinum attenuated total reflectance (ATR) single reflection diamond crystal accessory. Each sample was analyzed in the range of 4,000–400 cm⁻¹ with a resolution of 4 cm⁻¹.

Separate jar tests were carried out for coagulant suspensions derived from Moringa seeds with husk and without husk. Each jar test followed a specific sequence of steps. First, rapid mixing was initiated by adding the Moringa seed coagulant to the 1 L of cell suspension, and the mixture was agitated rapidly at 200 rpm for 1 min to ensure a uniform dispersion. Subsequently, the cell suspensions underwent a slow mixing phase at 50 rpm for 15 min, facilitating the coagulation and flocculation of cyanobacteria cells. After the slow mixing, the samples were left undisturbed for an additional 15 min, allowing the formed floc to settle. Through this process, the best Moringa seed coagulant extract and dosage were determined.

Additionally, a reference experiment was performed using a similar volume of 1 M NaCl corresponding to each dosage within the range of 0–500 mg/L to assess the potential effect of NaCl alone on cyanobacteria removal. The same jar test procedure was followed, but instead of Moringa seed coagulants, 1 M NaCl was added to the cell suspension at various dosages. The results from this reference experiment were compared to those obtained using Moringa seed coagulants to evaluate the performance of Moringa extracts against a standard NaCl treatment.

After the settling period, the water quality of the supernatant was measured. All water samples underwent physicochemical analyses following the standard methods for water and wastewater examination (AWWA-American Water Works Association 1995). The chemicals utilized in the experiments were procured from FUJIFILM Wako Pure Chemical Industries, Ltd, Osaka, Japan.

The pH of the samples was assessed with a portable digital meter (Orion 3 star), while turbidity was measured using a laboratory turbidity meter (2100N, HACH Company, Colorado, USA). Electrical conductivity (κ) was determined using a portable conductivity meter (AS710, AS ONE Corporation, Osaka, Japan).

The following parameters were assessed to evaluate the effectiveness of the proposed treatment method in removing M. aeruginosa cells from the suspension. Water samples were obtained from a depth of 5 cm below the surface using a pipette to minimize disturbance to floating flocs.

OD, turbidity, and zeta potential were used to recognize the removal of M. aeruginosa cells. Previous researchers demonstrated that there is a linear relationship between cell count and OD₇₃₀ for five cyanobacteria species (R² > 0.99) (Patel et al. 2018). Therefore, in this study, 730 nm (OD₇₃₀) was analyzed using a ultraviolet (UV)–vis spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan). Zeta potential analysis was conducted with a zeta potential and particle size analyzer (ELSZ-2000).

A one-way Analysis of Variance (ANOVA) was conducted for each dosage level to compare the removal efficiency across the four extraction types (Moringa seed with husk and without husk, extracted with 1 M NaCl or distilled water) using SPSS 20. Post-hoc Tukey Honestly Significant Difference tests were performed to identify the significant differences in removal efficiency at each dosage level. All statistical analyses used a significance level of 5% (p ≤ 0.05).

In the FTIR analysis of Moringa seed powders, whether they contain the husk or not, a nearly identical pattern of major absorption bands was detected (Figure 4). These findings suggest that the chemical compositions of Moringa seeds remain consistent regardless of the presence or absence of the husk.

A prominent feature observed was a broad band centered at 3,292 cm⁻¹, attributed to O–H stretching. This indicates the presence of hydroxyl functional groups, primarily found in the fatty acid and protein structures within Moringa seeds. Additionally, the high protein content contributes to the N–H stretching of amide groups in this region. Absorption bands were also noted at 2,919 and 2,851 cm⁻¹, indicative of C–H stretching in CH₂ groups. Furthermore, there were three distinct bands observed between 1,485 and 1,770 cm⁻¹ associated with C = O stretching. Specifically, the absorption band at 1,541 cm⁻¹ corresponds to the amide group in protein, while those at 1,653 and 1,745 cm⁻¹ are linked to fatty acids (Figure 4). Similar results were reported by Bouchareb et al. (2021); Kapse & Samadder (2021); and Magalhães et al. (2021).

When the Moringa coagulant dosage was further increased from 100 to 500 mg/L, it was observed that overdosage of coagulant resulted in low cell removal efficiency. This is due to the restabilization of the cells caused by the excess positive charge from the Moringa seed coagulants (Chales et al. 2022). Therefore, it is important to maintain the optimal dosage, as both insufficient dosage and overdosing would result in poor performance (Muhammad et al. 2021).

Our study demonstrates that optimal cyanobacteria removal occurs with minimal Moringa seed coagulant dosage when extracted with a 1 M NaCl solution, as compared to extraction with distilled water. An experiment conducted by Okuda et al. (1999) revealed that the removal of suspended kaolinite when using Moringa coagulants extracted by a 1 M NaCl solution was 7.4 times greater than that achieved with Moringa coagulant extracted by distilled water. This is due to the fact that saline solutions extract active coagulation compounds more effectively than water (Oladoja 2015; Ueda Yamaguchi et al. 2021), as the salt enhances the ionic strength and solubility of the soluble proteins in Moringa seeds (Kansal & Kumari 2014; Baptista et al. 2015).

Additionally, the results from the reference experiment with 1 M NaCl, designed to assess the influence of salt on cyanobacteria removal, showed a gradual increase in cell removal as the NaCl concentration was elevated (Figure 5). Specifically, the effect of 1 M NaCl at a dosage of 80 mg/L resulted in a 22% removal efficiency. Our findings suggest that the combined removal efficiency achieved using distilled water and 1 M NaCl alone did not exceed the removal efficiency observed when Moringa seed coagulants extracted with 1 M NaCl were used. This indicates that the coagulation properties of the Moringa seed extract contribute an additional removal effect beyond that provided by the NaCl solution alone. These results highlight the potential of Moringa seed coagulants, especially those extracted with NaCl, in enhancing cyanobacteria removal compared to the salt alone.

Our study demonstrated a 90% removal at a dosage of 80 mg/L. Another study conducted by Ribau Teixeira et al. (2017) showed M. oleifera natural coagulant removed almost 80% of M. aeruginosa cells at a dosage of 50 mg/L. The extraction method was similar to the current study 1 M NaCl for 30 min using a magnetic stirrer. Carvalho et al. (2016) showed 78.9% M. aeruginosa cell removal when applying a dosage of 50 mg/L, CaCl₂ coagulant extract.

Table 2 summarizes recent studies on the treatment of M. aeruginosa using plant-based bio-coagulants. Among the listed coagulants, pomegranate peel tannin demonstrated the highest removal efficiency at 94% (Wang et al. 2018), even at a low dosage of 2 mg/L. Senecio anteuphorbium also showed high removal efficiency (88%) but required a higher dosage (15 mg/L) at a pH of 4 (El Bouaidi et al. 2022). Other plant-based coagulants, such as Vicia faba seeds and Opuntia ficus indica cladodes, achieved 85% removal efficiency at relatively low dosages of 5 and 10 mg/L, respectively (El Bouaidi et al. 2020).

Based on the comparison above, different plant-based coagulants function in varying ways and to different extents in removing M. aeruginosa cells. Their effectiveness depends on several factors, including the initial cell concentration, the coagulant extraction method, and the pH of the solution. Some studies have combined plant-based coagulants with additional treatment methods, such as adsorption with activated carbon (Ribau Teixeira et al. 2017), nanofiltration (Camacho et al. 2015), and dissolved air flotation (Carvalho et al. 2016), to achieve maximum cell removal.

This study demonstrates the impact of extraction solvents and the presence or absence of Moringa seed husk on the coagulation properties for M. aeruginosa removal in water treatment. Our findings indicate that the dehusked Moringa seed coagulants required lower dosages than those with husk to achieve similar removal efficiencies, suggesting that the husk does not significantly contribute to coagulation activity. Moreover, dehusked seed coagulants extracted with 1 M NaCl exhibited superior performance compared to those extracted with distilled water, achieving up to 90% removal efficiency, as opposed to 53% with distilled water extraction. This improvement is attributed to the enhanced solubilization of active coagulation compounds in saline solutions and the additional contribution of residual NaCl, which accounted for approximately 22% of the observed removal efficiency. These findings suggest that NaCl extraction enhances the coagulation efficiency of Moringa seeds, making them a promising alternative for water treatment applications.

We express our gratitude to the Comprehensive Analysis Center for Science, Saitama University, for the analysis of FTIR spectra with the Tensor II FTIR spectrometer and for providing insight and expertise that greatly assisted the research. The authors are thankful to the Strategic Research Area for Sustainable Development in East Asia (SRASDEA) at Saitama University for the financial support.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.