Treatment of Pumping Water from the Engraulis ringens Fishmeal Industry Using Moringa oleifera Seed Coagulant and Chitosan

Abstract

The Peruvian anchoveta fishmeal industry generates wastewater (pumping water) during the transport of fish from boats to production plants. This study represents the first evaluation in Peru of Moringa oleifera (MOD) and chitosan as bio-coagulants specifically applied to the coagulation–flocculation treatment of pumping water, providing a direct comparative analysis against traditional ferric sulfate under identical experimental conditions. The effluent is characterized by an extreme turbidity of 5,683 NTU, total suspended solids (TSS) at 3359.3 mg/L, and oils and fats at 451.3 mg/L, and it was treated using optimized doses: 4.0 g/L for MOD and 0.2 g/L for chitosan. The results demonstrate that natural alternatives achieve turbidity removal exceeding 97.5%, matching the efficiency of inorganic salts. Notably, chitosan achieved 88.59% TSS removal with no significant statistical difference (p > 0.05 according to the Kruskal–Wallis test) from ferric sulfate, while MOD excelled in oil reduction (37.84%) compared with chitosan. Beyond treatment efficiency, this research fills a gap in circular economy data by identifying that the resulting sludge, containing >4% non-toxic nitrogen, is suitable for composting. These findings establish a new renewable benchmark for the Peruvian fishing industry’s transition toward sustainable, zero-waste water management.

1. Introduction

The global projection for fishmeal production in 2030 is 7583 thousand metric tons [1], with Peru producing approximately 30% [2]. Fishmeal processing plants generate wastewater from cleaning and maintenance, pumping water, sanguaza, and stickwater. Pumping water originates during the transfer of raw material from the vessel to the plant and uses large volumes of seawater as a transport fluid [3]. Between 2 and 15 m³ of pumping water is produced per Tm of anchoveta raw material [2,3,4], which contains between 6.9 and 12.4 kg/m³ of suspended solids, 4.0 to 8.3 kg/m³ of organic matter, and 0.56 kg/m³ of oils and fats [3,5]. These percentages depend on the time of capture, the quality of the raw material (higher or lower blood content), the seasonality of the fishery (higher or lower fat content), and the pump used to transfer the raw material (damage to the raw material) [2,3,5]. The pumping water is treated by various processes, including rotating filters, grease traps, a dissolved air flotation (DAF) system, and a clarifier, in which chemical agents are added during a Chemical Coagulation Process to promote coagulation and flocculation of suspended solids. The clarified water is then discharged into the sea in compliance with maximum permissible limits (MPL): 700 mg/L for total suspended solids (TSS) and 350 mg/L for fats, oils, and grease (D.S. 010-2018-MINAM).

The use of iron and aluminum coagulants in wastewater treatment has raised concerns due to their adverse effects on the ecosystem and public health [6,7]. Excessive iron levels can cause significant damage to aquatic fauna, affecting gills and other vital organs of fish and crustaceans [8,9], and reduce the filtering capacity of bivalves [10]. From a production perspective, treated wastewater generates sludge with trace iron that re-enters the production process, accelerating the oxidation of fishmeal and adversely affecting its organoleptic characteristics [11,12]. Therefore, it is necessary to explore safer and more sustainable coagulant alternatives, such as Moringa oleifera and chitosan, over conventional chemical coagulants due to their biodegradability and low toxicity [13,14]. The main removal mechanisms identified in Moringa oleifera are charge neutralization thanks to its low-molecular-weight cationic proteins, which destabilize the negative colloids in the effluent [15]. However, due to its long-chain polymeric structure, chitosan complements this process through a bridging mechanism between particles, facilitating the formation of denser, larger flocs [16,17].

The structural characteristics of Moringa oleifera, an evergreen tree native to the southern Himalayas, are directly linked to its chemical efficiency as a bio-coagulant. It belongs to the Moringaceae family, order Capparidales, class Magnoliopsida. It is a very fast-growing species. The seeds are dark brown, globular, and 1 cm in diameter, with wings and a papery consistency. The mature pods remain on the tree for several months before splitting and releasing the seeds, which are dispersed by wind, water, and probably animals. The importance of its use as a forage crop is due to its good nutritional characteristics and its high yield in fresh biomass production. The seed contains 33.6–40% oil. Studies conducted in Pakistan extracted oil from dry seeds with hexane. It contains 7.01% palmitic acid, 71.4% oleic acid, 1.92% linolenic acid, and 0.976% linoleic acid [18].

The ability of Moringa oleifera to reduce pollutant loads and turbidity in aquatic environments has been widely documented, and its potential to remove heavy metals—specifically iron and manganese—has also been highlighted [19,20,21,22]. Recent research highlights this versatility in the treatment of industrial and complex effluents; for example, Khattabi et al. [21] achieved 83% turbidity removal and 65.6% COD removal when treating wastewater from the olive oil industry, while Kenea et al. [20] reported an 87% reduction in turbidity and a 52.6% reduction in COD in surface water using a mixture of moringa and aloe vera. Similarly, in matrices containing anionic dyes, El Gaayda et al. [19] achieved 98.5% efficiency in turbidity removal, and Nkurunziza et al. [22] demonstrated a removal capacity exceeding 90.4% for metals such as iron. Even in effluents with high lipid loads, such as crude oil emulsions, Magalhães et al. [23] achieved 82.43% removal of oils and fats using these biopolymers. However, these investigations have been predominantly conducted using synthetic wastewater, surface water, oil-contaminated wastewater, and textile wastewater matrices, with limited attention paid to industrial effluents of high organic complexity, such as those from the fishing industry. To substantiate this trend, a structured bibliometric search was performed in the Scopus database. An initial query combining Moringa oleifera, chitosan, and coagulation–flocculation processes in wastewater treatment retrieved 803 documents for the period 1984–2026. A co-occurrence analysis of author keywords, conducted using VOSviewer version 1.6.20.0 (Leiden, Netherlands), revealed well-defined and strongly interconnected thematic clusters, indicating a mature and consolidated research field (Figure 1a). By contrast, when the search was refined to include fishery-related wastewater, only five documents were identified within the same timeframe. The resulting keyword network exhibited a sparse and weakly connected structure, dominated by generic terms such as fish and wastewater treatment, with no significant representation of natural coagulants or coagulation–flocculation processes (Figure 1b). This lack of thematic density highlights a clear research gap in the application of natural coagulants to fisheries-related effluents, justifying the need for the present study.

The general objective of this study was to evaluate the effectiveness of Moringa oleifera and chitosan in removing total suspended solids from pumping water at a fishmeal and fish oil processing plant. To achieve this, three specific objectives were established: (1) to characterize the pumping wastewater from the equalization tank of a processing facility located in Callao, Peru; (2) to determine the optimal dosage of each natural coagulant using jar test to maximize turbidity removal and comply with environmental regulations; and (3) to comparatively evaluate the performance of natural coagulants (Moringa oleifera and chitosan) against an inorganic coagulant (ferric sulfate) regarding TSS removal efficiency, optimal dosage, pH variation, and sludge generation.

2. Materials and Methods

2.1. Sampling and Characterization of Pumping Water

Samples were collected in January 2024 at the outlet of the grease trap treatment system and the DAF system at an industrial fishing establishment located in Callao. This sampling point corresponds to the wastewater stream conventionally subjected to physicochemical coagulation–flocculation treatment under industrial operating conditions. Therefore, the evaluated matrix represents partially clarified pumping water after primary removal of free oils, floatable materials, and coarse suspended solids. The collected samples were refrigerated until their characterization. Analyses for pH, oils and fats, and TSS were conducted according to SMEWW-APHA-AWWA-WEF (24th Ed. 2023) methods 4500-H+ B, 5520B, and 2540 D, respectively, and turbidity was assessed using Environmental Protection Agency Method 180.1 (1993).

2.2. Preparation and Characterization of Coagulants

Moringa oleifera seeds were obtained from a wholesale market in Lima. Subsequently, they were subjected to dehulling and crushing, resulting in a material referred to as crushed Moringa oleifera (MOT). The MOT defatting procedure was carried out using hexane as a dissolving agent using Soxhlet equipment [23,24]. Subsequently, the sample was subjected to a drying process at 70 °C for 24 h in a forced convection oven (UF110, Memmert, Büchenbach, Germany). This temperature is suitable for removing the solvent used in the extraction process (68 °C) and for preserving protein integrity at temperatures ranging from 70 to 80 °C [20]. This step was performed to ensure uniform dehydration in the sample. Once drying was complete, the sample was sieved using a 100-mesh screen. The resulting product was designated as defatted Moringa oleifera (MOD), which had a yield of 26.5%. Then, 1 L of sodium chloride solution (NaCl 1M) was added to 20 g of MOD and subjected to agitation at 350 rpm for 35 min [25]. This procedure favors the extraction of the cationic proteins responsible for the coagulant activity, as the cationic proteins in moringa seeds are globulin proteins with low water solubility; therefore, a saline medium is necessary to promote sodium ion exchange with the cationic proteins on the surface of the MOD [19,21,23].

Low-molecular-weight chitosan was obtained from Aoxin Biotechnology company (Lianyungang, China) with a degree of deacetylation ≥95% and an 80-micron granule size. Dissolution of 2 g of chitosan in 100 mL of a 2% acetic acid solution, with the pH adjusted to 3.0, was carried out by using the magnetic stirrer (YP5105, Ovan, Barcelona, Spain) [26,27].

The surface morphologies of crushed Moringa oleifera (MOT), defatted Moringa oleifera (MOD), protein-free Moringa oleifera after activation (SP-MOD), and chitosan were analyzed via scanning electron microscopy (SEM) (Q250 Analytical, Thermo Fisher Scientific, Middleton, WI, USA), where the samples were covered by a gold film, with a voltage acceleration of 25 kV and 6000 magnification [28]. The functional structure was performed via Fourier transform infrared spectroscopy (FTIR) (Nicolet iS10, Thermo Fisher Scientific, Middleton, WI, USA), with a spectrum between 700 and 3500 cm⁻¹ [29]. These techniques provide information on their potential mechanism of action in coagulation.

2.3. Coagulation–Flocculation Tests of Pumping Water

The measured pH of the pumping water was 6.36 ± 0.6; this value was maintained without adjustment for two primary reasons. First, it falls within the optimal operating range of 6–8 for Moringa oleifera, where the prevalence of positively charged active molecules remains stable and facilitates charge neutralization and flocculation, resulting in improved contaminant removal [30]. Similarly, previous studies on the use of chitosan as a natural coagulant have reported high removal efficiencies, including turbidity, biochemical oxygen demand, chemical oxygen demand, and TSS, under mildly acidic to near-neutral conditions, typically within a pH range of approximately 4–7, as its amino groups become protonated in this range, which improves binding to the particles [31]. Second, treating the enormous volumes of wash water typical of the fishmeal industry requires large quantities of chemical additives (acids or alkalis), making the process economically unfeasible and increasing the environmental impacts of treatment. Therefore, by operating at the effluent’s natural pH, the study ensures a more sustainable, environmentally friendly, and cost-effective treatment without compromising the chemical efficiency of natural biopolymers. Therefore, no pH adjustment was performed to maintain realistic treatment conditions and prevent excessive chemical use, which would be economically impractical on an industrial scale; furthermore, the use of extreme pH levels would affect the speciation of the amino groups in chitosan and the isoelectric point of the moringa proteins.

Coagulation–flocculation tests were performed using a jar test apparatus (JLT6, VELP Scientifica, Usmate Velate, Italy) to determine the optimal dosage of the coagulant by measuring turbidity removal. The following were tested: MOD at 500–5000 mg/L, chitosan at 100–400 mg/L, and ferric sulfate as a control at 125–1000 mg/L.

After determining the optimal dosages through turbidity reduction, the treated supernatant was analyzed to determine TSS, oils and fats, and residual turbidity to comprehensively assess treatment performance. This approach allowed us to use turbidity strictly as a preliminary operational parameter while considering TSS as the main response variable of the study.

The coagulation process consisted of rapid mixing for one minute at 150, 120, and 120 rpm for MOD, chitosan, and ferric sulfate, respectively, followed by slow mixing at 60, 40, and 40 rpm for 15 min to promote floc formation. The mixing speed ranges and durations used were based on the findings of Rodríguez et al. and Alvarez et al. [26,32], with a higher stirring speed for MOD due to the need for better dispersion of the viscous cationic proteins, thereby breaking the surface tension while keeping the mixing time constant. After mixing, the samples were allowed to settle for 30 min. The treated water was then carefully collected for physicochemical analysis (turbidity, TSS, and oils and fats).

The sludge generated during coagulation–flocculation was separated, dried, and stored in a refrigerator before characterization. Subsequently, sludge valorization was explored through total nitrogen analysis using the Official Methods of Analysis (19th edition, method 920.87) due to the sludge’s potential as a protein-rich byproduct. This viscous, protein-rich byproduct is often concentrated by evaporation and used in animal feed to enhance its nutritional value.

2.4. Statistical Analysis

The assays for each test were performed in triplicate (n = 3), reporting the average and standard deviation. To determine the appropriate statistical approach, the Shapiro–Wilk test was used to assess normality and Bartlett’s test for homogeneity of variances. As the data did not satisfy the assumptions for parametric testing, significant differences between coagulant doses and the control group were evaluated using the non-parametric Kruskal–Wallis test, followed by Dunn’s post hoc test for multiple comparisons. All statistical procedures were performed using XLSTAT version 2025.1.3 (Addinsoft, NY, USA), with a significance level of 5% (p < 0.05) in all cases.

3. Results and Discussion

3.1. Pumping Water Characterization

The results of the analyses of oils and fats, turbidity, TSS, and pH are presented in

Table 1. Characterization of pumping water from the fishmeal industry. Data reported as mean ± standard deviation, n = 3.

Parameter	Unit	Present Study	Aguilar-Ascón et al. [3,33]	Putra et al. [35]	Jayashree et al. [5]	Marti et al. [34]
Oils and fats	mg/L	451.3 ± 14.6	520	37,000	-	2180
Turbidity	NTU	5683.3 ± 402.7	-	-	-	-
TSS	mg/L	3359.3 ± 45.7	12,360	38,000	6962	45,250
pH	-	6.36 ± 0.01	6.13	6.0	9.0	-

. The measurements reported served as a starting point for evaluating the efficacy of both coagulants. The results obtained from the oil and fats analysis, as well as the TSS analysis, were lower than those reported by other authors [3,5,33,34,35]. These variations in concentration are primarily influenced by the specific industrial pre-treatment sequence. The evaluated wastewater had previously undergone primary separation through grease traps and dissolved air flotation (DAF), which explains the lower concentrations of suspended solids, oils and fats compared with untreated raw pumping water reported in some studies. However, this condition reflects the actual industrial treatment sequence commonly applied in fishmeal processing plants, where coagulation–flocculation is used as a secondary clarification stage to remove residual fine suspended solids, colloidal material, and remaining emulsified oils not efficiently removed during primary treatment. Therefore, the efficiencies obtained in this study should be interpreted within the context of post-DAF pumping water treatment under real operating conditions. In addition, the difference in collection time, raw material quality, seasonality of fishing, and type of pump used to transfer the raw material contributed to the variation in the results.

3.2. Characterization of Moringa and Chitosan as the Basis of Coagulants

MOD and chitosan were superficially characterized using SEM at magnifications of 6000× and 3000×, respectively, and a heterogeneous and porous matrix of small spherical agglomerates can be seen in Figure 2a [23]. The spherical agglomerates represent seed oil storage compartments. Oil extraction produces a greater fiber exposure and increases the surface area; this increase facilitates protein extraction during the activation of natural coagulants [23]. An increase in the amount of extracted proteins can increase their coagulant activity [23]. Furthermore, the porous and fibrous morphology is related to the high coagulation performance of Moringa oleifera, as it provides more active sites for the adsorption of organic matter and facilitates the capture of suspended solids during floc formation.

In the chitosan (Figure 2b), a particle distribution with a marked lack of homogeneity can be observed [36], which can be attributed to the characteristic branching of polysaccharides. The surface presents a rough and coarse texture, which agrees with the findings reported by Abdullah et al. [37]. These researchers used crab exoskeletons to extract chitin and subsequently prepare chitosan. The squamous layers had a dense and firm structure, lacking porosity, agreeing with the observations made by El Knidri et al. [38]. The lack of a porous structure in chitosan limits its available surface area, which could compromise its efficiency in adsorption processes [39]. The lack of porosity in chitosan allows the polymer, through its chains, to extend into the water like “arms”, which trap multiple particles simultaneously, binding them together.

The functional groups of moringa and chitosan were characterized with an FTIR spectrophotometer in a range of 650 to 4000 cm⁻¹ for both cases. MOT, MOD, and SP-MOD were evaluated to assess the difference in the absorbance of the functional groups and to determine how oil extraction impacts their coagulant activity.

The functional groups of moringa are shown in Figure 3a. A peak at 3284 cm⁻¹ is also identified in MOT, MOD, and SP-MOD, attributable to amine and OH- bond stretching [40] due to the high protein and fatty acid content. The peaks at 2924 and 2851 cm⁻¹ [23] correspond to the CH₃–CH₂ chains of the fatty acids in the seed lipids. A decrease in the absorption of these peaks is evidenced after oil extraction. The signal detected at 1744 cm⁻¹ is due to the stretching vibration of the C=O groups, indicating carbonyl groups associated with the protein amides and the fatty acids in the sample [23]. A slight increase in absorbance can be observed for MOD due to the higher protein concentration after the degreasing process. In the case of SP-MOD, the peak disappears due to the removal of proteins from the MOD surface during activation, which verifies the relevance of the proposed process. In addition, a peak can be observed at 1647 cm⁻¹, which undergoes a further decrease after oil extraction. The peaks between 1658 cm⁻¹ (amide I) and 1587 cm⁻¹ (amide II) correspond to the amide group of the protein, which confirms the presence of protein structures [41]. From a mechanistic perspective, the dibasic amino acids that make up these proteins contribute terminal and side-chain amino groups that become protonated in the aqueous medium. This net positive charge reduces the repulsive energy of the electric double layer of the pumping water colloids. These peaks increase their transmittance due to the removal of proteins in SP-MOD. Finally, a peak was found at 1035 cm⁻¹, similar to that reported by George et al. [42] at 1053.5 cm⁻¹, corresponding to the amine functional group, specifically to the bending of the C–O–H group.

Therefore, polar functional groups such as carboxyl, amine, or imine (O–H, C–O, and C–N) on the surface may be responsible for coagulation activity in wastewater. The protonation of the amine functional group in Moringa oleifera protein releases positively charged polyelectrolytes when the protein dissolves in water. These cationic sites enable the capture of negatively charged impurities, bacteria, and sediments, which promote floc formation and the subsequent clarification of the effluent [43].

The functional groups of chitosan are shown in Figure 3b. The characteristic band at 3354 cm⁻¹ can be attributed to the strain of the –OH group, and the 3289 cm⁻¹ band indicates the strain of the N-H group, while the 2871 cm⁻¹ band is assigned to the stretching of the C–H group. In addition, the peaks at 1648, 1589, 1375, and 1026 cm⁻¹ can be attributed to C–O stretching of amide-I, N–H bending, C–N stretching, and C–O stretching, respectively [44]; the 1589 cm⁻¹ band (–NH₂ group bending vibration) and the broad band around 3289 cm⁻¹ (N–H stretching superimposed with O–H) not only confirm the chemical structure of chitosan but also form the basis of its coagulant activity. At the effluent’s operating pH, these amino groups undergo significant protonation, transforming into cationic sites (–NH₃⁺). This high positive charge density is directly responsible for the charge neutralization process, electrostatically attracting the colloidal particles and suspended proteins from the anchoveta, which possess predominantly negative surface charges.

The spectrum shows signals at 1149 cm⁻¹ and 850 cm⁻¹, corresponding to the antisymmetric stretching of the C–O bonds and the vibration of the glycosidic bridge (C–O–C), respectively [45]; this polymeric chain complements the removal process through the interparticle bridging mechanism, allowing chitosan to act simultaneously in an electrostatic and structural manner. These characteristics, combined with the high density of amino and hydroxyl functional groups, give chitosan an exceptional capacity for adsorbing various contaminants [46].

The spectra show that the chemical nature of MOD is based on cationic proteins (amide I and II bands), which primarily act by neutralizing charges to clarify the water. By contrast, chitosan is a linear polysaccharide (C–O–C glycosidic bond bands) with abundant free amino groups, which allows it to combine charge neutralization with interparticle bridging. While moringa is more efficient at interacting with lipids due to its protein affinity, chitosan excels at capturing suspended solids thanks to its long-chain structure, which physically traps the particles.

Table 2. Proximate analysis of defatted Moringa oleifera (MOD), chitosan, and sludge generated by each coagulant after the coagulation–flocculation treatment. Data reported as mean ± standard deviation, n = 3.

Parameters	MOD	Chitosan	Sludge with MOD	Sludge with Chitosan	Sludge with Fe2(SO4)3
Moisture (%)	6.7 ± 0.01	9.4 ± 0.01	-	-	-
Total nitrogen (%)	8.0 ± 0.15	6.62 ± 0.14	4.39 ± 0.15	4.52 ± 0.14	4.14 ± 0.20
Fat (%)	6.5 ± 0.05	nd	-	-	-
Crude fiber (%)	4.4 ± 0.12	nd	-	-	-
Ash (%)	5.6 ± 1.40	1.4 ± 0.94	-	-	-
Nitrogen-free extract (%)	26.9 ± 0.05	47.8 ± 0.10	-	-	-

nd: not detected.

shows the results for moisture content, total nitrogen, fat, and crude fiber in natural coagulants such as MOD and chitosan, as well as in natural coagulants and ferric sulfate in the sludge.

The resulting moisture percentage (6.7%) coincides with the value indicated by Silva et al. [47]. The protein percentage was 50%, which is slightly lower than 63.7% [47] and higher than 45.86% [32]. The differences in the results can be attributed to differences in the oil extraction methods employed. The fat percentage was 6.5%, well below the 11% obtained by using the screw press (CA 59 G, Komet, Mönchengladbach, Germany) reported in previous research [32]. This result suggests that the Soxhlet method, employed in the present investigation, could be more efficient than screw press extraction. Removing oils increases the concentration of proteins, resulting in a higher efficiency in the coagulation–flocculation processes due to an increase in the concentration of solubilized coagulant proteins.

Proximal analysis of chitosan was performed before mixing with acetic acid. The moisture percentage was 9.4%, confirming the data provided in the data sheet that establishes a lower limit of ≤10%. In contrast to the above findings, Okafor et al. [48] reported moisture percentages of 1.65 in chitosan extracted from Scylla serrata shell, suggesting a variation in moisture conditions between different regions or products. The nitrogen percentage was 6.62%, which is higher than that recorded by Okafor et al. [48], who found protein contents of 0.62%. Notably, the nitrogen present in chitosan is found predominantly in the primary aliphatic amino groups [49], in agreement with the technical data sheet provided by Aoxin Biotechnology, which indicates minimum undetected protein values. The ash content was 1.4%, which was higher than that indicated in the technical data sheet (≤1%). Studies carried out by Okafor et al. [48] reported higher ash concentrations due to the conditions of production.

3.3. Coagulation–Flocculation Tests

Figure 4a–c show the maximum percentage of turbidity removal following the coagulation–flocculation of the feedwater; for this purpose, different doses of the coagulants MOD, chitosan, and ferric sulfate were used.

In the pumping water coagulation–flocculation test, a maximum turbidity removal of 97.5 ± 0.32% was obtained when using a dose of 4.0 g/L of MOD at pH 6.36 (Figure 4a), with an initial turbidity of 5683.33 ± 402.66 NTU. This removal rate was higher than that reported by Tong et al., Nonfodji et al., and Noor et al., which ranged from 47% to 64% [27,50,51]. This efficiency exhibits significant differences compared with the other doses used, ranging from 0.5 to 3.0 g/L MOD. Statistical analysis showed no significant differences between 4.0 and 5.0 g/L (p > 0.05), indicating a plateau effect in the coagulation performance at higher dosages. Therefore, 4.0 g/L was selected as the optimal dose considering treatment efficiency and operational economy.

The optimal dose found exceeds the values reported by other researchers for wastewater with high levels of organic matter, from 0.189 to 3.0 g of moringa/L [27,50,51,52]; the effectiveness of natural coagulants in this study was validated by optimizing direct physicochemical parameters (turbidity, TSS, and fats/oils). These results provide sufficient evidence that colloidal destabilization occurred effectively, which can be verified by measuring the zeta potential. However, the optimal dose found is lower than that reported by other authors, ranging from 6.7 to 65.0 g/L [21,53,54,55].

Lower doses are associated with wastewater with low turbidity contents between 15 and 273 NTU, while higher doses are associated with high turbidity contents between 1144 and 5846 NTU. The optimal dose found is similar to 7.5 g/L for feed lot water reported by Arias-Hoyos et al. [55], where a high protein content in the wastewater is also evident. When proteins from Moringa oleifera are dissolved in a medium with a pH lower than their isoelectric point, the amino groups become protonated. This phenomenon creates an attractive force toward various colloids and anionic contaminants—including microorganisms, fatty acid salts, and sediments—which catalyzes floc formation [43,56].

Although the optimal dose of 4.0 g/L of MOD is higher than the dose used in less complex waters, this is justified by the extremely high colloidal load in anchoveta pumping water. From an economic perspective, the low cost of moringa (as a byproduct of oil extraction) offsets the dosage volume. Furthermore, the increase in sludge production does not represent an environmental burden but rather an opportunity for recovery as fertilizer due to its high nitrogen content and absence of toxic metal residues, thereby complying with zero-waste principles.

In the coagulation–flocculation test of pumping water with chitosan, a maximum turbidity removal of 99.30 ± 0.10% was obtained when a dose of 0.2 g/L was used (Figure 4b); this result was higher than the 84% reported by Tong et al. [27]. This efficiency exhibits a significant difference compared with the 0.1 g/L dosage; however, it shows no difference with the other dosages used of 0.3 and 0.4 g/L. The optimal dose of chitosan found is in a range of 0.1–0.7 g of chitosan/L. These values were reported by other authors for wastewater rich in organic matter when working with turbidity values between 227 and 1000 NTU [26,27,57].

Due to the speed of this analytical method and its standardized use as an indicator of initial colloidal destabilization, the optimal doses of the bio-coagulants were preselected based on turbidity removal; however, the subsequent evaluation of TSS and oils and fats confirmed the validity of this selection. In the case of chitosan, the 0.2 g/L dose not only reduced the turbidity by 97.5% but also represented the optimal saturation point for the interparticle bridging mechanism, achieving maximum TSS removal (88.59%). Increasing the dose beyond this point did not result in significant increases in TSS or oil and fat removal due to the phenomenon of restabilization caused by excess positive charge and the nature of the fats.

Treating pumping water with coagulation–flocculation using ferric sulfate (the most commonly used coagulant in wastewater treatment for anchoveta fishmeal companies) yields a maximum turbidity removal of 99.76 ± 0.062% with a dose of 1 g/L (Figure 4c). The optimal dose with ferric sulfate was lower than that obtained with MOD and higher than that obtained with chitosan, similar to other studies [58,59].

Table 3. Pumping wastewater treatment results. Data reported with mean ± standard deviation, n = 3.

Parameter	Unit	Initial Concentration	MOD		Chitosan		Fe2(SO4)3		MPL D.S. 010-2018-MINAM
			Concentration After Treatment (mg/L)	Percent Removal (%)	Concentration After Treatment (mg/L)	Percent Removal (%)	Concentration After Treatment (mg/L)	Percent Removal (%)
Oils and fats	mg/L	451.33 ± 14.57	327.57 ± 8.17	37.84 ± 3.390 a,b	424.47 ± 6.67	6.34 ± 1.66 a	304.23 ± 6.18	48.39 ± 2.99 b	350
Turbidity	NTU	5683.33 ± 402.66	141.67 ± 18.58	97.51 ± 0.32 a	134 ± 22.33	97.64 ± 0.39 a	68.13 ± 9.93	98.80 ± 0.17 b	-
TSS	mg/L	3359.33 ± 45.74	551.67 ± 16.07	83.58 ± 0.48 a	383.33 ± 53.92	88.59 ± 1.61 b	318.33 ± 17.55	90.52 ± 0.52 b	700
pH	-	6.36 ± 0.01	6.30 ± 0.02	-	6.82 ± 0.03	-	5.87 ± 0.02	-	6–9

^a,b Different letters within the same series indicate the presence of statistically significant differences between treatments, as determined by the Kruskal–Wallis test (p < 0.05). MPL: maximum permissible limits.

shows the concentrations and removal percentages for oils and fats, turbidity, TSS, and pH for natural coagulants such as MOD and chitosan, as well as ferric sulfate. The pumping water treatment with the optimal doses of MOD, chitosan, and ferric sulfate removed 83.58 ± 0.48%, 88.59 ± 1.61%, and 90.52 ± 0.52% TSS, respectively. The treatment with chitosan does not differ significantly when compared with the ferric sulfate coagulant, making it an alternative treatment with lower environmental impact and greater potential for reusing the sludge generated for food use [33]. However, the results with MOD were lower than the values of 96% and 95% reported by Mera-Alegria et al. [54] and Bhatia et al. [60], respectively. These authors used higher doses of 6.7 g/L and 6 g/L at longer settling times of 48 h and 90 min, respectively. This treatment not only generates sludge but also generates sludge that can be reused in the food industry, as it is non-toxic due to the absence of metals. Finally, the three types of treatment can obtain treated wastewater that complies with the MPL of D.S. 010-2018-MINAM [61] for the TSS parameter (700 mg/L).

Regarding oil and grease removal, MOD (37.84%) showed a numerical increase of over 30 percentage points compared with chitosan (6.35%). Although the Kruskal–Wallis test resulted in a statistical overlap (sharing letters “a” and “b” in Table 3) due to the conservative nature of rank-based tests on small sample sizes (n = 3), this difference is chemically significant. The superior performance of MOD is mechanistically supported by the presence of specific hydrophobic amino acid residues, which facilitate the adsorption of nonpolar lipid droplets, a feature that the predominantly hydrophilic chitosan lacks. The values obtained for moringa and chitosan are lower than those reported (82.43% and 75.26%) by Magalhaes et al. and Castañeda et al. [23,62].

The removal capacity of oils and fats from pumping water was higher when using MOD than when using chitosan, similar to results obtained by other studies [23,62]. The mechanisms that remove oils and fats when MOD is applied include charge neutralization between negatively charged lipid droplets and positively charged protein fractions, inter-particle bridging through polymeric adsorption, emulsion destabilization, and sweep flocculation; fundamentally, the superior performance of MOD is linked to specific hydrophobic amino acid residues, such as leucine, valine, alanine, and phenylalanine. These residues promote London dispersion interactions with the long aliphatic carbon chains of fats, creating a hydrophobic effect that facilitates the agglomeration of oil droplets even at low concentrations [23,51,60,63,64]. This dual action (electrostatic attraction followed by hydrophobic entrapment) allows MOD to effectively capture nonpolar compounds within the floc structure.

By contrast, the lower oil and fat removal observed with chitosan may be associated with its higher degree of acetylation and lower molecular weight, which prevent particle aggregation. Chitosan primarily promotes charge neutralization for emulsion destabilization through its protonated amino groups (–NH₃⁺). Owing to the abundance of hydroxyl groups and protonated amino groups, the predominantly hydrophilic nature of chitosan favors interactions with the aqueous phase and polar suspended solids through hydrogen bonding and charge neutralization. Thus, chitosan lacks significant hydrophobic fractions necessary to effectively interact with and destabilize emulsified oil droplets. However, its rigid polysaccharide backbone and high degree of acetylation limit the flexibility needed to form dense hydrophobic agglomerates. A high degree of acetylation implies fewer free amino groups and a more polar structure, which restricts van der Waals interactions with lipid chains [16].

Using chitosan with a lower degree of acetylation (less than 70%) has been reported to enhance lipid removal efficiency by reducing polarity and improving hydrophobic interactions [17]. Therefore, while chitosan acts primarily as a bridging agent for suspended solids, MOD functions as a more versatile amphiphilic agent, capable of interacting with the charged and nonpolar fractions of the pumping water. The higher removal capacity of ferric sulfate is attributable to stronger emulsion destabilization and the formation of larger, denser flocs through metal salt hydrolysis.

Notably, the primary focus of this study was not the removal of oils and fats, as the evaluated pumping water was collected from an equalization tank downstream of grease traps and DAF systems, where most of the lipids had already been removed. Therefore, oils and fats were assessed as a secondary parameter to confirm that the coagulation–flocculation treatments did not cause re-emulsification or increase their concentration. Positive removal percentages were observed in all cases, and none of the treatments increased oil and fat levels. This confirms the stability of the process under post-DAF industrial conditions. Consequently, the treated pumping water obtained with MOD and ferric sulfate complied with MPL for oils and fats. However, chitosan showed worse performance. This suggests that higher lipid pre-removal during flotation could improve its efficiency, as suggested by Khattabi et al. [21].

Operational stability was further evidenced by the minimal pH fluctuations observed following coagulation–flocculation. The final pH values recorded were 6.30 ± 0.02 for MOD, 6.82 ± 0.03 for chitosan, and 5.87 ± 0.02 for ferric sulfate (Table 3). These results demonstrate that all evaluated treatments maintain the effluent within the maximum permissible limits (MPL) established by D.S. 010-2018-MINAM. Operating at the effluent’s natural pH not only ensures the chemical efficiency of the biopolymers by preserving the protonation of amino groups and protein stability but also confirms the process’s sustainability by eliminating the need for large-scale chemical neutralization.

3.4. Characterization of Sludge

Nitrogen levels in all sludge samples exceeded 4% (Table 2), confirming their high proteinaceous nature regardless of the coagulant used. Notably, in all cases, the total nitrogen content was analyzed mainly as protein. The value obtained using MOD was lower than the 5.60% and 7.06% reported by Aguilar-Ascón et al. [3]. This variation is technically attributable to the fact that the pumping water in this research was collected downstream of a pre-treatment stage consisting of aeration and grease traps, as well as a dissolved air flotation (DAF) system. These mechanical and flotation processes achieved a preliminary removal of organic matter and coarse solids containing significant nitrogenous fractions before the coagulation–flocculation step. A similar situation can be observed in the treatments with chitosan and ferric sulfate. These sludges have lower nitrogen content than 8–9.6% fishmeal [65]. The valorization of sludge resulting from MOD and chitosan treatment, as an input to be reused in fishmeal production, can negatively impact fishmeal quality if the doses are very high. However, given its high nitrogen content, it is possible to use it as an input in composting processes of carbon-rich lignocellulosic material, allowing for an appropriate C/N ratio within the recommended range of 20 to 25 [66,67].

4. Conclusions

This study demonstrates that MOD and chitosan are technically viable and sustainable alternatives for treating anchoveta pumping water in Peru, achieving turbidity removal rates exceeding 97.5% at optimal dosages of 4.0 and 0.2 g/L, respectively. While chitosan stood out for its high efficiency in removing total suspended solids (88.59%), our analysis revealed a critical limitation in its ability to remove oils and fats (6.35%) due to its predominantly hydrophilic nature, which hinders interaction with lipid fractions. By contrast, MOD showed superior performance in grease removal (37.84%) owing to hydrophobic amino acid residues in its cationic proteins, which facilitate lipid adsorption. Finally, the valorization of the generated sludge, with a nitrogen content greater than 4%, reinforces the viability of these bio-coagulants within a circular economy model, transforming industrial waste into useful byproducts for composting. Thus, the knowledge gap regarding the treatment of high-organic-load fishery effluents under local conditions is closed.