Moringa oleifera Seed Addition Prior to Sludge Thickening for Supernatant Quality Improvement: Analyses of Clarification Performance and Toxicity

Abstract

Low-cost and easily accessible sludge treatment technologies are necessary in low- and middle-income countries. This study aimed to evaluate the use of Moringa oleifera seed powder (MO) as a natural sludge conditioner for supernatant quality improvement prior to thickening as a result of gravity settling. The zone settling rate (ZSR) and sludge volume index (SVI) were used to evaluate the gravity settling capacity. Supernatant clarification was evaluated in terms of the capacity to remove turbidity, apparent colour, Escherichia coli, and organic matter associated with zeta potential evolution. The effects on the values of pH and electrical conductivity were also evaluated. Finally, the effects on the toxicity (chronic and acute) of the supernatant effluent were examined. A significant supernatant quality improvement was observed with the addition of MO. The ZSR (0.16 cm/min) and SVI (53 mL/g) results showed that the sludge had good sedimentability, and the addition of MO maintained these characteristics in a statistical manner. Increasing the MO dosage increased the zeta potential of the supernatant, resulting in an optimal dosage of 1.2 g/L, with a removal of 90% turbidity, 70% apparent colour, 99% E. coli, and 40% organic matter. The pH and electrical conductivity values did not change with increasing MO dosage, which is a competitive advantage of MO addition compared to iron and aluminium salt addition. A reduction in the ability to remove organic matter was observed at higher dosages of the natural coagulant due to the presence of residual MO in the final effluent. The optimal MO dosage of 1.2 g/L did not affect the acute or chronic toxicity of the supernatant. These results emphasized that M. oleifera seed powder can improve the supernatant quality and can potentially be a low-cost and easily accessible conditioner for wastewater sludge thickening.

1. Introduction

It is projected that there will be a significant increase in waste sludge generation, since the sustainable development goal SDG 6.3.1 includes increasing the proportion of safely treated domestic and industrial wastewater flows by 2030 [1]. In 2020, only 55.5% of the globally generated household wastewater was safely discharged, mainly due to low- and middle-income countries. For example, in 2020, only 33.0% and 48.3% of household wastewater in Brazil and Nigeria, respectively, were safely discharged [2]. This issue is imperative due to the health and environmental risks associated with the inadequate handling and disposal of sludge [3]. Furthermore, the sludge disposal cost accounts for more than half of the total sewage treatment system cost, and the efficient separation of liquids and solids in sludge is one of the key steps in reducing the costs of sludge treatment, transportation, and final disposal [4].

Usually, 94% to 99% of unthickened wastewater sludge is water, and the first step of sludge treatment is usually thickening, which is a procedure used to increase the solids content in sludge by removing a portion of the liquid fraction. Gravity settling is one of the most common thickening methods used due to its low cost, and it is widely used in various applications. The supernatant flow from gravity settling has typical BOD and total suspended solids (TSSs) values of 250 mg/L and 200 mg/L, respectively, which makes the reuse of this flow unfeasible; it is normally returned to either the primary settling tank or the influent of the treatment plant [5]. Coagulation/flocculation is one of the most commonly used sludge conditioning approaches in consideration of cost and efficiency. Small colloidal particles are destabilized and transformed into larger and stronger aggregates to improve the thickening process, increasing the zone settling rate (ZSR) and the supernatant quality [6].

Inorganic salt coagulants, such as ferric and aluminium salts, and synthetic organic polymeric flocculants, including polyacrylamide and derivatives, have been proven to be efficient in sludge thickening. However, these products are expensive and difficult to access for remote populations, mainly those in low- and middle-income countries. Furthermore, they might result in secondary pollution and may entail many health risks due to residual metal ions or the release of noxious polymeric monomers into the target water or due to the generation of toxic sludge [7]. Plant-based coagulants have been applied as alternatives to conventional synthetic chemical coagulants/flocculants, with the main benefits of being safe for humans, cost-effective, sustainable, and an abundant resource. The sludge produced after the coagulation process is biodegradable and rich in nutrients; therefore, it can be further processed to produce biofertilizer. Plant-based coagulants are natural, water-soluble, and organic coagulants derived from various plant parts and species, such as Opuntia ficus-indica, Abelmoschus esculentus, and Moringa oleifera [8]. Among plant-based coagulants, M. oleifera seeds have received attention for use as a coagulant for drinking water [9] and wastewater treatments [10] towards a green economy and cleaner production. M. oleifera seeds have water-soluble cationic proteins with an isoelectric point at pH 10–11 and a molecular weight ranging between 6.5 and 30 kDa that are capable of destabilizing negatively charged colloids and facilitating coagulation through the dominant mechanism of adsorption and charge neutralization [11]. M. oleifera seeds are effective in treating faecal sludge and produce substrates that can potentially increase biomethane yields in anaerobic digestion compared to those produced by sludge without plant-based coagulant treatment [12]. The M. oleifera tree has been well cultivated in the tropical belt [13] in countries, such as Brazil and Nigeria, and its seed could provide a more sustainable and affordable solution to the problem of sludge conditioning in low- and middle-income countries.

In this sense, the objective of this study was to evaluate the potential of M. oleifera seeds for supernatant quality improvement when used as a sludge conditioner prior to thickening via the gravity settling of sludge generated from an anaerobic baffled reactor (ABR). Gravity settling capacity was evaluated in terms of the zone settling rate (ZSR) and sludge volume index (SVI). Supernatant clarification was evaluated in terms of the capacity to remove turbidity, apparent colour, E. coli, and organic matter associated with zeta potential and particle size. The effects on the values of pH and electrical conductivity were also evaluated. Finally, the effects on toxicity (chronic and acute) of the supernatant effluent were examined. Although there are few studies in the literature indicating that M. oleifera seed can be used for sludge conditioning prior to thickening [14,15], there is no study on evaluating the hypothesis that the toxicity of the supernatant could be affected after using the seed.

2. Materials and Methods

2.1. Waste Sludge

Waste sludge samples used for the studies were collected from the pilot-scale wastewater plant installed in the Technological Park of São José dos Campos, Brazil (23°09′24.27″ S and 45°47′30.80″ W). The pilot-scale plant receives wastewater after preliminary (coarse screen and rectangular horizontal-flow grit chamber) and primary (gravity sedimentation) steps and treats 4 m³/day in a vertical anaerobic baffled reactor (ABR) with a series of 3 chambers, a hydraulic detention time of 6 h, and an organic loading rate of 1.6 kg COD/(m³day). Characterization and experimental sludge settling were performed immediately after sampling. This pilot-scale plant was evaluated as an alternative for decentralized domestic wastewater treatment for rural areas, peri-urban areas, and informal settlements, such as slums, which have deficient sanitation services in developing countries, as in the case in Brazil [3].

2.2. M. oleifera Seed Powder Preparation

M. oleifera seeds were purchased from Arbocenter, and they were harvested in the city of Araçatuba/SP, Brazil (20°56′19.72″ S and 50°40′6.17″ W). For sludge thickening in low- and middle-income countries, the simplest and most effective technique for preparing the natural coagulant, which consists of the direct use of seed powder, was chosen [16]. The seeds were manually shelled, and the kernels were ground in a domestic blender and sieved through a 14 mesh (1.18 mm) (Figure 1). The husks obtained from the shelling process were stored later to study their use as an adsorbent [17]. The obtained powder, henceforth referred to as MO, was kept at 4 °C in a closed flask to prevent the coagulant properties from deteriorating [18].

2.3. Gravity Settling Studies

To evaluate the efficiency of MO to be used as a sludge conditioner for thickening, 1000 mL of sludge was combined with MO dosages varying from 0.2 to 1.4 g/L and agitated at 40 rpm for 1 min. The rotation rate and agitation time adopted were selected to avoid breaking the biological flocs of the sludge [15], and the MO dosages were selected based on preliminary tests. The samples were immediately transferred into 1000 mL graduated cylinders, and the initial sludge heights were recorded. A control sludge sample with no MO applied was also placed in a graduated cylinder, and the initial sludge height was recorded. The height of the solid–liquid interface was recorded at intervals of 1 min until 30 min, and a sample from the supernatant was collected. The zone settling rate (ZSR) was calculated as a slope from the plot interface height (cm) vs. time (min) to investigate the settling behaviour of sludge at different settling times. The sludge volume index was calculated according to Equation (1) [19]:

$$\mathrm{SVI}=\frac{\mathrm{SLV}\times 1000}{\mathrm{TSS}}$$

(1)

where SVI is the sludge volume index (mL/g), SLV (settled sludge volume, mL/L) is the 30 min settled sludge volume, and TSS is the total suspended solids concentration (mg/L) of the sludge. The TSS value increased with MO dosage, and this variation was considered in the calculation of SVI. SVI is typically used to monitor the settling characteristics of activated sludge and other biological suspensions [19].

2.4. Physicochemical Properties

Characterization of the raw sludge and the supernatant samples after the gravity settling studies was conducted by measuring physicochemical parameters according to the methodologies from the Standard Methods for the Examination of Water and Wastewater [19]. Sludge characterization parameters included total solids (TSs), total fixed solids (TFSs), total volatile solids (TVSs), total suspended solids (TSSs), total fixed suspended solids (TFSSs), total volatile suspended solids (TVSSs), pH (Tecnopon mPa210 pH metre, Piracicaba, Brazil), and alkalinity (potentiometric titration curve). Supernatant characterization analysis included pH, turbidity (Policontrol AP2000 nephelometer, Diadema, Brazil), apparent colour (Policontrol AquaColor, Diadema, Brazil), conductivity (Tecnopon mCA150 Conductivity Meter, Piracicaba, Brazil), zeta potential and particle size distribution (Beckman Coulter’s Delsa Nano C Particle Analyzer, Brea, CA, USA), and biological oxygen demand (Velp Scientifica BOD EVO sensor system 6, Usmate Velate, Italy). Supernatant organic matter content monitoring was carried out according to MO dosage via ultraviolet absorbance at 254 nm (ThermoFisher Scientific Spectrophotometer UV—Vis Genesys 50, Lenexa, KS, USA), which was chosen due to the UV absorbance of many of the known organic components of wastewater and has been reported in the literature to have a high correlation with the total organic carbon (TOC) and chemical oxygen demand (COD) results of water and wastewater. This method has the advantages of being a simple, fast, and low-cost method when compared to conventional TOC, BOD, and COD analysis techniques [20,21].

2.5. Escherichia coli Removal and Toxicity Evaluation from the Supernatant

Escherichia coli in the sludge supernatant (after settling experiments) was enumerated at MO dosages of 0.0 (control), 0.8, 1.0, 1.2, and 1.4 g/L. The supernatant samples were diluted in a 10-fold series, and appropriate dilutions were used in the subsequent enumeration by using modified membrane-thermotolerant Escherichia coli Agar (modified mTEC) according to EPA method 1603 [22]. The results were expressed as colony forming units (CFUs) per 100 mL of sample.

The acute toxicity was assessed with Daphnia similis according to the methodology of the Brazilian standard NBR 12713 [23]. The results were expressed as effective concentration EC(I)50 48 h, which is the concentration of the sample at the beginning of the test that had an acute effect (immobility) on 50% of exposed organisms in 48 h. The pH, conductivity, hardness, and dissolved oxygen of the samples and controls were determined at the beginning and end of the toxicity tests.

The chronic toxicity was assessed with Ceriodaphnia dubia according to the methodology of the Brazilian standard NBR 13373 [24]. With this method, the chronic toxicity was measured using less than 24 h old neonates during a three-brood (seven-day) static renewal test. The effects included the synergistic, antagonistic, and additive effects of all the chemical, physical, and biological components that adversely affected the test organisms’ physiological and biochemical functions. For statistical calculations, the criteria of reproduction and mortality were considered, and test organism sensitivity was assessed in parallel to all toxicity assays. The results were expressed as the inhibition concentration IC(I)50, which is the nominal concentration of the sample that caused a 50% reduction in the reproduction of the test organisms compared to that of the control after seven days of exposure. The results were also expressed as the no-observed-effect concentration (NOEC), which is the highest nominal concentration of the toxic agent that does not cause a statistically significant deleterious effect on the survival and reproduction of the organisms after seven days of exposure under the test conditions [25].

Samples were classified according to their toxicity based on average EC(I)50 or IC(I)50 values using a modified relative toxicity scale (

Table 1. Toxicity data classification according to EC(I)50 or IC(I)50 values. Adapted from [26,27].

IC(I)50/EC(I)50 (%)	Toxicity
<25	highly toxic
25≥ and <75	toxic
75≥ and <100	slightly toxic
≥100	non-toxic

) [26,27].

2.6. Statistical Analysis

All experiments were conducted in triplicate to ensure the reproducibility of the results. Data obtained from experiments were compared by one-way ANOVA and Tukey’s post hoc multiple comparison test. The Shapiro—Wilk test was performed to examine if the data were normally distributed. The level of significance was set at 5%.

3. Results and Discussion

3.1. Unthickened Sludge Characterization

The characteristics of the raw wastewater sludge samples (

Table 2. Characteristics of unthickened sludge.

Parameter	Average	Standard Deviation
Total solids—TS (%)	1.9	±0.7
Total fixed solids—TFS (%)	0.5	±0.2
Total volatile solids—TVS (%)	1.2	±0.6
TVS/TS (%)	69	±4
Total suspended solids—TSS (g/L)	18	±7
Total fixed suspended solids—TFSS (g/L)	4.4	±2.2
Total volatile suspended solids—TVSS (g/L)	13.5	±4.4
TVSS/TSS (%)	76	±4
pH	7.4	±0.2
Alkalinity (mg/L)	1137	±201

) showed typical unthickened sludge concentrations [5]. The TS of 1.9% is in accordance with the typical concentrations for using a gravity thickener (1–6%), and the thickened sludge is expected to reach 3 to 10% of TS after settling [5].

3.2. Supernatant Characterization of Thickened Sludge (without MO Conditioner)

The characteristics of the supernatant of thickened sludge samples without MO addition are shown in

Table 3. Characteristics of the supernatant of thickened sludge samples without MO addition.

Parameter	Average	Standard Deviation
Turbidity (NTU)	180	±58
Apparent colour (CU)	343	±45
pH	7.6	±0.1
Electrical conductivity (µS/cm)	1147	±125
BOD (mg/L)	166	±63
Organic matter (UV254 cm−1)	1.290	±0.109
Escherichia coli (CFU/100 mL)	3.8 × 105	0.4 × 105

and Figure 2 and Figure 3. The high values of turbidity, apparent colour, BOD, and E. coli presented in Table 3 show that the discharge or reuse of the supernatant from sludge thickening is not recommended. It needs to be returned to the influent of the treatment plant or undergo a treatment that improves these properties. The particle size shown in Figure 2 indicates that the supernatant is composed of particles with a distribution range from 0.7 µm to 90 µm and a D50 of 6.7 ± 1.5 μm. Since the supernatant sample is collected only after 30 min of settling, the remaining suspended particles are expected to have colloidal size characteristics. The electrophoretic mobility (EM) of the particles in the sludge supernatant shown in Figure 3 has a mean of (−1.64 ± 0.07) × 10⁻⁴ cm²/Vs, which is indicative of a zeta potential of −21.0 ± 0.9 mV. Based on these results, it is supposed that the existence of colloidal negatively charged particles prevents the formation of larger flocs. Adding a coagulant can destabilize these particles, allowing larger flocs to form with a large enough size to settle, clarifying the supernatant [5].

3.3. Gravity Settling Properties

The sludge volume index (SVI) and zone settling rate (ZSR) results from the gravity settling of sludge with varying dosages of MO show that the treatments are not statistically significantly different at the 0.05 level, with an average, including all MO dosages and control, of 0.16 ± 0.09 cm/min and 53 ± 17 mL/g for ZSR and SVI, respectively (Table S1—Supplementary Material). The probable reason for the lack of improvement in these variables after MO addition is that the raw sludge already presents a very good SVI value (50 ± 16 mL/g), since values below 100 mL/g are considered to indicate well-settled sludge [5]. Anaerobic baffled reactors (ABRs) have shown potential to produce granular sludge, and, therefore, the sludge settling capacity is considered to be high [28]. Similar results were reported by [14], who evaluated the use of an aqueous extract of M. oleifera seed in the thickening of faecal sludge, and the results indicated that conditioning does not increase the ZSR but does enhance the removal of TSS from the supernatant.

3.4. Thickened Sludge Supernatant Evaluation

The turbidity and apparent colour removal and the zeta potential of the thickened sludge supernatant after the addition of different MO dosages are shown in Figure 4. The removal of turbidity and apparent colour increases with increasing MO dosage until a maximum and constant value is obtained at a dosage of 1.2 g/L MO, corresponding to 90.4 ± 3.8% turbidity and 69.9 ± 6.9% apparent colour removal. The reason for this profile of turbidity and apparent colour removal with the increase in MO dosage may be related to the increase in the zeta potential of the particles present in the supernatant, which changes from an initial value of −21.0 ± 0.9 mV (without the addition of MO) to −12.9 ± 0.5 mV (1.2 g/L MO). Increasing the dosage to 1.4 g/L increases the zeta potential to −11.5 ± 0.6 mV but does not significantly change the removal of turbidity and apparent colour. This result is in accordance with the adsorption and charge neutralization coagulation mechanism, as the zeta potential of the particles present in the supernatant is neutralized to the point that the attractive forces outweigh the repulsive forces, forming larger flocs and clarifying the supernatant [11]. The main coagulation mechanism of MO seeds has been reported to be flocculation due to the charge neutralization caused by cationic protein adsorption [29]. It is a challenge to compare the results with the literature, given the scarcity of studies using M. oleifera seeds to clarify the thickened sludge supernatant. Gold et al. [14] evaluated the use of an aqueous extract of the M. oleifera seed in the thickening of faecal sludge, and the settling was optimal at dosages of approximately 6–8 mL/g TS, with reductions in TSS in the supernatant of 81–95%.

Despite the increase in MO dosage, the sludge supernatant pH and electrical conductivity remain stable. Both do not show a statistically significant difference at the 0.05 level with different MO dosages. Including all MO dosages and the control, the average pH value is 7.6 ± 0.1, and the electrical conductivity is 1151 ± 122 µS/cm (Table S2—Supplementary Material). Similar results have been reported in the literature, and this is a competitive advantage of using M. oleifera seeds when compared to the use of synthetic aluminium and iron salt coagulants, which normally reduce the pH and increase the electrical conductivity as a function of the release of H⁺ protons [5,16].

The organic matter profile of the sludge supernatant thickened with the addition of different MO dosages is shown in Figure 5. An organic matter removal of 40.3 ± 5.9% is achieved at 1.2 g/L MO (optimal turbidity removal dosage). This ability to remove organic matter is due to the removal of suspended solids from the supernatant, as confirmed by the turbidity removal results. On the other hand, despite the values of organic matter in the supernatant being statistically equal (with 95% confidence) at dosages from 0.4 to 1.4 g/L, from dosages of 0.8 g/L and beyond, an increasing tendency of the organic matter with increasing MO dosage is observed. M. oleifera seeds contain approximately 36.7% proteins, 34.6% lipids, and 5% carbohydrates [11], which, when used without an advanced step of purification of cationic proteins, can lead to an increase in organic matter depending on the dosage applied [16,30]. Gold et al. [14] evaluated the use of an aqueous extract of M. oleifera seed in the thickening of faecal sludge, which reduced the COD of the supernatant at dosages below 6–8 mL/g TS, whereas this value increased at higher dosages. Therefore, crude M. oleifera seeds should be cautiously used at high dosages to prevent the presence of residual organic matter in the effluent.

Table 4. Influence of increasing MO dosage on Escherichia coli count and removal in the supernatant of thickened sludge.

MO Dosage (g/L)	Escherichia coli
	Count (CFU/100 mL)	Removal (%)
0.0 (Control)	3.8 ± 0.4 × 105	–
0.8	3.6 ± 0.4 × 104	90.5 ± 1.1%
1.0	3.1 ± 0.4 × 104	91.8 ± 0.9%
1.2	5.1 ± 0.6 × 103	98.7 ± 0.2%
1.4	5.4 ± 0.6 × 103	98.6 ± 0.2%

shows the influence of increasing the Moringa oleifera (MO) dosage on the Escherichia coli count and removal in the supernatant of thickened sludge. The removal profile of E. coli is similar to that demonstrated in the removal of turbidity and apparent colour (Figure 4) with increasing MO dosage. An optimal level of E. coli removal (98.7% ± 0.2%) is reached at 1.2 g/L MO. Along with turbidity, microorganisms present in water can also be removed during sedimentation after destabilization through the addition of M. oleifera seed powder and the formation of larger particles [11]. Vega Andrade et al. [30] reported 99.7% E. coli removal by adding 0.6 g/L M. oleifera aqueous extract in a wastewater tertiary treatment after sedimentation and rapid granular filtration, and the ability to remove E. coli was mainly attributed to physical processes since the MO dosage studied (0.3–0.75 g/L) did not show a significant antibacterial effect against E. coli. Higher MO dosages have resulted in microbial inhibition capacity, as in the case of Virk et al. [31], who reported a minimum inhibitory concentration of 12.5 g/L MO against E. coli, and Taiwo et al. [32], who demonstrated that 40 g/L MO had antimicrobial activity against five bacterial strains. Thus, considering the dosage applied in this study (1.4 g/L maximum), it is believed that the dominant mechanism of E. coli removal is physical processes (sedimentation) and not any antibacterial effect of the M. oleifera seed.

The use of MO at the ideal dosage of 1.2 g/L does not change the supernatant toxicity, which remains slightly toxic in terms of acute toxicity and toxic in terms of chronic toxicity (

Table 5. Acute and chronic toxicity of raw supernatant and after 1.2 g/L MO.

	Acute Toxicity (%)	Chronic Toxicity (%)
MO dosage (g/L)	EC(I)50 48 h	IC(I)50 7 days	NOEC 7 days
0.0 (Control)	83.33 ± 2.1	36.43 ± 2.92	20 ± 2.1
1.2	83.33 ± 2.1	39.59 ± 5.41	20 ± 2.1

). Silva et al. [33] evaluated the acute and chronic toxicity of treated sewage using a UASB (upflow anaerobic sludge blanket) and found that the sewage remained highly toxic to C. dubia (chronic) and toxic to D. rerio (acute) after treatment. Dupont and Lobo [34] evaluated the acute and chronic toxicity of treated sewage using a UASB followed by a biofilter system and a secondary settling tank and found that the sewage remained highly toxic to C. dubia (chronic) and toxic to D. magna (acute) after treatment. For both studies, posttreatment was indicated to reduce effluent toxicity.

4. Conclusions

This research work summarizes the performance of Moringa oleifera seed powder (MO) as a natural coagulant for supernatant quality improvement when used as a sludge conditioner prior to thickening through the gravity settling of sludge generated from an anaerobic baffled reactor. A significant quality improvement in the supernatant was observed with the addition of MO, and the zone settling rate, ZSR (0.16 ± 0.09 cm/min), and sludge volume index, SVI (53 ± 17 mL/g), results showed that the sludge had good sedimentability, and the addition of MO maintained these characteristics in a statistical manner. Increasing the MO dosage increased the zeta potential of the supernatant, resulting in a better dosage of 1.2 g/L, which resulted in a removal of 90.4 ± 3.8% turbidity, 69.9 ± 6.9% apparent colour, 98.7 ± 0.2% E. coli, and 40.3 ± 5.9% organic matter. The pH and electrical conductivity values did not change with increasing MO dosage, which is a competitive advantage of MO addition compared to iron and aluminium salt addition. A reduction in the ability to remove organic matter was observed at higher dosages of the natural coagulant due to the presence of residual MO seed in the final effluent. The addition of 1.2 g/L MO did not significantly change the supernatant toxicity, with an acute toxicity of 83.3 ± 2.1% EC(I)50 48 h average and an average chronic toxicity of 39.59 ± 5.41% IC(I)50 7 days, which are indicative of a slightly toxic and toxic effluent, respectively. These results emphasized that MO seed powder can improve the supernatant quality and has the potential to be used as a low-cost and easily accessible conditioner for domestic wastewater sludge thickening in low- and middle-income countries.