Phosphorus Removal from Piggery Wastewater Using Alginate-like Exopolymers from Activated Sludge

Abstract

The growing depletion of global phosphorus reserves underscores the urgent need for sustainable and circular nutrient recovery solutions. Rich in phosphorus, piggery wastewater represents not just a waste stream but a valuable resource. In this study, we explore an innovative approach by recovering alginate-like exopolymers (ALE) from activated sludge (AS) and utilizing them to produce biosorbent hydrogel beads capable of removing phosphorus directly from real piggery wastewater. The ALE extraction process yielded approximately 175 $\mathrm{m}\mathrm{g} {\mathrm{V}\mathrm{S}}_{\mathrm{A}\mathrm{L}\mathrm{E}}/\mathrm{g}{\mathrm{V}\mathrm{S}}_{\mathrm{s}\mathrm{l}\mathrm{u}\mathrm{d}\mathrm{g}\mathrm{e}}$, highlighting the potential of wastewater biomass as a source of functional biopolymers. Adsorption experiments revealed phosphorus removal efficiencies approaching 80%, with capacities ranging from 0.68 to 1.18 mgP/gVS_ALE. Structural and chemical characterizations confirmed both the successful adsorption of phosphorus and the stability of the biosorbent post-treatment. This work demonstrates a dual benefit: the recovery of critical nutrients and the transformation of wastewater-derived materials into value-added biosolids. By integrating phosphorus capture and biosorbent production, the approach offers a cost-effective and environmentally responsible pathway toward nutrient recycling and wastewater valorization.

1. Introduction

Phosphorus (P) is an essential nutrient for agricultural productivity, but is a non-renewable resource predominantly obtained from phosphate rock, with reserves concentrated in just a few regions worldwide. Morocco alone holds approximately 75% of the remaining global phosphorus reserves, creating a critical dependency and vulnerability in global food production systems [1]. The ongoing extraction of phosphorus for fertilizer manufacturing accelerates the depletion of these limited reserves, which are estimated to finish up to the next 100 years. In this scenario, the recovery of phosphorus from wastewater represents a sustainable and strategic alternative aligned with the principles of the circular economy. Among phosphorus-rich effluents, piggery wastewater is particularly significant due to its high phosphorus content [1,2].

Adsorption has gained attention as an effective method for nutrient recovery, particularly phosphorus, from wastewater. The recovered phosphorus can be reused in agricultural applications, potentially lowering dependence on mineral fertilizers [3,4]. The effectiveness of material to be considered a good adsorbent relies on key parameters as selectivity, adsorption capacity, costs, good stability, and long operational lifespan. Each adsorbent exhibits distinct characteristics, which are primarily determined by factors such as surface area, pore size and distribution, as well as the nature and abundance of surface functional groups [5].

Extracellular polymeric substances recovered from biological sludge have been extensively studied due to their possible uses, such as being used as cement-curing material, as fertilizer, as flame retardant, as seed coating, as water proof coating material, and adsorbent [6,7,8]. Composition, physic-chemical properties, the environmental factors affecting their production, and the recovery and applicability have all been revisited [7,9,10].

According to the bioproduct recovery value pyramid, residual sludge should be first recovered and converted into an added-value biomaterial before being directed toward final energy use (lower value), such as its conversion into biogas or other forms of energy. For these reasons, the recovery of ALE from biological sludge has greater application potential compared to other technologies [11].

Compared to aerobic granular sludge (AGS), the extraction of ALE from activated sludge (AS) has been relatively underexplored. Nevertheless, since its introduction, the AS process has become the dominant approach for biological wastewater treatment, leading to the large-scale production of excess sludge [12].

Despite its significant quantitative advantage, ALE extracted from AS tends to be somewhat inferior in both composition and functional properties compared to that derived from AGS. This limitation is a key factor behind the relatively few studies and the limited attention devoted to ALE recovery from AS [12,13].

Cabral et al. [14] studied a mixture of alginate and ALE beads structure (2.5%), which provided structural integrity and resulted in 70% phosphorus removal from a 100 mg/L synthetic phosphorus aqueous solution. However, a major limitation observed was the disintegration of the biosorbent, which hindered the possibility of nutrient desorption. The authors highlighted the potential for directly applying the biosorbent in agriculture, leveraging both the adsorbed phosphorus and the nutrient content of ALE itself. Nonetheless, they emphasized the need to improve the structural integrity of the biosorbent to prevent its breakdown during use, avoiding the leakage of residual organic matter and nutrients to the liquid phase.

While some authors highlight limitations in exploiting ALE from AS due to their complex compositional properties [12] this study pioneers a comprehensive approach to overcome these challenges. Specifically, we recovered ALE from AS biomass and engineered ALE-alginate hybrid beads as efficient biosorbents. These beads were applied to remove phosphorus from real piggery wastewater, and the resulting post-adsorption material was thoroughly characterized to assess its potential as a value-added biosolid for agricultural or environmental reuse. This integrated strategy not only addresses existing compositional constraints but also advances sustainable nutrient recovery and waste valorization technologies.

2. Materials and Methods

2.1. Biopolymers Recovery and Hydrogel Beads Preparation

Activated sludge (AS) was collected from a municipal wastewater treatment plant (WWTP), located in Florianopolis, Brazil. The WWTP has a treatment capacity of 278 L/s. The biological treatment system employed a continuous-flow, extended aeration activated sludge reactor, operating under a sludge age of 18 days and a hydraulic retention time (HRT) of 8 h. Following aerobic reactor, the effluent was directed to secondary clarifiers for solid–liquid separation. The resulting waste sludge was directed to a thickener and subsequently dewatered using a centrifuge prior to final disposal. The sludge sample was collected after centrifugation for sludge dewatering.

ALE extraction followed the protocol presented by [15], which contains the sequence for alkaline extraction (Na₂CO₃, 80 °C), centrifugation (2150 g for 25 min), and precipitation (HCl, 1 M). ALE recovery yield was estimated based on solids (total and volatile) analyses [16].

The adsorbent beads were prepared by mixing ALE and sodium alginate 1% (m/v) resulting in a 2.5% alginate/ALE ration. ALE previously obtained during the extraction step was then added to sodium alginate and dripped in ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$ solution (12%) to form the hydrogel beads.

2.2. Adsorption Experiments

Adsorption was performed using piggery wastewater sampled from a biodigester located in a pig farm in Santa Catarina state (Brazil). The samples were frozen until their use. Wastewater samples were previously diluted (25×) prior to the adsorption experiments, to set the initial phosphorus concentration around 100 mgP/L.

The hydrogel beads used in the adsorption experiments were tested at 3.95 and 0.38 g/L (dry weight, VS) to compare their effect on the adsorption results.

Adsorption experiments were performed in a thermostatically controlled stirred bath (MARQ LABOR, Brazil) under continuous agitation at 100 rpm and a constant temperature of 25 °C. Samples were collected (0, 5, 15, 30, and 60 min), centrifuged (2150× g for 2 min), and used for phosphorus, nitrogen, and chemical oxygen demand (COD) concentration analysis. The removal efficiency (RE) was calculated according to Equation (1) [5].

$$RE\left(\%\right)={\displaystyle \frac{\left(C_o-C_f\right)}{C_o}} \times 100$$

(1)

where RE is the removal efficiency (%), and $C_o$ and $C_f$ are the initial and final concentration of P (mgP/L), respectively. Adsorption capacity was calculated according to Equation (2) [5].

$$q_e={\displaystyle \frac{\left(C_o-C_f\right)V}{W}}$$

(2)

where $C_o$ and $C_t$ are the initial and final concentration of phosphorus in the sample (mgP/L), respectively; W is the biosorbent mass (g); and V is the volume of the solution (mL).

Adsorption experiments were performed in triplicate (n = 3), and the results were expressed as mean values. Statistical analyses were carried out using analysis of variance (ANOVA), and mean comparisons were conducted using Tukey’s test at a 5% significance level (p < 0.05). All statistical procedures were performed using Origin^® 2017 software.

2.3. Analytical Characterization Procedures

The piggery wastewater was characterized for pH, P, COD, and nitrogen. P was obtained using the vanadomolybophosphoric method, the COD determined using the colorimetric method, and the ammonium nitrogen (NH₄⁺) by the Nessler method [16].

The ALE hydrogel beads before and after the adsorption experiment were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and inductively coupled plasma optical emission spectroscopy (ICP-OS). A scanning electron microscope (SEM) equipped with energy-dispersive spectroscopy (EDS) was employed to analyze the morphology and chemical composition of the tested materials. To analyze the microstructure and its composition, samples were frozen before being lyophilized for 48 h in a benchtop lyophilizer (Liotop L101, Brazil). Lyophilized samples were affixed to an aluminum stub using silver conductive adhesive and subsequently sputter-coated with gold. SEM images were obtained using a scanning electron microscope (JEOL JSM-6390LV, USA) operating at 10 kV with magnifications ranging from 50× up to 1000× at a resolution of 1 μm to 500 μm.

Fourier-transform infrared (FTIR) spectroscopy was performed using a Cary 600 Series spectrometer (Agilent, USA) equipped with a horizontal attenuated total reflectance (ATR) accessory featuring a ZnSe crystal. Spectra were recorded in the 400–4000 cm⁻¹ range, with a resolution of 4 cm⁻¹ and 20 scans per sample. Prior to analysis, all samples were lyophilized.

The metals concentrations were quantified according to method 3120-B [16], using ICP-OS (ICP-OES 5800, Agilent, USA). Nitrite and nitrate were determined by ion chromatography according to the method 4110B [16], while solids were obtained with method 2540 [16].

3. Results and Discussion

3.1. Yield of ALE Extraction from AS

The average yield of ALE extraction was 175 ± 23$\mathrm{m}\mathrm{g} {\mathrm{V}\mathrm{S}}_{\mathrm{A}\mathrm{L}\mathrm{E}}/\mathrm{g}{\mathrm{V}\mathrm{S}}_{\mathrm{s}\mathrm{l}\mathrm{u}\mathrm{d}\mathrm{g}\mathrm{e}}$. Such a yield falls within the concentration range reported by Lin et al. [17]. Chen et al. [6] reported ALE recovery yields from activated sludge ranging from 90 to 300 $\mathrm{m}\mathrm{g} {\mathrm{V}\mathrm{S}}_{\mathrm{A}\mathrm{L}\mathrm{E}}/\mathrm{g}{\mathrm{V}\mathrm{S}}_{\mathrm{s}\mathrm{l}\mathrm{u}\mathrm{d}\mathrm{g}\mathrm{e}}$. The recovery yields were related to influent composition, sludge retention time, C:N ratio. Therefore, the results obtained herein suggest the potential use of AS for ALE recovery.

Recovery of ALE from AS has been compared to the recovery yields found for AGS. Due to the biofilm nature, AGS usually has higher ALE yields. Yield experiments of ALE reported by Rehman [9] showed that ALE from conventional AS presented different composition properties and extraction yield (130 $\mathrm{m}\mathrm{g} {\mathrm{V}\mathrm{S}}_{\mathrm{A}\mathrm{L}\mathrm{E}}/\mathrm{g}{\mathrm{V}\mathrm{S}}_{\mathrm{s}\mathrm{l}\mathrm{u}\mathrm{d}\mathrm{g}\mathrm{e}}$) when compared to that obtained from AGS (352 $\mathrm{m}\mathrm{g} {\mathrm{V}\mathrm{S}}_{\mathrm{A}\mathrm{L}\mathrm{E}}/\mathrm{g}{\mathrm{V}\mathrm{S}}_{\mathrm{s}\mathrm{l}\mathrm{u}\mathrm{d}\mathrm{g}\mathrm{e}}$) and membrane bioreactors (170 $\mathrm{m}\mathrm{g} {\mathrm{V}\mathrm{S}}_{\mathrm{A}\mathrm{L}\mathrm{E}}/\mathrm{g}{\mathrm{V}\mathrm{S}}_{\mathrm{s}\mathrm{l}\mathrm{u}\mathrm{d}\mathrm{g}\mathrm{e}}$). Besides lower recovery yields, the lower uronic and mannuronic acids composition play a critical role in the hydrogel properties, which are important for potential ALE applications [12]. However, lower hydrogel formation capacity could be improved by adding alginate to the ALE when using it as biosorbent, as demonstrated in this study.

3.2. Phosphorus Removal Experiments

The piggery wastewater analysis resulted in initial P, COD and N-NH₄⁺ concentrations of 3250 ± 445 mg/L, 24,620 ± 3970 mg/L and 7380 ± 650 mg/L, respectively. The pH was around 8.0.

Previous experiments using synthetic wastewater were conducted at pH 8.0 and using 100 mg/L of initial phosphorus concentration, which induced a 25-fold dilution of the real piggery effluent to meet similar conditions. The diluted effluent presented a pH close to 8.0, suitable for this adsorption process [14]. Thus, the real initial P, COD and N-NH₄⁺ concentrations were 130 ± 17 mg/L, 984 ± 160 mg/L, and 295 ± 26 mg/L, respectively, for the experiment using 150 g/L of biosorbent (Figure 1). The initial P, COD and N-NH₄⁺ concentrations using 15 g/L of biosorbent were 78 ± 3 mg/L, 1021 ± 136 mg/L, and 186 ± 25 mg/L, respectively (Figure 1).

The biosorbent concentrations showed significant differences in phosphorus removal efficiency. At 3.95 g/L biosorbent dosage, the P removal efficiency was approximately 79%, while at 0.38 g/L, the removal efficiency was 34%. These values corroborate the studies performed by Schambeck et al. [18] who found that PO₄³⁻ removal increased from 75% to 90.8% when ALE dosage was increased from 1.88 to 3.75 g/L.

The P concentration significantly decreased after 60 min. Tukey’s post hoc test revealed two statistically distinct groups: samples collected at time 0 showed significantly higher concentrations compared to those collected at 60 min, which exhibited significantly lower values. No statistically significant differences were observed within the same time group, as indicated by shared letters in the bar chart.

The adsorption capacity results were 0.68 mgP/gVS_ALE using 3.95 g/L of biosorbent dosage and 1.78 mgP/gVS_ALE using 0.39 g/L of biosorbent dosage on a dry basis. Studies have shown that smaller doses of adsorbent result in the complete attachment of phosphate to the active sites, whereas excessive adsorbent dosage may lead to available active sites, resulting in reduced adsorption capacities [5].

Kong et al. [19] achieved higher adsorption capacity results (15 mgP/gVS_ALE) when using a Fe-ALE biosorbent in synthetic phosphorus solution at 50 mg/L and an adsorbent dosage of 0.5 g/L.

Although adsorbent debris were observed after the adsorption tests, and considering that the adsorbent composition is mainly proteins, polysaccharides, humic acids and phosphate compounds [17], there was little variation in COD and N-NH₄⁺ during the tests. Statistical analysis revealed no significant differences among the concentration values across all groups, as determined by one-way ANOVA followed by Tukey’s test.

These results indicate that the mixture of ALE with sodium alginate, for the formation of the biosorbent, helps to maintain its integrity during the adsorption process avoiding beads disintegration. Contrarily, Schambeck et al. [18] observed an increase of 87% for COD and 118% of total nitrogen when using ALE recovered from AGS, but without alginate reinforcement. According to Figure 1B,C, the increase in the adsorbent dosage seems to have no effect over the removal efficiencies due to organic matter and/or nitrogen leakage to the aqueous medium.

The kinetic behavior of the adsorption is depicted in Figure 2. Total phosphorus was removed from the aqueous medium after 5 min. This result is in line with that obtained by Cabral et al. [14] testing synthetic wastewater with phosphorus concentration of 100 mg/L, pH 8.0, 25 °C, and, a biosorbent concentration of 0.38 g/L. The authors found the adsorption equilibrium after 10 min. According to the authors, the pseudo-first-order kinetic model fit with the experimental data, whereas this model suggests that adsorption occurs primarily through the initial surface contact between the ALE beads and the phosphorus in the aqueous solution.

According to the SEM images in Figure 3A, B the surfaces of the ALE beads are more uniform when compared to those after the adsorption (Figure 3C, D), where rough surfaces were observed, probably due to the partial disintegration of the beads. In addition, the energy spectrum (Figure 4) indicates an increase in the mass percentages of P after the adsorption. A reduction of Ca in ALE beads after adsorption was also observed in the ICP analysis (

Table 1. Results of the inorganic parameters quantified in the biosorbent before and after adsorption, in comparison with Resolution 498/2020—Criteria and procedures for application of biosolids in soils (Brazilian Environment National Council). (number of replicates: n = 1).

Parameter	Before Adsorption (mg/kg)	After Adsorption (mg/kg)	Brazilian Legislation (mg/kg)
Arsenic	0.042	0.049	41
Barium	0.367	0.671	1300
Cadmium	0.002	0.006	39
Calcium	7928	505	-
Chrome	0.106	0.088	1000
Copper	3.248	9.573	1500
Lead	0.098	0.037	300
Magnesium	1.710	166	-
Molybdenum	0.396	0.191	50
Nickel	0.128	0.093	420
Nitrate	<3	<3	-
Nitrite	<3	<3	-
Phosphorus	269	293	-
Potassium	5.993	58	-
Selenium	0.016	<0.010	36
Sodium	307	36	-
Sulfur	43	29	-
Zinc	2.845	11	2800

), suggesting that part of the P removal could be attributed to precipitation by the formation of calcium phosphates.

Figure 5 shows the spectra of ALE beads before and after piggery wastewater adsorption. The transmittance peaks from 3600 to 3000 cm⁻¹ can be assigned to stretching vibrations of O–H bonds, which are typical of polysaccharides [20]. Asadi and co-workers suggested a broad peak at 3452 cm⁻¹ to O–H stretching vibrations of calcium alginate. They also indicated that the peak at 2926 cm⁻¹, which was reported herein as well, can be attributed to symmetric C–H vibrations [21]. Around 1640 and 1540 cm⁻¹ are the peaks related to N–H bending that can be related to amide I and amide II bands for nitrogen compounds, respectively [10]. The peaks at 1083 and 1073 cm⁻¹ are related to the asymmetric stretching vibration of phosphate ions (P-O), often observed around 1100–1000 cm⁻¹. The characteristic peaks at 573 and 563 cm⁻¹ are related to the vibrations of the O-P-O [22]. Similarly, Kumar and co-workers, when evaluating zirconium (IV) cross-linked alginate/kaolin hybrid beads for PO₄³⁻ retention, reported that the asymmetric stretching and bending vibration modes of PO₄³⁻ at 1032 and 560 cm⁻¹ in the FTIR spectra of the PO₄³⁻-sorbed beads could confirm PO₄³⁻ adsorption [23].

The FTIR spectra revealed characteristic peaks at 2925 cm⁻¹ and 2926 cm⁻¹, corresponding to the asymmetric and symmetric stretching vibrations of C–H bonds, respectively. A distinct peak at 1635 cm⁻¹ was associated with the bending vibration of H–O–H bonds from interlayer water molecules, indicating the presence of adsorbed moisture. Furthermore, functional groups related to proteins were identified, including the amide I band (C=O stretching) at 1655 cm⁻¹ and the amide II band (N–H bending) at 1530 cm⁻¹. Polysaccharide-associated vibrations were also detected, such as the C–H stretching in CH₂ groups at 2930 cm⁻¹ and the in-plane C–H bending vibration at 1073 cm⁻¹ [24].

All parameters analyzed (Table 1) are within the limits set by Brazilian regulations for criteria and procedures for the application of biosolids in soils. According to these regulations, biosolid is a product from the treatment of sewage sludge that meets established microbiological and chemical criteria and is therefore suitable for application in soils. In addition, the regulations state that biosolids should meet the following criteria: have agronomic potential; reduce vector attractiveness and chemical substances; and improve microbiological quality.

The EPS extraction procedure used in this work met the minimal requirements when considering the treatment to convert sludge into biosolids. The extraction temperature of 80 °C, over 30 min in alkaline conditions (Na₂CO₃) is superior to the most restrictive treatment required for the sludge treatment (50 °C for 20 min and/or high pH). The harsh conditions applied to the EPS extraction could be considered as a treatment to prevent microbial contamination in the biosolids. Regarding the chemical composition, the biosorbents before and after adsorption were within the regulatory limits for Class A biosolid.

According to Wei [25], the proteins contained in the biopolymer extracted from the sludge can adsorb Cd²⁺ and Cu²⁺, while humic substances have a predilection for adsorbing Zn²⁺. An increase in the concentration of these metals in the adsorbent was observed after the adsorption process. Zinc concentration was significantly reduced from the biosorbent after the adsorption while copper suffered the inverse reaction. Copper from swine effluent was adsorbed by the biopolymer beads.

Calcium was reduced from 7928 to 505 mg/kg after the adsorption process. Calcium cations have previously been associated with competing with phosphate ions for the adsorption sites of biochar [26].

Bead disintegration may occur during the adsorption process, as debris were observed at the end of the experiments. This characteristic suggests the potential application of post-sorption beads as a slow-release material for metals and nutrients in agriculture. Both the adsorbed phosphorus and the constituents of ALE may serve as nutrient sources. It is probable that the concentration of copper increased due to the adsorption from the piggery wastewater. Piggery wastewater contains elevated levels of copper due to its abundance in feed [27]. About 60–70% of the copper is discharged with feces and urine, and then enter the piggery wastewater [22].

4. Conclusions

In this study, ALE were recovered from a full-scale activated sludge process and successfully applied to adsorb phosphorus from real piggery wastewater. Unlike ALE derived from AGS, which exhibits stronger hydrogel properties, the ALE biosorbent beads required reinforcement with commercial alginate to enhance their stability. The reinforced ALE beads effectively removed 79% of P from piggery wastewater without causing increases in organic matter or N-NH₄⁺ levels. Comprehensive characterization confirmed both the P removal efficiency and the structural integrity of the biosorbent after treatment. Importantly, the phosphorus-loaded material meets Brazilian regulatory standards for biosolid application in agriculture, highlighting the potential for sustainable nutrient recovery and the practical applicability of this biosorbent in real wastewater treatment scenarios.