Synthesis of a Bioflocculant Using Blood Plasma: A Promising Approach for Sustainable Water Treatment

Synthesis of a Bioflocculant Using Blood Plasma: A Promising Approach for Sustainable Water Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of a Bioflocculant Using Blood Plasma: A Promising Approach for Sustainable Water Treatment João Paulo Eckert, Dionatan Morandin Lima, Gabriele Caroline Thomas, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7131666/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Water Conservation Science and Engineering → Version 1 posted 4 You are reading this latest preprint version Abstract The exponential growth of urban centers and the scarcity of treated water underscore the urgent need for environmental stewardship. The rising demand for alternatives to replace inorganic compounds in water treatment presents a significant opportunity to develop sustainable methods. This study evaluates the use of blood plasma-derived bioflocculants as a promising alternative. The methodology involves the separation and chemical treatment of porcine, bovine, and poultry blood plasma through acid hydrolysis with 0.1 M sulfuric acid to denature proteins, followed by drying and grinding to obtain a fine powder. The clarifying agents were characterized using Point Zero Charge (PZC), Fourier Transform Infrared Spectroscopy (FTIR), and Thermogravimetric Analysis (TGA), while turbidity removal efficiency was assessed using Response Surface Methodology with Central Composite Rotational Design (RSM–CCRD). The clarifying agents exhibited isoelectric points of 6.41, 6.47, and 6.64 for porcine, bovine, and poultry plasma, respectively. FTIR analysis confirmed amide groups and functional groups –CH, –CO, –OH, and H–S(=O) 2 –OH, demonstrating potential for coagulation. TGA results indicated good thermal stability between 40 °C and 400 °C. The findings revealed turbidity removal efficiencies of 95%, 93%, and 90% for the agents derived from porcine, bovine, and poultry plasma, respectively. These results highlight the potential for innovation in developing water clarifiers from slaughterhouse by-products. Bovine porcine and poultry plasma natural coagulants protein-based flocculants water treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Water bodies, in general, play an extremely vital role in the economic development of a country, besides being essential for human activities and survival [1]. Human practices in the environment slowly and gradually cause alterations that, over time, become more pronounced in water sources, posing risks to public health, impacting the environment, and hindering economic and social growth [2]. Water treatment plays a crucial role in this scenario as it aims to eliminate pathogens, impurities, and harmful substances, ensuring that the water meets the standards set by health authorities [3]. Among the indicators of water quality, turbidity is an important criterion, usually related to the efficiency of physical treatments for solid removal, where suspended particles and dissolved algae hinder light penetration. Compliance with this parameter, which is an aesthetic criterion that impacts the acceptance or rejection of the product, can be ensured through clarification treatments, which involve water purification processes such as coagulation/flocculation and sedimentation [4]. These processes are controlled by the charge neutralization mechanism [5]. This neutralization occurs in the presence of a flocculating agent with a charge opposite to that of the colloidal particles. The added product adsorbs and neutralizes the charges of the suspended particles, reducing the zeta potential (ζ) and the repulsive forces between the colloidal particles. Thus, it facilitates the aggregation of the suspended material to form flocs [6]. In water treatment systems, both in Brazil and other countries, the most commonly used coagulants are derived from inorganic compounds, mainly trivalent iron and aluminum salts, as well as synthetic polymers, as indicated by Choy et al. [7] and Franco et al. [8]. However, there are growing concerns about the potential environmental impacts of these coagulants due to the presence of non-biodegradable and potentially ecotoxic residues in both the treated water and the sludge generated during coagulation and flocculation processes, as reported by Yegambaram et al. [9], Freitas et al. [10], and Michelan et al. [11]. In response to these challenges, interest has emerged using of natural coagulants, which are seen as a viable alternative due to their biodegradability and low toxicity [4]. According to Essandoh et al. [12] and Tannouri and Simmons [13], hemoglobin, the most abundant protein in blood, and serum albumins, which are the most abundant proteins in blood plasma, have been recognized for their potential to form agglutinations with other materials. Koul et al. [14] and Nath et al. [15] also identified that these protein molecules play a fundamental role in coagulation and flocculation processes. This is related to the fact that the net charge of a protein is affected by the pH of the medium in which it is found. Piazza and Garcia [16] describe that the protein acquires a negative charge when the pH exceeds the isoelectric point. However, when the pH is below this point, the protein's net charge becomes positive, and due to this, Quintapana [17] suggests its use as a possible clarifying agent. The work carried out by Koul et al. [14] and Nath et al. [15] aligns with projections for the production of bovine, porcine, and poultry meat in Brazil. Such projections suggest an approximate production of 33 million tons of meat by the end of 2029 [18]. This increase in production will result in a greater generation of hard-to-treat waste, such as blood. This amount will produce approximately 11 million tons of blood, which is usually disposed of without proper treatment [19, 20]. As cited by Wang et al. [21], blood accounts for 7% to 11% of slaughterhouse waste. The direct discharge of untreated blood into sewage systems increases the organic pollution load in wastewater by 35% to 50%, posing risks to the environment [20]. Thus, it is understood that the low utilization of this waste, especially in less qualified facilities, is a major concern. It is imperative to implement measures to avoid this waste, which results in significant environmental pollution, economic losses, and the diversion of raw materials [22]. Considering the aforementioned context, there arises a need to explore the feasibility of a clarifying agent based on animal blood plasma. In this context, this project aims to synthesize and evaluate the efficacy of bioflocculants derived from plasma extracted from bovine, porcine, and poultry blood in reducing the turbidity of aqueous solutions. This study will investigate the performance of different bioflocculant concentrations across various pH ranges, comparing their effects with one another. This approach seeks to provide sustainable and effective alternatives for water treatment, contributing to reducing environmental impact and promoting the responsible use of natural resources. 2. Methodology 2.1. Reagents The reagents used in the experimental procedures included: EDTA tetrasodium (C 10 H 12 N 2 Na 4 O 8 ·3H 2 O) of analytical grade, supplied by the brand Êxodo Científica; calcium carbonate (CaCO 3 ) of analytical grade, supplied by Dinâmica Química Contemporânea LTDA; sulfuric acid (H 2 SO 4 ) and sodium chloride (NaCl), both of analytical grade, supplied by Vetec Química Fina; sodium hydroxide (NaOH) of analytical grade, supplied by Reatec; and hydrochloric acid (HCl) 37% of analytical grade, supplied by ACS Científica. 2.2. Plasma Collection and Treatment Porcine, bovine, and poultry blood was collected from slaughterhouses located in the western region of Santa Catarina – Brazil, using EDTA 4Na as an anticoagulant at a concentration of 1.5 g/L of pure blood, following the methodology adapted from Kundu et al. [ 14 ]. As cited by Rakesh et al. [ 15 ], the most conventional process for plasma separation is centrifugation. The blood plasma was separated immediately after collection in a FANEM centrifuge, model 206 BL, at a rotation speed of 3600 rpm for 20 minutes. After phase separation, the supernatant plasma was removed, and the solid residue of blood cells was appropriately discarded for subsequent treatment. The bioflocculant was obtained using the acid hydrolysis technique, following the methodology adapted from Araújo et al. [ 16 ]. The plasma was placed in a jacketed reactor, and the pH was adjusted to 3.5 using 0.2 M sulfuric acid. Agitation was applied using a FISATOM − 713D agitator at a speed of 300 rpm. The solution remained in this state for 4 hours at a constant temperature of 60°C. Subsequently, the solution was neutralized to a pH of 6.0 using 0.1 M sodium hydroxide. The pH measurements were carried out using a KASVI pH meter, model K39-1420A. The remaining solution was placed in Petri dishes and dried in a LOGEN oven, model LSDH-9140A-220, for 20 hours at 60°C to remove moisture. After drying, an IKA grinder, model A11BS032, was used to grind the material into powder for subsequent analysis. The powder was stored in a sealed container at room temperature, protected from light. To standardize the nomenclature and facilitate the interpretation of the experimental data, the clarifying agents derived from bovine, porcine, and poultry plasma will be referred to as BCA (Bovine Clarifying Agent), PCA (Porcine Clarifying Agent), and PLCA (Poultry Clarifying Agent), respectively, throughout this study. 2.3. Point Zero Charge (PZC) The Point Zero Charge (PZC) parameter indicates the pH at which the net surface charge of a material is zero, providing crucial information about the electrostatic interactions between the material’s surface and charged species [ 17 ]. The methodology used for pHpzc determination was adapted from Hirendrasinh et al. [ 18 ]. A 0.01 M NaCl solution was prepared, and 100 mL of this solution was added to different Erlenmeyer flasks, adjusting each one to pH values ranging from 2 to 12 using 0.1 M HCl and 0.1 M NaOH solutions. Subsequently, 0.4 g of the flocculant was added to each flask. The solutions were allowed to react for 48 hours in a SOLAB Shaker SL-222 incubator at 130 rpm and room temperature. The pHpzc value was estimated from the intersection of the final pH in relation to the initial pH. 2.4. Fourier Transform Infrared Spectroscopy (FTIR) FTIR analysis is used to study the secondary structures and functional groups of proteins, allowing detailed analysis of molecular interactions by correlating them with the vibrations emitted by chemical bonds [ 19 ]. The FTIR spectra of the flocculants were obtained using an IRSpirit (Shimadzu) spectrophotometer at a wavelength range of 4000 to 400 cm⁻¹. The dry material samples were analyzed using the ATR (Attenuated Total Reflectance) method, with a prior background evaluation (blank). 2.5. Thermogravimetric Analysis (TGA) TGA is a technique used to determine the composition of materials, including the quantities of highly-volatile matter, medium-volatility materials, combustible material, and ash content (ASTM E1131-20). TGA was conducted on a mass of approximately 30 mg of pure plasma and bioflocculant from each type of blood, placed in an open alumina sample holder. The analysis conditions were carried out with a heating rate of 10°C/min, from 40°C to 600°C, under a nitrogen atmosphere at a flow rate of 50 mL/min. 2.6. Clarification Studies The analyses to determine the ideal bioflocculant and its optimal performance parameters were conducted through experimental design using Response Surface Methodology (RSM) with Central Composite Rotational Design (CCRD), which determined the variation of concentrations and pH of the plasma samples selected for the study. The methodology used for the clarification study was adapted from Wang et al. [ 20 ] and Pacheco et al. [ 21 ], in which a standardized solution of calcium carbonate (CaCO₃) in water with a concentration of 7 g/L was prepared under agitation for 5 minutes, until complete homogenization. Four hundred mL of the standardized calcium carbonate solution were transferred to 1000 mL beakers allocated in equipment specifically designed for the Jar Test method. The pH of the calcium carbonate solution was adjusted using 0.1 M sulfuric acid (H₂SO₄) and sodium hydroxide (NaOH) solutions. After the pH adjustment, a 15 mL aliquot of the standardized solution was collected to determine the initial turbidity using an AKSO Turbidity Max device. Next, the bioflocculant was added to the solution, and agitation was initiated at 160 rpm for 2 minutes to disperse the material in the solution. Subsequently, the system was maintained at 60 rpm for 5 minutes for floc formation. After this period, the agitation was turned off, and the samples were left undisturbed for 40 minutes to allow for the decantation of the particulate matter. After this decantation time, 80 mL aliquots were withdrawn for the final turbidity measurement. The process was performed using calcium carbonate samples adjusted to pH values of 4.2, 5.0, 7.0, 9.0, and 9.8, with varying concentrations of 0.3, 0.5, 1.0, 1.5, and 1.7 g/L, according to the experimental design. A sample of the calcium carbonate solution was maintained without the addition of bioflocculant to compare the effect of the obtained material with the natural decantation process of calcium carbonate. 3. Results and Discussion 3.1. Material Characterization 3.1.1. Point Zero Charge (PZC) The pHpzc values of porcine (PCA), bovine (BCA), and poultry (PLCA) clarifying agents were approximately 6.41, 6.47, and 6.64, respectively, as shown in Fig. 1. Figure 1 reveals that the pHpzc values are within a neutral range, with values close to 6.5. This result demonstrates that the PCA, BCA, and PLCA flocculants can be more efficient in removing anionic contaminants when the pH of the solution is above the pHpzc. This is because the surface of the flocculants under these conditions is positively charged, maximizing the attraction effects described by Coulomb's Law, as observed in Deng et al. [22], thus favoring the removal of anionic species. However, in acidic pH, flocculation efficiency is compromised due to the increase in electrostatic repulsion. 3.1.2. Fourier Transform Infrared Spectroscopy (FTIR) Figure 2 illustrates the FTIR spectra for porcine, bovine, and poultry blood plasmas, along with their respective clarifying agents. The general analysis of the infrared spectra reveals that the shape and position of the peaks remain consistent between different samples, indicating the structural stability of the protein components after acid hydrolysis treatment. This finding suggests that the process does not promote a complete breakdown of protein structures nor the significant formation of new functional groups, as noted by Wang et al. [19], with only a change in the intensity of the existing bands. Deeper analysis of the FTIR spectra, as illustrated in Figs. 2a, 2b, and 2c, reveal that wavelengths near 3300 cm − 1 indicate the presence of characteristic amide A bands, which are attributed to the stretching vibrations of –NH groups, as described by Singh et al. [23] and Kong et al. [17]. This evidence suggests the presence of partially preserved protein structures after treatment. Furthermore, additional studies by Garcia and Wilson [24] and Agbovi and Wilson [25] support these findings by reporting distinct oscillations around 2900 cm − 1 associated with the stretching of –NH 3 , –CH, –CH 2 , and –CH 3 groups. These results imply the formation of the amide B band, indicating spatial changes in the structure of the treated proteins. Studies by Wang et al. [19], Singh et al. [23], and Garcia and Wilson [24] also highlight the presence of multiple adsorption peaks in the infrared region, particularly within the amide I and amide II bands, which reveal significant details about the protein structures in question. Specifically, the amide I bands, located nearly 1650 cm − 1 , are primarily attributed to the stretching vibrations of the C = O, –CO, and –COO– groups. On the other hand, the amide II bands, around 1500 cm − 1 , are associated with the vibrations and shortening of the –CN groups. Both are observed with greater intensity in porcine and bovine plasmas, as well as in and clarifying agents, as illustrated in Figs. 2a and 2b. These findings are consistent with previous studies conducted by Wei et al. [26] and Kong et al. [17], further reinforcing the reliability and consistency of the obtained results. A significant increase in stretching around the 1100 cm − 1 wavelength was also observed in Fig. 2a, indicating specific alterations in the FTIR spectrum for CS. Recent studies by Mishra et al. [27], Safdar et al. [28], and Singh et al. [23] emphasize that this behavior is closely related to the vibrations of the –CO, –C–OH, –COC, and H–S(= O) 2 –OH groups. These findings suggest subtle structural modifications in plasma proteins. According to Saguer, Álvarez, and Ismail [29], the hydrolysis process at acidic pH, combined with exposure to high temperatures, promotes the partial denaturation of molecules. Meutter and Goormaghtigh [30] discuss how this process disrupts the secondary and tertiary conformations of the protein, altering its spatial arrangement. The presence of carboxyl, hydroxyl, and amine groups in bioflocculant molecules is critical for determining their flocculation effect, as they promote suitable binding sites that attract dispersed particles in solution, thereby enhancing the flocculants effective in binding mechanisms between the particles and the flocculants [31–34]. Another significant factor that may improve flocculant activity is the introduction of sulfonic acid functional groups on plasma surfaces after acid hydrolysis, as indicated by peaks observed around 1110 cm − 1 [35, 36]. According to Yu et al. [37], Feng et al. [26] and Artifon et al. [38], the presence of these groups in bioflocculants enhances their capacity to remove colloidal particles and suspended metals. 3.1.3. Thermogravimetric Analysis (TGA) Thermogravimetric analysis (TGA) was conducted on flocculants and pure plasmas to elucidate their thermal responses at different temperature ranges. Figure 3 illustrates the TGA curves, emphasizing the mass losses associated with thermal decomposition, oxidation, and volatilization [25]. Examining Figs. 3a and 3b, it is observed that the initial mass loss occurs between 40°C and 270°C, primarily due to water desorption and the volatilization of low-molecular-weight compounds [24]. Between 270°C and 400°C, significant mass loss is attributed to the removal of moisture associated with hydroxyl and carboxyl functional groups [39]. The final stage of mass loss, observed above 400°C, results from the thermal degradation of the polymer matrix and the decomposition of organic material [24]. The average mass loss percentages for the three thermal zones in the flocculants were 16.27%, 45.21%, and 26.38%, respectively. In contrast, for pure plasma, the mass losses in the same zones were 19.05%, 49.41%, and 29.48%, respectively. These data indicate that both the flocculants and pure plasmas exhibit three distinct stages of thermal degradation. The substantial mass loss observed between 270°C and 400°C suggests significant volatilization of components and structural dehydration. In contrast, the degradation occurring above 400°C reflects the disintegration of the polymer structure and the decomposition of organic constituents. The higher mass loss in pure plasma across all zones, compared to the flocculants, indicates a composition with a higher volatile content and a more thermally susceptible organic structure. 3.2. Clarification Studies 3.2.1. Mathematical modeling Given that the quality of effluent treatment is intrinsically linked to effluent parameters, such as pH, mathematical modeling emerges as a key resource for optimizing procedures and predicting the efficiency of these processes [40]. Based on the results obtained from the CCRD experimental design, an analysis was conducted using Minitab software for statistical evaluation and the development of a mathematical model that describes the relationship between bioflocculant dosage, its source, the characteristics of the effluent to be treated, and flocculation efficiency. The interactions between parameters, as determined through statistical analysis, are illustrated in Fig. 4 and Table 1. Table 1 – p-value for analysis of variance (ANOVA). Linear Square Two-factor interaction Model C pH δ C² pH² C x pH C x δ pH x δ 0.019 0.777 0.026 0.174 0.989 0.003 0.990 0.956 0.022 C – Concentration; pH – Hydrogen potential; δ – Blood type. Source: Elaborated By the authors. Using ANOVA statistical analysis with a significance level of 5%, it was determined that the parameters that significantly influence the model are pH in its linear form, pH in its squared form (pH²), and the combination of pH and blood type (δ) through a two-factor interaction. Consequently, the mathematical models 1, 2, and 3, which define the efficiency (η) of the bioflocculant, are presented in Equations 1 to 3 for porcine, bovine, and poultry, respectively. \(\:{\eta\:}_{porcine}=-0.659+\left(0.358\times\:pH\right)-(0.02306\times\:{pH}^{2})\) (1) \(\:{\eta\:}_{bovine}=-1.194+\left(0.443\times\:pH\right)-(0.02306\times\:{pH}^{2})\) (2) \(\:{\eta\:}_{poultry}=-0.472+\left(0.360\times\:pH\right)-(0.02306\times\:{pH}^{2})\) (3) 3.2.2. Evaluation of Efficiencies Using the Response Surface Methodology (RSM) The removal of suspended solids from the solution was evaluated through coagulation and flocculation processes using the Jar Test. Two primary effects were investigated to assess process efficiency: the variation in flocculant concentration and the pH of the medium. The results obtained through the RSM method are presented in Figs. 5, 6, and 7. Based on the results presented, distinct performance behaviors for each of the evaluated clarifying agents are evident. Current legislation in various countries, including Brazil's Portaria GM/MS Nº. 888, the European Parliament Directive (EU) 2020/2184 [50], and the U.S. Environmental Protection Agency (EPA) Primary Drinking Water Regulations [55], indicates that fresh surface water typically has pH values ranging from 6.5 to 9.0. This range aligns with the experimental pH levels that demonstrated good turbidity reduction efficiency. Analyzing Fig. 5, which illustrates the behavior of the PLCA, reveals a clear correlation between pH and product concentration. The optimal pH range for achieving efficiencies above 94% is between 7.5 and 8.2, with bioflocculant concentrations exceeding 1.3 g/L. Additionally, broader pH ranges, varying from 6.7 to 9.0, are noteworthy as they demonstrate efficiencies above 90% regardless of bioflocculant concentration. This behavior indicates that within this broader pH range, variations in clarifying agent concentration do not significantly affect treatment efficiency. Such robustness suggests valuable operational flexibility, allowing for adjustments in clarifying agent dosage without compromising process effectiveness. However, at pH levels below 6.5 or above 9.2, even changes in clarifying agent concentration fail to maintain the desired efficiency, which drops below 80%. In case of BCA, as illustrated in Fig. 6, the most promising results are observed within more alkaline pH ranges, specifically between 8.0 and approximately 11.0, with efficiencies exceeding 88% across various concentrations. The optimal range is identified as being between pH 9.0 and 10.0, where efficiencies surpass 93%. These findings suggest that more alkaline environments enhance the optimized performance of BCA. Additionally, similar to the observations made for PLCA, the concentration of the flocculant within the optimal pH range does not significantly affect efficiency, thereby ensuring the robustness of the process. Conversely, when the bioflocculant is applied at pH levels below 7.5, there is a notable decline in efficiency, with values dropping below 70%. In contrast to the previously presented results, the PCA emerges as an even more promising alternative, demonstrating higher efficiencies under similar pH conditions compared to other clarifying agents, while requiring lower concentration. As illustrated in Fig. 7(b), within the pH range of 7.2 to 9.5, variations in concentration do not significantly affect clarification efficiency, which consistently remains above 95%. However, it is noteworthy that within this pH range, concentrations exceeding 2.5 g/L achieve efficiency levels close to 100%. These findings are further supported by the analysis in Fig. 6(a), which highlights the relationship between pH and concentration in the clarification process, underscoring the importance of considering multiple factors when designing wastewater treatment strategies. Additionally, FTIR analyses validate these results, revealing more intense peaks for hydroxyl, carbonyl, and sulfonic acid functional groups in the bioflocculant derived from porcine plasma. From Table 2, one can compare the optimal results achieved for each clarifying agent with the standardized calcium carbonate solution, in relation to the sample that did not have a flocculant agent added. Table 2 – Efficiency of clarification as a function of medium pH, concentration and flocculant origin. Clarifying agent pH Concentration – g.L − 1 Initial Turbidity – NTU Final Turbidity – NTU Efficiency (η) – % Sample 0 8.2 – 453.0 412.0 9.10 Poultry 8.0 0.09 534.0 28.6 94.64 8.0 1.50 534.0 29.0 94.57 8.0 2.91 534.0 27.9 94.78 Bovine 8.0 0.09 452.0 23.5 94.80 8.0 1.50 452.0 22.3 95.07 8.0 2.91 452.0 27.9 93.83 Porcine 8.0 1.50 494.0 26.2 94.70 10.0 2.50 456.0. 22.2 95.13 8.0 2.91 439.0 26.8 94.03 The results obtained, as demonstrated above, reveal a promising performance of the proposed method in comparison to previous studies. For instance, the results from Medeiros [4], who investigated the efficacy of bioflocculants derived from Moringa oleifera , showed turbidity removal efficiencies of around 87%. Similarly, studies conducted by Amran et al. [52] reported turbidity removal rates of approximately 88% using products derived from Carica papaya seeds. Lee et al. [53] examined the clarification efficiency of kaolin/hematite using modified hemoglobin at different dosages and pH levels, discovering that the optimal dosage of hematite achieved a turbidity reduction of 99%. In another study, Lee et al. [54] analyzed native bovine blood and three types of chemically modified blood in comparison to kaolin and hematite suspensions, with polymerized bovine blood demonstrating an 81% precipitation rate with hematite suspensions. It has been also observed that the combination of adding a clarifying agent to the solution, along with pH adjustment, significantly enhances the sedimentation process of colloidal particles present in the initial solution compared to the same solution without the clarifying agent. The bioflocculant prepared from porcine plasma (PCA) demonstrated high removal efficiency across a broad pH spectrum, achieving removal rates exceeding 95%. These results underscore the synergy between the clarifying agent's action and environmental conditions, highlighting the effectiveness of this combined approach for efficiently removing suspended impurities from surface water. 4. Conclusion This study focused on the utilization of porcine, bovine, and poultry blood plasma for the synthesis of a bioflocculant applicable in water treatment. The characterization of the resulting clarifying agents was conducted using PZC, resulting in isoelectric points of 6.41, 6.47, and 6.64 for PCA, BCA, and PLCA, respectively. FTIR analyses indicate alterations in the spatial structure of the protein complexes, emphasizing the presence of amide groups such as and functional groups –CH, –CO, –OH, and H–S(=O) 2 –OH on the peripheral regions of the molecules, which enhance the capacity to remove colloidal particles. The thermal stability of the materials was confirmed by TGA, demonstrating the thermal robustness of the synthesized compounds within a temperature range of 40 °C to 400 °C. According to ANOVA analysis, it was determined that only the origin of the blood and pH significantly affect the process efficiency, with optimal performance observed at alkaline pH levels, where the electrostatic affinity of the molecules facilitates the formation of flocs and the sedimentation of particulate matter. The efficiency values obtained during the research exceeded 95%, 93%, and 90% for PCA, BCA, and PLCA, respectively. These results indicate the successful development of efficient bioflocculants derived from waste, produced through a simple and cost-effective method. Furthermore, the findings highlight the significant potential for research and innovation in the development of water clarifiers from plasma proteins, presenting a promising area for further research and process optimization. Declarations Acknowledgements The authors would like to thank the Community University of Chapecó Region (Unochapecó) for the institutional support provided during the development of this work. Funding sources This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Okumura, A. T. R., Silva, A. G., Silva, N. R. S., Lopes, E. R. N., Bifano, R. B. A., & Quilenato, R. V. (2020). Determinação da qualidade da água de um rio tropical sob a perspectiva do uso do solo e cobertura vegetal. Revista Brasileira de Geografia Física, 13 (4), 1835–1850. Coelho, F. R., De Rubin, J. C. R., & Silva, A. M. T. C. (2021). Análise de qualidade da água no alto curso do rio Meia Ponte entre 2013 e 2018. 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P., & Liang, C. (2022). Influence of methylation and polymerization on flocculant properties of bovine blood. ACS Omega, 7 (3), 3037–3043. https://doi.org/10.1021/acsomega.1c06126 U.S. Environmental Protection Agency. (2024). pH . Washington, DC. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.png Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Water Conservation Science and Engineering → Version 1 posted Editorial decision: Revision requested 21 Jul, 2025 Editor assigned by journal 15 Jul, 2025 Submission checks completed at journal 15 Jul, 2025 First submitted to journal 15 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7131666","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488883956,"identity":"56fdba1f-bb2b-4178-ac40-20d3e08d8458","order_by":0,"name":"João Paulo 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(UNOCHAPECÓ)","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"Lopes","lastName":"Colpani","suffix":""}],"badges":[],"createdAt":"2025-07-15 14:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7131666/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7131666/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s41101-025-00457-x","type":"published","date":"2025-11-18T15:58:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91622517,"identity":"c075c2da-da9b-4ccd-a613-cb98763c313a","added_by":"auto","created_at":"2025-09-18 11:42:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1024854,"visible":true,"origin":"","legend":"\u003cp\u003ePoint Zero Charge (PZC) as a function of varying initial pH for PCA, BCA, and PLCA.\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors, 2024.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/cc843dd7cd164ca92f1c924b.png"},{"id":91622519,"identity":"00788b5e-9b52-454b-b12b-0cea8526672b","added_by":"auto","created_at":"2025-09-18 11:42:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":250014,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of FTIR spectra of porcine (a), bovine (b), and poultry (c) blood plasmas with their respective clarifying agents.\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors, 2024.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/e3ebc1a6cd30d468b7617914.png"},{"id":91623000,"identity":"df325dcf-ae0e-4791-b8ea-6e95d406ed75","added_by":"auto","created_at":"2025-09-18 11:50:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1117602,"visible":true,"origin":"","legend":"\u003cp\u003eTGA curves for the clarifying agents (a) and pure plasmas (b).\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/c0b7414cb4bdc05953bf8358.png"},{"id":91622999,"identity":"5869c95a-7b23-4e91-9b2f-9dbf518cefa1","added_by":"auto","created_at":"2025-09-18 11:50:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104631,"visible":true,"origin":"","legend":"\u003cp\u003ePareto chart of standardized effects.\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors, 2024.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/35ce4863235729909955127e.png"},{"id":91622524,"identity":"f7ece637-a707-48b1-89cc-1b64899e9456","added_by":"auto","created_at":"2025-09-18 11:42:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":197583,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface for the variation of flocculant dosage and pH on the surface (a) and contour (b) for poultry clarifying agent (PLCA).\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/e69c55c5fceb63aebc0d0c58.png"},{"id":91622521,"identity":"717302f1-b59b-4a5e-bfd0-c4eb8a684ad4","added_by":"auto","created_at":"2025-09-18 11:42:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":263554,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface for the variation of flocculant dosage and pH on the surface (a) and contour (b) for bovine clarifying agent (BCA).\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/5f63f202c4c04da0d35612fd.png"},{"id":91622523,"identity":"e5813213-e04f-418c-b633-e84a91a28175","added_by":"auto","created_at":"2025-09-18 11:42:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":224490,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface for the variation of flocculant dosage and pH on the surface (a) and contour (b) for porcine clarifying agent (PCA).\u003c/p\u003e\n\u003cp\u003eSource: Elaborated by the authors.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/173c906094638839143cec99.png"},{"id":96650382,"identity":"ab861daf-ccaf-49bf-a56f-21e7d821650d","added_by":"auto","created_at":"2025-11-24 16:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3456621,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/d4165914-32e7-4ba3-9f72-7b7cd0bddb8e.pdf"},{"id":91622522,"identity":"fcc8106a-6459-4b9b-be1e-29f19d4b18b6","added_by":"auto","created_at":"2025-09-18 11:42:05","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":119113,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7131666/v1/3117681f26af11c0d605ac28.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of a Bioflocculant Using Blood Plasma: A Promising Approach for Sustainable Water Treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWater bodies, in general, play an extremely vital role in the economic development of a country, besides being essential for human activities and survival [1]. Human practices in the environment slowly and gradually cause alterations that, over time, become more pronounced in water sources, posing risks to public health, impacting the environment, and hindering economic and social growth [2].\u003c/p\u003e\n\u003cp\u003eWater treatment plays a crucial role in this scenario as it aims to eliminate pathogens, impurities, and harmful substances, ensuring that the water meets the standards set by health authorities [3]. Among the indicators of water quality, turbidity is an important criterion, usually related to the efficiency of physical treatments for solid removal, where suspended particles and dissolved algae hinder light penetration. Compliance with this parameter, which is an aesthetic criterion that impacts the acceptance or rejection of the product, can be ensured through clarification treatments, which involve water purification processes such as coagulation/flocculation and sedimentation [4].\u003c/p\u003e\n\u003cp\u003eThese processes are controlled by the charge neutralization mechanism [5]. This neutralization occurs in the presence of a flocculating agent with a charge opposite to that of the colloidal particles. The added product adsorbs and neutralizes the charges of the suspended particles, reducing the zeta potential (\u0026zeta;) and the repulsive forces between the colloidal particles. Thus, it facilitates the aggregation of the suspended material to form flocs [6].\u003c/p\u003e\n\u003cp\u003eIn water treatment systems, both in Brazil and other countries, the most commonly used coagulants are derived from inorganic compounds, mainly trivalent iron and aluminum salts, as well as synthetic polymers, as indicated by Choy et al. [7] and Franco et al. [8]. However, there are growing concerns about the potential environmental impacts of these coagulants due to the presence of non-biodegradable and potentially ecotoxic residues in both the treated water and the sludge generated during coagulation and flocculation processes, as reported by Yegambaram et al. [9], Freitas et al. [10], and Michelan et al. [11]. In response to these challenges, interest has emerged using of natural coagulants, which are seen as a viable alternative due to their biodegradability and low toxicity [4].\u003c/p\u003e\n\u003cp\u003eAccording to Essandoh et al. [12] and Tannouri and Simmons [13], hemoglobin, the most abundant protein in blood, and serum albumins, which are the most abundant proteins in blood plasma, have been recognized for their potential to form agglutinations with other materials. Koul et al. [14] and Nath et al. [15] also identified that these protein molecules play a fundamental role in coagulation and flocculation processes. This is related to the fact that the net charge of a protein is affected by the pH of the medium in which it is found. Piazza and Garcia [16] describe that the protein acquires a negative charge when the pH exceeds the isoelectric point. However, when the pH is below this point, the protein\u0026apos;s net charge becomes positive, and due to this, Quintapana [17] suggests its use as a possible clarifying agent.\u003c/p\u003e\n\u003cp\u003eThe work carried out by Koul et al. [14] and Nath et al. [15] aligns with projections for the production of bovine, porcine, and poultry meat in Brazil. Such projections suggest an approximate production of 33 million tons of meat by the end of 2029 [18]. This increase in production will result in a greater generation of hard-to-treat waste, such as blood. This amount will produce approximately 11 million tons of blood, which is usually disposed of without proper treatment [19, 20]. As cited by Wang et al. [21], blood accounts for 7% to 11% of slaughterhouse waste. The direct discharge of untreated blood into sewage systems increases the organic pollution load in wastewater by 35% to 50%, posing risks to the environment [20].\u003c/p\u003e\n\u003cp\u003eThus, it is understood that the low utilization of this waste, especially in less qualified facilities, is a major concern. It is imperative to implement measures to avoid this waste, which results in significant environmental pollution, economic losses, and the diversion of raw materials [22].\u003c/p\u003e\n\u003cp\u003eConsidering the aforementioned context, there arises a need to explore the feasibility of a clarifying agent based on animal blood plasma. In this context, this project aims to synthesize and evaluate the efficacy of bioflocculants derived from plasma extracted from bovine, porcine, and poultry blood in reducing the turbidity of aqueous solutions. This study will investigate the performance of different bioflocculant concentrations across various pH ranges, comparing their effects with one another. This approach seeks to provide sustainable and effective alternatives for water treatment, contributing to reducing environmental impact and promoting the responsible use of natural resources.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Reagents\u003c/h2\u003e\u003cp\u003eThe reagents used in the experimental procedures included: EDTA tetrasodium (C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eNa\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO) of analytical grade, supplied by the brand \u0026Ecirc;xodo Cient\u0026iacute;fica; calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) of analytical grade, supplied by Din\u0026acirc;mica Qu\u0026iacute;mica Contempor\u0026acirc;nea LTDA; sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) and sodium chloride (NaCl), both of analytical grade, supplied by Vetec Qu\u0026iacute;mica Fina; sodium hydroxide (NaOH) of analytical grade, supplied by Reatec; and hydrochloric acid (HCl) 37% of analytical grade, supplied by ACS Cient\u0026iacute;fica.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Plasma Collection and Treatment\u003c/h2\u003e\u003cp\u003ePorcine, bovine, and poultry blood was collected from slaughterhouses located in the western region of Santa Catarina \u0026ndash; Brazil, using EDTA 4Na as an anticoagulant at a concentration of 1.5 g/L of pure blood, following the methodology adapted from Kundu et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs cited by Rakesh et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the most conventional process for plasma separation is centrifugation. The blood plasma was separated immediately after collection in a FANEM centrifuge, model 206 BL, at a rotation speed of 3600 rpm for 20 minutes. After phase separation, the supernatant plasma was removed, and the solid residue of blood cells was appropriately discarded for subsequent treatment.\u003c/p\u003e\u003cp\u003eThe bioflocculant was obtained using the acid hydrolysis technique, following the methodology adapted from Ara\u0026uacute;jo et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The plasma was placed in a jacketed reactor, and the pH was adjusted to 3.5 using 0.2 M sulfuric acid. Agitation was applied using a FISATOM \u0026minus;\u0026thinsp;713D agitator at a speed of 300 rpm. The solution remained in this state for 4 hours at a constant temperature of 60\u0026deg;C. Subsequently, the solution was neutralized to a pH of 6.0 using 0.1 M sodium hydroxide. The pH measurements were carried out using a KASVI pH meter, model K39-1420A. The remaining solution was placed in Petri dishes and dried in a LOGEN oven, model LSDH-9140A-220, for 20 hours at 60\u0026deg;C to remove moisture. After drying, an IKA grinder, model A11BS032, was used to grind the material into powder for subsequent analysis. The powder was stored in a sealed container at room temperature, protected from light. To standardize the nomenclature and facilitate the interpretation of the experimental data, the clarifying agents derived from bovine, porcine, and poultry plasma will be referred to as BCA (Bovine Clarifying Agent), PCA (Porcine Clarifying Agent), and PLCA (Poultry Clarifying Agent), respectively, throughout this study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Point Zero Charge (PZC)\u003c/h2\u003e\u003cp\u003eThe Point Zero Charge (PZC) parameter indicates the pH at which the net surface charge of a material is zero, providing crucial information about the electrostatic interactions between the material\u0026rsquo;s surface and charged species [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The methodology used for pHpzc determination was adapted from Hirendrasinh et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A 0.01 M NaCl solution was prepared, and 100 mL of this solution was added to different Erlenmeyer flasks, adjusting each one to pH values ranging from 2 to 12 using 0.1 M HCl and 0.1 M NaOH solutions. Subsequently, 0.4 g of the flocculant was added to each flask. The solutions were allowed to react for 48 hours in a SOLAB Shaker SL-222 incubator at 130 rpm and room temperature. The pHpzc value was estimated from the intersection of the final pH in relation to the initial pH.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\u003cp\u003eFTIR analysis is used to study the secondary structures and functional groups of proteins, allowing detailed analysis of molecular interactions by correlating them with the vibrations emitted by chemical bonds [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The FTIR spectra of the flocculants were obtained using an IRSpirit (Shimadzu) spectrophotometer at a wavelength range of 4000 to 400 cm⁻\u0026sup1;. The dry material samples were analyzed using the ATR (Attenuated Total Reflectance) method, with a prior background evaluation (blank).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Thermogravimetric Analysis (TGA)\u003c/h2\u003e\u003cp\u003eTGA is a technique used to determine the composition of materials, including the quantities of highly-volatile matter, medium-volatility materials, combustible material, and ash content (ASTM E1131-20). TGA was conducted on a mass of approximately 30 mg of pure plasma and bioflocculant from each type of blood, placed in an open alumina sample holder. The analysis conditions were carried out with a heating rate of 10\u0026deg;C/min, from 40\u0026deg;C to 600\u0026deg;C, under a nitrogen atmosphere at a flow rate of 50 mL/min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Clarification Studies\u003c/h2\u003e\u003cp\u003eThe analyses to determine the ideal bioflocculant and its optimal performance parameters were conducted through experimental design using Response Surface Methodology (RSM) with Central Composite Rotational Design (CCRD), which determined the variation of concentrations and pH of the plasma samples selected for the study. The methodology used for the clarification study was adapted from Wang et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Pacheco et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], in which a standardized solution of calcium carbonate (CaCO₃) in water with a concentration of 7 g/L was prepared under agitation for 5 minutes, until complete homogenization.\u003c/p\u003e\u003cp\u003eFour hundred mL of the standardized calcium carbonate solution were transferred to 1000 mL beakers allocated in equipment specifically designed for the Jar Test method. The pH of the calcium carbonate solution was adjusted using 0.1 M sulfuric acid (H₂SO₄) and sodium hydroxide (NaOH) solutions. After the pH adjustment, a 15 mL aliquot of the standardized solution was collected to determine the initial turbidity using an AKSO Turbidity Max device. Next, the bioflocculant was added to the solution, and agitation was initiated at 160 rpm for 2 minutes to disperse the material in the solution. Subsequently, the system was maintained at 60 rpm for 5 minutes for floc formation. After this period, the agitation was turned off, and the samples were left undisturbed for 40 minutes to allow for the decantation of the particulate matter. After this decantation time, 80 mL aliquots were withdrawn for the final turbidity measurement. The process was performed using calcium carbonate samples adjusted to pH values of 4.2, 5.0, 7.0, 9.0, and 9.8, with varying concentrations of 0.3, 0.5, 1.0, 1.5, and 1.7 g/L, according to the experimental design. A sample of the calcium carbonate solution was maintained without the addition of bioflocculant to compare the effect of the obtained material with the natural decantation process of calcium carbonate.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e3.1. Material Characterization\u003c/h2\u003e\n \u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.1.1. Point Zero Charge (PZC)\u003c/h2\u003e\n \u003cp\u003eThe pHpzc values of porcine (PCA), bovine (BCA), and poultry (PLCA) clarifying agents were approximately 6.41, 6.47, and 6.64, respectively, as shown in Fig. 1.\u003c/p\u003e\n \u003cp\u003eFigure 1 reveals that the pHpzc values are within a neutral range, with values close to 6.5. This result demonstrates that the PCA, BCA, and PLCA flocculants can be more efficient in removing anionic contaminants when the pH of the solution is above the pHpzc. This is because the surface of the flocculants under these conditions is positively charged, maximizing the attraction effects described by Coulomb\u0026apos;s Law, as observed in Deng et al. [22], thus favoring the removal of anionic species. However, in acidic pH, flocculation efficiency is compromised due to the increase in electrostatic repulsion.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.1.2. Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\n \u003cp\u003eFigure 2 illustrates the FTIR spectra for porcine, bovine, and poultry blood plasmas, along with their respective clarifying agents.\u003c/p\u003e\n \u003cp\u003eThe general analysis of the infrared spectra reveals that the shape and position of the peaks remain consistent between different samples, indicating the structural stability of the protein components after acid hydrolysis treatment. This finding suggests that the process does not promote a complete breakdown of protein structures nor the significant formation of new functional groups, as noted by Wang et al. [19], with only a change in the intensity of the existing bands.\u003c/p\u003e\n \u003cp\u003eDeeper analysis of the FTIR spectra, as illustrated in Figs. 2a, 2b, and 2c, reveal that wavelengths near 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate the presence of characteristic amide A bands, which are attributed to the stretching vibrations of \u0026ndash;NH groups, as described by Singh et al. [23] and Kong et al. [17]. This evidence suggests the presence of partially preserved protein structures after treatment. Furthermore, additional studies by Garcia and Wilson [24] and Agbovi and Wilson [25] support these findings by reporting distinct oscillations around 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e associated with the stretching of \u0026ndash;NH\u003csub\u003e3\u003c/sub\u003e, \u0026ndash;CH, \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e, and \u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e groups. These results imply the formation of the amide B band, indicating spatial changes in the structure of the treated proteins.\u003c/p\u003e\n \u003cp\u003eStudies by Wang et al. [19], Singh et al. [23], and Garcia and Wilson [24] also highlight the presence of multiple adsorption peaks in the infrared region, particularly within the amide I and amide II bands, which reveal significant details about the protein structures in question. Specifically, the amide I bands, located nearly 1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, are primarily attributed to the stretching vibrations of the C\u0026thinsp;=\u0026thinsp;O, \u0026ndash;CO, and \u0026ndash;COO\u0026ndash; groups. On the other hand, the amide II bands, around 1500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, are associated with the vibrations and shortening of the \u0026ndash;CN groups. Both are observed with greater intensity in porcine and bovine plasmas, as well as in and clarifying agents, as illustrated in Figs. 2a and 2b. These findings are consistent with previous studies conducted by Wei et al. [26] and Kong et al. [17], further reinforcing the reliability and consistency of the obtained results.\u003c/p\u003e\n \u003cp\u003eA significant increase in stretching around the 1100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e wavelength was also observed in Fig. 2a, indicating specific alterations in the FTIR spectrum for CS. Recent studies by Mishra et al. [27], Safdar et al. [28], and Singh et al. [23] emphasize that this behavior is closely related to the vibrations of the \u0026ndash;CO, \u0026ndash;C\u0026ndash;OH, \u0026ndash;COC, and H\u0026ndash;S(=\u0026thinsp;O)\u003csub\u003e2\u003c/sub\u003e\u0026ndash;OH groups. These findings suggest subtle structural modifications in plasma proteins. According to Saguer, \u0026Aacute;lvarez, and Ismail [29], the hydrolysis process at acidic pH, combined with exposure to high temperatures, promotes the partial denaturation of molecules. Meutter and Goormaghtigh [30] discuss how this process disrupts the secondary and tertiary conformations of the protein, altering its spatial arrangement.\u003c/p\u003e\n \u003cp\u003eThe presence of carboxyl, hydroxyl, and amine groups in bioflocculant molecules is critical for determining their flocculation effect, as they promote suitable binding sites that attract dispersed particles in solution, thereby enhancing the flocculants effective in binding mechanisms between the particles and the flocculants [31\u0026ndash;34]. Another significant factor that may improve flocculant activity is the introduction of sulfonic acid functional groups on plasma surfaces after acid hydrolysis, as indicated by peaks observed around 1110 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [35, 36]. According to Yu et al. [37], Feng et al. [26] and Artifon et al. [38], the presence of these groups in bioflocculants enhances their capacity to remove colloidal particles and suspended metals.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.1.3. Thermogravimetric Analysis (TGA)\u003c/h2\u003e\n \u003cp\u003eThermogravimetric analysis (TGA) was conducted on flocculants and pure plasmas to elucidate their thermal responses at different temperature ranges. Figure 3 illustrates the TGA curves, emphasizing the mass losses associated with thermal decomposition, oxidation, and volatilization [25].\u003c/p\u003e\n \u003cp\u003eExamining Figs. 3a and 3b, it is observed that the initial mass loss occurs between 40\u0026deg;C and 270\u0026deg;C, primarily due to water desorption and the volatilization of low-molecular-weight compounds [24]. Between 270\u0026deg;C and 400\u0026deg;C, significant mass loss is attributed to the removal of moisture associated with hydroxyl and carboxyl functional groups [39]. The final stage of mass loss, observed above 400\u0026deg;C, results from the thermal degradation of the polymer matrix and the decomposition of organic material [24]. The average mass loss percentages for the three thermal zones in the flocculants were 16.27%, 45.21%, and 26.38%, respectively. In contrast, for pure plasma, the mass losses in the same zones were 19.05%, 49.41%, and 29.48%, respectively.\u003c/p\u003e\n \u003cp\u003eThese data indicate that both the flocculants and pure plasmas exhibit three distinct stages of thermal degradation. The substantial mass loss observed between 270\u0026deg;C and 400\u0026deg;C suggests significant volatilization of components and structural dehydration. In contrast, the degradation occurring above 400\u0026deg;C reflects the disintegration of the polymer structure and the decomposition of organic constituents. The higher mass loss in pure plasma across all zones, compared to the flocculants, indicates a composition with a higher volatile content and a more thermally susceptible organic structure.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.2. Clarification Studies\u003c/h2\u003e\n \u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e3.2.1. Mathematical modeling\u003c/h2\u003e\n \u003cp\u003eGiven that the quality of effluent treatment is intrinsically linked to effluent parameters, such as pH, mathematical modeling emerges as a key resource for optimizing procedures and predicting the efficiency of these processes [40]. Based on the results obtained from the CCRD experimental design, an analysis was conducted using Minitab software for statistical evaluation and the development of a mathematical model that describes the relationship between bioflocculant dosage, its source, the characteristics of the effluent to be treated, and flocculation efficiency. The interactions between parameters, as determined through statistical analysis, are illustrated in Fig. 4 and Table 1.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003e\u0026ndash; \u003cem\u003ep-value\u003c/em\u003e for analysis of variance (ANOVA).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLinear\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSquare\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eTwo-factor interaction\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026delta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u0026sup2;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u0026sup2;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC x pH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eC x \u0026delta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003epH x \u0026delta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.026\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eC \u0026ndash; Concentration; pH \u0026ndash; Hydrogen potential; \u0026delta; \u0026ndash; Blood type.\u003c/p\u003e\n \u003cp\u003eSource: Elaborated By the authors.\u003c/p\u003e\n \u003cp\u003eUsing ANOVA statistical analysis with a significance level of 5%, it was determined that the parameters that significantly influence the model are pH in its linear form, pH in its squared form (pH\u0026sup2;), and the combination of pH and blood type (\u0026delta;) through a two-factor interaction. Consequently, the mathematical models 1, 2, and 3, which define the efficiency (\u0026eta;) of the bioflocculant, are presented in Equations 1 to 3 for porcine, bovine, and poultry, respectively.\u003c/p\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\\(\\:{\\eta\\:}_{porcine}=-0.659+\\left(0.358\\times\\:pH\\right)-(0.02306\\times\\:{pH}^{2})\\)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(1)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\\(\\:{\\eta\\:}_{bovine}=-1.194+\\left(0.443\\times\\:pH\\right)-(0.02306\\times\\:{pH}^{2})\\)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\\(\\:{\\eta\\:}_{poultry}=-0.472+\\left(0.360\\times\\:pH\\right)-(0.02306\\times\\:{pH}^{2})\\)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e3.2.2. Evaluation of Efficiencies Using the Response Surface Methodology (RSM)\u003c/h2\u003e\n \u003cp\u003eThe removal of suspended solids from the solution was evaluated through coagulation and flocculation processes using the Jar Test. Two primary effects were investigated to assess process efficiency: the variation in flocculant concentration and the pH of the medium. The results obtained through the RSM method are presented in Figs. 5, 6, and 7.\u003c/p\u003e\n \u003cp\u003eBased on the results presented, distinct performance behaviors for each of the evaluated clarifying agents are evident. Current legislation in various countries, including Brazil\u0026apos;s Portaria GM/MS N\u0026ordm;. 888, the European Parliament Directive (EU) 2020/2184 [50], and the U.S. Environmental Protection Agency (EPA) Primary Drinking Water Regulations [55], indicates that fresh surface water typically has pH values ranging from 6.5 to 9.0. This range aligns with the experimental pH levels that demonstrated good turbidity reduction efficiency.\u003c/p\u003e\n \u003cp\u003eAnalyzing Fig. 5, which illustrates the behavior of the PLCA, reveals a clear correlation between pH and product concentration. The optimal pH range for achieving efficiencies above 94% is between 7.5 and 8.2, with bioflocculant concentrations exceeding 1.3 g/L. Additionally, broader pH ranges, varying from 6.7 to 9.0, are noteworthy as they demonstrate efficiencies above 90% regardless of bioflocculant concentration. This behavior indicates that within this broader pH range, variations in clarifying agent concentration do not significantly affect treatment efficiency. Such robustness suggests valuable operational flexibility, allowing for adjustments in clarifying agent dosage without compromising process effectiveness. However, at pH levels below 6.5 or above 9.2, even changes in clarifying agent concentration fail to maintain the desired efficiency, which drops below 80%.\u003c/p\u003e\n \u003cp\u003eIn case of BCA, as illustrated in Fig. 6, the most promising results are observed within more alkaline pH ranges, specifically between 8.0 and approximately 11.0, with efficiencies exceeding 88% across various concentrations. The optimal range is identified as being between pH 9.0 and 10.0, where efficiencies surpass 93%. These findings suggest that more alkaline environments enhance the optimized performance of BCA. Additionally, similar to the observations made for PLCA, the concentration of the flocculant within the optimal pH range does not significantly affect efficiency, thereby ensuring the robustness of the process. Conversely, when the bioflocculant is applied at pH levels below 7.5, there is a notable decline in efficiency, with values dropping below 70%.\u003c/p\u003e\n \u003cp\u003eIn contrast to the previously presented results, the PCA emerges as an even more promising alternative, demonstrating higher efficiencies under similar pH conditions compared to other clarifying agents, while requiring lower concentration. As illustrated in Fig. 7(b), within the pH range of 7.2 to 9.5, variations in concentration do not significantly affect clarification efficiency, which consistently remains above 95%. However, it is noteworthy that within this pH range, concentrations exceeding 2.5 g/L achieve efficiency levels close to 100%. These findings are further supported by the analysis in Fig. 6(a), which highlights the relationship between pH and concentration in the clarification process, underscoring the importance of considering multiple factors when designing wastewater treatment strategies. Additionally, FTIR analyses validate these results, revealing more intense peaks for hydroxyl, carbonyl, and sulfonic acid functional groups in the bioflocculant derived from porcine plasma.\u003c/p\u003e\n \u003cp\u003eFrom Table 2, one can compare the optimal results achieved for each clarifying agent with the standardized calcium carbonate solution, in relation to the sample that did not have a flocculant agent added.\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003e\u0026ndash; Efficiency of clarification as a function of medium pH, concentration and flocculant origin.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClarifying agent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration \u0026ndash; g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInitial Turbidity \u0026ndash; NTU\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFinal Turbidity \u0026ndash; NTU\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEfficiency (\u0026eta;) \u0026ndash; %\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample 0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e453.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e412.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003ePoultry\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e534.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e534.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e534.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eBovine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e452.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e452.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e452.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003ePorcine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e494.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e456.0.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e439.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eThe results obtained, as demonstrated above, reveal a promising performance of the proposed method in comparison to previous studies. For instance, the results from Medeiros [4], who investigated the efficacy of bioflocculants derived from \u003cem\u003eMoringa oleifera\u003c/em\u003e, showed turbidity removal efficiencies of around 87%. Similarly, studies conducted by Amran et al. [52] reported turbidity removal rates of approximately 88% using products derived from \u003cem\u003eCarica papaya\u003c/em\u003e seeds. Lee et al. [53] examined the clarification efficiency of kaolin/hematite using modified hemoglobin at different dosages and pH levels, discovering that the optimal dosage of hematite achieved a turbidity reduction of 99%. In another study, Lee et al. [54] analyzed native bovine blood and three types of chemically modified blood in comparison to kaolin and hematite suspensions, with polymerized bovine blood demonstrating an 81% precipitation rate with hematite suspensions.\u003c/p\u003e\n \u003cp\u003eIt has been also observed that the combination of adding a clarifying agent to the solution, along with pH adjustment, significantly enhances the sedimentation process of colloidal particles present in the initial solution compared to the same solution without the clarifying agent. The bioflocculant prepared from porcine plasma (PCA) demonstrated high removal efficiency across a broad pH spectrum, achieving removal rates exceeding 95%. These results underscore the synergy between the clarifying agent\u0026apos;s action and environmental conditions, highlighting the effectiveness of this combined approach for efficiently removing suspended impurities from surface water.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study focused on the utilization of porcine, bovine, and poultry blood plasma for the synthesis of a bioflocculant applicable in water treatment. The characterization of the resulting clarifying agents was conducted using PZC, resulting in isoelectric points of 6.41, 6.47, and 6.64 for PCA, BCA, and PLCA, respectively. FTIR analyses indicate alterations in the spatial structure of the protein complexes, emphasizing the presence of amide groups such as and functional groups \u0026ndash;CH, \u0026ndash;CO, \u0026ndash;OH, and H\u0026ndash;S(=O)\u003csub\u003e2\u003c/sub\u003e\u0026ndash;OH on the peripheral regions of the molecules, which enhance the capacity to remove colloidal particles. The thermal stability of the materials was confirmed by TGA, demonstrating the thermal robustness of the synthesized compounds within a temperature range of 40 \u0026deg;C to 400 \u0026deg;C. According to ANOVA analysis, it was determined that only the origin of the blood and pH significantly affect the process efficiency, with optimal performance observed at alkaline pH levels, where the electrostatic affinity of the molecules facilitates the formation of flocs and the sedimentation of particulate matter.\u003c/p\u003e\n\u003cp\u003eThe efficiency values obtained during the research exceeded 95%, 93%, and 90% for PCA, BCA, and PLCA, respectively. These results indicate the successful development of efficient bioflocculants derived from waste, produced through a simple and cost-effective method. Furthermore, the findings highlight the significant potential for research and innovation in the development of water clarifiers from plasma proteins, presenting a promising area for further research and process optimization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Community University of Chapec\u0026oacute; Region (Unochapec\u0026oacute;) for the institutional support provided during the development of this work. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOkumura, A. 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Washington, DC.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"water-conservation-science-and-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wcse","sideBox":"Learn more about [Water Conservation Science and Engineering](http://link.springer.com/journal/41101)","snPcode":"41101","submissionUrl":"https://submission.nature.com/new-submission/41101/3","title":"Water Conservation Science and Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bovine, porcine and poultry plasma, natural coagulants, protein-based flocculants, water treatment","lastPublishedDoi":"10.21203/rs.3.rs-7131666/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7131666/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe exponential growth of urban centers and the scarcity of treated water underscore the urgent need for environmental stewardship. The rising demand for alternatives to replace inorganic compounds in water treatment presents a significant opportunity to develop sustainable methods. This study evaluates the use of blood plasma-derived bioflocculants as a promising alternative. The methodology involves the separation and chemical treatment of porcine, bovine, and poultry blood plasma through acid hydrolysis with 0.1 M sulfuric acid to denature proteins, followed by drying and grinding to obtain a fine powder. The clarifying agents were characterized using Point Zero Charge (PZC), Fourier Transform Infrared Spectroscopy (FTIR), and Thermogravimetric Analysis (TGA), while turbidity removal efficiency was assessed using Response Surface Methodology with Central Composite Rotational Design (RSM–CCRD). The clarifying agents exhibited isoelectric points of 6.41, 6.47, and 6.64 for porcine, bovine, and poultry plasma, respectively. FTIR analysis confirmed amide groups and functional groups –CH, –CO, –OH, and H–S(=O)\u003csub\u003e2\u003c/sub\u003e–OH, demonstrating potential for coagulation. TGA results indicated good thermal stability between 40 °C and 400 °C. The findings revealed turbidity removal efficiencies of 95%, 93%, and 90% for the agents derived from porcine, bovine, and poultry plasma, respectively. These results highlight the potential for innovation in developing water clarifiers from slaughterhouse by-products.\u003c/p\u003e","manuscriptTitle":"Synthesis of a Bioflocculant Using Blood Plasma: A Promising Approach for Sustainable Water Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 11:42:00","doi":"10.21203/rs.3.rs-7131666/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-22T03:07:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T22:48:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-15T22:48:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Water Conservation Science and Engineering","date":"2025-07-15T14:25:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"water-conservation-science-and-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wcse","sideBox":"Learn more about [Water Conservation Science and Engineering](http://link.springer.com/journal/41101)","snPcode":"41101","submissionUrl":"https://submission.nature.com/new-submission/41101/3","title":"Water Conservation Science and Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"55507121-90c0-465a-bdba-61e9dd78653f","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:07:37+00:00","versionOfRecord":{"articleIdentity":"rs-7131666","link":"https://doi.org/10.1007/s41101-025-00457-x","journal":{"identity":"water-conservation-science-and-engineering","isVorOnly":false,"title":"Water Conservation Science and Engineering"},"publishedOn":"2025-11-18 15:58:52","publishedOnDateReadable":"November 18th, 2025"},"versionCreatedAt":"2025-09-18 11:42:00","video":"","vorDoi":"10.1007/s41101-025-00457-x","vorDoiUrl":"https://doi.org/10.1007/s41101-025-00457-x","workflowStages":[]},"version":"v1","identity":"rs-7131666","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7131666","identity":"rs-7131666","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}