The distribution of phosphorus forms in dewatered sludge with three classification analysis methods

The distribution of phosphorus forms in dewatered sludge with three classification analysis methods | 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 The distribution of phosphorus forms in dewatered sludge with three classification analysis methods Zhigang Liu, Junjie He, Siqi Zhou, Xiaohu Dai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7369225/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Determining the distribution of phosphorus forms in dewatered sludge is crucial, as it directly impacts the choice and effectiveness of recovery strategies. Analyses using the standards measurements and testing (SMT) method, soluble/insoluble fractionation and extracellular polymeric substances (EPS) /EPS residue extraction, revealed that sludge characteristics significantly influenced phosphorus speciation and content. Inorganic phosphorus (IP) was the dominant form in dewatered sludge, primarily regulated by the levels of Al, Fe, and Ca, while dissolved orthophosphate (ortho-P) constituted only 1% of the total phosphorus (TP). Notably, phosphorus exhibited a distinct distribution pattern between EPS and EPS residues: EPS comprised only 2.09% of TP, 74.19% of which was organic phosphorus (OP), whereas EPS residues contained 93.26% of TP, with a much lower OP proportion (15.04%). Phosphorus form dewatered sludge Standards Measurements and Testing (SMT) soluble/insoluble Extracellular Polymeric Substances (EPS)/EPS residue Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Unlike nitrogen, which cycles naturally in the biosphere, phosphorus behaves as a non-renewable resource in the biosphere because of its predominantly unidirectional flows (Venkiteshwaran et al., 2018 ). Also, phosphorus is an indispensable element for the survival of both humans and animals, and a major limiting element for primary producers such as plants and microorganisms (Darcy et al., 2018 ). However, global phosphate rock reserves-estimated by the United States Geological Survey and other studies-may only sustain current consumption for another 100 years, if no alternative phosphorus sources are secured (Cooper et al., 2011 ). Moreover, rising demand for phosphorus in advanced applications, such as black phosphorus (a novel semiconductor material) (Xu et al., 2018 ) and lithium iron phosphate (an advanced battery material) (Ciez and Whitacre, 2019 ), etc., could accelerate phosphorus resource depletion. Given these pressures, phosphorus has been declared a strategic resource in multiple regions (European commission, 2017 ). To mitigate scarcity, recovering phosphorus from wastewater, sludge, and livestock manure has become crucial. For instance, Germany has legislated that wastewater treatment plants (WWTP) with population equivalent of over 50,000 must recover phosphorus (Shi et al., 2018 ). Approximately 90% of phosphorus in wastewater is ultimately transferred to sludge (Balmer, 2004 ) due to the characteristics of current sewage treatment technologies. Consequently, sludge contains significantly higher phosphorus concentration than raw wastewater. Common waste activated sludge contains phosphorus at 1%-3% of total solids (Fischer et al., 2011 ), while sludge from enhanced biological phosphorus removal systems can reach 6%-12%, with some cases as high as15% (Yuan et al., 2012 ). Notably, the latter has exceeded the phosphorus content in the magmatic rock-type apatite of the three major phosphate ore types in China, which generally contains less than 10% phosphorus and can be as low as 2%-3%. This high phosphorus content positions sludge as a viable and sustainable alternative phosphate resource (Zhou et al., 2017 ). Phosphorus in sludge is mainly distributed across cell membranes, cell walls, intracellular spaces, and extracellular flocs (Hu et al., 2018 ). It exists in three primary forms: the first form is physiological phosphorus, also known as organic phosphorus (OP), which includes phytic acid phosphorus, DNA phosphorus, and monolipid phosphorus, etc.; the second form is polyphosphates stored within cells of the organisms present in sludge; the third form is phosphorus fixed by physical and chemical processes. Numerous studies have investigated the distribution of phosphorus forms in sludge. For instance, Xu et al. ( 2015 ) reported that polyphosphate and inorganic precipitated phosphorus accounted for approximately 40% and 50% of the total phosphorus (TP) in sludge, respectively. OP constituted 10% -35% of the TP content (Medeiros et al., 2015). In terms of solubility, only about 20% of the phosphorus was soluble, with the remaining 80% existing in solid form (Latif et al., 2017 ). Moreover, over half of phosphorus was classified as non-reactive, which included forms such as OP and accumulated polyphosphates etc. (Venkiteshwaran et al., 2018 ). These findings revealed that the current methods for analyzing phosphorus distribution were somewhat rudimentary, primarily relying on a single classification approach. However, the complex and varied distribution of phosphorus forms in sludge significantly influence the selection of recovery methods and the overall efficiency of phosphorus recovery. Thus, it is advantageous to compare distribution phosphorus forms in sludge using three distinct classification methods: Standards Measurements and Testing (SMT), the content of soluble versus insoluble fractions, and the ratio of Extracellular Polymeric Substances (EPS) to EPS residual solids. By determining the distribution of phosphorus forms through these classifications, we can gain insights into how phosphorus is released and can be recovered from sludge, which is beneficial for optimizing recovery strategies. 2. Materials and methods 2.1 Sludge sample The dewatered sludge used in the experiment was taken from dewatering facility at Shanghai A WWTP (SH-A) and was stored in the laboratory refrigerator at 4°C as soon as possible. This plant has a total wastewater treatment capacity of 150,000 m 3 /d, with over 90% of the wastewater being domestic wastewater. The biological treatment process employed is an anaerobic-anoxic-oxic system. The dewatered sludge had a solids content of 21.70±0.15%, VS/TS=55.06±0.19%, and pH=7.23±0.05. Prior to initiating the experimental procedures, the sludge was thoroughly mixed to guarantee the homogeneity and representativeness of the samples used in the study. 2.2 Phosphorus analytical classification method In this study, the distribution of phosphorus forms in sludge were categorized using three approaches: SMT method (Ruban et al., 1999), soluble/insoluble classification, and EPS/EPS residue classification, as illustrated in Fig.1. The SMT method employed to determine the concentrations of TP, IP, OP, non-apatite inorganic phosphorus (NAIP) and apatite inorganic phosphorus (AP), following the procedure described by Medeiros et al. (2005). The soluble/insoluble classification was used to investigate the distribution of phosphorus between the solid and liquid phases of sludge. Specifically, 10 g of dewatered sludge was diluted with 50 g of deionized water (a 5-fold dilution) and then separated by centrifugation at 12,000 r/min for 20 minutes. The supernatant was filtered through a 0.45 μm filter membrane. The residue was further diluted by 3 and 2 times, respectively, and subjected to the same separation and filtration process. The three filtrates were combined and analyzed for dissolved TP and ortho-P. The combined centrifugation and filtration residues were frozen at -20°C for 48 hours, followed by freeze-drying for 72 hours. The dried residues were then ground through a 100-mesh sieve and stored in a sealed bag for insoluble TP analysis. The EPS/EPS residue classification method was used to differentiate phosphorus forms within EPS extracts and their residues. EPS was extracted from a 20 g sludge sample using the formaldehyde-NaOH method (Liu and Fang, 2002). Subsequently, the SMT method was applied to determine the distinct phosphorus forms in both the EPS extract and the EPS residue. 2.3 Measurement methods The concentrations of TP and ortho-P in the liquid phase were determined according to the standard methods (APHA, 2005). The contents of Ca, Fe, Al, and TP in the solid phase were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 5110-OES, USA) after digestion with aqua regia using a microwave digestion instrument (PreeKem WX-7000HP, Shanghai, China). The total solids (TS) and volatile solids (VS) in the sludge were measured according to the standard methods (APHA, 2005). The pH value was measured using a pH meter (S210, METTLER, Switzerland). 3. Results and discussion 3.1 SMT method The distribution of phosphorus forms in sludge, as determined using the SMT method, was shown in Fig. 2. The TP content in this dewatered sludge sample was 19.41 mg/gDS (dry sludge), equivalent to 1.94% of the dry weight. This value fell within the typical range of phosphorus content in normal residual sludge (Fischer et al., 2011), but it was lower than that observed in sludge from enhanced biological phosphorus removal process (Yuan et al., 2012). This discrepancy might be attributed to the relatively low influent TP concentration of the plant, which was only about 4 mg/L. The content of IP was 15.71 mg/gDS, which accounted for 80.94% of the TP. Of this IP, 57.60% was identified as AP. Thus, the predominant form of phosphorus in the sludge was IP, which was consistent with the findings in the literature (Medeiros et al., 2005). The OP content was measured at 3.25 mg/gDS, accounting for 16.74% of the TP, which also consistent with the results reported by Medeiros et al. (2005). It is important to note that the content and form of phosphorus in dewatered sludge are highly dependent on its source. Even dewatered sludge from the same WWTP may exhibit variations in phosphorus content and forms due to factors such as changes in influent water quality, sludge dewatering techniques, seasonal variations, and differences in wastewater treatment processes. When considering the form of IP, the respective proportions of NAIP and AP can vary significantly. These variations are predominantly influenced by the concentrations of metals such as Ca, Fe, and Al in the sludge, as well as the solubility products of their phosphate precipitates (see Equations 1–3). 3Ca 2+ +2PO 4 3- ⇌Ca 3 (PO 4 ) 2 ↓ Ksp=2.0×10 -29 (1) Fe 3+ +PO 4 3- ⇌ FePO 4 ↓ Ksp=1.3×10 -22 (2) Al 3+ +PO 4 3- ⇌AlPO 4 ↓ Ksp=6.3×10 -19 (3) Equations 1–3 indicate that Ca forms more stable phosphorus complexes compared to Fe and Al. To better understand the relationship between the ratios of NAIP/IP and AP/IP and the metal content, the concentrations of Ca, Fe, Al, AP, NAIP, and IP were measured in dewatered sludge samples from three WWTPs (Fig. 3): SH-A, SH-B (solids content=17.45%±0.20%, VS/TS= 65.13%±0.09, pH=7.3±0.4), and JS-A (solids content= 20.82%±0.27%, VS/TS= 55.43%±0.21, pH=7.07±0.03). The ratio of AP/IP in SH-A sludge was significantly higher than that in the other two plants, which correlated with the substantially higher Ca content in SH-A sludge compared to SH-B and JS-A sludges. In contrast, the combined concentrations of Fe and Al in SH-A sludge were lower than those in SH-B and JS-A sludges. These findings provided a clearer explanation for the observed high AP/IP and low NAIP/IP ratios in SH-A sludge. 3.2 Soluble/Insoluble fractionation The results obtained using the soluble/insoluble classification method were presented in Table 1. Over 94% of the phosphorus in dewatered sludge was found in the solid phase, with ortho-P accounting for approximately 20% of the soluble TP and only 1% of the TP content. The soluble TP content was lower than that reported by Ding et al. (2022), where the soluble TP content exceeded 10%. This discrepancy could be attributed to several factors. First, the sludge samples were sourced from different WWTPs. Second, the tested sludge had a solids content of 2.45% and a pH value of 6.50. As is well known, low solids content and low pH values tend to promote the dissolution of phosphorus in sludge. Additionally, some soluble phosphorus may be lost with the supernatant during the dewatering process. However, the concentrations of Ca, Fe, and Al might increase, which could further reduce the soluble TP content. Based on the current state of phosphorus recovery methods, the primary target for phosphorus recovery is dissolved ortho-P, which is typically achieved through techniques such as calcium phosphate precipitation (Schott et al., 2023), struvite precipitation (Sun et al., 2023), vivianite precipitation (Chen et al., 2023), and adsorption methods (Li et al., 2023). It is important to note that dissolved ortho-P represents the most readily accessible form of phosphorus for recovery and utilization from sludge. As shown in Fig. 1, the IP content in the dewatered sludge accounted for over 80%, primarily in the form of NAIP and AP. However, the dissolved ortho-P content was only 1.00%, which was significantly lower than the levels required for effective phosphorus recovery. Therefore, it is imperative to enhance the transformation of phosphorus within the sludge and release it in the form of ortho-P. This step is essential to provide the necessary conditions for efficient phosphorus recovery and utilization. 3.3 EPS/ EPS residue method Fig. 4 illustrated the distribution of phosphorus forms in the EPS extracted from dewatered sludge (abbreviated as P-EPS). The TP content in the EPS extracts was 0.40 mg/gDS, which was significantly lower than the overall phosphorus content in the sludge. This result differed substantially from the findings reported by Ding et al. (2002), likely due to variations in EPS extraction methods and the different sources of sludge (specifically, sludge from secondary sedimentation tanks) used in their study. OP was the predominant form of phosphorus within the EPS extract, accounting for 74.19% of the TP in EPS. The remaining IP was primarily in the form of NAIP, comprising approximately 25.81% of the TP. AP was essentially undetectable in the EPS. Although the extraction efficiency of EPS might slightly influence the actual phosphorus content, it was generally observed that phosphorus in EPS extracted from dewatered sludge was minimal and predominantly existed as OP. This was related to the presence of phosphate, nucleic acids, and humic substances in the composition of EPS (Wingender et al., 1999). The distribution of different phosphorus forms in the EPS residue (abbreviated as P-EPSr) was shown in Fig. 5. The TP content in the EPS residue was 18.09 mg/gDS, with IP being the predominant form, accounting for 81.06% of the TP. The ratios of OP to TP and AP to TP in the EPS residue were 15.04% and 59.35%, respectively. This indicated that AP was the main component of the EPS residue. According to the EPS/ EPS residue classification method, the content of different phosphorus forms in dewatered sludge (P-DS) should be equivalent to the sum of P-EPS and P-EPSr. As shown in Table 2, the sum of P-EPS and P-EPSr exceeded 90% of the TP in dewatered sludge, indicating the reliability of this analysis method. Over 93% of IP and 83% of OP were found in the EPS residue, while the TP in the EPS accounted for only 2% of the dewatered sludge. However, significant differences were observed in the proportions of different phosphorus forms between the EPS and its residue. Specifically, OP was the main component in the EPS, while IP was the predominant form in the EPS residue. Additionally, NAIP was the primary form of IP in the EPS, whereas in the EPS residue, IP was mainly present in the form of AP. 4. Conclusion The results from the three different analysis methods indicated that the characteristics of sludge determine the distribution and content of phosphorus forms in dewatered sludge. Phosphorus in dewatered sludge primarily exists in the form of IP, which is influenced by the concentrations of metals such as Al, Fe, and Ca in the sludge. Dissolved ortho-P in dewatered sludge accounted for only about 1%, while insoluble TP made up approximately 95%. The TP content in the EPS and EPS residue accounted for 2.09% and 93.26% of the TP in dewatered sludge, respectively. OP constituted 74.19% of the TP in EPS, but only 15.04% of the TP in the EPS residue. Declarations Funding This study was financially supported by the National Natural Science Foundation of China (NSFC) (52070146). Author Contribution Zhigang Liu: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, funding acquisition.Junjie He: Writing-original draft, Formal analysis.Siqi Zhou: Formal analysis, Investigation, Data curation.Xiaohu Dai: Writing – review & editing, supervision, project administration. References APHA.2005. Standard methods for the examination of water and wastewater. American water works association and water environmental federation, Washington, DC, USA. Balmer, P., (2004). Phosphorus recovery- an overview of potentials and possibilities. Water Science & Technology , 49,185-190. Chen, T., Song, X., & Xing, M., (2023). Study on anaerobic phosphorus release from pig manual and phosphorus recovery by vivitanite method. Scientific Reports , 13,16095. Ciez, R. E., & Whitacre, J. F., (2019). Examining different recycling processes for lithium-ion batteries. Nature Sustainability , 2,148-156. Cooper, J., Lombardi, R., Boardman, D., & Carliell-Marquet, C., (2011). The future distribution and production of global phosphate rock reserves. Resources, Conservation & Recycling , 57,78-86. Darcy, J.L., Schmidt, S.K., Knelman, J. E., Cleveland, C.C., Castle, S.C., & Nemergut, D.R., (2018). Phosphorus, not nitrogen, limits plants and microbial primary producers following glacial retreat. Science Advances , 4, eaaq0942. Ding, Y., Dai, X., Wu, B., Liu, Z., & Dai, L., (2022). Targeted clean extraction of phosphorus from waste activated sludge: From a new perspective of phosphorus occurrence states to an innovative. Water Research , 215,118190. European commission. (2017). Communication from the commission to the European parliament, the council, the European economic and social committee and the committee of the regions on the 2017 list of critical raw materials for the EU 8. Fischer, F., Bastian, C., Happe, M., Mabillard, E., & Schmidt, N., (2011). Microbial fuel cell enables phosphate recovery from digested sewage sludge as struvite. Bioresource Technology , 102, 5824-5830. Hu, P., Liu, J.Y., Bao, H.X., Wu, L., Jiang, L., Zou, L.P., Wu, Y.J., Qian, G.R., & Li, Y.Y., (2018). Enhancing phosphorus release from waste activated sludge by combining high-voltage pulsed discharge pretreatment with anaerobic fermentation. Journal of Cleaner Production , 196,1044-1051. Latif, M.A., Mehta, C.M., & Batstone, D.J., (2017). Influence of low pH on continuous anaerobic digestion of waste activated sludge. Water Research , 113,42-49. Li, L., Zhu, Z., Ni, J., & Zuo, X., (2023). Sustainable phosphorus adsorption and recovery from aqueous solution by a novel recyclable Ca-PAC-C-C. According to TS. Science of the Total Environment , 897,165444. Liu, H., & Fang, H.H.P. (2002). Extraction of extracellular polymeric substances (EPS) of sludges. Journal of Biotechnology , 95,249-256. Medeiros, J.J.G., Cid, B.P., & Gomez, E.F., (2005). Analytical phosphorus fractionation in sewage sludge and sediment samples. Analytical and Bioanalytical Chemistry , 381,873-878. Ruban, V., López-Sánchez, J.F., Pardo, P., Rauret, G., Muntau, H., & Quevauviller, P., (1999). Selection and evaluation of sequential extraction procedures for the determination of phosphorus forms in lake sediment. Journal of Environmental Monitoring , 1,51-56. Schott, C., Yan, L., Gimbuttyte, U., Cunha, J. R., van der Weijden, R.D., & Busman, C., (2023). Enabling effective phosphorus recovery from cow manual: Liberation of phosphorus through oxidation and recovery of phosphorus as calcium phosphate grains. Chemical Engineering Journal , 460,141695. Shi, S., Xu, G., Yu, H., & Zhang, Z., (2018). Strategies of valorization of sludge from wastewater treatment, Journal of Chemical Technology and Biotechnology , 93,936-944. Sun, L., Yang, Y., Yuan, W., Wu, X., Cui, Z., Wang, H., Deng, H., Zhu, X., & Li, R., (2023). Struvite purity prediction by response surface methodology and chemical equilibrium modeling combination. Environmental Technology & Innovation , 29,103016 Venkiteshwaran, K., McNamara, P.J., & Mayer, B.K., (2018). Meta-analysis of non-reactive phosphorus in water, wastewater, and sludge, and strategies to convert it for enhanced phosphorus removal and recovery. Science of the Total Environment , 644,661-674. Wingender, J., Neu, T.R., & Flemming, H.C., (1999). Microbial Extracellular Polymeric Substances: Characterization, Structure and Function. Springer Berlin Heidelberg, Berlin, Heidelberg,1–19. Xu, Z.L., Lin, S., Onofrio, N., Zhou, L., Shi, F., Lu, W., Kang, K., Zhang, Q., & Lau, S. P., (2018). Exceptional catalytic effects of black phosphorus quantum dots in shuttling-free lithium sulfur batteries. Nature Communications , 2018, 9,1-11. Xu, Y., Hu, H., Liu, J., Luo, J., Qian, G., & Wang, A., (2015). pH dependent phosphorus release from waste activated sludge: contributions of phosphorus speciation. Chemical Engineering Journal , 267,260-265. Yuan, Z.G., Pratt, S., & Batstone, D.J., (2012). Phosphorus recovery from wastewater through microbial processes. Current Opinion in Biotechnology , 23,878-883. Zhou, K., Barjenbrush, M., Kabbe, G., Inial, G., & Remy, C., (2017). Phosphorus recovery from municipal and fertilizer wastewater: China’s potential and perspective. Journal of Environmental Sciences , 52,151-159. Tables Table 1 The distribution of phosphorus forms in sludge by soluble/insoluble classification method Phosphorus Forms Ortho-P Other soluble TP Insoluble TP Total Proportion (%) 1.00±0.05 3.46±0.06 94.75±0.37 99.21±0.42 Table 2 Comparison between P-EPS/P-DS and P-EPSr /P-DS P form TP/% IP/% OP/% AP/% NAIP/% P-EPS/P-DS 2.09±0.50 0.60±0.10 9.56±2.84 0.06±0.04 1.35±0.28 P-EPSr/P-DS 93.26±3.45 93.40±3.80 83.92±11.88 96.47±16.25 95.44±1.21 Sum 95.35±3.76 94.00±3.90 93.48±10.94 96.53±16.29 96.80±1.35 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7369225","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506468437,"identity":"7116a116-788c-4002-8e62-f93030f2f3e0","order_by":0,"name":"Zhigang 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different phosphorus forms in EPS extracted from dewatered sludge\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7369225/v1/f653a15792796bdfb427e6a6.png"},{"id":93418534,"identity":"5e6278ab-cee3-4736-8927-26359fa80b75","added_by":"auto","created_at":"2025-10-13 15:51:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22399,"visible":true,"origin":"","legend":"\u003cp\u003eThe content of different phosphorus forms in EPS residue\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7369225/v1/cbb4dfe904127fe221e891c0.png"},{"id":95527809,"identity":"cea2edb0-628b-4b71-bd58-22993065bfd1","added_by":"auto","created_at":"2025-11-10 10:14:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":491809,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7369225/v1/608293c6-ddbc-4d0c-84de-650182a16510.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The distribution of phosphorus forms in dewatered sludge with three classification analysis methods","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eUnlike nitrogen, which cycles naturally in the biosphere, phosphorus behaves as a non-renewable resource in the biosphere because of its predominantly unidirectional flows (Venkiteshwaran et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Also, phosphorus is an indispensable element for the survival of both humans and animals, and a major limiting element for primary producers such as plants and microorganisms (Darcy et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, global phosphate rock reserves-estimated by the United States Geological Survey and other studies-may only sustain current consumption for another 100 years, if no alternative phosphorus sources are secured (Cooper et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, rising demand for phosphorus in advanced applications, such as black phosphorus (a novel semiconductor material) (Xu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and lithium iron phosphate (an advanced battery material) (Ciez and Whitacre, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), etc., could accelerate phosphorus resource depletion. Given these pressures, phosphorus has been declared a strategic resource in multiple regions (European commission, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To mitigate scarcity, recovering phosphorus from wastewater, sludge, and livestock manure has become crucial. For instance, Germany has legislated that wastewater treatment plants (WWTP) with population equivalent of over 50,000 must recover phosphorus (Shi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eApproximately 90% of phosphorus in wastewater is ultimately transferred to sludge (Balmer, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) due to the characteristics of current sewage treatment technologies. Consequently, sludge contains significantly higher phosphorus concentration than raw wastewater. Common waste activated sludge contains phosphorus at 1%-3% of total solids (Fischer et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), while sludge from enhanced biological phosphorus removal systems can reach 6%-12%, with some cases as high as15% (Yuan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Notably, the latter has exceeded the phosphorus content in the magmatic rock-type apatite of the three major phosphate ore types in China, which generally contains less than 10% phosphorus and can be as low as 2%-3%. This high phosphorus content positions sludge as a viable and sustainable alternative phosphate resource (Zhou et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhosphorus in sludge is mainly distributed across cell membranes, cell walls, intracellular spaces, and extracellular flocs (Hu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It exists in three primary forms: the first form is physiological phosphorus, also known as organic phosphorus (OP), which includes phytic acid phosphorus, DNA phosphorus, and monolipid phosphorus, etc.; the second form is polyphosphates stored within cells of the organisms present in sludge; the third form is phosphorus fixed by physical and chemical processes. Numerous studies have investigated the distribution of phosphorus forms in sludge. For instance, Xu et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported that polyphosphate and inorganic precipitated phosphorus accounted for approximately 40% and 50% of the total phosphorus (TP) in sludge, respectively. OP constituted 10% -35% of the TP content (Medeiros et al., 2015). In terms of solubility, only about 20% of the phosphorus was soluble, with the remaining 80% existing in solid form (Latif et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, over half of phosphorus was classified as non-reactive, which included forms such as OP and accumulated polyphosphates etc. (Venkiteshwaran et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These findings revealed that the current methods for analyzing phosphorus distribution were somewhat rudimentary, primarily relying on a single classification approach. However, the complex and varied distribution of phosphorus forms in sludge significantly influence the selection of recovery methods and the overall efficiency of phosphorus recovery. Thus, it is advantageous to compare distribution phosphorus forms in sludge using three distinct classification methods: Standards Measurements and Testing (SMT), the content of soluble versus insoluble fractions, and the ratio of Extracellular Polymeric Substances (EPS) to EPS residual solids. By determining the distribution of phosphorus forms through these classifications, we can gain insights into how phosphorus is released and can be recovered from sludge, which is beneficial for optimizing recovery strategies.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003ch2\u003e2.1 Sludge sample\u003c/h2\u003e\n\u003cp\u003eThe dewatered sludge used in the experiment was taken from dewatering facility at Shanghai A WWTP (SH-A) and was stored in the laboratory refrigerator at 4\u0026deg;C as soon as possible. This plant has a total wastewater treatment capacity of 150,000 m\u003csup\u003e3\u003c/sup\u003e/d, with over 90% of the wastewater being domestic wastewater. The biological treatment process employed is an anaerobic-anoxic-oxic system. The dewatered sludge had a solids content of 21.70\u0026plusmn;0.15%, VS/TS=55.06\u0026plusmn;0.19%, and pH=7.23\u0026plusmn;0.05. Prior to initiating the experimental procedures, the sludge was thoroughly mixed to guarantee the homogeneity and representativeness of the samples used in the study.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.2 Phosphorus analytical classification method\u003c/h2\u003e\n\u003cp\u003eIn this study, the distribution of phosphorus forms in sludge were categorized using three approaches: SMT method (Ruban et al., 1999), soluble/insoluble classification, and EPS/EPS residue classification, as illustrated in Fig.1. The SMT method employed to determine the concentrations of TP, IP, OP, non-apatite inorganic phosphorus (NAIP) and apatite inorganic phosphorus (AP), following the procedure described by Medeiros et al. (2005).\u003c/p\u003e\n\u003cp\u003eThe soluble/insoluble classification was used to investigate the distribution of phosphorus between the solid and liquid phases of sludge. Specifically, 10 g of dewatered sludge was diluted with 50 g of deionized water (a 5-fold dilution) and then separated by centrifugation at 12,000 r/min for 20 minutes. The supernatant was filtered through a 0.45\u0026nbsp;\u0026mu;m filter membrane. The residue was further diluted by 3 and 2 times, respectively, and subjected to the same separation and filtration process. The three filtrates were combined and analyzed for dissolved TP and ortho-P. The combined centrifugation and filtration residues were frozen at -20\u0026deg;C for 48 hours, followed by freeze-drying for 72 hours. The dried residues were then ground through a 100-mesh sieve and stored in a sealed bag for insoluble TP analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe EPS/EPS residue classification method was used to differentiate phosphorus forms within EPS extracts and their residues. EPS was extracted from a 20 g sludge sample using the formaldehyde-NaOH method (Liu and Fang, 2002). Subsequently, the SMT method was applied to determine the distinct phosphorus forms in both the EPS extract and the EPS residue.\u003c/p\u003e\n\u003ch2\u003e2.3 Measurement methods\u003c/h2\u003e\n\u003cp\u003eThe concentrations of TP and ortho-P in the liquid phase were determined according to the standard methods (APHA, 2005). The contents of Ca, Fe, Al, and TP in the solid phase were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 5110-OES, USA) after digestion with aqua regia using a microwave digestion instrument (PreeKem WX-7000HP, Shanghai, China). The total solids (TS) and volatile solids (VS) in the sludge were measured according to the standard methods (APHA, 2005). The pH value was measured using a pH meter (S210, METTLER, Switzerland).\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003ch2\u003e3.1 SMT method\u003c/h2\u003e\n\u003cp\u003eThe distribution of phosphorus forms in sludge, as determined using the SMT method, was shown in Fig. 2. The TP content in this dewatered sludge sample was 19.41 mg/gDS (dry sludge), equivalent to 1.94% of the dry weight. This value fell within the typical range of phosphorus content in normal residual sludge (Fischer et al., 2011), but it was lower than that observed in sludge from enhanced biological phosphorus removal process (Yuan et al., 2012). This discrepancy might be attributed to the relatively low influent TP concentration of the plant, which was only about 4 mg/L. The content of IP was 15.71 mg/gDS, which accounted for 80.94% of the TP. Of this IP, 57.60% was identified as AP. Thus, the predominant form of phosphorus in the sludge was IP, which was consistent with the findings in the literature (Medeiros et al., 2005). The OP content was measured at 3.25 mg/gDS, accounting for 16.74% of the TP, which also consistent with the results reported by Medeiros et al. (2005).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is important to note that the content and form of phosphorus in dewatered sludge are highly dependent on its source. Even dewatered sludge from the same WWTP may exhibit variations in phosphorus content and forms due to factors such as changes in influent water quality, sludge dewatering techniques, seasonal variations, and differences in wastewater treatment processes. When considering the form of IP, the respective proportions of NAIP and AP can vary significantly. These variations are predominantly influenced by the concentrations of metals such as Ca, Fe, and Al in the sludge, as well as the solubility products of their phosphate precipitates (see Equations 1\u0026ndash;3).\u003c/p\u003e\n\u003cp\u003e3Ca\u003csup\u003e2+\u003c/sup\u003e+2PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e⇌Ca\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026darr; Ksp=2.0\u0026times;10\u003csup\u003e-29\u003c/sup\u003e (1)\u003c/p\u003e\n\u003cp\u003eFe\u003csup\u003e3+\u003c/sup\u003e+PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e⇌ FePO\u003csub\u003e4\u003c/sub\u003e\u0026darr; Ksp=1.3\u0026times;10\u003csup\u003e-22 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sup\u003e(2)\u003c/p\u003e\n\u003cp\u003eAl\u003csup\u003e3+\u003c/sup\u003e+PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e⇌AlPO\u003csub\u003e4\u003c/sub\u003e\u0026darr; Ksp=6.3\u0026times;10\u003csup\u003e-19 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/sup\u003e (3)\u003c/p\u003e\n\u003cp\u003eEquations 1\u0026ndash;3 indicate that Ca forms more stable phosphorus complexes compared to Fe and Al. To better understand the relationship between the ratios of NAIP/IP and AP/IP and the metal content, the concentrations of Ca, Fe, Al, AP, NAIP, and IP were measured in dewatered sludge samples from three WWTPs (Fig. 3): SH-A, SH-B (solids content=17.45%\u0026plusmn;0.20%, VS/TS= 65.13%\u0026plusmn;0.09, pH=7.3\u0026plusmn;0.4), and JS-A (solids content= 20.82%\u0026plusmn;0.27%, VS/TS= 55.43%\u0026plusmn;0.21, pH=7.07\u0026plusmn;0.03).\u003c/p\u003e\n\u003cp\u003eThe ratio of AP/IP in SH-A sludge was significantly higher than that in the other two plants, which correlated with the substantially higher Ca content in SH-A sludge compared to SH-B and JS-A sludges. In contrast, the combined concentrations of Fe and Al in SH-A sludge were lower than those in SH-B and JS-A sludges. These findings provided a clearer explanation for the observed high AP/IP and low NAIP/IP ratios in SH-A sludge.\u003c/p\u003e\n\u003ch2\u003e3.2 Soluble/Insoluble fractionation\u003c/h2\u003e\n\u003cp\u003eThe results obtained using the soluble/insoluble classification method were presented in Table 1. Over 94% of the phosphorus in dewatered sludge was found in the solid phase, with ortho-P accounting for approximately 20% of the soluble TP and only 1% of the TP content. The soluble TP content was lower than that reported by Ding et al. (2022), where the soluble TP content exceeded 10%. This discrepancy could be attributed to several factors. First, the sludge samples were sourced from different WWTPs. Second, the tested sludge had a solids content of 2.45% and a pH value of 6.50. As is well known, low solids content and low pH values tend to promote the dissolution of phosphorus in sludge. Additionally, some soluble phosphorus may be lost with the supernatant during the dewatering process. However, the concentrations of Ca, Fe, and Al might increase, which could further reduce the soluble TP content.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the current state of phosphorus recovery methods, the primary target for phosphorus recovery is dissolved ortho-P, which is typically achieved through techniques such as calcium phosphate precipitation (Schott et al., 2023), struvite precipitation (Sun et al., 2023), vivianite precipitation (Chen et al., 2023), and adsorption methods (Li et al., 2023). It is important to note that dissolved ortho-P represents the most readily accessible form of phosphorus for recovery and utilization from sludge. As shown in Fig. 1, the IP content in the dewatered sludge accounted for over 80%, primarily in the form of NAIP and AP. However, the dissolved ortho-P content was only 1.00%, which was significantly lower than the levels required for effective phosphorus recovery. Therefore, it is imperative to enhance the transformation of phosphorus within the sludge and release it in the form of ortho-P. This step is essential to provide the necessary conditions for efficient phosphorus recovery and utilization.\u003c/p\u003e\n\u003ch2\u003e3.3 EPS/ EPS residue method\u003c/h2\u003e\n\u003cp\u003eFig. 4 illustrated the distribution of phosphorus forms in the EPS extracted from dewatered sludge (abbreviated as P-EPS). The TP content in the EPS extracts was 0.40 mg/gDS, which was significantly lower than the overall phosphorus content in the sludge. This result differed substantially from the findings reported by Ding et al. (2002), likely due to variations in EPS extraction methods and the different sources of sludge (specifically, sludge from secondary sedimentation tanks) used in their study.\u003c/p\u003e\n\u003cp\u003eOP was the predominant form of phosphorus within the EPS extract, accounting for 74.19% of the TP in EPS. The remaining IP was primarily in the form of NAIP, comprising approximately 25.81% of the TP. AP was essentially undetectable in the EPS. Although the extraction efficiency of EPS might slightly influence the actual phosphorus content, it was generally observed that phosphorus in EPS extracted from dewatered sludge was minimal and predominantly existed as OP. This was related to the presence of phosphate, nucleic acids, and humic substances in the composition of EPS (Wingender et al., 1999).\u003c/p\u003e\n\u003cp\u003eThe distribution of different phosphorus forms in the EPS residue (abbreviated as P-EPSr) was shown in Fig. 5. The TP content in the EPS residue was 18.09 mg/gDS, with IP being the predominant form, accounting for 81.06% of the TP. The ratios of OP to TP and AP to TP in the EPS residue were 15.04% and 59.35%, respectively. This indicated that AP was the main component of the EPS residue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to the EPS/ EPS residue classification method, the content of different phosphorus forms in dewatered sludge (P-DS) should be equivalent to the sum of P-EPS and P-EPSr. As shown in Table 2, the sum of P-EPS and P-EPSr exceeded 90% of the TP in dewatered sludge, indicating the reliability of this analysis method. Over 93% of IP and 83% of OP were found in the EPS residue, while the TP in the EPS accounted for only 2% of the dewatered sludge. However, significant differences were observed in the proportions of different phosphorus forms between the EPS and its residue. Specifically, OP was the main component in the EPS, while IP was the predominant form in the EPS residue. Additionally, NAIP was the primary form of IP in the EPS, whereas in the EPS residue, IP was mainly present in the form of AP.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe results from the three different analysis methods indicated that the characteristics of sludge determine the distribution and content of phosphorus forms in dewatered sludge. Phosphorus in dewatered sludge primarily exists in the form of IP, which is influenced by the concentrations of metals such as Al, Fe, and Ca in the sludge. Dissolved ortho-P in dewatered sludge accounted for only about 1%, while insoluble TP made up approximately 95%. The TP content in the EPS and EPS residue accounted for 2.09% and 93.26% of the TP in dewatered sludge, respectively. OP constituted 74.19% of the TP in EPS, but only 15.04% of the TP in the EPS residue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was financially supported by the National Natural Science Foundation of China (NSFC) (52070146).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhigang Liu: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, funding acquisition.Junjie He: Writing-original draft, Formal analysis.Siqi Zhou: Formal analysis, Investigation, Data curation.Xiaohu Dai: Writing \u0026ndash; review \u0026amp; editing, supervision, project administration.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAPHA.2005. Standard methods for the examination of water and wastewater. American water works association and water environmental federation, Washington, DC, USA.\u003c/li\u003e\n\u003cli\u003eBalmer, P., (2004). Phosphorus recovery- an overview of potentials and possibilities. \u003cem\u003eWater Science \u003c/em\u003e\u0026amp; \u003cem\u003eTechnology\u003c/em\u003e, 49,185-190.\u003c/li\u003e\n\u003cli\u003eChen, T., Song, X., \u0026amp; Xing, M., (2023). Study on anaerobic phosphorus release from pig manual and phosphorus recovery by vivitanite method. \u003cem\u003eScientific Reports\u003c/em\u003e, 13,16095.\u003c/li\u003e\n\u003cli\u003eCiez, R. E., \u0026amp; Whitacre, J. F., (2019). Examining different recycling processes for lithium-ion batteries. \u003cem\u003eNature Sustainability\u003c/em\u003e, 2,148-156.\u003c/li\u003e\n\u003cli\u003eCooper, J., Lombardi, R., Boardman, D., \u0026amp; Carliell-Marquet, C., (2011). The future distribution and production of global phosphate rock reserves. \u003cem\u003eResources, Conservation \u0026amp; Recycling\u003c/em\u003e, 57,78-86.\u003c/li\u003e\n\u003cli\u003eDarcy, J.L., Schmidt, S.K., Knelman, J. E., Cleveland, C.C., Castle, S.C., \u0026amp; Nemergut, D.R., (2018). Phosphorus, not nitrogen, limits plants and microbial primary producers following glacial retreat. \u003cem\u003eScience Advances\u003c/em\u003e, 4, eaaq0942.\u003c/li\u003e\n\u003cli\u003eDing, Y., Dai, X., Wu, B., Liu, Z., \u0026amp; Dai, L., (2022). Targeted clean extraction of phosphorus from waste activated sludge: From a new perspective of phosphorus occurrence states to an innovative. \u003cem\u003eWater Research\u003c/em\u003e, 215,118190.\u003c/li\u003e\n\u003cli\u003eEuropean commission. (2017). Communication from the commission to the European parliament, the council, the European economic and social committee and the committee of the regions on the 2017 list of critical raw materials for the EU 8.\u003c/li\u003e\n\u003cli\u003eFischer, F., Bastian, C., Happe, M., Mabillard, E., \u0026amp; Schmidt, N., (2011). Microbial fuel cell enables phosphate recovery from digested sewage sludge as struvite. \u003cem\u003eBioresource Technology\u003c/em\u003e, 102, 5824-5830.\u003c/li\u003e\n\u003cli\u003eHu, P., Liu, J.Y., Bao, H.X., Wu, L., Jiang, L., Zou, L.P., Wu, Y.J., Qian, G.R., \u0026amp; Li, Y.Y., (2018). Enhancing phosphorus release from waste activated sludge by combining high-voltage pulsed discharge pretreatment with anaerobic fermentation. \u003cem\u003eJournal of Cleaner Production\u003c/em\u003e, 196,1044-1051.\u003c/li\u003e\n\u003cli\u003eLatif, M.A., Mehta, C.M., \u0026amp; Batstone, D.J., (2017). Influence of low pH on continuous anaerobic digestion of waste activated sludge. \u003cem\u003eWater Research\u003c/em\u003e, 113,42-49.\u003c/li\u003e\n\u003cli\u003eLi, L., Zhu, Z., Ni, J., \u0026amp; Zuo, X., (2023). Sustainable phosphorus adsorption and recovery from aqueous solution by a novel recyclable Ca-PAC-C-C. According to TS.\u003cem\u003eScience of the Total Environment\u003c/em\u003e, 897,165444.\u003c/li\u003e\n\u003cli\u003eLiu, H., \u0026amp; Fang, H.H.P. (2002). Extraction of extracellular polymeric substances (EPS) of sludges. \u003cem\u003eJournal of Biotechnology\u003c/em\u003e, 95,249-256.\u003c/li\u003e\n\u003cli\u003eMedeiros, J.J.G., Cid, B.P., \u0026amp; Gomez, E.F., (2005). Analytical phosphorus fractionation in sewage sludge and sediment samples. \u003cem\u003eAnalytical and Bioanalytical Chemistry\u003c/em\u003e, 381,873-878.\u003c/li\u003e\n\u003cli\u003eRuban, V., L\u0026oacute;pez-S\u0026aacute;nchez, J.F., Pardo, P., Rauret, G., Muntau, H., \u0026amp; Quevauviller, P., (1999). Selection and evaluation of sequential extraction procedures for the determination of phosphorus forms in lake sediment. \u003cem\u003eJournal of Environmental Monitoring\u003c/em\u003e, 1,51-56.\u003c/li\u003e\n\u003cli\u003eSchott, C., Yan, L., Gimbuttyte, U., Cunha, J. R., van der Weijden, R.D., \u0026amp; Busman, C., (2023). Enabling effective phosphorus recovery from cow manual: Liberation of phosphorus through oxidation and recovery of phosphorus as calcium phosphate grains. \u003cem\u003eChemical Engineering Journal\u003c/em\u003e, 460,141695.\u003c/li\u003e\n\u003cli\u003eShi, S., Xu, G., Yu, H., \u0026amp; Zhang, Z., (2018). Strategies of valorization of sludge from wastewater treatment, \u003cem\u003eJournal of Chemical Technology and Biotechnology\u003c/em\u003e, 93,936-944.\u003c/li\u003e\n\u003cli\u003eSun, L., Yang, Y., Yuan, W., Wu, X., Cui, Z., Wang, H., Deng, H., Zhu, X., \u0026amp; Li, R., (2023). Struvite purity prediction by response surface methodology and chemical equilibrium modeling combination. \u003cem\u003eEnvironmental Technology \u0026amp; Innovation\u003c/em\u003e, 29,103016\u003c/li\u003e\n\u003cli\u003eVenkiteshwaran, K., McNamara, P.J., \u0026amp; Mayer, B.K., (2018). Meta-analysis of non-reactive phosphorus in water, wastewater, and sludge, and strategies to convert it for enhanced phosphorus removal and recovery. \u003cem\u003eScience of the Total Environment\u003c/em\u003e, 644,661-674.\u003c/li\u003e\n\u003cli\u003eWingender, J., Neu, T.R., \u0026amp; Flemming, H.C., (1999). Microbial Extracellular Polymeric Substances: Characterization, Structure and Function. Springer Berlin Heidelberg, Berlin, Heidelberg,1\u0026ndash;19.\u003c/li\u003e\n\u003cli\u003eXu, Z.L., Lin, S., Onofrio, N., Zhou, L., Shi, F., Lu, W., Kang, K., Zhang, Q., \u0026amp; Lau, S. P., (2018). Exceptional catalytic effects of black phosphorus quantum dots in shuttling-free lithium sulfur batteries. \u003cem\u003eNature Communications\u003c/em\u003e, 2018, 9,1-11.\u003c/li\u003e\n\u003cli\u003eXu, Y., Hu, H., Liu, J., Luo, J., Qian, G., \u0026amp; Wang, A., (2015). pH dependent phosphorus release from waste activated sludge: contributions of phosphorus speciation. \u003cem\u003eChemical Engineering Journal\u003c/em\u003e, 267,260-265.\u003c/li\u003e\n\u003cli\u003eYuan, Z.G., Pratt, S., \u0026amp; Batstone, D.J., (2012). Phosphorus recovery from wastewater through microbial processes. \u003cem\u003eCurrent Opinion in Biotechnology\u003c/em\u003e, 23,878-883.\u003c/li\u003e\n\u003cli\u003eZhou, K., Barjenbrush, M., Kabbe, G., Inial, G., \u0026amp; Remy, C., (2017). Phosphorus recovery from municipal and fertilizer wastewater: China\u0026rsquo;s potential and perspective. \u003cem\u003eJournal of Environmental Sciences\u003c/em\u003e, 52,151-159.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 The distribution of phosphorus forms in sludge by soluble/insoluble classification method\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"589\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhosphorus Forms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOrtho-P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOther soluble TP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInsoluble TP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProportion (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.46\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e94.75\u0026plusmn;0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99.21\u0026plusmn;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2 Comparison between P-EPS/P-DS and P-EPSr /P-DS\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eP form\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTP/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIP/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOP/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAP/%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNAIP/%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eP-EPS/P-DS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.09\u0026plusmn;0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.60\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e9.56\u0026plusmn;2.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.06\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.35\u0026plusmn;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eP-EPSr/P-DS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e93.26\u0026plusmn;3.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e93.40\u0026plusmn;3.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e83.92\u0026plusmn;11.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e96.47\u0026plusmn;16.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e95.44\u0026plusmn;1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e95.35\u0026plusmn;3.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e94.00\u0026plusmn;3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e93.48\u0026plusmn;10.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e96.53\u0026plusmn;16.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e96.80\u0026plusmn;1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Phosphorus form, dewatered sludge, Standards Measurements and Testing (SMT), soluble/insoluble, Extracellular Polymeric Substances (EPS)/EPS residue","lastPublishedDoi":"10.21203/rs.3.rs-7369225/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7369225/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDetermining the distribution of phosphorus forms in dewatered sludge is crucial, as it directly impacts the choice and effectiveness of recovery strategies. Analyses using the standards measurements and testing (SMT) method, soluble/insoluble fractionation and extracellular polymeric substances (EPS) /EPS residue extraction, revealed that sludge characteristics significantly influenced phosphorus speciation and content. Inorganic phosphorus (IP) was the dominant form in dewatered sludge, primarily regulated by the levels of Al, Fe, and Ca, while dissolved orthophosphate (ortho-P) constituted only 1% of the total phosphorus (TP). Notably, phosphorus exhibited a distinct distribution pattern between EPS and EPS residues: EPS comprised only 2.09% of TP, 74.19% of which was organic phosphorus (OP), whereas EPS residues contained 93.26% of TP, with a much lower OP proportion (15.04%).\u003c/p\u003e","manuscriptTitle":"The distribution of phosphorus forms in dewatered sludge with three classification analysis methods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-13 15:51:45","doi":"10.21203/rs.3.rs-7369225/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e87ac4c-0723-4e9c-be4a-fab2426d394b","owner":[],"postedDate":"October 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-08T13:53:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-13 15:51:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7369225","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7369225","identity":"rs-7369225","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}