Effects of Water Hardness on the Microorganisms in Activated Sludge

Phosphorus (P), as a key limiting element in aquatic eutrophication, its excessive discharge can trigger algal blooms and ecosystem degradation (Jones R A, Lee G F., 1982). The Enhanced Biological Phosphorus Removal (EBPR) process, which relies on the metabolic cycle of Polyphosphate Accumulating Organisms (PAOs) under anaerobic/aerobic conditions to achieve phosphorus removal, is widely used in municipal wastewater treatment due to its low cost and environmental friendliness (Zhang et al., 2011). However, its operational stability is susceptible to environmental factors such as temperature, pH, carbon source ratio, and ionic composition. Among these, water hardness (primarily Ca2+ and Mg2+) also significantly influences phosphorus removal. Nevertheless, research on the transformation pathways of phosphorus under the influence of water hardness is currently limited, and studies on its regulatory mechanisms on microbial metabolism are relatively scarce (Ding et al., 2022).Similar findings have been verified in European karst areas, where groundwater Ca2+ and Mg2+ concentrations range from 22.7-313 mg/L and 6.7-72.0 mg/L, respectively (Kozisek F, 2020). In typical karst areas of southern China (e.g., Yunnan, Guizhou, Guangxi), groundwater hardness usually falls within the range of 250-500 mg/L as CaCO3, with the total concentration of Ca2+ and Mg2+ exceeding 150 mg/L (Baomin Z, Jingjiang L., 2009; Keqiang et al., 2011). Domestic empirical studies also indicate that groundwater in karst regions generally exhibits high Ca2+/Mg2+ characteristics, with total hardness significantly higher than in non-karst areas (Shen et al., 2019). In these high-hardness areas, EBPR systems commonly face influent characteristics such as high Ca2+/Mg2+ concentrations and high ionic strength, which differ markedly from conventional soft water environments.In recent years, the impact of water hardness on EBPR systems has gradually become a research focus. Existing studies have shown that high hardness conditions can directly promote the formation of phosphate minerals. Crutchik D and Garrido J M. (2011) found that high Ca2+ concentration not only improved phosphorus removal efficiency but also promoted the formation of recoverable phosphorus minerals like hydroxyapatite (HAP). Quantitative analysis of phosphorus speciation in full-scale wastewater treatment plant sludge by Petriglieri et al. (2022) further confirmed that the contribution of chemically bound phosphorus to total phosphorus removal under hard water conditions was significantly higher than that of biologically bound phosphorus. It has been pointed out that the synergy between Ca2+-induced crystallization and biological phosphorus removal in the EBPR process can significantly enhance phosphorus removal efficiency and the level of mineral fixation. Furthermore, Ca2+/Mg2+ can enhance the structural stability of extracellular polymeric substances (EPS) through ionic bridging, promote granulation and settling performance, thereby improving the adsorption and fixation of phosphorus at the sludge interface . Beyond chemical and physical effects, hardness also regulates the microbial community structure and metabolic network within EBPR systems. Multi-omics studies have revealed that under high Ca2+/Mg2+ conditions, functional genes related to organic phosphorus cleavage (phnP, phnX), polyphosphate synthesis (ppk1, ppk2), and phosphorus stress response (phoR) were significantly upregulated . This indicates complex coupling effects among chemical precipitation, microbial metabolism, and EPS structural reinforcement under the influence of hardness. However, research remains limited on how hardness fundamentally alters the phosphorus uptake and storage behaviors of PAOs, and on the ultimate fate of phosphorus in sludge under high hardness conditions.