Effects of phase separation on dewaterability promotion and heavy metal removal of sewage sludge during bioleaching

Bioleaching is of increasing interest because of its high efficiency in improving sludge dewaterability and removing heavy metals from sewage sludge. However, in traditional single-phase bioleaching, a high-efficiency level cannot be maintained continuously, wherein the microbial synergistic effect is disrupted at a low pH environment. Therefore, in this study, a series of multi-compartment–baffled flow trials were performed to assess the effects of phase separation on sludge bioleaching by comparing a two-phase trial with two single-phase trials. Energy substrate and part of the bioleached sludge were introduced separately into two compartments to form two phases, namely selection phase and bioleaching phase. The results show that phase separation apparently shortened the start-up duration of sludge bioleaching from 7 days in a single-phase bioleaching to 4 days in two-phase bioleaching. The dewaterability of bioleached sludge was also enhanced by phase separation with relative decreases of 25.0–33.3% for specific resistance to filtration and 14.2% for capillary suction time, which was attributed to lower pH values, zeta potential closer to zero, and less dissolved organic matter in bioleached sludge after two-phase bioleaching. Phase separation generally increased the removal ratios of heavy metals during sludge bioleaching by −0.79 to 2.60%, 11.06 to 15.04%, 4.45 to 11.03%, 17.98 to 23.46%, 7.20 to 9.28%, −9.22 to −2.46%, and −6.72 to −10.68% for As, Cd, Cr, Cu, Ni, Pb, and Zn, respectively. Phase separation also enriched the Acidithiobacillus spp. and reduced the inactivation of acid-tolerant fungi, which can be conducive to better synergistic effect, and therefore maintain long-term stable state in the bioleaching phase of the two-phase bioleaching process.


Introduction
Production of sewage sludge has increased rapidly owing to the enlargement of sewage treatment capacity in China (Li et al. 2013). Since sewage sludge still has a high value of moisture content after gravity thickening, dewatering is necessary to minimize sludge volume and facilitate its transportation and treatment. Moisture content in sewage sludge can decline to approximately 80% via mechanical dewatering (Lee and Liu 2000;Lo et al. 2001;Neyens et al. 2004). This moisture content is nonetheless high for subsequent sludge treatment, implying a considerable overall cost. To decrease the overall cost of sludge treatment (Lee and Liu 2000;Mahmoud et al. 2011), effective sludge preconditioning is necessary to improve dewaterability (Raynaud et al. 2012).
Generally, three main classes of conditioning methods exists, which promotes sludge dewaterability: physical methods such as sonication (Lippert et al. 2020), chemical methods, such as advanced oxidation processes (AOPs) (Masihi and Gholikandi 2018), and biological methods that include microbial flocculants and bioleaching (Liu et al. 2012a, b;Lu et al. 2019). Among these methods, AOPs and bioleaching are the most promising technologies. In recent years, AOPs (Fenton, O 3 , electro-Fenton, Fe 2+ -persulfate, nZVI-Fenton, nZVI-persulfate) have been extensively investigated, which demonstrates the highest efficiency in enhancing sludge dewaterability (Cao et al. 2020;Coha et al. 2021;Wu et al. 2020). However, the key disadvantage of AOPs is the highoperating costs due to the addition of many expensive oxidizers and pre-acidification with inorganic acids. Compared to AOPs, bioleaching has considerably lower operating costs because it achieves acidification and oxidation of sludge via the addition of cheap industrial chemicals, such as FeSO 4 · 7H 2 O or S 0 , and uses oxygen as an oxidizer by simple aeration (Benmoussa et al. 1998;Couillard and Mercier 1991;He et al. 2019;Kim et al. 2005;Liu et al. 2015). Additionally, bioleaching also has an added advantage; it removes heavy metals from sewage sludge.
In recent decades, the mechanism underlying how bioleaching improves sludge dewaterability and the related factors have been illustrated. During bioleaching, energy substrates, such as Fe 2+ and S 0 , are biologically oxidized by Acidithiobacillus spp., which results in a low pH via the hydrolysis of Fe 3+ and ferric biomineralization associated with several monovalent cations, such as K + (e.g., jarosite, ammoniojarosite, and schwertmannite) or oxidation of S 0 (He et al. 2019;Liu et al., 2012a, b;Tyagi et al. 1994). At a low pH, sludge dewaterability is promoted via neutralization of charged sludge flocs, destruction of extracellular polymer substances (EPS), and replacement of microorganisms Zhou et al. 2015), which has been observed based on the pronounced reductions in specific resistance to filtration (SRF) and capillary suction time (CST) (Liu et al., 2012a, b;Song and Zhou 2008). Many researchers reported that the bioleaching efficiency of the conditioned sludge is affected by several operating factors, such as energy substrate and pH of bioleached sludge (Ban et al. 2018;Ghavidel et al. 2018;Lin et al. 2020). Ban et al. (2018) observed that increasing the concentration of energy substrates accelerated sludge acidification. The acidic environment, particularly low pH, benefits greater sludge dewaterability. For example, Wong et al. (2015) observed the best conditioning of sludge at a low pH value of 2.67, with reductions in SRF, CST, and EPS of bioleached sludge by 96.0%, 88.0%, and 73.0%, respectively.
However, maintaining a stable low pH during long-periods of single-phase bioleaching is difficult, which is attributed to the instability of the microbial synergistic effect between acidtolerant heterotrophic microbes and Acidithiobacillus spp. (Wang et al. 2010). The growth of Acidithiobacillus spp. is inhibited by small-molecular-weight dissolved organic matter (DOM) at concentrations greater than 150 mg/L (Fang and Zhou 2006;Zheng et al. 2009), which originates from the degradation of macromolecular organic substances in sludge flocs in a highly acidic environment Wang et al. 2010). The presence of acid-tolerant heterotrophic microbes can relieve this negative effect by biodegrading DOM to lower concentrations, which is the supposed microbial synergistic effect between acid-tolerant heterotrophic microbes and Acidithiobacillus spp. However, although acid-tolerant heterotrophic microbes can survive at a low pH of 1 to 2 (Wang et al. 2010;Zheng et al. 2009), they gradually lose their physiological activity and are incapable of efficiently biodegrading small-molecular-weight DOM after long operating periods at a low pH, thereby weakening or even invalidating the microbial synergistic effect during bioleaching (Lin et al. 2020). The invalidation inhibits growth of Acidithiobacillus spp., decelerates acidification, which subsequently deteriorates bioleaching, implying a reduced improvement in sludge dewaterability (Zheng et al. 2016). Hence, it is important to maintain a high activity of both acid-tolerant heterotrophic microbes and Acidithiobacillus spp. to sustain a low pH during sludge bioleaching, which may be attained in theory via phase separation, producing optimal pH values specific for these two types of microorganisms.
However, few researches have reported on the methods of phase separation for sludge bioleaching, where the information about the effects of phase separation on bioleaching of sewage sludge is lacking. Therefore, in this study, two-phase (selection and bioleaching phase) baffled bioleaching was first designed by refluxing bioleached sludge and adding energy substrates in two different compartments. In the selection phase, a moderate pH value of approximately 4−7 is produced to recover the activity and biomass of acid-tolerant heterotrophic microbes, whereas a low pH value of 2−4 in the bioleaching phase assists in the rapid growth of Acidithiobacillus spp. to leach sewage sludge. A two-phase bioreactor was subsequently used to evaluate the feasibility of phase separation by investigating its effects on the physicochemical properties, dewaterability improvement, heavy metals removal, and sludge microbial community during bioleaching.

Sewage sludge and standard samples
Sewage sludge was collected from a sludge gravity thickener of the Yanshan Municipal Wastewater Treatment Plant in Guilin, China, which treats 20,000 m 3 /day of domestic wastewater with a cyclic activated sludge system combined with flocculation-disc filtration as the tertiary treatment. The sludge was pumped out from the sludge gravity thickener by placing the inlet of the suction pipe 2.5 m below the solid/liquid interface. Before use, the collected sludge was diluted with tap water to attain a solid content of approximately 2.0%. To be used as a raw sludge, the diluted sludge was sieved using a 10mesh nylon sieve (2 mm) to remove sand and fibers larger than 2 mm. Physicochemical properties of the raw sludge were pH 7.2 ± 0.2, zeta potential 15.4 ± 1.2 mV, soluble chemical oxygen demand (SCOD) 619.0 ± 18.8 mg/L, CST 31.5 ± 1.3 s, and SRF 3.9 ± 0.3 × 10 −13 m/kg. Mixed standard solution of heavy metals was purchased from Tmrm (Beijing, China), which included As, Cd, Cr, Cu, Pb, Ni, and Zn (100 mg/L of each metal).
Single-phase and two-phase baffled flow bioleaching reactor A bioleaching apparatus mainly consisted of a bioreactor, a mixing tank, and a storage tank (Fig. 1). The bioreactor is cuboid with total effective volume of 15 L, and length 300 mm, width 200 mm, and height 300 mm, and evenly divided into four compartments. Each compartment comprised of a down flow area (15 mm wide) and an upflow area (60 mm wide) partitioned by a 14-mm long baffle with 45°inclination angle. Air was supplied into the bioreactor using an air pump. After mixing in the tank, raw sludge without adding heavy metals was continuously transported to the 1 st compartment, which subsequently moved forward from the 1 st to the 4 th compartment. The bioleached sludge was transferred into the storage tank thrice per day (50% at 9:00 am, 13% at 12:00 pm, and 37% at 9:00 pm), a part of which was returned as an inoculum twice a day and the remaining was discarded from the storage tank after the second sludge refluxing. For singlephase bioleaching, both refluxed sludge and energy substrate were added into the 1 st compartment, whereas for two-phase bioleaching, they were added into the 1 st and 2 nd compartment, respectively.

Acclimation and enrichment of inoculum
A two-step procedure was applied to acclimate and enrich the bioleaching inoculum. First, 3.3 L of raw sludge was added into each compartment with 10 g/L (substrate mass / sludge volume) FeSO 4 ·7H 2 O as energy substrate. The sludge was inoculated to attain a stable pH value of approximately 2.0 −3.0 at 28 ± 2°C and an aeration rate of 3.8 L/min in each compartment. After inoculation, the cultivation continued for another 2-3 days to complete the acclimation. Second, a similar inoculation was completed to enrich bioleaching microbes in the raw sludge, using the acclimated sludge as the inoculum at a rate of 25.0% (volume of inoculum/volume of raw sludge). The enrichment procedure was repeated thrice, and the final enriched culture was applied as an inoculum for the subsequent bioleaching trials.

Bioleaching trials
Three trials were run for 15 days to investigate the effects of phase separation on bioleaching of sewage sludge. In each trial, the enriched sludge as an inoculum was introduced into the 1 st compartment at an inoculation rate of 40.0%. Raw sludge was then fed into the bioreactors and bioleached continuously at 28 ± 2°C and an aeration rate of 3.8 L/min in each compartment. The daily bioleached sludge was evenly refluxed from the storage tank twice a day (50% at 9:00 am and 50% at 9:00 pm), with a total proportion of 40% after Fig. 1 Schematic diagram of twophase baffled flow bioleaching apparatus: 1, 2, 3, and 4 represent the 1 st , 2 nd , 3 rd , and 4 th compartment, respectively adding FeSO 4 ·7H 2 O as an energy substrate into the bioreactor with a total dosage of 6 g/L (substrate mass/raw sludge volume). Before adding the energy substrate, sludge samples were collected daily at 9:00 am from each compartment to measure the pH. Three trials were considered as follows: trial 1 for two-phase bioleaching with four compartments (labeled as TP-4), trial 2 for single-phase bioleaching with four compartments (labeled as SP-4), and trial 3 for single-phase bioleaching with three compartments (labeled as SP-3). In both trial 1 and trial 2, a 7.5-L/day raw sludge was treated with the energy substrate added into the 2 nd and 1 st compartment for trial 1 and trial 2, respectively. For trial 3, a 5.6-L/day raw sludge was treated with energy substrate added into the 1 st compartment, with no aeration in the 4 th compartment, making it another storage tank. Each trial included two stages: start-up and stabilization. Start-up was considered complete when the pH value in each compartment did not vary for 2-3 days. During the start-up process, 110 mL of sludge samples were collected daily from each compartment to measure the pH. Each trial continued for another 7-10 days for stabilization, during which 700 mL of sludge sample was collected from each compartment every 3-5 days to analyze the physicochemical properties, dewaterability, and microbial community. Two replicates were performed for each trial.

Analysis methods
Sludge morphology and fluorescence distribution were observed under an optical microscope (N-10E, Novel, China) and a confocal laser scanning microscope (CLSM; Revolution XD, Andor, UK), respectively, both at 10 × 10 magnification. The observed sludge samples were collected from the last compartment of each bioreactor. For the CLSM analysis of the samples, blue spots were observed at ultraviolet excitation (380-420 nm). To the best of our knowledge, the blue light emitted from the sludge or bioleached sludge was most likely attributed to three groups of autofluorescent microorganisms: methanogenic archaea (Demirel and Orhan 2006;Stabnikova et al. 2006), anammox (anaerobic ammonium oxidizing) bacteria, such as Candidatus Brocadia fulgida (Böllmann et al. 2019) and Acidithiobacillus ferrooxidans (Bai et al. 2011). The first two groups are most likely unable to grow normally or survive at low pH and aerobic conditions prevailing during sludge bioleaching. Therefore, in this study, we attributed the blue fluorescence to A. ferrooxidans and analyzed the probable spatial distribution by observing the blue fluorescence under CLSM.
Sludge pH was measured using a pH meter (PB-10, Sartorius, China). Solid content in the sludge sample was determined by a gravimetric method. After settling in a centrifuge tube for 2 h, the supernatant of each sludge sample was collected to measure zeta potential using a particle size and zeta potential analyzer (Zetasizer Nano ZS90, Malvern, UK). The bioleached sludge samples were collected from each compartment to analyze SCOD, which was employed to represent DOM and indirectly reflect the amount of small-molecularweight DOM (Qiao et al. 2008). Sludge samples were centrifuged at 4000 rpm for 5 min, and then the SCOD of the supernatant was determined using a fast catalytic digestion method with a graphite furnace digester (SH220F, Hanon, China). Furthermore, SRF was measured by the Buchner funnelvacuum suction method with a neutral quantitative filter paper (8 μm) under a negative pressure of 0.03-0.04 MPa (Chen et al. 2010;Velmuzhov et al. 2020), and CST was measured by a CST Meter (304M, Triton, UK) with a 1.8-cm diameter funnel (Yu et al. 2010). After acid digestion with a mixture of nitric acid-perchloric acid, concentrations of heavy metals in sludge were determined using an inductively coupled plasma mass spectrometer (NexION 350X, PerkinElmer, America), and the recoveries of the heavy metal standard solution were 72.2%, 70.6%, 75.1%, 77.4%, 71.2%, 73.9%, and 108.1% for As, Cd, Cr, Cu, Pb, Ni, and Zn, respectively.
During the stabilization process, samples were collected from the raw sludge and from each compartment and stored at −20°C to measure the microbial community, which were labeled as A0 (raw sludge), A1, and A3 (1 st and 3 rd compartments in SP-3); B1, B2, and B4 (1 st , 2 nd , and 4 th compartments in SP-4); and C1, C2, and C4 (1 st , 2 nd , and 4 th compartments in TP-4). For each sludge sample, bacterial and fungal DNA was extracted using the kit from Novogene (Beijing, China) by CTAB and SDS methods, respectively. The extracted DNA was PCR-amplified with the primers 341F (5,-C C T A Y G G G R B G C A S C A G -3 ) a n d 8 0 6 R ( 5 , -GGACTACNNGGTATCTAAT-3) for 16S rDNA of bacteria, and ITS3-2024F (5,-GCATCGATGAAGAACGCAGC-3) and ITS4-2409R (5,-TCCTCCGCTTATTGATATGC-3) for ITS of fungi. The 16S rDNA/ITS amplicons were sequenced on the Illumina NovaSeq 6000 platform. The GreenGene database was applied to annotate taxonomic information of microorganisms.

Statistical analysis
Correlation analysis of the results was carried out to analyze the relationship between physicochemical properties and dewaterability indicators of bioleached sludge using R software (R Core Team 2018), at a significance level of p = 0.05.

Results and discussion
Morphology of sewage sludge and fluorescence distribution Figure 2 shows morphology of the raw and bioleached sludge in the three trials. In this figure, the shadow represents sludge flocs, reflecting the size and compactness of flocs. Compared with the raw sludge, the bioleached sludge had larger and aggregated flocs in each trial, which was enhanced by phase separation. The sludge gradually aggregated along the sludge flow direction. Changes in sludge morphology were due to the enhanced flocculation caused by hydrolysis and biomineralization of Fe 3+ , followed by Fe 2+ oxidation and the microbemediated EPS production during bioleaching (Li et al. 2012;Lin et al. 2020;Mohammadi et al. 2016;Yu et al. 2015). Since the blue spot was speculated to be excited by living Acidithiobacillus spp. (Fig. 3), more and larger spots in the sludge indicated a significant amount of highly active Acidithiobacillus spp. In each trial, bioleached sludge had more blue spots than raw sludge, which indicated that bioleaching enriched more Acidithiobacillus spp. in sludge (Bai et al. 2011;Lin et al. 2020). Additional blue spots were observed in compartments from two-phase bioleaching, especially in the 2 nd compartment of TP-4 ( Fig. 3(C4)), which indicated that phase separation benefitted the cultivation of additional Acidithiobacillus spp. in a bioleaching bioreactor. The higher abundance of Acidithiobacillus spp. in TP-4 could be illustrated by a relief in toxicity inhibition of SCOD towards Acidithiobacillus spp. caused by phase separation (Mohammadi et al. 2016). Figure 4 shows the changes in pH during the bioleaching process in the three trials. When pH value in each compartment varies no less or more than 0.2 per day, the start-up in the trial is complete. Phase separation markedly shortened the start-up period of sludge bioleaching from 7 days in two single-phase bioleaching trials (SP-3, SP-4) to 4 days in the two-phase bioleaching trial (TP-4). During start-up, pH values increased rapidly in the 1 st compartment, whereas relatively smaller variations were observed in the other compartments (Fig. 4a). This increase was due to the gradual dilution of high H + released from the inoculum by continuously adding raw sludge of a relatively neutral pH, as mentioned previously (Liu et al., 2012a, b;Misra et al. 2015). During the stabilization period, pH values generally declined along the sludge flow direction in each trial, with similar order as 1 st compartment > 2 nd compartment > 3 rd compartment ≈ 4 th compartment, which was caused by the growth of Acidithiobacillus spp. Meanwhile, a noticeable phenomenon in phase separation was observed in TP-4, which had higher pH values of 6.4-6.6 in 1 st compartment and low pH values of 2.7-2.9 in 3 rd~4th compartments than those of the two single-phase bioreactors. This could benefit a long period of synergistic effect between acid-tolerant heterotrophic microbes and Acidithiobacillus spp., because a slightly acidic environment in the selection phase (the 1 st compartment in TP-4) is suitable for the growth of acid-tolerant heterotrophic microbes Ye et al. 2021).

Physicochemical properties
In each trial, bioleaching significantly increased the sludge zeta potential in the last compartment compared with that of the raw sludge, indicating a higher flocculation capability (Fig. 5a). Zeta potentials in the last compartments were −2.6, −3.4, and −3.7 mV for TP-4, SP-4, and SP-3, respectively, implying that phase separation could improve dewaterability of bioleached sludge owing to zeta potential being closer to zero. Meanwhile, more subphases in SP-4 slightly increased the zeta potential of the bioleached sludge during single-phase bioleaching. This variation in zeta potential was associated with the pH trend among the three trials (Figs. 4, 5a). During bioleaching, SCOD of raw sludge was distinctly reduced from 619.0 ± 18.8 mg/L to (63.6 ± 7.5) -(135.3 ± 16.3) mg/L in all three trials (Fig.   5b), indicating that most SCOD from the raw sludge was biodegraded by acid-tolerant heterotrophic microbes. In these trials, SCOD gradually increased along the sludge flow direction, which was due to the production of SCOD derived from cell lysis and EPS decomposition, rather than its biodegradation. Furthermore, each compartment in TP-4 had a lower concentration of SCOD in the range of (63.6 ± 7.5) -(112.0 ± 26.2) mg/L than that in the other two trials, suggesting that phase separation could be considerably effective in biodegrading DOM of sludge and reducing the DOM inhibiting the growth of Acidithiobacillus spp. . A lower pH can be achieved, which results from improved growth of Acidithiobacillus spp., when DOM inhibition is alleviated (Fig. 4). Figures 5c and 5d show SRF and CST of sludge in the three trials during the stabilization period of bioleaching. In each trial, bioleaching markedly reduced the SRF from 3.9 × 10 −13 m/kg of raw sludge to 0.12-0.18 × 10 −13 m/kg of bioleached sludge in the last compartment (Fig. 5c), indicating a substantial improvement in sludge dewaterability (Huang et al. 2020;Liu et al., 2012a, b). Compared with the two trials of single-phase bioleaching, the bioleached sludge in TP-4 had a lower SRF, which indicated that phase separation enhanced the dewaterability of bioleached sludge. Meanwhile, more subphases in SP-4 slightly decreased the SRF of bioleached sludge during single-phase bioleaching. After bioleaching, sludge CST had a similar trend as SRF, which also sharply decreased from 31.5 s for the raw sludge to 9.4-13.9 s for the bioleached sludge in the last compartment (Fig.  5d). In TP-4, CST noticeably declined when sludge flowed from the 1 st compartment to the 2 nd compartment, but only a small reduction was observed in SP-4 and SP-3, implying that this difference was enlarged by phase separation. These variations in SRF and CST were related to the distinct pH values and zeta potentials among the three trials (Figs. 4, 5c), which was mainly attributed to denser floc structure caused by better flocculation performance, when the zeta potential was close to zero under high activity of H + and Fe 3+ (Citeau et al. 2011). Additionally, biodegradation also affected the flocculation and dewaterability of sludge. For example, biodegradation changed the EPS composition and the hydrophilic/ hydrophobic characteristics of the sludge floc surface, impacting the structure of sludge flocs (Wang et al. 2010;Wong et al. 2015).

Dewaterability
Phase separation also displayed a decrease in SRF by decreasing the sludge SCOD due to cell lysis in the bioleaching phase with acid-tolerant heterotrophic microbes. These results were consistent with those from the correlation coefficient matrix (Fig. 6). There was a strong positive correlation among SRF, CST, and pH at a significance level of p = 0.05, and a strong negative correlation between zeta potential and SRF or CST. As for SCOD, it was weakly positively related to SRF, but presented no significant relationship with CST. Therefore, the factors that affect the dewaterability of bioleached sludge follow the sequence: pH ≈ zeta potential > SCOD.

Removal of heavy metal from sludge
Generally, heavy metals in sludge can be reduced in an acidic environment, which is dissolved out in the form of an anion from the acid-soluble and oxidizable fractions Zheng et al. 2021). Throughout bioleaching, heavy metals were removed from raw sludge with the ratios of 13. 40-16.78%, 34.07-49.12%, 4.47-15.50%, 6.13-29.59%, 10.62-19.90%, 0.97-10.19%, and 45.57-56.25% for As, Cd, Cr, Cu, Ni, Pb, and Zn, respectively (Table 1). Furthermore, Pb was not essentially reduced in the sludge, which could be attributed to the chemical sedimentation of lead sulfate. Compared with single-phase bioleaching, twophase bioleaching increased the absolute removal ratios of heavy metals by −0.79 to 2.60%, 11.06 to 15.04%, 4.45 to 11.03%, 17.98 to 23.46%, 7.20 to 9.28%, −9.22 to −2.46%, and −6.72 to −10.68% for As, Cd, Cr, Cu, Ni, Pb, and Zn, respectively, suggesting that phase separation generally enhanced the removal of heavy metals from sludge during bioleaching. Zhu et al. (2013) also found that a lower pH value promotes heavy metal removal from sewage sludge during bioleaching process. Moreover, for the two-phase bioleaching, the bioleached sludge satisfied the requirement for the concentration limits of heavy metals in class A sludge for agricultural use (GB 4284-2018).

Microbial community
For each sludge sample, there were a high number of sequences, which indicated that the measured sample contained sufficient information to analyze the microbial community (Table 2). During bioleaching, specie numbers (OTU) generally decreased along the direction of sludge flow in each trial for both bacteria and fungi, where the decreases in fungi were more evident than bacteria. Similar trends also were investigated when considering the community diversity (Shannon index, Simpson index) and the community richness (Chao1 index, ACE index). Compared with single-phase bioleaching, sludge samples in two-phase bioleaching contained more fungal species, higher community diversities, and higher richness, implying that phase separation relieved the inactivation of acid-tolerant fungi. Considering that fungi is one key part of the acid-tolerant heterotrophic microbes (Yang et al. 2015), a relatively higher abundance of fungi could benefit by producing better synergistic effects in TP-4.
In each sludge sample, the four main bacterial phyla were Proteobacteria, Bacteroidetes, Acidobacteria, Firmicutes, Fig. 6 Pearson's correlation coefficient matrix with colorcoded correlation coefficients. "×" sign indicates a nonsignificant correlation defined by a p-value of > 0.01 among which Proteobacteria was dominant with a relative abundance of more than 50% (Fig. 7a). Similar phenomenon also was observed by Huang (Huang et al. 2020). At the genus level, Acidithiobacillus spp. had the largest abundance of the identified types in most bioleached sludge, other than the raw sludge and the bioleached sludge from the selection phase (C1) in TP-4, indicating that bioleaching improved the growth of Acidithiobacillus spp. in the bioleaching phase (Fig. 7b). Throughout the bioleaching process, the relative abundance of Acidithiobacillus spp. gradually increased in the bioleached sludge along the sludge-flow direction, and the values were 20.7%, 18.1%, and 13.8% in the corresponding last compartments for TP-4, SP-4, and SP-3, respectively. Higher relative abundances of Acidithiobacillus spp. were observed in the bioleaching phase (C2, C4) of TP-4 than that observed in the two trials of single-phase bioleaching, which indicated that p h a s e s e p a r a t i o n e n h a n c e d t h e e n r i c h m e n t o f Acidithiobacillus spp., benefitting a long-term and stable operation of the bioleaching reactor. These results were consistent with the change trends of physicochemical properties and dewaterability of sludge. Although, fungi are important in reducing DOM-mediated inhibition of Acidithiobacillus spp. activity by biodegrading DOM (Zheng et al. 2016), most fungi are unidentified in the sludge samples at the phylum and genus levels (Figs. 7c,7d). Based on the identified fungus, phase separation resulted in a dramatic succession, where the major phylum and genus shifted from Basidiomycota, and Candida to Chytridiomycota, and Boothiomyces, respectively, while the fungal succession only occurred at the genus level for two trials with single-phase bioleaching. This difference could be related to the different pH levels in these bioleaching reactors, resulting from the phase separation. Nonetheless, it still is necessary to obtain additional detailed information about fungal community to elucidate the relationship between phase separation, fungal community, physicochemical properties, and improvement in sludge dewaterability.

Conclusion
In this study, we investigated the effects of phase separation on physicochemical properties, dewaterability improvement, heavy metals removal, and sludge microbial community during bioleaching of sewage sludge. Phase separation evidently shortened the duration of start-up for sludge bioleaching, which was decreased from 7 days in single-phase bioleaching to 4 days in two-phase bioleaching, with a slighter lower pH 2.7-2.9 of the final bioleached sludge after two-phase bioleaching than 3.2-3.4 after the single phase. The results of SRF and CST showed that phase separation also markedly enhanced the dewaterability of bioleached sludge with relative decreases of 25.0-33.3% for SRF and 14.2% for CST, which it is attributed to lower pH values, zeta potentials closer to zero, and less DOM of bioleached sludge during the twophase bioleaching. Additionally, phase separation generally increased the absolute removal ratios of heavy metals from sludge by −0.79 to 2.60%, 11.06 to 15.04%, 4.45 to 11.03%, 17.98 to 23.46%, 7.20 to 9.28%, −9.22 to −2.46%, and −6.72 to −10.68% for As, Cd, Cr, Cu, Ni, Pb, and Zn, respectively. Phase separation enriched the Acidithiobacillus spp. and relieved the inactivation of acid-tolerant fungi, which could produce better synergistic effect in bioleaching phase during twophase sludge bioleaching and be conducive for maintaining long-term and stable operation of the bioleaching reactor. Future research on two-phase bioleaching is necessary to develop a more effective pretreatment technology of sewage sludge to improve its dewaterability and heavy metal removals.