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Our analyses of marine invertebrate communities at a regional scale and at two local sites revealed that taxonomic density (i.e. species density) was a sensitive index of marine sediment quality. However, although Hill–Simpson diversity and Pielou evenness were shown to respond to sediment variability in the regional dataset, they could be insensitive or respond falsely when a low number of individuals was observed, and when more than one community co-existed at a local site. These results from two local sites should serve as a point of caution when using diversity indices. Although these indices can provide a good understanding of how communities respond to sediment degradation, it is important to understand how these indices collapse when there is a small number of individuals observed or when the data span multiple co-existing communities. This emphasises the need for better strategies for the ecological assessment of sediment quality based on diversity indices at the local scale in marine areas.The analyses of the regional dataset show that WC had a larger impact than other variables on the taxonomic density of benthic invertebrate communities, although grain size and organic matter content are also thought to affect benthic invertebrate richnesse.g.17,18. The high contribution of WC likely reflects its physical effects on sediment structure. The optimal range of WC for the burrowing activity of benthic invertebrates is between around 25% WC at the densest (i.e. hardest) and around 40% WC at the loosest (i.e. softest) packing of sediment19,20. A WC value exceeding the upper limit of this optimal range could indicate sediment that is too soft for the burrowing activity of benthic invertebrates; this may explain the negative effect of WC on taxonomic density observed in this study.The relatively high standard deviation of random effects as compared against the effect size of WC in GLMMs suggests that unmeasured variables had a strong effect on taxonomic density. Salinity21 and anthropogenic impacts, such as dredging and trawling13, are well-known factors that could affect the diversity of benthic invertebrates. However, they were not considered in the regional dataset, because these factors are site- and sampling-location specific, and therefore it was impossible to identify which factors needed to be measured prior to investigation. Our study highlights one advantage of GLMMs, which is the ability to show the effects of these unmeasured factors. The effect size of WC was almost as large as that of the random effects in the low-frequency group (Table 1), which suggests that rare invertebrates were more sensitive to sediment degradation in this group, and that this sensitivity contributed to the overall response of taxonomic density.The analyses of the regional dataset also showed that an increase in WC caused a decrease in Hill–Simpson diversity and Pielou evenness in the reliable data. This result is consistent with previously identified responses to sediment degradation6. Because the low values of these indices occurred in communities with a few dominant species, this suggests that the benthic community was dominated by a few species in soft sediments (i.e. where WC was high).Similarly, increasing WC was associated with a significant decline in species density at the two local sites (Table 2), and the trend was significant for both reliable and unreliable data. This suggests that WC can be an indicator of benthic invertebrate species density at the local scale. However, it is likely that the trend in species density was not only caused by the effect of sediment softness (as is suggested by our analysis of the regional dataset) but also by other factors. One such factor is anoxia, which has been observed from August to October in the water column above the sediment in Nagoya Port at locations where no individuals were sampled (i.e., N5, N9, N10, and N12)22. Similarly, high organic-carbon and trace-metal concentrations have been reported in our study area23. These factors could have co-occurred with high WC, and thereby contributed to the decline in species density observed in our study. Because spatial correlations between variables tend to occur at local scales24, it is difficult to identify factors that affect species density at this scale. Species density is itself a sensitive indicator; however, if alternatives are needed, parameters that explain variations in species density, such as WC, are recommended for use as a representative variable in local assessment.WC did not always have a significant negative effect on Hill–Simpson diversity or Pielou evenness at the local scale (Table 2). The significant negative effect of WC on Hill–Simpson diversity identified in the reliable data from Nagoya Port indicates that community structure was dominated by a few species at higher WC, which mirrors the results obtained from the regional dataset. However, WC had no significant effect on Pielou evenness, and even the effect on Hill–Simpson diversity was only significant once locations that have different coexisting community structures (i.e., the Fujimae tidal flat, N8, and N20) were excluded from the analysis. These results mean that these diversity indices are not as sensitive to changes in WC as species density. Conversely, we found questionable significant negative effects of WC on both Hill–Simpson diversity and Pielou evenness when unreliable data were included in the analysis (Table 2). The low values of these indices obtained at high WC likely reflect artefacts in the unreliable data (Fig. 5c, d).It is important to find and exclude coexisting communities when analysing the effects of sediment degradation on indices of community structure (i.e., Hill–Simpson diversity and Pielou evenness) in a target community. In Matsunaga Bay, the river-mouth community on the intertidal flat was found to have a distinct sediment-particle-size composition compared to other communities in the bay based on multivariate analysis (Fig. 3c). In addition, because the polychaete Simplisetia erythraeensis that dominated the river-mouth community can be found in brackish environments (WoRMS: http://www.marinespecies.org/), low salinity (which was not measured in this study) may be a distinguishing feature of this location. Therefore, environmental characteristics such as sediment particle size, salinity, and the location of the intertidal flat likely underlie the spatial variability of community structure in this bay.Whereas we were able to predict the spatial variability of community structure prior to field sampling in Matsunaga Bay, this was not true in Nagoya Port. Our a priori expectation was that the benthic community on the Fujimae intertidal flat would have a distinct structure because of its location; although this was borne out by the data, we were unable to predict that there would also be distinct community structures at N8 and N20 because of the complex spatial patterning of benthic communities in this area. The explanatory variables we selected (salinity, C/N, WC, and D50) explained less than 11% of the total variance in community structure. This weak explanatory power indicates that unmeasured environmental variables may underlie the complex spatial patterning of benthic communities observed in our study, which is typical of the complexity often found in urbanised marine areas13.Although our results demonstrate that excluding distinct coexisting communities from the overall data is important when analysing species density (Fig. 5b) and Hill–Simpson diversity (Fig. 5c), such communities can be difficult to distinguish prior to field sampling. Therefore, post-hoc multivariate analysis is needed to distinguish between a target community and other communities. In addition, because diversity indices are affected by both species composition and the proportions of individuals in each taxon, the use of multiple distances between sampling points is recommended to assess how communities differ across space.The unreliability of Hill–Simpson diversity and Pielou evenness values calculated from small sample sizes can be explained by a theoretical framework for the effective number of species9. The effective number of species, which reflects the number of dominant species14, is predicted to decline or remain unchanged in response to low species density in cases where taxonomic density has a sensitive negative response (Fig. 6a). However, the effective number of species can be underestimated when there is a small sample size (Fig. 6b). This suggests that the questionable negative responses of Hill–Simpson diversity and Pielou evenness (which is calculated from the Shannon index) (Table 2) likely do not reflect real changes in community structure in Nagoya Port, but instead are caused by an artefact that negatively correlates with sediment degradation. However, low Pielou evenness was rarely associated with unreliable data in our study (Appendix S2). Pielou evenness tended to be high, approaching 1.0, in unreliable data from the regional dataset (Supplementary Fig. S2) and Matsunaga Bay (Supplementary Fig. S4). This bias can be explained as a possible result of small sample size. Our results should serve as a warning that false or insensitive responses in Hill–Simpson diversity and Pielou evenness may occur if sample size is insufficient to estimate these indices accurately.Figure 6Two mechanisms that can affect the effective number of species (which can be estimated with Hill–Simpson diversity). (a) The effective number of species becomes lower at low species density with sufficient sample size. When the degradation of sediment quality (SQ degradation) affects species density but not the density of individuals, the effective number of species decreases as a real response to community structure. However, as shown in (b), the effective number of species also becomes lower at small sample size n. When SQ degradation affects the density of individuals, the effective number of species might not reflect a real response in community structure.Full size imageIn this study, we used a sample size of 50 individuals as the threshold between reliable and unreliable data. Although a sample-size threshold can be useful when judging whether a sample accurately reflects community structure, the specific value we used was not based on any scientific evidence. In fact, our datasets included several data points classified as “reliable” that were not sufficiently saturated in Hill–Simpson diversity (Supplementary Figs. S2a, S4a, and S5a). Sample coverage is an index that standardises the number of taxa observed by the completeness of the sample15,25. The sample coverage of the reliable data was close to 1.0 (complete) and greater than that of the unreliable data in all three datasets used in this study. Although the rarefaction curve is a more direct way to show the estimation accuracy of Hill–Simpson diversity, the simplicity of the sample coverage index (as compared to drawing a rarefaction curve) is an advantage when judging data reliability. In addition, sample coverage is useful when plotting the degree of accuracy in two-dimensional figures, as was done in this study.When the number of individuals observed, n, is not sufficient to estimate the indices of community structure accurately, we can use an extrapolation technique that provides more reliable estimates by doubling the number of individuals observed to 2n9. Although we did not use this technique in this study because our objective was to explore how small sample sizes affect assessments of marine sediment quality, this technique is a useful solution for practical assessment when the number of individuals observed is not sufficient.In conclusion, our results show that species density responds sensitively to sediment degradation. By contrast, indices of community structure (i.e. Hill–Simpson diversity and Pielou evenness) were insensitive at the local scale because of masking by multiple coexisting communities, and sometimes produced misleading results because of inaccuracies associated with small sample sizes. Because indices for community structure provide a good understanding of how communities respond to sediment degradation, which cannot be provided by species density, ecological approaches using these indices have merits for assessing sediment quality because they are more realistic under field conditions3 and because they reduce uncertainties26,27. The potential for misleading and insensitive results must be avoided to keep from diluting these merits. We recommend that these diversity indices for community structure be used in local assessments only if it is possible to obtain a sufficient sample size for accurate estimation, and if co-existing communities can be differentiated before field sampling or by post-hoc analysis through sampling at multiple distances. More

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Bioleaching processVariation of pH and ORP during bioleaching processpH and ORP of the sludge are widely known to be the important parameters influencing heavy metal solubilization during bioleaching process, as well as the activity of iron-oxidizing microorganisms10,26,27. The variation of sludge pH and ORP during the single bioleaching process is presented in Fig. 1.Figure 1Variation of pH and ORP during bioleaching process.Full size imageAn appropriate pH could enhance the activities of microbes, affecting the release of metals and the stability of metal ions in the liquid phase5. As shown in Fig. 1, the pH value of sewage sludge quickly decreased from 6.44 to 3.07 in the first 6 days, due to the oxidation of Fe2+ and metal sulfides, the production of sulfuric acid, ferric hydroxide and jarosite from the hydrolysis of Fe3+18. Then the pH gradually decreased to 2.89 on the 10th day. The change of ORP followed an opposite trend. ORP value of the sludge rapidly increased from − 155.6 mV to 480.0 mV in the first 6 days, then to 505.0 mV in the following 4 days, due to the oxidation of Fe2+ to Fe3+ by leaching microorganisms.Heavy metals solubilization and chemical speciation distribution during bioleaching processThe removal of heavy metals during bioleaching process and the distribution of chemical fractions of heavy metals before and after bioleaching are presented in Figs. 2 and 3, respectively. The single bioleaching led to the removal of Zn, Cu, Cd, Cr, Mn, Ni, As and Pb of 67.28%, 50.78%, 64.86%, 6.32%, 56.15%, 49.83%, 20.78% and 10.52% in 10 days, respectively. The solubilization efficiency was highly related to the evolution of pH and ORP, the chemical fraction distributions and the nature of heavy metals.Figure 2Removal of heavy metals during bioleaching process.Full size imageFigure 3Chemical speciation distributions of heavy metals in raw sludge and bioleached sludge, total concentration of heavy metals in the raw sludge was set as 100% (RS raw sludge, BS bioleached sludge).Full size imageFigure 2 illustrated that Zn had the highest solubilization and removal efficiency. It was found that below the threshold pH of 6–6.5, Zn could be dissolved28. Thus, the dissolving out of Zn had started at the beginning of leaching experiment with a removal percentage of 10.15% on the 2nd day. Yet the quick solubilization of Zn was observed from the 4th day (pH 4.01). And until the 6th day (pH 3.00) when the solubilization percentage of Zn was 65.71%, the leaching rate of Zn was slowed down due to the stable pH. In the raw sludge, Zn mainly existed in mobile forms (exchangeable/acid soluble and reducible forms) as shown in Fig. 3. After bioleaching, the solubilization efficiencies of Zn in exchangeable/acid soluble form and reducible form was 58.66% and 87.93%, respectively. Meanwhile, 48.27% of Zn in oxidizable form was also dissolved out due to the oxidation of metal sulfide and loss of sludge organic matter. However, Zn in residual form remained almost unchanged in the bioleached sludge due to its high stability.It has been pointed out that Cu could be rapidly solubilized below pH of 3.7 or under a high ORP condition29. As shown in Fig. 2, in the first 4 days, the solubilization efficiency of Cu was relatively low (11.44%). The removal rate of Cu increased rapidly to 43.54% on the 6th day due to the increase of ORP (480 mV). The proportion of Cu in exchangeable/acid soluble form increased by 55.16% after bioleaching, probably because the solubilized Cu2+ was re-adsorbed on the EPS of sludge cells30,31. Most of Cu was present in reducible and oxidizable forms in the raw sludge as shown in Fig. 3, because the complexation of copper and organic materials was relatively stable30,32,33. The removal percentages of Cu in reducible and oxidizable forms were 71.11% and 61.83% after bioleaching, respectively, which was the main reason for Cu removal.Cd could be solubilized rapidly under acidic conditions as shown in Fig. 2, which is consistent with the previous study34. The solubilization of Cd could be finished in 6 days with the removal rate of 64.36%. Cd was mainly present in mobile forms (91.07%) as shown in Fig. 3, which agreed with the findings of Zeng et al.35 Thus, the acid dissolution was the main removal mechanism of Cd34. Due to the low pH of the bioleached sludge, the content of Cd in mobile forms decreased by 62.77% after bioleaching. Furthermore, Cd in immobile forms (oxidizable and residual forms) also reduced significantly.The previous study found that Cr was relatively stable with the dissolved pH threshold of 2.3–3.028. Although the percentage of Cr present in mobile forms was over 40%, the removal rate of Cr (6.32%) was the lowest among all the heavy metals investigated as shown in Fig. 2, because the lowest pH of the bioleached sludge was about 2.9, which was close to the dissolution threshold limit of Cr.As shown in Fig. 2, Mn and Ni were solubilized quickly in the first 4 days. The solubilization percentage of Mn and Ni were 56.14% and 49.83% after bioleaching, respectively. Mn and Ni mainly existed in the mobile forms (Mn 82.05%, Ni 76.08%). In the early stage of bioleaching, the removal rates of Mn and Ni were closely related to the variation of pH and displayed obvious acid dissolution mechanism. After bioleaching, the concentrations of Mn in exchangeable/acid soluble, reducible and oxidizable forms were reduced by 34.65%, 78.82% and 90.84%, respectively. As for Ni, the removal rates in such forms were 34.66%, 74.58% and 64.99%, respectively. Thus, the higher extraction efficiency of Mn and Ni arose from mixed bioleaching mechanisms, which contain acid dissolution, oxidation and reduction by Fe2+/Fe3+.Relatively low removal efficiency of As (20.78%) was observed in this study. One reason, as shown in Fig. 3, was that As was mainly distributed in residual form with high stability. The other reason was that the dissolved As3+ could be oxidized to As5+ (AsO43-) by Fe3+ generated from the metabolism of iron-oxidizing bacteria, and then insoluble FeAsO4 could be produced through the reaction of AsO43- and Fe3+, which resulted in the reprecipitation of As34.Pb in exchangeable/acid soluble form was not detected in the raw sludge, and mainly existed in reducible (59.20%) and oxidizable (23.19%) forms. The removal rates of Pb in reducible and oxidizable forms were 33.51% and 58.17% after bioleaching, respectively. However, the insoluble compounds such as PbSO4 (Ksp = 1.62 × 10–8) could be generated during the bioleaching process36, which resulted in a significant increase in the concentration of Pb in residual form (from 10.89 to 25.00 mg/kg), and thus led to the low removal ratio of Pb (10.52%).To summarize, the solubilization efficiencies of Zn, Cu, Cd, Mn and Ni, which mainly existed in mobile forms in the raw sludge, were relatively high due to the instability of these metals, while the removal rates of Cr, As and Pb, which mainly existed in immobile forms, were relatively low. However, the contents of most heavy metals in mobile forms decreased obviously after bioleaching and would lead to the corresponding reduction of the environmental risk of the sludge.Combined bioleaching/Fenton-like processEffect of H2O2 dosage on the removal of heavy metals under various pH conditionsPrevious studies have shown that the production ability of hydroxyl radical during the Fenton-like reaction process could be enhanced under pH range of 2.5–4.5, and meanwhile, the amount of H2O2 directly influences the production of hydroxyl radical10,18. Therefore, as shown in Fig. 4, the effects of H2O2 dosage on the solubilization efficiencies of heavy metals were investigated at different stages of the bioleaching process, when the pH values of the bioleached sludge were 4.5 (about 3.5th day), 4.0 (4th day) and 3.0 (6th day).Figure 4Effects of H2O2 dosage on the removal efficiency of heavy metals under various pH conditions.Full size imageWith the increasing concentrations of H2O2 (0.0–8.0 g/L), the solubilization efficiency of Zn increased significantly at pH of 4.5 (Fig. 4) due to the oxidation of metal sulfide and organics by hydroxyl radical10. However, the solubilization percentages of Zn barely changed with further increase of H2O2 dosage (from 8.0 to 15.0 g/L). The solubilization percentage of Zn at the H2O2 dosage of 8.0 g/L (pH of 4.5) was significantly higher than when only using single bioleaching (75.31% vs. 67.64%). The enhancement of solubilization efficiency of Zn at a pH of 4.0 and 3.0 was not very noticeable (Fig. 4), because most of the Zn in immobile forms was dissolved out by bioleaching. The highest solubilization percentages of Zn were 74.96% at a pH of 4.0 and 75.53% at a pH of 3.0, which were 7.32% and 7.89% higher than that of the single bioleaching process.Due to the lower dissolved pH threshold of Cu compared with Zn, the solubilization efficiency of Cu was significantly affected by the dosage of H2O2 at a pH of 4.5 and 4.0 as shown in Fig. 4, while when the reaction pH was 3.0, the subsequent Fenton treatment had a relatively small impact on the removal of Cu. The highest removal rate of Cu (52.17%) was obtained at pH of 3.0 and H2O2 dosage of 13.0 g/L, which was slightly higher than that of the single bioleaching (50.78%). The change in solubilization efficiency of Cd was similar to that of Cu. When the pH values were 4.5 and 4.0, the solubilization percentages of Cd with H2O2 dosage of 15.0 g/L were 4.59% and 1.23% higher than that of the single bioleaching process, respectively. Meanwhile, the highest solubilization percentage of Cd (71.91%) could be reached at a pH of 3.0 and H2O2 dosage of 13.0 g/L, which was higher than that of the single bioleaching process (64.86%).The addition of H2O2 did not increase the removal rate of Cr significantly as shown in Fig. 4. At a reaction pH of 4.5, the solubilization percentage of Cr was 7.59% with H2O2 dosage of 15.0 g/L, which was a little higher than that of the single bioleaching process (6.32%), while the highest solubilization percentages of Cr could reach 11.63% and 9.18% at pH of 4.0 and 3.0, respectively, with H2O2 dosage of 15.0 g/L.The solubilization process of Mn and Ni displayed similar trend as shown in Fig. 4. The solubilization percentage of Mn was not significantly improved when the H2O2 dosage was increased from 5.0 to 11.0 g/L at pH of 4.5 and 4.0, but a much faster increase of the removal rate was observed with the H2O2 dosage over 13.0 g/L. It could be due to the enhanced oxidizing ability of Fenton-like reaction with abundant H2O2. However, the solubilization efficiency of Mn under a pH of 3.0 began to increase with H2O2 concentration of 11.0 g/L, which could be attributed to the high efficiency of Fenton action under lower pH15. The highest removal percentage of Mn was 66.29% at pH of 3.0 and H2O2 dosage of 15.0 g/L, while the removal percentage of Mn in the single bioleaching process was 56.14%. The removal behavior of Ni at various pH was consistent with Mn. The highest removal rate of Ni (65.81%) was found at a pH of 3.0 with H2O2 dosage of 15.0 g/L, which was significantly improved, compared with the single bioleaching process (49.83%).On the contrary, the removal efficiency of As and Pb in the combined process was not promoted compared with the single bioleaching process. Due to the strong oxidizing capacity of Fenton-like process, the yield of SO42− and insoluble FeAsO4 could be improved. Correspondingly, Pb2+ could be transformed into residual form, such as insoluble PbSO410. Therefore, the removal efficiencies of As and Pb decreased in the combined process. The highest removal rates of As and Pb after Fenton-like treatment were 12.46% and 10.20%, respectively.In the combined process, higher solubilization efficiencies of most heavy metals (Zn, Cu, Cd, Mn, Ni, Cr) could be achieved in 6 days. The removal efficiency of heavy metals (except Cr, As and Pb) of combined process (pH of 3.0, H2O2 dosage of 15 g/L) is higher than that of the single bioleaching process. The removal rate of Zn, Cu, Cd, Mn and Ni increased by 7.89%, 0.38%, 5.56%, 10.15% and 15.35%, respectively. Meanwhile, the total concentrations of heavy metals measured in this study after treatment could meet the control standards of pollutants in sludge for agricultural use of China (National Standard GB 4284-2018). The removal of As and Pb was not improved by the combined process, other methods such as chemical leaching, electrokinetic remediation and phytoremediation could be considered as alternatives. However, their transformation into insoluble forms may also reduce the bioavailability of heavy metals and increase the environmental safety of the treated sludge. For that reason, the chemical speciation distributions of heavy metals in the combined process were further analyzed in detail.Chemical fraction distributions of heavy metals in the combined processIt can be seen in Fig. 4 that the solubilization efficiency of most heavy metals did not change significantly with H2O2 dosage below 8.0 g/L. Therefore, the chemical speciation changes of heavy metals after Fenton treatment under H2O2 dosage of 11.0, 13.0 and 15.0 g/L, as shown in Fig. 5, were discussed.Figure 5Change of chemical speciation distributions of heavy metals under different H2O2 dosage at a pH of 4.5, 4.0 and 3.0, total concentration of heavy metal in the raw sludge was set as 100%.Full size imageUnder various pH conditions, the contents of Zn in all of the four forms showed a downward trend along with the increasing H2O2 dosage (Fig. 5). After bioleaching, Zn mainly existed in exchangeable/acid soluble form under the final pH of 4.5 (64.89%), pH of 4.0 (73.33%) and pH of 3.0 (80.82%). The removal of Zn in exchangeable/acid soluble form showed good correlation to the dosage of H2O2, which might be attributed to the destruction of EPS, and the released heavy metals were transferred to the liquid phase. Meanwhile, the improvement of sludge dewaterability could also promote the removal of heavy metals. After Fenton-like reaction at a pH of 4.5, the percentages of Zn in exchangeable/acid soluble forms were reduced by 30.35%, 31.41% and 40.09% at H2O2 dosage of 11.0, 13.0 and 15.0 g/L, respectively, compared with the percentage of Zn in the sludge at the end of the single bioleaching process. However, the percentage of Zn in other forms did not change significantly after Fenton-like treatment. Therefore, the further removal of Zn in exchangeable/acid soluble form and the dewaterability improvement of sludge may be the main reasons for the higher removal efficiency of Zn in the combined process.Cu was still mainly associated with the oxidizable form after bioleaching ended at pH of 4.5, 4.0 and 3.0 (Fig. 5), which might be attributed to the preference of Cu for organic materials22. The addition of H2O2 at pH 4.5 significantly boosted the solubilization efficiency of Cu in exchangeable/acid soluble form. The percentages of Cu in exchangeable/acid soluble form in the sludge after Fenton treatment at pH 4.5 were 24.69% (11.0 g/L), 29.50% (13.0 g/L) and 38.15% (15.0 g/L), which were lower than that at the end of the single bioleaching process. Meanwhile, the content of Cu in reducible form was reduced by nearly 50% with H2O2 dosage of 13.0 and 15.0 g/L, compared with its content after bioleaching ended at pH 4.5. However, the highest removal rate of Cu in oxidizable form was only 33.20% with H2O2 dosage of 15.0 g/L. The removal efficiency of Cu in exchangeable/acid soluble and reducible forms increased with the increasing H2O2 dosage at pH 4.0 and 3.0, similar to the observation at pH 4.5. Under a reaction pH of 4.0, 47.2% of Cu in oxidizable form was removed after Fenton treatment with H2O2 dosage of 13.0 g/L, while only 28.6% was removed at H2O2 dosage of 15.0 g/L. In addition, the removal rates of Cu in oxidizable form were only 4.9–17.7% at various H2O2 dosage at a Fenton reaction pH of 3.0. The removal efficiency of Cu was reduced in despite of the increasing oxidation capacity of Fenton-like reaction. The macro-molecular organic matters could be degraded into small organic molecules during Fenton treatment process, releasing partial Cu. However, the generated small molecule organic matters had more undissociated carboxyl that would combine with released Cu31, which formed Cu in oxidizable form. Thus, it could explain the low removal efficiency of Cu in oxidizable form under stronger oxidizing condition. However, the highest removal rate of Cu (52.17%) was observed at pH 3.0 and H2O2 dosage of 15.0 g/L, due to the high reduction ratio of Cu in mobile forms at that condition.Cd mainly existed in mobile forms in the sludge after bioleaching and Fenton treatment, as shown in Fig. 5. The contents of Cd in mobile and oxidizable forms decreased with the increasing H2O2 dosage at pH 4.5. The content of Cd in exchangeable/acid soluble form after Fenton treatment at pH 4.5 and H2O2 dosage of 15.0 g/L was 29.10% lower than that at the end of the single bioleaching process. Meanwhile, the content of Cd in mobile form was decreased by 27.54% (11.0 g/L), 26.56% (13.0 g/L) and 36.72% (15.0 g/L) after Fenton treatment at pH 4.0. The removal of Cd in exchangeable/acid soluble form after Fenton treatment could be largely due to the improvement of sludge dewaterability. However, the reduction of Cd was not obvious after Fenton treatment at pH 3.0, because the solubilization threshold of most of Cd in various forms were reached after the bioleaching process ended at pH 3.0.The removal efficiency of Cr was not improved obviously by Fenton treatment in this study, as shown in Fig. 5. It was also reported that Cr was difficult to be removed by bioleaching or combined process due to its relatively high stability10. However, the content of Cr in oxidizable form after Fenton treatment at pH 4.5 was 4.76% (11.0 g/L), 9.20% (13.0 g/L) and 9.84% (15.0 g/L) lower than that at the end of the single bioleaching process, due to the strong oxidizing capacity of hydroxyl radical. And the lowest content of Cr in oxidizable form was observed after Fenton treatment at pH 4.0 and H2O2 dosages of 13.0 g/L, which was 39.4% lower than that in the bioleached sludge. Meanwhile, the highest Cr removal rate was also obtained at this condition after Fenton-like treatment. Thus, the improvement of Cr removal in combined process was mainly due to the release of Cr in oxidizable form. Furthermore, the released metals could be absorbed on the surface of oxides31, thus inevitably caused the increase of Cr in reducible form as shown in Fig. 5. The chemical speciation change of Cr after Fenton treatment at pH 3.0 was similar to that at pH 4.0.The removal efficiency and chemical speciation distribution of Mn varied obviously after Fenton treatment with different dosages of H2O2. The removal rate of Mn was improved with the increasing dosage of H2O2 at various pH values. Because most of the Mn in reducible form (over 80%) was removed by bioleaching process, the reduction of Mn in exchangeable/acid soluble form should account for the removal of a substantial part of Mn after Fenton treatment. The highest removal rate of Mn in exchangeable/acid soluble form under different pH conditions was 26.27% (pH 4.5), 25.06% (pH 4.0) and 42.18% (pH 3.0), all with H2O2 dosage of 15.0 g/L. Although nearly 30% of Mn in reducible and oxidizable forms was also removed after Fenton treatment with H2O2 dosage of 15.0 g/L at various pH values, it contributed little to the removal of Mn considering the low concentration of Mn in reducible and oxidizable forms in the raw sludge. Furthermore, the changes of Mn in residual form were not obvious under different pH.The chemical speciation change of Ni was similar to that of Mn after Fenton treatment. The contents of Ni in mobile and oxidizable forms decreased along with the increasing dosage of H2O2, as shown in Fig. 5. Meanwhile, the reduction of Ni in exchangeable/acid soluble form after the addition of H2O2 was the prime reason for the higher removal efficiency of Ni after the combined process than that after the single bioleaching process. The highest removal rate of Ni in exchangeable/acid soluble form was found with H2O2 dosage of 15.0 g/L at pH 4.0, which was 34.47% lower than that in the sludge after the signal bioleaching process. However, the highest removal efficiency of Ni (65.19%) was reached when the reaction pH was 3.0 with H2O2 dosages of 15.0 g/L due to the simultaneous reduction of Ni in reducible and oxidizable forms. The contents of Ni in reducible and oxidizable forms were reduced by 50.30% and 52.83% under this reaction condition, respectively, compared with that at the end of the single bioleaching process.As and Pb were mainly present in residual form before Fenton treatment as shown in Fig. 5. The content of As in exchangeable/acid soluble form decreased significantly due to the degradation of EPS at various pH values with the addition of H2O2. However, the content of As in residual form gradually rose with the increasing dosage of H2O2, probably because As3+ could be oxidized to As5+ by hydroxyl radical and/or Fe3+ with the formation of insoluble FeAsO434. The content of Pb in reducible form showed a trend of increase after Fenton treatment. SO42− was generated due to the oxidation of sulfur elements and/or sulfide in sludge by hydroxyl radicals with the production of insoluble PbSO410, and thus the content of Pb in residual form also increased after further Fenton treatment. Although the Fenton treatment had a negative impact on the removal of As and Pb as shown in Fig. 5, because of the formation of insoluble compounds under strong oxidizing condition, the environmental risk of these two heavy metals decreased to some extent under an appropriate condition, due to the increased proportion of immobile fractions, especially residual form. compared with the bioleached sludge.The content and proportion of most heavy metals (Zn, Cu, Cd, Mn, Ni, As) in mobile forms were lower in the treated sludge after the combined bioleaching and Fenton-like process, compared with the single bioleaching process, which was also the main reason for the high removal efficiency of these metals. Their bioavailability and toxicity were also reduced. However, Fenton treatment was found to have a negative impact on the removal of As, but the increased proportion of As in residual form also lowered its bioavailability and mobility in the environment. The increase in the content of Pb in both mobile forms (mainly in reducible form) and immobile forms (mainly in residual form) was observed under different conditions, so special attention should be paid to the chemical speciation distributions of Pb during sludge treatment process.The effect of H2O2 dosage on sludge dewaterability at different pH valuesThe changes of CST of treated sludge under various conditions are presented in Fig. 6. The CST of the raw sludge (98.7 s) was dramatically reduced by bioleaching and Fenton oxidation treatments. After bioleaching ended on the 10th day (pH 2.89), the 6th day (pH 3.0), the 4th day (4.0) and the 3.5th day (pH 4.5), CST values of 20.3 s, 24.2 s, 30.7 s and 35.0 s were observed. The decreased pH after bioleaching process could destroy the EPS and neutralize the negative charge of the sludge flocs, resulting in the release of bound water37. Moreover, sludge dewatering could also be improved by the coagulation effect of Fe2+ 10. Furthermore, hydroxyl radicals were essential to improve sludge dewatering performance by destroying EPS and porous structure during the Fenton treatment process35. Therefore, the CST value of treated sludge was reduced to 20.6 s after Fenton treatment with H2O2 dosage of 15 g/L at pH 4.5, which was comparable to the CST value at the end of the single bioleaching process. The CST values were further reduced along with the decreasing reaction pH (4.0 and 3.0) and the increasing H2O2 dosage. The lowest CST value of 12.4 s was observed at Fenton reaction pH 3.0 and H2O2 dosage of 15.0 g/L, which meant a reduction from the initial CST of 87.44%. Therefore, the combined process could lead to an obvious improvement of the sludge dewaterability and significantly reduced the treatment period.Figure 6Changes of CST under different H2O2 dosage and pH.Full size image More

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