Changes in Microbial and Metabolic Pathways of Solidifying Manganese and Removing Nitrogen from Electrolytic Manganese Residue by the Sulfate-Reducing Bacteria

Abstract

Electrolytic manganese residue (EMR) contains a large number of soluble manganese ions and ammonia nitrogen, which seriously endangers the surrounding environment. Solidifying manganese and removing nitrogen has become the primary method for controlling EMR. In this study, an EMR stacking yard in Guangxi was used as a study site to study the solidification of soluble manganese ions and the removal of ammonia nitrogen by mixed bacteria under natural conditions. Further, Illumina MiSeq high-throughput sequencing technology was used to analyze the difference in microbial community structure and function. The results showed that the solidification rate of soluble manganese ions in the remediation area reached more than 99%, and the removal effect of ammonia nitrogen in EMR was obvious. The mechanism showed that manganese in EMR was solidified into MnS. High-throughput sequencing results showed that the abundance of sulfate-reducing bacteria in the remediation area was significantly higher than that in the control area. The functional groups predicted by the FAPROTAX database showed the functional groups related to N and S reduction increased significantly in the remediation area, while the functional groups related to N and S oxidation decreased. Microorganisms in the remediation area promoted the circulation of N and S elements, and the vegetation on the surface of the residue field in the remediation area was also restored.

1. Introduction

Electrolytic manganese residue (EMR) is the acid-leaching filtrate produced in electrolytic manganese production. The leachate of EMR contains a high concentration of manganese and ammonia nitrogen [1,2]. China is the largest producer and exporter of electrolytic manganese in the world, and each ton of electrolytic manganese produces 10–12 tons of EMR [3]. China produces about 20 million tons of EMR annually, with an accumulated production of more than 160 million tons [4,5,6]. Manganese in EMR is often present in manganese sulfate, manganese dioxide, and manganese oxide [7]. Among them, manganese sulfate is dissolved easily in water, manganese dioxide is insoluble, and manganese oxide dissolves Mn²⁺ under acidic conditions. Therefore, the contaminations by ammonia nitrogen and manganese sulfate in EMR are critical problems. Excessive intake of manganese can lead to various diseases, such as liver and biliary dysfunction, renal dysfunction, and mental deterioration [8,9]. Ammonia nitrogen intake can cause liver damage, blue baby syndrome, and gastric cancer [10]. The rapid development of the manganese electrolysis industry brings benefits and severe environmental pollution. Manganese pollution is the most serious in EMR, and its downward leakage will cause water pollution. Contaminated drinking water is dangerous to human health. So far, the comprehensive utilization rate of EMR in China is less than 7% [11]. The untreated EMR is piled directly into the slag field, and the pollutants seep into the nearby soil and migrate to the groundwater, severely damaging the local ecological environment. As a result, there is a lot of environmental pressure on the electrolytic manganese industry, and the disposal of EMR is a growing issue.

The soluble manganese research in EMR mainly focused on solidifying repair, which can be divided into chemical and microbial curing methods according to the different action objects. The chemical curing method refers to the treatment of EMR by using chemical curing agents to reduce the solubility of manganese through the reaction of chemical curing agents with soluble manganese ions. Among them, the more widely used chemical curing agents include alkaline materials, phosphates, sulfides, carbonates, silicates, and other agents [3,12,13,14,15]. Although the chemical curing method can quickly and efficiently cure the EMR, there are problems such as the introduction of secondary pollution and high cost, which seriously limit the large-scale application of this technology in the electrolytic manganese slag dumps. Microbial remediation technology has shown an eco-friendly and green method that effectively overcomes the shortcomings of chemical curing methods in field engineering applications, and is a hot spot for site remediation research in soils, sediments, tailing, and residues polluted so far [16]. EMR is rich in sulfate and ammonia so anaerobic biological treatment can be used. Under anaerobic conditions, sulfate-reducing bacteria (SRB) reduce sulfates to sulfides and then combine with heavy metal ions to form metal sulfide precipitates, thereby reducing the toxicity and environmental fluidity of harmful heavy metals [17]. SRB is a common soil bacterium and is highly adaptable, which can be used to remove higher concentrations of Mn²⁺ when the electron donor supply is sufficient [18].

Multiple ecological relationships among microbial communities interact with each other, and the introduction of remediation bacteria will inevitably affect community relationships. Therefore, Illumina MiSeq high-throughput sequencing technology was used to determine the structure of bacterial communities in EMR leachate, analyze the dominant species under different repair times, and further analyze the differences in functional genes of species. Exploring the differences in microbial communities during the solidification process provides a basis for in situ microbial remediations of EMR.

2. Materials and Methods

2.1. EMR, SRB, and Medium

The EMR used in this study was collected from a rhodochrosite area, located in Guangxi Zhuang Autonomous Region, China. The sampling interval was set as 10 m, and a total of 6 samples were collected, with a depth of 3 m. The physicochemical properties of leachate are shown in Table 1, which shows that the main pollutants of EMR are manganese and ammonia nitrogen. The concentration of Mn and ammonia nitrogen are far above the thresholds of the Integrated Wastewater Discharge Standard (GB 8978-1996) [19], indicating that these concentrations are a significant risk to the surrounding environment.

The SRB used in this study was provided by the National Engineering Research Center for Environment-friendly Metallurgy in Producing Premium Non-ferrous Metals, and the main dominant groups are Desulfovibrio and Desulfobulbus. This study was conducted in a slag heap, and the SRB bacterial solution sprayed in this experiment was mixed bacteria.

Medium: yeast extract 2 g/L, sodium lactate 1 g/L, MgSO₄ 1.2 g/L, K₂HPO₄ 0.5 g/L, CaCl₂ 0.1 g/L.

2.2. Remediation Experiment of EMR by SRB

The SRB was gradually expanded at a rate of 10% to 20%, and hydrogen sulfide production was detected with lead acetate test paper. The next expansion occurred when a large amount of hydrogen sulfide was produced. The restoration test was started when the expansion reached 10 tons, and the amount of bacterial solution used was 8 tons each time, and the remaining 2 tons were re-expanded to 10 tons as seed solution.

Between September and November 2021, an area of approximately 600 m² was selected in the slag heap site, of which 300 m² is the remediation area (R) and the remaining 300 m² is the control area (C). The remediation area was regularly repaired with SRB injection and spray, with a total SRB injection and spraying volume of 180 tons and a spraying cycle of 3–5 days. In the control area, the same amount of water was injected and sprayed under the same conditions. Three monitoring wells were set up in the remediation and control areas. Fortnightly sampling and testing leachate from monitoring wells in the remediation and control areas was conducted. The leachate was taken from the monitoring wells in the remediation and control areas every two weeks after the spray remediation. The leachate samples were analyzed for physical and chemical properties. pH and ORP (oxidation-reduction potential) were detected by a pH-Eh meter. The concentration of Mn was determined by using an Inductively Coupled Plasma Emission Spectrometry (HJ 557-2015). Ammonia and nitrite nitrogen were determined with a UV spectrophotometry (TU-1810PC). The characteristics of stabilization samples were analyzed by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). The leachate samples were sent to Bioengineering Co., Ltd. (Shanghai) in time for 16S rRNA high-throughput sequencing.

2.3. High-Throughput Sequencing

The genomic DNA was extracted with the E.Z.N.A™ Mag-Bind Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) extraction kit and used as a template to amplify the V3-V4 region of the 16S rRNA gene. The primers used for the polymerase chain reaction were 341F (5′-CCTACGGGAGGCAGCAG-3′), 805R (5′-GACTACHVGGGTATCTAATCC-3′). Raw image data files obtained from Illumina Miseq™/Hiseq™ were transformed into raw sequences by base recognition analysis.

The primer splice sequences were first removed using Cutadapt, and the paired reads were spliced into one sequence based on the overlapping relationship between PE reads using FLASH software [20]. The samples are identified and differentiated according to the barcode tag sequence to obtain sample data. Finally, the quality control filtering of each sample data is performed to obtain valid data for each sample. Use PRINSEQ to excise the bases with quality values below 20 in the tail of the reads, set a window of 10 bp, truncate the back-end bases from the window if the average quality value in the window is below 20, filter the N-containing sequences, and short sequences after quality control. Finally, filter out the low-complexity sequences to obtain valid data [21].

Cluster analysis of operational taxonomic units was performed using research software, and sequences were clustered into OTUs with 97% agreement [22]. The sequences were analyzed by mothur alpha diversity analysis (Shannon index, Chao index, Ace index, Simpson index). The RDP classifier software was used to classify the processed sequences by species, community change analysis, and comparison with the NCBI 16S database to determine the sequence classification information [23]. Finally, FAPROTAX software was used to predict the function of the flora based on the FAPROTAX database.

2.4. Data Analysis

Data were processed and analyzed using Microsoft Excel 2019 (2302 Build 16.0.16130.20186) and SPSS software (IBM SPSS Statistics 25), and statistical graphs were made in R with OriginPro 2018C software.

3. Results

3.1. Changes in the Physical and Chemical Properties of Leachate

The pH of leachate is shown in Figure 1a. The pH of leachate in the remediation area increased and became less acidic with the extension of the remediation time and remained between 6.8 and 7.0. The SRB effectively improved the acidic environment of the EMR. In contrast, the leachate in the control area was acidic, and the acidity strengthened in the later period.

The ORP macroscopically reflects the redox characteristics of the manganese sludge leachate. Figure 1b shows the ORP changes of the leachate, as the ORP of the manganese sludge leachate in the remediation area continued to decrease. The potential decreased to about −300 mV in a reduced state after 12 weeks of remediation by SRB bacterial solution spraying. The remediation system maintained a relatively stable anaerobic environment, which is conducive to the growth of sulfate-reducing bacteria [24]. In contrast, the potential of the control area remained at about 100 mV in an oxidized state. It indicated that the introduction of functional flora caused changes in the basic physicochemical properties of EMR.

The solidification of soluble manganese ions is the main purpose of SRB to remediate EMR. The changes in soluble manganese ion concentration in the leachate of this experiment are shown in Figure 1c, the manganese concentration in the leachate of electrolytic manganese slag in the control area continued to migrate to the aqueous solution with time, and the manganese concentration in the leachate of the control area increased. There was a significant decrease in manganese concentration in the leachate of the remediation area, and the solidification rate of manganese in the leachate of the manganese slag area was restored by spraying with SRB solution reached more than 99% after 12 weeks of remediation. As shown in Figure 1d,e, the concentrations of ammonia and nitrite nitrogen in the leachate of the remediation area also decreased significantly, and the concentrations of ammonia and nitrite nitrogen were 238.33 mg/L and 0.15 mg/L, respectively, after 12 weeks of remediation. The concentration of ammonia nitrogen in the leachate at the beginning of the restoration was high, and the concentration of ammonia nitrogen in the control area also decreased, so it is presumed that the decrease in ammonia nitrogen concentration may be related to evaporation [25].

3.2. Remediation Mechanisms

As shown in Figure 2a, the mineralogical components of EMR were CaSO₄·2H₂O, SiO₂, (NH₄)₂Fe(SO₄)₂·6H₂O and KNa₄CaMn₁₄Al(PO₄)₁₂(OH)₂. The mineralogical components of ES were CaSO₄·2H₂O, SiO₂ and MnS. The result showed that Mn²⁺ in EMR was mainly stabilized by MnS. FTIR analysis was further carried out to verify the remediation mechanism. As shown in Figure 2b, the characteristic bands of crystal water appear at 3410–3550 cm⁻¹ and 1620–1680 cm⁻¹ in EMR, which were −OH stretching vibration and bending vibration of water, respectively, indicating that EMR contains crystal water [26]. The stretching vibration peak of SO₄²⁻ was observed at 673 cm⁻¹ and weakened in ES [27]. Combined with XRD analysis results, it is shown that ES still contains sulfate, as the sulfate in EMR has not been completely reduced. A stretching vibration of Si-O-Si appears at 797 and 1105 cm⁻¹ [28], and the peak of ES is weakened, presumably due to the microbial solubilization of silica. The bending vibration peak of N-H-N is at 1444 cm⁻¹, and the peak intensity of ES decreases, which is due to the removal of ammonia by SRB in the anaerobic environment [29]. ES showed a stretching vibration of PO₄³⁻ at 1012 cm⁻¹, indicating that phosphate was present in the sediments, which was speculated to be the composition of the medium [16].

The surface chemical composition and binding environment of the solidification product (ES) were further investigated by XPS. The full spectrum of ES is shown in Figure 2c with four elements Mn, S, N, P, O, and C detected. The S2p XPS spectra was shown in Figure 2d, the binding energies of S2p in ES were 161.9 and 163.8 eV, which are 2p3/2 and 2p1/2 of S, respectively [27]. Sulfur is mainly present in the form of SO₄²⁻ and S²⁻. The two characteristic peaks at 653.83 and 642.06 eV in the spectra of ES (Figure 2e) are ascribed to Mn 2p1/2 and Mn 2p3/2, combined with two split peaks at 90.16 and 83.66 eV in the Mn3s XPS (Figure 2f). The high-resolution spectrum of Mn3s showed a binding energy difference of ΔE = 6.5 eV between the two peaks, confirming the presence of the sample Mn element mainly in the form of Mn²⁺ [30].

3.3. Microorganism Community Diversity of Leachate

Microbial succession information is an important indicator for studying regional environmental changes, among which the α-diversity index is often used to describe the abundance and diversity of microbial communities. R0, R2, R4, R6, R8, R10, and R12 denote leachate samples taken from the remediation area every two weeks from 0 to 12 weeks. C0, C2, C4, C6, C8, C10, and C12 denote the leachate samples taken from the control area at the same time from 0 to 12 weeks, respectively. The present sequencing results with coverage values above 99% can ensure that the sequencing results represent the microbial composition of the samples. As shown in Figure 3a,b for the box line plots of Chao and Ace indices, the species richness in the remediation area appeared to decrease first, and the species richness rebounded in the later stage of restoration. The inoculation of SRB increased the systemic microbial species, leading to an increase in total microbial abundance. The total species richness plot of the control area showed a decreasing trend, presumably due to the influence of external disturbances. The Shannon index of leachate samples at different remediation times is shown in Figure 3c, and the microbial species diversity in the remediation area showed a decreasing trend. The Simpson index is shown in Figure 3d, which is more responsive to the homogeneity of the community, and the Simpson index in the SRB remediation area gradually increased, indicating that the homogeneity of species became higher, while the control area Shannon and Simpson indices did not vary much.

3.4. Analysis of Microbial Differences at Genus Level

Microbial community structure was different at the genus level. Changes in the abundance of SRB in the remediation area are shown in Figure 4a. In the bioremediation group, there are Desulfovibrio [31], Desulfobulbus [32], Desulfuromonadales, Sulfurovum [33], Sulfurospirillum, and Sedimentibacter [34], indicating that SRB survives well in EMR. Desulfovibrio, as most bacteria, can combine the oxidation of organic acids with the reduction of sulfate, sulfite, or thiosulfate. Sulfurovum can also achieve sulfate reduction and enrichment in high sulfate concentrations [33]. A large amount of SO₄²⁻ exists in the EMR, and the above SRB plays the most critical role in converting it to S²⁻, which combines with manganese in the aqueous state to achieve the solidification of heavy metals and the formation of stable sulfides [35]. The highest abundance of SRB was observed at week 8, and the abundance decreased in the late restoration period, probably due to the decrease in local temperature. The sulfide accumulation produced by SRB in the later stage of remediation and toxic effects on microorganisms also lead to reduced abundance [36].

As shown in Figure 4b, the nitrogen-transforming reactions are linked by microorganisms forming complex networks in restoration ecosystems. Nitrogentransforming microorganismsinclude Pseudomonas [37], Arcobacter [38], Nitrosospira [39], Burkholderia [40], and Bradyrhizobium [41]. Changes in the relative abundance of nitrogen-cycling microorganisms in the remediation area are shown in Figure 4c. Cai et al. [42] found that Bacillus was able to oxidize ammonia with sulfate as the electron acceptor in the absence of molecular state oxygen and had sulfate reduction driven ammonia anaerobic oxidation, suggesting that the this phenomenon was likely caused by Bacillus reproduction. The reaction equation is Equation (1). Some denitrification processes can be carried out by reducing nitrite to nitrogen while oxidizing sulfide by autotrophic denitrifying bacteria [43], as listed in Equation (2). The reaction in the traditional anammox process is in Equation (3).

3SO₄²⁻ + 4NH₄⁺ → 3S²⁻ + 4NO₂⁻ + 4H₂O + 8H⁺(1)

3S²⁻ + 2NO₂⁻ + 8H⁺ → N₂ + 3S + 4H₂O(2)

2NO₂⁻ + 2NH₄⁺ → 2N₂ + 4H₂O(3)

The key strains in control and remediation areas also differed. The chord diagram of species abundance changes at the level of microbial genera in the remediation and control areas after remediation is shown in Figure 4d. Comparative analysis revealed that the remediation area was mainly dominated by reducing microorganisms. In contrast, the control area was dominated primarily by oxidizing microorganisms. The reducing microorganisms in the remediation area were mainly Bacteroides [44], Veillonella [45], and Macellibacteroides [46]. The relative abundance of Parcubacteria_genera_incertae_sedis, Aquabacterium [47], and Sphingomonas were higher in the control area, with 22.32%, 17.82%, and 9.95%, respectively. Bacteroides are specialized anaerobic bacteria, and the anaerobic state of the remediation area creates a good environment for their growth. Bacteroides were also found to be a common denitrifying functional bacterium that promotes the cycling of nitrogen. These results indicated artificial inoculation of functional microorganisms regulated the structure of native microbial communities in the EMR so that competitive microorganisms occupy ecological niches [48]. It constructed a healthy microbial community structure for a perdurable and effective bioremediation system.

3.5. Differences in Microbial Metabolic Function

To compare the functional characteristics of microorganisms in the remediation and control areas, FAPROTAX was used for functional prediction. As shown in Figure 5, FAPROTAX functional predictions indicated that chemoheterotrophy was the main driver of leachate microbial metabolism. The relative abundance of aerobic chemoheterotrophic bacteria was higher in the control group, which also indicates an anaerobic remediation system in the remediation zone. Respiration of sulfur compounds, sulfur respiration, sulfate respiration, sulfite respiration, and other functional groups in the remediation area were higher than those in the control area, which indicated that SO₄²⁻ in the EMR in the remediation area was reduced to the main process of the sulfur cycle [49,50]. Regarding the functional groups of sulfur oxidation, the abundance of sulfur oxidation-related functional bacteria in dark oxidation of sulfur compounds, dark sulfide oxidation, dark thiosulfate oxidation, and dark sulfur oxidation in the control area was higher than that in the remediation area.

The abundance of nitrogen cycle-related functional genes such as nitrate reduction, nitrogen respiration, nitrate respiration, nitrite respiration, denitrification, nitrous oxide denitrification, nitrite denitrification, and nitrate denitrification in the remediation area was higher than that in the control area. The nitrification of the remediation area is lower than the control area. In the control area, ammonia nitrogen can be converted into nitrate nitrogen under the action of nitrite bacteria and nitrifying bacteria under aerobic conditions. The function of aerobic nitrite oxidation and aerobic ammonia oxidation in the remediation area was almost zero, whereas the relative abundance of the control area was higher. The high abundance of functional genes for aerobic ammonia oxidation in the control area and the possible oxidation of ammonia nitrogen to nitrite nitrogen in electrolytic manganese slag [51]. The aerobic nitrite oxidation function is also present in the control area microorganisms to continue the oxidation of NO₂⁻-N.

3.6. Metabolism Pathway

Figure 6a illustrates the differences in the sulfur metabolism pathway between the remediation and control areas. The expression products of k00956, k00957, k00958, and k00860 were isozymes, encoding 3′-phosphoadenosine 5′-phosphosulfate synthase, which catalyzes the reaction involving sulfate to produce adenosine 5′-phosphosulfate (APS) or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) [52,53]. There are differences in k00956, k00957, k00958, and k00860 between the remediation area and the control area. k00394 and k00395 encode adenylylsulfate reductase, which catalyzes APS to sulfite [54,55]. Furthermore, the genes k00380, k00381, and k00392 encode sulfite reductase, which can catalyze sulfite to sulfide. The abundance of k00394, k00395, k00381, and k00392 in the microorganisms in the remediation area was higher than that in the control area. This study speculates that the promotion of sulfur metabolism is mainly manifested in the stage of APS generating sulfite and sulfite generating sulfide.

The nitrogen cycle is inextricably linked to the role of microorganisms. As in Figure 6b, microorganisms in the remediation area mainly promoted denitrification and organic nitrogen synthesis in the nitrogen cycle. k00366 and k03385, encoding nitrite reductase, had a higher percentage in the microorganisms of the remediation area. k00266, k00265, k00284, and k01915, encoding glutamate synthase, also had a higher percentage in the microorganisms of the remediation area. During organic nitrogen synthesis, ammonia nitrogen is converted to L-glutamine by the action of glutamine synthetase (E.C.6.3.1.2), and then L-glutamine is converted to L-glutamate by the action of glutamate synthase (E.C.1.4.1.13 or E.C.1.4.1.14) for microbial growth [56].

3.7. Vegetation Restoration

Sulfur is one of the essential elements for plant growth and one of the main non-metallic elements in organisms. Sulfur in plants accounts for about 0.1% of plant dry weight [57]. Since sulfur in EMR mainly exists in the form of sulfate, the increase in sulfur metabolism by microorganisms promotes plant growth. The nitrogen cycle is the basic material cycle in the ecosystem. Dominant bacteria participated in the nitrogen cycle and promoted plant growth [58,59]. As shown in Figure 7, the surface ecology of the remediation area recovered rapidly after 12 weeks of curing. This experiment has achieved a good repair effect, and SRB repair EMR is green and eco-friendly.

4. Conclusions

The SRB can adjust the pH of EMR leachate, reduce the environmental potential, and effectively solidify soluble manganese ions. The solidification rate of soluble manganese ions reached over 99% after 12 weeks of remediation in the natural state. The surface vegetation of the slag site was restored, forming a demonstration project for on-site restoration. Microbial data showed that the SRB in the leachate of the remediation area after remediation were Desulfovibrio, Desulfobulbus, Desulfuromonadales, Sulfurovum, Sulfurospirillum, and Sedimentibacter. Microorganisms were involved in the nitrogen cycle, and ammonia nitrogen escapes as nitrogen gas or participates in the formation of organic nitrogen, which promotes plant growth. The nitrogen-transforming microorganisms are Pseudomonas, Arcobacter, Nitrosospira, Burkholderia, and Bradyrhizobium. Microbial nitrate reduction, sulfate respiration, and respiration of sulfur compounds were higher in the leachate of the remediation area. Functional genes for nitrogen and sulfur metabolism were increased in the remediation area, and enzymes for sulfur and nitrogen metabolism differed between the remediation and control areas.

Author Contributions

Conceptualization, G.M.; methodology, G.M.; software, G.M., Y.L. and X.Y.; validation, G.M., X.L. and M.Z.; formal analysis, G.M.; investigation, G.M.; resources, M.Z.; data curation, G.M.; writing—original draft preparation, G.M.; writing—review and editing, G.M.; visualization, G.M.; supervision, X.Z. and X.Y.; project administration, X.Y. and X.L.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

The authors are grateful for the financial support from the National Key Research and Development Program of China (grant numbers 2018YFC18027 and 2018YFC18018), the National Natural Science Foundation of China (grant numbers 51974279), and the Guangxi Scientific Research and Technology Development Plan [GuikeAB17129025].

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Table

Figure 1. Changes in physicochemical properties of electrolytic manganese residue leachate of remediation area (R) and control area (C): (a) pH; (b) ORP; (c) Mn; (d) NH4+-N; (e) NO2−.

Figure 2. Stabilization mechanisms: (a) XRD patterns analysis; (b) FTIR spectra; (c) whole pattern; (d) S2p; (e) Mn2p; (f) Mn3s XPS spectra of ES.

Figure 3. Alpha-diversity index of remediation area (R) and control area (C). (a) Chao; (b) Ace; (c) Shannon; (d) Simpson.

Figure 4. Microbial differences at genus level between remediation area (R) and control area (C): (a) relative abundance of SRB; (b) potential nitrogen-transforming microbial networks; (c) relative abundance of nitrogen cycling microorganisms; (d) differences of microorganism.

Figure 5. Difference in the microbial function of electrolytic manganese residue leachate after remediation in remediation area (R) and control area (C).

Figure 6. Differences in metabolic pathways between the remediation area (R) and control area (C), according to the KEGG database: (a) sulfur and (b) nitrogen metabolic pathways.

Figure 7. Comparison of repair effect of remediation area (R) and control area (C): (a) before remediation; (b) after remediation.

Table 1

Physicochemical properties of electrolytic manganese residue leachate.

Sample	Pb(mg/L)	Fe(mg/L)	Mn(mg/L)	As(mg/L)	NH4+-N(mg/L)	NO2−(mg/L)	Eh(mV)	pH
EMR leachate	0.23	7.15	4566.67	0.01	5245	1.805	241.88	6.76

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