A novel sulfidogenic process via sulfur reduction to remove arsenate in acid mine drainage: Insights into the performance and microbial mechanisms.

Biological sulfidogenic processes based on sulfate-reducing bacteria (SRB) are not suitable for arsenic (As)-containing acid mine drainage (AMD) treatment because of the formation of the mobile thioarsenite during sulfate reduction. In contrast, biological sulfidogenic processes based on sulfur-reducing bacteria (S0RB) produce sulfide without pH increase, which could achieve more effective As removal than the SRB-based process. However, the reduction ability and toxicity tolerance of S0RB to As remains mysterious, which may substantially affect the practical applicability of this process when treating arsenate (As(V))-containing AMD. Thus, this study aims to develop a biological sulfur reduction process driven by S0RB, and explore its long-term performance on As(V) removal and microbial community evolution. Operating under moderately acidic conditions (pH=4.0), the presence of 10 mg/L As(V) significantly suppressed the activity of S0RB, leading to the failure of As(V) removal. Surprisingly, a drop in pH to 3.0 enhanced the tolerance of S0RB to As toxicity, allowing for efficient sulfide production (396±102 mg S/L) through sulfur reduction. Consequently, effective and stable removal of As(V) (99.9 %) was achieved, even though the sulfidogenic bacteria were exposed to high levels of As(V) (42 mg/L) in long-term trials. Spectral and spectroscopic analysis showed that As-bearing sulfide minerals were present in the bioreactor. Remarkably, the presence of As(V) induced notable changes in the microbial community composition, with Desulfurella and Clostridium identified as predominate sulfur reducers. The qPCR result further revealed an increase in the concentration of functional genes related to As transport (asrA and arsB) in the bioreactor sludge as the pH decreased from 4.0 to 3.0. This suggests the involvement of microorganisms carrying asrA and arsB in an As transport process. Furthermore, metagenomic binning demonstrated that Desulfurella contained essential genes associated with sulfur reduction and As transportation, indicating its genetic potential for sulfide production and As tolerance. In summary, this study underscores the effectiveness of the biological sulfur reduction process driven by S0RB in treating As(V)-contaminated AMD. It offers insights into the role of S0RB in remediating As contamination and provides valuable knowledge for practical applications.

Groundwater chromate removal by autotrophic sulfur disproportionation.

Chromate [Cr(VI)] contamination in groundwater is a global environmental challenge. Traditional elemental sulfur-based biotechnologies for Cr(VI) removal depend heavily on the synthesis of dissolved organic carbon to fuel heterotrophic Cr(VI) reduction, a bottleneck in the remediation process. Here we show an alternative approach by leveraging sulfur-disproportionating bacteria (SDB) inherent to groundwater ecosystems, offering a novel and efficient Cr(VI) removal strategy. We implemented SDB within a sulfur-packed bed reactor for treating Cr(VI)-contaminated groundwater, achieving a notable removal rate of 6.19 mg L-1 h-1 under oligotrophic conditions. We identified the chemical reduction of Cr(VI) via sulfide, produced through sulfur disproportionation, as a key mechanism, alongside microbial Cr(VI) reduction within the sulfur-based biosystem. Genome-centric metagenomic analysis revealed a symbiotic relationship among SDB, sulfur-oxidizing, and chromate-reducing bacteria within the reactor, suggesting that Cr(VI) detoxification by these microbial communities enhances the sulfur-disproportionation process. This research highlights the significance of sulfur disproportionation in the cryptic sulfur cycle in Cr(VI)-contaminated groundwater and proposes its practical application in groundwater remediation efforts.

Elimination of methylmercury production potential in excessive sludge in wastewater treatment plants by sulfur addition.

Mercury ions (Hg(II)) in wastewater can accumulate and transform into the highly neurotoxic methylmercury (MeHg) in activated sludge. The release of MeHg can have severe environmental consequences, making the treatment of MeHg-contaminated sludge a pressing concern. In this study, we found that all the collected activated sludge samples, from different wastewater treatment plants in four cities, had the potential for Hg methylation. The Hg-methylating capacity reached a maximum level of 0.70-0.92 µg/g volatile suspended solids after 48 h of exposure to 5 µg/L Hg(II) and showed an average MeHg production rate of 4.8±0.5%. Accordingly, a sludge treatment method involving the addition of elemental sulfur (S0) for a short-term or long-term duration (3 or 180 days, respectively) was proposed. The results demonstrated that this treatment approach effectively mitigated and potentially eliminated MeHg formation by simultaneously reducing Hg bioavailability and Hg-methylating bioactivity. We found that bioavailable Hg(II) ions were converted to a secondary phase similar to insoluble HgS after S0 addition treatment, leading to a decrease in Hg bioavailability in sludge. The enhancement of Hg and extracellular polymeric substances (EPS) complexation via the increasing amount of thiol groups in EPS also reduced the Hg bioavailability after the long-term treatment. Furthermore, the long-term S0 addition significantly reduced the abundance of Hg-methylators with hgcA gene and promoted the growth of Hg-reducers with merA gene, which ensured the complete elimination of MeHg production potential of the excessive activated sludge. Our findings demonstrated that the proposed S0-addition sludge treatment is a promising and safe biotechnology for treating Hg-contaminated sludge. This approach has the potential to contribute significantly to the mitigation of MeHg pollution within environmental contexts.

Adaptability of sulfur-disproportionating bacteria for mine water remediation under the pressures of heavy metal ions and high sulfate content.

Biological sulfide production processes mediated by sulfate/sulfur reduction have gained attention for metal removal from industrial wastewater (e.g., mine water (MW) and metallurgical wastewater) via forming insoluble metal sulfides. However, these processes often necessitate the addition of external organic compounds as electron donors, which poses a constraint on the broad application of this technology. A recent proof of concept study reported that microbial sulfur disproportionation (SD) produced sulfide with no demand for organics, which could achieve more cost-benefit MW treatment against the above-mentioned processes. However, the resistance of SD bioprocess to different metals and high sulfate content in MW remains mysterious, which may substantially affect the practical applicability of such process. In this study, the sulfur-disproportionating bacteria (SDB)-dominated consortium was enriched from a previously established SD-driven bioreactor, in which Dissulfurimicrobium sp. with a relative abundance of 39.9 % was the predominated SDB. When exposed to the real pretreated acidic MW after the pretreatment process of pH amelioration, the sulfur-disproportionating activity remained active, and metals were effectively removed from the MW. Metal tolerance assays further demonstrated that the consortium had a good tolerance to different metal ions (i.e., Pb2+, Cu2+, Ni2+, Mn2+, Zn2+), especially for Mn2+ with a concentration of approximately 20 mg/L. It suggested the robustness of Dissulfurimicrobium sp. likely due to the presence of genes encoding for the enzymes associated with metal(loid) resistance/uptake. Additionally, although high sulfate content resulted in a slight inhibition on the sulfur-disproportionating activity, the consortium still achieved sulfide production rates of 27.3 mg S/g VSS-d on average under an environmentally relevant sulfate level (i.e., 1100 mg S/L), which is comparable to those reported in sulfate reduction. Taken together, these findings imply that SDB could ensure sustainable MW treatment in a more cost-effective and organic-free way.

Deep insight into oriented propionate production from food waste: Microbiological interpretation and design practice.

Using mixed microbial cultures (MMCs) for oriented volatile fatty acids (VFAs) refining in an open environment is a typical challenge due to the microbial diversiform and the process complexity. Especially for carbohydrate-rich waste (such as food waste), butyrate-type fermentation is usually dominant in a single-stage MMCs anaerobic process, while the production of odd-carbon VFAs (such as propionate) is difficult although it plays a significant role in chemicals industries. In this study, firstly, we gave a new perspective on the rationality of the oriented propionate production using MMCs with lactate as feedstock by conducting in-depth microbial informatics and reaction analysis. Secondly, we verified the feasibility of the "food waste-lactate-propionate" route to reverse the original butyrate-type fermentation situation and explore mechanisms for maintaining stability. In the first stage, a defined lactate fermentation microbiome was used to produce lactate-containing broth (80% of total chemical oxygen demand) at pH=4. In the second stage, an undomesticated undefined anaerobic microbiome was used to drive propionate production (45.26% ± 2.23% of total VFAs) under optimized conditions (C/N = 100:1-200:1 and pH=5.0). The low pH environment in the first stage enhanced the lactic acid bacteria to resist the invasion of non-functional flanking bacteria, making the community stable. In the second stage, the system maintained the propionate-type fermentation due to the absence of the ecological niche of the invasive lactic acid bacteria; The selection of propionate-producing specialists was a necessary but not sufficient condition for propionate-type fermentation. At last, this study proposed an enhanced engineering strategy framework for understanding elaborate MMCs fermentation.

Sodium Dynamics in the Cellular Environment.

Sodium ions are essential for the functions of biological cells, and they are maintained at the balance between intra- and extracellular environments. The quantitative assessment of intra- and extracellular sodium as well as its dynamics can provide crucial physiological information on a living system. 23Na nuclear magnetic resonance (NMR) is a powerful and noninvasive technique to probe the local environment and dynamics of sodium ions. However, due to the complex relaxation behavior of the quadrupolar nucleus in the intermediate-motion regime and because of the heterogeneous compartments and diverse molecular interactions in the cellular environment, the understanding of the 23Na NMR signal in biological systems is still at the early stage. In this work, we characterize the relaxation and diffusion of sodium ions in the solutions of proteins and polysaccharides, as well as in the in vitro samples of living cells. The multi-exponential behavior of 23Na transverse relaxation has been analyzed according to the relaxation theory to derive the crucial information related to the ionic dynamics and molecular binding in the solutions. The bi-compartment model of transverse relaxation and diffusion measurements can corroborate each other to quantify the fractions of intra- and extracellular sodium. We show that 23Na relaxation and diffusion can be used to monitor the viability of human cells, which offers versatile NMR metrics for in vivo studies.

Treatment and remediation of metal-contaminated water and groundwater in mining areas by biological sulfidogenic processes: A review.

Heavy metal pollution in the mining areas leads to serious environmental problems. The biological sulfidogenic process (BSP) mediated by sulfidogenic bacteria has been considered an attractive technology for the treatment and remediation of metal-contaminated water and groundwater. Notwithstanding, BSP driven by different sulfidogenic bacteria could affect the efficiency and cost-effectiveness of the treatment performance in practical applications, such as the microbial intolerance of pH and metal ions, the formation of toxic byproducts, and the consumption of organic electron donors. Sulfur-reducing bacteria (S0RB)-driven BSP has been demonstrated to be a promising alternative to the commonly used sulfate-reducing bacteria (SRB)-driven BSP for treating metal-contaminated wastewater and groundwater, due to the cost-saving in chemical addition, the high efficiency in sulfide production and metal removal efficiency. Although the S0RB-driven BSP has been developed and applied for decades, the present review works mainly focus on the developments in SRB-driven BSP for the treatment and remediation of metal-contaminated wastewater and groundwater. Accordingly, a comprehensive review for metal-contaminated wastewater treatment and groundwater remediation should be provided with the incorporation of the SRB- and S0RB-driven BSP. To identify the bottlenecks and to improve BSP performance, this paper reviews sulfidogenic bacteria presenting in metal-contaminated water and groundwater; highlight the critical factors for the metabolism of sulfidogenic bacteria during BSP; the ecological roles of sulfidogenic bacteria and the mechanisms of metal removal by sulfidogenic bacteria; and the application of the present sulfidogenic systems and their drawbacks. Accordingly, the research knowledge gaps, current process limitations, and future prospects were provided for improving the performance of BSP in the treatment and remediation of metal-contaminated wastewater and groundwater in mining areas.

Aerobic starvation treatment of activated sludge enhances the degradation efficiency of refractory organic compounds.

Many refractory organic compounds (ROCs) in wastewater are toxic to human and aquatic organisms. Here, we reported an aerobic starvation approach to improve the degradation efficiencies of ROCs in activated sludge systems. The highest degradation rates of bisphenol AF (BPAF) (11.4 mg/g VSS · h) and gabapentin (GBP) (8.9 mg/g VSS · h) were achieved on the second day of the starvation process. While, the degradation rate of bisphenol A (BPA) on the 43rd day reached the maximum value of 0.8 mg/g VSS ·h, which was significantly higher than that of the seeding sludge (0.01 mg/g VSS · h). To investigate the mechanisms of this finding, we applied magnetic-nanoparticle mediated isolation, 16S rRNA gene sequencing, metagenomic sequencing and metatranscriptomic sequencing to analyze the microbial community structures and functions during the starvation process. The results showed that the increase of the BPA degradation ability was caused by the increase of the relative abundance of BPA degrading bacteria (Sphingomonas, Achromobacter, etc.), while, the enhancement of BPAF and GBP degradation was attributed to the increase of the expression of ROC degrading genes. Overall, these results improve our understanding of the microbial ecology of starved activated sludge and provide useful information for the future development of ROC removal technologies.

Phenolic compounds promote the horizontal transfer of antibiotic resistance genes in activated sludge.

Phenolic compounds are common organic pollutants in wastewater. During the wastewater treatment process, these compounds may influence the microbial community structure and functions. However, the impact of the phenolic compounds in the wastewater treatment plants on the horizontal transfer of antibiotic resistance genes (ARGs) has not been well assessed. In this study, we investigated the horizontal transfer of ARGs under the stress of phenolic compounds. The results showed that in pure culture bacteria system, p-nitrophenol (PNP), p-aminophenol (PAP) and phenol (PhOH) (10-100 mg/L) can significantly increase the horizontal transfer frequency of ARGs by 2.2-4.6, 3.6-9.4 and 1.9-9.0 fold, respectively. And, the RP4 plasmid transfer from Escherichia coli HB101 (E. coli HB101) to the bacteria in activated sludge increased obviously under the stress of phenolic compounds. Further investigation revealed that the PNP and PhOH at the concentration of 10-100 mg/L increased the production of reactive oxygen species and the permeability of cell membrane in the donor and recipient, which could be the causes of horizontal transfer of RP4 plasmid. In addition, it was also found that PNP, PAP and PhOH stress inhibit the expression of the global regulatory genes korB and trbA in the RP4 plasmid, and increase the expression level of the traF gene, thereby promoting the conjugative transfer of the RP4 plasmid. Taken together, these results improved our understanding of the horizontal transfer of ARGs under the stress of phenolic compounds and provided basic information for management of the systems that treat wastewater containing phenolic compounds.

Ozone pretreatment of wastewater containing aromatics reduces antibiotic resistance genes in bioreactors: The example of p-aminophenol.

Aromatic matters are widely present in wastewater, especially industrial wastewater, and may lead to a high abundance of antibiotic resistance genes (ARGs) in wastewater treatment bioreactors and stimulate horizontal transfers of ARGs. Here, we investigated a practical approach that applying ozone pretreatment to mitigate ARGs in bioreactors treating wastewater containing a typical aromatic pollutant, p-aminophenol (PAP). The results showed that ozone pretreatment could effectively reduce the aromaticity of wastewater, and the relative abundance of ARGs in the bioreactor fed with ozone treated wastewater decreased by over 70% compared to the control reactor. Multidrug, quinolone, mupirocin, polymyxin, aminoglycoside, glycopeptide, beta-lactam, and trimethoprim resistance genes were all reduced in the bioreactors receiving wastewater pretreated by ozone. Metagenomic analysis suggested that the reduction of ARGs could be attributed to the co-occurrence of ARGs and aromatic degradation genes in bacteria. Furthermore, we expanded our analysis to investigate 71 metagenomes from different environments, and the results indicated that the impact of aromatics on ARG abundance widely occurs in various ecosystems and confirmed that high levels of aromatics could lead to high abundance of ARGs. Taken together, our work confirmed that the aromatics played critical roles in selecting ARGs and proposed a feasible approach to reduce ARGs in wastewater treatment bioreactors.

Aromatic compounds lead to increased abundance of antibiotic resistance genes in wastewater treatment bioreactors.

Various aromatic compounds in wastewater, especially industrial wastewater, are treated by biological processes in bioreactors which are regarded as hotspots and reservoirs of antibiotic resistance genes (ARGs). Yet, little is known about the relationship between the aromatic compound degradation process and antibiotic resistance. Here, we report on the co-occurrence of ARGs and aromatic degradation genes (ADGs) in bacteria in bioreactors. We confirmed this by bioreactor experiments and bioinformatics analysis of over 10,000 publicly available bacterial genomes. We observed a significant enrichment of ARGs in bioreactors treating wastewater that contained p-aminophenol and p-nitrophenol. The potential hosts harboring ARGs and ADGs were mainly Pseudomonas, Leucobacter, Xanthobacter, Acinetobacter, and Burkholderiaceae. Genome analysis revealed that 67.6% of the publicly available bacterial genomes harboring ADGs also harbor ARGs. Over 80% of Burkholderiales, Xanthomonales, Enterobacteriaceae, Acinetobacter, Pseudomonas, and Nocardiaceae genomes harbor both ARGs and ADGs, which strongly suggests the co-occurrence of these genes. Furthermore, bacteria carrying ADGs harbored more than twice the number of ARGs than bacteria only carrying ARGs. Network analysis suggested that multidrug, beta-lactam, aminoglycoside, macrolide-lincosamide-streptogramin, and polymyxin resistance genes are the major ARGs associated with ADGs. Taken together, the presented findings improve the understanding of ARG prevalence in biological wastewater treatment plants, and highlight the potential risk of the effect of regular aromatic compounds on the selection and spread of ARGs.

Microbial community structure and function in aerobic granular sludge.

Aerobic granular sludge (AGS), a self-immobilized microbial consortium containing different functional microorganisms, is receiving growing attention, since it has shown great technological and economical potentials in the field of wastewater treatment. Microbial community is crucial for the formation, stability, and pollutant removal efficiency of aerobic granules. This mini-review systematically summarizes the recent findings of the microbial community structure and function of AGS and discusses the new research progress in the microbial community dynamics during the granulation process and spatial distribution patterns of the microbiota in AGS. The presented information may be helpful for the in-depth theoretical study and practical application of AGS technology in the future.