Manganese sulfide-sulfur and limestone autotrophic denitrification system for deep and efficient nitrate removal: Feasibility, performance and mechanism.

Despite the great potential of sulfur-based autotrophic denitrification, an improvement in nitrate removal rate is still needed. This study used the desulfurized products of Mn ore to develop the MnS-S0-limestone autotrophic denitrification system (MSLAD). The feasibility of MSLAD for denitrification was explored and the possible mechanism was proposed. The nitrate (100 mg/L) was almost removed within 24 h in batch experiment in MSLAD. Also, an average TN removal of 98 % (472.0 mg/L/d) at hydraulic retention time of 1.5 h in column experiment (30 mg/L) was achieved. MnS and S0 could act as coupled electron donors and show synergistic effects for nitrate removal. γ-MnS with smaller particle size and lower crystallinity was more readily utilized by the bacterium and had higher nitrate removal efficiency than that of α-MnS. Thiobacillus and Sulfurimonas were the core functional bacterium in denitrification. Therefore, MnS-S0-limestone bio-denitrification provides an efficient alternative method for nitrate removal in wastewater.

Autotrophic denitrification by sulfur-based immobilized electron donor for enhanced nitrogen removal: Denitrification performance, microbial interspecific interaction and functional traits.

Sulfur-driven autotrophic denitrification (SdAD) is a promising nitrogen removing process, but its applications were generally constrained by conventional electron donors (i.e., thiosulfate (Na2S2O3)) with high valence and limited bioavailability. Herein, an immobilized electron donor by loading elemental sulfur on the surface of polyurethane foam (PFSF) was developed, and its feasibility for SdAD was investigated. The denitrification efficiency of PFSF was 97.3%, higher than that of Na2S2O3 (91.1%). Functional microorganisms (i.e., Thiobacillus and Sulfurimonas) and their metabolic activities (i.e., nir and nor) were substantially enhanced by PFSF. PFSF resulted in the enrichment of sulfate-reducing bacteria, which can reduce sulfate (SO42-). It attenuated the inhibitory effect of SO42-, whereas the generated product (hydrogen sulfide) also served as an electron donor for SdAD. According to the economic evaluation, PFSF exhibited strong market potential. This study proposes an efficient and low-cost immobilized electron donor for SdAD and provides theoretical support to its practical applications.

Enhancement of bio-promoters on hexavalent chromium inhibited sulfur-driven denitrification: repairing damage, accelerating electron transfer, and reshaping microbial collaboration.

Proposing recovery strategies to recover heavy-metal-inhibited sulfur-driven denitrification, as well as disclosing recovery mechanisms, can provide technical support for the stable operation of bio-systems. This study proposed an effective bio-promoter (mediator-promoter composed of L-cysteine, biotin, cytokinin, and anthraquinone-2,6-disulfonate) to recover Cr(VI) inhibited sulfur-driven denitrification, which effectively reduced the recovery time of NO3--N reduction (18-21 cycles) and NO2--N reduction (27-42 cycles) compared with self-recovery. The mediator-promoter repaired microbial damage by promoting intracellular chromium efflux. Moreover, the mediator-promoter reduced the accumulated reactive oxygen species by stimulating the secretion of antioxidant enzymes, reaching equilibrium in the oxidative-antioxidant system. To improve electron transmission, the mediator-promoter restored S2O32- oxidation to provide adequate electron donors and increased electron transfer rate by increasing cytochrome c levels. Mediator-promoter boosted the abundance of Thiobacillus (sulfur-oxidizing bacterium) and Simplicispira (denitrifying bacterium), which were positively correlated, facilitating the rapid denitrification recovery and the long-term stable operation of recovered systems.

Differential surface modification mechanism of chalcopyrite and pyrite by Thiobacillus ferrooxidans and its response to bioflotation.

Mineral processing encounters the challenge of separating chalcopyrite and pyrite, with the conventional high alkali process characterized by issues such as large dosages of reagents, complex procedures, and environmental pollution. This study addresses this challenge by isolating and enriching Thiobacillus ferrooxidans (T·f) from acidic mine drainage, employing it as a biosurfactant. The modification mechanism of T·f was thoroughly analyzed. Fe dissolution through biological oxidation formed a passivation layer (jarosite [KFe3(SO4)2(OH)6], elemental sulfur (S0), and metal sulfides (Cu/Fe-S) on the surface of minerals. Metal oxides, hydroxides, and sulfates were detected on the surface of two minerals, but the difference was that elemental sulfur (S0) and copper sulfide (Cu-S) were detected on the surface of chalcopyrite. elucidating the fundamental reason for the significant difference in surface hydrophobicity between chalcopyrite and pyrite. T·f has been successfully used as a biosurfactant to achieve copper-sulfur separation.

Removal capacities and varying characteristics of substrates and microbial community structure in simultaneous sulfide and nitrate biological removal process'.

Under the background of carbon neutrality, resource and energy utilization technologies have become the focus of future research. The paper investigated the removal efficiencies and varying characteristics of substrates and microbial community structure in the simultaneous sulfide and nitrate biological removal (SSNBR) process. The results showed that the sulfide and nitrate removal loads reached 2.998 kg m-3∙d-1 and 1.011 kg m-3∙d-1 respectively when HRT was 2.4 h. The sulfide and nitrate molar ratios (S/N ratios) hardly influenced the removal efficiencies of sulfide and nitrate. However, the reaction products sulfate and nitrite concentrations in the effluent became higher as the S/N ratios decreased. Under the S/N ratio of 5:5, when the influent sulfide and nitrate concentrations were improved from 100 mg L-1 to 600 mg L-1 and from 87.5 mg L-1 to 306.25 mg L-1, respectively, the sulfide removal efficiencies were all above 99%, but the nitrate removal efficiencies reduced from 95.53% to 55.54%. Sulfide removal effect was better than nitrate. HRT had great effect on the nitrate removal efficiencies, but hardly affected the sulfide removal. When HRT was shortened from 12 h to 2.4 h, the sulfide removal efficiencies were all above 99%, while the nitrate removal efficiencies decreased from 93.14% to 77.04%. The main functional genera included Exiguobacterium, Clostridium, Bacillus, Thiobacillus and Sphingomonas, all of which had the nitrogen and sulfur removal functions.

Impact of different sulfur sources on the structure and function of sulfur autotrophic denitrification bacteria.

Nitrate pollution in surface water has become a significant environmental concern. Sulfur autotrophic denitrification (SAD) technology is gaining attention for its cost-effectiveness and efficiency in nitrate removal. This study aimed to investigate the structure and function of sulfur autotrophic denitrification microbial communities in systems using sodium thiosulfate (Group A) and elemental sulfur (Group B) as the sole electron donors. Metagenomic amplicon sequencing and physicochemical analysis were performed to examine the microbial communities. The results revealed that on day 13, the nitrate nitrogen removal rate in Group A was significantly higher (89.2%) compared to Group B (74.4%). The dominant genus in both Groups was Thiobacillus, with average abundances of 34.15% and 16.34% in Groups A and B, respectively. ß-diversity analysis based on species level showed significant differences in bacterial community structure between the two Groups (P < 0.001). Group A exhibited a greater potential for nitrate reduction and utilized both thiosulfate and elemental sulfur (P < 0.01) compared to Group B. This study provides a sufficient experimental basis for improving the start-up time and operating cost of SAD system through sulfur source switching and offers new prospects for in-depth mechanistic analysis.

Dissimilatory sulfate reduction in an anaerobic biofilm reactor for tofu processing wastewater treatment: Bacterial community and their functional genes.

Dissimilatory sulfate reduction (DSR) is the key sulfur cycle that transforms sulfate to sulfide. This process leads to odour issues in wastewater treatment. However, few studies have focused on DSR during treating food processing wastewater with high sulfate. This study investigated DSR microbial population and functional genes in an anaerobic biofilm reactor (ABR) treating tofu processing wastewater. The tofu processing wastewater is a common food processing wastewater in Asia. The full-scale ABR was operated for over 120 days in a tofu and tofu-related products manufacturing factory. Mass balance calculations based on the reactor performance indicated that 79.6-85.1 % of the sulfate was transformed into sulfide irrelevant to dissolved oxygen supplementation. Metagenomic analysis revealed 21 metagenome-assembled genomes (MAGs) containing enzymes encoding DSR. The biofilm contained the complete functional genes of DSR pathway in the full-scale ABR, indicating that biofilm could process DSR independently. Comamonadaceae, Thiobacillus, Nitrosomonadales, Desulfatirhabdium butyrativorans, Desulfomonile tiedjei were the dominant DSR species in the ABR biofilm community. Dissolved oxygen supplementation directly inhibited DSR and mitigated HS- production. It was also found that Thiobacillus contained all the function genes encoding every necessary enzyme in DSR, and thus Thiobacillus distribution directly correlated to DSR and the ABR performance.

Thiobacillus as a key player for biofilm formation in oligotrophic groundwaters of the Fennoscandian Shield.

Biofilm formation is a common adaptation for microbes in energy-limited conditions such as those prevalent in the vast deep terrestrial biosphere. However, due to the low biomass and the inaccessible nature of subsurface groundwaters, the microbial populations and genes involved in its formation are understudied. Here, a flow-cell system was designed to investigate biofilm formation under in situ conditions in two groundwaters of contrasting age and geochemistry at the Äspö Hard Rock Laboratory, Sweden. Metatranscriptomes showed Thiobacillus, Sideroxydans, and Desulforegula to be abundant and together accounted for 31% of the transcripts in the biofilm communities. Differential expression analysis highlighted Thiobacillus to have a principal role in biofilm formation in these oligotrophic groundwaters by being involved in relevant processes such as the formation of extracellular matrix, quorum sensing, and cell motility. The findings revealed an active biofilm community with sulfur cycling as a prominent mode of energy conservation in the deep biosphere.

Nanoplastics impacts on Thiobacillus denitrificans: Effects of size and dissolved organic matter.

The widespread distribution of nanoplastics and dissolved organic matter (DOM) in sewage raises concerns about the potential impact of DOM on the bioavailability of nanoplastics. In this study, the effects of different sizes (100 nm and 350 nm) of polystyrene nanoplastics (PS-NPs, 50 mg/L) and combined with 10 mg/L or 50 mg/L DOMs (fulvic acid, humic acid and sodium alginate) on the growth and denitrification ability of Thiobacillus denitrificans were investigated. Results showed that 100 nm PS-NPs (50 mg/L) cause a longer delay in the nitrate reduction (3 days) of T. denitrificans than 350 nm PS-NPs (2 days). Furthermore, the presence of DOM exacerbated the adverse effect of 100 nm PS-NPs on denitrification, resulting in a delay of 1-4 days to complete denitrification. Fulvic acid (50 mg/L) and humic acid (50 mg/L) had the most significant adverse effect on increasing 100 nm PS-NPs (50 mg/L), causing a reduction of 20 mmol/L nitrate by T. denitrificans in nearly 7 days. It is noteworthy that the presence of DOM did not modify the adverse effect of 350 nm PS-NPs on denitrification. Further analysis of toxicity mechanism of PS-NPs revealed that they could induce reactive oxygen species (ROS) and suppressed denitrification gene expression. The results suggested that DOM may assist in the cellular internalization of PS-NPs by inhibiting PS-NPs aggregation, leading to the increased ROS levels and accelerated T. denitrificans death. This study highlights the potential risk of nanoplastics to autotrophic denitrifying bacteria in the presence of DOM and provides new insights for the treatment of nitrogen-containing wastewater by T. denitrificans.

Achieving nitrogen removal with low material and energy consumption through partial nitrification coupled with short-cut sulfur autotrophic denitrification in a single-stage SBR.

An innovative partial nitrification and short-cut sulfur autotrophic denitrification (PN-SSAD, NH4+-N â†’ NO2--N â†’ N2) coupled system in a single-stage SBR was proposed to treat low C/N wastewater with low material and energy consumption. Nearly 50 % alkalinity consumption and 40 % sulfate production were reduced in S0-SSAD compared with S0-SAD, whereas the autotrophic denitrification rate was increased by 65 %. In S0-PN-SSAD, the TN removal efficiency reached almost 99 % without additional organic carbon. Furthermore, pyrite (FeS2) rather than S0 served as the electron donor to optimize the PN-SSAD process. The practical sulfate production in S0-PN-SSAD and FeS2-PN-SSAD were about 38 % and 52 % lower than complete nitrification and sulfur autotrophic denitrification (CN-SAD), respectively. Thiobacillus was the major autotrophic denitrification bacteria in S0-PN-SSAD (34.47 %) and FeS2-PN-SSAD (14.88 %). Nitrosomonas and Thiobacillus played a synergistic effect in the coupled system. FeS2-PN-SSAD is expected as an alternative technology for nitrification and heterotrophic denitrification (HD) in treating low C/N wastewater.

Thiobacillus spp. and Anaeromyxobacter spp. mediate arsenite oxidation-dependent biological nitrogen fixation in two contrasting types of arsenic-contaminated soils.

As(III) oxidation-dependent biological nitrogen fixing (As-dependent BNF) bacteria use a novel biogeochemical process observed in tailings recently. However, our understanding of microorganisms responsible for As-dependent BNF is limited and whether such a process occurs in As-contaminated soils is still unknown. In this study, two contrasting types of soils (surface soils versus river sediments) heavily contaminated by As were selected to study the occurrence of As-dependent BNF. BNF was observed in sediments and soils amended with As(III), whereas no apparent BNF was found in the cultures without As(III). The increased abundances of the nitrogenase gene (nifH) and As(III) oxidation gene (aioA) suggest that an As-dependent BNF process was catalyzed by microorganisms harboring nifH and aioA. In addition, DNA-SIP demonstrated that Thiobacillus spp. and Anaeromyxobacter spp. were putative As-dependent BNF bacteria in As-contaminated soils and sediments, respectively. Metagenomic analysis further suggested that these taxa contained genes responsible for BNF, As(III) oxidation, and CO2 fixation, demonstrating their capability for serving as As-dependent BNF. These results indicated the occurrence of As-dependent BNF in various As-contaminated habitats. The contrasting geochemical conditions in different types of soil suggested that these conditions may enrich different As-dependent BNF bacteria (Thiobacillus spp. for soils and Anaeromyxobacter spp. for sediments).

Enhanced biological S0 accumulation by using signal molecules during simultaneous desulfurization and denitrification.

A high rate of elemental sulfur (S0) accumulation from sulfide-containing wastewater has great significance in terms of resource recovery and pollution control. This experimental study used Thiobacillus denitrificans and denitrifying bacteria incorporated with signal molecules (C6 and OHHL) for simultaneous sulfide (S2-) and nitrate (NO3-) removal in synthetic wastewater. Also, the effects on S0 accumulation due to changes in organic matter composition and bacteria proportion through signal molecules were analyzed. The 99.0% of S2- removal and 99.3% of NO3- was achieved with 66% of S0 accumulation under the active S2- removal group. The S0 accumulation, S2- and NO3- removal mainly occurred in 0-48 h. The S0 accumulation in the active S2- removal group was 2.0-6.3 times higher than the inactive S2- removal groups. In addition, S0/SO42- ratio exhibited that S0 conversion almost linearly increased with reaction time under the active S2- removal group. The proportion of Thiobacillus denitrificans and H+ consumption showed a positive correlation with S0 accumulation. However, a very high or low ratio of H+/S0 is not suitable for S0 accumulation. The signal molecules greatly increased the concentration of protein-I and protein-II, which resulted in the high proportion of Thiobacillus denitrificans. Therefore, high S0 accumulation was achieved as Thiobacillus denitrificans regulated the H+ consumption and electron transfer rate and provided suppressed oxygen environment. This technology is cost-effective and commercially applicable for recovering S0 from wastewater.

Improved sulfur autotrophic denitrification using supplementary bovine serum albumin.

Excess nitrate presented in natural water body and drinking water has been a challenge for maintaining safe ecosystem and human health. Sulfur autotrophic denitrification is proved a feasible technology to remove nitrate from water environment. However, comparatively low rate of sulfur autotrophic denitrification needs to be addressed before wide application of this technology, which is a result of the low solubility of elemental sulfur. Therefore, this study employed bovine serum albumin (BSA) as a supplementary material to modify the elemental sulfur for improved sulfur autotrophic denitrification rate. Artificial biofilm of Thiobacillus denitrificans was prepared and employed in experiments. By testing different amount of BSA applied in both elemental sulfur and the biofilm, including 1 %, 2 % and 4 % mass ratios, it was found that larger employment of BSA had significant effect in increasing the denitrification rate. Particularly when 4 % BSA was added into elemental sulfur, the highest denitrification rate reached 26.8 mg-N/(L·d), 3.7 times of the control group. Meanwhile, the largest reaction rate constant was achieved, 4.13 mg0.5/(L0.5·d), 2.78 times of the control group. This effect was attributed to promoted conversion of elemental sulfur to polysulfide that was easily utilized by sulfur-oxidizing bacteria. A long-term operation (14 days) of packed bed reactor filled with sulfur particles and 1 % BSA delivered a much faster start-up than the control and outperformed it with better denitrification performance all-through the experiment. This result evidenced again that BSA could make a highly effective supplement in sulfur autotrophic denitrification.

The nitrogen removal performance and microbial community on mixotrophic denitrification process.

Sulfur autotrophic denitrifiers and heterotrophic denitrifiers widely exist in aquatic ecosystem, however, the response of sulfide to the microbial community structure in mixotrophic denitrification ecosystem is unknown yet. In this study, the denitrification performance and microbial community were explored by changing the molar ratio of influent C/N/S. From the level of genus, the joint action of Thauera, Pacacoccus, Fusibacter Pseudoxanthomonas, Thiobacillus, Sulfurovum and Sulfurimonas brought about the efficient denitrification performance in the mixotrophic system. Thauera increased from from 0.97% to more than 13%, and the relative abundances of Thiobacillus and Sulfurimonas were about 4.14% and 3.89% separately after adding S2-. The results of this study showed that the denitrification performance could be indeed intensified in the mixotrophic system, among which provided a theoretical basis for establishing an efficient biological nitrogen removal system.

Sulfur enhances cadmium bioaccumulation in Cichorium intybus by altering soil properties, heavy metal availability and microbial community in contaminated alkaline soil.

Cadmium (Cd) contamination seriously threatens the soil health and food safety. Combination of amendment and accumulator plant is a green and effective technique to improve phytoremediation of Cd-contaminated alkaline soil. In this study, a potting experiment was conducted to investigate the effect of sulfur on Cd phytoextraction by Cichorium intybus (chicory). Soil chemical and microbial properties were determined to reveal the mechanism of sulfur-assisting Cd phytoremediation by chicory. Soil pH decreased from 7.77 to the lowest 7.30 with sulfur addition (0.6, 0.9 and 1.2 g kg-1, LS, MS and HS treatment); Electric conductivity, sulfate anion and available cadmium concentration increased gradually with increasing sulfur doses. Cd concentration of shoot and root significantly increased from 1.47 to 4.43 mg kg-1, 6.15 to 20.16 mg kg-1 by sulfur treatment relative to CK, which were attributed to increased available Cd concentration induced by decreased pH. Sulfur treatments significantly increased the Cd bioconcentration factor by 64.1%, 118.6%, 201.0% for shoot, 76.3%, 145.6% and 227.7% for root under LS, MS and HS relative to CK treatment, respectively (P < 0.05). However, only MS treatment significantly improved the Cd removal efficiency by 82.9% in comparison of CK treatment (P < 0.05). Microbial community diversity measured by 16SrRNA showed that Thiobacillus and Actinobacteria were the key and dominant strains of soil microbial communities after sulfur addition, which played a pivotal role in the process of sulfur oxidation involved in decrease of soil pH and the transformation of Cd forms. Correlation analysis and path analysis by structural equation model indicated that soil sulfate anion and Thiobacillus directly affected Cd removal efficiency by chicory in Cd-contaminated alkaline soil. This suggests that combination of sulfur and chicory may provide a way to promote Cd bioaccumulation for phytoremediation of Cd-contaminated alkaline soil.

The inhibitory effects and underlying mechanism of high ammonia stress on sulfide-driven denitrification process.

Sulfide-driven denitrification (SD) process has been widely studied for treating wastewater containing sulfate and ammonia in recent years. But influence of high ammonia stress on the SD process and microbial community remained unclear. In this work, a series of tests were conducted to investigate effects of different ammonia stress (200-3000 mg-total ammonia nitrogen (TAN)/L) on denitrification efficiency, byproduct accumulation and microbial community of the SD process. According to our results, the SD process was severely inhibited, and 32.67 ± 5.15 mg/L NO2--N was accumulated when ammonia stress reached 3000 mg TAN/L. But the inhibited SD process could recover in about 40 days when ammonia stress was decreased to 200 mg TAN/L. After analyzing the microbial community, Thiobacillus sp. (Thiobacillus sp. 65-29, Thiobacillus sp. SCN 64-317, Thiobacillus sp. 63-78 and Thiobacillus denitrificans) was confirmed as dominant bacteria responsible for the SD process. Further, expression of narG, napA, nirK and nirS were inhibited under high ammonia stress, thus making the SD process stuck in NO3- and NO2- reduction step. This study reveals the inhibitory effects of high ammonia stress on the SD process and its possible underlying mechanism with discussion in gene level.

Regulating intermediates to realize the coupled hydrion with biology polysulfide in wastewater treatment.

The purpose of this study is to clarify the mechanism of the coupled hydrion with biology polysulfide in the simultaneous denitrification and desulfurization process. The coupled hydrion with biology polysulfide, uncoupled hydrion with biology polysulfide and no polysulfide experiments were performed in wastewater with two kinds of sulfide loads (100 and 200 mg/L). When the concentration of thiosulfate was suitable, the free H+ concentration (74.2 and 91.0 mg/L) and the proportion of Thiobacillus denitrificans (85.4% and 59.7%) were both higher under the two kinds of sulfide loading conditions (100 and 200 mg/L), and coupled hydrion with biology polysulfide was realized (the production of elemental sulfur is as high as 33 and 101 mg/L). Further analysis shown that the way of coupled hydrion with biology polysulfide were both: 2.0S2-+6.4NO3-+30.1H++21.7e-→1.0S2-+1.0SO42-+3.2N2+15.0H2O. In addition, for the coupled hydrion with biology polysulfide, more nitrates could be utilized to produce elemental sulfur S0, and the lower ratio of H+/S0 and SO42-/S0 were observed (S2- = 100 mg/L: 2.3 and 0.9; S2- = 200 mg/L: 0.9 and 0.03), which could promote the growth of Thiobacillus denitrificans and increase the proportion of Thiobacillus denitrificans. This maybe one of the reasons why coupled hydrion with biology polysulfide could be achieved.

The interaction between Pseudomonas C27 and Thiobacillus denitrificans in the integrated autotrophic and heterotrophic denitrification process.

Compared to autotrophic and heterotrophic denitrification process, the integrated autotrophic and heterotrophic denitrification (IAHD) shows wider foreground of applications in the actual wastewaters with organic carbon, nitrogen and sulfur co-existing. The efficient co-removal of sulfur, nitrogen, and carbon in the IAHD system is guaranteed by the interaction between heterotrophic and autotrophic denitrificans. In order to further explore the interaction between functional bacteria, Pseudomonas C27 and Thiobacillus denitrifcans were selected as typical heterotrophic and autotrophic bacteria, and their characteristics metabolic responses to different sulfide concentrations were studied. Pseudomonas C27 had higher metabolic activity than T. denitrificans in the IAHD medium with sulfide concentration of 3.12-15.62 mmol/L. Moreover, the fastest sulfide removal rate (0.35 mmol/L·h) was achieved with a single inoculation of Pseudomonas C27. Meanwhile, in mixed inoculant conditions, the interaction between Pseudomonas C27 and T. denitrificans (P:T = 3:1, P:T = 1:1 and P:T = 1:3) yielded the highest sulfide removal efficiency (more than 85%) when sulfide concentration was 6.25-12.5 mmol/L. Additionally, the sulfide removal rate increased with the inoculation proportion of Pseudomonas C27. Thus, this apparent interaction provided a theoretical basis for further understanding and guidance on the efficient operation of IAHD system.

Metagenomic insights into mixotrophic denitrification facilitated nitrogen removal in a full-scale A2/O wastewater treatment plant.

Wastewater treatment plants (WWTPs) are important for pollutant removal from wastewater, elimination of point discharges of nutrients into the environment and water resource protection. The anaerobic/anoxic/oxic (A2/O) process is widely used in WWTPs for nitrogen removal, but the requirement for additional organics to ensure a suitable nitrogen removal efficiency makes this process costly and energy consuming. In this study, we report mixotrophic denitrification at a low COD (chemical oxygen demand)/TN (total nitrogen) ratio in a full-scale A2/O WWTP with relatively high sulfate in the inlet. Nitrogen and sulfur species analysis in different units of this A2/O WWTP showed that the internal sulfur cycle of sulfate reduction and reoxidation occurred and that the reduced sulfur species might contribute to denitrification. Microbial community analysis revealed that Thiobacillus, an autotrophic sulfur-oxidizing denitrifier, dominated the activated sludge bacterial community. Metagenomics data also supported the potential of sulfur-based denitrification when high levels of denitrification occurred, and sulfur oxidation and sulfate reduction genes coexisted in the activated sludge. Although most of the denitrification genes were affiliated with heterotrophic denitrifiers with high abundance, the narG and napA genes were mainly associated with autotrophic sulfur-oxidizing denitrifiers. The functional genes related to nitrogen removal were actively expressed even in the unit containing relatively highly reduced sulfur species, indicating that the mixotrophic denitrification process in A2/O could overcome not only a shortage of carbon sources but also the inhibition by reduced sulfur of nitrification and denitrification. Our results indicate that a mixotrophic denitrification process could be developed in full-scale WWTPs and reduce the requirement for additional carbon sources, which could endow WWTPs with more flexible and adaptable nitrogen removal.

Change in microbial profile and environmental conditions in a constructed wetland system treating greywater.

The objective of this work was to verify the relationships between environmental conditions and microbial processes along a raw-light greywater flow in an improved constructed wetland (CW) system. Physicochemical analysis and high-throughput DNA sequencing were performed in the different zones to investigate the environmental conditions and microbial communities. The results showed that the system operated predominantly under anaerobic conditions, with redox potential (Eh) increasing from the inlet (-342.9 mV) to the outlet (-316.4 mV). Conversely, the chemical oxygen demand (COD) decreased along the greywater flow, suggesting negative correlation between these characteristics. The zones of the evapotranspiration and treatment tank (CEvaT) were characterized by lower community diversity and richness and by the presence of specific groups: Proteobacteria and Synergistetes related to the first steps of the conversion of organic carbon, in the bottom layer inside the anaerobic chamber (AnC); methanogens (Methanosaeta and Methanobacterium) and sulphate-reducing bacteria (Desulfovibrio, Desulforhabdus and Desulfomonile) in the middle layer; and microorganisms associated with the nitrogen cycle and oxygen release (Acinetobacter, Novosphingobium, Candidatus Nitrososphaera) in the top layer. On the other hand, the increase of the ORP and decrease of organic matter concentrations were associated with higher community diversity and richness in the middle layer of the CW, which showed higher abundance of microorganisms involved in methane (Methylobacterium and Candidatus Koribacter) and sulphur (Rhodoblastus and Thiobacillus) oxidation.