Autotrophic Denitrifiers Research Articles

Simultaneous Mixotrophic Nitrate Removal and Phosphorus Removal in a Sponge-Iron Denitrifying Filter

Due to stricter municipal wastewater discharge standards, there is an increased need for further treatment of nitrate in the secondary effluent of wastewater treatment plants. This is achieved through denitrification by the addition of external carbon sources, which leads to increased costs in wastewater treatment. The aim of this study was to examine the possibility of simultaneous removal of nitrate and phosphorus from simulated secondary effluent by employing a sponge-iron-based denitrifying filter at room temperature. The results indicate that at hydraulic retention times of over 2 h, more than 60% of the nitrate was reduced to ammonia and nitrite via iron-based abiotic nitrate reduction. However, sponge iron easily scaled after two months of operation. Therefore, a little glucose was added to the influent, resulting in a final COD/N ratio of 1:1. Mixotrophic nitrate reduction was observed, and the rust of sponge iron was successfully dissolved. Batch test results demonstrate that biological nitrate denitrification accounted for 70.0% of the total nitrate reduction. Additionally, high-efficiency phosphorus removal through the chemical reaction of released iron and phosphorus was achieved throughout the entire experiment, with removal efficiencies of more than 90% at hydraulic retention times of over 2 h. Moreover, high-throughput sequencing data show that the species diversity obviously increased after adding organic carbon, suggesting the coexistence of heterotrophic and autotrophic denitrifiers. Hence, the sponge-iron denitrifying filter has considerable prospects in the field of secondary effluent treatment and is likely to be the future direction of zero-valent iron application in sewage treatment.

Pyrite coupled with steel slag to enhance simultaneous nitrogen and phosphorus removal in constructed wetlands

FeS2-based constructed wetland has garnered increasing interest as an efficient and eco-friendly method for nitrogen and phosphorus removal from carbon limitation wastewater. However, large-scale application is limited due to low nitrogen and phosphorus simultaneous removal performance, slow growth rate of autotrophic denitrifiers and high effluent acidity. This study proposes a novel constructed wetland by coupling with pyrite and steel slag (PSCW) as substrates. The results showed that steel slag addition in PSCW increased total nitrogen removal by 40% ∼ 54%. The higher removal of total phosphorus was mainly attributed to the strong substrate adsorption and metal ions precipitation, which was removed at the pyrite and steel slag layer. The genes involved in denitrification, glycolysis, oxidative phosphorylation and sulfur oxidation were notably enriched in PSCW, indicating the increasing contribution of FeS2-based autotrophic denitrification coupled with steel slag to NO3− removal. The XPS analysis identified the formation of SO42−, S2−, S0, and Fe(III) on the pyrite surface in PSCW. S0 formation and sulfate reducing bacteria enrichment in PSCW were all promoted due to the coupling of pyrite and steel slag, which also maintained higher NO3− removal efficiency. Thiobacillus was specifically enriched in PSCW due to steel slag addition. The toxicity effect of pyrite on root tip can be alleviated by steel slag, which was conducive to nutrients assimilation by roots. The findings of this study provide new insights into the molecular mechanism of carbon, nitrogen, and sulfur metabolism in the pyrite-steel slag based constructed wetland.

Study on the Treatment of Simulated Recirculating Mariculture Wastewater by Thiosulfate-Based Autotrophic Denitrification

In this study, a sulfur-based autotrophic denitrifying filter (SADF) was developed for the purpose of removing nitrate from simulated recirculating mariculture wastewater. Results showed that over 90% of the nitrate could be effectively eliminated by utilizing thiosulfate as the electron donor, with a molar ratio of thiosulfate-S to nitrate molar ratio of 2:1 or greater. Additional batch tests confirmed that thiosulfate was a suitable sulfur source for nitrate removal even without prior accumulation of the biomass to nitrite. Excess thiosulfate had a minor impact on N-removal efficiency, so an external sulfur source was not required for nitrate removal, however, it could still help to reduce nitrate accumulation and water replacement to some extent. High-throughput sequencing results illustrated that Thiomicrospira and Thioalkalivibrio were the dominant autotrophic denitrifying genera in the SADF, while Thiomicrospira was more significantly affected in the case of insufficient sulfur sources. As the issue of nitrate accumulation in the mariculture recirculating system has been resolved, only a small amount of water needs to be added to the system daily. Therefore, the thiosulfate-based SADF process has the potential to be implemented for nitrate removal in mariculture systems, which could present a promising sustainable solution to the nitrate pollution issue.

Performance, comparison and utilization of reduced sulfur (−2) compounds (S2−, FeS and SCN−) in autotrophic denitrification process by thiosulfate-driven autotrophic denitrifier

The coexistence of reduced sulfur (−2) compounds (S2−, FeS and SCN−) are found in some industrial wastewaters due to pre-treatment of Fe(II) salts. These compounds as electron donors have attracted increasing interest in autotrophic denitrification process. However, the difference of their functions still remain unknown, which limit efficient utilization in autotrophic denitrification process. The study aimed to investigate and compare utilization behavior of these reduced sulfur (−2) compounds in autotrophic denitrification process activated by thiosulfate-driven autotrophic denitrifiers (TAD). Results showed that the best denitrification performance was observed in SCN−; while the reduction of nitrate was significantly inhibited in S2− system and the efficient accumulation of nitrite was observed in FeS system with cycle experiments continuing. Additionally, intermediates containing sulfur were produced rarely in SCN− system. However, the utilization of SCN− was limited obviously in comparison with S2− in coexistence systems. Moreover, the presence of S2− increased the accumulation peak of nitrite in coexistence systems. The biological results indicated that the TAD utilized rapidly these sulfur (−2) compounds, in which genus of Thiobacillus, Magnetospirillum and Azoarcus might play main roles. Moreover, Cupriavidus might also participate in sulfur oxidation in SCN− system. In conclusion, these might be attributed to the characteristics of sulfur (−2) compounds including the toxicity, solubility and reaction process. These findings provide theoretical basis for regulation and utilization of these reduced sulfur (−2) compounds in autotrophic denitrification process.

The efficient utilization of thiocyanate on simultaneous removal of ammonium and nitrate through thiosulfate-driven autotrophic denitrifiers and anammox

The efficient utilization of thiocyanate remain be an important bottleneck in the low-cost nitrogen removal for wastewaters containing thiocyanate. The study aimed to investigate the feasibility of thiocyanate in removal of nitrate and ammonium through anammox (AN) and thiosulfate-driven autotrophic denitrifiers (TSAD). The results showed that removal of nitrate and ammonium were achieved rapidly utilizing thiocyanate, which was attributed to degradation of thiocyanate by TSAD and cooperation with AN. The utilization efficiency of thiocyanate in nitrogen removal was increased by 250% due to the microbial cooperation. Excess thiocyanate and ammonium did not influence the nitrogen removal amount. However, the nitrogen removal were affected obviously by the biomass ratio (XAN/XTSAD) between AN and TSAD Moreover, the dynamics related to removal of pollutants was described successfully by a modified Monod model with time constraints. These findings offer an insight for efficient utilization of thiocyanate in nitrogen removal via microbial cooperation.

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.

Denitrification performance and mechanism of a novel sulfur-based fiber carrier fixed-bed reactor: Co-existence of sulfur-based autotrophic denitrification and endogenous denitrification

Sulfur autotrophic denitrification becomes a research hotspot in recent years, but traditional packed bed reactor has several disadvantages, including small sulfur specific surface area, easy blockage, and sulfur loss when using small sulfur grains. A sulfur-based fiber carrier fixed-bed reactor (SFFR) was developed in the study, which had the advantage of higher denitrification efficiency due to the large sulfur specific surface area, and the nitrogen removal rate (NRR) of it could reach 0.304 kg (NO3−-N)/m3·d. Meanwhile, the sulfur-based fiber filler that used in SFFR had characteristics of high surface roughness and suitable for microbial attachment growth, which promoted the endogenous denitrification in the reactor. Therefore, SAD and endogenous denitrification were jointly involved in nitrate removal in SFFR, and the sulfate productivity gradual decreased to 5.58 mgSO42−/mgNO3−-N in this study, which decreased by 26 % compared with the theoretical value (7.54 mgSO42−/mgNO3−-N). The result of this research showed that compared with the traditional SAD process, the electron utilization rate of SFFR increased from 75.76 % to 91.8 %. Meanwhile, the higher electron utilization rate of SFFR indicated that the advantages of SFFR included lower elemental sulfur consumption, lower acid production and smaller sludge production. In addition, the maximum NRR of SFFR was further increased to 0.528 kg (NO3−-N)/m3·d with the assist of vibrating device because it could enhance the mass transfer during the process of wastewater treatment. Microbial analysis suggested that several autotrophic denitrifiers along with heterotrophs and endogenous denitrifiers, co-existed in the biofilm.

Understanding the role of polyurethane sponges on rapid formation of aerobic granular sludge and enhanced nitrogen removal

Long formation time and limited simultaneous nitrification–denitrification (SND) are still the problems for aerobic granular sludge (AGS) application. This study presents a novel strategy to simultaneously promote sludge granulation and enhance nitrogen removal. This new approach consists of adding polyurethane sponges (PUS) and applying intensive aeration periodically. To demonstrate the effectiveness of this strategy and understand the role of PUS, two sequencing batch reactors (R1: without PUS; R2: PUS addition) were used. The results show that the granulation time in R2 (14 days) was much shorter and the nitrogen removal (80.7 %) was much higher when compared to the R1 (75 days and 64.8 %). Biofilm was rapidly formed on PUS because of the porous network structure, resulting in the low suspended biomass concentration. Thus, a close to discrete non-flocculent settling type was created, which promoted the selection of well-settling aggregates rapidly and efficiently. Meanwhile, biofilm was detached from PUS by intensive aeration, providing the nucleus (fragments of biofilm) in-suit for microbial adhesion. Under the combined effects, the aerobic granulation process was significantly accelerated. The total biomass amount was maintained during initial granulation because of the rapid biofilm formation, resulting in the stable removal of NH4+-N at a rapid start-up period. The rapid formation of biofilm and fast granulation created more anoxic zones during aeration and thereby enhanced SND. The biofilm formation also increased the relative abundances of aerobic denitrifiers (Dokdonella and Denitratisoma), autotrophic denitrifiers (Sulfuritalea), and hydrolytic/fermentativebacteria (Ferruginibacter, Opitutus, and Lactobacillus), which enhanced the SND performance and endogenous denitrification.

Low pH induces simultaneous fungal bulking and nitrogen removal deterioration in an activated sludge reactor: Mechanisms based on microbial transboundary coevolution

Fungal bulking caused by the abnormal acidic pH in influents stimuli is always accompanied by the deterioration of nitrogen removal in conventional activated sludge process. This is probably the result of co-evolution between dominant fungi and nitrogen-cycling bacterial communities. However, little is known about how they interact across borders in low pH stimuli. In this work, we explored the effects of fungal bloom on the community networks and metabolic pathways of nitrogen-cycling bacteria using an activated sludge reactor. We induced Trichoderma (54%) sludge bulking by lowering the pH, leading to high SVI (301 mL/g) and ammonia nitrogen (10.6 mg/L) in effluent. The abundances and expressions of classic nitrogen cycle genes all decreased, involving in ammoxidation (amo and hao), nitrification (nirS and nirK), and denitrification (napA and nrfA). With the occurrence of Trichoderma bloom, the relative abundances of major N cycling bacteria, such as Nitrosomonas, Nitrospira, and Dechoromonas, significantly reduced by 72%, 60%, and 74%, respectively. As a result, the nitrogen cycling metabolic network collapsed. In contrast, dominant autotrophic denitrifying bacteria Rhodanobacter, clustered with Trichoderma on the phylogenetic tree, increased from 2% to 47%. Trichoderma exhibited symbiotic relationships with certain enriched denitrifiers. Herein, these findings demonstrate that the succession between eukaryotic-prokaryotic microbes can impair nitrogen metabolism functioning in activated sludge systems.

Effect of salinity on the denitrification of the sulfur-based autotrophic denitrification system

This study investigated the effect of gradual salt concentration increases on the nitrogen removal performance from a sulfur-based autotrophic denitrification (SAD) process. A continuous operation reactor was used to study the adaptability of sulfur-based autotrophic denitrifying bacteria to changes in salinity. The study focused specifically on the effects of SO42− and Cl− on the activity of SAD bacteria. When the concentration of SO42− was 0∼4 ​g/L, NO3−-N was removed at more than 97%. When the concentration of SO42− increased to 15 ​g/L, the removal of NO3−-N decreased to 51%. When the concentration of NaCl was 0∼30 ​g/L, the removal rate of NO3−-N was all greater than 97%. When the concentration of NaCl increased to 80 ​g/L, the removal rate of NO3−-N decreased to 34.6%. When the NaCl concentration was 50 ​g/L, the removal rate of NO3−-N decreased to 80.59% at first, and the removal rate of NO3−-N recovered to 98% after 16 days of operation. When the NaCl concentration increased to 80 ​g/L, the NO3−-N removal rate decreased to 50%, and the reactor still could not recover its performance after long-term operation. The growth of halophilic heterotrophic bacteria with the increase of NaCl concentration leads to an increase in the abundance of Proteobacteria. The higher concentration of Cl− seriously inhibits the growth of Sulfurimonas and Thiobacillus, decreasing the effectiveness of bacteria to remove nitrogen.

Nitrogen removal and metabolic pathways of nitrifying coal ash propelled by temperature change under high nitrogen loading

Coal ash, owing to its cost-effectiveness and greater adhesion with nitrifiers, has been used as a carrier for immobilizing nitrifiers when treating wastewater with a low C/N ratio and high nitrogen loading. Various environmental conditions significantly affect the nitrogen metabolic pathways of nitrifying coal ash. In this study, the nitrogen metabolic pathways of nitrifying coal ash propelled at different temperatures were investigated to evaluate the impact of seasonal variation on nitrifying coal ash. Results showed average NH<sub>4</sub><sup>+</sup>-N effluent concentrations increased with temperature, and reached 3.37, 5.16, and 76.06 mg N/L with 1158 mg N/L influent under 15 ℃, 25 ℃, and 35 ℃ systems. The total nitrogen removal rate at a cooling temperature of 15 ℃ was higher than that at 25 ℃ and 35 ℃. Further analysis showed <i>Nitrosomonas</i> and <i>Nitrobacter</i> were enhanced, whereas a significant evolution of autotrophic denitrifiers, <i>Acidovorax</i>, was enriched at 15 ℃. An in-depth investigation of N-metabolism pathways suggested higher temperatures of 25 ℃ and 35 ℃ reduced the abundance of key genes involved in ammonia oxidation (amoABC, <i>hao</i>) and nitrite oxidation (<i>nar</i>GH). Network analysis between N-metabolic functional genes and genera presented stronger relevance and richer sources between nitrifiers/denitrifiers and nitrification/denitrification-associated genes under cooler temperatures.

Insight into characteristics of sulphur-based autotrophic denitrifying microbiota in the nitrate removal

ABSTRACT Mariculture wastewater is characterized by low organic carbon to nitrogen ratio (C/N) but high nitrate concentration, which makes it difficult to remove nitrate by the completely heterotrophic denitrification. However, high nitrate discharge poses a threat to the natural environment and human health. Thus, we enriched sulphur-based autotrophic denitrifying (SAD) microbiota and optimized the nitrate removal under different environmental factors and electron donor conditions. The results showed that the dominant genera in the enriched microbiota were previously confirmed autotrophic denitrifiers, Sulfurovum, Thioalkalispira-Sulfurivermis, and Sedimenticola, with a high relative abundance of 41.14%, 21.01%, and 6.17%. Among the environmental factors, pH was the key factor affecting SAD microbiota, and pH 7–9 favoured nitrate removal. However, high pH led to nitrite accumulation (e.g. 10 mg/L at pH = 9), which should be strictly avoided. With regard to electron donors, the optimal concentrations of thiosulphate and nitrate were 50 and 5 mg/L, respectively. The best organic carbon is acetate with an optimal concentration of 10 mg/L. Meanwhile, the initial concentration of thiosulphate was proportional to the nitrate removal rate, while higher concentrations of organic carbon stimulated the heterotrophic denitrification potential of microbiota and thus benefited to dentrification. This study showed that the enriched SAD microbiota was able to achieve efficient nitrate removal under suitable environmental conditions and mixed electron donors and thus presented the potential for application in the treatment of mariculture wastewater.

Biofilm coupled micro-electrolysis of waste iron shavings enhanced iron and hydrogen autotrophic denitrification and phosphate accumulation for wastewater treatment

Autotrophic denitrification has attracted more and more attention because it does not require organic carbon sources. A circulation ditch filled with plastic filler and waste iron shavings was established to develop hydrogen and iron autotrophic denitrifying bacteria for nitrogen and phosphorus removal from wastewater. The results demonstrated that Hydrogenophaga (hydrogen autotrophic denitrifying bacteria), Unclassified_f__Comamonadaceae (iron autotrophic denitrifying bacteria), and Sphaerotilus (iron autotrophic denitrifying bacteria) were enriched in biofilms, and the relative abundances were 4.0%, 9.0% and 1.2% in aerobic biofilms; 2.9%, 17.8% and 7.7% in aerobic biofilms (Fe–C); 0.6%, 1.8% and 0.04% in anoxic biofilms, and 13.8%, 13.6% and 0.7% in anoxic biofilms (Fe–C). In the system, the removal rates of chemical oxygen demand, ammonia nitrogen, total nitrogen and total phosphorus reached 91.4%, 93.4%, 80.2%, and 79.2%, respectively. The hypothetic mechanism implies that iron-carbon micro-electrolysis produced Fe2+ and H2 to be electron donors for autotrophic denitrification and enriched autotrophic denitrifying bacteria and formed ferrous phosphate in this wastewater treatment system.

Insights into microbial interactive mechanism regulating dissimilatory nitrate reduction processes in riparian freshwater aquaculture sediments

Aquaculture can substantially alter the accumulation and cycling of nutrients in sediments. However, the microbial mechanisms mediating sediment dissimilatory nitrate (NO3−) reduction in freshwater aquaculture ponds are still unclear, which rule the removal and retention of N element. In the present study, three microbial NO3− reduction processes in riparian aquaculture pond sediments (i.e., crab, shrimp and fish ponds) and natural freshwater sediments (i.e., lakes and rivers) were investigated via isotopic tracing and molecular analyses. The potential rates of denitrification, anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA) significantly increased in the aquaculture ponds compared with the natural freshwaters. Denitrification contributed 90.40–94.22% to the total NO3− reduction (product as N2), followed by 2.49–5.82% of anammox (product as N2) and 2.09–5.18% of DRNA (product as NH4+). The availability of C and N substrates, rather than functional gene abundance, regulated the activities of NO3− reductions and microbiome composition. Microbial mechanism based on network analysis indicated that heterotrophic denitrifiers and DNRA bacteria (e.g., Bacillus, Micromonospora, Mycobacterium and Brachybacterium) determined the community structure and function for N conversions in aquaculture ponds, whereas the such microbial network in natural freshwater sediments was manipulated by autotrophic denitrifiers (e.g., Desulfuromonas, Polaromonas, Solitalea). Collectively, this study provides an in-depth exploration of microbial nitrogen removal in freshwater aquaculture areas and supports management strategies for N pollution caused by reclamation for aquaculture in riparian zones.

Deciphering the coupling of partial nitrification/anammox and sulfur autotrophic denitrification: Microbial metabolism and antibiotic resistance genes propagation

Partial nitrification/anaerobic ammonium oxidation (anammox) (PN/A) was considered as an economically beneficial and environmentally friendly biological nitrogen removal process. The elimination of nitrate produced by PN/A system was the key to improving the total nitrogen removal efficiency (TNRE) of the system under mainstream conditions. In this study, the feasibility of the coupling S(0)-driven autotrophic denitrification (SAD) with PN/A in a single sequencing moving bed biofilm reactor (SMBBR) at room temperature was explored. Additionally, biological treatment process is ideal environments for the emerge and spread of emerging pollutants antibiotic resistance genes (ARGs), therefore, the shifts of ARGs in the system were investigated. After the coupling of PN/A and SAD, SMBBR was steadily operated with high TNRE. Nitrite-oxidizing bacteria (Nitrospira and Candidatus Nitrotoga) and nitrite oxidoreductase enzyme were inhibited by SAD process. And anammox bacteria were enriched, especially oligotype GAGTTTAAT of Ca. Brocadia. Sulfurimonas became the predominant autotrophic denitrifier. Heterotrophic denitrifiers Thauera and Dechloromonas were elutriated out from the system during the coupling of PN/A and SAD process, and they might be potential hosts for ARGs, leading to the decrease of ARGs. Overall, this study proposed a new strategy that SAD could promote the TNRE of PN/A and cut the risks of ARGs propagation, and PN/A-SAD coupling system was a biological nitrogen removal technology with promising applications under mainstream conditions.

Insight into the shaping of microbial communities in element sulfur-based denitrification at different temperatures

Nitrate pollution is an important cause of eutrophication and ecological disruption. Recently, element sulfur-based denitrification (ESDeN) has attracted increasing attention because of its non-carbon source dependence, low sludge yield, and cost-effectiveness. Although the denitrification performance of sulfur autotrophic denitrifying bacteria at different temperatures has been widely studied, there are still many unknown factors about the adaptability and the shaping of microbial community. In this study, we comprehensively understood the shaping of ESDeN microbial communities under different temperature conditions. Results revealed that microbial communities cultivated at temperatures ranging from 10 °C to 35 °C could be classified as high-temperature (35 °C), middle-temperature (30, 25 and 20 °C), and low-temperature (15 and 10 °C) communities. Dissolved oxygen in water was an important factor that, in combination with temperature, shaped microbial community structure. According to network analysis, the composition of keystone taxa was different for the three groups of communities. Some bacteria that did not have sulfur compound oxidation function were identified as the “keystone species”. The abundances of carbon, nitrogen, and sulfur metabolism of the three microbial communities were significantly changed, which was reflected in that the high-temperature and middle-temperature communities were dominated by dark oxidation of sulfur compounds and dark sulfide oxidation, while the low-temperature community was dominated by chemoheterotrophy and aerobic chemoheterotrophy. The fact that the number of microorganisms with dark oxidation of sulfur compounds capacity was quite higher than that of microorganisms with dark sulfur oxidation capacity suggested that the sulfur bioavailability at different temperatures, especially low temperature, was the main challenge for the development of efficient ESDeN process. This study provided a biological basis for developing a high-efficiency ESDeN process to cope with temperature changes in different seasons or regions.

Pyrite-Based Autotrophic Denitrifying Microorganisms Derived from Paddy Soils: Effects of Organic Co-Substrate Addition.

The presence of organic co-substrate in groundwater and soils is inevitable, and much remains to be learned about the roles of organic co-substrates during pyrite-based denitrification. Herein, an organic co-substrate (acetate) was added to a pyrite-based denitrification system, and the impact of the organic co-substrate on the performance and bacterial community of pyrite-based denitrification processes was evaluated. The addition of organic co-substrate at concentrations higher than 48 mg L−1 inhibited pyrite-based autotrophic denitrification, as no sulfate was produced in treatments with high organic co-substrate addition. In contrast, both competition and promotion effects on pyrite-based autotrophic denitrification occurred with organic co-substrate addition at concentrations of 24 and 48 mg L−1. The subsequent validation experiments suggested that competition had a greater influence than promotion when organic co-substrate was added, even at a low concentration. Thiobacillus, a common chemolithoautotrophic sulfur-oxidizing denitrifier, dominated the system with a relative abundance of 13.04% when pyrite served as the sole electron donor. With the addition of organic co-substrate, Pseudomonas became the dominant genus, with 60.82%, 61.34%, 70.37%, 73.44%, and 35.46% abundance at organic matter concentrations of 24, 48, 120, 240, and 480 mg L−1, respectively. These findings provide an important theoretical basis for the cultivation of pyrite-based autotrophic denitrifying microorganisms for nitrate removal in soils and groundwater.

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.

Role of iron(II) sulfide in autotrophic denitrification under tetracycline stress: Substrate and detoxification effect

Autotrophic denitrification using inorganic compounds as electron donors has gained increasing attention in the field of wastewater treatment due to its numerous advantages, such as no need for exogenous organic carbon, low energy input, and low sludge production. Tetracycline (TC), a refractory contaminant, is often found coexisting with nutrients (NO3− and PO43−) in wastewater, which can negatively affect the biological nutrient removal process because of its biological toxicity. However, the performance of autotrophic denitrification under TC stress has rarely been reported. In this study, the effects of TC on autotrophic denitrification with thiosulfate (Na2S2O3) and iron (II) sulfide (FeS) as the electron donors were investigated. With Na2S2O3 as the electron donor, TC slowed down the nitrate removal rate, which decreased from 1.32 to 0.18 d−1, when TC concentration increased from 0 mg/L to 50 mg/L. When TC concentration was higher than 2 mg/L, nitrite reduction was seriously inhibited, leading to nitrite accumulation. With FeS as the electron donor, nitrate removal was much more efficient under TC-stressed conditions, and no distinct nitrite accumulation was observed when the initial TC concentration was as high as 10 mg/L, indicating the effective detoxification of FeS. The detoxification effects in the FeS autotrophic denitrification system mainly resulted from the rapid adsorption of TC by FeS and effective degradation of TC, as proven by a relatively higher living biomass area. This study offers new insights into the response of sulfur-based autotrophic denitrifiers to TC stress and demonstrates that the FeS-based autotrophic denitrification process is a promising technology for the treatment of wastewater containing emerging contaminants and nutrients.

Effect of electric current intensity on performance of polycaprolactone/FeS2-based mixotrophic biofilm–electrode reactor

In this study, a bioelectrochemical system consisting of pyrite-based autotrophic denitrification (PAD) and heterotrophic denitrification (HD) was established to polish nitrate wastewater. The loading of electric current (EC) could stimulate the dissolution of pyrite. Appropriate EC (I ≤ 30 mA) was conducive to nitrate removal, too high EC (I = 40 mA) would inhibit nitrate removal and lead to an obvious accumulation of NO2–-N and NH4+-N. Microbial analysis revealed that the increase of EC could inhibit the diversity of heterotrophic microbes, but appropriate EC (I = 10 mA) could increase the diversity of autotrophic microbes. The EC loading was conducive to the enrichment of iron autotrophic denitrifiers (Ferritrophicum), pyrite-oxidizing bacteria (Thiobacillus, Sulfurimonas), and sulfur autotrophic denitrifiers (Dechloromonas, Thiobacillus, and Arenimonas). The EC loading enlarged the contribution of PAD, making PAD a dominant pathway in denitrification.