Sulfide-driven Denitrification Research Articles

Novel Sulfate Reduction Coupled to Simultaneous Nitrification and Autotrophic Denitrification Process for Removing Nitrogen and Organics from Saline Wastewater.

Highly efficient sulfate reduction coupled to autotrophic denitrification plus nitrification is demonstrated by integrating an anaerobic membrane bioreactor (AnMBR) with a membrane aerated biofilm reactor (MABR). Concurrent chemical oxygen demand (COD) removal and sulfate reduction were accomplished in the AnMBR, while simultaneous nitrification and autotrophic denitrification were carried out in the MABR. Separate operation of the MABR achieved >90% total nitrogen (TN) removal when the N/S ratio was controlled at 0.4 gN/gS. The integrated AnMBR-MABR system efficiently resisted influent variability, realizing >95% COD removal in the AnMBR and >75% TN removal in the MABR when the influent COD/N ratio was above 4 gCOD/gN. Membrane fouling did not happen during ∼170 days of operation. Due to sulfide oxidation, a large amount of elemental sulfur (S0) accumulated in the MABR biofilm, where it served as an electron donor for denitrification. Microbial community analysis indicated that Nitrospira and Thiobacillus played key roles in nitrification and sulfide-driven denitrification, respectively, and that they occurred in different layers of the biofilm. This novel process offers advantages of a small land-area footprint, modular operation, and high efficiency electron-donor and oxygen utilizations, particularly for wastewater with a low COD/N ratio.

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 (200-3000mg-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±5.15mg/L NO2--N was accumulated when ammonia stress reached 3000mg TAN/L. But the inhibited SD process could recover in about 40 days when ammonia stress was decreased to 200mg 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.

Effect of feeding gas type and nitrogen: Sulfur ratio on a novel sulfide-driven denitrification methane oxidation (SDMO) system

To save treatment costs of nitrogen-polluted water, a novel sulfide-driven denitrification methane oxidation (SDMO) system, which integrated autotrophic denitrification (AD) and denitrification anaerobic methane oxidation (DAMO) to simultaneously realize wastewater denitrification and biogas utilization in situ, has been operated. In this work, nine groups V0—V8 were derived from a mature SDMO reactor which already enriched AD and DAMO microorganisms. Batch tests about them were proceeded to investigate the effect of feeding gas type and N/S ratio on kinetic performance, microorganism community structure, and functional genes. Results showed as N/S rising up from 2.3 to 23.3, the nitrate removal efficiency decreased from 100 % to 15.4 % but its contribution by DAMO/AD ratio increased from 0.5 to 2.2. Biogas serving as feeding gas enhanced nitrate removal efficiency to 100 % at most in group V3 (N/S = 2.3, H2S = 1.5 %) whereas DAMO and AD contribution for nitrate removal was 33.1 % and 65.6 % respectively. Low H2S content gave little effect on microorganism community structure but high hydrogen sulfide content decreased the abundance of DAMO bacteria Candidatus Methylomirabilis to ∼ 4 % and increase abundance of AD bacteria Thiobacillus to > 20 %. Comparing with V1 feeding with methane, nitrate reduction genes napA, narG, nirK, nirS, cnorB and sulfide oxidation gene soxB up-regulated to > 3.0 × 109 copies/g in HS groups (biogas with high H2S content > 1 %) while gene pmoA up-regulated to ∼ 1.0 × 109 copies/g in LS groups (biogas with low H2S content < 1 %). This study provided more comprehensive understanding for the effect of feeding gas type and N/S ratio on SDMO system.

A novel sulfide-driven denitrification methane oxidation (SDMO) system: Operational performance and metabolic mechanisms

Microbial denitrification is a crucial biological process for the treatment of nitrogen-polluted water. Traditional denitrification process consumes external organic carbon leading to an increase in treatment costs. We developed a novel sulfide-driven denitrification methane oxidation (SDMO) system that integrates autotrophic denitrification (AD) and denitrification anaerobic methane oxidation (DAMO) for cost-effective denitrification and biogas utilization in situ. Two SDMO systems were operated for 735 days, with nitrate and nitrite serving as electron acceptors, to explore the performance of sewage denitrification and characterize metabolic mechanisms. Results showed SDMO system could reach as high as 100% efficiency of nitrogen removal and biogas desulfurization without an external carbon source when HRT was 10 days and inflow nitrogen concentrations were 50–100 mgN·L−1. Besides, nitrate was a preferable electron acceptor for SDMO system. Biogas not only enhanced nitrogen removal but also intensified the DAMO, nitrogen removed through DAMO contribution doubled as original period from 2.9 mgN·(L·d)−1 to 6.2 mgN·(L·d)−1, and the ratio of nitrate removal through AD to DAMO was 1.2:1 with nitrate as electron acceptor. While nitrogen removed almost all through AD contribution and DAMO was weaken as before, the ratio of nitrate removal through AD to DAMO was 21.2:1 with nitrite as electron acceptor. Biogas introduced into SDMO system with nitrate inspired the growth of DAMO bacteria Candidatus Methylomirabilis from 0.3% to 19.6% and motivated its potentiality to remove nitrate without ANME archaea participation accompanying with gene mfnE upregulating ∼100 times. According to the reconstructed genome from binning analysis, the dramatically upregulated gene mfnE was derived from Candidatus Methylomirabilis, which may represent a novel metabolism pathway for DAMO bacteria to replace the role of archaea for nitrate reduction.

The Inhibitory Effects and Underlying Mechanism of High Ammonia Stress on Sulfide-Driven Denitrification Process

The Inhibitory Effects and Underlying Mechanism of High Ammonia Stress on Sulfide-Driven Denitrification Process

Sulfide-driven nitrous oxide recovery during the mixotrophic denitrification process

We propose a novel sulfide-driven process to recover N2O during the traditional denitrification process. The optimum initial sulfide concentration was 120 mg/L, and the N2O percentage in the gaseous products (N2O+N2) was up to 82.9%. Moreover, sulfide involved in denitrification processes could substitute for organic carbon as an electron donor, e.g., 1 g sulfide was equivalent to 0.5-2 g COD when sulfide was oxidized to sulfur and sulfate. The accumulation of N2O was mainly due to the inhibiting effect of sulfide on nitrous oxide reductase (N2OR), which was induced by the supply insufficiency of electrons from cytochrome c (cyt c) to N2OR. When the initial sulfide concentration was 120 mg/L, the N2OR activity was only 36.8% of its original level. According to the results of cyclic voltammetry, circular dichroism spectra and fluorescence spectra, significant changes in the conformations and protein structures of cyt c were caused by sulfide, and cyt c completely lost its electron transport capacity. This study provides a new concept for N2O recovery driven by sulfide in the denitrification process. In addition, the findings regarding the mechanism of the inhibition of N2OR activity have important implications both for reducing emissions of N2O and recovering N2O in the sulfide-driven denitrification process.

Exploration and verification of the feasibility of sulfide-driven partial denitrification coupled with anammox for wastewater treatment

Anaerobic ammonia oxidation (anammox) is a well-developed biotechnology for treating high-strength ammonium wastewaters. Recently, partial denitrification has been considered as an alternative to supply anammox with the required nitrite. In this study, a process of sulfide-driven partial denitrification and anammox (SPDA) was developed and operated continuously in an upflow anaerobic sludge blanket (UASB) reactor for 392 days. This reactor was fed with synthetic wastewater containing 100 mgN/L nitrate, 80 mgN/L ammonium and 20–80 mgS/L sulfide. After 160 days of operation, the reactor reached stable performance, and the nitrogen removal efficiency and rate were maintained at 80% and 0.29 kgN/(m³•d), respectively. The estimated nitrogen removal via anammox and sulfide-driven denitrification were 87.2% and 12.8%. Additional batch experiments were conducted to investigate the effects of sulfide on anammox and the mechanisms of nitrogen removal in the SPDA system. The following results were obtained: (1) sulfide had an inhibitory effect on the specific anammox activity with IC50 of 9.7 mgS-H2S/L. (2) The rapid oxidation of sulfide by sulfur-oxidizing bacteria (SOB) could relieve the toxic effects of sulfide on the anammox in the SPDA system. (3) Sulfide bio-oxidation was a two-step reaction with biologically produced elemental sulfur (BPS0) as the intermediate, and the second step using BPS0 as the electron donor, can efficiently produce nitrite via partial denitrification (NO3− → NO2−) as a supply for anammox. Finally, a high-throughput sequencing analysis identified Thiobacillus and Sulfurimonas as the dominant genera of SOB in the SPDA system, and Candidatus Kuenenia as the dominant anammox bacteria. Overall, this research gives the foundation for the practical application of sulfide-driven partial denitrification and anammox process in the future.

Long-term sulfide input enhances chemoautotrophic denitrification rather than DNRA in freshwater lake sediments

Partitioning between nitrate reduction pathways, denitrification and dissimilatory nitrate reduction to ammonium (DNRA) determines the fate of nitrate removal and thus it is of great ecological importance. Sulfide (S2-) is a potentially important factor that influences the role of denitrification and DNRA. However, information on the impact of microbial mechanisms for S2- on the partitioning of nitrate reduction pathways in freshwater environments is still lacking. This study investigated the effects of long-term (108d) S2- addition on nitrate reduction pathways and microbial communities in the sediments of two different freshwater lakes. The results show that the increasing S2- addition enhanced the coupling of S2- oxidation with denitrification instead of DNRA. The sulfide-oxidizing denitrifier, Thiobacillus, was significantly enriched in the incubations of both lake samples with S2- addition, which indicates that it may be the key genus driving sulfide-oxidizing denitrification in the lake sediments. During S2- incubation of the Hongze Lake sample, which had lower inherent organic carbon (C) and sulfate (SO42-), Thiobacillus was more enriched and played a dominant role in the microbial community; while during that of the Nansi Lake sample, which had higher inherent organic C and SO42-, Thiobacillus was less enriched, but increasing abundances of sulfate reducing bacteria (Desulfomicrobium, Desulfatitalea and Geothermobacter) were observed. Moreover, sulfide-oxidizing denitrifiers and sulfate reducers were enriched in the Nansi Lake control treatment without external S2- input, which suggests that internal sulfate release may promote the cooperation between sulfide-oxidizing denitrifiers and sulfate reducers. This study highlights the importance of sulfide-driven denitrification and the close coupling between the N and S cycles in freshwater environments, which are factors that have often been overlooked.

Sulfide-driven denitrification: detecting active microorganisms in fed-batch enrichment cultures by DNA stable isotope probing.

A microbial community was enriched in the anoxic compartment of a pilot-scale bioreactor that was operated for 180days, fed with sewage and designed for organic matter, nitrogen and sulfide removal by coupling anaerobic digestion, nitrification and mixotrophic denitrification. Denitrification occurred with endogenous electron donors, mainly sulfide and residual organic matter, coming from the anaerobic compartment. The microorganisms involved in denitrification with sulfide as electron donor were identified by DNA-stable isotope probing with [U-13C]-labelled CO2 and NaHCO3. Complete denitrification occurred every two days, and the applied NO3-/S2- ratio was 1.6. Bacteria belonging to the Sulfurimonas denitrificans was identified as a chemoautotrophic denitrifier, and those related to Georgfuchisa toluolica, Geothrix fermentans and Ferritrophicum radicicola were most probably associated with heterotrophic denitrification using endogenous cells and/or intermediate metabolites. This study showed that DNA-SIP was a suitable technique to identify the active microbiota involved in sulfide-driven denitrification in a complex environment, which may contribute to improve design and operation of bioreactors aiming for carbon-nitrogen-sulfur removal.

Effects of nitrate dosing on the migration of reduced sulfur in black odorous river sediment and the influencing factors

At present, nitrates were widely used to remediate sulfur-rich sediment in black odorous rivers. However, the optimal nitrate dosage and influencing factors during remediation have not been fully investigated. In this study, various nitrate dosages and possible influencing factors were investigated for their efficiency in treating sulfur-rich sediment. The results indicated that acceptable remedial efficiency (the percentage of acid volatile sulfide (AVS) oxidation and the residual ammonium production) can be achieved when 1.6 times the theoretical nitrate dosage is used. Large amounts of AVS cannot be oxidized if too little nitrate is used and will regenerate after several days of remediation. Conversely, high concentrations of ammonium will form if too much nitrate is used. The results of this study indicated that a rapid AVS oxidation rate will be achieved in higher temperatures; no significant treatment efficiency was found when temperatures were in the range of 15–35 °C. Organic matter slowed the rate of nitrate reduction but did not affect its remedial efficiency for AVS oxidation in sediment. A high concentration of ferrous iron primarily influenced nitrate treatment efficiency in the oxidation of AVS in sediment, simultaneously causing the production of a high ammonium concentration. This study once again proved that microbial mediation of the autotrophic sulfide-driven denitrification dominates AVS oxidation in sediment. This study indicated that a universal nitrate dosage calculation equation should be established, and that factors such as ferrous iron concentration should be considered in future remediation of black odorous rivers.

Integrating biogeochemistry with multiomic sequence information in a model oxygen minimum zone

Microorganisms are the most abundant lifeform on Earth, mediating global fluxes of matter and energy. Over the past decade, high-throughput molecular techniques generating multiomic sequence information (DNA, mRNA, and protein) have transformed our perception of this microcosmos, conceptually linking microorganisms at the individual, population, and community levels to a wide range of ecosystem functions and services. Here, we develop a biogeochemical model that describes metabolic coupling along the redox gradient in Saanich Inlet-a seasonally anoxic fjord with biogeochemistry analogous to oxygen minimum zones (OMZs). The model reproduces measured biogeochemical process rates as well as DNA, mRNA, and protein concentration profiles across the redox gradient. Simulations make predictions about the role of ubiquitous OMZ microorganisms in mediating carbon, nitrogen, and sulfur cycling. For example, nitrite "leakage" during incomplete sulfide-driven denitrification by SUP05 Gammaproteobacteria is predicted to support inorganic carbon fixation and intense nitrogen loss via anaerobic ammonium oxidation. This coupling creates a metabolic niche for nitrous oxide reduction that completes denitrification by currently unidentified community members. These results quantitatively improve previous conceptual models describing microbial metabolic networks in OMZs. Beyond OMZ-specific predictions, model results indicate that geochemical fluxes are robust indicators of microbial community structure and reciprocally, that gene abundances and geochemical conditions largely determine gene expression patterns. The integration of real observational data, including geochemical profiles and process rate measurements as well as metagenomic, metatranscriptomic and metaproteomic sequence data, into a biogeochemical model, as shown here, enables holistic insight into the microbial metabolic network driving nutrient and energy flow at ecosystem scales.

A model-based insight into the coupling of nitrogen and sulfur cycles in a coastal upwelling system.

The biogeochemical cycling in oxygen-minimum zones (OMZs) is dominated by the interactions of microbial nitrogen transformations and, as recently observed in the Chilean upwelling system, also through the energetically less favorable remineralization of sulfate reduction. The latter process is masked, however, by rapid sulfide oxidation, most likely through nitrate reduction. Thus, the cryptic sulfur cycle links with the nitrogen cycle in OMZ settings. Here, we model the physical-chemical water column structure and the observed process rates as driven by formation and sinking of organic detritus, to quantify the nitrogen and sulfur cycles in the Chilean OMZ. A new biogeochemical submodule was developed and coupled to the Regional Ocean Model System (ROMS). The model results generally agree with the observed distribution of reactive species and the measured process rates. Modeled heterotrophic nitrate reduction and sulfate reduction are responsible for 47% and 36%, respectively, of organic remineralization in a 150 m deep zone below mixed layer. Anammox contributes to 61% of the fixed nitrogen lost to N2 gas, while the rest of the loss is through canonical denitrification as a combination of organic matter oxidation by nitrite reduction and sulfide-driven denitrification. Mineralization coupled to heterotrophic nitrate reduction supplies ∼48% of the ammonium required by anammox. Due to active sulfate reduction, model results suggest that sulfide-driven denitrification contributes to 36% of the nitrogen loss as N2 gas. Our model results highlight the importance of considering the coupled nitrogen and sulfur cycle in examining open-ocean anoxic processes under present, past, and future conditions.