Hydrazine Dehydrogenase Research Articles

Monitoring the Biochemical Activity of Single Anammox Granules with Microbarometers.

Granule-based anaerobic ammonium oxidation (Anammox) is a promising biotechnology for wastewater treatments with extraordinary performance in nitrogen removal. However, traditional analytical methods often delivered an average activity of a bulk sample consisting of millions and even billions of Anammox granules with distinct sizes and components. Here, we developed a novel technique to monitor the biochemical activity of individual Anammox granules in real-time by recording the production rate of nitrogen gas with a microbarometer in a sealed chamber containing only one granule. It was found that the specific activity of a single Anammox granule not only varied by tens of folds among different individuals with similar sizes (activity heterogeneity) but also revealed significant breath-like dynamics over time (temporal fluctuation). Statistical analysis on tens of individuals further revealed two subpopulations with distinct color and specific activity, which were subsequently attributed to the different expression levels of heme c content and hydrazine dehydrogenase activity. This study not only provides a general methodology for various kinds of gas-producing microbial processes but also establishes a bottom-up strategy for exploring the structural-activity relationship at a single sludge granule level, with implications for developing a better Anammox process.

Potential Growth of Anammox Bacteria under Aerobic Conditions.

Anammox bacteria are obligate anaerobic bacteria that exist widely in nature with sufficient amounts of dissolved oxygen. However, whether anammox bacteria can grow under aerobic conditions remains unclear. In this study, we found that the production of nitrate in the anammox system under aerobic conditions was significantly higher than that under anaerobic conditions without total nitrogen loss. Anammox bacteria can grow by oxidizing nitrite and dehydrogenating hydrazine to produce electrons for carbon fixation. The hydrazine dehydrogenase in anammox bacteria was inhibited under aerobic conditions, and the nitrite oxidoreductase transcription expression of anammox bacteria increased by 2.7 times compared to that under anaerobic conditions, which was the main way for anammox bacteria perform carbon fixation. DNA-stable isotope probing with 13C bicarbonate found the existence of anammox bacteria with 13C isotopes in aerobic cultivation, further proving that anammox bacteria can grow under aerobic condition. More than half of the pathways in glycolysis, the Wood-Ljungdahl pathway, and the tricarboxylic acid cycle were upregulated in anammox bacteria in aerobic condition. Large amounts of bacterioferritins are the important antioxidative enzymes in anammox bacteria in the aerobic environment, which contributes to their stronger oxygen adaptation than other anaerobes. This study expands our understanding of the growth mechanism of anammox bacteria as well as the oxygen adaptation strategies of obligate anaerobic bacteria.

Characteristics and nontargeted metabolomics analysis of anammox granular sludge under short-term exposure to polypropylene and polylactic acid microplastics

A significant quantity of microplastics (MPs) is concealed within biological sludge. Current research predominantly examines the effects of non-biodegradable MPs on traditional sludge. However, the influence of biodegradable MPs on the functional microbial activity of anaerobic ammonium oxidation granular sludge (AnGS) remains inadequately explored. This study specifically investigated the effects of non-biodegradable polypropylene (PP) MPs and biodegradable polylactic acid (PLA) MPs on AnGS under short-term stress. The study found that PP MPs inhibited nitrogen removal performance, reducing nitrogen removal efficiency by 1.14% (100 mesh) and 5.77% (1000 mesh) respectively, along with decreased hydrazine dehydrogenase (HDH) activity. Conversely, PLA promoted denitrification performance, increasing efficiency by 8.21% (100 mesh) and 6.54% (1000 mesh). In response to MPs-induced environmental stress, the secretion of extracellular polymeric substances (EPS) increased in all experimental groups. Additionally, non-targeted metabolomic analysis revealed a decrease in the abundance of KEGG pathways related to carbon and amino acid metabolism in the experimental groups, while the enrichment of terephthalate and benzamide in the PLA groups significantly impacted its process. These findings offered valuable insights into the impact of MPs on anaerobic ammonium oxidation (anammox) and could potentially enhance its application.

Metagenomic insights into microbial metabolic mechanisms of a combined solid-phase denitrification and anammox process for nitrogen removal in mainstream wastewater treatment

To overcome the significant challenges associated with nitrite supply and nitrate residues in mainstream anaerobic ammonium oxidation (anammox)-based processes, this study developed a combined solid-phase denitrification (SPD) and anammox process for low-strength nitrogen removal without the addition of nitrite. The SPD step was performed in a packed-bed reactor containing poly-3-hydroxybutyrate-co-3-hyroxyvelate (PHBV) prior to employing the anammox granular sludge reactor in the continuous-flow mode. The removal efficiency of total inorganic nitrogen reached 95.7 ± 1.2% under a nitrogen loading rate of 0.18 ± 0.01 kg N·m3·d−1, and it required 1.02 mol of nitrate to remove 1 mol of ammonium nitrogen. The PHBV particles not only served as biofilm carriers for the symbiosis of hydrolytic bacteria (HB) and denitrifying bacteria (DB), but also carbon sources that facilitated the coupling of partial denitrification and anammox in the granules. Metagenomic sequencing analysis indicated that Burkholderiales was the most abundant HB genus in SPD. The metabolic correlations between DB (Betaproteobacteria, Rhodocyclaceae, and Anaerolineae) and anammox bacteria (Candidatus Brocadiac and Kuenenia) in the granules were confirmed through microbial co-occurrence networks analysis and functional gene annotations. Additionally, the genes encoding nitrate reductase (Nap) and nitrite reductase (Nir) in DB primarily facilitated nitrate reduction, thereby supplying nitric oxide to anammox bacteria for subsequent nitrogen removal with hydrazine synthase (Hzs) and hydrazine dehydrogenase (Hdh). The findings provide insights into microbial metabolism within combined SPD and anammox processes, thus advancing the development of mainstream anammox-based processes in engineering applications.

Revealing critical functional enzymes in anammox nitrogen removal and rate-limiting step in catalytic pathways: Insight into metaproteomics and density functional theory

To reveal the key enzymes in the nitrogen removal pathway and to further elucidate the mechanism of the catalytic reaction, this study utilized metaproteomics combined with molecular dynamics and density functional theory calculation. K. stuttgartiensis provided the proteins up to 88.37 % in the anammox-based system. Hydrazine synthase (HZS) and hydrazine dehydrogenase (HDH) accounted for 15.94 % and 3.45 % of the total proteins expressed by K. stuttgartiensis, thus were considered as critical enzymes in the nitrogen removal pathway. The process of HZSγ binding to NO with lowest binding free energy of −4.91 ± 1.33 kJ/mol. The reaction catalyzed by HZSα was calculated to be the rate-limiting catalyzing step, because it transferred the proton from NH3 to ·OH by crossing an energy barrier of up to 190.29 kJ/mol. This study provided molecular level insights to enhance the performance of nitrogen removal in anammox-based system.

Start-up and Recovery Performance of ANAMMOX Reactors Under Long-term Starvation

The application of ANAMMOX technology is constrained by sluggish growth and difficulty in enriching ANAMMOX bacteria. Long-term starvation of functioning bacteria due to limited substrate supply makes the steady operation of ANAMMOX reactors more difficult. Re-examining the start-up and recovery performance of the ANAMMOX reactor and identifying its resistance mechanism are important from the standpoint of long-term starvation. By inoculating nitrifying and denitrifying sludge under various operating circumstances, the ANAMMOX reactors were successfully started. Under various start-up procedures, the tolerance mechanism and recovery performance were examined. The outcomes demonstrated that the denitrifying sludge-inoculated reactor operated steadily with a high substrate concentration and low flow rate. After 85 days of operation, the removal efficiencies of NH4+-N, NO2--N, and total nitrogen reached 98.7%, 99.3%, and 89.3%, respectively. After 144 days of starvation and 30 days of recovery, the better nitrogen removal performance was achieved at a low substrate concentration and high flow rate, and the removal efficiencies were 99.8% （NH4+-N）, 99.8% （NO2--N）, and 93.6% （total nitrogen）. During the starvation, extracellular polymeric substances wrapped the ANAMMOX bacteria and kept them intact to resist long-term starvation stress. The expression of nirS, hzsA, and hdh genes ensured the synthesis of nitrite/nitric oxide oxidoreductase, hydrazine synthase, and hydrazine dehydrogenase to maintain ANAMMOX activity. There was no significant difference in the relative abundance of ANAMMOX bacteria before and after starvation recovery. Candidatus Kuenenia had better anti-hunger ability, and the relative abundance increased by more than 86% after 30 days of recovery, confirming its tolerance to long-term starvation.

Ferroheme/Ferriheme Directly Involved in the Synthesis and Decomposition of Hydrazine as an Electron Carrier during Anammox.

Anammox bacteria performed the reaction of NH4+ and NO with hydrazine synthase to produce N2H4, followed by the decomposition of N2H4 with hydrazine dehydrogenase to generate N2. Ferroheme/ferriheme, which serves as the active center of both hydrazine synthase and hydrazine dehydrogenase, is thought to play a crucial role in the synthesis and decomposition of N2H4 during Anammox due to its high redox activity. However, this has yet to be proven and the exact mechanisms by which ferroheme/ferriheme is involved in the Anammox process remain unclear. In this study, abiotic and biological assays confirmed that ferroheme participated in NH4+ and NO reactions to generate N2H4 and ferriheme, and the produced N2H4 reacted with ferriheme to generate N2 and ferroheme. In other words, the ferroheme/ferriheme cycle drove the continuous reaction between NH4+ and NO. Raman, ultraviolet-visible spectroscopy, and X-ray absorption fine structure spectroscopy confirmed that ferroheme/ferriheme is involved in the synthesis and decomposition of N2H4 via the core FeII/FeIII cycle. The mechanism of ferroheme/ferriheme participation in the synthesis and decomposition of N2H4 was proposed by density functional theory calculations. These findings revealed for the first time the heme electron transfer mechanisms, which are of great significance for deepening the understanding of Anammox.

Interspecies electron transfer and microbial interactions in a novel Fe(II)-mediated anammox coupled mixotrophic denitrification system

This study effectively coupled anammox and mixotrophic denitrification at a high nitrogen load rate of 6.84 g N/L/d with 40 mg/L Fe(II). Fe(II) enhanced the activity of nitrate reductase, nitrite reductase, and hydrazine dehydrogenase enzymes, facilitating accelerated ATP synthesis. Through electrochemical experiments, interspecies electron transfer processes in coupled system were explored. Fe(II) promoted flavin mononucleotide secretion, enhancing electron-donating and electron-accepting capacity by 2.8 and 1.3 times, respectively. Fe(II) triggered the enrichment of autotrophic denitrifying bacteria (Azospira and Hydrogenophaga), transitioning from single organic nutrient to mixotrophic denitrification. Meanwhile, Fe(II) increased Candidatus_Kuenenia abundance from 35.2 % to 49.0 %, establishing the competitive advantage of anammox bacteria over completed denitrifying bacteria (Comamonas). The synergistic interactions between anammox and various denitrification pathways achieved a nitrogen removal rate of 5.88 g N/L/d, with anammox contribution rate of 88.3 %. This study provides insights into broadening the application of partial denitrification /anammox and electron transfer in multi-bacterial coupling systems.

Microbial divergence and evolution. The case of anammox bacteria.

Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future.

Unraveling potential mechanism of different metal ions effect on anammox through big data analysis, molecular docking and molecular dynamics simulation

Heavy metals (HMs) have been widely reported to pose an adverse effect on anaerobic ammonia oxidation (anammox) bacteria, yet the underlying mechanisms remain unclear. This study provides new insights into the potential mechanisms of interaction between HMs and functional enzymes through big date analysis, molecular docking and molecular dynamics simulation. The statistical analysis indicated that 10 mg/L Cu(II) and Cd(II) reduced nitrogen removal rate (NRR) by 85% and 43%, while 5 mg/L Fe(II) enhanced NRR by 29%. Additionally, the results of molecular simulations provided a microscopic interpretation for these macroscopic data. Molecular docking revealed that Hg(II) formed a distinctive binding site on ferritin, while other HMs resided at iron oxidation sites. Furthermore, HMs exhibited distinct binding sites on hydrazine dehydrogenase. Concurrently, the molecular dynamics simulation results further substantiated their capacity to form complexes. Cu(II) displayed the strongest binding affinity with ferritin for −1576 ± 79 kJ/mol in binding free energy calculation. Moreover, Cd(II) bound to ferritin and HDH for −1052.67 ± 58.49 kJ/mol, −290.02 ± 49.68 kJ/mol, respectively. This research addressed a crucial knowledge gap, shedding light on potential applications for remediating heavy metal-laden industrial wastewater.

The roles of different Fe-based materials in alleviating the stress of Cr(VI) on anammox activity: Performance and mechanism

Hexavalent chromium Cr(VI) is widely present in industrial wastewater, which can inhibit anammox performance significantly. Iron is a critical element of anammox metabolism and also has the capacity to remove Cr(VI). In this study, batch experiments were conducted to investigate the effects of commercial zero-valent iron (Fe0) and ferroferric oxide (Fe3O4) in the nano-(n) and micron-(m) scales (mFe0, nFe0, mFe3O4, and nFe3O4, respectively) on recovering anammox activity with inhibition of Cr(VI). Their recovery efficiencies on the anammox process were in the order of mFe0, mFe3O4, nFe0, and nFe3O4 at their minimum effective dosages, with specific anammox activities being promoted by 95.2%, 57.0%, 45.0%, and 15.8%, respectively, in comparison to the control. More hydroxyl groups in extracellular polymeric substances (EPS) were observed with mFe0, and the electron transport performances of soluble and loosely-bound EPS were particularly enhanced. By contrast, Fe3O4 stimulated more production of EPS than mFe0, which helped remove Cr(VI) outside the cell membrane. Moreover, the contents of heme c and cytochrome c were improved most with mFe3O4 by 10.9% and 41.3%, respectively, and hydrazine dehydrogenase activity was enhanced by 47.7% with mFe0, at their optimal dosages. In addition, cytochrome c showed a significant positive correlation with recovery performance for all four materials (R2 > 0.82, P < 0.05), indicating its pivotal role in alleviation effectiveness. More FeOOH was found on the two micron-scale materials detected by X-ray diffractometry and Fourier transform infrared spectroscopy, which would help electron transfer between materials and anammox bacteria. In contrast, the fast corrosion and adsorption processes and biological toxicity of nanoscale materials may lead to their poor performance.

Efficient nitrogen removal from municipal wastewater by an autotrophic-heterotrophic coupled anammox system: The up-regulation of key functional genes

The metabolic pathways based on key functional genes were innovatively revealed in the autotrophic-heterotrophic coupled anammox system for real municipal wastewater treatment. The nitrogen removal performance of the system was stabilized at 88.40 ± 3.39 % during the treatment of real municipal wastewater. The relative abundances of the nitrification functional genes ammonia oxidase (amoA/B/C), hydroxylamine oxidoreductase (hao), and nitrite oxidoreductases (nxrA/B) were increased by 1.2–2.4 times, and these three nitrification functional genes were mostly contributed by Nitrospira that dominated the efficient nitrification of the system. The relative abundance of anammox bacteria Candidatus Brocadia augmented from 0.35 % to 0.75 %, accompanied with the increased expression of hydrazine synthase (hzs) and hydrazine dehydrogenase (hdh), resulting in the major role of anammox (81.24 %) for nitrogen removal. The expression enhancement of the functional genes nitrite reductase (narG/H, napA/B) that promoted partial denitrification (PD) of the system weakened the adverse effects of the sharp decline in the population of PD microbe Thauera (from 5.7 % to 2.2 %). The metabolic module analysis indicated that the carbon metabolism pathways of the system mainly included CO2 fixation and organic carbon metabolism, and the stable enrichment of autotrophic bacteria ensured stable CO2 fixation. Furthermore, the enhanced expression of the glucokinases (glk, GCK, HK, ppgk) and the abundant pyruvate kinase (PK) achieved stable hydrolysis ability of organic carbon metabolism function of the system. This study offers research basics to practical application of the mainstream anammox process.

Enhanced anammox through endogenous partial denitrification/anammox (EPDA) granular sludge: Granule formation, treatment performance, and multi-perspective microbial analysis

The direct application of anammox to wastewater treatment is valuable, but the enrichment of anammox bacteria (AnAOB) is complicated due to the malignant competition of heterotrophic bacteria. Here, we propose the use of endogenous partial denitrification/anammox (EPDA) granular sludge to enhance anammox while achieving simultaneous removals of nitrogen and phosphorus. After operation for 330 days, the EPDA granules were successfully cultured with the mean size of 0.89 mm. Nitrogen and phosphorus were efficiently removed with the effluent total nitrogen (TN) and PO43--P of 6.68±0.41 mg/L and 0.06±0.03 mg/L, respectively. Anammox took a leading role in the removal of TN with a contribution of 75±3%. Moreover, high-throughput sequencing (16S rRNA) results showed that AnAOB could coexist harmoniously with glycogen accumulating organisms (GAOs) and polyphosphate accumulating organisms (PAOs) in the EPDA granules, whereas mature EPDA granules could promote in situ enrichment of AnAOB. Microbial stratification analysis of the EPDA granule found that the inner layer was resided with AnAOB, while the outer layer was employed with GAOs and PAOs. Metagenomic and metatranscriptomic analyses found that the nitrate reductase (1150.56 RPM) was 4.6 times of nitrite reductase (249.98 RPM) at the transcriptome level. Moreover, the key enzymes related to anammox (hydrazine synthase and hydrazine dehydrogenase) were much higher than the other nitrogen conversion functional genes at the transcriptome level, confirming the main role of AnAOB in the removal of nitrogen. This study presents a novel strategy for the application of anammox from EPDA granular sludge.

Effects of reducing, stabilizing, and antibiotic agents on “Candidatus Kuenenia stuttgartiensis”

Anaerobic ammon ium oxidizing (anammox) bacteria oxidize ammonium and reduce nitrite, producing N2, and could play a major role in energy-optimized wastewater treatment. However, sensitivity to various environmental conditions and slow growth currently hinder their wide application. Here, we attempted to determine online the effect of environmental stresses on anammox bacteria by using an overnight batch activity test with whole cells, in which anammox activity was calculated by quantifying N2 production via headspace-pressure monitoring. A planktonic mixed culture dominated by “Candidatus Kuenenia stuttgartiensis” strain CSTR1 was cultivated in a 30-L semi-continuous stirring tank reactor. In overnight resting-cell anammox activity tests, oxygen caused strong inhibition of anammox activity, which was reversed by sodium sulfite (30 µM). The tested antibiotics sulfamethoxazole, kanamycin, and ciprofloxacin elicited their effect on a dose-dependent manner; however, strain CSTR1 was highly resistant to sulfamethoxazole. Anammox activity was improved by activated carbon and Fe2O3. Protein expression analysis from resting cells after anammox activity stimulation revealed that NapC/NirT family cytochrome c (KsCSTR_12840), hydrazine synthase, hydrazine dehydrogenase, hydroxylamine oxidase, and nitrate:nitrite oxidoreductase were upregulated, while a putative hydroxylamine oxidoreductase HAO (KsCSTR_49490) was downregulated. These findings contribute to the growing knowledge on anammox bacteria physiology, eventually leading to the control of anammox bacteria growth and activity in real-world application.Key Points• Sulfite additions can reverse oxygen inhibition of the anammox process• Anammox activity was improved by activated carbon and ferric oxide• Sulfamethoxazole marginally affected anammox activityGraphical abstract

Living on hydrazine: Metabolic protein complexes from an anaerobic ammonium oxidizer.

The discovery of anammox bacteria in the 1990s has dramatically changed our understanding of the global nitrogen cycle. Anammox bacteria are now believed to be responsible for up to 30 to 70% of the nitrogen removal from the oceans. These organisms have the unique metabolic ability to combine ammonium and nitrite to form dinitrogen gas, a process that takes place in a special cellular compartment, the anammoxosome. To elucidate how bacteria perform such extraordinary chemistry, we have determined the structures of the key enzymes in this process. Central to harvesting the energy from hydrazine is the hydrazine dehydrogenase (HDH), which converts hydrazine into dinitrogen gas, liberating four extremely low-potential electrons (−750 mV). Our crystal and cryo-EM structures reveal that this 1.7-MDa complex contains an extended system of 192 heme groups spanning the entire complex, which is only accessible via narrow holes in the side of the complex. Moreover, we identified an unexpected assembly factor for this complex. In addition, anammox bacteria obtain additional reducing equivalents through the oxidation of nitrite to nitrate, which is catalyzed by a nitrite oxidoreductase (NXR) related to the NXR from nitrifying bacteria. Despite its importance in the biogeochemical nitrogen cycle, essential issues on NXR functions remain unanswered, particularly due to the lack of structural information. To meet this challenge, we used a multiscale approach combining cryo-electron tomography, crystallography, single-particle cryo-EM together with reconstitution studies and enzyme kinetics to characterize NXR. We show that, in contrast to what was shown for NXR in NOB, NXR of anammox bacteria forms tubule-like structures inside the anammoxosome held together by a novel subunit NXR-T. As with the hydrazine dehydrogenase structure, our multiscale structure of the anammox NXR tubules suggest how electrons are passed on to redox partners.

Fe(Ⅱ) improving sulfurized Anammox coupled with autotrophic denitrification performance: Based on interspecies and intracellular electron transfer

Insufficient nitrite supply and slow metabolism of Anammox bacteria (AnAOB) impeded the application of Anammox process in low level ammonia (LLA) (≤50 mg/L) wastewater. At the initial concentration of 50 mg/L NH4+-N and 75 mg/L NO3–-N, Fe(Ⅱ) (10 mg/L) promoted the total nitrogen removal efficiency from 80.79 to 94.92 % by core–shell sulfurized AnAOB coupled with sulfur oxidizing bacteria (S0@AnAOB + SOB). AnAOB outcompeted SOB for nitrite, because the addition of Fe(Ⅱ) not only increased the nitrate reductase activity (37.54 %), but also enhanced the metabolism and electron capture ability of AnAOB, which was highly related with energy metabolic process: hydrazine dehydrogenase activity increased to 139.00 %. Particularly, Fe(Ⅱ) accelerated the interspecies electron transfer (INET) (from SOB to AnAOB) by stimulating the secretion of redox species and electron hopping in EPS. This study shed light on the mechanism of Fe(Ⅱ) promoting electron transfer in S0@AnAOB + SOB system, and provided basis for engineering practice.

Activity enhancement and the anammox mechanism under low temperature via PVA-SA and nano Fe2O3-PVA-SA entrapped beads

Anaerobic ammonia-oxidizing bacteria (AAOB) have a long growth time and low activity at low temperatures. In suspended systems, sludge is easily lost, which limits the mainstream application of anaerobic ammonia oxidation (anammox).Entrapment provides effective ideas for solving these problems. In this study, polyvinyl‑sodium alginate (PVA-SA) and nano Fe2O3-PVA-SA entrapment beads were prepared to discuss the effectiveness of entrapment enhanced anammox sludge at low temperatures. The differences in the entrapped beads and granules were compared to analyze the strengthening mechanism. The results show that the nitrogen removal performance of granules, PVA-SA and nano Fe2O3-PVA-SA entrapped beads, first decreased and then increased during the cooling and low-temperature operation. Nano Fe2O3-PVA-SA entrapped beads showed the smallest decline and the highest degree of recovery. Reaction metering ratio (△NO2−-N/△NH4+-N and △NO3−-N/△NH4+-N) showed that entrapment could realize Nitrite oxidizing bacteria (NOB) inhibition and improve the activity of denitrifying bacteria (DNB) to promote the removal of total nitrogen by providing a strict anaerobic environment. The results demonstrate that entrapment is beneficial for maintaining the content of heme c, specifically, nano Fe2O3 can stimulate its production, and is beneficial for alleviating the reduction of hydrazine dehydrogenase (HDH) enzyme activity. The extracellular polymeric substances (EPS) content and analysis showed that entrapment does not change the composition of EPS, and can maintain the EPS content. Nano Fe2O3 can stimulate AAOB to secrete more EPS to maintain sludge stability. From a molecular perspective, entrapment can maintain the expression of functional genes, promote the enrichment of AAOB, thus improving the nitrogen removal performance from the dual perspectives of “quality” and “quantity”.

Novel strategy for rapid start-up and stable operation of anammox: Negative pressure coupled with the direct-current electric field

An application challenge of anaerobic ammonia oxidation (anammox) is the slow proliferation rate of anaerobic ammonium oxidation bacteria (AnAOB). This study adopted negative pressure coupled with the direct-current electric field (NP–DCEF) to evaluate system nitrogen removal performance. Results showed that the total nitrogen removal rate (TNRR) of the NP–DCEF system was stable at 88.6% after seven days. Compared with that of the ordinary operating system (45.4%), the relative abundance of Candidatus–kuenenia considerably increased from 51.9% to 57.6%. Under transient and long-term influent fluctuation, the NP–DCEF system showed high nitrogen removal performance. The specific activity of AnAOB (SAA) reached 11.0 mg N∙g Vss−1 h−1 under load fluctuation, and it was 8.7 mg N∙g Vss−1 h−1 under ordinary operational conditions. In addition, the specific activities of hydrazine dehydrogenase (HDH) and hydrazine synthetase (HZS) reached 32.66 and 92.95 U∙L−1, which are considerably higher than those under the ordinary operating conditions (18.41 and 63.20 U∙L−1). These results indicated that the novel operation strategy has specific feasibility and potential for the start-up and long-term operation of anammox.

Autotrophic biological nitrogen removal in a bacterial-algal symbiosis system: Formation of integrated algae/partial-nitrification/anammox biofilm and metagenomic analysis

Bacterial and algal symbiosis systems have been gradually applied in wastewater treatment. In this study, an integrated algal/partial nitrification/anammox biofilm system (IAPNABS) was constructed through light cultivation. The diverse biofilm was formed under the light, which was demonstrated by metagenomic analysis. The external biofilm at the steady stage was dominated by algae and nitrifying bacteria. Specifically, Geitlerinema and Chlorella were the two main species of algae, accounting for 6.0% and 1.9%, respectively; Nitrosomonas_europaea was the dominant species of nitrifying bacteria (2.8%). Anammox bacteria were mainly found in the inner biofilm, with Candidatus Kuenenia Stuttgartiensis and Candidatus Brocadiaceae Bacterium B188 representing the dominant species (1.0% and 0.3%, respectively). This layered biofilm performed efficient nitrogen removal. When initial NH4+-N in the system was 100 ± 20 mg/L, the IAPNABS achieved highest NH4+-N and TN removal rate (8.9 mg/(L·h) and 6.3 mg/(L·h), respectively) after 8 h of light and 2 h of dark. The oxygen produced by the algae was consumed by nitrifying bacteria, which provided the anaerobic environment and the nitrite substrate to maintain the activity of anammox bacteria. The abundance of the key enzymes involved in nitrogen removal (e.g. ammonia monooxygenase (Amo), hydrazine dehydrogenase (Hdh) and hydrazine synthase (Hzs)) further demonstrated main nitrogen metabolic processes in the layered biofilm. This study provides a new idea for autotrophic nitrogen removal from wastewater, which can greatly reduce the additional oxygen supply requirement.

Influence of temperature on anammox reaction and microbial diversity in a bio-carriers reactor under mainstream conditions

The hypersensitive with seasonal temperature fluctuation of anaerobic ammonium-oxidizing bacteria has usually limited the actual implementation of the anammox process. Thus far, temperature influences on anammox reaction and microbial consortium structure under mainstream conditions was not thoroughly studied. In this study, hydrazine dehydrogenase (HDH) activity and specific activity of anammox bacteria were studied in a 10 L bench scale Continuous Anammox Bio-carrier Reactor (CABR) at nitrogen loading rates of 0.2–0.4 kg N/m3 ⋅ d and a temperature range from 18 °C to 35 °C. At 35 °C, CABR achieved nitrogen removal efficiency above 94% and the nitrogen removal rate of 0.25–0.3 kg N/m3 ⋅ d. When the temperature decreased to 18 °C, the nitrogen removal efficiency and nitrogen removal rate reduced 62.2% and 0.1 kg N/m3 ⋅ d, respectively due to 3.5-folds reduced HDH activity. At 10 °C, specific activity of anammox bacteria declined to 0.02 g N/gVSS ⋅ d, which was 1/7 of that at 35 °C. Anammox reaction could recover when temperature increased to 23 °C with NRE of 85%. It revealed that temperature directly impacted HDH activity was a key parameter influencing anammox metabolism. Activation energy (Ea) calculated for specific activity of anammox bacteria at 10–18 °C and 10–35 °C were approximately 120 kJ/mol and 57 kJ/mol, respectively. Also, 16S rDNA sequencing results revealed that C. Jettenia well adapted to temperature-varying conditions, which implied the CABR could be a favorable process for anammox applications with seasonal temperature change in sub-tropical and tropical climate regions.