Anammox bacteria drive fixed nitrogen loss in hadal trench sediments.

Benthic N2 production by microbial denitrification and anammox is the largest sink for fixed nitrogen in the oceans. Most N2 production occurs on the continental shelves, where a high flux of reactive organic matter fuels the depletion of nitrate close to the sediment surface. By contrast, N2 production rates in abyssal sediments are low due to low inputs of reactive organics, and nitrogen transformations are dominated by aerobic nitrification and the release of nitrate to the bottom water. Here, we demonstrate that this trend is reversed in the deepest parts of the oceans, the hadal trenches, where focusing of reactive organic matter enhances benthic microbial activity. Thus, at ∼8-km depth in the Atacama Trench, underlying productive surface waters, nitrate is depleted within a few centimeters of the sediment surface, N2 production rates reach those reported from some continental margin sites, and fixed nitrogen loss is mainly conveyed by anammox bacteria. These bacteria are closely related to those known from shallow oxygen minimum zone waters, and comparison of activities measured in the laboratory and in situ suggest they are piezotolerant. Even the Kermadec Trench, underlying oligotrophic surface waters, exhibits substantial fixed N removal. Our results underline the role of hadal sediments as hot spots of deep-sea biological activity, revealing a fully functional benthic nitrogen cycle at high hydrostatic pressure and pointing to hadal sediments as a previously unexplored niche for anaerobic microbial ecology and diagenesis.

Isolation and characteristics of two heterotrophic nitrifying and aerobic denitrifying bacteria, Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6.

In order to solve nitrogen pollution in environmental water, two heterotrophic nitrifying and aerobic denitrifying strains isolated from acid paddy soil were identified as Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6 respectively. Strain HNDS-1 and strain HNDS-6 exhibited amazing ability to nitrogen removal. When (NH4)2SO4, KNO3, NaNO2 were used as nitrogen resource respectively, the NH4+-N, NO3--N, NO2--N removal efficiencies of strain HNDS-1 were 93.31%, 89.47%, and 100% respectively, while those of strain HNDS-6 were 82.39%, 96.92%, and 100%. And both of them could remove mixed nitrogen effectively in low C/N (C/N = 5). Strain HNDS-1 could remove 76.86% NH4+-N and 75.13% NO3--N. And strain HNDS-6 can remove 65.07% NH4+-N and 78.21% NO3--N. A putative ammonia monooxygenase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein and nitric oxide reductase of strain HNDS-1, while hydroxylamine reductase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein, and nitric oxide reductase of strain HNDS-6 were identified by genomic analysis. DNA-SIP analysis showed that genes Nxr, narG, nirK, norB, nosZ were involved in nitrogen removal pathway, which indicates that the denitrification pathway of strain HNDS-1 and strain HNDS-6 was NO3-→NO2-→NO→N2O→N2 during NH4+-N removal process. And the nitrification pathway of strain HNDS-1 and strain HNDS-6 was NO2-→NO3-, but the nitrification pathway of NH4+→ NO2- needs further studies.

Impact of anodophilic biofilm bioelectroactivity on the denitrification behavior of air-cathode microbial fuel cells.

Generally, high bioelectroactivity of anodophilic biofilm favors high power generation of microbial fuel cell (MFC); however, it is not clear whether it can promote denitrification of MFC synchronously. In this study, we studied the impact of anodophilic biofilm bioelectroactivity on the denitrification behavior of air-cathode MFC (AC-MFC) in steady state and found that high bioelectroactivity of anodophilic biofilm not only favored high power generation of the AC-MFC, but also promoted the growth of denitrifers at the anodes and strengthened denitrification. Anodophilic biofilms of AC-MFC with various bioelectroactivity were acclimated at conditions of open circuit (OC), Rext of 1000 Ω and 20 Ω (denoted as AC-MFC-OC, AC-MFC-1000Ω, and AC-MFC-20Ω, respectively) and performed for over 100 days. Electrochemical tests and microbial analysis results showed that the anode of the AC-MFC-20Ω delivered higher current response of both oxidation and denitrification and had higher abundance of electroactive bacteria than the AC-MFC-OC, AC-MFC-1000Ω, demonstrating a higher bioelectroactivity of the anodophilic biofilms. Moreover, these electroactive bacteria favored the accumulation of denitrifers, like Thauera and Alicycliphilus, probably by consuming trace oxygen through catalyzing oxygen reduction. The AC-MFC-20Ω not only delivered a 61.7% higher power than the AC-MFC-1000Ω, but also achieved a stable and high denitrification rate constant (kDN ) of 1.9 h-1 , which was 50% and 40% higher than that of the AC-MFC-OC and AC-MFC-1000Ω, respectively. It could be concluded that the high bioelectroactivity of the anodophilic biofilms not only favored high power generation of the AC-MFC, but also promoted the enrichment of denitrifers at the anodes and strengthened denitrification. This study provided an effective method for enhancing power generation and denitrification performance of the AC-MFC synchronously.

Sulfur-driven autotrophic denitrification of nitric oxide for efficient nitrous oxide recovery.

Nitrous oxide (N2 O) was previously deemed as a potent greenhouse gas but is actually an untapped energy source, which can accumulate during the microbial denitrification of nitric oxide (NO). Compared with the organic electron donor required in heterotrophic denitrification, elemental sulfur (S0 ) is a promising electron donor alternative due to its cheap cost and low biomass yield in sulfur-driven autotrophic denitrification. However, no effort has been made to test N2 O recovery from sulfur-driven denitrification of NO so far. Therefore, in this study, batch and continuous experiments were carried out to investigate the NO removal performance and N2 O recovery potential via sulfur-driven NO-based denitrification under various Fe(II)EDTA-NO concentrations. Efficient energy recovery was achieved, as up to 35.5%-40.9% of NO was converted to N2 O under various NO concentrations. N2 O recovery from Fe(II)EDTA-NO could be enhanced by the low bioavailability of sulfur and the acid environment caused by sulfur oxidation. The NO reductase (NOR) and N2 O reductase (N2 OR) were inhibited distinctively at relatively low NO levels, leading to efficient N2 O accumulation, but were suppressed irreversibly at NO level beyond 15 mM in continuous experiments. Such results indicated that the regulation of NO at a relatively low level would benefit the system stability and NO removal capacity during long-term system operation. The continuous operation of the sulfur-driven Fe(II)EDTA-NO-based denitrification reduced the overall microbial diversity but enriched several key microbial community. Thauera, Thermomonas, and Arenimonas that are able to carry out sulfur-driven autotrophic denitrification became the dominant organisms with their relative abundance increased from 25.8% to 68.3%, collectively.

Microbial ecosystem dynamics drive fluctuating nitrogen loss in marine anoxic zones.

The dynamics of nitrogen (N) loss in the ocean's oxygen-deficient zones (ODZs) are thought to be driven by climate impacts on ocean circulation and biological productivity. Here we analyze a data-constrained model of the microbial ecosystem in an ODZ and find that species interactions drive fluctuations in local- and regional-scale rates of N loss, even in the absence of climate variability. By consuming O2 to nanomolar levels, aerobic nitrifying microbes cede their competitive advantage for scarce forms of N to anaerobic denitrifying bacteria. Because anaerobes cannot sustain their own low-O2 niche, the physical O2 supply restores competitive advantage to aerobic populations, resetting the cycle. The resulting ecosystem oscillations induce a unique geochemical signature within the ODZ-short-lived spikes of ammonium that are found in measured profiles. The microbial ecosystem dynamics also give rise to variable ratios of anammox to heterotrophic denitrification, providing a mechanism for the unexplained variability of these pathways observed in the ocean.

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa.

Many proteobacteria, such as Escherichia coli, contain two main types of quinones: benzoquinones, represented by ubiquinone (UQ) and naphthoquinones, such as menaquinone (MK), and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been discovered recently in E. coli Its physiological relevance is not yet understood, because MK and DMK are also present in E. coli Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV, and ubiU genes, respectively, are essential for UQ9 biosynthesis and, thus, for denitrification in P. aeruginosa These three genes here are called ubiTPa , ubiVPa , and ubiUPa We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for the pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a target for antibiotics development to manage P. aeruginosa infections.

nir gene-based co-occurrence patterns reveal assembly mechanisms of soil denitrifiers in response to fire.

Denitrification causes nitrogen losses from terrestrial ecosystems. The magnitude of nitrogen loss depends on the prevalence of denitrifiers, which show ecological differences if they harbour nirS or nirK genes encoding nitrite reductases with the same biological function. Thus, it is relevant to understand the mechanisms of co-existence of denitrifiers, including their response to environmental filters and competition due to niche similarities. We propose a framework to analyse the co-existence of denitrifiers across multiple assemblages by using nir gene-based co-occurrence networks. We applied it in Mediterranean soils before and during 1 year after an experimental fire. Burning did not modify nir community structure, but significantly impacted co-occurrence patterns. Bacteria with the same nir co-occurred in space, and those with different nir excluded each other, reflecting niche requirements: nirS abundance responded to nitrate and salinity, whereas nirK to iron content. Prior to fire, mutual exclusion between bacteria with the same nir suggested competition due to niche similarities. Burning provoked an immediate rise in mineral nitrogen and erased the signals of competition, which emerged again within days as nir abundances peaked. nir co-occurrence patterns can help infer the assembly mechanisms of denitrifying communities, which control nitrogen losses in the face of ecological disturbance.

A bet-hedging strategy for denitrifying bacteria curtails their release of N2O.

When oxygen becomes limiting, denitrifying bacteria must prepare for anaerobic respiration by synthesizing the reductases NAR (NO3- → NO2-), NIR (NO2- → NO), NOR (2NO → N2O), and NOS (N2O → N2), either en bloc or sequentially, to avoid entrapment in anoxia without energy. Minimizing the metabolic burden of this precaution is a plausible fitness trait, and we show that the model denitrifier Paracoccus denitrificans achieves this by synthesizing NOS in all cells, while only a minority synthesize NIR. Phenotypic diversification with regards to NIR is ascribed to stochastic initiation of gene transcription, which becomes autocatalytic via NO production. Observed gas kinetics suggest that such bet hedging is widespread among denitrifying bacteria. Moreover, in response to oxygenation, P. denitrificans preserves NIR in the poles of nongrowing persister cells, ready to switch to anaerobic respiration in response to sudden anoxia. Our findings add dimensions to the regulatory biology of denitrification and identify regulatory traits that decrease N2O emissions.

Sulphide addition favours respiratory ammonification (DNRA) over complete denitrification and alters the active microbial community in salt marsh sediments.

The balance between nitrate respiration pathways, denitrification and dissimilatory nitrate (NO3 - ) reduction to ammonium (DNRA), determines whether bioavailable nitrogen is removed as N2 gas or recycled as ammonium. Saltwater intrusion and organic matter enrichment may increase sulphate reduction leading to sulphide accumulation. We investigated the effects of sulphide on the partitioning of NO3 - between complete denitrification and DNRA and the microbial communities in salt marsh sediments. Complete denitrification significantly decreased with increasing sulphide, resulting in an increase in the contribution of DNRA to NO3 - respiration. Alternative fates of NO3 - became increasingly important at higher sulphide treatments, which could include N2 O production and/or transport into intracellular vacuoles. Higher 16S transcript diversity was observed in the high sulphide treatment, with clear shifts in composition. Generally, low and no sulphide, coupled with high NO3 - , favoured the activity of Campylobacterales, Oceanospirillales and Altermonadales, all of which include opportunistic denitrifiers. High ∑sulphide conditions promoted the activity of potential sulphide oxidizing nitrate reducers (Desulfobulbaceae, Acidiferrobacteraceae and Xanthomonadales) and sulphate reducers (Desulfomonadaceae, Desulfobacteraceae). Our study highlights the tight coupling between N and S cycling, and the implications of these dynamics on the fate of bioavailable N in coastal environments susceptible to intermittent saltwater inundation and organic matter enrichment.

Dynamics of nitric oxide controlled by protein complex in bacterial system.

Nitric oxide (NO) plays diverse and significant roles in biological processes despite its cytotoxicity, raising the question of how biological systems control the action of NO to minimize its cytotoxicity in cells. As a great example of such a system, we found a possibility that NO-generating nitrite reductase (NiR) forms a complex with NO-decomposing membrane-integrated NO reductase (NOR) to efficiently capture NO immediately after its production by NiR in anaerobic nitrate respiration called denitrification. The 3.2-Å resolution structure of the complex of one NiR functional homodimer and two NOR molecules provides an idea of how these enzymes interact in cells, while the structure may not reflect the one in cells due to the membrane topology. Subsequent all-atom molecular dynamics (MD) simulations of the enzyme complex model in a membrane and structure-guided mutagenesis suggested that a few interenzyme salt bridges and coulombic interactions of NiR with the membrane could stabilize the complex of one NiR homodimer and one NOR molecule and contribute to rapid NO decomposition in cells. The MD trajectories of the NO diffusion in the NiR:NOR complex with the membrane showed that, as a plausible NO transfer mechanism, NO released from NiR rapidly migrates into the membrane, then binds to NOR. These results help us understand the mechanism of the cellular control of the action of cytotoxic NO.

Iron as electron donor for denitrification: The efficiency, toxicity and mechanism.

For the treatment of low C/N wastewaters, methanol or acetate is usually dosed as electron donor for denitrification but such organics makes the process costly. To decrease the cost, iron which is the fourth most abundant element in lithosphere is suggested as the substitution of methanol and acetate. The peak volumetric removal rate (VRR) of nitrate nitrogen in the ferrous iron-dependent nitrate removal (FeNiR) reactor was 0.70 ± 0.04 kg-N/(m3·d), and the corresponding removal efficiency was 98%. Iron showed toxicity to cells by decreasing the live cell amount (dropped 56%) and the live cell activity (dropped 70%). The toxicity of iron was mainly expressed by the formation of iron encrustation. From microbial community data analysis, heterotrophs (Paracocccus, Thauera and Azoarcus) faded away while the facultative chemolithotrophs (Hyphomicrobium and Anaerolineaceae_uncultured) dominated in the reactor after replacing acetate with ferrous iron in the influent. Through scanning electron microscope (SEM) and transmission electron microscope (TEM), two iron oxidation sites in FeNiR cells were observed and accordingly two FeNiR mechanisms were proposed: 1) extracellular FeNiR in which ferrous iron was bio-oxidized extracellularly; and 2) intracellular FeNiR in which ferrous iron was chemically oxidized in periplasm. Bio-oxidation (extracellular FeNiR) and chemical oxidation (intracellular FeNiR) of ferrous iron coexisted in FeNiR reactor, but the former one predominated. Comparing with the control group without electron donor in the influent, FeNiR reactor showed 2 times higher and stable nitrate removal rate, suggesting iron could be used as electron donor for denitrification. However, further research works are still needed for the practical application of FeNiR in wastewater treatment.

Simultaneous nitrification-denitrification by phosphate accumulating microorganisms.

Nitrogen and phosphorous are important inorganic water pollutants that pose a major threat to the environment and health of both humans and animals. The physical and chemical ways to remove these pollutants from water and soil are expensive and harsh, so biological removal becomes the method of choice to alleviate the problem without any side effects. The identification of microorganisms capable of simultaneous heterotrophic nitrification and aerobic denitrification has greatly simplified the sequestration of nitrogen from ammonium (NH4+) into dinitrogen (N2). Further, the discovery of phosphorous accumulating organisms offers greater economic benefits because these organisms can favourably and simultaneously remove both nitrogen and phosphorous from wastewaters hence reducing the nutrient burden. The stability of the system and removal efficiency of inorganic pollutants can be enhanced by the use of immobilized organisms. However, limited work has been done so far in this direction and there is a need to further the efforts towards refining process efficiency by testing low-cost substrates and diverse microbial populations for the total eradication of these contaminants from wastewaters.

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes, with implications for their release of nitrogenous gases.

Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Three reported haloarchaeal denitrifiers, Haloferax mediterranei, Haloferax denitrificans and Haloferax volcanii, were characterized with respect to their denitrification phenotype. A semi-automatic incubation system was used to monitor the depletion of electron acceptors and accumulation of gaseous intermediates in batch cultures under a range of conditions. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N2 , while the other two released significant amounts of NO and N2 O, which affect tropospheric and stratospheric chemistries respectively. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation. This work is the first detailed physiological study of denitrification in haloarchaea, and as such a seed for our understanding of the drivers of nitrogen turnover in hypersaline systems.

A metatranscriptomics-based assessment of small-scale mixing of sulfidic and oxic waters on redoxcline prokaryotic communities.

Lateral intrusions of oxygen caused by small-scale mixing are thought to shape microbial activity in marine redoxclines. To examine the response of prokaryotes to such mixing events we employed a shipboard mixing experiment in the euxinic central Baltic Sea: oxic, nitrate containing and sulfidic water samples without detectable oxygenized substances were incubated directly or after mixing. While nitrate, nitrite and ammonium concentrations stayed approximately constant in all incubations, we observed a decrease of sulfide after the contact with oxygen in the sulfide containing incubations. The transcription of marker genes from chemolithoauthotrophic key players including archaeal nitrifiers as well as gammaproteobacterial and campylobacterial autotrophic organisms that couple denitrification with sulfur-oxidation were followed at four time points within 8.5 h. The temporally contrasting transcriptional profiles of gammaproteobacterial and campylobacterial denitrifiers that depend on the same inorganic substrates pointed to a niche separation. Particular archaeal and campylobacterial marker genes involved in nitrification, denitrification and sulfur oxidation, which depend on oxidized substrates, were highly upregulated in the anaerobic sulfidic samples. We suggest that, despite the absence of measurable oxygenated compounds in the sulfidic water, frequent intermittent small-scale intrusions stimulate the permanent upregulation of genes involved in nitrification, denitrification and sulfur oxidation.

Ectomycorrhizal fungi inoculation alleviates simulated acid rain effects on soil ammonia oxidizers and denitrifiers in Masson pine forest.

Acid rain can cause severe effects on soil biota and nutrient biogeochemical cycles in the forest ecosystem, but how plant-symbiotic ectomycorrhizal fungi will modulate the effects remains unknown. Here, we conducted a full factorial field experiment in a Masson pine forest by simultaneously controlling the acidity of the simulated rain (pH 5.6 vs. pH 3.5) and the ectomycorrhizal fungi Pisolithus tinctorius inoculation (non-inoculation vs. inoculation), to investigate the effects on ammonia oxidizers and denitrifiers. After 10 months, compared with the control (rain pH 5.6, and non-inoculation), simulated acid rain (pH 3.5) reduced soil nutrient content, decreased archaeal amoA gene abundance and inhibited denitrification enzyme activity. Also, simulated acid rain altered the community compositions of all the examined functional genes (archaeal amoA, bacterial amoA, nirK, nirS and nosZ). However, inoculation with ectomycorrhizal fungi under acid rain stress recovered soil nutrient content, archaeal amoA gene abundance and denitrification enzyme activity to levels comparable to the control, suggesting that ectomycorrhizal fungi inoculation ameliorates simulated acid rain effects. Taken together, ectomycorrhizal fungi inoculation - potentially through improving soil substrate availability - could alleviate the deleterious effects of acid rain on nitrogen cycling microbes in forest soils.

Using kinetics and modeling to predict denitrification fluxes in elemental-sulfur-based biofilms.

Elemental sulfur (S0 ) can serve as an electron donor for water and wastewater denitrification, but few researchers have addressed the kinetics of S 0 -based reduction of nitrate (NO 3- ), nitrite (NO 2- ), and nitrous oxide (N 2 O). In addition, S 0 -based denitrifying biofilms are counter-diffusional. This is because the electron donor (S 0 ) is supplied from the biofilm attachment surface while the acceptor, for example, NO 3- , is supplied from the bulk liquid. No existing mathematical model for S 0 -based denitrification considers this behavior. In this study, batch tests were used to determine the kinetic parameters for the reduction of NO 3- , NO 2- , and N 2 O. Additionally, a biofilm model was developed to explore the effects of counter-diffusion on overall fluxes, that is, the mass of NO 3- or NO 2- removed per unit biofilm support area per unit time. The maximum specific substrate utilization rates ( qˆ ) for NO 3- , NO 2- , and N 2 O were 3.54, 1.98, and 6.28 g N g COD -1 ·d -1 , respectively. The maximum specific growth rates ( µË† ) were 0.71, 1.21, and 1.67 d -1 for NO 3- to NO 2- , NO 2- to N 2 O, and N 2 O to N 2 , respectively. Results suggest that the observed NO 2- accumulation during S 0 -based denitrification results from a low qˆ for NO 2- relative to that for NO 3- . The high qˆ for N 2 O, relative to that for NO 3- and NO 2- , suggest that little N 2 O accumulation occurs during denitrification. A counter-diffusional biofilm model was used to predict trends for NO 3- fluxes, and confirmed NO 2- accumulation in S 0 -based denitrification biofilms. It also explains the observed detrimental effects of biofilm thickness on denitrification fluxes. This study allows a more accurate prediction of NO 3- , NO 2- , and N 2 O transformations in S 0 -based denitrification.

Nitrous oxide production in autotrophic nitrogen removal granular sludge: A modeling study.

The sustainability of autotrophic granular system performing partial nitritation and anaerobic ammonium oxidation (anammox) for complete nitrogen removal is impaired by the production of nitrous oxide (N2 O). A systematic analysis of the pathways and affecting parameters is, therefore, required for developing N 2 O mitigation strategies. To this end, a mathematical model capable of describing different N 2 O production pathways was defined in this study by synthesizing relevant mechanisms of ammonium-oxidizing bacteria (AOB), nitrite-oxidizing bacteria, heterotrophic bacteria (HB), and anammox bacteria. With the model validity reliably tested and verified using two independent sets of experimental data from two different autotrophic nitrogen removal biofilm/granular systems, the defined model was applied to reveal the underlying mechanisms of N 2 O production in the granular structure as well as the impacts of operating conditions on N 2 O production. The results show that: (a) in the aerobic zone close to the granule surface where AOB contribute to N 2 O production through both the AOB denitrification pathway and the NH 2 OH pathway, the co-occurring HB consume N 2 O produced by AOB but indirectly enhance the N 2 O production by providing NO from NO 2- reduction for the NH 2 OH pathway, (b) the inner anoxic zone of granules with the dominance of anammox bacteria acts as a sink for NO 2- diffusing from the outer aerobic zone and, therefore, reduces N 2 O production from the AOB denitrification pathway, (c) operating parameters including bulk DO, influent NH 4+ , and granule size affect the N 2 O production in the granules mainly by regulating the NH 2 OH pathway of AOB, accounting for 34-58% of N 2 O turnover, and (d) the competition between the NH 2 OH pathway and heterotrophic denitrification for nitric oxide leads to the positive role of HB in reducing N 2 O production in the autotrophic nitrogen removal granules, which could be further enhanced in the presence of a proper level of influent organics.

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 180 days, 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.

The co-existence of anammox genera in an expanded granular sludge bed reactor with biomass carriers for nitrogen removal.

Anaerobic ammonium oxidation (anammox)-based nitrogen removal saves aeration energy and organic carbon costs, attributed to its anaerobic and autotrophic nature. However, due to the slow growth of anaerobic ammonium oxidation bacteria (AnAOB), drawbacks including long startup time and sensitivity to toxins still hamper the application of anammox-based processes. To cope with the slow growth of AnAOB, various bioreactor configurations have been investigated for the capability of retaining anammox biomass, among which, the expanded granular sludge bed (EGSB) reactor is a promising option. In this study, two laboratory-scale EGSB reactors were used to gain insights of microbial population and their response to amending biofilm-carriers, aiming to enhance the biomass retention of AnAOB. The respective ammonium and nitrite removal efficiencies were up to over 90%, and the overall nitrogen removal efficiency (NRE) was stable at over 70%, in the EGSB reactor amended with carriers (CEGSB). Compared to the control EGSB, CEGSB's observed performance was more stable during the 236-day operational period. The abundance of AnAOB reached 22% in the EGSB and 49% in the CEGSB. It was also observed that Ca. Brocadia (14.25%) and Asahi BRW2 (33.19%) coexisted in the CEGSB. The dynamics of major metabolisms and functional genes involved in nitrogen conversion were further observed by FAPROTAX based on the taxonomic data, providing more insights into the functions of the microbial communities.

Analysis of enhanced nitrogen removal mechanisms in a validation wastewater treatment plant containing anammox bacteria.

Anammox bacteria have attracted attention due to their apparent importance in saving energy and reducing organic chemical demands. Here, we report the detection and quantification of anammox bacteria with an improved primer set in a validation wastewater treatment plant. The improved primer set was shown to detect a broad range of anammox bacteria (47.3%) facilitating more accurate analyses of nitrogen removal mechanisms. Nitrogen removal efficiency and dominant nitrogen removal mechanisms were compared in the modification-Johannesburg (Mod-JHB), modified Ludzack-Ettinger (MLE) single-feed, and anoxic-oxic-anoxic-oxic (AOAO) step-feed modes. In the Mod-JHB configuration, simultaneous nitrification and denitrification (SND) and anammox were found to be responsible for more than 80% of total inorganic nitrogen (TIN) removal (98.5 ± 0.8% of TIN removal). Decrease of anoxic SRT from 5 to 2.5 days did not have any obvious effect on nitrogen removal or the abundance of functional microorganisms. Microbial batch tests demonstrated that both partial nitrification and dissimilatory nitrate reduction to ammonium (DNRA) were responsible for maintaining the anammox process. Short SRT (2 days) in the aerobic zone may explain the presence of partial nitrification. This study provides insights to the analysis of nitrogen removal mechanisms in validation wastewater treatment plants (WWTPs) aiming for high nitrogen removal efficiency.