Globally, denitrification is commonly employed in biological nitrogen removal processes to enhance water quality. However, substantial knowledge gaps remain concerning the overall community ...structure, population dynamics and metabolism of different organic carbon sources. This systematic review provides a summary of current findings pertaining to the microbial ecology of denitrification in biological wastewater treatment processes. DNA fingerprinting-based analysis has revealed a high level of microbial diversity in denitrification reactors and highlighted the impacts of carbon sources in determining overall denitrifying community composition. Stable isotope probing, fluorescence in situ hybridization, microarrays and meta-omics further link community structure with function by identifying the functional populations and their gene regulatory patterns at the transcriptional and translational levels. This review stresses the need to integrate microbial ecology information into conventional denitrification design and operation at full-scale. Some emerging questions, from physiological mechanisms to practical solutions, for example, eliminating nitrous oxide emissions and supplementing more sustainable carbon sources than methanol, are also discussed. A combination of high-throughput approaches is next in line for thorough assessment of wastewater denitrifying community structure and function. Though denitrification is used as an example here, this synergy between microbial ecology and process engineering is applicable to other biological wastewater treatment processes.
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•Organic carbon source has significant impacts on denitrifying community structure.•New methodologies better link denitrifying community structure to function.•Microbial ecology and kinetic models can be integrated to improve prediction ability.•Alternative carbon sources and N2O control are some new frontiers.
Nitrosomonas europaea is a widely studied chemolithoautotrophic ammonia oxidizing bacterium. While significant work exists on the ammonia oxidation pathway of N. europaea, its responses to factors ...such as dissolved oxygen limitation or sufficiency or exposure to high nitrite concentrations, particularly at the functional gene transcription level are relatively sparse. The principal goal of this study was to investigate responses at the whole-cell activity and gene transcript levels in N. europaea 19718 batch cultures, which were cultivated at different dissolved oxygen and nitrite concentrations. Transcription of genes coding for principal metabolic pathways including ammonia oxidation (amoA), hydroxylamine oxidation (hao), nitrite reduction (nirK) and nitric oxide reduction (norB) were quantitatively measured during batch growth, at a range of DO concentrations (0.5, 1.5 and 3.0 mg O2/L). Measurements were also conducted during growth at 1.5 mg O2/L in the presence of 280 mg-N/L of externally added nitrite.
Several wide ranging responses to DO limitation and nitrite toxicity were observed in N. europaea batch cultures. In contrast to our initial hypothesis, exponential phase mRNA concentrations of both amoA and hao increased with decreasing DO concentrations, suggesting a mechanism to metabolize ammonia and hydroxylamine more effectively under DO limitation. Batch growth in the presence of 280 mg nitrite-N/L resulted in elevated exponential phase nirK and norB mRNA concentrations, potentially to promote utilization of nitrite as an electron acceptor and to detoxify nitrite. This response was in keeping with our initial hypothesis and congruent with similar responses in heterotrophic denitrifying bacteria. Stationary phase responses were distinct from exponential phase responses in most cases, suggesting a strong impact of ammonia availability and metabolism on responses to DO limitation and nitrite toxicity. In general, whole-cell responses to DO limitation or nitrite toxicity, such as sOUR or nitrite reduction to nitric oxide (NO) did not parallel the corresponding mRNA (nirK) profiles, suggesting differences between the gene transcription and enzyme translation or activity levels.
The results of this study show that N. europaea possesses specific mechanisms to cope with growth under low DO concentrations and high nitrite concentrations. These mechanisms are additionally influenced by the physiological growth state of N. europaea cultures and are possibly geared to enable more efficient substrate utilization or nitrite detoxification.
The overarching goal of this study was to determine the role of inorganic carbon (IC) in influencing the microbial ecology, performance and nitrogen turnover by individual microbial communities of a ...biofilm based combined nitritation–anammox process. IC limitation was transiently imposed by reducing the IC input from 350% to 40% of the stoichiometric requirement for 40 days. The principal impact observed during IC limitation was the overgrowth of nitrite oxidizing bacteria (NOB) at the expense of anaerobic ammonia oxidizing bacteria (AMX). On the other hand, the concentrations of ammonia oxidizing bacteria (AOB) were relatively stable during the imposition of and recovery from IC limitation. The resulting dominance of NOB, in terms of their concentration and contribution to nitrite consumption over AMX, resulted, in turn, in a decrease in overall nitrogen removal from 78 ± 2.0% before IC limitation to 46 ± 2.9% during IC limitation. Upon recovery back to non-limiting IC input, it took an inordinately long time (about 57*HRT) for the N-removal to recover back to pre-limitation conditions. Even after recovery, NOB were still persistent in the biofilm and could not be washed out to pre-limitation concentrations. The emission of nitrous oxide (N2O) and nitric oxide (NO), likely from AOB, transiently increased in concert with transient increases in ammonia and hydroxylamine concentrations during the period of IC limitation. Therefore, an unintended consequence of IC limitation in nitritation–anammox systems can be an increase in their greenhouse gas footprint, in addition to compromised process performance. Most emphasis to date on nitritation and anammox studies has been on the nitrogen cycle. The results of this study demonstrate that the differing strategies used by AOB, NOB and AMX to compete for their preferred assimilative carbon source can also significantly influence the microbial ecology, performance and carbon footprint of such processes.
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•Inorganic carbon availability can influence microbial interactions in nitritation–anammox.•AOB and NOB are more adept at overcoming IC limitation than AMX.•IC limitation can result in increased emissions of nitrous oxide (N2O) and nitric oxide (NO).•Increased emissions during IC limitation are likely due to an imbalance in electron fluxes in AOB.
The overall goal of this study was to develop an appropriate biological process for achieving autotrophic conversion of methane (CH4) to methanol (CH3OH). In this study, we employed ammonia-oxidizing ...bacteria (AOB) to selectively and partially oxidize CH4 to CH3OH. In fed-batch reactors using mixed nitrifying enrichment cultures from a continuous bioreactor, up to 59.89 ± 1.12 mg COD/L of CH3OH was produced within an incubation time of 7 h, which is approximately ten times the yield obtained previously using pure cultures of Nitrosomonas europaea. The maximum specific rate of CH4 to CH3OH conversion obtained during this study was 0.82 mg CH3OH COD/mg AOB biomass COD-d, which is 1.5 times the highest value reported with pure cultures. Notwithstanding these positive results, CH4 oxidation to CH3OH by AOB was inhibited by NH3 (the primary substrate for the oxidative enzyme, ammonia monooxygenase, AMO) as well as the product, CH3OH, itself. Further, oxidation of CH4 to CH3OH by AOB was also limited by reducing equivalents supply, which could be overcome by externally supplying hydroxylamine (NH2OH) as an electron donor. Therefore, a potential optimum design for promoting CH4 to CH3OH oxidation by AOB could involve supplying NH3 (needed to maintain AMO activity) uncoupled from the supply of NH2OH and CH4. Partial oxidation of CH4-containing gases to CH3OH by AOB represents an attractive platform for the conversion of a gaseous mixture to an aqueous compound, which could be used as a commodity chemical. Alternately, the nitrate and CH3 OH thus produced could be channeled to a downstream anoxic zone in a biological nitrogen removal process to effect nitrate reduction to N2, using an internally produced organic electron donor.
Despite recognition of the possible role of biological nitrogen removal (BNR) processes in nitrous oxide (N2O) emission, a measured database of N2O emissions from these processes at the national ...scale does not currently exist. This study focused on the quantification of N2O emissions at 12 wastewater treatment plants (WWTPs) across the United States using a newly developed U.S. Environmental Protection Agency (USEPA) reviewed protocol. A high degree of variability in field-scale measurements of N2O was observed, both across the WWTPs sampled and within each WWTP. Additionally, aerobic zones, which have hitherto not been considered in the USEPA approach of estimating N2O emissions, generally contributed more to N2O fluxes than anoxic zones from BNR reactors. These results severely qualify the conventional use of a single emission factor to “estimate” N2O emissions from BNR processes, solely by virtue of denitrification. Upon subjecting the nationwide data set to multivariate regression data mining, high nitrite, ammonium, and dissolved oxygen concentrations were positively correlated with N2O emissions from aerobic zones of activated sludge reactors. On the other hand, high nitrite and dissolved oxygen concentrations were positively correlated with N2O emissions from anoxic zones. Based on these results, it can be argued that activated sludge processes that minimize transient or permanent build up of ammonium or nitrite, especially in the presence of dissolved oxygen, are expected to have low N2O emissions.
Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms ...and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.
Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats' virus hosting capacities, including uniquely constitutive immune pathways, on ...cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our
system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats.
This work describes the development of an intermittently aerated pilot-scale process (V = 0.34 m(3)) operated without oxidized nitrogen recycle and supplemental carbon addition optimized for nitrogen ...removal via nitritation/denitritation. The aeration pattern was controlled using a novel aeration strategy based on set-points for reactor ammonia, nitrite and nitrate concentrations with the aim of maintaining equal effluent ammonia and nitrate + nitrite (NOx) concentrations. Further, unique operational and process control strategies were developed to facilitate the out-selection of nitrite oxidizing bacteria (NOB) based on optimizing the chemical oxygen demand (COD) input, imposing transient anoxia, aggressive solids retention time (SRT) operation towards ammonia oxidizing bacteria (AOB) washout and high dissolved oxygen (DO) (>1.5 mg/L). Sustained nitrite accumulation (NO2-N/NOx-N = 0.36 ± 0.27) was observed while AOB activity was greater than NOB activity (AOB: 391 ± 124 mgN/L/d, NOB: 233 ± 151 mgN/L/d, p < 0.001) during the entire study. The reactor demonstrated total inorganic nitrogen (TIN) removal rate of 151 ± 74 mgN/L/d at an influent COD/ Formula: see text -N ratio of 10.4 ± 1.9 at 25 °C. The TIN removal efficiency was 57 ± 25% within the hydraulic retention time (HRT) of 3 h and within an SRT of 4-8 days. Therefore, this pilot-scale study demonstrates that application of the proposed online aeration control is able to out-select NOB in mainstream conditions providing relatively high nitrogen removal without supplemental carbon and alkalinity at a low HRT.
•TiO2, CeO2, and ZnO nanoparticles showed increasing cytotoxicity in sequence.•Cell membrane integrity was not a sensitive indicator of nanoparticle cytotoxicity.•Intracellular metal content ...increased after cells’ exposure to its nanosized oxide.•Increase of nanoparticle concentration would not always elevate its cytotoxicity.
Nanosized TiO2 (n-TiO2), CeO2 (n-CeO2), and ZnO (n-ZnO) and bulk ZnO were chosen for a 4-h exposure study on a model ammonia oxidizing bacterium, Nitrosomonas europaea. n-ZnO displayed the most serious cytotoxicity while n-TiO2 was the least toxic one. The change of cell morphologies, the retardance of specific oxygen uptake rates and ammonia oxidation rates, and the depression of amoA gene expressions under NP stresses were generally observed when the cell densities and membrane integrities were not significantly impaired yet. The TEM imaging and the synchrotron X-ray fluorescence microscopy of the NPs impacted cells revealed the increase of the corresponding intracellular Ti, Ce or Zn contents and suggested the intracellular NP accumulation. The elevation of intracellular S contents accompanied with higher K contents implied the possible activation of thiol-containing glutathione and thioredoxin production for NP stress alleviation. The NP cytotoxicity was not always a function of NP concentration. The 200mgL−1 n-TiO2 or n-CeO2 impacted cells displayed the similar ammonia oxidation activities but higher amoA gene expression levels than the 20mgL−1 NPs impacted ones. Such phenomenon further indicated the possible establishment of an anti-toxicity mechanism in N. europaea at the genetic level to redeem the weakened AMO activities along with the NP aggregation effects.
A distinctive red biofilm was observed in a glycerol-fed digester liquid effluent treatment process coupling partial nitrification (nitritation) and partial denitrification (denitritation) processes. ...Based on initial phylogenetic screening using 16S rRNA clone libraries and quantitative polymerase chain reaction, the biofilm was enriched in novel anaerobic ammonium oxidizing bacteria (AMX/anammox) closely related to Candidatus “Brocadia caroliniensis”. The metabolic functionality of the C. “Brocadia caroliniensis” enrichment was further explored using high-throughput sequencing and de novo metagenome assembly. The population anammox genome that was binned from the metagenome consisted of 209 contigs with a total of 3.73 Mbp consensus sequences having 43.3% GC content, and 27.4 average coverage depth. The assembled metagenome bin was comprised of 3582 open reading frames (ORFs). Based on 16S rRNA similarity the binned metagenome was closely related with Candidatus “Brocadia caroliniensis”, Candidatus “Brocadia fulgida”, planctomycete KSU-1, and Candidatus “Kuenenia stuttgartiensis” with 99%, 96%, 92% and 93% similarity, respectively. Essential genes in anammox metabolic functions including ammonium and nitrite transport, hydrazine synthesis, electron transfer for catabolism, and inorganic carbon fixation, among several other anabolic pathways, were also observed in the population genome of the C. “Brocadia caroliniensis” related enrichment. Our results demonstrate the wider profusion of anammox bacteria in engineered nitrogen removal systems than expected. The utility of metagenomics approaches to deciphering such novel functionality in these systems is also highlighted.
•Anammox bacteria were fortuitously enriched in a full-scale nitritation-denitritation process.•The enriched anammox bacteria were closely related to Candidatus “Brocadia caroliniensis”.•The population-genome of the enrichment was constructed using next-generation sequencing.•Using the population-genome, the metabolic pathways of C. “Brocadia caroliniensis” were elucidated.
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