Search

Although nitrogen removal by partial nitritation and anammox is more cost-effective than conventional nitrification and denitrification, one downside is the production and accumulation of nitrous oxide (N2O). The potential exploitation of N2O-reducing bacteria, which are resident members of anammox microbial communities, for N2O mitigation would require more knowledge of their ecophysiology. This study investigated the phylogeny of resident N2O-reducing bacteria in an anammox microbial community and quantified individually the processes of N2O production and N2O consumption. An up-flow column-bed anammox reactor, fed with NH4+ and NO2- and devoid of oxygen, emitted N2O at an average conversion ratio (produced N2O: influent nitrogen) of 0.284%. Transcriptionally active and highly abundant nosZ genes in the reactor biomass belonged to the Burkholderiaceae (clade I type) and Chloroflexus genera (clade II type). Meanwhile, less abundant but actively transcribing nosZ strains were detected in the genera Rhodoferax, Azospirillum, Lautropia, and Bdellovibrio and likely act as an N2O sink. A novel 15N tracer method was adapted to individually quantify N2O production and N2O consumption rates. The estimated true N2O production rate and true N2O consumption rate were 3.98 ± 0.15 and 3.03 ± 0.18 mgN·gVSS-1·day-1, respectively. The N2O consumption rate could be increased by 51% (4.57 ± 0.51 mgN·gVSS-1·day-1) with elevated N2O concentrations but kept comparable irrespective of the presence or absence of NO2-. Collectively, the approach allowed the quantification of N2O-reducing activity and the identification of transcriptionally active N2O reducers that may constitute as an N2O sink in anammox-based processes.

Many enveloped animal viruses produce a variety of particle shapes, ranging from small spherical to long filamentous types. Characterization of how the shape of the virion affects infectivity has been difficult because the shape is only partially genetically encoded, and most pleomorphic virus structures have no selective advantage in vitro. Here, we apply virus fractionation using low-force sedimentation, as well as antibody neutralization coupled with RNAScope, single-particle membrane fusion experiments and stochastic simulations to evaluate the effects of differently shaped influenza A viruses and influenza viruses pseudotyped with Ebola glycoprotein on the infection of cells. Our results reveal that the shape of the virus particles determines the probability of both virus attachment and membrane fusion when viral glycoprotein activity is compromised. The larger contact interface between a cell and a larger particle offers a greater probability that several active glycoproteins are adjacent to each other and can cooperate to induce membrane merger. Particles with a length of tens of micrometres can fuse even when 95% of the glycoproteins are inactivated. We hypothesize that non-genetically encoded variable particle shapes enable pleomorphic viruses to overcome selective pressure and may enable adaptation to infection of cells by emerging viruses such as Ebola. Our results suggest that therapeutics targeting filamentous virus particles could overcome antiviral drug resistance and immune evasion in pleomorphic viruses.

ANAMMOX (anaerobic ammonium oxidation) represents an energy-efficient process for biological nitrogen removal, particularly from wastewater streams with low chemical oxygen demand (COD) to nitrogen (C/N) ratios. Its widespread application, however, is still hampered by a lack of access to biomass-enriched with ANAMMOX bacteria (AMX), slow growth rates of AMX, and their sensitivity to inhibition. Although the coupling of ANAMMOX processes with partial nitrification is already widespread, especially for sidestream treatment, maintaining a functional population density of AMX remains a challenge in these systems. Therefore, strategies that maximize retention of AMX-rich biomass are essential to promote process stability. This paper reviews existing methods of biomass retention in ANAMMOX-mediated systems, focusing on (i) granulation; (ii) biofilm formation on carrier materials; (iii) gel entrapment; and (iv) membrane technology in mainstream and sidestream systems. In addition, the microbial ecology of different ANAMMOX-mediated systems is reviewed.

Tertiary wastewater treatment could provide a reliable source of water for reuse. Amongst these types of wastewater treatment, deep-bed filtration of secondary effluents can effectively remove particles and organic matter, however, NH4+ and NO2&minus, are not easily removed. This study examined the feasibility of stimulating microbial activity using hydrogen peroxide (H2O2) as a bio-specific and clean oxygen source that leaves no residuals in the water and is advantageous upon aeration due to the solubility limitations of the oxygen. The performance of a pilot bio-filtration system at a filtration velocity of 5&ndash, 6 m/h, was enhanced by the addition of H2O2 for particle, organic matter, NH4+, and NO2&minus, removal. Hydrogen peroxide provided the oxygen demand for full nitrification. As a result, influent concentrations of 4.2 &plusmn, 2.5 mg/L N-NH4+ and 0.65 &plusmn, 0.4 mg/L N-NO2 were removed during the short hydraulic residence time (HRT). In comparison, filtration without H2O2 addition only removed up to 0.6 mg/L N-NH4+ and almost no N-NO2&minus, A DNA metagenome analysis of the functional genes of the media biomass reflected a significant potential for simultaneous nitrification and denitrification activity. It is hypothesized that the low biodegradability of the organic carbon and H2O2 addition stimulated oxygen utilization in favor of nitrification, followed by the enhancement of anoxic activity.

An intermittently aerated, pilot scale biological nitrogen removal process was operated in modified Ludzack Ettinger (MLE) and fully intermittent aeration (all reactors aerated) configurations. The process was fed both A-stage effluent (ASE), and primary clarifier effluent (PCE), which differ in chemical oxygen demand (COD) composition. The objective was to determine the effects of influent carbon fractionation and reactor configuration on nitrite oxidizing bacteria (NOB) out-selection and total inorganic nitrogen (TIN) removal during intermittent aeration. TIN removal was affected by both the type and amount of influent COD, with particulate COD (pCOD) having a stronger influence than soluble COD (sCOD). NOB out-selection was lowest in the MLE configuration, regardless of the feed, and highest in fully intermittent configuration with A-stage feed. During fully intermittent operation with A-stage effluent feed, nitrite accumulation correlated positively with influent particulate COD concentration, and correlated negatively with ex situ NOB activity rates. In addition, ex situ denitrification batch tests showed that nitrite was consumed faster than nitrate when NOB rates were low. These observations suggested that pCOD improved heterotrophic competition for nitrite, leading to ammonia oxidation rates higher than nitrite oxidation rates. Therefore, the influent COD fractions should be tailored to achieve the desired nitrogen removal goals downstream.

Denitratation, the selective reduction of nitrate to nitrite, is a novel process when coupled with anaerobic ammonium oxidation (anammox) could achieve resource-efficient biological nitrogen removal of ammonium- and nitrate-laden waste streams. Using a fundamentally-based, first principles approach, this study optimized a stoichiometrically-limited, glycerol-driven denitratation process and characterized mechanisms supporting nitrite accumulation with results that aligned with expectations. Glycerol supported selective nitrate reduction to nitrite and near-complete nitrate conversion, indicating its viability in a denitratation system. Glycerol-supported specific rates of nitrate reduction (135.3 mg-N/g-VSS/h) were at least one order of magnitude greater than specific rates of nitrite reduction (14.9 mg-N/g-VSS/h), potentially resulting in transient nitrite accumulation and indicating glycerol’s superiority over other organic carbon sources in denitratation systems. pH and ORP inflection points in nitrogen transformation assays corresponded to maximum nitrite accumulation, indicating operational setpoints to prevent further nitrite reduction. Denitratation conditions supported enrichment of Thauera sp. as the dominant genus. Stoichiometric limitation of influent organic carbon, coupled with differential nitrate and nitrite reduction kinetics, optimized operational controls, and a distinctively enriched microbial ecology, was identified as causal in glycerol-driven denitratation.

Two sizes of polystyrene (PS) were compared to investigate their impact on nitrification. The smaller PS (50 nm) had a higher impact than the larger PS (500 nm). Lower NO2- and NO3- accumulation was observed in the 50 nm PS treatment. There was no significant difference in DIN concentration between the control and 500 nm PS treatments. PS treatment did not have a significant influence on the specific ammonia oxidation rate, but the specific nitrite utilization rate was the lowest in the 50 nm PS treatment. The changes in transcript levels of amoA gene did not correspond well with the observed changes in DIN concentrations, suggesting that the effects of 50 nm PS treatment might be unrelated to biological phenomena, for which an actual uptake of PS is needed. The fluorescent images revealed that the smaller PS can easily access bacterial cells, which corroborated the results of inhibition of nitrification by the smaller PS. Notably, most of the PS particles did not penetrate bacterial cells, suggesting that the observed effects of 50 nm PS on nitrification might be due to disruption of the membrane potential of the cells.

In this study, concurrent operation of anammox and partial denitrification within a nonacclimated mixed culture system was proposed. The impact of carbon sources (acetate, glycerol, methanol, and ethanol) and COD/NO3- -N ratio on partial denitrification selection under both short- and long-term operations was investigated. Results from short-term testing showed that all carbon sources supported partial denitrification. However, acetate and glycerol were preferred due to their display of efficient partial denitrification selection, which may be related to their different electron transport pathways in comparison with methanol. Long-term operation confirmed results of batch tests by showing the contribution of partial denitrification to nitrate removal above 90% after acclimation in both acetate and glycerol reactors. In contrast, methanol showed challenges of maintaining efficient partial denitrification. COD/NO3- -N ratio mainly controlled the rate of nitrate reduction and not directly partial denitrification selection; thus, it should be used to balance between denitrification rate and anammox rate. PRACTITIONER POINTS: The authors aimed to investigate the impact of carbon sources and COD/NO3-N ratio on partial denitrification selection. All the carbon sources supported partial denitrification as long as the nitrite sink was available. 90% partial denitrification could be achieved with both acetate and glycerol in long-term operations. COD/NO3-N ratio did not directly control partial denitrification but can be used to balance between denitrification rate and anammox rate.

Soil columns simulating soil aquifer treatment (SAT), fed with synthetic secondary effluent by intermittent infiltration of flooding/drying cycles, were characterized for nitrogen and organic carbon removal, and microbial ecology and biokinetics. The columns differed in the concentration ratio of chemical oxygen demand (COD) to the summed NH 4 + , NO 2 − and organic nitrogen—2 (C/N2) or 5 (C/N5). Chemical profiles along the column demonstrated a preference for COD oxidation over nitrification and coupled denitrification, with higher nitrogen loss (57% vs. 16%) in the C/N5 column. Unexpectedly, significant dominance of the genus Nitrospira over the genus Nitrobacter and ammonia-oxidizing bacteria (AOB) was strongly correlated at column depths where NH 4 + removal occurred. Moreover, the Nitrospira profile had the strongest correlation to the profile of NH 4 + (positive) and NO 3 − (negative), strongly indicating complete ammonia oxidation. 16S sequencing analysis of the topsoil in C/N2 vs. C/N5 columns revealed double the abundance of microbial aerobic potential (64% vs. 32%) vs. one-third the denitrification potential (13% vs. 31%). The concentrations and degradability levels of organic carbon were the most influential parameters shaping community structure. Niche differentiation within the biofilm attached to the soil is suggested to have an important role in the process's anoxic activity and nitrogen removal.

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, a highly inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitroCRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to rapidly introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.Graphical AbstractOverview of MAVERICC. Conceptual overview of the approach outlined in this manuscript. To make VACV recombinants, the parental virus is first purified and vDNA is isolated with phenol:chloroform extraction. This purified vDNA is then treated with Cas9 enzyme and sgRNAs that are directed to a specific locus in the VACV genome. The cleaved vDNA is then transfected into FWPV-infected BSC-40 cells along with a transfer amplicon containing an insertion or mutation of interest flanked by homologous sequences. Recombination is allowed to occur for 5-7 days, during which time the cleaved vDNA is healed by the transfer amplicon, thus editing the VACV genome, and packaged into infectious viral particles. Individual plaques are grown up and rVACVs are isolated after a single round of plaque purification. Image was created with Biorender.com.

Bacteria are the driving force of the microbial fuel cell (MFC) technology, which benefits from their natural ability to degrade organic matter and generate electricity. The development of an efficient anodic biofilm has a significant impact on the power performance of this technology so it is essential to understand the effects of the inoculum nature on the anodic bacterial diversity and establish its relationship with the power performance of the system. Thus, this work aims at analysing the impact of 3 different types of inoculum: (i) stored urine, (ii) sludge and (iii) effluent from a working MFC, on the microbial community of the anodic biofilm and therefore on the power performance of urine-fed ceramic MFCs. The results showed that MFCs inoculated with sludge outperformed the rest and reached a maximum power output of 40.38 mW&middot, m&minus, 2anode (1.21 mW). The power performance of these systems increased over time whereas the power output by MFCs inoculated either with stored urine or effluent decreased after day 30. These results are directly related to the establishment and adaptation of the microbial community on the anode during the assay. Results showed the direct relationship between the bacterial community composition, originating from the different inocula, and power generation within the MFCs.

The application of metagenomics and metatranscriptomics to field-scale engineered biological nitrogen removal (BNR) processes revealed a complex N-cycle network (the meta-azotome) therein in terms of microbial structure,potentialandextantfunction. Autotrophic nitrification bore the imprint of well-documentedNitrosomonasandNitrospirain most systems. However, in select BNR processes, complete ammonia oxidizing bacteria, comammoxNitrospira, unexpectedly contributed more substantially to ammonia oxidation than canonical ammonia oxidizing bacteria, based on metatranscriptomic profiling. Methylotrophic denitrification was distinctly active in methanol-fed reactors but not in glycerol-fed reactors. Interestingly, glycerol metabolism and N-reduction transcript signatures were uncoupled, possibly suggesting the role of other carbon sources in denitrification emanating from glycerol itself or from upstream process reactors. In sum, the meta-azotome of engineered BNR processes revealed both traditional and novel mechanisms of N-cycling. Similar interrogation approaches could potentially inform better design and optimization of wastewater treatment and engineered bioprocesses in general.

Highlights • MFC cascade can withstand and degrade high concentrations of ampicillin. • Chloroxylenol caused loss of power and microbial community within MFC cascade. • MFC anodic biofilm responds differentially to different inhibitory chemical agents. • MFC cascade has the potential for bioremediation of certain chemical agents., This study evaluates the fate of certain bactericidal agents introduced into microbial fuel cell (MFC) cascades and the response of the microbial community. We tested the response of functioning urine fed MFC cascades using two very different bactericidal agents: a common antibiotic (Ampicillin, 5 g/L) and a disinfectant (Chloroxylenol 4.8 g/L) in concentrations of up to 100 times higher than the usual dose. Results of power generation showed that the established bacteria community was able to withstand high concentrations of ampicillin with good recovery after 24 h of minor decline. However, power generation was adversely affected by the introduction of chloroxylenol, resulting in a 99% loss of power generation. Ampicillin was completely degraded within the MFC cascade (>99.99%), while chloroxylenol remained largely unaffected. Analysis of the microbial community before the addition of the bactericidal agents showed a significant bacterial diversity with at least 35 genera detected within the cascade. Microbial community analysis after ampicillin treatment showed the loss of a small number of bacterial communities and proportional fluctuations of specific strains within the individual MFCs community. On the other hand, there was a significant shift in the bacterial community after chloroxylenol treatment coupled with the loss of at least 13 bacterial genera across the cascade.

Summary There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition., Graphical Abstract, Highlights • Highly infectious recombinant VSV expressing SARS-CoV-2 spike (S) was generated • rVSV-SARS-CoV-2 S resembles SARS-CoV-2 in entry and inhibitor/antibody sensitivity • rVSV-SARS-CoV-2 S affords rapid screens and forward-genetic analyses of antivirals, Surrogate systems are needed to evaluate COVID-19 vaccines and therapeutics rapidly and at scale. Dieterle & Haslwanter et al. describe a highly infectious recombinant vesicular stomatitis virus encoding the SARS-CoV-2 spike protein that is suitable for screening and mechanistic studies of small molecule inhibitors, recombinant biologics, and convalescent plasma.

Ebola virus (EBOV) entry into host cells comprises stepwise and extensive interactions of the sole viral surface glycoprotein GP with multiple host factors. During the intricate process, following virus uptake and trafficking to late endosomal/lysosomal compartments, GP is proteolytically processed to GPCLby the endosomal proteases cathepsin B and L unmasking GP’s receptor-binding site. Engagement of GPCLwith the universal filoviral intracellular receptor Niemann-Pick C1 (NPC1) eventually culminates in fusion between viral and cellular membranes, cytoplasmic escape of the viral nucleocapsid and subsequent infection. Mechanistic delineation of the indispensable GPCL:NPC1 binding step has been severely hampered by the unavailability of a robust cell-based assay assessing interaction of GPCLwith full-length endosomal NPC1.Here, we describe a novelin situassay to monitor GPCL:NPC1 engagement in intact, infected cells. Visualization of the subcellular localization of binding complexes is based on the principle of DNA-assisted, antibody-mediated proximity ligation. Virus-receptor binding monitored by proximity ligation was contingent on GP’s proteolytic cleavage, and was sensitive to perturbations in the GPCL:NPC1 interface. Our assay also specifically decoupled detection of virus-receptor binding from steps post-receptor binding, such as membrane fusion and infection. Testing of multiple FDA-approved small molecule inhibitors revealed that drug treatments inhibited virus entry and GPCL:NPC1 recognition by distinctive mechanisms. Together, here we present a newly established proximity ligation assay, which will allow us to dissect cellular and viral requirements for filovirus-receptor binding, and to delineate the mechanisms of action of inhibitors on filovirus entry in a cell-based system.IMPORTANCEEbola virus causes episodic but increasingly frequent outbreaks of severe disease in Middle Africa, as shown by a currently ongoing outbreak in the Democratic Republic of Congo. Despite considerable effort, FDA-approved anti-filoviral therapeutics or targeted interventions are not available yet. Virus host-cell invasion represents an attractive target for antivirals; however our understanding of the inhibitory mechanisms of novel therapeutics is often hampered by fragmented knowledge of the filovirus-host molecular interactions required for viral infection. To help close this critical knowledge gap, here, we report anin situassay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that ourin situassay based on proximity ligation represents a powerful tool to delineate receptor-viral glycoprotein interactions. Similar assays can be utilized to examine receptor interactions of diverse viral surface proteins whose studies have been hampered until now by the lack of robustin situassays.

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes . The temperature-dependent stability (thermostability) of the pre-fusion conformers of ‘Class I’ viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple ELISA-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of pre-fusion conformation at elevated temperatures, but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GPCL. Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.ImportanceThough a vaccine for Ebola virus has been approved by the FDA within the past year, no FDA-approved therapeutics are available to treat infections by Ebola virus or other filoviruses. The development of such countermeasures is challenged by our limited understanding of the mechanism by which Ebola virus enters cells, especially at the final step of membrane fusion. The sole surface-exposed viral protein, GP, mediates key steps in virus entry, including membrane fusion, and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these structural rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer forms of GP with enhanced stability that could be useful as part of an antiviral vaccine and to discover and improve drugs that act by modulating the stability of GP.

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. The hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimeric protomers associated into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the Gn head / Gc heterodimer, and of the homotetrameric Gn base. Docking them into an 11.4 A resolution cryo- electron tomography map of the hantavirus surface accounted for the complete extracellular portion of the viral glycoprotein shell and provided unprecedented detail of the surface organization of a pleomorphic virus. These results, which further revealed an in-built mechanism controlling Gc membrane-insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses

Crimean-Congo hemorrhagic fever virus (CCHFV), the only endemic biosafety level 4 pathogen in Europe, is the world’s most widely distributed tick-borne zoonotic virus with a 30% fatality rate in humans, underlining the need for specific therapeutics and vaccines. The CCHFV membrane fusion glycoprotein Gc is the main target of the host neutralizing antibody response. Here we describe the structure of pre-fusion Gc in complex with the antigen-binding fragments (Fabs) corresponding to a therapeutically potent bispecific antibody as well as the structure of unbound Gc in its post-fusion conformation. Our findings suggest that one Fab blocks insertion of the fusion loops into the target membrane, whereas the other blocks formation of the post-fusion trimer. Combined with yeast display of mutagenized Gc, the structures allowed epitope mapping of a large panel of neutralizing antibodies. These data provide the essential molecular underpinnings for developing vaccines and therapeutics against CCHFV.

Aquaponics is a technology for food production (fish and vegetables/fruits) with concomitant remediation of nitrogen-rich aquaculture effluent. There is, however, a critical need to improve the nitrogen use efficiency (NUE) in aquaponics. Here, we employed quantitative polymerase chain reactions and next-generation sequencing to evaluate the bacterial communities and their links to nitrogen transformations for improving NUEs in four bench-scale plant-based floating-raft aquaponics (pak choi, lettuce, chive, and tomato) and three pH levels (7.0, 6.0, and 5.2). Low relative abundance of nitrifiers in plant roots and biofilters suggested nitrogen loss, which decreased NUE in aquaponics. Low pH level was a major factor that shifted the microbial communities and reduced the relative abundance of nitrifiers in aquaponic systems, leading to total ammonia nitrogen accumulation in recirculating water. In plant roots, the abundance of nitrite-oxidizing bacteria (e.g., Nitrospira spp.) did not decrease at low pH levels, suggesting the benefit of growing plants in aquaponics for efficient nitrification and improving NUE. These findings on microbial communities and nitrogen transformations provided complementary strategies to improve the performance of the aquaponics regarding water quality and extent of nutrient recovery from aquaculture effluent.

A wide range of trace organic contaminants (TOrCs), including the endocrine-disrupting compound bisphenol A (BPA), are subject to microbial transformations during biological wastewater treatment. However, relatively little is known about the identity of organisms capable of assimilating emerging contaminants. Here, 13C-DNA stable isotope probing (DNA-SIP) was used to investigate biodegradation and assimilation of BPA by mixed microbial communities collected from two full-scale wastewater treatment plant bioreactors in New York City and subsequently enriched under two BPA exposure conditions. The four enrichment modes (two reactors with two initial BPA concentrations) resulted in four distinct communities with different BPA degradation rates. On the basis of DNA-SIP, bacteria related to Sphingobium spp. were dominant in the assimilation of BPA or its metabolites. Variovorax spp. and Pusillimonas spp. also assimilated BPA or its metabolites. Our results highlight that microbial communities originating from wastewater treatment facilities harbor the potential for addressing not only human-derived carbon but also BPA, a complex anthropogenic TOrC. While previous studies focus on microbial biodegradation of BPA, this study uniquely determines the "active" fraction of microorganisms engaged in assimilation of BPA-derived carbon. Ultimately, information on both biodegradation and assimilation can facilitate better design and operation of engineered treatment processes to achieve BPA removal.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies1. Viperin, a member of the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes. Viperin inhibits the replication of various viruses by catalysing the conversion of CTP to ddhCTP, which is a unique nucleotide that functions as replication-chain terminator of RNA-dependent RNA polymerases.

When investigating the N2O emissions by ammonia oxidizing bacteria, research has mainly focused on identifying and quantifying the biological pathways. This work evaluated previous studies with Nitrosomonas europaea (ATCC 19718) and assessed the role of the abiotic reaction of hydroxylamine with free nitrous acid during transient anoxia. In cultivations when transient anoxia is cyclically imposed, nitrous oxide and hydroxylamine peaked every time upon recovery to aerobic conditions. When using the same culture conditions abiotically (i.e., without biomass, but adding hydroxylamine and nitrite), the volumetric N2O emission rates were very comparable to those from the biological experiments, ranging from 0.04 to 0.08 mg-N/L/h in both abiotic and biotic conditions. These results demonstrate that at the culture conditions tested, abiotically produced N2O is likely the major source of emission. Therefore, for the correct investigation of the biological pathways, abiotic tests must always be performed and hydroxylamine should be added. To our knowledge there is no means to distinguish abiotic from biological N2O production in a biological system, even using N15 labelling. We suggest that the contribution of abiotic N2O emissions can be minimized by, for example, maintaining lower nitrite concentration and higher pH.

Anaerobic ammonium oxidation (ANAMMOX) represents an efficient, cost-effective, but sensitive nitrogen removal process for wastewater treatment. Different reactor configurations have been used in full-scale applications but each configuration has unique characteristics, which could influence process performance and community dynamics. The objective of this study is thus to analyze the impact of mixing conditions on nitrogen removal and community structures in a hybrid up-flow anaerobic sludge blanket reactor (H-UASB), moving bed biofilm reactor (MBBR) and gas-lift reactor (GLR). Community dynamics were studied through shotgun sequencing, while concentrations of NH4+, NO3- and NO2- were determined colorimetrically. During the study, MBBR displayed the highest average nitrogen removal efficiency (NRE) (67%) followed by H-UASB (63%), and then GLR (54%). The relative abundance of AMX in the suspended biomass was consistently higher in H-UASB than in MBBR and GLR, while that of nitrite-oxidizing bacteria (NOB) and complete ammonia-oxidizing bacteria (CMX) was higher in MBBR than in GLR and H-UASB. It was observed that the relative abundance of ammonia-oxidizing bacteria (AOB) in the suspended biomass fluctuated across the reactors. The relative abundance of CMX and Nitrospira in the biofilms in H-UASB and GLR was higher than in the suspended biomass, while comparable abundance was observed in MBBR. On the contrary, the relative abundance of AMX in the suspended biomass in H-UASB and MBBR was higher than in the biofilms, whereas it was comparable in the GLR. It was thus concluded that the mixing conditions in the three reactors influenced process performance and community dynamics.

To investigate the major triggers of nitrous oxide (N2O) production in a full-scale wastewater treatment plant, N2O emissions and wastewater characteristics (ammonia, nitrite, nitrate, total nitrogen, dissolved inorganic carbon, dissolved organic carbon, pH, temperature, dissolved oxygen and specific oxygen uptake rate), the results of variations in the cycling of a sequential batch reactor (SBR, where only full nitrification was performed), were monitored seasonally for 16 months. Major triggers of N2O production were investigated based on a seasonal measured database using a random forest (RF) model and sensitivity analysis, which was applied to identify important input variables. As the result of seasonal monitoring in the full-scale SBR, the N2O emission factor relative to daily total nitrogen removal ranged from 0.05 to 2.68%, corresponding to a range of N2O production rate from 0.02 to 0.70 kg-N/day. Results from the RF model and sensitivity analysis revealed that emissions during nitrification were directly or indirectly related to nitrite accumulation, temperature, ammonia loading rate and the specific oxygen uptake rate ratio between ammonia oxidizing bacteria and nitrite oxidizing bacteria (sOUR-ratio). However, changes in the microbial community did not significantly impact N2O emissions. Based on these results, the sOUR-ratio could represent the major trigger for N2O emission in a full-scale BNR system: a higher sOUR-ratio value with an average of 3.13 ± 0.23 was linked to a higher N2O production rate with an average value of 1.27 ± 0.12 kg-N/day (corresponding to 3.96 ± 1.20% of N2O emission factor relative to daily TN removal), while a lower sOUR-ratio with an average value of 2.39 ± 0.27 was correlated with a lower N2O production average rate of 0.17 ± 0.11 kg-N/day (corresponding to 0.74 ± 0.69% of N2O emission factor) (p-value = 0.00001, Mann-Whitney test).

Metschnikowia pulcherrima, a non-traditional oleaginous yeast, was investigated using 0.5–20 g/L of acetic, propionic, butyric and valeric acid (four model products of anaerobic acidification) respectively as carbon sources to accumulate microbial lipids so as to produce biodiesel. The results showed that acetic acid was the most efficient carbon source for improving cell growth, lipid content and biodiesel quality. After 8 cycles non-sterile batch cultivation, M. pulcherrima still dominated in bioreactor. Controlling C/P ratio, K+ and Na+ concentrations, and initial pH could effectively enhance the lipid content to 25.79 ± 0.51%. Under nitrogen starvation, M. pulcherrima performed higher cellular activity and lipid productivity than that of phosphorous limitation. The biomass yield was about twice and the lipid content was 3% higher. Besides, different from the lipid improvement strategy of traditional oleaginous yeast, acetate from ethanol served as the precursor of M. pulcherrima cytoplasmic acetyl-CoA instead of citric acid, and pentose phosphate pathway (PPP) was the main donor of reducing equivalent NADPH by transcriptomics sequencing analysis. These results demonstrate M. pulcherrima is a promising species for large-scale production of biofuel from organic wastes due to the low operating and maintenance costs of non-sterile cultivation, which is beneficial for reducing environmental pollution and energy shortages.

Methane-laden anthropogenic emissions are widely variable in terms of methane (CH4) concentration depending on their source, ranging from

Metabolomics provides insights into the actual physiology of cells rather than their mere “potential”, as provided by genomic and transcriptomic analysis. We investigate the modulation of nitrous oxide (N2O) accumulation by intracellular metabolites in denitrifying bacteria using metabolomics and genome-based metabolic network modeling. Profiles of metabolites and their rates of production/consumption were obtained for denitrifying batch cultures under four conditions: initial COD:N ratios of 11:1 and 4:1 with and without nitrite spiking (28 mg-N L–1). Only the nitrite-spiked cultures accumulated N2O. The NO2– spiked cultures with an initial COD:N = 11:1 accumulated 3.3 ± 0.57% of the total nitrogen added as N2O and large pools of tricarboxylic acid cycle intermediates and amino acids. In comparison, the NO2– spiked cultures with COD:N = 4:1 showed significantly higher (p = 0.028) N2O accumulation (8.5.3 ± 0.9% of the total nitrogen added), which was linked to the depletion of C11–C20 fatty acids. Metabol...

This paper quantifies the effect of varying solids retention time (SRT), hydraulic retention time (HRT) and dissolved oxygen (DO) concentrations on extracellular polymeric substances (EPS) production and subsequently effluent quality, carbon capture (bioflocculation) and carbon redirection (settling) in a high rate activated sludge A-stage system treating domestic wastewater. Two pilot-scale A-stage reactors were set-up with HRTs of 30 and 60 min. Cascade DO control was used to maintain 3 DO set-points of 0.5, 1.0 and 1.5 mg/L. A mixed liquor suspended solids (MLSS) concentration of 3000 mg/L was maintained and the waste activated sludge (WAS) flow was varied to achieve SRTs of 0.28 and 0.56 day. EPS fractions and the protein and polysaccharide concentrations of the mixed liquor were measured. Operation at the 0.56 day SRT and 1.0 mg/L DO resulted in the highest total suspended solids (TSS), total COD (tCOD), particulate COD (pCOD), and colloidal (cCOD) removal. The best overall performance in terms of bioflocculation (cCOD removal) and carbon capture (percent COD in the WAS) occurred at the 0.56 day SRT and coincided with decreasing total EPS concentrations but the settling characteristics of the sludge were better at the 0.28 day SRT. Overall, low correlations were found between EPS production and system performance. It is likely that at the high loading rate of the A-stage system, EPS production did not play a major role compared to the influence of operating parameters on effluent quality, carbon capture and redirection.

This paper quantifies the effect of varying solids retention time (SRT), hydraulic retention time (HRT) and DO concentrations on intracellular storage production and subsequently methane yield in a high rate activated sludge A-stage system treating domestic wastewater. Two 511 L pilot-scale A-stage reactors were set-up with HRTs of 30 (0.28 day SRT) and 60 (0.56 day SRT) minutes. Cascade DO control was used to maintain DO set-points of 0.5, 1.0 and 1.5 mg/L. Polyhydroxyalkanoates (PHA), triacylglycerides, and glycogen concentrations were measured. The shortest SRT and DO of 1.0 mg/L resulted in the highest fraction of soluble COD (sCOD) in the influent per day being captured as PHA per day (0.33 ± 0.02 kg PHA COD/ kg sCOD). At all scenarios, glycogen concentration decreased in the anaerobic sludge blanket in the clarifier and then increased in the aerobic reactors and the opposite trend was observed for PHA. When one of the aerated tanks was turned into an anaerobic selector, the same trend was observed in addition to uptake and release of orthophosphate (OP). Glyceride bound lipids increased in the anaerobic sludge blanket in the clarifier with the greatest increase at the 0.28 day SRT and 1.5 mg/L DO. This could have been due to microorganisms using the free fatty acids for growth or storing lipid droplets. Overall, the shorter SRT resulted in higher potential methane yield and this could be attributed to higher sludge yield and more intracellular storage products formation.

Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct VH and VL sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 µg of scFv-Fc on days −1, 1 and 3 demonstrated protective efficacies ranging from 75–100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.

Nitrospira spp. are chemolithoautotrophic nitrite-oxidizing bacteria (NOB), which are ubiquitous in natural and engineered environments. However, there exist few independent biokinetic studies on Nitrospira spp., likely because their isolation and selective enrichment from environmental consortia such as activated sludge can be challenging. Herein, planktonic Nitrospira spp. cultures closely related to Candidatus Nitrospira defluvii (Nitrospira lineage I) were successfully enriched from activated sludge in a sequencing batch reactor by maintaining sustained limiting extant nitrite and dissolved oxygen concentrations. Morphologically, the enrichment consisted largely of planktonic cells with an average characteristic diameter of 1.3 ± 0.6 μm. On the basis of respirometric assays, estimated maximum specific growth rate (μmax), nitrite half saturation coefficient (KS), oxygen half saturation coefficient (KO), and biomass yield coefficient (Y) of the enriched cultures were 0.69 ± 0.10 d–1, 0.52 ± 0.14 mg-N/L,...

In recent years, aquaponic systems have gained significant popularity as soilless agriculture systems for organic fruits and vegetables production with concomitant remediation of aquaculture effluent. Aquaponics is a potential sustainable food production system that integrates aquaculture with hydroponics in which nitrogen-rich effluent from the fish production is utilized for plant growth. Because nitrogen is one of the most important inputs in an aquaponic system, it is critical to investigate the nitrogen transformations in the system for enhanced recovery of resources. Since studies on nitrogen transformations and nitrogen utilization efficiency (NUE) in aquaponic systems have been very limited, this review critically examines the important fates of nitrogen from input to outputs (e.g., ammonia nitrogen generation, nitrification, nitrate assimilation and nitrogen loss) to improve NUE in aquaponic systems. Various factors affecting the nitrogen transformations are also discussed. Furthermore, an example of nitrogen imbalance between nitrate uptake and nitrate generation rates in an aquaponic system was demonstrated. This review aims to advance our current understanding of nitrogen transformations and outlines future research needs in aquaponic systems, a sustainable model for efficient water and nutrient managements, and food production.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function., Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

Many enveloped animal viruses, including emerging human pathogens Ebola, Nipah, and Hendra viruses, produce a mixture of virus-particle sizes including very long, filamentous members. However, the function of the filamentous particles is unknown. The main impediments to characterizing viral filaments are their phenotypic origin (a single infecting particle gives rise to a range of particle sizes), difficulty of purifying particles to homogeneity according to their size, and the apparent lack of filament advantage in vitro. Influenza virus particles range in length by three orders of magnitude (~55 nm to ~30 μm). All influenza particles package at most a single genome, but the total number of the surface-exposed viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and the HA/NA ratio scale with particle length. HA is the cell-entry protein of influenza, which mediates fusion between the viral and the endosomal membranes by the combined action of 3-5 active HA neighbors (fusion cluster). Here we identify influenza filaments as viral persisters increasing the probability of fusion-cluster formation and cell entry under HA-directed selective pressure. We fractionated viruses to enrich for spherical or filamentous particles and measured the single-particle kinetics of membrane fusion. As a surrogate for HA-directed selective pressure, we used a Fab fragment of a broadly neutralizing antibody that inactivates bound HA. In its presence, filamentous particles fuse more rapidly and more efficiently than do spherical ones. We show that the infectious advantage of filaments derives from their enhanced fusion efficiency rather than from rate effects. Filaments also offer universal protection from extreme HA inactivation. Our results show how the virus can adapt to any condition limiting HA function, and suggest targeting viral filaments as a strategy to prolong vaccine effectiveness or to thwart viral pandemic adaptation.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the β-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention., Author summary Chikungunya virus (CHIKV) is a globally emerging virus that can cause significant disease, including a prolonged and painful arthritis. The virus is spread by mosquitoes that circulate in many regions of the world including the United States. Currently, there are no available vaccines or therapies to treat CHIKV infection. In this report, we identified and characterized a large panel of antibodies against CHIKV from two donors that contracted the viral infection in the Dominican Republic. These antibodies target a number of different regions of the membrane proteins that coat the surface of the virus, and many can inhibit the ability of CHIKV to infect cells. Two of the antibodies were shown to protect mice from a lethal dose of CHIKV. These antibodies have therapeutic potential, and provide insight into the human immune response that may facilitate vaccine development.

Herpes simplex virus (HSV) glycoprotein D (gD) is required for virus entry and cell-to-cell spread, but also binds the host immunomodulatory molecule, HVEM, blocking interactions with its ligands. Natural infection primarily elicits neutralizing antibodies targeting gD, but subunit protein vaccines designed to induce this response have failed clinically. In contrast, preclinical studies demonstrate that an HSV-2 single-cycle strain deleted in gD, ΔgD-2, induces primarily non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC). The current studies were designed to test the hypothesis that gD interferes with ADCC through engagement of HVEM as an immune evasion strategy. Immunization of Hvem(−/−) mice with ΔgD-2 resulted in significant reduction in HSV-specific IgG2 antibodies, the subclass associated with FcγR activation and ADCC, compared to wild-type controls. This translated into a parallel reduction in active and passive vaccine protection. A similar decrease in ADCC titers was observed in Hvem(−/−) mice vaccinated with an alternative HSV vaccine candidate (dl5–29) or an unrelated vesicular stomatitis virus-vectored vaccine. Surprisingly, not only did passive transfer of immune serum from ΔgD-2-vaccinated Hvem(−/−) mice fail to protect wild-type mice, transfer of immune serum from ΔgD-2-vaccinated wild-type mice failed to protect Hvem(−/−) mice. Immune cells isolated from Hvem(−/−) mice were impaired in FcγR activation and, conversely, addition of gD protein or anti-HVEM antibodies to in vitro murine or human FcγR activation assays inhibited the response. Together, these findings uncover a previously unrecognized role for HVEM signaling in generating and mediating ADCC and an additional HSV immune evasion strategy.

Despite the increased research efforts, full-scale implementation of shortcut nitrogen removal strategies has been challenged by the lack of consistent nitrite-oxidizing bacteria out-selection. This paper proposes an alternative path using partial denitrification (PdN) selection coupled with anaerobic ammonium-oxidizing bacteria (AnAOB). A nitrate residual concentration (>2 mg N/L) was identified as the crucial factor for metabolic PdN selection using acetate as a carbon source, unlike the COD/N ratio which was often suggested. Therefore, a novel and simple acetate dosing control strategy based on maintaining a nitrate concentration was tested in the absence and presence of AnAOB, achieving PdN efficiencies above 80%. The metabolic-based PdN selection allowed for flexibility to move between PdN and full denitrification when required to meet effluent nitrate levels. Due to the independence of this strategy on species selection and management of nitrite competition, this novel approach will guarantee nitrite availability for AnAOB under mainstream conditions unlike shortcut nitrogen removal approaches based on NOB out-selection. Overall, a COD addition of only 2.2 g COD/g TIN removed was needed for the PdN-AnAOB concept showing its potential for significant savings in external carbon source needs to meet low TIN effluent concentrations making this concept a competitive alternative. PRACTITIONER POINTS: Nitrate residual is the key control parameter for partial denitrification selection. Metabolic selection allowed for flexibility of moving from partial to full denitrification. 2.2 g COD/g TIN removed was needed for partial denitrification-anammox process.

A new model for accounting carbon and energy footprint of wastewater treatment plants (WWTPs) is proposed. The model quantifies direct and indirect greenhouse gas (GHG) emissions related to biological and physical processes of a WWTP. The model takes into account several innovative aspects with respect to already available literature models: i. kinetic/mass-balances; ii. nitrification as a two-step process; iii. nitrous oxide (N2O) formation during nitrification and denitrification both in dissolved and off-gas forms. A full-scale application has been performed by adopting the case study of a real WWTP. A scenario analysis was performed to quantify the influence of: composition of inflow wastewater (scenario 1), operating conditions (scenario 2), and oxygen transfer efficiency (scenario 3). Results have underlined the key role of the ratio between influent biodegradable carbon and nitrogen concentration on influencing direct and indirect GHG emissions. Direct GHG emissions increase from 0.49 to 0.63 kgCO2eq m−3 with the decrease of the influent ratio of the readily biodegradable carbon and organic and ammonia nitrogen. The increase of the influent organic and ammonia nitrogen favours the daily production of active ammonia oxidization biomass. The simultaneous variation of the investigated factors has amplified direct and indirect GHG emissions to a maximum value of 0.94 and 0.24 kgCO2eq m−3, respectively.

The molecular mechanisms of microbial adaptation to repeated anoxic-aerobic cycling were investigated by integrating and whole community gene expression (metatranscriptomics) and physiological responses, including the production of nitric (NO) and nitrous (N2O) oxides. Anoxic-aerobic cycling was imposed for seventeen days in a lab-scale full-nitrification mixed culture system. Prior to cycling, NO and N2O levels were sustained at 0.097 ± 0.006 and 0.054 ± 0.019 ppmv, respectively. Once the anoxic-aerobic cycling was initiated, peak emissions were highest on the first day (9.8 and 1.3 ppmv, respectively). By the end of day seventeen, NO production returned to pre-cycling levels (a peak of 0.12 ± 0.007 ppmv), while N2O production reached a new baseline (a peak of 0.32 ± 0.05 ppmv), one order of magnitude higher than steady-state conditions. Concurrently, post-cycling transcription of norBQ and nosZ returned to pre-cycling levels after an initial 5.7- and 9.5-fold increase, while nirK remained significantly expressed (1.6-fold) for the duration of and after cycling conditions. The imbalance in nirK and nosZ mRNA abundance coupled with continuous conversion of NO to N2O might explain the elevated post-cycling baseline for N2O. Metatranscriptomic investigation notably indicated possible NO production by NOB under anoxic-aerobic cycling through a significant increase in nirK expression. Opposing effects on AOB (down-regulation) and NOB (up-regulation) CO2 fixation were observed, suggesting that nitrifying bacteria are differently impacted by anoxic-aerobic cycling. Genes encoding the terminal oxidase of the electron transport chain (ccoNP, coxBC) were the most significantly transcribed, highlighting a hitherto unexplored pathway to manage high electron fluxes resulting from increased ammonia oxidation rates, and leading to overall, increased NO and N2O production. In sum, this study identified underlying metabolic processes and mechanisms contributing to NO and N2O production through a systems-level interrogation, which revealed the differential ability of specific microbial groups to adapt to sustained operational conditions in engineered biological nitrogen removal processes.

Quantitative PCR (qPCR) is broadly used as the gold standard to quantify microbial community fractions in environmental microbiology and biotechnology. Benchmarking efforts to ensure the comparability of qPCR data for environmental bioprocesses are still scarce. Also, for partial nitritation/anammox (PN/A) systems systematic investigations are still missing, rendering meta-analysis of reported trends and generic insights potentially precarious. We report a baseline investigation of the variability of qPCR-based analyses for microbial communities applied to PN/A systems. Round-robin testing was performed for three PN/A biomass samples in six laboratories, using the respective in-house DNA extraction and qPCR protocols. The concentration of extracted DNA was significantly different between labs, ranged between 2.7 and 328 ng mg−1 wet biomass. The variability among the qPCR abundance data of different labs was very high (1−7 log fold) but differed for different target microbial guilds. DNA extraction caused maximum variation (3–7 log fold), followed by the primers (1–3 log fold). These insights will guide environmental scientists and engineers as well as treatment plant operators in the interpretation of qPCR data.