Intensive Microalgal Cultivation and Tertiary Phosphorus Recovery from Wastewaters via the EcoRecover Process.

Mixed community microalgal wastewater treatment technologies have the potential to advance the limits of technology for biological nutrient recovery while producing a renewable carbon feedstock, but a deeper understanding of their performance is required for system optimization and control. In this study, we characterized the performance of a 568 m3·day-1 Clearas EcoRecover system for tertiary phosphorus removal (and recovery as biomass) at an operating water resource recovery facility (WRRF). The process consists of a (dark) mix tank, photobioreactors (PBRs), and a membrane tank with ultrafiltration membranes for the separation of hydraulic and solids residence times. Through continuous online monitoring, long-term on-site monitoring, and on-site batch experiments, we demonstrate (i) the importance of carbohydrate storage in PBRs to support phosphorus uptake under dark conditions in the mix tank and (ii) the potential for polyphosphate accumulation in the mixed algal communities. Over a 3-month winter period with limited outside influences (e.g., no major upstream process changes), the effluent total phosphorus (TP) concentration was 0.03 ± 0.03 mg-P·L-1 (0.01 ± 0.02 mg-P·L-1 orthophosphate). Core microbial community taxa included Chlorella spp., Scenedesmus spp., and Monoraphidium spp., and key indicators of stable performance included near-neutral pH, sufficient alkalinity, and a diel rhythm in dissolved oxygen.

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations.

Lack of SARS-CoV-2 in environmental samples collected from September 2020-February 2021 in a university that followed CDC reopening guidance.

This study aimed to provide environmental surveillance data for evaluating the risk of acquiring SARS-CoV-2 in public areas with high foot traffic in a university. Air and surface samples were collected at a university that had the second highest number of COVID-19 cases among public higher education institutions in the U.S. during Fall 2020. A total of 60 samples were collected in 16 sampling events performed during Fall 2020 and Spring 2021. Nearly 9800 students traversed the sites during the study period. SARS-CoV-2 was not detected in any air or surface samples. The university followed CDC guidance, including COVID-19 testing, case investigations, and contact tracing. Students, faculty, and staff were asked to maintain physical distancing and wear face coverings. Although COVID-19 cases were relatively high at the university, the possibility of acquiring SARS-CoV-2 infections at the sites tested was low.

Diversity and Genetic Reassortment of Keystone Virus in Mosquito Populations in Florida.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected > 6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR-positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

Analysis of Zika Virus Sequence Data Associated with a School Cohort in Haiti.

Zika virus (ZIKV) infections occurred in epidemic form in the Americas in 2014-2016, with some of the earliest isolates in the region coming from Haiti. We isolated ZIKV from 20 children with acute undifferentiated febrile illness who were part of a cohort of children seen at a school clinic in the Gressier region of Haiti. The virus was also isolated from three pools of Aedes aegypti mosquitoes collected at the same location. On phylogenetic analysis, three distinct ZIKV clades were identified. Strains from all three clades were present in Haiti in 2014, making them among the earliest isolates identified in the Western Hemisphere. Strains from all three clades were also isolated in 2016, indicative of their persistence across the time period of the epidemic. Mosquito isolates were collected in 2016 and included representatives from two of the three clades; in one instance, ZIKV was isolated from a pool of male mosquitoes, suggestive of vertical transmission of the virus. The identification of multiple ZIKV clades in Haiti at the beginning of the epidemic suggests that Haiti served as a nidus for transmission within the Caribbean.

Spatial Video and EpiExplorer: A Field Strategy to Contextualize Enteric Disease Risk in Slum Environments.

Disease risk associated with contaminated water, poor sanitation, and hygiene in informal settlement environments is conceptually well understood. From an analytical perspective, collecting data at a suitably fine scale spatial and temporal granularity is challenging. Novel mobile methodologies, such as spatial video (SV), can complement more traditional epidemiological field work to address this gap. However, this work then poses additional challenges in terms of analytical visualizations that can be used to both understand sub-neighborhood patterns of risk, and even provide an early warning system. In this paper, we use bespoke spatial programming to create a framework for flexible, fine-scale exploratory investigations of simultaneously-collected water quality and environmental surveys in three different informal settlements of Port-au-Prince, Haiti. We dynamically mine these spatio-temporal epidemiological and environmental data to provide insights not easily achievable using more traditional spatial software, such as Geographic Information System (GIS). The results include sub-neighborhood maps of localized risk that vary monthly. Most interestingly, some of these epidemiological variations might have previously been erroneously explained because of proximate environmental factors and/or meteorological conditions.

Environmental Surveillance for SARS-CoV-2 in Two Restaurants from a Mid-scale City that Followed U.S. CDC Reopening Guidance.

Since mask use and physical distancing are difficult to maintain when people dine indoors, restaurants are perceived as high risk for acquiring COVID-19. The air and environmental surfaces in two restaurants in a mid-scale city located in north central Florida that followed the Centers for Disease Control and Prevention (CDC) reopening guidance were sampled three times from July 2020 to February 2021. Sixteen air samples were collected for 2 hours using air samplers, and 20 surface samples by using moistened swabs. The samples were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence of SARS-CoV-2 genomic RNA. A total of ~550 patrons dined in the restaurants during our samplings. SARS-CoV-2 genomic RNA was not detected in any of the air samples. One of the 20 surface samples (5%) was positive. That sample had been collected from a plastic tablecloth immediately after guests left the restaurant. Virus was not isolated in cell cultures inoculated with aliquots of the RT-PCR-positive sample. The likelihood that patrons and staff acquire SARS-CoV-2 infections may be low in restaurants in a mid-scale city that adopt CDC restaurant reopening guidelines, such as operation at 50% capacity so that tables can be spaced at least 6 feet apart, establishment of adequate mechanical ventilation, use of a face covering except while eating or drinking, and implementation of disinfection measures.

Isolation of a Novel Recombinant Canine Coronavirus From a Visitor to Haiti: Further Evidence of Transmission of Coronaviruses of Zoonotic Origin to Humans.

We isolated a novel coronavirus from a medical team member presenting with fever and malaise after travel to Haiti. The virus showed 99.4% similarity with a recombinant canine coronavirus recently identified in a pneumonia patient in Malaysia, suggesting that infection with this virus and/or recombinant variants occurs in multiple locations.

SARS-CoV-2 in residential rooms of two self-isolating persons with COVID-19.

Individuals with COVID-19 are advised to self-isolate at their residences unless they require hospitalization. Persons sharing a dwelling with someone who has COVID-19 have a substantial risk of being exposed to the virus. However, environmental monitoring for the detection of virus in such settings is limited. We present a pilot study on environmental sampling for SARS-CoV-2 virions in the residential rooms of two volunteers with COVID-19 who self-quarantined. Apart from standard surface swab sampling, based on availability, four air samplers positioned 0.3-2.2 m from the volunteers were used: a VIable Virus Aerosol Sampler (VIVAS), an inline air sampler that traps particles on polytetrafluoroethylene (PTFE) filters, a NIOSH 2-stage cyclone sampler (BC-251), and a Sioutas personal cascade impactor sampler (PCIS). The latter two selectively collect particles of specific size ranges. SARS-CoV-2 RNA was detected by real-time Reverse-Transcription quantitative Polymerase Chain Reaction (rRT-qPCR) analyses of particles in one air sample from the room of volunteer A and in various air and surface samples from that of volunteer B. The one positive sample collected by the NIOSH sampler from volunteer A's room had a quantitation cycle (Cq) of 38.21 for the N-gene, indicating a low amount of airborne virus [5.69E-02 SARS-CoV-2 genome equivalents (GE)/cm3 of air]. In contrast, air samples and surface samples collected off the mobile phone in volunteer B's room yielded Cq values ranging from 14.58 to 24.73 and 21.01 to 24.74, respectively, on the first day of sampling, indicating that this volunteer was actively shedding relatively high amounts of SARS-CoV-2 at that time. The SARS-CoV-2 GE/cm3 of air for the air samples collected by the PCIS was in the range 6.84E+04 to 3.04E+05 using the LED-N primer system, the highest being from the stage 4 filter, and similarly, ranged from 2.54E+03 to 1.68E+05 GE/cm3 in air collected by the NIOSH sampler. Attempts to isolate the virus in cell culture from the samples from volunteer B's room with the aforementioned Cq values were unsuccessful due to out-competition by a co-infecting Human adenovirus B3 (HAdVB3) that killed the Vero E6 cell cultures within 4 days of their inoculation, although Cq values of 34.56-37.32 were measured upon rRT-qPCR analyses of vRNA purified from the cell culture medium. The size distribution of SARS-CoV-2-laden aerosol particles collected from the air of volunteer B's room was >0.25 µm and >0.1 µm as recorded by the PCIS and the NIOSH sampler, respectively, suggesting a risk of aerosol transmission since these particles can remain suspended in air for an extended time and travel over long distances. The detection of virus in surface samples also underscores the potential for fomite transmission of SARS-CoV-2 in indoor settings.

Long-Term Exposure and Effects of rGO-nZVI Nanohybrids and Their Parent Nanomaterials on Wastewater-Nitrifying Microbial Communities.

Single nanomaterials and nanohybrids (NHs) can inhibit microbial processes in wastewater treatment, especially nitrification. While existing studies focus on short-term and acute exposures of single nanomaterials on wastewater microbial community growth and function, long-term, low-exposure, and emerging NHs need to be examined. These NHs have distinctly different physicochemical properties than their parent nanomaterials and, therefore, may exert previously unknown effects onto wastewater microbial communities. This study systematically investigated long-term [∼6 solid residence time [(SRT)] exposure effects of a widely used carbon-metal NH (rGO-nZVI = 1:2 and 1:0.2, mass ratio) and compared these effects to their single-parent nanomaterials (i.e., rGO and nZVI) in nitrifying sequencing batch reactors. nZVI and NH-dosed reactors showed relatively unaffected microbial communities compared to control, whereas rGO showed a significantly different (p = 0.022) and less diverse community. nZVI promoted a diverse community and significantly higher (p < 0.05) biomass growth under steady-state conditions. While long-term chronic exposure (10 mg·L-1) of single nanomaterials and NHs had limited impact on long-term nutrient recovery, functionally, the reactors dosed with higher iron content, that is, nZVI and rGO-nZVI (1:2), promoted faster NH4+-N removal due to higher biomass growth and upregulation of amoA genes at the transcript level, respectively. The transmission electron microscopy images and scanning electron microscopy─energy-dispersive X-ray spectroscopy analysis revealed high incorporation of iron in nZVI-dosed biomass, which promoted higher cellular growth and a diverse community. Overall, this study shows that NHs have unique effects on microbial community growth and function that cannot be predicted from parent materials alone.

Independent infections of porcine deltacoronavirus among Haitian children.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3-5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human-animal contact.

A Valve-Enabled Sample Preparation Device with Isothermal Amplification for Multiplexed Virus Detection at the Point-of-Care.

Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses at the point-of-care is crucial for reducing disease transmission during the current pandemic and future flu seasons. To prepare for potential cocirculation of these two viruses, we report a valve-enabled, paper-based sample preparation device integrated with isothermal amplification for their simultaneous detection. The device incorporates (1) virus lysis and RNA enrichment, enabled by ball-based valves for sequential delivery of reagents with no pipet requirement, (2) reverse transcription loop-mediated isothermal amplification, carried out in a coffee mug, and (3) colorimetric detection. We have used the device for simultaneously detecting inactivated SARS-CoV-2 and influenza A H1N1 viruses in 50 min, with limits of detection at 2 and 6 genome equivalents, respectively. The device was further demonstrated to detect both viruses in environmental samples.

Status and advances in technologies for phosphorus species detection and characterization in natural environment- A comprehensive review.

Poor recovery of phosphorus (P) across natural environment (water, soil, sediment, and biological sources) is causing rapid depletion of phosphate rocks and continuous accumulation of P in natural waters, resulting in deteriorated water quality and aquatic lives. Accurate detection and characterization of various P species using suitable analytical methods provide a comprehensive understanding of the biogeochemical cycle of P and thus help its proper management in the environment. This paper aims to provide a comprehensive review of the analytical methods used for P speciation in natural environment by dividing them into five broad categories (i.e., chemical, biological, molecular, staining microscopy, and sensors) and highlighting the suitability (i.e., targeted species, sample matrix), detection limit, advantages-limitations, and reference studies of all methods under each category. This can be useful in designing studies involving P detection and characterization across environmental matrices by providing insights about a wide range of analytical methods based on the end user application needs of individual studies.

Yield, profitability, and prospects of irrigated Boro rice cultivation in the North-West region of Bangladesh.

The North-West (NW) region of Bangladesh is pivotal for the country's agricultural development, mainly in producing irrigated Boro rice. However, increasing cost of irrigation water, fertilizers, labour and other inputs, and the spatio-temporal variation in actual yield, market price and profitability of rice, have added uncertainty to the sustainability of Boro rice cultivation. In this study, we evaluated the productivity, profitability, and prospect of Boro rice production using comprehensive field data collected directly from 420 farmers' fields over two consecutive seasons (2015-16 and 2016-17), across seven geographically distributed locations in the NW region. We also analyzed the risk and return trade of popular Boro rice cultivars using Monte-Carlo simulation. The results show that there were significant (p≤0.05) variations in rice yield between sites, irrigation pump-types, and rice varieties, with Hybrid rice and BRRI dhan29 producing highest yields (6.0-7.5 t/ha). Due to different pricing systems, the cost of irrigation water varied from site to site and from year to year, but always comprised the highest input cost (20-25% of total production). The total paid-out cost, gross benefit, and gross income of rice significantly (p≤0.05) differed between sites, type of irrigation pumps, rice varieties, transplanting dates, and two cropping years. The variations in observed yield and profitability reveal considerable scope to improve rice production systems. Market variation in the price of rice affected overall profitability significantly. Probability and risk analysis results show that Minikit and BRRI dhan29 are the most stable varieties for yield and profitability. Hybrid rice, which has the maximum attainable yield among the cultivated rice varieties, also has the risk of negative net income. Based on the analysis, we discussed ways to improve yield and profitability and the prospect of Boro rice cultivation in the region. The study provides valuable information for policy-makers to sustain irrigated rice cultivation in both the NW region and nationally.

Emergence of porcine delta-coronavirus pathogenic infections among children in Haiti through independent zoonoses and convergent evolution.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, coronavirus emergence in our species has been associated with zoonotic transmissions from animal reservoirs 1,2 , underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae - Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus , - human infections reported to date have been limited to alpha- and betacoronaviruses 3 . We identify, for the first time, porcine deltacoronavirus (PDCoV) strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the nsp15 and the spike glycoprotein genes by convergent evolution. In particular, structural analysis predicts that one of the changes in the Spike S1 subunit, which contains the receptor-binding domain, may affect protein's flexibility and binding to the host cell receptor. Our findings not only underscore the ability of deltacoronaviruses to adapt and potentially lead to human-to-human transmission, but also raise questions about the role of such transmissions in development of pre-existing immunity to other coronaviruses, such as SARS-CoV-2.

Earliest detection to date of SARS-CoV-2 in Florida: Identification together with influenza virus on the main entry door of a university building, February 2020.

In February and March, 2020, environmental surface swab samples were collected from the handle of the main entry door of a major university building in Florida, as part of a pilot surveillance project screening for influenza. Samples were taken at the end of regular classroom hours, between the dates of February 1-5 and February 19-March 4, 2020. Influenza A(H1N1)pdm09 virus was isolated from the door handle on four of the 19 days sampled. Both SARS-CoV-2 and A(H1N1)pdm09 virus were detected in a sample collected on February 21, 2020. Based on sequence analysis, the Florida SARS-CoV-2 strain (designated UF-11) was identical to strains being identified in Washington state during the same time period, while the earliest similar sequences were sampled in China/Hubei between Dec 30th 2019 and Jan 5th 2020. The first human case of COVID-19 was not officially reported in Florida until March 1st. In an analysis of sequences from COVID-19 patients in this region of Florida, there was only limited evidence of subsequent dissemination of the UF-11 strain. Identical or highly similar strains, possibly related through a common transmission chain, were detected with increasing frequency in Washington state between end of February and beginning of March. Our data provide further documentation of the rapid early spread of SARS-CoV-2 and underscore the likelihood that closely related strains were cryptically circulating in multiple U.S. communities before the first "official" cases were recognized.

Exploring convolutional neural networks and spatial video for on-the-ground mapping in informal settlements.

BACKGROUND: The health burden in developing world informal settlements often coincides with a lack of spatial data that could be used to guide intervention strategies. Spatial video (SV) has proven to be a useful tool to collect environmental and social data at a granular scale, though the effort required to turn these spatially encoded video frames into maps limits sustainability and scalability. In this paper we explore the use of convolution neural networks (CNN) to solve this problem by automatically identifying disease related environmental risks in a series of SV collected from Haiti. Our objective is to determine the potential of machine learning in health risk mapping for these environments by assessing the challenges faced in adequately training the required classification models. RESULTS: We show that SV can be a suitable source for automatically identifying and extracting health risk features using machine learning. While well-defined objects such as drains, buckets, tires and animals can be efficiently classified, more amorphous masses such as trash or standing water are difficult to classify. Our results further show that variations in the number of image frames selected, the image resolution, and combinations of these can be used to improve the overall model performance. CONCLUSION: Machine learning in combination with spatial video can be used to automatically identify environmental risks associated with common health problems in informal settlements, though there are likely to be variations in the type of data needed for training based on location. Success based on the risk type being identified are also likely to vary geographically. However, we are confident in identifying a series of best practices for data collection, model training and performance in these settings. We also discuss the next step of testing these findings in other environments, and how adding in the simultaneously collected geographic data could be used to create an automatic health risk mapping tool.

Environmental Surveillance and Transmission Risk Assessments for SARS-CoV-2 in a Fitness Center.

Fitness centers are considered high risk for SARS-CoV-2 transmission due to their high human occupancy and the type of activity taking place in them, especially when individuals pre-symptomatic or asymptomatic for COVID-19 exercise in the facilities. In this study, air (N=21) and surface (N=8) samples were collected at a fitness center through five sampling events from August to November 2020 after the reopening restrictions were lifted in Florida. The total attendance was ~2500 patrons during our air and environmental sampling work. Air samples were collected using stationary and personal bioaerosol samplers. Moistened flocked nylon swabs were used to collect samples from high-touch surfaces. We did not detect SARS-CoV-2 by rRT-PCR analyses in any air or surface sample. A simplified infection risk model based on the Wells-Riley equation predicts that the probability of infection in this fitness center was 1.77% following its ventilation system upgrades based on CDC guidelines, and that risk was further reduced to 0.89% when patrons used face masks. Our model also predicts that a combination of high ventilation, minimal air recirculation, air filtration, and UV sterilization of recirculated air reduced the infection risk up to 94% compared to poorly ventilated facilities. Amongst these measures, high ventilation with outdoor air is most critical in reducing the airborne transmission of SARS-CoV-2. For buildings that cannot avoid air recirculation due to energy costs, the use of high filtration and/or air disinfection devices are alternatives to reducing the probability of acquiring SARS-CoV-2 through inhalation exposure. In contrast to the perceived ranking of high risk, the infection risk in fitness centers that follow CDC reopening guidance, including implementation of engineering and administrative controls, and use of personal protective equipment, can be low, and these facilities can offer a relatively safe venue for patrons to exercise.

Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients.

OBJECTIVES: Because the detection of SARS-CoV-2 RNA in aerosols but failure to isolate viable (infectious) virus are commonly reported, there is substantial controversy whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be transmitted through aerosols. This conundrum occurs because common air samplers can inactivate virions through their harsh collection processes. We sought to resolve the question whether viable SARS-CoV-2 can occur in aerosols using VIVAS air samplers that operate on a gentle water vapor condensation principle. METHODS: Air samples collected in the hospital room of two coronavirus disease-2019 (COVID-19) patients, one ready for discharge and the other newly admitted, were subjected to RT-qPCR and virus culture. The genomes of the SARS-CoV-2 collected from the air and isolated in cell culture were sequenced. RESULTS: Viable SARS-CoV-2 was isolated from air samples collected 2 to 4.8 m away from the patients. The genome sequence of the SARS-CoV-2 strain isolated from the material collected by the air samplers was identical to that isolated from the newly admitted patient. Estimates of viable viral concentrations ranged from 6 to 74 TCID50 units/L of air. CONCLUSIONS: Patients with respiratory manifestations of COVID-19 produce aerosols in the absence of aerosol-generating procedures that contain viable SARS-CoV-2, and these aerosols may serve as a source of transmission of the virus.

Bis{4-[(2-hy-droxy-5-meth-oxy-3-nitro-benzyl-idene)amino]-phen-yl} ether.

The mol-ecule of the title compound, C28H22N4O9, exhibits crystallographically imposed twofold rotational symmetry, with a dihedral angle of 66.0 (2)° between the planes of the two central benzene rings bounded to the central oxygen atom. The dihedral angle between the planes of the central benzene ring and the terminal phenol ring is 4.9 (2)°. Each half of the mol-ecule exhibits an imine E configuration. An intra-molecular O-H⋯N hydrogen bond is present. In the crystal, the mol-ecules are linked into layers parallel to the ab plane via C-H⋯O hydrogen bonds. The crystal studied was refined as a two-component pseudomerohedral twin.