Editors' Choice-Perspective-Deciphering the Glycan Kryptos by Solid-State Nanopore Single-Molecule Sensing: A Call for Integrated Advancements Across Glyco- and Nanopore Science.

Glycans, or complex carbohydrates, are information-rich biopolymers critical to many biological processes and with considerable importance in pharmaceutical therapeutics. Our understanding, though, is limited compared to other biomolecules such as DNA and proteins. The greater complexity of glycan structure and the limitations of conventional chemical analysis methods hinder glycan studies. Auspiciously, nanopore single-molecule sensors-commercially available for DNA sequencing-hold great promise as a tool for enabling and advancing glycan analysis. We focus on two key areas to advance nanopore glycan characterization: molecular surface coatings to enhance nanopore performance including by molecular recognition, and high-quality glycan chemical standards for training.

Coupling Partial Nitritation, Anammox and n-DAMO in a membrane aerated biofilm reactor for simultaneous dissolved methane and nitrogen removal.

Anaerobic technologies with downstream autotrophic nitrogen removal have been proposed to enhance bioenergy recovery and transform a wastewater treatment plant from an energy consumer to an energy exporter. However, approximately 20-50 % of the produced methane is dissolved in the anaerobically treated effluent and is easily stripped into the atmosphere in the downstream aerobic process, contributing to the release of greenhouse gas emissions. This study aims to develop a solution to beneficially utilize dissolved methane to support high-level nitrogen removal from anaerobically treated mainstream wastewater. A novel technology, integrating Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (i.e. PNAM) was demonstrated in a membrane-aerated biofilm reactor (MABR). With the feeding of ∼50 mg NH4+-N/L and ∼20 mg/L dissolved methane at a hydraulic retention time of 15 h, around 90 % of nitrogen and ∼100 % of dissolved methane can be removed together in the MABR. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (n-DAMO bacteria and archaea) and aerobic methanotrophs co-existed in the established biofilm. Batch tests confirmed the active microbial pathways and showed that AOB, anammox bacteria and n-DAMO microbes were jointly responsible for the nitrogen removal, and dissolved methane was mainly removed by the n-DAMO process, with aerobic methane oxidation making a minor contribution. In addition, the established system was robust against dynamic changes in influent composition. The study provides a promising technology for the simultaneous removal of dissolved methane and nitrogen from domestic wastewater, which can support the transformation of wastewater treatment from an energy- and carbon-intensive process, to one that is energy- and carbon-neutral.

Comprehensive assessment of microplastics in Australian biosolids: Abundance, seasonal variation and potential transport to agroecosystems.

Striving towards a circular economy, the application of treated sewage sludge (biosolids) to land is an opportunity to improve the condition of the soil and add essential nutrients, in turn reducing the need for fertilisers. However, there is an increasing concern about microplastic (MP) contamination of biosolids and their transport to terrestrial ecosystems. In Australia, agriculture is the largest biosolids end-user, however, there is limited understanding of MPs in Australian biosolids. Also, while the method to isolate MPs from biosolid is established, a need to extract and analyse MPs more efficiently is still pressing. In this study, we comprehensively quantified and characterised MPs in 146 biosolids samples collected from thirteen wastewater treatment plants (WWTPs) including different seasons. We have optimised an oxidative-enzymatic purification method to overcome current limitations for MP identification in complex samples and accurately report MPs in biosolids. This method enabled removal of >93 % of dry weight of organic material and greatly facilitated the MPs instrumental analysis. The concentration of MPs (>20 µm) in all biosolids samples ranged from 11 to 150 MPs/g dry weight. Abundance of MPs was affected by seasons with higher abundance of MPs usually found during cold and wet seasons. Despite seasonal variations, polyethylene terephthalate, polyurethane and polymethyl methacrylate were the most abundant polymers. Smaller MPs (20 to 200 µm) comprised >70 % of all detected MPs with a clear negative linear relationship observed between MP size and abundance. Per capita concentration of MPs in biosolids across all studied WWTPs was 0.7 to 21 g MPs per person per year. Therefore, biosolids are an important sink and source of MPs to agroecosystems, emphasising the need to more comprehensively understand the fate, impact and risks associated with MPs on agricultural soils.

Probing nanopore surface chemistry through real-time measurements of nanopore conductance response to pH changes.

We developed a flow cell apparatus and method for streamlined, real-time measurements of nanopore conductance (G) in response to pH changes. By time-resolving the measurements of interfacial kinetics, we were able to probe nanopore surface coating presence and properties more thoroughly than in our previous work. Nanopores have emerged as a prominent tool for single-molecule sensing, characterization, and sequencing of DNA, proteins, and carbohydrates. Nanopore surface chemistry affects analyte passage, signal characteristics, and sensor lifetime through a range of electrostatic, electrokinetic, and chemical phenomena, and optimizing nanopore surface chemistry has become increasingly important. Our work makes nanopore surface chemistry characterizations more accessible as a complement to routine single-pH conductance measurements used to infer nanopore size. We detail the design and operation of the apparatus and discuss the trends in G and capacitance. Characteristic G vs pH curves matching those obtained in previous work could be obtained with the addition of time-resolved interfacial kinetic information. We characterized native and chemically functionalized (carboxylated) silicon nitride (SiNx) nanopores, illustrating how the method can inform of thin film compositions, interfacial kinetics, and nanoscale chemical phenomena.

Deep learning in wastewater treatment: a critical review.

Modeling wastewater processes supports tasks such as process prediction, soft sensing, data analysis and computer assisted design of wastewater systems. Wastewater treatment processes are large, complex processes, with multiple controlling mechanisms, a high degree of disturbance variability and non-linear (generally stable) behavior with multiple internal recycle loops. Semi-mechanistic biochemical models currently dominate research and application, with data-driven deep learning models emerging as an alternative and supplementary approach. But these modeling approaches have grown in separate communities of research and practice, and so there is limited appreciation of the strengths, weaknesses, contrasts and similarities between the methods. This review addresses that gap by providing a detailed guide to deep learning methods and their application to wastewater process modeling. The review is aimed at wastewater modeling experts who are familiar with established mechanistic modeling approach, and are curious about the opportunities and challenges afforded by deep learning methods. We conclude with a discussion and needs analysis on the value of different ways of modeling wastewater processes and open research problems.

N-Heterocyclic carbene-based gold etchants.

N-Heterocyclic carbenes (NHCs) are an emerging alternative to thiols for the formation of stable self-assembled monolayers (SAMs) on gold. We examined several different species that have been used to produce NHC-based monolayers on gold, namely 1,3-diisopropyl-5-nitrobenzimidazolium iodide, 1,3-diisopropyl-5-nitrobenzimidazolium hydrogen carbonate, bis(1,3-diisopropyl-5-nitrobenzimidazolium)gold(I) iodide, and 1,3-diisopropyl-5-nitrobenzimidazole-2-ylidene. Contrary to expectation, solutions containing the first two species in tetrahydrofuran and dichloromethane caused visible loss of gold from thin-film-coated glass slides. The use of toluene solutions of all species resulted in no apparent dissolution of gold. We present scanning electron micrographs and elemental imaging analyses by energy dispersive X-ray spectroscopy to examine the effect of solutions of each species on the gold film. This work highlights the risk of unwanted etching during some routes to NHC-based surface functionalization but also the potential for deliberate etching, with the outcome determined by choice of chemically synthesized organic species and solvent.

Biofouling potential of surface-enhanced Raman scattering-based seawater quality sensors by Ulva spp.

This study investigated the biofouling potential of surface-enhanced Raman scattering (SERS)-based sensor materials in the context of marine environments. Uncoated and monolithic commercial gold (Au) silicon nanopillar array SERS substrates, Au-coated carbon black nanoparticle (AuCB NP) substrates, uncoated and Au sputter-coated in-house SERS, and uncoated and Au sputter-coated glass controls were tested for biofouling potential using Ulva spp. as model biofouling organisms. The mean percentages of Ulva spp. zoospores that adhered per mm2 (×103) on the uncoated and coated Au silicon nanopillar array, AuCB NP, uncoated and Au sputter-coated in-house, and uncoated and Au sputter-coated glass substrates were 10.28%, 5.45%, 10.49%, 3.25%, 24.84%, 12.86% and 7.78%, respectively. Results indicated that surface properties such as hydrophobicity, roughness, Au sputter-coating and the presence of micro-refuges on nano- and microstructured substrates were critical to the biofouling formation.

Over One Million DNA and Protein Events Through Ultra-Stable Chemically-Tuned Solid-State Nanopores.

Stability, long lifetime, resilience against clogging, low noise, and low cost are five critical cornerstones of solid-state nanopore technology. Here, a fabrication protocol is described wherein >1 million events are obtained from a single solid-state nanopore with both DNA and protein at the highest available lowpass filter (LPF, 100 kHz) of the Axopatch 200B-the highest event count mentioned in literature. Moreover, a total of ≈8.1 million events are reported in this work encompassing the two analyte classes. With the 100 kHz LPF, the temporally attenuated population is negligible while with the more ubiquitous 10 kHz, ≈91% of the events are attenuated. With DNA experiments, the pores are operational for hours (typically >7 h) while the average pore growth is merely ≈0.16 ± 0.1 nm h-1 . The current noise is exceptionally stable with traces typically showing <10 pA h-1 increase in noise. Furthermore, a real-time method to clean and revive pores clogged with analyte with the added benefit of minimal pore growth during cleaning (< 5% of the original diameter) is showcased. The enormity of the data collected herein presents a significant advancement to solid-state pore performance and will be useful for future ventures such as machine learning where large amounts of pristine data are a prerequisite.

One-year stable pilot-scale operation demonstrates high flexibility of mainstream anammox application.

Mainstream nitrogen removal via anammox is widely recognized as a promising wastewater treatment process. However, its application is challenging at large scale due to unstable suppression of nitrite-oxidizing bacteria (NOB). In this study, a pilot-scale mainstream anammox process was implemented in an Integrated Fixed-film Activated Sludge (IFAS) configuration. Stable operation with robust NOB suppression was maintained for over one year. This was achieved through integration of three key control strategies: i) low dissolved oxygen (DO = 0.4 ± 0.2 mg O2/L), ii) regular free nitrous acid (FNA)-based sludge treatment, and iii) residual ammonium concentration control (NH4 + with a setpoint of ∼8 mg N/L). Activity tests and FISH demonstrated that NOB barely survived in sludge flocs and were inhibited in biofilms. Despite receiving organic-deficient wastewater from a pilot-scale High-Rate Activated Sludge (HRAS) system as the feed, the system maintained a stable effluent total nitrogen concentration mostly below 10 mg N/L, which was attributed to the successful retention of anammox bacteria. This study successfully demonstrated large-scale long-term mainstream anammox application and generated new practical knowledge for NOB control and anammox retention.

Achieving robust mainstream nitrite shunt at pilot-scale with integrated sidestream sludge treatment and step-feed.

As a promising energy- and carbon efficient process for nitrogen removal from wastewater, mainstream nitrite shunt has been extensively researched. However, beyond the laboratory it is challenging to maintain stable performance by suppressing nitrite-oxidising bacteria (NOB). In this study, a pilot-scale reactor system receiving real sewage was operated in two stages for >850 days to evaluate two novel NOB suppression strategies for achieving nitrite shunt: i) sidestream sludge treatment based on alternating free nitrous acid (FNA) and free ammonia (FA) and ii) sidestream FNA/FA sludge treatment integrated with in-situ NOB suppression via step-feed. The results showed that, with sidestream sludge treatment alone, NOB developed resistance relatively quickly to the treatment, leading to unstable nitrite shunt. In contrast, robust nitrite shunt was achieved and stably maintained for more than a year when sidestream sludge treatment was integrated with a step-feed strategy. Kinetic analyses suggested that sludge treatment and step-feed worked in synergy, leading to stable NOB suppression. The integrated strategy demonstrated in this study removes a key barrier to the implementation of stable mainstream nitrite shunt.

Photoswitchable Binary Nanopore Conductance and Selective Electronic Detection of Single Biomolecules under Wavelength and Voltage Polarity Control.

We fabricated photoregulated thin-film nanopores by covalently linking azobenzene photoswitches to silicon nitride pores with ∼10 nm diameters. The photoresponsive coatings could be repeatedly optically switched with deterministic ∼6 nm changes to the effective nanopore diameter and of ∼3× to the nanopore ionic conductance. The sensitivity to anionic DNA and a neutral complex carbohydrate biopolymer (maltodextrin) could be photoswitched "on" and "off" with an analyte selectivity set by applied voltage polarity. Photocontrol of nanopore state and mass transport characteristics is important for their use as ionic circuit elements (e.g., resistors and binary bits) and as chemically tuned filters. It expands single-molecule sensing capabilities in personalized medicine, genomics, glycomics, and, augmented by voltage polarity selectivity, especially in multiplexed biopolymer information storage schemes. We demonstrate repeatedly photocontrolled stable nanopore size, polarity, conductance, and sensing selectivity, by illumination wavelength and voltage polarity, with broad utility including single-molecule sensing of biologically and technologically important polymers.

A Closer Look at Delayed Primary Cleft Surgery and Unrepaired Cleft Lip and/or Palate in 5 UK Cleft Centers.

BACKGROUND: There may be many reasons for delays to primary cleft surgery. Our aim was to investigate the age of children undergoing primary cleft lip or primary cleft palate repair in 5 cleft centers within the United Kingdom. Identify the reasons for delayed primary cleft lip repair (beyond 6 months) and delayed primary palate repair (beyond 13 months). Identify children who had a cleft lip and/or palate (CL±P) that was intentionally unrepaired and the reasons for this. METHODS: A retrospective, multicenter review of patients born with a CL±P between December 1, 2012, and December 31, 2016. Three regional cleft centers, comprising of 5 cleft administrative units in the United Kingdom participated. RESULTS: In all, 1826 patients with CL±P were identified. Of them, 120 patients had delayed lip repair, outside the expected standard of 183 days. And, 178 patients in total had delayed palate repair, outside the expected standard of 396 days. Twenty (1%) patients had an unrepaired cleft palate. CONCLUSIONS: This large retrospective review highlights variations between centers regarding the timing of lip and palate surgery and details the reasons stated for delayed primary surgery. A small number of patients with an unrepaired cleft palate were identified. All had complex medical problems or comorbidities listed as a reason for the decision not to operate and 50% had a syndromic diagnosis. The number of patients receiving delayed surgery due to comorbidities, being underweight or prematurity, highlights the importance of the cleft specialist nurse and pediatrician within the cleft multidisciplinary team.

Targeting improved reproducibility in surface-enhanced Raman spectroscopy with planar substrates using 3D printed alignment holders.

Drop-casting is frequently used to deliver a sample for surface-enhanced Raman spectroscopy (SERS) and can result in inhomogeneous sample distribution during solvent evaporation. While soaking can provide better analyte homogeneity, it may require more sample than is available. Failure to optically sample analyte-rich substrate locations can compromise measurement outcomes. We developed and tested 3D printed SERS substrate holders that provided spatial registry of the dried sample droplet center for subsequent optical measurements. We found that deliberate and controlled spatial offsets (0-900 µm) between the analyte drop center and the laser excitation prevented signal intensity drops of as much as ∼3× and improved reproducibility. Thus, the use of offset-controlled 3D printed holders provided a quick and inexpensive way to improve the reliability of SERS measurements when using the convenient and popular choice of sample drop-casting.

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis.

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community: A temporal case study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus which causes coronavirus disease (COVID-19), has spread rapidly across the globe infecting millions of people and causing significant health and economic impacts. Authorities are exploring complimentary approaches to monitor this infectious disease at the community level. Wastewater-based epidemiology (WBE) approaches to detect SARS-CoV-2 RNA in municipal wastewater are being implemented worldwide as an environmental surveillance approach to inform health authority decision-making. Owing to the extended excretion of SARS-CoV-2 RNA in stool, WBE can surveil large populated areas with a longer detection window providing unique information on the presence of pre-symptomatic and asymptomatic cases that are unlikely to be screened by clinical testing. Herein, we analysed SARS-CoV-2 RNA in 24-h composite wastewater samples (n = 63) from three wastewater treatment plants (WWTPs) in Brisbane, Queensland, Australia from 24th of February to 1st of May 2020. A total of 21 samples were positive for SARS-CoV-2, ranging from 135 to 11,992 gene copies (GC)/100 mL of wastewater. Detections were made in a Southern Brisbane WWTP in late February 2020, up to three weeks before the first clininal case was reported there. Wastewater samples were generally positive during the period with highest caseload data. The positive SARS-CoV-2 RNA detection in wastewater while there were limited clinical reported cases demonstrates the potential of WBE as an early warning system to identify hotspots and target localised public health responses, such as increased individual testing and the provision of health warnings.

Chemically tailoring nanopores for single-molecule sensing and glycomics.

A nanopore can be fairly-but uncharitably-described as simply a nanofluidic channel through a thin membrane. Even this simple structural description holds utility and underpins a range of applications. Yet significant excitement for nanopore science is more readily ignited by the role of nanopores as enabling tools for biomedical science. Nanopore techniques offer single-molecule sensing without the need for chemical labelling, since in most nanopore implementations, matter is its own label through its size, charge, and chemical functionality. Nanopores have achieved considerable prominence for single-molecule DNA sequencing. The predominance of this application, though, can overshadow their established use for nanoparticle characterization and burgeoning use for protein analysis, among other application areas. Analyte scope continues to be expanded, and with increasing analyte complexity, success will increasingly hinge on control over nanopore surface chemistry to tune the nanopore, itself, and to moderate analyte transport. Carbohydrates are emerging as the latest high-profile target of nanopore science. Their tremendous chemical and structural complexity means that they challenge conventional chemical analysis methods and thus present a compelling target for unique nanopore characterization capabilities. Furthermore, they offer molecular diversity for probing nanopore operation and sensing mechanisms. This article thus focuses on two roles of chemistry in nanopore science: its use to provide exquisite control over nanopore performance, and how analyte properties can place stringent demands on nanopore chemistry. Expanding the horizons of nanopore science requires increasing consideration of the role of chemistry and increasing sophistication in the realm of chemical control over this nanoscale milieu.

Beyond nanopore sizing: improving solid-state single-molecule sensing performance, lifetime, and analyte scope for omics by targeting surface chemistry during fabrication.

Solid-state nanopores (SSNs) are single-molecule resolution sensors with a growing footprint in real-time bio-polymer profiling-most prominently, but far from exclusively, DNA sequencing. SSNs accessibility has increased with the advent of controlled dielectric breakdown (CDB), but severe fundamental challenges remain: drifts in open-pore current and (irreversible) analyte sticking. These behaviors impede basic research and device development for commercial applications and can be dramatically exacerbated by the chemical complexity and physical property diversity of different analytes. We demonstrate a SSN fabrication approach attentive to nanopore surface chemistry during pore formation, and thus create nanopores in silicon nitride (SiNx) capable of sensing a wide analyte scope-nucleic acid (double-stranded DNA), protein (holo-human serum transferrin) and glycan (maltodextrin). In contrast to SiNx pores fabricated without this comprehensive approach, the pores are Ohmic in electrolyte, have extremely stable open-pore current during analyte translocation (>1 h) over a broad range of pore diameters ([Formula: see text]3- ∼30 nm) with spontaneous current correction (if current deviation occurs), and higher responsiveness (i.e. inter-event frequency) to negatively charged analytes (∼6.5 × in case of DNA). These pores were fabricated by modifying CDB with a chemical additive-sodium hypochlorite-that resulted in dramatically different nanopore surface chemistry including ∼3 orders of magnitude weaker Ka (acid dissociation constant of the surface chargeable head-groups) compared to CDB pores which is inextricably linked with significant improvements in nanopore performance with respect to CDB pores.

Full-scale investigation of ferrous dosing in sewers and a wastewater treatment plant for multiple benefits.

This study demonstrates the full scale application of iron dosing in a metropolitan wastewater treatment plant (WWTP) and the upstream sewer system for multiple benefits. Two different dosing locations, i.e., the WWTP inlet works (Trial-1) and upstream sewer network (Trial-2) were tested in this study. Both dosing trials achieved multiple benefits such as sulfide control, phosphate removal and improved sludge dewaterability. During Trial-1, a sulfide reduction of >90% was achieved at high dosing rates (>19 kgFe ML-1) of ferrous chloride in the inlet works and in Trial-2 the in-sewer ferrous dosing had significant gas phase hydrogen sulfide (H2S) concentration reduction in the sewer network. The ferrous dosing enhanced the phosphate removal in the bioreactor up to 76% and 53 ± 2% during Trial-1 & 2, respectively. The iron ending up in the anaerobic sludge digester reduced the biogas H2S concentration by up to 36% and 45%, respectively. The dewaterability of the digested sludge was improved, with relative increases of 9.7% and 9.8%, respectively. The presence of primary clarifier showed limited impact on the downstream availability of iron for achieving the afore-mentioned multiple benefits. The iron dosing enhanced the total chemical oxygen demand removal in the primary clarifier reaching up to 49% at the high dose rates during Trial-1 and 42 ± 1% during Trial-2. This study demonstrated that multiple benefits could be achieved independent of the iron dosing location (i.e., at the WWTP inlet or in the network). Further, iron dosing at both locations enhances primary settling, beneficial for bioenergy recovery from wastewater.

Free nitrous acid pre-treatment enhances anaerobic digestion of waste activated sludge and rheological properties of digested sludge: A pilot-scale study.

In this study, the effects of free nitrous acid (FNA) pre-treatment on the rheological properties of digested sludge were investigated at a pilot-scale, along with the improvement in volatile solids (VS) destruction and biogas production. Two pilot-scale anaerobic sludge digesters were operated for one year, one receiving thickened waste activated sludge (TWAS) without pre-treatment (control) and one receiving TWAS pre-treated for 24 h at an FNA concentration of 4.9-6.1 mgN/L (nitrite = 250 mgN/L, pH = 5.0, T = 22-30 °C). The results confirmed the enhancing effect of FNA pre-treatment on methane production (37 ± 1%), consistent with previous laboratory studies. Equally importantly, FNA pre-treatment substantially reduced the shear viscosity of TWAS by 51 ± 8% at 100 s-1 and 49 ± 7% at 250 s-1, likely due to the solubilization of the TWAS (11.1 ± 0.8%). Similarly, FNA pre-treatment also reduced these viscosity parameters of the digested sludge by 80 ± 4% and 78 ± 4%, respectively, caused by both enhanced VS destruction and disintegration of the digested sludge. The dewaterability of digested sludge, assessed by dewatered solids content, capillary suction time and specific resistance to filtration, was not improved by FNA pre-treatment. The polymer requirement for dewatering was reduced by 24 ± 0.6% due to the lower solids concentration in the digested sludge achieved with FNA pre-treatment. The changes to sludge rheological properties revealed in this study further enhances the business case for the FNA pre-treatment technology.