Nitrite reduction using a membrane biofilm reactor (MBfR) in a hypoxic environment with dilute methane under low pressures.

Methane-based membrane biofilm reactors (MBfRs) can be an effective solution for nitrogen control in wastewater, but there is limited information on nitrite reduction for dilute wastewater (e.g., municipal wastewater) in hypoxic MBfRs. This study assessed the impacts of dilute (20 %), low-pressure methane (0.35-2.41 kPa) applied to MBfRs at hydraulic retention times (HRTs) of 2-12 h on nitrite removals, dissolved methane concentrations, and the resulting changes in the microbial community. High nitrite flux along with rapid and virtually complete (>99 %) nitrite removals were observed at methane pressures of 1.03-2.41 kPa at HRTs above 4 h, despite the use of diluted methane gas for the MBfR. The lowest methane pressure (0.35 kPa) was also able to achieve up to 98 % nitrite removals but required HRTs of up to 12 h. All scenarios had low dissolved methane concentrations (<10 mg/L), indicating that dilute methane at low supply pressures can effectively remove nitrite while meeting dissolved methane guidelines in treated effluent. Methylococcus genus was the key bacterium in MBfR biofilm grown at different HRTs and methane pressures, along with Methylocystis and other heterotrophic denitrifiers (Terrimonas and Hyphomicrobium). This study indicates that methane-based denitrification MBfRs can be a valuable tool to meet nitrogen limits for dilute wastewater coupled to partial nitrification, while limiting the release of methane to the environment.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

Examination of single-stage anaerobic and anoxic/aerobic and dual-stage anaerobic-anoxic/aerobic digestion to remove pharmaceuticals from municipal biosolids.

Many trace contaminants of emerging concern (CECs) including a number of pharmaceutically active compounds are not effectively removed during conventional wastewater treatment processes and instead accumulate in wastewater sludge. Unfortunately, many existing sludge stabilization treatments such as anaerobic digestion (AD) also have limited effectiveness against many of these CECs including the four pharmaceuticals ibuprofen, diclofenac, carbamazepine, and azithromycin which can then enter the environment through the disposal or land application of biosolids. Single-stage AD, single-stage cycling aerobic-anoxic (AERO/ANOX) and sequential digesters (AD followed by an AERO/ANOX digester) at sludge retention times (SRT) of 5 to 20-days were evaluated side-by-side to assess their effectiveness in removing pharmaceuticals and conventional organic matter. Single-stage ADs (35 °C) and AERO/ANOX (22 °C) digesters effectively removed total solids while sequential AD + AERO/ANOX digesters offered further improvements. Ibuprofen was not effectively removed during AD and resulted in up to a 23 ± 8% accumulation. However, ibuprofen was completely removed during AERO/ANOX digestion and in several sequential digestion scenarios. Each type of digestion was less effective against carbamazepine with slight (3 ± 2%) accumulations to low levels (14 ± 1%) of removals in each type of digestion studied. Diclofenac was more effectively removed with up 30 ± 3% to 39 ± 4% reductions in the single-stage digesters (AD and AERO/ANOX, respectively). While sequential digestion scenarios with the longest aerobic SRTs significantly increased diclofenac removals from their first-stage digesters, scenarios with the longest anaerobic SRTs actually decreased removals from first-stage digesters, possibly due to reversible biotransformation of diclofenac conjugates/metabolites. Up to 43 ± 6% of azithromycin was removed in AERO/ANOX digesters, while the best performing sequential-digester scenario removed up to 63 ± 7% of azithromycin. This study shows that different digester configurations can reduce the CEC burden in biosolids while also greatly reducing their volumes for disposal, although none can remove CECs completely.

A Review on the Fate of Legacy and Alternative Antimicrobials and Their Metabolites during Wastewater and Sludge Treatment.

Antimicrobial compounds are used in a broad range of personal care, consumer and healthcare products and are frequently encountered in modern life. The use of these compounds is being reexamined as their safety, effectiveness and necessity are increasingly being questioned by regulators and consumers alike. Wastewater often contains significant amounts of these chemicals, much of which ends up being released into the environment as existing wastewater and sludge treatment processes are simply not designed to treat many of these contaminants. Furthermore, many biotic and abiotic processes during wastewater treatment can generate significant quantities of potentially toxic and persistent antimicrobial metabolites and byproducts, many of which may be even more concerning than their parent antimicrobials. This review article explores the occurrence and fate of two of the most common legacy antimicrobials, triclosan and triclocarban, their metabolites/byproducts during wastewater and sludge treatment and their potential impacts on the environment. This article also explores the fate and transformation of emerging alternative antimicrobials and addresses some of the growing concerns regarding these compounds. This is becoming increasingly important as consumers and regulators alike shift away from legacy antimicrobials to alternative chemicals which may have similar environmental and human health concerns.

Comparison of anaerobic, cycling aerobic/anoxic, and sequential anaerobic/aerobic/anoxic digestion to remove triclosan and triclosan metabolites from municipal biosolids.

The antimicrobial triclosan (TCS) is a pervasive and persistent environmental micropollutant which can contaminate land, biota, and water through the land application of biosolids. Many existing sludge management techniques have limited effectiveness against TCS and TCS metabolites including triclosan-sulfate (TCS-SO4). The objective of this study was to evaluate the impacts of different digestion types (anaerobic, aerobic/anoxic, and sequential anaerobic + aerobic/anoxic), temperatures, and digester sludge retention times (SRTs) on the destruction of organic matter, and on TCS/TCS metabolites. Conventional mesophilic anaerobic digesters (AD), room temperature cycling aerobic/anoxic digesters (AERO/ANOX), and sequential AD + AERO/ANOX digesters were all effective in removing organic matter. The optimum single-stage AD, and AERO/ANOX scenarios were both 20-day SRTs which had 52.3 ± 1.4 and 47.1 ± 3.7% chemical oxygen demand (COD) removals, respectively. Sequential AD + AERO/ANOX digesters improved organic matter destruction, removing up to 68.2 ± 2.1% of COD at an 8-day AD + 12-day AERO/ANOX second-stage (mesophilic) SRTs. While AD showed modest levels of TCS removals (all <40%), TCS was substantially more degradable aerobically with AERO/ANOX removing up to 80.3 ± 2.5% of TCS and nearly all TCS-SO4 entering the digester at a 20-day SRT. Sequential AD + AERO/ANOX removed virtually all TCS-SO4 entering the system and improved TCS removals from first stage ADs. However, they were less effective than a single-stage AERO/ANOX digester operating at the same overall SRT. These results demonstrate that AERO/ANOX and sequential AD + AERO/ANOX processes could be used to reduce the amount of TCS, TCS-SO4 and TCS-related compounds in digested sludge, minimizing the environmental burden of the land application of biosolids.

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

Mitigation of recalcitrant nutrients and organic pollutants from small- to medium-scale biological nutrient removal plant sludge by digester optimization.

Digestion of biological nutrient removal (BNR) plant sludge can be challenging, particularly for small- to medium-sized wastewater treatment facilities (WWTF) which often lack the economies of scale, and/or expertise to make digestion feasible. This study compared various types of sludge digestion, sludge retention times (SRTs), and temperatures on the release of recalcitrant nutrients, digestion economics, and digester performance utilizing mixed primary and secondary sludge from a small- to medium-sized BNR facility. Mesophilic anaerobic digestion (AD), cycling aerobic/anoxic (AERO/ANOX) digestion, and sequential anaerobic/aerobic/anoxic (AD/AERO/ANOX) digestion at room and mesophilic temperatures were compared at SRTs between 5 and 20 days. AERO/ANOX digestion was very effective in treating recalcitrant forms of nitrogen and phosphorous by removing up to 87% of dissolved organic nitrogen (DON), up to 88 ± 2% of non-reactive dissolved phosphorous (NRDP). AERO/ANOX digestion also offered the lowest increase in sludge management costs versus the existing no-digestion baseline scenario. ADs removed up to 53 ± 1% of volatile solids (VS), whereas unheated AERO/ANOX digesters were less effective, removing up to 39 ± 1% of VS. Sequential AD/AERO/ANOX digesters with a mesophilic second-stage removed up to 61 ± 3% of VS but had the highest operational and capital costs. Experiments also indicated that significant amounts of orthophosphate (PO43-) may be released from digested AERO/ANOX sludge during on-site storage, with longer SRTs releasing PO43- more rapidly than shorter ones. These results are important as more WWTFs deploy BNR to meet increasingly stringent nutrient limits.

Occurrence of the Persistent Antimicrobial Triclosan in Microwave Pretreated and Anaerobically Digested Municipal Sludges under Various Process Conditions.

Treatment of emerging contaminants, such as antimicrobials, has become a priority topic for environmental protection. As a persistent, toxic, and bioaccumulative antimicrobial, the accumulation of triclosan (TCS) in wastewater sludge is creating a potential risk to human and ecosystem health via the agricultural use of biosolids. The impact of microwave (MW) pretreatment on TCS levels in municipal sludge is unknown. This study, for the first time, evaluated how MW pretreatment (80 and 160 °C) itself and together with anaerobic digestion (AD) under various sludge retention times (SRTs: 20, 12, and 6 days) and temperatures (35 and 55 °C) can affect the levels of TCS in municipal sludge. TCS and its potential transformation products were analyzed with ultra-high-performance liquid chromatography and tandem mass spectrometry. Significantly higher TCS concentrations were detected in sludge sampled from the plant in colder compared to those in warmer temperatures. MW temperature did not have a discernible impact on TCS reduction from undigested sludge. However, AD studies indicated that compared to controls (no pretreatment), MW irradiation could make TCS more amenable to biodegradation (up to 46%), especially at the elevated pretreatment and digester temperatures. At different SRTs studied, TCS levels in the thermophilic digesters were considerably lower than that of in the mesophilic digesters.

Occurrence and fate of antimicrobial triclocarban and its transformation products in municipal sludge during advanced anaerobic digestion using microwave pretreatment.

This study, for the first time, investigated the impact of microwave pretreatment on the fate of the pervasive antimicrobial triclocarban (TCC) that was already present in municipal sludge, before and during advanced anaerobic digestion (AD) under thermophilic and mesophilic conditions. A range of microwave temperature (80 and 160 °C) and exposure duration (1 and 30 min) configurations were studied by employing ten bench-scale anaerobic digesters fed with mixed sludge at three different solids retention times (SRTs) including 20, 12, and 6 days. Seasonal changes influenced the levels of TCC in municipal sludge sampled from a plant employing the biological nutrient removal. Initial batch pretreatment studies showed that microwave irradiation itself can achieve TCC removal efficiencies up to 30 ± 4 and 64 ± 5% at 80 and 160 °C, respectively. The control digesters utilizing un-pretreated mixed sludge showed limited TCC removals, between 18 and 32% and 11-26% respectively, under thermophilic and mesophilic temperatures. On the other hand, the highest TCC elimination (78 ± 2%) was obtained from the thermophilic digester utilizing microwaved sludge at 160 °C for 30 min at SRT of 12 days. The non-chlorinated carbanilide (a transformation product of TCC) was detected and quantified for the first time during conventional and microwave-pretreated anaerobic sludge digestion. The formation of carbanilide in biosolids through reductive dechlorination could be an indicator of efficient and complete TCC transformation. This research demonstrated that AD coupled with microwave pretreatment can be used to reduce environmental concentrations of TCC in municipal sludge and biosolids.

CRISPR-mediated live imaging of genome editing and transcription.

We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.

Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.

Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

Evolution at the Cutting Edge: CRISPR-Mediated Directed Evolution.

In a recent issue of Nature, Halperin et al. (2018) develop a new technology to continuously diversify specific genomic loci by combining CRISPR-Cas9 with error-prone DNA polymerases.

Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.

Assessing iron and aluminum-based coagulants for odour and pathogen reductions in sludge digesters and enhanced digestate dewaterability.

Anaerobic digestion (AD) is an effective way of recovering energy and nutrients from organic waste. However, several issues including the production of corrosive, highly odorous and toxic volatile sulfur compounds (VSCs) in digester biogas, and long digestion times to achieve sufficient pathogen reductions can limit its wider adoption. In this study, Kemira™ PIX-311 (ferric chloride), PAX XL-6 (aluminum chloride hydroxide sulfate), and PAX XL-19 (polyaluminum chlorohydrate) were added to the digester feeds to evaluate the effects on digester stability, organic removal, VSCs formation in digester headspace, pathogen removal and sludge dewaterability. After preliminary dose trials, two different doses of PIX-311, PAX XL-19, and a 1:1 mixture of PIX-311 and PAX XL-19 were selected. PAX XL-6 was removed from further study as dosing significantly increased VSC levels and the PAX XL-6 dosed digester exhibited signs of instability. During the total operation period of 100days, addition of PIX-311, PAX XL-19, a combination of PIX-311, PAX XL-19 at concentrations of 4000 and 4500mg/kg total solids (TS) to digester feed did not lead to process instability. Biogas yields of all metal added digesters were similar to that of the control (no metal addition) digester. PIX-311 achieved up to a 93% reduction in biogas VSCs, 82% better fecal coliform inactivation and exhibited improved dewaterability over the control digester. The PAX XL-19 dosed digester showed modest reductions in biogas VSC concentrations, pathogen levels and improved dewaterability versus the control. Metal addition can be an effective way to control odours from VSCs, pathogens and to improve dewaterability during AD.

Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators.

Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.

Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure-function relationship of two RNA mechanisms that regulate translation in Escherichia coli. We find that nucleotides that participate in RNA-RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA-RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell.

Effects of metal salt addition on odor and process stability during the anaerobic digestion of municipal waste sludge.

Anaerobic digestion (AD) is an effective way to recover energy and nutrients from organic waste; however, several issues including the solubilization of bound nutrients and the production of corrosive, highly odorous and toxic volatile sulfur compounds (VSCs) in AD biogas can limit its wider adoption. This study explored the effects of adding two different doses of ferric chloride, aluminum sulfate and magnesium hydroxide directly to the feed of complete mix semi-continuously fed mesophilic ADs on eight of the most odorous VSCs in AD biogas at three different organic loading rates (OLR). Ferric chloride was shown to be extremely effective in reducing VSCs by up to 87%, aluminum sulfate had the opposite effect and increased VSC levels by up to 920%, while magnesium hydroxide was not shown to have any significant impact. Ferric chloride, aluminum sulfate and magnesium hydroxide were effective in reducing the concentration of orthophosphate in AD effluent although both levels of alum addition caused digester failure at elevated OLRs. Extensive foaming was observed within the magnesium hydroxide dosed digesters, particularly at higher doses and high OLRs. Certain metal salt additions may be a valuable tool in overcoming barriers to AD and to meet regulatory targets.

Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.