Long-term efficacy of house dust mite sublingual immunotherapy on clinical and pulmonary function in patients with asthma and allergic rhinitis.

Background: A previous study reported that house dust mite (HDM) sublingual immunotherapy (SLIT) for 48 weeks was effective as add-on treatment for allergic asthma; however, data regarding its long-term efficacy are scarce. Objective: We sought to evaluate the effect of HDM SLIT on asthma control, pulmonary function, and airway inflammation and remodeling throughout the 5-year treatment period. Methods: A total of 140 patients with asthma and allergic rhinitis sensitized to HDM were randomized to receive either drugs alone or drugs plus SLIT for 5 years. The 5-item Asthma Control Questionnaire (ACQ-5), Asthma Quality of Life Questionnaire (AQLQ), Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ), spirometry, quantitative computed tomography, and type 2 biomarkers were assessed. Results: An improvement in the ACQ-5, AQLQ, and RQLQ scores was observed in the SLIT group compared with the control group. HDM SLIT increased lung function and reduced the percentage of airway wall area. The levels of fractional exhaled nitric oxide (Feno), blood eosinophil, serum specific IgE for HDM, and total IgE decreased and were sustained during the 5 years. The change in type 2 biomarkers correlated with change in the AQLQ score. On the basis of receiver-operating characteristic analysis for predicting responders, the area under the receiver-operating characteristic curve in FEV1% predicted, airway wall area, Feno, and specific IgE was high. Multivariate regression analysis showed that the strongest predictor of responders was Feno. Conclusions: HDM SLIT continued to provide sustained efficacy, improve lung function, and prevent progression of airway inflammation and remodeling in asthma throughout the 5-year treatment period.

Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems.

To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.IMPORTANCECulture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

Microbiological insights into anaerobic phenol degradation mechanisms and bulking phenomenon in a mesophilic upflow anaerobic sludge blanket reactor in long-term operation.

In this study, a long-term operation of 2,747 days was conducted to evaluate the performance of the upflow anaerobic sludge blanket (UASB) reactor and investigated the degradation mechanisms of high-organic loading phenol wastewater. During the reactor operation, the maximum chemical oxygen demand (COD) removal rate of 6.1 ± 0.6 kg/m3/day under 1,680 mg/L phenol concentration was achieved in the mesophilic UASB reactor. After a significant change in the operating temperature from 24.0 ± 4.1 °C to 35.9 ± 0.6 °C, frequent observations of floating and washout of the bloated granular sludge (novel types of the bulking phenomenon) were made in the UASB reactor, suggesting that the change in operating temperature could be a trigger for the bulking phenomenon. Through the metagenomic analysis, phenol degradation mechanisms were predicted that phenol was converted to 4-hydroxybenzoate via two possible routes by Syntrophorhabdaceae and Pelotomaculaceae bacteria. Furthermore, the degradation of 4-hydroxybenzoate to benzoyl-CoA was carried out by members of Syntrophorhabdaceae and Smithellaceae. In the bulking sludge, a predominant presence of Nanobdellota, belonging to DPANN archaea, was detected. The metagenome-assembled genome of the Nanobdellota lacks many biosynthetic pathways and has several genes for the symbiotic lifestyle such as trimeric autotransporter adhesin-related protein. Furthermore, the Nanobdellota have significant correlations with several methanogenic archaea that are predominantly present in the UASB reactor. Considering the results of this study, the predominant Nanobdellota may negatively affect the growth of the methanogens through the parasitic lifestyle and change the balance of microbial interactions in the granular sludge ecosystem.

A novel method for the whole-cell detection of environmental microorganisms using hemin and tyramide signal amplification (Hemin-TSA) with a desired fluorescent dye.

A method called hemin-tyramide signal amplification (Hemin-TSA) was developed for visualization of environmental microorganisms using hemin and tyramide signal amplification. In Hemin-TSA, hemin, which has peroxidase activity, is bound to microbial cells, and a desired fluorescent dye is deposited on the microbial cells by a hemin-catalyzed TSA reaction. The protocol was initially optimized in terms of hemin concentration, hemin binding time and repeated reaction times of TSA. Hemin-TSA showed a comparative or improved signal-to-noise ratio compared to DAPI staining. The shapes of fluorescent signals obtained from microbial cells were almost morphologically identical to those observed in phase contrast microscopy. Hemin-TSA staining provided more accurate cell counts than DAPI staining, especially for actively growing cells, for which two or three spotty DAPI signals were obtained from a single cell. In addition, microbial cells that were not detected by DAPI staining were detected by Hemin-TSA with fluorescein, which enabled us to avoid high non-specific fluorescence under UV excitation. The method developed in this study allows us to visually detect microbial cells in various environments with the characteristics of better cell morphological identification, improved enumeration accuracy and selectivity of fluorescent dyes.

Propidium monoazide - polymerase chain reaction reveals viable microbial community shifts in anaerobic membrane bioreactors treating domestic sewage at low temperature.

An anaerobic membrane bioreactor (AnMBR) treated domestic sewage at 15 °C under different hydraulic retention time (HRT) conditions (6, 12, 16, and 24 h). Propidium monoazide (PMA)-PCR excluded microorganisms without intact cell membranes, focusing on the viable microbial community in anaerobic digestion. The results showed that the 6-hour HRT had poor treatment performance: low chemical oxygen demand removal efficiency (below 80%) and high mean trans-membrane pressure and flux (15 kPa and 9.4 L/(m2 h)). Comparatively, PMA-PCR combined with next-generation sequencing improved the identification of microbial changes compared to conventional 16S rRNA gene sequencing. HRT influenced microorganisms in the hydrolysis and acid-production stages, including carbohydrate-degrading bacteria such as Bifidobacterium and Prevotella 1. Remarkably, a comparison with an AnMBR at 25 °C showed Proteobacteria to be the main cause of membrane fouling in the low-temperature AnMBR, with most operational taxonomic units negatively correlated with HRT and solids retention time.

Improved Properties and Enhancement Strategies of Hydroxyapatite-Based Functional Granular Sludge for a High-Rate Partial Nitritation/Anammox System.

Retaining sufficient anammox bacteria (AnAOB) while keeping the anammox-based process stable is the focus of the study of anammox technology, especially in a one-stage partial nitritation/anammox (PNA) process. The use of hydroxyapatite (HAP) granules in an anammox-based process is innovative for its potential to improve the nitrogen removal rate and achieve simultaneous removal of phosphorus. In this study, the HAP-based granular sludge was employed using enhancement strategies for an excellent nitrogen removal performance in a one-stage PNA process. Compared to those of other granular sludge PNA systems, a remarkable sludge volume index of 7.8 mL/g and an extremely high mixed liquor volatile suspended solids of 15 g/L were achieved under a low hydraulic retention time of 2 h. Consequently, an unprecedented nitrogen removal rate as high as 4.8 kg N/m3/d at 25 °C was obtained under a nitrogen loading rate of 6 kg N/m3/d. After a long-term operation of 870 days, the enhancement strategies underlying the superior performance of the granular sludge were identified. These findings clearly demonstrate that the enhancement strategies are crucial for the superior operating performance of the PNA process, and they can promote the application of the anammox-based process.

Anammox upflow hybrid reactor: Nitrogen removal performance and potential for phosphorus recovery.

Echoing to the call of recovering high-value-added chemicals from wastewater and achieving carbon-neutral operation in wastewater treatment, an anammox upflow hybrid reactor was successfully applied for nitrogen removal, and the potential for phosphorus recovery was put forward. Moreover, the spatial pattern of removal capacities, and distribution of biomass and HAP precipitates were recognized and demonstrated as height-oriented. The intensity of HAP precipitates was highly consistent with the amount of anammox biomass and the relative abundance of the Candidatus Kuenenia, indicating that HAP formation was encouraged by the anammox reaction itself and heterogeneous nucleation induced by organic matters (proteins and polysaccharides). The fixed bed also played an important role in immobilizing the anammox biomass, secreted organic matrix, and HAP precipitates. This finding also provoked the thought that in the anammox process, HAP precipitation was more achievable, effective and practicable using the fixed-carrier system.

Novel Cross-domain Symbiosis between Candidatus Patescibacteria and Hydrogenotrophic Methanogenic Archaea Methanospirillum Discovered in a Methanogenic Ecosystem.

To identify novel cross-domain symbiosis between Candidatus Patescibacteria and Archaea, we performed fluorescence in situ hybridization (FISH) on enrichment cultures derived from methanogenic bioreactor sludge with the newly designed 32-520-1066 probe targeting the family-level uncultured clade 32-520/UBA5633 lineage in the class Ca. Paceibacteria. All FISH-detectable 32-520/UBA5633 cells were attached to Methanospirillum, indicating high host specificity. Transmission electron microscopy observations revealed 32-520/UBA5633-like cells that were specifically adherent to the plug structure of Methanospirillum-like rod-shaped cells. The metagenome-assembled genomes of 32-520/UBA5633 encoded unique gene clusters comprising pilin signal peptides and type IV pilins. These results provide novel insights into unseen symbiosis between Ca. Patescibacteria and Archaea.

Symbiosis between Candidatus Patescibacteria and Archaea Discovered in Wastewater-Treating Bioreactors.

Each prokaryotic domain, Bacteria and Archaea, contains a large and diverse group of organisms characterized by their ultrasmall cell size and symbiotic lifestyles (potentially commensal, mutualistic, and parasitic relationships), namely, Candidatus Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) and DPANN archaea, respectively. Cultivation-based approaches have revealed that Ca. Patescibacteria and DPANN symbiotically interact with bacterial and archaeal partners and hosts, respectively, but that cross-domain symbiosis and parasitism have never been observed. By amending wastewater treatment sludge samples with methanogenic archaea, we observed increased abundances of Ca. Patescibacteria (Ca. Yanofskybacteria/UBA5738) and, using fluorescence in situ hybridization (FISH), discovered that nearly all of the Ca. Yanofskybacteria/UBA5738 cells were attached to Methanothrix (95.7 ± 2.1%) and that none of the cells were attached to other lineages, implying high host dependency and specificity. Methanothrix filaments (multicellular) with Ca. Yanofskybacteria/UBA5738 attached had significantly more cells with no or low detectable ribosomal activity (based on FISH fluorescence) and often showed deformations at the sites of attachment (based on transmission electron microscopy), suggesting that the interaction is parasitic. Metagenome-assisted metabolic reconstruction showed that Ca. Yanofskybacteria/UBA5738 lacks most of the biosynthetic pathways necessary for cell growth and universally conserves three unique gene arrays that contain multiple genes with signal peptides in the metagenome-assembled genomes of the Ca. Yanofskybacteria/UBA5738 lineage. The results shed light on a novel cross-domain symbiosis and inspire potential strategies for culturing CPR and DPANN. IMPORTANCE One highly diverse phylogenetic group of Bacteria, Ca. Patescibacteria, remains poorly understood, but, from the few cultured representatives and metagenomic investigations, they are thought to live symbiotically or parasitically with other bacteria or even with eukarya. We explored the possibility of symbiotic interactions with Archaea by amending wastewater treatment sludge samples that were rich in Ca. Patescibacteria and Archaea with an isolate archaeon that is closely related to a methanogen population abundant in situ (Methanothrix). This strategic cultivation successfully established enrichment cultures that were mainly comprised of Ca. Patescibacteria (family level lineage Ca. Yanofskybacteria/UBA5738) and Methanothrix, in which we found highly specific physical interactions between the two organisms. Microscopic observations based on transmission electron microscopy, target-specific fluorescence in situ hybridization, and metagenomic analyses showed evidence that the interaction is likely parasitic. The results show a novel cross-domain parasitism between Bacteria and Archaea and suggest that the amendment of host Archaea may be an effective approach in culturing novel Ca. Patescibacteria.

Long term operation performance and membrane fouling mechanisms of anaerobic membrane bioreactor treating waste activated sludge at high solid concentration and high flux.

High solid anaerobic membrane bioreactor (HSAnMBR) is widely applied in biomass treatment and energy regeneration, while membrane operation performance and membrane fouling control remain critical issues. In this study, a HSAnMBR was utilized for waste activated sludge (WAS) treatment at organic loading rates of 3.69-3.72 gCOD/L·d and biogas yield was ranged in 0.38-0.39 L/gVSfed with the COD conversion efficiency of 40 %. The membrane operated stably when the average flux was 9.6, 4.5 and 1.2 L/m2/h at mixed liquor total solid of 25, 30 and 40 g/L with a filtration: relaxation of 4:1, 1:1 and 1:2, respectively. The distinctive characteristics of membrane fouling at high solid condition were that the polysaccharides and proteins had high fouling propensity and were the main composition of the foulant layer. Furthermore, phosphorus and magnesium were the predominant causes of inorganic fouling. The minerals precipitated on the membrane and were embedded into membrane pores, contributing to cake layer formation and pore blocking. This research provided a comprehensive analysis of the membrane operation characterization and fouling mechanisms of HSAnMBR, which was expected to push forward HSAnMBR applications to WAS treatment.

Efficacy of a house dust mite sublingual immunotherapy tablet as add-on dupilumab in asthma with rhinitis.

BACKGROUND: HDM SLIT is one of the disease-modifying treatment for allergic asthma, and has demonstrated efficacy in clinical trials. Dupilumab, blocks IL-4 and IL-13 signaling, key drivers of type 2 inflammation, and is approved for patients with uncontrolled, moderate-to-severe asthma. The aim of this study was to evaluate outcomes after HDM SLIT initiation in asthma with rhinitis not optimally controlled with dupilumab in a real-world setting. METHODS: At baseline and 48 weeks after treatment, asthma control questionnaire (ACQ)-5, asthma quality of life questionnaire (AQLQ) and rhinoconjunctivitis quality of life questionnaire (RQLQ) were assessed. Spirometry, type 2 inflammatory biomarkers and quantitative computed tomographic parameters of airway remodeling were also collected. RESULTS: Of 47 patients received HDM SLIT and 41 completed the study. Combined HDM SLIT and dupilumab improved ACQ-5 (p < 0.05), AQLQ (p < 0.05), RQLQ (p < 0.05), and increased lung function and reduced FeNO (p < 0.05) and airway percentage wall area, and wall thickness (each, p < 0.05). The change in ACQ-5 and AQLQ score correlated with both changes in FeNO and FEV1 percent predicted. Multiple regression analysis showed that the change in FEV1 percent predicted was independent factor for improvement of AQLQ (r2 = 0.510, p = 0.012). Based on ROC analysis for predicting SLIT responders, the baseline area under the curves in serum HDM specific-IgE, total IgE and FEV1 percent predicted were high (>0.8). CONCLUSIONS: These results support the benefits of adding HDM SLIT to pharmacotherapy plus dupilumab in uncontrolled asthma with rhinitis.

Diversity of Candidatus Patescibacteria in Activated Sludge Revealed by a Size-|Fractionation Approach.

Uncultivated members of Candidatus Patescibacteria are commonly found in activated sludge treating sewage and are widely distributed in wastewater treatment plants in different regions and countries. However, the phylogenetic diversity of Ca. Patescibacteria is difficult to examine because of their low relative abundance in the environment. Since Ca. Patescibacteria members have small cell sizes, we herein collected small microorganisms from activated sludge using a filtration-based size-fractionation approach (i.e., 0.45-0.22| |µm and 0.22-0.1| |µm fractions). Fractionated samples were characterized using 16S rRNA gene amplicon and shotgun metagenomic sequence ana-lyses. The amplicon ana-lysis revealed that the relative abundance of Ca. Patescibacteria increased to 73.5% and 52.5% in the 0.45-0.22| |µm and 0.22-0.1| |µm fraction samples, respectively, from 5.8% in the unfractionated sample. The members recovered from the two size-fractionated samples included Ca. Saccharimonadia, Ca. Gracilibacteria, Ca. Paceibacteria, Ca. Microgenomatia, class-level uncultured lineage ABY1, Ca. Berkelbacteria, WS6 (Ca. Dojkabacteria), and WWE3, with Ca. Saccharimonadia being predominant in both fraction samples. The number of operational taxonomic units belonging to Ca. Patescibacteria was approximately 6-fold higher in the size-fractionated samples than in the unfractionated sample. The shotgun metagenomic ana-lysis of the 0.45-0.22| |µm fractioned sample enabled the reconstruction of 24 high-quality patescibacterial bins. The bins obtained were classified into diverse clades at the family and genus levels, some of which were rarely detected in previous activated sludge studies. Collectively, the present results suggest that the overall diversity of Ca. Patescibacteria inhabiting activated sludge is higher than previously expected.

Fast formation of anammox granules using a nitrification-denitrification sludge and transformation of microbial community.

A lengthy start-up period has been one of the key obstacles limiting the application of the anammox process. In this investigation, a nitrification-denitrification sludge was used to start-up the anammox EGSB process. The transformation process from nitrification-denitrification sludge to anammox granule sludge was explored through the aspects of nitrogen removal performance, granule properties, microbial community structure, and evolution route. A successful start-up of the anammox process was achieved after 94 days of reactor operation. The highest nitrogen removal rate (NRR) obtained was 7.25±0.16 gN/L/d at a nitrogen loading rate (NLR) of 8.0 gN/L/d, and the corresponding nitrogen removal efficiency was a high 90.61±1.99%. The results of the microbial analysis revealed significant changes in anammox bacteria, nitrifying bacteria, and denitrifying bacteria in the sludge. Notably, the anammox bacteria abundance increased from 2.5% to 29.0% during the operation, and Candidatus Kuenenia and Candidatus Brocadia were the dominant genera. Distinct-different successions on Candidatus Brocadia and Candidatus Kuenenia were also observed over the long-term period. In addition, the settling performance, anammox activity and biomass retention capacity of the granules were significantly enhanced during this process, and the corresponding granule evolution route was also proposed. The results in this study indicate the feasibility of using available seed sludge source for the fast-transformation of anammox granules, it is beneficial to the large-scale application of anammox process and the utilization of excess sludge.

Experimental Adaptation of Murine Norovirus to Calcium Hydroxide.

Slaked lime (calcium hydroxide) is a commonly used disinfectant for fecal sludge. Although viruses are inactivated by lime treatment, whether RNA viruses adapt to lime treatment has not yet been determined. Here, we show that murine norovirus developed higher tolerance during serial passages with lime treatment. We compared synonymous and non-synonymous nucleotide diversities of the three open reading frames of viral genome and revealed that virus populations were subjected to enhanced purifying selection over the course of serial passages with lime treatment. Virus adaptation to lime treatment was coincident with amino acid substitution of lysine to arginine at position 345 (K345R) on the major capsid protein VP1, which accounted for more than 90% of the population. The infectious clones with the K345R produced using a plasmid-based reverse genetics system exhibited greater tolerance in a lime solution, which indicated that the specific amino acid substitution was solely involved in the viral tolerance in lime treatment.

Sodium hypochlorite induced inhibition in anaerobic digestion and possible approach to maintain methane fermentation performance.

Since sodium hypochlorite (NaClO), a commonly used chemical to deal with membrane fouling, is toxic to microorganisms, it is a major concern in the membrane cleaning process. In this study, the concentration-dependent effects of NaClO (0-9 g/L) on the biodegradation performance and microbial activity were investigated via batch experiments. The methane production (obtained approximately 140 mL) and microbial community revealed by principal coordinates analysis were almost unaffected when the NaClO concentration ranged between 0 and 3 g/L. A follow-up batch experiment was conducted and revealed that the microbial products could help protect or recover the activity of anaerobic microorganisms at a high NaClO concentration of 10 g/L. Additionally, correlation analysis was used to investigate the associations between the 15 major bacterial genera. Moreover, the microbial analysis results indicated that the top 10 operational taxonomic units most affected by NaClO were primarily coryneform and filamentous bacteria.

Full-scale application of a down-flow hanging sponge reactor combined with a primary sedimentation basin for domestic sewage treatment.

The down-flow hanging sponge (DHS) reactor is advantageous for sewage treatment since it produces an effluent quality that complies with the standards for reuse and there is little excess sludge. A full-scale DHS module was efficiently employed for the treatment of domestic sewage (200 m3 day-1) flowing from a primary sedimentation basin (PSB), which was used to reduce the suspended solids loading rate and enhance the oxidation of organics by heterotrophs. The combined PSB-DHS was successfully operated at a total hydraulic retention time of 3.4 h (2.4 h for PSB and 1.0 h for DHS) for the relatively long period of 600 days at sewage temperatures of 10 °C to 32 °C. The PSB-DHS consistently produced an effluent quality with minimum values of chemical oxygen demand, biochemical oxygen demand, and suspended solids of 59 ± 15, 12 ± 3.0, and 15 ± 7 mg L-1, respectively. The proposed system performed exceptionally well at removing organics and particulate matter over a short hydraulic retention time.

Microbial characteristics in anaerobic membrane bioreactor treating domestic sewage: Effects of HRT and process performance.

Two anaerobic membrane bioreactors (AnMBRs) equipped with different membrane pore size (0.4 or 0.05 µm) were operated at 25˚C and fed with domestic wastewater. The hydraulic retention time (HRT) of the reactors was shortened. The microbial communities of the two AnMBRs were investigated by 16S rRNA gene amplicon sequencing to see the effects of HRT. The predominant Archaea was an aceticlastic methanogen Methanosaeta. The composition of hydrogenotrophic methanogens changed with the HRTs: the population of Methanobacterium was higher for longer HRTs, whereas the population of unclassified Methanoregulaceae was higher for shorter HRTs. The Anaerolineae, Bacteroidia and Clostridia bacteria were dominant in both of the reactors, with a combined relative abundance of over 55%. The relative abundance of Anaerolineae was proportional to the biogas production performance. The change in the population of hydrogenotrophic methanogens or Anaerolineae can be used as an indicator for process monitoring. The sum of the relative abundance of Anaerolineae and Clostridia fluctuated slightly with changes in the HRT in both AnMBRs when the reactor was stably operated. The co-occurrence analysis revealed the relative abundance of the operational taxonomic units belonging to Anaerolineae and Clostridia was functionally equivalent during the treatment of real domestic sewage. A principal coordination analysis revealed that the changes in the microbial community in each reactor were consistent with the change of HRT. In addition, both the HRT and the stability of the process are important factors for maintaining microbial community structures.

A successful start-up of an anaerobic membrane bioreactor (AnMBR) coupled mainstream partial nitritation-anammox (PN/A) system: A pilot-scale study on in-situ NOB elimination, AnAOB growth kinetics, and mainstream treatment performance.

In this pilot-scale study, an innovative mainstream treatment process that couples the anaerobic membrane reactor (AnMBR) with a one-stage PN/A system was proposed for advancing the concept of carbon neutrality in the municipal wastewater treatment plant. This work demonstrates the start-up procedure of a pilot-scale one-stage PN/A system for mainstream treatment. The 255-day start-up of the one-stage PN/A system involved the cultivation of ammonium-oxidizing bacteria (AOB) from the activated sludge, suppression of nitrite-oxidizing bacteria (NOB), investigation of in-situ growth kinetics of anammox bacteria (AnAOB), and the 50-day operation of the pilot-scale AnMBR-PN/A process for natural mainstream treatment. It is verified in the pilot-scale system for the first time that the in-situ free ammonia (FA) and free nitrous acid (FNA) exposure could effectively eliminate the Nitrospira (the NOB genus) while retaining the Nitosonomas (the AOB genus) community in the suspended sludge. NOB community rebounding was not detected even at the mainstream conditions with low nitrogen concentrations (Influent ammonium concentration=38±6 mg-NH4+-N/L) by intermittent aeration to control the system dissolved oxygen (DO) below 0.5 mg/L. The results of the mainstream treatment showed that the average effluent total nitrogen (TN) in the coupled process was generally lower than 10 mg-N/L, which meets the discharge limits of most prefectures in Japan. The investigated results of the in-situ anammox bacteria (AnAOB) growth kinetics suggested that the promoted start-up strategy of taking advantage of the warm months with higher mainstream temperature to achieve the rapid in-situ growth of the AnAOB is applicable in the investigated regions. From the perspective of the removal performance of the TN and organic substance, the AnMBR-PN/A process has great potential as the layouts of the carbon-neutral mainstream wastewater treatment plants.

Sequence-Specific Capture of Oligonucleotide Probes (SCOPE): a Simple and Rapid Microbial rRNA Quantification Method Using a Molecular Weight Cutoff Membrane.

A method named sequence-specific capture of oligonucleotide probes (SCOPE) was developed for quantification of microbial rRNA molecules in a multiplex manner. In this method, a molecular weight cutoff membrane (MWCOM) was used for the separation of fluorescence-labeled oligonucleotide probes hybridized with rRNA from free unhybridized probes. To demonstrate proof of concept, probes targeting bacteria or archaea at different taxonomic levels were prepared and were hybridized with rRNAs. The hybridization stringency was controlled by adjusting reaction temperature and urea concentration in the mixture. Then, the mixture was filtered through the MWCOM. The rRNA and hybridized probes collected on the MWCOM were recovered and quantified using a spectrophotometer and fluorospectrometer, respectively. The method showed high accuracy in detecting specific microbial rRNA in a defined nucleic acid mixture. Furthermore, the method was capable of simultaneous detection and quantification of multiple target rRNAs in a sample with sensitivity up to a single-base mismatch. The SCOPE method was tested and benchmarked against reverse transcription-quantitative PCR (RT-qPCR) for the quantification of Bacteria, Archaea, and some key methanogens in anaerobic sludge samples. It was observed that the SCOPE method produced more reliable and coherent results. Thus, the SCOPE method allows simple and rapid detection and quantification of target microbial rRNAs for environmental microbial population analysis without any need for enzymatic reactions. IMPORTANCE Microorganisms play integral roles in the Earth's ecosystem. Microbial populations and their activities significantly affect the global nutrient cycles. Quantification of key microorganisms provides important information that is required to understand their roles in the environment. Sequence-based analysis of microbial population is a powerful tool, but it provides information only on relative abundance of microorganisms. Hence, the development of a simpler and quick method for the quantification of microorganisms is necessary. To address the shortcomings of a variety of molecular methods reported so far, we developed a simple, rapid, accurate, and multiplexed microbial rRNA quantification method to evaluate the abundance of specific microbial populations in complex ecosystems. This method demonstrated high specificity, reproducibility, and applicability to such samples. The method is useful for quantitative detection of particular microbial members in the environment.

The impact of calcium supplementation on methane fermentation and ammonia inhibition of fish processing wastewater.

The effect of trace metal supplementation on the methane fermentation of fish processing wastewater (FPW) was studied in both batch and continuous experiments using a self-agitated anaerobic baffled reactor (SA-ABR). In the batch experiments, a single supplementation of Ca2+, Co2+ and Fe2+ was show to have a significant positive impact on the performance of methane fermentation. The continuous experiment results showed that supplementation with 1.5 g-Ca2+/L-substrate remarkably enhanced the performance of methane fermentation of the SA-ABR in treating FPW with the optimal organic loading rate achieved at 7.62 g-COD/L/d. During the steady states (stages 2 to 5), the average removal efficiencies of COD, protein, carbohydrate and lipid were 89, 85, 80 and 91%, respectively. The biogas conversion rates were in the range of 0.39 to 0.45 L-biogas/g-COD with a high methane content of 74%. Besides, Ca2+ supplementation also improved the resistance of the methane fermentation system to ammonia inhibition.