Nondestructive and three-dimensional visualization by identifying elements using synchrotron radiation microscale X-ray CT reveals microbial and cavity distributions in anaerobic granular sludge.

We developed a nondestructive three-dimensional microbial visualization method utilizing synchrotron radiation X-ray microscale computed tomography to better understand the relationship between microorganisms and their surrounding habitats. The method was tested and optimized using a mixture of axenic Escherichia coli and Comamonas testosteroni. The osmium-thiocarbohydrazide-osmium method was used to stain all the microbial cells, and gold in situ hybridization was used to detect specific phylogenetic microbial groups. The stained samples were embedded in epoxy resin for microtomographic analysis. Differences in X-ray absorbances were calculated by subtracting the pre-L3-edge images from the post-L3-edge images to visualize the osmium and gold signals. Although we successfully detected cells stained with osmium, those labeled with gold were not detected, probably because of the insufficient density of gold atoms in the microbial cells. We then applied the developed technique to anaerobic granules and visualized the distribution of microbial cells and extracellular polymeric substances. Empty spaces were highlighted to determine the cavity distribution in granules. Numerous independent cavities of different sizes were identified in the granules. The developed method can be applied to various environmental samples for deeper insights into microbial life in their habitats. IMPORTANCE: Microorganisms inhabit diverse environments and often form biofilms. One factor that affects their community structure is the surrounding physical environment. The arrangement of residential space within the formed biofilm plays a crucial role in the supply and transportation of substances, as well as the discharge of metabolites. Conventional approaches, such as scanning electron microscopy and confocal laser scanning microscopy combined with fluorescence in situ hybridization, have limitations as they provide information primarily from the biofilm surface and cross-sections. In this study, we developed a method for detecting microorganisms in biofilms using synchrotron radiation X-ray microscale computer tomography. The developed method allows nondestructive three-dimensional observation of biofilms at a single-cell resolution (voxel size of approximately 200 nm), facilitating an understanding of the relationship between microorganisms and their physical habitats.

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.

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.

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.

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.

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.

Association between biomarkers and house dust mite sublingual immunotherapy in allergic asthma.

BACKGROUND: House dust mite (HDM) sublingual immunotherapy (SLIT) has demonstrated efficacy in clinical trials of patients with asthma. Airway inflammation is a characteristic of respiratory allergy, but its relationship to SLIT remains unclear. OBJECTIVE: We evaluate the association between clinical outcomes with pulmonary function and biomarkers in before and after HDM SLIT (UMIN Number 000022390). METHODS: One hundred twelve patients with asthma sensitized to HDM were randomized to add-on 6 standardized quality (SQ)-HDM SLIT to pharmacotherapy or pharmacotherapy alone for 48 weeks. At baseline and end of study, biomarkers, blood eosinophils, serum IgE, serum periostin, fractional exhaled nitric oxide (FeNO), and spirometry and clinical symptoms were measured. Association between biomarkers and an increase in FEV1 of 120 mL or greater were analysed. RESULTS: Sublingual immunotherapy (SLIT) demonstrated a significant reduction of serum periostin (P < .001), FeNO (P < .01), and increase in HDM-specific IgE (P < .05), FEV1 (P < .001) and improvement of clinical symptom scores, when compared to pharmacotherapy. The change in FEV1 correlated with the changes in serum periostin (r = .696, P < .001) and the changes in FeNO (r = .682, P < .001). The independent predictor of improvement in airflow limitation was changed in serum periostin (r2  = .753, P = .013) and FeNO (P = .038). Based on cut-off values derived by receiver operating characteristic analysis (periostin 30.9 ng/mL, FeNO 28.0 ppb), patients were distinguished responders from non-responders, but with no predictive value for blood eosinophils or total IgE. The proportion of patients with both high periostin and FeNO levels was significantly higher in responder than in non-responder (P = .026). CONCLUSIONS AND CLINICAL RELEVANCE: Adding HDM SLIT to pharmacotherapy resulted in reduced serum periostin and FeNO, and improved pulmonary function. Serum periostin and FeNO may be useful biomarkers for prediction of SLIT.

Uncovering Viable Microbiome in Anaerobic Sludge Digesters by Propidium Monoazide (PMA)-PCR.

Use of anaerobic sludge digester is a common practice around the world for solids digestion and methane generation from municipal sewage sludge. Understanding microbial community structure is vital to get better insight into the anaerobic digestion process and to gain better process control. However, selective analysis of viable microorganisms is limited by DNA-based assays. In this study, propidium monoazide (PMA)-PCR with 16S rRNA gene sequencing analysis was used to distinguish live and dead microorganisms based on cell membrane integrity. Microbial community structures of PMA-treated and PMA-untreated anaerobic digester sludge samples were compared. Quantitative PCR revealed that 5-30% of the rRNA genes were derived from inactive or dead cells in anaerobic sludge digesters. This caused a significant decrease in the numbers of operational taxonomic units and Chao1 and Shannon indices compared with that of the PMA-untreated sludge. Microbial community analysis showed that majority of the viable microbiome consisted of Euryarchaeota, Bacteroidetes, Deltaproteobacteria, Chloroflexi, Firmicutes, WWE1, Spirochaetes, Synergistetes, and Caldiserica. On the other hand, after the PMA treatment, numbers of Alphaproteobacteria and Betaproteobacteria declined. These were considered residual microbial members. The network analysis also revealed a relationship among the OTUs belonging to WWE1 and Bacteroidales. PMA-PCR-based 16S rRNA gene sequencing analysis is an effective tool for uncovering viable microbiome in complex environmental samples.

Effect of treated sewage characteristics on duckweed biomass production and microbial communities.

Duckweed biomass production in a duckweed pond fed with three differently treated sewage (i.e. sewage treated by primary sedimentation (PS); conventional activated sludge process (CAS); and downflow hanging sponge process (DHS)) was evaluated in order to assess the effects of water quality on biomass yield. Higher and stable biomass production was observed when the duckweed pond was fed with PS or DHS-effluent than with CAS-effluent, evidently due to the difference in nutrient loads. Availability of nutrients, especially phosphorus, affected the biomass production rate: higher the nutrient, faster the production. Microbial community analysis revealed that the members of Rhizobiales were likely to contribute to stable and high biomass growth. From the results of the study, a sewage treatment system consisting of a primary sedimentation followed by a duckweed pond and a tertiary treatment unit can be proposed to maximize biomass production without compromising treatment objectives. Size and operational parameters of the duckweed pond should be determined primarily based on the nutrient availability in the influent water to maximize duckweed growth.

Characterization of downflow hanging sponge reactors with regard to structure, process function, and microbial community compositions.

The activated sludge (AS) process has been the most widely used process for wastewater treatment despite it has several limitations for its further application and adoption worldwide, owing to unsustainable properties such as high-energy consumption and the production of large amount of excess sludge. To overcome the drawbacks of the AS process, the downflow hanging sponge (DHS) has been developed as an energy-saving and easy-to-maintain alternative. To date, six types of different sponge configurations have been developed and their performances have been evaluated in practical- to full-scale DHS reactors. A large number of studies have been carried out in order to enhance the performance and expand the application fields of the DHS. Transition of this process to the deployment and diffusion stage from the research and development phase is now ongoing in India and Egypt as well as in Japan. Under this situation, concise and state-of-the art review is important for enhancing DHS research and future applications. Herein, we summarize and present the DHS concerning the history of development, the mechanism of treatment, recent studies on its use in the field of wastewater treatment, and the features of microbial community structure.

Defining microbial community composition and seasonal variation in a sewage treatment plant in India using a down-flow hanging sponge reactor.

The characteristics of the microbial community in a practical-scale down-flow hanging sponge (DHS) reactor, high in organic matter and sulfate ion concentration, and the seasonal variation of the microbial community composition were investigated. Microorganisms related to sulfur oxidation and reduction (2-27%), as well as Leucobacter (7.50%), were abundant in the reactor. Anaerobic bacteria (27-38% in the first layer) were also in abundance and were found to contribute to the removal of organic matter from the sewage in the reactor. By comparing the Simpson index, the abundance-based coverage estimator (ACE) index, and the species composition of the microbial community across seasons (summer/dry, summer/rainy, autumn/dry, and winter/dry), the microbial community was found to change in composition only during the winter season. In addition to the estimation of seasonal variation, the difference in the microbial community composition along the axes of the DHS reactor was investigated for the first time. Although the abundance of each bacterial species differed along both axes of the reactor, the change of the community composition in the reactor was found to be greater along the vertical axis than the horizontal axis of the DHS reactor.

Characterization of sludge properties for sewage treatment in a practical-scale down-flow hanging sponge reactor: oxygen consumption and removal of organic matter, ammonium, and sulfur.

The characteristics of sludge retained in a down-flow hanging sponge reactor were investigated to provide a better understanding of the sewage treatment process in the reactor. The organic removal and sulfur oxidation conditions were found to differ between the first layer and the following three layers. It was found that 63% and 59% of the organic matter was removed in the first layer, even though the hydraulic retention time was only 0.2 h. It is thought that the organic removal resulted from aerobic and anaerobic biodegradation on the sponge medium. The sulfate concentration increased 1.5-1.9-fold in the first layer, with almost no subsequent change in the second to fourth layers. It was shown that oxidation of sulfide in the influent was completed in the first layer. The result of the oxygen uptake rate test with an ammonium nitrogen substrate suggested that the ammonium oxidation rate was affected by the condition of dissolved oxygen (DO) or oxidation-reduction potential (ORP).

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

Metagenomic characterization of 'Candidatus†Defluviicoccus tetraformis strain TFO71', a tetrad-forming organism, predominant in an anaerobic-aerobic membrane bioreactor with deteriorated biological phosphorus removal.

In an acetate-fed anaerobic-aerobic membrane bioreactor with deteriorated enhanced biological phosphorus removal (EBPR), Defluviicoccus-related tetrad-forming organisms (DTFO) were observed to predominate in the microbial community. Using metagenomics, a partial genome of the predominant DTFO, 'Candidatus Defluviicoccus tetraformis strain TFO71', was successfully constructed and characterized. Examining the genome confirmed the presence of genes related to the synthesis and degradation of glycogen and polyhydroxyalkanoate (PHA), which function as energy and carbon storage compounds. TFO71 and 'Candidatus Accumulibacter phosphatis' (CAP) UW-1 and CAP UW-2, representative polyphosphate-accumulating organisms (PAO), have PHA metabolism-related genes with high homology, but TFO71 has unique genes for PHA synthesis, gene regulation and granule management. We further discovered genes encoding DTFO polyphosphate (polyP) synthesis, suggesting that TFO71 may synthesize polyP under untested conditions. However, TFO71 may not activate these genes under EBPR conditions because the retrieved genome does not contain all inorganic phosphate transporters that are characteristic of PAOs (CAP UW-1, CAP UW-2, Microlunatus phosphovorus NM-1 and Tetrasphaera species). As a first step in characterizing EBPR-associated DTFO metabolism, this study identifies important differences between DTFO and PAO that may contribute to EBPR community competition and deterioration.

Metaproteomic identification of diazotrophic methanotrophs and their localization in root tissues of field-grown rice plants.

In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.

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.

Biomethane recovery and prokaryotic shifts in anaerobic co-digestion of food waste and paper waste in organic fraction of municipal solid waste: Effect of paper content.

Biomethane recovery from paper waste (PW) was achieved by mesophilic co-digestion with food waste. The feeding material containing 0%, 20%, 40% and 50% of PW in total solids (TS) were investigated in the long-term continuous operation. The results showed that the biogas production, pH, alkalinity and biodegradation of volatile solids (79.8 ± 3.6%) were stable for PW contents no more than 50%. The PW = 50% condition was considered the critical limit for the reasons of pump clogging, sufficient alkalinity (2.0 ± 0.3 g-CaCO3/L) and depletion of ammonia. Prokaryotic diversity indices decreased with the increased PW contents. Great shifts were observed in the prokaryotic communities before and after the PW contents reaches 50% as TS (18.4% as total weights). Biomethane recovery yields were deceasing from 445 to 350 NL-CH4/kg-fed-volatile-solids. The PW contents as 40% as TS (13.1% as total weights) obtained the optimal performance among all the feeding conditions.

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.

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.