High-rate nitrification of electronic industry wastewater by using nitrifying granules.

Nitrifying granules have a high sedimentation property and an ability to maintain a large amount of nitrifying bacteria in a reaction tank. Our group has examined the formation process of nitrifying granules and achieved high-rate nitrification for an inorganic synthetic wastewater using these granules. In this research, a pilot-scale test plant with an 850-liter reaction tank was assembled in a semiconductor manufacturing factory in order to conduct a continuous water conduction test using real electronics industry wastewater. The aim was to observe the formation of nitrifying granules and determine the maximum ammonia removal rate. The average granule diameter formed during the experiment was 780 µm and the maximum ammonia removal rate was observed to be 1.5 kgN·m-3·day-1 at 20 °C, which is 2.5-5 times faster than traditional activated sludge methods. A fluorescence in situ hybridization analysis showed that ß-proteobacterial ammonia oxidizing bacteria and the Nitrospira-like nitrite-oxidizing bacteria dominate the bacteria population in the granules, and their strong aggregation capacity might confer some benefits to the formation of these nitrifying granules.

Tailoring Effective Phage Cocktails for Long-Term Lysis of Escherichia coli Based on Physiological Properties of Constituent Phages.

Background: Bacteriophage (phage) therapy has regained attention as an alternative to antimicrobial agents for eliminating bacteria; however, the emergence of phage-resistant bacteria during the therapy is a major concern. One method to control this emergence is to create a cocktail composed of multiple phages. Materials and Methods: In this study, we isolated 28 phages infecting Escherichia coli and evaluated their bacteriolysis (lysis) activity, lytic spectrum, adsorption rate constant, burst size, and titer of a 1-day incubation, followed by clustering of the phages based on these physiological characteristics. Results: The variation in lysis onset time and duration was more significant for cocktails of phages from different clusters than for phage cocktails from the same cluster. Conclusions: This suggests that a combination of phages with different physiological characteristics is necessary to create a cocktail that rapidly and continuously lyses bacteria over a prolonged duration while suppressing the emergence of resistant bacterial strains.

Cross-Reactivity of Antibodies in Intravenous Immunoglobulin Preparation for Protection against SARS-CoV-2.

Severe cases of COVID-19 continue to put pressure on medical operations by prolonging hospitalization, occupying intensive care beds, and forcing medical personnel to undergo harsh labor. The eradication of SARS-CoV-2 through vaccine development has yet to be achieved, mainly due to the appearance of multiple mutant-incorporating strains. The present study explored the utility of human intravenous immunoglobulin (IVIG) preparations in suppressing the aggravation of any COVID-19 infection using a SARS-CoV-2 pseudovirus assay. Our study revealed the existence of IgG antibodies in human IVIG preparations, which recognized the spike protein of SARS-CoV-2. Remarkably, the pretreatment of ACE2/TMPRSS2-expressing host cells (HEK293T cells) with IVIG preparations (10 mg/mL) inhibited approximately 40% entry of SARS-CoV-2 pseudovirus even at extremely low concentrations of IgG (0.16-1.25 mg/mL). In contrast, the antibody-dependent enhancement of viral entry was confirmed when SARS-CoV-2 pseudovirus was treated with some products at an IgG concentration of 10 mg/mL. Our data suggest that IVIG may contribute to therapy for COVID-19, including for cases caused by SARS-CoV-2 variants, since IVIG binds not only to the spike proteins of the virus, but also to human ACE2/TMPRSS2. An even better preventive effect can be expected with blood collected after the start of the COVID-19 pandemic.

Effect of inorganic carbon limitation on the nitrogen removal performance of the single-stage reactor containing anammox and nitritation gel carriers.

Herein, the effect of inorganic carbon (IC) limitation on the nitrogen removal performance of the single-stage reactor containing nitritation and anammox gel carriers was investigated. As a result of a continuous feeding test, the effluent ammonium concentration increased as the IC concentration decreased, indicating the deterioration of nitritation activity, not anammox. Furthermore, the sensitivity of IC to anammox and nitritation activity was investigated in anammox and nitritation reactors, respectively. Consequently, the relationship between the effluent IC concentration and nitritation rate was well described using the Michaelis-Menten equation. The apparent Km value of nitritation was calculated as 4.4 mg-C L-1. In anammox reactor, it was calculated as 1.7 mg-C L-1. These results revealed that the affinity of nitritation gel carriers to IC was lower than that of anammox, supporting that nitritation activity was easily deactivated by decrease in the IC concentration rather than anammox. Microbial community analysis revealed that Nitrosomonas europaea and Candidatus Jettenia asiatica were the dominant species of ammonium-oxidizing and anammox bacteria.

Long-Term Limitation Effects of Se(VI), Zn(II), and Ni(II) on Start-Up of the Anammox Process Using Gel Carrier.

Anaerobic ammonia oxidation (anammox) bacteria are inhibited by heavy metals at high concentrations but require trace amounts of some heavy-metal elements for growth and activity maintenance. The present study evaluates the long-term limitation effects of Se(VI), Zn(II), and Ni(II) on the start-up period of an anammox reactor. To strictly limit the levels of heavy metals in the reactor, all tests used ultrapure water as the influent synthetic wastewater and all reactors were installed in a clean booth. The anammox biomass was maintained through the gel entrapment technique. In the absence of Se(VI) and Ni(II), the anammox reactor start-up was 18.9 kg-N (m3-carrier d)-1 (nitrogen conversion rate (NCR) per gel carriers), indicating that Se(VI) and Ni(II) are not required or need not be continuously added to maintain the anammox process. Under Zn(II) limitation, the anammox process failed to start-up and the NCR tended to decrease rapidly. After readdition of 0.005 mg L-1 of Zn(II), the NCR did not decline further and instead partially recovered at a very slow rate. The NCR was completely recovered after adding 0.020 mg L-1 of Zn(II). These results reveal that Zn(II) limitation seriously affects the start-up of the anammox process while Se(VI) and Ni(II) are not required or need not be continuously added to the anammox process.

Transitions in oral and intestinal microflora composition and innate immune receptor-dependent stimulation during mouse development.

Commensal bacteria possess immunostimulatory activities that can modulate host responses to affect development and homeostasis in the intestine. However, how different populations of resident bacteria stimulate the immune system remains largely unknown. We characterized here the ability of intestinal and oral microflora to stimulate individual pattern recognition receptors (PRRs) in bone marrow-derived macrophages and mesothelial cells. The intestinal but not oral microflora elicited age- and cell type-specific immunostimulation. The immunostimulatory activity of the intestinal microflora varied among individual mice but was largely mediated via Toll-like receptor 4 (TLR4) during breast-feeding, whereas it became TLR4 independent after weaning. This transition was associated with a change from a microflora rich in TLR4-stimulatory proteobacteria to one dominated by Bacteroidales and/or Clostridiales that poorly stimulate TLR4. The major stimulatory activity of the intestinal microflora was still intact in NOD1-, NOD2-, TLR2-, TLR4-, TLR5-, TLR9-, TLR11-, ASC-, or RICK-deficient cells but still relied on the adaptor MyD88. These studies demonstrate a transition in the intestinal microflora accompanied by a dynamic change of its ability to stimulate different PRRs which control intestinal homeostasis.

Effect of Cholecalciferol in Food Allergy Mouse Model Is Associated with Decrease of CD69+ CD4+ T Cells.

Food allergy prevalence is increasing all over the world. Recent epidemiologic studies have shown the link between vitamin D3 insufficiency and food allergy occurrence. In this study, we investigated the effect of supplementation with cholecalciferol, a widely used form of vitamin D3, on food allergy using an experimental mouse model. In wild-type BALB/c mice which were sensitized and challenged with an experimental allergen, ovalbumin, a clinical symptom of food allergy, diarrhea, was significantly induced with the elevation of immunoglobulin E level and the increases of T helper 2 cytokine productions, such as interleukin-4, -5, and -13 (p<0.05), whereas no change in T helper 1 cytokine production, such as interferon-γ, was observed. It was also found that cell population of CD69+ CD4+ T cells was increased slightly in spleen and significantly in the mesenteric lymphnode with the diarrheal symptom (p<0.05). Treatment of cholecalciferol reduced the allergic diarrhea (p<0.05) with the decreasing tendency of CD69+ CD4+ T cells, suggesting that the cell population might be associated with the attenuating effect of cholecalciferol on diarrhea occurrence, although immunoglobulin E levels and cytokine productions were not significantly altered by the treatment of cholecalciferol. When given the mice anti-CD69 mAb treatment, significant improvement of allergic diarrhea symptom was observed (p<0.05), accompanying the decrease of CD69+ CD4+ T cells which suggested the contribution of these cells to the diarrhea symptom. Taken together, we suggest that administration of cholecalciferol might be useful to suppress symptomatic food allergy in association with the decrease of CD69+ CD4+ T cells.

Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing.

Methane is used as an alternative carbon source in the denitrification of wastewater lacking organic carbon sources because it is nontoxic and may be efficiently produced by anaerobic biological processes. Methane-dependent denitrification (MDD) in the presence of oxygen requires the co-occurrence of methanotrophy and denitrification. Activated sludge was incubated with 13C-labeled methane in either a nitrate-containing medium or a nitrate-free medium. Then, bacterial and methanotrophic populations were analyzed by cloning analysis and terminal restriction fragment length polymorphism analysis targeting 16S rRNA gene and cloning analysis targeting pmoA genes. DNA-based stable-isotope probing (DNA-SIP) analysis of the 16S rRNA gene revealed an association of the Methylococcaceae and the Hyphomicrobiaceae in a MDD ecosystem. Furthermore, supplementation of nitrate stimulated methane consumption and the activity of methanotrophic populations (i.e. the stimulation of uncultivated relatives of distinct groups of the Methylococcaceae). In particular, uncultured type-X methanotrophs of Gammaproteobacteria were dominant when nitrate was added, i.e. in the MDD incubations. On the other hand, most methanotrophs (types I, II, and X methanotrophs) were found to have been labeled with 13C under nitrate-free conditions. This DNA-SIP study identifies key bacterial populations involved in a MDD ecosystem.

Effects of carbon source on denitrification efficiency and microbial community structure in a saline wastewater treatment process.

Two different denitrifying reactors were monitored in order to evaluate the effects of carbon source on denitrification efficiency and microbial community structure under various saline conditions. Nitrogen removal performances were determined when salinity concentrations increase gradually in acetate- or methanol-fed denitrifying reactor. As a result, acetate-fed process attained high nitrate removal at 0-10% NaCl, while methanol was proven beneficial electron donors at 0-3% NaCl. A parallel analysis of T-RFLP and cloning in the acetate-fed sludge showed that a specialized microbial population (i.e. the genera Halomonas and Marinobacter) adapted to a high saline environment. Meanwhile, there were no major changes of bacterial populations in the methanol-fed reactor at 4% NaCl, although the relative abundances of the genera Azoarcus and Methylophaga increased when salinity concentration was at 1-3% NaCl, indicating that methanol-utilizing populations in activated sludge was unable to adapt to a high saline environments (>4% NaCl).

Meta-Analysis of Fecal Microbiota and Metabolites in Experimental Colitic Mice during the Inflammatory and Healing Phases.

The imbalance of gut microbiota is known to be associated with inflammatory bowel disease, but it remains unknown whether dysbiosis is a cause or consequence of chronic gut inflammation. In order to investigate the effects of gut inflammation on microbiota and metabolome, the sequential changes in gut microbiota and metabolites from the onset of colitis to the recovery in dextran sulfate sodium-induced colitic mice were characterized by using meta 16S rRNA sequencing and proton nuclear magnetic resonance (¹H-NMR) analysis. Mice in the colitis progression phase showed the transient expansions of two bacterial families including Bacteroidaceae and Enterobacteriaceae and the depletion of major gut commensal bacteria belonging to the uncultured Bacteroidales family S24-7, Rikenellaceae, Lachnospiraceae, and Ruminococcaceae. After the initiation of the recovery, commensal Lactobacillus members promptly predominated in gut while other normally abundant bacteria excluding the Erysipelotrichaceae remained diminished. Furthermore, ¹H-NMR analysis revealed characteristic fluctuations in fecal levels of organic acids (lactate and succinate) associated with the disease states. In conclusion, acute intestinal inflammation is a perturbation factor of gut microbiota but alters the intestinal environments suitable for Lactobacillus members.

Bacterial metabolites directly modulate farnesoid X receptor activity.

BACKGROUND: The farnesoid X receptor (FXR), a ligand-activated transcription factor belonging to the adopted orphan receptor, plays an important role in maintaining health of the liver and intestine. In this study, we identified individual bacterial strains that directly modulated the activation of intestinal FXR. METHODS: The FXR stimulatory potential of 38 bacterial strains was determined using a stable FXR reporter system derived from intestinal epithelial cells (IEC). The induction of FXR target genes by screened FXR stimulatory bacteria was determined by real-time PCR. In addition, a high fat diet (HFD)-induced obese mouse model was used to evaluate in vivo FXR stimulatory potential of bacterial metabolites screened in this study. RESULTS: A luciferase assay with the FXR reporter cell line demonstrated that the FXR-stimulatory activity of most bacterial cell samples was less than 2-fold. The culture supernatants of Bacteroides dorei and Eubacterium limosum induced FXR activity and selectively regulated FXR target expression in the FXR reporter system. Treatment with B. dorei-derived metabolites strongly induced ileal bile acid binding protein (IBABP) (8.4-fold) and organic solute transporter (OST) α (3.1-fold) compared with E. limosum-derived metabolites. Furthermore, administration of B. dorei derived metabolites showed significant reduction in body weight gain, and both two bacterial metabolites reduced liver weight in obese mice compared to PBS-treated controls. Administration of each bacterial metabolites improved in serum levels of obesity-related metabolic biochemical markers such as ALT, AST, total cholesterol, and triglyceride. Furthermore, two bacterial metabolites enhanced the Fxr gene expression in the intestine and liver, and ileal Shp gene expression tended to be increased by treatment with the metabolites derived from B. dorei. CONCLUSIONS: B. dorei and E. limosum secreted the bioactive substances that directly stimulate FXR in the intestinal epithelial cells. Administration of these bacterial FXR-stimulatory metabolites improves the obesity phenotype including body weight gain, liver damage, lipid metabolism in DIO mice.

Strengthening of the intestinal epithelial tight junction by Bifidobacterium bifidum.

Epithelial barrier dysfunction has been implicated as one of the major contributors to the pathogenesis of inflammatory bowel disease. The increase in intestinal permeability allows the translocation of luminal antigens across the intestinal epithelium, leading to the exacerbation of colitis. Thus, therapies targeted at specifically restoring tight junction barrier function are thought to have great potential as an alternative or supplement to immunology-based therapies. In this study, we screened Bifidobacterium, Enterococcus, and Lactobacillus species for beneficial microbes to strengthen the intestinal epithelial barrier, using the human intestinal epithelial cell line (Caco-2) in an in vitro assay. Some Bifidobacterium and Lactobacillus species prevented epithelial barrier disruption induced by TNF-α, as assessed by measuring the transepithelial electrical resistance (TER). Furthermore, live Bifidobacterium species promoted wound repair in Caco-2 cell monolayers treated with TNF-α for 48 h. Time course (1)H-NMR-based metabonomics of the culture supernatant revealed markedly enhanced production of acetate after 12 hours of coincubation of B. bifidum and Caco-2. An increase in TER was observed by the administration of acetate to TNF-α-treated Caco-2 monolayers. Interestingly, acetate-induced TER-enhancing effect in the coculture of B. bifidum and Caco-2 cells depends on the differentiation stage of the intestinal epithelial cells. These results suggest that Bifidobacterium species enhance intestinal epithelial barrier function via metabolites such as acetate.

Complete autotrophic denitrification in a single reactor using nitritation and anammox gel carriers.

A novel aerobic denitrification reactor, aerobic denitrification using nitrifying and anoxic ammonium-oxidizing (anammox) bacteria immobilized on gel carriers in a single stage (AIGES), was developed. Two types of gel carriers, a nitritation gel carrier and an anammox gel carrier, were installed in single reactor, and the denitrification performance of simultaneous nitritation and anammox was evaluated. The denitrification performance increased gradually with increased aeration rate, reaching a denitrification rate of 1.4 kg N m(-3) d(-1) 2 weeks after the nitritation and anammox gel carriers were mixed. A high average denitrification efficiency of 82% was confirmed. Stable aerobic denitrification performance was observed for more than half a year. In the startup period of AIGES operation, ammonia-oxidizing bacteria were shown by fluorescence in situ hybridization analysis to grow on the surface layer of anammox gel cubes. These results indicated that anammox gel carriers promptly adapted to an aerobic environment by altering the microbial ecosystem.

High-rate denitrification using polyethylene glycol gel carriers entrapping heterotrophic denitrifying bacteria.

This study evaluated the nitrogen removal performance of polyethylene glycol (PEG) gel carriers containing entrapped heterotrophic denitrifying bacteria. A laboratory-scale denitrification reactor was operated for treatment of synthetic nitrate wastewater. The nitrogen removal activity gradually increased in continuous feed experiments, reaching 4.4 kg N m(-3) d(-1) on day 16 (30 °C). A maximum nitrogen removal rate of 5.1 kg N m(-3) d(-1) was observed. A high nitrogen removal efficiency of 92% on average was observed at a high loading rate. In batch experiments, the denitrifying gel carriers were characterized by temperature. Nitrate and total nitrogen removal activities both increased with increasing temperature, reaching a maximum at 37 and 43 °C, respectively. Apparent activation energies for nitrate and nitrite reduction were 52.1 and 71.9 kJ mol(-1), respectively. Clone library analysis performed on the basis of the 16S rRNA gene revealed that Hyphomicrobium was mainly involved in denitrification in the methanol-fed denitrification reactors.

Temperature dependence for anammox bacteria enriched from freshwater sediments.

The anoxic ammonium oxidation (anammox) process has been regarded as an attractive alternative process to treat wastewater containing high ammonium concentrations. By the implementation of anammox process at moderately low temperatures (<25°C), the anammox process will be applied to more various industrial wastewater treatments. In this study, we established enrichment cultures of anammox bacteria from freshwater sediments by using an up-flow column reactor equipped with porous polyester nonwoven fabric at moderately low temperatures. Their nitrogen conversion rates reached 0.07-0.26 kg-N/m³/d. Phylogenetic analysis based on 16S rRNA gene from enrichment cultures revealed the presence of various anammox bacteria affiliated with unknown anammox bacteria as well as known anammox candidates, i.e., Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida, Candidatus Scalindua wagneri. Anammox bacterial populations were influenced by enrichment conditions, i.e., seed sediments and temperature.

Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing.

Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [(13)C]acetate or [(13)C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from (13)C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from (13)C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from (13)C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).