Effect of Lipopolysaccharide on the Progression of Non-Alcoholic Fatty Liver Disease in High Caloric Diet-Fed Mice.

The incidence of non-alcoholic steatohepatitis (NASH) is increasing. Because gut microbiota have been highlighted as one of the key factors in the pathogenesis of metabolic syndrome, we investigated the involvement of the bacterial component in the progression of non-alcoholic fatty liver (NAFL) to NASH. C57BL/6 mice were fed with maintenance food (MF, groups A and B) or a high caloric diet (HCD, groups C and D) for 1 month. Mice were then divided into four groups: Groups A and C were inoculated with PBS, while groups B and D were inoculated with lipopolysaccharide (LPS) plus complete Freund's adjuvant (CFA). The inoculations were performed a total of 3 times over 3 months. At 6 months, while hepatic steatosis was observed in groups C and D, cellular infiltration and fibrosis were less evident in group C than in group D. Inflammatory cytokines were upregulated in groups B and D. 16S rRNA pyrosequencing of whole colon homogenates containing faeces showed that certain bacterial groups, such as Bacteroidaceae, Peptostreptococcaceae and Erysipelotrichaceae, were increased in groups C and D. Although loading of bacterial components (LPS) resulted in hepatic inflammation in both MF- and HCD-fed mice, HCD feeding was more crucial in the progression of NAFL during the triggering phase.

Modeling the nutrient removal process in aerobic granular sludge system by coupling the reactor- and granule-scale models.

We developed a model for nutrient removal in an aerobic granular sludge system. This model can quantitatively describe the start-up of the system by coupling a model for studying the population dynamics of the granules in the reactor (reactor-scale model) and a model for studying the microbial community structure in the granules (granule-scale model). The reactor-scale model is used for simulation for 10 days from the start, during which the granule size is relatively small; the granule-scale model is used after Day 10. The present approach proposes the output data of the reactor-scale model after 10 days as initial conditions for the granule-scale model. The constructed model satisfactorily describes experimental data in various spatial and temporal scales, which were obtained in this study by performing the anaerobic-aerobic-anoxic cycles using a sequencing batch reactor. Simulations using this model quantitatively predicted that the stability of nutrient removal process depended largely on the dissolved oxygen (DO) concentration, and the DO setpoint adaptation could improve the nutrient removal performance.

Microbial community of anammox bacteria immobilized in polyethylene glycol gel carrier.

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway in the biological nitrogen cycle and a new cost-effective way to remove ammonium from wastewater. We have so far developed new immobilization technique that anammox bacteria entrapped in polyethylene glycol (PEG) gel carrier. However, fate and behavior of anammox bacteria in a gel carrier is not well understood. In the present study, we focused on the population changes of anammox bacteria in a gel carrier. Three specific primer sets were designed for real-time PCR. For quantification of anammox bacteria in a gel carrier, real-time PCR was performed. The anammox bacteria related to HPT-WU-N03 clone were increased the rate in anammox population, and found to be a major population of anammox bacteria in a gel carrier. Furthermore, from the results of nitrogen removal performance and quantification of anammox bacteria, the correlation coefficient between copy numbers of anammox bacteria and nitrogen conversion rate was calculated as 0.947 in total anammox population. This is the first report that population changes of anammox bacteria immobilized in a gel carrier were evaluated.

Real-time control strategy for simultaneous nitrogen and phosphorus removal using aerobic granular sludge.

To achieve stable and simultaneous removal of nitrogen and phosphorus using aerobic granular sludge in a sequencing batch reactor, a real-time control strategy was established, where time derivatives of electric conductivity (EC) and pH were monitored to facilitate the determinations of ends of phosphate release, nitrification and denitrification as well as corresponding optimum time-lengths of anaerobic, oxic, and anoxic phases in treatment cycles. Although biomass concentration in a reactor drastically fluctuated at the startup period because of very short sludge settling time for the formation of aerobic granular sludge, cycle length for proper treatment was automatically adjusted in this control system. Even when characteristics of influent wastewater markedly fluctuated, stable nitrogen and phosphorus removal was successfully attained both before and at pseudo-steady-state. Effluent concentrations of NH4-N, NOx-N and PO4-P were always lower than 0.3 mg/L. On the other hand, when time lengths of the anaerobic/oxic/anoxic phases were fixed, stable nitrogen and phosphorus removal was not accomplished. Therefore, it is clear that the designed control system is very effective to obtain stable treatment performance in simultaneous nitrogen and phosphorus removal by aerobic granular sludge.

Tertiary treatment of domestic wastewater using zeolite ceramics and aquatic plants.

In this study, we examined tertiary treatment of domestic wastewater using zeolite ceramics and aquatic plants, especially reeds, Phragmites australis. The experiment was made at real domestic wastewater treatment facilities, and comparison of treatment performance was made between the method with zeolite ceramics and that with pebble stones as conventional way. SEM observation of the ceramics' surface was also made to examine its possibility as the habitat of bacteria. The results obtained are as follows. Through the tertiary treatment experiment, it was suggested that the water purification system with zeolite ceramics and reeds could keep higher nitrogen removal efficiency for a long time. Zeolite ceramics would be useful when nitrogen compound, NH(4)-N in particular, in the influent was higher. Under SEM observation, bacteria-like objects were observed on the ceramics' surface. Appropriate operation and maintenance would be needed to keep long-term performance of both the NH(4) (+) absorption and nitrogen removal with use of zeolite ceramics.

Use of real-time PCR to examine the relationship between ammonia oxidizing bacterial populations and nitrogen removal efficiency in a small decentralized treatment system 'Johkasou'.

The aim of this study was to examine the relationship between ammonia oxidizing bacterial populations and biological nitrogen removal in a small on-site domestic wastewater treatment system "Johkasou". The population dynamics of ammonia oxidizing bacteria (AOB) in six full-scale advanced Johkasous was surveyed using real-time PCR assay over a period of one year. These Johkasous were selected to compare the AOB populations in different treatment performance. When the effluent NH4-N concentration was higher than 2 mg L(-1), it was difficult to meet the effluent standard of advanced Johkasous (T-N 10 mg L(-1)). In contrast, the nitrogen removal efficiency was hardly affected by nitrite oxidation and denitrification in these systems. In other words, ammonia oxidation was a rate-limiting step. Furthermore, we focused on the relationship between NH4-N loading per AOB cell and nitrogen removal. Real time PCR monitoring results demonstrated that it is important to regulate NH4-N loading per AOB cell below 210 pg cell(-1) day(-1) to meet the effluent standard of advanced Johkasou. It is considered that NH4-N loading per AOB cell is a useful parameter for determining suitable nitrogen loading and small decentralized system design.

Experimental and simulation analysis of community structure of nitrifying bacteria in a membrane-aerated biofilm.

Until now, only few attempts have been made to assess biofilm models simulating microenvironments in a biofilm. As a first step, we compare the microenvironment observed in a membrane aerated biofilm (MAB) to that derived from a two-dimensional computational model with individual ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) embedded in a continuum EPS matrix. Gradients of oxygen were determined by means of microelectrodes. The change in nitrifying bacterial populations with the biofilm depth was quantified using fluorescence in situ hybridization (FISH) in combination with a confocal laser scanning microscopy (CLSM). Microelectrode measurements revealed that oxic and anoxic or anaerobic regions exist within the MAB. The oxygen profile predicted by the model showed good agreement with that obtained by microelectrode measurements. The oxic part of the biofilm was dominated by NSO190 probe-hybridized AOB, which formed relatively large clusters of cells directly on the membrane surface, and by the NOB belonging to genus Nitrobacter sp. On the other hand, NOB belonging to genus Nitrospira sp. were abundant at the oxic-anoxic interface. The model prediction regarding AOB and Nitrobacter sp. distribution was consistent with the experimental counterpart. Measurements of AOB cluster size distribution showed that colonies are slightly larger adjacent to the membrane than at the inner part of the biofilm. The sizes predicted by the current model are larger than those obtained in the experiment, leading to the arguments that some factors not contained in the model would affect the cluster size.

High-rate nitrification using aerobic granular sludge.

The performance of nitrifying granules, which had been produced in an aerobic upflow fluidised bed (AUFB) reactor, was investigated in various types of ammonia-containing wastewaters. When pure oxygen was supplied to the AUFB reactor with a synthetic wastewater containing a high concentration of ammonia (500 g-N/m3), the ammonia removal rate reached 16.7 kg-N/m3/day with a sustained ammonia removal efficiency of more than 80%. The nitrifying granules possessing a high settling ability could be retained with a high density (approximately 10,000 g-MLSS/m3) in a continuous stirring tank reactor (CSTR) even under a short hydraulic retention time (44 min), which enabled a high-rate and stable nitrification for an inorganic wastewater containing low concentrations of ammonia (50 g-N/m3). Moreover, the nitrifying granules exhibited sufficient performance in the nitrification of real industrial wastewater containing high concentrations of ammonia (1000-1400 g-N/m3) and salinity (1.2-2.2%), which was discharged from metal-refinery processes. When the nitrifying granules were used in cooperation with activated sludge to treat domestic wastewater containing organic pollutants as well as ammonia, they fully contributed to nitrification even though a part of activated sludge adhered onto the granule surfaces to form biofilms. These results show the wide applicability of nitrifying granules to various cases in the nitrification step of wastewater treatment plants.

Evaluation of sludge reduction and phosphorus recovery efficiencies in a new advanced wastewater treatment system using denitrifying polyphosphate accumulating organisms.

A new biological nutrient removal process, anaerobic-oxic-anoxic (A/O/A) system using denitrifying polyphosphate-accumulating organisms (DNPAOs), was proposed. To attain excess sludge reduction and phosphorus recovery, the A/O/A system equipped with ozonation tank and phosphorus adsorption column was operated for 92 days, and water quality of the effluent, sludge reduction efficiency, and phosphorus recovery efficiency were evaluated. As a result, TOC, T-N and T-P removal efficiency were 85%, 70% and 85%, respectively, throughout the operating period. These slightly lower removal efficiencies than conventional anaerobic-anoxic-oxic (A/A/O) processes were due to the unexpected microbial population in this system where DNPAOs were not the dominant group but normal polyphosphate-accumulating organisms (PAOs) that could not utilize nitrate and nitrite as electron acceptor became dominant. However, it was successfully demonstrated that 34-127% of sludge reduction and around 80% of phosphorus recovery were attained. In conclusion, the A/O/A system equipped with ozonation and phosphorus adsorption systems is useful as a new advanced wastewater treatment plant (WWTP) to resolve the problems of increasing excess sludge and depleted phosphorus.

Deuteration kinetics of deoxyguanosine, deoxycytidine, and their polynucleotides by means of ultraviolet spectrophotometry.

The kinetics of the hydrogen-deuterium exchange reactions of deoxyguanosine (dG), deoxycytidine (dC), double-helical poly[d(G-C)] X poly[d(G-C], and double-helical poly(dG) X poly(dC) have been examined at 20 degrees C, pH 7.0, and in low-salt (0.15 M NaCl) medium by stopped-flow ultraviolet spectrophotometry, in the spectral region of 260 to 320 nm. The rate constant was found to be 78.9 s-1 for dG-NH, 2.2 s-1 for dG-NH2, 39.3 s-1 for dC-NH2, 2.4 s-1 (fast) and 0.94 s-1 (slow) for poly[d(G-C)] X poly[d(G-C)], and 2.2 s-1 (fast) and 0.92 s-1 (slow) for poly(dG) X poly(dC). From these values, the probability of base-pair opening of the G X C containing B-form double helix is estimated to be (3 +/- 1) X 10(-3). This is much greater than what is expected from an extrapolation of the van't Hoff plot at the helix-coil transition region, i.e. at about 110 degrees C. The mechanism of these base-pair openings at 20 degrees C (as well as the mechanism of base-pair reformation) is suggested to be totally different from those in the melting temperature range.

Ion exchange of lysozyme during permeation across a microporous sulfopropyl-group-containing hollow fiber.

A microporous hollow fiber containing a sulfopropyl (SP) group as a strongly acidic cation-exchange group was prepared by radiation-induced graft polymerization of glycidyl methacrylate, followed by hydrolysis of the resulting epoxide group into a diol, and then conversion of the diol into the SP group. The SP group density of the resulting hollow fiber ranged from 0.21 to 0.84 mol/kg of dry fiber with a pure water flux of 2.7 m/h at a filtration pressure of 0.1 MPa. Lysozyme adsorption was examined during permeation of the lysozyme solution (pH 6) through the pores across a microporous cation-exchange hollow fiber. The lysozyme concentration of the effluent penetrating the outside of the hollow fiber did not change irrespective of the residence time of the solution across the hollow fiber, which was indicative of the negligible diffusional resistance of lysozyme to the SP group. The binding capacity of lysozyme to the fiber was constant in this range of SP group density. For comparison, the adsorption characteristics of a cupric chloride solution during permeation were also determined. The binding capacity of Cu to the fiber increased linearly with increasing SP group density, because cupric ions of a smaller size than lysozyme can invade the depths of the grafted polymer branches formed in the amorphous domain of the polymer matrix.

Binding of lysozyme onto a cation-exchange microporous membrane containing tentacle-type grafted polymer branches.

Ion-exchange adsorption of lysozyme to the sulfonic acid (SO3H) group on polymer chains grafted onto microporous polyethylene hollow-fiber membranes was examined. The lysozyme solution was forced to permeate across the hollow fiber. Diversely anchored SO3H groups, i.e., SP and SS groups, were introduced into the membrane by reaction of the glycidyl methacrylate-grafted membrane with propanesultone and sodium sulfite, respectively. The resulting SP and SS group-containing membranes, designated as SP-T and SS-T fibers, respectively, had 95 and 77% water flux of the original membrane, respectively. The binding capacity of lysozyme as a function of the SO3H group density was compared between the SP-T and SS-T fibers from measurement of the ion-exchange breakthrough curves during the permeation of lysozyme solution across the SP-T and SS-T fibers. The binding capacity of lysozyme to the SP-T fiber remained constant, independent of the SP group density, whereas that to the SS-T fiber increased linearly with increasing SS group density. This difference was explained by means of a model whereby lysozyme adheres onto the SP group-containing grafted polymer branches, while the SS group-containing grafted polymer branches hold lysozyme in a tentacle-like manner.

Synthesis of 4'-deoxy-4'-fluorokanamycin A and B.

4'-Deoxy-4'-fluorokanamycins A (17) and B (25) have been prepared through fluorinative ring-opening of the D-galacto-3',4'-oxiranes (8 and 21) derived from kanamycin A and B with potassium hydrogenfluoride in ethane-1,2-diol. The mechanism of preponderant formation of the 4'-deoxy-4'-fluoro-D-gluco (9 and 22) over the 3'-deoxy-3'-fluoro-D-gulo derivatives was discussed. In the synthesis of 25, the unusual 3',6'-epimine (23) was the main product along with the 4'-deoxy-4'-fluoro derivative. The mechanism of this reaction is also discussed. Both 17 and 25 were active against resistant bacteria producing aminoglycoside-adenylylating enzymes for HO-4'.

Metabolic behavior of denitrifying phosphate-accumulating organisms under nitrate and nitrite electron acceptor conditions.

The effects of various types of electron acceptors on anoxic phosphorus uptake were investigated in detail to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms. Batch experimental tests under three different electron acceptor conditions, i.e., nitrate, nitrite and mixtures of nitrate and nitrite, were carried out using activated sludge cultivated in a sequencing batch reactor. The experimental results confirmed no inhibition of the utilization of nitrate or nitrite as an electron acceptor for anoxic phosphorus uptake. Anoxic phosphorus uptake occurred provided there was an electron acceptor present regardless of whether it was nitrate or nitrite. However, for nitrite a relatively small amount of anoxic phosphorus was taken up per nitrogen denitrified compared to nitrate. On the other hand, the amount of anoxic phosphorus taken up per nitrogen denitrified increased with an increase in the initial loading amount of electron acceptor in the case of nitrate, whereas it slightly decreased nitrite. Moreover, the amount of phosphorus taken up per nitrogen denitrified decreased with increasing mixed liquor suspended solid (MLSS) concentration in the case of nitrate, while it slightly increased for nitrite. From these results, it was confirmed that the activity of anoxic phosphorus uptake is strongly associated with the type and the initial loading amount of electron acceptor and the MLSS concentration under anoxic conditions.

Microbial ecology of nitrifying bacteria in wastewater treatment process examined by fluorescence in situ hybridization.

The microbial ecology of nitrifying bacteria in various types of wastewater treatment processes and the dynamic response of the microbial ecology in biofilms were investigated using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Nitrifying bacteria were found to exhibit various organizational forms under different conditions of substrate composition and concentration. Ammonia-oxidizing bacteria were dominant in ammonia-rich inorganic wastewater, while heterotrophic bacteria and ammonia-oxidizing bacteria were localized at different positions in the biofilm in organic wastewater. The dynamics of the microbial ecology in the biofilm with regard to the spatial distribution of ammonia-oxidizing bacteria and heterotrophic bacteria caused by a gradual change in substrate composition was successfully monitored by FISH analysis.

Microbial community analysis in the denitrification process of saline-wastewater by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA and the cultivation method.

The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds and salts. Biological nitrogen removal from this wastewater was attempted using a circulating bioreactor system equipped with an anaerobic packed bed or an anaerobic fluidized bed. The denitrification capability of the system with the anaerobic packed bed was more stable than that of the system with the anaerobic fluidized bed. The NOx removal rate of the anaerobic packed bed was as high as 97%. Microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments and the cultivation method revealed that the community diversity varied in accordance with wastewater composition such as the level of salinity and so on. Phylogenetic analysis suggested that the taxonomic affiliation of the dominant species in the anaerobic reactors was to the gamma-Proteobacteria including Halomonadaceae species. The PCR-DGGE method as a non-cultivation method was found to be a powerful tool for analysis of the microbial community, because the cultivation method could detect only a fraction of the microbial species present in these systems. The genetic diversity of the isolated bacteria belonging to the gamma-Proteobacteria which reduced both nitrate and nitrite in the anaerobic packed bed was higher than that of the bacteria in the anaerobic fluidized bed. This suggested that a genetically diverse microbial community stabilized the denitrifying performance in the anaerobic packed bed.

Evaluation of kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor for wastewater treatment.

This study evaluates the kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor from the steady state values of the response of the system to step changes in inlet concentration. It was observed from the outlet biological oxygen demand (BOD(5)) plot of the response of the system that as the inlet BOD(5) was increased, the outlet BOD(5) also increased, reached a peak value and then decreased until it leveled to a new steady state value corresponding to the new inlet concentration level. The increase in BOD(5) was attributed to the accumulation of substrate within the reactor as well as the decrease in biofilm substrate consumption rate as the microorganisms adjusted to the new environment. Using the substrate balance at steady state and assuming Monod kinetics, an equation relating the substrate consumption rate to substrate concentration (BOD(5)) and total biofilm surface area had been established. Monod kinetic parameters were found to be K=2.20g/m(2)/day, K(m)=17.41g/m(3) and K/K(m)=0.13m/day. The ratio K/K(m) can be taken as the indicator for biofilm substrate degradation effectiveness at low substrate concentrations.

Transition of bacterial spatial organization in a biofilm monitored by FISH and subsequent image analysis.

The dynamic transition of bacterial community structure in a biofilm was monitored by the fluorescence in situ hybridization (FISH) technique and subsequent image analysis. Heterotrophic bacteria that had occupied the outer layer were gradually decreased whereas ammonia-oxidizing bacteria (AOB) gradually increased their growth activity and extended their existence area to the outer layer of the biofilm through the gradual reduction of the C/N ratio. The spatial organization of AOB in the biofilm dynamically changed responding to the environmental conditions such as pH fluctuation and lack of dissolved oxygen (DO) and had great influence on the nitrification activity. The accumulation of nitrite was observed at lower DO concentration, which might be due to the property that nitrite-oxidizing bacteria (NOB) possess of higher Km values for oxygen than AOB.

Biological nitrogen removal from industrial wastewater discharged from metal recovery processes.

The wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of acids and alkalis such as nitric acid and aqueous ammonia, and of salts such as sodium chloride and sodium sulfate. Biological nitrogen removal from this wastewater was attempted by using a circulating bioreactor system equipped with an anaerobic packed bed and an aerobic three-phase fluidized bed. As a result of acclimating microorganisms with change of the hydraulic residence time, this system effectively removed nitrogen from diluted wastewater (T-N: from 2,000 to 4,000 g/m3), such that the total nitrogen concentration in the effluent met the sewage discharge control criteria in Japan (240 g/m3). The removal ratio of total nitrogen was 90% to 98% and that of ammonia was 80% to 92%. In addition, the characteristic equations for biological treatment were applied to this system on the assumption that both reactions of denitrification in the anaerobic reactor and nitrification in the aerobic reactor can be approximated to a first-order reaction. This simplified approach successfully led to a new analytical method for simulating the optimum volume ratio of anaerobic reactor to aerobic reactor for minimizing the total hydraulic residence time.

Formation mechanism of nitrifying granules observed in an aerobic upflow fluidized bed (AUFB) reactor.

The influences of trace metals in the wastewater and shear stress by aeration were particularly examined to clarify the formation mechanism of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor. It was found that Fe added as a trace element to the inorganic wastewater accumulated at the central part of the nitrifying granules. Another result obtained was that suitable shear stress by moderate aeration (0.07-0.20 L/min/L-bed) promoted granulation. Furthermore, it was successfully demonstrated that pre-aggregation of seed sludge using hematite promoted core formation, leading to rapid production of nitrifying granules. From these results, a nitrifying granulation mechanism is proposed: 1) as a first step, nitrifying bacteria aggregate along with Fe precipitation, and then the cores of granules are formed; 2) as a second step, the aggregates grow to be spherical or elliptical in form due to multiplication of the nitrifying bacteria and moderate shear stress in the reactor, and then mature nitrifying granules are produced. Fluorescence in situ hybridization (FISH) analysis successfully visualized the change in the spatial distribution of nitrifying bacteria in the granules, which supports the proposed granulation mechanism.