Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.

Members of the genus Nitrosomonas are major ammonia oxidizers that catalyse the first step of nitrification in various ecosystems. To date, six subgenus-level clades have been identified. We have previously isolated novel ammonia oxidizers from an additional clade (unclassified cluster 1) of the genus Nitrosomonas. In this study, we report unique physiological and genomic properties of the strain PY1, compared with representative ammonia-oxidising bacteria (AOB). The apparent half-saturation constant for total ammonia nitrogen and maximum velocity of strain PY1 were 57.9 ± 4.8 μM NH3  + NH4 + and 18.5 ± 1.8 μmol N (mg protein)-1  h-1 , respectively. Phylogenetic analysis based on genomic information revealed that strain PY1 belongs to a novel clade of the Nitrosomonas genus. Although PY1 contained genes to withstand oxidative stress, cell growth of PY1 required catalase to scavenge hydrogen peroxide. Environmental distribution analysis revealed that the novel clade containing PY1-like sequences is predominant in oligotrophic freshwater. Taken together, the strain PY1 had a longer generation time, higher yield and required reactive oxygen species (ROS) scavengers to oxidize ammonia, compared with known AOB. These findings expand our knowledge of the ecophysiology and genomic diversity of ammonia-oxidising Nitrosomonas.

Persisters are a subpopulation that exhibit growth suppression, antibiotic tolerance, and regrowth after antibiotic removal, without any genetic mutations, which causes the recalcitrance and recurrence of infectious diseases. Persisters are majorly induced through the repression of energy metabolism, but some exceptions have been reported. We have previously shown that ldhA, which encodes lactate dehydrogenase, induces Escherichia coli persisters, resulting in a state of high-energy metabolism. However, the detailed mechanism of persister formation upon ldhA expression remains elusive. In the present study, we focused on the SOS response pathway via the DNA repair pathway that consumes adenosine triphosphate and revealed that the SOS response pathway is activated upon ldhA expression even before antimicrobial treatment. Metabolome analysis of ldhA-overexpressing cells revealed that nucleotide metabolic pathways, such as de novo purine biosynthesis, were activated to prepare a nucleotide pool, as substrate for repairing ofloxacin-induced DNA damage. We provide a novel persister model that contributes to survival as a species by “accidentally” activating the SOS response even before receiving antimicrobial stress.

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.

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.

Droplet microfluidics has emerged as a powerful technology for improving the culturing efficiency of environmental microorganisms. However, its widespread adoption has been limited due to considerable technical challenges, especially related to identification and manipulation of individual growth-positive droplets. Here, we combined microfluidic droplet technology with on-chip “fluorescent nucleic acid probe in droplets for bacterial sorting” (FNAP-sort) for recovery of growth-positive droplets and droplet microdispensing to establish an end-to-end workflow for isolation and culturing of environmental microbes. As a proof-of-concept, we demonstrate the ability of our technique to yield high-purity cultures of rare microorganisms from a representative complex environmental microbiome. As our system employs off-the-shelf commercially available equipment, we believe that it can be readily adopted by others and may thus find widespread use toward culturing the high proportion of as-of-yet uncultured microorganisms in different biomes.

Pseudomonas aeruginosa harbours two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N-binding sites at single-nucleotide resolution and mapped more than 500 binding sites to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also verified the ANGGA sequence in apical loops skewed towards 5ʹUTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results suggested previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the RsmA/N-titrating RNAs RsmY/RsmZ may be positively regulated by the RsmA/N-mediated translational repression of their upstream regulators, thus providing a possible mechanistic explanation for homoeostasis of the Rsm system. Thus, our study provides a detailed view of RsmA/N-RNA interactions and a resource for further investigation of the pleiotropic effects of RsmA/N on gene expression in P. aeruginosa.

To simulate the cell dynamics within mouse and human colonic crypts, many computational models have been developed. While real colonic crypts are shaped like test tubes, crypts were assumed as cylinders in several such models. However, possible drawbacks by modeling the crypt as a cylinder have not been investigated. Here we constructed a model considering a test-tube-like-shaped crypt in three-dimensional space, and simulated cell dynamics within it. The simulation results were compared with those obtained with a model using a cylinder crypt. We found that these two models showed different simulation results for the dynamics of expansion of a mutation occurred in a stem cell at the crypt bottom, and that the model using a test-tube-like-shaped crypt simulated the stem cell expansion more accurately. To simulate cell dynamics occurred in the lower part of the crypt, such as apoptosis and initial expansion of mutation, using a test-tube-like-shaped crypt is preferable. In a model assuming a cylinder crypt, it is considered that reported values for the required time of monoclonal conversion in a colonic crypt and its occurrence rate were overestimated and underestimated, respectively.

The methanol tolerance level of the anammox process was investigated in continuous feeding tests. In the conducted pulse methanol addition tests of gel carrier-immobilized anammox bacteria, the nitrogen removal performance was not significantly affected at influent methanol concentrations of up to 30 mg L−1; however, irreversible inhibition occurred at 40 mg L−1. In the absence of methanol acclimation, the anammox process showed low methanol tolerance levels. Moreover, the stepwise addition of 5 mg L−1 of methanol resulted in no anammox inhibition even when the methanol concentration became 100 mg L−1. The effluent total organic carbon concentration did not increase with the increase in the influent methanol concentration; this indicates that methanol was consumed along with the nitrate or nitrite by heterotrophic denitrification. These results show that the increase in the methanol tolerance level was due to the increase in the methanol consumption ability during the anammox process because of the increased denitrifying bacteria and not because the anammox bacteria exhibited resistance to methanol. The microbial community analysis revealed the dominant species in the gel carriers (Candidatus Jettenia asiatica), existence of methylotrophic bacteria, and growth in the methanol-consuming bacterial population.

Isotopic fractionation factors against 15N and 18O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ18O of water to investigate the effects of water δ18O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (15εNXR of -77.8 to -65.9‰ and 15ΔNXR of -31.3 to -30.4‰) and possibly O isotopic effect (18εNXR of -20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ18O vs δ15N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ18O vs δ15N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses. Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis (ATP synthase and ribosomal proteins) and motility and adhesion (flagella, pilus, and chemotaxis). Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and inositol 1,4,5-trisphosphate receptor type 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF-FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

Bacterial small noncoding RNAs (sRNAs) play posttranscriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the Pseudomonas aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5' untranslated regions (UTRs) and sRNAs and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that the association of sRNAs with Hfq is primarily a function of their expression levels, strongly supporting the notion that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of posttranscriptional regulation in P. aeruginosa IMPORTANCE The Gram-negative bacterium P. aeruginosa is ubiquitously distributed in diverse environments and can cause severe biofilm-related infections in at-risk individuals. Although the presence of a large number of putative sRNAs and widely conserved RNA chaperones in this bacterium implies the importance of posttranscriptional regulatory networks for environmental fluctuations, limited information is available regarding the global role of RNA chaperones such as Hfq in the P. aeruginosa transcriptome, especially under different environmental conditions. Here, we characterize Hfq-dependent differences in gene expression and biological processes in two physiological states: the planktonic and biofilm forms. A combinatorial comparative CLIP-seq and total RNA-seq approach uncovered condition-dependent association of RNAs with Hfq in vivo and expands the potential direct regulatory targets of Hfq in the P. aeruginosa transcriptome.

A Corrigendum on Nitrosomonas europaea MazF Specifically Recognises the UGG Motif and Promotes Selective RNA Degradation by Miyamoto, T., Yokota, A., Ota, Y., Tsuruga, M., Aoi, R., Tsuneda, S., et al. (2018). Front. Microbiol. 9:2386. doi: 10.3389/fmicb.2018.02386 In the original article, there was a mistake in Figure 4 as published. Figure 4 shows the relationship between the actual number of TGG triplets (K) and Pvalues in each CDS. In this figure, we mistook hao for rbcL
the dot which represents hao corresponds to the rbcL transcript, while the dot which represents rbcL corresponds to the hao transcripts. The corrected Figure 4 appears below.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Assembling three-dimensional (3D) tissues from single cells necessitates the use of various advanced technological methods because higher-density tissues require numerous complex capillary structures to supply sufficient oxygen and nutrients. Accordingly, creating healthy culture conditions to support 3D cardiac tissues requires an appropriate balance between the supplied nutrients and cell metabolism. The objective of this study was to develop a simple and efficient method for low-temperature cultivation (< 37 °C) that decreases cell metabolism for facilitating the buildup of 3D cardiac tissues. We created 3D cardiac tissues using cell sheet technology and analyzed the viability of the cardiac cells in low-temperature environments. To determine a method that would allow thicker 3D tissues to survive, we investigated the cardiac tissue viability under low-temperature culture processes at 20-33.5 °C and compared it with the viability under the standard culture process at 37 °C. Our results indicated that the standard culture process at 37 °C was unable to support higher-density myocardial tissue; however, low-temperature culture conditions maintained dense myocardial tissue and prevascularization. To investigate the efficiency of transplantation, layered cell sheets produced by the low-temperature culture process were also transplanted under the skin of nude rats. Cardiac tissue cultured at 30 °C developed denser prevascular networks than the tissue cultured at the standard temperature. Our novel findings indicate that the low-temperature process is effective for fabricating 3D tissues from high-functioning cells such as heart cells. This method should make major contributions to future clinical applications and to the field of organ engineering.

Bacterial persisters are phenotypic variants that survive the treatment of lethal doses of growth-targeting antibiotics without mutations. Although the mechanism underlying persister formation has been studied for decades, how the persister phenotype is switched on and protects itself from antibiotics has been elusive. In this study, we focused on the lactate dehydrogenase gene (ldhA) that was upregulated in an Escherichia coli persister-enriched population. A survival rate assay using an ldhA-overexpressing strain showed that ldhA expression induced persister formation. To identify ldhA-mediated persister formation at the single-cell level, time-lapse microscopy with a microfluidic device was used. Stochastic ldhA expression was found to induce dormancy and tolerance against high-dose ampicillin treatment (500 μg/ml). To better understand the underlying mechanism, we investigated the relationship between ldhA-mediated persister formation and previously reported persister formation through aerobic metabolism repression. As a result, ldhA expression enhanced the proton motive force (PMF) and ATP synthesis. These findings suggest that ldhA-mediated persister formation pathway is different from previously reported persister formation via repression of aerobic metabolism.

Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2- and MPL-unmutated Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5-base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR-Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5-haematopoietic progenitor cells (Ins5-HPCs) than in WT-HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR-mutant ET patients. Ins5-HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3-hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.

Persisters are multidrug-tolerant cells that are present within antibiotic-sensitive populations. Persister formation is not induced by genetic mutations, but rather by changes in the degree of expression of some genes. High redundancy has been observed among the pathways that have been hypothesized to respond to specific stresses. In this study, we conducted RNA sequencing of Escherichia coli persisters under various stress conditions to identify common mechanisms. We induced stresses such as glucose or amino acid exhaustion, acid stress and anaerobic conditions, all of which are encountered during bacterial pathogenesis. We found that most genes are differentially expressed depending on the specific stress condition
however, some genes were commonly expressed in persisters in most stress conditions. Commonly expressed genes are expected to be promising therapeutic targets for combating persistent infections. We found that knockdown of aldehyde dehydrogenase (aldB), which was expressed in every condition except for acid stress, decreased persisters in the non-stressed condition. However, the same strain unexpectedly showed an increased number of persisters in the amino acid-limited condition. Because the increase in persister number is glycolytic metabolite-dependent, metabolic flow may play a crucial role in aldB-mediated persister formation. These data suggest that environmental stresses alter persister mechanisms. Identification of environmental influences on persister formation during pathogenesis is therefore necessary to enabling persister eradication.

To enhance the therapeutic effect of growth factors, a powerful strategy is to direct their localization to damaged sites. To treat skin wounds and myocardial infarction, we selected vascular endothelial growth factor (VEGF) carrying binding affinity to collagen. A simple conjugation of a reported collagen-binding sequence and VEGF did not increase the collagen-binding affinity, indicating that the molecular interaction between the two proteins abolished collagen binding activity. Here, we present a new molecular evolution strategy, "all-in-one" in vitro selection, in which a collagen-binding VEGF (CB-VEGF) was directly identified from a random library consisting of random and VEGF sequences. As expected, the selected CB-VEGFs exhibited high binding affinity to collagen and maintained the same growth enhancement activity for endothelial cells as unmodified VEGF in solution. Furthermore, the selected CB-VEGF enhanced angiogenesis at skin wounds and infarcted myocardium. This study demonstrates that "all-in-one" in vitro selection is a novel strategy for the design of functional proteins for regenerative medicine.

The genus Nitrospira represents a dominant group of nitrite-oxidizing bacteria in natural and engineered ecosystems. This genus is phylogenetically divided into six lineages, for which vast phylogenetic and functional diversity has been revealed by recent molecular ecophysiological analyses. However, the genetic basis underlying these phenotypic differences remains largely unknown because of the lack of genome sequences representing their diversity. To gain a more comprehensive understanding of Nitrospira, we performed genomic comparisons between two Nitrospira strains (ND1 and NJ1 belonging to lineages I and II, respectively) previously isolated from activated sludge. In addition, the genomes of these strains were systematically compared with previously reported six Nitrospira genomes to reveal their similarity and presence/absence of several functional genes/operons. Comparisons of Nitrospira genomes indicated that their genomic diversity reflects phenotypic differences and versatile nitrogen metabolisms. Although most genes involved in key metabolic pathways were conserved between strains ND1 and NJ1, assimilatory nitrite reduction pathways of the two Nitrospira strains were different. In addition, the genomes of both strains contain a phylogenetically different urease locus and we confirmed their ureolytic activity. During gene annotation of strain NJ1, we found a gene cluster encoding a quorum-sensing system. From the enriched supernatant of strain NJ1, we successfully identified seven types of acyl-homoserine lactones with a range of C10-C14. In addition, the genome of strain NJ1 lacks genes relevant to flagella and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated genes) systems, whereas most nitrifying bacteria including strain ND1 possess these genomic elements. These findings enhance our understanding of genomic plasticity and functional diversity among members of the genus Nitrospira.

Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia coli strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non-dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non-dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that persisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters.

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 (H-1-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, H-1-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.

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 mu m and the maximum ammonia removal rate was observed to be 1.5 kgN.m(-3).day(-1) at 20 degrees C, which is 2.5-5 times faster than traditional activated sludge methods. A fluorescence in situ hybridization analysis showed that beta-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.

Microbes are known to withstand environmental stresses by using chromosomal toxin-antitoxin systems. MazEF is one of the most extensively studied toxin-antitoxin systems. In stressful environments, MazF toxins modulate translation by cleaving single-stranded RNAs in a sequence-specific fashion. Previously, a chromosomal gene located at DR0417 in Deinococcus radiodurans was predicted to code for a MazF endoribonuclease (MazF(DR0417)); however, its function remains unclear. In the present study, we characterized the molecular function of MazF(DR0417). Analysis of MazF(DR0417)-cleaved RNA sites using modified massively parallel sequencing revealed a unique 4-nt motif, UACA, as a potential cleavage pattern. The activity of MazF(DR0417) was also assessed in a real-time fluorometric assay, which revealed that MazF(DR0417) strictly recognizes the unique tetrad UACA. This sequence specificity may allow D. radiodurans to alter its translation profile and survive under stressful conditions.

Nitrite-oxidizing bacteria (NOB) are responsible for the second step of nitrification in natural and engineered ecosystems. The recently discovered genus Nitrotoga belongs to the Betaproteobacteria and potentially has high environmental importance. Although environmental clones affiliated with Nitrotoga are widely distributed, the limited number of cultivated Nitrotoga spp. results in a poor understanding of their ecophysiological features. In this study, we successfully enriched the nonmarine cold-adapted Nitrotoga sp. strain AM1 from coastal sand in an eelgrass zone and investigated its physiological characteristics. Multistep-enrichment approaches led to an increase in the abundance of AM1 to approximately 80% of the total bacterial population. AM1 was the only detectable NOB in the bacterial community. The 16S rRNA gene sequence of AM1 was 99.6% identical to that of "Candidatus Nitrotoga arctica," which was enriched from permafrost-affected soil. The highest nitrogen oxidation rate of AM1 was observed at 16 degrees C. The half-saturation constant (K-m) and the generation time were determined to be 25 mu M NO2- and 54 h, respectively. The nitrite oxidation rate of AM1 was stimulated at concentrations of &lt;30 mM NH4Cl but completely inhibited at 50 mM NH4Cl. AM1 can grow well under specific environmental conditions, such as low temperature and in the presence of a relatively high concentration of free ammonia. These results help improve our comprehension of the functional importance of Nitrotoga.
IMPORTANCE Nitrite-oxidizing bacteria (NOB) are key players in the second step of nitrification, which is an important process of the nitrogen cycle. Recent studies have suggested that the organisms of the novel NOB genus Nitrotoga were widely distributed and played a functional role in natural and engineered ecosystems. However, only a few Nitrotoga enrichments have been obtained, and little is known about their ecology and physiology. In this study, we successfully enriched a Nitrotoga sp. from sand in a shallow coastal marine ecosystem and undertook a physiological characterization. The laboratory experiments showed that the Nitrotoga enrichment culture could adapt not only to low temperature but also to relatively high concentrations of free ammonia. The determination of as-yet-unknown unique characteristics of Nitrotoga contributes to the improvement of our insights into the microbiology of nitrification.

The first full-scale nitritation-anammox process using gel entrapment technology was evaluated for nitrogen removal. Ammonia plant effluents contained not only ammonium but also methanol, an anammox inhibitor. The nitrogen concentration and loading design were 690 mg/L and 400 kg-N/d, respectively. A nitritation reactor (170 m(3)) filled with nitrification gel carriers was started up on day 20, with a resulting nitrification efficiency of 58%. A stable nitritation performance was observed without significant nitrate production for &gt;1 year. A nitrogen conversion rate of 3.2 kg-N/m(3)/d was observed on the anammox reactor (100 m3) filled with anammox gel carrier on day 69. The full-scale anammox reactor could be started up in approximately 2 months. Subsequently, stable nitrogen removal performance was observed for &gt;1 year. The average nitrogen loading, nitrogen conversion rate, and total nitrogen removal on the anammox reactor were 3.6 kg-N/m(3)/d, 3.0 kg-N/m(3)/d and 330 kg-N/d, respectively. The nitrogen removal performance obtained in this study will facilitate further application of the anammox processes to many types of industrial wastewater treatment. (C) 2017 Elsevier B.V. All rights reserved.

Nitrite oxidation is an aerobic process of the nitrogen cycle in natural ecosystems, and is performed by nitrite-oxidizing bacteria (NOB). Also, nitrite oxidation is a rate-limiting step of nitrogen removal in wastewater treatment plants (WWTPs). Although Nitrospira is known as dominant NOB in WWTPs, information on their physiological properties and kinetic parameters is limited. Here, we report the kinetic parameters and inhibition of nitrite oxidation by free ammonia in pure cultures of Nitrospira sp. strain ND1 and Nitrospira japonica strain NJ1, which were previously isolated from activated sludge in a WWTP. The maximum nitrite uptake rate (V-max_NO2) and the half-saturation constant for nitrite uptake (K-m_NO2) of strains ND1 and NJ1 were 45 +/- 7 and 31 +/- 5 (mu mol NO2-/mg protein/h), and 6 +/- 1 and 10 +/- 2 (mu M NO2-), respectively. The V-max_NO2 and K-m_NO2 of two strains indicated that they adapt to low-nitrite-concentration environments like activated sludge. The half-saturation constants for oxygen uptake (K-m_O2) of the two strains were 4.0 +/- 2.5 and 2.6 +/- 1.1 (mu M O-2), respectively. The K-m_O2 values of the two strains were lower than those of other NOB, suggesting that Nitrospira in activated sludge could oxidize nitrite in the hypoxic environments often found in the interiors of biofilms and flocs. The inhibition thresholds of the two strains by free ammonia were 0.85 and 4.3 (mg-NH3 I-1), respectively. Comparing the physiological properties of the two strains, we suggest that tolerance for free ammonia determines competition and partitioning into ecological niches among Nitrospira populations. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

Recent progress in tissue engineering technology has enabled us to develop thick tissue constructs that can then be transplanted in regenerative therapies. In clinical situations, it is vital that the engineered tissues to be implanted are safe and functional before use. However, there is currently a limited number of studies on real-time quality evaluation of thick living tissue constructs. Here we developed a system for quantifying the internal activities of engineered tissues, from which we can evaluate its quality in real-time. The evaluation was achieved by measuring oxygen concentration profiles made along the vertical axis and the thickness of the tissues estimated from cross-sectional images obtained noninvasively by an optical coherence tomography system. Using our novel system, we obtained (i) oxygen concentration just above the tissues, (ii) gradient of oxygen along vertical axis formed above the tissues within culture medium, and (iii) gradient of oxygen formed within the tissues in real-time. Investigating whether these three parameters could be used to evaluate engineered tissues during culturing, we found that only the third parameter was a good candidate. This implies that the activity of living engineered tissues can be monitored in real-time by measuring the oxygen gradient within the tissues. The proposed measuring strategy can be applied to developing more efficient culturing methods to support the fabrication of engineered thick tissues, as well as providing methods to confirm the quality in real-time. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 855-864, 2017.

Bioassay-guided separation of a lipophilic extract of the crinoid Alloeocomatella polycladia, inhibiting the activity of HCV NS3 helicase, yielded two groups of molecules: cholesterol sulfate and four new aromatic sulfates 1-4. The structures of the aromatics were elucidated by spectroscopic analysis in addition to theoretical studies. The aromatic sulfates 1-4 showed moderate inhibition against NS3 helicase with IC50 values of 71, 95, 7, and 5 mu M, respectively.

Selective separation of specific microbial types from a heterogeneous microbial population, such as an environmental microbial community, is an important process for microbial research and biotechnological industries. In the present study, pH-gradient ion-exchange microbial cell chromatography (PIE-MCC) was developed as a new method for microbial separation. The proposed method enables target microorganisms to be separated from a microbial community based on differences in microbial surface characteristics, because these characteristics, such as the zeta (zeta)-potential, vary among microbial cells. PIE-MCC was conducted by controlling the adhesion and detachment of microbial cells to and from the carrier surface by manipulating the pH of the running buffer. As a proof of concept, microbial cell separation via PIE-MCC was demonstrated using pure-cultured strains, model mixtures of two different pure-cultured strains, and an environmental sample targeting uncultivated microorganisms; i.e., each pure-cultured strain showed unique chromatograms; specific single species were separated from the model mixture; and a specific, uncultivated target was separated from the environmental sample. The zeta-potential of several tested strains suggested that not only electrostatic interactions, but also other factors affected microbial adhesion to the carrier surface. The newly developed method has several potential advantages compared with other techniques, not only in terms of its microbial separation capability, but also in terms of its simplicity and ability to be scaled up. Thus, the method has the potential to be widely used for a variety of purposes in the microbiology and biotechnology fields. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

Bioassay-guided separation of a lipophilic extract of the crinoid Alloeocomatella polycladia, inhibiting the activity of HCV NS3 helicase, yielded two groups of molecules: cholesterol sulfate and four new aromatic sulfates 1-4. The structures of the aromatics were elucidated by spectroscopic analysis in addition to theoretical studies. The aromatic sulfates 1-4 showed moderate inhibition against NS3 helicase with IC50 values of 71, 95, 7, and 5 mu M, respectively.

Nitrification is an important reaction in the biological nitrogen removal process in wastewater treatment plants (WWTPs). As ammonia-oxidizing microbes are slow-growing and sensitive to environmental factors such as free ammonia, pure strains are hard to obtain, preventing our understanding of their physiological characteristics. To conquer this hurdle, we report a high-throughput isolation technique based on scattering signatures, which exploits the tendency of many ammonia-oxidizing bacteria (AOB) to form microcolonies in activated sludge. The AOB microcolonies were directly sorted from the activated sludge without long incubation and enrichment bias, and were sequentially inoculated into 96-well microtiter plates containing growth medium. Phylogenetic analysis of the pure strains isolated in this study revealed a deeply branching and unrecognized lineage and diversity within the genus Nitrosomonas, beyond our expectation. (C) 2016 Elsevier Ltd. All rights reserved.

Ammonia-oxidizing bacteria (AOB), which oxidize ammonia to nitrite in the first step of nitrification, play an important role in biological wastewater treatment systems. Nitrosomonas mobilis is an important and dominant AOB in various wastewater treatment systems. However, the detailed physiological and genomic properties of N. mobilis have not been thoroughly investigated because of limited success isolating pure cultures. This study investigated the key physiological characteristics of N. mobilis Ms1, which was previously isolated into pure culture from the nitrifying granules of wastewater treatment bioreactor. The pure culture of N. mobilis Ms1 was cultivated in liquid mineral medium with 30 mg-NL-1 (2.14 mM) of ammonium at room temperature under dark conditions. The optimum growth of N. mobilis Ms1 occurred at 27 degrees C and pH 8, with a maximum growth rate of 0.05-0.07 h(-1), which corresponded to a generation time of 10-14 h. The half saturation constant for ammonium uptake rate and the maximum ammonium uptake rate of N. mobilis Ms1 were 30.70 +/- 0.51 mu M NH4+ and 0.01 +/- 0.002 pmol NH4+ cells(-1) h(-1), respectively. N. mobilis Ms1 had higher ammonia oxidation activity than N. europaea in this study. The oxygen uptake activity kinetics of N. mobilis Ms1 were K-m(O2) = 21.74 +/- 4.01 it mu M O-2 and V-max(O2) = 0.06 +/- 0.02 pmol O-2 cells(-1) h(-1). Ms1 grew well at ammonium and NaCl concentrations of up to 100 and 500 mM, respectively. The nitrite tolerance of N. mobilis Ms1 was extremely high (up to 300 mM) compared to AOB previously isolated from activated sludge and wastewater treatment plants. The average nucleotide identity between the genomes of N. mobilis Ms1 and other Nitrosomonas species indicated that N. mobilis Ms1 was distantly related to other Nitrosomonas species. The organization of the genes encoding protein inventory involved in ammonia oxidation and nitrifier denitrification processes were different from other Nitrosomonas species. The current study provides a needed physiological and genomic characterization of N. mobilis-like bacteria and a better understanding of their ecophysiological properties, enabling comparison of these bacteria with other AOB in wastewater treatment systems and natural ecosystems.

It is important to understand the ecology and physiology of microbes in activated sludge of wastewater treatment plants. Recently, molecular based approaches such as 16S rRNA genes and environmental genomics have illuminated black boxes in nutrient removal process and expanded our knowledge. However, most microbes responsible for the removal of phosphate and nitrogen such as Accumulibacter and Nitrospira remain uncultured. This is because optimum methodologies to concentrate these uncultured microbes and to obtain pure cultures have not been established. Here, we report a novel approach for physical enrichment of uncultured Accumulibacter and Nitrospira from microbial communities in activated sludge by a cell sorting system. Two scattering signatures representing forward scatter and side scatter of this system allowed morphological characterization of microbial particles in activated sludge. The distribution and size of microbial particles consisting of single cells, microcolonies, and aggregates depended on the levels of scattering signatures. Next generation sequencer and principal component analysis revealed each microbial population fractionated according to the levels of scattering signatures, resulting that uncultured Accumulibacter and Nitrospira could be sorted as single cells or microcolonies. Finally, quantitative fluorescence in situ hybridization analysis determined optimum fractions to collect sufficiently these target microbes from activated sludge. Consequently, this method would be very useful as an enrichment technique prior to isolation, genomic analysis, and physiological investigation of uncultured bacteria. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

Nitrosomonas europaea carries numerous toxin-antitoxin systems. However, despite the abundant representation in its chromosome, studies have not surveyed the underlying molecular functions in detail, and their biological roles remain enigmatic. In the present study, we found that a chromosomally-encoded MazF family member, predicted at the locus NE1181, is a functional toxin endoribonuclease, and constitutes a toxin-antitoxin system, together with its cognate antitoxin, MazE. Massive parallel sequencing provided strong evidence that this toxin endoribonuclease exhibits RNA cleavage activity, primarily against the AAU triplet. This sequence-specificity was supported by the results of fluorometric assays. Our results indicate that N. europaea alters the translation profile and regulates its growth using the MazF family of endoribonuclease under certain stressful conditions.

Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.

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 1month. 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 3months. At 6months, 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.

Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three-dimensional (3-D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3-D culture conditions, contributing crucial data for an efficient 3-D culture system design. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:189-197, 2016

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) a (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.

Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB) or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representatives of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful subculturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member's ecophysiology in a variety of habitats.

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

Controlling local dissolved oxygen concentration (DO) in media is critical for cell or tissue cultures. Various biomaterials and culture methods have been developed to modulate DO. Direct measurement of local DO in cultures has not been validated as a method to test DO modulation. In the present study we developed a DO measurement system equipped with a Clarktype oxygen microelectrode manipulated with I p.m precision in three-dimensional space to explore potential applications for tissue engineering. By determining the microelectrode tip position precisely against the bottom plane of culture dishes with rat or human cardiac cells in static monolayer culture, we successfully obtained spatial distributions of DO in the medium. Theoretical quantitative predictions fit the obtained data well. Based on analyses of the variance between samples, we found the data reflected "local" oxygen consumption in the vicinity of the microelectrode and the detection of temporal changes in oxygen consumption rates of cultured cells was limited by the diffusion rate of oxygen in the medium. This oxygen measuring system monitors local oxygen consumption and production with high spatial resolution, and can potentially be used with recently developed oxygen modulating biomaterials to design microenvironments and non-invasively monitor local DO dynamics during culture. (C) 2015 Wiley Periodicals, Inc.

Understanding the dynamics of dormant cells in microbial biofilms, in which the bacteria are embedded in extracellular matrix, is important for developing successful antibiotic therapies against pathogenic bacteria. Although some of the molecular mechanisms leading to bacterial persistence have been speculated in planktonic bacterial cell, how dormant cells emerge in the biofilms of pathogenic bacteria such as Pseudomonas aeruginosa remains unclear. The present study proposes four hypotheses of dormant cell formation; stochastic process, nutrient-dependent, oxygen-dependent, and time-dependent processes. These hypotheses were implemented into a three-dimensional individual-based model of biofilm formation. Numerical simulations of the different mechanisms yielded qualitatively different spatiotemporal distributions of dormant cells in the growing biofilm. Based on these simulation results, we discuss what kinds of experimental studies are effective for discriminating dormant cell formation mechanisms in biofilms.

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.

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.

Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.

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. (C) 2014 Wiley Periodicals, Inc.

The representative DNA-labeling agent 5-ethynyl-2'-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxymethyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro. PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.

Nitrification is an important process in the biogeochemical nitrogen cycle and is widely exploited in biological wastewater treatment. Recently, Nitrospira has been recognized as the numerically dominant nitrite-oxidizing bacterial genus and is primarily responsible for the second step of aerobic nitrification. Nevertheless, the physiological properties of Nitrospira remain poorly understood because the organisms are difficult to isolate and culture. Here, we report a novel cultivation strategy for obtaining members of the Nitrospira sublineage I in pure culture. The method combines: (i) selective enrichment of Nitrospira using a continuous feeding reactor and (ii) purification followed by sub-cultivation via a cell sorting system by focusing on the unique characteristics of Nitrospira forming spherical micro-colonies. This strategy is potentially applicable to other uncultured or unisolated Nitrospira and could accelerate the physiological and biochemical understandings of this important group of organisms.

Phosphorylated gelatin was prepared for surface modification of titanium to enhance cell attachment. The modified gelatin was synthesized by coupling gelatin with phosphonobutyric acid with water-soluble carbodiimide. Circular dichroism revealed no significant change in the gelatin as a result of phosphorylation. The binding behavior of phosphorylated gelatin on the titanium surface was observed by quartz crystal microbalance. The modified titanium surface was analyzed by measuring the water contact angle. Enhancement of the attachment of osteoblast cells MC-3T3L1 was observed on the phosphorylated-gelatin-modified titanium. Phosphorylation of gelatin was effective for preparation of a cell-adhesive titanium surface. (C) 2013 Society of Chemical Industry

Objectives: Modelling the apoptotic process is essential for simulating and understanding tumour growth, as most tumour tissues carry mutations in apoptotic signalling pathways. Thus here, we have aimed to construct a mathematical model of colonic crypts that explicitly incorporates the apoptotic mechanism.
Methods: A murine colonic crypt was described as being a two-dimensional rectangular surface model. In this system, three types of cells with different proliferating and differentiating potentials migrate. Apoptosis was described as a process activated by irradiation that progresses in a stepwise manner. Parameter values in the model were determined to be consistent with experimental data for changes in the apoptotic cell ratio within murine transverse colonic crypts following irradiation.
Results: First, we constructed a model reproducing cell proliferation dynamics in normal murine colonic crypts; next, we applied the apoptotic mechanism to this model. As a result, we succeeded in simultaneous reproduction of both spatial and temporal changes in distribution of apoptotic cells in murine colonic crypts by determining parameter values in numerical simulations. Through this adjustment process, we were able to predict that stem cells and transit amplifying (TA) cells in each generation must react distinctly from each other, to apoptosis-inducing stimuli.
Conclusions: We constructed a mathematical model with which we could quantitatively describe cell proliferative and apoptotic dynamics in a murine colonic crypt. Using this model, we were able to make novel predictions that sensitivity to apoptosis-inducing stimuli is dependent on cell type.

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, is frequently detected in classical myeloproliferative neoplasms (MPNs). In the present study, we determined the JAK2V617F allele burden in Japanese MPN patients using alternately binding probe competitive-polymerase chain reaction, a highly quantitative method recently developed by our group. Although we observed strong similarities in terms of epidemiological parameters associated with the JAK2V617F allele burden between our cohort and others, we found a higher JAK2V617F allele burden in Japanese polycythemia vera (PV) patients and lower frequencies of thrombosis in Japanese MPN patients compared with previous reports. In addition, despite the presence of high red blood cell counts, some patients bearing the JAK2V617F mutation were not diagnosed as PV, as their hemoglobin values were lower than the WHO PV criterion. In these patients, the JAK2V617F allele burden was strikingly similar to that in PV patients fulfilling the 2008 WHO criteria, suggesting that these patients can be classified as PV. Although isotopic measurement of red cell mass (RCM) is required for definitive diagnosis of PV, our data suggest that precise measurement of the JAK2V617F allele burden may improve the diagnosis of PV when RCM has not been determined.

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 +/- 0.2 mu M with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3) is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE) 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1) on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1) against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

Several possible scenarios of cellular dynamics in human colonic crypts have been inferred from transgenic animal experiments. However, because of the discrepancy in size and physiology between humans and animals, quantitative predictions of tissue renewal and cancer development are difficult to execute.
A two-dimensional individual based model was developed for the first time to predict cellular dynamics in human colonic crypts. A simple scenario, in which stem cells were not fixed positionally, divide symmetrically and asymmetrically in a stochastic fashion in the lower part of the crypt, was proposed and implemented in the developed model. Numerical simulations of the model were executed in silico.
By comparing the results of computational simulations with available experimental data, the presented scenario was consistent with various experimental evidence. Using this scenario, we simulated and visualized monoclonal conversion in the human colonic crypt. We also predicted that the propensity for monoclonal expansion of a mutant cell was largely dependent on the phenotype, the cell type, the position and the state of the crypt.
Using the computational framework developed in this study, model users can verify possible scenarios of stem cell dynamics occurring in human colonic crypts and quantitatively predict cell behavior. Its applicability in scenario verification and predictability makes it a valuable tool for elucidation of stem cell dynamics in human colonic crypts.

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 mu M, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 mu M, and 7, 3, and 34 mu M, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 mu M. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

A sequencing batch membrane biofilm reactor (SBMBfR) was developed towards simultaneous carbon, nitrogen and phosphorous removals from a low carbon/nitrogen (C/N) ratio wastewater. The SBMBfR is composed of two functional parts in a single-reactor vessel: (1) a fibrous-composite matrix of a gas-permeable hollow-fiber membrane on which a nitrifying biofilm grows and (2) activated sludge in which denitrifying polyphosphate-accumulating organisms (DNPAOs) are predominant. The reactor was operated in a batch manner with the turning on and off of the membrane aeration. Anaerobic period (without membrane aeration) allowed consumption of organic carbon by DNPAOs. They further took phosphate in the bulk with nitrite/nitrate produced via nitrification at the nitrifying biofilm during membrane aeration period. A higher nitrogen removal efficiency was obtained with the nitrifying biofilm formed on a gas-permeable membrane at various C/N ratios in synthetic wastewaters than without the biofilm, corroborating the significance of the biofilm as a region for ammonia oxidation. Continuous operation of the SBMBfR achieved average nitrogen and phosphorus removal efficiencies of 90% and 92%, respectively, at a C/N ratio of 2.0 indicating the effectiveness of the SBMBfR for nutrient removal from low C/N ratio wastewater.

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. (C) 2013 Elsevier Ltd. All rights reserved.

Hepatitis C virus (HCV) is the causative agent of hepatitis C, a chronic infectious disease that can lead to development of hepatocellular carcinoma. The NS3 nucleoside triphosphatase (NTPase)/helicase has an essential role in HCV replication, and is therefore an attractive target for direct-acting antiviral strategies. In this study, we employed high-throughput screening using a photo-induced electron transfer (PET) system to identify an inhibitor of NS3 helicase from marine organism extracts. We successfully identified psammaplin A as a novel NS3 inhibitor. The dose-response relationship clearly demonstrates the inhibition of NS3 RNA helicase and ATPase activities by psammaplin A, with IC₅₀ values of 17 and 32 μM, respectively. Psammaplin A has no influence on the apparent Km value (0.4 mM) of NS3 ATPase activity, and acts as a non-competitive inhibitor. Additionally, it inhibits the binding of NS3 to single-stranded RNA in a dose-dependent manner. Furthermore, psammaplin A shows an inhibitory effect on viral replication, with EC₅₀ values of 6.1 and 6.3 μM in subgenomic replicon cells derived from genotypes 1b and 2a, respectively. We postulate that psammaplin A is a potential anti-viral agent through the inhibition of ATPase, RNA binding and helicase activities of NS3.

Nitrite oxidation is a key step in nitrogen removal in biological wastewater treatment plants. Recently, two phylogenetically different Nitrospira (sublineages I and II) have been recognized as the numerically dominant nitrite-oxidizing bacteria in wastewater treatment plants. However, Nitrospira sublineage II inhabiting activated sludge was not isolated and its detailed properties were unclear. In this study, we developed a new method for the isolation of Nitrospira forming micro-colonies using a cell sorter. We obtained a novel pure strain "Nitrospira japonica" from the activated sludge. Subsequently, phylogenetic and physiological analyses revealed that Nitrospira japonica belongs to sublineage II and grew in medium containing formate. This method has the potential to isolate other uncultured microorganisms forming micro-colonies.

Nitrification is an important step in nitrogen removal in biological wastewater treatment processes. Recently, Nitrospira have been recognized as the numerically dominant nitrite-oxidizing bacterial genus primarily responsible for the second step of aerobic nitrification
however, Nitrospira usually resist cultivation under laboratory conditions and only one species enriched from activated sludge has been described. In this study, a novel enrichment method for Nitrospira was successfully developed using continuous feeding bioreactors. By controlling nitrite concentrations strictly in the bioreactor at low levels below 10 mg-N L-1, coexisting members of sublineages I and II of the genus Nitrospira were enriched selectively. The maximum ratios of sublineages I and II to total microbial cells achieved 88.3% and 53.8%, respectively. This enrichment method is potentially applicable to other uncultured Nitrospira.

RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken delta-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process. (C) 2012 Elsevier Ltd. All rights reserved.

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 mu g/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 mu g/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

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 (&lt;25 degrees 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/m(3)/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. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

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(-1) d(-1) on day 16 (30 degrees 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 degrees 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. (C) 2012 Elsevier Ltd. All rights reserved.

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8?h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000?s-1), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications. Biotechnol. Bioeng. 2012; 109:17451754. (C) 2012 Wiley Periodicals, Inc.

Formation of aerobic granular sludge was investigated using a laboratory-scale continuous-flow reactor. Although it is generally thought that aerobic granular sludge can be formed only when a sequencing batch reactor (SBR) is used, formation of aerobic granular sludge was observed using a continuous-flow reactor in this study. The formation was observed only when nitrification well occurred in the reactor, and any indication of the aerobic granulation was not observed in the presence of nitrification inhibitor. Therefore, it was considered that existence of nitrifying bacteria is responsible for formation of the granular sludge. It has been reported that existence of slow growing organisms like nitrifying bacteria induces formation of granular sludge when an SBR is used. Hence, it was considered that formation of aerobic granular sludge is possible in the presence of certain population of nitrifying bacteria even when a continuous-flow reactor is used. To our best knowledge, this is the first study that indicates the possibility of forming aerobic granular sludge in a continuous-flow reactor where organic wastewater is fed. These findings will contribute to dissemination of aerobic granular sludge technology because continuous-flow reactors are more widely used all over the world than SBRs.

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC50 of 11.7 mu g/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC50 value of 23 to 44 mu g/mL, which is similar to the IC50 value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose dependent manner, with IC50 values of 15 and 70 mu M, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K-m stranded RNA was inhibited by 1. Monoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.

In this study, the formation characteristics of aerobic granular sludge (AGS) mainly composed of nitrifying bacteria (referred to as nitrifying granules) in a continuous stirred-tank reactor (CSTR) were investigated at several surface loading rates (equal to the liquid linear velocity at the sludge settling zone). The surface loading rate strongly affected the selection of larger granular sludge similarly to the sludge settling time in a sequencing batch reactor. By setting an appropriate surface loading rate (1.4 m³/m²/d), small particles were effectively washed out, and larger granular sludge selectively remained in the reactor. As a result, nitrifying granules were effectively formed even with keeping an identical flow condition from the startup. To the best of our knowledge, this is the first study that has succeeded in forming nitrifying granules with keeping an identical flow condition from the startup using a continuous-flow reactor. These findings will contribute to the dissemination of AGS technology because information on the formation of AGS in a continuous-flow reactor is limited.

We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms. (C) 2011 Elsevier Ltd. All rights reserved.

In this study, the effectiveness of aerobic granular sludge as seed sludge for rapid start-up of nitrifying processes was investigated using a laboratory-scale continuous stirred-tank reactor (CSTR) fed with completely inorganic wastewater which contained a high concentration of ammonia. Even when a large amount of granular biomass was inoculated in the reactor, and the characteristics of influent wastewater were abruptly changed, excess biomass washout was not observed, and biomass concentration was kept high at the start-up period due to high settling ability of the aerobic granular sludge. As a result, an ammonia removal rate immediately increased and reached more than 1.0 kg N/m(3)/d within 20 days and up to 1.8 kg N/m(3)/d on day 39. Subsequently, high rate nitritation was stably attained during 100 days. However, nitrite accumulation had been observed for 140 days before attaining complete nitrification to nitrate. Fluorescence in situ hybridization analysis revealed the increase in amount of ammonia-oxidizing bacteria which existed in the outer edge of the granular sludge during the start-up period. This microbial ecological change would make it possible to attain high rate ammonia removal.

We developed a simple, cost-effective, and accurate JAK2 allele burden quantification method named alternately binding probe competitive PCR (ABC-PCR). ABC-PCR can be performed to quantify target JAK2 allele burdens in a single reaction. The throughput and running cost of ABC-PCR are markedly improved compared with those of allele-specific quantitative PCR (AS-qPCR). The quantification of samples with known JAK2 allele burdens revealed that ABC-PCR had a small assay-to-assay variation. The JAK2 allele burdens in the patients with myeloproliferative neoplasms measured by ABC-PCR and AS-qPCR showed a good fitting. ABC-PCR would be a powerful tool for quantifying target JAK2 allele burdens. (C) 2011 Elsevier Ltd. All rights reserved.

RNA interference (RNAi) is one of the most promising new approaches for disease therapy. The design of a dumbbell-shaped nanocircular RNA allows it to act as a short interfering RNA (siRNA) precursor. To optimize the design, we studied the relationship between the nanostructure and RNAi activity by synthesizing various RNA dumbbells. An RNA dumbbell with a 23-bp stem and 9-nt loops was the most potent. Sequence analysis by mass spectrometry showed that Dicer could edit RNA dumbbells to siRNA species. The reaction offered the slow release of siRNA species, which conferred prolonged RNAi activity. Introduction of DNA into the loop position significantly stabilized the dumbbell in biological fluid without any loss of RNAi activity. In-depth pharmacological evaluation was performed by introducing dumbbells into HeLa cells that stably express the target luciferase gene. The dumbbells provided a rapid silencing effect and retained this effect for a longer time even at a lower concentration than that at which standard siRNA completely lost RNAi activity. We conclude that an RNA dumbbell with DNA loops is the most promising design for in vivo applications for RNA medicine. © 2011 American Chemical Society.

Nitrifying granules were applied to high-speed nitrification of wastewater discharged from an electronic industrial factory. The behavior of the formation of nitrifying granules and the start-up performance of nitrification process were examined with three types of reactors: a column with a small diameter, a column with a large diameter and a rectangular-type reactor. As a result, the performance of the three types of reactors did not give significant differences in terms of the formation of nitrifying granules and the start-up trend of the reactor. A nitrification rate of about 2.0 kgN/m³/day was achieved in all reactors.

Mature adult tissues contain stem cells that express many genes normally associated with the early stage of embryonic development, when maintained in appropriate environments. Cells procured from adult tissues representative of the three germ layers (spinal cord, muscle, and lung), each exhibiting the potential to mature into cells representative of all three germ layers. Cells isolated from adult tissues of different germ layer origin were propagated as nonadherent clusters or spheres that were composed of heterogeneous populations of cells. When the clusters or spheres were dissociated, the cells had the ability to reform new, nonadherent spheres for several generations. When implanted in vivo, in association with biodegradable scaffolds, into immunodeficient mice, tissue containing cells characteristic of the three germ layers was generated. These findings suggest the existence of a population of stem cells in adult tissues that is quite different and distinct from embryonic stem cells that demonstrate a greater potency for differentiation across germ lines than previously believed. Such cells could potentially be as useful as embryonic stem cells in tissue engineering and regenerative medicine.

The effect of external carbon (acetate) addition on nitrogen and phosphorus removal in a sequencing batch reactor (SBR) employing anaerobic/oxic/anoxic (AOA) regime was investigated. Two SBRs, receiving wastewater with different total organic carbon per nitrogen concentration (TOC/N) ratios (influent TOC/N ratio: 2.2 and 4.5 in Run 1 and Run 2, respectively) were operated by changing the amount of external carbon addition at the beginning of oxic phase. The operations in both reactors revealed that nitrogen and phosphorus removal efficiencies increased with an increase in external carbon addition, reaching more than 90% at an external carbon addition of 45.0 mg-C/L. Moreover, the ratio of anoxic/oxic phosphate uptake rate (PUR), an index reflecting a fraction of denitrifying phosphate-accumulating organisms (DNPAOs) in total PAOs, indicates that nitrogen and phosphorus removal efficiencies were enhanced as the PUR ratio increased. Quantitative fluorescence in situ hybridization (FISH) revealed that external carbon addition facilitated the activity of DNPAOs as well as that of glycogen-accumulating organisms (GAOs). The degree of nitrogen removal by denitrifying GAOs (DNGAOs) seemed to increase with an increase in the dosage of external carbon added.

Nitrifying granules were used to improve nitrification performance of activated sludge process under high organic load. After inoculation of nitrifying granules, nitrification activity immediately increased, and kept for at least 70 days. Fluorescence in situ hybridisation analysis confirmed that nitrifying bacteria were retained in the granules even though nitrifying granules had been served in the organic wastewater treatment reactor for 70 days. Nitrification performance strongly depended on C/N ratio of the influent wastewater. Nitrifying granules that had been statically stored for 100 days could completely recover ammonia removal rate within 15 days after inoculation to ammonia-rich solution. Copyright © 2011 Inderscience Enterprises Ltd.

Particulate and slowly biodegradable substrates form an important fraction of industrial wastewater and sewage. To study the influence of suspended solids and colloidal substrate on the morphology and performance of aerobic granular sludge, suspended and soluble starch was used as a model substrate. Degradation was studied using microscopy, micro-electrode measurements, batch experiments and long term laboratory scale reactor operation. Starch was removed by adsorption at the granule surface, followed by hydrolysis and consumption of the hydrolyzed products. Aerobic granules could be maintained on starch as sole influent carbon source, but their structure was filamentous and irregular. It is hypothesized that this is related to the low starch hydrolysis rates, leading to available substrate during the aeration period (extended feast period) and resulting in increased substrate gradients over the granules. The latter induces a less uniform granule development. Starch adsorbed and was consumed at the granule surface instead of being accumulated inside the granules as occurs for soluble substrates. Therefore the simultaneous denitrification efficiencies remained low. Moreover, many protozoa and metazoans were observed in laboratory reactors as well as in pilot- and full-scale Nereda(R) reactors, indicating an important role in the removal of suspended solids too. (C) 2010 Elsevier Ltd. All rights reserved.

Microbial population dynamics were investigated during the formation of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor fed ammonia as a sole energy source. Analyses of clone libraries of 16S rRNA gene and the ammonia monooxygenase subunit A gene (amoA) revealed that although the clones obtained from the seed sludge were widely distributed among the ammonia-oxidizing bacteria (AOB) isolates, the community structure of AOB shifted towards the Nitrosomonas mobilis lineage as granulation proceeded. Quantitative fluorescence in situ hybridization showed that changes in the bacterial population occurred concomitantly with changes in nitrification performance and the size of granules. AOB associated with the N. mobilis lineage were predominant in the early stages as nitrifying granules formed (average diameter, 126 mu m). In mature granules (average diameter, 270 mu m), at least three types of AOB, N. mobilis, Nitrosomonas oligotropha, and Nitrosomonas europaea, formed different niches and coexisted. Nitrite-oxidizing bacteria (NOB) affiliated with Nitrospira spp. were detected in the start-up period, but were replaced by NOB affiliated with Nitrobacter spp. after granules formed.

In this study, the formation characteristics of aerobic granular sludge in a continuous-flow reactor were investigated under several experimental conditions. Both surface loading rate (equal to liquid linear velocity at a sludge settling zone) and aeration rate strongly affected the selection of well-settling sludge in the same manner as sludge settling time in a sequencing batch reactor. By setting and controlling adequate surface loading and aeration rates, small particles were effectively washed out, and well-settling sludge selectively remained in the reactor. As a result, aerobic granular sludge was effectively formed. On the other hand, feeding pattern, i.e., continuous and intermittent feeding, did not affect the aerobic granulation when completely inorganic wastewater was fed. These findings will contribute to the dissemination of aerobic granular sludge technology because the information on the formation of aerobic granular sludge in a continuous-flow reactor is limited.

We have developed a new approach for monitoring the metabolic dynamics in microbial ecosystems using a combination of DNA fingerprinting and metabolome analysis based on stable-isotope-labeling technologies. Stable-isotope probing of DNA (DNA-SIP) has been used previously for the evaluation of cross-feeding in microbial communities. For the development and validation of our monitoring approach, fecal microbiota were analyzed with stable-isotope-labeled glucose used as the sole carbon source. In order to link the metabolic information and the microbial variability, we performed metabolic-microbial correlation analysis based on nuclear magnetic resonance (NMR) profiles and denaturing gradient gel electrophoresis (DGGE) fingerprints, which successfully identified the glucose-utilizing bacteria and their related extracellular metabolites. Moreover, our approach revealed information regarding the carbon flux, in that the "first" wave of extracellular metabolites secreted by the glucose-utilizing bacteria were incorporated into the "secondary" group of substrate-utilizing bacteria, and that this "secondary" group further produced their own secondary metabolized substrates. Thus, this approach is a powerful tool for monitoring the metabolic dynamics in microbial ecosystems and allows for the tracking of the carbon flux within a microbial community. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

Putative esterase genes were isolated from environmental DNA by using pre-amplified inverse PCR. The sequence analysis of the isolated genes showed 32-80% amino acid sequence identities to known esterases/lipases in public databases. The isolated genes were subsequently expressed in recombinant Escherichia coli. Insoluble proteins were noted in the expression of the majority of the isolated genes. The findings suggest that it is difficult to isolate these genes by using activity-based screening with construction of metagenome library. For the enzymes characterized, we examined substrate specificity, optimal temperature, optimal pH, and thermal stability. The substrate specificity of all the enzymes was high for p-nitrophenyl acetate, but almost undetectable for p-nitrophenyl decanoate. The results indicate that the obtained enzymes are defined as esterases. The enzymes were active in a broad range of temperature. The optimum activity was observed at 25-70 degrees C and at pH 8.0-9.0. Some enzymes have moderate thermostability and would be useful for industrial enzymes. This study illustrates that pre-amplified inverse PCR, which is one of the sequence-based approach, is potentially applicable to the isolation of diverse genes from environmental DNA. (C) 2010 Elsevier Inc. All rights reserved.

Dehalococcoides spp. are responsible for the reductive dehalogenation of environmental contaminants and are candidates for engineered bioremediation. The development of a sensitive, reliable, and rapid method for the quantification of Dehalococcoides spp. is required for the effective use of the organisms in bioremediation sites. Here, we describe the quantification of the 16S rRNA gene of Dehalococcoides spp. using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The primers and probe sets that were newly designed for ABC-PCR were found to have a high specificity for Dehalococcoides spp. The standard curve of ABC-PCR had a good fitting (R = 0.999), and the lower detection limit was 10 copies/mu l of template DNA. We also investigated the effects of inherent PCR-inhibiting compounds in an environmental sample on the quantification using ABC-PER or real-time PCR by adding the soil extraction solution to PCR mixtures. ABC-PCR was more robust against the PER amplification inhibitors than real-time PCR. The copy number of the 16S rRNA gene of Dehalococcoides spp. in soil and groundwater samples was successfully quantified using ABC-PER. In conclusion, ABC-PCR is useful for the quantification of Dehalococcoides spp. populations and dynamics at bioremediation sites. (C) 2009 Elsevier Ltd. All rights reserved.

We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5&apos; fluorophore-labeled strand hybridized to a 3&apos; quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore-quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput. (C) 2010 Elsevier Inc. All rights reserved.

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.

A new photocaged fluorescent compound, azidomethyl fluorescein, was successfully utilized to monitor the dynamics of oligonucleotides in living human cells.

We developed a new nucleic acid-based fluorescence probe for protein detection. The method is based on the scission of an aptamer into two probes, which are then attached with a chemically reactive fluorogenic compound. The protein-dependent association of the two probes accelerates a reduction-triggered fluorogenic reaction and indicates the presence of the target protein. which is detected using a fluorescence readout. The fluorescence signal is generated via the deprotection of the azidomethyl group of fluorescein. The arginine-rich motif peptide of the human immunodeficiency virus-1 Rev protein was targeted by this type of probe. Emission was detected at 522 nm and was enhanced by about 19.4-fold in the presence of the target peptide. An oligonucleotide-based reduction-1 triggered fluorescence probe was successfully applied to the defection of the Rev peptide ill solution.

In this study, the microbial community in the anaerobic/oxic/anoxic (A/O/A) process combined with sludge ozonation and phosphorus recovery was characterized by using phosphorus uptake rate (PUR) analysis and PCR-cloning analysis. Despite effective phosphorus removal, PUR analysis indicated a lower activity of both polyphosphate-accumulating organisms (PAOs) and denitrifying PAOs (DNPAOs) than in other systems utilizing DNPAOs. This result suggested that endogenous denitrifying bacteria actively contributed to denitrification. The PCR-cloning analysis revealed that Bacteroidetes was most prominent in the process, followed by Betaproteobacteria and Alphaproteobacteria. For Bacteroidetes, most of the sequences obtained in this study were not closely related to isolates. On the other hand, for the Alphaproteobacteria, the genera Amaricoccus, Aminobacter, Hyphomicrobium, and Paracoccus, which have the ability both to accumulate poly-β-hydroxybutyrate (PHB) and to reduce nitrate to nitrite, were detected. For the Betaproteobacteria, which are major denitrifying bacteria in wastewater treatment systems, the genera Dechloromonas and Zoogloea, were identified. Organisms belonging to the family Comamonadaceae, some of which have been reported as being primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria, also existed in the system. Major PAOs/DNPAOs, Rhodocyclus-related PAOs and Actinobacterial PAOs, were not detected, suggesting that unknown PAOs/DNPAOs could have played an important role for phosphorus removal.

We have developed a flexible, specific, and cost-effective real-time polymerase chain reaction (PCR) method. In this technique, a quenching probe (QProbe) and a nonfluorescent 3&apos;-tailed probe are used. The QProbe is a singly labeled oligonucleotide bearing a fluorescent dye that is quenched via electron transfer between the dye and a guanine base at a particular position. The nonfluorescent 3&apos;-tailed probe consists of two parts: one is the target-specific sequence on the 5&apos; side, and the other is complementary to the QProbe on the 3&apos; side. When the QProbe/nonfluorescent 3&apos;-tailed probe complex hybridizes with the target in PCR, the fluorescence of the dye is quenched. Fluorescence quenching efficiency is proportional to the amount of the target. We called this method the universal QProbe system. This method substantially reduces the cost of real-time PCR setup because the same QProbe can be used for different target sequences. Moreover, this method allows accurate quantification even in the presence of nonspecific PCR products because the use of nonfluorescent 3&apos;-tailed probe significantly increases specificity. Our results demonstrate that this method can accurately and reproducibly quantify specific nucleic acid sequences in crude samples, comparable with conventional TaqMan chemistry. Furthermore, this method is also applicable to single-nucleotide polymorphism (SNP) genotyping.

The effects of ozonation conditions on the performance of a continuous anaerobic/oxic/anoxic (A/O/A) process with sludge ozonation and phosphorus adsorption were investigated. In this system, excess sludge was ozonated by microbubble ozonation, and then the supernatant of the ozonated sludge was flowed into a phosphorus adsorption column packed with zirconium-ferrite adsorbent. The effluent from the column and the settlings of the ozonated sludge were recirculated in the A/O/A process. Long-term operation of a lab-scale system treating rural wastewater showed that ozonation affected not only the sludge reduction efficiency but also the nitrogen removal efficiency. When the amount of sludge to be ozonated was set at 16% of total MLSS per day, no excess sludge was withdrawn, but the nitrogen removal efficiency was deteriorated. Decreasing the amount of sludge to be ozonated (to 9.4% of total MLSS per day) resulted in efficient nitrogen removal, but the MLSS concentration increased slightly. Phosphorus accumulated in the sludge was re-solubilized by ozonation, and a large part of the solubilized phosphorus consisted of Pi. Almost all Pi was recovered in the phosphorus adsorption column.

A hollow-fiber membrane chamber (HFMC) was developed as an in situ cultivation device for environmental microorganisms. The HFMC system consists of 48 to 96 pieces of porous hollow-fiber membrane connected with injectors. The system allows rapid exchange of chemical compounds, thereby simulating a natural environment. Comparative analysis through the cultivation of three types of environmental samples was performed using this newly designed device and a conventional agar-based petri dish. The results show that the ratios of novel phylotypes in isolates, species-level diversities, and cultivabilities in HFMC-based cultivation are higher than those in an agar-based petri dish for all three samples, suggesting that the new in situ cultivation device is effective for cultivation of various environmental microorganisms.

We have developed a novel method for microbial community analysis of bacterial 16S rRNAs based on affinity capillary electrophoresis using 16S rRNA-conjugated magnetic beads. We called this method magnetic beads affinity capillary electrophoresis (MB-ACE) which can be used for sequential and quantitative analysis of 16S rRNA. In this method, RNA extracted from a microbial community is biotin-modified and mixed with streptavidin-modified paramagnetic beads. This mixture is then injected into a capillary and localized in the middle of the capillary using a magnet held adjacent to the capillary. Subsequently, a fluorescent-labeled probe to detect the target 16S rRNA is injected into the capillary, and voltage is applied. The probe trapped on the RNA is dissociated by formamide and detected at its anodic end by measuring the fluorescence. Next, another fluorescent probe is injected, and thus the target 16S rRNA in the sample is quantified one by one. MB-ACE was used for the quantification of the 16S rRNAs of Escherichia coli and Pseudomonas putida in samples that were prepared by mixing RNA extracted from activated sludge and 16S rRNAs prepared by in vitro transcription. The two types of 16S rRNAs were quantified, indicating that MB-ACE can be used for sequential quantitative analysis of bacterial 16S rRNAs. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and P-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.

Objectives: This Study developed a novel MRD monitoring method targeting Wilms&apos; tumor gene (WT1) mRNA Using reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Design and methods: A primer set for the assay was designed oil the basis of the sequences between the 17AA and KIN regions of WT1 mRNA. WT1 mRNA was quantified by real-time RT-LAMP and the accuracy of RT-LAMP was compared with that of real-time RT-PCR.
Results: The standard curve was expressed as a linear relationship between the log copy numbers of WT1 mRNA ranging from 6.8 x 10 to 6.8 x 10(9) copies and the threshold time with a correlation coefficient of R(2)&gt;0.994. The measured values obtained by RT-LAMP strongly correlated with those obtained by real-time RT-PCR.
Conclusion: RT-LAMP can be used to determine WT1 mRNA expression levels. This assay will contribute to a more specific, simple, and rapid MRD monitoring than conventional assays. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Aerobic granular sludge technology was applied to the simultaneous nitrogen and phosphorus removal from livestock wastewater that contains high concentrations of nitrogen and phosphorus (TN: 650 mg/L; TP: 125 mg/L). A lab-scale sequencing batch reactor was operated in an alternating anaerobic/oxic/anoxic denitrification mode. Granular sludge was first formed using synthetic wastewater. When livestock wastewater was diluted with tap water, the shape and settleability of aerobic granular sludge were maintained even though livestock wastewater contained suspended solids. Simultaneous nitrification, denitrification, and phosphate uptake were observed under an aerobic condition. However, when nondiluted livestock wastewater was used, the diameter of granular sludge and the denitrification efficiency under an oxic condition decreased. When the concentrations of nitrogen and phosphorus in wastewater increased, hydraulic retention time (HRT) increased resulting in a decrease in selection pressure for granular sludge. Therefore, the sustainment of granular sludge was difficult in livestock wastewater treatment. However, by applying a new excess sludge discharge method based on Stokes&apos; law, the shape of granular sludge was maintained in spite of the long HRT (7.5 days). To select large granular sludge particles, excess sludge was discharged from the upper part of settled sludge because small particles localized there after settling. Finally, excellent nitrogen and phosphorus removal was accomplished in practical livestock wastewater treatment. The effluent concentrations of NH(4)-N, NO(x)-N, and PO(4)-P were &lt; 0.1, 1.4, and 1.2 mg/L, respectively.

Enrichment of anammox bacteria from three types of seed sludge, sewage, digester, and nitrification sludges, was conducted using a nonwoven fabric carrier for immobilizing the anammox bacteria, and the microbial diversity of the enriched anammox culture was investigated. About four months later, simultaneous removals of ammonium and nitrite, and production of a small amount of nitrate, which is unique to the anammox reaction, were observed in all 3 sludge reactors. Results of 16S rRNA gene analysis indicated that anammox bacteria were cultivated and diversified in each sludge type. Moreover, the microbial diversity of anammox bacteria was higher in the enriched culture from sewage sludge compared to the other two types of seed sludge. Bacillus sp. coexisted in the anammox culture cultivated from sewage sludge. These results suggest that differences in the anammox community in the enriched culture were caused by differences in the type of seed sludge. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

The molecular diversity of bacterial chitinases in the bulk soils of arable land was investigated using culture-independent methods. The results demonstrate that bacterial chitinases in arable soils are highly diverse and comprise unique groups when their sequences were compared to those in public databases. The diversity of bacterial chitinases in arable soil was further evaluated using conventional phylogenetic analysis, the UniFrac analysis of the phylogenetic data, and the multidimensional scaling (MDS) analysis of T-RFLP profiles to elucidate the relationship between the diversity of bacterial chitinases and soil characteristics. These analyses indicate that environmental factors such as soil type and pH are responsible for shaping the composition of bacterial chitinases. (C) 2008 Elsevier Ltd. All rights reserved.

We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5&apos;-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3&apos;-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the Screening for NS3 helicase inhibitors from cell extracts Of Microorganisms, and found several cell extracts containing potential inhibitors. (C) 2009 Elsevier Inc. All rights reserved.

We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops(1). RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme, and are thus transformed into double-stranded RNA in cells, although this RNA is resistant to degradation in serum. The structure was optimized to maximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA.

We developed a new nucleic acid-based fluorescence probe for protein detection. The method is based on the scission of an aptamer into two probes, which are then attached with a chemically reactive fluorogenic compound. The protein-dependent association of the two probes accelerates a chemical reaction and indicates the presence of the target protein, which is detected using a fluorescence readout. The arginine-rich motif peptide was targeted by this type of probe. In presence of the peptide, the fluorescence signal at 450 nm increased, and no significant increase in fluorescence was observed in the absence of the peptide. An oligonucleotide-based fluorescence probe was successfully applied to the detection of the ARM peptide in solution.

We have developed a new red fluorogenic compound derived from naphthorhodamine for a reduction-triggered fluorescence probe to sense oligonucleotides. The fluorogenic reaction between naphthorhodamine azide derivatives and reducing reagents such as triphenylphosphine (TPP) on the DNA target does not use any enzyme or reagent, and fluoresces at 650 nm. The probes were used for dual color detection of a single nucleotide difference on the leukemia-related bcr/abl gene.

Nitrification stability and biofilm robustness were examined by comparing a fibrous support membrane-aerated biofilm reactor (FS-MABR), where a woven fibrous support was surrounded on a silicone tube, with an MABR. The overall mass transfer coefficient of oxygen for the FS-MABR, assuming no boundary layer between the fibrous material and bulk liquid, was 5.85 m/d at an air pressure of 27 kPa, which was comparable to that value of the MABR (5.54 m/d). The amount of biomass on the fibrous support with a silicone tube was 2.48 times larger than on the bare silicone. The biomass loss after a high liquid flow rate condition was 49% and 75% in the FS-MABR and MABR, exhibiting robust biofilms grown on the fibrous support. The FS-MABR provided more stable nitrification performance than the MABR irrespective of a high liquid flow rate. Both reactors have deteriorated ammonium (NH(4)(+)-N) removal without a high liquid flow rate condition to eliminate excessive biomass, indicating that regular maintenance is essential to eliminate excessive biofilm from a MABR for nitrification, which potentially acts as a NH(4)(+) diffusion barrier.

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying (NN)-N-14-N-15 (N-29) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.

The oral mucosa is an attractive cell source for autologous transplantation in human patients who require regenerative therapies of various epithelia. However, the tune-course of cellular changes in transplanted oral mucosal epithelia at ectopic sites remains poorly understood. By applying a rat model, we analyzed phenotypic changes in oral mucosal epithelial cell sheets after harvest from temperature-responsive culture dishes and subsequent autologous subcutaneous transplantation. We used monoclonal antibodies to identify epithelial-specific cytokeratins 4, 10, 13, and 14, the stem/progenitor cell marker p63, and proliferating cell nuclear antigen, within the regenerated tissues. Transplanted oral mucosal epithelial cell sheets proliferated during the first week after grafting in conjunction with host inflammation, but then began to degenerate afterward with complete disappearance after 3 weeks. Our findings suggest that host subcutaneous tissues support proliferation and differentiation of the oral mucosal epithelial cell sheets, but are unable to promote maintenance of stern and progenitor cells and therefore cannot produce long-term survivability. (c) 2007 Wiley Periodicals, Inc.

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 (&gt;4% NaCl). (C) 2008 Elsevier Ltd. All rights reserved.

We demonstrate a generation of tandem repeats of a malachite green (MG) RNA aptamer using rolling circle transcription. To keep the higher-order structure of each aptamer on long RNA, we designed a sequence of circular DNA with a 14-base linker. T7 RNA polymerase was superior to Escherichia coli RNA polymerase in the specific transcription of the MG RNA aptamer. Finally. the generation of the fluorescence signal was confirmed from aptamer repeats with MG. (c) 2008 Elsevier Ltd. All rights reserved.

A mathematical model based on the simulation software AQUASIM was developed to validate an anaerobic/aerobic/anoxic (AOA) process that enables simultaneous nitrogen and phosphorus removal in a single reactor by adding external organic carbon to preclude excess aerobic phosphate uptake by polyphosphate-accumulating organisms (PAOs) and provide phosphate for denitrifying PAOs (DNPAOs). Aerobic batch tests after anaerobic phosphate release with different chemical oxygen demand (COD) concentrations indicated that the effect of COD concentration on the phosphate uptake preclusion could be expressed by a simple formula. The reduction factor reflecting the formula, which retards the aerobic phosphate uptake in the presence of COD, was added to the process rates of aerobic polyphosphate storage and PAOs growth in the model. The improved model, which included the reduction factor, reasonably matched the experimental result regarding aerobic phosphate uptake behavior whereas the model without it did not; thus, the former precisely predicts the AOA process behavior. (C) 2008 Elsevier Ltd. All rights reserved.

We have developed a reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from Rhodamine for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and the reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The signal/background ratio of this fluorogenic compound reached 2100-fold enhancement in fluorescence intensity. Dithio-1,4-threitol or triphenylphosphine as reducing reagents were successfully utilized for this chemistry to be introduced into the DNA probe. The genetic detection requires that two strands of DNA bind onto target oligonticleotides, one probe carrying a reducible fluorogenic compound while the other carries the reducing reagents. The reaction proceeds automatically without any enzymes or reagents under biological conditions to produce a fluorescence signal within 10-20 min in the presence of target DNA or RNA. In addition, the probe was very stable tinder biological conditions, even such extreme conditions as pH 5 solution, pH 10 solution, or high temperature (90 degrees C) with no undesirable background signal. The probes were successfully applied to the detection of oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.

An anaerobic ammonium oxidation (anammox) process for ammonia-rich wastewater treatment has not been reported at temperatures below 15 degrees C. This study used a gel carrier with entrapped anammox bacteria to obtain a stable nitrogen removal performance at low temperatures. In a continuous feeding test, a high nitrogen conversion rate (6.2 kg N m(-3) day(-1)) was confirmed at 32 degrees C. Nitrogen removal activity decreased gradually with decreasing operation temperature; however, it still occurred at 6 degrees C. Nitrogen conversion rates at 22 and 6.3 degrees C were 2.8 and 0.36 kg N m(-3) day(-1), respectively. Moreover, the stability of anammox activity below 20 degrees C was confirmed for more than 130 days. In batch experiments, anammox gel carriers were characterized with respect to temperature. The optimum temperature for anammox bacteria was found to be 37 degrees C. Furthermore, it was clear that the temperature dependence changed at about 28 degrees C. The apparent activation energy in the temperature range from 22 to 28 degrees C was calculated as 93 kJ mol(-1), and that in the range from 28 to 37 degrees C was 33 kJ mol(-1). This value agrees with the result of a continuous feeding test (94 kJ mol(-1), between 6 and 22 degrees C). The nitrogen removal performance demonstrated at the low temperatures used in this study will open the door for the application of anammox processes to many types of industrial wastewater treatment.

Linear alkylbenzene sulfonates (LAS) constitute, quantitatively, the most important group of synthetic surfactants used today. We studied the gene expression of Nitrosomonas europaea in response to LAS using a DNA microarray because ammonia-oxidizers are thought to be more sensitive to LAS than other microorganisms. Our objective was to elucidate which genes are expressed for N. europaea in response to LAS exposure. Microarray analysis and real-time PCR assay revealed that c. 30 genes were significantly expressed after LAS exposure, in particular genes associated with energy production and conversion. Our findings demonstrate that physical disruption of membrane structures, which contain enzymes associated with energy production and conversion, might be an important explanation for the high sensitivity of N. europaea to LAS exposure.

We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modi. cation of the 2,4-dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells. (C) 2008 Elsevier Ltd. All rights reserved.

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10 000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

Most biofilms have redox stratification where aerobic and anaerobic zones are stratified. This inherent property can be potentially applied to removals of persistent organic pollutants and simultaneous carbon and nitrogen. Biofilm reactors, therefore, have been numerously developed; however, their downsides are long-term startup and difficulty to maintain stable reactor performance since the reactor might experience sloughing events. The engineering challenges are to reduce startup time and to create rigid biofilm resistant to such sloughing events. Given that initial bacterial adhesion is an important factor to govern biofilm cohesiveness, enhancement of initial bacterial adhesion to a substratum is required. Here, we applied radiation-induced graft polymerization (RIGP) in terms of modification of a substratum to enhance bacterial adhesion and finally to develop a novel biofilm reactor system. Polyethylene sheets and hollow-fibers were modified with either amino or sulfonic acid groups. RIGP provides precise degree of grafting and density of the functional groups. Bacterial adhesion test on surface-modified membrane has revealed that membrane potential, i.e., electrostatic interaction, mainly governs bacterial adhesion rate, indicating that positivelycharged surfaces are favorable for initial bacterial adhesion. On the contrary, these surfaces potentially decrease bacterial activity, which is probably dependent on cell wall structures of Gram-positive and -negative bacteria. Even though bacteria attaching to the surfaces decrease the activity, flow cell test has demonstrated that these surfaces enhanced and maintained E. coli biofilm growth whereas the biofilm on negatively-charged surfaces did not grow well, paving the way for the effectiveness of the positively charged surfaces for bacterial immobilization carriers. Membrane-aerated biofilm reactors with positively-charged surface have been developed for controllable nitritation (conversion from ammonium to nitrite) and for simultaneous nitrification and denitrification, which achieved high oxygen utilization efficiency and high nitrogen removal rates. Therefore, we conclude that surface-modification by RIGP provides a suitable surface where rigid biofilm grows rapidly, leading to development of the novel biofilm reactor system for wastewater treatment.

Nitritation (ammonium being oxidized to nitrite) is a cost-effective method for treating wastewater having high ammonium concentrations or low ON ratios. We developed a novel nitritation process based on the observation that nitrite-oxidizing bacteria (NOB) in sewage sludge can be killed by heat shock, but ammonium-oxidizing bacteria (AOB) may survive. The effects of maximum heat-shock temperature and heat-shock duration on populations of AOB and NOB in gel carriers were measured. No NOB were detected after a heat-shock treatment higher than 60 degrees C for 20 min. However, the population of AOB continued to exist at above 10(8) MPN/mL-carrier even after heat shock at 80 degrees C for 1 h. To evaluate the nitritation performance, continuous feeding tests were conducted using heat-shocked gel carriers treated at three temperatures. Stable nitritation was observed for 49 days when gel carriers were heat shocked at 60-90 degrees C for 1 h. However, because nitrate production, i.e., nitratation, was observed after 77 days, the gel carriers were heat shocked again. Consequently, nitratation stopped immediately and nitritation restarted after 14 days. These results clearly show that this technique is effective for suppressing nitratation. (C) 2007 Elsevier Ltd. All rights reserved.

The phylogenetic diversity and species richness of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were examined with aquarium biofiltration systems. Species richness, deduced from rarefaction analysis, and diversity indices indicated that the phylogenetic diversity and species richness of AOA are greater than those of AOB; the diversity of AOA and of AOB is minimized in cold-water aquaria. This finding implies that temperature is a key factor influencing the population structure and diversity of AOA and AOB in aquarium biofiltration systems.

Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping. (c) 2007 Elsevier B.V. All rights reserved.

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.

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 NH(4)-N, NO(x)-N and PO(4)-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.

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&apos; 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&apos; 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.

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.

To immobilize lipase for enzymatic reactions in organic solvent, various functional [epoxy (GMA-fiber), hydroxyl (OH-fiber) or diethyl amino (DEA-fiber)] groups were introduced onto porous hollow-fiber membranes by radiation-induced graft polymerization of glycidyl methacrylate and chemical modification. Lipase from Candida rugosa was immobilized on polymer brushes by permeation of lipase. The activities of immobilized lipase were measured by esterification reactions between lauric acid and benzyl alcohol in isooctane. The activity of immobilized lipase on GMA-fibers, DEA-fibers and OH-fibers was 0.70 mol/(h kg-lipase), 0.50 mol/(h kg-lipase), and 2.45 mol/(h kg-lipase), respectively. Immobilized lipase on DEA-fibers or OH-fibers was reused three times after it was used in the batch reactor for 24 h. It was found that lipase activity showed no signs of denaturation. However, when native lipase was used, lipase activity remarkably decreased after reusing. © 2007 Elsevier B.V. All rights reserved.

Protein adsorption was performed by a polymer brush prepared by atom-transfer radical polymerization (ATRP) to a porous inorganic membrane. The porous inorganic membrane, Shirasu Porous Glass made from silica, was modified with a halogen-containing compound to bind the active species for the polymerization. Glycidyl methacrylate was polymerized from the halogen compound by ATRP for a prescribed time, and subsequently chemically modified. The progression of the chemical modification allowed the membrane to lower the phosphate-buffer flux of the porous membrane due to the attachment of the polymer brush. Bovine serum albumin (BSA), as a model protein, was adsorbed at 12 mg per gram of the membrane in permeating BSA solution through the polymer-brush-attached porous membrane. © 2006 Springer Science+Business Media, LLC.

A technology to achieve stable and high rates of nitrification of landfill leachate at low temperatures has been desired. Nitrifying bacteria entrapped in a polyethylene glycol (PEG) gel carrier produced high nitrification rates of 0.71 kg N/m(3) /day at 10 degrees C for more than I year. As a characteristic of nitrification, ammonium nitrogen at 16-35 mg/L remained in effluent water irrespective of nitrogen load and nitrite accumulation was observed. Batch experiments clearly showed that the relationship between ammonium concentration and ammonium removal rate followed a Monod-type equation. It was also revealed that ammonium-oxidizing bacteria cultivated in a gel carrier had a low affinity for ammonium, leading to incomplete nitrification. Moreover, it was suggested that the remaining ammonium in the reactor produced free ammonium, which inhibited the activities of nitrite-oxidizing bacteria. Thus, only nitritation was observed. Molecular biological methods, such as denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH), revealed that Nitrosomonas sp. was the dominant ammonium-oxidizing bacteria in the gel carrier at low temperature. (C) 2007 Elsevier B.V. All rights reserved.

A multipopulation model of a membrane-aerated biofilm (MAB) considering heterotrophic bacteria (HB), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB) was constructed with the simulation software AQUASIM 2.1 to corroborate the process concept of the membrane-aerated biofilm reactor (MABR) and to reveal an operational range for high chemical oxygen demand (COD) and nitrogen removal efficiencies. The modeling results confirm that simultaneous nitrification and denitrification (SND) is feasible in the MAB but not in a top-down aerated biofilm (conventional biofilm) due to the absence of oxygen for AOB and NOB. The model precisely predicts the COD, NH4+-N, and T-N removal efficiencies and determines operating parameters like COD/nitrogen (C/N) ratio, biofilm, thickness and surface loading of oxygen, which significantly affect SND efficiency. High nitrogen removal efficiency (more than 70%) is attained at ranges of C/N ratio from 3.0 to 5.25 and of biofilm thickness from 600 to 1200 mu m. In addition, it was clearly demonstrated that nitrogen removal not via nitrate but via nitrite could be achieved by controlling the relative surface loadings of oxygen and ammonia, supporting the feasibility of short-cut SND with MABs. (C) 2007 Elsevier B.V. All rights reserved.

Anaerobic ammonium-oxidizing (anammox) bacteria were immobilized in polyethylene glycol gel carriers. A small amount of seed sludge [0.24% (w/v)] was entrapped in the carriers, and continuous feeding tests were performed. Nitrogen removal activity increased gradually, reaching 3.7 kg N/m(3) stop reactor per day on day 67. The average of nitrogen conversion rate was calculated as 3.4 kg N/m(3) reactor per day. Microscopic examination clearly showed that small red clusters formed in the gel carrier. Moreover, fluorescence in situ hybridization analysis revealed that these clusters consisted of anammox bacteria. From real-time polymerase chain reaction analysis, the growth of anammox bacteria in the gel carriers was clearly shown by increased concentration of 16S rRNA gene of planctomycete from 4.3 x 10(8) to 4.2 x 10(9) copies/ml between days 41 and 55. To determine the effects of inoculation on the start-up of the reactor, the amount of seed sludge in the gel carrier was varied and it was found that the start-up period could be reduced to as little as 25 days when a sludge concentration of 1.4% (w/v) was used. This is the first report of successful immobilization and cultivation of anammox bacteria in a gel carrier.

We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (1,AMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5' end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by 1,AMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.

"Candidatus Accumullibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumullibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH4+) is supplied form the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and HH4+ are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH4+ (J(O2)/J(NH4+)) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 mu m; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH4+. Under conditions of oxygen excess (J(O2)/J(NH4)(+) &gt; 3.98), almost all NH4+ was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidiers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.

We developed an RNA microarray protocol in which total RNA frorn a microbial community was attached to a slide glass, and rRNA was detected by fluorescently labeled oligonucleotide probes. The RNA microar-ray requires only 4 h for hybridization and enables double staining and estimating relative abundance of rRNA. (C) 2007 Elsevier B.V. All rights reserved.

High rates of nitrogen removal from wastewater have been reported using anammox bacteria at temperatures around 37 C, but not at moderately low temperatures. In this study, nitrogen removal performance of an anaerobic biological filtrated (ABF) reactor, filled with porous polyester nonwoven fabric carriers as a fixed bed for anammox bacteria, was tested at 37 C and at moderately low temperature (20-22 degrees C). To attain higher nitrogen removal performance, effects of influent nitrogen concentrations and hydraulic retention time (HRT) on nitrogen removal rates were investigated. Nitrogen removal rate increased with influent ammonium and nitrite concentrations, resulting in a removal rate of 3.3 kg-N/m(3)/d on day 32 for an HRT of 180 min at 37 degrees C. However, influent nitrite concentrations greater than 280 mg/l inhibited anammox activity. Therefore, the influent nitrite concentration was adjusted to be below 280 mg/l, and high-loading tests were performed for a shorter HRT. As a result, a nitrogen conversion rate of 11.5 kg-N/m(3)/d was achieved. Moreover, to evaluate long-term anammox activity at moderately low temperatures, ABF reactors were operated for 446 d. Anammox activity could be maintained at 20-22 degrees C, and stable nitrogen removal performance was observed. Furthermore, high nitrogen conversion rate of 8.1 kg-N/m(3)/d was attained. These results clearly show that an appropriate nitrite concentration in the influent and a shorter HRT resulted in high nitrogen conversion rates. The nitrogen removal performance we obtained at moderately low temperatures will open the door for application of anammox processes to many types of industrial wastewater treatment.

We have developed a simple, cost-effective, and accurate method for the quantification of specific nucleic acid sequences by the combined use of competitive PCR and a sequence-specific fluorescent probe that binds to either the gene of interest (target) or internal standard (competitor), referred to as alternately binding probe (ABProbe). In this method, the target and competitor were coamplified with the ABProbe, and then the fluorescence intensity was measured. The ratio of the target to the competitor can be calculated from the fluorescence intensity of the ABProbe using fluorescence quenching and fluorescence resonance energy transfer, that is, the starting quantity of the target is successfully calculated by end-point fluorescence measurement. Therefore, this method eliminates the complex post-PCR steps and expensive devices for real-time fluorescence measurement. We called this method alternately binding probe competitive PCR (ABC-PCR). We quantified amoA as a model target by ABC-PCR and real-time PCR. By comparison, the sensitivity, accuracy, and precision of ABC-PCR were similar to those of real-time PCR. Moreover, ABC-PCR was able to correctly quantify DNA even when PCR was inhibited by humic acid; therefore, this method will enable accurate DNA quantification for biological samples that contain PCR inhibitors.

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.

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.

Fifteen oligonucleotide probes, targeting a broad range of microorganisms, were spotted on a DNA microarray. Specificity and reproducibility were evaluated using fluorescently labeled rRNA from 16 bacterial and eukaryal strains. The oligonucleotide microarray was then used to analyze activated sludge samples of saline industrial wastewater in which microbial communities had been characterized previously using 16S rRNA clone libraries. The results obtained were partially consistent with the results of cloning, and demonstrate the possibility of using oligonucleotide microarrays for the monitoring and characterization of saline industrial wastewater populations.

To distinguish subgroups of Defluvicoccus-related glycogen-accumulating organisms (Defluvicoccus-related G-bacteria), new oligonucleotide probes were designed. Two of these probes, DF636 and DF827, were used successfully to detect each subgroup of Defluvicoccus-related G-bacteria. The morphological differences in clusters and cells between the DF636- and DF827-binding cells were confirmed. Interestingly, DF636-binding cells showed autofluorescence under UV light. Additionally, auto fluorescence was observed in some alphaproteobacterial G-bacteria, which did not bind to any of the probes designed in this study. The results suggested that the Defluvicoccus-related G-bacteria consist of several physiologically different subspecies.

Pregnancy-specific glycoproteins (Psgs) secreted by the placenta regulate the immune system to ensure the survival of the fetal allograft by inducing IL-10, an anti-inflammatory cytokine. However, it is unknown whether Psgs are involved in more general aspects of immune response other than maternal immunity. Here, we report that Psg18 is highly expressed in the follicle-associated epithelium (FAE) overlaying Peyer's patches (PPs). Bioinformatics analysis with Reference Database for Immune Cells (RefDIC) as well as RT-PCR data demonstrated that Psg18 is exclusively expressed in FAE in adult mice, in contrast to other Psg family members that are either not expressed or only slightly expressed in FAE. Psg18 expression was observed in FAE of germ-free-conditioned mice, and was slightly upregulated after bacterial inoculation. In situ hybridization analysis revealed that Psg18 is widely expressed throughout FAE. Furthermore, Psg18 protein is deposited on the extracellular matrix in the subepithelial dome beneath FAE, where antigen-presenting cells accumulate. These results suggest that Psg18 is an FAE-specific marker protein that could promote interplay between FAE and immune cells in mucosa-associated lymphoid tissues.

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying pbosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia cofiand a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E coli markedly increased at a membrane potential higher than -7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than -8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80 % of E coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60 % of B. subtilis cells were inactivated. Furthermore, E coli viability significantly decreased at a membrane potential higher than -7.8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E coli and B. subtilis.

A hydrogen-based membrane biofilm reactor (MBfR), employing fibrous slag as a bacterial carrier, was developed for rapid and stable autohydrogenotrophic denitrification. This reactor allows hydrogen to be supplied through a gas-permeable membrane to the biofilm supported by fibrous slag. The estimation of hydrogen supply rate clearly demonstrated that hydrogen flux (J(H2)) is dependent on the gas pressure, leading to a possibility to control J(H2) by adjusting the pressure. A startup experiment to investigate denitrification rate with time clarified that denitrification rate of 4.35 g N/m(2)/day was achieved on day 10, exhibiting rapid startup for autohydrogenotrophic denitrification. Continuous denitrification experiment obviously indicated the effectiveness of the fibrous slag as a bacterial support; concretely, mean denitrification efficiency and rate after 70-day operation reached 99% and 6.58 g N/m(2)/day at a hydrogen pressure of 50 kPa, respectively, which results in the accomplishment of stable and high-speed denitrification. However, hydrogen utilization efficiency (HUE) was approximately 40%. This low efficiency allowed autotrophic sulfate-reducing bacteria (SRB) to grow in the fibrous-membrane matrix; eventually the RUE for sulfate reduction increased up to 28% on day 74. This result clearly indicates the significance of J(H2) control through the gas-permeable membrane for suppressing the occurrence of sulfate reduction. (c) 2006 Elsevier B.V. All rights reserved.

A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the gamma-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4-10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.

The presence of cyanobacterial bloom in water supply reservoirs can cause potential health hazards. In this study, we aimed at the quantification of microcystin-producing cyanobacteria based on the microcystin synthetase A (mcyA) gene using real-time PCR. To perform a highly sensitive real-time PCR assay, the novel primer MSR-2R was designed and a coprecipitation DNA extraction method was used in this study. Cyanobacterial cells could be collected efficiently by coprecipitation with other bacteria suspended in solution even in the case of low concentrations of cyanobacteria. The detection limit of the method was found to be 8.8 cells per reaction. When cyanobacterial growth was monitored in pure culture, the cell concentration determined by real-time PCR positively correlated with the cell concentration determined from direct microscopic count. Furthermore, we could detect and quantify the mcyA gene in lake water samples using real-time PCR. It was concluded that the quantification of the mcyA gene based on real-time PCR is a powerful tool for the rapid quantification of microcystin-producing cyanobacteria in environmental samples.

In a biological nutrient removal (BNR) process, the utilization of denitrifying polyphosphate-accumulating organisms (DNPAOs) has many advantages such as effective use of organic carbon substrates and low sludge production. As a suitable process for the utilization of DNPAOs in BNR, an anaerobic/oxic/anoxic granular sludge (AOAGS) process was proposed in this study. in spite of performing aeration for nitrifying bacteria, the AOAGS process can create anaerobic/anoxic conditions suitable for the cultivation of DNPAOs because anoxic zones exist inside the granular sludge in the oxic phase. Thus, DNPAOs can coexist with nitrifying bacteria in a single reactor. In addition, the usability of DNPAOs in the reactor can be improved by adding the anoxic phase after the oxic phase. These characteristics enable the AOAGS process to attain effective removal of both nitrogen and phosphorus. When acetate-based synthetic wastewater (COD: 600 mg/L, NH4-N: 60 mg/L, PO4-P: 10mg/L) was supplied to a laboratory-scale sequencing batch reactor under the operation of anaerobic/oxic/anoxic cycles, granular sludge with a diameter of 500 Pin was successfully formed within 1 month. Although the removal of both nitrogen and phosphorus was almost complete at the end of the oxic phase, a short anoxic period subsequent to the oxic phase was necessary for further removal of nitrogen and phosphorus. As a result, effluent concentrations of NH4-N, NOx-N and PO4-P were always lower than 1 mg/L. It was found that penetration depth of oxygen inside the granular sludge was approximately 100 mu m by microsensor measurements. in addition, from the microbiological analysis by fluorescence in situ hybridization, existence depth of polyphosphate-accumulating organisms was further than the maximum oxygen penetration depth. The water quality data, oxygen profiles and microbial community structure demonstrated that DNPAOs inside the granular sludge may be responsible for denitrification in the oxic phase, which enables effective nutrient removal in the AOAGS process. (c) 2006 Elsevier Ltd. All rights reserved.

A porous anion-exchange hollow-fiber membrane was prepared by radiation-induced graft polymerization and chemical modification to immobilize lipase for enzymatic reaction in an organic solvent. The amount of anion-exchange group introduced to the porous hollow-fiber membrane was 2.5 mol/kgfiber. A lipase solution was allowed to permeate through the porous anion-exchange hollow-fiber membrane, and lipase molecules that adsorbed onto the grafted polymer brush were cross-linked with glutaraldehyde. The lipase was immobilized at a density of 0.14 kglipase/kgfiber, which was equivalent to a degree of multilayer binding of 20. Esterification was carried out by passing a solution of lauric acid and benzyl alcohol in anhydrous isooctane through the lipase-immobilized membrane, and lipase activity was determined. A reaction percentage of 50% was achieved at space velocity 68 h-1. The maximum immobilized lipase and native lipase activities were 8.9 and 0.38 mol/(h·kglipase), respectively. Thus, the activity of the immobilized lipase was 23.4 times higher than that of the native lipase. Copyright © 2006 by AOCS Press.

A ligand-containing porous membrane was prepared by radiation-induced graft polymerization of an epoxy-group-containing monomer of glycidyl methacrylate onto a polyethylene porous hollow-fiber membrane and by subsequent conversion of the epoxy group to an N-methylglucamino (NMG) group at a density of 0.78 mmol/g of the membrane. Sb(V) solution was permeated through the NMG-ligand-containing porous hollow-fiber membrane. Optimum pH for Sb(V) recovery was 3.0. Breakthrough curves of Sb (V) overlapped irrespective of residence times of Sb (V) in the membrane, due to negligible diffusional mass-transfer resistance. Maximum amount of Sb (V) adsorbed was 130 mg/g-membrane, which was equivalent to 1.3 binding molar ratio. Repeated usage of the membrane for adsorption and elution was possible. (c) 2006 Elsevier B.V. All rights reserved.

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.

The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1x10(6) and 1.7x10(7) cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (mu) was 0.39 day(-1). This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.

Aims: Denitrification efficiency at 10% salinity was compared with that at 2% salinity. The characteristics of bacterial strains isolated from the denitrification system, where an improvement of denitrification efficiency was observed at a high salinity were investigated.
Methods and Results: Two continuous feeding denitrification systems for saline solutions of 2% and 10% salinity, were operated. Denitrification efficiency at 10% salinity was higher than that at 2% salinity. The bacterial strains were isolated using the trypticase soy agar (TSA) medium at 30 degrees C. The phylogenetic analysis of 16S rRNA gene sequences of isolates indicated that halophilic species were predominant at 10% salinity.
Conclusions: The improvement of denitrification efficiency at a high salinity was demonstrated. The strains isolated from the denitrifying system with 10% salinity were halophilic bacteria, Halomonas sp. and Marinobacter sp., suggesting that these bacteria show a high denitrifying activity at 10% salinity.
Significance and Impact of Study: The long-term acclimated sludge used in this study resulted in high denitrification performance at a high salinity, indicating that the design of a high-performance denitrification system for saline wastewater will be possible.

We have developed a novel method for genotyping based on free solution affinity capillary electrophoresis. We prepared DNA-nail opal-tic le conjugates by mixing biotin-modified DNA and NeutrAvidin-modified polystyrene nanoparticles; this mixture was then injected into a capillary. Subsequently, we injected the fluorescent-labeled sample DNAs into the capillary, applied the voltage, increased its temperature after 7 min, and detected the fluorescence at its anodic end. This novel method was applied for genotyping human c-K-ras, and the three genotypes were definitely distinguishable with high reproducibility. This method can be easily automated, and it is useful for high-throughput gene mutation analysis. (c) 2006 Elsevier B.V. All fights reserved.

A feasibility of a membrane-aerated biofilm reactor (MABR) for controllable nitrification was examined. The estimation of oxygen supply rate (OSR) with three polyacrylonitrile membrane modules revealed that specific OSR was equivalent in these membrane modules and OSR affected only air pressure, thus enabling control of aeration simply by adjustment of air pressure. A continuous nitrification experiment consisting of three reactors differing in membrane surface area investigated the reactor performance of the MABR at an air pressure of 23 kPa. The results indicated that the ammonia removal rate at steady state was dependent on membrane surface area, at rates nearly equivalent to that predicted by the above OSR experiment. The amount of bacteria adhering to the membrane surface was not completely proportional to membrane surface area due to clogging in a reactor with high membrane surface area, which accompanies a decrease in specific ammonia removal rate per biomass with membrane surface area. Stable ammonia removal rates at air pressures of 23, 45 and 100 kPa corresponded to the predicted values from the OSR experiment. Further, more than 80% oxygen utilization efficiency (OUE) was achieved under all operational conditions. indicating effective oxygen uptake by nitrifying bacteria under oxygen-depleted conditions. Based on these experiments, the MABR was shown to be a controllable nitrification system, and to be able to provide a reaction space for nitrification in a membrane-attached biofilm without altering the bulk conditions. (c) 2005 Elsevier B.V. All rights reserved.

In order to improve the microbial reaction rate within a cell-immobilized space, this study experimentally investigated enhanced substrate transport by electrophoresis of a specific ion. Batch-mode and continuous feeding denitrification tests were carried out in an autotrophic denitrifying bioreactor using sulfur-oxidizing microbes as a model system. Seed sludge and powdered sulfur were immobilized on fibrous carrier medium, and an electric field was applied through the cell-immobilized zone via inert electrodes on both sides of vessel. In batch-mode denitrification tests, nitrogen removal rate reached 1.99 g-N·m-3·h-1 when the electric field of 18V was reversed at intervals of 30min, as compared with 0.40g-N·m -3·h-1 when the electric field was not applied. These findings demonstrated that the denitrification rate was increased by promoting the transport of NO3- ions across the cell-immobilized zone by electrophoresis. In addition, no reduction of NO 3- to NO2- occurred, indicating that decrease of nitrogen compound was due to biological reaction. Mathematical simulation analysis using a model involving Monod-type reaction kinetics and the Stokes formula for NO3- mobility was in general agreement with the experimental results. The results of the continuous feeding test also indicated that electrophoresis improved the nitrogen removal rate. Copyright © 2006 The Society of Chemical Engineers, Japan.

In this study, an anaerobic/aerobic/anoxic process (referred to as an AOA process) using a sequencing batch reactor (SBR) was proposed for simultaneous phosphorus and nitrogen removal from wastewater. The AOA process was stably operated over more than one year when a certain amount of carbon substrate (40 mg-C/L in a reactor) was supplemented to inhibit aerobic phosphate uptake. The average nitrogen and phosphorus removal efficiencies were 83% and 92%, respectively. It was demonstrated that phosphate-accumulating organisms (PAOs) capable of utilizing nitrite as an electron acceptor, the so-called denitrifying phosphate-accumulating organisms (DNPAOs), could exist in the AOA process. Moreover, the ratio of anoxic phosphate uptake rate (PUR) to aerobic PUR (anoxic/aerobic PUR ratio), which indicates the fraction of DNPAOs in total PAOs, was experimentally evaluated. The results indicate that the AOA process has a much larger anoxic/aerobic PUR ratio than the conventional A(2)O (anaerobic/anoxic/aerobic) and AO (anaerobic/aerobic) processes. In conclusion, the AOA process allows DNPAOs to take an active part in simultaneous nitrogen and phosphorus removal in an SBR when a suitable amount of carbon substrate is supplied at the start of aerobic conditions. (c) 2005 Elsevier B.V. All rights reserved.

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 (500g-N/m(3)), the ammonia removal rate reached 16.7 kg-N/m(3)/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/m(3)) 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/m(3)). Moreover, the nitrifying granules exhibited sufficient performance in the nitrification of real industrial wastewater containing high V concentrations of ammonia (1,000-1,400 g-N/m(3)) 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 CA 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.

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 an phosphorus adsorption systems is useful as a new advanced wastewater treatment plant (WWTP) to resolve the problems of increasing excess sludge and depleted phosphorus.

The simultaneous detection of different bacterial strains carrying a certain functional gene is absolutely imperative in order to detect bacteria on the basis of functional gene expression products (mRNA) in situ. Functional genes are possessed by bacterial strains with different types of cell walls
therefore, the optimization of conditions for digesting different types of cell walls is significant. In this study, we defined the optimal conditions for permeabilization in eight bacterial strains belonging to different phylogenetic divisions using solutions containing different concentrations of lysozyme and/or achromopeptidase, for conventional fluorescence in situ hybridization (FISH), digoxigenin (DIG)-FISH, and catalyzed reporter deposition (CARD)-FISH with a rRNA-targeting oligonucleotide probe. Most bacterial strains were successfully detected using CARD-FISH with lysozyme 10 mg/ml pretreatment. Additionally, achromopeptidase pretreatment combined with lysozyme pretreatment was a highly effective means of permeabilizing bacterial strains that were unable to be detected using lysozyme pretreatment. However, this additional pretreatment resulted in a loss of cell morphology in some bacterial strains due to excessive permeabilization. Consequently, permeabilizing conditions for applying highly sensitive FISH to the well-defined target bacterial strains used in this study were optimized. The results of this study will contribute to the optimization of permeabilizing conditions, which is one of the most important factors for the successful application of highly sensitive FISH. © 2006, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

The selective removal of low-concentration heavy metal ions from heavy-metal-contaminated water was investigated using the activity or sulfate-reducing bacteria (SRB) immobilized on a fibrous slag (FS), which was employed as a both biocarrier and a fine particle collector medium. An upflow-type bioreactor composed of an FS-packed column, where SRB were immobilized at a lower part, was used. In this process, sulfate ions in the liquid are biologically reduced to sulfide ions, which react with metal ionic species to produce ultrafine metal sulfide particles. Subsequently, formed particles adhere onto the FS surface in the upper part of the reactor, thereby accumulating heavy metal ions as their sulfide precipitates. Five different organic compounds were evaluated, and as a result, lactic acid was found to serve as the most suitable electron donor for SRB in this system. In the continuous treatment of water contaminated with several heavy metal ions (Cd2+, Cu2+, Ni2+, Zn2+, and Mn2+), almost complete removal was attained over an 80-day operation period, except for Mn2+ having a relatively high solubility product constant. Heavy metal sulfide precipitates gradually accumulated on an FS bed. These experimental results indicate that the proposed bioreactor, which is the sulfate-reducing bioreactor with a fibrous slag carrier (SRB-FS), enables the efficient recovery of heavy metal ions from heavy-metal-contaminated water.

We developed a novel single-stage autotrophic nitrogen-removal process comprised of two composite immobilized biomass layers-one of nitrifying bacteria and one of sulfar-denitrifying bacteria and elemental sulfur-in a Fe-Ni fibrous slag matrix. Nitrification and consumption of dissolved oxygen occurred in the outer part and sulfur denitrification in the anoxic inner part of the composite matrix, thus realizing autotrophic nitrogen removal in a single reactor. The complete conversion of ammonia into N-2 in a single reactor was demonstrated in both batch-mode incubation and continuous-feed operation. The spatial profiles of the ammonia-oxidizing bacteria and denitrifying bacteria were evaluated by real-time PCR, targeting their functional genes, and stratification of these two types was observed in the matrix after several months of incubation. This process does not require any specific reactor type or conditions and thus has the potential to be applied to many different wastewater treatment processes due to its simplicity in both operation and construction.

Surface-modified polyethylene (PE) membrane sheets were prepared by the radiation-induced graft polymerization (RIGP) of an epoxy-group-containing monomer, glycidyl methacrylate (GMA). The epoxy ring of GMA was opened by introducing diethylamine (DEA) or sodium sulfite (SS). We examined the properties of these sheets by measuring the amount of grafting polymer, surface roughness and membrane potential, and also investigated the adhesion of five Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens and Paracoccus denitrificans, onto the prepared sheet surfaces. A linear relationship between the degree of grafting (dg) and surface roughness was observed. Moreover, membrane potential was dependent on the amount of DEA or SS as the ionizable group. These results indicate that RIGP enables the control of the physicochemical properties of such a sheet surface by adjusting dg and the subsequent conversion of functional groups. A batch test on bacterial adhesion onto the sheets clarified that the DEA-containing sheet (DEA sheet) exhibited an adhesion rate constant, k, significantly greater than those of other types of sheet. Clearly, the adhesion rate constant of the DEA sheet increased with dg, indicating that electrostatic interaction is the most decisive factor for bacterial adhesion when it works as an attractive force. Furthermore, the densities of bacteria adhering onto the GMA-containing sheet (GMA sheet) and the SS-containing sheet (SS sheet) were almost the same as that onto a PE sheet, whereas that onto a DEA sheet significantly increased. Thus, the introduction of the GMA- and SS-containing graft chain did not have much influence on bacterial adhesion onto the surfaces, supporting the conclusion that the promotion of bacterial adhesion onto the GMA and SS sheets was due to an increase in surface area resulting from RIGP. Moreover, the scanning electron microscopy images of the sheet surfaces indicate that the conditions and morphologies of initial bacterial adhesion are dependent on surface properties, particularly membrane potential. (c) 2005 Elsevier B.V. All rights reserved.

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.

Nitrite reductase gene (nirS) fragments in the activated sludge obtained from a sequencing batch reactor (SBR) under anaerobic-aerobic condition were cloned and classified by restriction fragment length polymorphism (RFLP) analysis, and representative fragments were sequenced. One of the nirS clones was approximately 70% of all nirS clones in anaerobic/aerobic (existing oxygen and nitrate) cycle operation in which a large amount of anoxic phosphate uptake was observed. Although the activated sludge samples analyzed might contain bacteria that did not accumulate polyphosphate, it was likely that this nirS fragment sequence was that from denitrifying polyphosphate-accumulating organisms (DNPAOs) which can utilize both oxygen and nitrate as electron acceptors. The sequence was similar to the WrS sequences of Thauera mechemichensis (83% similarity) and Azoarcus tolulyticus (83% similarity) both of which belong to the Rhodocyclus group.

The effects of wastewater salinity on both nitrogen removal efficiency and N2O emission rate were investigated in a single nitrification process, a single denitrification process and an anoxic-oxic activated sludge process. In the single nitrification process, by increasing the salt concentration from 1.0 to 2.0 wt%, the N2O conversion ratio in the steady state increased by 2.2 times, from 0.22 to 0.48%. In the single denitrification process, a minimal change in the N2O conversion ratio was observed in the steady state even when the salt concentration was increased from 3.0 to 5.0 wt%. From the results of the anoxic-oxic activated sludge process, it was found that a salt concentration increase from 1.6 to 3.0 wt% significantly increases the N2O conversion ratio from 0.7 to 13%. It is suggested that an increase in salt concentration markedly influences N2O emission both directly and indirectly via the inhibition of N2O reductase activity. The indirect inhibition is due to the high concentration of dissolved oxygen which is transported from the oxic tank to the anoxic tank through the circulated liquid. Thus, the salt concentration should be maintained below 3.0% to suppress N2O emission in an anoxic-oxic activated sludge process. (c) 2004 Elsevier B.V. All rights reserved.

Chlorinated volatile organic compounds, e.g., chloroform (CF), carbontetrachloride (CTC), tri- and tetrachloroethylene (TCE and PCE) are pollutants commonly found in ground water and soil. Their oxidation in a novel, bubble column photochemical reactor, combining the mass transfer of pollutant from the gas phase followed by a free-OH radical reaction in a liquid phase is described mathematically and in terms of economic feasibility. The combined system eliminates the negative effects of direct oxidation in groundwater (reduced efficiency due to the presence of UV radiation absorbers and OH radical scavengers) and gas phase oxidations (production of stable reaction intermediates). For low initial concentrations of pollutant in a gas phase, reaction occurs mainly in the liquid phase (slow reaction regime). The absorption with reaction could be well predicted based on the ratio of the apparent rate constant of pollutant degradation to the volumetric mass transfer coefficient. The Electrical Energy per Order (EE/O) values depend mostly on the initial concentration of pollutant and hydrogen peroxide, as well as reactor radius. The levels of PCE and TCE in the order of 20 mg/L could be treated economically. However, the treatment of compounds with low affinity to OH radicals (CF and CTC) is economically unfavorable even for parts per billion level concentrations. This is an abstract of a paper presented at the 7th World Congress of Chemical Engineering (Glasgow, Scotland 7/10-14/2005).

The performance of charcoal bricks made from beer lees (MaltCeramics, MC) was investigated as the material for purification of water breeding goldfish in order to compare with gravel and activated charcoal. The goldfish used in this study discharges 0.409 mg/D-g of TOC, 0.26 mg/D-g of NH4-N, 0.024 mg/D-g of NO3-N and 0.007 mg/D-g of T-P. It was confirmed that MC is useful for the carrier of microorganisms and it is possible to reduce soluble organic matter and soluble nitrogen compound such as NH4-N, NO3-N and NO2-N as same as activated charcoal. In the case of MC, NH4-N is decomposed to form NO2-N and NO3-N within 50 h. NO2-N and NO3-N are the decomposition products of NH4-N substrate and NO2-N is denitrated for 50–80 h. NH4-N and NO2-N are rapidly decomposed, but the denitrification of NO3-N is a very slow reaction. TOC is decreased down to the initial concentration level for 20–30 h. MC contains phosphorus at the ratio of about 2 wt%, which is soluble in water, and some minerals. It is considered that they would activate bacteria in water. © 2005, The Resources Processing Society of Japan. All rights reserved.

The study of community structure in wastewater biofilms is made difficult by the slow growth rates and high environmental sensitivities of autotrophic nitrifiers. Simulations of such films can generate data quickly and without susceptibility to random environmental perturbation. This study uses a 2D cellular automaton model to compare the community structures of biofilms grown under top-down and membrane aeration conditions. This study found dramatic differences in community structure between the two approaches, most notably the emergence of a niche at the solid - biofilm interface that facilitates the growth of nitrite oxidizing bacteria.

Aims; The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification.
Methods and Results: cDNA reverse-transcribed from 16S rRNA was amplified with fluorescent labelled primer for terminal restriction fragment length polymorphism (T-RFLP) analysis and an unlabelled primer for cloning analysis. The terminal restriction fragments (T-RFs) that increased in association with the start of denitrification were determined. These T-RFs were identified by in silico analysis of 16S rRNA sequences obtained from cloning. As a result, it was clearly observed that the bacteria belonging to the genera Hydrogenophaga and Acidovorax increased in number after the start of denitrification.
Conclusions: It was demonstrated that T-RFLP analysis targeting 16S rRNA is appropriate for the daily monitoring of a bacterial community to control wastewater treatment processes. Combination of the results of T-RFLP analysis and 16S rRNA clone library indicated that the bacteria belonging to the genera Hydrogenophaga and Acidovorax play an important role in denitrification.
Significance and Impact of the Study: The results of this study provide new insight to the 16S rRNA level of active denitrifying bacteria in wastewater treatment processes.

Wastewater containing high nitrogen and high salinity is discharged from the processes of recovering precious metals from industrial waste. Because high salinity inhibits metabolic activity of microorganisms, biological nitrogen removal from such a wastewater has a lot of difficulties. The purpose of this study is to develop an effective nitrification denitrification process for industrial saline-wastewater. At first, nitrogen removal from this wastewater was attempted using a normal suspended nitrifying sludge system. In consequence of estimating tolerance of such a sludge system to salinity, nitrification progressed up to 5% of salt concentration. However, keeping MLSS at 5, 000 g/m³ is necessary to achieve practical nitrification rate. Therefore, nitrifying granules resulting from an aerobic upflow fluidized bed reactor for inorganic wastewater containing high concentration of NH₄-N were applied to this industrial saline-wastewater. In consequence of operating nitrification reactor at various salt concentrations, MLSS was kept at 12000g/m³ throughout the operation, and sufficient nitrogen removal rate was achieved. Finally, the nitrifying granules were applied to an aerobic reactor in the aerobic-anaerobic nitrogen removal system. As a result, nitrogen in the saline wastewater was completely removed for 150 days. Through these experiments, it is demonstrated that nitrifying granules are effectively applied to nitrogen removal from industrial saline-wastewater.

The microbial population dynamics at the start-up stage of a wastewater treatment reactor was investigated using terminal restriction fragment length polymorphism (T-RFLP) analysis based on 16S rDNA and rRNA gene sequences. The results of fragment peaks suggested that the number and activity of nitrifying bacteria increased in association with the start of nitrification, and the relative ratios of 16S rRNA of these bacteria changed prior to those of the 16S rDNA. Furthermore, multidimensional scaling (MDS) analysis revealed that the 16S rRNA exhibited wider dispersion than the 16S rDNA at the start-up stage, indicating that the diversity of 16S rRNA in the microbial communities was strongly affected by environmental changes.

The influence of extracellular polymeric substances (EPSs) on bacterial cell electrokinetic properties and on cell adhesion onto glass beads in connection with bacterial cell electrokinetic properties was investigated using 12 heterotrophic bacterial strains. Bacterial cell surface properties such as the softness 1/lambda and charge density ZN were determined by Ohshima's soft-particle analysis using the measured electrophoretic mobility as a function of ionic strength. In 10 of 12 strains, when EPSs covering the cell surface were removed, the softness of the cell decreased, indicating that EPS adsorption enhanced the ease of liquid fluid in the ion-penetrable layer on the cell surface. On the other hand, the negative charge density of the cell surface increased for 9 of 12 strains, suggesting that EPSs covering the cell surface decreased the negative charge density of the cell surface layer. In addition, the characteristics of bacterial cell adhesion onto glass beads were evaluated by the packed-bed method and the data were interpreted to indicate cell adhesiveness. As a result, the efficiency of cell adhesion onto glass beads increased as negative cell surface potential Psi(0) decreased, whereas there seemed to be no correlation between zeta potential and cell adhesiveness. Cell surface potential Psi(0), which was derived by taking the bacterial polymer layer with EPSs into consideration, provided a more detailed understanding of the electrokinetic properties of bacterial cells. (C) 2004 Elsevier Inc. All rights reserved.

A polyol-ligand-containing porous hollow-fiber membrane capable of removing antimony (III) from a liquid stream was prepared by radiation-induced graft polymerization of an epoxy-group-containing vinyl monomer, glycidyl methacrylate (GMA), and subsequent functionalization with N-methylglucamine (NMG) and 3-amino-1,2-propanediol (APD). The resultant chelate-forming group density was 1.6 mol per kg of the NMG-group-containing porous hollow-fiber membrane. An antimony (111) oxide solution (10 mg per L, pH 3.6-13) was forced to permeate through the submicron-diameter pores of the chelating porous hollow-fiber membrane. The antimony concentration of the effluent penetrating the outside surface of the hollow fiber was determined as a function of the effluent volume. The breakthrough or dynamic adsorption capacity for antimony was 54 g of Sb per kg of membrane at pH 11. Because of negligible diffusional mass-transfer resistance, the breakthrough curves overlapped irrespective of the permeation rate of the antimony solution across the chelating porous hollow-fiber membranes.

The expression of ammonia monooxygenase encoding mRNA (amoA mRNA) in a wastewater treatment process was analyzed in an attempt to propose an effective target for the monitoring of nitrifying bacteria in engineered systems or natural environments. The quick response (1-2 h) of amoA mRNA transcription to the recovery of ammonia oxidation activity induced by the sudden exposure to ammonia was observed in a short-time batch-mode incubation whereas the amount of amoA DNA did not markedly change during the incubation under any conditions. In the continuous feeding-operation, amoA mRNA level dynamically changed in response to the change in the surrounding environmental conditions and increase in ammonia oxidation rate. Although, amoA mRNA level did not quickly respond to the decrease in ammonia oxidation activity, it decreases over long time scales. These results suggest that the profiles of amoA mRNA expression can be used as an indicator of the ammonia oxidation activity. (C) 2004 Elsevier B.V. All rights reserved.

A polyol-ligand-containing porous hollow-fiber membrane for the recovery of antimony(III) was prepared by radiation-induced graft polymerization of an epoxy-group-containing vinyl monomer, glycidyl methacrylate (GMA), and by subsequent functionalization with N-methylglucamine (NMG) and 3-amino-1,2-propanediol (APD), that forma coordination complex with Sb(III). The structure of NMG-Sb(III) and APD-Sb(III) complexes in aqueous solution were determined by electron ionization-time-of-flight mass spectrometer (ESI-TOF-MS), and the binding ratio of NMG or APD to Sb(III) is 2: 1. An antimony(III) oxide solution (10 mg Sb/l, pH 11.4) was forced to permeate through the submicron-diameter pores of the polyol-ligand-containing porous hollow-fiber membranes. The equilibrium binding capacity for antimony(III) to the NMG-ligand-containing porous hollow-fiber membrane, 96 gSb/kg, was 10 times higher than that of the APD membrane. In a further study of the NMG membrane, the equilibrium binding ratios for antimony(III) to NMG groups were all approximately 0.5, illustrating that the NMG-Sb(III) complex on the fibers was in the ratio of 2:1. The results of computational structural analysis of the NMG-Sb(III) complex were in agreement with the experimental results of binding ratio. It was verified that an antimony(III) ion formed a coordination complex with two adjacent hydroxyl groups of two NMG moieties. The length of a functional group and the distance between functional groups on the polymer brush were significant factors to bind antimony(III) through the computational simulation. (C) 2004 Elsevier B.V. All rights reserved.

Batch experiments with varying initial substrate concentrations and biomass volumes were performed in a three-phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the simultaneous carbon oxidation and nitrification in the biofilm Process. A simplified mass balance equation for the biofilm was proposed and five different kinetic rate equations were used to match the actual data. The kinetic parameters were obtained by nonlinear regression analysis on a set of two differential equations representing the simultaneous carbon oxidation and nitrification. The competitive inhibition model incorporating the effects of total organic carbon (TOC) concentrations on nitrification rates was the best-suited model based on the average r(2). In this model, oxygen concentration and its affinity constants were not included. Instead, it was assumed that the rate of carbon oxidation is independent of the NH4+-N, while nitrification is affected by TOC. The number of parameters was successfully minimized without reducing its ability to accurately predict the bulk concentration time course, which would reduce computational complexity and possibly enhance the availability for an actual wastewater treatment process.

Three 16S rRNA-targeted oligonucleotide probes, namely, PSMg437 targeting several members of the genus Pseudomonas, Hlm474 targeting several members of the genus Halomonas, and Clw844 targeting several members of the genus Colwellia, were designed. The microbial community structure and nitrogen removal ability of nitrate-containing saline wastewater treatment systems with anaerobic packed bed and fluidized bed were monitored. Direct cell counting using fluorescence in situ hybridization (FISH) images revealed that various phylogenetic groups were evenly distributed in the anaerobic packed bed whereas members of the genus Halomonas were dominant particularly in the anaerobic fluidized bed. These results suggest that the microbial communities produced by different flow conditions correlated with denitrification ability in saline industrial wastewater treatment systems. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The genetic diversity and expression of amoA of autotrophic ammonia oxidizers in wastewater treatment processes were investigated by RT-PCR and denaturing gradient gel electrophoresis (DGGE) in order to identify active components of ammonia-oxidizer populations in a such processes. Ammonia oxidizers, evidenced by the presence of amoA mRNA, were regarded as metabolically active. The DGGE profiles derived from amoA mRNA and from its gene, which were amplified by RT-PCR or PCR using samples collected from a bench-scale reactor treating high concentration of inorganic ammonia, were similar. In contrast, RNA and DNA-derived DGGE profiles from three domestic wastewater treatment facilities were different from each other. These data indicate that the dominant ammonia oxidizers in the bench-scale reactor exhibited ammonia-oxidizing activity, whereas some ammonia oxidizers in the domestic wastewater treatment facilities apparently did not express high levels of amoA mRNA.

Investigation of the diversity of nirK and nirS in denitrifying bacteria revealed that salinity decreased the diversity in a nitrate-containing saline wastewater treatment system. The predominant nirS clone was related to nirS derived from marine bacteria, and the predominant. nirK clone was related to nirK of the genus Alcaligenes.

Rapid collection and removal of gram-negative heterotrophic bacterial strain, Escherichia coli, was investigated by utilizing the interfacial interaction between cell and solid substratum. Both E.coli collection from stable suspension and its successive removal were performed by a column bed packed with fibrous ferro-nickel slag, which was employed as the collector media of bacterial cells. In the cell collection tests, little cell recovery was obtained at neutral pH condition where both E.coli and FS had negative surface potential. On the other hand, E.coli collection gradually increased as the pH decreased. These findings were explained by the suppression of electrostatic repulsive interaction between E.coli and FS led to an improvement in cell attachment to FS surface. Cell collection capacity Γ_max exponentially increased as the interfacial interaction energy minimum V_min became lower, indicating that surface characteristics played crucial roles in cell attachment mechanism. Moreover, part of the E.coli cells that had adhered to FS surface were effectively removed when the eluting solutions were alkaline conditions; hence reversible cell detachment from FS was possible by controlling the electro-repulsive force. Cyclic E.coli collection/removal tests demonstrated that cell collection and successive removal were repeatedly carried out al least six times, although about 40 mg of E.coli cell made a firm and irreversible attachment on FS. E.coli collection/removal behaviors in the present experiments were generally in good agreement with electrokinetic properties of cell and FS, suggesting that cell recovery based on surface characteristics is a promising method, especially for stable bacterial suspension.

The dynamic transition of bacterial community structure in a biofilm was monitored by the fluorescence in situ hybridization (FISH) technique and subsequent image analysis. Heterotrbphic 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 K-m values for oxygen than AOB.

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.

Surface-modified hollow-fiber membranes were prepared by radiation-induced grafting of an epoxy-group-containing monomer, glycidylmethacrylate (GMA), onto a polyethylene-based fiber (PE-fiber). The epoxy ring of GMA was opened by introduction of diethylamine (DEA). The bacterial adhesivity to this material (DEA-fiber) was tested by immersion into a nitrifying bacterial suspension. The initial adhesion rates and the amount of attached bacteria of the DEA-fiber were 6-10-fold and 3-fold greater than those of the PE fiber, respectively. A membrane-aerated biofilm reactor (MABR) composed of DEA fibers was developed for partial nitrification with nitrite accumulation. Prior to the nitrification test, it was confirmed that the oxygen supply rate (OSR) was proportional to air pressure up to 100 kPa, allowing easy control of oxygen supply. Stable nitrite accumulation was observed in the partial nitrification test at a fixed oxygen supply throughout the operation period, indicating that oxygen was consumed only by ammonia oxidizers. Furthermore, it was demonstrated that oxygen utilization efficiency (OUE) in the ammonia oxidation process was nearly 100% after 300 h incubation.

Aims: To quantitatively analyse the changes to amoA mRNA (ammonia mono-oxygenase encoding mRNA) profiles in response to a change in ammonia oxidation activity in a complex microbial community.
Methods and Results: The amoA mRNA levels in both a batch-mode incubation and a continuously fed nitrification reactor were determined by real-time reverse transcription-PCR analysis. The amoA mRNA level changed rapidly in response to the change in environmental conditions which affect ammonia oxidation activity.
Conclusion: An increase in amoA mRNA level can be detected within 1-2 h in response to an initiation of cell activity whereas a decrease in amoA mRNA level is detected within 24 h in response to a cessation of activity.
Significance and Impact of the Study: amoA mRNA, which shows sensitive response to ammonia oxidation activity, can be used as a biomarker of ammonia oxidation activity in wastewater treatment processes where many bacterial species exist.

To establish an automatic control system of external carbon addition in biological nitrogen removal, a bench-scale sequencing batch reactor with real-time control strategy was designed in this study. An oxidation-reduction potential (ORP) profile as used for automatic control of external carbon addition. The mean removal efficiency of total organic carbon was over 98%. Complete denitrification in an anoxic phase and complete denitrification and nitrification in anoxic and oxic phases were accomplished, respectively, because the oxic and anoxic periods were also appropriately controlled with ORP and pH profiles, respectively. Mean removal efficiency of total nitrogen was over 95%. When concentration of influent wastewater was changed, volume of additional carbon was automatically changed with the influent, fluctuation, and flexible hydraulic retention time was achieved in this system.

An upflow anaerobic sludge blanket (UASB) reactor was successfully applied to continuous degradation of ferric ethylene diamine tetra acetate (Fe-EDTA) as a typical xenobiotic substance contained in photo-processing wastewater. The sludge in the UASB reactor had an abundance of sulfate-reducing bacteria (SRB), which had been anaerobically cultivated in a sulfate-rich culture medium including Fe-EDTA and yeast extract as the carbon sources. Since the prominent reductions of Fe-EDTA and sulfate ion were observed, the contribution of SRB to Fe-EDTA degradation in the UASB reactor was confirmed. The aggregated sludge in the UASB rector became gradually large, reaching steady state with an equivalent diameter of 60-90, μm after 92 days operation. An increase of the amount of yeast extract addition to feed solution, which contained 100 mg-C/l of Fe-EDTA, improved the Fe-EDTA removal efficiency up to 90%. Moreover, approximately 80% of intermediate compounds were mineralized by O₃/H₂O₂ oxidation process. When the combination of SRB treatment and O₃/H₂O₂ was applied to an actual fixing wastewater, approximately 80% of Fe-EDTA and 70% or more of dissolved organic carbon (DOC) in thefixing wastewater were degraded.

In this study, effects of phosphorus concentration on aquatic microcosms were evaluated in terms of microbial population dynamics and the photosynthetic activity that is the starting point of the material flux. Microcosms that consist of algae Chlorella vulgaris, bacteria Pseudomonas putida and ciliate Cyclidium glaucoma were incubated using media that contain various concentrations of KH₂PO₄. At 14-16 days after the incubation started, sodium [¹⁴C] bicarbonate was added to each microcosm, and then microbial uptake of radioactive substrate was monitored. The population density of C. uulgaris and C. glaucoma increased, as initial phosphorus concentration was higher, while that of P.putida was constant. The photosynthetic activity of C. vulgaris also increased, as initial phosphorus concentration becomes higher. In the microcosm, P. putida uses metabolites produced by C. vulgaris and is preyed by C. glaucoma. Therefore, the growth activity of P.putida increased as the photosynthetic activity of C. vulgaris increased, but predation pressure by C. glaucoma also increased. Consequently, the population of P. putida kept constant. These findings lead to the conclusion that phosphorus concentration strongly affects the microbial population dynamics and material flux in the microbial loop in the aquatic ecosystem. However, the mass balance of each organism should keep steady state in the environment where the population of predator such as protozoa, invertebrates, fish and so on was kept suitable.

The charcoal brick made from beer lees (CBBL) was investigated as the material for purification of lake water. The beer lees containing 67wt% water were first dried and then the dried beer lees were pressed at high temperature and pressure to make beer lees brick without any binder. The beer lees brick was carbonized in a low oxygen atmosphere to produce charcoal brick.<BR>The CBBL is useful for the carrier of microorganisms and it is possible to reduce soluble organisms and soluble nitrogen as same as charcoal. The CBBL contains phosphorus at the ratio of about 2wt%, which is soluble in water. The addition of 20wt% CaCO₃ to the dried beer lees in the pressing process is beneficial to prevent solubilization of phosphorus in the CBBL. The CBBL does not have a weak point that it is abased in the purification column compared with the charcoal used in usual water purification.

Expression of ammonia monooxygenase (amoA) gene in domestic wastewater treatment process was monitored by combination analysis of reverse transcription (RT) -PCR and denaturing gradient gel electrophoresis (DGGE) . Sludge samples were collected from a bench-scale reactor treating synthetic domestic wastewater. After total RNA was extracted, RT-PCR was performed. DGGE analysis was used to separate the amplified products according to their sequence and thereby to visualize individual community members. Ammonia oxidizers that were demonstrated by the presence of amoA mRNA were regarded as metabolically active ones. The DGGE profiles of amoA mRNA RT-PCR products obtained at the initial stage of the operation showed decrease of the diversity of amoA mRNA. When influent load was changed with reduction of hydraulic retention time (HRT) or increase of concentration of wastewater, new DGGE bands newly appeared on only the short HRT condition. The amoA sequences of DGGE bands disappeared in the beginning of the operation affiliated to the Nitrosomonas eutropha lineage. On the other hand, all the amoA sequences of new DGGE bands affiliated to the genus Nitrosomonas, and formed phylogenetic lineage for which no cultured representative exists.

Since nitrification is the rate-determining step in the biological nitrogen removal from wastewater, many research studies have been conducted on the immobilization of nitrifying bacteria. In this research, granulation of nitrifying bacteria in an aerobic upflow fluidized bed (AUFB) reactor in a nitrification process for inorganic wastewater containing 500 g/m(3) of NH(4)(+)-N was investigated. It was observed that spherical, pseudocubic and elliptical granules with a diameter of 346 mum were produced at the bottom of the reactor after 300 days. Denaturing gradient gel electrophoresis analysis revealed that Nitrosomonas-like bacteria were the dominant ammonia-oxidizing species in the granules. Many colonies of Nitrosomonas-like bacteria were found in the outer part of the granules based on the spatial distribution analysis by fluorescence in situ hybridization. By stepwise reduction of the hydraulic retention time, the ammonia removal rate of the AUFB reactor containing these nitrifying granules finally reached 1.5 kg-N/m(3)/day. Results suggested that the use of granules realizes the retention of a large amount of nitrifying bacteria in the reactor, which guarantees a highly efficient nitrification. (C) 2003 Elsevier Ltd. All rights reserved.

In this study, ammonia-oxidizing bacteria present in biofilms resulting from a nitrifying reactor were detected by both a conventional FISH technique and an original in situ PCR technique. Both techniques showed that ammonia-oxidizing bacteria were found near the surface of the biofilms. However, after the biofilm had been exposed to 2 weeks of ammonia starvation, ammonia-oxidizing bacteria present in the biofilm could not be detected by fluorescence in situ hybridization (FISH) because they did not have sufficient copies of rRNA. In contrast, ammonia-oxidizing bacteria could be detected by in situ PCR with strong signal. It was thus demonstrated that a cell possessing a specific functional gene is detectable by in situ PCR regardless of its activity. (C) 2003 Elsevier B.V. All rights reserved.

Recovery of heavy metal ions from their low concentrated water is developed using the activity of sulfate-reducing bacteria immobilized on a fibrous slag (FS), which was employed as both bio-carrier and particle collector medium. An up-flow type bioreactor was composed of FS packed column, where sulfate-reducing bacteria (SRB) was immobilized at a lower part. In this process, sulfate ion (SO₄^2-) in the liquid is biologically converted to hydrogen sulfide (H₂S), which reacts with metal ionic species to produce ultrafine metal sulfide particles. Subsequently, formed particles are collected onto the FS surface in the upper part of the reactor, thereby accumulating heavy metals ions as their sulfides precipitates. In the continuous treatment of water contaminated with 6 mg / l of cadmium, almost complete removal was carried out throughout the operating period, approximately 30 days. Cadmium sulfide precipitates gradually accumulated on the FS bed, and finally total amount of cadmium sulfide recovered reached 27.3 mg per unit gram of FS. The experimental result indicated that the proposed bioreactor, Sulfate-Reducing Bioreactor with Fibrous Slag carrier (SRB-FS), served as an efficient heavy metal recovery column with the aid of SRB.

Mass transfer of tetrachloroethylene (PCE) gas, followed by a free-OH radical reaction in the liquid phase, was studied in a bubble column reactor equipped with a UV light source and containing aqueous H2O2 as the reacting medium. Degradation of PCE in the liquid phase was found to follow pseudo-first-order kinetics, and the optimal ratio of H2O2/PCE leading to the highest oxidation rate could be successfully obtained from the analysis of the steady-state OH radical concentration. Absorption of PCE gas was in the kinetic slow regime and could be predicted based on the ratio of the apparent rate constant of PCE liquid degradation to the volumetric mass transfer coefficient (kapp/K(L)a). The presence of small amounts of PCE in the liquid bulk had positive effect on the mass transfer over the range studied and need to be, taken into account.

The influence of extracellular polymeric substances (EPS) on bacterial cell adhesion onto solid surfaces was investigated using 27 heterotrophic bacterial strains isolated from a wastewater treatment reactor. Cell adhesion onto glass beads was carried out by the packed-bed method and the results were discussed in terms of the amount of each EPS component produced and cell surface characteristics such as zeta potential and hydrophobicity. Protein and polysaccharides accounted for 75-89% of the EPS composition, indicating that they are the major EPS components. Among the polysaccharides, the amounts of hexose, hexosamine and ketose were relatively high in EPS-rich strains. For EPS-poor strains, the efficiency of cell adhesion onto glass beads increased as the absolute values of zeta potential decreased, suggesting that electrostatic interaction suppresses cell adhesion efficiency. On the other hand, the amounts of hexose and pentose exhibited good correlations with cell adhesiveness for EPS-rich strains, indicating that polymeric interaction due to the EPS covering on the cell surface promoted cell adhesion. It was concluded that, if the EPS amount is relatively small, cell adhesion onto solid surfaces is inhibited by electrostatic interaction, and if it is relatively large, cell adhesion is enhanced by polymeric interaction. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A porous hollow-fiber membrane containing an iminodiethanol (IDE) group as the chelate-forming group was applied to the recovery of antimony in the permeation mode. An antimony solution was forced to permeate through the pores of the chelating porous hollow-fiber membrane, driven by a transmembrane pressure. The membrane with a thickness of 0.7 mm and a porosity of 70% had an immodiethanol group of 1.6 mol/kg of the membrane and a water flux of 0.95 m/h at 0.1 MPa and 298 K. The breakthrough curves of antimony overlapped irrespective of the permeation rate of the antimony solution ranging from 2 to 20 ml/min, i.e. the residence time across the membrane thickness ranging from 3.4 to 0.34 s, because of negligible diffusional mass-transfer resistance of the ionic species of antimony to the iminodiethanol group. At antimony concentrations below 10 mg/l (pH 4.0), a linear adsorption isotherm was obtained. The adsorbed antimony was quantitatively eluted by permeation of 2 M hydrochloric acid through the pores of the membrane. (C) 2002 Elsevier Science B.V. All rights reserved.

Chlorinated volatile organic compounds (CVOCs) such as tri- and tetrachloroethylene (TCE and PCE) are common contaminants of ground water and soil. Numerous studies have been carried out with the long-term objective of the development of efficient, destructive on-site technologies for their removal. The so-called advanced oxidation processes (AOPs) were applied in the liquid and in the gas, but were shown to have limited application. In the liquid phase the efficiency was limited due to the presence of OH radical scavengers and UV light absorbers; and in the gas phase due to the production of stable intermediates.
A new photochemical reactor system is described, in which the polluted air (from the air stripper or SVE unit) is absorbed into a bubble column reactor equipped with the UV light (UV-BCR) containing only distilled water and H2O2 as a reacting medium. The experiments showed that the oxidation of model pollutant PCE in a liquid phase occurred approximately 6 times faster in an OH radical scavenger-free environment compared to the experiments in which the OH radical scavenger concentration was adjusted to a level usually found in ground waters. It was also observed, that for the certain PCE concentration, there exists an optimal hydrogen peroxide concentration above and below which the rate is reduced and could be predicted by the kinetic model under operational conditions of this work. For the experiments in which PCE gas was absorbed into the UV-BCR, the influences of the two critical parameters, gas flow rate and the hydrogen peroxide concentration, were investigated using the experimental design methodology. There has been observational evidence of the efficiency of the process (cca 75%-80% PCE gas removal efficiency in one flow through the UV-BCR) but the operational parameters still need to be optimized.

A membrane-aerated biofilm reactor (MABR) capable of simultaneous nitrification and denitrification in a single reactor vessel was developed to investigate the characteristics of nitrogen removal from high-strength nitrogenous wastewater, and biofilm analysis using microelectrodes and the fluorescence in situ hybridization (FISH) technique was performed. Mean removal percentages of total organic carbon (TOC) and nitrogen were 96% and 83% at removal rates of 5.76 g-C m(-2) d(-1) and 4.48 g-N m(-2) d(-1), respectively. For stable removal efficiency, constant washing of the biofilm was needed. Dissolved oxygen microelectrode measurement revealed that the biofilm thickness was about 1600 mum, and that oxygen penetrated about 300 to 700 mum from the outer surface of the membrane. Furthermore, FISH analysis revealed that ammonia-oxidizing bacteria (AOB) were located near the outer surface of the membrane, whereas other bacteria were located from the inner to the outer part of the biofilm. Combining these results demonstrated that simultaneous nitrification and denitrification occurred in the biofilm of the MABR system. In addition, stoichiometric analysis revealed that after 130 d the free ammonia (FA) concentration ranged within the concentration causing inhibition of the growth of nitrite oxidizing bacteria (NOB) and that AOB consumed 86% of the oxygen supplied through the intra-membrane. These results indicate that nitrogen removal not via nitrate but via nitrite was mainly achieved in the MABR system.

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor. (C) 2002 Elsevier Science B.V. All rights reserved.

The in situ fluorescent antibody and fluorescence in situ hybridization (FISH) methods are very useful in the in situ detection of specific bacteria like nitrifiers in a biofilm. In this study, simultaneous staining using the FISH and in situ fluorescent antibody methods was examined. As a result, no specific fluorescence was observed with either method when FISH was performed followed by the in situ fluorescent antibody method; however, when the in situ fluorescent antibody method was performed first followed by FISH, specific fluorescence was observed in both cases. Moreover, it was suggested that the detection specificities of FISH and the in situ fluorescent antibody method are almost identical.

Selective enrichment of phosphate-accumulating organisms (PAOs) and denitrifying phosphate-accumulating organisms (DNPAOs) was conducted using a sequencing batch reactor (SBR). To elucidate biomarkers for DNPAOs, quinone profiles were monitored during selective enrichment. As a result, a high correlation between the mole fraction of ubiquinone with eight isoprene units (Q-8) and anoxic phosphate uptake ability was observed, indicating that Q-8 was one of the biomarkers for DNPAOs. In addition, the mole ratio of ubiquinones to menaquinones (Q/MK) increased throughout this selective enrichment. © 2003, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

Nitrous oxide (N2O) is emitted from wastewater treatment processes, and is known to be a green house gas contributing to global warming. It is thus important to develop technology that can suppress N2O emission. The effects of sludge retention time (SRT) and dissolved oxygen (DO) on N2O emission in an anoxic-oxic activated sludge system were estimated. Moreover, the microbial community structure in the sludge, which plays an important role in N2O Suppression, was clarified based on nitrous oxide reductase (nosZ) gene analysis by molecular biological techniques. The results showed that under low SRT conditions, nitrification efficiency was reduced and the N2O emission rate in the oxic reactors was increased. It was also observed that N2O emission was enhanced under low DO conditions, where the available oxygen is insufficient for nitrification. Moreover, molecular analysis revealed that the clones identified in this study were closely related to Ralstonia eutropha and Paracoccus denitrificans. The fact that the identified sequences are not closely related to known culturable denitrifier nosZ sequences indicates a substantial in situ diversity of denitrifiers contributing to N2O suppression, which are not reflected in the cultivatable fraction of the population. The further application of these new molecular techniques should serve to enhance our knowledge of the microbial community of denitrifying bacteria contributing to N2O suppression in wastewater treatment systems.

An upflow anaerobic sludge blanket (UASB) reactor was successfully applied to continuous degradation of ferric ethylene diamine tetraacetate (Fe-EDTA) as a typical xenobiotic substance contained in photo-processing wastewater. The sludge in the UASB reactor had an abundance of sulfate-reducing bacteria (SRB), which had been anaerobically cultivated in a sulfate-rich culture medium including Fe-EDTA and yeast-extract as the carbon sources. Since the prominent reductions of Fe-EDTA and sulfate ion were observed, the contribution of SRB to Fe-EDTA degradation in the UASB reactor was confirmed. The aggregated sludge in the UASB reactor became gradually large reaching steady state with an equivalent diameter of 60-90 mum after 124 days operation. An increase of the amount of yeast extract addition to feed solution improved the Fe-EDTA removal efficiency up to 90%. Moreover, the combination of ozone treatment with SRB treatment further improved removal efficiency of total organic carbon (TOC) in an actual photo-processing wastewater composed of fixing and developing wastes.

Selective cultivation of denitrifying phosphate-accumulating organisms (DNPAOs) was conducted using a sequencing batch reactor (SBR) under different electron acceptor conditions. A new biological nutrient removal process configured with anaerobic/aerobic/anoxic conditions was successfully operated in a single SBR. The supplementation of carbon substrate at the start of the aerobic condition was effective for the simultaneous removal of phosphorus and nitrate by DNPAOs.

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors. (C) 2002 Wiley Periodicals, Inc.

Step changes in inlet concentration has been introduced into the completely mixed three-phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the dynamic behavior of the system and to establish the suitable kinetic model from the response curve. Three identical reactors having different biomass volumes were operated in parallel. It was found that the response curves showed second-order characteristics, and thus at least two first-order differential equations are necessary to simulate the substrate and biomass response curves. Nonlinear regression analysis was performed using different types of rate equations and their corresponding kinetic parameters were used to simulate the theoretical response curve using the Runge-Kutta numerical integration method. As a result, although various types of conventional biokinetic models such as Monod, Haldane and Andrew types were examined, all the theoretical substrate response curves underestimated time constants compared to the actual substrate response plots. On the other hand, the theoretical curve of the kinetic model that incorporates adsorption term has best fit to the actual response in most of the cases. Thus, it was concluded that adsorption of substrate onto biofilm and carrier particles has significant effect on he dynamic response in biofilm processes. (C) 2002 Elsevier Science B.V. All rights reserved.

A novel technique of immobilizing nitrifying bacteria using fibrous material, the three-dimensional immobilization with a fibrous network (3-D IFN), is proposed and its use in a high-rate nitrifying bioreactor is investigated. The fibrous carrier employed is ferro-nickel fibrous slag (FS), which is industrial solid waste from a ferro-nickel electrosmelting process. Since cell immobilization with FS is readily carried out within 90 seconds, this method is by far more rapid than any other cell immobilization techniques. A fixed-bed nitrifying reactor packed with cell-immobilizing FS is examined by both batch-mode and continuous feeding tests. By controlling the circulation flow rate, the transport of dissolved oxygen to the immobilized cells is improved, which is a distinct characteristic of this reactor. The continuous feeding test revealed that the ammonia removal rate of the reactor reached 6.5 kg-N/(m(3)-reactor)/d, which was extremely high compared with that of the conventional fixed-bed reactor for wastewater treatment. It is demonstrated that the 3-D IFN is a simple yet effective cell immobilization technique that can be successfully used in a high-rate nitrifying reactor.

To investigate the characteristics and the microbial diversity of denitrifying phosphate-accumulating organisms (DNPAOs) that are capable of conducting enhanced biological phosphorus removal (EBPR) using nitrate as electron acceptor, three sequencing batch reactors were operated under three different electron acceptor conditions, i.e., only oxygen, oxygen together with nitrate and only nitrate. Based on the chemical analysis concerning the biochemical transformation of each reactor, it was found that phosphate-accumulating organisms responsible for EBPR consisted of at least three populations including DNPAOs, and that the microbial community structure was changed according to the electron acceptor conditions. Also, the sludge cultivated with oxygen together with nitrate showed a drastic increase in the amount of phosphorus uptake under anoxic conditions, which suggested that a proportion of DNPAOs capable of utilizing nitrate under aerobic conditions were present.
On the other hand, the change in microbial community structure depending on the type of electron acceptor was demonstrated by the analysis of the results of denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal DNA fragments. It was found that the bacteria commonly contained in all the reactors were Rhodocyclus sp. (96% identity) and Dechlorimonas sp. (97% identity) that belonged to the beta subclass of Proteobacteria, on the basis of the analysis of the sequence excised from DGGE bands and the determination of phylogenetic affiliation. However, only the presence of Rhodocyclus sp. in all the reactors was demonstrated by fluorescent in situ hybridization analysis. (C) 2002 Elsevier Science Ltd. All rights reserved.

Ammonia monooxygenase encoding mRNA (amoA mRNA) transcription in the wastewater treatment process was investigated using reverse transcription PCR (RT-PCR) as the model indicating specific function and activity in nitrifying processes. The dynamic response of amoA mRNA transcription and ammonia-oxidizing activity to the change of environmental conditions such as pH and concentration of ammonia was examined to determine the inductive factor and the inhibitor for amoA mRNA expression. Furthermore, we semiquantitatively investigated the response of amoA mRNA transcription to the pH fluctuation in a continuous fed nitrifying reactor. AS a result, amoA mRNA oriented analysis enabled real-time assay of ammonia-oxidizing activity within 2 h as a response time. In contrast, rRNA and amoA encoding DNA were constantly detected at almost the same amount throughout the experiment. mRNA transcription was regulated by the many environmental conditions: ammonia seems to be one of the strong inducers for transcription of amoA mRNA, whereas low pH seems to be a strong inhibitor. These factors simultaneously affected the mRNA transcription and enzymatic activity leading to the complex phenomena of ammonia-oxidizing activity and amoA mRNA transcription in the continuous feeding reactors.

The wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of acids such as nitric acid and of salts. Biological nitrogen removal from this wastewater was attempted by using a circulating bioreactor system equipped with an anoxic packed bed or an anoxic fluidized bed and an aerobic three-phase fluidized bed. The system was found to effectively remove nitrogen from the diluted wastewater (T-N; 1,000-4,000 mg litre(-1)). The microbial population structure of activated sludge in an anoxic reactor was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. DGGE analysis under different operating conditions demonstrated the presence of some distinguishable bands in the separation pattern, which were most likely derived from many different species constituting the microbial communities. Furthermore, the population diversity varied in accordance with the nitrate-loading rate, water temperature and reactor condition. Some major DGGE bands were excised, reamplified and directly sequenced. It was revealed that the dominant population in the anoxic reactor were affiliated with the beta subclass of the class Proteobacteria.

Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria is an important step in the biological nitrogen removal process. The first conversion step, the oxidation of ammonia to hydroxylamine is catalyzed by ammonia monooxygenase (AMO). To investigate the activity of ammonia oxidation, mRNA (designated as amoA) encoding a subunit of AMO was quantified by competitive reverse transcription (RT)-PCR. As a result, it was possible to detect and quantify amoA expression in cultured Nitrosomonas europaea and even complex microbial communities such as nitrifying bacterial aggregates by competitive RT-PCR. It was estimated that amoA concentration in cultured N. europaea was 2.3 x 10(8) copies.ml(-1). Additionally, it was calculated that the copy number of amoA in nitrifying bacterial aggregates was 1.0 x 10(12) copies.ml(-1) (5.1 x 10(10) copies.mg(-1).dry weight). On the other hand, amoA expression in the natural activated sludge in a household Gappei-Johkaso was undetectable, whereas 16S rRNA of ammonia-oxidizing bacteria was detected by RT-PCR. Then, four days cultivation of this sludge in inorganic artificial wastewater resulted in increasing amoA expression to a quantifiable amount by competitive RT-PCR. In conclusion, the competitive RT-PCR was effective to investigate the expression of amoA as an indicator of ammonia oxidation activity by autotrophic ammonia-oxidizing bacteria.

UV-H2O2 process is widely used as an advanced oxidation process (AOP) for the treatment of chlorine volatile organic compounds (CVOCs) such as dichloromethane (DCM) with strong oxidativity of hydroxyl radical generated from photolysis of H2O2. The result of DCM degradation rate at different initial concentrations in UV-H2O2 processes indicated the inhibition effect of produced chlorine ions on DCM oxidation processes, because the first-order degradation rate constant increased with lower initial concentrations. A spin trapping adduct of hydroxyl radical with 5,5-dimethyl-1-pyrroline-n-oxide (DMPO) was quantified by ESR spectrometer after UV irradiation in the presence of different amounts of chlorine ion, and as a result, the chlorine ion was found to act as a hydroxyl radical scavenger, which resulted in decreasing DCM degradation rate. An UV-H2O2 reactor equipped with ion exchangers for removing chlorine ion achieved higher DCM degradation rate than that without ion exchangers.

Fluorescent in situ hybridization (FISH) method with 16S rRNA-targeted oligonucleotide probes was used for quantitative estimation of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in a Johkasou. Although the occupation ratios of AOB and NOB increased as nitrification progressed, about one month later, the occupation ratios decreased, despite showing good nitrification ability. Furthermore, even when urea was added to the feeding wastewater to raise the amount of T-N, the occupation ratios of both nitrifying bacteria remained constant. For further investigation, denaturing gradient gel electrophoresis (DGGE) was used to study the community structure of AOB in the Johkasou. As a result, DGGE band patterns and following sequence analysis revealed that the community structure of AOB was complicated and changed during this experiment. It was suggested that even if the occupation ratio of AOB to eubacteria was constant, the majorities of AOB were changed through temperature and load fluctuation. The combination of FISH and PCR-DGGE provides new information that was not available by conventional cultivation based methods.

The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds such as ammonia and nitric acid and of salts such as sodium chloride and sodium sulfate. Biological nitrogen removal from this wastewater was attempted by a circulating bioreactor system equipped with an anoxic packed bed and an aerobic fluidized bed. The anoxic packed bed of this system was found to effectively remove nitrite and nitrate from the wastewater by denitrification at a removal ratio of 97%. As a result of denitrification activity tests at various NaCl concentrations, the sludge obtained from the anoxic packed bed exhibited accumulation of nitrite at 5.0 and 8.4% NaCl concentrations, suggesting that the reduction of nitrite is the key step in the denitrification pathway under hypersaline conditions. The microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments revealed that the community diversity varied in accordance with water temperature, nitrate-loading rate and ionic strength. When particular major DGGE bands were excised, reamplified and directly sequenced, the dominant species in the anoxic packed bed were affiliated with the beta and gamma subclasses of the class Proteobacteria such as Alcaligenes defragrans and Pseudomonas spp., respectively.

A sequencing batch reactor under different electron acceptor conditions was operated serially to investigate the selection and dominance mechanisms of denitrifying phosphate-accumulating organisms (DNPAOs) in a biological nutrient removal process. The presence of a small amount of NO3- at the start of the anaerobic phase stimulated the selection of DNPAOs in an anaerobic/aerobic system, and switching O-2 to NO3- as an electron acceptor enhanced the activity of anoxic phosphate uptake.

The UV/H2O2 process is commonly used for the remediation of drinking and groundwater pollution with chlorinated volatile organic compounds. In direct treatment, its efficiency can be lowered in the presence of radical scavengers and/or UV light absorbers. This research is focused on the improvement of the UV/H2O2 photolytic process, due to the reduction of OH radical scavengers and UV absorbers in the reacting system. Degradation of tetrachloroethylene (PCE) gas, which was absorbed into a bubble column reactor equipped with UV light (UV-BCR), containing distilled water and H2O2, as the reacting medium, was studied in a one flow-through mode. Degradation of PCE in the liquid phase was found to follow pseudo-first-order kinetics and apparent rate constants in the order of 0.02 s(-1) have been observed. An absorption-reaction model based on slow reaction in the bulk liquid was proposed, and it fitted the experimental data reasonably well. However, when sequence making internal (designed as to prolong the gas pathway through the reactor) was in the system in addition to the relatively high PCE concentrations (500 ppm), production of the byproduct chloroform was observed, indicating that some reaction might have occurred in the gas phase. The addition of term for a reaction in the gas phase was able to account for the reaction due to direct absorption of PCE. (C) 2001 Elsevier Science Ltd. All rights reserved.

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.

Total organic carbon (TOC) removals of two synthetic wastewaters which contain diethylene glycol mono-n-hexyl ether (DIGME) and 1,4-butanediol diglycidyl ether (1,4-BDE) respectively as a main component, have been carried out by using hydrogen peroxide and ultraviolet irradiation (H2O2/UV). Experiments were carried out in a batch reactor with a low pressure UV lamp (500 W) irradiating ultraviolet at 254 nm and at 185 nm (5% of energy). The following results were obtained. (1) The complete TOC removals of the two synthetic wastewaters have been obtained. TOC removals of the two synthetic wastewaters are pseudo-first-order processes. (2) The removal rates of TOC of the two synthetic wastewaters were governed by the concentration of hydrogen peroxide added initially and the maximum pseudo-first-order rate constant and the optimum initial hydrogen peroxide concentration existed both in the two synthetic wastewaters. (3) The relation between the initial TOC concentration and the optimum initial hydrogen peroxide concentration exhibited linear in both synthetic wastewaters. The optimum initial hydrogen peroxide concentration in the TOC removal of the 1,4-BDE synthetic wastewater is higher than that of the DGME synthetic wastewater at the given initial TOC concentration. (4) The maximum pseudo-first-order rate constant increases with the decrease in the initial TOC concentration in both synthetic wastewaters. The maximum pseudo-first-order rate constant for the DGME synthetic wastewater is higher than that for the 1,4-BDE synthetic wastewater at the given initial TOC concentration. (5) The experimental results agree well with the theoretical consideration which has been previously proposed by authors (Hou et al., 2001c) while hydrogen peroxide concentration and TOC concentration in the wastewater were very high.

Total organic carbon (TOC) removals of the synthetic wastewater and of the raw industrial wastewater discharged from LSI photo-resist processing of which main component is 1,2-naphthoquinone-2-diazido-5-sulfonic acid sodium salt (abbreviation naphthoquinone-5), have been carried out by using hydrogen peroxide and ultraviolet irradiation (H2O2/UV). Experiments were carried out in a batch reactor with a low pressure UV lamp (500 W) irradiating ultraviolet of 254 nm and of 185 nm (5% of energy). The following results were obtained. (1) TOC removals of the synthetic wastewater and of the raw industrial wastewater are pseudo-first-order processes, and TOC shows zero in about ten and several hours. (2) The TOC removal rate of the wastewater was governed by the concentration of hydrogen peroxide initially added and by the maximum pseudo-first-order rate constant and the optimum initial hydrogen peroxide concentration both existed in the synthetic wastewater and in the raw industrial wastewater. (3) The relation between the initial TOC concentration and the optimum initial hydrogen peroxide concentration exhibited linear both in the synthetic wastewater and in the raw industrial wastewater. The optimum initial hydrogen peroxide concentration in TOC removal of the raw industrial wastewater is same as that in the synthetic wastewater at the given initial TOC concentration. (4) The maximum pseudo-first-order rate constant increases with the decrease in the initial TOC concentration both in the synthetic wastewater and in the raw industrial wastewater. (5) A theoretical consideration was carried out and could explain with experimental results.

2,4,5-Trichlorophenol (2,4,5-TCP) is one of the most toxic chlorophenols (CPs) and is very difficult to be biodegraded. The establishment of an appropriate treatment which is friendly to the earth environment for 2,4,5-TCP is an urgent matter to be solved. Total organic carbon (TOC) removal of the synthetic wastewater which contains 2,4,5-TCP at TOC of 405 g/m(3) has been carried out by using hydrogen peroxide and ultraviolet irradiation (H2O2/UV). Experiments were carried out in a batch reactor with a low pressure UV lamp (500 W) irradiating ultraviolet at 254 nm and at 185 nm (5%). The chemical compounds included in the synthetic wastewater have been completely removed in the presence of hydrogen peroxide. It was found that the optimum concentration of hydrogen peroxide, which was added initially, exists.

Structural examinations of a biofilm immobilized on small cement ball (CB) particles in a three-phase fluidized bed reactor for a highly concentrated nitrification process were carried out. With transmission and scanning electron microscopes, a diverse microbial arrangement was determined. The scanning electron micrographs showed that a large number of clustering bacteria was covered by extracellular polymers. Between the clusters, many open spaces were found. The transmission electron micrographs showed a regular arrangement of bacteria inside the biofilm. Most clusters of Nitrosomonas sp.-like bacteria were found near the surface of the biomm and the majority of Nitrobacter sp.-like bacteria were observed inside the biofilm. At least 14 types of bacteria were found in the mixed culture nitrifying biofilms.

The raw industrial wastewater from LSI photo-resist processing which contains 1,2-naphthoquinone-2-diazido-5-sulfonic acid sodium salt as a main component with high NaCl concentration (&gt;20 kg/m(3)), is very difficult to treat, The establishment of an appropriate treatment, which is friendly to the earth's environment for raw industrial wastewater is an urgent matter to be solved. Total organic carbon (TOC) removal of the raw industrial wastewater, which contains TOC at 8000 g-TOC/m(3), by using hydrogen peroxide and ultraviolet irradiation (H2O2/UV), has been carried out. Experiments were carried out in a batch reactor with a low pressure UV lamp (500 W) irradiating ultraviolet at 254 nm and at 185 nm (5 %), The chemical compounds included in the raw industrial wastewater have been removed completely in the presence of initial concentration of hydrogen peroxide of two times the stoichiometric ratio.

The possibility of enhancing nitrifying biofilm formation rate with the aid of selected EPS produced by heterotrophic bacteria was investigated. When EPS production characteristics were examined for four kinds of heterotrophs isolated from a domestic wastewater treatment reactor, two strains obtained from biofilms (B1, B2) exhibited a higher polysaccharide production rate than those from suspended flocs (A1, A2). Among EPS components, the concentration of uronic acids gave a good correlation with flocculation ability, which suggests that acidic polysaccharides play a major role in bioaggregate formation. Addition of 1g/L D-glucuronic acid as an EPS substitute enhanced the homocoagulation rate of autotrophic Nitrosomonas europaea and altered its zeta potential from (n) over tilde 30.4mV to + 4.3mV, which indicates a possibility that particular EPS components produced by heterotrophs are utilized as neutralising reagents for nitrifying biofilm formation. Moreover, when heterotrophic isolates with Nitrobacter winogradskyi were cultured in batch with fabric supports, biofilm formed on the substratum, These experimental results suggest the application of selected EPS for enhancing nitrifying biofilm formation.

The electrokinetic properties of two nitrifying strains, Nitrosomonas europaea and Nitrobacter winogradskyi, and three heterotrophic bacteria, Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa, were examined by electrophoretic mobility measurement and analyzed using the soft particle electrophoresis theory that is suitable for biological particles. The bacterial adhesion characteristics onto glass bead substratum were also evaluated by packed bed method. The mobility of the bacterial cells employed converged to a non-zero value as the ionic concentration increased, suggesting that the bacterial cells exhibited typical soft particle characteristics. Moreover, cell surface potentials based on the soft particle theory were lower than those estimated by the conventional Smoluchowski formula, i.e. zeta potential. Cell collision efficiencies onto glass beads (α0) were largely dependent on interfacial interaction, although almost electrically neutral P. aeruginosa did not follow that trend. From a comparison of α0 with DLVO interaction energy maximum (Vmax), it was assumed that heterocoagulation between cell and substratum at primary minimum potential took place under Vmax of 24-34 kT based on soft particle analysis. On the other hand, Vmax predictions using the Smoluchowski theory gave 81-223 kT, which indicated the possibility of overestimating electrostatic repulsive forces by the conventional Smoluchowski theory. Thus, the application of this new electrophoresis theory to several kinds of bacterial cells has led to the revision of the interpretation of bacterial mobility data and provided a more detailed understanding of the bacterial adhesion phenomenon. Copyright © 2001 Elsevier Science B.V.

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/m(3)), such that the total nitrogen concentration in the effluent met the sewage discharge control criteria in Japan (240 g/m(3)). 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.

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 γ-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 γ-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.

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.

The effects of oxygen supply conditions and specific biofilm interfacial area on the phenol removal rate in a three-phase fluidized bed bioreactor were evaluated. the experimental data were well-explained by the semi-theoretical equation based on the assumption that the reaction rate follows first-order reaction kinetics with respect to oxygen and zero-order one with respect to phenol. Two cases; biological reaction as rate-controlling step and oxygen absorption as rate-controlling step, were both explicable by this semi-theoretical equation. The maximum volumetric phenol removal rate was 27.4 kg.m(-3) d(-1).

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO 14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. whogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. it was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

Monoclonal antibodies against the two kinds of nitrifying bacteria Nitrosomonas europaea (IFO14298) and Nitrobacter winogradskyi (IFO14297) were raised and isotypes of these monoclonal antibodies, IgM and IgG(1), were successfully obtained. Cross reactivities of these monoclonal antibodies against various kinds of representative heterotrophic bacteria turned out to be relatively low by competitive ELISA. In contrast, these monoclonal antibodies were very specific for nitrifying bacteria used as antigens. By means of sandwich ELISA using different isotype monoclonal antibodies such as IgM and IgG(1), calibration curves were successfully developed for quantification of nitrifying bacteria. It was shown that the obtainable lower limit of quantification of N. europaea and N. winogradskyi were 7.0 x 10(6) N/ml and were 6.0 x 10(5) N/ml, respectively. Nitrifying bacteria in activated sludge of advanced domestic wastewater treatment johkaso were counted by sandwich ELISA and MPN methods. The bacterial number estimated by MPN method was lower than that estimated by sandwich ELISA, It was indicated that this monoclonal antibody method could be used asa quick and powerful tool for estimating and controlling the population of nitrifying bacteria in the advanced domestic wastewater treatment processes.

We proposed two tailoring methods for efficient nitrifying biofilms on particles which are expected to be used in fluidized bed in nitrogen removal processes for industrial wastewaters. The first method was examined with gradual reduction of the hydraulic retention time in continuous feeding reactor to form biofilm with high nitrification ability. As a result, nitrification rate was successfully improved mainly due to acclimation of nitrifying bacteria to higher loading. The second tailoring method for nitrifying biofilm started with the biofilm which had been previously constructed in synthetic domestic wastewater containing high concentration of NH4+-N as well as various biodegradable organic compounds. Stepwise reduction of CIN ratio in inlet wastewater was performed during one month simultaneously with observation of microbial population dynamics in the biofilm using fluorescent in situ hybridization (FISH) analysis. As a result, this acclimation process promoted occupation of the biofilm by ammonia-oxidizing bacteria and resulted in making suitable biofilm structure for nitrification of ammonia-rich industrial wastewater. Moreover, it is confirmed that this new tailoring method greatly shortened required time to obtain nitrifying biofilms.

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.

Degradation kinetics of dichloromethane in water were investigated in a rectangular quartz cell under combination of UV irradiation and hydrogen peroxide (H2O2) addition. The presence of H2O2 not only enhanced the degradation rate of dichloromethane but prevented the formation of other chlorinated organic compounds as transient intermediates to mineralization. Since H2O2 was consumed by UV at a much higher rate than dichloromethane, it was found that the consecutive addition of H2O2 was highly effective to maintain high-rate degradation of dichloromethane. When 2,000 mg·l-1 of H2O2 was added every 5 min, the decomposition rate of 1,500 mg·l-1 dichloromethane exhibited 9.3 times higher than that of one-time addition of H2O2. Moreover, we successfully demonstrated that the degradation rate of dichloromethane was almost proportional to the square of incident light intensity.

We have described a preparation scheme for immobilizing polymer chains at a uniformly high density onto a microfiltration membrane. Highly efficient protein recovery was demonstrated by the results of the determination of breakthrough and elution curves. The three requirements of high rare, high capacity, and repeated use for the protein recovery were satisfied by ensuring the occurrence of convection, multilayer binding, and hydrophilization, respectively. In addition, easy scale-up to fabrication of a membrane module was verified on a small scale. (C) 1999 Published by Elsevier Science Ltd, All rights reserved.

We investigated an effective pretreatment method for a polycrystalline gold film surface on which an ordered self-assembled monolayer (SAM) could form. The wetting properties of the SAM surface determined by the hysteresis between advancing and receding contact angles against water and hexadecane revealed that the ordered SAM was formed on the gold surface pretreated with a combination of UV/ozone exposure and ethanol rinsing prior to SAM formation. The change in optical properties of the gold film surface was observed by surface plasmon spectroscopy (SPS), indicating that pure ethanol rinsing makes the gold surface smooth by removing small gold grains oxidized by UV/ozone exposure. (C) 1999 Elsevier Science S.A. All rights reserved.

We have measured high-resolution X-ray photoelectron spectroscopy (XPS) spectra of 1-octadecanethiol (ODT) self-assembled monolayers (SAMs) on a Au(111) surface in order to investigate the Au-S binding properties at the initial stage of SAM growth and after the desorption of the ODT from the Au surface. For the SAM prepared by the 1 min immersion into the ODT solution, we found new sulfur species around 161 eV and assigned it to isolated sulfur without C-S cleavage. We also confirmed the presence of a similar 161 eV peak in the S(2p) spectra after desorption of the ODT molecules. Furthermore, we observed a peak shift in the carbon 1s (C(1s)) peak, depending on the surface coverage of the ODT. In addition to the C(1s) peak of 285.1 eV which might correspond to alkyl chains of densely packed ODT molecules, another lower binding energy peak at 284.3 eV appeared after annealing. This lower C(1s) binding energy peak formation suggested that some of the alkyl chains for the remaining ODT molecules might be oriented parallel to the Au surface after annealing.

The fluorescence probe technique was used to investigate the characteristics of an amino group-containing polymer chain grafted onto a polyethylene microfiltration (MF) membrane. The amino group-containing polymer chain labeled with a dansyl group that served as a fluorescence probe was grafted onto a polyethylene MF membrane by radiation-induced graft polymerization. The conformational changes of the grafted polymer chain (graft chain) in various solvents were monitored by considering that the steady-state fluorescence emission spectrum of the dansyl group was affected by the polarity of the solvent, the polyethylene, and the graft chain itself. The shift of the emission peak wavelength of graft chains with different lengths demonstrated that the graft chain containing amino groups stretched in water and methanol and shrank in dimethylformamide, acetone, and benzene. These results corresponded to changes in solvent permeability through the membrane pore to which the amino group-containing polymer chains were grafted.

Phenolic wastewater was treated in complete mixing type three-phase fluidized bed which contained both biofilm and suspended sludge. By considering the contributions of biofilm and suspended sludge to biodegradation of phenol separately, phenol removal rates with biofilm and with suspended sludge were evaluated both theoretically and experimentally. As a result, biodegradation of phenolic wastewater by biofilm process could be treated as a zero-order reaction. The volumetric biological removal rates with biofilm was proportional to the specific surface area of the biofilm with biodegradation coefficients (K) of 1.25x10(-2) kg-PhOH/m(2)-biofilm/d At a lower suspended sludge concentration, bioparticle diameter and bioparticle holdup in the three-phase fluidized bed reactor were decisive factors for the efficiency of phenol treatment, and it was also proved that almost 100 % of phenol removal could be attained at a larger specific biofilm surface area per volumetric phenol loading rate than 80 m(2)/(kg-PhOH/d) without suspended sludge. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.

The surface-conditioning effect of gold substrates on n-octadecanethiol (ODT) self-assembled monolayer (SAM) growth was studied by X-ray photoelectron spectroscopy (XPS). The gold substrates were immersed in an ethanol solution containing 1 mM ODT for 1 h. The C/Au ratios of the ODT SAMs estimated by XPS formed on gold substrates depended on the amount of contamination on the gold surface. A full-coverage ODT SAM was successfully obtained on the gold substrate after ultraviolet light (UV)/ozone treatment for 1 h and rinsing with pure ethanol for 1 h. XPS measurements indicated that low contamination levels and a sulfur-free gold surface were obtained using this pretreatment and, hence, the adsorption of ODT molecules on gold proceeded efficiently. On the other hand, the C/Au ratio of an ODT SAM on a contaminated gold substrate was less following immersion in an ODT solution for 1 h, because surface contamination may reduce the adsorption rate of ODT molecules on the gold surface. When the gold substrate was rinsed with a chloroform solution for 1 h before SAM formation, the lowest C/Au ratio was obtained. XPS results showed that sulfur atoms quickly adsorbed onto the gold surface after chloroform rinsing and prevented the adsorption of ODT molecules onto the gold surface.

The fluorescence depolarization method was used for investigating the local mobility of polymer chains grafted onto a porous polyethylene membrane. The real value of the rotational diffusion coefficient of a dansyl probe attached to the grafted polymer chain was obtained by using a correction method which eliminated the effect of multiple scattering on fluorescence anisotropy, The rotational mobility of the dansyl probe attached to the grafted polymer chain was sensitive to both degree of grafting and solvent polarity, which indicated that the conformation of the grafted polymer chain and the pore size of the base membrane strongly governed the dynamic parameters of the grafted polymer chain. (C) 1997 Elsevier Science B.V.

Two types of anaerobic-aerobic biofilm processes were applied to the treatment of the photographic processing wastewater. Two-phase fixed bed reactor packed with sponge cubic media and completely mixing three-phase fluidized bed reactor, respectively, were used as an anaerobic and aerobic biofilm reactors. One of the aerobic biofilm reactors was packed with cement balls (CB) made by crushed cement particles, and another packed with biological activated carbon (BAC). The fivefold diluted photographic processing wastewater, from which Ag had been removed previously, was used as an influent (BOD 5,700 g/m(3), CODcr 17,000 g/m(3), T-N 2,600 g/m(3)). During long-term continuous biological treatment, BOD values in effluent decreased gradually and reached 280 g/m(3), which could fulfill the sewage discharge control value in Japan (BOD &lt; 600 g/m(3)). It took more than one year to acclimatize the sludge and to get the effective microorganisms for degrading the compounds in the photographic processing wastewater. However, pH values in the aerobic biofilm reactors fell down to 3 similar to 4. This was possibly because thiosulfate (5,700 g/m(3)) in the photographic processing wastewater was almost oxidized to sulfate by sulfur-oxidizing bacteria. For the purpose of obtaining higher BOD removal efficiency, pH in the aerobic biofilm reactor was adjusted to 7 using pH controller. As a result, BOD removal ratio was gradually improved, and the sewage discharge control value was steadily achieved after 181 days. The number of bacteria in the anaerobic biofilm reactor and the aerobic biofilm reactor with pH controller were 6.0 x 10(9) N/mL and 1.1 x 10(8) N/mL, respectively. (C) 1997 IAWQ. Published by Elsevier Science Ltd.

Growth kinetics of self-assembled monolayers (SAMs) of n-alkanethiols on gold substrates was studied by X-ray photoelectron spectroscopy (XPS). We compared adsorption kinetics for three alkanethiol homologues: butanethiol (C4SH), dodecanethiol (C12SH), and octadecanethiol (C18SH). Results of-quantitative analysis by XPS suggested that the adsorption rate of short alkyl chain thiols is higher than that of long ones on gold substrates at the initial growth stage, in contrast to previous reports. For C4SH SAM formation, we have also confirmed the occurrence of a replacement effect from contamination to SAM on gold, based on our observation of a decrease in the amount of carbon and an increase in the amount of gold where as determined from XPS spectra.

An epoxy-group-containing polymer chain was grafted onto a porous polyethylene membrane of hollow-fiber form by applying radiation-induced graft polymerization, The produced epoxide was converted into a diol group, ion-exchange (diethylamino and sulfonic acid) and chelate-forming (ethylenediamine and iminodiacetic acid) groups, and pseudoaffinity ligands (phenylalanine and tryptophan), The conversion of the epoxide into the functional group was investigated as a function of the degree of GMA grafting and molecular weight of the reactants. A reactant with low molecular weight exhibited a high conversion. The high conversion of the epoxide into the ion-exchange group caused the extension of the polymer chain grafted onto the pore interface and decreased the water permeability through the hollow-fiber membrane.

A porous hollow-fiber membrane was modified with diethylamino (DEA) and ethanolamino (EA) groups by radiation-induced graft polymerization and subsequent chemical modifications. Adsorption behavior of bovine serum albumin (BSA) was investigated by permeating a BSA buffer solution through the membrane. A polymer chain grafted onto the pore surface of the membrane held BSA in multilayers, i.e., three-dimensionally in a tentacle-like manner. The membrane with a DEA group density of 2.5 mol/kg had a binding capacity for BSA of 443 g/kg, equivalent to 11 times the monolayer adsorption. Permeation of a buffer solution containing 0.5 M NaCl as an eluent caused the graft chain to shrink, resulting in restricted diffusion of BSA departing from the graft chain toward the pore interior while realizing a quantitative elution. Hydrodynamic analysis of the change in permeation pressure accompanying BSA binding to the graft chain showed that the BSA molecules layered in the graft chains form a closely packed structure. (C) 1995 Academic Press, Inc.

Novel ion-exchange membranes containing sulfonic acid (SO3H) and trimethyl ammonium [N(CH3)(3)] groups were prepared by a simple method of radiation-induced cografting of sodium styrenesulfonate (SSS) with acrylic acid (AAc) and vinyl benzyl trimethyl ammonium chloride (VBTAC) with 2-hydroxyethyl methacrylate (HEMA), onto a polyethylene film with a thickness of 50 mu m. The high density graft chain was introduced throughout the polyethylene film. The maximum cation- and anion-exchange capacities of the resultant membranes were 2.5 and 1.3 mol/kg, respectively. These membranes exhibited an electrical resistance one order lower than commercially available ion-exchange membranes; for example, 12 h cografting provided cation- and anion-exchange membranes whose electrical resistances in a 0.5 M NaCl solution were 0.25 and 0.85 Omega cm(2) respectively. From the evaluation of electrodialytic desalination in a batch mode, using a pair of the graft-type ion-exchange membranes, the time required to achieve 99.5% desalination of the initial 0.5 M NaCl solution was reduced to 85% comparing with that of the commercial ion-exchange membranes.

A polymer chain containing a diethylamino group was grafted onto the pore surface of a porous hollow-fiber membrane by radiation-induced graft polymerization. Dependence of the protein binding capacity of the membrane on environmental parameters such as salt concentration, pH and temperature was investigated. Saturation capacity of protein bound onto the graft chain containing ion-exchange group was governed by the conformation of the graft chain and the intensity of ion-exchange interaction. The conformation of the graft chain was investigated based on the pore radius of the membrane estimated from the permeation flux of a buffer solution through the membrane. By sufficiently permeating a bovine serum albumin (BSA) solution within the concentration range of 0.2-10 mg-BSA/ml through the membrane, the BSA binding capacity was determined. With increasing salt concentration or pH of the protein buffer solution, the graft chain shrank and BSA binding capacity decreased. On the other hand, the BSA binding capacity slightly increased with increasing temperature, and the conformation of the graft chain was insensitive to temperature in the range from 278 to 303 K. The bound BSA could be quantitatively eluted by permeating a buffer solution containing 0.5 M NaCl, and no deterioration in the BSA binding capacity was observed during five cycles of adsorption, elution and conditioning.

We describe a novel ''tentacle-type'' porous membrane that allows adsorption of enzymes in multilayers in amounts about 50-fold those permitted by monolayer adsorption. A diethylamino (DEA) group as an anion-exchanger was appended to a polymer chain, grafted onto the pores of a hollow-fiber membrane. A urease solution was forced to permeate through the pores of the anion-exchange membrane, which had a DEA group density up to 2.9 mmol per gram of membrane and a thickness of 0.8 mm. The grafted chain with a higher DEA group density provided a larger number of three-dimensional adsorption (tentacle-like binding) sites for urease. The binding capacity exceeded one gram of urease per gram of the membrane at DEA group densities higher than 1.6 mmol per gram, which amounted to a more than 37-fold greater amount of adsorbed enzyme compared to monolayer adsorption. In addition, urease captured by the DEA on the graft chain could be quantitatively eluted with retention of 90% of the feed urease activity.

A polymer chain containing a diethylamino group was grafted onto the pore surface of a porous hollow-fiber membrane by radiation-induced graft polymerization. Dependence of the protein binding capacity of the membrane on environmental parameters such as salt concentration, pH and temperature was investigated. Saturation capacity of protein bound onto the graft chain containing ion-exchange group was governed by the conformation of the graft chain and the intensity of ion-exchange interaction. The conformation of the graft chain was investigated based on the pore radius of the membrane estimated from the permeation flux of a buffer solution through the membrane. By sufficiently permeating a bovine serum albumin (BSA) solution within the concentration range of 0.2-10 mg-BSA/ml through the membrane, the BSA binding capacity was determined. With increasing salt concentration or pH of the protein buffer solution, the graft chain shrank and BSA binding capacity decreased. On the other hand, the BSA binding capacity slightly increased with increasing temperature, and the conformation of the graft chain was insensitive to temperature in the range from 278 to 303 K. The bound BSA could be quantitatively eluted by permeating a buffer solution containing 0.5 M NaCl, and no deterioration in the BSA binding capacity was observed during five cycles of adsorption, elution and conditioning. © 1995.

The immobilization of polymer chains containing a diethylamino (DEA) group on the pore surface of a porous hollow-fibre membrane is reported. This novel membrane can collect proteins at a high rate and high capacity because of convective transport and multi-layering of proteins. Overlapping of the breakthrough curves for different residence times of bovine serum albumin (BSA) solution demonstrates that the diffusional resistance of BSA to the DEA group anchored to the polymer chain was negligible. Membranes with a higher density of DEA groups exhibited a higher binding capacity for BSA. For example, a membrane with a DEA group density of 2.9 mol/kg had a BSA binding capacity of 490 g/kg, which was equivalent to eleven times the adsorption capacity of a monolayer. This vertical layering is due to holding of the BSA molecules in a tentacle-like manner by the graft chains extending from the pore surface towards the pore interior.

The sulfonic acid (SO3H) group was attached to polyethylene by radiation-induced graft polymerization of sodium p-styrenesulfonate and compared with the SO3H group introduced into the polystyrene chain cross-linked with divinylbenzene. The catalytic activities of the SO3H group were measured in a batch mode for the hydrolysis of methyl acetate and sucrose. The temperature dependence of the reaction rate constants for the hydrolysis was determined. For the hydrolysis of methyl acetate, the SO3H group anchored to the grafted polymer chain had the same activity as the SO3H group introduced into the cross-linked polymer chain. On the other hand, the graft-type acid catalyst had about 10 times the hydrolytic activity for sucrose as the cross-linked acid catalyst.

An attempt was made to introduce a strong base anion-exchange group by radiation-induced grafting of vinyl benzyltrimethylammonium chloride (VBTAC) onto polyethylene film. Both two-step grafting and comonomer grafting techniques were tried owing to the difficulty of direct graft polymerization of VBTAC onto polyethylene. 2-Hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA), having the same backbone structure except that the hydroxyl group of HEMA was grafted onto the polyethylene film and then VBTAC was grafted to examine the reactivity of VBTAC with each grafted polyethylene film. Cografting of the binary mixtures of VBTAC and HEMA, or EMA, onto polyethylene film was also carried out. (C) 1994 John Wiley & Sons, Inc.

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.

Sulfonic acid (SO3H) groups were attached to four different polymeric substrates by means of cografting sodium styrene sulfonate (SSS) with acrylic acid (AAc). The polymeric substrates were: microporous polyethylene (PE) hollow-fiber membrane, polypropylene (PP) tube, non-woven fabric made from PE and PP, and polytetrafluoroethylene (PTFE) film. The polymers were pre-irradiated by means of an electron beam source and immersed into monomer mixtures of SSS and AAc at 323 K. The molar ratio of the sulfonic acid groups to carboxyl (COOH) groups in the grafted polymers increased with reaction time. The salt-splitting capacities of the resultant ion exchangers, after 20 h of reaction, were 2.5, 1.4, 1.6 and 1.1 mol/kg for hollow fiber, tube, non-woven fabric and film, respectively. These values are in the range of salt-splitting capacities characteristic for conventional cation exchangers based on styrene-divinyl benzene copolymer beads.

The sulfonic acid (SO3H) group was readily introduced into a polyethylene (PE) membrane by radiation-induced cografting of sodium styrenesulfonate (SSS) with hydrophilic monomers such as acrylic acid (AAc) and hydroxyethyl methacrylate (HEMA). The density of SSS grafted onto the PE membrane was determined as a function of molar ratio of hydrophilic monomer to SSS in the monomer mixture. Immersion of the electron-beam-irradiated PE membrane into the mixture of SSS and HEMA for 5 h at 323 K provided the SO3H density of 2.5 mol/kg of the H-type product.

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.

Anion-exchange groups were introduced into an existing microfiltration membrane by radiation-induced grafting and subsequent chemical modification. The tertiary-amino-group-containing monomers, i.e. diethylaminoethyl methacrylate (DEAEMA) and vinyl pyridine (VP), and epoxy-group-containing monomer, i.e. glycidyl methacrylate (GMA), were grafted onto the porous polyethylene hollow-fiber membrane. The epoxy group produced in the GMA-grafted hollow fiber was converted into tertiary amino groups by reaction with diethylamine (DEA) and 4-aminopyridine (AP). Subsequently, the DEA- and AP-modified hollow fibers and the DEAEMA- and VP-grafted hollow fibers were all quaternized with benzyl chloride (BC). Measurements of pure water flux and ion-exchange capacity of the resulting membranes, which contained a strongly basic anion-exchange group, demonstrated that the quaternized membrane (DEA-BC-T fiber) originating from the DEA-modified membrane was the most suitable membrane for anion collection by permeation through its pores. The DEA-BC-T fiber had a pure water flux of 1.0 m/hr at a filtration pressure of 0.1 MPa and an ion-exchange capacity of 1 mol/kg.

Diethylamino (DEA) groups were introduced into a porous hollow-fiber membrane having a nominal pore size of 0.34 micrometer and porosity of 71% via two reactions: graft polymerization of glycidyl methacrylate (GMA) and subsequent functionalization. The graft chains were formed both on the pore surface and in the amorphous domain of the polymer matrix, and were distributed uniformly throughout the membrane. Water and acetone were forced to permeate across the porous membrane the DEA group density of which ranged up to 6.7 mol per kg of the starting hollow fiber membrane with a 100% constant degree of GMA grafting. The density of the DEA groups on the graft chains induced conformational changes which altered the permeability of the membrane. The pure water flux curve exhibited a sharp decrease at a conversion ratio of the epoxide group to the DEA group between 40 and 60%. The acetone flux curve had a maximum value near the 20% conversion point. The DEA-group-containing membrane, with a conversion below 20%, showed a higher swelling in acetone than in water. This is due to the swelling of the graft chains invading the amorphous domains.

Three kinds of functional groups, iminodiacetate (IDA), amidoxime (AO), and phosphoric acid (PA), were introduced into a microfiltration hollow fiber membrane by radiation-induced grafting and subsequent chemical modification. The chelating hollow fibers containing AO or IDA groups exhibited a 1.1 m3/(m2-hr) pure water flux at 100% grafting of each precursor monomer, while the hollow fiber containing the PA group had a negligible flux. Breakthrough curves were obtained by permeating a metal-ion-containing feed solution radially from inside to outside of the chelating hollow fiber. The IDA chelating hollow fiber was superior to the AO chelating hollow fiber with regard to the collection of cobalt ion. The IDA hollow fiber, whose degree of glycidyl methacrylate grafting ranged from 20 to 260%, had a saturation capacity of cobalt ion of 0.13-1.3 mole Co per kg of base polymer.