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

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

Recovery oriented phosphorus adsorption process in decentralized advanced Johkasou.

Decentralized advanced wastewater treatment using adsorption and desorption process for recovery and recycling oriented phosphorus removal was developed. Adsorbent particles made of zirconium were set in a column, and it was installed as subsequent stage of BOD and nitrogen removal type Johkasou, a household domestic wastewater treatment facility. The water quality of the effluent of adsorption column in a number of experimental sites was monitored. The effluent phosphorus concentration was kept below 1 mg l(-1) during 90 days at all the sites. Furthermore, over 80% of the sites achieved 1 mg l(-1) of T-P during 200 days. This adsorbent was durable, and deterioration of the particles was not observed over a long duration. The adsorbent collected from each site was immersed in alkali solution to desorb phosphorus. Then the adsorbent was reactivated by soaking in acid solution. The reactivated adsorbent was reused and showed almost the same phosphorus adsorption capacity as a new one. Meanwhile, the desorbed phosphorus was recovered with high purity as trisodium phosphate by crystallization. It is proposed as a new decentralized system for recycling phosphorus that paves the way to high-purity recovery of finite phosphorus.

Evaluation of constructed wetlands by wastewater purification ability and greenhouse gas emissions.

Domestic wastewater is a significant source of nitrogen and phosphorus, which cause lake eutrophication. Among the wastewater treatment technologies, constructed wetlands are a promising low-cost means of treating point and diffuse sources of domestic wastewater in rural areas. However, the sustainable operation of constructed wetland treatment systems depends upon a high rate conversion of organic and nitrogenous loading into their metabolic gaseous end products, such as N2O and CH4. In this study, we examined and compared the performance of three typical types of constructed wetlands: Free Water Surface (FWS), Subsurface Flow (SF) and Vertical Flow (VF) wetlands. Pollutant removal efficiency and N2O and CH4 emissions were assessed as measures of performance. We found that the pollutant removal rates and gas emissions measured in the wetlands exhibited clear seasonal changes, and these changes were closely associated with plant growth. VF wetlands exhibited stable removal of organic pollutants and NH3-N throughout the experiment regardless of season and showed great potential for CH4 adsorption. SF wetlands showed preferable T-N removal performance and a lower risk of greenhouse gas emissions than FWS wetlands. Soil oxidation reduction potential (ORP) analysis revealed that water flow structure and plant growth influenced constructed wetland oxygen transfer, and these variations resulted in seasonal changes of ORP distribution inside wetlands that were accompanied by fluctuations in pollutant removal and greenhouse gas emissions.

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

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

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

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

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process.

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.

Comparative analysis of genetic diversity and expression of amoA in wastewater treatment processes.

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.

Biodegradation of microcystis and microcystins by indigenous nanoflagellates on biofilm in a practical treatment facility.

The potential for degradation of Microcystis and microcystins by organisms in biofilm in a practical biological treatment facility combined with conventional treatment processes was examined. Viable cells of Microcystis viridis, which produced microcystin LR, RR and YR, were degraded in 6 to 10 days by the addition of biofilm collected in early summer, mid-summer and autumn. Monas sp. grew remarkably well compared with other organisms in the samples of all seasons. Therefore, we conclude that Monas sp. was indigenous animals in the biological treatment facility and was able to degrade Microcystis cells. Some previous studies reported the predation of Microcystis cells by Monas guttula in pure culture conditions (monoxenic culture). We clarified that Monas sp. can grow and prey on Microcystis cells even in mixed culture including many kinds of other organisms in biofilm in a practical treatment facility. Also, intracellular microcystin LR, RR and YR in Microcystis cells could be degraded simultaneously with high reduction of Microcystis cells by organisms in biofilm.

Comparison of detection specificity of nitrifying bacteria in biofilm using fluorescence in situ hybridization and in situ fluorescent antibody methods.

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.

Effects of SRT and DO on N2O reductase activity in an anoxic-oxic activated sludge system.

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.

Detection and quantification of expression of amoA by competitive reverse transcription-pCR.

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 x ml(-1). Additionally, it was calculated that the copy number of amoA in nitrifying bacterial aggregates was 1.0 x 10(12) copies x ml(-1) (5.1 x 10(10) copies x 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.

PCR-DGGE analysis of denitrifying bacteria in a metallurgic wastewater treatment process.

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.

Characterization of microbial community in nitrogen removal process of metallurgic wastewater by PCR-DGGE.

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.

Community analysis of nitrifying bacteria in an advanced and compact Gappei-Johkasou by FISH and PCR-DGGE.

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.

Degradation of microcystin by biofilm in practical treatment facility.

Potential for degradation of microcystin by biofilm was examined by some batch experiments using biofilm scraped from practical biological treatment facility combined with conventional treatment processes. The viable cells of Microcystis viridis, which produced microcystin LR, RR and YR were degraded at 6 to 10 days by the addition of biofilm. Biofilm collected in summer season had especially higher potential for degradation of Microcystis with complete degradation at 6 days. In all seasons, Monas spp. grew remarkably, accompanied with the higher decrease of the viable cells of Microcystis and the micro-animals were considered as a main predator for Microcystis cells. Intracellular microcystin LR, RR, YR were degraded simultaneously with high reduction of Microcystis cells. Dissolved microcystin LR of 1,000 microg l(-1) was effectively degraded by indigenous aquatic bacteria on biofilm during 5 days, the degradability became higher with the increase in the concentration of microcystin LR. From the results of our research, it was clarified that the aggregated microorganisms consisting of biofilm had high potential for degradation of intracellular and dissolved microcystin.

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

Direct detection by in situ PCR of the amoA gene in biofilm resulting from a nitrogen removal process.

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.

Biological activity of alpha-thujaplicin, the minor component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO.

Alpha-thujaplicin, a minor component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO, which was synthesized, showed the antibacterial activity, phytogrowth-inhibitory effect, inhibition of carboxypeptidase A and cytotoxic effect. Antibacterial activity of alpha-thujaplicin on Enterococcus faecalis IFO-12965 [minimum inhibitory concentration (MIC): 1.56 microg/ml] was higher than that of gentamicin (MIC: 6.25 microg/ml) used as a positive control. Inhibitory activity of alpha-thujaplicin on carboxypeptidase A [50% inhibitory concentration (IC50): 3.24 x 10(-5) M] was higher than that of 1,10-phenanthroline used as a positive control. Alpha-thujaplicin showed germination inhibition toward the seed of Echinochloa utilis Ohwi et Yabuno even at the low concentration of 10 ppm and its growth inhibitory effect was stronger than that of sodium 2,4-dichlorophenoxyacetate used as a standard. Alpha-thujaplicin at 1.25 microg/ml inhibited cell growth of human stomach cancer KATO-IIl by 86%, and Ehrlich's ascites carcinoma by 87%, respectively. This compound even at the low concentration of 0.32 microg/ml also inhibited cell growth of the former by 66%, and the latter by 75%, respectively. The acute toxicity of alpha-thujaplicin [50% lethal dose (LD50) value: 256 mg/kg] in mice was as strong as those of beta-dolabrin (LD50 value: 232 mg/kg) and gamma-thujaplicin (LD50 value: 277 mg/kg).

Nitrous oxide emissions from aerated composting of organic waste.

The composting of high organic content wastes has been shown to produce nitrous oxide (N2O). This study was initiated to investigate the mechanisms of N2O emissions from aerated composting and to determine the optimal operational conditions that minimize N2O emissions. The results of our experiment in laboratory-scale composters showed that more than 95% of N2O was produced during the later period of composting when readily available carbon sources had been depleted. Significant increases in N2O emission after nitrite (NO2-) addition, and good NO(2-)-N2O correlation, indicates that N2O was transformed from NO2-. Extremely high N2O generation was observed after NO2- addition in the presence and absence of composted cattle manure. This suggests an identical mechanism for N2O production in both treatments. However, the addition of composted cattle manure resulted in an earlier initiation of the main N2O generation period. Intermittent feeding of fresh food waste postponed the main N2O generation period, and reduced the mass-based N2O emissions by 20%.