Characteristics of compounds with strong or weak nitrification inhibition in sewage.

To clarify the characteristics of compounds with strong or weak nitrification inhibition in sewage, 64 organic compounds including compounds registered in Pollutant Release and Transfer Register (PRTR) were evaluated in terms of their chemical structures and molecular weights. Nineteen compounds showed strong nitrification inhibition by testing with Nitrosomonas europaea. Compounds with thioamide structures had the lowest median value of EC50 (0.017 mg/L), followed by those with alkyne structures (0.121 mg/L), chlorophenol structures (0.300 mg/L), and then azole structures (0.365 mg/L). In contrast, 33 of the 64 compounds showed weak nitrification inhibition at a concentration of 10 mg/L, 27 of which were categorized into three main groups: long-chain alcohol structures, alkyne structures with a phenyl group, and aromatic structures. Most compounds with strong nitrification inhibition had a low molecular weight (MW) from 50 to 200. Meanwhile, the proportion of compounds with weak nitrification inhibition tended to be greater with increasing MW and such compounds were predominant at higher molecular weights above 300. The correlations of results derived from tests of nitrification inhibition based on ISO 9509 and N. europaea showed that 24 out of 30 compounds provided results that were highly correlated between these tests (R = 0.85), while 4 compounds with chlorophenol structures and 2 compounds with alkyne structures showed weaker inhibition rates in the ISO 9509 test than in the N. europaea test. Our results indicate that the magnitude of nitrification inhibition depends on MW in addition to the chemical structure, which is helpful in the search for the cause of nitrification inhibition in wastewater treatment plants.

Comparative analyses of microbial structures and gene copy numbers in the anaerobic digestion of various types of sewage sludge.

Anaerobic co-digestion of various sewage sludges is a promising approach for greater recovery of energy, but the process is more complicated than mono-digestion of sewage sludge. The applicability of microbial structure analyses and gene quantification to understand microbial conditions was evaluated. The results show that information from gene analyses is useful in managing anaerobic co-digestion and damaged microbes in addition to conventional parameters like total solids, pH and biogas production. Total bacterial 16S rRNA gene copy numbers are the most useful tools for evaluating unstable anaerobic digestion of sewage sludge, rather than mcrA and total archaeal 16S rRNA gene copy numbers, and high-throughput sequencing. First order decay rates of gene copy numbers during pH failure were higher than typical decay rates of microbes in stable operation. The sequencing analyses, including multidimensional scaling, showed very different microbial structure shifts, but the results were not consistent.

Behavior of radioactive materials and safety stock of contaminated sludge.

The radioactive fallout from the Fukushima Dai-ichi nuclear power plant disaster in 2011 has flowed into and accumulated in many wastewater treatment plants (WWTPs) via sewer systems; this has had a negative impact on WWTPs in eastern Japan. The behavior of radioactive materials was analyzed at four WWTPs in the Tohoku and Kanto regions to elucidate the mechanism by which radioactive materials are concentrated during the sludge treatment process from July 2011 to March 2013. Furthermore, numerical simulations were conducted to study the safe handling of contaminated sewage sludge stocked temporally in WWTPs. Finally, a dissolution test was conducted by using contaminated incinerated ash and melted slag derived from sewage sludge to better understand the disposal of contaminated sewage sludge in landfills. Measurements indicate that a large amount of radioactive material accumulates in aeration tanks and is becoming trapped in the concentrated sludge during the sludge condensation process. The numerical simulation indicates that a worker's exposure around contaminated sludge is less than 1 µSv/h when maintaining an isolation distance of more than 10 m, or when shielding with more than 20-cm-thick concrete. The radioactivity level of the eluate was undetectable in 9 out of 12 samples; in the remaining three samples, the dissolution rates were 0.5-2.7%.

Leachate tests with sewage sludge contaminated by radioactive cesium.

The sewer systems of eastern Japan have transported radioactive fallout from the Fukushima Dai-ichi nuclear power plant accident to wastewater treatment plants, where the radioisotopes have accumulated. To better understand the potential problems associated with the disposal of contaminated sewage sludge in landfills, leachate tests were conducted with radioactive incinerator ash, cement solidification incinerator ash, and dewatered sludge cake. Radioactivity was undetectable in the eluate from incinerator ash and dewatered sludge cake, but about 30% of the radioactivity initially in cement solidification incinerator ash appeared in the eluate during the leaching experiments. Moreover, modification of test conditions revealed that the presence of Ca(2+) ions and strong alkali in the water that contacted the incinerator ash enhanced leaching of cesium. Lastly, the capacity of pit soil to absorb radioactive cesium was estimated to be at least 3.0 Bq/g (dry).

Microbial community structure and population dynamics of granules developed in expanded granular sludge bed (EGSB) reactors for the anaerobic treatment of low-strength wastewater at low temperature.

The anaerobic biological treatment of sucrose-based, low-strength wastewater was investigated in expanded granular sludge bed (EGSB) reactors at low temperatures over a 300-day trial period. During the trial, the operating temperature was lowered in a stepwise manner from 20 degrees C to 5 degrees C. As a result, the reactors exhibited sufficient performances until 10 degrees C operation. The COD removal rate was 3.1-3.8 kgCOD m(-3) day(-1) at 10 degrees C. In particular, the COD removal rate increased gradually through the low-temperature operation; indeed, the later stages of the 10 degrees C operation attained a rate similar to those achieved at 20 degrees C and 15 degrees C. This finding is especially practical for applications of psychrophilic methane fermentation. Additionally, the structure of the microbial community in the granular sludge was analyzed by clone analysis based on 16S rRNA genes and fluorescence in situ hybridization (FISH). As a result, the percentage of the phylum Firmicutes, which were assumed to be Anaerobivrio sp. and Lactococcus sp., greatly increased from 0.7% to 8.0% of the total cells, especially in the surface layer of the granular sludge. These bacteria would contribute to the degradation of the sucrose substrate anaerobically at ambient temperatures. Moreover, the results suggest that a Methanospirillum species, which is a H2-utilizing methanogen, increased from 0.5% to 6.7% during the low-temperature incubation, with a significant increase of methanogenic activity from H2/CO2 at 20 degrees C. Thus, the Methanospirillum species detected in this study may have a key role as hydrogen scavenger during hydrogen-metabolism in low-temperature conditions.

Influence of sugar content of wastewater on the microbial characteristics of granular sludge developed at 20 degrees C in the anaerobic granular sludge bed reactor.

The influence of the sugar content of wastewater on changes in the characteristics of the retained sludge was investigated by using two lab-scale granular sludge bed reactors at 20 degrees C. Both reactors were inoculated with granular sludge grown at 20 degrees C and were fed with synthetic wastewater containing sucrose and volatile fatty acids (VFAs). On day 70, the sucrose content of the wastewater was changed to 90% (based on wastewater COD value) for the first reactor and 0% (VFA 90%) for the second. After this change in feed composition, the COD removal efficiency became about 91% for the sucrose-fed reactor and 95% for the VFA-fed reactor. The growth yield (Yg) of the sucrose-fed sludge increased more than that of the VFA-fed sludge. Consequently, deterioration of the settleability of the sucrose-fed sludge was observed. The sucrose-degrading activity of the retained sludge obtained from the sucrose-fed reactor increased significantly from 3.7 g COD g VSS(-1) day(-1) on day 62 to 36.8 g COD g VSS(-1) day(-1) on day 230, in accordance with the predominant growth of sugar-degrading bacteria--namely, Lactococcus, Clostridium and Chloroflexi--in the retained sludge. The excessive growth of these sugar-degrading bacteria in the retained sludge caused unstable process performance in the sucrose-fed reactor at 20 degrees C.

Changes in process performance and microbial characteristics of retained sludge during low-temperature operation of an EGSB reactor.

A lab-scale expanded granular sludge bed (EGSB) reactor was seeded with granular sludge and operated to investigate the influence of temperature decrease on both process performance and the microbial community structure of the granular sludge. Synthetic wastewater containing sucrose and volatile fatty acids was used as feed. The EGSB reactor was brought online at a starting temperature of 15 degrees C and was reduced stepwise to a final temperature of 5 degrees C. The reactor exhibited sufficient COD removal efficiency between 10 degrees C and 15 degrees C. However at 5 degrees C serious deterioration of process performance was observed. The methane-producing activity of the retained sludge increased when it was compared to the activity of the seed sludge (day 0) during 10 degrees C to 15 degrees C operation. When hydrogen fed, sludge showed much higher methanogenic activity as compared with seed sludge activity at test temperatures of 15 degrees C and 20 degrees C on day 196 of reactor operation. At this time, proliferation of the genus Methanospirillum in the retained sludge was observed and a decrease in Methanobacterium species was also measured. Throughout the experiment, the genus Methanosaeta was detected in abundance and the community structure of the Domain Bacteria was stably maintained. The sugar-degrading acid-forming bacteria, Lactococcus and Anaerovibrio were detected in the retained sludge throughout the experiment as well and the propionate-degrading acetogen Syntrophobacter fumaroxidans was also detected, although its population size decreased at 5 degrees C.

Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules.

The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH(4), H(2), pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH(4), H(2), pH, and ORP revealed that acid and H(2) production occurred in the upper part of the granule, below which H(2) consumption and CH(4) production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H(2) was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH(4) in the inner layer. We determined the effective diffusion coefficient for H(2) in the anaerobic granules to be 2.66 x 10(-5) cm(2) s(-1), which was 57% in water.

Development of a super high-rate Anammox reactor and in situ analysis of biofilm structure and function.

The anaerobic ammonium oxidation (Anammox) process is a new efficient and cost effective method of ammonium removal from wastewater. Under strictly anoxic condition, ammonium is directly oxidised with nitrite as electron acceptor to dinitrogen gas. However, it is extremely difficult to cultivate Anammox bacteria due to their low growth rate. This suggests that a rapid and efficient start-up of Anammox process is the key to practical applications. To screen appropriate seeding sludge with high Anammox potential, a real-time quantitative PCR assay with newly designed primers has been developed. Thereafter, the seeding sludge with high abundance of Anammox bacteria (1.7 x 10(8) copies/mg-dry weight) was selected and inoculated into an upflow anaerobic biofilters (UABs). The UABs were operated for more than 1 year and the highest nitrogen removal rate of 24.0 kg-N m-3 day(-1) was attained. In addition, the ecophysiology of Anammox bacteria (spatial distribution and in situ activity) in biofilms was analysed by combining a full-cycle 16S rRNA approach and microelectrodes. The microelectrode measurement clearly revealed that a successive vertical zonation of the partial nitrification (NH4+ to NO2-), Anammox reaction and denitrification was developed in the biofilm in the UAB. This result agreed with the spatial distribution of corresponding bacterial populations in the biofilm. We linked the micro-scale information (i.e. single cell and/or biofilm levels) with the macro-scale information (i.e. the reactor level) to understand the details of Anammox reaction occurring in the UABs.

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms.

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors.

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-Nm(-3)day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH(4)(+) to NO(2)(-) molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH(4)(+)/NO(2)(-) molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH(4)(+) and NO(2)(-)) of 4.45 g-N of (NH(4)(+)+NO(2)(-))m(-2)day(-1) was detected in the upper 800 microm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.

Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR.

The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9x10(1) to 8.9x10(6) copies per PCR, corresponding to the detection limit of 3.6x10(3) target copies mL(-1). A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples.