Short- and long-term effects of copper on anammox under gradually increased copper concentrations.

This study aims to determine both short- and long-term response of enriched anammox culture to Cu. Assessment of short-term inhibition is based both on total applied Cu concentration and potential bioavailable fractions like intracellular, surface-bound, soluble and free Cu ion. The half maximal inhibitory concentration (IC50) values for total applied, soluble, intracellular and cell-associated concentrations were determined as 4.57 mg/L, 1.97 mg/L, 0.71 mg/L, 1.11 mg/L, respectively. Correlation between the surface-bound fraction of Cu and inhibition response was weak, suggesting that Cu sorbed to biomass was not directly responsible for the effects on anammox activity. There was a disparity between the results of short- and long-term experiments in terms of inhibition threshold concentration (i.e. short-term IC50 = 4.57 mg/L vs long-term IC50 = 6.74 mg/L). Candidatus Kuenenia (59.8%) and Candidatus Brocadia (40.2%) were the two main anammox genera within the initial biomass sample. One of the most interesting finding of the study is the demonstration that a complete wash-out of C. Brocadia genus at an applied Cu concentration of 6.5 mg/L. This strongly indicates that C. Brocadia were not able to tolerate high copper concentrations and all nitrogen conversion was carried out by C. Kuenenia during the Cu exposure period.

Transcriptomic analysis of nonylphenol effect on Saccharomyces cerevisiae.

Nonylphenol (NP) is a bioaccumulative environmental estrogen that is widely used as a nonionic surfactant. We have previously examined short-term effects of NP on yeast cells using microarray technology. In the present study, we investigated the adaptive response of Saccharomyces cerevisiae BY4742 cells to NP exposure by analyzing genome-wide transcriptional profiles using RNA-sequencing. We used 2 mg/L NP concentration for 40 days of exposure. Gene expression analysis showed that a total of 948 genes were differentially expressed. Of these, 834 genes were downregulated, while 114 genes were significantly upregulated. GO enrichment analysis revealed that 369 GO terms were significantly affected by NP exposure. Further analysis showed that many of the differentially expressed genes were associated with oxidative phosphorylation, iron and copper acquisition, autophagy, pleiotropic drug resistance and cell cycle progression related processes such as DNA and mismatch repair, chromosome segregation, spindle checkpoint activity, and kinetochore organization. Overall, these results provide considerable information and a comprehensive understanding of the adaptive response to NP exposure at the gene expression level.

Genome reprogramming in Saccharomyces cerevisiae upon nonylphenol exposure.

Bioaccumulative environmental estrogen, nonylphenol (NP; 4-nonylphenol), is widely used as a nonionic surfactant and can affect human health. Since genomes of Saccharomyces cerevisiae and higher eukaryotes share many structural and functional similarities, we investigated subcellular effects of NP on S. cerevisiae BY4742 cells by analyzing genome-wide transcriptional profiles. We examined effects of low (1 mg/l; <15% cell number reduction) and high (5 mg/l; >65% cell number reduction) inhibitory concentration exposures for 120 or 180 min. After 120 and 180 min of 1 mg/l NP exposure, 187 (63 downregulated, 124 upregulated) and 103 genes (56 downregulated, 47 upregulated), respectively, were differentially expressed. Similarly, 678 (168 repressed, 510 induced) and 688 genes (215 repressed, 473 induced) were differentially expressed in cells exposed to 5 mg/l NP for 120 and 180 min, respectively. Only 15 downregulated and 63 upregulated genes were common between low and high NP inhibitory concentration exposure for 120 min, whereas 16 downregulated and 31 upregulated genes were common after the 180-min exposure. Several processes/pathways were prominently affected by either low or high inhibitory concentration exposure, while certain processes were affected by both inhibitory concentrations, including ion transport, response to chemicals, transmembrane transport, cellular amino acids, and carbohydrate metabolism. While minimal expression changes were observed with low inhibitory concentration exposure, 5 mg/l NP treatment induced substantial expression changes in genes involved in oxidative phosphorylation, cell wall biogenesis, ribosomal biogenesis, and RNA processing, and encoding heat shock proteins and ubiquitin-conjugating enzymes. Collectively, these results provide considerable information on effects of NP at the molecular level.

Transcriptomic analysis displays the effect of (-)-roemerine on the motility and nutrient uptake in Escherichia coli.

Among the different families of plant alkaloids, (-)-roemerine, an aporphine type, was recently shown to possess significant antibacterial activity in Escherichia coli. Based on the increasing demand for antibacterials with novel mechanisms of action, the present work investigates the potential of the plant-derived alkaloid (-)-roemerine as an antibacterial in E. coli cells using microarray technology. Analysis of the genome-wide transcriptional reprogramming in cells after 60 min treatment with 100 µg/mL (-)-roemerine showed significant changes in the expression of 241 genes (p value <0.05 and fold change >2). Expression of selected genes was confirmed by qPCR. Differentially expressed genes were classified into functional categories to map biological processes and molecular pathways involved. Cellular activities with roles in carbohydrate transport and metabolism, energy production and conversion, lipid transport and metabolism, amino acid transport and metabolism, two-component signaling systems, and cell motility (in particular, the flagellar organization and motility) were among metabolic processes altered in the presence of (-)-roemerine. The down-regulation of the outer membrane proteins probably led to a decrease in carbohydrate uptake rate, which in turn results in nutrient limitation. Consequently, energy metabolism is slowed down. Interestingly, the majority of the expressional alterations were found in the flagellar system. This suggested reduction in motility and loss in the ability to form biofilms, thus affecting protection of E. coli against host cell defense mechanisms. In summary, our findings suggest that the antimicrobial action of (-)-roemerine in E. coli is linked to disturbances in motility and nutrient uptake.

Anammox-zeolite system acting as buffer to achieve stable effluent nitrogen values.

For a successful nitrogen removal, Anammox process needs to be established in line with a stable partial nitritation pretreatment unit since wastewater influent is mostly unsuitable for direct treatment by Anammox. Partial nitritation is, however, a critical bottleneck for the nitrogen removal since it is often difficult to maintain the right proportions of NO2-N and NH4-N during long periods of time for Anammox process. This study investigated the potential of Anammox-zeolite biofilter to buffer inequalities in nitrite and ammonium nitrogen in the influent feed. Anammox-zeolite biofilter combines the ion-exchange property of zeolite with the biological removal by Anammox process. Continuous-flow biofilter was operated for 570 days to test the response of Anammox-zeolite system for irregular ammonium and nitrite nitrogen entries. The reactor demonstrated stable and high nitrogen removal efficiencies (approximately 95 %) even when the influent NO2-N to NH4-N ratios were far from the stoichiometric ratio for Anammox reaction (i.e. NO2-N to NH4-N ranging from 0 to infinity). This is achieved by the sorption of surplus NH4-N by zeolite particles in case ammonium rich influent came in excess with respect to Anammox stoichiometry. Similarly, when ammonium-poor influent is fed to the reactor, ammonium desorption took place due to shifts in ion-exchange equilibrium and deficient amount were supplied by previously sorbed NH4-N. Here, zeolite acted as a preserving reservoir of ammonium where both sorption and desorption took place when needed and this caused the Anammox-zeolite system to act as a buffer system to generate a stable effluent.

Analysis of transcriptional profiles of Saccharomyces cerevisiae exposed to bisphenol A.

Bisphenol A (BPA), an endocrine disrupting chemical, is used as a monomer in the production of epoxy resins and polycarbonates, and as a plasticizer in polyvinyl chloride. As such, it is produced in large quantities worldwide and continuously leaches into the environment. To capture the genome reprogramming in eukaryotic cells under BPA exposure, here, we used Saccharomyces cerevisiae as model organism and analyzed the genome-wide transcriptional profiles of S. cerevisiae BY4742 in response to BPA, focusing on two exposure scenarios: (1) exposure to a low inhibition concentration (50 mg/L; resulting in <10 % inhibition in cell number) and (2) a high inhibition concentration (300 mg/L; resulting in >70 % inhibition in cell number). Based on the transcriptional profiling analyses, 81 genes were repressed and 104 genes were induced in response to 50 mg/L BPA. Meanwhile, 378 genes were downregulated and 606 genes were significantly upregulated upon exposure to 300 mg/L BPA. While similar processes were affected by exposure to distinct BPA concentrations, including mitochondrial processes, nucleobase-containing small molecule metabolic processes, transcription from the RNA polymerase II promoter, and mitosis and associated processes, the number and magnitude of differentially expressed genes differ between low and high inhibition concentration treatments. For example, exposure to 300 mg/L BPA resulted in severe changes in the expression levels of several genes involved in oxidative phosphorylation, the tricarboxylic acid cycle, ribosomal activity, replication, and chemical responses. Conversely, only slight changes were observed in the expression of genes involved in these processes in cells exposed to 50 mg/L BPA. These results demonstrate that yeast cells respond to BPA in a concentration-dependent manner at the transcriptional level via different genes and provide insight into the molecular mechanisms underlying the modes of action of BPA.

Preparation of poly(3-hydroxybutyrate-co-hydroxyvalerate) films from halophilic archaea and their potential use in drug delivery.

Halophilic archaea offer a potential source for production of polyhydroxyalkanoates (PHAs). Hence, the experiments were carried out with five extremely halophilic archaeal isolates to determine the highest PHA-producing strain. PHA production of each isolates was separately examined in cheap carbon sources such as corn starch, sucrose, whey, apple, melon and tomato wastes. Corn starch was found to be a fairly effective substrate for PHA production. Among the strains studied here, the strain with the highest capability for PHA biosynthesis was found to be 1KYS1. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that 1KYS1 closely related to species of the genus Natrinema. The closest phylogenetic similarity was with the strain of Natrinema pallidum JCM 8980 (99 %). PHA content of 1KYS1 was about 53.14 % of the cell dry weight when starch was used as a carbon source. The formation of large and uniform PHA granules was confirmed by transmission electron microscopy and the biopolymer was identified as poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV). PHBV produced by 1KYS1 was blended with low molar mass polyethylene glycol (PEG 300) to prepare biocompatible films for drug delivery. Rifampicin was used as a model drug and its release from PHBV films was investigated at pH 7.4, 37 °C. It was found that PHBV films obtained from 1KYS1 were very effective for drug delivery. In conclusion, PHBV of 1KYS1 may have a potential usage in drug delivery applications.

Spatial and temporal analysis of black carbon aerosols in Istanbul megacity.

Black carbon (BC) is an important component of particulate matter due to its effects on human health and climate. In this study, we present the first BC concentrations measured in the Istanbul megacity (~15 million inhabitants). Two measurement campaigns have been conducted to measure BC and fine particulate matter (PM2.5) concentrations at four locations, characterized by different traffic densities. In the first campaign, BC daily mean concentrations have been found to be between 4 µg/m(3) and 10 µg/m(3). In the second campaign, BC and PM2.5 have been measured at the site with the highest traffic density for an entire year. Annually averaged BC contributes by 38 ± 14% to the PM2.5 levels (annual average BC: 13 µg/m(3) and PM2.5: 36 µg/m(3)). Diurnal variations of BC concentrations followed those of traffic density (correlation coefficient of 0.87). These measurements are essential to identify the sources of BC and PM2.5 concentrations in Istanbul and develop mitigation measures.

Treatment of landfill leachate using UASB-MBR-SHARON-Anammox configuration.

Leachate treatment is a challenging issue due to its high pollutant loads. There are several studies on feasible treatment methods of leachate. In the scope of this study, high organic content of young leachate was eliminated using an upflow anaerobic sludge blanket (UASB) and a membrane bioreactor (MBR) in sequence and effluent of the system was given to single reactor for high activity ammonia removal over nitrite (SHARON) and anaerobic ammonia oxidation (Anammox) reactors to remove nitrogen content. All reactors were set up at lab scale in order to evaluate the usage of these processes in sequencing order for leachate treatment. COD and TKN removal efficiencies were over 90 % in the combined processes which were operated during the study. The biodegradable portion of organic matter was removed with an efficiency of 99 %. BOD5 concentration decreased to 50 mg/L by UASB and MBR in sequence even the influent BOD5 concentration was over 8,000 mg/L. Although high nitrogen concentrations were observed in raw leachate, successful removal of nitrogen was accomplished by consecutive operations of SHARON and Anammox reactors. The results of this study demonstrated that with an efficient pretreatment of leachate, the combination of SHARON-Anammox processes is an effective method for the treatment of high nitrogen content in leachate.

Solidification/stabilization of landfill leachate concentrate using different aggregate materials.

The application of reverse osmosis for the treatment of landfill leachate is becoming widespread in Turkey as well as in Europe. A major drawback of this process is the production of concentrate, which could be as much as 30% of the feed stream, and high concentrations of salts and contaminants. The reverse osmosis concentrate is disposed of by using several methods including re-infiltration, drying, incineration and solidification/stabilization. In this study, solidification/stabilization (S/S) technology was studied for the treatment of reverse osmosis concentrate produced from landfill leachate. In order to benefit from its capability to absorb heavy metals, ammonia and some other pollutants, zeolite and different aggregate materials were used in solidification experiments. Main pollutants in the leachate concentrate, TOC, DOC, TDS and ammonia were successfully solidified and approximately 1% of TOC, DOC, TDS and ammonia remained in the eluate water. The results indicated that the landfill disposal limits could be attained by solidification/stabilization process.

Inhibitory effects of free ammonia on Anammox bacteria.

Anammox bacteria can effectively treat high ammonia and nitrite concentrations under anoxic environments. However, the presence of high ammonia and nitrite concentrations may cause free ammonia and nitrous acid inhibition at high pH and temperature environments. In this study, the inhibitory effect of free ammonia on Anammox bacteria was investigated in a lab-scale upflow fixed-bed reactor with Kaldnes biofilm carriers. Results of continuous operation showed that inhibition was not observed in the Anammox reactor when the free ammonia concentration gradually increased up to 150 mg/L. However, Anammox activity suddenly dropped to 10 % when the free ammonia concentration reached to 190 mg/L. Nevertheless, high influent ammonia and nitrite concentrations up to 1,500 and 500 mg/L, respectively, did not noticeably inhibit the Anammox activity. Gradually decreasing Anammox activity was also supported by fluorescent in situ hybridization (FISH) analysis. FISH and 16S rRNA gene analysis results revealed that main Anammox organisms were phylogenetically related to Candidatus Kuenenia stuttgartiensis, Candidatus Jettenia asiatica and Candidatus Brocadia anammoxidans.

Investigation of nitrogen converters in membrane bioreactor.

In this study, the activity and diversity of nitrogen converters, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), ammonia-oxidizing archaea (AOA) and Anammox bacteria in a pilot-scale membrane bioreactor (MBR) were investigated and monitored using amoA and 16S rDNA-based molecular tools. The pilot-scale MBR (100 m(3)/day) was located inside the full-scale Pasakoy Advanced Wastewater Treatment Plant (WWTP), and operated for approximately 5 months without sludge purge. During 148 days of operation, volatile suspended solids (VSS) concentration increased from 2,454 mg/L to 10,855 mg/L and the average organic carbon and ammonia nitrogen removal rates were 92% and 99%, respectively. Real-time PCR results indicated that the fraction of AOB increased from 2.94% to 4.05% when VSS concentration reached to 3,750 mg/L throughout 148 days of operation. At higher VSS concentrations, the fraction of AOB declined gradually to 1.15% while the fraction of Nitrospira population was varied between 8.23 and 13.01%. However, significant change or any positive and negative correlations between VSS concentration and Nitrospira population were not observed in this period. The phylogenetic analysis revealed that MBR harbored diverse AOB community which was related to the Nitrosomonas and Nitrosospira lineage. Candidatus Nitrospira defluvii was the only detected NOB in this study.

Identification and quantitative evaluation of nitrogen-converting organisms in a full-scale leachate treatment plant.

The presence of ammonia nitrogen in landfill leachates poses a significant problem for treatment plant operators. The nitrification-denitrification process mostly carries out the nitrogen conversion in biological treatment systems. However, recent research shows that other processes by anaerobic ammonia-oxidizing bacteria (Anammox) and ammonia-oxidizing archaea (AOA) were also responsible for the removal of nitrogen in biological systems. In this study, the nitrogen-converting microorganisms in the Bursa Hamitler Leachate Treatment Plant were identified and monitored by using molecular tools. Fluorescent in situ hybridization (FISH) and slot-blot hybridization results showed that the Nitrosomonas and Nitrospira species were the dominant ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), respectively. Quantitative real-time PCR results indicated that AOB, NOB, AOA and Anammox bacteria exist in the leachate treatment plant. However, the removal of ammonia can be ascribed mainly to nitrification because AOB (1.5%) and NOB (11.3%) were predominant among all nitrogen-converting bacteria. The results of the phylogenetic analysis based on amoA and 16S rDNA gene revealed that the uncultured bacterium clone 4-24, Kuenenia stuttgartiensis genome fragment KUST_E and the uncultured Crenarchaeota clone NJYPZT-C1 belong to AOB, Anammox and AOA populations, respectively, and were the dominant species in their cluster.

Monitoring of population shifts in an enriched nitrifying system under gradually increased cadmium loading.

The changes in nitrifying bacterial population under cadmium loading were monitored and evaluated in a laboratory scale continuous-flow enriched nitrification system. For this purpose, the following molecular microbiological methods were used: slot-blot hybridization, denaturing gradient gel electrophoresis (DGGE), real-time PCR followed by melting curve analysis, cloning and sequence analysis. The initial cadmium concentration was incrementally increased from 1 to 10mg/l which led to a drop in ammonia removal efficiency from 99 to 10%. Inhibition was recovered when cadmium loading was stopped. During the second application of cadmium, nitrifying population became more tolerant. Even at 15mg/l Cd, only a minor inhibition was observed. To investigate the variations in ammonia and nitrite oxidizing bacteria populations in a period of 483 days, ammonia monooxygenase (amoA) and 16S rRNA genes-based molecular techniques were used. An obvious shift was experienced in the diversity of ammonia oxidizers after the first application of 10mg/l Cd. Metal-tolerant ammonia oxidizing species became dominant and the microbial diversity sharply shifted from Nitrosomonas and Nitrosococcus sp. to Nitrosospira sp. which were observed to tolerate higher cadmium loadings. This result indicated that the extent of nitrification inhibition was not only related to the metal concentration and quantity of microorganisms but also depended on the type of species.

Long-term assessment of nitrification in a full-scale wastewater treatment plant.

In this study, long term nitrification performances and microbial composition in a full-scale oxidation ditch type wastewater treatment plant (WWTP) were monitored and evaluated using different molecular methods; fluorescent in situ hybridization (FISH), slot-blot hybridization and quantitative real-time polymerase chain reaction (PCR) method. In situ and membrane hybridization results indicated that Nitrosomonas species were identified as the dominant ammonia oxidizing bacteria and Nitrospira related species were detected as the prevailing nitrite oxidizing bacteria in a full-scale wastewater treatment plant throughout 2 years of operation. Real-time PCR using the LightCyclerinstrument has been developed for the quantification of ammonia monooxygenase (amoA) and 16S rRNA genes. Results suggest that real-time PCR analysis, amoA/16S rRNA ratio, is an alternative method to understand nitrifying bacterial population and activity in wastewater treatment plant compared with the FISH and slot-blot hybridization assays. The autotrophic/heterotrophic bacterial ratio and their influence on reactor performances were investigated using real-time PCR amoA/16S rRNA gene copy ratios and the results showed that this ratio varied from 3.6% to 8.3% during operational period.

Effects of insufficient air injection on methanogenic Archaea in landfill bioreactor.

In this study, methanogenic Archaea diversity in an aerated landfill bioreactor filled with co-disposed incineration bottom ashes and shredded incombustible wastes was monitored and analyzed as a function of time using molecular techniques. Besides, the effects of insufficient air injection on the bioreactor performance and methanogenic diversity were evaluated thoroughly. Results indicated that rapid bio-stabilization of solid waste are possible with aerated landfill bioreactor at various oxygen and oxidation reduction potential levels. Slot-blot hybridization results of leachate samples collected from aerated landfill bioreactor showed that archaeal and bacterial activities increased as stabilization accelerated and bacterial populations constituted almost 95% of all microorganisms. The results of slot-blot hybridization and phylogenetic analysis based on 16S rRNA gene revealed that Methanobacteriales and Methanomicrobiales were dominant species at the beginning while substituted by Methanosarcina-related methanogens close to the end of the operation of bioreactor.

Comparative analysis of nitrifying bacteria in full-scale oxidation ditch and aerated nitrification biofilter by using fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE).

In this study, nitrification performances and composition of nitrifying populations in a full-scale oxidation ditch and a high-rate submerged media nitrification biofilter were comparatively analyzed. In addition to different reactor configurations, effects of differing operational conditions on the nitrification efficiency and bacterial diversity were also explored and evaluated thoroughly. In microbial analysis of sludge samples fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques were used complementary to each other. The extended aeration oxidation ditch subjected to the study is operated as a nitrogen and phosphorus removal system consisting of anaerobic, anoxic, and aerobic zones. The high-rate submerged media aerated filter is operated as nitrification step following the conventional activated sludge unit and the nitrified wastewater is discharged to the sea without complete nitrogen removal. In situ hybridization results have indicated that Nitrosomonas-like ammonia oxidizing and Nitrospira-related nitrite oxidizing bacteria were intensively present in vigorous flocs in nitrification biofilter while carbonaceous bacteria belong to beta subclass of Proteobacteria were considerably dominant in oxidation ditch. Low quantities of nitrifiers in oxidation ditch were also confirmed by the dissimilarity in intensive bands between two systems obtained with DGGE analysis.

Landfill leachate management in Istanbul: applications and alternatives.

Treatment alternatives for Istanbul, Komurcuoda Landfill (KL) leachate that is currently transported to the nearest central wastewater treatment plant were comprehensively investigated with laboratory scale experiments. As flow rate of leachate increases parallel to increment in landfilled solid waste, an individual treatment will be needed to reduce the transportation cost and pollution load on central treatment. However, if the leachate is separately treated and discharged to a brook, in that case more stringent discharge standards will be valid and therefore advanced processes in addition to conventional ones should be included. In laboratory scale experiments, the young landfill leachate having BOD5/COD ratio above 0.6 was successfully treated with efficiencies above 90% in upflow anaerobic reactors if pH is kept below free ammonia inhibition level. Subsequently, nitrification of anaerobically treated leachate was performed with rates of about 8.5 mg NH4+-Ng-1 VSS h-1 and efficiencies above 99% were provided with automated pH regulation by using sodium bicarbonate. Furthermore, denitrification rates as high as 8.1 mg NOx-N g-1VSS h-1 was obtained when carbon source was externally supplied. In addition to nitrification and denitrification, air stripping and struvite precipitation were also applied to remove ammonia in leachate and in average 94% and 98% efficiencies were achieved, respectively. Finally, in average 85% of biologically inert COD was successfully removed by using either ozone or Fenton's oxidation.

Molecular analysis of microbial communities in nitrification and denitrification reactors treating high ammonia leachate.

Molecular analysis of microbial populations in two bench-scale nitrification and denitrification reactors fed with high ammonia landfill leachate was conducted in this study by using DGGE, cloning, and FISH techniques in addition to classical efficiency control parameters. Nitrification tank was operated with a computer-controlled alkalinity dosing system to supply the alkalinity intermittently as consumed on the basis of on-line pH monitoring. By keeping the pH at 7.0 with this system, 99% nitrification efficiency and rates of about 0.14-0.18 mgNH4+-N/mgVSSday were obtained. Meanwhile, as ammonia oxidizing bacteria Nitrosomonas and Nitrosococcus mobilis-like cells and as nitrite oxidizing bacteria Nitrobacter-related cells were intensively indicated. Moreover, some aerobic denitrifiers as Thauera species were also identified. After the termination of pH adjustment in the preceding anaerobic reactors, nitrification tank was loaded with more biodegradable COD as a result of reduced COD removal in anaerobic reactors. Microbial diversity was immediately affected from this alteration and heterotrophic carbonaceous bacteria and aerobic denitrifiers have dominated. To provide the former high efficiencies, retention time has increased from 24 to 48 h and a second pump dosing HCl was included to the automatic control system. Subsequent to these precautions, numbers of ammonia (Nso190) and nitrite oxidizing bacteria (NIT3) were comparatively increased. In denitrification system, about 98% denitrification efficiencies were obtained at 2000 mg/L NOx-N concentrations if sodium acetate was supplied as carbon source. Meanwhile, with 20 gVSS/l biomass concentration, denitrification rates of about 1.34 mgNOx-N/mgVSSday were obtained. All sludge samples have represented similar DGGE patterns and Paraccoccus-related species were identified as dominant denitrifying bacteria.