Nitrogen removal in an upflow sludge blanket (USB) reactor combined by aerobic biofiltration systems.

A new nitrogen removal process (up-flow sludge blanket and aerobic filter, USB-AF) was proposed and tested with real sewage. In the USB reactor, the larger part of influent organic and nitrogen matters were removed, and ammonia was effectively oxidized in the subsequent aerobic filter. The role of the aerobic filter was to convert ammonia into nitrate, an electron acceptor that could convert soluble organic matters into volatile suspended solid (VSS) in the USB. The accumulated as well as influent VSS in the USB was finally degraded to fermented products that were another good carbon source for denitrification. Total COD, settleable COD and soluble COD in the raw sewage were 325, 80 and 140 mg/l, respectively. Most unsettleable COD as well as some SCOD in the influent was successfully removed in the USB. TCOD removal in the anoxic filter was by denitrification with the recycled nitrate. Low COD input to the aerobic filter could increase nitrification efficiency, reduce the start-up period and save the aeration energy in the USB-AF system. About 95% of ammonia was nitrified in the aerobic filter with no relation to the influent ammonia concentration. Denitrification efficiency of the recycled nitrate in the anoxic filter was about 85, 83, and 72% at recycle ratios of 100, 200, and 300%, respectively. T-N removal efficiency was 70% at recycle ratio of 300%.     

Granulation in an upflow anaerobic sequencing batch reactor treating disintegrated waste activated sludge.

An upflow anaerobic reactor operated with a sequencing batch mode to enhance high rate digestion of raw and thermally disintegrated waste activated sludge with formation of granules. The gas production rate doubled when disintegrated waste activated sludge was introduced. Gradual granulation took place and the dispersed particles become coarse granulation as the operation continued. The granular sludge showed relatively higher specific methanogenic activity than the dispersed sludge. Bacterial morphology by a scanning electron microscope showed diversity of bacteria such as filamentous, rod and spherical shape in the section of granules. Filamentous bacteria, which might support the frame of a granule, were observed as long chains at the outer surface. Meanwhile, rod and spherical bacteria, which might play a role in the initial stage of granule formation, were observed from the inner surface of the granule. High rate digestion of sludge along with efficient liquid-solids separation was achieved due mainly to development of sludge granules within the upflow reactor.     

Co-degradation of phenol and m-cresols by upflow anaerobic sludge blanket reactor.

The study was performed to assess the efficacy of an upflow anaerobic sludge blanket reactor for the degradation of mixtures of phenol and m-cresol. The experiments were performed in an upflow anaerobic sludge blanket reactor. The reactor was seeded with digested sewage sludge and was initially operated at 24 HRT. A phenol concentration of 200 mg/L was fed to the reactor to acclimatize the microorganisms to phenols. Subsequently the dosages of phenols were increased to 400 mg/L, 500 mg/L, and 600 mg/L. Cresols were introduced in the reactor when phenol removal efficiency of 77% was achieved at phenol concentration of 600 mg/L. Different phenol to m-cresol ratios were tried and the performance of the reactor was evaluated for each case. The result demonstrates that it is important to consider phenol/ m-cresol ratio to avoid toxic effects and both can be co-degraded successfully under anaerobic conditions provided proper acclimatization time is given.     

Evaluation of a rapid polymerase chain reaction based identification technique for Vibrio cholerae isolates.

Rapid and accurate identification of waterborne pathogens, such as Vibrio cholerae, in drinking-water sources is important to enable effective resource management and public health protection. Phenotypic systems currently being used for the identification of Vibrio cholerae isolates are time-consuming and the need exists for the development of suitable molecular techniques that can offer both fast and reliable identification. During this study, isolates identified as Vibrio cholerae by means of two different biochemical test systems (API 20E and VITEK 32) were analysed with the polymerase chain reaction (PCR) to compare the reliability of the various identification systems. The selected PCR technique amplified a sequence within the outer membrane protein of Vibrio cholerae, a gene specific for V. cholerae. It was found that out of 243 isolates biochemically identified as V. cholerae with either the API or VITEK system, 21 isolates did not give a positive result with the PCR detection method. Sequencing the 16S rDNA of more than half of these isolates and comparison of the sequences with Internet databases indicated that most of the isolates belonged to the genus Aeromonas. The results indicated that the rapid PCR procedure was more accurate than the API or VITEK systems currently being used for the phenotypic identification of Vibrio cholerae isolates.     

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

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

Development of a sensitive polymerase chain reaction method for the detection of Toxoplasma gondii in water.

Toxoplasma gondii is becoming a potential threat for public water supplies worldwide, as demonstrated by the occurrence of waterborne toxoplasmosis outbreaks in developing countries as well as industrialised countries. The aim of the present study was to develop a sensitive molecular approach (PCR) for the detection of Toxoplasma oocysts in water. Sporulated and unsporulated T. gondii oocysts (strains DX and AHC1) were isolated from faeces of laboratory-infected cats. After purification and enumeration, oocysts were spiked into 1 -L water replicates and concentrated using centrifugation, Al2(SO4)3 or Fe2(SO4)3 flocculation. DNA was extracted from the concentrated pellets, and a universal primer and a T. gondii-specific primer were selected to amplify a region at the small subunit ribosomal RNA gene. A theoretical detection limit of 0.1 oocysts was achieved for samples that had been concentrated using centrifugation or Al2(SO4)3 flocculation. No PCR products were generated for samples that had been pre-treated using Fe2(SO4)3 flocculation. The final target would be the development of a complete technique able to work as a diagnostic tool for the detection of Toxoplasma in environmental and drinking water.     

The effect of upflow air velocity on the structure of aerobic granules cultivated in a sequencing batch reactor.

The effect of upflow air velocity on the formation and structure of aerobic granules was studied in three column sequencing batch reactors. Upflow aeration would be the major cause of hydrodynamic shear force in the column reactor. Results showed that high upflow air velocity resulted in more compact, denser, rounder, stronger and smaller aerobic granules, while high biomass retention in the reactor was achieved. It was found that high upflow air velocity could induce granular sludge to secrete more cell polysaccharides which in turn contributed to the compact and strong structure. It appears from this study that the structure of aerobic granules could be controlled by manipulating the upflow air velocity.     

Biological nutrient removal in simple dual sludge system with an UMBR (upflow multi-layer bioreactor) and aerobic biofilm reactor.

In this study, a simple dual sludge process was developed for small sewage treatment. It is a hybrid system that consists of upflow multi-layer bioreactor (UMBR) as anaerobic and anoxic reactor with suspended growth microorganisms and post aerobic biofilm reactor with inclined plates. UMBR is a multifunction reactor that acts as primary sedimentation tank, anaerobic reactor, anoxic reactor, and thickener. The sludge blanket in the UMBR is maintained at a constant level by automatic control so that clear water (30 mg-SS/L) can flow into the post aerobic biofilm reactor. It leads to improving performance of the biofilm reactor due to preventing of excess microbial attachment on the media surface and no requirment for a large clarifier caused by low solid loading. The HRT in the UMBR and the aerobic biofilm reactor were about 5.8 h and 6.4 h, respectively. The temperature in the reactor during this study varied from 12.5 degrees C to 28.3 degrees C. The results obtained from this study show that effluent concentrations of TCOD, TBOD, SS, TN, and TP were 29.7 mg/L, 6.0 mg/L, 10.3 mg/L, 12.0 mg/L, and 1.8 mg/L, which corresponded to a removal efficiency of 92.7%, 96.4%, 96.4%, 74.9%, and 76.5%, respectively. The sludge biomass index (SBI) of the excess sludge in the UMBR was about 0.55, which means that the sludge in the UMBR was sufficiently stabilized and may not require further treatment prior to disposal.     

Characteristics of denitrifying granular sludge grown on nitrite medium in an upflow sludge blanket (USB) reactor

While inoculating pre-acclimatized floccular sludge, nitrite-denitrifying granular sludge was obtained after approximately 40 days of cultivation in a 10 L upflow sludge blanket (USB) reactor. The nitrite removal efficiency was approximately 95% when the nitrite concentration was 50 mg L(-1)at an influent flow rate of 20 L h(-1). The nitrite granular sludge had several notable features including good settleability (110 m h(-1)), high ash content (79%), and high density (1.248 g cm(-3)). The mixed liquor suspended solids (MLSS) of the sludge bed remained at 130.04 g L(-1), at a hydraulic upflow velocity of 2 m h(-1). These interesting characteristics were attributed to a high effluent pH (9.7) caused by the release of alkalinity during the nitrite denitrification process. The surfaces of the granules were dominated by cocci bacteria with a diameter of approximately 3 μm, which could be classi?ed as Nitrosomonas-like species based on our analysis of 16 S rDNA sequences.     

Characteristics of granular sludge in a single upflow sludge blanket reactor treating high levels of nitrate and simple organic compounds.

Simultaneous denitrification and methanogenesis were accomplished in a single upflow sludge blanket (USB) reactor. More than 99% and 95% of nitrate and chemical oxygen demand (COD) removal rates were obtained at a loading of 600 mg NO3-N/L x d and 3,300 mg COD/L x d, respectively. The specific denitrification rate (SDR) increased as COD/NO3-N ratios decreased. Maximum SDR with acetate could reach 1.05 g NO3-N/gVSS x d. Significant sludge flotation was observed at the top of the reactor due to the change of microbial composition and the formation of hollow granules. Granules became fluffy and buoyant due to the growth of denitrifiers. Microscopic examination showed that granules exhibited layered structure and they were mainly composed of Methanosarcina sp., Pseudomonas sp., and rod-shaped bacteria.     

Analysing mass balance of viruses in a coagulation-ceramic microfiltration hybrid system by a combination of the polymerase chain reaction (PCR) method and the plaque forming units (PFU) method.

Virus removal experiments using river water spiked with bacteriophages were conducted by an in-line coagulation-ceramic microfiltration hybrid system to investigate the effects of filtration flux (62.5 and 125 L/(m2 x h)) and type of virus (Qbeta and MS2) on virus removal. In addition, the mass balance of viruses through the hybrid system was analysed by quantifying the infectious and inactive viruses by a combination of the polymerase chain reaction (PCR) method and the plaque forming units (PFU) method. Even when the system was operated at high filtration flux (125 L/(m2 x h)), high virus removal (> 6 log) with short coagulation time (2.4 s) was successfully achieved by dosing polyaluminium chloride (PACI) at more than 1.08 mg-Al/L. Removal performances were different between Qbeta and MS2, although their diameters are almost the same: greater virus removal was achieved for MS2 at PACI dosing of 0.54 mg-Al/L, and for Qbeta at PACI dosing of more than 1.08 mg-Al/L. The combination of the PCR and PFU methods revealed that two phenomena, adsorption to/entrapment in aluminium floc and virucidal activity of PACI, partially account for the high virus removal in the coagulation-MF hybrid system.     

Real-time PCR quantification of nitrite reductase (nirS) genes in a nitrogen removing fluidized bed reactor.

Molecular approaches were applied to identify and enumerate denitrifying bacteria subsisting in a fluidized bed reactor (FBR). The FBR was continuously operated as a unit for the removal of nitrogen from the effluents of domestic sewage treatment plant, with an additional supply of methanol as a carbon source. By denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S ribosomal RNA genes, Thauera group was found to be dominant among the denitrifying bacteria in the FBR sludge. Oligonucleotide probe THA155 for fluorescence in situ hybridization (FISH) was newly designed for specifically targeting the Thauera group. However, the THA155 signal obtained from the sludge was only 0.9-5.7% of the DAPI-stained total cells. The real-time polymerase chain reaction (PCR) targeting the sequences of nitrite reductase (NIR) gene, a key enzyme of denitrification processes, was performed to quantify the cells of denitrifying bacteria cells including the Thauera group in FBR sludge. An excellent correlation was obtained between the numbers of nirS genes and the activity of denitrifiers in the FBR sludge.     

Evaluation of an optimal fill strategy to biodegrade inhibitory wastewater using an industrial prototype discontinuous reactor.

This work presents the results and discussions of the application of an optimally controlled influent flow rate strategy to biodegrade, in a discontinuous reactor, a synthetic wastewater constituted by 4-chlorophenol. An aerobic automated discontinuous reactor system of 1.3 m3, with a useful volume of 0.75 m3 and an exchange volume of 60% was used. As part of the control strategy influent is fed into the reactor in such a way as to obtain the maximal degradation rate avoiding inhibition of microorganisms. Such an optimal strategy was able to manage increments of 4-chlorophenol concentrations in the influent between 250 and 1000 mg/L. it was shown that the optimally controlled influent flow rate strategy brings savings in reaction time and flexibility in treating high concentrations of an influent with toxic characteristics.     

Treatment of a low concentration industrial chemicals mixture in an UASB reactor.

A wastewater containing a mixture of methanol, isopropyl alcohol, ethylene glycol, acetic anhydride, methyl, ethyl and isopropyl acetate, acrylic acid, butyl and methyl acrylate, o, m and p-xylene and styrene was fed to an UASB reactor. Isopropanol addition diminished the removal efficiency to 60% and required a long adaptation time for its total mineralization. When acrylic acid was added to the mixture, the removal dropped to 83% and recovered after 40 days. As for the rest of the substrates, p-m-o-xylene addition had no effect on reactor performance, although in batch assays it showed low mineralization. Also the effect of volumetric organic load on removal efficiency was followed up. After diminishing the HRT to 4 and 3 h yielding 4.8 and 6.5 gCOD L(-1) d(-1), removal efficiencies decreased to 79 and 74% respectively.     

Nitrification of high-strength ammonium wastewater by a fluidized-bed reactor.

A laboratory-scale fluidized-bed reactor with an external aeration loop was used for nitrification of high-strength ammonium wastewater (up to 500 mg NH4-N/L). The results demonstrated that the system is capable of handling ammonium removal rates of up to 2.5 kg NH4-N/m3 x d, while removal efficiencies were as high as 98% and independent of the applied ammonium loading rates. Ammonium loading rates higher than 2.5 kg NH4-N/m3 x d resulted in decreased ammonium removal efficiency. The data show that near complete ammonium removal occurred at DO concentrations as low as 0.3-0.5 mg/L. However, the nitrite-nitrogen fraction in the effluent increased from 3.5% to 23.2% when the DO dropped from 1.0 mg/L to approximately 0.4 mg/L, respectively. The high specific removal rates in this system are one order of magnitude higher than that of suspended-growth systems. This can reduce the supplementary reactor volumes required for nitrification to less than 10% of that needed in conventional activated sludge systems. These results clearly indicate the potential economic gains that could be achieved through implementation of this technology.     

Optimal biodegradation of phenol and municipal wastewater using a controlled sequencing batch reactor.

This work presents the results of the application of an optimally controlled influent flow rate strategy to biodegrade, in a discontinuous reactor, a mixture of municipal wastewater and different concentrations of phenol when used as a toxic compound model. The influent is fed into the reactor in such a way to obtain the maximal degradation rate avoiding the inhibition of the microorganisms. Such an optimal strategy was able to manage increments of phenol concentrations in the influent up to 7000 mg/L without any problem. It was shown that the optimally controlled influent flow rate strategy is a good and reliable tool when a discontinuous reactor is applied to degrade an industrial wastewater.     

Treatment of distillery vinasse in a high rate anaerobic reactor using low density polyethylene supports.

An anaerobic fixed bed reactor, filled with small floating supports of polyethylene material (Bioflow 30) as inert media, was operated for 6 months to treat vinasse (wine residue after distillation). Bioflow 30 has a density of 0.93 and a specific area of 320 m2/m3. The experimental results showed that the efficiency of the reactor in removal of soluble COD was very good with a maximum organic loading rate of more than 30 g of COD/L x d and a COD removal efficiency of more than 80%. Bioflow 30 showed a high capability of biomass retention with 4-6 g of dried solids per support. Thus, at the end of the experiment, the fixed biomass represented 57 g of solids/L of reactor. The visual observation of the supports and the specific activity (0.54 g COD/g solids x d) of the fixed solids, which remained close to the values obtained with suspended biomass, showed that entrapment was playing an important role in the retention of the biomass inside the reactor. It was then possible to operate the reactor with a very high loading rate as the result of the increase of the solids in the reactor and the maintaining of the specific activity. Bioflow 30 is then an excellent support for use in a high rate anaerobic fixed bed.     

Bioaugmentation of a sequencing batch biofilm reactor by horizontal gene transfer.

Bioaugmentation by introduction of catabolic genes residing on mobile genetic elements into the microbial community of a soil or wastewater environment might be an alternative to bioaugmentation by addition of bacterial cells with chromosomally encoded catabolic genes. This study investigates the possibility to enhance degradation of the xenobiotic model compound 2,4-dichlorophenoxyacetic acid in a sequencing batch biofilm reactor (SBBR) by using the conjugative plasmid pJP4 carrying genes for 2,4-D degradation. After introduction of a plasmid donor strain to a lab-scale SBBR operated without 2,4-D, the number of plasmid-carrying cells first dropped, and then increased after switching to 2,4-D as the sole carbon source. The donor cells were unable to grow in the applied synthetic wastewater with 2,4-D as the sole carbon source. Transconjugants could be detected both by culture-dependent and culture-independent methods in the 2,4-D degrading biofilm. In contrast to 90% 2,4-D degradation in the bioaugmented reactor within 40 h, a control reactor which had not received the plasmid still contained 60% of the initial 2,4-D concentration after 90 h. This experiment clearly demonstrates the introduction of 2,4-D degradative genes into a microbial biofilm and indicates that horizontal gene transfer is a promising tool for bioaugmentation of reactors treating wastewater.     

Bench-scale study using PVA gel as a biocarrier in a UASB reactor treating corn steep liquor wastewater.

PVA-gel beads were used as a biocarrier for treatment of corn steep liquor wastewater containing high levels of volatile fatty acids (VFA), where retention of biomass could be either solely in the porous microstructure of the gel or by granule formation using a gel bead as a nucleus. With stable COD removal efficiencies of 90% or greater, continuous treatment was demonstrated over a four month period, with organic loading rates being increased stepwise from 2.5 to 22.5 kg COD/m3 d. In addition, VFA in the effluent were, with few exceptions maintained close to zero. Gas production increased over the course of the study and reached a level of 0.38 m3/kg COD consisting of 65% methane with the remainder being mostly carbon dioxide. Biomass granules containing methane producing bacteria progressively formed around the PVA-gel beads during the study. In contrast, very few small natural granules developed apart from PVA-gel nuclei indicating that PVA gel may serve well as a seeding material to enhance granulation when natural occurrence is lacking.     

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.