Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment

Bacteria were detected at five stages of municipal wastewater treatment using TaqMan(R) real-time quantitative PCR (qPCR). Thirteen probe and primer sets were tested for diverse pathogens that may be present in wastewater, including Aeromonas hydrophila, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, E. coli O157:H7, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The sensitivity of the assay was 100 fg of genomic DNA (=22 gene copies), based on a standard curve generated using A. hydrophila purified DNA. Samples from five stages of wastewater treatment were collected, including raw wastewater, primary effluents, mixed liquor, waste activated sludge and final effluents. In duplicate samples, E. coli, K. pneumoniae, C. perfringens and E. faecalis were detected throughout the wastewater process, and their numbers decreased by 3.52-3.98, 4.23-4.33, 3.15-3.39, and 3.24 orders of magnitude respectively, between the raw wastewater and final effluent stage. This qPCR method was effective for the detection of pathogens in wastewater and confirmed that the risk of exposure to pathogens in the wastewater discharge was well within the Environment Canada guidelines.     

Direct quantification of bacterial biomass in influent, effluent and activated sludge of wastewater treatment plants by using flow cytometry

A rapid multi-step procedure, potentially amenable to automation, was proposed for quantifying viable and active bacterial cells, estimating their biovolume using flow cytometry (FCM) and to calculate their biomass within the main stages of a wastewater treatment plant: raw wastewater, settled wastewater, activated sludge and effluent. Fluorescent staining of bacteria using SYBR-Green I + Propidium Iodide (to discriminate cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was applied to count bacterial cells by FCM. A recently developed specific procedure was applied to convert Forward Angle Light Scatter measured by FCM into the corresponding bacterial biovolume. This conversion permits the calculation of the viable and active bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial biomass represented only a small part of particulate COD in raw wastewater (4.8 ± 2.4%), settled wastewater (10.7 ± 3.1%), activated sludge (11.1 ± 2.1%) and effluent (3.2 ± 2.2%). Active bacterial biomass counted for a percentage of 30-47% of the viable bacterial biomass within the stages of the wastewater treatment plant.     

Behaviour of pathogenic and indicator bacteria during urban wastewater treatment and sludge composting, as revealed by quantitative PCR

Two enteric pathogens, Salmonella spp. and Campylobacter jejuni, and two bacteria commonly used as indicators, Escherichia coli and Clostridium perfringens, were monitored using quantitative real-time PCR during municipal wastewater treatment and sludge composting. The results were compared with those obtained using standard culture methods. A reduction of all bacteria was observed during wastewater treatment and during the thermophilic phase of composting. However, the bacterial groups studied behaved differently during the process, and the main differences were observed during biological treatment in activated sludge basins. In particular, Salmonella spp. and C. jejuni survived better during activated sludge treatment than E. coli. C. jejuni was the most resistant to wastewater treatment among the four bacterial groups. Overall, differences in survival were observed for all bacteria studied, when submitted to the same environmental pressure. This holds both for differences between indicators and pathogenic bacteria and between pathogenic bacteria. These results show the difficulty in defining reliable indicators.     

Estimation of viable biomass in wastewater and activated sludge by determination of ATP, oxygen utilization rate and FDA hydrolysis

ATP content, oxygen utilization rate (OUR) and fluorescein diacetate (FDA) hydrolysis were tested for the ability to express the amount of viable biomass in wastewater and activated sludge.The relationship between biomass and these activity parameters was established in growth cultures made by inoculating a nutrient medium with either wastewater or activated sludge. Biomass was then determined directly by measurement of dry weight of growth culture (dw), and compared to data obtained by using the previously mentioned methods. In the exponential growth phase, ATP content showed the best correlation with biomass, while FDA hydrolysis in the sludge failed to show any such correlation. Conversion factors of 3 mg ATP/g dw, 300 mg O₂/h g dw and 0.4 A/h (mg dw/ml) for ATP, OUR and FDA methods, respectively, were calculated.When the methods were applied for in situ determinations in four different wastewater plants, it was found that ATP content and respiration rate estimated viable biomass to range from 81 to 293 mg dw/g SS for raw wastewater and from 67 to 187 mg dw/g SS for activated sludge with a rather weak correlation between ATP and respiration measurements. The FDA hydrolysis estimated viable biomass to be higher than suspended solids, for which reason this parameter could not be recommended for determination of viable biomass in wastewater and activated sludge.     

Optimization of PCR-based methods for rapid detection of Campylobacter jejuni, Campylobacter coli and Yersinia enterocolitica serovar 0:3 in wastewater samples

PCR-based methods were evaluated for their adequacy to assess the removal of pathogens from wastewater samples. For the development and optimization of the methods, samples were taken at two different sites from two different constructed wetlands. Campylobacter jejuni/coli and Yersinia enterocolitica serogroup 0:3 were selected as model pathogens and Enterococcus faecalis as a standard microbiological indicator. The chosen PCR protocols were optimized for wastewater DNA extracts in order to obtain high sensitivity and reproducibility independently of the background flora. All PCR protocols were successfully performed and reproducible with a background of up to 10(10) nontarget cells per reaction. Five cells of Y. enterocolitica, 50 cells of C. jejuni/coli, and 500 cells of E. faecalis per 100ml treated water could be detected. The method detection limit in the settled wastewater was higher: 200 cells per 100ml for Y. enterocolitica, 2000 cells per 100ml for C. jejuni/coli, and 20,000 cells per 100ml for E. faecalis. C. jejuni/coli and Y. enterocolitica PCRs were adapted to municipal wastewater, with higher loads of potential PCR inhibitors. Sensitivity was lower for this type of wastewater: 200 cells of Y. enterocolitica and 2000 cells of C. jejuni/coli were detected per 100ml treated wastewater, 2500 cells of Y. enterocolitica and 25,000 cells of C. jejuni/coli per 100ml settled wastewater. The developed PCR methods enable the detection of C. jejuni/coli, Y. enterocolitica serogroup 0:3 and E. faecalis within 12h. They show specificity, reproducibility and low detection limits for the investigated pathogens.     

Micro-profiles of activated sludge floc determined using microelectrodes

The microbial activity within activated sludge floc is a key factor in the performance of the activated sludge process. In this study, the microenvironment of activated sludge flocs from two wastewater treatment plants (Mill Creek Wastewater Treatment Plant and Muddy Creek Wastewater Treatment Plant, with aeration tank influent CODs of 60-120 and 15-35 mg/L, respectively) were studied by using microelectrodes. Due to microbial oxygen utilization, the aerobic region in the activated sludge floc was limited to the surface layer (0.1-0.2mm) of the sludge aggregate at the Mill Creek plant. The presence of an anoxic zone inside the sludge floc under aerobic conditions was confirmed in this study. When the dissolved oxygen (DO) in the bulk liquid was higher than 4.0mg/L, the anoxic zone inside the activated sludge floc disappeared, which is helpful for biodegradation. At the Muddy Creek plant, with its lower wastewater pollutant concentrations, the redox potential and DO inside the sludge aggregates were higher than those at the Mill Creek plant. The contaminant concentration in the bulk wastewater correlates with the oxygen utilization rate, which directly influences the oxygen penetration inside the activated sludge floc, and results in redox potential changes within the floc. The measured microprofiles revealed the continuous decrease of nitrate concentration inside the activated sludge floc, even though significant nitrification was observed in the bulk wastewater. The oxygen consumption and nitrification rate analyses reveal that the increase of ammonia flux under aerobic conditions correlates with nitrification. Due to the metabolic mechanisms of the microorganisms in activated sludge floc, which varies from one treatment plant to another, the oxygen flux inside the sludge floc changes accordingly.     

Microorganisms in bioaerosol emissions from wastewater treatment plants during summer at a Mediterranean site

Measurements were conducted at a Mediterranean site (latitude 35 degrees 31' north and longitude 24 degrees 03' east) during summer, to study the concentration of microorganisms emitted from a wastewater treatment plant under intensive solar radiation (520-840 W/m2) and at elevated air temperatures (25-31 degrees C). Air samples were taken with the Air Sampler MAS 100 (Merck) at each stage of an activated-sludge wastewater treatment (pretreatment, primary settling tanks, aeration tanks, secondary settling tanks, chlorination, and sludge processors). Cultivation methods based on the viable counts of mesophilic heterotrophic bacteria, as well as of indicator microorganisms of faecal contamination (total and faecal coliforms and enterococci), and fungi were performed. During air sampling, temperature, solar radiation, relative humidity and wind speed were measured. The highest concentrations of airborne microorganisms were observed at the aerated grit removal of wastewater at the pretreatment stage. A gradual decrease of bioaerosol emissions was observed during the advanced wastewater treatment from the pretreatment to the primary, secondary and tertiary treatment (97.4% decrease of mesophilic heterotrophic bacteria, and 100% decrease of total coliforms, faecal coliforms and enterococci), 95.8% decrease of fungi. The concentration of the airborne microorganisms at the secondary and tertiary treatment of the wastewater was lower than in the outdoor control. At the same time, the reduction of the microbial load at the waste sludge processors was 19.7% for the mesophilic heterotrophic bacteria, 99.4% for the total coliforms, and 100% for the faecal coliforms and the enterococci, 84.2% for the fungi. The current study concludes that the intensive solar radiation, together with high ambient temperatures, as well as optimal wastewater treatment are the most important factors for low numbers of microbes in the air.     

Bacterial population changes in hospital effluent treatment plant in central India

Hospital effluent with its high content of multidrug resistant (MDR) enterobacteria and the presence of enteric pathogens could pose a grave problem for the community. It was planned at our tertiary care hospital in central India to study the population changes at various steps of effluent treatment plant (ETP) like collection, aeration, clarification, liquid sludge, dried sludge, high-pressure filter and treated wastewater. The study included viable bacterial counts, coliform counts, staphylococcal, enterococcal, Pseudomonas and multiple drug resistant (MDR) gram negative bacterial counts in the different stages of ETP. In order to study the distribution of bacteria as free floating in liquid and adherent to suspended particles, enumeration of the bacteria in the filterate and the sediment was also carried out. The effluent input showed 55% of the 8.6 x 10(6)/ml bacteria as coliforms and E. coli which was a typical of fecal flora. The prevalence of MDR coliforms was 0.26%. The substantial reduction (> 3log) was seen for the effluent coming from the clarifier. The bulk of the bacteria in the hospital effluent remains firmly adhered to solid particles; aeration and clarification removes bulk of the bacteria by physical processes like flocculation. The treated liquid effluent still contains sizeable loads of MDR bacteria and inactivation by procedure such as chlorination is required. The bacteria get concentrated in sludge and a greater concentration of chlorine is required for decontamination.     

Phenotypic fingerprinting of microbial communities in wastewater treatment systems

Community-level characterization of microbial biomass from a municipal activated sludge plant, and two bleached kraft mill effluent (BKME) treatment systems, an oxygenated activated sludge system and an aerated lagoon system, was carried out using the BIOLOG redox-based carbon-substrate utilization assay. Several extraction procedures for separating microbial cells from the dark colloidal samples, including culture enrichment, homogenization, gravel agitation and sonication, were evaluated in order to overcome interference with colour development in the assay. Optimal microbial recovery was achieved by homogenization in the presence of deflocculating agents, sodium pyrophosphate and Tween 80. Principal component analysis was used to differentiate among the microbial communities in the wastewater treatment systems. Biomass obtained from each of the reactors displayed different composite metabolic profiles suggesting a unique indigenous microbial population in each system. Characterization of separate bacterial and protozoal components of one community also showed that each fraction displayed different substrate utilization patterns. Substitution of the already prepared BIOLOG panel substrates with individual chemicals typically associated with BKME allowed for the determination of biodegradation potential in wastewater treatment systems.     

自固定化高效菌种强化处理焦化废水研究

以喹啉为唯一碳源驯化高铲菌种,将其一部分附着在陶粒载体上,比较了自化前后菌种活性的变化,然后在用活性污泥处理焦化废水时,以三种投加高效菌种的方式强化处理焦化废水：（１）只投加悬浮高效菌种,（２）投加悬浮高铲菌种和空白陶粒,（３）投加附着有效高效菌种的陶粒,研究了不同投加方式对保持菌种高效降解特性的作用。     

Airborne enteric coliphages and bacteria in sewage treatment plants

The concentrations of airborne culturable microorganisms were determined in wastewater and sludge treatment processes of seven sewage treatment plants. Two types of coliphages, Salmonella and total viable bacteria were sampled by the BioSampler and the numbers of faecal coliforms and enterococci were obtained from the Andersen 6-stage impactor. The BioSampler recovered higher numbers of airborne coliphage viruses than has been measured with other liquid samplers in previous studies, suggesting that this sampler has improved efficiency for sampling airborne coliphages. Airborne coliphages were detected in many stages of the wastewater or sludge treatment process. The highest microbiological air contaminations were found in pre-treatment and aerated grit separation stages of the operation. This was attributed to aerosolisation of microorganisms by mechanical handling or forced aeration. Aeration and settling processes located outdoors caused low microbial concentrations, but the brush aerator released more microorganisms into the air. Our results emphasize the necessity for controlling the exposure of sewage workers to airborne microorganisms, especially in process areas that involve mechanical agitation or forced aeration of wastewater.     

Total nitrogen removal in a hybrid, membrane-aerated activated sludge process

The hybrid (suspended and attached growth) membrane biofilm process (HMBP) is a novel method to achieve total nitrogen removal from wastewater. Air-filled hollow-fiber membranes are incorporated into an activated sludge tank, and a nitrifying biofilm develops on the membranes, producing nitrite and nitrate. By suppressing bulk aeration, the bulk liquid becomes anoxic, and the nitrate/nitrite can be reduced with influent BOD. The key feature that distinguishes the HMBP from other membrane-aerated processes is that it is hybrid; heterotrophic bacteria are kept mainly in suspension by maintaining low bulk liquid BOD concentrations. We investigated the HMBP's performance under a variety of BOD and ammonium loadings, and determined the dominant mechanisms of nitrogen removal. Suspended solids increased with the BOD loadings, maintaining low bulk liquid BOD concentrations. As a result, nitrification rates were insensitive to the BOD loadings, remaining at 1gNm(-2)day(-1) for BOD loadings ranging from 4 to 17gBODm(-2)day(-1). Nitrification rates decreased during short-term spikes in bulk liquid BOD concentrations. Shortcut nitrogen removal was confirmed using microsensor measurements, showing that nitrite was the dominant form of oxidized nitrogen produced by the biofilm. Fluorescence in situ hybridization (FISH) showed that ammonia oxidizing bacteria (AOB) were dominant throughout the biofilm, while nitrite oxidizing bacteria (NOB) were only present in the deeper regions of the biofilm, where the oxygen concentration was above 2mg/L. Denitrification occurred mainly in the suspended phase, instead of in the biofilm, decreasing the potential for biofouling. When influent BOD concentrations were sufficiently high, full denitrification occurred, with total nitrogen (TN) removal approaching 100%. These results suggest that the process is well-suited for achieving concurrent BOD and TN removal in activated sludge.     

Determination of virus abundance, diversity and distribution in a municipal wastewater treatment plant

Procedures to extract and count sludge viruses-like particles from municipal sewage treatment plant were optimized by epifluorescence microscopy using SYBR Green I as a stain. The highest indigenous virus yields from the bulk of the anaerobic digestion sludge and influent (solid) were obtained by utilizing 10 mM sodium pyrophosphate as eluant solution with vortex and 1 min of sonication. The use of 1× phosphate buffered saline as eluant with vortex and 1 min of sonication yields highest indigenous virus from activated sludge. The efficiency of extracting indigenous viruses by sodium pyrophosphate-ultrasound treatment was about 62% of the extractable virus particles from activated sludge and 87% for anaerobic digestion sludge, respectively. Samples treated with DNase had decreased, but not significant, virus counts, suggesting a minor effect of extracellular DNA on virus count. Following the optimized procedure, we investigated the abundance and diversity of virus particles in the wastewater stream of a municipal treatment plant. The concentrations of virus particles ranged from 0.28 × 10⁹ ml⁻¹ to 27.04 × 10⁹ ml⁻¹. Transmission electron microscopy (TEM) showed a high variety of virus morphotypes in sludge. Pulsed-field gel electrophoresis (PFGE) revealed a diverse and dynamic viral community in different stages of the system with genome sizes ranging from 33 kb to >350 kb with most of the viral DNA in the 30-80 kb and 200-350 kb size ranges. Collectively, our study suggested that indigenous viruses are abundant and dynamic in the municipal wastewater treatment system and may play an important role in functioning of the system.     

Fecal coliform removal in wastewater treatment plants studied by plate counts and enzymatic methods

Twelve wastewater treatment plants (WWTPs) were sampled in France and Belgium in 1999 and 2000 in order to estimate the fecal coliform (FC) removal efficiency of various types of treatment. Only one of these WWTPs was equipped with a specific step to eliminate microorganisms (UV disinfection preceded by sand filtration). FC abundance was measured in raw and treated sewage by plate counts on selective medium and rapid beta-D-glucuronidase (GLUase)-based assays. Removal of culturable FC was the most efficient in treatments with high retention time (activated sludge process with nitrification and denitrification, lagooning), in biofiltration and in the treatment with a tertiary disinfection step. GLUase activity measurements showed the same removal pattern as plate counts except for UV disinfection, where no reduction of GLUase activity was measured. Specific loads of culturable FC and GLUase activity, i.e. daily amounts of culturable FC or GLUase activity in sewage per inhabitant-equivalent, were calculated in raw and treated wastewater for the different WWTPs.     

Bacteria and fungi in aerosols generated by two different types of wastewater treatment plants

Raw wastewater is a potential carrier of pathogenic microorganisms and may pose a health risk when pathogenic microorganisms become aerosolized during aeration. Two different types of wastewater treatment plants were investigated, and the amounts of cultivable bacteria and fungi were measured in the emitted aerosols. Average concentrations of 17,000 CFU m(-3) of mesophilic, 2,100 CFU m(-3) of TSA-SB bacteria (bacteria associated with certain waterborne virulence factors), 1700 CFU m(-3) of mesophilic and 45 CFU m(-3) of thermotolerant fungi, were found in the aerosol emitted by the aeration tank of the activated sludge plant. In the aerosol of the fixed-film reactor 3000 CFU m(-3) mesophilic and 730CFUm(-3) TSA-SB bacteria, and 180 CFUm(-3) mesophilic and 14 CFU m(-3) thermotolerant fungi were measured. The specific emissions per population equivalent between the two types of treatment plants differed by two orders of magnitude. The microbial flux based on the open water surface area of the two treatment plants was similar. The aerosolization ratios of cultivable bacteria (expressed as CFU m(-3) aerosol/m(-3) wastewater) ranged between 8.4 x 10(-11) and 4.9 x 10(-9). The aerosolization ratio of fungi was one to three orders of magnitude higher and a significant difference between the two types of treatment plants could be observed.     

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria were investigated in two large-scale municipal treatment plants during a period of six months. Total and relative numbers of resistant bacteria were determined in raw sewage, treated sewage and anaerobically digested sludge by bacteriological counts on media selective for coliforms (MacConkey agar) and Acinetobacter spp. (Baumann agar). In addition, the level of antimicrobial susceptibility was determined by the disc-diffusion method in 442 Acinetobacter isolates identified by colony hybridisation with a genus-specific DNA probe. Independent of the different antibiotics and media used, the total numbers of resistant bacteria in treated sewage were 10-1000 times lower than in raw sewage. Based on linear regression analysis of data on bacteriological counts, the prevalences of antimicrobial-resistant presumptive coliforms and Acinetobacter spp. in treated sewage and digested sludge were not significantly higher compared with raw sewage. On the contrary at one plant, statistically significant decreases were observed in the prevalence of ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0188) following sewage treatment, and in the prevalence of either ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0013) or ampicillin- and gentamicin-resistant presumptive coliforms (p = 0.0273 and p = 0.0186) following sludge treatment. The results obtained by bacteriological counts were confirmed by antimicrobial susceptibility testing of Acinetobacter isolates. Based on logistic regression analysis, isolates from treated sewage and digested sludge were generally not significantly more resistant compared with isolates from raw sewage. Based on these evidences, it was concluded that tertiary wastewater treatment did not result in a selection of antimicrobial resistant bacteria.     

Microbiological aspects of a bioreactor with submerged membranes for aerobic treatment of municipal wastewater

An aerobic membrane bioreactor treating municipal wastewater at complete biomass retention was studied in respect of microbiological parameters over a period of 380 days. The results were compared to those obtained from a conventional activated sludge wastewater treatment plant (WWTP) treating the same wastewater. Microscopically, significant changes in the structure of the flocs and of the ratio between free suspended and aggregated cells could be observed. The presence of filamentous bacteria varied from almost not present to very high numbers. With the exception of short periods after changes in operating conditions, protozoa and metazoa were rarely present in the sludge community. The rate of oxygen consumption and the cell detectability by fluorescence in situ hybridizatio (FISH) with rRNA-targeted oligonucleotide probes were used to assess the physiological state of the bacterial cells Oxygen consumption rates of sludge samples obtained from both the conventional and membrane filtration plant wer determined without and after addition of different energy and carbon sources. In contrast to the conventional activate sludge, a pronounced increase in respiration activity upon the addition of organic substrates could be observed in th membrane filtration sludge. In situ probing with the Bacteria-specific probe EUB338 visualized 40-50% of all DAPI stainable bacteria in the membrane bioreactor, compared to 80% cells detectable by FISH in the conventional activate sludge. These results suggest that bacteria present in the highly concentrated biomass of the membrane reactor use the energy supplied for their maintenance metabolism and were not in a physiological state characteristic for growth This assumption could explain the zero net biomass production observed in the reactor.     

Bacteriophages--potential for application in wastewater treatment processes

Bacteriophages are viruses that infect and lyse bacteria. Interest in the ability of phages to control bacterial populations has extended from medical applications into the fields of agriculture, aquaculture and the food industry. Here, the potential application of phage techniques in wastewater treatment systems to improve effluent and sludge emissions into the environment is discussed. Phage-mediated bacterial mortality has the potential to influence treatment performance by controlling the abundance of key functional groups. Phage treatments have the potential to control environmental wastewater process problems such as: foaming in activated sludge plants; sludge dewaterability and digestibility; pathogenic bacteria; and to reduce competition between nuisance bacteria and functionally important microbial populations. Successful application of phage therapy to wastewater treatment does though require a fuller understanding of wastewater microbial community dynamics and interactions. Strategies to counter host specificity and host cell resistance must also be developed, as should safety considerations regarding pathogen emergence through transduction.     

Effect of chlorine on filamentous microorganisms present in activated sludge as evaluated by respirometry and INT-dehydrogenase activity

Activated sludge technology is more used than any other for biological treatment of wastewater. However, filamentous bulking is a very common problem in activated sludge plants, chlorine being the chemical agent normally used to control it. In this work the effect of chlorine on microorganisms present in activated sludge flocs was assessed by a respirometric technique (oxygen uptake rate, OUR) and by the INT-dehydrogenase activity test (DHA) measured by two techniques: spectrophotometry (DHA(a)) and image analysis (DHA(i)). Both DHA tests were optimized and correlated with the respirometric technique (OUR) using pure cultures of a filamentous microorganism (Sphaerotilus natans) under chlorine inhibition. Using these correlations the tested methods were applied to determine the action of chlorine on respiratory activity in activated sludge. The OUR and the DHA(a) quantifies the action of chlorine on the total respiratory activity (RA) of flocs (filamentous and floc-forming bacteria); in contrast, the DHA(i) test evaluates specific action of chlorine on the RA of filamentous microorganisms. In activated sludge flocs containing filamentous microorganisms, a chlorine dose of 4.75 mgCl(2) (gVSS)(-1) with a contact time of 20 min reduced about 80% of the RA of filamentous bacteria while affecting only 50-60% of the total RA of flocs. Besides, a chlorine dose of 7.9 mgCl(2) (gVSS)(-1) produced the total respiratory inactivation of filamentous microorganisms after 10 min contact, however, with this dose the total RA of activated sludge flocs was reduced only about 45-65%; controlling filamentous bulking without affecting too much floc-forming bacteria. At the tested chlorine concentrations the inhibition of filamentous microorganisms was higher than in the whole activated sludge. Although floc-forming microorganisms were demonstrated to be more susceptible to chlorine than filamentous in pure cultures, results obtained in the present work confirmed that it is the location of the filamentous microorganisms in the flocs and the presence of extracellular polymer substances which largely determines their higher susceptibility to chlorine; consequently this feature plays a critical role in bulking control.     

Aerobic storage by activated sludge on real wastewater

Activated sludge processes are often operated under dynamic conditions, where the microbial response can include, besides of growth, several COD removal mechanisms, and particularly the storage in form of polymers. While abundant evidence of aerobic storage under dynamic conditions with synthetic substrates can be found (Majone et al., Water Sci. Technol. 39(1) (1999) 61), there is still little knowledge about COD removal mechanisms with real activated sludge and wastewater. The aim of the present paper is therefore to give a direct evidence of storage phenomena occurring when a real sludge is mixed with influent wastewater and of their influence onto OUR profiles in typical respirometric batch tests. For this purpose, respirometric batch tests were performed on the same sludge by using acetate, filtered wastewater and raw wastewater as carbon source along with determination of acetate uptake and storage polymer formation. Comparison of results obtained has shown that poly-3-hydroxybutyrate (PHB) storage gives always the main contribution to acetate removal and that in the case of wastewater PHB is also formed from other substrates. PHB formation clearly occurs during the high-rate RBCOD-phase, however for wastewater it accounts for only a fraction (18-22%) of overall RBCOD removal, so calling for other unidentified storage compounds or other non-storage phenomena. In the low-rate SBCOD phase of respirogram PHB is clearly utilised in tests with acetate as internal reserve material once the acetate is depleted. In tests with filtered and raw wastewater the PHB concentration decreases much slower, probably because more PHB is formed due to the availability of external SBCOD (soluble and not). Moreover, reported OUR in the SBCOD-phase from filtered or raw wastewater are quite higher than those reported in batch tests with acetate, so confirming a main contribution of external SBCOD. However, the respective contributions for utilisation of previously stored compounds and of external SBCOD cannot be easily separated by the comparison of tests on filtered and raw wastewater, because both substrates are simultaneously present also in tests with the filtered wastewater. As a side consequence, the chemical-physical method for evaluation of true soluble and biodegradable COD tends to overestimate the respirometry-based RBCOD, at least for the wastewater under observation. Even though modelling by ASM3 (Gujer et al., Water Sci. Technol. 39(1) (1999) 183) makes it possible to well describe the whole experimental behaviour, it requires that much more storage compounds are formed than the experimentally observed PHB. These compounds have still to be identified and quantified in order to confirm the conceptual structure of ASM3.