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Optimizing granules size distribution is critical for both reactor performance and stability. In this research, an optimal size range of 1800 to 3000 μm was proposed regarding mass transfer and granules stability based on granules developed at DO around 8.0 mg L−1 with the feed COD:N:P at 100:5:1. A height-adjustable influent strategy was applied to facilitate the nutrient storage of granules at optimum size range via microbial selective pressure. Results suggested insufficient hydraulic shear stress led to overgrowth of granules size. High abundance of filamentous bacteria (Thiothrix sp.) was observed in oversized granules, which detached and affected the remaining granules, resulting in severe sludge bulking. Strong hydraulic shear stress suppressed uncontrolled growth of granules. However, fewer abundance of simultaneous nitrification and denitrification (SND) bacterium was acquired, which led to unfavored SND effect and total nitrogen (TN) removal efficiency. The height-adjustable influent strategy facilitated the poly-β-hydroxybutyrate (PHB) storage of granules at optimum size range, while limiting the overgrowth of granules size. Additionally, more than 87.51% of total granules situated in optimal sizes range, which led to higher abundance of SND bacterium and higher TN removal efficiency.

Many sources of wastewater contain sulfides, which can cause excessive growth of filamentous bacteria such as Thiothrix sp. resulting in bulking sludge in conventional activated sludge systems. Granular sludge systems could potentially also suffer from the growth of filamentous bacteria. Uptake of easily degradable COD by the relatively slow growing Ca. Accumulibacter phosphatis bacteria and the absence of strong diffusion gradients due to plug flow feeding through the settled granular sludge bed are assumed to be the dominant factors for successful granulation. Sulfides will remain after this anaerobic phase and cause growth of sulfide-consuming bacteria such as Thiothrix sp. Here we observed the impact of growth of Thiothrix sp bacteria in a laboratory aerobic granular sludge reactor by feeding a mixture of acetate and thiosulfate in the influent. Thiothrix sp, proliferated when 18% of the influent COD was due to thiosulfate, forming 51.4 ± 8.3% of the total granular biomass. Despite the strong presence of these filamentous bacteria a well settling sludge was maintained (SVI10 equal to 13.3 mL/g). These results confirm that sludge morphology is not necessarily a reflection of the cell morphology of the bacteria, but is highly influence by reactor operation. It also reiterates the fact that compact biofilms are formed when the substrate consumption rate is lower than the substrate transport rate.

Though sludge foaming often occurs and thus causes serious membrane fouling in membrane bioreactors (MBRs), the fouling mechanisms related with the foaming phenomenon have not been well addressed, hindering better understanding and solving foaming problem. In this work, it was interestingly found that, the foulants during the foaming period possessed extremely high specific filtration resistance (SFR) (over 1016 m kg−1) and strong adhesion ability to membrane surface. Chemical characterization showed that the proteins (178.57 mg/L) and polysaccharides (209.21 mg/L) in the foaming sample were about 6.4 times and 5.4 times of those in the supernatant sample, suggesting existence of a mechanism permitting continuous production of these foulants in the MBR during the foaming period. It was revealed that the fouling caused by foams was associated with gel layer filtration process, and the extremely high SFR can be interpreted by chemical potential change in the gel filtration process depicted in Flory-Huggins theory. Meanwhile, analyses by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory showed that the strong adhesion ability stemmed from the high interaction energy between the foaming foulants and membrane surface. In addition, 16S rDNA gene sequencing identified that the abundance of the foaming related bacteria species in the sludge suspension during the foaming period was more than 10 times of that during the non-foaming period. This study offered new mechanism insights into foaming fouling in MBRs.

Targeted inactivation of bacteria using bacteriophages has been proposed in applications ranging from bioengineering and biofuel production to medical treatments. The ability to differentiate between desirable and undesirable organisms, such as in targeting filamentous bacteria in activated sludge, is a potential advantage over conventional disinfectants. Like conventional disinfectants, bacteriophages exhibit non-linear concentration-time (Ct) dynamics in achieving bacterial inactivation. However, there is currently no workable model for predicting these observed non-linear inactivation rates. This work considers an approach to predicting bacteriophage-induced inactivation rates by utilizing classical particle aggregation theory. Bacteriophage-bacteria interactions are represented as a two-step process of transport by Brownian motion, differential settling, and shear, followed by attachment. Modifying classical expressions for particle-particle aggregation to include bacterial growth, death, and bacteriophage reproduction, the model was calibrated and validated using literature data. The calibrated model captures much of the observed non-linearity in inactivation rates and reasonably predicts the final host concentration. This model was shown to be most useful in systems more likely to reflect an industrial setting, where the initial multiplicity of infection, or MOI (the ratio of bacteriophage to host organisms), was 1 or greater. For systems of an initial MOI of less than 1 the model showed increased sensitivity to changes in input parameters and a less pronounced ability to reasonably predict inactivation rates.

The positive roles of N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) in aerobic granular sludge (AGS) have been widely acknowledged. However, it is not feasible to manipulate granulation via direct addition of AHL chemicals or AHL-producing strains. Here, several strains with high AHL-producing capacity were successfully isolated from AGS. These QS strains were cultivated, mixed as a consortium, and then divided into two groups: AHLs supernatant and bacterial cells encapsulated in sodium alginate (CEBs). The potential of QS regulation, via doses of AHLs supernatant and CEBs, in accelerating granulation was evaluated. Results clearly indicated that short-term (days 21–70) addition of AHLs supernatant led to a rapid specific growth rate (0.08 d−1), compact structure without filamentous bacteria overgrowth, excellent settlement performance (SVI10 37.2 mL/g), and a high integrity coefficient (4.4%) of the granules. Sustainable release of AHLs (mainly C6- and C8-HSL) was induced by exogenous AHLs, possibly attributed to the enrichment of the genera Aeromonas and Pseudomonas. Further, tryptophan and aromatic protein substances were produced to maintain structural stability, suggesting that short-term QS regulation had long-term positive effects on the characteristics of AGS. By comparison, the addition of CEBs posed negligible or negative impact on the granulation, as evidenced by the rupture of smaller aggregates and poor characteristics of AGS. Overall, augmentation of the signaling content via addition of AHLs supernatant from QS strains is an economical and feasible regulation strategy to accelerate granulation and sustain long-term structural stability.

This study revealed that, Thiothrix eikelboomii, a well-known filamentous bacterium that causes sludge bulking, could also interfere oxygen transfer during wastewater treatment. The volumetric oxygen transfer coefficient (KLa) in filamentous-bulking sludge (FBS) was found to be 43% lower than that in floc-forming sludge (FFS) at similar biomass concentrations, partially because the filamentous bacteria had increased the sludge apparent viscosity. The KLa value for FBS, however, was still significantly lower than that for FFS even if both sludges had similar apparent viscosity. Numerous tiny and free-swimming filaments were observed to attach on the air bubble surface, presumably reducing the liquid film renewal and increasing the liquid film thickness. Moreover, the filaments were co-coated with extracellular polymeric substances of protein and polysaccharide, which could make them performing like “amphiphilic molecules” of surfactants to hinder oxygen transfer. Therefore, the particular surface property of filaments and their interaction with air bubbles could also impact oxygen transfer. Thiothrix eikelboomii was identified to be the responsible filamentous bacterium that lowered the KLa value, while other filamentous bacteria with short filaments did not interfere oxygen transfer. This study implies that controlling sludge bulking benefits not only sludge settling but also oxygen transfer.

The objective of this work is to identify relevant settling velocity and rheology model parameters and to assess the underlying filamentous microbial community characteristics that can influence the solids mixing and transport in secondary settling tanks. Parameter values for hindered, transient and compression settling velocity functions were estimated by carrying out biweekly batch settling tests using a novel column setup through a four-month long measurement campaign. To estimate viscosity model parameters, rheological experiments were carried out on the same sludge sample using a rotational viscometer. Quantitative fluorescence in-situ hybridisation (qFISH) analysis, targeting Microthrix parvicella and phylum Chloroflexi, was used. This study finds that M. parvicella - predominantly residing inside the microbial flocs in our samples - can significantly influence secondary settling through altering the hindered settling velocity and yield stress parameter. Strikingly, this is not the case for Chloroflexi, occurring in more than double the abundance of M. parvicella, and forming filaments primarily protruding from the flocs. The transient and compression settling parameters show a comparably high variability, and no significant association with filamentous abundance. A two-dimensional, axi-symmetrical computational fluid dynamics (CFD) model was used to assess calibration scenarios to model filamentous bulking. Our results suggest that model predictions can significantly benefit from explicitly accounting for filamentous bulking by calibrating the hindered settling velocity function. Furthermore, accounting for the transient and compression settling velocity in the computational domain is crucial to improve model accuracy when modelling filamentous bulking. However, the case-specific calibration of transient and compression settling parameters as well as yield stress is not necessary, and an average parameter set - obtained under bulking and good settling conditions - can be used.

Filamentous bacteria can impact on the physical properties of flocs in the activated sludge process assisting solid-liquid separation or inducing problems when bacteria are overabundant. While filamentous bacteria within the flocs are understood to increase floc tensile strength, the relationship between protruding external filaments, dewatering characteristics and floc stability is unclear. Here, a quantitative methodology was applied to determine the abundance of filamentous bacteria in activated sludge samples from four wastewater treatment plants. An automated image analysis procedure was applied to identify filaments and flocs and calculate the length of the protruding filamentous bacteria (PFB) relative to the floc size. The correlation between PFB and floc behavior was then assessed. Increased filament abundance was found to increase interphase drag on the settling flocs, as quantified by the hindered settling function. Additionally, increased filament abundance was correlated with a lower gel point concentration leading to poorer sludge compactability. The floc strength factor, defined as the relative change in floc size upon shearing, correlated positively with filament abundance. This influence of external protruding filamentous bacteria on floc stability is consistent with the filamentous backbone theory, where filamentous bacteria within flocs increase floc resistance to shear-induced breakup. A qualitative correlation was also observed between protruding and internal filamentous structure. This study confirms that filamentous bacteria are necessary to enhance floc stability but if excessively abundant will adversely affect solid-liquid separation. The tools developed here will allow quantitative analysis of filament abundance, which is an improvement on current qualitative methods and the improved method could be used to assist and optimize the operation of waste water treatment plants.

Rapid sand filtration (RSF), proceeded by chemical coagulation and flocculation, is a commonly used, effective pretreatment in the desalination industry. We designed and tested a novel, large pilot-scale, two-stage granular Rapid Bioflocculation Filter (RBF) based on a first-stage Bioflocculator (BF) unit followed by a mixed-media bed filter (MBF). The BF filter bed consisted of an extremely porous volcanic Tuff granular medium which provided an enlarged surface area for microbial development and biofilm proliferation. We compared the efficiency of the pilot RBF to that of a full-scale RSF, operating with upstream chemical coagulation, by measuring the removal from the same untreated seawater feed of key factors related to membrane clogging: SDI, turbidity, chlorophyll a (Chl a ) and transparent exopolymer particles (TEP). After 2 weeks of operation, the Tuff grains were colonized extensively by coccoid bacteria that formed biofilm along the entire BF. With bacterial colonization and biofilm development, numerous aggregates of bacteria and some algal cells embedded in an amorphous organic matrix were formed on and within the Tuff grains. By 1–3 months, the biotic diversity within the Tuff filter bed had increased to include filamentous bacteria, cyanobacteria, fungi, protista and even crustaceans and marine worms. During and for ∼24 h after each cleaning cycle (carried out every5 to 7 days by upward flushing with air and water), large numbers of floc-like particles, from ∼15 μm to ∼ 2 mm in size were observed in the filtrate of the BF unit. Microscopic examination of these flocs (stained with Alcian Blue and SYTO R 9) showed that they were aggregates of many smaller particles with associated bacteria and algae within a polysaccharide gel-like matrix. These biogenic flocs ( bioflocs ) were observed to form during normal operation of the RBF, accumulating as aggregates of inorganic and organic material on the Tuff surfaces. With each flush cleaning cycle, these bioflocs were released into the BF effluent but were retained by the second phase MBF unit. No flocs were seen in the MBF filtrate. Over a year-long study, both the pilot RBF and the full-scale RSF showed similar filtration efficiencies, measured as the percentage removal of Chl a , TEP, turbidity and SDI from the same seawater feed. These results indicate the potential of the bioflocculation approach with no chemical additives as an alternative to conventional RSF pretreatment for large SWRO facilities.

In this study, pyrosequencing combined with clone library analysis, qPCR, and fluorescent in situ hybridization (FISH) were performed to identify detailed changes of bacterial and filamentous bacterial communities in activated sludge (AS) in 3 types of typical AS samples: sludge bulking (B-AS), excessive bulking (EB-AS), and non-bulking (N-AS). Sludge bulking resulted in a decrease in total bacterial numbers from (6.4 ± 0.18) × 108 gene copies/mL in N-AS to (2.4 ± 0.22) × 108 in EB-AS and a decrease in bacterial diversity from 2757 OTUs in N-AS to 2217 OTUs in EB-AS. With the occurrence of sludge bulking, Actinobacteria and Firmicutes increased sharply, whereas Proteobacteria, which was the predominant phylum in N-AS, decreased markedly. In addition, Nitrospirae, a major lineage of the nitrite-oxidizing bacteria, had quite a low abundance in EB-AS (0.15%), while it was relatively high in N-AS (1.17%). On the other hand, filamentous bacteria accounted for 28.77% and 5.72% of total sequences in EB-AS and N-AS, respectively. More interestingly, 11 types of filamentous bacteria were always present in 3 types of typical AS samples from different stages of sludge bulking, and most of them enriched in EB-AS compared to N-AS. It is noteworthy that, in addition to the frequently reported filamentous bacteria such as Candidatus M. parvicella and Tetrasphaera, novel filamentous species of Trichococcus might exist in this bulking WWTP. Our results reveal that sludge bulking are derived from diverse taxa, which expands previous understanding and provides new insight into the underlying complications of the bulking phenomenon in AS.

Filamentous bulking sludge due to excessive growth of filamentous bacteria is a serious operational problem in activated sludge plants. The addition of chemicals is one of widespread ways to control filamentous bulking. In this study, filamentous bulking in a continuous activated sludge system was found to be mainly caused by Eikelboom Type 021N filamentous bacteria likely due to low substrate concentration gradients. These Type 021N bacteria were found to be resistant to chlorination, maintaining cell integrity at a dosage of up to 80 mg Cl/gSS. An alternative biocidal agent, cetyltrimethyl ammonium bromide (CTAB), exhibited a much stronger biocidal effect on these filaments, which significantly improved sludge settleability. Type 021N with filamentous index of 5 was selectively killed, but floc-formers recovery their activity after CTAB termination. The study implied that CTAB might have more penetration capacity to cell wall of chlorine-resistant Type 021N bacteria. We therefore suggest the penetration property of filament cell wall should be considered or tested before the selection of biocide type in practice.

This research demonstrates the first ever application of lytic bacteriophage (virus) mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium. Bacteriophages are viruses that specifically infect bacteria only. The lytic phage specifically infecting H. hydrossis was isolated from the mixed liquor of a local wastewater treatment plant. The isolated bacteriophage belongs to the Myoviridae family with a contractile tail (length-126 nm; diameter-18 nm) and icosahedral head (diameter-81 nm). Titer of the isolated phage with H. hydrossis was calculated to be 5.2 ± 0.3 × 10(5) PFU/mL and burst size was found to be 105 ± 7 PFU/infected cell. The phage was considerably stable after exposure to high temperature (42 °C) and pH between 5 and 8, emphasizing that it can withstand the seasonal/operational fluctuations under real-time applications. Phage to host (bacteria) ratio for the optimal infection was found to be 1:1000 with ∼54% host death. The isolated phage showed no cross infectivity with other bacteria most commonly found in activated sludge systems, thus validating its suitability for biocontrol of filamentous bulking caused by H. hydrossis. Following the phage application, successful reduction in sludge volume index (SVI) from 155 to 105 was achieved, indicating improved biomass settling. The application of phage did not affect nutrient removal efficiency of the biomass, suggesting no collateral damage. Similar to phage therapy in medical applications, phage-mediated biocontrol holds a great potentiality for large-scale applications as economic agent in the mitigation of several water, wastewater and environmental problems. Present study in this direction is a novel effort.

The main objective of this paper is to demonstrate how including the occurrence of filamentous bulking sludge in a secondary clarifier model will affect the predicted process performance during the simulation of WWTPs. The IWA Benchmark Simulation Model No. 2 (BSM2) is hereby used as a simulation case study. Practically, the proposed approach includes a risk assessment model based on a knowledge-based decision tree to detect favourable conditions for the development of filamentous bulking sludge. Once such conditions are detected, the settling characteristics of the secondary clarifier model are automatically changed during the simulation by modifying the settling model parameters to mimic the effect of growth of filamentous bacteria. The simulation results demonstrate that including effects of filamentous bulking in the secondary clarifier model results in a more realistic plant performance. Particularly, during the periods when the conditions for the development of filamentous bulking sludge are favourable – leading to poor activated sludge compaction, low return and waste TSS concentrations and difficulties in maintaining the biomass in the aeration basins – a subsequent reduction in overall pollution removal efficiency is observed. Also, a scenario analysis is conducted to examine i) the influence of sludge retention time (SRT), the external recirculation flow rate (Qr) and the air flow rate in the bioreactor (modelled as kLa) as factors promoting bulking sludge, and ii) the effect on the model predictions when the settling properties are changed due to a possible proliferation of filamentous microorganisms. Finally, the potentially adverse effects of certain operational procedures are highlighted, since such effects are normally not considered by state-of-the-art models that do not include microbiology-related solids separation problems.

A rapid quantification method for bacteria in activated sludge has been developed, based on fluorescence in situ hybridization (FISH) and epifluorescence microscopy. Samples are hybridized on slides and analyzed by direct microscopic observation. Abundance categories were designed based on digital images of the target organisms. These rating systems were developed for the filamentous bacteria Microthrix parvicella and for different morphotypes of nocardioform actinomycetes, but can easily be adapted to other types of microorganisms. Due to the quantification by direct microscopic observation, this method is suitable for samples that are difficult to be processed by semi-automated image analysis techniques, such as samples containing fluorescent debris, cells of different fluorescence intensities and target organisms that need partial enzymatic digestion prior to FISH. In contrast to commonly used rating systems consisting of photographs, the newly developed categories allow to quantitatively compare results of different categories and different organisms. The uncertainties of the results were calculated by a non-parametric bootstrap procedure; a thorough uncertainty analysis was performed including sample variability and operator subjectivity.

One of the most common problems in the operation of activated sludge plants is the inability to separate solids from treated effluents in the clarifiers caused by the overgrowth of filamentous micro-organisms (FM) with respect to floc-forming bacteria. In order to develop kinetic models that help to predict bulking events, growth kinetics of the FMs and non-filamentous bacteria (NFB) should be known. This paper addresses the competition of a FM and a NFB in a continuous stirred tank reactor. Experimental observations of the effect of the dilution rate on the composition of the mixed culture were compared to simulated results. Image analysis was used to measure NFB and FM fractions in order to evaluate the proposed mathematical model. Experimental results and numerical simulations showed that low D values favored the growth of FM; on the contrary, when high D values were applied a rapid overgrowth of the NFB were observed. Thus, high D values favored the growth of NFB minimizing the risk of filamentous bulking.

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.

The selection of filamentous bacteria is often assumed to be associated with specific microbial properties such as growth rate, substrate uptake rate, substrate affinity and potential for substrate storage. In this study we aimed to verify some of these factors. Sequencing batch reactor (SBR) systems were used to scale-down aerobic activated sludge systems with an aerobic selector. Adding acetate in different aerobic feeding periods allowed us to simulate a variable relative size of aerobic selector with different bulk liquid substrate concentrations. The experiments showed that as expected, the aerobic fill time ratio (FTR(ox)) and the corresponding feast period, which can be assumed similar to contact time in an aerobic selector, had a strong effect on the sludge settleability. Promoting a strong substrate gradient in the SBR (FTR(ox)5.4%) resulted in good sludge settleability (SVI120mLg(-1)). Whenever acetate was added in a limiting rate (FTR(ox)6.2%), a condition in which the acetate concentration in the reactor was always very low, the sludge settleability decreased (SVI150mLg(-1)). Sludge settleability could be improved by changing the feeding strategy to a pulse feed. The maximum specific acetate uptake rate and poly beta-hydroxybutyrate (PHB) production rate of bad settling sludge, including bulking sludge, was similar to well-settling sludge, which is not in accordance with the general assumptions that well settling sludge have a higher maximal substrate uptake rate and better storage capacities. An alternative hypothesis for the development of filamentous structures in biological flocs has been formulated. It is hypothesized that bulking sludge originates from the presence of substrate gradients in sludge aggregates. Whereas at low bulk liquid substrate concentration filamentous bacteria give easier access to the substrate at the outside of the flocs and thereby proliferate, at high bulk liquid substrate concentration there is no substrate advantage for filamentous organisms and smooth bacterial structures predominate. In this hypothesis there is no need for an intrinsic difference in kinetic parameters between floc and filamentous bacteria. Where presence of filamentous bacteria is related to process conditions, the presence of a specific filament is likely due to presence of a specific limiting substrate.

Disintegration of aerobic granular sludge (AGS) is a challenging issue in the long-term operation of an AGS system. Chemical oxygen demand (COD)-to-nitrogen (N) ratio (COD/N), often variable in industrial wastewaters, could be a destabilizing factor causing granule disintegration. This study investigates the impact of this ratio on AGS disintegration and identifies the key causes, through close monitoring of AGS changes in its physical and chemical characteristics, microbial community and treatment performance. For specific comparison, two lab-scale air-lift type sequencing batch reactors, one for aerobic granular and the other for flocculent sludge, were operated in parallel with three COD/N ratios (4, 2, 1) applied in the influent of each reactor. The decreased COD/N ratios of 2 and 1 strongly influenced the stability of AGS with regard to physical properties and nitrification efficiency, leading to AGS disintegration when the ratio was decreased to 1. Comparatively the flocculent sludge maintained relatively stable structure and nitrification efficiency under all tested COD/N ratios. The lowest COD/N ratio resulted in a large microbial community shift and extracellular polymeric substances (EPS) reduction in both flocculent and granular sludges. The disintegration of AGS was associated with two possible causes: 1) reduction in net tyrosine production in the EPS and 2) a major microbial community shift including reduction in filamentous bacteria leading to the collapse of granule structure.

International audience; Using oligonucleotide probes directed at the rRNA of filamentous bacteria, this study looks at the influence of the components of transient substrate overloads on the growth of the dominant filamentous bacteria of activated sludge fed by a synthetic sustrate. By dissociating the massive input of organic matter from the oxygen shortage that the latter generally induces, it is revealed that each of these factors applied alone, induces only transitory, small-scale growth of the filamentous Nostocoida limicola, Haliscomenobacter hydrossis, Thiothrix and of type 021N. In contrast, combining them during a reconstituted transient substrate overload with an artificially created oxygen deficit, induces very fast growth of H. hydrossis which is responsible for establishing major proliferation. This massive proliferation was easily reduced by chlorination.

Activated sludge bulking by the filamentous bacteria type 021N, was repeatedly detected in the mixed liquor of an urban wastewater treatment plant receiving paper mill effluents. The effect of chlorine-induced damage on type 021N was assessed using suitable fluorescent intracellular stains (BacLight™ viability kit, BL). This approach permitted the relatively rapid assessment of the extent and site of disinfection damage in the cells of type 021N. The method permits to see and probably even quantify the injured cells or flocs. The (BL) method was useful for assessing chlorine effect on filamentous microorganisms in mixed cultures such as activated sludge.

Production of excess biomass during biological treatment of wastewaters requires costly disposal. Also with environmental and legislative constraints limiting disposal options, considerable impetus exists for reducing the amount of biomass produced. Uncoupling metabolism in activated sludge may reduce biomass production and this approach is investigated in conjunction with consequences upon substrate removal and population dynamics. To induce uncoupled metabolism, para-nitrophenol (pNP) a known protonphoric uncoupler of oxidative phosphorylation, was introduced to a bench-scale activated sludge process. Microbial populations were monitored by both microscopic and by three methods of molecular analyses. Presence of the protonphore caused a shift in the microbial population with protozoa being washed out of the system and filamentous bacteria proliferating. The molecular composition of the microbial community was determined by PCR amplification of 16SrRNA genes and subsequent denaturing gradient gel electrophoresis (DGGE). Band Patterns obtained by both a direct and nested approach were similar. However, profiles derived from nested PCR contained more bands, indicative of the increased sensitivity of this approach. Analysis of the active biomass by Polyacrylamide Gel Electrophoresis (PAGE) of small molecular weight RNA (5mwRNA) showed that a sustained shift in the diversity of the predominant, metabolically active species present occurred within two days of the introduction of the protonphore. Biomass production was reduced by 49%, but the total substrate removal rate was also reduced by 25%. The combined effect was a 30% decrease in the biomass yield. Introduction of the protonphore caused substrate removal efficiency to decrease from a consistent value of 96% to 68.5% with considerable variance. This decline in overall process performance was attributed to a surmountable effect arising from the design of apparatus that resulted in a decrease in the reactor biomass concentration. Although the specific biomass volume was consistent throughout, decreased sedimentation resulted in solids being removed in the final effluent which decreased the amount of biomass which could be recycled. The catalytic efficiency of the biomass increased as reflected by a 3.3 fold increase in the specific substrate uptake rate.

In this paper a prototype mathematical model is introduced which describes the growth of flocs and filaments in activated sludge waste water treatment systems. The model is based on both kinetic selection and filamentous backbone theory. A stability analysis of the model is performed and it is shown that this model, in contrast with mathematical models only based on kinetic selection theory, is able to describe coexistence of both flocs and filaments for a wide range of dilution rates. Afterwards the model is extended in order to describe a standard activated sludge waste water treatment system.

One of the main reasons for failing of the sedimentation process in activated sludge waste water treatment systems is the phenomenon of filamentous bulking. This occurs when filamentous bacteria overgrow floc-forming bacteria. In this paper mathematical models are discussed which describe the competition of flocs and filaments based on the kinetic selection theory. It is proven with a stability analysis for a continuous reactor, that, in order to control filamentous bulking, the dilution rate plays a crucial role. Moreover, it is shown that coexistence of both organisms is generically not possible. Afterwards the continuous reactor model is extended in order to describe a standard waste water treatment system. Due to the complexity of the extended model it is difficult to perform the stability analysis analytically. It is proven that the model can be reduced without loss of stability properties. For the reduced model it is proven with a stability analysis that the transport terms play a crucial role in the survival of one or the other organism. As for the continuous reactor model it is shown that coexistence of both organisms is generically not possible.

In various activated sludge systems, the biomass grows under transient (unbalanced) conditions and the storage response (formation of internal polymers as the fastest adaptation to the changing environment) becomes important. The aim of the present work was to select mixed cultures with high storage response by cultivation on an acetate-limited medium in a completely mixed reactor operated with intermittent feed (CSTR-i) and to characterise their storage response by using batch experiments under different experimental conditions. Even though kinetic selection based on storage response should bring about predominance of floc-formers, in this work a bulking sludge was established steadily for long periods and starting from different inocula. Batch experiments showed that the mixed culture dominated by the filamentous bacteria presented immediate response to the substrate spike with both substrate removal and storage of it as PHB at high rates, in the range 740÷920 and 510÷610 COD mg/COD g h, respectively. According to the COD balance, storage of poly-3-hydroxybutyrate (PHB) was practically the only significant metabolism. Moreover, these filaments were able to store PHB about up to their dry weight and were highly resistant to long starvation (24 h). Both high storage response and resistance to starvation can contribute to competitive advantage of these filaments in a large range of operating conditions. Nevertheless, inactivity under anoxic conditions makes easy to control bulking by metabolic selection. Although better insight into implications for full-scale plants must be gained, these results confirm that population dynamics under transient conditions is mainly due to storage phenomena and indicate that a competitive advantage of floc-formers against filaments, under such a selective pressure, has not to be considered as an absolute rule.

Settling of the biomass produced during biological treatment of wastewater is a critical and often problematic process. Filamentous bacteria content is the best-known factor affecting biomass settleability in activated sludge wastewater treatment systems, and varying biomass density has recently been shown to play an important role as well. The objective of this study was to systematically determine how filament content and biomass density combine to affect microbial biomass settling, with a focus on density variations over the range found in full-scale systems. A laboratory-scale bioreactor system was operated to produce biomass with a range of filamentous bacterium contents. Biomass density was systematically varied in samples from this system by addition of synthetic microspheres to allow separation of filament content and density effects on settleability. Fluorescent in-situ hybridization indicated that the culture was dominated by Sphaerotilus natans, a common contributor to poor settling in full-scale systems. A simple, image-based metric of filament content (filament length per floc area) was linearly correlated with the more commonly used filament length per dry biomass measurement. A non-linear, semi-empirical model of settleability as a function of filament content and density was developed and evaluated, providing a better understanding of how these two parameters combine to affect settleability. Filament content (length per dry biomass weight) was nearly linearly related to sludge volume index (SVI) values, with a slightly decreasing differential, and biomass density exhibited an asymptotic relationship with SVI. The filament content associated with bulking was shown to be a function of biomass density. The marginal effect of filament content on settleability increased with decreasing biomass density (low density biomass was more sensitive to changes in filament content than was high density biomass), indicating a synergistic relationship between these factors. Consideration of both biomass density and filament content, as by the methods and model described herein, should improve operation and troubleshooting of settling processes for biological solids.

A total of 68 strains of filamentous bacteria belonging to the genera Sphaerotilus and Haliscomenobacter , Eikelbooms types 021N, 1701, some nocardioform organisms in addition to some unidentified, Gram-negative pink-pigmented isolates were studied qualitatively and quantitatively for their growth requirements and growth response to different nutrients. With the exception of type 021N, all isolates grew better with d -fructose and d -glucose, than with acetate as sole carbon source. Acetate concentrations > 1.0 g 1 −1 inhibited all isolates and Haliscomenobacter and the pink-pigmented strains did not grow at all with acetate. With the exception of Haliscomenobacter isolates, growth of the majority of strains was stimulated by low peptone and yeast extract concentrations (0.05–0.5 g 1 −1 ). Haliscomenobacter strains did not grow in the presence of yeast extract and peptone. For all isolates a strict requirement for calcium, and (to some extent) magnesium was observed. Below a concentration of 20 mg 1 −1 Ca and 0.1 mg 1 −1 Mg only few strains were able to show visible growth. The Haliscomenobacter isolates were not able to grow in media containing −1 Ca and 5 mg 1 −1 Mg. All isolates did not grow in media containing −1 NH 4 N, and 2.5 mg 1 −1 P. Often group or genus specific growth requirements were recorded, indicating that the occurrence of one type of filamentous organisms in bulking sludge can be associated with a specific nutrient mixture in the activated sludge.

Bulking and foaming are two frequently occurring operational problems in activated sludge wastewater treatment plants, and these problems are mainly associated with excessive growth of filamentous bacteria. In this study, a comprehensive investigation of the identity and population dynamics of filamentous bacteria in 28 Danish municipal treatment plants with nutrient removal has been carried out over three years. Fluorescence in situ hybridization was applied to quantify more than twenty probe-defined populations of filamentous bacteria that in total constituted a large fraction of the entire microbial community, on average 24%. Despite the majority being present within the flocs, they occasionally caused settling problems in most of the plants. A low diversity of probe-defined filamentous bacteria was found in the plants with Microthrix and various species belonging to phylum Chloroflexi (e.g., type 0803 and type 0092) as the most abundant. Few other filamentous probe-defined species were found revealing a large similarity between the filamentous populations in the plants investigated. The composition of filamentous populations was stable in each plant with only minor changes in relative abundances observed during the three-year study period. The relative composition of the different species was unique to each plant giving a characteristic "fingerprint". Comprehensive statistical analyses of the presence and abundance of the filamentous organisms did not reveal many correlations with a particular plant design or process parameter.

The changes in the microbial population of a two-stage anaerobic digestion system in terms of number and composition were studied during the start-up phase. The numbers of methanogens and non-methanogens slightly decreased in the upflow filter, whereas in the pre-acidification stage the number of acidogens was reasonably constant. Variations in the morphology of the methanogens occurred along the filter, for example the numbers of Methanosarcina and filamentous bacteria species decreased considerably in the drain, possibly becoming attached to the biofilm along the reactor, whereas in the pre-acidification stage only a few Methanosarcina species were identified. Slight variations within the bacterial populations occurred in terms of composition and numbers in the filter. The number of viable methanogens varied in the filter effluent depending on the OLR changes but this did not adversely affect the COD removal efficiency of the upflow filter. Among the autofluorescent methanogenic population, Methanococcus, Methanobacterium, Methanobrevibacteria , and Methanosarcina species were tentatively identified in the filter.

In a survey of activated sludge plants in Australia, where both foams and mixed liquor samples were examined, the organism most frequently dominant in both was Microthrix parvicella. Although Nocardia amarae-like organisms and Nocardia pinensis were often seen as dominant organisms in foams, they only rarely occurred as dominants in mixed liquor samples from the same plants. Instead, Types 0041/0675, 0092 and Haliscomenobacter hydrossis were more usually present. There appeared to be little relationship between filament community composition and plant operating parameters, and most filament types were seen in plants with diverse characteristics. Partial success was achieved in isolating a few of these filaments in pure culture, although in some cases, the media successfully used in other studies did not support their growth here. Their morphological properties are described.

Limited filamentous bulking has been proposed as a means to enhance floc size and make conditions more favorable for simultaneous nitrification/Denitrification (SND). Moreover a slightly heightened SVI is supposed to increase the removal of small particulates in the clarifier. Integrated nitrogen, phosphorus and COD removal performance under limited filamentous bulking was investigated using a bench-scale plug-flow enhanced biological phosphorus removal (EBPR) reactor fed with raw domestic wastewater. Limited filamentous bulking in this study was mainly induced by low DO levels, while other influencing factors associated with filamentous bulking (F/M, nutrients, and wastewater characteristics) were not selective for filamentous bacteria. The optimum scenario for integrated nitrogen, phosphorus and COD removal was achieved under limited filamentous bulking with an SVI level of 170-200 (associated with a DO of 1.0-1.5 mg/L). The removal efficiencies of COD, TP and NH4+-N were 90%, 97% and 92%, respectively. Under these conditions, the solid-liquid separation was practically not affected and sludge loss was never observed. A well-clarified effluent with marginal suspended solids was obtained. The results of this study indicated the feasibility of limited filamentous bulking under low DO as a stimulation of simultaneous nitrification/denitrification for enhancing nutrient removal and effluent quality in an EBPR process.

Activated sludge sewage treatment plants that were affected by biological foams attributed to the presence of actinomycetes were surveyed in Queensland, Australia.Nocardia amarae andNocardia pinensis were the predominant organisms found to accumulate selectively in the foam phase. Foaming was shown to be a significant problem in Queensland with 92% of surveyed plants being affected. A total of 60% of these plants suffered from bulking, however, no correlation between these two problems was noted. No operational, design, influent or weather feature was found to be consistent in all foaming plants. Apart from the fact that the plants were all variations of the activated sludge process, the only other aspect shared by them was that large numbers of filamentous bacteria were present in the foam. Field scale trials that evaluated control strategies against actinomycete foaming or scumming in activated sludge plants are described. The effects were assessed of: altering the rate of return activated sludge recycle, dosing of the aeration tank with anaerobic digester products, the addition of a commerical product specifically designed to control foaming, altering sludge age, varying levels of mixed liquor suspended solids (MLSS) and altering other operational features. These strategies did not efficaciously control scumming in activated sludge, however, some operational features were highlighted as exacerbating the problem.

In the sewage treatment plants of Berlin Ruhleben (R) and Marienfelde (M), chemical, physical and technical parameters of wastewater as well as the occurrence of filamentous bacteria in activated sludge (bulking sludge) were investigated. Over a period of 10 (R) and 9 (M) months the influent and effluent concentrations were recorded in order to find possible interactions with the growth of filamentous bacteria, if any. It was shown that the factors of influence were too complex to obtain specific information about the growth of filamentous bacteria in vivo. The frequency (%) of the major types in the treatment plants Ruhleben and Marienfelde were: (R) type 021 N (21%), type 0041 (19%), Microthrix parvicella (17%), nocardioforms (6%); (M) nocardioforms (49%), type 021 N (13%), type 0041 (12%), type 1701 (5%). Different isolation techniques and culture media were developed and used to isolate 54 (M) and 87 (R) axenic strains of filamentous bacteria from the bulking sludge samples collected. It was shown that the methods of centrifugation, direct streaking, ultra turrax, and whirl mix, and the I- and AcS-medium, followed by the GS-medium and standard media were the most successful to isolate filamentous bacteria. The isolates were identified by morphological and cytological criteria as strains of type 021 N, Sphaerotilus natans/type 1701, Haliscomenobacter hydrossis, nocardioforms and pigmented colonies. Because some groups are not easily distinguished on the basis of cytological and morphological features, further physiological and chemotaxonomical investigations must be done in order to acquire better knowledge on filamentous bacteria.

Nematode diversity and dynamics of a full-scale rotating biological contactor plant (RBC) has been studied. Analysis of biofilm composition showed a well-established zoning of microfauna among the three RBC sections analysed. Nematodes appeared to be the dominant group within the larger microfauna populations with average abundances between 200 and 300 ind/mg or 8000 and 17 000 ind/cm2. The most abundant nematode species were Diplogasteritus nudicapitatus and Paroigolaimella coprophages and, to a lesser extent, Paroigolaimella bernensis and Steinernema intermedia. The relationship between nematodes and filamentous bacteria (specifically the genus Beggiatoa) was the most significant biotic relationship found, and to a lesser extent, nematodes with ciliates. The relationship between the abundance of nematode species and the physical–chemical variables suggests that nematodes may be good indicators of low pollutant load levels in the entry of the RBC system. Finally, the results indicate that nematodes may have a relevant role for a good biofilm development.

Sludge deflocculation can cause, like excessive growth of filamentous bacteria, activated sludge wastewater treatment failure. Yet, unlike the latter cause, there exists no widely accepted tool to assess the flocculation level of activated sludge and to predict sludge deflocculation. In this study, a test procedure is proposed to assess the sensitivity of activated sludge flocs to shear. The test consists in subjecting sludge sample to a shear treatment followed by a sludge volume measurement (SV(30)), with reference to a control. The ratio (%) obtained by dividing the SV(30) of the sheared sample by the SV(30) of the control is used to express the shear sensitivity. In a first series of experiments using two types of sludge, the test was shown to be able of ranging sludge samples in a correct order of shear sensitivity. Applying this so-called shear-SV test procedure to a series of sludge samples allowed to distinguish amorphous sludges (SV(30) ratio> or =100%) from normal sludges (SV(30) ratio

Cases of low stress can frequently occur in a wastewater treatment plant (WWTP). As they have no visible impact on the plant operation, they generally go unnoticed. Nevertheless, it would appear that an accumulation of such cases can result in serious operational problems. The impact of the repeated application of oxygen deficiency on the proliferation of the filamentous bacteria Sphaerotilus natans, Haliscomenobacter hydrossis, Eikelboom type 021N and Thiothrix spp has been studied on WWTP pilots. Two different oxygen deficiencies series were tested. Filamentous bacteria evolution in activated sludge was monitored using fluorescent in situ hybridization. For the weak deficiencies, no significant variation in the filamentous bacteria response level was recorded, whereas with more severe deficiencies the filamentous bacteria response increased considerably with the increase in stress number. From the results obtained, it would appear that there is an intensity and frequency threshold beyond which the filamentous bacteria response levels increase when the stress is applied in series. This "oxygen deficiencies repetition" appears to be an important factor in the appearance of bulking.

Instantaneous improvement of the settling of bulking filamentous activated sludge can be achieved by the addition of a polymer or a large amount (up to 100% of the MLSS concentration) of talc powder to the sludge. Long-term improvement relies on repeated additions, as these additives have no adverse effects on the causative filaments. A multi-component additive was compared to the traditional additives in lab-scale activated sludge units using three highly filamentous sludges from different industrial treatment plants. The study demonstrated that the multi-component additive was superior to the traditional remedies. It was shown that, in the case of severe filamentous bulking, a single addition of the new additive immediately improved sludge settling and exerted a destructive effect on the causative filamentous bacteria. Thus, the latter additive also ensured a long-term sludge sedimentation improvement. The traditional additives exhibited an immediate and short-term effect. The novel additive also retarded sludge rising due to denitrification and it improved sludge dewaterability. The study revealed Nostocoido limicola II, with slightly hydrophobic cell wall, to be somewhat resistant to the quaternary ammonium salt present as biocide in the additive.

A method is presented for the identification and isolation of filamentous bacteria from activated sludge samples using the technique of micromanipulation. This employs a forged microneedle which is inserted into a drop of activated sludge either resting on an agar surface or as a smear on a glass slide. It enables a single filamentous bacterium to be removed and identified based on its morphological characteristics. The same organism can then be transfered into a growth medium for confirmation of its identity using biochemical characteristics. Use of this technique reduces the confusion which can arise in identification and ensures that prevalent filament species associated with specific bulking incidences can be isolated and accurately identified.

Haliscomenobacter hydrossis DSM 1100 and an orange-coloured filamentous bacterium isolated from a high rate algal pond are dependent on calcium ions for growth. In addition, the effect of calcium on the growth of the orange-coloured filamentous bacterium is enhanced by bicarbonate ions. Bicarbonate alone does not affect growth.

New developments of precipitation techniques in Sweden and other countries have been directed towards dual purpose chemical pre-precipitation. This implies not only the removal of phosphorus to avoid eutrophication but also the removal of organics in order to save energy. In this connection the optimization of chemical and biological treatment is particularly interesting. Too low concentrations of BOD and easily assimilated phosphates in the pre-precipitated sewage may have a detrimental effect on the activated sludge process, primarily the sludge characteristics. The experimental studies for optimization of chemical pre-precipitation in Enkoping in 1985 included as an important part the influence of low soluble phosphorus concentrations on the activated sludge process by extreme pre-preciptation in order to attain a lower total phosphorus concentration in the effluent than the standard value 0.5 mg l−1 P. During extreme pre-precipitation conditions the sedimentation characteristics of the activated sludge deteriorated with a very marked increase of sludge volume index and growth of filamentous organisms. Specialized laboratory studies showed that wide variations of pre-precipitation dosages used in the plant trial operation apparently caused shifts in microbial population. This was indicated by variations in the amount of filamentous bacteria. It seems clear that extreme pre-precipitation favours filamentous organisms, probably due to easily assimilated phosphate depletion in this study. Optimized preprecipitation followed by polishing precipitation in a second point is accordingly preferred over just extreme pre-precipitation alone. The findings are also in agreement with other studies, which show that activated sludge properties may be altered by major changes of some operational parameters such as oxygen and substrate concentration, or as in this case, pre-precipitation dosage.

Nutrient starvation of aerated activated-sludge in a batch reactor without recycle, caused a rapid utilization of the intracellular storage polymer poly-3-hydroxy butyrate (PHB) and a decline in sludge respiration rate. During this period sludge settleability, as determined by SSVI and ease of dewatering, as measured by specific resistance to filtration, declined markedly. There was no change in sludge exopolysaccharide concentration or composition during the starvation period and loss of settling ability was attributed to extensive cell lysis, producing changes in floc structure from firm, round, flocs to pin-point flocs. Appearance of filamentous bacteria during starvation suggests that these organisms have a growth rate advantage at low nutrient concentrations; the implications of this in the control of bulking sludges is discussed. Starvation of activated sludges appears to offer an ideal laboratory model to study environmental factors implicated in sludge flocculation.