Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n = 24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 °C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.     

Determination of nutrients limiting biofilm formation and the subsequent impact on disinfectant decay.

Understanding the contribution of both organic and inorganic nutrients to biofilm development and the subsequent impact of developed biofilms on disinfectant decay are important requirements for distribution system management strategies. Nutrient limitation may be one way to control biofilm development without increasing disinfectant dosing. Little is known, however, of the nutrient requirements of biofilms in distribution systems. Indeed, the effects on biofilm development due to the addition of nutrients to distribution systems and what impact biofilm development may have on disinfectant decay is still poorly understood. This study used annular reactors to determine the nutrients limiting for biofilm development in drinking water from two different Sydney sources and the subsequent effects of biofilm development on disinfectant decay. It was found that biofilm development in Sydney water was limited by organic carbon and that biofilm development promoted chloramine decay. Moreover, biofilm development occurred in the presence of chloramine. The ability of biofilms to respond to increases in disinfectant concentrations was dependent on the biomass of the biofilms. In a comparative study using chlorinated drinking water containing very low levels of organic carbon, biofilm development was not detected. Removal of organic carbon resulted in greater persistence of chlorine, which led to greater biofilm control. It was also shown that biofilms could contribute cells to the aqueous phase. The results of the study indicate that treatment and system management strategies should incorporate organic carbon removal to limit biofilm development through a combination of retarding bacterial growth and enhancing disinfectant persistence.     

Effect of mechanical stress on biofilms challenged by different chemicals

In this study a methodology was applied in order to ascertain the mechanical stability of biofilms, by using a stainless-steel (SS) rotating device immersed in a biological reactor where biofilms formed by Pseudomonas fluorescens were allowed to grow for 7 days at a Reynolds number of agitation of 2400. The biofilms developed with this system were characterised in terms of amount of total, extracellular and intracellular proteins and polysaccharides, amount of mass, metabolic activity and mechanical stability, showing that the biofilms were active, had a high content of extracellular constituents and an inherent mechanical stability. In order to assess the role of chemical agents on the mechanical stability, the biofilms were exposed to chemical agents followed by mechanical treatments by submission to increase Reynolds number of agitation. Seven different chemical agents were tested (two non-oxidising biocides, three surfactants and two oxidising biocides) and their effects on the biofilm mechanical stability were evaluated. The increase in the Reynolds number increased the biofilm removal, but total biofilm removal was not found for all the conditions tested. For the experiment without chemical addition (only mechanical treatment), the biofilm remaining on the surface was about 76%. The chemical treatment followed by the subsequent mechanical treatment did not remove all the biofilms from the surface. The biofilm remaining on the SS cylinder ranged from 3% to 62%, depending on the chemical treatment, showing that the chemical treatment is far from being a cause that induces massive biofilm detachment and even the synergistic chemical and mechanical treatments did not promote biofilm removal. Some chemical agents promoted an increase in the biofilm mechanical stability such as glutaraldehyde (GTA), benzalkonium chloride (BC), except for the lower concentration tested, and sodium dodecyl sulphate (SDS), except for the higher concentration tested. Treatments that promoted biofilm removal, to an extent similar to the control experiment (without chemical treatment), were BC, for the lower and the higher concentration of SDS. Cetyltrimethyl ammonium bromide (CTAB), ortho-phthalaldehyde (OPA), sodium hydroxide (NaOH) and sodium hypochlorite (SHC) promoted the weakening of the biofilm mechanical stability.     

Experimental and modeling approach to study sorption of dissolved hydrophobic organic contaminants to microbial biofilms

A biofilm reactor was developed to investigate the sorption of polycyclic aromatic hydrocarbons (PAH) as model compounds for hydrophobic organic contaminants (HOC) to intact microbial biofilms at environmentally realistic concentrations. When operated as a differential column batch reactor, the system can be used to study the thermodynamics as well as the kinetics of the exchange of HOC between an aqueous phase and microbial biofilms. Organic carbon normalized partition coefficients (K(oc)) for phenanthrene, fluoranthene and pyrene were at the lower end of those known for other organic sorbents. Intra-biofilm diffusion coefficients (D) were calculated from decrease in solute concentration over time using a model for diffusion through a plane sheet and ranged from 0.23 to 0.45x10(-9)cm(2)s(-1) for the three PAH. These diffusion coefficients are about four orders of magnitude lower than those reported in literature for free aqueous solution. These data and the experimental approach presented here are useful to assess the importance of microbial biofilms for exchange processes of HOC in heterogeneous systems such as water distribution systems, membranes and aquifers, sewer systems or surface soils.     

Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes

The action of the cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated to control biofilms (aged 7d) formed by Pseudomonas fluorescens on stainless-steel slides, using flow cells reactors, under turbulent and laminar flow. The effect of CTAB was also investigated using planktonic cells in the presence and absence of BSA, by measuring the cellular respiratory activity and the ATP released. The action of CTAB on biofilms was assessed by means of cellular respiratory activity and variation of biofilm mass, immediately and 3, 7 and 12h after the application of CTAB. The physical stability of the biofilm was also assessed using a rotating device, where the effect of the surfactant on the biofilm stability was evaluated through the variation of the mass remaining on the surface. CTAB significantly reduced the activity of the planktonic cells probably due to the rupture of the cells. This effect was significantly reduced in the presence of BSA. Planktonic cells were more easily inactivated than bacteria in biofilms. Biofilms formed under laminar flow were more susceptible than those formed under turbulent flow, but in both cases total inactivation was not achieved. Biofilm recovery was observed, in terms of respiratory activity, in almost all the cases studied. CTAB application by itself did not promote the detachment of biofilms. The physical stability tests showed that the synergistic action of the surfactant and the application of high shear stress to the biofilm increase its detachment.     

Phosphorus uptake into algal biofilms in a lowland chalk river

This paper examines the growth and uptake of phosphorus into algal biofilms in the River Kennet, a lowland chalk (Cretaceous-age) stream in southern England. Algal biofilms were grown on artificial plastic substrates (templates) placed (i) on the riverbed and (ii) within the mid-water column. Experiments were set up to examine differences in growth rates of newly colonising biofilms compared with biofilms left to accumulate for periods of up to 6 months. Rates of algal biofilm production were measured by the chlorophyll a concentration that had accumulated per cm² over the number of days that the biofilm template had been immersed in the river water. An algal biofilm bloom occurred in early spring, prior to peak suspended chlorophyll a concentrations within the water column. Biofilm samples collected in February and March had the highest chlorophyll a and total phosphorus concentrations. The biofilm bloom corresponded with increased solar radiation and declining river flow conditions. Periodic increases in soluble reactive phosphorus concentrations in the overlying river water did not correspond with any significant increase in biofilm production. These results suggests that light, rather than phosphorus is a key factor for biofilm growth in the River Kennet. Higher rates of chlorophyll a development in mid-water column biofilms may be linked to greater light exposure; however, maximum total-P concentrations were similar for both bed and water column biofilms. Newly colonising biofilms exhibited higher chlorophyll a and total-P concentrations than biofilms left to accumulate over longer terms, suggesting that fresh substrate availability promotes high rates of biofilm growth. Both ‘condensed and organic’ P (stored in biomass) and ‘inorganic’ (mineral) P fractions within the biofilms were present in varying proportions, although the early spring biofilm bloom resulted in maximum proportions and absolute concentrations of ‘condensed and organic’ P. Calcite was the only crystalline mineral detected within the biofilms. Ratios of Ca:inorganic P are largely consistent with the presence of CaCO₃–P co-precipitates, although one very low value suggested that there may also be additional sources of inorganic P, possibly P adsorbed to clays or organics within the biofilm. However, poor linkages between CaCO₃ and inorganic P concentrations suggest that, although the inorganic P fraction within the biofilm may be derived largely from CaCO₃–P co-precipitation, the subsequent processes controlling overall CaCO₃ and inorganic P concentrations in the biofilm are complex.     

Role of shear stress on composition, diversity and dynamics of biofilm bacterial communities

This article evaluates the effect of shear stress on the composition of biofilm bacterial communities. For the first time, a Conical Couette-Taylor Reactor (CCTR) was used to develop biofilms at varying shear stresses (from 0.055 to 0.27 Pa) and provided a useful model for studying the effect of hydrodynamics on biofilms. The composition, diversity and dynamics of biofilm bacterial communities were analysed using the PCR-SSCP fingerprint method. Results clearly demonstrate a link between shear stress and composition of the microbial communities. High shear stresses decrease biofilm diversity and the analysis of biofilm community dynamics suggests that shear stress would slow down biofilm maturation and tend to maintain a young biofilm.     

Demonstration of mass transfer and pH effects in a nitrifying biofilm

A bench-scale nitrifying trickling filter (surface AREA = 0.5 m²) was developed to permit evaluation of diffusion of oxygen within a biofilm, the pH dependence of ammonium oxidation and external mass transfer. In addition, a biofilm model was developed and verified for homogeneous nitrifying biofilms of varied thickness and for thin nitrifying biofilms covered by heterotrophic biofilms. The model uses literature values for the pH dependence of Monod coefficients for Nitrosomonas and Nitrobacter.

The diffusion coefficient of oxygen in the biofilm was found to be 40–80% of the value in pure water. Due to mass transfer resistance, the biomass ·sees” a lower pH than is measured in the water film passing over it. The surface uptake rate of ammonia is used as an indicator of pH gradients within the biofilm system. With the help of oxygen limitation experiments, the location of nitrifying biomass within mixed biofilms (heterotrophic, autotrophic) can be determined.

The biofilm model predicts ammonium uptake rate of a trickling filter as a function of the bicarbonate concentration in the water film.     

Influence of growth conditions on biofilm development and mass transfer at the bulk/biofilm interface

In a long-term study on heterotrophic biofilms in tube reactors, this investigation focused on mass transfer at the bulk/biofilm interface, biofilm density and substrate conversion rates. Several biofilms were cultivated under different substrate and hydrodynamic conditions. Oxygen concentration profiles were measured with microelectrodes in the biofilm and in the boundary layer directly in the biofilm tube reactors. The thickness of the concentration boundary layer was found to depend on the surface structure of the biofilm. The hydrodynamic conditions and the substrate load during the growth phase of the biofilm in biofilm systems are two key parameters that influence the biofilm growth, particularly the structure, density and thickness. The measured substrate conversion rates, biofilm densities and the boundary layer thickness were used to formulate an equation for the mass transfer in biofilm tube reactors.     

Effect of turbulence on nitrifying biofilms at non-limiting substrate conditions

The effect of turbulence on nitrifying biofilms was studied in five cylindrical PVC (polyvinyl chloride) reactors, each having ten biofilm sampling taps, over a period of 196 days. Bulk water in the reactors was stirred by paddles at 32, 92, 140, 278 and 500 rpm and the turbulent intensities measured at 10 mm from the wall were 0.6, 1.5, 2.6, 4.4 and 8.9 cm/s. Biofilms appeared as isolated colonies and continued to grow as filament-type biofilms. Higher turbulence resulted in higher NH₄-N flux and higher areal biomass density. Turbulent diffusion of substrates and by-products in the vicinity of filament-type biofilms must have resulted in the above phenomena. Photographic observation of the biofilm surfaces on sampling taps showed uniform biofilm filaments at higher turbulent intensities and large variation in the height of filaments at low turbulent intensities. Substrate flux and biofilm structure (areal density, filament height and cross-sectional area of filament) are inter-related parameters and are strongly affected by turbulence near the biofilm. Substrate flux is expressed as a power function of turbulent intensity, volumetric density and substrate concentration for filament-type biofilm when substrates are non-limiting.     

Interactions of 1 μm latex particles with pseudomonas aeruginosa biofilms

Fluorescently labelled latex microbeads were used to study the interaction of particles with Pseudomonas aeruginosa biofilms in a continuous flow annular reactor. Beads were readily distinguished and enumerated in both intact and disaggregated biofilm samples. The fraction of beads that attached to biofilm during a 24 h period ranged from 0.001 to 0.01 and was proportional to biofilm cell carbon and to the standard deviation of biofilm thickness. Microbeads added to biofilm of steady state thickness (30 μm) were observed to be located throughout the entire biofilm depth in 24 h. Many of the microbeads that attached to biofilm shortly after bacterial inoculation (thickness of 2 μm) remained near the substratum as cells grew past and covered them. Microbeads were observed near the biofilm-substratum interface for up to 5 days after bead addition. Beads formed aggregates on biofilms, but not in bulk water. Beads captured by biofilm remained in the reactor system longer than beads that never attached to biofilm.     

Effect of shear stress and growth conditions on detachment and physical properties of biofilms

Detachment is one of the major processes determining the physical structure and microbial functionalities of biofilms. To predict detachment, it is necessary to take the mechanical properties of the biofilm and the effect of both hydrodynamic and growth conditions into account. In this work, experiments were conducted with biofilms developed under various shear stresses and with various substrate natures. In addition, two cases were considered in order to differentiate between the effect of hydrodynamic factors and growth factors: the biofilms were directly grown under the targeted shear stress (τ) condition or they were precultivated under very low shear stress (0.01 Pa) and then exposed to high shear stress in the range of 0.1-13 Pa. An exponential and asymptotic decrease of the biofilm thickness and mass with increasing τ was observed in both cases. On contrary density, expressed as the biofilm dry mass on a known substratum divided by the average thickness increased with τ. Denitrifying biofilms always showed greater thickness and density than oxic biofilms. These results showed the presence of a compact basal layer that resisted shear stresses as high as 13 Pa whatever the culture conditions. Above this basal layer, the cohesion was lower and depended on the shear stress applied during biofilm development. The application of shear stress to the biofilms resulted in both detachment and compression, but detachment prevailed for the upper part of the biofilms and compression prevailed for the basal layers. A model of biofilm structure underlying the stratified character of this aggregate is given in terms of density and cohesion.     

Degradation of polymers in a biofilm airlift suspension reactor

The effect of degradation of polymeric substrates (starch and soy proteins mixture) on the structure of biofilms has been studied. The characteristics of the obtained biofilms were compared to those obtained on corresponding monomeric substrates (glucose and aspartic acid). Based on literature suggestions it was hypothesized that the polymeric substrates, which have a low diffusion rate in the biofilm matrix, would affect the biofilm structure if hydrolytic activity occurs in the biofilm. The obtained biofilm could be expected to present properties like low density and rough surface, facilitating transport and conversion of large polymeric molecules. From the present study it was concluded that the structure of the formed biofilms was influenced by the substrate degraded, however no unequivocal effect of degradation of a polymer on the biofilm structure could be observed. The hydrolytic activity with soy protein and starch as substrate was under stable conditions found to be mainly associated to the biofilm (more than 95% of the total activity). During unstable conditions or start-up significant hydrolytic activity occurred outside the biofilm.     

Species association increases biofilm resistance to chemical and mechanical treatments

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re_A). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re_A were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each other's survival to antimicrobials and the biofilm shear stress stability.     

Probing young drinking water biofilms with hard and soft particles

The aim of our study was to investigate, through the use of soft (Escherichia coli) and hard (polystyrene microspheres) particles, the distribution and persistence of allochthonous particles inoculated in drinking water flow chambers. Biofilms were allowed to grow for 7-10 months in tap water from Nancy's drinking water network and were composed of bacterial aggregates and filamentous fungi. Both model particles adhered almost exclusively on the biofilms (i.e. on the bacterial aggregates and on the filamentous structures) and not directly on the uncolonized walls (glass or Plexiglas). Biofilm age (i.e. bacterial density and biofilm properties) and convective-diffusion were found to govern particle accumulation: older biofilms and higher wall shear rates both increased the velocity and the amount of particle deposition on the biofilm. Persistence of the polystyrene particles was measured over a two-month period after inoculation. Accumulation amounts were found to be very different between hard and soft particles as only 0.03‰ of the soft particles inoculated accumulated in the biofilm against 0.3-0.8% for hard particles.     

Biofilm responses to ageing and to a high phosphate load in a bench-scale drinking water system

The effects of ageing and of phosphate load on drinking water biofilms developed on a polycarbonate substratum in the pseudo-equilibrium state have been evaluated. Phosphate was added in an amount higher than the stochiometric nutrient requirements of bacteria, at concentrations commonly applied in a drinking water distribution system for corrosion control. Multiple parameters were monitored: heterotrophic plate counts (HPCs), total direct counts (TDCs) and potential exoproteolytic activity (PEPA) in order to characterise changes in bacterial biofilms. The total carbohydrate, amino acid and phosphate contents of biofilms were analysed to characterise and monitor the biochemical composition of the biofilm.The three enumeration methods showed that a pseudo-equilibrium state was reached after 7 weeks of colonisation after which, the bacterial growth rate in the biofilm was 0.1 log per week on average. Bulk phosphate addition doubled the phosphate in the biofilm, but did not affect the other biological, physiological or chemical parameters measured.Polysaccharides increased in the biofilm with ageing and the dynamics of individual carbohydrate synthesis also varied with the age of the biofilm. Once pseudo-equilibrium, it was found that the total proteins were globally constant, whereas the spectra of some individual amino acids of the proteins had significantly changed.     

Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes

We studied the changes in water quality and formation of biofilms occurring in a pilot-scale water distribution system with two generally used pipe materials: copper and plastic (polyethylene, PE). The formation of biofilms with time was analysed as the number of total bacteria, heterotrophic plate counts and the concentration of ATP in biofilms. At the end of the experiment (after 308 days), microbial community structure, viable biomass and gram-negative bacterial biomass were analysed via lipid biomarkers (phospholipid fatty acids and lipopolysaccharide 3-hydroxy fatty acids), and the numbers of virus-like particles and total bacteria were enumerated by SYBR Green I staining. The formation of biofilm was slower in copper pipes than in the PE pipes, but after 200 days there was no difference in microbial numbers between the pipe materials. Copper ion led to lower microbial numbers in water during the first 200 days, but thereafter there were no differences between the two pipe materials. The number of virus-like particles was lower in biofilms and in outlet water from the copper pipes than PE pipes. Pipe material influenced also the microbial and gram-negative bacterial community structure in biofilms and water.     

Evaluation of some halogen biocides using a microbial biofilm system

A simple method for the formation of microbial biofilms of three species, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae, on a small glass slide was established, and its suitability for evaluation of disinfectant efficacy was examined. The biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal efficacy of several halogen biocides, such as hypochlorite (HOCl), bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite biocide named STABREX, and a mixed solution of NH4Br and HOCl, was evaluated. The formation of NHBrCl in the mixed solution was indicated by UV spectra analysis. Biofilm cells were more resistant to these biocides than planktonic cells and the extent of resistance varied with the biocide tested. Among the biocides tested, the biocidal potency of HOCl was the most susceptible to the change brought about by biofilm formation. By CLSM observation, differences in biofilm conformation were revealed between the microbial species. The efficacy of the biocide tested varied with the structure of biofilms formed. The assay method developed in the present study would be useful for further investigation on biofilm disinfection.     

Microbial community structures and in situ sulfate-reducing and sulfur-oxidizing activities in biofilms developed on mortar specimens in a corroded sewer system

Microbially induced concrete corrosion (MICC) caused by sulfuric acid attack in sewer systems has been a serious problem for a long time. A better understanding of microbial community structures of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) and their in situ activities is essential for the efficient control of MICC. In this study, the microbial community structures and the in situ hydrogen sulfide production and consumption rates within biofilms and corroded materials developed on mortar specimens placed in a corroded manhole was investigated by culture-independent 16S rRNA gene-based molecular techniques and microsensors for hydrogen sulfide, oxygen, pH and the oxidation-reduction potential. The dark-gray gel-like biofilm was developed in the bottom (from the bottom to 4 cm) and the middle (4–20 cm from the bottom of the manhole) parts of the mortar specimens. White filamentous biofilms covered the gel-like biofilm in the middle part. The mortar specimens placed in the upper part (30 cm above the bottom of the manhole) were corroded. The 16S rRNA gene-cloning analysis revealed that one clone retrieved from the bottom biofilm sample was related to an SRB, 12 clones and 6 clones retrieved from the middle biofilm and the corroded material samples, respectively, were related to SOB. In situ hybridization results showed that the SRB were detected throughout the bottom biofilm and filamentous SOB cells were mainly detected in the upper oxic layer of the middle biofilm. Microsensor measurements demonstrated that hydrogen sulfide was produced in and diffused out of the bottom biofilms. In contrast, in the middle biofilm the hydrogen sulfide produced in the deeper parts of the biofilm was oxidized in the upper filamentous biofilm. pH was around 3 in the corroded materials developed in the upper part of the mortar specimens. Therefore, it can be concluded that hydrogen sulfide provided from the bottom biofilms and the sludge settling tank was emitted to the sewer atmosphere, then oxidized to corrosive compounds in the upper and middle parts of the manhole, and only the upper part of the mortar specimens were corroded, because in the middle part of the manhole the generated corrosive compounds (e.g., sulfuric acid) was reduced in the deeper parts of the biofilm.     

Biofilm growth and characteristics in an alternating pumped sequencing batch biofilm reactor (APSBBR)

A novel biofilm reactor-alternating pumped sequencing batch biofilm reactor (APSBBR)-was developed to treat synthetic dairy wastewater at a volumetric chemical oxygen demand (COD) loading rate of 487 g COD m(-3) d(-1) and an areal loading rate of 5.4 g COD m(-2) d(-1). This biofilm reactor comprised two tanks, Tanks 1 and 2, with two identical plastic biofilm modules in each tank. The maximum volume of bulk fluid in the two-tank reactor was the volume of one tank. The APSBBR was operated as a sequencing batch biofilm reactor with five operational phases-fill (25 min), anoxic (9 h), aerobic (9 h), settle (6 h) and draw (5 min). The fill, anoxic, settle and draw phases occurred in Tank 1. In the aerobic phase, the wastewater was circulated between the two tanks with centrifugal pumps and aeration was mainly achieved through oxygen absorption by micro-organisms in the biofilms when they were exposed to the air. In this paper, the biofilm growth and characteristics in the APSBBR were studied in a 98-day laboratory-scale experiment. During the course of the study, it was found that the biofilm thickness (delta) in Tank 1 ranged from 1.2 to 7.2 mm and that in Tank 2 from 0.5 to 2.2 mm; the biofilm growth against time (t) can be simulated as delta=0.07t0.99 (R2 = 0.97, P = 0.002) in Tank 1 and delta = 0.08t0.66 (R2 = 0.81, P = 0.04) in Tank 2. The biomass yield coefficient, Y, was 0.18 g volatile solids (VS) g(-1) COD removal. The biofilm density in both tanks, X, decreased as the biofilm thickness increased and can be correlated to the biofilm thickness, delta .