Biofilm development in a membrane-aerated biofilm reactor: effect of intra-membrane oxygen pressure on performance

The effect on intra-membrane oxygen pressure at a constant carbon substrate loading rate on the development of biofilms of Vibrio natrigens in a membrane aerated biofilm reactor (MABR) was investigated experimentally and by mathematical modelling. A recently reported technique (Zhang et al., 1998. Biotechnol. Bioeng. 59: 80-89) for the in situ measurement of the substrate diffusion coefficients in a growing biofilm and the mass transfer coefficients in the boundary layer at the biofilm liquid interface was used. This aided the study of the effect of the heterogeneous biofilm structure and also improved the reliability of the model predictions. The different intra-membrane oxygen pressures used, 12.5, 25 and 50 kPa, with acetate as the carbon substrate, showed a marked effect on the initial biofilm growth rate, on acetate removal rate, particularly in thick biofilms and on biofilm structure. The model predicted the substrate limitation regimes, the location of the active biomass layer within the biofilms and the trends in oxygen uptake rate through the membrane into the biofilms. During the development of the biofilms, the biofilm thickness and the intra-membrane oxygen pressure were found to be the most important parameters influencing the MABR performance while the effect of biofilm structure was less marked.     

Biodegradation of acetonitrile by adapted biofilm in a membrane-aerated biofilm reactor

A membrane-aerated biofilm reactor (MABR) was developed to degrade acetonitrile (ACN) in aqueous solutions. The reactor was seeded with an adapted activated sludge consortium as the inoculum and operated under step increases in ACN loading rate through increasing ACN concentrations in the influent. Initially, the MABR started at a moderate selection pressure, with a hydraulic retention time of 16 h, a recirculation rate of 8 cm/s and a starting ACN concentration of 250 mg/l to boost the growth of the biofilm mass on the membrane and to avoid its loss by hydraulic washout. The step increase in the influent ACN concentration was implemented once ACN concentration in the effluent showed almost complete removal in each stage. The specific ACN degradation rate achieved the highest at the loading rate of 101.1 mg ACN/g-VSS h (VSS, volatile suspended solids) and then declined with the further increases in the influent ACN concentration, attributed to the substrate inhibition effect. The adapted membrane-aerated biofilm was capable of completely removing ACN at the removal capacity of up to 21.1 g ACN/m² day, and generated negligible amount of suspended sludge in the effluent. Batch incubation experiments also demonstrated that the ACN-degrading biofilm can degrade other organonitriles, such as acrylonitrile and benzonitrile as well. Denaturing gradient gel electrophoresis studies showed that the ACN-degrading biofilms contained a stable microbial population with a low diversity of sequence of community 16S rRNA gene fragments. Specific oxygen utilization rates were found to increase with the increases in the biofilm thickness, suggesting that the biofilm formation process can enhance the metabolic degradation efficiency towards ACN in the MABR. The study contributes to a better understanding in microbial adaptation in a MABR for biodegradation of ACN. It also highlights the potential benefits in using MABRs for biodegradation of organonitrile contaminants in industrial wastewater.     

Model-based comparative performance analysis of membrane aerated biofilm reactor configurations

The potential of the membrane aerated biofilm reactor (MABR) for high-rate bio-oxidation was investigated. A reaction-diffusion model was combined with a preliminary hollow-fiber MABR process model to investigate reaction rate-limiting regime and to perform comparative analysis on prospective designs and operational parameters. High oxidation fluxes can be attained in the MABR if the intra-membrane oxygen pressure is sufficiently high, however the volumetric oxidation rate is highly dependent on the membrane specific surface area and therefore the maximum performance, in volumetric terms, was achieved in MABRs with relatively thin fibers. The results show that unless the carbon substrate concentration is particularly high, there does not appear to be an advantage to be gained by designing MABRs on the basis of thick biofilms even if oxygen limitations can be overcome.     

Stratified growth in Pseudomonas aeruginosa biofilms

In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp(1) promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 microm wide in colony biofilms and 30 microm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 microm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.     

Oxygen mass transfer characteristics in a membrane-aerated biofilm reactor

Immobilization of pollutant-degrading microorganisms on oxygen-permeable membranes provides a novel method of increasing the oxidation capacity of wastewater treatment bioreactors. Oxygen mass transfer characteristics during continuous-flow steady-state experiments were investigated for biofilms supported on tubular silicone membranes. An analysis of oxygen mass transport and reaction using an established mathematical model for dual-substrate limitation supported the experimental results reported. In thick biofilms, an active layer of biomass where both carbon substrate and oxygen are available was found to exist. The location of this active layer varies depending on the ratio of the carbon substrate loading rate to the intramembrane oxygen pressure. The thickness of a carbon-substrate-starved layer was found to greatly influence the mass transport of oxygen into the active biomass layer, which was located close to, but not in contact with, the biofilm-liquid interface. The experimental results demonstrated that oxygen uptake rates as high as 20 g m-2 d-1 bar-1 can be achieved, and the model predicts that, for an optimized biofilm thickness, oxygen uptake rates of more than 30 g m-2 d-1 bar-1 should be possible. This would allow membrane-aerated biofilm reactors to operate with much greater thicknesses of active biomass than can conventional biofilm reactors as well as offering the further advantage of close to 100% oxygen conversion efficiencies for the treatment of high-strength wastewaters. In the case of dual- substrate-limited biofilms, the potential to increase the oxygen flux does not necessarily increase the substrate (acetate) removal rate.     

Successional development of sulfate-reducing bacterial populations and their activities in a wastewater biofilm growing under microaerophilic conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 microM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 microm from the surface during all stages of biofilm development. The first sulfide production of 0.32 micromol of H(2)S m(-2) s(-1) was detected below ca. 500 microm in the 3rd week and then gradually increased to 0.70 micromol H(2)S m(-2) s(-1) in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 x 10(9) cells cm(-3) and 3.6 x 10(9) cells cm(-3), respectively, in the surface 400 microm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter‐diffusion geometry

The link between nitritation success in a membrane‐aerated biofilm reactor (MABR) and the composition of the initial ammonia‐ and nitrite‐oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat‐sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO₂^‐‐N/NH₄⁺‐N) were obtained in the Inoculum B‐ (55.2–56.4%) versus the Inoculum A‐ (20.2–22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100–150 µm), but the AOB profiles [based on 16S rRNA gene targeted real‐time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B‐ versus A‐initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A‐ and B‐initiated reactors were Nitrosospira spp. (48.9–61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8–63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell^?1 h^?1 versus 0.79 fmol cell^?1 h^?1). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.     

Modeling of an aerobic biofilm reactor with double-limiting substrate kinetics: bifurcational and dynamical analysis

A mathematical model of an aerobic biofilm reactor is presented to investigate the bifurcational patterns and the dynamical behavior of the reactor as a function of different key operating parameters. Suspended cells and biofilm are assumed to grow according to double limiting kinetics with phenol inhibition (carbon source) and oxygen limitation. The model presented by Russo et al. is extended to embody key features of the phenomenology of the granular‐supported biofilm: biofilm growth and detachment, gas–liquid oxygen transport, phenol, and oxygen uptake by both suspended and immobilized cells, and substrate diffusion into the biofilm. Steady‐state conditions and stability, and local dynamic behavior have been characterized. The multiplicity of steady states and their stability depend on key operating parameter values (dilution rate, gas–liquid mass transfer coefficient, biofilm detachment rate, and inlet substrate concentration). Small changes in the operating conditions may be coupled with a drastic change of the steady‐state scenario with transcritical and saddle‐node bifurcations. The relevance of concentration profiles establishing within the biofilm is also addressed. When the oxygen level in the liquid phase is <10% of the saturation level, the biofilm undergoes oxygen starvation and the active biofilm fraction becomes independent of the dilution rate. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

The trade-offs and effect of carrier size and oxygen-loading on gaseous toluene removal performance of a three-phase circulating-bed biofilm reactor

We conducted a series of steady-state and short-term experiments on a three-phase circulating-bed biofilm reactor (CBBR) for removing toluene from gas streams. The goal was to investigate the effect of macroporous-carrier size (1-mm cubes versus 4-mm cubes) on CBBR performance over a wide range of oxygen loading. We hypothesized that the smaller biomass accumulation with 1-mm carriers would minimize dissolved-oxygen (DO) limitation and improve toluene removal, particularly when the DO loading is constrained. The CBBR with 1-mm carriers overcame the performance limitation observed with the CBBR with 4-mm carriers: i.e., oxygen depletion inside the biofilm. The 1-mm carriers consistently gave superior removal of toluene and chemical oxygen-demand, and the advantage was greatest for the lowest oxygen loading and the greatest toluene loading. The 1-mm carriers achieved superior performance because they minimized the negative effects of oxygen depletion, while continuing to provide protection from excess biomass detachment and inhibition from toluene.     

Conduction-based modeling of the biofilm anode of a microbial fuel cell

The biofilm of a microbial fuel cell (MFC) experiences biofilm-related (growth and mass transport) and electrochemical (electron conduction and charger-transfer) processes. We developed a dynamic, one-dimensional, multi-species model for the biofilm in three steps. First, we formulated the biofilm on the anode as a "biofilm anode" with the following two properties: (1) The biofilm has a conductive solid matrix characterized by the biofilm conductivity (kappa(bio)). (2) The biofilm matrix accepts electrons from biofilm bacteria and conducts the electrons to the anode. Second, we derived the Nernst-Monod expression to describe the rate of electron-donor (ED) oxidation. Third, we linked these components using the principles of mass balance and Ohm's law. We then solved the model to study dual limitation in biofilm by the ED concentration and local potential. Our model illustrates that kappa(bio) strongly influences the ED and current fluxes, the type of limitation in biofilm, and the biomass distribution. A larger kappa(bio) increases the ED and current fluxes, and, consequently, the ED mass-transfer resistance becomes significant. A significant gradient in ED concentration, local potential, or both can develop in the biofilm anode, and the biomass actively respires only where ED concentration and local potential are high. When kappa(bio) is relatively large (i.e., > or =10(-3) mS cm(-1)), active biomass can persist up to tens of micrometers away from the anode. Increases in biofilm thickness and accumulation of inert biomass accentuate dual limitation and reduce the current density. These limitations can be alleviated with increases in the specific detachment rate and biofilm density.     

Modeling of experiments on biofilm penetration effects in a fluidized bed nitrification reactor

A four-component, diffusion-reaction model with double Michaelis-Menten kinetics was used to describe the experimental data obtained from a laboratory biofilm, fluidized-bed nitrification reactor. Theory and experiment demonstrated that the stoichiometric ratio (3.5 mg O(2)/mg NH(4) (+)-N) can be employed as a criterion to determine whether the limiting substrate is oxygen or ammonia. For the present work, in the range of concentrations where limitation occurred, 4 mg/L NH(4) (+)-N and 14 mg/L O(2), the ratio of oxygen to ammonia in the bulk liquid determined which substrate was penetration-limiting-O(2) if <3.5 and NH(4) (+) if > 3.5. Halforder kinetics with respect to the limiting substrate described the apparent overall rates. Simulations provided biofilm concentration profiles which demonstrated the role of the oxygen-ammonia ratio. Experiments indicated that, generally, high NO(2) (-) concentrations can be expected. These depend on the residence time, biofilm area, and oxygen concentration. This dependency was investigated with the model, as was the parametric sensitivity with respect to the saturation constants. Particularly important for the NO(2) (-) levels were the ratios of the saturation constants for oxygen.     

Microbial community analysis of a methane-oxidizing biofilm using ribosomal tag pyrosequencing

Current ecological knowledge of methanotrophic biofilms is incomplete, although they have been broadly studied in biotechnological processes. Four individual DNA samples were prepared from a methanotrophic biofilm, and a multiplex 16S rDNA pyrosequencing was performed. A complete library (before being de-multiplexed) contained 33,639 sequences (average length, 415 nt). Interestingly, methanotrophs were not dominant, only making up 23% of the community. Methylosinus, Methylomonas, and Methylosarcina were the dominant methanotrophs. Type II methanotrophs were more abundant than type I (56 vs. 44%), but less richer and diverse. Dominant non-methanotrophic genera included Hydrogenophaga, Flavobacterium, and Hyphomicrobium. The library was de-multiplexed into four libraries, with different sequencing efforts (3,915-20,133 sequences). S?rrenson abundance similarity results showed that the four libraries were almost identical (indices > 0.97), and phylogenetic comparisons using UniFrac test and P-test revealed the same results. It was demonstrated that the pyrosequencing was highly reproducible. These survey results can provide an insight into the management and/or manipulation of methanotrophic biofilms.     

Sensitivity analysis of a biofilm model describing a one-stage completely autotrophic nitrogen removal (CANON) process.

A mathematical model for nitrification and anaerobic ammonium oxidation (ANAMMOX) processes in a single biofilm reactor (CANON) was developed. This model describes completely autotrophic conversion of ammonium to dinitrogen gas. Aerobic ammonium and nitrite oxidation were modeled together with ANAMMOX. The sensitivity of kinetic constants and biofilm and process parameters to the process performance was evaluated, and the total effluent concentrations were, in general, found to be insensitive to affinity constants. Increasing the amount of biomass by either increasing biofilm thickness and density or decreasing porosity had no significant influence on the total effluent concentrations, provided that a minimum total biomass was present in the reactor. The ANAMMOX process always occurred in the depth of the biofilm provided that the oxygen concentration was limiting. The optimal dissolved oxygen concentration level at which the maximum nitrogen removal occurred is related to a certain ammonium surface load on the biofilm. An ammonium surface load of 2 g N/m2. d, associated with a dissolved oxygen concentration level of 1.3 g O2/m3 in the bulk liquid and with a minimum biofilm depth of 1 mm seems a proper design condition for the one-stage ammonium removal process. Under this condition, the ammonium removal efficiency is 94% (82% for the total nitrogen removal efficiency) (30 degrees C). Better ammonium removal could be achieved with an increase in the dissolved oxygen concentration level, but this would strongly limit the ANAMMOX process and decrease total nitrogen removal. It can be concluded that a one-stage process is probably not optimal if a good nitrogen effluent is required. A two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.     

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi‐species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite‐oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia‐oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients. Biotechnol. Bioeng. 2010;105: 1115–1130. © 2009 Wiley Periodicals, Inc.     

Intra-particle oxygen diffusion limitation in solid-state fermentation

Oxygen limitation in solid-state fermentation (SSF) has been the topic of modeling studies, but thus far, there has been no experimental elucidation on oxygen-transfer limitation at the particle level. Therefore, intra-particle oxygen transfer was experimentally studied in cultures of Rhizopus oligosporus grown on the surface of solid, nutritionally defined, glucose and starch media. The fungal mat consisted of two layers--an upper layer with sparse aerial hyphae and gas-filled interstitial pores, and a dense bottom layer with liquid-filled pores. During the course of cultivation ethanol was detected in the medium indicating that oxygen was depleted in part of the fungal mat. Direct measurement of the oxygen concentrations in the fungal mat during cultivation, using oxygen microelectrodes, showed no oxygen depletion in the upper aerial layer, but revealed development of steep oxygen concentration gradients in the wet bottom layer. Initially, the fungal mat was fully oxygenated, but after 36.5 hours oxygen was undetectable at 100 microm below the gas-liquid interface. This was consistent with the calculated oxygen penetration depth using a reaction-diffusion model. Comparison of the overall oxygen consumption rate from the gas phase to the oxygen flux at the gas-liquid interface showed that oxygen consumption of the microorganisms occurred mainly in the wet part of the fungal mat. The contribution of the aerial hyphae to overall oxygen consumption was negligible. It can be concluded that optimal oxygen transfer in SSF depends on the available interfacial gas-liquid surface area and the thickness of the wet fungal layer. It is suggested that the moisture content of the matrix affects both parameters and, therefore, plays an important role in optimizing oxygen transfer in SSF cultures.     

The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.     

Evaluation of kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor for wastewater treatment

This study evaluates the kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor from the steady state values of the response of the system to step changes in inlet concentration. It was observed from the outlet biological oxygen demand (BOD(5)) plot of the response of the system that as the inlet BOD(5) was increased, the outlet BOD(5) also increased, reached a peak value and then decreased until it leveled to a new steady state value corresponding to the new inlet concentration level. The increase in BOD(5) was attributed to the accumulation of substrate within the reactor as well as the decrease in biofilm substrate consumption rate as the microorganisms adjusted to the new environment. Using the substrate balance at steady state and assuming Monod kinetics, an equation relating the substrate consumption rate to substrate concentration (BOD(5)) and total biofilm surface area had been established. Monod kinetic parameters were found to be K=2.20g/m(2)/day, K(m)=17.41g/m(3) and K/K(m)=0.13m/day. The ratio K/K(m) can be taken as the indicator for biofilm substrate degradation effectiveness at low substrate concentrations.