The relationship between instability of H2 production and compositions of bacterial communities within a dark fermentation fluidized-bed bioreactor

Microbial community composition dynamics was studied during H(2) fermentation from glucose in a fluidized-bed bioreactor (FBR) aiming at obtaining insight into the H(2) fermentation microbiology and factors resulting in the instability of biofilm processes. FBR H(2) production performance was characterised by an instable pattern of prompt onset of H(2) production followed by rapid decrease. Gradual enrichment of organisms increased the diversity of FBR attached and suspended-growth phase bacterial communities during the operation. FBR bacteria included potential H(2) producers, H(2) consumers and neither H(2) producers nor consumers, and those distantly related to any known organisms. The prompt onset of H(2) production was due to rapid growth of Clostridium butyricum (99-100%) affiliated strains after starting continuous feed. The proportion trend of C. butyricum in FBR attached and suspended-growth phase communities coincided with H(2) and butyrate production. High glucose loading rate favoured the H(2) production by Escherichia coli (100%) affiliated strain. Decrease in H(2) production, associated with a shift from acetate-butyrate to acetate-propionate production, was due to changes in FBR attached and suspended-growth phase bacterial community compositions. During the shift, organisms, including potential propionate producers, were enriched in the communities while the proportion trend of C. butyricum decreased. We suggest that the instability of H(2) fermentation in biofilm reactors is due to enrichment and efficient adhesion of H(2) consumers on the carrier and, therefore, biofilm reactors may not favour mesophilic H(2) fermentation.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

E. coli biofilm formation and its susceptibility towards T4 bacteriophages studied in a continuously operating mixing – tubular bioreactor system

A system consisting of a connected mixed and tubular bioreactor was designed to study bacterial biofilm formation and the effect of its exposure to bacteriophages under different experimental conditions. The bacterial biofilm inside silicone tubular bioreactor was formed during the continuous pumping of bacterial cells at a constant physiological state for 2 h and subsequent washing with a buffer for 24 h. Monitoring bacterial and bacteriophage concentration along the tubular bioreactor was performed via a piercing method. The presence of biofilm and planktonic cells was demonstrated by combining the piercing method, measurement of planktonic cell concentration at the tubular bioreactor outlet, and optical microscopy. The planktonic cell formation rate was found to be 8.95 × 10⁻³ h⁻¹ and increased approximately four-fold (4×) after biofilm exposure to an LB medium. Exposure of bacterial biofilm to bacteriophages in the LB medium resulted in a rapid decrease of biofilm and planktonic cell concentration, to below the detection limit within < 2 h. When bacteriophages were supplied in the buffer, only a moderate decrease in the concentration of both bacterial cell types was observed. After biofilm washing with buffer to remove unadsorbed bacteriophages, its exposure to the LB medium (without bacteriophages) resulted in a rapid decrease in bacterial concentration: again below the detection limit in < 2 h.     

Bacterial Community Dynamics during Start-Up of a Trickle-Bed Bioreactor Degrading Aromatic Compounds

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a car painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.Many branches of industry produce waste gases which contain odorous organic and inorganic components. Apart from the conventional thermal and physicochemical techniques for removal of pollutants from exhaust air, biological waste gas treatment is becoming more and more important. This kind of treatment is advantageous in cases in which the recovery of the components (e.g., absorption in liquids and adsorption in solids) or the utilization of a thermal process (thermal or catalytic combustion) is not economical. Today three different process variations for biological waste gas treatment are used: biofilters, bioscrubbers, and trickle-bed bioreactors. In biofilters and trickle-bed reactors, the pollutant-degrading microorganisms are immobilized on a carrier material, whereas in bioscrubbers the microorganisms are dispersed in the liquid phase. Biofilters and bioscrubbers are preferred in industry, while biofilters are common in compost production and sewage plants (10).Biological waste gas treatment has a long tradition. Already in 1953, a soil system was employed for the treatment of odorous sewer exhaust gases in Long Beach, Calif. (25), and although up to now a lot of efforts have been funneled into process engineering (14, 17, 18, 24), current knowledge of the involved microorganisms is still very limited. Diversity of the microbial communities in the bioreactors for the exhaust gas purification have mostly been analyzed by culture-dependent methods (9, 12, 28, 31). However, there is a large discrepancy between the total (direct) microscopic cell counts and viable plate counts in many ecosystems and every cultivation medium selects for certain microorganisms. Therefore, cultivation-based studies of bacterial populations may give wrong impressions of the actual community structure in an ecosystem (35). A possible means of avoiding qualitative and quantitative errors in the analysis of microbial community structure in complex ecosystems is the use of fluorescently labeled, rRNA-targeted oligonucleotides (5) for the in situ identification and enumeration of bacteria. This method has already been used successfully in complex microbial communities, such as multispecies biofilms (6, 22, 26), trickling filters (27), and activated sludge (37).The current study was performed with a laboratory-scale trickle-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the inoculation of the bioreactor was an enrichment prepared in a fermentor which was itself started with a wastewater sample from a car painting factory as the inoculum and Solvesso100 as the sole carbon source. The goal of our study was to use for the first time fluorescent in situ hybridization (FISH) to investigate the microbial community structure and dynamics in the fermentor and the bioreactor during start-up. One of the open questions was whether the fermentor enrichment, which is done in suspension, indeed selects for those bacteria that later are immobilized in the bioreactor. In the course of this study, new 16S as well as 23S rRNA-targeted probes for phylogenetic groups within the beta subclass of the class Proteobacteria have been developed and applied in order to obtain a higher taxonomic resolution of the molecular techniques. The molecular data were compared to those obtained by traditional cultivation-dependent techniques.     

Membrane bioreactor with a porous hydrophobic membrane as a gas-liquid contactor for waste gas treatment

A novel type of bioreactor for waste gas treatment has been designed. The reactor contains a microporous hydrophobic membrane to create a large interface between the waste gas and the aqueous phase. To test the new reactor, propene was chosen because of its high air/water partition coefficient, which causes a low water concentration and hampers its removal from air. Propene transfer from air to a suspension of propene-utilizing Xanthobacter Py2 cells in the membrane bioreactor proved to be controlled by mass transfer in the liquid phase. The resistance of the membrane was negligible. Simulated propene transfer rates agreed well with the experimental data. A stable biofilm of Xanthobacter Py2 developed on the membrane during prolonged operation. The propene flux into the biofilm was 1 x 10(-6) mol m(-2) s(-1) at a propene concentration of 9.3 x 10(-2) mol m(-3) in the gas phase. (c) 1995 John Wiley & Sons, Inc.     

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     

Membrane process for biological treatment of contaminated gas streams

A hollow fiber membrane bioreactor was investigated for control of air emissions of biodegradable volatile organic compounds (VOCs). In the membrane bioreactor, gases containing VOCs pass through the lumen of microporous hydrophobic hollow fiber membranes. Soluble compounds diffuse through the membrane pores and partition into a VOC degrading biofilm. The hollow fiber membranes serve as a support for the microbial population and provide a large surface area for VOC and oxygen mass transfer. Experiments were performed to investigate the effects of toluene loading rate, gas residence time, and liquid phase turbulence on toluene removal in a laboratory-scale membrane bioreactor. Initial acclimation of the microbial culture to toluene occurred over a period of nine days, after which a 70% removal efficiency was achieved at an inlet toluene concentration of 200 ppm and a gas residence time of 1.8 s (elimination capacity of 20 g m-3 min-1). At higher toluene loading rates, a maximum elimination capacity of 42 g m-3 min-1 was observed. In the absence of a biofilm (abiotic operation), mass transfer rates were found to increase with increasing liquid recirculation rates. Abiotic mass transfer coefficients could be estimated using a correlation of dimensionless parameters developed for heat transfer. Liquid phase recirculation rate had no effect on toluene removal when the biofilm was present, however. Three models of the reactor were created: a numeric model, a first-order flat sheet model, and a zero-order flat sheet model. Only the numeric model fit the data well, although removal predicted as a function of gas residence time disagreed slightly with that observed. A modification in the model to account for membrane phase resistance resulted in an underprediction of removal. Sensitivity analysis of the numeric model indicated that removal was a strong function of the liquid phase biomass density and biofilm diffusion coefficient, with diffusion rates below 10(-9) m2 s-1 resulting in decreased removal rates.     

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.     

A pilot study of bacteriological population changes through potable water treatment and distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

Characterization of high density monolayers of the biofilm bacterium Caulobacter crescentus: evaluating prospects for developing immobilized cell bioreactors

Caulobacters are biofilm-forming members of the natural flora of soil and aquatic environments, which exhibit several characteristics that make them attractive for development of high surface area microbial bioreactors or biosensors. Although caulobacters are well characterized genetically, little is known about their biofilm-forming characteristics as a monoculture, or their tolerance of bioreactor-like conditions. Here we investigated the ability of caulobacters to spontaneously form high-density monolayers on artificial surfaces under a variety of environmental conditions, using phase contrast image analysis to assess biofilm density, and epifluorescence with the vital stain DiBAC to assess viability. With adequate nutrition, extremely dense monolayers formed within 24-48 h, and maintained near 100% viability in experiments ranging up to 22 days. When areas were abraded to remove cells, repopulation occurred rapidly with characteristics similar to the population of a clean surface. When established monolayers were starved for nutrients, a significant fraction of the cells detached from the surface, and cells remaining on the surface no longer tested as viable. Within 4-6 h of nutrient restoration, however, cells in the monolayer again appeared normal and tested as 100% viable. This is the first demonstration that Caulobacter crescentus is stable and amenable to high density monolayer growth and resists starvation, though some cells may express a programmed response to detach from the surface under severe nutrient limitation.     

Antimicrobial GL13K Peptide Coatings Killed and Ruptured the Wall of Streptococcus gordonii and Prevented Formation and Growth of Biofilms

Infection is one of the most prevalent causes for dental implant failure. We have developed a novel antimicrobial peptide coating on titanium by immobilizing the antimicrobial peptide GL13K. GL13K was developed from the human salivary protein BPIFA2. The peptide exhibited MIC of 8 µg/ml against planktonic Pseudonomas aeruginosa and their biofilms were reduced by three orders of magnitude with 100 µg/ml GL13K. This peptide concentration also killed 100% of Streptococcus gordonii. At 1 mg/ml, GL13K caused less than 10% lysis of human red blood cells, suggesting low toxicity to mammalian cells. Our GL13K coating has also previously showed bactericidal effect and inhibition of biofilm growth against peri-implantitis related pathogens, such as Porphyromonas gingivalis. The GL13K coating was cytocompatible with human fibroblasts and osteoblasts. However, the bioactivity of antimicrobial coatings has been commonly tested under (quasi)static culture conditions that are far from simulating conditions for biofilm formation and growth in the oral cavity. Oral salivary flow over a coating is persistent, applies continuous shear forces, and supplies sustained nutrition to bacteria. This accelerates bacteria metabolism and biofilm growth. In this work, the antimicrobial effect of the coating was tested against Streptococcus gordonii, a primary colonizer that provides attachment for the biofilm accretion by P. gingivalis, using a drip-flow biofilm bioreactor with media flow rates simulating salivary flow. The GL13K peptide coatings killed bacteria and prevented formation and growth of S. gordonii biofilms in the drip-flow bioreactor and under regular mild-agitation conditions. Surprisingly the interaction of the bacteria with the GL13K peptide coatings ruptured the cell wall at their septum or polar areas leaving empty shell-like structures or exposed protoplasts. The cell wall rupture was not detected under regular culture conditions, suggesting that cell wall rupture induced by GL13K peptides also requires media flow and possible attendant biological sequelae of the conditions in the bioreactor.     

A Pilot Study of Bacteriological Population Changes through Potable Water Treatment and Distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

Bioreactor for the study of defined interactions of toxic metals and biofilms.

A novel bioreactor system constructed for studies of the interactions of heavy metals and microbial cells at the solid-solution interface is described. The applicability of this experimental system to meet the severe constraints imposed on such an apparatus by the requirements for an unambiguous interpretation of data and for mathematical modeling of these interactions was explored with the trace metal lead and with the marine bacterium Pseudomonas atlantica. A chemically defined medium composed of the major components of seawater, simple salts required for growth, glucose, and the single amino acid glycine was derived. It supported a maximum growth rate several times less than that in a complex medium, but provided growth to high cell densities and the formation of biopolymer and supported the development of a monolayer biofilm. The use of such a medium in conjunction with our bioreactor system minimized trace metal contamination while allowing quantification of the partitioning of lead onto various reactor surfaces. Lead adsorption by reactor walls and model surfaces was linear with equilibrium led concentration up to 6 X 10(-6) mol/liter. Equilibrium lead adsorption due to P. atlantica biofilm surfaces ranged from 20 to 40% at a total lead concentration of 10(-6) mol/liter depending upon solution pH and ionic composition, indicating that biofilms can play an important role in controlling toxic metal concentrations in natural systems.     

Bioreactor for the study of defined interactions of toxic metals and biofilms

A novel bioreactor system constructed for studies of the interactions of heavy metals and microbial cells at the solid-solution interface is described. The applicability of this experimental system to meet the severe constraints imposed on such an apparatus by the requirements for an unambiguous interpretation of data and for mathematical modeling of these interactions was explored with the trace metal lead and with the marine bacterium Pseudomonas atlantica. A chemically defined medium composed of the major components of seawater, simple salts required for growth, glucose, and the single amino acid glycine was derived. It supported a maximum growth rate several times less than that in a complex medium, but provided growth to high cell densities and the formation of biopolymer and supported the development of a monolayer biofilm. The use of such a medium in conjunction with our bioreactor system minimized trace metal contamination while allowing quantification of the partitioning of lead onto various reactor surfaces. Lead adsorption by reactor walls and model surfaces was linear with equilibrium led concentration up to 6 X 10(-6) mol/liter. Equilibrium lead adsorption due to P. atlantica biofilm surfaces ranged from 20 to 40% at a total lead concentration of 10(-6) mol/liter depending upon solution pH and ionic composition, indicating that biofilms can play an important role in controlling toxic metal concentrations in natural systems.     

The effects of starvation and re-feeding on glycogen metabolism in mouse tail skin.

Although the glycogen content of mouse tail skin was decreased during starvation and was restored on re feeding, the proportion of glycogen synthase in the I form remained constant throughout at about 10% of the total. During the phase of net glycogen synthesis 1.5h after access to food was restored, the concentration of UDP glucose was markedly increased and the proportion of phosphorylase in the a form was significantly decreased.     

Treatment of chromium(VI) solutions in a pilot-scale bioreactor through a biofilm of Arthrobacter viscosus supported on GAC

The aim of this work is to evaluate the applicability of a biofilm to the removal of chromium in solution, at a pilot scale. The effect of the initial concentration of metal on the biosorption behavior of an Arthrobacter viscosus biofilm supported on granular activated carbon, in batch and column essays was also analyzed. Six isotherm equations have been tested in the present study. The best fit was obtained with the Freundlich model. It was observed that as the initial chromium concentration increases, the uptake increases too, but the removal percentage decreases, with values between 95.20% (C(0)=5mg/l) and 38.28% (C(0)=1000 mg/l). The batch adsorption studies were used to develop a pilot bioreactor able to remove chromium from aqueous solutions. Data obtained in a pilot-scale reactor showed an average removal percentage of 99.9%, during the first 30 days, for the initial concentration of 10mg/l and an average removal percentage of 72%, for the same period and for the initial concentration of 100mg/l. Uptake values of 11.35 mg/g and 14.55 mg/g were obtained, respectively, for the initial concentration of 10 and 100mg/l. The results obtained are very promising and encourage the utilization of this biofilm in environmental applications.     

Evidence for multisite growth of Escherichia coli murein involving concomitant endopeptidase and transpeptidase activities.

During diaminopimelic acid starvation of Escherichia coli W7, a large fraction of the preexisting murein cross-links are opened by murein endopeptidase and the resulting uncross-linked material is degraded. This is reflected morphologically in a general loss of rigidity of the murein sacculus long before lysis occurs. In growing cells, a dynamic situation is demonstrable. When cells whose murein sacculi are uniformly labeled with [14C]diaminopimelic acid were chased with unlabeled DAP, a significant, rapid shift of [14C]diaminopimelic acid from the donor to the acceptor half of dimers was observed. The shift can be explained by the presence of about 100 separate sites where new murein strands were being inserted between old radioactive strands of murein. Thus, the gradual loss of rigidity of the murein sacculus as endopeptidase continues to function during starvation of E. coli W7 suggests an even distribution of the active endopeptidases. This is consistent with the kinetic data which suggest that endopeptidase, along with murein synthetase and transpeptidase, acts at about 100 distinct sites to elongate the murein sacculus.     

Dynamics of living and dead bacterial cells within a mixed-species biofilm during toluene degradation in a biotrickling filter

AIMS: To study the accumulation of the bacterial living cells (LC) and dead cells (DC) in a mixed-species biofilm developed in a 3 l biotrickling filter (BTF) challenged with toluene. METHODS AND RESULTS: The bacterial LC and DC within the biofilm developed on polypropylene Pall rings in a toluene-degrading BTF were enumerated as fluoro-microscopic counts during a 62-operating day period using nucleic acid staining and the direct epifluorescence filter technique. The biofilm development could be separated into three distinct phases: (i) cell attachment, (ii) biofilm establishment and (iii) biofilm maturation. The LC were always dominant (>/=72%) in the biofilm during the establishment phase whereas the average LC fraction decreased to 51% of the total cells in the maturation phase. The concentration of LC and DC was observed to level off after 41 days at 1010 cells per ring. The biofilm thickness and the dry weight increased independently of the cell number during the maturation phase. CONCLUSIONS: After the LC reached a maximum concentration in the biofilm, the biofilm proliferation was only characterized by the accumulation of DC and organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in the present study are of particular relevance for biofilm mathematical modelling and numerical simulations. They will also be useful to estimate the contribution of the living bacteria within the biofilm in bioprocesses.