Utilisation of a packed-bed biofilm reactor for the determination of the potential of biofilm accumulation in water systems

An experimental system has been developed that allows the monitoring of biofilm development on supports exposed to water of different characteristics. The system consists of a series of packed-bed reactors filled with glass beads, and by periodically removing biofilm attached to these beads for off-line analyses this provides a means for monitoring biofilm development. Despite its reduced dimensions (6.9 cm long and 1.58 cm in diameter), the experimental system used has a sampling surface of 90.3 cm2 (including only the surface of the glass beads). This allows reproducible and representative samples to be taken from different water systems, providing a reliable and economic method for evaluating in situ the formation of biofilms from different environments. The set-up of the entire experimental system was constructed to meet the demands of field experiments in a well-defined hydrodynamic environment and to allow easy removal of samples for biomass quantification and microscopic observation. Data obtained using this device can be used as an indicator of the risk of biofilm formation in different water systems. This indicator, "the biofilm accumulation potential", represents an effective and representative tool for the monitoring of biofilm development in an integrated antifouling strategy, in order to help keep biofouling, scaling and microbial risks under control. According to the experiments with the packed-bed reactors used with a high flow regime, the ratio TCN/HPC could provide an indication of the state of the biofilm, and lower ratios could indicate a higher biofilm accumulation potential.     

Adhesion and biofilm development on suspended carriers in airlift reactors: hydrodynamic conditions versus surface characteristics

Adhesion and biofilm formation by Pseudomonas putida was studied using suspended carriers in laboratory airlift reactors. Standard, roughened, hydrophobic, and positively charged glass beads, sand, and basalt grains were used as carriers. The results clearly show that in airlift reactors hydrodynamic conditions and particle collisions control biofilm formation. In the reactors, on surfaces subjected to different shear levels, biofilm formation differed considerably. This could be described by a simple growth and detachment model. Increased surface roughness promoted biofilm accumulation on suspended carriers. The physicochemical surface characteristics of the carrier surface proved to be less important due to the turbulent conditions in the airlift reactors. Adhesion of P. putida to glass beads was poor, and results of an adhesion test under quiescent conditions were not predictive for adhesion and subsequent biofilm formation under reactor conditions. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:880-889, 1997.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

Aerobic dechlorination of low-chlorinated biphenyls by bacterial biofilms in packed-bed batch bioreactors

Cells of an aerobic three-membered bacterial co-culture, designated as ECO3, capable of cometabolizing and aerobically dechlorinating low-chlorinated biphenyls in the presence of biphenyl, were immobilized on Manville silica beads, on frosted-glass beads and on polyurethane foam cubes in packed-bed bioreactors continuously fed with a biphenyl-saturated air stream. The ECO3 biofilm reactors were found to be capable of extensively mineralizing several pure dichlorobiphenyls (75 mg/l) and Aroclor 1221 (75 mg/l) in batch mode. Immobilized ECO3 cells could aerobically degrade and dechlorinate the dichlorobiphenyls tested more extensively than suspended ECO3 cells. Among the three biofilm reactors, the glass bead bioreactor and the polyurethane bioreactor exhibited the highest capability of mineralizing both dichlorobiphenyls and Aroclor 1221; the polychlorinated biphenyl availability in the bioreactors, more than the biomass availability, both depending on the nature of the support employed, significantly governed the efficiency of the treatment. These results are of interest for the possible development of a bioreactor system for continuous treatment of polychlorinated-biphenyl-contaminated wastewaters.     

Immobilization of Lactobacillus casei cells to ceramic material pretreated with polyethylenimine

Summary The cells of Lactobacillus casei were adsorbed to Poraver, foam glass particles pretreated with polyethylenimine (PEI). Exposure of cells for a relatively short period to Poraver beads coated with a high concentration of PEI resulted in maximal adsorption with good retention of metabolic activity. The immobilized cells were tested in packed-bed and stirred-tank reactors for lactic acid production. Stirred-tank operations were more effective in terms of productivity but the support was sensitive to attrition. The beads exhibited good mechanical stability to withstand pressure in the packed-bed reactor. Correspondence to: Bo Mattiasson     

Fast remediation of coal-tar-related compounds in biofilm bioreactors

The biological degradation of complex mixtures of recalcitrant substances is still a major challenge in environmental biotechnology and the remediation of coal-tar constitutes one such problem area. Biofilm bioreactors offer many advantages and may be successfully used for this purpose. Two stirred-tank reactors and one packed-bed reactor were tested in a continuous mode. Continuous cultivation allows microbial selection to take place whilst adhesive growth provides a high degradation capacity and process stability. The reactors were inoculated with mixed microbial populations to favour complete metabolism and to prevent metabolite accumulation and substrate inhibition effects. Phenol, o-cresol, quinoline, dibenzofuran, acenaphthene and phenanthrene were used as model contaminants and constituted the sole energy and carbon sources. The hydraulic retention time (HRT) was initially set to 2.5 days for a period of several months to allow the establishment of a stable biofilm and was then gradually decreased. All the compounds were found to be degraded by more than 90% at HRT of 3 h or more. Neither substrate inhibition nor metabolite accumulation effects were observed. The stirred-tank configuration was found to be the most efficient for use with high loads. No improvement in the degradation capacity could be achieved by increasing the biofilm surface in these reactors, illustrating that the limiting factor may be the mass transfer limitations rather than the availability of the biofilm surface. Finally, anaerobic treatment was successfully achieved, confirming the potential for remediation of contaminated sites under anaerobic conditions, providing that alternative electron acceptors are present. Received: 16 March 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Modeling of an anoxic/methanogenic biofilm: effect of pH calculation within the biofilm

The models of anoxic/methanogenic processes in biofilm reactors published until now have supposed that pH does not change between the bulk liquid and biofilm. These assumptions are not necessarily valid for processes in reactors with biofilms. The present work studied an anoxic/methanogenic biofilm reactor incorporating the pH variation in both bulk and biofilm. Two dynamic models, one including the calculation of pH throughout the biofilm, were solved numerically and compared with each other. The results showed that the inclusion of a pH algorithm calculation produces different profiles and efficiencies on an anoxic/methanogenic biofilm system. Values of C/N ratio higher than 20 mg TOC/mg NO₃–N and values of HRT lower than 4.5 h produce differences of up to 46 % with a traditional model that does not include pH calculation inside the biofilm. Thus, the assumption of a constant pH within the biofilm when using the traditional model does not accurately describe the performance of the system under these conditions, and pH calculation inside the biofilm should be included.     

A microfluidic gradient mixer-flow chamber as a new tool to study biofilm development under defined solute gradients

Understanding the dynamics of biofilm development in response to chemical cues and signals is required toward the development of controllable biofilm-mediated bioprocesses. In this study, we report a new biofilm growth system that integrates a microfluidic gradient mixer with a biofilm growth chamber. The biofilm growth system allows biofilms to grow under defined solute gradients and enables nondestructive monitoring of the biofilm development dynamics in response to the defined gradients. The solute gradients generated in the system were simulated and then validated experimentally. We then demonstrated the applicability of the biofilm growth system in studying biofilm development under defined solute gradients. Specifically, we examined biofilm development of Shewanella oneidensis and Comamonas testosteroni under a defined calcium and nitrate gradient, respectively. Using two C. testosteroni strains (WDL7 and I2), we further demonstrated the applicability of our biofilm growth system to study the development of coculture biofilms under a defined solute gradient. Our results show that the biofilm growth system we have developed here can be a promising tool to reveal the dynamics of biofilm development in response to chemical cues and signals as well as the interorganism interactions in coculture biofilms.     

Influence of surface topography on biofilm development: Experiment and modeling

The effect of surface topography on the long-term development (≈10 weeks) of biofilms has been investigated using a monitoring technique based on images produced by a flat-bed scanner and initially developed for flat surfaces. The biofilm response to rotation speed changes in lab-scale rotating biological contactors (RBCs) has been studied. Two RBCs, each containing five discs (two with flat surfaces and three with rough surfaces) were run initially at two different rotation speeds: 4 rpm for reactor I and 40 rpm for reactor II. After 47 days, the rotation speed was increased in reactor I to 40 rpm and decreased in reactor II to 4 rpm. Prior to the rotation speed change, the biofilm on the flat discs underwent large detachments in both reactors, but the biofilm on rough discs was less extensively damaged. The increase in rotation speed induced large detachments of the biofilm in reactor I on all discs, but the biofilm on the rough discs recovered more effectively with faster regrowth. In reactor II, the decrease in rotation speed favored the development of the biofilm. Wall stress distributions obtained from CFD simulations on flat and rough discs at different rotation speeds were well correlated with experimental observations.     

Potential application of monolith packed columns as bioreactors, control of biofilm formation

Monolith reactors combine good mass transfer characteristics with low-pressure drop, the principle factors affecting the cost effectiveness of industrial processes. Recently, these specific features of the monolith reactors have drawn the attention toward the application of the monolith reactor in multiphase reaction systems. In this study, we explore the potential application of monolith reactors as bioreactor requiring gas-liquid mass transfer for substrate supply. It is demonstrated on theoretical grounds that the monolith reactor is a competitive alternative to conventional gas-liquid bioreactors such as stirred tanks, packed beds, and airlift bioreactors because it allows for a significant reduction of the energy dissipation that is normally required for gas-liquid contacting. A potential problem of monolith reactors for biological processes is clogging due to biofilm formation. This paper presents experimental results of a study into the formation and possible removal of biofilms during operation of a monolith reactor as suspended cells bioreactor. The results indicate that biofilm formation may be minimized and postponed by a proper choice of operating conditions. Periodic biofilm removal could straightforwardly be achieved by rinsing with water at moderate pressures and allows for stable operation for prolonged periods of time.     

Viability of a marine microbial biofilm

The formation of a microbial biofilm on glass surfaces arranged in lamellar piles parallel with circulating sea water (3 cm·sec^–1) was studied. The increase in dry weight, protein content, nucleotide content (ATP, ADP), and diatoms was followed over a period of 62 days. Dry weight and protein were estimates of the total biofilm development, whereas the nucleotide measurements revealed the viability of the biofilm and reflected the dynamics in the community structure.     

Continuous acetonitrile degradation in a packed-bed bioreactor

A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l^–1 reactor day^–1, 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH₃, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l^–1 reactor day^–1 was achieved at a feeding rate of 1.6 g acetonitrile l^–1 reactor day^–1. The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.     

Early stages in biofilm development in methanogenic fluidized-bed reactors

Summary Biofilm development in methanogenic fluidized-bed reactors with sand as the carrier was studied on a laboratory scale. The microorganisms present in consecutive layers of the biofilm of mature sludge granules were preliminarily characterized on the basis of their morphology, element composition and adhesion capacity and were compared to bacteria which take part in the initial colonization of sand. The early phase of biofilm development was monitored with reactors receiving waste-waters containing different mixtures of volatile fatty acids and inoculated with fluidized-bed reactor effluent for different lengths of time. The results obtained indicate that facultative anaerobic bacteria abundantly present in the outermost biofilm layers of mature sludge granules are probably the main primary colonizers of the sand. Methanothrix spp. or other methanogens were rarely observed among the primary colonizers. The course of biofilm formation was comparable under the various start-up conditions employed including variations in waste-water composition, inoculation and anaerobicity. However, omission of waste-water and thus of substrate resulted in rapid wash-out of the attached biomass. Offprint requests to: W. Heinen     

Assessment of artificial substrates for evaluating groundwater microbial quality

Protection of groundwater resources requires the development of reliable ecological indicators. Microorganisms involved in ecological services or being associated with particular hosts or habitats could be used for this purpose. Nevertheless, their tracking remains limited because of sampling issues, and a lack of devices for their long term monitoring. In the present study, three artificial substrates (glass and clay beads, and gravel particles) were tested in terms of efficacy at favoring bacterial growth, and at capturing bacterial diversity of waters (i.e., groundwater, surface water and wastewater). Total proteins, total carbohydrates, dehydrogenase and hydrolytic activities were used to monitor biofilm development on these artificial substrates. Fingerprinting analyses based on rrs (16S rRNA) − rrl (23S rRNA) spacer analyses (ARISA) and next generation sequencing (NGS) of partial rrs DNA segments (V5-V6) were used to compare operating taxonomic units (OTUs), and infer bacterial genera trapped on these substrates. Glass beads were found less efficient than the other two artificial substrates at increasing protein contents and microbial activities (hydrolytic and dehydrogenase activities). ARISA showed a discrimination of bacterial communities developing on artificial substrates that was matching water types. An incubation period of 7 days allowed a reliable assessment of bacterial diversity. From this incubation period, around 75% of water genera with more than four V5-V6 rrs DNA sequences detected in a water type were recovered from biofilms growing on artificial substrates. Based on relative abundances of genera, clay beads and gravel particles were more efficient than glass beads to capture and obtain bacterial communities matching those of the initial waters. Between 45–67% of similarities were found for these artificial substrates while it was between 36 and 43% for glass beads. This study demonstrated clay beads and gravel particles as being efficient tools for capturing bacterial diversity and monitoring bacterial growth. Overall, clay beads appeared the best choice for field monitoring because of the ease of their size standardization in comparison with gravel particles.     

Evaluation of straw as a biofilm carrier in the methanogenic stage of two-stage anaerobic digestion of crop residues

Straw was evaluated as a biofilm carrier in the methanogenic stage of the two-stage anaerobic digestion of crop residues. Three reactor configurations were studied, a straw-packed-bed reactor, a glass packed-bed reactor and a reactor containing suspended plastic carriers. The reactor with the packed straw bed showed the best results. It had the highest methane production, 5.4 11(-1) d(-1), and the chemical oxygen demand (COD) removal ranged from 73-50% at organic loading rates from 2.4-25 g COD l(-1) d(-1). The degradation pattern of volatile fatty acids showed that the degradation of propionate and longer-chain fatty acids was limiting at higher organic loading rates. A stable effluent pH showed that the packed-bed reactors had good ability to withstand the variations in load and volatile fatty acid concentrations that can occur in the two-stage process. The conclusion is that straw would work very well in the intended application. A further benefit is that straw is a common agricultural waste product and requires only limited resources concerning handling and cost.     

A simple rotating annular reactor for replicated biofilm studies

The performance of two types of rotating annular reactors for the cultivation of river biofilms was compared qualitatively and quantitatively. One reactor was a commercially available system with a rotating inner solid cylinder and polycarbonate slides in the outer fixed cylinder. The other, a non-commercial system manufactured in the laboratory, had the polycarbonate slides positioned on a machined, rotating inner cylinder. Microscale comparison of the biofilms was carried out using confocal laser scanning microscopy techniques including, fluorescent nucleic acid staining, fluor conjugated lectins and autofluorescence imaging. The results obtained indicated that the reactors were similar in terms of biofilm development pattern, thickness, bacterial biomass, and exopolymer production. Significant differences were found in terms of photosynthetic biomass with the glass bodied non-commercial reactor providing more favourable conditions for algal growth than the opaque polycarbonate outer cylinder of the commercial reactor. The study indicated that a simple inexpensive reactor constructed from available components and materials, produced river biofilms similar to those obtained using a commercial system but at substantially lower cost. The availability of such inexpensive annular reactors should facilitate much needed replicated studies of biofilm development.     

Degradation of acenaphthene, phenanthrene and pyrene in a packed-bed biofilm reactor

Biofilm reactors are particularly suitable for the treatment of large amounts of diluted effluent, such as groundwater contaminated with scarcely soluble pollutants. A packed-bed column reactor was tested for the degradation of acenaphthene, phenanthrene and pyrene provided at their aqueous solubility concentrations. Acenapthene and phenanthrene were removed to more than 99% efficiency from this reactor whilst pyrene was removed to 90%. Pollutant disappearance was also recorded in the control reactor and was probably caused by the adsorption of pollutants into the reactor. The measurement of oxygen consumption in both reactors confirmed that microbial degradation of the pollutants was indeed occurring in the inoculated reactor. Physical adsorption is not however unwanted, as it could help with the formation of a biofilm at an early stage of the treatment. Received: 29 February 2000 / Received revision: 30 May 2000 / Accepted: 3 June 2000     

Fast Kinetics of Fe2+ Oxidation in Packed-Bed Reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe²⁺ oxidized per liter per h (1,400 mmol of Fe²⁺ oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Influence of dissolved oxygen on the nitrification kinetics in a circulating bed biofilm reactor

The influence of dissolved oxygen concentration on the nitrification kinetics was studied in the circulating bed reactor (CBR). The study was partly performed at laboratory scale with synthetic water, and partly at pilot scale with secondary effluent as feed water. The nitrification kinetics of the laboratory CBR as a function of the oxygen concentration can be described according to the half order and zero order rate equations of the diffusion-reaction model applied to porous catalysts. When oxygen was the rate limiting substrate, the nitrification rate was close to a half order function of the oxygen concentration. The average oxygen diffusion coefficient estimated by fitting the diffusion-reaction model to the experimental results was around 66% of the respective value in water. The experimental results showed that either the ammonia or the oxygen concentration could be limiting for the nitrification kinetics. The latter occurred for an oxygen to ammonia concentration ratio below 1.5–2 gO₂/gN-NH₄ ⁺ for both laboratory and pilot scale reactors. The volumetric oxygen mass transfer coefficient (k _L a) determined in the laboratory scale reactor was 0.017?s^?1 for a superficial air velocity of 0.02?m s^?1, and the one determined in the pilot scale reactor was 0.040?s^?1 for a superficial air velocity of 0.031?m?s^?1. The k _L a for the pilot scale reactor did not change significantly after biofilm development, compared to the value measured without biofilm.     

Atrazine biodegradation by a bacterial community immobilized in two types of packed-bed biofilm reactors

Through selective enrichment of atrazine-metabolizing microorganisms, a microbial community was selected from agricultural soil. Bacterial isolates, identified by their closest similarity with 16S rDNA sequences stored in NCBI GeneBank, belonged to the genera: Massilia, Stenotrophomonas, Klebsiella, Sphingomonas, Ochrobactrum, Arthrobacter, Microbacterium, Xanthomonas and Ornithinimicrobium. From these strains, only the first six used atrazine as nitrogen and carbon source. The microbial community attached to a non-porous support was evaluated for its atrazine biodegradation rate and removal efficiency under aerobic conditions in two types of packed-bed biofilm reactors fed with a mineral salt medium containing glucose plus atrazine, or atrazine as the sole carbon and nitrogen source. Removal efficiencies near 100% were obtained at loading rates up to 10 mg l⁻¹ h⁻¹. After long periods of continuous operation, the richness of microbial species in biofilm reactors diminished to only three bacterial strains; Stenotrophomonas sp., Ochrobactrum sp. and Arthrobacter sp. By PCR analysis of their DNA, the presence of atzABC genes codifying for the enzymes of the upper catabolic pathway of atrazine, was confirmed in the three strains. The gene atzD that encodes for the cyanuric acid amidohydrolase enzyme was detected only in Stenotrophomonas sp.