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.     

Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity

The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.     

Diffusion of phenol through a biofilm grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor

Diffusion of phenol through a biofilm attached to activated carbon particles was investigated. The biofilm was grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor operating in a fed-batch mode. It was found that phenol did not adsorb on the biofilm and that the diffusion coefficient of phenol within the biofilm varied from 13 to 39% of its corresponding value in water. The diffusion coefficient of phenol within the biofilm was reduced by increasing the biofilm density. An extensive literature review of diffusion of substrates through biofilms indicated that this conclusion could be extended to biofilms grown on flat surfaces, rotating cylinders, and even bioflocs.     

Optimization of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> (KU923381) for diesel oil degradation using response surface methodology (RSM)

Efficiency of Enterobacter cloacae KU923381 isolated from petroleum hydrocarbon contaminated soil was evaluated in batch culture and bioreactor mode. The isolate were screened for biofilm formation using qualitative and quantitative assays. Response surface methodology (RSM) was used to study the effect of pH, temperature, glucose concentration, and sodium chloride on diesel degradation. The predicted values for diesel oil degradation efficiency by the statistical designs are in a close agreement with experimental data (R ² = 99.66%). Degradation efficiency is increased by 36.78% at pH = 7, temperature = 35°C, glucose = 5%, and sodium chloride concentration = 5%. Under the optimized conditions, the experiments were performed for diesel oil degradation by gas chromatographic mass spectrometric analysis (GC-MS). GC-MS analysis confirmed that E. cloacae had highly degrade hexadecane, heptadecane, tridecane, and docosane by 99.71%, 99.23%, 99.66%, and 98.34% respectively. This study shows that rapid bioremoval of hydrocarbons in diesel oil is acheived by E. cloacae with abet of biofilm formation. The potential use of the biofilms for preparing trickling filters (gravel particles) for the degradation of hydrocarbons from petroleum wastes before their disposal in the open environment is highly suggested. This is the first successful attempt for artificially establishing petroleum hydrocarbon degrading bacterial biofilm on solid substrates in bioreactor.     

Biofilm population dynamics in a trickle-bed bioreactor used for the biodegradation of aromatic hydrocarbons from waste gas under transient conditions

The dynamics of a multispecies biofilm population in a laboratory-scale trickle-bed bioreactor for the treatment of waste gas was examined. The model pollutant was a VOC-mixture of polyalkylated benzenes called Solvesso 100. Fluorescence in-situ hybridization (FISH) was applied in order to characterise the population composition. The bioreactor was operated under transient conditions by applying pollutant concentration shifts and a starvation phase. Only about 10% of the biofilm mass were cells, the rest consisted of extracellular polymeric substances (EPS). The average fraction of Solvesso 100-degrading cells during pollutant supply periods was less than 10%. About 60% of the cells were saprophytes and about 30% were inactive cells. During pollutant concentration shift experiments, the bioreactor performance adapted within a few hours. The biofilm population exhibited a dependency upon the direction of the shifts. The population reacted within days after a shift-down and within weeks after a shift-up. The pollutant-degraders reacted significantly faster compared to the other cells. During the long-term starvation phase, a shift of the population composition took place. However, this change of composition as well as the degree of metabolic activity was completely reversible. A direct correlation between the biodegradation rate of the bioreactor and the number of pollutant-degrading cells present in the biofilm could not be obtained due to insufficient experimental evidence.     

Investigation of settleability of biologically produced solids and biofilm morphology in moving bed bioreactors (MBBRs)

The objective of this work is to investigate the effects of surface area loading rates (SALRs) and hydraulic retention times (HRTs) in moving bed bioreactor (MBBR) systems on the morphology and thickness of the attached biofilm along with subsequent effects on particle size distribution and the settling characteristics of the biologically produced solids. The morphology of biofilm attached to the MBBR carriers changed from a porous biofilm to a biofilm with a more filamentous structure throughout the study at various operating conditions without observable correlation with SALR and HRT. Although, biofilm morphology did not demonstrate an effect on the biologically produced solids observed in this study, the thinnest biofilms resulted in the highest concentration of solids in the effluent. Furthermore, the particle size distribution analysis demonstrated that both higher SALRs and longer HRTs resulted in a shift towards larger-sized particles. Increases in SALR and HRT, independent of each other, also showed increases in effluent solid concentration and lower settleability of the solids.     

An analysis of a trickle-bed bioreactor: carbon disulfide removal

An analysis of the local processes occurring in a trickle-bed bioreactor (TBB) with a first-order bioreaction shows that the identification of the TBB operating regime requires knowledge of the substrate concentration in the liquid phase. If the substrate liquid concentration is close to 0, the rate-controlling step is mass transfer at the gas-liquid interface; when it is close to the value in equilibrium with the gas phase, the controlling step is the phenomena occurring in the biofilm. CS2 removal rate data obtained in a TBB with a Thiobacilii consortia biofilm are analyzed to obtain the mass transfer and kinetic parameters, and to show that the bioreactor operates in a regime mainly controlled by mass transfer. A TBB model with two experimentally determined parameters is developed and used to show how the bioreactor size depends on the rate-limiting step, the absorption factor, the substrate fractional conversion, and on the gas and liquid contact pattern. Under certain conditions, the TBB size is independent of the flowing phases' contact pattern. The model effectively describes substrate gas and liquid concentration data for mass transfer and biodegradation rate controlled processes.     

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, mu(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.     

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     

Mathematical modeling of hydrolysate diffusion and utilization in cellulolytic biofilms of the extreme thermophile Caldicellulosiruptor obsidiansis

In this study, a hydrolysate diffusion and utilization model was developed to examine factors influencing cellulolytic biofilm morphology. Model simulations using Caldicellulosiruptor obsidiansis revealed that the cellulolytic biofilm needs to generate more hydrolysate than it consumes to establish a higher than bulk solution intra-biofilm substrate concentration to support its growth. This produces a hydrolysate surplus that diffuses through the thin biofilm structure into the bulk solution, which gives rise to a uniform growth rate and hence the homogeneous morphology of the cellulolytic biofilm. Model predictions were tested against experimental data from a cellulose-fermenting bioreactor and the results were consistent with the model prediction and indicated that only a small fraction (10-12%) of the soluble hydrolysis products are utilized by the biofilm. The factors determining the rate-limiting step of cellulolytic biofilm growth are also analyzed and discussed.     

Modeling of biofilm reactors with consecutive reactions

Modeling of biofilm reactors has been carried out by several authors. Most of the models use first-order or zero-order kinetics, because of the simplicity of the solution of the mathematical problem. However, the reaction kinetics for many practical situations is a non-linear Monod kinetics, which requires numerical solutions. This paper deals with the modeling of biofilm reactors and effectiveness factor calculations for a biofilm particle with Monod kinetics and two consecutive reactions. The model is applied to biological denitrification in a fluidized bed bioreactor, in which the liquid phase is assumed to be in plug flow. Effectiveness factors of biofilm are numerically calculated by solving the system of ODES by orthogonal collocation. Axial concentration profiles of nitrate and nitrite species are calculated and compared with experimental results.     

Development of a multispecies periodontal biofilm model within a stirred bioreactor

Abstract

The objective of this work was to develop a subgingival biofilm model using a stirred bioreactor. Discs of bovine teeth were adapted to a stirred bioreactor filled with a culture medium containing bacterial species associated with periodontal health or disease. After anaerobic incubation, the biofilms growing on the substratum surfaces were collected and analyzed. The mean number of Colony-forming Units (CFUs) varied, but with no difference between 3 and 7?days of biofilm formation (p?>?0.05). Scanning Electron Microscopy (SEM) analysis showed a uniform biofilm layer covering the cement layer of the root surface containing bacteria with diverse morphology. In checkerboard DNA-DNA hybridization, bacterial species were identified in both biofilms. In conclusion, a subgingival biofilm model was developed using a stirred bioreactor, allowing the in vitro reproduction of complex microbial communities. This is an advanced model that may be useful to mimic complex clinical periodontal biofilms.     

营养及水力条件影响光合细菌生物膜生长特性实验

对平板式生物膜反应器内,流量及底物浓度范围分别为37.8~1080ml/h、0.05~10g/L的不同生长条件下光合产氢细菌生物膜生长特性进行了实验研究,讨论了不同水力及营养条件对沼泽红假单胞菌生物膜表面覆盖率、膜厚、干重和密度的影响。实验结果表明,不同水力及营养条件对生物膜生长速率及结构具有重要影响。在相同的时间间隔内,在高流速条件下光合细菌菌落生长较快,但过高的液体流速会导致部分生物膜脱落;高流速条件易使生物膜形成薄而致密的结构。光合细菌生物膜在循环液底物浓度较高时生长较快,密度也最高;而贫营养条件可以促成结构疏松生物膜在固液界面的形成,这种生物膜结构有利于微生物在低底物浓度条件下底物在生物膜内的传输。     

Heterotrophic cultivation of Paracoccus denitrificans in a horizontal rotating tubular bioreactor

In this work, the heterotrophic cultivation of bacterium Paracoccus denitrificans has been studied in a horizontal rotating tubular bioreactor (HRTB). After development of a microbial biofilm on the inner surface of the HRTB, conditions for one-step removal of acetate and ammonium ion were created. The effect of bioreactor process parameters [medium inflow rate (F) and bioreactor rotation speed (n)] on the bioprocess dynamics in the HRTB was studied. Nitrite and nitrogen oxides (NO and N₂O) were detected as intermediates of ammonium ion degradation. The biofilm thickness and the nitrite concentration were gradually reduced with increase of bioreactor rotation speed when the medium inflow rate was in the range of 0.5–1.5 l h⁻¹. Further increase of inflow rate (2.0–2.5 l h⁻¹) did not have a significant effect on the biofilm thickness and nitrite concentration along the HRTB. Complete acetate consumption was observed when the inflow rate was in the range of 0.5–1.5 l h⁻¹ at all bioreactor rotation speeds. Significant pH gradient (cca 1 pH unit) along the HRTB was only observed at the highest inflow rate (2.5 l h⁻¹). The results have clearly shown that acetate and ammonium ion removal by P. denitificans can be successfully conducted in a HRTB as a one-step process.     

Shear stress effects on production of exopolymeric substances and biofilm characteristics during phenol biodegradation by immobilized Pseudomonas desmolyticum (NCIM2112) cells in a pulsed plate bioreactor

This article reports studies on a continuous pulsed plate bioreactor (PPBR) with the cells of Pseudomonas desmolyticum (NCIM2112) immobilized on granular activated carbon (GAC) used as a biofilm reactor for biodegradation of phenol. Almost complete removal of 200 ppm phenol could be achieved in this bioreactor. Biofilm structure and characteristics are influenced by hydrodynamic and shear conditions in bioreactors. In this article, the effect of shear stress induced by frequency of pulsation on biofilm characteristics during the startup period in the PPBR is reported. The startup time decreased with the increase in frequency of pulsation. The formation of biofilm in PPBR was found to have three phases: accumulation, compaction, and plateau. The effect of frequency on production of exoploymeric substances (EPS) such as, protein, carbohydrate, and humic substance is reported. An increase in shear stress induced by the frequency of pulsation increased the production of exopolymeric substances in the biofilm during startup of the bioreactor. Increase in shear stress caused a decrease in biofilm thickness and an increase in dry density of the biofilm. Increase in shear stress resulted in a smoother and thinner biofilm surface with more compact and dense structure.     

Biodegradation of Phenol Using Immobilized Nocardia hydrocarbonoxydans in a Pulsed Plate Bioreactor: Effect of Packed Stages,Cell Carrier Loading,and Cell Acclimatization on Startup and Steady-State Behavior

The effect of the number of stages and cell carrier loading on the steady-state and startup performance of a continuous pulsed plate bioreactor with glass beads as the cell carrier material for biodegradation of phenol in wastewater using immobilized Nocardia hydrocarbonoxydans has been studied. It was found that the performance of the pulsed plate bioreactor during startup and at steady state can be improved by an increase in cell carrier loading, number of stages, total plate stack height, and with a decrease in plate spacing. The startup time for the continuous bioreactor can be decreased by increasing the number of preacclimatization steps for the cells. The attainment of steady effluent phenol concentration can be considered as an indication of steady state of the continuous bioreactor, as when phenol concentration attained a steady value, biofilm thickness, and the attached biomass dry weight also attained a constant value.     

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.     

Effects of flow rate and substrate concentration on the formation and H2 production of photosynthetic bacterial biofilms

Photosynthetic bacteria (PSB), Rhodopseudomonas palustris CQK 01, were immobilized on the surface of a thin glass slide in a lab-scale flat panel photobioreactor under different flow rates and substrate concentrations. The morphology, dry weight and thickness of the mature PSB biofilms were determined to reveal the relationship between biofilm formation and hydrogen production performance. The mature biofilm formed at a low flow rate and a high substrate concentration showed a looser structure, these structures of the mature biofilm then affected the H₂ production performance of the bioreactor during mature stage. The biofilm formed at a flow rate of 228 ml/h and a substrate concentration of 60 mmol/l exhibited the highest dry weight and optimally porous structure, which is beneficial not only for hydrogen removal from the biofilm but also glucose diffusion into the biofilm, thus significantly boosting the photo-hydrogen production performance.     

Biofilm formation in attached microalgal reactors

The objective of this study was to investigate the fundamental question of biofilm formation. First, a drum biofilm reactor was introduced. The drums were coated with three porous substrates (cotton rope, canvas, and spandex), respectively. The relationships among the substrate, extracellular polymeric substances (EPS), and adhesion ratio were analyzed. Second, a plate biofilm reactor (PBR) was applied by replacing the drum with multiple parallel vertical plates to increase the surface area. The plates were coated with porous substrates on each side, and the nutrients were delivered to the cells by diffusion. The influence of nitrogen source and concentration on compositions of EPS and biofilm formation was analyzed using PBR under sunlight. The results indicated that both substrate and nitrogen were critical on the EPS compositions and biofilm formation. Under the optimal condition (glycine with concentration of 1 g l^?1 and substrate of canvas), the maximum biofilm productivity of 54.46 g m^?2 d^?1 with adhesion ratio of 84.4 % was achieved.     

Dynamics of a biological fixed film for phenol degradation in a fluidized-bed bioreactor

Experimental and modeling studies were conducted to analyze the dynamic response behavior of a phenol-oxidizing fixed film using a differential, fluidized-bed bioreactor in a recycle loop with a well-mixed reservoir. With the bioreactor at steady state, a pulse of phenol was added to perturb the system, and the phenol concentration was monitored continuously until steady state was again achieved.The experimental dynamics were compared with a dynamic mathematical model based on diffusion and reaction within the biofilm, liquid mixing, and biofilm growth. Constant-pH experiments could be adequately described using an unstructured, double-Monod kinetic expression with substrate inhibition by phenol.However, in dynamic experiments without pH control, the pH of the liquid phase dropped, and damped oscillations were observed in the phenol concentration and reaction rate trajectories. Oscillatory solutions could not be induced in the model, even when product inhibition was included, and a linear stability analysis did not reveal tendencies toward instability. The cause of the experimental oscillations remains unknown.