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.     

Modelling and simulation of steady-state phenol degradation in a pulsed plate bioreactor with immobilised cells of Nocardia hydrocarbonoxydans

A novel bioreactor called pulsed plate bioreactor (PPBR) with cell immobilised glass particles in the interplate spaces was used for continuous aerobic biodegradation of phenol present in wastewater. A mathematical model consisting of mass balance equations and accounting for simultaneous external film mass transfer, internal diffusion and reaction is presented to describe the steady-state degradation of phenol by Nocardia hydrocarbonoxydans (Nch.) in this bioreactor. The growth of Nch. on phenol was found to follow Haldane substrate inhibition model. The biokinetic parameters at a temperature of 30 ± 1 °C and pH at 7.0 ± 0.1 are μ _m = 0.5397 h⁻¹, K _S = 6.445 mg/L and K _I = 855.7 mg/L. The mathematical model was able to predict the reactor performance, with a maximum error of 2% between the predicted and experimental percentage degradations of phenol. The biofilm internal diffusion rate was found to be the slowest step in biodegradation of phenol in a PPBR.     

Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Detachment of multi species biofilm in circulating fluidized bed bioreactor

In this study, the detachment rates of various microbial species from the aerobic and anoxic biofilms in a circulating fluidized bed bioreactor (CFBB) with two entirely separate aerobic and anoxic beds were investigated. Overall detachment rate coefficients for biomass, determined on the basis of volatile suspended solids (VSS), glucose and protein as well as for specific microbial groups, i.e., for nitrifiers, denitrifiers, and phosphorous accumulating organisms (PAOs), were established. Biomass detachment rates were found to increase with biomass attachment on carrier media in both beds. The detachment rate coefficients based on VSS were significantly affected by shear stress, whereas for protein, glucose and specific microbial groups, no significant effect of shear stress was observed. High detachment rates were observed for the more porous biofilm structure. The presence of nitrifiers in the anoxic biofilm and denitrifiers in the aerobic biofilm was established by the specific activity measurements. Detachment rates of PAOs in aerobic and anoxic biofilms were evaluated.     

Production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an Expanded-Bed Bioreactor

Summary Continuous production of Manganese Peroxidase by free pellets ofPhanerochaete chrysosporium in an Expanded-Bed Bioreactor was successfully achieved for more than 30 days of operation. The stability in continuous production was attained because of two modifications in the operational policy: an adequate feed control and the supply of oxygen in a pulsing mode. Feed control allowed to stablish an equilibrium between primary and secondary metabolism, thus avoiding metabolic stress. The use of pulsation had two beneficial effects: 1) Increase of stability by avoiding bed compactation during operation performance; 2) Control of mycelial pellet size. The bioreactor operated without oxygen pulsation did not maintain enzyme production longer than 14 days.     

A novel membrane bioreactor for detoxifying industrial wastewater: I. Biodegradation of phenol in a synthetically concocted wastewater

A novel process has been used to biodegrade phenol present in an acidic (1 M HCI) and salty (5% w/w NaCl) synthetically bioreactor, in which the phenol present in the wastewater is separated from the inorganic components by means of a silicone rubber membrane. Transfer of the phenol from the wastewater and into a biological growth medium allows biodegradation to proceed under controlled conditions which are unaffected by the hostile inorganic composition of the wastewater. At a wastewater flow rate of 18 mL h(-1) (contact time 6 h), 98.5% of the phenol present in the wastewater at an inlet concentration of 1000 mg ( (-1) ) was degraded; at a contact time of 1.9 h, 65% of the phenol was degraded. Phenol degradation was accompanied by growth of a biofilm on the membrane tubes and by conversion of approximately 80% of the carbon entering the system to CO(2) carbon. Analysis of the transport of phenol across the membrane revealed that the major resistance to mass transfer arose in the diffusion of phenol across the silicone rubber membrane. A mathematical model was used to describe the transfer of phenol across the membrane and the subsequent diffusion and reaction of phenol in the biofilm attached to the membrane tube. This analysis showed that (a) the attached biofilm significantly lowers the mass transfer driving force for phenol across the membrane, and (b) oxygen concentration limits the phenol degradation rate in the biofilm. These conclusions from the model are consistent with the experimental results. (c) 1993 Wiley & Sons, Inc.     

Shear‐induced detachment of biofilms from hollow fiber silicone membranes

A suite of techniques was utilized to evaluate the correlation between biofilm physiology, fluid‐induced shear stress, and detachment in hollow fiber membrane aerated bioreactors. Two monoculture species biofilms were grown on silicone fibers in a hollow fiber membrane aerated bioreactors (HfMBR) to assess detachment under laminar fluid flow conditions. Both physiology (biofilm thickness and roughness) and nutrient mass transport data indicated the presence of a steady state mature biofilm after 3 weeks of development. Surface shear stress proved to be an important parameter for predicting passive detachment for the two biofilms. The average shear stress at the surface of Nitrosomonas europaea biofilms (54.5 ± 3.2 mPa) was approximately 20% higher than for Pseudomonas aeruginosa biofilms (45.8 ± 7.7 mPa), resulting in higher biomass detachment. No significant difference in shear stress was measured between immature and mature biofilms of the same species. There was a significant difference in detached biomass for immature vs. mature biofilms in both species. However, there was no difference in detachment rate between the two species. Biotechnol. Bioeng. 2013; 110: 525–534. © 2012 Wiley Periodicals, Inc.     

On the calculation of the elastic modulus of a biofilm streamer

Biofilm mechanical properties are essential in quantifying the rate of microbial detachment, a key process in determining the function and structure of biofilm systems. Although properties such as biofilm elastic moduli, yield stress and cohesive strength have been studied before, a wide range of values for the biofilm Young's modulus that differ by several orders of magnitude are reported in the literature. In this article, we use experimental data reported in Stoodley et al. [Stoodley et al., Biotechnol Bioeng (1999): 65(1):83-92] and present a methodology for the calculation of Young's modulus, which partially explains the large difference between the values reported in the literature.     

A statistical analysis of the effect of substrate utilization and shear stress on the kinetics of biofilm detachment

One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment.Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed.A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono- and mixed-population biofilms. (c) John Wiley & Sons, Inc.     

Simulation of growth and detachment in biofilm systems under defined hydrodynamic conditions

Detachment from biofilms was evaluated using a mixed culture biofilm grown on primary wastewater in a tube reactor. The growth of biofilms and the detachment of biomass from biofilms are strongly influenced by hydrodynamic conditions. In a long-term study, three biofilms were cultivated in a biofilm tube reactor. The conducted experiments of biofilm growth and detachment can be divided into three phases: 1) an exponential phase with a rapid increase of the biofilm thickness, 2) a quasi-steady-state with spontaneous fluctuation of the biofilm thickness between 500 and 1,200 microm in the investigated biofilm systems, and 3) a washout experiment with increased shear stress in three to four steps after several weeks of quasi-steady-state. Whereas the biofilm thickness during the homogeneous growth phase can be regarded constant throughout the reactor, it was found to be very heterogeneous during the quasi-steady-state and the washout experiments. Growth and detachment during all three phases was simulated with the same one-dimensional biofilm model. For each of the three phases, a different detachment rate model was used. During the homogeneous growth phase, detachment was modeled proportional to the biofilm growth rate. During the quasi-steady-state phase, detachment was described by random detachment events assuming a base biofilm thickness. Finally, the washout experiment was simulated with detachment being a function of the biofilm thickness before the increase of the shear stress.     

Textural fingerprints: a comprehensive descriptor for biofilm structure development

Automated tools to determine biofilm structure are necessary to interpret large time series of biofilm images. Image analysis based on the evaluation of Spatial Gray Level Dependence Matrices (SGLDM) enabled us to monitor biofilm structure development in response to external disturbances (i.e., periodic increases of wall shear stress) at a large scale (i.e., >1 mm). We applied our method to an experiment conducted in an annular reactor over a 10-week period. Six states of biofilm development were differentiated by their unique structure. Previous exposure to rapidly increased shear influenced the resulting biofilm structure after additional shear increases. In addition, on the scale of the biofilm images, the biofilm structure after a shear increase was spatially heterogeneous and resulted in spatially differentiated regrowth after detachment at different locations in the biofilm. SGLDM was developed further as an alternative to approaches based on image binarization as binarization leads to information loss for low-magnification and low-resolution images. During post-processing of image data, structural states of biofilm development were identified by K-means clustering and data display in Principal Component plots. Quantitatively selected representative images were used to illustrate the meaning of the clusters. Post-treatment of image data was essential for managing several thousands of raw biofilm images and therefore improved the usefulness of the image analysis.     

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.     

Determination of biomass detachment rate coefficients in anaerobic fluidized-bed-GAC reactors

In this study a simple analytical technique that determines the active biomass in a sample capable of degrading specific substrates anaerobically was developed and used to measure first-order detachment rate coefficients of acetate and phenol utilizing organisms. Different detachment rate coefficients were measured for the two microbial populations. A general relationship between the detachment rate coefficients and biomass accumulation was developed. The detachment rate coefficient was found to increase sharply after a certain biomass accumulation until it reaches a maximum, after which it decreases continuously with increasing biomass until it levels off at a constant value independent of biomass. Detachment rate coefficients measured in different reactors, operating at different conditions with respect to loading, biological SRT, and attachment media particle size, were in close agreement with each other after being normalized to the shear stress and daily methane production rate per unit biofilm area.     

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.     

Applying the digital image correlation method to estimate the mechanical properties of bacterial biofilms subjected to a wall shear stress

A digital image correlation (DIC) method was applied to characterize the mechanical behavior of Pseudomonas aeruginosa biofilms in response to wall shear stress using digital video micrographs taken from biofilm flow cells. The appearance of the biofilm in the transmitted light photomicrographs presented a natural texture which was highly conducive to random encoding for DIC. The displacement fields were calculated for two biofilm specimens. The DIC method concurred with previous analysis showing that biofilms exhibit viscoelastic behavior, but had the advantage over simple length measurements of longitudinal strain that it could precisely measure local strains in length (x) and width (y) within biofilm clusters with a 2 μm resolution as a function of time and wall shear stress. It was concluded that DIC was more accurate at measuring elastic moduli than simple length measurements, but that time-lapse 3D images would enable even more accurate estimates to be performed.     

Intracellular reactive oxygen species mediate suppression of sporulation in Bacillus subtilis under shear stress

Sporulation is an important cellular response to stress that is also significant from a bioreactor operation viewpoint. While sporulating organisms are known to show an enhanced sporulation response under several stress situations, the sporulation response to shear stress has not been investigated thus far. Such a study could be of interest since shear stress, to a greater or lesser degree, is always present in bioreactor operation. In this article, we investigate the sporulation extents of the Gram-positive bacteria Bacillus subtilis at various defined shear levels. We show that, contrary to expectations, shear inhibits sporulation. We found an inverse correlation between the shear rate-dependent specific intracellular reactive oxygen species level (siROS), and the sporulation extent. A 10-fold increase in siROS resulted in about 17-fold decrease in sporulation extent. The involvement of reactive oxygen species (ROS) in sporulation was unknown thus far. Further, through experiments that specifically increased and reduced intracellular ROS (iROS), we established that siROS is responsible for the inhibition of sporulation under shear stress. In addition, we found that shear induced siROS regulated the expression levels of the general stress proteins Ctc and sigma(B). Based on the above, we hypothesize that siROS may regulate suppression of sporulation under high shear by altering sigma(B) and Ctc expression levels, and a model for the same is presented.     

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.     

Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3–10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.     

Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Effect of shear on morphology,viability and metabolic activity of succinic acid-producing Actinobacillus succinogenes biofilms

Two custom-designed bioreactors were used to evaluate the effect of shear on biofilms of a succinic acid producer, Actinobacillus succinogenes. The first bioreactor allowed for in situ removal of small biofilm samples used for microscopic imaging. The second bioreactor allowed for complete removal of all biofilm and was used to analyse biofilm composition and productivity. The smooth, low porosity biofilms obtained under high shear conditions had an average cell viability of 79% compared to 57% at the lowest shear used. The maximum cell-based succinic acid productivity for high shear biofilm was 2.4 g g⁻¹DCW h⁻¹ compared to the 0.8 g g⁻¹DCW h⁻¹ of the low shear biofilm. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed higher cell metabolic activities for high shear developed biofilm compared to biofilm developed at low shear conditions. Results clearly indicated that high shear biofilm cultivation has beneficial morphological, viability, and cell-based productivity characteristics.