Design of immobilised dextransucrase for fluidised bed application

Immobilisation of dextransucrase from Leuconostoc mesenteroides NRRL B-512F in alginate is optimised for applications in a fluidised bed reactor with high concentrated sugar solutions, in order to allow a continuous formation of defined oligosaccharides as prebiotic isomalto-oligosaccharides. Efficient design of fluidised bed immobilised biocatalyst in high density solutions requires particles with elevated density, high effectiveness and both thermal and mechanical stability. Inert silica flour/sand (Mikrosil 300) as supplement turned out to be best suited for increasing the density up to 1400 kg m(-3) of the alginate beads and generating a stable expanded bed without diffusional restrictions. Kinetic investigations demonstrate that low effectiveness of immobilised enzyme due to close association to dextranpolymers (dextran content of enzyme preparation >90%) is compensated by reducing the particle size and/or by decreasing the dextran content. A low dextran content (5%) is sufficient to immobilise and stabilise the enzyme, thus diffusional limitation is reduced essentially while operational stability is maintained. Fluidisation behaviour and bed expansion proved to be appropriate for the intended application. Both calculated and measured expansion coefficients showed good agreement for different conditions.     

Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of Qs_rAb to .Abbreviations C concentration - D dilution rate - FBR fixed bed bioreactor - FIBR fluidised bed bioreactor - Gln glutamine - Qs cell specific rate - Qv volumetric rate - rAb recombinant antibody - X_v viable cell density - specific growth rate     

Hydrolysis,acidification and methanogenesis during low-temperature anaerobic digestion of dilute dairy wastewater in an inverted fluidised bioreactor

The application of low-temperature (10 °C) anaerobic digestion (LtAD) for the treatment of complex dairy-based wastewater in an inverted fluidised bed (IFB) reactor was investigated. Inadequate mixing intensity provoked poor hydrolysis of the substrate (mostly protein), which resulted in low chemical oxygen demand (COD) removal efficiency throughout the trial, averaging ~69 % at the best operational period. Overgrowth of the attached biomass to the support particles (Extendospheres) induced bed stratification by provoking agglutination of the particles and supporting their washout by sedimentation, which contributed to unstable bioprocess performance at the organic loading rates (OLRs) between 0.5 and 5 kg COD m^?3 day^?1. An applied OLR above 2 kg COD m^?3 day^?1 additionally promoted acidification and strongly influenced the microbial composition and dynamics. Hydrogenotrophic methanogens appeared to be the mostly affected group by the Extendospheres particle washout as a decrease in their abundance was observed by quantitative PCR analysis towards the end of the trial, although the specific methanogenic activity and maximum substrate utilisation rate on H₂/CO₂ indicated high metabolic activity and preference towards hydrogenotrophic methanogenesis of the reactor biomass at this stage. The bacterial community in the bioreactor monitored via denaturing gradient gel electrophoresis (DGGE) also suggested an influence of OLR stress on bacterial community structure and population dynamics. The data presented in this work can provide useful information in future optimisation of fluidised reactors intended for digestion of complex industrial wastewaters during LtAD.     

Minimum fluidization velocity and friction factor in a liquid-solid inverse fluidized bed reactor

In the present study the hydrodynamic characteristics (bed expansion and pressure drop) of low-density polyethylene (LDPE) and polypropylene (PP) are studied in a liquid-solid inverse fluidized bed reactor as a function of particle diameter, liquid viscosity and density. The bed expansion and pressure drop data are used to determine the minimum fluidization velocity, U_mf and friction factor, P. It was found that the U_mf increased with an increase in the particle diameter and a decrease in solid density and was independent of initial bed height (solid loading). In addition, the U_mf decreased with an increase in the CMC concentration. The friction factor Reynolds number plot was found similar to that of classical fluidization. Dimensionless equations were proposed for the. prediction of the friction factor and the U_mf.     

Organic Matter Degradation Kinetics in an Anaerobic Thermophilic Fluidised Bed Bioreactor

This paper describes the thermophilic anaerobic biodegradation of wine distillery wastewater (vinasses) in a laboratory fluidised bed reactor (AFB) with a porous support medium. The experimental protocol was defined to examine the effect of increasing organic loading rate on the efficiency of AFB and to report on its steady-state performance. Moreover, in order to evaluate treatment efficiency and to investigate fermentation kinetics in an AFB reactor, experimental data were used to estimate the ‘active biomass’ concentration using an autocatalytic kinetic model proposed in this paper, since viable biomass in AFB reactors is very difficult to measure experimentally. The AFB reactor was subjected to a program of steady-state operation over a range of hydraulic retention time (HRTs) of 2.5–0.37 days and organic loading rate (OLRs) up to 5.88 kgCOD/m³/day in order to evaluate its treatment capacity. The AFB reactor was initially operated with organic loading rate of 5.88 kgCOD/m³/day and HRT of 2.5 days. The chemical oxygen demand (COD) removal efficiency was found to be 96.5% in the reactor while the methane content of biogas produced in the digester reached 1.08 m³/m³digester/day. Over 94 days operating period, an OLR of 32 kgCOD/m³/day at a food-to-micro-organisms (F:M) ratio of 0.55 kgCOD/kgVS_att/day was achieved with 81.5% COD removal efficiency in the experimental AFB reactor. At this moment, the methane content of biogas produced in the digester reached 9.0 m³/m³digester/day. The proposed kinetic model is able to estimate kinetic constants of the biodegradation process: non-biodegradable substrate (S_nb) and active adhered biomass concentration (X_a). The parameters of the model were obtained by the curve-fitting method to the proposed kinetic model using the COD as substrate of the anaerobic process and assuming a maximum specific μ_max: 0.72 per day. The comparison of the measured concentration of volatile attached solids (VS_att) with the estimated ‘active’ biomass concentration indicated that extremely high ‘active biomass’ concentrations can be maintained in the system because biofilm thickness is limited by the liquid flow rate applied. This is due to the fact that the anaerobic fluidised bed system retains the growth support medium in suspension by drag forces exerted by upflowing wastewater, and the distribution of biomass holdup (in the form of a biofilm) is thus relatively uniform.     

Cultural and physiological factors affecting expression of recombinant proteins

The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physicochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR).Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture. The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode. Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate.The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line. The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min^–1) and dilution rates (1 d^–1) and glutamine concentration (2.5 mmol l^–1), plus the lowest values for bead size (2 mm) and inoculum density (10⁷ ml^–1). Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system.Abbreviations FIBR fluidised bed bioreactor - FBR fixed bed reactor - STR stirred tank reactor - SEAP secreted alkaline phosphatase - rAb recombinant antibody     

Mathematical model for the fluidized bed biofilm reactor

A mathematical model is proposed for the fluidized bed biofilm reactor (FBBR). For individual biofilm-covered particles (bioparticles) within the reactor, an analysis of intrabiofilm mass transfer and simultaneous intrinsic zero order reaction yields an effectiveness factor expression which is a function of the modified, zero order Thiele modulus, Φ_0,m. This expression is linked to a one-dimensional reactor flow model and a fluidization model to yield an overall reactor model describing convective transport and simultaneous biochemical conversion of substrate within a FBBR. For Φ_0,m<1.15, FBBR is mass transfer limited and 0.45 order kinetics are observed. For Φ_0,m<1.15, mass transfer limitations are insignificant and intrinsic zero order kinetics are observed. A sensitivity analysis using the proposed mathematical model indicates that biofilm thickness and media size are the two most important operating parameters. These two parameters can be optimized simultaneously for a specific application. The proposed model provides a rational approach for FBBR design.     

Evaluation of biological nutrient removal from wastewater by Twin Circulating Fluidized Bed Bioreactor (TCFBBR) using a predictive fluidization model and AQUIFAS APP

A two-phase and three-phase predictive fluidization model based on the characteristics of a system such as media type and size, flow rates, and reactor cross sectional area was proposed to calculate bed expansion, solid, liquid and gas hold up and specific surface area (SSA) of the biofilm particles. The model was subsequently linked to 1d AQUIFAS APP software (Aquaregen) to model biological nutrient removal in two phase (anoxic) and three phase (aerobic) fluidized bed bioreactors. The credibility of the proposed model for biological nutrient removal was investigated using the experimental data from a Twin Circulating Fluidized Bed Bioreactors (TCFBBR) treating synthetic and municipal wastewater.The SSA of bio-particles and volume of the expanded bed were simulated as a function of operational parameters. Two-sided t-tests demonstrated that simulated SCOD, NH₄-N, NO₃-N, TN, VSS and biomass yields agreed with the experimental values at the 95% confidence level.     

Optimization of food waste hydrolysis in leach bed coupled with methanogenic reactor: effect of pH and bulking agent

The effects of pH and bulking agents on hydrolysis/acidogenesis of food waste were studied using leach bed reactor (LBR) coupled with methanogenic up-flow anaerobic sludge blanket (UASB) reactor. The hydrolysis rate under regulated pH (6.0) was studied and compared with unregulated one during initial experiment. Then, the efficacies of five different bulking agents, i.e. plastic full particles, plastic hollow sphere, bottom ash, wood chip and saw dust were experimented under the regulated pH condition. Leachate recirculation with 50% water replacement was practiced throughout the experiment. Results proved that the daily leachate recirculation with pH control (6.0) accelerated the hydrolysis rate (59% higher volatile fatty acids) and methane production (up to 88%) compared to that of control without pH control. Furthermore, bottom ash improved the reactor alkalinity, which internally buffered the system that improved the methane production rate (0.182 l CH₄/g VS_added) than other bulking agents.     

Utilizing the macroporous media for insect cell/baculovirus expression

Due to their bio-compatibility and high porosity, cellulose foam and polyurethane foam (PUF) were selected as the media to immobilize insect cells for recombinant baculovirus expression. In packed beds, entrapment efficiency and axial cell distribution are two prime important parameters associated with reactor performance. These two parameters were found to be affected by initial cell loading density and liquid circulation rate. Based on the entrapment kinetic model, the entrapment parameters as K ₀, σ _m could be evaluated. The establishment of the correlation between K ₀, σ _m and Reynolds number, N _Re, described in this study provided convenient tools to achieve an efficient cell entrapment in the packed bed. The appropriated circulation velocity for uniform cell distribution was also determined.     

Glycerol production by immobilised cells of Pichia farinosa

Summary Cells ofPichia farinosa were immobilised in calcium alginate and K-Carrageenan and their ability to produce glycerol from glucose under aerobic conditions with acidic as well as alkaline pH was investigated. An average glycerol production rate of 0.07 g/l.h was obtained with immobilised cells (IMC) in shake flasks. Continuous glycerol production in a fluidised bed reactor (FBR) under steady state operation gave a glycerol concentration of 13.5 g/l in the product stream.     

Gas-phase influence on the mixing in a fluidized bed bio-reactor

Summary The liquid and solids mixing in fluidized bed bio-reactors containing particles with a density only slightly higher than water (1100 kg/m³) is generally consistent with the results found in previous studies for reactors with particles of higher density. The liquid mixing can be described by an axial dispersion model for a large variety of conditions while the solids follow the streamlines of the liquid. In the presence of a gas phase the degree of mixing of both the liquid and the solid phase increased. This effect became larger with increasing reactor diameter. In the extrapolation of laboratory data of three phase fluidized bed bio-reactors to pilot plant systems this effect should be taken into account. The liquid and solids mixing may have a substantial effect on overall conversion rates and on possible microbial stratification in the reactor.Nomenclature Bo Bodenstein number v L/D (-) - D _r diameter of the fluidized bed reactor (m) - D ₁ Dispersion coefficient of the liquid phase (m²/s) - D _g dispersion coefficient of the solid phase (m²/s) - E(in) normalized dye concentration function entering the ideally mixed tank reactor (-) - E(t) normalized dye concentration function as measured (-) - L length of the axial dispersed reactor (m) - t time after dye injection (s) - t _m time constant for microbial selection (s) - t _s solid mixing time constant (s) - t time interval in which a particle migrates within the bed (s) - v _t superficial gas velocity (m/s) - v _g superficial liquid velocity (m/s) - z migration distance of a particle in the bed (m) - ₁ in situ growth rate of a dominant organism (s^-1) - ₂ in situ growth rate of a recessive organism (s^-1) - average residence time in the axial dispersed reactor (s) - _t average residence time in the ideally mixed tank reactor (s)     

Hydrolysis of cellulose in a cellulase-bead fluidized bed reactor

Cellulase was immobilized in a collagen fibril matrix, and no leakage of cellulase from the collagen fibril matrix was observed. The immobilized cellulase was more stable than the native cellulase. The substrate cellulose was hydrolyzed quantitatively with immobilized cellulase. The final reaction product was identified as glucose. Immobilized cellulase was used in a fluidized bed reactor where the pressure drop of the fluidized bed reactor was low and constant. Cellulose was hydrolyzed to glucose by the cellulase-bead fluidized bed reactor. The minimum flow velocity (U_mf) was 0.5 cm/sec and the optimum flow velocity of the cellulose hydrolysis was 1 cm/sec.     

Evaluation of methanogenic kinetics in an anaerobic fluidized bed reactor (AFBR)

A kinetic study of the methanogenic phase was carried out on a pilot lab scale anaerobic fluidized bed reactor (AFBR) in batch mode. An examination of the effect of initial acetate concentration, bed expansion and bed segregation is presented.Experimental data observed for the acetate removal against time were adjusted to a zero-order kinetic equation, over the chemical oxygen demand (COD) range studied (1430–5340 mg litre⁻¹), independently of the bed expansion (11–37%). The kinetic constant was calculated using robust regression analysis. The zero-order kinetic constant, K₀ was between 1180–1380 mg COD litre⁻¹ h⁻¹ on the fixed bed volume basis, and the maximum specific substrate utilization rate, k, was between 145–198 mg COD g VS⁻¹ h⁻¹.The kinetic behaviour was found to be different throughout the reactor, on the fixed bed volume basis and the activity at the bottom of the bed was lower than the activity in the upper region. However, on an attached volatile solids basis, the activity at the bottom level was the greatest.     

Interaction of bovine serum albumin and long-chain imidazolium ionic liquid measured by fluorescence spectra and surface tension

The binding of long-chain imidazolium ionic liquid (IL), 1-tetradecyl-3-methylimidazolium bromide (C₁₄mimBr) to bovine serum albumin (BSA) was investigated by fluorescence spectra and surface tension. Fluorescence spectra show that tryptophan (Trp) residues, one of the intrinsic fluorophores in BSA, are buried in a hydrophobic microenvironment with the addition of C₁₄mimBr, which induces the denaturation of BSA. Moreover, the fluorescence quenching mechanism was determined to be static quenching. The equilibrium constant (K) and the number of binding sites (n) were calculated based on the results of fluorescence measurement. The critical aggregation concentration (CAC) and critical micelle concentration (CMC) under different BSA concentrations at various temperatures were investigated based on the surface tension plots. Surface tension indicates that C₁₄mimBr binds to BSA through electrostatic attraction at low C₁₄mimBr concentrations (below CMC) and through hydrophobic interaction at high C₁₄mimBr concentrations (above CMC). Additionally, the thermodynamic parameters of micelle formation were determined. This study provides an understanding of the binding of C₁₄mimBr to BSA.     

Characteristics of granular methanogenic sludge grown on glucose in a UASB reactor

The formation of granules grown on glucose in an upflow anaerobic sludge blanket (UASB) reactor was investigated. Total granular sludge concentration retained in the UASB reactor was 34.5 g MLSS/l (30.0 g MLVSS/l) during 240 d operation on glucose minimum medium with the supplementation of 1.07 g NaHCO₃ per 1 g glucose. This realized a high-rate methanogenic fermentation of glucose of 17.6 g COD/l-reactor-d at 3.4 d⁻¹ of space velocity. The granules formed were relatively small, ranging mainly from 0.4 to 0.5 mm, had a relatively low cell density of 0.0542–0.0560 g MLVSS/ml, and had low specific gravity (0.97–1.19) due to very low ash content (11–13%). Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by glucose, acetate, and propionate.     

Some Experiments and Conclusions about the Influence of Pure H2 Gason the Anaerobic Degradation of Propionate by Mixed Cultures

Reaction enthalpy for propionate degradationΔG₀ is only negative when the partial pressure ofhydrogen pH₂ is less than 10^—4 bar. This means that for pH₂ more than 10^—4 bar, a total anaerobic degradation of propionate is impossible for thermodynamic reasons. Therefore, with increasing pH₂, the anaerobic degradation rate of propionate via acetate is inhibited. There are two ways to investigate the inhibitory effect of pH₂: to keep the concentration of hydrogen consuming bacteria low or to increase the mass transfer by feeding the hydrogen at higher flow rates. The author used an extended fixed bed reactor filled with polyurethane particles as a carrier for the bacteria, aerated with pure H₂ gas. The results, compared with the literature by using model equations in order to standardize the data, correspond well: The addition of pure H₂ gas has no observable effects on propionate degradation.In the fixed bed reactor with immobilized bacteria, it was not possible to reach an inhibitory concentration of H₂ and high process stability could be maintained.     

Biofilm Growth and Bed Fluidization in a Fluidized Bed Reactor Packed with Support Materials of Low Density

Support materials of low‐density for fluidized bed reactors provide several operational advantages, including lower energy requirements and proper biofilm growth balance. The aim of this investigation was to study the extent of biofilm growth and bed fluidization in an experimental reactor, using polyester resin (ρ_pr = 1220 kg/m³) and vitrified expanded perlite (ρ_vep = 1710 kg/m³) as alternative support materials to conventional silica sand. A noteworthy amount of biofilm was observed to be attached to both support materials from the very beginning of the bioreactor operation. Nevertheless, there were significant variations in biofilm growth and activity over the course of the experimental trials. For both perlite and polyester beds, the highest biofilm mass and the highest total number of mesophilic bacteria were observed between the 7^th and the 10^th day, showing a steady state trend at the end of the experimental runs. The chemical oxygen demand (COD) removal levels were concomitant with biofilm mass and total mesophilic bacteria changes, although the polyester bed efficiency was slightly higher than that for the perlite bed. As expected, the polyester bed was fluidized at a lower re‐circulation flow compared to the perlite bed. Reactor back‐washing was not required for these support materials since biomass excess was adequately separated by means of a special internal device. The efficiencies of removal of organic matter achieved were acceptable (up to 78 %) despite the low volume of the support material (25 %) and the low hydraulic retention time (30 min).     

Modelling a solid-state fluidized bed fermenter for Ethanol production with S. cerevisiae

A model is proposed to describe the performance of a new type of fermenter for ethanol production, the fluidized bed gas-solid fermenter, with respect to scaling-up effects. Based on the fact that in the fluid bed the substrate is not supplied continuously to each particle, two scale-up parameters are derived, circulation time τ and specific substrate supply Δm _G,P , which are shown to influence reactor efficiency significantly. The validity of the model is checked by comparing the calculated yield coefficients for ethanol, cell mass and carbon dioxide to the results of fermentation experiments performed under aerobic conditions in a laboratory-scale reactor and a semi-technical fermenter.     

Reaction Engineering Aspects of Microbial Mercury Removal

Mercury‐resistant microorganisms are widespread in natural environments and can effectively be used to demercurize Hg(II)‐contaminated wastewaters as was already demonstrated on an industrial scale. The aim of this paper is to find the performance limits with regard to Hg(II) loadings D c_Hg,in (dilution rate × Hg(II) inlet concentration) and residual Hg(II) at the reactor outlet and to provide a reasonable basis for an optimal and safe process design. To this end, comprehensive studies were carried out with different single microbes (natural isolates and a genetically engineered strain) as well as microbial consortia in batch and continuous stirred reactors and fixed beds with microorganisms immobilized as films. The rate of the biotransformation (reduction of inorganically and organically bound Hg(II) to elemental Hg(0)) was found to follow a uniform mechanism with inhibition kinetics (Haldane type). Both reactor types are able to cope with high Hg(II) loadings and yield conversions up to 98 %. The stirred vessel is particularly suited for high c_Hg,in but restricted to low D (D < μ_max), while the fixed bed can be operated at high D, say 10 h^–1, but can only deal with c_Hg,in < 10 mg/L due to the limited Hg(II) tolerance of microorganisms. The loading limitations can be removed by appropriate recycle flows for both reactor types. However, irrespective of reactor type used, the residual Hg at the outlet cannot be reduced below the legal discharge limit (50 μg/L) mainly owing to the adsorption of Hg(II) on biomass. Therefore, a separation step following the reactor is required (sand bed, activated carbon filter). Comparing the reactor types exhibits the superiority of the fixed bed system due to its simpler construction, easier operation and higher cost effectiveness. Furthermore, the fixed bed shows better flexibility and robustness to extreme loadings. This justifies a posteriori the choice of a fixed bed reactor applied in the technical process.