Continuous ethanol production by flocculating yeast in the fluidized bed bioreactor

Abstract: Continuous fermentation by a highly flocculant strain of the yeast Saccharomyces cerevisiae was carried out in a tower fluidized-bed bioreactor. The synthetic and molasses media with a total sugar concentration of 17% (w/v) were used for fermentation. Different dilution rates were tested. Stable cell densities of 50 kg m^-3(dry weight) were maintained for all dilution rates. The ethanol productivity was increasing linearly with dilution rates up to 15—20 kg m^-3 h^-1. Aeration of the culture stabilized flocculating activity and viability of yeast and also permitted long-term operation of the bioreactor.     

Continuous production of acetic acid using immobilized acetobacter aceti in a three-phase fluidized bed bioreactor

Continuous operation of a three-phase fluidized bed bioreactor using immobilized cells showed that both immobilized and suspended cells contributed to the production of acetic acid. Unlike the rapid decrease in the productivity at dilution rates above 0.25 h⁻¹ in the free cell fermentation, the productivity was little affected by the dilution rate in the immobilized cell fermentation. Theoretical models were proposed for the continuous process. The models approximately agreed with the experimental results. Experimental results and/or theoretical calculations based on the kinetic models showed that suspended cells were important in the production of acetic acid if the solid holdup was small or if gel radius was large. Theoretical calculations showed that an optimal solid holdup or gel size existed at higher dilution rates because of the k_La dependence on solid holdup and particle diameter.     

Mathematical description of bikaverin production in a fluidized bed bioreactor

Summary  Growth of Gibberella fujikuroi in submerged cultures occurs as micelles or filamentous hyphae dispersed in fluid and pellets or stable, spherical agglomerations. Gibberella fujikuroi growth, substrate consumption and bikaverin production kinetics obtained from submerged batch fermentation were fitted to three different sigmoid models: two and three-parameter Gompertz models and one Logistic model. Growth fitting was used to compare between models and select the best one by means of an F test. The best model for describing growth was the two-parameter Gompertz model and was used for glucose consumption and bikaverin production fitting. Data from eight different schemes of fermentations were analysed and parameter estimation was carried out by means of minimization of residual sum of squares. Some characteristic values obtained with the two-parameter Gompertz model fit are: μ=0.028 h⁻¹, Y_x/s=0.1089 g substrate/g biomass, α =0.1384 g product/g biomass.     

Continuous operations of a fluidized bed bioreactor for anaerobic digestion : Residence time effect on degradation kinetics

Summary A fluidized bed fermentor is used for anaerobic digestion. The influence of Hydraulic Retention Time, inlet concentration and degree of colonization is investigated. The degradation kinetics seems to depend on the ratio between outlet and inlet concentration. Different kinetic regimes are identified, showing that mass transfer in the biofilms may play an important role on the degradation kinetics.     

Heterogeneous biofilm activity in a segregated anaerobic fluidized bed bioreactor

Bed segregation in a fluidized bed bioreactor profoundly influenced biofilm thickness and microbial activities of the biofilm along the bed height. Bioparticles coated with a thin biofilm, observed at the bottom of the reactor, had a higher specific activity in propylene glycol and n-propanol degradation than in thick biofilms developed at the top of the reactor. Although no significant difference was observed in specific activity for propionate and acetate along the reactor flow axis, more total propionate and acetate conversion occurred in regions of thicker biofilm accumulation.     

Biodegradation of dichloromethane in waste water using a fluidized bed bioreactor

Summary Biological treatment of a synthetic waste water containing 120 mM dichloromethane (10.2g/l) was carried out under aerobic conditions using dichloromethane-degrading bacteria as an inoculum. The bacteria were adsorbed to support particles and grown in a fluidized bed bioreactor. Charcoal and sand particles were compared as support materials with regard to abrasion, the maximum degradation rate for dichloromethane and the stability of the biological activity in the system.The use of charcoal led to the incorporation of coal dust into the biomass and to an uncontrollable thickness of the biofilm. Therefore the system became unstable and the biological activity decreased. In contrast sand as support material was indifferent to abrasion and allowed biofilm thickness to be controlled. The dichloromethane degrading capacity of the bioreactor increased during the first 30 days. It reached a steady state level of 1.6 g CH₂Cl₂/lxh. Dichloromethane concentration in the effluent was <0.01 mM (<0.85 mg/l) and consequently the degradation efficiency better than 99.99%.     

Feasibility study of degradation of phenol in a fluidized bed bioreactor with a cyclodextrin polymer as biofilm carrier

This work is focused on the evaluation of a beta-cyclodextrin polymer as a carrier medium in a fluidized bed bioreactor treating aqueous phenol as a model pollutant. The insoluble polymer support was obtained in the shape of spherical beads by crosslinking beta-cyclodextrin with epichlorohydrin. A batch of swollen polymer particles was loaded into the reactor and inoculated with a mixed bacterial culture. Bacterial growth on the polymer beads was initially stimulated by glucose addition to the medium, and then gradually replaced with phenol. The operational variables studied after the acclimation period included phenol load, hydraulic residence time and recirculation flow rate. Low hydraulic residence times and moderate phenol loads were applied. The elimination capacity was usually about 1.0 kg-phenol/m(3)d, although a maximum of 2.8 kg-phenol/m(3)d was achieved with a retention time of only 0.55 h. The depuration efficiency was not affected by the recirculation flow rate in the range studied. Neither operational nor support stability problems were detected during the operation. A high degree of expansion was achieved in the bioreactor due to the hydrogel nature of the cyclodextrin polymer and, consequently, a low energy requirement was necessary to fluidize the bed.     

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.     

Effects of stratification in a fluidized bed bioreactor during treatment of metalworking wastewater

During wastewater treatment, biofilm-coated sand particles stratified in a fluidized bed bioreactor (FBB); particles coated by thicker biofilm segregated toward the top of the bed. Stratification was so well developed that at least two co-existing regions of significantly different mean biofilm thickness were visually distinct within the operating FBB. The observed stratification is attributed to differences in forces of drag, buoyancy, shear, and collisional impact, as well as differences of collision rate within the different regions. Particles with thick biofilm (thickness >100 μm) near the top of the bed consumed substrate at significantly lower rates per unit biomass than particles with thin biofilm (10-20 μm) near the bottom of the bed, thereby suggesting that substrate mass-transfer resistance through biofilm may limit biodegradation rates in the upper portion of the FBB. Large agglomerates of biomass floc and sand, which formed at the top of the fluidized bed, and sand particles with thick biofilm were susceptible to washout from the FBB, causing operational and treatment instability. Radial injection of supplemental liquid feed near the top of the bed increased shear and mixing, thereby preventing formation and washout of agglomerates and thickly coated sand particles. Supplemental liquid injection caused the mean specific biomass loading on the sand to increase and also increased the total biomass inventory in the FBB. Rates of biodegradation in the FBB appeared to be limited by penetration of substrates into the biofilm and absorption of oxygen from air into the wastewater. Copyright 1999 John Wiley & Sons, Inc.     

Biodegradation of sodium benzoate by a Gram-negative consortium in a laboratory-scale fluidized bed bioreactor

Gram-negative bacteria with the potential to metabolize n-alkanes and cyclic hydrocarbons were isolated from local soils and identified using 16S rDNA sequence analysis. Three isolates (CS1CO, GL1CO, GCI1CO) were identified as strains of Pseudomonas (P.) aeruginosa and a further strain (DSS2) as P. putida. Isolates were co-cultured in a laboratory-scale fluidized bed biofilm bioreactor (FBBR) utilizing sodium benzoate as the sole carbon source, under two batch and/or one continuous growth conditions. Biofilm and planktonic bacterial growth dynamics were monitored by plate counts, and optical density measurements (230 nm) determined benzoate biodegradation. Overall higher attached and planktonic bacterial counts, and benzoate depletion, were determined under batch compared to continuous conditions, and the bioreactor performed better during the second batch phase when compared to the first batch phase. It thus appeared that both the planktonic and biofilm components of the system were necessary for the most successful sodium benzoate degradation in this system.     

Gas-liquid mass transfer in bioreactor with three-phase inverse fluidized bed

In the present study the oxygen mass transfer from the gas to the aqueous phase in a Three-Phase Inverse Fluidized Bed (TPIFB) has been studied. A pilot scale TPIFB has been designed and constructed. For determination of the volumetric oxygen mass transfer coefficient the elegant dynamic method, described by Dang et al. (1977) was used. The influence of hydrodynamic parameters, e.g., superficial velocities of the gas and liquid phases on the mass transfer rate was studied. In the range of variables covered, it was found that the superficial liquid velocity had a weak effect on the mass transfer whereas the gas flowrate affects the mass transfer positively. The results for the volumetric oxygen transfer coefficient in the TPIFB were compared to reported values of that coefficient, measured in a classic three-phase fluidised bed under similar hydrodynamic conditions and solid phase properties. The comparison demonstrated a two-fold increase of the oxygen transfer rate in the inverse bed over that in the classic one.     

Biodegradation of 3,4-dichloroaniline in a fluidized bed bioreactor and a steady-state biofilm Kinetic model

Mixed culture of microorganisms immobilized onto Celite diatomaceous earth particles were used to degrade 3,4-dichloroaniline (34DCA) in a three-phase draft tube fluidized bed bioreactor. Biodegradation was confirmed as the dominant removal mechanism by measurements of the concomitant chloride ion evolution. Degradation efficiencies of 95% were obtained at a reactor retention time of 1.25 h. A mathematical model was used to describe the simultaneous diffusion and reaction of 34DCA and oxygen in the biofilms on the particles in the reactor. The parameters describing freely suspended cell growth on 34DCA were obtained in batch experiments. The model was found to describe the system well for three out of four steady states and to predict qualitatively the experimentally observed transition in the biofilm kinetics from 34DCA to oxygen limitation.     

Plant cell culture using a novel bioreactor: the magnetically stabilized fluidized bed.

A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture.     

Evaluation of production of recombinant human interleukin-2 in fluidized bed bioreactor

The production of recombinant human interleukin-2 in a fluidized bed bioreactor containing porous glass carriers is described. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield could be maintained even when protein free medium was perfused, with less than 10% cell washout. Due to this effective immobilization of the cells in the reactor, continuous operation was easy to perform. Final cell densities on the order of 3.8 x 10(8) mL(-1) intrasphere volume were reached while the interleukin-2 production rate was 0.75 mg L(-1) d(-1). The production rate showed a maximum of a 1.9 fold decrease compared with a homogeneous stirred bubble-free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performance was obtained with the fluidized bed bioreactor. The situation may reflect the problems caused by the dense cell culture with adherent cells, as previously shown in a hollow-fiber bioreactor with the same cell line.     

Kinetics and modeling of GM-CSF production by recombinant yeast in a three-phase fluidized bed bioreactor

Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by Saccharomyces cerevisiae strain XV2181 (a/a, Trp 1) containing plasmid palphaADH2 and immobilized on porous glass beads in a fluidized bed bioreactor was studied. Kinetic models for plasmid stability, cell growth, and protein production in the three-phase fluidized bed bioreactor were developed and used to study the effects of solid loading or cell immobilization on plasmid stability and recombinant protein production. With increasing cell immobilization or solid loading in the bioreactor, plasmid stability and protein production improved significantly. The improvements could be attributed to the decreased theta value, which is the plasmid loss probability during cell division and is an indication of segregational instability of the recombinant cell, and the increased alpha value, which is the ratio of the specific growth rate of a plasmid-carrying cell to that of a plasmid-free cell and is indicative of competitive stability of the recombinant cell culture. theta decreased from 0.552 to 0.042 and alpha increased from 0.351 to 0.991 when solid loading in the bioreactor was increased from 5% (v/v) to 33%. The model simulation also showed that the specific growth rate of cells in the bioreactor was lower at higher solid loading. This indicated that there was significant mass transfer limitation, particularly for oxygen transfer, when the total cell density in the bioreactor was high at high solid loading. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 470-477, 1997.     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

In vitro assessment of encapsulated C3A hepatocytes functions in a fluidized bed bioreactor

In the present in vitro model, the authors intended to assess viability and functionality of hepatocytes encapsulated into alginate beads and submitted to a fluidized bed motion in a bioreactor. Human immortalized C3A line was chosen as cell model. Two controls consisting of (1) cells cultured on flasks and (2) cells encapsulated in alginate beads under static conditions were implemented. The cell functions studied were total protein, albumin, urea, and ammonia synthesis, as well as ammonia removal in the case of overdose. The comparison among the three cases studied showed that the three-dimensional structure of alginate offered a suitable environment for cell functions. In addition, the fluidized bed bioreactor enhanced the mass transfer and thus increased the amount of species released out of the beads, as compared with the static case. Ammonia detoxification only appeared reduced by encapsulation. The concept of a fluidized bed bioartificial liver was thus validated by this in vitro model, which indicated that cell functions could be efficiently retained. In addition, as far as urea and protein synthesis and release were concerned, the use of the C3A cell line, in combination with encapsulation and fluidization technology, offered a real potentiality for the purpose of extracorporeal liver supply.