Investigation of the structure of two-phase flow fermentation model media in bubble-column bioreactors

Summary Electrical conductivity microprobes have been used to estimate the transverse variation of bubble size, local gas holdup and local specific gas/liquid interfacial area in bench scale bubble column bioreactors containing fermentation model media. Inserted O₂-electrodes and plane parallel windows alter the structure of the two phase flow. Even slight tilting of the column strongly influences the transverse profiles of the bubble size and local gas holdup. The larger bubbles are collected at the wall, where they can be redispersed. These observations open up new possibilities for the construction of bubble column bioreactors.     

Investigation of the structure of two-phase flow model-media in bubble-column bioreactors

Summary By applying photographic, electrical conductivity, and electrooptical methods, the transverse variation of bubble size and velocity, the local gas holdup, and the local specific gas/liquid interfacial area were estimated in a bench scale bubble-column bioreactor containing distilled water. The liquid velocity profile, the transverse turbulence intensity variations, and the turbulence energy dissipation scale were also measured by a hot film turbulence probe and constant temperature anemometer technique.     

Investigation of the structure of two-phase flow model-media in bubble-column bioreactors

Summary By applying photographic, electrical conductivity, and electrooptical methods, the transverse variation of bubble size and velocity, the local gas hold up, and the local specific gas/liquid interfacial area were estimated in a bench scale bubble-column bioreactor containing model cultivation media. The liquid velocity profile, the transverse turbulence intensity variations, and the turbulence energy dissipation scale were also measured by a hot film turbulence probe and constant temperature anemometer technique.A significant relationship was found between the two-phase flow fluid dynamical state and the transverse variation of the various properties.Symbols M mass - L length - T time - a gas/liquid interfacial area L² - specific gas/liquid interfacial area with regard to the bubbling layer volume L^–1 - D transverse coordinate (measured from the wall of the column) L - d bubble diameter L - d mean bubble diameter L - d_e dynamic equilibrium (maximum stable) bubble diameter L - d_p primary bubble diameter L - d_s Sauter bubble diameter L - E specific energy dissipation rate with regard to the volume of the liquid ML^–1T^–3 - EV_L energy dissipation rate ML²T^–3 - , since =1 g/cm³, E has the same numerical value as E. Therefore, the symbol E is used everywhere in the present paper for E for simplicity and called energy dissipation rate (S.s^–2=Stokes.s^–2) L²T^–3 - E_G or local relative gas holdup - f (r) cross correlation function - g acceleration of gravity LT^–2 - h longitudinal distance from the aerator L - relative turbulence intensity - N_O number of u and crossings T^–1 - n_B bubble frequency T^–1 - r distance between two points 1 and 2 of the cross correlation function L - t time - u instantaneous liquid velocity LT^–1 - mean liquid velocity LT^–1 - mean square fluctuation velocity L²T^–2 - turbulence intensity LT^–1 - w_SG superficial gas velocity LT^–1 - w_SL superficial liquid velocity LT^–1 - or E_G local relative gas holdup LT^–1 - energy dissipation scale L - kinematic liquid viscosity L²T^–1 - liquid density M L^–3 - surface tension M T^–2 - dynamic turbulence pressure M L^–1T^–2 Indices p primary (at the aerator) - e equilibrium (far from the aerator)     

Characterization of the two-phase systems in airlift tower-loop bioreactors during the cultivation of E. coli

During the cultivation of E. coli in an airlift tower-loop bioreactor, the following properties were measured: transverse profiles of Sauter bubble diameter, d(S); local relative gas holdup, E(G); bubble rise velocity, u(BS); local mean velocity, ū turbulence intensity, u'; macrotime scale, T(EL); dissipation time scale, tau(E); power spectrum, E(n); and energy dissipation spectrum D(n) at different distances from the aerator. The influence, distance from the aerator, absence and/or presnece of cells, and batch and/or continuous-culture operation on the behavior of the two-phase system are discussed on the basis of these properties.     

Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors

An algal-bacterial consortium formed by Chlorella sorokiniana and a phenanthrene-degrading Pseudomonas migulae strain was able to biodegrade 200-500 mg/l of phenanthrene dissolved in silicone oil or tetradecane under photosynthetic conditions and without any external supply of oxygen. Phenanthrene was only removed when provided in organic solvent, which confirms the potential of two-phase systems for toxicity reduction. Phenanthrene was degraded at highest rates when provided in silicone oil rather than in tetradecane since this solvent probably sequestered the PAH, reducing its mass transfer to the aqueous phase. The influence of phenanthrene concentration, amount of inoculum and light intensity on pollutant removal was also investigated and, under the best conditions, phenanthrene was degraded at 24.2 g m(-3).h(-1). In addition to being cost-effective and mitigating the release of greenhouse gases into the atmosphere, photosynthetic oxygenation was especially beneficial to the use of two-phase partitioning bioreactors since it prevented solvent emulsification and/or volatilization and evidence was found that the microalgae release biosurfactants that could further enhance phenanthrene degradation.     

Recent advances in two-phase partitioning bioreactors for the treatment of volatile organic compounds

Biological processes are considered to be the most cost-effective technology for the off-gas treatment of volatile organic compounds (VOC) at low concentrations. Two-phase partitioning bioreactors (TPPBs) emerged in the early 1990s as innovative multiphase systems capable of overcoming some of the key limitations of traditional biological technologies such as the low mass transfer rates of hydrophobic VOCs and microbial inhibition at high VOC loading rates. Intensive research carried out in the last 5 years has helped to provide a better understanding of the mass transfer phenomena and VOC uptake mechanisms in TPPBs, which has significantly improved the VOC biodegradation processes utilizing this technology platform. This work presents an updated state-of-the-art review on the advances of TPPB technology for air pollution control. The most recent insights regarding non-aqueous phase (NAP) selection, microbiology, reactor design, mathematical modeling and case studies are critically reviewed and discussed. Finally, the key research issues required to move towards the development of efficient and stable full-scale VOC biodegradation processes in TPPBs are identified.     

Apparent viscosity for non-Newtonian fermentation media in bioreactors

The apparent viscosity of non-Newtonian fermentation media is examined. The present state of this subject is discussed. The energy dissipation rate concept is used for a new evaluation of the apparent viscosity in bioreactors, i.e. stirred tank and bubble column bioreactors. The proposed definition of the apparent viscosity is compared with the definitions available in the literature.List of Symbols A _d m ² downcomer cross-sectional area - A _r m ² riser cross-sectional area - a m^–1 specific surface area - C constant in eq. (13) - D m column diameter - D _I m impeller diameter - g m s^–2 gravitational acceleration - h J m^–2 s^–1 K^–1 heat transfer coefficient - K Pa s ⁿ consistency index in a power-law model - k constant in eq. (3) - k _L m s ^–1 liquid-phase mass transfer coefficient - N s^–1 impeller speed - n flow index in a power-law model - P W power input - Re Reynolds number ND _I ^/2 /(/) - U _sg m s ^–1 superficial gas velocity - (U _sg ) _r m s^–1 superficial gas velocity based on riser - V-m³ liquid volume - v ₀ m s^–1 friction velocity Greek Symbols s^–1 shear rate - _c s^–1 characteristic shear rate - W kg^–1 energy dissipation rate per unit mass - W kg^–1 characteristic energy dissipation rate per unit mass - Pa s viscosity - _app Pa s apparent viscosity - kg m^–3 density - Pa shear stress     

Biodegradation of phenol at high initial concentrations in two-phase partitioning batch and fed-batch bioreactors

A two-phase organic-aqueous system was used to degrade phenol in both batch and fed-batch culture. The solvent, which contained the phenol and partitioned it into the aqueous phase, was systematically selected based on volatility, solubility in the aqueous phase, partition coefficient for phenol, biocompatibility, and cost. The two-phase partitioning bioreactor used 500 mL of 2-undecanone loaded with high concentrations of phenol to deliver the xenobiotic to Pseudomonas putida ATCC 11172 in the 1-L aqueous phase, at subinhibitory levels. The initial concentrations of phenol selected for the aqueous phase were predicted using the experimentally determined partition coefficient for this ternary system of 47.6. This system was initially observed to degrade 4 g of phenol in just over 48 h in batch culture. Further loading of the organic phase in subsequent experiments demonstrated that the system was capable of degrading 10 g of phenol to completion in approximately 72 h. The higher levels of phenol in the system caused a modest increase in the duration of the lag phase, but did not lead to complete inhibition or cell death. The use of a fed-batch approach allowed the system to ultimately consume 28 g of phenol in approximately 165 h, without experiencing substrate toxicity. In this system, phenol delivery to the aqueous phase is demand based, and is directly related to the metabolic activity of the cells. This system permits high loading of phenol without the corresponding substrate inhibition commonly seen in conventional bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 155-162, 1997.     

Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors.

Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media. Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed. Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media. In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation. Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium. In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging. The mechanism of this damage effect was not clear. Pluronic F-68 provided protective effect to cells but not to the medium. A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth. Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.     

Investigation of the structure and proteolytic activity of papain in aqueous miscible organic media

The stability of papain was studied in aqueous-organic mixtures by means of residual proteolytic activity along with various spectroscopic analyses (fluorescence and ATR-FTIR combined with isotopic exchange with D₂O). The investigated systems contained 1 or 10% (v/v) of an aqueous buffered solution (pH 8.0) in acetonitrile (ACN), methanol (MeOH) or dimethyl formamide (DMF). The results evidenced that papain retained almost all its catalytic activity after 24 h of incubation in the presence of ACN, and a more compact conformation of the enzyme was detected. Papain suffered an important loss of enzymatic activity (ca. 80%) after 24 h incubation in MeOH although, no global conformational change and minor secondary structure rearrangements were detected. This observation suggests that somehow the active site region was altered. On the other hand, papain suffered a complete inactivation when exposed to those media containing DMF. Fluorescence analyses revealed that an irreversible conformational change took place after 24 h incubation, and a moderate increase in β-sheet and β-turn structures was the most relevant finding when secondary structure was analyzed. The evidences demonstrated that the organic solvents induce a more rigid and compact structure of papain regardless of the organic:buffer ratio investigated. In turn, these modifications affect the active catalytic site in the particular case of MeOH and DMF. These findings were in agreement with the thermo-stability of the enzyme performed after heating at 353 K in all the studied media, that is the presence of ACN did not substantially affect the secondary structure of papain. Nevertheless, the α-helix domain demonstrated to be less thermally stable than the β-sheet domain, turning into aggregated structures after heating, especially in the presence of MeOH and DMF.     

Large-scale production of monoclonal antibodies in defined serum-free media in airlift bioreactors

Forty- and ninety-liter airlift bioreactors have been used successfully to grow hybridoma cell lines in chemically defined serum-free media. In the airlift bioreactor, hybridoma cell growth and monoclonal antibody productivity are comparable to that obtained by conventional cell culture. At sparging rates of 0.60-1.20 vvh (volume of sparged gas per bioreactor volume per hour), the airlift bioreactor achieves rapid mixing and adequate oxygen mass transfer. Foaming is minimal and inconsequential for serum-free media and media supplemented with 5%-10% fetal bovine serum. The use of serum-free medium facilitates monoclonal antibody purification and enhances the purity of the final MAb product.     

Model of oxygen transport limitations in hollow fiber bioreactors

Axial and radial oxygen depletion are believed to be critical scale-limiting factors in the design of cell culture hollow fiber bioreactors. A mathematical analysis of oxygen depletion has been performed in order to develop effectiveness factor plots to aid in the scaling of hollow fiber bioreactors with cells immobilized in the shell-side. Considerations of the lumen mass transport resistances and the axial gradients were added to previous analyses of this immobilization geometry. An order of magnitude analysis was used to evaluate the impact of the shell-side convective fluxes on the oxygen transport. A modified Thiele modulus and a lumen and membrane resistance factor have been derived from the model. Use of these terms in the effectiveness factor plots results in a considerable simplification of the presentation and use of the model. Design criteria such as fiber dimensions and spacing, reactor lengths, and recycle flow rates can be selected using these plots. Model predictions of the oxygen limitations were compared to experimental measurements of the axial cell distributions in a severely oxygen limited hollow fiber bioreactor. Despite considerable uncertainty in our parameters and nonidealities in hollow fiber geometry, the cell distribution correlated well with the modeling results.     

Effect of light intensity on beta-carotene production and extraction by Dunaliella salina in two-phase bioreactors

Application of two-phase bioreactors is a useful technique for improvement of the productivity of fermentations. Fermentative extraction of the products in situ is performed in this technique. The effect of light intensity on the extraction of beta-carotene from Dunaliella salina, in the fermentative extraction, has been investigated. Three different average light exposures were applied: 1.5 x 10(-8) (low), 2.7 x 10(-8) (intermediate) and 4.5 x 10(-8) (high) micromol s(-1) per cell. Results show that beta-carotene content of the cells increases by increasing the light exposure. Increase in the beta-carotene content of the cells is not necessarily coupled with an increase in the volumetric production of beta-carotene. Final volumetric production is about the same for the three bioreactors. beta-Carotene extraction rate is enhanced by the increase in the light exposure. The results suggest that extraction rate is related to beta-carotene content of the cells and is not essentially related to the volumetric production of beta-carotene. Although the effectiveness of extraction with respect to the light input is comparable for all light intensities applied, increasing the light input per cell leads to a higher volumetric extraction rate. Moreover, extracted beta-carotene stays very pure even so the extraction increased by the increase of light intensity.     

Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors

Two-phase olive mill waste (TPOMW) is a semi-solid effluent that is rich in contaminating polyphenols and is produced in large amounts by the industry of olive oil production. Laboratory-scale bioreactors were used to investigate the biodegradation of TPOMW by its indigenous microbiota. The effect of nutrient addition (inorganic N and P) and aeration of the bioreactors was studied. Microbial changes were investigated by PCR-temperature time gradient electrophoresis (TTGE) and following the dynamics of polar lipid fatty acids (PLFA). The greatest decrease in the polyphenolic and organic matter contents of bioreactors was concomitant with an increase in the PLFA fungal/bacterial ratio. Amplicon sequences of nuclear ribosomal internal transcribed spacer region (ITS) and16S rDNA allowed identification of fungal and bacterial types, respectively, by comparative DNA sequence analyses. Predominant fungi identified included members of the genera Penicillium, Candida, Geotrichum, Pichia, Cladosporium, and Aschochyta. A total of 14 bacterial genera were detected, with a dominance of organisms that have previously been associated with plant material. Overall, this work highlights that indigenous microbiota within the bioreactors through stimulation of the fungal fraction, is able to degrade the polyphenolic content without the inoculation of specific microorganisms.     

Viscous media in tower bioreactors: Hydrodynamic characteristics and mass transfer properties

Studies in tower reactors with viscous liquids on flow regime, effective shear rate, liquid mixing, gas holdup and gas/ liquid mass transfer (k _La) are reviewed. Additional new data are reported for solutions of glycerol, CMC, PAA, and xanthan in bubble columns with diameters of 0.06, 0.14 and 0.30 m diameter. The wide variation of the flow behaviour index (1 to 0.18) allows to evaluate the effective shear rate due to the gas flow. New dimensionless correlations are developed based on the own and literature data, applied to predict k _La in fermentation broths, and compared to other reactor types.List of Symbols a(a) m^–1 specific interfacial area referred to reactor (liquid) volume - Bo Bond number (g D _c ² _L/) - c _L(c _L ^* ) kmol m^–3 (equilibrium) liquid phase oxygen concentration - C coefficient characterising the velocity profile in liquid slugs - C _s m^–1 coefficient in Eq. (2) - d _B(d_vs) m bubble diameter (Sauter mean of d _B) - d ₀ m diameter of the openings in the gas distributor plate - D _c m column diameter - D _L m²s^–1 diffusivity - E _L(E_W) m² s^–1 dispersion coefficient (in water) - E ² square relative error - Fr Froude number (u _G/(g D_c)^0.5) - g m s^–2 gravity acceleration - Ga Gallilei number (g D _c ³ _L ² / _eff ² ) - h m height above the gas distributor the gas holdup is characteristic for - k Pasⁿ fluid consistency index (Eq. 1) - k _L m s^–1 liquid side mass transfer coefficient - k _La(k_La) s^–1 volumetric mass transfer coefficient referred to reactor (liquid) volume - L m dispersion height - n flow behaviour index (Eq. 1) - P W power input - Re liquid slug Reynolds number ( _L(u _G +u _L) D _c/_eff) - Sc Schmidt number ( _eff/( _L D _L )) - Sh Sherwood number (k _La D _c ² /D_L) - t s time - u _B(u_sw) m s^–1 bubble (swarm) rise velocity - u _G(u_L) m s^–1 superficial gas (liquid) velocity - V(V_L) m³ reactor (liquid) volume Greec Symbols W m^–2 K^–1 heat transfer coefficient - y(y _eff) s^–1 (effective) shear rate - _G relative gas holdup - s relaxation time of viscoelastic liquid - _L(_eff) Pa s (effective) liquid viscosity (Eq. 1) - _L kg m^–3 liquid density - N/m surface tension     

Physico-chemical features of solvent media in the phases of aqueous polymer two-phase systems

Solvent polarity and pH in the coexisting aqueous phases of aqueous dextran-poly(ethylene glycol) and dextran-Ficoll two-phase systems of varied polymer concentrations were examined using the solvatochromic technique and potentiometric measurements, respectively. The relative solvent polarity of the phases, as measured by the solvatochromic technique, is suggested as a measure of the hydration power of water in the phases of aqueous polymer systems. Partitioning of a series of sulphonephthalein dyes in aqueous dextran-poly(ethylene glycol) and dextran-Ficoll two-phase systems of fixed polymer composition containing 0.01 mol/L universal buffer, pH 7.15, was studied. The results obtained are discussed together with those reported earlier on the physico-chemical features of aqueous media in the coexisting phases of the systems. It is suggested that the two phases of aqueous polymer systems should be viewed as two immiscible water-like solvents. The implications of the suggestion for the theoretical treatment of aqueous polymer two-phase systems are discussed.     

Long-term influence of the presence of a non-aqueous phase on the cell surface hydrophobicity of Pseudomonas in two-phase partitioning bioreactors

The long-term influence of silicone oil 200 cSt (SO200) and 2, 2, 4, 4, 6, 8, 8-heptamethylnonane (HMN) on the cell surface hydrophobicity (CSH) of a hexane-degrading Pseudomonas aeruginosa strain and a toluene-degrading Pseudomonas putida strain was assessed in two-phase partitioning bioreactors under batch and continuous operation. CSH was evaluated using a modified BATH method based on optical density (CSH_OD) and colony-forming unit (CSH_CFU) measurements. In the presence of HMN, P. aeruginosa turned hydrophobic over the time course as shown by the gradual increase in CSH_OD (61 ± 1%) and CSH_CFU (53 ± 3%) under batch degradation and in CSH_OD (49 ± 0%) under continuous operation. However, P. putida turned hydrophobic only under continuous operation (CSH_OD = 28 ±2% {\hbox{CS}}{{\hbox{H}}_{\rm{OD}}} = 28 \pm 2\% ). On the other hand, no significant CSH enhancement was observed in both Pseudomonas strains in the presence of SO200. These results suggested that CSH is species, non-aqueous phase, and cultivation mode dependant, and an inducible property of bacteria. Maximum hexane elimination capacities increased by 2 and 3 in the presence of SO200 and HMN, respectively. Based on the absence of CSH in P. aeruginosa in the presence of SO200, the higher elimination capacities recorded were likely due to an improved hexane mass transfer (physical effect). However, in the presence of HMN, a direct hexane uptake from the non-aqueous phase (biological effect) might have also contributed to this enhancement.     

Model of forced expiratory flows and airway geometry in infants.

Early measurements of autopsied lungs from infants, children, and adults suggested that the ratio of peripheral to central airway resistance was higher in infants than older children and adults. Recent measurements of forced expiration suggest that infants have high flows relative to lung volume. We employed a computational model of forced expiratory flow along with physiological and anatomic data to evaluate whether the infant lung is a uniformly scaled-down version of the adult lung. First, we uniformly scaled an existing computational model of adult forced expiration to estimate forced expiratory flows (FEF) and density dependence for an 18-mo-old infant. The values obtained for FEF and density dependence were significantly lower than those reported for healthy 18-mo-old infants. Next, we modified the model for the infant lung to reproduce standard indexes of expiratory flow [forced expiratory volume in 0.5 s (FEV(0.5)), FEFs after exhalation of 50 and 75% forced vital capacity, FEF between 25 and 75% expired volume] for this age group. The airway sizes obtained for the infant lung model that produced accurate physiological measurements were similar to anatomic data available for this age and larger than those in the scaled model. Our findings indicate that the airways in the infant lung model differ from those in the scaled model, i.e., middle and peripheral airway sizes are larger than result from uniform downscaling of the adult lung model. We show that the infant lung model can be made to reproduce individual flow-volume curves by adjusting lumen area generation by generation.     

Valuation of bioreactors for low viscous media and high oxygen transfer demand

A strategy of a systematic comparison is developed in which the valuation parameters and the influencing parameters are put in order in a three dimensional matrix. The performance of a bioreactor construction is then evaluated with a biological test system that has representative medium properties and where all important parameters of microbial growth are known. This strategy is applied to four bioreactor constructions and to low viscous bacteria and yeast systems with and without foam formation. On the basis of this data set the performances of the different bioreactors are compared.The financial support of the Kommission zur Förderung der Wissenschaftlichen Forschung, Bern, is gratefully acknowledged.