A novel centrifugal impeller bioreactor. II. Oxygen transfer and power consumption

The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus

Clavulanic acid (CA), a potent β-lactamase inhibitor, is produced by a filamentous bacterium. Here, the effect of DO and shear, expressed as impeller tip velocity, on CA production was examined. Cultivations were performed in a 4 L fermentor with speeds of 600, 800 and 1,000 rpm and a fixed air flow rate (0.5 vvm). Also, cultivation with automatic control of dissolved oxygen, at 50% air saturation, by varying stirrer speed and using a mixture of air and O₂ (10% v/v) in the inlet gas, and a cultivation with fixed stirrer speed of 800 rpm and air flow rate of 0.5 vvm, enriched with 10% v/v O₂, were performed. Significant variations in CA titer, CA production rate and O₂ uptake-rate were observed. It was also found that the DO level has no remarkable effect on CA production once a critical level is surpassed. The most significant improvement in CA production was related to high stirrer speeds.     

Growth characteristics of Pleurotus ostreatus in bioreactors

Pleurotus ostreatus was cultured in a bioreactor equipped with different impeller geometries under non-limiting nutrient conditions. With a Rushton turbine impeller the specific growth rate decreased 30% and pellet diameter was reduced 15% when the aeration rate was increased from 1 to 1.5 vvm. Agitation rate reduced the pellet diameter from 5.1 mm to 2.8 mm using 200 rpm and 400 rpm of agitation, respectively. Specific growth rates of 0.036, 0.020 and 0.041 h–1 were obtained with Roshton (disc turbine), Helical Ribbon and InterMIG impellers, respectively. Impeller geometry is important to control the pellet size and consequently growth rate of P. ostreatus.     

Agitation,aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus

Factorial design and response surface analyses were used to optimize the production of inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) by Kluyveromyces marxianus ATCC 16045, using sucrose as carbon source. Effects of aeration, agitation and type of impeller (disk turbine, marine, pitched blade) were studied in a batch stirred reactor. Two factorial designs 2² were carried out. Agitation speed varied from 50 to 550 rpm (revolution per minute), aeration rate from 0.5 to 2.0 vvm (air volume/broth volume·minute). It has been shown that the enzyme production was strongly influenced by mixing conditions, while aeration rate was shown to be less significant. Additionally, the increase in the agitation speed is limited by the death rate, which increases drastically at high speeds, lowering the enzyme production. Also, the impeller type has significant influence in the production, the disk impeller at 450 rpm and aeration at 1.0 vvm led to an activity of 121 UI/mL, while the pitched blade was shown to be the best impeller for this process, leading to the best production, 176 UI/mL, at 450 rpm and 1.0 vvm. The maximum shear stress for inulinase production was about 0.22 Pa, since higher values cause higher cell death rates, affecting the enzyme production. The same results were confirmed with another microorganism, which was also sensible to shear stress. Therefore, it has been concluded that in some cases, mainly when the microorganism is sensible to shear stress, the interaction between mass transfer and mechanical stress should be considered in scale up processes.     

The Effect of the Chopper on Granules from Wet High-Shear Granulation Using a PMA-1 Granulator

Chopper presence and then chopper speed was varied during wet high shear granulation of a placebo formulation using a PMA-1 granulator while also varying the impeller speed. The granules were extensively analyzed for differences due to the chopper. The effect of the chopper on the granules varied with impeller speed from no effect at a low impeller speed of 300 rpm to flow interruptions at an impeller speed of 700 rpm to minimal impact at very high impeller speeds as caking at the bowl perimeter obscured the effect of the chopper on the flow pattern. Differences in the granule flowability were minimal. However, it was concluded that the largest fraction of optimal granules would be obtained at an impeller speed of 700 rpm with the chopper at 1,000 rpm allowing balances between flow establishment, segregation, and centrifugal forces.     

High bioreactor production and emulsifying activity of an unusual exopolymer by Chromohalobacter canadensis 28

Unusual composition of an exopolymer (EP) from an obligate halophilic bacterium Chromohalobacter canadensis 28 has triggered an interest in development of an effective bioreactor process for its production. Its synthesis was investigated in 2‐L bioreactor at agitation speeds at interval 600‐1000 rpm, at a constant air flow rate of 0.5 vvm; aeration rates of 0.5, 1.0, and 1.5 vvm were tested at constant agitation rate of 900 rpm. EP production was affected by both, agitation and aeration. As a result twofold increase of EP yield was observed and additionally increased up to 3.08 mg/mL in a presence of surfactants. For effective scale‐up of bioreactors mass transfer parameters were estimated and lowest values of K_La obtained for the highest productivity fermentation was established. Emulsification activity of EP exceeded that of trade hydrocolloids xanthan, guar gum, and cellulose. A good synergism between EP and commercial cellulose proved its potential exploration as an enhancer of emulsifying properties of trade emulsions. A pronounced lipophilic effect of EP was established toward olive oil and liquid paraffin. Cultivation of human keratinocyte cells (HaCaT) with crude EP and purified γ‐polyglutamic acid (PGA) showed higher viability than control group.     

Effect of aeration on denitrification byOchrobactrum anthropi SY509

Aeration was found to affect the biological denitrification byOchrobactrum anthropi SY509. Although cell growth was vigorous under 1 vvm of aeration and an agitation speed of 400 rpm in a 3-L jar fermentor, almost no nitrate was removed. Yet under low agitation speeds (100, 200, and 300 rpm), denitrification occurred when the dissolved oxygen was exhausted shortly after the inoculation of the microorganism.Ochrobactrum anthropi SY509 was found to express highly active denitrifying enzymes under anaerobic conditions. The microorganism also synthesized denitrifying enzymes under aerobic conditions (1 vvm and 400 rpm), yet their activity was only 60% of the maximum level under anaerobic conditions and the nitrate removal efficiency was merely 15%. However, although the activities of the denitrifying enzymes were inhibited in the presence of oxygen, they were fully recovered when the conditions were switched to anaerobic conditions.     

Kinetic modelling of continuous submerged fermentation of cheese whey for single cell protein production

A mathematical model describing the kinetics of continuous production of single cell protein from cheese whey using Kluyveromyces fragilis was developed from the basic principles of mass balance. The model takes into account the substrate utilization for growth and maintenance and the effect of substrate concentration and cell death rate on the net cell growth and substrate utilization during the fermentation process. A lactose concentration below 1.91 g/L limited growth of yeast cells whereas a lactose concentration above 75 g/L inhibited the growth of the yeast. The model was tested using experimental data obtained from a continuous system operated at various retention times (12, 18 and 24 h), mixing speeds (200, 400 and 600 rpm) and air flow rates (1 and 3 vvm). The model was capable of predicting the effluent cell and substrate concentrations with R2 ranging from 0.95 to 0.99. The viable cell mass and lactose consumption ranged from 1.3 to 34.3 g/L and from 74.31% to 99.02%, respectively. A cell yield of 0.74 g cell/g lactose (close to the stoichiometric value of 0.79 g cell/g lactose) was achieved at the 12 h retention time-3 vvm air flow rate-600 rpm mixing speed combination. The total biomass output (viable and dead cells) at this combination was 37 g/L.     

Fluid mixing and oxygen transfer in cell suspensions ofTaxus chinensis in a novel stirred bioreactor

In high-density plant cell cultures, mixing and mass transfer are two key issues, which should be emphasized for process optimization. In this work, both mixing and oxygen transfer characteristics of cell suspensions ofTaxus chinensis were studied in a new centrifugal impeller bioreactor with a working volume of 1.2 L. The mixing time (t _M) and the volumetric oxygen transfer coefficient (K _L a) under different operational conditions were determined in both tap water and cell suspensions of 100–400 g fresh weight/L (i.e., 5.65–23.1 g DW/L). At an aeration rate of 0.1 L/min,t _M decreased from 10.6s at 30 rpm to 2.89 s at 200 rpm under 100 g FW/L, and from 9.63 s (120 rpm) to 4.05 s (300 rpm) under 400 g FW/L. Compared with the effect of agitation, aeration was less significant to the suspension mixing. At a relatively high agitation speed (e.g., 200 rpm),t _M remained almost the same even though aeration rate was changed from 0.1 to 0.4 L/min. Thet _M value increased slowly from 3.98 to 5.26 s at 120 rpm when the cell density was raised from 100 to 250 g FW/L. A rapid increase of botht _M and the suspension viscosity was observed at a cell density above 300 g FW/L. As expected, theK _L a value increased with an increase of aeration rate and agitation speed, but decreased with an increase of cell density. The quantitative data obtained here are useful to investigate the effect of mixing stress on the cell physiology and metabolism ofTaxus chinensis in the bioreactor. This paper is dedicated by JJZ to his colleague Prof. Jun-Tang Yu on the occasion of his 70 birthday.     

Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by a psychrophilic marine bacterium, <Emphasis Type="Italic">Psychrobacter aquimaris</Emphasis> LBH-10 using orthogonal array method

The optimal conditions for the production of cellulases by a marine bacterium, Psychrobacter aquimaris LBH-10, were established and their effects were compared using orthogonal array experiments based on the Taguchi method. The optimal conditions of rice bran, peptone and initial pH for the production of avicelase and CMCase by P. aquimaris LBH-10 were 50.0, 3.0, and 8.0 g/L, respectively, whereas those for filter paperase (FPase) were 100.0, 3.0, and 8.0 g/L, respectively. Rice bran was found to be the most important factor for the production of cellulases based on the calculated percentage of participation P (%) from an analysis of the variance (ANOVA). The optimal temperature for the cell growth of P. aquimaris LBH-10 was 25°C, whereas that for the production of avicelase, CMCase and FPase was 30°C. The optimal agitation speed and aeration rate for cell growth was 400 rpm and 1.5 vvm, respectively, whereas those for the production of CMCase were 300 rpm and 1.0 vvm, respectively. Aeration was found to be more important for cell growth and CMCase production than agitation. The maximum production of avicelase, CMCase and FPase in a 100 L bioreactor for 72 h under optimized conditions was 83.2, 388.7, and 75.4 U/mL, respectively.     

Dewatering of algal suspension using microfiltration with cross flow in the presence of magnetite as a filter aid

Dewatering algal suspensions is an important step in the extraction of oil and other useful substances from algae. In this study, spherical Nannochloropsis sp. and ellipsoidal Monoraphidium sp. suspensions were dewatered in the presence of different amounts of 350-nm magnetite particles using a microfiltration membrane with 360-nm pores in cross-flow mode. Magnetite functions as a filter aid by reducing the deformation of the cake of filtered algae on the membrane and providing paths for water to flow through the filtration cake of algae. In the case of Nannochloropsis sp., the highest dewatering rate was obtained when the number ratio, defined based on the size and ideal density, between Nannochloropsis sp. and magnetite was 1:12.5, but the addition of magnetite had no observable effect on the filtration of ellipsoidal Monoraphidium sp. suspensions through the membrane. After dewatering, magnetite was effectively separated from the concentrated algal suspension by the application of a magnetic field in an open flow system. Magnetite has the potential to enhance dewatering performance using a cross-flow membrane system.     

Biodesulphurization of mengen lignite by Rhodoccocus rhodochrous in a batch stirred and aerated tank fermenter

The biodesulphurization of Mengen lignite by a mesophilic bacterium, Rhodococcus rhodochrus ATCC 53968, was investigated in a batch stirred and aerated reactor. The experiments were carried out at 28°C with an inoculum percentage, initial pH, initial sodium acetate and lignite concentration of the biodesulphurization medium of 8% [v/v], 6.5 mM, 20 mM and 20 g/l, respectively. Variations in the sulphur contents of the lignite relative to the biodesulphurization period were monitored. The effects of the stirring and aeration rates on the removal of different sulphur forms from coal were investigated in the ranges 450–1,200 rpm and 0.1–0.53 vvm and the optimum values were found to be 500 rpm and 0.18 vvm, respectively. An increase in the total sulphur reduction with increasing biodesulphurization time was observed. The maximum total sulphur removal percentage was found to be 15.2% at 1,200 rpm after four days of incubation. The highest total sulphur removal rate was calculated on the second day of microbial desulphurization for each run. The total and organic sulphur contents of the coal after biodesulphurization were correlated with the stirring and aeration rates by using the non-linear least squares regression method. In the experimental runs lasting 8 days, the highest organic sulphur reducing percentage of 10.1% was obtained at a stirring rate of 500 rpm and an aeration rate of 0.40 vvm.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Protein fouling during constant-flux virus filtration: Mechanisms and modeling

As biomanufacturers consider the transition from batch to continuous processing, it will be necessary to re-examine the design and operating conditions for many downstream processes. For example, the integration of virus removal filtration in continuous biomanufacturing will likely require operation at low and constant filtrate flux instead of the high (constant) transmembrane pressures (TMPs) currently employed in traditional batch processing. The objective of this study was to examine the effect of low operating filtrate flux (5–100 L/m²/h) on protein fouling during normal flow filtration of human serum Immunoglobulin G (hIgG) through the Viresolve® Pro membrane, including a direct comparison of the fouling behavior during constant-flux and constant-pressure operation. The filter capacity, defined as the volumetric throughput of hIgG solution at which the TMP increased to 30 psi, showed a distinct minimum at intermediate filtrate flux (around 20–30 L/m²/h). The fouling data were well-described using a previously-developed mechanistic model based on sequential pore blockage and cake filtration, suitably modified for operation at constant flux. Simple analytical expressions for the pressure profiles were developed in the limits of very low and high filtrate flux, enabling rapid estimation of the filter performance and capacity. The model calculations highlight the importance of both the pressure-dependent rate of pore blockage and the compressibility of the protein cake to the fouling behavior. These results provide important insights into the overall impact of constant-flux operation on the protein fouling behavior and filter capacity during virus removal filtration using the Viresolve® Pro membrane.     

Effect of dissolved oxygen concentration and impeller tip speed on itaconic acid production by Aspergillus terreus

Summary Effect of aeration rate and impeller tip speed on mycelium growth and itaconic acid production was investigated in a batch culture of Aspergillus terreus IFO-6365. When impeller tip speed was 94.2 cm/sec at a fixed aeration rate of 0.5 vvm, itaconic acid concentration was 3.6 and 1.6 times higher than those in the impeller tip speed of 62.8 and 125.7 cm/sec, respectively. When an oxygen-enriched air was supplied at a fixed impeller tip speed of 94.2 cm/sec and dissolved oxygen concentration was maintained in the 20–60 % range, both itaconic acid concentration and mycelium growth were not affected by the dissolved oxygen concentration.     

Actinomycetes scale‐up for the production of the antibacterial,nocathiacin

An Amycolatopsis fastidiosa culture, which produces the nocathiacin class of antibacterial compounds, was scaled up to the 15,000 L working volume. Lower volume pilot fermentations (600, 900, and 1,500 L scale) were conducted to determine process feasibility at the 15,000 L scale. The effects of inoculum volume, impeller tip speed, volumetric gas flow rate, superficial gas velocity, backpressure, and sterilization heat stress were examined to determine optimal scale‐up operating conditions. Inoculum volume (6 vs. 2 vol %) and medium sterilization (R_o of 68 vs. 92 min^?1) had no effect on productivity or titer, and higher impeller tip speeds (2.1 vs. 2.9 m/s) had a slight effect (20% decrease). In contrast, higher backpressure, incorporating increased head pressure at the 15,000 L scale (1.2 vs. 0.7 kg/cm²) and low gas flow rates (0.25 vs. 0.8 vvm), appeared to be problematic (40–50% decrease). High off‐gas CO₂ levels were likely reasons for observed lower productivity. Consequently, air flow rate for this 25‐fold scale‐up (600–15,000 L) was controlled to match off‐gas CO₂ profiles of acceptable smaller scale batches to maintain levels below 0.5%. The 15,000 L‐scale fermentation achieved an expected nocathiacin I titer of 310 mg/L after 7 days. Other on‐line data (i.e., pH, oxygen uptake rate, and CO₂ evolution rate) and off‐line data (i.e., analog production, glucose utilization, ammonium production, and dry cell weight) at the 15,000 L scale also tracked similarly to the smaller scale, demonstrating successful fermentation scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.     

Production of anti‐inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti‐inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 μg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.     

Poly-β-hydroxyalkanoate production by halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> U7

Medium optimization for production of poly-β-hydroxyalkanoate (PHA) from Rhodobacter sphaeroides U7 cultivated in glutamate–acetate (GA) medium supplemented with 40 mM valeric acid as co-substrate under aerobic-dark condition was investigated. Studies on effect of nitrogen source and cultivation temperature by conventional and statistical methods illustrated that (NH₄)₂SO₄ (0.2 g/l) had no effect and the optimal temperature was at 30°C. The optimum environmental conditions were found to be anaerobic-light (3000 lux) cultivation with aeration rate of 1.0 vvm and agitation speed of 200 rpm for PHA production (2.5 g/l) with the highest PHA content (65.15%) at 0.5 vvm, and 200 rpm. Under this optimized medium and condition, PHA production from R. sphaeroides U7 increased 3.86-folds (from 0.69 to 2.66 g/l) (PHA content increased 1.5-folds). The biopolymer was purified and characterized by using ¹³C NMR, FTIR, DSC, X-ray diffraction and intrinsic viscosity techniques to be a copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) consisting of 84.8 mol% β-hydroxybutyric acid (HB) and 15.2 mol% β-hydroxyvaleric acid (HV).