Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: evaluation by CO2 pulse injection and autogenous CO2 methods

In order to see the effect of CO(2) inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O(2) were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO(2) concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O(2)), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O(2). We used two different experimental methods for evaluating CO(2) inhibition: CO(2) pulse injection and autogenous CO(2) methods. A 10 or 22% (v/v) CO(2) pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO(2) affects the synthesis of biomass and PHB. CO(2) inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO(2) concentration and pulse duration. The new proposed autogenous CO(2) method makes it possible to place microbial cells under different CO(2) level environments by varying the gas flow rate. Introduction of O(2) gas at a low flow rate of 0.42 vvm resulted in an increase of CO(2) concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O(2) flow rate. This new method measures the inhibitory effect of CO(2) produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO(2) inhibition without introducing artificial CO(2) into the fermentor.     

Critical assessment of gassing-in methods for measuring k(l)a in fermentors

The reliability of dynamic measurement methods of k(l)a in fermentors using a step oxygen concentration change in the feed gas was tested. The tests were performed both for the original variant using the nitrogen right harpoon over left harpoon air exchange and the newly presented variant using the oxygen-enriched air (27 vol % O(2)) --> air exchange. The testing consisted in comparing k(l)a values determined from these methods with values determined from the steady-state Na(2)SO(3) feeding method and the dynamic pressure method, the reliability of which was proven earlier. The measurements were done in water (coalescent batch) and in 0.5M Na(2)SO(4) solution with and without the addition of 1 wt % carboxymethylcellulose (noncoalescent batches). It was found that in noncoalescent liquids the methods tested give extremely low k(l)a values (as low as 15% of the correct value). The methods are defective in principle irrespective of the gases used for exchange.     

Dynamic pressure method for kla measurement in large-scale bioreactors

A dynamic method is proposed for k(l)a measurement in aerated and agitated reactors, in which a change in the total pressure in the reactor by approximately 20% leads to a simultaneous change in the oxygen concentration in all the bubbles in the dispersion. This procedure suppresses the influence of nonideal mixing of the gas phase on the k(l)a value. Other dynamic methods so far used do not possess this advantage. They are based on a step change in oxygen concentration in the entering gas, where the interfacial nitrogen transport and the finite rate of the concentration change propagation into the individual bubbles in the dispersion can cause an error in the reported k(l)a values of more than hundreds of percent. The reliability of the pressure method is tested by comparison both with the standard dynamic method, in which pure oxygen is absorbed in a liquid from which all other gas components were previously removed, and with the steady-state sulphite method. The signal of the oxygen probe used in the experiments must be independent of the pressure. A test for this in dependence is described. The pressure method is also suitable for large-scale reactors since the necessary pressure changes are sufficiently small and, morever, air can be used.     

Large scale production and semi-purification of kedarcidin in a 1000-L fermentor

Summary Actinomycete strain ATCC 53650 was grown in a 1000-L fermentor containing 680 L of medium and the production of kedarcidin was monitored by HPLC. The titers of kedarcidin in the fermentor cultures were 0.49–0.53 mg ml^–1. A quick and efficient purification method involving the use of anion exchange resin DE23 (batch adsorption-desorption) and an ultrafiltration system yielded high recovery (65% yield) of kedarcidin from the fermentor culture. Over 200 grams of lyophilized kedarcidin of 70% purity was recovered from each of two 1000-L fermentor cultures using this process.     

Bacterial cellulose production by Acetobacter xylinum in a 50-L internal-loop airlift reactor

Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal-loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water.     

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.     

Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7 was utilized in this study as a host strain in both 5-L and 30-L scale fermentors. To supply high purity oxygen into a bioreactor, nearly 100 % high purity oxygen from commercial bomb or higher than 93 % oxygen available in-situ from a pressure swing adsorption oxygen generator (PSA) was employed. Under the optimal fermentation of H. polymorpha with high purity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/L and 5.1 g/L in the 5-L fermentor, and 24.8 g/L and 4.5 g/L in the 30-L fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-L and 30-L fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-L fermentor. This study, therefore, proved the positive effect of high purity oxygen to enhance viable cell density as well as target recombinant protein production in microbial fermentations.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Laccase-induced C–N coupling of substituted <Emphasis Type="Italic">p</Emphasis>-hydroquinones with <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid in comparison with known chemical routes

Magnetotactic bacteria are difficult to grow under defined conditions in culture, which has presented a major obstacle to commercial application of magnetosomes. We studied the relationships among the cell growth, magnetosome formation, dissolved oxygen concentration (DO), and the ability to supply oxygen to the cells. Mass culture of Magnetospirillum gryphiswaldense MSR-1 for the production of magnetosomes was established in a 42-L fermentor under the following conditions: (1) sterile air was the sole gas supplied in the fermentor, and DO could be regulated at any level below 10% saturation by cascading the stir rate to DO, (2) to resolve the paradoxical situation that the cell growth requires higher DO whereas magnetosome formation requires low DO below the detectable range of regular oxygen electrode, DO was controlled to optimal level using the change of cell growth rate, rather than reading from the highly sensitive oxygen electrode, as the signal for determining appropriate DO, and (3) timing and rate of supplying the substrates were determined by measuring cell density and Na-lactate concentration. Under these conditions, cell density (OD565) of strain MSR-1 reached 7.24 after 60-h culture in a 42-L fermentor, and cell yield (dry weight) was 2.17 g/L, the highest yield so far being reported. The yield of magnetosomes (dry weight) was 41.7 mg/L and 16.7 mg/L/day, which were 2.8 and 2.7 times higher than the previously reported yields.     

Oxygen and carbon dioxide mass transfer and the aerobic, autotrophic cultivation of moderate and extreme thermophiles: a case study related to the microbial desulfurization of coal

Mass transfers of O(2), CO(2), and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high-pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, k(L)a measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H(2)SO(4) in water) phase as a function of the temperature has been studied in a laboratory-scale fermentor. At 15, 30, 45, and 70 degrees C, (k(L)a)(o) values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (k(L)a)(o) was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (k(L)a)(o) was plotted against the temperature. By multiplying the equilibrium concentration of O(2) in water with (k(L)a)(o) maximal, O(2) transfer capacities were calculated. It appeared that in finite of a decreased solubility of O(2) at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70 degrees C. However, in an air-mixed fermentor the transfer capacity of O(2) decreased slowly but steadily.Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO(2) transfer capacity, calculated as the product of the equilibrium CO(2) concentration times (k(L)a)(c), decreased slowly as the temperature increased over the range 15-70 degrees C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO(2) limitation is more likely to occur than O(2) limitation.     

Shaken helical track bioreactors: Providing oxygen to high-density cultures of mammalian cells at volumes up to 1000 L by surface aeration with air

A new scalable reactor was developed by applying a novel mixing principle that allows the large-scale cultivation of mammalian cells simply with surface aeration using air owing to increased liquid-gas transfer compared to standard stirred-tank bioreactors. In the cylindrical vessels (50 mL-1500 L) with a helical track attached to the inside wall, the liquid moved upward onto the track as the result of orbital shaking to increase the liquid-gas interface area significantly. This typically resulted in a 5-10-fold improvement in the volumetric mass transfer coefficient (k(L)a). In a 1500-L helical track vessel with a working volume of 1000 L, a k(L)a of 10h(-1) was obtained at a shaking speed of 39 rpm. Cultivations of CHO cells in a shaken 55-L helical track bioreactor resulted in improved cell growth profiles compared to control cultures in standard systems. These results demonstrated the possibility of using these new bioreactors at scales of 1000 L or more.     

Influence of oxygen adsorption on the dynamic K(L)a measurement in three-phase slurry reactors

To check for possible mass transfer limitations of oxygen and/or carbon dioxide in kinetic experiments on microbial desulphurization of coal, it is important to properly measure the volumetric mass transfer coefficient (k(L)a) especially at high slurry densities. Volumetric mass transfer coefficients of oxygen, at different solid hold-up values (epsilon(s) = 0 to 0.28) of coal slurries (d(par) < 100 * 10(-6) m), were measured in a lab scale fermentor and in a lab scale pachuca tank, using the dynamic gas-liquid absorption method. It was shown that serious errors could occur due to oxygen adsorption at the coal surface. Using the data of an independently measured adsorption isotherm, the real k(L)a could be calculated from the measured apparent k(L)a. The results show a k(L)a decrease of 40% to 50% at a volumetric solid hold-up of 28%. Estimation of the oxygen and carbon dioxide transfer rates, from the measured mass transfer coefficients, indicates that the stirred fermentor is suitable for kinetic experiments at high slurry densities, whereas the pachuca tank and shake flask are not. (c) 1992 John Wiley & Sons, Inc.     

Critical assessment of the steady-state Na2SO3 feeding method for kla measurement in fermentors

Important aspects of k(l)a measurement in agitated aerated vessels are briefly characterized from the standpoint of reliability of the measured data. It seems that most of the k(l)a data, based on a number of variants of the steady-state and dynamic methods in noncoalescent liquids, do not have a clear physical meaning, because they are affected by the differences between the actual driving force and the driving force assumed by the model used for its evaluation. A reliability test is given for the Na(2)SO(3) feeding steady-state method (FSM), by comparing the results of air and pure oxygen absorption in a noncoalescent liquid (0.5M Na(2)SO(4) solution) with the results obtained by the independent pressure step dynamic method (RDM). The RDM is one of a few variants of the dynamic method which gives correct k(l)a data unaffected by nonideal mixing of the gas phase in the reactor. It was found that the FSM yields correct k(l)a values only when pure oxygen is used for absorption. When air is absorbed, the FSM gives k(l)a values in the region of k(l)a > 0.1 s(-1) substantially (to 55%) lower than those for pure oxygen absorption.     

Wine vinegar production using a noncommercial 100-litre bubble column reactor equipped with a novel type of dynamic sparger

This paper describes batch and semicontinuous acetic acid fermentations for wine vinegar production carried out with Acetobacter pasteurianus, and an industrial strain using a noncommercial 100-L bubble column reactor equipped with a novel type of gas-liquid dynamic sparger. Results showed acetification rates with this fermentor (i.e., an overall acetic acid productivity of 1. 8 g/L/h and yield of 94%) similar to that of the Frings acetator and higher as compared to others fermentors in current industrial use in Spanish wine vinegar factories, and a linear relationship between overall productivity and kLa with different operating conditions and fermentation scales. Copyright 1999 John Wiley & Sons, Inc.     

Flow characteristics of a pilot-scale airlift fermentor

The effects of aeration on the flow characteristics of water in a glass pilot-scale airlift fermentor have been examined. The 55-L capacity fermentor consisted of a 15.2-cm-i.d. riser column with a 5.1-cm-i.d. downcomer tube. It was found that the average bubble size diminished with increased aeration. Typically, average bubble sizes ranged from 4.32 mm at a superficial gas velocity of 0.64 cm/s to 1.92 mm at 10.3 cm/s. A gas holdup of 0.19 was attained with superficial gas velocities (v_s) on the order of 10 cm/s, indicating the highly gassed nature of the fluid in the riser section of the fermentor. Circulation velocities of markers placed in the fermentor decreased with increasing aeration rates due to increased turbulence and axial liquid back mixing within the riser section. Actual volumetric liquid circulation rates remained relatively constant (0.36–0.49 L/s) for values of (v_s) up to 10 cm/s. Based on theoretical calculations, the ascending velocity of bubbles in a swarm reached 54 cm/s in the range of (v_s) values studied.     

Efficient production of cellulase in the culture of Acremonium cellulolyticus using untreated waste paper sludge

Cellulase was produced by Acremonium cellulolyticus using untreated waste paper sludge (PS) as the carbon source. The clay present in PS did not show any inhibitory effect on cellulase production but did alter the pH during fermentation. On the flask scale, the maleate buffer concentration and pH were key factors that affected the efficiency of cellulase production from PS cellulose. Optimum cellulase production in a 3-L fermentor of working volume 1.5 L was achieved by controlling the pH value at 6.0 using 2 M NaOH and 2 M maleic acid, and the productivity reached 8.18 FPU/mL. When 40.89 g/L PS cellulose, 2.2 g/L (NH(4) )(2) SO(4) , and 4.4 g/L urea were added to a 48-h culture, the cellulase activity was 9.31 FPU/mL at the flask scale and 10.96 FPU/mL in the 3-L fermentor. These values are ～80% of those obtained when pure cellulose is used as the carbon source. The method developed here presents a new route for the utilization of PS.     

氮源对重组大肠杆菌发酵产L-精氨酸的影响

考察有机氮源种类、蛋白胨用量以及(NH4)2SO4用量对重组E.coli发酵产L-精氨酸的影响.结果表明:以蛋白胨作为有机氮源且用量在10 g/L,( NH4 )2SO4用量在15 g/L时,摇瓶发酵产L-精氨酸产量最高,达到9.4g/L.在5L发酵罐进行补料分批培养,通过补加(NH4)2 SO4,L-精氨酸产量可以达到18.8 g/L,比未补加提高了108.9％.     

Characterization of oxygen transfer in miniature and lab-scale bubble column bioreactors and comparison of microbial growth performance based on constant k(L)a

This work describes the engineering characterization of miniature (2 mL) and laboratory-scale (100 mL) bubble column bioreactors useful for the cultivation of microbial cells. These bioreactors were constructed of glass and used a range of sintered glass gas diffusers with differently sized pores to disperse humidified air within the liquid biomedium. The effect of the pressure of this supplied air on the breakthrough point for gas diffusers with different pore sizes was examined and could be predicted using the Laplace-Young equation. The influence of the superficial gas velocity (u(g)) on the volumetric mass transfer coefficient (k(L)a) was determined, and values of up to 0.09 s(-1) were observed in this work. Two modeling approaches were considered in order to predict and provide comparison criteria. The first related the volumetric power consumption (P/V) to the k(L)a and a good correlation was obtained for differently sized reactors with a given pore size, but this correlation was not satisfactory for bubble columns with different gas diffusers. Values for P/V ranged from about 10 to 400 W.m(-3). Second, a model was developed predicting bubble size (d(b)), bubble rising velocity (u(b)), gas hold-up (phi), liquid side mass transfer coefficient (k(L)), and thus the k(L)a using established theory and empirical correlations. Good agreement was found with our experimental data at different scales and pore sizes. Values for d(b) varied from 0.1 to 0.6 mm, and k(L) values between 1.7 and 9.8 x 10(-4) m.s(-1) were determined. Several E. coli cultivations were performed in the miniature bubble column at low and high k(L)a values, and the results were compared to those from a conventional stirred tank operated under identical k(L)a values. Results from the two systems were similar in terms of biomass growth rate and carbon source utilization.     

A dense cell retention culture system using stirred ceramic membrane reactor

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the k(L)a to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed-batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. (c) 1994 John Wiley & Sons, Inc.     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.