Effects of rheological properties and mass transfer on plant cell bioreactor performance: production of tropane alkaloids

Volumetric mass transfer coefficients, K(L)a were measured over an aeration rate range from 0.1 to 1.0 vvm in a 1.2-L draft-tube-type airlift bioreactor for different Datura stramonium cell concentrations and correlated with superficial air velocity and rheological properties of the cell suspension. The measured K(L)a values (17-40 h(-1)) for a cell volume fraction of 0.2 (v/v) were approximately 2 times higher than those for the highest cell concentrations tested (cell volume fraction 0.7-0.8 v/v). Cell suspensions exhibited yield stress and pseudoplastic behavior. This behavior was described by the Casson model. The estimated yield stress values depended upon cell concentration with an exponent of 4.0. An empirical correlation based on the data for plant cell suspensions exhibiting yield stress was developed in order to determine aeration strategy for the plant cell cultivation in draft-tube-type airlift bioreactors: \documentclass{article}\pagestyle{empty}\begin{document}$$ {\rm K}_{\rm L} {\rm a} = {\rm A}({\rm U}_{{\rm gr}});{0.3} ({\rm \eta }_{{\rm eff}});{ - 0.4} $$\end{document} Aeration rates above 1.0 vvm caused a significant drop in cell yield and product content. Maximum growth and production were obtained at 0.6 vvm aeration. The cell and product yields obtained at 1.7 vvm were 2.8 times lower than the maximum values (25 g cell DW/L and 73.8 mg tropane alkaloid/L). The effects of the increased aeration rates on cell yield were also evaluated in terms of Reynolds stress. It was found that there was a relation between cell damage and the estimated Reynolds stress. The Reynolds stress estimated for the same aeration rate decreased with increasing cell concentration, suggesting that cells in the cultures at low cell concentrations are subjected to hydrodynamic damage. In the experiments with the cell cultures having a cell concentration of 0.3 (v/v), approximately 70% reduction in cell concentration was observed when the Reynolds stress was increased from 10 to 50 dyn/cm(2). (c) 1993 John Wiley & Sons, Inc.     

Enantioselective reduction of acetyldimethylphenylsilane by Trigonopsis variabilis (DSM 70714)

Summary Growing and resting cells of the yeast Trigonopsis variabilis (DSM 70714) can be used for the enantioselective reduction of the organosilicon compound acetyldimethylphenylsilane (1) to give optically active (R)-(1-hydroxyethyl)dimethylphenylsilane [(R)-2] in good yields. The enantiomeric purity of the isolated product was determined to be 62–86% ee depending on the substrate concentration used. Both substrate and product caused an inhibition of the reaction at concentrations higher than 0.35 and 0.5 g/l, respectively. Besides, higher substrate and product concentrations led to increased formation of the by-product 1,1,3,3-tetramethyl-1,3-diphenyldisiloxane. Considering the limiting substrate and product concentrations, it was possible to use the same biomass at least 5 times without significant loss of enzyme activity. 3-Methyl-3-phenyl-2-butanone (5) and acetyldimethylphenylgermane (7), which represent carbon and germanium analogues of 1, were also found to be accepted as substrates by Trigonopsis variabilis (DSM 70714). The reduction rates of the silicon (1) and germanium compound (7) were much higher than the transformation rate of the corresponding carbon analogue 5.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Effects of oxygen volumetric mass transfer coefficient on transglutaminase production by Bacillus circulans BL32

The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (k_La). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; k_La of 33.7 h⁻¹), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when k_La varied from 4.4 to 33.7 h⁻¹. The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation.     

Continuous production of gluconic acid by immobilized Gluconobacter oxydans cell bioreactor

Summary Living Gluconobacter oxydans cells were attached on fibrous nylon carrier. Free gluconic acid was directly continuously produced in an aerated tubular immobilized-cell bioreactor for at least 6 months, with a volumetric productivity of at least 5 g/lh at 100 g/l substrate glucose and about 80 g/l product gluconic acid concentrations. The highest volumetric productivity in respect to glucose concentration was obtained with 175 g/l glucose, with about 120 g/l product gluconic acid level. With self-directing optimization procedure in respect to maximum product gluconic acid level, productivities as high as about 12–15 g/lh were obtained at relatively high substrate feed rate of 0.166 l/lh and relatively low aeration rate of 0.5 l/lmin. The highest glucose conversion of about 96% was obtained with a long residence time, at the lowest substrate feed rate used at a relatively low aeration rate, resulting however in a significant increase in ketogluconic acid production.     

The effect of pH and oxygen concentration on the formation of 3-ketodisaccharides by Agrobacterium tumefaciens

The further optimization of 3-ketodisaccharide formation with sucrose, leucrose and iso maltutose was studied with special regard to pH and oxygen concentration in the reaction mixture with resting cells of Agrobacterium tumefaciens. It was found that the optimal pH values for the highest reaction rate and highest yield were different as the pH affected the stability of the 3-keto derivatives formed. A pH shift to 5.0 clearly reduced the enzymatic degradation of the 3-keto derivatives thus stabilizing them. The influence of constant oxygen concentrations on 3-ketosucrose formation was tested showing results not explicable with normal Michaelis-Menten kinetics. For each substrate a maximum of reaction rate and yield were obtained at very low oxygen concentrations.Dedicated to Professor Dr. Fritz Wagner on the occasion of his 65th birthday     

Production of l-serine from methanol and glycine by resting cells of a methylotroph under automatically controlled conditions

Serine production from methanol and glycine was tried using frozen-thawed resting cells of a methylotroph, Protomonas extorquens NR-1 under multi-variable controlled conditions. The conditions for l-serine formation were optimized at 30°C. The production of l-serine in 0.4% CaCl₂ solution (initial pH 8.2) was the same as in 0.1 M Tris-HCl buffer (initial pH 8.3). Increasing the initial glycine concentration promoted l-serine formation. A high aeration rate decreased l-serine production. The optimum concentrations of dissolved oxygen and methanol were 0.5 ppm and 10 g/l, respectively. The highest l-serine, 24.9 g/l, was obtained at 24 h from 30.94 gl (as dry weight) resting cells using 100 g/l initial glycine with controlled pH. The relationship between the initial rate of l-serine formation and cell concentration indicated an unusual curve due to the effects of the added NaOH which was used for controlling the pH. In similar experiments without control of pH, a normal profile was observed with respect to the relationship between the initial rate of l-serine formation and cell concentration. The highest l-serine, 54.5 g/l, was obtained at 48 h by 36.4 g/l (as dry weight) resting cells. The yield (mol of l-serine/mol of added substrate) of l-serine from methanol and glycine were 8.3% and 39.3%, respectively. The selectivity of l-serine (mol of l-serine/mol of glycine consumed) was 67.9%. The stoichiometry of the maximum l-serine formation showed that the resting cells carried the highly active methanol dehydrogenase while serine transhydroxymethylase was rather low. Serine glyoxalate aminotransferase was not completely inhibited by the high concentration of glycine (about 68% of synthesized l-serine was detected in the supernatant.     

Investigations of cephalosporin C production in an airlift tower loop reactor

Summary Cephalosporin C was produced by Cephalosporium acremonium in a 60 l airlift loop reactor on complex medium (with 30 kg/m³ peanut flour) in fed-batch operation. A final product concentration of 5 kg/m³ and a maximum productivity of 45 g/m³ h were attained. On-line analysis was used to determine ammonia, methionine, phosphate, reducing sugar and cephalosporin C by an autoanalyser, glucose by a flow injection analyser and cephalosporin C, penicillin N, deacetoxycephalosporin C, deacetylce-phalosporin C and methionine by HPLC. The volumetric productivity of the stirred tank reactor was higher than that of the airlift reactor because of differences in cell concentration. Specific productivities in relative to cell mass were similar in the two reactors. The substrate yield coefficient in the airlift reactor was twice that in the stirred tank reactor.Nomenclature E _o2 efficiency of oxygen transfer with regard to the specific power input - K _La volumetric mass transfer coefficient - OTR oxygen transfer rate - P power input - PR volumetric productivity of CPC - q _a volumetric aeration rate/broth volume (vvm) - SPR specific productivity with regard to RNA - V _L broth volume in reactor - z relative height of the aerated reactor     

Aldehyde production by alcohol oxidation with Gluconobacter oxydans

The microbial oxidation of various primary alcohols to the corresponding aldehydes has been investigated. A focused screening performed amongst some acetic acid bacteria showed that a newly isolated strain of Gluconobacter oxydans oxodizes various short-chain aliphatic alcohols to the corresponding aldehydes with negligible acid production. 3-Methyl-1-butanol (isoamyl alcohol) proved to be the better substrate with high yields (more than 90%) without by-product formation. This biotransformation also occurs with continuous or semicontinuous addition of substrate since the volatile product is removed from the medium under vigorous aeration conditions. Product recovery is attained either by the use of cold traps or by reversible complex formation.     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.     

Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula

Veillonella parvula strain 259 (=DSM 2007) was able to grow on a mineral salts medium supplemented with (per litre) 1 g yeast extract, 1 g Tween-80, and 3 mg putrescine. 2 HCl, with 6 mM thioglycolate as reductant and lactate as growth substrate. Succinate did not serve as a growth substrate, but when added in conjunction with lactate, it was decarboxylated to propionate and resulted in a measurable increase in growth yield, corresponding to the formation of 2.4 g cell dry mass per mol succinate. A growth yield increase linked to succinate metabolism occurred only while lactate was also being metabolised. Experiments with cell suspensions showed that succinate decarboxylating activity was constitutive. Addition of succinate produced clear increases in cellular ATP levels in ATP-depleted washed cells.     

Improving arachidonic acid fermentation by Mortierella alpina through multistage temperature and aeration rate control in bioreactor

Effective production of arachidonic acid (ARA) using Mortierella alpina was conducted in a 30-L airlift bioreactor. Varying the aeration rate and temperature significantly influenced cell morphology, cell growth, and ARA production, while the optimal aeration rate and temperature for cell growth and product formation were quite different. As a result, a two-stage aeration rate control strategy was constructed based on monitoring of cell morphology and ARA production under various aeration rate control levels (0.6–1.8 vvm). Using this strategy, ARA yield reached 4.7 g/L, an increase of 38.2% compared with the control (constant aeration rate control at 1.0 vvm). Dynamic temperature-control strategy was implemented based on the fermentation performance at various temperatures (13–28°C), with ARA level in total cellular lipid increased by 37.1% comparing to a constant-temperature control (25°C). On that basis, the combinatorial fermentation strategy of two-stage aeration rate control and dynamic temperature control was applied and ARA production achieved the highest level of 5.8 g/L.     

Influence of pH and malate-glucose ratio on the growth kinetics of Leuconostoc oenos

Summary Growth, substrate utilization and product formation were studied in batch cultures of a Leuconostoc oenos strain. The effect of various culture conditions, i.e. pH-control at different values and various initial concentrations of malate and glucose, on growth and metabolism were investigated. Addition of malate resulted in a marked stimulation of growth, with only a slight increase in final biomass but a high conversion yield of glucose. Under pH control this stimulation was much greater than could be accounted for from changes in pH profile resulting from malate utilization. The specific rate of malate utilization was maximal at pH 4.0 whereas the specific rate of glucose consumption was highest at pH 5.5. During co-metabolism of malic acid and glucose, substrate utilization and product formation agreed with the stoichiometric relationships of the malo-lactic reaction and the heterolactic fermentation of glucose. Offsprint requests to: A. Pareilleux     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P_i was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran^® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO₂ conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTR_max) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR_max was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Effect of inoculation strategies,substrate to biomass ratio and nitrogen sources on the bioconversion of wood sterols by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

4-Androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) are the main precursors in the production of steroidal drugs from phytosterols. To carry out the bioconversion, different inoculation strategies have been proposed. We compared the use of whole fermented broth and of free resting cells of two mutant strains of Mycobacterium sp. (DSMZ2966 and DSMZ2967) in shake flasks. Also the effect of the nitrogen source (ammonium sulfate, ammonium chloride and ammonium nitrate) and the sterol to biomass ratio at high substrate concentrations (19.2 g/l and 48.1 g/l) was evaluated. We found that the bioconversion with free resting cells (cell pellets) is more efficient than that with whole fermented broth, increasing both AD and ADD production. The use of ammonium nitrate in the culture medium and low substrate to biomass ratios (close to 1.0) increased the production yield. We also found that the bioconversion can be run at high substrate concentration under non-sterile conditions.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.