Baffled Flasks Research Articles

Production of 8-hydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by recombinant cells expressing H1004A-C1006S variant of Aspergillus nidulans diol synthase

An H1004A-C1006S variant of Aspergillus nidulans diol synthase was identified as an 8-dioxygenase. Whole recombinant Escherichia coli cells expressing the double-site variant showed the highest activity for converting linoleic acid to 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE), which in turn was reduced to 8-hydroxy-9,12(Z,Z)-octadecadienoic acid (8-HODE) by the addition of a reducing agent. The optimization of 8-HPODE production was performed by response surface methodology, and the optimal conditions were pH 8.3, 27.2°C, 22.7% dimethyl sulfoxide, 27.0gl−1 cells, and 16.5gl−1 linoleic acid in a 100ml baffled flask containing a 10ml reaction mixture with agitation at 236rpm. The reduction of 8-HPODE to 8-HODE was highest when Tris(2-carboxyethyl)phosphine hydrochloride (TECP-HCl) was added at a final concentration of 25mM. Under the optimized reaction and reduction conditions, whole cells expressing H1004A-C1006S variant of A. nidulans diol synthase produced 5.0gl−1 8-HODE for 1h without the formation of di-hydroxy fatty acids. This is the first report of the biotechnological production of 8-HODE.

5,8‐Dihydroxy‐9,12,15(Z,Z,Z)‐Octadecatrienoic Acid Production by Recombinant Cells Expressing Aspergillus nidulans Diol Synthase

AbstractWhole cells of recombinant Escherichia coli expressing diol synthase from Aspergillus nidulans produced 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid from α‐linolenic acid via 8‐hydroperoxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid as an intermediate. The optimal conditions for 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid production using whole recombinant cells were exhibited at pH 7.0, 40 °C, and 250 rpm with 40 g/L cells, 12 g/L, α‐linolenic acid, and 5 % (v/v) dimethyl sulfoxide in a 250‐mL baffled flask containing 50 mL reaction solution. Under these conditions, whole recombinant cells produced 9.1 g/L 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid for 100 min, with a conversion yield of 75 % (w/w), a volumetric productivity of 5.5 g/L/h, and specific productivity of 137 mg/g‐cells/h. As an intermediate, 8‐hydroperoxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid was observed at approximately 1.4 g/L after 100 min. With regard to dihydroxy fatty acid production, this is the highest reported volumetric and specific productivities thus far. This is the first report on the biotechnological production of 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid.

Recombination of thermo-alkalistable, high xylooligosaccharides producing endo-xylanase from Thermobifida fusca and expression in Pichia pastoris.

For xylooligosaccharide (XO) production, endo-xylanase from Thermobifida fusca was modified by error-prone PCR and DNA shuffling. The G4SM1 mutant (S62T, S144C, N198D, and A217V) showed the most improved hydrolytic activity and was two copies expressed in Pichia pastoris under the control of GAP promoter. The maximum xylanase activity in culture supernatants was 165 ± 5.5 U/ml, and the secreted protein concentration reached 493 mg/l in a 2-l baffled shake flask. After 6× His-tagged protein purification, the specific activity of G4SM1 was 2036 ± 45.8 U/mg, 2.12 times greater than that of wild-type enzyme. Additionally, G4SM1 was stable over a wide pH range from 5.0 to 9.0. Meanwhile, half-life of G4SM1 thermal inactivation at 70 °C increased 8.5-fold. Three-dimensional structures suggest that two amino acid substitutions, S62T and S144C, located at catalytic domain may be responsible for the enhanced activity and thermostability of xylanase. Xylobiose was the dominant end product of xylan hydrolysis by G4SM1. Due to its attractive biochemical properties, G4SM1 has potential value in commercial XO production.

Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp. LU310

A new isolated Schizochytrium sp. LU310 from the mangrove forest of Wenzhou, China, was found as a high producing microalga of docosahexaenoic acid (DHA). In this study, the significant improvements for DHA fermentation by the batch mode in the baffled flasks (i.e. higher oxygen supply) were achieved. By applied the nitrogen-feeding strategy in 1000mL baffled flasks, the biomass, DHA concentration and DHA productivity were increased by 110.4%, 117.9% and 110.4%, respectively. Moreover, DHA concentration of 21.06g/L was obtained by feeding 15g/L of glucose intermittently, which was an increase of 41.25% over that of the batch mode. Finally, an innovative strategy was carried out by intermittent feeding carbon and simultaneously feeding nitrogen. The maximum DHA concentration and DHA productivity in the fed-batch cultivation reached to 24.74g/L and 241.5mg/L/h, respectively.

Production of 5,8-dihydroxy-9(Z)-octadecenoic acid from oleic acid by whole recombinant cells of Aspergillus nidulans expressing diol synthase.

The optimal conditions for the production of 5,8-dihydroxy-9(Z)-octadecenoic acid (5,8-diHOME) from oleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans were 40 °C, pH 7.5, 10 % (v/v) dimethyl sulfoxide, 35 g cells l(-1), and 12 g oleic acid l(-1) at 250 rpm in a 250 ml baffled flask. Under these conditions, whole recombinant cells produced 5.2 g 5,8-diHOME l(-1) together with 1 g l(-1) of the intermediate 8-hydroperoxy-9(Z)-octadecenoic acid (8-HPOME) after 60 min. This corresponded to a conversion yield of 43 % (w/w), a volumetric productivity of 5.2 g l(-1 )h(-1), and a specific productivity of 148 mg g cells(-1 )h(-1). This is the first report of the biotechnological production of 5,8-diHOME from oleic acid.

Development of small scale cell culture models for screening poloxamer 188 lot-to-lot variation.

Shear protectants such as poloxamer 188 play a critical role in protecting cells during cell culture bioprocessing. Lot-to-lot variation of poloxamer 188 was experienced during a routine technology transfer across sites of similar scale and equipment. Cell culture medium containing a specific poloxamer 188 lot resulted in an unusual drop in cell growth, viability, and titer during manufacturing runs. After switching poloxamer lots, culture performance returned to the expected level. In order to control the quality of poloxamer 188 and thus maintain better consistency in manufacturing, multiple small scale screening models were developed. Initially, a 5L bioreactor model was established to evaluate cell damage by high sparge rates with different poloxamer 188 lots. Subsequently, a more robust, simple, and efficient baffled shake flask model was developed. The baffled shake flask model can be performed in a high throughput manner to investigate the cell damage in a bubbling environment. The main cause of the poor performance was the loss of protection, rather than toxicity. It was also suggested that suspicious lots can be identified using different cell line and media. The screening methods provide easy, yet remarkable models for understanding and controlling cell damage due to raw material lot variation as well as studying the interaction between poloxamer 188 and cells.

Cross-section perimeter is a suitable parameter to describe the effects of different baffle geometries in shaken microtiter plates

BackgroundBiotechnological screening processes are performed since more than 8 decades in small scale shaken bioreactors like shake flasks or microtiter plates. One of the major issues of such reactors is the sufficient oxygen supply of suspended microorganisms. Oxygen transfer into the bulk liquid can in general be increased by introducing suitable baffles at the reactor wall. However, a comprehensive and systematic characterization of baffled shaken bioreactors has never been carried out so far. Baffles often differ in number, size and shape. The exact geometry of baffles in glass lab ware like shake flasks is very difficult to reproduce from piece to piece due to the hard to control flow behavior of molten glass during manufacturing. Thus, reproducibility of the maximum oxygen transfer capacity in such baffled shake flasks is hardly given.ResultsAs a first step to systematically elucidate the general effect of different baffle geometries on shaken bioreactor performance, the maximum oxygen transfer capacity (OTRmax) in baffled 48-well microtiter plates as shaken model reactor was characterized. This type of bioreactor made of plastic material was chosen, as the exact geometry of the baffles can be fabricated by highly reproducible laser cutting. As a result, thirty different geometries were investigated regarding their maximum oxygen transfer capacity (OTRmax) and liquid distribution during shaking. The relative perimeter of the cross-section area as new fundamental geometric key parameter is introduced. An empirical correlation for the OTRmax as function of the relative perimeter, shaking frequency and filling volume is derived. For the first time, this correlation allows a systematic description of the maximum oxygen transfer capacity in baffled microtiter plates.ConclusionsCalculated and experimentally determined OTRmax values agree within ± 30% accuracy. Furthermore, undesired out-of-phase operating conditions can be identified by using the relative perimeter as key parameter. Finally, an optimum well geometry characterized by an increased perimeter of 10% compared to the unbaffled round geometry is identified. This study may also assist to comprehensively describe and optimize the baffles of shake flasks in future.

The role of volumetric power input in the growth, morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks

The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51kW/m3, followed by coiled flasks (CF) with 0.44kW/m3 and normal Erlenmeyer flasks (NF) with 0.20kW/m3 (flasks volume of 250mL, filling with 50mL and agitated at 150rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15mL, 150rpm (15mL-NF), and 25mL, 168rpm (25mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.

Production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans

Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l(-1) linoleic acid, 23 g l(-1) cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l(-1) 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l(-1) h(-1). This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.

Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications.

BackgroundA useful heterologous production system is required to obtain sufficient amounts of recombinant therapeutic proteins, which are often necessary for chemical characterization and engineering studies on the development of molecules with improved properties. Human Fas ligand extracellular domain (hFasLECD) is an agonistic death ligand protein that has potential applications for medical purposes. Site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. This study aimed to enhance the yield of hFasLECD using a Pichia pastoris secretory expression system suitable for efficient production on a small laboratory scale, and further to provide procedures for its site-specific chemical modification without impairing the biological functions based on the developed production system.ResultsA convenient cultivation system using a disposable plastic bag provided a three-fold increase in purification yield of tag-free hFasLECD as compared with the conventional system using a baffled glass flask. The system was further applied to the production of a mutant, which contains an additional reactive cysteine residue in the N-terminal tag-sequence region. Site-specific conjugations and cross-linking without impairing biological functions were achieved by reaction of the mutant hFasLECD with single maleimide group containing compounds and a linear polyethylene glycol derivative containing two maleimide groups at either end, respectively. All purified tag-free and chemically modified hFasLECDs showed an evident receptor binding activity in co-immunoprecipitation experiments mediated by wild-type and N-glycosylation site deficient mutant human Fas receptor extracellular domain derivatives. An N-Ethylmaleimide conjugated hFasLECD derivative demonstrated a significant cytotoxic activity against human HT-29 colorectal cancer cells.ConclusionsA new, efficient cultivation system for enhanced secretory production of hFasLECD using P. pastoris and an effective strategy for site-specific chemical modifications of hFasLECD were devised. The results obtained constitute the basis for biomedical applications including developments of novel therapeutic proteins and diagnostic tools targeted to related diseases and their biomarkers.

Deglycosylation of Isoflavones in Isoflavone-Rich Soy Germ Flour by Aspergillus oryzae KACC 40247

Aspergillus oryzae KACC 40247 was selected as an efficient daidzein-producing fungus from strains of the genus Aspergillus by using 5% (w/v) soy germ flour (SGF) as an isoflavone-glycoside-rich medium. The culture conditions, including SGF concentration, agitation speed, initial pH, temperature, and time, were optimized as follows: 7% (w/v) SGF, initial pH 6.0, 33 °C, 300 rpm, and 24 h in a 100 mL baffled flask. The determined amount of isoflavone aglycons in SGF using 50% ethyl acetate was the highest among the solvent systems tested and it was 3.7-fold higher than that using 70% methanol. Under the optimized conditions, the content and concentration of daidzein were 134 mg/g of SGF and 9.4 g/L, respectively, with a productivity of 391 ± 2.8 mg/L/h, and those of isoflavone aglycons were 165 mg/g of SGF and 11.5 g/L, respectively, with a productivity of 479 mg/L/h. Optimization of culture conditions increased the content, concentration, and productivity of isoflavone aglycons by 3.1-, 3.0-, and 3.7-fold, respectively. To our knowledge, this is the highest production of isoflavone aglycons reported to date.

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28°C, pH 4.5, an agitation speed of 600rpm and a dissolved oxygen concentration of ~25% of air saturation, the laccase productivity in the bioreactor exceeded 19UL(-1)days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325UL(-1).

출아효모(Saccharomyces cerevisiae)에서 Bacillus sp. HY-20균주의 재조합 endoxylanase의 효율적 분비 발현

Bacillus sp. HY-20균주 유래 endoxylanase를 코드하는 XylP 유전자를 효모에서 발현시키기 위해 두 개의 발현 플라스미드 pG-xylP와 pGMF-xylP를 구축하였다. 이들 플라스미드는 endoxylanase의 분비발현을 위해 각각 다른 분비서열인 XylP 유전자의 자체 분비서열(XylP s.s)과 최적화된 <TEX>$MF{\alpha}$</TEX> 분비서열(<TEX>$MF{\alpha}_{opt}$</TEX> s.s)을 가지고 있으며, S. cerevisiae SEY2102와 FY833균주에 형질전환되어 그 분비활성이 비교 조사되었다. 재조합 endoxylanase는 분비발현시스템과 숙주세포에 따라 23.7~70.1 unit/ml의 활성으로 효모 세포에서 성공적으로 발현되었고, 그 중 SEY2102/pGMF-xylP 형질전환주를 이용해 baffled-flask 배양을 실시한 결과 최대 88.1 unit/ml의 endoxylanase 활성을 보임을 확인하였다. 대부분의 재조합 endoxylanase는 세포 외 분획에 효율적으로 분비 생산되었으며, <TEX>$MF{\alpha}_{opt}$</TEX> 분비서열이 XylP 유전자의 자체 분비서열보다 endoxylanase를 더 효율적으로 분비시킴을 확인할 수 있었다. 그러므로 본 연구에서 개발된 발현시스템은 효모를 숙주세포로 하여 많은 양의 세포 외 endoxylanase의 생산을 가능하게 하고, 바이오에탄올 생산 및 산업적 응용에도 유용하게 사용 될 수 있으리라 기대된다. The XylP gene, which encodes endoxylanase in Bacillus sp. HY-20, was subcloned, and two expression plasmids, pG-xylP and pGMF-xylP were constructed. These plasmids, which contain different signal sequences, XylP s.s and <TEX>$MF{\alpha}_{opt}$</TEX> s.s, respectively, for the secretory expression of endoxylanase, were transformed into Saccharomyces cerevisiae SEY2102 and FY833, respectively. The recombinant endoxylanases were successfully expressed, with a total activity range of 23.7-70.1 unit/ml according to the expression system and host strain. The endoxylanase activity in SEY2102/pGMF-xylP reached a maximum of 88.1 unit/ml in baffled flask culture. Most of the recombinant endoxylanase was efficiently secreted in the extracellular fraction, and the <TEX>$MF{\alpha}_{opt}$</TEX> s.s was more efficient for secreting endoxylanase in yeast than the XylP s.s. Therefore, the expression system developed in this study produces large extracellular amounts of endoxylanase using S. cerevisiae as the host strain, and it could be used in bioethanol production and industrial applications.

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.

Improvement of docosahexaenoic acid production on glycerol by Schizochytrium sp. S31 with constantly high oxygen transfer coefficient

Volumetric mass transfer coefficient (kLa) is a key fermentation parameter for the production of docosahexaenoic acid (DHA) from glycerol by Schizochytrium sp. S31. In order to elucidate the effects of kLa on the fermentations, both baffled and unbaffled flask cultures and fed-batch cultures were developed in present work. The results showed that high kLa could effectively increase the DHA concentration, DHA productivity and conversion yield (Yx/s, g/g). When kLa was set at 1802 ± 105 h(-1) in the fed-batch culture, DHA concentration was achieved at 28.93 g/L, DHA productivity at 301 mg/L/h and Yx/s at 0.44 ± 0.02 g/g, all of which were significantly higher than those in the previous similar studies.

Scale-up from shake flasks to bioreactor, based on power input and Streptomyces lividans morphology, for the production of recombinant APA (45/47 kDa protein) from Mycobacterium tuberculosis

Culture conditions in shake flasks affect filamentous Streptomyces lividans morphology, as well the productivity and O-mannosylation of recombinant Ala-Pro-rich O-glycoprotein (known as the 45/47 kDa or APA antigen) from Mycobacterium tuberculosis. In order to scale up from previous reported shake flasks to bioreactor, data from the literature on the effect of agitation on morphology of Streptomyces strains were used to obtain gassed volumetric power input values that can be used to obtain a morphology of S. lividans in bioreactor similar to the morphology previously reported in coiled/baffled shake flasks by our group. Morphology of S. lividans was successfully scaled-up, obtaining similar mycelial sizes in both scales with diameters of 0.21 ± 0.09 mm in baffled and coiled shake flasks, and 0.15 ± 0.01 mm in the bioreactor. Moreover, the specific growth rate was successfully scaled up (0.09 ± 0.02 and 0.12 ± 0.01 h(-1), for bioreactors and flasks, respectively), and the recombinant protein productivity measured by densitometry, as well. More interestingly, the quality of the recombinant glycoprotein measured as the amount of mannoses attached to the C-terminal of APA was also scaled- up; with up to five mannose residues in cultures carried out in shake flasks; and six in the bioreactor. However, final biomass concentration was not similar, indicating that although the process can be scaled-up using the power input, others factors like oxygen transfer rate, tip speed or energy dissipation/circulation function can be an influence on bacterial metabolism.

Cultivation conditions and the diffusion of oxygen into culture media: The rationale for the flask-to-medium ratio in microbiology

Bacterial cultivation requires consideration of three things: The bacterial strain, cultivation medium, and cultivation conditions. Most microbiologists dutifully report their choice of strains and cultivation media in manuscripts; however, these same microbiologists often overlook reporting cultivation conditions. Without this information, it is difficult to determine if cultures were grown aerobically, microaerobically, or anaerobically. To cultivate bacteria aerobically, it is necessary to understand that oxygen does not readily diffuse into culture media; it needs help to get in. Microbiologists can do this by altering the flask-to-medium ratio, rpm of agitation, and/or the concentration of atmospheric oxygen, or by using baffled flasks.

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

BackgroundFab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions.ResultsSmall-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume.ConclusionsYield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.

CFD analysis of the turbulent flow in baffled shake flasks

In this work, computational fluid dynamics (CFD) technique is used to simulate the complicated unsteady-state turbulent flow field formed in baffled flask. The baffled flask shows advantages both in mass transfer capacity and in shear formation in comparison with unbaffled flasks. Detailed investigations of power consumption, mass transfer and shear rate are carried out in baffled flasks under shaking frequencies ranging from 100rpm to 250rpm, and filling volumes from 50mL to 150mL. The results show that the specific power input and specific interface area are both greatly influenced by shaking frequency and filling volume. For the positive effect of shaking frequency on both mass transfer coefficient (kL) and specific interface area (a), the volumetric mass transfer coefficient (kLa) increases greatly with shaking frequency. Results also show that filling volume has no significant effect on kL but negative effect on specific interface area. Shear force formed in baffled flask shows great dependent on shaking frequency, but it is insensitive to the filling volume. Based on these investigations, correlations linking these parameters are proposed. Finally, cultivations of filamentous fungus conducted in unbaffled and baffled flasks validated the simulating results.

Highly accumulative production of l(+)-lactate from glucose by crystallization fermentation with immobilized Rhizopus oryzae

In order to produce microbiologically large amount of l(+)-lactic acid (LA) from glucose, batch and fed-batch (intermittent addition of sterilized glucose powder aseptically) cultures of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) whose mycelia were immobilized in situ within sponge-like cubic particles (3.5 mm edge long) were carried out at 37°C in a three baffled shake flask. Appropriately calculated amount of fine powdery calcium carbonate (CaCO(3)) was added initially or intermittently to control pH of the culture liquids. High accumulations of LA (145 g/L and 231 g/L, in reality 176 g/L and 280 g/L as anhydrous calcium lactate) were achieved by a batch (glucose concentration = 150 g/L) and a fed-batch cultures (the initial glucose concentration = 150 g/L and the intermittent addition of glucose equivalent to 100 g/L). In these cultures the yields and productivities of LA were, 95.0%, 1.42 g/L·h and 92.5%, 1.83 g/L·h, respectively. Existence of considerable amounts of calcium lactate (Ca(LA)(2)) as crystals in the culture broth was experimentally proved by two evidences: (i) heating up (70°C) followed by quick low centrifugal force to remove remaining CaCO(3) solids from culture broth and then cooling down (37°C) followed by incubation of the culture supernatant at 37°C to observe recrystallization of Ca(LA)(2), and (ii) the measurement of solubility of Ca(LA)(2) in the culture media. It was conceptually discussed to be able to avoid the product inhibition by means of crystallization fermentation for the high accumulation of LA by R. oryzae.