A simple and inexpensive method for investigating microbiological, enzymatic, or inorganic catalysis using standard histology and microbiology laboratory equipment: assembly, mass transfer properties, hydrodynamic conditions and evaluation

We introduce a generic, simple, and inexpensive method for performing microbiological, enzymatic, or inorganic catalysis with solids using standard histology and microbiology laboratory equipment. Histology cassettes were used to standardize hydrodynamic conditions and to protect the catalysts and their solid supports. Histology cassettes have the following advantages: they are readily available, inexpensive, solvent and acid resistant, automatable, and the slots in the cassette walls allow liquid to circulate freely. Standard Erlenmeyer flasks were used as reaction vessels. We developed a new camera to observe the movement and position of the histology cassettes as well as the liquid in the Erlenmeyer flasks. The camera produces a stable image of the rotating liquid in the Erlenmeyer flask. This visualization method revealed that in a 250 ml Erlenmeyer flask, stable operating conditions are achieved at a shaking frequency of 300 rpm and a fill volume of 30 ml. In vessels with vertical walls, such as beakers or laboratory bottles, the movement of the histology cassette is not reproducible. Mass transfer characterization using a biological model system and the chemical sulfite-oxidation method revealed that the histology cassette does not influence gas-liquid mass transfer.     

A simple and inexpensive method for investigating microbiological, enzymatic, or inorganic catalysis using standard histology and microbiology laboratory equipment: assembly, mass transfer properties, hydrodynamic conditions and evaluation.

We introduce a generic, simple, and inexpensive method for performing microbiological, enzymatic, or inorganic catalysis with solids using standard histology and microbiology laboratory equipment. Histology cassettes were used to standardize hydrodynamic conditions and to protect the catalysts and their solid supports. Histology cassettes have the following advantages: they are readily available, inexpensive, solvent and acid resistant, automatable, and the slots in the cassette walls allow liquid to circulate freely. Standard Erlenmeyer flasks were used as reaction vessels. We developed a new camera to observe the movement and position of the histology cassettes as well as the liquid in the Erlenmeyer flasks. The camera produces a stable image of the rotating liquid in the Erlenmeyer flask. This visualization method revealed that in a 250 ml Erlenmeyer flask, stable operating conditions are achieved at a shaking frequency of 300 rpm and a fill volume of 30 ml. In vessels with vertical walls, such as beakers or laboratory bottles, the movement of the histology cassette is not reproducible. Mass transfer characterization using a biological model system and the chemical sulfite-oxidation method revealed that the histology cassette does not influence gas-liquid mass transfer.     

装液量和接种量对红发夫酵母生长和虾青素积累的影响

在摇瓶中研究了不同接种量和装液量对红发夫酵母PhaffiarhodozymaAs2 .15 5 7生长及虾青素积累的影响。结果表明在 5 0 0ml摇瓶中装液量为 5 0ml时对红发夫酵母生长及虾青素合成有利 ,接种量为 10 %有利于菌体生长和虾青素合成。     

Suspension culture of drosophila cells employing a gyratory shaker

Summary To develop a method for culturing a large number of small-scale suspension cultures ofDrosophila melanogaster cells simultaneously, basic conditions were studied using a cell line GM₂ and a gyratory shaker. Under gyration at more than 180 rpm, a majority (>80%) of the cells still remained as suspension and grew normally. Lower speed of gyration caused adhesion of the cells to a substratum. Furthermore, size of the culture vessels was found to affect the pattern of cell growth. Five- or 10-ml Erlenmeyer flasks gave satisfactory results, but the growth curves in 30-ml flasks differed from flask to flask and the saturation level was lower. Besides, the growth curves in the latter case were quite different depending on the volume of the medium. A preliminary experiment showed that the type of flask might affect the pattern of a growth curve. Initial cell densities has to be more than 6×10⁴ cells per ml. Lower densities resulted in the longer doubling time or no increase in the cell number. Therefore the following conditions are recommended as a standard for gyration culture ofD. melanogaster cell, GM₂: speed of gyration, 180 rpm; culture vessel, 5- or 10-ml Erlenmeyer flask of a certain type; initial cell density, 1 to 5×10⁵ per ml. Both D20 and modified Schneider’s medium could be utilized as the medium.     

Real-time monitoring of shake flask fermentation and off gas using triple disposable noninvasive optical sensors

Bioprocess development is a data-driven process requiring a large number of experiments to be conducted under varying conditions. Small-scale upstream bioprocess development is often performed in shake flasks because they are inexpensive and can be operated in parallel. However, shake flasks are often not equipped to accurately monitor critical process parameters such as pH, dissolved oxygen, and CO2 concentrations. Therefore, there is no definitive information on oxygen supply of growing cells, CO2 formation, and pH changes. Here we describe several shake flask fermentations where all three parameters are monitored by disposable noninvasive optical sensors. The sensitive element of these sensors is a thin, luminescent patch affixed inside the flask. Small electronic devices for excitation and fluorescence detection are positioned outside the shake flask for noninvasive monitoring. By measuring the process parameters throughout the course of the E. coli fermentations, we obtain information that is not routinely available in shake flask fermentations. For example, for cultures with only a few millimeters liquid depth, oxygen limitation can occur at relatively low agitation speeds. Under certain conditions oscillations in dissolved oxygen can occur. An increase in shaker speed and a decrease in culture volume can increase the oxygen availability and reduce the duration of oxygen limitation.     

Reassessing the out-of-phase phenomenon in shake flasks by evaluating the angle-dependent liquid distribution relative to the direction of the centrifugal acceleration

Shake flasks are still the most relevant experimental tool in the development of viscous fermentation processes. The phase number, which defines the onset of the unfavorable out-of-phase (OP) phenomenon in shake flasks, was previously defined via specific power input measurements. In the OP state, the bulk liquid no longer follows the orbital movement of the imposed centrifugal force, which is for example, detrimental to oxygen transfer. In this study, an optical fluorescence technique was used to measure the three-dimensional liquid distribution in shake flasks. Four new optically derived evaluation criteria for the phase transition between the in-phase and OP condition were established: (a) thickness of the liquid film left on the glass wall by the rotating bulk liquid, (b) relative slope of the leading edge of bulk liquid (LB) lines, (c) trend of the angular position of LB, and (d) very high angular position of the leading edge. In contrast to the previously applied power input measurements, the new optical evaluation criteria describe the phase transition in greater detailed. Instead of Ph = 1.26, a less conservative value of Ph = 0.91 is now suggested for the phase transfer, which implies a broader operating window for shake flask cultivations with higher viscosities.     

Noninvasive oxygen measurements and mass transfer considerations in tissue culture flasks

Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were monitored. Oxygen levels were measured noninvasively by interrogating an oxygen-sensitive patch mounted on the interior surface of the tissue culture flask with an optrode from outside the tissue culture flask. Readings were made in tissue culture flasks with caps both cracked open and completely closed. Although the oxygen in the gas phase remained near atmospheric oxygen levels in both flasks, over time the liquid-phase oxygen tension at the bottom of the flasks reached zero during cell growth in both the open and closed tissue culture flasks. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and probably unnecessary.The mass transfer characteristics of the tissue culture flask were also studied. The dominant resistance to oxygen mass transfer to the sensor and the cells was through the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone. This suggests that mixing at a microscale occurs. Volumetric and specific oxygen consumption rates were also calculated from the sensor data. These consumption rates were comparable with values published elsewhere. (c) 1996 John Wiley & Sons, Inc.     

Combined dissolved oxygen tension and online viscosity measurements in shake flask cultivations via infrared fluorescent oxygen-sensitive nanoparticles

Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the dissolved oxygen tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric oxygen transfer coefficient (k_La) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Characterisation of the gas-liquid mass transfer in shaking bioreactors

The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium. The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask. This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters. The model agrees well with the experimental results. It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR).     

Influence of size of culture vessel on in vitro proliferation of grape in a liquid medium

The in vitro proliferation of Vitis vinifera L. cv. Liemberger in a liquid medium was compared in 125 and 250 ml Erlenmeyer flasks and in 473 ml (pint) Mason jars. After 6 weeks of culture the jars yielded a significantly greater number of shoots 3 mm or longer than the flasks. Jars yielded the greatest number of shoots 7 mm or longer, followed by 250 ml, then 125 ml flasks. The mean length of shoots in the 250 ml flasks was significantly greater than that of shoots in 125 ml flasks. The final mean fresh weight of the cultures in jars was significantly less than that of the cultures in flasks. Thus the size of vessel used influenced the in vitro proliferation of grapevines in liquid culture.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Determination of CO₂ sensitivity of micro-organisms in shaken bioreactors. I. Novel method based on the resistance of sterile closure

Influence of carbon dioxide on growth and product kinetics of industrially important micro-organisms is essential for the interpretation of a bioprocess. In this research, the CO? effects on productivity and growth rate of micro-organisms have been studied by using a variety of kplug. The applied method is based on a different concentration of CO? in the headspace of ventilation flasks. The presented method is simple, inexpensive and shows similar results compared to large-scale fermentation regarding the evolution of CO? in a batch system. For the investigation of the proposed method, experiments employing Arxula adeninivorans LS3, Corynebacterium glutamicum (DM1730 and ATCC WT13032) and Hansenula polymorpha DSM70277 as model organisms in the ventilation flasks are performed. The fermentations in the RAMOS (respiratory activity monitoring system) device were carried out with a normal aeration rate (1 vvm) and under the same operating conditions as the ventilation flask f1. The modified unsteady-state model was used to predict the operation conditions of a biological system in the ventilation flasks. In the present study, a novel and easy method for the quantification of CO? sensitivity of micro-organisms in shaken bioreactors (called ventilation flask) was achieved.     

Improved shake-flask test for the screening of xanthan-producing microorganisms

Baffled 500 ml Erlenmeyer flasks were compared with conventional 2800 ml Fernbach flasks forXanthomonas campestris to produce xanthan. Bacterial growth rates were similar in both types of flask although the Fernbach flasks gave higher biomass concentrations. Xanthan production was similar in both types of flasks but different viscosities were attained. On a weight basis, the xanthan produced in baffled flasks was up to three times more viscous and more pseudoplastic or shear thinning. For screening purposes, baffled flasks are better because the rheological quality of the gum produced in them is more like that obtained in stirred fermentors than the gum from Fernbach flasks and considerably less shaker space is required, thus allowing a larger number of tests to be performed.     

Use of immobilized Candida cells on xylitol production from sugarcane bagasse

In this study we used the yeast Candida guilliermondii FTI 20037 immobilized by entrapment in Ca-alginate beads (2.5-3 mm diameter) for xylitol production from concentrated sugarcane bagasse hemicellulosic hydrolysate in a repeated batch system. The fermentation runs were carried out in 125- and 250-ml Erlenmeyer flasks placed in an orbital shaker at 30 degrees C and 200 rpm during 72 h, keeping constant the proportion between work volume and flask total volume. According to the results, cell viability was substantially high (98%) in all fermentative cycles. The values of parameters xylitol yield and volumetric productivity increased significantly with the reutilization of the immobilized biocatalysts. The highest values of xylitol final concentration (11.05 g/l), yield factor (0.47 g/g) and volumetric productivity (0.22 g/lh) were obtained in 250-ml Erlenmeyer flasks containing 80 ml of medium plus 20 ml of immobilized biocatalysts. The support used in this study (Ca-alginate) presented stability in the experimental conditions used. The results show that the use of immobilized cells is a promising approach for increasing the xylitol production rates.     

Stable and strictly controlled expression of LTR-flanked autoregulated expression cassettes upon adenoviral transfer

An autoregulatory bidirectional expression cassette encoding all components necessary for regulated gene expression in a one-step gene transfer was evaluated for use in adenoviral vectors. Adenoviral vectors transducing this cassette provide about 1000-fold regulation. Regulation could be further improved by integrating the cassette as a retroviral vector into the adenoviral backbone. Moreover, with these adeno/retroviral hybrid vectors, the frequency of chromosomal integration is enhanced and about 1% of infected cells show stable chromosomal integration of the autoregulated cassette. In these stably transduced cells high regulation capacity is maintained. To elucidate the molecular mechanism underlying this unexpected observation we investigated the regulation capacity of these cassettes in a viral and non-viral vector background after stable integration into the host's DNA. While naked cassettes show regulated expression that is strongly influenced by the chromosomal surrounding sequences the regulatory capacity of LTR flanked cassettes is highly comparable amongst different cell clones. This strict regulation with little influence from the flanking sequences is obtained when LTR-flanked cassettes are transduced as DNA, by retroviral or by adenoviral infection.     

New Methods for Cultivating,Harvesting, and Purifying Mass Cultures of the Hypotrich Ciliate Euplotes aediculatus1

A new method is described for mass cultivation of Euplotes aediculatus, a hypotrich ciliate containing omikron-symbionts. The ciliate cultures, continuously aerated in Erlenmeyer flasks (5000 ml) with 4500 ml medium, yield densities of 2300 cells/ml which are four to five times higher than cell densities of cultures grown in unaerated Fernbach flasks. Harvesting such cultures involves the application of 25-μm mesh sieves. Cells so concentrated can be purified by using columns or special chambers in which Euplotes migrates towards the cathodes in an electric field (field strength 7 V/cm).     

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k_La value of the Tubespin at standard conditions was 24.3 h⁻¹, while that of a spinner flask was only 2.7 h⁻¹. The maximum cell density in the Tubespin bioreactor reached 6 × 10⁶ cells mL⁻¹, which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.     

Noninvasive online biomass detector system for cultivation in shake flasks

A novel online sensor system for noninvasive and continuous monitoring of cell growth in shake flasks is described. The measurement principle is based on turbidity measurement by detecting 180°‐scattered light and correlation to OD by nonlinear calibration models. The sensor system was integrated into a commercial shaking tablar to read out turbidity from below the shake flasks bottom. The system was evaluated with two model microorganisms, Escherichia coli K12 as prokaryotic and Saccharomyces cerevisiae as eukaryotic model. The sensor allowed an accurate monitoring of turbidity and correlation with OD₆₀₀ ≤ 30. The determination of online OD showed relative errors of about 7.5% for E. coli K12 and 12% for S. cerevisiae. This matches the errors of the laborious offline OD and thus facilitates to overcome the drawbacks of the classical method as risk of contamination and decreasing volumes through sampling. One major challenge was to ensure a defined, nonvarying measurement zone as the rotating suspension in the shake flask forms a liquid sickle which circulates round the flasks inner bottom wall. The resulting alteration of liquid height above the sensor could be compensated by integration of an acceleration sensor into the tablar to synchronize the sensor triggering.     

胀果甘草悬浮培养细胞合成甘草总黄酮

比较了胀果甘草(Glycyrrhiza inflata)悬浮细胞在逐级放大摇瓶中的生长、黄酮产量以及营养消耗过程,以便了解其放大规律。结果表明,在250和500mL摇瓶中,细胞的最大生物量、黄酮产量以及最大比生长速率没有显著性差异,但是在1L的摇瓶中,这三种参数都显著地降低,分别比250mL摇瓶中降低了27%,30%和27%。在逐级放大的摇瓶中,氮、磷、铵浓度都随着培养时间延长而逐渐降低,尽管在1L的摇瓶中磷消耗得最慢,但三种摇瓶中磷在细胞生长对数期基本都被消耗尽了。此外,硝态氮在第18天时基本被消耗完,而铵态氮在细胞收获时仍能维持在100mg/L。因此在反应器中培养时,主要的培养条件还需进一步优化。