Measuring kLa with randomly pulsed dynamic method

This reports on the determination of the overall oxygen transfer coefficient in a mechanically agitated vessel using a randomly pulsed dynamic method. This method consists in exciting the system by randomly switching the inlet gas stream with air or nitrogen with an identical volumetric flow rate. A pseudo-random binary sequence was used. This procedure is routinely used in process control for the identification of system's transfer function. The pulsed dynamic method gives good reliability (as compared with the traditional gassing-out method) and reproducibility in water. However, further improvement is needed before it can be used to monitor on-line the k(L)a during a fermentation.     

Enhanced in situ dynamic method for measuring KLa in fermentation media

The overall oxygen mass transfer coefficient (K_La) is often used as scale-up factor of fermentation systems. In fermenter scale-up, it is desired to achieve the same K_La values at the larger scale than the one that was obtained at a smaller scale during the development stage. It is therefore important to be able to measure K_La in situ during fermentation and to also determine the action to be taken to maintain its value at its design set point. These objectives can be obtained by measuring K_La using the dynamic method and enhancing the K_La information by immediately conducting a series of changes in agitation speed and/or aeration rate to determine the influence of these variables on K_La. This enhanced dynamic method is demonstrated with two filamentous microorganisms: Trichoderma reesei for the production of cellulase and Aspergillus niger for the production of citric acid. Two different types of bioreactor were used: a reciprocating plate bioreactor and a stirred (Rushton) bioreactor. It is shown that the proposed method can provide a simple way to measure the local variation of K_La and to adjust its value to its set point during the course of fermentation.     

Winter anoxic layer in Lake Hibara

The concentration of dissolved oxygen in waters 0.5–0.6 m above the bottom of Lake Hibara, a dimictic lake, was zero in early spring of 1994 and 1997. The concentrations in early spring of 1992, 1993, 1995, 1996, and 1998 ranged from 3.75 to 10.1 mg l⁻¹. The depth profiles of water temperature suggest that water had not circulated prior to the sample collections of 1994 and 1997, but it had done so in the cases of the other years, suggesting that winter conditions were well preserved in the former years. On the other hand, the dissolved oxygen in the same strata decreased severely in summer. However, more or less titratable amounts of dissolved oxygen still remained (0.01–0.73 mg l⁻¹) at the final stages of summer stratification from 1992 to 1998. These facts show that a completely anoxic condition is not formed in this lake in summer but is sometimes formed in winter. It is interesting to note that in spite of unfavorable winter conditions for oxygen consumption, i.e., shorter duration and lower water temperature, oxygen is exhausted. These facts suggest that ventilation to the depths is much greater in summer than in winter. Received: March 5, 1999 / Accepted: October 18, 1999     

Online monitoring of oxygen in spinner flasks

We show the application of a novel optical on-line sensor fixed in spinner flasks for the online monitoring of dissolved O₂ concentrations during mammalian cell growth. Using this sensor that requires only minute changes to the flask to be made, we could determine the volumetric O₂ transfer coefficient as well as O₂ consumption rates. Under normal growth conditions the cells did not undergo O₂ limitation. Also, the transfer of O₂ from the atmosphere to the spinner flasks is influenced by the use of screw caps. The on-line measurement was further applied to determine the O₂ uptake rates which can then be used to monitor the metabolic state of the cells and also for online process monitoring.     

以摇瓶所得摄氧率为基准进行发酵放大

通过设计特殊摇瓶,用亚硫酸盐法测出摇瓶口纱布层氧通透率的基础上,在实际发酵情况下通过测定瓶内气、液相氧的变化得出其发酵过程中的摄氧率(OUR)及氧传递系数(K_La)。以特制摇瓶取得的菌体CUR为基准进行发酵过程和发酵罐的放大。通过质谱仪在线检测及采样分析,研究了3种不同供气流量及搅拌转速下的放大结果。摇瓶与发酵罐在菌体OUR、菌体产量方面吻合很好,而在整个放大过程中,发现摇瓶与发酵罐内的氧传递系数(K_La)、溶解氧(C_L)差异较大。     

A device for simultaneously controlling multiple treatment levels of dissolved oxygen in laboratory experiments

Hypoxia is common in freshwater, estuarine, and shallow or enclosed marine systems. Research on the sub-lethal effects of hypoxia on feeding and growth includes both field and laboratory studies. Extended regulation of dissolved oxygen (DO) is difficult to precisely control in the laboratory. Here we describe a device that is capable of simultaneously monitoring and controlling DO at multiple treatment levels, in replicate experimental tanks, for indefinite periods of time. Each treatment level may be static or fluctuate on a diel cycle. The device consists of a series of independent aquarium systems, each of which is monitored and adjusted by a computer program. A data file and chart of DO levels in each treatment level is recorded. A computer program that repeatedly measures and adjusts DO controls the device. Because this apparatus is software-controlled, the user has the flexibility of altering treatment parameters at any time without interrupting experiments.     

Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates

A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing.     

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.     

摇瓶的体积氧传递系数和氧通透率的测定

通过设计特殊摇瓶,用亚硫酸钠法测出摇瓶的体积氧传递系数和氧透过纱布层的通透率。以氧电极测其内外氧的分压降后,可以算出摇瓶表观体积氧传递系数(k_La)app及真实体积氧传递系数k_La,并进一步求出氧通透率。由实验得出：氧分压降低6．1％,氧传递系数增加一倍;在37c、220r／min、500m1摇瓶(内盛液50m1)8层纱布的氧通透率Pm=43．7moI／m²·h·arm;并且关联出摇瓶容积V、装液量VL、转速n、摄氏温度t之间的模型式：(k_La)_app=1．84×10^-7[t]1.8479·[n]2.3906.(VLV]-0.6360(kLa)=2．02×10^-7[t]1.8525·[n]2.39441·[VLV]-0.6370     

Diurnal O2 and carbohydrate levels in wheat kernels during embryony

In vitro zygotic and somatic embryogenesis procedures for wheat have been improved by simulating in ovulo nutritional, hormonal and dissolved oxygen (dO₂) conditions. However, diurnal fluctuations in these conditions during early embryony are not well characterized. In this study, dO₂ and water-soluble carbohydrate levels in wheat kernels were determined after 8 h of light and 8 h of dark at approximately 6, 12 and 18 day post anthesis (DPA). Clark style O₂ microelectrodes, having a tip diameter of approximately 115 μm, were inserted into intact kernels immediately distil to the developing embryo, and dO₂ levels were recorded at 50 μm intervals into the center of kernels. High-performance anion exchange chromatography with pulsed amperometric detection was used to quantify carbohydrate levels in endosperm sap. dO₂ levels in the chlorophyllous layer of the pericarp reached 190 mmol m⁻³ during the day, which probably represents, because of photosynthesis, a supersaturated O₂ condition relative to the external environment (21% O₂). At the embryo surface, dO₂ levels at 6 DPA ranged from 135 to 170 mmol m⁻³. At 12 and 18 DPA, dO₂ levels at the embryo axis ranged from 100 to 150 mmol m⁻³. At all three stages, dO₂ levels in the center of the endosperm were below 13 mmol m⁻³. Extreme fluctuations in carbohydrate levels were observed diurnally during rapid seed fill (12 DPA). Levels of sucrose and short-chain fructans were much higher during the day than during the night. In contrast, fructose, glucose, and myo-inositol levels were much higher during the night than during the day. By 18 DPA (hard dough stage), carbohydrate levels tended to be similar during the day and night. These dynamic fluctuations may assist in regulating embryony in ovulo, and their simulation might improve the development of somatic and zygotic embryos in vitro.     

Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures

Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol l(-1), which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid l(-1), reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply.