Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics

<p>Abstract</p> <p>Background</p> <p>There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na₂CO₃. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms.</p> <p>Results</p> <p>The controlled-release discs presented here were successfully used to cultivate <it>E. coli </it>K12 and <it>E. coli </it>BL21 pRSET eYFP-IL6 in mineral media with glucose and glycerol as carbon (C) sources, respectively. With glucose as the C-source it was possible to reduce the required buffer concentration in shake flask cultures by 50%. Moreover, with glycerol as the C-source, no buffer was needed at all.</p> <p>Conclusions</p> <p>These novel polymer-based controlled-release discs allowed buffer concentrations in shake flask media to be substantially reduced or omitted, while the pH remains in the physiological range of the microorganisms during the whole cultivation time. Therefore, the controlled-release discs allow a better control of the pH, than merely using high buffer concentrations. The conditions applied here, i.e. with significantly reduced buffer concentrations, enhance the comparability of the culture conditions used in screening experiments and large-scale fermentation processes.</p

Magnetron deposition of TCO films using ion beam

Thin films of tin oxide (TO) were deposited on the glass substrates at room temperature using reactive magnetron sputtering at various oxygen partial pressures. After the deposition the films were irradiated with argon ions beam. The change of the optical and electrical properties of the films depending on the irradiation time was studied. Films optical properties in the range of 300-1100 nm were investigated by photometry as well as their structural properties were studied using X-ray diffraction. Diffractometric research showed that the films, deposited on a substrate, have a crystal structure, and after argon ions irradiation they become quasi-crystalline (amorphous). It was found that the transmission increases proportionally with the irradiation time, but the surface resistance -disproportionally

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions

Combination of On-line pH and Oxygen Transfer Rate Measurement in Shake Flasks by Fiber Optical Technique and Respiration Activity MOnitoring System (RAMOS)

Shake flasks are commonly used for process development in biotechnologyindustry. For this purpose a lot of information is required from the growth conditions duringthe fermentation experiments. Therefore, Anderlei et al. developed the RAMOS technology[1, 2], which proviedes on-line oxygen and carbondioxide transfer rates in shake flasks.Besides oxygen consumption, the pH in the medium also plays an important role for thesuccessful cultivation of micro-organisms and for process development. For online pHmeasurement fiber optical methods based on fluorophores are available. Here a combinationof the on-line Oxygen Transfer Rate (OTR) measurements in the RAMOS device with anon-line, fiber optical pH measurement is presented. To demonstrate the application of thecombined measurement techniques, Escherichia coli cultivations were performed and on-line pH measurements were compared with off-line samples. The combination of on-lineOTR and pH measurements gives a lot of information about the cultivation and, therefore, itis a powerful technique for monitoring shake flask experiments as well as for processdevelopment

Optimizing microbial screenings using controlled-release systems

Microbial screening experiments are of utmost importance for developing biotechnological processes. The cultivation parameters for selecting the most suited microorganisms during these screening experiments should match the parameters for the subsequent production process as exact as possible. It is absolutely necessary, to apply comparable cultivation conditions in small-scale screenings and large-scale production processes, to ensure a meaningful analysis of the screening experiments as well as a successful scale-up of its results. In the presented work, different facets of screening processes were analyzed and solutions for their optimization regarding the aforementioned general principles were investigated. One key factor to improve the comparability of screening and production-scale experiments is to establish an online monitoring of cultivation parameters not only in large- but also in small-scale cultivations. To enhance the online information obtained during screening and process development in shake flasks, the RAMOS device for measuring respiration activities in shake flaks and a fiber optical, online pH-measurement technique were successfully combined. To further improve the comparability between the different scales and to enable a more reliable scale-up of experiments, the cultivation strategy (i.e. the progression of pH-value and substrate concentration during the cultivation) has to be comparable in small- and in large-scales. Thus, the in large-scale applied pH-control and fed-batch operational mode have to be adapted to small scale screening experiments. A disc-shaped polymer-based controlled-release system for pH-control in shake flasks was developed and applied in this thesis. It consists of a polymer matrix in which sodium carbonate as pH-control reagent is encased. When applied in cultivation media, this system releases sodium carbonate in pre-defined kinetics. With this system, it was possible to substantially reduce the buffer concentrations in shake flask cultivations of Escherichia coli, while the pH-values remained in the physiological range of microbial growth. An additional physiological effect of the pH-value is its influence on the growth behavior of the microorganisms and thereby especially on the duration of the lag-phase. Different lag times of the microorganisms considerably affect the outcome of screening processes. In this work it could be shown that the initial pH-value of the cultivation media has an enormous strain dependent effect on the lag time of E. coli cultures. For three E. coli strains a lower initial pH-value resulted in a shorter lag phase and one strain showed the opposite behavior. This parameter should be considered in the design of production processes as well as of screening experiments. Another analyzed facet of the screening process was the microbial growth in precultures. It could be demonstrated, that differences in the inoculum from precultures in shaken bioreactors have a tremendous effect on the microbial growth and thus on rational design of screening processes. Therefore, a new technique applying fed-batch mode in high-throughput precultivations for equalizing the initial parameters of subsequent screening experiments was introduced. For fed-batch cultivation in shake flasks, glucose containing polymer-based controlled-release discs were applied. For high-throughput applications a new fed-batch microtiter plate, with immobilized polymer-based controlled-release systems at the bottom of each well were presented. The newly developed fed-batch precultivation method enables equalized growth of all screened strains and will generate, therefore, more relevant and reliable data in subsequent main screening experiments. The feasibility of the presented concept has been proven for cultivations of E. coli and Hansenula polymorpha. All these results demonstrate the importance of choosing the correct cultivation parameters for a successful microbial screening. Especially the control of the pH-value and the controlled-release of substrate are important for several aspects of screening experiments. The systems and methodologies described in the current work significantly improve screening procedures and the meaningful analysis and scale-up of the obtained results

St. John's Daily Star, 1921-03-10

The St. John's Daily Star was published daily except Sunday between 17 April 1915 - 23 July 1921