Characterization of antibacterial polyethersulfone membranes using the Respiration Activity Monitoring System (RAMOS).

Membranes with antibacterial properties were developed using surface modification of polyethersulfone ultrafiltration membranes. Three different modification strategies using polyelectrolyte layer-by-layer (LbL) technique are described. The first strategy relying on the intrinsic antibacterial properties of poly(diallyldimethylammonium chloride) (PDADMAC) and poly(ethylenimine) (PEI) exhibits only little antibacterial effects. The other two strategies contain silver in both ionic (Ag(+)) and metallic (Ag(0)) form. Ag(+) embedded into negatively charged poly(sodium 4-styrene sulfonate) (PSS) layers totally inhibits bacterial growth. Ag(0) nanoparticles were introduced to the membrane surface by LbL deposition of chitosan- and poly(methacrylic acid) - sodium salt (PMA)-capped silver nanoparticles and subsequent UV or heat treatment. Antibacterial properties of the modified membranes were quantified by a new method based on the Respiration Activity Monitoring System (RAMOS), whereby the oxygen transfer rates (OTR) of E. coli K12 cultures on the membranes were monitored online. As opposed to colony forming counting method RAMOS yields more quantitative and reliable data on the antibacterial effect of membrane modification. Ag-imprinted polyelectrolyte film composed of chitosan (Ag(0))/PMA(Ag(0))/chitosan(Ag(0)) was found to be the most promising among the tested membranes. Further investigation revealed that the concentration and equal distribution of silver in the membrane surface plays an important role in bacterial growth inhibition.

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

BACKGROUND: 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. RESULTS: The controlled-release discs presented here were successfully used to cultivate E. coli K12 and E. coli 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. CONCLUSIONS: 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.

High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale.

Most large-scale production processes in biotechnology are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode, which results in the microorganisms being subjected to different physiological conditions. This significantly affects strain selection. To demonstrate differences in ranking during strain selection depending on the operational mode, screenings were performed in batch and fed-batch modes. Two model populations of the methylotrophic yeast Hansenula polymorpha RB11 with vector pC10-FMD (P(FMD)-GFP) (220 clones) and vector pC10-MOX (P(MOX)-GFP) (224 clones) were applied. For fed-batch cultivations in deep-well microtiter plates, a controlled-release system made of silicone elastomer discs containing glucose was used. Three experimental set-ups were investigated: batch cultivation with (1) glucose as a substrate, which catabolite represses product formation, and (2) glycerol as a carbon source, which is partially repressing, respectively, and (3) fed-batch cultivation with glucose as a limiting substrate using the controlled-release system. These three experimental set-ups showed significant variations in green fluorescent protein (GFP) yield. Interestingly, screenings in fed-batch mode with glucose as a substrate resulted in the selection of yeast strains different from those cultivated in batch mode with glycerol or glucose. Ultimately, fed-batch screening is considerably better than screening in batch mode for fed-batch production processes with glucose as a carbon source.

Equalizing growth in high-throughput small scale cultivations via precultures operated in fed-batch mode.

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non-parallel and non-equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag-phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed-batch mode in precultures in microtiter plates and shake flasks. Fed-batch operation in precultures is realized through a slow-release system for glucose. After differently growing cultures turn to glucose-limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow-release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high-throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha.

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.