Interaction of levitated ionic liquid droplets with water.

The influence of a humid or dry atmosphere on acoustically levitated ionic liquid droplets was studied by volumetric analysis and vibrational spectroscopy. Imidazolium-based ionic liquids with two types of anions, fluorinated (BF(4) and PF(6)) and alkylsulfate anions, were investigated. The amount of absorbed water was correlated with structural differences of the ionic liquids and analyzed in terms of equilibrium mole fraction as well as absorption rate. The type of anion had the most significant influence on the amount of adsorbed water from the atmosphere. Furthermore, spectral changes in the in situ Raman spectra due to absorbed water were studied for all investigated ionic liquids. For 1-ethyl-3-methylimidazolium ethylsulfate, an exemplary detailed analysis of the intermolecular interactions between cations, anions and water was carried out based on the spectroscopic data. The observed band shifts were explained with a hydrogen bond between the anion and water.

On-line monitoring of nine different batch cultures of E. coli by mid-infrared spectroscopy, using a single spectra library for calibration.

A single spectra library was used to monitor on-line, by mid-infrared spectroscopy, nine different batch cultures of Escherichia coli performed with various medium compositions, including chemically complex formulations. Whereas the classic chemometrics approach would have required the preparation and measurement of hundreds of standards, only six spectra were included in the library. These included the molar absorbance of the four main metabolites (i.e. glucose, glycerol, ammonium and acetate), and the remaining two were drift spectra found by factor analysis. The accuracy of the prediction was not altered by a change of the carbon source, the ammonium concentration or even the addition of chemically undefined compounds, such as yeast extract and peptone. The standard errors of prediction averaged over the nine experiments were 8.0, 12.3, 5.9 and 5.6 mM for glucose, glycerol, ammonium and acetate, respectively. Inclusion of two drift spectra in the library provided an estimation of how noisy an experiment was. This also allowed detection of batch cultures that require further investigation, namely runs which were subject to large signal drift or during which an unexpected compound was produced, without having to carry out time-consuming off-line analyses.

Influence of specific growth rate on specific productivity and glycosylation of a recombinant avidin produced by a Pichia pastoris Mut+ strain.

A recombinant avidin-producing Mut+ Pichia pastoris strain was used as a model organism to study the influence of the methanol feeding strategy on the specific product productivity (q(p)) and protein glycosylation. Fed-batch cultivations performed at various specific growth rates (micro) and residual methanol concentrations showed that the specific avidin productivity is growth-dependent. The specific productivity increases strongly with the specific growth rate for micro ranging from 0 to 0.02 h(-1), and increases only slightly with the specific growth rate above this limit. N-terminal glycosylation was also found to be influenced by the specific growth rate, since 9-mannose glycans were the most abundant form at low growth rates, whereas 10-mannose carbohydrate chains were favored at higher micro. These results show that culture parameters, such as the specific growth rate, may significantly affect the activity of glycoproteins produced in Pichia pastoris. In terms of process optimization, this suggests that a compromise on the specific growth rate may have to be found, in certain cases, to work with an acceptable productivity while avoiding the addition of many mannoses.

pH prediction and control in bioprocesses using mid-infrared spectroscopy.

An on-line pH monitoring method based on mid-infrared spectroscopy relevant to bioprocesses is presented. This approach is non-invasive and does not require the addition of indicators or dyes, since it relies on the analysis of species of common buffers used in culture media, such as phosphate buffer. Starting with titrations of phosphoric and acetic acid solutions over almost the entire pH range (2-12), it was shown that the infrared spectra of all samples can be expressed as a linear combination of the molar absorbance of the acids and their deprotonated forms. In other words, pH had no direct influence on the molar infrared spectra themselves, but only on deprotonation equilibria. Accurate prediction (standard error of prediction for pH < 0.15 pH units) was achieved by taking into account the non-ideal behavior of the solutions, using the Debye-Hückel theory to estimate the activity coefficients. Batch cultures of E. coli were chosen as a case study to show how this approach can be applied to bioprocess monitoring. The discrepancy between the spectroscopic prediction and the conventional electrochemical probe never exceeded 0.12 pH units, and the technique was fast enough to implement a feedback controller to maintain the pH constant during cultivation.

A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris.

The advantages of mixed feeds of sorbitol and methanol for the production of recombinant proteins with Pichia pastoris were analyzed quantitatively. The influence of the methanol-sorbitol ratio in the feed medium was investigated on growth stoichiometry and recombinant protein productivity with a P. pastoris Mut(+) strain secreting avidin by performing a transient nutrient gradient in continuous cultivation at a dilution rate of 0.03h(-1). Results showed that mixed feeds of sorbitol and methanol instead of methanol as sole carbon source can improve the productivity in recombinant avidin due to increased biomass yields during mixed substrate growth. The highest volumetric avidin productivity was achieved at a methanol fraction of 43%C-molC-mol(-1) in the feed medium: the volumetric avidin productivity was 1.3-fold higher than during chemostat culture on methanol. The heat production and the oxygen consumption rates were reduced by about 38% for a given dry cell weight concentration at this methanol fraction, features which are very useful for high cell density cultures. Results were in good agreement with a high cell density fed-batch culture performed with a mixed feed of 43% methanol and 57% sorbitol C-molC-mol(-1) at a specific growth rate of 0.03h(-1) during the induction phase. Moreover, it was confirmed that sorbitol accumulation in the culture medium does not affect recombinant avidin productivity, which can especially be advantageous during large-scale cultures where transient substrate accumulation can result from imperfect mixing.

Simplified Fourier-transform mid-infrared spectroscopy calibration based on a spectra library for the on-line monitoring of bioprocesses.

In order to significantly reduce the time involved in mid-infrared spectroscopy calibrations, a novel approach based on a library of pure component spectra was developed and tested with an aerobic Saccharomyces cerevisiae fermentation. Instead of the 30-50 standards that would have been required to build a chemometric model, only five solutions were used to assemble the library, namely one for each compound (glucose, ethanol, glycerol, ammonium and acetate). Concentration profiles of glucose, ethanol and ammonium were monitored with a fair accuracy, leading to standard error of prediction (SEP) values of 0.86, 0.98 and 0.15 g L(-1). Prediction of the two minor metabolites, acetate and glycerol, was less accurate and presented a detection limit of around 0.5 g L(-1). The overall performance of the library-based method proved to be very similar to a 49-standard chemometrics model. The model was shown to be very robust and uncorrelated, since it was able to predict accurately the concentration changes during a spiking experiment. Even though simple, this method allows more advanced calculations, such as determination of the explained variance and detection of unexpected compounds using residuals analysis.

A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy.

Mid-infrared FTIR spectroscopy is an efficient tool for the monitoring of bioprocesses, since it is fast and able to detect many compounds simultaneously. However, complex and time-consuming calibration procedures are still required, and have inhibited the spreading of these instruments. A simple and quick method to calibrate a FTIR instrument was developed for the control of fed-batch fermentations of the methylotrophic yeast Pichia pastoris. Based on the assumptions that (1) only substrate concentration may change significantly during a fed-batch process and (2) absorbance can be considered as proportional to concentration, a linear two-point calibration was implemented. Long-term instability of the instrument had to be addressed in order to get accurate results: two fixed points, on both sides of substrate absorbance peak, were used to perform on-line a linear correction of the signal drift. Fed-batch experiments at constant methanol (substrate) concentration ranging from 0.8 to 15gl(-1) were carried out. Off-line HPLC control analysis showed a good agreement with on-line FTIR data, with standard error of prediction values < 0.12gl(-1). Even though methanol acts both as carbon source and inducer of protein expression, no significant effect was observed on the level of protein expression in the recombinant strain used.