Ramon

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess.

In this work a systematic study of the influence of methanol set-point and sorbitol feeding rate in fed-batch operation with a Pichia pastoris Mut(s) strain producing Rhizopus oryzae lipase is presented. Different experiments were made at a constant methanol set-point of 0.5, 2 and 4gl(-1), controlled by a predictive algorithm at two different sorbitol feeding rates to assure a constant specific growth rate of 0.01 and 0.02h(-1), by means of a pre-programmed exponential feeding rate strategy. Lipolytic activity, yields, productivity and specific productivity, but also specific growth, consumption and production rates were analyzed showing that the best values were reached when the methanol set-point was 2gl(-1) with a low influence of the constant specific growth rate tested. The sorbitol addition as a co-substrate during the induction phase avoids the severe decrease of the specific production rate obtained when methanol was used as a sole carbon source and it permits to achieve higher ROL production.

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris.

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastorisbased on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensors interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g ⋅ l−1 and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.

Substrate feeding strategies in Pichia pastoris fed-batch cultivation processes: Analysis of key parameters influencing recombinant protein production

has been studied in fed-batch bioprocesses witha manual (off-line) methanol concentration control [4].These studies demonstrated that variations of the residualmethanol concentration influence drastically in the spe-cific consumption and production rates. To avoid thisproblem a predictive control algorithm coupled with a PIfeedback controller has been satisfactorily implemented[5].This set-up has allowed for further analysis of several keyparameters influencing heterologous protein productionin

Use of a Focused Microwave System for the Determination of Kjeldahl Nitrogen in Industrial Wastewaters

Abstract This paper demonstrates the use of a focused microwave method involving chemical digestion in two steps, mineralization and oxidation with H2O2, for the determination of Kjeldahl nitrogen in industrial wastewaters. The most influential variables of the process (viz., microwave power, mineralization time, and oxidation time) were optimized by using an experimental design in conjunction with acetanilide as the reference substance. Tests revealed microwave power to have little effect on the extent of digestion in the range studied. The optimum mineralization time and oxidation time were both in the region of 10 min. However, the latter two must be adjusted as a function of the concentration and ease of degradation of the Kjeldahl nitrogen present in each sample. The proposed method was applied to industrial wastewaters of variable origin and found to provide results on a par with those of the standard method for this purpose, with digestion times shorter than this by a factor ranging from 6 to 60 depending on the ease of degradation of the particular sample.