RESUMO
Various bio-based processes depend on controlled micro-aerobic conditions to achieve a satisfactory product yield. However, the limiting oxygen concentration varies according to the micro-organism employed, while for industrial applications, there is no cost-effective way of measuring it at low levels. This study proposes a machine learning procedure within a metabolic flux-based control strategy (SUPERSYS_MCU) to address this issue. The control strategy used simulations of a genome-scale metabolic model to generate a surrogate model in the form of an artificial neural network, to be used in a micro-aerobic fermentation strategy (MF-ANN). The meta-model provided setpoints to the controller, allowing adjustment of the inlet air flow to control the oxygen uptake rate. The strategy was evaluated in micro-aerobic batch cultures employing industrial Saccharomyces cerevisiae yeast, with defined medium and glucose as the carbon source, as a case study. The performance of the proposed control scheme was compared with a conventional fermentation and with three previously reported micro-aeration strategies, including respiratory quotient-based control and constant air flow rate. Due to maintenance of the oxidative balance at the anaerobiosis threshold, the MF-ANN provided volumetric ethanol productivity of 4.16 g·L-1 ·h-1 and a yield of 0.48 gethanol .gsubstrate-1 , which were higher than the values achieved for the other conditions studied (maximum of 3.4 g·L-1 ·h-1 and 0.35-0.40 gethanol ·gsubstrate-1 , respectively). Due to its modular character, the MF-ANN strategy could be adapted to other micro-aerated bioprocesses.
Assuntos
Reatores Biológicos/microbiologia , Fermentação/fisiologia , Aprendizado de Máquina , Oxigênio/metabolismo , Anaerobiose , Técnicas de Cultura Celular por Lotes , Etanol/análise , Etanol/metabolismo , Análise do Fluxo Metabólico , Saccharomyces cerevisiae/metabolismoRESUMO
The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32⯰C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.
RESUMO
The impact of cultivation strategy on the cost of recombinant protein production is crucial for defining cost-effective bioreactor operation conditions. This paper presents a methodology to estimate and compare cost impacts related to utilities as well as medium composition, using simple design equations and accessible data. Data from batch bioreactor cultures were used as case study involving the production of pneumococcal surface protein A, a soluble recombinant protein, employing E. coli BL21(DE3). Cultivation strategies and corresponding process costs covered a wide range of operational conditions, including different media, inducers, and temperatures. The core expenses were related to the medium and cooling. When the price of peptone was above the threshold value of US$ 30/kg, defined medium became the best choice. IPTG and temperatures around 32°C led to shorter cultures and lower PspA4Pro production costs. The procedure offers a simple, accessible theoretical tool to identify cost-effective production strategies using bioreactors.
RESUMO
New conjugated vaccines against Streptococcus pneumoniae are being developed using pneumococcal surfaceproteins as carriers. The pneumococcal surface protein A (PspA) was selected as carrier because it is indispensablefor virulence of S. pneumoniae. The PspA can be classified into 3 families according to the homology of proteinsequences, within each family there is immunological cross-reactivity and PspA from family 1 or 2 are present in99% of strains associated with pneumococcal invasive disease. Hence, the purpose of this work was to develop an industrial production and purification process of His-tagged recombinant fragment of PspA in E. coli BL21 (DE3),rfPspA245 from family 1. Fed-batch cultivations in 5-L bioreactors with defined medium were carried out using glycerol as carbon source. Itwas obtained circa 60 g/L of dry cell weight and 3.0 g/L of rfPspA. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. The clarification step was done by centrifugation. The results ofchromatographic steps were analyzed by densitometry of SDS-PAGE protein bands. Using the chromatographicsequence anion exchange (Q-Sepharose) followed by metal affinity (IMAC-Sepharose), the rfPspA245 was obtained with 67% and 97% of purity respectively for each step and final recovery of 23%. In conclusion, the purification process was developed and rfPspA245 was obtained with high purity, but the recovery should still be improved.