Anaerobiosis revisited: growth of Saccharomyces cerevisiae under extremely low oxygen availability

The budding yeast Saccharomyces cerevisiae plays an important role in biotechnological applications, ranging from fuel ethanol to recombinant protein production. It is also a model organism for studies on cell physiology and genetic regulation. Its ability to grow under anaerobic conditions is of interest in many industrial applications. Unlike industrial bioreactors with their low surface area relative to volume, ensuring a complete anaerobic atmosphere during microbial cultivations in the laboratory is rather difficult. Tiny amounts of O2 that enter the system can vastly influence product yields and microbial physiology. A common procedure in the laboratory is to sparge the culture vessel with ultrapure N2 gas; together with the use of butyl rubber stoppers and norprene tubing, O2 diffusion into the system can be strongly minimized. With insights from some studies conducted in our laboratory, we explore the question ‘how anaerobic is anaerobiosis?’. We briefly discuss the role of O2 in non-respiratory pathways in S. cerevisiae and provide a systematic survey of the attempts made thus far to cultivate yeast under anaerobic conditions. We conclude that very few data exist on the physiology of S. cerevisiae under anaerobiosis in the absence of the anaerobic growth factors ergosterol and unsaturated fatty acids. Anaerobicity should be treated as a relative condition since complete anaerobiosis is hardly achievable in the laboratory. Ideally, researchers should provide all the details of their anaerobic set-up, to ensure reproducibility of results among different laboratories. A correction to this article is available online at http://eprints.whiterose.ac.uk/131930/ https://doi.org/10.1007/s00253-018-9036-

Animal cell cultures aiming high cell concentrations: perfusion process and amino acids supplementation.

Células animais são alvo de pesquisas visando sua utilização como plataforma para a expressão de proteínas recombinantes, desde vacinas veterinárias até fatores de coagulação para hemofílicos. Exemplos incluem células de inseto Drosophila melanogaster S2 e células de mamífero BHK-21, que vêm sendo estudadas visando a sua utilização para a produção da glicoproteína do vírus da raiva. Independentemente da estratégia de cultivo utilizada, altas concentrações celulares são em geral associadas a uma maior produção da proteína de interesse. O objetivo deste trabalho foi o de investigar estratégias que possibilitariam o cultivo de células animais em altas concentrações celulares. Células de inseto Drosophila melanogaster S2 produtoras da glicoproteína do vírus da raiva foram cultivadas em frascos agitados a 100 rpm e 28, em meio livre de soro SF 900 II suplementado com os aminoácidos asparagina, cisteína, prolina e serina. A adição dos quatro aminoácidos no meio de cultura refletiu em um aumento da concentração celular máxima (XV MÁX) em 16%. Cisteína, quando adicionada isoladamente no meio de cultura, refletiu em uma velocidade específica máxima de crescimento celular (MÁX) 56% maior. Nessa condição, o fator de conversão glicose a célula (YX/GLC) foi 47% maior, indicando um metabolismo de glicose mais eficiente na geração de células. Esses resultados indicam que cisteína é provavelmente substrato limitante do cultivo de células S2AcGPV em meio SF 900 II. Já células de mamífero BHK-21 (C13), adaptadas ao crescimento em suspensão, foram cultivadas em processo de perfusão, processo contínuo em que há retenção celular, o que permite alcançar concentrações celulares mais altas que processos em batelada ou em modo contínuo sem retenção celular. Foi utilizado um biorreator do tipo tanque agitado com 1,5 L de volume de trabalho e spin-filter interno, com poro de diâmetro igual a 10 m, acoplado ao eixo do impelidor. Durante o cultivo, o pH foi controlado em 7,2, a agitação em 80 rpm, a temperatura em 37 e o oxigênio dissolvido em 50% da saturação com o ar. A concentração celular máxima alcançou 15,7 x 106 céls mL-1, muito superior à do cultivo em batelada (aproximadamente 5 x 106 céls mL-1). A viabilidade celular foi superior a 90% durante os 48 dias de cultivo. Na fase batelada do cultivo em perfusão, as velocidades de consumo de glicose (qGLC) e de glutamina (qGLN) foram 84% e 32% maiores, respectivamente, em relação às velocidades observadas no cultivo em batelada. Analogamente, as velocidades de produção de lactato (qLAC) e de amônio (qNH4) foram 78% e 102% maiores, respectivamente. Ainda, o coeficiente de manutenção celular não foi desprezível, e o consumo de glicose associado à manutenção celular foi de 83%. Esses dados indicam que a presença do spin-filter interno pode estar associada a estresse celular. Na perfusão, a concentração celular foi cerca de 3 vezes maior do que no cultivo contínuo sem reciclo de células. Provou-se que é possível cultivar células BHK-21 adaptadas a crescimento em suspensão em altas concentrações celulares em escala laboratorial, utilizando biorreator de bancada e spin-filter interno como sistema de retenção celular.Animal cells have been under research as a platform for the expression of recombinant proteins, ranging from veterinary vaccines to blood coagulation factors for treating hemophilia. Examples include insect Drosophila melanogaster S2 and hamster BHK-21 cells, currently being studied for the production of rabies virus glycoprotein. Regardless of the cultivation strategy, high cell concentrations are usually associated to a higher protein production. Thus, the aim of this research was to investigate animal cell cultivation strategies that would allow higher cell concentrations than those previously reported. Cells of Drosophila melanogaster S2 expressing the rabies virus glycoprotein (S2AcGPV) were cultivated in shake flasks at 100 rpm and 28 , in SF 900 II serum-free medium supplemented with the following amino acids: asparagine, cysteine, proline, and serine. The addition of the four amino acids to the medium increased the maximum cell concentration (XV MAX) in 16%. When only cysteine was added to the medium, the maximum specific growth rate (ÊMAX) was 56% higher. In this condition, the cell yield on glucose (YX/GLC) was 47% higher, indicating a more efficient glucose metabolism. These results show that cysteine is likely a limiting substrate of S2AcGPV cells growing in SF 900 II medium. In turn, baby hamster kidney cells (BHK-21/C13), adapted to growth in suspension culture, were cultivated in perfusion, a continuous process with cell retention that allows higher cell concentration than batch or continuous cultures without cell retention. A stirred tank bioreactor with a working volume of 1.5 L was used, with an internal spin-filter with 10 µm diameter pores attached to the impeller shaft. Temperature was controlled at 37 , pH at 7.2, agitation at 80 rpm and dissolved oxygen at 50% of air saturation. The maximum cell concentration reached 15.7 x 106 cells mL-1, much higher than the cell concentration achieved in a standard batch cultivation (5 x 106 cells mL-1). Cell viability was above 90% during the 48-day cultivation period. During the batch phase of the perfusion cultivation, specific rates of glucose (qGLC) and glutamine (qGLN) consumption were 84% and 32% higher, respectively, when compared to the batch cultivation. Similarly, the specific rates of lactate (qLAC) and ammonium (qNH4) formation were 78% and 102% higher, respectively. During perfusion, the cell maintenance coefficient was not negligible and represented 83% of total glucose consumption. These data indicate that the presence of an internal spin-filter may be associated to cell stress. In perfusion, cell concentration was about 3 times higher than that in continuous culture without cell recycle. In conclusion, it was proved that suspension-adapted BHK-21 cells can be cultivated in a laboratory-scale bioreactor with an internal spin-filter, in order to achieve high cell concentrations

Forever panting and forever growing : physiology ofsaccharomyces cerevisiae at extremely low oxygen availability in the absence of ergosterol and unsaturated fatty acids

We sought to investigate how far the growth of Saccharomyces cerevisiae under full anaerobiosis is dependent on the widely used anaerobic growth factors (AGF) ergosterol and oleic acid. A continuous cultivation setup was employed and, even forcing ultrapure N-2 gas through an O-2 trap upstream of the bioreactor, neither cells from S. cerevisiae CEN.PK113-7D (a lab strain) nor from PE-2 (an industrial strain) washed out after an aerobic-to-anaerobic switch in the absence of AGF. S. cerevisiae PE-2 seemed to cope better than the laboratory strain with this extremely low O-2 availability, since it presented higher biomass yield, lower specific rates of glucose consumption and CO2 formation, and higher survival at low pH. Lipid (fatty acid and sterol) composition dramatically altered when cells were grown anaerobically without AGF: saturated fatty acid, squalene and lanosterol contents increased, when compared to either cells grown aerobically or anaerobically with AGF. We concluded that these lipid alterations negatively affect cell viability during exposure to low pH or high ethanol titers196COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2015/14109-0; 2018/17172-

Semliki Forest Virus as a Vector: Pros and Cons for Its Use in Biopharmaceuticals Production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 109 BHK-21 cells from 4 × 106 cells in 13 day with 1,051 mL culture medium.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Strain dynamics of contaminating bacteria modulate the yield of ethanol biorefineries

Abstract Bioethanol is a sustainable energy alternative and can contribute to global greenhouse-gas emission reductions by over 60%. Its industrial production faces various bottlenecks, including sub-optimal efficiency resulting from bacteria. Broad-spectrum removal of these contaminants results in negligible gains, suggesting that the process is shaped by ecological interactions within the microbial community. Here, we survey the microbiome across all process steps at two biorefineries, over three timepoints in a production season. Leveraging shotgun metagenomics and cultivation-based approaches, we identify beneficial bacteria and find improved outcome when yeast-to-bacteria ratios increase during fermentation. We provide a microbial gene catalogue which reveals bacteria-specific pathways associated with performance. We also show that Limosilactobacillus fermentum overgrowth lowers production, with one strain reducing yield by ~5% in laboratory fermentations, potentially due to its metabolite profile. Temperature is found to be a major driver for strain-level dynamics. Improved microbial management strategies could unlock environmental and economic gains in this US $ 60 billion industry enabling its wider adoption

A Multivariate Calibration Procedure for UV/VIS Spectrometric Monitoring of BHK-21 Cell Metabolism and Growth

Monitoring mammalian cell culture with UV-vis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off-line UV-vis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH were used to calibrate and validate PLS models. Glutamine, glutamate, glucose, and lactate concentrations were suitably predicted by means of this strategy. Especially for glutamine and glucose concentrations, the prediction error averages were lower than 0.50 +/- 0.10 mM and 2.21 +/- 0.16 mM, respectively. These values are comparable with those previously reported using near infrared and Raman spectroscopy in conjunction with PLS. However, viable cell concentration models need to be improved. The present work allows for UV-vis at-line sensor development, decrease cost related to nutrients and metabolite quantifications and establishment of fed-batch feeding schemes. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:241-248, 2014Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq