Differential effects of major inhibitory compounds from sugarcane-based lignocellulosic hydrolysates on the physiology of yeast strains and lactic acid bacteria.

OBJECTIVES: Major lignocellulosic inhibitory compounds found in sugarcane-based industrial hydrolysate samples were tested in laboratory and industrial yeast strains, as well as in lactic acid bacteria, in order to verify their effects on important physiological parameters. RESULTS: Saccharomyces cereviaise SA-1, an industrial strain, stood out as compared to the remaining strains for virtually all inhibitors investigated. This strain presented the highest growth rate and the lowest lag-phase in the presence of acetic acid, levulinic acid, p-coumaric acid, ferulic acid, and HMF, when compared to the other strains. In sugarcane-based hydrolysate fermentations, both SA-1 and CEN.PK113-7D presented similar fermentation performances. Industrial isolates of contaminating lactic acid bacteria were evaluated in the presence of an inhibitory cocktail, containing a mixture of 76.6 mM acetic acid, 1.3 mM HMF, 7.1 mM furfural, and 1.9 mM p-coumaric acid. Whilst all yeast strains were unable to grow under such conditions, bacteria had an average inhibition of roughly 50% on their growth rates. CONCLUSIONS: Overall, industrial strain SA-1 might be a promising microbial chassis for second generation ethanol production and for future metabolic and evolutionary engineering strategies, and for strain robustness understanding.

Forever panting and forever growing: physiology of Saccharomyces 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 N2 gas through an O2 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 O2 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 titers.

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

The published online version contains mistake in Figure1. In the x-axis, instead of "1000", the number should be "100".

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.

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 × 10(9) BHK-21 cells from 4 × 10(6) cells in 13 day with 1,051 mL culture medium.

Culturing Drosophila melanogaster (S2) in a chemostat.

Insect cells are used for the expression of complex proteins in products such as vaccines and biopharmaceuticals. Physiology of a Drosophila melanogaster (lineage S2), transfected to stably express rabies virus glycoprotein (RVGP), was studied in batch culture and in a chemostat with serum-free medium. In batch mode, the system reached 3 × 10(7) cells mL(-1) with specific growth rate of 1.5 d(-1) with RVGP at 2.50 µg L(-1). When operated continuously, three well-defined steady states were achieved at dilution rates (D) of 0.8, 0.5 and 0.2 d(-1). The residual glucose and glutamine concentrations and the cell yields on glucose and glutamine decreased at lower D. High values of substrate consumption for maintenance may explain this variation in yields. The results indicated that glucose is not the limiting substrate of this process.

Influence of aeration-homogenization system in stirred tank bioreactors, dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism.

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO2 and NaHCO3 solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO3 solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10(6) cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation-aeration systems.