Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.

Sticky Bacteria: Understanding the Behavior of a D-Galactose Adapted Consortium of Acidophilic Chemolithotroph Bacteria and Their Attachment on a Concentrate of Polymetallic Mineral.

Various strategies to accelerate the formation of biofilms on minerals have been studied, and one of them is the use of D-galactose as an inducer of EPS production in planktonic cells of biooxidant bacteria. With the aim to evaluate the influence on the attachment and the effect over the solubilization of a polymetallic mineral concentrate, the behavior of a microbial consortium formed by Acidithiobacillus thiooxidans DSM 14887T and Leptospirillum ferrooxidans DSM 2705T previously induced with D-galactose for the early formation of EPS was studied. These microorganisms were previously adapted to 0.15 and 0.25% of D-galactose, respectively; afterward, different proportions of both strains were put in contact with the particle surface of a concentrate of polymetallic mineral. Also, to evaluate the affinity of each bacterium to the mineral, attachment tests were carried out with one of these species acting as a pre-colonizer. The same consortia were used to evaluate the solubilization of the polymetallic mineral. The results obtained show that the induction by D-galactose increases the microbial attachment percentage to the mineral by at least 10% with respect to the control of non-adapted consortia. On the other hand, the tests carried out with pre-colonization show that the order of inoculation also affects the microbial attachment percentage. From the different proportions tested, it was determined that the use of a consortium with a proportion of 50% of each species previously adapted to D-galactose and inoculated simultaneously, present a microbial attachment percentage to the mineral greater than 95% and better solubilization of a polymetallic mineral, reaching values of 9.7 and 11.7mgL-1 h-1 of Fe3+ and SO4 2-, respectively. Therefore, the use of D-galactose in small concentrations as inducer of EPS in acidophilic cells and the selection of an adequate strategy of inoculation can be beneficial to improve biooxidation since it would allow this process to develop in a shorter time by achieving a greater number of attached cells in a shorter time accelerating the solubilization of a sulfide mineral. Graphical AbstractEPS production using D-galactose as inducer and its influence in the attachment of consortia formed by differents proportions of A. thiooxidans and L. ferrooxidans inoculated at the same time and when one of them acting as a pre-colonizer.

Kinetic model of Clostridium beijerinckii's Acetone-Butanol-Ethanol fermentation considering metabolically diverse cell types.

Clostridium beijerinckii population branches into metabolically diverse cell types in batch cultures. Here, we present a new kinetic model of C. beijerinckii's Acetone-Butanol-Ethanol fermentation that considers three cell types: producers of acids (acidogenic), consumer of acids and producers of solvents (solventogenic), and spores cells. The model accurately recapitulates batch culture data. Also, the model estimates cell type-specific kinetic parameters, which can be helpful to improve the operation of the ABE fermentation and give a framework to study acidogenic and solventogenic metabolic pathways. To exemplify the latter, we used a constraint-based model to study how the ABE pathways are used among acidogenic and solventogenic cell types. We found that among both cell types, glycolytic production of ATP and consumption of NAD+ varies widely during the fermentation, with their maximum production/consumption rates happening when acidogenic and solventogenic growth rates were at their highest. However, acidogenic cells use the ABE pathway to contribute with an extra 12.5% of the total production of ATP, whereas solventogenic cell types use the ABE pathway to supply more than 75% of the demand for NAD+, alternating between the production of lactate and butyrate, being both coupled to the production of NAD+.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

BACKGROUND: Methanol can be effectively removed from air by biofiltration (Shareefdeen et al., 1993; Babbitt et al., 2009 [1,2]). However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms (Negruta et al., 2010 [3]), and it can be released out of the cell constituting a secondary emission. RESULTS: The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m−3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1 . Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. CONCLUSIONS: Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment.

Release of formaldehyde during the biofiltration of methanol vapors in a peat biofilter inoculated with Pichia pastoris GS115

Background: Methanol can be effectively removed from air by biofiltration. However, formaldehyde is one of the first metabolic intermediates in the consumption of methanol in methylotrophic microorganisms, and it can be released out of the cell constituting a secondary emission. Results: The total removal of methanol was achieved up to input loads of 263 g m−3 h−1 and the maximum elimination capacity of the system was obtained at an empty bed residence times of 90 s and reached 330 g m− 3 h−1 at an input methanol load of 414 g m−3 h−1 and 80% of removal efficiency. Formaldehyde was detected inside the biofilter when the input methanol load was above 212 g m−3 h−1 . Biomass in the filter bed was able to degrade the formaldehyde generated, but with the increase of the methanol input load, the unconsumed formaldehyde was released outside the biofilter. The maximum concentration registered at the output of the system was 3.98 g m−3 when the methanol load was 672 g m−3 h−1 in an empty bed residence times of 60 s. Conclusions: Formaldehyde is produced inside a biofilter when methanol is treated in a biofiltration system inoculated with Pichia pastoris. Biomass present in the reactor is capable of degrading the formaldehyde generated as the concentration of methanol decreases. However, high methanol loads can lead to the generation and release of formaldehyde into the environment

Making sticky cells: effect of galactose and ferrous iron on the attachment of Leptospirillum ferrooxidans to mineral surfaces.

The purpose of this study was to compare the efficacy of galactose and high initial ferrous iron concentrations as inducers for extracellular polymeric substances (EPS) production in planktonic cells of Leptospirillum ferrooxidans and to study cell attachment to a mineral surface in comparison to cells not exposed to such substances. L. ferrooxidans was successfully adapted to grow in a modified 9K medium at different concentrations of galactose (0.15, 0.25, 0.35%) and also at different initial ferrous iron concentrations (18, 27, 36 g/L), which are higher than 9K medium (9 g/L). The experiments were done in shake flasks using ferrous iron as energy source. A comparison of growth kinetics showed a decreasing of maximum specific growth rate of L. ferrooxidans with increasing concentrations of galactose and initial ferrous iron. The EPS content increased and the EPS chemical composition (relative abundance of carbohydrates, proteins and ferric iron) changed with increasing concentrations of galactose and initial ferrous iron. Results revealed that the increase of the bacterial adhesion rather depended on the chemical composition, i.e. relative abundance of the constituents of the EPS, than on the total amount of EPS. The EPS induced by galactose seemed to be "stickier" than the one induced by ferrous iron. Based on the results of this study it is proposed that galactose might enhance biooxidation processes which needs to be tested in future studies.

Comparison on the removal of hydrogen sulfide in biotrickling filters inoculated with Thiobacillus thioparus and Acidithiobacillus thiooxidans

Emissions of hydrogen sulfide (H2S) by industrial activities is frequent cause of corrosion and unpleasant odours. Treatment of gaseous emissions contaminated with H2S by biotrickling filters inoculated with single cultures of sulfur oxidizer bacteria exhibit several advantages over physicochemical methods, such as shorter adaptation times and higher removal ability. Biofilms of Thiobacillus thioparus and Acidithiobacillus thiooxidans have proved to exhibit high removal capacities, yet no comparative studies between them have been reported. This article reports the efficiency of biotrickling filters inoculated with T. thioparus and A. thiooxidans under similar conditions excepting the pH, that was the optimal for the bacterial growth, for the removal of H2S. The support was selected by determining the respirometric coefficients of the biomass. The maximum removal capacity of the biofilter inoculated with T. thioparus, operating within the range of pH (5.5-7.0) was 14 gS m-3 h-1, lower the value obtained for the biotrickling filter inoculated with A. thiooxidans; 370 gS m-3 h-1. Therefore, it is concluded that acid biotrickling filter inoculated with A. thiooxidans constitute the best strategy to remove H2S, with the advantage that the system not require an exhaustive pH control of the liquid media.