Batch and fed-batch cultures of E. coli TB1 at different oxygen transfer rates: Effect of stirring and oxigen partial pressures on cell growth and cytochrome b5 production

Batch cultures of E. coli TB1/pUC13 were carried out at different oxygen transfer rates (OTR) enhanced by the increase of stirring rate and by the increase of air total pressure of the bioreactor. These two variables showed to have little effect on cell growth but a negative effect on cytochrome b5 (recombinant protein) production. However, this effect was more significant of high stirring rates than for values of pressure up to 0.4 MPa. The effects of stirring and pressure were also investigated for fed-batch mode operation. In this type of cell cultivation high cell densities are reached, thus a high capacity of oxygen supply of the system is required. To compare the two ways of improving OTR, cell behaviour was followed in two bioreactors at different operational conditions giving the same maximum OTR value. The first one operated at a high stirring rate (500 rpm) and at atmospheric pressure (0.1 MPa) and the other one at high air pressure (0.48 MPa) and low stirring rate. The increased pressure seemed to be a better way of ensuring an adequate oxygen supply to a culture of E. coli TB1 cells than an increased stirring rate. For the high pressure experiment a higher cellular density was reached, as well as a higher cyt.b5 expression which led to a 4-fold increase in final productivity. These experiments showed that bioreactor pressurization can be successfully used as a means of enhancing oxygen mass transfer to shear sensitive cell cultures.Fundação para a Ciência e a Tecnologia (FCT) – PRAXIS XXI

Morphological and physiological changes in Saccharomyces cerevisiae by oxidative stress from hyperbaric air

Increase in air or oxygen pressure in microbial cell cultures can cause oxidative stress and consequently affect cell physiology and morphology. The behaviour of Saccharomyces cerevisiae grown under hyperbaric atmospheres of air and pure oxygen was studied. A limit of 1.0 MPa for the air pressure increase (i.e. 0.21 MPa of oxygen partial pressure) in a fed-batch culture of S. cerevisiae was established. Values of 1.5 MPa air pressure and 0.32 MPa pure oxygen pressure strongly inhibited the metabolic activity and the viability of the cells. Also, morphological changes were observed, especially cell-size distribution and the genealogical age profile. Pressure caused cell compression and an increase in number of aged cells. These effects were attributed to oxygen toxicity since similar results were obtained using air or oxygen, if oxygen partial pressure was equal to or higher than 0.32 MPa. The activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD) (cytosolic and mitochondrial isoformes) indicated that the enzymes have different roles in oxidative stress cell protection, depending on other factors that affect the cell physiological state

Citric acid production by Yarrowia lipolytica from crude glycerol: Influence of oxygen mass transfer rate (OTR)

Glycerol from biodiesel industry, which is available in high amounts nowadays, is a renewable low-cost substrate that can be used for many biotechnological applications. Yarrowia lipolytica is a strictly aerobic yeast, known for the ability to use several carbon sources and to produce several high value compounds. Citric acid, an intermediate of tricarboxylic acids cycle, is extensively used in food and pharmaceutical industry. This organic acid can be produced by Y. lipolytica from glycerol under specific growth conditions, namely under nitrogen limitation. Oxygen availability in the culture medium is also an important factor that influences these aerobic bioprocesses. In this work, the influence of Oxygen Transfer Rate (OTR) from air to the culture on citric acid production by Y. lipolytica W29 (ATCC 20460:CLIB89) using crude glycerol was evaluated. Batch cultures with 50 g L1 of glycerol were performed at OTR values from 52mgL1 h1 to 878mgL1 h1 obtained varying aeration and agitation rates, from 1vvm to 3 vvm, and from 200rpm to 600 rpm, respectively. The increase of OTR up to 408mgL1 h1 leaded to a 7.7-fold increase of citric acid production (10.8±0.5gL1). Further increase of OTR values above 408mgL1 h1 did not improve citric acid concentration and productivity. In fact, for these OTR values oxygen dissolved concentration in the medium was kept above 50% of the saturation value, during the production of citric acid, meaning that the process control by oxygen limitation was prevented

Enhanced growth of Pichia pastoris under increased air pressure on different carbon sources

Pichia pastoris has many biotechnological applications. Two aspects of the species have contributed to its utility: (1) fermentation techniques were developed for maintaining extremely high cell densities in excess of 100 g/L dry weight, and (2) because P. pastoris assimilates methanol, the expression system is linked with alcohol oxidase, which is abundantly produced in the presence of methanol. The high oxygen demand of methanol metabolism and cultivation at very high-cell-density makes oxygen supply a major parameter in Pichia pastoris cultivation. Previous work demonstrated that hyperbaric air could be successfully applied to yeast cultivation, as a way of improving the oxygen transfer rate (OTR) to aerobic cultures [1]. In the present work, we investigate whether increasing air pressures may lead to increasing biomass yields of P. pastoris, growing with four carbon sources, without giving rise to unbalance oxidative stress. Pichia pastoris strain was grown in glucose, pure glycerol, crude glycerol from biodiesel industry and methanol media under total air pressure from 1 bar to 5 bar. In all the experiments, the cultures reached maximum cell density at 5 bar of total air pressure. A 4fold increase on specific growth rate was obtained at 5 bar on glycerol and crude glycerol compared to the value at atmospheric pressure. Biomass yield was also enhanced by air pressure rise, for all carbon sources. With 5 bar air pressure biomass yield (g cells/g carbon) was 0.97 and 1.86 whereas at 1 bar was 0.67 and 0.77, respectively in methanol and glycerol media

Effects of hyperbaric air on the Saccharomyces cerevisae morphology and viability

Fed-batch cultivation of Saccharomyces cerevisae is the dominating technique in high cell density cultures of processes such as the production of Baker's yeast [l] and recombinant proteins [2]. Due to the high oxygen demand of these cultures, the oxygen supply to the culture is an important and difficult task. The use of hyperbaric air, e.g., air at increased pressure, for oxygen mass transfer improvement has been proved to be applicable to several microbial strains [3,4]. In this study, the effects of hyperbaric air up to 1.5 MPa on the viability and morphology of S. cerevisiae cells grown in fed-batch cultures were investigated. Fed-batch experiments were performed in a stainless steel stirred tank reactor. Exponential feeding at dilution rates up to 0.1 h-1 was used, in order to ensure full respiratory metabolism. The ethanol production due to oxygen _limitation at atmospheric pressure was reduced by the bioreactor pressurization up to 1.0 MPa. No differences on the fraction of viable cells, size of the cells and genealogical age were observed in this range of total pressure. Moreover, best results were obtained for experiments where pressure was increased gradually throughout time. This observation indicates the existence of an adaptation period of the cells to hyperbaric conditions. However, a strong inhibition of cell activity was observed for the operation at 1.5 MPa total air pressure. This effect was due to the increase on the oxygen partial pressure because similar cell behaviour was found using pure oxygen at the same partial pressure (0.32 MPa). Oxygen toxicity resulted in a drastic decrease of cell viability, inhibition of ATP synthesis and morphologic changes, mainly, cell size decrease

Utilization of mycelial growth to study the tolerance of some white rot fungi to phenolic compounds

Phanerochaete chrysosporium, Aureobasidium pu/lulans, Coriolus versicolor, Pleurotus ostreatus and Dichomitus sqoo/ens were some white-rot fungi selected to study the effect of increasing concentrations of phenol, catechol and resorcinol on the mycelial growth on solid media. The increasing concentrations of the phenolic compounds added to a mineral Czapec-Dox agar medium were progressively inhibitory up to a certain maximum value beyond which growth became impossible. P. ostreatu.s was the most affected fungus by the high concentrations used, following by C. versicolor, while P. chrysosporium, A. pul/ulans, and D. squalens tolerated better the presence of the phenolic compounds. It was observed that the toxicity of these compounds towards the microorganisms progressed in accordance to the following order: resorcinol< phenol<cathecol. Furthermore, the capability of growing at the expense of agar, agarose and gelatine as sole carbon and energy source was demonstrated by all the fungi

Production of citric acid from glycerol by Yarrowia lipolytica – optimization of culture conditions

The global expansion of biodiesel production turned the glycerol (the main byproduct of this industry) into an excess product, consequently available at low cost on the market. Despite the well-known applications of pure glycerol, it is important to look for new ways to valorize the raw glycerol; it can be through biotechnological alternatives, by its use to produce high added value compounds. Several microorganisms are able to use glycerol and convert it into different compounds. Yarrowia lipolytica is the non-conventional yeast more extensively studied; it is not pathogenic to humans and was classified as GRAS by FDA to produce citric acid. This yeast can use several different carbon sources like glucose, glycerol, alcohols, acetate and hydrophobic substrates as fatty acids or alkanes, moreover it can produce important metabolites having an intense secretory activity. Citric acid is one of these metabolites that this yeast can produce and secrete. The citric acid is an intermediate of the tricarboxylic acid cycle, that are used as a food ingredient and used in the production of wines, jams, cheese, ice creams, kellies and ciders, is also used in the pharmaceutical industry and recently in the manufacture of detergent. Most of citric acid has been produced by Aspergillus niger in submerged fermentation, but because of the constant increased of the amount of citric acid needed is important to find alternative processes and the use of high-yield yeast could be this alternative. The production of citric acid by Y. lipolytica is influenced by the culture conditions: the carbon source and its concentration, the concentration of nitrogen, pH value and others factors that change the metabolism to produce other organic acids instead of citric acid. The principal aim of this work was to select the better conditions to stimulate and maximize the production of citric acid by Yarrowia lipolytica W29 (ATCC20460:CLIB89). Using the Taguchi method through the experimental design software (Qualitek-4), the influence of carbon/nitrogen ration (C/N), the pH, quantity of salts and the aeration in the production of citric acid was evaluated. These four factors were varied at three different levels resulting in a total of 9 experiments that were preformed in flasks. The optimal conditions to maximize the concentration of citric acid (9.6 g/L) were a C/N ratio of 50/0.1, a pH of 5, presence of salts at middle concentration and aeration of 200 rpm in flasks with baffles. The optimum conditions were validated in flasks and in 2 L bioreactor operating either with pure or crude glycerol

Physiological behaviour of saccharomyces cerevisiae under increased air and oxygen pressures

Saccharomyces cerevisiae, in a pressure batch reactor, coped with higher air (1.2–3 bar) pressures better than with pure oxygen pressures (1.2–3 bar) for an equivalent dissolved oxygen concentration. However, pure oxygen pressure enhanced ethanol production. Both pressures did not influence the type of metabolism followed by the organism which was always oxidoreductive. Growth was inhibited with the increase of air and pure oxygen pressure and almost completely inhibited with 8 bar of pure oxygen. Above 3 bar activities of mitochondrial superoxide dismutase and glutathione reductase increased with air pressure, but cytosolic superoxide dismutase and catalase increased activity only in pure oxygen pressure