Immobilization of Yarrowia lipolytica for aroma production from castor oil

The main aim of this study was to compare different materials for Y. lipolytica immobilization that could be used in the production of γ-decalactone (a peach-like aroma) in order to prevent the toxic effect both of the substrate and the aroma upon the cells. Therefore, cells adsorption onto pieces of methyl polymethacrylate and of DupUM® was studied and further used in the biotransformation of castor oil into γ-decalactone. The highest aroma concentration was obtained with immobilized cells in DupUM®, where reconsumption of the aroma by the cells was prevented, contrarily to what happens with free cells. This is a very promising result for γ-decalactone production, with potential to be used at an industrial level since the use of immobilized cells system will facilitate the conversion of a batch process into a continuous mode keeping high cell density and allowing easier recovery of metabolic products.The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/63701/2009)

Enhancement of castor oil biotransformation into aroma by Yarrowia lipolytica mutants

The food industry has a great interest in biotechnological production of γ- decalactone by Yarrowia lipolytica, due to its increasing consumers acceptability in comparison with similar products obtained by chemical synthesis. This yeast is able to produce γ-decalactone by transformation of a hydroxylated C18 fatty acid. However, lower yields of γ-decalactone were obtained (up to 4–5 gL-1), mainly due the degradation of newly synthesized lactone and the partial use of ricinoleic acid or intermediate at the C10 level, which is simultaneously the precursor for other γ-lactones. Thus, the purpose of this work is to enhance the biotransformation of castor oil, source of ricinoleic acid, into γ-decalactone exploring different operation mode strategies in bioreactor (batch and fed-batch) and compare the yields obtained with wild type strain with those achieved by mutant strains. Different experiments were conducted in a 3.7-L bioreactor using an aeration rate of 5.1 L min-1, agitation 650 rpm and pH 6.0 (previously optimized conditions [1]). The influence of castor oil concentration and cell density on γ-decalactone production was investigated. Two different cell and castor oil concentrations (30 g L-1 and 60 gL-1) were used for the biotransformation. In the expectation of achieving higher γ-decalactone concentrations, a step-wise fed-batch strategy was also attempted. In a first approach, this study was conducted with Yarrowia lipolytica W29 (ATCC20460) and the highest γ-decalactone productivity of 215.4 mg L-1 h-1 was obtained in a batch mode of operation with 60 g L-1 of cells and 60 g L-1 of castor oil. After that, γ-decalactone production with two Yarrowia lipolytica mutants was studied. Experiments performed with Y. Lipolytica MTLY40-2P, with a deletion of all the POX 3–5 genes and a multicopy insertion of POX2 [2], resulted in an increased accumulation and an inhibition of γ-decalactone degradation. Since this yeast is also known to be a lipase producer and these enzymes catalyze the hydrolysis of triacylglycerides into glycerol and free fatty acids, a Y. lipolytica JMY3010 mutant, that overexpress extracellular lipase by the LIP2 gene (encoded the main extracellular lipase activity) cloned under the control of the TEF promoter [3], as also used. With these different approaches is possible to increase aroma productivity and a greater enhance in γ-decalactone production was achieved (up to 7-9 gL-1) through conjugation of a bioprocess optimization and genetic engineering approach

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

Stochastic Partial Differential Equations and Applications in Bayesian Inference

This work is devoted to the study of stochastic partial differential equations and its applications in Bayesian inference. Essentially it is composed by two essential parts: In the first part, we study a stochastic non-linear partial differential equation from the theoretical point of view, and in the second one we perform a Bayesian analysis based on a specific stochastic linear partial differential equation. The former problem to be addressed has its roots in fluid dynamics. More precisely, we consider the equation which governs the time evolution of a third grade non-Newtonian fluid, filling a two-dimensional non axisymmetric bounded domain, perturbed by a multiplicative white noise. We recall that the stochastic third grade fluid equation can be considered as a generalization of the stochastic Navier-Stokes equations, so we are faced with a strongly nonlinear stochastic partial differential equation and its analysis is not an easy issue. Considering initial conditions in the Sobolev space H2, and a Navier slip boundary condition, we show the existence and the uniqueness of the strong solution (in the stochastic sense). To show the existence of the solution, we first construct a sequence of solutions for the finite dimensional approximate problems, by using the finite dimensional Galerkin approximation method. Next, we pass to the limit by using a conjugation of compactness results and a uniqueness type argument. Let us mention that the study of stochastic fluid dynamics equations, where the solutions correspond to stochastic processes defined on some probability space, with sample paths on appropriate functional spaces is crucial for the statistical description of turbulent flows. In contrast to the usual deterministic individual solutions, in this framework each solution should correspond to a collection of possible realizations, and a probability of certain occurrences should be determined. As many fluids used in the industry are classified as third grade non- Newtonian fluid, we hope that our result will have practical consequences in the analysis of turbulence flows. As far as we know, this is the first time that the stochastic third grade fluid equation is being studied in the literature. The second problem to be studied consists on the application of the INLA methodology to perform Bayesian inference, considering a certain linear stochastic partial differential equation, which has a solution with a Matérn covariance. We recall that the Matérn covariance has a central role in spatial statistics, since it successfully captures the spatial behaviour of a wide number of phenomena. We consider a Gaussian vector field modelling the velocity of the wind and perform a Bayesian analysis to approximate the mean of the wind velocity field through the INLA methodology, combined with stochastic partial differential equations (SPDE). We emphasize that the behaviour of the wind velocity field is crucial in the weather forecast. We expect that this new statistical method will improve the classical methods mainly based on the numerical analysis of complex fluid dynamic equations.O presente trabalho é dedicado ao estudo de equações diferenciais parciais estocásticas e à sua aplicação na inferência Bayesiana. É composto essencialmente por duas partes. Na primeira parte estudamos uma equação diferencial parcial estocástica não-linear do ponto de vista teórico, e na segunda parte aplicamos os princípios da inferência Bayesiana à estimação usando uma equação diferencial parcial estocástica linear. O primeiro problema a ser estudado tem as suas origens na dinâmica dos fluidos. Mais precisamente, consideramos a equação que descreve a evolução de um fluido não- Newtoniano de terceiro grau, num domínio bi-dimensional limitado e não axissimétrico, perturbada por um ruído branco. A equação estocástica de fluidos de terceiro grau pode ser considerada como uma generalização da equação de Navier-Stokes, estamos perante uma equação diferencial parcial estocástica fortemente não linear, cuja análise é uma tarefa difícil. Considerando a condição inicial no espaço de Sobolev H2 , e uma condição de fronteira de deslizamento do tipo Navier, mostramos a existência e unicidade de solução forte (no sentido estocástico). Para mostrar a existência de solução, construímos primeiro uma sucessão de soluções para o problema aproximado em dimensão finita, usando o método de aproximação de Galerkin. A seguir é feita a passagem ao limite, através de resultados de compacidade, e um argumento de unicidade. Referimos também que o estudo de equações estocásticas de fluidos, cujas soluções correspondem a processos estocásticos definidos num determinado espaço de probabilidade, com trajetórias em espaços funcionais apropriados, é crucial na descrição de fluxos turbulentos. No contexto estocástico, cada solução da equação corresponde a uma coleção de possíveis realizações, pelo que a probabilidade de ocorrência de certas realizações pode ser determinada. Uma vez que muitos fluidos usados na indústria são classificados como fluidos não-Newtonianos de terceiro grau, esperamos que os nossos resultados venham a ter aplicação na análise da turbulência de fluidos. Tanto quanto pudemos constatar, esta é a primeira vez que a equação estocástica de fluidos de terceiro grau é estudada na literatura. O segundo problema a ser estudado consiste na aplicação da metodologia INLA, que é especialmente adequada para fazer inferência Bayesiana, combinada com a utilização de determinada equação diferencial parcial estocástica, cuja solução apresenta covariância de Matérn. A covariância de Matérn tem um papel central na estatística espacial, uma vez que descreve significativamente bem vários fenómenos de natureza espacial. Neste trabalho, consideramos que a velocidade do vento é modelada por um campo vetorial aleatório Gaussiano, e aproximamos a média do campo de velocidades aplicando os princípios da inferência Bayesiana, através da metodologia INLA combinada com SPDE. Salientamos que a velocidade do vento é crucial na previsão do tempo, então esperamos que esta nova abordagem estatística, venha a melhorar os métodos usuais de previsão, baseados na análise numérica das equações de fluidos

Yarrowia lipolytica as a microbial host for flavors and fragrances production

This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and of the project TUBITAK/0009/2014 and by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Fermentative capacity of baker’s yeast exposed to hyperbaric stress

Baker’s yeast suspensions were incubated at different pressures (from 1 bar to 6 bar) and different gases [air, O2 and a mixture of 8% (v/v) CO2, 21% O2 and N2]. Raising the air pressure from 1 bar to 6 bar stimulated cell growth but had no effect on leavening ability or viability of the cells. A 50% reduction of the CO2 produced in dough occurred with 6 bar O2 which also stopped growth. The fermentative capacity of the cells was stimulated by the cells exposure to increased CO2 partial pressure up to 0.48 bar

Valorisation of wastes for single cell oil production by Yarrowia lipolytica

Various strains of oleaginous microorganisms, mainly fungi and yeast, have been widely used for the production of single cell oil (SCO) rich in polyunsaturated fatty acids or having an exceptional triacylglycerol structure. Difficulties for industrial scale production are related to the high cost of fermentation and oil extraction. The quantity of oil accumulated per unit of dry cellular mass is a critical factor that influences the final cost of SCO. The economics of these bioprocess become more favourable when zero or negative value waste substrates are utilized as carbon or nitrogen sources. Although utilisation of crude glycerol in the fermentation medium without prior purification offers a remarkable advantage against the traditional use of pure glycerol as substrate only few reports have appeared in the literature on the use of this substrate as sole carbon sources. The aim of current investigation was to assess the potentialities of valorisation of crude glycerol and lard, residues used as a carbon source by Yarrowia lipolytica strain in order to the production of SCO. Batch fermentations in 1-L Erlenmeyer flasks were performed using pure glycerol, crude glycerol and lard as carbon sources, with different concentrations (20 g/L, 50 g/L and 80 g/L). Y. lipolytica W29 was pre-grown overnight in YPD medium, centrifuged and resuspended in each carbon source medium, supplemented with yeast extract (0.5 g/L). The production of SCO was carried out during 168 h at 27 ºC and 185 rpm. Yeast cells were able to grown on all carbon sources, although a slight inhibition with 80 g/L of pure and crude glycerol was observed. No significant differences on final cell dry weight were noted between the carbon sources, reaching approximately 5 g/L. The increase in carbon source concentration leads to an improvement in lipid accumulation inside the cells. The highest amount of reserve lipid was observed in medium with lard 80 g/L (21.3 % of cell dry weight). The strain showed the tendency to degrade its storage lipids when grown on 20 g/L of each carbon sources, probably due to the early consumption of substrate. Crude glycerol batch fermentation at 50 g/L in a 2-L bioreactor led to an accumulation of lipid content inside the cells of 37 % cell dry weight. The results of this study suggest that SCO could be produced by Y. lipolytica W29 using low-cost substrates, such as crude glycerol and lard

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

Lard as a suitable and cheap substrate for microbial lipids production by Yarrowia lipolytica

[Excerpt] Current agricultural and industrial practices have led to the generation of large amounts of various low-value or negative cost crude wastes, which are difficult and economically notattractive to treat and valorize. One important example of waste generation is animal fat, commonly found in tanning process and slaughterhouses. These wastes, in which the lipids are often the main and most problematic components, are not currently used effectively and there are almost no application methods to recover the respective value. (...