Alcoholic fermentation of lactose by engineered flocculent Saccharomyces cerevisiae

The construction of Saccharomyces cerevisiae strains with the ability to ferment lactose has biotechnological interest, particularly for cheese whey fermentation to ethanol. Direct fermentation of whey to ethanol is generally not economically feasible because the low lactose content (ca. 5% w/v) results in low ethanol titre (2 – 3% v/v), making the distillation process too expensive. Concentration of whey lactose (e.g. by ultrafiltration) prior to fermentation is an option to obtain higher ethanol titres. Microbial strains are therefore needed that can efficiently convert high concentrations of lactose into ethanol. We describe here the engineering of a S. cerevisiae strain for efficient lactose fermentation, involving genetic and evolutionary engineering strategies. The evolved strain obtained fermented efficiently lactose concentrations up to 150 g L-1, including 3-fold concentrated cheese whey, producing ethanol titres up to 8% v/v. The strain is highly flocculent, a property that makes it particularly suitable for the development of high cell density fermentation processes

Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey

Cheese whey, the main dairy by-product, is increasingly recognized as a source of many bioactive valuable compounds. Nevertheless, the most abundant component in whey is lactose (ca. 5% w/v), which represents a significant environmental problem. Due to the large lactose surplus generated, its conversion to bio-ethanol has long been considered as a possible solution for whey bioremediation. In this review, fermentation of lactose to ethanol is discussed, focusing on wild lactose-fermenting yeasts, particularly Kluyveromyces marxianus, and recombinant Saccharomyces cerevisiae strains. The early efforts in the screening and characterization of the fermentation properties of wild lactose-consuming yeasts are reviewed. Furthermore, emphasis is given on the latter advances in engineering S. cerevisiae strains for efficient whey-to-ethanol bioprocesses. Examples of industrial implementation are briefly discussed, illustrating the viability of whey-to-ethanol systems. Current developments on strain engineering together with the growing market for biofuels will likely boost the industrial interest in such processes.Fundação para a Ciência e a Tecnologia (FCT) - Projecto ProBioethanol PTDC/BIO/66151/2006 ; bolsa SFRH/BD/13463/2003 and SFRH/BPD/44328/200

Inconsistency in the Diagnosis of Functional Heartburn: Usefulness of Prolonged Wireless pH Monitoring in Patients With Proton Pump Inhibitor Refractory Gastroesophageal Reflux Disease

Background/Aims The diagnosis of functional heartburn is important for management, however it stands on fragile pH monitoring variables, ie, acid exposure time varies from day to day and symptoms are often few or absent. Aim of this study was to investigate consistency of the diagnosis of functional heartburn in subsequent days using prolonged wireless pH monitoring and its impact on patients' outcome. Methods Fifty proton pump inhibitotor refractory patients (11 male, 48 years [range, 38-57 years]) with a diagnosis of functional heartburn according to Rome III in the first 24 hours of wireless pH monitoring were reviewed. pH variables were analysed in the following 24-hour periods to determine if tracings were indicative of diagnosis of non-erosive reflux disease (either acid exposure time > 5% or normal acid exposure time and symptom index >= 50%). Outcome was assessed by review of hospital files and/or telephone interview. Results Fifteen out of 50 patients had a pathological acid exposure time after the first day of monitoring (10 in the second day and 5 in subsequent days), which changed their diagnosis from functional heartburn to non-erosive reflux disease. Fifty-four percent of non-erosive reflux disease vs 11% of functional heartburn patients (P < 0.003) increased the dose of proton pump inhibitors or underwent fundoplication after the pH test. Outcome was positive in 77% of non-erosive reflux disease vs 43% of functional heartburn patients (P < 0.05). Conclusions One-third of patients classified as functional heartburn at 24-hour pH-monitoring can be re-classified as non-erosive reflux disease after a more prolonged pH recording period. This observation has a positive impact on patients' management

Translocating the blood-brain barrier using electrostatics

Copyright © 2012 Ribeiro,Domingues, Freire,Santos and Castanho. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Mammalian cell membranes regulate homeostasis, protein activity, and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells (EC) are more negatively charged than human umbilical cord cells, using zeta-potential measurements by dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier (BBB) and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components—red blood cells (RBC), platelets, and peripheral blood mononuclear cells (PBMC).To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and those having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.Fundação para a Ciência e Tecnologia — Ministério da Educação e Ciência (FCT-MEC, Portugal) is acknowledged for funding (including fellowships SFRH/BD/42158/2007 to Marta M.B. Ribeiro, SFRH/BD/41750/2007 to Marco M. Domingues and SFRH/BD/70423/2010 to João M. Freire) and project PTDC/QUI-BIQ/119509/2010. Marie Curie Industry-Academia Partnerships and Pathways (European Commission) is also acknowledged for funding (FP7-PEOPLE-2007-3-1-IAPP, Project 230654)

Fermentation of high concentrations of lactose to ethanol by engineered flocculent saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l-1 in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l-1 h-1, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.Fundação para a Ciência e a Tecnologia (FCT

An open and parallel multiresolution framework using block-based adaptive grids

A numerical approach for solving evolutionary partial differential equations in two and three space dimensions on block-based adaptive grids is presented. The numerical discretization is based on high-order, central finite-differences and explicit time integration. Grid refinement and coarsening are triggered by multiresolution analysis, i.e. thresholding of wavelet coefficients, which allow controlling the precision of the adaptive approximation of the solution with respect to uniform grid computations. The implementation of the scheme is fully parallel using MPI with a hybrid data structure. Load balancing relies on space filling curves techniques. Validation tests for 2D advection equations allow to assess the precision and performance of the developed code. Computations of the compressible Navier-Stokes equations for a temporally developing 2D mixing layer illustrate the properties of the code for nonlinear multi-scale problems. The code is open source

Performance fermentativa de uma estirpe recombinante de Saccharomyces cerevisiae consumidora de lactose e floculante

In recent years, there has been a growing interest in bioreactors utilizing immobilized or flocculating cells in continuous process in order to improve the bioprocess productivity. One of possible promising implementations of continuous flocculation yeast system is bioremediation of cheese whey by means of alcoholic fermentation of lactose. The aim of this work was to carry out a kinetic analysis of alcoholic fermentation of lactose using strain NCYC869-A3/T1, a recombinant Saccharomyces cerevisiae flocculent strain expressing both the LAC4 (coding for b-galactosidase) and LAC12 (lactose permease) genes of Kluyveromyces lactis. Fermentations were performed in a 600 mL bubble column bioreactor, with different initial lactose concentrations. The lactose was completely consumed in all the fermentations. The maximum specific growth rate was found to increase with initial lactose concentration, reaching its maximum at 20 g L-1 initial lactose (doubling time of about 2 h). At higher initial lactose concentrations, specific growth rate decreased, indicating that the effect of substrate inhibition had become significant. The maximum ethanol concentration produced increased linearly when the initial lactose concentration was increased between 5 and 200 g L-1. However, the ethanol yields obtained were low (45 60% of the theoretical value), probably because of the high aeration rates used. In shake-flask fermentations, in conditions of micro aeration, the yeast was unable to completely consume 200 g L-1 initial lactose, producing a maximum of 57 g L-1 ethanol (which is about the same concentration produced in the bioreactor from complete consumption of 200 g L-1 lactose). Probably, the yeast has low ethanol tolerance and the ethanol produced inhibits further lactose fermentation. Ethanol productivity increased with increasing initial lactose concentration up to 150 g L-1 (1.23 g L-1 h-1). Further increase in initial lactose to 200 g L-1 led to a slight decrease in ethanol productivity.Recentemente, tem havido um interesse crescente em biorectores que utilizam células imobilizadas ou floculantes em processos contínuos, para melhorar a productividade dos bioprocessos. Uma das possíveis e promissoras aplicações de sistemas contínuos com células de levedura floculante é a bioremediação do soro do queijo, através da fermentação alcoólica da lactose. O objectivo deste trabalho foi fazer uma análise cinética da fermentação alcoólica da lactose utilizando a estirpe NCYC869-A3/T1, uma estirpe recombinante de S. cerevisiae floculante que expressa os genes LAC4 (codifica a b-galactosidase) e LAC12 (permease da lactose) de Kluyveromyces lactis. As fermentações foram feitas numa coluna de bolhas de 600 mL, com diferentes concentrações iniciais de lactose. A lactose foi consumida completamente em todas as fermentações. A taxa específica de crescimento máxima ampliou com o aumento da concentração inicial de lactose, atingindo o valor máximo para uma concentração inicial de lactose de 20 g L-1 (tempo de duplicação de aproximadamente 2 h). Com concentrações iniciais de lactose mais elevadas, a taxa específica de crescimento diminuiu, indicando que o efeito da inibição pelo substrato se tornou significativo. A concentração máxima de etanol produzido ampliou linearmente com o aumento da concentração incial de lactose entre 5 e 200 g L-1. Contudo, os rendimentos em etanol obtidos foram baixos (45 60 % do valor teórico), provavelmente devido às elevadas taxas de arejamento utilizadas. Em fermentações realizadas em matrazes agitados, em condições de micro-arejamento, a levedura foi incapaz de consumir completamente uma concentração inicial de lactose de 200 g L-1, produzindo um máximo de 57 g L-1 de etanol (aproximadamente a mesma concentração produzida no biorector a partir do consumo completo de 200 g L-1 de lactose). Provavelmente, a levedura tem uma baixa tolerância ao etanol, e o etanol produzido inibiu a fermentação da lactose que restava. A productividade em etanol ampliou com o aumento da concentração inicial de lactose até 150 g L-1 (1.23 g L-1 h-1). O aumento da concentração inicial de lactose para 200 g L-1 conduziu a um ligeiro decréscimo da productividade em etanol.Fundação para a Ciência e a Tecnologia (FCT)

Ethanol production from high-glucose industrial substrates using ethanol-tolerant Saccharomyces cerevisiae strains

Ethanol is well known as a toxic metabolite for yeast cells. Thus, strains that can grow well under high ethanol stress condition are highly desirable. This work aims to select and characterize Saccharomyces cerevisiae strains with improved ethanol tolerance. Moreover, it aims to evaluate the feasibility of industrial residues as fermentation media and to optimize the composition of such media. The ethanol production and tolerance of the yeast strains have been evaluated, carrying out batch alcoholic fermentations with high-glucose YP medium. The most ethanol-tolerant strain was able to ferment 300 g/L glucose producing up to 17.4 % (v/v) of ethanol in trials carried out in anaerobic shake-flasks. Aiming to develop a fermentation medium based in industrial substrates, corn steep liquor (CSL) has been tested as medium supplement, in order to replace nutrients that are needed to allow both cellular growth and fermentation. Supplementation of 300 g/L glucose medium with CSL concentrations around 90 - 110 g/L has resulted in fermentation performance similar to that observed in YP medium with the same glucose concentration, thus confirming the feasibility of CSL as peptone and yeast extract substitute