CFD predicted pH gradients in lactic acid bacteria cultivations.

The formation of pH gradients in a 700 L batch fermentation of Streptococcus thermophilus was studied using multi-position pH measurements and computational fluid dynamics (CFD) modeling. To this end, a dynamic, kinetic model of S. thermophilus and a pH correlation were integrated into a validated one-phase CFD model, and a dynamic CFD simulation was performed. First, the fluid dynamics of the CFD model were validated with NaOH tracer pulse mixing experiments. Mixing experiments and simulations were performed whereas multiple pH sensors, which were placed vertically at different locations in the bioreactor, captured the response. A mixing time of about 46 s to reach 95% homogeneity was measured and predicted at an impeller speed of 242 rpm. The CFD simulation of the S. thermophilus fermentation captured the experimentally observed pH gradients between a pH of 5.9 and 6.3, which occurred during the exponential growth phase. A pH higher than 7 was predicted in the vicinity of the base solution inlet. Biomass growth, lactic acid production, and substrate consumption matched the experimental observations. Moreover, the biokinetic results obtained from the CFD simulation were similar to a single-compartment simulation, for which a homogeneous distribution of the pH was assumed. This indicates no influence of pH gradients on growth in the studied bioreactor. This study verified that the pH gradients during a fermentation in the pilot-scale bioreactor could be accurately predicted using a coupled simulation of a biokinetic and a CFD model. To support the understanding and optimization of industrial-scale processes, future biokinetic CFD studies need to assess multiple types of environmental gradients, like pH, substrate, and dissolved oxygen, especially at industrial scale.

Fsy1, the sole hexose-proton transporter characterized in Saccharomyces yeasts, exhibits a variable fructose:H(+) stoichiometry.

In the model yeast Saccharomyces cerevisiae, hexose uptake is mediated exclusively by a family of facilitators (Hxt, hexose transporters). Some other Saccharomyces species (e.g. Saccharomyces bayanus and Saccharomyces pastorianus) possess, in addition, a specific fructose transporter (Fsy1, fructose symporter) that has been previously described to function as a proton symporter. In the present work, we compared growth of a yeast strain in which FSY1 occurs naturally in anaerobic, fructose- and glucose-limited chemostat cultures. Especially at low specific growth rates, fructose-proton symport was shown to have a strong impact on the biomass yield on sugar. We subsequently employed energized hybrid plasma membrane vesicles to confirm previous observations concerning the mode of operation and specificity of Fsy1 mediated transport. Surprisingly, these experiments suggested that the carrier exhibits an unusual fructose:H(+) stoichiometry of 1:2. This energetically expensive mode of operation was also found consistently in vivo, in shake flask and in chemostat cultures, and both when Fsy1 is the sole transporter and when the Hxt carriers are present. However, it is observed only when Fsy1 is operating at higher glycolytic fluxes, a situation that is normally prevented by downregulation of the gene. Taken together, our results suggest the possibility that fructose symport with more than one proton may constitute an energetically unfavorable mode of operation of the Fsy1 transporter that, in growing cultures, is prevented by transcriptional regulation.

Performance of a two-stage anaerobic digestion system treating fruit pulp waste: The impact of substrate shift and operational conditions.

Food and beverage industry wastes present high amounts of organic matter, which may cause water quality degradation if not treated. Two-stage anaerobic digestion is a promising and efficient solution for the treatment of this type of wastes whilst producing bioenergy. The composition of fruit pulp waste varies throughout the different harvesting seasons, which may impact the process performance. In this study, a two-stage anaerobic digestion system was operated to assess the effect of substrate shift from peach to apple pulp wastes (obtained from a fruit juice company) on the microbial community activity and performance. During acidogenesis, the sugar conversion was higher than 95% for all operational conditions tested, obtaining a degree of acidification up to 89%. Principal Component Analysis was used to evaluate the relationship between the initial fermentation state of the residues in each operational condition and the obtained effluent. Methanogenic activity resulted in high organic carbon consumption (89%) and high methane productivities, achieving a maximum of 4.33 [Formula: see text] for peach waste influent. Overall, the results showed that the microbial community activity was not affected by the substrate shift, converting the sugars into biogas rich in methane (>70% CH4). Microbial analysis showed that the communities present in the acidogenic and methanogenic reactors were highly enriched in bacteria and archaea, respectively. The observed stability of the process, also demonstrated in pilot scale, confirmed the robustness of the process and thus, was suitable for implementation in companies producing seasonally different fruit wastes in a continuous operation.

Improving succinic acid production by Actinobacillus succinogenes from raw industrial carob pods.

Carob pods are an inexpensive by-product of locust bean gum industry that can be used as renewable feedstock for bio-based succinic acid. Here, for the first time, unprocessed raw carob pods were used to extract a highly enriched sugar solution, afterwards used as substrate to produce succinic acid using Actinobacillus succinogenes. Batch fermentations containing 30g/L sugars resulted in a production rate of 1.67gSA/L.h and a yield of 0.39gSA/g sugars. Taking advantage of A. succinogenes' metabolism, uncoupling cell growth from succinic acid production, a fed-batch mode was implemented to increase succinic acid yield and reduce by-products formation. This strategy resulted in a succinic acid yield of 0.94gSA/g sugars, the highest yield reported in the literature for fed-batch and continuous experiments, while maintaining by-products at residual values. Results demonstrate that raw carob pods are a highly efficient feedstock for bio-based succinic acid production.

Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments.

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks.

Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains.

Two industrial effluents, a pre-fermentation effluent and a post-fermentation effluent from a wheat starch production plant, were used as substrates for fuel ethanol production in anaerobic batch cultures using minimal nutritional amendment. The performances of three metabolically engineered xylose-utilizing Saccharomyces cerevisiae strains: TMB 3001 expressing XYL1, XYL2 and XKS1, redox metabolism modulated CPB.CR1 and glucose de-repressed CPB.CR2, as well as a reference strain CEN.PK 113-7D not fermenting xylose, were evaluated. For the recombinant strains a glucose consumption phase preceded the xylose consumption phase. In both effluents, biomass and ethanol production occurred predominantly during the glucose consumption phase, whereas xylitol and glycerol formation were predominant in the xylose consumption phase. Total specific ethanol productivities on glucose were 6-fold higher than on xylose in the pre-fermentation effluent and 15-fold higher than on xylose in the post-fermentation effluent. CPB.CR1 showed impaired growth compared to the two other xylose-utilizing strains, but displayed 18% increased ethanol yield in the post-fermentation effluent.

Exopolysaccharides enriched in rare sugars: bacterial sources, production, and applications.

Microbial extracellular polysaccharides (EPS), produced by a wide range of bacteria, are high molecular weight biopolymers, presenting an extreme diversity in terms of chemical structure and composition. They may be used in many applications, depending on their chemical and physical properties. A rather unexplored aspect is the presence of rare sugars in the composition of some EPS. Rare sugars, such as rhamnose or fucose, may provide EPS with additional biological properties compared to those composed of more common sugar monomers. This review gives a brief overview of these specific EPS and their producing bacteria. Cultivation conditions are summarized, demonstrating their impact on the EPS composition, together with downstream processing. Finally, their use in different areas, including cosmetics, food products, pharmaceuticals, and biomedical applications, are discussed.

Carob pod water extracts as feedstock for succinic acid production by Actinobacillus succinogenes 130Z.

Carob pods are a by-product of locust bean gum industry containing more than 50% (w/w) sucrose, glucose and fructose. In this work, carob pod water extracts were used, for the first time, for succinic acid production by Actinobacillus succinogenes 130Z. Kinetic studies of glucose, fructose and sucrose consumption as individual carbon sources till 30g/L showed no inhibition on cell growth, sugar consumption and SA production rates. Sugar extraction from carob pods was optimized varying solid/liquid ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Batch fermentations containing 10-15g/L total sugars resulted in a maximum specific SA production rate of 0.61Cmol/Cmol X.h, with a yield of 0.55Cmol SA/Cmol sugar and a volumetric productivity of 1.61g SA/L.h. Results demonstrate that carob pods can be a promising low cost feedstock for bio-based SA production.

Succinic acid production from glycerol by Actinobacillus succinogenes using dimethylsulfoxide as electron acceptor.

Glycerol, a highly abundant byproduct of the biodiesel industry, constitutes today a cheap feedstock for biobased succinic acid (SA) production. Actinobacillus succinogenes is one of the best SA producers. However, glycerol consumption by this biocatalyst is limited because of a redox imbalance during cell growth. The use of an external electron acceptor may improve the metabolism of SA synthesis by A. succinogenes in glycerol. In this study, the effect of dimethylsulfoxide (DMSO), an electron acceptor, on glycerol consumption and SA production by A. succinogenes under controlled fermentation conditions was investigated. Concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by A. succinogenes. During fed-batch cultivation, SA concentration reached 49.62 g/L, with a product yield of 0.87 gSA/gGLR and a maximum production rate of 2.31 gSA/Lh, the highest values so far reported in the literature for A. succinogenes using glycerol as carbon source. These results show that using DMSO as external electron acceptor significantly promotes glycerol consumption and succinic acid production by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst.

A powerful yeast-based screening assay for the identification of inhibitors of indoleamine 2,3-dioxygenase.

Activation of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) underlies the course of several human pathological conditions and, to date, no efficacious therapeutic IDO inhibitors are available. We proposed to develop a robust screening system based on the use of yeast cells to identify new lead compounds for the pharmacological inhibition of IDO-the BLOCKADE platform. Yeast combines the advantages of a relevant surrogate model for eukaryotic cell processes with the amenity to miniaturization and automation. We brought added value to the system by increasing the stringency of our assay, as the BLOCKADE strain was not deleted for any efflux pump, thus creating additional challenges for test compounds to be identified as hits. Screening of a library of 50 080 small molecules led to the identification of 101 potential IDO inhibitors, a low hit rate of 0.2%, reflecting the stringent assay conditions imposed. Most important, secondary pharmacology assays in mammalian cells confirmed activity for 76% of the hits, whereas hepatotoxicity testing indicated that 87% of them displayed a safe profile. The high predictivity rates obtained using the BLOCKADE platform clearly validate our system as a powerful tool for drug discovery.

CreA influences the metabolic fluxes of Aspergillus nidulans during growth on glucose and xylose.

The physiological phenotype of Aspergillus nidulans was investigated for different genetic and environmental conditions of glucose repression through the quantification of in vivo fluxes in the central carbon metabolism using (13)C-metabolic-flux analysis. The particular focus was the role of the carbon repressor CreA, which is the major regulatory protein mediating carbon repression in many fungal species, in the primary metabolism of A. nidulans. Batch cultivations were performed with a reference strain and a deletion mutant strain (creADelta4) using [1-(13)C]glucose as carbon source. The mutant strain was also grown on a mixture of [1-(13)C]glucose and unlabelled xylose. Fractional enrichment data were measured by gas chromatography-mass spectrometry. A model describing the central metabolism of A. nidulans was used in combination with fractional enrichment data, and measurements of extracellular rates and biomass composition for the estimation of the in vivo metabolic fluxes. The creA-mutant strain showed a lower maximum specific growth rate than the reference strain when grown on glucose (0.11 and 0.25 h(-1), respectively), whereas the specific growth rate of the mutant strain grown on the glucose/xylose mixture was identical to that on glucose (0.11 h(-1)). Different patterns and increased levels of extracellular polyols were observed both upon deletion of the creA gene and upon addition of xylose to the growth medium of the mutant strain. Concerning metabolic fluxes, the major change observed in the flux distribution of A. nidulans upon deletion of the creA gene was a 20 % decrease in the flux through the oxidative part of the pentose-phosphate pathway. Addition of xylose to the growth medium of the mutant resulted in an increase of about 40 % in the activity of the oxidative part of the pentose-phosphate pathway, as well as decreases in the fluxes through the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle (in the range of 20-30 %). The derepression of key pathways leads to alterations in the demands for cofactors, thereby imposing changes in the central metabolism due to the coupling of the many different reactions via the redox and energy metabolism of the cells.

Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production.

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.