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Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory. Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.

International audience; The production of 2-phenylethanol (2-PE), a high-valued aroma, has been performed naturally through the bioconversion of Lphenylalanine by the Ehrlich pathway in yeast. Nevertheless, one of the main limitations of this bioconversion is the inhibition of yeast growth by 2-PE. This work evaluated the intensification of 2-PE production in a batch culture using a conventional bioreactor coupled to an in-situ product removal (ISPR) process, by means of membrane-based solvent extraction (MBSE). The culture of the native yeast Kluyveromyces marxianus ITD0090, using a shaker flask stirred at 150 rpm, produced a maximum concentration of 2-PE of 0.7 g L −1 after 24 hours. When the yeast was cultivated in a controlled aerated-stirred bioreactor operated at 0.8 vvm and 350 rpm, the maximum production of 2-PE after 48 h reached 1.49 g L −1. Intensification of the bioprocess was achieved by coupling the controlled bioreactor with a MBSE system, which allowed the continuous recovery of the 2-PE produced, reaching a final titre of 3.02 g L −1 after 56 h.

Microbial production of aroma compounds is a promising alternative to extracting plants or chemical synthesis. The yeast Pichia fermentans ITD-00165 has demonstrated a high potential to produce isoamyl acetate. The yeast was cultured in a bioreactor on a chemically defined medium to evaluate the influence of airflow (0.5, 0.75, and 1.0 vvm) and agitation speed (150, 175, and 200 rpm) on the isoamyl acetate production. Isoamyl acetate was retained using an in situ entrapping system, which was obtained, adding a cap of n-decane in a ratio of 80:20 (medium:decane). This work demonstrated the relevance of well-controlled aeration to enhance isoamyl acetate production using isoamyl alcohol as a precursor. Culture operating near to 0.71 vvm and 168 rpm (kLa = 0.16 min-1) achieved production of 2.14 g L-1, which means an almost 2.5-fold increase in respect to the higher value previously reported. On the other hand, the minimum production occurred at the best aeration conditions (kLa = 0.22 min-1), meaning that excessive aeration could negatively affect the aroma production. Nevertheless, it appears that the concomitant ethanol production negatively affects aroma production.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low β-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized β-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The β-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the β-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, β-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

We evaluated the effects of fermentation time and acid casein content on the microbial rennet obtained by solid-state fermentation using wheat bran as the carbon source. The experiments used two fermentation times (72 and 96 h), while acid casein content was 1.5, 2.0, 2.5, and 3.0 g. Rennet strength from eight enzymatic extracts was measured using pasteurized whole milk. Rennet strength of samples from 72 h of fermentation showed an increase when acid casein content increased. The rennet strength increased at 96 h of fermentation with increasing amount of casein (up to 2.5 g), and then decreased with the largest addition (3.0 g) of casein. Coagulation time for the sample with highest rennet strength was 420 s.

A solid standard mixture (SSM) representing the annual composition of fresh fruits and vegetables residues generated at the Supply Center in Mexico City was used for bioethanol production. This type of residues allows bioethanol production with a single thermal pre-treatment instead of hard thermochemical or enzymatic treatments. The release of fermentable carbohydrates from the SSM by a mild thermal pretreatment was firstly optimized. After that, mixed and single cultures of Saccharomyces cerevisiae, Scheffersomyces stipitis, and Schwanniomyces occidentalis were evaluated for bioethanol production. The maximum ethanol production, 282.61 ± 13.09 L ethanol per ton of dry matter (DM), was reached using a severity factor (SF) of 2.35 and a mixed culture composed of Saccharomyces cerevisiae, Scheffersomyces stipitis, and Schwanniomyces occidentalis. The improved lab scale conditions were evaluated in a pilot scale (18 Kg) stirred bioreactor with an SF of 2.35 and the mixed culture, obtaining 245.72 ± 17.76 L ethanol per ton DM. The obtained results demonstrate for the first time the use of fresh fruits and vegetables residues for bioethanol production under solid-state culture conditions without any thermochemical or enzymatic pre-treatment.

Yeast cells are subjected to diverse environmental conditions during the alcoholic fermentation of agave juice, causing different kinetic behaviors. Agave juice was used as culture medium to evaluate the kinetic behavior of Saccharomyces cerevisiae ITD-00185 and Torulaspora delbrueckii ITD-00014a, as pure and mixed cultures, under different inoculum sizes (1×10 5 , 1×10 6 , 1×10 7 and 1×10 8 cells mL -1 ), and combined pH levels (3.5, 4.0 and 4.5) and temperatures (18 °C, 28 °C and 38 °C). Saccharomyces cerevisiae displayed high fermentation capacities at all inoculum concentrations assayed. However, T. delbrueckii required a high inoculum concentration (≥1×10 7 cells mL -1 ) to perform at fermentation levels similar to S. cerevisiae . Low temperatures (18 °C) slowed fermentation, while high temperatures (38 °C) adversely affected the development of the yeast strains, especially T. delbrueckii . The best temperature was 28 °C in all fermentations. The pH level had a strong effect on the performance of the coculture, since the fermentation kinetics suggested a synergistic effect at pH 4.5, while an antagonistic effect was postulated at pH 3.5. In all of the mixed culture cases, a positive effect at 28 °C, especially at pH 4.0 and 4.5, was demonstrated by greater levels of sugar consumption and ethanol production (~20%, p

Recently, biotechnological opportunities have been found in non-Saccharomyces yeasts because they possess metabolic characteristics that lead to the production of compounds of interest. It has been observed that Kluyveromyces marxianus has a great potential in the production of esters, which are aromatic compounds of industrial importance. The genetic bases that govern the synthesis of esters include a large group of enzymes, among which the most important are alcohol acetyl transferases (AATases) and esterases (AEATases), and it is known that some are present in K. marxianus, because it has genetic characteristics like S. cerevisiae. It also has a physiology suitable for biotechnological use since it is the eukaryotic microorganism with the fastest growth rate and has a wide range of thermotolerance with respect to other yeasts. In this work, the enzymatic background of K. marxianus involved in the synthesis of esters is analyzed, based on the sequences reported in the NCBI database.

The interaction between nanostructures and yeast cells, as well as the description of the effect of nanoparticles in ethanol production are open questions in the development of this nanobiotechnological process. The objective of the present study was to evaluate the ethanol production by Saccharomyces cerevisiae in the free and immobilized state on chitosan-coated manganese ferrite, using cane molasses as a carbon source. To obtain the chitosan-coated manganese ferrite, the one-step coprecipitation method was used. The nanoparticles were characterized by X-ray diffraction obtaining the typical diffraction pattern. The crystal size was calculated by the Scherrer equation as 15.2 nm. The kinetics of sugar consumption and ethanol production were evaluated by HPLC. With the immobilized system, it was possible to obtain an ethanol concentration of 56.15 g/L, as well as the total sugar consumption at 24 h of fermentation. Productivity and yield in this case were 2.3 ± 0.2 g/(L * h) and 0.28 ± 0.03, respectively. However, at the same time in the fermentation with free yeast, 39.1 g/L were obtained. The total consumption of fermentable sugar was observed only after 42 h, reaching an ethanol titer of 50.7 ± 3.1, productivity and yield of 1.4 ± 0.3 g/(L * h) and 0.25 ± 0.4, respectively. Therefore, a reduction in fermentation time, higher ethanol titer and productivity were demonstrated in the presence of nanoparticles. The application of manganese ferrite nanoparticles shows a beneficial effect on ethanol production. Research focused on the task of defining the mechanism of their action and evaluation of the reuse of biomass immobilized on manganese ferrite in the ethanol production process should be carried out in the future.

In this work we studied two approaches for the revalorization of corn stover (CS): Feedstock for bioethanol generation and substrate for the production of biocatalysts. The practical application of two of these biocatalysts (laccases and cellulases) was also evaluated. The hydrolysis of CS was improved through a pretreatment with the thermotolerant fungus Fomes sp. EUM1; simultaneously, enzymatic extracts enriched with laccases or cellulases were obtained and applied for the pretreatment or hydrolysis of untreated CS. After 7 days, the fungal pretreatment promoted a significant increase in the release of reducing sugars (60%) and in the hydrolysis rate (50%). Lignin content decreased 61.2%, while holocellulose remained practically unchanged; a significant positive correlation (r = 0.948, p = 0.01) was found between laccase production and lignin degradation. Regarding biocatalysts application, the cellulases of Fomes sp. EUM1 showed catalytic properties similar to those of commercial enzymes; in addition, the pretreatment with laccases reduced 70% the processing-time (compared to the fungal treatment), and improved the release of reducing sugars and the hydrolysis rate by 26 and 29%, respectively. The pretreatment of CS with Fomes sp. EUM1 produces two valuable products: saccharificable biomass useful for bioethanol production and highly active enzymes applicable in bioconversion processes.

Opuntia ficus-indica is used in animal feed but has a low protein content (above 4%) in comparison to high-quality forage (above 16%). It isnecessary to develop technologies that improve the nutrimental value of Opuntia ficus-indica through the process of solid-state fermentation (SSF). Therefore, the objective of this study was to evaluate the changes in the chemical composition of Opuntia ficus-indica during the SSF with the yeast Kluyveromyces marxianus ITD00262. Kluyveromyces marxianus ITD00262 was grown in glucose, peptone, yeast (GPY) broth, at 28 oC and pH 4.8 for 12 h. Opuntia ficus-indica was incubated in SSF at 28 °C without inoculation (T1) or inoculated with K. marxianus ITD00262 (T2). Fermented samples of Opuntia ficus-indica were dried, and the dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin (LIGN) and in vitro dry matter digestibility (IVDMD) contents were ascertained. Data were analyzed with a completely randomized design with a factorial arrangement of 2×6. A decrease of 11% in DM was observed for T1 compared with T2 at 48 h of fermentation. A 300% increase in PC content was observed for T2 at 120 h of fermentation. The DMIVD was increased by 3%, whereas the values of NDF and ADF decreased by 18% in T2. According to our results, it is confirmed that the use of K. marxianus in the SSF of Opuntia ficus-indica has a positive effect and improves the nutrimental quality of this forage.

The goal of this study was to evaluate the effect of high Tween 80 concentrations on hydrocarbon contaminants and hydrocarbonoclastic microorganisms present in contaminated mining soil during a bioremediation process. The independent variables included Tween 80 concentration and process time. The elected response variables included concentration of total petroleum hydrocarbons (TPH), the chemical composition of contaminants, viable microbial count, and CO2 production. These were measured at various time points during the bioremediation process, which was conducted at room temperature. Higher removal of pollutants occurred at low Tween 80 concentrations. An analysis of the normalized variables shows that the rate of TPH degradation depended on microbial growth rate, which depended on Tween 80 concentration. The addition of Tween 80 changed the concentration and type of the residual hydrocarbon contaminants present, as well as the count and genus of the hydrocarbonoclastic microorganisms that grew during the bioremediation process. Higher concentrations of Tween 80 increased the levels of the chemical compounds present in the bioremediated soil while reducing the viable count and microbial diversity during the bioremediation process. Conversely, low Tween 80 concentrations produced only monoethylhexyl phthalate, which is not a petroleum hydrocarbon derivative. Overall, these results prove that the removal of TPH is dependent on the count and genus of active indigenous hydrocarbonoclastic microorganisms in soil, and these conditions may be controlled by modulating the concentration of Tween 80 that is applied.

International audience; This study deals with the production of isoamyl acetate (IAA) by fermentation of sugar cane molasses with the strain Pichia fermentans ITD00165, using L-leucine as precursor. A mathematical model that describes the experimental data from fermentation was developed for its use as a tool for further process optimization. The fermentation system was constantly aerated and coupled to liquid–liquid in situ extraction with decane as the recovery solvent. Thus, the model integrates the biological production of IAA, its partition coefficient in the two liquid phase system and the stripping effect of aeration. A productivity of 26 mg L−1 h−1 was obtained with addition of 4 g L−1 of L-leucine at 12 h of fermentation. The use of the model for process optimization was explored. According to it, the maximum theoretical productivity that can be obtained is 63 mg L−1 h−1. The model was used to determine that 1.6 g L−1 is the minimum concentration of L-leucine that can be added without significantly reducing IAA production. Also, it makes possible to propose an adequate decane/culture medium ratio, to have a desired final concentration and amount of recovered IAA. This value can be adjusted based on the needs of further purification steps and is useful to define a global economic optimum of the process.

Processing option such as fungal fermentation (tempeh) improves legume nutraceutical properties. The aim of this work was to evaluate the physicochemical and antioxidant potential of common bean tempeh flour from two varieties: Bayo victoria (BV) and Pinto durango (PD) processed at two different temperature and fermentation times. Results showed differences between cultivars followed by changes in temperature and fermentation times, being more significant at 35C for 40 h. The phenolic content in both cultivars varied considerably after cooking, being higher in raw flour (0.4–3.0-fold for BV and 0.35–0.5-fold for PD). The highest phenolic content was for BV fermented at 35C and 40 h, whereas for PD was at 30C and 40 h. Antioxidant capacity was evaluated by 2,2-diphenyl-1-picrylhydrazyl, low-density lipoprotein oxidation and hydroxyl radical-scavenging assays. Results showed different antioxidant capacity for each test. The major differences in results were shown between cultivars than the processing variations of temperature and fermentation time. Practical Applications Phaseolus vulgaris is one of the most important grain legumes for human consumption attributable to its nutritional properties, low cost and health promoter effects. However, the nutraceutical properties of this legume have changed due to process variables such as temperature and time of cooking. It has been demonstrated experimentally that fungal fermentation or tempeh production can be an efficient strategy to improve the phenolic content and antioxidant activity of common beans, becoming a favored alternative as an ingredient/supplement development for the prevention and control of degenerative diseases. However, little information is available on the effect of temperature, fermentation time and common bean cultivars in relation with their nutraceutical properties. The present investigation demonstrated that the major effects were between cultivars rather than processing variations of temperature and fermentation time. Therefore, considering cultivars and processing variables, common bean tempeh flour may be an efficient strategy to enhance the antioxidant activity of this seed.

Agave plants are members of the Agavaceae family and utilize crassulacean acid metabolism (CAM) for CO2 fixation. Fructans are the main photosynthetic products produced by Agave plants, and are their principal source of storage carbohydrates. The aim of this work was to determine the chemical and molecular characterization of fructans from Agave durangensis. Fructans were extracted from 10 year old A. durangensis plants. Trimethylsilyl derivatization was employed to determine the monomer composition. The linkage types in these carbohydrates were determined by methylation followed by reduction and O-acetylation, and finally analysis by gas chromatography-mass spectrometry (GC-MS). Samples were shown to contain t-β-D-Fruf, t-α-D-Glup, i-α-D-6-Glup and 1,6-di-β-D-Fruf linkages. The analysis of the degree of polymerization (DP) was confirmed by MALDI-TOF-MS, showing a wide DP ranging from 2 to 29 units. The analyses performed revealed that fructans from A. durangensis are formed of 97.11% fructose and 2.89% glucose, and are a complex mixture of fructooligosaccharides of the neo-fructan type containing principally β(2-1) and β(2-6) linkages, with branch moieties. Key words: Degree of polymerization (DP), GC-MS, MALDI-TOF-MS.

The effect of pH, C/N ratio, addition of a microbial consortium (MC) and temperature upon mineralisation of Organic Fraction of Municipal Solid Waste (OFMSW) was studied; mineralisation was measured through the CO2 production rate and total CO2 formation. Through this process up to 432.9mg of CO2g(-1) initial dry matter (IDM) after 2days of treatment was obtained. It was found that under a slightly acidic pH (5-6) and C/N of 30, the mineralisation process was accelerated. Moreover, temperature (27-50°C) had no effect on the total CO2 produced. The highest CO2 production rate (5.28d(-1)) was observed at 27°C, C/N ratio of 30 and 8% of microbial consortium; it is at least 3.52 times higher than that reported (1.5d(-1)). The highest release of reducing sugars was determined at 50°C, possibly due to an increase in hydrolytic enzymes. Results suggest the potential use of rapid mineralisation of OFMSW for further friendly environmental processes.

Agave is a good source of polysaccharides for the production of fermentable sugars as sustainable bioenergy feedstock solutions for semi-arid and arid lands. This plant grows in arid areas, which correspond to a large territory in northern Mexico. Having lignocellulose as the polysaccharide of interest, the information for the enzymatic saccharification of this kind of material is limited. Agave cell walls have a unique recalcitrant nature, but having a high cellulose content, makes this plant material an interesting research subject. In this work, acidic, alkaline and aqueous pretreatments were evaluated to generate a biomass rich in cellulose. The saccharification of pretreated Agave leaves-residue was evaluated under experimental designs to identify the most suitable conditions for enzymatic hydrolysis. Maximum value obtained was 31% glucose, which further increased to 41.4% at extended hydrolysis time of 96 h. The highest cellulose-saccharification reached was up to 61.81%, making Agave atrovirens an alternative for bioethanol production in its geographical area of cultivation.

Batch denitrification of a synthetic saline medium at 37 °C, pH 7.5 was studied. A kinetic expression and an inhibition function which adequately expresses inhibition and cross-inhibition by nitrate and nitrite in denitratation and denitritation steps occurring in high organic and nitrate load wastewater treatment was determined. Denitratation and denitritation rates were measured independently. Monod- and Haldane-type kinetics were used for denitratation and denitritation, respectively. The maximum specific rate, k N O 3 = 12.7 mg N O 3 -N / g VSS h , and the saturation constant, K S N O 3 = 0.47 mg N O 3 -N / L were calculated for denitratation. The maximum specific rate, k N O 2 = 13.8 mg N O 2 -N / g VSS h , the saturation constant K S N O 2 = 0.36 mg N O 2 -N / L and the inhibition constant K I-N O 2 = 906 mg N O 2 -N / L were calculated for denitritation. Among other functional forms the Levenspiel inhibition model for denitratation and denitritation kinetics was proposed. The Levenspiel constants were S N O 2 -M = 1149.4 mg N O 2 -N / L , S N O 3 -M = 34.7 mg N O 3 -N / L , β 1 = 1.578 and β 2 = 1.005, respectively. The fitted experimental values show a good representation of the denitrification of effluents with high nitrogen load containing both nitrate and nitrite. Parametric sensitivity analysis show that inhibition functions ( f i ) and dimensionless constants ( β i ) greatly affect predicted nitrite and nitrate consumption rates.

Use of the Pirt and Luedeking-Piret equations permits the determination of the effect of medium composition on the metabolic patterns of Megasphaera elsdenii grown in minimal and complex media with lactate as the major carbon source. To establish the significance of the parameters involved in the Pirt and Luedeking-Piret equations, a quantitative statistical criterion was proposed. In the complex medium, lactate was completely used for growth and product formation, whereas in the minimal medium a fraction of the energy obtained from lactate was used for maintenance purposes. Modeling of VFA production by the Luedeking-Piret equation showed that, independent of the type of medium, acetate and propionate are growth-associated products, while butyrate and valerate are only partially growth-associated. The growth-associated products are related to energy-yielding metabolism and the non-growth-associated products are related to the consumption of reducing equivalents.

Continuous culture study of the ruminal bacterium Megasphaera elsdenii using lactate as carbon source is reported. Butyrate, the minor product in batch culture, was the main product in continuous culture. Under steady state conditions the biomass concentration was increased from 0.46 to 0.72 g l-1. Addition of Yea Sacc did not result an increased availability of carbon. The produced amounts of valerate and CO2 were calculated by using a combined carbon and degree of reduction balances. According to these results, the lactate catabolism of M. elsdenii was changed by the presence of yeast additive, yielding more butyrate and diminishing the production of valerate, while acetate and propionate production was not affected. Results reported in this work suggested that soluble compounds present in the yeast additive filtrate enhanced the anabolism of M. elsdenii and modified the carbon fluxes through its pathways by increasing butyrate and decreasing valerate production in continuous culture.Estudo em cultura contínua da bactéria ruminal Megasphaera elsdenii que utiliza lactato como fonte de carbono foi realizada. Butirato, foi o produto produzido em menor concentração na cultura em batelada, porém em cultura contínua foi o produto de maior concentração. Sob condições definidas a concentração de biomassa aumentou de 0.46 a 0.72 g l-1. A adição do aditivo Yea Sacc não resultou num aumento da disponibilidade do carbono. As quantidades produzidas de valerato e CO2 foram calculadas pelo uso combinado do balanço e grau de redução do carbono. De acordo com esses resultados, o catabolismo de lactato de M. elsdenii foi mudado pela adição de aditivo a base de levedura. Maior rendimento em butirato e redução na produção de valerato foi observada, por outro lado a produção deacetato e propionato não foi afetada. Os resultados desse trabalho sugerem que os componentes solúveis presentes no aditivo a base de levedura aumentam o anabolismo de M. elsdenii modificando o fluxo de carbono através de suas vias de metabólicas, pelo aumento do butirato e redução do valerato. Em cultura contínua.

The effect of the composition of a mixture containing, oat straw (OS), oat bran (OB) and copra cake (CC), on the mycelial growth of Pleurotus ostreatus was studied using mixture and response surface methodologies. The applied constraints to the mixtures were: moisture content higher than 70%, C/N ratio less than 30 and total mixture cost less than 2% of the retail cost of fruiting bodies of P. ostreatus. The maximum observed value of apical growth rate (0.50±0.02 cm day−1) was obtained using 0.633, 0.284 and 0.083 (g g−1 mixture, dry basis) for OS, CC and OB mass fractions, respectively. Under these conditions the C/N ratio was 22.4–23.2. Loss of dry matter decreased from 16.9 to 8.5% as the OS fraction (lignin and cellulose source) was increased from 0.55 to 0.80 (g g−1 mixture, dry basis). The utilisation of mixture and response surface methodologies was an useful approach to evaluate the relationship between substrate composition and mycelial development of P. ostreatus.

In this work, fifty yeast strains, isolated from the spontaneous alcoholic fermentation of Agave duranguensis to produce mezcal, were tested using the double coupling system. These yeasts were from the genera Pichia, Torulaspora, Saccharomyces, Kluyveromyces, Deckera, Hanseniaspora, and Candida. P. fermentans ITD00165 was the best isoamyl acetate producer, yielding 0.38 g/L of ester after incubation for 24 h, while K. marxianus ITD00211 produced 0.32 g/L of ester. Thus P. fermentans TD00165 could be considered as an excellent choice for use in optimization studies of the culture medium and bioreactor operating conditions to develop a process for biotechnological production of isoamyl acetate.

Alpha-synuclein is a major component of Lewy bodies (LBs) in the substantia nigra and cortex in Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and in glial inclusions in multiple systems atrophy (MSA). Mutations in alpha-synuclein have been associated with autosomal dominant forms of PD. We investigated the clinical and neuropathological effects of overexpression of human alpha-synuclein, alpha-synuclein A30P, and alpha-synuclein A53T under the control of the hamster prion protein (PrP) promoter; 5-15x endogenous levels of protein expression were achieved with widespread neuronal, including nigral, transgene expression. High expression of alpha-synuclein A30P in the Tg5093 line was associated with a progressive motor disorder with rigidity, dystonia, gait impairment, and tremor. Histological analysis of this line showed aberrant expression of the protein in cell soma and progressive CNS gliosis, but no discrete Lewy body-like alpha-synuclein inclusions could be identified. Biochemical analysis demonstrated alpha-synuclein fragmentation. Despite strong expression of the transgene in the nigra, there was no specific deterioration of the nigrostriatal dopaminergic system as assessed by quantitation of nigral tyrosine hydroxylase (TH) containing neurons, striatal TH immunoreactivity, dopamine levels, or dopamine receptor number and function. Lower expressing lines had no specific behavioral or histopathological phenotype. Thus, high expression of mutant human alpha-synuclein resulted in a progressive motor and widespread CNS gliotic phenotype independent of dopaminergic dysfunction in the Tg5093 line.

Restarter yeast strains are used to consume residual sugar (mainly fructose) in stuck wine fermentations. Forty-three yeast strains were evaluated initially. Strain ITD-00068 showed the highest values for the maximum growth rate, maximum fructose consumption rate, and maximum ethanol production rate (0.143, 0.268, and 0.231 h−1, respectively). Therefore, it was selected for further molecular and kinetic analyses. RFLP analysis identified this isolate as Saccharomyces cerevisiae. Strain ITD-00068 consumed 100% and 36% of the fructose present (at 20°C and 30°C, respectively), when cultured in medium with 12% (v/v) ethanol. The fructose consumption rate was reduced by 97% at 30°C, when ethanol was initially present in the media. S. cerevisiae ITD-00068 was tested in stuck fermentation conditions and was able to complete fermentation in 144 h in a commercial red wine, demonstrating that S. cerevisiae ITD-00068 is a potential candidate for use as a restarter in wine stuck fermentation at 20°C.