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A quick fed-batch saccharification (QFBs) strategy of pretreated wheat straw at high solid loadings (HSL) was developed to achieve high sugar concentrations and increase lignocellulosic ethanol productivities in the fermentation stage. Pretreatment time, number of feedings, and the time between them were evaluated using a Box-Behnken design. A surface response model was employed to maximize sugars released at 20% (w/w) HSL. Total sugar concentrations after 60 h with 20% (w/w), 25% (w/w), and 30% (w/w) HSL were 128.5 g/l, 142.4 g/l, and 163.5 g/l, respectively. These were 14.6–42% higher than their batch saccharification counterparts. The QFBs was implemented as part of sequential (SHF) and simultaneous saccharification-fermentation (SSF) strategies as well as the presaccharification stage of a semi-simultaneous saccharification-fermentation (SSSF) strategy. In the latter strategy, the highest ethanol concentration achieved was 31.7 g/l. The ethanol volumetric productivity was 2.2- and 3-fold their SHF and SSF counterparts.

Background: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by β-galactosidases. Results: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with β(1 → 6) and β(1 → 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. Conclusions: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides. How to cite: Yañez-Ñeco CV, Cervantes FV, Amaya-Delgado L, et al. Synthesis of β(1→3) and β(1→6) galactooligosaccharides from lactose and whey using a recombinant β-galactosidase from Pantoea anthophila. Electron J Biotechnol 2020;49. https://dx.doi.org/10.1016/j.ejbt.2020.10.004, This work was supported by the bilateral project CONACYT-ANUIES/ECOS-NORD M14A01, CONACYT project CB-2012-01/000000000181766 and CONACYT and the FODECIJAL project7907-2019. This work was also supported by a grant from the Spanish Ministry of Economy and Competitiveness (GrantBIO2016-76601-C3-1-R). We thank CONACYT for Ph.D. grants to Fadia Cervantes and Claudia Yañez

Microbial physiology is an essential characteristic to be considered in the research and industrial use of microorganisms. Conventionally, the study of microbial physiology has been limited to carrying out qualitative and quantitative analysis of the role of individual components in global cell behaviour at a specific time and under certain growth conditions. In this framework, groups of observable cell physiological variables that remain over time define the physiological states. Recently, with advances in omics techniques, it has been possible to demonstrate that microbial physiology is a dynamic process and that, even with low variations in environmental culture conditions, physiological changes in the cell are provoked. However, the changes cannot be detected at a macroscopic level, and it is not possible to observe these changes in real time. As an alternative to solve this inconvenience, dielectric spectroscopy has been used as a complementary technique to monitor on-line cell physiology variations to avoid long waiting times during measurements. In this review, we discuss the state-of-the-art application of dielectric spectroscopy to unravel the physiological state of microorganisms, its current state, prospects and limitations during fermentation processes. Key points • Summary of the state of the art of several issues of dielectric spectroscopy. • Discussion of correlation among dielectric properties and cell physiological states. • View of the potential use of dielectric spectroscopy in monitoring bioprocesses.

Optimum conditions for the enzymatic hydrolysis of non-pretreated agave bagasse using two binary mixtures of commercial enzymatic preparations with cellulase and hemicellulase activities (Celluclast 1.5 L/Viscozyme L and Cellic® Ctec2/Cellic® Htec2) were determined. The optimization was carried out by using the response surface methodology (RSM). Two response variables were analyzed, hydrolysate reducing sugar concentration (RS; mg/mL) and saccharification yield (SY; mg RS/g bagasse). Higher RS concentration (27.58 ± 3.68 mg/mL) and SY (312.54 ± 46.89 mg RS/g bagasse) were obtained with the mixture Celluclast 1.5 L and Viscozyme L, even when compared with lignocellulosic biomass specialized enzyme mixture of Cellic® Ctec2 and Cellic® Htec2. The RS concentration in the hydrolysate and the SY obtained under optimal conditions for both enzymatic mixtures were higher when compared with previous results. In the case of the mixture Cellic® Ctec2 and Cellic® Htec2, an increase of 5.4- and 2.2-times for the RS concentration and SY were respectively observed, while in the case of the mixture Celluclast 1.5 L and Viscozyme L, an increase of 2- and 1.6-times were detected. The degree of synergy (DS) for the enzymatic hydrolysis was 1.52 (with SY as response variable) and 1.65 (with RS concentration as response variable) for the mixture of Celluclast 1.5 L and Viscozyme L, which indicates the synergism between the enzymes present in this binary mixture.

Fructans are the main sugar in agave pine used by yeasts during mezcal fermentation processes, from which Candida apicola NRRL Y-50540 and Torulaspora delbrueckii NRRL Y-50541 were isolated. De novo transcriptome analysis was carried out to identify genes involved in the hydrolysis and assimilation of Agave fructans (AF). We identified a transcript annotated as SUC2, which is related to β-fructofuranosidase activity, and several differential expressed genes involved in the transcriptional regulation of SUC2 such as: MIG1, MTH1, SNF1, SNF5, REG1, SSN6, SIP1, SIP2, SIP5, GPR1, RAS2, and PKA. Some of these genes were specifically expressed in some of the yeasts according to their fructans assimilation metabolism. Different hexose transporters that could be related to the assimilation of fructose and glucose were found in both the transcriptomes. Our findings provide a better understanding of AF assimilation in these yeasts and provide resources for further metabolic engineering and biotechnology applications.

Cell physiology parameters are essential aspects of biological processes; however, they are difficult to determine on-line. Dielectric spectroscopy allows the on-line estimation of viable cells and can provide important information about cell physiology during culture. In this study, we investigated the dielectric property variations in Kluyveromyces marxianus SLP1 and Saccharomyces cerevisiae ERD yeasts stressed by 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde during aerobic growth. The dielectric properties of cell permittivity, specific membrane capacitance (Cm), and intracellular conductivity (σIn) were considerably affected by furan aldehydes in the same way that the cell population, viability, cell size, substrate consumption, organic acid production, and respiratory parameters were. The yeasts stressed with furan aldehydes exhibited three physiological states (φ): adaptation, replicating, and nonreplicating states. During the adaptation state, there were small and stable signs of permittivity, Cm, and σIn; additionally, no cell growth was observed. During the replicating state, cell growth was restored, and the cell viability increased; in addition, the permittivity and σIn increased rapidly and reached their maximum values, while the Cm decreased. In the nonreplicating state, the permittivity and σIn were stable, and Cm decreased to its minimum value. Our results demonstrated that knowing dielectric properties allowed us to obtain information about the physiological state of the cells under control and stressed conditions. Since the permittivity, Cm, and σIn are directly associated with the physiological state of the yeast, these results should contribute to a better understanding of the stress response of yeasts and open the possibility to on-line monitor and control the physiological state of the cell in the near future.

This work proposes a mathematical model for describing the dynamics of xylan depolymerisation of lignocellulosic biomass in a pilot-scale pretreatment continuous tubular reactor. Xylan depolymerisation, modelled as pseudo-homogeneous first order reactions and mass flow dynamics, are described as a set of ordinary differential equations. The model accurately describes the dynamics of xylooligosaccharides and other depolymerisation products at the reactor output. Experimental validations of residence time distribution, mass flow rates and kinetics were carried out at 18 and 37 min of mean residence time for wheat straw, corn stover and sugar cane bagasse. Wheat straw exhibited the highest production of xylooligosaccharides (approximately 70%) without degradation at 37 min of residence time.

Protein display approaches have been useful to endow the cell surface of yeasts with new catalytic activities so that they can act as enhanced whole-cell biocatalysts. Despite their biotechnological potential, protein display technologies remain poorly developed for filamentous fungi. The lignocellulolytic character of some of them coupled to the cell surface biosynthesis of valuable molecules by a single or a cascade of several displayed enzymes is an appealing prospect. Cell surface protein display consists in the co-translational fusion of a functional protein (passenger) to an anchor one, usually a cell-wall-resident protein. The abundance, spacing, and local environment of the displayed enzymes-determined by the relationship of the anchor protein with the structure and dynamics of the engineered cell wall-are factors that influence the performance of display-based biocatalysts. The development of protein display strategies in filamentous fungi could be based on the field advances in yeasts; however, the unique composition, structure, and biology of filamentous fungi cell walls require the customization of the approach to those microorganisms. In this prospective review, the cellular bases, the design principles, and the available tools to foster the development of cell surface protein display technologies in filamentous fungi are discussed.

Volatile esters are formed during alcoholic fermentation, particularly that performed by yeasts. These compounds confer fruity attributes to drinks. Fermented beverages contain namely two groups of esters: acetate esters formed by alcohol acetyltransferase enzymes (Atf1 and Atf2) and ethyl esters produced by the acyl-CoA ethanol O-acyltransferase enzymes (Eht1 and Eeb1), encoded by the ATF1, ATF2, EHT1 and EEB1 genes. The purpose of this study was to evaluate the effect of nitrogen addition, aeration and low temperatures on ester production during fermentation on Agave tequilana juice in a continuous system. Low temperatures increased ethyl esters production (4.5-fold) and enhanced the expression of the involved genes (more than 3.5-fold). Conversely, when compared to aeration, the effect was negative as esters production decreased (75%) whereas the ATF1, ATF2 and EHT1 genes were repressed (0.6-, 0.8- and 0.4-fold, respectively). The results obtained during a continuous cultivation on Agave tequilana juice showed that production of esters occurred concomitantly with an increased gene expression, except for ethyl acetate. Therefore, the studied genes are not the only ones involved in its synthesis.

Este libro presenta un diagnóstico que da cuenta del potencial natural mexicano para impulsar el estudio, formulación y ejecución de proyectos sustentables en el ámbito energético y transferir conocimiento no sólo en áreas de amplio desarrollo, sino también en zonas de vulnerabilidad socioeconómica. Se exponen las nuevas formas de generar energías limpias con el apoyo de los mismos recursos naturales y la tecnología, una combinación que abre la oportunidad de mejorar la calidad del medio ambiente, la salud y la modernización que permite un despunte económico y desarrollo. El esfuerzo de los investigadores para contribuir en la construcción de la cultura científica y tecnológica en materia energética del país va a permitir que se alcance un análisis del problema global y se involucre a la sociedad en este gran tema que se refleja desde una óptica técnico-legal, en especial para que existan las formas de convivir en un planeta al que se le ha deteriorado de manera preocupante en menos de cien años.

International audience; Enzymatic glycosylation is an efficient way to increase the water solubility and the bioavailability of flavonoids. Levansucrases from Bacillus subtilis (Bs_SacB), Gluconacetobacter diazotrophicus (Gd_LsdA), Leuconostoc mesenteroides (Lm_LevS) and Zymomonas mobilis (Zm_LevU) were screened for puerarin (daidzein-8-C-glucoside) fructosylation. Gd_LsdA transferred the fructosyl unit of sucrose onto the glucosyl unit of the acceptor forming beta-D-fructofuranosyl-(2 -> 6)-puerarin (P1a), while Bs_SacB, Lm_LevS and Zm_LevU synthesized puerarin-4'-O-beta-D-fructofuranoside (P1b) and traces of P1a. The Gd_LsdA product P1a was purified and assayed as precursor for the synthesis of puerarin polyfructosides (PPFs). Bs_SacB elongated P1a more competently forming a linear series of water-soluble PPFs reaching at least 21 fructosyl units, as characterized by HPLC-UV-MS, HPSEC and MALDI-TOF-MS. Simultaneous or sequential Gd_LsdA/Bs_SacB reactions yielded PPFs directly from puerarin with the acceptor conversion ranging 82-92 %. The bi-enzymatic cascade synthesis of PPFs in the same reactor avoided the isolation of the intermediate product P1a and it is appropriate for use at industrial scale.

The production of tequila in Mexico generates a large amount of agave bagasse per year. However, this biomass can be considered as a potential source for biofuel production. In this study, it is described how the hydrothermal pretreatment was scaled in a bench scale, considering the severity index as a strategy. The best condition was at 180°C in isothermal regime for 20min with 65.87% of cellulose content and high concentration of xylooligosaccharides (15.31g/L). This condition was scaled up (using severity factor: [logR0]=4.11), in order to obtain a rich pretreated solid in cellulose to perform the enzymatic hydrolysis, obtaining saccharification yields of 98.5 and 99.5% at high-solids loading (10 and 15%, respectively). The pre-saccharification and fermentation strategy was used in the bioethanol production at 10 and 15% of total pretreated solids, obtaining 38.39 and 55.02g/L of ethanol concentration, corresponding to 90.84% and 87.56% of ethanol yield, respectively., Financial support is gratefully acknowledged from the Energy Sustainability Fund 2014-05 (CONACYT-SENER), Mexican Centre for Innovation in Bioenergy (Cemie-Bio), Cluster of Bioalcohols (Ref. 249564). We gratefully acknowledge support for this research by the Mexican Science and Technology Council (CONACYT, Mexico) for the Infrastructure Project – INFR201601 (Ref. 269461) and Basic Science Project – 2015-01 (Ref. 254808). Author Daniela L. Aguilar would like to thank the Mexican Science and Technology Council (CONACY, Mexico) for their master fellowship support (grant number: 582801/ 414478) andauthor ElisaZanuso thanksthe EnergySustainability Fund 2014-05 (CONACYT-SENER, Ref. 249564) for the undergraduate fellowship granted. Also, the authors thank Prof. Lucília Domingues and José Teixeira (University of Minho, Portugal) for kindly providing the yeast strain., info:eu-repo/semantics/publishedVersion

The gene Cap inv32 from Candida apicola and Tde inv32 from Torulaspora delbrueckii were identified as β-fructofuranosidase belonging to glycoside hydrolases family 32, with an opening reading frame of 1593 base pairs and 1743 base pairs, and a molecular weight of 59 kDa and 65 kDa, for Cap Inv32 and Tde Inv32 correspondingly. These enzymes contained the motif characteristics of the GH32 and showed an identity of 40 % (Cap Inv32) and 52.4 % (Tde Inv32) with an invertase of S. cerevisiae. The fructosyltransferase activity was tasted obtaining for Cap Inv32 the quantity of FOS 12.4 gL-1 and 9.03 gL-1 with 420 gL-1 and 700 gL-1, the production of FOS with Tde Inv32 was 14.61 gL-1 (420 gL-1) and 13.3 gL-1 (700 gL-1). Cap Inv32 and Tde Inv32 are β-fructofuranosidase with fructosyltransferase activity, with potential to industrial applications, focused in FOS production.

Las levaduras utilizadas en la produccion de etanol de segunda generacion estan afectadas por compuestos inhibidores como el 5-hidroximetilfurfural (HMF) y el furfural que se liberan durante la etapa de hidrolisis; estos compuestos afectan la capacidad fermentativa de la levadura. Para encontrar nuevas levaduras con una capacidad excepcional para ser utilizadas en la produccion de etanol de segunda generacion; en este estudio se evaluo la respuesta fisiologica al estres por derivados de furano en la levadura nativa Kluyveromyces marxianus (SLP1), y se comparo con la levadura comercial Saccharomyces cerevisiae etanol red (ERD). Utilizamos un medio quimicamente definido anadido con HMF y furfural a diferentes concentraciones; una condicion de control sin inhibidores, y cuatro condiciones de estres, HMF 7 gL -1 , furfural 3gL -1 , HMF 3,5 gL -1 con furfural 1,5 gL -1 y HMF 7 gL -1 con furfural 3 gL -1 . K. marxianus exhibio una mayor capacidad para asimilar los compuestos inhibidores en menor tiempo que S. cerevisiae ERD; tambien, K. marxianus SLP1 mostro un mejor comportamiento para producir etanol en condiciones de inhibicion. A pesar de los efectos provocados por los compuestos inhibidores, las levaduras podrian producir etanol en un 80% de conversion. En conclusion, la levadura K. marxianus SLP1 puede ser una opcion para producir etanol de segunda generacion a nivel industrial.

espanolEl objetivo de este estudio fue evaluar la capacidad de transformacion de hesperidina a partir de cascara de lima por Aspergillus saitoi en fermentacion solida. La disminucion de hesperidina durante la fermentacion se cuantifico durante 8 dias utilizando Cromatografia Liquidia de Alta Presion (HPLC). La actividad de atrapamiento de los radicales DPPH• y ABTS•+ incremento de 5.8 a 11 veces, respectivamente, despues de 6 dias de fermentacion comparado con la cascara de lima sin fermentar. El contenido fenolico de cascara de lima fermentada fue 8.66 veces mas alta que sin fermentar, mientras que los flavonoides aumentaron 5.14 veces. Despues de 6 dias de fermentacion, A. saitoi biotransformo hesperidina de la cascara de lima e incremento su actividad antioxidante. Estos resultados proporcionan informacion importante concerniente al potencial de la fermentacion solida en los desechos de la industria de los citricos a traves de la biotransformacion de flavonoides, los cuales dan una solucion efectiva para modificar la esctructura de estos mejorando su actividad inicial. EnglishThe aim of this study was to assess the ability of the biotransformation of hesperidin from lime peel by Aspergillus saitoi in solid fermentation. The decrease of hesperidin by High-Performance Liquid Chromatography (HPLC) was monitored during 8 days of fermentation. The radical scavenging activity of DPPH· and ABTS·+ increased to 5.8 and 11 times, respectively, after 6 days of fermentation compared with unfermented lime peel. Similarly, the phenolic content of the extract of fermented lime peel was 8.66 times higher than unfermented lime peel, while flavonoids increased 5.14 times. After 6 days of fermentation, A. saitoi biotransformed hesperidin from lime peels and increased its antioxidant activity. These results provide important information concerning the potential of the solid fermentation in the citrus industry waste through the biotransformation of flavonoids, which provides an effective solution for modifying the flavonoid structure and improving their initial bioactivity.

The yeast Saccharomyces cerevisiae is commonly employed in industrial ethanol production, regardless of the capability of Kluyveromyces marxianus strains to produce ethanol at similar or higher levels and on inhibitory conditions. Therefore, in this work strains of S. cerevisiae (ethanol RED and AR5) and K. marxianus (SLP1 and OFF1) were compared for ethanol production from sugarcane bagasse (SCB) and wheat straw (WS) hydrolysates. As it is known, during the lignocellulosic hydrolysis not only free sugars were obtained (SCB, g L−1: glucose 7.64, xylose 8.38, arabinose 2.43; and WS, g L−1: glucose 6.07, xylose 6.36, arabinose 2.09) but also growth inhibitors of yeast such as hydroxymethylfurfural and furfural that could modify the fermentation capability. The volumetric ethanol productivity (Q p) was evaluated, and it was observed that the K. marxianus SLP1 was the most efficient for ethanol production reaching a Q p of 0.292 and 0.250 g L−1 h−1 on SCB and WS hydrolysates, respectively. In contrast, S. cerevisiae AR5 and ethanol RED exhibited a reduced Q p on SCB, but similar values of Q p to K. marxianus OFF1 on WS. The results obtained show that it is possible to select K. marxianus yeast strains for ethanol production using SCB and WS hydrolysates obtaining higher Q p than S. cerevisiae yeast strains. Considering the efficiency of ethanol production and the tolerance to inhibitors, K. marxianus strain SLP1 possesses a great potential as an industrial yeast for lignocellulosic ethanol production.

The transition from an economy dependent on nonrenewable energy sources to one with higher diversity of renewables will not be a simple process. It requires an important research effort to adapt to the dynamics of the changing energy market, sort costly processes, and avoid overlapping with social interest markets such as food and livestock production. In this review, we analyze the desirable traits of raw plant materials for the bioethanol industry and the molecular biotechnology strategies employed to improve them, in either plants already under use (as maize) or proposed species (large grass families). The fundamentals of these applications can be found in the mechanisms by which plants have evolved different pathways to manage carbon resources for reproduction or survival in unexpected conditions. Here, we review the means by which this information can be used to manipulate these mechanisms for commercial uses, including saccharification improvement of starch and cellulose, decrease in cell wall recalcitrance through lignin modification, and increase in plant biomass.

In this work, the white-rot fungus Pleurotus djamor was used for the first time to determine the degradation kinetics of the nonsteroidal anti-inflammatory drugs diclofenac, naproxen and, ketoprofen, either individually or in mixtures, in submerged cultures. Removal of 93% individual diclofenac and 99% diclofenac in mixtures with naproxen and ketoprofen at 6 h of incubation with the fungus was achieved. The elimination levels of naproxen and ketoprofen individually were 90% and 87%, respectively, after 48 h of incubation. However, the removal levels of these compounds in mixtures were 85% and 83%, respectively. On the other hand, during the degradation kinetics analysis, the enzymatic activities of laccases, manganese peroxidases, and lignin peroxidases were evaluated, yielding values of 3700, 270 and 31 U/L, respectively. Additionally, it was demonstrated that during degradation of diclofenac or the three drugs mixed in the submerged cultures, the enzymatic activity of extracellular laccases expressed by P. djamor increased by 200% and 300%, respectively. The activity of manganese peroxides increased by 126% with diclofenac and 138% when the mixture of drugs was added to the cultures. On the other hand, lignin peroxidase only increased activity by 123% with the drug mixture.

Fructosylation can significantly improve the solubility, stability and bioactivity of phenolic compounds, increasing their health benefits. Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) was found to transfer the fructosyl unit of sucrose to different classes of phenolic compounds. Among the various acceptors tested, the isoflavone puerarin and the phenol coniferyl alcohol were the most efficiently fructosylated compounds, with conversion rates of 93% and 25.1%, respectively. In both cases, mono-, di-, and trifructosides were synthesized at a ratio of 37:14:1 and 32:8:1, respectively. Structural characterization of the puerarin mono-fructoside revealed that the enzyme transferred the fructosyl moiety of sucrose to the O6-position of the glucosyl unit of puerarin. The water solubility of fructosyl-β-(2→6)-puerarin was increased 23-fold, up to 16.2 g L−1, while its antioxidant capacity was only decreased 1.25-fold compared with that of puerarin.

Highlights • Yeast species influenced the de novo synthesis of 2-phenylethylacetate. • Inhibitory compounds showed a strong influence on cell growth and 2-phenylethylacetate production for the evaluated yeasts. • More than a 50 % reduction in the chemical and biochemical oxygen demand was achieved by yeast fermentation., Vinasses from the tequila industry are wastewaters with highly elevated organic loads. Therefore, to obtain value-added products by yeast fermentations, such as 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEA), could be interesting for industrial applications from tequila vinasses. In this study, four yeasts species (Wickerhamomyces anomalus, Candida glabrata, Candida utilis, and Candida parapsilosis) were evaluated with two different chemically defined media and tequila vinasses. Differences in the aroma compounds production were observed depending on the medium and yeast species used. In tequila vinasses, the highest concentration (65 mg/L) of 2-PEA was reached by C. glabrata, the inhibitory compounds decreased biomass production and synthesis of 2-PEA, and biochemical and chemical oxygen demands were reduced by more than 50 %. Tequila vinasses were suitable for the production of 2-phenylethylacetate by the shikimate pathway. A metabolic network was developed to obtain a guideline to improve 2-PE and 2-PEA production using flux balance analysis (FBA).

A consolidated two-stage bioprocessing scheme integrates the hydrogen and acetone–butanol–ethanol (ABE) production from wheat straw (WS). In the first stage, hydrogen production was performed from the xylan fraction of native WS with synthetic microbial consortia integrated by epiphytic species of Enterococcus. Three species showed a synergic effect between them attaining the maximum production of 79.5 mL H2 per gram of added wheat straw xylose. In the second stage, ABE production was carried out from biologically treated WS enriched in cellulose with a coculture of Clostridium beijerinckii 10132 with Clostridium cellulovorans 35296. The maximum ABE production was 23.3 g/L obtained in 5 days (3.7 g/L ethanol, 14.2 g/L butanol, and 5.4 g/L acetone). The structural changes in the WS were analyzed by FTIR and X-ray diffraction. The biological treatment applied to the native WS reduced the contents of lignin, hemicellulose and amorphous cellulose, increasing the cellulose availability for ABE production. This work proposes a biorefinery for hydrogen and ABE production from native wheat straw using two synthetic microbial consortia with hydrolytic and fermentative capacities. This configuration obviates the need for dedicated units for exhaustive pretreatments, enzyme production, and cellulose saccharification, which could reduce the operational costs considerably.

Two Pantoea anthophila bacterial strains were isolated from “tejuino”, a traditional Mexican beverage, and studied as β-galactosidase producers for galactooligosaccharides synthesis. Using 400 g/L of lactose, 50 °C, and 15 U/mL of β-galactosidase activity with ethanol-permeabilized cells, the maximum galactooligosaccharides (GOS) yield determined by High performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD) was 136 g/L (34% w/w of total sugars) at 96% of lactose conversion for Bac 55.2 and 145 g/L (36% w/w of total sugars) at 94% of lactose conversion for Bac 69.1. The main synthesized products were the disaccharides allolactose [Gal-β(1 → 6)-Glc] and 6-galactobiose [Gal-β(1 → 6)-Gal], as well as the trisaccharides 3′-galactosyl-lactose [Gal-β(1 → 3)-Gal-β(1 → 4)-Glc], 6-galactotriose [Gal-β(1 → 6)-Gal-β(1 → 6)-Gal], 3′-galactosyl-allolactose [Gal-β(1 → 3)-Gal-β(1 → 6)-Glc], and 6′-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc]. The β-galactosidases present in both strains showed a high transgalactosylation activity and formed principally β(1 → 3) and β(1 → 6) linkages. Considering the stability and bifidogenic properties of GOS containing such types of bonds, P. anthophila strains Bac 55.2 and Bac 69.1 possess a high potential as novel biocatalysts for prebiotic industrial production., This research was supported by the CONACYT project CB-2012-01/000000000181766, and by the Spanish Ministry of Economy and Competitiveness (projects BIO2013-48779-C4-1/3/4 and BIO2016-76601-C3-1-R/2-R/3-R).

The goal of this study was to isolate, select and characterize bacteria with cellulolytic activity from two different coffee residue composting piles, one of which had an internal temperature of 57°C and pH 5.5 and the other, a temperature of 61°C, and pH 9.3. Culture media were manipulated with carboxymethylcellulose and crystalline cellulose as sole carbon sources. The enzyme activity was assessed by hydrolysis halo formation, reducing sugar production and zymograms. Three out of twenty isolated strains showed higher enzymatic activity and were identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and based on the sequence analysis of 16S rDNA regions. The enzymatic extracts of the three selected strains showed exocellulase and endocellulase maximum activity of 0.254 and 0.519 U/ml, respectively; the activity of these enzymes was maintained even in acid pH (4.8) and basic (9.3) and at temperatures of up to 60°C. The enzymatic activities observed in this study are within the highest reported for cellulose produced by bacteria of the genus Bacillus. Endocellulase activity was shown in the zymograms from 24h until 144h of incubation. Furthermore, the pH effect on the endocellulase activity is reported for the first time by zymograms. The findings in this study entail the possibility to use these enzymes in the procurement of fermentable substrates for the production of energy from the large amount of residues generated by the coffee agroindustry.

Enzymatic fructosylation of organic acceptors other than sugar opens access to the production of new molecules that do not exist in nature. These new glycoconjugates may have improved physical-chemical and bioactive properties like solubility, stability, bioavailability, and bioactivity. This review focuses on different classes of acceptors including alkyl alcohols, aromatic alcohols, alkaloids, flavonoids, and xanthonoids, which were tested for the production of fructoderivatives using enzymes from the glycoside hydrolase (GH) families 32 and 68 that use sucrose as donor substrate. The enzymatic strategies and the reaction conditions required for the achievement of these complex reactions are discussed, in particular with regard to the type of acceptors. The solubility and pharmacokinetic and antioxidant activity of some of these new beta-D-fructofuranosides in comparison is reviewed and compared with their glucoside analogs to highlight the differences between these molecules for technological applications.

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

High throughput screening (HTS) is a powerful tool in biotechnology. The search for new or improved enzymes with suitable biochemical properties for industrial processes, has resulted in high efforts and research activities to develop new methodologies for activity screening. In this context, important advances have been achieved for the screening of cellulases and xylanases activities from wild and recombinant microorganisms, and from sequence databases. These enzymes have a wide range of industrial applications, including food, animal feed, textile, pulp and paper industries and detergents. Cellulases and xylanases along with pectinases, represent 20% of the world enzyme market. Recently, cellulases and xylanases have been used on fermentable sugars recovered from lignocellulosic biomass for second-generation biorefineries, aimed to produce chemical and biofuel platforms. As a result, HTS methods for biomass or biomass-degrading enzymes are gaining importance. This article presents evidence of the studies carried out for HTS of cellulase and xylanase activities.

A multiplex gas sampler for monitoring CO2 and O2 for column-type bioreactors used in solid-state fermentation was designed. The sampler mechanically couples up to 24 bioreactors to a central set o...

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

The reaction of boron heterocycles 1 and 2 with n-butyl lithium and alkyl halides led to (N→B) phenyl[N-alky-N-(2-alkyl)aminodiacetate-O,O′,N]boranes 3–6(a–b), 7(b) and 9(b), where alkyl can be in exo and/or endo position, and phenyl[N-alkyl-N-(2-alkyl)aminodiacetate-O,O′,N]boranes 7(c) and 8(c) isomers, which do not display the intramolecular N→B coordination bond. The existence of steric interactions between N-benzyl and the alkyl group at 2 position was indicated by 1H and 13C NMR, while, the δ(11B) values confirm the tetrahedral and trigonal environment of the 11B nucleus in these compounds. Moreover, the compounds were characterized by COSY, HETCOR and homonuclear proton decoupling experiment. The study of the intramolecular N→B coordination by dynamic NMR afforded a ΔG‡ value of 81.09 kJ/mol for compound 6(b).

Candida apicola, a highly osmotolerant ascomycetes yeast, produces sophorolipids (biosurfactants), membrane fatty acids, and enzymes of biotechnological interest. The genome obtained has a high-quality draft for this species and can be used as a reference to perform further analyses, such as differential gene expression in yeast from Candida genera.

Torulaspora delbrueckii presents metabolic features interesting for biotechnological applications (in the dairy and wine industries). Recently, the T. delbrueckii CBS 1146 genome, which has been maintained under laboratory conditions since 1970, was published. Thus, a genome of a new mezcal yeast was sequenced and characterized and showed genetic differences and a higher genome assembly quality, offering a better reference genome.

Candida apicola , a highly osmotolerant ascomycetes yeast, produces sophorolipids (biosurfactants), membrane fatty acids, and enzymes of biotechnological interest. The genome obtained has a high-quality draft for this species and can be used as a reference to perform further analyses, such as differential gene expression in yeast from Candida genera.

The effect of cell density on xylanolytic activity and productivity of Cellulomonas flavigena was evaluated under two different culturing conditions: fed-batch culture with discontinuous feed of sugar cane bagasse (SCB; condition 1) and glycerol fed-batch culture followed by addition of SBC as xylanases inducer (condition 2). The enzymatic profile of xylanases was similar in both systems, regardless of the initial cell density at time of induction. However, the xylanolytic activity changed with initial cell density at the time of induction (condition 2). The maximum volumetric xylanase activity increased with increased initial cell density from 4 to 34 g l(-1) but decreased above this value. The largest total volumetric xylanase productivity under condition 2 (1.3 IU ml(-1) h(-1)) was significantly greater compared to condition 1 (maximum 0.6 IU ml(-1) h(-1)). Consequently, induction of xylanase activity by SCB after growing of C. flavigena on glycerol at intermediate cell density can be a feasible alternative to improve activity and productivity of xylanolytic enzymes.

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: Vmax values were 1.37 and 1.06 mmol min−1 mg−1, respectively. The Km and Ki values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.

Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application.

Este trabajo tuvo como objetivo principal estudiar la fisiología de la levadura Kluyveromyces marxianus por efecto de la temperatura durante el cultivo, así como también evaluar el efecto de diversas concentraciones de calcio y magnesio en el medio de cultivo, teniendo como respuesta de análisis la actividad fructanasa. Se realizó un primer análisis para observar el efecto de diferentes fuentes de carbono como inductores en la actividad fructanasa obteniendo como resultado que los fructanos de agave fueron el mejor inductor, posteriormente se probaron dos medios químicamente definidos, también se analizaron diferentes temperaturas de cultivo (20-45° C). Por otro lado se implementaron técnicas para evaluar el estado fisiológico de la cepa, estas técnicas fueron, la microscopia de fluorescencia y la espectroscopia dieléctrica, esto para conocer la viabilidad del cultivo, otras técnicas utilizadas fueron la cuantificación del tamaño celular y el cultivo en placa (UFC). Una vez establecidas las técnicas de análisis se procedió con la evaluación de la temperatura y las diferentes concentraciones de calcio y magnesio sobre la expresión de fructanasas, para cual se realizó un diseño experimental multifactorial. Por último se realizó un cultivo en continuo para observar el efecto del calcio y el magnesio durante la producción de la fructanasa, también se observó el efecto del aumento de temperatura sobre la fisiología celular