Adaptive evolution and selection of stress-resistant Saccharomyces cerevisiae for very high-gravity bioethanol fermentation

Background: In industrial yeasts, selection and breeding for resistance to multiple stresses is a focus of current research. The objective of this study was to investigate the tolerance to multiple stresses of Saccharomyces cerevisiae obtained through an adaptive laboratory evolution strategy involving a repeated liquid nitrogen freeze­thaw process coupled with multi-stress shock selection. We also assessed the related resistance mechanisms and very high-gravity (VHG) bioethanol production of this strain. Results: Elite S. cerevisiae strain YF10-5, exhibiting improved VHG fermentation capacity and stress resistance to osmotic pressure and ethanol, was isolated following ten consecutive rounds of liquid nitrogen freeze­thaw treatment followed by plate screening under osmotic and ethanol stress. The ethanol yield of YF10-5 was 16% higher than that of the parent strain during 35% (w/v) glucose fermentation. Furthermore, there was upregulation of three genes (HSP26, HSP30, and HSP104) encoding heat-shock proteins involved in the stress response, one gene (TPS1) involved in the synthesis of trehalose, and three genes (ADH1, HXK1, and PFK1) involved in ethanol metabolism and intracellular trehalose accumulation in YF10-5 yeast cells, indicating increased stress tolerance and fermentative capacity. YF10-5 also showed excellent fermentation performance during the simultaneous saccharification and fermentation of VHG sweet potato mash, producing 13.40% (w/ v) ethanol, which corresponded to 93.95% of the theoretical ethanol yield. Conclusions: A multiple-stress-tolerant yeast clone was obtained using adaptive evolution by a freeze­thaw method coupled with stress shock selection. The selected robust yeast strain exhibits potential for bioethanol production through VHG fermentation.

Improvement of ethanol production from birch and spruce pretreated with 1-H-3-methylmorpholinium chloride

Background: Pretreatment is the critically important step for the production of ethanol from lignocelluloses. In this study, hardwood birch (Betula pendula) and softwood spruce (Norway spruce) woods were pretreated with a newly synthesized morpholinium ionic liquid, 1-H-3-methylmorpholinium chloride ([HMMorph][Cl]), followed by enzymatic hydrolysis and fermentation to ethanol. Results: [HMMorph][Cl] was synthesized using inexpensive raw materials, i.e., hydrochloric acid and N-methyl morpholine, following a simple process. The influence of pretreatment time (2, 3, 5, and 8 h) and temperature (120 and 140°C) in terms of hydrolysis efficiency was investigated. Glucose yields from enzymatic hydrolysis were improved from 13.7% to 45.7% and 12.9% to 51.8% after pretreatment of birch and spruce woods, respectively, under optimum pretreatment conditions (i.e., at 140°C for 3 h) as compared to those from pristine woods. Moreover, the yields of ethanol production from birch and spruce were increased to 34.8% and 44.2%, respectively, while the yields were negligible for untreated woods. Conclusions: This study demonstrated the ability of [HMMorph][Cl] as an inexpensive agent to pretreat both softwood and hardwood.

Kinetic modeling of the simultaneous production of ethanol and fructose by Saccharomyces cerevisiae

Background: Ethanol and fructose are two important industrial products that enjoy many uses. In this contribution, their production via selective fermentation of date extract using Saccharomyces cerevisiae was studied. Scaling up the process for possible commercialization was investigated in three fermentors with working volume ratio of 1:40:400. Results: Higher ethanol concentration was obtained in the larger fermentor due to conversion of fructose. Fructose yields in the 0.5-L, 7.5-L and 80-L fermentors were 99, 92 and 90%, respectively. Good fitting was obtained with the modified Monod kinetics; however, a better fit of cell mass was obtained with the modified Ghose­Tyagi model which accounts for ethanol inhibition. Conclusions: The modified Gompertz model was expanded to facilitate prediction of products' formation and fructose fractions in all three fermentors. Such expansion will be beneficial in industrial applications.

Balance de Flujos Metabólicos en Saccharomyces cerevisiae basado en Compartimentalización Intracelular / Metabolic Flows Balance in Saccharomyces cerevisiae based on Intracellular Compartmentalization

Una de las técnicas más utilizadas para la predicción de producción de bioproductos y distribución intracelular de flujos de microorganismos es el Análisis de Balance de Flujos - FBA por sus siglas en inglés. El FBA requiere de una función objetivo que represente el objetivo biológico del microorganismo estudiado. En este trabajo se propone un nuevo tipo de funciones objetivo basada en la combinación de objetivos de compartimentos físicos presentes en el microorganismo estudiado. Este tipo de funciones objetivo son examinadas junto con un modelo estequiométrico extraído de la reconstrucción iMM904 del microorganismo S. cerevisiae. Su desempeño se compara con la función objetivo más usada en la literatura, la maximización de biomasa, en condiciones experimentales anaeróbicas en cultivos continuos y aeróbicas en cultivos tipo lote. La función objetivo propuesta en este trabajo mejora las predicciones de crecimiento en un 10% y las predicciones de producción de etanol en un 75% respecto a las obtenidas por la función objetivo de maximización de biomasa, en condiciones anaeróbicas. En condiciones aeróbicas tipo lote la función objetivo propuesta mejora en un 98% las predicciones de crecimiento y en un 70% las predicciones de etanol con respecto a la función objetivo de biomasa.

Flux Balance Analysis - FBA - is one of the most used techniques in prediction of microorganism bioproducts. It requires an objective function that represents biological objective of the studied microorganism. This paper presents a new kind of objective functions based on individual physical compartment objetives in the studied microorganism. These kind of functions was tested with a stoichiometric model extracted from iMM904 reconstruction of S. cerevisiae and its performance is compared with the most used objective function in literature, growth maximization, in anaerobic and aerobic batch conditions. The presented objective function outperform growth predictions in 10% and ethanol predictions in 75% compared with obtained by maximization of growth objective function, in anaerobic conditions. In aerobic batch conditions the presented objective function outperforms in 98% growth preditions and 70% ethanol predictions compared with growth maximization.

Estudio comparativo de los efectos de hidrato de cloral y etanol en ratas expuestas a fluoracetato de sodio / Comparative study of the effects of cloral hydrate and ethanol in rats exposed to sodium fluoracetate

El fluoroacetato de sodio, es un raticida prohibido en algunos países y permitido en otros, que causa severas intoxicaciones humanas y animales. Actúa por inhibición del ciclo de Krebs e interfiere con la producción de energía, lo cual conduce a disfunción celular irreversible, especialmente en sistema nervioso central y corazón. El alcohol etílico, debido a su oxidación a acido acético y a su amplia disponibilidad, es uno de los fármacos usados en esta intoxicación, lo que podría causar controversias éticas y legales. La biotransformación del hidrato de cloral a tricloroetanol y a ácido tricloroacético, el efecto anticonvulsivante y su amplio uso en Pediatría, fueron las razones para su evaluación en la intoxicación por fluoroacetato. Se realizó un estudio experimental, para comparar los efectos de hidrato de cloral y alcohol en ratas intoxicadas con fluoroacetato de sodio. El análisis estadístico aplicado fue la prueba de chi cuadrado. Los resultados mostraron que el hidrato de cloral a dosis bajas, permite la sobrevivencia en 100% de los animales expuestos. Se confirmó igualmente la efectividad del alcohol etílico a dosis altas. Este resultado sugiere que el hidrato de cloral puede ser una opción tan útil como el etanol y que podría ser el fármaco de elección en aquellos pacientes que no puedan recibir monoacetin o etanol o porque haya mayor accesibilidad al hidrato de cloral.

Sodium fluoroacetate is a banned rodenticide in some countries and allowed in others, which causes severe human and animal poisonings. It acts for inhibition of Krebs's cycle and interferes with energy production leading to irreversible cellular dysfunction, specially in nervous central system and heart. Ethyl alcohol, because oxidation to acetic acid and to its wide availability, is one of the drugs used in this poisoning, which should can cause ethical and legal controversies. Biotransformation of chloral hydrate to trichloroethanol and trichloroacetic acid, the anticonvulsant effect and its widespread use in Pediatrics, were the reasons for its evaluation in fluoroacetate poisoning. An experimental study was conducted, to compare effects of chloral hydrate and ethanol in poisoned rats with sodium fluoroacetate. The statistical analysis applied was the chi square test. The results showed that chloral hydrate in low doses allows survival in 100 % of the exposed animals. It also confirms the effectiveness of ethyl alcohol at high doses. This result suggests that chIoral hydrate may be an option as useful as ethanol and could be the choice drug in those patients who could not receive monoacetin or ethanol or because there is greater accessibility to chloral hydrate.

Evaluación del pretratamiento con ácido sulfúrico diluido del pasto maralfalfa (Pennisetum glaucum x Pennisetum purpureum) para la producción de etanol / Dilute sulfuric acid pretreatment of goliath grass (Pennisetum glaucum x Pennisetum purpureum) for ethanol cellulosic

Evaluar la producción de etanol a partir de cultivos lignocelulósicos, específicamente pastos de rápido crecimiento en la región, constituye una alternativa a la demanda de biocombustibles. En la presente investigación se seleccionó el pasto Maralfalfa (Pennisetum glaucum x Pennisetum purpureum) utilizando el pretratamiento con ácido sulfúrico diluido a diferentes temperaturas (110, 130, 150, 170 y 190 °C) y concentraciones de ácido (0.8, 1.2 y 2.0% (p/p)), seguido de un proceso de hidrólisis enzimática utilizando celulasas y celobiosas comerciales y un proceso de hidrólisis y fermentación simultanea. La máxima producción de etanol obtenido fue 117 mg etanol/ g biomasa pretratada a 190 °C y 1,2 %(p/p) de ácido sulfúrico. El líquido hidrolizado fue caracterizado calculando el porcentaje de glucosa, xilosa y lignina solubilizadas y degradadas durante el pretratamiento.

The goliath grass (Pennisetum glaucum x Pennisetum purpureum) was pretreated with different sulfuric acid concentrations (0.8, 1.2 y 2.0% (w/w)) from low to high temperatures (110, 130, 150, 170 y 190 °C) followed by enzymatic hydrolysis and SSF of remaining solids. The maximum yield was 117 mg of ethanol/g biomass to 190 °C and 1.2 % (w/w) of sulfuric acid.

Efecto de diámetro de esfera y densidad celular en la producción de etanol con levadura inmovilizada en alginato / Effect of pellet diameter and cell density on ethanol production with alginate immobilized yeast

Las células inmovilizadas tienen aplicación potencial en la producción de biocombustibles posibilitando la reutilización de biomasa, el empleo de diversas configuraciones de reactores y sistemas de cultivo, el manejo de altas densidades celulares alcanzando altas productividades volumétricas, y la simplificación de operaciones de procesamiento de salida. El objetivo del presente estudio fue evaluar la influencia del diámetro de las perlas y la densidad celular en la producción de etanol con Saccharomyces uvarum inmovilizada en alginato al 2% (p/v). Para ello se evaluaron tres diámetros de perlas de 2, 2,5 y 3 mm. Las células inmovilizadas fueron cultivadas en medio con 12% (p/v) de glucosa en biorreactores de columna sin agitación a 28 ºC, y se operaron cuatro lotes consecutivos de 48 horas cada uno. En cada lote se cuantificó el consumo de glucosa y se determinó la cantidad de etanol producido. Los rendimientos máximos de etanol para las esferas de 2, 2,5 y 3 mm de diámetro fueron 81, 83 y 97% del rendimiento teórico. La máxima productividad volumétrica de etanol fue 1,2 g/L-1/h-1 con un consumo de glucosa de 99,8% al término del lote, correspondiente a las columnas con perlas de 3 mm y con una producción de 0,017 g de etanol por esfera. La producción de etanol acumulada en cada sistema fue 178, 189 y 200 g/L-1 para 2, 2,5 y 3 mm respectivamente, encontrándose una relación directa con el diámetro de perla e inversa respecto a la densidad celular. Los rendimientos de etanol obtenidos son superiores a los reportados para la misma especie.

Immobilized cells have a potential use in biofuel production. They also allow re-using biomass, using diverse reactor configurations and culture systems, handling high cell densities to obtain high volumetric productivities and to simplify the downstream processing. The purpose of this work was to evaluate the influence of bead diameter and cell density on ethanol production using immobilized Saccharomyces uvarum in 2% (w/v) alginate. For that, three bead diameters (2, 2.5 and 3 mm) were evaluated. Immobilized cells were cultured on a 12% (w/v) glucose medium in column bioreactors without agitation at 28 °C for four 48 h–repeated batches. For each batch, both glucose consumption and ethanol produced were measured. Maximum yields for 2, 2.5 and 3 mm bead diameters were 81, 83 and 97% of theoretical yield. Maximum volumetric productivity of ethanol was 1.2 g/L-1/h-1 with 99.8% glucose consumption at the end of the batch, corresponding to the 3 mm bead diameter and the ethanol production per bead was 0.017 g. Accumulated ethanol production for each system was 178, 189 and 200 g/L-1 for 2, 2.5 y 3 mm bead diameter, respectively, being this directly related to bead diameter and inversely related to cell density. Ethanol yields were higher than those reported for the same species.

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products / Avaliação da suplementação do meio de sacarose na formação de bio–produtos por Zymomonas mobilis

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

A influência das variáveis: ácido pantotênico, extrato de levedura, cloreto de sódio, e a técnica de permeabilização celular foram investigadas na formação de levana, sorbitol, etanol e biomassa de Zymomonas mobilis utilizando um delineamento estatístico fatorial fracionado 24-1. A biomassa foi determinada por turbidimetria, Os açúcares redutores foram quantificados por Somogy e Nelson, açúcar total por Fenol Sulfúrico, sorbitol por HPLC e etanol por micro-destilação. A levana produzida foi precipitada com etanol absoluto e determinada como unidade de frutose. Na biossíntese de levana, a variável que mais contribuiu foi a condição celular. Os resultados sugerem que, para a formação da biomassa e etanol, os fatores que mais interferiram foram a concentração de cloreto de sódio e a condição celular que influencia negativamente a produção. Para o sorbitol, a variável que teve efeito significativo foi a permeabilização celular que atuou diminuindo a sua síntese. Estudos que ampliam a faixa de variação dos fatores estabelecidos são interessantes.

Avaliação in vitro do extrato hidroalcoólico (EHA) de alecrim pimenta (Lippia sidoides Cham. ) sobre o desenvolvimento de ovos de nematódeos gastrointestinais (Trichostrongylidae) / In vitro evaluation of alecrim pimenta (Lippia sidoides Cham. ) hydroalcoholic extract (HAE) on the development of gastrointestinal nematode (Trichostrongylidae) eggs

Atualmente a fito medicina vem sendo usada no controle de diversas doenças parasitárias particularmente as parasitoses gastrointestinais. Objetivou-se com este estudo avaliar a eficácia do extrato hidroalcoólico (EHA) de Lippia sidoides Cham sobre o desenvolvimento de ovos de nematódeos gastrointestinais. O efeito ovicida foi determinado através de análise probabilística, modelo logístico, utilizados os softwares R versão 2.2.1 e EPI6. Foram obtidos ovos embrionados em fezes de cabras naturalmente infectadas com nematóides Trichostrongylidae e o número de ovos fecais foi determinado usando-se a técnica modificada de McMaster. Cinquenta μL da suspensão contendo 40 ovos foram transferidas a placas de poliestireno e incubadas com 12 concentrações diferentes do EHA sendo avaliada durante 72 horas a temperatura ambiente. Os resultados demonstraram diferentes eficácias para o fito medicamento com menor taxa de desenvolvimento de ovos na concentração de 500 mg mL-1 . Conclui-se que o EHA pode ter papel importante sobre o desenvolvimento in vitro de ovos de nematóides gastrintestinais, revelando atividade ovicida.

Phytomedicine has been currently used in the control of several parasitic diseases, particularly gastrointestinal ones. The aim of this study was to evaluate the efficacy of the hydroalcoholic extract (HAE) from Lippia sidoides Cham. on the development of gastrointestinal nematode eggs. The ovicidal effect was determined through probabilistic analysis, logistic model, by using the programs R version 2.2.1 and Epi InfoTM 6. Embryonated eggs were obtained from feces of goats naturally infected with Trichostrongylidae nematodes and the fecal egg count was determined by using the modified McMaster technique. Fifty microliters of the suspension containing 40 eggs were transferred to polystyrene plates and incubated with 12 different HAE concentrations, and evaluations were performed during 72h at room temperature. The results demonstrated different efficacy of extracts, with lower egg development rate at 500 mg mL-1. In conclusion, HAE may play an important role on the in vitro development of gastrointestinal nematode eggs, indicating ovicidal activity.

Utilization and biodegradation of starch stillage (distillery wastewater)

Stillage (distillery wastewater) is the main by-product originating in distilleries, and its volume is approximately 10 times that of ethanol produced. It is not surprising that the utilization of the stillage raises serious problems, and that many attempts have been made all over the world to solve them. In Poland most of the ethanol (about 90 percent) is produced from starch-based feedstocks, i.e. grains and potatoes. Starch feedstocks are widely used for spirit production also in other European countries, as well as outside Europe. The manuscript provides an overview of global fuel ethanol production and information on methods used for starch-based stillage biodegradation and utilization. The methods presented in this paper have been classified into two major groups. One of these includes the mode of utilizing starch stillage, the other one comprises methods, both aerobic and anaerobic, by which the stillage can be biodegraded.

Utilization of bee (Apis mellifera) honey for vinegar production at laboratory scale

Vinegar was obtained from bee (Apis mellifera) honey. The wort was prepared by diluting honey in distilled water to 21 per cent total solids and by adding ammonium sulfate and ammonium phosphate. Saccharomyces cerevisiae was inoculated to the wort (4 g/L). Ethanol production was carried out at room temperature during 84 hours. In this study, 1 Kg of honey yielded about 5 L of wine, containing 8 per cent alcohol (v/v), from a wort with 17.11 per cent total sugars (w/v). The efficiency of the alcoholic fermentation was 81.34 per cent. The acetic fermentation with an inoculum of mixed acetic microorganisms was performed by quick process in a 15 L vertical fermenter. This resulted in a vinegar containing up to 9 per cent of acetic acid (w/v) and about 1 per cent of alcohol (v/v). The acetic fermentation yielded between 91.24 and 97.21 per cent. Approximately 5 L of honey vinegar with 9 per cent acetic acid (w/v) were obtained from 1 Kg of bee honey. All attributes of honey vinegar showed acceptability index over 70 per cent: 95.37 per cent for appearance, 94.81 per cent for color, 79.07 per cent for odor and 75.56 per cent for flavor, indicating it would show good consumer acceptability.

Comparative study of two raw media and two fermentation methods for ethanol production by local and imported strains of saccharomyces cerevisiae

Two fermentation media and two fementation methods were compared for their efficiency for ethanol production by local and imported strains of Saccharomyces cerevisiae The result appeared that using stil culture method for alcohol production from date juice extract [24 prix] The yield was 13.5% alcohol and 4% sugar residue after 12 hrs for local strain. Whereas 12.1% alcohol and 5.4% sugar residue for imported strain. In lab fermenter produced alcohol was 15% and sugar residue 0.4% for local strain, and 13.3% alcohol 3.6% sugar residue for imported strain. Using molasses as fermentation medium [23% sugar] in still culture method, produced alcohol was 9.6%, 7.8% and sugar residue was 7.8%, 8.2% for local and imported strains, respectively. While in lab-fermenter, produced alcohol was 8.5%, 7.9% and sugar residue was 11.6%, 12.2% for local and imported strains, respectively. It was stated in this work that Date juice was better than molasses and lab-fermenter better than still culture method for ethanol production. In all these cases isolated local strain was more efficient than imported european strain for ethanol production. Other fermentation products methanol, propanol, and acetylaldehyde was determined by gas chroniatography in both fermention media

Production of invertase and ethyl alcohol from cane molasses by immobilized cells of saccharomyces cerevisiae y-1347

In order to improve the metabolic activities, the immobilization technique was adapted using Ca-alginate as an entrapping gel and a 3% gel concentration was found to be the most adequate gel level to attain higher metabolic activities by immobilized culture of S. cerevisiae Y-1347 upon using static flask cultures. The entrapped culture could be repeatedly used in batch-wise fermentation successfully for at least 3 times as suspended in distilled water

Importancia del polimorfismo en el metabolismo del etanol / Importance of polimorphism in ethanol metabolism

En este trabajo se presenta la influencia de la variabilidad genética sobre el metabolismo el etanol, hecho que puede generalizarse a prácticamente todos los procesos porque, en mayor a menor proporción, existen variaciones genéticas que influyen de manera importante en la respuesta individual a un estímulo dado; sólo que en la respuesta metabólica a la ingestión aguda o crónica de etanol esto es particularmente claro, porque las principales enzimas que participan en su degradación presentan formas polimórficas con actividades diferentes. Factores como la edad, sexo, raza, etc., contribuyen en la obsorción, la distribución y el metabolismo del etanol. la deshidrogenasa alcohólica y el sistema microsomal oxidante del etanol son los principales sistemas de oxidación del etanol a acetaldehído. formas múltiples de la deshidrogenasa alcohólica han sido caracterizadas y permiten explicar la base molecular del polimorfismo enzimático. La siguiente oxidación del acetaldehído a acetato está catalizada por la deshidrogenasa aldehídica, cuyo polimorfismo influye en el control del alcoholismo, saobre todo en poblaciones de origen monogoloide (chinos, japoneses). Resta determinar el papel de un perfil específico de deshidrogenasa aldehídica en el desarrollo de una hepatopatía