The isolation of pentose-assimilating yeasts and their xylose fermentation potential

ABSTRACT For the implementation of cellulosic ethanol technology, the maximum use of lignocellulosic materials is important to increase efficiency and to reduce costs. In this context, appropriate use of the pentose released by hemicellulose hydrolysis could improve de economic viability of this process. Since the Saccharomyces cerevisiae is unable to ferment the pentose, the search for pentose-fermenting microorganisms could be an alternative. In this work, the isolation of yeast strains from decaying vegetal materials, flowers, fruits and insects and their application for assimilation and alcoholic fermentation of xylose were carried out. From a total of 30 isolated strains, 12 were able to assimilate 30 g L-1 of xylose in 120 h. The strain Candida tropicalis S4 produced 6 g L-1 of ethanol from 56 g L-1 of xylose, while the strain C. tropicalis E2 produced 22 g L-1 of xylitol. The strains Candida oleophila G10.1 and Metschnikowia koreensis G18 consumed significant amount of xylose in aerobic cultivation releasing non-identified metabolites. The different materials in environment were source for pentose-assimilating yeast with variable metabolic profile.

Inhibitor tolerance of a recombinant flocculating industrial Saccharomyces cerevisiae strain during glucose and xylose co-fermentation

ABSTRACT Lignocellulose-derived inhibitors have negative effects on the ethanol fermentation capacity of Saccharomyces cerevisiae. In this study, the effects of eight typical inhibitors, including weak acids, furans, and phenols, on glucose and xylose co-fermentation of the recombinant xylose-fermenting flocculating industrial S. cerevisiae strain NAPX37 were evaluated by batch fermentation. Inhibition on glucose fermentation, not that on xylose fermentation, correlated with delayed cell growth. The weak acids and the phenols showed additive effects. The effect of inhibitors on glucose fermentation was as follows (from strongest to weakest): vanillin > phenol > syringaldehyde > 5-HMF > furfural > levulinic acid > acetic acid > formic acid. The effect of inhibitors on xylose fermentation was as follows (from strongest to weakest): phenol > vanillin > syringaldehyde > furfural > 5-HMF > formic acid > levulinic acid > acetic acid. The NAPX37 strain showed substantial tolerance to typical inhibitors and showed good fermentation characteristics, when a medium with inhibitor cocktail or rape straw hydrolysate was used. This research provides important clues for inhibitors tolerance of recombinant industrial xylose-fermenting S. cerevisiae.

Isolation of fungi from dung of wild herbivores for application in bioethanol production

ABSTRACT Producing biofuels such as ethanol from non-food plant material has the potential to meet transportation fuel requirements in many African countries without impacting directly on food security. The current shortcomings in biomass processing are inefficient fermentation of plant sugars, such as xylose, especially at high temperatures, lack of fermenting microbes that are able to resist inhibitors associated with pre-treated plant material and lack of effective lignocellulolytic enzymes for complete hydrolysis of plant polysaccharides. Due to the presence of residual partially degraded lignocellulose in the gut, the dung of herbivores can be considered as a natural source of pre-treated lignocellulose. A total of 101 fungi were isolated (36 yeast and 65 mould isolates). Six yeast isolates produced ethanol during growth on xylose while three were able to grow at 42 °C. This is a desirable growth temperature as it is closer to that which is used during the cellulose hydrolysis process. From the yeast isolates, six isolates were able to tolerate 2 g/L acetic acid and one tolerated 2 g/L furfural in the growth media. These inhibitors are normally generated during the pre-treatment step. When grown on pre-treated thatch grass, Aspergillus species were dominant in secretion of endo-glucanase, xylanase and mannanase.

Xylitol production and furfural consumption by a wild type Geotrichum sp

Background: Xylitol is a five carbons polyol with promising medical applications. It can be obtained from chemical D-xylose reduction or by microbial fermentation of Sugarcane Bagasse Hemicellulosic Hydrolysate. For this last process, some microbial inhibitors, as furfural, constitute severe bottleneck. In this case, the use of strains able to produce xylitol simultaneously to furfural neutralization is an interesting alternative. A wild-type strain of Geotrichum sp. was detected with this ability, and its performance in xylitol production and furfural consumption was evaluated. Furthermore, were analyzed its degradation products. Results: Geotrichum sp. produced xylitol from D-xylose fermentation with a yield of 0.44 g-g-1. Furfural was fully consumed in fermentation assay and when provided in the medium until concentration of 6 g-L-1. The furfural degradation product is not an identified molecule, presenting a molecular weight of 161 g-mol-1, an uncommon feature for the microbial metabolism of this product. Conclusion: This strain presents most remarkable potential in performing furfural consumption simultaneous to xylitol production. Subsequent efforts must be employed to establish bioprocess to simultaneous detoxification and xylitol production by Geotrichum sp.

High levels of ß-xylosidase in Thermomyces lanuginosus: potential use for saccharification

ABSTRACT A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.

Sugarcane straw as a feedstock for xylitol production by Candida guilliermondii FTI 20037

Abstract Sugarcane straw has become an available lignocellulosic biomass since the progressive introduction of the non-burning harvest in Brazil. Besides keeping this biomass in the field, it can be used as a feedstock in thermochemical or biochemical conversion processes. This makes feasible its incorporation in a biorefinery, whose economic profitability could be supported by integrated production of low-value biofuels and high-value chemicals, e.g., xylitol, which has important industrial and clinical applications. Herein, biotechnological production of xylitol is presented as a possible route for the valorization of sugarcane straw and its incorporation in a biorefinery. Nutritional supplementation of the sugarcane straw hemicellulosic hydrolyzate as a function of initial oxygen availability was studied in batch fermentation of Candida guilliermondii FTI 20037. The nutritional supplementation conditions evaluated were: no supplementation; supplementation with (NH4)2SO4, and full supplementation with (NH4)2SO4, rice bran extract and CaCl2·2H2O. Experiments were performed at pH 5.5, 30 °C, 200 rpm, for 48 h in 125 mL Erlenmeyer flasks containing either 25 or 50 mL of medium in order to vary initial oxygen availability. Without supplementation, complete consumption of glucose and partial consumption of xylose were observed. In this condition the maximum xylitol yield (0.67 g g-1) was obtained under reduced initial oxygen availability. Nutritional supplementation increased xylose consumption and xylitol production by up to 200% and 240%, respectively. The maximum xylitol volumetric productivity (0.34 g L-1 h-1) was reached at full supplementation and increased initial oxygen availability. The results demonstrated a combined effect of nutritional supplementation and initial oxygen availability on xylitol production from sugarcane straw hemicellulosic hydrolyzate.

Genetic analysis of D-xylose metabolism by endophytic yeast strains of Rhodotorula graminis and Rhodotorula mucilaginosa

Two novel endophytic yeast strains, WP1 and PTD3, isolated from within the stems of poplar (Populus) trees, were genetically characterized with respect to their xylose metabolism genes. These two strains, belonging to the species Rhodotorula graminis and R. mucilaginosa, respectively, utilize both hexose and pentose sugars, including the common plant pentose sugar, D-xylose. The xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes were cloned and characterized. The derived amino acid sequences of xylose reductase (XR) and xylose dehydrogenase (XDH) were 32 percent~41 percent homologous to those of Pichia stipitis and Candida. spp., two species known to utilize xylose. The derived XR and XDH sequences of WP1 and PTD3 had higher homology (73 percent and 69 percent identity) with each other. WP1 and PTD3 were grown in single sugar and mixed sugar media to analyze the XYL1 and XYL2 gene regulation mechanisms. Our results revealed that for both strains, the gene expression is induced by D-xylose, and that in PTD3 the expression was not repressed by glucose in the presence of xylose.

Sugar cane bagasse as feedstock for second generation ethanol production: part II: hemicellulose hydrolysate fermentability

Sugar cane bagasse is produced in Brazil as waste of the sugar and ethanol industries. This lignocellulosic material is a potential source for second-generation ethanol production; however a pretreatment stage is essential, which aims at removing the hemicellulose component by disorganizing the lignocellulosic complex. In this work sugar cane bagasse was pretreated by diluted acid hydrolysis resulting in xylose-rich hydrolysates, which could be fermented to ethanol by a strain of the yeast Pichia stipitis. Statistical approach was used to investigate the effects of factors associated with the diluted acid hydrolysis process (acid concentration, solid:liquid ratio and time of exposure) on the fermentability of different hydrolysates. The statistical analysis was useful for determining the effects of the individual factors and their interactions on the response variables. An acid concentration of 1.09 percent (v/v), a solid:liquid ratio of 1:2.8 (g:ml), and an exposure time of 27 min were established and validated as the optimum pretreatment conditions for ethanol production from hemicellulose hydrolysates of sugar cane bagasse. Under these conditions, a hydrolysate with 50 g/l of xylose, 6.04 g/l of acetic acid, 0.55 g/l of hydroxylmethylfurfural and 0.09 g/l of furfural was obtained and its fermentation yielded roughly 20 g/l of ethanol in 40 hrs.

Aislamiento de Candida dubliniensis en distintos materiales clínicos: Análisis de métodos fenotípicos de diferenciación con Candida albicans / Isolation of Candida dubliniensis in different clinical samples: Analysis of phenotypical methods to differenciate from Candida albicans

Con el objeto de estimar la frecuencia de aislamientos de Candida dubliniensis en materiales clínicos en el Hospital de Infecciosas F. J. Muñiz, se identificaron 388 levaduras entre setiembre de 2005 y agosto de 2007. Doscientos doce aislamientos presentaban color verde en CHROMagar® y producían tubos germinativos y clamidoconidias en agarleche. Para diferenciar cuales de ellos correspondían a Candida albicans o a C. dubliniensis, se utilizaron distintos métodos fenotípicos y se evaluó la utilidad de cada técnica a fin de proponer un algoritmo de identificación simple, económico y confiable. Se estudió el color en 2 medios con sustratos cromogénicos, la producción de clamidoconidias en medios de Staib, agar tomate-zanahoria y agar-tabaco; en este último medio también se evaluaron las características macromorfológicas de las colonias; se evaluó la presencia de actividad lipolítica (medio-opacidad), capacidad de desarrollo a 45 °C y asimilación de D-xilosa. El 6,1% (13/212 aislamientos) correspondió a C. dubliniensis (3,3% del total de levaduras). No se pudo diferenciar entre ambas especies por el color en los medios cromogénicos usados. Las pruebas que resultaron más sensibles y específicas fueron crecimiento a 45 °C, asimilación de D-xilosa, color y desarrollo en agar-tabaco. C. albicans produjo clamidoconidias en los 3 medios diferenciales, entre 11,6% y 15,1% de los casos. La presencia de lipasas se evidenció en el 95,6% de C. albicans pero 2 de las 13 cepas de C. dubliniensis también presentaron halo de opacidad. Consideramos que se deben usar, al menos, 3 métodos diferentes para discriminar entre estas levaduras ya que ninguna prueba es absolutamente sensible o específica.

In order to estimate the frequence of Candida dubliniensis in clinical samples in F. J. Muñiz Infectious Diseases Hospital, a total of 388 yeasts from September 2005 to August 2007. There were 212 isolates which presented a green color on CHROMagar® Candida medium and produced germ tubes and chlamidoconidiae in milk-agar; so as to distinguish whether they corresponded to Candida albicans or C. dubliniensis, different phenotypical methods were utilized. It was also evaluated the usefulness of each one in order to suggest a simple, economic and reliable identification algorithm. Each isolate was subcultured in two chromogenic media and then, the following determinations were done: chlamidospores production in Staib-agar, tomato-carrot-agar and tobacco-agar, colonies macromorphology was also studied in the last medium; opacity-test in Tween 80-CaCl2 agar (lipase activity), growing capacity at 45 °C, and D-xylose assimilation. Thirteen strains (6.1%) corresponded to C. dubliniensis. The difference in color between both species on chromogenic media was not so stressed as it is pointed out in some works. The more specific and sensitive tests were the ability to grow at 45 °C, D-xylose assimilation, color and macroscopic appearance in tobacco-agar. Between 11.6% and 15.1% of C. albicans strains produced chlamidoconidiae in the 3 differential media tested. The opacity halo (lipase) was evident in 95.6% of C. albicans isolates but 2 out of 13 C. dubliniensis also presented precipitation halo. We consider that at least 3 different phenotypical methods should be used to distinguish properly these two species since none of the tests is absolutely sensitive or specific.

Effect of protein malnutrition on the intestinal absorption of monosaccharides in rats in vivo.

The present study was planned to elucidate the role of protein malnutrition on the intestinal absorption of monosaccharides particularly--glucose and xylose, in inbred female albino rats. The experimental rats were fed with protein deficient diet containing 3% protein, whereas the control rats were given a diet containing 18% protein. The study on intestinal absorption of monosaccharides was conducted on both the groups of rats after the 7th and 15th day of receiving respective diets. The results indicated no significant impairment of glucose absorption of experimental rats fed 3% diet for 7 days as compared to the controls. However a 42% decrease in glucose absorption was observed when the animals were fed with the same diet for 15 days. The impairment was significant in all segments of intestine suggesting diminution in the absorption capacity of small intestine in malnutrition perhaps as a result of some permanent injury to mucosal cells of small intestine. Regarding xylose absorption, in experimental rats an increase of intestinal uptake was noticed in most of the segments of small intestine as compared to control rats.

Effect of fasting on the intestinal absorption of D-glucose and D-xylose in rats in vivo.

The present study was planned to elucidate the role of fasting on the intestinal absorption of monosaccharides particularly--glucose and xylose in inbred female albino rats. Rats (weighing 250-300 grams) were divided into three groups. One group of rats served as control while the other two were experimental. One of the experimental groups was starved for 48 hours while the other for a period of 72 hours. It was found that fasting for 72 hours causes an overall increase in absorption of glucose from small intestine. Forty-eight hours of fasting caused a significant increase in glucose absorption from distal ileum only. Increase in the glucose absorption in fasting from small intestine can well be explained on the basis of a reduction in glucose metabolism in general as an adaptation to starvation so as to leave more glucose for cerebral metabolism. No significant changes, whatsoever, were encountered with xylose absorption in fasting animals.

Obtenção e cinética da xilose redutase e xilitol desidrogenase de Candida guilliermondii cultivada em hidrolisado hemicelulósico de bagaço de cana-de-açúcar / Obtainment and kinetics of xylose reductase and xylitol dehydrogenase of Candida guillirmondii cultivated in sugarcane bagasse hemicellulosic hydrolysate

O metabolismo da D-xilose em leveduras inicia-se com a redução da D-xilose a xilitol pela xilose redutase (XR), a qual requer como cofator o NADPH e/ou NADH. O xilitol é oxidado a xilulose pela xilitol desidrogenase (XD), a qual emprega NA`D IND. +ï ou NAD`P IND. +ï como cofator. No presente trabalho, a bioconversão de D-xilose em xilitol por Candida guilliermondii FTI 20037 foi avaliada sob os aspectos enzimático e fermentativo, durante o cultivo em hidrolisado hemicelulósico de bagaço de cana-de-açúcar sob variadas condições de pH inicial, temperatura e aeração. Frente aos resultados obtidos nos cultivos em frascos agitados, encontrou-se a máxima atividade da XR (874,2 U.`mg IND. prot POT. -1ï) em pH inicial 6,0 e temperatura de 35ºC, condição esta na qual foram também verificados os máximos parâmetros fermentativos da produção de xilitol...

Efecto de la caseína en la absorción de d-xilosa medida mediante la prueba de H2 espirado / Effect of casein on d-xylose absorption measured by expired H2 test

Different experimental studies suggest that the presence of food in the alimentary tract, promote small intestinal absorption. The mechanism involved are not completely understood and might be related to motility changes or to humoral factors. Since studies have shown a decrease of small bowel motility after casein administration, the aim was to analyze the effect of this protein on small intestinal absorption and orocecal transit time. The hydrogen breath test was used to estimate d-xylose absorption. H2 production is dependent on the amount of this carbohydrate reaching the colon and therefore inversely proportional to d-xylose absorption. Six normal volunteers ingested 25 g d-xylose and 25 g de-xyllose + 30 g casein, and alveolar breath samples were obtained at 15 min intervals. Results: by adding casein to d-xylose solution a statistically significant decrease of maximal H2 concentration was observed from a mean of 40ñ11 ppm to a mean of 26ñ8 ppm. Similarly the area under the curve (which reflects the amount of xylose that was not absorbed) was also significantly decreased from a mean of 3281ñ1399 ppm to mean of 1394ñ700 ppm of H2. The orocecal transit time was significantly prolonged from 85.5ñ40 min to 112ñ38 min. Our results suggest that casein increased d-xylose absorption in normal subjects and that this effect might be related to a slower transit time

Lack of correlation between jejunal mucosal morphology and intestinal absorption in the hepatosplenic form of Schistosomiasis mansoni

Onze indivíduos portadores da forma hepatoesplênica da esquistossomose mansônica foram estudados. Em todos os pacientes a absorçäo intestinal de xilose e de gorduras foi normal. Apesar disto, o estudo histológico da mucosa jejunal mostrou graus variados de hipotrofia das vilosidades e aumento das células inflamatórias na lâmina própria. Através de análise morfométrica, foi observado que as vilosidades eram mais baixas e que a relaçäo altura das vilosidades/profundidade das criptas era maior nos esquistossomóticos do que nos controles. Em cinco havia ovos e granulomas na lâmina própria

Efecto de la irradiación aguda sobre la morfometria del intestino delgado y la absorción de D-Xilosa en ratas / Effect of radiation on morphometry of small bowel and D-Xilose absorption in rats

En estudios previos en nuestro laboratorio, hemos demostrado el efecto de la radiación sobre la morfometría del intestino delgado de ratas, luego de 7 días de una única dosis abdominal externa de 10000 rad. Nuestra intención fue determinar el efecto del mismo esquema de radiación gamma, en ratas, sobre la morfometría del yeyuno y la absorción de D-Xilosa, 7, 14 y 21 días luego de la injuria, en comparación con un grupo control. La absorción de la pentosa fue medida a través de la xilosemia, 60 minutos luego de una dosis intragástrica (0,5 grs/kgr de pseo corporal) del azúcar. Resultados: En el grupo control (n: 22) el espesor de la mucosa fue de 880 ñ 40 micrones (X ñ DS), la altura vellositaria de 640 ñ 37.6 micrones y la xilosemia 15.91 ñ 11.39 mg%. Enel grupo irradiado a los 7 días (n: 11) el peso se redujo en 33.36 ñ 6.29% (vs. control p < 001), el espesor mucoso 666 ñ 97 micrones (p < 001), la altura vellositaria 466 ñ 66.9 micrones (p < 001), mientras que la xilosemia fue de 6.16 ñ 2.3 mg% (p < 05). Los valores a los 14 días (n:16) expresan una mejoría generalizada y una recuperación completa a los 21 días (n:14). En conclusión, las ratas irradiadas demostraron estar afectadas en su peso, morfometría y absorción de D-Xilosa, ésta alteración es máxima a los 7 días y se recupera completamente a los 21 días. En cambio, nosotros hemos hallado una absorción normal de D-Xilosa a los 21 días. Es de destacar que entre ese trabajo (3) y el presente existen diferencias metodológicas, lo que no los hace comparables. Hemos observado recientemente que, la correspondencia entre la absorción y las alteraciones morfométricas del yeyuno, se mantienen aún cuando se utiliza una sustancia radioprotectora como las prostaglandinas (8). Sin embargo esto no agrega luz a la discusión patogénica...

Absorción intestinal en escolares asintomáticos portadores de Giardia lamblia / Intestinal absorption in asymptomatic school children with Giardia lamblia

En 14 escolares con edades entre 6,2 y 13,6 años, portadores asintomáticos de Giardia lamblia se hicieron evaluaciones antropométricas y mediciones de hematocrito, hemoglobinemia, proteinemia total, albuminemia, carotinemia basal y prueba de absorción de vitamina A y D-xilosa, antes y después de recibir tratamiento antiparasitario con Tinidazol y Mebendazol. Se demonstraron aumentos significativos en pliegue cutáneo tricipital, hematocrito, hemoglobinemia y proteinemia. La xilosemia fue de 38,8 + ou - 8,9 y de 57,5 + ou - 10,8 mg% (p <0,005) antes y después del tratamiento respectivamente. Las concentraciones de vitamina A después de sobrecarga oral fueron 66,5 + ou - 26,0 y 213,0 + ou - 113,0 microng% (p <0,005) y el delta vitamina A de 32,7 + ou - 25,3 y de 152,7 + ou - 115,8 microng% (p <0,005) para las etapas previa y posterior al tratamiento respectivamente. Se concluye que en escolares asintomáticos, portadores de Giardia lamblia, podría incrementar la absorción intestinal luego de erradicar al parásito

Colonización bacteriana del duodeno y absorción de d-xilosa en recién nacidos alimentados a través de sonda transpilórica / Bacterial colonization in the duodenum and intestinal absorption of d-xylose in newborn infants receiving transpyloric feedings

Los altos requerimientos calóricos del recién nacido, ya sea prematuro o de peso bajo al nacer, han llevado a la aceptación cada vez mayor de su alimentación mediante una sonda colocada al duodeno. A partir de la introducción del método, han surgido varios estudios que mencionan algunas complicaciones encontradas. Se ha propuesto que las alteraciones de la flora intestinal secundarias al empleo de una sonda transpilórica pueden repercutir sobre la capacidad de absorción del intestino; sin embargo, no han aparecido estudios que evalúen la función de absorción intestinal en estos casos. En el presente trabajo se investigó la influencia de la presencia de una sonda transpilórica en 13 pacientes recién nacidos (edades entre 1 y 30 días de vida) alimentados mediante sonda duodenal, correlacionándola con el conteo bacteriano del líquido aspirado del duodeno. En seis de estos pacientes se estudio la absorción de hidratos de carbono mediante la prueba de absorción a la hora de una carga de d-xilosa administrada por vía enteral. Se formó un grupo control con seis pacientes recién nacidos alimentados por vía oral, para determinar la absorción normal de la d-xilosa. En conclusión, se señala que a pesar de los altos conteos bacterianos encontrados en los aspirados duodenales en algunos de los pacientes del grupo experimental, durante el período de estudio no se encontró mala absorción al hidrato de carbono secundaría al empleo de una sonda transpilórica para la alimentación