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.

New cultive medium for bioconversion of C5 fraction from sugarcane bagasse using rice bran extract

The use of hemicellulosic hydrolysates in bioprocesses requires supplementation as to ensure the best fermentative performance of microorganisms. However, in light of conflicting data in the literature, it is necessary to establish an inexpensive and applicable medium for the development of bioprocesses. This paper evaluates the fermentative performance of Scheffersomyces (Pichia) stipitis and Candida guilliermondii growth in sugarcane bagasse hemicellulosic hydrolysate supplemented with different nitrogen sources including rice bran extract, an important by-product of agroindustry and source of vitamins and amino acids. Experiments were carried out with hydrolysate supplemented with rice bran extract and (NH4)2SO4; peptone and yeast extract; (NH4)2SO4, peptone and yeast extract and non-supplemented hydrolysate as a control. S. stipitis produced only ethanol, while C. guilliermondii produced xylitol as the main product and ethanol as by-product. Maximum ethanol production by S. stipitis was observed when sugarcane bagasse hemicellulosic hydrolysate was supplemented with (NH4)2SO4, peptone and yeast extract. Differently, the maximum xylitol formation by C. guilliermondii was obtained by employing hydrolysate supplemented with (NH4)2SO4 and rice bran extract. Together, these findings indicate that: a) for both yeasts (NH4)2SO4 was required as an inorganic nitrogen source to supplement sugarcane bagasse hydrolysate; b) for S. stipitis, sugarcane hemicellulosic hydrolysate must be supplemented with peptone and yeast extract as organic nitrogen source; and: c) for C. guilliermondii, it must be supplemented with rice bran extract. The present study designed a fermentation medium employing hemicellulosic hydrolysate and provides a basis for studies about value-added products as ethanol and xylitol from lignocellulosic materials.

Evaluation of oat hull hemicellulosic hydrolysate fermentability employing Pichia stipitis

Oat hull hemicellulosic hydrolysate obtained by diluted acid hydrolysis was employed as fermentation medium for Pichia stipitis cultivation. A comparison between the use of treated hydrolysate with 1% activated charcoal to reduce the toxic compounds generated during the hydrolysis process and untreated hydrolysate as a control was conducted. In the cultures using treated hydrolysate the total consumption of glucose, low xylose consumption and ethanol and glycerol formation were observed. The medium formulated with untreated hydrolysate showed morphological cell modifications with consequently cell death, no ethanol formation and formation of glycerol as byproduct of fermentative process, probably as a response to stressful conditions to yeast due to presence of high concentration of toxic compounds. Thus, further studies are suggested in order to determine the best conditions for hydrolysis and detoxification of the hydrolysate to improve the fermentative performance of P. stipitis.

Obtaining partial purified xylose reductase from Candida guilliermondii / Obtenção de xilose redutase de Candida guilliermondii parcialmente purificada

The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40 percent, 7 percent and 67 percent, respectively.

A bioconversão enzimática da xilose em xilitol pela xilose redutase (XR) é uma alternativa para as vias química e microbiológica. Avaliouse a purificação parcial da XR, utilizando os três seguintes procedimentos: uma coluna de agarose, um reator com membrana ou tubos de ultracentrifugação Amicon Ultra-15 50K, com rendimento de 40 por cento, 7 por cento ou 67 por cento, respectivamente.

Improvement of biotechnological xylitol production by glucose during cultive of Candida guilliermondii in sugarcane bagasse hydrolysate

The effect of glucose on xylose-to-xylitol bioconversion by Candida guilliermondii was examined by adding it to sugarcane bagasse hydrolysate medium to obtain different glucose:xylose ratios (1:25, 1:12, 1:5 and 1:2.5). Under experimental conditions, increasing glucose:xylose ratio improved the assimilation of the xylose present in the hydrolysate by yeast, resulting in biomass increase, and in the formation of xylitol and glycerol/ethanol by-products. Maximum values of xylitol yield (0.59 g g-1) and volumetric productivity (0.53 g l-1.h-1) were obtained with glucose:xylose ratio of 1:5, resulting in the higher conversion efficiency (64.3 percent).

O efeito da glicose na bioconversão de xilose em xilitol por Candida guilliermondii foi avaliado em hidrolisado hemicelulósico de bagaço de cana com diferentes relações glicose:xilose (1:25, 1:12, 1:5 and 1:2,5). Sob as condições experimentais, o aumento da relação glicose:xilose favoreceu a assimilação da xilose presente no hidrolisado, resultando em aumento da biomassa celular e aumento da formação de xilitol e dos sub-produtos glicerol e etanol. Os valores máximos do fator de conversão de xilose em xilitol (0,59 g g-1) e da produtividade volumétrica de xilitol (0,53 g l-1.h-1) foram obtidos com a relação glicose:xilose 1:5, resultando na maior eficiência de conversão (64,3 por cento).

Caracterização da xilose redutase extraída por micelas reversas-CTAB a partir de homogenato de Candida guilliermondii / Characterization of xylose reductase extracted by CTAB-reversed micelles from Candida guilliermondii homogenate

Xylosereductase (XR) (E.C.1.1.1.21), produced by Candida guilliermondii, grown in sugar cane bagasse hydrolysate, was separated directly from the cell homogenate by reversed micelles of cetyl trimethyl ammonium bromide (CTAB), attaining a recovery yield of 100% and enrichment factor of 5.6 fold. The extraction conditions were: pH=7.0, electrical conductivity= 14 mS/cm, T=5 degrees C, 5% (w/w) of hexanol, 22% (w/w) of butanol and 0.15 M CTAB. The XR after extraction was stable in pH interval of 6.0-6.5 and its heat inactivation constant was about 6.5 fold higher than that before extraction. The 'V IND. max' values against both xylose and NADPH for XR before and after extraction by reversed-micelles differed about 6%, whereas the difference on KM values were more pronounced. The ('K IND. m') 'IND. xylose' for XR after extraction was about 35% higher than before extraction, meanwhile ('K IND. m') 'IND. NADPH' was about 30% lower after than before extraction. As the KM variations indirectly signaled, the XR affinity simultaneously diminishes for xylose and increases for NADPH. This could probably explain why the 'V IND. max' values for XR before and after extraction were quite similar

A xilose redutase (XR) (E.C.1.1.1.21), produzida por Candida guilliermondii cultivada em hidrolisado de bagaço de cana de açúcar, foi separada diretamente do homogenato livre de células através da técnica de micelas reversas feitas com cetil trimetil brometo de amônio (CTAB). Obteve-se um rendimento de recuperação da enzima de 100% e um fator de enriquecimento de 5,6 vezes. As condições de extração foram: pH=7,0, condutividade elétrica = 14 mS/cm, T= 5 ºC, 5% (w/w) de hexanol, 22% (w/w) de butanol e 0.15M CTAB. A XR após a extração manteve-se estável no intervalo de pH entre 6.0 e 6.5, sendo a constante de inativação térmica cerca de 6,5 vezes maior do que aquela antes da extração. Os valores de Vmax da XR frente à xilose e NADPH antes e após a extração por micelas reversas diferiram cerca de 6%, enquanto que as diferenças nos valores de KM foram mais pronunciadas. O (KM)xilose para a XR após a extração foi cerca de 35% maior do que antes da extração, enquanto que (KM)NADPH foi 30% menor após do que antes da extração. As variações nos valores de KM indicam, indiretamente, que a afinidade da XR simultaneamente diminui para a xilose e aumenta para o NADPH. Este resultado poderia explicar a razão pela qual os valores de Vmax antes e após a extração terem sido praticamente iguais

Reclassification of Candida guilliermondii FTI 20037 as Candida tropicalis based on molecular phylogenetic analysis

As leveduras do gênero Candida possuem tanto importância clínica como diversas aplicações industriais, principalmente na indústria de alimentos. A levedura Candida guilliermondii FTI 20037 tem sido exaustivamente estudada pois pretende-se utilizá-la no estabelecimento de um processo biotecnológico para a produção de xilitol. O objetivo deste trabalho foi verificar a classificação taxonômica desta levedura por análise de sequências do rDNA e do gene xyl1. Fragmentos correspondentes a estas regiões foram amplificados por PCR e a análise destas sequências por BLAST revelou alta identidade com sequências correspondentes de Candida tropicalis. Estes resultados nos levam a propor que C. guilliermondii FTI 20037 deva ser reclassificada como C. tropicalis.

Reclassification of Candida guilliermondii FTI 20037 as Candida tropicalis based on molecular phylogenetic analysis

Yeasts of the genus Candida are of clinical importance and also have many industrial applications, mainly in the food industry. The yeast Candida guilliermondii FTI 20037 has been extensively studied in order to establish a biotechnological process for the production of xylitol. The goal of this study was to verify the taxonomic classification of this strain based on the analysis of rDNA sequences and the xyl1 gene. DNA fragments from these sequences were amplified by PCR and BLAST analysis revealed strong identity with the corresponding sequences from Candida tropicalis. Based on these results, we propose that C. guilliermondii FTI 20037 must be reclassified as C. tropicalis.

As leveduras do gênero Candida possuem tanto importância clínica como diversas aplicações industriais, principalmente na indústria de alimentos. A levedura Candida guilliermondii FTI 20037 tem sido exaustivamente estudada pois pretende-se utilizá-la no estabelecimento de um processo biotecnológico para a produção de xilitol. O objetivo deste trabalho foi verificar a classificação taxonômica desta levedura por análise de sequências do rDNA e do gene xyl1. Fragmentos correspondentes a estas regiões foram amplificados por PCR e a análise destas sequências por BLAST revelou alta identidade com sequências correspondentes de Candida tropicalis. Estes resultados nos levam a propor que C. guilliermondii FTI 20037 deva ser reclassificada como C. tropicalis.

Otenção biotecnológica de xilitol a partir de cavacos de eucalipto / Xylitol production from eucalyptus chips by biotechnological means

Cavacos de eucalipto foram submetidos à hidrólise ácida resultando em um hidrolisado hemicelulósico rico em acúcares fermentescíveis. O hidrolisado foi usado como meio de cultivo para o crescimento da levedura Candida guilliermondii FTI 20037 para avaliar a influência da suplementação do mesmo com os nutrientes sulfato de amônio, cloreto de cálcio e farelo de arroz, bem como a concentração de xilose no hidrolisado, o pH e o tempo de fermentação na produção de xilitol. A formação de xilitol a partir do hidrolisado hemicelulósico de cavacos de eucalipto foi influenciada pela presença de sulfato de amônio e farelo de arroz, pela concentração de xilose no hidrolisado e pH inicial de fermentação...

Obtencao do xilitol por fermentacao do hidrolisado hemicelulosico de bagaco de cana-de-acucar / Obtainment of the xylitol by fermentation of the sugar cane bagasse hemicellulosic hydrolyzate

Com o objetivo de viabilizar a obtencao de xilitol por via biotecnologica a partir de hidrolisado hemicelulosico do bagaco de cana-de-acucar, foram conduzidos experimentos utilizando-se a levedura C. guilliermondii FTI 20037 e avaliados os parametros fermentativos, pH, concentracao e idade do inoculo, concentracao do hidrolisado, aeracao e adaptacao da levedura no hidrolisado. Os resultados obtidos revelaram a necessidade do tratamento do hidrolisado antes da sua utilizacao como meio de fermentacao, ja que a suplementacao do hidrolisado hemicelulosico acido com nutrientes, nao proporcionou o crescimento de C. guilliermondii, causando a morte celular ao longo do tempo