Integrated bioinformatics, modelling, and gene expression analysis of the putative pentose transporter from Candida tropicalis during xylose fermentation with and without glucose addition.

The transport of substrates across the cell membrane plays an essential role in nutrient assimilation by yeasts. The establishment of an efficient microbial cell factory, based on the maximum use of available carbon sources, can generate new technologies that allow the full use of lignocellulosic constituents. These technologies are of interest because they could promote the formation of added-value products with economic feasibility. In silico analyses were performed to investigate gene sequences capable of encoding xylose transporter proteins in the Candida tropicalis genome. The current study identified 11 putative transport proteins that have not yet been functionally characterized. A phylogenetic tree highlighted the potential C. tropicalis xylose-transporter proteins CtXUT1, CtXUT4, CtSTL1, CtSTL2, and CtGXT2, which were homologous to previously characterized and reported xylose transporters. Their expression was quantified through real-time qPCR at defined times, determined through a kinetic analysis of the microbial growth curve in the absence/presence of glucose supplemented with xylose as the main carbon source. The results indicated different mRNA expression levels for each gene. CtXUT1 mRNA expression was only found in the absence of glucose in the medium. Maximum CtXUT1 expression was observed in intervals of the highest xylose consumption (21 to 36 h) that corresponded to consumption rates of 1.02 and 0.82 g/L/h in the formulated media, with xylose as the only carbon source and with glucose addition. These observations indicate that CtXUT1 is an important xylose transporter in C. tropicalis. KEY POINTS: • Putative xylose transporter proteins were identified in Candida tropicalis; • The glucose concentration in the cultivation medium plays a key role in xylose transporter regulation; • The transporter gene CtXUT1 has an important role in xylose consumption by Candida tropicalis.

A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid.

Experiments based on a 2(3) central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar-alcohol mill. The independent variables selected for study were temperature, varied from 112.5°C to 157.5°C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.

Fermentation of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing.

This work aims to evaluate the fermentability of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing using Candida guilliermondii FTI 20037 yeast. The inoculum was obtained from yeast culture in a medium containing glucose as a carbon source supplemented with rice bran extract, CaCl(2)·2H(2)O and (NH(4))(2)SO(4) in 50 mL Erlenmeyer flasks, containing 20 mL of medium, initial 5.5 pH under agitation of an orbital shaker (200 rpm) at 30°C for 24 h. The cellulosic hydrolysates, prior to being used as a fermentation medium, were autoclaved for 15 min at 0.5 atm and supplemented with the same nutrients employed for the inoculum, except the glucose, using the same conditions for the inoculum, but with a period of 48 h. Preliminary results showed the highest consumption of glucose (97%) for all the hydrolysates, at 28 h of fermentation. The highest concentration of ethanol (20.5 g/L) was found in the procedure of sugarcane bagasse pretreated by hydrothermal processing (195°C/10 min in 20 L reactor) and delignificated with NaOH 1.0% (w/v), 100°C, 1 h in 500 mL stainless steel ampoules immersed in an oil bath.

Role of glycerol addition on xylose-to-xylitol bioconversion by Candida guilliermondii.

The effect of glycerol on xylose-to-xylitol bioconversion by Candida guilliermondii was evaluated by its addition (0.7 and 6.5 g/l) to semidefined media (xylose as a substrate). The glycerol concentrations were chosen based on the amounts produced during previous studies on xylitol production by C. guilliermondii. Medium without glycerol addition (control) and medium containing glycerol (53 g/l) in substitution to xylose were also evaluated. According to the results, the addition of 0.7 g/l glycerol to the fermentation medium favored not only the yield (Y(P/S)=0.78 g/g) but also the xylitol productivity (Q(P)=1.13 g/l/h). During the xylose-to-xylitol bioconversion, the formation of byproducts (glycerol and ethanol) was observed for all conditions employed. In relation to the cellular growth, glycerol as the only carbon source for C. guilliermondii was better than xylose or xylose and glycerol mixtures, resulting in a maximum cellular concentration (5.34 g/l).

Enhanced xylitol production by precultivation of Candida guilliermondii cells in sugarcane bagasse hemicellulosic hydrolysate.

The present work evaluated the key enzymes involved in xylitol production (xylose reductase [XR] and xylitol dehydrogenase [XDH]) and their correlation with xylose, arabinose, and acetic acid assimilation during cultivation of Candida guilliermondii FTI 20037 cells in sugarcane bagasse hemicellulosic hydrolysate. For this purpose, inocula previously grown either in sugarcane bagasse hemicellulosic hydrolysate (SBHH) or in semidefined medium (xylose as a substrate) were used. The highest xylose/acetic acid consumption ratio (1.78) and the lowest arabinose consumption (13%) were attained in the fermentation using inoculum previously grown in semidefined medium (without acetic acid and arabinose). In this case, the highest values of XR (1.37 U mg prot(-1)) and XDH (0.91 U mg prot(-1)) activities were observed. The highest xylitol yield (approximately 0.55 g g(-1)) and byproducts (ethanol and glycerol) formation were not influenced by inoculum procedure. However, the cell previously grown in the hydrolysate was effective in enhancing xylitol production by keeping the XR enzyme activity at high levels (around 0.99 U.mg(prot) (-1)), reducing the XDH activity (34.0%) and increasing xylitol volumetric productivity (26.5%) with respect to the inoculum cultivated in semidefined medium. Therefore, inoculum adaptation to SBHH was shown to be an important strategy to improve xylitol productivity.

Sugarcane bagasse as raw material and immobilization support for xylitol production.

Xylose-to-xylitol bioconversion was performed utilizing Candida guilliermondii immobilized in sugarcane bagasse and cultured in Erlenmeyer flasks using sugarcane bagasse hydrolysate as the source of xylose. Fermentations were carried out according to a factorial design, and the independent variables considered were treatment, average diameter, and amount of bagasse used as support for cell immobilization. By increasing the amount of support, the xylitol yield decreased, whereas the biomass yield increased. The diameter of the support did not influence xylitol production, and treatment of the bagasse with hexamethylene diamine prior to fermentation resulted in the highest amount of immobilized cells.

Inhibitory effect of acetic acid on bioconversion of xylose in xylitol by Candida guilliermondii in sugarcane bagasse hydrolysate

Hidrolisado de bagaço de cana-de-açúcar contendo uma concentração inicial de ácido acético de 3,5g/L foi utilizado como meio de fermentação para a bioconversão de xilose em xilitol pela levedura Candida guilliermondii FTI 20037. Ácido acético (2,0g/L) foi adicionado ao meio em diferentes tempos de fermentação, com o objetivo de avaliar o efeito deste ácido neste bioprocesso. O maior efeito inibitório deste ácido na bioconversão de xilose em xilitol pela levedura ocorreu quando este foi adicionado ao meio após 12h de fermentação. Nesta condição observou-se uma redução de 23,22 porcento e 11,24 porcento, respectivamente, no consumo de xilose e no crescimento celular em relação à fermentação em que a adição deste ácido ocorreu no tempo inicial de incubação. Como conseqüência do efeito inibitório, observou-se os menores valores de rendimento (0,39g/g) e produtividade (0,22g/L.h) de xilitol, correspondendo a uma redução de 36 e 48 porcento, respectivamente, em relação aos valores obtidos com a adição de ácido acético nos outros tempos de fermentação. Os resultados obtidos permitem concluir que, nas condições experimentais empregadas neste trabalho, o efeito inibitório do ácido acético sobre a bioconversão de xilose em xilitol é dependente do tempo de fermentação em que a adição do ácido foi feita e não apenas de sua concentração no meio.

Batch xylitol production from wheat straw hemicellulosic hydrolysate using Candida guilliermondii in a stirred tank reactor.

Batch production of xylitol from the hydrolysate of wheat straw hemicellulose using Candida guilliermondii was carried out in a stirred tank reactor (agitation speed of 300 rpm, aeration rate of 0.6 vvm and initial cell concentration of 0.5 g l(-1)). After 54 h, xylitol production from 30.5 g xylose l(-1) reached 27.5 g l(-1), resulting in a xylose-to-xylitol bioconversion yield of 0.9 g g(-1) and a productivity of 0.5 g l(-1) h(-1).

Use of immobilized Candida yeast cells for xylitol production from sugarcane bagasse hydrolysate: cell immobilization conditions.

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate. A full factorial design was employed to determine whether variations in the immobilization conditions would have any effects on the beads, chemical stability and on the xylitol production rates. Duplicate fermentation runs were carried out in 125-mL Erlenmeyer flasks maintained in a rotatory shaker at 30 degrees C and 200 rpm for 72 h. Samples were periodically analyzed to monitor xylose and acetic acid consumption, xylitol production, free cell growth, and bead solubilization. Concentrations of sodium alginate at 20.0 g/L and calcium chloride at 11.0 g/L and bead curing time of 24 h represented the most appropriate immobilization conditions within the range of conditions tested.

Improvement in xylitol production from sugarcane bagasse hydrolysate achieved by the use of a repeated-batch immobilized cell system.

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate during five successive fermentation batches, each lasting 48 hours. The bioconversion efficiency of 53.2%, the productivity of 0.50 g/l x h and the final xylitol concentration of 23.8 g/l obtained in the first batch increased to 61.5%, 0.59 g/l x h and 28.4 g/l, respectively, in the other four batches (mean values), with variation coefficients of up to 2.3%.