Xylitol production from corn fiber and sugarcane bagasse hydrolysates by Candida tropicalis

A natural isolate, Candida tropicalis was tested for xylitol production from corn fiber and sugarcane bagasse hydrolysates. Fermentation of corn fiber and sugarcane bagasse hydrolysate showed xylose uptake and xylitol production, though these were very low, even after hydrolysate neutralization and treatments with activated charcoal and ion exchange resins. Initial xylitol production was found to be 0.43 g/g and 0.45 g/g of xylose utilised with corn fiber and sugarcane bagasse hydrolysate respectively. One of the critical factors for low xylitol production was the presence of inhibitors in these hydrolysates. To simulate influence of hemicellulosic sugar composition on xylitol yield, three different combinations of mixed sugar control experiments, without the presence of any inhibitors, have been performed and the strain produced 0.63 g/g, 0.68 g/g and 0.72 g/g of xylose respectively. To improve yeast growth and xylitol production with these hydrolysates, which contain inhibitors, the cells were adapted by sub culturing in the hydrolysate containing medium for 25 cycles. After adaptation the organism produced more xylitol 0.58 g/g and 0.65 g/g of xylose with corn fiber hydrolysate and sugarcane bagasse hydrolysate respectively.     

Microbial production of xylitol from D-xylose and sugarcane bagasse hemicellulose using newly isolated thermotolerant yeast Debaryomyces hansenii

A thermotolerant yeast capable of fermenting xylose to xylitol at 40°C was isolated and identified as a strain of Debaryomyces hansenii by ITS sequencing. This paper reports the production of xylitol from D-xylose and sugarcane bagasse hemicellulose by free and Ca-alginate immobilized cells of D. hansenii. The efficiency of free and immobilized cells were compared for xylitol production from D-xylose and hemicellulose in batch culture at 40°C. The maximum xylitol produced by free cells was 68.6 g/L from 100 g/L of xylose, with a yield of 0.76 g/g and volumetric productivity 0.44 g/L/h. The yield of xylitol and volumetric productivity were 0.69 g/g and 0.28 g/L/h respectively from hemicellulosic hydrolysate of sugarcane bagasse after detoxification with activated charcoal and ion exchange resins. The Ca-alginate immobilized D. hansenii cells produced 73.8 g of xylitol from 100 g/L of xylose with a yield of 0.82 g/g and volumetric productivity of 0.46 g/L/h and were reused for five batches with steady bioconversion rates and yields.     

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 (∼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.     

Metabolic engineering of Saccharomyces cerevisiae for increased bioconversion of lignocellulose to ethanol

The absence of pentose-utilizing enzymes in Saccharomyces cerevisiae is an obstacle for efficiently converting lignocellulosic materials to ethanol. In the present study, the genes coding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) from Pichia stipitis were successfully engineered into S. cerevisae. As compared to the control transformant, engineering of XYL1 and XYL2 into yeasts significantly increased the microbial biomass (8.1 vs. 3.4 g/L), xylose consumption rate (0.15 vs. 0.02 g/h) and ethanol yield (6.8 vs. 3.5 g/L) after 72 h fermentation using a xylose-based medium. Interestingly, engineering of XYL1 and XYL2 into yeasts also elevated the ethanol yield from sugarcane bagasse hydrolysate (SUBH). This study not only provides an effective approach to increase the xylose utilization by yeasts, but the results also suggest that production of ethanol by this recombinant yeasts using unconventional nutrient sources, such as components in SUBH deserves further attention in the future.     

Evaluation of ethanol production by a new isolate of yeast during fermentation in synthetic medium and sugarcane bagasse hemicellulosic hydrolysate

A new xylose fermenting yeast was isolated from over-ripe banana by enrichment in xylose-containing medium. The phylogenetic analysis of ITS1-5.8S-ITS2 region sequences of ribosomal RNA of isolate BY2 revealed that it shows affiliation to genus Pichia and clades with Pichia caribbica. In batch fermentation, Pichia strain BY2 fermented xylose, producing 15 g l^?1 ethanol from 30 g l^?1 xylose under shaking conditions at 28°C, with ethanol yield of 0.5 g g^?1 and volumetric productivity of 0.31 g l^?1 h^?1. The optimum pH range for ethanol production from xylose by Pichia strain BY2 was 5–7. Pichia strain BY2 also produced 6.08 g l^?1 ethanol from 30 g l^?1 arabinose. Pichia strain BY2 can utilize sugarcane bagasse hemicellulose acid hydrolysate for alcohol production, efficiency of fermentation was improved by neutralization, and sequential use of activated charcoal adsorption method. Percent total sugar utilized and ethanol yield for the untreated hydrolysate was 17.14% w/v and 0.33 g g^?1, respectively, compared with 66.79% w/v and 0.45 g g^?1, respectively, for treated hemicellulose acid hydrolysate. This new yeast isolate showed ethanol yield of 0.45 g g^?1 and volumetric productivity of 0.33 g l^?1 h^?1 from sugarcane bagasse hemicellulose hydrolysate detoxified by neutralization and activated charcoal treatment, and has potential application in practical process of ethanol production from lignocellulosic hydrolysate.     

Yeast Immobilization in LentiKats®: A New Strategy for Xylitol Bioproduction from Sugarcane Bagasse

Summary A new PVA-hydrogel matrix for yeast cell immobilization for xylitol bioproduction from sugarcane bagasse was studied. Five repeated-batch fermentation runs were carried out in medium based on sugarcane bagasse hemicellulosic hydrolysate with reuse of the entrapped biocatalyst. The system performance as well as the metabolic behaviour of cells entrapped into the matrix were evaluated. The biocatalyst remained stable and exhibited a similar fermentative profile in all the successive batches, demonstrating the viability of the system. At the end of the run, an average xylitol production was observed of 35.1 g l⁻¹ and average xylitol yield and productivity of 0.58 g g⁻¹ and 0.49 g l⁻¹ h⁻¹, respectively.     

Effects of sulfuric acid loading and residence time on the composition of sugarcane bagasse hydrolysate and its use as a source of xylose for xylitol bioproduction

A 2(2) full factorial design was employed to evaluate the effects of sulfuric acid loading and residence time on the composition of sugarcane bagasse hydrolysate obtained in a 250-L reactor. The acid loading and the residence time were varied from 70 to 130 mg acid per gram of dry bagasse and from 10 to 30 min, respectively, while the temperature (121 degrees C) and the bagasse loading (10%) were kept constant. Both the sulfuric acid loading and the residence time influenced the concentrations of xylose and inhibitors in the hydrolysate. The highest xylose concentration (22.71 g/L) was achieved when using an acid loading of 130 mg/g and a residence time of 30 min. These conditions also led to increased concentrations of inhibiting byproducts in the hydrolysate. All of the hydrolysates were vacuum-concentrated to increase the xylose concentration, detoxified by pH alteration and adsorption into activated charcoal, and used for xylitol bioproduction in a stirred tank reactor. Neither the least (70 mg/g, 10 min) nor the most severe (130 mg/g, 30 min) hydrolysis conditions led to the best xylitol production (37.5 g/L), productivity (0.85 g/L h), and yield (0.78 g/g).     

Fermentation of hexoses and pentoses from sugarcane bagasse hydrolysates into ethanol by Spathaspora hagerdaliae

The present study evaluated 13 strains of yeast for ethanol and xylitol production from xylose. Among them, Spathaspora hagerdaliae UFMG-CM-Y303 produced ethanol yields (Y_P/S) of 0.25 g g^− 1 and 0.39 g g^− 1 under aerobic and microaerophilic conditions, respectively, from a mixture of glucose and xylose in flasks. A pH of 5.0 and an inoculum of 3.0 × 10⁸ cells mL^− 1r resulted in the highest ethanol yields. These conditions were tested in a bioreactor for fermenting a medium containing an enzymatic hydrolysate of sugarcane bagasse with 15.5 g L^− 1 of glucose and 3 g L^− 1 of xylose, and achieved a Y_P/S of 0.47 g g^− 1, in relation to total available sugar. These results suggest that S. hagerdaliae UFMG-CM-Y303 has potential for use in second-generation ethanol studies.

     

Evaluation of fermentative potential of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> ATCC 36907 in cellulosic and hemicellulosic sugarcane bagasse hydrolysates on xylitol and ethanol production

This paper evaluates the fermentative potential of Kluyveromyces marxianus grown in sugarcane bagasse cellulosic and hemicellulosic hydrolysates obtained by acid hydrolysis. Ethanol was obtained from a single glucose fermentation product, whereas xylose assimilation resulted in xylitol as the main product and ethanol as a by-product derived from the metabolism of this pentose. Fermentation performed in a simulated hydrolysate medium with a glucose concentration similar to that of the hydrolysate resulted in ethanol productivity (Qp?=?0.86 g L^?1 h^?1) that was tenfold higher than the one observed in the cellulosic hydrolysate. However, the use of hemicellulosic hydrolysate favored xylose assimilation in comparison with simulated medium with xylose and glucose concentrations similar to those found in this hydrolysate, without toxic compounds such as acetic acid and phenols. Under this condition, xylitol yield was 53.8 % higher in relation to simulated medium. Thus, the total removal of toxic compounds from the hydrolysate is not necessary to obtain bioproducts from lignocellulosic hydrolysates.     

Effect of acetic acid present in bagasse hydrolysate on the activities of xylose reductase and xylitol dehydrogenase in <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The first two steps in xylose metabolism are catalyzed by NAD(P)H-dependent xylose reductase (XR) (EC 1.1.1.21) and NAD(P)-dependent xylitol dehydrogenase (XDH) (EC 1.1.1.9), which lead to xylosexylitolxylulose conversion. Xylitol has high commercial value, due to its sweetening and anticariogenic properties, as well as several clinical applications. The acid hydrolysis of sugarcane bagasse allows the separation of a xylose-rich hemicellulosic fraction that can be used as a substrate for Candida guilliermondii to produce xylitol. However, the hydrolysate contains acetic acid, an inhibitor of microbial metabolism. In this study, the effect of acetic acid on the activities of XR and XDH and on xylitol formation by C. guilliermondii were studied. For this purpose, fermentations were carried out in bagasse hydrolysate and in synthetic medium. The activities of XR and XDH were higher in the medium containing acetic acid than in control medium. Moreover, none of the fermentative parameters were significantly altered during cell culture. It was concluded that acetic acid does not interfere with xylitol formation since the increase in XR activity is proportional to XDH activity, leading to a greater production of xylitol and its subsequent conversion to xylulose.     

Biotechnological production of xylitol in a three-phase fluidized bed bioreactor with immobilized yeast cells in Ca-alginate beads

Cells of Candida guilliermondii entrapped in Ca-alginate beads were used for xylitol production, from concentrated hemicellulose hydrolyzate of sugarcane bagasse, in a fluidized bed bioreactor (FBR). The maximum xylitol concentration 28.9 g xylitol/L was obtained at a high aeration rate of 600 mL/min after 70 h of fermentation, indicating that the use of high aeration rate in this system is favored for better oxygen transfer into the immobilized cells. The specific xylitol productivity and the xylitol yield were of 0.4 g xylitol/L.h and 0.58 g xylitol/g xylose respectively. The immobilization efficiency at the end of the fermentation was of 65 %. After 90 h of fermentation xylitol productivity and yield decreased to 0.25 g xylitol/L.h and 0.47 g xylitol/g xylose respectively, indicating the beginning of xylitol consumption by the yeast. The use of FBR system with immobilized cells presented high xylitol yield and productivity.     

A strain of <Emphasis Type="Italic">Meyerozyma guilliermondii</Emphasis> isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.     

酵母发酵蔗渣半纤维素水解物生产木糖酶

采用二次正交旋转组合设计研究了蔗渣半纤维素水解过程中硫酸浓度与液 固比对木糖收率的影响。回归分析表明 ,这两个因素与木糖的收率之间存在显著的回归关系。通过回归方程优化水解条件 ,当硫酸浓度 2 .4g L ,液 固 =6 .2 ,在蒸汽压力 2 .5× 10 4Pa的条件下水解 2 .5h ,10 0g蔗渣可水解生成木糖约 2 4g。大孔树脂吸附层析处理蔗渣半纤维素水解物 ,能有效地减少其中的酵母生长抑制物含量 ,显著改善水解物的发酵性能。用大孔树脂在pH 2条件下处理过的蔗渣半纤维素水解物作基质 ,含木糖 2 0 0g L ,产木糖醇酵母菌株CandidatropicalisAS2 .1776发酵 110h耗完基质中的木糖 ,生成木糖醇 12 7g L ,产物转化率 0 .6 4(木糖醇g 木糖g) ,产物生成速率 1.15g L·h .     

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L⁻¹), the main ethanol producers were Scheffersomyces stipitis (14.08 g L⁻¹), Scheffersomyces sp. (7.94 g L⁻¹) and Spathaspora boniae (7.16 g L⁻¹). Sc. stipitis showed the highest ethanol yield (0.42 g g⁻¹) and the highest productivity (0.39 g L⁻¹h⁻¹). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g⁻¹, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg⁻¹) and Saitozyma podzolica (0.384 U mg⁻¹). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.     

Xylitol formation by Candida guilliermondii grown in a cane bagasse hemicellulosic hydrolysate: Effect of aeration and inoculum adaptation

The xylose conversion into by Candida guilliermondii was evaluated in sugar cane bagasse hemicellulosic hydrolysate. The effect of air flow rates of 0.4, 0.6 and 0.8 vvm cn xylitol formation was studied. In addition, inoculum previously adapted to the hydrolysate was also tested in the fermentation carried out at 0.6 vvm. The results showed that xylitol production depends markedly on the aeration rate and on the previous adaptation of the yeast to the hydrolysate. When the highest productivity of xylitol was 0.39 g/l × h. However, during the fermentation carried out at an air flow rate of 0.6 vvm with adapted inoculum, the productivity increased to 0.65 g/l × h. Furthermore, the adapted cells performed quite well in the presencel of acetic concentrations of about 4.5 g/l in the medium.     

Critical factors affecting ethanol production by immobilized Pichia stipitis using corn cob hemicellulosic hydrolysate

Fermentation of xylose from hydrolysate of acid-treated corn cob by Pichia stipitis is inhibited by acetic acid and lignin derivatives. In the present study, we have designed and implemented an immobilized cell culture for xylose to ethanol conversion from acid-treated corn cob hydrolysate without the removal of fermentation inhibitors. In this study, cultivations of suspended and immobilized Pichia were compared in terms of ethanol yield and productivity to investigate whether the cell immobilization could improve resistance to inhibitors. Cell immobilization clearly favored the fermentative metabolism in nondetoxified corn cob hydrolysate leading to an improvement of twofold ethanol productivity as compared to that achieved with suspension culture. Calcium alginate as an immobilization matrix was selected to immobilize Pichia cells. Concentrations of sodium alginate, calcium chloride, and fermentor agitation speed were optimized for ethanol production using statistical method. Statistical analysis showed that agitation speed had maximum influence on ethanol production by immobilized Pichia cells. In comparison to suspension culture, immobilization had a positive impact on the fermentative metabolism of Pichia, improving the ethanol yield from 0.40 to 0.43?g/g and productivity from 0.31 to 0.51?g/L/h for acid-treated corn cob hydrolysate.     

Variables that affect xylitol production from sugarcane bagasse hydrolysate in a zeolite fluidized bed reactor

The operational conditions for xylitol production by fermentation of sugarcane bagasse hydrolysate in a fluidized bed reactor with cells immobilized on zeolite were evaluated. Fermentations were carried out under different conditions of air flowrate (0.0125-0.0375 vvm), zeolite mass (100-200 g), initial pH (4-6), and xylose concentration (40-60 g/L), according to a 2(4) full factorial design. The air flowrate increase resulted in a metabolic deviation from product to biomass formation. On the other hand, the pH increase favored both the xylitol yield (Y(P/S)) and volumetric productivity (Q(P)), and the xylose concentration increase positively influenced the xylitol concentration. The best operational conditions evaluated were based on the use of an air flowrate of 0.0125 vvm, 100 g of zeolite, pH 6, and xylose concentration of 60 g/L. Under these conditions, 38.5 g/L of xylitol were obtained, with a Y(P/S) of 0.72 g/g, Q(P) of 0.32 g/L.h, and cell retention of 25.9%.     

Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

Summary The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.Nomenclature max maximum specific growth rate (per hour) - qSmax maximum specific rate of xylose consumption (g xylose per g dry biomass per hour) - qpmax maximum specific productivity of xylitol (g xylitol per g dry biomass per hour) - Qp average volumetric productivity of xylitol (g xylitol per liter per hour) - Y_P/S xylitol yield (g xylitol per g substrate utilized) - Y_P'/S glycerol yield (g glycerol per g substrate utilized) - Y_X/S biomass yield (g dry biomass per g substrate utilized)     

The influence of pH and dilution rate on continuous production of xylitol from sugarcane bagasse hemicellulosic hydrolysate by C. guilliermondii

Continuous fermentation of sugarcane bagasse hemicellulosic hydrolysate by the yeast Candida guilliermondii FTI 20037 was used for xylitol production from xylose. Experiments were carried out in a reactor with 1.25 l of treated hydrolysate, at 30 °C and 300 rpm. A 2² full-factorial central composite design was employed for experimental study and analysis of the results. A statistical analysis of the results showed that the effects of the pH and dilution rate (D), the interactions between these variables and the second-order effect of D on the xylitol volumetric productivity (Q_p) were significant at a 95% confidence level. The second-order effect of pH was also significant at a 90% confidence level. The k_La effect on the Q_p was not significant. A volumetric productivity of 0.68 g/l h, representing 95.8% of the predicted value (0.72 g/l h), was obtained.