Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.     

Supplementation requirements of brewery’s spent grain hydrolysate for biomass and xylitol production by Debaryomyces hansenii CCMI 941

The effect of nutrient supplementation of brewery’s spent grain (BSG) hydrolysates was evaluated with respect to biomass and xylitol production by Debaryomyces hansenii. For optimal biomass production, supplementation of full-strength BSG hydrolysates required only phosphate (0.5 g l⁻¹ KH₂PO₄), leading to a biomass yield and productivity of 0.60 g g⁻¹ monosaccharides and 0.55 g l⁻¹ h⁻¹, respectively. Under the conditions studied, no metabolic products other than CO₂ and biomass were identified. For xylitol production, fourfold and sixfold concentrated hydrolysate-based media were used to assess the supplementation effects. The type of nutrient supplementation modulated the ratio of total polyols/total extracellular metabolites as well as the xylitol/arabitol ratio. While the former varied from 0.8 to 1, the xylitol/arabitol ratio reached a maximum value of 2.6 for yeast extract (YE)-supplemented hydrolysates. The increase in xylitol productivity and yield was related to the increase of the percentage of consumed xylose induced by supplementation. The best xylitol yield and productivity were found for YE supplementation corresponding to 0.55 g g⁻¹ and 0.36 g l⁻¹ h⁻¹, respectively. In sixfold concentrated hydrolysates, providing that the hydrolysate was supplemented, the levels of xylitol produced were similar or higher than those for arabitol. Xylitol yield exhibited a further increase in the sixfold hydrolysate supplemented with trace elements, vitamins and minerals to 0.65 g g⁻¹, albeit the xylitol productivity was somewhat lower. The effect of using activated charcoal detoxification in non-supplemented versus supplemented sixfold hydrolysates was also studied. Detoxification did not improve polyols formation, suggesting that the hemicellulose-derived inhibitor levels present in concentrated BSG hydrolysates are well tolerated by D. hansenii.     

Improvement of xylitol production by Candida guilliermondii FTI 20037 previously adapted to rice straw hemicellulosic hydrolysate

AIMS: To evaluate a simple and economical technique to improve xylitol production using concentrated xylose solutions prepared from rice straw hemicellulosic hydrolysate. METHODS AND RESULTS: Experiments were carried out with rice straw hemicellulosic hydrolysate containing 90 g l-1 xylose, with and without the addition of nutrients, using the yeast Candida guilliermondii previously grown on the hydrolysate (adapted cells) or on semi-defined medium (unadapted cells). By this method, the yield of xylitol increased from 17 g l-1 to 50 g l-1, and xylose consumption increased from 52% to 83%, after 120 h of fermentation. The xylitol production rates were very close to that (0.42 g l-1 h-1) attained in a medium simulating hydrolysate sugars. CONCLUSION: Yeast strain adaptation to the hydrolysate showed to be a suitable method to alleviate the inhibitory effects of the toxic compounds. Adapted cells of Candida guilliermondii can efficiently produce xylitol from hydrolysate with high xylose concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeast adaptation helps the bioconversion process in hydrolysate made from lignocellulosic materials. This low-cost technique provides an alternative to the detoxification methods used for removal of inhibitory compounds. In addition, the use of adapted inocula makes it possible to schedule a series of batch cultures so that the whole plant can be operated almost continuously with a concomitant reduction in the overall operation time.     

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.     

Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii

A detoxification method using activated charcoal with concentrated rice straw hemicellulosic hydrolysate improved the conversion of xylose to xylitol by the yeast Candida guilliermondii by 22%. This was achieved when the hydrolysate:charcoal ratio was 40 g g^–1, resulting in removal of 27% of phenolic compounds. Under this condition, the xylitol yield factor (0.72 g g^–1) and volumetric productivity (0.61 g l^–1 h^–1) were close to those attained in a semi-defined medium simulating hydrolysate sugars.     

Xylitol production from high xylose concentration: evaluation of the fermentation in bioreactor under different stirring rates

AIMS: To investigate the production of xylitol by the yeast Candida guilliermondii FTI 20037, in a bioreactor, from rice straw hemicellulosic hydrolysate with a high xylose concentration. METHODS AND RESULTS: Batch fermentation was carried out with rice straw hemicellulosic hydrolysate containing about 85 g xylose l(-1), in a stirred-tank bioreactor at 30 degrees C, under aeration of 1.3 vvm (volume of air per volume of medium per min) and different stirring rates (200, 300 and 500 rev min(-1)). The bioconversion of xylose into xylitol by the yeast depended on the stirring rate, the maximum xylitol yield (YP/S = 0.84 g g(-1)) being achieved at 300 rev min-1, with no need to pretreat the hydrolysate for purification. CONCLUSIONS: To determine the most adequate oxygen transfer rate is fundamental to improving the xylose-to-xylitol bioconversion by C. guilliermondii. SIGNIFICANCE AND IMPACT OF THE STUDY: For the microbial production of xylitol to be economically viable, the initial concentration of xylose in the lignocellulosic hydrolysate should be as high as possible, as with high substrate concentrations it is possible to increase the final product concentration. Nevertheless, there are few reports on the use of high xylose concentrations. Considering a process in bioreactor, from rice straw hemicellulosic hydrolysate, this is an innovator work.     

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.     

Eucalyptus hydrolysate detoxification with activated charcoal adsorption or ion-exchange resins for xylitol production

Eucalyptus hemicellulosic hydrolysate used for xylitol production by Candida guilliermondii FTI20037 was previously treated either with ion-exchange resins or with activated charcoal adsorption combined with pH adjustment, in order that acetic acid, furfural and hydroxymethylfurfural could be removed. The best results for xylitol yield factor (0.76 g/g) and volumetric productivity (0.68 g/(l h) were attained when a three-fold concentrated hydrolysate was treated with ion-exchange resins. Using activated charcoal combined with pH adjustment for treating a three-fold concentrated hydrolysate resulted in a xylitol yield factor of 0.40 g/g and a volumetric productivity of 0.30 g/(l h). This same treatment applied to a six-fold concentrated hydrolysate resulted in a xylitol yield factor of 0.66 g/g and a volumetric productivity of 0.50 g/(l h).     

Production of ethanol from soybean hull hydrolysate by osmotolerant Candida guilliermondii NRRL Y-2075

In this research, we studied the use of soybean hull hydrolysate (SHH) as a substrate for ethanol and xylitol production using an osmotolerant strain of Candida guilliermondii. The best acid hydrolysis of soybean hull achieved a recovery of 85 and 62% of xylose and mannose, respectively. Among detoxification treatments, activated charcoal 10% (w/v) showed the best results. Kinetic parameters obtained from the cultivation on four-fold concentrated SHH have shown that the osmotic pressure of this medium is higher than that supported by most osmophilic yeasts, revealing the osmotolerant characteristic of C. guilliermondii NRRL Y-2075. When cultivations were carried out on two times concentrated SHH, we obtained high yields of ethanol production, showing the prospect of SHH as a candidate for this biofuel production. Although xylose was present in high concentrations, no xylitol was produced, probably due to the presence of furfural acting as external electron acceptor or some varying cofactor preference of xylose reductase in this yeast strain.     

Production of xylitol from concentrated wood hydrolysates by Debaryomyces hansenii: Effect of the initial cell concentration

Summary Eucalyptus globulus wood hydrolysates were concentrated by vacuum evaporation to increase their xylose contents, treated with activated charcoal, supplemented with nutrients and used as culture media for xylitol production by Debaryomyces hansenii NRRL Y-7426. The susceptibility of hydrolysates to fermentation was strongly dependent on the initial cell concentration: media containing 58–78 g xylose/l were hardly consumed in batch experiments starting with 16 g cells/l, whereas 39–41 g xylitol/l were achieved in fermentations carried out with similar concentration of the carbon source and initial cell concentrations of 50–80 g/l).     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from highly concentrated hydrolysate

Rice straw hemicellulosic hydrolysate containing a high xylose concentration was used as fermentation medium to evaluate the kinetic behavior of Candida guilliermondii yeast (FTI 20037) during the bioconversion of xylose into xylitol. Assays were conducted first with detoxified and non-detoxified (raw) hydrolysates and semi-synthetic medium in agitated flasks, and second with detoxified hydrolysate in a stirred-tank bioreactor at a given oxygen transfer rate. The results for the agitated flasks showed that in detoxified hydrolysate the xylose-to-xylitol bioconversion by the yeast was as effective as in synthetic medium and 47% higher than in raw hydrolysate. In the stirred-tank bioreactor, the kinetic behavior of the yeast in detoxified hydrolysate was slower, resulting in smaller values of fermentative parameters, probably due to unsuitability of the oxygen transfer rate employed (K_La=22 h⁻¹).     

The behavior of key enzymes of xylose metabolism on the xylitol production by <Emphasis Type="Italic">Candida guilliermondii</Emphasis> grown in hemicellulosic hydrolysate

A variety of raw materials have been used in fermentation process. This study shows the use of rice straw hemicellulosic hydrolysate, as the only source of nutrient, to produce high added-value products. In the present work, the activity of the enzymes xylose reductase (XR); xylitol dehydrogenase (XD); and glucose-6-phosphate dehydrogenase (G6PD) during cultivation of Candida guilliermondii on rice straw hemicellulosic hydrolysate was measured and correlated with xylitol production under different pH values (around 4.5 and 7.5) and initial xylose concentration (around 30 and 70 g l(-1)). Independent of the pH value and xylose concentration evaluated, the title of XD remained constant. On the other hand, the volumetric activity of G6PD increased whereas the level of XR decreased when the initial xylose concentration was increased from 30 to 70 g l(-1). The highest values of xylitol productivity (Q (P) approximately 0.40 g l(-1)) and yield factor (Y (P/S) approximately 0.60 g g(-1)) were reached at highest G6PD/XR ratio and lowest XR/XD ratio. These results suggest that NADPH concentrations influence the formation of xylitol more than the activity ratios of the enzymes XR and XD. Thus, an optimal rate between G6PD and XR must be reached in order to optimize the xylitol production.     

Effect of nutrient supplementation of crude or detoxified concentrated distilled grape marc hemicellulosic hydrolysates on the xylitol production by Debaryomyces hansenii

Biosynthesis of xylitol using the yeast Debaryomyces hansenii NRRL Y-7426 was carried out using distilled grape marc (DGM) hemicellulosic hydrolysates directly concentrated by vacuum evaporation or after detoxification with activated charcoal. The effect of nutrient supplementation with vinasses, corn steep liquor (CSL) or commercial nutrients was explored. Using crude concentrated hemicellulosic hydrolysates, the maximum xylitol concentration, 11.3?g/L, was achieved after 172?hr (Q ( xylitol )?=?0.066?g/L-hr; Y ( xylitol ) (/SC)?=?0.21?g/g); meanwhile, using detoxified concentrated hydrolysates, the concentration increased up to 19.7?g/L after 72?hr (Q ( xylitol )?=?0.274?g/L-hr; Y ( xylitol ) (/SC)?=?0.38?g/g). On the other hand, using crude or detoxified hydrolysates, the xylose-to-xylitol bioconversion was strongly affected by the addition of nutrients, suggesting that these hydrolysates present essential nutrients favouring the growth of D. hansenii.     

Optimization of the pretreatment of rice straw hemicellulosic hydrolyzates for microbial production of xylitol

Hemicellulosic hydrolyzate obtained from rice straw was evaluated to determine if it was a suitable fementation medium for the production of xylitol byCandida mogii ATCC 18364. To obtain xylose selectively from rice straw, it is important to establish rapid hydrolysis conditions that yield xylose-rich substrates. The results of hydrolysis experiments indicated that the optimal reaction conditions for the recovery of xylose from rice straw hemicellulose were obtained using a sulfuric acid concentration of 1.5%, a reaction temperature of 130°C, a reaction time of 20 min and a solid to liquid ratio of 1∶10. Because the fermentation of concentrated acid hydrolyzates can be inhibited by compounds present in the raw material or produced during the hydrolysis process, various methods were tested to determine if they could detoxify the hydrolyzates and thus improve xylitol production. The greatest xylitol yield (0.53 g/g) and volumetric productivity (0.38 g/L·h) were obtained when an overlimed hydrolyzate was treated with activated charcoal.     

Xylitol production from non-detoxified corncob hemicellulose acid hydrolysate by Candida tropicalis

The interest on use of lignocellulose for producing chemicals is increasing as these feedstocks are low cost, renewable and widespread sources of sugars. Corncob is an attractive raw material for xylitol production due to its high content of xylan. In this study, hemicellulose hydrolysate from corncobs without detoxification was used for xylitol production by Candida tropicalis CCTCC M2012462. Compared with prepared xylose medium, xylitol production with dilute acid hydrolysate medium does not seem to influence specific xylose reductase activity. The decrease in xylitol productivity with dilute acid hydrolysate medium is a result of a lower biomass concentration and lag-phase time. It appears that biomass growth rate is essential for xylitol production. In xylitol fermentation with a low initial inhibitors concentration and substrate feeding strategy, a maximal xylitol concentration of 38.8 g l⁻¹ was obtained after 84 h of fermentation, giving a yield of 0.7 g g⁻¹ xylose and a productivity of 0.46 g l⁻¹ h⁻¹.     

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.     

Inhibitory action of toxic compounds present in lignocellulosic hydrolysates on xylose to xylitol bioconversion by <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The inhibitory action of acetic acid, ferulic acid, and syringaldehyde on metabolism of Candida guilliermondii yeast during xylose to xylitol bioconversion was evaluated. Assays were performed in buffered and nonbuffered semidefined medium containing xylose as main sugar (80.0 g/l), supplemented or not with acetic acid (0.8–2.6 g/l), ferulic acid (0.2–0.6 g/l), and/or syringaldehyde (0.3–0.8 g/l), according to a 2³ full factorial design. Since only individual effects of the variables were observed, assays were performed in a next step in semidefined medium containing different concentrations of each toxic compound individually, for better understanding of their maximum concentration that can be present in the fermentation medium without affecting yeast metabolism. It was concluded that acetic acid, ferulic acid, and syringaldehyde are compounds that may affect Candida guilliermondii metabolism (mainly cell growth) during bioconversion of xylose to xylitol. Such results are of interest and reveal that complete removal of toxic compounds from the fermentation medium is not necessary to obtain efficient conversion of xylose to xylitol by Candida guilliermondii. Fermentation in buffered medium was also considered as an alternative to overcome the inhibition caused by these toxic compounds, mainly by acetic acid.     

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.     

Production of xylitol by recombinant Saccharomyces cerevisiae containing xylose reductase gene in repeated fed-batch and cell-recycle fermentations

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20 g/l xylose and 18 g/l glucose resulted in 9.52 g/l dry cell mass, 20.1 g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200 g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7 g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0 g/l, 116 g/l xylitol concentration, 2.34 g/l h overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters.     

Fermentation performance of Candida guilliermondii for xylitol production on single and mixed substrate media

Semidefined media fermentation simulating the sugar composition of hemicellulosic hydrolysates (around 85 g l^-1 xylose, 17 g l^-1 glucose, and 9 g l^-1 arabinose) was investigated to evaluate the glucose and arabinose influence on xylose-to-xylitol bioconversion by Candida guilliermondii. The results revealed that glucose reduced the xylose consumption rate by 30%. Arabinose did not affect the xylose consumption but its utilization by the yeast was fully repressed by both glucose and xylose sugars. Arabinose was only consumed when it was used as a single carbon source. Xylitol production was best when glucose was not present in the fermentation medium. On the other hand, the arabinose favored the xylitol yield (which attained 0.74 g g^-1 xylose consumed) and it did not interfere with xylitol volumetric productivity (Q _P=0.85 g g^-1), the value of which was similar to that obtained with xylose alone.