Ethanol Production from Sugarcane Bagasse Hydrolysate Using Pichia stipitis

The objective of this study was to evaluate the ethanol production from the sugars contained in the sugarcane bagasse hemicellulosic hydrolysate with the yeast Pichia stipitis DSM 3651. The fermentations were carried out in 250-mL Erlenmeyers with 100 mL of medium incubated at 200 rpm and 30 A degrees C for 120 h. The medium was composed by raw (non-detoxified) hydrolysate or by hydrolysates detoxified by pH alteration followed by active charcoal adsorption or by adsorption into ion-exchange resins, all of them supplemented with yeast extract (3 g/L), malt extract (3 g/L), and peptone (5 g/L). The initial concentration of cells was 3 g/L. According to the results, the detoxification procedures removed inhibitory compounds from the hemicellulosic hydrolysate and, thus, improved the bioconversion of the sugars into ethanol. The fermentation using the non-detoxified hydrolysate led to 4.9 g/L ethanol in 120 h, with a yield of 0.20 g/g and a productivity of 0.04 g L(-1) h(-1). The detoxification by pH alteration and active charcoal adsorption led to 6.1 g/L ethanol in 48 h, with a yield of 0.30 g/g and a productivity of 0.13 g L(-1) h(-1). The detoxification by adsorption into ion-exchange resins, in turn, provided 7.5 g/L ethanol in 48 h, with a yield of 0.30 g/g and a productivity of 0.16 g L(-1) h(-1).

Evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii on the key enzymes for xylitol production in sugarcane hemicellulosic hydrolysate

The evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii FTI 20037 yeast on xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes activities was performed during fermentation in sugarcane bagasse hemicellulosic hydrolysate. The xylitol production was evaluated by using cells previously growth in 30.0 gl(-1) xylose, 30.0 gl(-1) glucose and in both sugars mixture (30.0 gl(-1) xylose and 2.0 gl(-1) glucose). The vacuum evaporated hydrolysate (80 gl(-1)) was detoxificated by ion exchange resin (A-860S; A500PS and C-150-Purolite(A (R))). The total phenolic compounds and acetic acid were 93.0 and 64.9%, respectively, removed by the resin hydrolysate treatment. All experiments were carried out in Erlenmeyer flasks at 200 rpm, 30A degrees C. The maximum XR (0.618 Umg (Prot) (-1) ) and XDH (0.783 Umg (Prot) (-1) ) enzymes activities was obtained using inoculum previously growth in both sugars mixture. The highest cell concentration (10.6 gl(-1)) was obtained with inoculum pre-cultivated in the glucose. However, the xylitol yield and xylitol volumetric productivity were favored using the xylose as carbon source. In this case, it was observed maximum xylose (81%) and acetic acid (100%) consumption. It is very important to point out that maximum enzymatic activities were obtained when the mixture of sugars was used as carbon source of inoculum, while the highest fermentative parameters were obtained when xylose was used.

PVA-Hydrogel Entrapped Candida Guilliermondii for Xylitol Production from Sugarcane Hemicellulose Hydrolysate

Viable cells of Candida guilliermondii were immobilized by inclusion into polyvinyl alcohol (PVA) hydrogel using the freezing-thawing method. Entrapment experiments were planned according to a 2(3) full factorial design, using the PVA concentration (80, 100, and 120 g L(-1)), the freezing temperature (-10, -15, and -20 degrees C), and the number of freezing-thawing cycles (one, three, and five) as the independent variables, integrated with three additional tests to estimate the errors. The effectiveness of the immobilization procedure was checked in Erlenmeyer flasks as the pellet capability to catalyze the xylose-to-xylitol bioconversion of a medium based on sugarcane bagasse hemicellulosic hydrolysate. To this purpose, the yield of xylitol on consumed xylose, xylitol volumetric productivity, and cell retention yield were selected as the response variables. Cell pellets were then used to perform the same bioconversion in a stirred tank reactor operated at 400 rpm, 30 degrees C, and 1.04 vvm air flowrate. At the end of fermentation, a maximum xylitol concentration of 28.7 g L(-1), a xylitol yield on consumed xylose of 0.49 g g(-1) and a xylitol volumetric productivity of 0.24 g L(-1) h(-1) were obtained.

Determination of furanic aldehydes in sugarcane bagasse by high-performance liquid chromatography with pulsed amperometric detection using a modified electrode with nickel nanoparticles

A glassy carbon electrode chemically modified with nickel nanoparticles coupled with reversed-phase chromatography with pulsed amperometric detection was used for the quantitative analysis of furanic aldehydes in a real sample of sugarcane bagasse hydrolysate. Chromatographic separation was carried out in isocratic conditions (acetonitrile/water, 1:9) with a flow rate of 1.0 mL/min, a detection potential of -50 mV vs. Pd, and the process was completed within 4 min. The analytical curves presented limits of detection of 4.0 × 10(-7) mol/L and 4.3 × 10(-7) mol/L, limits of quantification of 1.3 × 10(-6) and 1.4 × 10(-6) mol/L, amperometric sensitivities of 2.2 × 10(6) nA mol/L and 2.7 × 10(6) nA mol/L for furfural and 5-hydroxymethylfurfural, respectively. The values obtained in this sample by the standard addition method were 1.54 ± 0.02 g/kg for 5-hydroxymethylfurfural and 11.5 ± 0.2 g/kg for furfural. The results demonstrate that this new proposed method can be used for the quick detection of furanic aldehydes without the interference of other electroactive species, besides having other remarkable merits that include excellent peak resolution, analytical repeatability, sensitivity, and accuracy.

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.

Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol-producing yeasts, low oxygen transfer volumetric coefficient (K(L)a) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K(L)a values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol clehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 +/- 0.21 U mg(protein)(-1)) was achieved during the experiment with the lowest K(L)a value (12 h(-1)), while the highest XD specific activity (0.19 +/- 0.03 U mg(protein)(-1)) was observed with a K(L)a value of 25 h(-1). Xylitol production was enhanced when K(L)a was increased from 12 to 50 h(-1), which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 +/- 0.08 g(xylitol) L(-1) h(-1) and an efficiency of 71 +/- 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. (C) 2008 Society of Chemical Industry

Biological detoxification of different hemicellulosic hydrolysates using Issatchenkia occidentalis CCTCC M 206097 yeast

This work had as its main objective to contribute to the development of a biological detoxification of hemicellulose hydrolysates obtained from different biomass plants using Issatchenkia occidentalis CCTCC M 206097 yeast. Tests with hemicellulosic hydrolysate of sugarcane bagasse in different concentrations were carried out to evaluate the influence of the hydrolysate concentration on the inhibitory compounds removal from the sugarcane bagasse hydrolysate, without reduction of sugar concentration. The highest reduction values of inhibitors concentration and less sugar losses were observed when the fivefold concentrated hydrolysate was treated by the evaluated yeast. In these experiments it was found that the high sugar concentrations favored lower sugar consumption by the yeast. The highest concentration reduction of syringaldehyde (66.67%), ferulic acid (73.33%), furfural (62%), and 5-HMF (85%) was observed when the concentrated hydrolysate was detoxified by using this yeast strain after 24 h of experimentation. The results obtained in this work showed the potential of the yeast Issatchenkia occidentalis CCTCC M 206097 as detoxification agent of hemicellulosic hydrolysate of different biomass plants.

Contribution of Tris Buffer on Xylitol Enzymatic Production

Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L(-1) h(-1) xylitol volumetric productivity with 99% xylose-xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.

Produção de bioetanol a partir de pedúnculo de caju (Anacardium occidentale L.) por fermentação submersa

Recently, global demand for ethanol fuel has expanded very rapidly, and this should further increase in the near future, almost all ethanol fuel is produced by fermentation of sucrose or glucose in Brazil and produced by corn in the USA, but these raw materials will not be enough to satisfy international demand. The aim of this work was studied the ethanol production from cashew apple juice. A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentration (from 24.4 to 103.1 g.L-1). Maximal ethanol, cell and glycerol concentrations (44.4 g.L-1, 17.17 g.L-1, 6.4 g.L-1, respectively) were obtained when 103.1 g.L-1 of initial sugar concentration were used, respectively. Ethanol yield (YP/S) was calculated as 0.49 g (g glucose + fructose)-1. Pretreatment of cashew apple bagasse (CAB) with dilute sulfuric acid was investigated and evaluated some factors such as sulfuric acid concentration, solid concentration and time of pretreatment at 121°C. The maximum glucose yield (162.9 mg/gCAB) was obtained by the hydrolysis with H2SO4 0.6 mol.L-1 at 121°C for 15 min. Hydrolysate, containing 16 ± 2.0 g.L-1 of glucose, was used as fermentation medium for ethanol production by S. cerevisiae and obtained a ethanol concentration of 10.0 g.L-1 after 4 with a yield and productivity of 0.48 g (g glucose)-1 and 1.43 g.L-1.h-1, respectively. The enzymatic hydrolysis of cashew apple bagasse treated with diluted acid (CAB-H) and alkali (CAB-OH) was studied and to evaluate its fermentation to ethanol using S. cerevisiae. Glucose conversion of 82 ± 2 mg per g CAB-H and 730 ± 20 mg per g CAB-OH was obtained when was used 2% (w/v) of solid and loading enzymatic of 30 FPU/g bagasse at 45 °C. Ethanol concentration and productivity was achieved of 20.0 ± 0.2 g.L-1 and 3.33 g.L-1.h-1, respectively when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g.L-1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g.L-1), ethanol concentration and productivity was 8.2 ± 0.1 g.L-1 and 2.7 g.L-1.h-1, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 g/g glucose and 0.47 g/g glucose, with pretreated CABOH and CAB-H, respectively. The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated too in this work. First, the yeast CE025 was preliminary cultivated in a synthetic medium containing glucose and xylose. Results showed that it was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pre-treatment. The fermentation of CABH was conducted at pH 4.5 in a batch-reactor, and only ethanol was produced by K. marxianus CE025. The influence of the temperature in the kinetic parameters was evaluated and best results of ethanol production (12.36 ± 0.06 g.L-1) was achieved at 30 ºC, which is also the optimum temperature for the formation of biomass and the ethanol with a volumetric production rate of 0.25 ± 0.01 g.L-1.h-1 and an ethanol yield of 0.42 ± 0.01 g/g glucose. The results of this study point out the potential of the cashew apple bagasse hydrolysate as a new source of sugars to produce ethanol by S. cerevisiae and K. marxianus CE025. With these results, conclude that the use of cashew apple juice and cashew apple bagasse as substrate for ethanol production will bring economic benefits to the process, because it is a low cost substrate and also solve a disposal problem, adding value to the chain and cashew nut production

Otimização das condições de cultivo do fungo Aspergillus nidulans para produção de melanina usando resíduos agroindustriais

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diversity and Physiological Characterization of D-Xylose-Fermenting Yeasts Isolated from the Brazilian Amazonian Forest

Background: This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. Methodology/Principal Findings: A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L.h to 0.75 g/L.h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L.h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. Conclusions/Significance: This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Scale-up of diluted sulfuric acid hydrolysis for producing sugarcane bagasse hemicellulosic hydrolysate (SBHH)

Sugarcane bagasse was pretreated with diluted sulfuric acid to obtain sugarcane bagasse hemicellulosic hydrolysate (SBHH). Experiments were conducted in laboratory and semi-pilot reactors to optimize the xylose recovery and to reduce the generation of sugar degradation products, as furfural and 5-hydroxy-methylfurfural (HMF). The hydrolysis scale-up procedure was based on the H-Factor, that combines temperature and residence time and employs the Arrhenius equation to model the sulfuric acid concentration (100 mg(acid)/g(dm)) and activation energy (109 kJ/mol). This procedure allowed the mathematical estimation of the results through simulation of the conditions prevailing in the reactors with different designs. The SBHH obtained from different reactors but under the same H-Factor of 5.45 +/- 0.15 reached similar xylose yield (approximately 74%) and low concentration of sugar degradation products, as furfural (0.082 g/L) and HMF (0.0071 g/L). Also, the highest lignin degradation products (phenolic compounds) were rho-coumarilic acid (0.15 g/L) followed by ferulic acid (0.12 g/L) and gallic acid (0.035 g/L). The highest concentration of ions referred to S (3433.6 mg/L), Fe (554.4 mg/L), K (103.9 mg/L), The H-Factor could be used without dramatically altering the xylose and HMF/furfural levels. Therefore, we could assume that H-Factor was directly useful in the scale-up of the hemicellulosic hydrolysate production. (C) 2009 Published by Elsevier Ltd.

A novel use for sugarcane bagasse hemicellulosic fraction: Xylitol enzymatic production

The excess of sugarcane bagasse (SCB) from the sugar-alcohol industry is considered a by-product with great potential for many bioproducts production. This work had as objective to verify the performance of sugarcane bagasse hemicellulosic hydrolysate (SCBHH) as source of sugars for enzymatic or in vitro xylitol production. For this purpose, xylitol enzymatic production was evaluated using different concentrations of treated SCBHH in the commercial reaction media. The weak acid hydrolysis of SCB provided a hydrolysate with 18 g L(-1) and 6 g L(-1) of xylose and glucose, respectively. Considering the reactions, changes at xylose xylitol conversion efficiency and volumetric productivity in xylitol were not observed for the control experiment and using 20 and 40% v.v (1) of SCBHH in the reaction media. The conversion efficiency achieved 100% in all the experiments tested. The results showed that treated SCBHH is suitable as xylose and glucose source for the enzymatic xylitol production and that this process has potential as an alternative for traditional xylitol production ways. (C) 2011 Published by Elsevier Ltd.

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a d-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-a dagger 1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. d-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose) l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

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 112.5A degrees C to 157.5A degrees 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.