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)center dot 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 30A degrees 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 (195A degrees C/10 min in 20 L reactor) and delignificated with NaOH 1.0% (w/v), 100A degrees C, 1 h in 500 mL stainless steel ampoules immersed in an oil bath.

Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer`s spent grain

Enzymatic hydrolysis of brewer`s spent grain in three different forms: original (untreated), pretreated by dilute acid (cellulignin), and pretreated by a sequence of dilute acid and dilute alkali (cellulose pulp), was studied to verify the effect of hemicellulose and lignin on cellulose conversion into glucose. The hydrolysis was carried out using a commercial cellulase concentrate (Celluclast 1.5 L) in an enzyme/substrate ratio of 45 FPU/g, 2% (w/v) substrate concentration, 45 degrees C for 96 h. According to the results, the cellulose hydrolysis was affected by the presence of hemicellulose and/or lignin in the sample. The cellulose conversion ratio (defined as glucose yield + cellobiose yield) from cellulignin was 3.5-times higher than that from untreated sample, whereas from cellulose pulp such value was 4-times higher, correspondent to 91.8% (glucose yield of 85.6%). This best result was probably due to the strong modification in the material structure caused by the hemicellulose and lignin removal from the sample. As a consequence, the cellulose fibers were separated being more susceptible to the enzymatic attack. It was concluded that the lower the hemicellulose and lignin contents in the sample, the higher the efficiency of cellulose hydrolysis. (C) 2007 Elsevier Inc. All rights reserved.

Technological advances and mechanistic basis for fungal biopulping

Biopulping fundamentals, technology and mechanisms are reviewed in this article. Mill evaluation of Eucalyptus grandis wood chips biotreated by Ceriporiopsis subvermispora on a 50-tonne pilot-plant demonstrated that equivalent energy savings can be obtained in lab- and mill-scale biopulping. Some drawbacks concerning limited improvements in pulp strength and contamination of the chip pile with opportunist fungi have been observed. The use of pre-cultured wood chips as inoculum seed for the biotreatment process minimized contamination problems related to the use of blended mycelium and corn-steep liquor in the inoculation step. Alkaline wash restored part of the brightness in biopulps and marketable brightness values were obtained by one-stage bleaching with 5% H2O2 when bio-TMP pulps were under evaluation. Considering the current scenario, the understanding of biopulping mechanisms has gained renewed attention because more resistant and competitive fungal species could be selected with basis on a function-directed screening project. A series of studies aimed to elucidate structural changes in lignin during wood biodegradation by C. subvermispora had indicated that lignin depolymerization occurs during initial stages of wood biotreatment. Aromatic hydroxyls did not increase with the split of aryl-ether linkages, suggesting that the ether-cleavage-products remain as quitione-type structures. On the other hand, cellulose is more resistant to the attack by C subvermispora. MnP-initiated lipid peroxidation reactions have been proposed to explain degradation of non-phenolic lignin substructures by C subvermispora, while the lack of cellobiohydrolases and the occurrence of systems able to suppress Fenton`s reaction in the cultures have explained non-efficient cellulose degradation by this biopulping fungus. (C) 2007 Elsevier Inc. All rights reserved.

Effects of medium supplementation and pH control on lactic acid production from brewer`s spent grain

A cellulose pulp obtained by chemical pre-treatment of brewer`s spent grain was saccharified by a commercial cellulase preparation and the produced hydrolysate (50 g/l glucose) was fermented to lactic acid by Lactobacillus delbrueckii. The effects of pH control and nutrient supplementation of the hydrolysate on fermentation performance were investigated. Addition of 5g/l yeast extract enhanced the lactic acid volumetric productivity that attained 0.53 g/l h, value 18% higher than that obtained from non-supplemented hydrolysate. Addition of the MRS broth medium components (except the carbon source) was still better, providing a productivity of 0.79 g/l h. In all the cases, the lactic acid yield factor was of 0.7 g/g glucose consumed, but the fermentations stopped after 24 h due to the pH drop from 6.0 to 4.2, resulting in large amounts of residual glucose (38-41 g/l). Fermentation runs pH-controlled at 6.0 gave better results than those where the initial pH was not further controlled. The best result, 35.54 g/l lactic acid (0.99 g/g glucose consumed) was obtained during the pH-controlled fermentation of hydrolysate medium supplemented with MRS components. The volumetric productivity at the end of this fermentation was 0.59 g/l h, with a maximum of 0.82 g/l h during the first 12 h. (c) 2008 Elsevier B.V. All rights reserved.

Sequential production of amylolytic and lipolytic enzymes by bacterium strain isolated from petroleum contaminated soil

Amylases and lipases are highly demanded industrial enzymes in various sectors such as food, pharmaceuticals, textiles, and detergents. Amylases are of ubiquitous occurrence and hold the maximum market share of enzyme sales. Lipases are the most versatile biocatalyst and bring about a range of bioconversion reactions such as hydrolysis, inter-esterification, esterification, alcoholysis, acidolysis, and aminolysis. The objective of this work was to study the feasibility for amylolitic and lipolytic production using a bacterium strain isolated from petroleum contaminated soil in the same submerged fermentation. This was a sequential process based on starch and vegetable oils feedstocks. Run were performed in batchwise using 2% starch supplemented with suitable nutrients and different vegetable oils as a lipase inducers. Fermentation conditions were pH 5.0; 30 degrees C, and stirred speed (200 rpm). Maxima activities for amyloglucosidase and lipase were, respectively, 0.18 and 1,150 U/ml. These results showed a promising methodology to obtain both enzymes using industrial waste resources containing vegetable oils.

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.

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to UP of Phanerochaete chrysosporium and a total inhibition of UP was observed. (C) 2007 Elsevier Ltd. All rights reserved.

Clarification of a wheat straw-derived medium with ion-exchange resins for xylitol crystallization

BACKGROUND: Xylitol bioproduction from lignocellulosic residues comprises hydrolysis of the hemicellulose, detoxification of the hydrolysate, bioconversion of the xylose, and recovery of xylitol from the fermented hydrolysate. There are relatively few reports on xylitol recovery from fermented media. In the present study, ion-exchange resins were used to clarify a fermented wheat straw hemicellulosic hydrolysate, which was then vacuum-concentrated and submitted to cooling in the presence of ethanol for xylitol crystallization. RESULTS: Sequential adsorption into two anion-exchange resins (A-860S and A-500PS) promoted considerable reductions in the content of soluble by-products (up to 97.5%) and in medium coloration (99.5%). Vacuum concentration led to a dark-colored viscous solution that inhibited xylitol crystallization. This inhibition could be overcome by mixing the concentrated medium with a commercial xylitol solution. Such a strategy led to xylitol crystals with up to 95.9% purity. The crystallization yield (43.5%) was close to that observed when using commercial xylitol solution (51.4%). CONCLUSION: The experimental data demonstrate the feasibility of using ion-exchange resins followed by cooling in the presence of ethanol as a strategy to promote the fast recovery and purification of xylitol from hemicellulose-derived fermentation media. (c) 2008 Society of Chemical Industry.

Establishment of the optimum initial xylose concentration and nutritional supplementation of brewer`s spent grain hydrolysate for xylitol production by Candida guilliermondii

The effects of initial xylose concentration and nutritional supplementation of brewer`s spent grain hydrolysate on xylitol production by Candida guilliermondii were evaluated using experimental design methodology. The hydrolysate containing 55, 75 or 95 g/l xylose, supplemented or not with nutrients (calcium chloride, ammonium sulfate and rice bran extract), was used as fermentation medium. The increase in xylitol yield and productivity was related to the increase of initial xylose concentration, but up to a certain limit. above of which the yeast performance was not improved. The hydrolysate supplementation with nutrients did not interfere with xylose-to-xylitol conversion. By using the statistic tool the best conditions for maximum xylitol production were found. which consisted in using the non-supplemented hydrolysate containing 70 g/l initial xylose concentration. Under these conditions, a xylitol yield of 0.78 g/g and productivity of 0.58 g/(l h) were achieved. (C) 2008 Elsevier Ltd. All rights reserved.

Evaluation of hydrodynamic parameters of a fluidized-bed reactor with immobilized yeast

The fluidized bed reactor has successfully been used to perform biotechnological processes addressed to the production of high added value. The present work evaluates hydrodynamic parameters of a bench-scale fluidized bed reactor with cells of the yeast Candida guilliermondii immobilized either in calcium alginate beads or in polyvinyl alcohol (PVA). The effects of the following variables on cell immobilization were evaluated at 30 degrees C and feeding a synthetic medium containing 50 g L-1 xylose: total particle density (cells plus support), terminal velocity, particle drag force, minimum fluidization velocity and bed porosity. According to the results obtained, the reactor was shown to operate like a fixed-bed bioreactor at xi < 0.5 and a fluidized bed bioreactor at xi > 0.5. The maximum flow rate needed to obtain maximum bed fluidization in the reactor was equal to the terminal velocity of the immobilized cell particles. Particles of cells immobilized within these supports showed values of drag coefficient lower than those reported for other high-density supports. The evaluation of these hydrodynamic characteristics lead to an adequate bed fluidization inside the reactor, thus improving oxygen transference and availability in the fermentation medium, making the process more viable for future scale-up. (c) 2008 Society of Chemical Industry.

Relevance of extractives and wood transformation products on the biodegradation of Pinus taeda by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a promising white-rot fungus for biopulping. However, the underlying biochemistry involved in lignin removal and insignificant cellulose degradation by this species is not completely understood. This paper addresses this topic focusing on the involvement of ethanol-soluble extractives and wood transformation products in the biodegradation process. Cultures containing ethanol-extracted or in natura wood chips presented similar levels of extracellular enzymes and degradation of wood components. Fe3+-reducing compounds present in undecayed Pinus taeda were rapidly diminished by fungal degradation. Lignin-degradation products released during biodegradation restored part of the Fe3+-reducing activity. However, Fe3+ reduction was ineffective in presence of 0.5 mM oxalate at pH 4.5. Fungal consumption of Fe3+-reducing compounds and secretion of oxalic acid minimized the significance of Fenton`s reaction in the initial stages of wood biotreatment. This would explain limited polysaccharide degradation by the fungus that also lacks a complete set of hydrolytic enzymes. Scientific relevance of the paper: Ceriporiopsis subvermispora is a white-rot fungus suitable for biopulping processes because it degrades lignin selectively and causes significant structural changes on the wood components during the earlier decay stages. However, the intricate mechanism to explain lignin transformation and insignificant cellulose degradation by this species remains poorly understood. Some recent evidences pointed out for lipid peroxidation reactions as all initiating process explaining lignin degradation. On the other hand, alkylitaconic acids produced by the fungus via transformations of fatty acids occurring in wood showed to prevent polysaccharide degradation in Fenton reactions. In this context, one may conclude that the involvement of native wood substances or their transformation products in the overall wood biodegradation process induced by C subvermispora is still a matter of discussion. While free and esterified fatty acids present in wood extractives may be involved in the biosynthesis of alkylitaconic acids and in lipid peroxidation reactions, some extractives and lignin degradation products can reduce Fe3+, providing Fe2+ species needed to form OH radical via Fenton`s reaction. The present study focuses on this topic by evaluating the relevance of ethanol-soluble extractives and wood transformation products on the biodegradation of P. taeda by C subvermispora. For this, solid-state cultures containing ethanol-extracted and in natura wood chips were evaluated in details for up to 4 weeks. (C) 2007 Elsevier Ltd. All rights reserved.

Fermentation kinetics for xylitol production by a Pichia stipitis D-Xylulokinase mutant previously grown in spent sulfite liquor

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3 Delta) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h(-1)). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g(xylose)/g(cel) h) and xylitol production (0.059 g(xylitol)/g(cel) h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

An alternative application to the Portuguese agro-industrial residue: Wheat straw

The effects of alkaline treatments of the wheat straw with sodium hydroxide were investigated. The optimal condition for extraction of hemicelluloses was found to be with 0.50 mol/l sodium hydroxide at 55C for 2 h. This resulted in the release of 17.3% of hemicellulose (% dry starting material), corresponding to the dissolution of 49.3% of the original hemicellulose. The yields were determined by gravimetric analysis and expressed as a proportion of the starting material. Chemical composition and physico-chemical properties of the samples of hemicelluloses were elucidated by a combination of sugar analyses, Fourier transform infrared (FTIR), and thermal analysis. The results showed that the treatments were very effective on the extraction of hemicelluloses from wheat straw and that the extraction intensity (expressed in terms of alkali concentration) had a great influence on the yield and chemical features of the hemicelluloses. The FTIR analysis revealed typical signal pattern for the hemicellulosic fraction in the 1,200-1,000 cm(-1) region. Bands between 1,166 and 1,000 cm(-1) are typical of xylans.

Response surface methodology as an approach to determine optimal activities of lipase entrapped in sol-gel matrix using different vegetable oils

The conditions for maximization of the enzymatic activity of lipase entrapped in sol-gel matrix were determined for different vegetable oils using an experimental design. The effects of pH, temperature, and biocatalyst loading on lipase activity were verified using a central composite experimental design leading to a set of 13 assays and the surface response analysis. For canola oil and entrapped lipase, statistical analyses showed significant effects for pH and temperature and also the interactions between pH and temperature and temperature and biocatalyst loading. For the olive oil and entrapped lipase, it was verified that the pH was the only variable statistically significant. This study demonstrated that response surface analysis is a methodology appropriate for the maximization of the percentage of hydrolysis, as a function of pH, temperature, and lipase loading.

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production

Aims: The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability. Methods and Results: W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe3+-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe3+-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible. Conclusions: W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators. Significance and Impact of the Study: This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.