Chemical composition and enzymatic digestibility of sugarcane clones selected for varied lignin content

<p>Abstract</p> <p>Background</p> <p>The recalcitrance of lignocellulosic materials is a major limitation for their conversion into fermentable sugars. Lignin depletion in new cultivars or transgenic plants has been identified as a way to diminish this recalcitrance. In this study, we assessed the success of a sugarcane breeding program in selecting sugarcane plants with low lignin content, and report the chemical composition and agronomic characteristics of eleven experimental hybrids and two reference samples. The enzymatic digestion of untreated and chemically delignified samples was evaluated to advance the performance of the sugarcane residue (bagasse) in cellulosic-ethanol production processes.</p> <p>Results</p> <p>The ranges for the percentages of glucan, hemicellulose, lignin, and extractive (based on oven-dry biomass) of the experimental hybrids and reference samples were 38% to 43%, 25% to 32%, 17% to 24%, and 1.6% to 7.5%, respectively. The samples with the smallest amounts of lignin did not produce the largest amounts of total polysaccharides. Instead, a variable increase in the mass of a number of components, including extractives, seemed to compensate for the reduction in lignin content. Hydroxycinnamic acids accounted for a significant part of the aromatic compounds in the samples, with <it>p</it>-coumaric acid predominating, whereas ferulic acid was present only in low amounts. Hydroxycinnamic acids with ester linkage to the hemicelluloses varied from 2.3% to 3.6%. The percentage of total hydroxycinnamic acids (including the fraction linked to lignin through ether linkages) varied from 5.0% to 9.2%, and correlated to some extent with the lignin content. These clones released up to 31% of glucose after 72 hours of digestion with commercial cellulases, whereas chemically delignified samples led to cellulose conversion values of more than 80%. However, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.</p> <p>Conclusion</p> <p>Some of the experimental sugarcane hybrids did have the combined characteristics of high biomass and high sucrose production with low lignin content. Conversion of glucan to glucose by commercial cellulases was increased in the samples with low lignin content. Chemical delignification further increased the cellulose conversion to values of more than 80%. Thus, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.</p

Evaluation of lignin degradation initiated by extracellular metabolites recovered from Ceriporiopsis subvermispora cultures

Ceriporiopsis subvermispora é um fungo filamentoso, muito seletivo na degradação de lignina e, por isso, tem sido uma das espécies mais estudadas no processo de biopolpação. A biopolpação consiste em um tratamento biológico da madeira que antecede etapas convencionais de polpação, proporcionando níveis de economia de energia elétrica no processo que podem atingir valores de 30 a 40%. Para degradar a lignina, esse fungo secreta a enzima manganês-peroxidase (MnP), a qual requer um ácido carboxílico para quelar e transportar íons Mn3+ oriundos do seu ciclo catalítico. O complexo quelante-Mn3+ degrada apenas frações fenólicas da lignina, porém pode também iniciar a peroxidação de lipídeos e com isso gerar radicais peroxila que apresentam capacidade oxidativa suficiente para degradar estruturas não-fenólicas da lignina. Com base nesses aspectos, o presente trabalho teve o objetivo de avaliar a degradação de lignina por reações que envolvem a peroxidação de ácido linoléico iniciadas por metabólitos extracelulares extraídos de cultivos de C. subvermispora. Também foram avaliados sistemas miméticos baseados nos íons Fe2+ e Mn3+ como iniciadores das mesmas reações. Essencialmente, foi estudada a degradação de lignina in vitro em reações iniciadas por sistemas compostos que incluíram, MnP/Mn+2/H2O2, Fe3+/agentes redutores de Fe3+ produzidos durante a biodegradação da madeira, íons Mn+3 ou íons Fe+2, todos adicionados ao ácido linoléico. Para realizar esse estudo foi necessário preparar, tanto MnP, quanto compostos redutores de Fe3+, em cultivos de C. subvermispora. Também foram preparados e caracterizados dois substratos para as reações em estudo. Esses substratos compreenderam um complexo lignina-carboidrato (CLC) e um modelo de um material lignocelulósico completo, porém moído e livrado de toda a fração de extrativos. Nos dois casos, o material de partida foi à madeira de Eucalyptus grandis. A caracterização química desses substratos indicou um teor de lignina de 44,8% e 29,0%, respectivamente. Reações de peroxidação de ácido linoléico iniciadas pelos sistemas em estudo mostraram que todos foram efetivos para esse propósito, sendo que as maiores taxas de consumo de oxigênio durante essas reações foram observadas nos meios reacionais que continham Fe2+ em solução. O CLC inibiu as reações de peroxidação quando adicionado ao meio reacional em concentraçãoes maiores do que 0,3 mg/mL. Entretanto, reações prolongadas por 72 h com o CLC numa concentração inicial de 1 mg/mL indicaram que ele sofreu despolimerização. As vias de degradação da lignina contida no CLC ou na madeira de E. grandis moída envolveram a despolimerização da molécula, ou simplesmente a oxidação das cadeias laterais e das estruturas fenólicas livres. Quando o sistema foi baseado na ação de MnP/Mn+2/H2O2/ácido linoléico, foram comprovadas vias de degradação da lignina que envolveram desde a simples oxidação do carbono-&#945; até a quebra de ligações do tipo &#946;-O-4 e/ou entre os carbonos &#945; e &#946;. Os resultados obtidos corroboraram dados anteriormente publicados para a ação de C. subvermispora in vivo. Uma exceção foi à observação da reação de simples oxidação do C&#945; nos sistemas in vitro, que havia sido descartada em trabalhos anteriores que se basearam na caracterização de lignina contida em madeira biotratada por C. subvermispora (sistema in vivo). Os resultados permitiram concluir que vários sistemas miméticos podem iniciar a peroxidação de ácido linoléico in vitro. Quando essas reações foram conduzidas na presença de lignina (CLC ou E. grandis moído) foi possível observar transformações importantes na estrutura da lignina que eventualmente poderiam ser exploradas, por exemplo, em etapas de processos de branqueamento de polpas kraft.The white-rot fungus Ceriporiopsis subvermispora degrades lignin selectively, being one of the most studied species in biopulping. Biopulping consists of a biological treatment of wood that precedes conventional pulping stages. The process can provide up to 30-40% of energy savings in mechanical pulping. To degrade lignin, this fungus secretes the enzyme manganese-peroxidase (MnP), which needs carboxylic acids to chelate and transport Mn3+ ions formed in the catalytic cycle of the enzyme. The chelate-Mn3+ complex is able to degrade phenolic structures of lignin; however, can also initiate lipid peroxidation reactions generating peroxyl radicals that are able to degrade nonphenolic lignin structures. Based on this background, the aim of this work was to evaluate lignin degradation through linoleic acid peroxidation reactions initiated by extracellular metabolites recovered from C. subvermispora cultures. Some biomimetic systems based on Fe2+ and Mn3+ ions were also evaluated as initiators of such reactions. The lignin degradation was studied in reaction systems composed of MnP/Mn+2/H2O2, Fe3+-reducing compounds produced during wood biodegradation by C. subvermispora, Mn+3 or Fe+2 ions, all of them in the presence of linoleic acid. To perform this study, MnP and Fe3+-reducing compounds were initially produced in C. subvermispora cultures. Two different reaction substrates were also prepared. One was a lignin-carbohydrate complex (LCC) and, the other, was a complete lignocellulosic material that was milled and extracted to remove the extractive fraction. Both substrates were prepared from Eucalyptus grandis wood. The chemical characterization of the substrates showed 44.8 % and 29.0 % of total lignin, respectively. Linoleic acid peroxidation reactions initiated by the studied systems showed that all of them were efficient on this purpose. The highest oxygen consumption rates during these reactions were observed in the Fe2+ initiated reactions. The LCC inhibited the peroxidation reactions when added to the reaction medium at concentrations higher than 0.3 mg/mL. However, prolonging the reactions up to 72h with LCC at 1 mg/mL showed that it was depolymerized. The lignin degradation routes involved depolymerization or simple side chain and free-phenolic structure oxidations. When the reactive system was based on the use of MnP/Mn+2/H2O2/linoleic acid, some lignin degradation routes were demonstrated and they included C&#945;-oxidation, as well as &#946;-O-4 and/or C&#945;-C&#946; cleavages. These results corroborate previous findings published for the action of C. subvermispora in vivo. One exception was the simple C&#945; oxidation that was observed for the in vitro reactions, but was ruled out by previous works that were based on the characterization of residual lignins extracted from wood samples biotreated by C. subvermispora (in vivo system). The current results permitted to conclude that several mimetic systems were able to initiate linoleic acid peroxidation in vitro. When these reactions were performed in the presence of lignin (LCC or milled E. grandis) it was possible to show the occurrence of several lignin transformation reactions that could be exploited, for example, in pulp bleaching processes

Evaluation of lignin degradation initiated by extracellular metabolites recovered from Ceriporiopsis subvermispora cultures

Ceriporiopsis subvermispora é um fungo filamentoso, muito seletivo na degradação de lignina e, por isso, tem sido uma das espécies mais estudadas no processo de biopolpação. A biopolpação consiste em um tratamento biológico da madeira que antecede etapas convencionais de polpação, proporcionando níveis de economia de energia elétrica no processo que podem atingir valores de 30 a 40%. Para degradar a lignina, esse fungo secreta a enzima manganês-peroxidase (MnP), a qual requer um ácido carboxílico para quelar e transportar íons Mn3+ oriundos do seu ciclo catalítico. O complexo quelante-Mn3+ degrada apenas frações fenólicas da lignina, porém pode também iniciar a peroxidação de lipídeos e com isso gerar radicais peroxila que apresentam capacidade oxidativa suficiente para degradar estruturas não-fenólicas da lignina. Com base nesses aspectos, o presente trabalho teve o objetivo de avaliar a degradação de lignina por reações que envolvem a peroxidação de ácido linoléico iniciadas por metabólitos extracelulares extraídos de cultivos de C. subvermispora. Também foram avaliados sistemas miméticos baseados nos íons Fe2+ e Mn3+ como iniciadores das mesmas reações. Essencialmente, foi estudada a degradação de lignina in vitro em reações iniciadas por sistemas compostos que incluíram, MnP/Mn+2/H2O2, Fe3+/agentes redutores de Fe3+ produzidos durante a biodegradação da madeira, íons Mn+3 ou íons Fe+2, todos adicionados ao ácido linoléico. Para realizar esse estudo foi necessário preparar, tanto MnP, quanto compostos redutores de Fe3+, em cultivos de C. subvermispora. Também foram preparados e caracterizados dois substratos para as reações em estudo. Esses substratos compreenderam um complexo lignina-carboidrato (CLC) e um modelo de um material lignocelulósico completo, porém moído e livrado de toda a fração de extrativos. Nos dois casos, o material de partida foi à madeira de Eucalyptus grandis. A caracterização química desses substratos indicou um teor de lignina de 44,8% e 29,0%, respectivamente. Reações de peroxidação de ácido linoléico iniciadas pelos sistemas em estudo mostraram que todos foram efetivos para esse propósito, sendo que as maiores taxas de consumo de oxigênio durante essas reações foram observadas nos meios reacionais que continham Fe2+ em solução. O CLC inibiu as reações de peroxidação quando adicionado ao meio reacional em concentraçãoes maiores do que 0,3 mg/mL. Entretanto, reações prolongadas por 72 h com o CLC numa concentração inicial de 1 mg/mL indicaram que ele sofreu despolimerização. As vias de degradação da lignina contida no CLC ou na madeira de E. grandis moída envolveram a despolimerização da molécula, ou simplesmente a oxidação das cadeias laterais e das estruturas fenólicas livres. Quando o sistema foi baseado na ação de MnP/Mn+2/H2O2/ácido linoléico, foram comprovadas vias de degradação da lignina que envolveram desde a simples oxidação do carbono-&#945; até a quebra de ligações do tipo &#946;-O-4 e/ou entre os carbonos &#945; e &#946;. Os resultados obtidos corroboraram dados anteriormente publicados para a ação de C. subvermispora in vivo. Uma exceção foi à observação da reação de simples oxidação do C&#945; nos sistemas in vitro, que havia sido descartada em trabalhos anteriores que se basearam na caracterização de lignina contida em madeira biotratada por C. subvermispora (sistema in vivo). Os resultados permitiram concluir que vários sistemas miméticos podem iniciar a peroxidação de ácido linoléico in vitro. Quando essas reações foram conduzidas na presença de lignina (CLC ou E. grandis moído) foi possível observar transformações importantes na estrutura da lignina que eventualmente poderiam ser exploradas, por exemplo, em etapas de processos de branqueamento de polpas kraft.The white-rot fungus Ceriporiopsis subvermispora degrades lignin selectively, being one of the most studied species in biopulping. Biopulping consists of a biological treatment of wood that precedes conventional pulping stages. The process can provide up to 30-40% of energy savings in mechanical pulping. To degrade lignin, this fungus secretes the enzyme manganese-peroxidase (MnP), which needs carboxylic acids to chelate and transport Mn3+ ions formed in the catalytic cycle of the enzyme. The chelate-Mn3+ complex is able to degrade phenolic structures of lignin; however, can also initiate lipid peroxidation reactions generating peroxyl radicals that are able to degrade nonphenolic lignin structures. Based on this background, the aim of this work was to evaluate lignin degradation through linoleic acid peroxidation reactions initiated by extracellular metabolites recovered from C. subvermispora cultures. Some biomimetic systems based on Fe2+ and Mn3+ ions were also evaluated as initiators of such reactions. The lignin degradation was studied in reaction systems composed of MnP/Mn+2/H2O2, Fe3+-reducing compounds produced during wood biodegradation by C. subvermispora, Mn+3 or Fe+2 ions, all of them in the presence of linoleic acid. To perform this study, MnP and Fe3+-reducing compounds were initially produced in C. subvermispora cultures. Two different reaction substrates were also prepared. One was a lignin-carbohydrate complex (LCC) and, the other, was a complete lignocellulosic material that was milled and extracted to remove the extractive fraction. Both substrates were prepared from Eucalyptus grandis wood. The chemical characterization of the substrates showed 44.8 % and 29.0 % of total lignin, respectively. Linoleic acid peroxidation reactions initiated by the studied systems showed that all of them were efficient on this purpose. The highest oxygen consumption rates during these reactions were observed in the Fe2+ initiated reactions. The LCC inhibited the peroxidation reactions when added to the reaction medium at concentrations higher than 0.3 mg/mL. However, prolonging the reactions up to 72h with LCC at 1 mg/mL showed that it was depolymerized. The lignin degradation routes involved depolymerization or simple side chain and free-phenolic structure oxidations. When the reactive system was based on the use of MnP/Mn+2/H2O2/linoleic acid, some lignin degradation routes were demonstrated and they included C&#945;-oxidation, as well as &#946;-O-4 and/or C&#945;-C&#946; cleavages. These results corroborate previous findings published for the action of C. subvermispora in vivo. One exception was the simple C&#945; oxidation that was observed for the in vitro reactions, but was ruled out by previous works that were based on the characterization of residual lignins extracted from wood samples biotreated by C. subvermispora (in vivo system). The current results permitted to conclude that several mimetic systems were able to initiate linoleic acid peroxidation in vitro. When these reactions were performed in the presence of lignin (LCC or milled E. grandis) it was possible to show the occurrence of several lignin transformation reactions that could be exploited, for example, in pulp bleaching processes

Evaluation of Eucalyptus grandis Hill ex Maiden biopulping with Ceriporiopsis subvermispora under non-aseptic conditions

In biopulping, efficient wood colonization by a selected white-rot fungus depends on previous wood chip decontamination to avoid the growth of primary molds. Although simple to perform in the laboratory, in large-scale biopulping trials, complete wood decontamination is difficult to achieve. Furthermore, the use of fungal growth promoters such as corn steep liquor enhances the risk of culture contamination. This paper evaluates the ability of the biopulping fungus Ceriporiopsis subvermispora to compete with indigenous fungi in cultures of fresh or poorly decontaminated Eucalyptus grandis wood chips. While cultures containing autoclaved wood chips were completely free of contaminants, primary molds grew rapidly when non-autoclaved wood chips were used, resulting in heavily contaminated cultures, regardless of the C. subvermispora inoculum/wood ratio evaluated (5, 50 and 3000 mg mycelium kg(-1) wood). Studies on benomyl-amended medium suggested that the fungi involved competed by consumption of the easily available nutrient sources, with C. subvermispora less successful than the contaminant fungi. The use of acid-washed wood chips decreased the level of such contaminant fungi, but production of manganese peroxidase and xylanases was also decreased under these conditions. Nevertheless, chemithermomechanical pulping of acid-washed samples biotreated under non-aseptic conditions gave similar fibrillation improvements compared to samples subjected to the standard biodegradation process using autoclaved wood chips

Linoleic acid peroxidation initiated by Fe(3+)-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work work evaluates linoleic acid peroxidation reactions initiated by Fe(3+)-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe(3+) ions from freshly prepared solutions. The compounds responsible for the Fe(3+)-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe(3+) ions and the Fe(3+)-reducing compounds showed that the rate of O(2) consumption during peroxidation was proportional to the Fe(3+)-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe(3+)-reducing compounds formed during wood biodegradation by C subvermispora can mediate lignin degradation through linoleic acid peroxidation. (C) 2010 Elsevier Ltd. All rights reserved.FAPESPCNPqCAPES in BrazilFONDECYT in ChileCAPE

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment. (C) 2009 Elsevier Inc. All rights reserved.FAPESPCNPqCAPES in BrazilFONDECYT in Chil

Fruit and Restaurant Waste Polysaccharides Recycling Producing Xylooligosaccharides

A significant part of fruit production is wasted annually, a material of high value without use, causing environmental and social damage. These residues from agro-industrial processes, or those that can no longer be used in the market, can be recycled and generate value-added products by pretreatments/hydrolysis. One of the important pretreatments is acid hydrolysis, which can produce xylooligosaccharides (XOS) from biomass, a product of great commercial value in the food and pharmaceutical markets, mainly due to its prebiotic potential. Bananas, oranges, and guava generate a large volume of waste and represent much of Brazil’s fruit production. The dilute acid hydrolysis resulted in XOS production of 37.69% for banana peel, 59.60% for guava bagasse, 28.70% for orange bagasse, and 49.64% for restaurant residue. XOS were quantified by a liquid chromatograph system with a Bio-Rad Aminex HPX-87C column. The results show that, for this type of material and hydrolysis, the ideal conditions to produce XOS are high temperature, low time, and high acid concentration for banana peel residue (160 °C, 15 min, and 3% H2SO4), low temperature, low time, and high acid concentration for guava bagasse (100 °C,15 min and 3% H2SO4), high temperature and acid concentration with low time for orange bagasse (160 °C,15 min and 3% H2SO4) and high temperature and time and high acid concentration for restaurant waste (160 °C, 55 min and 3% H2SO4). This study identified acid hydrolysis conditions that maximized XOS production with a low amount of xylose production using agro-industrial and food residues, also showing the high potential of the chosen residues through the high yields of XOS production

Lignin chemistry and topochemistry during kraft delignification of Eucalyptus globulus genotypes with contrasting pulpwood characteristics

Eucalyptus globulus Labill. is a short-fibre resource for pulp and paper production. Ten different E. globulus genotypes with varied pulpwood quality and chemical composition were evaluated under kraft pulping conditions. Characterisation of the wood and pulp samples by thioacidolysis indicated that the content of syringyl units in beta-O-4 linkages (S-beta-O-4) was distinct for the studied genotypes. The highest S-beta-O-4 levels were detected in the samples with the lowest original lignin and highest glucan levels. This group of samples provided the pulps with the lowest final lignin content at higher yields. UV microspectrophotometric (UMSP) evaluation of the wood chips revealed that the samples with the lowest lignin levels have the lowest UV absorbances at 278 nm (A(278 nm)) in the secondary walls (S-2). During kraft pulping, lignin from the S-2 was dissolved, whereas lignins from the middle lamella and cell corner lignin was not removed not even for prolonged reaction periods, independently of the evaluated genotype. The A(278 nm) values of the S-2 were significantly lower in the pulps from the genotypes with less original lignin content.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

The enzymatic recalcitrance of internodes of sugar cane hybrids with contrasting lignin contents

The recalcitrance in grasses varies according to cell type and maturation. The origin of the recalcitrance in different regions from sugar cane internodes with varied lignin contents was evaluated. The efficiency of enzymatic hydrolysis was correlated with the chemical, micromorphological and microspectrophotometric characteristics of the samples. The internodes of three sugar cane hybrids were dissected into four different fractions. The outermost fraction and the rind were the most recalcitrant regions, whereas the pith-rind interface and the pith were less recalcitrant. Cellulose conversion reached 86% after 72h of enzymatic digestion of the pith from the hybrid with the lowest lignin content. There was an inversely proportional correlation between the area occupied by vascular bundles and the efficiency of cellulose hydrolysis. High cellulose and low lignin or hemicellulose contents enhanced the efficiency of enzymatic hydrolysis of the polysaccharides. The critical evaluation of the results permitted to propose an empirical parameter for predicting cellulose conversion levels that accounts for the positive effect of high cellulose and low lignin plus hemicellulose and the detrimental effect of abundant vascular bundles. The cellulose conversion levels fit well to this calculated parameter, following a second order polynomial with an r2 value of 0.96. © 2013 Elsevier B.V