Evidence for Lignin Oxidation by the Giant Panda Fecal Microbiome

The digestion of lignin and lignin-related phenolic compounds from bamboo by giant pandas has puzzled scientists because of the lack of lignin-degrading genes in the genome of the bamboo-feeding animals. We constructed a 16S rRNA gene library from the microorganisms derived from the giant panda feces to identify the possibility for the presence of potential lignin-degrading bacteria. Phylogenetic analysis showed that the phylotypes of the intestinal bacteria were affiliated with the phyla Proteobacteria (53%) and Firmicutes (47%). Two phylotypes were affiliated with the known lignin-degrading bacterium Pseudomonas putida and the mangrove forest bacteria. To test the hypothesis that microbes in the giant panda gut help degrade lignin, a metagenomic library of the intestinal bacteria was constructed and screened for clones that contained genes encoding laccase, a lignin-degrading related enzyme. A multicopper oxidase gene, designated as lac51, was identified from a metagenomic clone. Sequence analysis and copper content determination indicated that Lac51 is a laccase rather than a metallo-oxidase and may work outside its original host cell because it has a TAT-type signal peptide and a transmembrane segment at its N-terminus. Lac51 oxidizes a variety of lignin-related phenolic compounds, including syringaldazine, 2,6-dimethoxyphenol, ferulic acid, veratryl alcohol, guaiacol, and sinapinic acid at conditions that simulate the physiologic environment in giant panda intestines. Furthermore, in the presence of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringic acid, or ferulic acid as mediators, the oxidative ability of Lac51 on lignin was promoted. The absorbance of lignin at 445 nm decreased to 36% for ABTS, 51% for syringic acid, and 51% for ferulic acid after incubation for 10 h. Our findings demonstrate that the intestinal bacteria of giant pandas may facilitate the oxidation of lignin moieties, thereby clarifying the digestion of bamboo lignin by the animal.     

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam gave maximum activities of 220 U lignin peroxidase ml^–1, 150 U manganese peroxidase ml^–1, 50 U laccase ml^–1 and 6.2 U protease ml^–1 at the retention time of 24 h for 60 days. Protease secretion destabilized the produced lignin peroxidase, manganese peroxidase and laccase.     

Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification

Agricultural by-products such as wheat straw are attractive feedstocks for the production of second-generation bioethanol due to their high abundance. However, the presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of cellulose. The purposes of this work are to study the ability of a laccase-mediator system to remove lignin improving saccharification, as a pretreatment of wheat straw, and to analyze the chemical modifications produced in the remaining lignin moiety. Up to 48 % lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as mediator, followed by alkaline peroxide extraction. The lignin removal directly correlated with increases (～60 %) in glucose yields after enzymatic saccharification. The pretreatment using laccase alone (without mediator) removed up to 18 % of lignin from wheat straw. Substantial lignin removal (37 %) was also produced when the enzyme-mediator pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw material swollen in dimethylsulfoxide-d ₆ revealed modifications of the lignin polymer, including the lower number of aliphatic side chains involved in main β-O-4′ and β-5′ inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-hydroxyphenyl, guaiacyl, and syringyl lignin units and of p-coumaric and ferulic acids, as well as a moderate decrease of tricin units, was observed without a substantial change in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-oxidized lignin units during the enzymatic treatment.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

Biochemical characterization of laccase from hairy root culture of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. and role of redox mediators to enhance its potential for the decolorization of textile dyes

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg⁻¹ of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg⁻¹ specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.     

Metabolite secretion,Fe3+-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869

Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m⁻¹). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m⁻¹) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m⁻¹). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe³⁺-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe³⁺-reducing activity, which was restored in the cultures after the first week of biodegradation.     

Biochemical and molecular characterization of a novel laccase from selective lignin-degrading white-rot fungus Echinodontium taxodii 2538

A novel laccase was isolated and characterized from a new selective lignin-degrading white-rot fungus Echinodontium taxodii 2538, in which a high yield of laccase was obtained. No laccase isoenzyme was detected in the synthetic liquid media. The purified laccase (designated as EtL2538) had an apparent molecular mass of 56 kDa, pI value of 3.1, and N-terminal amino acid sequence of GIGPVTDLHIVNAAV. EtL2538 showed optimum pH at 3.0 and optimum temperature at 60 °C using ABTS as the substrate. EtL2538 revealed superior thermostability, and retained over 80% of its original activity after incubation for 2 h at 50 °C. The laccase gene, etl2538, was also cloned and sequenced. This gene encoded a mature laccase protein containing 499 amino acids (aa) preceded by a signal peptide of 21 aa, and the deduced protein sequence contained four copper-binding conserved domains of typical laccase protein. EtL2538 was further used in lignin oxidation and dye decolorization. Even without the existence of redox mediators, EtL2538 could cleave the methoxyl groups and β-O-4 ether linkages in lignin from bamboo, and significantly decolorize malachite green and RBBR. These novel properties of EtL2538 may render it as a potential biocatalyst for biotechnological and environmental applications.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.     

Production and characterization of laccase from Cyathus bulleri and its use in decolourization of recalcitrant textile dyes

Many fungi (particularly the white rot) are well suited for treatment of a broad range of textile dye effluents due to the versatility of the lignin-degrading enzymes produced by them. We have investigated decolourization of a number of recalcitrant reactive azo and acid dyes using the culture filtrate and purified laccase from the fungus Cyathus bulleri. For this, the enzyme was purified from the culture filtrate to a high specific activity of 4,022 IU mg⁻¹ protein, produced under optimized carbon, nitrogen and C/N ratio with induction by 2,6-dimethylaniline. The protein was characterized as a monomer of 58±5.0 kDa with carbohydrate content of 16% and was found to contain all three Cu(II) centres. The three internal peptide sequences showed sequence identity (80–92%) with laccases of a number of white rot fungi. Substrate specificity indicated highest catalytic efficiency (k _cat/K _M) on guaiacol followed by 2,2′-azino-bis(3-ethylthiazoline-6-sulfonic acid) (ABTS). Decolourization of a number of reactive azo and acid dyes was seen with the culture filtrate of the fungus containing predominantly laccase. In spite of no observable effect of purified laccase on other dyes, the ability to decolourize these was achieved in the presence of the redox mediator ABTS, with 50% decolourization in 0.5–5.4 days.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Correlation between anatomical characteristics of ethanol organosolv pretreated Buddleja davidii and its enzymatic conversion to glucose

Buddleja davidii is a unique biomass that has many attractive agroenergy features, especially its wide range of growth habitat. The anatomical characteristics of B. davidii were investigated before and after ethanol organosolv pretreatment (one of the leading pretreatment technologies) in order to further understand the alterations that occur to the cellular structure of the biomass which can then be correlated with its enzymatic digestibility. Results showed that the ethanol organosolv pretreatment of B. davidii selectively removes lignin from the middle lamella (ML), which does not significantly disrupt the crystalline structure of cellulose. The removal of ML lignin is a major factor in enhancing enzymatic cellulose‐to‐glucose hydrolysis. The pretreatment also causes cell deformation, resulting in cracks and breaks in the cell wall. These observations, together with characterization analysis of the cell wall polymer material, lend support to the hypothesis that the physical distribution of lignin in the biomass matrix is an important structural feature affecting biomass enzymatic digestibility. Biotechnol. Bioeng. 2010;107: 795–801. © 2010 Wiley Periodicals, Inc.     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.     

Laccase activities of Penicillium chrysogenum in relation to lignin degradation

 An extracellular laccase capable of oxidizing ABTS (the diammonium salt of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was detected in ligninolytic cultures of Penicillium chrysogenum. By contrast, no lignin peroxidase, manganese-dependent peroxidase or aryl-alcohol oxidase was detected at any time during culturing. Both ABTS laccase activity and mineralization of dehydrogenative polymerizate of coniferyl alcohol were regulated by the C/N ratio in the medium and partially inhibited in the presence of thioglycolic acid, suggesting that both events are associated. In the presence of several known laccase inducers neither ABTS laccase activity nor mineralization rates were enhanced. However, a new laccase was detected in P. chrysogenum, able to oxidize 2,6-dimethoxyphenol but not involved in lignin mineralization. Studies with the known ligninolytic basidiomycete Trametes villosa suggest that lignin degradation by this fungus also involves the action of laccase. Received: 6 July 1995/Received revision: 28 October 1995/Accepted: 6 November 1995     

Phenol degradation and colour removal in submerged culture of Pleurotus sajor-caju with paper mill effluents

The fungus Pleurotus sajor-caju secretes phenol-oxidases that enable the use of recalcitrant compounds as substrates. The residues of paper manufacture contain high lignin levels, which gives the effluents a characteristic brownish colour. To test the potential of P. sajor-caju cultures on reducing these parameters, we used 90% of raw effluents from medium consistency oxygen delignification and bleaching stages plus 10% of mineral solution and different levels of glucose (5–15 g L^?1) as substrate. We observed a greater fungal biomass in cultures using effluent than in controls. Cultures containing 10 to 15 g L^?1 of glucose resulted in about 42% colour reduction. The polyphenol content was also reduced by 58.9% by the 13th day of culture. In addition, we observed the secretion of laccases (211.44 U mL^?1 and 45.98 U mL^?1 using ABTS and syringaldazine, respectively) and peroxidases (6.11 U mL^?1-ABTS) both peaking at the 7th day of culture and with similar kinetics of production in different glucose concentrations.     

Production of laccase, manganese peroxidase and lignin peroxidase by Brazilian marine-derived fungi

Marine-derived fungi are a potential for the search of new compounds with relevant features. Among these, the ligninolytic enzymes have potential applications in a large number of fields, including the environmental and industrial sectors. This is the work aimed to evaluate the enzymatic activities of three marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 and Mucor racemosus CBMAI 847) under different carbon sources and salinity conditions by using statistical experimental design. MnP, LiP and laccase were detected when these fungi were cultured in malt extract, however when grown on basal medium containing glucose and wheat bran LiP was not detected and yet an increase in MnP and laccase was observed. Statistical analysis through surface responses was performed and results showed high values of MnP and laccase activities under 12.5% and 23% (w/v) salinity, highlighting the potential use of these fungi for industrial applications and in bioremediation of contaminated sites having high salt concentrations. The highest values for LiP (75376.34 UI L⁻¹), MnP (4484.30 IU L⁻¹) and laccase (898.15 UI L⁻¹) were obtained with the fungus M. racemosus CBMAI 847 and it is the first report concerning ligninolytic enzymes production by a zygomycete from this genus.     

Expression,characterization and 2,4,6-trichlorophenol degradation of laccase from <Emphasis Type="Italic">Monilinia fructigena</Emphasis>

A novel laccase gene from Monilinia fructigena was synthesized chemically according to the yeast bias codon and integrated into the genome of Pichia pastoris GS115 by electroporation. The expressed enzyme was recovered from the culture supernatant and purified. The result of enzyme activity assay and SDS-PAGE demonstrated that the recombinant laccase was induced and extracellularly expressed in P. pastoris. Main biochemical properties of this laccase, such as thermodependence and thermostability, optimal pH and pH stability, and the effect of metal ions and inhibitors, were characterized. With 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate (ABTS) as the substrate, MfLcc had its optimal pH at 3.5 and optimal temperature at 45°C. The Km values of the ABTS, guaiacol were 0.012 and 0.016 Mm, respectively, and the corresponding V _max values are 243.9 and 10.55 Um min⁻¹ mg⁻¹, respectively. The recombinant laccase degraded 80% 2,4,6-trichlorophenol after 8 h under the optimal conditions. The recombinant strain and its laccase can be considered as candidate for treating waste water polluted with trichlorophenols.     

Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969

Laccase is a widespread group of multi-copper enzymes which can catalyze the oxidation of a variety of organic compounds, with concomitant reduction of molecular oxygen to water. It has a wide application in industrial processes, particularly in renewable bio-energy industry. In this study, Pleurotus ostreatus strain 10969 with high yield of laccase, previously isolated from edible fungus growing on Juncao, was applied for optimization of fermentation media and growth parameters for the maximal enzyme production through response surface methodology and further characterization of the laccase activity. The results show that glucose and Mg²⁺ are the key ingredients for laccase production with the optimum concentration of 0.0988 g/mL and 7.3 mmol/L respectively. Compared to the initial medium, the highest laccase yield observed is approximately increased by 2.5 times under the optimized conditions. Extracellular laccase was then purified and its characters were analyzed. The molecular weight of the laccase is about 40 kDa, and the optimum pH and temperature for its activity is 4.0 and 50 °C with the corresponding Km and Vmax of 0.31 mmol/L and 303.25 mmol/min respectively. DTT, β-mercaptoethanol and NaN₃ nearly inhibit all activity of the laccase, as well as the metal ions especially Ag⁺. In summary, our results will facilitate the utilization of plant lignin in biomass energy industry.     

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200°C and 5–20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R ² = 0.8861) and for ethanol production (R ² = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190°C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.