Species Of White-rot Fungi Research Articles

Screening, Production and Optimization of Extracellular Laccase Enzyme from Pleurotus Ostreatus

The enzyme laccase (p-diphenol: oxygenoxidoreductase; EC 1.10.3.2) is known to degrade many phenolic aromatic compounds. Many species of white rot fungi are well–known producers of the enzyme laccase. The first phase was the isolation of eight different strains of white rot fungi and screening them for laccase activity. Then highest laccase producing white rot fungal strains was identified. This strain Pleurotus ostreatus produced high amount of laccase compared to rest of the strains. In the second phase, the culture conditions for Pleurotus ostreatus were standardized to maximize the laccase production. The further steps involved the isolation, purification, and characterization of the enzyme laccase produced by Pleurotus ostreatus and in the final phase, the project deals with the textile industrial application of dyes and sludge treatment.

Effects of culturing rapeseed meal, soybean meal, macrophyte meal, and algal meal with three species of white-rot fungi on their in vitro and in vivo digestibilities evaluated using rainbow trout

The present study was conducted in order to improve the nutritive value of plant feed ingredients. Rapeseed meal (RM), soybean meal (SM), algal meal (AM; made from Gelidiaceae algae), and macrophyte meal (MM; made from Egeria densa) were cultured (fermented) with three species of white-rot fungi — Trametes coccinea (+T), Lentinula edodes (+L), and Pleurotus sajor-caju (+P) — for 6weeks at 38°C (+T) or 28°C (+L, +P). The controls were not cultured. The neutral detergent fiber (NDF) content of RM decreased significantly in RM+T (25%) and RM+L (26%) compared with RM control (35%). NDF content of SM decreased significantly in SM+T (5.0%) and SM+L (5.2%) compared with SM control (8.1%). NDF content of AM decreased significantly in AM+T (20%), AM+L (59%), and AM+P (33%) compared with AM control (62%). However, NDF content of MM did not decrease by fungal culture compared with MM control (30%). In vitro degradability of NDF was high in RM+T (49%), RM+L (41%), SM+T (50%), and AM+T (78%) compared with respective controls (0%). Yield rates of the fermented ingredients ranged from 57% (RM+L) to 84% (AM+L). Crude protein (CP) content of RM tended to increase in RM+T (51%) and RM+L (56%) compared with RM (46%). CP content of SM increased significantly in SM+T (62%) compared with SM (51%). CP content of AM tended to increase in AM+T (40%) compared with AM (27%). Among these fermented materials, four with low NDF and high CP content were selected (i.e., RM+T, RM+L, SM+T, and AM+T). These, together with respective controls, were evaluated for their in vivo digestibility using rainbow trout. Digestibilities of CP, organic matter, and dry matter tended to be lower in fungal cultured than control samples. However, the amount of CP absorbed was higher with cultured ingredients than the controls due to the increased CP content by fermentation. Digestibility of phosphorus (P) in RM increased significantly in RM+T (100%) and RM+L (86%) compared with RM (52%). Digestibility of P in SM was 100% for SM+T and SM control. Digestibility of P in AM tended to increase in AM+T (100%) compared with AM (89%). Digestibilities (absorption) of Mg, Zn, and Cu in RM tended to increase by fungal fermentation due presumably to the decrease in phytate content. These results collectively suggest that fungal fermentation of plant feed ingredients is a cost-effective approach to increasing the nutritive value and decreasing the environmental burden of fish feeds.

English

Identifying the most suitable lithium compounds for fungal growth is important for the lithium (Li) enrichment of mushrooms. However, enrichment thus far has been carried out using LiCl. In an effort to identify an alternative source of lithium, we investigated the effect of five lithium compounds on lag phase, growth rate and biomass of ten species of white-rot fungi. The objective of this work was to make a screening of promising lithium compounds for further studies of Li enrichment of mushrooms. The fungal growth was affected depending on the species and lithium form tested. Lithium sulfate and lithium chloride were found to be the most promising candidate compounds for the Li enrichment of mushrooms. Key words: Li-enriched fungi, mycelial growth, Lithium compounds.

Evaluación del crecimiento y compatibilidad de hongos de la podredumbre blanca

<p>El uso de hongos de la podredumbre blanca (HPB) ha generado un gran interés debido a su potencial<br />enzimático, permitiendo a estos organismos degradar una amplia gama de compuestos xenobióticos. Los<br />consorcios fúngicos pueden mejorar dichos procesos de degradación con respecto a los monocultivos y para<br />su asociación debe establecerse previamente su compatibilidad. En este trabajo se evaluó la influencia de dos condiciones nutricionales sobre el crecimiento y la compatibilidad entre tres especies de los HPB, Trametes versicolor, Pleurotus ostreatus y Pleurotus pulmonarius, para su futura utilización como consorcios en el tratamiento de contaminantes orgánicos. Discos de dos especies de los hongos evaluados crecieron por siete días sobre los medios de cultivo agar papa dextrosa (PDA) y agar salvado de trigo (AST). Se evaluaron tres tratamientos, T1: T. versicolor vs P. ostreatus, T2: T. versicolor vs P. pulmonarius y T3: P. ostreatus vs P. pulmonarius con sus respectivos controles (crecimiento libre). El porcentaje de inhibición micelial obtenido (< 70%) mostró compatibilidad entre los hongos evaluados en los distintos tratamientos bajo las condiciones nutricionales utilizadas. Con respecto a la tasa de crecimiento, se observó que el medio AST favoreció un rápido desarrollo para los tres hongos en comparación con PDA.</p>

English

Three species of white rot-fungi (Pleurotus ostreatus, Coriolus versicolor, and Lentinula edodes) were grown on 12 solid media based on several lignocellulosic materials (oak sawdust, coconut husks, coffee husks and corn bran) during 49 days. The media had varied carbon/nitrogen ratios and CuSO4 content. The objective of the work was to evaluate the effect of the media formulation on the production of lignocellulolytic enzymes and degradation of lignocellulosic components by the three fungal species. C. versicolor exhibited the highest ability to degrade the three main polymers of the lignocellulosic waste materials employed and to produce ligninases with titers as high as 107 U/g solid substrate in the case of laccase. In addition, a mathematical model describing the fermentation kinetics of the cell biomass growth, degradation of lignocellulosic components, and lignocellulolytic enzyme production for the fungal species/medium combination exhibiting the best performance under solid-state fermentation conditions was proposed and validated in the case of C. versicolor. The mathematical model could be used to provide valuable information on the process itself as well as to contribute to the development of a future commercial process for lignocellulolytic enzyme production. Key words: White-rot fungi, cellulases, ligninases, degradation of lignocellulosic materials, fermentation kinetics.

Trametes meyenii possesses elevated dye degradation abilities under normal nutritional conditions compared to other white rot fungi.

Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn3+ chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation.

Chemical alteration of banana pseudostems by white rot fungi

The ability of fungi to degrade lignocellulosic materials is due to their highly efficient enzymatic system. However, many of these fungi have the ability to not only depolymerize and metabolize lignin but also to degrade cellulose and hemicellulose. This study aims to investigate the differences between white rot fungi species, namely, Pycnoporus sanguineus, Oxyporus latemarginatus, Coriolus versicolor, and Rigidoporus vinctus, in chemical constituent changes on the pseudostem of Musa acuminata var. truncata. The sterilized banana pseudostem chips were inoculated with each fungus separately and incubated for 1, 2 and 4 weeks. P. sanguineus was found to be the best fungus for banana pseudostem among 4 species because it degraded mainly on lignin and extractives but less on holocellulose and alpha-cellulose. On the other hand, O. latemarginatus was found to degrade all the chemical contents, while C. versicolor and R. vinctus appeared less efficient in banana degradation. It appeared that the suitable pre-treatment duration was two weeks, which was due to the lower amounts of degraded holocellulose and alpha-cellulose.

Toxicity of organic and inorganic nanoparticles to four species of white-rot fungi

The rapid development of nanoparticles (NP) for industrial applications and large-volume manufacturing, with its subsequent release into the environment, raised the need to understand and characterize the potential effects of NP to biota. Accordingly, this work aimed to assess sublethal effects of five NP to the white-rot fungi species Trametes versicolor, Lentinus sajor caju, Pleurotus ostreatus, and Phanerochaete chrysosporium. Each species was exposed to serial dilutions of the following NP: organic-vesicles of SDS/DDAB and of Mo/NaO; gold-NP, quantum dot CdSe/ZnS, and Fe/Co. Fungi growth rate was monitored every day, and at the end of assay the mycelium from each replicate was collected to evaluate possible changes in its chemical composition. For all NP-suspensions the following parameters were characterized: hydrodynamic diameter, surface charge, aggregation index, zeta potential, and conductivity. All tested NP tended to aggregate when suspended in aqueous media. The obtained results showed that gold-NP, CdSe/ZnS, Mo/NaO, and SDS/DDAB significantly inhibited the growth of fungi with effects on the mycelium chemical composition. Among the tested NP, gold-NP and CdSe/ZnS were the ones exerting a higher effect on the four fungi. Finally to our knowledge, this is the first study reporting that different types of NP induce changes in the chemical composition of fungi mycelium.

Bioremediation of Direct Blue 14 and Extracellular Ligninolytic Enzyme Production by White Rot Fungi:PleurotusSpp.

In the present investigation, four species of white rot fungi (Pleurotus), that is, P. flabellatus, P. florida, P. ostreatus and P. sajor-caju were used for decolorization of direct blue 14 (DB14). Among all four species of Pleurotus, P. flabellatus showed the fastest decolorization in petri plates on different concentration, that is, 200 mg/L, 400 mg/L, and 600 mg/L. All these four species were also evaluated for extracellular ligninolytic enzymes (laccase and manganese peroxidase) production and it was observed that the twelve days old culture of P. flabellatus showed the maximum enzymatic activity, that is, 915.7 U/mL and 769.2 U/mL of laccase and manganese peroxidase, respectively. Other three Pleurotus species took more time for dye decolorization and exhibited less enzymatic activities. The rate of decolorization of DB14 dye solution (20 mg/L) by crude enzymes isolated from P. flabellatus was very fast, and it was observed that up to 90.39% dye solution was decolorized in 6 hrs of incubation.

Biosynthesis and structural characterization of Ag nanoparticles from white rot fungi

Five species of white rot fungi were screened for their capability to synthesize Ag nanoparticles (AgNPs). Three modes of AgNP bioreduction were developed. Pycnoporus sanguineus is found as a potential candidate for the synthesis of AgNPs with a yield at 98.9%. The synthesized AgNPs were characterized using UV–vis spectroscopy, DLS, FTIR, TEM, and SEM. Results showed that AgNP absorption band was located at a peak of 420nm. Both the SEM and TEM confirmed that the formation of AgNPs were mainly spherical with average diameters of 52.8–103.3nm. The signals of silver atoms' presence in the mycelium were observed by SEM-EDS spectrum.

Differences in crystalline cellulose modification due to degradation by brown and white rot fungi

Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and 13C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.

Biomodification of kenaf using white rot fungi

White rot fungi can be used as a pretreatment of biomass to degrade lignin. It also alters the structure of the lignocellulosic matter, thus increasing its accessibility to enzymes able to convert polysaccharides into simple sugars. This study compares the ability of two species of white rot fungi, Pycnoporous sanguineus and Oxyporus latemarginatus FRIM 31, to degrade lignin in kenaf chips. The white rot fungi were originally isolated from the tropical forest in Malaysia. Kenaf chips were first inoculated with each fungus separately using corn steep liquor as a fungal growth promoter. The kenaf chips were inoculated with white rot fungus for a period of 1, 2, 4, 8 and 16 weeks, after which they were observed under the scanning electron microscope (SEM). Chemical analyses were conducted following TAPPI Standard Methods and Fourier Transmission Infra Red (FTIR). SEM observations showed evidence of fungal colonization. When calculating weight loss, both P. sanguineus and O. latemarginatus FRIM 31 showed the greatest reduction. Amounts by mass of cellulose, hemicelluloses, extractives, and lignin in the treated kenaf chips all were lowered. The results show that O. latemarginatus FRIM 31 had a greater ability to degrade lignin when compared to P. sanguineus .

BIOMODIFICATION OF KENAF USING WHITE ROT FUNGI

White rot fungi can be used as a pretreatment of biomass to degrade lignin. It also alters the structure of the lignocellulosic matter, thus increasing its accessibility to enzymes able to convert polysaccharides into simple sugars. This study compares the ability of two species of white rot fungi, Pycnoporous sanguineus and Oxyporus latemarginatus FRIM 31, to degrade lignin in kenaf chips. The white rot fungi were originally isolated from the tropical forest in Malaysia. Kenaf chips were first inoculated with each fungus separately using corn steep liquor as a fungal growth promoter. The kenaf chips were inoculated with white rot fungus for a period of 1, 2, 4, 8 and 16 weeks, after which they were observed under the scanning electron microscope (SEM). Chemical analyses were conducted following TAPPI Standard Methods and Fourier Transmission Infra Red (FTIR). SEM observations showed evidence of fungal colonization. When calculating weight loss, both P. sanguineus and O. latemarginatus FRIM 31 showed the greatest reduction. Amounts by mass of cellulose, hemicelluloses, extractives, and lignin in the treated kenaf chips all were lowered. The results show that O. latemarginatus FRIM 31 had a greater ability to degrade lignin when compared to P. sanguineus.

Degradation of endosulfan and endosulfan sulfate by white-rot fungus Trametes hirsuta

Endosulfan, an organochlorine insecticide, and its metabolite endosulfan sulfate are persistent in environments and are considered toxic. We investigate the possible nontoxic bioremediation of endosulfan. An endosulfandegrading fungus that does not produce endosulfan sulfate was selected from eight species of white-rot fungi. High degradation of endosulfan and low accumulation of endosulfan sulfate were found in cultures of Trametes hirsuta. A degradation experiment using endosulfan sulfate as the substrate revealed that T. hirsuta is able to further degrade endosulfan sulfate following the oxidative conversion of endosulfan to endosulfan sulfate. Endosulfan and endosulfan sulfate were converted to several metabolites via hydrolytic pathways. In addition, endosulfan dimethylene, previously reported as a metabolite of the soil bacterium Arthrobacter sp., was detected in T. hirsuta culture containing endosulfan sulfate. Our results suggest that T. hirsuta has multiple pathways for the degradation of endosulfan and endosulfan sulfate and thus has great potential for use as a biocatalyst in endosulfan bioremediation.

Experimental calcium-oxalate crystal production and dissolution by selected wood-rot fungi

Twenty-six species of white-rotting Agaricomycotina fungi ( Basidiomycota) were screened for their ability to produce calcium-oxalate (CaOx) crystals in vitro. Most were able to produce CaOx crystals in malt agar medium in the absence of additional calcium. In the same medium enriched with Ca 2+, all the species produced CaOx crystals (weddellite or whewellite). Hyphae of four species ( Ganoderma lucidum, Polyporus ciliatus, Pycnoporus cinnabarinus, and Trametes versicolor) were found coated with crystals (weddellite/whewellite). The production of CaOx crystals during the growth phase was confirmed by an investigation of the production kinetics for six of the species considered in the initial screening ( Pleurotus citrinopileatus, Pleurotus eryngii, Pleurotus ostreatus, P. cinnabarinus, Trametes suaveolens, and T. versicolor). However, the crystals produced during the growth phase disappeared from the medium over time in four of the six species ( P. citrinopileatus, P. eryngii, P. cinnabarinus, and T. suaveolens). For P. cinnabarinus, the disappearance of the crystals was correlated with a decrease in the total oxalate concentration measured in the medium from 0.65 μg mm −2 (at the maximum accumulation rate) to 0.30 μg mm −2. The decrease in the CaOx concentration was correlated with a change in mycelia morphology. The oxalate dissolution capability of all the species was also tested in a medium containing calcium oxalate as the sole source of carbon (modified Schlegel medium). Three species ( Agaricus blazei, Pleurotus tuberregium, and P. ciliatus) presented a dissolution halo around the growth zone. This study shows that CaOx crystal production is a widespread phenomenon in white-rot fungi, and that an excess of Ca 2+ can enhance CaOx crystal production. In addition, it shows that some white-rot fungal species are capable of dissolving CaOx crystals after growth has ceased. These results highlight a diversity of responses around the production or dissolution of calcium oxalate in white-rot fungi and reveal an unexpected potential importance of fungi on the oxalate cycle in the environment.

Efficient production of Al(OH)3-immobilized laccase with aHeterobasidion annosumstrain selected by microplate screening

Wild-type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. By using a newly developed 96-well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l−1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger-scale liquid cultures. Aeration with a sparger lead to a 3·8-fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l−1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short-range ordered Al(OH)3 particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)3-adsorbed laccase was suitable for repeated decolourization of indigo carmine. Microplate cultivation allowed a large-scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)3 proved to be suitable as direct immobilization technique.

Metabolism of organochlorine pesticide heptachlor and its metabolite heptachlor epoxide by white rot fungi, belonging to genus Phlebia

White rot fungi of the genus Phlebia have demonstrated a high capacity to degrade organic pollutants, including polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls. In this study, we evaluated the ability of 18 white rot fungi species of genus Phlebia to degrade heptachlor and heptachlor epoxide, and described the metabolic pathways by selected white rot fungi. Phlebia tremellosa, Phlebia brevispora and Phlebia acanthocystis removed about 71%, 74% and 90% of heptachlor, respectively, after 14 days of incubation. A large amount of heptachlor epoxide and a small amount of 1-hydroxychlordene and 1-hydroxy-2,3-epoxychlordene were detected as metabolic products of heptachlor from most fungal cultures. The screening of heptachlor epoxide-degrading fungi revealed that several fungi are capable of degrading heptachlor epoxide, which is a recalcitrant metabolite of heptachlor. Phlebia acanthocystis, P. brevispora, Phlebia lindtneri and Phlebia aurea removed about 16%, 16%, 22% and 25% of heptachlor epoxide, respectively, after 14 days of incubation. Heptachlor diol and 1-hydroxy-2,3-epoxychlordene were produced in these fungal cultures as metabolites, suggesting that the hydrolysis and hydroxylation reaction occur in the epoxide ring and in position 1 of heptachlor epoxide, respectively.

Avaliação do potencial de degradação de fungos causadores de podridão branca

The objective of this work was to evaluate the in vitro degrading potential of white-rot fungi in sawdust of eucalyptus clones. The white-rot fungi species tested were: Pycnoporus sanguineus, Phellinus gilvus and Schizophyllum commune. The experiment was carried out in factorial arrangement 3 x 3, displayed in a completed randomized design. The analysed parameters were: diameter of the mycelial growth (cm), speed of growth (cm day-1), mycelial density of the fungal isolate and loss of sawdust mass (%). The isolated Pycnoporus sanguineus and Phellinus gilvus presented higher speed growth and resulted in higher loss of sawdust mass when compared to Schizophyllum commune. Pycnoporus sanguineus presented the highest mycelial density in all clones tested. Clone 2361 resulted in the smallest fungal isolate growth and smaller loss of sawdust mass, but it favored the formation of moderately and highly dense mycelial in all isolates. The most efficient in in vitro degradation of eucalyptus sawdust was achieved with Pycnoporus sanguineus and Phellinus gilvus.

English

Treatment of crop residues with some species of white-rot fungi can enhance the nutritive value. After the fungal treatment of sorghum (Sorghum bicolor) stover with two white-rot fungi in a solid state fermentation, the chemical composition and in vitro digestibility of the resultant substrate was determined. The results show a significant (p < 0.05) increase in crude protein contents from 2.54% for the control to 4.51% for Pleurotus ostreatus (POS) and 4.59% for Pleurotus sajor pulmonarius(PPT). The nitrogen free extract (NFE) content also increased significantly (p < 0.05). The crude fibre decreased significantly from 31.65% for the control to 27.49% for POS and 23.54% for PPS. There were also consistent significant decreases (p < 0.05) in the values obtained for NDF, ADF ADL. Significant differences were also observed in the hemicellulose and cellulose contents. Fermentation of the insoluble fraction (b) was enhanced by the fungal treatment. Wide variations were also observed in the mineral contents of the different substrates The estimated organic matter digestibility (OMD) ranged from 42.99 to 57.75%, short chain fatty acid ranged from 0.56 to 0.94 µM and metabolisable energy (ME) ranged from 5.97 to 8.21 MJ/Kg DM. This result suggests that fungal treatment of sorghum stover resulted in improved CP and digestibility, hence its potential in ruminant nutrition. Key words: Chemical composition, crop residues, in vitro digestibility, solid state fermentation, sorghum stover, white-rot fungi.

Effect of biopretreatment on thermogravimetric and chemical characteristics of corn stover by different white-rot fungi

The thermogravimetric and chemical characterization of corn stover biopretreated by three different species of white-rot fungi have been studied in this research. Results indicated that biopretreatment can optimize the thermal decomposition, decrease the reaction temperature and reduce the gas contamination (SO x ), making the biomass pyrolysis more efficient and environmentally friendly. Biopretreatment can decrease the activation energy and reacting temperature of the hemicellulose and cellulose pyrolysis (up to 36 °C), shorten the temperature range of the active pyrolysis (up to 14 °C), and increase the thermal decomposition rate, greatly promoting the reaction and making the biomass pyrolysis easier to start and carry on. On the other hand, by biopretreatment, the sulphur content can decrease up to 46.15%, which can considerably reduce the inventory of SO x emission. Moreover, the mechanism of the biopretreatment was also explored that the deconstruction and depolymerization of the recalcitrant linkages of lignin and cellulose by biopretreatment can make the structure of biomass incompact and easier to be pyrolyzed.