Biological Pretreatment With White-rot Fungi And Enzymatic Hydrolysis Of Poplar Wood

Lignocellulosic biomass is composed of three major components:cellulose, hemicelluloses, and lignin. This abundant renewable resource can serve as ideal feedstock for the biofuels production. However, many crucial obstacles should be overcome due to the complicated structure of the cell wall during the pretreatment process. To overcome these obstacles, thorough investigations on improving the performance of biological pretreatment with white-rot fungi, effects of residues from white-rot fungi pretreatment on enzymatic hydrolysis, and how do ultrostructure changes from white-rot fungal decay improve enzymatic hydrolysis, were carried out in this thesis.Based on agar plate tests,18isolates from23white rot fungi were selected to a subsequent wood block decay test. According to the ratio of Klasson lignin losses to holocellulose losses, three isolates, Perenniporia ochroleucaD9597, Funalia trogiiC697S and Trametes orientalisC6320, showed selective delignification on Populus nigra. Based on primary component analysis, three different types of degradation were found during the fermentation of P. nigraby18white rot fungi:type A represents selective delignification; types B and C can selectively decompose holocelluloses, but type C has a stronger capacity than type B. Investigation was carried out on the UV absorbance spectra of ethanol extracts of wood particles treated by P. ochroleucaD9597, F. trogiiC6978and T. orientalisC6320for20days, and lignin in the treated wood was found to be decomposed markedly.Threewhite rot fungi (Lenzites betulinus, Trametes orientalis, and Trametes velutina) as well as their respective paired cultures were used to pretreat Populus tomentosa for enhanced lignocellulosic degradation and enzymatic hydrolysis. Hemicellulose and cellulose were slightly degraded, while a maximum lignin degradation of58.1%was caused by T. velutinaduring the12-week cultivation. After the pretreated samples were subjected to enzymatic hydrolysis for96h, the reducing sugar released by T. orientalis at week12was as high as41.3%, which was in line with the lignin loss at2.2times the control sample. Overall, the monocultures of white-rot fungi exhibited better degradation and saccharification of woody biomass than their co-culture. This can be attributed to the partial removal of lignin and hemicellulose, with an associated increase of cellulose accessibility to enzymes. In addition, different nutrients were added into the solid fermentation of woody biomass, Populus tomentosa, to improve pretreatment by a white rot fungus, Trametes velutina. Fungal pretreatment supplemented with trace elements resulted in large amount of lignin loss but low degradation of carbohydrate. Only12.6%of Klason lignin was left in the residues pretreated by T. velutina for8weeks supplemented with1%trace elements (TE group). When fungal-pretreated residues were subjected to enzymatic hydrolysis for96h, a maximum reducing sugar yield of44%was obtained from the TE group at the8th week,2.3times higher than that of untreated samples. In addition, the highest ethanol yield of22%was observed by the fermentation of8-week pretreated residues from the basic medium plus trace element group, which was five times more than that of untreated samples.Fomitopsis palustris, screened from11wood rotting fungi, was optimized with a sequential optimizationstrategy to produce the largest amount of cellulase, and the efficiency of the enzyme was evaluated. Based on the Plackett-Burman and Box-Behnken designs, the most significant variables, time, urea, and Tween80were varied for optimizing cellulase production. An optimized result for FPase activity with130.45FPU/mL was achieved for an8-day culture containing4.46g/L of urea and27.83μL/L of Tween80, which experimentally matched well with the predicted value from the model. The obtained crude cellulase was subsequently employed in the saccharification of the poplar wood, Populus tomentosa, which was pretreated with liquid hot water (LHW) at different temperatures. A maximum release of25.15%of reducing sugars was observed after a72-h enzymatic hydrolysis of the180℃-LHW-pretreated poplar wood, which is1.72times higher than that from untreated wood (14.66%), indicating that F. palustrishas a potential to produce cellulase for woody biomass hydrolysis.A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa. The results showed that lignin and cellulose increased with the elevating temperature, while significant amount of hemicellulose was degraded during the LHW pretreatment. A highest hemicellulose removal of92.33%was observed by combination of Lenzites betulina C5617with LHW treatment at200℃, which was almost2times higher than that of sole LHW treatment at the same level. Saccharification of poplar co-treated with L. betulina C5617and LHW at200℃resulted in a2.66-fold increase of glucose yield than that of sole LHW treatment, and an increase (2.25-fold) of glucose yield was obtained by the combination of Trametes ochracea C6888with LHW. The combination pretreatment performed well at accelerating the enzymatic hydrolysis of poplar wood. Fungal treatment followed by FeCl3treatment was also used to improve saccharification of wood ofP. tomentosa. Combined treatments accumulated lignin and slightly degraded cellulose, whereas almost all hemicelluloses were removed. The white rot fungus, Trametes orientalis, and the brown rot fungus, Fomitopsis palustris, both accompanied by FeCl3post-treatment resulted in98.8and99.7%of hemicelluloses loss at180℃, respectively. In addition, the solid residue from the T. orientalis-assisted and F. palustrisassisted FeC13treatment at180℃released84.5and95.4%of reducing sugars, respectively. Combined treatments disrupted the intact cell structure and increased accessible surface area of cellulose therefore enhancing the enzymatic digestibility, as evidenced by XRD and SEM analysis data. Wood residues from fungal cultivation with white-rot fungus were further dissolved in ionic liquid (IL) to mitigate the biomass recalcitrance for enhanced bioconversion. Firstly,4-week fungus-pretreated residues were subjected to IL pretreatment at100℃and120℃, respectively. Synergistic pretreatment at100℃can achieve a similar enzymatic digestibility to sole IL pretreatment and synergistic pretreatment at120℃. Therefore, prolonged fungal fermentation followed by IL dissolution at100℃was further investigated. There was no substantial improvement on saccharification of co-treated samples when bio-pretreatment exceeded8weeks. As high as96%of cellulose conversion was achieved by co-treatment with4-week bio-pretreatment and IL pretreatment at100℃, which was3fold,1.3fold and1.2fold higher than that of untreated samples, sole IL pretreatment at100℃and120℃, respectively. This fungi-assisted IL pretreatment would gain enhanced bioconversion at lower severity with minimal costs.Selective delignification and hemicellulose removal were performed on white rot fungus-pretreated residuesto investigate the effects of lignin and hemicellulose removal on enzymatic hydrolysis.43.66-77%of lignin with small part of hemicellulose were degraded by chlorite treatment, while79.97-95.09%of hemicellulose with little lignin were degraded by dilute acid treatment, indicating that cross effect between lignin and hemicellulose was minimized. In subsequent enzymatic digestion, regardless of the cellulase loading, residues from series-grade delignification released more glucose and xylose than that from hemicellulose removal, suggesting that lignin rather than hemicellulose in fungi-pretreated residues played a dominant role in hindering enzymatic hydrolysis. Based on the fundamental mechanisms of acidic/alkaline pretreatments in literature, it is proposed that fungal pretreatment prefers to integrate with alkaline pretreatment rather than acidic pretreatment to maximize the synergy. This indication would be helpful to optimize and renovate the integrated pretreatment.Multi-scale visualization and characterization of poplar wood cell walls were carried out to elucidate the mechanism behind fungal pretreatment with white-rot fungus, Trametes orientalis C6320. During16-week cultivation, cellulose decreased slightly but hemicellulose and total lignin gradually reduced from18.7%to11%and27.7%to12.5%, respectively. Cellulose conversion increased gradually from39%in4-week pretreatment to50.6%in16-week pretreatment, being consistent with the degradation of hemicellulose and lignin. XRD analysis showed thatthe fungal pretreatment had a negligible effect on the cellulose crystalline, indicating that crystallinity is not responsible for the improved enzymatic digestion. Revealed byultrastructural analysis,especiallyby TEM,it can be concluded that the white-rot fungus pretreatmentachieved the improved enzymatic digestibility bycreating extensive new surface area via etching away cell wall matrix and leaving microfibrils exposed on cell wall structures, in addition to partial hemicellulose and lignin removal.