(411a) Comparative Biochemical Analysis of Biological Pretreatment of Wood Biomass With White Rot Fungi Using Sum Frequency Generation (SFG) Spectroscopy and Secondary Ion Mass Spectrometry (SIMS)

Advanced analytical methods of Sum Frequency Generation (SFG) spectroscopy, Secondary Ion Mass Spectrometry (SIMS), InfraRed microspectroscopy are used to quantify difference in the dynamics of degradation of lignocellulosic substrates by two different wood-degrading fungi. Wood is a composite that undergoes natural degradation in different ways depending on the genetics of the degrading microorganism. This provides a means to further refine these methods of characterization while learning more about the underlying biological process. We are particularly interested in the degradation process because of its particular utility for pretreatment of biomass for the production of biofuels, but this approach to study provides a validation of the logic of depolymerization of lignocellulosic biomass that has its basis in the genetics of the organisms under study. 

Wood is composite of cellulose and lignin and the lignin is the toughest biopolymer to degrade and chemical methods to remove lignin has been very difficult and often incomplete. Phanerochaete chrysosporium and Ceriporiopsis subvermispora are well characterized white rot fungi known to systematically degrade lignocellulosic biomass leaving some or most of the cellulose behind; of which P. chrysosporium has been used in biopulping in paper and pulp industries. Delignification is the initial process, as the recalcitrant lignin is found on the surface, coating the cellulose and hemicelluloses, through complex sequence of biochemical reactions. Both the organisms are known as efficient delignifier with varying mechanistic cellulolytic activity. P. chrysosporium simultaneously degrade cellulose, hemicellulose and lignin whereas C. subvermispora  preferentially remove lignin. Number of studies have been performed to understand the expression profile - at both transcript and enzymatic levels - of lignocellulosic and carbohydrate active enzymes (CAZymes) secreted during degradation of lignocellulose by P. chrysosporium and C. subvermispora. The comparative genomic and proteomic analyses of the expression profile of P. chrysosporium and C. subvermispora suggest a differentially ordered mechanism of degradation by both the organisms reflecting the diversity and presence of some CAZymes such as CBH1/GH7, EG/GH5 and EG/GH 12 families in both these organisms.

A comparison of two fungi is undertaken in this work based on the genomic comparison where the hypothesis is that C. subvermispora will be more selective for lignin consumption, and P. chrysosporium will leave more cellulose behind. The wood chips of all the four biomass studied - Poplar, Oak, Pine and Switch grass - show differential degradation pattern with respect to the change in the orderliness of the cellulose crystals detected using SFG analysis. The orderliness could potentially reflect exposure of the underlying crystalline cellulose after delignification; the extend of lignin removed simultaneously studied by analysing the depleting lignin content through the depth of the wood sample using SIMS analysis. Preferential lignin degradation and differential pattern of degradation by each organism changes the cellulose:lignin ratio which is studied using IR-microspectroscopy. The study is perfored using 1cmx 1cm square of wood chip of 5 mm thickness from all the woody biomass and a unit leaf blade of grassy member, placed on freshly grown fungal mycelium on a culture plate. The samples were removed and sectioned into equal portion for each of the anlaysis that are collected every week for four weeks. The SFG signals are generated from; (1) the CH2 stretch from the exocyclic hydroxymethylene group (6CH2OH), and (2) the 3OH and 2OH stretch vibrations from intra-chain hydrogen bonding along the cellulose chain. In lignocellulose, the SFG signal is only generated from crystalline cellulose; there is no signal contribution from any amorphous wall polymers or absorbed water. The change in CH:OH signal reflects the long range ordering in crystalline cellulose.  The data from SFG analysis of wood degradation suggest that the CH2 and OH signal ratio is consistently changing in all the wood samples while degraded by both the fungi. The hetetrogenity of differential enzymative degradation of both the fungi and the way the fibers are lignified in hardwood, softwood and grasses marks the difference in SFG signal.

SIMS analysis could chemically image the presence of the fungi and the chemical changes which occur with distribution and depth as a function of time. This may permit a chemical depth profile of the chemistry as  the intrusion of fungi progresses. The SIMS analysis is used to trace the chemical signatures of lignin, cellulose, fungi, and other chemicals along the gradient. 2D and 3D Chemical imaging is being conducted with of a physically cut cross section of the sample and after etching by the primary ion beam, C60+, respectively. This is because an advanced degradation (including approx. 70% delignification) is anticipated to have gone several millimeters deep. The change in ratio of lignin:cellulose while either one is preferentially consumed in each case and during the time course of the degradation is measured using IR microspectroscopy. We anticipate a change in lignin:cellulose ratio during the course of degradation. The lignin is found only in the secondary cell walls and therefore confocal imaging is performed to image the change in the cell wall morphology and thickness in each of the condition. The confocal imaging is peformed to image the autoflourescent lignin with an excitation of laser line 514 nm, emission filter BP 530 to 600 nm. The images are being analysed to understand the physiology of delignification and resultant change in cell wall thickness and morphology. 

In light of the importance of the biomass pretreatment of lignocellulose for various industrial application including making of composite from cellulosic material; establishing SFG, SIMS and IR microspectroscopy as a tool to quantify constituents of the material should find application beyond depolymerization for biofuels.