To investigate the variation in biodegradability of lignocellulose under anaerobic degradation, extended anaerobic bacterial degradation experiments with four stages were conducted for both untreated and dilute-acid pretreated rice straw. In situ biodegradability visualization of the interior and exterior lignocellulosic biomass surfaces was carried out by fluorescent protein GFP-CBM3 labeling with or without bovine serum albumin blocking and observation with confocal laser scanning microscopy. The amount of fermentation products and the degradation rate of cellulose and hemicellulose decreased significantly along the extended experiments, suggesting a decrease of biodegradability under anaerobic degradation, although 50% (untreated) and 63% (pretreated) of the cellulose left at the end of the experimental period. In contrast, the quantity of cellulose-GFP-CBM3 points on plant cell walls increased slightly and the spatial distribution obviously changed, suggesting an increase in accessible cellulose and revealing that most of the cellulose exposed on the surface could not be hydrolyzed effectively under anaerobic degradation. From the combined results of SEM, AFM and FTIR, the increased accessibility and decreased biodegradability were attributed to the cell wall deconstruction by hydrolysis and the reaggregation, redistribution and redeposition of lignin and non-productive cellulose with increasing crystallinity. Furthermore, the initially conducted dilute-acid pretreatment of raw biomass did not enhance the successive biodegradability of residual biomass, even though the accessibility of pretreated biomass was higher than that of untreated biomass. Hence, the biodegradability cannot be immediately predicated by accessibility; rather, it mainly depends on the crystallinity of cellulose combined with enzymes, as well as the spatial structure of lignocellulose.
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