Abstract
The wide-scale production of renewable fuels from lignocellulosic feedstocks continues to be hampered by the natural recalcitrance of biomass. Therefore, there is a need to develop robust and reliable methods to characterize and quantify components that contribute to this recalcitrance. In this study, we utilized a method that incorporates pyrolysis with successive gas chromatography and mass spectrometry (Py-GC/MS) to assess lignification in cell suspension cultures. This method was compared with other standard techniques such as acid-catalyzed hydrolysis, acetyl bromide lignin determination, and nitrobenzene oxidation for quantification of cell wall bound phenolic compounds. We found that Py-GC/MS can be conducted with about 250 µg of tissue sample and provides biologically relevant data, which constitutes a substantial advantage when compared to the 50–300 mg of tissue needed for the other methods. We show that when combined with multivariate statistical analyses, Py-GC/MS can distinguish cell wall components of switchgrass (Panicum virgatum) suspension cultures before and after inducing lignification. The deposition of lignin precursors on uninduced cell walls included predominantly guaiacyl-based units, 71% ferulic acid, and 5.3% p-coumaric acid. Formation of the primary and partial secondary cell wall was supported by the respective ~15× and ~1.7× increases in syringyl-based and guaiacyl-based precursors, respectively, in the induced cells. Ferulic acid was decreased by half after induction. These results provide the proof-of-concept for quick and reliable cell wall compositional analyses using Py-GC/MS and could be targeted for either translational genomics or for fundamental studies focused on understanding the molecular and physiological mechanisms regulating plant cell wall production and biomass recalcitrance.
Highlights
Growing efforts to achieve energy sustainability have led to considering lignocellulosic feedstocks as a promising source for the production of renewable fuels and value-added chemicals and products
Variations in phenolics were observed across the cell wall, as indicated by the twodimensional spectroscopic images of adjacent switchgrass cells which were constructed by integrating the intensity around the phenolic band at 1,580 cm−1 (Figures 1A, C, E)
Spectra acquired in regions approaching the cell wall of the induced cells revealed bands at 1,580, 1,600, and 1,630 cm−1, suggesting an increase in concentration of lignin derivatives and phenolic acids when compared to the uninduced sample (Figure 1F) (Wang et al, 2012)
Summary
Growing efforts to achieve energy sustainability have led to considering lignocellulosic feedstocks as a promising source for the production of renewable fuels and value-added chemicals and products. Considerable interest has emerged in developing genetically modified biomass such as hybrid poplar and switchgrass with altered cell wall chemistry These genetic modifications have been achieved through silencing genes involved in the lignin synthetic pathway or overexpressing the transcription factors that repress the pathway. The overexpression of the R2-R3 MYB4 transcription factor has demonstrated a significant reduction in lignin and increase in saccharification efficiency, without the need for acid pretreatment (Shen et al, 2012; Shen et al, 2013a; Shen et al, 2013b) While this modification has shown promise in biomass conversion, only one of eight lines survived the first winter in field trials
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