Quantification of Lignin and Its Structural Features in Plant Biomass Using ¹³C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS

Abstract

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12C and 13C lignin were isolated from nonlabeled and uniformly 13C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12C lignin analogue and was shown to be extremely accurate (>99.9%, R2 > 0.999) and precise (RSD < 1.5%). Third, 13C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

Evaluation of fungal degradation of wheat straw cell wall using different analytical methods from ruminant nutrition perspective

“…Prior to Py‐GC/MS, ground wheat straw (1 mm) was ball‐milled in a MM200 mixer mill (Retsch, Haan, Germany). Detailed procedures have been described previously . Pyrolysis was performed with an EGA/PY‐3030D Multi‐shot pyrolyzer (Frontier Laboratories, New Ulm, MN, USA) equipped with an AS‐1020E Autoshot auto‐sampler.…”

“…For semi‐quantitative analysis (samples of weeks 0, 1, 3 and 7), pyrograms were processed with Xcalibur 2.2 software. Areas were normalized by dividing by corresponding relative response factors (RRFs), multiplied with the molecular weight of the respective compound and summed . Lignin content was estimated on the basis of the total area of lignin‐derived pyrolysis products and compared to a wheat straw reference sample with known total Klason lignin content (20.5% w / w ) …”

“…Only samples of weeks 0 and 7 were subjected to the quantitative Py‐GC/MS, using the previously described procedures . Briefly, 10 µL of a 13 C lignin internal standard (IS) solution (1 mg mL −1 ) was mixed with ∼80 µg of sample and dried before analysis.…”

“…The RRF values were updated to system performance by recalculation to obtain an identical relative abundance of lignin‐derived pyrolysis products of the 13 C IS added to the wheat straw reference sample. Lignin content (% w / w ) was determined as the sum of lignin‐derived pyrolysis products by the established formula . Relative abundances of lignin‐derived pyrolysis products were based on areas normalized for the 13 C analogues from the IS present in the same sample to distinguish matrix and treatment effects.…”

“…Pyrolysis coupled to gas chromatography with mass spectrometry (Py‐GC/MS) has been shown to be a powerful tool in estimating the lignin content, while simultaneously providing information on the lignin structural features . Recent development of a highly accurate Py‐GC/MS technique, which uses carbon‐13 ( 13 C) lignin as internal standard, allows an accurate quantification of the lignin content . This precise technique has also been used to study the mechanisms of different fungal species in modifying lignin structure .…”