Lignocellulose pretreatment in a fungus-cultivating termite

Abstract

Depolymerizing lignin, the complex phenolic polymer fortifying plant cell walls, is an essential but challenging starting point for the lignocellulosics industries. The variety of ether- and carbon-carbon interunit linkages produced via radical coupling during lignification limit chemical and biological depolymerization efficiency. In an ancient fungus-cultivating termite system, we reveal unprecedentedly rapid lignin depolymerization and degradation by combining laboratory feeding experiments, lignocellulosic compositional measurements, electron microscopy, 2D-NMR, and thermochemolysis. In a gut transit time of under 3.5 h, in young worker termites, poplar lignin sidechains are extensively cleaved and the polymer is significantly depleted, leaving a residue almost completely devoid of various condensed units that are traditionally recognized to be the most recalcitrant. Subsequently, the fungus-comb microbiome preferentially uses xylose and cleaves polysaccharides, thus facilitating final utilization of easily digestible oligosaccharides by old worker termites. This complementary symbiotic pretreatment process in the fungus-growing termite symbiosis reveals a previously unappreciated natural system for efficient lignocellulose degradation.

Keywords: NMR; age polyethism; carbohydrate; lignin; symbiosis.

Fig. 1.

The plant material food-processing pathway in fungus-cultivating termites. As dominant decomposers, an O. formosanus colony forms in subtropical China with massive fungus combs underground (A), which are made of various plant materials foraged by worker termites (B). Age-related labor division in food processing of fungus-cultivating termites (C). Old workers forage outside and transport plant materials back to nest and form a food store (i); young workers ingest the food store (ii), imbue them with the fungal nodules (that are rich in asexual Termitomyces spores) (iii), and use their feces to build fresh fungus combs (iv); old workers gain their nutrition by consuming the mature fungus comb (v). (D) The development of Termitomyces fungi in fungal comb: the poplar fungal comb has a cycle time of ∼45 d. The Termitomyces spores and plant materials rapidly pass through the gut of young worker (∼3.5 h) and form as fresh comb with the fast spores germination (i); after 15 d of growth, the middle-aged comb with dense fungal mycelia and fungal nodules (ii); and after 30 d more growth, the comb materials complete maturation and containing little recognizable mycelia (iii).

Fig. 2.

¹H–¹³C HSQC NMR spectra of cell wall gels from samples. Lignin aromatic regions are shown for the original poplar wood (A), fresh fungus comb (B), middle-aged fungus comb (C), and mature fungus comb (D). Correlations from the major aromatic ring unit types are well dispersed and color coded by their indicated structures. See SI Appendix, Table S2 for signal assignments.

Fig. 3.

¹H–¹³C HSQC NMR spectra of cell wall gels from samples. Lignin aliphatic regions are shown for: (A) original poplar wood, (B) fresh fungus comb, (C) middle-aged fungus comb, and (D) mature fungus comb. Correlation from the major structural units are well dispersed and color coded by their indicated structures with characteristic interunit bonding. See SI Appendix, Table S2 for signal assignments.

Fig. 4.

Thermochemolysis profiles of cell walls from samples. (A) Py-GC-MS profiles. Poplar wood, fresh fungus comb, middle-aged fungus comb, and mature fungus comb with added internal standard (I.S.). The peaks labeled are identified phenolics-derived compounds. See SI Appendix, Table S5 for peak identification and relative molar areas. The structures of the labeled peaks are shown in SI Appendix, Fig. S7. (B) TMAH-Py-GC-MS profiles. Poplar wood, fresh fungus comb, middle-aged fungus comb, and mature fungus comb. The peaks labeled are identified phenolics-derived compounds. See SI Appendix, Table S6 for peak identification and relative molar areas. The structures of the labeled peaks are shown in SI Appendix, Fig. S8.