Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that "Anaerocellum thermophilum" DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first-and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.

BackgroundAs a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement.ResultsWe analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis.ConclusionThe research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.

The three major components of plant biomass, cellulose, hemicellulose and lignin, are highly recalcitrant and deconstruction involves thermal and chemical pretreatment. Microbial conversion is a possible solution, but few anaerobic microbes utilize both cellulose and hemicellulose and none are known to solubilize lignin.Herein, we show that the majority (85%) of insoluble switchgrass biomass that had not been previously chemically treated was degraded at 78 C by the anaerobic bacterium Caldicellulosiruptor bescii.Remarkably, the glucose/xylose/lignin ratio and physical and spectroscopic properties of the remaining insoluble switchgrass were not significantly different than those of the untreated plant material. C. bescii is therefore able to solubilize lignin as well as the carbohydrates and, accordingly, lignin-derived aromatics were detected in the culture supernatants. From mass balance analyses, the carbohydrate in the solubilized switchgrass quantitatively accounted for the growth of C. bescii and its fermentation products, indicating that the lignin was not assimilated by the microorganism. Immunoanalyses of biomass and transcriptional analyses of C. bescii showed that the microorganism when grown on switchgrass produces enzymes directed at key plant cell wall moieties such as pectin, xyloglucans and rhamnogalacturonans, and that these and as yet uncharacterized enzymes enable the degradation of cellulose, hemicellulose and lignin at comparable rates. This unexpected finding of simultaneous lignin and carbohydrate solubilization bodes well for industrial conversion by extremely thermophilic microbes of biomass that requires limited or, more importantly, no chemical pretreatment. Broader contextThe three major components of plant biomass are cellulose (a glucose polymer), hemicellulose (a polymer of xylose and a variety of other sugars) and lignin (a complex polymer of aromatic units). The sugar polymers are potential feedstocks for the production of biofuels by anaerobic microorganisms. However, plant biomass is highly recalcitrant and harsh and inefficient chemical treatments are required to solubilize the biomass and release the sugars. Moreover, no anaerobic microorganism is known that can degrade the highly recalcitrant lignin. Herein it is shown that switchgrass, a model plant for bioenergy production, can be degraded at moderate temperatures (78 C) by an anaerobic bacterium that solubilizes lignin as well as cellulose and hemicellulose. The microorganism produces a range of both known and as yet uncharacterized enzymes that degrade at comparable rates all of the major components of the plant cell wall. Such thermophilic microbes could potentially be developed to enable the direct conversion of plant biomass to biofuels without the need for any chemical pretreatment.