Abstract
BackgroundBiological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples.ResultsOf the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii.ConclusionsFeedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.
Highlights
Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment
The other major carbohydrate components, hemicelluloses, are heterogeneous polymers of primarily xylose along with smaller amounts of arabinose, mannose, rhamnose, galactose, glucose and glucuronic acid [9]. While these substrates are rich in carbohydrate content, the barrier to biomass conversion of the carbohydrate content is the recalcitrance of renewable feedstocks [6], which has been shown to be a strong function of lignin content [16]
C. bescii fermentation of transgenic lines of P. trichocarpa Based on previous work [28], 17 transgenic samples of P. trichocarpa (Additional file 1: Table S1), along with the wild-type control, were fermented without pretreatment with a metabolically engineered strain of C. bescii in which the adhE gene from Clostridium thermocellum was inserted to enable the generation of ethanol, in addition to its natural fermentation products: acetate, H2 and CO2 [29]
Summary
Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. The other major carbohydrate components, hemicelluloses, are heterogeneous polymers of primarily xylose along with smaller amounts of arabinose, mannose, rhamnose, galactose, glucose and glucuronic acid [9] While these substrates are rich in carbohydrate content, the barrier to biomass conversion of the carbohydrate content is the recalcitrance of renewable feedstocks [6], which has been shown to be a strong function of lignin content [16]. In attempts to reduce recalcitrance, transgenic trees and grasses have been generated through a variety of molecular strategies [24], the efficacy of microbial conversion of these biomasses to fermentation products is highly variable [18, 19] and significantly dependent on pretreatment conditions. Striking a balance between reducing feedstock recalcitrance, often by lowering lignin content, and achieving excellent growth and fitness under field conditions is a key challenge for developing renewable transgenic biomasses
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