Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

Abstract

Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR-down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼ 20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

Keywords: GM; bioethanol; second-generation bioenergy.

Fig. 1.

Saccharification yields of xylem. Saccharification yields after 48 h, without and with acid (HCl) pretreatment, of scrapings of greenhouse-grown WT poplar (n = 2) and white and red xylem of greenhouse-grown FAS13 (n = 7). Error bars represent SDs. *P < 0.01. Dark gray, WT; white, white xylem of FAS13; light gray, red xylem of FAS13.

Fig. 2.

Pictures of the Belgian and French field trials. (A) Field trial in Belgium (July 2009). (B) Field trial in France just before harvest (March 2010). (C) Classification and illustration of the variegated red phenotype observed for the transgenic trees in the Belgian field trial. (D) Illustration of the variegated red coloration in cross-sections for WT (Top), FS3 (Middle), and FAS13 (Bottom) from the French field trial.

Fig. 3.

Ethanol yield (g/L) for the Belgian and French field trials. For both locations, SSF was performed on pooled trees (Table S1) (Belgian trial: WT, n = 6; FS3, n = 5; FS40, n = 6; French trial: WT, n = 10; FS3, n = 10; FAS13, n = 10). Ethanol yields were measured after 31 h and 28 h of SSF for the Belgian and French field trials, respectively. Error bars represent SDs. Bold, significantly increased compared to WT. *P = 0.08.

Fig. 4.

Saccharification yields of wood and bark analyzed separately and of wood–bark mixtures of transgenic trees and WT of the Belgian field trial. The same trees that were selected for SSF and wood compositional analysis were used (WT, n = 6; FS3, n = 6; FS40, n = 7). A pretreatment with 6.25 mM NaOH was applied. Error bars represent SDs. Dark gray, WT; light gray, FS3; white, FS40. ^aSignificantly different compared with WT within the same tissue; ^bsignificantly different compared with wood within the same line; ^csignificantly different compared with bark within the same line.