Douyong Min

Down-regulation of glycosyltransferase 8D genes in Populus trichocarpa caused reduced mechanical strength and xylan content in wood

Members of glycosyltransferase protein families GT8, GT43 and GT47 are implicated in the biosynthesis of xylan in the secondary cell walls of Arabidopsis. The Arabidopsis mutant irx8 has a 60% reduction in xylan. However, over-expression of an ortholog of Arabidopsis IRX8, poplar PoGT8D, in Arabidopsis irx8 mutant could not restore xylan synthesis. The functions of tree GT8D genes remain unclear. We identified two GT8 gene homologs, PtrGT8D1 and PtrGT8D2, in Populus trichocarpa. They are the only two GT8D members and are abundantly and specifically expressed in the differentiating xylem of P. trichocarpa. PtrGT8D1 transcript abundance was >7 times that of PtrGT8D2. To elucidate the genetic function of GT8D in P. trichocarpa, the expression of PtrGT8D1 and PtrGT8D2 was simultaneously knocked down through RNAi. Four transgenic lines had 85-94% reduction in transcripts of PtrGT8D1 and PtrGT8D2, resulting in 29-36% reduction in stem wood xylan content. Xylan reduction had essentially no effect on cellulose quantity but caused an 11-25% increase in lignin. These transgenics exhibit a brittle wood phenotype, accompanied by increased vessel diameter and thinner fiber cell walls in stem xylem. Stem modulus of elasticity and modulus of rupture were reduced by 17-29% and 16-23%, respectively, and were positively correlated with xylan content but negatively correlated with lignin quantity. These results suggest that PtrGT8Ds play key roles in xylan biosynthesis in wood. Xylan may be a more important factor than lignin affecting the stiffness and fracture strength of wood.

Improved Protocol for Alkaline Nitrobenzene Oxidation of Woody and Non-Woody Biomass

The protocol of alkaline nitrobenzene oxidation was investigated to improve its ability to identify the different lignin structures for both woody and non-woody biomass. The survival factors of all six oxidation products—syringaldehde (Sr), vanillin (V), p-hydroxybenzaldehyde (B) and their corresponding acids, syringic acid (Sa), vanillic acid (Va), and p-hydroxybenzoic acid (Ba)—were studied at 170, 180, and 190°C for several residence times. Under similar conditions, various lignin model compounds—a softwood (loblolly pine), a hardwood (red maple), and a non-wood raw material (corn stover)—were oxidized. Molar yields of oxidation products were determined and the ratios of (Sr+Sa)/(V+Va), (Sr/V), and B/(V+Va) (B/V) were calculated. All oxidation products were relatively stable at 170 and 180°C but showed some degradation at 190°C, especially at long residence time. In all cases, p-hydroxybenzoic acid was barely detectable. While yields of oxidation products reach a maximum at 170°C for pine and maple, maximal yields of corn stover require 190°C. Consequently, we recommend that nitrobenzene oxidation be carried out at 170°C for 2.5 h for softwood and hardwood, but at 190°C and 4 h with correction for the survival factors for corn stover and other non-woody biomass. Alternatively, a protocol of oxidation at two temperatures is recommended for non-woody biomass.

The structural changes of lignin and lignin–carbohydrate complexes in corn stover induced by mild sodium hydroxide treatment

Non-woody biomass such as corn stover is a very abundant and sustainable biofuel feedstock in the US whose technical hurdles for enzymatic hydrolysis have not been adequately addressed. There is very little useful data on the lignin and the lignin–carbohydrate complexes of corn stover and the impacts of them on bioconversion to fermentable sugars. The following principal tasks were addressed, which will help to develop the roadmap of effective saccharification of corn stover: (1) corn stover was separated into stem, cob, and leaf; (2) lignin (cellulolytic enzyme lignin, CEL) and lignin–carbohydrate complexes (milled wood lignin, MWLc) were isolated from the extractive-free and the alkaline-treated samples, respectively; and (3) the structural changes of lignin and lignin–carbohydrate complexes (LCCs) were characterized by alkaline nitrobenzene oxidation, 13C, and 1H–13C HSQC NMR. The results indicated: (1) a significant amount of p-coumarate and ferulate esters was identified and quantified; (2) lignin of the alkaline-treated sample was more condensed; (3) an unanticipated amount of LCCs was quantified in the extractive-free sample, however, the amount of LCCs decreased significantly with the alkaline treatment. Therefore, lignin and LCCs of the treated sample should be characterized to elucidate their effects on the enzymatic saccharification.

Structural characteristics of milled wood lignin (MWL) isolated from green liquor (GL) pretreated poplar (Populus deltoides)

Abstract Pretreatment is one of the key steps for the utilization of lignocellulosic biomasses via biorefinery. Green liquor (GL) pretreatment has been considered as an effective approach to improve the subsequent enzymatic saccharification. For the better understanding of the structural changes of lignin in GL pretreatment, milled wood lignin (MWL) samples isolated from untreated and GL-pretreated poplar by the Björkman method were characterized by means of gel permeation chromatography (GPC), alkaline nitrobenzene oxidation (NBO), Fourier transform-infrared (FT-IR) spectroscopy, and quantitative 13C and 2D heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR). The results indicate that the average molecular weight of MWLs decreased after GL pretreatment. Surprisingly, more guaiacyl-propane units are extracted under mild alkaline conditions than syringyl-propane units, which results in a higher condensation degree and higher S/G ratios of MWLs isolated from GL-pretreated poplars. The amount of β–O–4 structures decreased, while the β–β and β-5 structures increased after GL pretreatment. The structure of esterified p-hydroxybenzoic acid was detected in poplar MWL sample and it degraded obviously after GL pretreatment.