Background

Wood represents the majority of the biomass on land and constitutes a renewable source of biofuels and other bioproducts. However, wood is recalcitrant to bioconversion, raising a need for feedstock improvement in production of, for instance, biofuels. We investigated the properties of wood that affect bioconversion, as well as the underlying genetics, to help identify superior tree feedstocks for biorefining.

Results

We recorded 65 wood-related and growth traits in a population of 113 natural aspen genotypes from Sweden. These traits included three growth and field performance traits, 20 traits for wood chemical composition, 17 traits for wood anatomy and structure, and 25 wood saccharification traits as indicators of bioconversion potential. Glucose release after saccharification with acidic pretreatment correlated positively with tree stem height and diameter and the carbohydrate content of the wood, and negatively with the content of lignin and the hemicellulose sugar units. Most of these traits displayed extensive natural variation within the aspen population and high broad-sense heritability, supporting their potential in genetic improvement of feedstocks towards improved bioconversion. Finally, a genome wide association study (GWAS) revealed 13 genetic loci for saccharification yield (on a whole tree biomass basis), with six of them intersecting with associations for either height or stem diameter of the trees.

Conclusions

The simple growth traits of stem height and diameter were identified as good predictors of wood saccharification yield in aspen trees. GWAS elucidated the underlying genetics, revealing putative genetic markers for bioconversion of bioenergy tree feedstocks.

Plant material

The Swedish Aspen (SwAsp) collection consists of 113 Populus tremula aspen genotypes from 12 locations across Sweden (Luquez et al., 2008). The genotypes represent potential sub-populations (Fig. 2), but whole-genome sequencing and sequence comparisons have shown that these genotypes are mostly unrelated (Wang et al., 2018a).

The genotypes were clonally propagated in 2003 from root cuttings and grown in a randomized block experiment in a plantation in southern Sweden (Ekebo, 55.9°N). Three to five trees per genotype were successfully established in 2004 (Luquez et al., 2008; Wang et al., 2018a).

After ten years of growth, tree height and diameter at breast height (DBH) were measured, and wood samples were collected from the stem. At 79 cm above ground, a 1-cm-thick section of the stem was collected, and the south-western facing quarter of the stem section was aliquoted for wood chemical composition analyses. In addition, 80-90 cm above ground, another piece of stem was harvested for analysis of wood anatomical and structural properties from the south-western facing quarter of the stem section. We obtained a full set of successful phenotypic measurements for a total of 418 trees, representing two to five replicates per genotype (Additional file 1).

 Analyses of wood chemical composition

The wood quarters selected for compositional analyses were manually debarked, cut into roughly match-stick-sized wood pieces and freeze dried (CoolSafe Pro 110-4, LaboGene A/S, Denmark). This material was homogenized by coarse milling (Retsch ZM 200 centrifugal mill, Retsch GmbH, Germany) and sieved (Retsch AS 200) into two particle size fractions. The fraction of particle size between 0.1 mm and 0.5 mm was aliquoted for subsequent saccharification experiments (see below), while the fraction of particle size under 0.1 mm was aliquoted for pyrolysis coupled with gas chromatography followed by mass spectrometry analysis (pyrolysis-GC/MS) and monosaccharide composition analysis. Both analyses were performed as technical duplicates for each tree.

Carbohydrate content, lignin content, lignin composition, and content of other phenolics were determined by pyrolysis-GC/MS as previously described (Gerber et al., 2016). Briefly, 40 µg - 80 µg of homogenized wood powder was loaded into an autosampler (PY‐2020iD and AS‐1020E, Frontier Labs, Japan), allowing a sub-sample (~1 µg) into the pyrolizer of the GC/MS apparatus (Agilent, 7890A/5975C, Agilent Technologies AB, Sweden). Following pyrolysis, the samples were separated along a DB‐5MS capillary column (30 m × 0.25 mm i.d., 0.25‐µm‐film thickness; J&W, Agilent Technologies), and scanned by the mass spectrometer along the m/z range 35 – 250. The GC/MS data were processed as previously described (Gerber et al., 2012). Results were normalized by expressing the area of each peak as a percentage of the total peak area considering all peaks.

Cell wall monosaccharide units were quantified following the acidic methanolysis and trimethylsilyl (TMS) derivatization method as described previously (Gandla et al., 2015). Briefly, wood powder was washed with HEPES buffer (4 mM, pH 7.5) containing 80% ethanol, as well as methanol:chloroform 1:1 (V:V) and acetone to generate alcohol insoluble residues (AIRs) which were then dried. To avoid contamination with glucose from starch, the AIRs were treated with 1 unit per AIR mg of type I α-amylase (Roche 10102814001, Roche GmbH, Germany). The de-starched AIRs, and inositol as an internal standard, were methanolysed using 2 M HCl/MeOH at 85°C for 24 h. Following repeated washes with methanol, the samples and standard were silylated using Tri-sil reagent (3-3039, SUPELCO, Sigma-Aldrich, Germany) at 80°C for 20 min. The solvent was evaporated under a stream of nitrogen and pellets were dissolved in 1 mL hexane and filtered through glass wool. The filtrates were evaporated until 200 μL remained, of which 0.5 μL were analysed by GC/MS (7890A/5975C; Agilent Technologies AB, Sweden) according to Sweeley et al. (1966). The levels of the sugars and sugar acids are presented in the hydrous form.

Saccharification assays and total wood glucose yield (TWG)

Saccharification assays without or with acid pretreatment of the biomass were performed following an established methodology (Gandla et al., 2015). In short, 50 mg of dry wood powder (moisture measured with an HG63 moisture analyser, Mettler-Toledo, USA) with particle size between 0.1 mm and 0.5 mm were pretreated with 1% (w/w) sulphuric acid (fraction of sulphuric acid based on the mass of the whole reaction mixture) during 10 min at 165°C in a single-mode microwave system (Initiator Exp, Biotage, Sweden), or remained untreated. The pretreated samples were centrifuged to separate the solid fraction from the pretreatment liquid. The solid fraction was washed with ultrapure water and sodium citrate buffer (50 mM, pH 5.2). The washed, pretreated solid fraction as well as the untreated samples were enzymatically hydrolysed 72 h at 45°C under agitation, using 25 mg of a 1:1 (w/w) mixture of liquid enzyme preparations Celluclast 1.5 L (measured CMCase activity of 480 units per gram of liquid enzyme preparation, following Ghose (1987)) and Novozyme 188 (measured β-glucosidase activity of 15 units per gram liquid enzyme preparation, following Mielenz (2009) (Sigma-Aldrich). Sodium citrate buffer (50 mM, pH 5.5) was added to reach 1 g of final reaction mixture. During enzymatic saccharification, samples were collected at 2 h and 72 h. Glucose production rates were determined at 2 h using an Accu-Chek ®Aviva glucometer (Roche Diagnostics Scandinavia AB, Sweden). Monosaccharide (arabinose, galactose, glucose, xylose and mannose) yields in pretreatment liquids and enzymatic hydrolysates collected at 72 h were determined using high-performance anion-exchange chromatography with pulsed amperometric detection (Ion Chromatography System ICS-5000, Dionex, USA) as previously described (Wang et al., 2018b). Saccharification was performed on technical duplicates for each tree.

The total-wood glucose yield from an entire tree trunk (TWG) was calculated using the formula TWG = 1/3 × π × height × (diameter/2)² × wood density × glucose release_{(AFTER PRETREATMENT)}, as previously described (Escamez et al., 2017), assuming a conical shape of the tree stem.

Anatomical and structural characterisation

Anatomical and structural features were determined on parallelepipedal wood pieces across the stem diameter using the SilviScan®  instrument (CSIRO, Australia) which consists of three separate units: (i) a cell scanner with a video microscope for measurement of the numbers and sizes of fibres and vessels, (ii) a density scanner recording X-ray absorption images for measuring wood density, and (iii) a diffraction scanner recording X-ray diffraction images for measuring the microfibril angle. The measurements on these parallelepipedal wood pieces were then projected onto the entire wood section to reflect the average values for the entire wood section of each tree. 

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