Douglas-fir core samples used to determine recalcitrance variability.
Douglas-fir core samples used to determine recalcitrance variability.

Douglas-fir trees are not all alike. Some grow faster, some adapt better to harsher climates and they all differ somewhat in their chemical makeup. These distinctions are due to variations in their genetic makeup. Those interested in breeding plantation trees for timber value have capitalized on genetic variation to select softwoods with characteristics (traits) that provide better timber products and reduce production costs.

NARA was established to facilitate development of a new industry that uses the residual wood in slash piles to produce chemical products like biojet fuel. As this industry develops, softwood breeders will be interested in bringing seedlings to the marketplace that not only enhance timber production but also allow improved chemical conversion from the residuals.

Measuring carbohydrates, lignin and recalcitrance

Forest residuals that provide high carbohydrate yields at low cost would be an ideal feedstock for biojet fuel production. NARA researchers in the Feedstock Development Team wanted to know if carbohydrate (simple sugars) yields varied among Douglas-fir samples.  If variation did exist, then the genetic causes for the variation could potentially be identified and used to breed softwood trees that produced superior residuals for conversion to biojet fuel and other chemical products.

To test carbohydrate yield variation among Douglas-fir trees, a research team lead by Xiao Zhang and Keith Jayawickrama sampled 150 plantation-grown Douglas-fir trees, representing 40 Douglas-fir families, for variation in carbohydrate yield and published their results in the journal Bioenergy Research.

To view A Multi-Level Analysis Approach to Measuring Variations in Biomass recalcitrance of Douglas Fir Tree Samples view here.

Carbohydrate yields are determined by carbohydrate content and, more importantly, by the level of recalcitrance. Recalcitrance, in this case, refers to the resistance wood has to deconstruction. A high level of recalcitrance would inhibit carbohydrate extraction more than a lower level. For this study, the authors developed a high throughput screening process to determine the chemical composition and level of recalcitrance for each sample.

Douglas-fir trees exhibit variation in chemical composition and recalcitrance

There was a large degree of chemical variation evident among the samples tested. Extractives (fatty acids, resin acids, waxes and terpenes) ranged from 0.25 to 5.1% of the wood composition. Lignin ranged from 23 to 39.9%, and total polysaccharides ranged from 62.1 to 78.8%.

To measure the level of recalcitrance, two steps were required. The first step involved subjecting the samples to pretreatment, used to remove the lignin and hemicelluloses from the carbohydrates stored as cellulose. Enzymatic hydrolysis represented the second step, where enzymes were added to the pretreated material to liberate the individual simple sugars from the cellulose. The simple sugars released were measured; the simple sugar yield compared to the total amount of carbohydrates available represented a level of recalcitrance.

Two pretreatment methods were used in this study: dilute acid (DA) and alkaline peroxide (AP). After pretreatment, a smaller degree of carbohydrate variation existed between pretreated samples (9%) compared to the carbohydrate variation observed in the raw non-pretreated biomass (16%). The authors point out that pretreatment has a “normalizing” effect on the carbohydrate levels; because pretreated material contains all of the carbohydrates available for further downstream release, the carbohydrate variation detected in the raw biomass is an unlikely indicator of how much carbohydrate will be available to convert to biofuels and other products.

When the pretreated samples were subjected to hydrolyzing enzymes, the resulting carbohydrate (sugar) yields showed the high degree of variation. Sugar yields from DA pretreated biomass after enzymatic hydrolysis ranged from 12.6 to 46.1%; yields from the AP pretreated material after hydrolysis ranged from 6.31 to 24.2. These yields will vary depending on the severity of the pretreatment, but the wide degree of variation is the significant factor.

Because pretreatment and enzymatic hydrolysis can cause variation in carbohydrate yield, the authors developed a “recalcitrance factor” that incorporates both pretreatment and hydrolysis results into a yield value for the overall conversion process.

Future direction

Breeding softwood trees with improved sugar yields is a long-term prospect, well beyond NARA’s 5-year project duration. The results from this study indicate that recalcitrance variation does exist among Douglas-fir individuals. This data will be paired with genomic data taken from the same samples and used to identify identifying genetic markers that influence the level of recalcitrance. Those genetic markers should allow breeders to develop a future generation of plantation trees that produce residual material more suitable to chemical conversion.