Large-scale production of transportation fuels from woody biomass will result in expansion of land dedicated to biomass production. Improved hybrid poplar, along with other high-yielding energy crops such as switchgrass and hybrid willow, have the potential to supply feedstocks to an emerging biofuels industry with lower land footprints than wild undomesticated species. Lignocellulosic ethanol, derived from woody biomass, is an alternative to conventional gasoline that can be produced from fermentation of sugars derived from woody biomass. One of the preferred methods for the production of fermentable sugars from biomass is a dilute acid hydrolysis pretreatment followed by an enzymatic hydrolysis. The goal of these processes is to obtain the highest conversion possible, thus leading to the greatest ethanol yield. A venue for increasing ethanol yields is to genetically modify the biomass to either increase total sugar content or alter the structure in a way to make the sugars more accessible under less aggressive pretreatment conditions. Genetic modifications targeted at improving biomass properties involve testing large amount of progenies and/or genetically modified transgenics. This requires high throughput and a small amount of sampling material. The results presented here demonstrate “mini-reactors” for dilute acid and enzymatic hydrolysis which can be set up for medium to high throughput studies.

The experiments were conducted with hybrid poplars modified for rate and extent of secondary growth. Eight different samples were studied including three wild-type (Control 3C, Control 4D, and Control 5E) and five transgenic poplars (BL 121 (1), BL 121 (2), BL 12, BL 161, and BL 204). The goal of the experiments was to determine whether the genetic modifications will result in higher total sugar yields from dilute acid hydrolysis pretreatment followed by enzymatic hydrolysis and test the feasibility using the mini-reactor for these studies. A reduction in size of 200x for each experiment was necessary to accommodate the extremely small size of the sample (less than 5 grams total for each hybrid poplar sample).

Two mini-reactors were used; each consisted of a stainless steel Swagelok Fitting with a ˝ inch tube outer diameter and two Stainless Steel Swagelok Plugs; and a volume of approximately 3 ml. 0.12g solids were added to each reactor with 2.4 ml of 0.5% sulfuric acid. The optimum maximum temperature for these experiments was determined to be 170 degrees Celsius. The mini-reactors were secured to the cooling coils on the top of a 1-L well-mixed batch reactor (Parr Instruments, Model 4570 reactor) using stainless steel wire. The bottom of the Parr reactor was filled with approximately 900 ml of distilled water and the top was secured in place. Reactor temperature was increased from room temperature to 170 degrees Celsius at a rate of approximately 3 degrees C/min, and upon reaching the maximum temperature the reactor was cooled at a rate of 10 degrees C/min using tap water. One sample of the hydrosylate was taken from each reactor at the end of the experiment and analyzed by High Performance Liquid Chromatography (HPLC) for sugar content.

The hydrosylated biomass was removed from the mini-reactors, washed, and subjected to enzymatic hydrolysis. This procedure was also scaled back, more than 100x compared to the procedure previously used. Enzyme loadings of 60FPU Spezyme CP (Genencor) / dry gram substrate and 2 CBU/FPU of beta-glucosidase (Novozyme, Novozym 188) were used. Enzymatically digested samples were taken at the end of 72 hours and analyzed by HPLC for sugar content.

Results from the three control samples indicate that the small scale pretreatment experiments were highly reproducible with respect to xylose yield and yields of the minor sugars, both from trial to trial in the mini-reactors, and also compared to larger volume trials at the 0.5 L scale. BL204 transgenic modification showed enhanced xylose yield compared to the controls, whereas the other transgenics exhibited smaller xylose yields. The overall yield of total sugars (glucose, xylose, galactose, arabinose, and mannose) generated by both the pretreatment and enzymatic hydrolysis expressed as percent of the weight of the starting raw material ranged from 52-62%. Two of the genetically-modified poplars, BL 161 and BL 204, appear to have statistically significant higher total sugar yields (61-62%) compared to controls and other hybrid poplar samples (e.g., yields ranging from 52-56%). We are currently investigating the structural and chemical properties of the modified materials to better understand the changes resulting in the observed higher sugar yields.

The small scale pretreatment and enzymatic hydrolysis experimental procedures developed for this work allow for reproducible analysis of samples on a much smaller scale. With the experimental setup previously used, it could take years for enough wood biomass to be produced for testing the effect of the genetic modifications. Here we demonstrate a device and procedure that achieves significant reduction (less than five grams) in sample size needed. This coupled with reproducibility of the results allows for more convenient, intensive and high throughput studies of lignocellulosic conversion properties.