High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The overall goal of this procedure is to measure the thermochemical pretreatment and enzyme hydrolysis recalcitrants between related biomass variants. This is accomplished by first preparing the biomass samples for testing by size, reduction, and extraction of starch and soluble sugars. The second step is to dispense the biomass with high precision into thermo chemically compatible high throughput reactor plates.

The samples are pretreated in a steam reactor. Next, the samples are digested with a standard commercial cellulase for three days to hydrolyze the cellulose and hemi cellulose. The final step is determination of glucose and xylose released during enzyme hydrolysis using GO pod and XDH assays.

Ultimately, high throughput glucose and xylose quantitation is used to determine the relative sugar release between samples on a gram sugar per gram starting biomass basis. The main advantage of this technique over other bench scale methods, such as bench scale, pre-treatment, and enzymatic hydrolysis, is that this technique can be performed on hundreds or even thousands of samples simultaneously. This method can help answer key questions in the biomass conversion field, such as which cultivars of variants are more amenable to conversion to sugars and subsequent products.

So the implications of this technique can be extended towards understanding cell wall structure and function because the mutations that we generate in the cell wall biosynthetic pathway can be screened for structural chemical changes. Well, this method can provide insight into plant cell wall recalcitrants. It can also be applied to other components of the biomass conversion, such as enzyme activity and synergy studies.

Generally, individuals new to this method will struggle with it because it involves large sample sets of biomass variants that have to be handled identically and simultaneously on the milligram scale. So it requires specialized robotics and equipment To begin the procedure at approximately 250 milligrams of previously ground biomass from a barcoded antistatic bag to a numbered teabag, carefully roll up the teabag being sure to fold the ends over the biomass to prevent loss during d starching and extraction. Next, wrap Each teabag closed using tin coated copper wire record the teabag number for each barcoded sample.

Add 500 milliliters of a previously prepared de starching enzyme solution to a plastic container. Then add 120 teabags to the buffer enzyme solution for a batch batching protocol. After incubation in a shaker, rinse and soak the biomass in several liters of deionized water for 30 minutes following exhaustive rinsing of the des starched biomass.

To remove buffer salts, place the teabags into a sock lit column for extraction. Set up the sock lit reflux using 95%ethanol, and extract the samples for 24 hours. Once the extraction is complete, unroll the teabags and return the dried biomass to the original barcoded.

Antistatic bags Transfer at least 50 milligrams of the dried biomass to a barcoded hopper. Scan the barcodes of the antistatic bags and the receiving hopper to ensure accurate sample tracking. After loading the hoppers onto a solid weighing robot robotically weigh five milligrams of the dried samples into acid resistant stainless steel.

96 well plates weigh out three replicates of each sample distributed around the plate to minimize any localized variations. Next, add 250 microliters of deionized water to each well and seal the plates with silicone adhesive backed POLYTETRAFLUOROETHYLENE or PTFE tape. Using a one eighth inch soldering iron tip, pierce the PTFE tape at each steam port.

For each plate, clamp the plates tightly with 0.031 inch thick glass reinforced PTFE gaskets between the plates and empty plates on the top and bottom of the stack. Pretreat the samples using a steam reactor set to 180 degrees Celsius for 17.5 minutes. When finished, cool the reactor plates to 50 degrees Celsius by flooding with deionized water.

When the plates are cool, centrifuge them in a swinging bucket. Rotor at 1700 GS for 20 minutes. Then remove the ceiling film.

Add 40 microliters of previously prepared 8%enzyme stock solution to each well after resealing With new PTFE tape, place the sealed plate into a magnetic plate clamp. Gently mix the samples by inversion at least 15 times and incubate at 50 degrees Celsius for 70 hours. After incubation, the reactor plates are mixed and centrifuged.

Then the liquid handling robot is used to carry out quantitation of glucose and xylose through enzyme linked colormetric assays, xylose dehydrogenase, or XDH for xylose and glucose oxidase, peroxidase, or go pod for glucose. The deck is set up with reservoirs of GO POD and XDH reagent, as well as water for dilution and 96 channel 200 microliter tip racks for each reagent. These tips are reused for multiple reactor plates.

The deck also contains single use, 200 microliter tips for water removal and sugar standard edition, as well as pre-made sugar standards. In HPLC vials from the storage carousel, the robot brings in three clear micro titer plates, one for making dilution of the hydrolysis mix and one each for the XDH and GoPod assays. The gripper moves all four plates to positions on the deck.

Single use 96 channel 100 microliter tips are brought on deck from the carousel for use in transferring from reactor to dilution plate and from dilution to XDH and go pod plates. Using the variable span eight channel pipetter, the water that was added before pretreatment is removed from the three wells in each corner of the reactor plate to prevent carry over to the dilution plate. As these 12 wells are used for sugar standards using the 96 channel pipetting head.

With the three tips in each corner removed, 180 microliters of water is added to the wells of the dilution plate. After changing tips, XD, H and go pod reagents are transferred from their respective reservoirs to their respective assay plates. Using the 96 channel pipetting head.

While this is occurring, the sugar standards are added to the three wells of each corner of the dilution plate. Using the eight channel pipetting head After changing to the smaller tips, the 96 channel pipetting head is used to transfer 20 microliters of hydrolysis supernatant to the dilution plate, and the plate is mixed by tri. The sample should be aspirated near the top of the liquid to limit fouling of the tips by biomass particles.

After mixing 20 microliters is transferred sequentially. Using the 96 channel pipetting head to the XDH and go pod plates. With each being mixed by trier, the sample tips reactor and dilution plates are returned to a waste rack in the carousel, the XDH and go pod plates are removed to a separate carousel rack and allowed to sit for one hour before reading in the spectrophotometer, using an ultraviolet visible 96 well plate reader.

Set the measured wavelength to 510 nanometers and record the absorbance against a reagent blank. Following this, set the measured wavelength on the plate reader to 340 nanometers and record the absorbance using a small spatula. Dispense approximately four milligrams of the prepared biomass into an 80 microliter stainless steel cup designed for the autos sampler of the mass spectrometer.

Using a standard hole punch, manually produce glass filter discs from type ad glass fiber sheets with no binder load samples into the autos sampler racks using tweezers randomly load samples into the cups to avoid bias due to spectrometer drift over time. After performing the calibration and inputting the appropriate parameters in the mass spectrometer, begin data acquisition and wait at least 60 seconds to obtain sufficient data for background spectra collection. Finally, start the automated autos sampler method using a helium carrier gas flow rate of 0.9 liters per minute.

An example of trends is a survey of recalcitrants of 755 natural variants of poplar sampled in the Pacific Northwest. The results indicate that as the syringe to guil ratio rises, recalcitrant decreases until a ratio of around two or the recalcitrant improvement levels off. Outliers can be seen here where the PT four CL one gene was downregulated.

In poplar, several of the hundred cultivar screened are clearly increased in recalcitrants as evidenced by the decreased sugar release. Different wheat straw varieties grown on several sites and harvested after several variations in growth conditions resulted in 20 distinct populations based on combinations of the above variables. These sample sets are readily distinguished and indicate positive and negative variables.

For recalcitrants pyrolysis, molecular beam mass spectrometry can be used to evaluate changes in cell wall composition. Mass spectral fragments are assigned to different lignin monomers. When comparing a sample with high lignin versus high carbohydrate content, the disparities in the spectral data are apparent.

The use of mass spectrometry data, coupled with a principle component analysis is illustrated here. The principle component loadings plot can aid in identifying which chemical traits are being explained in the scores classification plot, as well as elucidating, which traits are changing between the sample clusters Once mastered, this technique can be used to screen thousands of samples per week if performed properly. While attempting this procedure, it's important to be as consistent as possible and only compare samples within a related set.

So after its development, this technique paved the way for other researchers to work on high throughput screening methods and other areas for plant cell wall recalcitrant, using things like ionic liquids and alkaline pretreatment. After watching this video, you should have a good understanding of how to properly prepare, handle, and analyze large sample sets of biomass at the microscale for differences in recalcitrant using size reduction and extraction, thermochemical pretreatment and enzyme hydrolysis, Please remember that working with high pressure, steam and automated equipment is extremely hazardous and that all safety precautions should be taken, such as wearing personal protective equipment, as well as having safety interlocks on all of the equipment while performing these procedures.