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

Summary

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

Abstract

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Introduction

As the global supply of non-renewable fuels and their associated products declines, scientists have been challenged to create similar fuels and chemicals from plant-derived sources¹. A key aspect of this work is determining which species of plants may be suitable for the production of biofuels and biomaterials^2,3. Typically, these feedstocks are evaluated for lignin, cellulose, and hemicellulose content; as well as their susceptibility to deconstruction (recalcitrance) through thermal, mechanical, and/or chemical pretreatment with or without subsequent enzyme saccharification. More detailed analyses are used to determine the specific composition ....

Protocol

1. High-throughput Determination of Glucose and Xylose Yields Following Enzymatic Saccharification^9,29

1. Sample Preparation (Grinding, De-starching, Extraction, Pretreatment)
   1. Grind at least 300 mg of each biomass sample using a Wiley mill, such that the particles pass through a 20 mesh (850 µm) screen. Transfer to anti-static zip-top bags (typically bar-coded) and record sample information to the barcode database.
   2. Add approximately 250 mg or more of the ground biomass from anti-static bag to a numbered (with pencil, do not use ink or marker) tea-bag, carefully roll up the teabags, being sure to fold the ends over the bi....

Results

The combined effect of the thermochemical pretreatment and subsequent enzyme saccharification is measured as a function of the mass of glucose and xylose released at the end of the assay. The results are reported in terms of milligrams of glucose and xylose released per gram of biomass. This is in stark contrast to data reported from bench-scale assays, which is usually reported as percent theoretical yield based on compositional analysis of the starting material. As it is not yet practical to carry out compositional ana.......

Discussion

The key sample preparation steps for obtaining accurate and reproducible data when conducting high-throughput screening experiments are as follows:

Sugar Release Assay:

In general, samples are prepared in lots ranging from a few dozen to several thousand at a time. Each major step is typically carried out for all samples prior to moving forward in order to minimize variations in preparation between samples. De-starching was not originally part of the protocol and ca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biomek FX Automated Workstation	Beckman Coulter	Biomek FX	Automated Liquid Handler
Wiley mill	Thomas Scientific	3375E15 (Model 4), or 3383L20 (Mini-mill)
anti-static bags	Minigrip*	MGST4P02503	2.5x3", multiple suppliers available
tin-coated copper wire	McMaster-Carr	8871K84	0.016" diameter, bend-and-stay wire
tea-bags	Herbco	press n' brew teabags	3.5x5 inches
gluco-amylase	Novozymes	Spirizyme Fuel
alpha-amylase	Novozymes	Liquozyme SC DS
sodium acetate trihydrate	any chemical supplier		reagent grade
acetic acid	any chemical supplier		reagent grade
190 proof (95%) ethanol	any chemical supplier		reagent grade
hoppers	Freeslate
96-well C-276 Hastelloy plates	Aspen Machining (Lafayette, Colorado)	N/A (custom built)
1/8” soldering iron tip	Sears
silicone-adhesive backed Teflon tape	3M	5180	3" wide (36-yard rolls)
enzyme solution	Novozymes		Cellic CTec2
citric acid monohydrate	any chemical supplier
trisodium citrate dihydrate	any chemical supplier
disposable, polystyrene 96-well plates	Greiner Bio-One	655101	or equivalent; multiple suppliers available
glucose oxidase/peroxidase	Megazyme	K-Gluc	Megazyme D-glucose assay kit
xylose dehydrogenase	Megazyme	K-Xylose	Megazyme D-xylose assay kit
glucose standard solution	Megazyme	K-Gluc	Megazyme D-glucose assay kit
xylose standard solution	Megazyme	K-Xylose	Megazyme D-xylose assay kit
stainless steel sample cups	Frontier Laboratories	PY1-EC80F
glass fiber sheets	Pall	66227	8x10" sheets; circles punched with standard hole punch
Sugarcane Bagasse Whole Biomass Feedstock	NIST	8491
Eastern Cottonwood (poplar) Whole Biomass Feedstock	NIST	8492
Monterey Pine Whole Biomass Feedstock	NIST	8493
Wheat Straw Whole Biomass Feedstock	NIST	8494