A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.
Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90°C. The pretreatment solution is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme mixture for a variable length of time at 50°C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH colorimetric method.
Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.
The authors have nothing to disclose.
The authors would like to thank A Viksø-Nielsen (Novozymes) for the gift of the cellulolytic enzymes. This work was funded by FP7 RENEWAL and by BBSRC projects BB/G016178 and BB/G016194.
Name of the reagent | Company | Catalogue number |
---|---|---|
Grinding & weighing robot | Labman Automation | |
2 ml micro tube with caps | Sarstedt Ltd | 72.694 |
5 mm stainless steel beads | Qiagen Ltd | 69989 |
1.2ml Abgene square well storage plates | Fisher Scientific Ltd | TUL-050-050C |
Whatman cap mat for 96 square well plates | Fisher Scientific Ltd | 7704-0104 |
Liquid hanling robot | Tecan Group Ltd. | Freedom Evo 200 |
Sulphuric acid | Fisher Scientific Ltd | S/9231/PB17 |
Sodium hydroxide | Fisher Scientific Ltd | BPE359-500 |
Sodium acetate | Sigma-Aldrich | S8750-500G |
Acetic acid | Fisher Scientific Ltd | A/0420/PB17 |
Novozyme 188 | Novozyme | DCN00214 |
Celluclast 1.5L | Novozyme | CCN03122 |
96 well PCR full skirt plates | Sarstedt Ltd | 72.1980.202 |
D-Glucose | Fisher Scientific Ltd | G/0450/53 |
3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate | Sigma-Aldrich | 129739-25G |
DL-dithiothreitol | Sigma-Aldrich | D9163-1G |
Corning microplate 96 well flat bottom | Fisher Scientific Ltd | TKT-521-050H |
Ammonium iron (III) sulfate dodecahydrate | Sigma-Aldrich | F1668-250G |
Sulfamic acid | Sigma-Aldrich | 242772-500G |
Hydrochloric acid | Fisher Scientific Ltd | 12462-0026 |