Article source technical application “Liquid-Liquid Extraction (LLE) vs. Novum™ Simplified Liquid Extraction (SLE) Case Study: Analysis of Monomeric Phenolic Fermentation Inhibitors in Dilute-Acid Plant Hydrolysate”
Pretreatment of lignocellulosic biomass is a prerequisite in order to maximize glucose release from cellulose for subsequent fermentation or chemical conversions. A leading pretreatment involves dilute acid (e.g., 0.3 – 2% sulfuric acid) at elevated temperatures (140 to 200 °C) with different holding times and biomass/acid loadings1. A drawback of this procedure is the partial degradation of lignin, a polyphenolic macromolecule, to smaller phenolics that are released together with the sugars into the so-called hydrolysate. These phenolic compounds are toxic to microorganisms and are potential fermentation inhibitors 2-5.
Analyzing this phenolic profile is important when optimizing pretreatment or detoxification methods aiming to minimize inhibitor levels or to engineer more resistant microorganisms. In a widely employed sample preparation and cleanup step, the phenolic compounds undergo a liquid-liquid extraction using an organic solvent (e.g., ethyl acetate) and are then analyzed via GC/MS6.
While effective, the liquid-liquid extraction can be time-consuming, requiring 4 separate extractions and multiple centrifugation steps. In an effort to reduce time and sample handling steps, a simplified liquid extraction (SLE) using Novum SLE was employed, saving a significant amount of time and reducing the amount of required pipetting and vial handling steps.
Materials and Methods
Lignocellulosic biomass was prepared using two different methods, a traditional liquid-liquid extraction and simplified liquid extraction (SLE) using a Novum SLE MAX 96-well plate (part no. 8E-S138-5GA). Prior to each extraction method, the samples were pretreated as follows:
Pretreatment
A dilute-acid pretreatment hydrolysate (1.5% sulfuric acid, 190 °C for 1 minute, 25% biomass loading) from Miscanthus X giganteus was filtered using a PES (polyethersulfone) 0.45 µm filter and was spiked with 20 µg/mL iso-propylphenol as an internal standard (IS). The pretreated samples were then extracted by either liquid-liquid extraction or SLE (below).
Liquid-Liquid Extraction Procedure
1 mL aliquots of the spiked hydrolysate from the pretreatment step were extracted 4x each with 0.5 mL of ethyl acetate. After each extraction, a centrifugation step (1500 g for 1 minute) was applied for phase separation. The combined extracts of each sample were dried over sodium sulfate6 (samples A).
Simplified Liquid Extraction (SLE) Procedure
400 µL aliquots of the spiked hydrolysate from the pretreatment step were loaded into the wells of a Novum SLE MAX 96-well plate. The sample was allowed to soak into the sorbent for 5 minutes and then the ethyl acetate was applied to elute the analytes of interest. Three different volumes were applied to determine the most effective elution volume. Either 1x 0.4 mL (samples B), 1x 0.8 mL (samples C), or 2x 0.4 mL (samples D) of ethyl acetate was applied using a short pulse of vacuum. No further drying of ethyl acetate was performed after the elution. GC/MS Conditions
Prior to GC analysis, 100 µL aliquots of the extracts (samples A – D) were incubated with 50 µL BSTFA at 70 °C for 30 minutes and then analyzed by GC/MS6. All extractions were performed in triplicate and replicates (1-3) were run in the following order: samples A1 – D1, then A2 – D2, then A3 – D3 to avoid any bias because of potential changes in sample composition of unprocessed samples due to the long run time (67 minutes). 1 µL was injected in splitless mode onto a 5-ms capillary GC column (30 m x 0.25 mm x 0.25 µm).
An Agilent® 7890A gas chromatograph coupled to an Agilent 5975C single quadrupole mass spectrometer was used for analysis with the following settings: Injector and transfer line temperature 280 °C, carrier gas: helium at 1 mL/min, temperature program: 75 °C for 3 min isocratic, 5 °C/min to 150 °C, 0.5 °C/min to 160 °C, 2 °C/min to 190 °C, 5 °C/min to 240 °C, 70 °C/min to 325 °C, 3 min isocratic. Ions were detected in full scan mode m/z 35-500.
Results and Discussion
Figure 1 shows the relative recoveries of 25 selected phenolic compounds from hydrolysate eluted from the Novum SLE MAX 96-well plate using the 3 elution procedures (0.4 mL, 0.8 mL and 2x 0.4 mL) compared to the values obtained by the conventional liquid-liquid extraction (=100% on the representative chart).
Overall the Novum SLE extraction produced recoveries that were comparable to the traditional liquid-liquid extraction procedure in a fraction of the time (a five-fold decrease in the amount of sample preparation time). The 0.4 mL ethyl acetate elution resulted in a general lower recovery of the analytes compared to the 0.8 mL and 2x 0.4 mL volume extractions. Exceptions were 4-hydroxy-3-methoxy-cinnamaldehyde (Figure 1, analyte 9) with almost identical recovery levels observed (95% and 93%, respectively) for all elution volumes and sinapaldehyde (Figure 1, analyte 12) with a higher recovery for the 0.4 mL extraction volume (93 %) compared to the other extraction volumes applied (89 % and 85 %, respectively). However, a 0.4 mL extraction volume was generally deemed to be suboptimal. Increasing the elution volume to 0.8 mL improved the recovery with most recoveries reaching >90%. Slightly lower yields were observed for 4-hydroxybenzaldehyde (87%) (Figure 1, analyte 1) and vanillin (89%) (Figure 1, analyte 2). Only the homologs 2-guaiacylacetaldehyde (Figure 1, analyte 14) and 2-syringacetaldehyde (Figure 1, analyte 16) exhibited lower recovery levels of 75-76% and 73-75%, respectively. Applying the 0.8 mL elution volume in two steps (2x 0.4 mL) did not improve the recovery as compared to a single aliquot of 0.8 mL. TN-0082 Page 2 of 4 APPLICATIONS
Conclusion
When extracting 0.4 mL of a dilute-acid plant hydrolysate using a Novum™ SLE MAX 96-well plate, a 0.8 mL elution volume of ethyl acetate resulted in high recoveries of >90% for most phenolic analytes. No further drying of the ethyl acetate phase was necessary to ensure successful derivatization for subsequent GC/MS analysis. Compared to the conventional liquid-liquid extraction, we estimate an up to a five-fold decrease in sample preparation time.
Furthermore, the Novum SLE extraction also greatly simplifies sample preparation by reducing the amount of required pipetting and vial handling steps. Therefore, sample preparation using the Novum SLE resulted in a phenolic inhibitor profile that was very similar and comparable to the conventional liquid-liquid extraction but at a fraction of the time and labor.
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