The characterization of the conversion processes of lignocellulosic biomass to biofuels requires a large array of methods and analytical systems to extract the meaningful parameters necessary to describe the solid materials and the conversion liquors. The crucial point is also to develop robust, reliable and high-throughput methods that allow the analysis of large number of samples of various sizes (from mg to kg) and of high heterogeneity. The Walker lab has been developing new and cutting-edge methods ranging from single molecule analysis, microplate assays for the studies of cellulases and cellulase cocktails, and non destructive vibrational spectroscopy methods for biomass and bioprocesses analysis, among others. The Biofuel Research Laboratory has been equipped with analytical systems that meet these needs and expand the analytical capabilities for our group and the broader community involve in biofuel R&D. These systems include HPLCs for fluorescence, UV-VIS and Refractive Index (RI) detection, Liquid Chromatography coupled with a Mass Spectrometer (LC-MS) for metabolites characterization and quantification, UV-Vis and fluorescence plate readers, Fourier Transform Infrared and Near-Infrared spectrometers, gas chromatograph, UV-Vis spectrometers, automated protein purification system (FPLC). Some of these methods and instrumentation are described below.
Using FT-MIR, fundamental vibrations of complex organic samples can be analyzed and sample specific fingerprints can be obtained. FT-MIR exploits the mid-infrared region (4,000 - 400 wavenumber, cm-1). A wild range of samples can be analyzed, categorized and quantified by FT-MIR, ranging from microbes to proteins and biomass. Applied to lignocellulosic biomass conversion for sugar production, the prominent peaks within the spectra are at 1033, 1059, 1112, and 1163 cm-1 where 1033 and 1059 cm-1 correspond to C-O stretching vibration, 1112 cm-1 corresponds to asymmetric glucose ring stretch, and 1163 cm-1 corresponds to C-O-C asymmetric vibrations within cellulose (Fig. 1B). Another important peak pertinent to enzymatic hydrolysis of cellulose is the peak at 897 cm-1 corresponding to beta-glucosidic bonds of amorphous cellulose. Therefore a direct analysis of conversion efficiency can be performed using FT-IR. The most advanced FT-MIR can be coupled with automated platforms for high throughput analysis in a 96-well microplate format.
Figure 1: (A) Fourier Transform Mid Infrared spectrometer (Vertex 70, Bruker) with high-throughput attachment (HTS-XT, Bruker). (B) Attenuated Total Reflectance spectra (3600-550 cm-1) of hardwood (blue) and hydrothermal pretreated hardwood (red) and assigned vibrations for cellulose and lignin.
Chemometric models associated with Near Infrared (NIR) spectral analysis lends themselves handily to the high throughput, off-line or on-line, monitoring and process control industry, where fast and inexpensive systems are needed to test, predict and make decisions about product quality, or real-time adjustments with online process monitoring. These methods have been developed for agricultural and industrial applications mainly to assess the quality of feeds. FT-NIR is based on the quantification of the vibration overtone intensities in the near-infrared region (12,000-4,000 wavenumber, cm-1). Fourier Transform Near Infrared spectroscopy (FT-NIR) and multivariate modeling are the core of new analytical methods that can be applied for the high-throughput, fast, online or offline analysis of biomass throughout the logistics of the harvest, storage, conversion and assessing chemical composition of biomass. Indeed, the new generations of FT-NIR spectrometers have gained in improved accuracy and reliability compared to diffusive NIR spectrometers (NIRS). The most recent advances include the use of high quality optic fibers and process-resistant optic probes for the online and real-time monitoring of chemical and biochemical processes (Fig. 2A). The construction of robust chemometric models (Fig. 2B) associated with the speed and accuracy of FT-NIR spectrometers (FT-NIRS) is used for compositional analysis of feedstock, and analytical monitoring of conversion of feedstocks to the final products, to ultimately develop quality analysis (QA) of the feedstocks and quality control (QC) of the conversion processes.
Figure 2: (A) Fourier Transform Near Infrared Spectrometer (MPA, Bruker) with four sampling ports. (B) Scheme or the development of multivariate chemometric models based on FT-NIR spectral data.
Fluorescently tagging functional molecules such as enzymes is key to develop single molecule applications and fluorescence based assays for example. However, it is not trivial as enzyme functions and activities rely upon their conformation, charges, hydrophobicity, all of which can be hindered by the addition of the signaling molecules. Applied to the study of any enzymes, fluorescent labeling has to ensure that the catalytic activity remain unchanged. We have developed an analytical and preparative method based on FPLC purification (Fig. 3A) for the production of enzymes (Fig. 3B) for which we can assess and sort by their degree of labeling and their catalytic activities ensuring optimal fluorescence and activities compared to the native ones. This polishing purification method allows reaching defined and homogenous biochemical reagents.
Figure 3: (A) Fast Protein Liquid Chromatography (AKTA Explorer, GE). (B) Elution chromatograms of fluorescently labeled cellulases (Cel9A, alexafluor647) with pure population with a defined degree of labeling for single molecule microscopy studies.
An important aspect for the conversion of lignocellulosic biomass to biofuels is the saccharification step which consists in using enzymes to convert the cellulose polymer into sugars. Characterizing and quantifying these sugars is necessary as they will be later fermented into ethanol or other biofuels. HPLC systems (Fig. 4A) based on appropriate separation columns are used to measure these sugars (Fig. 4B) and to measure the specific activities of enzymes, engineered enzymes and cocktails of these. Other methods have been implemented by the Walker lab to measure other compounds and metabolites from lignocellulosic biomass pretreatment and fermentation such as organic acids and alcohols during anaerobic fermentation for hydrogen production.
Figure 4: (A) Automated High Performance Liquid Chromatography system (Shimadzu) for carbohydrate analysis. (B) HPLC chromatogram of the hydrolysis products of cellulose (micro crystalline cellulose) by 3 different thermophilic cellulases ( Cel9A, Cel5A and Cel6B) from T. fusca.