Synchrotron radiation Fourier transform infrared (SR-FTIR) spectra of cellulose undergoing hydrolysis by Trichoderma reesei Cel7A in buffer were collected. IR spectra were recorded at 64 locations every ~ 10 min in an 8x8 grid within a 70 x 70 μm sample area. 

Enzyme

• Trichoderma reesei Cel7A purified from Celluclast 1.5L (Sigma C2730)

Cellulose

• Algal cellulose fibrils from Cladophora aegagropila

Reaction conditions

• 37^oC
• 50 mM sodium acetate buffer, pH 5

Synchrotron Radiation Fourier Transform Infrared (SR-FTIR) spectromicroscopy of cellulose undergoing enzymatic hydrolysis

Experimental samples for monitoring cellulose hydrolysis by SR-FTIR were prepared by depositing and air-drying 60 mL of algal cellulose (0.025mg/mL) fibrils onto the SiNx membrane in an open-channel microfluidics (OMM) device, followed by an infiltration of 20 mL of Cel7A (0.5 µmole/g) through the cellulose. The enzyme and substrate was equilibrated in air at ambient conditions (~20^oC) for ~ 40 minutes at room temperature, then mounted onto the FTIR microscope for optical alignment and location of regions of interest (ROI). The experiment was initiated by flowing 1 mM sodium acetate buffer at 0.3 mL/min into the fluid distribution channel connected to the bottom microchannel, and raising the temperature of the FTIR microscope stage to 37°C. The acetate solution wicked upwards by capillary forces through the SiNx pores maintained a buffered reaction at the membrane surface. At 37 ^oC the system maintained a relative humidity of ~80%.  With the initiation of reaction, infrared absorption measurements were collected and monitored to evaluate the quality of the experiment in real-time, followed by detailed post-experiment analysis.

SR-FTIR spectral measurements

All SR-FTIR measurements were made at the infrared beamline at the Advanced Light Source (ALS) (Lawrence Berkeley National Laboratory, CA, USA) using a Nicolet Magna 760 FTIR spectrometer equipped with a liquid nitrogen cooled Mercury Cadmium Telluride (MCT) detector, and an all-reflective optics infrared microscope (Nicolet Nic-Plan, Thermo Scientific, Inc., MA, USA). Mid-infrared photons emitted from the synchrotron were focused with a numerical aperture (NA = 0.65) objective through the cellulose fibrils onto the gold-coated surface inside the OMM device, and reflection SR-FTIR measurements by the transflection mode over a mid-infrared wavenumber range of 4000 – 650 cm^-1 were made over time. Each real-time spectrum represents an average of 32 scans at a spectral resolution of 4 cm^-1 with an absorption peak position accuracy of 1/100 cm^-1.

To image the spatiotemporal dynamics of cellulose during enzymatic hydrolysis, we typically divided the entire view-field of the cellulose fibrils into equal-sized 5×5-µm squares before raster scanning, collecting full SR-FTIR spectra at each pixel. A 70 x 70 μm sample area was divided into 8x8 grid such spectra were collected at 64 points every ~ 10 minutes. Time-elapsed mapping of sample areas were conducted using in-house software written in the Python programming language (version 3.6). The master program controlled the movement of the microscope stage, spectra collection of FTIR spectrometer and data storage in the OMNIC software program (version 9.8, Thermo Fisher Scientific, Inc.) using the Dynamic Data Exchange (DDE) protocol. The source code of our python programs for controlling the microscope and FTIR bench is available upon request.