Master of Science in Biological and Agricultural Engineering (MSBAE)


Biological and Agricultural Engineering

Document Type



Advances in enzymatic hydrolysis have developed new methods for conversion of lignocellulosic biomass into fermentable sugars for various applications, mainly ethanol production. The present study involves immobilization of a cellulase enzyme complex on a solid support which can be recovered for subsequent use in multiple reactions. The supports of interest include Fe3O4 nanoparticles (~13 nm) and polystyrene-coated particles containing a Fe3O4 core (1-2 µm). Each support contains amine functional groups based on the surface that allow covalent attachment of enzymes via carbodiimide activation. The nanoparticles were characterized using transmission electron microscopy (TEM) and immobilization was confirmed using Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The nanoparticles were successfully recycled 6 times and the polystyrene-coated microparticles 4 times before their corresponding activity levels had fallen below 10%. Activity was determined using a dinitrosalicylic acid (DNS) assay, which detected the total reducing sugars present. Sugar production was confirmed by high performance liquid chromatography (HPLC) with the highest concentration of sugars detected as glucose with minimal amounts of xylose and cellobiose also present. Activity retention was 30.2% of the free enzymes activity following immobilization on the magnetite nanoparticles and 26.5% after immobilization on the polystyrene-coated particles. A performance evaluation over all recycles indicated that 78% of the free enzyme sugars were produced by magnetite nanoparticles and 42% produced by polystyrene-coated particles following 96 hours of hydrolysis. Further characterization of the magnetite nanoparticles revealed that maximum protein attachment (~90%) occurred at low enzyme loadings (1-2 mg). The enzyme-to-support saturation point occurred at a weight ratio of 0.02. Thermal measurements for the nanoparticles indicated increased stability over a broader range of temperatures with a peak temperature of 50˚C. Ionic forces between the enzyme and support surface caused a shift in pH from 4.0 to 5.0, and stability was assessed over 72 hours of hydrolysis with the free enzyme losing 49% +/- 0.05% of its activity while the activity of the immobilized enzyme complex had dropped by only 42% +/- 0.53%. An activity comparison was assessed to compare the performance of Fe3O4 nanoparticles and polymeric microparticles, noting the advantages and disadvantages of each.



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Committee Chair

Theegala, Chandra S.



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Engineering Commons