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Document Type

Dissertation - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)


Agricultural and Biosystems Engineering

First Advisor

K. Muthukumarappan


The current raw materials for the production of bioethanol are starch-based materials as well as sugar cane and molasses. However lignocellulosic biomass (e.g. agricultural residues and herbaceous grasses) hold a great potential to be used as carbohydrtae source for fermentation to ethanol. Between the two routes developed for hydrolysis of biomass to carbohydrate monomers, acid and enzymatic hydrolysis, the latter is favored. However, a foremost concern for economically efficient ethanol production from biomass through enzymatic hydrolysis pathway is the large volume and high cost of the Cellulolytic enzymes used in this process. Due to the recalcitrant nature of biomass a typical dose of 15 FPU/g glucan is used for enzymatic hydrolysis. This level of enzyme is equivalent to ~30 g enzyme/l ethanol produced, a costly dose (at an enzyme price of $10/kg) that cause the cellulolytic enzyme to account for 27-40% of the process cost when ethanol sold for ~$0.94/l. Thus, enzyme costs must either be reduced to lower than $2/kg protein or strategies developed to substantially reduce loadings (<5 FPU/g glucan) so that cellulolytic enzymes would be comparable with enzymes currently used in corn to ethanol industry. It has been shown that cellulolytic enzymes are deactivated due to a variety of reasons such as deformation due to thermal, shear effect, high surface tension of solution that would expose the enzyme to air-liquid interface and irreversible adsorption to lignin and crystalline cellulose that inhibit their catalytic action. In this dissertation, the potential role of novel protein-based micelles and surfactant or protein-based polymeric micelles (PMs) was tested on enzyme activity, hydrolysis, fermentation and enzyme recycling. And the potential mechanisms of action of these amphiphiles and their economical viability were inspected. Formation of irreversible-bound monolayers of casein, on hydrophobic surfaces was shown to alleviate the de-activation of protein of interest. Application of colloidal proteins of casein on the surface of SiO2 has been used to prevent from deactivation of kinesin. Also a variety of blocking buffers ranging from casein, milk or normal serum to highly purified proteins is commonly used to block the free sites on a microcellulose membrane on which the antibodies (proteins) has been transferred from a gel, to prevent nonspecific binding of the detection antigens (protein) during the subsequent steps. Casein, a phospho-protein, that makes the 80% of the bovine milk containing substructures of αs1-casein, β-casein, k-casein, αs2-casein was tested in this work for the first time as a lignin-blocker for biomass and potential cellulase stabilizer in hydrolysate. A variety of caseins blocking buffers, such as complete commercial casein, freeze-dried acid casein, gluten and lactose-free casein and liquid casein (containing 0.01-0.45% lactose) and ultrafiltered casein were applied to dilute acid, lime, alkali, extrusion and AFEX pretreated corn stover (3% solid loading) at above critical micelles concentration (CMC), 0.15-7.5 g/g glucan. Two strategies were used for application of these additives followed by enzymatic hydrolysis with 25, 35, 50 FPU/g glucan cellulase and 2.5 CBU:FPU β-glucosidase. Application of 0.5 g/g glucan of glucose/lactose-free casein was found to effectively increase the glucose yield of corn stover pretreated with dilute acid, lime, alkali, extrusion and AFEX by 31.9%, 17.0%, 22.7%, 29.5%, and 17.4%, respectively with no positive impact on Avicel. Consequently 96 h simultaneous saccharification and fermentation (SSF) of these hydrolysates with native S. cereviseae reduced the fermentation period by up to 48 h and increased the theoretical yield of ethanol by 8.48– 33.7% compared to control. It is suspected that lignin play a major role in cellulase irreversible adsorption and consequently deactivation specially at extended incubation time. Hence the majority of enzyme in hydrolysate from Avicel (lacking lignin) can be recycled and re-used successfully, resulting in release of the 74% of the first cycle glucose in the second cycle. However in control, the recycled hydrolysate from corn stover (20.2% lignin) resulted in 29% of the sugar yield that was obtained in the first cycle, a drastic reduction that was improved to 36% when casein was used as additive. To understand the potential mechanism of action behind casein, we have utilized analytical tools such as scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays to evaluate the adsorption of commercial cellulase and casein polypeptides to lime, alkali, dilute acid, extrusion and AFEX pretreated corn stover. As a result, the adsorption of casein to biomass was observed with all of the analytical techniques utilized and varied depending on the pretreatment techniques of biomass. It is rational to expect that casein molecules interweave into biomass particles based on the surface area, compactness of the biomass particles, exposed functional groups of biomass and many other factors. Kjeldahl and BSA analyses suggested that 45-68.4% of casein was adsorbed to corn stover after 24 h incubation at 50°C that was irreversible and increased to >90% after 72 h. The qualitative analysis (SEM, FT-IR) of the same samples confirmed the adsorption of casein on biomass. Analysis of liquid phase of corn stover hydrolysate showed that with no additive, the majority of one of the cellulase monocomponent, 97.1 ± 1.1, was adsorbed to corn stover within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein blocking buffer reduced the adsorption of the same mono-component of cellulase to 32.9% ± 6.0 and 82.8% ± 6.0, respectively. It was concluded that casein may improve the enzyme activity through several mechanisms of action. The hydrated layer(s) formed on biomass through interaction between hydrophobic parts of the lignin (e.g. phenyl, methy groups) and CH2 groups of casein, act as a steric hindrance blocking the cellulase from unproductively binding to lignin. Also super activity of cellulase at the pseudo-phase of the casein micelles-liquid phase may occur due to activation of certain amino acids in cellualse or the re-formation of enzyme sub-structure. Moreover the reduction in surface tension of the enzymecontaining liquid through the loose network of casein proteins in solution can reduce the enzyme deactivation by air-liquid interface. We also developed a new sequential technique for pretreatment of corn stover and prairie cordgrass, which utilized an initial extrusion pretreatment (155 or 65 rpm screw speed and temperatures of 70–180°C or 90-180°C corresponding to feed, barrel and die zones for prairie cordgrass and corn stover, respectively) followed by treatment with poly ethylene glycol 6000 (PEG) or Tween 20. In order to fully characterize the response for sugar yield over the range of surfactant treatment conditions assessed, response surface methodology was used. Treatment temperature, incubation time and PEG or Tween concentration were varied between 45-55°C, 1-4 h, 0.15-0.6 g additive/g glucan, respectively. Statistical analysis was conducted by fitting the glucose and xylose yields to a quadratic polynomial model. PEG, Tween concentration and temperature were found to be the most significant factors in surfactant treatment with time of incubation having no significant impact on product yield. The optimum condition found resulted in improvement in glucose and xylose recovery by up to 27.5% and 33%, respectively in corn stover and prairie cord grass. Improved enzyme specific activity between 10.5- 18.5%, increased maximum forward velocity (vm) and decreased irreversible adsorption of cellulase to non-productive sites of biomass evaluated by nitrogen combustion technique and affinity constant (km) are some of the potential reasons behind the induced cellulase activity. Later we utilized a newer approach for the improvement in conversion of biomass. Previously it was found that polymeric micelles (PMs) of polymer-surfactants retain unique properties. This includes improved interfacial properties such as increased solubilization of colloidal carriers (e.g. surfactant or protein micelles) or improved rheological properties of the solution (reduced surface tension) and reduced hydrophobicity of micelles (highly hydrophile groups of ethylene oxide (EO) in PEG shown to reduce the hydrophobicity of reverse micelles). We found that enzymatic hydrolysis of corn stover (CS) in the presence of polymeric-surfactant micelles (PMs) improved saccharification yield to a greater extent than using only surfactant micelles. Application of 0.06 g/g glucan of Poly ethylene glycol (PEG) with casein, Tween 20 and Triton X-100 at above of the critical micelles concentrations (CMC) improved the glucose yield of CS containing high bound-lignin (extrusion pretreated) by up to 70.7%, 10.6%, 2.2% above that obtained with only micelles of amphiphiles, respectively. These PMs were not effective for enzymatic hydrolysis of biomass lacking lignin or alkali pretreated CS (7.2% lignin). Main reason for the enhanced cellulase activity observed due to PEG-casein, PEG-Tween and PEG-Triton was found to be potentially due to the enhanced cellulase availability in liquid phase (PEG-casein), reformation of α-helix substructure (PEG-Tween), and combination of induced cellulase solubilization, α-helix reformation and chemical changes in micro-structure of biomass (PEG-Triton). Then the efficacy of enzyme recycling in simultaneous (SSF) and separate (SHF) hydrolysis and fermentation of corn stover was evaluated with and without the use of novel PMs of PEG-casein and PEG-Tween 20. These stabilizers were added to maximize the percentage of enzyme remaining in fermented liquor that could be recycled twice back into the system with new biomass. Application of PMs of PEG-Casein in one cycle of SHF significantly improved the theoretical ethanol yield from 0.48±0.00 to 0.91±0.00 compared to when only casein (0.66±0.00), Tween 20 (0.52±0.00) and Tween 20-PEG (0.77±0.08) were applied. Combination of PEG was most effective with commercial casein for which increased the enzyme recycling by 127% and 150% compared to only casein treated and control (no additives) sample, respectively. Also neither Tween 20 nor PEG showed toxicity impact on yeasts used in this study. Our economical evaluation showed that if casein or Tween, or PMs are going to be used as additives in biomass to ethanol industry, their dose of application and cost of enzyme plays a determining role in their viability. A permissible cost of ¢3.1/kg and ¢3.4/kg for additives compared to their market price of ~$4-6/kg could not justify the economically use of Tween at 0.47 g/g and Casein at 2.5 g/g glucan during SHF of corn stover. Even reduction of the Tween and casein utilization to 0.1 g/g glucan would potentially yield in a permissible cost of ¢16.0/kg and ¢78.0/kg, respectively. In order for the casein application to be justifiable as additives, a recycling strategy or addition of 0.06 g/g glucan PEG would be necessary to increase the permissible cost by up to $2.5/kg. However, even with these strategies, the permissible cost of Tween 20 was only $1.3/kg which wouldn’t permit the application of this additive as enzyme stabilizer. As a result of the sensitivity analysis it was found that, the permissible cost of the additive is increased with the increase in cost of enzyme. The extent of improvement in ethanol yield obtained and the level of additives used, along with the price of enzyme were the determinative factors in permissible cost of additive.

Library of Congress Subject Headings

Ethanol fuel industry


Includes bibliographical references (pages 289-314)



Number of Pages



South Dakota State University


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