Impact of Amphiphiles in Cellulase Stabilization, Recycling and Ethanol Production from Pretreated Biomass

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$0.94/l. Thus, enzyme costs must either be reduced to lower than $2/kg protein or strategies developed to substantially reduce loadings (\u3c5 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$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

A Review of the Role of Amphiphiles in Biomass to Ethanol Conversion

One of the concerns for economical production of ethanol from biomass is the large volume and high cost of the cellulolytic enzymes used to convert biomass into fermentable sugars. The presence of acetyl groups in hemicellulose and lignin in plant cell walls reduces accessibility of biomass to the enzymes and makes conversion a slow process. In addition to low enzyme accessibility, a rapid deactivation of cellulases during biomass hydrolysis can be another factor contributing to the low sugar recovery. As of now, the economical reduction in lignin content of the biomass is considered a bottleneck, and raises issues for several reasons. The presence of lignin in biomass reduces the swelling of cellulose fibrils and accessibility of enzyme to carbohydrate polymers. It also causes an irreversible adsorption of the cellulolytic enzymes that prevents effective enzyme activity and recycling. Amphiphiles, such as surfactants and proteins have been found to improve enzyme activity by several mechanisms of action that are not yet fully understood. Reduction in irreversible adsorption of enzyme to non-specific sites, reduction in viscosity of liquid and surface tension and consequently reduced contact of enzyme with air-liquid interface, and modifications in biomass chemical structure are some of the benefits derived from surface active molecules. Application of some of these amphiphiles could potentially reduce the capital and operating costs of bioethanol production by reducing fermentation time and the amount of enzyme used for saccharification of biomass. In this review article, the benefit of applying amphiphiles at various stages of ethanol production (i.e., pretreatment, hydrolysis and hydrolysis-fermentation) is reviewed and the proposed mechanisms of actions are described

Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively