Measuring Mixing, Diffusion and Suspension Rheology to Enable Efficient High Solids Enzymatic Saccharification

Transcription

1 Measuring Mixing, Diffusion and Suspension Rheology to Enable Efficient High Solids Enzymatic Saccharification M.J. McCarthy, E.J.Tozzi, S.P. Shoemaker, D.M. Lavenson, R.L. Powell, and T. Jeoh University of California, Davis

2 Introduction Lignocellulosic Biomass Pretreatment Cellulases Hemicellulases Hydrolysis Sugars (Hexoses, Pentoses) Fermentation Micro organism (wild or recombinant) Separation Main Product Alcohols Acids Amino acids Industrial chemicals By-products (Fuel for boiler, Animal feed, Industrial chemicals)

3 Mass Transfer Limitations Above 20% insoluble solids (w/w) Decrease in rate of hydrolysis High solids inhibits diffusion of enzyme High solids inhibits mixing Rate becomes diffusion limited Expect higher rates at higher solids Hodge et al., (2008), Soluble and Insoluble Solids Contributions to High Solids Enzymatic Hydrolysis of Lignocellulose

4 Physical Mixing Mechanisms Intensity of Segregation Molecular diffusion Solid mixing Scale of Segregation

5 Yield Stress Mixing Issues Herschel Bulkley fluid CFD model Saeed et al, Ind. Eng. Chem. Res Pseudoplastic fluid CFD model Pakzad et al, Chem. Eng. And Proc. 2007

6 Mixing non-newtonian Fluids Need to use non-conventional methods Stretching and folding (chaotic advection) Split and recombine (SAR) mixer Generates striations/layers each mixing element

7 Key Design Issues for SAR Mixing quality is a function of the number of layers and characteristic diffusion time scales Energy requirement for pumping related to pressure drop Pressure drop a function of rheological behavior Mixing profiles C1, C2, C3, and C4 represent the concentrations distributions after 1, 2, 3 and 4 periods of mixing M.K. Singh et al. AIChE Journal, 55(9): , 2009.

8 Experimentation & Computational modeling

9 Mass transport challenges in high-solids enzymatic hydrolysis Well-mixed condition needed to proceed at acceptable rates. Homogenization dominated by slow convective flow and diffusion. Designing energy efficient mixing processes requires quantitative information on the rheology and diffusion properties of the materials.

10 Goals Measure effective diffusion coefficients in different types of cellulosic fiber beds Compare results with predictions using a model of diffusion-adsorption in porous media Determine the influence of adsorption properties on effective diffusivity

11 Industrial cellulose fibers studied C EZ 0.5 mm 0.5 mm Fiber type Length (LW) Length (NW) Width (LW) a) Solka Floc 200EZ mm mm 26.4 um b) Solka Floc C mm mm 31.7 um

12 Imaging System 1 Tesla permanent-magnet-based imaging spectrometer (Aspect Magnet Technologies Netanya, Israel).

13 One-dimensional diffusion At time T=0 bottom half of fiber bed contains MnCl 2

14 Evolution of concentration profiles Effective diffusivity computed by matching numerical (1-D Diffusion) and experimental concentration profiles C t = D eff 2 C 2 Z M. S. Olson, R. M. Ford,J.A.Smith,and E. J. Fernandez. Environ. Sci. Technol., 39: , 2005.

15 Adsorption

16 Simplified effective diffusivity model Effective diffusivity Porosity D eff εd = τ (1 + 0 R) Bulk diffusivity Tortuosity Adsorption constant P. B. Weisz. Trans. Faraday Soc., 63: , 1967.

17 Diffusivity Comparison Bulk Experiment Simplified model

18 Parameter Comparison C EZ 0.5 mm 0.5 mm Fibers C EZ K ads. (l/g) R = K ads. (l/g) * C s (g/l) Tortuosity factor Void fraction D eff exp. (m 2 /s) 4.39 x x10-11 D eff model. (m 2 /s) 3.06x x10-11

19 Enzymatic hydrolysis Before After Y. Lu et al., Appl. Biochem. Biotechnol., DOI: /s , 2008.

20 Rheology of Biomass Substrate Model Reference Pretreated corn stover Pretreated barley straw Power Law, H-B, Bingham, Casson Power Law N. V. Pimenova and T. R. Hanley, Appl. Biochem. Biotechol , 2003 L. Rosgaard, P. Andric, K. Dam-Johansen, S. Pedersen, and A. S. Meyer.. Appl. Biochem. Biotechnol., 143:27 40, 2007 Solka Floc H-B, Bingham, Casson B. Um and T. R. Hanley. Appl. Biochem. Biotechno., 145, 2008 Untreated & pretreated corn stover Pretreated corn stover Pretreated corn stover Casson S. Viamajala, J. D. McMillan, D. J. Schell, and R. T. Elander. Bioresource Technology, 100, Bingham M. R. Ehrhardt, T. O. Monz, T. W. Root, R. K. Connely, C. T. Scott, and D. J. Klingenberg. Appl. Biochem. Biotechnol., DOI /s z, Yield stress = f (Mass fraction) J. S. Knutsen and M. W. Liberatore. Journal of Rheology, 53(4), Corn stover undergoing hydrolysis Yield stress = f (Volume fraction) C. M. Roche, C. J. Dibble, J. S. Knutsen, J. J. Stickel, and M. W. Liberatore. Biotechnology and Bioengineering, DOI /bit.22381, 2009.

21 Rheology of Biomass Generalized Newtonian models predict pipe flows with continuous, symmetric velocity profiles When is this valid?

22 Flow imaging and Fiber Settling

23 Velocity Profiles: Short Fibers C = 0.05% (w/w) SolkaFloc 200EZ Length (LW) = mm Width (LW) = 26.4 um C = 2.01% (w/w)

24 Velocity Profiles: Short Fibers C = 13.7% (w/w) SolkaFloc 200EZ Length (LW) = mm Width (LW) = 26.4 um C = 16.4% (w/w)

25 Velocity Profiles: Medium Fibers C = 3.14% (w/w) Solka Floc C100 Length (LW) = mm Width (LW) = 31.7 um C = 7.05% (w/w)

26 Velocity Profiles: Long Fibers C = 0.47% (w/w) Wood pulp Length (LW) = mm Width (LW) = 22.5 um C = 1.01% (w/w)

27 Jeoh Lab Research Activities The structural components of the cell walls of plants are composed of the polysaccharides cellulose, hemicellulose and pectin. The complex matrix structure of the cell wall, the interactions of the polysaccharides within the structure and the chemical and structural complexities of the polysaccharides themselves are such that plant cell walls are highly resistant to being broken down. The mass fraction of the polysaccharides account for up to 60 80% of the dry weight of the plant. Access to the sugars that make up the polysaccharides can provide a renewable and sustainable resource for conversion to fuels and chemicals. In the Jeoh lab, we study the mechanisms by which enzymes produced in nature interact with and hydrolyze (breakdown) the cell wall polysaccharides to fermentable sugars. Our research ranges from fundamental mechanistic studies at the molecular scale through applied research into strategies to overcome saccharification limitations at high solids loadings. The three on-going projects (from fundamental through applied) are: Molecular-scale investigations to elucidate the mechanism of cellulose hydrolysis by cellulases Investigating the Effects of Water Interactions in Lignocellulosic Biomass on High Solids Enzymatic Saccharification Efficiency Using an encapsulation strategy to incorporate synergistic ratios of cellulolytic enzymes in high solids saccharification reactions Contact: Tina Jeoh Assistant Professor Biological and Agricultural Engineering UC Davis tjeoh@ucdavis.edu (530)

28 Molecular-scale investigations to elucidate the mechanism of cellulose hydrolysis by cellulases The mechanism of cellulose hydrolysis by cellulases has not been solved. The heterogeneous reaction requires productive binding of soluble cellulases to specific substrate reactive sites on the insoluble cellulose. One key piece of missing information to solve the mechanism is to define and measure S in the reaction mechanism shown below. E E f b + S k cat E k a kd f E b +αp Left: confocal image of cellulose microfibrils bound by fluorescence-labeled cellulases. Right: AFM height data on the same group of microfibrils. This project seeks to define and measure the reactive substrate by conducting experiments on the molecular scale. The approach taken is to integrate AFM/confocal microscopy and biochemical assays. The following 2 slides show some results. Jeoh Lab Research Activities

29 Microstructural changes in cellulose during hydrolysis Jeoh Lab Research Activities

30 Microstructural changes in cellulose during hydrolysis Jeoh Lab Research Activities

31 Investigating the Effects of Water Interactions in Lignocellulosic Biomass on High Solids Enzymatic Saccharification Efficiency Minimizing process water use and wastewater generation from the conversion of lignocellulosic material to biofuels/biocommodities is essential for the economics and sustainability of the overall process. However, increasing solids loading in the saccharification decreases saccharification rates (shown below): Saccharification, % % % 80.00% 60.00% 40.00% 20.00% 0.00% 0% 5% 10% 15% 20% 25% Solids Content, % 24 hours 72 hours 120 hours We hypothesize that one key reason for the decrease in saccharification efficiency is due to increasing diffusion resistance due to increasing constraint of the water at higher solids loadings. We are using NMR to study the effect of water interactions with the substrate on diffusion and saccharification. Jeoh Lab Research Activities

32 Investigating the Effects of Water Interactions in Lignocellulosic Biomass on High Solids Enzymatic Saccharification Efficiency T2 Relaxation Time, ms 25% 20% 15% 10% 5% 0% Solids Content, % Cellulose Only 1% Glucose 1% Mannose Saccharification, % 100% 80% 60% 40% 20% 0% Increasing solids loading resulted in increased constraint of the water in the system. The addition of solutes (1% glucose or 1% mannose) also increased constraint of the water in the system. The saccharification extent at 24 hours for a system with 5% solids is reduced in the presence of either 1% glucose or 1% mannose. Cellulolytic enzymes can be product inhibited (i.e glucose can inhibit cellulase activity). However, mannose is not known to inhibit cellulase activity per se (currently being confirmed in our experiments). Thus we conclude that the reduction in saccharification extent observed is likely not due simply to product inhibition. We speculate that saccharification reduction is due to diffusion limitations in the system due to water constraint. Jeoh Lab Research Activities

33 Using an encapsulation strategy to incorporate synergistic ratios of cellulolytic enzymes in high solids saccharification reactions In nature, a fungus deconstructs lignocellulosic biomass in high solids reactions by secreting optimal ratios of an array of cell wall degrading enzymes at the hyphae tip. In practice, the enzymes secreted by such fungi are collected in the supernatant and applied and mixed into a saccharification reaction. The enzymes are assumed to distribute on the biomass surface homogenously, and at the optimal ratio (as secreted by the fungus). if We are exploring a means to overcome this reliance on mass transfer to homogenously distribute and co-locate the synergistic enzymes throughout the reaction by first encapsulating the secreted enzyme product. Encapsulation serves as a means to deliver aliquots of the enzyme mixture, to protect the enzymes against shear during initial mixing, and delay the release of the enzymes in the reaction until after incorporation with the biomass. Jeoh Lab Research Activities

34 Using an encapsulation strategy to incorporate synergistic ratios of cellulolytic enzymes in high solids saccharification reactions exocellulase endocellulase xylanase esterases etc Cell Wall Degrading Enzymes in Solution Cellulolytic fungi express and secrete cell wall degrading enzymes of varying substrate specificities that synergistically breakdown plant cell walls. Encapsulation of Cell Wall Degrading Enzymes by Spray Drying Microliter-scale aliquots of the enzyme solution are encapsulated and stabilized by spray-drying. Co-location of Synergistic Ratios of Cell Wall Degrading Enzymes in High-Solids Saccharification Synergistic ratios of enzymes are co-located throughout the reaction, minimizing the reliance on mass transfer to maximize synergistic action. Jeoh Lab Research Activities

35 Summary Measured effective diffusivities consistent with simplified diffusion-adsorption model that accounts for system porosity, adsorption constant and tortuosity. Adsorption constant has a large effect on the diffusivity of dilute solutes. Highly adsorbing systems will diffuse slowly and require more intense convective mixing to achieve a desired level of homogeneity in a practical timescale. Newtonian, Non-Newtonian and asymmetric velocity profiles are observed for fiber suspensions. Features of velocity profiles explained by fibers sedimentation and entanglement Smaller fibers have more water-like behavior Important settling effect for medium fibers Long fibers tend to form networks at relatively low concentration Range of length scales being investigated range from molecular to macroscopic. Initial characterization of microstructural changes to cellulose due to hydrolysis by a cellobiohydrolase shows untwisting of microfibrils during the rapid hydrolysis phase and extensive thinning and formation of channels at high hydrolysis extents. Water is shown to be increasingly constrained with increasing solids loadings, as well as by the addition of solutes. Decrease in saccharification rates appear to be due at least in part to water constraints in the reaction system. Cell wall degrading enzymes have been encapsulated in a cross-linked alginate matrix by spray-drying. Studies are currently on-going to determine the efficacy of applying the spraydried enzymes for saccharification of pretreated switchgrass.