measuring mixing, diffusion and suspension rheology to...

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, [email protected]

IntroductionLignocellulosic

Biomass

HydrolysisCellulasesHemicellulases

Sugars (Hexoses, Pentoses)

Fermentation Micro organism (wild or recombinant)

Separation Main Product

By-products

Pretreatment

•Alcohols

•Acids

•Amino acids

•Industrial chemicals

(Fuel for boiler, Animal feed, Industrial chemicals)

Mass Transfer Limitations

Above 20% insoluble solids (w/w)

Decrease in rate of hydrolysis High solids inhibits diffusion of enzymeHigh solids inhibits mixing

Rate becomes diffusion limitedExpect higher rates at higher solids

Hodge et al., (2008), “Soluble and Insoluble Solids Contributions to High‐Solids Enzymatic Hydrolysis of Lignocellulose”

Physical Mixing Mechanisms

Scale of Segregation

Inte

nsity

of S

egre

gatio

n

Solid mixing

Mol

ecul

ar d

iffus

ion

Yield Stress Mixing Issues

Herschel‐Bulkley fluid CFD modelSaeed et al, Ind. Eng. Chem. Res. 2008

Pseudoplastic fluid CFD modelPakzad et al, Chem. Eng. And Proc. 2007

Mixing non-Newtonian Fluids

Need to use non-conventional methodsStretching and folding (chaotic advection)

Split and recombine (SAR) mixerGenerates striations/layers each mixing element

Key Design Issues for SARMixing quality is a function of the number of layers and characteristic diffusion time scalesEnergy 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):2208-2216, 2009.

Experimentation & Computational modeling

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.

Mass transport challenges in high-solids enzymatic hydrolysis

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

Goals

Industrial cellulose fibers studied

Fiber type Length (LW) Length (NW) Width (LW)

a) Solka Floc 200EZ 0.207 mm 0.183 mm 26.4 um

b) Solka Floc C100 0.349 mm 0.273 mm 31.7 um

200EZC100

200EZ200EZC100

0.5 mm0.5 mm 0.5 mm0.5 mm

Imaging System

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

One-dimensional diffusion

At time T=0 bottom half of fiber bed contains MnCl2

Evolution of concentration profiles

Effective diffusivity computed by matchingnumerical (1-D Diffusion) and experimental concentration profiles

M. S. Olson, R. M.Ford, J. A. Smith, andE. J. Fernandez.Environ. Sci. Technol.,39:149-154, 2005.

2

2

eff ZCD

tC

∂∂

=∂∂

Adsorption

Simplified effective diffusivity model

)1(0

eff RDD+

=τε

Effective diffusivity

Bulk diffusivity

Tortuosity Adsorptionconstant

Porosity

P. B. Weisz. Trans. Faraday Soc., 63:1801-1805, 1967.

Diffusivity Comparison

Bulk

Simplified model

Experiment

Parameter Comparison

Fibers C100 200EZKads. (l/g) 2.710 0.197R = Kads. (l/g) * Cs (g/l) 360.84 35.23Tortuosity factor 1.04 1.06Void fraction 0.92 0.89Deff exp. (m2/s) 4.39 x10-12 4.37x10-11

Deff model. (m2/s) 3.06x10-12 2.91x10-11

200EZC100

200EZ200EZC100

0.5 mm0.5 mm 0.5 mm0.5 mm

Enzymatic hydrolysis

Y. Lu et al., Appl. Biochem. Biotechnol., DOI:10.1007/s12010-008-8306-0, 2008.

Before After

Rheology of BiomassSubstrate Model Reference

Pretreated corn stover

Power Law, H-B, Bingham, Casson

N. V. Pimenova and T. R. Hanley, Appl. Biochem. Biotechol. 105-108, 2003

Pretreated barley straw

Power Law 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

Casson S. Viamajala, J. D. McMillan, D. J. Schell, and R. T. Elander. Bioresource Technology, 100, 2009.


Bingham M. R. Ehrhardt, T. O. Monz, T. W. Root, R. K. Connely, C. T. Scott, and D. J. Klingenberg. Appl. Biochem. Biotechnol., DOI 10.1007/s12010-009-8606-z, 2009.


Yield stress = f (Mass fraction)

J. S. Knutsen and M. W. Liberatore. Journal of Rheology, 53(4), 2009.

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 10.1002/bit.22381, 2009.

Rheology of Biomass

Generalized Newtonian models predict pipe flows with continuous,

symmetric velocity profiles

When is this valid?

Flow imaging and Fiber Settling

Velocity Profiles: Short Fibers

C = 2.01% (w/w)

C = 0.05% (w/w)

SolkaFloc 200EZ

Length (LW) = 0.207 mm

Width (LW) = 26.4 um

Velocity Profiles: Short Fibers

C = 16.4% (w/w)

C = 13.7% (w/w)

SolkaFloc 200EZ



Velocity Profiles: Medium Fibers

C = 7.05% (w/w)

C = 3.14% (w/w)

Solka Floc C100



Velocity Profiles: Long Fibers

C = 0.47% (w/w)

C = 1.01% (w/w)

Wood pulp



Jeoh Lab Research ActivitiesThe 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 cellulasesInvestigating the Effects of Water Interactions in Lignocellulosic Biomass on High Solids Enzymatic Saccharification EfficiencyUsing an encapsulation strategy to incorporate synergistic ratios of cellulolytic enzymes in high solids saccharification reactions

Contact: Tina JeohAssistant ProfessorBiological and Agricultural EngineeringUC [email protected](530)752-1020

mailto:[email protected]

Molecular-scale investigations to elucidate the mechanism of cellulose hydrolysis by cellulases

PEE

ESE

f

k

b

b

k

kf

cat

a

d

α+

+

→

↔

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.

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.

Left: confocal image of cellulose microfibrils bound by fluorescence-labeled cellulases. Right: AFM height data on the same group of microfibrils.

Jeoh Lab Research Activities

Microstructural changes in cellulose during hydrolysis


Investigating the Effects of Water Interactions in Lignocellulosic Biomass on High Solids Enzymatic Saccharification Efficiency

0.00%20.00%40.00%60.00%80.00%

100.00%120.00%

0% 5% 10% 15% 20% 25%Sacc

harif

icat

ion,

%

Solids Content, %

24 hours 72 hours 120 hours

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):

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.


Investigating the Effects of Water Interactions in Lignocellulosic Biomass on High Solids Enzymatic Saccharification Efficiency

0%

5%

10%

15%

20%

25%

0.00 200.00 400.00 600.00 800.00

T2 R

elax

atio

n Ti

me,

ms

Solids Content, %

Cellulose Only 1% Glucose 1% Mannose

0%20%40%60%80%

100%

Sacc

harif

icat

ion,

%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.


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).

http://www.biomatnet.org/secure/images/f0805a.gif

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.


Using an encapsulation strategy to incorporate synergistic ratios of cellulolytic enzymes in high solids saccharification reactions

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.

Cell Wall Degrading Enzymes in SolutionCellulolytic fungi express and secrete cell wall degrading enzymes of varying

substrate specificities that synergistically breakdown plant cell walls.

endocellulaseexocellulase

xylanaseesterases

etc

Encapsulation of Cell Wall Degrading Enzymes by Spray Drying

Microliter-scale aliquots of the enzyme solution are encapsulated and stabilized by spray-drying.


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 spray-dried enzymes for saccharification of pretreated switchgrass.

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