Roller bottle reactors (RBR) are loaded by mass to half of the volume

Christine M. Roche* and Jonathan J. StickelAbstract Enzymatic Saccharification Shake Flasks VS Roller Bottles

C diti Ob tiThe ability to screen new biomass pretreatments and advanced

High‐Solids Enzymatic Saccharification Screening Method for Lignocellulosic Biomass

Operated by the Alliance for Sustainable Energy, LLC

Roller bottle reactors (RBR) are loaded by mass to half of the volumeof the vessel, assuming a density of 1g/ml. RBRs are charged withwashed pretreated biomass, buffer and anti‐microbial agent, andthen brought to temperature while mixing before adding enzyme.Enzyme is distributed on the surface of the biomass slurry across thelength of the vessel. Mixing is achieved through gravitationaltumbling by rotating the RBRs horizontally on a Wheaton MiniRoller Apparatus. Temperature control is achieved by housing theroller apparatus in a general purpose floor‐standing incubator.

Experimental Conditions:• 15, 20, or 30% insoluble solids (IS) loading (mass basis)

• 5, 10, 15, 20, or 30 mg protein/ g cellulose enzyme loading

• Genencor GC 220 cellulase (Lot # 4900759448, 220 mg protein/mL) or Genencor Spezyme CP cellulase (Lot# 301 05021 011 134

Conditions:

• High severity PCS

• GC220 (20 mg/g cellulose)

• Shake Flasks – 130 rpm• Roller Bottles – 2 rpm

Observations:

• No material movement in 20% or 30%

IS Shake Flasks while shaking

• 30% IS Shake Flask never fully wet

• 30% IS Roller Bottle fully wet in 4 hr

The ability to screen new biomass pretreatments and advancedenzyme systems at process‐relevant conditions is key to developingeconomically viable lignocellulosic ethanol. While much research isbeing invested in developing pretreatment technologies and enzymesystems that will more efficiently convert cellulosic biomass tosugars, the current standard reactor vessel, a shake flask, that is usedfor screening enzymatic saccharification of cellulosic biomass isinadequate at high‐solids conditions. Shake flasks do not provideadequate mixing at high solids conditions. In this work, a rollerbottle reactor was identified as a small‐scale high‐solidssaccharification reaction vessel, and a method was developed foruse in screening both pretreated biomass and enzyme systems atprocess‐relevant conditions. This new method addresses mixingissues observed in high‐solids saccharifications. In addition, yield

mL) or Genencor Spezyme CP cellulase (Lot# 301‐05021‐011, 134mg protein/ mL)

• pH – 4.8

• 10 μg/mL tetracycline

• Temperature ‐ 48 ̊C• 2 – 4 rpm

• 7 days

Yield Calculations

c fi: mass fraction [g i/g slurry]

• Concentrations are converted from g/L to g/g slurry

Note: Constant uncertainty was assumed for each experimental condition over theentire enzymatic saccharification process. Error bars are for a 95% confidence interval.

• Roller Bottles give 2.4 times the total biomass conversion asShake Flasks at 30% insoluble solids loadings

calculations from sugar concentrations on a mass basis were usedto account for the two‐phase nature of the saccharification slurry,which eliminates discontinuities in comparing high‐solids to low‐solids saccharifications that occur when using concentrations on avolume basis. The roller bottle reactors out‐performed the shakeflasks by 5% for an initial insoluble solids loading of 15% and 140%for an initial soluble solids loading of 30%. The reactor system andmethod was compared at bench and floor scales and determined tobe scalable for initial insoluble solids loading in the range of 15% to30%. Pretreatment and enzyme screening results indicate that midseverity pretreated biomass is more digestible than the low and highseverity biomass and GC220 is a superior enzyme to Spezyme CP.

)1( isl

ii f

cf 





fi g g yci: liquid concentration [g i/L liquid]ρl: liquid density [g/L]fis: [g insoluble solids/g slurry]







 M

j jis

N

i iij

xf

fr

1 0,0,

1 /

rj/i : MW ratio of polysaccharide component j to mono‐ or oligosaccharide i

Δfi: increase in i over timexj: mass fraction [g j/g insoluble solids]

General Conversion Equation

• Yields are calculated based on maximum theoretical yield

G: glucan

Cellulose Conversion

// cbcbGggG frfr 

Introduction

Ethanol produced from lignocellulosic biomass has been noted as aclean and renewable domestic source of energy that can feasiblydisplace a significant fraction of petroleum usage in the UnitedStates.1 However, significant challenges exist with breaking downbiomass to fermentable sugars Research indicates that a chemical

Pretreatment

LignocellulosicBiomass

Enzymatic Hydrolysis

FermentationOf Sugars

Ethanol Recovery

Pretreatment and Enzyme Screening

Conditions:

• 20% IS loading

• GC220 (20 mg/g cellulose)

• Roller Bottles – 125 mL; 4 rpm

Conditions:

• 20% IS loading high severity PCS

• GC220 and Spezyme CP

• Roller Bottles – 125 mL; 4 rpm

• Shake Flasks exhibit lesser efficiency and greater variability inconverting biomass at all insoluble solids loadings

• Glucose concentrations were observed as high as 170 g/L at 30%insoluble solids loading

g: glucosecb: cellobioseX: xylanx: xylose0:  initial condition

)( 0,0,0,

///

XGis

xxXcbcbGggGTB xxf

frfrfr





Total Biomass Conversion

0,0,

//

Gis

cbcbGggG

C xf

ffbiomass to fermentable sugars. Research indicates that a chemical

pretreatment followed by enzymatic hydrolysis increases the overallefficiency of saccharification.2,3 One promising approach toimproving process economics is to increase biomass concentrationduring pretreatment and enzymatic hydrolysis, thereby resulting inhigher product concentrations. In order to exploit the synergism ofpretreatment and enzymatic hydrolysis at process‐relevantconditions, it is necessary to be able to effectively screen pretreatedbiomass and enzyme preparations at high‐solids loadings in small‐scale reactors.

Biomass Substrate – Dilute Acid PretreatmentSteam Digester: 4‐L steam explosion batch reactor

d /d l l l

Scalability

Conditions:

• 20% IS loading high severity PCS

• Spezyme CP (20 mg/g cellulose)

• Mid severity pretreated corn stover has the highest digestibilityof the three pretreatment severities for given conditions tested

• GC220 out‐performs Spezyme CP. At all loadings tested After 7days of hydrolysis with loadings of 20% IS and 20 mg/g

Note: Uncertainty error bars of 95% confidence interval are included but typically are not larger than the data point size.

Summary• Roller Bottles perform significantly better than traditionalShake Flasks as enzymatic saccharification pretreatment andenzyme screening reaction vessels, likely due to more effectivemixing by horizontal tumbling than shaking

• Roller Bottle vessels are scalable between bench and floorscales and for insoluble solids loadings between 15% and 30%

• High‐solids enzymatic saccharification screening method can beapplied to a variety of biomass feedstocks and enzymes

Low Severity

• 33A14 Corn Stover •1⁄4 inch milled

• Steam Digester • 7.25 mg H2SO4/       g dry biomass

Mid Severity

• 33A14 Corn Stover •1⁄4 inch milled

• Steam Digester • 7.25 mg H2SO4/       g dry biomass

High Severity

• 34M95 Corn Stover •3⁄4 inch milled

• Sunds• 48 mg H2SO4/           g dry biomass

Sunds Reactor : Continuous 1 ton/day pilot‐scale vertical reactorp y ( g/g )

• Roller Bottles – Size Rotation

• Enzymatic saccharification reactions do not appreciably differfor conditions tested

• All conditions reached about 70% of theoretical total biomassconversion after one week

Conditions:

• High severity PCS

• GC220 (20 mg/g cellulose)

cellulose enzyme, GC220 converts 81% cellulose while SpezymeCP converts 68%

125 mL 4 rpm250 mL 2 rpm2000 mL 2‐20 rpm

2 mm

y y

• Using this high‐solids enzymatic saccharification screeningmethod, the mid severity pretreatment and GC220 enzyme wereidentified as top performers of the conditions tested

g y

• 150°C – 30 min

• Composition (wt/wt):

g y

• 170°C – 30 min

• Composition (wt/wt):

• 190°C – 1 min

• Composition (wt/wt):

References1. Jørgensen et al. (2007) Biofuels, Bioprod Biorefin, 1(2):119‐1342. Mosier et al. (2005) Bioresour Technol, 96(6), 673‐6863. Rosgaard et al. (2007) Biochem Biotechnol, 143(3), 284‐296 

Note: Uncertainty error bars of 95% confidence interval are included but are not larger than the data point size

• Roller Bottles – Size Rotation

• No significant difference in conversion after one week betweenroller bottle sizes (125 mL to 250 mL) at insoluble solidsloadings ranging from 15% to 30%.

AKNOWLEDGEMENTS: This work is supported by the U.S. Department ofEnergy (DOE), The Office of the Biomass Program. We would like to thankour NREL colleagues Gary McMillen for his help with enzymaticsaccharifications and Clare Dibble for her assistance in imaging the PCS.

NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC. 

125 mL 4 rpm250 mL 2 rpm

The information contained in this poster is subject to a government license.31st Symposium on Biotechnology for Fuels and ChemicalsMay 2‐6, 2009 in San Francisco, CA • NREL/PO‐510‐45751

- Slide Number 1