Impact of Recycling Stillage on Conversion of Dilute Sulfuric Acid Pretreated Corn Stover to Ethanol

Ali Mohagheghi and Daniel J. Schell

National Renewable Energy Laboratory, Golden, CO 80401

Objective

A national laboratory of the U.S. Department of Energy

Office of Energy Efficiency & Renewable Energy

Operated by the Alliance for Sustainable Energy, LLC

Introduction Objective

Materials and Methods

Assess impact of process water recycle on process performance

Goal: Achieve 25% process water recycle 

with no negative impact on process performance 

Process Configuration

Conditioning Method: Overliming (OL) Process

•The amount and the nature of inhibiting compounds depends on:

 Raw material

 Prehydrolysis & enzymatic hydrolysis procedure

 Extent of recirculation

•Large quantities of water needed in biomass-to-ethanol process (Approx. 6 Gal /Gal of EtOH produced)

•Recycle waste water reduces fresh water requirements and downstream treatment needs

•Consequence of recycling is accumulation of non-metabolizable compounds that may inhibit fermentation

•Knowledge of inhibitors and how to minimize their effect on fermentation performance is key

Introduction

Hydrolysate: Overlimed pretreated corn stover liquor

Separation

Mixing (50°C, pH 10)

Mixing

Pretreated 

Feedstock

Ca(OH)2

H2SO4

Liquor

Conditioned Liquor

Solids

Filtration Gypsum

Conditioning Method: Overliming (OL) Process Pretreatment Solid/Liq.

Separation Conditioning

Fermentation

Distillation Solid/Liq.

Separation

Feed

Solids

Slurry

Solids

Stillage

Nutrient/Inoculum

Overlimed

Hydrolysate

Ethanol

Fresh Hydrolysate + Other

Experimental Procedure

Distillation: In rotary evaporator, replacing evaporation with

DI water afterward

Recycle ratio (RR): 10% and 25%, defined as the amount of 

stillage water recycled

Q lit C t l F t ti d i t i li t ith

•Pretreated at 30.0% total solids (TS) loading (34.2% after flash step)

•Adjusted to 15% to 20% TS loading

•Initial component concentrations (g/L): Glucose: 33.6, Xylose: 81.3, 

Acetic acid: 16.1, HMF: 3.3, and Furfural: 4.1

•OL Liquor supplemented with glucose to 55 and 74 g/L, respectively 

at 15% and 20% TS 

Strain: Zymomonas mobilis 8b 

Growth Media: Rich Medium (RM) (10 g/L Yeast Extract +

2 g/L KH2PO4) plus hydrolysate

Fermentation: BioStatQ with 340 mL working volume,

pH = 5.8, Temperature = 35°C, Initial OD = 1 @600nm Recycled Water

Fresh Water

Analytical

•Sugar concentrations measured by HPLC using the Shodex SP0810 

carbohydrate column (55oC, 0.6 mL/min, water mobile phase, RI detection) 

and ethanol concentration measured using BioRad HPX-85

•Liquor densities measured using an Anton-Paar densitometer

Rotary 

Vacuum 

Flask Ethanol + 

Water Water Discard

Fresh Hydrolysate + Other

Media + Water

Bioreactor

Baseline 1st Recycle 2ndRecycle 3rd Recycle

Triplicate runs performed at 

each condition

Quality Control: Fermentations done in triplicate, with pure 

sugar control

Procedure: Three recycles were done at each condition to 

achieve a steady state concentration of acetic acid

Total of 12 sets of fermentation performed, each set included 

4 fermentation

For each Condition:

•Total of 4 Fermentors; Three with recycling of stillage at one condition in 

triplicate. One for pure sugar control with Acetate 

•A shake flask control to check for culture viability

•Initial OD =1 @600nm

•Initial OD =4 @600nm to evaluate initial cell concentration

Experimental Setup

Summary of the Results

•Acetate inhibition of Zymomonas:

– At lower pH is major problem

– At higher pH as acetate concentration builds up inhibition becomes an 

issue

•Other inhibitors may become an issue

Issues:

2-factor, 2-level factorial design

3 to 4 iterations required to achieve 

steady state concentrations

Experimental Design

Recycle Rate

15%

20%

10% 25%

Performance Results

• With 15% Solids loading & 25% RR there was little impact

– Ethanol Yield: 75%

– Glucose & Xylose Utilization: 95% & 60%

With 20% S lid l di & 10% RR i t ti d

Results

6

7

8

9

10

11

12

13

14

Acetic Acid Concentration (g/L)

Hyd./15%/10%

Hyd./15%/25%

Hyd./20%/10%

Hyd./20%/25%

PS/15%/10%

PS/15%/25%

PS/20%/10%

PS/20%/25%

Legend:

Feed/Solids/RR

Achieving Steady State

0

10

20

30

40

50

60

70

80

90

100

Yield (%) or Conversion (%)

PS + Acetate

Hyd., Baseline

Hyd., 3rd Recycle

15% Solids

10% RR

20% Solids

25% RR

20% Solids

10% RR

15% Solids

25% RR

Ethanol Yield

Glucose Conversion

Xylose Conversion

Performance Results • With 20% Solids loading & 10% RR some impact was noticed

– Ethanol Yield: 65%

– Glucose & Xylose Utilization: 85% & 37% 

• With 20% Solids loading & 25% RR, impact was very significant

– Ethanol Yield: 10%

• High acetate at 20% solids level in pure sugar did not affect performance

• Under 20% solids & 25% RR conditions in pure sugar,

– Ethanol yield: 75%

– % Gluc & Xyl utilization: 95% & 50%

• Increasing initial cell mass improved pure sugar control but not in hydrolysate 

process

– Confirms other inhibitors besides acetic acid have effect on the process

Conclusions Acknowledgements

Funding for this work was provided by the Office of Biomass Program 

in the Department of Energy’s Office of Energy Efficiency and 

Renewable Energy

We wish to thank Jody Farmer, Bob Lyons, and Wes Hjelm for performing 

the pilot scale pretreatment that produced the hydrolysate used in this study. 

The information contained in this poster is 

subject to a government license.

Baseline 1st Recycle 2nd Recycle 3rd Recycle Confirms other inhibitors besides acetic acid have effect on the process

•Higher yield and sugar utilization in pure sugar compared to hydrolysate shows:

•Other inhibitors besides acetic acid are producing negative impact on the 

process

•With 20% Solids loading and 25% RR, impact was very significant

•Ethanol Yield: 10%

Recent Advances in Fermentation Technology VIII • San Diego, CA • November 8-11, 2009 • NREL/PO-510-47183

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.