ETHANOL PRODUCTION FROM CORN

STOVER HEMICELLULOSIC
HYDROLYSATE USING IMMOBILIZED
RECOMBINANT YEAST CELLS
BY: JING ZHAO AND LIMING XIA

ERIC R. HALABASO
CHE326

OVERVIEW

INTRODUCTION
MATERIALS AND
METHODS
RESULTS AND
DISCUSSION
CONCLUSIONS

OVERVIEW

INTRODUCTION
MATERIALS AND
METHODS
RESULTS AND
DISCUSSION
CONCLUSIONS

FOSSIL FUELS VS. RENEWABLE

ENERGY
FOSSIL
FUELS
Finite natural resources
Caused serious global climate
and environmental problems

FOSSIL FUELS VS. RENEWABLE

ENERGY
Renewable
Energy
Clean
Safe
Reduce Fossil Fuels Production
BIOETHAN
OL

BIOETHANOL RAW MATERIALS

Sugarcane
Presently used as raw material
for commercial bio-ethanol
Compete against the limited
agricultural land needed

BIOETHANOL RAW MATERIALS

Lignocellulosic
Materials
Attractive renewable sources
Great availability
Relatively low cost
Corn
Stover

BIOETHANOL RAW MATERIALS

Corn Stover
Low commercial valueagricultural residue
Most favored lignocellulosic
resource for large scale ethanol
production
Lowers production
cost by 20-30%

BIOETHANOL RAW MATERIALS

Corn Stover
Pretreated with dilute acid
hydrolysis to produce glucose
readily fermented
Problem!
XYLOSE

BIOETHANOL RAW MATERIALS

Xylose
Not so easily utilized due to
varying amounts of toxic
substances in it
Intolerance of yeast toward this
inhibitors

RECOMBINAT YEAST CELLS

Saccharomyces
cerevisiae yeast
Employed for the yeast to
readily ferment xylose to
ethanol

IMMOBILIZED VS FREE
CELLS
Immobilized Cells
Enhancement of productivity
Easy cell recovery
Prevention of cell washout
Protection of cells against toxic
substances
Cell recycling

OVERVIEW

INTRODUCTION
MATERIALS AND
METHODS
RESULTS AND
DISCUSSION
CONCLUSIONS

MATERIALS AND METHODS

Raw Material

milling

screeni
ng

washin
g

drying

filtratio
n

Corn
Stover

stored

MATERIALS AND METHODS

Preparation of Hemicellulosic
Hydrolysate
Dried
Milled
Corn
Stover

STEEPING
10% ammonia (24
h)

WASHING

HYDROLYSIS
1.5% H2SO4
(6h, 108C, 1:10
w/v)

STORED

FILTRATION

MATERIALS AND METHODS

Detoxification of Hemicellulosic
Hydrolysate
Hemicellulo
sic
Hydrolysate

EVAPORATION

MIXING
(WATER BATH
SHAKER)

ANALYZED

FILTRATION

MATERIALS AND METHODS

Microorganism and Inoculum
Preparation
The recombinant
xylose-utilizing yeast
strain S. cerevisiae ZU-10 expresses
xylose reductase (XR) and xylitol
dehydrogenase (XDH) from
chromosomally integrated Pichia stipites
genes XYL1 and XYL2, respectively, and
over-expresses the homologous XKS1
gene encoding xylulokinase (XK).
S. cerevisiae ZU-10 was maintained on
YPX-agar slants containing 10g/L yeast
extract, 20g/L peptone, 20g/L xylose and
20g/L agar

MATERIALS AND METHODS

Microorganism and Inoculum
Preparation
50 mL MEDIUM
5 g/L yeast
extract
10 g/L peptone
10 g/L xylose
10 g/L glucose
LOOPFU
L OF
CELLS

INCUBATED
in Orbital Shaker
(24 h, 180 rpm,
30C)

INCUBATED
in Orbital Shaker
(36 h, 180 rpm,
30C)

10 mL of
inoculum is
FURTHER
PROPAGATED
Containing 100
mL medium:
5 g/L yeast
extract
10 g/L peptone
20 g/L xylose
20 g/L glucose
2.5 g/L
KH2PO4
0.25 g/L CaCl2
0.25 g/L MgCl2

MATERIALS AND METHODS

2% (w/v)
sodium alginate
solution
1% (w/v)
diatomite

Cell
Immobilization
Cells grown
in
Propagation
Medium

CENTRIFUGATION
(10 min, 4000 rpm,
4C)

Extruded through
syrince into 2%
(w/v) CaCl2
solution

MATERIALS AND METHODS

Fermentation

30 mL
immobilized
cells
150 mL detoxified
hemicellulose
hydrolysate

5 g/L yeast
extract
10 g/L peptone
20 g/L xylose
20 g/L glucose
2.5 g/L KH2PO4
0.25 g/L CaCl2
0.25 g/L MgCl2

MATERIALS AND METHODS

Analytical
Methods
CENTRIFUGATION
(10 min, 10000
rpm)

Samples
from E. Flask
at different
time interval

FILTRATION
(0.45 m
membrane filters)

Sugar
Ethanol
Byproducts

Analyzed using

HPLC

OVERVIEW

INTRODUCTION
MATERIALS AND
METHODS
RESULTS AND
DISCUSSION
CONCLUSIONS

RESULTS AND DISCUSSION

Concentration and
Detoxification of Corn Stover
Hemicellulose Hydrolysate

RESULTS AND DISCUSSION

Ethanol Production from Detoxified Hemicellulosic
Hydrolysate by Immobilized Recombinant S.
cerevisiae
ZU-10
cells
1. Influence of
initial pH

RESULTS AND DISCUSSION

Ethanol Production from Detoxified Hemicellulosic
Hydrolysate by Immobilized Recombinant S.
cerevisiae ZU-10 cells
2. Influence of different xylose concentration in the
hemicellulosic hydrolysate

RESULTS AND DISCUSSION

Ethanol Production from Detoxified Hemicellulosic
Hydrolysate by Immobilized Recombinant S.
cerevisiae
ZU-10 cells
3. Time course
of ethanol
production from detoxified
hemicellulose hydrolysate

RESULTS AND DISCUSSION

Ethanol Production from Detoxified Hemicellulosic
Hydrolysate by Immobilized Recombinant S.
cerevisiae
ZU-10
cellsfermentation by
4. Repeated
batch
immobilized recombinant S. cerevisiae
ZU-10 cells

OVERVIEW

INTRODUCTION
MATERIALS AND
METHODS
RESULTS AND
DISCUSSION
CONCLUSIONS

CONCLUSIONS
It is feasible to use Ca-alginate
immobilized cells of recombinant S.
cerevisiae ZU-10 for ethanol
production from detoxified corn stover
hemicellulosic hydrolysate
After detoxification, the concentration
of fermentation inhibitors was
effectively controlled within an
acceptable level.

CONCLUSIONS
Detoxified hemicellulosic hydrolysate
could be converted to ethanol by
immobilized cells both efficiently and
economically.
The immobilized cells were also proved
to be reusable in five batches of
fermentation.

CONCLUSIONS
At the initial pH of 5.5 and xylose
concentration 71.8 g/L, 97.1 % xylose
and 100% glucose in the hydrolysate
were utilized within 72 h; 31.1 g/L
ethanol was produced with the
ethanol yield on fermentable sugars
0.406 g/g.
More than 92.1% xylose was utilized in
each batch by the same immobilized
cells, and ethanol yield on fermentable
sugar could maintain above 0.393 g/g.

ABOUT THE JOURNAL

PARTS
INTRODUCTION

MATERIALS AND
METHODS

RESULTS AND
DISCUSSION

CONCLUSIONS

OBSERVATIONS
The purpose of the article made clear
All the discussions are relevant and only pertinent
authors were cited
The subject of recombinant yeast cells was
underemphasized. Discussion of the function of the
recombinant yeast cells to the study should be
included.
Clarity of the order of procedures were presented
except for the microorganism and inoculum
preparation
All the tables and figures are in accordance with
the standards
Some of the important details were omitted in the
presentation of data
All the statements are concise, clear and relevant
with respect to the objectives of the study.

END

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