ENHANCED SUGARCANE BAGASSE CONVERSION TO SUGARS BY OZONOLYSIS AND LIQUID HOT WATER PRETREATMENTS

Sidnei Bordignon1, 2, Eduardo Ximenes2*, Roberto Da-Silva1, Haley Ross2, Michael Ladisch2

(1)Laboratório de Bioquímica e Microbiologia Aplicada, IBILCE - UNESP, São Jose do Rio Preto, Brazil (2) Agricultural & Biological Engineering, Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN

ABSTRACT

Global concerns about renewable ways to produce energy have motived the

development of biorefineries based in lignocellulosic biomasses. Sugarcane

bagasse is one of the most abundant carbon resources that can be converted in

liquid biofuels and other high-added value bio-products. A well-established

technology using Liquid Hot Water (LHW), and an emergent approach based

on Ozonolysis to pretreat lignocelluloses were combined in the current work,

which generate a solid content with more than 70% cellulose, and a liquid

phase (liquor) composed of phenolic compounds from lignin, and xylose and

arabinose solubilized from hemicellulose. High cellulose conversion to sugars

was obtained when the liquor was separated and phenolics were removed by

sequential washing at room temperature. Ozonolysis showed a strong

potentiality to oxide lignin without use of any other chemical reagent. On the

other hand, LHW process is required to solubilize and clean up the fibers

from the by-products after ozone attack, allowing appropriate conditions the

following steps of enzymatic hydrolysis and fermentation of sugars to ethanol

by industrial yeasts.

BACKGROUND

CONCLUSIONS

• Combining Ozonolysis and LHW pretreatments increases twice the

cellulose availability in sugarcane bagasse compared to untreated

biomass, and through enzymatic hydrolysis this bagasse releases more

than 50 g.L-1 of glucose (around 75% of glucan conversion);

• Ethanol is produced from hydrolysate media with 100% of efficiency

for single pretreatments, and 71% for combined pretreatment in 30 h.

RESULTS

ACKNOWLEDGEMENTS• CAPES/Brazilian Government by financial support

• Novozymes for enzymes supply; AEB Latin-American for yeast supply

• LORRE students and staff

*eximenes@purdue.edu

METHODOLOGY

RESULTS

• Ozonolysis conditions: batches with 25.0g SCB, 50% moisture, room

temperature, 60 minutes, dosage: 76 mg O3.g-1bagasse.

• Liquid Hot Water conditions: batches with 3.5 g SCB, 10% (w/v) total

solids, 190 ºC, 15 minutes, severity factor: log Ro: 4.40 (Kim et al., 2014).

• Washing procedures: 5% (w/v) total solids in distilled water, room

temperature, 30 minutes under agitation, separation by filtration.

• Enzymatic hydrolysis conditions: 10% (w/v) total solids, Sodium Citrate

buffer 0.05M pH 5.0, 50 ºC, 150 RPM, 96 hours, commercial cocktail Cellic®

CTEC2 and HTEC2 (4:1), dosage range from 9.1 to 17.5 mg protein.g-1 glucan.

• Alcoholic fermentation conditions: S. cerevisiae JP1 strain, 0.5 g.L-1

inoculum, 32 ºC, 150 rpm, 48 hours.

• Phenolics are strongly released from lignin after pretreatments;

• Simple room temperature washes can efficiently remove them from pretreated

bagasse and improve the conversion yield in enzymatic hydrolysis:

• Combining Ozonolysis and LHW pretreatments double and increases 5

times the amount of glucose in enzymatic hydrolysis in comparison to

single pretreatments, and untreated bagasse, respectively.

• Separating the liquor of solids from combined pretreatment, the cellulose

conversion yield reaches around 75% with 11 mg protein / g glucan;

• All the three hydrolysate media are fermentable by industrial yeast JP1:Pretreatment I:OZONOLYSIS

ENZYMATIC HYDROLYSIS

ALCOHOLIC FERMENTATIONSaccharomyces cerevisiae

Pretreatment II:LIQUID HOT WATER

Cellic® Ctec 2

Cellic® Htec 2

Cellulose 36%

45%47%

Cellulose 73%

Hemicellulose 19%14%

17%

Hemicellulose 4%

Lignin 21%

13%

22%

Lignin 13%

In natura Ozone-pretreated LHW-pretreated Oz+LHW pretreated

• Combination of Ozonolysis and LHW pretreatments improve the cellulose

availability in 100% in comparison to the untreated sample.

• The delignification rate is around 40%, and 80% of hemicellulose is

solubilized in the liquor phase.

(NREL- National Renewable Energy Laboratory: Sluiter et al., 2008; 2012).

Dry

su

garc

an

e b

aga

sse

Ozonolysis LHW

Washing

En

zym

ati

c h

yd

roly

sis

Ozonolysis

Ozonolysis

LHW

LHW

Drying

Drying

Liquor

Bagasse

Liquor

Bagasse

Washing Drying

Alc

oh

oli

c fe

rmen

tati

on

Washing

(Bagasse + Liquor)

(Bagasse)

+buffer

+buffer

+buffer

44%

62%66%

71%75% 73%

0

100

200

300

400

500

600

700

800

900

1,000

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Ph

eno

lics con

centra

tion

(mg

.L-1

GA

E)

Con

ver

sion

yie

ld (

%)

SCB pretreated by ozonolysis

69.15

936.69

857.08

681.35

0 1,000 2,000 3,000

In

natura

Ozonol

ysis

LHW

Oz+L

HW

Concentration (GAE mg.L-1)

Bagasse

Liquor

25%

37%

47%

8%

59%

0%

10%

20%

30%

40%

50%

60%

70%

Non

-tre

ated

LH

W

Ozo

ne

Oz+

LH

W (

+li

quor)

Oz+

LH

W(-

liquor)

Con

ver

sion

yie

ld (

%)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

0 24 48 72 96

Glu

cose

con

cen

tra

tio

n (

g. L

-1)

Time of hydrolysis (hours)

Non-treated Ozone treated LHW treated

Oz+LHW treated Oz+LHw (w/ liquor)

8.0%

74.1% 75.9%

0

250

500

750

1,000

1,250

1,500

1,750

2,000

2,250

2,500

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Ph

eno

lics (GA

E m

g.L

-1)

Glu

cose

con

cen

tra

tio

n (

g. L

-1)

Phenolics

Cellulose

yield

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0 6 12 18 24 30

Eth

an

ol co

ncen

tratio

n (g

.L-1)

Glu

cose

con

cen

tra

tio

n (

g. L

-1)

Time of fermentation (hours)

Ozone (S) LHW (s) Oz+LHW (s)

Ozone (p) LHW (p) Oz+LHW (p)

17.5 mg protein.g-1 glucan

9.1 mg protein.g-1 glucan

11 mg protein.g-1 glucan