Second-generation bioethanol of hydrothermally pretreated

stover biomass from maize genotypes Jaime Barros-Rios1, Aloia Romaní2, Gil Garrote2, and Bernardo Ordás1

1Misión Biológica de Galicia (CSIC), 36080, Pontevedra, Spain. 2Departamento de Ingeniería Química (Universidad de Vigo), 32004, Ourense, Spain

Introduction ~manufacture biofuels from renewable resources~

Enzymatic hydrolysis and bioethanol yields

Significant differences were found between genotypes for enzymatic hydrolysis

parameters and total polysaccharide recovery (Fig. 4a,b). All genotypes had high

cellulose-to-glucose conversions (>87%). Harsher autohydrolysis conditions resulted

in substrates leading to higher glucose concentrations (>34 g/L). Both the hydrolysis

rate and the fraction susceptible of hydrolysis increased with TMAX (Fig 4a,c), but the

overall bioethanol yield decreased for substrates pretreated at TMAX above 220 C

owing mainly to losses in hemicellulose fraction from solid phase (Figs. 4e, 3b).

Profound differences between extreme genotypes were found in bioethanol production

(L/ha) when stover biomass yield (ton/ha) was considered in the calculation (Fig. 4d)

(1) Kim et al.,2009. Biotechnolog. Prog. 25, 340–348; (2) Garrote et al., 2008. Ind. Eng. Chem Res. 47, 1336–1345; (3)

Lorenzana et al., 2010. Crop Sci. 50:541–555; (4) Lewis et al., 2010. Crop Sci. 50:516–523; (5) Lundvall et al., 1994.

Crop Sci. 34:1672–1678.

References

• From the farmer s point of view. Grain production for food/fed industries and stover

biomass for the nascent bioenergy industry are compatible productions in maize.

• From the processing point of view. Glucose yield increased steadily with the severity

of the pretreatment. However, the overall bioethanol yield decreased for substrates

pretreated at TMAX above 220 C because of the degradation of the hemicellulose

(mainly arabinan) in the solid fraction.

• The presence of lignin and acetyl groups in the spent solids explained part (40-50%)

of the genetic variation observed in bioconversion of corn stover into ethanol.

Therefore, future research is needed to understand additional biochemical and

anatomical factors that influence recalcitrance to sugar release.

Summary

Results 1. Field biomass, material balance data,

and composition of spent solids and liquors

Composition of spent solids and liquors

In contrast to the raw corn stover, genotypes showed significant differences for

composition of the liquid and solid phase from autohydrolysis, with the exception of

non-volatile compounds, arabinan, and acetyl groups of spent solids, and volatile

compounds, and furfural concentrations of the liquid phase (Figs. 2c, 3a,c). Solid

yield, xylan, arabinan and acetyl groups decreased steadily with the severity of the

treatment. Cellulose and Klason lignin increased due to condensation reactions. The

major components of the liquors were hemicellulose-derived products (Figs. 3b,d,e)

Results 2. Saccharification, polysaccharide

recovery, and potential bioethanol production

0

50

100

150

200

250

0

5

10

15

20

25

Pla

nt

Heig

ht

(cm

)

Pla

nt

pa

rt y

ield

s (

ton

/ha

)

Grain Cob Stover Plant height

Genotype Cellulose Xylan Arabinan Acetyl

groups

Klason

Lignin ------ g/100g spent solids, oven dry basis ------

B73xMo17 58.7a 9.4bcd 0.48 0.76 24.1d

PR34G13 59.1a 6.2e 0.33 1.02 26.1bc

PR36B08 57.8ab 8.3cde 0.46 0.98 25.1cd

BS17 59.2a 7.5de 0.50 0.86 23.3d

BSL 52.1de 7.5de 0.66 1.04 28.4a

Minnesota No.13 53.6cd 11.4ab 0.84 1.14 23.9d

Aranga 55.6bc 7.9de 0.76 1.03 27.4ab

Lazcano 56.5abc 8.1cde 0.45 1.11 26.4bc

Posada 53.9cd 8.4cde 0.74 1.03 27.5ab

Faro 56.5abc 7.1de 0.49 0.86 27.0ab

Rastrojero 50.8de 12.1a 0.83 1.11 26.3bc

Vejer 49.6e 10.6abc 0.62 0.74 26.1bc

LSD (P < 0.05) 3.3 2.5 - - 1.9

0%

20%

40%

60%

80%

100%

Ma

teri

al

ba

lan

ce

(g

/100

g d

ry s

tove

r)

Volatile Non-volatile Solid

0

0.2

0.4

0.6

0.8

1

1.2

0

2

4

6

8

10

12

210 215 220 225

Gluco-o Xylo-o Arabino-o Acetyl-o

Glucan 36.1 ± 2.7

Xylan 21.1 ± 3.3

Arabinan 3.6 ± 1.8

Acetyl groups 2.4 ± 2.3

Klason lignin 18.2 ± 1.2

Extracts 12.9 ± 1.8

Ashes 5.0 ± 0.2

Total cell wall 81.4 ± 5.3

Objective of our project is to provide an assessment on the manufacture of second-generation ethanol from stover of Zea maize L.

genotypes. Nine local maize populations from Europe and North-America and three elite commercial hybrids that represent a wide range of the

genetic base for temperate hybrid development were evaluated in two locations in Spain in 2011. Maize is a strategic crop for Europe, in 2009

Europe produced 58 MT (8Mha- the size of Austria). Polysaccharides from lignocellulosic materials (LCM) may provide a sustainable alternative

to the problems derived from the extensive utilization of fossil fuels. In comparison with starchy seeds used in first-generation bioethanol, LCM not

compete with fed production. However, posses a complex heterogeneous nature that makes the hydrolysis of the polysaccharide fraction difficult.

The conversion of LCM to ethanol is a three step process that involves pretreatment followed by polysaccharide hydrolysis to simple

sugars and sugar fermentation to ethanol (Figure 1). The pretreatment needed to render the native LCM is one of the most important stages. The

pretreatment with hot compressed water (autohydrolysis or hydrothermal processing) is simple, economical, has low generation of residues, and

low energy requirements (1, 2). Stover biomass from corn genotypes pretreated under several operational conditions were used as substrates for

enzymatic hydrolysis, and the experimental data enabled the interpretation of the reaction yields as a function of the major operational variables.

Genotype Gluco-o Xylo-o Arabino-o Acetyl-o Glucose Xylose Arabinose Acetic

acid

HM-

Furfural Furfural

------------------------------------- g/100g spent solids, oven dry basis ----------------------------------------------------------

B73xMo17 0.83f 10.0de 0.72bcd 0.62bcd 0.48de 0.67ef 0.12d 1.64a 0.04e 0.28

PR34G13 1.84de 11.2ab 0.91a 0.76ab 0.67abc 1.04abc 0.18abc 1.36bcd 0.14a 0.33

PR36B08 1.86de 11.9a 0.86ab 0.86a 0.80a 1.14a 0.22a 1.38bcd 0.12ab 0.30

BS17 2.97abc 11.7ab 0.80abc 0.62bcd 0.37e 0.70def 0.14bcd 1.59a 0.06de 0.27

BSL 2.55bcd 9.5de 0.55de 0.72abc 0.84a 0.89bcde 0.18abc 1.06ef 0.10abc 0.27

Minnesota No.13 1.62def 9.9de 0.86ab 0.72abc 0.48de 0.65f 0.22a 1.25cd 0.03e 0.20

Aranga 3.47ab 9.0e 0.65cde 0.72abc 0.61bcd 0.86cdef 0.20a 0.98f 0.11abc 0.25

Lazcano 2.47cde 10.2cd 0.70bcd 0.77ab 0.47de 0.83cdef 0.20ab 1.40bc 0.08bcd 0.29

Posada 3.54a 10.6bcd 0.72bcd 0.75ab 0.58cd 0.87bcdef 0.20a 1.22de 0.08cd 0.27

Faro 1.52ef 9.9de 0.50e 0.61bcd 0.78ab 1.11ab 0.18abcd 1.39bc 0.08bcd 0.31

Rastrojero 2.13cde 11.1abc 0.86ab 0.54cd 0.57cd 0.92abcd 0.13cd 1.47ab 0.12abc 0.26

Vejer 1.79def 8.8e 0.84ab 0.50d 0.55cde 0.67ef 0.14cd 1.39bc 0.06de 0.22

LSD (P < 0.05) 0.98 1.15 0.18 0.20 0.19 0.23 0.06 0.17 0.04 -

Figure 2. Biomass yields of different plant parts and plant height in maize genotypes evaluated (a). Composition of the corn

stover (wt% SD, N = 24) (b). Material balance data of liquid and solid phase from autohydrolisys (c).

(a)

(b)

(c)

Figure_3. Composition of the spent solids for maize genotypes evaluated (a) and operational conditions assayed (b), and

composition of the liquid phase by same genotypes (c) and conditions (d, e) resulting from the autohydrolysis of stover biomass.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

210 215 220 225

g m

on

om

er

eq

uiv

. /

L

Glucose Arabinose HM-Furfural

Furfural Xylose Acetic acid

Figure_4. Time courses of glucose concentration during enzymatic hydrolysis (LSR 20g/g; ESR 10.3FPU/g) (a). Polysaccharide

recovery by maize genotype (b) and operational condition (c), and bioethanol yield by genotype (d), and pre-treatment severity (e).

0

500

1000

1500

2000

2500

0

50

100

150

200

250

300

350

400

450

500

Bio

eth

an

ol yie

ld (

L/h

a)

Bio

eth

an

ol yie

ld (

L/t

on

)

Cellulose Hemicellulose

Liquid phase Total ethanol (L/ha)

1000

1100

1200

1300

1400

1500

1600

1700

1800

1900

0

50

100

150

200

250

300

350

400

450

210 215 220 225

Bio

eth

an

ol yie

ld (

L/h

a)

Bio

eth

an

ol yie

ld (

L/t

on

)

Hemicellulose Cellulose Total (L/Ton) Total (L/Ha)

Biomass yield

Genetic variation was observed for biomass traits indicating that

maize improvement for both grain and stover yield is feasible. The

elite hybrids B73xMo17 and PR34G13, along with the local

populations Faro and BSL had good aptitude for both traits and

would be good candidates to develop new maize hybrids

improved for both uses (Fig. 2a).

Figure 1. Conceptual flowchart of the biorefinery

scheme considered in this work

HYDROTHERMAL PROCESSING

Corn stover

Water

Stream A - Simple sugars - Oligomers

Buffer / Enzymes

Stream B

- Simple sugars

Spent solids enriched in cellulose

HYDROLYTIC DEGRADATION

Aqueous

phase

Second generation bioethanol

SUGAR FERMENTATION

(a)

(b)

(c)

Raw corn stover composition

Maize genotypes evaluated showed no significant differences for cell wall composition

of the raw stover- before the hydrothermal treatment. The average composition among

genotypes (Fig. 2b) agree with previous work (3, 4). Less genetic variation has been

detected for stover than seen in separated corn plant parts (4, 5).

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

0

10

20

30

40

50

60

210 215 220 225

g/1

00g

dry

sp

en

t s

oli

d

Cellulose Klason lignin Xylan Solid yield Arabinan Acetyl groups

(d) (e)

b a a a

b b cd

b b b cd

d

0

10

20

30

40

50

60

70

80

g p

oly

mer

eq

uiv

./1

00

g s

tove

r

Oligomers Sugars Glucose at 48h

0

10

20

30

40

50

60

70

80

210 215 220 225

g p

oly

mer

eq

uiv

./1

00

g s

tove

r

Sugars

Oligomers

Glucose

Total polysaccharides

C o m m e r c i a l h y b r i d s

A t l a n t i c - E u r o p e l o c a l p o p u l a t i o n s

M e d i t e r r a n e a n - E u r o p e l o c a l p o p u l a t i o n s

U S - c o r n b e l t p o p u l a t i o n s

(a)

(b)

(c)

(d) (e)

81% higher 17% higher

Severity of the pretreatment (°C) Severity of the pretreatment (°C)

Severity of the pretreatment (°C)

Severity of the pretreatment (°C)

Severity of the pretreatment (°C)