Effect of pretreatment dilute acid - peroxide on napier grass
(pennisetum purpureum schumach) to enhance reducing
sugar yield by enzymatic hydrolysis
Cristian Bohórquez1, Eliseo Amado-González2, Marlon Martínez-Reina3
Ing.Químico, M.Sc, IBEAR, Universidad de Pamplona, [email protected]
Químico,.Ph.D., IBEAR, Universidad de Pamplona, [email protected]
3
Ing.Materiales, M.Sc, IBEAR, Universidad de Pamplona, [email protected]
1
2
Fecha de recepción: 21/05/2014 Fecha de aceptación del artículo: 10/02/2015
Abstract
1. Introduction
The napier grass (Pennisetum purpureum Schumach.)
is a potential feedstock biomass for conversion to
ethanol, bio-energy or high added value products.
Variation in prehydrolysis parameters (time, acid
strength, biomass %, particle size) and enzymatic
saccharification conditions were examined for conversion of napier grass into fermentable sugars.
A pretreatment of the water insoluble substrate
(WISH2SO4) at 15.1 % of reducing sugar yield
(RSY) was done with peroxide (H202) at 122 °C.
The relationship between RSY and the acid process
parameters are described by a mathematical model
derived from the experimental data.The peroxide
pretreatment at 0.7 % (w/w), 4.75 mm particle
size, 8% (w/v) biomass concentration, improved
the RSY production from enzymatic hydrolysis
in 97.6 % after 75 min. The enzymatic hydrolysis
produced 287.81 mg/(g initial dry sample) of
glucose and 245.81 mg/(g initial dry sample) of
Xylose. Therefore, it was concluded that combination peroxide - acid pretreatment is an effective and
environmentally friendly method for the enzyme
hydrolysis of napier grass.
The chance to develop a sustainable economic bioethanol project implies fast growing crop adapted
to the climatic and soils conditions of a specific
area and a lignocellulosic biomass efficient technology conversion to fermentable sugars at lower
costs [1,2]. It means to compete with gasoline; the
cost production of ethanol from biomass should
be competitive [3].
Keywords
Dilute acid, hydrolysis, reducing sugars, napier
grass, Pennisetum purpureum, peroxide.
Napier grass (P. purpureum) can be considered as a
potential feedstock of biomass for conversion to
ethanol. P. purpureum is crop for animal feed and the
excess may be used as feedstocks for bioethanol
production to avoid direct competition between
bioethanol and food productions. The optimization of pre-treatment process parameters of the
lignocellulosic biomass dilute acid pretreatment is
a necessary condition to get the highest glucose
and hemicellulose sugars release [4]. Pretreatment
of lignocellulosic materials is done to remove
lignin and enhance the hydrolysis of cellulose.
Even dilute acid pretreatment of lignocellulosic
biomass has been researched for several materials
[5]. The pretreatment of biomass at high temperature (150 - 240 °C) and the quantity of acid
used becomes an uneconomical cost production
[6]. However, dilute acid pretreatment has been
shown to successfully hydrolyze hemicelluloses
and disrupt the ligno-cellulosic structure for a
wide range of feedstocks [7,12].
The pretreatment of potential biomass to increase
RSY gives the opportunity to develop new pro-
48 AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953
cedures with the environmental requirements,
low concentration of inhibitory substances for
saccharification and fermentation [13,14]. Peroxide (H2O2) an oxidative agent under appropriate
conditions reacted readily with lignin and related
phenolics [15,18]. Besides, alkaline peroxide pretreatment combination with steam explosion and
hydrothermal process of lignocellulose biomass
showed to improve enzymatic saccharification [1921].
Different pretreatment variations of alkali peroxide procedure especially with acids have been
investigated for lignin removal to enhance hydrolysis [22-29]. A variation peroxide-sulphuric acid
pretreatment has not received enough attention.
However, a pretreatment peroxide 1% -acid 1 % on
sawdust produced between 47 – 56 % of fermentable sugars [23]. The high cost of peroxide do not use
feasible for pretreatment. This paper describes the
effect of peroxide pretreatment on washed at 15.1
% RSY. A factorial surface response methodology
was employed to describe the relationship between
RSY and the major process variables, namely acid
concentration, pre-treatment temperature, size particle and time. The procedure improves the RSY by
enzymatic hydrolysis about 81.1 %. This research
can give new clues to improve lignocellulosic pretreatment procedure on the commercial use of P.
purpureum for large-scale ethanol production.
2. Material and methods
Samples of napier grass (P. purpureum) were
provided by Pharmavicola. Four random samples
of 16-wk-old material were gathered and weighed
immediately after cutting. The grass samples were
a mixture of leaf and stem. The material was dried
at 55 ºC by 12 hours. Then it was milled to a particle size smaller than 0.5 mm using a laboratory
mill, homogenized and stored in a desiccator at
room temperature until used. A sieve shaker was
used. The chemical composition of raw material
and water insoluble fraction were performed using
standards [30].
Ash was determined as the percentage of dry
residue remaining after the oxidation between 550
ºC - 600 ºC. The liquor was analyzed for total RSY
using a DNS assay [31,21].
The concentrations of main components were as
follows: Cellulose 35.5±0.801 %, lignin 11.3±0.638,
xylose 14.3±0.302, extractives 16.0± 0.456, ash
5.84± 0.076.These values are in the range found
for this kind of materials [33-38].
To reducing sugars (in prehydrolyzate), pretreatments samples were autoclaved in a 10-L stainless
steel reactor at 105 °C and 20 psi. Biomass samples
were loaded into the reactor at the different solid (2,
4, 6, and 8 g of biomass)/liquid ratios containing
0.5 to 1 g H2SO4/100 g liquor on dry basis. Samples
were collected from the reaction media at several
reaction times in the range 30–90 min. A factorial
design based on the variation of the pretreatment
conditions acid concentration, size particle, biomass concentration and residence time was used to
identify the best pretreatment conditions for RSY
in the aqueous fraction are summarized in Figure 1.
The hemicellulose from washed water insoluble
substrate (WISH2SO4) at 15.1 % RSY was solubilized with peroxide (0.4 – 0.7) % (w/w) at 30 °C
with agitation at 130 rpm for 72 h. The parameters
used for acid pretreatment are shown on Table 1.
Table 1. Parameters used for acid pretreatment of napier grass.
Code
Parameter
K1
Acid, % (w/w)
K2
Biomass, % (w/w)
K3
Size (mm)
K4
Residence time (min)
Low level
High level
Relation
F
R2
0,5
1
k1-k2
0.76
0.86
2
8
k1-k3
0.67
0.69
2.36
4.75
k1-k4
1
0.89
30
90
k1-k4
0.99
0.76
AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953(2014) 49
RSY Production (%) =100 (RSY /Total theoretical
RSY)
RSY)
(1)(1)
elution (flow rate 0.4 ml/min; mobile phase: H2SO
0.005N).N).Standard
Standardsamples
sampleswere
wereprepared
preparedf
0.005
determination.
determination.
Totaltheoretical
theoretical
RSY
(Biomass
concentration)
Total
RSY
= =(Biomass
concentration)
RSY Production
(%) =100
(RSY
/Total
theoretical
elution (flow rate 0.4 ml/min; mobile phase: H2SO4
(dry
biomass)
(hemicellulose)
Theeffect
effectofofthe
theparameters
parameters
acidconcentratio
concentra
biomass)
(hemicellulose)
(2)(2)0.005 The
RSY) (dry
Standard
samples
were acid
prepared
for
of N).
the
parameters
acid
concentration,
The reduction
sugar yield production (arabinose, (1)The effect
size
particle,
biomass
concentration
on
the
RSY
size
particle,
biomass concentration
on the RSY
determination.
For
enzyme
hydrolysis
filter
paper
procedures
were
size
particle,
biomass
concentration
on
the
RSY
glucoseTotal
and For
xylose)
was
calculated
by
Eq.
1
and
2.
enzyme
hydrolysis
filter
paper
procedures
were
acidpretreatment
pretreatmentwas
wasfound
foundto tobebenotnotlinear.
linear
theoretical RSY = (Biomass concentration)
acid
by acidand
pretreatment
was found to be
not linear.
used
to
determine
the
activities
of
the
cellulase
and
used
to
determine
the
activities
of
the
cellulase
factorial
surface
response
methodology w
biomass)
(2)
The effect
of the
parameters
acid concentration,
factorial
surface
response
methodology
RSY (dry
Production
(%)(hemicellulose)
= 100
(RSY /Total
theoretical
A multifect
factorial surface
response
methodology
was
cellobiase.
Comercial
cellulose
1500and
and
cellobiase.
Comercial
cellulose
1500
multifect
employed
to
setup
the
experiment.
were
size
particle,
biomass
concentration
on
the Data
RSY by
RSY) (1)
employed
to setup theData
experiment.
employed
to setup
the experiment.
were usedData were us
CXB
enzymes
were
used
for
the
enzymatic
For enzyme
hydrolysis
filter
paper
procedures
were
CXB enzymes were used for the enzymatic
toconstruct
constructwas
a polynomial
model
acid pretreatment
found to
bemathematic
not
A wi
amathematic
polynomial
mathematic
model
to
construct
atopolynomial
model
withlinear.
Total used
theoretical
RSY
=
(Biomass
concentration)
hydrolysis
step.
to
determine
the
activities
of
the
cellulase
and
hydrolysis step.
the
following
form
factorial
surface
response
the
following
form
byby
EqEq
4: 4: was
the following
form
presented
by presented
Eqpresented
4: methodology
(dry biomass)
(hemicellulose)
(2) and multifect
cellobiase.
Comercial
cellulose 1500
employed to setup the experiment. Data were used
Accellerase1500
(Genencor,
USA),
endoglucanase
!
Accellerase1500
(Genencor,
USA),
endoglucanase
CXB
enzymes
were
used
for
the
enzymatic
RSY %
=! !
!!! B!!X (4)
to construct
a= polynomial
mathematic
model with(4)(4
RSY %
For enzyme hydrolysis
filter CMCase
paper procedures
were
!!! B! X (2200�2800
units/g),
and
�-glucosidase
(2200�2800
CMCase
units/g),
and
�-glucosidase
step.
used tohydrolysis
determine
the activities
of
the cellulase
and
the
form presented by Eq 4:
(525�775
pNPG
units/g)
enzyme
were
used
thisfollowing
Where𝐵𝐵 𝐵𝐵is
adepends
polynomial
that
depends
biom
(525�775
pNPG
units/g)
enzyme
were
used
in in
this
! is
Where
is a polynomial
ofthat
biomass
Where
a polynomial
depends
ofof
bioma
cellobiase. Comercial
cellulose
1500
and
multifect
! that
study.
Enzymes
were
kindly
provided
by
GenencorAccellerase1500
(Genencor,
USA),
endoglucanase
!
(w/w)
5:
study.
Enzymes
kindly provided
(w/w) by
eq 5:
(w/w)
byby
eq
RSY %
= !!!
B!eq
X5:! (4)
CXB enzymes
were
used forwere
the enzymatic
hydro-by GenencorMerquiant
LTDA.
Multifect
CX
B,
�-Gluconase
(2200�2800
CMCase
units/g),
and
�-glucosidase
Merquiant
LTDA.
Multifect
CX
B,
�-Gluconase
lysis step.
!
(5)that depends of biomass(5)(5
C! (2250
BGL
U/mL)
obtained
from
Trichoderma
reesei.
(525�775
pNPG
units/g)
enzyme
were
used
in this reesei.
WhereB!B𝐵𝐵=!! =
is !
a !!!
polynomial
B!"BC!"! (2250
BGL
U/mL)
obtained
from
Trichoderma
!!!
Accellerase1500
(Genencor,
USA),
endoglucanase
Adosis
dosis
of0.05
0.05
enzyme/
wasused,
used,
study.AEnzymes
kindly
provided
bysubtracts
Genencor(w/w) by eq 5:
ofwere
g genzyme/
g gsubtracts
was
The
lowest
RSYbybywere
aciddeterpretreatmentwe
Theinsoluble
lowest RSY
bylowest
acid
pretreatment
(2200−2800
CMCase
units/g),
and
β-glucosidase
RSY
acid
pretreatment
specific
gravity
1.3
g/mL.
The
washed
Merquiant
LTDA.
Multifect
CX
B,
�-Gluconase
specific gravity 1.3 g/mL. The washed insoluble The
!
determinated.
Themodel
model
analyzed
usinga
minated.
The model
was analyzed
usingwas
awas
95
% (5)using
(525−775
units/g)
enzyme
were
used were
Bfor
subtracts
after
peroxide
pretreatment
used
The
analyzed
(2250pNPG
BGL
U/mL)
obtained
from
Trichoderma
reesei.
! = determinated.
!!! B!" C! subtracts
after
peroxide
pretreatment
were
used
for
confidence
and theF coefficient,
r2,
was
in thisAstudy.
Enzymes
kindlyg provided
%confidence
confidence
Ftest,
test,and
andthe
thecoefficient,
coefficient,r2,r2w,
enzymatic
hydrolysis.
Enzymatic
hydrolysis
was F%test,
dosis
of 0.05 hydrolysis.
g were
enzyme/
subtracts by
was
used,
enzymatic
Enzymatic
hydrolysis
was
checked.each
Genencor- Merquiant
LTDA.100mL
Multifect
CX B, flasks,
checked.
The lowest
RSY by acid pretreatment were
performed
Erlenmeyer
specific
gravity 1.3in in
g/mL.
The
washed insoluble
performed
100mL
Erlenmeyer
flasks,
each checked.
β-Gluconase (2250 BGL U/mL) obtained from
determinated. The model was analyzed using a 95
25
mL
buffer
of 0.029
sodium
citrate
subtractscontaining
after peroxide
pretreatment
were
used
forcitrate
mL
buffer
MM
sodium
Results
and
discussion
Trichodermacontaining
reesei. A25dosis
of
0.05ofg0.029
enzyme/
3.
Results
and
discussion
% confidence
F test,
and
the coefficient, r2, was
3.3.Results
and
discussion
buffer
(pH
4.8)
at
8
%
(w/v)
dry
pretreated
enzymatic
hydrolysis.
Enzymatic
hydrolysis
was
(pHspecific
4.8) atgravity
8 % 1.3
(w/v)
dry pretreated
g subtracts buffer
was used,
g/mL.
checked.
The100
usemin.
of bioethanol
supposes a reduction our
substrate
loading
at
50
°C
at
150
pmr
performed
in
100mL
Erlenmeyer
flasks,
each
loading
at 50 °C
pmr forfor
100 min.
The washedsubstrate
insoluble
subtracts
afterat 150
peroxide
dependence
expensive
oil and
reduces a reduction
Samples
were
taken
attimed
timed
intervals,boiled
boiled
2 upon
Theforeign
bioethanol
supposes
containing
25used
mL
buffer
of
Mintervals,
sodium
citrate
Samples
were
taken
at 0.029
forfor
The
useuse
ofofbioethanol
supposes
a reduction o
pretreatment
were
for
enzymatic
hydrolysis.
3.2 Results
and
discussion
greenhouse
gas
emissions
[13].
Napier
grass
(Pen- oil and red
min
denature
enzymes,
and
analyzed
RSY dependence
dependence
upon
foreign
expensive
buffermin
(pH
4.8)
at performed
8 the
%
(w/v)
dry
pretreated
Enzymatic
hydrolysis
was
inand
100mL
to to
denature
the
enzymes,
analyzed
forfor
RSY
upon foreign expensive oil and reduc
nisetum were
purpureum
Schumach.)gas
biomass
can be
an Napier g
concentrations
[39].
experiments
greenhouse
emissions
[13].
substrate
loading
50
°CAll
atAll
150
pmrbuffer
for
100 min.
Erlenmeyer
flasks,
eachatcontaining
25hydrolytic
mL
concentrations
[39].
hydrolytic
experiments
were
greenhouse
gas
emissions
[13].
Napier gra
adequate
substrate
for ethanol
production
due to
performed
in
triplicate
and
the
average
results
are
(Pennisetum
purpureum
Schumach.)
biomass
can
Samples
were citrate
taken
at
timed
intervals,
boiled
for
2
The
use
of
bioethanol
supposes
a
reduction
our
of 0.029
M performed
sodium
buffer
(pH
4.8)
at
8
%
in triplicate and the average results
arecarbohydrate
(Pennisetumcontent
purpureum
can
beb
the high
of Schumach.)
60.0 % of biomass
the
showninsubstrate
inFigure
3.Data
Data
of
enzymatic
hydrolysis
adequate
substrate
forethanol
ethanol
production
du
toshown
denature
theFigure
enzymes,
andof
(w/v) min
dry pretreated
atanalyzed
50
°C atfor RSY
dependence
upon
foreignfor
expensive
oilproduction
and
reducesdue
3.loading
enzymatic
hydrolysis
adequate
substrate
dry weight. The
hemicellulose
fraction
comprises
150 pmr
foryield
100
min.
Samples
were
takenexperiments
atin
timed
yield
(EHY)
was
calculated
100g ggreenhouse
of the
high
carbohydrate
content
of
60.0
concentrations
[39].
All
hydrolytic
were
gas
emissions
[13].
Napier
grass
(EHY)
was
calculated
in
mgmgperper
100
high
carbohydrate
60.0
%%
ofof
thethe
d
24.7
% of rawthe
material,
xylose
being content
the
mainofsugar
intervals,
boiled
for
2
min
to
denature
the
enzymes,
pretreated
substrate
by
Eq
3:
weight.
The
hemicellulose
fraction
comprises
performed
in
triplicate
and
the
average
results
are
(Pennisetum
purpureum
Schumach.)
biomass
can
be
an
pretreated substrate by Eq 3:
weight.
fraction
(80%). Cellulose
and The
ligninhemicellulose
content is around
35.3comprises 24
and analyzed
RSY 3.concentrations
[39]. All
%
of
raw
material,
xylose
being
main
shown inforFigure
Data of enzymatic
hydrolysis
adequate
substrate
for
ethanol
production
due
to sugs
% and 8.0 %,%respectively.
From literature
review,thethe
of raw material,
xylose being
main
hydrolytic
experiments
were
performed
in
triplicate
Cellulose
and
lignin
content
isaround
around
yield (EHY) was calculated in mg per 100 gparticle
of the
carbohydrate
content
ofimportant
60.0
% ofis the
dry 35
sizehigh
has(80%).
notCellulose
considered
aslignin
an
(80%).
and
content
and thepretreated
average results
are
shown
in
Figure
3.
Data
%
and
8.0
%,
respectively.
From
literature
rev
substrate
by Eq 3:
weight.
The
fraction
comprises
24.7
!"#"$%"& !"#$%&'( !"#$%! ! !.!
parameter
in %pretreatment
lignocellulose
bioand hemicellulose
8.0 %, of
respectively.
From
literature
revie
!"#"$%"& !"#$%&'( !"#$%! ! !.!
EHY
=
(3
of enzymaticEHY
hydrolysis
yield
(EHY)
was
calculated
= !"#$%&' !"#$ !"##$#%&" (!"#)
(3 [33,
particle
sizehashas
considered
impo
% 38,
ofparticle
raw
material,
xylose
being
the main
sugar
mass
39].
!"#$%&' !"#$ !"##$#%&" (!"#)
size
notnotconsidered
as asanan
importa
in mg per 100 g of pretreated
substrate by Eq 3:
parameter
in
pretreatment
of is
lignocellulose
biom
(80%). Cellulose
lignin content
around 35.3
inand
pretreatment
bioma
In our study,parameter
a dilute acid
pretreatmentofoflignocellulose
napier
[33,
38,
39].
% and[33,
8.0
respectively.
From literature review,
!"#"$%"& !"#$%&'( !"#$%! ! !.!
38,%,
39].
grass
(P. purpureum)
biomass
was done under differ(3)fraction
The
water-insoluble
phenolic
analyzed
EHY =
(3was
The
water-insoluble
phenolic
fraction
was
analyzed
!"#$%&' !"#$ !"##$#%&" (!"#)
study,
aconsidered
dilute
acid
na
particle
size
has
nota dilute
aspretreatment
an important
InIn
ourourstudy,
acid
pretreatment
ofofnap
ent process
temperature
(122
°C),
solid
UV–visvisspectroscopy
spectroscopyat at280
280nm.
nm.Glucose
Glucose
andparameters:
bybyUV–
and
(P.purpureum)
purpureum)
biomass
wasdone
done und
u
parameter
in(P.
pretreatment
ofbiomass
lignocellulose
biomass
concentration
ingrass
the
reactor
(2-6%,
w/v),
sulfuric
was
xyloseyield
yieldfrom
from
peroxide-enzymatic
hydrolysis grass
The water-insoluble
phenolic
fraction
was analyzed hydrolysis
xylose
peroxide-enzymatic
different
process
parameters:
temperature
(122
[33, 38,
39]. (0.5–1.0
acid
concentration
%, parameters:
w/w)
and size
pardifferent
process
temperature
(122
°C
wereanalized
analized
byHPLC-Trangenomic
HPLC-Trangenomic
ION-300
The
fraction
by UV–
viswater-insoluble
spectroscopy
atphenolic
280
nm.
Glucosewas
andanalyzed
were
by
ION-300
solid
concentration
in
the
reactor
(2-6%,
w
In– our
study,
a
dilute
acid
pretreatment
of
napier
ticle
(2.0
5.0
mm).
column
(oventemperature
temperature
at4545
with
isocratic solid concentration in the reactor (2-6%, w/
UV–
vis spectroscopy
at 280 at
nm.
Glucose
and
xylosebyyield
from
peroxide-enzymatic
hydrolysis
column
(oven
°C)°C)with
isocratic
grass (P. purpureum) biomass was done under
On Figure 1, shows the estimated response surface
xylose yield
from peroxide-enzymatic
hydrolysis
were analized
by HPLC-Trangenomic
ION-300
different process parameters: temperature (122 °C),
(R2 = 0.79) for acid pretreatment of napier grass
column
(ovenanalized
temperature
at 45 °C) with isocratic
were
by HPLC-Trangenomic
ION-300
solidincreases
concentration
in thebiomass
reactorfeed.
(2-6%,
where RSY
with decrease
A w/v),
elutioncolumn
(flow rate
0.4 ml/min;
mobile
phase:
SO4isocratic
(oven
temperature
at 45
°C) Hwith
2
0.005 N). Standard samples were prepared for low RSY production of 15.1 % for a biomass feed
of 8.0 %.
determination.
50 AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953
/w) and
r acid
id and
e surface
ier grass
s feed. A
mass feed
at 2.0%
%. The
d at high
mical and
d charge
ndustrial
sulfuric acid concentration (0.5–1.0 %, w/w) and
size particle (2.0 – 5.0 mm).
around 3 % (w/w). But for biomass concentration
above 6 % (w/w) the RSY is around 10 %. RSY %
varies from 77 - 98% from 30 - 60 min residence
time. Pretreated WISH2SO4 were washed 3 volumes
of water prior to enzymatic hydrolysis to remove
inhibitors [39]. The WISH2SO4 obtained from the
peroxide pretreatment was carefully washed and
used to enzymatic hydrolysis for 120 min.
Figure
1. Estimated
response
surface
for acid
Figure
1. Estimated
response
surface for
acid pretreatment
showing the influence
acidinfluence
and biomass
RSY.
pretreatment
showingofthe
of on
acid
and
biomass on RSY.
On Figure 1, shows the estimated response surface
(R2 = 0.79) for acid pretreatment of napier grass
where RSY increases with decrease biomass feed. A
low RSY production of 15.1 % for a biomass feed
of 8.0 %.
From the above results it was decided to use
washed WISH2SO4 at 15.1% RSY (2.455±0.02
g/L) dilute acid (0.5 %, w/w), biomass feed (8 %,
w/v), size particle (4.75 mm) and residence time
2. the
Estimated
response surface
acid
(30 Figure
min) for
next pretreatment
with for
a peroxpretreatment
showing
the
influence
of
size
particle
ide solution (0. 4 - 0.7 %,w/w). The experimental
and11,4
biomasa
RSY. pretreatment
conditions at pH
for on
second
were biomass feed (8 %, w/v), residence time (72
h),
mm),2,130
at 30 °C
was done
As particle
shown in(4.5
Figure
tworpm
important
groups
of data
on
WIS
.
H2SO
with high
RSY
production at 2.5 mm and 4.5 mm
4
2
are found.
the results
particlethesize
(r =0.81)
From
results
on Figure
3, the From
best RSY
was
on Figure
the best
was3 found
at 60But
minfor
and
found
at 603, min
and RSY
around
% (w/w).
around 3concentration
% (w/w). Butabove
for biomass
concentration
biomass
6 % (w/w)
the RSY
isabove
around
%. the RSY is around 10 %. RSY %
6 %10(w/w)
variesperoxide
from 77 pretreatment
- 98% from has
30 -showed
60 mintoresidence
Even
be an
time.
Pretreated
𝑊𝑊𝑊𝑊𝑊𝑊
were
washed
efficient agent procedure. However,
the cost of 3
!! !"!
peroxide,
andwater
the high
of reagentto
volumes of
prior concentration
to enzymatic hydrolysis
used,
deeply
affects
the
bioethanol
production.
remove inhibitors [39]. The 𝑊𝑊𝑊𝑊𝑊𝑊!! !! obtained from
For
this reason,
new studies
to carefully
decreasewashed
peroxide
the peroxide
pretreatment
was
and
concentration
without
affecting
RSY
are
useful
used to enzymatic hydrolysis for 120 min.
[15].
From the above results it was decided to use
washed 𝑊𝑊𝑊𝑊𝑊𝑊!! !"! at 15.1% RSY (2.455±0.02 g/L)
60 min and
dilute acid (0.5 %, w/w), biomass feed (8 %, w/v),
% (w/w)
size particle (4.75 mm) and residence time (30 min)
explosion (
for the next pretreatment with a peroxide solution
mg/(g initia
(0. 4 - 0.7 %,w/w) . The experimental conditions at
initial dry s
pH 11,4 for second pretreatment were biomass feed saccharificat
(8 %, w/v), residence time (72 h), particle (4.5 mm), treatments o
130 rpm at 30 °C was done on 𝑊𝑊𝑊𝑊𝑊𝑊!! !"! .
produced xy
The maximum of RSY of 98 % was found at 2.0%
(w/v) biomass concentration, and acid 0.8 %. The
results suggest that RSY is negative affected at high
biomass concentration. From the economical and
environment perspectives low biomass feed charge
implies an important limitation to the industrial
Figure 2. Estimated response surface for acid pretreatment
process
[40,41].
showing the influence of size particle and biomasa on RSY.
Figure 2. Estimated response surface for acid
pretreatment showing the influence of size particle
and biomasa on RSY.
The maximum of RSY of 98 % was found at 2.0%
(w/v) biomass concentration, and acid 0.8 %. The
As
shown
in Figure
2, two
groupsatofhigh
data
results
suggest
that RSY
is important
negative affected
with
highconcentration.
RSY production
at 2.5
and 4.5 and
mm
biomass
From
the mm
economical
2
particle
size (rperspectives
=0.81) are low
found.
Fromfeed
thecharge
results
environment
biomass
on
Figure
3,
the
best
RSY
was
found
at
60
min
and
implies an important limitation to the industrial
230 mg/10
around
% (w/w). But for biomass concentration
process3[40,41].
alkaline hyd
above 6 % (w/w) the RSY is around 10 %. RSY %
glucose yield
As shown in Figure 2, two important groups of data
varies from 77 - 98% from 30 - 60 min residence
pretreated
with high RSY production at 2.5 mm and 4.5 mm
time.
Pretreated
were the
washed
!! !"! From
are found.
results3 Figure 3. Estimated response surface for acid pretreatment pretreated f
particle size
(r2 =0.81)𝑊𝑊𝑊𝑊𝑊𝑊
volumes
prior
enzymatic
on Figureof3,water
the best
RSYtowas
found athydrolysis
60 min andto showing the effect of biomass and time residence on RSY.
peroxide [1
Figure
3.
Estimated
response
surface
for
acid
remove inhibitors [39]. The 𝑊𝑊𝑊𝑊𝑊𝑊!! !! obtained from
material (18
pretreatment
the2 effect
of biomass
and
AVANCES Investigación
en Ingenieríashowing
Vol. 11 - No.
(2014) ISSN:
1794-4953(2014)
51
°C for 24 h
the peroxide pretreatment was carefully washed and
time residence on RSY.
raw materia
used to enzymatic hydrolysis for 120 min.
[42]. These
From the above results it was decided to use
ned from
de 0.7 %
min. The
differences
n samples
nd 0.7 %.
.81 mg/g
81 mg/(g
tively, by
dilute acid
d 146 mg
) and, on
5% (w/w),
peroxide [15]. Also, a combined steam-pretreated
material (184 °C for 4min) and 2% peroxide at 60
°C for 24 h, obtain a glucose yield of 503.5mg/g
raw material with a substrate concentration of
3.3% [42]. These pretreatment are very expensive
because the peroxide high concentration and the
low substrate concentration used.
300
250
200
150
100
50
0
0
20
40
60
80
100
120
Time (min)
Figure 4.
Figure
4. Experimental
Experimental dependence
dependence of
of Glucose
Glucoseand
andXylose
Xylose
production
on
time
at
several
peroxide
concentrations
by by
production on time at several peroxide concentrations
enzymatic
hydrolysis.
Peroxide:
0.3
%(w/w),
Glucose
(
�
),
enzymatic hydrolysis. Peroxide: 0.3 %(w/w), Glucose (□),
Xylose
� ); 0.5
( �(○),
), Xylose
Xylose( (■);
0.5 %%(w/w),
(w/w),Glucose
Glucose
Xylose( �
(•); 0.5
0.5 %
%
(w/w),
( � ), Xylose ( ◊ ).
(w/w), Glucose
(♦),Glucose
Xylose (◊).
The peroxide pretreatment of 𝑊𝑊𝑊𝑊𝑊𝑊!! !"! , released
glucose
proceed
from
Figure should
4 shows
97.6 %
of hemicelluloses
RSY obtainedmainly.
from
From
literature,
it
was
found
that
pretreatment
of
washed WISH2SO4 treated with peroxide 0.7 %
sawdust
with
acid 1%,hydrolysis
sawdust-to-acid
(w/w) by
enzymatic
at 75 (w/v)
min. ratio
The
The peroxide pretreatment of WISH2SO4, released
glucose should proceed from hemicelluloses
mainly. From literature, it was found that pretreatofment
1:10, of
120°C
at 15with
psi for
min.,
then the acid
sawdust
acid401%,
sawdust-to-acid
(w/v) ratio of
of 1:10,
120°C
at 15 psi
for peroxide
40 min.,
hydrolyzate
sawdust
treated
with
then
the acid47hydrolyzate
sawdust treated with
1%
produced
- 56 % RSYof[43,44].
peroxide
1%
produced
47
56
RSY [43,44].and
The grass P. purpureum with %cellulose
hemicellulose
40%cellulose
and 30%,and
andhemilow
The grass P.fractions
purpureumof with
lignin
content
(23.89
%)
respectively
showed
to
be
cellulose fractions of 40% and 30%, and lowa
promising
biomass
for %)
highrespectively
RSY production
lignin content
(23.89
showedand
to
low
inhibitors
substances
[33].The
analysis
of
the
be a promising biomass for high RSY production
liquor
afterinhibitors
peroxidesubstances
pretreatment
showed
low
and low
[33].The
analysis
of the liquor after
peroxide
pretreatment
concentrations
of 0.33
g furfural/L
and showed
0.73 g
low concentrations of 0.33
g furfural/L
and 0.73
hidroximetilfurfural/L.
Furfural
is considered
g
hidroximetilfurfural/L.
Furfural
is
considered
affect hydrolysis above 5 g/L only after 48 hours
affect hydrolysis above 5 g/L only after 48 hours
[45].
[45].data of the RSY resulted from the enzymatic
The
hydrolysis
napierresulted
grass pretreated
found to
The data of
of the
the RSY
from the enzymatic
depend
on the
concentration.
hydrolysis
of peroxide
the napier
grass pretreated found to
depend on the peroxide concentration.
compositional analysis shows significant differences in glucose and xylose production between
samples treated with peroxide at 0.3 %, 0.5 % and
0.7 %. The peroxide treatment produced 287.81
mg/g (initial dry sample) of glucose and 245.81
mg/(g initial dry sample) of Xylose, respectively,
by enzymatic hydrolysis after 80 min. In the dilute
acid pretreatment, on rye straw it was found 146
mg xylose/g (acid 1.5%, 90 min, 121 °C) and, on
100
Bermuda grass 137 mg xylose/g, acid 1.5% (w/w),
90
0.7 %
80
60 min and 121 °C. 37 But also, alkaline peroxide
70
10 % (w/w) pretreatment combined with steam
60
explosion (20 atm) on bamboo produced 456
0.5 %
50
mg/(g initial dry sample) of glucose and 460 mg/
40
(g initial dry sample) of reducing sugar by enzy30
matic saccharification at 20 h. [19]. The peroxide
20
0.3 %
treatments of sucarcane bagasse at 70 °C for 48
10
h produced xylose and glucose as 250 mg/100 g
0
0
20
40
60
80
100
120
and 230 mg/100 g respectively [22]. However, by
Time alkaline hydrogen peroxide treatment the higher
glucose yield: 691 mg/ g of glucose were found Figura 5. Experimental dependence of RSY (%) on time
5. Experimental dependence of RSY (%) on
for pretreated bagasse after hydrolysis of bagasse Figura
at several peroxide concentrations by enzymatic hydrolysis.
time
at(w/w):
several
Peroxide %
0.3peroxide
%, (○), 0.5concentrations
% (□); 0.7 % (◊). by
pretreated for 1 h at 25 °C with 7.35% (v/v) of
enzymatic hydrolysis. Peroxide % (w/w): 0.3 %,
(�), 0.5 % (�); 0.7 % (◊).
52 AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953
RSY (%)
to be an
e cost of
of reagent
ction. For
peroxide
seful [15].
[42]. These pretreatment are very expensive because
the peroxide high concentration and the low
substrate concentration used.
Glucose and Xylose production (mg/g)
RSY was
). But for
he RSY is
Conclusions
%, w/w),
(2.36 - 4.7
on RSY p
done. Th
± 0.255 g
biomass f
residence
In this s
napier gr
enhance
substrate
combinat
dramatica
substrate
peroxide
productio
substrate
yield (R
relationsh
paramete
mathema
data. On
% (w/w)
concentra
enzymatic
hydrolysi
sample)
sample) o
peroxide
environm
hydrolysi
Acknow
The auth
this resea
Merquian
Industria
Conclusions
References
Our objective was to combine the effect of
peroxide acid pretreatment to enhance RSY by
enzymatic hydrolysis. A factorial design based
on the variation of the pretreatment conditions
of the dilute sulfuric acid pretreatment at loading (0.5–1.0 %, w/w), residence time (30 – 90
min), particle size (2.36 - 4.76 mm) and solid/
liquid ratio (2–8%, w/v) on RSY production in a
batch reactor at 122 °C was done. The best RSY
production of 99.71 % (7.001 ± 0.255 g/L) was
found at dilute acid (1 %, w/w), biomass feed (2
% w/v), size particle (2.36 mm) and residence
time (90 min).
1. Sheehan, J., Himmel, M. (1999) Enzymes, energy,
and the environment: Cellulose development in the
emerging bioethanol industry. Biotechnol Progr, 15,
817-827
In this study, peroxide - acid pretreatment of
napier grass (P. purpureum) were investigated to
enhance RSY enzymatic hydrolysis as a potential
substrate for bioethanol production. The combination of peroxide acid pretreatment dramatically
improved the hydrolyzability of the substrate
(WISH2SO4). This study aims to combine peroxide
–acid dilute pretreatment to low cost production.
A pretreatment of the water insoluble substrate
(WISH2SO4) at 15.1 % of reducing sugar yield
(RSY) was done with peroxide. The relationship
between RSY and the acid process parameters
could be well described by a mathematical model
derived from the experimental data. On Figure
5, peroxide acid pretreatment at 0.7 % (w/w),
4.75 mm particle size, 8% (w/v) biomass concentration, improved the RSY production from
enzymatic hydrolysis in 97.6 %. After enzymatic
hydrolysis, this produced 287.81 mg/(g initial dry
sample) of glucose and 245.81 mg/(g initial dry
sample) of Xylose. Therefore, it was concluded
that peroxide acid pretreatment is an effective and
environmentally friendly method for the enzyme
hydrolysis of napier grass.
Acknowledgments
The authors are grateful for the financial support of
this research from Pharmavicola – Unipamplona,
Merquiant-Genencor, Colombia, to the project of
Industrial alcohol production.
2. Sakai, S., Tsuchida, Y., Nakamoto, I., Okino,
S., Ichihashi, O., Kawaguchi, H., et al. (2007).
Effect of lignocellulose-derived inhibitors on
growth of and ethanol production by growtharrested Corynebacterium glutamicum R. Appl
Environ Microbiol 73, 2349-2353.
3. Anderson, W., Peterson, J., Akin, D., Morrison W. (2005). Enzyme Pretreatment of
Grass Lignocellulose for Potential High-Value
Co-products and an Improved Fermentable
Substrate. Applied Biochem Biotechnol, 121, 303310.
4. Ballesteros, I., Ballesteros, M., Manzanares, P.,
Negro, J., Oliva, J, Sáez, F. (2008). Dilute sulfuric acid pretreatment of cardoon for ethanol
production. Biochem Eng. J., 42, 84-91.
5. Mosier, N., Wyman, C., Dale, B., Elander, R.,
Lee, Y., Holtzapple, M., Ladisch, M. (2005).
Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour.
Technol, 96, 673-686.
6. El-Zawawya, W., Ibrahima, M., Abdel-Fattahb,
Y., Nadia, A., Solimanb, N., Mahmoudc, M.
(2011). Acid and enzyme hydrolysis to convert
pretreated lignocellulosic materials into glucose
for ethanol production. Carbohydr Polymers, 84,
865-871.
7. Chen, W., Tu, Y., Sheen, H. (2010). Impact of
dilute acid pretreatment on the structure of
bagasse for bioethanol production. Int J Energy
Res, 34, 265-274 (2010).
8. Jung. S., Foston, M., Sullards, M., Ragauskas
A. (2010). Surface characterization of dilute
acid pretreated Populus deltoides by ToF-SIMS.
Energy Fuels, 24,1347-1357.
9. Mao, J., Holtman, K., Franqui-Villanueva D.
(2010). Chemical structures of corn stover and
its residue after dilute acid prehydrolysis and
AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953(2014) 53
enzymatic hydrolysis: Insight into factors limiting enzymatic hydrolysis. J. Agric. Food Chem, 58,
11680-11687.
10.Pingali, S., Urban, V., Heller, W., McGaughey,
J., O’Neill, H., Foston, M., et al. (2010). Breakdown of cell wall nanostructure in dilute acid
pretreated Biomass. Biomacromol, 11, 2329-2335.
11.Sannigrahi, P., Ragauskas, A., Miller, S. (2008).
Effects of two-stage dilute acid pretreatment
on the structure and composition of lignin and
cellulose in loblolly pine. Bioenergy Res, 1, 205214.
12.Zhang, J., Ma, X., Yu, J., Zhang, X., Tan, T.
(2011). The effects of four different pretreatments on enzymatic hydrolysis of sweet
sorghum bagasse. Bioresour. Technol. 102, 45854589.
13.Elander, R., Dale, B., Holtzapple, M., Ladisch,
M., Lee, Y., Mitchinson, C., et al. (2009). Summary of findings from the Biomass Refining
Consortium for Applied Fundamentals and
Innovation (CAFI): corn stover pretreatment.
Cellulose 16,649-659.
14. Quintero, J., Montoya, M., Sánchez, O., Giraldo,
O., Cardona, C. (2008). Fuel ethanol production
from sugarcane and corn: comparative analysis
for a Colombian case. Energy, 33, 385-399.
15.Rabelo, S., Amezquita-Fonseca, N., Andrade,
R., Filho, M., Costa, A. (2011). Ethanol production from enzymatic hydrolysis of sugarcane
bagasse pretreated with lime and alkaline hydrogen peroxide. Biomass Bioenergy, 35, 2600-2607.
16.Gould, J. (1985). Studies on the mechanism of
alkaline peroxide delignification of agricultural
residues. Biotechnol Bioeng, 27, 225-231.
17.Lachenal, D., De Choudens, C., Monzie, P.
(1980). Hydrogen peroxide as a delignifying
agent. Tappi J, 63, 119-122 .
18.Azzam, A. (1989). Pretreatment of cane
bagasse with hydrogen peroxide for enzymatic
hydrolysis of cellulose and ethanol fermentation. J. Environ. Sci. Health B., 24, 421-433.
19.Yamashita, Y., Shono, M., Sasaki, Ch., Nakamura, Y. (2010). Alkaline peroxide pretreatment
for efficient enzymatic saccharification of
bamboo. Carbohydr Polym, 79 , 914–920.
20.Ayeni, A., Hymore, F., Mudliar, S., Deshmukh,
S., Satpute, D., Omoleye, J., Pandey, R. (2013).
Hydrogen peroxide and lime based oxidative pretreatment of wood waste to enhance
enzymatic hydrolysis for a biorefinery: Process
parameters optimization using response surface
methodology. Fuel, 106, 187-194.
21.Yáñez, R., Alonso, J., Parajó, J. (2006). Enzymatic
saccharification of hydrogen peroxide-treated
solids from hydrothermal processing of rice
husks. Process Biochem, 41,1244-1252.
22.Gao, M., Yano, S., Inoue, H., Sakanishi, K.
(2012). Combination of wet disk milling and
hydrogen peroxide treatments for enhancing
saccharification of sugarcane bagasse. Biochem
Eng J., 68,152-158 .
23.Tan, H., Yang, R., Sun, W., Wang, S. (2010).
Peroxide Acetic Acid Pretreatment to Remove
Bagasse Lignin Prior to Enzymatic Hydrolysis.
Ind Eng Chem Res, 49, 1473-1479.
24.Sun, R., Tomkinson, J., Zhu, W., Wang, S.
(2000). Delignification of maize stems by
peroxy-mono-sulfuric acid, peroxy-formic
acid, per-acetic acid, and hydrogen peroxide.
1. Physicochemical and structural characterization of the solubilized lignins. J Agric Food Chem,
48,1253-1262.
25.Nimz, H., Schwind, H. (1979). Oxidation of
monomeric lignin model compounds with peracetic acid. Cellulose Chem Techno,13, 35-46.
26.Yuan, Z., Ni, Y., Heiningen, A. (1998). An
improved peracetic acid bleaching process.
Appita J., 51, 377-380.
27.Teixeira, L., Linden, J., Schroeder, H. (2000).
Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass. Appl
Biochem Biotechnol, 84-86,111-127.
28. Teixeira, L., Linden, J., Schroeder, H. (1999).
Optimizing peracetic acid pretreatment conditions for improved simultaneous saccharification
and co-fermentation (SSCF) of sugar can bagasse
to ethanol fuel. Renew Ener, 16,1070-1073.
54 AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953
29.Khama, L., Bigot, Y., Delmas, M., Avignon, G.
(2005). Delignification of wheat straw using a
mixture of carboxylic acids and peroxoacids Ind
Crops Prod, 21, 9-15.
37.Sun, Y., Cheng, J. (2005). Diluted acid pretreatment of rye straw and Bermuda grass for
ethanol production. Bioresource Technol., 96,15991606.
30.
National Renewable Energy Laboratory
(NREL). (2008). Chemical Analysis and
Testing Laboratory Analytical Procedures:
TP-510-42620, TP-510-42619, TP-510-42621,
TP-510-42622, TP-510-42623, TP- 222 om-98,
Golden, USA. www.ott.doe.gov/biofuels/analytical methods.html.
38.Anderson, W., Adien, B., Brandon, S., Peterson
J. (2008).Assesment of Bermuda grass and
bunch grasses as feedstock for conversion to
ethanol. Appl. Biochem. Biotechnol, 145, 13-21.
31.Miller, G.(1959). Glucose DNS Metodology.
Anal Chem, 31, 426-428.
32.Ghose, T. (1987). Measurement of cellulase
activities. Pure Appl Chem, 59, 257-268.
33.Madakadze, C., Masamvu, T., Radiotis, T., Li, J.,
Smith, D. (2010). Evaluation of pulp and paper
making characteristics of elephant grass (Pennisetum purpureum Schum) and switchgrass
(Panicum virgatum L.). African J Environ Sci
Technol, 4,465-470.
34.López , A., Ortegón, G., Robles, F. (2010).
Obtaining of Reducing Sugars from Kikuyu
Grass (Pennisetum Clandestinum). Avances
Invest Ing., 13:98-101.
35.Prinsen, P., Gutiérrez, A., Del Río, J. (2012).
Lipophilic Extractives from the Cortex and
Pith of Elephant Grass (Pennisetum purpureum
Schumach.) Stems. J Agric Food Chem., 60, 64086417.
36.Del Río, J., Prinsen, P., Rencoret, J., Nieto, L.,
Jiménez-Barbero, J., Ralph, J., et al. (2012).
Structural Characterization of the Lignin in
the Cortex and Pith of Elephant Grass (Pennisetum purpureum) Stems. J Agric Food Chem.,
60, 3619–3634.
39.Hodge, D., Karim, M., Schell, D., Macmillan, J.
(2008). Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of
lignocellulose. Bioresour. Technol, 99, 89408948.
40.Magcale – Macandog, D., Predo, C., Menz, K.
(1998). Napier grass strips and livestock: a bioeconomic analysis. Agrofores Sys., 40, 41-58.
41. Somerville, Ch., Youngs, H., Taylor, C., Davis,
S., Long, S. (2013). Feedstocks for lignocellulosic biofuels. Scien., 329, 790-792.
42.Su, Z., Bu, L., Zhao, D., Sun, R., Jiang, J. (2012).
Processing of Lespedeza stalks by pretreatment
with low severity steam and post-treatment
with alkaline peroxide. Ind Crops and Prod., 36,
1-8.
43.Ogundipe, A., Lu, Y. (1989). Ethanol production from dilute acid hydrolyzate of sawdust
treated with hydrogen peroxide. Biomass., 20,
291-299.
44.Kupiainen, L., Ahola, J., Tanskanen, J. (2012).
Distinct Effect of Formic and Sulfuric Acids
on Cellulose Hydrolysis at high temperatures.
Ind Eng Chem Res., 51,3295-3300.
45.Arora, A., Martin, E., Pelkki, M., Carrier, D.
(2013). Efect of Formic Acid and Furfural on
the Enzymatic Hydrolysis of Cellulose Powder
and Dilute Acid-Pretreated Poplar Hydrolysates. ACS sustainable Chem Eng., 1, 23-28.
AVANCES Investigación en Ingeniería Vol. 11 - No. 2 (2014) ISSN: 1794-4953(2014) 55