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Influence of heating source on the efficacy of lignocellulosicpretreatment e A cellulosic ethanol perspective

Jagdish Gabhane, S.P.M. Prince William*, Atul Narayan Vaidya, Kalyani Mahapatra,Tapan Chakrabarti

Solid and Hazardous Waste Management Division, National Environmental Engineering Research Institute, Nehru marg, Nagpur 440020,

Maharashtra, India

a r t i c l e i n f o

Article history:

Received 8 October 2009

Received in revised form

23 July 2010

Accepted 4 August 2010

Available online 9 September 2010

Keywords:

Heating devices

Autoclave

Hot plate

Microwave

Pretreatment

Lignocellulosic biomass

a b s t r a c t

Three different heating devices (Hot plate (HP), Autoclave (AC) & Microwave (MW)) were

tested for their efficiency to pretreat garden biomass (GB), a lignocellulosic substrate.

Effectiveness of different modes of heat on pretreatment was assessed taking into

consideration the yield of reducing sugar (RS), recovery of cellulose after pretreatment,

conversion of hemicellulose into reducing sugar, changes in the ultra structure of GB

tissues, changes in the crystallinity of GB etc. The results indicated that all three heating

devices are useful for pretreatment, however, the efficacy of MW on GB was found be better

than AC and HP. A maximum of 53.95% of cellulose recovery was obtained in case of MW

heating along with 46.97% of reducing sugar yield. This when compared to AC and HP is

significantly higher (more than 10% increase) and time saving (only 15 min reaction time)

as well.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Increasing demand for fossil fuel has forced the global scien-

tific community to intensify research on alternative and

renewable energy sources. Bioethanol is the green and clean

fuel tried world wide for different energy purposes. Lignocel-

lulosic materials can be used as substrates for the production

of ethanol through microbial intervention because they are

abundant, cheap and renewable [1]. However, the recalcitrant

structure of lignocellulosic biomass makes the hydrolysis to

monomeric sugars more difficult as compared with starch [2].

Although different kinds of pretreatment processes have been

proposed the major challenge remains is the development of

cost-effective pretreatment technology, which can help

cellulosic ethanol production economically viable.

Pretreatment is an important tool for practical cellulose

conversion process, which is required to alter the structure of

cellulose and to make it more accessible to the enzymes that

convert carbohydrate polymers into fermentable sugars [3,4].

Thus, the general idea of pretreatment is to remove or alter the

hemicellulose or lignin, decrease the crystallinity of cellulose

and increase thesurfacearea. Pretreatmentmethodsareeither

physical or chemical. Some methods incorporate both effects

[5].However,most of thepretreatment technologies are energy

intensive in nature. Among the chemical pretreatment

methods, dilute acid pretreatment has been widely studied

because it is effective and inexpensive [6]. However, it also

needs some kind of thermal energy for the hydrolytic process.

Supply of heat (thermal energy) is crucial for pretreatment

process as it mediates bond breaking between molecules

* Corresponding author. Tel./fax: þ91 712 2249752.E-mail address: prince_environment@yahoo.com (S.P.M. Prince William).

Avai lab le at www.sc iencedi rect .com

ht tp : / /www.e lsev ier . com/ loca te /b iombioe

b i om a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 9 6e1 0 2

0961-9534/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biombioe.2010.08.026

Author's personal copy

during chemical action. A perusal of literature survey showed

the utility of different kind of heating devices for the

pretreatment of lignocellulosic feed stocks. Generally,

pretreatment studies utilize conventional heating devices

such as hot plate, heating mantles, and burners etc. for the

supply of heat. These techniques are rather slow and create

a temperature gradient within the sample. Steam pretreat-

ment (with pressure) is also equally preferred for the hydro-

lysis of lignocellulosic feed stocks. Steam pretreatment

supplies moist heat under pressure which results in

substantial breakdown of the lignocellulosic structure,

hydrolysis of the hemicellulosic fraction, depolymerization of

the lignin components and defibration [7].

In addition, microwave irradiation has also been used for

lignocellulosic pretreatments since long ago [8,9] Microwave

generates thermal energy through dielectric heating and the

energy is introduced into the reactor remotely without any

contact between the energy source and the reaction mixture

[10]. Microwave irradiation could change the ultra structure of

cellulose, degrade lignin and hemicellulose and increase the

enzymic susceptibility of RS [11].

The mode of heat generation and transfer varies

according to the device used. In case of hot plate heat is

generated through heating coils and gets transferred to the

substrate (lignocellulose) through convective mode of heat

transfer. Whereas, in case of steam pretreatment moist

heat is employed with pressure to facilitate disruption of

lignocelluloses. Microwave on the other hand causes

cellulosic breakdown mainly through molecular collision

due to dielectric polarization. Although different kind of

heating devices were employed for the pretreatment of

different lignocellulosic substrates, hardly any study

address the comparison of these heating devices for their

effectiveness in pretreatment. Further, it is well known

that the optimization of pretreatment parameter for each

different feed stock is an important issue when enhancing

the conversion efficiency during lignocellulosic biomass to

ethanol process [12].

Therefore, in the present investigation an attempt has been

made to find out the influence of different modes of heating on

thepretreatmentofGB.Theefficacyofdifferentmodesofheating

was assessed through studying the rate of hemicellulose degra-

dation, removal of lignin, changes in the crystallinity of cellulose

and impacts on the ultra structure of GB tissues.

2. Materials and methods

2.1. Collection and processing of feed stock

GB consisting mostly (70e80%) of grasses and fallen leaves

along with small (10e20%) portion of weeds was collected

from NEERI garden and sun dried for 2e3 days followed by

oven drying at 70 �C for about 92 h in hot air oven. The

oven-dried material was powdered in a pulverizer and

sieved through a 1 mm sieve. This powder was subjected to

compositional analysis using standard protocols as per

Section 2.5. All experiments were carried out with

a minimum of three replicates and the mean values are

reported.

2.2. Mild acid pretreatment using hot plate (HP)

500mg of powdered substrate (GB) was taken in a 250ml conical

flask and was added with 50ml of 5% H2SO4 (V/V). The contents

weremixedwell andboiled at 100 �C for 30minusing ahot plate.

After cooling the content was neutralized using NaOH.

The neutralized mixture was filtered through Whatman filter

paper e 42 and the filtrate was analyzed for reducing sugar

concentration using DNS method [13] and the yield (%) of

reducing sugar (mostly the pentoses as a result of hemicellulose

degradation and small amount of glucose from cellulose

degradation) was calculated according to the formula:

Reducing sugaryieldð%Þ¼Reducing sugar inmg=ml�VolðmlÞSubstrate inmg

�100

The residue was carefully removed and subjected to

compositional analysis using standard protocols as per

Section 2.5.

2.3. Mild acid pretreatment using autoclave (AC)

500mg of powdered substrate (GB) was taken in a 250ml conical

flask and was added with 50ml of 5% H2SO4 (V/V). The contents

were mixed well and kept in an autoclave for heating. The

temperature of the autoclavewas set at 121 �Cwith a pressure of

15 lbs and the contents were autoclaved for 30 min and pro-

cessed further as per Section 2.2 using standard protocols.

2.4. Mild acid treatment using microwave (MW)

The microwave digester used for the present study was Mile-

stone (Model-Ethos 900). 500mg of powdered substrate (GB) was

taken in a 250 ml conical flask and was added with 50 ml of 5%

H2SO4 (V/V). The contentswere placed in specializedmicrowave

sample holders made of Teflon. Experiments were conducted at

two (100 �C and 200 �C, 700W) different temperatures usingMW

with 15min as reaction time. The reaction conditionswere fixed

based on our preliminary experiments, wherein we tested an

array of conditions involving different concentrations of acid

and MW temperatures to fix the maximum effective concen-

tration (Data not provided). The contents were then processed

and analyzed as per Section 2.2.

2.5. Compositional analysis of feed stock and itshydrolyzed residues

Known quantity (mg) of substrate (GB) and its hydrolyzed

residue after pretreatment was taken and analyzed for cellulose

by HNO3eethanol method [14]. The lignin content of samples

was estimated by 72% (w/w) H2SO4 method [14] and the hemi-

cellulose by Liu method [14]. The reducing sugar concentration

of the hydrolysate was measured using DNS method [13].

2.6. SEM studies on the pretreated feed stock

SEM studies were conducted using JEOL JED-2300 scanning

electron microscope to analyze the impact of different modes

of heating on the ultra structure of GB tissues.

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2.7. XRD and crystallinity measurements

Samples of pretreated GB were examined by XRD using

a powder X-ray diffractometer (Model e Rigaku (Miniflex-II)).

Approximately 50 mg of sample was pressed in a specified

sample holder and scanned at 2�/min from 2^ ¼ 10�e30�. Thebiomass crystallinity index (Cr) was calculated from the XRD

patterns by an empirical method [15] using the following

equation:

Cr ¼ lcr� lamlcr

Where Cr is the crystallinity index, Icr is maximumdiffraction

intensity at peak position 2^ w22.6� and Iam is the intensity at

2 ^ ¼ 18.7�

3. Results and discussion

3.1. Optimization of mild acid concentration forpretreatment

Garden biomass (GB) was subjected to different (0.25e10 (%,

v/v)) concentrations of H2SO4. The effectiveness of different

concentrations of acid to disrupt lignocellulose was

assessed taking into consideration the yield of reducing

sugar (RS) after mild acid treatment. A perusal of results

(Fig. 1) showed an increase in the yield of RS with

increasing concentrations of H2SO4 tested. However, this

increase was found only upto 6% of acid concentration,

above which a steady decline in the yield of RS was noticed.

Based on these results, an acid concentration of 5% was

used for further experiments in the present investigation.

Generally mild acid pretreatment is effective that it solu-

bilize hemicellulose into monomeric sugars and soluble

oligomers and thereby improving cellulose conversion [6].

Many other workers [[16e19]] have also suggested mild acid

treatment as an effective option for the pretreatment of

lignocellulosic feed stocks.

3.2. Optimization of reaction time for mild acidpretreatments

Fig. 2 illustrates the influence of reaction time on the effec-

tiveness of pretreatment. Results clearly indicated that

increasing reaction time over 30 min was not favorable for

mild acid hydrolysis as it reduced RS yield. A maximum of

38.06% of RS yield was obtained at 30 min reaction time.

Increasing the reaction time to 60e90min reduced the yield of

RS to 35.4 and 25.21%, respectively. This could have been

attributed as a result of further degradation of cellulose to

furfural, 5-hydroxymethyl furfural (HMF) levulinic acid and

formic acid together with other substances. This phenomena

has already been documented for severe conditions during

pretreatments which causes greater degradation of hemi-

cellulosic sugars [20,21]. Based on these results, reaction time

for further experiments was fixed as 30 min in the present

investigation.

3.3. Combined effect of different heating devices onlignocellulosic pretreatments

In another experiment, combination of different heating

devices was tested for their combined effect upon ligno-

cellulosic disruption. A perusal of the results (Fig. 3) did not

show any improvement in the yield of RS as a result of

combination of different heating devices, instead a decrease

was noticed. This may be due to severe conditions of

pretreatment, such as temperature and duration (more than

30 min in case of combination of different heating

0

5

10

15

20

25

30

35

40

45

0.25 0.5 1 2 4 6 8 10

Conc of acid (% v/v)

ragusgnicuderfo

dleiy%

GB

Fig. 1 e Effectiveness of different concentrations of acid on

pretreatment of GB wrt yield of reducing sugar.

0

5

10

15

20

25

30

35

40

30min 60min 90min

Time (min)

ragusgnicuderfo

dleiy%

GB

Fig. 2 e Influence of reaction time on pretreatment of GB

wrt yield of reducing.

05

10152025303540

CA PH WMA

+CHP

A+C

WMPH+

WM WM+PH+CAHeating devices

ragusgnicuderfo

dleiy%

GB

Fig. 3 e Effectiveness of individual and combination of

different heating devices on pretreatment of GB (at

100e121 �C) wrt yield of reducing sugar.

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processes) which can cause greater degradation of hemi-

cellulosic sugars leading to the conversion of RS into other

compounds such as HMF etc. Therefore, no plausible

conclusions can be drawn on the combined effect of

different heating devices for lignocellulosic disruption

based on the yield of RS.

3.4. Comparison between different heating devices fortheir pretreatment efficiency

Table .1 shows comparison of different heating devices for their

efficiency in the pretreatment of selected feed stock (GB) mate-

rial. Comparisonwasmade taking into consideration the yield of

Table 1 e Comparison between different heating devices for their efficiency in pretreatment of Garden biomass.

Parameters Initial Concentration (% w/w) Final Concentration (% w/w)

AC HP MW

(121 �C) (100 �C) (200 �C)

Cellulose 39.57 48.12 (21.60) 48.60 (22.82) 53.95 (36.29)

Hemicellulose 26.55 15.59 (�41.28) 12.86 (�51.56) 11.62 (�56.30)

Lignin 24.46 31.93 (30.53) 29.98 (22.56) 27.03 (10.50)

Reducing sugars 3.69 38.06 (931.43) 37.01 (909.98) 46.97 (1172.89)

Values are the average three replications.

Figures in parenthesis indicate � increase or decrease.

Fig. 4 e SEM images of GB after pretreatment (with and without acid) using HP. a) GB D Water (5003) b) GB D Water (50003)

c) GB D Water D HP (5003) d) GB D Water D HP (50003) e) GB D Acid D HP (5003) f) GB D Acid D HP (50003).

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RS, change inhemicelluloseand ligninconcentration, changes in

the ultra structure of GB tissues, and the crystallization index.

3.4.1. Reducing sugar yieldAmong the three different heating devices (HP, AC and MW)

evaluated for pretreatments at 100 �C (excepting AC, where it

was 121 �C), RS yield was comparatively higher (Fig. 3) in case

of AC (38.06) followed by HP (37.01). In autoclave, heat is

delivered with pressure making the substrate fragile for

further breakdown. Steam pretreatment of lignocellulosic

feed stocks constitutes an effective way of hydrolyzing

hemicellulose and softening the structure of cellulose to

Fig. 5 e SEM images of GB after pretreatment (with and without acid) using AC and MW a) GB D Water D AC (5003) b)

GB D Water D AC (50003) c) GB D Acid D AC (5003) d) GB D Acid D AC (50003) e) GB D Water D MW (5003) f)

GB D Water D MW (50003) g) GB D Acid D MW (5003) h) GB D Acid D MW (50003).

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facilitate enzymatic attack [16]. However, during steam

pretreatment parts of the hemicellulose hydrolyze and forms

acids, which could catalyze the further hydrolysis of hemi-

cellulose [22]. Heating through a hot plate represents

conventional heating (open boiling) which is so far accepted

as the most preferred method of heating in lignocellulosic

pretreatments. It is commonly available and inexpensive.

Pretreatment through HP heating could have been attributed

as a result of localized over heating of the hot surface of hot

plate[10], which facilitated fast and effective means of ligno-

cellulosic disruption. HP also yielded equally good amounts of

RS. Interestingly, yield of RS under MW pretreatment (Fig. 3)

was very low (9.14) compared to AC and HP, could be because

of the low (100 �C) temperature used for MW treatment in our

investigation. Normally, MW mediated pretreatments are

carried out at higher temperatures >160 �C [8,11].

Therefore, to further study the effectiveness of MW on

pretreatment processes, experiments were carried out at high

(200 �C) temperature using MW and the results clearly indi-

cated that microwave could be effective for pretreatment if

the temperature is increased to 200 �C. At a condition of 5%

H2SO4, 200 �C of temperature at 700 W power with 15 min of

reaction time the RS yield obtained was 46.97% (Table. 1). This

when compared to other treatments (AC and HP) is signifi-

cantly high (around 10% increase) and time saving (15 min) as

well. Microwave radiation, different from the conventional

heatingmethods, has been successfully applied inmany fields

because of either non-thermal or thermal effects which arise

from the heating rate, hot spots, acceleration of ions and

collision with other molecules and rapid rotation of dipoles

such as water with an alternating (2450 million times/s)

electric field [23].

3.4.2. Change in cellulose, hemicellulose and ligninconcentrationThe recovery (%) of cellulose (Concentration of pure cellulose

after the removal of hemicellulose and lignin) after pretreat-

ment showed an overall increase irrespective of the heating

device used (Table. 1). However, it varied significantly among the

different heating devices. Only in case of MW pretreatment

(at 200 �C) amaximumof 53.94% of cellulosewas recovered after

pretreatment. Heating through AC and HP showed comparably

low (48.12 and 48.60, respectively) percentage of cellulose

recovery. Similarly, the concentration of lignin also showed

a marginal increase after pretreatment. Although there could

not be any further increase in the total or original concentration

of cellulose and lignin after pretreatment, the relative increase

in the concentration of cellulose and lignin after pretreatment

could be because of the removal of hemicellulose from ligno-

cellulosic moiety; wherein hemicelluloses are probably con-

verted into sugars [24].

3.4.3. Changes in the ultra structure of GB tissuesScanning electron microscope (SEM) studies revealed the

details on tissue damage and ultra structural changes in GB

tissues as a result of pretreatment using different heating

devices. The response of GB to different modes of heating

varied greatly. HP heating without acid affected the smooth

(Fig. 4a and b) surface of substrate (GB) and turned it into

a rough and cracked surface (Fig. 4c and d). Here, the cracks

developed are surface, uniform and mostly linear in nature.

However, HP heating with acid (H2SO4) developed cracks that

are deep, heterogeneous and scattered in nature (Fig. 4e and f).

This kind of cracks can increase the surface area and porosity

to facilitate disruption of lignocellulose, a prerequisite for

enzymatic hydrolysis of cellulose. Similar observations were

reported by [25e27].

Heating through AC, on the other hand, developed lumps

on the surface of substrate and this condition was observed

only in case of heating through AC without acid (Fig. 5a and

b). With the addition of acid, AC heating resulted in

collapsing of cellular structure (Fig. 5c and d). Formation of

lumps could have been attributed as a result of heating

under pressure in presence of water. Lump formation as

a result of imbibition of water into tissues can dissociate

microfibrilar assembly of cellulose and making it suscep-

tible for hydrolysis. Impact of MW heating without acid on

the surface of substrate resembled that of AC heating (Fig. 5e

and f). However, the density of lumps formed was more in

case of MW than AC. On the other hand, MW treatment with

acid (Fig. 5g and h) caused a total collapse of cellular struc-

ture along with the removal of hemicellulose and lignin

layers, which could possibly make the tissue vulnerable for

rapid hydrolysis.

3.4.4. Changes in the crystallinity of celluloseThe effectiveness of different heating devices for the

pretreatment of GB was also evaluated (Table. 2) through

studying the changes in crystallinity of cellulose. The crys-

tallinity is influenced by the composition of the biomass, in

which hemicellulose and lignin are considered to be amor-

phous while cellulose is considered to be crystalline [28].

Although the results did not show any significant change in

crystallinity due to the difference in the mode of heating,

a marginal increase in crystallinity was noticed in case of AC

(with and without acid). Similarly, HP heating (with acid) also

recorded an insignificant increase in crystallinity. Increase in

crystallinity can be viewed as the indication of effective

pretreatment which by the way of removing hemicellulose

and lignin expose all crystalline cellulose available and

increase the rate of enzymatic hydrolysis. These results are in

accordance with many previous studies [4,29e31]. The lowest

value for crystallinity was found in case of raw material (RM)

which was not subjected to any kind of heating. The order of

crystallinity after heating through different heating devices

was AC > HP > MW > RM.

Table 2 e . Changes in the crystallinity of cellulose afterpretreatment using different heating devices.

Sample Crystallinityindex (CrI)

Raw material (RM) 0.7290

RM þ Water þ AC (without acid) 0.8033

RM þ Water þ HP (without acid) 0.7535

RM þ Water þ MW (without acid) 0.7467

RM þ Acid þ AC 0.8243

RM þ Acid þ HP 0.8184

RM þ Acid þ MW 0.7976

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4. Conclusion

The present investigation clearly demonstrated that mode of

heating is an important issue in the pretreatment of ligno-

celluloses. Among the three (MW, AC &HP) differentmodes of

heating tested, efficacy of MW heating was comparatively

better than HP and AC, but only at a higher temperature

(200 �C). Performance of HP and AC was almost same;

however, AC heating produced bulging effects on the feed

stock that might favour further enzymatic hydrolysis of the

material. Considering the above, it may be concluded thatMW

may be the effective means of heating for the pretreatment of

GB and other lignocellulosic substrates resembling GB.

Acknowledgements

We thank the Council of Scientific and Industrial Research

(CSIR) for financially supporting this project through SIP-MES

(Activity 4.8). Thanks are also due to Environmental Material

Unit, NEERI and VisvesvarayaNational Institute of Technology

(VNIT), Nagpur for their kind support in XRD and SEM anal-

ysis, respectively.

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