Biochemical Engineering Journal 63 (2012) 1 9

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Biochemical Engineering Journal

journa l h omepage: www.elsev ier .c

Regular article

Sono-a gaproduc

RajendraDepartment of ai, Ch

a r t i c l

Article history:Received 28 JuReceived in re22 December 2Accepted 3 JanAvailable onlin

Keywords:EthanolBioconversionCellulaseSono-assisted Fermentation

pretrol. Thom Sisted as fo

cell the mglucoof celzate hano

1. Introduction

The steady increase in energy consumption and the depletion offossil fuels have reawakened the interest in developing alternativeenergy sourcellulosic mpast [2]. Sesugarcane bhyacinth [6of bioethanavailable anbioethanol tion and fecompositiolulose digesmost widelsion [7], diluand wet oxto their draperature remajor probof metabolifurfural and

CorresponE-mail add

(K. Muthukum

Nikolic et al. [11] have reported the benecial effect of ultra-sound pretreatment on the production of bioethanol from cornmeal. The application of ultrasound produces cavitation in theaqueous solution and it generates microbubbles at various nucle-

1369-703X/$ doi:10.1016/j.ces [1]. The production of fuels from renewable ligno-aterials has been given much attention in the recentveral industrial and agricultural byproducts such asagasse [3], bermudagrass [4], soft wood [5] and water] are proposed as potential sources for the productionol. Among these, sugarcane bagasse (SCB) is abundantlyd is also rich in polysaccharides [3]. The production offrom SCB requires suitable pretreatment, saccharica-rmentation [7]. The pretreatment alters the structuraln of the lignocellulosic materials to increase the cel-tibility by removing hemicellulose and lignin [5]. They used pretreatment techniques include steam explo-ted acid hydrolysis [8], diluted alkali pretreatment [8,9]idation [10]. These methods need improvement owingwbacks such as concentrated chemical and high tem-quirements for effective removal of lignin. The otherlems associated with these methods are the formationc inhibitory products such as furfural, hydroxymethyl

acetic acid [8].

ding author. Tel.: +91 44 22359153; fax: +91 44 22352642.resses: muthukumar@annauniv.edu, chemkmk@gmail.comar).

ation sites in the uid. The implosion and collapsing of bubblesrelease violent shock waves that propagate through the medium.The collapse of bubbles produced during cavitation decomposeswater into radicals, which helps for the cleavage of lignin linkagesand xylan meshwork [12].

Saccharication of pretreated lignocellulosic materials can becarried out with acid hydrolysis [13], alkaline treatment [14] andenzymatic hydrolysis [15,16]. Saccharication breaks the hydrogenbonds present in cellulose and hemicellulose fractions and pro-duces sugars such as hexoses and pentoses respectively. Enzymatichydrolysis is more specic but not a cost-effective method becauseof the high cost associated with the isolation of pure enzymes[3]. Hence, the organisms currently employed for the commer-cial cellulase production produce very less quantity of extracellular-glucosidase compared with the other cellulase enzymes [17].Moreover, Stoppok et al. studied the formation and location of -glucosidase in Cellulomonas sp. and reported that 75% of the enzymewas present in the cell wall [18]. Gokhale and Deobagkar observedmuch carboxymethyl cellulase (CMCase) activity in cells of Cellu-lomonas sp. and Rajoka reported the presence of cell associatedexoglucanase [19,20]. Therefore, to make the process economical,saccharication was carried out using Gram-positive bacteriumCellulomonas avigena. The availability of C. avigena in hydroly-sis medium may also produce xylanases. However, the presence of

see front matter 2012 Elsevier B.V. All rights reserved.bej.2012.01.001ssisted enzymatic saccharication of sution

n Velmurugan, Karuppan Muthukumar

Chemical Engineering, Alagappa College of Technology Campus, Anna University Chenn

e i n f o

ly 2011vised form011uary 2012e 10 January 2012

saccharication

a b s t r a c t

This study presents the sono-assisted(SCB) for the production of bioethanremoval of hemicellulose and lignin frand 90.6% of lignin removal. Sono-assavigena (MTCC 7450) and the yield wpH. The optimum reaction time, LSR,respectively. At optimum conditions, yield and the maximum amount of may be correlated with the swelling application of ultrasound. The hydrolyand about 91.22% of the theoretical etom/ locate /be j

rcane bagasse for bioethanol

ennai 600025, India

eatment and enzymatic saccharication of sugarcane bagassee effect of sono-assisted alkali (NaOH) pretreatment on theCB was studied and the results showed 80.8% of hemicelluloseenzymatic saccharication was performed with Cellulomonasund to be affected by liquid-to-solid ratio (LSR), cell mass andmass and pH were found to be 360 min, 15:1, 15 g/L and 6.0aximum glucose yield obtained was 91.28% of the theoreticalse obtained was 38.4 g/L. The enhancement in performancelulose and accelerated enzymatic saccharication due to theobtained was fermented using Zymomonas mobilis (MTCC 89)l yield was observed in 36 h of fermentation.

2012 Elsevier B.V. All rights reserved.

2 R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9

Nomenclature

[M] monomers concentration (g/L)[M]nk1k2P Rme LSR R2

X0X XCK(E)H

S I

other enzymtate the celand cell boultrasound.etrability ttransportat[22]. Therefmance of ulSCB for the maximizedthe inuential cell confermented u

2. Materia

2.1. Microo

MicroorMTCC 7450Culture ColChandigarhgrowth medpeptone 5.0was cultureand was ke

2.2. Cultiva

C. avige0.05% (w/veral solutioCaCl22H2Obation wasfor 48 h, unwere ltereto remove obtained afat 4 C and

2.3. Sonoly

A probe frequency o

used in this study. The amplitude maintained was 100% and thetemperature was maintained at 40 2 C by using water bath [12].

2.4. Sono-assisted alkali pretreatment of SCB

arcad aftand wdeeyer

ted tanc

rried

no-a

cult anding 10.0

sepae ce

108

forn ofricaC [2stemerati0:1, char

at r andmentepor

avaitical

rviva

cell d, sp

were witas inounted aspolymer concentration (g/L)rate of pentose generation (min1)rate of pentose decomposition (min1)ethanol production potential (g/L)maximum ethanol production rate (g/L h)exponential constant 2.72lag-phase time, tliquid-to-solid ratio (mL/g)regression coefcientinitial biomass concentration (g/L)biomass concentration (g/L)carrying capacity (g/L)carrying capacity coefcient (h1)hydrolysis efciencysum of sugar concentration (g/L)sum of inhibitor concentration (g/L)

es may hydrolyze other polysaccharides which facili-lulose availability to hydrolysis [21]. The intracellularund enzymes can be excreted by the application of

The low frequency ultrasound also improves the pen-hrough cell membrane, which in turn improves theion of substrates and enzymes through the cell wallore, the aim of this study was to evaluate the perfor-trasound assisted pretreatment and saccharication ofproduction of bioethanol. The production of sugars was

in sono-assisted enzymatic hydrolysis by optimizingcing parameters such as substrate concentration, ini-centration and initial pH. The hydrolyzate obtained wassing Zymomonas mobilis MTCC 89.

ls and methods

rganisms and culture conditions

ganisms such as Z. mobilis MTCC 89 and C. avigena, used in this study, were obtained from Microbial Typelection, Institute of Microbial Technology (IMTECH),, India. C. avigena MTCC 7450 was maintained in aium containing (g/L): beef extract 1.0, yeast extract 2.0,, NaCl 5.0 and agar 15.0 (if needed). Z. mobilis MTCC 89d in Yeast Extract Glucose Salt Agar (YEGSA) mediumpt in incubator without shaking at 25 C [23].

Sugair driedered SCB poErlenmsubjecperformwas ca

2.5. So

The(15 g)contain13.32, minedand thof 2.4 appliedduratiosaccha40 2trol syThe op15:1, 2the sacdrawn10 minexperiwere rsion oftheore

2.6. Su

Themetho(1 mL)dilutedtion wwere creporttion of C. avigena

na strain was inoculated in mineral medium containing) yeast extract supplemented with 1% SCB. The min-n consisted of (L1): 5.5 g NaCl, 2.5 g (NH4)2SO4, 0.1 g, 0.1 g MgSO47H2O, 5.3 g K2HPO4 and 2 g KH2PO4. Incu-

carried out at 30 C in an orbital shaker (150 rpm)der aerobic condition. After incubation, the samplesd through GD-120 glassber lter disks (Whatman)the residual insoluble SCB. Then, the liquid contentter ltration was centrifuged at 10,000 g for 10 minpellet was used as cell mass for further studies.

zer

type sonolyzer (Hielscher, Germany) with an operatingf 24 kHz and a rated maximum power of 400 W was

2.7. Isolatio

C. avigepreviously.culture broand the supwere resussubjected tapplied for sonic energfrom this dienzyme. Thtrifugation 5.8) and thethe suspensered in thewere used fne (Saccharum ofcinarum) bagasse obtained locally waser rinsing with distilled water. The dried SCB was pow-sieved to 0.27 mm particle size (ASTM standard). Ther was added into 200 mL of 2% NaOH solution in an

ask in the ratio of 20:1 (mL/g) and the mixture waso continuous ultrasound irradiation [9]. To analyze thee of sono-assisted pretreatment, alkaline pretreatment

out at the same conditions as control.

ssisted enzymatic saccharication of SCB

ivated C. avigena cells were weighed on wet weight inoculated aseptically into an 250 mL Erlenmeyer ask200 mL of pretreated SCB with distilled water (20.00,0 and 8.00 g). The initial number of cells was deter-rately for ve different cell concentrations (525 g/L)ll count was found to be approximately in the range1.2 109 CFU/mL. Then, the ultrasonic irradiation was

a period of 5 min in the interval of 5 min and the total the ultrasound course was 3 h. During sono-assistedtion, the temperature was maintained as constant at4]. The ultrasonic energy was pulsed using cycle con-

and the cycle was set on 50% in all experiments [12].ng parameters such as liquid-to-solid ratio (LSR) (10:1,25:1), biomass loading (525 g/L), initial pH (48) andication time were optimized [25]. The samples with-egular time intervals were centrifuged at 8000 g for

the supernatant was subjected to sugar analysis [6]. Alls were performed in triplicate and the average valuested. The % glucose yield was calculated as the % conver-lable cellulose in pretreated SCB to sugars assuming ayield of 1.11 g sugar/g cellulose [26].

lity analysis

viability of C. avigena was determined using pour plateread plate method and colony counts [27]. Samples

taken from the inoculated medium and were seriallyh sterile saline solution. Then, 1 mL from each dilu-oculated in agar plates and incubated at 30 C. Coloniesed after 24 h of incubation and the bacterial count was

colony-forming units per mL of sample [28].

n of enzymes

na were cultivated in mineral medium as mentioned After cell growth reached early stationary phase, theth was ltered and centrifuged at 8000 g for 15 min,ernatant was used as extracellular enzyme. The cells

pended in 0.07 M phosphate buffer (pH 5.8) and it waso sonication for 15 min. The ultrasonic irradiation wasa period of 1 min in the interval of 1 min and the ultra-y was pulsed using cycle control (50%). Supernatantsruption mixture was used as the intracellular source ofe membranous fraction obtained as a pellet after cen-was washed three times in 0.07 M phosphate buffer (pH

cell walls were brought into autolysis by incubation ofion at 30 C with gentle agitation. The enzymes recov-

supernatant after centrifugation (8000 g for 15 min)or cell bound enzyme study.

R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9 3

2.8. Partial purication and electrophoresis of enzymes

The crude enzyme extract obtained (intracellular, extracellularand cell wall bounded) was precipitated using solid (NH4)2SO4 at80% saturatprecipitated20 min at 4dialyzed. Thdiameter anrespectivelytrophoresiswas carriedon 10% gelof the enzystandards. Fglucosidasecellulose (0d-glucopyrarespectivelystrate for th

The crud(125 mM Trjected to elwashed foutaining 25%renatured b(pH 5) cont-glucosidadetecting thcanase, andwith 0.5% (wNaCl to reve

2.9. Fermen

Fermentsaccharicathetic mediand the synKH2PO4, 0.2extract andadjusted to89 seed culcentration oinoculum a48 h [23]. Tand centrifujected to et(weight) es

2.10. Analy

The amodinitrosaliccellulose wmethod anmatographyprotocol [3acetic acid achromatogrtory protocowere estimGoering andbasis and boxymethy-glucosida

dened as the amount (mol) of product formed per minute understandard assay conditions [36].

2.11. Models

Kine kineicatedels

ption des

cchar

k1 isomeed ons [40

0ek

G antivelyFrom

coef mots thalysientsinat

Groistic ial gt plaobilisgiven

(X0 X0 istratiximustan

Prod etha

mod

P exp

E(t) n pot, iseters

Hyd efcusin

SI

(E)Htionsion at 4 C with continuous stirring and left for 3 h. The enzyme was collected by centrifugation (10,000 g,

C) and was resuspended in 0.1 M phosphate buffer ande available dialysis membrane (Himedia) at width,d capacity were 29.31 mm, 17.5 mm and 2.41 mL cm1

. Sodium dodecyl sulfatepolyacrylamide gel elec- (SDSPAGE) analysis of partially puried enzymes

out according to the method described by Laemmli containing 0.1% SDS [29,30]. The molecular weightme was determined by comparing with the proteinor the zymogram analysis of CMCase, exoglucanase, -

and xylanase, the substrates used were carboxymethyl.1%, w/v), avicel (0.1%, w/v), 4-methylumbelliferyl--noside (2 mmol/L) and birch wood xylan (0.25%, w/v),. The electrophoresis was run separately with the sub-e enzyme to be analyzed.e enzyme was treated with 2 SDSPAGE sample bufferisHCl buffer, pH 6.8) at 37 C for 5 min and then sub-ectrophoresis. After the electrophoresis, the gels werer times at 4 C for 15 min in 50 mM acetate buffer con-

isopropanol (pH 5.0). The enzyme components werey maintaining the gel overnight at 4 C in acetate bufferaining 5 mM -mercaptoethanol and 1 mM EDTA. These zymogram was visualized under ultraviolet light bye uorescence of 4-methylbelliferone. CMCase, exoglu-

xylanase activities were visualized by staining the gel/v) Congo red solution for 30 min and destaining in 1 Mal the zones of clearing [30].

tation

ation of hydrolyzate obtained from sono-assistedtion of SCB was compared with the fermentation of syn-um to analyze the fermentability. The SCB hydrolyzatethetic medium were supplemented with salts (2 g/L of5 g/L of MgSO4, 0.01 g/L of FeSO47H2O), 10 g/L of yeast

38.4 g/L of glucose (in synthetic medium). The pH was 5.7 with 2.0 M NaOH and/or 1.0 N HCl. Z. mobilis MTCCture of 10% (v/v) corresponding to an initial cell con-f approximately 0.1 g dried cells per liter was used asnd the inoculated medium was incubated at 30 C forhe samples were withdrawn at regular time intervalsged at 8000 g for 10 min. The supernatant was sub-

hanol analysis and the pellet was subjected to biomasstimation.

tical methods

unt of total reducing sugars was estimated by 3,5-ylic acid (DNS) method [31]. The concentration ofas estimated spectrophotometrically by Anthrone

d the ethanol content was analyzed using gas chro- (GC-2014 Shimadzu) as outlined in NREL Laboratory2,33]. Concentrations of glucose, xylose, arabinose,nd furfural were analyzed by high performance liquidaphy LC-10AD (Shimadzu) as outlined in NREL Labora-l [34]. Hemicellulose, cellulose, lignin and ash contentsated by detergent extraction methods described by

Vansoest [35]. Biomass was quantied on wet weightthe enzyme extracts were used to determine car-l cellulase (CMCase), exo-glucanase, lter paper (FPase),se and xylanase activities [36]. One unit of activity (U) is

2.11.1.The

complous moassumused to

Polysa

whereof mon

Basgiven a

[G] = G

whererespecvalue. kineticsis. Theaccounsion ancoefcdeterm

2.11.2.Log

microbproduct Z. mand is

X =1

whereconcenthe mais a con

2.11.3.The

using a

E(t) =

whereductio(g/L h)paramware.

2.11.4.The

dicted

(E)H =

wherecentratic modeling of hydrolysistic analysis of hydrolysis of cellulosic materials is veryd due to structural complexities [37]. There are vari-

available in the literature based on pseudo rst order and the model proposed by Saeman was successfullycribe the decomposition of polysaccharides [38,39].

idek1Monomers k2degradatives (1)

the rate of monomer release (min1) and k2 is the rater degradation (min1).

Eq. (1), the two fraction model was proposed and is]:

2t + G [Gnp] k1

k2 k1 (ek1t ek2t) (2)

d Gnp are glucose and cellulose concentrations (w/v), t is the time and the subscript 0 denotes initial

the potential concentration of monomers (g/L), thecients k1 and k2 can be obtained by regression analy-del is considering part of glucan being unreactive and e fraction susceptible to hydrolysis. Nonlinear regres-es were performed to obtain the kinetic parameters and. The results were evaluated based on the co-efcient ofion (R2) to values.

wth kineticsequation was developed to describe the kinetics ofrowth when the inhibitory effects of substrates andy no role. In this study, logistic equation was used to

growth data obtained from fermentation experiments as [41]:

X0 ekt/XC )(1 ekt)

(3)

the initial biomass concentration (g/L), X is the biomasson at any time t (g/L), t is the incubation time (h), XC ism of bacterial growth or carrying capacity (g/L) and kt resembling the specic growth rate (h1).

uction kineticsnol production in a batch experiment was describedied Gompertz equation and is given as [42]:{

exp[

Rm eP

( t) + 1]}

(4)

is the ethanol concentration (g/L), P is the ethanol pro-ential (g/L), Rm is the maximum ethanol production rate

the lag-phase time (h) and e is 2.72 and the kinetic P, Rm and were determined using the MATLAB soft-

rolysis efciencyiency of sono-assisted saccharication of SCB was pre-g the following equation [43]:

(5)

is the hydrolysis efciency,

S is the sum of con- of all sugars in the hydrolyzate (glucose, xylose and

4 R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9

arabinose, g/L) and

I is the sum of concentrations of inhibitor inthe hydrolyzate (furfural and acetic acid, g/L).

The glucose yield per pretreated SCB (g/g) was calculated byusing the following equation [44].

Y(G/SCB) =[

where [G] iis the globequation, thydrolysis m

2.11.5. EneThe ener

the followin

E = (P t)Rm

where E is tthe sonicati(g).

3. Results

3.1. Sono-a

The comlose and 17was 56.96%treatment wand lignin experimentand 12.28%ultrasound lignin contwith 10% NCai [9] pretand reportethat sono-adelignicatAlthough thpre-treatmetial. The enebased on Eq(23.3 104for 1 h. The1 h also reqsono-assistpower requment was don lignin re

The comSCB (2% Naication) anglucose yiefrom SCB wpretreated cal glucosepre-treatmestructure, wrify cellulos

3.2. Sono-a

In sonoSCB using

0

5

10

15

20

25

30

35

40

45

/l)

Pretreated SCB

Raw SCB

ffect tic sacanism

und , intrd the, micrediumh the

incre prarriesentlanasxo-g1), wcose.d -

celle ex

ctionse acer, ad xylar wine wf cellrried out for the enzyme extract obtained after sono-assistedrication. The results showed the presence of three endoglu-s, one exoglucanase, one -glucosidase and one xylanase). The multiple enzymes secreted in turn apparently coop-o enhance the hydrolysis of lignocelluloses. To assess theance, the products like glucose, xylose, arabinose, acetic

d furfural were analyzed. The inuences of saccharicationiquid-to-solid ratio, organism dose and pH on the sacchari-were studied and the results are discussed in the followings in detail.

Effect of saccharication time effect of saccharication time on glucose production is

in Fig. 3. The concentration of glucose released was found toe with an increase in saccharication time and there werefferent periods, exponential and stationary phases, found to

in the course of production of glucose while the unsonicatedrication had lag and exponential phases. This improvedtion was due to the combined effect of substrate alterationG] (LSRG + 1)1000

(6)

s the concentration of glucose expressed as g/L, LSRGal liquid-to-solid ratio (g/g). In order to deduce thehe total solubilization of the solid and density of the

edium equal to 1 g/mL was assumed.

rgy calculationgy required for pre-treatment was calculated based ong equation [45].

(7)

he required energy (J/g), P is the applied power (J/s), t ison time and Rm is the amount of treated raw material

and discussion

ssisted alkali pretreatment of SCB

position of SCB was 35.6% cellulose, 26.63% hemicellu-.1% lignin and after the pretreatment the composition

cellulose, 8.06% hemicellulose and 1.6% lignin. The pre-as found to reduce the hemicellulose (80.8% reduction)(90.6% reduction) contents signicantly. The controls (without ultrasound) showed 19.17% hemicellulose

lignin reduction, which indicates that the presence ofimproved the alkaline pretreatment. The reduction inent reported during the pretreatment of corm stoveraOH for 1 h in an autoclave was 95% [46]. Zhang andreated the corn stover with 2% NaOH at 85 C for 1 hd 36.24% reduction in lignin. The results concludedssisted alkaline pretreatment is more effective for

ion than steam explosive alkaline treatment technique.e lignin removal was high in sono-assisted alkalinent, the comparison of energy requirement is essen-rgy requirement for each pretreatment was calculated. (7) and the results showed higher power requirementJ/g) in case of autoclave (in case 1800 W) pre-treatment

steam explosive pre-treatment (in case 250 W) foruired higher energy (9.9 104 J/g) when compared toed alkaline pre-treatment. In this method the maximumirement was 7.2 104 J/g. This reduced energy require-ue to combined action of NaOH and cavitation reactionmoval with reduced reaction time [45].parison among glucose yield obtained with pretreatedOH, 24 kHz, 30 min), alkaline pretreated (without son-d SCB without pretreatment is shown in Fig. 1. Theld for pretreated SCB was 91.28% while the glucoseithout pretreatment was only 48.4% and the alkalineSCB (without sonication) produced 54.9% of theoreti-

yield. The results showed that sono-assisted alkalinent opened the complex lignin sheath and hemicellulosehich increased the accessibility of enzymes to saccha-e.

ssisted enzymatic saccharication

-assisted enzymatic saccharication of pretreatedC. avigena cells, the application of low intensity

Glu

cose

concentr

atio

n (

g

Fig. 1. Eenzyma15:1, org

ultrasoicationinduceHencethe mthrouggreatlyrm thwere care preand xythree e3.2.1.2to glushowe(PF). Inand onlar fraxylanaHowevlase anmolecube in lence owas casacchacanase(Lane 4erate tperformacid antime, lcation section

3.2.1. The

shownincreastwo diappearsacchaproduc0 10 0 20 0 30 0 40 0 50 0

Time (min)

Control

of saccharication time on glucose concentration in sono-assistedcharication of pretreated and unpretreated SCB (conditions LSR:

dose: 15 g/L, pH: 6).

enhanced the mass transfer within cells. During son-acellular micro-streaming was reported to occur that

rotation of organelles and circulation within vacuoles.obial products synthesized are secreted effectively into

[4750]. Apart from enhancing enzyme secretion uid boundary layer around the cells, ultrasound canease the rates of enzyme-catalyzed reactions. To con-esence of enzyme, SDSPAGE and zymogram analysisd out (Fig. 2I and II) and the molecular weights obtaineded in Table 1. C. avigena was found to secrete cellulaseses, including at least four endo-glucanases (EC 3.2.1.4),lucanases (EC 3.2.1.74) and three -glucosidases (EChich act synergistically for the conversion of cellulose

The electrophoresis of intracellular extract (Lane 2)glucosidase activity in three pooled protein fractions-wall bounded fraction (Lane 3), two endo-glucanaseo-glucanase activities were observed. The extracellu-

(Lane 5) showed endo-glucanase, exo-glucanase andtivities but did not show the presence of -glucosidase.

fraction from extracellular enzyme showed both cellu-lanase activities (Fig. 2II). Most of the secreted enzymeeights were determined and the values were found toith the reported data (Table 1). To conrm the pres-

ulases in the saccharication medium, electrophoresis

R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9 5

Fig. 2. SDSPAGE (I) and zymogram (II) of puried cellulases of C. avigena: Lane 1, protein molecular mass standards; Lane 2, intracellular; Lane 3, cell wall bounded; Lane4, after sonication; Lane 5, extracellular (a: endo-glucanase, b: exoglucanase, c: -glucosidase, d: bifunctional endo--1-4-glucanase, e: xylanase).

Table 1Molecular weight of cellulase and xylanase obtained from C. avigena.

S. no. Enzymes Molecular weight (kDa) Relevant references

Intracellularfraction

Extracellularfraction

Cell-wall boundedfraction

Fraction from sono-assistedhydrolysis

1 Xylanase 45 45 2 Endo-glucanase 49 49 [51]3 -Glucosidase 52 [52]4 Endo-glucanase 55 5 Exo-glucanase 57 6 -Glucosidase 71 [52]7 -Glucosidase 89 89 [52]8 Exo-glucanase 90 [53]9 Exo-glucanase 94 94

10 Endo-glucanase 106 106 [54]11 Endo-glucanase 110 110

Fig. 3. Effect of saccharication time on glucose production during sono-assistedsaccharication and unsonicated saccharication (conditions LSR: 15:1, organismdose: 15 g/L, pH: 6).

0

2

4

6

8

10

0

1

2

3

4

0 10 0 20 0 30 0 40 0 50 0

Cell

via

bili

ty (

CF

U/m

l)

10

8

Cellu

lase

activity

(F

PU

/ml)

Time (min)

SdUt

Cellula se activityCell viabili ty

Fig. 4. Effect of sonication time on cell viability and cellulase production duringsono-assisted enzymatic saccharication of pretreated SCB (conditions LSR: 15:1,organism dose: 15 g/L, pH: 6).

6 R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9

0

5

10

15

20

25

30

35

40

45G

lucose

concentr

atio

n (

g/l)

10:1 15 :1 20 :1 25 :1

Fig. 5. Effect omatic saccharipH: 6).

and the imassisted saccount and shown in Fity and enzthe cell disrthe releasethe Cellulomcanase, EC 33.2.1.21) bo-glucosidaof carbon swas increasity was obsmolecules. process doehas been clever, sonicaalkaline propower was 2.82 FPU/mafter 360 m

3.2.2. EffectThe effe

ent LSRs is sand furfuralin Table 2. TLSR of 10:1which is inwere releasboth celluloarabinose aAt high LSRcant, but thefound to occdividing the(5)) and theindicate thasugar and le

Acetic alinked to hfound to be

0

5

10

15

20

25

30

35

40

45

(g/l)

5 (g/l) 10 (g/l) 15 (g/l)20 (g/l) 25 (g/l)

ffect tic sac

trati micraner meacid rganncenffector, be proe kines wnd 3l, va

up tsed t4 g/grrivetratidrolytion acid 15:1ose 0 100 20 0 300 40 0

Time (min )

f liquid to solid ratio on glucose production in sono-assisted enzy-cation of sono pretreated SCB (conditions organism dose: 15 g/L,

proved enzyme release. To analyze the effect of sono-charication on cell viability and enzyme release, cellenzyme activity were determined and the results areig. 4. The sonication time inuenced the cell viabil-yme activity. The decrease in cell viability indicatesuption and the increase in enzyme activity conrmed

of enzymes from C. avigena cells. It is known thatonas sp. produces cellulase complex (i.e., endoglu-.2.1.4; exoglucanase EC 3.2.1.91, and -glucosidase ECth in extracellular and cell associated fractions, whereasse was found to be associated with cells, regardlessource [1820]. However, when the sonication timeed beyond a certain value, decrease in enzyme activ-erved and it may be due to the inactivation of enzymeUnlike conventional thermal denaturation, sonications not obliterate the active site of an enzyme and this

early reported for -amylase and laccase [55,56]. How-tion-mediated deactivation of some enzymes, includingtease and trypsin, was reported when high ultrasoundused [56]. The maximum observed enzyme activity wasL and the obtained glucose concentration was 38.4 g/Lin of saccharication.

of liquid-to-solid ratio (LSR)

Glu

cose c

oncentr

atio

n

Fig. 6. EenzymapH: 6).

concenfor themembcellulaacetic microoacid cotoxic einhibitglucosand thk1 valuis arougenerain LSRdecreato 0.61were aconcenthe hycentraacetic LSR of of gluc15:1.ct of saccharication time on glucose released at differ-hown in Fig. 5. The glucose, xylose, arabinose, acetic acid

concentrations obtained at different LSRs are presentedhe glucose yield of 24.6 g/L was observed at the lowest

and the corresponding glucose conversion was 38.9%,signicant. During saccharication, other sugars thated are xylose and arabinose. Glucose originates fromsic and hemicellulosic fractions, whereas xylose andre pentose, which are present only in hemicelluloses.s (25:1 and 20:1), the glucose conversion was signi-

formation of inhibitors like acetic acid and furfural wasur. Hydrolysis efciency (E)H values were calculated by

sugar concentration with inhibitor concentration (Eq. values obtained for all LSRs were higher than 1.0, whicht hydrolyzate obtained contains high concentration ofss inhibitors.cid is produced from the hydrolysis of acetyl groupsemicellulosic sugars. The prole of acetic acid was

similar to that of arabinose, establishing a maximum

3.2.3. EffectThe effe

ent initial ctions of gluin sono-assfrom the tacell concenthe yield dithat the gluand at the almost the cose concentrations arecoefcientscoefcient and predictzero or verglucose dec4003002001000

Time (min)

of initial cell concentration on glucose production in sono-assistedcharication of sono-alkali pretreated SCB (conditions LSR: 15:1,

on of 1.42 g/L at 10:1 LSR. Acetic acid is an inhibitorrobial growth because it enters through the cellulars and decreases the intracellular pH, which affects thetabolism [57,58]. However, it has been reported thatconcentration of 910 g/L stimulates the growth of theism [59]. In the present study, the maximum acetictrations observed were below the lower limit of the

(0.65 g/L). Furfural has also been reported as a growthut its production was observed with a LSR of 10:1. Theduction kinetics was analyzed using two fraction modeletic values are tabulated in Table 3. The comparison ofith k2 concluded that the rate of glucose generation1-fold higher than the rate of glucose degradation. Inlues of kinetic coefcients increased with an increaseo a value of 20:1 and further increase in value to 25:1he values. The value of G was found to vary from 0.271

(Table 3). In the present study, the optimum valuesd based on higher concentrations of sugar and lowerons of the inhibitors. From Table 2, it can be seen thatzate obtained with a LSR of 15:1 showed glucose con-of 38.4 g/L, xylose concentration of around 3.93 g/L andacetic concentration of 0.7 g/L. On the other hand the

showed higher (E)H value (20.55) and theoretical yield(91.28%). Therefore, the optimum LSR was arrived as of initial cell concentrationct of saccharication time on glucose released at differ-ell concentrations is shown in Fig. 6 and the concentra-cose, xylose, arabinose, acetic acid and furfural obtainedisted hydrolysis are presented in Table 4. As can be seenble, the concentration of glucose increased as the initialtration was increased from 5 to 15 g/L and beyond whichd not increase signicantly. From Fig. 6, it can be seencose concentration increased with an increase in timeend of saccharication, the glucose concentration wassame for 15, 20 and 25 g/L of cell concentration. The glu-tration data obtained with different initial cell concen-

tted with two fraction model and the values of kinetic obtained are presented in Table 3. The determination(R2) showed a good agreement between experimentaled values. The values of k2 obtained are either close toy much lower than k1, which indicates the negligibleomposition. The value of Y(G/SCB) was found to increase

R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9 7

Table 2Composition of SCB hydrolyzate obtained at different liquid to solid ratios.

Liquid to solid ratio (LSR) Concentration (g/L) Theoretical glucose yield (%) (E)H

Initial cellulose Glucose Xylose Arabinose Acetic acid Furfural

10:1 57.0 24.6 5.34 1.3 1.42 0.1 38.88 20.5515:1 37.9 38.4 3.93 0.7 0.8 Nc 91.28 53.7920:1 28.5 28.6 2.95 0.5 0.65 Nc 90.41 49.3125:1 22.8 21.5 2.34 0.4 0.56 Nc 84.95 43.29

Nc not considerable.

Table 3Kinetic values of glucose released during enzymatic saccharication of SCB obtained by tting the double fraction model.

k1 102 (min1) k2 103 (min1) R2 G (g/g) Y(G/SCB)Effect of liq10:115:1 20:1 25:1 Effect of org5 10 1520 25Effect of pH4.0 5.06.0 7.0 8.0

Y(G/SCB) gluco

with an incvary from 0was very lowhereas thenzyme aclose to its mwith initial degrading reected frothe hydrolyin lignocellincreases thof the releasugar consuwas increasthe value (krelease of encentration yield obtainSCB.

Effect effe

Table 4Composition o

Cell mass (g

5 10 15 2025

Nc not consiuid to solid ratio (LSR)0.594 0.781 0.805 0.257 0.768 0.213 0.243 0.142

anism dose (g/L)0.383 0.106 0.403 0.203 0.805 0.257 0.913 0.193 0.711 1.011

0.490 0.335 0.520 0.242 0.805 0.257 0.726 0.100 0.601 0.149

se yield per pretreated sugarcane bagasse.

rease in initial cell concentration and it was found to.262 g/g to 0.624 g/g. At low dosage, the sugar yield

3.2.4. Thew due to insufcient release of enzyme (0.7 FPU/mL),e increase in initial cell concentration increased thetivity and sugar yield. The conversion of hemicellu-onomers like xylose and arabinose was also increasedcell concentration due to the release of hemicellulose-enzymes. The breakdown of hemicellulose was alsom the yield of acetic acid, which is formed mainly bysis of acetylated -d-xylanopyranose residue presentulosic materials. Although the organism concentratione enzyme concentration, high degrees of degradationsed sugars were observed, which is due to increase inmption for cell growth and maintenance. The k2 valueed with an increase in initial cell concentration and2 = 1.011 103) was maximum at 25 g/L. The sufcientzymes and less sugar consumption at an organism con-

of 15 g/L favored the maximum glucose yield and theed with this concentration was 0.614 g/g of pretreated

ent initial pxylose, arabin Table 5. enced the gdecrease asattributed tmum produpH 8.0 gavewith an indown (k2) The resultsenvironmendecrease inand acetic tion was noonly at acidincreased e[60] report

f SCB hydrolyzate obtained at different concentrations of cell mass.

/L) Concentration (g/L)

Glucose Xylose Arabinose Acetic acid

16.4 2.10 0.6 0.7 29.6 2.74 0.65 0.75 38.4 3.93 0.7 0.8 39.0 3.90 1.12 1.2 39.0 3.80 1.1 1.25

derable.0.828 0.392 0.2710.952 0.917 0.6140.979 0.678 0.4470.882 0.569 0.372

0.936 0.392 0.2620.897 0.707 0.4740.952 0.917 0.6140.929 0.931 0.6240.998 0.931 0.624

0.921 0.743 0.4980.919 0.800 0.5360.952 0.917 0.6140.921 0.797 0.5360.952 0.616 0.413

of initial pHct of saccharication time on glucose released at differ-

H is shown in Fig. 7 and the concentrations of glucose,inose, acetic acid and furfural obtained are presented

The results showed that the change in initial pH inu-lucose production signicantly. The pH was found to

the reaction proceeds and this phenomenon could beo the formation of acidic metabolites [37]. The maxi-ction was observed at an initial pH 6.0 while the initial

the lowest yield. The value of k1 was found to increasecrease in pH (up to 6.0), whereas the rate of break-was increased when the pH was decreased (Table 3).

indicated that the slightly acidic condition as a bettert for the saccharication. On the other hand, further

pH below 5.0 supported the formation of furfuralacid. At neutral or alkaline conditions, furfural forma-t observed and sugar monomer degradation occurredic conditions. The xylose production was considerablyven at alkaline conditions. Santiago-hernandez et al.ed that xylanases were stable and active at alkaline

Theoretical glucose yield (%) (E)H

Furfural

Nc 38.98 27.29Nc 70.36 43.99Nc 91.28 53.790.1 92.70 33.860.2 92.70 30.28

8 R. Velmurugan, K. Muthukumar / Biochemical Engineering Journal 63 (2012) 1 9

Table 5Composition of SCB hydrolyzate obtained at different pH.

pH Concentration (g/L) Theoretical glucose yield (%) (E)H

Glucose Xylose Arabinose Acetic acid

4.0 31.1 2.74 1.28 1.21 5.0 33.5 3.05 1.30 0.95 6.0 38.4 3.93 0.70 0.80 7.0 33.5 3.81 0.80 0.80 8.0 25.8 3.93 0.82 0.50

Nc not considerable.

conditions. An increase in initial pH decreased the G suggest-ing that low initial pH was better for sono-assisted enzymaticsaccharication. The optimum susceptible fraction of 0.917 g/g cel-lulose was obtained at pH 6.0, which corresponds to 0.614 g ofsusceptible cellulose per gram of raw material. The results showedhigher sugar yield at pH 6.0 and hence, further studies were carriedout at pH 6.0.

3.3. Fermentation

The SCB hydrolyzate containing glucose (38.4 g/L) was fer-mented using Z. mobilis and the ethanol prole obtained is shownin Fig. 8. As can be seen from the gure, the ethanol produc-tion obtained was around 17.9 g/L after 36 h of fermentation andis equivalewere ttedethanol prosynthetic mproductionthe syntheproductionmatic sacchhemicellulotion of penttted with lwas found duced the mfor the hydmedium hasaccharicalignocellulo

0

5

10

15

20

25

30

35

40

45

0

Glu

cose

concentr

atio

n (

g/l)

Fig. 7. Effect otion of sono-a

0

5

10

15

20

0

ce

ntr

atio

n o

f e

tha

no

l a

nd

bio

ma

ss (

g/l)

ffect oCB hy

clus

s study presented the sono-assisted pretreatment andatic saccharication of sugarcane bagasse to enhance theanol production using Z. mobilis. The results showed that thisd can be successfully used for the conversion of polysaccha-o reactive intermediates. Based on the results, the followingsions were arrived:

o-assisted pretreatment is a better alternative method for structural modication of SCB.

application of low intensity ultrasound enhanced theyme release and intensied the enzyme-catalyzed reaction.

optimum LSR, cell mass, pH and saccharication time werend to be 15:1, 15 g/L, 6.0 and 360 min for the better glucoseld (91.28% of theoretical yield).o-assisted enzymatic saccharication of cellulose was found

decrease the reaction time. maximum ethanol yield obtained was 91.22% of theoreticalld after 36 h of fermentation.

wledgementsnt to 91.22% of theoretical yield. The data obtained with modied Gompertz model and the maximumduction rate was found to be 0.497 g/L h whereas theedium showed a the maximum of 0.613 g/L h but the

potential was 24.11 g/L which is 5.9 g/L higher thantic medium, which would explore the feasibility of

of bioethanol from SCB. Although sono-assisted enzy-arication released pentoses due to the presence ofse-degrading enzymes, there is no further consump-oses for fermentation. The growth data obtained wereogistic equation and the maximum growth for Z. mobilisto be 11.84 g/L whereas the synthetic medium pro-aximum of 12.07 g/L. The specic growth rate obtainedrolytic product was 0.189 g/L h while the syntheticd 0.64 g/L h. The results showed that the sono-assistedtion as a suitable method for ethanol production fromsic wastes.

pH 4 pH 5 pH 6

pH 7 pH 8

Co

n

Fig. 8. Etion of S

4. Con

Thienzymbioethmethorides tconclu

(i) Sonthe

(ii) Theenz

(iii) Thefouyie

(iv) Sonto

(v) Theyie

Ackno100 200 30 0 400

Time (min)

f pH on glucose production in sono-assisted enzymatic saccharica-lkali pretreated SCB (conditions LSR: 15:1, organism dose: 15 g/L).

The authing nanciascheme. OnNew Delhi,would like

References

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0.12 73.93 26.410.10 79.63 36.05Nc 91.28 53.79Nc 79.63 47.64Nc 61.33 61.10

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Ethanol (Control) Biomass (Control)

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Sono-assisted enzymatic saccharification of sugarcane bagasse for bioethanol production1 Introduction2 Materials and methods2.1 Microorganisms and culture conditions2.2 Cultivation of C. flavigena2.3 Sonolyzer2.4 Sono-assisted alkali pretreatment of SCB2.5 Sono-assisted enzymatic saccharification of SCB2.6 Survivality analysis2.7 Isolation of enzymes2.8 Partial purification and electrophoresis of enzymes2.9 Fermentation2.10 Analytical methods2.11 Models2.11.1 Kinetic modeling of hydrolysis2.11.2 Growth kinetics2.11.3 Production kinetics2.11.4 Hydrolysis efficiency2.11.5 Energy calculation

3 Results and discussion3.1 Sono-assisted alkali pretreatment of SCB3.2 Sono-assisted enzymatic saccharification3.2.1 Effect of saccharification time3.2.2 Effect of liquid-to-solid ratio (LSR)3.2.3 Effect of initial cell concentration3.2.4 Effect of initial pH

3.3 Fermentation

4 ConclusionsAcknowledgementsReferences