XVIII Congresso Brasileiro deEngenharia Qufmica

r:}C~M~V CONGRESSO BRASILEIRO DETERMODINAMICA APLICADA

8° Encontro 8rasileiro deAdsorção

A 22 DE SETE BRO 2010FOZ DO'rG AÇU/PR

XVIII Con9res~o IIral.ile-iro de-En~enhiJrliJ QvlmitiJ

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PRETREATMENTS EFFECTS ON SUGARCANE BAGASSE FORHIGH YIELD OF CELLULLASE PRODUCTION

U. RODRÍGUEZ-ZúNIGA1,2, C.S. FARINAS2

, F.N. GONÇALVEZ2, V. BERTUCCI

NET02, S. COURT3

, S. CRESTANA2.

IEESC-USP Pós-graduação em Ciências da Engenharia Ambiental, São Carlos, SP

e-mail: ursula@cnpdia.embrapa.br

2Embrapa Instrumentação Agropecuária, São Carlos, SP.

3Embrapa Tecnologia de Alimentos, Rio de Janeiro, RJ.

RESUME - Sugarcane bagasse is a low-cost, abundant Brazilian feedstock and apotential substrate for producing high specificity cellulases tailored to allow theviability of 2nd generation ethanol production. Due to its poor digestibility, it needssuitable pretreatments to obtain high yields for biological conversion. The present workaims a comparison of physicochemical pretreatments (sulfuric acid, sodium hydroxideand its combination) and its effects on cellulases production using solid statefermentation (SSF) of Aspergillus niger. The acidic/alkaline pretreatment resulted inmaximum concentration of cellulose (46.6% to 85.8%). Micrographs showed structuralcells loosening with partial exposition of cellulose network and lignin remova!.Crystalline degree increased with alkaline pretreatment indicating the loosing ofamorphous components or formation of microcrystalline cellulose. Natural bagasseshowed maxirnum FPase and xylanase production (0.3 and 23.20UJ/g respectively)Lower enzymatic production of acidic, alkaline and acidic/alkaline substrates (indecreasing order) suggest formation and recalcitrance of toxins/inhibitors hinderingfungal uptake.

Keywords: Agroenergy, cellulases, solid state fermentation, Aspergillus niger.

1. INTRODUCTIONIn last 15 years, an increasing effort hasmade towards developing efficient andsustainable technologies to attend thegrowing world energy demands.

Lignocellulose shows enormouspotential for conversion into bioethanol,

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this feedstock is made up predominantlyof cellulose, hemicellulose, and lignin.These fractions must be hydrolyzed inorder to produce fermentable sugars.There are numerous structural factors(e.g., crystallinity, available fibersurface area, pore structure anddistribution) and enzymatic mechanisticfactors that lead enzymatic hydrolysis

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(Weng et. ai., 2008; Hendriks andZeeman, 2009). The factors mentionedabove a11 need to be addressed force11ulosic ethanol to emerge on anindustrial scale. Constraints in the largescale process summarize in three criticalsteps: pretreatment, enzymatichydrolysis, and fermentation. Anadditional associated factor has been thehigh price of cellulase enzymes capableofhydrolyzing lignocellulose.

Efforts on cost reduction have beendirected towards increasing enzymeproduction by developing bettermicrobial strains, efficient fermentationtechnologies and recovery systems (Xuet. ai., 2005). Solid state fermentation(SSF) is one such technique; it enablesfungi growth in the absence of freewater and in a relative short time.

The use of SSF for production ofenzymes has many advantages like noneed for complex and sophisticatedmachinery, easy product recovery,simple and inexpensive substrates forthe fermentation, low energy demand,high volumetric productivity and often ahigh yield of products (Shah andMadamwar, 2005; Gessesse and Mamo,1998).

However a11these opportunities shouldbe supported by an essential operationof pretreatment if we are to achievehigh yields from biological operations.

In this context, the present work airns acomparison of physicochernicalpretreatments (sulfuric acid, sodiumhydroxide and its combination) and itseffects on ce11ulases production. Thesubstrate chosen, sugarcane bagasse(SCB) is an abundant and availablebyproduct from Brazilian industry andsuitable for the solid state culture of

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Aspergillus niger. Resultant low-costand highly specific celIuloytic enzymecomplex wilI expect to efficientlyhydrolyze abundant SCB in sugars forthe viable bioethanol production.

2. MATERIALSMETHODS

AND

2.1 MicroorganismThe microorganism used in this studywas a wild-type strain of A. Niger fromthe Embrapa Food TechnologycolIection. The culture was kept on drysand and activated in basic agar slants,formulated by Couri and Farias, 1995.Conidia were suspended in sterilizedTween 80 solution 0.3% (v:v).

2.2 Bagasse PretreatmentsA mass of 50 g of dried sugar canebagasse was grinded to attain 0.5 mmsize of mesh powder and then treatedwith 2% (w/v) concentrations ofNaOH,H2S04 and NaOH + H2S04, with 1:5(v/w) ratio in an autoclave at 121°C for30 mino After the treatment, alI thesamples were washed three times with3L (per wash) of distilIed wateracidified to pH 2 with H3P04. The finalpH of the wet solids was approximately5. Then the substrates were dried in anoven at 60 DCfor 5 h.

2.3 Solid State FermentationsSolid state fermentations (SSF) werecarried out in 500 mL flask with 5 g ofthe sterile pretreated substratesupplemented with Mandels & Webermedium (Mandels and Weber, 1969).The medium was inoculated with 1 x107 conidia/g and incubated at 32°C for72 h 111 a temperature-controlledchamber.

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At the end ofthe process each flask wassupplemented with 100 ml of an acetatebuffer 200 ruM (pH 4.5). The mixturewas stirred for 1 h at 120 rpm and 32°C.The solid residue was separated byfiltration through Whatman 40 filterpaper.

2.4 Analytical Methods2.4.1 Chemical composition

Van Soest's methodology (Van Soestand Wine, 1967) was used to determineeellulose, hemieellulose and lignin asthey eorrelate with neutral-detergentfiber (NDF), acid-detergent fiber (ADF)and lignin as follows:

Cellulose = ADF-Lignin

Hem ieelluloseCellulose

Dry matter and ash were determined byAOAC methodology (AOAC, 1980).

NDF Lignin-

2.4.2 Fourier transform infrared (FT-IR)

Samples speetra were obtained on anFT -IR speetrophotometer (SpeetrumOne Perkin Elmer) in the range 4000 e400 em-I using a KBr di se eontaining1% of finely ground pretreated sample.

2.4.3 X-ray diffraetion analysis

Crystallinity of the untreated andpretreated SCB samples were measuredusing a Shimadzu, 6000 X-raydiffraetometer with Cu Tg (À = 1,5418A). The erystallinity index (IC%) wasea\culated based on the method of Segalet al. (1959).

2.4.4 Seanning eleetron mieroseopy(SEM)

Samples were prepared for SEMinspeetion by stieking them on carbonglue and allowed to Au-eoated (CoatingSystem BAL-TEC MED 020).

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The eoated samples were examined byscanning eleetron microseopy (LEO 440model) with OXFORD deteetor at 20kV).

2.4.5 Enzyme assays

Eaeh filtrate of the enzymes produeedwas monitored for filter paperase(FPase), earboxymethyl eellulase(CMCase) and xylanase aetivity.

FPase and CMC aetivities weredetermined aeeording Mandels et aI.(1974). For FPase, assay mixture (3 ml)eonsisted of2 ml eitrate buffer (50 ruM,pH 4.5), filter paper Whatman NO.l(100 mg, 1 x 6 crrr') and 1 ml ofenzyme. The reaetion mixture wasineubated at 50°C for 60 min.

For CMCase, the total reaction volumeof I ml (0.5 ml sample of suitablydiluted enzyme and 0.5 ml of 4% CMCsolution in eitrate buffer) was ineubatedat 50°C for 10 mino

Xylanase aetivity was assayed in 3 mlof a reaction mixture eontaining 1 ml ofdiluted enzyme solution and 2 ml of1.0% (w/v) xylan (Sigma) in 0.05 Meitrate buffer (pH 4.8). The mixture wasineubated at 50°C for 30 min aeeordingto Bailey (1992).

After all the incubation periods, theredueing sugars were determined by thedinitrosalieylie acid method (Miller,1959) with glueose as standard.

One unit of enzyme activity is definedas the amount of enzyme whieh releases1 umol of glueose in 1 min under theassay eonditions.

3.RESULTS3.1 Chemical composition

The Table 1 presents the chemicalcharacterization of untreated and

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pretreated SCB samples and the massyield associated with each pretreatment.

Table 1. Relative chemical compositionof SCB before and after pretreatments.

ComponentSCB NaOH H2S04

NaOH+(%) H2S04

Cellulose 46,62 76,44 72,72 85,78

Hemicellulos 26,51 15,48 2,89 4,69

Lignin 21,7 2,42 19,98 2,57

Ash 2,51 1,87 2,22 1,12

Extracti ves 2,44 - -Mass Yield - 60 52 41

lt can be observed that cellulose valuesincreased in comparison to the untreatedmaterial indicating a relativeeoneentration of this fraetion due to thehemieellulose and lignin elimination.

Results in Table 2 show the totaleomposition of the pretreated samplesin relation of the final mass yieldobtained by eaeh pretreatment (Table1). These infonnation permit toealculate effieieneies in relation to eaehbiomass eomponent.

Table 2. Quantification of chemicalcomponents in the pretreated samples.

Biomass component NaOH H2S04NaOH+

(%) H2S04

Cellulose 45,87 37,82 35,17

Hemicellulose 9,29 1,50 1,92

Lignin 1,45 10,39 1,05

Ash 1,12 1,15 0,46

As suggested by the values, thepretreatments used were not seleetivefor the lignin and hemieellulose, smeethey promoted the eellulose and

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earbohydrates solubilization (Fengel eWegener,1989).

The final eoneentration of ligninindieates an effieieney of 93% ineonsideration of this eomponent. Asreported by Silvertein et al., 2007 andCardona et aI., 2010; the main effeet ofNaOH is delignifieation by breaking theester eross-linking lignin and xylan,thus inereasing the porosity of biomass.It resulted in lesser eellulose andhemieellulose solubilization than aeidand eombined pretreatrnents.

On the other hand, the main objeetive ofthe aeid pretreatments is to solubilizethe hernieellulosie fraetion in sugars(xylose, arabinose, ete.) to make theeellulose more aeeessible (Alvira et al.,2010). A deerease of 94% mhemieellulose eontent after aeidtreatrnent eonfinns this statement.However it resulted toa in 18,8% ofeellulose remotion.

After the combination of the acid andalkaline pretreatments both reduetion inlignin and hernieellulose was 95% and93%, respeetive1y. This reduetioninflueneed in the deereasing of eelluloseeoneentration of approximately 25%.

In general, it is expeeted a bettereffieieney in fungal growth and uptakewith the resultant major eoneentrationsof eellulose.

3.2 Fourier transfonn infrared (FT-IR)

FT-IR teehnique was perfonned to inferstruetural ehanges in ehemieal groupsderived by the pretreatmentsapplieation.

Figure 1 shows the resultant speetra ofthe analysis of untreated and pretreatedSCB. It ean be seen that the earbonylband at 1735 em-I, whieh has been

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ascribed to hemicelluloses (Zhao et ai.,2008) is reduced in all pretreated SCB.As well as, lignin bands atapproximately 1595 and 1510 em"(aromatic ring stretch) are stronglyreduced in the alkaline and acid/alkalinepretreated samples compared with bothacid treated and untreated SCB(Kristensen et ai., 2008).

-lnnatureBH2S04

-H2S04NaOHNaOH

2500 2000 1000 500

Figure 1. FTlR spectra of untreated,acid, alkaline and its combinationpretreated SCB. Excerpt of spectra. Allspectra are separated to easecomparison. The vertical lines in red,blue and green mark the positions of thebands ascribed to hemicellulose, ligninand cellulose respectively.

Differences between samples withregard to the relative amounts ofamorphous and crystalline cellulosehave earlier been described throughinfrared peak ratios at 1429 cm-I

(crystalline) and 893 cm-I (amorphous)(Hulleman et al., 1994; Wistara et ai.,1999).

Tough the FTlR spectra in this studywere not quantitative; comparisonbetween cited bands will allow inferringqualitative alterations. As observed theintensification in 1429 cm-I shouldcorrespond to the concentration ofcrystalline cellulose. To complementthis information, X-ray spectroscopy

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was performed in order to quantify thecellulose crystallinity.

3.3. X-ray diffraction (DRX) analysis

Crystallinity indexes (%IC) of thesamples in Table 3 were calculated fromDRX spectra (data not shown) as citedin methodology. %IC of a biomassmeasures the relative quantity ofcrystalline cellulose in the whole solid;therefore it should be corrected by themass yield of each pretreatment to havea net result of each pretreatment.

Table 3. Comparison of crystallinityindexes, between untreated and

pretreated SCB.Pre-

IC (%)Mass yield Corrected

treatment (%) IC (%)SCB 52,83 52,83NaOH 88,40 60 53,04

H2S04 60,17 52 31,29NaOH+

73,53 41 30,15H2S04

As can be observed alkalinepretreatment caused a slight increase in%IC. This can be explained as therelative concentration of crystallinecellulose after the massive removal ofamorphous components (Zhao et ai.,2008).

But then, acid and combinedpretreatments caused a reduction in%IC of about 40.8% and 42.9%respectively. This fact suggests asignificant recalcitrance reduction in theremaining cellulose and a rupture of thenatural physical barrier Lin biomass(Himmel et aI., 2007).

3.4 Scanning electron microscopyobservation (SEM)

Morphological modifications caused bypretreatments were determined fromSEM (Fig. 2).

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Figure 2. SEM micrographs ofuntreatedand pretreated SCB. A) untreated SCB500x. B) acid treated SCB IOOOx.C)alkaline treated SCB IOOOx andD)acid/alkaline treated SCB 2000x.

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An entire comparison betweenmicrographs of untreated SCB 2A and2B, 2C and 2D reveals in generalstructural loosening of cells includingepidermis and parenchyma tissue.Untreated SCB micrograph (Fig 2A)shows a disrupted structure derivedfrom previous operations of milling andwashing, original from sugarcaneprocessing.

Although the whole pretreated materialis heterogeneous and contains largerpieces easily recognized as SCB, Fig 2Bdisplays a partial defibration and theexposition of vascular bundles (phloemand xylem) after acid pretreatment.

Moreover, in relation to alkalinepretreatment (Fig 2C), it can beobserved a highlighted exposure ofmycrofibrilar cellulose structure derivedfrom the solubilization of lignin andhemicellulose.

Finally the acid/alkaline pretreatment(Fig 2D) promoted a partial removal ofmycrofibrils and the appearance ofamorphous cellulose aggregates.

3.5 Enzyme production

Table 4 summarizes the enzyme activityobtained at the end of the solid statefermentations (SSF).

Table 4. Mean final values of enzymaticactivities of A. Niger SSF.

Pre-treatment CMCase I Xilanase I Fpase

(UIIg)SCB 9,32 23,20 0,30

NaOH 2,34 12,30 0,07

H2S04 13,70 14,86 0,16NaOH+ 1,72 9,87 0,03H2S04

Highest FPase and xylanase activmesindicate the more efficient fungal

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utilization of natural SCB, yielding 2-fold mcrease of these enzymeproductions when compared to thesecond better substrate (acid pretreatedSCB).

On the other hand, CMCase activitieswere the highest in SSF of acidpretreated SCB. As established bychemical compositions, the acidpretreatment solubilized hemicellulosefavoring its conversion to easily usefulsugars for the fungi growth andendoglucanase production.Nevertheless, the lower FPase andxylanase fungal expression incomparison with natural SCB could beattributed to the specific inhibition bysome other sugars formed too as aconsequence of the pretreatrnent(Pathak and Ghose, 1973; Holtzapple etaI., 1990).

The lowest activities in alkali andcombined pretreated SCB could be dueto absorption of cellulase on cellulosewith high porosity (Sutcliffe andSaddler, 1986; Chernoglazov et ai.,2008) and/or to inhibition by thepresence of sugars degradationcompounds. It has been documentedthat furfural, hydroxymethylfurfural andaromatic lignin degradation compoundsinhibit fungi growing (Adsul et aI.,2005).

However combined treatment leads tosignificant concentration of cellulose.The difference in cellulase activitiesmay be due to variation in the amountsof utilizable amorphous componentspresent in treated samples andexpressed as an increasing in the % ICof the remaining cellulose. Theseobservations led to the conclusion thatphysical structure of bagasse cellulose

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is an important factor in the SSF for theproduction of cellulases.

4. CONCLUSSIONSBetween the substrates evaluated;untreated SCB represent the mostefficient and economical substrate forFSS and a feasible altemative to savecosts on the enzyme production processo

Although, partial hemicellulose andlignin removal is an important factor inincreasing the digestibility of FSSsubstrates, it is shown that the morecellulose remains undegraded the moresuitability for fungal uptake.

Finally, the study shows the relevanceof the washing stage in the pretreatmentprocess in order to extract fungal andyeast toxic substances that may havebeen formed.

5. REFERENCESADSUL, M.G.; GHULEB, J.E.;SINGHB, R.; SHAIKHB, H. Enzymatichydrolysis of delignified bagassepolysaccharides. Carbohyd Polym., V.

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BAILEY, M.J.; BlELY, P.;POUTANEN, K., Interlaboratorytesting of methods for assay of xylanase

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HENDRIKS, A. T. W. M.; ZEEMAN,G. Pretreatments to enhance thedigestibility of Iignocellulosic biornass-Review. Bioresource Technol., v. 100 p.10-18,2009.

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