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Particle geometry affects differentially substrate composition and enzyme profiles by Pleurotus ostreatus growing on sugar cane bagasse

Isabel Membrillo 't, Carmen Sánchez b, Marcos Meneses d, Ernesto Favela e, Octavio Loera e,fo

"Divisíón de Quimica y Bioquímica, Tecnológico de Estudios Superiores Ecatepec" Ecarepec 55210, Mexico "Universidad Autónoma de T/axca/a, C/CB, Laboratorio de Biorecn%gía. c.P. 90000, T/axca/a, Mexico , Departamento de Biorecn%gía. Universidad Aurónoma Meeropolicana-Izcapalapa, 09340 Mexico D.F., Mexica d Programa en Ganadería. Colegio de Posgrad(lados. Km. 36.5. Carro Mexico-Texcoco, Montecillo 56230. Mexico

ARTICLE INFÜ A B S T R A e T

ArtieJe history: Received 20 May 2010 Received in revised form 22 August 2010 Acceptecl 24 Augllst 2010 Available online 16 September 2010

{(eywords: Paniele size FUl1gal growth Enzymatic selectivity C1assinecl substrate

The growth of Pleurotlls ostreatus was analyzed on three particle sizes of sugar eane bagasse: 0.92 mm and1.68 mm in diameter, in addition to heterogeneous fibers (average 2,9 mm in diameter). Speeifie growth rate on heterogeneous particles was lower (Jl = 0,043 h- 1), although soluble protein produetion was maximal (809 ¡.¡gjg dry wt). Higher Je values were reaehed on the other two panieles sizes (0.049-0.05 h-1

) with less soluble protein (500 ¡.¡gjg dry wt). Xylanases and laceases were favored in het­erogeneous partieles; while the highest seleetivity for xylanases over eellulases was observed in 1.68 mm particles, eorresponding with the maximal hemieellulose breakdown. Lignin and eellulose were preferen­tially c1egraded in smallest particles. This study shows that the geometrieal ratio, shape and size of sugar eane bagasse fibers strongly influenee paeking density for SSF substrate, with an impaet in the produetion of extracellular enzymes, growth rates and composition changes in substrate,

© 2010 Elsevier Ltd, AII rights reserved.

1. Introduction

Sinee the miel-90's, many of the studies related to the applica­tion of soJid-substrate fermentation (SSF) were focused on increas­ing the added value of agro-industrial residues (Pandey et al., 2000), Various processes have been developed to enhance the pro­tein content in starchy fruit wastes (Smail et al.. 1995). synthesis of metabolites (Paliares et al., 1996) and enzyme production (Mazutti et al.. 2006). In most cases the source of nutrients acts also as a support for microbial growth, Thus, the degradation of substrate in SSF is attributed to the release of cell bound enzymes or extra­cellular enzymes to the external environment (Nandakumar et al., 1994),

Jn SSF processes there are three physical phases: gas, Iiquid and solid. Aqueous phase absorbed to solid surfaces is also in contact with the gas phase; the gas-liquid interface represents a frontier for the gaseous exchange oxygen-carbon dioxide and heat transfer. Enzymatic action on the substrate depends upon the size of this frontier, which is determined by the physical properties of the materials inclucling the crystalline 01' amorphous nature, accessible and surface area, porosity and mainly particle size (Knapp and Ho­well, 1980; Viniegra-González et al., 2003; Roclríguez ancl San­

román, 2005). Thus, structure and size of substrates allow different materials to be available for microbial degradation (Pan­cley, 2003), Hence mechanical separation of any substrate can be useful for specific purposes, such as the cultivation of edible fungi, feed production (Zadrazil and Puniya, 1995) or more specific pro­duction of extracellular enzyme extracts (Mazulti el al., 2006).

The effect of particle size on growth and product formation has been studied by different authors (Roul<as, 1994; Recldy et el l., 2003). Recently, Membrillo et al. (2008) using two Pleurotus ostre­atus strains grown on sllgar cane bagasse, reported differences in the production of lignoceJluJolytic enzymes (xylanases, laccases and celllllases) ancl protein production, as a response to particle geometry; however, that worl< focused on variation between indi­vidual strains. The aim of the present work was to analyze the growth of P. ostreatus on sugar cane bagasse lIsing three particle sizes, relating these reslIlts to lignocelllllolytic enzymes patterns and protein profiles, in acldition to degradation of each component (lignin. cellulose ancl hemicelllllose) as a function of particle geom­etry in SSF,

2. Methods

2,1. Microorganisms and inoculum produetion

* Corresponcling author. Tel.: +52 55 58 04 64 08; fax: +52 55 58 04 64 07. The strain P. ostreatus IE-8 belongs to the fungal collection of the E-mair address: loera@xanum.uam,mx (O. Loera), Colegio de Posgraduados in Texcoco, Mexico, This strain was grown

0960-8524/$ - see front maner © 2010 Elsevier Ltcl. AI\ rights reselved. do;:l 0.1 016/.1.1>iorcech.201 0.08.091

1582 I. Membrillo ee a/.jBioresource Technology 102 (2011) /581-1586

in plates of malt extract agar (MEA, DIFCOTM) containing sterile fil­ter paper strips (1 x 6.5 cm). After the strips were covered by mycelium, they were collected and submerged in 5 mI of sterile water in glass vials. These vials were kept at room temperature for up ro 1 year. Before starting SSF cultures, MEA was entirely dis­solved in wheat straw extract (WSE) which was obtained as previ­ously clescribed (Membrillo et al., 2008). AII culture media were autoclaved at 15 PSI for 15 mino In order to prepare inoculum for SSF, three plates with MEA-WSE medium were inoculated with a 6 mm mycelium-agar plug, obtained from simple MEA plates, and incubated at 29.5 ± 0.5 oC for 5 days. This fresh mycelium was taken as inoculum for SSF kinetics as described below.

2.2. 511bstrate characterization

Sugar cane bagasse was obtained from the Emiliano Zapata su­gar mili in Zacatepec, Morelos, Mexico. Dry material was subjected to a sieving procedure employing mesh-size sieves of: 4, 6, 8, 12, 16,20,24,35,48,50.60 (Mc Cabe et al., 2002). Then, bagasse par­ticles were classifiecl according to three diameter sizes and were usecl as substrates for SSF. The smallest partic\es (0.92 mm) were collected from fractions between meshes 16 and 20 (- 16, +20); intermediate particles (1.68 mm) were collected from fractions be­tween meshes 8 and 12 (-8. +12); heterogeneous bagasse (0.25­5.5 mm, average 2.9 mm diameter) was also used as a substrate. The geometrical ratio (fiber length: diameter or L/D) was deter­mined for each particle size as an average value from 100 fibers per fraction. These L/D values correspondecl to 12.3 ± 0.44, 8.7 ±1.1 and 18 ±0.55 for particles sizes of 0.92, 1.68 and 2.9 mm, respectively. In the present work, these L/D values will be denoted as 12,9 and 18, respectively. For all three selected frac­tions, chemicaJ composition was determinecl according to the methoclology proposed by Bassi (2005) and Van Soest et al. (1991).

2.3. 5a/id-sllbstrate fermentatian

Sugar cane bagasse was rehydrated with hot water (90 OC) for 30 min; then excess moisture was drained at room temperature and the final moisture content of 80% was determined in the sam­pies by weight difference after drying samples for 24 h at 60 oc. Five grams of this substrate were placed in 250 mi flasks and were autoc\aved at 121°C for 15 mino Each flask was inoculated with four mycelium plugs (6 mm diameter) obtained from MEA-WSE meclium as described aboye, the plugs were uniformly covered by the substl'ate with a sterile spatula. Cultures were kept at 29.5 ± 0.5 oC and samples were taken periodically in triplicates, dissolving the content of each flask in 50 mI of sodium citl'ate buf­fer (50 mM, pH 5.0) for 30 min in an ice bath. Solids were separated by filteríng through a gauze c\oth, and filtrate was then centrifuged (4 oC, 12,1 OOg, 30 min). Supernatant was used for measurements of enzyme activity and soluble protein. Additionally at the beginning of each culture, measurements of bed density (package density when a column is filled with substrate), fractional voidage (p.), sphericity (1),), and specific surface (a) were done for each rehy­drated particle size fraction according Mc Cabe et al. (2002).

2.4. Ana/ytica/ procedllres

Protein was determined according to Bradford (1976) using 80­vine Serum Albumin (40 mg/I) as a standard; xylanase activity was estimated by DNS method (Miller et al., 1960) using a 0.5% solution of Birchwoocl xylan as substrate, previously dissolved in a sodium citrate buffer (50 mM, pH 5.3) according to Loera ancl Córdova (2003); laccase activity was determined registering oxidation of 2,2-azo-bis-( ethylbenzothiazoline-6-sulfonic acjd) (ABTS) in an aceta te buffer (0.5 mM, pH 5) at 420 nm during 2 min (Wolfenden

and Willson. 1982); carboxymethyl cellulase (CMCase) activity was estimated by DNS method (J. = 575 nm) using a solution of 1% car­boxymethyl cellulose as a sllbstrate, c1issolved in a sodillm citrate buffer (50 mM, pH 4.8) according to Miller et al. (1960); filter papel' activity (FPA) in the filtrates was measured also at pH 4.8 employ­ing a piece ofWhatman 41filter paper (1 x 6 cm) according to the procedure described by and Decker et al. (2003). AII activities were calculated from triplica tes and vallles were expressed in interna­tional units (IU), where one unit (IU) of enzyme activity is defined as the amount of enzyme required to release 1 pmol of prodllct per minute under the given assay conditions. Activities were referred to initial substrate dry weight (IU/g dry wt). Alternatively, when activities were lower than 1 IU these vallles were expressed as mU (1000 mU = 1 IU).

2.5. Growth and biamass estimatian

Biomass production was determineclusing measurement of glu­cosamine as a specific cell wall constitllent (Scottí et al., 2001) in order to estimate the fungal biomass. In SSF, growth patterns can be fitted to non-linear profiles such as the logistic equation whose integrated form is (Mitchell et al .. 2004):

X= Xm 1 + ((Xl71/Xo) - 1)exp-1iC

In this work the adjllstment of experimental data was achieved with Origin 7.0 for Windows software.

3. Results and discussion

3.7. 511bstrate charaeterization

Dried sugar cane bagasse was classified after sieving, subse­qllently composition on dry basis for every size fraction was deter­mined to be the same: lignin 19% ± 1.9, hemicellulose 20% ± 1.8, cellulose 57% ± 3.3, and ashes 3.2% ±0.7. These results were in agreement with Pancley et al. (2000), who also reported a major proportion of cellulose in sugar cane bagasse and equal parts of lig­nin and hemicellulose, with an ashes content of 2.4%; in fact the hemicellulose/lignin ratio was the same as our reslllts. Once rehy­drated as mentionecl aboye, substrate or individual particle density was estimated to be 1131 ± 107 kg/m 3 for the three analyzed frac­tions, showing a similar water retention capacity in all fibers sizes. Although substrate density of individual particles was the same. packing density (bed density) of the SSF did vary for every particle size (Table 1). Intermediate particles showecl higher density than smaller particles, this can be explained taking into account both geometrical ratio L/O and sphericity factor, since particles with smallest diameter were also less spherical. reslllting in a less com­pact substrate bed.

Small particles offer greater area/volume ratios 01' specific sur­faces, and corresponding vallles were 2.56 and 1.85 mm- 1 for parti ­cles witl1 diameter 0.92 and 1.68 mm, respectively. Heterogeneolls particles (2.9 mm díameter) contain a considerable fraction of par­ticutate smaller than 1.68 mm (45.5%) and also 70% particles with

Table 1 SlIbstrare characterization for rehydrated sugar cane bagasse particles.

Diameter Geometrical Packing Sphericity Specific Fractional (mm) ratio (LID) density ('/J,) slIrface voidage

(kg/m3 ) (mm- I ) (¡;)

0.92 12 244 26 0.55 2.56 0.78 168 9 349 37 0.60 185 0.69 2.9 18 392 50 0.54 2.55 0.65

1583 l. Membrillo er al./ 8ioresollfce Technology 102 (2011) 1581-1586

a L/O ratio higher than 10; thus, the specific surface in these particles (2.55 mm- I

) is a global measure of the substrate (Table 1).

3.2. Plant material degradation

Degradation of plant material for the three particle sizes was very similar, diminishing from 92% to around 81% after 15 days of cultivation; however, composition of particular fractions was af­fected in a different manner. Balances for each component in sl1gar cane bagasse were achieved considering the amount of fermented material (dry basis), neutral detergent fiber content (NDF, repre­senting the amOl1nt of celll1lar plant material) and each material fraction. Variations in the utilization of each component are sum­marized in Table 2. Notably, in 1.68 mm particles (L/O; 9) half of hemicellulose and lignin were modified, suggesting that as break­down takes place the availability of both components showed a synergistic effect in the degradation. On the other hand, he te roge­neous substrate (2.9 mm diameter, L/O ~ 18) showed similar levels of hydrolysis for three materials: 35.9% for Jignin, 43.5% for hemi­celll1lose and 40.1% for celll1lose. In the case of small particles (0.92 mm diameter), cellulose was degraded preferentially over hemicelJulose, in fact cellulose degradation was the highest in comparison to other substrate sizes. As mentioned, this would cause lignin to be more accessible within the plant strl1cture, thus degradation (67.4%) is improved. These results are consistent with a stl1c1y by Reddy et al. (2003), who reported a redl1ction of lignin content in banana leaves (52%) and psel1dostems (23%) after grow­ing P. ostreatus, supporting that lignocelll1loJytic enzymes produc­tion depends on plant material strl1cture. In terms of hemicelll1lose modification, our results are comparable to those reportecl by Oka­no et al. (2007), who described that Pleurotus eryngii grown on su­gar cane bagasse and rice bran was able to reduce hemicelll1lose up to 46%; although lignin degradation was only 6% with no substan­tial changes in cellulose content.

3.3. Growth and soluble protein profiles

Growth profiles for P. ostreatus on each particle sizes are shown in Fig. l. It is remarkable that at the end of the culture, biomass reached a similar value (8 mg/g dry wt) independently of substrate size. Growth profiles were very similar on medium and small par­ticles (0.92 and 1.68 mm in diameter), whereas on heterogeneol1s particles (average size 2.9 mm) growth pattern was modified. Spe­cific surface of heterogeneous particles (2.55 mm- 1

) was compara­ble to that of small particles (2.56 mm .1), which would provide a good plant material availability for microorganismo It is possible that in heterogeneous particles, both bed density (392 kg/m3

)

and fractional voidage (e ~ 0.65) caused a slower biomass formation.

The data adjl1strÍ1ent to logistic equation allowed to determined specific growth rates (¡.¡.) values of 0.05, 0.049 ancl 0.043 h- 1 for particles of 0.92, 1.68 and 2.9 mm, respectively. These estimated growth rates were superior by over 100% than those measl1red

labIe 2 Plant material changes in sugar cane bagasse after growth of Pleurotus ostreatus for 1S days.

Diameter LID NDF(%) Degradation percentage FM" (mm) (%)

Lignin Hemicellulose Cellulose

0.92 12 80.26 67.4 37.5 85.9 76.1 168 9 79.65 470 50.7 39.3 76.4 2.9 18 83.34 35.9 43.5 40.1 73.1

Average 81.08 ± 198

• Final moisture.

10

9 ..:;+,--",.. -.~-:.~::-:.~

8 . ,~ e ..."O 7 01 1Cñ

6E (f) (f)

ro 5 1

E o

4 • 0.92 mm (ñ • 1.68 mm

3 4 2.90 mm

o 3 6 9 12 15

Cultivation time (days)

Fig. 1. Biomass prodllction by Plcurotus ostreatus on sllgar cane bagasse with dífferent particle sizes. Dotted lines: 0.92 mm (e) and 1.68 mm (.) particles. 50lid line: hecerogeneolls particles (").

for P. ostreatus grown in liquid medium (Márquez-Rocha et al., 1999); thus in des pite of the low nitrogen content (CN ~ 142: 1), sugar cane bagasse is a suitable substrate for biomass formation. Specific growth rate on heterogeneous particles was lower (¡.¡.; 0.043 h- 1

), although soluble protein production was favored (Fig. 2) with a maximul1l of 809 ~lg/g dry wt. On the contrary, for homogeneous particles, highest growth rates were reachecl (0.049-0.05 h- I

) with around 500 ¡.¡g/g dry wt of soluble protein. These results show rhat protein production by P. ostreatus shows different profiles depending on the size of particles. In adclition, these findings show that soluble protein can be used as an estima­tion of fungal growth in SSF; nevertheless, both protein content and biomass are not at all times proportionally related (Figs. 1 and 2).

3.4. Enzymes produetion

3.4.1. Enzymatic aetivities Enzyme production was determined for different activities:

xylanases, laccases and cellulases, since these enzymatic families are involved in the breakdown of ceH waH components. Xylanase

1000 -O.92mm

~

-.-1.68 mm~ >- 800 -4-2.90 mm .....

"O ...--------~ ...01 Cñ 2- 600 e <lJ e ~ -=.+1 ü o 400 e CD

e 200eL

12 156 93 Cultivation time (days)

Fig. 2. Protein production by Plellrotus ostreneus on sllgar cane bagasse with different particlo sizes.

1584

-O.92mm -.-1.68 mm - ... ­ 2.9 mm

15

.. 6 9 123

5

1, Membrillo el ol./BioreSOllrce Technology 102 (2011) 1581-1586

activity produced by P. ostreatus is shown in Fig. 3: a peal< of activ­iry was reached on heterogeneous bagasse (11 IU!g dry wt) after 3 days of culture, followed by a decline with an abrupt rise at the end of cultivation (20 IU/g dry wt). The peak production was also observed at day 14 on the other partide sizes: 17 and 10 IU/g dry wt, for 0.92 and 1.68 mm diameter partides, respectively, although a rapid decrease was observed. These production levels were substantially higher than those titres reported by Reddy et ,]1. (2003), who found xylanase activity of 0.94 IU/g dry wt grow­ing P. Dsrreatus on banana leaves cut into 2 cm size particles: although the xylanolytic system of P. ostreatlts responded accord­ing to the substrate since Salmones amI Mata (2002) reponed up to 2501U/g dry wt using coffee wastes. However, sugar cane bagasse added wi th ammonium salts proved to be a suitable sub­strate for Tilermoascus aurial1cicus, chis microorganism reached up to 110 lUjg dry wt (da Silva et al., 2005).

Similarly, on heterogeneous material the highest lacease pro­dllccion was achieved (24.5 mU/g dry wt) since the second day (Fig. 4), the activity was suscained for 8 days and decreased dra­matieatly afterwards. On the contrary, lacease activity was lower

in particles with diameter 0.92 and 1.68 mm. with a peak around 5 mU/g dry wt at day 9. For other P. ostreatus strains cultivated un­der SSF, laceases production was substantially higher. I<rishna Pra­sad et al. (2005) showed that levels of lacease acrivity up to 750 IU/ g dry wt can be achieved after optimizing culture conditions (glu­cose, wheat bran, urea, yeast extracr) in the presence of inducers and nitrogen sources. Reddy et al. (2003) reported laccase titres of 7.52 and 40.8 IU/g dry wt using banana leaves and pseudostems as sllbstrates; Salmones and Mata (2002) found 27 IU/g dry wt on coffee pulpo Sugar cane bagasse added with veratrylic alcohol and wheat bran allowed a lacease activity between 3.1 and 4,2IU!g dry wt before P. ostreatus fruity boclies formation (Suguimoto el al., 2001).

In the case of cellulases, both maximal CM Case (250 mU!g c1ry wt) and FPA activity (130 mU!g dry wt) were obtained on small particles (0.92 mm diameter) during first days of cultivarion (Figs. 5 and 6); whereas on heterogeneous substrate, CMease activity reached a maximal level after 5 days (85 mU/g c1ry wt) with a

25

20 ~

3' e -o 15O'>

:3 (/J Q) 10(/J ro e ro >. X

Cultivatian time (days)

250.----------------------..,

--0.92 mm -.-1.68 mm

200 -"'-2.9 mm

~ e

'O 150 O'>

:3 E

e¡ 100 ro o 2 U 50

3 6 9 12 15 Cultivation time (days)

Fig. 5. Endoglucanasc activiry by pleurocus ostrenrus 0\1 sugar calle b,1gasse wirh different particle siles.

Fig. 3. Xylanases activity by Plellroll/S ostreoll/s on sugar cane bagassc with differenr partiele sizes.

25 ~ ·------.1 ­ 0,92 mm ~

4-

l\o-16Bmm1 - ... - 2.9 mmtzo O'>

:3 E ~ 15 e 'S; 1u m 10 l~Q) Ul

ü

ro

m

/·-li_" •-l

ü 5 .---. . O

O 3 6 9 12 15 Cultivation time (days)

Fig.4. Laccase accivity by Pleurorus oslreotus on sugar cane b.1gasse wirh different particle sizes.

150 -,.-------------------.,

-O.92mm 120 -.-1.68 mm

-"'-2.9 mm

30 ,

o~ O 3 6 9 12 15

Cultivatian time (days)

Fig. 6. Filter paper activity by Plcurows osrrea/llS on slIgar cane bag¿lsse wirh different particle sizes.

1585 l. Membrillo el al.fBioresource Technology 102 (2011) 1581-1586

smoother loss at the end of the culture. On intermediate size par­ticles this activity was detected only at very low values (2 mU/g dry wt). OUT results were in agreement with others reports show­ing cellulases by P. ostreatus on agricultural wastes, for instance Reddy et al. (2003) reported that CMCase activlty on banana waste reached 0.81 IU/g dry wt and FPA was practically undetected after 20 days: [(umaran et al. (1997) found a CMCase activity of0.56 ¡U/ g dry wt on sago hampas. Other fungi are better producers of cellulases on sugar cane bagasse: Aspergillus niger and Aspergillus terrells reached 10 and 2 IU/g dry wt, respectively (Gawande and l<amJt, 1999): T auriantiws grown on sugar cane bagasse supple­mented with ammonium salts produced 30 IU/g dry wt (da Silva el: aL, 2005). The analyses of these resulcs confirm that sugar cane bagasse is a pOOl' substrate for FPA production; additionally P ostreatus has not proved to be a suitable organism to produce cel­IlIlases. Sugar cane bagasse is a good alternative for this propose when mixed with other substrates and are used in co-cultures of Trichoderma reseei and A niger (Gutierrez-Correa <lne! Tengercly, 1998; Massacleh et al.. 2001).

In the present study, parcicle had an effect on CMCase and Frase. Blandino et al. (2002) have showed rllat both particle size and chemical eomposition of blends containing wheat grains and milled wheat affeet the rate of microbial growth and, therefore, the patterns observed by polysaceharidases production in Aspergi/­tus awamori after 7 days of cultivatíon at 30 oc Nandakumar et al. (1994) showed rhat the type of substrate available in wheat bran (st<lrch or cellulose) causes sequential production of enzymes. These authors propose d mathematical model which gives the pro­gression of substrate degradarion (size reductíon) during fermen­tarion in terlns of particle dimensions: our results agree with this point, since levels and proportions of each enzyme are affected by the exposure to variable marerials in sugar cane bagasse with dlfferent particles size and geometry. even though the initial com­position was rhe same.

3.42. Seleetivity in enzyme productiol1 As a general rule, fungal xylanases and cellulases occur simulra­

ncously dllring SSF on agricultural substr.:ltes: however, it is inter­esting ro evaluate xylanase/CMC.:lse (X/C) acrivity ratios in ssr­proeesses design. In this stucly, this r<ltio was estimated to be be­rween 15 and 184 for small partieles (L/O = 12); the value X/C var­ied between 54 and 250 for heterogeneous bagasse (L/O = 18); interestingly, high selectivity for xylanase aetivity was achieved in intennediate size particles (L/O = 9), with values ranging from 4560 (after 2 and 3 days) up to 11,560 at the end of the culture. These findings are important since there are specific applications demanding higher xylanolytic activities (Loera and Villaseiior, 2006). The present reslllts show that particles with sm.:lll L/O ratio. favor xylanolityc activity production. chus further studies could determine a L/O limir value for selectiviry in xylanases procluction. However, our selectivity values (high X/C) for elongated particles ( L/O> 10) are comparable to a report by Gutierrez-Correa and Ten­gerdy (1998) in a ca-culture of A. niger and T reseei (X/C=270). Additionally, our results were almost seven times higher than those calculated from A niger and A rerreus (36 and 35, respec­tively) (Gawande and [(amat, 1999), and <lbout 50 times than those estímated for Thermoasc1I5 auranliiac1l5 (X/C = 3.7) (Da Silva et aL. 20(5) or those calculated from P. ostreatlls stlldies reported by Red­dy et aL (2003) and Kumaran et al. (1997), with X/C ratios of 6 and 3.6, respeceively.

Degradation of the sllbstr<lte occurs in stages depending on the exposed material substrate: Jignin degradarion (secondary walls material) at the beginning. shortly after cellulose consumption linked to CMCase, fol1owed by xylan<lses action on hemicelluloses. NandakumM et al. (1994) sllggest ,1 seqllential release of extracel­lular enzymes by A niger in response to the type of substrate avail­

..~_.--,..

able in wheat bran (starch or cellulose): <x-amylase, glucoamylase. followed by cellulase and xyl<lnase: our results were similar to those patterns, since enzyme levels are affected according to expo­sure to different materials of sugar cane bagasse in particles with different geometry.

Substrate composition (Table 2) and prodllction of enzymes by P. ostreatus show that in 0.92 mm particles. maximum productioll of CMCase and FPA was obtained, which corresponds wirh the maximllm cellulose degradatíon. However. hemicellulose degrada­tion cannot be associated with an increasc in xylanolytic activity; this can be explained rather by the constant presence of these en­zymes along fermentatíon in 1.68 mm particles. As for lignin deg­radation, maximum degradatíon occurrecl in small particles with the lower production of laceases, probably lignin degradarion is as­sisted by other types of phenoloxidases, in addition Co non soluble enzymes associated to biomass. This can also be facilitated by the high levels of xylanases and celllllases in the smallest partieles, whose action makes Iignin more accessíble to laceases. This has practical implication since sugar cane bagasse is used in manufac­turing feed for livestock, and lignin content shoulcl be redllced. without compromising the amount of celluJose: then, it seems interesting to assay pre-treatment of bagasse varying size particles wirh L/O ratios lower than 10, due to the fact that chis relation re­duces preferentially lignin over cellulose.

4. Conclusion

As final remarks. the present study shows that the geometrical ratio, shape and size of sllgar cane bagasse ftbers strongly inflllence packing density for SSf substrate, with an impact in the production of extracellular enzymes, growth rates and composition changes in substrate. Xylanases and laccases are favored in heterogeneous and elongated substrate; although high selectivity for xylanases induc­tion is observed in short particles. Fiber components such as lignin and cel111lose are preferentially degraded in smallest particles, while hemicellulose breakdown reached the highest level in med­ium size particles. Thus, substrate classification can be used as a criterion to direct the synthesis of specific enzymes, tiber modifiea­tion and protein enrichment by P. ostreatus in salid cultures with particular techniea[ applications.

Aclmowledgements

The authors thanl< the financial support of Red-PROMEP and National Council of Scíence and Technology (CONACyT: Grant 61395 for L Membrillo) during this research.

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