BIOPROCESS DEVELOPMENTS FOR CELLULASE PRODUCTION BY Aspergillus oryzae CULTIVATED UNDER SOLID-STATE FERMENTATION

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1 Brzilin Journl of Chemicl Engineering ISSN Printed in Brzil Vol. 33, No. 1, pp , Jnury - Mrch, 216 dx.doi.org/1.159/ s352 BIOPROCESS DEVELOPMENTS FOR CELLULSE PRODUCTION BY spergillus oryze CULTIVTED UNDER SOLID-STTE FERMENTTION R. D. P. B. Pirot 1,2, M. Tonelotto 1,2, P. S. Delon 1,3, R. F. Fonsec 1,2, D... Pixão 3, F. C. F. Bleeiro 1,2, V. Bertucci Neto 2 nd C. S. Frins 1,2* ¹Universidde Federl de São Crlos, CEP: , São Crlos - SP, Brzil. ²Emrp Instrumentção, Ru XV de Novemro 1452, CEP: , São Crlos - SP, Brzil. Phone: + (55) (16) ; Fx: + (55) (16) E-mil: cristine.frins@emrp.r ³Lortório Ncionl de Ciênci e Tecnologi do Bioetnol (CTBE), Cmpins - SP, Brzil. (Sumitted: My 25, 214 ; Revised: Decemer 8, 214 ; ccepted: Ferury 8, 215) strct - Bioprocess development studies concerning the production of cellulses re of crucil importnce due to the significnt impct of these enzymes on the economics of iomss conversion into fuels nd chemicls. This work evlutes the effects of solid-stte fermenttion (SSF) opertionl conditions on cellulse production y novel strin of spergillus oryze using n instrumented l-scle iorector equipped with n on-line utomted monitoring nd control system. The use of SSF cultivtion under controlled conditions sustntilly improved cellulse production. Highest production of FPse (.4 IU g -1 ), endoglucnse ( IU g -1 ), nd -glucosidse (18.32 IU g -1 ) ws chieved t 28 C, using n initil sustrte moisture content of 7%, with n inlet ir humidity of 8% nd n irflow rte of 2 ml min -1. Further studies of kinetic profiles nd respirometric nlyses were performed. The results showed tht these dt could e very useful for ioprocess development of cellulse production nd scle-up. Keywords: spergillus oryze; Cellulse; Solid-stte fermenttion; Biorector; Respirometric nlysis; Instrumenttion; mzon Forest. INTRODUCTION Cellulse is complex of enzymes whose components hve synergistic ction during degrdtion of the polymeric chins of cellulose. Studies of cellulse production re of criticl importnce due to the influence of these enzymes on the economics of iomss ioconversion into fuels nd other chemicls (Klein-Mrcuschmer et l., 212). The cellulolytic enzymtic complex is produced y wide vriety of microorgnisms (cteri nd fungi); however, the eroic fungi re known for their high growth nd protein secretion rtes (Lynd et l., 22). Most commercil cellulses re produced y filmentous fungi of the gener Trichoderm nd spergillus (Bht, 2; Guskov, 211). mong the spergillus gener,. niger long with. oryze re the two most importnt fungi worldwide for iotechnologicl pplictions (Hu et l., 211). Nevertheless, recent findings on the genomics of. oryze hve reveled tht it is highly enriched with genes involved in iomss degrdtion (Koyshi et l., 27). Besides the well-estlished pplictions of. oryze in the food industry, this fungus hs gret potentil for the production of industril enzymes, which needs to e further investigted. The use of solid-stte fermenttion (SSF) for the development of industril ioprocesses hs een re- *To whom correspondence should e ddressed

2 22 R. D. P. B. Pirot, M. Tonelotto, P. S. Delon, R. F. Fonsec, D... Pixão, F. C. F. Bleeiro, V. Bertucci Neto nd C. S. Frins ceiving significnt ttention over the pst 2 yers (Brrios-Gonzlez, 212; Thoms et l., 213). SSF is prticulrly dvntgeous for enzyme production y filmentous fungi, since it simultes the nturl hitt of these microorgnisms (Holker nd Lenz, 25). However, the chrcteriztion of individul microorgnisms in terms of the influence of temperture nd moisture content of the sustrte on the kinetics of growth nd product formtion is essentil for SSF process scle-up. Previous studies hve shown the importnce of evluting the influence of process opertionl prmeters on cellulse production y spergillus niger cultivted under SSF using controlled conditions of forced ertion nd inlet ir reltive humidity (Frins et l., 211; Vitcosque et l., 212). Studies concerning the production of industril enzymes y. oryze cultivted under SSF hve een descried in the literture. The enzymes considered include -mylse (Bogr et l., 22; Frid nd Sht, 211; Kreem et l., 29; Pengthmkeerti et l., 212; Sivrmkrishnn et l., 27; Xu et l., 28), protese (Chutmnop et l., 28), nd xylnse (Pirot et l., 213; Szendefy et l., 26), mongst others. However, to the est of our knowledge, there hve een only few studies concerning the production of cellulse y. oryze cultivted under SSF, from the ioprocess development point of view (Begum nd limon, 211; Lio nd Wng, 212; Sndhu et l., 212). Given the potentil of. oryze nd its recognized ppliction for industril enzyme production, there is gret interest in the chrcteriztion of this fungus in terms of cellulse production using SSF under controlled opertionl conditions. The present work investigtes the development of ioprocess for cellulse production (FPse, endoglucnse, nd -glucosidse) y new mzon Forest strin of spergillus oryze cultivted under SSF using n instrumented l-scle iorector. The influence of temperture nd initil sustrte moisture content on the efficiency of cellulse production ws evluted, nd comprisons were mde with sttic ertion conditions. The selected conditions were used for chrcteriztion of kinetic profiles nd in respirometric nlyses. MTERILS ND METHODS Instrumented Biorector The iorector used ws l-scle system consisting of 16 columns (2.5 cm dimeter, 2 cm length) plced in wter th. The iorector ws equipped with n on-line system to control the ir flow rte nd the inlet ir reltive humidity, s descried previously (Frins et l., 211). For this study, the ir flow rte nd inlet ir reltive humidity were kept constnt during ll cultivtions, t 2 ml min -1 nd 8%, respectively. Microorgnism Screening n initil screening of 4 fungl strins isolted from the mzon Forest (Delon et l., 212) ws crried out in terms of endoglucnse production under SSF. In this preliminry step, set of SSF cultivtions ws crried out in 25 ml flsks for 24 hours, using solid sustrte composed of whet rn moistened t 6% with nutrient medium (Mndels nd Sternerg, 1976). Enzymes were extrcted t 24 hour intervls nd nlyzed s descried elow. The selection criterion for this study ws sed on endoglucnse productivity vlues (IU g -1 h -1 ) of the different strins, clculted in terms of ctivity units per mss of initil dry solid sustrte (IU g -1 ), s function of time (in hours). Identifiction of the Selected Strin The microorgnism selected for further use in this study ws strin of spergillus oryze (P27C3) deposited in the Emrp Food Technology microorgnism collection (Rio de Jneiro, Brzil). The culture ws mintined on PD slnts t 32 C for 5 dys efore inocultion for SSF cultivtion studies. The identifiction of the selected strin ws performed y extrction of the genomic DN nd mplifiction of the Internl Trnscried Spcer (ITS) regions, using the sme procedure descried in previous work (Pirot et l., 213). The sequence of the ITS region corresponding to rdn ws sumitted for registrtion t the Ntionl Center for Biotechnology Informtion (NCBI) nd received the ccess numer JX Selection of Opertionl Conditions for SSF Cultivtions Biorector cultivtions were crried out for 72 hours using whet rn s solid sustrte. The solid medium ws sterilized y utoclving t 121 C for 2 minutes efore inocultion. spore suspension volume corresponding to 1 7 conidi per g of dry solid medium ws inoculted into the solid medium y gently stirring with glss rod until uniform mixture ws otined. The strtegy dopted for se- Brzilin Journl of Chemicl Engineering

3 Bioprocess Developments for Cellulse Production By spergillus oryze Cultivted Under Solid-Stte Fermenttion 23 lection of SSF opertionl conditions ws to evlute ech vrile individully nd then select the est vlue for incorportion in the next vrile selection step. Initilly, cultivtions were crried out t 35 C with different moisture levels of 5, 6, 7, nd 8%. The moisture content ws djusted with nutrient medium solution (Mndels nd Sternerg, 1976). fter selection of the most fvorle moisture content, different tempertures (28, 3, 32, 35, nd 37 C) were evluted using n initil moisture content of 7%. ll cultivtions were crried out in 25 ml flsks under similr conditions to enle comprison of the results otined using forced ertion (iorector) with those chieved using the sttic ertion system. fter the cultivtion period of 72 hours, the enzymes were extrcted nd nlyzed s descried elow. Mens for ech condition were sujected to sttisticl nlysis using the Origin (version 8.) softwre. Cellulse Production Kinetic Profiles The kinetic profiles of cellulse production were evluted during 12 hour cultivtion period using the selected SSF opertionl conditions (28 C, ir flow rte of 2 ml min -1, inlet ir reltive humidity of 8%, nd initil sustrte moisture content of 7%). Cultivtions were crried out in 25 ml flsks under similr conditions in order to compre forced ertion (iorector) with the sttic ertion system. Smples were withdrwn t 24 hour intervls, nd the enzymes were extrcted nd nlyzed s descried elow. Enzyme Extrction fter the cultivtion period, the solid medium ws trnsferred to flsks (in the cse of column cultivtions) nd the enzymes were extrcted y dding sufficient volume of.5 mol L -1 citrte/ cette uffer, t ph 4.8, to chieve solid/liquid rtio of 1:1. The suspension ws stirred t 12 rpm for 3 minutes t room temperture, nd the enzymtic solution ws recovered y filtrtion. The enzyme extrcts were stored t -18 C prior to the nlyses. Respirometric nlyses Respirometric nlyses were crried out y mesuring CO 2 in the outlet ir strem from the columns of the iorector system, using GMM 22 instrument (Visl, Finlnd). The cumultive mount of CO 2 produced ws clculted from the re under the CO 2 vs. cultivtion time curve. The dt otined for CO 2 production up to the end of the exponentil phse (pproximtely 15 hours fter the end of the lg phse) were fitted to two different functions of cell growth ehvior for comprison. These were the logistic nd the decelertion equtions, nd the fitting ws performed y mens of non-liner regression using numericl softwre (Origin, version 8.). The logistic eqution (Eqution (1)) ws chosen for estimtion of the specific growth velocity (µ) for ech experimentl condition vrying the initil sustrte moisture content nd the temperture, s well s the cultivtion crried out for estimtion of the kinetic profile under the selected opertionl conditions. Cm C Cm 1 1e C t (1) In Eqution (1), C is the iomss content (in terms of mol CO 2 ), C m is the mximum iomss content (in terms of mol CO 2 ), C is the initil iomss content (in terms of mol CO 2 ), µ is the specific growth rte (h -1 ), nd t is the time. Here, the CO 2 produced ws used to descrie cell growth. This ssumption is resonle, since in the exponentil period the CO 2 derived from mintennce is much lower thn the CO 2 produced from growth, which mkes it possile to fit the CO 2 production during this period with logistic curve, s for iomss profile. The mount of initil CO 2 (C ) ws ssumed to e the sme ( mol of CO 2 ) in ech experiment, since the inoculum concentrtion ws fixed for these studies. The mximum CO 2 (C m ) ws estimted s the totl mount of CO 2 produced up to the end of the exponentil phse (which corresponded to pproximtely 15 hours fter the end of the lg phse). Enzyme ctivity ssys The ctivities of FPse nd endoglucnse were mesured ccording to the methodology descried y Ghose (1987). One unit of ctivity corresponds to 1 mol of glucose relesed per minute per ml, under the rection conditions. The quntifiction of the reducing groups ws performed using the dinitroslicylic cid (DNS) method (Miller, 1959). The - glucosidse ctivity ws determined using celloiose (Sigm, St. Louis, US) s sustrte nd quntifying the sugrs relesed y mens of n enzymtic kit for glucose mesurement (Lorl, São Pulo, Brzil). Brzilin Journl of Chemicl Engineering Vol. 33, No. 1, pp , Jnury - Mrch, 216

4 24 R. D. P. B. Pirot, M. Tonelotto, P. S. Delon, R. F. Fonsec, D... Pixão, F. C. F. Bleeiro, V. Bertucci Neto nd C. S. Frins The results were expressed s ctivity units per mss of initil dry solid sustrte (IU g -1 ). RESULTS ND DISCUSSION Screening of Cellulse-Producing Fungl Strins The efficiency of cellulse production in terms of endoglucnse productivity is presented in Figure 1 for 4 fungl strins isolted from the mzon Forest nd cultivted under SSF on whet rn. Figure 1: Screening of 4 strins of mzon Forest fungi for endoglucnse production under SSF. The two strins tht presented the highest endoglucnse productivity vlues were P4M2 (12.3 IU g -1 h -1 ), followed y P27C3 (1.1 IU g -1 h -1 ). The P4M2 strin hs een previously identified s spergillus fumigtus (Delon et l., 212). Since some strins of. fumigtus cn exhiit degree of pthogenesis (Meyer et l., 211), the P27C3 strin ws selected for identifiction. The ITS region of the fungl rrn ws mplified nd sequenced. The ITS rdn sequence of isolte P27C3 ws sumitted to lignment using the Bsic Locl lignment Serch Tool (BLSTN) progrm, nd showed stisfctory homology of 1% with the spergillus oryze species. Since there hve een very few ioprocess development studies of cellulse production y. oryze, the P27C3 strin ws selected for further chrcteriztion of its potentil s new source of industril enzymes. Effect of Initil Moisture Content on Cellulse Production y. oryze Bioprocess development for cellulse production y. oryze P27C3 cultivted under controlled SSF opertionl conditions ws crried out using the instrumented iorector. In order to compre the effectiveness of this system, similr set of cultivtions ws conducted under sttic ertion conditions. The effect of initil moisture content on cellulse production ws investigted y cultivting the fungus for 72 hours t 35 C using different initil sustrte moisture contents (5, 6, 7, nd 8%). Comprisons of the influence of moisture content on endoglucnse, -glucosidse, nd FPse production under forced nd sttic ertion conditions re presented in Figures 2, 2, nd 2c, respectively. s cn e oserved, there ws mrked positive effect of cultivtion under controlled conditions of forced ertion, where cellulse production ws significntly higher thn in cultivtions crried out under sttic conditions. Sttisticl nlysis indicted tht the popultion mens of ctivities otined using the instrumented l-scle iorector nd flsks were significntly different fter Tukey test comprison (p <.5). In these experiments, endoglucnse, -glucosidse, nd FPse ctivities were up to 48, 29, nd 336% higher, respectively, using forced rther thn sttic ertion. Lower enzyme production under sttic conditions is proly relted to oxygen limittion during fungl growth, since O 2 is trnsferred minly y diffusion. Besides oxygen trnsfer, het trnsfer nd CO 2 dissiption re lso fvored under dynmic conditions of forced ertion (Pirot et l., 213). n initil sustrte moisture content of 7% ws most fvorle for endoglucnse production (Figure 2). Nevertheless, endoglucnse production ws not significntly ffected y the initil moisture content used, for either of the ertion systems, s indicted y the sttisticl nlysis. Endoglucnse ctivity vlues vried from 39.8 to 5.4 IU g -1 when using forced ertion nd from 26.9 to 38.2 IU g -1 when using sttic ertion. Brzilin Journl of Chemicl Engineering

5 Bioprocess Developments for Cellulse Production By spergillus oryze Cultivted Under Solid-Stte Fermenttion 25 Endoglucnse ctivity (U g -1 ) B-glucosidse ctivity (U g -1 ) FPse ctivity (U g -1 ) B B Instrumented l-scle iorector Sustrte moisture (%) () Sustrte moisture (%) () (c) Figure 2: Effect of initil moisture content on () endoglucnse, () -glucosidse, nd (c) FPse production y. oryze cultivted under SSF using forced nd sttic ertion. Lower cse nd cpitl letters compre results for ctivities otined using either the instrumented l-scle iorector or flsks, respectively. Mens with different letters re significntly different fter Tukey test comprison (p <.5). The popultion mens of ctivities otined using the instrumented l-scle iorector nd flsks were significntly different fter Tukey test comprison (p <.5). more pronounced effect ws therefore oserved for sttic ertion, since modulting the initil moisture B Instrumented l-scle iorector Sustrte moisture (%) Instrumented l-scle iorector content from 5 to 7% resulted in 42% increse in endoglucnse ctivity, compred to 27% increse when using forced ertion. This cn e explined y the ility of the iorector system to mintin the moisture content of the medium y humidifiction of the ir pssing through the columns. This is very positive result in terms of ioprocess development; since it implies tht endoglucnse iosynthesis y. oryze P27C3 ws not very sensitive to vritions in sustrte initil moisture content when using iorector system with control of ertion nd inlet ir reltive humidity. similr trend ws oserved for -glucosidse, since n initil sustrte moisture content of 7% lso fvored production of this enzyme (Figure 2). -Glucosidse ctivity vlues vried from 23.1 to 31.2 IU g -1 when using forced ertion nd from 17.9 to 25.4 IU g -1 when using sttic ertion. s oserved for endoglucnse, there ws more pronounced effect of moisture content on -glucosidse ctivity using sttic ertion, with 42% increse in ctivity chieved y modulting the initil moisture content from 5 to 7%, s compred to 35% increse when using forced ertion. In terms of FPse ctivity, higher vlues were chieved using n initil sustrte moisture content of 8%. FPse ctivity vlues vried from.22 to.48 IU g -1 when using forced ertion nd from.11 to.14 IU g -1 when using sttic ertion. more pronounced effect of moisture content ws oserved for forced ertion, with 118% increse in FPse ctivity chieved y modulting the initil moisture content from 5 to 8%, s compred to 27% increse when using sttic ertion. This finding cn e explined y restricted production of the cellulse enzymes tht ccount for FPse ctivity due to oxygen limittion during fungl growth, together with reduced het trnsfer, when using the sttic ertion system. In ddition, since totl cellulse ctivity ssys re mesured using insolule sustrtes, prolems cn result from the heterogeneity of insolule cellulose nd the complexity of the cellulse system, especilly regrding reproduciility when the vlues re of the mgnitude encountered here. Moisture content is one of the most importnt fctors tht ffect SSF process efficiency. The effect of the initil sustrte moisture content on the production of cellulse y other spergillus strins cultivted using SSF hs een descried previously. Mmm et l. (28) evluted enzyme production under SSF, using the fungus. niger with ornge peel s sustrte, nd were le to significntly increse enzyme ctivities fter optimizing the initil moisture content of the solid medium. Go et l. Brzilin Journl of Chemicl Engineering Vol. 33, No. 1, pp , Jnury - Mrch, 216

6 26 R. D. P. B. Pirot, M. Tonelotto, P. S. Delon, R. F. Fonsec, D... Pixão, F. C. F. Bleeiro, V. Bertucci Neto nd C. S. Frins (28) found tht n increse in the initil moisture content enhnced enzyme production y the thermocidophilic fungus spergillus terreus M11, cultivted under SSF using corn stover s sustrte. Our results demonstrted tht the initil moisture content lso plyed n importnt role in cellulse production y. oryze P27C3, especilly for cultivtions crried out using sttic ertion. Nevertheless,. oryze P27C3 ws not very sensitive to vritions in sustrte moisture content when using iorector system with controlled forced ertion. Bsed on these results, n initil sustrte moisture content of 7% ws selected infurther studies to evlute the effect of temperture on cellulse production. Effect of Temperture on Cellulse Production y. oryze Comprisons of the effect of temperture on endoglucnse, -glucosidse, nd FPse production, using forced nd sttic ertion, re presented in Figures 3, 3, nd 3c, respectively. Cultivtions using tempertures of 28, 3, 32, 35, nd 37 C were crried out using oth forced nd sttic ertion systems, with fixed initil sustrte moisture content of 7%. In this set of cultivtions, there ws lso lrge positive effect of controlled forced ertion, with cellulse production eing significntly higher thn under sttic conditions. Sttisticl nlysis indicted tht the popultion mens of ctivities otined using the instrumented l-scle iorector nd flsks were significntly different fter Tukey test comprison (p <.5). Endoglucnse, - glucosidse, nd FPse ctivities were up to 87, 281, nd 21% higher, respectively, using forced ertion. This cn e explined y the more fvorle environmentl conditions encountered in the iorector system, such s efficient het dissiption nd voidnce of temperture grdients cused y the het generted during microil ctivity (Pirot et l., 213). The most fvorle temperture for endoglucnse production y. oryze ws 28 C (Figure 3). Production of the enzyme ws significntly influenced y temperture, in oth cultivtion systems. Endoglucnse ctivity vlues vried from 36.7 to 79.4 IU g -1 using forced ertion nd from 3.2 to 7.3 IU g -1 using sttic ertion. There ws therefore more pronounced effect of temperture for sttic ertion, since 133% increse in endoglucnse ctivity ws chieved y modulting the temperture, s compred to 116% increse when using forced ertion. In terms of ioprocess development, it is importnt to highlight tht endoglucnse iosynthesis y. oryze P27C3 ws significntly sensitive to vritions in temperture. FPse ctivity (U g -1 ) Endoglucnse ctivity (U g -1 ) B-glucosidse ctivity (U g -1 ) C 3 BC c Instrumented l-scle iorector 32 C () () (c) Figure 3: Effect of temperture on () endoglucnse, () -glucosidse, nd (c) FPse production y. oryze cultivted under SSF using forced nd sttic ertion. Lower cse nd cpitl letters compre results for ctivities otined using either the instrumented l-scle iorector or flsks, respectively. Mens with different letters re significntly different fter Tukey test comprison (p <.5). The popultion mens of ctivities otined using the instrumented l-scle iorector nd flsks were significntly different fter Tukey test comprison (p <.5). 35 Temperture ( o C) B BC Temperture ( o C) B 37 Instrumented l-scle iorector Temperture ( o C) Instrumented l-scle iorector C Brzilin Journl of Chemicl Engineering

7 Bioprocess Developments for Cellulse Production By spergillus oryze Cultivted Under Solid-Stte Fermenttion 27 different trend ws oserved for -glucosidse, with higher tempertures (35 nd 37 C) fvoring production of the enzyme (Figure 3). -glucosidse ctivity vlues vried from 9.7 to 29.5 IU g -1 using forced ertion nd from 2.9 to 26.5 IU g -1 using sttic ertion. Such different ehvior could e possily relted to different mechnism for -glucosidse production, given the different fungl ccessiility to the solule sugrs which ct s inducers for such enzymes. Nevertheless, s oserved for endoglucnse, there ws more pronounced effect of temperture on -glucosidse ctivity when sttic ertion ws employed, with temperture modultion resulting in n increse of 813% in enzyme ctivity, compred to 24% increse when forced ertion ws used. Using forced ertion, cellulse production in terms of FPse ctivity ws highest t 37 C, followed y 28 C (Figure 3c). FPse ctivity vlues vried from.19 to.31 IU g -1 using forced ertion nd from.1 to.12 IU g -1 using sttic ertion. more pronounced effect of temperture ws oserved for forced ertion, with 63% increse in FPse ctivity chieved y modulting the temperture, s compred to 2% increse when sttic ertion ws used. Temperture is n importnt vrile tht ffects microil growth under SSF, therey influencing product formtion, while the time t which mximum enzyme ctivity level is reched will lso vry ccording to the cultivtion temperture. The influence of temperture on cellulse production hs een reported for other spergillus strins cultivted under SSF. Jecu (2) studied the effect of tempertures etween 25 nd 37 C on endoglucnse production y. niger nd found tht the optimum for endoglucnse production ws in the rnge 28 to 34 C. Jsingh nd Nchiyr (211) studied the influence of different vriles on cellulse production y. nidulns MTCC344 using response surfce methodology. The effect of temperture ws evluted in the rnge 2 to 6 C, nd the optimum vlue for cellulse production ws found to e 37.5 C. s with the effect of moisture content, the chrcteriztion of ech prticulr microorgnism in terms of the influence of temperture on the kinetics of growth nd product formtion is essentil for SSF ioprocess development. Bsed on the results, n initil moisture content of 7% nd temperture of 28 C were selected for evlution of the kinetic profiles of cellulse production over cultivtion period of 12 hours. Kinetic Profiles of Cellulse Production Under the Selected Conditions The kinetic profiles of cellulse production over period of 12 hours, using the selected opertionl conditions (temperture of 28 C, initil sustrte moisture content of 7%, inlet ir humidity of 8%, nd flow rte of 2 ml min -1 ), re illustrted in Figures 4, 4, nd 4c for endoglucnse, -glucosidse, nd FPse, respectively. In order to compre the effectiveness of the system used, similr set of cultivtions ws conducted under sttic ertion conditions. Both endoglucnse nd FPse ctivities reched mximum vlues (123.64nd.4 IU g -1, respectively) fter 48 hours of cultivtion, wheres the mximum vlue for -glucosidse (18.32 IU g -1 ) ws only chieved fter round 96 hours of cultivtion, for oth cultivtion systems. Nevertheless, the dvntges of performing SSF under forced ertion conditions cn e oserved y compring the results with those otined under sttic conditions (Figures 4, 4, nd 4c). The dynmic system used here ws therefore suitle for controlling SSF opertionl conditions to chieve higher enzyme production efficiency. Zhng et l. (23) nd Mo et l. (24) lso found tht forced ertion hd positive effect on cellulse production y Penicillium decumens under SSF. In previous work, endoglucnse production of up to 56.1 IU g -1 ws chieved using whet rn s solid sustrte nd selected strin of spergillus niger (Frins et l., 211). The vlues for endoglucnse production chieved here were more thn two times higher, demonstrting the importnce of the chrcteriztion of new fungl strins in terms of their ility to produce iomss-degrding enzymes. Respirometric nlyses The evolution of CO 2 during the SSF process ws monitored using sensor connected to the gs strem exiting the columns of the iorector system. CO 2 dt cn provide n importnt mens of understnding the reltionship etween fungl growth nd enzyme production, since it is difficult to mesure iomss in SSF due to the prolem of seprting the iomss from the sustrte (Rimult, 1998). Figures 5, 5, nd 5c show CO 2 evolution during the cultivtions, investigting the initil moisture content, temperture, nd kinetic profiles, respectively, under the selected opertionl conditions. The CO 2 evolution curves shown in Figure 5 re very similr, which could imply tht fungl growth Brzilin Journl of Chemicl Engineering Vol. 33, No. 1, pp , Jnury - Mrch, 216

8 28 R. D. P. B. Pirot, M. Tonelotto, P. S. Delon, R. F. Fonsec, D... Pixão, F. C. F. Bleeiro, V. Bertucci Neto nd C. S. Frins Endoglucnse ctivity (U g -1 ) () Instrumented l-scle iorector B-glucosidse ctivity (U g -1 ) () Instrumented l-scle iorector.6 Instrumented l-scle iorector.5 FPse ctivity (U g -1 ) (c) Figure 4: Cellulse profile in terms of () endoglucnse, () -glucosidse, nd (c) FPsefor.oryze cultivted under SSF over period of 12 hours, using temperture of 28 C, n initil sustrte moisture content of 7%, n inlet ir humidity of 8%, nd flow rte of 2 ml min -1. CO 2 concentrtion (%) % Moisture 6% Moisture 7% Moisture 8% Moisture CO 2 concentrtion (%) o C 3 o C 32 o C 35 o C 37 o C () () 2. CO 2 concentrtion (%) (c) Figure 5: CO 2 evolution during cellulse production y.oryze cultivted using () different initil sustrte moisture contents, () different tempertures, nd (c) the selected opertionl conditions (temperture of 28 C, initil sustrte moisture content of 7%, inlet ir humidity of 8%, nd flow rte of 2 ml min -1 ). Brzilin Journl of Chemicl Engineering

9 Bioprocess Developments for Cellulse Production By spergillus oryze Cultivted Under Solid-Stte Fermenttion 29 ws not very much ffected y the different sustrte moisture contents used. This is in greement with the previous oservtion tht endoglucnse production ws not significntly ffected y the initil moisture content when using the iorector system with controlled conditions of ertion nd inlet ir reltive humidity (Figure 2). In order to further evlute the significnce of the CO 2 evolution curves otined using different initil moisture contents, the specific growth rte (µ) ws otined y regression nlysis of Eqution (1). The growth profile ws fitted very well y the logistic eqution, s reflected y the good correltion coefficients chieved, which were ll ove.99 (Tle 1). The decelertion eqution ws lso evluted nd resulted in similr good correltion (dt not shown). However, the logistic eqution ws chosen due to the fmilirity of its prmeters nd its wider use in the literture concerning SSF. The clculted specific growth rtes were similr for ll conditions of initil moisture content investigted (Tle 1). Nevertheless, the highest vlue of µ (.532 h -1 ) ws otined using n initil moisture content of 7%. This ws in greement with the moisture condition selected sed on cellulse production. There ws no significnt effect of the initil sustrte moisture content on the durtion of the lg phse. The CO 2 evolution curves reveled distinct influence of temperture on fungl growth (Figure 5), which ws fvored t 28 C, in greement with the condition identified for higher endoglucnse production (Figure 3). Similrly, there were distinct temperture-relted differences in totl cumultive CO 2 evolution, s well s the clculted specific growth rtes (Tle 2). The highest vlue of µ (.43h -1 ) ws chieved using temperture of 28 C, in greement with the temperture selected sed on cellulse production. Figure 5c illustrtes the evolution of CO 2 over period of 12 hours during the cultivtion crried out to otin the kinetic profiles of cellulse production under the selected opertionl conditions (temperture of 28 C, initil sustrte moisture content of 7%, inlet ir humidity of 8%, nd flow rte of 2 ml min -1 ). The totl CO 2 produced during the exponentil phse ( ± mol), s well s the vlue of µ(.4 ± h -1 ), re in greement with the results descried previously (Tle 2). Good correltion with enzyme production ws shown y oth the totl mount of CO 2 produced nd the clculted specific growth rtes, indicting the potentil of these prmeters for use in ioprocess development for cellulse production. Tle 1: Effect of initil sustrte moisture content on fermenttion prmeters otined using the logistic eqution. Initil moisture content (%) CO 2 produced in exponentil phse (mol) C C m 1 Lg phse (h) µ (h -1 ) R² 5 (1.14±.8) ± ± (1.9 ±.6) ± ± (1.18 ±.2) ± ± (1.15 ±.3) ± ± Tle 2: Effect of temperture on fermenttion prmeters otined using the logistic eqution. Temperture (C) CO 2 produced in exponentil phse (mol) C C m 1 Lg phse (h) µ (h -1 ) R² 28 (1.5±.3) ± ± (3.3 ±.4) ± ± (5.13 ±.13) ± ± (3.82 ±.11) ± ± Brzilin Journl of Chemicl Engineering Vol. 33, No. 1, pp , Jnury - Mrch, 216

10 3 R. D. P. B. Pirot, M. Tonelotto, P. S. Delon, R. F. Fonsec, D... Pixão, F. C. F. Bleeiro, V. Bertucci Neto nd C. S. Frins CONCLUSIONS The performnce of new mzon Forest strin of spergillus oryze P27C3 in cellulse production using solid-stte fermenttion under controlled opertionl conditions ws chrcterized using lscle instrumented iorector. The results otined enled selection of the vriles tht could e djusted in order to improve cellulse production. Highest production of FPse (.4 IU g -1 ), endoglucnse ( IU g -1 ), nd -glucosidse (18.32 IU g -1 ) ws chieved t 28 C, using n initil sustrte moisture content of 7%, n inlet ir humidity of 8%, nd n ir flow rte of 2 ml min -1. The results of respirometric nlyses were in good greement with enzyme production vlues, showing tht this informtion could contriute to ioprocess development for cellulse production. CKNOWLEDGMENTS The uthors would like to thnk Emrp nd the Brzilin gencies CNPq nd Cpes for finncil support. REFERENCES Brrios-Gonzlez, J., Solid-stte fermenttion: Physiology of solid medium, its moleculr sis nd pplictions. Process Biochemistry, 47(2), (212). Begum, M. F., limon,. R., Bioconversion nd scchrifiction of some lignocellulosic wstes y spergillus oryze ITCC for fermentle sugr production. Electronic Journl of Biotechnology, 14(5), 3-3 (211) Bht, M. K., Cellulses nd relted enzymes in iotechnology. Biotechnology dvnces, 18(5), (2). Bogr, B., Szkcs, G., Tengerdy, R., Linden, J., Pndey,., Production of lph-mylse with spergillus oryze on spent rewing grin y solid sustrte fermenttion. pplied Biochemistry nd Biotechnology, 12, (22). Chutmnop, J., Chuichulcherm, S., Chisti, Y., Sirinophkun, P., Protese production y spergillus oryze in solid-stte fermenttion using groindustril sustrtes. Journl of Chemicl Technology nd Biotechnology, 83(7), (28). Delon, P., Pirot, R., Codim, C., Tremcoldi, C., Rodrigues,., Frins, C., Using mzon forest fungi nd griculturl residues s strtegy to produce cellulolytic enzymes. Biomss nd Bioenergy, 37, (212). Frid, M., Sht, H., mylse production from spergillus oryze LS1 y solid-stte fermenttion nd its use for the hydrolysis of whet flour. Irnin Journl of Biotechnology, 9(4), (211). Frins, C., Vitcosque, G., Fonsec, R., Neto, V., Couri, S., Modeling the effects of solid stte fermenttion operting conditions on endoglucnse production using n instrumented iorector. Industril Crops nd Products, 34(1), (211). Go, J., Weng, H., Zhu, D., Yun, M., Gun, F., Xi, Y., Production nd chrcteriztion of cellulolytic enzymes from the thermocidophilic fungl spergillus terreus M11 under solid-stte cultivtion of corn stover. Bioresource Technology, 99(16), (28). Ghose, T., Mesurement of cellulse ctivities. Pure nd pplied Chemistry, 59(2), (1987). Guskov,. V., lterntives to Trichoderm reesei in iofuel production. Trends in Biotechnology, 29(9), (211). Holker, U., Lenz, J., Solid-stte fermenttion - re there ny iotechnologicl dvntges? Current Opinion in Microiology, 8(3), (25). Hu, H., vn den Brink, J., Gruen, B., Wosten, H., Gu, J., de Vries, R., Improved enzyme production y co-cultivtion of spergillus niger nd spergillus oryze nd with other fungi. Interntionl Biodeteriortion nd Biodegrdtion, 65(1), (211). Jsingh, S., Nchiyr, C., Utiliztion of pretreted gsse for the sustinle ioproduction of cellulse y spergillus nidulns MTCC344 using response surfce methodology. Industril Crops nd Products, 34(3), (211). Jecu, L., Solid stte fermenttion of griculturl wstes for endoglucnse production. Industril Crops nd Products, 11(1), 1-5 (2). Kreem, S., kpn, I., Oduntn, S., Cowpe wste: novel sustrte for solid stte production of mylse y spergillus oryze. fricn Journl of Microiology Reserch, 3(12), (29). Klein-Mrcuschmer, D., Oleskowicz-Popiel, P., Simmons, B.., Blnch, H. W., The chllenge of enzyme cost in the production of lignocellulosic iofuels. Biotechnology nd Bioengineering, 19(4), (212). Koyshi, T., e, K., si, K., Gomi, K., Juvvdi, P., Kto, M., Kitmoto, K., Tkeuchi, M., Mchid, Brzilin Journl of Chemicl Engineering

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