Steam-Exploded Sugar Cane Bagasse for On-Site Production of

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Steam-Exploded Sugar Cane Bagasse for On-Site Production of Cellulases and Xylanases by Penicillium echinulatum Marli Camassola* and Aldo José Pinheiro Dillon Institute of Biotechnology, University of Caxias do Sul, Rua Francisco Getúlio Vargas, 1130, 95070-560 Caxias do Sul, Rio Grande do Sul (RS), Brazil ABSTRACT: The high cost of cellulases and xylanases for the hydrolysis of lignocellulosic wastes is a bottleneck in the production of second-generation ethanol. The use of a low-cost carbon source, such as steam-exploded sugar cane bagasse (seSCB), is an alternative to reduce the costs in cellulase production. Enzyme supernatants produced by the mutant Penicillium echinulatum 9A02S1 on seSCB as a substrate have good filter paper activity (0.48 IU/mL) values combined with endoglucanases (3.24 IU/mL), β-glucosidase (0.48 IU/mL), and xylanases (0.64 IU/mL) activities. It was found that the enzymes produced on seSCB showed activity at the same hydrolysis potential of the enzymes produced with cellulose as a carbon source. In conclusion, the results presented in this paper showed that it is possible to obtain efficient enzymes by P. echinulatum using very low-cost substrates, such as seSCB, and, therefore, contributing to the development of on-site enzyme production to produce secondgeneration ethanol.

1. INTRODUCTION Interest in commercial-scale production of alternative transportation fuels chiefly emanates from issues relating to the use, impacts, and rising demand of traditional fossil fuels. Growing dependency upon oil and the inability to protect supply lines from global political intrigues, projected declines in worldwide petroleum reserves, and record crude oil prices afford major incentives for pursuing the development of alternative fuels.1 Unlike fossil fuels, ethanol is a renewable energy source produced through fermentation of sugars. Ethanol is widely used as a partial gasoline replacement in many countries, such as Brazil. A dramatic increase in ethanol production using the current cornstarch or sugar cane sucrose-based technology may not be practical, because the available land needed to cultivate these important food crops is limited. In this context, the production of second-generation ethanol is an attractive alternative. The lignocellulosic material sugar cane bagasse is an ideal raw material for ethanol production, because of its abundant availability in Brazil.2 There are three technological routes for the conversion of lignocellulosic materials into fermentable sugars: (1) concentrated acid hydrolysis, (2) dilute acid hydrolysis, and (3) enzymatic hydrolysis after biomass pretreatment. The enzymatic route is advantageous for energy consumption, because it operates under mild temperature, pH, and pressure conditions. Moreover, the selective reaction conditions prevent the formation of sugar degradation products, such as furfural and hydroxymethylfurfural, which decrease the sugar concentration in the resulting biomass syrups and inhibitors of the fermentation process.3 Enzyme production is an important step in the biomass-to-ethanol process. Because the realization of the process is highly sensitive to the cost of the raw material, on-site enzyme production on part of the lignocellulose substrate available in the ethanol plant would be advantageous.4 Kovacs et al.5−8 reported good extracellular production of cellulase and β© 2012 American Chemical Society

glucosidase by Trichoderma atroviride mutants on steampretreated willow and steam-pretreated spruce in submerged cultures. The cellulase and xylanase production of Penicillium echinulatum 9A02S1 was studied in submerged cultures using steam-exploded sugar cane bagasse (seSCB) as a carbon source. The enzyme complex of cellulases by P. echinulatum shows stability at 50 °C, a relevant condition for application of these enzymes in enzymatic hydrolysis, and also contains βglucosidases in greater proportion than Trichoderma reesei. This process can lead to economic production of secondgeneration ethanol by contributing toward a decrease in the production costs of enzymatic complexes, which are capable of hydrolyzing lignocellulose residues to simple sugars.

2. MATERIALS AND METHODS 2.1. Microorganism. The cellulolytic mutant P. echinulatum strain 9A02S1 (DSM 18942) was used in this study. This strain was obtained by exposing wild-type P. echinulatum strain 2HH to ultraviolet (UV) light and hydrogen peroxide (H2O2).9 These strains are stored in the culture collection of the Enzymes and Biomass Laboratory, Institute of Biotechnology, Caxias do Sul, Rio Grande do Sul (RS), Brazil. The strain 9A02S1 was grown and maintained on cellulose agar9 consisting of distilled water containing 1% (v/v) swollen cellulose [Celuflok, Cotia, São Paulo (SP), Brazil], 0.1% (w/v) proteose peptone (Oxoid L85), 2% (w/v) agar, and 10% (v/v) 10× concentrated Mandels and Reese solution (MS)10 containing (in w/v) 20 g/L KH2PO4, 13 g/L (NH4)2SO4, 3 g/L CO(NH2)2, 3 g/L MgSO4·7H20, 3 g/L CaCl2, 0.050 g/L FeSO4·7H2O, 0.0156 g/L MnSO4·H2O, 0.014 g/L ZnSO4·7H2O, and 0.0020 g/L CoCl2. The reagents used, when not mentioned, are from Sigma-Aldrich. 2.2. Pretreated Sugar Cane Bagasse. Pretreated sugar cane bagasse was kindly provided by the sugar and ethanol industry through Usina Vale do Rosário (Santa Elisa Vale Conglomerate), Morro Received: May 29, 2012 Revised: July 27, 2012 Published: July 30, 2012 5316

dx.doi.org/10.1021/ef3009162 | Energy Fuels 2012, 26, 5316−5320

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Figure 1. Variation of (A) FPA, (B) endoglucanase, (C) β-glucosidase, and (D) xylanase in submerged cultures using 1, 2, and 3% (w/v) utSCB and seSCB as substrates in flasks kept under reciprocal agitation, using P. echinulatum 9A02S1. Supplemented medium with 1% (w/v) cellulose as a control was used. Cel, cellulose; utSCB, untreated sugar cane bagasse; and seSCB, steam-exploded sugar cane bagasse. The values shown in the legend indicate the percentage of substrate employed. The values (averages) with the same letter for the same day are not significant by Tukey’s test (p < 0.05). Agudo, São Paulo (SP), Brazil. The sugar cane bassage used in this study was from the city of Piracicaba, São Paulo (SP), Brazil. It consists of approximately 49.8% cellulose, 22.1% hemicellulose, and 23.5% lignin. During the harvest season, this plant generally processes 250 tons of bagasse per day, which is later used as cattle feed. The sugar cane bagasse is pretreated with steam at 200 °C for 7 min (average pressure of 15 atm), followed by gradual decompression. In the laboratory, the bagasse was dried for 48 h at room temperature. The seSCB was not washed to avoid the loss of sugars. 2.3. Enzyme Production. Submerged cultures were carried out in 500 mL Erlenmeyer flasks with 100 mL of production medium containing 10% (v/v) 10× MS, 0.2% (w/v) soy meal, and 0.1% (v/v) Tween 80. Cultivation was performed using a concentration of 1, 2, and 3% (w/v) untreated sugar cane bagasse (utSCB) and seSCB. A medium with cellulose was used as the control. The flasks were inoculated with sufficient conidial suspension to give a final concentration of 1 × 105 conidia/mL and then shaken at 28 °C and 180 rpm for 5 days. Samples were removed at various times and centrifuged at 3000 rpm for 10 min. The supernatant was analyzed for extracellular enzyme activity. Experiments were carried out in triplicate. 2.4. Enzymatic Assays. The enzymatic activity was analyzed on filter paper [filter paper activity (FPA)].10 The β-glucosidase activity was dosed using salicin as the substrate.11 Endoglucanase activity was determined according to the study by Ghose,12 using 2% (w/v) carboxymethylcellulose solution in citrate buffer. The xylanase activity was determined in the same way as the endoglucanase activity, except that 1% xylan from an oat spelt solution was used as the substrate in place of carboxymethylcellulose. The reducing sugar was estimated as either xylose or glucose equivalent by the dinitrosalicylic acid (DNS) method.13

A total of 1 international unit (IU) of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of reducing sugar from the appropriate substrate per minute and per milliliter of crude filtrate under the assay conditions. 2.5. Analytical Methods. The quantity of mycelial mass estimated was estimated by N-acetylglucosamine, according to the method described by Reissig et al.14 The total soluble protein was determinated according to the study by Bradford.15 The pH was determinated in a pH-meter Orion 420A. 2.6. Statistical Tests. The results were statistically analyzed using the PrismGraphPad program to perform analysis of variance with Tukey’s post-hoc test for a p < 0.05.

3. RESULTS AND DISCUSSION Because the price of commercial enzymes used for the saccharification of pretreated lignocellulosic materials represents a significant part in the overall cost of the biomass-toethanol process, a lower cost alternative is highly desirable. One way to reduce the cost of the enzymes needed for the hydrolysis might be on-site production of enzymes on part of the lignocellulosic substrate present in the ethanol process, such as seSCB. During this assay, seSCB was used as the only carbon source for the production of cellulases and xylanases by P. echinulatum 9A02S1. The results of enzymatic analyses are shown in Figure 1. Cultures at concentrations of 1, 2, and 3% (w/v) seSCB were carried out. The control with microcrystalline cellulose was used. Cultures with higher concentrations of these substrates were employed to provide media with cellulose concentrations 5317


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Table 1. Comparison of Enzymatic Activities of Different Fungi Grown on Lignocellulosic Materials in Submerged Cultures enzymatic activities (IU/mL) production time substrate used in the enzymatic analysis microorganism

substrate and concentration

FPUa

30 g/L steam-pretreated SCB (corresponding to 13 g/L cellulose)

0.48 48 h

P. echinulatum 9A02S1 −1

productivity (IU mL Penicillium brasilianum IBT 20888

h )

35.8 g/L steam-pretreated spruce 20 g/L cellulose −1

productivity (IU mL T. reesei Rut-C30

−1

−1

h )

15 g/L steam-pretreated spruce productivity (IU mL−1 h−1)

T. atroviride TUB F-1663





15 g/L pretreated willow (whole slurry) −1

productivity (IU mL T. atroviride TUB F-1724

h )

15 g/L pretreated willow (whole slurry) −1

productivity (IU mL T. reesei Rut-C30

−1

−1

h )

10 g/L steam-pretreated spruce −1

productivity (IU mL

−1

h )

0.01 0.59 165 h 0.004 0.53 96 h 0.006 0.38 96 h 0.004 0.31 96 h 0.003 0.68 72 h 0.009 0.61 72 h 0.008 0.79 92 h 0.045

βglucosidase 0.48 48 h salicin 0.01 3.5 165 h pNPGc 0.02 0.2 96 h pNPG 0.002 7.1 96 h pNPG 0.074 4.9 96 h pNPG 0.051 9.83 72 h 0.14 11.70 72 h 0.16 0.18 92 h cellobiose 0.002

endoglucanaseb

xylanase

0.045 19 165 h azo-carboxymethylcellulose 0.12

0.64 48 h oat spelt xylan 0.013 0.4 165 h birchwood xylan 0.002

d

d

d

d

d

d

d

d

d

d

d

d

d

d

d

d

d

d

d

d

3.24 72 h

reference

this study

18

5

5

5

4

4

19

Measured using filter paper (Whatman number 1). bMeasured using carboxymethylcellulose (CMC). cpNPG = 4-nitrophenyl β-D-glucopyranoside. d ND = not determinated. a

similar to the control culture, with 1% (w/v) cellulose. The normalized seSCB composition contains about 43.6% cellulose, 8.75% hemicellulose, and 33.75% lignin and ashes. As expected for steam-pretreated material, it showed a low hemicellulose content. In Figure 1A, it appears that the control culture with cellulose (Cel) showed higher enzymatic activities than the utSCB and seSCB cultures, except on the second day, when the culture supplemented with 3% seSCB showed a higher average activity (0.48 ± 0.01 FPU/mL) than the control culture Cel (0.42 ± 0.01 FPU/mL). This result suggests that, for the production of FPA with 3% seSCB, the second day of culture corresponds to the ideal time for the process interruption, because it allows for higher productivity. After 24 h of fermentation, FPA was not detected. In addition, it is noteworthy that this culture (3% seSCB) had the highest concentration of mycelial mass, as determined on the 6th day of process, and also presented the greatest reductions in pH, thus suggesting that these factors contributed to the increased FPA early in the process. On the 3rd and 5th days, the control culture with Cel showed the highest activities. However, the culture supplemented with 1% utSCB (w/v) showed higher results than those from cultures with the highest utSCB concentration and seSCB cultures on the 3rd, 4th, and 5th days; this probably reflects the

catabolite repression caused by the greater availability of easily hydrolyzable sugars in seSCB. There was induction of endoglucanase activity in cultures supplemented with utSCB and seSCB. However, the greatest activities of endoglucanases, from the 2nd to the 4th days, were obtained in the control culture with Cel (Figure 1B). This suggests that pure or partially purified cellulose has a higher capability of inducing the endoglucanases than seSCB cellulose. The activities of β-glucosidases shown in Figure 1C reveal that, on the 2nd day, the 3% seSCB culture had the highest productivity, while the cellulose culture had the highest activities. The highest activity observed in the culture supplemented with 3% seSCB in relation to culture with lower concentrations of substrate is contrary to those found in the literature. Previous research has indicated that, at high substrate content, β-glucosidase is influenced by end-product inhibition.16 It has also been shown that glucose has a significant effect on not only β-glucosidases but also celobiohydrolase and endoglucanase activities during hydrolysis.17 Figure 1D shows data on xylanase activity. It is apparent that the cultures with cellulose and utSCB have the highest activity of this enzyme, indicating that the use of seSCB is not as efficient for the production of xylanases as utSCB or cellulose. 5318


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Maybe in the case of xylanases, it is possible that simultaneous secretion and regulation of glucanases occurred. As shown in Table 1, the data obtained in this work are comparable and often superior to the data referred to other microorganisms in submerged cultures with substrate pretreated by steam explosion. For FPA, the yield was higher than other fungi on substrates pretreated by steam explosion. Although the substrates used for the determination of βglucosidades were not the same, in some works, 4-nitrophenyl β-D-glucopyranoside was used and, in the others, salicin was used (Table1), it was found that seSCB can be used by P. echinulatum. The activities of endoglucanases and xylanases also indicate the possibility of employing seSCB or utSCB as the substrate for the production of these enzymes. Figure 2 shows the variations of pH measurements during the cultivations. It appears that, with the exception of the 1%

Figure 3. Concentration of mycelial mass produced by P. echinulatum strain 9A02S1 in submerged cultures using 1, 2, and 3% (w/v) utSCB and seSCB as the substrate in flasks kept under reciprocal agitation on the 6th day of cultivation. Cel, cellulose; utSCB, untreated sugar cane bagasse; and seSCB, steam-exploded sugar cane bagasse. The values shown in the legend indicate the percentage of substrate employed. The values (averages) with the same letter for the same day are not significant by Tukey’s test (p < 0.05).

had the highest concentration of mycelial mass on the 6th day of cultivation. These results may be related to the greater amount of easily metabolizable sugars, resulting from the pretreatment step. Although the mycelial mass degradation is already happening on the 6th day of culture, these data indicate that the production of cellulases and xylanases were not associated with fungal growth. Figure 4 illustrates the concentration of protein detected in the submerged cultures. The Cel culture presented the highest Figure 2. pH variation in submerged cultures with 1, 2, and 3% (w/v) utSCB and seSCB as the substrate, in flasks kept under reciprocal agitation, using the strain of P. echinulatum 9A02S1. The medium supplemented with 1% (w/v) cellulose was used as a control. Cel, cellulose; utSCB, untreated sugar cane bagasse; and seSCB, steamexploded sugar cane bagasse. The values shown in the legend indicate the percentage of substrate employed.

utSCB culture, which maintained the pH value of the beginning of the process until the 2nd day and even on the 3rd and 4th days. The other cultures showed decreases in pH values until the 2nd day of culture and, subsequently, increases in pH values. However, the cultures supplemented with seSCB had a longer acid phase compared to the culture supplemented with cellulose. This increased acid phase can be related to increased amounts of monosaccharides present in the medium, which may have contributed to a more intense metabolism. This assumption is consistent with the proposal by Sternberg and Dorval20 that the fungus T. reesei growing on medium containing (NH4)2SO4 has its metabolism followed by the use of NH3, resulting in the release of H+ in the medium, with a consequent drop in pH values. Furthermore, in accordance with Blandino et al.21 and Botella et al.22 on the production of enzymes by Aspergillus awamori in solid medium cultures, the pH drop in the initial stage of culture possibly occurs because of the production of organic acids. When the concentration of reducing sugars decreases, the pH increases, probably because of microbial assimilation of organic acids. Besides, the lower pH observed for seSCB may be because this substrate was not washed before steam explosion results in the partial hydrolysis of esters liberating organic acids, such acetic acid. Figure 3 shows the concentration values of mycelial mass, estimated by the determination of the N-acetylglucosamine concentration. These data showed that the 3% seSCB culture

Figure 4. Protein concentration measured in the enzymatic broth produced by P. echinulatum strain 9A02S1 in submerged cultures using 1, 2, and 3% (w/v) utSCB and seSCB as the substrate in flask kept under reciprocal agitation on the 6th day of cultivation. Cel, cellulose; utSCB, untreated sugar cane bagasse; and seSCB, steam-exploded sugar cane bagasse. The values shown in the legend indicate the percentage of substrate employed.

levels and also the highest enzymatic activities of FPA (3rd and 5th days), β-glucosidase (6th day), and endoglucanase (2nd, 3rd, and 4th days), suggesting that the dosed proteins may be the analyzed enzymes. According to the results obtained in the enzymatic analyzes, it is possible to verify that supplementation with higher concentrations of utSCB and seSCB, except for the FPA of the 3% seSCB culture, on the 2nd day, which did not show positive results. The production of lignocellulolytic enzymes using lignocellulosic materials pretreated by steam explosion as the carbon source is reported by Khan and Lamb.23 These authors employed T. reesei Rut-C30 for the production of cellulases and xylanases in aspen wood. Low cellulase activity was obtained. In 5319


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addition, the enzyme complex produced is deficient in exoglucanase and β-glucosidase activities. However, mixtures of the substrate pretreated by steam explosion with 10−20% pure cellulose decreased deficiency and increased enzymatic activity. This enzyme complex, produced with the substrate mixture, was rich in xylanases and showed hydrolytic capacity equal to the complex made from pure cellulose. The hydrolytic potential of the enzyme complex produced with seSCB as an inducer source was evaluated by comparing it to enzymes produced in a cellulose medium (Figure 5). It was

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(1) Vancov, T.; McIntosh, S. Energy Fuels 2011, 25, 2754−2763. (2) Camassola, M.; Dillon, A. J. P. Appl. Biochem. Biotechnol. 2010, 162, 1889−1900. (3) Wyman, C. E. Bioresour. Technol. 1994, 50, 3−15. (4) Kovacs, K.; Megyeri, L.; Szakacs, G.; Kubicek, C. P.; Galbe, M.; Zacchi, G. Enzyme Microb. Technol. 2008, 43, 48−55. (5) Kovacs, K.; Szakacs, G.; Zacchi, G. Bioresour. Technol. 2009, 100, 1350−1357. (6) Delabona, P. S.; Farinas, C. S.; Silva, M. R.; Azzoni, S. F.; Pradella, J. G. Bioresour. Technol. 2012, 107, 517−521. (7) Barta, Z.; Kovacs, K.; Reczey, K.; Zacchi, G. Enzyme Res. 2010, 1−8. (8) Buaban, B.; Inoue, H.; Yano, S.; Tanapongpipat, S.; Ruanglek, V.; Champreda, V.; Pichyangkura, R.; Rengpipat, S.; Eurwilaichitr, L. J. Biosci. Bioeng. 2010, 110, 18−25. (9) Dillon, A. J. P.; Zorgi, C.; Camassola, M.; Henriques, J. A. P. Appl. Microbiol. Biotechnol. 2006, 70, 740−746. (10) Camassola, M.; Dillon, A. J. P. J. Anal. Bioanal. Tech. 2012, DOI: 10.4172/scientificreports.125. (11) Chahal, D. S. Appl. Environ. Microbiol. 1985, 49, 205−210. (12) Ghose, T. K. Pure Appl. Chem. 1987, 59, 257−268. (13) Miller, G. Anal. Chem. 1959, 31, 426−428. (14) Reissig, J. L.; Strominger, J. L.; Leloir, L. F. J. Biol. Chem. 1955, 27, 959−966. (15) Bradford, M. Anal. Biochem. 1976, 72, 248−254. (16) Breuil, C.; Chan, M.; Gilbert, M.; Saddler, J. N. Bioresour. Technol. 1992, 39, 139−142. (17) Xiao, Z. Z.; Zhang, X.; Gregg, D. J.; Saddler, J. N. Appl. Biochem. Biotechnol. 2004, 113−116, 1115−1126. (18) Jørgensen, H.; Olsson, L. Enzyme Microb. Technol. 2006, 38, 381−390. (19) Szengyel, Z.; Zacchi, G.; Varga, A.; Reczey, K. Appl. Biochem. Biotechnol. 2000, 84−86, 679−691. (20) Sternberg, D.; Dorval, S. Biotechnol. Bioeng. 1979, 21, 181−191. (21) Blandino, A.; Iqbalsyah, T.; Pandiella, S. S.; Cantero, D.; Webb, C. Appl. Microbiol. Biotechnol. 2002, 58, 164−169. (22) Botella, C.; Diaz, A.; de Ory, I.; Webb, C.; Blandino, A. Process Biochem. 2007, 42, 98−101. (23) Khan, A. W.; Lamb, K. A. Biotechnol. Lett. 1984, 6, 663−666.

Figure 5. Yield of reducing sugars released from enzymatic hydrolysis of sugar cane bagasse pretreated by steam explosion by cellulases produced in medium containing sugar cane bagasse pretreated by steam explosion (seS + seE) or in medium containing cellulose (seS + celE) using the fungus P. echinulatum 9A02S1. A total of 20 FPU/g sugar cane bagasse pretreated was used by steam explosion. The values (averages) with the same letter for the same day are not significant by Tukey’s test (p < 0.05).

found that, after 48 h of hydrolysis, the enzymatic broth produced from seSCB showed the same yield in comparison to the broth produced with cellulose, suggesting that, during growth, the fungus produces a set of enzymes for the hydrolysis of substrate where the microorganism was developed for the production of hydrolytic enzymes.

4. CONCLUSION The fungus P. echinulatum, grown in submerged culture with a simple medium based on agricultural byproduct and a cheap mineral source, proved to be a promising microorganism for the simultaneous production of cellulases and xylanases. The incorporation of cheap sources, such as sugar cane bagasse, into the media for the production of lignocellulose enzymes should contribute to a decrease in the production costs of enzymatic complexes capable of hydrolyzing lignocellulose residues for the formation of fermented syrups. Consequently, it can lead to the economic production of second-generation ethanol, helping to minimize the competition with the food chain while increasing overall yields in comparison to first-generation biofuels. Moreover, it was found that the enzymes produced on seSCB showed activity at the same potential hydrolysis of the enzymes produced with cellulose as the carbon source.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: 55-54-3218-2149. E-mail: mcamassola@ gmail.com. Notes

The authors declare no competing financial interest. 5320


Steam-Exploded Sugar Cane Bagasse for On-Site Production of

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