Evaluation of plant cell wall degrading enzyme production by Clostridium thermocellum B8 in the presence of raw agricultural wastes

International Biodeterioration & Biodegradation 105 (2015) 97e105

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Evaluation of plant cell wall degrading enzyme production by Clostridium thermocellum B8 in the presence of raw agricultural wastes Pedro Ricardo V. Hamann a, Dayane L. Serpa a, Amanda Souza Barreto da Cunha a, Brenda R. de Camargo a, Karen Ofuji Osiro a, Marcelo Valle de Sousa b, Carlos R. Felix a, Robert N.G. Miller c, Eliane F. Noronha a, * a b c

Laboratory of Enzymology, Department of Cell Biology, University of Brasília, Brasilia, DF, Brazil Laboratory of Biochemistry and Protein Chemistry, Department of Cellular Biology, University of Brasília, Brasília, DF, Brazil Department of Cellular Biology, University of Brasília, Brasília, DF, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 March 2015 Received in revised form 19 August 2015 Accepted 20 August 2015 Available online xxx

Plant cell wall represents an important source of fermentable sugars for second generation bioethanol production. However, cellulosic biomass hydrolysis still is a bottleneck to bioethanol production in an efficient and low cost process. Thermophilic bacteria have been studied as a source of cellulolytic enzymes for cellulosic biomass deconstruction, as their enzymes present unique features compatible with current industrial process conditions. The present study was carried out to evaluate the use of different agro-industrial wastes as suitable carbon sources for growth and enzyme secretion by a strain of Clostridium thermocellum isolated from goat rumen. C. thermocellum B8 was able to grow on/degrade microcrystalline cellulose, Sugar cane bagasse/Straw and Cotton waste, and produced different sets of cellulases and hemicellulases in their presence. The enzymatic mixtures produced by C. thermocellum (B8) showed activities over a broad range of temperatures (50e70
C). Highest values were obtained between 60 and 70
C, at a pH range from 5 to 7, decreasing at alkaline pH values. In addition, enzymes displayed thermostability, with CMCase and xylanase activities maintaining maximum values over 12 days at 50
C. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Clostridium thermocellum Holocellulases Agro-industrial wastes Biofuel

1. Introduction The possibility of exhaustion of fossil fuels sources, together with associated environmental pollution is driving world energy policies towards the development of alternative energy sources (Goldemberg, 2008). In this context, there is a global interest in the development of industrial processes to produce biofuels from vegetal biomasses such as natural oils, starch, sucrose and lignocelluloses. The use of lignocellulosic biomass as a fuel source has emerged from a neglected potential energy source to a promising alternative for bioethanol production (second generation). Several tons of this type of biomass, which includes sugar cane straw/ bagasse, cotton waste, corn stover and rice straw, are produced daily and accumulated as waste by different agricultural activities.

* Corresponding author. Enzymology Laboratory, University of Brasilia (UnB), 70910-900, Brazil. E-mail address: [email protected] (E.F. Noronha). http://dx.doi.org/10.1016/j.ibiod.2015.08.013 0964-8305/© 2015 Elsevier Ltd. All rights reserved.

However, in order to develop an industrial process based on lignocellulosic biomass with a competitive price in the fuel market, bottlenecks need to be overcome. Raw lignocellulosic materials are composed of cellulose, hemicellulose, pectin and lignin (45e60%, 20e40%, 5e10% and 10e40%, respectively), which are mainly found in plants as cell wall components (Hadar et al., 1993; Howard et al., 2004; Kumar et al., 2008). Their complex organization has resulted in a remarkable resistance against mechanical forces and recalcitrance to enzymatic hydrolysis. As such, the complete deconstruction of the plant cell wall requires a set of glycosyl hydrolases (cellulases, xylanases, pectinases) and lignin modifying enzymes, together with accessory proteins such as disbranching enzymes and swollenin. This resistant nature of raw lignocellulosic materials hampers its complete hydrolysis and has a direct negative impact on yield and production costs of second generation bioethanol. Historically, bacteria haven't been explored as a source of lignocellulolytic enzymes, although more recently some genera

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have been described as promising producers of these enzymes, notably, Clostridium, Bacillus and Paenibacillus (Maki et al., 2009; Blumer-Schuette et al., 2014). Members of the phylum Paenibacillus/Bacteriodetes and the genera Clostridium have been described as the main taxonomic units of gut microbiomes in ruminant animals, where they are known to play a central role in plant fiber degradation (Cunha et al., 2011; Hess et al., 2011; Dai et al., 2012). These bacteria also occur in soil, hot springs and decaying plant material, presenting saprophytic activity and playing a role in carbon and nitrogen recycling (Gao et al., 2013; Lv and Yu, 2013). A large percentage of these bacteria are thermophile and thus could potentially be explored as a source of robust enzymes with kinetic parameters suitable to harsh bioprocessing conditions. Among Clostridium species, the most widely studied is Clostridium thermocellum, which is a thermophilic, anaerobic, grampositive bacterium widely known for displaying true cellulolytic activity. Its ability to efficiently degrade crystalline cellulose is due to the secretion of a highly organized enzymatic complex called cellulossome. Celulossomal proteins and enzymes have previously been purified and characterized as b-glucosidase, CelS (Family 48 cellulase) and XynX (xylanase) (Ait et al., 1979; Jung et al., 1998), with much of the knowledge of these enzymes originating from proteomic, genomic and transcriptomic analysis (Raman et al., 2009; Riederer et al., 2011; Wilson et al., 2013). The celulossome secreted by C. thermocellum has been completely characterized, with recent studies showing that its architecture can be switched depending on the carbon source and bacterial strain (Stevenson and Weimer, 2005; Raman et al., 2009; Riederer et al., 2011). Although these bacteria also secrete a set of holocellulolytic enzymes not associated in an enzymatic complex, their properties and diversity have been poorly described to date. As such, C. thermocellum's potential as a source of robust industrial enzymes remains an open research topic for further investigation. The present study was carried out to evaluate the use of different agro-industrial wastes (cotton wastes, sugar cane straw and bagasse) as suitable carbon sources for growth and enzyme secretion in a strain of C. thermocellum isolated from goat rumen, focusing specifically on the less well characterized noncellulossomal enzymes with potential application in biotechnological processes, including those involved in production of cellulosic ethanol.

2. Methodology 2.1. Growth conditions and culture maintenance Bacterial growth was carried out under anaerobic conditions using reducing liquid culture medium (yeast extract (3 g l1), NH2PO4 (5 g l1), Na2HPO4 (2.5 g l1), NH4Cl (0.5 g l1), (NH4)2SO4 (0.5 g l1), NaHCO3 (0.5 g l1), MgCl2 (0.09 g l1), mineral solution (5.0 ml), vitamin solution (0.5 ml), NaOH (0.04 g l1), Na2S9H2O (0.125 g l1), cysteine (0.05 g l1)) or solid medium (with agar 2%, m/v) at 60
C, as previously described in Blume et al. (2013). Liquid cultures were maintained at 4
C for two weeks or 80
C for at least 6 months, until re-culture. Commercial cellulose and agro-industrial residues were used as carbon sources. Agro-industrial residues (cotton waste, sugar cane straw and bagasse) were previously autoclaved at 120
C for 40 min, then vigorously washed to remove soluble sugars and post harvested impurities, dried at 75
C until constant weight was reached, and finally blended. The resulting powder was used to supplement the growth medium.

2.2. Bacterium isolation
breed) Solid and liquid samples of goat rumen contents (Moxoto were transferred to culture flasks containing 100 ml of liquid medium supplemented with 1% microcrystalline cellulose (w/v) as a carbon source (softwood substrate (Sigma Aldrich®, MO-US)). After 48 h of growth, an aliquot of 1 ml of each enriched culture (solid and liquid rumen samples) was transferred to a new culture flask containing 30 ml of the culture medium, and used in serial dilutions (1:10; 1:100 and 1:1000). Subsequently, 0.5 ml of each sample was placed on solid medium containing carboxymethyl cellulose as carbon source and incubated at 60
C under anaerobic conditions. After 48 h of growth isolated colonies were collected and re-plated. This procedure was repeated three times. The isolated colonies were collected and re-inoculated into a culture flask containing 100 ml of culture medium supplemented with 1% microcrystalline cellulose (w/v) as a carbon source, and incubated at 60
C for 48 h. The isolated colony able to grow on liquid medium containing cellulose as a carbon source, C. thermocellum B8, was further identified by 16S rDNA analysis and genome comparison. 3. Enzyme production 3.1. Time course of enzymatic activity and production Culture flasks containing 100 ml of reducing liquid medium supplemented with agro-industrial residue powder (cotton waste, sugar cane straw and sugar cane bagasse) or crystalline cellulose as a carbon source were inoculated with 5 ml of a fresh culture of C. thermocellum B8. These flasks were incubated at 60
C. Five ml samples were removed after 2, 4, 8 and 10 days of growth. These were vacuum filtered and centrifuged at 10,000 g for 10 min at 4
C to obtain the supernatants. For the enzymatic assays, the cell-free culture supernatants were dialyzed against 2 l of distilled water overnight (12 kDa cut-off) and concentrated 10-fold by lyophilization followed by rehydration with deionized water. Concentrated samples were stored at 4
C until their use as enzyme sources for hollocelulolytic activity determination and electrophoretic analysis. 3.2. Enzymatic assays The reducing sugar content of cell-free culture supernatants (non-dialyzed and non-concentrated) was determined using the method of Miller (1959). Cell-free dialyzed and concentrated supernatants were used as enzyme source in enzymatic assays. Enzymatic activities against the following polysaccharides: carboxymethyl cellulose (CMC) (2% w/v), avicel (2% w/v), xylan oat spelts or birchwood (2% w/v), mannan (1% w/v) and pectin (2% w/v), were evaluated by mixing 50 ml of the enzymatic samples with 100 ml of one of these substrates in sodium phosphate buffer, 50 mM pH 6.0. Hydrolysis was carried out for 30e60 min at 65
C and the reaction was stopped by adding 300 ml of dinitrosalicylic reagent (Miller, 1959). The absorbance of the mixture was read at 540 nm. One unit of enzymatic activity was defined as the amount of enzyme required to produce 1 mmol of reducing sugar per minute of reaction. Glucose, xylose, mannose and galacturonic acid were used for construction of standard curves. All tests were performed at least in triplicate. b-Glucosidase activity was determined using p-Nitrophenyl glucopyranoside (4 mM) as a substrate at 65
C. After 30 min of incubation, reactions were stopped by adding 300 ml of 1 M sodium carbonate, and p-Nitrophenyl production was determined at 400 nm. One unit of enzymatic activity was defined as the amount

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of enzyme required to produce 1 mmol of p-Nitrophenyl per minute of reaction. Protein concentration was determined by the Bradford assay using bovine serum albumin as standard (Bradford, 1976). 3.3. Effect of temperature and pH on enzymatic activities The effects of temperature and pH on the enzymatic activities were evaluated over a temperature range from 40 to 80
C and pH 3 to 9. pH effects were evaluated using the following buffer systems: 100 mM sodium citrate, pH 3 to 7 and 100 mM TriseHCl, pH 7 to 9. The effect of temperature was evaluated at pH 6. Cell free supernatant from cultures containing cotton waste as a carbon source was chosen to assess the thermal stability of enzymatic blends secreted by C. thermocellum B8. Enzymatic samples were incubated at 50 and 70
C, and then residual activities of xylanases and CMases were periodically evaluated. 3.4. Electrophoresis For SDS-PAGE analysis the samples were precipitated using trichloroacetic acid (TCA) at a final concentration of 10%, as previously described by Blume et al. (2013), and applied to SDS-PAGE and zymogram gels. Enzyme preparations were submitted to denaturing (SDS-PAGE) electrophoresis on 10% gels as described by Laemmli (1970). Twenty mg of precipitated protein dissolved in 10 ml of sample buffer were applied to the gel, which was subsequently stained with silver nitrate (Blum et al., 1987). Molecular mass standards from Sigma (USA) were used as markers. Zymogram analysis was performed using denaturing gels as described for SDS-PAGE, except for their copolymerization with CMC, xylan and mannan. The resulting gels were washed with triton X-100 (2.5% w/v), soaked in sodium phosphate buffer at pH 6.0 and incubated at 65
C. After 90 min they were stained with Congo Red (0.1% w/v) to visualize the protein bands as a representation of activity. 3.5. Scanning electron microscopy Lignocellulosic residues and cellulose fibers degradation were also analyzed through Scanning Electron Microscopy. Residual substrates from C. thermocellum's, 10 days grown cultures, carried as described above in enzyme production, were vacuum filtered and then dried at 50
C until reaching constant weight. Residual biomass preparation and visualization by SEM was carried as previously described in Blume (2011). Non-inoculated liquid media containing the different residues and cellulose were use as source of non-degraded biomasses. 4. Results and discussion Clostridium species have been isolated from different microbiomes, especially from landfills, soil, sediment, manure and sludge, where they carry out the first step in anaerobic degradation of cellulosic material providing substrates which could be further metabolized by the microbial community (Shiratori et al., 2006). Members of this genus are also found as dominant species in anaerobic communities participating in decomposition of yellow poplar wood chips (van der Lelie et al., 2012). Nowadays, in the genomics era, the structure and diversity of a bacterial community can be accurately assessed using metagenomic approaches. Recent advances using these technologies have contributed to a dramatic increase in the description of bacterial communities in different microbiomes (van der Lelie et al., 2012). Indeed, such work has enabled detailed description of anaerobic communities in rumens of goats, bovines and other

99

ruminants. Clostridium species have been detected as members of these communities, playing a central role in cellulose fiber degradation, increasing digestibility and the availability of nutrients for the host animals (Hess et al., 2011; Dai et al., 2012; Gao et al., 2013). In this context, our research group has isolated novel Clostridium isolates from previously underexplored microbiomes, aiming the obtainment and characterization of enzymes applicable to biotechnological processes. C. thermocellum strain B8, was able to grow in the presence of crystalline cellulose, sugarcane bagasse/straw and cotton waste as carbon sources, causing tissue breakdown as showed by scanning electron microscopy images (Fig. 1) and reducing sugar production (Fig. 2). A different degradation pattern was observed for microcrystalline cellulose/cotton in comparison to sugar cane bagasse/ straw, for cellulose/cotton it was noticed more intense cellulosic fiber degradation (Fig. 1). Reducing sugar production was time course-dependent, with the highest values detected after 8 days of growth in the presence of crystalline cellulose and 8e10 days of growth in the presence of agricultural residues. The reducing sugar production was also substrate-dependent, with the highest values obtained with crystalline cellulose and sugar cane straw as carbon source (Fig. 2). As C. thermocellum strains were previously described as capable of degrading crystalline cellulose, so degradation of this substrate by C. thermocellum B8 was expected (Yue et al., 2013). A higher degradation rate of crystalline cellulose by strain B8 in comparison to agro-industrial residues was also observed, which is in agreement to the lower recalcitrant feature of cellulose and the lack of inhibitors in comparison to sugar cane straw/bagasse and cotton waste (Duarte et al., 2012). Pretreatment of lignocellulosic materials has been described as a fundamental step to increase saccharification and fermentable sugars, which are then used in fermentation steps to produce cellulosic ethanol (Kumar et al., 2010; Singh et al., 2010). Instead of previous reports, isolate B8 was able to degrade recalcitrant raw materials, such as the non-pretreated cotton waste and sugar cane bagasse substrates. This result suggests a potential application of isolate B8 to degrade raw agro-industrial residues in order to obtain products presenting higher commercial interest without pretreatment steps. These data are therefore relevant to consolidated bioprocessing (CBP), which combines the steps of pretreatment, hydrolysis and fermentation in a single bioreactor. This result is in agreement with previous studies which showed C. thermocellum as able to use raw banana stem as sole carbon source and Anaerocellum thermophilum as able to degrade a set of non-pretreated lignocellulosic biomasses. However, in both these previous studies, no attention was given to enzymes involved in their degradation (Vieira et al., 2007; Yang et al., 2010). C. thermocellum B8 produced CMCases, xylanases, mannanases and pectinases during growth in the presence of crystalline cellulose and cellulosic residues. Production of hemicellulases and pectinases by C. thermocellum isolates in culture media containing only cellulose as carbon source was previously reported by our research group (Blume et al., 2013). Cellulases, hemicellulases and pectinase-encoding genes would be organized in a cluster being under a unique promoter and therefore under the same expression control mechanism. This has been already described for cellulossomal enzymes encoding genes. Time course curves showed increased levels of all enzymatic activities assayed for all growth conditions evaluated; highest activities values were detected after 2 days of growth in the presence of cellulose and 4 days in agro-industrial residues. Highest values of xylanase and mannanase activities were detected when the bacteria was grown in the presence of sugar cane straw as carbon

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Fig. 1. Scanning Electron microscopy images of agroindustrial residues and cellulose fibers after ten days degradation by C. thermocellum B8. (a) raw Sugar cane bagasse, (b) (c) degraded Sugar Cane Bagasse; (d) Cellulose fibers, (e) (f) degraded Cellulose fibers; (g) raw Cotton waste fibers (h) (i) degraded Cotton waste fibers; (j) raw Sugar Cane straw (k) (l) degraded Sugar Cane Straw. White arrows indicate places where degradation was more representative.

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Fig. 2. Time-course production of reducing sugars (mg ml1) by C. thermocellum B8 during growth in liquid medium containing cellulose or agro-industrial residues as a carbon source. (a) Cellulose ( ) and Cotton waste ( ). (b) Sugar cane straw ( ), Sugar cane bagasse ( ).

Fig. 3. Time-course production of CMCases, xylanases, mannanases and Pectinases by C. thermocellum B8 in liquid medium containing cellulose or agro-industrial residues as a carbon source. Cellulose ( ), Sugar cane bagasse ( ), Sugar cane straw ( ) and Cotton waste ( ).

source (Fig. 3). C. thermocellum (B8) produced Avicelase, CMCase, b-Glucosidase and hemicellulases (xylanases, pectinases and mannanases) after growth in the presence of crystalline cellulose, sugar cane straw/ bagasse and cotton waste as carbon sources. Avicelase activity wasn't detected for cultures containing cotton waste as a carbon source (Table 1). Pectinase and mannanase activities presented the highest values for growth in the presence of cotton waste as a carbon source. Indeed, these agro-industrial residues could be used as substrates for the production of enzymes with potential applications as industrial biocatalysts. The highest values of b-glucosidase were obtained in the presence of crystalline cellulose. This activity was secreted by C. thermocellum isolate B8, unlike previously described for other isolates that showed intracellular activity (Ait et al., 1979). The

presence of b-Glucosidase activity concomitant to (Exo/endo)glucanases in an enzymatic blend is fundamental to complete the degradation of cellulose fibers. Previous reports have described the production of cellulases and hemicellulases by thermophilic bacteria and filamentous fungi grown in the presence of corn stover, soy bran, crushed corncob and wheat straw (Chandel and Singh, 2011; Facchini et al., 2011). C. thermocellum has been described as a strong producer of cellulases and hemicellulases mainly during growth on cellulose, Switchgrass and Populus pretreated biomasses (Shao et al., 2011; Wilson et al., 2013; Dykstra et al., 2013). However, so far there has been no report of production of these activities by Clostridium species using raw agro industrial residues as substrates. The particular features reported in this study can be an important key to the development of new saccharification processes.

Table 1 Enzymatic activities produced by C. thermocellum B8 after 4 days of growth in liquid medium containing sugar cane bagasse, crystalline cellulose, sugar cane straw and cotton wastes as carbon sources. Enzymatic Activities (UI mL1)

Cellulose

Cotton's waste

Sugar cane straw

Sugar cane bagasse

CMCase Xylanase Oatspelt Birchwood Mannanase Pectinase Avicelase b-Glucosidase

0.497 ± 0.010

0.354 ± 0.006

0.732 ± 0.009

0.590 ± 0.056

0.439 0.482 0.303 0.139 0.037 0.163

± ± ± ± ± ±

0.004 0.010 0.009 0.044 0.006 0.041

0.400 0.163 0.214 0.364 e 0.100

± ± ± ±

0.040 0.04 0.018 0.018

± 0.042

0.780 1.295 0.428 0.097 0.082 0.134

± ± ± ± ± ±

0.033 0.126 0.015 0.051 0.003 0.030

0.736 0.881 0.338 0.104 0.054 0.045

± ± ± ± ± ±

0.080 0.038 0.017 0.008 0.008 0.024

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Fig. 4. Protein profile (a) and enzymatic activity in gel (b, c, d and e) of samples from cell-free supernatants of C. thermocellum grown for 4 days in the presence of cellulose or agroindustrial residues as carbon sources. (b) CMCase, (c) Mannanase, (d) Xylanase activity against oat spelt, (e) Xylanase activity against birchwood. M: Molecular markers, (1) Crystalline Cellulose, (2) Sugar Cane Bagasse, (3) Sugar Cane Straw, (4) Cotton Waste.

Fig. 5. Graphical representation of temperature effect on holocellulolytic activities produced by C. thermocellum B8. (a) CMCase ( Xylanase against Birchwood ( ) and Pectinase ( ), (c) b-Glucosidase ( ) and Mannanase ( ).

A different set of CMCases, xylanases and mannanases were produced by C. thermocellum in the presence of crystalline cellulose and agro-industrial residues (Fig. 5). Enzyme molecular masses ranged from 35 to 116 kDa, and the number of isoforms detected also varied between enzymatic samples from cultures containing crystalline cellulose or agro-industrial residues as carbon source. The highest number of enzyme isoforms was observed on zymograms containing mannan or xylan as substrates and after 4 days of growth. SDS-PAGE protein profiles presented similar patterns of secreted proteins for the different enzymatic samples (Fig. 5). Therefore, C. thermocellum B8 could be used as a source of different enzymatic blends containing cellulases and hemicellulases suitable for application in industrial processes.

) and Xylanase against Oat Spelt (

), (b)

Our results are in agreement with previously reported data, which demonstrated that Clostridium species switch their gene expression and protein production patterns according to the carbon source used to supplement the culture growth media, such as cellulose, celobiose, xylan, avicel, pretreated biomass of Switchgrass or Populus (Rydzak et al., 2012; Blume et al., 2013; Wilson et al., 2013; Wei et al, 2014). The enzymatic mixtures produced by C. thermocellum (B8), independent of growth conditions, showed higher enzymatic activity values over a pH range from 5 to 7, decreasing at alkaline pH values (Fig. 6), as previously described for cellulases and hemicellulases produced in the presence of agro-industrial residues by filamentous fungi (de Siqueira et al., 2010). A broad pH range of enzymatic

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Fig. 6. Graphical representation of pH effect on holocellulolytic activities produced by C. thermocellum B8. (a) CMCase ( against Birchwood ( ) and b-Glucosidase ( ), (c) Pectinase ( ) and Mannanase ( ).

Table 2 Temperature effect on plant cell wall degrading enzymes produced by C. thermocellum B8 in the presence of different carbon sources after 4 days on liquid cultivation. Temperature (
C)

Carbon source

Cellulose Relative activity (%)

Sugar cane straw Relative activity (%)

Sugar cane bagasse Relative activity (%)

Cotton's waste Relative activity (%)

40

50

60

70

80

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

7.22 10.82 27.60 55.44 33.45 46.85

85.19 25.33 51.53 100 74.83 93.23

88 82.52 78 45.51 100 95

100 100 100 54.11 76 100

46.05 27.87 36.46 44 10.43 44.41

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

39.29 31.03 42.93 30.12 24.37 50.80

79.76 73.05 81.18 39.28 48.07 88.78

94 89.67 86 100 66.31 100

100 100 100 50 100 99.00

49.44 37.48 39.23 20.45 12.29 39.75

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

50.12 34.41 42.08 84.33 21.91 56.80

70.95 55.46 73.97 91.17 48.55 73.77

100 93 100 67.04 100 100

57 100 98.18 47 41 83.51

39.78 34.26 36.05 100 0.00 38.29

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

18.15 29.33 37.79 44.90 16.34 35.66

36.47 52.99 59.74 39.37 26.04 72.31

76 78 91 100 100 100

100 100 100 37.38 55 98.95

15.51 12.72 24.72 30 20.16 28.11

* ¼ Birchwood xylan as substrate.

activity for all enzymatic blends could be attributed to the presence of multiple enzymes, as shown on zymograms. The above mentioned enzymatic mixtures also showed activity over a broad range of temperatures (50e70
C) (Fig. 4). The highest values were obtained between 60 and 70
C, except for pectinases

) and Xylanase against Oat Spelt (

103

), (b) Xylanase

produced in the presence of sugar cane bagasse, which presented the highest activity value at 80
C (Table 2). These results of optimal temperature for hollocelulolytic activities are directly correlated to C. thermocellum optimal growth temperature and in agreement with optimal temperatures, as previously described for enzymes produced by others thermophile bacteria (Frederick et al., 1981; Jiang et al., 2006; Singh et al., 2012). In addition, they are quite higher from those described for hollocelulolytic enzymes produced by well known plant cell wall degrading fungi strains as Trichoderma spp. and Aspergillus spp (Frederick et al., 1981; Stålbrand et al., 1993). Regardless the thermal stability of C. thermocellum B8's plant cell wall degrading enzymes, xylanase activity was maximal and endoglucanase (CMcase) was about 50% for over12 days at 50
C (Fig. 7). Activity under high temperatures and thermo stability are both desirable enzymatic features for enzymes applied to industrial processes. Therefore, our results showed that enzymatic blends produced by C. thermocellum B8 presented desirable kinetics parameters for industrial purposes. (Table 3).

5. Conclusion Enzymes from thermophilic microorganisms have unique characteristics that are compatible with existing industrial processes and conditions, and because of this there is a growing interest in these microorganisms as a source of enzymes that can be applied in industries instead of chemical catalysts. The present work is an important report regarding the use of sugar cane bagasse/straw and cotton waste by C. thermocellum in production of enzymes with potential applications in lignocellulosic biomass degradation. Our results attest that C. thermocellum B8 was able to degrade and use non chemical-pretreated agro industrial residues as carbon source. In addition, the bacteria secrete a different set of hollocelulolytic enzymes according to the biomass used as carbon source. Enzymes secreted by C. thermocellum B8 showed activity in a broad range of pH and temperature, presenting a remarkable thermal stability at 50
C. These results set this microorganism as

Fig. 7. Thermal stability at 50 and 70
C of CMCase and xylanase from enzymatic blends produced by C. thermocellum B8 after growth in liquid medium containing cotton waste as a carbon source. ( ) CMCase and ( ) xylanase activity against oat spelt.

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Table 3 pH effect on plant cell wall degrading enzymes produced by C. thermocellum B8 in the presence of different carbon sources after 4 days on liquid cultivation. Carbon source

pH

Buffer system Sodium citrate

Cellulose Relative activity (%)

Sugar cane straw Relative activity (%)

Sugar cane bagasse Relative activity (%)

Cotton's waste Relative activity (%)

TriseHCl

3

4

5

6

7

7

8

9

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

13.55 8.09 20.65 26.58 0.21 _

35.56 39.01 51.74 16.53 11.05 41.04

100 100 99.60 68.41 73.81 100

84.38 92.06 100 98.61 100 86.47

63.90 32.24 78.73 100 16.22 68.27

83.41 92.32 95.77 _ 65.71 70.84

59.10 78.43 74.09 29.08 18.72 66.77

37.02 49.18 51.65 48.33 2.98 46.93

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

18.45 15.19 16.89 31.46 2.04 13.74

36.71 41.44 29.03 _ 10.52 41.64

100 100 91.35 46.57 66.77 100

87.12 81.03 73.70 100 100 82.22

72.65 44.82 82.24 89.70 47.61 70.37

78.62 89.76 100 _ 60.79 87.53

59.67 63.73 81.83 42.81 26.62 62.63

48.90 46.43 56.31 86.92 21.02 51.04

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

10.96 8.09 12.45 68.24 2.80 12.84

30.75 39.01 29.88 _ 2.70 37.35

97.19 100 93.75 81.16 54.62 100

100 92.06 92.82 100 100 96.26

76.47 32.24 70.55 92.00 25.13 79.46

84.11 92.32 100 38.50 57.31 84.93

63.10 78.43 72.83 67.29 15.60 56.38

50.89 49.18 56.15 78.89 3.50 52.56

CMCase Xylanase* Xylanase Pectinase b-Glucosidase Mannanase

30.18 53.78 35.37 43.90 0.80 30.34

40.03 65.96 41.10 15.29 6.70 39.04

100 100 100 66.27 92.68 100

93.54 94.01 85.91 46.86 100 96.83

80.08 58.41 73.77 100 18.57 73.58

94.79 97.62 87.86 55.43 77.47 97.79

71.90 79.61 73.29 34.32 57.13 84.07

56.50 53.04 50.46 68.97 18.52 66.95

* ¼ Birchwood xylan as substrate. _ No activity detected.

candidate as a source of enzymes suitable for industrial processes. We are currently working to develop new bioconsolidate processes utilizing Clostridiuma thermocelum B8 and other thermophile bacteria. Acknowledgments E.F.N. is recipient of a research fellowship from the Brazilian Research Council (CNPq). P.R.V.H. was recipient of maintenance scholarship from CAPES. This work was supported by research grants from the University of Brasilia e UnB, CAPES and Foundation to support research from the Federal District (FAPDF) (193000584/ 2009). References Ait, N., Creuzet, N., Cattaneo, J., 1979. Characterization and purification of thermostable b-glucosidase from Clostridium thermocellum. Biochem. Biophys. Res. Commun. 90 (2), 537e546. Blum, H., Beier, H., Gross, H.J., 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8 (2), 93e99. Blume, L.R., Noronha, E.F., Leite, J., Queiroz, R.M., Ricart, C.A.O., de Sousa, M.V., Felix, C.R., 2013. Characterization of Clostridium thermocellum isolates grown on cellulose and sugarcane bagasse. BioEnergy Res. 6 (2), 763e775. ~o e caracterizaça ~o da atividade holocelulolítica de isoBlume, L.R., 2011. Avaliaça lados de Clostridium thermocellum. A dissertation submitted for the degree of Master of Molecular Biology. University of Brasíla, BR. Blumer-Schuette, S.E., Brown, S.D., Sander, K.B., Bayer, E.A., Kataeva, I., Zurawski, J.V., Kelly, R.M., 2014. Thermophilic lignocellulose deconstruction. FEMS Microbiol. Rev. 38 (3), 393e448. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1), 248e254. Chandel, A.K., Singh, O.V., 2011. Weedy lignocellulosic feedstock and microbial metabolic engineering: advancing the generation of ‘Biofuel’. Appl. Microbiol. Biotechnol. 89 (5), 1289e1303.

Cunha, I.S., Barreto, C.C., Costa, O.Y., Bomfim, M.A., Castro, A.P., Kruger, R.H., Quirino, B.F., 2011. Bacteria and archaea community structure in the rumen microbiome of goats (Capra hircus) from the semiarid region of Brazil. Anaerobe 17 (3), 118e124. Dai, X., Zhu, Y., Luo, Y., Song, L., Liu, D., Liu, L., Dong, X., 2012. Metagenomic insights into the fibrolytic microbiome in yak rumen. PLoS One 7 (7), e40430. de Siqueira, F.G., de Siqueira, E.G., Jaramillo, P.M.D., Silveira, M.H.L., Andreaus, J., Couto, F.A., Ferreira Filho, E.X., 2010. The potential of agro-industrial residues for production of holocellulase from filamentous fungi. Int. Biodeterior. Biodegrad. 64 (1), 20e26. Duarte, G.C., Moreira, L.R.S., Jaramillo, P.M.D., F Filho, E.X., 2012. Biomass-derived inhibitors of holocellulases. BioEnergy Res. 5 (3), 768e777. Dykstra, A.B., St Brice, L., Rodriguez Jr., M., Raman, B., Izquierdo, J., Cook, K.D., Hettich, R.L., 2013. Development of a multipoint quantitation method to simultaneously measure enzymatic and structural components of the Clostridium thermocellum cellulosome protein complex. J. Proteome Res. 13 (2), 692e701. Facchini, F.D.A., Vici, A.C., Reis, V.R.A., Jorge, J.A., Terenzi, H.F., Reis, R.A., de Moraes, M.D.L.T., 2011. Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed. Bioprocess Biosyst. Eng. 34 (3), 347e355. Frederick, M.M., Frederick, J.R., Fratzke, A.R., Reilly, P.J., 1981. Purification and characterization of a xylobiose-and xylose-producing endo-xylanase from Aspergillus niger. Carbohydr. Res. 97 (1), 87e103. Gao, W., Navarroli, D., Naimark, J., Zhang, W., Chao, S.H., Meldrum, D.R., 2013. Microbe observation and cultivation array (MOCA) for cultivating and analyzing environmental microbiota. Microbiome 1 (1), 1e8. Goldemberg, J., 2008. The Brazilian biofuels industry. Biotechnol. Biofuels 1 (6), 1e7. Hadar, Y., Kerem, Z., Gorodecki, B., 1993. Biodegradation of lignocellulosic agricultural wastes by Pleurotus ostreatus. J. Biotechnol. 30 (1), 133e139. Hess, M., Sczyrba, A., Egan, R., Kim, T.W., Chokhawala, H., Schroth, G., Rubin, E.M., 2011. Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331 (6016), 463e467. Howard, R.L., Abotsi, E., Van Rensburg, E.J., Howard, S., 2004. Lignocellulose biotechnology: issues of bioconversion and enzyme production. Afr. J. Biotechnol. 2 (12), 602e619. Jiang, Z., Wei, Y., Li, D., Li, L., Chai, P., Kusakabe, I., 2006. High-level production, purification and characterization of a thermostable b-mannanase from the newly isolated Bacillus subtilis WY34. Carbohydr. Polym. 66 (1), 88e96. Jung, K.H., Lee, K.M., Kim, H., Yoon, K.H., Park, S.H., Pack, M.Y., 1998. Cloning and expression of a Clostridium thermocellum xylanase gene in Escherichia coli.

P.R.V. Hamann et al. / International Biodeterioration & Biodegradation 105 (2015) 97e105 IUBMB Life 44 (2), 283e292. Kumar, R., Singh, S., Singh, O.V., 2008. Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. J. Ind. Microbiol. Biotechnol. 35 (5), 377e391. Kumar, S., Gupta, R., Lee, Y.Y., Gupta, R.B., 2010. Cellulose pretreatment in subcritical water: effect of temperature on molecular structure and enzymatic reactivity. Bioresour. Technol. 101 (4), 1337e1347. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259), 680e685. Lv, W., Yu, Z., 2013. Isolation and characterization of two thermophilic cellulolytic strains of Clostridium thermocellum from a compost sample. J. Appl. Microbiol. 114 (4), 1001e1007. Maki, M., Leung, K.T., Qin, W., 2009. The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int. J. Biol. Sci. 5 (5), 500. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31 (3), 426e428. Raman, B., Pan, C., Hurst, G.B., Rodriguez Jr., M., McKeown, C.K., Lankford, P.K., Mielenz, J.R., 2009. Impact of pretreated switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis. PLoS One 4 (4), e5271. Riederer, A., Takasuka, T.E., Makino, S.I., Stevenson, D.M., Bukhman, Y.V., Elsen, N.L., Fox, B.G., 2011. Global gene expression patterns in Clostridium thermocellum as determined by microarray analysis of chemostat cultures on cellulose or cellobiose. Appl. Environ. Microbiol. 77 (4), 1243e1253. Rydzak, T., McQueen, P.D., Krokhin, O.V., Spicer, V., Ezzati, P., Dwivedi, R.C., Sparling, R., 2012. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression. BMC Microbiol. 12 (1), 214. Shao, X., Jin, M., Guseva, A., Liu, C., Balan, V., Hogsett, D., Lynd, L., 2011. Conversion for avicel and AFEX pretreated corn stover by Clostridium thermocellum and simultaneous saccharification and fermentation: insights into microbial conversion of pretreated cellulosic biomass. Bioresour. Technol. 102 (17), 8040e8045.

105

Shiratori, H., Ikeno, H., Ayame, S., Kataoka, N., Miya, A., Hosono, K., Ueda, K., 2006. Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor. Appl. Environ. Microbiol. 72 (5), 3702e3709. Singh, A., Pant, D., Korres, N.E., Nizami, A.S., Prasad, S., Murphy, J.D., 2010. Key issues in life cycle assessment of ethanol production from lignocellulosic biomass: challenges and perspectives. Bioresour. Technol. 101 (13), 5003e5012. Singh, R., Dhawan, S., Singh, K., Kaur, J., 2012. Cloning, expression and characterization of a metagenome derived thermoactive/thermostable pectinase. Mol. Biol. Rep. 39 (8), 8353e8361. Stålbrand, H., Siika-aho, M., Tenkanen, M., Viikari, L., 1993. Purification and char^-mannanases from Trichoderma reesei. J. Biotechnol. 29 acterization of two a (3), 229e242. Stevenson, D.M., Weimer, P.J., 2005. Expression of 17 genes in Clostridium thermocellum ATCC 27405 during fermentation of cellulose or cellobiose in continuous culture. Appl. Environ. Microbiol. 71 (8), 4672e4678. van der Lelie, D., Taghavi, S., McCorkle, S.M., Li, L.L., Malfatti, S.A., Monteleone, D., Tringe, S.G., 2012. The metagenome of an anaerobic microbial community decomposing poplar wood chips. PloS One 7 (5), e36740. Vieira, W.B., Moreira, L.R.D.S., Monteiro Neto, A., Ferreira Filho, E.X., 2007. Production and characterization of an enzyme complex from a new strain of Clostridium thermocellum with emphasis on its xylanase activity. Braz. J. Microbiol. 38 (2), 237e242. Wilson, C.M., Rodriguez Jr., M., Johnson, C.M., Martin, S.L., Chu, T.M., Wolfinger, R.D., Brown, S.D., 2013. Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated populus and switchgrass. Biotechnol. Biofuels 6 (1), 179. Yang, S.J., Kataeva, I., Wiegel, J., Yin, Y., Dam, P., Xu, Y., Adams, M.W., 2010. Classification of ‘Anaerocellum thermophilum’ strain DSM 6725 as Caldicellulosiruptor bescii sp. nov. Int. J. Syst. Evol. Microbiol. 60 (9), 2011e2015. Yue, Z.B., Li, W.W., Yu, H.Q., 2013. Application of rumen microorganisms for anaerobic bioconversion of lignocellulosic biomass. Bioresour. Technol. 128, 738e744.