Biotechnology Letters

Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol.
--Manuscript Draft-Manuscript Number: Full Title: Article Type: Section/Category: Keywords: Corresponding Author: Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol. Manuscript Microbial and Enzyme Technology BGL gene; -glucosidase; hydrolyzed bagasse; lignocellulose Marcos Antonio de Morais Jr, Ph.D. Federal University of Pernambuco Recife, Pernambuco BRAZIL

Corresponding Author Secondary Information: Corresponding Author's Institution: Corresponding Author's Secondary Institution: First Author: First Author Secondary Information: Order of Authors: Alexandre LS Reis, MSc Rochane RNB Torres, MSc Fernanda CB Leite, Ph.D. Raquel FR de Souza, BSc Thiago H Napoleo, Ph.D. Patrcia Maria G Paiva, Ph.D. Marcos Antonio de Morais Jr, Ph.D. Order of Authors Secondary Information: Abstract: In the present work we confirmed the potential of the yeast Dekkera bruxellensis to produce ethanol from cellobiose both in synthetic medium, which has been recently reported, as well as in enzyme-treated steam-exploded sugarcane bagasse. It is shown the main features of the purified cellobiase (-glucosidase, E.C. 3.2.1.21). Additional in silico analysis identified the corresponding BGL gene and revealed the main structural characteristics of the coded intracellular enzyme, which is similar to Kluyveromyces marxianus counterpart. Physiological and enzyme data pointed that low assimilation capacity maybe the limiting step for the complete and fast conversion of cellobiose towards ethanol, besides to already known negative Custer effect for disaccharide fermentation. However, the overpowering capacity of this yeast to settle and stay in industrial environments such as the ethanol fermentation process makes it a promising yeast to ferment lignocellulosic substrates. Ken Tokuyasu, Ph.D. Researcher, National Agriculture and Food Research Organization tokuyasu@affrc.go.jp Dr. Tokuyasu works on fermention of plant biomass and has been recently publishig in the subject, such as: Guan et al (2012) Sequential incubation of Candida shehatae and ethanol-tolerant yeast cells for efficient ethanol production from a mixture of glucose, xylose and cellobiose. Bioresour Technol 132:419-422. Alexandre LS Reis, MSc Federal University of Pernambuco

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Badal C Saha, Ph.D. Researcher, National Center for Agricultural Utilization Research sahabc@ncaur1.ncaur.gov Dr. Saha works on the fermentation of xylose and with production of hydrolytic enzymes and enzymes involved in the metabolism of alternative sugars for second generation etahnol production. Lisbeth Olsson, Ph.D. Professor, Technical University of Denmark lisbeth.olsson@chalmers.se Dr. Olsson works on the production and characterization of oligosaccharides-degrading enzymes for convertion of plant biomass to second generation ethanol: Krogh et al (2010) Characterization and kinetic analysis of a thermostable GH3 betaglucosidase from Penicillium brasilianum. Appl Microbiol Biotechnol 86:143-154.

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*Manuscript Click here to download Manuscript: Reis et al 2013 Cellobiose fermentation by Dekkera [Biotech Lett].doc

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Journal: Biotechnology Letters Section: Microbial and Enzyme Technology

Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol

Alexandre Libanio Silva Reis1, Rochane Regina Neves Baptista Torres2, Fernanda Cristina Bezerra Leite2, Raquel de Ftima Rodrigues de Souza1,2, Thiago Henrique Napoleo 3, Patrcia Maria Guedes Paiva3 and Marcos Antonio de Morais Jr1,3,4*

Laboratory of Bioprocessing, CETENE. 50740-540 Recife, PE, Brazil. Interdepartmental Research Group on Metabolic Engineering, 3Department of Genetics and Department of Biochemistry, Federal University of Pernambuco. 50670-901 Recife, PE, Brazil.

*Author for correspondence: Marcos A de Morais Jr Departamento de Gentica Universidade Federal de Pernambuco Av. Moraes Rego, 1235, Cidade Universitria 50670-901 Recife PE Brasil E-mail: marcos.morais@pesquisador.cnpq.br Phone: 55-81-21267817 Fax: 55-81-21268522

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Abstract In the present work we confirmed the potential of the yeast Dekkera bruxellensis to produce ethanol from cellobiose both in synthetic medium, which has been recently reported, as well as in enzymetreated steam-exploded sugarcane bagasse. It is shown the main features of the purified cellobiase (-glucosidase, E.C. 3.2.1.21). Additional in silico analysis identified the corresponding BGL gene and revealed the main structural characteristics of the coded intracellular enzyme, which is similar to Kluyveromyces marxianus counterpart. Physiological and enzyme data pointed that low assimilation capacity maybe the limiting step for the complete and fast conversion of cellobiose towards ethanol, besides to already known negative Custer effect for disaccharide fermentation. However, the overpowering capacity of this yeast to settle and stay in industrial environments such as the ethanol fermentation process makes it a promising yeast to ferment lignocellulosic substrates.

Keywords: BGL gene, -glucosidase, hydrolyzed bagasse, lignocellulose.

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Introduction

The potential of the yeast Dekkera bruxellensis to ferment sucrose from sugarcane juice has been recently reported (Pereira et al. 2012; Leite et al. 2013) which, in connection to its high fitness in industrial production plants (De Souza Liberal et al. 2007), makes of this yeast a promising microorganism for first generation fuel ethanol production. Besides, another putative use of this yeast is for ethanol production from biomass hydrolysates, using resources that are inaccessible for the fermenting yeasts (Blomqvist et al. 2011). Assimilation of cellobiose, a disaccharide generated by the incomplete hydrolysis of cellulose, is a welcome feature for yeasts that are intended to be used for this purpose. This trait have been reported long time ago for D. bruxellensis (Blondin et al. 1982) despite the fact that this is not the characteristic described for the species from its type strain CBS 74 and many other strains (Blomqvist et al. 2010; Galafassi et al. 2011). Sequential fermentation approach was proposed for conversion the mixture of glucose, xylose and cellobiose after enzymatic hydrolysis of cellulosic biomass. This included the combination of Candida shehatae (for glucose and xylose fermentation) with D. bruxellensis (for cellobiose fermentation), with complete consumption of sugars and ethanol yield near to 0.4 g ethanol per gram of consumed sugar (Guan et al. 2013). In the present report we show the ability of D. bruxellensis strain GDB 248 to ferment cellobiose for ethanol production. The advantages and constraints regarding to the biotechnological use of this yeast for production of second generation ethanol are discussed in the text.

Materials and Methods

Yeast strain and cultivation The strain Dekkera bruxellensis GDB 248 was maintained in YPD medium. Cells were pre-grown on liquid YPD at 30C and 150 rpm for 24 h. Aerobic growth experiments in different carbon 15

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sources and analytical determination of extracellular metabolites by HPLC were performed as described (Leite et al. 2013). The experiments were performed in biological duplicate with two technical replicates for each point.

Bagasse hydrolysis and fermentation assays Steam-exploded sugarcane bagasse was suspended in 100 mM Tris-Acetate pH 4.5 buffer to 20 g/l and treated with Fibrenzyme LWT commerc ial preparation (Dyadic International Inc., Jupiter, USA), with 1 ml of enzyme preparation for each 10 g of bagasse, at 50C for 72 h with gentle agitation. The hydrolysate was centrifuged at 1,200 x g for 5 minutes and the liquid part was used for fermentation assays. The initial sugar composition was evaluated by HPLC (Leite et al. 2013). Fermentation assays were performed as described by Pereira et al. (2012) with 10% (w/v) yeast biomass in synthetic fermentation medium (YNB at 1.7 g/l) containing sucrose or cellobiose at 20 g/l or in the bagasse hydrolysate. The suspensions were incubated at 30C and 120 rpm in orbital shaker. Samples were collected every hour for cell density determination and metabolites analyses by HPLC. The experiments were performed in three biological replicates with two technical replicates for each point.

Cellobiase (-glucosidase) production Yeast cells were cultivated in synthetic cellobiose medium (1.7 g YNB/l and 1.0 g cellobiose/l) until 0.6 A600nm and diluted 1/1000 to 250 ml fresh synthetic medium containing glucose, cellobiose or sucrose at 1.0 g/l. The flasks were incubated for 24 h at 33C and 130 rpm in orbital shaker. Afterwards, the cells were collected by centrifugation, re-suspended in 250 ml corresponding medium and cultivated as above. This recycling process was repeated by four times and after the last cycle the final cell density was determined (Leite et al. 2013). All cells were collected by centrifugation at 3,800 x g and 4C for 30 min. Cell pellet was re-suspended in two volumes of 10 mM Acetate buffer pH 4 containing 1 mM -mercaptoethanol and lysed by maceration in liquid 16

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nitrogen. The lysates were centrifuged at 21,000 x g for 15 minutes at 4C and the supernatant was recovered. Enzyme reaction were performed by mixing 100 g of protein from cell free extract and sugar solution diluted in 100 mM sodium citrate buffer pH 4.8 for final volume of one ml. The reaction was incubated 10 min at 30C and stopped by transferring the tubes to ice bath. Release glucose was measured by glucose oxidase kit (LabLabor, Brazil). Specific activity was recorded as the amount of glucose equivalent released per minute from the amount of protein in one gram of yeast cells. For testing the presence of extracellular enzyme, supernatant of synthetic media containing sucrose or cellobiose was used for enzyme reactions.

Cellobiase (-glucosidase) purification Yeast cells grown in synthetic-cellobiose medium were lysed as above and subjected to fractioning in ammonium sulfate from 0% to 60% saturation and the fraction were dialyzed in 100 mM sodium citrate buffer pH 5. Protein concentration was measured by Commasie Blue method. Fractions were tested for -glucosidase activity using the chromogenic substrate pNPG as below, and those containing enzyme activity were pooled (fraction EF1) and subjected to molecular exclusion chromatography in Sephadex G75 (26 mm diameter, 10 cm height columns equilibrated with 100 mM citrate-phosphate buffer pH 5 at 6 ml/h). Fractions containing -glucosidase activity were pooled and subjected to ion exchange chromatography in CM-cellulose (15 mm diameter, 10 cm height columns equilibrated with 10 mM citrate-phosphate buffer pH 3.8 at 10 ml/h). Proteins linked to the matrix were eluted with 0.5 M NaCl solution at 10 ml/h flux and the fraction containing -glucosidase activity were pooled (fraction EF2). Protein concentration was measured and protein purity was checked by standard SDS-PAGE method in 12% acrilamide gel. Isoelectrofocusing was performed to determine the isoelectric point of the protein. Immobilized pH Gradient (IPG) strips with pH ranging from 3 to 10 were equilibrated for 30 min with solution containing 6.5 mM DTT and 134 mM IAA and the protein was submitted to electrophoretic run at

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200 V (2 mA) for two hours followed 3,500 V (2mA) for 1.25 h. The strips were used for second dimension run in 12% acrylamide gel and revealed by Comassie Blue staining.

Enzyme kinetics assays Substrate specificity for disaccharides was evaluated as described above. Kinetic profile of EF2 fraction was evaluated by using the chromogenic substrate p-nitrophenyl--D-glucopyranoside (pNPG). Standard reactions used a volume of enzyme fraction containing 100 g protein, equal volume of 100 mM pNPG solution and 100 mM sodium citrate buffer pH 4.8 to one ml final volume. The reaction were incubated at 30C for 10 minutes and stopped by adding 100 L of 1 M sodium bicarbonate solution and the yellow color of pNP release was quantified at 410 nm. A standard curve was prepared with pNP to correlate the absorbance with the amount of product released and the specific activity was calculated as the amount of enzyme that release one mol pNP per minute per milligram of protein in the sample. Optimal pH was evaluated by using citratephosphate buffer adjusted for different pHs and the reactions were incubated at 30C for 10 minutes (see Figure 3A). For testing optimal temperature, pH was adjusted to 4.0 and the reactions were incubated in different temperatures for 10 minutes (see Figure 3B). Thermal stability of the enzyme was evaluated by incubating EF2 fraction for 10 minutes at temperatures ranging from 20C to 60C. Afterwards, enzyme preparation was left at room temperature (c.a. 25C) for 10 min and then used for enzyme activity using pNPG at optimal pH and temperature. Maximum conversion rate (Vmax) and affinity constant (KM) were calculated from Lineweaver-Burk plot by varying pNPG concentration in the reactions, and were used to calculate catalytic constant (Kcat) of the purified enzyme. Inhibitory activity was measured by adding disaccharides or pNPGal at 10 mM in reactions containing pNPG and expressed as the percentage of pNPG cleavage. All those measurements were performed under optimal pH and temperature.

Gene identification and in silico analysis 18

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Searches by the keywords hydrolase, amylase, glucosidase and amyloglucosidase were performed in the Dekkera bruxellensis Genomic Database (http://www.lge.ibi.unicamp.br/dekkera/) and the nucleotide sequences of the retrieved contigs were used for BLASTx analysis at GenBank. The D. bruxellensis contig with higher similarity to -glucosidase encoding genes was recovered and the ORF determined by the ORF Finder tool at NCBI. The partial sequence of the -glucosidase protein was used for BLASTp analysis in the D. bruxellensis genome database of the Joint Genome Initiative-JGI (http://genome.jgi.doe.gov/Dekbr1/Dekbr1.home.html) to recover the complete protein sequence. Phylogenetic analysis of the amino acids sequences coded by -glucosidase genes of D. bruxellensis and other fungi was performed as previously reported (De Souza Liberal et al. 2012). Sequence of the bacterial Thermotoga neapolitana -glucosidase was used as outgroup. Functional domains of the putative -glucosidase of D. bruxellensis were identified by using the structural analysis tools available online at European Bioinformatic institute (http://www.ebi.ac.uk/) and SIB Bioinformatic Resource Portal (http://www.expasy.org/).

Results and Discussion

Aerobic growth and fermentation Aerobic growth curves (Fig. 1A) showed that GDB 248 strain can grow on cellobiose as carbon source at 0.13 h-1 that is lower than the growth rate of 0.19 h-1 calculated for sucrose. No glycerol was detected in the course of cell growth on both disaccharides (data not shown), indicating that the cultures were aerobic (Pereira et al. 2012; Leite et al. 2013). In this yeast most of the sucrose hydrolysis occurs inside the yeast cells since the invertase activity is mainly intracellular (Leite et al. 2013). Similarly, no cellobiase (-glucosidase) activity was observed in the culture supernatant (see below), indicating that this enzyme is intracellular too. Therefore, differences for growth rates might be related to differences observed in the uptake of sucrose or cellobiose (Fig. 1B). Ethanol production was higher in cellobiose than in sucrose, although ethanol consumption was observed 19

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even in the presence of this sugar (Fig. 1C). This feature remains to be nvestigated. Acetate production during aerobic growth was similar in both sugars (Fig. 1D) and reached almost one third in mass (one forth in molar ratio) of the ethanol production in aerobic cultures as previously reported (Aguilar-Uscanga et al. 2003; Leite et al. 2013). Figure 1

Fermentation of cellobiose was assayed under high cell density condition in order to simulate the industrial processes, a situation that differed from the previous reports. However, the experiments were performed under gently agitation to ensure minimal oxygenation of the cultures due to the Custer effect, in which D. bruxellensis cells stopped ethanol production when oxygen is depleted from the medium (Leite et al. 2013). This situation of oxygen supply was attested by the production of acetate (Table 1). No variation in cell mass was observed (data not shown). Only 44% of the cellobiose was consumed after six hour of fermentation, while no sucrose was left in the medium (Table 1), once again pointing out for differences in sugar uptake as probably the limiting step in cellobiose assimilation. Sugar consumption rate (qS) and ethanol production rate (qE) were higher in sucrose medium, while acetate production rate (qA) was higher in cellobiose medium (Table 1). Mass balance was closed for sucrose medium, with almost all carbon being recovered from the fermentation products (Table 1). This was not observed when cellobiose was the carbon source. Since no other fermentation metabolites were detected, we supposed that CO 2 production was underestimated in cellobiose medium. As the fermentation continues to 24 h, all cellobiose was consumed and ethanol production reached 3.7 g/l, even though ethanol yield still in the range of 0.18 g/g. Cellobiose fermentation by D. bruxellensis CBS 11269 was reported to be performed for long period of incubation (Blomqvist et al. 2010), although ethanol yield with that strain of 0.29 g/g was higher than the strain in the present study (Table 1). High ethanol yield on cellobiose by the strain CBS 5512 of Brettanomyces custersii (the former epithet of D. bruxellensis) in the range of 0.40 g/g was reported (Spindler et al. 1992). Table 1

Steam-exploded enzyme-treated sugarcane bagasse hydrolysates used in the present work contained 5 times more glucose than cellobiose. Under fermentation with agitation, glucose was rapidly 20

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consumed by D. bruxellensis cells while cellobiose was uptake slowly (Fig. 2). Acetate but not glycerol was detected, attesting the presence of oxygen in the fermentation. Ethanol and acetate production and yield (0.25 g/g) were similar. The stoichiometric CO2 yield was calculated as 0.48 g/g, closing the mass balance at 98%. Similar production of ethanol and acetate corroborates the tendency of D. bruxellensis for respiration (Leite at al. 2013). Figure 2

Production and purification of D. bruxellensis cellobiase (-glucosidase, E.C. 3.2.1.21) Yeast cells cultivated in synthetic media containing different sugars were tested for the cross production of the main gluco-hydrolases: invertase, cellobiase and maltase. None of the tested enzymes were detected in the supernatant of the growth of fermentation cultures (data not shown), confirming their intracellular localization. Cellobiase as well as invertase were highly produced when yeast cells were cultivated on glucose, while they were more produced when the cells were cultivated on the respective sugars (Table 2). In Kluyveromyces marxianus the production of cellobiase was highly induced by cellobiose (Rajoka et al. 2004). In order to decrease the background of invertase, yeast cells were cultivated in cellobiose medium for preparation of cellfree extract to purify cellobiase (Table 3). Enzyme purification was confirmed by the presence of a band of 93 KDa in SDS-PAGE and the isoelectric point of 6.13 was calculated from isoelectrofocusing method (data not shown), similar to the enzyme from K. marxianus (96 KDa) (Yoshida et al. 2009). Table 2 Table 3 Kinetics and optimal activity of the D. bruxellensis cellobiose Purified cellobiase (EF2) from D. bruxellensis displayed a narrow range of pH with its optimal activity pH 4.2 (Fig. 3A). On the other hand, it showed a wider range of optimal temperature (Fig. 3B). At pH 5 the enzyme preparation EF2 maintained 100% of the activity after 60 minutes of incubation at 40C (data not shown). Thus, the optimal conditions for enzyme activity were established as 30C and pH 4.2 in phosphate-citrate buffer and all experiments hereafter were 21

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performed in this condition. The specificity assays showed that D. bruxellensis cellobiase has broad substrate specificity (Table 4), while no activity was detected for pNP--D-galactopyranoside (Table 4) neither for carboxymethyl cellulose (data not shown). By using the chromogenic substrate pNPG it was observed that disaccharides competitively inhibited cellobiase activity, while the presence of pNPGal did not prevent the interaction of pNPG with the active site of the enzyme (Table 4). The following kinetics parameters were determined using pNPG: KM = 0.58 mM; Vmax = 154 mol/min.mgProtein; Kcat = 12.84 min-1. Although the kinetics parameters were not high than reported for other yeasts, a clear advantage concerning to D. bruxellensis enzyme is its stability at high temperatures allied to the capacity of the cells to settle the industrial process to produce ethanol (De Souza Liberal et al. 2007). Figure 3 Table 4 Gene identification and protein structure Computational analysis identified a nucleotide sequence in the genome of D. bruxellensis whose theoretical protein showed similarity to -glucosidase of the yeasts K. marxianus and Schwanniomyces etchellsii (Fig. 4). The theoretical protein contained 840 amino acids with predicted molecular weight of 93 KDa, compatible with the experimental data above of the purified enzyme. Its predicted structure presents three major domains. The N-terminus presented a glucosylhydrolase family 3 motif followed by the PA14 -barrel, this last one being thought to be involved in carbohydrate binding in the K. marxianus enzyme (Yoshida et al. 2010). At C-terminus there was a fibronectin type III-like domain that is also present in the structure of K. marxianus cellobiase (Yoshida et al. 2010), whose function still unknown. It may be possible that such domain is involved in protein-protein interaction to form a homodimer structure of the enzyme. Neither transmembrane nor signal peptide domain or glycosylation sites were identified, which corroborates with the results above showing the intracellular localization of the enzyme. Theoretical pI was calculated as 6.1, which corresponded to the experimentally determined pI value using IEF procedure. These results confirmed the identification of the BGL gene that codes for cellobiose (22

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glucosidase) of D. bruxellensis (for complete nucleotide and amino acids sequences consult http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Dekbr2&id=34222). Figure 4 Conclusion and Perspectives It has been recently reported the increasing number of yeasts capable of hydrolyzing cellobiose for the production of ethanol, for example Candida queiroziae, Clavispora sp., Spathaspora passalidarum (Long et al. 2012; Liu et al. 2012; Santos et al. 2011), and D. bruxellensis (Blomqvist et al. 2010; Galafassi et al. 2011; Leite 2013). However, only the last species has been reported as able to settle and survive in industrial environments (Passoth et al. 207; De Souza Liberal et al. 2007; Pereira et al., 2012). Thus, the great challenging for its use as fermenting yeast is regarded to the low conversion rates when facing simulated industrial conditions. Studies are being undertaken in our laboratory in order to identify the main metabolic bottlenecks of this feature and to evaluate its capacity towards the conversion hydrolysates of sugarcane and sweet sorghum bagasse into ethanol at high industrial yields.

Acknowledgements This work was sponsored by the Bioethanol Research Network of the State of Pernambuco (CNPqFACEPE/PRONEM program, grant n APQ-1452-2.01/10), by CNPq-Universal program (grant n 472106/2012-0) and by the Ministry of Science and Technology of Brazil (SIGTEC number PRJ03.33).

References Blondin B, Ratomahenina R, Arnaud A, Galzy P (1982) A study of cellobiose fermentation by a Dekkera strain. Biotechnol Bioeng 24:2031-2037. doi: 10.1002/bit.260240910. 23

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Blomqvist J, Eberhard T, Schnrer J, Passoth V (2010) Fermentation characteristics of Dekkera bruxellensis strains. Appl Microbiol Biotechnol 87:1487-1497. doi: 10.1007/s00253-0102619-y. Blomqvist J, South E, Tiukova L, Momeni MH, Hansson H, Sthlberg J, Horn SJ, Schnrer J, Passoth V (2011) Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis. Lett Appl Microbiol 53:73-8. doi: 10.1111/j.1472765X.2011.03067.x. de Souza Liberal AT, Baslio ACM, do Monte Resende A, Brasileiro BTV, da Silva-Filho EA, de Morais JOF, Simes DA, de Morais Jr MA (2007) Identication of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation. J Appl Microbiol 102: 538-547. doi:10.1111/j.1365-2672.2006.03082.x. de Souza Liberal AT, Carazzolle MF, Pereira GA, Simes DA, de Morais Jr MA (2012) The yeast Dekkera bruxellensis genome contains two orthologs of the ARO10 gene encoding for phenylpyruvate decarboxylase. World J Microbiol Biotechnol 28:2473-2478. doi: 10.1007/s11274-012-1054-x. Galafassi S, Merico A, Pizza F, Hellborg L, Molinari F, Pikur J, Compagno C (2011) Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions. J Ind Microbiol Biotechnol 38:1079-1088. doi: 10.1007/s10295-010-0885-4. Guan D, Yuan L, Shiroma R, Mazakazu I, Tokuyasu K (2013) Sequential incubation of Candida shehatae and ethanol-tolerant yeast cells for efficient ethanol production from a mixture of glucose, xylose and celobiose. Biores Technol 132: 419-422. doi:

10.1016/j.biortech.2012.12.040.

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Leite FC, Basso TO, de Barros Pita W, Gombert AK, Simes DA, de Morais Jr MA (2013) Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants. FEMS Yeast Res 13:34-43. doi: 10.1111/1567-1364.12007. Pereira LF, Bassi AP, Avansini SH, Neto AG, Brasileiro BT, Ceccato-Antonini SR, de Morais Jr MA (2012). The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae. Antonie van Leeuwenhoek 101: 529-539. doi: 10.1007/s10482-011-9662-2. Rajoka MI, Khan S, Latif F, Shahid R (2004) Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant beta-glucosidase from synthetic medium by Kluyveromyces marxianus. Appl Biochem Biotechnol 117:75-92. doi: 10.1385/ABAB:117:2:075. Spindler DD, Wyman CE, Grohmann K, Philippidis GP (1992) Evaluation of the cellobiosefermenting yeast Brettanomyces custersii in the simultaneous saccharification and fermentation of cellulose. Biotechnol Lett 14:403 407. doi: 10.1007/BF01021255 Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, Kitaoka M, Katayama T, Kumagai H (2009) Purification, crystallization and preliminary X-ray analysis of betaglucosidase from Kluyveromyces marxianus NBRC1777. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:1190-1192. doi: 10.1107/S1744309109042948. Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, Kitaoka M, Katayama T, Kumagai H (2010) Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 -glucosidase from Kluyveromyces marxianus. Biochem J 431:39-49. doi: 10.1042/BJ20100351.

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Legend to figures

Figure 1. Shake flask growth curve of Dekkera bruxellensis GDB 248 in synthetic medium containing sucrose () or cellobiose (). Variation of cell density (A), consumption of sugar (B) and production of ethanol (C) and acetate (D) were plotted.

Figure 2. Fermentation of steam-exploded enzyme-treated sugarcane bagasse by Dekkera bruxellensis GDB 248. The following parameters were measured: sugar consumption (total: , straight line; glucose: , dotted line; cellobiose: , dotted line) and ethanol () and acetate () production.

Figure 3. Optimal pH (A) and temperature (B) of the -glucosidase (cellobiase) purified from Dekkera bruxellensis GDB 248 using pNPG as substrate.

Figure 4. Phylogenetic analysis of the Dekkera bruxellensis -glucosidase (cellobiase) from yeasts and filamentous fungi. Amino acids sequences were used to prepare the p-distance matrix and clustered by maximum likelihood method.

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Figure 1 Click here to download high resolution image

Figure 2 Click here to download high resolution image

Figure 3 Click here to download high resolution image

Figure 4 Click here to download high resolution image

table 1 Click here to download table: Reis et al - Table 1.doc

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Reis et al. Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol

Table 1. Global physiological and kinetics data for the eight-hour fermentation of cellobiose or sucrose in agitated flasks by Dekkera bruxellensis GDB 248. Parameter Residual sugar (g/l)a qSugar (mmol/gCel.h) qEthanol (mmol/gCel.h) qAcetate (mmol/gCel.h) Y Ethanol (g/g) Y Acetate (g/g) Y CO2 (g/g)b Y Biomass (g/g) Mass balance (%) Cellobiose 11.5 0.31 (0.08) 0.66 (0.09) 0.53 (0.03) 0.18 (0.03) 0.25 (0.04) 0.41 (0.01) 0.0 84 Sucrose 0.00 1.22 (0.01) 2.62 (0.08) 0.38 (0.03) 0.34 (0.02) 0.12 (0.02) 0.45 (0.02) 0.06 (0.02) 97

7 8 9 10

Initial sucrose 25 g L-1 and initial cellobiose 20 g L-1. Calculated on the basis of stoichiometry of ethanol and acetate production.

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Reis et al. Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol

Table 2. Enzyme activity in the cell-free extracts from Dekkera bruxellensis GDB 248 cultivated in different sugars. Sugar in the medium Glucose Substrate of enzyme reaction Sucrose Cellobiose Maltose Sucrose Sucrose Cellobiose Maltose Cellobiose Sucrose Cellobiose Maltose Enzyme assayed Invertase -glucosidase -glucosidase Invertase -glucosidase -glucosidase Invertase -glucosidase -glucosidase Specific activity (U/gCel) 0.998 0.835 0.000 0.598 0.024 0.000 0.295 0.460 0.000

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table 3 Click here to download table: Reis et al - Table 3.doc

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Reis et al. Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol

Table 3. Purification of cellobiase from Dekkera bruxellensis GDB 248 grown on cellobiose medium. Specific activity Enzyme fraction Cell-free extract EF1 EF2 Protein (mg/ml) 0.95 1.19 0.11 (mol EqGlucose/min.mgProtein) 0.104 0.113 0.848 Purification factor 1.00 1.09 8.16

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Reis et al. Cellobiose fermentation by the yeast Dekkera bruxellensis and implications for production of second generation ethanol

Table 4. Effect of disaccharides on the activity of the purified cellobiase from Dekkera bruxellensis GDB 248. Substrate Cellobiose Maltose Sucrose pNPG pNPGal Glucosyl link Glucose-14)-Glucose Glucose-(14)-Glucose Glucose-(12)-Fructose Glucose-14)-phenyl Galactose-14)-phenyl Relative activity (%) 100.0 27.7 90.0 100.0 0.0 Inhibitory activity (%)a 100.0 94.9 95.2 n.a.b 0.0

8 9 10

Dissacharides were added to reactions with the chromogenic substrate pNPG. n.a.: not applicable

13

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April 2013