How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis
- Gernot Jäger1Email author,
- Michele Girfoglio2Email author,
- Florian Dollo1,
- Roberto Rinaldi3,
- Hans Bongard3,
- Ulrich Commandeur2,
- Rainer Fischer2, 4,
- Antje C Spiess5 and
- Jochen Büchs1Email author
© Jäger et al; licensee BioMed Central Ltd. 2011
Received: 7 July 2011
Accepted: 23 September 2011
Published: 23 September 2011
In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.
After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.
Recombinant swollenin can be easily produced with the robust yeast K. lactis. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.
List of abbreviations
- a :
non-linear Gaussian cumulative function parameter (-)
- A :
adsorbed protein per g cellulose (μmol/g)
- A max :
maximum protein adsorption per g cellulose at equilibrium (μmol/g or mg/g)
- b :
non-linear Gaussian cumulative function parameter (-)
bovine serum albumin
- c :
non-linear Gaussian cumulative function parameter (-)
- CrI :
crystallinity index (%)
- d :
non-linear Gaussian cumulative function parameter (-)
- d 0 :
shaking diameter (mm)
- e :
non-linear Gaussian cumulative function parameter (-)
- E :
free protein concentration (μmol/L)
- f :
non-linear Gaussian cumulative function parameter (-)
- g :
non-linear Gaussian cumulative function parameter (-)
- I 002 :
maximum intensity of the crystalline plane (002) reflection (1/s)
- I AM :
XRD scattering for the amorphous component at 18o in cellulose-I (1/s)
- K A :
association constant (L/μmol)
- K D :
dissociation constant (μmol/L)
- λ :
- n :
shaking frequency (rpm)
nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate
- P :
probability for significant scores (protein matching) (-)
phosphate buffered saline containing Tween-20
- R 2 :
coefficient of determination (-)
- T :
- θ :
diffraction angle (°)
- V L :
filling volume (mL)
medium containing yeast extract, peptone and galactose.
Naturally occurring lignocellulose is a promising starting material for the sustainable production of platform chemicals and fuels [1–6]. The hydrolysis of its main component cellulose to glucose necessitates a cellulase system consisting of cellobiohydrolase (CBH, E.C. 18.104.22.168), endoglucanase (EG, E.C. 22.214.171.124) and β-glucosidase (E.C. 126.96.36.199) [7–9]. Besides enzyme-related factors (for example, enzyme inactivation and product inhibition) , the enzymatic hydrolysis of cellulose is limited by its physical properties [11–14]. These properties, in particular, are the degree of polymerization, accessibility and crystallinity [15–18]. Cellulose accessibility, which is determined by cellulose particle size (external surface area) and porosity (internal surface area) [15, 19], is the most important factor for hydrolysis [15, 18, 20–24]. This accessibility reflects the total surface area available for direct physical contact between cellulase and cellulose and, therefore, influences cellulase adsorption as well as the rate and extent of cellulose hydrolysis [21, 25]. Furthermore, crystallinity is a relevant factor for cellulose hydrolysis, since it influences the reactivity of adsorbed cellulases . Here, it should be noted that crystallinity may also affect cellulase adsorption [26, 27] and, therefore, cellulose accessibility [15, 21, 28]. Up to now, the relationship between crystallinity and accessibility has not been clearly understood [15, 29]. However, for high cellulose hydrolysis rates and yields, cellulose accessibility needs to be increased and, conversely, its crystallinity reduced [30, 31]. To achieve this and accordingly improve subsequent hydrolysis, pretreatment techniques are essential [6, 14, 16, 32].
Since pretreatment can be expensive, there is a prime motivation to screen and improve it [33–37]. Over time, many pretreatment technologies have been developed: physical (for example, milling or grinding), physicochemical (for example, steam explosion or ammonia fiber explosion), chemical (for example, acid or alkaline hydrolysis, organic solvents or ionic liquids), biological or electrical methods, or combinations of these methods [33, 35]. Some of these techniques entail expensive equipment, harsh conditions and high energy input . By contrast, in the past years, non-hydrolyzing proteins have been investigated that pretreat cellulose under mild conditions [17, 20]. After regular lignocellulose pretreatment, these non-hydrolyzing proteins can be added during cellulose hydrolysis  or they can be utilized in a second pretreatment step in which cellulose is the substrate .
During this second pretreatment step, cellulose is incubated under mild conditions with non-hydrolyzing proteins that bind to the cellulose. As a result, cellulose microfibrils (diameter around 10 nm [39, 40]) are dispersed and the thicker cellulose macrofibrils or fibers (diameter around 0.5 to 10 μm, consisting of microfibrils [39–41]) swell, thereby decreasing crystallinity and increasing accessibility [20, 42–44]. This phenomenon was named amorphogenesis [20, 42]. Furthermore, cellulose-binding proteins can lead to deagglomeration of cellulose agglomerates (diameter > 0.1 mm, consisting of cellulose fibers) [45, 46], thereby separating cellulose fibers from each other and additionally increasing cellulose accessibility. Ultimately, amorphogenesis as well as deagglomeration promote cellulose hydrolysis .
Various authors have described hydrolysis-promoting effects when pretreating cellulose with single cellulose-binding domains , expansins from plants [38, 47–49] or expansin-related proteins from Trichoderma reesei , Bacillus subtilis , Bjerkandera adusta  or Aspergillus fumigatus . A prominent expansin-related protein is swollenin from the fungus T. reesei. In contrast to cellulases, the expression levels of swollenin in T. reesei are relatively low (1 mg/L) . Thus, swollenin from T. reesei has been heterologously expressed in Saccharomyces cerevisiae , Aspergillus niger  and Aspergillus oryzae . The expression levels in S. cerevisiae, however, are also low (25 μg/L)  and only A. oryzae produces swollenin in higher concentrations (50 mg/L) . According to Saloheimo et al. , swollenin can disrupt the structure of cotton fiber or the cell wall of the algae Valonia macrophysa. Since swollenin shows a high sequence similarity to plant expansins , it may have a similar function and lead to the disruption of cellulosic networks within plant cell walls . Thus, swollenin may have an important role in the enzymatic degradation of lignocellulose by T. reesei . Up to now, however, there is no systematic and quantitative analysis of the effects of swollenin on cellulosic substrates and their hydrolysis.
First, this study presents an alternative way of producing recombinant swollenin in order to generate sufficient swollenin for industrial applications. Second, the main objective is to show how recombinant swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects their subsequent hydrolysis.
Results and discussion
Production and analysis of recombinant swollenin
To clearly identify the protein band at about 80 kDa (Figure 1A and 1B), its amino acid sequence was determined by using mass spectrometry  and the Mascot search engine . Figure 1C shows the results of mass spectrometry and the expected amino acid sequence of the recombinant swollenin. As shown by a high Mascot score of 502 (Figure 1C), the protein at 80 kDa was clearly identified to be a variant of swollenin from T. reesei. Regarding the native swollenin sequence, a protein score of greater than 57 (homology threshold) indicates identity or extensive homology (P < 0.05). In addition, potential N- and O-glycosylation sites were detected by using the NetNGlyc 1.0 and NetOGlyc 3.1 servers  (Figure 1C). Here, it should be noted that the native swollenin contains almost no N-glycosylation . Therefore, the difference between the calculated molecular mass of 49 kDa, based on the primary sequence of swollenin, and the observed molecular mass of 80 kDa (Figure 1A and 1B) may be explained by O-glycosylation and other post-translational modifications. Proofs are given as follows: (i) the linker region of cellulases or cellulase-related proteins is highly O-glycosylated ; (ii) swollenin contains potential O-glycosylation sites within the linker region (Figure 1C); (iii) no peptides of the linker region were identified by mass spectrometry, since glycosylation alters the mass/charge ratio of the peptides (Figure 1C).
Adsorption of swollenin
Pretreatment of filter paper with swollenin
The non-hydrolytic deagglomeration or amorphogenesis of cellulose was also described for single cellulose-binding domains of cellulases [17, 45, 67] and for other expansin-related proteins from B. subtilis , A. fumigatus  or B. adusta . However, there is no detailed and quantitative analysis of different cellulosic substrates after pretreatment with non-hydrolyzing proteins, especially with regard to swollenin.
Effect of swollenin pretreatment on the physical properties of cellulosic substrates
To analyze in detail the effect of recombinant swollenin on cellulose, different cellulosic substrates were pretreated with buffer, BSA or recombinant swollenin. After pretreatment and removal of bound proteins, the physical properties of the pretreated cellulosic substrates were analyzed by laser diffraction, cellulase adsorption studies and crystallinity measurements.
Maximum cellulase adsorption onto cellulosic substrates after pretreatment with swollenin.
Pretreatment with buffer
Pretreatment with swollenin
A max (cellulase) (mg/g)
A max (cellulase) (mg/g)
Whatman filter paper No.1
Hydrolysis of cellulosic substrates pretreated with swollenin
Recombinant swollenin was easily produced with the yeast K. lactis and purified by affinity chromatography. Additionally, the adsorption of swollenin onto cellulose was quantified for the first time, and its adsorption parameters were comparable to those of individual cellulases. The pretreatment with swollenin caused a significant decrease in particle size as well as in crystallinity of the cellulosic substrates, thereby substantially increasing maximum cellulase adsorption. Moreover, pretreatment of the cellulosic substrates with swollenin - even in non-saturating concentrations - significantly accelerated the hydrolysis. By correlating particle size and crystallinity with initial hydrolysis rates, it could be shown that high initial hydrolysis rates resulted from the swollenin-induced reduction in particle size and crystallinity. Consequently, this study shows an efficient means to produce recombinant swollenin with the robust yeast K. lactis. Moreover, this study shows that swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects cellulosic substrates and their hydrolysis.
A pretreatment of cellulosic substrates has been presented here which is simply based on the incubation of recombinant swollenin under mild conditions. Since the enzymatic hydrolysis of cellulose is a rate-limiting processing step in biorefineries , this pretreatment could significantly improve hydrolysis rates. To exclude possible side effects between swollenin and cellulase, swollenin pretreatment was performed as a separate step within this study. In future studies, swollenin should be directly added during cellulose hydrolysis. Since standard assays are missing for deagglomeration, amorphogenesis and for the comparison of different non-hydrolyzing proteins, this study may serve as an initial means to establish such assays.
Cellulosic substrates and cellulases
The cellulosic substrates Whatman filter paper No.1, α-cellulose, Avicel PH101 and Sigmacell 101 were purchased from Sigma-Aldrich (MO, USA). Physical properties and product information have been summarized by various authors [10, 64]. Agglomerates of Whatman filter paper No.1 were prepared by using a hole-punch and quartering the resulting filter paper discs. The final filter paper agglomerates had an average diameter of approximately 3 mm. The cellulase preparation Celluclast® 1.5 L (Novozymes, Bagsværd, DK) - a filtrated culture supernatant of T. reesei  - was used for the hydrolysis of the pretreated cellulosic substrates. According to various authors, Celluclast® contains CBHs (Cel7A and Cel6A), EGs (for example, Cel7B and Cel5A) as well as β-glucosidases [81, 82]. To remove salts, sugars and other interfering components, Celluclast® was previously rebuffered with an Äkta FPLC (GE Healthcare, Little Chalfont, UK). Celluclast® was loaded on Sephadex G-25 Fine (2.6 cm × 10 cm, GE Healthcare), and 0.05 M sodium acetate (pH 4.8) was used as a running buffer at 110 cm/h. Sephadex G-25 Fine exhibits an exclusion limit of 1-5 kDa which is comparable to the molecular mass cut-off of dialysis membranes for protein desalting. Since cellulases have a molecular mass of > 25 kDa [62, 83, 84], the mixture of cellulases was not changed during rebuffering. Chromatography was conducted at room temperature, and the automatically collected fractions were directly cooled at 4°C. To determine specific filter paper activities, different dilutions of Celluclast® and the rebuffered Celluclast® - applied for all hydrolysis experiments - were tested according to Ghose . Here, the following specific filter paper activities (per g protein) were measured: 201 U/g (Celluclast®) and 279 U/g (rebuffered Celluclast®).
Genetic engineering for recombinant swollenin
The below-mentioned cloning procedure was designed for secreted protein expression according to the K. lactis Protein Expression Kit (New England Biolabs, MA, USA). The cDNA of the swollenin-coding region was synthesized by reverse-transcription PCR using mRNA isolated from T. reesei QM9414 (swo1 gene [GenBank: AJ245918], protein sequence [GenBank: CAB92328]) and reverse transcriptase (M-MLV, Promega, WI, USA) according to the manufacturer's protocol. Specific primers were applied to synthesize a cDNA starting from the 19th codon of the swollenin-coding region and, therefore, missing the secretion signal sequence of T. reesei . By using the aforementioned primers, SalI and SpeI restriction sites were added upstream and downstream of the swollenin-coding region, respectively. The amplified cDNA was cloned into the pCR2.1-TOPO vector (Invitrogen, CA, USA) according to the manufacturer's protocol. After DNA sequencing and isolation of a correct clone, the DNA was excised from pCR2.1-TOPO and cloned into the pKLAC1-H vector using XhoI and SpeI restriction enzymes (New England Biolabs, MA, USA) according to the manufacturer's protocol. The pKLAC1-H is a modified version of the integrative pKLAC1 vector (New England Biolabs; [GenBank: AY968582]). The pKLAC1 - developed by Colussi and Taron  - exhibits the α-mating factor signal sequence and can be used for the expression and secretion of recombinant proteins in K. lactis . The pKLAC1-H was constructed by including an additional SpeI restriction site directly followed by a His-tag coding sequence (6xHis) between the XhoI and AvrII restriction sites of pKLAC1. The DNA sequence of the final pKLAC1-H construct (containing the DNA coding for recombinant swollenin) is shown in Additional file 1. Moreover, the final amino acid sequence of recombinant swollenin (without the α-mating factor signal sequence) is given in Figure 1C.
Expression and purification of recombinant swollenin
All below-mentioned transformation, selection and precultivation procedures - developed by Colussi and Taron  - were performed according to the manufacturer's protocol (K. lactis Protein Expression Kit, New England Biolabs). After cloning, K. lactis GG799 cells were transformed with pKLAC1-H (containing the DNA coding for recombinant swollenin), and transformed clones were selected (acetamide selection). One clone was precultivated in YPGal (yeast extract, peptone and galactose) medium, consisting of 20 g/L galactose, 20 g/L peptone and 10 g/L yeast extract - all media components were purchased from Carl Roth (Karlsruhe, Germany). After inoculation with 2.5 mL of the preculture, the main culture was cultivated in triplicates in 2 L Erlenmeyer flasks with YPGal medium under the following constant conditions: temperature T = 30°C, total filling volume V L = 250 mL, shaking diameter d 0 = 50 mm, shaking frequency n = 200 rpm. Additionally, a non-transformed K. lactis wild type was cultivated as a reference. After incubation for 72 h, the main cultures were centrifuged (6000 g, 20 min, 4°C), and the pooled supernatants of the triplicates were treated with endoglycosidase Hf by using 20 U per μg protein for 12 h  according to the manufacturer's protocol without denaturation (New England Biolabs). Afterwards, the protein solution was concentrated 100-fold at 4°C using a Vivacell 100 ultrafiltration system with a molecular mass cut-off of 10 kDa (Sartorius Stedim Biotech, Göttingen, Germany). For affinity chromatography, the recombinant swollenin was previously rebuffered using Sephadex G-25 Fine (2.6 cm × 10 cm, GE Healthcare) at 110 cm/h with a running buffer (pH 7.4) consisting of 0.05 M sodium dihydrogen phosphate, 0.3 M sodium chloride and 0.01 M imidazole. The rebuffered sample was loaded on Ni Sepharose 6 Fast Flow (1.6 cm × 10 cm; GE Healthcare) at 120 cm/h. The bound swollenin was eluted with the aforementioned running buffer, containing 0.25 M imidazole.
SDS-PAGE and Western blot analysis
SDS-PAGE and Western blot analysis were applied to analyze the purity and to identify the recombinant swollenin. Novex 12% polyacrylamide Tris-Glycine gels (Invitrogen), and samples were prepared according to the manufacturer's protocol. The Plus Prestained Protein Ladder (Fermentas, Burlington, CA, USA) was used as a molecular mass marker. Finally, the proteins were stained with Coomassie Brilliant Blue and analyzed densitometrically  using the scanner Perfection V700 (Epson, Suwa, Japan). The molecular mass and purity of swollenin was determined using the software TotalLab TL100 (Nonlinear Dynamics, Newcastle, UK). For Western blot analysis, gels were blotted onto a nitrocellulose membrane (Whatman, Springfield Mill, UK) according to the manufacturer's protocol (Invitrogen). The membranes were blocked at room temperature with 50 g/L skim milk dissolved in phosphate buffered saline containing 0.5 g/L Tween-20 (PBST) for 30 min. To detect the recombinant swollenin, the membranes were incubated at room temperature for 1.5 h with a rabbit polyclonal antibody against His-tag (Dianova, Hamburg, Germany) diluted 1:10,000 in PBST. After the membrane was washed thrice with PBST, it was incubated with alkaline phosphatase conjugated goat anti-rabbit IgG (Dianova) diluted 1:5,000 in PBST at room temperature for 1 h. Finally, bound antibodies were visualized by incubating the membrane for 5 min with nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) diluted 1:100 in phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.6).
Measurement of protein concentration
Protein concentrations were analyzed with the bicinchoninic acid assay  using the BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) and BSA as a standard. Depending on the protein concentration of the samples, the standard procedure (working range: 0.02 to 2 g/L) or the enhanced procedure (working range: 0.005 to 0.25 g/L) was performed according to the manufacturer's protocol. The absorbance at 562 nm was measured with a Synergy 4 microtiter plate reader (BioTek Instruments, VT, USA). To quantify swollenin in the culture supernatant of K. lactis, the bicinchoninic acid assay was combined with the aforementioned SDS-PAGE (including densitometric analysis). Here, total protein concentrations were determined and multiplied with the ratio of swollenin to total protein (purity).
Mass spectrometry and glycosylation analysis
Mass spectrometry was applied to identify the expressed and purified recombinant swollenin. The protein band (approximately 80 kDa) was excised from the SDS-polyacrylamide gel, washed in water, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin . Peptide analysis was carried out using a nanoHPLC (Dionex, Germering, Germany) coupled to an ESI-QUAD-TOF-2 mass spectrometer (Waters Micromass, Eschborn, Germany) as previously described . The Mascot algorithm (Matrix Science, London, UK) was used to correlate the mass spectrometry data with amino acid sequences in the Swissprot database. Thereby, the sequences of the analyzed peptides could be identified, and, ultimately, protein matches could be determined. The Mascot score is derived from the ions scores of the detected peptides matching the peptides in the database and reflects a non-probabilistic basis for ranking protein hits . By using this database, the peptide mass tolerance was set at ± 0.3 Da. Additionally, the following modifications to the amino acids in brackets were allowed: carbamidomethyl (C), carboxymethyl (C), oxidation (M), propionamide (C). Moreover, potential areas for N-glycosylation and O-glycosylation were identified by using the NetNGlyc 1.0 and NetOGlyc 3.1 servers http://www.cbs.dtu.dk/services/.
in which A denotes the amount of adsorbed protein per g cellulose (μmol/g), A max , the maximum protein adsorption per g cellulose at equilibrium (μmol/g), E, the free protein concentration (μmol/L), and K D , the dissociation constant (μmol/L). Within the literature , the association constant K A (L/μmol) is sometimes used instead of the dissociation constant K D .
To analyze the effect of swollenin pretreatment (see below) on cellulase adsorption [44, 93], the maximum cellulase adsorption was also determined by incubating various concentrations (0.7 to 2.5 g/L) of rebuffered Celluclast® with 10 g/L pretreated cellulosic substrates. Here, all incubations were conducted under the aforementioned conditions for 1 h, 1.5 h and 2 h.
Pretreatment with swollenin
Pretreatment experiments were performed with 20 g/L cellulosic substrates and various concentrations of swollenin in 0.05 M sodium acetate buffer (pH 4.8). The mixtures were incubated as triplicates in 2 mL Eppendorf tubes on a thermomixer under the following constant conditions: T = 45°C, V L = 1 mL, d 0 = 3 mm, n = 1000 rpm. To exclude a sole mechanical effect on cellulosic substrates due to shaking and to verify a specific effect of swollenin, blanks without swollenin (buffer) or with 0.4 g/L BSA instead of swollenin were incubated similarly. To detect a possible hydrolytic activity of recombinant swollenin, the sensitive p-hydroxy benzoic acid hydrazide assay  was applied by using glucose as a standard. After incubation for 48 h, the supernatants of the pretreatment solution were analyzed and the absorbancies were measured at 410 nm in a Synergy 4 microtiter plate reader. Subsequently, all cellulosic samples were washed to remove adsorbed proteins. Therefore, the mixtures were centrifuged (14,000 × g, 10 min, 4°C), and the cellulosic pellets were washed four times with 800 μL 0.05 M citrate buffer (pH 10) , and once with 800 μL distilled water. Finally, the triplicates were pooled. According to Zhu et al. , citrate buffer (pH 10) is an appropriate washing solution, and a single washing step with 0.05 M citrate buffer (pH 10) leads to a desorption efficiency of 61% in case of fungal cellulases and Avicel. Since no acids or bases are formed during the washing procedure, the weak buffer capacity of citrate buffer at pH 10 can be neglected. In this study, the washing procedure was conducted four times to ensure a high desorption of swollenin. The measurements of protein concentration in the washing supernatants - by applying the aforementioned bicinchoninic acid assay (working range starting from 0.005 g/L) - showed that swollenin desorbed almost completely. Already after three washing steps, a total swollenin desorption efficiency of > 90% was achieved.
Photography and microscopy
Photography and microscopy were applied to visualize the effect of swollenin pretreatment on filter paper. After pretreatment with buffer, BSA or swollenin, the different filter paper solutions were transferred into petri dishes, the particles were evenly distributed and images were taken with an Exilim EX-FH100 camera (Casio, Tokyo, Japan). Afterwards, the number of filter paper agglomerates (> 0.5 mm) was determined by image analysis using the software UTHSCSA ImageTool 3.0 (freeware) and a ruler as a reference. Light microscopic pictures were taken with an Eclipse E600 (Nikon, Tokyo, Japan). Additionally, scanning electron microscopy was performed using a Hitachi S-5500 (Hitachi, Tokyo, Japan) and a field emission of 5 kV. All washed filter paper samples were covered with a layer of carbon (3 nm) and, subsequently, with a layer of PtPd (3 nm, 80% to 20%). The images were taken by using secondary electrons.
Laser diffraction and X-ray diffraction
where I 002 is the maximum intensity of the crystalline plane (002) reflection (2θ = 22.5°) and I AM is the intensity of the scattering for the amorphous component at about 18° in cellulose-I . Here, it should be noted that there are several methods for calculating CrI from XRD data and these methods can provide significantly different results [28, 70]. Although the applied peak height method produces CrI values that are higher than those of other methods, it is still the most commonly used method and ranks CrI values in the same order as the other methods .
Hydrolysis experiments and dinitrosalicylic acid assay
Hydrolysis experiments with 10 g/L pretreated cellulosic substrate and 1 g/L rebuffered Celluclast® were conducted in 0.05 M sodium acetate buffer (pH 4.8). The mixtures were incubated as triplicates in 2 mL Eppendorf tubes on a thermomixer under the following constant conditions: T = 45°C, total filling V L = 1 mL, d 0 = 3 mm, n = 1000 rpm. In general, attention has to be paid to cellulase inactivation, which would reduce the final yield of cellulose hydrolysis . In this current study, however, a shaken system with relatively low shear forces was applied. According to Engel et al. , rebuffered Celluclast® is stable under the applied incubation conditions, so that cellulase inactivation could be neglected. The shaking frequency was chosen to ensure the complete suspension of cellulose particles [64, 91]. Thus, mass transfer limitations are excluded, and the whole cellulose particle surface becomes accessible to the cellulases, thereby optimizing cellulase adsorption and activity . Three different blanks were incubated similarly: (i) without cellulase, (ii) without substrate, or (iii) without substrate and without cellulase. The dinitrosalicylic acid assay  was applied to quantify the reducing sugars released during hydrolysis by using glucose as a standard. After defined time intervals, samples were taken, and the hydrolysis was stopped (10 min, 100°C). According to Wood and Bhat , low reducing sugar concentrations were quantified by adding 1.25 g/L glucose to the samples. The absorbancies were measured at 540 nm in a Synergy 4 microtiter plate reader. Since the dinitrosalicylic acid assay exhibits a lower sensitivity towards cellobiose than glucose, reducing sugar concentrations may be underestimated when glucose is used as a standard and β-glucosidase is not in excess . However, under the applied hydrolysis conditions, cellobiose did not accumulate (the highest cellobiose to glucose ratio was measured in the case of Sigmacell after 10 h at 0.12) and, therefore, this underestimation was minimal and the addition of β-glucosidase was not needed. Initial hydrolysis rates (g/(L*h)) were calculated by applying a linear fit to the reducing sugar concentration data from 0 to 6 h.
in which a, b, c, d, e, f and g denote the various fitting parameters of the non-linear Gaussian cumulative function (-).
This work was performed as part of the Cluster of Excellence "Tailor-Made Fuels from Biomass", which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities.
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