TY - JOUR
AU - Filho, Edivaldo Ximenes Ferreira
AB - Abstract Holocellulase production by Aspergillus niger using raw sugarcane bagasse (rSCB) as the enzyme-inducing substrate is hampered by the intrinsic recalcitrance of this material. Here we report that mild hydrothermal pretreatment of rSCB increases holocellulase secretion by A. niger. Quantitative proteomic analysis revealed that pretreated solids (PS) induced a pronounced up-regulation of endoglucanases and cellobiohydrolases compared to rSCB, which resulted in a 10.1-fold increase in glucose release during SCB saccharification. The combined use of PS and pretreatment liquor (PL), referred to as whole pretreated slurry (WPS), as carbon source induced a more balanced up-regulation of cellulases, hemicellulases and pectinases and resulted in the highest increase (4.8-fold) in the release of total reducing sugars from SCB. The use of PL as the sole carbon source induced the modulation of A. niger’s secretome towards hemicellulose degradation. Mild pretreatment allowed the use of PL in downstream biological operations without the need for undesirable detoxification steps. Electronic supplementary material The online version of this article (10.1007/s10295-019-02207-0) contains supplementary material, which is available to authorized users. Introduction Enzymes account for a significant percentage of the overall costs associated with the refining of lignocellulosic biomass into renewable fuels and chemicals. Currently, most of the existing lignocellulose biorefineries purchase enzymes from suppliers. Costs can be reduced if enzymes are produced on-site in a production unit attached to the biorefinery plant. In both cases, the current modus operandi is to employ simple sugars (primarily glucose) and carbon catabolite de-repressed filamentous fungi to produce crude enzyme preparations. It has been proposed that enzyme production costs can be further reduced by employing an integrated enzyme production approach, in which cheap incoming lignocellulosic biomass is used as feedstock for on-site enzyme manufacture in biorefineries [14, 24]. The use of biomass as a carbon source for enzyme-producing fungi is particularly interesting since it is likely to induce a tailor-made enzyme arsenal especially suited for the saccharification of that specific material, in contrast to broad-spectrum commercial enzyme preparations. The recalcitrance of raw lignocellulose, however, limits fungal growth and enzyme production, and biomass pretreatment can be a useful tool to make feasible the integrated enzyme production approach [24]. Although several pretreatment technologies have been developed to reduce biomass recalcitrance (usually aiming maximum enzymatic conversion of cellulose to glucose), little attention is given to the effect of pretreatment on the production of enzymes when lignocellulose is offered as a carbon source to filamentous fungi. Among the leading technologies, hydrothermal pretreatment stands out for its ability to generate low levels of microbial inhibitory compounds (such as furans, phenolic compounds and organic acids), allowing the use of both aqueous and solid pretreated fractions in downstream biological operations without the need for undesirable detoxification steps [29]. Reports have shown that hydrothermal pretreatment of biomass significantly affects enzyme induction in filamentous fungi and, depending on pretreatment severity, holocellulases (i.e. enzymes involved in the degradation of plant cell wall polysaccharides, including cellulose, hemicellulose and pectin) are differentially expressed [2, 7]. The primary goal of this work is to evaluate the effect of mild hydrothermal pretreatment of sugarcane bagasse on the production of holocellulases by the model ascomycete Aspergillus niger when using both solid and liquid fractions arising from pretreatment as the carbon sources. Here, we characterize and compare the A. niger secretomes produced in response to raw sugarcane bagasse (rSCB), pretreated solids (PS), pretreatment liquor (PL) and whole pretreated slurry (WPS, i.e. the combination of pretreated solids and liquor) by measuring enzyme activities, biomass saccharification yields and relative enzyme abundance levels by label-free quantitative proteomics. Although previous works have investigated the effect of sugarcane bagasse pretreatment on enzyme induction [2], the present report is unprecedented in using both solid and liquid fractions, together or separately, in comparison to untreated biomass as the enzyme-inducing substrate. Materials and methods Hydrothermal pretreatment of biomass Raw sugarcane bagasse (rSCB) was pretreated at 1% solids loading (w/w, g solids per g total) following the procedure described by Silva et al. [23]. Briefly, 2.25 g of rSCB (milled and dried) and 222.75 mL of distilled water were added to stainless steel cylindrical reactors of internal volume of 300 mL (Swagelok, Cleveland, OH, USA), sealed at both ends, and incubated in a pre-heated fluidized sand bath at 170 ± 2 °C for 30 min (Tecam SBL-2, Cole Parmer, Vernon Hills, IL, USA). Worthy of note, biomass heat-up phase (not determined) was accounted for in the total incubation period of pretreatment. After pretreatment, reactors were quenched in cold water. The whole pretreated slurry (WPS) was vacuum filtered through filter paper, generating a solid fraction (hereafter called pretreated solids, or PS) and a liquid fraction (pretreatment liquor, or PL). PS were washed with distilled water at 5% (w/w) for 5 min under agitation, filtered and dried at 65 °C, while PL was centrifuged at 2739 g for 10 min before both were used as carbon sources for A. niger and/or subjected to compositional analyses. Biomass compositional analysis and scanning electron microscopy For the compositional analysis of raw sugarcane bagasse (rSCB) and pretreated solids (PS), the biomasses were sequentially washed with ethanol 95% and distilled water for 24 h using a Soxhlet apparatus [26]. The extractable-free materials were subjected to a two-step sulfuric acid hydrolysis (as described in [27]) and the released monosaccharides were quantified by high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) using a Dionex ICS-3000DC System equipped with a CarboPac PA-1 column (2 × 250 mm) and guard-column (2 × 50 mm) (Dionex Co., Sunnyvale, CA, USA). The chromatographic elution gradient was the same as described by Silva et al. [23]. Glucose, xylose, arabinose, galactose and mannose (Sigma, St. Louis, MO, USA) were used as standards. The acid-soluble lignin (ASL) content in the acid hydrolysate was determined spectrophotometrically and the solids remaining after acid hydrolysis were muffled to obtain the contents of acid-insoluble lignin (AIS) and ash according to Sluiter et al. [27]. Regarding the composition of PL, free monosaccharides were determined directly by HPAEC-PAD analyses. For the quantification of oligosaccharides, PL was subjected to dilute acid hydrolysis to decompose oligosaccharides into their monomeric units [25]. For such, 174 μL of 72% sulfuric acid solution was added to 5 mL of liquor (initial pH 4.16), bringing the concentration of sulfuric acid to 4%, and the mixture was autoclaved at 121 °C for 1 h in sealed glass bottles. The dilute acid hydrolysate was then neutralized to pH 5–6 with sodium carbonate and analysed by HPAEC-PAD as described above. To calculate the concentration of polymeric (in solid biomasses) and oligomeric sugars (in liquor) from the concentration of the corresponding monomeric sugars, we used an anhydro correction factor of 0.88 (or 132/150) for pentoses and 0.90 (or 162/180) for hexoses [27]. For biomass surface morphology images, rSCB and PS samples were coated with gold in an SCD 050 sputter coater (Balzers, Germany) and examined in a JMS 7001F (JEOL, Akishima, Japan) scanning electron microscope [15]. Microorganism and enzyme production Aspergillus niger DCFS11 was isolated from soil samples collected from the Brazilian Cerrado biome. The strain is deposited at the Laboratory of Enzymology, University of Brasilia, Brazil (genetic heritage number 010237/2015-1), and in the Bank of Microorganisms for Control of Plant Pathogens and Weeds of the Brazilian Agricultural Research Corporation (EMBRAPA) registered at the World Data Centre for Microorganisms (WDCM) under the code MCPPW 1128. Raw sugarcane bagasse (rSCB) and the three fractions arising from hydrothermal pretreatment of rSCB (i.e. WPS, PS and PL) were offered as carbon sources for A. niger DCFS11 under submerged fermentation, aiming the comparison of their ability to induce holocellulase production. The carbohydrate content of each carbon source employed in the cultivations is shown in the results section. The rSCB and PS growth conditions were prepared by adding rSCB (0.75 g) and PS (0.55 g, calculated as the amount of pretreated solids that would be obtained after hydrothermal pretreatment of 0.75 g of rSCB), respectively, to 75 mL of liquid minimum medium formulated in distilled water containing KH2PO4 (7.0 g L−1), K2HPO4 (2.0 g L−1), MgSO4 (0.5 g L−1), (NH4)2SO4 (1.0 g L−1) and yeast extract (0.6 g L−1), with initial pH adjusted to 7.0 with NaOH. In the case of the PL growth condition, 75 mL of PL were enriched with KH2PO4, K2HPO4, MgSO4, (NH4)2SO4 and yeast extract in the same concentrations as mentioned above, and adjusted to initial pH 7.0 with NaOH before being used as a cultivation medium. At last, the WPS growth condition was prepared by adding 0.55 g of PS to 75 mL of the above mentioned nutrient-enriched PL. After sterilization through autoclavation (121 °C, 20 min), the four cultivation media were inoculated with 7.5 × 106 A. niger spores and incubated at 28 °C and 120 rpm for five days. Enzyme production experiments were carried out in 250-mL conical flasks and in triplicate. Secretomes were obtained by vacuum filtration on filter paper of culture broths. Enzyme assays and total protein quantification Xylanase, endoglucanase, mannanase and pectinase activities were determined by mixing 10 μL of enzyme broth with 5 μL of 1% (w/v) solution of oat spelt xylan, carboxymethyl cellulose, mannan (locust bean gum) and apple pectin, respectively, for 30 min at 50 °C. Filter paper activity was determined by mixing 100 μL of enzyme broth with 200 μL of 100 mM sodium acetate buffer pH 5.0 and 10 mg of filter paper (Whatman No 1) for 60 min at 50 °C. The released reducing sugars were quantified by dinitrosalicylic acid reagent (DNS) [17]. For the determination of β-glucosidase, β-xylosidase, α-l-arabinofuranosidase, β-mannosidase and α-galactosidase activities, 5 μL of enzyme broth were incubated with 45 μL of 5 mM pNP-β-d-glucopyranoside, pNP-β-d-xylopyranoside, pNP-α-l-arabinofuranoside, pNP-β-d-mannopyranoside, pNP-α-galactopyranoside, respectively, for 30 min at 50 °C. The reaction was quenched by the addition of 50 μL of 1 M sodium carbonate and the release of p-nitrophenol was quantified spectrophotometrically at 430 nm. The enzymatic activities were expressed as micromoles of product formed per minute (IU) and per milliliter of enzyme solution. All natural and synthetic substrates were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Total protein in secretomes was quantified by Quick Start™ Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, USA) using bovine serum albumin as standard. Interfering pigments were removed from secretomes by acetone–NaCl protein precipitation [6] before protein quantification. Enzyme assays and protein determination were performed in quintuplicate. Biomass saccharification assays Enzymatic saccharification assays of rSCB and PS were conducted using 10 g L−1 biomass loading (dry weight) and 5 mg of total protein per g dry biomass in 250-mL conical flasks with a working volume of 50 mL in 100 mM sodium acetate buffer pH 5.0 containing sodium azide (0.1 g L−1), at 40 °C and 120 rpm for 120 h, in triplicate. The released total reducing sugars and glucose were determined by DNS reagent [17] and glucose-oxidase/peroxidase assay kit (Doles, Goiânia, Brazil), respectively. Identification and relative quantification of proteins by label-free proteomics Sample preparation Secretome aliquots containing 50 µg of total protein were precipitated [6] vacuum-dried, reconstituted in 150 µL of 8 M urea, 7.5 mM NaCl, 50 mM triethylammonium bicarbonate (TEAB), 5 mM dithiothreitol (DTT), pH 8.2, and incubated at 55 °C for 25 min. Iodoacetamide was added to a final concentration of 14 mM followed by 40 min incubation at 25 °C in the dark. DTT was added to a final concentration of 10 mM and the reaction mixture was then diluted five-fold in 25 mM TEAB, pH 7.9, followed by CaCl2 addition to a final concentration of 1 mM. Samples were then digested with modified porcine trypsin (1 µg per 50 µg of total protein) overnight at 37 °C, followed by addition of trifluoroacetic acid (TFA) to a final concentration of 0.5% (v/v). Samples were vacuum-dried, reconstituted in TFA 0.1% (v/v) and desalted using StageTips (filled with Empore C18 membrane) [20]. Tryptic peptides were quantified by fluorometry (Qubit, Thermo Fisher Scientific, Waltham, MA, USA). Instrumentation An UltiMate® 3000 Nano LC system (Dionex, Amsterdam, The Netherlands) equipped with Reprosil-Pur 120 C18-AQ analytical column (particle size 3 μm, length 15 cm, inner diameter 75 μm, outer diameter 360 μm) and trap column (particle size 5 μm, length 5.0 cm, inner diameter 100 μm, outer diameter 360 μm) (Dr. Maish, Ammerbuch, Germany) was used, coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via a nanospray probe. Chromatographic and MS conditions were performed as described by Gomes et al. [11]. Data analysis The predicted proteins from the genomes of A. niger CBS 513.88 (ASPN_A), A. niger ATCC 1015 (ASPN_C) and A. niger An76 (ASPN_G) (http://www.uniprot.org) were used in database searching for protein identification using PEAKS® software (Version 7.1; Bioinformatics Solutions Inc., Waterloo, Ontario, Canada). At least one unique peptide was considered, and the false discovery rate (FDR) was set to 1%. Carbamidomethylation of cysteines and acetylation of aminoterminal ends were taken as fixed modifications. Tolerance filters of 0.5 Da to parent spectra and 10 ppm to tandem MS spectra were employed. Proteins were analyzed for the presence of carbohydrate-active enzyme (CAZyme) conserved domains using hmmscan software (HMMER3, version 3.1) and dbCAN database release 4.0 [30] and for the presence of secretion signal using SignalP 4.1 [18]. Relative protein quantification was performed with Progenesis QI for Proteomics software (Nonlinear Dynamics, Durham, USA). Normalized protein abundance values in WPS-, PS- and PL-induced secretomes were compared to the rSCB condition by ANOVA (p < 0.05) and Student’s t test (p < 0.05). Data were subjected to Principal Component Analysis (PCA) using MetaboAnalyst platform [5]. Results and discussion Effect of hydrothermal pretreatment on biomass composition and structure Figure 1a brings the carbohydrate and lignin contents of sugarcane bagasse before and after pretreatment. A more detailed composition of PL is shown in Fig. 1b. Mild hydrothermal pretreatment of rSCB led to 44.5% of xylan solubilization, mainly into xylooligosaccharides (XOS), which made up the majority of PL’s total carbohydrate content. Approximately 70% of arabinan and galactosan (structural components of arabinoxylan and rhamnogalaturonan I) were solubilized, mostly as free arabinose and galactooligosaccharides (GalOS). Only 3.7% of glucan was solubilized, mainly in the form of glucooligosaccharides (GlcOS), likely derived from β-1,3-1,4-glucan (lichenan) or xyloglucan hydrolysis. Fig. 1 Open in new tabDownload slide a Carbohydrate and lignin contents of raw sugarcane bagasse (rSCB), whole pretreated slurry (WPS), pretreated solids (PS) and pretreatment liquor (PL) used as the carbon sources for enzyme production by Aspergillus niger. Carbohydrates in rSCB and PS correspond to insoluble polysaccharides while in PL they correspond to soluble mono- and oligosaccharides. WPS is comprised of insoluble polysaccharides and soluble mono- and oligosaccharides. b Detailed mono- and oligosaccharide composition of PL. c, d Scanning-electron micrographs of rSCB and PS, respectively. AIL acid insoluble lignin, ASL acid-soluble lignin, XOS xylooligosaccharides, GlcOS glucooligosaccharides, AOS arabinooligosaccharides, GalOS galacto-oligosaccharides In summary, the majority of hemicellulose (represented mainly by xylan and arabinan) and a minor fraction of pectin (represented mainly by galactosan and arabinan) were retained in PS together with cellulose (glucan) after pretreatment. The retention of these polysaccharides in pretreated solids is typical of mild hydrothermal pretreatment (low temperature and/or short treatment period). These results, on the other hand, contrast those usually observed when severe hydrothermal pretreatments (usually performed at ≥ 190 °C for ≥ 20 min) are applied onto biomass, which can lead to near-complete solubilization of pectin and xylan to the liquid fraction [7, 15, 21], but at the expense of sugar loss (as decomposition products) and high generation of microbial and enzymatic inhibitory compounds (e.g. acetic acid, formic acid, furfural, hydroxymethyl-furfural and phenolic compounds). Mild pretreatment has the advantage of generating liquors with little or no toxicity, which can be offered as carbon sources for enzyme-producing microorganisms as it is or in the form of whole pretreated slurry. Silva et al. [23] have found a suitable hydrothermal pretreatment severity range of sugarcane bagasse which allowed the use of pretreatment liquor as a substrate for the production of xylan-degrading enzymes by A. niger without the need for detoxification steps. The biomass pretreatment parameters used here were selected after the work of Silva et al. [23]. Scanning electron micrographs revealed a shift from a smooth and orderly appearance in rSCB (Fig. 1c) to a rough, less organized and corrugated one in PS (Fig. 1d). Although not measured in this work, hydrothermal pretreatment is also known to reduce particle size and increase biomass surface area and porosity [15]. These factors combined likely favor fungal growth and enzyme production by increasing the accessibility of polysaccharides in PS to A. niger mycelium and enzymes. Holocellulase activity profile of A. niger secretomes Increases in protein secretion and holocellulase activities were achieved when pretreated carbon sources, i.e. WPS, PS and PL, were offered to A. niger in place of rSCB (Table 1). As shown above, PL contains only 20% of the total carbohydrate content found in untreated biomass (rSCB) and yet it induced a higher production of proteins and enzymes than rSCB. This is possibly due to the presence of soluble sugars in PL, mainly XOS, which are easy to hydrolyze and consume, favoring fungus growth and enzyme induction. This suggests that the accessibility of carbohydrates in the substrate, and not only the quantity, is determinant for holocellulase induction in A. niger [23]. The panel of enzymatic activities and total protein concentration in Aspergillus niger secretomes induced by raw sugarcane bagasse (rSCB), whole pretreated slurry (WPS), pretreated solids (PS) and pretreatment liquor (PL) as carbon sources . Enzyme activities (IU mL−1) . rSCB . WPS . PS . PL . FPase 0.030 ± 0.003 0.107 ± 0.007 0.092 ± 0.008 0.078 ± 0.007 Endoglucanase 0.066 ± 0.009 0.225 ± 0.003 0.211 ± 0.006 0.168 ± 0.022 β-Glucosidase 0.209 ± 0.011 0.355 ± 0.112 0.150 ± 0.007 0.242 ± 0.020 Xylanase 1.808 ± 0.081 4.244 ± 0.153 4.233 ± 0.121 3.700 ± 0.030 β-Xylosidase 0.018 ± 0.004 0.245 ± 0.010 0.100 ± 0.017 0.193 ± 0.010 α-l-Arabinofuranosidase 0.008 ± 0.002 0.148 ± 0.013 0.034 ± 0.001 0.101 ± 0.018 Mannanase 0.031 ± 0.003 0.200 ± 0.002 0.218 ± 0.011 0.074 ± 0.012 β-Mannosidase 0.000 ± 0.004 0.000 ± 0.001 0.001 ± 0.001 0.000 ± 0.002 α-Galactosidase 0.114 ± 0.003 0.258 ± 0.015 0.103 ± 0.017 0.267 ± 0.060 Pectinase 0.000 ± 0.008 0.100 ± 0.014 0.065 ± 0.025 0.012 ± 0.008 Total protein concentration (µg mL−1) 15.946 ± 2.300 31.539 ± 0.345 28.623 ± 0.281 22.993 ± 0.493 . Enzyme activities (IU mL−1) . rSCB . WPS . PS . PL . FPase 0.030 ± 0.003 0.107 ± 0.007 0.092 ± 0.008 0.078 ± 0.007 Endoglucanase 0.066 ± 0.009 0.225 ± 0.003 0.211 ± 0.006 0.168 ± 0.022 β-Glucosidase 0.209 ± 0.011 0.355 ± 0.112 0.150 ± 0.007 0.242 ± 0.020 Xylanase 1.808 ± 0.081 4.244 ± 0.153 4.233 ± 0.121 3.700 ± 0.030 β-Xylosidase 0.018 ± 0.004 0.245 ± 0.010 0.100 ± 0.017 0.193 ± 0.010 α-l-Arabinofuranosidase 0.008 ± 0.002 0.148 ± 0.013 0.034 ± 0.001 0.101 ± 0.018 Mannanase 0.031 ± 0.003 0.200 ± 0.002 0.218 ± 0.011 0.074 ± 0.012 β-Mannosidase 0.000 ± 0.004 0.000 ± 0.001 0.001 ± 0.001 0.000 ± 0.002 α-Galactosidase 0.114 ± 0.003 0.258 ± 0.015 0.103 ± 0.017 0.267 ± 0.060 Pectinase 0.000 ± 0.008 0.100 ± 0.014 0.065 ± 0.025 0.012 ± 0.008 Total protein concentration (µg mL−1) 15.946 ± 2.300 31.539 ± 0.345 28.623 ± 0.281 22.993 ± 0.493 Mean values are presented with their respective standard deviations Open in new tab The panel of enzymatic activities and total protein concentration in Aspergillus niger secretomes induced by raw sugarcane bagasse (rSCB), whole pretreated slurry (WPS), pretreated solids (PS) and pretreatment liquor (PL) as carbon sources . Enzyme activities (IU mL−1) . rSCB . WPS . PS . PL . FPase 0.030 ± 0.003 0.107 ± 0.007 0.092 ± 0.008 0.078 ± 0.007 Endoglucanase 0.066 ± 0.009 0.225 ± 0.003 0.211 ± 0.006 0.168 ± 0.022 β-Glucosidase 0.209 ± 0.011 0.355 ± 0.112 0.150 ± 0.007 0.242 ± 0.020 Xylanase 1.808 ± 0.081 4.244 ± 0.153 4.233 ± 0.121 3.700 ± 0.030 β-Xylosidase 0.018 ± 0.004 0.245 ± 0.010 0.100 ± 0.017 0.193 ± 0.010 α-l-Arabinofuranosidase 0.008 ± 0.002 0.148 ± 0.013 0.034 ± 0.001 0.101 ± 0.018 Mannanase 0.031 ± 0.003 0.200 ± 0.002 0.218 ± 0.011 0.074 ± 0.012 β-Mannosidase 0.000 ± 0.004 0.000 ± 0.001 0.001 ± 0.001 0.000 ± 0.002 α-Galactosidase 0.114 ± 0.003 0.258 ± 0.015 0.103 ± 0.017 0.267 ± 0.060 Pectinase 0.000 ± 0.008 0.100 ± 0.014 0.065 ± 0.025 0.012 ± 0.008 Total protein concentration (µg mL−1) 15.946 ± 2.300 31.539 ± 0.345 28.623 ± 0.281 22.993 ± 0.493 . Enzyme activities (IU mL−1) . rSCB . WPS . PS . PL . FPase 0.030 ± 0.003 0.107 ± 0.007 0.092 ± 0.008 0.078 ± 0.007 Endoglucanase 0.066 ± 0.009 0.225 ± 0.003 0.211 ± 0.006 0.168 ± 0.022 β-Glucosidase 0.209 ± 0.011 0.355 ± 0.112 0.150 ± 0.007 0.242 ± 0.020 Xylanase 1.808 ± 0.081 4.244 ± 0.153 4.233 ± 0.121 3.700 ± 0.030 β-Xylosidase 0.018 ± 0.004 0.245 ± 0.010 0.100 ± 0.017 0.193 ± 0.010 α-l-Arabinofuranosidase 0.008 ± 0.002 0.148 ± 0.013 0.034 ± 0.001 0.101 ± 0.018 Mannanase 0.031 ± 0.003 0.200 ± 0.002 0.218 ± 0.011 0.074 ± 0.012 β-Mannosidase 0.000 ± 0.004 0.000 ± 0.001 0.001 ± 0.001 0.000 ± 0.002 α-Galactosidase 0.114 ± 0.003 0.258 ± 0.015 0.103 ± 0.017 0.267 ± 0.060 Pectinase 0.000 ± 0.008 0.100 ± 0.014 0.065 ± 0.025 0.012 ± 0.008 Total protein concentration (µg mL−1) 15.946 ± 2.300 31.539 ± 0.345 28.623 ± 0.281 22.993 ± 0.493 Mean values are presented with their respective standard deviations Open in new tab The use of washed PS as a carbon source also strongly increased total protein and holocellulase production in comparison to the rSCB substrate, likely a result of biomass particle size reduction, increased biomass porosity, rearrangement of the lignocellulosic matrix, partial removal of hemicellulose and pectin and a consequent increase in the accessibility of A. niger mycelium and secreted enzymes to cellulose. The highest activity titers were obtained using WPS as the carbon source, likely due to the combined presence of readily available soluble sugars in the liquid fraction and more accessible polysaccharides in the pretreated solid fraction. The protein secretion levels (15.9–31.5 µg/mL) observed here for A. niger DCFS11 (a new wild-type strain isolated from soil samples) were not impressive compared to other strains that have been genetically optimized from holocellulase secretion, such as Trichoderma reesei RUT-C30, which can reach protein levels above 500 µg/mL when cultivated in the presence of lignocellulosic substrates [1]. Despite this, A. niger is a model organism for the study of holocellulases and other industrial enzymes. The complete genome of different strains of A. niger is publicly available and well annotated and the holocellulolytic system of this species is very well characterized. This enabled accurate protein identification in A. niger DCFS11 secretomes by mass spectrometry-based proteomic analysis and a detailed evaluation of the dynamics of holocellulase expression in different carbon sources, as shown below. Protein profile of A. niger secretomes In total, 154 identified proteins, 86 of them classified as carbohydrate-active enzymes (CAZymes), were shared by the four A. niger secretomes analysed (Fig. 2a). Fifty-nine CAZymes were holocellulases and 52 of them presented signal peptide that leads proteins towards the secretory pathway (Fig. 2a–c). Other 64 proteins of known and unknown functions also presented signal peptide (Fig. 2c). Forty-two proteins with no signal peptides were detected (Fig. 2b), which may correspond to extracellular proteins secreted by alternative secretion pathways or to intracellular leaked proteins (Online Resource 1). Fig. 2 Open in new tabDownload slide Overview of the proteomic analysis of Aspergillus niger secretomes induced by raw sugarcane bagasse and pretreatment-derived materials. a Categorization of all identified proteins, regardless of the presence of signal peptide. b Categorization of proteins according to the presence of signal peptide that leads proteins towards the secretory pathway. c Categorization of proteins that carry a signal peptide for secretion A total of 18 cellulose-degrading enzymes with signal peptide were shared by all secretomes (Table 2), including endoglucanases (belonging to CAZy families GH5, GH6 and GH12), cellobiohydrolases (GH6 and GH7), β-glucosidases (GH1 and GH3) and lytic polysaccharide monooxygenases (LPMOs) from family AA9 (Table 2). The expression of the majority of cellulase encoding-genes in A. niger is activated by XlnR and/or AraR transcriptions factors, which are induced by d-xylose and l-arabinose, respectively [10, 13]. It was recently demonstrated by Gong et al. [12] that XOS also induce the expression of the XlnR transcription factor in A. niger and at higher levels than xylose. This partially explains why rSCB and all pretreated fractions, including PL (devoid of crystalline cellulose but rich in XOS, xylose and arabinose), induced the complete set of enzymes necessary for the breakdown of cellulose into glucose. Transcriptomic and proteomic data obtained by Gong et al. [12] suggest that A. niger possesses yet uncharacterized sugar transporters that are able to import XOS (both branched and unbranched), which would allow XOS internalization and intracellular signaling. It has also been shown that Neurospora crassa has cello-dextrin transporters that internalize cello-dextrins and trigger downstream activation of (hemi)cellulolytic gene expression [4, 31]. An analogous mechanism might occur for XOS import and induction of (hemi)cellulases in A. niger. List of holocellulases identified in Aspergillus niger secretomes induced by raw sugarcane bagasse (rSCB), pretreated solids (PS), pretreatment liquor (PL) and whole pretreated slurry (WPS) as carbon sources Accession number . Signal peptide . CAZy family . Protein name . Description . Predicted substrate . log2 (fold-change) . PS . PL . WPS . A2QVN9_ASPNC Y GH1 Putative β-glucosidase Cellulose, xyloglucan 4.133 3.639 4.955 G3Y786_ASPNA N GH1 β-Glucosidase Cellulose, xyloglucan 1.324 − 9.620 2.559 A0A100IJJ3_ASPNG N GH3 β-Glucosidase Cellulose, xyloglucan − 0.212 1.818 1.207 C7C4Z9_ASPNG Y GH3 Bgl β-Glucosidase Cellulose, xyloglucan 0.610* 0.959* 2.126* A0A1V1FQ75_ASPNG Y GH3 XlnD β-Xylosidase Xylan 2.722* 3.248* 3.069* A0A100IKN9_ASPNG Y GH3 α-Galactosidase Galactomannan 3.913* 1.820 3.919* A0A1V1G264_ASPNG Y GH3 XlsV Putative β-xylosidase Xylan 1.076 0.252 1.653* G3Y4E8_ASPNA N GH3 Bgl1 β-Glucosidase Cellulose, xyloglucan 3.022* 1.117 2.383 A2R2S3_ASPNC Y GH3 Putative β-glucosidase Cellulose, xyloglucan − 2.806* − 4.150* − 3.369* G3YBE0_ASPNA Y GH3 β-Glucosidase Cellulose, xyloglucan − 1.125 − 0.014 0.708 A0A023UH08_ASPNG Y GH5 Eg1 Endo-β-1,4-glucanase Cellulose 11.226* 6.836 9.979* H2E6Y8_ASPNG Y GH5 ManA Endo-β-1,4-mannanase Mannan 1.644* 4.951 1.536 A2QAI8_ASPNC Y GH5, CBM1 Endo-β-1,4-glucanase Cellulose 7.143* 3.467* 5.969* G3Y873_ASPNA Y GH5 Putative endo-β-1,6-galactanase Pectin − 1.886 9.460 − 0.792 G3XZI3_ASPNA Y GH5 EglB Endo-β-1,4-glucanase Cellulose 5.098* 1.926 4.134* A2QQ99_ASPNC Y GH6 Cellobiohydrolase Cellulose 6.098* 2.849* 4.249* A2QYR9_ASPNC Y GH6, CBM1 CbhC Cellobiohydrolase Cellulose 2.932* 2.493* 2.728* A0A100IHS6_ASPNG Y GH6 Glucanase Cellulose 2.959* 3.775 1.072 Q9UVS9_ASPNG Y GH7 CbhA Cellobiohydrolase Cellulose 4.299* 3.076* 2.858* A2QAI7_ASPNC Y GH7, CBM1 CbhB Cellobiohydrolase Cellulose 3.904* 2.473* 2.690* G3Y866_ASPNA Y GH10 XynC Endo-β-1,4-xylanase Xylan 3.813* 2.639* 3.763* Q6QA21_9EURO Y GH11, CBM60 XynB Endo-β-1,4-xylanase Xylan 3.303* 2.852* 3.653* U6C3R6_ASPNG Y GH11 XynV Endo-β-1,4-xylanase Xylan 0.966 3.852 2.325* E3UN71_ASPNG Y GH11, CBM60 Endo-β-1,4-xylanase Xylan 1.387* 2.016* 2.007* O74705_ASPNG Y GH12 EglA Endo-β-1,4-glucanase Cellulose 4.710* 2.624 4.016* G3XRM3_ASPNA Y GH12 XgeA Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 1.025 2.897* 2.455* G5D7B5_ASPNG N GH27, CBM13 AglA α-Galactosidase Galactomannan 2.134* − 0.166 2.364* A0A0U5AE32_ASPNG Y GH27 AglB α-Galactosidase Galactomannan 1.370 − 1.150 2.716* G3XQY4_ASPNA Y GH28 Pgal Endo-polygalacturonase I Pectin 5.988 − 0.645 6.924* A2QK83_ASPNC Y GH28 XghA Putative endo-xylogalacturonan hydrolase Pectin 3.906* − 0.945 2.448* A2QTU5_ASPNC Y GH31 AxlA α-Xylosidase A Xyloglucan 1.474* 4.071* 3.671* B6HYI9_ASPNG Y GH35, CBM37 LacZ β-Galactosidase Pectin, xyloglucan 0.519 2.056 2.494* A0A1D8MQA0_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 1.114 2.328* 3.323* A0A1D8MQF6_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 2.897* 2.599 4.262* G3XM01_ASPNA Y GH36 AglC α-Galactosidase Galactomannan 0.819 1.434 2.101* A0A0S2CVZ9_ASPNG Y GH43 Xylosidase:arabinofuranosidase Xylan 4.076* 2.516* 4.161* G3XY38_ASPNA Y GH43 Uncharacterized protein Xylan − 1.920* 1.176 0.674 U6C191_ASPNG N GH43 Xilanase putative Xylan − 2.256 1.530 0.059 A2R511_ASPNC Y GH54, CBM42 AbfB α-l-Arabinofuranosidase Xylan, pectin 3.099* 2.750* 4.872* A0A100I6G0_ASPNG Y GH62 α-l-Arabinofuranosidase Xylan, pectin 2.762 2.001 2.524* A2QFV9_ASPNC Y GH62 AxhA α-l-Arabinofuranosidase Xylan, pectin 2.893* 1.861* 2.865* Q96WX9_ASPNG Y GH67 AguA α-Glucuronidase Xylan 2.522* 2.699* 3.253* Q8TFP1_ASPNG Y GH74, CBM1 EglC Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 5.085* 1.040* 3.387* G3XSS5_ASPNA Y GH78 α-l-Rhamnosidase Pectin − 3.347* 0.197 0.259 G3XVM1_ASPNA Y CE1 AceA Acetyl xylan esterase A Xylan 4.921* 3.329 4.718* G3Y471_ASPNA Y CE3 Acetyl xylan esterase Xylan 5.611* 1.631 6.521 A2QSY5_ASPNC Y CE5 FeaA Probable feruloyl esterase A Xylan, pectin 4.697* 4.201* 5.153* G3YAH8_ASPNA Y CE12 RgaeB Rhamnogalacturonan acetyl esterase Pectin 3.367* 3.874 5.002* G3XZI2_ASPNA N CE16 Uncharacterized protein (acetylesterase) 3.457* 4.059* 4.041* A2QPC2_ASPNC N CE16 PaeB Putative pectin acetyl esterase Pectin 4.150* 2.663* 3.697* G3Y497_ASPNA Y CE16 Uncharacterized protein (acetylesterase) 3.633* 1.488* 3.647* G3Y478_ASPNA Y PL1 PlyA Pectate lyase Pectin 2.252* − 0.217 2.558* A5ABH4_ASPNC Y PL4 RglB Rhamnogalacturonate lyase B Pectin 0.757 3.788* 5.843* Q8WZI8_ASPNG Y – FaeB Feruloyl esterase B Xylan, pectin 4.896* 3.841 5.270* A0A117E071_ASPNG Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 2.755* 2.602* 4.512* G3XY89_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 1.634 1.533 2.978* G3XPC9_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 4.962* 3.118 5.772* G3YHB3_ASPNA Y AA9 Lytic polysaccharide monooxygenase Cellulose 2.544 0.578 6.559 A2QZE1_ASPNC Y AA9 Lytic polysaccharide monooxygenase Cellulose 1.558* 1.367* 1.989* Accession number . Signal peptide . CAZy family . Protein name . Description . Predicted substrate . log2 (fold-change) . PS . PL . WPS . A2QVN9_ASPNC Y GH1 Putative β-glucosidase Cellulose, xyloglucan 4.133 3.639 4.955 G3Y786_ASPNA N GH1 β-Glucosidase Cellulose, xyloglucan 1.324 − 9.620 2.559 A0A100IJJ3_ASPNG N GH3 β-Glucosidase Cellulose, xyloglucan − 0.212 1.818 1.207 C7C4Z9_ASPNG Y GH3 Bgl β-Glucosidase Cellulose, xyloglucan 0.610* 0.959* 2.126* A0A1V1FQ75_ASPNG Y GH3 XlnD β-Xylosidase Xylan 2.722* 3.248* 3.069* A0A100IKN9_ASPNG Y GH3 α-Galactosidase Galactomannan 3.913* 1.820 3.919* A0A1V1G264_ASPNG Y GH3 XlsV Putative β-xylosidase Xylan 1.076 0.252 1.653* G3Y4E8_ASPNA N GH3 Bgl1 β-Glucosidase Cellulose, xyloglucan 3.022* 1.117 2.383 A2R2S3_ASPNC Y GH3 Putative β-glucosidase Cellulose, xyloglucan − 2.806* − 4.150* − 3.369* G3YBE0_ASPNA Y GH3 β-Glucosidase Cellulose, xyloglucan − 1.125 − 0.014 0.708 A0A023UH08_ASPNG Y GH5 Eg1 Endo-β-1,4-glucanase Cellulose 11.226* 6.836 9.979* H2E6Y8_ASPNG Y GH5 ManA Endo-β-1,4-mannanase Mannan 1.644* 4.951 1.536 A2QAI8_ASPNC Y GH5, CBM1 Endo-β-1,4-glucanase Cellulose 7.143* 3.467* 5.969* G3Y873_ASPNA Y GH5 Putative endo-β-1,6-galactanase Pectin − 1.886 9.460 − 0.792 G3XZI3_ASPNA Y GH5 EglB Endo-β-1,4-glucanase Cellulose 5.098* 1.926 4.134* A2QQ99_ASPNC Y GH6 Cellobiohydrolase Cellulose 6.098* 2.849* 4.249* A2QYR9_ASPNC Y GH6, CBM1 CbhC Cellobiohydrolase Cellulose 2.932* 2.493* 2.728* A0A100IHS6_ASPNG Y GH6 Glucanase Cellulose 2.959* 3.775 1.072 Q9UVS9_ASPNG Y GH7 CbhA Cellobiohydrolase Cellulose 4.299* 3.076* 2.858* A2QAI7_ASPNC Y GH7, CBM1 CbhB Cellobiohydrolase Cellulose 3.904* 2.473* 2.690* G3Y866_ASPNA Y GH10 XynC Endo-β-1,4-xylanase Xylan 3.813* 2.639* 3.763* Q6QA21_9EURO Y GH11, CBM60 XynB Endo-β-1,4-xylanase Xylan 3.303* 2.852* 3.653* U6C3R6_ASPNG Y GH11 XynV Endo-β-1,4-xylanase Xylan 0.966 3.852 2.325* E3UN71_ASPNG Y GH11, CBM60 Endo-β-1,4-xylanase Xylan 1.387* 2.016* 2.007* O74705_ASPNG Y GH12 EglA Endo-β-1,4-glucanase Cellulose 4.710* 2.624 4.016* G3XRM3_ASPNA Y GH12 XgeA Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 1.025 2.897* 2.455* G5D7B5_ASPNG N GH27, CBM13 AglA α-Galactosidase Galactomannan 2.134* − 0.166 2.364* A0A0U5AE32_ASPNG Y GH27 AglB α-Galactosidase Galactomannan 1.370 − 1.150 2.716* G3XQY4_ASPNA Y GH28 Pgal Endo-polygalacturonase I Pectin 5.988 − 0.645 6.924* A2QK83_ASPNC Y GH28 XghA Putative endo-xylogalacturonan hydrolase Pectin 3.906* − 0.945 2.448* A2QTU5_ASPNC Y GH31 AxlA α-Xylosidase A Xyloglucan 1.474* 4.071* 3.671* B6HYI9_ASPNG Y GH35, CBM37 LacZ β-Galactosidase Pectin, xyloglucan 0.519 2.056 2.494* A0A1D8MQA0_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 1.114 2.328* 3.323* A0A1D8MQF6_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 2.897* 2.599 4.262* G3XM01_ASPNA Y GH36 AglC α-Galactosidase Galactomannan 0.819 1.434 2.101* A0A0S2CVZ9_ASPNG Y GH43 Xylosidase:arabinofuranosidase Xylan 4.076* 2.516* 4.161* G3XY38_ASPNA Y GH43 Uncharacterized protein Xylan − 1.920* 1.176 0.674 U6C191_ASPNG N GH43 Xilanase putative Xylan − 2.256 1.530 0.059 A2R511_ASPNC Y GH54, CBM42 AbfB α-l-Arabinofuranosidase Xylan, pectin 3.099* 2.750* 4.872* A0A100I6G0_ASPNG Y GH62 α-l-Arabinofuranosidase Xylan, pectin 2.762 2.001 2.524* A2QFV9_ASPNC Y GH62 AxhA α-l-Arabinofuranosidase Xylan, pectin 2.893* 1.861* 2.865* Q96WX9_ASPNG Y GH67 AguA α-Glucuronidase Xylan 2.522* 2.699* 3.253* Q8TFP1_ASPNG Y GH74, CBM1 EglC Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 5.085* 1.040* 3.387* G3XSS5_ASPNA Y GH78 α-l-Rhamnosidase Pectin − 3.347* 0.197 0.259 G3XVM1_ASPNA Y CE1 AceA Acetyl xylan esterase A Xylan 4.921* 3.329 4.718* G3Y471_ASPNA Y CE3 Acetyl xylan esterase Xylan 5.611* 1.631 6.521 A2QSY5_ASPNC Y CE5 FeaA Probable feruloyl esterase A Xylan, pectin 4.697* 4.201* 5.153* G3YAH8_ASPNA Y CE12 RgaeB Rhamnogalacturonan acetyl esterase Pectin 3.367* 3.874 5.002* G3XZI2_ASPNA N CE16 Uncharacterized protein (acetylesterase) 3.457* 4.059* 4.041* A2QPC2_ASPNC N CE16 PaeB Putative pectin acetyl esterase Pectin 4.150* 2.663* 3.697* G3Y497_ASPNA Y CE16 Uncharacterized protein (acetylesterase) 3.633* 1.488* 3.647* G3Y478_ASPNA Y PL1 PlyA Pectate lyase Pectin 2.252* − 0.217 2.558* A5ABH4_ASPNC Y PL4 RglB Rhamnogalacturonate lyase B Pectin 0.757 3.788* 5.843* Q8WZI8_ASPNG Y – FaeB Feruloyl esterase B Xylan, pectin 4.896* 3.841 5.270* A0A117E071_ASPNG Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 2.755* 2.602* 4.512* G3XY89_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 1.634 1.533 2.978* G3XPC9_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 4.962* 3.118 5.772* G3YHB3_ASPNA Y AA9 Lytic polysaccharide monooxygenase Cellulose 2.544 0.578 6.559 A2QZE1_ASPNC Y AA9 Lytic polysaccharide monooxygenase Cellulose 1.558* 1.367* 1.989* Asterisks (*) show significantly different abundances (Student’s t test, p < 0.05) relative to the control condition (rSCB) Open in new tab List of holocellulases identified in Aspergillus niger secretomes induced by raw sugarcane bagasse (rSCB), pretreated solids (PS), pretreatment liquor (PL) and whole pretreated slurry (WPS) as carbon sources Accession number . Signal peptide . CAZy family . Protein name . Description . Predicted substrate . log2 (fold-change) . PS . PL . WPS . A2QVN9_ASPNC Y GH1 Putative β-glucosidase Cellulose, xyloglucan 4.133 3.639 4.955 G3Y786_ASPNA N GH1 β-Glucosidase Cellulose, xyloglucan 1.324 − 9.620 2.559 A0A100IJJ3_ASPNG N GH3 β-Glucosidase Cellulose, xyloglucan − 0.212 1.818 1.207 C7C4Z9_ASPNG Y GH3 Bgl β-Glucosidase Cellulose, xyloglucan 0.610* 0.959* 2.126* A0A1V1FQ75_ASPNG Y GH3 XlnD β-Xylosidase Xylan 2.722* 3.248* 3.069* A0A100IKN9_ASPNG Y GH3 α-Galactosidase Galactomannan 3.913* 1.820 3.919* A0A1V1G264_ASPNG Y GH3 XlsV Putative β-xylosidase Xylan 1.076 0.252 1.653* G3Y4E8_ASPNA N GH3 Bgl1 β-Glucosidase Cellulose, xyloglucan 3.022* 1.117 2.383 A2R2S3_ASPNC Y GH3 Putative β-glucosidase Cellulose, xyloglucan − 2.806* − 4.150* − 3.369* G3YBE0_ASPNA Y GH3 β-Glucosidase Cellulose, xyloglucan − 1.125 − 0.014 0.708 A0A023UH08_ASPNG Y GH5 Eg1 Endo-β-1,4-glucanase Cellulose 11.226* 6.836 9.979* H2E6Y8_ASPNG Y GH5 ManA Endo-β-1,4-mannanase Mannan 1.644* 4.951 1.536 A2QAI8_ASPNC Y GH5, CBM1 Endo-β-1,4-glucanase Cellulose 7.143* 3.467* 5.969* G3Y873_ASPNA Y GH5 Putative endo-β-1,6-galactanase Pectin − 1.886 9.460 − 0.792 G3XZI3_ASPNA Y GH5 EglB Endo-β-1,4-glucanase Cellulose 5.098* 1.926 4.134* A2QQ99_ASPNC Y GH6 Cellobiohydrolase Cellulose 6.098* 2.849* 4.249* A2QYR9_ASPNC Y GH6, CBM1 CbhC Cellobiohydrolase Cellulose 2.932* 2.493* 2.728* A0A100IHS6_ASPNG Y GH6 Glucanase Cellulose 2.959* 3.775 1.072 Q9UVS9_ASPNG Y GH7 CbhA Cellobiohydrolase Cellulose 4.299* 3.076* 2.858* A2QAI7_ASPNC Y GH7, CBM1 CbhB Cellobiohydrolase Cellulose 3.904* 2.473* 2.690* G3Y866_ASPNA Y GH10 XynC Endo-β-1,4-xylanase Xylan 3.813* 2.639* 3.763* Q6QA21_9EURO Y GH11, CBM60 XynB Endo-β-1,4-xylanase Xylan 3.303* 2.852* 3.653* U6C3R6_ASPNG Y GH11 XynV Endo-β-1,4-xylanase Xylan 0.966 3.852 2.325* E3UN71_ASPNG Y GH11, CBM60 Endo-β-1,4-xylanase Xylan 1.387* 2.016* 2.007* O74705_ASPNG Y GH12 EglA Endo-β-1,4-glucanase Cellulose 4.710* 2.624 4.016* G3XRM3_ASPNA Y GH12 XgeA Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 1.025 2.897* 2.455* G5D7B5_ASPNG N GH27, CBM13 AglA α-Galactosidase Galactomannan 2.134* − 0.166 2.364* A0A0U5AE32_ASPNG Y GH27 AglB α-Galactosidase Galactomannan 1.370 − 1.150 2.716* G3XQY4_ASPNA Y GH28 Pgal Endo-polygalacturonase I Pectin 5.988 − 0.645 6.924* A2QK83_ASPNC Y GH28 XghA Putative endo-xylogalacturonan hydrolase Pectin 3.906* − 0.945 2.448* A2QTU5_ASPNC Y GH31 AxlA α-Xylosidase A Xyloglucan 1.474* 4.071* 3.671* B6HYI9_ASPNG Y GH35, CBM37 LacZ β-Galactosidase Pectin, xyloglucan 0.519 2.056 2.494* A0A1D8MQA0_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 1.114 2.328* 3.323* A0A1D8MQF6_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 2.897* 2.599 4.262* G3XM01_ASPNA Y GH36 AglC α-Galactosidase Galactomannan 0.819 1.434 2.101* A0A0S2CVZ9_ASPNG Y GH43 Xylosidase:arabinofuranosidase Xylan 4.076* 2.516* 4.161* G3XY38_ASPNA Y GH43 Uncharacterized protein Xylan − 1.920* 1.176 0.674 U6C191_ASPNG N GH43 Xilanase putative Xylan − 2.256 1.530 0.059 A2R511_ASPNC Y GH54, CBM42 AbfB α-l-Arabinofuranosidase Xylan, pectin 3.099* 2.750* 4.872* A0A100I6G0_ASPNG Y GH62 α-l-Arabinofuranosidase Xylan, pectin 2.762 2.001 2.524* A2QFV9_ASPNC Y GH62 AxhA α-l-Arabinofuranosidase Xylan, pectin 2.893* 1.861* 2.865* Q96WX9_ASPNG Y GH67 AguA α-Glucuronidase Xylan 2.522* 2.699* 3.253* Q8TFP1_ASPNG Y GH74, CBM1 EglC Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 5.085* 1.040* 3.387* G3XSS5_ASPNA Y GH78 α-l-Rhamnosidase Pectin − 3.347* 0.197 0.259 G3XVM1_ASPNA Y CE1 AceA Acetyl xylan esterase A Xylan 4.921* 3.329 4.718* G3Y471_ASPNA Y CE3 Acetyl xylan esterase Xylan 5.611* 1.631 6.521 A2QSY5_ASPNC Y CE5 FeaA Probable feruloyl esterase A Xylan, pectin 4.697* 4.201* 5.153* G3YAH8_ASPNA Y CE12 RgaeB Rhamnogalacturonan acetyl esterase Pectin 3.367* 3.874 5.002* G3XZI2_ASPNA N CE16 Uncharacterized protein (acetylesterase) 3.457* 4.059* 4.041* A2QPC2_ASPNC N CE16 PaeB Putative pectin acetyl esterase Pectin 4.150* 2.663* 3.697* G3Y497_ASPNA Y CE16 Uncharacterized protein (acetylesterase) 3.633* 1.488* 3.647* G3Y478_ASPNA Y PL1 PlyA Pectate lyase Pectin 2.252* − 0.217 2.558* A5ABH4_ASPNC Y PL4 RglB Rhamnogalacturonate lyase B Pectin 0.757 3.788* 5.843* Q8WZI8_ASPNG Y – FaeB Feruloyl esterase B Xylan, pectin 4.896* 3.841 5.270* A0A117E071_ASPNG Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 2.755* 2.602* 4.512* G3XY89_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 1.634 1.533 2.978* G3XPC9_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 4.962* 3.118 5.772* G3YHB3_ASPNA Y AA9 Lytic polysaccharide monooxygenase Cellulose 2.544 0.578 6.559 A2QZE1_ASPNC Y AA9 Lytic polysaccharide monooxygenase Cellulose 1.558* 1.367* 1.989* Accession number . Signal peptide . CAZy family . Protein name . Description . Predicted substrate . log2 (fold-change) . PS . PL . WPS . A2QVN9_ASPNC Y GH1 Putative β-glucosidase Cellulose, xyloglucan 4.133 3.639 4.955 G3Y786_ASPNA N GH1 β-Glucosidase Cellulose, xyloglucan 1.324 − 9.620 2.559 A0A100IJJ3_ASPNG N GH3 β-Glucosidase Cellulose, xyloglucan − 0.212 1.818 1.207 C7C4Z9_ASPNG Y GH3 Bgl β-Glucosidase Cellulose, xyloglucan 0.610* 0.959* 2.126* A0A1V1FQ75_ASPNG Y GH3 XlnD β-Xylosidase Xylan 2.722* 3.248* 3.069* A0A100IKN9_ASPNG Y GH3 α-Galactosidase Galactomannan 3.913* 1.820 3.919* A0A1V1G264_ASPNG Y GH3 XlsV Putative β-xylosidase Xylan 1.076 0.252 1.653* G3Y4E8_ASPNA N GH3 Bgl1 β-Glucosidase Cellulose, xyloglucan 3.022* 1.117 2.383 A2R2S3_ASPNC Y GH3 Putative β-glucosidase Cellulose, xyloglucan − 2.806* − 4.150* − 3.369* G3YBE0_ASPNA Y GH3 β-Glucosidase Cellulose, xyloglucan − 1.125 − 0.014 0.708 A0A023UH08_ASPNG Y GH5 Eg1 Endo-β-1,4-glucanase Cellulose 11.226* 6.836 9.979* H2E6Y8_ASPNG Y GH5 ManA Endo-β-1,4-mannanase Mannan 1.644* 4.951 1.536 A2QAI8_ASPNC Y GH5, CBM1 Endo-β-1,4-glucanase Cellulose 7.143* 3.467* 5.969* G3Y873_ASPNA Y GH5 Putative endo-β-1,6-galactanase Pectin − 1.886 9.460 − 0.792 G3XZI3_ASPNA Y GH5 EglB Endo-β-1,4-glucanase Cellulose 5.098* 1.926 4.134* A2QQ99_ASPNC Y GH6 Cellobiohydrolase Cellulose 6.098* 2.849* 4.249* A2QYR9_ASPNC Y GH6, CBM1 CbhC Cellobiohydrolase Cellulose 2.932* 2.493* 2.728* A0A100IHS6_ASPNG Y GH6 Glucanase Cellulose 2.959* 3.775 1.072 Q9UVS9_ASPNG Y GH7 CbhA Cellobiohydrolase Cellulose 4.299* 3.076* 2.858* A2QAI7_ASPNC Y GH7, CBM1 CbhB Cellobiohydrolase Cellulose 3.904* 2.473* 2.690* G3Y866_ASPNA Y GH10 XynC Endo-β-1,4-xylanase Xylan 3.813* 2.639* 3.763* Q6QA21_9EURO Y GH11, CBM60 XynB Endo-β-1,4-xylanase Xylan 3.303* 2.852* 3.653* U6C3R6_ASPNG Y GH11 XynV Endo-β-1,4-xylanase Xylan 0.966 3.852 2.325* E3UN71_ASPNG Y GH11, CBM60 Endo-β-1,4-xylanase Xylan 1.387* 2.016* 2.007* O74705_ASPNG Y GH12 EglA Endo-β-1,4-glucanase Cellulose 4.710* 2.624 4.016* G3XRM3_ASPNA Y GH12 XgeA Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 1.025 2.897* 2.455* G5D7B5_ASPNG N GH27, CBM13 AglA α-Galactosidase Galactomannan 2.134* − 0.166 2.364* A0A0U5AE32_ASPNG Y GH27 AglB α-Galactosidase Galactomannan 1.370 − 1.150 2.716* G3XQY4_ASPNA Y GH28 Pgal Endo-polygalacturonase I Pectin 5.988 − 0.645 6.924* A2QK83_ASPNC Y GH28 XghA Putative endo-xylogalacturonan hydrolase Pectin 3.906* − 0.945 2.448* A2QTU5_ASPNC Y GH31 AxlA α-Xylosidase A Xyloglucan 1.474* 4.071* 3.671* B6HYI9_ASPNG Y GH35, CBM37 LacZ β-Galactosidase Pectin, xyloglucan 0.519 2.056 2.494* A0A1D8MQA0_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 1.114 2.328* 3.323* A0A1D8MQF6_ASPNG Y GH35, CBM67 LacB β-Galactosidase Pectin, xyloglucan 2.897* 2.599 4.262* G3XM01_ASPNA Y GH36 AglC α-Galactosidase Galactomannan 0.819 1.434 2.101* A0A0S2CVZ9_ASPNG Y GH43 Xylosidase:arabinofuranosidase Xylan 4.076* 2.516* 4.161* G3XY38_ASPNA Y GH43 Uncharacterized protein Xylan − 1.920* 1.176 0.674 U6C191_ASPNG N GH43 Xilanase putative Xylan − 2.256 1.530 0.059 A2R511_ASPNC Y GH54, CBM42 AbfB α-l-Arabinofuranosidase Xylan, pectin 3.099* 2.750* 4.872* A0A100I6G0_ASPNG Y GH62 α-l-Arabinofuranosidase Xylan, pectin 2.762 2.001 2.524* A2QFV9_ASPNC Y GH62 AxhA α-l-Arabinofuranosidase Xylan, pectin 2.893* 1.861* 2.865* Q96WX9_ASPNG Y GH67 AguA α-Glucuronidase Xylan 2.522* 2.699* 3.253* Q8TFP1_ASPNG Y GH74, CBM1 EglC Xyloglucan-specific endo-1,4-β-glucanase Xyloglucan 5.085* 1.040* 3.387* G3XSS5_ASPNA Y GH78 α-l-Rhamnosidase Pectin − 3.347* 0.197 0.259 G3XVM1_ASPNA Y CE1 AceA Acetyl xylan esterase A Xylan 4.921* 3.329 4.718* G3Y471_ASPNA Y CE3 Acetyl xylan esterase Xylan 5.611* 1.631 6.521 A2QSY5_ASPNC Y CE5 FeaA Probable feruloyl esterase A Xylan, pectin 4.697* 4.201* 5.153* G3YAH8_ASPNA Y CE12 RgaeB Rhamnogalacturonan acetyl esterase Pectin 3.367* 3.874 5.002* G3XZI2_ASPNA N CE16 Uncharacterized protein (acetylesterase) 3.457* 4.059* 4.041* A2QPC2_ASPNC N CE16 PaeB Putative pectin acetyl esterase Pectin 4.150* 2.663* 3.697* G3Y497_ASPNA Y CE16 Uncharacterized protein (acetylesterase) 3.633* 1.488* 3.647* G3Y478_ASPNA Y PL1 PlyA Pectate lyase Pectin 2.252* − 0.217 2.558* A5ABH4_ASPNC Y PL4 RglB Rhamnogalacturonate lyase B Pectin 0.757 3.788* 5.843* Q8WZI8_ASPNG Y – FaeB Feruloyl esterase B Xylan, pectin 4.896* 3.841 5.270* A0A117E071_ASPNG Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 2.755* 2.602* 4.512* G3XY89_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 1.634 1.533 2.978* G3XPC9_ASPNA Y AA9, CBM1 Lytic polysaccharide monooxygenase Cellulose 4.962* 3.118 5.772* G3YHB3_ASPNA Y AA9 Lytic polysaccharide monooxygenase Cellulose 2.544 0.578 6.559 A2QZE1_ASPNC Y AA9 Lytic polysaccharide monooxygenase Cellulose 1.558* 1.367* 1.989* Asterisks (*) show significantly different abundances (Student’s t test, p < 0.05) relative to the control condition (rSCB) Open in new tab Fifteen xylan-degrading enzymes with secretion signal were also detected, including endo-β-1,4-xylanases (GH10 and GH11), β-xylosidases (GH3), α-l-arabinofuranosidases (GH54 and GH62), one bifunctional xylosidase:arabinofuranosidase (GH43), one α-glucuronidase (GH67), acetyl xylan esterases (CE1 and CE3) and feruloyl esterases (CE5) (Table 2). The high number of xylanolytic enzymes reflects the chemical complexity of the acetylated glucuronoarabinoxylan found in sugarcane bagasse [8]. Xyloglucan-specific endoglucanases (GH74 and GH12) and xyloglucan-debranching enzymes (α-xylosidase, GH31) were also secreted, a consequence of xyloglucan being an important component in sugarcane cell walls [9]. Despite the low galacto(gluco)mannan content in sugarcane, one endo-1,4-β-mannanase (GH5) and α-galactosidases (GH 27, GH36 and GH3) were detected in all growth conditions. Like celulases, most genes encoding for xylan-, xyloglucan- and mannan-degrading enzymes in A. niger are induced by the pentose-responsive XlnR and AraR transcription factors, confirming sugarcane bagasse and its pretreated fractions (having high oligomeric and monomeric pentose contents) as potential substrates for the induction of a wide variety of holocellulases. Several pectin-degrading enzymes with signal peptide were present in all secretomes, including endo-polygalacturonase (GH28), β-galactosidase (GH35), α-l-rhamnosidase (GH78), pectate lyase (PL1), rhamnogalacturonate lyase (PL4), rhamnogalaturonan acetyl esterase (CE12), endo-β-1,6-galactanase and endo-xylogalacturonan hydrolase (Table 2). Moreover, the α-l-arabinofuranosidases and feruloyl esterases mentioned above as hemicellulases also display activity toward pectin, i.e. they show relaxed specificity, and likely act synergistically with other pectinases on the degradation of rhamnogalacturonan I, the main pectic component in sugarcane cell walls [9]. Despite the low starch content in sugarcane bagasse, starch-degrading enzymes (α-amylase GH13, glucoamylase GH15 and α-glucosidases GH31) were also secreted in the presence of rSCB and pretreated fractions (Online Resource 1). These enzymes are possibly induced by glucose released from biomass via the glucose-responsive AmyR regulator [13]. CAZymes involved in the organization and remodeling of fungal cell wall polysaccharides were also produced, including chitin-degrading (GH18 and GH20) and β-1,3-glucan-active enzymes (GH16, GH17 and GH72). It is possible that some of the detected β-1,3-glucanases are active towards lichenan present in sugarcane cell walls. The enzyme A2Q8J5 (GH16), in particular, is predicted to have both β-1,3- and β-1,4-glucanase activities, being possibly described as lichenase. Catalase was also secreted in all growth conditions. Frequently produced by filamentous fungi during growth on lignocellulose [3], it has been reported that catalases can increase the efficiency of biomass conversion by protecting enzymes from inactivation by free radicals [19, 22], such as those released during biomass pretreatment or indirectly generated by H2O2-producing enzymes. The up-regulation of catalase in PL and WPS growth conditions (Online resource 1) supports that catalase expression might have been induced by soluble free radicals generated during biomass pretreatment. Proteases were also detected in all growth conditions and, although assumed as necessary, their role in lignocellulose degradation is still unclear. Relative quantification of holocellulases Among the 52 holocellulases with signal peptide, 23, 34 and 42 enzymes were significantly up-regulated in the presence of PL, PS and WPS, respectively, while only 3 or less were significantly down-regulated compared to the use of rSCB as the carbon source (t test, p < 0.05) (Table 2, Online Resource 2). PS, being enriched in cellulose, induced a pronounced up-regulation of cellulose-degrading enzymes (Table 2). Indeed, through PCA analysis, it could be clearly visualized that the PS group was characterized by a pronounced presence of cellobiohydrolases and endoglucanases, as seen in the upper-left regions of Fig. 3a, b. Among the top-10 up-regulated holocellulases in PS condition, four endoglucanases (especially the endoglucanase Eg1, GH5), one cellobiohydrolase and one LPMO were included (Fig. 3d). Despite the strong modulation toward cellulose degradation, the majority of the pectin- and hemicellulose-degrading enzymes were also significantly up-regulated in PS condition, which can be attributed to the partial retention of hemicellulose and pectin in PS in a more accessible form after mild pretreatment of rSCB. The overall up-regulation of holocellulases (and not only cellulases) in PS growth condition observed here is not usually observed, however, when severely pretreated biomasses (having very low hemicellulose and pectin contents) are employed as carbon sources. In the works of Daly et al. [7] and Borin et al. [2], for example, even though cellulases were highly up-regulated, the expression of pectinolytic enzymes by A. niger was drastically reduced when wheat straw or sugarcane bagasse subjected to high severity hydrothermal pretreatments were offered as the carbon sources in place of untreated biomasses. Fig. 3 Open in new tabDownload slide Principal component analysis (PCA) of proteomic data obtained from Aspergillus niger secretomes induced by raw sugarcane bagasse (rSCB), pretreated solids (PS), pretreatment liquor (PL) and whole pretreated slurry (WPS). Scores and loadings plots are shown in a and b, respectively. Holocellulase regulation number and top-10 up-regulated holocellulases induced by pretreated substrates in comparison to rSCB are shown in c–e The WPS-induced secretomes, on the other hand, had a more balanced up-regulation of cellulases, hemicellulases and pectinases. Six pectin-degrading enzymes (Pgal, RglB, RgaeB, AbfB, FaeA and FaeB), four xylan-degrading enzymes (AceA, AbfB, FaeA and FaeB) and three cellulose-degrading enzymes (two endoglucanases and one LPMO) were among the top-10 up-regulated holocellulases in WPS condition (Fig. 3c). In PCA plots, the WPS cluster was discriminated from the other groups by a prominent presence of hemicellulases (e.g. xylanases, α-l-arabinofuranosidases, β-xylosidase and acetyl-xylan esterase), as seen in the lower-left regions of Fig. 3a, b. Although endoglucanases and cellobiohydrolases were highly induced by pretreated solids (PS condition), the presence of pretreatment liquor in WPS condition reduced the cellulase-inducing ability of the solid fraction. These enzymes followed a repeated pattern of abundance, i.e. PS > WPS > PL > rSCB (see Online Resource 2), and this was possibly caused by soluble pretreatment-derived inhibitors (such as phenolic compounds, furans and organic compounds) or monosaccharides (via the carbon-catabolite repression mechanism) found in liquor. CAZyme up-regulation in the PL condition was lower. Most holocellulases in the PL-induced secretome showed statistically the same abundance levels as observed in the rSCB condition and those that were up-regulated had a lower fold-change than observed in PS or WPS growth conditions. In contrast to PS and WPS groups, the PL group was not characterized by the conspicuous presence of any particular enzyme group in PCA plots (Fig. 3a, b). The PL-induced secretome was slightly shifted toward hemicellulose degradation, which was expected in view of liquor being mainly composed of hemicellulose-derived sugars. The top-10 up-regulated holocellulases included six hemicellulose-degrading enzymes (FeaA, AxlA, XlnD, XgeA, XynB, AbfB), three pectin-degrading enzymes (RglB, FaeA and AbfB) and three cellulose-degrading enzymes (two cellobiohydrolases and one endoglucanase) (Fig. 3e). Lastly, the rSCB-induced secretome cluster in PCA analysis was characterized by the pronounced presence of enzymes that were down-regulated when pretreated substrates was offered as carbon sources, including two β-glucosidases (A2R2S3 and G3YBE0) and one α-l-rhamnosidase (G3XSS5), as seen in the right regions of PCA plots (Fig. 3a, b). It is not surprising that enzyme expression is modulated according to the given substrate. Changes in holocellulase expression in response to different carbon sources have been demonstrated through transcriptomic and proteomic analyses in several fungi, including A. niger. Much of the published work, however, compares simple sugars (such as glucose, xylose and arabinose), isolated polysaccharides (such as Avicel and xylan) or different lignocellulosic biomasses (such as sugarcane bagasse, wheat straw and soybean hulls) as carbon sources [12, 16, 28]. The novelty of this work lies in the evaluation of the enzyme-inducing abilities associated with different fractions arising from hydrothermal pretreatment of biomass. The effect of biomass pretreatment on enzyme induction is not a frequently studied topic, especially regarding the use of pretreatment liquor. Each fraction from pretreated biomass induces a particular enzyme arsenal, which may have different applications. The PS- and WPS-induced secretomes, for having higher levels of cellulases, present greater potential for saccharification of biomass. The hemicellulase-rich secretome induced by PL, on the other hand, may be suited for other applications such as pulp biobleaching, textile, baking and wine industries, for example. Saccharification of pretreated sugarcane bagasse by A. niger secretomes Higher biomass saccharification yields were achieved by A. niger secretomes produced in the presence of pretreated carbon sources (WPS, PS and PL) in comparison to the secretome produced using raw biomass (rSCB) (Fig. 4). The PS- and WPS-induced secretomes achieved the highest holocellulose-to-reducing sugars conversion yields (4.6- and 4.8-fold increase, respectively, compared to the rSCB-induced secretome). The PS-induced secretome achieved the highest glucan-to-glucose conversion (10.1-fold increase), which is likely a result of the strong modulation of this secretome towards cellulose hydrolysis, as noted earlier. Regarding the PL-induced secretome, although it had a lower saccharification performance compared to the WPS- and PS-induced enzymes, it also represented a substantial increase in the release of total reducing sugars and glucose (4.4- and 8.5-fold, respectively) over the rSCB-induced secretome, reaffirming the potential of this residual stream as a carbon source for the production of enzymes. The glucan conversion of pretreated bagasse (0.081 g of released glucose, or 1.54 mg mL−1) did not exceed 30% of the theoretical maximum (which in this case would correspond to 0.271 g of released glucose, or 5.14 mg mL−1), and this is far below the > 90% conversion pursued for the viability of industrial biorefining. This can be attributed to the retention of hemicellulose and pectin in mildly pretreated solids, resulting in incomplete exposure of cellulose to enzymes. Higher conversion yields would likely be achieved if more severe hydrothermal pretreatment were applied to biomass and higher enzyme loadings were employed. Fig. 4 Open in new tabDownload slide Saccharification of hydrothermally pretreated sugarcane bagasse by Aspergillus niger secretomes produced in the presence of raw sugarcane bagasse (rSCB), whole pretreated slurry (WPS), pretreated solids (PS) and pretreatment liquor (PL) as carbon sources. a The release of total reducing sugars and the holocellulose conversion yields. b The release of glucose and the glucan conversion yields Conclusions Mild hydrothermal pretreatment of sugarcane bagasse is proposed here as a useful tool to enhance the integrated production of holocellulases by A. niger in biorefineries. Increases in enzyme activity titers, biomass saccharification yields and overall abundance of holocellulases in A. niger secretomes were achieved when the solid and liquid fractions arising from mild pretreatment were used as a carbon source in place of untreated sugarcane bagasse. Mild biomass hydrothermal pretreatment offered the possibility of using pretreatment liquor (PL), either alone or in the form of whole pretreated slurry (WPS), as a carbon source for enzyme production without the need for detoxification or dilution steps. In contrast to high severity pretreatments that generate liquors which are toxic for microorganisms, mild pretreatment generates low concentrations of microbial inhibitors, preventing inhibition of enzyme production [14]. Furthermore, the utilization of liquor contributes to the full exploitation of lignocellulose components. It is noteworthy that mild hydrothermal pretreatment of biomass is not compatible with maximum cellulose saccharification yields of pretreated solids, which requires harsh pretreatment conditions to expose cellulose fibers. For integrated enzyme production in biorefineries, we propose that a fraction of the incoming biomass feedstock is treated at mild conditions before being offered as the enzyme-inducing substrate for the enzyme-producing microorganism, while the remaining is severely treated aiming efficient biomass-to-monosaccharides conversion by on-site produced enzymes. Acknowledgements This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel (CAPES) and the Foundation for Research Support of the Federal District (FAPDF, Grant PRONEX 0193.001195/2016). Funding was also provided by the Funding Authority for Studies and Projects (FINEP/CT-INFRA, Grants 0439/2011 and 0694/2013) to MVS. References 1. Bigelow M , Wyman CE Finkelstein M, McMillan JD, Davison BH Cellulase production on bagasse pretreated with hot water Biotechnology for fuels and chemicals 2002 Totowa, NJ Humana Press Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 2. Borin GP , Sanchez CC, de Souza AP, de Santana ES, de Souza AT, Leme AFP, Squina FM, Buckeridge M, Goldman GH, de Castro Oliveira JV Comparative secretome analysis of Trichoderma reesei and Aspergillus niger during growth on sugarcane biomass PLoS One 2015 10 e0129275 10.1371/journal.pone.0129275 Google Scholar Crossref Search ADS PubMed WorldCat 3. Borin GP , Sanchez CC, Santana ES, Zanini GK, Santos RAC, Pontes AO, Souza AT, Dal RMMTS, Riaño-Pachón DM, Goldman GH Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei BMC Genom 2017 18 501 10.1186/s12864-017-3857-5 Google Scholar Crossref Search ADS WorldCat 4. Cai P , Gu R, Wang B, Li J, Wan L, Tian C, Ma Y Evidence of a critical role for cellodextrin transporte 2 (CDT-2) in both cellulose and hemicellulose degradation and utilization in Neurospora crassa PLoS One 2014 9 e89330 10.1371/journal.pone.0089330 Google Scholar Crossref Search ADS PubMed WorldCat 5. Chong J , Soufan O, Li C, Caraus I, Li S, Bourque G, Wishart DS, Xia J MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis Nucleic Acids Res 2018 46 W486 W494 10.1093/nar/gky310 Google Scholar Crossref Search ADS PubMed WorldCat 6. Crowell AM , Wall MJ, Doucette AA Maximizing recovery of water-soluble proteins through acetone precipitation Anal Chim Acta 2013 796 48 54 10.1016/j.aca.2013.08.005 Google Scholar Crossref Search ADS PubMed WorldCat 7. Daly P , van Munster JM, Blythe MJ, Ibbett R, Kokolski M, Gaddipati S, Lindquist E, Singan VR, Barry KW, Lipzen A, Ngan CY, Petzold CJ, Chan LJG, Pullan ST, Delmas S, Waldron PR, Grigoriev IV, Tucker GA, Simmons BA, Archer DB Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments Biotechnol Biofuels 2017 10 35 10.1186/s13068-017-0700-9 5294722 Google Scholar Crossref Search ADS PubMed WorldCat 8. de Carvalho DM , Martínez-Abad A, Evtuguin DV, Colodette JL, Lindström ME, Vilaplana F, Sevastyanova O Isolation and characterization of acetylated glucuronoarabinoxylan from sugarcane bagasse and straw Carbohydr Polym 2017 156 223 234 10.1016/j.carbpol.2016.09.022 Google Scholar Crossref Search ADS PubMed WorldCat 9. de Souza AP , Leite DC, Pattathil S, Hahn MG, Buckeridge MS Composition and structure of sugarcane cell wall polysaccharides: implications for second-generation bioethanol production BioEnergy Res 2013 6 564 579 10.1007/s12155-012-9268-1 Google Scholar Crossref Search ADS WorldCat 10. de Souza WR , Maitan-Alfenas GP, de Gouvêa PF, Brown NA, Savoldi M, Battaglia E, Goldman MHS, de Vries RP, Goldman GH The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in d-xylose, l-arabinose and steam-exploded sugarcane bagasse Fungal Genet Biol 2013 60 29 45 10.1016/j.fgb.2013.07.007 Google Scholar Crossref Search ADS PubMed WorldCat 11. Gomes HAR , da Silva AJ, Gómez-Mendoza DP, dos Santos Júnior ACM, di Cologna NDM, Almeida RM, Miller RNG, Fontes W, de Sousa MV, Ricart CAO, Filho EXF Identification of multienzymatic complexes in the Clonostachys byssicola secretomes produced in response to different lignocellulosic carbon sources J Biotechnol 2017 254 51 58 10.1016/j.jbiotec.2017.06.001 Google Scholar Crossref Search ADS PubMed WorldCat 12. Gong W , Dai L, Zhang H, Zhang L, Wang L A highly efficient xylan-utilization system in Aspergillus niger An76: a functional-proteomics study Front Microbiol 2018 9 430 10.3389/fmicb.2018.00430 Google Scholar Crossref Search ADS PubMed WorldCat 13. Gruben BS , Mäkelä MR, Kowalczyk JE, Zhou M, Benoit-Gelber I, De Vries RP Expression-based clustering of CAZyme-encoding genes of Aspergillus niger BMC Genom 2017 18 900 10.1186/s12864-017-4164-x Google Scholar Crossref Search ADS WorldCat 14. Johnson E Integrated enzyme production lowers the cost of cellulosic ethanol Biofuels Bioprod Biorefin 2016 10 164 174 10.1002/bbb.1634 Google Scholar Crossref Search ADS WorldCat 15. Li X , Lu J, Zhao J, Qu Y Characteristics of corn stover pretreated with liquid hot water and fed-batch semi-simultaneous saccharification and fermentation for bioethanol production PLoS One 2014 9 e95455 10.1371/journal.pone.0095455 Google Scholar Crossref Search ADS PubMed WorldCat 16. Liu L , Gong W, Sun X, Chen G, Wang L Extracellular enzyme composition and functional characteristics of Aspergillus niger An-76 induced by food processing byproducts and based on integrated functional omics J Agric Food Chem 2018 66 1285 1295 10.1021/acs.jafc.7b05164 Google Scholar Crossref Search ADS PubMed WorldCat 17. Miller G Modified DNS method for reducing sugars Anal Chem 1959 31 426 428 10.1021/ac60147a030 Google Scholar Crossref Search ADS WorldCat 18. Nielsen H Kihara D Predicting secretory proteins with SignalP Protein function prediction 2017 New York, NY Humana Press Google Scholar Crossref Search ADS Google Preview WorldCat COPAC 19. Peciulyte A , Samuelsson L, Olsson L, McFarland K, Frickmann J, Østergård L, Halvorsen R, Scott BR, Johansen KS Redox processes acidify and decarboxylate steam-pretreated lignocellulosic biomass and are modulated by LPMO and catalase Biotechnol Biofuels 2018 11 165 10.1186/s13068-018-1159-z Google Scholar Crossref Search ADS PubMed WorldCat 20. Rappsilber J , Mann M, Ishihama Y Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips Nat Protoc 2007 2 1896 10.1038/nprot.2007.261 Google Scholar Crossref Search ADS PubMed WorldCat 21. Rocha GJ , Silva VF, Martín C, Gonçalves AR, Nascimento VM, Souto-Maior AM Effect of xylan and lignin removal by hydrothermal pretreatment on enzymatic conversion of sugarcane bagasse cellulose for second generation ethanol production Sugar Tech 2013 15 390 398 10.1007/s12355-013-0218-9 Google Scholar Crossref Search ADS WorldCat 22. Scott BR , Huang HZ, Frickman J, Halvorsen R, Johansen KS Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation Biotech Lett 2016 38 425 434 10.1007/s10529-015-1989-8 Google Scholar Crossref Search ADS WorldCat 23. Silva CdOG , de Aquino Ribeiro JA, Souto AL, Abdelnur PV, Batista LR, Rodrigues KA, Parachin NS, Ferreira Filho EX Sugarcane bagasse hydrothermal pretreatment liquors as suitable carbon sources for hemicellulase production by Aspergillus niger BioEnergy Res 2018 11 316 10.1007/s12155-018-9898-z Google Scholar Crossref Search ADS WorldCat 24. Silva CdOG , Ferreira Filho EX A review of holocellulase production using pretreated lignocellulosic substrates BioEnergy Res 2017 10 592 602 10.1007/s12155-017-9815-x Google Scholar Crossref Search ADS WorldCat 25. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D (2006) Determination of sugars, byproducts, and degradation products in liquid fraction process samples. NREL Laboratory Analytical Procedure NREL/TP-510-42623. National Renewable Energy Laboratory, Golden, CO 26. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2005) Determination of extractives in biomass. NREL Laboratory Analytical Procedure NREL/TP-510-42619. National Renewable Energy Laboratory, Golden, CO 27. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. NREL Laboratory Analytical Procedure NREL/TP-510-42618. National Renewable Energy Laboratory, Golden, CO 28. Souza WR , Maitan-Alfenas GP, Gouvêa PF, Brown NA, Savoldi M, Battaglia E The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in d-xylose, l-arabinose and steam-exploded sugarcane bagasse Fungal Genet Biol 2013 10.1016/j.fgb.2013.07.007 Google Scholar OpenURL Placeholder Text WorldCat Crossref 29. Yang B , Tao L, Wyman CE Strengths, challenges, and opportunities for hydrothermal pretreatment in lignocellulosic biorefineries Biofuels Bioprod Biorefin 2017 12 1 125 138 10.1002/bbb.1825 Google Scholar Crossref Search ADS WorldCat 30. Yin Y , Mao X, Yang J, Chen X, Mao F, Xu Y dbCAN: a web resource for automated carbohydrate-active enzyme annotation Nucleic Acids Res 2012 40 W445 W451 10.1093/nar/gks479 Google Scholar Crossref Search ADS PubMed WorldCat 31. Znameroski EA , Li X, Tsai JC, Galazka JM, Glass NL, Cate JH Evidence for transceptor function of cellodextrin transporters in Neurospora crassa J Biol Chem 2014 289 2610 2619 10.1074/jbc.M113.533273 Google Scholar Crossref Search ADS PubMed WorldCat Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. © Society for Industrial Microbiology 2019 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © Society for Industrial Microbiology 2019
TI - Mild hydrothermal pretreatment of sugarcane bagasse enhances the production of holocellulases by Aspergillus niger
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-019-02207-0
DA - 2019-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mild-hydrothermal-pretreatment-of-sugarcane-bagasse-enhances-the-fxqPxpbE09
SP - 1517
EP - 1529
VL - 46
IS - 11
DP - DeepDyve
ER -