C. fimi B-402 was obtained from NRRL Agricultural Research Service Culture Collection (IL, USA). Cellulomonas sp. B6 was isolated from a bacterial consortium obtained from a preserved native subtropical forest soil sample (Piccinni et al. 2016). Strain Cellulomonas sp. B6 has been deposited in public collections DSMZ and NCIMB, as DSM 107934 and NCIMB 15124, respectively. The strains were maintained for long term in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) (Bertani 1951) with 20% w/w glycerol at – 80 °C. Before use, a loopfull was spread in LB agar plates (LB supplemented by 15 g/L agar) for single colonies isolation.
Model substrates and biomass feedstocks
Different carbon sources were tested for cell growth and enzyme secretion. Solka-floc (SF, kindly donated by Lund University, Lund, Sweden) and carboxymethyl cellulose of low viscosity (CMC, Sigma-Aldrich, STL, USA) were used as model substrates. The selection of CMC was due to its high purity and solubility in aqueous solutions. SF is a model substrate obtained from milled pinewood through several extraction steps and it has around 76% w/w cellulose and 12% w/w hemicellulose, in terms of dry matter content, and which is insoluble in water (Sipos et al. 2010). Wheat bran (WB), pre-treated sweet corn cob (PSCC) and pre-treated waste paper (PWP) were used as lignocellulosic carbon sources. Sweet corn cob pre-treated by alkali extrusion was provided by INRA (Toulouse, France). Waste paper consisted of corrugated cardboard pieces, which were cut into small pieces (smaller than 0.5 cm), mixed with distilled water to set 10% w/w dry matter content, homogenised with a hand blender for 10 min and autoclaved (121 °C for 30 min). Wheat bran was purchased at a dietary shop.
For enzymatic hydrolysis experiments, extruded barley straw (EBS) was used. EBS was obtained from the Babetreal5 project (https://www.babet-real5.eu/) and provided by CIEMAT (Madrid, Spain). The extrusion conditions were 100 °C, 4.5% NaOH/dry barley straw and neutralisation with H3PO4 (Duque et al. 2017).
Structural carbohydrates (glucan, xylan and arabinan) and the acid insoluble solids of WB and PWP were determined as described by the National Renewable Energy Laboratory (DEN, USA) (Sluiter et al. 2012). Relative composition of PSCC was provided by INRA. The structural carbohydrate analyses of WB and PWP were carried out in triplicates. Starch content of WB was also determined by using enzymatic treatment with α-amylase as detailed by Bedő et al. (2019). Composition of PSCC, WB and PWP is detailed in Table 1.
Enzyme production in shake flask
Bacteria (C. fimi and Cellulomonas sp. B6) were grown on minimal medium (MM) (1.67 g/L dipotassium hydrogen phosphate (K2HPO4), 0.87 g/L potassium dihydrogen phosphate (KH2PO4), 0.05 g/L sodium chloride (NaCl), 0.1 g/L magnesium sulphate (MgSO4×7H2O), 0.04 g/L calcium chloride (CaCl2), 0.004 g/L iron(III) chloride (FeCl3), 0.005 g/L sodium molybdate (NaMoO4×2H2O), 0.01 g/L biotin, 0.02 g/L nicotinic acid, 0.01 g/L pantothenic acid, 1 g/L ammonium chloride (NH4Cl)) supplemented with 1 g/L yeast extract and 1% w/w of different model substrates (SF, CMC) or 1% w/w of different types of biomass as carbon source (WB, PWP or PSCC as indicated). The MM culture media (without microelements) with the substrates was sterilised in autoclave at 121 °C for 20 min, after which microelements (FeCl3, NaMoO4×2H2O, biotin, nicotinic acid and pantothenic acid) were added to the indicated concentration. Starter cultures were obtained by inoculating single colonies (from fresh agar plates) in 10 mL LB medium and incubating at 30 °C and 220 rpm for 24 h, in the case of Cellulomonas sp. B6, and for 72 h, in the case of C. fimi. Cultures were inoculated from the starter cultures to obtain an initial cell concentration that corresponds to an optical density (OD) of 0.05. Cultures were carried out in 20 mL culture media filled in 100-mL shake flasks, at 30 °C and 220 rpm for 72 h. Cultures were performed in triplicates.
Enzyme production in bench-top bioreactor
Enzyme production of C. fimi and Cellulomonas sp. B6 was performed in 500-mL bench-top bioreactors (JFermi Ltd., Jenő, Hungary) filled with 300 mL of MM medium supplemented with 1% w/w PWP and WB, respectively. Bacteria were first grown as starter cultures in LB medium and then inoculated into the MM supplemented with biomass to get an initial cell concentration that corresponds to an OD of 0.05. Enzyme productions, in duplicates, were accomplished at 30 °C for 72 h with daily sampling. The bioreactors were equipped to be able to control dissolved oxygen (DO) level by adjusting the agitation and air flow rate, and to monitor the pH in the culture medium. DO level was kept at 20% of saturation in order to ensure that the culture was maintained in aerobic conditions.
Production of extracellular enzyme fraction and intracellular enzyme fraction
When the culture of enzyme production reached late exponential phase (72 h), the remaining solid biomass and the obtained cell mass were separated from the supernatant by centrifugation (6000×g, 10 min). The supernatants are referred to as extracellular enzyme (EE) fractions. EE fractions were supplemented with 0.04% w/w sodium azide and kept at 4 °C until use. The cells and remaining biomass (centrifugation pellet) were resuspended in citrate buffer (100 mM, pH 6) in a ratio of 1:10 regarding the initial culture volume, ultrasonicated on ice (six pulses of 10 s, 28% amplitude) and centrifuged (10000×g, 30 min). After centrifugation, filtered supernatants were used as intracellular enzyme (IE) fractions.
Lyophilisation of EE and IE fractions
EE and IE fractions were frozen at – 80 °C. Then, they were taken into the freeze-dryer (EDWARDS Super Modulyo Freeze Dryer, Thermo Electron Corp., Waltham, MA, USA), treated at – 30 °C under 1 × 10−1 mbar vacuum for 48 h and stored at 4 °C. The lyophilised enzyme fractions were referred to as LEE and LIE in the cases of extracellular and intracellular enzyme fractions, respectively. For enzymatic assays, both fractions were resuspended with sterile water to the original volume. Ten times concentrated enzyme fraction of LEE (LEE (10×)) was also produced by dissolving LEE in appropriate amount of sterile water. Recovery of the xylanase activity was calculated as the ratio between xylanase activity after and before lyophilisation and expressed as a percentage. Lyophilisation experiments were performed in triplicates.
Enzymatic activity measurements
Xylanase and CMCase activities were assayed in microtubes using beechwood xylan (1% w/w) or CMC (2% w/w) as substrates, respectively (Ghio et al. 2012). For these assays, 0.1 mL of appropriately diluted extracts (EE, IC, LEE, LEE (10×) or LIE) were added to 0.1 mL of each substrate prepared in citrate buffer (pH 6). Hydrolysis reactions were carried out at 40 °C, 400 rpm for 10 min. These conditions were established in a previous work (Piccinni et al. 2019). Reducing sugars released from the reactions were measured by dinitrosalicylic acid (DNS) method (Miller 1959) using glucose or xylose standard curves.
β-Glucosidase, cellobiohydrolase, β-xylosidase and α-l-arabinofuranosidase activities were assayed, using 5 mM p-nitrophenyl-β-d-glucopyranoside (pNPG), p-nitrophenyl-β-d-cellobioside (pNPC), p-nitrophenyl-β-d-xylopyranoside (pNPX) and p-nitrophenyl-α-l-arabinofuranoside (pNPA) (Sigma-Aldrich, STL, USA) as substrates, according to previously established protocols (Ontañon et al. 2018). In brief, reactions were performed by combining 100 μL of LEE (10×) or LIE fraction with 100 μL of 2.5 mM substrate in citrate buffer (pH 6, 100 mM), and incubating at 40 °C for 20 min. Reactions were stopped with 500 μL of 2% w/w sodium carbonate and absorbance was determined at 410 nm. A p-nitrophenol (pNP) curve was used as a standard.
All enzymatic assays were conducted in triplicates and controls of enzyme without substrate and substrate without enzyme were included. In all cases, one international unit (U) was defined as the amount of enzyme required to release 1 μmol of product per minute under the assay conditions.
Analysis of secretome
Secretomic analysis was performed using flasks cultures of Cellulomonas sp. B6 in WB and C. fimi in PWP, in duplicate according to the protocol previously described by Piccinni et al. (2019). Briefly, total proteins contained in cell-free supernatants were quantified by Bradford assay (Promega, Biodynamics, CABA, Argentina), precipitated with 10% w/w trichloroacetic acid and then resuspended in ultrapure water (resistivity of 18.2 MΩ × cm) to a final concentration of 1 mg/mL. Protein digestion and mass spectrometry analysis were performed at CEQUIBIEM (http://cequibiem.qb.fcen.uba.ar/). Proteins were reduced with dithiotreitol 10 mmol/L for 45 min at 56 °C, alkylated with iodoacetamide (55 mmol/L) for 45 min in the dark and digested with trypsin (PromegaV5111; Promega, Fitchburg, WI, USA) overnight at 37 °C. The digests were analysed by nano LC-MS/MS in a Thermo Scientific Q-Exactive Mass Spectrometer coupled with a nano HPLC EASY-nLC 1000 (ThermoFisher Scientific, Waltham, MA, USA). The MS equipment has a high collision dissociation cell (HCD) for fragmentation and an Orbitrap analyser (ThermoFisher Scientific; Q-Exactive, Waltham, MA, USA). XCALIBUR 3.0.63 (ThermoFisher Scientific, Waltham, MA, USA) software was used for data acquisition and equipment configuration to allow peptides identification at the same time of their chromatographic separation. Full-scan mass spectra were acquired in the Orbitrap analyser. The scanned mass range was 400–2000 m/z, at a resolution of 70,000 at 400 m/z, and the 12 most intense ions in each cycle were sequentially isolated, fragmented by HCD and measured in the Orbitrap analyser. Peptides with a charge of + 1 or with unassigned charge state were excluded from fragmentation for MS2. Q-Exactive raw data were processed using PROTEOME DISCOVERER software (ver. 220.127.116.11; ThermoFisher Scientific, Waltham, MA, USA) and searched against Cellulomonas sp. B6 or C. fimi ATCC 484 UniProt sequences database (based on genome full sequences GenBank access LNTD00000000.1 and GCA_000212695.1, respectively), with trypsin specificity and a maximum of one missed cleavage per peptide. Proteome Discoverer searches were performed with a precursor mass tolerance of 10 ppm and product ion tolerance to 0.05 Da. Static modifications were set to carbamidomethylation of Cys, and dynamic modifications were set to oxidation of Met and N-terminal acetylation. Protein hits were filtered for high confidence peptide matches with a maximum protein and peptide false discovery rate of 1% calculated by employing a reverse database strategy. A minimum of two unique peptides was considered as confident detection. For the estimation of relative abundance, we used the protein abundance index emPAI calculated by Sequest using protein identification data. The equation emPAI/Σ (emPAI) × 100 was used to calculate the protein content in mol.% (emPAI%) (Ishihama et al. 2005; Shinoda et al. 2009). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022718.
Enzymatic hydrolysis of EBS and analysis of the hydrolysates
Enzymatic hydrolysis experiments on EBS were performed by using LEE fraction and a combination of LEE and IE fractions with different mixing ratios and dilutions. Enzymatic hydrolysis experiments were carried out in a reaction mixture containing 5% w/v dry EBS in citrate buffer (pH 6, 100 mM). The hydrolysis reactions were carried out at 45 °C, 220 rpm for 72 h and stopped by boiling for 5 min. The supernatant was clarified by centrifugation (6000×g, 15 min) and then used to measure the xylose released from the biomass employing d-xylose assay kit (Megazyme, Bray, Ireland). The hydrolysis products were analysed by thin layer chromatography (TLC) in silica gel plates using butanol/acetic acid/water (2:1:1) as solvents and revealed by water/ ethanol/sulfuric acid (20:70:3) with 1% v/v orcinol solution over flame.