Barrios et al. Biotechnology for Biofuels and Bioproducts
(2025) 18:48
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units per milliliter (U/mL). β-glucosidase activity, which represents the enzyme’s ability to hydrolyze cellobiose into two glucose molecules, is also quantified U/mL [35]. Protein content was determined using a bicinchoninic acid assay kit (BCA ™ assay, Thermo Scientific, USA) with bovine serum albumin as the standard [36]. CATO ® 237 modified corn starch, a cationic additive provided by Ingredion (Westchester, IL, USA), was used to enhance wet and dry strength and retention in papermaking (degree of substitution 0.053). The starch was prepared by dissolving it in boiling deionized water with continuous stirring. Solutions of 0.001 N Polydiallyldimethylammonium chloride (polyDADMAC, Mw = 200–350 kg/mol) and 0.001 N potassium polyvinyl sulfate (PVSK, Mw ~170 kg/mol) from BTG Americas Inc. (Alpharetta, GA, USA) were used as polyelectrolyte titrants. Sodium citrate buffer, sugar standards (glucose, xylose, galactose, mannose, arabinose, cellobiose), hydrochloric acid, sodium hydroxide, sodium azide, 3,5-dinitrosalicylic (DNS) acid, and Rochelle salt (sodium potassium tartrate tetrahydrate), analytical grade mercuric chloride (HgCl 2 ) and sodium acetate trihydrate (CH 3 COONa•3H 2 O) were purchased from Fisher Scientific (Waltham, MA, USA). Deionized water was used for all steps requiring water unless stated otherwise. All chemicals were used without further purification.
results for fiber characterization, such as morphological and structural analysis, water interaction and retention, chemical composition and reactivity, and analysis of fiber components, such as fines, obtained after the proposed cell-free enzyme treatments. By employing techniques such as scanning electron microscopy (SEM) and fiber quality analysis, this study assesses the extent of fibrillation, fiber surface modifications, and any structural alterations resulting from the enzymatic treatments. The colloidal titration and streaming potential measurements were utilized to quantify the changes in surface charge, and the results were correlated with the dewatering efficiency. Moreover, this study investigates the enzymatic treatment effect on the different forms of water in the fibers, which is crucial for understanding the drying behavior of cellulosic materials.
Materials and methods Raw materials, enzymes, and chemicals
Never-dried northern bleached hardwood kraft (BHW) pulp was provided by Sappi North America (Boston, MA, USA). The chemical composition of the wood fibers was determined by the NREL procedure for structural carbohydrates and lignin in biomass quantification [30]. The compositional results were cellulose—75.9%, hemicellulose—22.3%, lignin—0.74%, extractives—0.27%, and ash content—0.26%. Detailed physical properties of fibers are presented in Table S1. An enzyme blend (11 FPU/mL, 1125 U/mL, 0.09 g/mL protein content) containing cellulases (5% cellulase 1, 5% cellulase 2) and xylanases (45% xylanase 1, 45% xylanase 2) was used in this study. Cellulase activities (FPU, CMC, and β-glucosidase) were measured via the DNS method [31] at pH 5.0 and 50 °C with filter paper as the substrate. Xylanase activity was measured with beechwood xylan as the substrate [32]. Enzyme activity was expressed as micromoles of reducing sugars released per minute (U) and filter paper activity in FPU [33]. FPU is a standard measure of cellulase activity, specifically indicating the enzyme’s ability to hydrolyze filter paper. The activity is expressed as FPU/mL, representing the number of filter paper units released per milliliter of enzyme solution [34]. On the other hand, xylanase activity is measured by the release of reducing sugars from xylan, a major hemicellulose component in plant cell walls. The activity is expressed as units per milliliter (U/mL), where one unit represents the release of 1 μmol of reducing sugars per minute [32]. CMCase and β-glucosidase activities were also quantified to assess the broad spectrum of cellulase activity and its role in fiber modification. CMCase activity refers to the ability of cellulases to hydrolyze carboxymethylcellulose (CMC) and is measured in
Pulp processing Pulp refining
The never-dried pulp sample (219 g dry weight) was soaked in water and adjusted to a 10% consistency (oven- dried (OD) weight basis) following the standard TAPPI Method T205 [37]. Refining was conducted using a PFI laboratory mill at 1000 revolutions, following the TAPPI standard T248 [38]. Each refining batch was carried out with 30 g (dry basis) of pulp at 10% consistency. A simplified schematic flow diagram detailing the pulp pretreatment process and subsequent pulp and paper properties measurements adapted from our previous publication [16] is available in Figure S1 in the supplementary material. Cell-free enzyme pretreatment After refining, the BHW pulp samples at 10 wt.% consist- ency were pretreated with the enzyme cocktail. Various enzyme concentrations, ranging from 0 to 1.0 wt.% based on the OD weight of the pulp, were added to the samples, as detailed in Table 1. The enzyme-treated mixtures were then incubated in an incubator shaker at 45 °C with gen- tle shaking (60 rpm) for 30 min. The 30-min incubation period was selected based on previous studies on pulp treatments [39] and was designed to minimize reten- tion times for potential industrial-scale applications.
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