PAPER making! The e-magazine for the Fibrous Forest Products Sector
Produced by: The Paper Industry Technical Association
Publishers of: Paper Technology International ®
Volume 11 / Number 2 / 2025
PAPER making! FROM THE PUBLISHERS OF PAPER TECHNOLOGY INTERNATIONAL ® FROM THE PUBLISHERS OF PAPER TEC Volume 11, Number 2, 2025 CONTENTS:
FEATURE ARTICLES: 1. Enzymes : For enhanced properties, performance, and decarbonization. 2. Papermaking : Investigation of pulp dewatering by high vacuum suction boxes. 3. Testing : Neural network and image processing for measuring fibrils. 4. Wood Panel : Properties of OSB produced using green adhesives. 5. Decarbonisation : Energy efficient alternatives – the case of linerboard production. 6. Modelling : Mathematical modelling and optimization of stock preparation unit. 7. Tissue : Decorating sanitary-hygienic products by flexographic and digital printing. 8. Moulded Fibre : Moldable and plastic-free nonwoven aerosolized particulate filters. 9. Physics : Origins of out-of-plane auxetic response in paper. 10. Artificial Intelligence : AI hacks to supercharge your daily work life.
11. Health & Safety : 20 reasons to wear and use your PPE. 12. LinkedIn : How to improve your LinkedIn networking.
SUPPLIERS NEWS SECTION: News / Products / Services : x
Section 1 – PITA CORPORATE MEMBERS ABB / VALMET Section 2 – PITA NON-CORPORATE MEMBERS ANDRITZ / STORA ENSO / VOITH Section 3 – NON-PITA SUPPLIER MEMBERS BTG / Fagus-GreCon / Tasowheel / Tietoevry Advertisers: ABB / KEMIRA / VALMET
x
x
DATA COMPILATION: Events : PITA Courses & International Conferences / Exhibitions
Installations : Overview of equipment orders and installations between Mar. and Jun. Research Articles : Recent peer-reviewed articles from the technical paper press. Technical Abstracts : Recent peer-reviewed articles from the general scientific press.
The Paper Industry Technical Association (PITA) is an independent organisation which operates for the general benefit of its members – both individual and corporate – dedicated to promoting and improving the technical and scientific knowledge of those working in the UK pulp and paper industry. Formed in 1960, it serves the Industry, both manufacturers and suppliers, by providing a forum for members to meet and network; it organises visits, conferences and training seminars that cover all aspects of papermaking science. It also publishes the prestigious journal Paper Technology International ® and the PITA Annual Review , both sent free to members, and a range of other technical publications which include conference proceedings and the acclaimed Essential Guide to Aqueous Coating .
Contents
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PAPER making! FROM THE PUBLISHERS OF PAPER TECHNOLOGY INTERNATIONAL ® FROM THE PUBLISHERS OF PAPER TEC Volume 11, Number 2, 2025 Synergistic cell Ǧ free enzyme cocktails for enhanced fiber matrix development: improving dewatering, strength, and decarbonization in the paper industry NELSON BARRIOS, MARÍA GONZALEZ, RICHARD VENDITTI & LOKENDRA PAL Background The pulp and paper industry is under increasing pressure to adopt sustainable solutions that address its substantial energy consumption and environmental impact. One of the most energy-intensive operations is the thermal drying, which presents significant opportunities for efficiency improvements. This study evaluates a cell-free mild enzyme pretreatment, utilizing a cocktail of cellulases and xylanases, combined with cationic starch, to enhance dewatering efficiency and improve paper strength utilizing bleached hardwood pulp fibers. Life cycle and economic analysis were also conducted to quantify the environmental impact and economic benefits, with a particular focus on direct greenhouse gas emissions. Enhanced water removal during pressing can significantly reduce energy consumption during thermal drying, facilitating the decarbonization of the paper industry. Results The cell-free enzyme pretreatment, applied with mild refining and cationic starch, achieved significant improvements in dewatering efficiency and paper strength. The treatment led to an 11% point increase in solids and a 25% improvement in tensile strength. Morphological analyses revealed no changes in fiber length and width; however, reductions in kink and curl indexes indicated enhanced fiber flexibility and bonding potential. Furthermore, the enzyme–starch combination decreased water retention value by 27%, including substantial reductions in bound and hard-to-remove water content. Environmental assessments estimated a 12% reduction in global warming potential (GWP), with the technology yielding net savings of $11.29 per air- dried ton of paper through reduced natural gas consumption. Conclusions This study demonstrates the technical feasibility and economic viability of incorporating enzyme and cationic starch treatments into papermaking. The treatment improves paper quality while reducing energy consumption, costs, and carbon emissions. These findings support the broader adoption of enzyme-based innovations for sustainable manufacturing, aligning with decarbonization goals and industry demands for greater efficiency. The results highlight a promising avenue for achieving significant environmental and economic benefits in the pulp and paper sector. Contact information: Department of Forest Biomaterials, North Carolina State University, 431 Dan Allen Dr., Raleigh, NC 27695, USA. Biotechnology for Biofuels and Bioproducts (2025) 18:48 https://doi.org/10.1186/s13068-025-02646-1 Creative Commons Attribution 4.0 License The Paper Industry Technical Association (PITA) is an independent organisation which operates for the general benefit of its members – both individual and corporate – dedicated to promoting and improving the technical and scientific knowledge of those working in the UK pulp and paper industry. Formed in 1960, it serves the Industry, both manufacturers and suppliers, by providing a forum for members to meet and network; it organises visits, conferences and training seminars that cover all aspects of papermaking science. It also publishes the prestigious journal Paper Technology International ® and the PITA Annual Review , both sent free to members, and a range of other technical publications which include conference proceedings and the acclaimed Essential Guide to Aqueous Coating .
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Article 1 – Enzyme Technology
Biotechnology for Biofuels and Bioproducts
Barrios et al. Biotechnology for Biofuels and Bioproducts https://doi.org/10.1186/s13068-025-02646-1
(2025) 18:48
Open Access
RESEARCH
Synergistic cell-free enzyme cocktails for enhanced fiber matrix development: improving dewatering, strength, and decarbonization in the paper industry Nelson Barrios 1 , María Gonzalez 1 , Richard Venditti 1 and Lokendra Pal 1* Abstract Background The pulp and paper industry is under increasing pressure to adopt sustainable solutions that address its substantial energy consumption and environmental impact. One of the most energy-intensive operations is the ther- mal drying, which presents significant opportunities for efficiency improvements. This study evaluates a cell-free mild enzyme pretreatment, utilizing a cocktail of cellulases and xylanases, combined with cationic starch, to enhance dewatering efficiency and improve paper strength utilizing bleached hardwood pulp fibers. Life cycle and economic analysis were also conducted to quantify the environmental impact and economic benefits, with a particular focus on direct greenhouse gas emissions. Enhanced water removal during pressing can significantly reduce energy con- sumption during thermal drying, facilitating the decarbonization of the paper industry. Results The cell-free enzyme pretreatment, applied with mild refining and cationic starch, achieved significant improvements in dewatering efficiency and paper strength. The treatment led to an 11% point increase in solids and a 25% improvement in tensile strength. Morphological analyses revealed no changes in fiber length and width; however, reductions in kink and curl indexes indicated enhanced fiber flexibility and bonding potential. Furthermore, the enzyme–starch combination decreased water retention value by 27%, including substantial reductions in bound and hard-to-remove water content. Environmental assessments estimated a 12% reduction in global warming poten- tial (GWP), with the technology yielding net savings of $11.29 per air-dried ton of paper through reduced natural gas consumption. Conclusions This study demonstrates the technical feasibility and economic viability of incorporating enzyme and cationic starch treatments into papermaking. The treatment improves paper quality while reducing energy consumption, costs, and carbon emissions. These findings support the broader adoption of enzyme-based innova- tions for sustainable manufacturing, aligning with decarbonization goals and industry demands for greater efficiency. The results highlight a promising avenue for achieving significant environmental and economic benefits in the pulp and paper sector. Keywords Cell-free enzymes, Dewatering, Energy sustainability, Decarbonization, Cellulose fibers, Cationic polymers, Tensile strength
*Correspondence: Lokendra Pal lpal@ncsu.edu
© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
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Graphical Abstract
Introduction Cell-free enzyme systems, typically derived from cell-free extracts or purified enzymes, have been increasingly adopted in the pulp and paper industry (P&PI) to enhance the efficiency and sustainability of various processes [1]. These systems enable the controlled application of specific enzymatic activities without the complications associated with living cells, such as the need for growth, metabolic byproducts, or the regulation of cellular pathways that may interfere with the desired reactions [2]. By focusing on purified enzymes or enzyme cocktails, the industry can achieve targeted modifications to pulp fibers, such as reducing lignin content, enhancing fiber flexibility, or increasing fibrillation, thereby improving the
overall quality of the final paper products. One of the primary applications of cell-free systems in the pulp and paper industry is in biobleaching, where enzymes such as laccases, xylanases, and lignin peroxidases are employed to reduce the use of harmful chemicals, such as chlorine and chlorine dioxide [3]. Xylanases have been used to degrade hemicellulose, which helps remove lignin from the fibers, making the bleaching process more environmentally friendly and cost- effective. Laccases, often used with mediators, can oxidize phenolic compounds in lignin, facilitating its breakdown and removal during subsequent washing stages [4]. These enzymatic treatments can significantly lower the chemical oxygen demand (COD) and
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point due to capillary effects, while NFBW is strongly bound to cellulose at the molecular level and does not undergo phase transitions [19]. Bound water is held tightly within the fiber matrix due to hydrogen bonding with cellulose, making it difficult to remove, especially during the falling rate drying period [20]. As fibers undergo refining, their ability to retain bound water increases due to more significant fibrillation and the exposure of more hydroxyl groups on the fiber surface [21]. This bound water significantly contributes to the energy required for thermal drying. The economic and environmental implications of dewatering processes in the P&PI are significant; however, comprehensive analyses addressing both decarbonization potential and economic implications of dewatering technologies are limited [22–24]. The P&PI stands as a significant contributor to greenhouse gas emissions, primarily from natural gas boilers and lime kiln operations during chemical recovery [25]. Among the industry’s processes, the dryer section of the paper machine is one of the most energy-intensive units, consuming 20–30% of total steam demand [26]. Innovative technologies, such as impulse dryers, shoe presses, and enzymatic treatments, have shown promise in enhancing dewatering efficiency and decreasing energy use [27]. For example, impulse dryers have demonstrated up to 10% decarbonization potential in the Austrian pulp and paper industry [23]. Enzymatic processes, in particular, offer additional environmental and economic advantages by reducing water retention in fibers and minimizing steam consumption [13, 16]. By integrating enzymatic treatments into production lines, mills can lower fossil fuel reliance, cut direct CO ᔐ emissions, and achieve cost savings, addressing both decarbonization potential and the economic trade-offs essential for sustainable pulp and paper manufacturing. Building on the findings reported from our previous work [16], this study aims to investigate the application of the cell-free enzyme pretreatment, comprising mild mechanical refining, the use of a commercial enzyme formulation, and treatment with a cationic biopolymer, on bleached hardwood pulp to evaluate its efficacy in enhancing press dewatering during papermaking. Hardwood pulps, which differ from softwood pulps in their higher hemicellulose content and smaller fiber dimensions, present unique challenges and opportunities in water removal and paper strength enhancement. The differences in hemicellulose content, particularly the presence of hexenuronic acid (HexA) groups, and the typically lower lignin content in hardwoods may impact the efficiency and outcomes of enzymatic treatments [28, 29]. In addition to measuring the moisture content after pressing and paper properties, this study includes
adsorbable organic halides (AOX) in effluents, reducing environmental impact. Cell-free enzymatic systems have also been used in fiber modification and refining processes. Applying cellulases, hemicellulases, and pectinases can selectively hydrolyze components of the fiber cell wall, thereby enhancing fiber swelling, flexibility, and fibrillation. This results in better fiber bonding and improved paper strength properties, such as tensile and tear strength [5, 6]. Moreover, the use of these enzymes can reduce energy consumption during the refining process, as enzymatically treated fibers require less mechanical energy to achieve the desired level of fibrillation [7–9]. The selectivity of these enzymes also minimizes fiber degradation, preserving the length and integrity of the fibers, which is crucial for maintaining the strength of the final paper products [10]. Enzymatic treatments are increasingly integrated into processes designed to convert lignocellulosic biomass into valuable bioproducts, such as bioethanol, bioplastics, and other chemicals, alongside traditional pulp and paper production [11, 12]. In addition to quality improvements, enzymatic treatments offer significant potential for energy savings, particularly in water removal during papermaking [13]. Water removal is a critical step, as thermal drying is one of the most energy-intensive processes in paper production [14, 15]. Previous research has shown the potential of enzymatic treatments, particularly those involving cellulases and xylanases, to enhance dewatering and improve the strength properties of paper made from southern bleached softwood kraft pulp, leading to significant energy reductions during papermaking [16]. Water in cellulosic fibers exists in different forms, each with distinct impacts on the dewatering and drying stages in paper manufacturing. Understanding these forms of water—free, bound, and hard-to-remove water (HRW)—is essential for optimizing energy consumption and enhancing the efficiency of the papermaking process. Mechanical dewatering and thermal drying are key stages where water removal significantly affects paper mills’energy demands and overall productivity. Changes in Water Retention Value (WRV) reflect how easily water can be extracted during mechanical dewatering, particularly in the forming and press sections of the paper machine. Enzymatic treatments, which modify the fiber structure and surface characteristics, have been shown to reduce WRV, enhancing dewatering efficiency [17, 18]. Mechanical dewatering is typically followed by thermal drying, where bound water, including freezing-bound water (FBW) and non-freezing-bound water (NFBW), becomes the focus. FBW, which exists in the outer layers of the fiber hydration structure, has a depressed melting
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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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sputter-coated with a gold (Au) layer for 2 min and imaged at 15 kV. Equilibrium moisture content (EMC) The TAPPI standard for handsheet formation (T 205) was modified to measure the equilibrium moisture content (EMC) of handsheets immediately after pressing, as detailed in our previous work [16]. Following the couching step, handsheets were carefully removed from the blotter paper and placed on a metal plate within the press, with a dry blotter and additional plate layered above, following the setup illustrated in our prior publication. The combined weight of the handsheet and plate was recorded post-pressing, then both were oven-dried at 105 ± 1 °C for 30 min before reweighing. Calculations for handsheet weight after pressing and moisture content were conducted, with full calculation details available in our earlier publication. Charge demand titrations The cationic or anionic demand of samples diluted to approximately 1 mg/L and dispersed using a British disintegrator for 15,000 revolutions (typically sampled as 10 mL aliquots) was measured using a CAS Touch streaming current detector (emtec Electronic GmbH, Leipzig, Germany). This device features a polytetrafluoroethylene (PTFE) piston, approximately 15 mm in diameter, which moves up and down at a frequency of around 4 Hz within a loosely fitted PTFE boot, with a gap width of less than 1 mm. The detector utilizes electrode probes near the boot’s base and above the annular region to detect the presence and polarity of the electrical double layer formed at the PTFE surfaces. As the PTFE surfaces become coated with polyelectrolytes and colloidal material from the aqueous sample, the device can effectively detect the endpoint of a titration involving known polyelectrolytes. A 0.001 molar solution of polyDADMAC was used as the cationic titrant for these measurements. In contrast, the potassium salt of PVSK served as the anionic titrant (0.001 M). Hexenuronic acid (HexA) determination HexA hydrolysis was performed according to Chai et al. [41]. A solution of 6 g HgCl 2 and 7 g CH 3 COONa•3H 2 O was prepared in 500 mL distilled water, then diluted in a 1-L flask to achieve 0.6% HgCl 2 and 0.7% CH 3 COONa. A sample of 0.05 g pulp with known moisture content was added to 10 mL of this solution in a 20-mL vial, which was sealed and shaken. The vial was heated for 30 min in a 60 °C–70 °C water bath, then cooled and filtered using a 0.2-μm syringe filter. The filtrate was placed in a 10-mm silica cuvette for 260 and 290 nm UV absorption
Table 1 Experimental design to evaluate the effect of refining, enzymatic, and chemical pretreatment on fiber and strength properties of paper Parameter Range
Enzyme (% OD pulp basis)
0, 0.5, 1.0 0, 0.5, 1.0
Cationic starch (% OD pulp basis)
Refining, PFI revs
0 (control), 1000
Following incubation, the enzymes were inactivated by heating the samples to 60–70 °C. The pulp samples were then cooled at room temperature, thoroughly washed with deionized water, and drained three times using a standard handsheet mold [37]. The white water collected during drainage was analyzed for fines content to ensure no fines were lost through the standard handsheet mold screen. After inactivating the enzymes, the pulp samples were diluted with distilled water and subjected to 15,000 revolutions in a laboratory propeller pulp disintegrator (TMI disintegrator, 400 Bayview Ave., Amityville, NY 11701) following the TAPPI T205 standard [37]. This mechanical treatment effectively separates fibers without significantly altering their structural properties. Following disintegration, the appropriate dose of cationic starch (as indicated in Table 1) was added, and the pulp was diluted to a 0.30% consistency with distilled water, following the TAPPI T205 standard to obtain handsheets of 60 g/m 3 basis weight [37]. Each experimental condition, whether control or enzymatically pretreated, was evaluated 2 to 4 times to ensure reproducibility. The results were highly consistent, with standard deviations for moisture content after pressing and tensile strength never exceeding 2% of the mean.
Fiber characterization Fiber morphology and imaging
Fiber dimensional properties, including weighted average fiber length, fiber width, and related metrics, were measured per TAPPI standards T232, T233, T234, and T261 [40] using a HiRes fiber quality analyzer (FQA) (OpTest Equipment Inc., Canada). Samples were diluted to ~ 1 mg/L and dispersed using a British disintegrator for 15,000 revolutions. Each FQA run analyzed 5000 particles from 0.03 mm to 10.0 mm in size; fiber width was recorded for particles > 0.2 mm, and particle length was calculated as contoured length, reported as length- weighted (Lw). Morphological analysis was performed on handsheets using a JEOL JEM-6000Plus scanning electron microscope (SEM) at 400 × –800 × . Handsheets were
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measurements. HexA content was calculated using the following equation:
isothermally at 90 °C for 45 min until the sample mass stabilized. Bound water (BW) TGA and differential scanning calorimetry (DSC) were employed to determine the bound water content [45]. The analyses were performed using a TGA-550 thermogravimetric analyzer and a DSC-Discover differential scanning calorimeter (TA Instruments, New Castle, DE, USA). Approximately 100 mg of the sample with ~ 10 wt.% solid content was placed in a 100 μL platinum pan. The nitrogen gas flow rates were set to 40 mL/min for the balance gas and 60 mL/min for the sample gas. Isothermal heating at 90 °C was applied and interrupted just before the falling rate zone (as described elsewhere [46]) to obtain fully saturated fibers at a moisture ratio of 1.4, corresponding to approximately 58 wt.% water content. The samples were then rapidly removed from the TGA furnace, placed into DSC T-zero pans (TA Instruments, New Castle, DE, USA), and sealed. The samples were equilibrated at room temperature (23 ± 1 °C) for 1 h before analysis using the modulated DSC mode. The DSC thermal procedure included a temperature drop to − 30 °C, isothermal cooling for 10 min, and a heating ramp at 3 °C/min until reaching 15 °C. After the analysis, the pans were perforated and returned to the TGA furnace for solid content determination, using isothermal heating at 90 °C until a constant weight was achieved. The non-reversing melting curves obtained were Gaussian deconvoluted to determine the melting enthalpy of each peak, corresponding to free and freezing-bound water [47]. Non-freezing bound water was calculated by subtracting the amounts of freezing- bound water and unbound water (detected by DSC) from the total water measured by TGA, according to Eqs. 2–4 [47, 48]:
g
C HexA
( A 260 − 1.2 A 290 ) • V ( mL ) w ( g )
μ mol
= 0.287 ×
(1)
with a calibration constant of 0.287 and a correction factor of 1.2 for lignin absorption [42], where V is the hydrolysis solution volume (mL), and w is the o.d. pulp sample weight (g). Water retention value (WRV) WRV tests were conducted using the TAPPI Useful Method 256 [43]. Treated pulp samples were first disintegrated for 5 min (15,000 revolutions) following TAPPI Method 205. The pulp was then thoroughly washed with excess deionized water and soaked overnight. Although some loss of fines might occur, the impact was expected to be minimal in this study due to the low refining level and the fact that the pulp had already been washed. After additional washing, the pulp was collected on a vacuum filter and dewatered to approximately 25% solids content. For the WRV test, moist pulp samples equivalent to 0.16 g of dry mass were placed into sintered centrifuge tubes (pore size 0.22 μm, volume 3 mL). The samples were centrifuged at 900 g for 30 min. Post-centrifugation, the moisture content was determined by weighing the samples immediately, followed by drying at 105 °C for 2 h. The dried samples were then cooled in a desiccator for 30 min before a final weighing to assess moisture content accurately. Hard-to-remove water (HRW) Thermogravimetric analysis (TGA) was employed to determine the HRW content [44]. The study was performed using a TGA-550 thermogravimetric analyzer (TA Instruments, New Castle, DE, USA) operating isothermally at 90 °C, after ramping the temperature from room temperature to 90 °C at a rate of 90 °C/min. The HR water content is defined as the moisture content of the fibers at the transition point between the constant rate drying zone and the falling rate zone and is calculated as the ratio of the water weight at the onset point of this transition (determined thermogravimetrically) to the dry weight of the fibers. During the analysis, nitrogen gas was used as purge gas, with flow rates set at 40 mL/min for the balance and 60 mL/min for the sample. Wet pulp samples containing approximately 10 mg of solid mass and 90 mg of water were prepared for the experiments. The temperature was increased from 25 °C to 90 °C at a rate of 90 °C/min, and the drying process was continued
H peak H f *w dry
g / g
(2)
W water =
(3)
W NFBW = M ratio − W FBW − W FW
(4)
W BW = W NFBW + W FBW
where W water is the water mass fraction, g/g, H f is the specific heat of fusion (334 J/g), H peak is the enthalpy peak of each curve, (J), and W dry is the dry weight of sample, (g):
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Fig. 1 System boundary for the manufacturing of bleached hardwood kraft pulp and paper and board production
mill producing bleached fiber and employs a cradle-to- gate system boundary encompassing scopes 1, 2, and 3 for determining global warming potential (GWP). Life cycle assessment (LCA) The LCA was conducted to estimate the environmental impact of reducing energy consumption in paper manu- facturing through enhanced dewatering. The study was framed within a cradle-to-gate system boundary, as depicted in Fig. 1, to conduct an attributional LCA based on standard ISO 14040-44 [49]. The functional unit was one air-dried ton of paper product (ADt). Open-source openLCA software and the Ecoinvent 3.10 database were used [50]. The environmental impact methodology applied was TRACI 2.1, focused on reporting the GWP impact category for the assessment [51]. The life cycle inventory was obtained through process simulation using WinGems software, in which bleached hardwood fiber and paper and board production was modeled [52]. Mass and energy balance were compared with standard values reported in the technical informa- tion paper (TIP) 0404-47 (2022) for paper machine per- formance guidelines [53]. Energy consumption for virgin cartonboard production, one of the paper grades with
where NFBW is the non-freezing bound water, FBW is the freezing bound water, BW is the bound water, and FW is the free water.
Paper properties Grammage and tensile index
Handsheets were formed using a 159-mm-diameter sheet machine with a stirrer, following the TAPPI T205 standard [37]. After conditioning the handsheets following the TAPPI T402 standard (at 50% relative humidity and 23 °C), the following paper properties were measured: grammage, as per TAPPI T410; bulk, as per TAPPI T220; and tensile index, as per TAPPI T494. Only handsheets with a grammage of 60 ± 1 g/m2 were used to evaluate paper properties. Economic and environmental evaluation Life cycle assessment (LCA) and techno-economic analysis (TEA) were conducted to estimate the energy savings, environmental impact, and economic benefits of applying cell-free enzyme technology. The analysis includes a mass and energy balance of a virgin integrated
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• In the enhanced dewatering scenario, the only vari- able is the change in solids content after pressing due to enzyme treatment and starch addition. • The dryer consumes medium pressure (MP) steam, which was adjusted to control the natural gas consumed in the NG. • Steam savings were reflected as a reduction in fuel usage. The steam energy produced by the RB and BB is assumed to remain constant. • The electricity demand is assumed to remain constant. Techno-economic analysis (TEA) The economic analysis evaluates the financial implica- tions of implementing enzymatic treatment and starch addition to enhance dewatering and increase solids con- tent after pressing in bleached hardwood kraft pulp and board production. Figure 2 illustrates the process flow used to estimate costs for both the baseline and enhanced dewatering scenarios. A reduction in natural gas con- sumption, resulting from energy savings, can significantly reduce costs for a pulp and paper mill. The potential eco- nomic benefits are estimated by integrating the cell-free enzyme technology to increase solids content after press- ing. Changes in natural gas costs due to change in con- sumption, along with procurement costs for enzymes and starch in the enhanced dewatering scenario, were con- sidered, as shown in Fig. 2. Key inputs for the analysis, including costs for natural gas, enzymes, and starch, were sourced to assess the savings from reduced natural gas consumption against the added expenses of enzyme and starch procurement, providing a comprehensive assess- ment of the economic feasibility of the enhanced dewa- tering strategy [55, 56]. The primary parameters influencing cost savings are enzyme usage, natural gas fuel, and starch costs. A sensitivity analysis was conducted to evaluate the impact of cost variability on overall savings. For this analysis, a ± 25% variation was applied to each parameter. Natural gas prices, which are the most significant contributor to cost savings, were varied between 4.35 to 7.25 USD/GJ, with results ranging from − 7.27 to − 15.30 USD/ADt. Enzyme and starch costs were also varied by ± 25%; enzyme cost savings remained largely stable, while changes in starch costs had a smaller impact on total savings. More details on the variation in savings due to changes in these primary cost parameters are provided in Figure S2 and Table S3.”
Table 2 Specifications of the simulated mill, including baseline and enhanced dewatering scenarios, used to evaluate the impact of the cell-free enzyme treatments on solids content increase after press
Parameter
Baseline
Enhanced dewatering
Fiber type
Bleached hardwood Bleached hardwood
Paper grade
Cartonboard
Cartonboard
Production (ADt/day)
1356
1356
Powerhouse configuration
RB, NB, BB
RB, NB, BB
Electricity demand (kWh/ ADt)
980
980
Power self-sufficiency % 93 Solids before dryer % 39 Solids after dryer % 95
93 50 95 0.5 0.5
Enzyme (wt.% OD) Starch (wt.% OD)
– –
ADt: air-dried ton, RB: recovery boiler, NB: natural gas boiler, BB: biomass boiler
the highest share of bleached hardwood fiber in its fur- nish, was used as a reference for comparison. The speci- fications of the simulated mill, baseline scenario, and enhanced dewatering scenarios are shown in Table 2. The uncertainty in the reported reduction in GWP was estimated using a sensitivity analysis (SA) approach [54]. The primary variable influencing fossil fuel consumption and direct emissions in the LCA was the solids content after pressing, which directly affects the final GWP. Therefore, the SA was based on the experimental variation of solids, with a standard deviation of ± 1%. However, to account for expected variability at a larger scale, a range of ± 2% was applied to capture potential process fluctuations. The simulation was run with solids after pressing varying between 48% (minimum) and 52% (maximum). The uncertainty in the GWP reduction was expressed as the mean ± half the range. The method, which uses a range based on standard deviation and scales it for larger-scale variability, provides a straightforward estimate of uncertainty, with clear results for GWP reduction. More details on the uncertainty calculations can be found in Table S2.
Considerations of the simulation process
• The powerhouse consists of the recovery boiler (RB), biomass boiler (BB), and natural gas boiler (NB). • CO 2 emissions from the RB, BB, and part of the limekiln (due to the calcination process) are considered biogenic and thus carbon neutral. • Fossil CO 2 emissions are released by gas combustion at the limekiln and NB.
Barrios et al. Biotechnology for Biofuels and Bioproducts
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Fig. 2 Process diagram illustrating the cost analysis of the cell-free enzyme treatment to increase solids content after pressing in bleached hardwood kraft pulp and board production
Table 3 Enzyme activity and protein content determined for the commercial enzyme Enzyme preparations Enzyme activities
Protein concentration (mg mL − 1 )
FPA (FPU mL − 1 )
β-glucosidase (U mL − 1 )
Xylanase (U mL − 1 )
CMCase (U mL − 1 )
Enzyme blend
11
0.9
0.1
1125
90
Table 4 Effect of enzyme pretreatment on paper’s freeness, EMC, and tensile properties
Enzyme
Dose (%)
Duration of pretreatment (min)
Freeness (mL)
EMC (%)
Tensile index (Nm/g)
Avg
Std
Avg
Std
Avg
Std
Refined to 1000 revs Control
n/a 0.5
n/a
510 600 602
5 3 9
61.11 53.76 56.68
0.91 0.97 0.84
61.22 59.27 56.03
4.45 6.38 3.79
30 30
Enzyme blend
1
Values are presented as the average and standard deviations based on five replicates
Results and discussion Enhanced dewatering and paper strength Enzymatic activity
mL is within the range commonly seen in commercial cellulase preparations, indicating that this blend is com- petitive with other products for cellulose degradation [57, 58]. However, the CMCase and β-glucosidase activi- ties are on the lower side, with 0.9 U/mL and 0.1 U/mL, respectively [59, 60]. These values suggest that while the enzyme preparation is robust in its xylanase func- tion, it may be more specialized or intended to be com- bined with other enzymes or additives to achieve optimal results.
The results for activity quantification for the enzyme blend indicate a strong profile in xylanase activity, which was 1125 U/mL, as shown in Table 3. This level of xyla- nase activity is high compared to typical values reported in the literature, positioning the blend as highly effec- tive for applications that require extensive hemicellulose breakdown. The filter paper activity (FPA) of 11 FPU/
Barrios et al. Biotechnology for Biofuels and Bioproducts
(2025) 18:48
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Table 5 Combined effect of enzyme and cationic starch dose on EMC and tensile strength of paper for BHW-1k PFI mill refining
Tensile index (Nm/g) Bulk (cm 3 /g)
Enzyme dose (%)
Cationic starch dose (%)
Duration of pretreatment (min)
Freeness (mL)
EMC (%)
Avg
Std
Avg
Std
Avg
Std
Avg
Std
Refined to 1000 rev 0 0
n/a n/a n/a
510 550 570 600 628 629 602 629 630
5 9 6 8 5 6 4 6 5
61.11 58.12 57.69 53.76 50.05 56.73 56.68 55.94 55.56
0.90 1.14 0.76 0.98 1.01 0.90 1.10 1.07 0.73
61.22 73.73 71.83 59.27 76.69 66.45 56.03 71.33 65.13
4.45 3.69 3.60 6.38 4.24 3.11 3.79 4.41 1.94
1.60 1.40 1.42 1.88 1.82 1.79 1.54 1.55 1.54
0.01 0.01 0.02 0.00 0.02 0.02 0.03 0.02 0.02
0.5
1 0
0.5
30 30 30 30 30 30
0.5
1 0
1
0.5
1
Averages and standard deviation with five replicates
timing and method of additive addition is crucial for opti- mizing dewatering rates while maintaining or enhancing paper strength [62]. The results obtained in the present study support such claims. The cell-free enzyme pretreatment was also evaluated at various refining levels on a laboratory scale. After extensive screening of refining levels, temperature, and dosages of chemicals and enzymes (data not shown), the optimal conditions are presented in Table 5. A refining level of 1,000 PFI mill revolutions was identified as optimal. The ideal enzyme and cationic starch dosage combination should achieve minimum EMC, maximum freeness, and maximum tensile strength in the handsheets from the pretreated pulp. The results indicate that the cell-free enzyme pretreatment with 0.5 wt.% enzyme combined with 0.5 wt.% cationic starch yielded the best outcomes. This condition resulted in the lowest EMC (50.05%), a significant increase in tensile strength (~ 25.0%) to 76.69 Nm/g, and an improvement in freeness (~ 23.0%) to 628 mL compared to the control (no enzyme or starch). This demonstrates a positive impact on reducing EMC and enhancing both paper strength and freeness. Notably, the reduction in EMC (~ 11.0% total solids increase) under these conditions suggests an improved dewatering process, which is crucial for efficient paper production. However, increasing the enzyme dosage beyond 0.5 wt.% led to less favorable results, with increased EMC and decreased tensile strength. Specifically, at 1.0 wt.% enzyme dosage, despite a slight increase in freeness, the tensile strength dropped to 66.45 Nm/g, and EMC increased to 56.73%, indicating a trade-off between dewatering and mechanical properties. This finding aligns with the trend observed in previous studies, where increased cationic starch dosage improved freeness but
EMC and paper properties Preliminary tests were conducted at a laboratory scale to determine the optimal dose of the enzyme cocktail to achieve a significant reduction in moisture content after pressing, as shown in Table 4. The pulp samples were refined to 1000 PFI revs before enzymatic treatment. The optimal enzyme dosage was determined by identifying the condition that resulted in the lowest EMC and the highest tensile strength in the paper handsheets. As presented in Table 4, at a 0.5 wt.% enzyme dosage (on an oven-dry pulp basis), there was a significant reduction in EMC from 61.11% (control) to 53.76%, alongside a slight decrease in tensile strength from 61.22 Nm/g to 59.27 Nm/g. This dosage also improved the drainage rate, as evidenced by increased freeness from 510 mL (control) to 600 mL. Increasing the enzyme dosage to 1.0 wt.% resulted in a slight further increase in freeness accompanied by a less desirable outcome: a reduction in EMC (to 56.68%) and a decrease in tensile strength (to 56.03 Nm/g). These results suggest that while a 0.5 wt.% enzyme dosage effectively improves drainage and reduces EMC, higher dosages may negatively impact the paper’s mechanical strength. The enzyme dosages applied in this study, spe- cifically 0.5 and 1.0 wt.% based on dry pulp, correspond to protein concentrations of 0.9 mg and 0.18 mg protein per gram of dry pulp, respectively. These concentrations are similar to those reported in other articles assessing the impact of enzyme treatments on press dewatering with various commercial enzymes [61]. However, differ- ences in the consistency and treatment times between the mentioned study and this one may account for vari- ations in the effectiveness of the enzyme treatments on paper properties observed here. A balance between refin- ing conditions, enzymatic reaction temperatures, and the
Barrios et al. Biotechnology for Biofuels and Bioproducts
(2025) 18:48
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Table 6 Effect of cell-free enzyme pretreatment on 1k-refining BHW fibers’ dimensional characteristics Enzyme dose (%) Cationic starch dose (%) Mean weighted fiber length (μm) Mean fiber width (μm) Mean Kink Index (1/mm)
Mean Curl Index (L w ) Fines content (L w %)
Avg
Std
Avg
Std
Avg
Std
Avg
Std
Avg
Std
Refined to 1000 revs 0 0
997 994 974 994 991 981 973 987 996
21 28 16 19 20 15
18.90 19.35 18.65 18.35 19.14 19.35 18.65 18.45 18.36
0.14 0.21 0.07 0.35 0.19 0.31 0.06 0.18 0.07
1.57 1.48 1.43 1.36 1.53 1.44 1.32 1.62 1.53
0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.01
0.081 0.080 0.077 0.070 0.071 0.080 0.087 0.086 0.077
0.002 0.001 0.005 0.004 0.002 0.001 0.004 0.004 0.005
5.23 4.89 3.92 9.93 3.56 3.38
0.06 0.18 0.09 0.13 0.47 0.37 0.31 0.08 0.45
0.5
1 0
0.5
0.5
1 0
1
7
10.93
0.5
23 21
4.65 3.72
1
wet-pressing, allowing quicker water removal without compromising the paper’s mechanical properties [66]. However, it is essential to carefully control enzyme dos- age and refining conditions to prevent over-hydrolysis of fibers, which could negatively impact the paper’s strength and runnability [67]. Cell-free enzymatic fiber modification for enhanced dewatering Fiber dimensions The effect of enzyme cocktail and cationic starch dosage on the dimensional properties of 1k-refining bleached hardwood (BHW) pulp fibers is presented in Table 6. No significant change in the mean weighted fiber length was detected when comparing the control sample (0 wt.% enzyme, 0 wt.% cationic starch) with the best performing condition (0.5 wt.% enzyme, 0.5 wt.% cationic starch). The average fiber length for all conditions remained close to 990 μm, with the standard deviation indicating minimal variability. This stability in fiber length suggests that the 1k mild refining conditions, in conjunction with the enzymatic and cationic starch treatments, did not cause excessive fiber shortening, which is crucial for maintaining the paper’s tensile strength [64]. Similarly, the cell-free enzyme pretreatment caused only minor changes in fiber width. The width values before and after pretreatment were around 18.9 μm for all conditions, with only slight variations observed. This consistency in fiber width indicates that the enzymes did not significantly contribute to the fibers’degradation, which could otherwise compromise the dewatering and physical properties of the paper [68]. More pronounced effects were observed in the kink and curl indexes of the fibers. The enzymatically treated samples consistently exhibited lower kink and curl
required careful balancing to avoid compromising other paper properties [63]. The significant enhancement in tensile strength suggests improved fiber bonding, probably due to the enzyme’s ability to modify the fiber surfaces, facilitating better interaction with the added cationic starch. It can be attributed to the enzymatic action that likely involves a controlled modification of the hemicellulose and cellulose components of the fibers. Cellulases and xylanases act synergistically to selectively degrade specific fiber components, leading to a more favorable fiber morphology for bonding and a better surface area for interaction with cationic starch. This synergy between cellulase and xylanase enzymes is critical in enhancing fiber flexibility and reducing fiber stiffness, which contribute to improved inter-fiber bonding and, consequently, higher tensile strength [64]. Furthermore, the increase in tensile strength observed in this study aligns with findings from previous research, which demonstrated that enzyme treatments could significantly improve paper strength when combined with refining and appropriate chemical additives [65]. The effectiveness of enzyme treatment also depends on achieving a delicate balance between refining conditions and enzymatic pretreatment. Refining is known to increase the fiber surface area and promote fibrillation, which can be further enhanced by enzymatic treatments to optimize fiber bonding and reduce the need for excessive refining, which could damage fibers and reduce paper strength. In addition to the increase in tensile strength, the observed changes in bulk indicate that the enzyme treat- ment effectively modified the fiber structure, potentially leading to a more open and porous fiber network. This is crucial for enhancing dewatering efficiency during
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