Synergistic cell-free enzyme cocktails for enhanced fiber matrix development: improving dewatering, strength, and decarbonization in the paper industry

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 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.

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 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 [7,8,9]. 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 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 [22,23,24]. 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 CO₂ 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 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 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 (HgCl₂) and sodium acetate trihydrate (CH₃COONa•3H₂O) 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.

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.% consistency 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 gentle 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 retention times for potential industrial-scale applications. 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.

Table 1 Experimental design to evaluate the effect of refining, enzymatic, and chemical pretreatment on fiber and strength properties of paper

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/m³ 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 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 HgCl₂ and 7 g CH₃COONa•3H₂O was prepared in 500 mL distilled water, then diluted in a 1-L flask to achieve 0.6% HgCl₂ and 0.7% CH₃COONa. 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 measurements. HexA content was calculated using the following equation:

$${C}_{HexA}\left(\frac{\mu mol}{g}\right)=0.287\times \frac{({A}_{260}-1.2{A}_{290})\bullet V(mL)}{w(g)}$$

(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 900g 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 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]:

$${\text{W}}_{{{\text{water}}}} = \frac{{\Delta {\text{H}}_{{{\text{peak}}}} }}{{{\text{H}}_{{\text{f}}} {\text{*w}}_{{{\text{dry}}}} }}\left( {{\text{g}}/{\text{g}}} \right)$$

(2)

$${\text{W}}_{\text{NFBW}}={\text{M}}_{\text{ratio}}-{\text{W}}_{\text{FBW}}-{\text{W}}_{\text{FW}}$$

(3)

$${\text{W}}_{\text{BW}}= {\text{W}}_{\text{NFBW}}+{\text{W}}_{\text{FBW}}$$

(4)

where ${\text{W}}_{\text{water}}$ is the water mass fraction, g/g, ${\text{H}}_{\text{f}}$ is the specific heat of fusion (334 J/g), $\Delta {\text{H}}_{{{\text{peak}}}}$ is the enthalpy peak of each curve, (J), and ${\text{W}}_{\text{dry}}$ is the dry weight of sample, (g):

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 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 manufacturing 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].

Fig. 1

System boundary for the manufacturing of bleached hardwood kraft pulp and paper and board production

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 information paper (TIP) 0404-47 (2022) for paper machine performance guidelines [53]. Energy consumption for virgin cartonboard production, one of the paper grades with the highest share of bleached hardwood fiber in its furnish, was used as a reference for comparison. The specifications of the simulated mill, baseline scenario, and enhanced dewatering scenarios are shown in Table 2.

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

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).

• CO₂ emissions from the RB, BB, and part of the limekiln (due to the calcination process) are considered biogenic and thus carbon neutral.

• Fossil CO₂ emissions are released by gas combustion at the limekiln and NB.

• In the enhanced dewatering scenario, the only variable 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 implications of implementing enzymatic treatment and starch addition to enhance dewatering and increase solids content 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 consumption, resulting from energy savings, can significantly reduce costs for a pulp and paper mill. The potential economic benefits are estimated by integrating the cell-free enzyme technology to increase solids content after pressing. Changes in natural gas costs due to change in consumption, along with procurement costs for enzymes and starch in the enhanced dewatering scenario, were considered, 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 assessment of the economic feasibility of the enhanced dewatering strategy [55, 56].

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

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.”

Results and discussion

Enhanced dewatering and paper strength

Enzymatic activity

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 xylanase activity is high compared to typical values reported in the literature, positioning the blend as highly effective for applications that require extensive hemicellulose breakdown. The filter paper activity (FPA) of 11 FPU/mL is within the range commonly seen in commercial cellulase preparations, indicating that this blend is competitive with other products for cellulose degradation [57, 58]. However, the CMCase and β-glucosidase activities 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 function, it may be more specialized or intended to be combined with other enzymes or additives to achieve optimal results.

Table 3 Enzyme activity and protein content determined for the commercial enzyme

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.

Table 4 Effect of enzyme pretreatment on paper's freeness, EMC, and tensile properties

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, specifically 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, differences in the consistency and treatment times between the mentioned study and this one may account for variations in the effectiveness of the enzyme treatments on paper properties observed here. A balance between refining conditions, enzymatic reaction temperatures, and the timing and method of additive addition is crucial for optimizing 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.

Table 5 Combined effect of enzyme and cationic starch dose on EMC and tensile strength of paper for BHW-1k PFI mill refining

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 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 treatment effectively modified the fiber structure, potentially leading to a more open and porous fiber network. This is crucial for enhancing dewatering efficiency during wet-pressing, allowing quicker water removal without compromising the paper's mechanical properties [66]. However, it is essential to carefully control enzyme dosage 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].

Table 6 Effect of cell-free enzyme pretreatment on 1k-refining BHW fibers’ dimensional characteristics

More pronounced effects were observed in the kink and curl indexes of the fibers. The enzymatically treated samples consistently exhibited lower kink and curl indexes than untreated fibers. For instance, the mean kink index decreased from 1.57 1/mm in the control sample to 1.48 1/mm and 1.43 1/mm in the 0.5 wt.% and 1.0 wt.% enzyme-treated samples, respectively. Similarly, these samples reduced the curl index from 0.081 to 0.077. The reduction in kink and curl indexes is associated with increased fiber flexibility, enhancing the bonding potential between fibers during paper formation and improving paper strength and quality [69]. Increased flexibility, as indicated by lower kink and curl indexes, is desirable as it allows for better fiber-to-fiber contact during the papermaking process, which is critical for developing strong paper products [70].

A significant reduction in fines content was observed for the enzymatically treated samples with increasing addition of cationic starch. For the 0.5 wt.% enzyme-treated condition, the fines content was reduced from 9.93% in the control sample to 3.56%, representing a reduction of approximately 64%. This significant decrease in fines content can be attributed to the coagulating and flocculating effects of the cationic starch, which facilitates the agglomeration of fine particles within the pulp suspension [71]. Reducing fines is beneficial, since it improves drainage during papermaking, leading to more efficient water removal and faster production times. In addition, the fines content analysis of the white water collected after drainage during standard handsheet formation (data not shown) confirmed that no loss of fines occurred during the drainage process, suggesting that all fines were effectively retained within the paper handsheet, further supporting the efficacy of the cationic starch in fines retention.

Electrostatic properties

Maximum dewatering rates are closely associated with the neutralization of fiber surface charges, which is thought to be more influential than neutralizing charges buried within the cell wall or under layers of fibrillation. The charges on fiber surfaces are crucial, because they dominate the colloidal interactions between fibers, cellulosic fines, and water molecules. This assumption holds if dewatering effects are indeed dominated by colloidal forces between individual fibrils on fibers and fines [72]. Effective dewatering, therefore, can be optimized by targeting the surface charge neutralization of fibers.

A reliable technique for determining the charge of a fiber suspension is measuring the cationic/anionic demand using a streaming potential device. This volumetric method is based on the reaction between positively and negatively charged polyelectrolytes. The start potential obtained from this instrument represents the initial voltage or potential applied to the system before titration begins, and it varies depending on the experimental setup and the nature of the sample. In this study, the start potential for fiber suspensions after cell-free enzyme pretreatment is detailed in Table 7. All start potential values for the suspensions, without adding cationic starch, were negative and became less negative with increasing dosage of cationic polymer. The charge analysis revealed that cell-free enzyme pretreatments significantly altered the surface charge of the fibers.

Table 7 Effect of cell-free enzyme pretreatment on the charge of fiber surfaces

Refining and enzymatic pretreatment increased the negative surface charge of the fibers. The enzyme cocktail, rich in xylanases, likely reduced the presence of hydrophilic xylan groups on the fibers, contributing to the increased negative charge. As negatively charged polyelectrolytes, these fibers interact with cationic starch, a positively charged polyelectrolyte. This interaction likely reduces the affinity between water molecules and hydrophilic hydroxyl groups on the fiber surfaces, enhancing dewatering rates. In addition, the cationic starch promotes fiber bonding and fines retention, further increasing paper strength. The results showed that close to the neutralization point of the fiber suspension, where the fiber surface is close to saturation with cationic starch, the best conditions for dewatering (lowest EMC) and paper strength (highest tensile strength) were achieved. All treated systems exhibited a cationic demand, as shown in Table 7, indicating that polyDADMAC was used as the titrant for neutralization. Previous studies have shown that polyDADMAC, with a molecular mass higher than 100 kg/mol, adsorbs primarily on the external surface of cellulosic fibers without penetrating the micropores of the cell wall [73]. Given that the polyDADMAC used in this study has a molecular mass between 200 and 350 kg/mol, the measured charge corresponds to surface charge rather than total charge. This study is the first to directly relate charge analysis to moisture content reduction after wet-pressing, building on the established understanding that charge neutralization is critical in dewatering enhancement.

Fiber morphology

Various authors have visually assessed the hydrolytic effect of cellulases on cellulose model substrates, revealing significant morphological changes [74]. However, detecting these changes on the more complex surfaces of wood pulp fibers presents a substantial challenge [75,76,77]. For instance, Suchy et al. demonstrated that high enzyme dosages can induce notable morphological −alterations, such as cell wall dislocations and surface disruptions in the form of cracks [78]. These microstructural changes are critical to understanding the underlying mechanisms of fiber modification during enzyme treatment.

Scanning electron micrographs of handsheets formed from cell-free enzyme-treated fibers are presented in Fig. 3. The surface of non-enzymatically treated fibers (Fig. 3a) appeared relatively smooth, with low levels of fibrillation and delamination, alongside several structural irregularities. This smoothness suggests limited surface area exposure and minimal mechanical bonding potential, which may result in weaker inter-fiber adhesion in the final paper product. In contrast, fibers treated with cationic starch (Fig. 3b) showed increased smoothness, flexibility, and better adhesion between adjacent fibers. The cationic starch likely acts as a bonding agent, promoting closer contact between fibers and improving the overall strength of the paper. Enzymatically treated fibers (Fig. 3c, d) displayed a significant increase in surface roughness, improved fibrillation, and greater delamination, which indicate effective enzyme action. The enhanced fibrillation observed in Fig. 3c suggests that the enzyme treatment has facilitated the partial hydrolysis of the fiber surface, exposing more cellulose microfibrils and increasing the fiber's ability to bond with other fibers. This is consistent with findings from other studies, where enzyme treatments have been shown to enhance fibrillation and surface area, leading to stronger paper products [79].

Fig. 3

SEM images of 1k-PFI refined bleached hardwood fibers treated with different enzyme (E) and cationic starch (S) doses: a Untreated control (0E, 0S); b 0E, 0.5S—increased smoothness; c 0.5E, 0S—increased fibrillation; d 0.5E, 0.5S—increased fiber entanglement. Arrows indicate key morphological changes

Furthermore, the combination of enzyme and cationic starch treatment (Fig. 3d) results in increased fiber entanglement and more pronounced cell wall dislocations and cracks. These morphological changes contribute to improved fiber bonding and entanglement, which are critical for enhancing the mechanical properties of the paper. The visible disruptions on the fiber surface may also indicate areas where the enzyme has penetrated the fiber structure, further contributing to the improved inter-fiber bonding.

Enzymatic cleavage of HexA groups

Removing hexenuronic acid (HexA) groups is critical to secure efficient and cost-effective bleaching operations during papermaking [80]. These are harmful to kraft bleaching systems in the papermaking process as they reduce bleaching efficiency by consuming a disproportionate amount of bleaching chemicals. Hardwood pulps are characterized by a higher xylan content [81]. Degradation of HexA dominates over its formation only in the last part of the hardwood pulping and not in the beginning, as is the case for softwoods. Removal of HexA can be carried out by bleaching at acidic conditions or by acidic treatment at high temperatures without bleaching chemicals. The reactive double bond in the hexenuronic acid reacts with several bleaching chemicals such as chlorine, chlorine dioxide, ozone, and peracid but not with alkaline oxygen and hydrogen. Enzymes—xylanase and laccase-mediator systems—also effectively remove hexenuronic acid from kraft pulp [82]. Although the removal of HexA is typically conducted before or during the bleaching process to reduce the consumption of bleaching chemicals and improve the brightness and stability of the pulp, it is not 100% efficient. HexA groups are hydrophilic due to their carboxylic acid functionality, which can interact with water molecules, as shown in Fig. 4.

Fig. 4

Hardwood xylan a before and b after kraft pulping

Before bleaching, hardwood kraft pulps typically contain HexA in the range of 40–100 μmol/g [83, 84]. After undergoing oxygen delignification, a common pre-bleaching step, this content is usually reduced to 20–60 μmol/g [85]. However, it is after the complete bleaching process—whether through elemental chlorine-free (ECF) or total chlorine-free (TCF) sequences—that the HexA content is significantly lowered, typically ranging from 5 to 20 μmol/g [86]. The results for the untreated fibers indicate extensive or very aggressive bleaching sequences, as shown in Fig. 5. The slight reduction in HexA content following mechanical refining aligns with the understanding that refining can impact HexA levels by removing hemicellulose [87]. HexA levels below 5 μmol/g require specific treatments targeting HexA, such as using xylanase enzymes or additional oxidative bleaching stages [88]. In that sense, the enzyme treatment led to a significant decrease in the HexA content of the fibers. This reduction is likely due to the enzymatic cleavage of the HexA groups, particularly by xylanase enzymes, which target the xylan chains in the fiber and break down the hemicellulose portion that contains the HexA groups [89, 90]. The mechanism involves the selective hydrolysis of xylan and associated components, facilitating the removal of these hydrophilic groups, leading to an enhancement in paper web dewatering.

Fig. 5

Effect of cell-free enzyme treatment on HexAs content of the fibers

Mechanical dewatering and thermal drying

Water retention value (WRV)

After moving across the forming section, passing through foils and vacuum boxes, the paper web goes through a couch roll and enters the press section of the paper machine. As an initial approach to understanding the effect of the cell-free enzyme process on fiber dewatering in the press section, measurements of water retention value were conducted; the results are shown in Fig. 6. Previous studies have shown that enzymatic treatments can reduce WRV by modifying the fiber surface and internal structure, improving dewatering [17, 18]. The WRV is a critical parameter that measures the ability of cellulosic fibers to retain water after being subjected to centrifugal force [91]. Its importance cannot be overstated, as it is closely related to the fiber's internal structure, surface area, and chemical composition, making it an essential indicator of the fiber's behavior during the papermaking process.

Fig. 6

Effect of cell-free enzyme pretreatment on WRV, HRW, FBW, and NFBW content of the fibers. The cationic starch (S) and enzyme (E) dosages are indicated in the x-axis

WRV is used as an indicator of how easily water can be removed from the pulp in the forming and press sections. For unrefined fibers, the WRV was found to be 1.98 g water/g dry sample, which is typical for unrefined fibers, as their surface area and exposure of hydroxyl groups are limited compared to refined fibers [17]. Refining, which involves mechanical treatment to fibrillate the fibers and increase their surface area, significantly increases the WRV. This increase is consistent with previous findings, where refining enhances the fiber's ability to retain water due to increased fiber swelling and creating more internal spaces within the fiber structure [92].

When examining the effects of the cell-free enzyme pretreatment, the WRV for the sample treated with 0.5% enzyme without starch (0.5E, 0S) was slightly reduced to 2.21 g water/g dry sample, compared to the refined fibers without enzyme treatment. This reduction suggests that the enzyme pretreatment might have cleaned the fiber surfaces by removing fines and partially hydrolyzing the fiber walls, thereby reducing the fiber's capacity to hold water. This aligns with results reported elsewhere, where enzyme treatments were shown to modify the fiber surface, leading to changes in water retention behavior [93]. Interestingly, the combination of 0.5% enzyme and 0.5% starch (0.5E, 0.5S) resulted in the lowest WRV among the refined samples, with a 1.89 g water/g dry sample value. This significant reduction indicates a synergistic effect between the enzyme and starch treatments, where the enzyme likely facilitates the adsorption of the cationic starch onto the fiber surface. The starch then shields the hydrophilic groups on the fibers, reducing their interaction with water [94]. This result supports the hypothesis that cationic starch can effectively reduce water retention when combined with appropriate enzymatic pretreatments. This strategy can be beneficial in enhancing the efficiency of the papermaking process by reducing the energy required for drying.

Hard-to-remove water

After moving across the press section and passing through several press rollers, the paper web enters the dryer section of the paper machine. As an initial approach to understanding the effect of the cell-free enzyme process on fiber dewatering in the dryer section, measurements of different thermal properties, such as HRW and BW, were conducted; the results are shown in Fig. 6. The HRW content of cellulosic fibers is a critical parameter that describes the amount of water strongly bound to fibers, which remains difficult to remove during drying [44]. It is defined as the water content present during the transition from the constant rate drying zone to the falling rate drying zone [46]. This transition point, identified through the second derivative curve during TGA, allows for the precise calculation of HRW by dividing the mass at the transition stage by the mass of the dried fiber [45, 95].

Refining is crucial in increasing the HRW content due to changes in fiber structure, such as internal and external fibrillation, fiber shortening, and fines formation. These structural modifications increase the fiber surface area and expose more hydroxyl groups, which can hold more water molecules. The data presented in Fig. 6 show that refining significantly increases HRW content from 1.48 g water/g dry sample in unrefined fibers to 2.02 g water/g dry sample in fibers without enzyme or starch treatment (0E, 0S). This increase aligns with the expected outcome, as refining exposes more of the fiber surface area to water, enhancing its water retention capacity [21]. The effect of enzymatic pretreatment on HRW content was also significant. For example, when 0.5% enzyme (0.5E) was added without starch (0S), the HRW content decreased slightly to 1.91 g water/g dry sample. However, the combination of 0.5% enzyme and 0.5% cationic starch (0.5 A, 0.5S) resulted in the lowest HRW content at 1.49 g water/g dry sample. This suggests a synergistic effect between the enzyme and starch treatments in reducing water retention, possibly due to the enzyme's ability to clean fiber surfaces, making them more receptive to cationic starch, which then acts to reduce fiber–water interactions [96, 97].

Bound water

Identifying different types of water in cellulose fibers is challenging when relying solely on water retention value experiments or other dynamic dewatering methods. Specific spectroscopic and calorimetric techniques are necessary to accurately distinguish between these water types, which can detect the interactions between water and cellulose. Modulated Differential Scanning Calorimetry (MDSC), compared to conventional DSC, allows for precise quantification of first-order phase transitions, such as melting and crystallization, which are observed in the non-reversing curve of the thermal response. Within the fiber structure, two primary types of water exist: free water and bound water, which result from the strength of interaction between cellulose and water molecules [98]. These distinct types of water are classified based on their thermodynamic response and are separated by a thermodynamic boundary known as the capillary condensation point or fiber saturation point (FSP) in the case of cellulosic fibers [99, 100]. Bound water is absorbed below the capillary condensation point, which is characteristic of porous and hydrophilic materials [20]. Capillary condensation occurs in narrow capillaries at pressures lower than bulk water's normal saturation vapor pressure [101]. Consequently, the interaction of water with cellulosic surfaces leads to two subcategories of bound water: FBW and NFBW [102].

NFBW is tightly associated through hydrogen bonding with the fiber matrix, particularly within the first hydration layers, preventing water first-order phase transitions, such as crystallization or melting [19]. As the hydration layers across the fiber matrix increase, the interaction strength between water layers is reduced, allowing water molecules to move, accommodate, and condense [103]. On the other hand, the FBW exhibits a depressed melting point response due to the lower capillary pressure in the fiber's matrix. As the hydration layers extend beyond the fiber saturation point, the cellulose–water interactions decrease, and water exhibits bulk-like behavior [99]. The effect of enzymatic treatments on bleached softwood fibers has previously been reported [45]. However, no significant changes in bound water content were observed with mild treatments using cellulases. The effects of enzymatic treatments have shown to be highly dependent on the type of enzyme and the nature of the substrate employed [104].

The effect of cell-free enzyme treatments on the bound water content of the hardwood fibers is shown in Fig. 6. As expected, refining (Refined 1k) increases the amount of bound water (BW) due to the enhanced exposure of fiber surfaces. The increase in surface area leads to a higher association of water molecules with the fibers [105]. Interestingly, the application of enzymes (0.5E, 0S) and the independent addition of cationic starch (0E, 0.5S) both resulted in a decrease in FBW content, while NFBW remained unchanged. The lack of effect of the enzymes on NFBW can likely be attributed to the nanopore dimensions within the fiber matrix, where NFBW is typically located. These nanopores are too small for the enzymes used in this study to access and modify, explaining why NFBW was unaffected by the treatment [106]. Computational studies have shown that the water layers corresponding to bound water are inside microfibril bundles, highlighting the uniqueness and complexity of structural hierarchy and chirality of natural fibers [107, 108].

On the other hand, a synergistic effect was observed when enzyme and starch treatments were combined (0.5E, 0.5S), leading to a reduction in the overall BW content by approximately 27% compared to the untreated fiber (0E, 0S). However, the FBW content was reduced, and the NFBW content remained unaltered. Specific details on the different forms of water in the fibers are presented in Table S4. The promising results observed here lay the foundation for scaleup testing of this technology.

Economic and environmental impacts

In an integrated mill, the dryer section of the paper machine is one of the most energy-intensive units. In the baseline scenario used to assess economic and environmental impacts, the dryer section accounts for approximately 31% of the total steam demand. Detailed energy flow values for the baseline and enhanced dewatering scenarios are provided in Table S5 in the supplementary material. The powerhouse generates steam and electricity for various mill operations. In the simulation, high pressure steam at 855 psi and 440 °C is produced and sent to back pressure and condensing turbines, where 93% of the mill’s electricity demand is met. The steam pressure is then reduced to medium pressure (160 psi) and low pressure (60 psi) for delivery to various processes. Steam from the natural gas boiler accounts for 29% of the total steam production.

Energy savings and emissions reduction

The results for the enhanced dewatering scenario (Table S5) indicate that increasing the solids content after pressing, from 39 to 50% by the cell-free enzyme treatment and cationic starch addition reduces steam demand in the dryer section of the paper machine by up to 37%, as shown in Fig. 7a. This reduction in water content directly correlates with a decrease in steam consumption in the dryer section of the paper machine, as less thermal energy is required to remove the remaining moisture [109, 110]. Since other unit operations, such as the digester, the chlorine dioxide (ClO₂) bleach plant, and air heater system, also consume medium-pressure (MP) steam, a 25% energy reduction in MP steam can be achieved. When accounting for total steam demand (MP and low-pressure (LP)), overall energy savings of 12% are possible. The total CO₂ emissions released on-site by the process are 3560 kg CO₂-eq/ADt, with 79% classified as biogenic emissions. Input and output flows are detailed in the life cycle inventory for both the baseline and enhanced dewatering scenarios in Tables S6 and S7. In the cradle-to-gate analysis, the GWP for the baseline is 963 kg CO₂-eq/ADt, compared to 844 kg CO₂-eq/ADt for the enhanced dewatering scenario, as shown in Table S8.

Fig. 7

a Steam demand relative to the production of one ADt of paper for the baseline and enhanced dewatering scenario, b GWP breakdown by scopes 1, 2 and 3 for the baseline scenario, c direct emissions breakdown and contribution, and d total GWP variation and distribution by activities within the scopes

Figure 7b provides a breakdown of scope emission for the baseline scenario, showing that direct emissions account for 50% of the total GWP. External operations such as forest activities, transportation, and the procurement of chemicals and fuels represent 46% of the total GWP, while carbon emissions from electricity purchases make up 4%, as the mill is 93% self-sufficient in power. The GWP for bleached hardwood kraft pulp has been reported in the range of 572–1200 kg CO₂-eq/ADt, with variations primarily attributed to scope 1 emissions due to differences in fuel distribution and energy usage within the mill [54, 56]. In the simulation, natural gas consumption in the boiler was controlled by the MP steam demand. Thus, energy reductions in the dryer directly affect natural gas combustion and result in variations in fuel consumption.

The reduction in GWP obtained in the enhanced dewatering scenario is attributed to decreased natural gas combustion. Steam demand and emissions in the dryer are reduced by 37%, and CO₂ emissions directly released from the boiler decreased by 27%, as shown in Fig. 7c and Table S9. The reductions resulted in a 23% decrease in total direct emissions. Furthermore, when considering total emissions across scopes 1, 2, and 3, the cell-free enzyme technology for enhanced dewatering achieved a net GWP reduction of 12.3% ± 2.5% (Fig. 7d).

Techno-economic calculations

The adoption of enzyme technologies in the pulp and paper industry is increasing, driven by advancements in enzyme production and continuous efforts to optimize manufacturing costs. However, information on the economic evaluation of enzyme applications in the paper industry remains limited. Cost analysis estimates for the application of the cell-free enzyme technology are presented in Table 8. The change in consumption is defined as the difference between the baseline and enhanced dewatering scenarios. By increasing the solids content after pressing, from 39 to 50%, 2.77 GJ of fossil fuel energy per ADt of product can be saved. The increase in solids content after the pressing reduces steam demand in the drying section, leading to lower natural gas consumption in the boiler. Since the boiler is controlled to supply only the required amount of steam, the reduction in steam demand directly translates to a decrease in purchased fuel, resulting in cost savings. A detailed breakdown of the energy cost savings from reduced natural gas consumption is provided in the supplementary material under section Breakdown of Energy Cost Savings. Considering an average cost of the natural gas in the U.S at 5.8 USD $/GJ [56], this translates to savings of 16.05 USD $/ADt in fuel procurement.

Table 8 Cost analysis of implementing the enzymatic and starch treatment to increase solids content after pressing in the paper manufacturing process for bleached hardwood fiber and board production

However, the purchase of enzymes and starch is required for the treatment. Based on the analysis, the following were considered: the enzyme and starch usage per OD ton (0.5%), the experimentally quantified protein content in the enzyme cocktail (90 mg/mL), the cost of 4.25 USD $/kg protein, and the cost of 617 USD $/ton for wet end strength additive starch [55, 56]. These inputs resulted in an additional cost of 1.82 USD$/ADt for the enzyme cocktail and 2.94 USD$/ADt for starch. A summary of the costs and quantities used for the economic analysis is presented in Table S10. Calculations on enzyme costs are provided in the supplementary material under section Calculations of enzyme dose per protein content.

The cost of enzyme is identified as a key barrier to the application and commercialization of enzyme technologies in the pulp and paper industry. Estimates for bioethanol production suggest that enzyme costs range from 0.10 $ to 1.47$ per gallon of ethanol [55, 111]. In this study, the economic calculation estimated an enzyme cost of 1.82 USD $/ADt of paper product. However, the additional costs of enzyme and starch are offset by the significant reduction in fuel usage, leading to a total net cost change of − 11.29 USD $/ADt, as shown in Table 8.

Conclusions and perspectives

This study demonstrates the technical, environmental, and economic potential of cell-free enzyme technology to enhance press dewatering efficiency and paper mechanical properties. By reducing energy intensity in paper thermal drying processes, this technology contributes to decarbonization and net-zero goals in the pulp and paper industry. The synergistic integration of mild refining, cell-free enzyme treatments, and cationic starch proved highly effective in improving paper quality while simultaneously lowering the energy required for drying. The application of enzyme treatment, after mild refining, at a concentration of 0.5 wt.% combined with 0.5 wt.% cationic starch resulted in a significant 11% increase in total solids content after pressing and a 25% improvement in tensile strength. Morphological analysis revealed that the enzyme and starch treatments did not significantly alter the mean weighted fiber length and fiber width. However, the reductions in kink and curl indexes indicate increased fiber flexibility and improved inter-fiber bonding, leading to enhanced mechanical properties. The enzyme treatment was shown to reduce hexenuronic acid groups, which influences the water adsorption properties of fibers, as evidenced by a decrease in the water retention value and reductions in hard-to-remove and bound water content. Economic and life cycle assessment results demonstrate the potential of the cell-free treatment as an effective strategy for improving the sustainability and economic viability of paper production. The cost-effectiveness and decarbonization potential of this technology were demonstrated with a total net cost savings of − 11.29 USD/ADt of paper produced, a 12% reduction in GWP, a 37% reduction in emissions from the dryer, and a 23% reduction in direct emissions (scope 1).

While this study demonstrates the significant potential of enzyme-based pretreatments to improve press dewatering and paper strength, several challenges remain for industrial-scale adoption. Key concerns include enzyme stability in different pulp formulations, integration with existing mill operations, and potential regulatory and cost barriers. Variations in pulp composition, temperature, and pH, can affect enzyme performance, requiring further research. Scaling up may also necessitate adjustments in enzyme dosing and refining to fit current workflows. Finally, while environmentally beneficial, the economic and regulatory aspects of enzyme and biopolymer use must be carefully assessed.