Barrios et al. Biotechnology for Biofuels and Bioproducts
(2025) 18:48
Page 5 of 23
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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