3.1 Production of purified and functional SamLPMO10C
Functional SamLPMO10C was expressed in Escherichia coli BL21 (DE3) star and purified following the method developed previously (Valenzuela et al. 2017). A protein of approximately 40 kDa was obtained after the final step of purification (Fig. 1), in agreement with the theoretical molecular weight of the enzyme. The yield of the purified protein was 6 mg per liter of culture. The activity of the enzyme was initially verified by MALDI-TOF using PASC as substrate under standard conditions. This analysis showed peaks corresponding to oxidized cello-oligosaccharides of 4–7 degrees of polymerization (Fig. 1). Each product of the reaction can form adducts with Na +, K +, or both, so there is more than one peak for the same degree of polymerization. This is in concordance with the results of previous works for SamLPMO10C, confirming that this enzyme has a C1-oxidation pattern (Valenzuela et al. 2017) .
3.2 Enzymatic treatment of eucalyptus cellulose and bacterial cellulose
BC and eucalyptus pulps were treated with SamLPMO10C as summarized in S1 for 36 and 72 h in shaking conditions. The reactions stopped after the addition of EDTA.
The activity of the enzyme for each substrate was first verified by MALDI-TOF and then by HPAEC PAD analysis. The enzyme was active on both substrates as detected by the peaks observed by MALDI-TOF analysis (Fig. 2). For BC, oxidized cello-oligosaccharides of 5 to 7 degrees of polymerization can be observed. Meanwhile, for eucalyptus pulp, oxidized cello-oligosaccharides of 5 and 6 degrees of polymerization were found.
The second activity verification consisted of the quantification of aldonic acids by HPAEC-PAD, and the results are listed in Table 1 (chromatograms are attached in S2). For both substrates, the maximum amount of aldonic acids was found at 72 hours of reaction, which yielded an increase of 2 and 1.7 times for BC pulp and eucalyptus pulp respectively, regarding 36 hours of reaction. Consequently, the time of reaction of 72 hours was chosen for further investigations. The values of total aldonic acids were in the same magnitude order as the cellobionic acid found for cotton linters (Valls et al. 2019). SamLPMO10C showed more activity on BC than in eucalyptus, as expected since previous works described that the rate of oxidation was proportional to the crystalline degree of the substrate (Valenzuela et al. 2019).
The carboxyl content in the insoluble fraction of SamLPMO10C-oxidized pulps was also determined (Titration plots are attached in S3). BC and eucalyptus pulps increased their carboxyl groups 2.7 and 2.4 times, respectively, after the enzymatic treatment (Table 2). It is noteworthy that SamLPMO10C showed more activity on BC than on eucalyptus, in accordance with the quantification of soluble oligosaccharides shown above. Previous results evaluating the enzyme activity on cotton linters reported that carboxyl groups increased 1.6 times with respect to the initial carboxyl pulp content (Valls et al. 2019). SamLPMO10C successfully created carboxyl groups by oxidation of the hydroxyl group in the C1 position of the cellulose chain. Therefore, this enzyme is suitable to increase the carboxyl content in different cellulose substrates as this and previous studies have shown.
Table 1
Aldonic acid release produced by the enzymatic pretreatment
Pulp treatment | Enzymatic Pretreatment (h) | Aldonic acid release (mg/g pulp ± SD) |
BC-ox | 36 | 9.38 ± 0.10 |
BC-ox | 72 | 19.03 ± 2.81 |
eu-ox | 36 | 5.86 ± 1.64 |
eu-ox | 72 | 9.87 ± 0.88 |
Table 2
Carboxyl content of substrates before and after SamLPMO10C oxidation
Pulp treatment | Enzymatic Pretreatment (h) | COO− (mmol/Kg pulp) |
BC | 0 | 127 ± 12 |
BC-ox | 72 | 347 ± 23 |
eu | 0 | 153 ± 12 |
eu-ox | 72 | 373 ± 46 |
3.3 Addition of silver and characterization of paper supports
After the oxidation of the cellulosic substrates, the addition of silver and the production of paper supports were carried out. The functionalized supports were analyzed by scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS), and characterized in terms of silver content, leaching, and antibacterial capacity.
SamLPMO10C oxidized pulps were treated with 10 mM AgNO3 as a source of silver ions. Non-oxidized pulps were used as controls. As described before, the hydroxyl groups present in the cellulose structure can attract the silver cations through dipole-ion interaction (Barud et al. 2008). Furthermore, the new negatively charged carboxyl groups (COO−) created by the action of the enzyme were expected to attract Ag cations (Ag+) by electrostatic interactions.Musino et al. (2021) showed that hydroxyl groups were the nucleation centers that attract the positively charged silver, and the COO- groups helped the dispersion state of the fibers and thereby, increased the access of the silver to the cellulose. After the treatment of pulps with AgNO3, paper supports were produced and then subjected to thermal reduction. The heat is expected to cause a reduction of silver ions and triggers the formation of elemental silver nanoparticles (de Santa Maria et al. 2009; Morena et al. 2019).
The morphology of the surface of the paper supports was evaluated by scanning electron microscope (Fig. 3). Figure 3A shows the typical network structure of bacterial cellulose fibers, approximately 70–100 nm wide and several micrometers long. These BC nanofibers have a high surface area which could help to stabilize the particles avoiding agglomerations. In Fig. 3C, corresponding to oxidized and silver-treated BC (BC-ox/Ag/TT), spherical particles randomly distributed on the surface were observed with the BED-C filter in the same area as Fig. 3B, suggesting that these particles were composed of a high atomic weight element as was the case of silver. The size of these particles was quite homogeneous with a diameter of 26 ± 3.21 nm. For oxidized and silver-treated eucalyptus paper supports (eu-ox/Ag/TT) a similar type of nanoparticles was observed (Fig. 3B and 3C). In this case, the diameter of the particles was 152 ± 43 nm, a larger diameter probably attributed to the also larger pore diameter in the matrix of eucalyptus cellulose compared to bacterial cellulose.Garza-Cervantes et al. (2020) also found a similar average size for their silver-cellulose composite. However, no nanoparticles were found in the non-oxidized samples of eucalyptus and bacterial cellulose, suggesting that the formation of carboxyl groups by SamLPMO10C was necessary for the generation of silver nanoparticles (image not shown). As envisaged, no silver nanoparticles were found in controls without added silver.
The composition of these nanoparticles was analyzed by EDS. The EDS analysis (Fig. 4) shows a signal corresponding to silver and the peaks of carbon and oxygen that also appear in the spectrum corresponding to the components of the cellulose molecule.
To assess the content of silver incorporated into the paper supports Inductively Coupled Plasma (ICP) analysis was carried out. When comparing the BC and eucalyptus matrices, it was evident that eucalyptus incorporated more silver than BC (Fig. 5) which could be attributed to the higher size of eucalyptus pores observed by SEM images. These results agree with those obtained for the size of the particles. When oxidizing the cellulose pulp, it can be expected that by incorporating more carboxyl groups, the negative charges on the surface will improve the dispersion state as suggested by Musino et al. (2021) so the hydroxyl groups, the real nucleation point for metallic nanoparticles, can attract more metal ions. This occurred with BC-ox/Ag/TT where this support had incorporated, on average, 2.3 times more silver than the unoxidized silver-treated BC paper supports (BC/Ag/TT). On the contrary, in the case of silver functionalized eucalyptus, the oxidized eu-ox/Ag/TT supports contained less silver than the non-oxidized support (eu/Ag/TT), in this case, oxidation has not favored the incorporation of more silver ions probably due to the variability of pores sizes. However, the oxidation of cellulose was necessary for the generation of silver nanoparticles as previously explained.
The diffusion of silver from the different functionalized paper supports was also analyzed. For that purpose, 1 cm2 of paper samples were immersed in an aqueous medium and shaking conditions for 24 h. Then, the silver content in the aqueous medium was analyzed by ICP-MS before and after the addition of HNO3. When no HNO3 was added to the cellulose substrates, only silver cations released were measured. When HNO3 was added, the elemental silver was oxidized hence the total amount of silver released was measured. Table 3 shows the amount of silver ions and the total silver that migrated from the matrices to the aqueous media. The difference between the total amount of released silver and silver ions released corresponded to nanoparticles of elemental silver released to the aqueous medium, however statistical analysis (t Student) showed that there were no significant differences, meaning that silver migrated from the matrices as ions and not as nanoparticles. These results agree with the previous work of (Morena et al. 2019), where is described that silver also migrated from a bacterial cellulose matrix in form of ions.
Table 3
Silver leaching from the paper supports
Paper support | Ag+ release Ag+ng /mg of paper ± SD | Total silver release Ag ng/mg of paper |
BC/Ag/TT | 4.3 ± 0.1 | 5.9 ± 0.8 |
BC-ox/Ag/TT | 4.7 ± 0.1 | 6.4 ± 0.2 |
eu/Ag/TT | 18.0 ± 12.9 | 18.9 ± 12.9 |
eu-ox/Ag/TT | 64.5 ± 64.4 | 69.5 ± 70.1 |
When comparing the total amount of silver in the paper supports with that released from them after leaching evaluation, it can be concluded that for both matrices the thermal treatment played an important role in terms of the stabilization of silver within the matrix. Most of the silver incorporated into functionalized paper supports had been retained (Fig. 5), especially in bc supports where the ligand diffusion was as low as 0.9% for BC-ox/Ag/TT and 2% for BC/Ag/TT, the fine network of BC fibers and thermal treatment helped stabilize and trap the NPs embedded in the matrix. For the eu supports the variability of silver uptake was higher and so was the diffusion of it, 1% for eu/Ag/TT and 7.4% for eu-ox/Ag/TT. As pointed out above, eu has higher and more variable pore sizes so this feature can influence silver diffusion. The diffusion of the ligand incorporated into the cellulosic supports is an important characteristic for their future applications, on some occasions, the diffusion of the ligand is going to be a desirable feature (e.g., drug delivery) but on other occasions, the low diffusion is going to be necessary (e.g., sensors).
3.5 Antibacterial properties against Staphylococcus aureus of the paper supports
To determine the effect of Ag-NPs paper supports on cell viability, BC and eucalyptus papers were incubated at room temperature with a suspension of a known concentration of Staphylococcus aureus. Then, viable cells (CFU/mL) were determined after different times of incubation, and the percentage of inhibition was calculated with respect to the control of the untreated paper (Table 4).
After one hour of incubation, there was 100% of inhibition for eu-ox/Ag/TT meanwhile, for BC-ox/Ag/TT, there was 83.3% of inhibition. The slower reduction in viability for BC-ox/Ag/TT may be because in this functionalized paper support the Ag NPs are trapped in the cellulose matrix more efficiently, BC-ox/Ag/TT showed only 0.9% leaching of silver against 7,4% for eu-ox/Ag/TT. The faster effect of the eucalyptus functionalized paper supports can also be attributed to the higher silver content in the paper as well as to the higher amount of silver migrated to the buffer media. The diffusion of silver from the matrix favored the contact between silver and the microorganism, enhancing its toxic effect. Interestingly, there were no viable cells after 24 hours of contact with BC and eucalyptus functionalized papers. Silver salts have long been investigated for their inhibitory and bactericidal effects and potential application for burns (Fox, 1968; Moyer et al. 1965) but their mechanism of action is not quite elucidated. It has been reported that silver ions cause the loss of DNA´s replication ability (Feng et al. 2000). Also, silver ions bind to proteins´ thiol groups promoting the inactivation of bacterial proteins (Feng et al. 2000; Jung et al. 2008)
It is eye-catching that non-treated eucalyptus also presented a decrease in the viable cell count indicating the antimicrobial activity of eucalyptus cellulose itself. This characteristic was reported previously for flax cellulose, where a reduction of 17% of S. aureus viability after 1 h of incubation was observed following the same standard method (Fillat et al. 2018). This antibacterial property of plant cellulose is due to the residual lignin, which contains natural phenols that cannot be found in bacterial cellulose (Spasojević et al. 2016).
Table 4
Viable count (CFU/mL) and viability reduction percentage of Staphylococcus aureus in contact with paper supports containing AgNPs. BC and eucalyptus non-treated samples were used as controls
| | UFC/mL | % Reduction | | UFC/mL | % Reduction |
t0 | BC | 2.4·106 | 0 | eu | 2.94·106 | 0 |
t1 | 2.4·106 | 0 | 1,73·106 | 32 |
t24 | 2.4·106 | 0 | 7.0·105 | 72 |
t0 | BC-ox/Ag/TT | 2.4·106 | 0 | eu-ox/Ag/TT | 2.94·106 | 0 |
t1 | 3·105 | 83.3 | | 100 |
t24 | 0 | 100 | | 100 |
To test the effect of the functionalized paper supports on the metabolism of S. aureus, the BC and eucalyptus papers were immersed in a suspension of known concentration of the bacteria. The tubes were incubated at RT while shaking and the metabolic activity of the cells was determined after 0, 30, 60, and 120 minutes. The samples were incubated with resazurin, and then the fluorescence was measured as a measure of metabolic activity. For eu-ox/Ag/TT paper supports the reduction of metabolic activity occurred faster than for BC-ox/Ag/TT paper supports (Fig. 6). At 30 min there was a reduction of 89% of metabolic activity of S. aureus in contact with eu-ox/Ag/TT and a 24% reduction in the case of BC-ox/Ag/TT, in accordance with the higher silver content and leaching of eucalyptus matrices (Fig. 5). These results indicate that the amount of incorporated silver in the paper supports was toxic to S. aureus. On the other hand, for BC with silver NPs, the reduction of metabolic activity showed to be gradual over time and, only after 2 hours, a 74% of reduction in microbial activity was reached. This slower antimicrobial activity of BC/AgNPs fits with the cellular viability test previously explained.
Eucalyptus control samples showed antibacterial capacity that increased with time even though they did not contain silver, in good agreement with the previous result of reduction of cell viability. It is noteworthy that for BC-ox/Ag/TT there is an 83% of reduction of viability after 60 minutes but only a 35% reduction of metabolic activity. This apparent difference in antibacterial capacity is because the two antimicrobial tests used provide different but complementary, information. The results indicated that in the viability test, the cells were in an active but nonculturable state. For eu-ox/Ag/TT the same thing occurred, the viability was rapidly reduced to 100% after 60 minutes but the cells still showed metabolic activity, although reduced by 89%. This suggests that the silver caused Staphylococcus aureus cells to reach an active but nonculturable state first and then eventually die, as suggested by (Jung et al. 2008).
The two antibacterial tests assayed demonstrated that the eucalyptus and BC paper supports functionalized with silver had strong antibacterial capacity against Staphylococcus aureus due to the contact of the paper support with the bacteria as well as the diffusion of silver in the aqueous media.