A turning point in the bacterial nanocellulose production employing low doses of gamma radiation

The global concerns about the rising environmental hazards have increased year after year. Conceiving sustainable and green materials to substitute the non-eco-friendly synthetic polymers and the derivatives of the fossil oil has become a cosmic priority²¹. Cellulose is the most abundant natural polymer, where it comprises almost 30% of the worldwide plant matters. The universal cellulose production -mainly from plant source- estimated to be 10¹⁰–10¹¹ tons annually, and about 6 × 10⁹ tones has its way to the paper, textile, materials and chemical industries²².

Bacterial nanocellulose (BNC) represents a revolutionary shift to more sustainable processes, where it can be produced utilizing wide ranges of wastes and with fair effluents; constituting a high value-added and green product. Moreover, BNC is a cellulose in a fully pure form; there is no effort-, money-, or energy-wasting extraction methods are demanded, contrary to the plant cellulose²³.

Identification BNC-producing strain

In the present study, the bacterial strain Komagataeibacter hansenii KO28 was employed to produce BNC. The strain isolated and identified through morphological and physiological examinations, in addition to the partial sequencing of the 16 s rRNA gene ²⁴. More data about isolation and identification protocols can be found in the methodology part and supplementary information.

Gamma irradiation scheme

Figure 1 depicts the irradiation scheme we implemented to induce genetic mutation to the strain Komagataeibacter hansenii KO28. Briefly, the plan comprised treating the strain cultures with gamma ray viz. 0.5, 1, 2, 3 kGy with singular and dual doses in parallel, prior the BNC production was conducted from the irradiated cultures versus the control one (non-irradiated) on Hestrin-Schramm (HS) medium for 10 days to inspect the impact of irradiation doses on the BNC productivity.

It seemed that the gamma irradiation with doses ≥ 2 kGy (singular or dual) were either not supportive for the BNC production or even lethal for the culture cells; where no BNC was observed in the cultivation vessels.

Meanwhile, the doses 0.5, 0.5D, 1, and 1D kGy exerted distinctive enhancing influence on the BNC productivity (g/L), where the BNC yield escalated to reach 4.3, 10.5, 5.5, and 7 g/L, respectively, (Fig. 2c); versus only 2.2 g/L by the control culture. The digital images in Fig. 2 compare the cultures and the resulted BNC films of the control and irradiated bacterial cultures.

Cheng et al. reported that gamma ray-driven mutagenesis can trigger genetic or metabolic alterations in the treated cells, where the gamma ray interacts with the intracellular molecules, especially water molecules, emerging free radical flood. These plenty of free radicals strike the key functional genes and cause gene devastation and recombination¹⁹. We assumed that further examinations were necessary to configure the reasons beyond excessive BNC yield synthesized by the irradiated cultures.

Time course of BNC production

We monitored the BNC production by the control and irradiated cultures of the bacterial strain Komagataeibacter hansenii KO28 throughout ten days, recording the BNC yield of each culture every 24 h.

The outcomes of this investigation indicated that the general BNC production rate is relatively following comparable tendency for both the control and the irradiated cultures (Fig. 3a). The primary BNC composition emerged after 24 h of starting incubation and kept rising until the 8th day and the 9th day for the control and irradiated cultures, respectively.

Remarkably, The BNC production exhibited by the culture treated by dual 0.5 kGy doses appeared in a uniquely climbing rate, where more than 92% of the whole BNC yield was generated in the first six days, prior the BNC production rate had slowed down in the next four days until the incubation terminated.

We supposed that the irradiation dose either enhanced the proliferation rate of the culture, and subsequently increase the producing units (i.e. number of cells), or immensely improves the efficiency of the singular bacterial cell to produce the BNC by polymerizing every available glucose residue into nanocellulose chains.

Regarding the hypothesis of the impact of irradiation scheme on the number of cells, there were no serious variation in the turbidity of the control and irradiated cultures or the shape of the produced films after ending the production time course. Moreover, the cells entrapped in the thick films of the irradiated cultures made the precise comparison of the CFU comparison for the five cultures quantitatively is impractical.

Alternatively, we speculated that concluding the assimilated glucose (ΔS) for the five cultures could be a good proof about the irradiation-relied metabolic modifications. (ΔS) value for any culture can be estimated from the difference between the medium glucose concentration at the beginning and end of the incubation period (g/L). Accounting the (ΔS) values for the five cultures revealed that the irradiated cultures exerted higher competency of glucose assimilation than the control culture in general. Furthermore, the proportions of the exploited glucose were fully relevant to that of the BNC yield for the five cultures (Fig. 3b).

Another clue could be deduced from the pH trend of the five cultures throughout the whole incubation time course. Figure 3c declares the major variation of the pH tendency of the control and irradiated cultures of the strain K. hansenii KO28 throughout 240 h of incubation. The pH of the control culture showed accelerated and constant declining trend continued to the end of the incubation period, reaching 2.9. Meanwhile, the irradiated cultures followed the same trend but with more flattened pH decrease, to record pH values ranged between 3.5 and 3.9.

Worthy to mention that the pH decrease associated with the cultivation of the genus Komagataeibacter is correlated with a distinctive metabolic feature of excessive gluconic acid synthesis once the cells begin to assimilate glucose residues, hence the culture pH turns more acidic. This metabolic activity is always corresponding with the bacterial propagation, and the consumed glucose units is certainly subtracted from the glucose capital which would be polymerized into BNC¹¹, together with the impairing effect of the intense acidic environments on the BNC productivity²⁵. Herein, the pH trendlines of the irradiated cultures in general were not compatible with the higher glucose assimilation rates exhibited in Fig. 3b. Therefore, we presumed that the gamma ray treatment in our case was capable of redirecting the metabolic system of the irradiated cultures, in varied extents, to maximize the glucose consumption for the BNC production pathway rather than increasing the multiplication rate, and gluconic acid generation consequently.

The hereditary stability of any microorganism is a critical issue considering their extraordinary rate of multiplication. It was broadly reported that the nanocellulose-producing genus Komagataeibacter may irreversibly discontinue the production process (termed Cel^– mutants) as a result of experiencing a physical or chemical unusual conditions such as vigorous agitation⁸.

Herein, we pursued the impact of the irradiation scheme on the potentiality of nanocellulose production of the strain Komagataeibacter hansenii KO28 for nine generations of the control and the four irradiated cultures, where the cultures undergone the irradiation treatment was designated (generation 0). Figure 3d reveals that all the examined bacterial cultures exhibited accountable steady BNC yield trendlines by their first seven generations, while the BNC productivity declined starting from the 8th generation. This impairment was affirmed by the lower BNC outcomes by the 9th generation of the control and irradiated cultures, suggesting that the applied irradiation doses had no serious influence on the hereditary stability of the studied cultures from one generation to another, where the subsequent generations of the four irradiated cultures exposed a BNC production tendency almost similar to that of the control culture.

According to the United States Pharmacopeia (USP) recommendations for the best microbiological procedures, the original strain should be preserved in multiple copies, where the number of sub-culturing should not exceed five times to avoid the spontaneous phenotypic alterations or mutations in the bacterial cells²⁶. Therefore, we assume that the irradiated bacterial cultures will exhibit no susceptibility regarding the BNC productivity as long as such recommendations are appreciated.

Microstructure by FESEM

The fibrous morphology of the BNC products of the control and the four irradiated cultures were compared by FESEM inspection.

The FESEM imagery in Fig. 4 shows a typical shape of the 3D nanofibrous web, where the thickness of the BNC nanofibers ranged between 5 and 85 nm. The examination of the five BNC specimens manifested no significant contradistinction between the control and irradiated BNC products from one side, or even among the four irradiated specimens regarding the fiber’s diameters, or the branching patterns.

Florea and his coworkers reported that the strain Komagataeibacter hansenii 53,582 regulates the BNC production via main four systems: acsA, B, C, and D, where acsA and acsB responsible for polymerizing UDP-glucose residues into cellulose chains. Meanwhile, the acsC is an outer membrane pore system acts for extruding the formed cellulose chains, where their crystallinity is regulated by the periplasmic system acsD²⁷. Aside from influencing the BNC yield, we suppose that the irradiation doses had a mild or no impact on the acsC pore systems, where the fibers microstructure of the control and irradiated BNCs showed no considerable variations.

Surface area investigation

The BET surface area of each BNC product was determined to inspect the influence of the culture irradiation on the corresponding microstructure of the specimen.

Herein, the examined BNC samples showed a variable values of specific surface area (Table 1), where the highest value was shown by the BNC-1, while the control BNC product came second, recording 62.3 and 54.4 m²/g, in respective order. Regarding the mean size of pores, the BNC-1 showed the largest pore size (27.2 nm), while the BNC-05D and BNC-1D came in the second and third extents with comparable values (22.16 and 21.3 nm, respectively). In terms of the pore volumes, the whole BNC specimens showed relevant values, except the BNC-1D and the BNC-control which recorded 0.29 and 0.26 cm³/g. However, the proportion of the nitrogen adsorption by each BNC sample is directly proportional to its surface area (m²/g). Ashrafi et al. elucidated how the gas adsorption–desorption inspects the operating pores, in addition to the closed channels and blind pores which have no actual association with the material permeability²⁸.

Water retaining properties

The capability of the BNC to retain water has a substantial character for multiple applications such as; biomedical applications, food and food additives, adsorption, and not ending to the cosmetics and pharmaceutical emulsions⁶.

The water holding capacity (WHC) of the BNC is mainly owing to the multiplicity of the hydroxyl groups projected all through its adjacent glucan chains, which contribute with the elevated porosity and surface area per mass unit to endow the BNC with its prominent WHC¹⁰.

Here, the results manifested that the employed irradiation plan not only affected the BNC yield, but the ability of the BNC products to retain water as well. The BNC-05D exhibited the highest WHC improvement; recording about 116 g/g compared to the BNC generated by the control culture which showed only about 95 g/g (Fig. 5a). The BNC-05 and BNC-1D products came in the second and third ranks with comparable WHC values; 110 and 109 g/g, respectively. Finally, the BNC-1 film showed the lowest WHC improvement with only 100 g/g.

Referring to the water release rates (WRR) of the BNC products, it seems that the irradiation treatment had an impact on the potentiality of each BNC film to retain water, where all the four BNC products, comparing to the BNC-control, exerted higher invulnerability against dryness. Figure 5b depicts how the BNC-control film lost almost all its water content throughout only 40 h, while the BNC films produced by the irradiated cultures, particularly the BNC-05D, BNC-1, and BNC-1D, revealed prolonged water evaporation rates taking up to 72 h. At the test end, all the tested films exhibited fully dry texture, which was confirmed by constant weights even with the extended maintaining in air as a sign of the full excluding of moisture.

Rebelo and coworker reported the impact of the thickness and diameter of the BNC film on its dehydration time; postulating that the higher the thickness and/or diameter of the film, the slower the water evaporation rate²⁹. Hence, in our investigation we examined films of the same diameter to avoid the resulting overlapping. Nevertheless, we predicate that the WHC and WRR of the BNCs were affected by the varied thickness of films.

Additively, the impact of the irradiation scheme probably extends to affect the crystalline and chemical composition (namely the density of the hydroxyl group). Therefore, we assumed that extra examinations of the physical and chemical features of the produced BNCs would be inevitable.

Mechanical properties

We evaluated the stress–strain performance of the produced five BNC films to investigate the impact of the applied irradiation scheme on the mechanical properties of the produced BNC. Worthy to mention that the mechanical properties of the produced BNC are significant for a wide range of applications such as textile fabrication³⁰, food packaging³¹, cementitious compositing³², and not ending with the biomedical applications³³.

We summarized the values of tensile (MPa), elongation at break (%), and Young’s modulus for the five BNC products in Table 2. Regarding the tensile aspect, the BNC-05D film exhibited both the highest mean values of the tensile and Young’s modulus; recording an improvement of approximately 12 and 18%, respectively, if compared with the corresponding values of the BNC-control.

The BNC-1 attained the second highest tensile improvement comparing to the BNC-control, while its ductility was superior out of the whole tested films; representing about 1% improvement in the elongation at break comparing to the BNC-control.

Cellulose polymer is composed of self-assembled chains of β-1,4-linked anhydrous D-glucose units where the intermolecular hydrogen bonding and the Van der Waals forces promote the stacking of the adjacent chains to nanofibrils, which are further gathered to form larger fibrils. Furthermore, the molecular structure of these fibrils varies between crystalline and amorphous regions, where many factors govern which type is dominant and the proportion of each one. This unique crystalline structure is the main character which confers on the cellulose nanofibers some of their unparalleled properties such as the mechanical properties³⁴. Molnàr et al. studied in detail the correlation between the crystalline structure and the mechanical behavior of the cellulose nanofibers³⁵.

Martin-Martinez explained how the understanding of the behavior of the cellulose crystals is significant for designing up-to-demand nanocellulose products through a “bottom-up” process. Subsequently, this will make the manipulation of the nanocelluloses more facile and provide broader application fields³⁶.

X-ray diffraction

We accomplished a broad study of the diffractometry of the BNC produced by the control and irradiated cultures of the strain K. hansenii KO28 to scrutinize the impact of the applied irradiation doses on the crystalline structure of the BNC artifacts, in terms of the crystallite size (CrS), inter-planar distances (d-spacing), and crystallinity index (CrI %).

Figure 6 represents a comparison of the five X-ray diffraction portraits, where they showed up in the characteristic diffraction profile of cellulose I, including three main peaks; d1, d2, and d3.

The peak d1 appeared at 2θ = 14.66° ± 0.3°, as a mean value of the peaks of the BNC products, and ascribed to (100) plane of I_α or to the plane (1–10) of I_β. The peak d2 spiked at 2θ = 16.7° ± 0.2°, and accredited to (010) plane of I_α or (110) plane of I_β. The d3 at 2θ = 22.4° ± 0.1°, and could be a contribution of (110) plane of I_α, the (200) plane of cellulose I_β.

Moreover, the percentage of the crystalline allomorph in the produced five BNCs appeared to be dissimilarly influenced by the irradiation treatment. Whilst, the BNC-control comprised about 74%, the doses of 0.5 kGy and dual 1 kGy relatively had a mild impact on the percent of the crystalline allomorph; recording about ± 1% change.

On the other hand, the doses of dual 0.5 and 1 kGy appeared to exhibit a distinctive effect on the crystalline proportion in the produced BNC, where their products’ crystallinity attained about 80% and 77%, respectively.

The quantitative estimation of the crystallite sizes (CrS) and the d-spacing (d) for each peaking lattice are enumerated in Table 3. Out of the inspected five BNC products, the irradiation dose 0.5 kGy appeared to have a dramatic impact on the crystallite size. When the culture treated with a single 0.5 kGy dose, the BNC exhibited the highest crystallite sizes in the planes (100) and (1–10), while treating the culture with a dual 0.5 kGy dose plummeted the crystallite size to record the lowest values out of the whole five products.

This obviously proposed that applying the second irradiation 0.5 kGy dose had a distinctive impact on the periplasmic acsD system commanding the cellulose chain crystallization, resulting in relatively tinier crystallites. However, the case was dissimilar to irradiation by the 1 kGy or the dual 1 kGy doses, suggesting that the irradiation doses > 0.5 kGy promoted some amendments in the cellular metabolism or the BNC yield, but it does not extend to the crystalline structure such as the crystallite size or the d-spacing.

Thermogravimetric analysis

The outcomes of the thermogravimetric analysis showed that the five BNC products exhibited almost the same decomposition pattern throughout the scanned temperature range (Fig. S2).

The derivative thermogravimetry (DTG), plotted in Fig. 7a and listed in Table 4, declares that the decay temperature degrees for the produced BNCs ranged from 328 to 336 ℃. Comparing to BNC-control, it can be concluded that the applied irradiation scheme for the bacterial culture had a mild or no impact on the BNC structural bonds responsible for fastening the cellulose nanofibers and microfibers to each other, keeping them firmly attached to grant the BNC its extraordinary mechanical properties.

Fourier transform infra-red analysis

The results of the IR investigation for the five BNC products were depicted in Fig. 7b, and the band assignments were listed in Table 5¹⁰. Moreover, the two bands emerged at 1540 and 1640 cm^-1 for the five examined samples are assigned to the amide bond, which are most probably correlated to the protein remnants and cellular debris which kept attached to the BNC samples even after the washing steps. However, we assume that it is anticipated considering the elevated thickness of the BNC films produced by the irradiated cultures.

The IR profiles of the five BNC products were correlated with cellulose I, without clear evidence for existence of cellulose II. Moreover, the bands manifested at 3240 and 3270 cm^-1 affirm the existence of both I_α and I_β allomorphs in the tested BNC. The crystalline I_α fraction (F_α^IR) was estimated for the five BNC products Table 6 as an attempt to configure the influence of the irradiation application on the crystalline properties of the BNC products. It is clearly evident that the I_α amount in the produced BNC samples did not greatly affected by the applied irradiation scheme to the bacterial cultures.

Interestingly, the band at 3340 cm^-1, assigned for the hydroxyl group, proved higher hydroxyl group density for the irradiated samples rather than that of the control. These outcomes are consistent with the previously revealed WHC results, explaining how the BNC-control possesses inferior capability of water retaining, out of the five studied BNC products.

We presume that this data can stand beside those concluded from the XRD to give a clear figure for the structural characters of the produced BNCs.

Ultimately, we believe that the outcomes of our research are a massive leap forward in defining the best technique for feasible BNC mass production. Examining the capability of the irradiated strain to produce high yielded BNC via assimilating low-cost substrates, as well as revealing the impact of irradiation dosages on BNC yield and characteristics through a genetic perspective are listed on our future workplan.