Novel research on nanocellulose production by a marine Bacillus velezensis strain SMR: a comparative study

Bacterial nanocellulose (BNC) is a nanofibrillar polymer that possesses unique characteristics such as high chemical purity, mechanical strength, flexibility, and absorbency. In addition, different bacterial strains can form nanocellulose (NC) in multiple shapes and sizes. This study describes the first report of a marine Bacillus strain that is able to synthesize NC. The strain identified as B. velezensis SMR based on 16S rDNA sequencing, produced highly structured NC, as confirmed by X-ray diffraction (XRD) and Scanning Electron Microscopic Analysis (SEM). In Hestrin-Schramm (HS) medium, B. velezensis SMR produced twice the quantity of BNC in comparison to the reference strain, G. xylinus ATCC 10245. The ability of B. velezensis SMR to produce NC using different industrial waste materials as growth media was tested. Growth in Ulva seaweed extract supported a 2.5-fold increase of NC production by B. velezensis SMR and a threefold increase in NC production by G. xylinus ATCC 10245. As proof of principle for the usability of NC from B. velezensis SMR, we successfully fabricated a BNC-based polyvinyl alcohol hydrogel (BNC-PVA) system, a promising material used in different fields of application such as medicine, food, and agriculture.

www.nature.com/scientificreports/ and hemicellulosic materials 25,26 . Moreover, it has been shown that B. velezensis strain 157 is able to degrade various agro-industrial byproducts including soybean meal, wheat bran, sugarcane bagasse, wheat straw, rice husk, maize flour and maize straw utilized in biofuel production. B. velezensis strains were also investigated for their ability to depolymerize various types of lignocelluloses into fermentable sugars 27 . Nair et al. 27 isolated B. velezensis ASN1 that could synthesize cellulase used in food, textile, animal feed, petroleum, waste management, biosurfactant, and pulp/paper industries. With respect to BNC production, B. velezensis SMR was compared to the reference strain G. xylinus. Both strains were cultured in HS broth at pH 5 and 30 °C under static growth conditions for ten days. The produced BNC appeared as a white highly flexible membrane with artificial-looking leather (Fig. 3a,b). The nanocellulose product was purified and estimated as dry weight. It was noticed that after 10 days of incubation, B. velezensis SMR produced an average of 5.2 g/L NC compared to 2.6 g/L biosynthesized by G. xylinus. The residual glucose concentration in g/L was measured every two days for ten days to monitor the glucose consumption (see Supplementary Fig. S1). In the case of G. xylinus, a major problem in using glucose as a carbon source is the formation of by-products including gluconic acid that decreases medium pH, which inhibits BC production 28 . Several options to tackle this problem would be to optimize glucose feeding strategy and/or to use alternative carbon sources that do not trigger the production of by-products 28 . Fourier-transform infrared spectroscopy (FTIR). BNC samples of G. xylinus and B. velezensis SMR were analyzed by FTIR in comparison to plant cellulose (derived from dried rice husk) (Fig. 4). The observed hydroxyl groups (OH) in C1, C3 and C6 mainly contribute to the formation of various kinds of inter-and intramolecular hydrogen bonds and reflect the tendency of all NC samples to be hydrophilic. It has been shown that hydrogen bonds formation between cellulosic fibers and other materials gives rise to great benefits for the research on all other aspects of natural fibers and related materials as stated by Fan et al. 29 . The FTIR spectra and the peak positions of the major IR bands were compared to data in the literature [30][31][32][33][34] (Table 1).
Scanning electron microscopy (SeM). SEM micrographs of BNC (Fig. 5a,b) show the compact nanocellulose network structure consisting of a random assembly of fibrils (see Supplementary Fig. S2). Both purified BNC pellicles exhibited a reticulated structure consisting of ultrafine nanofibrils (Fig. 5c) (see Supplementary  Fig. S3). Nanocellulose fibers created by B. velezensis SMR had a diameter range from 1 to 60 nm with mean particle size xc1 = 9.389 nm as obviously observed from the nanoparticle size distribution curve (Fig. 5d). Previous  www.nature.com/scientificreports/ study concerning the production of BNC excreted by G. xylinus in HS medium declared that the obtained fibers diameter was in the range of 1 nm to 120 nm with an average of 60 nm 35 .
X-ray diffraction. X-ray diffractometer (XRD) was applied to determine the grain size and crystallinity of the produced NC. XRD analysis of G. xylinus-BNC (Fig. 6a) 35 . These observed peaks correspond to the crystal planes <110> , <110> , <200> and <004> , respectively. The grain sizes calculated from Scherrer equation Eq. (1) were 42, 42, 17, and 43 nm, respectively with a major peak observed at 30.003° with 17 nm.   www.nature.com/scientificreports/ The crystallinity of rice husk fibers was typical to cellulose with three well-defined crystalline peaks around 2θ (11.996°-18.995°) with peak position at 16.006°, (18.995°-24.994°) 21.994°, and (32.003°-36.002°) 34.406° (Fig. 6c). These characteristic peaks for cellulose are corresponding to the lattice planes <110> , <200> and <0 04> , respectively. The grain sizes calculated from Eq. (1) were 21, 28, and 22 nm, respectively 30 . According to Eq. (2), the Crystallinity Index (CrI) of BNC produced by G. xylinus, B. velezensis SMR, and rice husk-NC was  thermogravimetric analysis (tGA). Thermogravimetric analysis was performed to study the thermal degradation behavior of the three NC samples (Fig. 7). The primary alteration was attributed to the vaporization of water content due to the hydrophilic feature of the cellulosic fibers, which appeared for BNC samples from G. xylinus (a), B. velezensis SMR (b), and rice husk-NC (c) at 177.6 °C (43.85% wt loss), 141.5 °C (60.61%) and 217.5 °C (9.86%), respectively. The weight loss was dependent on the primary moisture content of the examined materials. The NC samples a, b, and c exhibited a major degradation process at 540 °C (54.56% wt loss), 170 °C (70.15%) and 481.4 °C (81.30%) leaving 45.44%, 29.85% and 18.7%, respectively, as remaining mass residues (Table 3) (see Supplementary Fig. S4a-c). BNC of G. xylinus was estimated to be stable at temperature 540 °C before complete degradation, while the stability of B. velezensis SMR-BNC and rice husk-NC was until 170 °C and 481.4 °C, respectively. This shows that marine B. velezensis SMR-BNC had less stability but higher water content which makes it a good biomaterial for  Formation of bacterial nanocellulose /polyvinyl alcohol (BNC/PVA) hydrogel. Polyvinyl alcohol (PVA) hydrogel containing BNC was prepared by direct dispersion of the nanofibers in an aqueous PVA solution (20%) as illustrated in the schematic diagram (Fig. 8). Polyvinyl alcohol (PVA) is a water-soluble polymer that www.nature.com/scientificreports/ has been extensively investigated because of its good biocompatibility and mechanical properties. PVA solution can form rigid hydrogel through freeze-thaw cycles. During freezing, the PVA chains interact to form a matrix which acts as physical cross-links, maintaining the insolubility of the material in water. Similar to the BNC-PVA hydrogel system fabricated in this work, Millon et al. 38 produced a PVA hydrogel reinforced with BNC with mechanical properties analogous to cardiovascular tissues. Castro et al. 39 percolated BNC with PVA; the BNC-PVA systems were subjected to the freeze-thaw technique to promote the physical cross-linking of PVA. Li et al. 40 investigated the effect of the amount of freezable bound water on the BNC hydrogels and physically cross-linked PVA. There was a significant increase in the amount of freezable bound water with more than 20% PVA. In this state, the water molecules were more strongly attached to the hydrogel, which hindered the loss of water and crack formation under compression. production of Bnc using industrial and agricultural wastes. In this study, we used several industrial and agricultural wastes, which included sugarcane bagasse, molasses, rice bark, rice husk, palm fronds, paper  www.nature.com/scientificreports/ waste, and dried algal biomass. The dry weight of BNC produced by the two examined strains using various wastes in g/L was assessed ( Table 4). The use of the green alga Ulva sp. in seawater based medium was the most promising substrate yielding 13 and 9.6 g/L with B. velezensis SMR and G. xylinus, respectively. Previous studies successfully used various wastes for BNC production 28,35,41 . Sugarcane molasses was used in seawater-based media for production of bioethanol using a marine yeast strain 42,43 . Using molasses, BNC levels were estimated as 5.6 and 5.1 g/L using G. xylinus, and B. velezensis SMR, respectively. These amounts are much higher than that obtained by G. saccharivorans MD1 3.9 g/L after incubation for 168 h 31 . Moosavi-Nasab and Yousefi 20 studied the feasibility of using low quality date syrup a fruit largely produced in the hot arid regions of Southwest Asia and North Africa, for the production of BNC using G. xylinus.
conclusion Bacterial nanocellulose is a promising material in nearer future for its unique properties and wide range of application in industry, technology, biotechnology and medicine. We herein report an economical approach for the production of nanocellulose by a marine Bacillus species using alga biomass as growth medium, which is a cheap substrate widely found in the seashore of the Mediterranean Sea. Moreover and to reduce the environmental footprint of the bio-production of NC, we demonstrate the potential of using seawater instead of distilled water in the growth medium. To the best of our knowledge, this is the first report to show that a bacterial isolate belong to the genus Bacillus is able to synthesize NC. In addition, we highlight the ability of B. velezensis SMR to produce NC at a comparable or significantly higher level in comparison to G. xylinus, which is traditionally used as a reference strain for NC production. Morphological, physical and structural analysis of NC produced by B. velezensis SMR show good mechanical stability, tensile strength, and crystallinity. As proof of principle for the usability of NC from B. velezensis SMR, we successfully fabricated a BNC-based polyvinyl alcohol hydrogel (BNC-PVA) system, a promising material used in different fields of application such as medicine, food, and agriculture.

Materials and methods
Samples. Samples of acidic rotten fruits such as pomegranate, tomatoes, grapes, strawberries, guava, and lemon utilized for the isolation of NC-producers were collected from markets, juice-bars, and used after incubation at room temperature in sealed glass jars for 1 week. Mediterranean seawater was also utilized as a source of isolation.
Bacteria. The marine bacterium used throughout this study was isolated from seawater and identified using Screening for nanocellulose producers. Isolation was performed using serial dilution technique 45 . One milliliter of each sample was used to inoculate agar plates of HS medium and incubated for 24-48 h at 30 °C. Colonies were picked up and purified. A loopful of each isolate was inoculated into 50 mL of the same medium in100 mL conical flasks and incubated statically at 30 °C for 7 days. Flasks with white pellicle on the surface of the growth medium were selected for further analysis. The pellicles formed at the air-liquid interface were collected by centrifugation at 6,000 rpm for 10 min, treated with 1 N NaOH at 80 °C for 15 min, and washed 3 to 4 times with distilled water 6 . Fehling test was used after acidic hydrolysis to confirm the carbohydrate nature of the products to facilitate selection of exopolysaccharide producers. Table 4. Mean values of BNC produced (g/L) of B. velezensis SMR and G. xylinus. Yield was calculated as % based on the total glucose (20 g).

BNC (g/L) Yield (%) Productivity (g/L/time) BNC (g/L) Yield (%) Productivity (g/L/time)
Glucose (control) 20  production of bacterial nanocellulose. Seed cultures of both strains were prepared after 2 days of incubation at 30 °C. One milliliter of each was used to inoculate 50 mL HS medium (2% v/v), in 100 mL conical flask, followed by incubation statically for 10 days at 30 °C. The thin film of BNC formed on the surface of each flask was collected, treated with 1 N NaOH, washed with distilled water and dry weight was determined in g/L. The remaining glucose in the media was measured every 2 days over an incubation period of 10 days using ANA-LYZER A25 Biosystem 30 to study the correlation between BNC production and glucose consumption.
Scanning electron microscopy (SeM). Nanocellulose produced by both strains were examined by scanning electron microscope JEOL JSM-5300 in the Electronic Microscope Unit in Alexandria University.

X-ray diffraction (XRD).
The XRD diffractogram of samples was obtained on X-ray powder diffraction-XRD-D2 Phaser (Bruker, Germany) in the Central Laboratory, Faculty of Science, Alexandria University using copper X-ray source operating at 30 kV and 10 mA. Scans were collected at 2° per min in the 2θ range of 10°-100°. The crystallite size was estimated using Scherrer's Eq. 49 : where (k) is the dimensionless Scherrer constant = 0.94, (λ) is the X-ray wavelength = 1.54184 nm, (β) is the peak full width at half maximum in radians, and (θ) is the diffraction angle in radians. The Crystallinity Index CrI (%) was calculated according to the method reported by Zheng et al. (2019) 50 as follows: where CrI was calculated from the ratio of the height of the maximum (I max ) and the height of the minimum (I am ). thermogravimetric analysis (tGA). TGA is a thermo-analytical technique used to determine the thermal stability of a material and its fraction of volatile components by monitoring the weight change that occurs when a sample is heated at a constant rate. TGA analysis was performed with a LINSEIS STA PT1000 (Germany) Thermal Analyser in the Central Laboratory, Faculty of Science, Alexandria University. For the thermal decomposition behavior test, cellulose samples were dried at 50 °C for 48 h. Water content was calculated by the following equation 51 : where W 0 and W t represent the weight of dried and wet NCs, respectively.
Bacterial nanocellulose (Bnc) hydrogel preparation. Polyvinyl alcohol (PVA) hydrogel containing BNC was prepared by direct dispersion of the nanofibers in an aqueous PVA solution (20%). The PVA/BNC suspension was subjected to freeze-thaw cycles at − 20 °C. The decrease in the crystallinity of BNC reinforced PVA was compensated by the strong interaction and miscibility between the components. The strength of the gels increased in the order of 1.5 wt% > 0.75 wt% ∼ 3.0 wt% > pure PVA 52 . production of bacterial nanocellulose using industrial and agricultural wastes. Waste products such as Palm frond, Sugarcane bagasse, Paper waste, Rice husk, Rice bark, and Ulva sp. (Fig. 9) were dried and grounded into powder and subjected to heat treatment followed by acid H 2 SO 4 -heat treatment (see Supplementary Fig. S5) according to Annamalai et al. 53 . Molasses was diluted five-fold with distilled water and centrifuged at 6,000 rpm for 20 min to separate solid materials before the acid H 2 SO 4 -heat treatment.