Green Immobilization of Glucanobacter xylinum onto Natural Polymers to Sustainable Bacterial Cellulose Production

Bacterial cellulose (BC) has profound applications in different sectors of biotechnology due to its unique properties which made it preferring it about plant cellulose. Although this polymer is extremely important in various applications, many problems still hinder the sustainable production in terms of increasing productivity and low-cost production. In order to overcome these problems, the continuous production potentiality of cellulose using immobilized Glucanobacter xylinum cells onto Sugar cane bagasse (SCB) and Ca-alginate beads will be evaluated. Comparatively, adsorption of Glucanobacter xylinum cells to the cavum of stalk cells of SCB could be efficiently stable while, entrapment of cells onto Ca-alginate has drawback observed by the rapid disruption and instability of the beads in the Potato Peel Waste (PPW) culture medium. Furthermore, the FT-IR, XRD and SEM analysis of the BC derived from immobilized cells on SCB observed a higher crystallinity (86%) than that produced from immobilized cells on alginate beads. Consequently, BC production was statistically optimized by SCB-immobilized cells using Plackett–Burman Design. Among seven selected variables, incubation period and pH value were found to be the highest significant parameters. Reusability of immobilized biomass was studied and showed continuous BC production even after five cycles without losing their activity. Our findings demonstrate that a combination between alternative low-cost medium with continuous production mode by immobilization onto inexpensive natural polymer can promote a sustainable bioprocess and reduction the production cost.


Introduction
Cellulose is one of important biopolymer on the earth. Although many bio-applications depending on cellulose as raw material [1][2][3], but some bio-fields cannot use the traditional cellulose [4,5]. Traditional cellulose produced mainly from tree fibers or wood pulping followed by many chemical process reactions to produce pure cellulose with some traces of contaminants [1,6]. On the other hand, the bacterial cellulose (BC) can be offered pure type of cellulose use, in all biofields without any restrictions [4,5]. Moreover, the BC produced overall via green methods without any environmental hazardous. However, the production of BC has many problems must be overcome to involve into the industrial levels e.g. contact performance of microorganism with media, productivity value, features of produced BC, economics factors and production strategy applicability [7]. The specific features of BC can be summarized as high biocompatibility, biodegradable, mouldability, easy handling, low equipment's required and free from contaminated ions [8]. The BC can be considered as the new generation material in many applications especially medical, pharmaceutical, biomaterials industries. Furthermore, according to the high safety profile of BC it can be considered as edible biopolymer with platform of bio-field uses. However, the biggest drawback of BC production is the productivity manipulation [7,9]. Since the production scheme can be controlled the productivity as well as characterization of BC. But, the BC production suffers from many inhibitory effects such as high operating cost, rapid consumption of the substrate, rapid alteration in cultural pH value, thus low BC productivity [4,10]. The inevitability of BC production from agro-industrial natural waste has been recommended by many authors because of its economic and environmental viability [4]. The potentiality to use the various wastes resulting from the food-processing in the hydrolysate form is effectively attractive for scalable BC biosynthesis [11][12][13]. Among the several agricultural waste advised by many studies for bacterial cellulose production, Potato peel wastes (PPW) have been shown to be practical, economical and environmental friendly [4]. Traditional industrial BC production is generally performed using a free-cell system. Several approaches have been suggested to improve BC production efficiency, involving supplementation of the cultural medium with some regulators such as ethanol or organic acids in order to inhibit the accumulation of the basic metabolic by-product (gluconic acid) and at the same time stimulate the synthesis of substances necessary for the cell stabilization [14,15]. However, these additives are not suitable, due to metabolic process in the bacterial cellulose cells comprised of series of enzymes which are extensively active in the presence of cyclic diguanidine monophosphate (c-di-GMP) [16]. As discussed by [14,17] c-di-GMP is a major metabolic molecule called as "quorum factor," since the highly cells concentrations were correlated with quorum state. Therefore, cells that produce BC should be stimulated to come into a quorum state, which the cells become genetically programmed to their increased population. The cellimmobilization system in case of BC producers could allow obtaining highly concentrated populations of cells since BC synthesis would be regulated by a quorum sensing phenomenon as described before. Interestingly, the immobilization of BC cells opens a way to improve cell stabilization and thus led to increase BC productivity. Comparatively, the immobilized cells have various benefits more than free cells in the production process, such as increased the cell population density, improved operational stability by protection the cells from the adverse environmental conditions, prevent the inhibition effect of the end product, enhance the cell resistance to high substrate concentration via diffusional constrains and afford microbial cells to reusability it in several bioprocess which reduce the production costs [14,18]. Unfortunately, in spite of the overall advantages of immobilization process the current reports concerning the production of BC by immobilized-cell system are very rare. In this regard, PVA cryogel was used for employing Komagataeibacter xylinum cellsin an immobilized system to increase the biosynthesis of BC [14]. Acetobacter xylinum ATCC 700,178 cells was successfully immobilized on a plastic composite support (PCS) to improve the BC production on the basis of polypropylene [19]. However, the severe mass transfers restrictions, low mechanical strength, non-biodegradability and highly toxicity of these synthetic polymers displaying a big problem in the operational stability of the immobilized cells [18,20]. Therefore, we tried to find out a renewable, easily prepared, inexpensive, biodegradable, nontoxic, and available naturally carrier. Limitations correlated with the use of synthetic polymers can be avoided by the use of Sugarcane bagasse (SCB) as immobilization carrier. SCB considers as a lignocellulosic material derives from the processing of sugarcane which naturally in abundance, easy to use, cheaper and non-toxic [20][21][22]. Selectivity of SCB in several studies due to its having various unique characteristics such as chemical stability, highly porous, high surface area, remained unchanged under different pH and temperature values [20]. In addition, alginate beads was frequently used in the immobilization technology, which proved to be an efficient support for entrapment of microbial cells due to their biodegradability, low toxicity, prepared easily, and low cost efficiency [23]. On the other hand, bioprocess production frequently needs to optimize its nutritional and cultural conditions by statistical experimental designs. The important of these designs are attributed to its reduction in time consumption and a reduction in operating costs due to fewer experimental units [24]. To the best of our knowledge, there are no studies have yet been established on the statistical optimization of BC production using immobilized-cell system despite its high industrial applications. Therefore, we investigated the enhancement of bacterial cellulose production by immobilized G. xylinum ATCC 10245 by using statistical analysis in PPW medium via continuous production of BC. The comparative study of fibrous SCB and nonfibrous alginate as an immobilization carrier was carried out as well as the BCs produced from both free and immobilized cells were also characterized. Moreover, studies have been done to perform reuse experiments with the immobilized cells and storage stability.

Materials
Bagasse fibers were delivered from integrated sugar industrial company, Quena, Egypt. Na-alginate purchase from molekula (U.K.), Potato peel waste (PPW) was resulting from potatoes processing, and collected from the disposal of free markets. The bright PPW without disease symptoms were selected then washed thoroughly with distilled water. All reagents, solvents, medium and its components used in this study were of analytical grade.

Microorganism and Culture Condition
The cellulose producing bacterium Glucanobacter xylinum ATCC 10245 used in this study was donated from the American Type Culture Collection (ATCC), Manassas, VA, USA. The bacterial strain was maintained by bimonthly transfer to fresh HS medium (glucose, 1%; peptone, 0.5%; yeast extract, 0.5%; K 2 HPO 4 , 0.27%; MgSO 4 , 0.05%; citric acid, 0.115%); and stored at 4 °C, after incubation at 30 °C. Preculture of the strain was carried out at 30 °C on a rotary shaker at 180 rpm for 24 h.

Adsorption of Bacterial Cells in SCB Particles
Sugar cane bagasse was sieved to remove fine (pith) and larger particles. The pieces of SCB were sieved to obtain particle sizes of 1 mm × 1 mm × 1 mm, 2.5 mm × 2.5 mm × 2.5 mm, 5 mm × 5 mm × 5 mm, and 10 mm × 10 mm × 10 mm. The crushed and classified SCB was washed several times with sterilized distilled water and dried at 105 °C. This untreated material was sterilized in autoclave and then used as support for cell immobilization. Cells were immobilized in situ in Erlenmeyer flasks by natural adsorption onto the untreated SCB according to the method described by [22]. 5 g SCB with different sizes was autoclaved and then mixed with 50 ml fresh pre-culture HS medium at 28 °C under static condition which was previously inoculated with 10 ml cell suspension (1.9 × 10 9 CFU/ ml) and incubated for 24 h. Then, SCB particles were collected aseptically and combined with 50 ml Potato Peel Waste hydrolysate medium.

Entrapment of Bacterial Cells in Alginate Beads
The cellulose-producing bacterium Glucanobacter xylinum ATCC 10245 was harvested after 24 h of growth (early stationary phase preculture) from 250 ml of HS culture medium. The cell pellet (0.8 g wet weight containing 4 × 10 9 CFU) was obtained by centrifugation at 5000 rpm for 10 min and subsequently re-suspended in 10 ml phosphate buffered saline (PBS). A stock of 2-5% (w/v) sodium alginate was prepared and autoclaved at 121 °C for 15 min. Ten milliliters of bacterial cell suspension (4 × 10 9 CFU) was added to 50 ml of sterilized alginate solution and mixed by stirring on a magnetic stirrer. This alginate cell mixture was extruded drop by drop into a cold sterile 0.1 M calcium chloride solution (CaCl 2 ). The drops of alginate cell solution were gelled to form a uniform and defined-sized sphere upon contact with CaCl 2 solution. The immobilized beads were left in 0.2 M CaCl 2 solution at room temperature for 1 h to harden and complete the gel formation. The beads were then rinsed with sterilized bi-distilled water several times to remove residual CaCl 2 . Blank alginate beads without bacterial cells were also prepared in the same way for control experiments [23]. 5 g of wet alginate beads containing entrapped cells were added to PPW culture medium and then replaced by a fresh 50 ml medium after 7 days.

Analytical Procedures
At the end of cultivation period, the produced BC is collected from the surface of the liquid medium by the sterilized spatula under aseptic condition, rinses in distilled water, and immerses in NaOH 0.1 N at 60 °C for 90 min to remove attached cells and impurities. Later, pellicles are rinsed in methanol solution and then washed with the deionized water and dry at 60 °C for 24 h to evaluate the BC yield concentration in g l −1 (mass (g) of BC/volume (l) of culture medium) [4]. The carrier SCB or alginate beads which retain in the bottom of the liquid medium can be separated from the BC pellicles without any interaction between them. The carrier matrix (SCB particles and alginate beads) and the immobilized cells were examined using a scanning electron microscope. The samples for electron microscopy were prepared according to the method described by [22]. In all BC production experiments, the reducing sugar concentration of in the PPW culture medium was measured by DNS according to the procedure reported in our previous work [25]. As well as, cell retention (Cr, CFU g −1 ) onto the SCB particle and alginate beads were measured as the ratio of total number of CFU immobilized onto the carrier to the carrier mass (g). Log CFU was determined as adapted by [4].
The immobilization efficiency (Yi, %) was calculated as follows: where Ci is the concentration of immobilized cell calculated as the ratio of total number of CFU immobilized onto the SCB carrier to the total medium volume.
Ct is the ratio of concentration of total cell in the flask, i.e., suspended plus immobilized, to the medium volume) [26].

Release of Adsorbed and Entrapped Cells
In order to determine the counting of bacterial cells onto the carriers, the carrier containing BC cells were released by citrate-phosphate buffer (pH 6.0, 1%) as reported by [20]. One gram of the alginate beads or SCB particles was transferred to 9 mL buffer. The solution was stirred on a shaker for 25 min vigorously until bacteria released from matrix completely. The counts (Log CFU/g) were determined by plating on HS agar plates and incubating for 48 h at 37 °C. The free bacteria were treated similarly.

Statistical optimization of BC production by immobilized Glucanobacter xylinum
The production and statistical optimization of BC using Glucanobacter xylinum immobilized onto SCB carrier has been performed in four sequential steps; Plackett-Burman experimental design, doing the experiment, data analysis and validation of the results [27]. Prior to statistical modeling, the different factors were tested for the optimum maximum and minimum levels of study based on One-factor-at-a-time method. Initially, the cultural conditions before optimization were evaluated for BC production, and then were considered for further optimization studies. Modeling of BC production by the immobilized Glucanobacter xylinum has been conducted using Plackett-Burman factorial design (PBD) to select the major factors influencing BC production. Table 1 shows the PBD with seven numeric factors namely; Sugar concentration of the PPW hydrolysate, Medium volume ratio, Spore concentration in the carrier, pH, Incubation time, Incubation temperature, and carrier quantity. The experimental design composed of 21 experimental trials; among these, one run was carried out at the center point values, while each remaining runs will conduct at 2-levels by combinations of upper ('high, +') and lower ('low, −') levels of all variables. In the PBD, two levels were used to determine whether the maximum production was obtained at lower or higher concentration of the variables by comparing them with the experimental results obtained from center point values.
Experimental responses were measured by first order model by the following equation: where Y is the response for BC production, B o is the model intercept and β i is the linear coefficient, and x i is the level of the independent variable. According to the Stat-Ease analysis, a first-order model could be obtained from the regression results of fractional factorial experiment. This model describes the interaction among factors and it is used to screen and evaluate important factors that influence the response. The main effect of easch variable was determined according to the following equation: where E xi is the variable main effect, ΣM i+ is the summation of the response value at high level; ΣMi − is the summation of the response value at low level, and Nis the number of experiments. For Plackett-Burman design and analysis of variance (ANOVA), Minitab 17-software (version 17.0.0) has been used.

Reusability and Storage Stability of Immobilized Cells
In order to study the reusability of the SCB immobilized cells, after every batch of BC production, the whole SCB were still existence in the bottom of the Erlenmeyer flask and the BC was formed over the broth medium. Thus, the BC was firstly collected by sterilized forceps and then the SCB were collected aseptically from the spent medium and washed three times with sterile bi-distilled water. Then, SCB particles were used again separately for BC production with fresh PPW medium under the same experimental conditions. This cycle was repeated for ten times to evaluate the BC production capacity of reused immobilized cells. For comparative purposes, fermentation with free cells in flasks without the immobilization matrix was also carried out under the same culture condition. Unless otherwise noted, all batch fermentations were duplicated and averaged data are reported. The operational stability of the immobilized system was determined by the following equation: Operational efficiency (%) = Cx C1 × 100, where C1 is the BC yield produced in the first (1st) fermentation cycle and Cx is the BC yield produced in the (Xth) fermentation cycle.
To identify the efficiency of SCB in the immobilization process, the fermentation kinetics during BC production is measured [4]. The efficiency of BC production is evaluated after 7 days of cultivation and the substrate conversion ratio, BC production rate and BC production yield are calculated, respectively, as described by [28]: where Si is the initial concentration of substrate (g/l) (i.e. corresponding to the reducing sugar content of the PPW culture medium), Sf is the final concentration (g/l), mBC is the amount of BC produced (g), V is the reactional volume (l) and t is time of reaction (h).
On the other hand, the effect of storage on the BC production capability of the immobilized cells was also investigated. The SCB and alginate beads containing the adsorbed and entrapped cells were stored for varying periods (up to 60 days) at 4 °C.

Characterization of BC Membranes
Fourier Transform Infrared (FTIR) The structure change between different produced BC was studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy (Spectrum Two IR Spectrometer-Perki-nElmer, Inc., Shelton, USA). All spectra were obtained by 32 scans and 4 cm −1 resolution in wave numbers ranging from 4000 to 450 cm −1 . The FTIR calculations were including crystallinity index (Cr.I.) and main hydrogen bond strength (MHBS).

X-ray Diffraction (XRD)
The crystalline structure of samples was characterized using X-ray (XRD) diffractometer (Schimadzu 7000, Japan) operating with Cu Kα radiation (λ = 0.154060 nm) generated at 30 kV and 30 mA with scanning rate of 4° min −1 for 2θ values between 10 and 80 degrees.

Scan Electron Microscope (SEM)
The micrographs of the prepared samples were analyzed by scanning electron microscopy (SEM, Quanta FEG 250, FEI). To prepare the SEM sample, a thin layer of Au was coated onto the sample by sputtering coating device.

Immobilization Efficiency onto SCB Particles and Alginate Beads
Immobilization of Glucanobacter xylinum cells onto fibrous and non-fibrous carriers could be an interesting technology to develop an efficient, low-cost and continuous process for cellulose productivity. In our previous report, Potato Peel waste-nitric acid hydrolysate culture medium was proved to be an excellent alternative medium for BC production and that due to its having high buffering capacity [4]. Therefore, in order to increase the efficiency of cellulose production, immobilization of bacterial cells was carried out onto SCB (adsorption method) and alginate beads (entrapment method) in comparison with free cells under the same culture condition. Firstly, the immobilization efficiency of bacterial cells onto SCB particles and alginate beads were confirmed using scanning electron microscope (SEM) compared with the non-inoculated matrices (Fig. 1). As shown in (Fig. 1A, B), the bacterial cells were success attached in the alveolate of the stalk cells of the SCB definitely, and high cell concentration was observed. Electrostatic forces or covalent bonding between bacterial cells and carrier surface of SCB could be discussed the good bacterial cell adherence in the alveolate of the stalk cells [20,26]. Another reason that clearly indicated to natural adsorption obtained due to the large surface area of the SCB structure, which could provided an easily attached and grow of the bacterial cells within the porous stalk cells which in turn increased and enhanced the biomass concentration. This ideally surface area contributing to the stability of the microenvironment for cell metabolism by greatly increasing the adherence of the bacterial cells [20]. On the other hand, alginate beads were also analyzed by SEM microscopy before and after bacterial cells inoculation. Scanning electron micrographs confirmed the effective immobilization of Glucanobacter xylinum into alginate beads. Alginate-entrapped bacterial cells were visually evaluated on the bead surface as seen in (Fig. 1C, D), since bacterial cells were restricted into the inner surface of the alginate beads via the entrapment process [29]. In this way, the immobilization efficiency (Yi) and bacterial cell retention (Cr) by the carrier was measured up to 36 h. As shown from (Fig. 2), SCB particles demonstrated increasing in the immobilization efficiency and bacterial cell retention with maximum values of 90.6% ± 2.8 and 8.2 ± 0.12 Log CFU/g SCB respectively after 16 h. and then decreased notably. In contrast, as evident from alginate beads results (Fig. 2), the high immobilization efficiency and bacterial cell retention obtained after 4 h. with 82.6% ± 1.4 and 6.5 ± 0.08 Log CFU/g alginate beads and then decreased slightly. Obviously, this high immobilization efficiency of SCB is more than alginate beads can be attributed to the large amount of vacuous and porous stalk cells which might be responsible for maximum cell adsorption [20,22]. Additionally, the retention of bacterial cells could be more coherent in alginate beads than SCB particles and that may be due to the nature of entrapment method which protects the cells from leaking into the environment better than the adsorption method [30].

Effect of Matrix Size
Almost of literature reported that use of alginate with 2-4% being optimally for immobilized cells production [23]. Therefore, preparation of different alginate concentrations (1-4%) were used for evaluating the optimal one. Results depict that the maximum production rate of BC was showed with 3% alginate concentration and further concentration caused decreasing gradually in the production rate (data not shown). Apparently, the production rate is found to be decreased at lower alginate concentration (1-2%) which attributed to rapid ruptured of beads at lower concentrations, while the production rate of BC observed was more reduced at higher alginate concentration (4%) and that could be related to the difficult diffusivity of the nutrients and bacterial cells through the rigid beads produced at higher concentrations [23]. Subsequently, 3% alginate was utilized for preparing different sizes of beads (2, 3, 4, and 6 mm) to determine its effects on the cellulose production. As illustrated in (Table 2), the rate of cellulose yield was increased with bead size increase up to reaching to its maximum value (4.8 ± 0.44 g/l) at 4 mm bead diameter and further increase in the alginate beads results in a slightly reducing cellulose yield. However, the bacterial growth was clearly increasing as beads size increased. Although the number of CFU/g alginate beads were increased in larger beads, but the cellulose production rate was decreased and that related to the high difficult diffusion as beads become larger which contributing in lowering the cellulose productivity [23,31]. In addition, the main disadvantage of Ca-alginate beads with each size display by the instability and rapid disruption at the end of cultivation period and that may be correlated with the PPW composition. On the other hand, the effect of SCB particle sizes was also established. As mentioned above, SEM profile proved that the immobilized cells are adherence in the alveolate of the stalk cells of the SCB particles. As shown in (Table 2), the cell retention was increased as a SCB particle size was increased. In fact, a larger SCB particle can be carrying more bacterial cells than a smaller one, and that because it has more intact stalk cells [20]. However, the highest cellulose production (4.9 ± 0.18 g/l) was achieved with smaller SCB particles (1 mm × 1 mm × 1 mm). This phenomenon could be related to the diffusion limitations in larger sizes of

Fig. 2 Immobilization efficiency (Yi) (A) and cell retention (Cr) of G. xylinum cells (B)
onto SCB and Ca-alginate as function of time SCB particle, since the mass transfer in the interior of this carrier will become more poor and difficult due to the increasing inner mass transfer resistance [21]. On the other hand, we can also noticed that as SCB become small in size the immobilization efficiency would be increased and the cell retention in the carrier being also increased. Conversely, in case of alginate beads the increase of size could be increased the immobilization efficiency of the carrier and that was directly proportional with the cellulose productivity. Thus, from these results we can concluded that, diffusion limitations could be avoided by using the optimum size of each of matrix. In comparison of SCB and Ca-alginate the obtained results are cleared that the SCB exhibited the higher stability of cells than Ca-alginate which indicated to easily recover from the PPW medium. Since, alginate beads proved to be inefficient to reuse in the PPW culture medium attributed to the rapid disruption of beads in the second cycle which may be due to the deformation or weakening of the alginate matrix in the PPW medium. Therefore, further experiments were desired in order to characterize the nature of the BC-production from each matrix in comparison with free cells and the cellulose producing from the standard medium (HS medium) separately.

Characterization of BC-Producing from Each Matrix
The used instrumental tools are useful in characterization of produced BC which included FTIR, XRD, SEM. The FTIR spectra are showed in Fig. 3. The BCs IR spectra are fit with cellulose (type I). Moreover, the main characteristic bands of cellulose are observed in all produced BCs where the OH starching band is observed at around 3400 cm −1 in all produced BCs. Additionally, C-H starching band is assigned at around 2930 cm −1 in all BCs. Similar, the asymmetric deformation vibration of methyl and methylene is around 1400 cm −1 . In addition, the cellulose produced from HS media, PPW media, bagasse immobilized and alginate immobilized are slightly similar. However, the crystallinity may be changed from type to other with lowest crystallinity for HS media cellulose and the higher crystallinity is bagasse immobilized cellulose. On the hand, the FTIR calculations including Cr.I. and MHBS are cleared significant differences between produced celluloses. The Cr.I. of HS, PPW, alginate, bagasse is 1.   with significant different. The HS media BC recorded less crystallinity which equal 77.5%. In contrary, The PPW, Alginate immobilized and bagasse immobilized cleared improve in crystallinity which recorded 82, 84 and 86 for PPW, alginate and bagasse, respectively. The topography study is carried out on the produced bacterial celluloses show significant differences in topography study in Fig. 5. The HS BC appears as spongy-like as shown in Fig. 5A. The PPW appears as dark spots cellulose with high contacted fibers together as shown in Fig. 5B [4]. On the other hand, the immobilized result celluloses appear changes in surface morphology. The BC produced from bagasse immobilization observed in Fig. 5C appears as slid-like cellulose. The BC produced from alginate immobilization in Fig. 5D appears as cracked surface and this may be referred to the nature of bacteria in present of alginate as immobilization material. Over all the SEM data are emphasized that the growth conditions are affected the surface structure and morphology. Additionally, the FT-IR calculations as well as XRD pattern are emphasized these different. Accordingly, SCB particles proved to be the efficient carrier for BC immobilization in terms of durability of the matrix and the unique producing-cellulose. Therefore, further optimization of cultural medium conditions using the SCB needs to be studied.

Optimization of the SCB-Immobilized Cell System Using Plackett-Burman Design (PBD)
Plackett-Burman design (PBD) has been proposed as one of the statistical approaches to improve the bioprocessing efficiency, since it provides several advantages. Perior to applied PBD, all factors and its levels were firstly selected according to the preliminary studies of one-factor-at-a-time (OFAT) on cellulose production by immobilized G. xylinum (data not shown). In consequence of these results, the independent variables of the cultural conditions that are significantly influence the BC production by SCB immobilized cells were studied by PBD with their respective high and low levels. Results of PBD signified that, there were variations from the BC production which ranges from 1.6 to 5.2 g/l in 21 runs as shown in Table 3. This variation involving the obtained units emphasizes obviously the significance of medium optimization to accomplish high BC production by immobilized cells when compared with the BC production by free cells (4.4 g/l) after statistical optimization previously [4]. Although the cellulose production efficiencies between the SCB-immobilized cells and the free cells were not significantly different, the cellulose yield produced by the SCB-immobilized cells was slightly higher than those of the free cells. Considering the sugar consumption in this study, the residual sugar concentrations in the supernatant of the immobilized cell culture were also less than those in the fermentation broth of the free cell cultures. In addition, the SCB-immobilized cells displayed a lower CFU number than the free cells in the fermentation broth culture. These findings indicated that the immobilized cell system proved to be higher cellulose productivity than the free cell cultures and that implies to the importance of the SCB carrier to protect the bacterial cells from the external stress conditions which allowing them to perform better than the free ones. As shown in Fig. 6, the Pareto chart indicated the order of significance for the variables affecting BC production. Among these variables, the static incubation period and pH value showed the highest significance through exhibiting its higher positive effect, and then medium volume ratio and sugar concentration in the PPW hydrolysate medium. On contrast, cellulose yield was not influenced by incubation temperature, matrix quantity and spore concentration in the carrier (P value < 0.05). Overall, the results of contribution of the different variables demonstrate that incubation period has the maximum contribution percent (42.05%) followed by pH (30.2%), medium volume ratio (22.5%) and sugar concentration (5.6%) and the significant medium components showing P values < 0.05 significance level obtained by regression analysis. The rest of the terms have contribution values of less than 1%. Although a very negligible effect of spore concentration was observed on cellulose yield, moderately significant interactive effect of spore concentration and incubation period was noted on cellulose productivity. Insignificant interactive effects were observed for yield and productivity. The optimum levels for the variables obtained by use of stat graphics software were sugar concentration (10% w/v), SCB quantity (2.0% w/v), and spore concentration (8% v/v) at 37 °C and at pH9.0 with 25 ml medium volume for 7 days. The analysis of variance (ANOVA) for the experiment design of SCB showed that, the Model F-value of 6.64 implies the model is significant. In such cases A, B, D, E, are significant model terms where "Prob > F" is less than 0.0500. The "Pred R-Squared" of 1.0000 is reasonable agreement with all the "Adj R-Squared" of 1.000 (Table 4). The initial order model equation created by PB design showed the dependence of BC production on the medium constituents: The three dimensional (3D) response surface plots-generated by Minitab-17 software is shown in Fig. 7, represents the relationships and effects of different experimental variables (factors) on BC productivity. Best experimental variables levels for maximizing BC production were predicted through analysis of these plots in combination with numerical optimization for each variable and desirability analysis.
As shown in Fig. 6, pareto chart demonstrated the most significant variables. Since, Incubation period and pH were the most significant parameters, followed by medium volume ratio (aeration) and sugar concentration in PPW hydrolysate. After numerically optimization of the BC production by immobilized G. xylinum, the optimum conditions have been applied practically to validate the Plackett-Burman model. The results reveal the validation of the model at different conditions (data not shown). The optimum conditions showed the maximum BC productivity as follows: sugar conc. (10 g/l), medium volume (25 ml/250 ml flask), Spore conc. (8%), pH (9), Incubation time (7 days), and temperature (37 °C).
Interestingly, BC production by SCB immobilized cells after statistical optimization is approximately 5 times higher  than of those observed in the HS medium (1.21 g/l). As reported before, generation of gluconic acid from the catabolism of glucose during the BC biosynthesis is regarded as the most common drawback due to the rapid decrease in the pH value of the medium which results in feedback inhibition of the BC synthesis [4,5,10]. Therefore, our previous studies attendance to the PPW medium is a successful hydrolysate waste to regular production of BC without any influence with the pH value and that due to its having high buffering capacity and also has a good impact on the formation of biopolymer. According to the statistical bioprocess optimization, we can notice, direct proportional between the sugar consumption and pH value. From these results we concluded that, immobilization of Glucanobacter xylinum onto a low-cost abundant natural byproduct (i.e. SCB) and optimization the BC production using the PPW hydrolysate medium opening an effective way to sustainability of BC biosynthesis. Comparatively, the yield capacity of BC by G. xylinum when cultivated on HS medium (reference medium) not reached to the half amount (1.2 g/l) that obtained by the immobilized G. xylinum cultured on PPW (5 g/l). In addition to that, the recycle and sustainable of SCB up to 7 cycles without any disruptions of the stalk cells provide an efficient and continuous production system.

Reusability and Storage Stability of Immobilized Cells
The storage and reusability of the cell-adsorbed SCB considered a significant parameters for successful application of this system in practical and industrial sectors. In order to evaluate the efficiency and stability of the SCB particles to immobilized BC cells, experiments were performed to reuse the SCB particles as inoculums for repeated batch production of BC.SCB immobilized cells were cultivated in the PPW culture medium based on the optimization process as described previously. The cycle's number of repeated batch cellulose production by the SCB-immobilized cells and the main fermentation kinetic parameters are summarized in Table 5. As can be seen, reuse of the SCB particles could be exactly carried out for four sequential times without any significant decrease in the operational efficiency of the BC yield. It was observed that the BC production rate was initially affected at the 7th cycle and that may be related to a limited amount of adsorbed bacterial cells in the carrier. The enhanced cell stability of the immobilized cells as observed in the present study suggests that the SCB carriers may protect the bacterial cells from severe conditions during the fermentation process. The increased cell stability and cell productivity of the immobilized system demonstrated in this study are in agreement with the reports by [20,21].
With respect to the kinetic studies, the SCB-immobilized cells exhibited slightly higher cellulose productivity rate (0.043 g/l h) through five repeated batch fermentation than the free cells (0.0401 g/l h). After that, the cellulose productivity was starting to reduced and this might be due to the fact that the immobilized cells was reduced in the carrier and that was clearly appeared in the substrate conversion rate which decreased with 10%. To deeply understand these changes, it should be noted that the utilization of sugars by the SCB-immobilized cells was not restricted with the carrier system, suggested that the diffusion of the substrates was not prevented by the carriers, which were highly porous and  thus, facilitated the mass transfer of the system. However, the decreasing in the loaded bacterial cells under repeated batch condition contributes in the lowering substrate conversion rate which led to reducing in the BC production efficiency. In addition, an increase in the number of viable cells adsorbed on the inner surfaces and in the micro-porous structure of the matrix suggesting that high sugar concentrations in the fermentation broth had no effect on the bacterial growth. We propose, from these findings, that the regeneration and protection of immobilized cells by the SCB are the main factors that work synergistically to prevent cell activity.
Storage stability of SCB immobilized Glucanobacter xylinum at 4 °C and room temperature with long term was investigated through 28 day. Viability of bacterial cells were expressed as remaining total number of Log CFU per gram of SCB, along with determined the operational efficiency of BC yield on every 7th day as a function time. Results are shown in Fig. 8, clearly indicate that at 4 °C the immobilized cells were still retain in the SCB with 44.3% up to the full period with high operational efficiency reached to 21 days. However, the storage stability of SCB at room temperature was not suitable to retain the cells during 28 days, only a small amount of immobilized cells was remained in the carrier (7.7%) correlated with sharply reduced in the BC production. This phenomenon may be due to the change in the storage temperature which increases the mass transfer barrier in the SCB at the room temperature caused the bacterial cells to die during the long-term storage. This study shows reasonable effectiveness for practical use of the SCB carrier which capable to retaining the immobilized cells without any expensive storage conditions.

Conclusion
Utilization of waste from the food industry as raw materials for both immobilized the bacterial cells and prepared the culture medium promotes economic advantages because they reduce environmental pollution and stimulate new research for science sustainability. The observed study was carried out to produce bacterial cellulose via immobilization onto fibrous and non-fibrous bio-polymers.The foregoing results justify the applicability of SCB as carrier matrix for immobilization of BC in biosynthesis of cellulose from Potato Peel Waste hydrolysate culture medium. Reused immobilized biomass indicated sustained cellulose production even after 6 cycles. The instrumental analysis of BC produced from fibrous biopolymer showed excellent characters with high crystal structure and homogenous network as illustrated from SEM topography. These results demonstrate the feasibility of the proposed immobilization system to be used in future industrial BC production from low-cost raw materials.