Immobilization of Glucanobacter xylinum onto natural polymers to enhance the 1 bacterial cellulose productivity 2

11 Bacterial cellulose (BC) has profound applications in different sectors of 12 biotechnology due to its unique properties preferring it about plant cellulose. 13 Although this polymer is extremely important in various applications, many problems 14 still hinder the sustainable production in terms of increasing productivity and low-cost 15 production. In order to overcome these problems, this study will focuses on the 16 continuous production of cellulose using immobilized Glucanobacter xylinum cells 17 onto Sugar Cane Bagasse (SCB) and Ca-alginate beads. Comparatively, adsorption of 18 Glucanobacter xylinum cells to the cavum of stalk cells of SCB could be efficiently 19 stable while, entrapment of cells onto Ca-alginate has drawback observed by the rapid 20 disruption and instability of the beads in the Potato Peel Waste (PPW) culture 21 medium. Our findings demonstrate that a combination between alternative low-cost 22 medium with continuous production mode by immobilization onto inexpensive 23 natural polymer can promote a sustainable bioprocess and reduction the production 24 cost. 25 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.

limitations, such as a high operating cost, rapid consumption of the substrate, low in 64 pH stability, and a low BC productivity. Several approaches have been suggested 65 to improve BC production efficiency, involving supplementation of the cultural 66 medium with some regulators such as ethanol or organic acids in order to inhibit the 67 accumulation of the basic metabolic byproduct (gluconic acid) and at the same time 68 stimulate the synthesis of substances necessary for the cell stabilization (Lu et al.,69 2016, Stepanov and Efremenko, 2018). However, these additivesare not suitable, due turn caused an inhibitory effect on BC biosynthesis (Morgan et al., 2014). Therefore, 76 to improve the BC yield it should be foster the key molecule c-di-GMP in the 77 metabolic process of the cells. As discussed by (Srivastava and Waters, 2012, 78 Stepanov and Efremenko, 2018) c-di-GMP is a major metabolic molecule called as 79 "quorum factor," since the highly cells concentrations was correlated with quorum 80 state. In this state the BC production was increased by the expression of "silent genes" 81 and the synthesis of exopolysaccharides with a simultaneous decrease in the rate of 82 active cell growth. Therefore, cells that produce BC should be stimulated to come 83 into a quorum state, which the cells become genetically programmed to their 84 increased population. The cell-immobilization system in case of BC producers could 85 allow obtaining highly concentrated populations of cells since BC synthesis would be 86 regulated by a quorum sensing phenomena as described before.Interestingly, the 87 immobilization of BC cells opens a way to improve cell stabilization and thus led to 88 increase BC productivity. Comparatively, the immobilized cells have various benefits 89 more than free cells in the production process, such as increased the cell population of the overall advantages of immobilization process the current reports concerning the 96 production of BC by immobilized-cell system are very rare. In this regard, PVA 97 cryogel was used for employing Komagataeibacterxylinum cells in an immobilized 98 system to increase the biosynthesis of BC (Stepanov and Efremenko, 2018). 99 Acetobacter xylinum ATCC 700178 cells was successfully immobilized on a plastic 100 composite support (PCS) to improve the BC production on the basis of polypropylene 101 (Cheng et al., 2009). However, the severe masstransfers restrictions, low mechanical 102 strength, non-biodegradability and highly toxicity of these synthetic polymers 103 displaying a big problem in the operational stability of the immobilized cells (Basak et 104 al., 2014, Nuanpeng et al., 2018). Therefore, we tried to finding out a renewable, easily 105 prepared, inexpensive, biodegradable, non-toxic, and available naturally carrier. studies have yet been established on the statistical optimization of BC production 125 using immobilized-cell system despite its high industrial applications. Therefore, we 126 investigated the enhancement of bacterial cellulose production by immobilized G.  Egypt. Na-alginate purchase from molekula (U.K), Potato peel waste (PPW) was 136 resulting from potatoes processing, and collected from the disposal of free markets.

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The bright PPW without disease symptoms were selected then washed thoroughly 138 with distilled water. All reagents, solvents, medium and its components used in this 139 study were of analytical grade.

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Sugar cane bagasse (SCB) was obtained from a sugarcane juice local market 152 in Cairo, Egypt,after the skin and the outside fiber were removed; SCB was chopped 153 into small particles using a food processor. The chopped SCB was then dried, and 154 approximately 50 mL moisture was vaporized from 100 g raw SCB. Bagasse which 155 was obtained after drying was sieved to remove fine and larger particles. The pieces 156 of SCB were sieved to obtain particle sizes of 1 mm x1 mm x 1 mm, 2.5 mm x 2.5 157 mm x 2.5 mm, 5 mm x 5 mm x 5 mm, and 10 mm x 10 mm x 10mm. The crushed and   scanning electron microscope. The samples for electron microscopy were prepared 195 according to the method described by (Yu et al., 2007). In all BC production 196 experiments, the reducing sugar concentration of in the PPW culture medium was measured by DNS according to the procedure reported in our previous work (Miller,198 1959). As well as, cell retention (Cr, CFU g -1 ) onto the SCB particle and alginate 199 beads were measured as the ratio of total number of CFU immobilized onto the carrier 200 to the carrier mass (g). Log CFU was determined as adapted by (Abdelraof et al.,201 2019a).

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The immobilization efficiency (Yi, %) was calculated as follows:   In order to study the reusability of the SCB immobilized cells, after every 254 batch BC production, the whole SCB contents of the flasks were collected aseptically 255 from the spent medium and washed three times with sterile bi-distilled water. Then,

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SCB particles were used again separately for BC production with fresh PPW medium 257 under the same experimental conditions. This cycle was repeated for ten times to  bacterial cells onto SCB particles and alginate beads were confirmed using scanning 309 electron microscope (SEM) compared with the non-inoculated matrices (Fig. 1). As 310 shown in (Fig. 1A, B), the bacterial cells were success attached in the alveolate of the 311 stalk cells of the SCB definitely, and high cell concentration was observed. measured up to 24 h. As shown from (Fig. 1E, F) (Table 1), the cell retention was increased as a SCB particle 378 size was increased. In fact, a larger SCB particle can be carrying more bacterial cells 379 than a smaller one, and that because it has more intact stalk cells (Basak et al., 2014).    406 The used instrumental tools are useful in characterization of produced BC 407 which included FTIR, XRD, SEM. The FTIR spectra are clearfield in Fig. (2A). The  respectively.

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The topography study is carried out on the produced bacterial celluloses 433 show significant differences in topography study in Fig. (3). The HS BC appears as 434 spongy-like this may be referring to low crystallinity as shown in Fig. (3A). The PPW 435 appears as dark spots cellulose with enhance in crystal appearance at     The three dimensional (3D) response surface plots-generated by Minitab-17 software 507 is shown in Fig. (4), represents the relationships and effects of different experimental 508 variables (factors) on BC productivity. Best experimental variables levels for 509 maximizing BC production were predicted through analysis of these plots in 510 combination with numerical optimization for each variable and desirability analysis.  Therefore, our previous studies attendance to the PPW medium is a successful 520 hydrolysate waste to regular production of BC without any influence with the pH 521 value and that due to its having high buffering capacity and also has a good impact on 522 the formation of biopolymer. According to the statistical bioprocess optimization, we 523 can noticed that the direct proportional between the sugar consumption and pH value.

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From these results we can conclude that, immobilization of Glucanobacter xylinum 525 onto a low-cost abundant natural byproduct (i.e. SCB) and optimization the BC 526 production using the PPW hydrolysate medium opening an effective way to 527 sustainability of BC biosynthesis.   cycle's number of repeated batch cellulose production by the SCB-immobilized cells 537 and the main fermentation kinetic parameters are summarized in Table 5. As can be 538 seen, reuse of the SCB particles could be exactly carried out for four sequential times 539 without any significant decrease in the operational efficiency of the BC yield. It was 540 observed that the BC production rate was initially affected at the 7 th cycle and that   With respect to the kinetic studies, the SCB-immobilized cells exhibited slightly 554 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 556 was starting reduced and this might be due to the fact that the immobilized cells was 557 reduced in the carrier and that was clearly appeared in the substrate conversion rate 558 which decreased with 10%. To deeply understand these changes, it should be noted 559 that the utilization of sugars by the SCB-immobilized cells was not restricted with the 560 carrier system, suggested that the diffusion of the substrates was not prevented by the 561 carriers, which were highly porous and thus, facilitated the mass transfer of the suggesting that high sugar concentrations in the fermentation broth had no effect on 567 the bacterial growth. We propose, from these findings, that the regeneration and