Accepted Manuscript Title: Optimization of Bacterial Cellulose Production by Gluconacetobacter xylinus using Carob and Haricot Bean Author: Eyup Bilgi Ece Bayir Aylin Sendemir Urkmez E.Esin Hames PII: DOI: Reference:

S0141-8130(16)30183-0 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.02.052 BIOMAC 5865

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

27-6-2015 3-2-2016 17-2-2016

Please cite this article as: Eyup Bilgi, Ece Bayir, Aylin Sendemir Urkmez, E.Esin Hames, Optimization of Bacterial Cellulose Production by Gluconacetobacter xylinus using Carob and Haricot Bean, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.02.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Optimization of Bacterial Cellulose Production by Gluconacetobacter xylinus using Carob and Haricot Bean Eyup Bilgi1, Ece Bayir1,2, Aylin Sendemir Urkmez1-3, E. Esin Hames3,* 1

Ege University, Graduate School of Natural and Applied Science, Department of Biomedical Technologies, Izmir, Turkey 2 Ege University, Application and Research Center for Testing and Analysis (EGE-MATAL), Izmir, Turkey 3 Ege University, Department of Bioengineering, Faculty of Engineering, Izmir, Turkey

*Corresponding Author E. Esin Hames Department of Bioengineering, Faculty of Engineering, Ege University, 35100, Bornova, Izmir, Turkey Tel: +90 232 388 4955 E-mail: [email protected]; [email protected]

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Abstract Bacterial cellulose (BC) can be used in medical, biomedical, electronic, food, and paper industries because of its unique properties distinguishing it from plant cellulose. BC production was statistically optimized by Gluconacetobacter xylinus strain using carob and haricot bean (CHb) medium. Eight parameters were evaluated by Plackett-Burman Design and significant three parameters were optimized by Central Composite Design. Optimal conditions for production of BC in static culture were found as: 2.5 g/L carbon source, 2.75 g/L protein source, 9.3 % inoculum ratio, 1.15 g/L citric acid, 2.7 g/L Na2HPO4, 30 °C incubation temperature, 5.5 initial pH, and 9 days of incubation. This study reveals that BC production can be carried out using carob and haricot bean extracts as carbon and nitrogen sources, and CHb medium has higher buffering capacity compared to Hestrin and Schramm media. Model obtained from this study is used to predict and optimize BC production yield using CHb medium.

Keywords: Bacterial cellulose, Gluconacetobacter xylinus, Statistical optimization, Carob, Haricot bean.

1. Introduction In biomedical area, synthesis of many synthetic or natural polymers is carried out for different purposes [1, 2]. Although synthetic polymer technology has developed substantially, manufactured products can cause tissue irritation due to low biocompatibility of such polymers [3, 4]. Natural polymers are the most suitable candidates instead of synthetic polymers, with the appropriate characteristics and high biocompatibility to overcome this problem [5]. Although plant cellulose, which is the most produced natural polymer on earth (1.5x1012 tones/year) [6] can be used widely in several fields from textile to food [7], it cannot be employed for medical or cosmetic uses because of its impurities such as hemicellulose, lignin and pectin [8]. The cellulose synthesized by bacteria is named bacterial cellulose (BC) or 2   

microbial cellulose [9]. Despite the fact that BC has the same molecular formula (C6H10O5)n with plant cellulose, it has additional physical and mechanical properties like i. high purity, ii. nanofibrous network structure (50-120nm), iii. high crystallinity, iv. high degree of polymerization, v. high water absorption and holding capacity, vi. high tensile strength and vii. biocompatibility [10]. BC can also be loaded with different bioactive compounds owing to its micro/nano sized pores to implement functionality for different purposes [11]. BC is a promising polymer as a replacement of plant cellulose in different sectors due to these unique properties. BC is an extracellular polymer that is formed by linear coupling of glucopyranose sugar monomers, and synthesized widely by Gluconacetobacter xylinus (also named as Acetobacter xylinum) strains but also synthesized by some other bacteria [12]. Acetobacter strains are prominent organisms because of the fact that they can easily be cultivated in the laboratory, can be commonly found in fruits and fruit products, are not pathogenic and produce relatively high yield and stable BC [13, 14]. Several methods and systems have been patented, and a lot of research has been published about BC in the last decades. The production parameters including temperature, pH, surface area to volume ratio of air-liquid interface of the culture medium (S/V), inoculum ratio and incubation time [15, 16] should be optimized using readily available and cheap raw materials for the production of high quality and cost effective BC with high yield. Decline of BC production in static culture due to formation of gluconic acid and subsequent pH decrease is one of the major problems that researchers must overcome. Optimum pH is 5.5 for Acetobacter strains but gluconic acid can decrease the pH even below 3.5 during the cultivation [17, 18]. Although there are some patents and papers about the cost effective BC production methods using cheap raw materials like agricultural wastes, wheat straw, dry olive mill residue, molasses, cotton-based waste, the sugar content of all of these substrates are relatively low [193   

23]. Many of these works have not focused on developing new culture media to overcome the pH decrease during cultivation and there is a substantial need to optimize the BC production process for cost effectiveness, and high yield. In traditional process optimization methods, experiments have been carried out by one-variableat-a-time (OVAT) approach. In these techniques, all parameters have been kept constant while only one parameter of input has been changed. Chance is an important factor to find actual optimum values of production conditions in these approaches, which inherently ignore the interaction between the parameters [24]. Design of experiment (DOE) approaches, which are also called statistical optimization, can be used to obtain much more reliable data for process optimization [25]. In this study, Plackett-Burman Design and Central Composite Design techniques were used to facilitate carob and haricot bean extracts as alternative carbon and nitrogen source, respectively, to develop cost effective BC production method using Gluconacetobacter xylinus. It has been demonstrated that haricot bean (Phaseolus vulgaris) contains 18.5-22 % protein by dry weight [26, 27]. Research also showed that besides protein, haricot bean also contains minerals (e.g calcium, magnesium) and carbohydrates [28]. Carob (Ceratonia siliqua L.) production is approximately 15 000 tones/year in Turkey and annual production in world is over 400 000 tones. It is shown that carob contains over 50 % sugar its dry weight [29] with about of 75 % those sugars is sucrose [30] and the rest are fructose, maltose and glucose [31] and it also contains approximately 8% protein (w/dry weight) and an important amount of minerals [29, 32]. All these properties make carob and haricot bean good candidates for industrial microbial production.

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2. Materials and Method 2.1.

Activation and cultivation of microorganism

Hestrin & Schramm (H&S) medium, which comprises following substances: 2-5 glucose (w/v %) (Merck, 108342, USA), 0.5 (w/v %) peptone (Merck, 107214, USA), 0.5 (w/v %) yeast extract (Oxoid, LP0021, England), 0.115 (w/v %) citric acid monohydrate (Merck, 100244, USA) and 0.27 (w/v %) Na2HPO4.2H2O (Merck, 106580, USA) was used for activation of Gluconacetobacter xylinus (ATCC 700178) at 30 °C for 150 rpm in agitated culture. 2.2.

Preparation of carob and haricot bean extract

Carobs and haricot beans were milled or broken into small pieces independently, then hydrolyzed in five volumes of distilled water (w:v) using an autoclave (Hirayama, HG-80, Japan) at 121 °C for 30 min. Then hydrolysate was centrifuged at 9000 rpm for 10 min to remove particles, and supernatant was collected as stock solution and sterilized in autoclave at 121 C for 20 min. These extracts were used for the preparation of Carob-haricot bean (CHb) medium. 2.3.

Total sugar & protein analysis

Total sugar analysis of the carob extract was made by a phenol-sulphuric acid method according to Nielsen [33]. Total protein or nitrogen analysis of haricot bean extracts was performed by Kjeldahl method [34]. 2.4.

Plackett-Burman Design

Plackett-Burman Design (PBD) was used to determine the most effective parameters for the production of BC using alternative raw materials. PBD is a useful method to screen high number of parameters reliably, and practically eliminate non-effective parameters by investigating the effects of each parameter without doing numerous experiments [35]. Design parameters for PBD, sugar content of carob (carbon source), protein content of haricot bean 5   

(nitrogen source), initial pH, S/V, inoculum ratio, incubation time, citric acid content and temperature, were chosen according to literature and our preliminary work. Parameters were tested in 12-run PBD at two levels (maximum and minimum value) to determine key factors of the BC production. Dry weight of BC was used to analyze PBD results. All experiments were done in triplicate. 2.5.

Central composite design (CCD)

CCD experiments were performed with significant parameters determined by PBD to find optimum level for each parameter and their mutual interaction on BC production. 23 full factorial CCD was used to investigate individual and synergetic effect of the three significant parameters (protein amount, incubation time and inoculum ratio) determined by PBD. In CCD experiments, the other parameters were kept constant; carbon source 2.5 % (w/v), initial pH 5.5, S/V 1.5 (1/cm), citric acid 0.115 % (w/v), temperature 30 °C and 2.7 g/L Na2HPO4 were added to culture medium. The CCD was designed based on carrying out 20 experiments, which comprises 6 axial points, 8 factorial points and 6 replicates at the center point [36]. Dry weight of BC was used to analyze CCD results. All experiments were done in triplicate. Additional to statistical analysis of dry weight, morphological and structural properties of harvested BC were also taken into consideration in both PBD and CCD experiments. 2.6.

Purification of BC

The purification of BC samples was performed by an alkali treatment method [38]. Harvested BC sheets were rinsed with distilled water to remove culture medium residues. Samples were boiled in 0.1 M NaOH (BC:NaOH, 1:5 w/v) (Merck, 106498, USA) for 20 min. Following neutralization step by keeping samples in 5 % (v/v) acetic acid (J.T. Baker, 6052, Holland) for 3-5 seconds, samples were boiled in distilled water repeatedly until BC was semi-transparent. 6   

This method was also implemented with minor modification for NaOH step. BC samples were kept in 0.1 M NaOH solution at 80 C for overnight instead of boiling with this solution. BC samples were dried by keeping at 60 °C until weights of the samples were constant for further statistical analysis. 2.7.

Characterization of BC

2.7.1. Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR analysis of dried BC samples was performed by FT-IR spectrophotometer (Perkin Elmer, Frontier FT-IR Spectrophotometer L1280044, USA). Scans were obtained in 650-4000 cm-1 spectral region. 2.7.2. X-ray Diffraction (XRD) Lyophilized pure BC samples were X-rayed using Philips X’Pert Pro (Netherlands) and data were collected at reflection mode 0-70. Crystallinity index (CI) was calculated as: CI= (I002-IAM) / (I002) where, I002 is the maximum diffraction intensity of the 002 lattice and IAM is the intensity at 2=18. In this notation, (I002-IAM) donates the intensity of the crystalline peak [39]. 2.7.3. Scanning Electron Microscope (SEM) Scanning electron microscope (Fei Quanta 250 FEG, USA) was used to take SEM images of nanofiber structure of the purified BC at an accelerated voltage of 5 kV. 2.8.

Statistical Analysis

Statistical experimental designs were constituted and analyzed using Design Expert 7.0 (StatEase, Inc. USA). Three-dimensional plots were drawn for visualization of individual effects and interaction between significant parameters. All experiments were done randomly and

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independently according to DOE’s run order in triplicate and average value of responses were used. 3. Results 3.1.

Total sugar and protein analysis

According to total sugar and protein analysis, carob contains 55±3 % sugar and haricot bean contains 19.3±0.4 % protein. Values were used to adjust the carbon and nitrogen content of the CHb medium. 3.2.

Plackett-Burman Design

Results from 12-run PBD in order to determine which culture parameters led to a higher yield of BC production among eight parameters are represented in Table 1a. All experiment were done in petri dishes, and S/V ratio were adjusted by changing cultivation volume; and responses were converted to bacterial cellulose dry weight per liter to compare the results. The most effective parameters for BC production were detected as incubation time, protein amount and inoculum ratio with contribution of 63.50 - 12.23 and 9.57 %, respectively (Table 1b). Total contribution of these parameters was calculated as 85.3 %; and the other parameters had no significant effect on BC production. 3.3.

Response Surface Methodology – Central Composite Design

The parameters and results of CCD experiments are given in Table 2. According to CCD experiments, BC production was fit to a second-order polynomial equation (Eq1.), (Eq1.) 0.016

1.71 .90.028 – 0. 18

– 0.164

0.215

0.069

4.375

10

– 0.042



– 0.264

where Y represents BC production (g/L), A, B, and C are inoculum ratio (v/v), incubation time (day) and protein concentration from haricot bean extract (w/v), respectively.

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The variance and regression analysis are represented in Table 3. Analysis of variance (ANOVA) statistical analysis was carried out followed by Fisher's Least Significant Difference (LSD) test. F and p values of the model indicate the model’s success for the prediction of BC production. Values of F was 31.79 and p was F” less than 0.05 are considered as significant, and values greater than 0.1 are nonsignificant model terms. In this case, B, C, A2, B2, and C2 are significant terms according to Table 3. The closer R2 value to 1 indicates better correlation between the predicted and experimental values and R2 was calculated as 0.9662 in our model. The signal to noise ratio was measured by Adeq Precission value (13.943), and greater than 4 indicates that this model can be used to navigate the design space. The individual and interactive effect of model terms on BC production can be interpreted by the 3D graphs of CCD given in Fig. 1, which represents a saddle-like curve obtained for essential parameters. In order to validate CCD model, seven experiments were selected to maximize production yield with short incubation time considering industrial process. The experimental conditions and results were presented in Table 4. 3.4.

FT-IR

FT-IR spectroscopy allows evaluating the efficiency of the purification method and investigating the structure of the BC samples. FT-IR spectra of BC produced with H&S medium and CHb medium are given in Fig. 2. Typical bonds of cellulose can be seen in both samples.

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The peaks of 3350 cm-1 and around in 3400 to 3500 cm-1 indicates O-H stretching for pure cellulose, strong absorption peak at 2900 cm-1 represent C-H bonding. The peak at 1162 cm-1 is assigned to C-O-C stretching (just same as pure cellulose), 1032 and 1055 cm-1 correspond to C-O bonds. Other bands at 1277 cm-1, 1335 cm-1, 1400 cm-1 correspond to C-H bending, OH bending and CH2 stretching, respectively, and indicates the presence of crystalline region and purity of BC [39-41]. As shown in Fig. 2, BC produced either with H&S standard medium or CHb medium show the same characteristic bonds in FT-IR spectra. 3.5.

XRD

X-ray diffraction pattern of the BC samples are shown in Fig. 3. BC from CHb medium (a) and H&S medium (b) shows peaks around 2θ=14.4, 16.3 and 22.6, which represent the typical cellulose I structure and correspond to diffraction planes 101, 101, and 002 respectively, which is the most abundant form of cellulose found in nature [42-44]. The results indicate that both BC samples were in cellulose Iα-Iβ form. The only difference between the samples was relative intensity of the 002 lattice diffraction, indicating higher cellulose crystallinity in the CHb sample (78 %) compared to H&S sample (61 %). 3.6.

SEM

SEM images of BC samples purified by two methods are given in Fig. 4. The method containing 0.1 M NaOH treatment for overnight at 80 °C removes bacterial cells effectively (Fig 4b). 4. Discussion Selection of the microorganisms with high yield cellulose production capacity is the most crucial parameter in BC production. Especially Gluconacetobacter strains, which are also used in this work, are well characterized and model microorganisms for this purpose [45].

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In addition to selection of strains, development of the most suitable media for production is very important, because cultivation parameters can significantly affect BC production. Another important point to consider in large-scale production is the cost. Most of the previous works on increasing cost effective BC production yield have concentrated on finding alternative carbon sources [19-23]. However, our results presenting the contribution of each parameter show that carbon source -the main medium component- is not a significant model term (p>0.05) for production optimization. The low significance of the carbon source is most probably because the carbon concentration in the media was chosen in the range between 1-3 % (w/v) (Table 1a) according to our OVAT experiments (data not shown) carried out to determine the high and low limits of each parameter. This range was chosen since high carbon concentration was previously reported to decrease BC production yield [46]. pH drop caused by production of gluconic acid is another important challenge in BC production. pH can drop even below 3.5, which is the range that microorganisms can proliferate but cannot produce BC with high yield [47]. Krystynowicz et al., found that after 7 days of incubation, pH decreases from 5.7 to 3.68 in H&S medium and production yield of BC is 0.47 g/L. After 14 days of incubation, pH value decreases to 3.22 and production yield increases to only 0.52 g/L although the cultivation time was doubled [48]. To overcome this challenge, some chemicals are added to the culture medium like tri-sodium citrate and corn step liquor in addition to citric acid [49]. In BC production, controlling the pH is difficult not only in static culture, but also in agitated culture conducted with bioreactors because BC attached on pH probes results in wrong or late measurement of pH and makes it difficult to maintain pH precisely [12]. As seen in Table 1a and Table 2 (initial pH 5.5), CHb does not require additional chemical supplements to prevent sharp pH drop. It has been shown that fructose and disaccharides that are present in CHb medium show only slight decrease in pH values after incubation, as opposed to glucose

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that shows high decrease in pH caused by the rapid conversion of glucose to gluconic acid [51]. Buffering capacity of carob and bean has also been shown in Al Dabbas et al.’s work [51]. Embuscado et al.’s research reported that bacterial growth increased until 8th day of incubation period and remain almost constant until the end of incubation period of 46th day [52]. It has been expected that inoculum ratio and incubation time have significant effect on BC production in 14 days of incubation period. In addition to these parameters, protein amount is found to be another significant term by PBD. The significance of this parameter was already expected since 8-14 % of dry weight of bacteria is composed of proteins, and nitrogen sources are essential for metabolism of G. xylinus. BC can absorb water 1-7 hundredfold of its dry weight, and >99 % of this water is captured physically, while the rest of the water is bound chemically [53, 54]. Physically bound water can easily leave BC, because it is in free form, so the wet weight of BC can change even with small manipulation while weighting. Dry weight of samples was used in our analyses as response in both PBD and CCD. BC production can be carried out either in static or agitated culture method. It is possible to produce over 20 g/L cellulose in agitated culture [55]. For static culture, BC sheets are produced in air liquid interface of culture medium, and while BC sheets get thicker, microorganisms on the surface have difficulties to reach the media components, and those below the sheet have difficulties to reach oxygen. Mass transfer limitations in static culture results in decreased BC production to even below 1 g/L [56]. From Table 1a and 2, it is seen that BC production in static culture CHb medium can reach 3.2 g/L. In this study, one of the main purposes was developing a cost effective BC sheet production technique. According to PBD results, limits of parameters were revised to produce high yield BC with better morphological and mechanical properties. In optimal conditions of 9.3 % inoculum ratio, 9 incubation days and 2.75 % (w/v) protein amount, the best production reached 1.8 g/L (Table 2) yield. 12   

FT-IR is a technique to detect functional groups present in the structure of organic compounds, to reveal the bonds in the sample structure, and to characterize molecular bonding properties using bond vibration frequency [57]. It is seen from Fig. 2 that FT-IR spectra of BC produced with standard medium and CHb medium are very similar to each other. It is possible to say that BC production with alternative raw materials used in this work do not change the structural properties of BC. Additionally, XRD results (Fig. 3) also support this statement. The only difference observed was in crystallinity of the BC obtained from the two different media. BC was produced with higher crystallinity in CHb medium, most likely due to high pH buffering capacity and the higher production yield of the CHb medium. High crystallinity of bacterial cellulose is due to the hydroxyl groups that are capable of generating intra- and intermolecular hydrogen bonds [38]. As the production yield is increased, the bacteria have more constrained movement, and therefore the molecular packing of BC in a more orderly fashion is observed [58]. Fructose, highly present in CHb medium, was claimed to be the optimal carbon source for the Acetobacter strains for higher crystallinity index (CI) as opposed to glucose that is present in H&S medium [59]. Although CI was not chosen as the selection parameter in this work, it is important to note that it can be considered as a design criteria for further studies when evaluating alternative raw materials. The effects of alkali treatment on the purification of BC has been demonstrated in previous studies [60]. Gea et al. reported that using NaOCl is more effective than one step NaOH treatment which could not be effective in removing organic (including bacteria itself) and inorganic compounds [61]. As seen in Fig. 4b, our method shows that an effective purification with only NaOH is obtained without the need of other bleaching agents such as NaOCl. Purification of BC with high concentration of NaOH can result in degradation of samples, and also can alter the mechanical and morphological properties [62]. It is clearly seen in Fig. 4, our purification method did not affect the microstructure of BC due to low concentration of NaOH. 13   

Obtaining a saddle-like curve (Fig. 1) in optimization process for each parameter means that the range of parameters was chosen correctly [63] and the accuracy of the model is high because narrow ranges were chosen according to our preliminary works. CCD can optimize all the affecting parameters collectively to eliminate the limitations of OVAT process. Interactions between the parameters can be seen in Fig. 1. Incubation time is the most important parameter in an industrial process to decrease production cost and yield more products. It is also possible to choose optimal conditions by decreasing incubation time and adjusting other parameters (Table 4) such as 7.98 % inoculum ratio and 2.55 % protein amount to obtain relatively high BC production (1.367 g/L) in a short time (7 days). The value of parameters can also be determined using Eq1 to predict BC production. It is possible that high mineral (calcium, iron, zinc, potassium and magnesium) and vitamin (thiamin, riboflavin, niacin and folacin) content of haricot bean extracts might have caused an increase in G. xylinus metabolism, and therefore contributed to increased BC yield in CHb medium. Oxygen transfer is an important limitation in static culture, so by using agitated culture, the production yield can be increased too much higher levels. But the quality and the characteristics of the produced BC by agitated culture must be carefully examined, since there may be differences in the morphology and the crystallinity of the final product [64]. Conclusions Statistical optimization approaches have many advantages in optimization of microbial bioprocesses such as obtaining more reliable data from processes, allowing scientists to screen high numbers of parameters without doing excessive experiments and finding optimum culture conditions without ignoring interaction between parameters. In this study we implemented PBD for the optimization of BC production and screened eight cultivation parameters. Optimum

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points of three significant parameters (i.e. incubation time, protein amount and inoculum ratio) were investigated by CCD. Our results indicate that BC can be produced with a yield of 1.8 g/L using carob and haricot bean as alternative raw materials, with culture conditions of 9 days of incubation time, 10 % v/v inoculum, 2.75 % w/v protein, 0.115 % w/v citric acid, 0.27% w/v Na2HPO4 and 30°C incubation temperature. If the morphological and mechanical properties of the final product is not important, it is possible to reach up to 3.2 g/L production yield. Although BC has its unique properties over plant cellulose and other synthetic polymers, and can be used in many different fields, it cannot be used widespread due to its low yield and high production cost. This study reveals that cost-effective BC production can be carried out using alternative raw materials and provides a model to implement this production into industrial processes. Statistical optimization of BC production is conducted using static culture condition in this study. It should be noted that when compared to static culture, BC productivity is higher in agitated culture which means that this approach can be further applied to agitated culture to obtain even higher yields. Acknowledgements This work is supported by Republic of Turkey, Ministry of Science, Industry, Technology and BioRed Laboratory Products Company (SANTEZ Project Number: 0198-STZ-2013-1 and Ege University Science and Technology Center (EBILTEM Project Number: 2014/BIL/015).

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References [1] H. Tian, Z. Tang, X. Zhuang, X. Chen, X. Jing, Biodegradable synthetic polymers: Preparation, functionalization and biomedical application, Prog. Polym. Sci. 37 (2012) 237280. [2] A. Sionkowska, Current research on the blends of natural and synthetic polymers as new biomaterials: Review, Prog. Polym. Sci. 36 (2011) 1254-1276. [3] R.L. Reis, N.M. Neves, J.F. Mano, M.E. Gomes, A.P. Marques, H.S. Azevedo, NaturalBased Polymers for Biomedical Applications, Woodhead Publishing, Cambridge, 2008. [4] P.A. Gunatillake, R. Adhikari, Biodegradable synthetic polymers for tissue engineering, Eur Cell Mater. 5 (2003) 1-16. [5] A. Aravamudhan, D.M. Ramos, A.A. Nada, S.G. Kumbar, Chapter 4 - Natural polymers: Polysaccharides and their derivatives for biomedical applications, in: S.G.K.T.L. Deng (Ed.) Natural and synthetic biomedical polymers, Elsevier, Oxford, 2014, pp. 67-89. [6] J.M. Moran-Mirabal, E.D. Cranston, Cellulose nanotechnology on the rise, Ind. Biotechnol. 11 (2015) 14-15. [7] D. Klemm, B. Heublein, H.-P. Fink, A. Bohn, Cellulose: Fascinating biopolymer and sustainable raw material, Angew. Chem. Int. Ed. 44 (2005) 3358-3393. [8] M. Gama, P. Gatenholm, D. Klemm, Bacterial Nanocellulose: A Sophisticated Multifunctional Material, CRC Press, Florida, 2012. [9] L.R. Lynd, P.J. Weimer, W.H. Van Zyl, I.S. Pretorius, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Rev. 66 (2002) 506-577. [10] L. Fu, J. Zhang, G. Yang, Present status and applications of bacterial cellulose-based materials for skin tissue repair, Carbohydr. Polym. 92 (2013) 1432-1442. 16   

[11] W. Hu, S. Chen, J. Yang, Z. Li, H. Wang, Functionalized bacterial cellulose derivatives and nanocomposites, Carbohydr. Polym. 101 (2014) 1043-1060. [12] M. Shoda, Y. Sugano, Recent advances in bacterial cellulose production, Biotechnol. Bioprocess Eng. 10 (2005) 1-8. [13] D. Klemm, D. Schumann, F. Kramer, N. Heßler, D. Koth, B. Sultanova, Nanocellulose materials-different cellulose, different functionality, Macromol. Symp. 280 (2009) 60-71. [14] M. Moosavi-Nasab, A. Yousefi, Biotechnological production of cellulose by Gluconacetobacter xylinus from agricultural waste, Iran J Biotechnol, 9 (2011) 94-101. [15] P.R. Chawla, I.B. Bajaj, S.A. Survase, R.S. Singhal, Microbial cellulose: Fermentative production and applications, Food Technol. Biotech. 47 (2009) 107-124. [16] R. Jonas, L.F. Farah, Production and application of microbial cellulose, Polym. Degrad. Stab. 59 (1998) 101-106. [17] E.J. Vandamme, S. De Baets, A. Vanbaelen, K. Joris, P. De Wulf, Improved production of bacterial cellulose and its application potential, Polym. Degrad. Stab. 59 (1998) 93-99. [18] Y. Kojima, A. Seto, N. Tonouchi, T. Tsuchida, F. Yoshinaga, High rate production in static culture of bacterial cellulose from sucrose by a newly isolated Acetobacter strain, Biosci. Biotechnol. Biochem. 61 (1997) 1585-1586. [19] F.P. Gomes, N.H. Silva, E. Trovatti, L.S. Serafim, M.F. Duarte, A.J. Silvestre, C.P. Neto, C.S. Freire, Production of bacterial cellulose by Gluconacetobacter sacchari using dry olive mill residue, Biomass Bioenergy. 55 (2013) 205-211. [20] S. Bae, M. Shoda, Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor, Appl. Microbiol. Biotechnol. 67 (2005) 45-51.

17   

[21] P. Carreira, J.A. Mendes, E. Trovatti, L.S. Serafim, C.S. Freire, A.J. Silvestre, C.P. Neto, Utilization of residues from agro-forest industries in the production of high value bacterial cellulose, Bioresour. Technol. 102 (2011) 7354-7360. [22] L. Chen, F. Hong, X.X. Yang, S.F. Han, Biotransformation of wheat straw to bacterial cellulose and its mechanism, Bioresour. Technol. 135 (2013) 464-468. [23] F. Hong, X. Guo, S. Zhang, S.F. Han, G. Yang, L.J. Jönsson, Bacterial cellulose production from cotton-based waste textiles: Enzymatic saccharification enhanced by ionic liquid pretreatment, Bioresour. Technol. 104 (2012) 503-508. [24] S. Arockiasamy, R.M. Banik, Optimization of gellan gum production by Sphingomonas paucimobilis ATCC 31461 with nonionic surfactants using central composite design, J Biosci Bioeng, 105 (2008) 204-210. [25] V.Y. Yatsyshyn, D.V. Fedorovych, A.А. Sibirny, Medium optimization for production of flavin mononucleotide by the recombinant strain of the yeast Candida famata using statistical designs, Biochem. Eng. J. 49 (2010) 52-60. [26] G.E. de Almeida Costa, K. da Silva Queiroz-Monici, S.M.P.M. Reis, A.C. de Oliveira, Chemical composition, dietary fibre and resistant starch contents of raw and cooked pea, common bean, chickpea and lentil legumes, Food Chem. 94 (2006) 327-330. [27] E.A. Shimelis, M. Meaza, S.K. Rakshit, Physico-chemical properties, pasting behavior and functional characteristics of flours and starches from improved bean (Phaseolus vulgaris L.) varieties grown in East Africa, Agric. Eng. Int.: CIGR J. 8 (2006) 1-19. [28] W.R. Aykroyd, J. Doughty, Legumes in Human Nutrition, Food and Agriculture Organization of the United Nations, Rome, 1982. [29] M. Khlifa, A. Bahloul, S. Kitane, Determination of chemical composition of carob pod (Ceratonia siliqua. L.) and its morphological study, J. Mater. Env. Sci. 4 (2013) 348-353. 18   

[30] S. Naghmouchi, M. Khouja, A. Khaldi, M. Rejeb, S. Zgoulli, P. Thonart, M. Boussaid, Biochemical diversity of wild carob tree populations and its economic value, in: T. Povilitis, (Ed), Topics in Concervation Biology, Intech, Rijeka, 2012. [31] M.K.E. Youssef, M.M. El-Manfaloty, H.M. Ali, Assessment of proximate chemical composition, nutritional status, fatty acid composition and phenolic compounds of carob (Ceratonia siliqua L.), Food Public Health, 3 (2013) 304-308. [32] L. Bravo, F. Saura Calixto, Composition and potential uses of mesquite pods (Prosopis pallida L.): Comparison with carob pods (Ceratonia siliqua L.), J. Sci. Food Agric. 65 (1994) 303-306. [33] S.S. Nielsen, Phenol-sulfuric acid method for total carbohydrates, in: S.S. Nielsen (Ed.) Food Analysis Laboratory Manual, Springer, 2010, pp. 47-53. [34] J. Kjeldahl, Neue methode zur bestimmung des stickstoffs in organischen körpern, Z. Anal. Chem. 22 (1883) 366-382. [35] R.L. Plackett, J.P. Burman, The design of optimum multifactorial experiments, Biometrika, 33 (1946) 305-325. [36] A. Salihu, M.Z. Alam, M.I. AbdulKarim, H.M. Salleh, Optimization of lipase production by Candida cylindracea in palm oil mill effluent based medium using statistical experimental design, J. Mol. Cat. B: Enzym. 69 (2011) 66-73. [37] M. Roman, W.T. Winter, Effect of sulfate groups from sulfuric acid hydrolysis on the thermal degradation behavior of bacterial cellulose, Biomacromolecules, 5 (2004) 1671-1677. [38] S. Park, J. Baker, M. Himmel, P. Parilla, D. Johnson, Cellulose crystallinity index: Measurement techniques and their impact on interpreting cellulase performance, Biotechnol. Biofuels. 3 (2010) 3-10.

19   

[39] N. Halib, M.C.I.M. Amin, I. Ahmad, Physicochemical properties and characterization of nata de coco from local food industries as a source of cellulose, Sains Malays. 41 (2012) 205211. [40] C. Castro, R. Zuluaga, C. Álvarez, J.-L. Putaux, G. Caro, O.J. Rojas, I. Mondragon, P. Gañán, Bacterial cellulose produced by a new acid-resistant strain of Gluconacetobacter genus, Carbohyd. Polym. 89 (2012) 1033-1037. [41] Y.Z. Wan, L. Hong, S.R. Jia, Y. Huang, Y. Zhu, Y.L. Wang, H.J. Jiang, Synthesis and characterization of hydroxyapatite–bacterial cellulose nanocomposites, Compos. Sci. Technol. 66 (2006) 1825-1832. [42] L. Hong, Y.L. Wang, S.R. Jia, Y. Huang, C. Gao, Y.Z. Wan, Hydroxyapatite/bacterial cellulose composites synthesized via a biomimetic route, Mater. Lett. 60 (2006) 1710-1713. [43] C. Castro, R. Zuluaga, J.-L. Putaux, G. Caro, I. Mondragon, P. Gañán, Structural characterization of bacterial cellulose produced by Gluconacetobacter swingsii sp. from colombian agroindustrial wastes, Carbohyd. Polym. 84 (2011) 96-102. [44] E. Trovatti, L.S. Serafim, C.S.R. Freire, A.J.D. Silvestre, C.P. Neto, Gluconacetobacter sacchari: An efficient bacterial cellulose cell-factory, Carbohydr. Polym. 86 (2011) 1417-1420. [45] V. Nguyen, B. Flanagan, M. Gidley, G. Dykes, Characterization of cellulose production by a Gluconacetobacter xylinus strain from kombucha, Curr. Microbiol. 57 (2008) 449-453. [46] S. Masaoka, T. Ohe, N. Sakota, Production of cellulose from glucose by Acetobacter xylinum, J. Ferment. Bioeng. 75 (1993) 18-22. [47] D. Klemm, D. Schumann, U. Udhardt, S. Marsch, Bacterial synthesized cellulose — artificial blood vessels for microsurgery, Prog. Polym. Sci. 26 (2001) 1561-1603.

20   

[48] A. Krystynowicz, W. Czaja, A. Wiktorowska-Jezierska, M. Gonçalves-Miśkiewicz, M. Turkiewicz, S. Bielecki, Factors affecting the yield and properties of bacterial cellulose, J. Ind. Microbiol. Biotechnol. 29 (2002) 189-195. [49] N. Noro, Y. Sugano, M. Shoda, Utilization of the buffering capacity of corn steep liquor in bacterial cellulose production by Acetobacter xylinum, Appl. Microbiol. Biotechnol. 64 (2004) 199-205. [50] S. Keshk, K. Sameshima, Evaluation of different carbon sources for bacterial cellulose production, Afr. J. Biotechnol. 4 (2005) 478-482. [51] M.M. Al-Dabbas, K. Al-Ismail, R.A. Taleb, S. Ibrahim, Acid-base buffering properties of five legumes and selected food in vitro, Am. J. Agric. Biol. Sci. 5 (2010) 154-160. [52] M.E. Embuscado, J.S. Marks, J.N. BeMiller, Bacterial cellulose. I. Factors affecting the production of cellulose by Acetobacter xylinum, Food Hydrocolloid. 8 (1994) 407-418. [53] W.-S. Chang, H.-H. Chen, Physical properties of bacterial cellulose composites for wound dressings, Food Hydrocolloid. 53 (2014) 75-83. [54] G. Gayathry, G. Gopalaswamy, Production and characterization of microbial cellulosic fibre from Acetobacter xylinum, Indian J. Fibre Text. 39 (2014) 93-96. [55] T. Kouda, T. Naritomi, H. Yano, F. Yoshinaga, Effects of oxygen and carbon dioxide pressures on bacterial cellulose production by Acetobacter in aerated and agitated culture, J. Ferment. Bioeng. 84 (1997) 124-127. [56] X. Zeng, D.P. Small, W. Wan, Statistical optimization of culture conditions for bacterial cellulose production by Acetobacter xylinum BPR2001 from maple syrup, Carbohydr. Polym. 85 (2011) 506-513.

21   

[57] A. Ulman, An introduction to ultrathin organic films: From langmuir--blodgett to self-assembly, Academic Press, London, 2013. [58] X. Zeng, J. Liu, J. Chen, Q. Wang, Z. Li, H. Wang, Screening of the common culture conditions affecting crystallinity of bacterial cellulose, J. Ind. Microbiol. Biotechnol. 38 (2011) 1993-1999. [59] H. Toyosaki, T. Naritomi, A. Seto, M. Matsuoka, T. Tsuchida, F. Yoshinaga, Screening of bacterial cellulose-producing Acetobacter strains suitable for agitated culture, Biosci. Biotechnol. Biochem. 59 (1995) 1498-1502. [60] J. George, V. Sajeevkumar, R. Kumar, K. Ramana, S. Sabapathy, A. Bawa, Enhancement of thermal stability associated with the chemical treatment of bacterial (Gluconacetobacter xylinus) cellulose, J. Appl. Polym. Sci. 108 (2008) 1845-1851. [61] S. Gea, C.T. Reynolds, N. Roohpour, B. Wirjosentono, N. Soykeabkaew, E. Bilotti, T. Peijs, Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process, Bioresource Tecchnol. 102 (2011) 9105-9110. [62] B.A. McKenna, D. Mikkelsen, J.B. Wehr, M.J. Gidley, N.W. Menzies, Mechanical and structural properties of native and alkali-treated bacterial cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, Cellulose, 16 (2009) 1047-1055. [63] D. Baş, İ.H. Boyacı, Modeling and optimization I: Usability of response surface methodology, J. Food Eng. 78 (2007) 836-845. [64] W. Czaja, D. Romanovicz, R. Malcolm Brown, Structural investigations of microbial cellulose produced in stationary and agitated culture, Cellulose, 11 (2004) 403-411.

22   

FIGURE CAPTIONS Figure 1. Response surface curve for BC production a) as a function of protein amount (P) and incubation time (IT), b) as a function of protein (P) and inoculum ratio (IR), and c) as a function of incubation time (IT) and inoculum ratio (IR). Figure 2. FT-IR spectra of BC sheets produced with different culture media, a) H&S medium, and b) CHb medium. Figure 3. X-ray diffraction patterns of (a) BC produced with CHb medium and (b) produced with H&S medium. Figure 4. SEM images of BC, a) boiling in 0.1 M NaOH for 20 min b) holding overnight in 0.1 M NaOH at 80 C

23   

a

b

A: IR

c

Figure 1.

A: IR 

24   

a 106

100 2896.10cm-1

1427.42cm-1

95

90

1315.48cm-1

3346.71cm-1

%T

1161.29cm-1

85 664.57cm-1

80 1109.09cm-1

612.76cm-1

559.19cm-1

75

70

65 4000

1055.81cm-1

3500

3000

2500

cm-1

2000

1500

1031.99cm-1

1000

500

105

b

1205.60cm-1

100 95 2896.38cm-1

90

1427.78cm-1

1360.55cm-1 1315.24cm-1

85 1335.90cm-1

%T

80 3344.95cm-1

1161.50cm-1

75 519.52cm-1

70

664.43cm-1 558.97cm-1

1108.69cm-1

65

612.23cm-1

60 55

1055.67cm-1 1032.27cm-1

50 49 4000

3500

3000

2500

cm-1

Figure 2.

25   

2000

1500

1000

500

002 101 101

101 101

Figure 3.

a 

b 

Figure 4.

26   

002

Table 1. Plackett-Burman Design (a) matrix and results and (b) % contribution of the parameters

a. Run 1 2 3 4 5 6 7 8 9 10 11 12

pH

C

P

w/v %

w/v %

1 1 1 3 3 1 3 3 3 3 1 1

0.5 2 2 0.5 0.5 0.5 2 2 0.5 2 0.5 2

4.5 6.2 4.5 6.2 4.5 6.2 4.5 4.5 6.2 6.2 4.5 6.2

S/V

IR

IT

CA

1/cm

v/v %

day

w/v %

1 2 2 2 1 1 2 1 2 1 2 1

5 5 15 15 15 15 15 5 5 5 5 15

4 4 4 4 4 10 10 10 10 4 10 10

0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.05 0.15 0.15 0.15 0.15

T C 25 30 30 25 30 30 25 30 30 25 25 25

R

pH*

D1

D2

D3

g/L

-1 -1 1 1 -1 1 -1 1 -1 1 1 -1

-1 1 -1 -1 1 1 1 -1 -1 1 1 -1

-1 1 -1 1 1 -1 -1 1 -1 -1 1 1

0.093 0.834 1.332 0.366 0.295 0.954 3.270 0.950 1.442 0.155 1.172 0.827

4.41 5.87 4.3 5.61 4.15 6.66 6.55 4.74 5.24 5.85 5.52 7.46

b. Cont.% p-value

C 0.022 0.8790

P 12.23 0.0296#

pH 2.13 0.2005

S/V 4.27 0.1036

IR 9.57 0.0405#

IT 63.50 0.0030#

CA 1.02 0.3412

T 4.86 0.0902

D1 0.29 -

D2 0.95 -

D3 1.15 -

C: sugar amount (carbon source), P: protein amount (nitrogen source), pH: initial pH, S/V: surface area – volume ratio, IR: inoculum ratio, IT: incubation time, CA: citric acid amount, T: temperature, D1, D2, D3: dummy factors, R: response, average values of three samples-BC dry weight, *: pH value of culture medium after incubation, Cont.%: contribution %, #: statistically significant.

Table 2. CCD matrix and with experimental responses Run

Inoculum (v/v %)

Incubation Time (day)

Protein (w/v %)

Response (g/L)

pH*

1

14.3

9

2.75

1.123

4.62

2

12.3

12

3.5

1.273

4.87

3

12.3

6

3.5

0.913

4.58

4

9.3

9

2.75

1.553

4.41

5

4.3

9

2.75

1.287

4.53

6

9.3

9

2.75

1.727

4.47

7

6.3

12

3.5

1.462

4.59

8

9.3

9

4

1.153

4.7

9

6.3

6

2

0.913

4.4

10

9.3

4

2.75

0.847

4.7

11

6.3

6

3.5

0.947

5.0

12

9.3

9

1.5

0.757

4.37

13

12.3

6

2

0.873

4.4

14

6.3

12

2

1.190

4.82

15

9.3

9

2.75

1.753

4.47

16

9.3

9

2.75

1.733

5.78

17

9.3

9

2.75

1.793

5.74

18

9.3

9

2.75

1.710

6.09

19

9.3

14

2.75

1.630

5.5

20

12.3

12

2

1.340

5.19

pH*; pH value of culture medium after incubation.

27   

Table 3. ANOVA of CCD experiments Source Model A-Inoculum Ratio B-Incubation Time C-Protein concentration AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor Total R2 Adj R-Squared Pred R-Squared Adeq Precision

Sum of Squares 2280000

9

11069.63 631100 65388.85 153.12 13861.13 2145.13 444700 386800 100500 79690.61 45499.11 34191.50 2359000

1 1 1 1 1 1 1 1 1 10 5 5 19

df

Mean Square 253300

F Value 31.79

p>F