Production and Characterization of Bacterial Cellulose with Different Nutrient Source and Surface – Volume Ratios

In this research, commercially available, carrot juice was explored as alternative feedstock for production of bacterial cellulose (BC) by Gluconacetobacter hansenii (ATCC® 23769TM). Two types of culture media were used: Hestrin–Schramm (HS) and the carrot juice medium and these culture media were incubated statically for 10 days. The effect of different volumes of media on the microbial process and the utilization of substrates by the bacteria, were also examined. The produced BC was analyzed using X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FT-IR). The water holding capacity (WHC) did not vary greatly with 210 mL (38.6 %), 310 mL (35.4 %), 360 mL (36.4 %) and 410 mL (37.3 %) of carrot juice media, however the WHC of 310 mL HS media (77.1 %), actually achieved a greater WHC, compared to 410 mL of HS media (55.8 %). BC produced in the carrot juice media showed higher yields than cellulose produced in HS media, with values of 1.19 g, 1.35 g, 1.33 g and 1.21 g for media with 210 mL, 310 mL, 360 mL and 410 mL, respectively. According to XRD and TGA results, there were no signifi cant differences in the crystallinity and thermal stability of cellulose produced between HS and the carrot juice medium. FT-IR of BC from HS and carrot juice medium also demonstrated a similar spectrum to alpha cellulose and microcrystalline cellulose.


INTRODUCTION 1. UVOD
Bacterial cellulose (BC) is a bio-nanomaterial with unique properties.This material is produced by several species of bacteria.The most notable of this group is Acetobacter xylinum, renamed nowadays as Gluconacetobacter xylinus and it is found wherever the fermentation of sugars and plant carbohydrates occur (Gama et al., 2013).BC is similar to plant cellulose.However, it is purer and does not contain hemicelluloses and lignin.On the other hand, BC has higher crystallinity, degree of polymerization, water absorbing and holding capacity, mechanical strength in the wet state and stronger biological adaptability (Castro et al., 2011;Wan et al., 2007).BC has a wide application in medicine (artifi cial blood vessels, skin tissue repair), cosmetics, food industry (Nata de Coco) and in the production of magnetic aerogels and magnetic nano papers (Klemm et al., 2001;Olsson et al., 2010;Halib et al., 2012;Fu and Yang, 2013).Many studies were carried out to decrease the high production costs of bacterial cellulose, which is the main problem for industrial scale production.Alternative carbon sources, such as olive oil residues, molass, corn steep liquor and fruits, were used and evaluated (El-Saied et al., 2008;Gomes et al., 2013;Castro et al., 2011).

Metode
The culture media used were sterilized at 121 °C in an autoclave for 20 min and poured into Erlenmeyer fl asks.Experiments were prepared by adding 10 % (v/v) inoculums to the Carrot and HS media in different surface-volume ratios, namely 17.5 x 11.5 x 1.5 cm = 210 ml (A), 17.5 x 11.5 x 2.0 cm = 310 ml (B), 17.5 x 11.5 x 2.2 cm = 360 ml (C), 17,5 x 11,5 x 2,5 cm = 410 ml (D) and (KA), (KB), (KC) and (KD), respectively, and they were statically incubated at 30 °C for 14 days.The collected pellicles were boiled in water for 1h and treated for 12 h in a 0.5 M NaOH solution, rinsed overnight with tap water and followed by washing with deionized water to neutral pH and weighed (wet weight).Freeze dried samples were prepared after a pre-treatment for 15 °C (48 h) under 0.454 mBar and -55 °C conditions and weighted (dry weight).
The water holding capacity (WHC) and yield (Y) were calculated as follows (Shezad et  The freeze-dried samples were coated with gold (Quorum, UK).Analysis of the BC structure was performed by using a SEM (Quanta FEG 450, Netherlands) at 5 kV.Images were taken with 50000 x SEM micrograph magnifi cations.

X-Ray diffraction (XRD) 2.2.2. Rendgenska difrakcija (XRD)
XRD was performed with a high resolution Xray diffractometer (Model Rigaku Smartlab, Made in Japan) with a Ni-fi ltered Cu Kα (2 kW, kα: 1.54 Å) radiation source operated at voltage of 40 kV and 30 mA.The samples were scanned from 10°-40° 2θ range with a step of 10 °/min.Crystallinity index (C.I.) of BC samples were calculated from the refl ected intensity data using Segal method (Keshk, 2014;Terinte et al., 2011): Where; I 020 is the maximum intensity of lattice diffraction (2θ of 16º to 17º) and I non-cr is that of the amorphous material between 2θ of 14º to 15º where the intensity is minimum.

FT-IR spectroscopy 2.2.3. FT-IR spektroskopija
Fourier-Transform InfraRed (ATR-FTIR) spectroscopy analysis of the BC sample was carried out on a Shimadzu IRAffi nity-One FTIR spectrometer (Japan), equipped with a Universal ATR accessory, using 200 scans and a resolution of 4 cm −1 , over the range 4000-800 cm −1 .

Thermogravimetric analysis (TGA) 2.2.4. Termogravimetrijska analiza (TGA)
TGA analysis was evaluated with an SII Model TG/DTA 7200 EXSTAR (Made in Japan) analyser.Each sample (5 mg) was scanned from 30° to 450 °C at a heating rate of 10 °C/min in the presence of nitrogen with a fl ow rate of 20 ml/min to avoid sample oxidation.

REZULTATI I RASPRAVA
Some data such as the yield, wet-dry weight values, and water holding capacity (WHC) of BC are given in Table 1, 2 and 3.
The composition of the nutrient medium, pH, temperature and the interaction of the surface area to the volume of substrate, as well as strain activity, are the fundamental factors affecting BC production and the profi tability of the biotechnological process (Krystynowicz et al., 2002;Poyrazoglu and Biyik, 2011;Ruka et al., 2012).The yield increased in Carrot-BC (1.27 g/L) compared to HS-BC (0.60 g/l).The water holding capacity (WHC) is considered one of the most important physical characteristics of BC, which is directly involved in the biomedical applications of BC as wound dressing material (Ul-Islam et al., 2012; Tsouko et al., 2015).The variations between the WHC are related to the porosity and surface area of each BC and it is also known that the greater the surface area and the larger the pore size, the greater will be the WHC of the BC sample (Tsouko et al., 2015).The results showed that the HS-BC absorbed 68 times its dry weight of water.The WHC decreased to 37 in Carrot-BC medium compared to HS-BC.
Scanning electron micrographs (SEM) of freezedried Carrot-BC pellicle and of the reference medium HS-BC were evaluated.The fracture surface morphology of the Carrot-BC pellicles exhibits a slightly smaller and narrower diameter distribution in comparison with HS-BC.The SEM micrographs indicate that most of the fi bers are in the range of 60 to 70 nm for Carrot-BC and 80-100 nm for HS-BC pellicles.The Carrot-BC pellicles showed much thinner and better network structure than the HS-BC pellicles (Figure 1).
Thermal stabilities of BCs obtained from different nutrition resources and surface-volume ratios were investigated by thermogravimetric analysis (TGA).According to TGA curves in Figure 2, it was observed that Carrot-BC showed lower thermal stability compared to HS-BC.
For TGA curves of the carrot-BC and HS-BC, low weight loss was detected over the temperature range of 50-200 ℃ because of evaporation of water bounded in the BCs, and the T onsets were generally found to be 265-290 °C for all BCs.According to TGA curves, all TGA results are summarized in Table 4.
As seen in Table 4, the best stability of the BCs was determined as KA.T 10% and T 50% of KA were found as 273.2 °C and 427.9 °C, respectively.T 10% and T 50% of the others were found to be lower than KA except for D in T 10% .According to the weight loss, the maximum degradation was found as 83.4 % for H, and the minimum degradation was measured as 52.6 % for KA.DTG curves showed maximum degradation at 354.2 °C for F. As seen in TGA-DTG curves of the BCs, they exhibited three different degradation stages: (1) in the range of 50-200 °C with weight loss of small percentage (%).This mass loss may be attributed to vaporiza-tion of water; the free water is evaporated below 100 °C, while linked water that forms physical bounds with polymers is only evaporated above 100 °C.(2) in the range of 200-370 °C as a result of thermal degradation of cellulose main chains, and (3) at 370-500 °C due to thermal degradation of BCs.
The FT-IR spectra demonstrated a similar spectrum to cellulose (alpha cellulose and microcrystalline cellulose), which proved that the material produced by G. hansenii was cellulose (HS-BC).The band at 1045 .The bands at 2900 and 3300 to 3400 cm -1 are attributed to the CH 2 stretching and the intramolecular hydrogen bonding, respectively, and the band at 3300 to 3400 cm -1 is important for elucidating hydrogen-bonding patterns (Sturcova et al., 2004).
There is no signifi cant difference between HS-BC and Carrot-BC and the results confi rmed that both BC samples exhibited similar chemical binding (Table 5, 6 and Figure 3).XRD analysis of BC from Carrot-BC and HS-BC medium showed three major characteristic peaks around 2θ = 14º, 16º and 25º, indicating the typical cellulose I structure.The only difference between the samples is a slight intensity change in the peaks.The crystalline indices (CI) of Carrot-BC (83 %) were also   slightly lower than those of HS-BC (84 %) (Figure 4).Similar results were also found for the utilization of dry olive mill residues for the production of BC (Gomes et al., 2013).Comparison of XRD results of different cellulose structures are given in Table 7.

ZAKLJUČAK
It is generally accepted that fruits containing suffi cient glucose can be used as a nutrient source for BC production.The results of this research demonstrated the possibility to produce BC in carrot juice instead from Hestrin-Schramm as a nutrient source.The fracture surface morphology of the carrot-BC medium pellicles provided a smaller cellulose fi bril diameter and a better network in comparison with the BC pellicle from the Hestrin-Schramm medium (HS-BC).The HS-BC absorbed 68 times its dry weight of water.The WHC decreased to 37 in Carrot-BC medium compared to HS- BC.The average yield of Carrot-BC was found to be 112 % higher than that of control samples (HS-BC).The yield also increased in both media with volume ratio.The crystalline indices (CI) of Carrot-BC (83 %) were similar to those of HS-BC (84 %).The FT-IR spectra showed no signifi cant difference between Carrot-BC and HS-BC.The results confi rmed that BC samples exhibited similar chemical binding.Carrot-BC showed lower thermal stability compared to HS-BC.