Morphological Characteristics of Bacterial Cellulose Thin Films
A Complementary Microscopy Study
Bacterial cellulose due to its high purity and properties presents a potential biomaterial in several industries such as for biomedical/cosmetics, electronic/smart materials applications and reinforcement agent in composites. This article describes the morphological changes, induced by alkali and ultrasound treatments on a commercially available bacterial cellulose obtained from nata de coco.
Bacterial cellulose (BC), also known as microbial cellulose, is composed of pure cellulose nanofibers mainly synthesized by certain bacteria belonging to the genus Gluconoacetobacter xylinus. BC exhibits unique properties, including high crystallinity, mechanical properties, water holding capacity and the ability to obtain various shapes and forms .
Several researchers have used Nata de Coco as a source of bacterial cellulose for their experiments. Nata de Coco, is a dessert originated from east Asian countries, mainly Philippines, Indonesia and Thailand . According to Halib et al.  the BC extracted from Nata de Coco meets the specifications of pure cellulose and is suitable for further research.
The objectives of this study were to examine the most favorable purification BC method and subsequently the effect of HIUS on the separation and individualization of BC nanofibrils of obtaining self-assembled BC films exhibiting high superior structural and physical properties.
Materials and Methods
BC (nata de coco, PT Cocomas, Indonesia) cubes was washed and soaked several times in distilled water to remove the citric acid and other components added to nata de coco syrup. Then a portion of distilled water washed crude BC (WBC) was further purified by different alkaline treatments to remove any remaining bacterial cell debris and successively was rinsed until neutral pH conditions. The one step (OSP) and two step purification (TSP) methods were conducted by the procedure reported by Gea et al. . Briefly, nata de coco cubes were immersed overnight in 2.5 m/m NaOH solution (OSP). Another sample was prepared in the same way and subsequently treated in 2.5 m/m NaOCl solution (TSP).
A third sample was prepared by warming nata de coco in 0.01 M NaOH at 70 °C for 2 h under continuous stirring (NaOHP).
Both water and alkaline BC samples were homogenized within a blender and dried via solution casting at 50°C. After drying, BC films were cut and redispersed (0.1% w/w) and alkaline treated BC dispersions were further subjected to high intensity ultrasonication (HIUS). HIUS was directly applied in a cold water bath for 30 min, with a frequency of 20 kHz and output power of 25 W cm-2. The ultrasonic horn (Tesla 150 WS) with a tip diameter of 18 mm was immersed near the bottom of the container. The supernatant of the ultrasound treated colloid was subjected to drying at 50 °C in order to obtain films with the finest bacterial cellulose nanofibrils.
The morphology of BC samples was observed using a Field Emission Scanning Electron Microscope (FE-SEM) Zeiss ULTRA Plus (Oberkochen, Germany), an Atomic Force Microscopy (AFM) MultiMode 8 with a Nanoscope Veeco V controller (Bruker Nano Surfaces, Santa Barbara, CA, USA) and a Transmission Electron Microscopy (SAED-TEM) JEOL JEM 2100 (Jeol Ltd., Akishima, Tokyo, Japan). The BC cellulose films were further analyzed by optical microscopy analysis with a Polarized Optical Microscope (POM) Olympus BX51 (Olympus Corporation, Tokyo, Japan), fitted with an Olympus DP12 (6V/2.5Å) digital camera.
Results and Discussion
Alkaline Purification Treatment
Even though, bacterial cellulose is free from hemicelluloses and lignin contaminants, the ferment broth contains other impurities such as bacterial cellulose debris and culture medium remaining. The most commonly used purification processes in the culture medium is the alkaline treatment . FE-SEM images were used to examine the efficiency of purification treatments on BC (fig. 1). From figure 1, it can be observed that NaOHP, displayed the most ideal purified BC samples.
High Intensity Ultrasound (HIUS)
The effect of HIUS conditions on morphology of 0.01 M NaOH BC samples was further investigated by FE-SEM, AFM, TEM and POM analysis (fig. 2). Microscopy results showed relevant differences in the morphology and degree of aggregation. The typical dense, 3D network of randomly oriented BC nanofibrils on all samples was more evident by AFM microscopy. TEM images allowed the identification of a highly entangled network of BC nanofibrils. However, HIUS treatment assisted the separation of aggregated BC fibrils and enhanced their individualization, as it was shown by FE-SEM. The surface roughness of BC films was examined by POM microscopy. POM images indicated that HIUS treatment carried out in alkaline purified BC samples eliminated air bubbles, and presented smooth films with a uniform dispersed, homogeneous films. Similar images were taken of the non-ultrasound alkaline purified BC. Results displayed that NaOH caused the aggregation of BC nanofibrils, yet their obtained films exhibited a less rough surface area compared to crude BC samples.
Bacterial cellulose is an alternative of native cellulose, thus its utilization without severe degradation or chemical treatments is considered to be essential to specific applications. FE-SEM morphological analysis displayed that removal of this bacterial cellulose debris depends on the purification method. It was found that, in this bacterial cellulose obtained from this type of nata de coco source, even such low amounts of NaOH were capable to remove any bacterial cellulose impurities. High intensity ultrasound (HIUS) is an easy, environmental mechanical treatment. Morphological analysis showed that HIUS resulted in the separation of bacterial cellulose nanofibrils and in the configuration of new reformed, smoother, more homogeneous films with improved properties.
 Nancy J. Petersen and Paul Gatenholm: Bacterial cellulose-based materials and medical devices: current state and perspectives. Appl Microbiol Biotechnol, 91, 1277-1286 (2011) DOI 10.1007/s00253-011-3432-y
 Faezah Esa, Siti Masrinda Tasirin, Norliza Abd Rahman: Overview of Bacterial Cellulose Production and Application, Agriculture and Agricultural Science Procedia, 2, 113-119 (2014) DOI10.1016/j.aaspro.2014.11.017
 Nadia Halib, Mohd Cairul Iqbal Mohd Amin, Ishak Ahmad: Physicochemical Properties and Characterization of Nata de Coco from Local Food Industries as a Source of Cellulose, Sains Malaysiana, 41 (2), 205-211 (2012)
 Saharman Gea, Christopher T. Reynolds, Nima Roohpour: Investigation into the structural, morphological, mechanical and thermal behaviour of bacterial cellulose after a two-step purification process, Bioresource Technology, 102 (19), 9105-9110 (2011) DOI 10.1016/j.biortech.2011.04.077
 Prashant R. Chawla, Ishwar B. Bajaj, Shrikant A. Survase and Rekha S. Singhal: Microbial Cellulose: Fermentative Production and Applications, Food Technol. Biotechnol, 47 (2), 107-124 (2009)
Dimitrios Tsalagkas1, Aránzazu Sierra Fernández2, Ida Poljanšek3, Primož Oven3, Rastislav Lagańa4 and Csóka Levente5
1 Inha University, Department of Mechanical Engineering, Incheon, Korea
2 University Carlos III of Madrid, Instituto de Geociencias (CSIC, UCM), Madrid, Spain
3 University of Ljubljana, Department of Wood Science, Ljubljana, Slovenia
4 Technical University in Zvolen, Department of Wood Science, Zvolen, Slovakia
5 University of West Hungary, Institute of Wood Based Products and Technologies, Sopron, Hungary
Prof. Dr. Csoka Levente
University of West Hungary
Institute of Wood Based Products and Technologies