Chapter 2

Bacterial Nanocellulose: Synthesis, Properties and Applications

M.L. Foresti, P. Cerrutti and A. Vazquez*

Institute of Polymer Technology and Nanotechnology (ITPN), University of Buenos Aires, Argentina

*Corresponding author: avazquez@fi.uba.ar; mforesti@fi.uba.ar

Abstract

Cellulose nanoparticles (i.e., cellulose elements having at least one dimension in the 1-100 nm range) have received increasing attention during the last decade due to their attractive properties, such as renewability, abundance and low cost of the raw material, large surface-to-volume ratio, high strength and stiffness, very low coefficient of thermal expansion, low weight, low density, and biodegradability. Cellulose nanoparticles of different aspect ratio can be obtained from lignocellulose by different routes, such as acid hydrolysis or intensive mechanical treatment. On the other hand, in certain culture medium and under proper fermentation conditions it is now well-established that some bacteria can also secrete cellulose microfibrils as primary metabolite. Although it has the same molecular formula as plant cellulose, bacterial nanocellulose (BNC) is fundamentally different because of its nanofiber architecture, which confers it special properties. In the current chapter, the synthesis, properties and applications of BNC will be described in detail.

Keywords: Bacterial nanocellulose, Gluconacetobacter xylinus, production conditions, applications

2.1 Introduction

It is now well-known that under proper conditions a number of bacteria can secrete cellulose microfibrils with nanometric widths as an extracellular primary metabolite. As such it is considered a nanomaterial, since it has at least one of its dimensions in the order of the nanometers (10-9 m). Bacteria-produced ribbons typically show rectangular cross-sections with thicknesses around 3-10 nm, 30-100 nm in width, and 1-9 µm in length. Bacteria able to produce bacterial nanocellulose (BNC) belong to the genera Acetobacter, Agrobacterium, Alcaligenes, Pseudomonas, Rhizobium, Aerobacter, Achromobacter, Azotobacter, Salmonella and Sarcina. Among these, one of the more efficient producer-and the one that has been studied most-is Acetobacter xylinum, an acetic acid bacteria (AAB) now reclassified and included within the novel genus Gluconacetobacter, as G. xylinus.

While AAB are mesophilic microorganisms and their optimum growth temperature is between 25-30°C, BNC biosynthesis is usually carried out in static or agitated conditions at temperatures around 28-30°C. Since AAB are strictly aerobic, the cellulose pellicles are formed only in the vicinity of the air-liquid interface, and they conform to its shape. In static fermentations the overlapping and intertwisted bacterial cellulose ribbons form a 3D dense reticulated structure stabilized by extensive hydrogen bonding containing up to 99% of water (pellicle). On the other hand, in agitated fermentations, bacterial cellulose nanofibers interconnect less frequently forming, instead of a pellicle, granules, stellate and fibrous strands well dispersed in culture broth.

The effectiveness of microbial cellulose production depends mainly on the strain, the composition of the culture medium (the carbon source used being very significant), the fermentation temperature and pH, and the oxygen supply. Moreover, the implementation of a static or an agitated process results in differences not only in terms of BNC yields; but also in the biopolymer properties. In reference to the composition of the culture medium, the most used carbon source for BNC production has been D-glucose, although in the last few years the costs associated for large-scale production have triggered the search for alternative substrates, mainly agroforestry and industrial residues.

Even if the first report of the synthesis of bacterial cellulose was done by A.J. Brown in 1886 [1,2], in the mid-1980s reports of the remarkable mechanical properties of bacterial cellulose pellicles brought a resurgence in the area [3,4], and its use as composite materials reinforcement grew rapidly after that [5,6]. Nowadays, the microbial route appears as a very promising eco-friendly source of cellulose microfibrils. Although chemically identical to plant cellulose, microbial cellulose is characterized by a unique fibrillar nanostructure which determines its extraordinary physical and mechanical properties. Well-separated nano- and microfibrils of bacterial cellulose create an extensive surface area which allows it to hold a large amount of water while maintaining a high degree of conformability. The hydrogen bonds between these fibrillar units stabilize the whole structure and confer its high mechanical strength. Moreover, and different to wood and plant cellulose sources, the high chemical purity of bacterial cellulose avoids the need of chemical treatments devoted to the removal of hemicellulose and lignin, which would imply extra isolation costs.

Referring to microbial cellulose applications, bacterial nanocellulose has proven to be a remarkably versatile biomaterial with use in paper products, electronics, acoustic membranes, reinforcement of composite materials, membrane filters, hydraulic fracturing fluids, edible food packaging films, and due to its unique nanostructure and properties, in numerous medical and tissue-engineered applications (tissue-engineered constructs, wound healing devices, etc).

In the current chapter, the synthesis (with particular focus on static versus agitated processes), properties and applications of bacterial nanocellulose, which have been herein briefly introduced, will be reviewed in detail.

2.2 Bacterial Nanocellulose Synthesis

2.2.1 Producer Strains

Under proper conditions a number of bacteria are known to secrete cellulose microfibrils with nanometric widths as an extracellular primary metabolite. Bacteria known to be able to synthetize BNC are those belonging to the genera Acetobacter (now Gluconacetobacter), Agrobacterium, Alcaligenes, Pseudomonas, Rhizobium, Aerobacter, Achromobacter, Azotobacter, Salmonella and Sarcina [7,8]. Currently, the AAB Gluconacetobacter xylinus (previously Acetobacter xylinum) is recognized as one of the more efficient producers of BNC and the one that has been studied most. AAB are gram-negative or gram-variable, aerobic, non-spore forming, ellipsoidal to rod-shaped cells that can occur singly, in pairs or chains. Their sizes vary between 0.4-1 µm wide and 0.8-4.5 µm long. They are catalase positive and oxidase negative. The optimum pH for their growth is 5-6.5 units, although they can grow at lower pH values between 3 and 4 units [9]. The AAB are heterogeneous assemble, comprising both peritrichously and polarly flagellated organisms. Altough their taxonomic classification has not been fully established yet, nowadays the family Acetobacteraceae accommodates twelve genera for the AAB: Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swaminathania, Saccharibacter, Neoasaia, Granulibacter, Tanticharoenia and Ameyamaea [10]. They are found wherever the fermentation of sugars and plant carbohydrates takes place, e.g., on damaged fruits, flowers, unpasteurized or unsterilized juice, beer, and wine.

2.2.2 BNC Biosynthesis

The synthesis of BNC is a precisely and specifically regulated multi-step process, involving a large number of both individual enzymes and complexes of catalytic and regulatory proteins. This process includes the synthesis of uridine diphosphoglucose (UDPGlc), which is the cellulose precursor, followed by glucose polymerization into the ß-1,4-glucan chain, and nascent chain association into characteristic ribbon-like structure, formed by hundreds or even thousands of individual cellulose chains [11]. Figure 2.1 schematizes bacterial cellulose biosynthesis from glucose via uridine diphosphate glucose (UDPGlc) in Acetobacter xylinum.

Figure 2.1. Pathways of carbon metabolism in A. xylinum. CS, cellulose synthase (EC 2.4.2.12); FBP, fructose-1, 6-biphosphate phosphatase (EC 3.1.3.11); FK, glucokinase (EC 2.7.1.2); G6PDH, glucose -6-phosphate dehydrogenase (EC 1.1.1.49); 1PFK, fructose-1-phosphate kinase (EC 2.7.1.56); PGI, phosphoglucoisomerase; PMG, phosphoglucomutase (EC 5.3.1.9); PTS, system of phosphotransferases; UGP, pyrophosphorylase UDPGlc (EC 2.7.7.9); Fru-bi-P, fructose -1,6-bi-phosphate; Fru-6-P, fructose-6-phosphate; Glc-6(1)-P, glucose- 6(1)-phosphate; PGA, phosphogluconic acid; UDPGlc, uridine diphosphoglucose.

Reprinted with permission of [11].

During fermentation, it is believed that cellulose molecules are synthesized in the interior of the cell and spun out to form protofibrils of ca. 2-4 nm diameter, which are crystallized into microfibrils, these into bundles and the latter into ribbons [11,12]. Macroscopically, the fermentation medium in static systems initially becomes turbid and after a few days a gelatinous pellicle appears on the air-liquid surface of the fermentation vessel, conforming to its shape. The pellicle entrapps CO2 bubbles generated from the bacterial metabolism. The reason why the bacteria generate cellulose is unclear, but it has been suggested that it is a means of maintaining their position close to the surface of the culture solution, a mechanism to guard bacteria from ultraviolet radiation, and/or a barrier to bacteria enemies such as...