Bacterial cellulose production under different culture conditions
Currently some principles of sustainability, eco-efficiency and green chemistry are guiding the development of a new generation of materials as an alternative to conventional polymers based on petroleum. Then, in the field of biodegradable polymers one of the most promising investigations is focused on the use of microbial cellulose (MC), biocellulose or bacterial cellulose. MC has received substantial interest since it is synthesized from the bacterium Gluconacetobacter genus from a variety of carbon sources such as glucose, fructose, galactose, etc. MC is an interesting emerging biomaterial, with no toxicity, and since its discovery has shown tremendous potential in various fields, because the structural aspect of MC is far superior to those of plant cellulose. Thus, the main focus of the chapter review involves detailed aspects about the biosynthesis and recent advances on microbial production, including mechanism for the biochemistry of the cellulose synthesis, new sources for culture medium, main aspects about static and air-reactor productions and genetic modifications. We also revised microbial cellulose devices for biomedical applications: artificial skin, artificial blood vessels and microvessels, wound dressing of second- or third-degree burn ulcers, scaffolds for tissue engineering, drug delivery systems, dental implants, among others.
- Microbial cellulose
- cellulose synthesis
- medical applications
Microbial cellulose (MC) presents the same chemical formula as plant cellulose, however with the fibers in nanometer dimensions; confer different properties to MC . The MC is a type of exopolysaccharides composed of glucose monomers bound by glycosidic β (1-4) linkages, with the chemical formula (C6H10O5)n, as can be seen in Figure 1. [2,3]. This biopolymer is produced extracellularly into nanofibers by several genera of bacteria, such as
Historical data show that the MC has been used for a long time in the manufacture of a traditional food in the Philippines, known as the coconut cream . Currently, the MC still remains widely utilized as food in various parts of the world however on the other hand aroused great academic and industrial interest due to its unique properties and diverse opportunity of applications. MC produced by
As proposed by Yamada and colleagues (1997)  and subsequently validated by International Journal of Systematic Bacteriology,
This bacterial species belonging to the family Acetobacteriaceae, being morphologically classified as a bacillus Gran-negative, strictly aerobic, no pathogenic which may be found singly arranged, in pairs or in small sets of chain formation of colonies shiny and smooth in Mannitol Agar. Bacteria belonging to this family are able to oxidize fully various carbon sources such as glucose, fructose, galactose, sucrose, mannitol, glycerol, inositol, among others [3,8] and alcohols such as ethanol  and is capable of extracellularly producing pulp at temperatures between 25 and 30 °C and pH 3 to 7. The bacterial cellulose may provide arrangements in parallel via hydrogen bonds and to form a tridimensional network. The morphology of the membrane depends directly on the environment and the interface culture medium / air where MC form a thick film, which can be easily manipulated according to the size of the vial used for cultivation [1,8].
Researchers have sought new bacterial strains capable to produce biopolymers with potential industrial application . Although many organisms are capable to produce cellulose,
In nature, microorganisms which produce cellulose are usually found in symbiosis with other microorganisms. In the fermentation of Kombucha, for example,
2. Biochemical and molecular mechanisms of bacterial cellulose biosynthesis by
Although several species of microorganisms are capable to produce cellulose,
One proposed hypothesis is that the UDP-glucose binds to lipids of the plasma membrane [1,21]. Another one considers that the soluble precursor interacts directly with the CS . The CS is a protein complex consisting of three (AxCcSAB, AxCcSC and AxCcSD) or four (AxCcSA, AxCcSB, AxCcSC and AxCcSD) protein subunits encoded by genes exist in an operon chromosomal called
The two conserved Asp residues (D) invariably are found in loops at the Carboxyl-terminal (C-terminal) ends of predicted strands, a position frequently observed for catalytic residues [21, 39]. The hydrophobic clusters in domain B are more difficult to interpret in terms of secondary structure. AxCcSA and AxCcSAB have a motif consisting of domain, a single conserved residues Asp (D-D-D), presumably important for catalysis, identified along with the conserved sequence motif Gln (Q) Arg-Trp (R-W) in glucotransferases [1, 29]. Through a functional analysis of CS, it appears that the A subunit of this complex with 83kDa, shows catalytic activity. The B subunit of 90kDa, increases the rate of cellulose synthesis by joining a positive allosteric regulator, cyclic diguanosine monophosphate (c-di-GMP). The C subunit (138kDa) and D (17kDa) appear to structural activity. It has been hypothesized that C subunit related to pore formation and extrusion of the cellulose D subunit appears related to the process decrystalization since mutant strains of the gene which encodes the D subunit production are still able to produce cellulose II .
3. Cellulose Synthase (CS)
Two bacterial cellulose synthase operons (
In cells actively producing cellulose, approximately 50 cellulose-synthesizing multienzyme complexes are organized in a single row along the longitudinal axis of the bacterial rod whereby each complex secretes approx. 12 to 25 glucan chains which assemble into larger microfibrils at the site of synthesis. This so-called linear terminal complex can be visualized by electron microscopy using freeze fracture as 35 Å pores in the outer membrane or as pits when the outer leaflet is fractured away [1,33].
The gene corresponding to cellulose synthase BcsA is constituted by a long string that presents between 723-880 amino acid residues, as represented in Figure 3, being the most conserved gene of the operon
The BcsB protein [Figure 3], related to indirect interaction with c-di-GMP  is less well conserved among the species. However, direct comparisons of the MCsB proteins with CelB from
The proposed model considers that after the transfer and addition of glycosyl terminal residue, the glucose molecule rotates around acetyl glucan binding to align the channel as shown Figure 4. It is believed that allosteric interactions guide the direction of rotation, causing rotation feature 180⁰ connecting β-1,4 glucan-glucan between the individual glucose units and intramolecular hydrogen bond between oxygen atoms of the hydroxyl groups of the neighboring unit [40,43]. This phenomenon may be sufficient to allow the polymer to move into the channel [Figure 4]. Alternatively, for the translocation of the elongated glucan occurs, replacement is required to UDP-glucose by UDP. Past the channel, induced glucan chain in the BcsA - twist BcssB interface, interaction with BcsB of CBDs (periplasmic carbohydrate binding domains), or aggregation with other glucans may additionally contribute to a unidirectional motion of the polymer .
4. Cultivation conditions for production of bacterial cellulose
The MC production depends of the appropriate cultivation conditions, which include the composition of the culture medium (synthetic and natural media), temperature, pH and methods agitated or static cultivation. The choice of condition cultivation or another depend on the purpose, once these conditions have significant influence on the properties of structure, physical and mechanical MC.
The current methods of MC production are static culture , submerged fermentation through aerated or agitated cultivation , and the airlift bioreactor . Large scale, semi-continuous and continuous fermentation are dominant to meet commercial demand. In all cases, the main objective is to achieve maximum production of MC with optimum form and suitable properties for the application for which it is intended. After all, a wider application of this versatile biopolymer depends on the practical considerations such as the scale-up capability and production costs.
Static cultivation is a relatively simple and widely used method of cellulose production. The medium is placed into shallow tray or bottles, inoculated, and cultivated for several days until the cellulose nearly fills the tray.
Nutrients required for the growth of these microorganisms are carbon and nitrogen sources, phosphorus, Sulphur, potassium and magnesium salts . Sometimes a complex medium supplying amino acids and vitamins is also used to enhance the cell growth and production . Between the years 1940 to 1960, researchers at the Hebrew University in Jerusalem intensively investigated the biochemistry of simplified production and quantification of cellulose cellulose for produced by
Attempts to enhance MC production by adding different additives in the fermentation medium have been made. The possible mechanisms of these various additives to enhance MC were also proposed such as reduction of the shear force by increasing the viscosity of medium . Different chemical compounds including alcohols , glycerol [60,68], organic acids , polysaccharides  thin stillage from rice wine distillery  and thin stillage from beer culture broth [47,53] have been used as additives to the fermentation medium with the aim of increasing MC production.
||Saccharified food wastes||airlift-type bioreactor||3||5.6||Song et al. (2009)|
and Yousefi (2011) 
|Glucose||Static||15||15.28||Ha and Park (2012)|
5. Microbial cellulose for biomedical applications
As previously described, intense research has focused on the use of natural biopolymers in a variety of biomedical materials and devices, including wound dressings, medical implants, drug delivery, vascular grafts, and scaffolds for tissue engineering . Consequently, continual efforts from many researchers, led to novel systems that closely mimic the complex and hierarchical structures inherent to the native tissue are sure to emerge.
In the last decade, several nanocellulose-based materials have been created for a diversity of biomedical applications. Some review articles have highlighted the potential applications of cellulose materials [72,79,80,81,82].
MC represents an interesting emerging nanomaterial, with no toxicity, and since its discovery has shown tremendous potential as an effective biopolymer which offers a wide range of applications, especially the biomedical ones, including the use as biomaterial for artificial skin, artificial blood vessels and microvessels, wound dressing of second- or third-degree burn ulcers and dental implants. Other studies with endothelial, smooth muscle cells and chondrocytes have shown that these cells present good adhesion to bacterial cellulose. 
5.1. Pristine MC based biomaterials
One of the main direct applications of MC membranes in biomedical field is related to wound dressing. Fontana et al.  were the pioneers in describing the use of bacterial cellulose to replace burned skin. Since then, literature shows a great number of papers related to wound dressing. Cellulose dressings are recommended as a temporary covering for the treatment of wounds, including pressure sores, skin tears, venous stasis, ischemic and diabetic wounds, second-degree burns, skin graft donor sites, traumatic abrasions and lacerations, and biopsy sites by the manufacturers .
MC based wound dressings are commonly available on the market nowadays, for example: BioFill®, Bioprocess®, XCell® and Gengiflex® (for periodontal diseases reconstruction . The biomembrane BioFill® was one of the first commercial product that fulfills the main prerequisites of an ideal wound dressing, including: low cost, good adherence to the wound, water vapor permeability, elasticity, transparency, durability, it constitutes a physical barrier for bacteria, is hemostatic, it presents easy handling and application with minimum exchanges. BioFill® effectiveness has been proven in more than 300 cases in accelerating the healing process, pain relief, etc. [86,87,88,89].
Despite the analgesic mechanism of action of these dressings has not been fully elucidated, some authors suggest that the healing mechanism involves the capture of ions by means of cellulose hydrogen bonds, or the nano MC 3-D network mimics the skin surface creating optimal conditions for healing or regeneration [87,88,89].
It is important to point out that MC wound dressing clearly shortened the time to heal or wound closure over standard care when applied to non-healing lower extremity ulcers, as observed by many researchers [88,89,90]. As can be seen in Figure 5, novel applications of wet MC as wound dressing in the treatment partial thickness burns were applied by Czaja et al. [88,89] presenting excellent results suggesting that MC as a wound dressing promotes a favorable moist environment for a fast wound cleansing, and consequently for rapid healing.
Other interesting biomedical applications for MC films have emerged. A Brazilian research group had designed and patented a device to manufacture MC contact lens for therapy in cases of regeneration of cornea .
In terms of vascular applications, a German group created BASYC® (Bacterial Cellulose Synthetised), consisting of a tubular biomaterial for applications in microsurgery of arteries and veias  and  applied vascular stents in animals arteries. Bodin et al.  obtained MC tubes by modifying the fermentation process of
Lately, Nimeskern et al.  designed and fabricated an ear-shaped pristine MC prototype material applying a Magnetic Resonance Imaging (MRI) scanning technique. This study was extremely important to confirm that MC is a promising tissue engineering material with appropriate mechanical properties for ear cartilage replacement. Thereby, it may be used to create patient-specific ear shapes.
5.2. Microbial cellulose nanocomposites for biomedical applications
Beyond the direct uses, MC can be widely and effectively utilized as either functional reinforcements or excellent matrices due to excellent mechanical properties and biocompatibility which allows it to be engineered in various forms from nano to macro scale. Thus, MC based nanocomposites can be manipulated to improve their properties and/or functionalities becoming one of the reasons that makes MC so exceptional material for biomedical applications.
In relation to biomaterial applications for wound dressing and skin tissue repair several MC based nanobiocomposites were fabricated. Here are some examples: membranes of MC/collagen , MC/gelatin , MC/ aloe vera films , MC/alginate for temporary dressing material .
Further, MC composite with kaolin was proved as short-term and long term wound healing materials .
Freeze-drying techniques allowed the preparation MC/poly(ethylene glycol) (PEG) composites of by immersing wet MC pellicle in PEG aqueous solution . This same technique was applied by some of us  to obtain MC/ silk fibroin (SF) sponge scaffolds.
Generally, a scaffold provides a foundation for cell attachment, and several materials have been tested as scaffolds to support growth of cells. The need for bio-mimicking scaffolds has led to the exploration of MC as a scaffold material. There is an increased interest in developing adipose tissue as an
Bäckdahl et al.  also developed MC scaffolds with controlled microporosity by placing paraffin wax and starch particles during culture and removing these particles once the cultivation process was finished. The MC scaffolds were then seeded with smooth muscle cells for investigating the potential tissue engineered blood vessel application.
A variety of surface functionalization through biosynthetic or chemical modification was also investigated. Various approaches to the preparation of functional MC-based nanocomposites by incorporating different guest substrates including small molecules, inorganic nanoparticles or nanowires, and polymers on the surfaces of MC nanofibers are exemplified which can improve the functionality of MC nanomaterials and expand its potential application in the biomedical fields.
Nanocomposites were obtained by the association of nanoparticles presenting antimicrobial activities, including silver nanoparticles [105,106,107,108,109,110]. Additionally, Barud et al.  also prepared MC/propol membranes that presented good antimicrobial activities to be used as wound dressing material.
In recent years, several controlled release systems based on nanocellulose material for various pharmaceutical applications have been also investigated to delivery Tetracycline , benzalkonium chloride , topical release of lidocaine  and release of proteins with serum albumin .
With respect to bone regeneration in defects of rat tibiae, MC-hydroxyapatite (MCHA) nanobiocomposite were prepared to evaluate the biological properties and performance of the material . The MCHA membranes were effective for bone regeneration and accelerated new bone formation. In addition, reabsorption of the membranes was slow, suggesting that this composite takes time to be completely reabsorbed.
Microbial cellulose is a natural renewable polymer synthesized from the bacterium
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