A Review Article of Unraveling the Potential; Exploring Application and Synthesis of Bacterial Cellulose

Cellulose is a natural and the most abundant biopolymer on the earth. It is derived from various sources, such as, microbes, naturally occurring chemical reactions, plants and enzymatic synthesis [1,2]. Bacterial cellulose should be produced in the laboratory or should be produced in the preparation of food for example nata de coco, a local dessert confectionary used in East Asia. Nata de coco was one of the most interesting foods produced by using the same methods in the laboratory. And become a famous food and gets a high commercial value. The word nata is derived from a native language of Philippines which means cellulose and it was named nata de coco because it is cultured in coconut milk or water-based media enriched with sugar [3]. Cellulose is a linear polysaccharide and is the most efficient organic materials with a wide range of beneficial application. Cellulose is a structural component in the cell wall of algae, plants and also in certain lower animals and it is typically combined to other biopolymers such as pectin, hemicellulose, lignin etc [4]. Just a small number of bacterial species, synthesize and release cellulose extracellularly in the form of fiber, which are taxonomically closely related to the genus Acetobacter [5]. Acetobacter xylinum produce bacterial cellulose which have unique characteristics which include hydrophilicity, high tensile strength, high water holding capacity, high crystallinity and extremely pure and ultra-fine fiber network structure [6-8]. Members of kingdoms Animalia and Plantae along with Eubacteria domain produce cellulose. Urochordates (tunicates) such as Microcosmus fulcatus member of the animal kingdom have the distinctive characteristics to synthesize rodshaped cellulose crystals, and hence enhance production of cellulose [9]. Cellulose is the most important biodegradable materials on earth and hence the topic of comprehensive study and great interest in macromolecular chemistry. In the previous 30 years, advancement in application of cell systems in laboratory and molecular biology had provide a comprehensive understanding about a mechanism necessary for the production of cellulose in nature [10]. Biopolymer derived from cellulose had extensive utilization in blood purification, sensor, agriculture, controllable delivery system, tissue engineering, and furthermore in water purification [11].

Louis Pasteur defined extracellular cellulose initially as a kind of moist skin, swollen, gelatinous and slippery and this product is also eaten in the past by people of Philippine as a conventional sweet after food and called Nata de coco [12]. It is a 1-cm thick gel sheet fermented from coconut water. In the meantime, the first scientific statement on this type of cellulose “on an acetic ferment which forms cellulose” [13]. He recognized a pellicle which has a chemically similar structure like ordinary cellulose and under favorable conditions its growth is heavily increases until the gelatinous membrane coated the entire surface of the liquid, and a thickness of 25mm may obtained. He deduced that there is no particular scientific name for “Vinegar plant”. After a careful attention, hence he proposed that it is a potential to produce cellulose, for this fermentation Bacterium xylinum could be an appropriate name [14].

Bacterial cellulose possesses a fundamental structure of fibril which has molecular formula (C6H10O5) n and comprises of β-1→4 glucan chain, Inter and intra-hydrogen bonding is required to join glucan chain together [15] (Figure 1) Bacterial cellulose is approximately 100 times smaller than plant cellulose and microfibrils [16].

Figure 1:Inter-and intra-hydrogen bonding of bacterial cellulose (created via Microsoft PowerPoint).

Difference between plant cellulose and bacterial cellulose

Earlier the plant cellulose had been utilized in different types of medical and as well as for clinical applications ranging from suture to cotton for hemostatic wound covering and renal dialysis membranes [17]. Cell wall of plant cells is the main source of cellulose, but cellulose derive from plant cell wall contain numerous contaminants which include lignin, pectin, hemicellulose etc. so for this reason additional decontamination is necessary before the acquired cellulose can be utilized practically [18]. Moreover, some specific bacterial genera and few members of algae secrete cellulose during their normal metabolic processes which include Agrobacterium, Rhizobium, Achromobacter, Salmonella, Sarcina, Aerobacter, Acetobacter, Escherichia and Azotobacter [19-21]. Microbial cellulose is the purest form cellulose and it holds numerous characteristics that are different from other types of cellulose [22,23]. Especially its physio-chemical, structural and mechanical characteristic is higher than that of ordinary cellulose [24]. Cellulose have highly order structure and one of the differences existed between this structure is; that a plant cellulose is naturally present in the cell wall and develops a complicated structure with pectin, lignin and hemicellulose and others additional impurities and its structure form when a number of cellulose molecular chains together constitute microfibrils and afterwards bunch and packets of high order structure such as fibril lamella and fiber cells [25]. While Gluconacetobacter xylinus release bacterial cellulose in the form of ribbon which consist of cluster of microfibrils. A ribbon which is secreting by a bacterium is finer which have a breadth of nearly one-hundredth of that plant cellulose. Ribbon cellulose more enlarges and can be clearly seen as a complex structure and forming a uniform structure which is different from plant cellulose [26]. Bacterial cellulose has unique properties different from plant cellulose which are as follows;
A. High purity, both hemicellulose and lignin are absent
B. High crystallinity
C. Excellent biodegradability
D. Water holding capacity is high, up to one hundred times its weight.
E. High biological affinity
F. Sheets of bacterial cellulose form, 15∼30 of GPa greater Young’s modulus, which is greater of all the twodimensional organic materials.

Microbial cellulose properties are previously mentioned. And microbial cellulose is expected to have useful applications which are very different from cellulose derived from plants. And it is a novel biodegradable product which is freely usable in chemical industries, food industry and in the field of medical sciences [16, 27,28].

Synthesis of bacterial cellulose

Acetobacter xylinum have the capability to polymerize 20 thousand glucose molecules per second. In two steps the assembly of cellulose microfibrils occurs such as polymerization and crystallization. In first step the glucan chain form from the polymerization of glucose which take place in membrane, during 2^nd step the glucan chains crystallization occur and form cellulose Ӏ, which take place in extracellular matrix. For cellulose production uridine diphosphate glucose gives the sugar nucleotide precursor. The production of cellulose from glucose by Acetobacter xylinum passing through a metabolic pathway which consists of four enzymatic steps. Phosphorylation of glucose by glucokinase occur in 1^st step, in 2^nd step the phosphoglucomutase enzyme take place which do the isomerization of glucose-6-phophate to glucose- 1-phosphate. While the production of UDP-glucose occurs in 3^rd step by UDPG-pyro phosphorylase enzyme, and the last step enzyme cellulose synthase enzyme takes part which consist of three subunits (BcsA, BcsB and BcsC) and consider as a specific enzyme for cellulose synthesis. After UDP phosphorylation the cellulose form as a product, which has been define chemically and enzymatically to be β-1, 4- linked glucan. For the synthesis of cellulose two genes are important such as CMCax which encode for endo-β-1-4-glucanase and CcpAx encodes for cellulose completing protein. BcsA and BcsB complex crystal structure consist of a translocating polysaccharide that prolong the polysaccharide by one glucose unit by producing a cellulose-conducting complex. In the form of ribbon, the cellulose secretes from the bacterium at a rate of 2μm per minute [29-32]; (Figure 2).

Figure 2:Schematic illustration of bacterial cellulose biogenesis and fibril formation (created via biorenders.com).

Meanwhile, bacterial cellulose has unique properties as compared to plant cellulose which include high density, high purity, high crystallinity, high water binding capacity, higher surface area and good shape retention. And it can be used in several industries such as textile, pharmaceutical, food, paper, waste water treatment, mining, refinery, and broadcasting. Following is some application of bacterial cellulose

Food applications

Pure cellulose can be utilized chemically as a thickening and stabilizing agent in processed food [33]. Bacterial cellulose was initially utilized in food industries in Philippines as a nata de coco. Bacterial cellulose has similar properties like gel and it is perfectly indigestibleness in the human intestinal tract made this one of the most attractive food base [34]. In the 1970s, one of the most important metabolites of Monascus specie monacolin K (mevinolin) was recognized and has the ability to prevent the production of cholesterol. A product of bacterial cellulose synthesized from Acetobacter xylinum such as nata, which is the most famous food in Philippines and other countries. Bacterial cellulose is extensively utilized in food processing because it is high fiber content and distinctly soft texture. The functional properties of monacolin K and bacterial cellulose has combined to form Monascus-nata complex, hence is likely a new functional foodstuff [35]. Bacterial cellulose was added into diet drinks in Japan. Acetobacter and yeast are culture together in the tea extract and sugar. This is use up as a kombucha, or Manchurian tea for enhancing health needs [36]. Gelatinous cellulose consists of cellulose fibrils (0.9%), bound water (0.3%) and free water (98.8%) is synthesized by fermentation with Acetobacter aceti AJ 12368. About 0.5-1.0 microns of water is taking up timidly in capillaries by cellulose network. The gel is deformed without cracks if it is stressed to release its water. Further processing is performed either with alginate or sugar alcohol and calcium chloride to fit the gel for human consumption because before processing it is hard to chew. The texture is similar to fruits, e.g. mollusks and grapes, especially squids. The gel becomes soft to break off with the teeth after processing of gel in a mechanism in which the water is immobilized in the gelatinous cellulose by gel or viscous forming materials. This mechanism demonstrates that gelatinous cellulose with its porous structure can be a novel material for fabricated food, low calorie desserts and salad [37].

Pharmaceutical and medical application

Microbial cellulose has unique characteristics such as extremely porous, high tensile strength, and micro fibrillar structure. Some chronic wounds are hard to heal which include bedsores, venous leg ulcers and diabetic ulcer, and they exhibit an important clinical challenge both for the diseased person as well as for the physician. The use of several products such as hydrogel, hydrocolloids and biological or synthetic membranes, which provide a good moist condition for wound healing which is required for normal healing, is important for the treatment of chronic wounds [38]. According to the current methodologies in the field of healing of wound, both structurally as well as functionally artificial skin must show resemblance to an ideal wound dressing system. Latest wound dressing product has unique properties including; nontoxicity, nonpyrogenicity and biocompatibility. It also has the capability to ease pain throughout the treatment, capability to control loss of fluid, provide protection against infection, capability to sustain control moist condition in the wound, provide comfortable covering which are resistant to potential damage, during inflammatory stage ability to absorb exudates, facilitate transfer or introduction of antibiotic into the wound, high comfortability, elasticity and mechanical strength, and allow comfortable healing of wound without pain. Bacterial cellulose permits an efficient transfer of medicine and other antibiotic into the wound as well as providing as an effective protection against any external infection. It fulfills the need of modern wound dressing product [39].

Skin therapy

The gelatinous membrane of microbial cellulose was usable as an artificial skin for covering of wound temporarily due to its unique characteristics such as high mechanical power in the moist state, extensive permeability for liquid and gases and causes less irritation to skin. Microbial cellulose products such as Gengiflex® and Biofll® which have widespread applications in dental implants and surgery and actually in the health care sectors of human. Biofll® provide covering for human skin wounds for a brief period of time and effectively treated cases of second and third degree of burns and ulcers. Biofll® have several benefits for more than three hundred treatment and the researchers documented some of them which include; decrease rate of infection, diminished postsurgery discomfort, closely sticking to the wound bed, easiness of wound inspection, rapid relief of pain, rapid healing, spontaneous detachment following re-epithelization, better exudates retention and decrease duration of treatment as well as expenses. Just one disadvantage was found that in area of excessive mobility partial elasticity was found. Another Microbial cellulose product was produce called Gengiflex® which heals periodontal tissues. Different researchers further documented more applications and results of Gengiflex® as well as Biofll®. Microbial cellulose application in veterinary medicine was describe that microbial cellulose is used for the treatment of large surface wound on dogs and horses [40,41].

Cartilage replacement

Complete replacement of joints by prostheses was carried out to treat advanced degeneration of articular cartilage. Lifetime of these prostheses is become short as a result of osteolysis and wear. Several reports have determined that bacterial cellulose composites may be exploiting as a material for cartilage replacement [42]. It is inferred that mechanical properties of cartilage are similar to mechanical properties of bacterial cellulose double network poly dimethyl acrylamide gel. So that could be suitable for the essential requirements of artificial cartilage. But the test performed in the living organisms that could approved that the bacterial cellulosebased cartilage replacements have no harmful effects on human and has been biocompatible are not still documented. Bacterial cellulose poly vinyl alcohol composites also have the same elastic modulus values which have been documented for natural articular cartilage when estimated using unrestricted compression testing [39].

Artificial cornea

About 10 million of people globally loss their ability of eye as a result of corneal disease and thus corneal disease is the major agent to cause sightlessness. Therefore, a broad variety of biomaterial is used as a bioengineered cornea to fulfill the need of corneal transplant. The characteristics of bacterial cellulose which include; nano-porous structure, excellent mechanical properties that provide a definite placidness and assist to keep the intraocular pressure and make it a desirable material to be utilized as an artificial cornea. Bacterial cellulose such as polyvinyl alcohol hydrogel with high light transmittance and high-water content which are similar to that of natural cornea was synthesized. Both biopolymers have hydroxyl group which supplied the important interfacial interaction in the composite. Improved mechanical strength, light transmission, water holding capacity and excellent thermal ability shown that the hydrogel composite is considering very favorable optically functional material to be used for corneal transplant. Due to these unique properties further studies would be greatly useful for achieving success in artificial corneas [43,44].

Dental implants

The ability of bacterial cellulose to be used for a dental canal treatment material for intra canal asepsis was identified in a recent report. In this exceptional analysis pointed cellulose is used which is isolated from bacterial cellulose membrane synthesis by two strains of Acetobacter hansenii (ATCC700178 and ATCC35059). In contrast to commercially available plant cellulose-based paper point, pointed bacterial cellulose exhibit different properties such as greater liquid absorption capacity and expansion capacity. Wet bacterial cellulose has high tensile strength and drug release capability and these characteristics are higher under normal condition which clearly shows that bacterial cellulose has a high ability to utilize in dental root canal treatment [45,46].

Bacterial cellulose in paper industry

Microbial cellulose is made up of very small bundles of cellulose microfibrils, and has been used as a binding in papers, and this characteristic greatly improves the stability and strength of pulp when incorporated into papers. Many industries in Japan are now a days busy in synthesizing a high-quality paper product from microbial cellulose such as Mitsubishi paper mills and Ajinomoto Co. Furthermore, once static microbial cellulose as well as agitated microbial cellulose was attached at moist end, then filler retention as well as tensile strength of the hand sheets become enhance. Especially, the microbial cellulose derived by agitated culture had strong impact on filler retention as compared to static one. Therefore, microbial cellulose can be suitable as a moist-end additive for manufacturing of paper. Moreover, nutrient medium containing suspended solid for Acetobacter xylinum in a rotating disk bioreactor become integrated into the gelatinous mat of microbial cellulose as it produced. Ordinary cellulose which has embedding fibers and forms composites with improves durability and intensity of microbial cellulose. Refined cellulose and extended fibers from paper are differently integrated than circular particles e.g. silica gel. Plain bacterial cellulose is much weaker than dried composite sheet per unit area and approximately 90% of the final cellulose can be derived from scrap paper. During paper sheet production integrating 5% of bacterial cellulose with wood pulp can effectively enhanced strength, fire resistance and kaolin retention characteristics which are different from paper sheets manufactured from integrating bacterial cellulose [47,48].

Artificial blood vessels

Microbial cellulose has different property which makes it unique from the cellulose of plants. Bacterial cellulose provides an exceptional material for making of artificial blood vessel, due to its beneficial characteristic of high permeability of oxygen. G. Xylinum biosynthesized tubular template materials called polydimethylsiloxane. Its characteristics were scanned through (scanning electron microscope), demonstrated that Bacterial cellulose tubes are best materials artificial blood vessel with high thermal stability qualified mechanical elaborated nano-fiber architecture, properties. In addition, that Bacterial cellulose tubes are greatly adjustable in vivo environment [49-51].

Potential scaffold for tissue engineering

It is not described yet that tissue constructs for cartilage with mechanical properties. To identify and fulfill this need microbial cellulose produced by G. xylinus and A. xylinum were explored as a material for scaffolding due to its exceptional characteristics [52]. Bacterial cellulose materials which are chemically modified were assessed by using bovine chondrocytes. Advance biomaterial of bacterial cellulose which is produced by the fermentation of bacteria such as Acetobacter xylinum. It is unique because of high mechanical properties and its biocompatibility despite the fact that it is high water content. Microbial cellulose has been utilized as scaffolds for tissue. Studies show that chondrocytes smooth muscle endothelial cells show that both cells stick to the microbial cellulose. However, a definite disadvantage with bacterial cellulose is that the Nano-fibrils form a dense mesh that can reduce cell infiltration. To increase cell infiltration into bacterial cellulose scaffolds, the material was become porous by introducing porogen into the process of fermentation by A. xylinum [53-55].

Wound care products

Bacterial cellulose makes a protective layer on the wounds which enhance healing of wounds and also protect wounds from entering bacteria into the wounds. Bacteria which is present already in wounds however can, increase in the damp environment produced by the Bacterial cellulose dressing which can exaggerate process of the healing. It is highly effected that the BC antimicrobial property without affecting the mechanical and structural properties of wounds. In some studies, it is recommended methods for the function of Bacterial cellulose with ε-poly-L-Lysine (ε-PLL), it has a broad spectrum antimicrobial activities.it inhibited growth of S. epidermidis on the membranes but did not affect the compatibility to cultured human fibroblasts as compared to native BC [56,57]. It has possibility to functionalize BC with ε-PLL is a green promising, and versatile to improve the performance of Bacterial cellulose in wound care and other applications of biomedical [58].

Tablet modification

Microcrystalline cellulose is prepared by new preparation techniques from kenaf (KF) and Gluconacetobacter xylinus (BC) is documented. These improve cellulose (DBC and DKF) products indicated various crystalline structures. DKF displayed cellulose II lattice with high crystallinity (70%) whereas DBC displayed cellulose I lattice with high crystallinity (85%). DBC particle size was 1-5mm while DKF particle size was 5-20mm. commercially available microcrystalline cellulose Avicel® PH 101 physical properties were compared with those of DBC and DKF materials. DKF and Avicel® PH 101 have high density than the DBC. Both Avicel PH 101 and microcrystalline DBC exhibit same behavior during flow and binding procedure. Thermo-gravimetric analysis was carried out to measure the thermal properties of DKF and DBC. Thermo-gravimetric analysis represents that thermal stability of DBC is increased as compared to DKF. Due to decomposition of cellulose DBC weight loss in one step degradation process from about 320 °C to 380°C. Both cellulose materials have difference in their thermal properties and this is generally due to difference in their crystallinity. DKF displayed 77% degree of crystallinity while DBC displayed 85% of crystallinity according to the XRD data. This shows a link between thermal degradation and crystal structure of cellulose. A greater crystalline structure required a higher degradation temperature and hence, DKF at 215 °C whereas DBC degraded at 320 °C [59-61].

Bacterial Cellulose (BC) as a transdermal drug delivery method

BC may be able to enter the domain of drug delivery systems using transdermal methods. It is evident that BC has been extremely helpful in wound healing including skin-substitute products. Due to its ability to keep moisture from evaporating, prevent external contamination, and maintain close touch with the exposed, inflammatory, or diseased area, the characteristics of such a woundhealing system are easily orientated towards transdermal drug administration [62]. This makes it easier to deliver drugs locally to the intended location. Following immersion in benzalkonium chloride (an antibacterial agent; Merck KGaA, Darmstadt, Germany), BC dry films were obtained. Drug effects persisted for at least 24 hours, and the drug-loading capacity per unit surface area was determined to be 0.116mg/cm2 against bacteria including Bacillus subtilis and Staphylococcus aureus that are flora in contaminated wounds [63]. BC fibers containing silver nanoparticles have demonstrated antibacterial efficacy against S. aureus and Escherichia coli of up to 99.99% [64]. When antibacterial qualities are needed, the usage of silver nanocomposites with BC has been recommended as a viable strategy [65]. An antihypertensive drug, propranolol’s S-enantiomer was released from a methacrylatebased BC membrane composite layer and used in transdermal applications where the parent BC membrane provided the main control over drug release.

A gel reservoir and molecularly Imprinted Polymer (MIP) membrane were used to illustrate how a transdermal patch could function for enantiomeric release. For in-vitro drug release, MIP membrane alone was compared to cellulose and Non-Imprinting Polymer (NIP) membranes. To clearly identify the control given upon release, the gel reservoir was put through in-vitro skin permeation tests using the dorsal skin removed from male Wistar rats. The BC membrane that made up NIP had reactive pore filling from S-propranolol. It was discovered that this procedure gave MIP a better enantioselective release profile than NIP-cellulose membranes.

Additionally, a cumulative release of 48% by poloxamer and 60% by chitosan was noted. The greater degree of ionization of the functional monomer, which boosted binding of the S-propranolol enantiomer at the imprint site, was responsible for the NIP composite membrane’s improved enantioselectivity. Furthermore, the enantioselective transport of propranolol was found to be influenced by the type of gel utilized; the more structured poloxamer gel formulation did not provide any selective release of S-propranolol. After 8 hours, the Cmax of S-propranolol (8.0±1.0ng/ ml) was obtained from a reservoir of 1.5mg of racemic propranolol, according to in-vivo studies with transdermal patch gel reservoirs made of chitosan and poloxamer [66]. These remarkable results demonstrated that BC, a base for molecular imprinting networks, is amenable to reactive pore filling and other treatments, making it appropriate for chiral applications.

Bacterial cellulose has a great importance in market in the field of cotton and forest industry. Bacteria like A. xylinum can produce a large number of fabrics then the expectations which has not reached to its peak point yet. Bacteria that produce acetic acid have the potential to produce cellulose that is the alternate source of plants through oxidative fermentation inspired and static conditions. But due to inefficient production it is high cost and its use is restricted at industrial and commercial levels the researchers are now focusing on waste of agriculture by-products of industry could be effective source of carbon. Along with this the modification in bacterial cellulose and its chemical properties enhances chances for its new applications.

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