Biopolymers are a large group of polymers that are produced from living organisms. Bacterial cellulose (BC) is a unique material made in surface culture by such rod-shaped Gram-negative bacteria as Gluconacetobacter xylinum. However, the extraction of cellulose from other components found naturally in the plants has many drawbacks because hazardous chemicals are used in this process. Some bacterial strains can produce bacterial cellulose (BC) with a high purity degree. In this process, bacteria are used as the nutrients to produce cellulose. Hence, the cost of the nutrients plays an important role in the production process economics. Additionally, is one of the most abundant macromolecule on Earth [1, 2]. Although its production is mostly by vascular plants, an alternative route that used to produce from another resource such as bacterial systems [2–5]. Some kinds of bacteria can produce cellulose with the same as cellulose produced by higher plants and algae, but overall, it exhibits extra chemical pure. Due to its unique physical, chemical, and mechanical properties that involve high crystallinity, high water holding capacity, large surface area, elasticity, mechanical strength, and biocompatibility, thus BC has potential applications in edible packing as a food contact packaging material. In medicine, as wound dressing materials, artificial skin, vascular grafts, scaffolds for tissue engineering, artificial blood vessels, medical pads, dental implants . In other industries products, such as sponges to collect leaking oil in drug delivery agents, capsule shells, oil spill cleanup sponge; mineral and oil recovery; leather products, sports items; ultra-filters for water purification; audio speaker diaphragm; plywood laminates; specialty papers and polyesters; automotive and aircraft bodies and materials for absorbing toxins and optoelectronics materials (liquid crystal displays) .
Several genera that have shown the ability to synthesize cellulose include Sarcina, Agrobacterium, Rhizobium, and Acetobacter also known as Gluconacetobacter. The most efficient producer of BC, a Gram negative and acetic acid bacterium Gluconacetobacterxylinus is the model microorganism for basic and applied studies on cellulose. G. xylinus produces an extra-cellular gel-like material or pellicle, which comprises a random assembly of cellulose ribbons, composed of several micro-fibrils. This organism and its product were first identified and characterized over a century ago . A three-dimensional interconnected reticular pellicle is produced under static fermentation, whereas agitated and stirred conditions to produce irregularly shaped, sphere-like cellulose particles [9, 10]. However, the high cost of fermentation media has limited the industrial production of BC, as such media account for 30% of the total production cost . Thus, finding new cost-effective culture media to achieve the highest yield of BC in large-scale industrial applications is paramount and requires new carbon and nitrogen sources. The Hestrin– Schramm (HS) medium is commonly used in the cultivation of BC. However, this medium is expensive and requires additional products, such as glucose, yeast, peptone, etc. Thus, wider applications of BC depend on practical considerations concerning scale-up capability and production costs [12–14].
In recent years, studies have focused on a variety of cellulose-producing bacterial strains, inexpensive nutrient sources, and supplementary materials for the production of inexpensive BC . Different waste products from agricultural and industrial activities have been investigated as a means to improve the yield and decrease the cost of BC production, such as dry olive mill residue , sugarcane molasses[7, 17], waste beer yeast , wastewater from candy processing , wood sugars , waste from fruit processing , lipid fermentation wastewater , rice bark, konjac powder , cotton-based textile waste , and coffee bean husks . The use of such materials could improve the sustainability of BC production as well as reduce environmental pollution associated with the disposal of industrial wastes. The potential of BC goes beyond existing applications, especially if produced in large amounts from the inexpensive feedstock. Such applications may include specialty textiles, packaging, and advanced functional materials . In this way, the high-value utilization of agricultural waste is regarded as beneficial in terms of economics, environment, and practicality .
Each year huge amounts of the eggs are consumed into foods such as cakes, mayonnaise, and fast foods . about 150,000 tons of eggshell waste is generated from the US only every year . The eggshell waste can be reused as fertilizer, animal feed constituents, and others . If high amounts of this kind of waste are kept in landfills they will attract rats and vermin due to the organic membrane attached to the shells , therefore, most landfill owners do not like to dispose of eggshell wastes. Consequently, the complete recycling of eggshell waste is considered the best choice to solve this environmental and economic problem. The eggshells are considered a cheap, abundant, and natural source of CaCO3 compound which can be utilized in the synthesis of hydroxyapatite (HAp) which is one kind of bioceramic material. It has been reported that HAp is mostly preferred bioceramics, especially for fabrication of bone engineering scaffolds, due to their similarity of composition with the inorganic part of human bone [29, 30]. Synthetic HAp materials have been prepared by different methods, such as sol-gel, wet precipitation, solid-state reaction, and hydrothermal method, and they are usually prepared from chemicals. The natural precursors for the preparation of HAp are considered a versatile approach to obtaining pure final products free from toxic impurities and they give more advantageous properties, such as the pore structure carbonated constituent. Coral , bovine bone , and fishbone  are examples of natural raw materials for HAp synthesis. However, the mean problems of those materials are changeability in their physical and chemical properties . Eggshell is considered the most natural raw material of the invariable chemical composition. Therefore, there have been numerous previous works that prepared HAp from eggshells as a natural source [34–42]. Biocompatibility is an important quality of biomaterials and cytotoxicity tests are primary biocompatibility tests which measure the capacity of a material to impact on cellular viability. While a cell is in contact with a biomaterial, many reactions can occur, and a sensing Phenomenon will launch between this cell and the biomaterial  Cells adhere to surfaces through adhesion proteins (i.e. fibronectin, collagen, laminin, vitro- nectin) using specific cell receptors, called integrins, attached to the cell membrane. Cell adhesion is the ability of a single cell to stick to another cell or an extracellular matrix (ECM). . Cell adhesion is involved in stimulating signals that regulate cell differentiation, cell cycle, cell migration, and cell survival .
The main objective of this work is in situ preparation of new bioactive composites based on BC synthesized by G. xylinum by using agricultural residues as an alternative culture media without any supplementation and hydroxyapatite (HAp) derived from eggshell or HAp derived from chemicals, as well as, eggshell wastes were used added directly to the culture media for comparison. Accordingly, these bioactive composites were prepared almost from natural raw materials (eggshells and bacterial cellulose) which expectedly are as pure as to be used safely in different biomedical applications.