STUDIES ON PRODUCTION AND APPLICATION OF BACTERIAL CELLULOSE

Abstract

Bacterial cellulose (BC) or Bacterial Nanocellulose (BNC), a polymer formed by linear coupling of glucopyranose units using microbial processes, has primed a great deal of attention worldwide due to its unique physico-structural properties. It is identical in chemical composition to plant cellulose, however, its physico-structural features such as ultra-fine nanofibrous network, mechanical strength, ability to be molded in any shape, and extra purity (i.e. devoid of pectin, lignin, hemicellulose and other metabolic products); bestow BNC with plenteous advantages over plant cellulose and make it a potential precursor for breakthrough technologies in several vital arenas leading to cutting edge products like optoelectronic materials, acoustic membranes, ultrafiltration membranes, supercapacitors, fuel cell, polymer matrices reinforcement, cosmetics, wound dressing materials, surgical implants, tissue engineering scaffolds, drug delivery systems and other biomedical devices. Despite its multidimensional applicability, the widespread usage of this promising polymer hinge on the practical considerations such as the scale-up capability and production costs, as if comparing with other popular commercial organic products, BNC is still expensive, therefore, its use is limited. Consequently, the reduction of production costs and scaling up the production process are technological prerequisites for BNC to be adopted at wider scale. One of the ways to make the production process economically feasible is to search for low- or no-cost, abundant and easily available carbon sources (as during BNC production, the culture medium itself represents approximately 30 % of the total cost) and to design a simple, less labor intensive cultivation strategy for higher productivity of BNC. Moreover, the low conversion yield of BNC, in terms of high input of raw materials, is another major economic constraint to the commercialization of BNC at a “low” cost. In addition, there are very few microorganisms proficient in producing BNC up to an extent where its industrial use is possible. Only a few bacteria of the genus Komagataeibacter (preferably Komagataeibacter xylinus: the most referenced and used strain worldwide) were found to produce significant amount of BNC. Since, BNC synthesis has been strictly linked to the bacterial cell metabolism; the strain type and the culture conditions have a crucial influence on BNC production, in particular regarding factors such as carbon and nitrogen sources, metabolic stimulants, temperature and pH. In this context, the present work was undertaken with a view to search for a potential cellulose producing strain(s) and to optimize the metabolic status of the selected strain(s) towards Abstract Swati Dubey Ph.D. Thesis 2017 Page ii enhanced cellulose synthesis by ameliorating the physiological dynamics to achieve a high conversion yield of BNC along with developing inexpensive culture media using low- or no-cost feedstocks to make the production process cost effective, followed by designing a simple and less labor intensive production process strategy to scale-up the production process for higher productivity of BNC. This may thus enable wider applicability of this value product and open up new avenues to deliver multifarious products to the market at competitive price. The thesis has been divided into six chapters. Chapter 1 and 2 includes the introduction and detailed literature review of the bacterial nanocellulose with respect to its properties, production, purification and applications. Chapter 3 embodies isolation of cellulose producing bacterial strain(s) and reprogramming of culture parameters for enhanced cellulose production. A total of 46 bacterial strains were isolated from different natural sources based on their colony size, shape and morphology. Amongst these, 4 bacterial isolates were found positive for cellulose production. All of these strains were isolated from black rotten grapes (Vitis vinifera) and no cellulose producers were obtained from other natural sources. Out of these 4 strains, isolate SGP37 was competent to produce notable amounts of BNC (5.61 ± 0.11 g L-1, after 16 days of cultivation), hence selected as the most potent BNC producer for further studies. The isolate was identified as a strain of Komagataeibacter europaeus (formerly Gluconacetobacter europaeus). The strain was kinetically analyzed to evaluate BNC production under different physiological conditions. The stagnant cultivation of the strain in HS medium resulted into the production of 5.61 g L-1 cellulose after 2 weeks of fermentation, with conversion yield of 0.36 g cellulose/g sugar, at initial production rate of 0.95 g L-1 d-1. Amelioration of physiological dynamics of the strain by devising preeminent culture conditions, enhanced the production rate of cellulose by ~1.65 fold (1.55 g L-1 d-1) and attained 9.98 g L-1 cellulose with initial sugar consumption of 12.08 g L-1, resulting into a very high conversion yield (0.82 g cellulose/g sugar) ever reported. Chapter 4 focuses on the development of inexpensive production media and designing the production process for low-cost and scaled-up production of bacterial nanocellulose. Sweet lime pulp waste (SLPW) and banana peel waste (BPW) were utilized as a low- or no-cost feedstock for the production of bacterial nanocellulose (BNC) alone and in amalgamation with other nutritional supplements by the isolate K. europaeus SGP37 under static batch and static intermittent fed-batch cultivation. The highest yield (26.2 ± 1.50 g L-1) was obtained in the hot water extract of SLPW supplemented with the components of HS medium, which got further boosted to 38 ± 0.85 g L-1 as Abstract Swati Dubey Ph.D. Thesis 2017 Page iii the cultivation strategy was shifted from static batch to static intermittent fed-batch. BNC obtained from various SLPW and BPW medium was similar or even superior to that obtained with standard HS medium in terms of its physicochemical properties. The production yields of BNC thus obtained are significantly higher and fit well in terms of industrial scale production. Chapter 5 depicts the effect of various purification approaches on purity and physicochemical properties of BNC. The study was carried out to evaluate the impact of various treatment approaches onto the purity and physicochemical properties of BNC in order to find out precisely a process that can remove the bacteria from BNC pellicle but, at the same time, prevents the polymorphic transformation of cellulose I to cellulose II. The BNC pellicles were purified using 0.5 M NaOH, 0.5 M KOH, 10% SDS, 0.5 M NaOCl and 0.5 M H2O2 separately and the purity of the membranes was monitored using solid-state 13C-NMR. Only the BNC treated with NaOH and KOH had shown the pure fingerprints of cellulose while the other treated samples were found to be contaminated by proteins which may be due to the presence of bacterial smidgens left after purification. Atomic force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FE-SEM) analyses also revealed the presence of bacterial cells and some other cloudy aggregations in all the BNC membranes except the BNC purified using NaOH and KOH. However, the BNC membrane purified using KOH method was more crystalline and thermally stable than the membrane purified using NaOH. Together, these results suggested that the KOH treatment of the pellicle was effectively able to remove the bacterial cells and other contaminants from the BNC membrane and at the same time was able to maintain the physicochemical properties of BNC. Chapter 6 focuses on the preparation of 3-D microporous-nanofibrous BNC scaffolds and evaluation of their potential for bone tissue engineering. Microporous-nanofibrous BNC scaffolds (mBNC) were prepared using freeze-dry method and thoroughly characterized in terms of their morphology, chemical structure, crystallinity and biodegradability which were then followed by culturing C3H10T1/2 mesenchymal stem cells on these scaffolds to assess the cell attachment, proliferation, infiltration and osteoblastic differentiation for effective regeneration of bone tissue. The prepared scaffolds revealed a highly porous microarchitecture compared to native BNC membrane. The in vitro biocompatibility of the mBNC scaffolds was analyzed based on the adhesion, growth and proliferation of C3H10T1/2 mesenchymal stem cells. Results indicated strong cell adhesion with extended morphology of the cells on the surface as well as inside the pores of mBNC scaffold. The scaffold exhibited very good biocompatibility with hardly any Abstract Swati Dubey Ph.D. Thesis 2017 Page iv detectable cell death and the cells continued to proliferate with respect to time. Cell ingress into mBNC scaffolds was observed by DAPI stained cell nuclei in scaffold cross sections and found that C3H10T1/2 cells had infiltrated and homogeneously distributed throughout the entire depth of scaffold; indicating the potential of mBNC scaffold for tissue in-growth. Alizarin red staining (ARS) and energy-dispersive X-ray spectroscopy (EDS) analysis revealed osteogenic differentiation of C3H10T1/2 on the scaffolds, which demonstrate the potential of mBNC scaffolds for bone tissue engineering applications.