TO ASSESS BACTERIAL CELLULOSE AS AN ALTERNATIVE TO PLANT CELLULOSE IN TEXTILE INDUSTRIES IN KENYA BY MARERI BRENDA MORAA 1 DECLARATION I declare that this is my own research work presented to the future ideas forum. DEDICATION I dedicate this project to my family and friends for their support and encouragement. 2 ABSTRACT Bacterial cellulose is an alternative to using plant cellulose due to its high level of purity due to the absence of hemicelluloses and lignin and does not require extensive processing. Bacterial cellulose is an exopolysaccharide produced by Acetobacter xylinum inoculated in suitable medium consisting glucose, yeast, citric acid referred to as Hestrin-schramm media. The concentration of glucose was varied to determine at what concentration there is maximum production of cellulose. Acetobacter xylinum was isolated from rotting apples and confirmed by biochemical and morphological tests shown in Plate 6, 7 and 8. A bacterial cellulose pellicle formed on the surface of the media as shown in Plate 9. There was a greater yield of bacterial cellulose at greater concentration of glucose as seen in Table 1. Plant cellulose was obtained from crushed maize seeds, hydrolyzed and compared with the bacterial cellulose. The method used for comparison was high performance liquid chromatography method. Plant cellulose contained hemicelluloses and lignin as shown by Graph 3 compared to bacterial cellulose shown in Graph 2 which lacked the impurities. It is a cheap alternative since less processing is required for removal of impurities as it is pure cellulose. 3 TABLE OF CONTENTS DECLARATION.............................................................................................................. i DEDICATION................................................................................................................. i ABSTRACT................................................................................................................... ii CHAPTER ONE.............................................................................................................. 1 INTRODUCTION........................................................................................................... 1 1.1 Background.......................................................................................................... 1 1.1 Statement of problem................................................................................................... 2 1.2 Justification............................................................................................................... 2 1.3 Objectives................................................................................................................. 3 1.3.1 Main objective......................................................................................................... 3 1.3.2 Specific objectives.................................................................................................... 3 1.4 Null hypothesis........................................................................................................... 3 CHAPTER TWO............................................................................................................. 4 LITERATURE REVIEW................................................................................................... 4 2.1 Description and Ecology of Acetobacter xylinum.................................................................4 2.2 Bacterial cellulose....................................................................................................... 5 2.3 Production of bacterial cellulose...................................................................................... 6 CHAPTER THREE........................................................................................................ 11 MATERIALS AND METHODS........................................................................................ 11 3.1 Preparation of fermentation media and culture media conditions............................................11 4 3.2 Isolation of Acetobacter xylinum................................................................................... 11 3.3 Initial culture and Subculture of diluted sample.................................................................11 3.4 Morphological tests................................................................................................... 12 3.5 Gram negative test of Acetobacter xylinum and Biochemical tests..........................................12 3.6 Growth of Bacterial cellulose....................................................................................... 12 3.7 Purification of the bacterial cellulose pellicle....................................................................12 3.8 Lignin determination.................................................................................................. 13 3.9 Extraction of plant cellulose and bacterial cellulose............................................................13 3.1.1 Dissolution in NaOH............................................................................................... 13 3.1.2 Percentage yield of bacterial cellulose..........................................................................14 3.1.3 Data Analysis........................................................................................................ 14 CHAPTER FOUR.......................................................................................................... 15 RESULTS.................................................................................................................... 15 4.1 Initial culture of diluted sample..................................................................................... 15 Plate 3: Colonies after initial culture at 10-4 and 10 -3 dilution factor.............................................15 4.2 Gram negative test, Endospore staining and Biochemical test................................................16 4.3 Morphological tests................................................................................................... 17 4.4 Growth of Bacterial Cellulose....................................................................................... 18 4.5 Dissolution in NaOH (aq)............................................................................................ 19 4.6 Lignin Determination................................................................................................. 19 4.7 Bacterial cellulose dry weights...................................................................................... 19 5 4.8 Percentage yield of Bacterial Cellulose........................................................................... 19 4.9 Data Analysis........................................................................................................... 20 4.1.0 Analysis of purity of the cellulose extracted..................................................................................22 CHAPTER FIVE........................................................................................................... 24 DISCUSSION............................................................................................................... 24 5.1 Acetobacter xylinum characterization..............................................................................24 5.2 Bacterial Cellulose growth........................................................................................... 24 5.3 Analysis of purity...................................................................................................... 26 CONCLUSION............................................................................................................. 28 RECOMMENDATION................................................................................................... 29 Recommendations put forward after analysis of the project results to obtain a greater yield of glucose and obtain more information about the bacterial glucose include;.....................................................29 References................................................................................................................... 30 6 List of plates, tables and grap Plate 1: Scanning electron micrograph of bacterial cellulose (Moosavi-Nasab et al., 2011)........17 Plate 2: FTIR spectra of bacterial cellulose (Gor et al., 2012)......................................................17 Plate 3: Colonies after initial culture at 10-4 and 10 -3 dilution factor............................................23 Plate 4: Pure colonies after subculturing.......................................................................................24 Plate 5: Green dots of the cream colony and lack of green dots of translucent colony.................24 Plate 6: Rod like bacteria x10........................................................................................................25 Plate 7: Pink rods after gram staining............................................................................................26 Plate 8: Gas bubbles in hydrogen peroxide...................................................................................26 Plate 9: Bacterial cellulose after 7 days of incubation...................................................................27 Y Table 1: Table of dry weights of bacterial cellulose and different glucose concentrations replicates........................................................................................................................................27 Table 2: Table of percentage yield of bacterial cellulose and different glucose concentrations....28 Table 3: Table of bacterial cellulose weights and square roots of the weights..............................28 Graph 1: Chromatogram showing peaks for pure cellulose.............................................30 Graph 2: Chromatogram showing peaks for bacterial cellulose........................................31 Graph 3: Chromatogram showing peaks for plant cellulose............................................31 CHAPTER ONE INTRODUCTION 1.1 Background Bacterial cellulose, a biopolymer produced by several strains of acetic acid bacteria, has the same chemical structure compared to plant-derived cellulose, which is a homogeneous polymer composed of β-1, 4-glycosidic linkages between the glucose molecules. Paper and textile 7 industries require a significant amount of the plant derived cellulose, which leads to a considerable demand on wood biomass. The bacterial cellulose is distinguished from the plant- derived cellulose by its high degree of polymerization, high purity, and high water-holding capacity free from lignin and hemicellulose. In addition, bacterial cellulose has high polymer crystallinity and excellent physicochemical characteristics superior to the plant derived cellulose (Wee et al., 2011). Some species of Acetobacter, recently named as Gluconacetobacter, are known to produce bacterial cellulose exhibiting superior features over plant cellulose although being chemically identical. The unique features of this material such as extreme purity, high crystallinity and degree of polymerization have gained considerable commercial and scientific interest (Aydin, 2009). Bacterial cellulose is a suitable biomaterial instead of plant cellulose because of the high tensile strength, insolubility in most solvents, non-toxicity and good shape retention. The bacterial cellulose can be dried upon synthesis and used as raw material for textile industry as a cheaper alternative and requires minimal processing. Main disadvantage is the high water retention capacity which can be dealt with by adding water repellent biomolecules in the media which is still being researched 1.1 Statement of problem Use of bacterial cellulose in Kenya as an alternative raw material in textile industry has not been exploited. There has hardly been any study done to establish purity of bacterial cellulose and its tensile strength due to its ultrafine fibril structure. Bacterial cellulose has been used in other countries as food additive, in paper and textile industries and has proven to be a better 8 alternative. By determination of the purity of bacterial cellulose it can be incorporated into certain industries as a raw material for textiles. There is need to sensitize the community on the use of bacterial cellulose as an eco-friendly and cheaper alternative due to little need for further processing and high rate of biodegradability. Bacterial cellulose will reduce the need for wood and plant fibres in industries hence preserving the environment. 1.2 Justification Bacterial cellulose can be synthesized at lab scale using locally available chemicals and can be more cost effective option due to the little processing required. Bacterial cellulose has higher level of purity than plant cellulose which contains hemicelluloses and lignin. Bacterial cellulose deals with environmental concerns of production, consumption and disposal of textiles that is most related to using plant cellulose and fibres. Bacterial cellulose is a suitable biomaterial instead of plant cellulose because of the high tensile strength, insolubility in most solvents, non- toxicity and good shape retention. The bacterial cellulose can be dried upon synthesis and used as raw material for textile industry as a cheaper alternative and requires minimal processing. 1.3 Objectives 1.3.1 Main objective To determine purity of bacterial cellulose compared to plant cellulose in textile industries. 1.3.2 Specific objectives 1. To isolate Acetobacter xylinum from rotting apples 2. To characterize Acetobacter xylinum from morphological and biochemical tests 3. To extract plant and bacterial cellulose 9  4. To determine purity of bacterial cellulose and plant cellulose 1.4 Null hypothesis Bacterial cellulose derived from Acetobacter xylinum in suitable media is not purer than plant cellulose. 10  CHAPTER TWO LITERATURE REVIEW 2.1 Description and Ecology of Acetobacter xylinum Acetobacter xylinum is a microorganism found in symbiotic relationships with plants such as coffee and sugarcane. It is Gram negative and anaerobic. It has been used as model organism in the research on synthesis of bacterial cellulose. The microorganism can be isolated from rotting fruits such as apples and grapes. By performing suitable biochemical tests and morphological tests the microorganism can be identified. Although synthesis of an extracellular gelatinous mat by Acetobacter xylinum was reported for the first time in 1886 by Brown using Acetobacter xylinum as a model bacterium, practical work was started by Hestrin and Schramm in 1954, who proved that resting and lyophilized Acetobacter cells synthesized cellulose in the presence of glucose and oxygen (Bielecki et al.,2010). Acetobacter xylinum is an aerobic soil bacterium in the family of bacteria that ferments carbohydrates to vinegar (Acetobacter aceti). Acetobacter xylinum, a Gram negative bacterium found in the soil can frequently be isolated from decaying fruit such as apples and grapes. A. xylinum is an unusual member of this family because it synthesizes and extrudes fibrils of cellulose as part of the metabolism of glucose. The glucose subunits that form the cellulose micro fibrils are extruded through pores in the cell wall of the bacteria. In standing laboratory culture of the bacteria, the cellulose fibrils bundle together to form a mat or pellicle within which 11 the bacteria are held. The pellicle floats on the surface of the medium allowing the bacteria to obtain plenty of oxygen, which they require for growth, multiplication, and more cellulose synthesis (Cannon and Skinner, 2000). 2.2 Bacterial cellulose Extensive research on bacterial cellulose revealed that it is chemically identical to plant cellulose, but its macromolecular structure and properties differ from plant cellulose (Bielecki et al., 2010). Researchers have tried to increase the productivity of cellulose from Acetobacter xylinum using various biochemicals. Recently different carbon sources, such as monosaccharides, oligosaccharides, alcohol and organic acids, were used in bacterial cellulose production (Kesh and Sameshima, 2005). Bacterial cellulose has gained attention in the research realm for the favorable properties it possesses; such as its remarkable mechanical properties in both dry and wet states, porosity, water absorbency, moldability, biodegrability and excellent biological affinity. Because of these properties, bacterial cellulose has a wide range of potential applications including use as a separation medium for water treatment, a specialty carrier for battery fluids and fuel cells, a mixing agent, a viscosity modifier, immobilization matrices of proteins or chromatography substances (Marzieh and Yousefi, 2010). The first scientific paper was written by Brown in 1886 on a peculiar fermentative substance. Under pure cultivation in carbohydrate media, it was observed that the whole surface of the liquid is covered with a gelatinous membrane, which may attain a thickness of 25 mm under favorable circumstances. On removing the membrane from the liquid, it was found to be very tough, especially if an attempt is made to tear it across its plane of growth. From chemical 12 analysis and various reactions, the substance was concluded without doubt to be cellulose, although microscopy at that time only gave a picture of living bacteria embedded in a transparent structureless film (Amano et al., 2005). In recent research work bacterial cellulose has been modified to be used as electronic paper. Due to the nanostructured nature and paper-like optical properties when completely dried, microbial cellulose has been used instead of commercial paper for creating certain devices (Shah and Brown, 2005). There have been some studies on breeding bacterial cellulose producing bacteria. Previously, bacterial cellulose producers with increased bacterial cellulose synthase activity have been bred by genetic engineering, with branches of their metabolic pathway blocked to decrease the amounts of by-products (Tsuchida and Yoshinaga, 1997). Bacterial cellulose (BC) is chemically pure, free of undesirable components such as lignin and hemicellulose (there is no need for chlorine chemical bleaching) and has high polymer crystallinity and high degree of polymerization that distinguishes it from other forms of cellulose (Marzieh and Yousefi, 2011) 2.3 Production of bacterial cellulose Usually, glucose and sucrose are used as carbon sources for cellulose production, although other carbohydrates such as fructose, maltose, xylose, starch and glycerol have also been tried. The effect of initial glucose concentration on cellulose production is also important, since the formation of gluconic acid as a byproduct in the medium decreases the pH of the culture and ultimately decreases the production of cellulose. The addition of acetic acid in the media has proven to decrease the production of gluconic acid. 13 Shihara and colleagues in 1945 used xylose as a carbon source for the production of cellulose by A. xylinum and obtained a yield of 3.0 g/L. Sucrose, mannitol and glucose were found to be the optimal carbon sources for cellulose production by A. xylinum (Chawla et al., 2009). Components of sugarcane molasses were added such as sucrose, fructose, glucose, nitrogenous compounds, non-nitrogenous acids, nucleic acids, vitamins, other carbohydrates, minerals and black colour substances individually or in combined forms into Hestrin-Schramm medium. Their effect on bacterial cellulose production by Acetobacter xylinum was investigated. They concluded that the addition of vitamins, amino acids, other carbohydrates, minerals and black colour substances to the molasses in the Hestrin-Schramm medium with a mixture of sucrose and fructose as the carbon source increased the bacterial cellulose yield. The effect of the addition of sodium alginate on bacterial cellulose production by Acetobacter xylinum was studied. It was observed that sodium alginate hindered the formation of large clumps of bacterial cellulose, and enhanced cellulose yield. The effect of green tea on cellulose production by a G. xylinus strain isolated from kombucha was also investigated. Green tea at a level of 3 g/L gave the highest cellulose yield of 3.34 g/L after 7 days of incubation (Chawla, 2009). The results of various carbon sources for maximum cellulose production were performed by Panesar and colleagues. It was observed that the maximum cellulose production was obtained in mannitol followed by glucose. Similar trend of cellulose production has also been reported by other researchers. Mannitol gave best yield but due to the cost factor glucose was selected as 14 carbon source for further experimentation. Peptone, sodium nitrate and methionine were found to be most effective among these nitrogen sources. Panesar reported that vitamins and amino acids played important role for cell growth and cellulose production. Considering the cost factor sodium nitrate was screened as nitrogen source for further experimental studies (Panesar et al., 2009). Cellulose was isolated for gas liquid analysis by extraction procedure involving acetic acid and nitric acid modified to recover product. Materials such as corn were proven to have high levels of lignin by hydrolyzation with 72% sulphuric acid. Complete dissolution will not be achieved fully due to presence of lignin (Sloneker, 1971). It was concluded that the synthesis of cellulose involved several enzymatic processes that Acetobacter xylinum is involved in. Microbial cellulose have wide application in various fields such as food, healthcare, cosmetics and beauty, clothing and shoes, outdoor sports, baby care products, audio products like speaker diaphragms (Cannon and Skinner, 2000). It is also being used in paper industry for making electronic paper display packaging industry for building materials; pharmaceuticals, cosmetics, gelling agents and medicines for wound dressing (Panesar et al., 2009). In the Institute of Chemical Fibres, Poland, an ecological method has been developed for manufacturing of bacterial cellulose materials suitable for medical applica- tions. Being similar to human skin, bacterial cellulose can be applied as skin substitute in treating extensive burns (Ciechanska, 2004). 15 2.4 Analysis methods To determine the physical structure of the bacterial cellulose and standard cellulose fibers, scanning electron microscopy (SEM) was carried out. The results revealed more delicacy in structure of bacterial cellulose (Moosavi-Nasab et al., 2011). Scanning electron micrograph of microbial cellulose, which is characterized by an ultrafine network structure and the microbial cellulose layer, constituted by a compact cellulose network structure. Plate 1: Scanning electron micrograph of bacterial cellulose (Moosavi-Nasab et al., 2011) The analysis of crystallinity for the microbial cellulose was carried out by Fourier transformed infrared spectroscopy (FTIR). The position and intensity of absorption bands of FTIR spectrometer of a substance are extremely specific to that substance (Gor et al., 2012). 16  Plate 2: FTIR spectra of bacterial cellulose (Gor et al., 2012) FTIR spectra showed that microbial cellulose is free from contaminants such as lignin or hemicellulose, which is often present in plant cellulose. According to Gor et al.,2012 a weak and broad band centered at 891.59 cm-1, and a strong band centered at 1424.18 cm-1 were present in the spectra of the microbial cellulose samples, defining them as true cellulose (Gor et al., 2012). The characteristic wave numbers of cellulose, hemicelluloses and lignin were used to determine the peak height for individual components in different mixtures. Lignin was identified to absorb at maximum wavelength of 1514cm-1, cellulose at 1427cm-1 and hemicellulose 1044cm-1 (Adapa et al., 2009).Analysis of hemicellulose fractions was done by Karaaslan et al., 2010 from plant matter and results were obtained from gas liquid chromatography(Karaaslan et al., 2010). 17  CHAPTER THREE MATERIALS AND METHODS 3.1 Preparation of fermentation media and culture media conditions Hestrin-schramm media was used as the basic media. It was composed of 20g glucose, 5g yeast, 5g peptone, 2.7g Na2HPO4 and 1.15g citric acid. The materials were measured using an analytical balance and placed in a beaker. Small amount of water was added and the contents mixed until dissolution. This was topped up to 1 litre and the pH will be adjusted to 6.0 using citric acid and sodium hydroxide at room temperature. Agar was be added to 500ml of the media which was heated for 12minutes until boiling point then autoclave. The media was allowed to cool then dispensed to petri dishes. After solidification the dishes were sealed with parafilm strips. 3.2 Isolation of Acetobacter xylinum A sample of a rotting apple was crushed and serial diluted from dilution factor of 10 -0 to 10-4 using sterile water. 3.3 Initial culture and Subculture of diluted sample The diluted sample was inoculated onto solid Hestrin Schramm media and incubated for 24hours at 300C.The colonies that appeared were subcultured further into solid Hestrin Schramm and incubated at 300C for 24hours. The resulting pure colonies were tested for presence of Acetobacter xylinum. 18 3.4 Morphological tests Pure colonies were picked from the culture and spread on a glass slide and a cover slip was placed. Simple staining was done using safranin to determine shape and configuration of the colonies, observations were recorded. 3.5 Gram negative test of Acetobacter xylinum and Biochemical tests Pure colony was picked from the culture and spread on a glass slide, a drop of crystal violet dye was placed on the sample on the slide. Counter stain safranin dye was added after crystal violet dye. A cover slip was placed on the glass slide. The sample was observed under the microscope at magnification of x10 to x100, observations were recorded. Catalase test was performed and endospore test, observations were recorded. 3.6 Growth of Bacterial cellulose The colonies of Acetobacter xylinum were picked and inoculated into liquid Hestrin Schramm media followed by incubation at 370C for 7 days. The observations were made after 7 days. 3.7 Purification of the bacterial cellulose pellicle Cellulose pellicle obtained was be washed with distilled water to remove medium components and treated with 4%NaOH at 800C for 1 hr to eliminate bacterial cells. The pellicle was weighed and results were recorded. 19 3.8 Lignin determination Maize seeds were finely ground using a pestle and mortar. The ground seeds were placed in a glass beaker and 72% sulphuric acid added while stirring. The reaction was observed and recorded. Small amount of bacterial cellulose was placed in a glass beaker. 72% sulphuric acid was added while stirring and the reaction was observed. 3.9 Extraction of plant cellulose and bacterial cellulose Maize seeds was ground finely and 30-150mg used. 3ml Acetic –nitric acid reagent consisting 150ml of 80% acetic acid and 15ml of concentrated nitric acid added slowly to the ground powder while mixing. The test tubes were capped and heated at 100 0C for 30 minutes, cooled then centrifuged. Fibrous precipitate was washed twice with 3ml of the acetic-nitric acid reagent and twice with 2ml of acetone. The residue was analyzed using High performance liquid chromatography (HPLC). A portion of bacterial cellulose was placed in a beaker and 72% sulphuric acid added until dissolution. A solution of pure microcrystalline cellulose was used as a standard for comparison. The resulting mixture was analyzed by HPLC. Pure microcrystalline cellulose was also analyzed for comparison. 3.1.1 Dissolution in NaOH Maize seeds were crushed in a pestle and mortar and the powder placed in a beaker. 17.5% NaOH (aq) at 20oC was added to the ground maize seeds and reaction observed and recorded. A 20 portion of the bacterial cellulose was placed in a beaker and 17.5% NaOH (aq) at 20 0C added to the bacterial cellulose and the reaction observed and recorded. 3.1.2 Percentage yield of bacterial cellulose The percentage yield of bacterial cellulose from the different concentrations of glucose was determined by a formula after drying the bacterial cellulose pellicles. The formula was used by Wee et al., 2011 Percentage yield = Dry weight of bacterial cellulose pellicle x 100 Weight of carbon source used in media 3.1.3 Data Analysis Different concentrations of glucose were used; 20g/l, 30g/l, 40g/l representing the treatments. There were 3 replicates for each treatment. The experimental units used were petri dishes. The parameter being evaluated was the percentage yield of bacterial cellulose with the different concentrations of glucose. The completely randomized block design was used because there was no extraneous variable. Non-destructive method was used to weigh the weight of the resultant bacterial cellulose pellicles. The data obtained was subjected to analysis using significance level of 0.05 using ANOVA statistical method. 21  CHAPTER FOUR RESULTS 4.1 Initial culture of diluted sample After 24hours of incubating the diluted rotten apple sample at 30 0C two distinct colonies were observed. Cream and translucent colonies were observed. Both colonies appeared raised in elevation and smooth in appearance. Plate 3: Colonies after initial culture at 10-4 and 10 -3 dilution factor Subculturing Subculture was performed to attaining pure colonies as observed below. The translucent colonies and cream colonies were most prominent colonies observed after 24hours upon subculture. 22  . Transluce nt colony Crea m colon Plate 4: Pure colonies after subculturing Translucent colonies and cream colonies were observed after 24 hours of incubation at 300C. 4.2 Gram negative test, Endospore staining and Biochemical test Endospore staining performed on the cream and translucent colonies showed that the cream colonies were positive for endospores by retention of green colour of malachite green. Translucent colonies were negative by retention of pink colour of safranin. Plate 5: Green dots of the cream colony pink dots of translucent colony 23 4.3 Morphological tests Morphological tests were carried out on the translucent colonies and the bacteria viewed under the microscope appeared rod like after simple staining with safranin. They appeared singly, in pairs and chains. Rod like bacteria Plate 6: Rod like bacteria x10 Gram Staining Gram negative test was performed on the translucent colonies and pink rods were observed under the microscope confirming that the bacteria are Gram negative by retention of the colour of safranin. Plate 7: Pink rods after gram staining 24 Catalase test Catalase test performed in hydrogen peroxide was positive as bubbling was seen when Acetobacter xylinum colony was dipped inside the hydrogen peroxide. Gas bubbles observed Plate 8: Gas bubbles in hydrogen peroxide 4.4 Growth of Bacterial Cellulose After incubation for 7 days at 37 0C bacterial cellulose pellicle was observed floating on the surface of the Hestrin Schramm media. Bacteria l cellulos Plate 9: Bacterial cellulose after 7 days of incubation 25 4.5 Dissolution in NaOH (aq) The maize sample showed dissolution and precipitation when NaOH (aq) was added to the ground maize. The bacterial cellulose did not dissolve in NaOH (aq). 4.6 Lignin Determination Upon addition of 72% Sulphuric acid the ground maize seeds did not dissolve but the bacterial cellulose dissolved in the 72% sulphuric acid. 4.7 Bacterial cellulose dry weights The trend in Table 1 revealed that there was gradual increase in the amount of bacterial cellulose yield with increase in the concentration of glucose. Table 1: Dry weights of bacterial cellulose replicates and different glucose concentrations replicates Glucose concentration(g/l) 20g/l 30g/l 40g/l Dry weight of R1 replicates(g) 0.375 0.536 0.971 bacterial cellulose R2 0.406 0.589 0.956 R3 0.460 0.570 0.932 KEY R1- Replicate 1 R2- Replicate 2 R3- Replicate 3 4.8 Percentage yield of Bacterial Cellulose The trend in Table 2 reveals that the percentage yield of the bacterial cellulose increases with increase in the glucose concentration. 26 Inconsistency was observed at 20g/l where the percentage yield was higher than percentage yield at higher glucose concentrations. Table 2: Percentage yield of bacterial cellulose and different glucose concentrations Glucose concentration(g/l) 20g/l 30g/l 40g/l R1 1.88 1.79 2.43 replicates (%) Percentage weight of bacterial cellulose R2 2.03 1.96 2.39 R3 2.3 1.9 2.33 KEY R1- Replicate 1 R2- Replicate 2 R3- Replicate 3 The trend in the table indicates there is an increase in bacterial cellulose yield upon increase of the glucose concentration. 4.9 Data Analysis The data in Table 3 was analyzed using the ANOVA statistical method at 0.05 significance level. Table 3: Bacterial cellulose weights and square roots of the weights x x2 x x2 x x2 Bacterial cellulose RI 0.38 0.1444 0.54 0.2916 0.97 0.9409 weights replicates R2 0.40 0.16 0.59 0.3481 0.96 0.9216 (X) R3 0.46 0.2116 0.57 0.3249 0.93 0.8649 X: dry weight of bacterial cellulose X2: square of x R1- Replicate 1 R2- Replicate 2 R3- Replicate 3 Grand total of x (GT) = 5.8 Sum of x 2 (∑x2) = 4.208 27 Samples (N) = 9 Stage 2: Correction factor Stage 3: total sum of squares (SST) C= GT2 = 3.74 C= 3.74 (∑x2) – C = 0.47 N Stage 4: sum of squares between groups (SSB) Stage 5: sum of squares within group (SSW) ∑ Tc - C = 0.46 ∑(x2) - ∑ Tc = 0.01 nc nc Stage 6: degrees of freedom Degree of freedom for SST = 8 Degree of freedom for SSB = 2 Degree of freedom for SSW = 6 Stage 7: Mean squares Mean square between groups (MSB) = 0.23 Mean square within groups (MSW) = 0.002 Stage 8: calculated F value Tabulated F value F = MSB = 11.5 F value = 11.5 F = 5.14 MSW Comparison of tabulated and calculated F value Calculated statistic < tabulated statistic Reject null hypothesis Conclusion There is significant difference between mean weights of bacterial cellulose pellicles in different glucose concentrations at 5% significance level. 28 4.1.0 Analysis of purity of the cellulose extracted The chromatographs generated after HPLC analysis of plant, bacterial and pure microcrystalline cellulose. The peaks represented different retention times of the components in each sample. Graph 1: Chromatograph showing peaks for pure cellulose The chromatograph for pure cellulose above displayed one distinct peak at retention time of 6.593 minutes. 29 Graph 2: Chromatograph showing peaks for bacterial cellulose The bacterial cellulose chromatogram above displayed three distinct peaks at 6.593minutes, 8.13 minutes and 11.44 minutes. Graph 3: Chromatograph showing peaks for plant cellulose The plant cellulose chromatograph above displayed 4 distinct peaks at 6.63 minutes, 8.17 minutes, 11.53 minutes and 13.618 minutes. 30  CHAPTER FIVE DISCUSSION 5.1 Acetobacter xylinum characterization Acetobacter xylinum was successfully isolated from rotten apples and this was confirmed by morphological and biochemical tests. From the results, the translucent colonies isolated were found to be motile rod shaped, non spore forming catalase positive, and Gram negative growing at pH 6.0. According to the eighth edition of Bergeys manual of determinative Bacteriology, these strains should be classified into the genera Acetobacter. Acetobacter xylinum strain showed positive growth at 300C in Hestrin Schramm media (Kadere et al., 2008).The endospore test was able to confirm the colonies were non-sporing by observation of pink spots. The cream colonies when flooded with malachite green appeared green hence not considered being in the Acetobacter genera (Bielecki et al., 2010). 5.2 Bacterial Cellulose growth The yield of cellulose, relative to the amount of glucose consumed, increased with increase in the initial glucose concentration (Table 1). Bacterial cellulose production was enhanced with increasing amount of glucose (Coban and Biyik, 2011). This was as a result of the presence of more glucose for the bacteria to breakdown to form the bacterial cellulose. The trend in Table 2. showed inconsistencies in percentage yield of bacterial cellulose as there was lower yield at higher glucose concentrations of glucose. This was attributed to conversion of glucose to gluconic acid by glucose dehydrogenase which lowered the Hestrin Schramm media pH hence lowering yield of the bacterial cellulose (Wee et al., 2011). 31 Yeast extract added contained abundant nitrogen compounds as well as many growth factors, its addition stimulated cellulose production by Acetobacter (Son et al., 2001). A. xylinum converts glucose into cellulose from direct cellulose precursor UDPGlc to glucose-6-phosphate, catalyzed by glucokinase, followed by isomerization of this intermediate to Glc-1-P, catalyzed by phosphoglucomutase, and conversion of the latter metabolite to UDPGlc by UDPGlc pyrophosphorylase (Bielecki et al., 2010). The glucose subunits that form the cellulose micro fibrils extruded through pores in the cell wall of the bacteria. The cellulose fibrils bundled together to form a mat or pellicle within which the bacteria are held. The pellicle floated on the surface of the medium allowing the bacteria to obtain plenty of oxygen, which they require for growth, multiplication, and more cellulose synthesis (Cannon and Skinner, 2000).The synthesis of cellulose involved several enzymatic processes that Acetobacter xylinum is involved in. Glucose (glucokinase) Glucose-6-Phosphate (Phosphoglucomutase) Glucose-1-Phosphate (UDP-glucose pyrophosphorylase) UDP-Glucose (Cellulose synthase) Cellulose 32 5.3 Analysis of purity Cellulose is insoluble in most of the organic solvents due to its crystalline nature hence insoluble in sodium hydroxide proving it is a α cellulose which is true cellulose. Plant cellulose from the maize seeds dissolved and precipitated when sulphuric acid was added proving that it contained lignin which is an impurity while bacterial cellulose completely dissolved proving there was no lignin (Abbot et al., 1988). The results from the statistical analysis of bacterial cellulose dry weights showed that there was significant difference between the mean weights of bacterial cellulose at different glucose concentrations at 5% significance level. This was determined by the ANOVA analysis of the results obtained which showed that the calculated statistic < tabulated statistic. Chemical nature of the bacterial cellulose was defined by performing high performance liquid chromatography (Graph 1). Graph 2 shows the chromatograph for the bacterial cellulose while Graph 3 shows the chromatograph for plant cellulose. The pure cellulose chromatogram displayed a distinct peak at retention time of 6.593 minutes which was the cellulose. The bacterial cellulose displayed peaks at retention time of 6.593 minutes representing cellulose, 8.13 minutes representing glucose and 11.44 minutes representing sucrose. The plant extract showed several peaks on the chromatogram which represented 6.63 minutes retention time for cellulose, 8.177minutes for glucose, 11.53 minutes for hemicellulose and 13.618 minutes for lignin. The peaks were interpreted by referring to carbohydrate retention time’s table for HPLC with mobile phase as Acetonitrile: Water (75:25), using column c18, run time 15minutes and flow rate of 0.6ml/min (Adapa et al., 2009). 33 The only difference between the peaks of the pure cellulose and the bacterial cellulose is the presence of glucose and sucrose in the bacterial cellulose. Graph 2 showed that microbial cellulose is free from contaminants such as lignin or hemicellulose (Gor et al., 2012). Plant cellulose was showed to contain hemicellulose and lignin contaminants. By comparing the plant cellulose and bacterial cellulose chromatographs it can be concluded that plant cellulose contains contaminants lignin and hemicellulose. The presence of glucose and sucrose in bacterial cellulose (Graph 2) is as a result of the enzymatic activities that resulted in formation of the bacterial cellulose pellicle. 34  CONCLUSION From the findings, it was concluded that Acetobacter xylinum can be isolated from rotten apples successfully which was confirmed by morphological and biochemical tests. Lignin test carried out and dissolution in NaOH (aq) proved that bacterial cellulose is true cellulose and did not contain contaminants. Greater amount of bacterial cellulose was yield from greater concentration of glucose in the Hestrin Schramm media. Bacterial cellulose was proven to be purer than plant cellulose by comparison of the HPLC chromatographs. The results are of great significance since they indicate that bacterial cellulose is purer than plant cellulose hence can be chosen as an alternative to plant cellulose. Due to the high degree of purity it is easily purified by washing in acetic acid. Growth of bacterial cellulose was fast taking only two weeks. Therefore this will reduce the destruction of the plant vegetation to obtain fibres hence conserving the environment. To conclude, high purity bacterial cellulose can be successfully produced by Acetobacter xylinum in Hestrin Schramm media and can be used in the textile industry as an alternative since it requires minimal processing and no chemical bleaching since it is free of contaminants. 35  RECOMMENDATION Recommendations put forward after analysis of the project results to obtain a greater yield of glucose and obtain more information about the bacterial glucose include; 1. The effect of initial glucose concentration on cellulose production is also important, since the formation of gluconic acid as a byproduct in the medium decreases the pH of the culture and ultimately decreases the production of cellulose. The addition of acetic acid in the media is recommended to decrease the production of gluconic acid. 2. For a higher yield commercial production of bacterial cellulose it is recommended that agitated shaking culture techniques be employed. 3. The tensile properties of bacterial cellulose could be determined for further information on the bacterial cellulose mechanical strength and use of the scanning electron microscope to determine physical structure is recommended. 4. To determine the optimum concentration of glucose for maximum bacterial cellulose yield, higher glucose concentrations are recommended to be used in further experimentation. 36 References 1. Adapa.P.K, C. Karunakaran, L.G Tabil and G.J Schoenau (2009), Qualitative and Quantitative Analysis of Lignocellulosic Biomass using Infrared spectroscopy, CSBE/SCGAB 2009 Annual conference, Paper No. CSBE09-307 2. Amano.Y, F.Ito and T.Kanda,( 2009), Novel cellulose producing system by microorganisms such as Acetobacter sp., Journal of Biology and Macromology, Vol.5 (1), pp3-10 3. Aydin A.Y and N.D Aksoy, (2009), Isolation of cellulose producing bacteria from wastes of vinegar fermentation, World Congress on Engineering and Computer Science, Vol.1 4. Bielecki .S, A. Krystynowicz, M.Turkiewicz and H.Kalinowska, (2010), Bacterial cellulose, Institute of technical biochemistry of Lodz Stefanowskiego, pp90-924 5. Cannon R.E and P.O Skinner, (2000), Acetobacter xylinum: An inquiry into cellulose biosynthesis, The American Biology Teacher, Vol.62, No.6 6. Chawla P.R, I.B Bajaj, S.A Survase and R.S Singhal, (2009), Microbial cellulose: Fermentative production and Applications, Journal of Food Technology and Biotechnology, Vol.47 (2), pp 107-124. 7. Ciechanska .D, (2004), Multifunctional bacterial cellulose, Institute of Chemical Fibres, Vol.12, No.4, pp48 8. Goh W.N, A.Rasma, B.Kaur, A.Fazilah, A.A.Karim and B.Rajeev,(2012), Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (Kombucha).II, International Food research Journal 19(1):153-158 9. Karaaslan A.M, M.A.Tshabalala and G.Buschle-Diller , (2010), Wood hemicellulose/chitosan based semi-interpenetrating network hydrogels: Mechanical swelling and controlled drug released properties, Bioresources 5(2), 1036-1054 10. Kesk M.A and K.Sameshima, (2005), Evaluation of different carbon sources for bacterial cellulose production, African Journal of Biotechnology, Vol.4, pp 478-482 37 11. Moosavi-Nasab.M and A.R Yousefi, (2010), Investigation of physicochemical properties of the bacterial cellulose produced by Glucenobacter xylinus from Date syrup, World Academy of Science Engineering and Technology, Vol.68, pp893-899. 12. Moosavi-Nasab .M and A.R Yousefi, (2011), Biotechnology production of cellulose by Glucenobacter xylinus from agricultural waste, Iranian Journal of Biotechnology, Vol.9, No.2 13. Panesar P.S, Y.V Chavan, M.B.Bera, O. Chand and H. Kumar, (2009), Evaluation of Acetobacter strain for the production of Microbial cellulose, Asian journal of chemistry, Vol. 21, No.10, SO99-102. 14. Shah. J and M. Brown, (2005), towards electronic paper displays made from microbial cellulose, Applied microbiology and Biotechnology, Vol.66, pp 352-355. 15. Sloneker J.H, (1971), Determination of cellulose and Apparent Hemicellulose in Plant tissue by Gas-liquid chromatography, Analytical biochemistry, Vol.43, No.2, pp 539- 546 16. Thomas.P.Abbot, Doris.M.Palmer, Sherald.H.Gordon and Marvin.O.Barghy, (1988), Solid state analysis of Plant polymers, Journal of Wood Chemistry and Technology, Vol.8 (1), pp 1-28. 17. Tsuchida. T and F. Yoshinaga, (1997), Production of bacterial cellulose by agitation culture systems, Pure and applied Chemistry, Vol.69, No.11, pp2453-2458. 18. T. T. Kadere, T. Miyamoto, R. K. Oniang`o, P. M. Kutima and S. M. Njoroge, (2008), Isolation and identification of the genera Acetobacter and Gluconobacter in coconut toddy (mnazi), African Journal of Biotechnology Vol. 7 (16), pp. 2963-2971 19. Wee.Y, S. Kim, S. Yoon and H. Ryu, (2011), Isolation and characterization of bacterial cellulose producing bacterium derived from the persimmon vinegar, African journal of biotechnology, Vol.10 (72), pp 16267-16276. 38