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 theirsupport and encouragement.

i

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

ii

lacked the impurities. It is a cheap alternative since less

processing is required for removal of impurities as it is pure

cellulose.

iii

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

iv

CHAPTER THREE.......................................................11

MATERIALS AND METHODS...............................................11

3.1 Preparation of fermentation media and culture media conditions. .11

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

v

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

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

vi

References..........................................................30

vii

List of plates, tables and graphs

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

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

viii

ix

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 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

1

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

2

to be a better 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

3

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 plantcellulose 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

4. To determine purity of bacterial cellulose and plant

cellulose

1.4 Null hypothesisBacterial cellulose derived from Acetobacter xylinum in suitable

media is not purer than plant cellulose.

4

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).

5

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 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

6

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

7

found to be very tough, especially if an attempt is made to tear

it across its plane of growth. From chemical 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

8

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.

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).

9

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).

10

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 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).

11

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 applications. Being similar to human skin, bacterial

cellulose can be applied as skin substitute in treating extensive

burns (Ciechanska, 2004).

2.4 Analysis methods

12

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

13

spectrometer of a substance are extremely specific to that

substance (Gor et al., 2012).

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

14

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).

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

15

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.

3.4 Morphological tests

16

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.

17

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.

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

18

of concentrated nitric acid added slowly to the ground powder

while mixing.

The test tubes were capped and heated at 1000C 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 portion

of the bacterial cellulose was placed in a beaker and 17.5% NaOH

19

(aq) at 200C 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.

20

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.

CHAPTER FOUR

RESULTS

4.1 Initial culture of diluted sample

After 24hours of incubating the diluted rotten apple sample at300C two distinct colonies were observed. Cream and translucentcolonies were observed. Both colonies appeared raised inelevation and smooth in appearance.

21

Plate 3: Colonies after initial culture at 10-4 and 10 -3 dilutionfactor

Subculturing

Subculture was performed to attaining pure colonies as observedbelow. The translucent colonies and cream colonies were mostprominent colonies observed after 24hours upon subculture.

22

.

Plate 4: Pure colonies after subculturing

Translucent colonies and cream colonies were observed after 24hours 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.

23

Translucent

Cream colony

Plate 5: Green dots of the cream colony pink dots of translucentcolony

4.3 Morphological tests

Morphological tests were carried out on the translucent coloniesand the bacteria viewed under the microscope appeared rod likeafter simple staining with safranin. They appeared singly, inpairs and chains.

Plate 6: Rod like bacteria x10

Gram Staining

Gram negative test was performed on the translucent colonies andpink rods were observed under the microscope confirming that the

24

Rod like bacteria

bacteria are Gram negative by retention of the colour ofsafranin.

Plate 7: Pink rods after gram staining

Catalase test

Catalase test performed in hydrogen peroxide was positive asbubbling was seen when Acetobacter xylinum colony was dipped insidethe hydrogen peroxide.

Plate 8: Gas bubbles in hydrogen peroxide

25

Gas bubbles observed

4.4 Growth of Bacterial Cellulose

After incubation for 7 days at 370C bacterial cellulose pelliclewas observed floating on the surface of the Hestrin Schrammmedia.

Plate 9: Bacterial cellulose after 7 days of incubation

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 didnot dissolve in NaOH (aq).

4.6 Lignin Determination

Upon addition of 72% Sulphuric acid the ground maize seeds didnot dissolve but the bacterial cellulose dissolved in the 72%sulphuric acid.

26

Bacterial cellulo

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 anddifferent glucose concentrations replicates

Glucoseconcentration(g/l) 20g/l 30g/l 40g/l

Dry

weight

of bacteria

l cellulos

R1 0.375 0.536 0.971R2 0.406 0.589 0.956R3 0.460 0.570 0.932

KEY

R1- Replicate 1 R2- Replicate 2R3- 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.

Inconsistency was observed at 20g/l where the percentage yield was higher than percentage yield at higher glucose concentrations.

27

Table 2: Percentage yield of bacterial cellulose and differentglucose concentrations

Glucoseconcentration(g/l)

20g/l 30g/l 40g/l

Percentage

weight of

bacterial

cellulose

replicates

(%)

R1 1.88 1.79 2.43

R2 2.03 1.96 2.39

R3 2.3 1.9 2.33

KEY

R1- Replicate 1 R2- Replicate 2R3- Replicate 3

The trend in the table indicates there is an increase inbacterial cellulose yield upon increase of the glucoseconcentration.

4.9 Data AnalysisThe 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 theweights

x x2 x x2 x x2

Bact

erial

cell

ulose

weig

hts

repl

icates

RI 0.38 0.1444 0.54 0.2916 0.97 0.9409

R2 0.40 0.16 0.59 0.3481 0.96 0.9216

R3 0.46 0.2116 0.57 0.3249 0.93 0.8649

28

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 ( x∑ 2) = 4.208

Samples (N) = 9

Stage 2: Correction factorStage 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: sumof 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) = 0.00229

Stage 8: calculated F valueTabulated F value

F = MSB = 11.5 F value = 11.5F = 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 bacterialcellulose pellicles in different glucose concentrations at 5%significance level.

4.1.0 Analysis of purity of the cellulose extracted

The chromatographs generated after HPLC analysis of plant,bacterial and pure microcrystalline cellulose. The peaksrepresented different retention times of the components in eachsample.

30

Graph 1: Chromatograph showing peaks for pure cellulose

The chromatograph for pure cellulose above displayed one distinctpeak at retention time of 6.593 minutes.

31

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

32

The plant cellulose chromatograph above displayed 4 distinct peaks at 6.63 minutes, 8.17 minutes, 11.53 minutes and 13.618 minutes.

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).

33

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).

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,

34

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)

35

Cellulose

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.

36

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).

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 et37

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.

38

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

39

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.

40

RECOMMENDATIONRecommendations put forward after analysis of the project resultsto 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

41

concentrations are recommended to be used in further

experimentation.

42

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