(Đồ án HCMUTE) charaterization of bacterial cellulose produced by gluconacetobacter xylinus using rice extract as a nutrient source

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, August, 2022 SKL 0 0 9 1 5 5 SUPERVISOR:

MINISTRY OF EDUCATION AND TRAINING

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

Ho Chi Minh City, August, 2022

SKL 0 0 9 1 5 5

SUPERVISOR: VU TRAN KHANH LINH STUDENT: NGUYEN THUY THANH HIEN NGUYEN PHAM HUYEN PHUONG

USING RICE EXTRACT AS A NUTRIENT SOURCE

CHARACTERIZATION OF BACTERIAL CELLULOSE PRODUCED BY GLUCONACETOBACTER XYLINUS

GRADUATION THESIS FOOD TECHNOLOGY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

CODE: 2022-18116015

CHARACTERIZATION OF BACTERIAL CELLULOSE

PRODUCED BY GLUCONACETOBACTER XYLINUS

USING RICE EXTRACT AS A NUTRIENT SOURCE

NGUYEN THUY THANH HIEN Student ID: 18116015

Major: FOOD TECHNOLOGY NGUYEN PHAM HUYEN PHUONG Student ID: 18116030

Major: FOOD TECHNOLOGY Supervisor: VU TRAN KHANH LINH, PhD.

Ho Chi Minh City, August 2022

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

CODE: 2022-18116015

CHARACTERIZATION OF BACTERIAL CELLULOSE

PRODUCED BY GLUCONACETOBACTER XYLINUS

USING RICE EXTRACT AS A NUTRIENT SOURCE

NGUYEN THUY THANH HIEN Student ID: 18116015

Major: FOOD TECHNOLOGY NGUYEN PHAM HUYEN PHUONG Student ID: 18116030

Major: FOOD TECHNOLOGY Supervisor: VU TRAN KHANH LINH, PhD.

Ho Chi Minh City, August 2022

APPENDIX 3: (Graduation Project Assignment)

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

-

GRADUATION THESIS ASSIGNMENT

Student name: Nguyen Thuy Thanh Hien Student ID: 18116015

Student name: Nguyen Pham Huyen Phuong Student ID: 18116030

Supervisor: Vu Tran Khanh Linh, PhD Email: linhvtk@hcmute.edu.vn

Date of assignment: 14/02/2022 Date of submission: 08/08/2022

1 Thesis title: Characterization of bacterial cellulose produced by Gluconacetobacter xylinus using

rice extract as a nutrient source

2 Thesis assignment:

The research topic “Characterization of bacterial cellulose produced by Gluconacetobacter xylinus using rice extract as a nutrient source” includes 3 main parts:

- Determination of the chemical composition of coconut juice and rice extract

- Effects of rice extract as a nutrient source on film-like cellulosic biomass production

- Characterization of BC films obtained from growth culture containing different ratios of rice extract and coconut juice

The content and requirements of the graduation thesis have been approved by the Chair of the Food

Technology program

CHAIR OF THE PROGRAM

(Sign with full name)

Ho Chi Minh City, August 08, 2022

SUPERVISOR

(Sign with full name)

ACKNOWLEDGEMENTS

First and foremost, we would like to thank Ms Vu Tran Khanh Linh who is a lecturer at the Faculty of Chemical and Food Technology for her enthusiastic guidance, instruction, imparting valuable experience, expertise as well as skills required for us to execute experiments efficiently and scientifically Secondly, our team would like to thank the seniors who assisted us in instructing the microbiological tactics

To successfully finish the research process and the thesis, we would like to send many thanks

to all the lecturers at the Department of Food Technology, the Faculty of Chemical and Food Technology, the Ho Chi Minh City University of Technology and Education had actively taught and conveyed basic knowledge to us throughout the four-year course and made it possible for us

to study and do research at the institution

Finally, we'd like to thank our family and friends for their constant encouragement and support throughout the years

The thesis cannot escape flaws due to a lack of experience and knowledge We look forward

to receiving comments from the committee so that we can enhance our topic

Ho Chi Minh City, 08th August, 2022

We hereby certify that the whole material of this thesis is our own original work We declare

that the contents of the graduation thesis have been accurately and completely cited in line with

the requirements

Ho Chi Minh City, 08th August, 2022

Sign

TABLE OF CONTENTS

GRADUATION THESIS ASSIGNMENT iii

ACKNOWLEDGEMENTS iv

DISCLAIMER v

LIST OF FIGURES xx

LIST OF TABLES xxi

LIST OF ABBREVIATIONS xxii

ABSTRACT xxiii

Chapter 1: INTRODUCTION 24

1.1 Rationale 24

1.2 Research objectives 25

1.3 Research object and scope 25

1.4 Research content 25

1.5 Scientific objective 26

1.6 Practical objective 26

Chapter 2: LITERATURE REVIEW 27

2.1 Bacterial cellulose 27

2.1.1 Introduction 27

2.1.2 Gluconacetobacter xylinus 27

2.1.3 Characteristics of bacterial cellulose 28

2.2 Effects of growth conditions on biosynthesis of bacterial cellulose 30

2.2.1 Growing medium 30

2.2.2 Biosynthesis of BC 33

2.2.3 Cultivation modes 34

2.2.4 Applications of bacterial cellulose 36

2.3 Overview of food packaging from bacterial cellulose 39

2.3.1 The potential of BC in food packaging application 39

2.3.2 BC films produced by impregnation 40

2.3.3 Films with disassembled BC 42

2.3.4 Future expectation 44

2.4 Summary 45

Chapter 3: MATERIALS AND METHODS 46

3.1 Materials 46

3.1.1 Microorganism and culture medium 46

3.1.2 Chemicals 47

3.2 Production of bacterial cellulose 48

3.3 Experimental design 49

3.3.1 Experiment 1: Determination of the chemical composition of coconut juice and rice extract 49

3.3.2 Experiment 2: Effects of rice extract on film-like cellulosic biomass production 50

3.3.3 Experiment 3: Film characterization 51

3.4 Analytical methods 52

3.4.1 Dertermination of total solids of rice extract and coconut juice 52

3.4.2 pH measurement 52

3.4.3 Determination of total carbohydrate 52

3.4.4 Determination of total nitrogen 54

3.4.5 BC production yield 56

3.4.6 Film thickness 56

3.4.7 Field Emission – Scanning Electron Microscopy (FE – SEM) 57

3.4.8 Fourier – transform Infrared Spectroscopy (FT-IR) 57

3.4.9 Film color 58

3.4.10 Film light transmission 58

3.4.11 Moisture content (MC) 58

3.4.12 Moisture absorption (MA) 59

3.4.13 Water solubility 59

3.4.14 Water absorptivity 60

3.4.15 Water vapor permeability (WVP) 60

3.4.16 Porosity measurement 61

3.4.17 Oil permeability (PO) 62

3.4.18 Tensile strength (TS) and elongation at break (E) 62

3.4.19 Puncture resistance 63

3.4.20 Reusability testing 64

3.5 Statistical data analysis 64

Chapter 4: RESULTS AND DISCUSSION 65

4.1 Determination of the chemical composition of coconut juice and rice extract 65

4.2 Effects of rice extract on film-like cellulosic biomass production 65

4.2.1 Effects of rice extract on total sugar consumption 65

4.2.2 Effects of rice extract on bacterial cellulose film production 67

4.2.3 Changes of pH during BC production 70

4.3 Film characterization 71

4.3.1 Film thickness 71

4.3.2 Morphology 71

4.3.4 Film color 75

4.3.5 Film light transmission 77

4.3.6 Barrier properties 78

4.3.7 Mechanical properties 83

4.3.8 Reusability testing 86

Chapter 5: CONCLUSION AND RECOMMENDATIONS 87

REFERENCE 89

LIST OF FIGURES

Figure 2.1 A typical bacterial cellulose G xylinus: (a) The cellulose pellicle formed in the broth;

(b) The morphologies of the Cel+ and Cel- (right) G xylinus colonies [50, 51] 28

Figure 2.2 Bacterial cellulose inter- and intra-hydrogen bonding [60] 29

Figure 2.3 Production of cellulose microfibrils by Acetobacter xylinum [63] 30

Figure 2.4 Biosynthetic process of Gluconacetobacter xylinus [98] 33

Figure 2.5 Glucose oxidation pathways in G oxydans [100] G oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae, Gluconobacter strains [101] 34

Figure 2.6 Schematic diagram of BC culture (A) Conventional static culture and (B) Intermittent fed-batch culture [105] 35

Figure 2.7 Schematic process to obtain Microfibrils, Nanofibrils and Nanocrystals from Bacterial Cellulose (BC) 43

Figure 3.1 Production of BC film 48

Figure 3.2 Summary of research contents 49

Figure 3.3 Glucose standard curve 54

Figure 3.4 Dry cell weight standard curve 56

Figure 3.5 Linear regression of water vapor permeability 61

Figure 4.1 Effects of rice extract on total sugar concentration 66

Figure 4.2 Effects of rice extract on (a) suspended bacterial biomass concentration, (b) cellulosic biomass content at 192h (D1) and (c) bacterial cellulose yield coefficient 69

Figure 4.3 Result of pH values during fermentation of G.xylinus (0 – 192h) 70

Figure 4.4 Purified BC and FE-SEM images of BC samples utilizing 5 fermentation medium: (a) C100; (b) C75R25; (c) C50R50; (d) C25R75; (e) R100 72

Figure 4.5 Infrared spectra of bacterial cellulose 74

Figure 4.6 Five different BC samples 75

Figure 4.7 Light transmission of BC film samples from 200 to 800 nm 77

Figure 4.8 Results of porosity 79

Figure 4.9 Results of water absorption and water solubility 80

Figure 4.10 Results of water vapor permeability 82

Figure 4.11 Results of puncture strength 85

LIST OF TABLES

Table 2.1 Analyzing the differences between cellulose made by bacteria and plant [46] 29 Table 2.2 Bacterial cellulose composites and application 40

Table 3.1 Maitaining and inoculating medium of G xylinus 46

Table 3.2 Methods for analyzing the chemical characteristics of rice extract and coconut juice 49 Table 3.3 BC production medium (GM3) 50 Table 3.4 Glucose standard curve preparation 53 Table 4.1 Chemical composition of rice extract and coconut juice 65 Table 4.2 Results of films thickness 71 Table 4.3 Characterization of some cellulose unique peaks [219, 220] 74 Table 4.4 Results of colorimetry for BC samples 75 Table 4.5 Results of moisture content and moisture absorption 80 Table 4.6 Results of thickness, tensile strength and elongation at break 84 Table 4.7 Results of reusability of BC films 86

LIST OF ABBREVIATIONS

Gluconacetobacter xylinus G.xylinus

ABSTRACT

Bacterial cellulose (BC) is a nanostructured material that is mostly produced by the bacteria

Gluconacetobacter Traditionally, coconut juice has been applied for BC production growing

medium In this study, rice extract or rice wastewater was utilized to investigate the effects of rice

extract on BC production in static culture and film characteristics using Gluconacetobacter xylinus

(JCM 9730) The rice extract (20.81 g/L total sugar and 0.19% total nitrogen) was replaced the coconut juice (18.86 g/L total sugar and 0.08% total nitrogen) at different ratios of 0:1 (C100), 1:3 (C75R25), 1:1 (C50R50), 3:1 (C25R75), and 1:0 (R100) After 8 days of fermentation, a BC yield (YBC/S g/g) of 0.16 g/L (C75R25), 0.12 g/L (C50R50), 0.15 g/L (C25R75) and 0.11 g/L (C25R75 and R100) were achieved lower than that of C100 (0.18 g/L) Throughout the fermentation process, total sugar consumption and pH dramatically declined due to carbon catabolism and acidic metabolic substances biosynthesis The obtained BC membranes from all studied cultures medium were then characterized The light absorption values of all samples in the UV region were less than 8%, and less than 20% for the visible spectrum, which shows that all BC films were good UV resistant All samples had a tensile strength value greater than 30 MPa and high values of elongation at break, reflecting good mechanical properties and great elasticity of all BC films Hydrophilic properties such as moisture, water absorption, solubility, and water vapor permeability were more strongly performed by the BC films produced in mediums with high content of coconut water According to the reusability test, the C100 membrane can be reused up

to 19 times, while the R100 membrane can only be reused 3 times Besides, all BC specimens can prevent oil permeability because they exhibited no sign of oil absorption during the testing time All the above results indicate that the films obtained from the medium with the percentage of rice water from 25% to 75% were best suited for preserving food containing oil with their excellent properties

Keywords: Gluconactobacter xylinus, G.xylinus, bacterial cellulose, rice extract, rice wastewater, coconut juice.

Chapter 1: INTRODUCTION

1.1 Rationale

In today's industrial world, it is undeniable that the relationship between the packaging and food industries is indispensable Additionally, packaging also plays a crucial role in many other sectors where almost commodities must be meticulously enclosed in certain shapes before being distributed to the market [1] The fundamental functions of food packaging are not only to avoid food from being affected by external influences and spoiling agents but also to give customers detailed information about the product as well as the nutritional components in the product [2] Generally, food packaging aims to cost-effectively contain foods, satisfy industry standards and consumer expectations, guarantee food safety and nutritional values, and minimize adverse effects impact on the environment

Plastics have remained the preferred material in food packaging, where they have attributed to the majority of plastic waste contaminating the environment [3] Therefore, there is a need for the creation of alternative materials that can perform the same function as conventional plastics With advanced science and technology, various new types of packaging materials have been born, including bio-based and biodegradable packaging materials They are produced from natural, renewable, and recyclable resources such as bio-polymers (polysaccharides, protein, etc) [4], bioplastics (polylactic acid (PLA), biopolyethylene (bioPE), polyhydroxyalkanoates, etc) [5], and biomass (bacterial cellulose) These ‘green’ packaging materials can be self-degradable and diminish environmental issues [6] Among these, cellulose-based packaging and wrapping films and coatings are commercially appealing due to their compatibility with a variety of food items [7, 8] Many studies have recently proven that bacterial cellulose (BC)– a type cellulose originated

from Gluconacetobacter xylinus bacteria - offers numerous advantages in food packaging

applications [9-11]

Compared to plant cellulose, BC has smaller diameters (approximately 25 nm – 100 nm), hence

it is considered as cellulose-based nano-fiber [9] Despite having the same molecular formula (C6H10O5)n as plant celluloses, BC lacks lignin, hemicelluloses, and pectin; hence, it’s purification

is a simple, low-energy procedure, whereas plant cellulose purification typically requires harsh chemicals [9] Furthermore, with remarkable properties such as high degree of polymerization, good barrier and tensile features, biodegradability, and biocompatibility with different food product kinds [12, 13], BC is expected to become an eco-friendly packaging material in the near future Bio-cellulose application research has been steadily gaining traction in Vietnam in recent

for years [14, 15] However, there are currently no commercially available BC films for food packaging [12], as a consequence of costly production and low yield [16] The media and process parameters have been regularly optimized by researchers in an effort to increase BC yields and facilitate effective BC production Several studies have shown that BC can be produced from a variety of carbon and nitrogen sources, including fruit juices (coconut, pineapple, oranges, apples, etc) [17, 18] and vegetal extracts (tea, lychee, etc) [19, 20] Currently, coconut water is the top-notch choice as a substrate for the production of BC in Vietnam and other nations in a plethora of studies [21-25] However, the area of coconut is confined in a certain region and suffered shortages from climate changes Hence, there is a need of an alternative source, for instance, molasses [26-29], fruit wastes [29-32], tea [19, 33-35], rice extract [36-38], etc Therein, rice extract or rice waste water which is a mixture after washing or soaking the rice by a rice-wash machine collecting from industrial meal kitchens Rice extract has milky color and is rich in carbohydrates and vitamins that has not been widely exploited as a medium for BC production despite having suitable qualities and being a low-cost by-product Additionally, there were limited researches related to rice extract that had been conducted and only some properties like chemical structure, morphology, mechanic, thermogravimetry had been tested in medical application Therefore, the research

“Characterization of bacterial cellulose produced by Gluconacetobacter xylinus using rice extract

as a nutrient source” aims to replace coconut juice by rice extract in growing media to produce BC

as well as heading to characterize BC features resulting in assessing the applicability of BC film

as a food packaging Furthermore, this study not only utilized rice extract as industrial food waste but also collected BC serving for food packaging field

1.2 Research objectives

The research topic “Characterization of bacterial cellulose produced by

Gluconacetobacter xylinus using rice extract as a nutrient source” aimed to investigate the effects of growing media containing rice extract on the production of bacterial cellulose films by

G xylinus Furthermore, the characteristics of the films, and also their reusability were analyzed,

providing technical parameters that are compatible with packaging for oil and oily food

1.3 Research object and scope

The research object is bacterial cellulose films produced by Gluconacetobacter xylinus (JCM

9730) using media containing different ratios of rice extract and coconut juice

1.4 Research content

The study consists of 3 main parts:

 Determination of the chemical composition of coconut juice and rice extract

 Effects of rice extract as a nutrient source on film-like cellulosic biomass production

 Characterization of BC films obtained from growth culture containing different ratios of rice extract and coconut juice

1.5 Scientific objective

The research achievements are expected to play an elemental role in intensive studies In other words, they will contribute to authorizing optimal conditions which result in desirable BC collection and cellulose-based film properties

1.6 Practical objective

The project anticipates opening up the possibilities of bacterial cellulose film, an environmentally friendly material that can be made into biodegradable food packaging for a particular type of food

Chapter 2: LITERATURE REVIEW

2.1 Bacterial cellulose

2.1.1 Introduction

The most prevalent, reasonably priced, and easily accessible carbohydrate polymer in the world is cellulose, which is conventionally derived from plants or their byproducts [39] To produce the pure product, this polymer's natural branching with hemicellulose and lignin has to undergo hazardous chemical operations involving strong alkali and acid treatment [40] Growing demand for plant cellulose derivatives has increased the usage of wood as raw material, leading to deforestation and a global environmental issue [41] Various bacteria can manufacture cellulose

as a substitute source, even though plants are the primary producer of this material The discovery

of bacterial cellulose (BC) dates back to Dr Brown (1886), who noticed the development of like structure in acetic fermentation medium with a chemically similar structure to plant cellulose

jelly-from Bacterium aceti such as a network structure made of ultrapure cellulose nanofibers (BC

crystallinity is up to 95%, while that of plant cellulose is only 65%), non-toxic, biodegradable, and chemically stable [42] Following Brown's research, Teodula K Africa produced the first bacterial cellulose, nata de coco, as a replacement to the ancient Philippine Nata de pina in 1949 [43] The study by Lapuz et al provided scientific proof that cellulose produced by bacteria is used to make the gelatinous pellicle that makes up Nata As of present, the only marketed variety of bacterial cellulose is found in the Philippines' nata de coco [44] The following notable research on

Acetobacter sp.-derived bacterial cellulose synthesis is done by Schramm and Hestrin [45] The

media created by Schramm and Hestrin became the standard medium for the A xylinum as a result

of their important findings

2.1.2 Gluconacetobacter xylinus

Diverse types of bacterial cellulose will form cellulose with different morphologies, structures,

characteristics, and uses Due to its high yield, Gluconacetobacter xylinus (G.xylinus) is one of the

bacteria that can produce cellulose and is employed in industrial cellulose manufacturing [46]

According to the Bergey taxonomy (2005), the genus Gluconacetobacter belongs to the family

Acetobacteraceae, with about 17 species [47] However, according to Yamada et al., in this genus,

there are genotype and phenotypic differences between species, the representative group

Gluconacetobacter liquefaciens can motile and produce brown pigmentation while the

representative group Gluconacetobacter xylinus does not [48] Therefore, in 2012, Yamada et al divided the genus Gluconacetobacter into two new genera, Komagataeibacter (represented by

Komagataeibacter xylinus) and Gluconacetobacter genus (represented by Gluconacetobacter liquefaciens) G.xylinus is a gram-negative, obligate aerobic bacteria that can survive in severely

acidic settings [48] As a result, the culture conditions for obtaining BC have a temperature of 25 – 30 oC and a pH of 3 – 7 [49] G xylinus grows on the surface of a liquid media, generating transparent thin layers of varying thicknesses, whereas G.xylinus colonies on solid medium are

spherical, with an average diameter of 1 – 3 mm [49] shown in Figure 2.1

Figure 2.1 A typical bacterial cellulose G xylinus: (a) The cellulose pellicle formed in the broth;

(b) The morphologies of the Cel+ and Cel- (right) G xylinus colonies [50, 51]

2.1.3 Characteristics of bacterial cellulose

Bacterial cellulose (BC), a found naturally polymer generated by the acetic acid-producing bacterium, has attracted considerable attention in recent years due to its potential applications in biotechnology and medicine [52] Both plant and bacterial cellulose have the same molecular formula in terms of chemical structure (C6H10O5)n However, there are huge differences in their physical characteristics (Table 2.1) BC is completely pure and is extruded by cells as nanofibrils,

in contrast to plant-derived cellulose nanofibres that need pretreatment to disintegrate the recalcitrant lignocellulosic network These nanofibrils can also be transformed into macro fibers with superior material properties that are stronger than steel and can be used as alternatives to synthetic fibers made from fossil fuels Because BC is comprised entirely of D-glucopyranose sugar units are connected to each other by β-1,4 glycoside linkages [53], As depicted in Figure 2.2, the fibrous network of BC is made up of three-dimensional nanofibers that are organized by hydrogen bonds that exist both within and between the cellulose molecules This results in the formation of a film with a high surface area and high porosity [54] Therefore, it possessed several remarkable properties such as a unique nanostructure [55], a high water holding capacity [56], a

high degree of polymerization [57], a high mechanical strength [58], and a high percentage of

crystallinity [59]

Figure 2.2 Bacterial cellulose inter- and intra-hydrogen bonding [60]

Prior studies had clearly demonstrated that BC and its derivatives have significant potential and a promising future in a variety of disciplines such as the biological, electronic and food industry [61, 62]

Table 2.1 Analyzing the differences between cellulose made by bacteria and plant [46]

Young’s modulus (MPa) Sheet: 20,000

Single fibre:130,000

2.5 – 0.170

Total surface area (m2/g) > 150 < 10

As can be seen in Figure 2.3, Acetobacter xylinum generates the thermodynamically stable

polymer cellulose II and the ribbon-like polymer cellulose I [63] Protofibrils of the glucose chain are released through the bacteria cell wall during the production process and clump together to form nanofibrils cellulose ribbons [64] With a highly porous matrix, these ribbons construct BC's web-shaped network structure [65, 66] The cellulose produced has an abundance of hydroxyl groups on its surface, which explains its hydrophilicity, biodegradability, and chemical-modifying capacity [67] Chawla et al (2009) provided a detailed explanation of the mechanism of BC synthesis [63]

Figure 2.3 Production of cellulose microfibrils by Acetobacter xylinum [63]

2.2 Effects of growth conditions on biosynthesis of bacterial cellulose

2.2.1 Growing medium

The selection of bacterial sources is not the only element that influences BC synthesis; the culture medium is also important [13] The different ingredients employed in culture media are the elements that directly affect both the quantity and quality of cellulose production The most important ingredients for the culture media utilized for BC production are a carbon and nitrogen supply, as well as salts to buffer the pH [68] However, optimal conditions for cellulose production with BC have yet to be determined [69] Indeed, it is widely known that metabolic preferences for microorganisms within the same genus might change between species

Sugar, a carbon source, is one of the key components for cellulose synthesis in bacterial cellulose biosynthesis The three main sugars that are frequently employed in the formation of bacterial cellulose are sucrose, glucose, and fructose Varying bacterial strains have different needs for carbon sources Similar to this, each bacterial strain's fermentation output of bacterial cellulose

will vary When employing A.xylinum species to produce cellulose, glucose is frequently

employed [70] Numerous studies have revealed that the type of carbon source used greatly affects the formation of cellulose [70-72] However, from an industrial (bulk production) standpoint, the

HS medium by Schramm & Hestrin (1954) (glucose 2%; peptone (Difco Bactopeptone) 0.5%; yeast extract (Marmite), 0.5% disodium phosphate (anhydrous), 0.27% citric acid (monohydrate), 0.115%, pH 6.0) or sugar-based medium is more expensive to create the cellulose [73] Coconut water is a naturally isotonic liquid due to its specific mineral makeup and high reasonable total sugar level The "Nata de coco" bacterium, in particular, was recognized to thrive in coconut water

as early as the 1960s [74] BC is known as nata de coco is produced spontaneously at the coconut water/air interface Nata de coco, a Filipino dish, has gained popularity throughout many other

Asian nations The "Nata" bacteria was later identified as Acetobacter xylinum [75] As a result,

numerous research has also used coconut water as a BC culture medium, especially for the

Acetobacter xylinum strain [22, 76, 77] However, since coconut water is the primary medium for

biomass cultivation, the yield is not high and greatly depends on natural conditions [14] Coconut water or coconut juice is a sweet refreshing drink extracted directly from the interior section of coconut fruits [78] In addition to being a refreshing tropical drink, coconut water is also used as

a traditional medicine [79], a medium for the growth of microorganisms [80], a present for ceremonies [81], and it can be converted into vinegar [82] or wine [82] Futhermore, rice wastewater or rice extract was used by Rohaeti et al [83] as a potential source for BC production Starch, protein, minerals, and vitamin B are all present in rice wastewater and can be utilized as a

source of nutrients by the cellulose film-making bacteria A.xylinum As a result, it may be

employed as a source of both carbon and nitrogen The researcher used this together with some sucrose to produce the bacterial cellulose The yield of cellulose was 7.6 g/L according to the data Through the investigation, a higher percentage of crystalline (73%), mechanically and thermally stable cellulose was found [83] Therefore, a variety of materials have been investigated in an endeavor to find a BC culture medium that gives high biomass, has a stable price over time, or is less expensive than coconut water

Since then, other researchers have attempted to replace the carbon source used in the formation

of BC by using various alternate mediums using waste products from the food and agriculture industries It is worthwhile to investigate lower-cost culture media for BC production [84], therefore, the yield of BC has recently been increased by using diverse cellulosic wastes from

renewable agro-forestry residues or industrial by-products as carbon sources in addition to conventional coconut juice, for instance, food processing effluents, hemicelluloses in waste liquor from atmospheric acetic acid pulping [85], molasses [26], etc have been the subject of numerous investigations, which can also lower the economic cost [86-88] There has been some research of rice extract or rice waste water usage [83, 89, 90] as a substrate for BC growth Rohaeti et al characterized the properties of films obtained from rice water through several method such as Fourier Transform Infra Red (FTIR), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Thermogravimetric Analysis and Differential Thermal Analysis (TGA-DTA) and tensile tester [83] The antibacterial activity of BC and its composites was examined d against Staphylococcus aureus ATCC 25923 by the clear region method However, the study did not assess the solubility, water absorption, moisture transmittance, light transmittance, or oil permeability of

BC coatings Pham T K D also studied the production of BC films from rice water, but the amount of sugar added in the medium was still high and BC was applied as a drug loading material and controlled delivery of ranitidine [90] In another study, N X Thanh evaluated the curcumin release of BC cultured from rice water [89] Trinh V.T et al studied the application of BC cultured

in rice water as packaging for preserving Ham Yen oranges, however, only a few properties were measured such as chemical structure, mechanical strength and antibacterial properties [91] One of the crucial nutrients for the formation of bacterial cellulose is nitrogen, as it promotes the development and proliferation of the microorganism's cells The medium created by Hestrin and Schramm [92], which comprises 0.5% each of yeast extract and peptone, serves as the foundation for the majority of research projects Regarding the percentage of those partially defined nitrogen sources, including yeast extract, peptone, tryptone, etc., several research teams made minor adjustments Corn steep liquor (CSL) appeared to be the most productive of all the sources used [93, 94] CSL's impact on media used in various biotechnological processes has been documented in many previous studies [95, 96]

Few amino acids are frequently mentioned as essential when discussing defined nitrogen sources: methionine [93, 94, 97] and glutamate [97] Methionine alone accounted for 90% of cell growth and cellulose formation when compared to medium without this amino acid, as demonstrated by Matsuoka et al [94]

The production of cellulose was found to be aided by the vitamins pyridoxine, nicotinic acid, p-aminobenzoic acid, and biotin, whereas findings on the effects of pantothenate and riboflavin were conflicting [93, 94] Vitamins have no beneficial effects on the synthesis of cellulose, according to Fiedler et al [97] Fontana et al employed plant extract infusions, particularly those

with the quality of the cellulose generated led to its abandonment for the development of Biofill@ (Podlech and Jonas, unpublished results) [96]

2.2.2 Biosynthesis of BC

All obligate aerobic bacteria of the genera Acetobacter and Komagataeibacter spp both have

two major metabolic pathways: the pentosephosphate pathway for oxidizing carbohydrates and the less active glycolysis pathway There are enzymes of the Krebs cycle for the oxidation of organic acids and their biosynthetic substances The biochemical pathways and regulatory models

of cellulose biosynthesis from the A.xylinum strain have been well studied [42, 64]

Beginning in the bacterial cytoplasm of G.xylinus, nucleotide-activated glucose molecules

initiate the production of cellulose At the laboratory scale, glucose is frequently used as a substrate

during fermentation by G xylinus This BC biosynthesis will take place in a sequence of 4 primary

steps, with the associated biocatalysts (Figure 2.4): (1) glucokinase phosphorylates glucose to create glucose-6-phosphate from glucose; (2) phosphoglucomutase isomerizes glucose-6-phosphate to glucose-1-phosphate; (3) UDP - glucose pyrophosphorylase catalyzes the conversion

of glucose-1-phosphate into uridine diphosphate glucose (UDP - glucose); (4) glucan chains produced by cellulose synthase, which is catalyzed by UDP-glucose Fibrils are exported outside

of the cell by cellulose export components leading to cellulosic chain assembly and crystallization

Figure 2.4 Biosynthetic process of Gluconacetobacter xylinus [98]

In addition, the enzyme glucose oxidase transforms glucose into gluconate, which is then

produced as the 2- and 5-ketogluconate acids, when G xylinus is cultivated in glucose- or

sucrose-based media (Figure 2.5) The production of ketogluconate acids and gluconic acid by glucose dehydrogenase (GDH) results in a reduction in pH and lower BC yield [99]

Figure 2.5 Glucose oxidation pathways in G oxydans [100] G oxydans is a gram-negative

bacterium belonging to the family Acetobacteraceae, Gluconobacter strains [101]

2.2.3 Cultivation modes

a Static cultivation

Static cultivation is a straightforward and frequently utilized way of producing BC The culture medium (often placed in shallow trays) is inoculated, the BC pellicles form gradually, which float due to the bacteria's captured CO2 bubbles [102] Since the area of the air/liquid contact determines the efficiency of BC production, the duration of cultivation is about 5 – 20 days until the BC sheet almost fills the tray [102] Using vessels of various sizes and shapes, conventional static cultivation allows the BC membrane to take on the shape of the material on which it was formed [103] This

is advantageous if a material with a predetermined shape is needed, as is frequently the case in regenerative medicine Although this feature has not been thoroughly investigated for food applications, it may be used to create products that resemble nata but have diverse shapes, such as those intended for children

As seen in Figure 2.6, this method involves bacterial culture in shallow bottles or trays that contain the liquid growth media A floating layer of a gelatinous BC pellicle eventually covers the surface of the media during the cultivation period [104] Eventually, the BC pellicle will enclose the bacteria themselves [104]

Figure 2.6 Schematic diagram of BC culture (A) Conventional static culture and (B)

Intermittent fed-batch culture [105]

The typical static culture, on the other hand, is time-consuming and has low productivity, which could impede its industrial applicability [102] The cellulose membrane created in the medium has a propensity to cage the bacteria, limiting their access to oxygen, and the nutrients are continuously eaten, causing their concentration to gradually decline over time, limiting the production of BC [54] Fed-batch culture is an intriguing method for solving this issue According

to a study by Shezad et al (2009), fed-batch cultivation yields were raised by two to three times compared to batch cultivation when new alternative medium cultures were added during cultivation in a continuous process regime In this intermittent feeding method, there would be a crucial distance between the old air-liquid contact and the BC pellicle The newly created BC pellicles would prefer to form at the new air-liquid interface after the distance exceeds this critical value rather than piling up directly on top of the preexisting pellicles As a result, BC pellicles could develop layer by layer [106]

In recent years, bioreactors have been created to manufacture BC with greater yields sheets under almost static conditions, such as the Horizontal Lift Reactor [107] and rotary biofilm contactor [108]

b Agitated cultivation

The BC is produced with increased yield compared to static culture in agitated cultivation because oxygen is continuously infused into the medium, which lowers production costs [109] According to Esa et al (2014), the application of rotating speed during the agitated fermentation process can produce cellulose in a variety of shapes and sizes, including fiber suspension, spheres, and pellets [54, 109]

A significant disadvantage of agitation is the increased likelihood that cellulose-producing cells will mutate into cellulose-negative mutants as a result of the high turbulence and shear stress, despite the fact that the BC yield in agitated culture is typically thought to be higher than in static culture [41, 108] As a result, many reactor designs have been suggested to increase BC productivity while avoiding the development of cellulose-negative subpopulations [109] The accumulation of such mutants has also been reported to be prevented by adding more ethanol to the culture medium, which results in an increase in BC production [110].BC in fibrous form has frequently been produced in stirred-tank bioreactors However, because of shear stress during agitation, the crystallinity, elastic modulus, and degree of polymerization of fibrous BC were observed to be lower than those of pellicular BC [111] The fibrous BC suspension with a high cell density creates a high viscosity in stirred tank bioreactors, which limits the oxygen transport and necessitates a higher agitation power, resulting in significant energy consumption.An airlift bioreactor is another type of fermentation reactor in which oxygen is continually transported from the bottom of the reactor to the growth medium, ensuring a sufficient oxygen supply [109] Since then, various airlift bioreactor configurations have been suggested, such as Wu and Li's (2015) modified airlift bioreactor with a series of net plates to produce BC in pellicular form [112] The resulting membranes had a higher water-holding capacity than BC generated from static cultivation, and the elastic modulus was reported to be adjustable by altering the number of net plates.

2.2.4 Applications of bacterial cellulose

BC has a wide range of physical and mechanical properties, making it useful in tissue engineering, biomedicine, nanofluidics, wearable devices, functional foods, cosmeceuticals, and biocomposites

Pharmaceutical application

Numerous biomedical applications, such as artificial skin, dental implants, drug delivery, hemostatic materials, vascular grafts, tissue engineering scaffolds, biosensors, and diagnosis, are among several in which BC offers considerable potential [113-115] For all biomedical applications, excellent purity and biocompatibility of BC are essential The FDA's criteria for in vivo usage is met by the endotoxin in BC, which is tightly controlled at 20 endotoxin units/device [116] In addition, BC has a distinctive 3D reticulated network that offers a number of benefits, including huge surface areas, great water holding capacity, well-developed liquid/gas permeability, outstanding mechanical qualities, and transparency [117, 118] BC is a particularly special material that shows its supremacy in biomedical applications because of these

distinguishing qualities Devices for BC-based wound dressing have been successfully marketed, and a number of items relating to drug delivery, contact lenses, vascular grafts, and tympanic membrane replacement are in the process of commercialization [119]

The intricate interplay of different cell types, extracellular matrix (ECM) components, and soluble substances occurs throughout the dynamic process of wound healing [120] Winter (1962) [121] found that keeping the wound moist sped up healing, specifically re-epithelialization BC has been demonstrated to be a highly effective wound dressing material due to its special characteristics [121-124] The effectiveness of BC as a temporary skin substitute known as

"Biofill" was studied by Jonas and Farah in 1998 In relation to burns and other skin injuries, they talked about several clinical findings Positive results using Biofill included less post-operative discomfort, quicker healing, instant pain relief, lower infection rates, enhanced exudate retention, and, most importantly, shorter treatment times and lower costs According to Meftahi et al (2010),

a novel cellulose film coated with cotton gauze has a 30% higher water absorption and wicking performance than native cellulose film, making it better suited for use in wound dressings [125]

In addition to commercial wound care, BC has considerable potential for use in other biological applications [126, 127] BC as a drug carrier to deliver an anticancer agent, Kusano Sakko Inc declares that they have discovered that BC can enhance the controlled release of pharmaceuticals (Kusano Sakko Inc, 2020) In order to enhance the functions of drug delivery, they intend to utilise

BC as a fundamental material in medicines in the future Additionally, according to Axcelon Dermacare Inc., they are creating an oral vaccine employing BC as a drug carrier to keep the vaccine active while traveling through the stomach (Axcelon Dermacare Inc, 2020) The British Columbia-based company Axcelon Dermacare Inc also reports that it is working on prosthetic tympanic membranes, vascular grafts, and contact lenses, among other BC-based medical devices (Axcelon Dermacare Inc, 2020)

Additionally, BC has received much research for the development of vascular grafts [127] It

is the perfect choice for vascular grafts due to its great biocompatibility and outstanding wet mechanical strength Vascular grafts with the trade name Basyc were created by Jenpolymer Materials Ltd & Co for coronary artery bypass surgery [126, 128] The device pipeline for BC-based vascular grafts is also developed by other businesses including Innovatec and Axcelon Dermacare Inc [124, 129]

Paper industry

Due to its incredibly small clusters of cellulose microfibrils, microbial cellulose has been studied as a potential paper binder [130] This characteristic significantly increases the strength

and durability of pulp when incorporated into paper [130] The development of microbial cellulose for paper products is now being pursued by Ajinomoto Co and Mitsubishi Paper Mills in Japan (JP patent 63295793 and [131]) Additionally, the tensile strength and filler retention of the hand sheets were enhanced when the agitated bacterial cellulose and static bacterial cellulose were introduced at the wet end [132] Particularly, bacterial cellulose from an agitated culture had a greater impact on filler retention than bacterial cellulose from a static culture [130] The bacterial cellulose could therefore be beneficial as a wet-end ingredient for papermaking Furthermore, as the gelatinous mat of bacterial cellulose grows, suspended particles in the nutritional medium for

A xylinium in a rotating disk bioreactor become absorbed into it [133]

Composites with increased strength and bacterial cellulose's toughness can be made by including fibers of ordinary cellulose [130] Elongated paper fibers and purified cellulose are integrated in a different way than spherical particles like silica gel Paper scraps can provide about 90% of the final cellulose, and dried composite sheets were significantly more resilient per unit area than plain bacterial cellulose In comparison to paper sheets made by introducing BC [132, 133] Basta and Elsaied [134] discovered that adding 5% of BC to wood pulp during paper sheet formation considerably improved kaolin retention, strength, and fire resistance qualities [133]

Cosmetics

Bacterial cellulose has long been employed in a variety of industries, including paper production, food production, pharmaceuticals, and cosmetics A useful paradigm for bacterial cellulose in the cosmetic industry may result from the addition of bacterial cellulose to the cosmetic composition According to a prior study, the use of cellulose fibrils in cosmetic applications to create stable oil in water emulsions without the inclusion of extra surfactants offers the benefit of non-irritating skin [135] Excellent spreadability and adherence are generated by one of the early uses of cellulose powder in powdery cosmetics, whether in a loose or pressed state [135] In a recent discovery, bacterial cellulose was combined with various powdered cosmetic compounds [135]

However, there hasn't been any attempt to research how the bacterial cellulose addition affects the rheological behavior of the facial scrub The possibility of using the bacterial cellulose-containing face scrub as a natural facial scrub in the future exists [136] This is due to the natural components utilized, which made it safe for the skin and had a viscosity profile similar to that of commercial facial scrubs Another treatment that has an intriguing quality is the cellulose face mask, which, with only one application, improved facial skin moisture absorption The mask has been proven to be both secure and reliable [135, 137]

Food industry

BC is a dietary fiber that has been designated by the US Food and Drug Administration (FDA)

as a "generally regarded as safe" (GRAS) food [138] As a food ingredient, one of BC's key benefits as a food ingredient is its attractiveness for dietetic cuisine [136] In addition, it facilitates intestinal transit (like other dietary fibers) and improves mouthfeel [139]

BC has long been used to manufacture nata de coco, an indigenous South-East Asian dietary fiber presented as a gelatinous cube [140] Nata de coco has a chewy, soft, and smooth surface texture It contains no cholesterol, is low in fat, and is low in calories [141] BC was synthesized

in a static culture of coconut water during the manufacturing process and Acetobacter xylinum used coconut water as a carbon source, which was later transformed to extracellular cellulose [142] Natural unregulated pre-fermentation methods are becoming more popular, however, they pose a food safety issue due to the possibility of dangerous bacteria growing concurrently [143]

In addition, BC can plays as a fat replacer in meatballs [144] that resulting in cooking losses and softening, impairing the product acceptance Another research of S B LIN et al on surimi product resulted in increased gel strength and water-holding capacity due to its enhanced network structure [145]

Recent studies have shown that BC has various benefits for usage in food packaging [146, 147] All the research of [148, 149] when taken as a whole showed that BC and its derivative are potential materials for food packaging

2.3 Overview of food packaging from bacterial cellulose

2.3.1 The potential of BC in food packaging application

In the current state of the world market, consumers are demanding more natural foods that meet strict standards for excellent quality and safety To store fresh fruits and vegetables, new food packaging has been developed (Oliveira et al., 2015) The most common materials used to produce films are often alginate, cellulose, chitosan, carrageenan, or pectins and their derivatives (Sabina Galus & Kadzińska, 2015) Because they are compatible with a variety of food products, cellulose-based packaging and wrapping films and coatings are of particular commercial importance (C Chang & Zhang, 2011) Additionally, it has been demonstrated that these films and coatings significantly lower moisture loss and the amount of oil that fried foods absorb (Wang et al., 2018) Recent studies have shown that BC has numerous benefits for usage in food packaging However, using pure BC to manufacture packaging has significant drawbacks As a result, numerous modifications have emerged to enhance the desired qualities