Charaterization of bacterial cellulose produced by gluconacetobacter xylinus using rice extract as a nutrient source

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITYUNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION THESIS FOOD TECHNOLOGY CHARACTERIZATION OF BACTERIA

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY

UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR

HIGH QUALITY TRAINING

GRADUATION THESIS FOOD TECHNOLOGY

CHARACTERIZATION OF BACTERIAL CELLULOSE PRODUCED BY GLUCONACETOBACTER XYLINUS USING RICE EXTRACT AS A NUTRIENT SOURCE

SUPERVISOR: VU TRAN KHANH LINH STUDENT: NGUYEN THUY THANH HIEN

NGUYEN PHAM HUYEN PHUONG

SKL009155

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

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

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 Ho Chi Minh City, August 08, 2022SUPERVISOR

First and foremost, we would like to thank Ms Vu Tran Khanh Linh who is a lecturer at theFaculty of Chemical and Food Technology for her enthusiastic guidance, instruction, impartingvaluable experience, expertise as well as skills required for us to execute experiments efficientlyand scientifically Secondly, our team would like to thank the seniors who assisted us ininstructing 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 FoodTechnology, the Ho Chi Minh City University of Technology and Education had actively taughtand 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 andsupport 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 declarethat 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

4.3.3 Chemical structure 73

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 3.2 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

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 of0:1 (C100), 1:3 (C75R25), 1:1 (C50R50), 3:1 (C25R75), and 1:0 (R100) After 8 days offermentation, 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 membranesfrom all studied cultures medium were then characterized The light absorption values of allsamples in the UV region were less than 8%, and less than 20% for the visible spectrum, whichshows that all BC films were good UV resistant All samples had a tensile strength value greaterthan 30 MPa and high values of elongation at break, reflecting good mechanical properties andgreat elasticity of all BC films Hydrophilic properties such as moisture, water absorption,solubility, and water vapor permeability were more strongly performed by the BC filmsproduced in mediums with high content of coconut water According to the reusability test, theC100 membrane can be reused up to 19 times, while the R100 membrane can only be reused 3times Besides, all BC specimens can prevent oil permeability because they exhibited no sign ofoil absorption during the testing time All the above results indicate that the films obtained fromthe medium with the percentage of rice water from 25% to 75% were best suited for preservingfood 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 andfood industries is indispensable Additionally, packaging also plays a crucial role in many othersectors where almost commodities must be meticulously enclosed in certain shapes before beingdistributed to the market [1] The fundamental functions of food packaging are not only to avoidfood from being affected by external influences and spoiling agents but also to give customersdetailed 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 andconsumer expectations, guarantee food safety and nutritional values, and minimize adverseeffects 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 forthe 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 beenborn, including bio-based and biodegradable packaging materials They are produced fromnatural, 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-degradableand diminish environmental issues [6] Among these, cellulose-based packaging and wrappingfilms and coatings are commercially appealing due to their compatibility with a variety of fooditems [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 harshchemicals [9] Furthermore, with remarkable properties such as high degree of polymerization, goodbarrier 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 years althoughproducts from BC have been manufactured and applied in a wide range of fields

for years [14, 15] However, there are currently no commercially available BC films for foodpackaging [12], as a consequence of costly production and low yield [16] The media and processparameters have been regularly optimized by researchers in an effort to increase BC yields andfacilitate effective BC production Several studies have shown that BC can be produced from avariety 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-notchchoice 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 fromclimate changes Hence, there is a need of an alternative source, for instance, molasses [26-29], fruitwastes [29-32], tea [19, 33-35], rice extract [36-38], etc Therein, rice extract or rice waste waterwhich is a mixture after washing or soaking the rice by a rice-wash machine collecting fromindustrial meal kitchens Rice extract has milky color and is rich in carbohydrates and vitamins thathas not been widely exploited as a medium for BC production despite having suitable qualities andbeing a low-cost by-product Additionally, there were limited researches related to rice extract thathad 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 tocharacterize 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 BCserving 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 25

 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 Inother words, they will contribute to authorizing optimal conditions which result in desirable BCcollection and cellulose-based film properties

1.6 Practical objective

The project anticipates opening up the possibilities of bacterial cellulose film, anenvironmentally friendly material that can be made into biodegradable food packaging for aparticular 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 theworld is cellulose, which is conventionally derived from plants or their byproducts [39] Toproduce the pure product, this polymer's natural branching with hemicellulose and lignin has toundergo hazardous chemical operations involving strong alkali and acid treatment [40] Growingdemand 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 manufacturecellulose as a substitute source, even though plants are the primary producer of this material Thediscovery of bacterial cellulose (BC) dates back to Dr Brown (1886), who noticed thedevelopment of jelly-like structure in acetic fermentation medium with a chemically similarstructure to plant cellulose from Bacterium aceti such as a network structure made of ultrapurecellulose 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 ancientPhilippine Nata de pina in 1949 [43] The study by Lapuz et al provided scientific proof thatcellulose produced by bacteria is used to make the gelatinous pellicle that makes up Nata As ofpresent, 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-producingbacterium, has attracted considerable attention in recent years due to its potential applications inbiotechnology and medicine [52] Both plant and bacterial cellulose have the same molecularformula in terms of chemical structure (C6H10O5)n However, there are huge differences in theirphysical characteristics (Table 2.1) BC is completely pure and is extruded by cells asnanofibrils, in contrast to plant-derived cellulose nanofibres that need pretreatment todisintegrate the recalcitrant lignocellulosic network These nanofibrils can also be transformedinto macro fibers with superior material properties that are stronger than steel and can be used asalternatives 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], Asdepicted in Figure 2.2, the fibrous network of BC is made up of three-dimensional nanofibersthat 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 apromising 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]

Properties Bacterial cellulose Plant-based cellulose

Tensile strength (MPa) 20 – 300 25 – 200

Young’s modulus (MPa)

Sheet: 20,000 2.5 – 0.170Single fibre:130,000

Water holding capacity (%) > 95 25 – 35

Size of fibers (nm) 20-100 micrometer scale

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 glucosechain are released through the bacteria cell wall during the production process and clumptogether to form nanofibrils cellulose ribbons [64] With a highly porous matrix, these ribbonsconstruct 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, andchemical-modifying capacity [67] Chawla et al (2009) provided a detailed explanation of themechanism 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; theculture medium is also important [13] The different ingredients employed in culture media arethe elements that directly affect both the quantity and quality of cellulose production The mostimportant ingredients for the culture media utilized for BC production are a carbon and nitrogensupply, as well as salts to buffer the pH [68] However, optimal conditions for celluloseproduction with BC have yet to be determined [69] Indeed, it is widely known that metabolicpreferences 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 cellulosebiosynthesis The three main sugars that are frequently employed in the formation of bacterialcellulose are sucrose, glucose, and fructose Varying bacterial strains have different needs for carbonsources 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 ofcellulose [70-72] However, from an industrial (bulk production) standpoint, the HS medium bySchramm & Hestrin (1954) (glucose 2%; peptone (Difco Bactopeptone) 0.5%; yeast extract(Marmite), 0.5% disodium phosphate (anhydrous), 0.27% citric acid (monohydrate), 0.115%, pH6.0) or sugar-based medium is more expensive to create the cellulose [73] Coconut water is anaturally 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 the1960s [74] BC is known as nata de coco is produced spontaneously at the coconut water/airinterface 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 sweetrefreshing drink extracted directly from the interior section of coconut fruits [78] In addition tobeing a refreshing tropical drink, coconut water is also used as a traditional medicine [79], a mediumfor the growth of microorganisms [80], a present for ceremonies [81], and it can be converted intovinegar [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 inrice 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 7.6g/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 havebeen investigated in an endeavor to find a BC culture medium that gives high biomass, has a stableprice 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 agricultureindustries 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 toconventional coconut juice, for instance, food processing effluents, hemicelluloses in waste liquorfrom atmospheric acetic acid pulping [85], molasses [26], etc have been the subject of numerousinvestigations, which can also lower the economic cost [86-88] There has been some research of riceextract or rice waste water usage [83, 89, 90] as a substrate for BC growth Rohaeti et alcharacterized the properties of films obtained from rice water through several method such as FourierTransform 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 aureusATCC 25923 by the clear region method However, the study did not assess the solubility, waterabsorption, 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 themedium was still high and BC was applied as a drug loading material and controlled delivery ofranitidine [90] In another study, N X Thanh evaluated the curcumin release of BC cultured fromrice water [89] Trinh V.T et al studied the application of BC cultured in rice water as packaging forpreserving Ham Yen oranges, however, only a few properties were measured such as chemicalstructure, mechanical strength and antibacterial properties [91]

One of the crucial nutrients for the formation of bacterial cellulose is nitrogen, as it promotesthe development and proliferation of the microorganism's cells The medium created by Hestrinand Schramm [92], which comprises 0.5% each of yeast extract and peptone, serves as thefoundation for the majority of research projects Regarding the percentage of those partiallydefined nitrogen sources, including yeast extract, peptone, tryptone, etc., several research teamsmade minor adjustments Corn steep liquor (CSL) appeared to be the most productive of all thesources used [93, 94] CSL's impact on media used in various biotechnological processes hasbeen documented in many previous studies [95, 96]

Few amino acids are frequently mentioned as essential when discussing defined nitrogensources: methionine [93, 94, 97] and glutamate [97] Methionine alone accounted for 90% of cellgrowth and cellulose formation when compared to medium without this amino acid, asdemonstrated 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 riboflavinwere 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 thosemade from black tea, as a stimulant for the formation of cellulose [96] However, interferences

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 andthe less active glycolysis pathway There are enzymes of the Krebs cycle for the oxidation oforganic 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 createglucose-6-phosphate from glucose; (2) phosphoglucomutase isomerizes glucose-6-phosphate toglucose-1-phosphate; (3) UDP - glucose pyrophosphorylase catalyzes the conversion of glucose-1-phosphate into uridine diphosphate glucose (UDP - glucose); (4) glucan chains produced by cellulosesynthase, which is catalyzed by UDP-glucose Fibrils are exported outside of the cell by celluloseexport 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 byglucose 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,

As seen in Figure 2.6, this method involves bacterial culture in shallow bottles or trays thatcontain the liquid growth media A floating layer of a gelatinous BC pellicle eventually coversthe surface of the media during the cultivation period [104] Eventually, the BC pellicle willenclose 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 themedium has a propensity to cage the bacteria, limiting their access to oxygen, and the nutrientsare continuously eaten, causing their concentration to gradually decline over time, limiting theproduction 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 tothree times compared to batch cultivation when new alternative medium cultures were addedduring cultivation in a continuous process regime In this intermittent feeding method, therewould be a crucial distance between the old air-liquid contact and the BC pellicle The newlycreated BC pellicles would prefer to form at the new air-liquid interface after the distanceexceeds this critical value rather than piling up directly on top of the preexisting pellicles As aresult, BC pellicles could develop layer by layer [106]

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

b Agitated cultivation

The BC is produced with increased yield compared to static culture in agitated cultivationbecause 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 fermentationprocess 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-producingcells will mutate into cellulose-negative mutants as a result of the high turbulence and shearstress, 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 BCproductivity while avoiding the development of cellulose-negative subpopulations [109] Theaccumulation of such mutants has also been reported to be prevented by adding more ethanol tothe culture medium, which results in an increase in BC production [110] BC in fibrous form hasfrequently been produced in stirred-tank bioreactors However, because of shear stress duringagitation, the crystallinity, elastic modulus, and degree of polymerization of fibrous BC wereobserved to be lower than those of pellicular BC [111] The fibrous BC suspension with a highcell density creates a high viscosity in stirred tank bioreactors, which limits the oxygen transportand necessitates a higher agitation power, resulting in significant energy consumption An airliftbioreactor is another type of fermentation reactor in which oxygen is continually transportedfrom 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 fromstatic cultivation, and the elastic modulus was reported to be adjustable by altering the number ofnet plates

2.2.4 Applications of bacterial cellulose

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

Pharmaceutical application

Numerous biomedical applications, such as artificial skin, dental implants, drug delivery,hemostatic materials, vascular grafts, tissue engineering scaffolds, biosensors, and diagnosis, areamong 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 materialthat 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 tympanicmembrane replacement are in the process of commercialization [119].

The intricate interplay of different cell types, extracellular matrix (ECM) components, andsoluble 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 hasbeen 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 byJonas and Farah in 1998 In relation to burns and other skin injuries, they talked about several clinicalfindings Positive results using Biofill included less post-operative discomfort, quicker healing, instantpain relief, lower infection rates, enhanced exudate retention, and, most importantly, shorter treatmenttimes and lower costs According to Meftahi et al (2010), a novel cellulose film coated with cottongauze has a 30% higher water absorption and wicking performance than native cellulose film, making itbetter suited for use in wound dressings [125]

In addition to commercial wound care, BC has considerable potential for use in otherbiological applications [126, 127] BC as a drug carrier to deliver an anticancer agent, KusanoSakko Inc declares that they have discovered that BC can enhance the controlled release ofpharmaceuticals (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 drugcarrier 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 isworking on prosthetic tympanic membranes, vascular grafts, and contact lenses, among otherBC-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 wetmechanical strength Vascular grafts with the trade name Basyc were created by JenpolymerMaterials 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 AxcelonDermacare Inc [124, 129]

Paper industry

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

and durability of pulp when incorporated into paper [130] The development of microbialcellulose 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 ofthe hand sheets were enhanced when the agitated bacterial cellulose and static bacterial cellulosewere introduced at the wet end [132] Particularly, bacterial cellulose from an agitated culturehad a greater impact on filler retention than bacterial cellulose from a static culture [130] Thebacterial cellulose could therefore be beneficial as a wet-end ingredient for papermaking.Furthermore, as the gelatinous mat of bacterial cellulose grows, suspended particles in thenutritional 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 includingfibers of ordinary cellulose [130] Elongated paper fibers and purified cellulose are integrated in adifferent way than spherical particles like silica gel Paper scraps can provide about 90% of the finalcellulose, and dried composite sheets were significantly more resilient per unit area than plainbacterial cellulose In comparison to paper sheets made by introducing BC [132, 133] Basta andElsaied [134] discovered that adding 5% of BC to wood pulp during paper sheet formationconsiderably improved kaolin retention, strength, and fire resistance qualities [133]

Cosmetics

Bacterial cellulose has long been employed in a variety of industries, including paperproduction, food production, pharmaceuticals, and cosmetics A useful paradigm for bacterialcellulose in the cosmetic industry may result from the addition of bacterial cellulose to thecosmetic composition According to a prior study, the use of cellulose fibrils in cosmeticapplications to create stable oil in water emulsions without the inclusion of extra surfactantsoffers the benefit of non-irritating skin [135] Excellent spreadability and adherence aregenerated 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 variouspowdered cosmetic compounds [135]

However, there hasn't been any attempt to research how the bacterial cellulose additionaffects the rheological behavior of the facial scrub The possibility of using the bacterialcellulose-containing face scrub as a natural facial scrub in the future exists [136] This is due tothe natural components utilized, which made it safe for the skin and had a viscosity profilesimilar to that of commercial facial scrubs Another treatment that has an intriguing quality is thecellulose 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'skey benefits as a food ingredient is its attractiveness for dietetic cuisine [136] In addition, itfacilitates 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 dietaryfiber presented as a gelatinous cube [140] Nata de coco has a chewy, soft, and smooth surfacetexture 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 xylinumused 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 lossesand softening, impairing the product acceptance Another research of S B LIN et al on surimiproduct resulted in increased gel strength and water-holding capacity due to its enhancednetwork 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 arepotential 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 thatmeet strict standards for excellent quality and safety To store fresh fruits and vegetables, newfood packaging has been developed (Oliveira et al., 2015) The most common materials used toproduce 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 commercialimportance (C Chang & Zhang, 2011) Additionally, it has been demonstrated that these filmsand 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 foodpackaging However, using pure BC to manufacture packaging has significant drawbacks As aresult, numerous modifications have emerged to enhance the desired qualities