Production, Modification, and Characterization of Natural Bamboo

PRODUCTION, MODIFICATION, AND CHARACTERIZATION OF NATURAL BAMBOO

FIBER

AMK BAHRUM PRANG ROCKY

AMANDA J. THOMPSON, COMMITTEE CHAIR MARK E. BARKEY SELVUM PILLAY KEVIN H. SHAUGHNESSY RYAN M. SUMMERS

A DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of interdisciplinary Doctor of Philosophy in the Department of Metallurgical and Materials Engineering in the graduate school of The University of Alabama

TUSCALOOSA, ALABAMA

2019

ABSTRACT

In an effort to extract natural bamboo fiber (NBF) from bamboo for textiles and other uses, four bamboo species Bissetii, Giant Gray, Moso, and Red Margin were chosen for investigation.

Conventional fibers such as cotton, polyester, regular rayon, and 12 commercial bamboo viscose were included for comparative study. By using different chemicals and routes, 144 types of

NBFs were produced. Assessments on fiber yield percentages (40-77%), average lengths (1.50-

37.10 cm), fineness (9.68—93.3 Tex), and overall qualities, determined at least 47 sets were prospective for commercial use. Hand-spinning was executed on three sets of NBFs after blending with cotton fibers. Investigation on moisture regain (푀푅) and moisture content (푀퐶), revealed that bamboo plants and NBFs had 푀푅 = 8.0% and 푀푅 = 7.5% which was lower than rayon and bamboo viscose fiber (~11% and ~10%) but higher than raw cotton fibers (~5.7% and

5.4%). Among tensile properties, breaking tenacity of longer NBFs (33-37 cm) was 64-140

N/Tex and elongation at break was 2.0-2.5%; these values were 1.50-2.50 N/Tex and 8.0-11.0% respectively for blended yarns.

Elemental, chemical, and crystallographic investigations were accomplished by energy- dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), ATR-FTIR

(Attenuated Total Reflectance Fourier Transform Infra-Red), Raman spectroscopy (RS) and

X-ray diffraction (XRD) techniques. Bamboo plants were estimated to be 70-78% carbon (C) and 20-30% oxygen (O) atoms, and O/C ratio of 0.26-0.33. The NBFs had a higher O/C ratio of

0.58-0.70. Comparisons of the spectra revealed the differences between bamboo-NBF and other fibers. Some distinct lignocellulosic peaks were in NBFs that could be responsible for unique

properties. The crystallinity index (CI) of bamboo plants was 63-67% but CI of NBFs was higher

69-73% with crystallite sizes of 35-39 Å (3.5-3.9 nm). Four reflection planes and other properties are also documented.

A suitable antibacterial test method was modified for quantitative estimation of bacterial reduction. Results suggest that though 11 out of 12 bamboo viscose products failed to exhibit inhibition against the bacteria, most of the bamboo and NBF specimens successfully showed a bacterial reduction of 8-95% against Klebsiella pneumoniae and 3-50% against Staphylococcus aureus.

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DEDICATION

I would like to dedicate this dissertation —

To my mother who used to say “if you fail and stop, that means you are unfit on earth” and encourage me to work hard for fulfilling my own dreams. She was my heaven and the loveliest person in my life. I miss her every moment.

To my father who sacrificed the last penny for me and my siblings hiding his own sickness and sufferings for years and never complained to anybody. I learned how to work hard and sacrifice own happiness for others. He used to encourage me, “always try to do good for humanity even if it is very insignificant to make the world safe and peaceful for all people, it will at least encourage other people to do good things. You can never be really happy seeing your neighbors unhappy.”

To my siblings, and family who are the source of my energy and inspiration.

To my advisor, Dr. Amanda Thompson, who has always been more than an advisor. She has been a guardian, a guide, a patron, a well-wisher and a best teacher throughout my MS and Ph.D. studies and research activities.

To all homeless and destitute people who make me feel how lucky I am with so many privileges and benefits. I could be one of them. This feeling always reenergizes me in my tiredness and failures.

To all people who think about and work for all mankind.

LIST OF ABBREVIATIONS AND SYMBOLS

NBF Natural bamboo fiber w/w Weight to Weight concentration torr A unit of pressure; 1 torr = 133.32 Pascals

CH3COOH Acetic acid

AG ACS grade

AATCC American Association of Textile Chemists and Colorists

ACS American Chemical Society

ASTM American Society for Testing and Materials

ATCC American Type Culture Collection

Å Angstrom; 1 Å = 10−10 m

AR Ash & Rose

At% Atomic percentage

ATR Attenuated total reflectance

BV Bamboo viscose

BVC Bamboo viscose and cotton blend

BVS Bamboo viscose and spandex blend

BSA Bamboosa® LLC

BB Bambu Batu

BBB Big Boy Bamboo

B or BS Bissetii bamboo

BCF Bleached cotton fiber

BCF Bleached cotton fibers

BVCS Blend of bamboo viscose, cotton, and spandex

Ca Calcium

C Carbon

Cari Cariloha® Bamboo cm Centimeter

Co Cobalt

COD Coefficient of determination

CFU Colony-forming unit

Co. Company

Ne Cotton count/Number in English system

CI Crystallinity index

Cu Cuprum, copper dtex Decitex

°C Degree Celsius

°F Degree Fahrenheit

DSC Differential scanning calorimetry

DP Double-ply eV Electron volt, a unit of energy; 1 eV = 1.602 177×10-19 Joules

EDS Energy-dispersive X-ray spectroscopy

E. coli Escherichia coli et al. et alia, and others

FD Faerie's Dance

FE-SEM Field Emission Scanning Electron Microscope

FTIR Fourier Transform Infra-Red spectroscopy

FF Free Fly Apparel®

FWHM Full-width half maximum

VWR George Van Waters and Nat Rogers company

G or GG Giant Gray bamboo g Gram

Tex Gram weight per 1000 m, Tex count

GA Green Apple Active

HiVac High vacuum h Hour

H2O2 Hydrogen peroxide

Inc. Incorporated

IR Infrared

ISO International Standards Organization

K. pneumoniae/KP Klebsiella pneumoniae lb Libra, pound-mass

LLC Limited liability company

LR Liquor ratio

L Liter

M:L Material : liquor

μg Microgram

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μL Microliter

μm Micrometer mL Milliliter mm Millimeter

Min. Minute

푀퐶 Moisture content

푀푅 Moisture regain

M or MS Moso bamboo nm Nanometer

Na Natrium, sodium

NCC Natural Clothing Company

B-16 NBF from Bissetii bamboo through route-16

B-21 NBF from Bissetii bamboo through route-21

B-22 NBF from Bissetii bamboo through route-22

B-23 NBF from Bissetii bamboo through route-23

B-25 NBF from Bissetii bamboo through route-25

B-28 NBF from Bissetii bamboo through route-28

G-10 NBF from Giant Gray bamboo through route-10

G-13 NBF from Giant Gray bamboo through route-13

G-14 NBF from Giant Gray bamboo through route-14

G-16 NBF from Giant Gray bamboo through route-16

G-21 NBF from Giant Gray bamboo through route-21

G-22 NBF from Giant Gray bamboo through route-22

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G-23 NBF from Giant Gray bamboo through route-23

G-25 NBF from Giant Gray bamboo through route-25

G-28 NBF from Giant Gray bamboo through route-28

M-14 NBF from Moso bamboo through route-14

M-16 NBF from Moso bamboo through route-16

M-21 NBF from Moso bamboo through route-21

M-22 NBF from Moso bamboo through route-22

M-23 NBF from Moso bamboo through route-23

M-25 NBF from Moso bamboo through route-25

M-28 NBF from Moso bamboo through route-28

R-1 NBF from Red Margin bamboo through route-1

R-10 NBF from Red Margin bamboo through route-10

R-11 NBF from Red Margin bamboo through route-11

R-12 NBF from Red Margin bamboo through route-12

R-14 NBF from Red Margin bamboo through route-14

R-15 NBF from Red Margin bamboo through route-15

R-16 NBF from Red Margin bamboo through route-16

R-17 NBF from Red Margin bamboo through route-17

R-18 NBF from Red Margin bamboo through route-18

R-19 NBF from Red Margin bamboo through route-19

R-2 NBF from Red Margin bamboo through route-2

R-20 NBF from Red Margin bamboo through route-20

R-21 NBF from Red Margin bamboo through route-21

R-22 NBF from Red Margin bamboo through route-22

R-23 NBF from Red Margin bamboo through route-23

R-24 NBF from Red Margin bamboo through route-24

R-25 NBF from Red Margin bamboo through route-25

R-26 NBF from Red Margin bamboo through route-26

R-27 NBF from Red Margin bamboo through route-27

R-3 NBF from Red Margin bamboo through route-3

R-4 NBF from Red Margin bamboo through route-4

R-5 NBF from Red Margin bamboo through route-5

R-6 NBF from Red Margin bamboo through route-6

R-7 NBF from Red Margin bamboo through route-7

R-8 NBF from Red Margin bamboo through route-8

R-9 NBF from Red Margin bamboo through route-9

NY New York

N Nitrogen

NMR Nuclear magnetic resonance

No. Number

OD Optical density

O Oxygen

O/C Oxygen to carbon ratio

% C Percentage of cotton

% P Percentage of polyester

% S Percentage of spandex

%R Percentage reduction

Piko Piko Clothing pH Power/potential of hydrogen, measure of acidity or alkalinity

RS Raman spectroscopy

RCF Raw cotton fiber

RCF Raw cotton fibers

RG Reagent grade

R or RM Red Margin bamboo

RR/RT/CR Regular rayon/rayon twill/conventional rayon

RSF Relative Sensitivity Factor

RT Room temperature

SEM Scanning electron microscope s Second

Si Silicon

SP Single-ply

NaHCO3 Sodium bicarbonate

NaOH Sodium hydroxide

Na2SiO3 Sodium silicate

Na2SO3 Sodium sulfite

Na5P3O10 Sodium triphosphate

S. aureus/SA Staphylococcus aureus

H2SO4 Sulfuric acid

TAPPI Technical Association of the Pulp and Paper Industry

TG Technical grade

TM Test method hν The energy of light

3D Three dimensional

TSA Tryptic soy agar

TSB Tryptic soy broth

UV Ultra-violet

UCF Unbleached cotton fabric

N Unit of force, Newton

USA United States of America

UCF Untreated cotton fabrics

H2O Water

λ Wavelength

Wt% Weight percentage

WAXD Wide-angle X-ray diffraction

XRD X-ray diffraction

XPS X-ray photoelectron spectroscopy

Yala Yala Designs

Zn(NO3)2 Zinc nitrate

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ACKNOWLEDGMENTS

Though this dissertation is designated to my name as the author, this was far more than a sole endeavor. My biggest thank and credit goes to my advisor Dr. Amanda J. Thompson. Thank you for instructions, guides, supports, ideas, and collaborations all the times, training me and keeping me on track throughout my dissertation. I would like to acknowledge financial (graduate council fellowships and scholarships) and all other supports and facilities provided by the graduate school, The University of Alabama (UA). I would also like to acknowledge financial support, lab facilities, and different academic assistance from the Department of Clothing, Textiles and

Interior Design (CTID) in College of Human Environmental Science and Department of

Metallurgical and Materials Engineering (MTE) in College of Engineering, The University of

Alabama. I thank all of the faculty and officials of the departments.

I am thankful to Dr. Gregory B. Thompson (Professor, Departmental Graduate Program

Director, MTE, UA) for your supports in funding and lab facilities. Thank you for providing training and expenses for X-ray diffraction (XRD), scanning electron microscopes (SEM) and energy-dispersive spectroscopy (EDS) in FEI Quanta, EDAX, JEOL 7000F, Apreo S and X-ray photoelectron spectroscopy experiments. Your instructions and advice in crystallography helped me a lot in my dissertation.

I am grateful to each of the member in my dissertation committee. Thank you, Dr. Mark

E. Barkey (Professor, Interim Department Head of Aerospace Engineering and Mechanics, UA) for instruction in finite element analysis and theory of elasticity courses, training, guiding and providing me lab facilities with tensile tests. Thank Dr. Kevin H Shaughnessy (Professor,

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Department of Chemistry and Biochemistry, UA) for your instructions in organic chemistry and guiding me in chemical characterization, processing, and tests. Thanks for your advice, discussions, ideas and connecting me with different labs and experts. I am pleased to Dr. Selvum

Pillay (Chair and Professor, Department of Materials Science and Engineering, School of

Engineering, The University of Alabama at Birmingham, UAB) for serving in the committee and for your advice and suggestions. I would like to thank Dr. Ryan Summers (Assistant Professor

Department of Chemical and Biological Engineering, UA) for your instructions in bioprocessing engineering, discussions, guides and advice about working with bacteria and the antibacterial tests.

I would like to express my gratitude to Ms. Patricia McCaffrey (MBY Lab Supervisor) and Elizabeth Kitchens (Culture Curator) in the Department of Biological Sciences, UA for their supports, guides, instructions, and advice for antibacterial tests.

My special thanks go to Mr. Rob Holler (Instrumentation Specialist, Central Analytical

Facility, CAF, UA) for training, instructions, assistances with XPS, XRD, EDS, and SEM instruments. I will never forget your cordiality in teaching me all the instruments and working with their data. I would also like to thank Mr. Johnny Goodwin (Instrumentation Specialist,

CAF, UA) and Mr. Richard Martens (Manager, CAF, UA) for their supports and assistances with

SEM instruments.

I am thankful to Dr. Kenneth Belmore (Nuclear Magnetic Resonance Facility Manager,

Department of Chemistry and Biochemistry, UA) for your advice, instructions, and training with

FTIR. I would also like to express my gratefulness to Dr. Jair Lizarazo-Adarme (Research

Engineer, Adjunct Faculty, Department of Chemical and Biological Engineering, UA) for training me with Raman spectroscopy and Freeze dryer and giving me access to the labs. My

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special thanks go to Catherine Scott, hand spinner and fiber artist, for her support in the spinning of bamboo and cotton fiber blends. I would like to thank all the faculty, fellow students, friends, family and everyone for guiding and encouraging me throughout the course of my dissertation.

CONTENTS

ABSTRACT ...... ii

DEDICATION ...... iv

LIST OF ABBREVIATIONS AND SYMBOLS ...... v

ACKNOWLEDGMENTS ...... xiii

LIST OF TABLES ...... xx

LIST OF FIGURES ...... xxii

CHAPTER 1. INTRODUCTION ...... 1

1.1 Production of Natural Bamboo Fibers ...... 4

1.2 Characterization of Different Properties of Bamboo and NBF ...... 6

1.3 Antibacterial Investigation and Comparison ...... 10

CHAPTER 2. PRODUCTION AND MODIFICATION OF NATURAL BAMBOO FIBERS FROM FOUR BAMBOO SPECIES, AND THEIR PROSPECTS IN TEXTILE MANUFACTURING ...... 12

2.1 Abstract ...... 12

2.2 Introduction ...... 13

2.3 Methodology ...... 16

2.3.1 Materials ...... 16

2.3.2 Apparatus ...... 17

2.3.3 Initial Treatments ...... 17

2.3.4 Delignification ...... 18

2.3.5 Modification ...... 20

2.3.6 Final Treatments ...... 22

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2.3.7 Spinning ...... 22

2.3.8 Tests and Assessments ...... 22

2.4 Results and Discussions ...... 25

2.4.1 Discussions on Natural Bamboo Fibers Extraction ...... 25

2.5 Moisture Regain and Content ...... 27

2.6 Incineration Behavior (Burning Test) ...... 28

2.7 The Solubility of Bamboo and NBF ...... 29

2.8 Microscopic Analyses ...... 30

2.8.1 The diameter of the Single Natural Bamboo Fibers ...... 30

2.8.2 Morphology...... 30

2.9 Fiber Length and Yield Percentage ...... 32

2.10 Fiber Fineness ...... 35

2.11 Tensile Properties ...... 36

2.12 Conclusions ...... 37

2.13 References ...... 38

CHAPTER 3. CHARACTERIZATION OF THE CRYSTALLOGRAPHIC PROPERTIES OF BAMBOO PLANTS, NATURAL AND VISCOSE FIBERS BY X-RAY DIFFRACTION METHOD 44

3.1 Abstract ...... 44

3.2 Introduction ...... 45

3.3 Methodology ...... 47

3.3.1 Materials and machines...... 47

3.3.2 Extraction of Natural Bamboo Fibers (NBFs) ...... 47

3.3.3 Crystallographic Analyses by XRD ...... 49

3.4 Results and Discussion ...... 53

3.5 Crystallographic Analyses...... 53

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3.5.1 Crystallinity Indexes (CIs), Diffraction Angles, and Lattice Planes ...... 53

3.5.2 Crystallite Dimensions and Interplanar Spacings ...... 60

3.6 Conclusions ...... 60

3.7 References ...... 61

CHAPTER 4. ANALYSES OF THE CHEMICAL COMPOSITIONS AND STRUCTURES OF FOUR BAMBOO SPECIES AND THEIR NATURAL FIBERS BY INFRARED, LASER AND X-RAY SPECTROSCOPIES ...... 66

Abstract ...... 66

4.1 Introduction ...... 67

4.2 Methodology ...... 70

4.2.1 Materials and machines...... 70

4.2.2 Extraction of Natural Bamboo Fibers (NBFs) ...... 71

4.2.3 Elemental analyses by XPS & EDS ...... 71

4.2.4 FTIR and Raman: Analyses of Chemical Groups...... 72

4.3 Results and Discussion ...... 73

4.3.1 Elemental analyses ...... 73

4.3.2 X-ray Photoelectron Spectroscopy (XPS) ...... 75

4.4 FTIR and Raman: Identifying Chemical Groups ...... 80

4.5 Conclusions ...... 91

4.6 References ...... 92

CHAPTER 5. INVESTIGATION AND COMPARISON OF ANTIBACTERIAL PROPERTY OF BAMBOO PLANTS, NATURAL BAMBOO FIBERS AND COMMERCIAL BAMBOO VISCOSE TEXTILES ...... 97

5.1 Abstract ...... 97

5.2 Introduction ...... 98

5.2.1 Methods for Antibacterial Activity Assessment ...... 100

5.3 Methodology ...... 103

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5.3.1 Materials ...... 103

5.3.2 Equipment ...... 104

5.3.3 Extractions of Natural Bamboo Fibers ...... 104

5.3.4 Sample Preparation for antibacterial tests...... 106

5.3.5 Antibacterial Tests and Assessments ...... 106

5.4 Results and Discussions ...... 109

5.4.1 Assessment by Spectrophotometric technique...... 109

5.4.2 Assessment by Viable Plate Counting Technique ...... 110

5.4.3 Antibacterial Property of Bamboo, NBF and Bamboo Viscose ...... 113

5.5 Conclusions ...... 118

5.6 References ...... 119

CHAPTER 6. CONCLUSIONS ...... 125

REFERENCES ...... 129

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LIST OF TABLES

Table 1. Properties of the natural bamboo fibers from different studies ...... 7

Table 2. List of all recipes used in different processes of natural bamboo fiber (NBF) extraction...... 18

Table 3. Processing routes of the selected natural bamboo fiber (NBF) samples for different tests and use of NaOH in each route ...... 21

Table 4. Moisture/water content (%) of culms from four fresh bamboo plants ...... 25

Table 5. Burning tests and observations of different conventional fibers, raw bamboo, and natural bamboo fiber (NBF) samples ...... 28

Table 6. Solubility test of different conventional fibers, raw bamboo and natural bamboo fiber (NBF) samples in sulfuric acid ...... 29

Table 7. Lengths and yield percentages of natural bamboo fibers (NBFs) of four bamboo species extracted through different techniques and routes ...... 33

Table 8. The fineness of the natural bamboo fibers (NBFs) extracted from four bamboo plants 35

Table 9. Breaking tenacity and elongation of natural bamboo fibers and cotton-blended yarns… ...... 36

Table 10. Estimated crystallinity indexes (CIs) of different bamboo species, NBFs, bamboo viscose products, and conventional fibers by using Segal and Deconvolution methods on X-ray diffraction data ...... 54

Table 11. Size of the crystallites, reflection planes, diffraction angles, and interplanar distances of four bamboo species and natural bamboo fibers (NBFs) from X-ray (Co Kα source) diffraction technique ...... 56

Table 12. Recipes, chemicals and processing conditions for NBF extraction ...... 71

Table 13. Elemental compositions and oxygen-carbon ratios of different bamboo species and their fibers ...... 73

Table 14. Concentration, O/C ratios and binding energies of different elements in four bamboo specimens from XPS investigation ...... 75

Table 15. Binding energies, atomic percentages of Carbon and Oxygen with their components in XPS analysis of different bamboo species ...... 80

Table 16. Chemical bonds and band characteristics of ATR-FTIR and Raman spectra of four bamboo species, natural bamboo fibers (NBFs), bamboo viscose, regular rayon, and cotton fibers ...... 87

Table 17. Names and compositions of different commercial textile products and relevant information ...... 103

Table 18. Different chemicals and conditions applied to different stages of natural bamboo fiber (NBF) extraction ...... 105

Table 19. Percentage reduction of the optical density (OD) of different sample inoculated bacterial solutions after incubation of 24 h at 37°C ...... 109

Table 20. Percentage reduction of bacteria by different commercial textile products ...... 113

Table 21. Percentage reduction of bacteria by four bamboo species and their natural fibers .... 115

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LIST OF FIGURES

Figure 1. a general flowchart of natural bamboo fiber (NBF)extraction from four bamboo species using varying conditions...... 19

Figure 2. Photographs of the natural bamboo fibers (NBFs) from four bamboo plants and hand- carded yarn blended with cotton fiber...... 26

Figure 3. Comparison of moisture regain (%) and moisture content (%) of conventional fibers, bamboo, and natural fibers...... 27

Figure 4. The diameter of the single natural bamboo fibers from four bamboo plants...... 30

Figure 5. SEM images of four raw bamboo strips and natural bamboo fibers (NBFs) taken by JEOL 7000 (a—d, h) and Apreo S (e—g, i)...... 31

Figure 6. The relationship among fiber lengths, yield percentages and total use of NaOH in natural bamboo fiber (NBF) extraction...... 34

Figure 7: Bamboo sliced strips prepared for X-ray diffractometry in Bruker AXS...... 49

Figure 8. Deconvolution method applied to the X-ray (from Cobalt source, Co K) diffraction pattern of natural bamboo fiber (NBF) M-25 from Moso bamboo sample by using FitPeak processing...... 51

Figure 9. X-ray diffraction patterns of four bamboo species showing different peaks for determining the crystallinity indexes and the crystal sizes...... 55

Figure 10. Comparison of X-ray (from Co K source) diffraction patterns among four bamboo species, natural bamboo fibers (NBFs) and cotton...... 58

Figure 11. Change in crystallinity indexes as natural bamboo fibers (NBFs) were extracted into finer forms or count (Ne) through different stages of processing...... 59

Figure 12. X-ray photoelectron spectroscopic survey spectra of four bamboo species ...... 77

Figure 13. Deconvoluted peaks of the components (C1-C4) of C 1s peak from XPS spectra of 4 bamboo plants: (a) Bissetii, (b) Giant Gray, (c) Moso, and (d) Red Margin bamboo...... 78

Figure 14. Deconvoluted peaks of the components (O1 and O2) of O 1s peak from XPS spectra of 4 bamboo plants: (a) Bissetii, (b) Giant Gray, (c) Moso, and (d) Red Margin bamboo...... 79

Figure 15. Comparisons of ATR-FTIR and Raman spectra of raw bamboo samples and natural bamboo fibers (NBFs)...... 81 xxii

Figure 16. Comparisons of Raman spectra of four bamboo species, natural bamboo fiber (NBF) and cotton fiber...... 82

Figure 17. Comparisons of Raman spectra of Red Margin bamboo, natural bamboo fibers (NBFs) from four species and bamboo viscose fiber...... 83

Figure 18. Different peaks and their positions in the ATR-FTIR spectrum of Red Margin bamboo specimen...... 84

Figure 19. Different peaks and their positions in the Raman spectrum of Red Margin bamboo specimen...... 85

Figure 20. Unexpected bacterial growth on TSA plates inoculated with raw Bissetii bamboo (BS) and raw cotton fibers (RCF) when samples were non-sterilized and exposed to the environment vs. bacterial growth by sterilized samples...... 111

Figure 21. Sizes of the bacterial colony of S. aureus inoculated with natural bamboo fibers (NBFs) after 24 h vs 60 h with almost unchanged counts...... 112

Figure 22. Growth of the bacterial colony on TSA plates inoculated with different samples. .. 114

Figure 23. Growth of bacterial colony on TSA plates inoculated with different natural bamboo fiber (NBF) samples...... 116

Figure 24. Microscopic (SEM) images of natural bamboo fibers (NBFs) from four different species showing non-fibrous contents responsible for natural properties...... 117

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CHAPTER 1. INTRODUCTION

Increased demand for eco-friendly and sustainable textiles and clothing is driving textile scholars to investigate the fiber production and modification from fast-growing bamboo plants. In recent years, many scholarly works have been published on natural bamboo fiber production and characterization. The research on this topic is becoming very popular in modern eco-friendly textile production. Though production of natural bamboo fiber and its application in textile and clothing is very promising, the lack of successful research on extracting suitable natural bamboo fiber (NBF) for textiles has inhibited the promotion of its use in industrial textiles production.

Recently, with the advantage of faster growth and a larger cellulosic biomass, bamboo has been widely used in pulp production for paper and regenerated celluloses (Batalha et al., 2011;

ChaoJun, ShuFang, ChuanShan, & DaiQi, 2014; Correia, Santos, Mármol, Curvelo, &

Savastano, 2014; He, Cui, & Wang, 2008; Junior, Teixeira, & Tonoli, 2018; Ma, Huang, Cao,

Chen, & Chen, 2011; Sugesty, Kardiansyah, & Hardiani, 2015; Xie et al., 2015). Although bamboo viscose, a regenerated cellulosic fiber that can begin as a pulp product, is being produced and is commercialized globally, natural bamboo fiber is not as common because of the complexity of processing and costs. Since bamboo is a native plant in Asia and grows abundantly, most of the bamboo viscose fiber production facilities are in China. There are very few companies interested in producing natural bamboo fibers and it is also unclear the numbers and identities of such companies, if any, of the natural bamboo textiles are in the market

(Steffen, Marin, & Müggler, 2013).

Research has begun in recent years on natural bamboo fiber extraction. The interest of producing natural bamboo fibers comes from its properties which are not common in traditional textile fibers as a cluster along with the bamboo plant’s green status. The claimed features include soft hand and comfort, durability, breathability, antibacterial properties, moisture absorbency, protection against ultraviolet rays, better heat management (heat retention), anti- itching, antistatic, hypoallergenic, anti-odorous, and ease of dyeability (Afrin, Kanwar, Wang, &

Tsuzuki, 2014; Afrin, Tsuzuki, & Wang, 2009; Bambrotex, 2007; Rocky, 2017; Tanaka et al.,

2014; Zupin & Dimitrovski, 2010). Although these properties are theoretically described, there is not enough scientific research to support all the claims for all the properties (Hardin, Wilson,

Dhandapani, & Dhende, 2009; McLarren, 2010; Michael, 2008). Moreover, these properties are also not confirmed by standardization authorities on currently available commercial bamboo clothing (regenerated bamboo fiber). If the properties are present, some of the features may be artificially introduced to the viscose fibers by chemical means. While the manufacturers and sellers are taking advantage of this confusion by making unproven claims about the properties of their products, most of the research has failed to prove all of these properties as inherently part of commercial viscose products (Hardin et al., 2009; Nayak & Mishra, 2016; Xi, Qin, An, & Wang,

2013). This incongruity is further stretched when compared to the production processes. An in- depth study suggests that the currently available literature fails to introduce a successful methodology that could be applied in the commercial or large-scale production of natural bamboo fibers. However, the conventional chemical-dependent viscose process is already widely applied for regenerated bamboo viscose production which is an ecologically hazardous process and may fail to retain natural properties of bamboo fibers (Nayak & Mishra, 2016). There is no official classification of bamboo textiles according to the US federal trade commission. Retailers

have claimed viscose projects as bamboo clothing and have suffered penalties (13-cv-00002-

TFH, 2013; 15-cv-02130-APM, 2015; FTC, 2010b, 2010a; Hardin et al., 2009; McLarren, 2010).

So it is very crucial to design an effective production process of natural bamboo fibers.

Moreover, investigation and examination of the properties of the fiber will bolster the understanding of natural bamboo fibers. Thus, research to establish suitable methods of natural bamboo fiber production and characterizing different properties of that fiber is very timely in the textile sector.

Since bamboo is a fast-growing plant which is capable of producing a high amount of lignocellulosic biomass, it has been studied for utilization for multiple purposes. As a consequence, the viscose process for the production of regenerated cellulosic fibers has taken advantage of the cellulose content of the bamboo plant. This process was previously applied to cotton linter and wood pulp (Fu, Li, et al., 2012). In the past few years, scholarly circles have begun investigating bamboo for naturally produced fiber as opposed to viscose fibers. The main reasons are- the viscose process requires a huge amount of water, energy and toxic chemicals, and removes the distinctive properties of bamboo fibers. Since most of the existing textile processes have a significant impact on environmental pollution and may prove unsustainable for profitability, researchers and manufacturers are interested in focusing on sustainable production processes and raw materials to develop the next materials for textiles (Abdul Khalil et al., 2012;

Afrin et al., 2014; Parisi, Fatarella, Spinelli, Pogni, & Basosi, 2015; Rocky, 2017; Steffen et al.,

2013; Waite, 2009). Bamboo provides the possibility to produce eco-friendly and sustainable textiles products if processed through non-hazardous techniques. Reasonably, a myriad of efforts has been employed to produce natural bamboo fibers for textiles, clothing, composites and other products.

1.1 Production of Natural Bamboo Fibers

Though the research-interest in natural bamboo fibers is growing, the difficulties of fiber separation due to strongly bonded lignin has been discouraging. As a result, most researchers have discontinued their studies on the endeavor. The biggest challenge to NBF production is delignification which is defined as the process of separating fibers from lignin and other binding components in bast materials. The amount of lignin and other undesired components in fibers are different for different bamboo species (Muhammad, Rahman, Hamdan, & Sanaullah, 2019;

Nayak & Mishra, 2016). Almost 1200-1500 bamboo species under 70-91 genera are documented worldwide (Abdul Khalil et al., 2012; Chaowana, 2013; Correia et al., 2014; Hardin et al., 2009;

Kweon, Hwang, & Sung, 2001; Morton, 2005; Rai, Ghosh, Pal, & Dey, 2011; Rocky, 2017;

Zakikhani, Zahari, Sultan, & Majid, 2014a). However, only around 50 species have been studied for bamboo natural and viscose fibers production. For the natural bamboo fiber production studies, some identical initial steps to viscose production such as cutting bamboo culms in- between nodes, splitting, retting or crushing or braising were employed followed by different processes.

The enzymatic and the microbial or bacterial processes are considered the eco-friendliest processes for natural fiber production. However, these processes are extremely slow and not effective for bamboo fiber extraction for textiles. Moreover, the cost of the equipment, microbes/bacteria and enzymes are very high, which would not be commercially competitive.

None of the current enzymatic/microbial process research clearly mentioned successful results on producing bamboo fibers followed by spinning or weaving for textiles (Afrin et al., 2014; Fu,

Li, et al., 2012; Fu, Zhang, Yu, Guebitz, & Cavaco-Paulo, 2012; Hanana et al., 2015; Liu,

Cheng, Huang, & Yu, 2012; Rocky, 2017). Therefore, investigation on extracting the natural

bamboo fibers by solely using the enzymatic or bacterial treatment is not a productive idea for textile production. Enzymes or bacteria are found to be very effective when applied after the initial extraction of fibers in bundle form. One such study mentioned spinning of 100% natural bamboo yarn and blended yarn (Steffen et al., 2013). However, successive research or commercial implementation was not reported perhaps due to the expense of the process.

Some of the mechanical investigations were carried out by applying a steam explosion at an elevated temperature and pressure, while others were executed at high-temperature boiling.

Although the process is eco-friendlier than the chemical process, except for the use of higher heat energy, it was reported as unable to separate fibers completely (Biswas, Shahinur, Hasan, &

Ahsan, 2015; Kim, Okubo, Fujii, & Takemura, 2013; Ogawa, Hirogaki, Aoyama, & Imamura,

2008; Okubo, Fujii, & Yamamoto, 2004; Phong, Fujii, Chuong, & Okubo, 2012; Rao & Rao,

2007; Trujillo et al., 2014; Witayakran, Haruthaithanasan, Agthong, & Thinnapatanukul, 2013;

Zakikhani, Zahari, Sultan, & Majid, 2014b; Zakikhani et al., 2014a). Studies, where mechanical and chemical processes were combined, were focused on fiber production for purposes other than the textile application. The produced fibers were in bundle forms rather than in individual form, which was not applicable for spinning purposes. Therefore, no spinning and subsequent textile production were reported.

The chemical extraction of natural bamboo fibers was reported far more successful than other processes (Deshpande, Rao, & Rao, 2000; Kim et al., 2013; Phong et al., 2012; Prasad &

Rao, 2011; Rao & Rao, 2007; Rocky, 2017; Zakikhani et al., 2014b). However, most of the studies involved using a high concentration of alkali, acids or other non-ecofriendly chemicals which is better than the viscose process but not highly eco-friendly (Liu et al., 2012; Liu, Wang,

Cheng, Qian, & Yu, 2011). Additionally, the excessive use of chemicals can destroy the natural

unique properties of bamboo fibers. It is possible to reduce the amount of chemicals if different mechanical processes are applied before delignification by chemicals (Rocky, 2017).

A review of the biological or enzymatic, the mechanical or steam explosion, the chemical and the retting processes suggests that a one-way approach would not be successful. However, approachable guidelines could be deduced from the findings of different investigations and pilot experiments that the combinations of mechanical, enzymatic or microbial and chemical processes would be more effective and successful (Liu et al., 2012; Phong et al., 2012; Rocky,

2017; Steffen et al., 2013; Zakikhani et al., 2014a, 2014b). So, different combined approaches were mainly projected in this study. Based on the information in the articles of the past research, the most effective and available items were inspected such as different chemicals, raw materials, machines, equipment and technologies of NBFs extraction.

1.2 Characterization of Different Properties of Bamboo and NBF

The structural, physical, chemical and mechanical properties of natural bamboo fibers produced through different techniques have been reported in several investigations (Table 1).

Unfortunately, no standard has been formed due to the extreme incongruity of the results.

Different properties of natural bamboo fibers were reported with a wide range of values. Since no standard method for natural bamboo fiber processing has been established, fibers produced in different studies were different in length and diameter. In most of the cases, the properties were determined by testing the fiber bundles rather than the individual fibers. However, some of the properties are independent of the form (individual or bundle form) if the fibers are extracted with natural constituents intact. It should be noted the standardized value of the different properties of viscose (from bamboo) have already been established.

Table 1

Properties of the natural bamboo fibers from different studies Property and unit value Reference (Afrin et al., 2014; Liu et al., 2011; Rocky, 2017; Average length (mm) 2-270 Steffen et al., 2013; Zakikhani et al., 2014b) Average diameter (µm) of (Afrin et al., 2014; Komuraiah, Kumar, & Prasad, 4-20 single fiber 2014; Rocky, 2017; Steffen et al., 2013) (Liu et al., 2011; Rocky, 2017; Steffen et al., 2013; Linear density (dtex) 2-35 Yueping et al., 2010) (Jain, Kumar, & Jindal, 1992; Prasad & Rao, 2011; Specific density (g/cm3) 0.6-1.4 Zakikhani et al., 2014b) Moisture regain (%) Not available - Water absorbance (%) Not available - UV absorbance (%) 55.7 (UVA), 24.3 (UVB) (Afrin et al., 2014) (Afrin et al., 2014; He et al., 2008; Yueping et al., Crystallinity (%) 52~89 2010) Diffraction peaks at 15-16 and 22 angle (degree) (He et al., 2008; Jiang, Miao, Yu, & Zhang, 2016; Diffraction plane (101), (101̅), (002) Yueping et al., 2010) Interplanar distance (nm) -, 5.683, 4.003

Crystal Size (Å) 35.5 (Afrin et al., 2014) Tenacity (cN/tex) 8-35 (Liu et al., 2011; Steffen et al., 2013; Waite, 2010) (Liu et al., 2011; Prasad & Rao, 2011; Steffen et al., Breaking elongation (%) 1.4-2.9 2013) Tensile strength (MPa) 440-600 (Prasad & Rao, 2011) Tensile Modulus (MPa) 35-46 (Prasad & Rao, 2011) Aryl, conjugated and non- Chemical bonding conjugated carbonyl, (Afrin et al., 2014; Yueping et al., 2010) hydroxyl, hydrocarbon

The surface morphology of the natural bamboo fibers has been studied in many investigations (Afrin et al., 2014; Cho, Kim, & Kim, 2013; He et al., 2008; Ogawa et al., 2008;

Phong et al., 2012; Shih, 2007; Tokoro et al., 2008; Y. L. Yu, Huang, & Yu, 2014; Y. Yu, Wang,

& Lu, 2014; Yueping et al., 2010; Zakikhani et al., 2014b; Zou, Jin, Lu, & Li, 2009). The surface of the fibers has been reported as very smooth. Lignin or other components are sometimes shown on the surface which are capable of reflecting a higher fraction of incident light. As a result, the

fibers look lustrous and can exhibit higher dyeing yield (darker color-shade) at a lower concentration than that of other fibers. The cross-section of the natural bamboo fibers is circular- shaped consisting of different layers. From the center to the outermost surface are lumen, secondary walls, middle lamella, and primary cell walls.

The structural and the chemical composition of natural bamboo fibers are important factors needed to understand the behavior of the fibers under different conditions. The major components have been determined as cellulose, hemicellulose, lignin, pectin and other organic and/or inorganic soluble and insoluble components. Different chemical groups have also been analyzed by x-ray spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR).

Some of the identified chemical groups associated with the natural bamboo fibers are listed in

Table 1 under Chemical Bonding.

The physical and the crystallographic properties have also been investigated for natural bamboo fibers of 2-3 bamboo species only, which is not sufficient to establish a standard. So, further study on different species (as selected in this study) will help standardization. A wide

Gaussian range of the length of 2-270 mm and the diameter of 4-20 µm (Table 1) have been reported. The linear density (2-35 dtex*) of the fibers have not been determined with fair accuracy as the fiber-bundles were investigated rather than individual fibers. The specific density

(0.6-1.4 g/cm3) of the fibers has been established independent of the form (single or bundle).

However, the extraction process and the type of species influence the specific density. The moisture regain or the moisture content percentage in standard atmospheric conditions and the absorbance of water have not been found to be determined of standardized. The crystallinity,

* Tex is a unit of linear density of fiber or yarn used in textile industries which is the weight of the fiber/yarn in 푔푟푎푚푠 1 grams per 1000 m length: 1 푡푒푥 = and 1 푑푡푒푥 = 푡푒푥. 1000푚 10

size of the crystallites, diffraction planes and the interplanar distances that have been studied so far are listed in Table 1.

Different mechanical properties such as breaking strength or tenacity, breaking elongation, tensile strength and tensile modulus have been investigated (Table 1). Since the natural bamboo fibers were not extracted in individual form, the bundle of fibers was used for the tests in most of the studies. Thus, the tabulated values (Table 1) might not represent standardized values for the natural bamboo fibers.

In this study, different structural or morphological properties such as surface contour, cross-section, average diameter, average length, and other relevant analyses were selected to be executed by microbalance, scanning electron microscope (SEM), and optical microscopy. Since most of the previous studies were limited to measuring the length and the diameter of the fiber bundle, this study was focused on single/individual fiber from four bamboo species for those measurements. Thus, this study is expected to provide a better understanding of individual natural bamboo fibers.

Apart from surface morphology and dimensions, a crystallographic study is very important to understand mechanical properties, coloration behavior and luster of the natural bamboo fibers. In some studies, the modern x-ray diffractometric (XRD) technique was employed to determine the degree of amorphousness or crystallinity, and crystal size. So, a systematic investigation with XRD would establish crystallographic properties of the natural bamboo fibers. Moreover, incineration behavior, moisture Regain (%) and solubility are important properties of textiles. So, the standard AATCC (The American Association of Textile

Chemists and Colorists) /ASTM (The American Society for Testing and Materials) test methods were chosen to measure these properties.

The information about the chemical/elemental composition of bamboo and its NBFs would provide knowledge to scientifically interpret the reasons for the fibers showing unique properties. The presence of different chemical components or chemical bonding were studied by

X-ray Spectroscopy (XPS) and Fourier Transform Infrared Spectroscopy (FTIR) (Afrin et al.,

2014; Fuentes et al., 2011; Shih, 2007; Tran, Nguyen, Thuc, & Dang, 2013). So, further studies on the chemical composition of selected bamboo species would enrich the data and accuracy.

1.3 Antibacterial Investigation and Comparison

One of the biggest controversies about bamboo fiber/textile is the antibacterial activity. This debate is further muddied by manufacturers and retailers claiming/labeling viscose products as natural bamboo fibers (Hardin et al., 2009; McLarren, 2010; Rocky, 2017). While several investigations found traces of antibacterial activity in the bamboo plant or fibers (Afrin et al.,

2014; Afrin, Tsuzuki, Kanwar, & Wang, 2012; Rocky, 2017; Singh et al., 2010; Tanaka, Kim,

Oda, Shimizu, & Kondo, 2011; Tanaka, Shimizu, & Kondo, 2013; Zheng, Song, Wang, & Wang,

2014), others did not (Hardin et al., 2009; Rocky, 2017; Xi et al., 2013). The trends in the modern markets of textiles and clothing demand the necessity of antibacterial/antimicrobial activity. Hence, a detailed study on antibacterial activity of the plants, natural bamboo fibers and comparisons with the bamboo viscose commercial fabrics/products (12 brands), cotton fibers and fabrics, and other mainstream fibers would help to resolve the debate.

According to the proposal, this study was based on four main focuses relating to NBF in textiles. In part-1, production and modification of natural bamboo fibers (NBFs) from four widely-grown bamboo species was expected to be conducted. Through in-depth investigation, it was projected some accomplishments such as appropriate chemicals and ingredients for delignification, a series of experiments to design specific routes with a proper sequence that will

be able to extract expected NBFs, identifying morphological, physical, and mechanical properties, financial viability through yield percentages, production costs and other factors. In part-2 and 3, a detailed investigation on different elemental compositions, chemical bonds and behaviors, and crystallographic properties was anticipated to characterize bamboo plants and

NBFs along with different conventional and commercial fibers such as bamboo viscose, rayon, cotton, and polyester. For characterization, modern instruments and techniques were chosen by using X-ray photoelectron spectroscopy (XPS), Fourier Transform Infra-Red (FTIR) spectroscopy with Attenuated Total Reflectance (ATR), Raman spectroscopy (RS), and X-ray diffraction (XRD). In part-4, comprehensive trials and experiments were expected to modify a suitable method for the antibacterial test of textile materials in any form. Since the current standard methods are not suitable for all kinds of textile materials, this would help to solve difficulties related to antibacterial tests of fibers, yarns, fabrics, powders, and materials with other forms. Another major target of part 4 was to investigate and compare the antibacterial property of bamboo plants, NBFs, 12 commercial bamboo viscose, and some other conventional fibers.

This study was mainly focused on natural bamboo fiber extraction in the purpose of using the fibers in textile production. Thus, all of the investigation, modification, and characterization were executed to advance the fiber for textiles and comparing results with traditional fibers.

Correspondingly, results were reported and compared that are significant in textile sector.

CHAPTER 2. PRODUCTION AND MODIFICATION OF NATURAL BAMBOO

FIBERS FROM FOUR BAMBOO SPECIES, AND THEIR PROSPECTS IN TEXTILE

MANUFACTURING

2.1 Abstract

Four widely grown bamboo species, Bissetii, Giant Gray, Moso, and Red Margin, were studied for natural bamboo fiber (NBF) extraction using 36 routes with respective fiber yield percentages where total use of NaOH was less than 24 g/L in any specific processing route. The Red Margin species was found to have the most potential for NBF extraction. This study provides reports on moisture regain (average 8.0%) and moisture content (average 7.5%), incineration behavior, solubility properties, surface morphology, length and diameter of single NBF (10—13 μm), and fineness of NBFs from four plants and comparative results with conventional fibers. The spinning of yarns of NBFs blended with cotton and their tensile properties were tested. NBFs that were longer and coarser in initial stages, with high breaking tenacity of 63.74—138.63

N/Tex and lower elongation of 2.06-2.46% were judged to be good for some textile applications.

Finer and shorter NBFs provide a lower strength to the blended yarns than cotton fibers.

KEYWORDS: Natural Bamboo fibers; Natural fiber extraction; Properties of NBF; Eco-friendly fibers; Solubility; Incineration behavior; NBF morphology.

2.2 Introduction

Due to its growth, sustainability, eco-friendliness, and many other advantages, bamboo has been studied with the purpose of extracting fibers for apparel manufacturing (Nayak & Mishra, 2016;

Phong, Fujii, Chuong, & Okubo, 2012; Rocky & Thompson, 2018a, 2018c; Waite, 2010) and as a reinforcing material in composites (Chaowana, 2013; Hebel et al., 2014; Hu & Yu, 2014; K.

Liu, Takagi, Osugi, & Yang, 2012; W. Liu, Xie, Qiu, & Fan, 2015; Phong et al., 2012; Yu,

Wang, & Lu, 2014). Though bamboo viscose is commercially available, there are numerous efforts to extract the natural fibers from bamboo to maintain natural properties and for environmental concerns (Nayak & Mishra, 2016). Some studies have been conducted by applying different techniques such as enzymatic, microbial, chemical and mechanical, for natural bamboo fiber (NBF) extraction to retain the properties of bamboo. Delignification or degumming, a process of removing lignin and non-fibrous materials, if done by enzymes or microbes are very slow processes that take several weeks and still may not be effective for industrial textile fiber production (J. Fu, Zhang, Yu, Guebitz, & Cavaco-Paulo, 2012). Chemical and mechanical/steam explosion processes were not found to be effective, as these processes may weaken the fiber during delignification resulting in weak and rough fibers (J. Fu, Zhang, et al., 2012; Rao & Rao, 2007; Rocky & Thompson, 2018a; Witayakran, Haruthaithanasan,

Agthong, & Thinnapatanukul, 2013). The combination of multiple techniques has been found to be more effective in NBF extraction (Hanana et al., 2015; L. Liu, Cheng, Huang, & Yu, 2012;

Rocky & Thompson, 2018a).

Only a very few bamboo species have been studied for natural fiber extraction. Moso bamboo (Phyllostachys edulis/pubescens/heterocycla) is the most studied bamboo plant (Afrin,

Kanwar, Wang, & Tsuzuki, 2014; Afrin, Tsuzuki, & Wang, 2009; Chaowana, 2013; Erdumlu &

Ozipek, 2008; J. J. Fu et al., 2017; J. Fu, Zhang, et al., 2012; Kim, Okubo, Fujii, & Takemura,

2013; K. Liu et al., 2012; W. Liu et al., 2015; Phong et al., 2012; Sharma, Gatóo, & Ramage,

2015; Tokoro et al., 2008; F. Wang, Shao, Keer, Li, & Zhang, 2015; H. Wang et al., 2015; Xu,

Wang, Liu, & Wu, 2013; Yu et al., 2014). Large size, excellent reproducibility, and higher growth are some of the major factors that place this species as a top choice. Moso is also widely used in viscose fiber production (Afrin et al., 2014; Erdumlu & Ozipek, 2008). However, most of the studies have reported only natural fiber extraction, rather than assessing Moso fiber applicability in the subsequent processes of apparel manufacturing, such as spinning, weaving or knitting (Rocky & Thompson, 2018a, 2018c; Yueping et al., 2010). Actually, subsequent processes with different bamboo species are most often unfeasible because of the poor quality of the natural bamboo fibers (L. Liu et al., 2012). The natural fibers that have been extracted from different bamboo plants are reported as very coarse and rough and are not easy to spin even after blending with other fibers. Only a very few investigations reported yarn and fabric production

(Steffen, Marin, & Müggler, 2013) but as the processes were not commercially feasible, they were not continued.

Though investigations on Moso bamboo for NBF were not considered entirely successful, other species may offer better potential for fiber extraction as the quantities of the plant constituents are not the same in all species. The compositions may even vary with geographic locations (J. Fu, Li, et al., 2012; Komuraiah, Kumar, & Prasad, 2014; Nayak & Mishra, 2016).

Though there are over 1500 bamboo species of 70-91 genera (Abdul Khalil et al., 2012;

Chaowana, 2013; Correia, Santos, Mármol, Curvelo, & Savastano, 2014; Hardin, Wilson,

Dhandapani, & Dhende, 2009; Kweon, Hwang, & Sung, 2001; Morton, 2005; Nayak & Mishra,

2016; Rai, Ghosh, Pal, & Dey, 2011; Rocky & Thompson, 2018b; Waite, 2010), only a few have

been studied for NBF extraction. Therefore, an investigation on several bamboo species that are also easily available and reproducible for the purpose of NBF extraction is advisable.

It was found that most of the studies started with splitting of the culms, rasping/scraping, pressing-crimping/crushing/pounding followed by soaking and delignification (Ibrahim et al.,

2015; Kim et al., 2013; L. Liu et al., 2012; Murali Mohan Rao, Mohana Rao, & Ratna Prasad,

2010; Nayak & Mishra, 2016; Rocky & Thompson, 2018a; Steffen et al., 2013; Waite, 2009; Xi,

Qin, An, & Wang, 2013). Steffen et al. (2013) extracted NBF fibers (length = 2 mm—150 mm; diameter = 4 μm—150 μm; fineness = 5.8 dtex) by mechanical splitting and rasping of culms followed by repeated enzymatic delignification washing where peroxide was used for bleaching.

Liu et al. (2012) experimented with mechanical and chemical pretreatment using H2SO4 and

NaOH following enzymatic treatment using different enzymes (L. Liu et al., 2012). On the other hand, Xi et al. (2013) applied alkali degumming followed by acid and water rinsing (Xi et al.,

2013). Extensive mechanical and chemical processes were also studied for NBF extraction for the purpose of composite structures (Deshpande, Rao, & Rao, 2000; Rao & Rao, 2007). Kim et al. (2013) investigated the steam explosion, alkali, and chemical extraction of NBF using different conditions with the result that chemical treatment was found to be better for delignification (Kim et al., 2013). Zakikhani et al. (2014) extracted NBF for bamboo fiber- reinforced composites by steam explosion, water and chemical retting (Zakikhani, Zahari,

Sultan, & Majid, 2014). It was concluded that steam explosion produces charred fibers rather than spinnable natural fibers needed for textiles manufacture (Rocky & Thompson, 2018a;

Zakikhani et al., 2014). Chemical retting with NaOH, zinc nitrate [Zn(NO3)2], and hydrogen peroxide (H2O2) was reported to be very effective for delignification up to a certain degree

(Zakikhani et al., 2014). Liu et al. (2011) conducted pretreatment by acid (H2SO4) soaking and

heating, NaOH treatment and then chemical delignification by using variable concentrations of different chemicals and conditions. They concluded optimum parameters for NBF extraction by applying NaOH (20 g/L), Na5P3O10 (3 g/L), Na2SO3 (5 g/L, and a penetrating agent (3 g/L) at

100 °C for 2 h in a liquor ratio of 1:10 (L. Liu, Wang, Cheng, Qian, & Yu, 2011). Therefore, it can be concluded from previous investigations that a combination of different techniques along with different chemicals in suitable conditions and processing in multiple steps and repetition of some steps may help to extract NBF that would be useable in textile and apparel applications.

Although many studies have been performed to extract NBF, only a few studies attempted a comparative study on extracting fibers from different bamboo species. Phyllostachys edulis/pubescens/heterocycle (Moso bamboo), Bambusa emeiensis, Bambusa stenostachyum,

Bambusa stenostachyum, Bambusa beecheyama and Dendrocalamus latiflorus are among the different species considered in different studies (Ibrahim et al., 2015; L. Liu et al., 2012; Tran,

Nguyen, Thuc, & Dang, 2013; Waite, 2010; Witayakran et al., 2013). But none of the experiments compared more than two species in a single study. This study includes a comparative study on NBF extraction of four widely available bamboo species and assessment of yarn and fiber properties.

2.3 Methodology

2.3.1 Materials

Four bamboo species of 1—4 years old Bissetii (Phyllostachys Bissetii), Giant Gray

(Phyllostachys nigra 'henon'), Moso (Phyllostachys edulis/ pubescens) and Red Margin

(Phyllostachys rubromarginata) were provided by Lewis Bamboo, Inc., Oakman, Alabama. Raw cotton fibers directly from the field were provided by Alabama Cooperative Extension System

(Alabama A&M and Auburn Universities). Chemicals collected from Sigma-Aldrich, USA were:

sodium hydroxide/NaOH (beads, AG*), sodium bicarbonate/NaHCO3 (AG), sodium silicate/Na2SiO3 (RG*), sodium triphosphate/Na5P3O10 (85%, TG*), sodium sulfite/Na2SO3 (≥

98%), acetic acid/CH3COOH (≥ 99%, AG), buffer solution/ [CH3COOH+ CH3COONa] (AG, pH

= 4.6), and zinc nitrate hexahydrate/[Zn(NO3)2•6H2O] (98%, RG). Chemicals provided by VWR

International, LLC, USA: hydrogen peroxide/H2O2 (30% w/w, AG), and sulfuric acid/H2SO4

(95-98%, AG). Chemicals and solutions provided by Consos, Inc. USA: textile softener (Consoft

GPS), scouring agent (Consoscour GSRM), wetting/penetrating agent (Consamine JDA), and bleaching agent (Consobleach CIL). Fabric washing softener was collected from local Publix

Super Market, Inc., USA (Free & Clear Ultra Fabric Softener).

Note: *AG = ACS (American Chemical Society) grade; *RG = reagent grade; *TG = technical grade.

2.3.2 Apparatus

The major apparatuses used in the study were milling machine/rolling mill (Rolling Mill

Division, Stanat Manufacturing Co. Inc., NY, USA), Reactor and reactor controller (Parr

Instrument Company), Launderometer (AATCC Launder-Ometer, Atlas material testing

Technology LLC), micro-analytical balance (Mettler Toledo 0.00001g XP105DR Dual Range

Semi Micro Analytical Balance Scale), Dryer (GCA corporation, Thelco Precision Scientific

Laboratory Oven, Model 18), conditioning cabinet/Temperature and Humidity Environmental

Test Chamber (Associated Environmental Chamber), Motorized Drum Carder (DCM-405,

Strauch Fiber Equipment Co.), tensile tester (MTS Qtest 25), Ion sputter coater (Leica, MCM-

200 Ion Sputter Coater), SEM (Apreo S HiVac, Thermo Fisher Scientific), and EDS/EDX (JEOL

JSM-7000F, Field Emission Scanning Electron Microscope).

2.3.3 Initial Treatments

All of the samples were processed by splitting of the fresh bamboo culms by 1st crushing,

nd soaking in either water, NaOH or H2SO4 for 3—5 days at room temperature (RT), and 2 17

crushing, and combing with steel combs of various teeth density. Acid soaking with 2 mL/L of

H2SO4 (95—98%) at room temperature for 1-5 days proved to have limited potential. Therefore, the acid step was discarded and alkali soaking (Recipe 0, Table 2) was applied to all the approaches during initial processes. All four bamboo species samples were treated separately.

Pretreatments include mechanical and chemical processes (Figure 1).

2.3.4 Delignification

Table 2

List of all recipes used in different processes of natural bamboo fiber (NBF) extraction

(mL/L)

: L

(mL/L)

(2/g owm)

(g/L)

(% owm)(%

(g/L)

(mL/L)

(g/L)

(mL/L)

(Material (Material : Liquor)

Recipe NaOH NaHCO₃ ᵃH₂O₂ Na₅P₃O₁₀ Na₂SO₃ Na₂SiO₃ Zn(NO₃)₂ ᵇSoftener ᶜScour. agent ᵈBleaching agent (mL/L) ᵉpenetrant ball Steel Temperature X Time 0* 6 1 : 10 RT x 3-5 days 1 6 6 ------—20 90 °C X 2 h 2 20 ------90 °C X 3 h 3 6 - - 3 5 - - - - - 3 - 1 : 10 4 ------2 - - - - - 1 : 20 90 °C X 2 h 5 5 - - 20 20 20 ------6 6 ------3 - 1 : 15 7 6 6 4-6 ------90 °C X 2-3 h 8 4 4 4 ------1 : 10 90 °C X 3 h 9 6 - 6 ------10 10-20 - - 5-8 8-10 ------2 90 °C X 2.5 h 11 6 ------6 4 6 4 2 90 °C X 3.5 h 12 - - 6 - - - - - 3 3 4 - 1 : 15 13 3-5 - 4-9 - - - - 10-20 3-4 3-4 4-5 - 90 °C X 3-8 h 14 2-4 ------10 3 3 4 - 90 °C X 3.5 h 15 5-6 - - 10-20 10-15 10-20 ------90 °C X 6 h 16 6 6 6 - - - - 20 - - - - 1 : 30 90 °C X 3 h 17 4 4 4 - - - - 15 - - - - 1 : 15 18 3 6 6 - - - - - 3 3 4 - 90 °C X 6-7 h —20 19 5 - - 10 10 10 - 20 4 4 5 - 1 : 15 90 °C X 8 h 20 5 10 - 5 - 10 ------1 : 15 90 °C X 1-1.5 21 2 6 4-6 ------—40 h Notes: RT = room temperature (~21 ± 2)°C; h = hour; owm = on the weight of materials; *involves initial processes; ᵃ30% H₂O₂ ; ᵇFor 2-6 mL/L Consoft GPS and for 10-20 mL/L local fabric washing softener; ᶜConscour GSRM; ᵈConsobleach CIL; ᵉConsamine JDA.

Twenty-two different recipes were formulated as shown in Table 2. The M : L or material to solution ratios or liquor ratios (LR) were between 1:10 to 1:40 depending on the process, shape or volume of the fiber, size of the container, and effectiveness of the particular recipe. The setting of temperature and time for processing was varied to find out the better conditions. Either a reactor controller or Launderometer was employed for delignification by heat treatment using concentration. A list of the routes of selected samples and total use of NaOH for different formulae (Table 2). Different chemicals were chosen based on the literature review. Sodium hydroxide (NaOH) was most often used for delignification in identified studies and it was found

Fresh Raw bamboo culms Red Margin (Phyllostachys rubromarginata) Moso (Phyllostachys edulis) Giant Gray (Phyllostachys nigra henon) Bissetii (Phyllostachys bissetii)

Mechanical Pretreatments Splitting, scraping woody or noncellulosic parts, and crushing/ Soaking and/or heating: pressing H2SO4, NaOH, water, RT, drying, combing

Chemical: NaOH, Na2SiO3, Na2CO3, Chemical Pretreatments NaHCO3, [Zn(NO3)26H2O], Na5P3O10, Biological: water retting Na2SO3, wetting agent, softener, (3-7 days) liquor ratio (M:L), heat treatment, combing Delignification Bleaching: H O , NaOH, 2 2 Sorting, mixing, carding, Na CO , softener, scouring 2 3 combing agent, wetting agent, bleaching agent Modification

Removing residues & waste fibers Test & Analysis Fiber separation Mixing/blending with cotton with different ratios Spinning Blending (hand spinning) Carding, drawing, doubling Lap/Sliver preparation

Figure 1. a general flowchart of natural bamboo fiber (NBF)extraction from four bamboo species using varying conditions.

to be the most effective for this purpose. The selection of an appropriate concentration was extremely crucial, and the higher concentration was not always the best choice for NBF extraction. A higher concentration could have an adverse effect on fiber length and strength.

Proper LR, processing temperature and time, concentration of other chemicals, mechanical processes and manual techniques were also very important in the decision of NaOH each route is given in Table 3. It should be noted here that all four bamboo species samples were treated for each route separately to compare the suitability of NBF extraction and different properties.

Different NBFs produced from four bamboo samples were tracked using the first letter of each species (R = Red Margin; B = Bissetii; G = Giant Gray; and M = Moso) followed by numbers.

Each bamboo species was processed for 36 different NBFs extractions (say, R-1 to R-36; B-1 to

B-36). Therefore, all conditions were the same for a particular number with each bamboo initial.

For example, B-25, G-25, M-25, and R-25 NBFs were extracted from four bamboo species through the same route (Table 3). A general flowchart for all the formulae routes is presented in

Figure 1. The weight of each fiber sample was taken at the beginning and at the end of the route.

As shown in Table 3, only 47 samples out of 144 NBFs were chosen for continued assessments.

After each major treatment with a formula, each sample was subjected to water-washing, drying and discarding non-spinnable fibers (less than 1.5 cm long). Delignification by water retting for

3-7 days did not prove to be effective.

2.3.5 Modification

The commercial bleaching agent used for textile processing (Consobleach CIL) and/or hydrogen peroxide was included in different recipes and they were applied more than once in some cases.

When the bleaching agent or peroxide was applied more than one time for some routes, the

NBFs are found to be brighter and whiter. It was also observed that delignification was also

better in such cases. The modifications include the use of industrial textile softener and fabric softener to improve the softness of the NBFs. The addition of softener in different processes improved delignification but the strength of the fibers seemed to decrease as gauged by subjective assessment. Hand carding/combing was used to separate coarser fibers from their bundles as needed.

Table 3

Processing routes of the selected natural bamboo fiber (NBF) samples for different tests and use of NaOH in each route

Total Use of NaOH, Fiber Samples Route g/L* R-1 0 6 R-2 0→1→2 23 R-3 0→1→7 14 R-4 0→1→5 17 R-5 0→1→6 16 R-6 0→1→8 13 R-7 0→1→3 16 R-8 0→4→3 12 R-9 0→2 18 R-10, G-10 0→2→6 24 R-11 0→2→6→7→10 38 R-12 0→4→9 10 G-13 0→3 12 R-14, M-14, G-14 0→3→11 18 R-15 0→11→17 20 R-16, M-16, G-16, B-16 0→10 16-26 R-17 0→1 10 R-18 0→12→16 18 R-19 0→13→16 21 R-20 0→14→16 20 R-21, M-21, G-21, B-21 0→15 11 R-22, M-22, G-22, B-22 0→19 11 M-23, G-23, B-23 0→19→18 14 R-24 0→19→18→14 18 R-25, M-25, G-25, B-25 0→19→18→21 16 R-26 0→20→18→14 18 R-27 0→20→18→21 16 M-28, G-28, B-28 0→15→17 19 *The amounts are converted for an M : L of 1 : 15. In the Table “R” represents Red Margin bamboo; similarly, B = Bissetii; G = Giant Gray; M = Moso bamboo.

2.3.6 Final Treatments

For each of the NBF samples, washing with either warm or cold water, neutralizing with 1—2 mL/L of H2SO4 (95—98%) at 60—80 °C for 30 minutes, and drying were carried out. The fibers were not neutralized until the last step of the NBF production.

2.3.7 Spinning

Since all NBFs from Bissetii, Giant Gray and Moso had a wide variety of length distribution, only 3 NBFs (R-23, R-25, and R-27) from Red Margin bamboo were chosen for blending with raw cotton fibers (RCF). These 3 samples were blended with cotton with different ratios 30%,

40%, 50%, and 60%. The samples were mixed by hand and then carding was done twice to mix and align the fibers uniformly with a motorized carder to form slivers of 11-15 cm (4.4"-6") width and 91 cm long (36"). Very coarse, dust, short, damaged and rough fibers were cleaned and discarded during carding. The NBFs were coarser than RCF and volume of the same mass of

NBFs was lower, that is, the density of bamboo fibers was higher than that of cotton. Since mixing of fibers is not highly uniform, a coarser count and 2-ply yarn/thread was formed after hand-spinning of single-ply yarns.

2.3.8 Tests and Assessments

Unless mentioned, all the samples were prepared by preconditioning at standard atmosphere

[temperature (20±2) °C/ (68±4) °F; relative humidity = (65±5) %] as instructed in ASTM D1776 using a conditioning cabinet (ASTM D1776/D1776M−15, 2015).

Measuring Moisture Regain and Content (%)

For moisture regain and content percentages measurement, two similar methods, AATCC TM

20A-2017 and ASTM D629−15 were followed (AATCC 20A-2017, 2018; ASTM D629−15,

2015). By measuring conditioned (wet) weight (푊푤) and oven-dry weight (푊푑) of the

fibers/fabrics/bamboo, moisture regain percentage (푀푅) and moisture content percentage (푀퐶) were calculated by using equation (i) and (ii). The process was repeated at least two times to acquire consistent values.

퐴푚표푢푛푡 푤푎푡푒푟 푊푤 − 푊푑 푀퐶 = × 100% = × 100% … … … … (푖) 푊푡. 표푓 푚푎푡푒푟푖푎푙 푏푒푓표푟푒 표푣푒푛 푑푟푦푖푛푔 푊푤

퐴푚표푢푛푡 푤푎푡푒푟 푊푤 − 푊푑 푀푅 = × 100% = × 100% … … … … (푖푖) 푊푡. 표푓 푚푎푡푒푟푖푎푙 푎푓푡푒푟 표푣푒푛 푑푟푦푖푛푔 푊푑

Burning test

The incineration behavior of the raw bamboo and NBFs were tested by the burning test as

AATCC TM 20-2013 (AATCC TM 20-2013, 2018). For this test, a small tuft of the sample (0.1-

2.0 g) was moved by tweezers close to the flame for melting or shrinking, moving to flame for burning with or without flame, removing from flame for continuous burning or afterglow, and blowing out the flame for burning, smelling, color, odor and ash properties were observed.

Solubility test

For the solubility test, all samples were tested in test-tubes with 59.5% H2SO4 at 20 °C for 20 min and 70 % H2SO4 at 38 °C for 20 min. For the test, 0.1 g fiber sample was first taken into a test tube and 10 mL of sulfuric acid solution was added. The sample was shaken and allowed to set 20 min before observations. This test is AATCC method 20-2013 (AATCC TM 20-2013,

2018). Only H2SO4 was chosen as the fibers were mainly cellulose-based. The test was repeated twice to check the consistency of the measurement.

Microscopy for Morphology and Diameter

The diameter of the single fibers rather than the average diameter of NBF fiber-bundles samples were observed. An ion sputter coater was used for gold-coating on fiber samples before taking images and diameter measurement in SEM (Apreo S) and/or EDS (JEOL 7000) by applying 2

kV and 5 kV acceleration voltages respectively. The main purpose of the observation was to identify the diameter of the natural fibers from different bamboo plants. Since all fibers were circular or nearly circular in cross-section, at least 100 fibers of each type were measured for average calculation as directed by standard AATCC and ASTM methods (AATCC 20A-2017,

2018; ASTM D629−15, 2015).

Measuring Fiber Length and Fiber Yield (%)

An average length of each fiber sample was measured manually. For this, a small tuft of fiber was taken after mixing all fibers uniformly of a specimen and at least 100 fibers were chosen to measure length by using a line gauge. The average length and standard deviation were calculated for each sample. The fibers with a length smaller than 0.5 cm were discarded (not measured).

Any fiber above 1.5 cm was considered as spinnable.

The yield percentage was defined as the amount of cellulose-rich fibrous materials that was extracted from raw bamboo samples by measuring oven-dry weight in each of the cases. The oven-dry weight of raw bamboo or the specimen at the beginning of the step-process (푊푏) and the oven-dry weight of the finished fiber at the end of the process (푊푓) were measured to calculate the percentage of fiber yield (퐹푌) by using equation (iii).

푊푏 − 푊푓 퐹푌 = × 100% … … … … … … … … … … … … . . (푖푖푖) 푊푏

Measuring Fiber Fineness

A micro-analytical balance was employed to measure microgram (μg) mass of fibers. Since the balance was not highly sensitive to small masses, several fibers were weighted at a time and total length of the fibers was used as the length for that particular mass. Then fineness was measured in terms of Tex (direct count) and Ne (indirect/cotton/English count) where Tex is a gram mass of fiber/yarn per 1000 m length and Ne is a number of hanks (1 hank = 840 yards) of 1 lb

fiber/yarn. The conversion was calculated as mentioned in ASTM D6587 (ASTM D6587−12,

2012).

Testing Tensile Properties

Though ASTM D2256M was employed for measuring tensile properties of selected longer NBF fibers and yarns by using MTS Qtest tensile tester, there were some changes in the procedure and parameters (ASTM D2256/D2256M, 2015). A constant rate of elongation was applied with a crosshead speed of 10-30 cm/min for yarns and 10 mm/min for fibers to maintain minimum time-to-break no less than 20 seconds. The gauge length or fiber length was measured from grip to grip point of the tester and the length was 25 cm for yarns and 20 cm for fibers. Different load cells were chosen for fiber (500 lb) and yarns (5 or 500 lb).

2.4 Results and Discussions

2.4.1 Discussions on Natural Bamboo Fibers Extraction

Bissetii was the smallest bamboo plant among the four species when considering length and diameter (Table 4). The Red Margin bamboo culms had the greatest length and highest moisture content. The length and diameter did not have a direct relationship with extraction processes, however, the fresh culms with higher moisture proved to be easier in splitting, crushing and

Table 4

Moisture/water content (%) of culms from four fresh bamboo plants Bamboo Length of culms, The diameter of Moisture content Dry weight plant cm culms, cm (%) (%) Bissetii 16.5—25.4 1.5—2.3 32.16 67.84 Giant Gray 18.4—27.9 4.8—5.1 23.65 76.35 Moso 17.3—30.5 1.8—5.3 30.35 69.65 Red Margin 31.8—43.8 2.5—3.6 37.91 62.09 immediate combing. In this respect, the Giant Gray was inferior among the 4 species in preprocessing. After splitting, the length of the Giant Gray fibers gradually decreased as it went

through different extraction processes. Again, the rate of breakage and shorter fiber production were higher for the culms with lower moisture content. Therefore, as the extraction processes

Raw Bamboo (Red Margin) B-23 (from Bissetii) G-23 (from Giant Gray

M-23 (from Moso) R-9 (from Red Margin) R-23 (from Red Margin)

R-25 (from Red Margin) lap/sliver NBF/cotton (40/60) Hand spun yarn NBF/cotton (50/50)

Figure 2. Photographs of the natural bamboo fibers (NBFs) from four bamboo plants and hand- carded yarn blended with cotton fiber.

progressed in a particular route, Bissetii, Giant Gray and Moso produced shorter fibers than that of Red Margin and the NBFs were also more inconsistent in these 3 species (Figure 2). For example, though extracted through the same route, the NBFs B-23, G-23, and M-23 were almost the same (coarser, shorter, rough fibers) but R-23 was finer, longer, softer and had more consistent fiber (Figure 2). It was also possible to produce long (~33 cm) and flax-like fiber (R-

9) from the Red Margin. A shiny, highly uniform, spinnable fiber was also extracted from Red

Margin as shown R-25 in Figure 2.

2.5 Moisture Regain and Content

Figure 3. Comparison of moisture regain (%) and moisture content (%) of conventional fibers, bamboo, and natural fibers. [Note: BV = bamboo viscose; 70% BVC = 70% BV and 30% cotton;

95% BVS = 95% BV and 5% spandex; 70% BVCS = 70% BV, 25% cotton and 5% spandex;

40% BVCS = 40% BV, 55% cotton and 5% spandex; RF = 100% regular rayon fabric; UCF = untreated 100% cotton fabric; RCF = 100% raw cotton fiber; R, M, G and B = natural bamboo fibers from Red Margin, Moso, Giant Gray and Bissetii bamboo plants.]

MR% or MC% affects comfortability and mechanical properties of the fibers. A fabric with higher MR% is more comfortable. Moreover, a higher MR% decreases the flammability of the materials and promotes some processes such as scouring, bleaching and dye absorption.

Bamboo fiber had a round shape and there are many void spaces in-between where the fibers are

packed together. These voids promote a larger amount of moisture retention showing higher content. Bamboo viscose and conventional viscose both are reported to be more moisture absorbent than tencel (Waite, 2010). Figure 3 shows a comparison of moisture regain percentages (MR%) and content percentages (MC%) of some conventional fibers, bamboo strips, and NBF fibers. It was found that rayon and bamboo viscose were similar and the highest average MR% (~11%) and MC% (~10%) among all samples. All four bamboo dried strips and

NBFs showed similar values of MR% and MC% among themselves but higher than raw or bleached cotton fibers. The raw dried bamboo plants showed an average of about 8% MR%

(8.16%) and of about 7.5 MC% (7.55%). Similarly, the NBFs showed an average of about 8%

MR% (8.02%) and of about 7.5 MC% (7.42%). Though the moisture content of fresh bamboo plants had different values, dried bamboo plants and NBFs showed similar values.

2.6 Incineration Behavior (Burning Test)

Table 5

Burning tests and observations of different conventional fibers, raw bamboo, and natural bamboo fiber (NBF) samples

Sample Near Flame in flame removed from flame odor ash/residue black feathery ash burns quickly, continues to burn, Bamboo viscose with some white yellow flame afterglow spots burns the Burns quickly, burning surface, continues to burn, black feathery ash Rayon yellow flame, lots paper ignites quickly slight afterglow mottled with grey of white smoke Untreated/ burns quickly, continues to burn, light and feathery, Raw cotton yellow flame afterglow grayish, no black fuses and melts and burns chemical Hard, black, round Polyester burns with difficulty shrinks away slowly smell bead Burns the Bissetii Raw burning Gray and slightly surface, paper black Red Margin Raw ignites slowly continues to burn for burns quickly, Burns the few moments and then Moso Raw yellow flame surface when stops, afterglow Biomass Black Giant Gray Raw very close, ash ignites slowly All NBF fibers Burns the burns quickly, either stops after few burning Dark-grayish to black from four surface, yellow flame, moments or continues paper or grayish to black bamboo plants ignites quickly white smoke to burn, afterglow 28

Incineration behavior of different conventional fibers along with bamboo and NBFs are summarized in Table 5. Almost all of the fibers ignite quickly near the flame and the surfaces were burnt gradually with yellow flames. Most of the fibers emitted white smoke during or after incineration. When removed from the flame, the fibers with coarser forms or a higher non- fibrous content stopped burning after a shorter period than the fibers with finer forms or with less non-fibrous content. There was afterglow in almost all cases. The odor of the burnt fibers smelt like burning paper or burning ashes of biomass. As the non-fibrous contents were lower or removed in NBFs, the ash/residue was black for coarser fibers to dark grayish and then to grayish in case of finer fibers. So, a higher amount of non-fibrous content provided a darker blackish appearance to the ashes. The weight of the ashes was higher for the fibers with a greater diameter or higher non-fibrous content.

2.7 The Solubility of Bamboo and NBF

The observations on the solubility of bamboo viscose, rayon, cotton, polyester, dried raw bamboo specimens, and NBFs in sulfuric acid are summarized in Table 6. Though bamboo

Table 6

Solubility test of different conventional fibers, raw bamboo and natural bamboo fiber (NBF) samples in sulfuric acid

Sample 59.5 % H2SO4×20 °C×20' 70 % H2SO4×38 °C×20' Bamboo viscose Completely soluble Dissolved completely Rayon Cotton Mostly soluble with some small particles Dissolved completely Polyester Completely insoluble Completely insoluble Red Margin Raw Moso Raw Partially dissolved forming darker black/brown solution and Partially dissolved, black undissolved mass Giant Gray Raw turn the masses into dark black and brown solution Bissetii Raw Slightly dissolved and turned the mass into black and Mostly dissolved but there was a little mass All NBFs formed black solution left undissolved, darker black solution viscose and rayon dissolved completely in 59.5% and 70% H2SO4 solutions, none of the raw bamboo specimens or NBFs were completely dissolved. There was always black/dark brown

mass left undissolved with a black/dark brown solution for raw bamboo and NBF specimens.

Such behaviors were also unlike raw or bleached cotton and polyester.

2.8 Microscopic Analyses

2.8.1 The diameter of the Single Natural Bamboo Fibers

Mean, maximum and minimum diameters of the single bamboo fibers from four bamboo plants are represented in Figure 4. Though microscopic images showed some fibers were in bundle form, diameters of the single fibers were observed to identify standard values for bamboo fibers.

22 20 R-9 R-27 M-22

18 17.17

G-23 B-23 20.7

16 14.5

14 13.10

21.55

12.25 11.42

12 10.45 10.41 10 9.51

5.67

5.16 4.71

6 4.54 4.04 4 2 0 Mean diameter (μm) Maximum diameter (μm) Minimum diameter (μm) Figure 4. The diameter of the single natural bamboo fibers from four bamboo plants.

It was found that fibers from Moso were larger in diameter than that of other plants. The average values for Moso were within 10—13 μm. These values were consistent with other observations of 4-20 μm (Afrin et al., 2014; Komuraiah et al., 2014; Rocky & Thompson, 2018c; Steffen et al., 2013). Though there was a wide variety in maximum diameters, minimum diameters were within 4-6 μm. The standard deviation was between 2.00-4.73 μm for all observations.

2.8.2 Morphology

When raw bamboo strips were analyzed as in Figure 5 (a-d), it was found that all four bamboo species have similar structures where fibers are bonded and covered by lignin

(a) Raw Bissetii (1000X) (b) Raw Giant Gray (1000X) (c) Raw Moso (1000X)

(d) Raw Red Margin (1000X) (e) B-23 NBF (2000X) (f) G-23 NBF (1000X)

(g) M-22 NBF (2000X) (h) R-9 NBF (950X) (i) R-25 NBF (2000X)

Figure 5. SEM images of four raw bamboo strips and natural bamboo fibers (NBFs) taken by

JEOL 7000 (a—d, h) and Apreo S (e—g, i).

and other non-fibrous materials. However, investigation of chemical compositions would confirm if they have similar composition and structures. When fibers were extracted by removing lignin and other non-fibrous elements as shown in Figure 5 (e-i), NBFs are almost round or cylindrical in shape. The cross-section of the bamboo fibers were found to be round and circular solid in shape but some cross-sections had broken lines or cracks resulted from different

processes. It is also clear that there was an amount of non-fibrous materials in the NBFs (Figure

5g-5i) that can provide some natural properties of bamboo plants.

2.9 Fiber Length and Yield Percentage

Since 36 different routes were executed for NBFs extraction from four bamboo species, a list of selected fibers with their respective mean, maximum, minimum length along with fiber yield percentages are represented in Table 7. Since the different spans of fiber length can be selected for different purposes, such as spinning type, type of yarn (coarse or fine, based on spinning processes), composite application, and so on, this list can provide us with information for a prospective route for NBF extraction. Some NBFs in Table 7 had higher standard deviation values, meaning that fiber of such type had wide Gaussian distribution and would not be good for spinning of high-quality yarns. NBFs from Red Margin showed smaller standard deviation values which indicated that such fibers were more uniform. The longest NBFs produced after the

9th and 10th routes from Red Margin were 33.38±10.08 cm (13.14±3.97 inches) and 37.10±12.22 cm (14.61±4.81 inches) where fiber yield percentages were also very high: 77.08% and 70.14% respectively. Any use of such fibers would have excellent potential for textile production based on these measured factors. A moderate delignification process such as enzymatic or microbial may help to produce more uniform, strong and long NBFs. These fibers became finer in subsequent processing but lost length, strength, and uniformity along with fiber yield percentages, for example, R-11 (Table 7). NBFs with a prospect of textile uses had no less than

40% yield. NBFs extracted by routes 23rd and 25th were shorter but within the spinnable limit.

The lengths of NBFs (R-23 and M-23) and yield % (52.69% and 53.24%) were higher for Red

Margin and Moso than that of Giant Gray and Bissetii.

Table 7

Lengths and yield percentages of natural bamboo fibers (NBFs) of four bamboo species extracted through different techniques and routes

Min. length Max. length Avg. Length Total Fiber Yield, Total NaOH, Sample cm inch cm inch cm inch steps % g/L R-1 3.00 1.18 44.00 17.32 27.94±14.8 11±5.83 2 90.21 6 R-4 1.50 0.59 22.50 8.86 12.46±9.07 4.91±3.57 3 - 17 R-5 1.50 0.59 19.50 7.68 4.90±3.56 1.93±1.40 3 - 16 R-6 1.50 0.59 15.00 5.91 4.39±8.23 1.73±3.24 3 69.09 13 R-7 2.50 0.98 26.00 10.24 11.7±7.27 4.61±2.86 3 72.80 16 R-8 2.00 0.79 14.00 5.51 5.82±3.17 2.29±1.25 3 38.33 12 R-9 10.00 3.94 43.00 16.93 33.38±10.08 13.14±3.97 3 77.08 18 R-10 13.50 5.31 50.00 19.69 37.10±12.22 14.61±4.81 4 70.14 24 R-11 2.50 0.98 21.00 8.27 8.08±4.37 3.18±1.72 6 14.74 38 R-12 2.50 0.98 18.00 7.09 7.42±4.75 2.92±1.87 4 70.98 10 R-14 1.50 0.59 9.00 3.54 5.52±2.19 2.17±0.86 4 47.29 18 R-15 2.00 0.79 8.00 3.15 4.01±1.63 1.58±0.64 4 0.00 20 R-16 2.30 0.91 19.00 7.48 14.03±5.04 5.52±1.98 2 57.87 16 R-17 3.50 1.38 21.00 8.27 10.23±5.17 4.03±2.04 3 62.53 10 R-18 1.50 0.59 30.00 11.81 9.20±4.98 3.62±1.96 4 56.46 18 R-19 1.50 0.59 34.00 13.39 6.94±6.62 2.73±2.61 4 24.59 21 R-20 2.50 0.98 18.00 7.09 7.06±3.46 2.78±1.36 4 23.21 20 R-21 1.50 0.59 45.00 17.72 20.33±18.7 8.00±7.36 3 61.04 11 R-22 4.00 1.57 32.00 12.60 11.44±8.16 4.50±3.21 3 59.87 11 R-23 1.50 0.59 22.50 8.86 6.90±5.40 2.72±2.13 4 52.69 14 R-25 2.00 0.79 11.00 4.33 4.41±2.78 1.74±1.09 5 46.54 16 R-26 0.50 0.20 3.50 1.38 1.72±0.92 0.68±0.36 5 20.39 18 R-27 1.50 0.59 21.50 8.46 6.68±6.00 2.63±2.36 5 44.97 16 M-16 1.50 0.59 11.00 4.33 5.27±2.96 2.08±1.17 2 51.60 20 M-21 1.50 0.59 31.30 12.32 7.00±5.40 2.76±2.13 2 68.15 11 M-22 3.00 1.18 20.00 7.87 8.89±4.29 3.5±1.69 2 68.97 11 M-23 1.50 0.59 23.00 9.06 5.05±5.22 1.99±2.06 3 53.24 14 M-25 0.50 0.20 4.00 1.57 1.54±0.86 0.61±0.34 4 45.63 16 M-28 1.50 0.59 8.00 3.15 3.71±1.97 1.46±0.78 3 48.73 19 G-10 2.00 0.79 13.00 5.12 4.68±2.75 1.84±1.08 4 66.29 24 G-13 1.20 0.47 23.80 9.37 7.25±5.56 2.85±2.19 3.00 62.48 12 G-14 Almost all fibers were less than 1.5 cm 4 48.24 18 G-16 1.50 0.59 8.00 3.15 3.40±1.56 1.34±0.61 3 46.36 26 G-21 1.50 0.59 12.00 4.72 3.72±2.77 1.46±1.09 3 62.26 11 G-22 1.90 0.75 14.50 5.71 5.56±3.94 2.19±1.55 3 56.00 11 G-23 1.00 0.39 6.50 2.56 2.83±1.80 1.12±0.71 4 42.67 14 G-25 0.50 0.20 3.50 1.38 1.19±0.68 0.47±0.27 5 37.51 16 G-28 1.00 0.39 5.00 1.97 1.60±0.91 0.63±0.36 4 41.46 19 B-16 1.50 0.59 17.00 6.69 4.22±3.33 1.66±1.31 2 40.82 26 B-21 1.50 0.59 4.50 1.77 2.6±0.83 1.02±0.33 2 60.97 11 B-22 2.50 0.98 21.00 8.27 6.72±4.83 2.65±1.9 2 59.14 11 B-23 1.50 0.59 6.00 2.36 3.57±1.51 1.41±0.59 3 43.06 14 B-25 0.50 0.20 4.00 1.57 1.66±0.87 0.66±0.34 4 35.74 16 B-28 0.50 0.20 3.00 1.18 1.52±0.83 0.6±0.33 3 41.34 19

NBFs from Red Margin was overall better in length, uniformity and yield percentages.

Though the finest diameter fibers were extracted by the 25th route for all four bamboo plants, R-

25 from Red Margin was only spinnable with an average length of 4.41±2.78 cm (1.74±1.09 inches) and yield of 46.54%. Though the length of the NBFs was not directly related to the overall use of NaOH as shown in Figure 6, it was observed that high use of NaOH in a particular process would either damage the fibers or produce very short but non-uniform fibers. Since

Figure 6. The relationship among fiber lengths, yield percentages and total use of NaOH in natural bamboo fiber (NBF) extraction.

NaOH was applied in multiple steps and at different stages of the NBFs extraction, the NBFs treated with less overall NaOH performed better in length, strength, and uniformity, than NBFs with higher usage of NaOH in some cases. Yield percentages were weakly related to the overall application of NaOH such as a higher NaOH usage led to a lower yield of final fibers (Figure 6).

2.10 Fiber Fineness

Since the fiber fineness (linear density) was measured by manual methods using a microbalance with some difficulty, the NBF samples that appeared prospective for different uses were selected for assessments. A summary of fiber fineness of different NBFs in Tex and Ne is presented in

Table 8. Though listed linear densities of NBFs of 9.68—93.3 Tex are higher than cotton fibers of 0.05—0.20 Tex (Altaş & Kadoǧlu, 2006; Y. Liu, Thibodeaux, & Rodgers, 2014), they can be used for coarser yarns or can be modified into finer counts by further modifications. Moreover,

Table 8

The fineness of the natural bamboo fibers (NBFs) extracted from four bamboo plants Fiber count in Ne Fiber count in Tex Sample Average Maximum Minimum Average Maximum Minimum R-1 16.82±11.41 34.84 1.02 35.09±23.81 577.68 16.95 R-9 21.75±11.18 38.61 6.65 27.15±13.96 88.74 15.29 R-10 35.48±22.52 92.51 9.58 16.65±10.57 61.62 6.38 R-21 39.29±23.43 91.53 14.36 15.03±8.96 41.11 6.45 R-27 42.55±31.81 110.72 3.13 13.88±10.38 188.78 5.33 M-21 6.33±4.63 15.86 0.32 93.3±68.24 1825.40 37.22 M-22 17.4±16.82 49.21 0.73 33.94±32.81 803.84 12.00 M-25 32.47±25.64 74.56 2.95 18.19±14.36 200.40 7.92 G-21 35.17±25.90 83.65 6.58 16.79±12.37 89.75 7.06 G-23 46.68±34.34 138.40 12.42 12.65±9.31 47.53 4.27 G-25 61.03±45.36 119.29 9.84 9.68±7.19 60.00 4.95 B-16 27.24±25.15 78.15 0.71 21.68±20.02 836.25 7.56 B-23 28.66±24.13 73.81 3.36 20.61±17.35 175.69 8.00 B-25 32.68±17.00 61.33 8.27 18.07±9.40 71.43 9.63

most of the NBFs from Red Margin were longer than cotton fibers. So, they can be further modified at the expense of losing some length or they can be used as they are for specific purposes. It should also be mentioned that specialized machines for carding, combing and other processes were not employed for NBFs extraction. Hence it might be possible to extract even better fiber for fineness, length, strength, and spinnability with advanced specialized technologies. 35

2.11 Tensile Properties

The breaking tenacity of NBFs was found to be very high when the maximum length was retained (22—36 cm) with 28—37 Tex of linear density. For example, breaking tenacity of R-9 and R-10 was 138.63±56.80 N/Tex and 63.74±64.27 N/Tex respectively (Table 9). The NBFs exhibited a breaking elongation of 2.06—2.46% which is comparable with other studies of 1.4—

2.9% (L. Liu et al., 2011; Prasad & Rao, 2011; Sanjay et al., 2018; Steffen et al., 2013) but lower than cotton fibers 4—8% (Fiori, Sands, Little, & Grant, 1956; Yang & Gordon, 2016). It should

Table 9

Breaking tenacity and elongation of natural bamboo fibers and cotton-blended yarns

Breaking Composition Fineness Breaking tenacity elongation Sample Combination (M ± σ) (M ± σ) (M ± σ) (M ± σ) (M ± σ) %/% Ne Tex Kg/Tex N/Tex % NBF-1 R-9 100 22±11 37±19 141.31±57.90 138.63±56.80 2.46±0.52 NBF-2 R-10 100 36±23 28±19 64.97±65.51 63.74±64.27 2.06±0.76 SP yarn R-23/RCF 50/50 0.69±0.05 428±31 2.51±0.67 2.46±0.65 7.76±1.94 DP yarn 1 R-27/BCF 40/60 0.76±0.06 786±58 2.40±0.62 2.36±0.61 10.24±2.00 DP yarn 2 R-25/BCF 40/60 0.67±0.04 888±55 1.99±0.56 1.95±0.55 9.76±2.26 DP yarn 3 R-27/RCF 50/50 0.55±0.02 1066±43 2.29±0.54 2.24±0.53 10.78±1.44 DP yarn 4 R-23/RCF 50/50 0.69±0.05 855±61 2.13±0.46 2.09±0.45 10.89±2.53 DP yarn 5 R-25/BCF 60/40 0.75±0.04 795±43 1.62±0.33 1.58±0.32 8.51±1.15 DP yarn 6 R-25/BCF 70/30 0.56±0.02 1057±30 1.54±0.45 1.51±0.44 9.29±1.29 Note: R = natural bamboo fiber (NBF) from Red Margin; SP =single-ply; DP = double-ply; RCF/BCF = raw/bleached cotton fiber; M = mean value; σ = standard deviation. be noted that tensile properties of NBF of the other three species and shorter fibers were not determined as they were out of available machine capacity. But it was observed that breaking tenacity and elongation of the yarns decreased when the amount of NBF increased in the blend.

This means that the NBFs shared lower strength in the yarns than that of cotton fibers. Since the yarns were not produced by industrial techniques that would provide a higher number of twists, and uniform mixing, the current yarn might not convey all the potential of the fibers in yarn

usage. It was noticeable that yarns (7.76—10.89%) showed higher breaking elongations than

NBFs (2.06-2.46%) and double-ply yarns, such as DP yarn 4 with 10.89%, showed higher than single-ply (SP yarn with 7.76%) for similar blends (Table 9).

2.12 Conclusions

This study provides a list of different routes for natural bamboo fiber (NBF) extraction where total use of chemicals was within 6-38 g/L on the weight of initial raw bamboo throughout the particular route. The 25th formula was found to be most effective for extracting spinnable fibers, especially from Red Margin (R-25). The NBFs of this group were produced using overall 16 g/L of NaOH and the fibers were uniform, fine and spinnable with cotton blend. However, several routes were effective to yield of NBFs that were useable for textiles. The selection of the best route will depend on what kind of NBFs and their properties are required. Some routes were good for long but coarse fiber, some were good for fine but shorter fiber, some were good for retaining high strength, and some were only good for overall fiber yield. Different chemicals were studied where some chemicals were found to be very useful for NBF extraction such as

NaOH, NaHCO3, H2O2, and penetrating agent; some were moderately useful such as Na5P3O10,

Na2SO3, and Na2SiO3; and some were either not useful or used for neutralization such as

CH3COOH, H2SO4, and Zn(NO3)2. Yarns were produced by hand spinning after blending with cotton at different ratios. Advantages of extraction processes, moisture regain and content, incineration behavior, solubility, the effect of NaOH, length, diameter fiber yield, fineness, spinnability, and tensile properties were compared. Moisture regains and contents of the natural bamboo fibers (NBFs) were not found in the literature. So, this study on four bamboo species and NBFs provides some much needed information. Moisture regain of the NBFs was found to be higher than cotton; this is indicative of better comfortability in clothing. Similarly, reports on

diameter (10—13 μm), fiber yield for prospective NBFs (40-77%), breaking tenacity and elongation of NBFs and yarns in this study provide a baseline for future research. The analyses on the surface morphology of four bamboo species and their NBFs by two microscopic instruments (SEM and EDS) are also summarized where an explanation is provided why the

NBFs may have natural properties from bamboo plants. Moreover, this study provides a comparison of some properties such as moisture regain, moisture contents, incineration behavior, and solubility among bamboo, NBFs, bamboo viscose, cotton, and some conventional fibers.

Future research of more species, different age-group of species, introducing enzymes or microbes in intermediate steps of extraction, and developing special equipment and technologies specific to bamboo fiber extraction would be necessary to establish textiles from natural bamboo fibers.

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CHAPTER 3. CHARACTERIZATION OF THE CRYSTALLOGRAPHIC PROPERTIES

OF BAMBOO PLANTS, NATURAL AND VISCOSE FIBERS BY X-RAY

DIFFRACTION METHOD

3.1 Abstract

A comprehensive crystallographic investigation was performed by using X-ray (from cobalt source) diffraction (XRD) technique on different bamboo species, natural bamboo fibers (NBFs), commercial bamboo viscose products and different conventional fibers. Crystallinity indexes

(CI) were estimated as 61-67% of bamboo plants, 69-73% of NBFs, 35-40% of bamboo viscose and 77-80% of cotton fibers in this study. Results suggests that CI gradually increased during the delignification process to create NBFs up to a certain point and then decreased with further processes. Knowing this behavior informs decisions of the appropriate chemicals or enzymes for further modification processes and continuing to maintain expected strength of the fiber.

Therefore, delignifying raw bamboo increased the strength of the fibers until the maximum CI was achieved but further extraction of lignin reduced the strength of the NBF resulting in a higher number of fiber breakage and short fibers. Red Margin was found to have lower CI that hinted at easier NBF extraction. With overall crystallite size of 35-39 Å, four crystalline peaks were detected in all bamboo and NBF specimens as (101), (101̅), (002) and (040) at 17.0-

18.6°, 18.5-19.4°, ~25.5°, and ~40.5° respectively. Moreover, this study provides a list of lattice planes, interplanar spacings, reflection angles, and crystallite dimensions of the four targeted bamboo and NBF materials.

KEYWORDS: Bamboo Crystallinity; Crystallinity index; Crystal size; X-ray diffraction; Fiber lattice indices; Crystallinity of Fibers.

3.2 Introduction

Recent research interest in bamboo for fiber production is leading to investigations for identifying its structures and characteristics that can promote fiber extraction and modification processes. The major three components of bamboo are cellulose, hemicellulose, and lignin.

Cellulose and hemicellulose are naturally-occurring polymers, namely polysaccharides, of 1,4--

D glucopyranose (glucose) monomers containing crystalline (orderly) and amorphous

(disorderly) regions (Mohamed & Hassabo, 2015; Odian, 2004; Oudiani, Chaabouni, Msahli, &

Sakli, 2011). The presence of crystallinity in polymers determines properties, such as strength, elasticity, toughness, brittleness, stiffness, rigidity, chemical resistance, absorption, melting temperature, density, optical and dyeing property (Carraher, 2003; Chen, Yu, Zhang, & Lu,

2011; Hindeleh, 1980; Mohamed & Hassabo, 2015; Stana-Kleinschek, Ribitsch, Kreže, Sfiligoj-

Smole, & Peršin, 2003). Accordingly, knowing the degree of crystallinity or crystallinity index

(CI) of cellulose-based plants provide us with information about whether the material has the potential for fiber application and how it can be modified for suitable uses.

The CI is a very important distinct property that determines the uniqueness of a polymer or a fiber. It is a challenging and complex process to determine the CIs of cellulosic polymer materials (Ju, Bowden, Brown, & Zhang, 2015; Park, Baker, Himmel, Parilla, & Johnson, 2010).

Most of the polymers have a certain degree of crystallinity; only a very few of them have a CI of

100%. The CI of polymers generally ranges from 10-80% (Carraher, 2003; Ju et al., 2015). The

CI of natural fibers were reported between 30-90% (Sanjay et al., 2018). Unlike amorphous polymers, polymers with a certain CI have a definite melting point and glass transition temperature, and they display clear X-ray diffraction patterns. The crystallinity can be modified 45

or changed through production processes or by treatments (Carraher, 2003; Odian, 2004). The polymer structure or orientation and the intermolecular forces are two major factors responsible for crystal formation in polymers. The greater the secondary attraction forces, the higher the degree of ordering and therefore the crystallinity of a polymer (Carraher, 2003; Mohamed &

Hassabo, 2015; Odian, 2004).

There are several methods to consider when determining crystallinity of polymers such as differential scanning calorimetry (DSC), X-ray diffraction (XRD), solid-state nuclear magnetic resonance NMR, infrared (IR) spectroscopy, Raman spectroscopy, density measurement, and heat fusion (Carraher, 2003; Ju et al., 2015; Junior, Teixeira, & Tonoli, 2018; Moly, Radusch,

Androsh, Bhagawan, & Thomas, 2005; Odian, 2004; Park et al., 2010; Pe´rez & Mazeau, 2005;

Wada, Heux, & Sugiyama, 2004). Though DSC is most common, XRD, a non-destructive method, has recently been found to be the most direct, widely used and easier method to determine CI and crystal size of polymers. But it requires researchers to separate crystalline and amorphous phases from the diffracted graph in order to use it (Carraher, 2003; French & Cintrón,

2013; Hindeleh, 1980; Moly et al., 2005; Odian, 2004; Park et al., 2010).

The main purpose of this crystallographic study was to determine the degree of crystallinity (CI) and the crystallite sizes of four abundantly grown bamboo plants by X-ray diffraction (XRD) technique with Co K as an X-ray source instead of the routinely used Cu

K. Comparisons of the crystallographic properties such as crystal sizes, CIs, reflection planes, diffraction angles and interplanar distances among different bamboo plants, NBFs, cotton, and different commercial viscose fibers measured. This study also aimed to investigate Segal (Segal,

Creely, Martin, & Conrad, 1959), a frequently used method and deconvolution (Rotaru et al.,

2018), a more accurate but complex method for determining the CIs.

3.3 Methodology

3.3.1 Materials and machines

This study was conducted on four bamboo species of 2-4-year-old plants that are abundantly available: Red Margin (Phyllostachys rubromarginata), Moso (Phyllostachys edulis/ pubescens),

Giant Gray (Phyllostachys nigra henon), and Bissetii (Phyllostachys Bissetii) were collected from Lewis Bamboo, Inc., Alabama, USA. Sodium hydroxide (NaOH) beads, sodium bicarbonate (NaHCO3), sodium silicate (Na2SiO3), sodium triphosphate (85% Na5P3O10), and sodium sulfite (Na2SO3) were purchased from Sigma-Aldrich, USA. Hydrogen peroxide/H2O2

(30% w/w) was provided by VWR International, LLC, USA. A scouring agent (Consoscour

GSRM), bleaching agent (Consobleach CIL), and penetrating agent (Consamine JDA) were purchased from Consos, Inc. USA and fabric softener was collected from a local Publix Super

Market, Inc., USA.

The following machines and equipment were employed: Milling machine (Rolling Mill

Division, Stanat Manufacturing Co. Inc, NY.), Launderometer (AATCC Launder-Ometer, Atlas material testing Technology LLC), Dryer (Model 18, GCA corporation), Conditioning cabinet

(Associated Environmental Chamber), and X-ray diffractometer (D8 Discover, Bruker AXS) with Vantec 500 2D detector,

3.3.2 Extraction of Natural Bamboo Fibers (NBFs)

The details of the NBF extraction processes were documented in Part-1. A brief description is included here. Raw fresh/green bamboo culms were subjected to splitting, repeated pressing in a milling machine, steel-brushing, and removing short non-fibrous particles by washing and drying successively. Then samples were soaked in a 6 g/L NaOH solution with a material-to-liquor ratio

(M:L) of 1:10-20 at room temperature for 3-5 days. These soaked samples were washed, combed with a steel brush and dried in a dryer for 1 hour at 100-120 °C followed by conditioning in a 47

conditioning chamber. This yielded the initial fibers (R-1, B-1, G-1, and R-1) from each bamboo specimen, such as B-1 from Bissetii, G-1 Giant Gray, M-1 from Moso, and R-1 from Red

Margin bamboo.

The NBF R-9 was produced by using a high concentration solution of NaOH (20 g/L) with a liquor ratio (LR) of 1:10-20 at 90 °C for 3 hours followed by washing and drying. Initial fibers (R-1, and B-1) were treated with 10-20 g/L NaOH, 5-8 g/L Na5P3O10, and 8-10 g/L

Na2SO3 solution (LR = 1:15) at 90 °C for 2.5 hours followed by washing and drying to yield R-

16 and B-16. Similarly, B-1, G-1, M-1 and R-1 were treated with a 5-6 g/L NaOH, 10-20 g/L

Na5P3O10, 10-15 g/L Na2SO3, and 10-20 mL/L Na2SiO3 solution (LR = 1:15) at 90 °C for 6 h to yield G-21, M-21 and R-21. B-1, G-1, M-1 and R-1 were also treated with 5 g/L NaOH, 10 g/L

Na5P3O10, 10 g/L Na2SO3, 10 mL/L Na2SiO3, 4 mL/L scouring agent, 4 mL/L bleaching agent,

20 mL/L fabric softener, and 5 mL/L penetrating agent solution (LR = 1:15) at 90 °C for 8 h to yield B-22, G-22 and M-22. Another treatment of B-22, G-22 and M-22 with a 3 g/L NaOH, 6 g/L NaHCO3, 6 mL/L H2O2, 3 mL/L scouring agent, 3 mL/L bleaching agent, and 4 mL/L penetrating agent solution (LR = 1:15-20) at 90 °C for 6-7 h yielded B-23, G-23 and M-23.

Further treatment of B-23, G-23, M-23 and R-23 with a 2 g/L NaOH, 6 g/L NaHCO3, and 4-6 mL/L H2O2 solution (LR = 1:15-40) at 90 °C for 60-90 minutes yielded B-25, G-25, M-25 and

R-25. The NBF R-27 was extracted by treating R-1 with a solution of 5 g/L NaOH, 10 g/L

NaHCO3, 10 g/L Na5P3O10, and 10 mL/L Na2SiO3 (LR = 1:15) at 90 °C for 8 h, then final treatments of R-23 and R-25 were applied respectively. The details of recipes and routes are mentioned in part 1. All the heat treatments were executed in a Launderometer.

3.3.3 Crystallographic Analyses by XRD

Sample Preparation and Conditions

Raw bamboo specimens: A knife and a low-speed abrasive precision cutting machine with a rotary blade were used to make thin bamboo slices with dimensions of 2~3 cm long ×

1~2 cm wide × 1~3 mm. The exodermis and endodermis were removed to provide smooth surfaces where the distribution of fibers was uniform as shown in Figure 7. No polishing or metal-coating was applied to strip samples. Both sides of the bamboo strips were used for scanning in all analysis. Natural bamboo fiber (NBF) samples were prepared by gold-coating in an ion sputter coater. All the specimens were stored in a desiccator before using in a spectrometer.

Figure 7: Bamboo sliced strips prepared for X-ray diffractometry in Bruker AXS.

In XRD, for the raw bamboo samples, the bamboo culms were dried and cut into small square or rectangular thin strips as mentioned above. Different fabrics were directly used as a single-layered or a double-layered specimen to scan in the diffractometer. The NBFs and other fiber samples were cut into very small pieces, ground in a grinder into the powder form as much as possible, and collected by using a multiple layered micro-sieve containing 80, 120, 170, and

230 phosphor-bronzed meshes. The ground fibers were placed in a specimen holder in the diffractometer. The X-ray from cobalt source (Co K1 and K2 x-rays) with wavelengths of 49

1.7890 Å was run at a beam voltage of 40 kV and a filament current of 35 mA using wide-angle

X-ray diffraction (WAXD) method. The XRD data collection time was 4-15 minutes with 360- degree rotation around Z-axis between 4-96° scanning angle (2θ).

Determination of Crystallinity Index (CI)

The CIs of the four bamboo plants were determined by separating the crystalline and amorphous phases from the diffracted graph (Figure 8). It should be noted that copper is mostly used as an

X-ray source for fibers or polymers that has a lower wavelength (1.5406 Å). Therefore, it is expected that cobalt X-ray source with a higher wavelength would give bigger reflection angle in the diffraction patterns. There are two methods that are reported to determine the degree of crystallinity or crystallinity index (CI): 1) Segal method by using the height of the crystalline and amorphous peaks, and 2) deconvolution method by calculating the area under crystalline and amorphous peaks. The Segal method is direct and makes it easier to find CI by using equation

(2).

퐼 − 퐼 퐶퐼 = 002 푎푚 × 100% … … … … . (2) 퐼002

Where 퐼002 = the maximum intensity of the peak on 002 plane, and Iam = the minimum height of the amorphous phases of the diffracted beam between (002) and (101) planes (Afrin,

Kanwar, Wang, & Tsuzuki, 2014; French & Cintrón, 2013; Ju et al., 2015; Lionetto, Del Sole,

Cannoletta, Vasapollo, & Maffezzoli, 2012; Morais et al., 2013; Nam, French, Condon, &

Concha, 2016; Oudiani et al., 2011; Park et al., 2010; Rambo & Ferreira, 2015; Segal et al.,

1959). The values of 퐼002 and Iam were measured as shown in Figure 8. The deconvolution

Figure 8. Deconvolution method applied to the X-ray (from Cobalt source, Co K) diffraction pattern of natural bamboo fiber (NBF) M-25 from Moso bamboo sample by using FitPeak processing.

method is reported as more accurate but requires complex work to fit the peaks to calculate areas under crystalline and amorphous peaks with careful data processing. Both of these methods can be used to compare values obtained from each technique. Equation (3) is used to estimate CI in method 2.

퐴 푆 퐶퐼 = 푐푟 × 100% = [1 − 푎푚 ] × 100% … … … . (3) 퐴푐푟 + 퐴푎푚 푆푐푟 + 푆푎푚

Here, 퐴푐푟 = the integrated area of the crystalline phase/peak; 퐴푎푚 = the integrated area of the amorphous phase/peak; 푆푐푟 푎푛푑 푆푎푚 are the sum of the area of the crystalline peaks and 51

amorphous regions respectively (French & Cintrón, 2013; Junior et al., 2018; Malenab, Ngo, &

Promentilla, 2017; Park et al., 2010; Rambo & Ferreira, 2015; Rotaru et al., 2018; Segal et al.,

1959).

The Segal method was applied to the raw data and to the data after subtracting the background. The deconvolution method was used in Origin Pro 2018 64 bit to the XRD data for

CIs estimation. In this method, crystalline and amorphous regions were separated by utilizing maximum data fitting in the XRD spectrum as mentioned in the literature (Garvey, Parker, &

Simon, 2005; Ju et al., 2015; Junior et al., 2018; Malenab et al., 2017; Park et al., 2010; Polizzi,

Fagherazzi, Benedetti, Battagliarin, & Asano, 1990). Both Gaussian and Pseudo-Voigt 2 functions were applied to the XRD spectrograms and the best shape fitting function was chosen using least square fitting. The XRD raw data were treated with the maximum number of iterations to achieve the Coefficient of determination (COD), 푅2. The regression predictions were achieved above 97% (>97%) fit with raw data in all the cases after creating a baseline by subtracting the background as shown in Figure 8. The amorphous peak was assigned between

24-26° 2θ-angle as it fitted well within that range.

Determination of Crystallite Size (푫ퟎퟎퟐ)

If D002 = grain diameter or crystallite size using diffraction pattern on 002 lattice plane, β002 = the true peak broadening at full-width half maximum (FWHM) in radian of the peak on the (002) plane where instrumental broadening may need to be considered, θ = Bragg’s angle or the angle of the diffracted beam, and λ = wavelength of the X-ray source, then equation (4) can be used to determine the crystallite size of a polymer.

푘휆 퐷002 = … … … … (4) 훽002 푐표푠휃

Where k is a dimensionless number with a value of around unity, also called Scherrer constant. It depends on crystal shape, FWHM, and crystal orientation. The value of 푘 generally varies from

0.8 to 1.0 for polymer and cellulosic materials (Afrin et al., 2014; French & Cintrón, 2013; Hui

& Li, 2016; Kılınç et al., 2018; Langford & Wilson, 1978; Lionetto et al., 2012; Nam et al.,

2016; Tan, Hamid, & Lai, 2015; Yueping et al., 2010). The instrumental broadening was zero in this experiment. In this study, the values of k = 1 and λ = 1.7890 Å (Co Kα1) were taken for calculations.

3.4 Results and Discussion

3.5 Crystallographic Analyses

3.5.1 Crystallinity Indexes (CIs), Diffraction Angles, and Lattice Planes

The CIs were estimated as 63-66% and 70-72% in the Segal method and, 61-67% and 69-73% in deconvolution method for all four bamboo species and NBFs respectively (Table 10). In 4-8 different XRD scans on different samples of a certain species and NBFs, the CIs were very close providing <2% standard deviations. This was also the same for the two methods. This provides us with the conclusion that the selected four bamboo plants and NBFs had the CIs between 63-

67% and 69-73% which were consistent with both methods (Table 10). The reported CIs for bamboo and its natural fiber were reported as 52-89% (Afrin et al., 2014; He, Cui, & Wang,

2008; Junior et al., 2018; Y. Xu, Lu, & Tang, 2007; Yueping et al., 2010). Since a large fraction of other compounds are grown along with fibers in the bamboo plant and the crystalline orientation is not freely developed, Yueping et al. (2010) stated that the CI of bamboo fiber

(52.54%) was lower than cotton, ramie, and flax which is in good agreement with this study

(Yueping et al., 2010). Moreover, a higher content of non-crystal lignin compounds reduces the crystallinity of the cellulosic materials (Wang, Cui, & Zhang, 2012). Summarized in Table 10, is the investigation of the crystallinity of raw and mildly bleached 100% cotton fiber and fabric 53

(76-79%), several bamboo viscose products (35-40%), conventional rayon (40%), cotton/bamboo viscose blend (63-66%) and polyester (62-65%). This comparison revealed that intact bamboo and NBFs have a lower CI than cotton but a higher CI than all kinds of viscose products. It was also noticed that bamboo viscose and conventional rayon showed similar XRD patterns with close CI values. Understandably, specimen-BB showed a higher CI than bamboo viscose but a lower value than cotton as it was a blend of cotton and viscose (Table 10).

Table 10

Estimated crystallinity indexes (CIs) of different bamboo species, NBFs, bamboo viscose products, and conventional fibers by using Segal and Deconvolution methods on X-ray diffraction data

Segal Method, Deconvolution Sample Description CI% Method, CI% Bissetii Raw Bamboo 66.4 66.67 B-25 NBF: 33 Ne/17.9 Tex 69.54 71.24 Giant Gray Raw Bamboo 62.56 63.51 G-25 NBF: 61 Ne/9.7 Tex 72.2 69.06 Moso Raw Bamboo 62.97 64.3 M-25 NBF: 33 Ne/23.6 Tex 70.79 69.92 Red Margin Raw Bamboo 62.65 60.8 R-25 NBF: 43 Ne/13.7 Tex 72.09 73.2 RCF 100% Raw cotton fiber 78.9 76.51 BCF Mildly bleached cotton (100%) fiber 77.89 75.88 UC Untreated (sized) 100% cotton fabric 78.02 79.16 BV 100% Bamboo viscose 38.67 35.72 Yala Commercial 100% bamboo viscose apparel 40.41 35.33 CR Conventional 100% rayon fabric 39.79 40.15 Commercial (95% bamboo viscose, 5% Piko 40 37.91 spandex) apparel Commercial (70% bamboo viscose, 30% BB 65.73 62.87 cotton) apparel Polyester Polyester fiber 61.73 65.47

Three peaks were clearly found in the diffraction graphs of all bamboo and NBF specimens at 18-20°, ~25.5° and ~40.5° angles as shown in Figure 8 and Figure 9. In literature,

the reflection on (101), (101̅), (002)and (040) lattice planes were reported for cellulose-based materials (Chen et al., 2011; Ju et al., 2015; Malenab et al., 2017; Nam et al., 2016; Oudiani et al., 2011; Polizzi et al., 1990). Through peak fitting analysis, it was found that the peak at 18-20° was a combined peak of two peaks of (101), (101̅) planes at around 17.0-18.6° and 18.5-19.4°.

These two peaks were overlapped in most of the scans and they were treated as a single peak to

Figure 9. X-ray diffraction patterns of four bamboo species showing different peaks for determining the crystallinity indexes and the crystal sizes.

Table 11

Size of the crystallites, reflection planes, diffraction angles, and interplanar distances of four bamboo species and natural bamboo fibers (NBFs) from X-ray (Co K source) diffraction technique Lattice plane Diffraction angle, Interplanar Crystallite Sample (hkl) 2θ (°) distance (Å) Dimension (Å) 101 17.6 5.85 30.87 101̅ 19.2 5.37 30.49 Bissetii 002 25.6 4.04 36.50 040 40.6 2.58 51.79 101 17.7 5.80 29.11 101̅ 19.0 5.41 29.19 B-25 (NFB from Bissetii) 002 25.6 4.04 37.22 040 40.5 2.58 52.64 101 17.1 6.01 27.86 101̅ 18.8 5.49 28.00 Giant Gray 002 25.7 4.03 34.86 040 40.5 2.58 51.78 101 17.7 5.81 29.42 G-25 (NFB from Giant 101̅ 18.6 5.55 30.69 gray) 002 25.7 4.02 36.88 040 40.0 2.61 43.12 101 17.6 5.84 29.31 101̅ 18.5 5.56 29.73 Moso 002 25.5 4.05 34.92 040 40.5 2.59 61.03 101 17.5 5.90 32.06 101̅ 19.0 5.41 32.13 M-25 (NFB from Moso) 002 25.5 4.05 38.64 040 40.5 2.59 56.90 101 17.0 6.06 30.13 101̅ 19.2 5.37 29.37 Red Margin 002 25.7 4.03 37.28 040 40.6 2.58 33.94 101 18.6 5.53 32.87 R-25 (NFB from Red 101̅ 19.4 5.31 33.01 Margin) 002 25.5 4.06 38.49 040 40.5 2.58 52.03 101 17.3 5.96 44.31 101̅ 19.1 5.38 52.77 Cotton (Untreated 100% 002 26.4 3.92 69.72 cotton fabric) 040 39.6 2.64 107.87 021 24.4 4.24 90.40 estimate the crystallinity index. Thus, a thorough analysis of data in this study showed that the lattice planes (101), (101̅), (002) and (040) were detected at 17.0-18.6°, 18.5-19.4°, ~25.5° and

~40.5° respectively (Table 11). However, the most intense crystalline peak was at ~25.5° on the

(002) lattice plane for all the samples. Most of the reported investigations also found such clear peaks within different scanning angles (2θ) (Afrin et al., 2014; Lionetto et al., 2012; Yueping et al., 2010). The peaks on planes (101), (101̅), (002) and (040) are reported at 14-15.5°, 16-17°,

21.7-23° and 34.0-35.1° respectively when Cu K X-ray source with a wavelength, 휆 =

1.5406 Å was used (Besbes, Alila, & Boufi, 2011; Chen et al., 2011; Ciolacu, Gorgieva, Tampu,

& Kokol, 2011; French & Cintrón, 2013; Garvey et al., 2005; Hindeleh, 1980; Hu & Hsieh,

1996; Junior et al., 2018; Lionetto et al., 2012; Malenab et al., 2017; Oudiani et al., 2011; Park et al., 2010; Segal et al., 1959; Tan et al., 2015; Y. Xu et al., 2007; Yueping et al., 2010). So, by using Bragg’s law, these peaks were expected at 16.3-18.1°, 18.7-19.9°, 25.4-27.0° and 40.5-

41.9° angles for Co K X-ray source with a wavelength, 휆 = 1.7890 Å which was observed in this investigation.

Although the shape and the position of the amorphous regions of different samples were slightly different, an overlapping of the peak on (101) and (101̅) planes and all other peaks with their distribution were almost identical (Figure 9). It should be noted that similar patterns were observed in the X-ray spectra of NBFs specimens (Figure 9, Figure 10). Presented in Figure 10, the diffraction patterns of Bissetii, Giant Gray, Moso, Red margin, and their respective natural fibers B-25, G-25, M-25, and R-25 respectively revealed that bamboo and its fibers produced similar patterns. However, the amorphous area, the position and the height of peaks were different that produced unequal CIs (Table 11). Though the peak (101) and (101̅) were clear and in different positions, and a peak at 24.4° on (021) plane was identified in the diffraction pattern of cotton, this was not the cases for bamboo and NBFs (Figure 10). It was also noticeable in the diffraction spectra when plotted altogether that the intensity of the Bissetii plant sample,

Figure 10. Comparison of X-ray (from Co K source) diffraction patterns among four bamboo species, natural bamboo fibers (NBFs) and cotton.

compared with its amorphous peak and the greatest CI (66-67%), was greater than the other three plant samples (Figure 9). Similarly, the CIs were directly related to the intensity of the corresponding diffraction patterns. A higher CI is indicative of increased brittleness of the fibers in the plant (Carraher, 2003). This indicates that the Red Margin plant with the lowest CI (61-

63%) would be better for fiber production as it will promote a lower amount of fiber breakage

during mechanical and other processes. Such a result agrees with the finding in Part-1 that NBFs extraction was easier from Red Margin than the other three species. A lower crystallinity may also come from a younger-aged bamboo plant of the same species.

Figure 11. Change in crystallinity indexes as natural bamboo fibers (NBFs) were extracted into finer forms or count (Ne) through different stages of processing.

It was noticed that almost in all cases of four bamboo species, crystallinity indexes were decreased from raw bamboo to NBFs up to a certain stage of delignification in the production process (Figure 11) which was observed in other studies as well (G. Xu, Wang, Liu, & Wu,

2013). But CIs increased from that point to the final production when fibers were in the finest form from a sample. Probably, the first few steps of delignification caused the NBFs to become more disordered but later steps reorganized as non-crystalline lignin and other components were removed. Thus the CI of the final NBF from each of four bamboo species was the maximum. 59

However, though the range of the CIs of the raw bamboo from four species was 62-66%, the range of the CIs of their final NBFs in this study was 68-72% (Figure 11).

3.5.2 Crystallite Dimensions and Interplanar Spacings

The size of the crystallites with respect to (002) plane were 36.50, 34.86, 34.92 and 37.28 Å for the four species Red Margin, Moso, Giant Gray, and Bissetii respectively with very small standard deviations (Table 11). The results were similar for multiple scans on different samples from each species. The crystallite sizes were not too different when estimated from raw data or from background-subtracted data. The sizes of the crystallites of NBFs were equivalent (37-39

Å). So, the techniques were consistent and suitable in multiple repeats to estimate the results.

Therefore, the estimated overall size of the crystals based on the most intense peak on (002) lattice plane was within the range of 35-39 Å (3.5-3.9 nm) which is almost half of the crystal’s size (69.72 Å) of experimented cotton fibers (Table 11). These results are supported by other investigations as 36.5 Å for bamboo raw biomass (Afrin et al., 2014; Thomas et al., 2015).

In Table 11, the interplanar distances of the lattice structures of different crystals obtained from different bamboo species and NBFs were 5.8-6.0 Å for (101) plane, 5.4-5.6 Å for (101̅) plane, ~4.0 Å for (002) plane, and 2.6 Å for (040) planes. These value of the spacings were similar to cotton fibers as observed in this study.

3.6 Conclusions

There are over 1500 fast-growing bamboo species in the world. However, only a few species of bamboo have been studied for fiber applications and thus its properties. Only very few studies have been dedicated to the characterization of bamboo and its fibers. Knowing different chemical and structural properties would promote bamboo's benefits by matching it to different purposes, especially the production of textiles and apparel by using natural bamboo fiber (NBF) which could provide some special characteristics. 60

Though the crystallinity is one of the most important factors of fibers, it has not been studied in terms of crystalline properties of different bamboo species in previous studies. This investigation focused on four of the most common bamboo species and on extracted natural bamboo fibers (NBFs) from the plants to determine crystallinity indexes, their respective crystallite sizes and other crystallographic analyses by X-ray diffraction (XRD) technique using a Cobalt K (Co K) X-ray source. Four peaks were identified on (101), (101̅), (002) and (040) lattice planes at 17.0-18.6°, 18.5-19.4°, 25.5°, and 40.5° respectively in all bamboo and NBF samples where the peak on (002) corresponded to the most intense peak. Both Segal and deconvolution methods confirmed that the approximate crystallinity indexes (CIs) were 63-67% for the raw bamboo specimens of four species Red Margin, Moso, Giant Gray, and Bissetii, and

69-73% for NBFs from the plants. The CIs values of NBFs were lower than cotton fibers which may indicate lower strength of NBF fibers than cotton. Also, a lower CI is related to softer feel and perceived comfort in the clothing products of the fiber. The sizes of the crystallites as 35-39

Å (3.5-3.9 nm) were almost the same for all four bamboo plants and NBFs considering the most intense peak only. The crystallographic information of bamboo and NBFs about lattice planes, interplanar spacings, reflection angles, and crystallite dimensions are also reported. The results and observations of this study would be useful to advance crystallographic knowledge on bamboo plants and to guide fiber production and application from certain species.

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CHAPTER 4. ANALYSES OF THE CHEMICAL COMPOSITIONS AND STRUCTURES

OF FOUR BAMBOO SPECIES AND THEIR NATURAL FIBERS BY INFRARED,

LASER AND X-RAY SPECTROSCOPIES

Abstract

Four bamboo species, natural bamboo fibers (NBF) from each species, commercial bamboo viscose, and other conventional fibers were investigated in this study to determine and compare elemental compositions, chemical bonds and behaviors. Elemental analyses with energy- dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) were executed to identify elemental compositions in terms of atomic and weight/mass concentration,

O/C ratios of raw bamboo (0.26-0.42) and NBFs (0.44-0.70), binding energies, and components of C 1s and O 1s peaks and corresponding chemical bonds: C-C, C-H, C-O, C=O, O-C-O, and

O-C=O. It was revealed that the O/C ratio increased with the fineness of NBFs which is an indication of higher moisture regain or content. Since some chemical groups are not detected by

Fourier Transform Infra-Red (FTIR) spectroscopy and others are not detected by Raman spectroscopy (RS), the importance of an investigation using both methods for identifying chemical groups, bonds, and characteristics was verified in this study. FTIR with Attenuated

Total Reflectance (ATR) and Raman spectroscopies were employed to make a broad list of identified characteristic peaks and their chemicals information of different bamboo species,

NBFs, cotton, and regenerated cellulosic fibers. The investigation discovered that NBFs may possess most of the chemical groups and behaviors from the bamboo plants from which they were extracted, and thus the properties. It was observed that bamboo viscose had similar bands to regular viscose rayon, and many bands found in raw bamboo and NBFs spectra were not present 66

in the bamboo viscose. Results also revealed that bamboo and its NBF had some unique peaks that were not detected in cotton, rayon of bamboo viscose spectra.

KEYWORDS: EDS; XPS; Fiber chemistry; FTIR; Raman spectroscopy; NBF compositions;

Bamboo properties.

4.1 Introduction

Bamboo is a highly studied plant, especially in investigations on manufacturing fibers for textiles, fiber-reinforced composite materials, and other applications of the fiber. The major constituents of bamboo biomass are cellulose (45-55%), hemicellulose (15-25%), lignin (15-

30%), pectin (0.5-1.5%) and other organic and inorganic compounds (Liu, Wang, Cheng, Qian,

& Yu, 2011; Nayak & Mishra, 2016; Rocky & Thompson, 2018; Tolessa, Woldeyes, & Feleke,

2017). Knowing chemical compositions and functional groups, different properties of the bamboo plant and its fibers can be identified. Moreover, such information can help with selecting proper chemicals or enzymes for fiber extraction by removing non-fibrous components and enhancing existing properties of the extracted fibers (Fu et al., 2012; Thumsorn, Srisawat, On, &

Hamada, 2014; Xu, Wang, Liu, & Wu, 2013).

X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray spectroscopy

(EDS) are tools that are used frequently for quantification of elemental surface compositions

(Belgacem, Czeremuszkin, Sapieha, & Gandini, 1995; Herzele, van Herwijnen, Edler, Gindl-

Altmutter, & Konnerth, 2018; Johansson et al., 2005; Kılınç et al., 2018; Kyropoulou, 2013;

Migneault, Koubaa, Perré, & Riedl, 2015; Ouyang, Huang, & Cao, 2014; Popescu, Tibirna, &

Vasile, 2009; Truss, Wood, & Rasch, 2016; Wang et al., 2018; Zafeiropoulos, Vickers, Baillie,

& Watts, 2003). EDS, generally performed in a scanning electron microscope (SEM), has some limitations on elemental quantification and is less precise for organic materials. EDS has proven to be a tool capable of estimating some primary information about elemental composition of an 67

organic material (Hui & Li, 2016; Kyropoulou, 2013; Li, Bi, Song, Qu, & Liu, 2019; Nasser,

Mansour, Salem, Ali, & Aref, 2017; Ouyang et al., 2014). EDS, does not need special sample preparation, though fibers are difficult to use in X-ray spectroscopy because of the sensitivity issues and uneven/rough surface of the fiber that scatters reflected or transmitted rays out of the detectors, it can still provide useful information (Pakdel, Cyr, Riedl, & Deng, 2008). Notably, high sensitivity of XPS to a surface of a carbon-rich material can help to explore the atomic nature and organic functional groups along with elemental bonding (Wang et al., 2018). An example of a high carbon surface in which XPS is a highly used tool for characterizing is wood pulp. XPS can be used to collect information about the surface of a specimen within 0.1—10 nm

(Kazayawoko, Balatinecz, & Sodhi, 1999; Truss et al., 2016). However, there is no standard or systematic techniques to analyze fibers, polymers, or insulating fiber materials for such analysis

(Johansson et al., 2005). Determining of O/C atomic ratio and CC components are the two methods used for XPS analysis of cellulose-based materials where cellulose, hemicellulose, lignin, and other trace elements are the main components (Johansson et al., 2005; Kazayawoko et al., 1999; Xu et al., 2013). Though there are some limitations, elemental analysis with EDS and

XPS can provide us with approximate elemental compositions, and the nature of Carbon-Carbon and Carbon-Oxygen bonding (Belgacem et al., 1995; Kılınç et al., 2018; Migneault et al., 2015;

Pakdel et al., 2008; Popescu et al., 2009; Wang et al., 2018; Zhang, Zhang, & Lu, 2016; Zhou,

Guo, Yan, & Di, 2017).

The focuses of the EDS and XPS surveys were to identify and compare the elemental composition, changes of O/C ratios during NBF extraction, binding energies, and components of

C 1s and O 1s along with their nature of bonding of four bamboo species and natural bamboo fiber (NBF) from each of the plants.

As FTIR (Fourier Transform Infra-Red) and Raman spectroscopy (RS) have different degrees of sensitivity to different functional groups, a combination of the tools can be useful for chemical analysis. For example, FTIR has higher sensitivity towards hetero-functional groups such as –OH, C-O and C-O-C but Raman spectroscopy is more sensitive in identifying homo- functional groups such as C≡C, C=C, and C-C groups. For chemical groups and compositional investigations, different cellulose-based materials such as wood pulps, biomass, fibers and carbohydrate polymers were studied by using FTIR and Raman spectroscopies (Alves et al.,

2016; Cao, Shen, Lu, & Huang, 2006; Cho, 2007; Dai & Fan, 2011; Herzele et al., 2018; Hui &

Li, 2016; Kavkler & Demšar, 2011; Kılınç et al., 2018; Lee et al., 2015; Migneault et al., 2015;

Ouyang et al., 2014; Sepe, Bollino, Boccarusso, & Caputo, 2018; Zafeiropoulos et al., 2003;

Zhang et al., 2016). For convenience, FTIR, sometimes with an ATR (Attenuated Total

Reflectance) accessory, is extensively used for characterizing different natural or cellulose-based fibers to identify chemical groups and nature of bonding (Carrillo, Colom, Suñol, & Saurina,

2004; Comnea-Stancu, Wieland, Ramer, Schwaighofer, & Lendl, 2017; Garside & Wyeth, 2016;

Lee et al., 2015; Migneault et al., 2015; Sanjay et al., 2018; Zhou et al., 2017).

Raman spectroscopy, is a non-destructive technique requiring simple or no sample preparation and a very small sample can be used to collect information of micro- and macro- levels (Cho, 2007; Kavkler & Demšar, 2011). Since natural fibers are long-chain polymers of low dipole moments and FTIR is less sensitive to such materials, Raman spectroscopy can produce a higher number of peaks in the spectra to exhibit more chemical information than

FTIR. FTIR is more commonly used for characterization of textiles materials than Raman spectroscopy, even though Raman actually offers more advantages (Abbott, Batchelor, Smith, &

Moore, 2010; Alves et al., 2016; Cao et al., 2006; Cho, 2007; Kavkler & Demšar, 2011). Spectra of the textile fibers and materials can be characterized into 3 regions: 150-610, 800-1750 and 69

2700-3350 cm-1 (Kavkler & Demšar, 2011; Schenzel & Fischer, 2001). Though the last two regions are visible in both FTIR and Raman spectra, the first region is only detectable in Raman spectra. However, Raman spectroscopy is also unable to detect some functional groups, such as

OH bond stretch of water. Therefore, an investigation with both spectroscopies can be regarded as better than a single-tool observation. The only studies reporting the use of these techniques for characterizing bamboo and its fibers can be found in G. Xu et al. (2013) and Yueping et al.

(2010).

The major purposes of the study were to identify chemical groups, bonds, behaviors and other properties of different bamboo species and natural bamboo fibers (NBFs) by using FTIR and Raman spectroscopies. Targets of the investigation also included comparing IR and Raman spectra of bamboo and NBFs specimens with cotton, regular rayon viscose, and bamboo viscose fibers.

4.2 Methodology

4.2.1 Materials and machines

Four bamboo plants— Red Margin (Phyllostachys rubromarginata), Moso (Phyllostachys edulis/ pubescens), Giant Gray (Phyllostachys nigra henon), and Bissetii (Phyllostachys Bissetii) were used in this study. All the relevant information about materials and machines are mentioned in Part-2. Additionally, the following instruments were used: Ion Sputter Coater (Leica, MCM-

200 Ion Sputter Coater), JEOL 7000 (JEOL JSM-7000F, FE-SEM/Field Emission Scanning

Electron Microscope) for SEM and EDS, X-ray photoelectron spectrometer (Kratos Axis 165) for XPS, FTIR spectrometer (Jasco FT/IR-4100) with ATR (MiracleTM ATR, PIKE

Technologies, Inc.), and Raman Spectrometer (LabRam HR800, Horiba Jobin Yvon, Horiba

Scientific).

4.2.2 Extraction of Natural Bamboo Fibers (NBFs)

The primary samples B-1, G-1, M-1 and R-1 were produced from Bissetii, Giant Gray, Moso, and Red Margin respectively by applying initial treatments as mentioned in Part-1. The processing sequences of different NBF specimens were as follows—

 R-9: recipe 0  recipe 1 (Table 12).

 M-22: recipe 0  recipe 3 (Table 12).

 B/G-23: recipe 0  recipe 3  recipe 2 (Table 12).

 B/G/M/R-25: recipe 0  recipe 3  recipe 2  recipe 5 (Table 12).

 R-27: recipe 0  recipe 4  recipe 2  recipe 5 (Table 12).

The final sample of each set was subjected to neutralizing, washing, and drying.

Table 12

Recipes, chemicals and processing conditions for NBF extraction NaOH NaHCO₃ H₂O₂ Temperature X Recipe Other chemicals M : L (g/L) (g/L) (mL/L) Time

0 6 - 1 : 1020 RT x 35 days

1 20 - - - 90 °C X 3 h 3 mL/L scouring agent (Consoscour GSRM), 3 mL/L bleaching agent 2 3 6 6 1 : 1520 90 °C X 67 h (Consobleach CIL), and 4 mL/L penetrating agent (Consamine JDA) 10 g/L Na5P3O10, 10 g/L Na2SO3, 10 mL/L Na2SiO3, 20 mL/L fabric softener, 4 mL/L 3 5 - - scouring agent, 4 mL/L bleaching agent, 1 : 15 90 °C X 8 h and 5 mL/L penetrating agent 4 5 10 - 5 g/L Na5P3O10, 10 mL/L Na2SiO3 5 2 6 46 - 1 : 1540 90 °C X 11.5 h

4.2.3 Elemental analyses by XPS & EDS

Bamboo culms were split and undried strips were cut into small rectangular thin slices a low- speed abrasive precision cutting machine and then the thin strips were dried before using in a spectrometer. The details of the raw bamboo sample preparation are documented in Part-2. A 71

JEOL 7000 was used for EDS analysis of raw bamboo strips and gold-coated NBFs. Several small spot areas and the bulk area were chosen for elemental spectra collection. The spectrum and statistics from small spots were used to identify the presence of different elements and the bulk areas were employed for compositional investigation. The EDS investigation was repeated

2-3 times for all samples.

Only raw bamboo strips were observed in XPS spectrometer. A monochromatic Al Kα was used as the X-ray source (hν = 1486.7 eV) at a high vacuum of 10−9 to 10−8 torr. All XPS spectra were collected between 0-1000 eV binding energy with a step size of 0.05-0.10 eV. The

CasaXPS software was used for XPS data analysis. CasaXPS software allowed the C 1s and O 1s peaks and other components to be analyzed by curve fitting and deconvolution methods. The

O/C ratios of different spectra were calculated by using respective atomic percentage (At%), area

(A) and height (H) of C 1s and O 1s peaks from CasaXPS analysis.

AOxygen HOxygen O/C ratio = = … … . . (1) ACarbon × RSF HCarbon × RSF

In equation (1), AOxygen and ACarbon are the areas, and ACarbon and HCarbon are the heights of O

1s and C 1s peaks respectively. The Relative Sensitivity Factor (RSF) of Oxygen was taken from

CasaXPS library which was considered 2.93 for Oxygen.

4.2.4 FTIR and Raman: Analyses of Chemical Groups

Raw bamboo strips were directly placed in spectrometers for spectra and data collection. Both sides of each strip were scanned. The fiber samples were ground and collected through a micro- sieve containing 4 layers of mesh size 80, 120, 170, and 230. All the fibers were scanned in fiber and ground powder forms. In Raman spectroscopy, a single fiber was focused using a 100X objective lens and proved to be suitable. Fiber-form specimens were challenging in ATR-FTIR spectra collection.

For ATR-FTIR spectroscopy, spectra were collected for each sample at least 3 times (3-

-1 -1 5) within a range of 600—4000 cm with a resolution of 2-4 cm from 25-50 scans. Collected spectra were normalized with respect to the most intense peak after baseline correction.

Raman spectrometer was used to scan from 100 cm-1 to 4000 cm-1 with 950 grating and a laser of 785 nm wavelength (monochromatic light source) as it helps to avoid heating effects, fluorescence, works well with the low sensitivity of the detector, and absorption of the laser light. The acquisition time was chosen between 10-50 seconds with accumulations of 8-15 scans that made a total scanning time of at least 30-60 minutes. Each sample was scanned 5-8 times to confirm the consistency of the measurement and to achieve the best spectra. Some spectra required smoothing, but most of the spectra were correct without smoothing and were baseline corrected and normalized as needed.

4.3 Results and Discussion

4.3.1 Elemental analyses

Energy-Dispersive X-ray Spectroscopy (EDS)

Table 13

Elemental compositions and oxygen-carbon ratios of different bamboo species and their fibers

Sample Weight % Atomic % Other Fineness Fineness O/C from C O Other C O Other Elem. (Tex) (Ne) At% Red Margin 64.01 35.99 - 70.32 29.68 - - - - 0.42 Moso 64.87 35.13 - 71.1 28.9 - - - - 0.41 Giant Gray 64.23 35.77 - 70.52 29.48 - - - - 0.42 Bissetii 65.19 34.23 0.58 71.49 28.18 0.33 Na - - 0.39 R-9 Fiber 62.50 36.98 0.52 69.03 30.67 0.30 Na 27 22 0.44 R-27 Fiber 56.56 43.44 - 63.43 36.57 - - 13 47 0.58 M-22 Fiber 51.80 47.33 0.87 59.03 40.49 0.48 Na, Si 34 17 0.69 G-23 Fiber 51.12 47.7 1.18 58.57 41.03 0.40 Ca 13 47 0.70 B-23 Fiber 51.98 47.1 0.92 59.33 40.36 0.31 Ca 21 29 0.68 Yala* 52.11 47.29 0.60 59.3 40.40 0.30 Si - - 0.68 *100% commercial bamboo viscose cloth product.

From the energy-dispersive X-ray spectroscopic (EDS) investigations summarized in

Table 13, it was revealed that raw bamboo specimens were mainly composed of carbon (C) and oxygen (O). The proportions of C and O of four bamboo species in multiple specimens were found to be consistently 64-65% and 34-36% by weight %, and 70-72% and 28-30% by atomic

% respectively. The investigation also revealed that a fractional amount of carbon reduced as the

NBFs were extracted from raw bamboo and fineness (Tex) of the fibers reduced. For example, when raw Red Margin was subjected to processing of NBF extraction R-9 (27 Tex) and R-27 (13

Tex), the fraction of C reduced from 64.01% in raw to 62.50% in R-9 and then to 56.56% in R-

27 by weight%; similarly, 70.32% in raw to 69.03% in R-9 and then to 63.43% in R-27 by atomic%. This was the same trend for all bamboo species and the respective NBFs. It was also noticed that amount of C and O in commercial 100% bamboo viscose apparel product (Yala) and in finer NBFs (R-27, B-23, G-23, and M-22) from four bamboo species were similar (Table 13).

The O/C ratios from atomic% of the raw bamboo plants were found to be almost same,

0.39-0.42. However, there are some instrumental factors such as sensitivity, calibration, and contamination that can influence O/C ratios (Johansson et al., 2005). The O/C ratios were increased as the NBFs were extracted to finer forms and the finer fibers showed similar ratios

(~0.70) to bamboo viscose (Table 13). This is in agreement with the reduction of the quantity of carbon in NBFs as mentioned earlier. Since lignin is a compound with high C content, the amount of C reduces as the raw bamboo goes through delignification. Similar results were also observed for other fibers in previous studies (Nasser et al., 2017; Truss et al., 2016). An increase in the O/C ratio as a result of higher oxygen concentration or the reduction in carbon means that extracted NBFs will be more water-absorbent since it will have less hydrophobic lignin compounds. Because of the low sensitivity, some other elements might not be detected.

However, sodium (Na), silicon (Si), nitrogen (N) and calcium (Ca) were detected as nominal 74

portions (0-0.6%) as the other elements. Assignments of these elements were based on the assumption that the shared portion of 0-0.6% in the elemental composition of specimens was from other elements rather than from C and O. These elements were assigned based on a prediction of what is relevant to a fiber. So, a list of elements was selected from the Periodic

Table and the software detected the most likely elements. Though data and results from EDS are not highly accurate, they may provide us with an understanding of elemental distribution, presence, and changes of composition during processes.

4.3.2 X-ray Photoelectron Spectroscopy (XPS)

Like EDS, the XPS examination revealed that carbon is the most abundant element, outside hydrogen, in bamboo and oxygen is the next. In Table 14, atomic% of carbon in four bamboo species can be presented as 76-78% and oxygen as 20-24%. Nitrogen was detected as the third element with atomic% of 1-4% from different XPS observations. It can be noticed from Figure

12 that N 1s peak was detected on Giant Gray and Red Margin bamboo specimens but it was not detected on the other two bamboo specimens. Table 14 represents the amount of nitrogen in Red

Table 14

Concentration, O/C ratios and binding energies of different elements in four bamboo specimens from XPS investigation Binding O/C from O/C from Orbital Area FWHM Height At% O/C Sample Energy (eV) peak area peak height XPS EDS XPS C 1s 289 34972 4.47 13069 77.70 Red O 1s 535 26364 4.15 10364 19.99 0.42 0.26 0.27 Margin N 1s 402 1874 5.22 689 2.31 C 1s 288 25017 4.89 9021 76.29 Moso 0.41 0.31 0.33 O 1s 535 22781 4.40 8667 23.71 C 1s 283 40429 3.89 19216 77.37 Giant O 1s 531 31906 3.37 16325 20.84 0.42 0.27 0.29 Gray N 1s 397 1685 3.54 919 1.79 C 1s 287 27521 5.18 10167 76.37 Bissetii 0.39 0.31 0.33 O 1s 534 24957 4.58 9818 23.63

Margin (2.31%) and Giant Gray (1.79%) species only. Probably, nitrogen from the environment was detected in Giant Gray and Red Margin samples and it was not present on the scanned surfaces of Moso and Bissetii. Since two surfaces of each strip were slightly different with respect to fiber content, the presence of elements and quantities probably varied by the surface of the bamboo strip that was observed. The C 1s peaks in the XPS spectra of all bamboo plants were observed in the range of 283-289 eV binding energy and O 1s in the range of 531-535 eV.

These ranges are also reported by many other studies with cellulose-based materials (Belgacem et al., 1995; Herzele et al., 2018; Johansson et al., 2005; Kazayawoko et al., 1999; Migneault et al., 2015; Popescu et al., 2009; Truss et al., 2016; Wang et al., 2018; Zafeiropoulos et al., 2003).

The binding energy of N 1s was within 397-402 eV as reported by different studies (Truss et al.,

2016; Xu et al., 2013; Zhang et al., 2016). Although C 1s and O 1s peaks were clearly identified as intense peaks in all samples, the N 1s peak was not seen in all spectra and it was not intense as well (Figure 12). It could be environmental nitrogen on the surface of the specimens that were detected by the spectrometer.

In Table 14, the estimation of O/C ratios from atomic%, area, and height revealed that all three methods were consistent with each other as the values were approximately equal. The O/C ratios of all bamboo species were equivalent which was in the range of 0.26-0.33. This value was more accurate as it matches previous investigations of cellulose-based materials (Herzele et al.,

2018; Kazayawoko et al., 1999; Popescu et al., 2009; Truss et al., 2016; Xu et al., 2013). The

O/C ratios obtained from EDS (0.39-0.42) was slightly higher than XPS values (0.27-0.33).

Figure 12. X-ray photoelectron spectroscopic survey spectra of four bamboo species

From the deconvolution of C 1s and O 1s peaks using CasaXPS software as shown in

Figure 13 and Figure 14, four components of C 1s (C1-C4) and two components of O 1s (O1-O2)

2 were identified. Using more than 99.5% (푅 > 99.5%) data of C 1s and O 1s, C1-C4 and O1-O2 components were separated. Though C1- C3 components were in all spectra, C4 was not identified in Moso bamboo (Figure 13). The sample’s surface selection or the software’s precision and accuracy could be the reason it was not identified. However, it can be concluded that bamboo is composed of C 1s with C1- C4 components where binding energies were estimated as 283-287 eV for C1, 285-289 eV for C2, 287-289 eV for C3, and 290-292 eV for the

C4 component (Table 15). This provides us with the understanding that C1 corresponds to C—C and C—H bonding of aliphatic and aromatic carbon in lignin, extractives substances, and fatty acids; C2 corresponds to C—O (primary and secondary) alcohol and ether-type (non-carbonyl 77

group) bonding mainly in cellulose, C3 corresponds to C═O and/or O—C—O bonding of carbonyl and acetal groups in cellulose, C4 corresponds to O—C═O bonding of carboxyl group of esters and carboxylic acids (Belgacem et al., 1995; Johansson et al., 2005; Kazayawoko et al.,

1999; Popescu et al., 2009; Truss et al., 2016; Xu et al., 2013; Zafeiropoulos et al., 2003; Zhou et al., 2017).

Figure 13. Deconvoluted peaks of the components (C1-C4) of C 1s peak from XPS spectra of 4 bamboo plants: (a) Bissetii, (b) Giant Gray, (c) Moso, and (d) Red Margin bamboo.

Though O 1s spectra are often disregarded, they are very important to investigate surface composition as the carbon atoms need to be balanced in a substance (Truss et al., 2016). In

Figure 14, O1 and O2 components of O 1s are identified in all four bamboo species but with different concentration, as seen in C 1s (Table 15). The binding energies of peaks were equivalent in all species; examples are O1 with 531-534 eV and O2 with 532-536 eV. The O 1s peaks with such binding energies are reported to be O1 as an oxygen atom double-bonded to a carbon atom (O—C═O, C═O) and O2 as an oxygen atom single-bonded to a carbon atom (C—

O) (Popescu et al., 2009; Truss et al., 2016). The presence of N 1s peak may be from amide bonding in bamboo specimens.

Figure 14. Deconvoluted peaks of the components (O1 and O2) of O 1s peak from XPS spectra of

4 bamboo plants: (a) Bissetii, (b) Giant Gray, (c) Moso, and (d) Red Margin bamboo.

Table 15

Binding energies, atomic percentages of Carbon and Oxygen with their components in XPS analysis of different bamboo species C O C1 C2 C3 C4 O1 O2 Binding energy (eV) 286.92 288.64 289.44 291.80 533.73 535.91 Red margin Atomic% 22.03 22.03 52.01 3.93 45.49 54.51 Binding energy (eV) 284.18 286.41 288.83 290.98 533.19 535.67 Moso Atomic% 11.72 18.61 58.64 11.03 48.12 51.88 Binding energy (eV) 283.09 284.71 286.58 - 530.72 531.88 Giant gray Atomic% 59.60 32.45 7.95 0.00 90.46 9.54 Binding energy (eV) 284.15 287.30 288.01 289.54 532.70 535.03 Bissetii Atomic% 15.21 68.03 3.48 13.28 52.76 47.24

As listed in Table 15, atomic percentages of different components of C 1s and O 1s were dissimilar for different bamboo species. A combination of the selection of the surfaces for XPS scanning, data processing, species type and age of bamboo plants are plausible reasons for such results. Since the inner surface of the bamboo strips contain a higher amount of lignin and this gradually decreases to the outer surfaces, the differences in the concentration of different components of C 1s and O 1s in Table 15 are possibly due to the selection of scanned surface of bamboo strips. The percentage concentration of C4 was the lowest carbon component is all cases.

This suggests that there is a smaller quantity of carboxylic or ester-containing groups in bamboo than other chemical groups that contain C2 or C3 carbon atoms.

4.4 FTIR and Raman: Identifying Chemical Groups

A comparative presentation of infrared (IR) and Raman spectra obtained for untreated raw bamboo specimens and NBFs is displayed in Figure 15. It can be seen in the spectra that only a range between 700-4000 cm-1 of IR spectra could be investigated for peak analysis to determine the presence of different chemical groups and behaviors. Raman spectra could exhibit identifiable peaks within 100-4000 cm-1, giving a wider range. Moreover, within the same range, two types of spectra could provide different peaks or the same peak with different intensity. This 80

was because IR and Raman have different sensitivity to different chemical bonding and functional groups. For example, as FTIR is more sensitive to –OH bond stretching of water, a wide peak between 3100-3600 cm-1 is visible but Raman spectra were unable to detect such a peak in the range. Thus, Figure 15 can provide us with an understanding that both of these spectroscopies would be good for chemical analysis for a material. Moreover, it should also be mentioned that though it is said that sample preparation for Raman spectroscopy is simpler than for FTIR, it was found to be more difficult to use some fiber samples in the Raman spectrometer than in ATR-FTIR.

Figure 15. Comparisons of ATR-FTIR and Raman spectra of raw bamboo samples and natural bamboo fibers (NBFs).

A comparative Raman spectroscopic study of the four bamboo species Bissetii, Giant

Gray, Moso, and Red Margin revealed that they have almost similar spectral patterns to each other as shown in Figure 16. The spectra from IR were also similar to each other. This suggests that the different bamboo species may have the same type of chemical groups, behaviors, and

Figure 16. Comparisons of Raman spectra of four bamboo species, natural bamboo fiber (NBF) and cotton fiber.

bonds. But the spectra changed in natural bamboo fibers (NBFs), especially in the regions between 1500- 1800 cm-1 where the intensities of different peaks in NBF (R-25) spectra were lower than that of raw bamboo specimens (Figure 16). Though intensities of the peaks were very

low, they were definitely identified in NBFs specimens. There was a major difference in the spectra between cotton fibers and NBFs as shown in Figure 16, specially in the 1500-1800 cm-1 region. There are also some peaks in NBFs which could not be matched in cotton spectra. This establishes a reason why NBFs would exhibit some unique behaviors as compared to cotton.

Figure 17. Comparisons of Raman spectra of Red Margin bamboo, natural bamboo fibers

(NBFs) from four species and bamboo viscose fiber. [B-25, G-25, M-25, and R-25 are NBFs from Bissetii, Giant Gray, Moso, and Red Margin bamboo plants].

Again, a comparison of the Raman spectra of four NBF specimens B-25, G-25, M-25, and R-25 in Figure 17 exhibits that NBFs extracted from different bamboo species are almost the same with respect to their spectral peaks and intensities. Though the peaks between 1500-1800 83

cm-1 are less intense, all other peaks were more intense and clearer in the spectra of NBFs than raw Red Margin’s spectrum (Figure 17). However, almost all of the peaks in the spectra of NBFs were present in the spectra of each bamboo specimens. It was also noticeable that bamboo viscose has fewer peaks in the spectrum than NBFs’ peaks. The characteristic peaks of NBFs within 1500-1800 cm-1 that are mainly from lignin compounds were not observed in bamboo viscose. So, this was a clear difference between bamboo viscose and NBFs.

Since any of the spectra of the studied bamboo specimens would represent almost all peaks in both raw bamboo specimens or NBFs, identified peaks of a Red Margin specimen were chosen in a ATR-FTIR spectrum in Figure 18 to represent bamboo and NBFs, and a Red Margin specimen for Raman spectrum in Figure 19. Based on the clustering of peaks because of the presence of different chemical groups, the regions in the IR and Raman spectra can be divided

Figure 18. Different peaks and their positions in the ATR-FTIR spectrum of Red Margin bamboo specimen.

Figure 19. Different peaks and their positions in the Raman spectrum of Red Margin bamboo specimen.

into 3 portions in combination: 150-610 cm-1 band region, 630-1800 cm-1 band region, and 2700-

3600 cm-1 band region. It was observed that peaks in the IR spectra were not clearly detected below 800 cm-1 (Figure 18) but many peaks were clearly identified between 200-800 cm-1 in

Raman spectra (Figure 19). In contrast, though some clear peaks between 2700-3600 cm-1 band region were observed in IR spectra, only a few or no peaks in that region were detected by

Raman spectra. Moreover, peaks between 900-1160 cm-1 in IR spectra were of high intensity

(Figure 18) but these peaks were of low intensity in Raman spectra (Figure 19). The opposite patterns were observed between IR and Raman spectra in the 1500-1800 cm-1 band region. The above-mentioned differences explain the importance of investigating chemical behaviors of a material by both ATR-FTIR and Raman spectroscopies.

Different chemical groups, bonds, and characteristics of raw bamboo specimens, NBFs, cotton, bamboo viscose, and regular rayon from ATR-FTIR and Raman spectra are presented in Table

16. As mentioned earlier, some peaks were only observed in IR spectra, some are in Raman spectra, and other peaks were observed in both spectra. The first region of the band 150-610 cm-1 was detected in Raman spectra of all samples. It was observed that spectra of all samples, mainly cellulose-based, exhibited some peaks in the region. The peaks within this region were described as due to skeletal bending, glycosidic linkage and ring deformation of C-O-H, C-C-H, C-C-O, C-

C-C, C-O, and C-O-C groups in cellulose and hemicellulose (Cabrales, Abidi, & Manciu, 2014;

Cho, 2007; Kavkler & Demšar, 2011). Though a peak within 375-379 cm-1 observed in cotton,

NBFs and bamboo specimens which is related to the crystallinity of cellulose, and C-C-C, C-O,

C-C-O bonds and ring deformation, this peak was not identified in rayon or bamboo viscose.

Moreover, some peaks in the range of 630-890 cm-1 were detected but they were not identified in the literature. Peaks in 630-890 cm-1 can be assigned as C-H and O-H out-of-plane bending, mono-substituted benzenes, (N-H) primary amide, C-C stretch, CH=CH (cis), and

C=C-H (vinyl compounds) bending/twisting (Socrates, 2001).

In the second 630-1800 cm-1 band region, the most number (30-40 except rayon and bamboo viscose) of peaks were detected in all samples (Table 16, Figure 18, Figure 19). Peaks in this region were detected either by IR or by Raman or by both spectra. A peak at ~1060 cm-1 defined as C-O-H secondary alcohol was observed in cotton and bamboo viscose but it was not found in the spectra of bamboo and NBFs specimens. Similarly, many other peaks such as 1120-

1124, 1235-1247, 1335, 1484-1489, 1508-1514, 1660-1683, 1728-1745 cm-1 in the cotton, NBFs and bamboo specimens were detected but they were not present in rayon or bamboo viscose spectra. These peaks are mainly related to lignin, hemicellulose, and cellulose. Some peaks in the second region were also identified in NBFs and untreated bamboo specimens such as 1322-1330, 86

Table 16.

Chemical bonds and band characteristics of ATR-FTIR and Raman spectra of four bamboo species, natural bamboo fibers (NBFs), bamboo viscose, regular rayon, and cotton fibers (Alves et al., 2016; Cabrales et al., 2014; Carrillo et al., 2004; Comnea-Stancu et al., 2017; Dai & Fan, 2011; Kavkler & Demšar, 2011; Kılınç et al., 2018; Lee et al., 2015; Migneault et al., 2015; Sanjay et al., 2018; Schenzel & Fischer, 2001; Xu et al., 2013; Yueping et al., 2010)

B-25 Giant G-25 M-25 Red R-25 Bamb. Literature Cotton Bissetii Moso Rayon Functional Group/Assignments NBF Gray NBF NBF Margin NBF Viscose

cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ cmˉᴵ 170- 182- 206- 187- 203- 210- 154- 202- 195- 159- 172-252 164-233 C-O-H bending 287 277 267 280 282 277 288 272 284 221 304-308 307 308 311 309 309 307 304 309 309 309 309 C-C-O bending 331, 327, 318-350 - 352 331 331 324 330 350 353 354 C-C-C, C-O, C-C-O, ring deformation 347 350 377-383 379 378 379 375 378 376 379 377 377 - - C-C-C, C-O, C-C-O, ring deformation, crystalline peak 430, 413, 426, 418-439 436 435 433 414 435 434 419 420 CCC, CCO ring deformation 439 439 434 C-C-O and C-C-C bending, ring deformation, skeletal 457-461 458 457 456 457 459 458 459 460 460 460 462 bending 488, 489-496 487 492 491 493 492 - 494 492 489 491 C-O-C bending, glycosidic linkage; xylan (hemicellulose) 495 518-523 518 521 521 524 519 525 518 523 510 521 519 C-O-C glycosidic linkage, C-C-C ring deformation

540-542 537 539 540 537 539 - - C-O-C bending in-plane, glycosidic ring 569, 565-575 566 575 576 581 569 567 563 578 577 578 C-O-C bending, ring 574 600, 602-609 611 605 607 608 600 609 602 602 - 607 C-C-H bending 607 C-H and O-H out-of-plane bending, mono-substituted 704- 710, 692- 720- 675- 715- 643- 643- 708- 630-890 665-827 benzenes, (N-H) primary amide, C-C stretch, CH=CH 856 863 842 805 827 864 866 742 831 (cis), and C=C-H bending/twisting 895, 898- 897, 895, 894, 895, C-H deformation in cellulose, H-C-C bending, H-C-O 896-900 896 898 898 900 899 899 901 899 899 897 897 bending, 1° methine bending 910 - - - 923 915 918 - 922 911 - 918 C-O-C in plane, symmetric

B-25 Giant G-25 M-25 Red R-25 Bamb. Literature Cotton Bissetii Moso Rayon Functional Group/Assignments NBF Gray NBF NBF Margin NBF Viscose 984- 969- 983- 974- 983, 975, 971, 969, 983, 972-999 982 981 CH , skeletal 1002 999 988 995 988 988 992 995 998 2 C-O stretching, primary alcohol, guaiacyl (lignin), 1032- 1030, 1032- 1032, 1031, 1032, 1030, 1035-1039 1036 1038 1032 1031 circular C–O–C stretching and contracting symmetric 1037 1017 1037 1039 1038 1038 1041 motion 1054- 1047- 1051, 1052, 1052, 1051, 1052, 1050-1055 1052 1052 - - C-O-C symmetric stretching 1057 1050 1055 1055 1055 1053 1056 1060 1060 ------1063 - C-O-H 2° alcohol 1093, 1105, 1094- 1094- 1095, 1093, 1094, 1094, C–O–C antisymmetric stretching, C-O-C glycosidic, ring 1099-1105 1097 1096 1097 1103 1095 1103 1103 1103 1103 1103 1104 breathing 1121- 1120- 1120, 1120, C-H guaiacyl and syringyl (lignin), C-O-C glycosidic 1120-1124 1120 1122 1122 1120 1120 - - 1126 1123 1123 1122 linkage, symmetric stretching 1151, 1162, 1161- 1150, 1158- 1150- 1156- 1151- C-C, C-O ring breathing, C–O–C antisymmetric 1150-1163 1172 1156 1158 1161 1176 1170 1161 1170 1160 1172 1161 stretching, carbohydrate 1199- 1202, 1202, 1201, 1199, 1199, 1200-1205 1204 1205 1203 1204 1202 O–H in plane bending 1205 1197 1204 1204 1202 1203 1246- 1232- 1244- 1233, 1244- 1242, C–C, C–O, C=O stretching, guaiacyl ring breathing 1235-1247 1238 1237 1241 - - 1251 1238 1247 1247 1249 1248 (lignin and hemicelluloses), esters 1279- 1268- 1265, 1265- 1267, 1266, 1279-1295 1269 1266 1267 1268 1266 C-H bending, CH twisting 1293 1281 1281 1282 1280 1275 2 1314- 1312- 1311, 1312, 1314, 1317 1317 1311 1316 1316 1313 1313 C–H wagging 1315 1317 1319 1316 1319 1328, O-H phenol group (cellulose), bending vibration in 1322-1328 - 1328 1328 1326 1329 1326 1330 1325 - - 1330 plane 1334, 1334, 1335, 1335 1335 1338 1337 1336 1335 1336 - - O–H in plane bending 1336 1337 1338 1338- 1338, 1332-1355 1337 1336 - 1339 - 1341 - 1342 1339 CH wagging, -C-O-H, lignin 1340 1341 2 1362- 1365- 1365-1366 1365 1367 1366 1365 1365 1370 1365 1365 - Symmetric CH3 deformation 1369 1367 1373, 1372- 1372- 1373, 1373, 1374, 1374, 1371, CH , H-C-C, H-C-O, C-H deformation (cellulose and 1375-1379 1381 1374 1375 2 1377 1379 1379 1379 1380 1378 1379 1380 hemicellulose) H–C–H, O–C–H in plane bending, aromatic skeletal 1420- 1421, 1420- 1421, 1422, 1410-1425 1424 1423 1421 1420 1417 - vibrations (lignin) and C-H deformation in plane 1427 1425 1426 1424 1425 (cellulose) 1474- 1456- 1457, 1454, 1456, O–H in plane bending, correlated to amorphous 1458-1478 1457 1457 1455 1460 1462 1462 1279 1460 1460 1463 1467 cellulose, C-H symmetric bending in CH3 (lignin) 1481 1481 ------1481 - - Correlated to cellulose crystalline

B-25 Giant G-25 M-25 Red R-25 Bamb. Literature Cotton Bissetii Moso Rayon Functional Group/Assignments NBF Gray NBF NBF Margin NBF Viscose

1484 1489 1488 1489 - 1485 - 1487 - 1489 - - CH2 stretch 1508- 1508- 1510- C=C, aromatic ring (lignin), stronger guaiacyl element 1511 1503 1512 1515 1513 1506 1508 - - 1511 1513 1514 than syringyl 1576- 1578- 1578 1577 1560 1580 1580 1577 1594 - - - C=C stretch 1578 1580 1603- 1603, 1601 1601 1603 1601 1605 1601 1605 1603 1601 1601 C=C, aromatic ring (lignin) 1605 1605 1636- 1628- 1631- 1635-1644 1635 1637 1631 1638 1633 1631 - - H-O-H bending of absorbed water 1640 1644 1639 1646- 1650 - 1650 1651 1650 1650 1651 1651 1650 - - C=O, quinines and quinine methides, adsorbed water 1650 1666- 1662 1683 1657 1660 1661 1658 1667 1660 1667 - - C=O, lignin 1670 1700- 1700 - 1702 1700 1699 1700 1697 1700 1702 1699 - C=O or C=C stretch, carboxylic acid, secondary amides 1708 C=O stretching of an ester, free carbonyl groups, 1733- 1733, 1733- 1733, stretching of acetyl or carboxylic acid (-COOH, 1735-1740 1732 1732 1728 1732 1738 - - 1742 1748 1745 1741 hemicelluloses), non-conjugated carbonyl vibration from lignin 2734- 2723, 2736 - - 2731 - 2739 2736 2735 - - CH methine 2742 2743 2850- 2844- 2850 2850 2855 2850 2850 2855 2850 2845 2849 2849 CH symmetrical stretching 2851 2855 2 2892- 2901, 2893, 2890- 2921- 2891, CH , C–H stretching (expanding and contracting 2897-2944 2897 2897 2932 2895 2895 2 2948 2948 2916 2932 2939 2937 vibration) 3100- 3000- 3000- 3000- 3000- 3000- 3100- 3100- 3100- 3100- 3100- 3100-3600 O–H stretching, intra- and intermolecular H bonds; 3600 3600 3600 3600 3600 3601 3600 3600 3600 3600 3600 3286- 3242- 3249, 3242- 3242- 3242- 3242- 3242- 3256, 3240-3285 - - Strongly h-bonded OH 3292 3290 3281 3290 3290 3291 3292 3293 3288 3331- 3330 3335 3343 3344 3337 3341 3335 3344 3325 3330 3330 Moderately h-bonded OH 3334 3369- 3364, 3370 3385 3365 3365 3365 3366 3366 3375 - - Weakly h-bonded OH 3384 3385 Note: B-25, G-25, M-25, and R-25 are the natural bamboo fibers (NBFs) from Bissetii, Giant gray, Moso and Red Margin bamboo species respectively. Different peak centers/band values in green fonts were only detected by Raman, orange fonts were only detected by ATR-FTIR and black fonts were detected by both spectroscopies.

1646-1651 cm-1 related phenolic –OH and quinines (C=O) groups but they were not found in cotton, rayon or bamboo viscose specimens. A peak at ~1700 cm-1 was identified in the spectra of bamboo specimens which was not found to be described in the literature. This peak can be defined as C=O or C=C stretch, carboxylic acid, secondary amides (Socrates, 2001).

The third 2700-3600 cm-1 band region is mainly detected by ATR-FTIR spectra and the peaks in this region were mainly related to stretch due to weakly, moderately or strongly hydrogen-bonded –OH group (Table 16). However, peaks at 3240-3293 and 3364-3385 cm-1, which are related to intermolecular and intramolecular H-bond stretching, were present in all specimens except bamboo viscose and rayon. Another distinct characteristic peak (within 2723-

2742 cm-1) was also noticed in the spectra of bamboo and NBFs specimens in this region which was described as stretch due to –CH- or methine group.

Thus, the investigation of chemical groups, bonds and behaviors provide us with an insight that though cotton, bamboo species, NBFs, rayon and bamboo viscose are cellulose-based materials, there were some definite distinctions between the regenerated cellulose materials

(conventional rayon and bamboo viscose) and natural cellulosic materials such as cotton, raw bamboo, and NBFs. It was also observed that bamboo viscose and conventional rayon had similar peaks in the spectra to each other and this was reasonable as they are generally pure cellulose. Furthermore, it was revealed that there were some unique characteristic peaks providing information on chemical behavior in raw bamboo specimens and NBFs. It should be noted that Table 16 represents a comprehensive list of characteristic peak and relevant chemical information of different materials but since some peaks with low intensities are also listed, there might be some inexactly identified peaks. It is also possible that some peaks from unknown chemical groups may be unidentified.

4.5 Conclusions

Elemental analyses of four bamboo species and their fibers by energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) revealed that the materials are mainly composed of carbon (C) and oxygen (O) along with hydrogen elements. Some other elements were also identified such as nitrogen (N), sodium (Na), calcium (Ca) and silicon (Si).

The composition of bamboo species by EDS was estimated as 64-65% C and 34-36% O by weight %, and 70-72% C and 28-30% O by atomic %. However, XPS produced slightly different composition: 76-78% C and 20-24% O by atomic %. The O/C ratio was found to be increased as the NBFs are separated from lignin. The O/C ratios were determined as 0.39-0.42 for bamboo species in EDS but this was lower in XPS as 0.26-0.33. The ratio was found to be 0.58-0.70 in

NBFs as assessed by EDS. The higher O/C value of NBF suggests that it is more moisture- wicking than raw bamboo. XPS survey on four bamboo species also revealed that C 1s and O 1s peaks with different components. After deconvolutions, C 1s was identified with four components (C1-C4) of four types of chemical bonding at different binding energies: C1 as C—C and C—H, C2 as C—O, C3 as C═O and/or O—C—O, and C4 as O—C═O bonding. The analysis of O 1s peak also revealed O1 as O—C═O and/or C═O and O2 as C—O type chemical bonding.

The N 1s peak was also detected in some specimens. Continuing studies would confirm the presence of other elements if any. Results from this investigation on EDS and XPS would help to determine chemical natures of bamboo and its fibers.

The investigation of raw bamboo specimens and NBFs from four bamboo species proved that using ATR-FTIR and Raman spectroscopies, a better chemical analysis of a material such as bamboo can be executed since each spectroscopy has the ability to detect some unique peaks or the same peak with different sensitivities. A series of both types of spectra of four bamboo plants

also revealed that they have an almost-similar pattern in spectra. Though different in intensities, patterns of spectra of NBFs were also found to be comparable. This provides us a conclusion that both raw bamboo plants and their NBFs may carry analogous chemical groups, bonds, and behaviors which was shown in the analysis of characteristic peaks. There were some obvious differences in the spectra of cotton, regular rayon, and bamboo viscose when compared with the spectra of each bamboo and its NBF. Bamboo and NBF had some unique characteristic peaks that were not identified in either cotton or viscose/rayon. These unique peaks were mainly from lignin, hemicellulose, cellulose, and its crystallinity. Moreover, some peaks were detected in both IR and Raman spectra which were not found in previously reported studies. This study provides a comprehensive list of chemical information and behaviors of bamboo plants and their natural fibers. Further in-depth investigation of more species using other spectroscopies with larger samples would be required to strengthen the results of this study.

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CHAPTER 5. INVESTIGATION AND COMPARISON OF ANTIBACTERIAL

PROPERTY OF BAMBOO PLANTS, NATURAL BAMBOO FIBERS AND

COMMERCIAL BAMBOO VISCOSE TEXTILES

5.1 Abstract

This study was conducted with 12 commercial bamboo viscose textiles, conventional rayon fabric, raw cotton fibers and fabric, four bamboo plant species and 12 natural bamboo fiber

(NBF) samples for antibacterial activity using Staphylococcus aureus and Klebsiella pneumoniae. The accuracy and efficacy of test methods were investigated and modified for antibacterial assessment. The spectrophotometric method was found to be less effective when bacterial reduction is not very high as bamboo and NBF samples with low antibacterial/antimicrobial activity were tested. The revised viable plate counting technique was very consistent and effective when tested materials were in fiber, fabric or crushed forms and tested in sterilized and non-sterilized states. Results revealed that only one bamboo viscose showed antibacterial activity. A majority of the specimens from bamboo plant species and NBFs showed a measurable percentage reduction of bacteria against K. pneumoniae (8-95%) but had poor results against S. aureus (3-50%). These results are interpreted to be due to the presence of both bacteria-killing and promoting compounds found in the bamboo plant specimens and the

NBFs. Overall, NBFs showed higher reduction of bacteria than raw bamboo specimens.

However, due to the presence of bacteria from the environment, some NBF specimens showed a

greater number of colonies and thus a negative reduction of bacteria when sterilization of the fiber specimens was not performed prior to the experiment protocol.

KEYWORDS: Antibacterial fibers; Antibacterial test; Viable plate counting; Antibacterial activity of fibers; Bamboo viscose textiles; Bacterial reduction.

5.2 Introduction

With the advancement of textile technology, the uses of textiles are not limited to apparel, clothing, coverings, or protection against the weather. Technical textiles such as medical textiles, geotextiles, and smart textiles are being developed for specialized uses. Textiles because of their nature are ideal substrates for providing antibacterial property. Currently, textiles and apparel with antibacterial/antimicrobial, antifungal and anti-mildew properties are very important for use in medical clothing, therapeutic clothing, wound healing, and protective clothing as well as regular daily use. The necessity and demand for such products has led to innovation and development of antibacterial textiles (Abdel-Mohsen et al., 2013; Budama, Çakir, Topel, &

Hoda, 2013; Burnett-Boothroyd & McCarthy, 2011; Cheng, Ma, Li, Ren, & Huang, 2014;

Chung, Lee, & Kim, 1998; Comlekcioglu, Aygan, Kutlu, & Kocabas, 2017; Gokarneshan,

Nagarajan, & Viswanath, 2017; Ibrahim, El-Zairy, El-Zairy, Eid, & Ghazal, 2011; Ma, Sun, &

Sun, 2003; Prusty, Das, Nayak, & Das, 2010; Ren et al., 2017; Shahid et al., 2012; Simoncic &

Tomsic, 2010; Tanaka, Kim, Oda, Shimizu, & Kondo, 2011; Vroman & Tighzert, 2009;

Williams et al., 2006). Accordingly, the research and the production of antibacterial textiles has grown to help meet the demand (Gupta & Bhaumik, 2007). Properties can be developed in the products by special treatment during dyeing, wet processing, finishing or any other process stages (Bhuiyan, Hossain, Zakaria, Islam, & Uddin, 2017; Comlekcioglu et al., 2017; Gupta &

Bhaumik, 2007; Ibrahim et al., 2011; Manickam & Thilagavathi, 2015; Morais, Guedes, &

Lopes, 2016; Patel & Tandel, 2005; Shalini & Anitha, 2016; Zhang, Chen, Ji, Huang, & Chen,

2003). A challenge has been to develop chemical compounds that can provide antibacterial activity in industrial processing, as currently they are most often toxic and harmful to the human body and health (Budama et al., 2013; Cheng et al., 2014; Morais et al., 2016; Shalini & Anitha,

2016). In contrast, natural compounds that provide antibacterial property are considered more human-friendly (Gokarneshan et al., 2017). Consequently, there are numerous efforts to develop antibacterial properties from natural materials and processes, such as natural antibacterial activity from plants or microorganisms in fibers, pigments or dyes (Burnett-Boothroyd & McCarthy,

2011; Cheng et al., 2014; Comlekcioglu et al., 2017; Manickam & Thilagavathi, 2015; Shahid et al., 2012). Moreover, if a material has natural antibacterial activity it could prove more durable as part of the material that would not wash away as opposed to artificially added antibacterial compounds.

Interestingly, bamboo has been reported as naturally antibacterial and this property is expected to be retained if the fibers are extracted in their natural form along with some non- fibrous trace elements. Since most of the existing natural and synthetic fibers do not have antibacterial activity, but rather some promote bacteria growth (Budama et al., 2013;

Comlekcioglu et al., 2017; Gupta & Bhaumik, 2007; Ibrahim et al., 2011; Morais et al., 2016;

Shalini & Anitha, 2016), extraction of NBFs with such activity is of great interest. Studies have concluded that extracts from bamboo leaves have shown antibacterial activity against some bacteria (Singh et al., 2010). Tanaka et al. (2011) investigated the inhibition of extract from

Moso bamboo shoot skins against Staphylococcus aureus and reported that it exhibited antibacterial activity. Moreover, different parts of bamboo plants (roots, leaves, skins) have already been used for manufacturing preservatives and medicinal products (Keski-Saari et al.,

2008; Nirmala, Bisht, & Laishram, 2014; Quitain, Katoh, & Moriyoshi, 2004; Tanaka et al.,

2011; Tanaka, Shimizu, & Kondo, 2013; Tanaka et al., 2014). These studies on multiple bamboo species confirmed that the plants possess antibacterial activity. It was also shown that bamboo culms exhibited very high antibacterial property and fibers are mainly extracted from the culms

(Tanaka et al., 2014). Therefore, it can be proposed that natural fibers from bamboo have the potential to retain antibacterial compounds, for example, phenolic compounds and lipids

(Gokarneshan et al., 2017; Mishra, Behera, & Pal, 2012; Nirmala et al., 2014; Quitain et al.,

2004), and thus antibacterial properties. Some studies have indicated that NBFs extracted from the bamboo will have antibacterial properties (Afrin, Kanwar, Wang, & Tsuzuki, 2014; Afrin,

Tsuzuki, Kanwar, & Wang, 2012; Chen, Wang, Xu, & Sun, 2015; Waite, 2009; Zupin &

Dimitrovski, 2010).

5.2.1 Methods for Antibacterial Activity Assessment

Though there are different methods developed by AATCC, ISO, ASTM, and many other organizations, most of the methods are either qualitative or less precise quantitative methods and results vary by different factors such as sample preparation, size, shape, technique, and other parameters. Consequently, there are misunderstandings in industry and the market place about what is being measured (Burnett-Boothroyd & McCarthy, 2011; Williams et al., 2006).

The Agar disc diffusion method or parallel streak method (AATCC 147-2016, 2018;

AATCC 90-2016, 2018; ISO 20645, 2004) has been employed for many years relying on a qualitative assessment. Since a clear inhibition zone around the specimens is required to be measured, the quantitative assessment with this method has a limited accuracy when the antibacterial activity of the specimen is low. An example is a result of zero antibacterial activity which could be misleading (Abdel-Mohsen et al., 2013; Budama et al., 2013; Cheng et al., 2014;

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Comlekcioglu et al., 2017; Ibrahim et al., 2011; Manickam & Thilagavathi, 2015; Sarkar &

Appidi, 2009; Shahid et al., 2012; Xue, Chen, Yin, Jia, & Ma, 2012). In fact, the lack of a zone of inhibition or unclear inhibition zone is not always indicative of the lack of antibacterial property (Monticello & Askew, 2013). Thus, a specimen with very low antibacterial properties may sometimes be inappropriately graded as a non-antibacterial (Hardin, Wilson, Dhandapani, &

Dhende, 2009) or a reverse problem may arise. If the number of bacteria is less than the antibacterial compounds in the specimen, an inhibition zone may be clear or seen. Moreover, a material with low activity may show a wide zone of inhibition if it is subjected to a lower number of bacteria by the higher amount of antibacterial compounds/agents or by quick deactivation/death of bacteria that are less durable (Monticello & Askew, 2013). On the other hand, some materials may promote bacterial growth rather than inhibition. So, there may be growth of colony underneath the plated sample (Budama et al., 2013; Sarkar & Appidi, 2009;

Shateri Khalil-Abad & Yazdanshenas, 2010) or a higher number of colonies then the control which leads to negative percentage of bacterial reduction (Afrin et al., 2012; Ma et al., 2003; Xi,

Qin, An, & Wang, 2013). Most of the investigation described the negative results as no antibacterial property (Xi et al., 2013). Such results may occur due to inconsistent sample preparation, presence of some test-bacteria or other microorganisms already on the sample, or promoted growth by the specimens. Though the percentage reduction of bacteria by bacterial counting is quantitatively measured exposing fabric specimens to the bacterial culture for a certain time (0—24 h) in standard methods, testing of non-fabric samples such as fibers, small particle samples (dyes, pigments) are not covered well by these AATCC, ASTM or ISO methods.

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In Optical density (OD) or turbidity, bacteria in an inoculated culture (bacteria with nutrient broth) is measured by a spectrophotometer after incubating the culture with samples mixed into the liquid (Comlekcioglu et al., 2017; Prusty et al., 2010; Tanaka et al., 2011, 2013).

Again, a significant change in optical density or turbidity is required to estimate a difference in the reduction of bacteria. When a small number of bacteria are killed, the change in turbidity is almost “zero”. So, it is difficult to identify if the sample has any antibacterial property.

The core purposes of this study were 1) practicing and modifying antibacterial techniques that may be applied for textile material in any form, 2) assessing the activity of four bamboo species and their respective fibers (NBFs), 3) comparison of the results with 15 commercial fibers and/or fabrics, and 4) assessing different conditions and factors that may affect the tests.

Bacteria (microbes) for Assessment

Generally, a gram-positive and a gram-negative bacterium are recommended for antibacterial activity tests for textile materials. Staphylococcus aureus, a gram-positive facultative bacterium that can grow even without oxygen, is one of the most common pathogens that live on human skin and causes skin infections, food poisoning, respiratory infections and many others issues

(Tanaka et al., 2011). It has been repor ted that half a million people are hospitalized due to S. aureus, with 50, 000 deaths each year caused in the USA (Schlecht et al., 2015). Therefore, testing with S. aureus is highly recommended, especially with products that interact with the human body. Klebsiella pneumoniae (K. pneumoniae) is one of the most common gram- negative, facultative anaerobic, bacterium that lives on skin, in the mouth and intestine, and may cause many serious diseases such as pneumonia, urinary tract infections and liver diseases

(Paczosa & Mecsas, 2007; Vading, Nauclér, Kalin, & Giske, 2018). Thus, S. aureus and K. pneumoniae bacteria were selected for this study.

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5.3 Methodology

5.3.1 Materials

Fresh culms of four bamboo species directly from the field were provided by Lewis Bamboo,

Inc., Oakman, Alabama. The species were Bissetii (Phyllostachys Bissetii), Giant Gray

Table 17

Names and compositions of different commercial textile products and relevant information

Sample Type of the product Composition of sample Price Retailer/supplier initials

RT Regular rayon, twill fabric 100% RR - Dharma Trading Co. BV Bamboo viscose cloth 100% BV - Dharma Trading Co. AR Bamboo Sweater Tank (Magenta) 100% BV $19.99 Ash & Rose Bamboo Dreams Pillowcases® Yala 100% BV $20.00 Yala Designs (White) BSA Men's T-Shirts (white) 95% BV+5% S $29.99 Bamboosa® LLC BB Bamboo V-Neck Tee (White) 70% BV+30% C $19.95 Bambu Batu Bamboo Short-Sleeve T-Shirts BBB 70% BV+30% C $39.00 Big Boy Bamboo (Black) Bamboo Athletic V-Neck Cari 40% BV+55% C+5% S $12.00 Cariloha® Bamboo (Caribbean Breeze) FD Spun Bamboo Tee (Cream) 70% BV+30% C $22.00 Faerie's Dance Women's Bamboo Motion-V FF 68% BV+29% P+3% S $29.95 Free Fly Apparel® (Pacific Blue) GA Bamboo Yoga Pants (True Black) 60% BV+26% C+14% S $24.99 Green Apple Active NCC Men's Turtleneck shirt (Black) 70% BV+25% C+5% S $19.00 Natural Clothing Company Off the Shoulder Short Sleeve (Off Piko 95% BV+5% S $24.99 Piko Clothing White) UCF Untreated cotton fabrics 100% Cotton fabric - Testfabrics Inc. Raw cotton fibers from Alabama Cooperative RCF Raw cotton fibers - plants Extension System Mildly bleached cotton BCF Bleached cotton fibers - RCF bleached in lab fibers Note: RR = regular rayon; BV = bamboo viscose; S = spandex; and C = Cotton; P = Polyester.

(Phyllostachys nigra 'henon'), Moso (Phyllostachys edulis/ pubescens) and Red Margin

(Phyllostachys rubromarginata). Sigma-Aldrich, USA was used as the source for: sodium hydroxide/NaOH (beads, AG*), sodium bicarbonate/NaHCO3 (AG), sodium silicate/Na2SiO3

(RG), sodium triphosphate/Na5P3O10 (85%, TG), sodium sulfite/Na2SO3 (≥ 98%), and acetic acid/CH3COOH (≥ 99%, AG); hydrogen peroxide/H2O2 (30% w/w, AG) was purchased from

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VWR International, LLC, USA; from Consos, Inc. USA: scouring agent (Consoscour GSRM), wetting/penetrating agent (Consamine JDA), and bleaching agent (Consobleach CIL) were collected. Additionally, fabric softener was collected from a local Publix Super Market, Inc.,

USA (Free & Clear Ultra Fabric Softener). Details about different commercial fabrics, apparel, and fibers used in this study are reported in Table 17.

Note: *AG = ACS (American Chemical Society) grade; *RG = reagent grade; *TG = technical grade.

5.3.2 Equipment

The following equipment was used: milling machine/rolling mill (Rolling Mill Division, Stanat

Manufacturing Co. Inc, NY, USA), Launderometer (AATCC Launder-Ometer, Atlas material testing Technology LLC), Dryer (GCA corporation, Thelco Precision Scientific Laboratory

Oven, Model 18), conditioning cabinet/Temperature and Humidity Environmental Test Chamber

(Associated Environmental Chamber), Autoclave (Consolidated Stills & Sterilizers machine),

Spectrophotometer (Genesys 10 UV Spectrophotometer, Thermo Electron Corporation), Vortex mixer (Vortex-Genie, Scientific Industries, Inc.), Ion sputter coater (Leica, MCM-200 Ion

Sputter Coater), SEM— Apreo S HiVac (Thermo Fisher Scientific) and FEI Quanta 200 3D.

5.3.3 Extractions of Natural Bamboo Fibers

The NBFs were extracted from four bamboo species followed by pre-processing, delignification and modifications as discussed in Part 1. Pre-processing or initial treatments involved splitting of fresh culms, crushing/pressing multiple times in milling machine by applying gentle pressure to avoid excess breakage of fibers, soaking with the chemical reactants in recipe 0 (as in Table 18), combing by steel combs and pressing in the milling machine. The nomenclature of the extracted

NBFs are as follows; the initial of the name of the bamboo species is followed by the specific route (number) where “R” is for Red Margin, “B” is for Bissetii, “M” is for Moso and “G” is for

Giant Gray. Since some NBF specimens exhibited positive and prospective results in different 104

physical, mechanical and chemical tests, the following fibers were selected for antibacterial investigation. The extraction processes of the selected NBFs are discussed below:

R-1: this was longest NBFs from Red Margin after initial treatment and with recipe 0.

R-9: a treatment with recipe 1 (Table 18) was applied to R-1 followed by finishing treatments: washing, neutralizing, warm and cold washing, and drying.

B-16: after initial processing, recipe 0 and recipe 2 were applied consecutively and then finishing treatments were applied.

Table 18

Different chemicals and conditions applied to different stages of natural bamboo fiber (NBF) extraction

M-21/G-21: initial treatments, recipe 0, recipe 3, and finishing treatment were executed one after another.

M-22: initial treatments, recipe 0, recipe 5 along with 20 g/L of fabric softener, and then the finishing treatment were performed respectively.

G-23/B-23: initial treatments, recipe 0, recipe 5 along with 20 g/L of fabric softener, recipe 4 were applied respectively followed by finishing steps.

M-25/G-25/B-25: recipe 7 was applied to G-23/B-23/M-23 before finishing treatments.

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R-27: initial treatments, recipe 0, recipe 6, recipe 4 and then recipe 7 were applied one-by-one and fibers were yielded after the finishing steps.

5.3.4 Sample Preparation for antibacterial tests

Each bamboo species was crushed separately in a milling machine including their exodermis and endodermis. These specimens were dried and raw bamboo samples were prepared. Each fiber and fabric sample (Table 17) was cut into small pieces by using scissors and then stored separately in either glass vials or standard reclosable polyethylene Ziploc bag. For sterilization, to remove any in situ bacteria, specimens were inserted into sterilization pouches of 3.5” ×

10” or 90 푚푚 × 260 푚푚 sizes and autoclaved by applying the steam method of at 121 °C for

20 minutes in Consolidated Stills & Sterilizers m/c.

5.3.5 Antibacterial Tests and Assessments

This study on antibacterial tests, preparation, methods and other relevant information used from the standard methods of AATCC, ASTM, ISO, and TAPPI (AATCC 100-2012, 2018; AATCC

147-2016, 2018; AATCC 90-2016, 2018; ASTM E2149-13a, 2013; ISO 20645, 2004; TAPPI

T205 sp-02, 2006). The gram-positive bacterium Staphylococcus aureus, as referenced in ATCC

(American Type Culture Collection) No. 6538, and the gram-negative bacterium Klebsiella pneumoniae, as referenced in ATCC No. 4352 were used for the study.

Spectrophotometric technique (Optical Density, OD600)

A 1mL culture of approximate concentration of 109 CFU/mL from each bacterium was added to

9 mL tryptic soy broth (TSB) in a test tube. The McFarland equivalence turbidity standard (3~4) and visual comparison card (Thermo Scientific™ Remel) were used to match turbidity of the cultures to achieve an approximate concentration of 109 CFU/mL. Then 1 g of fiber or fabric sample was added to each test tube. This was done separately for both bacteria. All the samples

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were incubated for 20 h at 37 °C. A control sample was also incubated that contained no fiber samples. The percentage reduction of bacteria (%푅) was determined by optical density (OD), turbidity or absorbance using equation (1). The optical density (OD) is defined as the amount of light that passes through a bacterial solution and it estimates the concentration of bacteria in a suspension. A lower OD value means a higher reduction in bacteria. In this method, the optical density of the control culture solution of bacteria (푂퐷푐표푛푡푟표푙), and optical density of the culture solution of bacteria that is in contact with a sample for 18-24 h time (푂퐷푠푎푚푝푙푒) were measured by spectrophotometer (light with 600 nm wavelength).

(푂퐷푐표푛푡푟표푙 − 푂퐷푠푎푚푝푙푒) % 푅푒푑푢푐푡푖표푛 = × 100% … … … … (1) 푂퐷푐표푛푡푟표푙

This technique was employed in previous investigations for textiles fibers, yarns, fabrics, dyes and other materials (Afrin et al., 2012; Shahid et al., 2012). The test was performed separately with small-cut fiber/fabric (less than 1 cm long) and large-cut (1~2 cm long) samples to see if the size of the specimen affects test results. The optical density of the culture was measured by taking 1 mL culture solution from each sample in cuvettes carefully so that no fiber/fabric portions were in a cuvette and then each cuvette was measured by the spectrophotometer. Each specimen culture was shaken (20-60 seconds) on vortex properly before transferring from test tubes to cuvettes.

Viable Plate Counting Technique

Overnight-grown (12-14 hours) S. Aureus and K. pneumoniae in TSB (Tryptic soy broth) were diluted in sterile water until the turbidity of the culture was between 3 and 4 McFarland standards, providing a concentration of approximately 109 CFU/mL. Then the serial dilution of the cultures was done as follows in 3 steps [CFU/mL values are approximate] —

1. 99 mL Water + 1 mL of 109 CFU/mL  107 CFU/mL

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2. 99 mL water + 1 mL of 107 CFU/mL  105 CFU/mL

3. 99 mL water + 1 mL of 105 CFU/mL  103 CFU/mL

Then 0.1 g of fiber or fabric specimen from each sample were taken in separate sterile test tubes, and 10 mL of 103 CFU/mL culture was added. Test tubes were incubated for 1 h, and 18-24 h before plating. Tryptic Soy Agar (TSA) plate 15 × 100 푚푚 monoplate from Hardy Diagnostics was used for this study. A micro-pipetter was used to collect 100 μL (0.1 mL) of solution from each test tube, shaking it well, and then placing it on 3 TSA plates (triplicate), followed by spreading the sample uniformly by inoculating loops. One pipetter tip and an inoculating loop were used for each specimen and then they were disposed of in germicidal disinfectant (Lophene solution). A vortex mixer was used for shaking 20-60 seconds before transferring each specimen culture from one container to another or before plating. Incubation of the TSA plates was done for 20-24 h and 60 h at 37 °C. After incubating, photographs of each plate were taken to count the number of bacterial colonies in a computer. The used TSA plates were refrigerated before disposing and destroying the bacteria. The percentage reduction of bacteria was calculated for antibacterial activity assessment by using equation (2).

(푁푐표푛푡푟표푙 − 푁푠푎푚푝푙푒) % 푅 = × 100% … … … … (2) 푁푐표푛푡푟표푙

Here, %R = % Reduction of bacterial colony, 푁푐표푛푡푟표푙 = Number of the bacterial colony on the control plate, 푁푠푎푚푝푙푒 = Number of the bacterial colony on sample inoculated plate. The percentage reduction of bacteria was also calculated as mentioned in equation (1) as was standardly done in other studies (Manickam & Thilagavathi, 2015; Mishra et al., 2012; Ren et al., 2017; Xi et al., 2013; Zhang et al., 2003).

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5.4 Results and Discussions

5.4.1 Assessment by Spectrophotometric technique

This technique was not effective because the antibacterial activity or inhibition to bacterial growth of the samples was very low. As a result, the differences in the OD readings were very small. In most of the cases, the values of OD were found to be the same when measuring before or after inoculation followed by incubation. In most cases, the percentage of bacterial reduction was incorrectly determined. For example, though “Yala’ showed very good antibacterial activity against both bacteria in later experiments, it was found to be very poor by this technique (Table

19). It was especially difficult to assess %R of NBFs samples when antibacterial activity is not very high. Observations suggested that the size of the fiber/fabric specimen was not clearly influential on the results (Table 19). This was later confirmed by the viable plate counting method.

Table 19

Percentage reduction of the optical density (OD) of different sample inoculated bacterial solutions after incubation of 24 h at 37°C K. pneumoniae S. aureus Sample Size Sterilized OD %R OD %R Control - - 0.816 0.00 0.877 0.00 Larger yes 1.131 -38.60 1.025 -16.88 100% Bamboo Smaller yes 1.075 -31.74 1.112 -26.80 Viscose Larger no 0.836 -2.45 1.064 -21.32 Smaller no 1.106 -35.54 1.060 -20.87 Larger yes 0.781 4.29 0.910 -3.76 Yala (100% Smaller yes 0.821 -0.61 0.815 7.07 bamboo Larger no 0.889 -8.95 0.794 9.46 viscose) Smaller no 0.698 14.46 0.819 6.61 Raw Red yes 1.291 -58.21 1.232 -40.48 Mixed Margin no 1.297 -58.95 1.106 -26.11

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5.4.2 Assessment by Viable Plate Counting Technique

The test with K. pneumoniae was found to work in the direct plate counting technique where the sample-inoculated bacterial culture was shaken and incubated for 20-24 hours. The growth of colonies was evident but with a lower number of colonies. On the other hand, there was no colony in the case of S. aureus. Probably, the S. aureus bacteria were already dead and ineffective before they were plated after a 20-24-h incubation of sample-inoculated culture. So, there was no bacterial growth on any of the TSA plates (including control plate). This technique was repeated 3 times to confirm the result were consistent but still, no growth of S. aureus was evident. A probable reason is that the bacteria were dead in the water-diluted culture as there was not enough food for them for a long period of time. So, a shorter time (1 h) of shaking and incubation of sample-inoculated culture was considered before plating on TSA plates.

After shaking and incubating of sample-inoculated bacterial culture for 1 hour, both of the bacteria worked well in this technique. The number of colonies on the controlled TSA plates after 20-24 hours of incubation was achieved within 80-150. So, by plating cultures of S. aureus and K. pneumoniae after inoculating with different samples, the resulted bacterial colonies were usable for counting and the calculation of reduction of bacterial growth (%R). The results were pretty consistent and comparable. Since existing standard test methods are mainly suitable for fabrics or require fabric-like (strip) sample preparation and results are only comparable for textiles with a high degree of antibacterial activity, this method will be suitable for even a sample with very low antibacterial activity and in any form (fiber, yarn, fabric or other forms). The natural bamboo fibers, produced in eco-friendly ways, seem to have a certain degree of antibacterial activity, that can provide some protection against bacteria naturally. Some fibers may not have very high natural antibacterial activity but they are at least useful for a certain

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degree of protection that comes without any additional cost. The assessment by employing some existing test methods may estimate a result of zero or no antibacterial activity of textiles with low antibacterial activity as they need either clear inhibition zones or a substantial change in the concentration of bacterial culture. Thus, this method will be applicable for identifying the antibacterial activity in a sample if any.

Another finding of this research is that though it is mainly recommended to test antibacterial activity of textile materials without sterilization, it may sometime be biased when raw materials or untreated natural fibers are tested. It was found that there were numerous unexpected bacterial colonies on TSA plates when raw cotton fibers directly collected from cotton-balls or raw bamboo samples exposed to damp weather for few days were used for antibacterial tests (Figure 20a, 20c, 20e, 20g). But unexpected growth was not observed when

(a) Non-sterilized BS, KP (b) Sterilized BS, KP (c) Non-sterilized BS, SA (d) Sterilized BS, SA

(e) Non-sterile. RCF, KP (f) Sterilized RCF, KP (g) Non-sterile. RCF, SA (h) Sterilized RCF, SA

Figure 20. Unexpected bacterial growth on TSA plates inoculated with raw Bissetii bamboo

(BS) and raw cotton fibers (RCF) when samples were non-sterilized and exposed to the environment vs. bacterial growth by sterilized samples [KP = K. pneumoniae; SA = S. aureus].

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sterilization was done to the samples before they were added to the broth (Figure 20b, 20d, 20f,

20h). Since K. pneumoniae grows faster than many types of bacteria, they might be possible to separate in some cases (Figure 20a). Smaller size bacteria like S. aureus is almost impossible to separate (Figure 20c). It was also observed that %R of the bacteria was almost the same for sterilized and non-sterilized materials (Table 20).

(a) Control, SA, 24 h (b) G-21, SA, 24 h (c) M-25, SA, 24 h (d) R-27, SA, 24 h

(e) Control, SA, 60 h (f) G-21, SA, 60 h (g) M-25, SA, 60 h (h) R-27, SA, 60 h Figure 21. Sizes of the bacterial colony of S. aureus inoculated with natural bamboo fibers

(NBFs) after 24 h vs 60 h with almost unchanged counts. [SA = S. aureus; G-21, M-25, and R-

27 are NBFs from Giant gray, Moso, and Red Margin bamboo species respectively.]

Another issue was counting bacterial colonies when the size of the colony is very small and hard to recognize. This happens when test bacteria grow very slow like S. aureus even after

24 hours. In such a case, counting may be incorrect when there are overlapping or too many bacteria. Therefore, two observations were made for S. aureus bacteria: one was after 24 h and another was after 60 hours (Figure 21). It was difficult to count K. pneumoniae after 60 hours as the colonies overlapped. However, the number of the bacterial colonies after 24 h and 60 h were

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almost the same for S. aureus and it was easy to see and count them after 60 hours as shown in

Figure 21. So, the incubation can be allowed for a longer time for slow-growing bacteria when a viable plate counting technique is employed.

5.4.3 Antibacterial Property of Bamboo, NBF and Bamboo Viscose

Out of 11 bamboo viscose textiles, only one sample (Yala) exhibited antibacterial activity against both bacteria by at least 80% reduction of bacteria. Some of the samples showed nominal inhibition against one or both bacteria. However, most of the bamboo viscose products did not show any reduction of bacteria (Table 20). Similar behaviors were also observed for regular rayon and cotton fiber/fabric. BV and AR were other 100% bamboo viscose textiles showed

(35% and 43%) bacterial reduction against K. pneumoniae when non-sterilized samples were tested but sterilized samples did not show such activity. Otherwise, almost all other samples exhibited a similar percentage reduction of bacteria in both sterilized and non-sterilized cases. It

Table 20

Percentage reduction of bacteria by different commercial textile products

% Reduction, S. aureus % Reduction, K. pneumoniae Sample Non-sterilized Sterilized Non-sterilized Sterilized Control 0.00 0.00 0.00 0.00 RT -12.70 12.35 2.70 -12.32 BV 20.63 -13.58 35.14 -2.17 AR 4.76 2.06 43.24 7.25 Yala 90.48 80.25 100.00 98.55 BSA -19.05 14.81 -51.35 -27.54 BB -7.94 5.76 -10.81 -40.58 BBB -11.11 16.46 5.41 -44.20 Cari -4.76 -2.47 -64.86 -44.57 FD -7.94 7.00 -43.24 -50.36 FF -9.52 5.76 -10.81 6.30 GA 6.35 15.23 10.81 -43.12 NCC -14.29 4.94 -27.03 -33.70 Piko 4.76 -4.12 5.41 -25.00 UCF -17.53 9.53 -46.38 9.80 RCF 11.11 -5.10 -75.68 -16.73 BCF 30.16 17.35 37.84 11.84

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was observed that some sterilized materials showed a slight reduction of S. aureus growth (Table

20). Since there might be an unequal number of bacteria in plating due to a slight difference in concentration of CFU/mL from sample to sample test tubes, %R with a smaller difference bypositive or negative numbers may not be indicative of inhibition against or support of a bacterium. As we can see in Figure 22a, 22d, 22e, and 22h, Yala, a commercial apparel specimen, had antibacterial activity and reduced bacterial growth significantly for both bacteria as compared with the control. But %R of both bacteria by 100% bamboo viscose was not noticeable and was lower than even mildly bleached cotton fibers, BCF (Table 20). The BCF was prepared from raw cotton fiber (RCF) by treating with 4 mL/L Conscour GSRM (scouring agent), 4 mL/L Consobleach CIL (bleaching agent) and 5 mL/L Consamine JDA (penetrating agent) at 90 °C for 1 h following by washing and drying.

(a) Control, KP, 24 h (b) BV, KP, 24 h (c) BCF, KP, 24 h (d) Yala, KP, 24 h

(e) Control, SA, 60 h (f) BV, SA, 60 h (g) BCF, SA, 60 h (h) Yala, SA, 60 h Figure 22. Growth of the bacterial colony on TSA plates inoculated with different samples. [KP

= K. pneumoniae; SA = S. aureus; BV = 100% Bamboo viscose; BCF = mildly bleached cotton fibers; Yala = 100% bamboo viscose apparel.]

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As indicated from previous studies discussed earlier, bamboo has some compound that is responsible for showing antibacterial activity against some bacteria. This study also found bacterial inhibition by bamboo species and NBFs in most of the cases but the degree of such activity was not high for all samples (Table 21). Some samples had higher % reduction of bacteria by sterilized specimens. Probably, unexpected non-cultured environmental bacteria

Table 21

Percentage reduction of bacteria by four bamboo species and their natural fibers

% Reduction, S. aureus % Reduction, K. pneumoniae Sample Non-sterilized Sterilized Non-sterilized Sterilized Control 0.00 0.00 0.00 0.00 Bissetii 15.56 -9.55 24.32 52.65 B-16 12.70 -7.14 16.22 55.92 B-23 -1.59 0.00 32.43 61.22 B-25 -9.52 0.00 48.65 66.53 Giant Gray 1.00 19.39 -72.97 61.22 G-21 12.70 3.06 24.32 54.69 G-23 -4.76 10.20 8.11 55.92 G-25 -3.17 15.31 83.78 79.59 Moso 1.59 9.14 -40.54 51.84 M-21 -20.63 4.08 -5.41 53.47 M-22 -6.35 0.00 45.95 56.73 M-25 15.87 22.45 78.38 62.86 Red margin -3.17 8.64 -86.49 -18.37 R-1 -6.35 2.04 -24.32 22.45 R-9 3.17 3.06 32.43 94.69 R-27 49.21 14.29 27.03 -12.65 influenced non-sterilized samples tests as indicated by the previous study (Rocky & Thompson,

2019). It was also noticeable that most of the sterilized NBFs exhibited bacterial inhibition and

% reduction of bacteria against K. pneumoniae was more than 50% by most of the samples

(Table 21, Figure 23). Results indicated that Giant Gray and Moso bamboo showed higher bacterial inhibition against K. pneumoniae bacteria when samples were tested after sterilization.

Such results were stated in other studies (Nirmala et al., 2014). This was opposed to some studies that bamboo and its extracts showed less antibacterial property against gram-negative bacteria such as Escherichia coli (E. coli) (Tanaka et al., 2013). However, this study was not with E. coli

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as a gram-negative bacterium. So, perhaps, bamboo and NBFs may have lower inhibition against

E. coli.

Many studies have also reported that bamboo and its extracts showed antibacterial activity against S. aureus (Singh et al., 2010; Tanaka et al., 2011). However, S. aureus bacterial reduction percentages by raw bamboo specimens and NBFs were lower. In all of the cases, raw bamboo and NBFs exhibited higher inhibition against K. pneumoniae than that of against S. aureus (Table 21). There was not a big difference in antibacterial activity among different bamboo species. Though % reduction of bacterial colonies by different NBFs were not significantly high, there was an obvious reduction of bacteria by some samples (Figure 23) and the reduction was higher when fibers were extracted to a certain stage, especially finer or single fiber stages. It was not clear whether the activity resulted from remnant non-fibrous materials

(a) Control, SA, 60 h (b) B-25, SA, 60 h (c) M-25, SA, 60 h (d) R-9, SA , 60 h

(e) Control, KP, 24 h (f) B-25, KP, 24 h (g) M-25, KP, 24 h (h) R-9, KP, 24 h

Figure 23. Growth of bacterial colony on TSA plates inoculated with different natural bamboo fiber (NBF) samples [KP = K. pneumoniae; SA = S. aureus; B-25, M-25, and R-9 are NBFs from

Bissetii, Moso, and Red Margin bamboo species respectively].

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(a) B-23, 10000X (b) M-22, 2000X (c) R-27, 5000X

(d) M-25, 2900X (e) G-23, 1400X (f) R-9, 1700X Figure 24. Microscopic (SEM) images of natural bamboo fibers (NBFs) from four different species showing non-fibrous contents responsible for natural properties [Images a—c from

Apreo S and images d—f from Quanta; B = Bissetii; G = Giant Gray; M = Moso; R =Red

Margin].

on extracted NBFs as shown in Figure 24 or from processing chemicals, like NaOH. However, the SEM images of the fibers with antibacterial property revealed that there was a leftover non- fibrous material attached to or lying on different NBFs (Figure 24).

Larger negative numbers of % reduction (up to -86%) of bacteria were found for some raw bamboo samples or NBFs with high non-fibrous contents (Table 21). As presented in Figure

20 that unexpected, environmental or non-test bacterial growth may occur when a material has bacteria-promoting substances. So, it is crucial to test the antibacterial activity of a material in both sterilized and non-sterilized conditions.

It is also important that testing antibacterial activity against only two bacteria may not be enough to assess the property of NBFs as there are more than 650 organisms that can cause

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disease to the human body (Budama et al., 2013). So, the NBFs may also have effectiveness against some other disease-causing bacteria and it has not been proven yet.

There is speculation that viscose fibers or regenerated cellulose from bamboo are antibacterial (Afrin et al., 2014; Mishra et al., 2012; Waite, 2009). But it was reported either nominal or with no such activity (Hardin et al., 2009; Xi et al., 2013) in tests. The inconsistency of the antibacterial property by bamboo viscose may raise a question about its authenticity. Since viscose fibers are extracted cellulose, no natural non-fibrous elements may be retained and such property may come from processing chemicals or through provided antibacterial treatments or modifications (Afrin, Tsuzuki, & Wang, 2009; Gokarneshan et al., 2017; Mishra et al., 2012;

Sarkar & Appidi, 2009; Xi et al., 2013; Zheng, Song, Wang, & Wang, 2014).

5.5 Conclusions

To test the antibacterial property of natural fibers or plants, it is important to have suitable methods that can be conducted appropriately to assess even when the activity is very low. Since the zone of inhibition technique is not suitable and highly precise for non-fabric specimens, this study was conducted on spectrophotometric and viable plate counting techniques to assess the antibacterial property of different types of specimens. Since natural property as bacterial inhibition is generally not very high and the change in optical density very low, the spectrophotometric technique was found to not be suitable for such tests. The viable plate counting was revealed to be very suitable for almost all types of samples such as fabrics, fibers, and crushed raw bamboo samples. Results were found to be consistent and repeatable if conducted carefully. Sterilization of the samples before the experiment is recommended as any contaminating bacteria is eliminated. A longer period of time may be allowed for slow-growing bacteria for better counting precision.

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Percentage reduction of bacteria by bamboo viscose textiles was tested where antibacterial activity was found in only one out of 12 products. This provides an understanding that antibacterial properties by bamboo viscose may not be a property from the bamboo starting material, rather it comes from special treatments or through processing chemicals. Without these additional treatments, products from bamboo viscose would not offer bacterial protection. On the other hand, microscopic images revealed that there were some non-fibrous elements with natural bamboo fibers (NBFs) from four bamboo species. All four bamboo species and their NBFs exhibited antibacterial activity to a certain degree, at least at a low level. The activity was stronger against K. pneumoniae than against S. aureus. Results also suggested that though raw bamboo has antibacterial compounds, it may have bacteria-promoting substances at the same time that caused higher growth of bacteria if non-sterilized samples were tested. The bacterial inhibition was higher for sterilized bamboo and NBF samples. However, further investigations are needed to confirm if bamboo species and its NBFs have antibacterial activity and such activity is not contributed to processing chemicals. Moreover, the antibacterial property of such materials needs to be tested against other bacteria species.

5.6 References

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CHAPTER 6. CONCLUSIONS

The focus of this proposed research was to investigate and model a systematic-achievable production process of natural bamboo fibers, examination and characterization of the properties of the fibers in terms of physical, mechanical, chemical, crystallographic, and antibacterial properties. The extraction processes included production and modification of the natural bamboo fibers, blending with cotton fibers, carding-combing for sliver preparation, and spinning the fibers into yarns. The production of the fibers included the well-known bamboo species such as

Bissetii (Phyllostachys Bissetii), Giant Gray (Phyllostachys nigra 'henon'), Moso (Phyllostachys edulis/ pubescens) and Red Margin (Phyllostachys rubromarginata). The investigation on expected NBF extraction was successfully attained through different routes. The usage of the total amount of NaOH (6-38 g/L) in different routes and NBF yield percentages in each route are reported to assess financial viability. The assessment and identification of the physical and mechanical properties included dimensions of the fibers such as average length and diameter, fineness, moisture content, tenacity, elongation, incineration behavior, and solubility. This dissertation also includes reports on chemical composition, functional groups, behaviors and crystallographic data on bamboo plants and NBFs that were not determined and not available in prior literature. Designing and examining a suitable technique, a detailed investigation of the antibacterial properties, as well as the degree of the activity of the four bamboo plants, natural bamboo fibers, and 15 commercial textiles, was conducted.

By using 21 formulas with different chemicals and concentrations, 36 different routes were followed to extract 144 types of NBF samples from four bamboo species. It was observed

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that at least 47 NBF specimens had good prospects to promote for different uses, especially fibers for textiles and composites. Different properties of selected NBFs were assessed and compared with selected conventional fibers. A few sets of NBFs were blended with raw cotton fibers and spun into yarns. Moisture regain (%) and moisture content (%) of NBFs were found to be around 8% and 7.5% respectively which was lower than bamboo viscose (10.71% and 9.67%) or regular rayon (10.83% and 9.77%) but higher than cotton fibers (6.0-6.5% and 5.0-6.0%) and polyester fibers (0.82% and 0.81%). Incineration properties of different raw bamboo samples and

NBFs were investigated by the standard burning test method. Though some incineration behaviors were similar to cotton, most of the behaviors were found to be distinctive to some degree. Similarly, solubility tests also revealed that bamboo biomass and NBFs have almost the same solubility but they have different appearance in colors and residues in the solutions.

Microscopic examination was employed with scanning electron microscope (SEM) to inspect surface morphology of the raw bamboo specimens and NBFs. The diameter of the NBFs in terms of the single fibers was measured which was found to have an average of 10-13 μm and the range was between 4-22 μm. Yield percentages of the selected fibers were between 40-77%. The average length of the fibers varied with the routes. The NBF with the highest average length was extracted from Red Margin bamboo which was 33-37 cm. The extracted NBFs from the other bamboo species were shorter but within the average spinnable length 1.5-10 cm. The fineness, in terms of linear density, of the NBFs was 9.68—93.3 Tex and were found to be higher than cotton fibers. However, none of these fibers were processed or modified with precise machines which could produce finer fibers. The tensile properties of the spun yarns and some longer fibers were tested. While the longer but coarser NBFs showed a very high breaking tenacity of 60-140 N/Tex and a lower breaking elongation of 2.06-2.46%, the NBF yarns of shorter and finer fibers

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blended with cotton showed a very low breaking tenacity of 1.50-2.50 N/Tex and a higher breaking elongation of 8.5-11.0%.

The investigation on the characterization of the bamboo plants and NBFs revealed information that is not previously identified or published. The energy-dispersive X-ray spectroscopic (EDS) and the X-ray photoelectron spectroscopy (XPS) were used to determine the elemental composition of bamboo as 70-72% carbon (C) and 28-30% oxygen (O) in EDS and

76-78% C and oxygen 20-24% O in XPS by atomic %. The O/C ratio was found to be 0.26-0.33 for bamboo plants and 0.40-0.70 for NBFs. The major peaks C 1s and O 1s were analyzed to describe their components which revealed some common chemical bonds and functional groups such as C-C, C-H, C-O, C=O, O-C-O, and O-C=O. Chemical analyses by using ATR-FTIR

(Attenuated Total Reflectance Fourier Transform Infra-Red) and Raman spectroscopy (RS) were unveiled important information about bamboo and its fiber. A list of all possible peaks and their definitions are documented for bamboo plants, NBFs, cotton, rayon, and bamboo viscose fibers.

The importance and differences among spectra of FTIR and Raman spectroscopies of different materials were assessed. It was found that there were some differences in the peaks and chemical groups, bonds or behaviors. Specifically, some peaks were found in the spectra of bamboo plants and NBFs that were not detected in either cotton, rayon or bamboo viscose. This infers that there may be some unique properties by such chemical properties of bamboo and its fibers that may not be present in other investigated conventional fibers.

The study of crystallographic properties also revealed some very useful information that will advance the knowledge about different bamboo plants and their NBFs. The assessment with

X-ray diffraction (XRD) technique using cobalt X-ray source (Co K) was able to identify crystallinity indexes (CIs), diffraction angles (2θ), lattice planes (hkl), crystallite dimensions and

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interplanar spacings. The CIs of bamboo plants (61-67%) and NBFs (69-73%) were higher than both rayon (40%) and bamboo viscose (35-38%) fibers but lower than cotton fibers (76-79%).

These results suggest how different properties such as strength, elasticity, toughness, brittleness, stiffness, rigidity, chemical resistance, absorption, melting temperature, density, optical and dyeing property of NBFs will be. The investigation identified four peaks on (101), (101̅), (002) and (040) lattice planes at 17.0-18.6°, 18.5-19.4°, 25.5°, and 40.5° respectively in all samples where the peak on (002) corresponded to the most intense peak. The crystallite size of the bamboo plants and NBFs was determined as 35-39 Å (3.5-3.9 nm).

The development of an assessment of antibacterial activity was very critical to measure non-fabric materials. This study examined different techniques and modeled a method that was effective to quantify the antibacterial activity of all kinds of specimens. The reassessment of the techniques several times proved to be consistent to determine the percentage reduction of bacteria by the specimens. The effects of different conditions and preparation samples, such as non-sterilization and without sterilization, were also assessed. The antibacterial tests revealed that bamboo plants and NBFs had a different degree of inhibition against Staphylococcus aureus

(3-50%) and Klebsiella pneumoniae (8-95%) bacteria. However, except for one product, all 12 bamboo viscose products failed to exhibit bacterial reduction.

This study provides a list of routes for NBF extraction from four bamboo species by using different chemicals and ingredients, results on different physical, mechanical, chemical and structural properties, and a detailed assessment of antibacterial activity test methods along with the antibacterial property of different samples. Thus, the results of this study will be valuable to execute and continue further research on exploring NBF extraction for textiles.

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