Chemometric optimisation of cellulose 

extraction from Hemp: Removal of 

Synthetic Dyes from Aqueous Solutions 

Using Micro-Cellulose 

By  

Jessica Tsakani Mhlongo  

Student number: 844298  

BSc (Hons)  

A dissertation submitted to the Faculty of Science at the University of the  

Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of  

Master of Science in Chemistry.  

Supervisor: Dr Anita Etale  

Co-supervisor: Dr Yannick Nuapia  

Johannesburg, January 2023 

 



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DECLARATION  

I, Jessica Tsakani Mhlongo (844298), declare that this thesis “Chemometric optimisation of cellulose 

extraction from Hemp: Removal of Synthetic Dyes from Aqueous Solutions Using Micro-Cellulose” is 

my own, unaided work.  It is being submitted for the Degree of Master of Science at the University of 

the Witwatersrand, Johannesburg.  It has not been submitted before for any degree or examination at 

any other University.  

 

Jessica Tsakani Mhlongo  

Date: 20/01/2023  

  

 

  



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ABSTRACT  

Due to increasing awareness of the environmental impacts and costs of materials used in everyday 

products, industries are increasingly seeking more ecologically and environmentally friendly materials 

that are renewable, biodegradable, economically feasible, and have lower energy demands. As a result, 

recent research has focused on the extraction of natural materials such as cellulose, from plants, 

including agricultural biomass residue. As a cellulose source, agricultural biomass presents such 

advantages as being low-cost, widely available, environmentally friendly, and biodegradable. This 

study investigated the optimisation of cellulose microfibre extraction from Hemp (Cannabis sativa L.) 

bast fibres by organic acids via response surface methodology (RSM). The goal was to determine 

appropriate conditions under which organic acids could be applied, in order to replace the commonly 

used sulphuric acid process, thus providing a greener route for cellulose extraction. Upon extraction, 

surface treatment by cationisation was then performed in order to demonstrate their application in the 

remediation of water contaminated by various synthetic dyes.  

For the extraction of cellulose microfibres, hemp bast fibres were first subjected to alkali (4 wt% NaOH) 

and bleaching treatments using acetate buffer in aqueous chlorite. RSM was used to determine 

combinations of three processing conditions including acid concentration (45 – 64%), hydrolysis time  

(30 – 90 minutes), and temperature (45 – 65 ℃), using sulfuric acid, formic acid, and maleic acid. A 

central composite design model with 21 experimental runs was optimised using MODDE 13.1 software. 

Characterisation of cellulose and cellulose microfibres included surface morphology analysis by 

scanning electron microscopy (SEM), functional group analysis with Fourier Transform Infrared 

(FTIR) spectroscopy, crystallinity degree with X-ray diffraction (XRD) analysis and thermal stability 

analysis with thermogravimetric analysis (TGA). SEM confirmed that hydrolysis produced cellulose 

microfibres of varying size and morphology. FTIR spectra showed that the main chemical structure of 

cellulose was not altered during the hydrolysis process. TGA also showed that microfibres with high 

crystallinity resulted in good thermal stability, which is a favourable property for high temperature 

applications. The model suitably described the data (R2 =0.99; R2adj = 0.96). Microfibres with an 

average width of 6.91 µm, degree of crystallinity range between 40% and 75% and good thermal 

stability were produced with acid hydrolysis processes with assisted ultrasonic treatment. The optimum 

degree of crystallinity 83.21% was achieved with formic acid concentration of 62 wt%, hydrolysis time 

of 36 minutes, and hydrolysis temperature of 47 ℃ as predicted by the model. The optimisation results 

were validated to confirm the accuracy of the model. The data suggests that formic acid can be used as 

an alternative to sulfuric acid for synthesis of cellulose microfibres from biodegradable hemp waste 

fibres.  



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Using hemp plant fibres as a cellulose source, cationised hemp cellulose was synthesized and applied 

as an adsorbent for the removal of methyl orange (MO), and sunset yellow (SY) from aqueous solutions. 

For cellulose extraction, the previous method was utilised. Extracted cellulose fibres were 

functionalised using Glycidytrimethylammonium chloride (GTMAC) to synthesize cationised cellulose 

(GT-cellulose). Raw plant fibre, bleached cellulose, and GT-cellulose were characterised using SEM, 

FTIR, XRD, and TGA techniques. SEM showed long finger-like morphologies for all fibres and 

displayed that cationisation did not endorse any major modifications to the size and shape of the fibres. 

The FTIR spectra of raw hemp fibres, bleached cellulose, and GT-cellulose displayed functional group 

attributed to epoxy moieties of GTMAC, confirming cationisation. The crystallinity degree (CrI) of the 

fibres obtained from hemp bast material was 60%, which was improved following alkali and bleaching 

treatment extracting cellulose (CrI = 73%). XRD and TGA showed GT-cellulose with lower 

crystallinity degree and reduced thermal stability. For synthetic dye adsorption studies, the influence of 

pH, dosage of adsorbent, initial dye concentration, contact time, and temperature were investigated in 

batch experiments using GT-cellulose as an adsorbent. From the obtained results, the equilibrium 

processes were best described by Langmuir isotherm model for both dyes of interest, showing a 

monolayer adsorption. From the kinetic experiments, the adsorption processes for MO and SY dyes 

followed the pseudo first-order kinetic model indicating that the overall rate of dye adsorption could be 

governed by one of the reactants. The thermodynamic study showed that the adsorption processes for 

MO and SY were both endothermic and spontaneous in nature.   

Hemp bast fibres can therefore be regarded as a green and sustainable waste material for the preparation 

of cellulose microfibres with improved crystallinity, enhanced thermal stability and can be cationically- 

modified to act as an adsorbent for uptake of anionic dyes from aqueous solutions 



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DEDICATION  

To my late grandmother SOPHIE NKHENSANI MHLONGO. Etlela hi kurhula N’wa Mhlongo.



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ACKNOWLEDGEMENTS  

First and foremost, I would like to thank GOD for this opportunity and journey.   

I would like to sincerely thank Dr. Anita Etale and Dr. Yannick Nuapia at the University of the 

Witwatersrand for the supervisory role provided throughout this research. With their guidance and 

constructive feedback, I was able to conduct my masters research project effectively. Thank you for 

granting me this opportunity of growth and for your guidance, insights, advice, and encouragement 

throughout the seasons.  

A special mention to Mbongiseni Dlamini and Kgomotso Maiphetlho thank you for your kindness, 

assistance, and input in throughout this journey has been highly appreciated. To all the postgraduate 

students from the Environmental Analytical Chemistry group, thank you for your contribution in the 

form of positive feedbacks during group presentations, assistance with lab equipment and a peaceful 

working environment. To Dr. Mxolisi Motsa and Dr. Oranso Mahlangu from the University of South 

Africa (UNISA), thank you for immersing contributing to the success of this dissertation. To Boitumelo 

Tlhaole, thank you for always pouring out positive energy since first contact in 2019.  

To the Elite Master’s Squad- thank you for the friendship and support always. To The Girls (UJ) thank 

for the emotional support during the years, since 2014 you have played such important roles individually 

and as a group. To Dzunisani Mbedhli, and Moditsa Cordelia Rampya, I would thank you for carrying 

me through my good and worst days.   

To my aunt, Khanyisa Mhlongo, you have been there supporting and encouraging me from the very 

beginning of my tertiary studies and for that I will forever be grateful. Finally, my mother, Patricia 

Phamela Mhlongo, thank you so much for believing in me, you have always given me every opportunity 

to be great and for our endless conversations, encouragements, prayers in times of despair, and I am 

very grateful for you Mhana Nhunhu.  

Importantly, I would like to thank National Research Funding (NRF) of South Africa and Royal society 

for their financial support towards my studies.  



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PRESENTATIONS  

1. Mhlongo, J.T., Nuapia, Y., and Etale. A. ‘Synthesis of cellulose nanocrystals by mineral and organic 

acids: the influence of acid strength, exposure duration and temperature on structure of nanocrystals’ 

Poster presentation. 2021#RSC Poster Twitter Conference, Virtual, March 2021.   

2. Mhlongo, J.T., Nuapia, Y., and Etale. A. ‘Optimisation of cellulose nanocrystal production using 

sulfuric acid and maleic acid’ Poster presentation. Chemical Nanosciences and Nanotech Early Career 

Virtual Poster Symposium, March 2021.   

3. Mhlongo, J.T., Dlamini, M.L., Nuapia, Y., and Etale. A. ‘Development and application of cellulosic 

materials for treatment of dye-contaminated water’ Poster Presentation, 12th Wits Cross faculty 

Postgraduate Symposium 2021, Virtual, July 2021.   

4. Mhlongo, J.T., Dlamini, M.L., Nuapia, Y., and Etale. A. ‘Adsorption of methyl orange, sunset yellow 

FCF, and Coomassie brilliant blue R dyes from aqueous solutions using cationised cellulose’ Poster 

presentation. National Young Chemist’s Symposium 2021, Virtual, July 2021.   

5. Etale, A., Nhlane, D.S., Mosai, A. K., Mhlongo, J.T(Presenter)., Khan, A., Rumbold, K., and Nuapia, 

Y. B. ‘Synthesis and application of cationised cellulose for the removal of Cr(VI) from acid mine 

drainage contaminated water’ Oral presentation. AQUA 360 Conference- Water for all: Emerging 

Issues and Innovations, Virtual, August/September 2021.  

6. Mhlongo, J.T., Dlamini, M.L., Nuapia, Y., and Etale. A. ‘Adsorption of anionic dyes using 

functionalised cellulose as adsorbent’ Poster presentation. 2nd Commonwealth Chemistry Posters-

Building Networks to Address the Goals, Virtual, September 2021.   

7. Mhlongo, J.T., Nuapia, Y.B., Motsa, M.M., Mahlangu, O.T., and Etale. A. ‘Green chemistry 

approaches for the extraction of cellulose nanofibres (CNFs): A comparison of mineral and organic 

acids’ Oral presentation. Nanoscience’s Young Researcher’s Symposium (NYRS) 2020/2021, Virtual, 

October 2021.   



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PUBLICATIONS   

1. Mhlongo, J.T., Dlamini, M.L., Nuapia, Y., and Etale. A. (2022). Synthesis and application of 

cationized cellulose for adsorption of anionic dyes. Materials Today: Proceedings, 62(3), pp 1-8, 

https://doi.org/10.1016/j.matpr.2022.02.100 

2. Mhlongo, J.T., Nuapia, Y., Motsa, M.M., Mahlangu, O.T., and Etale. A. (2022). Green chemistry 

approaches for the extraction of cellulose nanofibres (CNFs): A comparison of mineral and organic 

acids. Materials Today: Proceedings, 62(1), pp 1-6,  https://doi.org/10.1016/j.matpr.2022.02.088 

3. Mhlongo, J.T., Nuapia, Y., Tlhaole, B., Mahlangu, O.T., and Etale. A. (2022). Optimization of Hemp 

Bast Microfibre Acid Hydrolysis Using Response Surface Modelling. Processes, 10(6):1150, 

DOI: 10.3390/pr10061150 



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TABLE OF CONTENTS   

DECLARATION ..................................................................................................................................... i 

ABSTRACT ............................................................................................................................................. i 

ACKNOWLEDGEMENTS .................................................................................................................... ii 

PRESENTATIONS .................................................................................................................................. i 

PUBLICATIONS ..................................................................................................................................... i 

TABLE OF CONTENTS ......................................................................................................................... i 

LIST OF FIGURES ................................................................................................................................ v 

LIST OF TABLES ............................................................................................................................... viii 

CHAPTER 1 INTRODUCTION ............................................................................................................ 1 

1.1 Background and motivation .......................................................................................................... 1 

1.2 Study aims and objectives ............................................................................................................. 3 

1.3 Research justification .................................................................................................................... 3 

1.4 Dissertation outline ....................................................................................................................... 4 

1.5 References ..................................................................................................................................... 5 

CHAPTER 2: LITERATURE REVIEW .............................................................................................. 11 

2.1 Hemp fibres (Cannabis Sativa L.) .............................................................................................. 11 

2.2 Cellulose and its hierarchical structure ....................................................................................... 11 

2.2.1 Cellulose structure and properties ........................................................................................ 14 

2.2.2 Cellulose microfibres (CMFs), cellulose nanofibres (CNFs), and cellulose nanocrystals 

(CNCs) .......................................................................................................................................... 16 

2.3 Isolation techniques and their effects on crystallinity index and the surface morphology. ......... 17 

2.3.1 Chemical methods ................................................................................................................ 17 

2.3.2 Mechanical methods ............................................................................................................ 23 

2.4 Response Surface Methodology .................................................................................................. 34 

2.4.1 Box-Behnken design (BBD) ................................................................................................ 34 

2.5 Cellulose surface modifications .................................................................................................. 37 

2.5.1 TEMPO-mediated Oxidation ............................................................................................... 37 

2.5.2 Cationisation ........................................................................................................................ 39 



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2.6 Environmental pollutants ............................................................................................................ 46 

2.6.1 Synthetic Dyes ..................................................................................................................... 46 

2.6.2 Azo dyes, environmental fate, and human health ................................................................ 46 

2.7 Conventional wastewater treatment methods .............................................................................. 49 

2.7.1 Chemical method ................................................................................................................. 49 

2.7.2 Biological methods .............................................................................................................. 49 

2.7.3 Physical methods .................................................................................................................. 50 

2.8 Adsorption process ...................................................................................................................... 54 

2.8.1 Adsorbents for dye removal ................................................................................................. 55 

2.9 References ................................................................................................................................... 66 

CHAPTER 3: RESEARCH METHODOLOGY ................................................................................. 106 

3.1 Chemicals .................................................................................................................................. 106 

3.2 Equipment ................................................................................................................................. 106 

3.3 References ................................................................................................................................. 107 

CHAPTER 4: CHEMICALLY TREATED CELLULOSE AND ITS CELLULOSE MICROFIBRES 

FROM HEMP FIBRES: ISOLATION AND CHARACTERISATION............................................. 108 

4.1 Introduction ............................................................................................................................... 108 

4.2 Methods ..................................................................................................................................... 109 

4.2.1 Extraction and purification of cellulose ............................................................................. 109 

4.2.2 Synthesis of cellulose microfibres ...................................................................................... 109 

4.3 Results and discussion .............................................................................................................. 110 

4.3.1. Pretreatment of cellulose ................................................................................................... 110 

4.3.2 Acid hydrolysis followed by ultrasonic treatment ............................................................. 111 

4.3.3 Scanning Electron Microscope analysis ............................................................................. 112 

4.3.4 Fourier Transform Infrared spectroscopy .......................................................................... 115 

4.3.5 X-ray diffraction analysis ................................................................................................... 118 

4.3.6 Thermogravimetric (TG) and Derivative Thermogravimetric (DTG) analysis.................. 121 

4.4 Conclusion ................................................................................................................................ 125 

4.5 References ................................................................................................................................. 126 



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CHAPTER 5: OPTIMISATION OF ACID HYDROLYSIS OF HEMP FIBRES USING RESPONSE 

SURFACE METHODOLOGY .......................................................................................................... 134 

5.1 Introduction ............................................................................................................................... 134 

5.2. Methods .................................................................................................................................... 134 

5.2.1 Response Surface Methodology experimental design ........................................................ 135 

5.3 Results and discussion ............................................................................................................... 136 

5.3.1 Interpretation of residual and coefficient plots .................................................................. 136 

5.3.2 ANOVA analysis and lack of fit ........................................................................................ 138 

5.3.3 Interpretation of Response Contour and Surface Contour plots......................................... 139 

5.3.4 Optimisation and model verification .................................................................................. 142 

5.4 Conclusion ................................................................................................................................ 143 

5.5 References ................................................................................................................................. 143 

CHAPTER 6: SYNTHESIS AND APPLICATION OF CATIONISED CELLULOSE FOR 

ADSORPTION OF ANIONIC DYES ................................................................................................ 146 

6.1 Introduction ............................................................................................................................... 146 

6.2. Methods .................................................................................................................................... 147 

6.2.1 Cationisation of cellulose fibres ......................................................................................... 147 

6.2.2 Methyl orange and sunset yellow dye adsorption studies .................................................. 148 

6.2.3 Adsorption isotherms, kinetics, and thermodynamic parameters ....................................... 148 

6.2.3.3 Thermodynamic analysis................................................................................................. 151 

6.3 Results and discussion .............................................................................................................. 151 

6.3.1 Characterisation of bleached treated and cationised cellulose ........................................... 151 

6.3.2 Scanning Electron Microscope analysis ............................................................................. 152 

6.3.3 Fourier Transform Infrared spectroscopy .......................................................................... 153 

6.3.4 X-ray diffraction analysis ................................................................................................... 155 

6.4 Adsorption experiments ............................................................................................................ 157 

6.4.1 Effect of pH ........................................................................................................................ 157 

6.4.2 Effect of adsorbent dosage ................................................................................................. 158 

6.4.3 Effect of initial concentration ............................................................................................. 159 



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6.4.4 Effect of temperature ......................................................................................................... 160 

6.10.5 Effect of contact time ....................................................................................................... 161 

6.5 Adsorption isotherms, kinetics, and thermodynamic parameters .............................................. 162 

6.5.1 Adsorption isotherm models .............................................................................................. 162 

6.5.2 Adsorption kinetic models ................................................................................................. 165 

6.5.3 Thermodynamic analysis.................................................................................................... 168 

6.6 Adsorption comparison studies between cellulose and GT-cellulose. ...................................... 169 

6.7 Application of real water samples. ............................................................................................ 170 

6.8 Conclusion ................................................................................................................................ 171 

6.9 References ................................................................................................................................. 171 

CHAPTER 7: CONCLUSION AND RECOMMENDATIONS ......................................................... 180 

7.1 General conclusion .................................................................................................................... 180 

7.2 Recommendations and future work .......................................................................................... 182 

 

 



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LIST OF FIGURES  

 
Figure 2.1: Sources of cellulose of various plant and animal species ................................................... 12  

Figure 2.2: Applications of cellulose. ................................................................................................... 14  

Figure 2.3: Molecular structure of cellulose showing the non-reducing ends, terminal reducing ends and 

cellobiose unit. ...................................................................................................................................... 15  

Figure 2.4: Schematic of showing cellulosic fibres, cellulose microfibres, cellulose nanofibres, and 

cellulose nanocrystals with their respective sizes. ................................................................................ 17 

Figure 2.5: Cellulose acid hydrolysis mechanism. ............................................................................... 19  

Figure 2.6: SEM images for the CNC at different magnification (300, 100 and 50 µm), .................... 20 

Figure 2.7: Schematic diagram of FeCl3-catalyzed FA hydrolysis for the integrated production of CNCs  

and CNFs ............................................................................................................................................. 21  

Figure 2.8: Schematic diagram for the MA hydrolysis of bleached eucalyptus pulp to produce CNFs,  

along with acid crystallization, acid recovery, and acid reuse ............................................................. 22 

Figure 2.9: Diagrammatic representation of the effects of cellulase on cellulose ................................ 23 

Figure 2.10: Schematic diagram showing the production of individualized CNFs using ultrasonic  

treatment. ............................................................................................................................................. 24  

Figure 2.11: Schematic diagram of a high-pressure homogenizer used for the isolation of CNFs....... 26 

Figure 2.12: Geometry of 3k factor Box-Behnken design a) cube defined by the midpoints of the edges 

and a centre point; (b) three interlocking 22 factorial designs and a centre point. ................................ 35 

Figure 2.13: Geometry of central composite design in three factors. ................................................... 36  

Figure 2.14: Schematic diagrams of a TEMPO-mediated oxidization process for nanocellulose isolation 

 .............................................................................................................................................................. 39  

Figure 2.15: Mechanism of reaction between (a) epoxides and cellulose fibres, (desirable)(b) showing 

multiple substitution, and (c) alkaline hydrolysis of EPTMAC (undesirable) ...................................... 41 

Figure 2.16: Schematic for physical and chemical adsorption mechanism ......................................... 57  

Figure 4.1: Schematic showing extraction and preparation of cellulose microfibres from hemp bast 

fibres through acid hydrolysis process coupled with ultrasonic treatment……………………………110  

Figure 4.2: Schematic showing the production of a) sulfuric acid, b) formic acid and c) maleic acid 

hydrolysis process from cellulose derived from hemp bast fibres. ..................................................... 112 

Figure 4.3: SEM images of bleached cellulose a) 50 µm magnification and b) 10 µm magnification. 

 ............................................................................................................................................................ 113  

Figure 4.4: SEM images of hemp cellulose microfibres after acid hydrolysis ................................... 114 

Figure 4.5: FTIR spectra of a) raw hemp fibre and bleached cellulose, b) sulfuric acid hydrolysed CMFs, 

c) formic acid hydrolysed CMFs, and d) maleic acid hydrolysed CMFs. .......................................... 116 

Figure 4.6: X-ray diffractogram patterns of a) fibres (raw hemp fibre-red and bleached cellulose 



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fibreblack), b) sulfuric acid hydrolysed CMFs, c) formic acid hydrolysed CMFs, and d) maleic acid 

hydrolysed CMFs produced from hemp fibres. .................................................................................. 119 

Figure 4.7: TG a) and DTG b) curves of raw hemp fibre and bleached cellulose fibres extracted from 

hemp fibre waste. ................................................................................................................................ 123  

Figure 4.8: TG a) and DTG b) curves of sulfuric acid hydrolysed microfibres extracted from hemp fibre 

waste. .................................................................................................................................................. 123  

Figure 4.9: TG a) and DTG b) curves of formic acid hydrolysed microfibres extracted from hemp fibre 

waste. .................................................................................................................................................. 124  

Figure 4.10: TG a) and DTG b) curves of maleic acid hydrolysed microfibre extracted from industrial  

hemp fibre waste. ................................................................................................................................ 125  

Figure 5.1: Residual plot of predicted versus observed crystallinity indexes (%) of CMFs extracted from  

hemp fibres.......................................................................................................................................... 140  

Figure 5.2: Coefficients plot for acid hydrolysed CMFs crystallinity index (%). .............................. 142 

Figure 5.3: Response contour plots for the response between a) hydrolysis temp vs acid conc., b) 

reaction time vs acid conc., c) reaction time vs hydrolysis temp and d) response surface plot from  

sulfuric acid hydrolysis. ...................................................................................................................... 144  

Figure 5.4: Response contour plots for the response between a) hydrolysis temp vs acid conc., b) 

reaction time vs acid conc., c) reaction time vs hydrolysis temp and d) response surface plot from formic  

acid hydrolysis. ................................................................................................................................... 145  

Figure 5.5: Response contour plots for the response between a) hydrolysis temp vs acid conc., b) 

reaction time vs acid conc., c) reaction time vs hydrolysis temp and d) response surface plot from maleic 

acid hydrolysis. ................................................................................................................................... 146  

Figure 6.1: Schematic showing the preparation of cationised cellulose fibres extracted from hemp 

(Cannabis sativa L.) bast fibres………............................................................................................... 147 

Figure 6.2: Desirable reaction scheme for the cationisation of cellulose using GTMAC................... 152 

Figure 6.3: SEM images of a) bleached and c) cationised cellulose, and histograms of b) bleached and  

d) cationised cellulose. ........................................................................................................................ 153  

Figure 6.4: a) FTIR spectrum of raw plant fibre, bleached cellulose, and GT-cellulose and b) FTIR 

spectrum of GTMAC solution. ........................................................................................................... 154 

Figure 6.5: XRD specrums of a) raw plant fibre, b) bleached cellulose and c) GT-cellulose. ........... 156  

Figure 6.6: a) TGA and b) DTA curves of raw plant fibre, bleached cellulose, and GT-cellulose. ... 157 

Figure 6.7: a) Point of zero charge graph and b) Effect of solution pH of dyes (MO and SY) adsorbed  

onto GT-cellulose adsorbent. .............................................................................................................. 158  

Figure 6.8: Effect of adsorbent dosage on the uptake of MO onto GT-cellulose adsorbent and b) Effect 

of adsorbent dosage on the uptake of SY onto GT-cellulose adsorbent ............................................. 159 



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Figure 6.9: Effect of initial dye concentration on the uptake of MO and SY onto GT-cellulose adsorbent 

at 298.15 K. ......................................................................................................................................... 160  

Figure 6.10: a) Effect on temperature on the uptake of MO onto GT-cellulose adsorbent and b) Effect 

on temperature on the uptake of SY onto GT-cellulose adsorbent. ......................................................161 

Figure 6.11: a) Effect of contact time of MO dye onto GT-cellulose adsorbent over a period of 630 min 

and b) Effect of contact time of SY dye onto GT-cellulose adsorbent over a period of 630 min. ...... 162 

Figure 6.12: Effect of contact time on MO adsorption and kinetic data fit to (a) pseudo first order and 

pseudo second order non-linear models. (b) Intra-particle diffusion for adsorption of MO onto GT- 

cellulose at 298.15 K. .......................................................................................................................... 165  

Figure 6.13: Effect of contact time on SY adsorption and kinetic data fit to (a) pseudo first order and 

pseudo second order non-linear models. (b) Intra-particle diffusion for adsorption of SY onto GT- 

cellulose at 298.15 K. .......................................................................................................................... 166  

Figure 6.14: Vant’ Hoff plot of MO and SY adsorption onto GT-cellulose ....................................... 168  

Figure 6.15: Adsorption of MO and SY onto cellulose and GT-cellulose. ......................................... 170 

Figure 6.16: Application of GT-cellulose to tap water, and sea water spiked with MO and SY (Initial 

dye concentration = 20 mg g-1, adsorbent mass = 10 mg, temperature 298.15 K, contact time for MO  

(120 min) and SY (270 min)). ............................................................................................................. 170  

   



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LIST OF TABLES  

Table 2.1: Cellulose, hemicellulose, and lignin as major components in the chemical composition of 

various plants materials. ........................................................................................................................ 12 

Table 2.2: Various sources of cellulose and their degree of polymerization (DP) ............................... 15  

Table 2.3: Type of nanocellulose with their respective average particle sizes ..................................... 18  

Table 2.4: Cellulose isolation techniques extracted from different sources. ........................................ 27  

Table 2.5: Cellulose modifications from various sources ..................................................................... 43  

Table 2.6: Chemical characteristics of Methyl Orange monoazo dye .................................................. 48  

Table 2.7: Chemical characteristics of Sunset Yellow FCF monoazo dye .......................................... 49  

Table 2.8: Conventional water treatment techniques for dye removal. ................................................ 53  

Table 2.9: Various materials used as adsorbents for removal of dyes via adsorption processes. ......... 63  

Table 4.1: Chemical functional groups of cellulose and hydrolysed cellulose derivatives. ............... 121 

Table 4.2: The crystallinity index (CrI %), thermal degradation onset temperature (Tonset), and max 

degradation temperature (Tmax) of cellulose microfibres obtained from XRD, TG, and DTG curves  

respectively. ........................................................................................................................................ 125  

Table 5.1: Experimental conditions and the types of acids to achieve optimum cellulose microfibres  

hydrolysis. ........................................................................................................................................... 135 

Table 5.2: Results from the RSM-CCD experimental design and response data for sulfuric, formic, and 

maleic acid hydrolysis of cellulose extracted from Cannabis sativa L. bast fibres. ........................... 137 

Table 5.3: Analysis variance estimated by ANOVA and statistical information for the crystallinity index  

(%) as a response from the optimisation of sulfuric, formic, and maleic acid hydrolysis 
treatment……………………………………………………………………………………………..139  

Table 5.4: Observed vs predicted results of the optimum crystallinity index (%) .............................. 142 

Table 6.1: The main functional groups observed on raw plant fibre, bleached cellulose, and GT-

cellulose by FTIR spectroscopy and their corresponding wavenumbers. 

............................................................................................................................................................. 155 

 Table 6.2: Crystallinity Index (CrI), Onset Temperature (Tonset), Maximum Degradation Temperature  

(Tmax), and Char Residuals at 900 °C Obtained from XRD, TGA and DTG plots. ............................ 156  

Table 6.3: Adsorption isotherm parameters for the adsorption of MO and SY onto GT-cellulose. ... 164  

Table 6.4: Comparison of other adsorbents adsorption isotherm parameters. .................................... 166 

Table 6.5: Maximum experimental adsorption capacity of MO and SY onto GT-cellulose (adsorbent 

dosage = 10 mg, initial dye concentration= 75 mg L-1, contact time = 24 hrs). ................................. 169 

Table 6.6: Kinetic adsorption model parameters of MO and SY onto GT-cellulose. ......................... 169  

Table 6.7: Thermodynamic parameters for adsorption of MO and SY onto GT-cellulose. ................. 173



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LIST OF ABBREVIATION  

AC  Activated carbon   

AB 24  Acid Black 24  

ADI  Acceptable daily intake  

ADHD  Attention deficient hypersensitivity disorder  

AGU  Anhydroglucose units  

AM  Acrylamide   

APSP  Aminated pumpkin seed powder  

AOP  Advanced oxidation processes  

BBD  Box-Behnken design   

BC  Bacterial cellulose   

BEP  Bleached eucalyptus pulp  

BET  Brunauer-Emmett-Teller  

BNFC  Bacterial nanofibrillated cellulose  

[Bmim]Cl  1-Butyl-3-methylimidazolium chloride  

BSKP  Bleached softwood kraft pulp  

CBH  Cellulogluconase   

CC  Coconut coir  

CCD  Central-composite design  

CCR  Corncob residue  

c-DAC  Cationic dialdehyde nanocellulose  

CF  Coagulation/flocculation   

CHPTAC  3-chloro-2-hydroxypropyltrimethylammonium chloride  

CMFs  Cellulose microfibres  

CNCs  Cellulose nanocrystals  

CNFs  Cellulose nanofibres  

CNWs  Cellulose nanowhiskers   

CR  Congo Red  

CRHC  Cationised rice husk cellulose  

CrI  Crystallinity index  

CV  Coefficient of variance   

DG B  Diamine green B  

DP  Degree of polymerization  

DPF  Date palm fibres  

D-R isotherm  Dubinin-Radushkevich isotherm  

DTG analysis  Derivative thermogravimetric analysis  



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EBSE  Eriochrome blue SE  

EG  Endogluconases  

EMPH  Ethylenediamine-modified peanut husk  

EPTMAC  2,3-epoxypropyl trimethylammonium chloride  

FA  Formic acid  

FAO  Food and Agricultural Organization   

FTIR  Fourier-transform infrared spectroscopy  

GTMAC  Glycidyl trimethylammonium chloride  

HDTMA  Hexadecyltrimethylammonium  

HIUS  High intensity ultrasonication   

HPH  High-pressure homogenisation   

IPD  Intraparticle diffusion   

JLP  Jackfruit leave powder  

MA  Maleic acid  

MCC  Microcrystalline cellulose  

MO  Methyl orange  

MMWS  Modified methanol walnut shell  

NCC  Nanocellulose crystals  

OPEFB  Oil palm empty fruit brunch  

OPW  Orange peel waste  

PAM-g-QC  Polyacrylamide grafted quatenised cellulose  

PEI  Polyethyleneimine  

PCC  Purified coir cellulose  

PFO  Pseudo-first order  

PSO  Pseudo-second order  

p-TsOH  p-toluene sulfonic acid  

Q-CNF  Quatenised cellulose nanofibres  

RHC  Rice husk cellulose  

RSM  Response surface methodology  

SA  Sulfuric acid  

SD  Standard deviation  

SDG  Sustainable development goals  

SEM  Scanning electron microscope  

SMBAC  Swietenia mahagoni bark activated carbon  

SY  Sunset yellow  

TEM  Transmission electron microscope  

TEMPO  2,2,6,6-tetramethylpiperidinyl-1-oxyl radical  



iii 
 

TGA  Thermogravimetric analysis  

Tmax  Maximum thermal degradation temperature  

Tonset  Thermal degradation onset temperature  

UIPAC  International Union of Pure and Applied Chemistry  

UV-vis  Ultraviolet visible  

WHO  World Health Organization  

XRD  X-ray diffraction  

 
  



1 
 

CHAPTER 1 INTRODUCTION  

1.1 Background and motivation  

According to the United Nations, in 2019 the global population was estimated to be 7.7 billion, with a 

projected rise to be 8.5 billion by 2030, and with this increase, the need for sustainable food sources 

and safe and clean drinking water will rapidly increase causing a decrease in water quality and quantity 

due to increase water pollution (Sharma et al., 2019) (World Population Prospects, 2019). Water 

pollution is the contamination of water bodies such as groundwater, surface waters, lakes, rivers, seas, 

and oceans leading to poor water quality (Sharma et al., 2019). Pollution leads to a significant 

deterioration in water quality resulting in close to 675 million people living without access to safe 

drinking water. This can lead to contacting sickness from waterborne diseases such as cholera, guinea 

worm and these mostly occur in the rural areas in underdeveloped countries (Sharma et al., 2020). 

According to some statistics:  

• Over a decade ago, it was estimated that 1.2 billion people worldwide more especially in developing 

countries did not have access to safe drinking water and this figure is expected to be triple by 2025 

(Mukheibir, 2010)  

• In 2019, 785 million people did not have access basic safe clean drinking-water service and more than 

2 billion people worldwide used a drinking water source contaminated with feces (World Health 

Organization, 2019).  

• Contaminated drinking water is estimated to cause 485 000 diarrheal deaths each year (World Health 

Organization, 2019).  

• In least developed countries, 22% of health care facilities have no water service, 21% no sanitation 

service, and 22% no waste management service (World Health Organization, 2019)   

• On global scale, 26.3 million people per year die from drinking water contaminated with inorganic toxic 

heavy metals such as arsenic, chromium, lead, and cadmium, and organics such as dyes, pesticides, and 

surfactants (Jain, Varshney and Srivastava, 2016).  

• Due to the rapid rise in population growth, industrialization, climate change and water pollution, it is 

estimated that by 2050 the global population will be 9.7 billion people and over  

40% of the world’s population will be living in water-stressed areas (Guppy and Anderson, 2017).  

 

Synthetic dyes are frequently used in many industries e.g. textile, cosmetics, leather, paper dyeing, 

printing and colour photography, pharmaceutical, food, etc., (Hashem and El-Shishtawy, 

2001)(Konicki et al., 2015). When released into water systems, synthetic dyes may result in harmful 



2  

  

effects to humans and aquatic life such as increased heart rate, diarrhea, vomiting, shock, cyanosis, 

jaundice, and tissue necrosis even when present at low concentrations. Dyes can have carcinogenic and 

mutagenic effects in humans and animals (Mahida and Patel, 2016). They can be categorized as cationic, 

non-ionic, or anionic. Cationic dyes have a net positive charge when in water due to the presence of 

chemical groups such as amine whereas anionic dyes have a net negative charge due to sulfonate groups 

(Fernandes et al., 2020). The continued use of synthetic dyes has resulted in a growing challenge to 

develop efficient, reliable, environmentally friendly, and low-cost technologies to reduce contamination 

of water resources.   

Various technologies exist for the treatment of contaminated water including membrane separation  

(Liu et al., 2015), electrocoagulation (Dermentzis et al., 2011)(Kobya et al., 2011), electrodeposition 

(Verduzco et al., 2019), chemical oxidation(Mohan and Pittman, 2007), ozone treatment,, reverse 

osmosis (Mahdavi and Rahimi, 2018), co-precipitation (Mohan and Pittman, 2007), ion exchange (El 

Torky et al., 2016), simple adsorption (Ballav, Maity and Mishra, 2012) and a combination of two or 

more processes (Ibrahim et al., 2019).  

Adsorption is a simple and reliable method as it has been extensively used on a variety of contaminants. 

It is a process that uses adsorbents for the removal of substances from liquid or gaseous solutions 

followed by adsorption of the substances onto the surface of the adsorbent. This technique has been 

used extensively owing to its great advantages ease of use and operation, financial feasibility, wide 

availability, little to no sludge generation and high removal efficiencies, therefore it is applicable in 

wastewater treatment on a large-scale. For adsorption to work effectively, the experiments conditions 

such as pH range, metal concentration, adsorbent type, ligand concentration, particle size, and 

competing for ions are to be optimised (Nicomel et al., 2015)(Li et al., 2019)(Ahmad et al., 

2015)(Awang et al., 2018). The adsorption process strongly depends on the type of adsorbent used and 

commonly used adsorbents include chitosan (Neeraja et al., 2016), bottom ash (Jarusiripot, 2014), 

natural zeolites (Sannino et al., 2012), nanomaterials such as activated carbon, graphene, carbon 

nanotubes (Sadegh et al., 2017)(Ilavský, Barloková and Marton, 2020)(Sujitha and Ravindhranath, 

2016), activated alumina(Adegoke et al., 2017), Hydrous zirconium oxide (Kumar et al., 2018), rice 

husk ash(Vieira et al., 2014), fly ash(Adegoke et al., 2017), and agriculture by-products (Batmaz et al., 

2014).   

Recently, the focus has increasingly been on the exploration of greener technological solutions, leading 

to increased interest in the use of cellulosic materials for applications in various fields. Cellulose is the 

most abundant natural polymer being a major component of higher plants, alongside lignin and 

hemicellulose (George and Sabapathi, 2015). The global production of cellulose has been estimated to 

be 1.5 × 1012 tons (Tavakolian, Jafari and Ven, 2020). To date, much of the cellulose in use has been 



3  

  

from wood sources (Pennells et al., 2020) but other sources, particularly agricultural wastes e.g., 

sugarcane bagasse, corn husks, hemp fibres, banana rachis etc are attracting significant attention.  

Cellulose is suitable for organic and inorganic pollutant remediation due to the presence of hydroxyl 

functional groups which can also be modified to improve adsorption (Jamshaid et al., 2017)(Tavakolian, 

Jafari and Ven, 2020). Through various treatments, cellulose nanomaterials can be extracted various 

techniques. Acid hydrolysis using most often, mineral acids e.g., sulphuric acid, is used to synthesize 

nanocrystals (Sartika et al., 2019). Work by Isogai and colleagues also showed that TEMPO-catalysed 

oxidation can be used to generate nanofibres with lengths up to 3 µm (Isogai, Saito and Fukuzumi, 

2011). Importantly, both cellulose nanocrystals and nanofibres can be further modified with cationic or 

anionic surface groups in order to improve the adsorption capacity for organic and inorganic 

contaminants (Singh, Sinha and Srivastava, 2015)(Carpenter, de Lannoy and Wiesner, 2015)  

1.2 Study aims and objectives   

In South Africa, hemp production results of wastes rich in cellulose which is available at lower cost due 

to their wide local availability and renewability. For this reason, the use of these agricultural wastes in 

science research has become of great interest. Therefore, the current work aimed to use of hemp fibres 

as cellulose sources renewable and biodegradable material for applications in water remediation studies. 

The following objectives were pursued to achieve this goal:  

i. To investigate green approaches for the extraction of cellulose from hemp waste using organic 

and mineral acids using response surface methodology for the selection of reaction conditions 

i.e., acid type, acid concentration, reaction temperature and duration.   

ii. To characterise extracted cellulose for their physical and chemical properties using scanning 

electron microscope (SEM), X-ray diffraction (XRD). thermogravimetric analysis (TGA), and 

Fourier-transform infrared spectroscopy (FTIR).  

iii. To modify the surface of extracted cellulose fibres by cationic moieties using glycidyl 

trimethylammonium chloride (GTMAC). 

iv. To determine the removal efficiency of anionic dyes from water using cationised cellulose.   

1.3 Research justification   

Natural Cellulose is a renewable, low-cost, and biodegradable material that can be extracted from a 

variety of living and non-living organisms. Hemp bast fibres is a great source of cellulose (78%) and 

with legal hemp farming in South Africa increased production of hemp waste could result. Further 

resulting in a surge of under-utilised waste material that are burnt unused. Adsorption is a simple, low-

cost, and reliable process that has been vastly used for remediation of organic and in organic 



4  

  

contaminants using cellulose materials as a natural adsorbent. To our best knowledge, there is no 

literature that has covered the use cationised cellulose extracted from hemp bast fibres for remediation 

of anionic dyes (methyl orange and sunset yellow) from aqueous solutions  

1.4 Dissertation outline  

Chapter 1: Introduction to study  

This chapter gives a brief background introduction into the world of water scarcity on a global scale, 

pollution caused by synthetic dyes, extraction techniques and use of agricultural cellulose residue and 

cellulose material as adsorbents. It also highlights information of the main research aim and objectives 

for the work to be conducted, and the research justification.  

Chapter 2: Literature review   

This chapter is the core component of the dissertation in which phrases are defined and elaborated. 

Hemp bast fibre as a cellulose source is discussed with focus on preparation and extraction techniques. 

Response surface methodology (RSM) as an optimisation method and cellulose modification techniques 

are discussed. Furthermore, synthetic dye water remediation using cationised cellulose fibres is 

discussed in the following chapter.  

Chapter 3: Research methodology   

i. The major intention of this chapter is to clarify on experimental protocol of the research. It 

brings attention to all chemical reagents, equipment, Characterisation techniques, and 

adsorption experimental procedures. The experimental procedures were divided into a series of 

activities outlined below:  

ii. Preparation and extraction of cellulose fibres from Industrial Hemp (Cannabis sativa L.) bast 

fibres waste through alkali and bleaching treatments.  ii. Green chemistry synthesis of cellulose 

microfibres (CMFs) through mineral and organic acid hydrolysis coupled with ultrasonication 

method.  

iii. Preparation of cationised cellulose fibres using GTMAC, as cationising agent.   

iv. Characterisation techniques employed for all cellulose derived fibres were Scanning Electron 

Microscope (SEM), Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD) 

analysis and Thermogravimetric analysis (TGA).  



5  

  

v. Adsorption parameters included solution pH, initial concentrations, adsorbent dosage, 

temperature effects, and contact time. Ultraviolet-visible (UV-vis) spectroscopy was used to 

measure the dye concentration after adsorption  

Chapter 4: Chemically treated cellulose and its cellulose microfibres extracted from hemp fibres: 

Isolation and Characterisation  

This chapter focuses on the acid hydrolysis extraction and characterisation of cellulose microfibres 

extracted from industrial hemp (Cannabis Sativa L.) plant fibres  

Chapter 5: Optimisation of acid hydrolysis of hemp fibres using response surface methodology  

In this chapter a response surface methodology with 33 full factorial experimental design was adapted 

to optimise organic and mineral hydrolysis in respect to acid concentration, temperature, and reaction 

time to obtain fibres high in crystallinity.  

Chapter 6: Synthesis and application of cationised cellulose for adsorption of anionic dyes  

This chapter presents the results of experiments conducted to examine the removal of azo dyes, methyl 

orange and sunset yellow by cationised cellulose. The chemical and physical characteristics of modified 

and unmodified cellulose are fully compared and discussed here. The adsorption efficiencies of raw 

plant fibre treated cellulose and cationised cellulose for remediation azo dyes are compared and 

discussed, primarily focusing on the influence of the following adsorption parameters: solution pH, 

mass of adsorbent, initial adsorbate concentration, contact time and reaction temperature. The 

applicability of the adsorbent on tap and seawater were also investigated. Adsorption isotherms and 

kinetic modelling were used to describe the adsorption behavior between adsorbate-adsorbent 

interactions.  

Chapter 7: Conclusions and Recommendations  

The general concluding remarks on the work covered in the study and future recommendations are made 

in this chapter.  

1.5 References   

Adegoke, K. A., Oyewole, R. O., Lasisi, B. M., and Bello, O. S. (2017) 'Abatement of organic pollutants 

using fly ash-based adsorbents', Water Science and Technology, 76(10), pp. 2580–2592. doi:  

10.2166/wst.2017.437.  



6  

  

Ahmad, M., Ahmed, S., Swani, B. L., and Ikram, S. (2015) 'Adsorption of heavy metal ions: Role of 

chitosan and cellulose for water treatment’, International Journal of Pharmacognosy, 2(6), pp. 280– 

289. doi: 10.13040/ijpsr.0975-8232.ijp.2(6).280-89.  

Awang, N. A., Salleh, W. N. W., Ismail, A. F., Yusof, N., Aziz, F., and Jaafar, J. (2018) 'Adsorption 

Behavior of Chromium (VI) onto Regenerated Cellulose Membrane', Industrial and Engineering 

Chemistry Research, pp. 1–30. doi: 10.1021/acs.iecr.8b02366  

Basheer, A. A. (2018) 'New generation nano-adsorbents for the removal of emerging contaminants in 

water’, Journal of Molecular Liquids, 261, pp. 583–593. doi: 10.1016/j.molliq.2018.04.021.  

Batmaz, R., Mohammed, N., Zaman, M., Minhas, G., Berry, R. M., and Tam, K. C. (2014) 'Cellulose 

nanocrystals as promising adsorbents for the removal of cationic dyes’, Cellulose, 21(3), pp. 1655– 

1665. doi: 10.1007/s10570-014-0168-8.  

Carpenter, A. W., de Lannoy, C. F. and Wiesner, M. R. (2015) 'Cellulose nanomaterials in water 

treatment technologies', Environmental Science and Technology,49(9), pp. 5277–5287. doi: 

10.1021/es506351r.  

Dermentzis, K., Christoforidis, A., Valsamidou, E., Lazaridou, A., and Kokkinos, N. (2011) 'Removal 

of Hexavalent Chromium From Electroplating Wastewater By Electrocoagulation With Iron Electrodes' 

, Global NEST Journal, 13(4), pp. 412–418.  

El-Torky, A. M. M., Mostafa, H. Y., El-Masry, A. M. M., and Mahdi, M. E. (2016) 'Purification of 

Heavy Metals from Industrial Wastewater Using Grafted Corn Husks Pulp Ion Exchanger', Sohag 

Journal of Sciences, 1(1), pp. 53–64. doi: 10.18576/sjs/010107.  

Fernandes, J. V., Rodrigues, A. M., Menezes, R. R., and de Araújo Neves, G. (2020) 'Adsorption of 

anionic dye on the acid-functionalized bentonite', Materials, 13(16), pp. 1–19. doi: 

10.3390/MA13163600.  

George, J. and Sabapathi, S. N. (2015) 'Cellulose nanocrystals: Synthesis, functional properties, and 

applications', Nanotechnology, Science and Applications, 8, pp. 45–54. doi: 10.2147/NSA.S64386.  

Guppy, L. and Anderson, K. (2017) 'Global Water Crisis Report: the Facts', United Nations University 

for Wter,Environment and Health. Hamilton, Canada. Available at: http://inweh.unu.edu.  



7  

  

Hashem, A. and El-Shishtawy, R. M. (2001) 'Preparation and characterization of cationized cellulose 

for the removal of anionic dyes', Adsorption Science and Technology, 19(3), pp. 197–210. doi: 

10.1260/0263617011494088.  

Ibrahim, Y., Abdulkarem, E., Naddeo, V., Banat, F., and Hasan, S. W. (2019) ‘Synthesis of super 

hydrophilic cellulose-alpha zirconium phosphate ion exchange membrane via surface coating for the 

removal of heavy metals from wastewater’, Science of the Total Environment, 690, pp. 167–180. doi:  

10.1016/j.scitotenv.2019.07.009.  

Ilavský, J., Barloková, D. and Marton, M. (2020) ‘Triazine herbicides removal from water with granular 

activated carbon’, in IOP Conference Series: Earth and Environmental Science. IOP Publishing Ltd. 

doi: 10.1088/1755-1315/609/1/012088.  

Isogai, A., Saito, T. and Fukuzumi, H. (2011) ‘TEMPO-oxidized cellulose nanofibers’, Nanoscale, 3(1), 

pp. 71–85. doi: 10.1039/c0nr00583e.  

Jain, P., Varshney, S. and Srivastava, S. (2016) ‘Functionalized nanobiomaterials : high-performance 

sorbents for chromium remediation from water streams’, International Journal of Environmental 

Science and Technology, 13(12), pp. 2893–2904. doi: 10.1007/s13762-016-1115-z.  

Jamshaid, A., Hamid, A., Muhammad, N., Naseer, A., Ghauri, M., Iqbal, J., Rafiq, S., Shah, N. S. (2017)  

‘Cellulose-based Materials for the Removal of Heavy Metals from Wastewater – An Overview’, 

ChemBioEng, 4(4), pp. 1–18. doi: 10.1002/cben.201700002.  

Jarusiripot, C. (2014) ‘Removal of Reactive Dye by Adsorption over Chemical Pretreatment Coal based 

Bottom Ash’, Procedia Chemistry, 9, pp. 121–130. doi: 10.1016/j.proche.2014.05.015.  

Kaur, Y., Bhatia, Y., Chaudhary, S., and Chaudhary, G. R. (2017) ‘Comparative performance of bare 

and functionalize ZnO nanoadsorbents for pesticide removal from aqueous solution’, Journal of 

Molecular Liquids, 234, pp. 94–103. doi: 10.1016/j.molliq.2017.03.069.  

Kobya, M., Gebologlu, U., Ulu, F., Oncel, S., and Demirbas, E. (2011) ‘Removal of arsenic from 

drinking water by the electrocoagulation using Fe and Al electrodes’, Electrochimica Acta, 56(14), pp. 

5060–5070. doi: 10.1016/j.electacta.2011.03.086.  

Konicki, W., Cendrowski, K., Bazarko, G., and Mijowska, E. (2015) ‘Study on efficient removal of 

anionic, cationic and nonionic dyes from aqueous solutions by means of mesoporous carbon 

nanospheres with empty cavity’, Chemical Engineering Research and Design, 94, pp. 242–253. doi:  



8  

  

10.1016/j.cherd.2014.08.006.  

Kumar, R., Kim, S., Kim, K., Lee, S., Park, H., Jeon, B. (2018) ‘Removal of hazardous hexavalent 

chromium from aqueous phase using zirconium oxide-immobilized alginate beads’, Applied 

Geochemistry, 88, pp. 113–121. doi: 10.1016/j.apgeochem.2017.04.002.  

Li, Z. J., Xue, H. D., Zhang, Y. Q., Hu, H., S., Zheng, X. D. (2019) ‘Construction of a cationic organic 

network for highly efficient removal of anionic contaminants from water’, New Journal of Chemistry, 

43(29), pp. 11604–11609. doi: 10.1039/c9nj00886a.  

Liu, Y., Ma, H., Liu, B., Hsiao, B. S., Chu, B. (2015) ‘High-performance nanofibrous membrane for 

removal of Cr(VI) from contaminated water’, Journal of Plastic Film and Sheeting, 31(4), pp. 379–

400. doi: 10.1177/8756087915590645.  

Mahdavi, H. and Rahimi, A. (2018) ‘Zwitterion functionalized graphene oxide / polyamide thin film 

nanocomposite membrane: Towards improved anti-fouling performance for reverse osmosis’, 

Desalination, 433, pp. 94–107. doi: 10.1016/j.desal.2018.01.031.  

Mahida, V. P. and Patel, M. P. (2016) ‘Removal of some most hazardous cationic dyes using novel poly 

(NIPAAm/AA/N-allylisatin) nanohydrogel’, Arabian Journal of Chemistry, 9(3), pp. 430–442. doi: 

10.1016/j.arabjc.2014.05.016.  

Mohan, D. and Pittman, C. U. (2007) ‘Arsenic removal from water/wastewater using adsorbents-A 

critical review’, Journal of Hazardous Materials, 142(1–2), pp. 1–53. doi:  

10.1016/j.jhazmat.2007.01.006.  

Mukheibir, P. (2010) ‘Water access, water scarcity, and climate change’, Environmental Management, 

45(5), pp. 1027–1039. doi: 10.1007/s00267-010-9474-6.  

Neeraja, G., Raghunandana, S. K., Kumar, P. S., Cabana, H., Kumar, V. V. (2016) ‘Adsorptive potential 

of dispersible chitosan coated iron-oxide nanocomposites toward the elimination of arsenic from 

aqueous solution’, Process Safety and Environmental Protection, 104, pp. 185–195. doi: 

10.1016/j.psep.2016.09.006.  

Nicomel, N. R., Leus, K., Folens, K., Van Der Voort, P., and Laing, G. D. (2015) ‘Technologies for  

Arsenic Removal from Water: Current Status and Future Perspectives’, Environmental Research and 

Public Health, 13(62), pp. 1–24. doi: 10.3390/ijerph13010062.  



9  

  

Pennells, J., Godwin, I. D., Amiralian, N., Martin, D. J. (2020) ‘Trends in the production of cellulose 

nanofibers from non-wood sources’, Cellulose, 27(2), pp. 575–593. doi: 10.1007/s10570-019-02828-9.  

Sadegh, H., Ali, G. A. M., Gupta, V. K., Makhlouf, A. S. H., Shahryari-ghoshekandi, R., Nadagouda,  

M. N., Sillanpää, M., and Megiel, E. (2017) ‘The role of nanomaterials as effective adsorbents and their 

applications in wastewater treatment’, Journal of Nanostructure in Chemistry, 7(1), pp. 1–14. doi: 

10.1007/s40097-017-0219-4.  

Sannino, F., Ruocco, S., Marocco, A., Esposito, S., and Pansini, M. (2012) ‘Cyclic process of simazine 

removal from waters by adsorption on zeolite H-Y and its regeneration by thermal treatment’, Journal 

of Hazardous Materials, 229–230, pp. 354–360. doi: 10.1016/j.jhazmat.2012.06.011.  

Sartika, D., Syamsu, K., Warsiki, E., and Fahma, F. (2019) ‘Optimization of Sulfuric Acid 

Concentration and Hydrolysis Time on Crystallinity of Nanocrystalline Cellulose: A Response Surface  

Methodology Study’, in IOP Conference Series: Earth and Environmental Science, pp. 1–9. doi: 

10.1088/1755-1315/355/1/012109.  

Sharma, A., Tomer, A., Singh, J., Chhikara, B.S. (2019) ‘Biosorption of metal toxicants and other water 

pollutants by Corn (Maize) plant: A comprehensive review’, Journal of Integrated Science and  

Technology,  7(2),  pp.  19–29.  Available  at: 

http://pubs.iscience.in/journal/index.php/jist/article/view/918.  

Sharma, P. R., Sharma, S. K., Lindström, T., Hsiao, B. S. (2020) ‘Nanocellulose-Enabled Membranes 

for Water Purification: Perspectives’, Advanced Sustainable Systems, 4(5), pp. 1–28. doi:  

10.1002/adsu.201900114.  

Singh, K., Sinha, T. J. M. and Srivastava, S. (2015) ‘Functionalized nanocrystalline cellulose: Smart 

biosorbent for decontamination of arsenic’, International Journal of Mineral Processing, 139, pp. 51– 

63. doi: 10.1016/j.minpro.2015.04.014.  

Sujitha, R. and Ravindhranath, K. (2016) ‘Removal of Coomassie brilliant blue dye from waste waters 

using active carbon derived from barks of Ficus racemosa plant’, Der Pharmacia Lettre, 8(10), pp. 72– 

83.  

Szewczyk, R. and Ku, A. (2018) ‘Chemosphere Ametryn removal by Metarhizium brunneum:  

Biodegradation pathway proposal and metabolic background revealed’, 190, pp. 174–183. doi: 

10.1016/j.chemosphere.2017.10.011.  



10  

  

Tavakolian, M., Jafari, S. M. and Ven, T. G. M. Van De (2020) ‘A Review on Surface ‑ Functionalized 

Cellulosic Nanostructures as Biocompatible Antibacterial Materials’, Nano-Micro Letters, 12(73), pp. 

1–23. doi: 10.1007/s40820-020-0408-4.  

Verduzco, L E., Oliva, J., Oliva, A I., Macias, E., Garcia, C R., Herrera-trejo, M., Pariona, N., and 

Mtzenriquez, A I. (2019) ‘Enhanced removal of arsenic and chromium contaminants from drinking 

water by electrodeposition technique using graphene composites’, Materials Chemistry and Physics, 

229, pp. 197–209. doi: 10.1016/j.matchemphys.2019.03.006.  

Vieira, M. G. A., De Almeida Neto, A. F., Silva, M. G. C., Carneiro, C. N., and Melo Filho, A. A.  

(2014) ‘Adsorption of Lead and Copper Ions from Aqueous Effluents on Rice Husk Ash In A Dynamic 

System’, Brazilian Journal of Chemical Engineering, 31(02), pp. 519–529.  

World Health Organization (2019) Drinking-water. Available at: https://www.who.int/news-

room/factsheets/detail/drinking-water (Accessed: 22 May 2021).  

World Population Prospects (2019).  

 .  



11  

  

CHAPTER 2: LITERATURE REVIEW  

2.1 Hemp fibres (Cannabis Sativa L.)  

Schultes wrote:  

“Hemp is a green, very abundant and ubiquitous plant, economically valuable, a versatile and 

multipurpose product, possibly dangerous and certainly in many ways mysterious” (Schultes 1970)  

Hemp (Cannabis sativa L.) is one of the most cultivated and used industrial crops worldwide. Hemp 

has an   estimated world production of 214×103 tons per year (Rahman et al., 2018). Hemp produces 

two types of natural fibres namely, bast fibres which are the fibrous form and woody core fibres (also 

known as Hurds) which is the granular form, and both are abundant in nature (Morin-Crini et al., 2018).  

Hemp bast fibres are mainly composed of 76% cellulose, 14% hemicellulose, 5% lignin, 1% pectin’s, 

and 6% of other substances, whereas Hurds are composed of 48% cellulose, 24% hemicellulose, 19% 

lignin, 2% ash and less than 5% of other substances (Väisänen et al., 2018)(Salami et al., 2020). Hemp 

fibres are low cost, biodegradable, environmentally friendly, durable, ease of processing, low density, 

and have good mechanical properties compared to synthetic fibres (Abraham, Wong and Puri, 

2016)(Jarabo et al., 2012). Furthermore hemp fibres have several industrial uses such as reinforcements 

for building materials, textile, paper processing, medicine, cosmetics, pharmaceuticals food 

manufacturing, detergent and bio-composites (Salami et al., 2020)(Jarabo et al., 2012)(Kassab et al., 

2020).   

2.2 Cellulose and its hierarchical structure  

Cellulose was first isolated in 1839 by the French chemist, Anselme Payen while studying various wood 

sources (Devabaktuni Lavanya, P.K.Kulkarni, Mudit Dixit, Prudhvi Kanth Raavi, 2011). Cellulose is a 

vital structural component of the cell walls of higher plants. It contributes up to 90 % of cotton, 80 % 

of hemp, 75 % of flax and 40-50% of wood (see Table 2.1) (Hokkanen, Bhatnagar and Sillanpää, 2016)( 

Huang et al., 2020a). Cellulose can be found in a number of living organisms such as amoeba, algae, 

fungi and bacterium acetobacter xylinum and even in some aquatic animals e.g tunicates (Figure 2.1) 

(Dong, Roman and Long, 2014)(Mansoori et al., 2020). Cellulose is the most abundant and renewable 

natural polymer on Earth. The global production of cellulose has been estimated to be 1.5 × 1012 tons 

each year (George and Sabapathi, 2015)(Tavakolian, Jafari and Ven, 2020). Cellulose has a wide range 

of physical and physiochemical properties such as high stiffness, high strength, biocompatibility, 

renewability, excellent thermal stability, high sorption capabilities and non-toxicity (Ratajczak and 

Stobiecka, 2020). Furthermore, due to previously mentioned properties and low cost, cellulose and other 

biopolymer materials attract more attention for applications in a variety of areas such as reinforcement 



12  

  

agents, water treatment, food packaging, textiles, bio-composite, medicine, and many more as shown 

in Figure 2.2(Abouzeid et al., 2019) (Di Giorgio et al., 2020)(Abiaziem et al., 2019). Cellulose as a 

main component of biomass has been used for decades for a wide variety of applications and will 

continue to be investigated and applied in the future to contribute to environmental and economic 

improvement.   

  

Figure 2.1: Sources of cellulose of various plant and animal species   

Table 2.1: Cellulose, hemicellulose, and lignin as major components in the chemical composition of 

various plants materials.  

Source Percentage composition (wt %) Ref  

Cellulose  Hemicellulose  Lignin  

 Softwood  42 40 32 (Tarasov et al., 2018) 

Hardwood 51 38 31 (Tarasov et al., 2018) 

Hemp fiber 74 14 5 (Väisänen et al., 2018) 

Corn husk  42 41 13 (De Carvalho Mendes et 

al., 2015) 

Sugarcane bagasse 46 24 24 (Candido and Gonçalves, 

2019) 

Bamboo 35 27 11 (Wijaya et al., 2019) 

Tea stalk 35 20 28 (Guo et al., 2020) 



13  

  

Rice husk  35 25 20 (Motaung and Linganiso, 

2018) 

Rice straw  37 29 23 (Ratnakumar et al., 2022) 

Sorghum 46 15 17 (Motaung and Linganiso, 

2018) 

Maize stem 39 28 15 (Longaresi et al., 2019) 

Banana trunks (Musa 

acuminata) 

33 39 27 (Merais et al., 2022) 

Banana rachis 34 8 16 (Gabriel et al., 2020) 

Calotropis procera fiber 64.1 19.5 9.7 (Song et al., 2019) 

Corn stalk 38 46 16 (Chen et al., 2020) 

Corn stover  40 28 10 (Hassan et al., 2016) 

Garlic straw 41 18 6 (Kallel et al., 2016) 

Yerba mate fiber  29 25 29 (Ju et al., 2020) 

Peanut shell 30 19 29 (Chen et al., 2020) 

Pineapple waste (roots) 42 32 19 (Maisyarah et al., 2019) 

Kans grass (Saccharum 

spontaneum) 

48 32 17 (Baruah et al., 2020) 

Date seed (Phoenix 

dactylifera L.) 

25 25 31 (Abu-thabit et al., 2020) 

Eggplant plant (Solanum 

melongena L.) 

63 7 23 (Bahloul, Kassab, El, et 

al., 2021) 

Sugar palm fibers  37-54 4-8 17-25 (Ilyas et al., 2021) 



14  

  

  

Figure 2.2: Applications of cellulose.  

2.2.1 Cellulose structure and properties   

Cellulose is a long linear polysaccharide chain consisting of repeating units of D-glucopyranose also 

commonly known as anhydroglucose units (AGU) in a chair conformation with molecular formula of 

(C6H11O5)n. The AGU are joined together by β-1,4-glycosidic linkages formed between C1 and C4 

position to form a dimer of glucose known as cellobiose (Figure 2.3) (Mansoori et al., 2020)(Hokkanen, 

Bhatnagar and Sillanpää, 2016)(Abouzeid et al., 2019). Each repeating unit of AGU has three highly 

reactive hydroxyl (OH-) groups which contribute to cellulose’s properties (hydrophilicity, degradability, 

chirality), each one at position C2, C3, and C6 (Dong, Roman and Long, 2014). The hydroxyl groups 

at position C2 and C3 are secondary alcohols and the hydroxyl group at position C6 is a primary alcohol 

making them hydrophilic in nature. Moreover, cellulose does not dissolve in water and most common 

solvents (acetone, ethanol, methanol) due to strong hydrogen bonding interactions between cellulose 

chains (Jasmani and Thielemans, 2018). Within the cellulose chains, crystalline domains are formed 

due to van der Waals forces formed between the glucose units and hydrogen bonds formation between 

the cellulose chains (Hokkanen, Bhatnagar and Sillanpää, 2016). As shown in Figure 2.3, cellulose  

chains are made up of two chemically different ends: the non-reducing end is the one end with a closed 

ring structure and the reducing end is the opposite end which has a free anomeric carbon atom of 

hemiacetal nature in equilibrium with an aldehyde (Jasmani and Thielemans, 2018)(Eyley and 

Thielemans, 2014). The degree of polymerization (DP) as the number of glucose unit in a 

polysaccharide chain molecule given as n, strongly depends on the cellulose source and treatment 



15  

  

conditions applied for the isolation of cellulose (Jasmani and Thielemans, 2018)(Seddiqi et al., 2021). 

The DP of cellulose materials can range from hundreds to a few thousands (Table 2.2).   

 

Figure 2.3: Molecular structure of cellulose showing the non-reducing ends, terminal reducing ends and 

cellobiose unit.  

Table 2.2: Various sources of cellulose and their degree of polymerization (DP)  

Source Type  Degree of 

polymerization (DP) 

Ref  

Wood Hardwood/softwood 1200-10,000 (Seddiqi et al., 2021)(Pei et al., 2013)  

Wood pulp 300-1700 (Dong et al., 2014)(Jasmani and 

Thielemans, 2018) 

Wood CNF 250-3500 (Seddiqi et al., 2021) 

Plants Cotton  800-10,000 (Jasmani and Thielemans, 

2018)(Ioelovich, 2014) 

Sugarcane bagasse 974–1039 (Bian et al., 2014) 

Corn husk 50-300 (El-Torky et al., 2016) 

Hemp  200-1300 (Ji et al., 2021) 

Bacteria  2000-16,000 (Yang et al., 2019)(Danafar, 

2020)(Wang, Tavakoli and Tang et al., 

2019) 

Algae  Up to 10,000 (Santmarti and Lee, 2018) 

Tunicate   700-3500 (Zhao and Li, 2014) 

 



16  

  

2.2.2 Cellulose microfibres (CMFs), cellulose nanofibres (CNFs), and cellulose nanocrystals (CNCs)  

Over the years in order to improve cellulose application in various industries, cellulose fibres have been 

derived from several sources with the aim to improve cellulose's mechanical and physical properties 

such as low density, high tensile strength and stiffness, high aspect ratio, and  high specific surface area 

for applications in water and wastewater treatment, paper making, biomedical engineering, and energy 

production (Abouzeid et al., 2019)(Shak, Pang and Mah, 2018)(Xie et al., 2018). Cellulose materials 

are thought of as emerging readily available biomass materials that is cost-effective, renewable, 

biocompatible, biodegradable, and causes little to no toxicity towards the environment.The Hierarchal 

nature cellulose is that it can be converted into different structures like microcrystalline cellulose 

(MCC), cellulose microfibres (CMFs), cellulose nanofibres (CNFs), cellulose nanocrystals (CNCs) 

using various treatments and approaches and Table 2.3 can be used to distinguish cellulose using their 

varying sizes (Figure 2.4).  

Purified CMFs can isolated from raw biomass sources by chemical treatment methods such as alkaline 

treatment using NaOH to remove hemicellulose followed by bleaching treatment using sodium chlorite 

to remove lignin material to bleach the fibres (Puttaswamy, Srinikethan and Shetty, 2017)(Ait 

Benhamou et al., 2022). Due to strong intra- and inter-molecular hydrogen bonds and van der Waals 

forces interactions within the cellulose molecular chain, the extraction and isolation of pure cellulose 

from the source is influenced by pretreatment methods that are used to aid the removal hemicellulose, 

lignin, pectin, ash, wax and other non-cellulosic materials from biomass materials (Wang, 

2019)(Shamsabadi, Behzad and Bagheri, 2015). CMFs usually exhibit several micrometer-wide fiber 

diameters and are composed of both crystalline and amorphous regions (Risite et al., 2022). Several 

other methods have also been used such as enzymatic hydrolysis, high-pressure homogenizer 

(SanchezSalvador et al., 2019), organosolv pretreatment (Ferreira et al., 2018), and steam explosion 

(Sonia and Dasan, 2013)(Tanpichai, Witayakran and Boonmahitthisud, 2019). CNFs are webbed-like 

cellulose particles composed of both crystalline and amorphous regions (Feng et al., 2018). CNFs have 

diameters less than<100 nm and lengths of 500 nm or even longer (Du et al., 2017). CNFs can be 

isolated from various cellulosic sources via mechanical treatments such as microfluidizer (Pacaphol and 

Aht-Ong, 2017), high-intensity ultrasonication (Kusumaningrum et al., 2020), high-pressure 

homogenisation (Li et al., 2014)(Wang et al., 2015), cryocrushing (Alemdar and Sain, 2008), grinding 

process (Xie et al., 2018), enzymatic hydrolysis (Tibolla, Pelissari and Menegalli, 2014), TEMPO 

oxidation method (Yang et al., 2017), or a combination of  two or more processes (Hu et al., 2017)(Soni, 

Hassan and Mahmoud, 2015). CNCs are rod-like shaped cellulose particles of highly crystalline nature 

with diameter size of 5 - 30 nm and length size of 100 nm up to several 𝜇m (Wijaya et al., 2019). CNCs 

can be isolated from cellulose using several methods for example, acid hydrolysis (Wijaya et al., 2019), 

enzymatic hydrolysis (Cui et al., 2016), high-pressure homogenisation (Lee et al., 2018), high-sheer 



17  

  

homogenisation (Zhao et al., 2013), microfluidisation (Khan et al., 2014), and/or a combination of two 

or more methods (Tang et al., 2014).   

 

Figure 2.4: Schematic of showing cellulosic fibres, cellulose microfibres, cellulose nanofibres, and 

cellulose nanocrystals with their respective sizes.  

Table 2.3: Type of nanocellulose with their respective average particle sizes  

Type of nanocellulose Sources of cellulose Average particle size  Ref 

Cellulose microfibers 

(CMFs) 

Doum tree, pineapple 

leaves, hemp 

Diameter of several 

micrometres  

(Bahloul, Kassab, 

Aziz, et al., 

2021)(Tanpichai et 

al., 2019) 

Cellulose nanofibers 

(CNFs) 

Wood, cotton, hemp, 

flax, straw, tunicin, 

algae, bacteria 

Width: 5–60 nm. 

Length: few microns 

(Wang, 2019)(Shak 

et al., 2018) 

Cellulose nanocrystals 

(CNCs) 

Wood, cotton, hemp, 

flax, straw, tunicin 

Width: 5–70 nm 

Length: 100 nm to 

several micrometres 

(Hokkanen et al., 

2016)(Xie et al., 

2018) 

 

2.3 Isolation techniques and their effects on crystallinity index and the surface morphology.  

2.3.1 Chemical methods   

Chemical methods such as acid hydrolysis and enzymatic hydrolysis have been of great interest for the 

isolation of nanocellulose from various sources.  



18  

  

2.3.1.1 Acid hydrolysis  

Acid hydrolysis treatment breaks apart the 𝛽-glycosidic bonds of the amorphous regions and crystalline 

regions of cellulose. This removes amorphous regions resulting in a distinct single crystalline region 

called CNCs with rod-like configuration and high crystallinity (Figure 2.5) (Yang et al., 

2019)(Zimmermann et al., 2016). During acid hydrolysis, the tightly packed cellulose chain undergoes 

an acid attack on the amorphous (disordered) regions during which the hydronium ions invade these 

regions encouraging the hydrolytic cleavage of glycosidic linkages in the process in order to isolate the 

acid attack resistant crystalline structures resulting in CNCs (Zimmermann et al., 2016)(Pereira and 

Arantes, 2018). Acid hydrolysis is the most frequently used method to yield CNCs due to its high 

efficiency for CNC preparation, and this can be achieved by using sulfuric acid (Lu and Hsieh, 2010)(de 

Andrade et al., 2019), hydrochloric acid (Huntley et al., 2015), p-toluenesulfonic acid (p-TsOH) (Wang 

et al., 2019), formic acid (Liu et al., 2016), maleic acid (Seta et al., 2020), phosphoric acid (Risite et 

al., 2022), hydrobromic acid (Sadeghifar et al., 2011), and other mixed acids (Wang et al., 2019)(Frost 

and Johan Foster, 2020).   

  

Figure 2.5: Cellulose acid hydrolysis mechanism.  

2.3.1.2 Sulfuric acid hydrolysis  

Sulfuric acid (SA) is a strong mineral acid (pKa -3.0 and 2.0) and has the chemical formula H2SO4 

(Almashhadani et al., 2022). SA is most frequently used in lignocellulosic residues hydrolysis, as it 

produces negatively charged surface charges to produce more stable CNC suspensions with good 

dispersibility in water. Consequently, because of the sulfate groups on the crystalline surface, this 

reduces the thermal stability of the fibres (Xie et al., 2018)(Jung, Choi and Yang, 2013). Shaheen et al, 

investigated the potential of commercially non-recyclable wood waste (sawdust) for the isolation of 

CNCs via acid hydrolysis together with ultrasonication technique. Alkaline treatment using 1.0 M 

NaOH was used to delignify sawdust material followed by sodium chlorite bleaching treatment for the 

removal of any non-cellulosic materials. Acid hydrolysis conditions of 65 wt% H2SO4 at 60℃ for 60 



19  

  

min were followed and CNCs were collected by centrifugation to remove traces of sulfate salts followed 

by dialysis to remove free acid then freeze-dried to produce CNC powder. From the results, the 

combination of sonication and chemical treatment had been effective for extraction of CNC with rodlike 

shape of the highly stiffened cellulose crystals like sticks and average diameter of 35.2 ± 7.4 nm were 

confirmed from SEM (Figure 2.6) and TEM, respectively. The CNC crystal structure is in accordance 

with cellulose type I with crystallinity index ⁓90%, according to the XRD data. (Shaheen and Emam, 

2018). 

  

Figure 2.6: SEM images for the CNC at different magnification (300, 100 and 50 µm), (Image adapted 

from (Shaheen and Emam, 2018)).  

Liu et al, also studied the extraction of CNCs from a waste material namely corncob residue (CCR). 

Prior to acid hydrolysis, alkaline and bleaching treatments were employed to treatment the CCR. 

Hydrolysis conditions of 64 wt% H2SO4 with 45℃ for 60 min was used and produced rod-like 

nanocrystals with an average diameter of 5.5 ± 1.9 nm. The nanocrystals produced seemed to be well 

dispersed and individualized due to the presence of negatively charged sulfate groups on the surface of 

the CNCs and the crystallinity of the CNC was found to be 55.9% decreasing from raw CCR (61.5%). 

Furthermore, this could be explained by the fact that the strong acid distorted the amorphous portion of 

cellulose as well as the crystalline portion during hydrolysis (Liu et al., 2016). In addition, Van Pham 

et al, studied the extraction of thermally stable CNCs from waste newspaper by sulfuric acid hydrolysis. 

The results showed that CNCs with rod-shaped structures were obtained with average diameters of 

about 12.3 ± 2.8 nm, high crystallinity index of 80.15%, and improved thermal stability with the 

crystalline regions of cellulose allowing for high temperature applications (Van Pham et al., 2020) 

Although the use strong mineral acids such as sulfuric acid produces high crystallinity cellulose 

particles, there are a few drawbacks (non-environmentally friendly, high operational and maintenance 

costs, highly corrosive to equipment and not easily recovered). Organic acids as alternatives to improve 

cellulose acid hydrolysis has been studied. For this study, formic and maleic acid were compared with 



20  

  

sulfuric acid in terms of surface morphology, chemical analysis, crystallinity degree and thermal 

stability.   

2.3.1.3 Formic acid hydrolysis  

Formic acid (FA) is a weak organic acid with the chemical formula HCOOH. FA can be used to prepare 

CNC with longer rod-like structures under controlled conditions. As a result of its low boiling point 

(about 100.8 ℃), FA is readily recoverable and reusable in multiple reactions, less corrosion to 

equipment and is environmentally friendly (Li et al., 2015) Liu et al, used 88 wt% formic acid to extract 

CNC from corncob residue and produced long rod-like nanocrystals with an average diameter of 6.5  

2.0 nm thereby forming agglomerated CNCs with high crystallinity of ~64% and good thermal stability 

(Liu et al., 2016). In addition, Lv’s et al, group demonstrated the tailored and integrated production of 

functional CNCs and CNFs through formic acid hydrolysis. CNCs and CNFs produced were tailored 

with sustainable characteristics appropriate for use in polymeric materials owing to the hydrophobic 

surfaces (see Figure 2.7). CNCs and CNFs exhibited high crystallinity index values of 79.1% and 

61.0%, respectively. The surface morphology of the CNCs and CNFs was shown to be differentiable 

where CNCs had the average diameter of 11 nm, and average length of 141 nm, whereas CNFs 

microfibrils bundles of several length (Lv et al., 2019). Formic acid hydrolysis was found to be able to 

isolate CNCs from oil palm empty fruit bunch (OPEFB). The resultant CNCs were found have high 

crystallinity index (69.82%) with needle-like structures (bin Jumhuri et al., 2017).  

  

Figure 2.7: Schematic diagram of FeCl3-catalyzed FA hydrolysis for the integrated production of CNCs 

and CNFs (Image adapted from (Lv et al., 2019)).  

2.3.1.4 Maleic acid hydrolysis  

To overcome challenges faced with mineral acid hydrolysis, solid acids (maleic acid, oxalic acid, 

phosphotungstic acid) have been applied in hydrolysis as hydrolysis catalysts. Maleic acid (MA) is a 

solid organic acid and has chemical formula HO2CCH=CHCO2H. MA has considerable advantages 



21  

  

compared to mineral acids such as that it causes less corrosive to equipment, safe for storage, low 

transportation costs, cheap, environmentally friendly, easy to recover, uses milder reaction conditions, 

possible surface modification during hydrolysis, and higher boiling points (Wang et al., 2019)(Seta, An 

and Liu, 2021)(Nurhadi et al., 2022). Seta et al, used a green approach to isolate and extract CNCs 

through MA hydrolysis from bamboo fibres. To increase the accessibility of MA molecules and reveal 

more hydroxyl groups on the surface of bamboo fibers, ball-mill pre-treatment were done to the 

cellulose fibres. MA hydrolysis resulted in improved crystallinity (CrI = 85-91%) due to the removal 

of amorphous region in cellulose (Seta et al., 2020). Wang et al, used MA to produce CNC and CNF 

extracted from bleached eucalyptus kraft pulp (BEP). Hydrolysis experiments were conducted using 

MA concentrations between 15–75 wt%, hydrolysis temperature ranges between 60–120℃, and 

hydrolysis reaction time between 5–300 min. The average length of CNCs produced was around 

450−650 nm whereas CNFs produced were several micrometers longer and could not be deduced by 

AFM analysis (Wang et al., 2017a). In addition, a research group by Bian et al, found that carboxylated  

CNFs extracted from bleached pulp fibres using recyclable MA have average height of about 6−20 nm 

with entangled fibril networks and average crystallinity index of about 80% (higher than the original 

BEP). It is worth noting that as the severity of hydrolysis conditions increased, CNF with a shorter 

length and smaller diameter resulted, and CrI value was slightly reduced (Bian et al., 2019).  

  

Figure 2.8: Schematic diagram for the MA hydrolysis of bleached eucalyptus pulp to produce CNFs, 

along with acid crystallization, acid recovery, and acid reuse (Image adapted from (Bian et al., 2019)).  

2.3.1.5 Enzymatic hydrolysis  

Cellulase is a multicomponent enzyme system that is widely utilised in enzymatic hydrolysis process, 

and it can be divided into three components namely endoglucanases (EG), cellobiohydrolases (CBH), 



22  

  

and β-glucosidase enzymes (Xie et al., 2018)(Tibolla, Pelissari and Menegalli, 2014). These enzymes 

have targeted reactivity and selectivity, thus the hydrolysis occurs in three parts i) firstly, the EG enzyme 

decomposes the amorphous region of cellulose by randomly hydrolysing the β-1,4-glycosidic linkages 

in a cellulose chain generating smaller fibres with new terminal chains, ii) secondly, by removing the 

crystalline region of cellulose, the CBH enzyme primarily targets the terminal chains of cellulose to 

form cellobiose and iii) lastly, β-glucosidase enzymes are used to hydrolyse cellobiose into glucose (Yi 

et al., 2020)(Tibolla et al., 2017)(Kargarzadeh et al., 2017). See mechanism for enzymatic hydrolysis 

in Figure 2.9.  

 

Figure 2.9: Diagrammatic representation of the effects of cellulase on cellulose (Image adapted from 

(Lynd et al., 2002)).  

Zhang et al, reported on production of CNFs using enzyme-assisted mechanical techniques and the 

results revealed that synthetized CNFs had diameters <100 nm, however the thermal stability of CNFs 

diminished as enzyme dosage was increased. This reduction in thermal stability of CNFs compared to 

original pulp reduces the applicability of CNFs in high temperature applications (Zhang et al., 2018a). 

Additionally, Tao and colleagues conducted research on enzymatic pretreatment to separate cellulose 

nanofibrils from bagasse pulp. using cellulase, alkali pretreatment and combination of ultrafine grinding 

and high-pressure homogenisation techniques. It was discovered that the produced CNFs had a diameter 

of approximately 30 nm, reduced crystallinity, and the cellulose crystal structure transitioned from type 

I to type II. Furthermore, CNFs prepared had lower thermal stability and this was ascribed to the 

removal of amorphous regions of cellulose by pretreatment, grinding and high-pressure homogenisation 

methods leading smaller fibre dimensions with each treatment (Tao et al., 2019). Long et al, stated that 

CNFs produced by xylanase-aided enzymatic pretreatment showed increased crystallinity. This increase 



23  

  

was attributed to the cleavage of the β-1,4-glucosidic linkages in the disordered region of cellulose fibre 

hence a decrease of polymerisation and an increase in crystallinity (Long et al., 2017). Although 

enzymatic hydrolysis has been utilised for isolation of CNCs/CNFs, it has been combined with other 

processes (high-pressure homogenisation, ultrafine grinding, alkali pretreatment processes) to extract 

effectively. In addition, Li and associates, reported the considerable evidence in a review supporting 

the challenges such as expensive equipment needed for extraction, high investment costs, secondary 

contamination, and production of inhibitors limit large-scale application of enzyme hydrolysis in 

industries (Li et al., 2022)   

2.3.2 Mechanical methods  

Mechanical methods including high intensity ultrasonication (HIUS), high-pressure homogenisation 

(HPH), cryocrushing, microfluidisation, and grinding have gained interest for CNFs extraction over the 

past decades   

2.3.2.1 High Intensity Ultrasonication (HIUS)  

Recently, the high-intensity ultrasonication (HIUS) technique has been vastly applied for the successful 

isolation and preparation of CNFs from various sources and it has attracted significant attention 

(Bracone, Luduena and Alvarez, 2022)(Dilamian and Noroozi, 2019)(Lee et al., 2020)(Chen, Yu and 

Liu, 2011). During the HIUS process, the effect of ultrasonic energy is applied to the polysaccharide 

chains of cellulose via a cavitation process which involves the formation, growth and rapid collapse of 

cavities in water through intense shear forces, shockwaves, and microjets (Chen et al., 2011)(Hu et al., 

2017)(Kargarzadeh et al., 2017). Sonochemistry is the energy provided by the cavitation process which 

is between 10−100 kJmol-1 of hydrogen bond energy scale, capable of gradually decomposing cellulose 

weak interfibrillar hydrogen linkages thus forming CNFs from micron-sized cellulose fibres as shown 

in Figure 2.10 (Chen et al., 2011)(Ishak et al., 2020)(Lu et al., 2013)( Ji, Yu, Yagoub and Chen, 2021a). 

The characteristics of isolated CNFs by HIUS strongly depend on ultrasonic conditions such as process 

time, frequency, amplitude and solvent chemistry thus influencing the crystallinity degree, nanofibre 

size and thermal stability of nanofibres (Mazela et al., 2020)(Ji et al., 2020)( Ji, Yu, Yagoub, Chen, et 

al., 2021b)(Lee et al., 2020).   



24  

  

  

Figure 2.10: Schematic diagram showing the production of individualized CNFs using ultrasonic 

treatment (Imaged adapted from (Chen et al., 2011)).  

During the isolation of CNFs from culinary banana peel using HIUS with assisted chemical treatment,  

Khawas and Deka reported that increasing the ultrasonication output power reduced the size of CNFs 

forming thinner and needle-like structural fibrils compared to banana plant fibres. In addition, high 

crystallinity index values of about 63.64% and improved thermal stability was attained with higher 

power output during HIUS process, hence CNFs can be employed as a bio-nanocomposites due to its 

reinforcing properties. (Khawas and Deka, 2016). Abral et al, investigated the bacterial cellulose for 

the isolation of nano-sized bacterial cellulose (BC) particles via ultrasonication method and it was 

reported that nano-sized BC particles were successfully isolated with HIUS process. However, a 

decrease in crystallinity index values from 80% (no ultrasonication) to 68% (with ultrasonication) was 

reported which was attributed to reduced nano-sized BC particles isolated from the scission of the 

micro-length BC fibre by the high kinetic energy of a jet of liquid from acoustic cavitation produced by 

the ultrasonic equipment (Abral et al., 2018). Quite recently, Szymańska-Chargot et al, reported on the 

production of CNFs extracted from Hop stems by HIUS treatments and how HIUS influences the 

properties of extracted CNFs. Extracted CNFs showed a decrease in crystallinity degree from 67% to 

60% and a decrease in the fibre diameter up to 4 nm as longer HIUS treatments were applied. In 

addition, CNFs with higher thermal stability were produced, suitable for applications as natural 

reinforced or packaging biocomponents (Szymańska-Chargot et al., 2022).   

2.3.2.2 High-pressure homogenisation   

High-pressure homogenisation (HPH) is one of the most widely used mechanical methods applied for 

preparation of CNFs as it is a simple, economically feasible, and high efficiency technique and it lacks 

organic solvents (Li et al., 2012). The fundamental role of the HPH process is to cause high pressure 

(>150MPa), high shear, turbulence, and cavitation to cellulose pulp suspensions that is continuously 



25  

  

flowing through the homogenisation chamber (see Figure 2.11). Hence, this causes the disintegration 

of amorphous regions in cellulose, decreases size of cellulose fibres, results in dispersion of 

brokendown cellulose fractions thereby resulting in total cell disruption and consequently producing 

CNFs (Li et al., 2014)(Pacaphol and Aht-Ong, 2017)(Yi et al., 2020). In the past decade, most research 

shows that the cellulose fibre dimensions typically become smaller and more uniform as the number of 

homogenisation cycles increases. Therefore, high energy consumption can result due to increase in 

number of cycles, along with tedious equipment maintenance and low reliability (Yi et al., 2020). 

Furthermore, as cellulose is insoluble in most organic solvents including water, homogenisation can 

result in valve clogging because of the numerous extensive cellulose networks of intra and/or 

intermolecular hydrogen bonds and therefore needs assistance by inorganic solvents or other 

pretreatment methods (Wang et al., 2015)(Li et al., 2012).  

  

Figure 2.11: Schematic diagram of a high-pressure homogenizer used for the isolation of CNFs (Image 

adapted from (Kargarzadeh et al., 2017))  

Previous studies reported by Tibolla et al, have shown that CNFs obtained by acid hydrolysis and high-

pressure homogenisation processes have potential to be applied as reinforcing agents of polymeric 

matrixes in food packaging industries. The study extracted CNFs from banana peel bran through 

chemical and mechanical treatments and it was reported that CNFs with mechanical treatments showed 

a higher crystallinity index (average CrI ~66%) than those without mechanical treatments. In addition, 

as the number of cycles increases, the better the crystallinity index (Tibolla et al., 2018). Bacterial 

nanofibrillated cellulose (BNFC) was isolated from bacterial (Gluconacetobacter xylinus) cellulose by 

varying levels of high-pressure homogenisation (Kawee, Lam and Sukyai, 2018). The crystal 

characteristics of the fibres showed that homogenisation had no effect of the crystal structure of 



26  

  

cellulose dispute different applied pressures. However, the crystal size and crystallinity index of BNFC 

decreased as the pressure increased due to the shear forces destroying inter and/or intramolecular 

hydrogen bonds of cellulose and leading to the breakdown of the crystal structure.  



27  

  

Table 2.4: Cellulose isolation techniques extracted from different sources.  

Main process Cellulose 

source 

Pre-treatment Main Treatment  Post-treatment Crystallinity Ref  

Acid hydrolysis Sugarcane 

bagasse waste 

Bleaching and alkaline 

treatment 

Acid hydrolysis 

using sulfuric acid. 

Washing, 

centrifugation, dialysis, 

and sonication 

~69% (Wulandari et al., 

2016) 

Waste office 

paper 

Mechanical pre-treatment, 

alkaline, and bleaching 

treatment. 

Maleic acid 

hydrolysis  

Washing, centrifuging, 

dialysis, sonication, 

filtration and drying. 

~81% (Yeganeh et al., 

2017) 

Sugarcane 

bagasse waste 

Not applicable Phosphoric acid 

hydrolysis with 

hydrogen peroxide 

treatment then 

homogenization. 

Washing and dialysis 64% (Wang et al., 2020) 

Brewery spent 

grain (BSG) 

Acid and alkali treatment 

followed by bleaching step.

Acid hydrolysis 

using sulfuric acid 

Washing, 

centrifugation, 

sonication, and freeze-

drying. 

76.3% (Matebie et al., 

2021) 

Ramie fibers Chemical treatments (de-

waxing, alkaline and 

bleaching treatments) 

Acid hydrolysis 

using sulfuric acid 

Washing, 

centrifugation, dialysis, 

and ultra-sonication. 

80-91% (Kusmono et al., 

2020) 



28  

  

Corn husk Alkaline and bleaching 

treatment. 

Acid hydrolysis 

using 64% sulfuric 

acid. 

Washing, 

centrifugation, dialysis, 

and sonication 

68.33% (Kampeerapappun, 

2015) 

 Bleached 

softwood kraft 

pulp (BSKP) 

FeCl3 catalyst  Formic acid 

hydrolysis  

CNFs−Centrifugation 

and high-pressure 

homogenization. 

CNCs with HPH 

CNFs−52.91% 

CNCs−75.21%

(Du et al., 2017) 

 Bleached 

softwood kraft 

pulp (BSKP) 

Not applicable  Formic acid 

hydrolysis  

Centrifugation and 

high-pressure 

homogenization. 

49.0%−52.9% (Du et al., 2016) 

Enzymatic 

hydrolysis 

Banana peel 

bran 

Alkaline treatment, 

washing and centrifugation

Enzymatic 

hydrolysis using 

xylanase 

Washing and 

centrifugation. 

61.0% (Tibolla et al., 

2017) 

 Cotton fibres Fenton’s pre-treatment Enzymatic 

hydrolysis using 

cellulase. 

Not applicable ~85% (Jain and 

Vigneshwaran, 

2012) 

 Cotton fibres DMSO and NaOH, 

ultrasonic treatments 

Enzymatic 

hydrolysis with 

buffer solution of 

cellulose at 45℃ 

Centrifugation. 78.1% (Chen et al., 2012) 



29  

  

 Flax and hemp 

fibres 

Washing, drying, chemical 

/ultrasonic/microwave pre-

treatment. 

Enzymatic treatment 

in acetate buffer 

supplemented with 

endoglucanase and 

incubated in a shaker 

at 50℃. 

 

Centrifugation, 

washing, ultrafiltration, 

freeze drying. 

Not applicable (Xu et al., 2013) 

High-Intensity 

Ultrasonication 

(HIUS) 

Apple pomace Not applicable Precipitate calcium 

carbonate was used 

to prepare 0.2 wt% 

cellulose 

suspension. 

Ultrasonication 

treatment (t = 30/60 

min, P = 40, 60, or 

80% of Pmax). 

Vacuum filtration, and 

drying 

50-58% (Szymańska-

Chargot et al., 

2018) 

Hop stems 

(Humulus 

lupulus L.) 

Shredding, acid pre-

treatment, alkaline, and 

bleaching treatment. 

Ultrasonication 

treatment (power 

output 130W for 60 

min) in an ice bat