Biomimetically
improved materials comprising
microfibrillated cellulose
Cornelia Byström Spring 2021
Degree Project in Master of Science Program in Bioresource Engineering, 30 credits
Examiner: Patrik Andersson
Supervisor: Leif Jönsson, Adrianna Svensson Pass:
I
Abstract
Microfibrillated cellulose (MFC) is a renewable, cellulosic material mainly produced from wood fibers, which are found in the secondary cell walls of plant cells. With the increased demand for renewable biopolymer films in packaging, MFC has emerged as a potential alternative to non-renewable polymer films. However, some obstacles for achieving a highly ductile material that also possesses sufficient barrier properties remain. Bacterial cellulose (BC) is a source of nanocellulose that has been reported to have higher purity, higher flexibility, and better water-absorption capacity and tensile strength than plant-derived cellulose. One source of BC is from the production of Kombucha, where BC is produced by acetic acid bacteria in a "Symbiotic Culture Of Bacteria and Yeast", generally referred to as SCOBY. During SCOBY fermentation, a multi-layered biofilm will form on the air-medium interface. The film consists of microfibrillated bacterial cellulose of high purity and mechanical strength. In this study, the objective was to find out how mechanical properties of a film made of microfibrillated cellulose can be improved by learnings obtained from investigating the properties and composition of a film made from SCOBY cellulose.
Characterization of the intrinsic properties of washed and unwashed SCOBY was performed by using field emission scanning electron microscopy (FE-SEM), Fourier- transform infra-red (FTIR) spectroscopy, and moisture uptake analysis. Gas
chromatography - mass spectrometry (GC-MS) was utilized to examine the presence of potential plasticizing compounds from the fermentation. Uniaxial tensile testing was performed on MFC films plasticized with discovered fermentation products to analyze their impact on the mechanical properties of MFC, such as strain-at-break and E modulus. Additionally, to evaluate how washed and unwashed BC from SCOBY potentially could be incorporated into MFC films to improve ductility, a study on the effect of fluidization was performed.
The characterization confirmed the high purity of washed SCOBY and the high water- absorption capacity of unwashed SCOBY. GC-MS of dry SCOBY revealed the presence of hydrophilic compounds from the fermentation with potential to act as bio- based plasticizing agents in SCOBY cellulose. Glycerol and another compound found in dry SCOBY were tested for their plasticizing properties in MFC films. Glycerol was found to increase the strain-at-break for MFC films with 181%. The other studied compound also improved the ductility of the MFC material. Thereby, a new
application of this compound was discovered. From the fluidization study, uniaxial tensile testing revealed a strain-at-break of (10.5 ± 0.4) % and E modulus of (10500 ± 1140) MPa for MFC films containing 10% washed SCOBY material. This
corresponds to an increase in strain-at-break of 402% compared to pure MFC films (2.09 ± 0.42), successfully improving the ductility. The results from this study are planned to be used as a basis for further studies in the area of bio-based packaging.
II
III
List of abbreviations
AAB Acetic acid bacteria BC Bacterial cellulose
DW Dry weight
MFC Microfibrillated cellulose RCK Research Centre Karlstad
RH Relative Humidity
SCOBY Symbiotic culture of bacteria and yeast
UW Unwashed
W Washed
Author contribution
Washing, fluidization, film preparation, GC-MS analysis, and tensile testing has been performed by the author.
FE-SEM images of MFC and SCOBY films was performed by Robert Wissler at RCK, Stora Enso. Moisture uptake analysis data was received from Claes Åkerblom at RCK, Stora Enso. FTIR was performed by Camilla Olsson at RCK, Stora Enso.
IV
V Table of contents
Abstract ... I
1. Introduction ... 1
1.1 Microfibrillated cellulose from wood fibers ... 1
1.1.1 Microfibrillated cellulose as a biopolymer ... 2
1.1.2 Challenges for MFC films as barrier material in liquid packaging ... 3
1.2 Microbial cellulose – SCOBY ... 4
1.2.1 Microfibrillated cellulose from SCOBY fermentation ... 5
1.2.2 Interaction of microorganisms in SCOBY ... 6
1.2.3 Utilizing SCOBY cellulose for bio-based packaging ... 6
1.3 Aim of degree project ... 7
2. Popular scientific summary including social and ethical aspects ... 9
2.1 Popular scientific summary ... 9
2.2 Social and ethical aspects ... 10
3. Experimental ... 10
3.1 Materials ... 10
3.2 Washing of SCOBY material ... 10
3.3 Characterization of potential inherent plasticizer and properties of SCOBY .... 11
3.3.1 Sample preparation ... 11
3.3.2 Characterization of SCOBY samples ... 11
3.3.3 Film casting with plasticizing agents ... 12
3.3.4 Uniaxial tensile testing ... 12
3.4 Incorporation of SCOBY cellulose in MFC films ... 12
3.4.1 Fluidization experiments ... 12
3.4.2 Film casting of MFC films with fluidized material ... 13
3.4.3 Analysis of fluidization experiments ... 13
4. Results ... 13
4.1 Material structure and effect of washing ... 13
4.2 Characterization of plasticizer ... 15
4.2.1 Tensile testing plasticized films ... 16
4.3 Incorporation of SCOBY cellulose in MFC films ... 17
5. Discussion ... 19
6. Conclusions and outlook ... 23
Acknowledgements ... 24
References ... 25
Appendix ... 30
Appendix 1 – SCOBY cellulose before/after washing process ... 30
Appendix 2 – Complementary data from GC-MS ... 30
Appendix 3 – Complementary data FTIR SCOBY ... 31
Appendix 4 – Data from uniaxial testing of MFC films with fluidized SCOBY material ... 31
VI
1
1. Introduction
In the face of the threat of climate change, the industries of today must shift their production towards a more sustainable, circular economy. This is relevant also for the packaging industry, with plastic being one of the most commonly used materials in packaging applications1. In 2019, the global consumption of plastic exceeded 320 million tons/year, with predictions of higher numbers in the coming years2. With the wide range of products where fossil-based packaging is applied and the increased consumption of packaged products, the packaging industry needs to their products to meet market needs by investing resources in the development of sustainable
packaging.
An essential part of food-safe liquid packaging is a structure of barrier layers on the inside of the package, with the purpose of protecting the product from oxygen, water vapor and other gases to prevent oxidative degradation and light damage3. Polyolefin coatings are commonly used on paperboard packaging as a barrier layer and as adhesive to improve the mechanical and barrier properties of liquid packaging4. To further improve the gas barrier in polymer-coated paperboard, one or more layers of aluminum foil is included into the barrier layer. A problem that arises with
introducing a fossil-based polymer and a metal coating is the recyclability of the packaging, due to the polymer coating being hard to separate from the fibres4.
Additionally, the aluminum foil is also an obstacle for creating sustainable packaging due to its high carbon footprint. In order to provide consumers with fully recyclable and sustainable packaging, alternatives to this barrier system need to be investigated.
1.1 Microfibrillated cellulose from wood fibers
MFC is a renewable cellulosic material mainly produced from cellulosic wood fibers, found in the secondary cell walls of plant cells. The smallest component in a
cellulosic fiber is the cellulose polymer, or the cellulose chain, which is a linear homopolysaccharide of β-1,4-linked anhydro-D-glucose units5. Each anhydroglucose unit in the native polymer holds three hydroxyl groups with the ability to form strong intermolecular bonds. These functional groups gives cellulose its characteristic traits of partial crystallinity, multi-component structure and amphiphilic nature6. Going up the structural hierarchy of cellulose, cellulose chains are covalently bound together to form elementary fibrils of cellulose I, displayed in Fig. 1. Cellulose I is the
conformation of cellulose where cellulose chains exist in parallel strands, whereas cellulose II exist as antiparallel strands7.
The elementary fibrils are furthermore bound together to form microfibrils. The microfibrils are the largest component in MFC, however defined not as rod-like fibers, but rather as partially detached microfibrils. They are conformed in a web-like structure of partially crystalline cellulose, giving MFC its flexibility8. The dimensions of microfibrils in MFC vary, but the general definition is a width around 20 nm and lengths varying between 100 nm to 1 µm6,9. MFC also have been presented as nanofibrillated cellulose (NFC) which is synonymous due to its definition being based on the general width of microfibrils being in nanoscale i.e., diameter lower than 100 nm.10 However, this definition may vary in literature10-12.
2 Figure 1: Hierarchical structure of cellulose fibers.
When the development of MFC was first introduced in the early 1980s, mechanical processing of wood fibers by pressure-drop homogenization was the primary method utilized11. However, due to the high energy demand of purely utilizing mechanical processing, the production shifted towards a combination of chemical or enzymatic pretreatment of the raw material prior to mechanical processing. Chemical and enzymatic pretreatments that have been developed to reduce the energy demand include using 2,2,6,6-tetramethylpiperidine (TEMPO) mediated oxidation, acid hydrolysis, partial carboxymethylation and enzymatic depolymerization13,14. The mechanical production of MFC today includes methods of high-pressure valve homogenization (HPVH), steam explosion, cryo-crushing, grinding and
microfluidization15, all with the purpose of reducing the particle size of fibers to the nanoscale. Microfluidization is a high-pressure homogenization method in which the diluted fiber slurry is fed into a narrow, z-shaped pressure-treatment chamber, subjecting the fibers to a large pressure drop at high velocity to promote particle collision in order to split the fibers into fibrils16,17. By changing to narrower
chambers, the shear and impact forces can be increased to further promote the degree of fibrillation12. The final product of homogenization results in a gel of web-like structured microfibrils with high aspect ratio6,11.
1.1.1 Microfibrillated cellulose as a biopolymer
With the increased demand for renewable biopolymer films in packaging,
microfibrillated cellulose (MFC) has emerged as a potential alternative material to non-renewable polymer films. MFC has since its discovery been utilized in many ways due to the intrinsic properties of the cellulose microfibrils. This includes low density, high crystallinity, high aspect ratio, biodegradability, and renewability18,19. The degree of fibrillation of MFC exposes a high number of hydroxyl groups over the fibril surface providing potential for derivatization and functionalization20. This opens the door for MFC to be utilized in many different applications, e.g., dispersion
stabilizers, novel bionanocomposite reinforcement and in antimicrobial films19,21-23. Hubbe et al. have discussed the process of preparing pure MFC cellulose films8. They have suggested that casting from either aqueous or non-aqueous solutions and
evaporating to promote strong hydrogen bonding would be superior to extrusion, since that would eliminate the issue of poor flow characteristics at high solid content8. As earlier discussed, the large crystalline content of MFC in combination with the high number of exposed hydroxyl groups on the surface enable the fibrils to form strong interfibrillar hydrogen bonds in films with low porosity, which furthermore
3 can act as a strong barrier towards gas and oil18,24. Oxygen permeability in dry
conditions have been extensively researched, with reports of promising air barrier properties for pure MFC films as well as MFC-coated paper20,25,26.
1.1.2 Challenges for MFC films as barrier material in liquid packaging
In the paper and packaging industry, specific mechanical properties of solid materials are significant for commercialization. Mechanical properties of materials are the physical properties that a material demonstrate when a force is applied to it. Several terms related to mechanical properties are discussed when talking about paper and packaging materials. Mechanical testing is performed with a tensile tester, where a longitudinal load is applied to the sample, most commonly a strip of paper, until a break occurs. The load is measured as a function of elongation of the sample,
generating a stress-strain curve (Fig. 2). From this test, the following information can be obtained:
§ Tensile strength (N): Maximum tensile force developed in a specimen before rupture. Presented as force per unit width of sample in the paper industry.
Dependent on the degree of bonding of fibers27.
§ Strain-at-break (%): Maximum tensile strain in sample before rupture.
Percentage elongation of initial length28. General definition of the material’s ductility. Strain-at-break <5% is generally considered brittle8, and this definition will be used in this thesis.
§ Young’s Modulus / E Modulus (MPa): Ratio of tensile force per unit width to tensile strength within the elastic region of the stress-strain curve (linear portion of load-elongation curve)28. Explains stiffness of the sample. High E modulus, stiff material, small elastic region.
Figure 2: Stress-strain curve displaying how Young’s modulus and strain-at-break can be derived from uniaxial tensile testing.
Figure 2 displays how the different parameters can be derived from the stress-strain curve. Another concept that is introduced with the stress-strain curve is the elastic and plastic region. This explains the materials behavior when stress is applied. The elastic
4 region is the specific amount of force applied to the sample in which the material will be able to return to its original shape once the applied strength is released. Beyond the elastic region is the plastic region, in which the material will irreversibly deform27. Although MFC films are great candidates for renewable barrier packaging, many obstacles need to be addressed before it can be fully commercialized. The film forming process, drying conditions and raw material for MFC have shown to highly contribute to the mechanical and barrier properties of MFC films, compromising the performance of the material29,30. Additionally, there is a conflict between having sufficient barrier properties for packaging and sufficient ductility for MFC films.
Although films that consist of pure MFC have a high fraction of crystalline regions, the amorphous regions and the exposed functional groups are susceptible to water absorption, both on the surface of microfibrils and in the amorphous regions20. The absorbed water leads to a large plasticization of the cellulose matrix due to swelling of the films, which enhances the gas permeability by increasing distance between fibrils and water aiding the transport of gas31,32. In order to counteract this behavior, the cross-linking of the fibrils must be increased, resulting in brittle films which are not suitable for packaging33 . Hence, the main obstacle with implementing MFC films as a barrier material in packaging is that the material should possess sufficient barrier properties and still exhibit ductile behavior.
Extensive research has been conducted regarding improving the mechanical properties of MFC films. Deng et al. presented a new method where the MFC was simultaneously exfoliated and acetylated by ball milling in the presence of hexanoyl chloride34. This resulted in highly fibrillated, hydrophobic MFC, a tensile strength of 140 MPa, and an elongation-at-break of 21.3%34. This method introduced the concept of surface modification to improve the ductility. Another approach to improve the ductility of MFC is the introduction of plasticizers to reduce the amount of hydrogen bonds between fibrils. As the main functional group of cellulose is the hydroxyl group, polyols (compounds with several hydroxyl groups) have been found to be the compounds mainly tested for MFC plasticization35. Vinay et al. studied the
incorporation of glycerol into a MFC film formulation in concentrations of 5-10-20%
by dry weight of MFC30. They observed that the strain-at-break was improved by the addition of glycerol, while the tensile strength and the E modulus deteriorated with increased concentration of glycerol. Glycerol appears to plasticize MFC films efficiently. However, it may compromise the fibril network, resulting in flexible but weak films30. Similarly, sorbitol and citric acid have been utilized to improve the mechanical properties of MFC, presenting similar results33.
1.2 Microbial cellulose – SCOBY
Plant material is not the only source of cellulose. Cellulose synthesized by bacteria has in recent years emerged as an interesting material36. Bacterial cellulose (BC), or bacterial nanocellulose as it is commonly called, has been reported to have high purity, higher flexibility, and better water-absorption capacity and tensile strength in comparison to plant-derived cellulose37-39. BC is structurally similar to plant
cellulose, but produced by gram-negative bacterial species such as
Gluconoacetobacter, Agrobacterium, Aerobacter, Azotobacter and Salmonella 40. BC has been shown to have application potential in fields such as biomedical science, bio-based packaging, pharmacy, and biotechnology. However, its potential to be commercialized is unfortunately hindered by high production costs41.
5 There is an alternative path towards obtaining bacterial cellulose. Kombucha, a
fermented tea beverage, is obtained by the fermentation of sugars by a Symbiotic Culture Of Bacteria and Yeast, generally referred to as SCOBY42. SCOBY is based on a unique symbiosis of acetic acid bacteria (AAB, commonly species of
Komagataeibacter, Gluconobacter, and Acetobacter) and osmophilic yeasts
(Brettanomyces, Zygosaccharomyces, and Saccharomyces). During the fermentation process, SCOBY will form a multi-layered biofilm at the air-medium interface. The biofilm consists of microfibrillated bacterial cellulose acting as support and protection structure for the microbial culture43,44. The pellicle possesses a high content of BC.
However, impurities like peptides, proteins, polyphenols, residual bacteria, yeast cells, and nucleic acids are still present in the pellicle43. Another impurity is
melanoidins responsible for Maillard reactions, giving the SCOBY its characteristic brown color. A commonly used method for lab-scale purification of this material is alkaline treatment with an aqueous solution of sodium hydroxide at elevated
temperature, which will remove residual organic matter41,43,45. Although the SCOBY pellicle is today considered to be a waste product of the fermentation process, the potential to obtain BC of high purity from SCOBY to prepare high-value biomaterials is something that should not be overlooked37.
1.2.1 Microfibrillated cellulose from SCOBY fermentation
The production of BC in the SCOBY is the result of a biochemical multi-step process performed by the present bacteria and involving many enzymes and regulatory proteins, which is presented in Fig. 339. In broad terms, glucose from the growth medium is transformed into uridine diphospho-glucose (UDP-glucose) via glucose-6- phosphate and glucose-1-phosphate44,46. The UDP-glucose is then sequentially added to the growing end of the polymer chain by cellulose synthase, forming the β-1-4 glucan chain47. The polymer chains are then extruded out of the cell wall of the bacteria into the extracellular environment47. The polymer chain is categorized as type I cellulose. However, cellulose II is also formed and extruded separately and arranged differently. Cellulose II are amorphous and more thermodynamically stable48.
Figure 3: Visual representation of the formation of bacterial cellulose nanoribbons.
6 The second step of BC formation is the crystallization of the polymer chains outside of the bacterial cells. Each bacterium can extrude up to 80 individual cellulose chains simultaneously, which is self-assembled outside the bacteria into a ribbon-shaped microfibril approximately 40-60 nm wide by strong hydrogen bonding and van der Waal forces41,47. The ribbon fibrils are then further assembled into a complex 3-D web-like structure of BC, forming the pellicle layer of the SCOBY at the air-medium interface. The microfibrillar structure of this polymer matrix is what gives rise to the characteristic traits of high tensile strength, high porosity, and a high degree of
polymerization, which also is observed for dried films of SCOBY46,49,50. Additionally, the large surface area with the presence of exposed hydroxyl groups makes it a good candidate for surface modification40.
1.2.2 Interaction of microorganisms in SCOBY
As previously mentioned, the combination of acetic acid bacteria and yeast in the SCOBY pellicle acts in symbiosis to produce the fermented tea and the cellulose biofilm. This symbiosis is visualized in Fig. 4. In the beginning of fermentation occurs the hydrolysis of sucrose into glucose and fructose, which are then converted by the yeast to produce ethanol and carbon dioxide through glycolysis. In this
process, glycerol is formed as a by-product from yeast51. The glucose produced from the yeast is utilized by the AAB to form cellulose, as previously discussed. Finally, the AAB converts the ethanol into acetic acid which further acts as a stimulant for the yeast to produce more ethanol44,48,52.
Figure 4: Visual representation of interactions between yeast and acetic acid bacteria in Kombucha SCOBY.
1.2.3 Utilizing SCOBY cellulose for bio-based packaging
With BC from Kombucha SCOBY being a fairly new topic of research, not many commercialized products from SCOBY BC have yet been presented. Many different approaches towards reinforcement of nanocomposites and novel packaging with bacterial cellulose have been studied. For instance, chitosan along with SCOBY cellulose have been assessed for packaging of fruit and vegetables53, bacterial
cellulose in combination with poly(vinyl alcohol)54, and bacterial cellulose produced from fiber sludge hydrolysate used as an additive in paper of chemithermomechanical pulp (CTMP)55. The general consensus from each study concluded an enhancement in mechanical strength and tear resistance, all derived from bacterial cellulose unique
7 structure and bonding site availability to bind to other polymers47. With this, it is of great interest to examine whether BC from SCOBY could act as a reinforcement in MFC films to improve ductility of the films.
In a study by Lee et al., the mechanical properties of BC and MFC were evaluated to assess their ability to be used as reinforcement in poly(L-lactic acid)
nanocomposites50. They argued that whilst the theoretical and experimental studies published today indicate MFC and BC possessing similar mechanical properties, intrinsic properties of BC may be superior to MFC regarding reinforcement50. One might argue that the great mechanical strength of BC in the SCOBY could be the result of a plasticizing compound. By-products from fermentation by SCOBY might be a key player in plasticizing the SCOBY cellulose, which would be of interest to study further. If a compound is found to plasticize SCOBY, this might also be applicable to MFC films.
1.3 Aim of degree project
To find out how mechanical properties of a film made of microfibrillated cellulose can be improved by learnings obtained from investigating the properties and composition of a film made from SCOBY cellulose. Questions addressed in this thesis includes:
§ Is the ductility of the SCOBY film derived from BC or is this due to an intrinsic plasticizer?
§ Does the addition of fluidized SCOBY cellulose into the film formulation containing MFC improve the mechanical properties of the film?
§ How should the SCOBY cellulose be used in MFC films to improve their ductility?
§ Can any plasticizer found in SCOBY be used for increasing ductility of MFC films?
The outcome of the thesis will be information about whether SCOBY cellulose could be utilized for improving the mechanical properties of MFC.
8
9
2. Popular scientific summary including social and ethical aspects
2.1 Popular scientific summary
Today, there is a wide range of products where fossil-based packaging is used.
Considering the impact of fossil-based materials on carbon dioxide emissions and the increased consumption of packaged products, the packaging industry needs to assure that their products meet market needs by investing resources in the development of sustainable packaging. An essential part of food-safe liquid packaging is a structure of barrier layers on the inside of the package, with the purpose of protecting the product from oxygen, water vapor and other gases to prevent spoilage of the product.
Unfortunately, the barrier layers that are utilized today consists of aluminum and fossil-based plastics. They are both hard to replace and hard to separate from the paperboards, which creates an obstacle in recyclability of the packaging.
Microfibrillated cellulose (MFC) is a renewable material mainly produced from wood fibers, which, when formed into a film, have potential to replace the current barrier layers in liquid packaging. It has great gas barrier properties. However, the film itself is not very flexible, which is an obstacle that must be resolved before
commercialization.
Bacteria can produce bacterial cellulose, and the film produced by the bacteria has better flexibility than MFC films. A source of BC is from the production of
Kombucha, a fermented tea drink, where BC is produced by acetic acid bacteria in a Symbiotic Culture Of Bacteria and Yeast, in a general term called SCOBY, which during its fermentation process will form a multi-layered biofilm which could potentially be utilized. In this study, the objective was to find out how the brittleness of a film made of MFC could be improved by learnings obtained from investigating the properties and composition of a film made from SCOBY material.
This study included the characterization of compounds in the SCOBY which might help to improve film flexibility, called plasticizers. Several plasticizers were investigated. Two plasticizers were tested with MFC, which helped to improve the flexibility of MFC films. A method called microfluidizing is often utilized to reduce the size of the molecules present in materials by letting it flow through a very narrow chamber under high pressure, which also was evaluated. The SCOBY material was passed through the microfluidizer different number of times, and then incorporated into MFC film formulations in different concentrations and by different methods. The incorporation of SCOBY material in the MFC film improved the flexibility of the films by 402%.
10 2.2 Social and ethical aspects
Considering the wide range of products where fossil-based packaging is utilized and the increased consumption of packaged products, the study of new environmentally friendly materials is essential to reduce the impact of packaging materials on carbon footprint and climate change. By utilizing natural resources, such as MFC and SCOBY, the topic of this degree project is connected to several of the global Sustainable Development Goals (SDGs) for a better world by 2030:
§ Goal 12 – Responsible consumption and production to ensure sustainable consumption and production patterns. The present study aids in the search to obtain materials which could be implemented into packaging, reducing the use of fossil-based materials. This thesis can especially be associated to goals 12.2 and 12.5.
§ 12.2 - By 2030, achieve the sustainable management and efficient use of natural resources. By investigating the applicability of SCOBY in production of bio-based plastic replacements, the efficiency of utilizing the MFC fully can be increased.
§ 12.5 - By 2030, substantially reduce waste generation through prevention, reduction, recycling and reuse. By obtaining a bio-based material which can successfully be included into liquid packaging, recyclability of packaging can be improved, thereby reducing waste.
§ Goal 13 – Climate action to combat change and its impacts. The present study contributes to the research needed in order to protect our planet by focusing on finding solutions with lower carbon footprint than the existing solutions.
In this project, no testing has been done on any human or animal. The Kombucha used is a byproduct that cannot be utilized for human consumption and does therefore not compete with food production.
3. Experimental
3.1 Materials
SCOBY pellicle and MFC used for these experiments were provided by Stora Enso (Finland). SCOBY pellicle and MFC used for these experiments were obtained from Stora Enso. NaOH (K ≤ 0,02%, ≥ 98%, pellets) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was used to prepare aqueous solvent used for washing of SCOBY. Compound X (50% solution in water, Sigma-Aldrich Canada Co, Canada) and glycerol (85% solution in water, AB Unimedic Pharma) were used as plasticizing agents in film formulations. Acetone (≥99.5%) and BSTFA [70% N,O-bis(trimethyl- silyl) trifluoroacetamide, Sigma-Aldrich] was used for GC-MS sample preparation.
3.2 Washing of SCOBY material
To obtain the pure bacterial cellulose from SCOBY, the pellicle was subjected to a washing process to ensure sufficient removal of other organic matter. This was performed to study where the essential mechanical properties of SCOBY films might originate from. The SCOBY pellicle was washed in deionized water to remove residual fermentation media and weighed. In order to ensure efficient washing of solvent, the pellicle was defibrillated for 5 minutes (Wet defibrillator, 3000 r/min) until smaller pieces was achieved. The pellicle was then submerged into a 1 M aqueous solution of NaOH (ratio of 2:1 ml NaOH/g SCOBY) in a 2-L glass reaction vessel with magnetic stirring. This was left stirring overnight, resulting in a total
11 washing time between 16 and 18 h. When the reaction was complete, the pellicle was transferred to a Büchner funnel (30 cm diameter) with a 3 mm thick wire filter and washed with deionized water until the pH of the filtrate was neutral. Another indication of a clean product was the color transition of brown to white during washing (see Appendix 1).
3.3 Characterization of potential inherent plasticizer and properties of SCOBY This part of the experiments explored what properties or potential plasticizer of the SCOBY might be the cause of the great mechanical properties found in SCOBY films. Furthermore, tensile testing of films prepared with the suggested plasticizer was performed.
3.3.1 Sample preparation
Three different samples were prepared for characterization. The first sample (ST1) was prepared by taking a thin piece of SCOBY and leave it to air dry at room temperature in a Petri dish. To include the effect on washing and fluidization, one sample of unwashed (SMF100) and one sample of washed (SWMF100) SCOBY were fluidized and casted. Samples of unwashed and washed SCOBY material were initially mixed prior to fluidization using a hand mixer (Multiquick 7, Braun) and further mixed using a dispersion instrument (Ultra-Turrax, Ika Werk) until a
homogenous mixture was obtained. The shredded SCOBY material was diluted to 2.0 wt % and fibrillated in an M-110EH Microfluidizer (M-110EH, Microfluidics, USA).
The material was initially fluidized once trough two serially connected chambers with diameters of 400/200 μm (1500 psi) and then twice through two serially connected chambers of 200/100 μm. Following fluidization, the formulations were diluted with deionized water to 1 wt % and poured into 145 mm Petri dishes and left to dry in a controlled climate (RH 50%, 23°C).
3.3.2 Characterization of SCOBY samples
Field emission scanning electron microscopy (FE-SEM) was used to explore the morphology of the SCOBY film surfaces and fibers. Film samples were coated with platinum for approximately 1 minute before examination with a Zeiss Sigma 300 VP FE-SEM instrument (Karl-Zeiss SMT AG, Germany). The image processing was performed with the integrated software.
The effects of washing and fluidization on the SCOBY samples were studied by FT- IR spectroscopy in its dried film form (Nicolet Summit FTIR Spectrometer, Thermo Scientific) and baseline corrected with the OMNIC Paradigm Software (Thermo Scientific).
Moisture uptake analysis was performed for two types of films of 1% wt MFC with the addition of washed and unwashed SCOBY (5% of cellulose dry weight) with a Controlled Moisture Generator, 100% MFC films as reference. The weight was measured over time during a moisture program of 50-15-30-50-65-80-50 % RH at 23°C and analyzed using MS Excel.
Qualitative analysis of the presence of plasticizing agents in the SCOBY was performed by single-quadrupole GC-MS (GCMS-QP2020, Shimadzu) with an HP- 5MS column (30 m × 0.250 i.d. × 0,25 μm, Agilent Technologies). Prior to analysis, the compounds of interest in the dried SCOBY sample (ST1) and the washed sample
12 as reference (SW100) were extracted in acetone (≥99.5%, Sigma-Aldrich Chemie GmbH) by ultrasonication for 30 min. Following extraction, the acetone was
evaporated by a gentle nitrogen stream at room temperature. Siliation of the samples was carried out by the addition of 100 μl BSTFA to the residue. The samples were then heated at 150 °C for 20 minutes. A total of 1 μl of the derivatized mixture was injected into the GC-MS for each sample, with a split ratio of 1:20. Temperatures of column oven, injector and ion source were set at 120, 250, 220°C, respectively. The carrier gas was helium, which was applied using a constant flow rate (3.0 ml/min).
The temperature program is presented in Appendix 2.
3.3.3 Film casting with plasticizing agents
The supplied MFC was diluted to 1 dw % with deionized water in order to obtain a viscosity suitable for casting. Glycerol and Compound X were added to the MFC suspensions in amounts calculated from the desired film density, 20 g/m2, at
concentrations varying between 0.1 – 0.3 % of the total film dispersion weight. After addition of plasticizer, the suspensions were simultaneously deaerated and stirred with a magnetic stirrer for about 30 minutes, and then approximately 30 g was poured onto 13.5 cm diameter Petri dishes. The films were dried without the lid in a
controlled climate of 50% relative humidity (RH) and 23 °C.
3.3.4 Uniaxial Tensile testing
With thickness of the dry films varied over the surface of the films, the films were tested for thickness on five points and then applied for each sample strip (Lorentzen
& Wettre Micrometer). Five replicates of each film (15mm wide, 55 mm long) were measured and arithmetically averaged. Elongation at break (%) and the E modulus of the dried films were tested and analyzed with a Zwick material tester (Zwick Roell Z010 tensile testing machine, testXpert III). Settings for tensile testing: Pre-load 0.2 N, grip-to-grip separation 20 mm, and sample speed 0.2 mm/min. The E modulus was calculated based on the steepest slope of the stress-strain curve. Conditions in the test room were 50% RH (relative humidity) and 23°C.
3.4 Incorporation of SCOBY cellulose in MFC films
This part of the experiments explored how the SCOBY material, both washed and unwashed, should be incorporated into the MFC film formulation in order to improve the mechanical properties of the dried films.
3.4.1 Fluidization experiments
The preparation prior to fluidization follows the same process as described in 3.2.1.
Similarly, the material was initially fluidized once trough two serially connected chambers with diameters of 400/200 μm (1500 psi) and then through two serially connected chambers of 200/100 μm. In this study, the number of passes through the 200/100 μm chambers varied between batches, in order to study the effect of
fluidization on the mechanical properties of films. The batch names correspond to SCOBY (S), washed or unwashed (W/UW), microfluidization (MF) and the total number of passes through the chambers, assigned as a and b. The batches ending with x corresponds to a different method of incorporation, assigned Method X. The
variation of passes for each batch is presented Table 1.
Batch name WMFa UWMFa SWMFb UWMFb WMFbx UWMFbx
13
Wt% 1.0 1.0 1.0 1.0 2.0 2.0
Passes
400/200μm 1 1 1 1 1 1
Passes 200/100μm
y y z z z z
Table 1: Number of passes through each set of microfluidization chambers for each batch. Numbers are coded according to an agreement with Stora Enso.
3.4.2 Film casting of MFC films with fluidized material
Following microfluidization of the SCOBY material a set of MFC film types was prepared for each batch with different concentrations of SCOBY material, varying between 1 – 30 dw %. The supplied MFC and the fluidized SCOBY material was diluted to 1 dw % with deionized water. The amount of SCOBY material added to the MFC for each concentration was calculated to achieve a film density of 20 g/m2 for each film. The suspensions were then simultaneously deaerated and with a magnetic stirrer stirred for about 30 minutes, and then approximately 30 g was poured onto 13.5 cm diameter Petri dishes. The films were dried without the lid in a controlled climate of 50% RH and 23 °C.
3.4.3 Analysis of fluidization experiments
Mechanical properties of the dried films were evaluated by uniaxial tensile testing as described in 3.2.4. Morphology and degree of fibrillation of films and fibers were evaluated by FE-SEM as described in 3.2.3.
4. Results
4.1 Material structure and effect of washing
FE-SEM images from pure SCOBY, fluidized SCOBY, and washed/fluidized SCOBY are shown in Fig. 5. Pure SCOBY displays a relativley homogeneous fibrillar network of BC, with implications of possibly entrapped bacteria under the long and thin fibrils. The precence of bacteria in the SCOBY is made more apparent in the fluidized SCOBY, where the bacteria can be seen as rod-shaped three-
dimensional objects. The shape of the bacteria has been confirmed by Stora Enso and other sources to be similar to acetic acid bacteria56.
14 Figure 5: FE-SEM images of A: Dried SCOBY, B: Fluidized SCOBY film, C: Washed fluidized SCOBY film. All viewed with 10 000x magnification.
Table 2: Average fibril width for each sample from FE-SEM.
The effect of washing is clearly seen in the images of washed and unwashed samples (Fig. 5 B,C), where the washed sample displays more distinct fibrils without the
“glue” which is present in the unwashed samples with no present bacteria. The effect of fluidization on the fibril size can be observed from Table 2, where the average fibril width decreases from (178 ± 23) nm to (65.0 ± 5.5) and (64.0 ± 7.5) nm.
Figure 6: Moisture uptake analysis for a 100% MFC film (red), 95% MFC film with unwashed SCOBY cellulose (5% of MFC dry) (blue), 95% MFC film with washed SCOBY cellulose (5% of MFC dry ) (green) with Controlled Moisture Generator, (50-15-30-50-65-80-50 % RH, 23 °C). Weight normalized to same starting weight after 1 h in 50% RH.
The effect of moisture absorption is presented in Fig. 6. Up to 50% RH the sample weight followed the same pattern for all samples. At relative humidity above 65%, the MFC film with the addition of UW SCOBY material show higher absorption of moisture compared to 100% MFC and the film with W SCOBY.
FTIR spectra in the range 4000-400 cm-1 for pure SCOBY, fluidized SCOBY, and washed/fluidized SCOBY showing the effect of washing are shown in Fig. 6.
Transmittance band assignments are presented in Appendix 2. Observed from all samples: characteristic transmittance around 3000 to 3600 cm-1 assigned to the
1,56 1,58 1,6 1,62 1,64 1,66 1,68 1,7 1,72 1,74
0 5 10 15 20 25 30 35
Sampleweight, g
Conditioning time, h
Reference MFC Unwashed scoby Washed scoby
15 hydroxyl groups on cellulose or possibly water absorbed in the film from
surroundings37,57, transmittance bands between 2896-2916 cm-1 corresponding to C-H stretching58, water bending vibrations between 1632-1652 cm-1, C-O stretching at C3;
C-C stretching; and C-O stretching at C6 around 1055-1058 cm-1, and out of plane C- O-H bending at 664-666 cm-159.
Figure 6: Transmittance spectra of A: Dried SCOBY, B: Fluidized SCOBY film, C:
Washed fluidized SCOBY film. Highlighted area shows the increase in transmittance around 1800-1500 cm-1 with washing.
The transmittance band at 1540 cm-1 (Fig 6, A) corresponds to stretching of N-H groups in amide II, confirming the precence of proteins and amino acids in the pure SCOBY pellicle60. The effect of washing can then be observed from Fig. 6 C, where this peak disappears. Another effect of washing is the disappearance of the
transmission band at 1727-1732 cm-1 for the washed sample. This is assigned to hydrogen-bonded carbonyl stretching vibration61. Compound X was analysed with FTIR as a reference and its presence was confirmed in dried SCOBY. Further details concerning Compound X are kept unrevealed in this thesis, as they are expected to be described in a patent application.
4.2 Characterization of plasticizers
Qualitative analysis of the presence of organic plasticizing compounds in the SCOBY pellicle by GC-MS is presented in Table 3. Mass spectra can be observed in Appendix 1. Published plasticizer application research for each compound is also included.
Chemical structure Compound Retention time (min)
SI Molecular weight (g/mol)
Studied
plasticizer/polymer applications Glycerol 3.570 96 92.092 Plasticizer in
biodegradable
polysaccharide films 62-64
16
Succinic acid (butanedioi c acid),
3.970 97 118.09 Diesters of succinic acid for plasticization of PVC films/composites65,66 methyl
alpha-D- glucofuran oside
19.03 87 194.18 Methyl glucoside for starch film plasticization67
Glucono- delta- lactone
20.10 95 178.14 Biodegradable polyesters from Gluconolactone and citric acid68
Gallic acid 23.40 96 170.12 Phenolic plasticizing agent in zein films69
Carbohydrate degradation products
D-(-)-Ribofuranose, , D-glucose, D-galactose alpha-D-mannopyranose, D- fructose, deoxyribopyranose
150.13 - 214.11
Glucose, fructose, mannose with polyols as plasticizing agents in Pea starch films70, oat starch films71
Other organic acids Quininic acid, galactaric acid, palmitic acid, mannoic acid
192.17 - 256.4
Mannoic and Galactaric acids as cement water retarder agent72
Table 3: Summarized Similarity search results from GC-MS of dried SCOBY sample.
Organic acids which have not been investigated for plasticization of polymer films are presented as one group. Carbohydrate degradation products which might originate from residual growth medium are also presented as one group. The results imply the presence of a variety of different compounds which might act as a plasticizer for the SCOBY material.
4.2.1 Tensile testing of plasticized films
From the GC-MS results, 2 compounds were chosen to further study their effect as plasticizers in MFC films. Table 4 shows the result from uniaxial tensile testing of plasticized MFC films and 100% MFC film used as a reference.
Sample code
Plasticizer Plasticizer content in film dispersion (%)
Film thickness (µm)
Strain- at-break (%)
E modulus (MPa)
GLY01 Glycerol 0.10 20.6 (±2.8) 5.59 (±0.6)
4490 (±941) GLY015 Glycerol 0.15 22.5 (±3.1) 5.87
(±0.5)
4440 (±554)
17 GLY025 Glycerol 0.25 28.3 (±1.1) 3.90
(±2.3)
3160 (±800) GLY03 Glycerol 0.30 29.4 (±1.2) 3.33
(±1.0)
4260 (±1340)
X01 X 0.10 21.5 (±2.6) 3.08
(±0.3)
6120 (±732)
X015 X 0.15 23.8 (±1.8) 2.84
(±0.5)
5930 (±977)
X025 X 0.25 20.4 (±1.9) 4.50
(±0.6)
3710 (±636)
X03 X 0.30 19.6 (±1.3) 4.07
(±0.7)
3350 (±286)
MFC100 - - 24.4 (±1.5) 2.09
(±0.4)
8460 (±970) Table 4: Tensile properties of MFC films with plasticizer (glycerol/compound X).
Film thickness, Strain-at-break, and E modulus with standard deviation are calculated averages from 6-10 samples obtained from two films per batch.
The largest thickness for the plasticized films, 29.4 µm, was observed in glycerol- plasticized MFC films with 0.3% glycerol in the MFC dispersion. The remaining films varied in thickness between 19.6 to 28.3 µm, which as earlier mentioned in the experimental section is a result of the thickness varying over the film surface.
The non-plasticized MFC film obtained strain-at-break of (2.09 ± 0.4) %. For films plasticized with Compound X, an increase in strain-at-break was observed for all samples in comparison with pure MFC films. The highest increase in strain-at-break for Compound X was observed at 0.25% plasticizer content, with values of (4.50 ± 0.6) %. That corresponds to an increase of 115%. The highest increase in strain at break was observed for 0.15% glycerol of (5.87 ± 0.5) %, corresponding to an increase of 181%.
Regarding the observed results of E modulus, the modulus decreased significantly for all plasticized samples. The observed E modulus for the non-plasticized MFC film, (8460 ± 970) MPa, was not reached for any plasticized sample. The highest achieved E modulus for plasticized samples could be observed for 0.1% of Compound X of (6120 ± 732) MPa.
4.3 Incorporation of SCOBY material in MFC films
Figure 7 presents the result of uniaxial testing of films composed of different fluidization grades of SCOBY. Films prepared varied in thickness over the surface and between film duplicates, explaining the high standard deviation for certain batches. All results from tensile testing are presented in Appendix 4. Due to confidentiality, FE-SEM images of the samples are not included.
18 Figure 7: A: E modulus (MPa) and B: Strain-at-break (%) for MFC films prepared with SCOBY of different numbers of fluidization cycles, with incorporation of W and UW SCOBY material in concentrations 0-1-5-10-20-30%. Results and standard deviations presented are calculated as arithmetic averages from 3-10 samples obtained from two films per batch. For batch names see chapter 3.4.1.
From Fig. 8: A, one can observe the effect of a number of fluidization cycles and concentrations on the E modulus of the MFC films with W and UW SCOBY. Results are compared to reference film of 100% MFC, exhibiting a E modulus of (8460 ± 970) MPa. With fluidization a, the E modulus of samples with 1% W/UW displays higher or equal values to 100% MFC films, which furthermore decreases with
10500
12100
8460
0 2000 4000 6000 8000 10000 12000 14000
0% SCOBY
Mat. 1% SCOBY
Mat. 5% SCOBY
Mat. 10% SCOBY
Mat. 20% SCOBY
Mat. 30% SCOBY Mat.
E Modulus (MPa)
WMFaWMFb WMFbx UWMFa UWMFb UWMFbx MFC
A
10,5
7,63
2,09
0 2 4 6 8 10 12
0% SCOBY
Mat. 1% SCOBY
Mat. 5% SCOBY
Mat. 10% SCOBY
Mat. 20% SCOBY
Mat. 30% SCOBY Mat.
Strain at break (%)
WMFaWMFb WMFbx UWMFa UWMFb UWMFbx MFC
B
19 increased concentration of SCOBY material. Similar behavior is observed for UW samples at fluidization b; however, the modulus has increased in value compared to fluidization a with increasing concentrations. Samples W at fluidization b do not follow the same behavior at higher concentrations, but rather keep relatively constant at concentrations between 1 to 20 % SCOBY material. Comparing the W/UW
batches, the films containing washed SCOBY material appear to exhibit a greater E modulus for all concentrations tested.
The largest increase in E modulus can be observed for the samples prepared with Method X. Batch UWMFbx improves the E modulus for MFC films at 5 and 10%
SCOBY material. However, at higher concentrations the modulus decreases to values lower than 100% MFC films. The batch displaying the highest modulus of (12100 ± 330) MPa was WMFbx with 20% of SCOBY material. This batch also exhibited other behavior than the other batches, where the modulus seemingly increased with higher concentrations, and then decreasing at 30% SCOBY material.
In Figure 8: B, the effect of fluidization and concentrations of SCOBY material on strain-at-break is observed. Here it is made apparent that fluidization grades a and b do improve the strain-at-break for MFC films at higher concentrations of W/UW SCOBY, however Method X gives significantly improved values of strain-at-break.
UWMFbx reached values >5% strain at break for all concentration tested, with the highest value, (7.62 ± 0.6) % observed at 30% SCOBY material. The batch which performed the best was WMFbx, where the highest strain observed reached (10.5 ± 0.4) % at 10% SCOBY material.
To conclude; the highest improvement in E modulus was observed for WMFbx at 20% SCOBY material of (12200 ± 330) MPa, corresponding to an increase of 44%
compared to pure MFC films (8460 ± 970) MPa, and the highest increase in strain-at- break was observed WMFbx at 10% SCOBY material of (10.5 ± 0.4) %,
corresponding to an increase of 402% compared to pure MFC films [(2.09 ± 0.4) %].
5. Discussion
The aim of this degree project was to find out how mechanical properties of a film made of microfibrillated cellulose can be improved by learnings obtained from investigating the properties and composition of a film made from SCOBY cellulose.
This was done by initially characterizing the properties of SCOBY cellulose, and then studying the effects of fluidization when introducing SCOBY cellulose in MFC films.
5.1 Fibril structure of SCOBY and effects of washing
The FE-SEM images clearly show that the pure SCOBY sample in its dried form exhibited a closely packed nanofiber network BC, with fibril morphology resembling the characteristic ribbon shape produced from AAB (Fig. 5: A). The width of the fibrils present in the pure SCOBY (178 ± 22 nm, Table 2) represents the bundeled cellulose nanofibrils forming microfibrils, which is to be expected73. Comparing with the UW and fludized SCOBY film, it can be observed that the homogenity of the film is decreased (Fig 5: B). The film appears however to still possess a form of
homogeneity, which could potentially be the effect of remaning fermentation by- products that surround the fibrils when dried. This homogenity appears to disappear with washing with clear images of cellulose fibrils, as presented in Fig. 5: C, indicating that the removal of residual fermentation products has been efficient. It would be interesting to take images at higher magnification. However, due to highly
20 charged samples this was not possible. Furthermore, the effect of fluidization of the morphology and width of the fibrils is made apparent by the FE-SEM images. For pure SCOBY, fibril bundles of larger widths can be observed in the cellulose matrix.
Fluidization of SCOBY appears to reduce the size and distribution of larger fibril bundles for both W and UW film samples. Observing the average width of pure SCOBY of (178 ± 23) nm, this is reduced by fluidization to approximately 65 nm (Table 2) for both the W and UW sample. With the W/UW samples being passed the same amount of times through the microfluidizer and obtaining similar average fibril width, one could argue that washing will not affect the width of the final product.
5.1.1 Moisture uptake analysis
Moisture uptake analysis revealed that UW SCOBY incorporated into MFC films absorbed a greater volume of water at relative humidity 65% and 80% compared to 100% MFC films and films with W SCOBY material. The 100% MFC films and films with W SCOBY showed almost identical absorptions patterns. While one would assume that the absorption of moisture would be greater for W films due to the
increase in porosity after washing (observed with FE-SEM), these results indicate that incorporation of washed SCOBY BC in MFC films hold similar moisture absorption properties to pure MFC. Why the film with UW showed this behavior at high RH, might be the presence of hydrophilic fermentation products that remain in the UW film which aid the absorption of moisture.
5.1.2 FTIR analysis
The effect of washing was also studied with FTIR. As expected, characteristic
transmittance bands for cellulose were displayed for all three samples (Fig. 6, A,B,C).
The presence of amino acids and/or proteins in the pure SCOBY sample was determined by the transmittance band at 1540 cm-1 (Fig. 6, A), which vanishes after the washing or/and fluidization process (Fig. 6, B,C). One would expect this
transmittance band to be present in the fluidized UW film. However, it is not present there. This might be the result of dilution prior to fluidization, reducing the
concentration of present organic acids following fluidization in the film. Similarly, the transmittance band at 1727-1732 cm-1 assigned to hydrogen-bonded carbonyl stretching vibration61 vanishes after washing but is, however, still present in the fluidized UW film. This carbonyl stretching reveals the presence of an ester bond, which might imply that there is some cross-linking between cellulose chains in the SCOBY which is removed with washing. More specifically, the carbonyl stretching is related to anhydride formation, which furthermore is an intermediary reaction of polysaccharide crosslinking by di- and polycarboxylic acids74. This suggest that crosslinking of cellulose fibrils might occur by the fermentation products in the SCOBY. In order to fully determine what compounds might disappear in the washing process, qualitative analysis by GC-MS would be most suitable, which will now be further discussed.
5.2 Characterization of SCOBY with GC-MS
As expected from both literature and the FTIR results, the GC-MS results revealed a variety of different organic acids and fermentation products present in the dry SCOBY (Table 3). Compounds such as glycerol, Compound X and carbohydrate degradation products are from the background of Kombucha fermentation expected to be found in the SCOBY and fermentation media48. With small knowledge of the growth medium and composition of the SCOBY, the compounds discovered which
21 cannot be directly related to the biosynthetic pathway of AAB and yeast could
possibly be assigned to degradation products of the synthesized acids and saccharide monomers.
Nevertheless, the compounds present in the SCOBY from GC-MS display properties that might be applicable for plasticizing of MFC films. The compounds found in the SCOBY, glycerol, organic acids and monosaccharides all exhibit more than one hydroxyl group (polyols) with relatively low molecular weights, which are compatible to the hydrophilic nature of cellulose and could potentially plasticize MFC64 and other polysaccharide films (as presented in Table 3). It is however worth to consider that the plasticizer must be compatible to the polymer in other parameters such as polarity, concentration of hydrogen bonding, and conformation64. Regarding improving the mechanical properties of MFC, the compound must efficiently
plasticize the cellulose fibrils while still maintaining the properties of low gas permeation. For instance, larger hydrophilic compounds might then plasticize MFC efficiently, but increase the water diffusion of the polymer films. This would be interesting to further explore with the present compounds in the SCOBY.
As earlier discussed, the results from FTIR presented the possibility of crosslinking between the cellulose fibrils. Di- and polycarboxylic acids such as citric acid has been reported to be used as a non-toxic crosslinking agent for polysaccharides, which have been tested for MFC films to improve mechanical strength34,74. Citric acid was not discovered in the pure SCOBY, however another dicarboxylic acid, succinic acid, was found. Succinic acid has also been suggested to be able to act as a natural crosslinking agent for polysaccharide films, however the mechanism of polymer crosslinking often require thermal treatment which might not be synonymous with the process of cellulose formation by AAB74.
Regarding the applicability of these molecules to plasticize the BC in the SCOBY, one aspect is how the process of incorporation between the cellulose chain is
performed. During the formation of the SCOBY pellicle, the biofilm grows layer by layer in the growth medium, which potentially could increase the possibility of fermentation products with plasticizing properties to occupy the intermolecular
spaces between polymer chains, enhancing the mechanical properties of the dried film by increasing the free volume between the polymer chains. However, this is only hypothetical, and cannot be concluded from these results. Quantitative analysis would be suggested to be done in order to determine if the present compounds have
concentrations that might have any significant impact on the BC.
5.3 Mechanical testing of plasticized film
Glycerol and one other compound from the GC-MS results was chosen to be studied as a plasticizer in MFC films, presented in Table 4. The process of choosing the concentrations of plasticizers in the MFC dispersion used for film making was explorative, since no quantitative analysis of the compounds in SCOBY was performed.I Initially concentrations up to 1% dw was tested. Above 0,50 % dw of plasticizer, the films would not dry and form uniform films, hence concentrations varying between 0,1-0,3 % dw was instead explored. Although the concentrations were lowered there was still issues with the drying of the films, where the films curled up on themselves. This may have attributed to the variation in film thickness between films, where the accumulation of the film formulation was shifted over the surface as the film dried. Furthermore, since the presented results of tensile testing are an arithmetic average of 5 sample strips for each film, variation in thickness over the film might have contributed to the standard deviations.
22 Nevertheless, an increase in strain-at-break could be observed for MFC films with both glycerol and compound X for all concentrations. A decrease in E modulus was observed for all samples, which is expected when the plasticizer decreases the hydrogen bonding between fibrils which reduces film stiffness.
The presence of glycerol in the MFC film gave the highest increase in strain-at-break of 5,87 ± 0,47 % (Table 4) at 0,1% dw plasticizer in the MFC dispersion. This implies the successful plasticization of the MFC fibrils, in which the glycerol molecules have been able to occupy the space between fibrils, generating a less brittle MFC film. This increase in strain could also be in combination of water absorption aided by the hygroscopic nature of glycerol, which further aids the plasticization of MFC. This behavior of glycerol in MFC was also observed in previous studies where glycerol was added in concentrations between 5-20 by weight of dry MFC30. At higher plasticizer concentration, the compatibility limit of the plasticizer with MFC might have been exceeded, causing phase separation in which the interaction of chains is too low to achieve a material with ductile behavior75. Therefore, if glycerol is to be
utilized for MFC film plasticization, lower concentrations and different methods of incorporation should investigated. However, in the light of the high water absorption capacity of glycerol, the application of MFC films with glycerol as a barrier material might not be relevant.
Observing the effect of compound X on strain-at-break also implied the successful plasticization of MFC. For this compound, concentrations below 0,25 % dw only increased the strain-at-break marginally compared to the original MFC film. The highest yielded strain-at-break of 4,50 (±0,64) % was achieved at 0,25 % dw, with E modulus of 3710 (±636) MPa. This implies that this compound found in SCOBY can in fact be utilized as a bioplasticizer for MFC improving its ductility, something that have not earlier been studied. Further studies are needed to determine the optimal concentrations and methods of incorporation in the MFC film formulation to obtain the best mechanical properties.
5.3 Mechanical testing of fluidization experiments
The last results answer the question whether if and how SCOBY cellulose should be introduced in order to improve the mechanical properties of MFC films. Here, the effect of fluidization of W and UW SCOBY material and incorporated concentration was studied (Fig. 8). One important difference that is to be made between W and UW SCOBY material is that it is not possible to denote UW SCOBY as pure cellulose.
The W SCOBY have been proven to contain very pure BC, whereas it was revealed from GC-MS that UW SCOBY still contain a variety of other degradation products.
The ratio of cellulose to degradation products in the UW SCOBY have not been determined, therefore the concentration of SCOBY material incorporated that truly is BC in the MFC films is not known.
MFC films with UW SCOBY display similar behavior in E modulus independently of number of fluidization passes with the modulus decreasing at higher concentrations (Fig. 8, A). As previously mentioned, the UW films still contained a certain amount of fermentation products which may have exerted a negative impact on the stiffness of the MFC film. At higher concentrations, one could assume that a higher
concentration of fermentation products also is present in the film, which according to the GC-MS, might act similarly as glycerol in MFC films and aiding the adsorption of water, thus producing films that are brittle and low in strength. This behavior is confirmed by moisture uptake analysis (Fig 7). The films containing W SCOBY
23 material displayed a E modulus similar to or higher than the modulus of MFC, which further proves the point of residual fermentation products disturbing the hydrogen bonding between fibrils in UW films.
W/UW films at fluidization grades a and b improve the strain-at-break of MFC films marginally. The difference in fluidization grades only seem to differ in E modulus, where W performed better. For these films, the SCOBY material was added by magnetic stirring following fluidization, which might imply that the interaction of BC and MFC have not been sufficient to improve the mechanical properties of the films.
The real difference for both W and UW films is when Method X is applied. By utilizing this method, a dramatic increase in strain-at-break is observed for batch UWMFbx and WMFbx, all exceeding 5% elongation. While UWMFbx displayed significant improvement in strain-at-break for all concentrations, the highest value of strain-at-break was observed WMFbx, the highest being at 10% SCOBY material, i.e., 10.5 ± 0.4 %. The E modulus was also improved for this batch, reaching of 10500 ± 1140 MPa. This might be the result of a higher concentration of nanostructured BC in W SCOBY material without the discrepancy caused by plasticizing fermentation products.
In conclusion, these results imply that this method successfully incorporated the BC to the degree of which the MFC fibrils with high probability have started to form bonds between the networks, integrating the 3D-network of BC into MFC and thereby improving mechanical properties. The incorporation of BC in MFC films depends highly on the interaction between fibrils. This is also confirmed by FE-SEM, where Method X generated very homogeneous films with seemingly low porosity for all concentrations tested. In the FE-SEM images one can see are also larger fibrils of BC, interwoven in the MFC matrix. In order to determine the applicability of BC- plasticized MFC films, parameters such as oxygen transmission rates, moisture absorption, fluidization grades, and concentration of SCOBY material in MFC films need to be studied.
It should be mentioned that although the production and the incorporation of SCOBY itself does not require any harmful chemicals, the process of washing the SCOBY requires large amounts of highly concentrated NaOH at high temperatures. In these experiments, a ratio of 2:1 ml NaOH/ g SCOBY was utilized without heating, which from an upscaling perspective is not as applicable due to the large volumes required.
Thus, the more environmentally friendly alternative is instead to utilize a
bioplasticizer to improve the mechanical properties of MFC. If a safer or cheaper process of washing the SCOBY can be developed, SCOBY have great potential to be utilized.
6. Conclusions and outlook
In this study, the properties and composition of a film made from SCOBY cellulose were investigated. The learnings were used for improving ductility of implemented on a film made of microfibrillated cellulose. Analysis of SCOBY with FE-SEM revealed a closely bound network of homogenous BC, where fibril diameter decreasing with alkali washing. Moisture content analysis revealed that UW SCOBY absorbs a higher amount of water, discussed to be the result of hydrophilic fermentation products that remain in the UW film which aids the absorption of moisture. FTIR confirmed the efficiency of washing.
