Uniaxial Testing

Problem Overview

In order to explore bacterial cellulose (BC) as a viable alternative to existing packaging materials and provide valuable feedback to the wetlab design, mechanical properties of BC needed to be quantitatively analyzed. Through uniaxial tensile testing, the characteristic stress-strain curves of BC were generated to assess its competitive strength compared to existing packaging materials. The uniaxial tensile testing was integral to the wetlab design considerations, as BC behaved similarly to paper but was significantly weaker than plastic. This conclusion informed the need to integrate polyhydroxybutyrate (PHB) into the final product. BC samples prepared using different purification methods, samples grown in varying types and ratios of fruit waste media, and existing packaging materials were tested and analyzed.

Integration of Human Practices

Our HP contact, Chris Clark from Star Produce, emphasized that our material needs to be strong enough to be stacked and shipped.

More specifically, Chris advised us that boxes are an ideal packaging design, as this prevents the addition of extra labor or machinery and individual coating usually requires heat to shape and apply them. BC shaped into a box will leverage the existing tools and equipment in present facilities while making our product more applicable to a wider variety of fruits and vegetables. Chris shared with us that a box design would serve approximately 95% of produce, while an individual wrap may only apply to about 5%.

Chris shared with us that boxes limit the damage that is experienced by the produce during transport. He advised that very fragile products are packaged in hard plastic containers. Boxes also maximize the efficiency of transport on the truck itself as shipping companies stray from possible “air space” at the top of the truck. In addition to its negative effects on the environment by lacking efficiency, it also poses a challenge in keeping the truck cold.

For the designing of the packaging, Chris encouraged us to consider what type of produce it is used for, as harder produce is usually placed in bags, and softer produce is transported using boxes. He also said to consider how much produce would go into the box, and how heavy the produce is.

Based on this feedback from our HP contact, the mechanical properties of BC, specifically the properties that are directly relevant to the design of a box and its strength in withstanding the applied load when stacked, were necessary to validate our product. As a result, performing a uniaxial tensile test became an essential contribution to the project to assess the viability of our BC as a competitive packaging material.

Design

To improve our understanding of the strength and quantify the behavior of BC, uniaxial tensile testing was conducted until the sample’s failure. Similar to how biological tissues and materials are characterized, we used an identical mechanical testing protocol to conduct failure tests to extract useful information on its properties. The extracted mechanical properties such as its brittleness and flexibility then directly informed wetlab on the necessary design modifications to increase the level of force that can be applied to be competitive as a fruit packaging.

Multiple uniaxial tensile tests were conducted to explore:
a) Varying types and ratios of fruit waste media as a potential growth medium for BC

b) Effect of integrating BC with PHB

c) Effect of different purification methods on mechanical properties of BC

d) BC’s strength to that of existing packaging material of paper

As mechanical testing is conducted to determine the properties of materials by measuring the material’s response to external loads, uniaxial tensile test was chosen for this purpose. The reason for choosing this method of testing is because it is one of the most common tests that can be used to determine valuable properties such as ultimate tensile strength, yield strength, Young's modulus, and maximum elongation (1). An important consideration for the designing of the mechanical test is the orientation of the fiber network within the BC pellicle. When the BC seed is grown on the petri dish in our lab, the BC grows outwards, increasing in diameter. As a result, the uniformity of the properties, in terms of the orientation of the fiber network, needed to be assessed through uniaxial testing. Following safety training and a lab orientation, we gained access to an engineering teaching lab in the University of Calgary’s Schulich School of Engineering to independently conduct uniaxial testing of our samples. In the engineering lab, we used the CellScale UniVert Uniaxial tension/compression tester and its software that is commonly used for biological tissue testing at forces up to 200 N (2). We ensured the samples were tested on the same equipment to ensure comparison across the samples were valid.

Figure 1a: CellScale UniVert uniaxial tension/compression tester

Figure 1b: Key components of uniaxial tensile testing machine

Figure 1c: CellScale UniVert software

Each uniaxial test was conducted based on the lab manual provided through the University of Calgary’s Schulich School of Engineering. After moving the actuator to a specified size and zeroing the load cell, the specimen was mounted on to the machine’s grips. With the sample placed in the top grips first, the top grip was lowered and the sample was clamped between the bottom grip faces.

Before the specimen was mounted, the width and thickness of the samples were measured using a digital caliper. Using calipers, the grip-to-grip distance between the two grips (L₀) was measured and recorded once the specimen was mounted in the testing machine. The cross-sectional area (A₀) of the specimen is calculated using the width and thickness of the sample.

Figure 2: Geometrical properties of the sample measured for analysis

Through uniaxial tensile testing, we were able to quantify the relationship between the applied force and the displacement experienced by the sample. For our purposes, the specimen was subjected to uniaxial tension to measure the resultant lengthening. The raw data outputted from the testing machine are time, displacement, and force.

For the purpose of our project, we chose to subject the samples to constant increase in strain until failure. Therefore, data was collected until fracture was observed on the sample. Failure occurs when the stress on the test specimen exceeds the strength of the material.

Figure 3a: BC sample following failure during a uniaxial tensile test

Figure 3b: BC samples dismounted from the uniaxial tensile testing machine following failure

The test protocol parameters set for BC sample and paper material testing were at a stretch magnitude of 5.0 mm and stretching for the duration of 200 seconds. The test was terminated when failure was observed in the sample.

A force-displacement graph was generated based on the raw output. To generate a stress-strain curve, the change in length (ΔL), stress (σ), and strain (ε) were determined from the values collected, using the equations below:

ΔL = x_n − x₀

σ = ^F ⁄ _A0

ε = ^ΔL ⁄ _L0

To determine the ultimate tensile strength, the maximum value on the stress-strain curve was chosen as this point signifies maximum tensile stress that the material is expected to endure without failure. Maximum elongation, or strain at failure, was also recorded for each sample. Young’s modulus (E) signifies material’s stiffness. As the stiffness of the material increases, the higher the elastic modulus will be. This property can be extracted from the linear elastic portion of a uniaxial deformation. As a result, we found the slope of the stress-strain curve in its linear regions to extract its Young’s modulus using the equation below:

E = ^σ ⁄ _ε

For data presentation, the 2014 Imperial College team’s mechanical testing results was used as a guideline (3).

 

Sample Preparation

After consulting with a biomedical engineering graduate student that is highly experienced with biological tissue testing, we clarified our understanding of methods of sample preparation and how to analyze the data to accurately quantify the material properties. We cut the samples into a “dog-bone" shape and attempted to maintain an ideal 4:1 ratio of length-to-width. The dog-bone shape is necessary in ensuring the sample’s area of interest is subjected to only pure tensile load as there is tensile, compressive, and shear load near the ends of the samples that are in contact with the grips.

Figure 4: BC samples cut into dog-bone shapes for uniaxial tensile testing

The strength of BC cultured in Hestrin–Schramm (HS) media were compared to BC grown in varying types and ratios of fruit waste media (FWM) (peel, pulp, and juice of an orange), to ensure the FWM did not compromise its mechanical properties. The mechanical properties of BC were also compared to BC integrated with PHB. In addition, the strength of BC samples purified using sodium hydroxide (NaOH) was also compared to samples purified with sodium bicarbonate (NaHCO₃) through uniaxial tensile testing. Finally, the strength of BC cultured in HS media and purified using NaOH was compared to existing paper packaging materials.

For each of the samples that were produced in the lab, BC samples were prepared for uniaxial tensile testing by autoclaving after it is grown, purifying with 0.1 M NaOH or NaHCO₃, boiling in distilled water, and air drying at room temperature (25°C) for 4-5 days on a parchment paper sheet.

Figure 5: Autoclaved, treated with 0.1 M NaOH, and boiled BC left to air dry for 4-5 days

We also prepared paper material that is commonly found in packaging by cutting it into horizontal and vertical directions to explore if the mechanical properties vary in the two directions.

Figure 6a: Paper packaging that was tested for comparison in terms of mechanical properties to BC

Figure 6b: The direction in which the paper packaging samples were cut to explore varying behavior in the 2 directions

Similar to BC, samples from paper packaging were also cut into dog-bone shapes for uniaxial tensile testing.

Figure 7: Paper packaging sample following failure during a uniaxial tensile test

Uniaxial Results

Effect of Fruit Waste Media on BC

The purpose of assessing the effect of FWM on BC strength is to explore if mechanical properties are compromised when compared to BC conventionally grown in HS media. The results of the conducted mechanical tests of BC grown in varying types and ratios of FWM are shown in Table 1.

Table 1: Mechanical properties of BC cultured in different types and ratios of fruit waste media

The FWM consisted of whole oranges which were processed into peel (PL), pulp (PU), and juice (JU). To explore if mechanical properties are compromised when varying ratios of FWM are substituted as a growth medium, uniaxial tensile tests were conducted. The stress-strain curves generated based on the collected data are shown in Figure 8 to 17.

Figure 8: Average tensile stress-strain curves of BC samples cultured in PL 15 media

Figure 9: Average tensile stress-strain curves of BC samples cultured in PL 30 media

Figure 10: Average tensile stress-strain curves of BC samples cultured in PL 45 media

Figure 11: Average tensile stress-strain curves of BC samples cultured in PU 15 media

Figure 12: Average tensile stress-strain curves of BC samples cultured in PU 30 media

Figure 13: Average tensile stress-strain curves of BC samples cultured in PU 45 media

Figure 14: Average tensile stress-strain curves of BC samples cultured in JU 15 media

Figure 15: Average tensile stress-strain curves of BC samples cultured in JU 30 media

Figure 16: Average tensile stress-strain curves of BC samples cultured in JU 45 media

Figure 17: Average tensile stress-strain curves of BC samples cultured in pure HS media

The FWM BC samples collected for each ratio were grown in the same tube and conditions, and the samples were treated, purified, and dried in the same manner. The samples for uniaxial tensile testing were cut in varying directions to account for potential differences in the orientation of the fibers, which will result in varying mechanical properties. The control that is used for comparison was also grown in the same conditions and prepared in the same manner. The large standard deviation can be attributed to a lack of uniformity in the orientation of the fibers, resulting in varying mechanical properties in each direction.

As shown in Table 1, the ultimate tensile strength, Young’s modulus, and maximum elongation of BC samples grown in varying ratios and types of FWM were analysed and recorded. As shown in Figure 8 to Figure 17, the samples from each media behaved differently, confirming our assumption that there is a lack of uniformity in the orientation of the fibers within the BC.

For comparison of the mechanical properties for each FWM ratio, bar graphs were generated to easily compare the quantified properties. In terms of ultimate tensile strength, the JU 30 ratio yielded the highest value (14.67 MPa higher than control BC). Amongst the FWM samples, JU 45 yielded the lowest value (1.121 MPa higher than control BC). An important note to make is that the ultimate tensile strength of all the samples grown using fruit waste media yielded values that are higher than the control BC grown in pure HS media. Based on these comparisons, JU 30 BC showed the highest potential in withstanding higher stress without failure. Control BC showed the lowest potential in this capability.

Figure 18a: Bar graph comparing ultimate tensile strength of BC cultured in different types and ratios of fruit waste media, compared to BC cultured in pure HS media

In terms of the Young’s modulus, all FWM BC samples with the exception of the PU 15 sample were stiffer than the control BC. PU 15 was 167.2 MPa lower than the control BC. The highest value of Young’s modulus was yielded by JU 30, being 2908 MPa higher than the control BC. Majority of the FWM BC samples showed higher stiffness, meaning it is only able to alter its initial shape to a small extent under a load. In comparison to the control BC, PU 15 showed lower stiffness.

Figure 18b: Bar graph comparing Young’s modulus of BC cultured in different types and ratios of fruit waste media, compared to BC cultured in pure HS media

Finally, based on maximum elongation, the control BC yielded the highest maximum elongation compared to the FWM samples. PL 15 resulted in a lowest maximum elongation, being 0.01516 lower than control BC. The maximum elongation, which signifies the elongation at the time of fracture, is highest in the control BC. This signifies that it failed at a large deformation before fracture under a tensile load, compared to the FWM samples.

Figure 18c: Bar graph comparing maximum elongation of BC cultured in different types and ratios of fruit waste media, compared to BC cultured in pure HS media

As one of the targets of our BC packaging is to make it customizable, the type of media and the desired mechanical property can be chosen to customize the packaging depending on the purpose of the packaging and the target fruit. As we were mainly focused on validating our BC material based on HP feedback from Chris Clark, in that our material needs to be strong enough to be stacked and shipped, we placed emphasis on the material’s ultimate tensile strength. Since FWM BC samples yielded ultimate tensile strength that were all higher than the control BC, we can state that FWM does not compromise the BC strength, when substituted for a ratio of HS media. However, future work should focus on improving the overall mechanical properties so it is competitive compared to existing packaging materials.

 

BC Integrated with PHB Results

As our packaging material needs to be strong enough to be stacked and shipped, the incorporation of PHB in BC aimed to achieve this objective. We attempted to quantify the extent of the change in mechanical properties through uniaxial tensile testing.

Table 2: Mechanical properties of BC compared to BC incorporated with PHB

Samples of BC made in a monoculture with the PHB added post-production were cut from the same petri dish to allow for consistent comparison.

Figure 19: BC samples cut into dog-bone shapes for uniaxial tensile testing for comparison to BC incoporated with PHB

The samples were again cut in different directions to explore variation in the behavior due to the orientation of the fibers within the BC. The same procedure applied for the pure BC samples. The large standard deviation can be attributed to the lack of uniformity in the orientation of the fibers, resulting in varying mechanical properties in each direction.

Figure 20: Average tensile stress-strain curve of BC samples for comparison to BC incorporated with PHB

Figure 21: Average tensile stress-strain curve of BC incorporated with PHB

Based on these results, BC showed the ultimate tensile strength that was approximately 3.780 MPa higher than that of BC incorporated wirth PHB.

Figure 22a: Bar graph comparing ultimate tensile strength of BC relative to BC+PHB

Its Young’s modulus is 758.2 MPa higher than that of BC incorporated with PHB and its maximum elongation is 0.02868 lower.

Figure 22b: Bar graph comparing Young’s modulus of BC relative to BC+PHB

Based on these comparisons, BC incorporated with PHB showed potential in improving a property that was of concern with pure BC--its stiffness. Although this method of post-production PHB incorporation shows its limitation in improving BC in terms of tensile strength, it showed its potential in giving BC the ability to alter its initial shape to a larger extent under a load. The maximum elongation, which signifies the elongation at the time of fracture, is higher in BC incorporated with PHB which signifies that it failed after a larger deformation before fracture under a tensile load, thus reinforcing this conclusion.

Figure 22c: Bar graph comparing maximum elongation of BC relative to BC+PHB

In addition, this uniaxial testing reveals the importance of producing PHB as a co-culture so that there is an improved distribution of PHB through the matrix. Integrating this understanding into wetlab design, the uniaxial testing data shows us the potential improvement in mechanical properties that can come from designing the PHB constructs to favor the production of smaller PHB particles to foster a more even distribution.

BC incorporated with PHB resists failure for a longer period of time as it was able to alter its initial shape to a larger extent under the load. This suggests that the presence of PHB is introducing an elasticity to the material which is a valuable factor in packaging development, as it increases its resistance to damage or rupture by deformation.

 

Effect of Different Purification Methods on BC

The purpose of assessing the effect of different purification methods on BC strength is to explore which method is ideal in preparing BC samples for varying packaging designs. The results of the conducted mechanical tests of BC prepared using different purification methods are shown in Table 3.

Table 3: Mechanical properties of BC prepared using different purification methods

BC grown and autoclaved in the same conditions were purified with either NaOH or NaHCO₃. To explore if mechanical properties differ between the two purification methods, uniaxial tensile tests were conducted. The stress-strain curves generated based on the collected data are shown in Figure 23 and 24.

Figure 23: Average tensile stress-strain curve of BC samples purified with NaOH

Figure 24: Average tensile stress-strain curve of BC samples purified with NaHCO₃

As is reflected in Table 3, the ultimate tensile strength, Young’s modulus, and maximum elongation of BC samples purified with NaOH are approximately 3.267 MPa, 400.7 MPa, and 0.06171, respectively. On the other hand, BC purified with NaHCO₃ yielded the ultimate tensile strength, Young’s modulus, and maximum elongation of approximately 9.796 MPa, 2202 MPa, and 0.03287, respectively. For comparison of the mechanical properties resulting from each FWM ratio, bar graphs were generated to easily compare the quantified properties.

Samples of NaOH purified BC were again prepared from the same petri dish, and cut in different directions to explore variation in the behavior of the BC due to the orientation of the fibers within the BC. The samples of NaHCO₃ purified BC were prepared in the same manner. Again, the large standard deviation can be attributed to a lack of uniformity in the orientation of the fibers.

Based on these results, BC purified with NaHCO₃ showed the ultimate tensile strength that was approximately 6.5287 MPa higher than that of BC purified with NaOH.

Figure 25a: Bar graph comparing ultimate tensile strength of BC prepared using different purification methods

Its Young’s modulus is 1801 MPa higher than that of BC purified with NaOH and its maximum elongation is 0.02884 lower.

Figure 25b: Bar graph comparing Young’s modulus of BC prepared using different purification methods

Based on these comparisons, NaHCO₃ purified BC showed potential in withstanding higher stress without failure. However, it comes at a compromise as it is stiffer, and is only able to alter its initial shape to a small extent under a load. The maximum elongation, which signifies the elongation at the time of fracture, is lower in NaHCO₃ purified BC. This signifies that it failed at a small deformation before fracture under a tensile load.

Figure 25c: Bar graph comparing maximum elongation of BC prepared using different purification methods

Again, as one of the targets of our BC packaging is to make it customizable, the type of purification method can be chosen to yield its characteristic mechanical properties depending on the purpose of the packaging and the target fruit.

 

Comparison of BC to Existing Packaging Materials

To understand the relative strength of BC to existing packaging materials, we cannot analyze the mechanical properties of BC on its own, but must also compare its competitiveness to other packaging materials.

Paper material that is commonly found in packaging, was tested on the same equipment to analyze its mechanical properties.

Table 4: Mechanical properties of paper packaging

The samples were analyzed according to the direction it was cut (horizontal and vertical) to examine if the mechanical properties vary between the 2 directions. From the data collected, stress-strain curves were generated for analysis.

Figure 26: Average tensile stress-strain curves of paper packaging samples

For better comparison, control BC from the fruit waste media analysis was compared to the paper material’s mechanical properties through a bar graph. The average of the properties in the horizontal and vertical direction of paper packaging was taken for comparison. In terms of ultimate tensile strength, BC is 3.051 MPa weaker than paper packaging. From this comparison, BC is able to withstand less force than paper packaging.

Figure 27a: Bar graph comparing ultimate tensile strength of BC relative to paper packaging

In terms of Young’s modulus, BC is 11.36 MPa less stiff than paper packaging. Based on this comparison alone, BC is able to alter its initial shape under a load to a greater extent than paper packaging.

Figure 27b: Bar graph comparing Young’s modulus of BC relative to paper packaging

In terms of maximum elongation, BC is 0.006164 less than paper packaging. This signifies that the BC failed at a smaller deformation before fracture under a tensile load, compared to paper packaging.

Figure 27c: Bar graph comparing maximum elongation of BC relative to paper packaging

 

Conclusion

As the purpose of the uniaxial tensile testing was to explore BC as a viable alternative as a packaging material, we can conclude from the collected data and analyzed mechanical properties that BC’s mechanical properties are customizable depending on the conditions of its culture and how it is treated.

As we were mainly focused on validating our BC material based on HP feedback from Chris Clark, in that our material needs to be strong enough to be stacked and shipped, we placed emphasis on the material’s ultimate tensile strength. Since FWM BC samples yielded ultimate tensile strength that were all higher than the control BC, we can state that FWM does not compromise the BC strength when substituted for a ratio of HS media.

As one of the targets of our BC packaging was to make it customizable, the type of purification method (either NaOH or NaHCO₃ treatment) can be chosen to yield its characteristic mechanical properties depending on the purpose of the packaging and the target fruit.

Overall, BC showed potential in replacing paper packaging, as its mechanical properties showed most similarity to that of paper. We were also able to understand the necessity in integrating PHB to give our BC mechanical properties that are comparable to that of conventional plastics.

BC incorporated with PHB resists failure for a longer period of time as it was able to alter its initial shape to a larger extent under the load. This suggests that the presence of PHB is introducing an elasticity to the material which is a valuable factor in packaging development, as it increases its resistance to damage or rupture by deformation.

Future Directions

For future direction of this project, a comment from our HP, Chris Clark from Star Produce, needs to be addressed. He shared with us that for fruits like peaches or apples, box design is ideal, as opposed to individual coating. However, he also mentioned how some harder produce is usually placed in bags, and that few fruits require wrapping as their respiration rate is so fickle. With this consideration, the project will need to explore and place more emphasis on mechanical properties such a maximum elongation and its ability to stretch to be applicable to these fruits.

Another important consideration is to validate our BC material using different data analysis. Instead of solely testing its behavior by subjecting it to constant increase in strain until failure, we could also explore stress relaxation and cyclic loading to simulate different loading conditions. Cyclic loading sets a maximum strain and runs a number of cycles where the sample is loaded to that strain and then unloaded. Stress relaxation involves setting a strain and holding it constant over a period of time, then measuring the change in stress during a period of time. By utilizing different data analysis, we can investigate how the material will behave under different conditions, predict its failures, and improve the mechanical properties.

 

References

1. Schulich School of Engineering. Mechanical Testing of Tissue. Introduction to Biomedical Engineering. Calgary: University of Calgary; [date unknown].
2. CellScale Biomaterials Testing. UniVert – Uniaxial Tension/Compression Tester [Internet]. [Place unknown]: CellScale Biomaterials Testing; [date unknown]. Available from: https://www.cellscale.com/univert-cellscale-biomaterials/
3. Aqualose [Internet]. London, England: Imperial College London 2014 iGEM team; 2014. Mechanical Testing; 2014 [cited 2022 Sep 23]. Available from: https://2014.igem.org/Team:Imperial/Mechanical_Testing#appendix