@@ -88,15 +88,13 @@ While preparing our first round of BC mats to test in Aachen, we also built our
Once the first small Bacterial Cellulose mats had been grown in the lab, our first goal was to make bigger versions that could be used for mechanical testing. With this, we first wanted to explore their potential for the future of our project and get used to handling them right. This way, we grew our first round of mats in a baking dish and a 15x15 cm glass container at room temperature due to limited incubator space [^15]. In the process of growing the mats, we could already see that they grew better on YPD Media [^16] than on DSMZ [^17] Media.
We then tested how they would react to autoclaving and washing as we feared that they would not survive the harsh conditions of the autoclave [^18]. The extraction of the mat grown on YPD before autoclaving can be seen in picture 1.
*Picture 1: Bacterial Cellulose mat, grown in YPD media for six days, being extracted.*
The autoclaving process did not destroy the mats. The only visible change was that the edges appeared to be a little bit dried out.
After later also extracting, washing, and drying the mat grown on DSMZ [^19], we could see that it was a lot thinner and less robust than the one grown on YPD, as it already ripped apart in the cleaning process. On the flip side though it wasn’t stained by the media like the one grown on YPD and was more transparent (Picture 2).
*Picture 2: Bacterial Cellulose mat, grown in DSMZ media (left) and YPD media (right), after drying. The mats were washed repeatedly with cold deionized water.*
In the meantime, a big Bacterial Cellulose mat was grown for an extended time to see if it would change its properties [^20]. The result was that the mat got thicker and stiffer with the extended cultivation time.
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@@ -110,26 +108,22 @@ The problem that then faced us, was that DWI cut our cooperation, meaning that w
The first time we went to HSN, we performed Martindale Abrasion tests and Tensile Strength tests on the mats from the experiment with differing amounts of glycerol and xyloglucan as well as on mats from other experiments (Pic. 3).
Additionally, we tested a cotton cloth we chose as our standard of comparison and pieces of Aramid/Kevlar.
*Picture 3: Tensile strength test (left) and Martindale abrasion test (middle) performed at HSN, and tear test performed on self-built property testing machine (right).*
The remnants of the tested BC mats were then sent to iGEM Dresden [^26] as they work on textile degradation and could find out if the textile can be recycled in a meaningful way.
Something that was also interesting to look at, was a Bacterial Cellulose mat, which had been unknowingly contaminated with a fungus that was washed and dried for the means of testing possible property changes.
Looking into the data, we couldn't draw any definite conclusions, as the standard deviation was too big, or in the case of cotton not calculable (Fig. 1).
*Figure 1: Tensile test data from the first round of tests at HSN. The graph shows the standardforce by thickness in N/mm (blue) and the elongation by thickness in %/mm (orange) for different textiles. Those textiles include 100% Cotton, Aramid, and BC Mat grown on YPD media, YPS media, and YPD media contaminated with fungus. The standard deviation is shown in red/black. For cotton, no standard deviation could be calculated, as there was only one sample.*
Before the tests at HSN, we performed a first round of HPAEC [^27], to find out how the concentrations of glycerol and xyloglucan affected the composition of our mat, and to find out if xyloglucan was integrated at all. After the HPAEC we could see from the peak in Xylose that xyloglucan was incorporated, but Ribose which we used as our standard, was also found in the BC mat, meaning that we would have to change our standard. The problem with the standard also made a statistical analysis of the data impossible, as we couldn't reliably calculate the amounts of our sugars. The problem led to us growing new mats for two more rounds of HPAEC [^28] [^29]. This time we grew mats with less variation, as the powdered xyloglucan we used is quite expensive. The standard was changed to Fucose, as we knew from the previous round, that it wasn't present in the BC mat. The results are summarized in Figures 2 and 3.
*Figure 2: Sugar composition of Bacterial Cellulose mats, grown/washed with different amounts of xyloglucan (XG). The concentrations have been measured via HPAEC with Fucose as standard.*
*Figure 3: Glucose and Xylose concentrations of Bacterial Cellulose mats, grown/washed with different amounts of xyloglucan (XG). The concentrations have been measured via HPAEC with fucose as standard.*
The results of the HPAEC were as follows. No Xylose could be found in the BC mat grown without xyloglucan (0% XG), while there was an intermediate amount in the mat washed with 0,5% (w/v) XG solution (0% XG washed in 0,5% XG) and a relatively high amount in the mat grown with 0,5% xyloglucan (0,5% XG), which means, that xyloglucan was incorporated in the Bacterial Cellulose mat (Fig. 3), as the Xylose could only come from the xyloglucan and is therefore not present in mats grown/washed without xyloglucan. We can additionally say, that the effect of growing the mat with XG is greater than washing with XG solution.
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@@ -140,16 +134,13 @@ Additionally, we performed a test intending to find the perfect amount of media
The second time we went to HSN, we prepared BC mats, grown with different amounts of xyloglucan, to evaluate possible property changes (Figure 4) [^33]. In addition, we tested the effects of different washing and drying methods on different textiles (Fig. 5 & 6).
*Figure 4: Effects of different concentrations of xyloglucan (XG) on the normalized tensile strength (tensile strength by thickness) and elongation (elongation by thickness -> elasticity) of Bacterial Cellulose mats.*
*Figure 5: Effects of different drying conditions on the normalized tensile strength (tensile strength by thickness) and elongation (elongation by thickness -> elasticity) of Bacterial Cellulose mats.*
*Figure 6: Effects of washing Bacterial Cellulose (BC) mat with 1% (w/v) NaOH solution. The graph shows the normalized tensile strength (tensile strength by thickness) and elongation (elongation by thickness -> elasticity) of BC mats, grown with different concentrations of xyloglucan (XG).*
Looking at the data from HSN round 2, it shows that a 0,25% (w/v) xyloglucan concentration increases the tensile strength of the BC mat, while higher concentrations decrease it (Fig. 4). Meanwhile, the elongation, which directly correlates with the elasticity, seems to decrease with the addition of xyloglucan to the BC mat.
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@@ -160,8 +151,7 @@ When it comes to washing methods, additionally washing the BC mat with a 1% (w/v
The creation of the first version of our self-built property testing machine started by making a frame out of upcycled wooden planks left from construction work [^34]. We tried to replicate Universal Testing machines like this one by Nextgen [^35]. The clamps of the machine were improvised with modular metal construction materials like were sold by Eitech [^36] (Pic. 4).
*Picture 4: Wooden property testing machine prototype, also nicknamed “DER GERÄT”. The version in the picture has been modified, to be used as an interactive game to present the basic principles of tear testing*
When we then presented the machine to our PI, Prof. Markus Pauly, who advised us that if we wanted to use the machine in the lab, we would have to either paint to machine or switch to another material as wood is porous, making disinfection a problem.
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@@ -169,21 +159,18 @@ This inspired us to build a new and improved property testing machine [^37] out
In the first tests with the motor, we ran into the problem that the string we used couldn’t be wound up properly (Pic. 5). After doing some research, we found that iGEM Team GreatBay_SZ 2019 had designed a spinning mechanism [^38] that solved our problem after being adapted to our system.
After solving this problem, the motor was integrated into the machine to do further tests, to also measure the maximum force output of the motor. There we only reached about 6 N which was barely enough to rip apart cardboard. This led us to redesign the mechanism, this time integrating a double motor system with stronger motors (Pic. 5). After writing the necessary code [^39], to get the motors to spin in the right direction, the first tests with this system could be performed. It turned out that with the changes made, the force output got beyond what we could measure with our 30 N Newton meter.
*Picture 5: Evolution of the LEGO winding mechanism of the property testing machine. A graphic of the iGEM engineering cycle was added digitally.*
The next change that was made was to screw legs onto the construction so that when it was flipped upside down a simplified tensile test could be performed (Pic. 6). The test was modelled after a simplified tensile test described by Michigan Tech [^40]. To make the machine flippable, the LEGO motor was integrated in a way, that made it possible to easily take it off the machine.
*Picture 6: Property testing machine mark 2 (“DAS MASCHIN”) in tear test configuration (left) and tensile strength configuration (right).*
The machine was then used to get tear strength data on our 100% cotton cloth which we wanted to use as our standard of comparison (Fig. 7) [^41]. This worked relatively well in weft direction, but failed in warp direction, as the clamps and Newton meter were not strong enough to hold the textile / measure the force at a point, where the textile would tear. Even though the standard deviation for the data in weft direction was relatively low, conclusions on how well the machine works compared to professional Universal Testing machines [^35], as a comparative test with such a machine and with the same cotton cloth was not possible, due to organizational constraints.
Later on, the machine was also used for doing the same in the context of a tensile test [^42]. Throughout these experiments, we realized that many improvements could be made for more reliable tests. These include stronger Newton meters or, as we discussed with a member of iGEM Münster, knowledgeable in the field, we could switch to an Arduino or Raspberry Pi based system, implementing a stepper motor, giving us a digital readout in terms of the voltage change of the motor over time. With this data, we could then properly calculate the applied force at a given time.
*Figure 7: Tear strength of a 100% cotton cloth in different directions (weft & warp) measured with the property testing machine “DAS MASCHIN”.*
A further modification that was made on the first property testing machine was to repurpose it, creating an interactive game teaching others about the basic principles of our tests. This was already used at the sustainability day at our University [^43], as well as at the JuniorJam in Münster [^44].
