Team:SZU-China/Proof Of Concept
Proof of concept
Blue is the essence of jeans, and they all come from a long-standing dye called indigo, first extracted from plants, such as wood blue, Isatis india, indigo blue, etc. These days, jeans have been popular all over the world, and the annual output of jeans has reached several billion pieces. Most of the jeans dyes are chemically synthetic indigo which brings many environmental problems. What's worse, a variety of toxic and harmful chemicals, such as hydrogen cyanide, formaldehyde, are involved in the dyeing processes. But many factories directly discharge sewage without treatment to reduce costs. That is why textile dyeing has become one of the most polluting industries in the world.
Aiming at the two most sticky problems in jeans processing, we proposed corresponding solutions based on synthetic biology.
As for dyeing, we expressed thermostable beta-glucosidase via recombinant E.coli to synthesize Gardenia Blue, a stable, environmentally-friendly pigment with high dyeing efficiency. As for fraying, we used recombinant cellulase (endo-glucanase) as an alternative of sandblasting to hydrolyze surface cellulose and form the faded worn-look.
Besides, we designed hardware to adjust our products to the jeans manufacturing process. And we created a BP neural network and other models to help manufacturers predict enzyme yields or adjust the number of raw materials added according to their set enzyme yield, so as to achieve the goal of obtaining the highest profit with the lowest cost.
In order to better connect the two parts of our design and make it more convenient for use, we designed a machine. With the machine, all you need to do is place your trousers onto the trousers clamp and it can help in transferring, dyeing, fraying and washing at last.
To start with, an internally integrated sensor detects the concentration of Gardenia Blue and temperature, and automatically adds dyes or heats if they are not met the threshold, guaranteeing for the follow-up dyeing to be carried out smoothly.
Besides, Gardenia Blue pigment has a wildly used, because, with the addition of different amino acids, it can be shown in different colors from green, blue to purple that meets the basic needs of customers for jeans.
After dyeing for the set time, users can order the machine to transfer the trousers to the fraying platform, where using sprayers of cellulase to replace the pollutive stone-washing in most factories. When these are all done, the machine will help the users to wash and dry the jeans and now you get a new pair of jeans!
Fig.1 Hardware
We first follow our best formula: add 440μl geniposide, 6μl glycine, 50μl beta glucosidase enzyme solution, and 504μl PBS buffer to the 1ml reaction system. And we prepared 250ml gardenia blue pigment solution. The color and output are within our expectations.
Fig.2 Homemade Gardenia Blue Solution
We first compared the difference in dyeing effect between bio-indigo blue and gardenia blue. The gardenia blue solution was prepared according to the best formula. Sample C represents the unstained control group, Sample G represents the experimental group stained with gardenia blue, and Sample I represents the experimental group stained with biological indigo.
Fig.3 Comparison of indigo dyeing and gardenia dyeing
Due to beautiful blue color showed in phenylalanine reaction, we tried to use phenylalanine to prepare a bottle 250ml of gardenia blue pigment solution. The mother liquor of phenylalanine was a saturated solution, and the dosage is 6μl. We used these two gardenia blue solutions (Glycine-Gardenia Blue and Phenylalanine-Gardenia Blue) to dye fabrics referring to the direct dyeing method. Our dyeing system was 25ml enzyme-pigment mixture with 100ml deionized water. The dyeing bath abounded at 1:30.
In addition, we used the gardenia blue pigment produced by glycine as the raw material to explore the difference in the dyeing effect between direct dyeing and post-mordant dyeing. The medium involved in pre-meddling dyeing was ferrous sulfate. The dyeing system was 25ml enzyme-pigment mixture with 100ml deionized water. The dyeing bath abounded at 1:30.
We also used the two gardenia blue solutions we produced to explore the influence of different liquor ratios on the dyeing effect. We set up three experimental groups of bath ratio: 1:10, 1:30 and 1:50.
Fig.4 Control group: Denim not dyed. P: Iron ion post mordant group (bath ratio 1:30). DG-10: Direct dyeing group with glycine, bath ratio 1:10. DG-30: Direct dyeing group with glycine, bath ratio 1:30. DG-50: Direct dyeing group with glycine, bath ratio 1:50. DP-10: Direct dyeing group with phenylalanine, bath ratio 1:10. DP-30: Direct dyeing group with phenylalanine, bath ratio 1:30. DP-50: Direct dyeing group with phenylalanine, bath ratio 1:50.
It shows from the Control group, group DP-30, and group DG-30 that the glycine-gardenia blue has a better and deeper coloration, which verified the superiority of glycine.
It proves from Control group, group DG-10, group DG-30, group DG-50 that different liquor ratios have significant effects on the dyeing effect. The effect of bath ratio of 1:30 and 1:50 is obviously better than 1:10. On the contrary, for Phenylalanine-Gardenia Blue, the color difference between the experimental group with a bath ratio of 1:30 and the experimental group with a bath ratio of 1:50 is not obvious.
We have explored the influence of the dyeing time after holding the salt to promote dyeing in the direct dyeing method. We made 6 experimental groups with a bath ratio of 1:50, and kept them for 10 minutes, 20 minutes, 30 minutes, and so on. After dyeing and drying, the result is as shown below. It can be seen that the dyeing effect becomes better and better with the extension of the heat preservation dyeing time, and the dyeing effect at 40mins is the best.
Fig.5 Influence of the dyeing time
For the post-mordant dyeing method, we set the temperature as a variable, using ferrous sulfate as the medium in the bath ratio of 1:30. Obviously, the holding temperature has a great influence on the dyeing effect. The 30°C group has a very large color difference compared to the 80°C group. There are obvious color differences between each set of temperature gradients.
Fig.6 The influence holding dyeing temperature
We also explored the effects of different metal fixative on dyeing. We used the four metal ions as mediators, which were ferrous ion, lanthanum ion, aluminum ion and cerium ion.
The control group was the dyeing group without any metal ions. It can be seen that the dyeing effect of the post-mordant dyeing group with metal ions is better than that of the control group. Among them, aluminum ion has the best dyeing effect, and ferrous ion is the second, and lanthanum ion is the third, and cerium ion is the worst which is almost the same as the control group.
Fig.7 The influence of different metal mediators
We sent four post-mordant dyed denim fabrics with different metal ions as fixative and a control group to the testing center to test their color fastness to washing, soaping and perspiration.
Taking soaping as an example, we sewed our dyed cloth with a standard lining fabric. After common soaping, we use the original sample as a reference to evaluate the discoloration of the sample and the discoloration of the lining fabric. The corresponding parameters are colorfastness and staining fastness, which are divided into 5 grades. Grade 5 is the best and grade 1 is the worst.
At first we thought that adding a metal ion fixative would make the color fastness better, but the data returned by the test report showed that the fastness between the experimental group using 4 metal ion fixatives and the control group without metal ion fixation was not Significant differences. In addition, in the sweat resistance test section, the control group scored higher.
This shows that our enzyme-pigment mixture does not rely on metal ions to improve colorfastness. In addition, according to the national standard used in the test report, our dyed fabrics have reached the wearing standard, which once again reflects our high efficiency and environmental protection advantages.
This shows that the gardenia blue pigment itself has the ability to be applied to the dyeing process. But the conditions of the dyeing process.
We took four cloths of equal mass, soaked them in the IARI-SP-2 crude enzyme solution and added an equal volume of pH=7 PBS buffer to dilute. The quality ratio of enzyme solution and denim was controlled at 80:1.
We put them in a flask and placed them in a shaker at room temperature (22°C) for shaking. After 48 hours, they were taken out for inspection, and the surface was photographed with an electron microscope to observe the destruction of the cellulose structure on the surface as shown below.
The comparison shows that the fiber structure on the denim fiber bundle is basically intact before the enzyme treatment is added. After 48 hours of enzymatic treatment, the denim fiber bundle structure was obviously damaged, and significant faults and traces left by the cellulose chains were visible. The result was within our expectations.
Limited by equipment reserves, we cannot control the constant temperature and keep shaking. If a higher mass ratio is used, the purified enzyme solution is not diluted and placed in a constant temperature shaker at 38°C for reaction, the treatment effect will be more impressive.
Fig.8 Fiber bundles destroyed by cellulase under electron microscope
