Team:Paris Bettencourt/Textile
Textile dyeing
As a response to our Integrated Human Practices we decided to try the conventional method of indigo dyeing and to compare it to our method. As presented in the Integrated Human Practices page, indigo production and indigo dyeing require a large number of chemicals. Our challenge was to dye textile without using additional chemicals for the coloring process.
Step 1: Indigo production by bacteria
First, a plasmid coding for trimethylamine monooxygenase (TMM) was transformed into wild-type E. coli. This enzyme allows for the production of indoxyl from indole. Indoxyl is then oxidized by air to indigo. The part from iGem registry was used (1).
To increase the yield of indoxyl production, supplementation of media with tryptophan (2g/L) is required (2). Additionally, a sufficient amount of oxygen has to be present to increase the oxidation of indoxyl. Bacteria were grown overnight.
Figure 1) Production of indigo by bacteria
Then, the pigment indigo from bacteria was purified based on the method of Hill et al., (1989). With an equivalent proportion of chloroform, the entire culture broth was mixed and shaken in a separating funnel. The mixture was centrifuged at 10,000 g for 20 min. A rotary evaporator was used to dry the pure indigo and to separate it from the chloroform layer.
Figure 2) Indigo extracted from bacteria resuspended in chloroform
The presence of indigo synthesized by bacteria was confirmed with TLC (Thin Layer Chromatography). This method is a chromatography method that relies on the separation principle. The stationary phase is a sheet coated with a thin layer of adsorbent material. The mobile phase is a solvent that is applied on the sheet and is drawn up the plate via capillary action taking up the sample at the same time. Here, the running solvent used was chloroform-methanol (15:1). The results are then visualized under normal light since indigo is a colored molecule.
Figure 3) TLC of standard indigo dye and indigo extracted from bacteria
Our sample presents a single spot corresponding to the indigo molecule. We can conclude the produced molecule is indeed
indigo and that no other colored impurities are present in the sample.
At the end of the procedure, 0,14 g of dry indigo was obtained for 150 ml of bacterial culture.
Figure 4) Yield of indigo
Step 2: Dyeing textile
Indigo is a challenging pigment because it is not soluble in water. To be dissolved, it must undergo a reduction which converts indigo to "white indigo" (leuco-indigo).
Figure 5) Oxidation-reduction reaction of indigo
In the current industrial process, sodium dithionite is the preferred reducing agent because of its low cost and short reduction time. However, sodium dithionite decomposes to form sulfate and sulfite, which can corrode equipment and pipes in dye mills and wastewater treatment facilities (2). This is why we decided to use a more sustainable reducing agent - sugar. Glucose was successfully employed as a reducing sugar in an indigo alkaline reduction (4). An aldehyde group of glucose can be oxidized to carboxylic acid as shown in Figure 6.
Figure 6) Oxidation of glucose in alkaline solution
The dyebath conditions at high temperatures are stable thanks to the combination of glucose and NaOH solution. The redox potential in the dyebath is sufficiently negative to keep indigo in its reduced form (5). Glucose is eco-friendly, non-toxic, biodegradable and inexpensive (6).
For the dyeing process we used the method that was described by Saikhao et al., 2018. The liquor ratio was 50:1.
The cotton fabric of 0,58 g was dyed with 5 g/L indigo, 12 g/L NaOH (8)
and 10 g/L glucose in 28 ml of water. The reduction temperature was set to 70°C for 10 min. After that the dyebath was cooled down to 30°C and the fabric was dipped into the dyebath for 30 seconds after which the fabric was oxidized in the air for 2 min and washed in distilled water until the water was neutral. Finally, the textile was left to dry.
Figure 7) Dyeing with indigo produced by bacteria
Results
We obtained a deep blue stained fabric which is stable and resistant to washing. (Fig 8)
The process of indigo extraction from bacteria demands the use of a large amount of chloroform. To color this fabric
we used 150 ml of chloroform. On average, a pair of blue jeans requires 3 grams to 12 grams of indigo dye. It means, we
would use more that 10 000 L of chloroform for one pair only. This is why our next challenge was to escape the purification
process and dye directly with bacterial culture.
Following the same previously described protocol but without indigo purification, a light blue fabric was obtained.
Figure 8) Dyeing with indigo produced by bacteria without purification
Despite the use of the same amount of indigo, the difference in color can clearly be seen. We suggest that such a difference is due to the absence of pigment purification.
Figure 9) A white cotton textile(a), dyed with indigo produced with bacteria without purification (b), dyed with indigo produced with bacteria with purification (c)
However, glucose and NaOH are still used for the reduction of indigo. To dye fabric with no chemicals we decided to use another approach. The pathway that is responsible for indigo production in bacteria is the following:
Figure 10) Metabolic pathway of indoxyl production
So, bacteria produce indoxyl which is oxidized to indigo in the air. This is why the oxygen concentration during the incubation should be high to increase the yield. We decided to put the fabric directly in the culture in order to initiate the indigo oxidation on the surface of the fabric, in which case there is no need for reducing indigo to its leuco-form. (3).
Figure 11) Metabolic pathway of indoxyl production
Bacteria expressing TMM were grown overnight with cotton thread and supply of Tryptophan.
The change from white to purple can clearly be observed. The purple color of the thread is stable and resistant to washing.
Figure 12) A white cotton thread (a), dyed with indigo produced with bacteria without purification and reduction (b)
Conclusion
As a result, we managed to dye the textile with our new method with indigo without using any additional chemicals for extraction or reduction. The fabric is dyed only thanks to the indigo production by the bacteria and the constant oxidation of the culture. Using minicells expressing TMM in this process instead of bacteria is the next step of our research. We suggest that it will assure biosafety and adapt this dyeing process to local no GMO use.
See more about the future of our project on the Proposed Implementation page.
REFERENCES
(1) iGEM Parts Registry. Part:BBa_K1403015: Trimethylamine moonoxygenase (tmm) expression cassette. URL: http://parts.igem.org/Part:BBa_K1403015
(2) Liu, C., Xu, J., Gao, S.-Q., He, B., Wei, C.-W., Wang, X.-J., Wang, Z., & Lin, Y.-W. (2018). Green and efficient biosynthesis of indigo from indole by engineered myoglobins. In RSC Advances (Vol. 8, Issue 58, pp. 33325–33330). Royal Society of Chemistry (RSC). https://doi.org/10.1039/c8ra07825d
(3) Hill et al. (1989) Hill, R., Hart, S., Illing. N., Kirby. R., Woods, D.R. (1989) Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli. Journal of General Microbiology 135, 1507 1513.
(4) Shin, Y., Son, K., & Yoo, D. I. (2016). Indigo dyeing onto ramie fabric via microbial reduction: Reducing power evaluation of some bacterial strains isolated from fermented indigo vat. In Fibers and Polymers (Vol. 17, Issue 7, pp. 1000–1006). Springer Science and Business Media LLC. https://doi.org/10.1007/s12221-016-6353-7
(5) Saikhao, L., Setthayanond, J., Karpkird, T., & Suwanruji, P. (2017). Comparison of sodium dithionite and glucose as a reducing agent for natural indigo dyeing on cotton fabrics. In H. F. Abdul Amir & P. S. Khiew (Eds.), MATEC Web of Conferences (Vol. 108, p. 03001). EDP Sciences. https://doi.org/10.1051/matecconf/201710803001
(6) Blackburn, R. S., & Harvey, A. (2004). Green Chemistry Methods in Sulfur Dyeing: Application of Various Reducing d-Sugars and Analysis of the Importance of Optimum Redox Potential. In Environmental Science & Technology (Vol. 38, Issue 14, pp. 4034–4039). American Chemical Society (ACS). https://doi.org/10.1021/es0498484
(7)Saikhao, L., Setthayanond, J., Karpkird, T., Bechtold, T., & Suwanruji, P. (2018). Green reducing agents for indigo dyeing on cotton fabrics. Journal of Cleaner Production, 197, 106–113. https://doi.org/10.1016/j.jclepro.2018.06.199
(8) Hossain, Md. D., Khan, Md. M. R., & Uddin, Md. Z. (2016). Fastness Properties and Color Analysis of Natural Indigo Dye and Compatibility Study of Different Natural Reducing Agents. In Journal of Polymers and the Environment (Vol. 25, Issue 4, pp. 1219–1230). Springer Science and Business Media LLC. https://doi.org/10.1007/s10924-016-0900-6