Difference between revisions of "Team:Paris Bettencourt/Project/Indigo"

Making patterns on denim

Abstract

The main focus of our project this year was enzyme design and discovery. We chose to engineer enzymes because they are a practical technology for removing stains, already known to be safe and economic. However, in our search for microbes that produce stain-removing enzymes we discovered something else: microbes are beautiful. When nutrients are added to fabric samples, bacterial and fungal colonies bloom in every size, shape and color. These microbes grow in beautiful patterns, remove pigments from fabric and leave other pigments behind. When the microbes were gone, the color and texture of the fabric was changed in ways that looked cool to us. What we describe below is not necessarily practical and only somewhat scientific. We are playing around with microbes on ordinary denim blue jeans and trying to create beauty.

Motivation and Background

Blue Jeans: Structure and Function

Jeans are trousers made with denim, a cotton textile. Blue jeans are dyed with indigo, a plant-derived pigment that is now mostly produced synthetically. In a classic blue denim weave, the warp thread is dyed with indigo while the weft threat is left white. The resulting fabric is blue on one surface, white on the other, with a grainy texture where the fibers cross. The blue threads are stained only on the outside and stay white at the core. As a result, the fabric lightens with wear, producing a characteristic fading pattern (Figure 1).

Denim Washes

The popular appeal of faded denim has lead to the development of many industrial processes that simulate or accelerate the fading process. New denim may be chemically treated, sandblasted, rubbed with sandpaper, or tumbled with abrasive stones prior to sale. These "wash" processes soften the fabric, fade the indigo, and produce a distinctive pattern and appearance. Especially with the trend toward high fashion and premium jeans, designers are motivated to discover new and innovative wash processes. The global jeans market was predicted to be 56 billion USD in 2014 (Agarwal, 2009).

Enzymatic Biowashing

In recent years, enzymes have become a popular tool for producing denim washes. Treatment with cellulases can degrade the external cellulose fibers under mild conditions (Montazer, 2008). As a result, it requires less energy, produces fewer chemical by-products, and causes less damage than chemical or abrasive washes.
Enzymatic treatment with laccase enzymes can be used to fade denim (Campos, 2001). Laccases (EC 1.10.3.2) are copper-containing oxidases that act on a variety of substrates. Acting on indigo, laccase abstracts one electron from each aromatic amine, leaving behind an unstable radical that quickly degrades.
Whole live cells with laccase activity have been used as bioremediation agents, in particular for indigo-containing wastewater from textile factories (Conceição, 2013). This activity was also the inspiration for the Bielefeld 2012 iGEM team. Thus, as we began this project, we knew that live cells and pure enzymes could act on the key components of blue jeans.

Results

We cultured microorganisms on denim covered with minimal M9, isolated the strains and identified them. Then, we tested their capacity at indigo’s degradation. At the end we have three microorganisms that degrade indigo: 2 Streptomyces and Pantoea.

Isolation of Microbes on Denim Media

Quantification of Indigo Degradation

Microbe Wash Denim

Methods

Preparation of Denim and Indio Media

We used denim pieces cut out of a pair of jeans. We put them in minimal M9 media without glucose (only M9 salts, CaCl2 and MgSO4, with agar). One piece was left in a bottle with the lid almost closed to avoid dehydration, the others were put in square plates with minimal M9. After a few days different colonies were taken with an inoculating loop and inoculated on LB plates for growth.

Microbial Identification with 16s rRNA Sequencing

Bacteria were identified with 16S rRNA Sequencing. For each, one colony was taken out of LB plate and used for 16S rRNA PCR (protocol can be find here ).

Fungi were identified with 18S rRNA Sequencing. For each, one colony from LB plate was inoculated on Sabouraud media and left to grow for 3 days. Genomic DNA was extracted, and it was then used for 18S rRNA PCR (protocol can be find here).

Quantification of Indigo Consumption

Indigo plates: we used minimal M9 media and added indigo dissolved in DMSO, until we could see a blue color on the plates. Then colonies from LB plates were inoculated and left to grow at 30°C.

Liquid M9 with indigo: 700µL of indigo in DMSO at 50mM was first added to 500mL Minimal liquid M9. 50mL falcon tubes were used, we put 5mL M9 with indigo in each of them, then put 20µL 50mM indigo in DMSO to have a deep blue color. Colonies from LB plates were used for inoculation. Tubes were left in a rotor at 30°C for a few days. Indigo Consumption is quantified using absorbance. Indigo strongest absorbance was determined at 680nm. Measurements were made for every strains every day for 6 days: 500µL media from previous 50mL falcon tubes were collected, vortexed, and 200µL were used in 96 well-plates. Absorbance was also measured at 450nm, for which indigo as a weak absorbance, to measure absorbance of cells and other things that can interfere with the absorbance at 680nm. Absorbance’s measurements were performed using a

Stained cotton: small pieces of cotton were cut out of a cotton fabric. They are stained with around 100µL 50mM indigo dissolved in DMSO (sometimes more indigo was needed or some parts of the cotton would have been unstained and white). They are washed at high temperatures, then cleaned with ethanol to sterilize them. These pieces are then used in minimal M9 plates and liquid culture to asses indigo consumption when on cotton.

Attributions

This project was done mostly by Mislav and Elisa. We would like to thank Jake for sacrifying his jeans for science, and XX for their collaboration.

References

• Agarwal, S. (2009). World Denim Market Production and Consuption Report 2012, Denimsandjeans.com.
• Montazer, M., & Maryan, A. S. (2008). Application of laccases with cellulases on denim for clean effluent and repeatable biowashing. Journal of Applied Polymer Science, 110(5), 3121–3129.
• Conceição, V., Freire, F. B., & Carvalho, K. Q. de. (2013). Treatment of textile effluent containing indigo blue dye by a UASB reactor coupled with pottery clay adsorption. Acta Scientiarum. Technology, 35(1), 1–6
• Campos, R., Kandelbauer, A., Robra, K. H., Cavaco-Paulo, A., & Gübitz, G. M. (2001). Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii. Journal of Biotechnology, 89(2-3), 131–139.
• Dubé, E., Shareck, F., Hurtubise, Y. et al. Appl Microbiol Biotechnol (2008) 79: 597. Homologous cloning, expression and characterization of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye.
• J Margot, C Bennati-Granier, J Maillard, P Blánquez, D.A Barry, C Holliger (2013). Bacterial versus fungal laccase : potential for micropollutant degradation. ABM Express ; 3 :63.
• Woodhead Publishing Series in Textile: Number 164; Denim, Manufacture, Finishing and Applications; edited by R. Paul