How do we avoid harsh chemicals when coloring denim?

A quick glance around the classroom reveals that denim jeans remain a popular choice among teenagers.

– Quite a few of you are wearing jeans today, Rasmus Ree, a microbiology biotechnology researcher from NORCE, notes.

– It is very likely they are colored with indigo, he adds.

Rasmus and fellow researcher Antonio García-Moyano are the main characters in today’s ‘meet a scientist’-event at the International School of Bergen, while indigo enzymes are the main topic.

Indigo has originally been isolated from plants growing in Africa and India, Rasmus explains, and for centuries indigo dye has been used to color textiles. Nowadays it is synthetically produced using harsh chemicals.

– Obviously, this is not very environmentally friendly. Alternatively, we could be using enzymes, small molecular machines that can achieve this in a more eco-friendly way. So today we are doing an experiment trying to make our own indigo, Rasmus tells his captive audience.

A future insight

The experiment is a mini-scale version of an idea arising from the Horizon Europe project OXIPRO. Both Rasmus and Antonio are involved in the project, in which 15 partners from 11 countries are collaborating to develop new enzymes – specifically oxidoreductases – for environmentally-friendly consumer products.

Oxidoreductases are a type of enzymes that can replace fossil-based chemicals. Today, fossil-based chemicals are used for producing everyday products like laundry detergent, cosmetics, nutritional supplements, and, not least, textiles. These fossil-based chemicals generate toxic pollution in wastewater and can harm the health of textile workers. Furthermore, chemicals contribute to a high percentage of climate emissions, and with an increasing textile production, it is crucial to find a greener way to colour denim. Other than being more environmentally-friendly than fossil-based chemicals, enzymes also add functionality to products, transform waste into value, and simultaneously enhance the quality of the product.

Today, the case in point is indigo. It is mainly used to colour denim, and the denim industry uses 50,000 tons of synthetic indigo annually. It is produced using harsh chemicals, and so it could be environmentally beneficial to produce it using enzymes and microbes instead, because this would require less chemical use. Biotechnologists have been trying for years to produce indigo using enzymes and microbes in an economically sustainable way.

The enzyme Rasmus brought today is produced by the researchers in the OXIPRO project. Enzymatic indigo production is not a major focus of OXIPRO – a side-reaction, really – but it allows us to visualize how an enzyme works because it produces an easily seen colour change. Now, let’s put on lab coats and see how it works.

Time to experiment

– Did you change the measurements, a girl asks a fellow student holding a micropipette?

– Yeah, is the short reply from the male student.

– Do you remember what it was? It is important that we use the right measurements.

The girl is right.

– We have two micropipettes that are different in size since they measure different volume. You want the exact volume when we are making the experiment, Antonio underlines to one of the four groups.

– First thing we do is label the tubes from 1 to 6, Rasmus tells another group.

Actually, the students are doing six small experiments within the experiment itself, to see how important it is to add the components in the correct order and amount.

Now they are getting ready to add the enzyme, cofactor, reaction buffer and lastly indole.

– It is very important that the indole is added as the last component, Rasmus explains and adds:

– Before you add the last component, that is when you start the reaction.

The enzyme the students use is called ‘mFMO’ (Methylophaga Flavin-containing monooxygenase) and has been developed by researchers in the OXIPRO project. An enzyme is a biological catalyst and is almost always a protein. It speeds up the rate of a specific chemical reaction in a biological process. It needs a cofactor called NADPH, which provides energy for the reaction. It also needs a substrate – indole. If the reaction works, it turns blue.

Timing is important

– Do you have everything, Rasmus asks a group of students?

– Yeah, we started with the enzyme and then added the cofactor, then the buffer and finally the indole, one of them replies.

Another student carefully explains how to handle a used micropipette to another student.

– You take it up, push it all the way down, be careful, and then you drop it in the glass.

– Remember to note the time of every single of them, Rasmus says.

– What are we supposed to time again, one of the male students asks?

– The indole, when you add it. Remember it.

This is because chemical reactions like this occur gradually over time and not instantaneously. The longer the time, the stronger the change in color will appear. Knowing how long such a reaction has gone on for is important for interpreting the results.

– Perfect timing, a student says to a fellow student.

– Remember to measure the full amount before you finish. It should be close to 200 microliter, Antonio says before Rasmus wraps it up ahead of the lunch break.

– Shake the tube a little bit so the liquid collects at the bottom, and then we should leave them in the dark, he says and points to a cardboard box where the test tubes will be kept during the 45 minutes lunch break.

Did it work?

– This one is actually quite blue. This one not so much, a student holding two tubes up against each other says.

– We got three that are blue, a student from another group says.

Rasmus hasn’t started talking yet before the students share their results from the experiment.

– No experiment is complete without analyzing it. What happened to the tubes? Did you see any color, he then asks?

– Yes, the groups are sporadically replying.

– Tube number one turned blue, two and three nothing, same with four, but five and six also turned blue, a student sums up.

– Why is that, Rasmus asks?

– If you’re missing one of the components, it doesn’t really work, a student replies.

The results correspond to the tests where number one, five and six contained all the components, while two, three and four missed one of the four components.

– If we want a stronger blue color, what should we have done, Rasmus asks?

– Adding more cofactor and less of the other components, one student says.

– Leave them even longer in the dark, another says.

– Correct.

Researchers from around the world have been working on indigo-producing bacteria for some years now. However, the production is still on a small-scale and not economically profitable. But this morning at least, a small group of students from the International School of Bergen got to try out a different way of producing an important commodity chemical. If the biotechnology revolution delivers on its promise of replacing polluting chemical processes with more environmentally friendly, enzyme-catalyzed ones, the students may have gotten a small glimpse of the future.