Enzyme researcher Ditte Hededam Welner uses genetic engineering to exploit E. coli bacteria in the production of indigo dye. Photo: Bax Lindhardt

Blue Jeans in a green way

Biotechnology Innovation and product development Bacteria and microorganisms Enzymes and proteins Chemistry
Researchers exploit E. coli bacteria for new, greener production of indigo to dye jeans.

By Anders Østerby Mønsted and Anne Wärme Lykke

The invention has the potential to revolutionize denim production and make it far more sustainable than it is today.

Blue jeans are among the world’s most widely used pieces of clothing. They’re produced in the billions, but staining denim with the coveted indigo dye is not particularly sustainable. 

Currently, up to 50,000 tonnes of indigo blue colour are produced every year by chemical synthesis—a production method which is environmentally hazardous, as it involves chemicals, heavy metals, acid waste—and high water consumption.

Transatlantic collaboration

Now, researchers from UC Berkeley, The Joint BioEnergy Institute (JBEI), a US Department of Energy research centre, and DTU Biosustain have developed a more sustainable approach to producing indigo using engineered bacteria. Bacteria and other microorganisms can be manipulated—for example, by adding exogenous material—to enable them to express a desired molecule. The researchers hope the method could help the clothing industry to pollute less:

“We hope this biological method can help speed up the green transformation and complement chemical production,” says Ditte Hededam Welner, a researcher at DTU Biosustain.

She was working at JBEI when she and her collaboration partners discovered a plant enzyme, PtUGT1, capable of overcoming one of the most difficult steps in the biosynthesis of indigo.

Inspiration from nature

Indigo is an insoluble dye produced in large blocks by chemical synthesis. This requires a ‘reducing chemical’ to make the indigo water-soluble so that it can be used for staining.

In order to produce indigo in bacteria, the researchers investigated how they could mimic nature’s way of producing the blue colour. Indigo plant leaves are green, but form blue pigment if damaged. First, the leaves produce an unstable precursor molecule, indoxyl, which, if not stabilized, quickly oxidizes into insoluble indigo pigment.

The researchers found it relatively straightforward to make E. coli bacteria produce indoxyl and thus indigo, as this method was already described in the literature. However, the indigo would still be insoluble and would require chemical processing. This did not, therefore, solve the problem of using harsh chemicals. The next step in the plant’s indigo production gave the researchers an idea for stabilizing indoxyl, however. Instead of converting indoxyl directly into indigo pigment, the plant cell takes a detour and converts unstable indoxyl into the colourless—but stable—substance, indican.

In the Japanese indigo plant, researchers found the enzyme that could convert indoxyl into indican. The enzyme—glucosyltransferase 1 (PtUGT1)—can stabilize indoxyl by adding it to a sugar molecule to create indican.

The researchers then added the PtUGT1 gene to E. coli bacteria, producing indican.

The researchers subsequently tested the method on small pieces of fabric and a scarf. The white fabric was first sprayed with biologically produced indican from the bacteria. Subsequently, the fabric was soaked in a well-known enzyme that can convert indican back to indoxyl. Finally, the fabric was hung out to dry to oxidize the indoxyl into blue indigo directly on the garment. And voilà—the blue colour appeared!

Photo: Bax Lindhardt
Chemicals, acid waste, and heavy metals used in staining denim today are a burden on the environment. Photo: Bax Lindhardt

A new way to reach the same end

The question is whether a pair of blue Levi’s jeans will look the same whether they are manufactured by the new or the conventional method. And they will, according to Ditte Hededam Welner.

“If they don’t look exactly the same, people won’t buy the product. In our studies, we dyed different cotton products, and there is virtually no difference because the dye is exactly the same,” she says.

The new method for the manufacture of indigo—which has not yet been tested on a large scale—currently yields about between four and seven grams per litre of ‘bacterial soup’. In order to warrant industrial application, the researchers will have to produce at least 100 grams of dye per litre.

Partners standing by

Research into sustainable indigo production is supported by the Levi’s company, which is focusing on making clothes production greener and more sustainable. And as more and more big names rebrand as sustainable, sustainable indigo production will gain ground, according to Richard Jones, Professor of Brand Management and Brand Governance at Copenhagen Business School

“Generally speaking, name brand companies are keen to communicate that they are not solely interested in profit, but equally keen to be part of a value chain that helps to address societal problems. Being able to tell a story about being sustainable and eco-friendly has a very strong symbolic value. So, many brands will find it interesting to be frontrunners in a large market like this,” Richard Jones says.

Photo: Bax Lindhardt

What is indigo?

More than 7,800 years ago, a tribe in Peru found that they could dye cotton blue with pigment from the leaves of the indigo plant.

About 6,000 years ago, people in India began extracting the dark blue dye on a large scale. Soon after, people all over the world began asking for the precious dye. Demand was so high in the 19th century that it was impossible to grow sufficient indigo plants.

In the 1870s, a physicist, chemist, and amateur mathematician—Johann Friedrich Wilhelm Adolf von Baeyer—discovered how to produce indigo using chemical synthesis. In 1905, he received the Nobel Prize in Chemistry for advancing organic chemistry and the chemical industry through his work with dyes and hydroaromatic substances.