Your favorite pair of jeans began as strings of cotton yarn, bobbing and weaving through scalding hot water and vats of indigo dye. The cotton runs through the dyeing cycle nearly a dozen times; the cotton fibers first take a yellow, then green, and finally a rich, blue color.This process has remained pretty much the same for over a hundred years. But it’s about to change.Producing indigo relies on toxic chemicals and reducing agents. Tinctorium Bio, currently in the eighth Indie Bio Accelerator class, have created a method to produce indigo by leveraging synthetic biology – not chemicals.“In the industry, the widely accepted standard is to use indigo powder to dye denim, which is heavily petroleum based…On top of that, you also need other toxic chemicals, like formaldehyde and cyanide, to make this indigo powder,” says Michelle Zhu, co-founder and CEO of Tinctorium.

Cotton fibers running through the denim dyeing process. The indigo oxidizes on the cotton, turning into a deep blue after repetitive cycles. Image courtesy of Tinctorium, Inc.Historically, indigo was harvested in India from specific plants, which store the chemical in their leaves. A recent study from the Joint BioEnergy Institute at UC Berkeley also demonstrated that indigoidine, another blue pigment, can be produced from engineered fungi. But the production of indigo and its derivatives via biology only addresses part of the issue. The toxic chemical compounds that make indigo water soluble are still required regardless of where the indigo comes from.“Part of the problem currently is that indigo crystallizes really quickly and it’s not water-soluble, so you have to apply equal parts of a water-polluting chemical reducing agent to actually use indigo and apply it as a dye,” says Zhu. “Our solution basically tackles both pieces of the problem and provides this holistic solution where we are biosynthesizing the dye precursor and, since we’re controlling the pathway to form the indigo, we bypass the need for any reducing agents.”Specifically, Tinctorium uses E. coli to produce indican, the chemical precursor to indigo, in large bioreactors (a process first reported in Nature Chemical Biology). To make the indican water-soluble, an enzyme called β-glucosidase is added, which produces leucoindigo, the actual chemical used to dye cotton yarn. The traditional process relies on sodium dithionite, a cheap but toxic reducing agent, to convert the indigo extracted from plants into leucoindigo.Tinctorium’s process, though different from the canonical approach, provides a completely equivalent result. Jeans look the same, but their dyeing requires far less chemicals. The leucoindigo that Tinctorium produces can even be dropped into existing dyeing facilities, without the need for new machines or equipment.But while the denim industry seems ripe for change, and a synthetic biology-based solution for indigo production seems like a natural progression, the route to form Tinctorium was completely unexpected.

A denim scarf dyed with Tinctorium’s unique indigo process. Image courtesy of Tinctorium, Inc.“There was this project started by an iGEM group, back in 2013, out of John Dueber’s lab. The idea was to use the formation of indigo to track certain processes in the cell. When indigo is formed, it is visible to the naked eye, which is useful when you’re screening a library or something like that. As the team looked into it, they realized it would be impactful to make indican for the denim and textile industry,” says Dr. Tammy Hsu, co-founder and CSO of Tinctorium. Hsu recently completed her PhD in John Dueber’s group at UC-Berkeley.Despite the unexpected start, the team has ambitious plans. After completing the Indie Bio program, they will raise a Seed Round to help develop a first line product launch - high-end, designer blue jeans dyed with bio-based indigo (you can even add your name to the waitlist here). To reach their goal, the team has partnered with notable industry experts like David Breslauer, the CSO and co-founder of Bolt Threads, and the internationally-renowned denim designer, Adriano Goldschmied.While Tinctorium has opted to provide solutions to specific problems – namely, indigo production and denim dyeing – other companies have been inspired by the brilliant colors found in nature, aiming to recreate them in the laboratory, offer a broader palette of dyes, and reduce waste in the process.

Hijacking Nature’s Color Palette to Produce Sustainable Dyes

Peer into the ocean’s reefs and you may see the dazzling orange of a Parrot fish and the deep blue of a Regal Tang. On land, a vast army of insects offer a similar retinue of colors, with glassy shades of blue and green. Could these colors be replicated in the laboratory, or even replace a portion of the 2 million tons of dyes produced annually?PILI, impressed by nature’s color palette, aims to do precisely that. The core of their technology leverages biological enzymes to convert carbon (from renewable sources) into molecules used to produce textile dyes, reducing waste and byproducts throughout the process.“The dyeing process in the fashion industry is energy and water-intensive and uses dyes that are polluting to produce and harmful for the environment,” says Guillaume Boissonnat , co-founder and Scientific Director at PILI. “Today, the production of one ton of dyes requires the use of 1000 cubic meters of water, 100 tons of heavy petroleum compounds, and 10 tons of toxic and corrosive chemicals - notwithstanding the energy required (at least 200 MJ/ton).”That’s a lot of waste.PILI circumvents the chemicals traditionally used in dye production – including heavy metals and formaldehyde – with fermentation. DNA encoding an enzyme, or a series of enzymes, is expressed in a microorganism. The cells are grown in a bioreactor and convert carbon into a specified dye or pigment, which is then extracted and purified. A range of colors can be produced by swapping out enzymes and optimizing the metabolic engineering process.The work is not simple, but the engineered cells (which PILI calls “Color Cell Factories”) produce bright colors, which can be screened with colorimetric assays to optimize titers and refine the process. The pigments produced by PILI are also compatible with existing equipment at dye houses, which means they can be deployed quickly and at scale.

A sample of colors produced with PILI’s bio-based dyes. Image courtesy of PILI.According to Marie-Sarah Adenis, co-founder and Creative Director, PILI's route to sustainable dye production began in an unusual way.“ started in 2012 in a discussion I had with a synthetic biologist, Thomas Landrain,” says Adenis. “We realized how polluting color production was, and had the idea to try to produce dyes with microbes. We tested the first proof-of-concept in a Biohacker space.”After this initial idea, Adenis says, outreach and public engagement were pivotal aspects of their initiative for bio-based dyes. The impetus to form a biotechnology company would not come until much later.“We made a kit so that people could grow their own ink at home, and we hosted lots of workshops both for adults and children in the Science Gallery in Dublin, the Opera Bastille in Paris, and at the V&A in London and places in New York. Public engagement is key to what we do,” says Adenis.“We eventually realized that we were criticizing petrochemistry, but we were not creating any serious alternatives…so we decided to build a technology that would be a true alternative to petrochemistry at the industrial scale. That’s why we started PILI, in 2015, taking this project to the entrepreneurial and industrial levels,” says Adenis.The team quickly expanded their expertise to develop their proof-of-concepts. Jérémie Blache, the CEO, brought business perspectives and Guillaume Boissonnat, the scientific director, helped the company unify green chemistry and synthetic biology to refine their production process.Today, the team boasts over a dozen metabolic engineers, designers, and other scientists. After a recent $4 million funding round, PILI aims to rapidly advance their bio-based dye production.“This funding will allow the company to strengthen its technological lead, fermentation and green chemistry processes, to produce high-performance biobased dyes and pigments and take its technology to pilot scale, as well as to validate the performance of our dyes with industry partners” says Blache.

A pigment meteor shower, made with PILI’s blue dye. Image courtesy of PILI.Blache estimates that PILI’s production will reach the kilogram scale in 2020, with initial products sold one or two years later, when production is expected to reach industrial scale.“Products made with our bio-based dyes will drastically reduce the environmental impact of textile chemistry by cutting down the use of water and energy and getting rid of toxic chemicals and fossil resources utilized in the current production of colors,” says Blache.PILI’s progress comes at a critical junction in the fashion industry. The EPA has long monitored certain waste products from the dyes and pigments industry, labeling some as hazardous based on specific thresholds. Dyes and pigments that use azo, triarylmethane, and anthraquinone dyes are particularly toxic. Water soluble dyes have a tendency to seep out of clothing and can pollute water in countries where no water treatment is available. To address this problem, sustainable, bio-based dyes are definitely useful, but the textile dyeing process itself will also need to change.Fortunately, there is a company that wants to radically alter how textiles are dyed – with biology again leading the charge.

Rethinking the Dyeing Process with Synthetic Biology

20% of the earth’s water pollution is caused by textile processing and 1,800 gallons of water are required to make a single pair of blue jeans. That is a staggering amount of fresh water required to make a single piece of clothing - but the industry is about to change.UK-based Colorifix wants to reduce water waste throughout the dyeing process by leveraging the biological capabilities of microbes.“As part of a University of Cambridge-led arsenic biosensor project, our team went to South Asia with our prototype biosensor, explained to people how it worked, and then asked people for a list of chemicals that they thought were worth monitoring,” says Dr. Orr Yarkoni, co-founder and CEO of Colorifix.“When we came back and did our homework, we discovered that most of these concerning chemicals were from the textile industry, and dyeing was the major culprit for both water use and chemical use,” says Yarkoni, who then decided to use synthetic biology to produce the dyes and reduce contaminants, rather than simply monitor them.After a few years of research with Dr. James Ajioka and Dr. David Nugent, Colorifix was spun out in 2016. Scientific progress was swift, and the team quickly looked at ways to reduce the environmental damage caused by the textile industry, beyond just the production of dyes.

Drs. Nugent (left) and Ajioka (right) in the Colorifix lab in Norwich, England. Image courtesy of Colorifix.“Normally, is done with chemicals or biologically-produced compounds, but without a biological agent. We are using the cells themselves to both produce and deposit the pigment into the fiber, so we are using biology for the entire process. Doing that is what allows us to save on water and energy and remove chemicals. We are using biology to transfer those pigments and dyes into the fiber,” explains Yarkoni.Thus far, Colorifix has developed a suite of colors, each derived from natural pigments.“Most of our pigments today are from insects, some microbiological ones, birds, and so forth. So yeah, we are looking everywhere for pigments right now. We’ve done a few pigments from underwater organisms as well,” says Yarkoni, who is quick to point out that the real benefit of Colorifix is their unique dyeing process, more so than the production of bio-based pigments.In a previous interview with Vogue Australia, Yarkoni emphasized that their dyeing process uses 10 times less water and consumes 20 percent less energy, mainly because the company uses molasses, a sugar by-product, to feed the pigment-producing microbes, and replaces fixing chemicals with biology itself. The normal textile dyeing process often happens at very high temperatures – well over 100 degrees Celsius – which consumes a great deal of energy. Colorifix’s microbes can dye at 37 degrees and then be heat-inactivated at well under 100 degrees, which saves energy. But the company does not plan to stop there.

A sample of colored textiles from Colorifix’s bio-based dyeing process. Image courtesy of Colorifix.“With regards to water, we’re excited to say that we’ve now successfully piloted both fermentation and dyeing using exclusively saltwater, so we now don’t need to touch fresh water in the process,” announced Yarkoni.That’s a huge leap for the textile industry and could save thousands of gallons of freshwater throughout the dyeing process. The best part is that, despite these energy and water savings, the process for dyeing a textile remains largely unchanged.“Our dye liquor essentially goes into their existing dye machine – they are all compatible - so all they need to do is change the dye liquor going into their machine,” says Yarkoni, referring to the aqueous solution of chemicals used to stain a garment. “The only difference is that can get rid of all of their other chemicals, since the organism is fixing the dye and staining the fabric without those chemicals.”From denim to dyes and everything in between, synthetic biology companies are answering the calls from a wasteful industry. Companies like Tinctorium, PILI and Colorifix are finding alternative ways to produce the same vibrant colors that appeal to consumers without the added toxicity, chemicals, and pollution.We are entering a pivotal moment in the fashion industry, where bio-based, sustainable alternatives will be the new vogue.