Dyeing for change

Around 700,000t of dyes are produced annually, over 90% of which are synthetics derived from petrochemicals. The remainder come from insects and plants. Whether synthetic or natural, all dyes need to be fixed to the various fabrics using chemicals – resulting in copious quantities of often hazardous waste.

‘The [dyestuffs] industry uses over 8000 different chemicals, including phenols, formaldehyde and heavy metal salts, all of which have a negative environmental impact,’ says Ore Yarkoni, CEO and founder of Colorifix, speaking at the recent Synbiobeta 2017 conference in London. The dye industry also consumes enormous amounts of water – around 6 trillion litres each year, he continues, making it the third largest consumer of water after the agriculture and energy industries.

Cambridge-based Colorifix has developed a new way of dyeing fabrics using synthetic biology that reduces the industry’s environmental impact. Yarkoni enthuses: ‘We borrow the pathways that Nature uses to make pigments, and we adapt microorganisms to produce, deposit and fix them directly onto fabrics in an environmentally and cost effective way. We do this without the need to add heavy metals, organic solvents or acids. And while ratios of water: final product are commonly 50:1, and 10:1 is considered a substantial achievement, our process uses a ratio of 3:1.’

Nature abounds with colourful pigments, from lycopene in tomatoes, through yellow in turmeric, to carotene in carrots. Yarkoni says they can produce any colour naturally found in Nature. With the latest genetic tools, like CRISPR, they target the DNA that an organism uses to encode for each chemical pathway that leads to the production of its dye molecules. They then modify their microorganism with the same DNA so that it too can produce the dye.

Scientists at Colorifix use a laboratory strain of E. coli bacteria, which has been modified so that it doesn’t produce some of its amino acids and will die outside of the laboratory. They make a few further ‘proprietary’ modifications using the tools of synthetic biology – namely DNA assembly and transformation – to introduce changes into the bacteria’s DNA to ensure it produces the pigments they want at a high enough density for dye transfer to take place efficiently.

‘These modifications make the bacteria less viable as an organism, which is an added benefit because it will be safer to use in the industrial process,’ explains Yarkoni.

The bacteria are first grown in standard culture medium at 37°C, and the resulting broth is then added to a transfer medium containing the fabric to be dyed. The transfer medium includes a different sugar source to the original growth medium, which causes the bacteria to behave differently. ‘They become much longer in length, upwards of 20µm in contrast to the normal 3.5 to 5µm. This has the advantage of increasing the contact surface area with the fabric,’ says Yarkoni.

As the bacteria grow, they concentrate the naturally occurring micronutrients, including metal salts, from the growth medium, as well as producing the dye molecules. The cells become entrenched within the fabric, which is then removed and sterilised using heat. The cells burst open, releasing their complex chemical brew directly onto the fabric. ‘To this day nobody understands fully the single cell,’ Yarkoni says, ‘but what we do know is that there is a rich complex chemical microenvironment conducive to the colour transfer process on a range of different fabrics – silk, wool, cotton, polyester, nylon, rayon and Lycra all stick to the dye. And the dye is colourfast on these fabrics.’

The only fabric that has eluded the team is polyurethane, probably because it is very hydrophobic and the tight-knit sheets don’t allow the bacteria to become entrenched as with the other fabrics.
There is another environmental advantage of the process. Over 200m t/year of fabric end up in landfill, leaching dyes and other chemicals into the environment. Since the Colorifix process doesn’t use such chemicals, Yarkoni says it should have a much-reduced environmental impact.

Sweet success
Some US food giants, meanwhile, have already come out against the use of synthetic dyes and are committed to finding natural alternatives. In February 2016, for example, confectionery major Mars announced that by 2021 it would remove all artificial colours from its processed foods for human consumption and replace them with pigments from natural substances. The industry is driven by consumers’ growing health concerns associated with artificial colours.

But while many colours have well-established natural-dye alternatives to pursue, blue is a rarity among plants and insects – even a blueberry is more red than blue when squashed. Currently, the preferred natural blue dye for the food industry is derived from the cyanobacteria Spirulina. The microorganism produces the blue C-phycocyanin pigment-protein (C-PC), which is relatively stable and water soluble making it a good candidate for food products.

Japanese chemical company DIC currently produces 90% of the world’s supply of the Spirulina-based pigment, granted US Food and Drug Administration (FDA) approval back in 2013. However, the market for the natural blue dye in the food industry alone is expected to increase 10-fold to around £350m in the next two years as the industry moves to replace the synthetic Blue No1. Apart from blue M&Ms and confectionery generally, the pigment also has other uses – as a fluorescent marker in clinical imaging and as a potential antioxidant in food supplements and in anticancer treatments. According to DIC, demand for natural blue dye is presently out-performing supply.

One again, however, synthetic biology may be on hand to help. Earlier in 2017, the UK Industrial Biotechnology Innovation Centre (IBioIC) awarded £200,000 to a collaborative project between biotech company Scottish BioEnergy and scientists at the University of Edinburgh to develop a large-scale process to extract C-PC from Spirulina. The collaboration is expected to identify and optimise techniques for extracting the pigment-protein and develop economically feasible methods for producing large volumes of C-PC. They also aim to engineer strains of cyanobacteria to produce high yield and high purity C-PC.

Alistair McCormick, whose research at the university focuses on photosynthesis in plants and cyanobacteria, and who is taking part in the project, comments: ‘The C-PC is very good at capturing light, which is great for the growing cyanobacteria in Nature, but presents a problem in a bioreactor because the cyanobacterial cells in the middle are shaded from the light by the cells further out, and this inhibits the yield of the final dye product. We are trying to genetically manipulate the organism to produce the dye under selected conditions of, for example, light, temperature and the use of dynamic sensing and regulating molecules to develop robust culture strains and get the maximum yield.’

This ‘gene circuitry’ approach to modulate gene expression is an exciting new research area, he adds – allowing researchers to coordinate production outputs with multiple environmental inputs.
There are several ways of bursting the cells open after the bacterial growth phase and isolating the dye. Vibration, osmotic shock, freezing and thawing, and chemical methods are commonly used. The Edinburgh scientists will be looking to open the cells when yields are at their highest. They will also be investigating the best and most economical ways to purify the compound at scale. ‘There is a lot of research to be done,’ explains McCormick, ‘unlike E. coli, which has a long history of genetic manipulation, cyanobacteria are relatively new kids on the block and many of the tools needed for manipulating these bacteria still need to be developed. But the potential advantage of having access to myriad pigments in the future make it well worth the challenge.’
Ultimately the team, working with Scottish Bioenergy, aims to develop a new commercial model organism to produce C-PC.