MEDIEVAL BLUE GENES: Reducing Industrial Indigo Dye Pollution of the Environment
Lead Research Organisation:
University of Nottingham
Department Name: School of Life Sciences
Abstract
Indigo is the oldest source of blue dye in the world. In use since 6000 BC, prior to the 20th century, it was sustainably isolated from the leaves of plants. It is now almost entirely derived from fossil oil and is one of the world's most important synthetic chemicals. Thus, >50,000 tons are annually synthesised. Of this, 95% is used to dye the over 4 billion denim garments that are produced every year. It is the mainstay of the denim industry.
Unfortunately, synthetic indigo creates substantial environmental and sustainability issues during both its manufacture and its exploitation as a dye. Its synthesis is reliant on the toxic petrochemical benzene and involves many other hazardous chemicals. During the dyeing process itself, the insoluble blue indigo is reduced by an excess of sodium dithionite - 2 kg for every 3 kg of indigo. The sulfate and sulfite by-products generated are corrosive and problematic to remove from wastewater. This has led to the dumping of spent dye materials into rivers by unscrupulous dye mill owners and alarming ecological impacts, e.g., rivers of blue or black in China and blue dogs in India.
In medieval times, cloth was dyed in a vat in which indigo was provided by adding leaves of woad (Isatis tinctoria) and a natural fermentation process brought about its biological reduction. A return to the natural plant-based process would have real environmental benefits particularly if its effectiveness and reproducibility were improved. Rational changes are, however, not possible because the mechanism by which the bacteria reduce indigo is unknown. Scientific recreation of a woad vat from a medieval recipe has enabled the identification of the bacterium responsible as Clostridium isatidis. To improve its performance it is important to understand how it reduces indigo. The most effective strategy is to use gene tools to isolate mutant bacteria that no longer reduce indigo, and then use DNA sequencing to identify which gene/protein is affected. However, although gene tools are available, until now no means of transferring them into C. isatidis had been reported.
We have now demonstrated, for the first time, DNA transfer into this bacterium. It is, therefore, now possible to generate mutants. In parallel, we have determined the entire sequence blueprint of the bacterium's chromosome, a prerequisite for identifying the nature of any mutants made. These will be made by randomly inserting a small piece of DNA, called a transposon, into the chromosome, disrupting the gene into which it has inserted. If that gene, and the protein it encodes, is involved in the indigo reduction process, then bacterial colonies will no longer be able to solubilise indigo and turn blue. By screening thousands of random mutants, all of the genes/ proteins involved in indigo reduction will be identified.
The factors involved will be rationally altered to improve the productivity of the organism in dyeing process. Until these factors are identified the nature of the changes to be made cannot be stated. Aside from their modification, a likely strategy will be to bring about their overproduction. Alternatively, factors that may be interfering with the identified process could be inactivated, through mutation. The performance of rationally modified strains in indigo reduction will be compared to the parental strain in dyeing trials of cotton and wool yarn samples that essentially recreate a medieval vat in the laboratory. Colour fastness of dyed samples will also be assessed for wash, light and rubbing fastness. To ensure success we will directly engage with industrial dyers and colourists and develop a greater understanding of the dyeing processes and industrial constraints and needs. The longer-term output of the project will be the development of an economic and sustainable process for a biological denim dyeing.
Unfortunately, synthetic indigo creates substantial environmental and sustainability issues during both its manufacture and its exploitation as a dye. Its synthesis is reliant on the toxic petrochemical benzene and involves many other hazardous chemicals. During the dyeing process itself, the insoluble blue indigo is reduced by an excess of sodium dithionite - 2 kg for every 3 kg of indigo. The sulfate and sulfite by-products generated are corrosive and problematic to remove from wastewater. This has led to the dumping of spent dye materials into rivers by unscrupulous dye mill owners and alarming ecological impacts, e.g., rivers of blue or black in China and blue dogs in India.
In medieval times, cloth was dyed in a vat in which indigo was provided by adding leaves of woad (Isatis tinctoria) and a natural fermentation process brought about its biological reduction. A return to the natural plant-based process would have real environmental benefits particularly if its effectiveness and reproducibility were improved. Rational changes are, however, not possible because the mechanism by which the bacteria reduce indigo is unknown. Scientific recreation of a woad vat from a medieval recipe has enabled the identification of the bacterium responsible as Clostridium isatidis. To improve its performance it is important to understand how it reduces indigo. The most effective strategy is to use gene tools to isolate mutant bacteria that no longer reduce indigo, and then use DNA sequencing to identify which gene/protein is affected. However, although gene tools are available, until now no means of transferring them into C. isatidis had been reported.
We have now demonstrated, for the first time, DNA transfer into this bacterium. It is, therefore, now possible to generate mutants. In parallel, we have determined the entire sequence blueprint of the bacterium's chromosome, a prerequisite for identifying the nature of any mutants made. These will be made by randomly inserting a small piece of DNA, called a transposon, into the chromosome, disrupting the gene into which it has inserted. If that gene, and the protein it encodes, is involved in the indigo reduction process, then bacterial colonies will no longer be able to solubilise indigo and turn blue. By screening thousands of random mutants, all of the genes/ proteins involved in indigo reduction will be identified.
The factors involved will be rationally altered to improve the productivity of the organism in dyeing process. Until these factors are identified the nature of the changes to be made cannot be stated. Aside from their modification, a likely strategy will be to bring about their overproduction. Alternatively, factors that may be interfering with the identified process could be inactivated, through mutation. The performance of rationally modified strains in indigo reduction will be compared to the parental strain in dyeing trials of cotton and wool yarn samples that essentially recreate a medieval vat in the laboratory. Colour fastness of dyed samples will also be assessed for wash, light and rubbing fastness. To ensure success we will directly engage with industrial dyers and colourists and develop a greater understanding of the dyeing processes and industrial constraints and needs. The longer-term output of the project will be the development of an economic and sustainable process for a biological denim dyeing.
Technical Summary
Objectives progressed by 4 WPs:-
[WP1] Establish technologies for generation of random (mariner Tn) & directed (RiboCas) mutants.
1. IMPLEMENT RIBOCAS: Most suitable clostridial promoter identified and used to inactivate C. isatidis catA & pyrE genes.
2. EXEMPLIFY MARINER: Suicide delivery of transposon under the control of inducible promoter (aTc) in catA mutant (above). Inverse PCR to quantify transposition, Sanger sequencing to identify sites of insertion.
[WP2] Forward & reverse genetic approaches to identify the genes/proteins involved in indigo reduction.
2.2. FORWARD GENETICS: Indigo-containing plates used to screen Tn insertions for defects in indigo reduction. Insertions sites identified by Sanger sequencing. Mutant defects confirmed by RiboCas deletion & bookmark complementation.
2.3. REVERSE GENETICS: Genes encoding enzymes predicted to be involved in indigo reduction deleted using RiboCas & bookmark.
[WP3] Optimisation of biological indigo reduction process
3.1. FERMENTATION OF WT: Fermentation conditions for evaluating indigo reduction in laboratory bioreactors established and dyeing trials undertaken. Colour fastness of dyed samples assessed using standard ISO methods for wash, light and rubbing fastness [ISO 105-C06:2010, ISO 105-B02:2013 & ISO 105-X12:2016].
3.2. FERMENTATION OF MODIFIED STRAINS
Rationally modified strains created in which genes involved in indigo reduction are overexpressed, replaced with rationally modified variants or in which genes that may be interfering with the identified process are deleted. Their performance in indigo reduction and the dyeing process compared to the parental strain.
[WP4] Ensuring the technology is fit for purpose
4.1. KE WORKSHOPS: Industrial dyers and colourists invited to KE workshops
4.2. ENGAGE WITH INDUSTRIAL DYERS AND COLOURISTS: Develop a greater understanding of the dyeing processes and industrial constraints and needs.
[WP1] Establish technologies for generation of random (mariner Tn) & directed (RiboCas) mutants.
1. IMPLEMENT RIBOCAS: Most suitable clostridial promoter identified and used to inactivate C. isatidis catA & pyrE genes.
2. EXEMPLIFY MARINER: Suicide delivery of transposon under the control of inducible promoter (aTc) in catA mutant (above). Inverse PCR to quantify transposition, Sanger sequencing to identify sites of insertion.
[WP2] Forward & reverse genetic approaches to identify the genes/proteins involved in indigo reduction.
2.2. FORWARD GENETICS: Indigo-containing plates used to screen Tn insertions for defects in indigo reduction. Insertions sites identified by Sanger sequencing. Mutant defects confirmed by RiboCas deletion & bookmark complementation.
2.3. REVERSE GENETICS: Genes encoding enzymes predicted to be involved in indigo reduction deleted using RiboCas & bookmark.
[WP3] Optimisation of biological indigo reduction process
3.1. FERMENTATION OF WT: Fermentation conditions for evaluating indigo reduction in laboratory bioreactors established and dyeing trials undertaken. Colour fastness of dyed samples assessed using standard ISO methods for wash, light and rubbing fastness [ISO 105-C06:2010, ISO 105-B02:2013 & ISO 105-X12:2016].
3.2. FERMENTATION OF MODIFIED STRAINS
Rationally modified strains created in which genes involved in indigo reduction are overexpressed, replaced with rationally modified variants or in which genes that may be interfering with the identified process are deleted. Their performance in indigo reduction and the dyeing process compared to the parental strain.
[WP4] Ensuring the technology is fit for purpose
4.1. KE WORKSHOPS: Industrial dyers and colourists invited to KE workshops
4.2. ENGAGE WITH INDUSTRIAL DYERS AND COLOURISTS: Develop a greater understanding of the dyeing processes and industrial constraints and needs.
