Microbial bioprocesses and synthetic biology for fostering circularity in the textile industry

The textile industry is a key player in our current economy, being responsible for the production of the almost ubiquitous fabrics surrounding us. Regrettably, this sector is currently guided by an almost completely linear take-make-use-dispose paradigm, with huge environmental and social implications: large amounts of resources are used to produce clothes that after a short time are destined to be incinerated or landfilled, dispersed, downcycled and only in a minor fraction (1%) recycled back into new clothing. A transition to a circular economy paradigm is essential to fully adopt the EU Strategy for Sustainable and Circular Textiles. Unfortunately, the textiles recycling technologies currently available are limited not only in the quantity of the treated material, but also in the possibility of obtaining products with comparable value, quality or resources investment of the virgin counterpart. In this thesis, which was carried out in collaboration with the textile company Albini Group, I propose and provide examples of a strategy based on the biomanufacturing concept to help in closing the loop between waste and resources in the textile industry. The first chapter describes the proof of concept of using textile waste as a feedstock to produce molecules of interest by microbial fermentation. First, we developed a protocol for the hydrolysis of cotton waste, by coupling mechanical and chemical (with NaOH) pretreatment before the enzymatic hydrolysis. For the chemical pretreatment we successfully exploited a second residue of the textile industry, alkaline mercerising wastewater. After having fine-tuned the enzymatic hydrolysis in terms of enzyme loading and catalytic parameters, we used the glucose-enriched hydrolysate in a fermentation with a Saccharomyces cerevisiae strain engineered to produce L-lactic acid. We show that it is possible to obtain titre and yield comparable to those obtained with medium formulated with pure substrates. In the following chapters we explore the use of the same strategy, but engineering yeasts for the production of natural dyes as an example of higher added-value bioproducts of interest for the textile industry. Firstly, we explored the possibility of producing hydroxyanthraquinones (HAQ) in Saccharomyces cerevisiae. In Chapters 3 and 4 we present the development of novel synthetic biology tools as simpler and faster alternatives to those already existing – the pCEC-red and the Easy-MISE toolkit – for CRISPR-Cas9 editing heterologous pathway expression. These tools were then applied in Chapter 5 to the construction of strains engineered for the biosynthesis of the HAQ precursor emodin, which was hindered by challenges in the expression of a key enzyme of the pathway. To facilitate solving this issue, the design-build-test-learn cycle led to the development of an expansion of the Easy-MISE toolkit to bring the modularity at the strain level (Chapter 6). In Chapter 7 we engineered the yeast Kluyveromyces marxianus for the production of prodeoxyviolacein and deoxycrhomoviridans, performed a fermentation to obtain the colour, and demonstrated the possibility of directly dyeing cotton and wool. We then combined the hydrolysis of cotton textile waste with a yeast fermentation in a separate hydrolysis and fermentation setup. Also the colours obtained with this procedure were successfully used in dyeing, with an unexpected advantage over the use of pure glucose. With the aim of future optimisation of the strains, in Chapter 8 we constructed a series of K. marxianus strains capable of overproducing tryptophan, the precursor of these colours, by repurposing existing chassis strains for the production of aromatics. Overall, the work presented in this thesis wants to pose itself as a practical example of how microbial bioprocesses and synthetic biology can be used as powerful tools to foster the circularity in the textile industry, closing the loop between waste and the production of new resources.