The Development of Textile Industry and Its Impact on our World
The textile industry is growing rapidly. At the same time, the amount of textile waste that harms the environment is also increasing. The invention of the spinning jenny in 1764 laid the foundations for the mass production of textiles (Watt, 2004). The invention of the steam engine 11 years later made it a reality (Watt, 2004). Although the prices for clothes dropped so the working class could afford them, the environmental impact of this industry was not considered. In addition, the people had no idea of how the textile industry would look nowadays.
Existing Solutions
Mixed textiles, such as cotton-polyester blends, cannot be recycled efficiently nowadays. Hence, the development of new recycling approaches is necessary.
As our team learnt about the impact of textile waste, we started looking for recycling strategies. We found that clothes are mainly made from polyester and cotton, which can be structurally regarded as PET (polyethylene terephthalate) and cellulose, respectively (Egan & Salmon, 2021). There are different approaches to recycle these materials, including the production of cleaning rags or melting and re-spinning thermoplastic fibers as well as many others (Piribauer et al., 2021). However, the degradation of mixed textiles, e.g. cotton-polyester blends, remains challenging and typically requires the separation of each composite fiber type (Loo et al., 2023).
Therefore, we decided to develop our own solution to this problem using the principles of synthetic biology. To this end, we conducted an extensive literature research on biological approaches for textile recycling. The advantage of biological approaches over others is that they can be applied to selectively degrade each type of fiber in blended materials (Kaabel et al., 2023; Li et al., 2019; Shen et al., 2013).
Furthermore, we searched for iGEM projects that tried to tackle the textile waste issue. Many iGEM projects from previous years were focused on the production of biodegradable polymer materials for fiber production, but, surprisingly, only a few projects were specifically dedicated to the degradation of the existing textile waste (GreatBay SCIE, 2022; Team Evry, 2016; Team Gothenburg, 2020; Team Imperial College London, 2013; Team LINKS China, 2021).
Our Solution
With our project "ReFiBa" we aim to degrade cotton and polyester clothing using recombinant bacterial cellulases and PETases produced by Bacillus subtilis.
Our project, ReFiBa (Enzyme-based Recycling of Textile Fibers using B. subtilis, is dedicated to developing an innovative solution for the degradation of cotton/polyester fibers and thus promoting a sustainable future. To achieve this, cellulases and PETases of bacterial origin have been selected and B. subtilis has been chosen as an expression host for enzyme production.
Whereas PETases catalyze the formation of terephthalic acid (TPA) and ethylene glycol (EG) from PET, three types of cellulases (β-1,4-glucosidases, exo- and endoglucanases) are required to degrade cellulose to glucose (Behera et al., 2017; Egan & Salmon, 2021). TPA can be removed from the solution by acidic precipitation, while the remaining EG and glucose can be applied for further fermentation processes to gain high value chemicals (Sandhwar & Prasad, 2017; Wagner et al., 2023). Hence, a circular economy concept could be developed.
Bacillus subtilis belongs to the phylum Firmicutes and is the best-studied Gram-positive bacterium. Due to its rapid growth, easy cultivation, non-pathogenic (generally regarded as safe) and probiotic properties as well as powerful genetic tools, it serves as a model organism in numerous studies (Su et al., 2020). The cells are rod-shaped, occur ubiquitously in the soil and exhibit an aerobic lifestyle. Additionally, B. subtilis is able to form highly resistant endospores and possesses a very efficient secretion capacity, making it an interesting host organism for heterologous protein expression (Cui et al., 2018; Errington & van der Aa, 2020; Harwood, 1992; Schumann, 2007).
Two Approaches for Protein Production
In our project, polyester/cotton degrading enzymes were displayed on the spore surface of B. subtilis. Additionally, B. subtilis was engineered to secrete these enzymes or to produce them intracellularly.
Two different approaches for protein production were explored in our project. Our main goal was to immobilize PETases and cellulases of bacterial origin on the spore surface of B. subtilis using a technology called spore surface display, in which the target protein is fused to a protein out of the spore coat (Zhang et al., 2019). Spores can be used under harsh environmental conditions, making them suitable for industrial applications (Lin et al., 2020). Furthermore, spores are relatively small (1.2 μm long and 0.8 μm wide) in comparison to other immobilization platforms (Chada et al., 2003). This is an important factor influencing the ability of spores to access dense textile fibers and is crucial for efficient degradation of apparels. Additionally, immobilization of enzymes on the spore surface eliminates the need for enzyme purification and might increase the stability and reusability of proteins (Lin et al., 2020; Zhang et al., 2019).
Prior to spore surface immobilization, selected enzymes, either secreted or intracellular were investigated to find suitable candidates for spore surface display. For protein secretion, PETases and cellulases were fused to signal peptides which either are naturally used for secretion of the respective enzymes or were derived from B. subtilis. In general, the secretion of enzymes into their surrounding makes their downstream processing easier and more cost-effective compared to intracellular enzyme expression (Neef et al., 2021). The disadvantage of freely diffusing enzymes in comparison to immobilized proteins is that their stability is typically more limited (Maghraby et al., 2023).
In general, spores are promising candidates for immobilization of proteins, as they can survive harsh environments and do not lose their resistance properties after genetic modification (Hinc et al., 2013). Particularly, B. subtilis endospores are frequently used due to their probiotic properties, for example in development of vaccines or animal feed production (Cutting, 2011; Hong et al., 2005; Isticato et al., 2001). The spore achieves its resistance to environmental stress (chemicals, heat, ultraviolet radiation) mainly through its spore coat (Driks & Eichenberger, 2016). It serves as a protective barrier, protecting the spore from toxic substances and predatory microbes (Balassa & Yamamoto, 1970; Klobutcher et al., 2006). Nevertheless, the envelope is permeable, for example to nutrients that pass through to receptors in the inner membrane, which then trigger a signal for germination (Moir & Cooper, 2015).
The composition of the spore coat is very complex. It contains at least 80 proteins, which are arranged in four layers: basement layer, inner coat, outer coat and crust (Driks & Eichenberger, 2016). The aim of displaying proteins on the spore surface is to improve their stability and functionality by anchoring them in the coat (Zhang et al., 2019). Moreover, this strategy enables the protection of target proteins from heat-induced denaturation and organic solvents. Another advantage is the easy recovery and reusability of the modified endospores for multiple reactions. Thus, the spore display technology represents a sustainable and cost-effective tool in biotechnology (Bartels et al., 2018; Sheldon, 2007).