Our Project


Nominated for Best Fashion and Cosmetics Project

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 allowing the working class to 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.

Today, fast fashion is a well-established business model based on selling fashionable clothes at a low price (Caro & Martìnez-De-Albèniz, 2015). You may be wondering how much clothing a person can consume when, in fact, clothes are not like food that your body needs to stay alive. This contradiction is exactly the result of fast fashion. In 2020, global clothing production reached 109 million tons, and it is projected to rise to 145 million tons by 2030 (European Parliament, 2024). The growing textile industry thereby leads to the accumulation of textile waste. Every year, the world produces 92 million tons of textile waste (Ruiz & Arabella, 2024). 87% of this waste is either incinerated or landfilled, contributing to 10 % of global carbon emissions (Ruiz & Arabella, 2024). Most shockingly, only 1 % of textile waste is actually recycled into new garments (European Parliament, 2024).

Figure 1: Textile Waste Picture provided by Freepik.com
Figure 2: Picture of clothing hills provided by Freepik.com

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 (Team LINKS China 2021, Team Evry 2016, Team Imperial College London 2013, Team GreatBay SCIE 2022, Team Edinburgh 2021, Team Gothenburg 2020).

Our Solution


With our project "ReFiBa" (Enzyme-Based Recycling of Textile Fibers Using Bacillus subtilis) 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 Bacillus 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 (polyethylene terephthalate hydrolases, EC 3.1.1.101) 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).

Endoglucanases (endo-1,4-β-D-glucanases, EC 3.2.1.4) cleave internal β-1,4-glycosidic bonds in amorphous regions of cellulose, thereby releasing reducing and non-reducing chain ends. Exoglucanases, also called 1,4-β-cellobiosidases, remove disaccharides (cellobiose) from the exposed ends in the crystalline region. Some exoglucanases work only on reducing ends (EC 3.2.1.176) while others cleave disaccharides only from non-reducing ends (EC 3.2.1.91). β-Glucosidases (1,4-β-glucosidases, EC 3.2.1.21) hydrolyze cellobiose or cello-oligosaccharides to glucose monomers (Akthar et al., 2016; Andlar et al., 2018).

Among the degradation products, 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.

Figure 3: Circular economy concept.
Infobox Bacillus subtilis

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, called anchor protein (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).



Infobox on Bacillus subtilis Spores for Protein Immobilization

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).

Figure 4: Immobilization of target proteins on B. subtilis endospores. The interior of the endospore consists of the core which contains DNA and is protected by the cortex. The spore coat, consisting of the basement layer, the inner and outer coat and especially the crust, can be used to anchor target proteins.

References

  • Akhtar N., Aanchal, Goyal D., Guyal A. (2016): Biodiversity of cellulase producing bacteria and their applications. Cellulose Chemistry and Technology 50 (9-10), 983-995.
  • Andlar M., Rezić T., Marđetko N., Kracher D., Ludwig R., Šantek, B. (2018): Lignocellulose degradation: An overview of fungi and fungal enzymes involved in lignocellulose degradation. Engineering in life sciences 18(11), 768-778.
  • Balassa, G., & Yamamoto, T. (1970). Biochemical genetics of bacterial sporulation. 3. Correlation between morphological and biochemical properties of sporulation mutants. Molecular & General Genetics : MGG, 108(1), 1–22. https://doi.org/10.1007/BF00343179
  • Bartels, J., López Castellanos, S., Radeck, J., & Mascher, T. (2018). Sporobeads: The Utilization of the Bacillus subtilis Endospore Crust as a Protein Display Platform. ACS Synthetic Biology, 7(2), 452–461. https://doi.org/10.1021/ACSSYNBIO.7B00285
  • Behera, B. C., Sethi, B. K., Mishra, R. R., Dutta, S. K., & Thatoi, H. N. (2017). Microbial cellulases – Diversity & biotechnology with reference to mangrove environment: A review. Journal of Genetic Engineering & Biotechnology, 15(1), 197. https://doi.org/10.1016/J.JGEB.2016.12.001
  • Caro, F., & Martìnez-De-Albèniz, V. (2015). Fast Fashion: Business Model Overview and Research Opportunities. International Series in Operations Research and Management Science, 223, 237–264. https://doi.org/10.1007/978-1-4899-7562-1_9
  • Chada, V. G. R., Sanstad, E. A., Wang, R., & Driks, A. (2003). Morphogenesis of Bacillus Spore Surfaces. Journal of Bacteriology, 185(21), 6255. https://doi.org/10.1128/JB.185.21.6255-6261.2003
  • Cui, W., Han, L., Suo, F., Liu, Z., Zhou, L., & Zhou, Z. (2018). Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond. World Journal of Microbiology & Biotechnology, 34(10). https://doi.org/10.1007/S11274-018-2531-7
  • Cutting, S. M. (2011). Bacillus probiotics. Food Microbiology, 28(2), 214–220. https://doi.org/10.1016/J.FM.2010.03.007
  • Driks, A., & Eichenberger, P. (2016). The Spore Coat. Microbiology Spectrum, 4(2). https://doi.org/10.1128/MICROBIOLSPEC.TBS-0023-2016
  • Egan, J., & Salmon, S. (2021). Strategies and progress in synthetic textile fiber biodegradability. SN Applied Sciences 2021 4:1, 4(1), 1–36. https://doi.org/10.1007/S42452-021-04851-7
  • Errington, J., & van der Aa, L. T. (2020). Microbe Profile: Bacillus subtilis: model organism for cellular development, and industrial workhorse. Microbiology, 166(5), 425. https://doi.org/10.1099/MIC.0.000922
  • European Parliament. (2024). The impact of textile production and waste on the environment (infographics) | Topics | European Parliament. https://www.europarl.europa.eu/topics/en/article/20201208STO93327/the-impact-of-textile-production-and-waste-on-the-environment-infographics
  • GreatBay SCIE. (2022). FabRevivo. https://2022.igem.wiki/greatbay-scie
  • Harwood, C. R. (1992). Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends in Biotechnology, 10(7), 247–256. https://doi.org/10.1016/0167-7799(92)90233-L
  • Hinc, K., Iwanicki, A., & Obuchowski, M. (2013). New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microbial Cell Factories, 12(1). https://doi.org/10.1186/1475-2859-12-22
  • Hong, H. A., Le, H. D., & Cutting, S. M. (2005). The use of bacterial spore formers as probiotics. FEMS Microbiology Reviews, 29(4), 813–835. https://doi.org/10.1016/J.FEMSRE.2004.12.001
  • Isticato, R., Cangiano, G., Tran, H. T., Ciabattini, A., Medaglini, D., Oggioni, M. R., De Felice, M., Pozzi, G., & Ricca, E. (2001). Surface display of recombinant proteins on Bacillus subtilis spores. Journal of Bacteriology, 183(21), 6294–6301. https://doi.org/10.1128/JB.183.21.6294-6301.2001
  • Kaabel, S., Arciszewski, J., Borchers, T. H., Therien, J. P. D., Friščić, T., & Auclair, K. (2023). Solid-State Enzymatic Hydrolysis of Mixed PET/Cotton Textiles. ChemSusChem, 16(1), e202201613. https://doi.org/10.1002/CSSC.202201613
  • Klobutcher, L. A., Ragkousi, K., & Setlow, P. (2006). The Bacillus subtilis spore coat provides “eat resistance” during phagocytic predation by the protozoan Tetrahymena thermophila. Proceedings of the National Academy of Sciences of the United States of America, 103(1), 165–170. https://doi.org/10.1073/PNAS.0507121102/SUPPL_FILE/INDEX.HTML
  • Li, X., Hu, Y., Du, C., & Lin, C. S. K. (2019). Recovery of Glucose and Polyester from Textile Waste by Enzymatic Hydrolysis. Waste and Biomass Valorization, 10(12), 3763–3772. https://doi.org/10.1007/S12649-018-0483-7/FIGURES/9
  • Lin, P., Yuan, H., Du, J., Liu, K., Liu, H., & Wang, T. (2020). Progress in research and application development of surface display technology using Bacillus subtilis spores. Applied Microbiology and Biotechnology 2020 104:6, 104(6), 2319–2331. https://doi.org/10.1007/S00253-020-10348-X
  • Loo, S. L., Yu, E., & Hu, X. (2023). Tackling critical challenges in textile circularity: A review on strategies for recycling cellulose and polyester from blended fabrics. Journal of Environmental Chemical Engineering, 11(5), 110482. https://doi.org/10.1016/J.JECE.2023.110482
  • Moir, A., & Cooper, G. (2015). Spore Germination. Microbiology Spectrum, 3(6). https://doi.org/10.1128/MICROBIOLSPEC.TBS-0014-2012
  • Neef, J., van Dijl, J. M., & Buist, G. (2021). Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential. Essays in Biochemistry, 65(2), 187. https://doi.org/10.1042/EBC20200171
  • Piribauer, B., Bartl, A., & Ipsmiller, W. (2021). Enzymatic textile recycling - best practices and outlook. Waste Management & Research : The Journal of the International Solid Wastes and Public Cleansing Association, ISWA, 39(10), 1277–1290. https://doi.org/10.1177/0734242X211029167
  • Ruiz, & Arabella. (2024). 17 Most Worrying Textile Waste Statistics & Facts. https://theroundup.org/textile-waste-statistics/
  • Sandhwar, V. K., & Prasad, B. (2017). Terephthalic acid removal from aqueous solution by electrocoagulation and electro-Fenton methods: Process optimization through response surface methodology. Process Safety and Environmental Protection, 107, 269–280. https://doi.org/10.1016/J.PSEP.2017.02.014
  • Schumann, W. (2007). Production of recombinant proteins in Bacillus subtilis. Advances in Applied Microbiology, 62, 137–189. https://doi.org/10.1016/S0065-2164(07)62006-1
  • Sheldon, R. A. (2007). Enzyme Immobilization: The Quest for Optimum Performance. Advanced Synthesis & Catalysis, 349(8–9), 1289–1307. https://doi.org/10.1002/ADSC.200700082
  • Shen, F., Xiao, W., Lin, L., Yang, G., Zhang, Y., & Deng, S. (2013). Enzymatic saccharification coupling with polyester recovery from cotton-based waste textiles by phosphoric acid pretreatment. Bioresource Technology, 130, 248–255. https://doi.org/10.1016/J.BIORTECH.2012.12.025
  • Su, Y., Liu, C., Fang, H., & Zhang, D. (2020). Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microbial Cell Factories 2020 19:1, 19(1), 1–12. https://doi.org/10.1186/S12934-020-01436-8
  • Team Evry. (2016). Let’s PLAy - Bioproduction of Poly-Lactic acid. https://2016.igem.org/Team:Evry/Description
  • Team Gothenburg. (2020). No Time To Waste. https://2020.igem.org/Team:Chalmers-Gothenburg/Description Team Imperial College London. (2013). Plasticity. https://2013.igem.org/wiki/index.php?title=Team:Imperial_College/tour
  • Team LINKS China. (2021). Neoleathic age. https://2021.igem.org/Team:LINKS_China
  • Wagner, N., Wen, L., Frazão, C. J. R., & Walther, T. (2023). Next-generation feedstocks methanol and ethylene glycol and their potential in industrial biotechnology. Biotechnology Advances, 69, 108276. https://doi.org/10.1016/J.BIOTECHADV.2023.108276
  • Watt, M. (2004). Nineteenth-Century European Textile Production | Essay | The Metropolitan Museum of Art | Heilbrunn Timeline of Art History. https://www.metmuseum.org/toah/hd/txtn/hd_txtn.htm
  • Zhang, G., An, Y., Zabed, H. M., Guo, Q., Yang, M., Yuan, J., Li, W., Sun, W., & Qi, X. (2019). Bacillus subtilis Spore Surface Display Technology: A Review of Its Development and Applications. Journal of Microbiology and Biotechnology, 29(2). https://doi.org/10.4014/JMB.1807.06066