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polyester fabric blends
Waste Management 102 (2020) 149–160
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Closing the textile loop: Enzymatic fibre separation and recycling of wool/polyester fabric blends Laura Navone a, Kaylee Moffitt a, Kai-Anders Hansen a, James Blinco a, Alice Payne b, Robert Speight a,⇑ a b
Science and Engineering Faculty, Queensland University of Technology, Brisbane, Queensland, Australia Creative Industries Faculty, Queensland University of Technology, Brisbane, Queensland, Australia
a r t i c l e
i n f o
Article history: Received 24 May 2019 Revised 8 September 2019 Accepted 12 October 2019
Keywords: Wool Keratinase Recycling Polyester Enzyme Textile
a b s t r a c t Textile waste presents a serious environmental problem with only a small fraction of products from the fashion industry collected and re-used or recycled. The problem is exacerbated in the case of postconsumer waste by the mixture of different natural and synthetic fibres in blended textiles. The separation of mixed fibre waste, where garments are often multicomponent, presents a major recycling problem as fibres must be separated to single components to enable effective recycling. This work investigates the selective digestion of wool fibres from wool/polyester blended fabrics using an enzymatic approach. Complete degradation of wool fibres was achieved by application of a keratinase in a two-step process with addition of reducing agent and undigested polyester fibres were recovered. Electron microscopy showed complete breakdown of the natural fibres in the fabric blends, while spectroscopic and mechanical analysis of the recovered synthetic fibres confirmed that the enzymatic treatment had no significant impact on the properties of the polyester compared to virgin samples. The polyester fibres are therefore suitable to be recycled to polyester yarn and re-used in the manufacture of new garments or other products. The nutrient rich keratin hydrolysate could be used in microbial growth media or incorporated into bio-fertilisers or animal feed, contributing to the development of the circular economy. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Textile waste is a serious environmental issue that will continue to grow if practical and economic solutions cannot be found that either reduce the production of waste or that close the loop and turn the waste into new products. Textile production processes require high volume of water, energy and chemicals (Pensupa et al., 2017). Globally, 64% percent of textile fibres are derived from petrochemicals and the remaining 36% is dominated by cotton (24%), manufactured cellulosic fibre (6%), wool (1%) and other natural fibres. The fashion industry with its current fast fashion business model, characterised by mass production, variety, agility and affordability, has greatly contributed to the volumes of waste and the rate at which this waste is generated. Fast fashion creates a demand for 80 billion new garments each year (Cline, 2012), yet garments typically follow a linear life cycle in which after use, garments are disposed to landfill rather than reused or recycled. This ‘cradle-to-grave’ (Braungart and McDonough, 2002) life cycle means the significant resources associated with textile production ⇑ Corresponding author at: Queensland University of Technology, 2 George Street, Brisbane, QLD 4000, Australia. E-mail address: [email protected] (R. Speight). https://doi.org/10.1016/j.wasman.2019.10.026 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
are wasted. In contrast, a circular economy model would mean keeping resources in use as long as possible through strategies such as reuse, repair, remanufacturing and recycling of products (Ghisellini et al., 2016). The growth of the textile industry is rapidly increasing the volumes of textile waste that predominantly end up in landfill or incinerators (WRAP, 2017), necessitating an immediate need for efficient recycling technologies to ‘close the loop’ and generate new value from textile waste streams. Current types of textiles recovery involve re-use for the same application, re-purposing for different applications and recycling. However, the major barrier to effective recycling is the large variety of materials used in apparel, and how they are blended (Payne, 2016). While collection of pre-consumer waste to generate products for insulation, carpet padding or industrial rags does occur, post-consumer waste is typically a diverse mixture of many fabric materials, which increases the level of complexity of recycling processes and reduces the opportunities for new products (Payne, 2016). There is an absolute requirement to separate blended fabrics into their components to allow effective recycling. For example polyester must be separated from cotton and wool to be able to be melted and reformed into polyester yarn (Jeihanipour et al., 2010). The challenge and complexity of separating and collecting the con-
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stituent fibres is currently the major technical and practical hurdle limiting recycling and landfill avoidance. There are currently no commercially viable separation and recycling technologies for these blended fabrics (Payne, 2016). While some advances have been made on mixed fibre separation technologies for cotton-synthetic blends (HKRITA, 2017; Schimper et al., 2008; Vasconcelos and Cavaco-Paulo, 2006), wool-synthetic blends are a waste-stream that requires new processes for recycling and removal of 1.1 million tons of waste from landfill (ICAC, 2013). Yet, only a small number of studies have been performed with limited success on the dissolution of wool in mixed fibre fabrics (Sandin and Peters, 2018). Studies attempting to dissolve wool fibres from garments have used extreme conditions that affect the mechanical properties of the synthetic fibres and can chemically modify the keratin peptides, thus reducing nutritional value if they are to be used as feed supplements (Brandelli et al., 2010; Cardamone et al., 2009; Gupta et al., 2013; Hou et al., 2014; Łaba and Szczekala, 2013; Sweetman, 1967). Investigations on the biological degradation of wool waste used 100% wool samples and selective degradation of wool fibres and recovery of synthetic polymers from wool blends has not yet been achieved (Eslahi et al., 2013; Fang et al., 2013a, b; Gousterova et al., 2005). The difficulties in degrading wool materials arise from the recalcitrant structure of the keratin protein, which is the main component of wool, and the overall need for sufficiently mild treatment conditions that maintain the mechanical properties of the synthetic polymers so that the recycling material meets performance specifications. Keratin accounts for 95% of wool weight and exhibits a high degree of disulfide bonding, which confers rigidity and chemical resistance (Cardamone et al., 2009). Selective and environmentally benign methods like the one presented here are necessary for efficient wool-synthetic textile blend recycling. We investigate the enzymatic treatment of wool/polyester fabric blends to selectively degrade wool fibres while recovering the synthetic fibres. A protease that was investigated previously for keratinolytic activity (Navone and Speight, 2018) was applied to fabric blends together with reducing agents. A two-step enzymatic process in the presence of sodium thioglycolate showed best results for degradation of wool fibres. Weight loss analysis and scanning electron microscopy (SEM) of the treated fabrics confirmed complete removal of the wool component. The purity and mechanical properties of the resulting polyester fibres were analysed by nano-indentation and infra-red spectroscopy. The polymer fibres obtained after enzymatic treatment retain the integrity and physical properties of virgin fibres and could be used in the manufacture of new garments or other textile and polyesterderived products. 2. Materials and methods 2.1. Fabric samples The fabric samples used in this work were supplied by Frost Textiles. The fabric types were 100% wool knit, 100% wool woven, 70% wool-30% polyester knit (note, this fabric was purchased on the basis of it being wool-polyester but IR and SEM analyses suggested that this particular fabric could be a wool-nylon blend), 45% wool-55% polyester woven, 100% polyester knit and 100% polyester woven fabrics.
dilution was added to 0.01 g of keratin azure in 2.4 mL of 100 mM Tris-HCl buffer pH 8 or pH 10. Samples were incubated at 37 °C for 1 h at 200 rpm. After incubation, samples were centrifuged at 4000 rpm for 10 min and the absorbance of the clarified supernatants was determined at 595 nm. Determinations for each enzyme dilution were performed in triplicate. For the negative control, 0.01 g of keratin azure in 2.5 mL of reaction buffer was incubated at 37 °C for 1 h with shaking at 200 rpm and the absorbance was measured at 595 nm. One keratin unit (KU) was arbitrarily defined as an increase of 0.1 in absorbance at 595 nm after incubation for 1 h at 37 °C according to previously reported methods (Navone and Speight, 2018). For keratinolytic activity determination in the presence of reducing agents, the keratin azure assay was performed as indicated with the addition of 1, 1.5 or 2% of sodium sulfite or 2, 3, 4 or 5% of sodium thioglycolate. Total protein was determined by Bradford assay (Bradford, 1976). Handling of sodium thioglycolate buffers was conducted according to reported safety indications (Burnett et al., 2009). 2.3. Enzymatic treatment of fabric samples, determination of soluble peptides and percentage degradation after treatment Ronozyme ProAct was the protease used in all experiments (provided by DSM Nutritional Products, Wagga Wagga, Australia). Fabric samples of 0.1 g were cut with scissors into pieces of approximately 5 5 mm or ground into pieces using a Rocklabs Ring Mill and fractioned into sample sizes of 0.6 mm using size sieves. Samples were treated with 2, 4 and10 KU/mL of protease in 5 mL of 100 mM Tris-HCl buffer pH 10 for 16 h at 37 or 50 °C at 200 rpm. When indicated, 1% sodium sulfite, or 2% or 3% sodium thioglycolate was added. Control fabric samples were incubated in reaction buffer without enzyme for 16 h at 50 °C at 200 rpm. Reducing agent was added where indicated. For the two step treatment, fabric samples of 0.1 g (5 5 mm pieces or ground pieces 0.6 mm) were treated with 2 or 4 KU/ mL of protease in 5 mL of 100 mM Tris-HCl buffer at pH 10 with 2% sodium thioglycolate for 6 h at 50 °C, 200 rpm. After 6 h, samples were centrifuged at 4000 rpm for 10 min and washed once with water, and 2 or 4 KU/mL of protease in 5 mL of 100 mM Tris-HCl buffer at pH 10 with 2% sodium thioglycolate was readded. Incubation proceeded for another 14–16 h at 50 °C, 200 rpm. Soluble peptides after enzymatic treatment were quantified by Bradford assay after 16 h of incubation (Bradford, 1976). Only peptides >3 KDa were quantified due to the lower mass limit of the Bradford assay. The percentage degradation was determined by weight loss. Remaining fabric samples after treatment were centrifuged, the supernatant removed, and completely dried to constant weight at 65 °C in an oven. The final weight was measured and % degradation calculated after subtracting the initial weight. Weight loss calculations were performed considering total available wool in each fabric type so that 100% weight loss corresponded to 100% loss of available wool in each fabric type. Statistical analysis was performed using one-tailed distribution t-test. The differences among means with P 0.01 were accepted as representing statistically significant differences. For analysis of the polyester component of fabric samples, the enzymatic treatment of fabric samples (5 5 mm pieces or ground pieces 0.6 mm) with 2 KU/mL of protease was scaled up to 4 g in a final volume of 200 mL. 2.4. Scanning electron microscopy
2.2. Keratinase activity determination Keratinolytic activity was determined using keratin azure (Sigma Aldrich) as the substrate following the manufacturer’s instructions with some modifications. Briefly, 100 lL of enzyme
Fabric samples treated in the two-step process at 50 °C with 2% sodium thioglycolate, with or without 4 KU/mL of protease, were centrifuged and air dried for several days, fixed in a sample holder stub and gold coated using a Leica EM SCD005 Gold Coater (to
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~10 nm). Secondary electron images were obtained with a Zeiss Rigma Field Emission Scanning Electron Microscope. Images were obtained under vacuum using 2 kV accelerating voltage. 2.5. Infra-Red spectroscopy Infra-red spectra were collected on a Nicolet Nexus 870 Fourier transform infrared (FTIR) spectrometer equipped with a Smart Endurance single bounce horizontal diamond ATR and a TEcooled deuterated triglycine sulfate (DTGS) detector (Nicolet Instrument Corp. Madison, WI). Spectra were recorded using 64 scans, 0.6329 cm s 1 mirror velocity and 4 cm 1 resolution over the range 4000–525 cm 1. Spectra were collected in absorbance mode and an ATR correction applied. Final processing was performed using GRAMS software (Thermo Galactic, Woburn, MA). To assess reproducibility of the spectra, 6 spots (3 on the exposed side and 3 on the unexposed side) of the 5 samples were analysed. Wool: IR (ATR): v = 3300 (NAH s), 1630 (Amide I, C@O s) and 1510 (Amide II, NAH b) cm 1. Polyester: IR (ATR): v = 1710 (C@O s) cm 1. 55/45 blend: Before: IR (ATR): v = 3300 (NAH s), 1710 (C@O s), 1630 (Amide I, C@O s) and 1510 (Amide II, NAH b) cm 1. After: IR (ATR): v = 1710 (C@O s) cm 1. 30/70 blend: IR (ATR): v = 3300 (NAH s), 1630 (Amide I, C@O s) and 1510 (Amide II, NAH b) cm 1. 2.6. Nano-indentation For sample preparation of 100% polyester (treated and untreated) and 45/55% wool/polyester blend (treated), 125 mg of polymer sample was dissolved in 1.75 mL of 1,1,1,3,3,3-hexa fluoro-2-propanol (HFIP) and filtered through a silica column. The solution was left to dry under air. The dried polymer was redissolved in 1.75 mL of HFIP and drop-cast onto a silicon wafer. For sample preparation for 45/55% wool/polyester blend (untreated), 227 mg of sample was treated with 3.5 mL of HFIP to extract the polyester. The resulting solution was separated and collected in a vial. The remaining fabric was washed with further 1.75 mL of HFIP. The extract was filtered through a silica plug and the collected clear solution dried under air. The dried polymer was redissolved in 1.75 mL of HFIP and drop-cast onto a silicon wafer. The dried polymer samples were subsequently analysed by nano-indentation. 3. Results and discussion 3.1. Enzymatic degradation of fabric blends in the presence of reducing agents Our previous studies investigated a protease with keratinolytic activity towards cattle hair and feather keratin with activity greatly enhanced in the presence of reducing agents (Navone and Speight, 2018). Fabric however, represents a greater challenge due to the structure and thickness of the fibres following the spinning process and the potential presence of dyes and other auxiliary chemicals. In this work, wool fabric samples were treated with protease in the presence of reducing agents to study the degradation of keratin from wool. Wool and polyester fabric blend samples (knit and woven) cut into 5 5 mm pieces were treated with 2, 4 and 10 KU/mL of protease at 37 °C. Wool breakdown was followed by determination of soluble peptides concentration and weight loss after treatment. Weight loss was calculated as a percentage of wool in the fabric. Fabric samples composed of 100% wool (knit and woven) were also treated under the same conditions (Fig. 1A and B). Fabric degradation without reducing agent ranged between 19 and 45% for all fabric types (Table A.3). The addition of sodium sul-
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fite did not show a significant (P 0.01) improvement in degradation for any of the fabric types, obtaining the most weight loss for 45/55% wool/polyester woven fabric (52 ± 4%). On the other hand, the presence of sodium thioglycolate significantly (P 0.01) improved degradation compared to treatment with no reducing agent or in the presence of sodium sulfite, reaching 76 ± 1% for 100% wool woven, 73 ± 2% for 45/55% wool/polyester woven, 52 ± 7% for 100% wool knit and 55 ± 1% for 70/30% wool/polyester knit fabric (Fig. 1). An increase in soluble peptide concentration was only observed when sodium thioglycolate was added (Fig. 1). The addition of reducing agent has been shown to be crucial for keratinase access to the peptide bonds in keratin in hair and feather (Navone and Speight, 2018). The results presented here confirm that the reduction of the disulfide bonds is equally important for wool degradation. Furthermore, sodium thioglycolate was found to be a better reducing agent for hair and feather degradation than sodium sulfite in previous studies (Navone and Speight, 2018). Sodium thioglycolate has higher reducing capacity than other reducing agents, including sodium sulfite (unpublished data). Using a reducing agent with a stronger reducing capacity gives a higher degree of reduction of disulfide bonds, allowing increased access of the protease to the keratin fibre and improved degradation. Previous research on the mechanism of keratin breakdown from hair at different time points during enzymatic treatment showed that lifting of the hair cuticle is the first step of degradation, followed by its complete removal and initial damage of the cortex (Navone and Speight, 2018). Fracturing of the cortex continues until the generated small keratin fragments are finally converted to soluble peptides along with residual insoluble protein. The dynamics of wool degradation are likely to be very similar to those observed for hair degradation due to the similar hierarchical structure between wool and hair (Fueghelman, 1997). 3.2. Temperature optimisation of fabric treatment The inability to obtain complete degradation of fabrics at 37 °C with or without reducing agents suggested that temperature optimisation studies of the enzymatic process should be conducted. The specific activity of the protease was determined using the keratin azure assay at 37 °C, 50 °C and 60 °C (Table 1). Since keratinase activity was highest at 50 °C, this temperature was selected for activity studies in the presence of the reducing agents sodium sulfite and sodium thioglycolate. Table 1 shows the specific activity of the protease in the presence of different concentrations of reducing agents. Increased keratinase activity was obtained with 1% sodium sulfite and 2 or 3% of sodium thioglycolate. Wool and wool/polyester fabric samples cut into 5 5 mm pieces were treated with 0, 2, 4 and 10 KU/mL of protease at 50 °C. Wool breakdown was followed by determination of soluble peptides concentration and weight loss after treatment (Fig. 2). In the case of the 45/55% blend woven fabrics, the presence of reducing agent in the enzymatic treatment increased the concentration of soluble peptides. However, the measured percent of degradation by weight loss did not improve in the presence of reducing agents. The opposite effect was observed for 100% wool woven fabric where soluble peptide concentration after treatment did not increase with reducing agents but weight loss was improved, particularly when using sodium thioglycolate. Relatively high values for soluble peptides were obtained for the control samples without enzyme in the presence of the reducing agents for both 100% wool fabrics and 45/55% wool/polyester blend but with no significant weight loss. A fabric component released during degradation at 50 °C (e.g. dye used for colouring) could generate a false positive result in the Bradford assay for the no enzyme controls.
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Fig. 1. Enzymatic degradation of wool fabric at 37 °C. Soluble peptides (mg peptides/g of wool) and weight loss (%) after treatment of wool woven fabrics (A) and wool knit fabrics (B) with 2, 4 or 10 KU/mL of protease with or without reducing agents. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). Data are represented as mean values ± standard deviation (n = 3).
Table 1 Keratinase activity of the protease determined by the keratin azure assay in the presence of reducing agents. Assay temperature (°C)
Reducing agent
Specific activity (103 KU/g)
37 50 60 50 50 50 50 50 50 50
– – – 1% sulfite 1.5% sulfite 2% sulfite 2% thioglycolate 3% thioglycolate 4% thioglycolate 5% thioglycolate
178 ± 13a 584 ± 38 432 ± 16 701 ± 87 389 ± 82 266 ± 25 940 ± 32 1014 ± 38 451 ± 39 236 ± 26
One keratinase unit (KU) is defined as an increase of 0.1 in absorption units at 595 nm after incubation with keratin azure for 1 h at 37, 50 or 60 °C. Data are represented as mean values ± standard deviation (n = 3). a Navone and Speight (2018).
Fabric degradation reached 95 ± 1% and 66 ± 1% in the presence of sodium thioglycolate for 100% wool knit or 70/30% wool/polyester knit blend, respectively. Significant statistical difference (P 0.01) was observed between 37 °C and 50 °C treatment without reducing agents or in the presence of sodium sulfite or sodium thiolgycolate for 100% wool or 70/30% wool/polyester knits (Table A.3). Degradation improvement at the higher temperature was less pronounced for woven fabrics. A statistical difference (P 0.01) between the 37 °C and 50 °C treatment was observed for 45/55% wool/polyester woven fabric without reducing agent
or in the presence of sodium thioglycolate, and for 100% woven fabric in the presence of sodium sulfite (Table A.3). Results showed best degradation in the presence of sodium thioglycolate obtaining 69 ± 1% and 60 ± 1% weight loss for 100% wool woven or 45/55% wool/polyester woven fabrics, respectively. Taken together, results suggest that the temperature increase from 37 °C to 50 °C improved enzymatic breakdown of keratin from wool. Fig. 2C shows a comparison of weight loss between 37 °C and 50 °C for the woven and knit fabrics in the presence of sodium thioglycolate. The only fabric type that reached almost complete degradation at 50 °C was the 100% wool knit fabric, 94 ± 1% with 2 and 4 KU/mL of protease, and 95 ± 1% with 10 KU/mL of protease. No significant difference (P 0.01) was observed between the degradation of 100% wool woven (66 ± 1% with 4 KU/mL), 45/55% woven blend (56 ± 5% with 4 KU/mL) or 70/30% knit blend (64 ± 1% with 4 KU/mL). Increasing the enzyme concentration from 2 to 10 KU/mL did not improve degradation under any of the conditions tested at 50 °C.
3.3. Size effect on the enzymatic treatment of fabric Each fabric type was ground and fractioned using a size sieve into pieces of 0.6 mm to test size effect on degradation. The addition of reducing agent to the enzymatic treatment of ground fabric had a very strong interfering effect on Bradford determination of soluble peptides (data not shown) and only weight loss determinations are presented for the following treatments.
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Fig. 2. Enzymatic degradation of wool fabric at 50 °C. Soluble peptides (mg peptides/g of wool) and weight loss (%) after treatment of wool woven fabrics (A) and wool knit fabrics (B) with 2, 4 or 10 KU/mL of protease with or without reducing agents. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). Comparison of weight loss (%) between treatment temperature of woven and knit fabrics with 2, 4 or 10 KU/mL of protease in the presence of 2% sodium thioglycolate (C). The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). 100% weight loss corresponds to complete removal of the available wool in each fabric type. Data are represented as mean values ± standard deviation (n = 3).
Fig. 3 shows weight loss (%) comparison between sample sizes 5 5 mm and 0.6 mm for 100% wool and 45/55% wool/polyester blend woven (Fig. 3A and B) and 100% wool and 70/30% wool/ polyester blend knit fabrics (Fig. 3C and D). No significant difference (P 0.01) on degradation was observed for 100% wool or
70/30% wool/polyester blend knit fabrics between the two sample sizes when treated without reducing agent or in the presence of sodium sulfite. However, degradation was significantly improved (P 0.01) in the presence of sodium thioglycolate for 5 5 mm sample sizes compared to 0.6 mm samples for 100% wool or
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Fig. 3. Enzymatic treatment of different size fabric samples with or without reducing agents. Weight loss (%) after treatment of 100% wool woven (A), 45/55% wool/polyester woven (B), 100% wool knit (C) and 70/30% wool/polyester knit fabrics (D) with 2, 4 or 10 KU/mL of protease with or without reducing agents. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). 100% weight loss corresponds to complete removal of available wool in each fabric type. Data are represented as mean values ± standard deviation (n = 3).
70/30% wool/polyester blend knit fabrics (Fig. 3C and D). No significant difference (P 0.01) was observed for 100% wool woven between sample sizes when treated with or without reducing agent (Fig. 3A). For the 45/55% woven blend, the 0.6 mm sample showed significantly (P 0.01) improved degradation compared to the 5 5 mm pieces in the presence of sodium sulfite or sodium thioglycolate (Fig. 3B). The woven blend almost reached complete degradation when the fabric was ground to 0.6 mm in the presence of 1% sodium sulfite (90 ± 4%) or 2% sodium thioglycolate (93 ± 13%). Yet the blended fabric only reached 50 ± 18% degradation with 1% sodium sulfite and 62 ± 5% degradation with 2% sodium thioglycolate when the sample size was 5 5 mm. Taken together, the results suggest that the sample size (5 5 mm or 0.6 mm) does not greatly influence the degradation capability of the enzyme towards wool keratin. The difference observed for the 45/55% wool/polyester blend 0.6 mm sample may be due to the specific nature of the fabric. 3.4. Enzymatic treatment with sodium thioglycolate Sodium thioglycolate was selected as the reducing agent for the following experiments since it showed increased degradation compared to sodium sulfite for all the fabrics tested. The concentration
of sodium thioglycolate was increased from 2 to 3% to test for degradation improvement. Enzymatic treatments were conducted with 2 or 4 KU/mL of protease only as the higher concentration of protease (10 KU/mL) did not appear to further improve degradation (Fig. 2). Fig. 4A shows weight loss for 5 5 mm or ground 0.6 mm samples of wool/polyester woven and knit blends treated with 2 and 4 KU/mL of protease with 3% sodium thioglycolate. No significant difference (P 0.01) in degradation between the 5 5 mm pieces and the ground 0.6 mm samples was observed for the 100% wool knit, 70/30% wool/polyester knit or 45/55% wool/polyester woven fabrics with 3% thioglycolate. A significant improvement in degradation (P 0.01) was only observed for 100% wool woven 0.6 mm samples compared to the 5 5 mm samples in the presence of 3% sodium thioglycolate. The sample size effect obtained here for the 100% wool woven fabric was not observed when the treatment was performed with the two sample sizes in 2% sodium thioglycolate (Fig. 3A). When comparing fabric treatment with the two different concentrations of sodium thioglycolate, degradation of the 5 5 mm pieces of the 45/55% wool/polyester blend was significantly (P 0.01) improved from 60 ± 1% in 2% sodium thioglycolate to 80 ± 5% in 3% sodium thioglycolate. On the other hand, the 0.6 mm sample of the same fabric blend reached 91 ± 4% in 2%
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Fig. 4. Enzymatic treatment with 3% sodium thioglycolate and two-step treatment of fabrics. Weight loss (%) after treatment of 100% wool woven, 45/55% wool/polyester woven, 100% wool knit and 70/30% wool/polyester knit fabrics with 2 or 4 KU/mL of protease in the presence of 3% sodium thioglycolate (A) and after two-step treatment with 2 or 4 KU/mL of protease in the presence of 2% sodium thioglycolate (B). The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). 100% weight loss corresponds to complete removal of available wool in each fabric type. Data are represented as mean values ± standard deviation (n = 3).
sodium thioglycolate and 80 ± 3% in 3% sodium thioglycolate (Figs. 3B and 4A), however, the difference in degradation percent was not significant (P 0.01). For the 70/30% wool/polyester knit blend, the increased concentration of sodium thioglycolate did not significantly (P 0.01) improve degradation for the 5 5 mm or the ground 0.6 mm samples (Figs. 4A and 3D). For the 100% wool knit fabric, the 5 5 mm samples showed a significant (P 0.01) decrease in degradation percent from 94 ± 0.6% in 2% sodium thioglycolate to 83 ± 0.4% in 3% sodium thioglycolate. The ground 0.6 mm samples showed no significant difference between the two concentrations of reducing agent (Figs. 4A and 3C). For the 100% wool woven fabric, significant improvement of degradation was observed for the 0.6 mm sample where degradation reached 95 ± 0.8% in 3% sodium thioglycolate compared with 61 ± 6.3% degradation in 2% sodium thioglycolate (Fig. 3A). No significant (P 0.01) degradation was difference was observed for the 5 5 mm pieces of 100% wool woven fabric.
No clear trend could be concluded from the increased concentration of sodium thioglycolate from 2 to 3%, for some of the fabric types and sample size degradation improved while in other cases degradation was decreased. Accordingly, the next experiments were conducted with 2% sodium thioglycolate.
3.5. Two-step treatment of fabric samples The inability to obtain complete degradation of some fabric samples could be related to an inactivation of the enzyme during several hours of incubation at 50 °C. To test this hypothesis, fabric samples cut into 5 5 mm pieces were treated in a two-step process, where each fabric was incubated with 2 or 4 KU/mL of protease with 2% sodium thioglycolate for 6 h, then centrifuged, washed with water and enzyme re-added (2 or 4 KU/mL) with 2% sodium thioglycolate and further incubated for 16 h. A first step of 6 h was chosen based on previous degradation experiments with
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hair and feather keratin (Navone and Speight, 2018). Weight loss percentages for all fabric types after a second treatment step are shown in Fig. 4B. All fabrics reached more than 90% degradation indicating that previous incomplete degradation could be related to enzyme inactivation (Table A.4). Enzyme activity was determined after 6 and 16 h of fabric treatment to investigate enzyme inactivation (Table A.1). Keratinase activity of the protease markedly decreased for all fabric types after 6 h of treatment at 50 °C. The remaining activity after 16 h of incubation was found to be very low, between 2 and 4% remaining activity for all fabric types. Such low enzymatic activity after 16 h of treatment suggests that keratin breakdown had been completed in a shorter period of time. According to the remaining activities showed in Table A.1, complete keratin degradation from wool most likely occurs within 6– 16 h, making the development of a shorter treatment feasible for industrial application. The presence of fabric increased the degree of enzyme inactivation, which was more evident for the 100% wool fabrics (Table A.1). The inactivation effect by the fabric could be related to chemicals associated with the fabric being released during treatment and that these chemicals are different between wool and polyester fibres. Unfortunately, due to the observed enzyme inactivation, the fabric treatment proposed in this work would not allow recovery of the enzyme for re-use. Optimisation of the process could include
a pre-treatment step with reducing agent to decrease the required time or temperature of the enzymatic treatment to obtain active enzyme at the end of the process for re-use. The pre-treatment could also get remove chemicals present in the fabric that might affect enzyme activity. Most studies on wool enzymatic degradation have been performed with 100% wool and usually from pre-consumer material. Wool has usually been treated with microbial whole cell cultures and in fewer cases with isolated enzymes (Fang et al., 2013b). One example of wool waste treatment with a commercial protease and a reducing agent used 100% wool fibres from spinning processes (Eslahi et al., 2013). The study reported 26% degradation by weight loss of wool waste after 4 h treatment at 55 °C and selective degradation of wool from synthetic fibres was not attempted. Enzymatic treatment of wool/cotton/polyester blended fabrics was recently conducted using a commercial protease and a cellulase cocktail (Quartinello et al., 2018). The fabric components were 2% wool, 61% cotton and the remaining fibres were polyester and polyamide. The authors reported a step-wise enzymatic extraction of textile fractions resulting in 95% and 85% extraction efficiency of wool and cotton constituents respectively. A protease treatment of the fabric blend was conducted for 2 days at 50 °C in the presence of sodium bisulfite as reducing agent, followed by filtration and drying of the fabric at high temperatures, with a final cellulase
Fig. 5. Scanning electron microscopy of two-step enzymatic treatment of fabric. Images correspond to 100% wool knit (A), 100% wool woven (B), 45/55% wool/polyester woven (C), and 70/30% wool/polyester knit (D) fabrics after two-step treatment with 4 KU/mL of protease in the presence of 2% sodium thioglycolate. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). White arrows indicate polyester fibres present in the woven fabric labelled as ‘‘100% wool”. Two different images and magnifications (higher magnification on the left side of each set) are shown for each treatment.
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treatment for 5 days at 50 °C (Quartinello et al., 2018). The process we present in this work shows a complete degradation of wool fibres within 16 h of treatment in a two-step process. 3.6. Scanning electron microscopy of treated fabrics Fabric samples enzymatically treated in a two-step process at 50 °C with 4 KU/mL of protease in the presence of 2% sodium thioglycolate were analysed by scanning electron microscopy (SEM) (Fig. 5). This treatment condition was selected based on it delivering the highest level of breakdown for all fabric types. Complete breakdown of 100% wool fabrics (knit and woven) after 16 h of treatment was observed. Fig. 5A and B show remaining amorphous protein aggregates after treatment of 100% wool fabrics. In the case of 100% woven wool a small percentage of synthetic fibres was observed during SEM imaging. The synthetic fibres were present in the completely digested sample and were also detected in the control sample without enzyme (Fig. 5B). Complete decomposition of wool fibres in the fabric blends (knit and woven) was also determined by SEM. Fig. 5C shows 45/55% wool/polyester samples treated in a two-step process with 4 KU/mL of protease in the presence of 2% sodium thioglycolate and control samples without enzyme. Complete disappearance of the wool fibres in the polyester fabric blend was observed from enzymatically treated samples compared to control samples where polyester was still visible. Fig. 5D shows
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knit blend fabric treated with 4 KU/mL of protease in the presence of 2% thioglycolate and control samples without enzyme treatment. Unexpectedly, the 70/30% wool/polyester blend fabric that was purchased did not actually appear to be composed of polyester fibres, but a different synthetic fibre that could be nylon based on the infra-red spectra (Fig. 6). Nevertheless, the wool fibres in the 70/30% fabric blend were completely degraded after enzymatic treatment, while the synthetic fibres appeared to be intact. The recycling method presented here could also be applied for blends composed of other synthetic fibres rather than polyester. The presence of synthetic fibres in the 100% wool woven fabric shows another example of mislabelling and/or fibre contamination during fabric manufacture (Fig. 5B). If inaccurate labelling of fibres in textiles and garments is widespread then the successful application of recycling processes are at risk, as labelling will be important for determining the appropriate recycling process technology. Alternatively it will be necessary to employ in situ spectroscopic sorting methods (Wang et al., 2016). 3.7. Studies of polyester component of fabric blends after enzymatic treatment by infra-red spectroscopy and nano-indentation Wool/polyester blends and 100% polyester fabric samples (knit and woven) were characterised by attenuated total reflectance Fourier-transform-infrared spectroscopy (ATR FT-IR) before and
Fig. 6. Fourier-transform-infrared spectroscopy of treated and untreated fabric samples. ATR FT-IR spectrum of 100% wool sample (A), ATR FT-IR spectra of 100% polyester fabric sample before (red) and after (black) enzymatic treatment (B), ATR FT-IR spectra of 45/55% wool/polyester sample before (red) and after (black) enzymatic treatment (C) and ATR FT-IR spectra of a fabric sample labelled as ‘‘70/30% wool/polyester” before (red) and after (black) enzymatic treatment (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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after the two-step enzymatic treatment (4 KU/mL of protease, 2% sodium thioglycolate), to identify structural and compositional changes to the polyester. Weight loss was determined after the two stage enzyme treatment and confirmed to have reached 100% degradation for the wool/polyester blends and to have no weight loss on the 100% polyester fabrics (data not shown). The ATR FTIR spectrum of 100% wool is shown in Fig. 6A. It displays a broad signal at 3300 cm 1 corresponding to the amide NAH stretch. The
characteristic Amide I band (C@O stretch) is visible at 1630 cm 1 and the Amide II band (NAH bend) at 1530 cm 1 (McGregor et al., 2018). The ATR FT-IR spectra of 100% polyester before (red) and after (black) enzymatic treatment are shown in Fig. 6B. The characteristic carbonyl band (C@O stretch) is visible at 1710 cm 1. No significant changes are discernible post-treatment. The ATR FT-IR spectra of a 45/55% wool/polyester blend are shown in Fig. 6C. The spectrum before treatment (red) features
Fig. 7. Enzymatic treatments and analyses performed in this work. Two different temperatures, 37 °C and 50 °C, were tested for enzymatic degradation, with complete degradation of wool fibres achieved in a two-step process at 50 °C in the presence of the reducing agent sodium thioglycolate. Percentages correspond to weight loss percent for each fabric type. Results correspond to 5 5 sample size and 2% sodium thioglycolate where not indicated otherwise. SEM image corresponds to enzymatically treated 45/ 55% wool/polyester blend (A). Wool/polyester garment linear life cycle (B) and wool/polyester circular garment life cycle (C).
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the characteristic bands of both materials with the NAH stretch band at higher wavenumbers and the Amide I and II bands belonging to the wool as well as the polyester carbonyl group. After enzymatic removal of the wool component the characteristic amide bands are no longer visible and a pure polyester spectrum remains, confirming the complete removal of wool from the sample. The 70/30% wool/polyester knit fabric blend was also investigated before and after enzyme treatment (Fig. 6D). The ATR FT-IR spectra before and after treatment lack the dominant and characteristic carbonyl band of a polyester. Both spectra resemble a typical wool or polyamide spectrum without significant changes posttreatment (Ardanuy et al., 2012; McGregor et al., 2018; Wang et al., 2018). Based on this observation it was concluded that this sample was mislabelled and did not contain any polyester, but was either pure wool with incomplete degradation or contained a synthetic polyamide, such as nylon, as its blending partner. The results from ATR FT-IR spectra confirm our previous SEM observations, where synthetic fibres in the 70/30% knit blend appeared to be most likely nylon fibres. The polymers were further analysed by nano-indentation to investigate their mechanical properties before and after enzyme treatment. In the case of the 45/55% wool/polyester blend, the polyester was first extracted from the fabric by dissolving in HFIP. The weight of the dried extracted polyester matched the expected mass fraction of 55%. The sample was further processed as above. Nano-indentation of 100% polyester gave an E-modulus of 7.08 ± 0.8199 GPa before treatment, which dropped slightly to 6.05 ± 0.1529 GPa after treatment. The hardness was measured as 103.4 ± 19.3 MPa before treatment and 89.3 ± 4.5 MPa after treatment. Measurement of the polyester from the 45/55% sample yielded an E-modulus of 7.71 ± 0.1465 GPa before treatment, while the modulus was 8.02 ± 0.1118 GPa after treatment. The hardness was measured at 110.1 ± 6.9 MPa and 115.5 ± 3.6 MPa before and after treatment respectively (Table A.2). Analysis of enzymatically treated fabrics by ATR FT-IR confirmed the complete removal of the wool component from blends after treatment. The measurement of the physical properties of the treated and untreated polymers via nano-indentation revealed Emoduli and hardness values that are generally in the same range and confirmed that the mechanical properties of the polymers are not affected by the enzymatic treatment. Contrary to more severe chemical or thermal treatments that commonly decrease the quality of the recycled material, the recovered fibres from this process could in principle be integrated back into the production cycle for textiles or in other industries (Fig. 7B and C). In addition to the recovery of synthetic fibres, the keratin hydrolysate generated from the enzymatic breakdown of wool fibres has improved nutritional value compared to the chemically modified hydrolysate obtained from common thermochemical processes and could be used for bio-fertilisers, microbial growth media, animal feed or in the cosmetics industry (Brandelli et al., 2010). Chemical modification of keratin decreases the proteogenic amino acid content of the hydrolysate. For example, during alkaline hydrolysis important amino acids, such as asparagine, arginine, serine, and glutamine, can be destroyed while others are racemised (Dalev, 1990). Protease cleavage of keratin peptides bonds, on the other hand, generates a complex mix of peptides containing non-modified amino acids (Gousterova et al., 2005). Keratin hydrolysate has been reported as a source of bioactive peptides, like antioxidants, due to the high amount of hydrophobic amino acid residues like proline, histidine, tyrosine and tryptophan (Brandelli et al., 2015; Fontoura et al., 2014). Peptides in protein hydrolysates from several sources – e.g. milk casein, soybean, rice bran, canola, egg yolk protein, quinoa seed protein – have been reported to have diverse biological activities such as being antihypertensive, antimicrobial, antioxidant, wound-healing, osteogenic, and haemostatic, suggest-
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ing wool keratin hydroxylate as a possible source of functional byproducts (Ferraro et al., 2016; Lasekan et al., 2013). Furthermore, keratin hydrolysate has also been incorporated as a foaming agent in dyeing processes for cotton and wool fabrics (Bhavsar et al., 2017) or in biocomposite materials (Arslan et al., 2017; Fortunati et al., 2015). Fig. 7C illustrates the recovery process of the natural and synthetic components of a garment as part of a circular lifecycle were original materials are looped back into manufacturing of the same or a new product. 4. Conclusions The results obtained in this work provide an example of future sustainable textile practices and demonstrate for the first time an effective recycling strategy for wool fabric blends. Such blends are part of increasing environmental and economic problems associated with textile waste. Effective recycling of textiles through separation of constituent fibres will drive the fashion industry towards a circular economy model. Further research towards innovative applications of the recovered polyester and solubilised keratin in a range of industrial applications, and the selective separation of wool fibres from other synthetic fibres and postconsumer textile wastes could bring further insights and contribute further to closing the loop on fashion waste. Funding This work was supported by Queensland University of Technology, through the Institute for Future Environments Catapult funding scheme. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements The authors would like to acknowledge the Central Analytical Research Facility, operated by the Institute for Future Environments (QUT). Access to CARF is supported by funding from the Science and Engineering Faculty (QUT). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.10.026. References Ardanuy, M., Antunes, M., Velasco, J.I., 2012. Vegetable fibres from agricultural residues as thermo-mechanical reinforcement in recycled polypropylene-based green foams. Waste Manage. 32, 256–263. Arslan, Y.E., Arslan, T.S., Derkus, B., Emregul, E., Emregul, K.C., 2017. Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: From waste to regenerative medicine products. Colloids Surf. B: Biointerfaces 154, 160–170. Bhavsar, P.S., Zoccola, M., Patrucco, A., Montarsolo, A., Mossotti, R., Giansetti, M., Rovero, G., Maier, S.S., Muresan, A., Tonin, C., 2017. Superheated water hydrolyzed keratin: a new application as a foaming agent in foam dyeing of cotton and wool fabrics. ACS Sustain. Chem. Eng. 5, 9150–9159. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brandelli, A., Daroit, D.J., Corrêa, A.P.F., 2015. Whey as a source of peptides with remarkable biological activities. Food Res. Int. 73, 149–161. Brandelli, A., Daroit, D.J., Riffel, A., 2010. Biochemical features of microbial keratinases and their production and applications. Appl. Microbiol. Biotechnol. 85, 1735–1750.
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Braungart, M., McDonough, W., 2002. Cradle to Cradle: Remaking the Way We Make Things. North Point Press, New York. Burnett, C.L., Bergfeld, W.F., Belsito, D.V., Klaassen, C.D., Marks, J.G., Shank, R.C., Slaga, T.J., Snyder, P.W., Andersen, F.A., 2009. Final amended report on the safety assessment of ammonium thioglycolate, butyl thioglycolate, calcium thioglycolate, ethanolamine thioglycolate, ethyl thioglycolate, glyceryl thioglycolate, isooctyl thioglycolate, isopropyl thioglycolate, magnesium thioglycolate, methyl thioglycolate, potassium thioglycolate, sodium thioglycolate, and thioglycolic acid. Int. J. Toxicol. 28, 68–133. Cardamone, J.M., Nuñ, A., Garcia, R.A., Aldema-Ramos, M., 2009. Characterizing wool keratin. Adv. Mat. Sci. Eng. 2009, 1–5. Cline, E.L., 2012. Overdressed: The Shockingly High Cost of Cheap Fashion. Portfolio/ Penguin, Portfolio, New York. Dalev, P., 1990. An enzyme-alkaline hydrolysis of feather keratin for obtaining aprotein concentrate for fodder. Biotechnol. Lett. 12, 71–72. Eslahi, N., Dadashian, F., Nejad, N.H., 2013. An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis. Prep. Biochem. Biotechnol. 43, 624–648. Fang, Z., Zhang, J., Liu, B., Du, G., Chen, J., 2013a. Biochemical characterization of three keratinolytic enzymes from Stenotrophomonas maltophilia BBE11-1 for biodegrading keratin wastes. Int. Biodeterior. Biodegrad. 82, 166–172. Fang, Z., Zhang, J., Liu, B., Du, G., Chen, J., 2013b. Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Stenotrophomonas maltophilia BBE11-1. Bioresour. Technol. 140, 286–291. Ferraro, V., Anton, M., Santé-Lhoutellier, V., 2016. The ‘‘sisters” a-helices of collagen, elastin and keratin recovered from animal by-products: functionality, bioactivity and trends of application. Trends Food Sci. Technol. 51, 65–75. Fontoura, R., Daroit, D.J., Correa, A.P., Meira, S.M., Mosquera, M., Brandelli, A., 2014. Production of feather hydrolysates with antioxidant, angiotensin-I converting enzyme-and dipeptidyl peptidase-IV-inhibitory activities. New Biotechnol. 31, 506–513. Fortunati, E., Aluigi, A., Armentano, I., Morena, F., Emiliani, C., Martino, S., Santulli, C., Torre, L., Kenny, J., Puglia, D., 2015. Keratins extracted from Merino wool and Brown Alpaca fibres: thermal, mechanical and biological properties of PLLA based biocomposites. Mater. Sci. Eng.: C 47, 394–406. Fueghelman, M., 1997. Mechanical Properties and Structure of Alpha-Keratin Fibers: Wool, Human Hair and Related Fibers. University of New South Wales Press, Sydney. Ghisellini, P., Cialani, C., Ulgiati, S., 2016. A review on circular economy: the expected transition to a balanced interplay of environmental and economic systems. J. Clean. Prod. 114, 11–32. Gousterova, A., Braikova, D., Goshev, I., Christov, P., Tishinov, K., Vasileva-Tonkova, E., Haertle, T., Nedkov, P., 2005. Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Lett. Appl. Microbiol. 40, 335–340.
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Closing the textile loop: Enzymatic fibre separation and recycling of wool/polyester fabric blends Laura Navone a, Kaylee Moffitt a, Kai-Anders Hansen a, James Blinco a, Alice Payne b, Robert Speight a,⇑ a b
Science and Engineering Faculty, Queensland University of Technology, Brisbane, Queensland, Australia Creative Industries Faculty, Queensland University of Technology, Brisbane, Queensland, Australia
a r t i c l e
i n f o
Article history: Received 24 May 2019 Revised 8 September 2019 Accepted 12 October 2019
Keywords: Wool Keratinase Recycling Polyester Enzyme Textile
a b s t r a c t Textile waste presents a serious environmental problem with only a small fraction of products from the fashion industry collected and re-used or recycled. The problem is exacerbated in the case of postconsumer waste by the mixture of different natural and synthetic fibres in blended textiles. The separation of mixed fibre waste, where garments are often multicomponent, presents a major recycling problem as fibres must be separated to single components to enable effective recycling. This work investigates the selective digestion of wool fibres from wool/polyester blended fabrics using an enzymatic approach. Complete degradation of wool fibres was achieved by application of a keratinase in a two-step process with addition of reducing agent and undigested polyester fibres were recovered. Electron microscopy showed complete breakdown of the natural fibres in the fabric blends, while spectroscopic and mechanical analysis of the recovered synthetic fibres confirmed that the enzymatic treatment had no significant impact on the properties of the polyester compared to virgin samples. The polyester fibres are therefore suitable to be recycled to polyester yarn and re-used in the manufacture of new garments or other products. The nutrient rich keratin hydrolysate could be used in microbial growth media or incorporated into bio-fertilisers or animal feed, contributing to the development of the circular economy. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Textile waste is a serious environmental issue that will continue to grow if practical and economic solutions cannot be found that either reduce the production of waste or that close the loop and turn the waste into new products. Textile production processes require high volume of water, energy and chemicals (Pensupa et al., 2017). Globally, 64% percent of textile fibres are derived from petrochemicals and the remaining 36% is dominated by cotton (24%), manufactured cellulosic fibre (6%), wool (1%) and other natural fibres. The fashion industry with its current fast fashion business model, characterised by mass production, variety, agility and affordability, has greatly contributed to the volumes of waste and the rate at which this waste is generated. Fast fashion creates a demand for 80 billion new garments each year (Cline, 2012), yet garments typically follow a linear life cycle in which after use, garments are disposed to landfill rather than reused or recycled. This ‘cradle-to-grave’ (Braungart and McDonough, 2002) life cycle means the significant resources associated with textile production ⇑ Corresponding author at: Queensland University of Technology, 2 George Street, Brisbane, QLD 4000, Australia. E-mail address: [email protected] (R. Speight). https://doi.org/10.1016/j.wasman.2019.10.026 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
are wasted. In contrast, a circular economy model would mean keeping resources in use as long as possible through strategies such as reuse, repair, remanufacturing and recycling of products (Ghisellini et al., 2016). The growth of the textile industry is rapidly increasing the volumes of textile waste that predominantly end up in landfill or incinerators (WRAP, 2017), necessitating an immediate need for efficient recycling technologies to ‘close the loop’ and generate new value from textile waste streams. Current types of textiles recovery involve re-use for the same application, re-purposing for different applications and recycling. However, the major barrier to effective recycling is the large variety of materials used in apparel, and how they are blended (Payne, 2016). While collection of pre-consumer waste to generate products for insulation, carpet padding or industrial rags does occur, post-consumer waste is typically a diverse mixture of many fabric materials, which increases the level of complexity of recycling processes and reduces the opportunities for new products (Payne, 2016). There is an absolute requirement to separate blended fabrics into their components to allow effective recycling. For example polyester must be separated from cotton and wool to be able to be melted and reformed into polyester yarn (Jeihanipour et al., 2010). The challenge and complexity of separating and collecting the con-
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stituent fibres is currently the major technical and practical hurdle limiting recycling and landfill avoidance. There are currently no commercially viable separation and recycling technologies for these blended fabrics (Payne, 2016). While some advances have been made on mixed fibre separation technologies for cotton-synthetic blends (HKRITA, 2017; Schimper et al., 2008; Vasconcelos and Cavaco-Paulo, 2006), wool-synthetic blends are a waste-stream that requires new processes for recycling and removal of 1.1 million tons of waste from landfill (ICAC, 2013). Yet, only a small number of studies have been performed with limited success on the dissolution of wool in mixed fibre fabrics (Sandin and Peters, 2018). Studies attempting to dissolve wool fibres from garments have used extreme conditions that affect the mechanical properties of the synthetic fibres and can chemically modify the keratin peptides, thus reducing nutritional value if they are to be used as feed supplements (Brandelli et al., 2010; Cardamone et al., 2009; Gupta et al., 2013; Hou et al., 2014; Łaba and Szczekala, 2013; Sweetman, 1967). Investigations on the biological degradation of wool waste used 100% wool samples and selective degradation of wool fibres and recovery of synthetic polymers from wool blends has not yet been achieved (Eslahi et al., 2013; Fang et al., 2013a, b; Gousterova et al., 2005). The difficulties in degrading wool materials arise from the recalcitrant structure of the keratin protein, which is the main component of wool, and the overall need for sufficiently mild treatment conditions that maintain the mechanical properties of the synthetic polymers so that the recycling material meets performance specifications. Keratin accounts for 95% of wool weight and exhibits a high degree of disulfide bonding, which confers rigidity and chemical resistance (Cardamone et al., 2009). Selective and environmentally benign methods like the one presented here are necessary for efficient wool-synthetic textile blend recycling. We investigate the enzymatic treatment of wool/polyester fabric blends to selectively degrade wool fibres while recovering the synthetic fibres. A protease that was investigated previously for keratinolytic activity (Navone and Speight, 2018) was applied to fabric blends together with reducing agents. A two-step enzymatic process in the presence of sodium thioglycolate showed best results for degradation of wool fibres. Weight loss analysis and scanning electron microscopy (SEM) of the treated fabrics confirmed complete removal of the wool component. The purity and mechanical properties of the resulting polyester fibres were analysed by nano-indentation and infra-red spectroscopy. The polymer fibres obtained after enzymatic treatment retain the integrity and physical properties of virgin fibres and could be used in the manufacture of new garments or other textile and polyesterderived products. 2. Materials and methods 2.1. Fabric samples The fabric samples used in this work were supplied by Frost Textiles. The fabric types were 100% wool knit, 100% wool woven, 70% wool-30% polyester knit (note, this fabric was purchased on the basis of it being wool-polyester but IR and SEM analyses suggested that this particular fabric could be a wool-nylon blend), 45% wool-55% polyester woven, 100% polyester knit and 100% polyester woven fabrics.
dilution was added to 0.01 g of keratin azure in 2.4 mL of 100 mM Tris-HCl buffer pH 8 or pH 10. Samples were incubated at 37 °C for 1 h at 200 rpm. After incubation, samples were centrifuged at 4000 rpm for 10 min and the absorbance of the clarified supernatants was determined at 595 nm. Determinations for each enzyme dilution were performed in triplicate. For the negative control, 0.01 g of keratin azure in 2.5 mL of reaction buffer was incubated at 37 °C for 1 h with shaking at 200 rpm and the absorbance was measured at 595 nm. One keratin unit (KU) was arbitrarily defined as an increase of 0.1 in absorbance at 595 nm after incubation for 1 h at 37 °C according to previously reported methods (Navone and Speight, 2018). For keratinolytic activity determination in the presence of reducing agents, the keratin azure assay was performed as indicated with the addition of 1, 1.5 or 2% of sodium sulfite or 2, 3, 4 or 5% of sodium thioglycolate. Total protein was determined by Bradford assay (Bradford, 1976). Handling of sodium thioglycolate buffers was conducted according to reported safety indications (Burnett et al., 2009). 2.3. Enzymatic treatment of fabric samples, determination of soluble peptides and percentage degradation after treatment Ronozyme ProAct was the protease used in all experiments (provided by DSM Nutritional Products, Wagga Wagga, Australia). Fabric samples of 0.1 g were cut with scissors into pieces of approximately 5 5 mm or ground into pieces using a Rocklabs Ring Mill and fractioned into sample sizes of 0.6 mm using size sieves. Samples were treated with 2, 4 and10 KU/mL of protease in 5 mL of 100 mM Tris-HCl buffer pH 10 for 16 h at 37 or 50 °C at 200 rpm. When indicated, 1% sodium sulfite, or 2% or 3% sodium thioglycolate was added. Control fabric samples were incubated in reaction buffer without enzyme for 16 h at 50 °C at 200 rpm. Reducing agent was added where indicated. For the two step treatment, fabric samples of 0.1 g (5 5 mm pieces or ground pieces 0.6 mm) were treated with 2 or 4 KU/ mL of protease in 5 mL of 100 mM Tris-HCl buffer at pH 10 with 2% sodium thioglycolate for 6 h at 50 °C, 200 rpm. After 6 h, samples were centrifuged at 4000 rpm for 10 min and washed once with water, and 2 or 4 KU/mL of protease in 5 mL of 100 mM Tris-HCl buffer at pH 10 with 2% sodium thioglycolate was readded. Incubation proceeded for another 14–16 h at 50 °C, 200 rpm. Soluble peptides after enzymatic treatment were quantified by Bradford assay after 16 h of incubation (Bradford, 1976). Only peptides >3 KDa were quantified due to the lower mass limit of the Bradford assay. The percentage degradation was determined by weight loss. Remaining fabric samples after treatment were centrifuged, the supernatant removed, and completely dried to constant weight at 65 °C in an oven. The final weight was measured and % degradation calculated after subtracting the initial weight. Weight loss calculations were performed considering total available wool in each fabric type so that 100% weight loss corresponded to 100% loss of available wool in each fabric type. Statistical analysis was performed using one-tailed distribution t-test. The differences among means with P 0.01 were accepted as representing statistically significant differences. For analysis of the polyester component of fabric samples, the enzymatic treatment of fabric samples (5 5 mm pieces or ground pieces 0.6 mm) with 2 KU/mL of protease was scaled up to 4 g in a final volume of 200 mL. 2.4. Scanning electron microscopy
2.2. Keratinase activity determination Keratinolytic activity was determined using keratin azure (Sigma Aldrich) as the substrate following the manufacturer’s instructions with some modifications. Briefly, 100 lL of enzyme
Fabric samples treated in the two-step process at 50 °C with 2% sodium thioglycolate, with or without 4 KU/mL of protease, were centrifuged and air dried for several days, fixed in a sample holder stub and gold coated using a Leica EM SCD005 Gold Coater (to
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~10 nm). Secondary electron images were obtained with a Zeiss Rigma Field Emission Scanning Electron Microscope. Images were obtained under vacuum using 2 kV accelerating voltage. 2.5. Infra-Red spectroscopy Infra-red spectra were collected on a Nicolet Nexus 870 Fourier transform infrared (FTIR) spectrometer equipped with a Smart Endurance single bounce horizontal diamond ATR and a TEcooled deuterated triglycine sulfate (DTGS) detector (Nicolet Instrument Corp. Madison, WI). Spectra were recorded using 64 scans, 0.6329 cm s 1 mirror velocity and 4 cm 1 resolution over the range 4000–525 cm 1. Spectra were collected in absorbance mode and an ATR correction applied. Final processing was performed using GRAMS software (Thermo Galactic, Woburn, MA). To assess reproducibility of the spectra, 6 spots (3 on the exposed side and 3 on the unexposed side) of the 5 samples were analysed. Wool: IR (ATR): v = 3300 (NAH s), 1630 (Amide I, C@O s) and 1510 (Amide II, NAH b) cm 1. Polyester: IR (ATR): v = 1710 (C@O s) cm 1. 55/45 blend: Before: IR (ATR): v = 3300 (NAH s), 1710 (C@O s), 1630 (Amide I, C@O s) and 1510 (Amide II, NAH b) cm 1. After: IR (ATR): v = 1710 (C@O s) cm 1. 30/70 blend: IR (ATR): v = 3300 (NAH s), 1630 (Amide I, C@O s) and 1510 (Amide II, NAH b) cm 1. 2.6. Nano-indentation For sample preparation of 100% polyester (treated and untreated) and 45/55% wool/polyester blend (treated), 125 mg of polymer sample was dissolved in 1.75 mL of 1,1,1,3,3,3-hexa fluoro-2-propanol (HFIP) and filtered through a silica column. The solution was left to dry under air. The dried polymer was redissolved in 1.75 mL of HFIP and drop-cast onto a silicon wafer. For sample preparation for 45/55% wool/polyester blend (untreated), 227 mg of sample was treated with 3.5 mL of HFIP to extract the polyester. The resulting solution was separated and collected in a vial. The remaining fabric was washed with further 1.75 mL of HFIP. The extract was filtered through a silica plug and the collected clear solution dried under air. The dried polymer was redissolved in 1.75 mL of HFIP and drop-cast onto a silicon wafer. The dried polymer samples were subsequently analysed by nano-indentation. 3. Results and discussion 3.1. Enzymatic degradation of fabric blends in the presence of reducing agents Our previous studies investigated a protease with keratinolytic activity towards cattle hair and feather keratin with activity greatly enhanced in the presence of reducing agents (Navone and Speight, 2018). Fabric however, represents a greater challenge due to the structure and thickness of the fibres following the spinning process and the potential presence of dyes and other auxiliary chemicals. In this work, wool fabric samples were treated with protease in the presence of reducing agents to study the degradation of keratin from wool. Wool and polyester fabric blend samples (knit and woven) cut into 5 5 mm pieces were treated with 2, 4 and 10 KU/mL of protease at 37 °C. Wool breakdown was followed by determination of soluble peptides concentration and weight loss after treatment. Weight loss was calculated as a percentage of wool in the fabric. Fabric samples composed of 100% wool (knit and woven) were also treated under the same conditions (Fig. 1A and B). Fabric degradation without reducing agent ranged between 19 and 45% for all fabric types (Table A.3). The addition of sodium sul-
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fite did not show a significant (P 0.01) improvement in degradation for any of the fabric types, obtaining the most weight loss for 45/55% wool/polyester woven fabric (52 ± 4%). On the other hand, the presence of sodium thioglycolate significantly (P 0.01) improved degradation compared to treatment with no reducing agent or in the presence of sodium sulfite, reaching 76 ± 1% for 100% wool woven, 73 ± 2% for 45/55% wool/polyester woven, 52 ± 7% for 100% wool knit and 55 ± 1% for 70/30% wool/polyester knit fabric (Fig. 1). An increase in soluble peptide concentration was only observed when sodium thioglycolate was added (Fig. 1). The addition of reducing agent has been shown to be crucial for keratinase access to the peptide bonds in keratin in hair and feather (Navone and Speight, 2018). The results presented here confirm that the reduction of the disulfide bonds is equally important for wool degradation. Furthermore, sodium thioglycolate was found to be a better reducing agent for hair and feather degradation than sodium sulfite in previous studies (Navone and Speight, 2018). Sodium thioglycolate has higher reducing capacity than other reducing agents, including sodium sulfite (unpublished data). Using a reducing agent with a stronger reducing capacity gives a higher degree of reduction of disulfide bonds, allowing increased access of the protease to the keratin fibre and improved degradation. Previous research on the mechanism of keratin breakdown from hair at different time points during enzymatic treatment showed that lifting of the hair cuticle is the first step of degradation, followed by its complete removal and initial damage of the cortex (Navone and Speight, 2018). Fracturing of the cortex continues until the generated small keratin fragments are finally converted to soluble peptides along with residual insoluble protein. The dynamics of wool degradation are likely to be very similar to those observed for hair degradation due to the similar hierarchical structure between wool and hair (Fueghelman, 1997). 3.2. Temperature optimisation of fabric treatment The inability to obtain complete degradation of fabrics at 37 °C with or without reducing agents suggested that temperature optimisation studies of the enzymatic process should be conducted. The specific activity of the protease was determined using the keratin azure assay at 37 °C, 50 °C and 60 °C (Table 1). Since keratinase activity was highest at 50 °C, this temperature was selected for activity studies in the presence of the reducing agents sodium sulfite and sodium thioglycolate. Table 1 shows the specific activity of the protease in the presence of different concentrations of reducing agents. Increased keratinase activity was obtained with 1% sodium sulfite and 2 or 3% of sodium thioglycolate. Wool and wool/polyester fabric samples cut into 5 5 mm pieces were treated with 0, 2, 4 and 10 KU/mL of protease at 50 °C. Wool breakdown was followed by determination of soluble peptides concentration and weight loss after treatment (Fig. 2). In the case of the 45/55% blend woven fabrics, the presence of reducing agent in the enzymatic treatment increased the concentration of soluble peptides. However, the measured percent of degradation by weight loss did not improve in the presence of reducing agents. The opposite effect was observed for 100% wool woven fabric where soluble peptide concentration after treatment did not increase with reducing agents but weight loss was improved, particularly when using sodium thioglycolate. Relatively high values for soluble peptides were obtained for the control samples without enzyme in the presence of the reducing agents for both 100% wool fabrics and 45/55% wool/polyester blend but with no significant weight loss. A fabric component released during degradation at 50 °C (e.g. dye used for colouring) could generate a false positive result in the Bradford assay for the no enzyme controls.
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Fig. 1. Enzymatic degradation of wool fabric at 37 °C. Soluble peptides (mg peptides/g of wool) and weight loss (%) after treatment of wool woven fabrics (A) and wool knit fabrics (B) with 2, 4 or 10 KU/mL of protease with or without reducing agents. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). Data are represented as mean values ± standard deviation (n = 3).
Table 1 Keratinase activity of the protease determined by the keratin azure assay in the presence of reducing agents. Assay temperature (°C)
Reducing agent
Specific activity (103 KU/g)
37 50 60 50 50 50 50 50 50 50
– – – 1% sulfite 1.5% sulfite 2% sulfite 2% thioglycolate 3% thioglycolate 4% thioglycolate 5% thioglycolate
178 ± 13a 584 ± 38 432 ± 16 701 ± 87 389 ± 82 266 ± 25 940 ± 32 1014 ± 38 451 ± 39 236 ± 26
One keratinase unit (KU) is defined as an increase of 0.1 in absorption units at 595 nm after incubation with keratin azure for 1 h at 37, 50 or 60 °C. Data are represented as mean values ± standard deviation (n = 3). a Navone and Speight (2018).
Fabric degradation reached 95 ± 1% and 66 ± 1% in the presence of sodium thioglycolate for 100% wool knit or 70/30% wool/polyester knit blend, respectively. Significant statistical difference (P 0.01) was observed between 37 °C and 50 °C treatment without reducing agents or in the presence of sodium sulfite or sodium thiolgycolate for 100% wool or 70/30% wool/polyester knits (Table A.3). Degradation improvement at the higher temperature was less pronounced for woven fabrics. A statistical difference (P 0.01) between the 37 °C and 50 °C treatment was observed for 45/55% wool/polyester woven fabric without reducing agent
or in the presence of sodium thioglycolate, and for 100% woven fabric in the presence of sodium sulfite (Table A.3). Results showed best degradation in the presence of sodium thioglycolate obtaining 69 ± 1% and 60 ± 1% weight loss for 100% wool woven or 45/55% wool/polyester woven fabrics, respectively. Taken together, results suggest that the temperature increase from 37 °C to 50 °C improved enzymatic breakdown of keratin from wool. Fig. 2C shows a comparison of weight loss between 37 °C and 50 °C for the woven and knit fabrics in the presence of sodium thioglycolate. The only fabric type that reached almost complete degradation at 50 °C was the 100% wool knit fabric, 94 ± 1% with 2 and 4 KU/mL of protease, and 95 ± 1% with 10 KU/mL of protease. No significant difference (P 0.01) was observed between the degradation of 100% wool woven (66 ± 1% with 4 KU/mL), 45/55% woven blend (56 ± 5% with 4 KU/mL) or 70/30% knit blend (64 ± 1% with 4 KU/mL). Increasing the enzyme concentration from 2 to 10 KU/mL did not improve degradation under any of the conditions tested at 50 °C.
3.3. Size effect on the enzymatic treatment of fabric Each fabric type was ground and fractioned using a size sieve into pieces of 0.6 mm to test size effect on degradation. The addition of reducing agent to the enzymatic treatment of ground fabric had a very strong interfering effect on Bradford determination of soluble peptides (data not shown) and only weight loss determinations are presented for the following treatments.
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Fig. 2. Enzymatic degradation of wool fabric at 50 °C. Soluble peptides (mg peptides/g of wool) and weight loss (%) after treatment of wool woven fabrics (A) and wool knit fabrics (B) with 2, 4 or 10 KU/mL of protease with or without reducing agents. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). Comparison of weight loss (%) between treatment temperature of woven and knit fabrics with 2, 4 or 10 KU/mL of protease in the presence of 2% sodium thioglycolate (C). The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). 100% weight loss corresponds to complete removal of the available wool in each fabric type. Data are represented as mean values ± standard deviation (n = 3).
Fig. 3 shows weight loss (%) comparison between sample sizes 5 5 mm and 0.6 mm for 100% wool and 45/55% wool/polyester blend woven (Fig. 3A and B) and 100% wool and 70/30% wool/ polyester blend knit fabrics (Fig. 3C and D). No significant difference (P 0.01) on degradation was observed for 100% wool or
70/30% wool/polyester blend knit fabrics between the two sample sizes when treated without reducing agent or in the presence of sodium sulfite. However, degradation was significantly improved (P 0.01) in the presence of sodium thioglycolate for 5 5 mm sample sizes compared to 0.6 mm samples for 100% wool or
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Fig. 3. Enzymatic treatment of different size fabric samples with or without reducing agents. Weight loss (%) after treatment of 100% wool woven (A), 45/55% wool/polyester woven (B), 100% wool knit (C) and 70/30% wool/polyester knit fabrics (D) with 2, 4 or 10 KU/mL of protease with or without reducing agents. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). 100% weight loss corresponds to complete removal of available wool in each fabric type. Data are represented as mean values ± standard deviation (n = 3).
70/30% wool/polyester blend knit fabrics (Fig. 3C and D). No significant difference (P 0.01) was observed for 100% wool woven between sample sizes when treated with or without reducing agent (Fig. 3A). For the 45/55% woven blend, the 0.6 mm sample showed significantly (P 0.01) improved degradation compared to the 5 5 mm pieces in the presence of sodium sulfite or sodium thioglycolate (Fig. 3B). The woven blend almost reached complete degradation when the fabric was ground to 0.6 mm in the presence of 1% sodium sulfite (90 ± 4%) or 2% sodium thioglycolate (93 ± 13%). Yet the blended fabric only reached 50 ± 18% degradation with 1% sodium sulfite and 62 ± 5% degradation with 2% sodium thioglycolate when the sample size was 5 5 mm. Taken together, the results suggest that the sample size (5 5 mm or 0.6 mm) does not greatly influence the degradation capability of the enzyme towards wool keratin. The difference observed for the 45/55% wool/polyester blend 0.6 mm sample may be due to the specific nature of the fabric. 3.4. Enzymatic treatment with sodium thioglycolate Sodium thioglycolate was selected as the reducing agent for the following experiments since it showed increased degradation compared to sodium sulfite for all the fabrics tested. The concentration
of sodium thioglycolate was increased from 2 to 3% to test for degradation improvement. Enzymatic treatments were conducted with 2 or 4 KU/mL of protease only as the higher concentration of protease (10 KU/mL) did not appear to further improve degradation (Fig. 2). Fig. 4A shows weight loss for 5 5 mm or ground 0.6 mm samples of wool/polyester woven and knit blends treated with 2 and 4 KU/mL of protease with 3% sodium thioglycolate. No significant difference (P 0.01) in degradation between the 5 5 mm pieces and the ground 0.6 mm samples was observed for the 100% wool knit, 70/30% wool/polyester knit or 45/55% wool/polyester woven fabrics with 3% thioglycolate. A significant improvement in degradation (P 0.01) was only observed for 100% wool woven 0.6 mm samples compared to the 5 5 mm samples in the presence of 3% sodium thioglycolate. The sample size effect obtained here for the 100% wool woven fabric was not observed when the treatment was performed with the two sample sizes in 2% sodium thioglycolate (Fig. 3A). When comparing fabric treatment with the two different concentrations of sodium thioglycolate, degradation of the 5 5 mm pieces of the 45/55% wool/polyester blend was significantly (P 0.01) improved from 60 ± 1% in 2% sodium thioglycolate to 80 ± 5% in 3% sodium thioglycolate. On the other hand, the 0.6 mm sample of the same fabric blend reached 91 ± 4% in 2%
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Fig. 4. Enzymatic treatment with 3% sodium thioglycolate and two-step treatment of fabrics. Weight loss (%) after treatment of 100% wool woven, 45/55% wool/polyester woven, 100% wool knit and 70/30% wool/polyester knit fabrics with 2 or 4 KU/mL of protease in the presence of 3% sodium thioglycolate (A) and after two-step treatment with 2 or 4 KU/mL of protease in the presence of 2% sodium thioglycolate (B). The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). 100% weight loss corresponds to complete removal of available wool in each fabric type. Data are represented as mean values ± standard deviation (n = 3).
sodium thioglycolate and 80 ± 3% in 3% sodium thioglycolate (Figs. 3B and 4A), however, the difference in degradation percent was not significant (P 0.01). For the 70/30% wool/polyester knit blend, the increased concentration of sodium thioglycolate did not significantly (P 0.01) improve degradation for the 5 5 mm or the ground 0.6 mm samples (Figs. 4A and 3D). For the 100% wool knit fabric, the 5 5 mm samples showed a significant (P 0.01) decrease in degradation percent from 94 ± 0.6% in 2% sodium thioglycolate to 83 ± 0.4% in 3% sodium thioglycolate. The ground 0.6 mm samples showed no significant difference between the two concentrations of reducing agent (Figs. 4A and 3C). For the 100% wool woven fabric, significant improvement of degradation was observed for the 0.6 mm sample where degradation reached 95 ± 0.8% in 3% sodium thioglycolate compared with 61 ± 6.3% degradation in 2% sodium thioglycolate (Fig. 3A). No significant (P 0.01) degradation was difference was observed for the 5 5 mm pieces of 100% wool woven fabric.
No clear trend could be concluded from the increased concentration of sodium thioglycolate from 2 to 3%, for some of the fabric types and sample size degradation improved while in other cases degradation was decreased. Accordingly, the next experiments were conducted with 2% sodium thioglycolate.
3.5. Two-step treatment of fabric samples The inability to obtain complete degradation of some fabric samples could be related to an inactivation of the enzyme during several hours of incubation at 50 °C. To test this hypothesis, fabric samples cut into 5 5 mm pieces were treated in a two-step process, where each fabric was incubated with 2 or 4 KU/mL of protease with 2% sodium thioglycolate for 6 h, then centrifuged, washed with water and enzyme re-added (2 or 4 KU/mL) with 2% sodium thioglycolate and further incubated for 16 h. A first step of 6 h was chosen based on previous degradation experiments with
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hair and feather keratin (Navone and Speight, 2018). Weight loss percentages for all fabric types after a second treatment step are shown in Fig. 4B. All fabrics reached more than 90% degradation indicating that previous incomplete degradation could be related to enzyme inactivation (Table A.4). Enzyme activity was determined after 6 and 16 h of fabric treatment to investigate enzyme inactivation (Table A.1). Keratinase activity of the protease markedly decreased for all fabric types after 6 h of treatment at 50 °C. The remaining activity after 16 h of incubation was found to be very low, between 2 and 4% remaining activity for all fabric types. Such low enzymatic activity after 16 h of treatment suggests that keratin breakdown had been completed in a shorter period of time. According to the remaining activities showed in Table A.1, complete keratin degradation from wool most likely occurs within 6– 16 h, making the development of a shorter treatment feasible for industrial application. The presence of fabric increased the degree of enzyme inactivation, which was more evident for the 100% wool fabrics (Table A.1). The inactivation effect by the fabric could be related to chemicals associated with the fabric being released during treatment and that these chemicals are different between wool and polyester fibres. Unfortunately, due to the observed enzyme inactivation, the fabric treatment proposed in this work would not allow recovery of the enzyme for re-use. Optimisation of the process could include
a pre-treatment step with reducing agent to decrease the required time or temperature of the enzymatic treatment to obtain active enzyme at the end of the process for re-use. The pre-treatment could also get remove chemicals present in the fabric that might affect enzyme activity. Most studies on wool enzymatic degradation have been performed with 100% wool and usually from pre-consumer material. Wool has usually been treated with microbial whole cell cultures and in fewer cases with isolated enzymes (Fang et al., 2013b). One example of wool waste treatment with a commercial protease and a reducing agent used 100% wool fibres from spinning processes (Eslahi et al., 2013). The study reported 26% degradation by weight loss of wool waste after 4 h treatment at 55 °C and selective degradation of wool from synthetic fibres was not attempted. Enzymatic treatment of wool/cotton/polyester blended fabrics was recently conducted using a commercial protease and a cellulase cocktail (Quartinello et al., 2018). The fabric components were 2% wool, 61% cotton and the remaining fibres were polyester and polyamide. The authors reported a step-wise enzymatic extraction of textile fractions resulting in 95% and 85% extraction efficiency of wool and cotton constituents respectively. A protease treatment of the fabric blend was conducted for 2 days at 50 °C in the presence of sodium bisulfite as reducing agent, followed by filtration and drying of the fabric at high temperatures, with a final cellulase
Fig. 5. Scanning electron microscopy of two-step enzymatic treatment of fabric. Images correspond to 100% wool knit (A), 100% wool woven (B), 45/55% wool/polyester woven (C), and 70/30% wool/polyester knit (D) fabrics after two-step treatment with 4 KU/mL of protease in the presence of 2% sodium thioglycolate. The control was defined as a fabric sample treated with buffer solution only (0 KU/mL protease). White arrows indicate polyester fibres present in the woven fabric labelled as ‘‘100% wool”. Two different images and magnifications (higher magnification on the left side of each set) are shown for each treatment.
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treatment for 5 days at 50 °C (Quartinello et al., 2018). The process we present in this work shows a complete degradation of wool fibres within 16 h of treatment in a two-step process. 3.6. Scanning electron microscopy of treated fabrics Fabric samples enzymatically treated in a two-step process at 50 °C with 4 KU/mL of protease in the presence of 2% sodium thioglycolate were analysed by scanning electron microscopy (SEM) (Fig. 5). This treatment condition was selected based on it delivering the highest level of breakdown for all fabric types. Complete breakdown of 100% wool fabrics (knit and woven) after 16 h of treatment was observed. Fig. 5A and B show remaining amorphous protein aggregates after treatment of 100% wool fabrics. In the case of 100% woven wool a small percentage of synthetic fibres was observed during SEM imaging. The synthetic fibres were present in the completely digested sample and were also detected in the control sample without enzyme (Fig. 5B). Complete decomposition of wool fibres in the fabric blends (knit and woven) was also determined by SEM. Fig. 5C shows 45/55% wool/polyester samples treated in a two-step process with 4 KU/mL of protease in the presence of 2% sodium thioglycolate and control samples without enzyme. Complete disappearance of the wool fibres in the polyester fabric blend was observed from enzymatically treated samples compared to control samples where polyester was still visible. Fig. 5D shows
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knit blend fabric treated with 4 KU/mL of protease in the presence of 2% thioglycolate and control samples without enzyme treatment. Unexpectedly, the 70/30% wool/polyester blend fabric that was purchased did not actually appear to be composed of polyester fibres, but a different synthetic fibre that could be nylon based on the infra-red spectra (Fig. 6). Nevertheless, the wool fibres in the 70/30% fabric blend were completely degraded after enzymatic treatment, while the synthetic fibres appeared to be intact. The recycling method presented here could also be applied for blends composed of other synthetic fibres rather than polyester. The presence of synthetic fibres in the 100% wool woven fabric shows another example of mislabelling and/or fibre contamination during fabric manufacture (Fig. 5B). If inaccurate labelling of fibres in textiles and garments is widespread then the successful application of recycling processes are at risk, as labelling will be important for determining the appropriate recycling process technology. Alternatively it will be necessary to employ in situ spectroscopic sorting methods (Wang et al., 2016). 3.7. Studies of polyester component of fabric blends after enzymatic treatment by infra-red spectroscopy and nano-indentation Wool/polyester blends and 100% polyester fabric samples (knit and woven) were characterised by attenuated total reflectance Fourier-transform-infrared spectroscopy (ATR FT-IR) before and
Fig. 6. Fourier-transform-infrared spectroscopy of treated and untreated fabric samples. ATR FT-IR spectrum of 100% wool sample (A), ATR FT-IR spectra of 100% polyester fabric sample before (red) and after (black) enzymatic treatment (B), ATR FT-IR spectra of 45/55% wool/polyester sample before (red) and after (black) enzymatic treatment (C) and ATR FT-IR spectra of a fabric sample labelled as ‘‘70/30% wool/polyester” before (red) and after (black) enzymatic treatment (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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after the two-step enzymatic treatment (4 KU/mL of protease, 2% sodium thioglycolate), to identify structural and compositional changes to the polyester. Weight loss was determined after the two stage enzyme treatment and confirmed to have reached 100% degradation for the wool/polyester blends and to have no weight loss on the 100% polyester fabrics (data not shown). The ATR FTIR spectrum of 100% wool is shown in Fig. 6A. It displays a broad signal at 3300 cm 1 corresponding to the amide NAH stretch. The
characteristic Amide I band (C@O stretch) is visible at 1630 cm 1 and the Amide II band (NAH bend) at 1530 cm 1 (McGregor et al., 2018). The ATR FT-IR spectra of 100% polyester before (red) and after (black) enzymatic treatment are shown in Fig. 6B. The characteristic carbonyl band (C@O stretch) is visible at 1710 cm 1. No significant changes are discernible post-treatment. The ATR FT-IR spectra of a 45/55% wool/polyester blend are shown in Fig. 6C. The spectrum before treatment (red) features
Fig. 7. Enzymatic treatments and analyses performed in this work. Two different temperatures, 37 °C and 50 °C, were tested for enzymatic degradation, with complete degradation of wool fibres achieved in a two-step process at 50 °C in the presence of the reducing agent sodium thioglycolate. Percentages correspond to weight loss percent for each fabric type. Results correspond to 5 5 sample size and 2% sodium thioglycolate where not indicated otherwise. SEM image corresponds to enzymatically treated 45/ 55% wool/polyester blend (A). Wool/polyester garment linear life cycle (B) and wool/polyester circular garment life cycle (C).
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the characteristic bands of both materials with the NAH stretch band at higher wavenumbers and the Amide I and II bands belonging to the wool as well as the polyester carbonyl group. After enzymatic removal of the wool component the characteristic amide bands are no longer visible and a pure polyester spectrum remains, confirming the complete removal of wool from the sample. The 70/30% wool/polyester knit fabric blend was also investigated before and after enzyme treatment (Fig. 6D). The ATR FT-IR spectra before and after treatment lack the dominant and characteristic carbonyl band of a polyester. Both spectra resemble a typical wool or polyamide spectrum without significant changes posttreatment (Ardanuy et al., 2012; McGregor et al., 2018; Wang et al., 2018). Based on this observation it was concluded that this sample was mislabelled and did not contain any polyester, but was either pure wool with incomplete degradation or contained a synthetic polyamide, such as nylon, as its blending partner. The results from ATR FT-IR spectra confirm our previous SEM observations, where synthetic fibres in the 70/30% knit blend appeared to be most likely nylon fibres. The polymers were further analysed by nano-indentation to investigate their mechanical properties before and after enzyme treatment. In the case of the 45/55% wool/polyester blend, the polyester was first extracted from the fabric by dissolving in HFIP. The weight of the dried extracted polyester matched the expected mass fraction of 55%. The sample was further processed as above. Nano-indentation of 100% polyester gave an E-modulus of 7.08 ± 0.8199 GPa before treatment, which dropped slightly to 6.05 ± 0.1529 GPa after treatment. The hardness was measured as 103.4 ± 19.3 MPa before treatment and 89.3 ± 4.5 MPa after treatment. Measurement of the polyester from the 45/55% sample yielded an E-modulus of 7.71 ± 0.1465 GPa before treatment, while the modulus was 8.02 ± 0.1118 GPa after treatment. The hardness was measured at 110.1 ± 6.9 MPa and 115.5 ± 3.6 MPa before and after treatment respectively (Table A.2). Analysis of enzymatically treated fabrics by ATR FT-IR confirmed the complete removal of the wool component from blends after treatment. The measurement of the physical properties of the treated and untreated polymers via nano-indentation revealed Emoduli and hardness values that are generally in the same range and confirmed that the mechanical properties of the polymers are not affected by the enzymatic treatment. Contrary to more severe chemical or thermal treatments that commonly decrease the quality of the recycled material, the recovered fibres from this process could in principle be integrated back into the production cycle for textiles or in other industries (Fig. 7B and C). In addition to the recovery of synthetic fibres, the keratin hydrolysate generated from the enzymatic breakdown of wool fibres has improved nutritional value compared to the chemically modified hydrolysate obtained from common thermochemical processes and could be used for bio-fertilisers, microbial growth media, animal feed or in the cosmetics industry (Brandelli et al., 2010). Chemical modification of keratin decreases the proteogenic amino acid content of the hydrolysate. For example, during alkaline hydrolysis important amino acids, such as asparagine, arginine, serine, and glutamine, can be destroyed while others are racemised (Dalev, 1990). Protease cleavage of keratin peptides bonds, on the other hand, generates a complex mix of peptides containing non-modified amino acids (Gousterova et al., 2005). Keratin hydrolysate has been reported as a source of bioactive peptides, like antioxidants, due to the high amount of hydrophobic amino acid residues like proline, histidine, tyrosine and tryptophan (Brandelli et al., 2015; Fontoura et al., 2014). Peptides in protein hydrolysates from several sources – e.g. milk casein, soybean, rice bran, canola, egg yolk protein, quinoa seed protein – have been reported to have diverse biological activities such as being antihypertensive, antimicrobial, antioxidant, wound-healing, osteogenic, and haemostatic, suggest-
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ing wool keratin hydroxylate as a possible source of functional byproducts (Ferraro et al., 2016; Lasekan et al., 2013). Furthermore, keratin hydrolysate has also been incorporated as a foaming agent in dyeing processes for cotton and wool fabrics (Bhavsar et al., 2017) or in biocomposite materials (Arslan et al., 2017; Fortunati et al., 2015). Fig. 7C illustrates the recovery process of the natural and synthetic components of a garment as part of a circular lifecycle were original materials are looped back into manufacturing of the same or a new product. 4. Conclusions The results obtained in this work provide an example of future sustainable textile practices and demonstrate for the first time an effective recycling strategy for wool fabric blends. Such blends are part of increasing environmental and economic problems associated with textile waste. Effective recycling of textiles through separation of constituent fibres will drive the fashion industry towards a circular economy model. Further research towards innovative applications of the recovered polyester and solubilised keratin in a range of industrial applications, and the selective separation of wool fibres from other synthetic fibres and postconsumer textile wastes could bring further insights and contribute further to closing the loop on fashion waste. Funding This work was supported by Queensland University of Technology, through the Institute for Future Environments Catapult funding scheme. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements The authors would like to acknowledge the Central Analytical Research Facility, operated by the Institute for Future Environments (QUT). Access to CARF is supported by funding from the Science and Engineering Faculty (QUT). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.10.026. References Ardanuy, M., Antunes, M., Velasco, J.I., 2012. Vegetable fibres from agricultural residues as thermo-mechanical reinforcement in recycled polypropylene-based green foams. Waste Manage. 32, 256–263. Arslan, Y.E., Arslan, T.S., Derkus, B., Emregul, E., Emregul, K.C., 2017. Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: From waste to regenerative medicine products. Colloids Surf. B: Biointerfaces 154, 160–170. Bhavsar, P.S., Zoccola, M., Patrucco, A., Montarsolo, A., Mossotti, R., Giansetti, M., Rovero, G., Maier, S.S., Muresan, A., Tonin, C., 2017. Superheated water hydrolyzed keratin: a new application as a foaming agent in foam dyeing of cotton and wool fabrics. ACS Sustain. Chem. Eng. 5, 9150–9159. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brandelli, A., Daroit, D.J., Corrêa, A.P.F., 2015. Whey as a source of peptides with remarkable biological activities. Food Res. Int. 73, 149–161. Brandelli, A., Daroit, D.J., Riffel, A., 2010. Biochemical features of microbial keratinases and their production and applications. Appl. Microbiol. Biotechnol. 85, 1735–1750.
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