Comparative Study on the Sequential Effects of Ozone and Single-Bath-Single-Stage Enzymatic Treatments in Sustainable Denim Processing: Enzyme-Only, Post-Ozone, and Pre-Ozone Treatments

Comparative Study on the Sequential Effects of Ozone and Single-Bath-Single-Stage Enzymatic Treatments in Sustainable Denim Processing: Enzyme-Only, Post-Ozone, and Pre-Ozone Treatments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Study on the Sequential Effects of Ozone and Single-Bath-Single-Stage Enzymatic Treatments in Sustainable Denim Processing: Enzyme-Only, Post-Ozone, and Pre-Ozone Treatments Jannatuz Faria, A J M Kamruzzaman Shakey This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7250614/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Nowadays, all age groups have shown a renewed interest in denim clothing due to its comfortable, worn-out style and altered appearance. Numerous technological advancements, including significant advancements in spinning, washing, and finishing, contribute to denim's status as one of the most iconic fashion items. In the textile sector, the traditional denim washing method is thought to be the most polluting. This industry produces an abundance of wastewater with an increased pollutant load and utilizes a lot of energy, water, and chemicals. So, this research investigates the combination of ozone treatment with single-bath-single-stage enzymatic treatment at 65°C temperatures on denim garments to not only achieve the desired aged or faded appearance but also save the environment, water, and time. Where three types of enzymes were used, including amylase (for desizing), cellulase (for removing producing fibers), and laccase (for removing indigo dyes). On the other hand, ozone works as a bleaching agent for denim garments, and it is a waterless treatment that makes our denim sector more sustainable. The samples from three washing recipes were analyzed properly for tensile strength, seam strength, color changes, and back staining. The highest fading of denim garments was obtained when first applying enzymatic treatment and then applying ozone treatment. This method adds a new dimension to denim textiles. Enzymatic Treatment Ozone Treatment Denim washing Sustainability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Denim, a popular dress material for over 30 years, is embraced by children, women, and men for its comfort and versatility. Initially created in the 1980s using 100% polyester threads, it is now one of the most popular cotton dress fabrics (Hassabo et al., 2023 ). So, it is a durable cotton warp-faced twill weave fabric that is thought to be extremely strong, stiff, and hard-wearing. It is distinguished from cotton duck by the diagonal ribbing created when the weft flows under more than one warp thread (Elmogahzy, 2019 ; Razzaque, 2004 ). Indigo is used to dye the warp of traditional blue denim. Numerous denim washing techniques, including the use of sand, stones, cellulase enzymes, potassium permanganate or sodium hypochlorite, hydrogen peroxide, and caustic soda, can remove the pigment from the surface and give denim a distinctive or faded or aged appearance (Choudhury, 2017 ). Denim washing was traditionally accomplished with a lot of chemicals, such as hydrogen peroxide, hypochlorite, and sodium permanganate, to achieve that popular aged and faded look. In a consistent way, they would diminish the clothes' true color. However, the reality that these chemicals pose serious environmental concerns is a major disadvantage (Halim et al., 2021 ). Also, these traditional methods of denim washing generate a lot of contaminated effluents and consume a lot of water (Hasanbeigi & Price, 2015 ). In this sense, enzymatic treatment in denim washing is both environmentally beneficial and efficient for producing sustainable denim clothing. Once more, consumers' attitudes toward the environment and their purchasing habits have grown, and the aesthetic qualities of the product also have an impact on their decisions (Butler & Francis, 1997 ; Eckman, 1997 ). Enzymes, which are classified as biocatalysts because they can speed up biochemical processes without being used, are created by the metabolism of microbes, especially bacteria. They are extensively employed in home washing, pharmaceutics, genetically modifying plants and animals, and figuring out the chemical design of DNA. Amylase is the most commonly used enzyme in textiles, and it is typically used to hydrolyze starch and reduce fabric size (Jegannathan & Nielsen, 2013 ). Traditionally, denim fabric is treated with diluted hypochlorite to partially bleach or remove environmentally harmful stains. Cellulase, either by itself or in combination with pumice stone, causes denim clothing to abrasively age. Additionally, laccase is employed to darken the indigo-dyed substance (Liu et al., 2013 ). On the other hand, ozone treatment is an environmentally friendly method that conserves electricity and water. "Ozone" (O₃) is the term for laundry, a textile care idea that uses a unique combination of electricity and oxygen in place of many of the chemicals commonly utilized in a traditional washing operation (Hoque & Faysal, 2019 ). Water Resources Ozone washing systems greatly lessen the environmental impact of traditional washing processes. The clothes can be bleached using this method. Denim clothing is bleached in a machine for washing by dissolving ozone in water. Additionally, denim clothing can be bleached or faded in a controlled environment using ozone gas (Eren et al., 2020 ). By lowering rinse times, water usage, and energy consumption, ozone finishing denim jeans has both economic and environmental advantages. Textile wastewater can be decolored by ozone, a specialist oxidant with a high oxidation potential that has a unique affinity for indigo (Sarker et al., 2021 ). As an alternative to traditional denim washing, Islam, Mohammad Tajul, et al. conducted an experiment using a single-bath-single-stage enzymatic treatment of denim. Here, denim is treated with a mixture of cellulase, amylase, and laccase at varying temperatures to get the desired aged or faded look. You can change the amount of laccase enzyme and the washing temperature to give denim cloth an aged appearance. The denim garment that faded the most was washed with 1.5 g/L of cellulase, 1.5 g/L of amylase, and 2 g/L of laccase enzyme for 45 minutes at 65°C. Since the resulting wastewater needed nearly four times as much oxygen to decompose as the wastewater from the conventional process, the enzymatic approach was determined to be far more environmentally friendly. The effectiveness of enzymatic treatments was highlighted by the fact that this temperature choice had no detrimental effects on the fabric's tensile strength or abrasion resistance (Islam et al., 2024 ). Our study aims to following this single-stage-single-bath enzymatic treatment combined with ozone treatment to create a sustainable process for denim sector and reducing use of toxic chemical, water and time. In addition to improving the findings' scalability and applicability, this practical implementation offers insightful information on how well textile treatments function in environments that closely resemble industrial processes. Additionally, a wide range of tests were included in our analysis, such as tensile strength, back staining, abrasion, and seam strength testing. Through this method, we not only get a good denim fading effect but also protect our environment from pollution and save water and time. 2. Materials and methods 2.1. Materials The denim fabric utilized for the study was provided by NZ TEX GROUP (Code: NZ-10217 HWS), Gulshan, Bangladesh. Since the cloth was non-desized and in its raw state, it was washed straight away without any additional processing. The fabric weighed 10.75 oz per square meter and was made entirely of 100% cotton. Its GSM was 369. With a structure of 61 ends per inch (EPI) and 38 picks per inch (PPI), it had a 3/1 twill weave. The warp and weft yarn counts were 6 Ne and 10 Ne, respectively. To test the pocketing fabric's ability to stain the back of denim after the enzymatic washing process was finished, it was affixed to the denim. Sunshine Textile, where the pocketing cloth was gathered. The cloth had a 1/1 plain weave and was composed entirely of 100% TC fabric. It was 118 (ends/inch) by 68 (picks/inch) in size. 100% polyester sewing thread that was bought from the Coats Company was used to sew the pocket. 20/2 was the sewing thread count. DW 16LT (an amylase enzyme) made in TURKEY, LAVA CELL NDF (a cellulase enzyme) made in INDIA, and LAVA ZYME LITE NEU (a laccase enzyme) were collected from Bangladesh. Folosen NOF (a non-ionic wetting agent) was collected from CHT Germany GMBH, IMACOL X JET LIQ (a non-ionic anti-creasing agent) was collected from Archroma Singapore PTE Ltd, and Anti Back Stainer K-300 (a non-ionic dispersing agent) was collected from Guangdong Kesida New Material Technology Co., Ltd. 2.2. Methods 2.2.1. Preparation of denim A high-speed lockstitch machine (Brother, China) was used to stitch denim fabric with a superimposed seam and 1 cm allowance and 8 stitches per inch in order to prepare the denim leg sample before washing Fig. 1 . In order to test the fabric sample's back staining and seam strength, a piece of pocketing fabric was also sewn to the main denim samples using the lock stitch on the same machine. The sewing thread was composed of 100% polyester collected from Coats Company, and the pocketing cloth was 100% TC fabric. Also, an overlock machine (JUKI, China) was used to overlock the denim cloth for safety purposes. The sewing thread was composed of 100% polyester collected from RUNNER Company; 40/2 was the sewing thread count. 2.2.2. Ozonition Method The Jeanlogia G2 ozone machine was used for ozonation treatment of denim fabric. Ozone, a gas formed naturally by photochemical reactions with solar ultraviolet radiation, can be generated artificially through corona discharge, a method involving electrical discharges from an ozone generator. A three-atom oxygen molecule (O₃) is created when the "O" atoms link with the O₂ molecules in Eq. (1). O 2 + O⋅ ↔ O 3 (1) Since ozone is unstable and cannot be transported or stored, it must be created as needed. The drum that holds the fabrics is injected with the resultant ozone. Similar to how sunshine ages textiles, ozone organically ages denim (Ben Fraj & Jaouachi, 2021 ). 2.2.3. Washing Treatment The single-bath-single-stage enzymatic washing was carried out with three different experiments based on the ozone process to find out the optimum washing recipe for which the best fading effect can be achieved. The amounts of amylase, cellulase, and laccase enzymes were fixed at 1.5 g/L, 1.5 g/L, and 2 g/L based on established industry recommendations for their typical application in denim washing processes. For the first experiment, to complete the washing process in one step, the sample washer extractor machine (Tolker, made in Turkey) was filled up with 30 L of water for each recipe. Here time is 45 m and temperature is 65°C. Wetting agent, dispersing agent, and anti-creasing agent were added at 25°C temperature. The drum was then rotated for 2 min, followed by the addition of the enzymes—amylase, cellulase, and laccase. The liquid amylase enzyme was directly prepared to achieve a concentration of 1.5 g/L (V/V). In contrast, cellulase and laccase powders were dissolved in water using a magnetic stirrer, resulting in concentrations of 1.5 g/L (W/V) for cellulase and 2 g/L (W/V) for laccase. After adjusting the pH to 4.5, the raw denim sample was added to the drum, and the temperature of the bath was raised to 65°C. That temperature was maintained for 45 min. After this the temperature was raised to 90°C, and the machine was run for 5 min to deactivate the enzyme. Then the rinse wash was given two times with 40 L of water. The temperature was dropped down to 25°C, and the sample was removed. Lastly, the sample was hydroid by a hydro extractor machine for 2m and dried for 20m Fig. 2 . And the 1st system was done (Islam et al., 2024 ). For the 2nd experiment (post-ozone treatment), we first followed the single-bath-single-stage enzymatic treatment like in the 1st experiment. Then did the ozone treatment by the Jeanlogia G2 machine, where gas power was 50% and cycle time was 10 min Fig. 3 . For the second experiment (pre-ozone treatment), we first did ozone treatment by the Jeanlogia G2 machine, where gas power was 50% and cycle time was 10 min. And then followed the single-bath-single-stage enzymatic treatment, like in the 1st experiment Fig. 4 . 2.2.4. Visual test Each washed sample's color variation from the raw sample was visually assessed. Based on how closely the color of each sample matched that of the main sample, each sample was assigned one of three color ratings: high, medium, and low. Three separate observers evaluated the samples' color differences, and each sample's highest vote rating was recorded. 2.2.5. Spectrophotometer test A Datacolor 1050 spectrophotometer (Dual-Beam Spectrophotometer by Datacolor, USA) was used to measure the reflectance of the cleaned samples under the following circumstances: Pulsed Xenon with a wavelength range of 300 nm to 700 nm, filtered to roughly D65 light, and a 10° viewing measurement geometry. With the aid of pertinent software, the color strength values (K/S) of the dyed samples were determined using Datacolor 1050 and the Kubelka-Munk Eq. ( 2 ) as follows: $$\:\frac{K}{S}=\frac{(1-{R}_{\lambda\:\:\text{m}\text{a}\text{x}}{)}^{2}}{{2R}_{\lambda\:\:max}}$$ 2 Where K is the absorption coefficient, S is the scattering coefficient, and Rλmax is the sample's reflectance value at a certain wavelength where a given dye or color component absorbs the most. 2.2.6. Back Staining Using a spectrophotometer (Datacolor, USA), the back staining of the pocketing fabrics was assessed as a whiteness index. The process is the same as that which was previously described for shade change. 2.2.7. Tensile Strength The stripe test, using the BS EN ISO 13934-2:2014 method, was used to assess the samples' tensile strength in warp directions. The tensile strength was assessed using the B.N. Titan Tester (UK), in which each sample was cut to a size of 200 mm by 50 mm, placed between the top and bottom grippers of the tester independently, and then pushed to break at a pace of 100 mm per minute. 2.2.8. Seam strength B.N. Titan Tester (UK) was used to assess the samples' seam strength in accordance with the BS EN ISO 13935-2:2014 standard. Here, a 200 mm by 50 mm specimen was fastened between the top and bottom grippers of the B.N Titan Tester and dragged at a pace of 50 mm per minute until it broke. 2.2.9. Abrasion Using the BS EN ISO 12947-2:2016 technique, the B.N. Martindale abrasion and pilling tester (UK) evaluated the samples' abrasion. Using this approach, a specimen with a diameter of 38 mm was abraded using an abrading cloth with a diameter of 140 mm under 9 kPa weight in order to assess the mass loss following particular cycles. Here, the sample's mass loss from abrasion was calculated for cycles of 5000. 3. Result and discussion 3.1. Color fading test 3.1.1. Visual assessment test The 3 different images in Fig. 5 show the samples derived from the 3 different processing methods, like enzyme-only, post-ozone, and pre-ozone treatments. The sample SC-2 showed the highest fading of color compared to the raw denim sample. And sample SC-3 had the next best fading result. Sample SC-1 showed the least fading of color. This result can be linked to the ozone process in those samples. SC-2 was applied using an ozone-based process after a single-bath-single-stage enzymatic treatment; for this reason, SC-2 had the highest fading effect. On the other hand, SC-3 was applied using an ozone-based process before a single-bath-single-stage enzymatic treatment. For this reason, the ozone process was not working properly, and SC-3 had the next highest fading effect. SC-1 had no ozone-based process, so it had the lowest fading effect. 3.1.2. Spectrophotometer assessment The spectrophotometer assessment, which involved evaluating each sample under a spectrophotometer, was the second method of color fading test used in this investigation. The color value results and changes in those values for samples treated with various washing procedures are shown in Table 1 . The degree of fading effect of the enzymatically treated samples can be assessed using the DL* value, which shows how light or dark the colored substrate is. Sample SC-1 has the lowest DL* value in this case, while sample SC-2 has the greatest value. The sample with the highest DL* value has a more faded effect than the others. Table 1 Spectrophotometer assessment of three treated sample Recipe no Sample code Process DL* Da* Db* 1 SC-1 Only Enzymatic treatment 1.16 -0.35 -8.22 2 SC-2 Post-ozone process 6.35 -0.99 -9.75 3 SC-3 Pre-ozone Process 3.42 -0.36 -8.38 Like the DL* number above, the difference between Da* and Db* values can also be taken into account when assessing the degree of fading. Accordingly, greater fading is shown by more shifting in the treated samples' Da* and Db* values as compared to the grey sample. In terms of Da*, sample SC-2 is either more greenish or less reddish than the other samples. Yellowish and bluish values are indicated by the Db* values. In this case, sample SC-2 is less yellowish and more bluish than the other samples. 3.2. Back staining test Figure 6 shows the whiteness of three pocketing samples that were subjected to different washing procedures. The pocketing fabric of sample SC-2 had a greater whiteness rating, indicating less indigo staining during the sample's washing process. This suggests less color migration, which causes more fading, as the treated denim sample's visual and spectrophotometric evaluations also verified. However, sample SC-1 showed the least amount of whiteness in the pocketing fabric, indicating that this sample had more indigo staining throughout the procedure. Because without significantly reducing the textiles' strength, ozone was capable of bleaching the denim jeans and lessening back-staining (Kamppuri & Mahmood, 2019 ). 3.3. Tensile strength test The tensile strength of several bio-washed samples is displayed in warp-wise directions in Fig. 7 , as previously mentioned. Looking at the tensile strength of the warp-wise yarns, the figure shows that the sample SC-1 washed only with enzymatic treatment has the medium tensile strength compared to the other two samples. The reason behind this is that the cellulase enzymes used to eliminate cellulosic protruding fibers might have the maximum activation at 65°C temperature. As a result, after eliminating cellulosic protruding fibers, it might attack the surface cotton fibers of the yarns, hydrolyze the fibers to some extent, and make the yarns weaker while treated at 65°C (Montazer & Sadeghian Maryan, 2010 ). It is also observed that samples that were washed with post-ozone treatment have minimum warp yarn tensile strength. 3.4. Abrasion test Figure 8 shows that the samples SC-1 & SC-3 have a similar amount of weight loss after the abrasion test. Because the cellulase enzyme used to remove protruding fibers might attack surface fibers and make them weaker, as it probably shows maximum activation at 65°C temperature. In addition to that, the amylase enzyme also shows higher efficiency at this temperature and eliminates maximum-size materials, making the yarns softer and susceptible to abrasion. Which in turn leads to higher weight loss (Panwar et al., 2020 ). On the other hand, sample SC-2 indicates the maximum weight loss compared to the other two samples. Because The enzyme acts on the cellulose fiber, hydrolyzing it and weakening it. Then, when ozone is added, this weakened fiber is further oxidized, so more breakdown occurs and more weight loss. 3.5. Seam strength test The seam strength of various bio-washed samples is shown in Fig. 9 . All of the cleaned samples showed no discernible difference in seam strength. As previously mentioned, the sewing thread was made entirely of polyester, which is completely unaffected by temperature or any of the chemicals utilized. The denim portion lost its strength as a result of all the chemical processing, which explains the modest loss in seam strength observed in the samples (Islam et al., 2016 ). 4. Conclusion Usually the process of washing denim is very long because it has different steps. Each step requires a lot of water, time, and chemicals. That is why the process of washing denim is not suitable for our environment, and its production cost is also high. But its demand is increasing day by day. This is why researchers have come up with an innovative method that combines ozone (post and pre) treatment with enzymatic treatment. This innovative method uses three enzymes, including 1.5 g/L amylase, 1.5 g/L cellulase, and 2 g/L laccase, at a temperature of 65°C for 45 minutes. Also uses 50% ozone gas for 10 minutes. This study showed that single-bath-single-stage enzymatic treatment was applied for the first step, and then ozone treatment was used, and a satisfactory fading effect was obtained. Also, it showed enhanced back staining, which suggests that the indigo dyes were better washed off the denim fabric, giving the garment a more aged or faded appearance. However, fading varied with the variation of applied ozone treatment. Future studies should examine the suitability of this single-bath-single-stage enzymatic treatment, with different ozone gas treatment options for different blue and sulfur-colored denim, to further broaden the scope of this study. Various sustainable processes such as laser, ultraviolet, ultrasound, plasma, and waterjet treatment should also be added to save water and time and make the environment safer. Positive outcomes would further support the implementation of this novel strategy in industrial practice, possibly displacing the traditional denim washing procedure and promoting more economical and environmentally friendly production techniques. Declarations Acknowledgements This is not applicable. Funding The authors affirm that no funding, money, or other assistance was obtained in order to prepare this work. Authors contribution Jannatuz Faria: Writing – original draft, review & editing, Supervision, Investigation, Conceptualization. A J M Kamruzzaman shakey: Writing – original draft, Visualization, Formal analysis. Ethical Approval This is not applicable. Consent to Participate This is not applicable. Consent to Publish This is not applicable. Competing interest The authors state that none of the work described in this study could have been influenced by any known competing economic interests or personal relationships. 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Int J Curr Eng Technol 11(05):516–621. https://doi.org/10.14741/ijcet/v.11.5.4 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 19 Dec, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers invited by journal 19 Aug, 2025 Editor assigned by journal 08 Aug, 2025 First submitted to journal 04 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Faria","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PsYrCMBjA8S8EkuU7u7bovUNF6KJcXiVQiEu3m+SGq7j6AG6+ReaUgDfdfuBiFXS5pcOBwg02t93S1k0w/yEJIT+SAPh8dxjPAYxbkPWp2NUz9toIGkckAM2zNHYbrAsBR3guk9Ct2wn/LM3lbMVTbtTsJ3sZMKDl/quJ4DQultLSaD7fbJ91Wj+MjUZZAxGgwKC0bLgAtY00rQmyfhPB4AjFr7QoNpC8Rvq9AwkV2PqWkCwhIZW2XcgR7EBNY7LCtE/0BzLa8hcMFK2+J2NBVryoLvpNBHxRHprIvyj+jV2Pu8j5ltM+n8/3MF0BdhRElRHArmIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0004-8626-1275","institution":"Bangladesh University of Textiles","correspondingAuthor":true,"prefix":"","firstName":"Jannatuz","middleName":"","lastName":"Faria","suffix":""},{"id":502513983,"identity":"2b7584e1-4d75-4cb4-9875-dd5d5d743acf","order_by":1,"name":"A J M Kamruzzaman Shakey","email":"","orcid":"","institution":"Bangladesh University of Textiles","correspondingAuthor":false,"prefix":"","firstName":"A","middleName":"J M Kamruzzaman","lastName":"Shakey","suffix":""}],"badges":[],"createdAt":"2025-07-30 08:41:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7250614/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7250614/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90043316,"identity":"bcd093a6-2e6e-4f6e-a891-ee90cc7b8168","added_by":"auto","created_at":"2025-08-27 17:33:17","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142039,"visible":true,"origin":"","legend":"\u003cp\u003eUsed superimposed seam (A) for making denim leg sample (B).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/85309c4119df3c6824254f60.jpeg"},{"id":90042822,"identity":"a22afc2f-b1c0-43e1-ad00-c18a94f26323","added_by":"auto","created_at":"2025-08-27 17:25:17","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99299,"visible":true,"origin":"","legend":"\u003cp\u003eTime-temperature profile that was followed for 1\u003csup\u003est\u003c/sup\u003e experiment\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/3a584e350f7ab6cfdad1059a.jpeg"},{"id":90042826,"identity":"c508f5aa-22f7-420b-b406-75c354dffcbb","added_by":"auto","created_at":"2025-08-27 17:25:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86873,"visible":true,"origin":"","legend":"\u003cp\u003eTime-temperature profile then the Ozone treatment that was followed for 2nd experiment.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/29e2f92fa8dc2daeb4d28b6b.png"},{"id":90043802,"identity":"002a886c-6fe2-47c6-9b15-5d0eb5095e4c","added_by":"auto","created_at":"2025-08-27 17:41:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94173,"visible":true,"origin":"","legend":"\u003cp\u003eOzone treatment then time-temperature profile that was followed for 3nd experiment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/e2b16bba766a632ba85a2f1d.png"},{"id":90042830,"identity":"7ac42253-6b38-4e94-9882-0261507913b2","added_by":"auto","created_at":"2025-08-27 17:25:17","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230749,"visible":true,"origin":"","legend":"\u003cp\u003eDigital image of 3 experimental denim sample along with raw denim fabric.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/dd49bdfee41b7453ef43fead.jpeg"},{"id":90043323,"identity":"dddae59a-2af3-4279-be4c-c045a3c5c53b","added_by":"auto","created_at":"2025-08-27 17:33:17","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":51368,"visible":true,"origin":"","legend":"\u003cp\u003eInformation from the pocketing fabric's whitening index.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/50e6196127e12bd61582569f.jpeg"},{"id":90042828,"identity":"d6e19dd3-addb-4ad9-9fea-12369ae9d537","added_by":"auto","created_at":"2025-08-27 17:25:17","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":40525,"visible":true,"origin":"","legend":"\u003cp\u003eTensile strength of three washing sample in warp direction.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/300db4e42388187b434e94ec.jpeg"},{"id":90044203,"identity":"ad5990c5-f7a7-40b7-b937-f5affa9fd538","added_by":"auto","created_at":"2025-08-27 17:49:17","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":39038,"visible":true,"origin":"","legend":"\u003cp\u003eInformation of weight loss of three washing sample due to abrasion.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/52ae8f55f4957e49574f55d3.jpeg"},{"id":90043319,"identity":"6e097b8b-d75f-4c49-973a-214965c8ddc3","added_by":"auto","created_at":"2025-08-27 17:33:17","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":45230,"visible":true,"origin":"","legend":"\u003cp\u003eInformation of seam strength of the 3 experimental sample.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/35369d8c999d165c675ff2c1.jpeg"},{"id":90044701,"identity":"bb25e407-6b71-4208-955b-04bb48e5f47c","added_by":"auto","created_at":"2025-08-27 17:57:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1478591,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7250614/v1/c65523a7-d083-4577-a334-9a0f9fdaa714.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eComparative Study on the Sequential Effects of Ozone and Single-Bath-Single-Stage Enzymatic Treatments in Sustainable Denim Processing: Enzyme-Only, Post-Ozone, and Pre-Ozone Treatments\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDenim, a popular dress material for over 30 years, is embraced by children, women, and men for its comfort and versatility. Initially created in the 1980s using 100% polyester threads, it is now one of the most popular cotton dress fabrics (Hassabo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). So, it is a durable cotton warp-faced twill weave fabric that is thought to be extremely strong, stiff, and hard-wearing. It is distinguished from cotton duck by the diagonal ribbing created when the weft flows under more than one warp thread (Elmogahzy, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Razzaque, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Indigo is used to dye the warp of traditional blue denim. Numerous denim washing techniques, including the use of sand, stones, cellulase enzymes, potassium permanganate or sodium hypochlorite, hydrogen peroxide, and caustic soda, can remove the pigment from the surface and give denim a distinctive or faded or aged appearance (Choudhury, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Denim washing was traditionally accomplished with a lot of chemicals, such as hydrogen peroxide, hypochlorite, and sodium permanganate, to achieve that popular aged and faded look. In a consistent way, they would diminish the clothes' true color. However, the reality that these chemicals pose serious environmental concerns is a major disadvantage (Halim et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Also, these traditional methods of denim washing generate a lot of contaminated effluents and consume a lot of water (Hasanbeigi \u0026amp; Price, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this sense, enzymatic treatment in denim washing is both environmentally beneficial and efficient for producing sustainable denim clothing. Once more, consumers' attitudes toward the environment and their purchasing habits have grown, and the aesthetic qualities of the product also have an impact on their decisions (Butler \u0026amp; Francis, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Eckman, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Enzymes, which are classified as biocatalysts because they can speed up biochemical processes without being used, are created by the metabolism of microbes, especially bacteria. They are extensively employed in home washing, pharmaceutics, genetically modifying plants and animals, and figuring out the chemical design of DNA. Amylase is the most commonly used enzyme in textiles, and it is typically used to hydrolyze starch and reduce fabric size (Jegannathan \u0026amp; Nielsen, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Traditionally, denim fabric is treated with diluted hypochlorite to partially bleach or remove environmentally harmful stains. Cellulase, either by itself or in combination with pumice stone, causes denim clothing to abrasively age. Additionally, laccase is employed to darken the indigo-dyed substance (Liu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the other hand, ozone treatment is an environmentally friendly method that conserves electricity and water. \"Ozone\" (O₃) is the term for laundry, a textile care idea that uses a unique combination of electricity and oxygen in place of many of the chemicals commonly utilized in a traditional washing operation (Hoque \u0026amp; Faysal, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Water Resources Ozone washing systems greatly lessen the environmental impact of traditional washing processes. The clothes can be bleached using this method. Denim clothing is bleached in a machine for washing by dissolving ozone in water. Additionally, denim clothing can be bleached or faded in a controlled environment using ozone gas (Eren et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). By lowering rinse times, water usage, and energy consumption, ozone finishing denim jeans has both economic and environmental advantages. Textile wastewater can be decolored by ozone, a specialist oxidant with a high oxidation potential that has a unique affinity for indigo (Sarker et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs an alternative to traditional denim washing, Islam, Mohammad Tajul, et al. conducted an experiment using a single-bath-single-stage enzymatic treatment of denim. Here, denim is treated with a mixture of cellulase, amylase, and laccase at varying temperatures to get the desired aged or faded look. You can change the amount of laccase enzyme and the washing temperature to give denim cloth an aged appearance. The denim garment that faded the most was washed with 1.5 g/L of cellulase, 1.5 g/L of amylase, and 2 g/L of laccase enzyme for 45 minutes at 65\u0026deg;C. Since the resulting wastewater needed nearly four times as much oxygen to decompose as the wastewater from the conventional process, the enzymatic approach was determined to be far more environmentally friendly. The effectiveness of enzymatic treatments was highlighted by the fact that this temperature choice had no detrimental effects on the fabric's tensile strength or abrasion resistance (Islam et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur study aims to following this single-stage-single-bath enzymatic treatment combined with ozone treatment to create a sustainable process for denim sector and reducing use of toxic chemical, water and time. In addition to improving the findings' scalability and applicability, this practical implementation offers insightful information on how well textile treatments function in environments that closely resemble industrial processes. Additionally, a wide range of tests were included in our analysis, such as tensile strength, back staining, abrasion, and seam strength testing. Through this method, we not only get a good denim fading effect but also protect our environment from pollution and save water and time.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eThe denim fabric utilized for the study was provided by NZ TEX GROUP (Code: NZ-10217 HWS), Gulshan, Bangladesh. Since the cloth was non-desized and in its raw state, it was washed straight away without any additional processing. The fabric weighed 10.75 oz per square meter and was made entirely of 100% cotton. Its GSM was 369. With a structure of 61 ends per inch (EPI) and 38 picks per inch (PPI), it had a 3/1 twill weave. The warp and weft yarn counts were 6 Ne and 10 Ne, respectively. To test the pocketing fabric's ability to stain the back of denim after the enzymatic washing process was finished, it was affixed to the denim. Sunshine Textile, where the pocketing cloth was gathered. The cloth had a 1/1 plain weave and was composed entirely of 100% TC fabric. It was 118 (ends/inch) by 68 (picks/inch) in size. 100% polyester sewing thread that was bought from the Coats Company was used to sew the pocket. 20/2 was the sewing thread count.\u003c/p\u003e\u003cp\u003eDW 16LT (an amylase enzyme) made in TURKEY, LAVA CELL NDF (a cellulase enzyme) made in INDIA, and LAVA ZYME LITE NEU (a laccase enzyme) were collected from Bangladesh. Folosen NOF (a non-ionic wetting agent) was collected from CHT Germany GMBH, IMACOL X JET LIQ (a non-ionic anti-creasing agent) was collected from Archroma Singapore PTE Ltd, and Anti Back Stainer K-300 (a non-ionic dispersing agent) was collected from Guangdong Kesida New Material Technology Co., Ltd.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Preparation of denim\u003c/h2\u003e\u003cp\u003eA high-speed lockstitch machine (Brother, China) was used to stitch denim fabric with a superimposed seam and 1 cm allowance and 8 stitches per inch in order to prepare the denim leg sample before washing Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In order to test the fabric sample's back staining and seam strength, a piece of pocketing fabric was also sewn to the main denim samples using the lock stitch on the same machine. The sewing thread was composed of 100% polyester collected from Coats Company, and the pocketing cloth was 100% TC fabric. Also, an overlock machine (JUKI, China) was used to overlock the denim cloth for safety purposes. The sewing thread was composed of 100% polyester collected from RUNNER Company; 40/2 was the sewing thread count.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Ozonition Method\u003c/h2\u003e\u003cp\u003eThe Jeanlogia G2 ozone machine was used for ozonation treatment of denim fabric. Ozone, a gas formed naturally by photochemical reactions with solar ultraviolet radiation, can be generated artificially through corona discharge, a method involving electrical discharges from an ozone generator. A three-atom oxygen molecule (O₃) is created when the \"O\" atoms link with the O₂ molecules in Eq.\u0026nbsp;(1).\u003c/p\u003e\u003cp\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;O\u0026sdot; \u0026harr; O\u003csub\u003e3\u003c/sub\u003e (1)\u003c/p\u003e\u003cp\u003eSince ozone is unstable and cannot be transported or stored, it must be created as needed. The drum that holds the fabrics is injected with the resultant ozone. Similar to how sunshine ages textiles, ozone organically ages denim (Ben Fraj \u0026amp; Jaouachi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Washing Treatment\u003c/h2\u003e\u003cp\u003eThe single-bath-single-stage enzymatic washing was carried out with three different experiments based on the ozone process to find out the optimum washing recipe for which the best fading effect can be achieved. The amounts of amylase, cellulase, and laccase enzymes were fixed at 1.5 g/L, 1.5 g/L, and 2 g/L based on established industry recommendations for their typical application in denim washing processes. For the first experiment, to complete the washing process in one step, the sample washer extractor machine (Tolker, made in Turkey) was filled up with 30 L of water for each recipe. Here time is 45 m and temperature is 65\u0026deg;C. Wetting agent, dispersing agent, and anti-creasing agent were added at 25\u0026deg;C temperature. The drum was then rotated for 2 min, followed by the addition of the enzymes\u0026mdash;amylase, cellulase, and laccase. The liquid amylase enzyme was directly prepared to achieve a concentration of 1.5 g/L (V/V). In contrast, cellulase and laccase powders were dissolved in water using a magnetic stirrer, resulting in concentrations of 1.5 g/L (W/V) for cellulase and 2 g/L (W/V) for laccase. After adjusting the pH to 4.5, the raw denim sample was added to the drum, and the temperature of the bath was raised to 65\u0026deg;C. That temperature was maintained for 45 min. After this the temperature was raised to 90\u0026deg;C, and the machine was run for 5 min to deactivate the enzyme. Then the rinse wash was given two times with 40 L of water. The temperature was dropped down to 25\u0026deg;C, and the sample was removed. Lastly, the sample was hydroid by a hydro extractor machine for 2m and dried for 20m Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. And the 1st system was done (Islam et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the 2nd experiment (post-ozone treatment), we first followed the single-bath-single-stage enzymatic treatment like in the 1st experiment. Then did the ozone treatment by the Jeanlogia G2 machine, where gas power was 50% and cycle time was 10 min Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the second experiment (pre-ozone treatment), we first did ozone treatment by the Jeanlogia G2 machine, where gas power was 50% and cycle time was 10 min. And then followed the single-bath-single-stage enzymatic treatment, like in the 1st experiment Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. Visual test\u003c/h2\u003e\u003cp\u003eEach washed sample's color variation from the raw sample was visually assessed. Based on how closely the color of each sample matched that of the main sample, each sample was assigned one of three color ratings: high, medium, and low. Three separate observers evaluated the samples' color differences, and each sample's highest vote rating was recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. Spectrophotometer test\u003c/h2\u003e\u003cp\u003eA Datacolor 1050 spectrophotometer (Dual-Beam Spectrophotometer by Datacolor, USA) was used to measure the reflectance of the cleaned samples under the following circumstances: Pulsed Xenon with a wavelength range of 300 nm to 700 nm, filtered to roughly D65 light, and a 10\u0026deg; viewing measurement geometry. With the aid of pertinent software, the color strength values (K/S) of the dyed samples were determined using Datacolor 1050 and the Kubelka-Munk Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) as follows:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\frac{K}{S}=\\frac{(1-{R}_{\\lambda\\:\\:\\text{m}\\text{a}\\text{x}}{)}^{2}}{{2R}_{\\lambda\\:\\:max}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere K is the absorption coefficient, S is the scattering coefficient, and Rλmax is the sample's reflectance value at a certain wavelength where a given dye or color component absorbs the most.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6. Back Staining\u003c/h2\u003e\u003cp\u003eUsing a spectrophotometer (Datacolor, USA), the back staining of the pocketing fabrics was assessed as a whiteness index. The process is the same as that which was previously described for shade change.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7. Tensile Strength\u003c/h2\u003e\u003cp\u003eThe stripe test, using the BS EN ISO 13934-2:2014 method, was used to assess the samples' tensile strength in warp directions. The tensile strength was assessed using the B.N. Titan Tester (UK), in which each sample was cut to a size of 200 mm by 50 mm, placed between the top and bottom grippers of the tester independently, and then pushed to break at a pace of 100 mm per minute.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8. Seam strength\u003c/h2\u003e\u003cp\u003eB.N. Titan Tester (UK) was used to assess the samples' seam strength in accordance with the BS EN ISO 13935-2:2014 standard. Here, a 200 mm by 50 mm specimen was fastened between the top and bottom grippers of the B.N Titan Tester and dragged at a pace of 50 mm per minute until it broke.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.9. Abrasion\u003c/h2\u003e\u003cp\u003eUsing the BS EN ISO 12947-2:2016 technique, the B.N. Martindale abrasion and pilling tester (UK) evaluated the samples' abrasion. Using this approach, a specimen with a diameter of 38 mm was abraded using an abrading cloth with a diameter of 140 mm under 9 kPa weight in order to assess the mass loss following particular cycles. Here, the sample's mass loss from abrasion was calculated for cycles of 5000.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Color fading test\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1. Visual assessment test\u003c/h2\u003e\u003cp\u003eThe 3 different images in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e show the samples derived from the 3 different processing methods, like enzyme-only, post-ozone, and pre-ozone treatments.\u003c/p\u003e\u003cp\u003eThe sample SC-2 showed the highest fading of color compared to the raw denim sample. And sample SC-3 had the next best fading result. Sample SC-1 showed the least fading of color. This result can be linked to the ozone process in those samples. SC-2 was applied using an ozone-based process after a single-bath-single-stage enzymatic treatment; for this reason, SC-2 had the highest fading effect. On the other hand, SC-3 was applied using an ozone-based process before a single-bath-single-stage enzymatic treatment. For this reason, the ozone process was not working properly, and SC-3 had the next highest fading effect. SC-1 had no ozone-based process, so it had the lowest fading effect.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2. Spectrophotometer assessment\u003c/h2\u003e\u003cp\u003eThe spectrophotometer assessment, which involved evaluating each sample under a spectrophotometer, was the second method of color fading test used in this investigation. The color value results and changes in those values for samples treated with various washing procedures are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The degree of fading effect of the enzymatically treated samples can be assessed using the DL* value, which shows how light or dark the colored substrate is. Sample SC-1 has the lowest DL* value in this case, while sample SC-2 has the greatest value. The sample with the highest DL* value has a more faded effect than the others.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSpectrophotometer assessment of three treated sample\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRecipe no\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSample code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProcess\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDL*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDa*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eDb*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSC-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOnly Enzymatic treatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-8.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSC-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePost-ozone process\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-9.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSC-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePre-ozone Process\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e-0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-8.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eLike the DL* number above, the difference between Da* and Db* values can also be taken into account when assessing the degree of fading. Accordingly, greater fading is shown by more shifting in the treated samples' Da* and Db* values as compared to the grey sample. In terms of Da*, sample SC-2 is either more greenish or less reddish than the other samples. Yellowish and bluish values are indicated by the Db* values. In this case, sample SC-2 is less yellowish and more bluish than the other samples.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Back staining test\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the whiteness of three pocketing samples that were subjected to different washing procedures. The pocketing fabric of sample SC-2 had a greater whiteness rating, indicating less indigo staining during the sample's washing process. This suggests less color migration, which causes more fading, as the treated denim sample's visual and spectrophotometric evaluations also verified. However, sample SC-1 showed the least amount of whiteness in the pocketing fabric, indicating that this sample had more indigo staining throughout the procedure. Because without significantly reducing the textiles' strength, ozone was capable of bleaching the denim jeans and lessening back-staining (Kamppuri \u0026amp; Mahmood, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Tensile strength test\u003c/h2\u003e\u003cp\u003eThe tensile strength of several bio-washed samples is displayed in warp-wise directions in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, as previously mentioned. Looking at the tensile strength of the warp-wise yarns, the figure shows that the sample SC-1 washed only with enzymatic treatment has the medium tensile strength compared to the other two samples. The reason behind this is that the cellulase enzymes used to eliminate cellulosic protruding fibers might have the maximum activation at 65\u0026deg;C temperature. As a result, after eliminating cellulosic protruding fibers, it might attack the surface cotton fibers of the yarns, hydrolyze the fibers to some extent, and make the yarns weaker while treated at 65\u0026deg;C (Montazer \u0026amp; Sadeghian Maryan, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt is also observed that samples that were washed with post-ozone treatment have minimum warp yarn tensile strength.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Abrasion test\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows that the samples SC-1 \u0026amp; SC-3 have a similar amount of weight loss after the abrasion test. Because the cellulase enzyme used to remove protruding fibers might attack surface fibers and make them weaker, as it probably shows maximum activation at 65\u0026deg;C temperature. In addition to that, the amylase enzyme also shows higher efficiency at this temperature and eliminates maximum-size materials, making the yarns softer and susceptible to abrasion. Which in turn leads to higher weight loss (Panwar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOn the other hand, sample SC-2 indicates the maximum weight loss compared to the other two samples. Because The enzyme acts on the cellulose fiber, hydrolyzing it and weakening it. Then, when ozone is added, this weakened fiber is further oxidized, so more breakdown occurs and more weight loss.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Seam strength test\u003c/h2\u003e\u003cp\u003eThe seam strength of various bio-washed samples is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. All of the cleaned samples showed no discernible difference in seam strength. As previously mentioned, the sewing thread was made entirely of polyester, which is completely unaffected by temperature or any of the chemicals utilized. The denim portion lost its strength as a result of all the chemical processing, which explains the modest loss in seam strength observed in the samples (Islam et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eUsually the process of washing denim is very long because it has different steps. Each step requires a lot of water, time, and chemicals. That is why the process of washing denim is not suitable for our environment, and its production cost is also high. But its demand is increasing day by day. This is why researchers have come up with an innovative method that combines ozone (post and pre) treatment with enzymatic treatment. This innovative method uses three enzymes, including 1.5 g/L amylase, 1.5 g/L cellulase, and 2 g/L laccase, at a temperature of 65\u0026deg;C for 45 minutes. Also uses 50% ozone gas for 10 minutes. This study showed that single-bath-single-stage enzymatic treatment was applied for the first step, and then ozone treatment was used, and a satisfactory fading effect was obtained. Also, it showed enhanced back staining, which suggests that the indigo dyes were better washed off the denim fabric, giving the garment a more aged or faded appearance.\u003c/p\u003e\u003cp\u003eHowever, fading varied with the variation of applied ozone treatment. Future studies should examine the suitability of this single-bath-single-stage enzymatic treatment, with different ozone gas treatment options for different blue and sulfur-colored denim, to further broaden the scope of this study. Various sustainable processes such as laser, ultraviolet, ultrasound, plasma, and waterjet treatment should also be added to save water and time and make the environment safer. Positive outcomes would further support the implementation of this novel strategy in industrial practice, possibly displacing the traditional denim washing procedure and promoting more economical and environmentally friendly production techniques.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that no funding, money, or other assistance was obtained in order to prepare this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJannatuz Faria: Writing \u0026ndash; original draft, review \u0026amp; editing, Supervision, Investigation, Conceptualization. A J M Kamruzzaman shakey: Writing \u0026ndash; original draft, Visualization, Formal analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that none of the work described in this study could have been influenced by any known competing economic interests or personal relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBen Fraj A, Jaouachi B (2021) Effects of ozone treatment on denim garment properties. 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Int J Curr Eng Technol 11(05):516\u0026ndash;621. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14741/ijcet/v.11.5.4\u003c/span\u003e\u003cspan address=\"10.14741/ijcet/v.11.5.4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Enzymatic Treatment, Ozone Treatment, Denim washing, Sustainability","lastPublishedDoi":"10.21203/rs.3.rs-7250614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7250614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNowadays, all age groups have shown a renewed interest in denim clothing due to its comfortable, worn-out style and altered appearance. Numerous technological advancements, including significant advancements in spinning, washing, and finishing, contribute to denim's status as one of the most iconic fashion items. In the textile sector, the traditional denim washing method is thought to be the most polluting. This industry produces an abundance of wastewater with an increased pollutant load and utilizes a lot of energy, water, and chemicals. So, this research investigates the combination of ozone treatment with single-bath-single-stage enzymatic treatment at 65\u0026deg;C temperatures on denim garments to not only achieve the desired aged or faded appearance but also save the environment, water, and time. Where three types of enzymes were used, including amylase (for desizing), cellulase (for removing producing fibers), and laccase (for removing indigo dyes). On the other hand, ozone works as a bleaching agent for denim garments, and it is a waterless treatment that makes our denim sector more sustainable. The samples from three washing recipes were analyzed properly for tensile strength, seam strength, color changes, and back staining. The highest fading of denim garments was obtained when first applying enzymatic treatment and then applying ozone treatment. This method adds a new dimension to denim textiles.\u003c/p\u003e","manuscriptTitle":"Comparative Study on the Sequential Effects of Ozone and Single-Bath-Single-Stage Enzymatic Treatments in Sustainable Denim Processing: Enzyme-Only, Post-Ozone, and Pre-Ozone Treatments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 17:25:13","doi":"10.21203/rs.3.rs-7250614/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-12-20T00:01:40+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-25T04:37:38+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T11:01:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-08T04:39:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2025-08-04T10:21:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4640c29d-50b2-42ba-b6a6-863b25c28d33","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T00:47:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-27 17:25:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7250614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7250614","identity":"rs-7250614","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}