Combatting synthetic dye toxicity through exploring the potential of lignin peroxidase from Pseudomonas fluorescence LiP RL5

Combatting synthetic dye toxicity through exploring the potential of lignin peroxidase from Pseudomonas fluorescence LiP RL5 | 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 Combatting synthetic dye toxicity through exploring the potential of lignin peroxidase from Pseudomonas fluorescence LiP RL5 Ranju Kumari Rathour, Nidhi Rana, Vaishali Sharma, Nitish Sharma, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3958055/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract Untreated disposal of toxic synthetic dyes is a serious threat to the environment. Every year, several thousand gallons of dyes are being disposed into the water resources without any sustainable detoxification. The accumulation of hazardous dyes in the environment poses a severe threat to the human health, flora, fauna, and microflora. Therefore, in the present study, a lignin peroxidase enzyme from Pseudomonas fluorescence LiP-RL5 has been employed for the maximal detoxification of selected commercially used dyes. The enzyme production from the microorganism was enhanced ~ 20 folds using statistical optimization tool response surface methodology. Four different combinations (pH, production time, seed age, and inoculum size) were found to be crucial for the higher production of LiP. The crude enzyme showed decolorization action on commonly used commercial dyes such as Crystal violet, Congo red, Malachite green, and Coomassie brilliant blue. Successful toxicity mitigation of these dyes culminated in the improved seed germination in three plant species, Vigna radiate (20–60%), Cicer arietinum (20–40%), and Phaseolus vulgaris (10–25%). The LiP treated dyes also exhibit reduced bactericidal effects against four common resident microbial species, Escherichia coli (2–10 mm), Bacillus sp. (4–8 mm), Pseudomonas sp. (2–8 mm), and Lactobacillus sp. (2–10 mm). Therefore, apart from the tremendous industrial applications, the LiP from Pseudomonas fluorescence LiP-RL5 could be a potential biocatalyst for the detoxification of synthetic dyes. Lignin peroxidase Pseudomonas fluorescence Synthetic dyes Detoxification Phytotoxicity Microbial toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The fundamental aspect of lignolytic enzymes is in delignification. Ligninases inherently act as a part of microbial catalytic weaponry to neutralize the phenolic/non-phenolic compounds of complex, rigid, and defense accreting lignin structure of plants (Samuel et al. 2011 ; Sainsbury et al. 2013 ; Chandel et al. 2022 ; Lin et al. 2023 ). The simpler compounds generated can be further utilized by microorganisms for their various metabolic activities (Wang et al. 2013 ). Nature has a vast repository of different lignin modifying enzymes encoded in the genomes of organisms. However, in recent times, lignin peroxidases have gained much more attention than other ligninases due to their mechanistic uniqueness (Arora and Gill 2001 ; Ding et al. 2022 ; Bhatt et al. 2022 ). Lignin peroxidases have been classified as oxidoreductase enzymes known to oxidize the lignin molecules, and form the aryl-cations from their constituents. Consecutively, the enzyme-treated molecules further oxidize their Cα-Cβ-bonds, ether linkages, and aromatic rings (Wong 2009 ). Lignin peroxidases are generally metal independent biocatalysts possess many other benevolences such as high redox potential and oxidation of complex polymer etc. (Nousiainen et al. 2014 ). In most of the instances, these enzymes are produced extracellularly, which broaden their substrate utilization ability and make lignin peroxidases a potential biocatalyst for various industrial processes (Patil 2014 ; Sasidhara 2014 ). The fiber processing industry enjoys a significant turnover of world trade. It is estimated to touch around 842.6 billion USD in 2020, with 26.2% from 2015 (Sheng 2017 ). Despite their undeniable significance, these industries are also one of the biggest polluters since the industrial revolution (Thiry 2011 ; Desore & Narula 2018 ). The effluents of textile industries mainly constitute toxic dyes and their byproducts, which are generally mutagenic and carcinogenic (Umesh et al. 2016 ; Ji et al. 2020 ). These effluents cause water, soil, and air pollution when spilt over into the environment (Lellis et al. 2019 ). Moreover, the toxic dyes threaten the flora, fauna, and microbial composition of both terrestrial and aquatic lives when spared untreated and detoxified (Sadek 2016 ). The untreated textile effluents when accumulates in water bodies, avert the sunlight to penetration through water (Shoiful et al. 2020 ; Wu et al. 2022 ), and ultimately reduce the rate of photosynthesis (Lellis et al. 2019 ). Apart from this, textile dyes were also reported to cause phytotoxicity in terms of delayed or inhibited seed germination, retardation of plant growth, reduction in the concentration of total sugars like starch, and malfunctioning of chlorophyll etc. (Sadek 2016 ; Ariaeenejad et al. 2021). Moreover, synthetic dyes are also known to halt the normal microflora of the environment as these are reactive compounds that exert specific bactericidal effects on plant growth-promoting microbes (Moawad et al. 2003 ; Kahraman and Yalcin 2005). Microorganisms play a significant role in nature assisted bioremediation processes. Sophisticated genes encoded in microbial genomes help them to neutralize the recalcitrant toxic molecules such as synthetic dyes, long chain hydrocarbons, and plastics. Many microbial enzymes have been reported to have toxicity mitigation properties. However, with high redox potential and ability to degrade both phenolic and non-phenolic constituents of robust lignin hetero-polysaccharide, lignin peroxidases are critical players in detoxification of textile effluents and xenobiotic compounds (Rathour et al. 2020 ; Kiran et al. 2020; Kumar et al. 2024 ). There have been reports of several microbial systems to produce lignin peroxidases especially white-rot fungi. However, due to certain limitations of fungal systems, bacteria like Pseudomonas aeruginosa, Serratia marcescens, Nocardia sp., Arthobacter sp., Flavobacterium sp., Micrococcus sp., Xanthomonas sp., Bacillus sp., Cellulomonas sp., Agaricus sp., Erwinia sp., Copricus sp., Mycema sp., Sterium sp., Rhodococcus sp., Sphingomonas paucimobilis and Streptomyces viridosporus have been preferentially identified as lignin peroxidase producers (Ahmad et al. 2010; Kalyani et al. 2011 ; Rathour et al. 2020 ; Kiran et al. 2020; Preethi et al. 2023). The ability of microorganisms in maintaining environmental processes such as bio-geochemical cycling and bioremediation could be channelized towards mitigation of dye toxicity. Therefore, in the present study, the yield of lignin peroxidase enzyme from previously isolated P. Fluorescence has been enhanced through statistical optimization methods. Further, the applicability of crude lignin peroxidase in degradation of synthetic dyes has been explored. The synthetic dyes are known to have microbicidal effects, and can cause certain level of phytotoxicity in plants (Kahraman and Yalcin 2005; Saratale et al. 2011). Therefore, the phytotoxicity and microbicidal effects of LiP treated and untreated dyes have been evaluated on seed germination of plant sp. and some common soil microbial sp., respectively. The LiP enzyme extracted in the present study could be a potential biocatalyst in paper and pulp industry, cosmetics, biorefineries, bioremediation, and food industry. 2. Materials and methods 2.1 Inoculum preparation and enzyme production The lignin peroxidase producing microorganism P. fluorescence LiP-RL5 was previously isolated from the soil sample of Shimla, India. 12 h old inoculum was used as seed culture for enzyme production. The media containing K 2 HPO 4 (4.55 g/L), KH 2 PO 4 (0.53 g/L), MgSO 4 (0.50 g/L), NH 3 NO 3 (0.50 g/L), Glucose (5.0 g/L), and meat extract (10 g/L), and growth conditions of 30°C and incubation time 24 h determined for the maximal production of the enzyme. After optimum incubation time, LiP was harvested from the fermented broth by centrifugation at 10,000 rpm for 10 min. Further, the collected supernatant was used for determining enzyme activity and degradation of dyes. 2.2 Enzymatic assay of Lignin peroxidase (LiP) Lignin peroxidase activity was measured by the veratryl alcohol method (Tien and Kirk 1988 ). The initial rate of oxidation of veratryl alcohol to veratraldehyde was followed by absorption at 310 nm. The assay mixture of 1 ml contained: 50 µl of the pre-diluted supernatant, 250 µl of 50 mM veratryl alcohol, 0.6 ml of 0.1 M citrate buffer (pH 5.5) and 0.1 ml of 10 mM H 2 O 2 . One enzyme activity unit was considered the amount of enzyme that oxidizes one micromole of veratryl alcohol per minute under standard assay conditions. 2.3 Statistical optimization of LiP production using Response Surface Methodology Optimization of growth parameters for enhanced production of LiP was done using Response surface methodology (RSM). The effect of an individual parameter, as well as their cumulative effect, was statistically analyzed. Each variable's effect checked on three levels, low, medium and high, in the range between − 1, 0, and + 1. The median value usually adjusted according to the values generated from manual optimization. In two-step optimization, firstly, Plackett-Burman design was used with seven different production variables (conc. of glucose, conc. of meat extract, substrate conc., inoculum size, inoculum age, incubation temperature and production time) to check their inter-dependent effect on enzyme production. The latest version of Design expert ™ (V11.0) was used for analysis. The independent variables predicted to be positively impacting lignin peroxidase production during first step optimization (pH, inoculum age, inoculum size, and production) were further optimized using Central Composite Design (CCD). Perturbation plot and predicted versus experimental values were also compared using the software. The software has automatically done variation of the data (ANOVA), the significance of the analysis model (p-value; <0.05), standard deviation, and R 2 value on data. Statistically normalized values were utilized for interactive graphs. 2.4 Enzyme assisted degradation of commercially used dyes The commercially used synthetic dyes, such as Crystal violet, Coomassie brilliant blue, Malachite green, and Congo red were used to study the enzyme's effect on them. For this, a 50 µM concentration solution of each dye was prepared. The enzyme assay was performed with crude enzyme using respective dye as substrate replacing methylene blue in sodium potassium tartrate buffer (pH 5.0) and H 2 O 2 as an inducer 8:1:1 (buffer: enzyme: inducer). The reaction was then incubated at 30 ºC for 40 min. After completion reaction was scanned on a spectrophotometer (VIS spectrophotometer) from 380–780 nm to record the λ max , and percentage dye decolorization was studied at λ max using the following formula (Equation I) (Roy et al. 2018 ): \(\text{D}\text{e}\text{g}\text{r}\text{a}\text{d}\text{a}\text{t}\text{i}\text{o}\text{n} \left(\text{%}\right)=\frac{\text{O}.\text{D}. \text{o}\text{f} \text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{O}.\text{D}. \text{o}\text{f} \text{t}\text{e}\text{s}\text{t} }{\text{O}.\text{D}. \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) X 100 …….. Equation I 2.5 Comparative analysis of LiP treated and untreated dyes on seed germination The effect of different treated and untreated synthetic textile dye on germination of Vigna radiate (green gram), Cicer arietinum (Chickpea) and Phaseolus vulgaris (kidney beans) has been evaluated. The seeds were germinated on sterile 10 cm Petri dishes, layered with the cotton bed. Seeds were sterilized before transferring to the surface of cotton in the Petri dishes (10 seeds per plate). The phytotoxicity bioassay was evaluated using the seed germination technique. This method involves incubating the synthetic dye effluent with seeds at room temperature for 5 days in the dark and then measuring the number of seeds germinated and root growth after that using the following equations (Equation II&III) (Zucconi 1981 ) \(\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{s}\text{e}\text{e}\text{d} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n} \left(\text{%}\right)=\frac{ \text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{s}\text{e}\text{e}\text{d}\text{s} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d} \text{i}\text{n} \text{s}\text{y}\text{n}\text{t}\text{h}\text{e}\text{t}\text{i}\text{c} \text{d}\text{y}\text{e} }{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{s}\text{e}\text{e}\text{d}\text{s} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) X 100 ……..Equation II \(\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{r}\text{o}\text{o}\text{t} \text{g}\text{r}\text{o}\text{w}\text{t}\text{h} \left(\text{%}\right)=\frac{\text{M}\text{e}\text{a}\text{n} \text{r}\text{o}\text{o}\text{t} \text{l}\text{e}\text{n}\text{g}\text{t}\text{h} \text{i}\text{n} \text{s}\text{y}\text{n}\text{t}\text{h}\text{e}\text{t}\text{i}\text{c} \text{d}\text{y}\text{e} }{\text{M}\text{e}\text{a}\text{n} \text{r}\text{o}\text{o}\text{t} \text{l}\text{e}\text{n}\text{g}\text{t}\text{h} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) X 100 ……..Equation III The seed germination and root length of all plants in the pure and LiP treated dyes were determined. The seed germination percentage and root elongation of the plants in tap water were also measured and used as the control. Seeds were considered germinated when the radical and hypocotyl together appeared. The percent seed germination and root shoot ratio were recorded at a regular interval of 24 h for five days continuously. The relative seed germination, relative root elongation and germination index (GI, the product of relative seed germination and relative root elongation) were calculated as follows (Equation IV) (Tiquia 2010 ). $$\text{G}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n} \text{i}\text{n}\text{d}\text{e}\text{x} \left(\text{G}\text{I}\right)=\frac{\left(\text{%} \text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{s}\text{e}\text{e}\text{d} \text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n}\right) \text{X} \left(\text{%} \text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e} \text{r}\text{o}\text{o}\text{t} \text{g}\text{r}\text{o}\text{w}\text{t}\text{h}\right)}{100}$$ ……..Equation IV 2.6 Microbicidal effects of LiP treated dyes The microbial toxicity test was carried out to check the untreated and LiP treated dyes' effect on the growth of soil microorganism’s viz., E. coli, Pseudomonas sp. and Lactobacillus sp. pure cultures suspension (0.1 ml ) of all these microorganisms was spread over the sterile Muller Hinton agar plates separately. 50 µl of pure dye (1%) and 50 µl Lip treated dyes were used. Treated and untreated dye sample was aseptically added in the wells and plates were incubated at 37°C for 24 h after that zone of inhibition was measured. 3. Results and Discussion 3.1 Statistical optimization for enhanced LiP production from P. fluorescence LiP-RL5 Statistical validation was done to optimize the design, reduce cost and time, human efforts, and minimize errors. Twelve different investigational combinations of variables (Table 1 , Supplementary data) were obtained using the Plackett-Burman design, and the study was carried out. Results of combinations revealed variation in activity ranging from 0.47 ± 0.21 to 18.07 ± 0.78 U/ml, and run 4 was found to be the most suitable combination and gave maximum activity of 18.07 ± 0.78 U/ml. Pareto chart was constructed using results of Plackett-Burman analysis (Fig. 1 , Supplementary data) that showed pH, production time, seed age, and inoculum size have a positive effect on enzyme activity and thus has a significant impact on lignin peroxidase production from P. fluorescence LiP-RL5.A total of 30 experimental combinations (Table 2, Supplementary data) were obtained using CCD for four positive variables (pH, production time, seed age and inoculum size). The ANOVA was generated by the data obtained after CCD, which showed that the lack of fit was non-significant and experimental data was highly reliable (Table 3, Supplementary data). Table 1 Dye decolourization study by lignin peroxidase Sr. No. Dyes O.D. (nm) % Decolorization 01. Crystal Violet 590 81.60 ± 0.91 02. Congo Red 490 77.24 ± 0.85 03. Malachite Green 610 57.18 ± 1.27 04. Commassie Brillant Blue 560 35 ± 0.74 Previously, (Vandana et al. 2018 ) also used statistical modelling to optimize lignin peroxidase from white-rot fungi and found six variables, i.e. incubation time, temperature, pH, substrate concentration, glucose and H 2 O 2 , having a significant effect on LiP activity. In another study by (Kaur et al. 2015 ), three variables, i.e. pH, incubation time, and dyes concentration, were significant. In the present study, a higher concentration of glucose as a carbon source leads to a decrease in LiP activity, similar to findings (Stewart et al. 1992 ). It suggested a high concentration of glucose inhibited lignin peroxidase LiP when genes were expressed under limited carbon media. 3.1.1 Statistical validation of the optimization model The cubic process order proved best, and ANOVA demonstrated that the model was highly significant (p < 0.05). The 3D response graphs were generated for regression analysis of CCD design data using a pairwise combination of four positive factors for lignin peroxidase (Fig. 2 , Supplementary data). The response surface plots were more or less dome-shaped and showed interactions among variables with an optimum activity around pH 7.5, seed age 8 h, inoculum size 4% and production time 48 h. ANOVA of the cubic regression model suggested that the model was very significant, and it is evident from the coefficient of variation (CV%=43.83), standard deviation (12.71), high determination coefficient R 2 of 0.7081 (p < 0.05), high adj. R-squared value (0.43) (Table 4, Supplementary data). The R 2 value indicated goodness of fit of the model, but high R 2 may be due to other non-significant variables and adj. The R-squared value must be considered to manage the R 2 . The RSM competence was shown by comparing the experimental data, and the predicted values, which are performed by generating a fitted line plot (with experimental values on X-axis and predicted values on Y-axis) form the obtained results, showing its closeness or deviation from the fitted line. There was a good agreement between the experimental and predicted values. The maximum activity obtained by performing RSM was 54.87 ± 0.58 U/ml, close to the predicted value of 50.44 ± 0.34 U/ml as calculated by the ANOVA test. The selected variables' overall closeness, indicating that the response surface model is adequate for predicting the lignin peroxidase characteristics. A perturbation plot was also obtained (Fig. 1 ) that showed optimum values of variables for lignin peroxidase production from P. fluorescence LiP-RL5. A similar study (Tuncer et al. 2009 ) reported 24–72 h of incubation time optimum for LiP activity as extracellular enzymes and culture are in its growth phase. The pH optimum was found to be 7.5 (Patil 2014 ). The LiP production was increased from pH 5.0 to 7.0, and after that, production was decreased. After statistical optimization there is 19.26 fold increase in activity from 0.47 ± 0.78 U/ml to 54.87 ± 0.58 U/ml. 3.2 Degradation of selected dyes study Lignin peroxidase of P. fluorescence LiP-RL5 used in the present study was able to degrade selected synthetic Azo dyes, i.e. Congo red, Malachite green, Crystal violet and Coomassie brilliant blue to 77.24 ± 0.85%, 57.18 ± 1.27%, 81.60 ± 0.91% and 35 ± 0.74%, respectively of their initial value within 60 min in the liquid assays as shown Table 1 . Congo red is most commonly used in textiles and having two azo bonds (-N = N-) in its molecular structure. The visible (380–750) spectra of dye showed a broad peak with a lambda maximum at 490 (Fig. 2 A), which is due to a double bond between azo groups. After treatment with LiP, the decrease in peak intensity was observed, which indicates that chromophores groups transformed due to degradation of the azo bond. (Asses et al. 2018 ) also attended the azo group's degradation after 96 h treatment with Aspergillus niger . They reported that this is due to the presence of lignin-degrading peroxidases in the fungus. Crystal violet, Malachite green and Coomassie brilliant blue are triphenyl methane dye, the visible spectra of dyes depicted their lambda maximum at 590 nm (Fig. 2 B), 610 nm (Fig. 2 C) and 570 nm (Fig. 2 D), respectively. After treatment with lignin peroxidase, the intensity of peak decreases in both the cases might be due to alternation in the conjugation between the benzene nuclei (Cheriaa et al. 2012 ), while shift at 615–620 nm indicated N-demethylation of malachite green (Bharagava et al. 2018 ) reported that lignolytic enzyme is responsible for the degradation of crystal violet. (Jadhav and Govindwar 2006 ) reported 84% degradation of malachite green in 7 h from Saccharomyces cerevisiae . Umesh et al., ( 2016 ) reported 33% degradation of Coomassie brilliant blue after 12 h with Bacillus sp. which is further characterized for the production of lignin peroxidase. According to obtained results and literature study, the possible degradation pathway of lignin peroxidase action on selected dyes was shown in Fig. 3 . The LiP used to degrade the textile dyes showed remarkable decolorization of dyes. A study (Ollikka et al. 1993 ) showed 54% decolorization of Congo red in the presence of crude preparation of lignin peroxidase. Selvam et al., ( 2003 ) observed a 10% reduction in Congo red with 15 U/ml LiP, while (Krishnan et al. 2019 ) used bacterial consortia to biodegrade azo dyes. The degradation percentage they get after three days were approximately similar to the degradation observed in the present study after 60 minutes. Saratale and co-workers (Saratale et al. 2011) stated that lignin peroxidase has better efficiency for dye degradation than intact cells. 3.3 Phytotoxicity of LiP treated and untreated dyes When released untreated to the environment, the synthetic dyes were reported to cause a negative impact on the normal microbial flora of soil and leads to a slow rate of seed germination. Therefore, in the present study, these dyes' injuriousness impact was studied on different crops and microorganisms. The effect of other treated and untreated dyes on germination of V. radiate (green gram), C. arietinum (Chickpea) and P. vulgaris (kidney beans) was evaluated (Fig. 4 a). The length of radicals and plumule was calculated in mm for five days; from this, mean % seed germination and root growth was calculated. Percentage of related seed germination (Fig. 4 b), root growth (Fig. 4 c) and germination index (Fig. 4 d) was obtained in both cases (Table 5–7, Supplementary data). Phytotoxicity analysis of LiP treated and untreated textile dyes indicated that the untreated dyes lead to delay in seed germination and affect the length of the radicle and plumule. Successful toxicity mitigation of these dyes culminated in the improved seed germination in three plant species, V. radiate (20–60%), C.arietinum (20–40%), and P. vulgaris (10–25%). The germination index of selected plants was also reported to increase from 25–50%. The present study's findings are similar to the study conducted by (Mahmood et al. 2015 ; Bilal et al. 2016 ). They reported that textile dyes negatively affect seed germination and plant growth.(Umesh et al. 2016 ) also found similar results when five different plants, i.e. C. arietinum, V. Sinensis, V. aconitifolia, V. radiata and T. aestivum , were treated with synthetic textile dyes. 3.4 Microbial toxicity studies The microbial toxicity studies of pure and LiP treated dyes was carried out against E. coli, Bacillus sp., Pseudomonas sp. and Lactobacillus sp. (Table 8 and Fig. 3 , Supplementary data). It was observed that the pure/untreated dyes were toxic to the microbial cells and showed inhibitory action on the microbial growth (Fig. 5 ). The LiP treated dyes exhibit reduced bactericidal effects (approximately 2–10 mm reduction in inhibitory zone) against four common resident microbial species, E.coli (2–10 mm), Bacillus sp. (4–8 mm), Pseudomonas sp. (2–8 mm), and Lactobacillus sp. (2–10 mm). However, after decolourization of the dyes, the product is significantly less harmful to the microorganisms than untreated ones. Congo red, Crystal violet, Coomassie brilliant blue and Malachite green showed an inhibitory effect on all tested microorganisms' growth. So it was concluded from the microbial toxicity test that the dyes which are toxic to the microbial cells and were converted into the nontoxic product by Lignin peroxidase. Besides phytotoxicity, these azo dyes also affect the microbial flora of soil and water (Pokharia 2015 ). (Moawad et al. 2003 ) has been reported that azo dyes eliminate microbial colonies due to the high frequency of mutation. Moreover, (Kahraman and Yalcin 2005) has also revealed the negative effect of the textile dyes on P. aeruginosa to assess the toxicity after decolorization. 4. Conclusions The production of enzyme lignin peroxidase from P. fluorescence LiP-RL5 has been enhanced ~ 20 folds using statistical methodology RSM. The optimization resulted in approx. 20-fold increase in LiP activity. The enzyme successfully detoxified the different commercially used textile dyes through decolorization. Therefore, this study strengthens the bioremediation potential of the LiP enzyme. Our findings further proved that, the enzyme-treated textile dyes possess relatively less hazardous impact on seed germination of different plant species. Moreover, the soil's normal microflora also falls short of the devastating effects of LiP treated dyes. Therefore, this enzyme could be a boon for the detoxification of hazardous textile industry effluents and help restore the fertility of contaminated soil by promoting the growth of native plants and microbial communities of the environment. Declarations Supplementary Information The online version contains supplementary material available at………………….. Acknowledgements RKR acknowledges fellowship provided by the Department of Environment Science and Technology, Govt. of Himachal Pradesh. RKB sincerely acknowledges the University Grant Commission (UGC), New Delhi, India, for providing financial assistance in the form of post-doctoral fellowship. Department of Biotechnology, Himachal Pradesh University Shimla, India, thankfully acknowledged for providing all the facilities for this study. Author contribution RKR: Conceptualization, Data curation, Formal analysis, Validation, Visualization, and Writing-original draft, Writing- review & editing, RKB: Conceptualization, Methodology, Writing-review & editing, NR: Methodology, Formal analyses, Investigation, VS: Methodology, Formal analyses, Investigation, NS: Formal analyses, Writing-review & editing, and AKB: Formal analysis, Funding acquisition, Investigation. Statements and Declarations Funding This work was supported by Grant numbers [No. Env. S&T (F/)/5-1)/2019-20] & [No. F./PDFSS201415SCHIM8434]. Author Ravi Kant Bhatia has received research support from Company UGC-New Delhi. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data availability All the data of this study has been provided. Corresponding author Correspondence to Dr. Ravi Kant Bhatia Ethics declarations Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. References Arora DS, Gill PK (2001) Comparison of two assay procedures for lignin peroxidase. Enzyme Microb Technol 28:602–605. https://doi.org/10.1016/S0141-0229(01)00302-7 Asses N, Ayed L, Hkiri N, Hamdi M (2018) Congo red decolorization and detoxification by Aspergillus Niger : Removal mechanisms and dye degradation pathway. Biomed Res Int 2018:. https://doi.org/10.1155/2018/3049686 Bhatt P, Tiwari M, Parmarick P, Bhatt K, Gangola S, Adnan M, Singh Y, Bilal M, Ahmed S, Chen S (2022) Insights into the catalytic mechanism of ligninolytic peroxidase and laccase in lignin degradation. Bioremediation J 15;26(4):281-91. Bharagava RN, Mani S, Mulla SI, Saratale GD (2018) Degradation and decolourization potential of an ligninolytic enzyme producing Aeromonas hydrophila for crystal violet dye and its phytotoxicity evaluation. Ecotoxicol Environ Saf 156:166–175. https://doi.org/10.1016/j.ecoenv.2018.03.012 Bilal M, Iqbal M, Hu H, Zhang X (2016) Mutagenicity, cytotoxicity and phytotoxicity evaluation of biodegraded textile effluent by fungal ligninolytic enzymes. Water Sci Technol 73:2332–2344. https://doi.org/10.2166/wst.2016.082 Chandel H, Kumar P, Chandel AK, Verma ML (2022) Biotechnological advances in biomass pretreatment for bio-renewable production through nanotechnological intervention. Biomass Convers Biorefinery 2022 1:1–23. https://doi.org/10.1007/S13399-022-02746-0 Cheriaa J, Khaireddine M, Rouabhia M, Bakhrouf A (2012) Removal of triphenylmethane dyes by bacterial consortium. Sci World J 2012:1–9. https://doi.org/10.1100/2012/512454 Desore A, Narula SA (2018) An overview on corporate response towards sustainability issues in textile industry. Environ Dev Sustain 20:1439–1459. https://doi.org/10.1007/s10668-017-9949-1 Ding Y, Cui K, Liu X, et al (2022) Lignin peroxidase-catalyzed direct oxidation of trace organic pollutants through a long-range electron transfer mechanism: Using propranolol as an example. J Hazard Mater 431:. https://doi.org/10.1016/J.JHAZMAT.2022.128544 Jadhav JP, Govindwar SP (2006) Biotransformation of malachite green by S accharomyces cerevisiae MTCC 463. Yeast 23:315–323. https://doi.org/10.1002/yea.1356 Ji J, Kulshreshtha S, Kakade A, et al (2020) Bioaugmentation of membrane bioreactor with Aeromonas hydrophila LZ-MG14 for enhanced malachite green and hexavalent chromium removal in textile wastewater. Int Biodeterior Biodegrad 150:104939. https://doi.org/10.1016/j.ibiod.2020.104939 Kahraman S, Yalcin P (2005a) Antibacterial Effect of Untreated and Treated (Decolorized) Dyes by Agricultural Wastes. Am J Biochem Biotechnol 1:50–53. https://doi.org/10.3844/ajbbsp.2005.50.53 Kahraman S, Yalcin P (2005b) Antibacterial Effect of Untreated and Treated (Decolorized) Dyes by Agricultural Wastes. Am J Biochem Biotechnol 1:50–53. https://doi.org/10.3844/ajbbsp.2005.50.53 Kalyani DC, Phugare SS, Shedbalkar UU, Jadhav JP (2011) Purification and characterization of a bacterial peroxidase from the isolated strain Pseudomonas sp. SUK1 and its application for textile dye decolorization. Ann Microbiol 61:483–491. https://doi.org/10.1007/s13213-010-0162-9 Kaur B, Kumar B, Garg N, Kaur N (2015) Statistical optimization of conditions for decolorization of synthetic dyes by Cordyceps militaris MTCC 3936 using RSM. Biomed Res Int 2015:. https://doi.org/10.1155/2015/536745 Kiran, Rathour RK, Bhatia RK, et al (2020) Fabrication of thermostable and reusable nanobiocatalyst for dye decolourization by immobilization of lignin peroxidase on graphene oxide functionalized MnFe 2 O 4 superparamagnetic nanoparticles. Bioresour Technol 317:124020. https://doi.org/10.1016/j.biortech.2020.124020 Krishnan S, Din MFM, Taib SM, et al (2019) Process constraints in sustainable bio-hythane production from wastewater: Technical note. Bioresour Technol Reports 5:359–363. https://doi.org/10.1016/j.biteb.2018.05.003 Kumar V, Pallavi P, Sen SK, Raut S (2024) Harnessing the potential of white rot fungi and ligninolytic enzymes for efficient textile dye degradation: A comprehensive review. Water Environ Res 96(1):e10959. Lellis B, Fávaro-Polonio CZ, Pamphile JA, Polonio JC (2019) Effects of textile dyes on health and the environment and bioremediation potential of living organisms. Biotechnol Res Innov 3:275–290. https://doi.org/10.1016/j.biori.2019.09.001 Lin J, Ye W, Xie M, Seo DH, Luo J, Wan Y, Van der Bruggen B (2023) Environmental impacts and remediation of dye-containing wastewater. Nat Rev Earth Environ 4(11):785-803. https://doi.org/10.1038/s43017-023-00489-8 Mahmood R, Sharif F, Ali S, Hayyat MU (2015) Enhancing the decolorizing and degradation ability of bacterial consortium isolated from textile effluent affected area and its application on seed germination. Sci World J 2015:. https://doi.org/10.1155/2015/628195 Moawad H, Abd El-Rahim WM, Khalafallah M (2003) Evaluation of biotoxicity of textile dyes using two bioassays. J Basic Microbiol 43:218–229. https://doi.org/10.1002/jobm.200390025 Nousiainen P, Kontro J, Manner H, et al (2014) Phenolic mediators enhance the manganese peroxidase catalyzed oxidation of recalcitrant lignin model compounds and synthetic lignin. Fungal Genet Biol 72:137–149. https://doi.org/10.1016/j.fgb.2014.07.008 Ollikka P, Alhonmäki K, Leppänen V-M, et al (1993) Decolorization of Azo, triphenyl methane, heterocyclic, and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium . Appl Environ Microbiol 59:4010 Patil S. (2014) Production and purification of lignin peroxidase from Bacillus megaterium and its application in bioremidation. J Microbiol 2:22–28 Pokharia A. ASS. (2015) Toxicological effect of textile dyes and their metabolites: A review. . 1: Preethi PS, Vickram S, Das R, Hariharan NM, Rameshpathy M, Subbaiya R, Karmegam N, Kim W, Govarthanan M 2023 Bioprospecting of novel peroxidase from Streptomyces coelicolor strain SPR7 for carcinogenic azo dyes decolorization. Chemosphere. 1;310:136836. Rathour RK, Sharma V, Rana N, et al (2020) Bioremediation of simulated textile effluent by an efficient bio-catalyst purified from a novel Pseudomonas fluorescence LiP-RL5. Curr Chem Biol 14:128–139. https://doi.org/10.2174/2212796814666200406100247 Roy DC, Biswas SK, Saha AK, et al (2018) Biodegradation of Crystal Violet dye by bacteria isolated from textile industry effluents. PeerJ 2018:e5015. https://doi.org/10.7717/peerj.5015 Sadek MB. HJ. SHB. BA. SS (2016) Experiment Findingspdf available toxic effect of textile dyeing effluents on germination, growth, yield and nutritional quality of Okra ( Abelmoschus esculentus ). Int J Ecotoxicol Ecobiol 1:82–87 Sainsbury PD, Hardiman EM, Ahmad M, et al (2013) Breaking Down lignin to high-value chemicals: the conversion of lignocellulose to vanillin in a gene deletion mutant of Rhodococcus jostii RHA1. ACS Chem Biol 8:2151–2156. https://doi.org/10.1021/cb400505a Samuel R, Foston M, Jiang N, et al (2011) Structural changes in switchgrass lignin and hemicelluloses during pretreatments by NMR analysis. Polym Degrad Stab. https://doi.org/10.1016/j.polymdegradstab.2011.08.015 Saratale RG, Saratale GD, Chang JS, Govindwar SP (2011a) Bacterial decolorization and degradation of azo dyes: A review. J. Taiwan Inst. Chem. Eng. 42:138–157 Saratale RG, Saratale GD, Chang JS, Govindwar SP (2011b) Bacterial decolorization and degradation of azo dyes: A review. J Taiwan Inst Chem Eng 42:138–157. https://doi.org/10.1016/j.jtice.2010.06.006 Sasidhara R. TT (2014) . Lignolytic and lignocellulosic enzymes of Ganoderma lucidum in liquid medium. . Eur jouranal Exp Biol 4:375–379 Selvam K, Swaminathan K, Chae K-S (2003) Decolourization of azo dyes and a dye industry effluent by a white rot fungus Thelephora sp. Bioresour Technol 88:115–119. https://doi.org/10.1016/S0960-8524(02)00280-8 Sheng L (2017) Market Size of the Global Textile and Apparel Industry: 2015 to 2020. Shoiful A, Kambara H, Cao LTT, et al (2020) Mn(II) oxidation and manganese-oxide reduction on the decolorization of an azo dye. Int Biodeterior Biodegradation 146:104820. https://doi.org/10.1016/j.ibiod.2019.104820 Stewart P, Kersten P, Vanden Wymelenberg A, et al (1992) Lignin peroxidase gene family of Phanerochaete chrysosporium: Complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome. J Bacteriol 174:5036–5042. https://doi.org/10.1128/jb.174.15.5036-5042.1992 Thiry MC Staying alive: Making textiles sustainable. AATCC Review . 2011 Tien M, Kirk TK (1988) Lignin peroxidase of Phanerochaete chrysosporium . pp 238–249 Tiquia SM (2010) Reduction of compost phytotoxicity during the process of decomposition. Chemosphere 79:506–512. https://doi.org/10.1016/j.chemosphere.2010.02.040 Tuncer G, Tekkaya C, Sungur S, et al (2009) Assessing pre-service teachers’ environmental literacy in Turkey as a mean to develop teacher education programs. Int J Educ Dev 29:426–436. https://doi.org/10.1016/j.ijedudev.2008.10.003 Umesh JU, Rhushikesh DN, Vishal D V, et al (2016) phytotoxic effect of synthetic textile dye effluent on growth of five plant species Trends Biotech. Res. 5(2), 1-6. Vandana TG, Rao R, Ashish Kumar S, et al (2018) Enhancing production of lignin peroxidase from white rot fungi employing statistical optimization and evaluation of its potential in delignification of crop residues. Int J Curr Microbiol Appl Sci 7:2599–2621. https://doi.org/10.20546/ijcmas.2018.701.312 Wang H, Tucker M, Ji Y (2013) Recent development in chemical depolymerization of lignin: A Review. J Appl Chem 2013:1–9. https://doi.org/10.1155/2013/838645 Wong DWS (2009) Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 157:174–209. https://doi.org/10.1007/s12010-008-8279-z Wu K, Shi M, Pan X, et al (2022) Decolourization and biodegradation of methylene blue dye by a ligninolytic enzyme-producing Bacillus thuringiensi s: Degradation products and pathway. Enzyme Microb Technol 156:109999. https://doi.org/10.1016/J.ENZMICTEC.2022.109999 Zucconi F, Bertoldi DE 1981) Biological evaluation of compost maturity. Biocycle 22:27–29 Supplementary Files Supplementrydata.docx Cite Share Download PDF Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted Editorial decision: Minor Revision 12 May, 2024 Reviewers agreed at journal 24 Mar, 2024 Reviewers invited by journal 24 Mar, 2024 Editor invited by journal 19 Mar, 2024 Editor assigned by journal 01 Mar, 2024 First submitted to journal 26 Feb, 2024 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3958055","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283365296,"identity":"238739f0-8d3d-4177-92b2-93da94473cf4","order_by":0,"name":"Ranju Kumari Rathour","email":"","orcid":"","institution":"Himachal Pradesh University","correspondingAuthor":false,"prefix":"","firstName":"Ranju","middleName":"Kumari","lastName":"Rathour","suffix":""},{"id":283365297,"identity":"1193d245-f2ac-435b-8453-99cd6566ce84","order_by":1,"name":"Nidhi Rana","email":"","orcid":"","institution":"Himachal Pradesh University","correspondingAuthor":false,"prefix":"","firstName":"Nidhi","middleName":"","lastName":"Rana","suffix":""},{"id":283365298,"identity":"3583fece-b7f0-46be-be0e-a9a2f61fff4d","order_by":2,"name":"Vaishali Sharma","email":"","orcid":"","institution":"Himachal Pradesh University","correspondingAuthor":false,"prefix":"","firstName":"Vaishali","middleName":"","lastName":"Sharma","suffix":""},{"id":283365299,"identity":"cb1ae7a8-5ffd-40ff-ab81-29512aebb92f","order_by":3,"name":"Nitish Sharma","email":"","orcid":"","institution":"Center of Innovative and Applied Bioprocessing","correspondingAuthor":false,"prefix":"","firstName":"Nitish","middleName":"","lastName":"Sharma","suffix":""},{"id":283365300,"identity":"50882c50-024e-471e-aa78-fb8a2ea6d677","order_by":4,"name":"Arvind Kumar Bhatt","email":"","orcid":"","institution":"Himachal Pradesh University","correspondingAuthor":false,"prefix":"","firstName":"Arvind","middleName":"Kumar","lastName":"Bhatt","suffix":""},{"id":283365301,"identity":"b5570d9b-e42c-4931-965a-696c2089bd0e","order_by":5,"name":"RAVI KANT BHATIA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYJCCwwwGIIqH4fCfCiDNzNxAlBYJoBbGBzxnQFoYCWthBmKQFmYD3jYQn4AW8/YzhocLCmzq+NnPHpOQnFcbzd8O1PKhZhtOLTJncgwOzzBIk5DsyUuTMNx2PHfGYcYGxhnHbuPUIsGQlnCYx+CwhMENHjOJxG3HchuAWph5G/Bo4X8G0WIP0nJwzrHc+QS1SCQfgNgiwWNs2NhQk7uBsJbHB0B+kZxxJsfwMcOxA7kbgVoO4vULf2Lz54I/Nvz87WcMDjPU1OXOO3/44IMPNbi1oIPDYPIA0eqBoI4UxaNgFIyCUTBCAACykFjzFzgfDwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5223-3541","institution":"Himachal Pradesh University","correspondingAuthor":true,"prefix":"","firstName":"RAVI","middleName":"KANT","lastName":"BHATIA","suffix":""}],"badges":[],"createdAt":"2024-02-15 08:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3958055/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3958055/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-34400-9","type":"published","date":"2024-08-05T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53559428,"identity":"598ea490-ea2f-4620-b690-00195317e115","added_by":"auto","created_at":"2024-03-27 13:17:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":306627,"visible":true,"origin":"","legend":"\u003cp\u003eA) A graph showing compared actual responses, B) Perturbation graph showing impact of all variables considered in CCD.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/8df43b420afaf68d1783fba5.png"},{"id":53559426,"identity":"db9085cd-5428-46bf-a53e-e45cda10a035","added_by":"auto","created_at":"2024-03-27 13:17:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55552,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Visible spectra of selected dyes before and after LiP treatment: A) Crystal Violet, B) Congo Red, C) Malachite green and D) Coomassie Brilliant Blue.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/c18d7d1b13ec39bf6c5c1fbb.png"},{"id":53559427,"identity":"733ff2de-5314-4da0-a62f-d060a16969b2","added_by":"auto","created_at":"2024-03-27 13:17:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":975800,"visible":true,"origin":"","legend":"\u003cp\u003ePossible pathway for degradation of different dyes by lignin peroxidase.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/3e427a1949a4a394a95d5fc9.png"},{"id":53559429,"identity":"6604f148-28f0-4c0f-ac69-cdfbac2f32a7","added_by":"auto","created_at":"2024-03-27 13:17:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2273521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4a:\u003c/strong\u003e Effects of LiP detoxified dyes on seed germination of three different plants viz., \u003cem\u003eVigna radiate \u003c/em\u003e(green gram), \u003cem\u003eCicer arietinum \u003c/em\u003e(Chickpea) and \u003cem\u003ePhaseolus vulgaris \u003c/em\u003e(kidney beans) A) Malachite Green, B) Congo Red, C) Coomassie Brilliant Blue, D) Crystal Violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4b: \u003c/strong\u003eRelative seed germination (%) of selected plant with LiP treated and untreated dyes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4c: \u003c/strong\u003eRelative root germination (%) of selected plant with LiP treated and untreated dyes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4d: \u003c/strong\u003eGermination index of selected seeds with LiP treated and untreated dyes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/c63297c5875e95e7bc1f09dd.png"},{"id":53559430,"identity":"e26866b2-9337-4eae-9f24-32badb075d84","added_by":"auto","created_at":"2024-03-27 13:17:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":786304,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial toxicity studies of pure and LiP treated dyes against selected bacterial isolates.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/079325025e71b13810e7ce11.png"},{"id":62299121,"identity":"a5075488-88b2-4e2e-a74f-34a8e7758a1e","added_by":"auto","created_at":"2024-08-12 16:18:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6817299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/ba98d529-e7e0-43fb-ab47-4c92f1fce293.pdf"},{"id":53559431,"identity":"c48ec566-5542-4538-b751-3ea355ff5848","added_by":"auto","created_at":"2024-03-27 13:17:49","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2695955,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementrydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-3958055/v1/a4b60df689dbdd8e7e8f0016.docx"}],"financialInterests":"","formattedTitle":"Combatting synthetic dye toxicity through exploring the potential of lignin peroxidase from Pseudomonas fluorescence LiP RL5","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe fundamental aspect of lignolytic enzymes is in delignification. Ligninases inherently act as a part of microbial catalytic weaponry to neutralize the phenolic/non-phenolic compounds of complex, rigid, and defense accreting lignin structure of plants (Samuel et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sainsbury et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chandel et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The simpler compounds generated can be further utilized by microorganisms for their various metabolic activities (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Nature has a vast repository of different lignin modifying enzymes encoded in the genomes of organisms. However, in recent times, lignin peroxidases have gained much more attention than other ligninases due to their mechanistic uniqueness (Arora and Gill \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bhatt et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lignin peroxidases have been classified as oxidoreductase enzymes known to oxidize the lignin molecules, and form the aryl-cations from their constituents. Consecutively, the enzyme-treated molecules further oxidize their Cα-Cβ-bonds, ether linkages, and aromatic rings (Wong \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Lignin peroxidases are generally metal independent biocatalysts possess many other benevolences such as high redox potential and oxidation of complex polymer etc. (Nousiainen et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In most of the instances, these enzymes are produced extracellularly, which broaden their substrate utilization ability and make lignin peroxidases a potential biocatalyst for various industrial processes (Patil \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sasidhara \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe fiber processing industry enjoys a significant turnover of world trade. It is estimated to touch around 842.6\u0026nbsp;billion USD in 2020, with 26.2% from 2015 (Sheng \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Despite their undeniable significance, these industries are also one of the biggest polluters since the industrial revolution (Thiry \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Desore \u0026amp; Narula \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The effluents of textile industries mainly constitute toxic dyes and their byproducts, which are generally mutagenic and carcinogenic (Umesh et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These effluents cause water, soil, and air pollution when spilt over into the environment (Lellis et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, the toxic dyes threaten the flora, fauna, and microbial composition of both terrestrial and aquatic lives when spared untreated and detoxified (Sadek \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The untreated textile effluents when accumulates in water bodies, avert the sunlight to penetration through water (Shoiful et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and ultimately reduce the rate of photosynthesis (Lellis et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Apart from this, textile dyes were also reported to cause phytotoxicity in terms of delayed or inhibited seed germination, retardation of plant growth, reduction in the concentration of total sugars like starch, and malfunctioning of chlorophyll etc. (Sadek \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ariaeenejad et al. 2021). Moreover, synthetic dyes are also known to halt the normal microflora of the environment as these are reactive compounds that exert specific bactericidal effects on plant growth-promoting microbes (Moawad et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kahraman and Yalcin 2005).\u003c/p\u003e \u003cp\u003eMicroorganisms play a significant role in nature assisted bioremediation processes. Sophisticated genes encoded in microbial genomes help them to neutralize the recalcitrant toxic molecules such as synthetic dyes, long chain hydrocarbons, and plastics. Many microbial enzymes have been reported to have toxicity mitigation properties. However, with high redox potential and ability to degrade both phenolic and non-phenolic constituents of robust lignin hetero-polysaccharide, lignin peroxidases are critical players in detoxification of textile effluents and xenobiotic compounds (Rathour et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kiran et al. 2020; Kumar et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). There have been reports of several microbial systems to produce lignin peroxidases especially white-rot fungi. However, due to certain limitations of fungal systems, bacteria like \u003cem\u003ePseudomonas aeruginosa, Serratia marcescens, Nocardia\u003c/em\u003e sp., \u003cem\u003eArthobacter\u003c/em\u003e sp., \u003cem\u003eFlavobacterium\u003c/em\u003e sp., \u003cem\u003eMicrococcus\u003c/em\u003e sp., \u003cem\u003eXanthomonas\u003c/em\u003e sp., \u003cem\u003eBacillus\u003c/em\u003e sp., \u003cem\u003eCellulomonas\u003c/em\u003e sp., \u003cem\u003eAgaricus\u003c/em\u003e sp., \u003cem\u003eErwinia\u003c/em\u003e sp., \u003cem\u003eCopricus\u003c/em\u003e sp., \u003cem\u003eMycema\u003c/em\u003e sp., \u003cem\u003eSterium\u003c/em\u003e sp., \u003cem\u003eRhodococcus\u003c/em\u003e sp., \u003cem\u003eSphingomonas paucimobilis\u003c/em\u003e and \u003cem\u003eStreptomyces viridosporus\u003c/em\u003e have been preferentially identified as lignin peroxidase producers (Ahmad et al. 2010; Kalyani et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rathour et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kiran et al. 2020; Preethi et al. 2023).\u003c/p\u003e \u003cp\u003eThe ability of microorganisms in maintaining environmental processes such as bio-geochemical cycling and bioremediation could be channelized towards mitigation of dye toxicity. Therefore, in the present study, the yield of lignin peroxidase enzyme from previously isolated \u003cem\u003eP. Fluorescence\u003c/em\u003e has been enhanced through statistical optimization methods. Further, the applicability of crude lignin peroxidase in degradation of synthetic dyes has been explored. The synthetic dyes are known to have microbicidal effects, and can cause certain level of phytotoxicity in plants (Kahraman and Yalcin 2005; Saratale et al. 2011). Therefore, the phytotoxicity and microbicidal effects of LiP treated and untreated dyes have been evaluated on seed germination of plant sp. and some common soil microbial sp., respectively. The LiP enzyme extracted in the present study could be a potential biocatalyst in paper and pulp industry, cosmetics, biorefineries, bioremediation, and food industry.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Inoculum preparation and enzyme production\u003c/h2\u003e\n \u003cp\u003eThe lignin peroxidase producing microorganism \u003cem\u003eP. fluorescence\u003c/em\u003e LiP-RL5 was previously isolated from the soil sample of Shimla, India. 12 h old inoculum was used as seed culture for enzyme production. The media containing K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (4.55 g/L), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (0.53 g/L), MgSO\u003csub\u003e4\u003c/sub\u003e (0.50 g/L), NH\u003csub\u003e3\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e (0.50 g/L), Glucose (5.0 g/L), and meat extract (10 g/L), and growth conditions of 30\u0026deg;C and incubation time 24 h determined for the maximal production of the enzyme. After optimum incubation time, LiP was harvested from the fermented broth by centrifugation at 10,000 rpm for 10 min. Further, the collected supernatant was used for determining enzyme activity and degradation of dyes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Enzymatic assay of Lignin peroxidase (LiP)\u003c/h2\u003e\n \u003cp\u003eLignin peroxidase activity was measured by the veratryl alcohol method (Tien and Kirk \u003cspan class=\"CitationRef\"\u003e1988\u003c/span\u003e). The initial rate of oxidation of veratryl alcohol to veratraldehyde was followed by absorption at 310 nm. The assay mixture of 1 ml contained: 50 \u0026micro;l of the pre-diluted supernatant, 250 \u0026micro;l of 50 mM veratryl alcohol, 0.6 ml of 0.1 M citrate buffer (pH 5.5) and 0.1 ml of 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. One enzyme activity unit was considered the amount of enzyme that oxidizes one micromole of veratryl alcohol per minute under standard assay conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Statistical optimization of LiP production using Response Surface Methodology\u003c/h2\u003e\n \u003cp\u003eOptimization of growth parameters for enhanced production of LiP was done using Response surface methodology (RSM). The effect of an individual parameter, as well as their cumulative effect, was statistically analyzed. Each variable\u0026apos;s effect checked on three levels, low, medium and high, in the range between \u0026minus;\u0026thinsp;1, 0, and +\u0026thinsp;1. The median value usually adjusted according to the values generated from manual optimization. In two-step optimization, firstly, Plackett-Burman design was used with seven different production variables (conc. of glucose, conc. of meat extract, substrate conc., inoculum size, inoculum age, incubation temperature and production time) to check their inter-dependent effect on enzyme production. The latest version of Design expert\u003csup\u003e\u0026trade;\u003c/sup\u003e (V11.0) was used for analysis. The independent variables predicted to be positively impacting lignin peroxidase production during first step optimization (pH, inoculum age, inoculum size, and production) were further optimized using Central Composite Design (CCD). Perturbation plot and predicted versus experimental values were also compared using the software. The software has automatically done variation of the data (ANOVA), the significance of the analysis model (p-value; \u0026lt;0.05), standard deviation, and R\u003csup\u003e2\u003c/sup\u003e value on data. Statistically normalized values were utilized for interactive graphs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Enzyme assisted degradation of commercially used dyes\u003c/h2\u003e\n \u003cp\u003eThe commercially used synthetic dyes, such as Crystal violet, Coomassie brilliant blue, Malachite green, and Congo red were used to study the enzyme\u0026apos;s effect on them. For this, a 50 \u0026micro;M concentration solution of each dye was prepared. The enzyme assay was performed with crude enzyme using respective dye as substrate replacing methylene blue in sodium potassium tartrate buffer (pH 5.0) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as an inducer 8:1:1 (buffer: enzyme: inducer). The reaction was then incubated at 30 \u0026ordm;C for 40 min. After completion reaction was scanned on a spectrophotometer (VIS spectrophotometer) from 380\u0026ndash;780 nm to record the \u0026lambda;\u003csub\u003emax\u003c/sub\u003e, and percentage dye decolorization was studied at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e using the following formula (Equation I) (Roy et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e):\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\text{D}\\text{e}\\text{g}\\text{r}\\text{a}\\text{d}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\left(\\text{%}\\right)=\\frac{\\text{O}.\\text{D}. \\text{o}\\text{f} \\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{O}.\\text{D}. \\text{o}\\text{f} \\text{t}\\text{e}\\text{s}\\text{t} }{\\text{O}.\\text{D}. \\text{o}\\text{f} \\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003eX 100 \u0026hellip;\u0026hellip;.. Equation I\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Comparative analysis of LiP treated and untreated dyes on seed germination\u003c/h2\u003e\u003cp\u003eThe effect of different treated and untreated synthetic textile dye on germination of \u003cem\u003eVigna radiate\u003c/em\u003e (green gram), \u003cem\u003eCicer arietinum\u003c/em\u003e (Chickpea) and \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e (kidney beans) has been evaluated. The seeds were germinated on sterile 10 cm Petri dishes, layered with the cotton bed. Seeds were sterilized before transferring to the surface of cotton in the Petri dishes (10 seeds per plate). The phytotoxicity bioassay was evaluated using the seed germination technique. This method involves incubating the synthetic dye effluent with seeds at room temperature for 5 days in the dark and then measuring the number of seeds germinated and root growth after that using the following equations (Equation II\u0026amp;III) (Zucconi \u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{s}\\text{e}\\text{e}\\text{d} \\text{g}\\text{e}\\text{r}\\text{m}\\text{i}\\text{n}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\left(\\text{%}\\right)=\\frac{ \\text{N}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{s}\\text{e}\\text{e}\\text{d}\\text{s} \\text{g}\\text{e}\\text{r}\\text{m}\\text{i}\\text{n}\\text{a}\\text{t}\\text{e}\\text{d} \\text{i}\\text{n} \\text{s}\\text{y}\\text{n}\\text{t}\\text{h}\\text{e}\\text{t}\\text{i}\\text{c} \\text{d}\\text{y}\\text{e} }{\\text{N}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{s}\\text{e}\\text{e}\\text{d}\\text{s} \\text{g}\\text{e}\\text{r}\\text{m}\\text{i}\\text{n}\\text{a}\\text{t}\\text{e}\\text{d} \\text{i}\\text{n} \\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003eX 100\u003c/p\u003e\n \u003cp\u003e\u0026hellip;\u0026hellip;..Equation II\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(\\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{r}\\text{o}\\text{o}\\text{t} \\text{g}\\text{r}\\text{o}\\text{w}\\text{t}\\text{h} \\left(\\text{%}\\right)=\\frac{\\text{M}\\text{e}\\text{a}\\text{n} \\text{r}\\text{o}\\text{o}\\text{t} \\text{l}\\text{e}\\text{n}\\text{g}\\text{t}\\text{h} \\text{i}\\text{n} \\text{s}\\text{y}\\text{n}\\text{t}\\text{h}\\text{e}\\text{t}\\text{i}\\text{c} \\text{d}\\text{y}\\text{e} }{\\text{M}\\text{e}\\text{a}\\text{n} \\text{r}\\text{o}\\text{o}\\text{t} \\text{l}\\text{e}\\text{n}\\text{g}\\text{t}\\text{h} \\text{i}\\text{n} \\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003eX 100 \u0026hellip;\u0026hellip;..Equation III\u003c/p\u003e\u003cp\u003eThe seed germination and root length of all plants in the pure and LiP treated dyes were determined. The seed germination percentage and root elongation of the plants in tap water were also measured and used as the control. Seeds were considered germinated when the radical and hypocotyl together appeared. The percent seed germination and root shoot ratio were recorded at a regular interval of 24 h for five days continuously. The relative seed germination, relative root elongation and germination index (GI, the product of relative seed germination and relative root elongation) were calculated as follows (Equation IV) (Tiquia \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\text{G}\\text{e}\\text{r}\\text{m}\\text{i}\\text{n}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{i}\\text{n}\\text{d}\\text{e}\\text{x} \\left(\\text{G}\\text{I}\\right)=\\frac{\\left(\\text{%} \\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{s}\\text{e}\\text{e}\\text{d} \\text{g}\\text{e}\\text{r}\\text{m}\\text{i}\\text{n}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\right) \\text{X} \\left(\\text{%} \\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e} \\text{r}\\text{o}\\text{o}\\text{t} \\text{g}\\text{r}\\text{o}\\text{w}\\text{t}\\text{h}\\right)}{100}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u0026hellip;\u0026hellip;..Equation IV\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Microbicidal effects of LiP treated dyes\u003c/h2\u003e\u003cp\u003eThe microbial toxicity test was carried out to check the untreated and LiP treated dyes\u0026apos; effect on the growth of soil microorganism\u0026rsquo;s viz., \u003cem\u003eE. coli, Pseudomonas\u003c/em\u003e sp. and \u003cem\u003eLactobacillus\u003c/em\u003e sp. pure cultures suspension (0.1 ml ) of all these microorganisms was spread over the sterile Muller Hinton agar plates separately. 50 \u0026micro;l of pure dye (1%) and 50 \u0026micro;l Lip treated dyes were used. Treated and untreated dye sample was aseptically added in the wells and plates were incubated at 37\u0026deg;C for 24 h after that zone of inhibition was measured.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Statistical optimization for enhanced LiP production from \u003cem\u003eP. fluorescence\u003c/em\u003e LiP-RL5\u003c/h2\u003e \u003cp\u003eStatistical validation was done to optimize the design, reduce cost and time, human efforts, and minimize errors. Twelve different investigational combinations of variables (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary data) were obtained using the Plackett-Burman design, and the study was carried out. Results of combinations revealed variation in activity ranging from 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 to 18.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 U/ml, and run 4 was found to be the most suitable combination and gave maximum activity of 18.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 U/ml. Pareto chart was constructed using results of Plackett-Burman analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary data) that showed pH, production time, seed age, and inoculum size have a positive effect on enzyme activity and thus has a significant impact on lignin peroxidase production from \u003cem\u003eP. fluorescence\u003c/em\u003e LiP-RL5.A total of 30 experimental combinations (Table\u0026nbsp;2, Supplementary data) were obtained using CCD for four positive variables (pH, production time, seed age and inoculum size). The ANOVA was generated by the data obtained after CCD, which showed that the lack of fit was non-significant and experimental data was highly reliable (Table\u0026nbsp;3, Supplementary data).\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\u003e\u003cb\u003eDye decolourization study by lignin peroxidase\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDyes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eO.D. (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Decolorization\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e01.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrystal Violet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e81.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e02.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCongo Red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e03.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMalachite Green\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e57.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e04.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommassie Brillant Blue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\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\u003ePreviously, (Vandana et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) also used statistical modelling to optimize lignin peroxidase from white-rot fungi and found six variables, i.e. incubation time, temperature, pH, substrate concentration, glucose and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, having a significant effect on LiP activity. In another study by (Kaur et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), three variables, i.e. pH, incubation time, and dyes concentration, were significant. In the present study, a higher concentration of glucose as a carbon source leads to a decrease in LiP activity, similar to findings (Stewart et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). It suggested a high concentration of glucose inhibited lignin peroxidase LiP when genes were expressed under limited carbon media.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Statistical validation of the optimization model\u003c/h2\u003e \u003cp\u003eThe cubic process order proved best, and ANOVA demonstrated that the model was highly significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The 3D response graphs were generated for regression analysis of CCD design data using a pairwise combination of four positive factors for lignin peroxidase (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary data). The response surface plots were more or less dome-shaped and showed interactions among variables with an optimum activity around pH 7.5, seed age 8 h, inoculum size 4% and production time 48 h. ANOVA of the cubic regression model suggested that the model was very significant, and it is evident from the coefficient of variation (CV%=43.83), standard deviation (12.71), high determination coefficient R\u003csup\u003e2\u003c/sup\u003e of 0.7081 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), high adj. R-squared value (0.43) (Table\u0026nbsp;4, Supplementary data). The R\u003csup\u003e2\u003c/sup\u003e value indicated goodness of fit of the model, but high R\u003csup\u003e2\u003c/sup\u003e may be due to other non-significant variables and adj. The R-squared value must be considered to manage the R\u003csup\u003e2\u003c/sup\u003e. The RSM competence was shown by comparing the experimental data, and the predicted values, which are performed by generating a fitted line plot (with experimental values on X-axis and predicted values on Y-axis) form the obtained results, showing its closeness or deviation from the fitted line. There was a good agreement between the experimental and predicted values. The maximum activity obtained by performing RSM was 54.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 U/ml, close to the predicted value of 50.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 U/ml as calculated by the ANOVA test. The selected variables' overall closeness, indicating that the response surface model is adequate for predicting the lignin peroxidase characteristics. A perturbation plot was also obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) that showed optimum values of variables for lignin peroxidase production from \u003cem\u003eP. fluorescence\u003c/em\u003e LiP-RL5. A similar study (Tuncer et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported 24\u0026ndash;72 h of incubation time optimum for LiP activity as extracellular enzymes and culture are in its growth phase. The pH optimum was found to be 7.5 (Patil \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The LiP production was increased from pH 5.0 to 7.0, and after that, production was decreased. After statistical optimization there is 19.26 fold increase in activity from 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78 U/ml to 54.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 U/ml.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Degradation of selected dyes study\u003c/h2\u003e \u003cp\u003eLignin peroxidase of \u003cem\u003eP. fluorescence\u003c/em\u003e LiP-RL5 used in the present study was able to degrade selected synthetic Azo dyes, i.e. Congo red, Malachite green, Crystal violet and Coomassie brilliant blue to 77.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85%, 57.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27%, 81.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91% and 35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74%, respectively of their initial value within 60 min in the liquid assays as shown Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Congo red is most commonly used in textiles and having two azo bonds (-N\u0026thinsp;=\u0026thinsp;N-) in its molecular structure. The visible (380\u0026ndash;750) spectra of dye showed a broad peak with a lambda maximum at 490 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which is due to a double bond between azo groups. After treatment with LiP, the decrease in peak intensity was observed, which indicates that chromophores groups transformed due to degradation of the azo bond. (Asses et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) also attended the azo group's degradation after 96 h treatment with \u003cem\u003eAspergillus niger\u003c/em\u003e. They reported that this is due to the presence of lignin-degrading peroxidases in the fungus. Crystal violet, Malachite green and Coomassie brilliant blue are triphenyl methane dye, the visible spectra of dyes depicted their lambda maximum at 590 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), 610 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and 570 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), respectively. After treatment with lignin peroxidase, the intensity of peak decreases in both the cases might be due to alternation in the conjugation between the benzene nuclei (Cheriaa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), while shift at 615\u0026ndash;620 nm indicated N-demethylation of malachite green (Bharagava et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that lignolytic enzyme is responsible for the degradation of crystal violet. (Jadhav and Govindwar \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported 84% degradation of malachite green in 7 h from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Umesh et al., (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported 33% degradation of Coomassie brilliant blue after 12 h with \u003cem\u003eBacillus\u003c/em\u003e sp. which is further characterized for the production of lignin peroxidase. According to obtained results and literature study, the possible degradation pathway of lignin peroxidase action on selected dyes was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The LiP used to degrade the textile dyes showed remarkable decolorization of dyes. A study (Ollikka et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) showed 54% decolorization of Congo red in the presence of crude preparation of lignin peroxidase. Selvam et al., (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) observed a 10% reduction in Congo red with 15 U/ml LiP, while (Krishnan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) used bacterial consortia to biodegrade azo dyes. The degradation percentage they get after three days were approximately similar to the degradation observed in the present study after 60 minutes. Saratale and co-workers (Saratale et al. 2011) stated that lignin peroxidase has better efficiency for dye degradation than intact cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Phytotoxicity of LiP treated and untreated dyes\u003c/h2\u003e \u003cp\u003eWhen released untreated to the environment, the synthetic dyes were reported to cause a negative impact on the normal microbial flora of soil and leads to a slow rate of seed germination. Therefore, in the present study, these dyes' injuriousness impact was studied on different crops and microorganisms. The effect of other treated and untreated dyes on germination of \u003cem\u003eV. radiate\u003c/em\u003e (green gram), \u003cem\u003eC. arietinum\u003c/em\u003e (Chickpea) and \u003cem\u003eP. vulgaris\u003c/em\u003e (kidney beans) was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The length of radicals and plumule was calculated in mm for five days; from this, mean % seed germination and root growth was calculated. Percentage of related seed germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), root growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and germination index (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) was obtained in both cases (Table\u0026nbsp;5\u0026ndash;7, Supplementary data).\u003c/p\u003e \u003cp\u003ePhytotoxicity analysis of LiP treated and untreated textile dyes indicated that the untreated dyes lead to delay in seed germination and affect the length of the radicle and plumule. Successful toxicity mitigation of these dyes culminated in the improved seed germination in three plant species, \u003cem\u003eV. radiate\u003c/em\u003e (20\u0026ndash;60%), \u003cem\u003eC.arietinum\u003c/em\u003e (20\u0026ndash;40%), and \u003cem\u003eP. vulgaris\u003c/em\u003e (10\u0026ndash;25%). The germination index of selected plants was also reported to increase from 25\u0026ndash;50%. The present study's findings are similar to the study conducted by (Mahmood et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bilal et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). They reported that textile dyes negatively affect seed germination and plant growth.(Umesh et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) also found similar results when five different plants, i.e. \u003cem\u003eC. arietinum, V. Sinensis, V. aconitifolia, V. radiata and T. aestivum\u003c/em\u003e, were treated with synthetic textile dyes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Microbial toxicity studies\u003c/h2\u003e \u003cp\u003eThe microbial toxicity studies of pure and LiP treated dyes was carried out against \u003cem\u003eE. coli, Bacillus\u003c/em\u003e sp., \u003cem\u003ePseudomonas\u003c/em\u003e sp. and \u003cem\u003eLactobacillus\u003c/em\u003e sp. (Table\u0026nbsp;8 and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary data). It was observed that the pure/untreated dyes were toxic to the microbial cells and showed inhibitory action on the microbial growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The LiP treated dyes exhibit reduced bactericidal effects (approximately 2\u0026ndash;10 mm reduction in inhibitory zone) against four common resident microbial species, \u003cem\u003eE.coli\u003c/em\u003e (2\u0026ndash;10 mm), \u003cem\u003eBacillus\u003c/em\u003e sp. (4\u0026ndash;8 mm), \u003cem\u003ePseudomonas\u003c/em\u003e sp. (2\u0026ndash;8 mm), and \u003cem\u003eLactobacillus\u003c/em\u003e sp. (2\u0026ndash;10 mm). However, after decolourization of the dyes, the product is significantly less harmful to the microorganisms than untreated ones. Congo red, Crystal violet, Coomassie brilliant blue and Malachite green showed an inhibitory effect on all tested microorganisms' growth. So it was concluded from the microbial toxicity test that the dyes which are toxic to the microbial cells and were converted into the nontoxic product by Lignin peroxidase. Besides phytotoxicity, these azo dyes also affect the microbial flora of soil and water (Pokharia \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). (Moawad et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) has been reported that azo dyes eliminate microbial colonies due to the high frequency of mutation. Moreover, (Kahraman and Yalcin 2005) has also revealed the negative effect of the textile dyes on \u003cem\u003eP. aeruginosa\u003c/em\u003e to assess the toxicity after decolorization.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe production of enzyme lignin peroxidase from \u003cem\u003eP. fluorescence\u003c/em\u003eLiP-RL5 has been enhanced\u0026thinsp;~\u0026thinsp;20 folds using statistical methodology RSM. The optimization resulted in approx. 20-fold increase in LiP activity. The enzyme successfully detoxified the different commercially used textile dyes through decolorization. Therefore, this study strengthens the bioremediation potential of the LiP enzyme. Our findings further proved that, the enzyme-treated textile dyes possess relatively less hazardous impact on seed germination of different plant species. Moreover, the soil's normal microflora also falls short of the devastating effects of LiP treated dyes. Therefore, this enzyme could be a boon for the detoxification of hazardous textile industry effluents and help restore the fertility of contaminated soil by promoting the growth of native plants and microbial communities of the environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRKR acknowledges fellowship provided by the Department of Environment Science and Technology, Govt. of Himachal Pradesh. RKB sincerely acknowledges the University Grant Commission (UGC), New Delhi, India, for providing financial assistance in the form of post-doctoral fellowship. Department of Biotechnology, Himachal Pradesh University Shimla, India, thankfully acknowledged for providing all the facilities for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRKR:\u003c/strong\u003e Conceptualization, Data curation, Formal analysis, Validation, Visualization, and Writing-original draft, Writing- review \u0026amp; editing,\u0026nbsp;\u003cstrong\u003eRKB:\u003c/strong\u003e Conceptualization, Methodology, Writing-review \u0026amp; editing,\u0026nbsp;\u003cstrong\u003eNR:\u0026nbsp;\u003c/strong\u003eMethodology, Formal analyses, Investigation,\u0026nbsp;\u003cstrong\u003eVS:\u003c/strong\u003e Methodology, Formal analyses, Investigation,\u0026nbsp;\u003cstrong\u003eNS:\u003c/strong\u003e Formal analyses, Writing-review \u0026amp; editing, and\u0026nbsp;\u003cstrong\u003eAKB:\u003c/strong\u003e Formal analysis, Funding acquisition, Investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grant numbers [No. Env. S\u0026amp;T (F/)/5-1)/2019-20] \u0026amp; [No. F./PDFSS201415SCHIM8434]. Author Ravi Kant Bhatia has received research support from Company UGC-New Delhi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data of this study has been provided.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Dr. Ravi Kant Bhatia\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArora DS, Gill PK (2001) Comparison of two assay procedures for lignin peroxidase. Enzyme Microb Technol 28:602\u0026ndash;605. https://doi.org/10.1016/S0141-0229(01)00302-7\u003c/li\u003e\n \u003cli\u003eAsses N, Ayed L, Hkiri N, Hamdi M (2018) Congo red decolorization and detoxification by \u003cem\u003eAspergillus Niger\u003c/em\u003e: Removal mechanisms and dye degradation pathway. Biomed Res Int 2018:. https://doi.org/10.1155/2018/3049686\u003c/li\u003e\n \u003cli\u003eBhatt P, Tiwari M, Parmarick P, Bhatt K, Gangola S, Adnan M, Singh Y, Bilal M, Ahmed S, Chen S (2022) Insights into the catalytic mechanism of ligninolytic peroxidase and laccase in lignin degradation. Bioremediation J 15;26(4):281-91.\u003c/li\u003e\n \u003cli\u003eBharagava RN, Mani S, Mulla SI, Saratale GD (2018) Degradation and decolourization potential of an ligninolytic enzyme producing Aeromonas hydrophila for crystal violet dye and its phytotoxicity evaluation. Ecotoxicol Environ Saf 156:166\u0026ndash;175. https://doi.org/10.1016/j.ecoenv.2018.03.012\u003c/li\u003e\n \u003cli\u003eBilal M, Iqbal M, Hu H, Zhang X (2016) Mutagenicity, cytotoxicity and phytotoxicity evaluation of biodegraded textile effluent by fungal ligninolytic enzymes. Water Sci Technol 73:2332\u0026ndash;2344. https://doi.org/10.2166/wst.2016.082\u003c/li\u003e\n \u003cli\u003eChandel H, Kumar P, Chandel AK, Verma ML (2022) Biotechnological advances in biomass pretreatment for bio-renewable production through nanotechnological intervention. Biomass Convers Biorefinery 2022 1:1\u0026ndash;23. https://doi.org/10.1007/S13399-022-02746-0\u003c/li\u003e\n \u003cli\u003eCheriaa J, Khaireddine M, Rouabhia M, Bakhrouf A (2012) Removal of triphenylmethane dyes by bacterial consortium. Sci World J 2012:1\u0026ndash;9. https://doi.org/10.1100/2012/512454\u003c/li\u003e\n \u003cli\u003eDesore A, Narula SA (2018) An overview on corporate response towards sustainability issues in textile industry. Environ Dev Sustain 20:1439\u0026ndash;1459. https://doi.org/10.1007/s10668-017-9949-1\u003c/li\u003e\n \u003cli\u003eDing Y, Cui K, Liu X, et al (2022) Lignin peroxidase-catalyzed direct oxidation of trace organic pollutants through a long-range electron transfer mechanism: Using propranolol as an example. J Hazard Mater 431:. https://doi.org/10.1016/J.JHAZMAT.2022.128544\u003c/li\u003e\n \u003cli\u003eJadhav JP, Govindwar SP (2006) Biotransformation of malachite green by S\u003cem\u003eaccharomyces cerevisiae\u003c/em\u003e MTCC 463. Yeast 23:315\u0026ndash;323. https://doi.org/10.1002/yea.1356\u003c/li\u003e\n \u003cli\u003eJi J, Kulshreshtha S, Kakade A, et al (2020) Bioaugmentation of membrane bioreactor with \u003cem\u003eAeromonas hydrophila\u003c/em\u003e LZ-MG14 for enhanced malachite green and hexavalent chromium removal in textile wastewater. Int Biodeterior Biodegrad 150:104939. https://doi.org/10.1016/j.ibiod.2020.104939\u003c/li\u003e\n \u003cli\u003eKahraman S, Yalcin P (2005a) Antibacterial Effect of Untreated and Treated (Decolorized) Dyes by Agricultural Wastes. Am J Biochem Biotechnol 1:50\u0026ndash;53. https://doi.org/10.3844/ajbbsp.2005.50.53\u003c/li\u003e\n \u003cli\u003eKahraman S, Yalcin P (2005b) Antibacterial Effect of Untreated and Treated (Decolorized) Dyes by Agricultural Wastes. Am J Biochem Biotechnol 1:50\u0026ndash;53. https://doi.org/10.3844/ajbbsp.2005.50.53\u003c/li\u003e\n \u003cli\u003eKalyani DC, Phugare SS, Shedbalkar UU, Jadhav JP (2011) Purification and characterization of a bacterial peroxidase from the isolated strain \u003cem\u003ePseudomonas\u003c/em\u003e sp. SUK1 and its application for textile dye decolorization. Ann Microbiol 61:483\u0026ndash;491. https://doi.org/10.1007/s13213-010-0162-9\u003c/li\u003e\n \u003cli\u003eKaur B, Kumar B, Garg N, Kaur N (2015) Statistical optimization of conditions for decolorization of synthetic dyes by \u003cem\u003eCordyceps\u003c/em\u003e \u003cem\u003emilitaris\u003c/em\u003e MTCC 3936 using RSM. Biomed Res Int 2015:. https://doi.org/10.1155/2015/536745\u003c/li\u003e\n \u003cli\u003eKiran, Rathour RK, Bhatia RK, et al (2020) Fabrication of thermostable and reusable nanobiocatalyst for dye decolourization by immobilization of lignin peroxidase on graphene oxide functionalized MnFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e superparamagnetic nanoparticles. Bioresour Technol 317:124020. https://doi.org/10.1016/j.biortech.2020.124020\u003c/li\u003e\n \u003cli\u003eKrishnan S, Din MFM, Taib SM, et al (2019) Process constraints in sustainable bio-hythane production from wastewater: Technical note. Bioresour Technol Reports 5:359\u0026ndash;363. https://doi.org/10.1016/j.biteb.2018.05.003\u003c/li\u003e\n \u003cli\u003eKumar V, Pallavi P, Sen SK, Raut S (2024) Harnessing the potential of white rot fungi and ligninolytic enzymes for efficient textile dye degradation: A comprehensive review. Water Environ Res 96(1):e10959.\u003c/li\u003e\n \u003cli\u003eLellis B, F\u0026aacute;varo-Polonio CZ, Pamphile JA, Polonio JC (2019) Effects of textile dyes on health and the environment and bioremediation potential of living organisms. Biotechnol Res Innov 3:275\u0026ndash;290. https://doi.org/10.1016/j.biori.2019.09.001\u003c/li\u003e\n \u003cli\u003eLin J, Ye W, Xie M, Seo DH, Luo J, Wan Y, Van der Bruggen B (2023) Environmental impacts and remediation of dye-containing wastewater. Nat Rev Earth Environ 4(11):785-803. https://doi.org/10.1038/s43017-023-00489-8\u003c/li\u003e\n \u003cli\u003eMahmood R, Sharif F, Ali S, Hayyat MU (2015) Enhancing the decolorizing and degradation ability of bacterial consortium isolated from textile effluent affected area and its application on seed germination. Sci World J 2015:. https://doi.org/10.1155/2015/628195\u003c/li\u003e\n \u003cli\u003eMoawad H, Abd El-Rahim WM, Khalafallah M (2003) Evaluation of biotoxicity of textile dyes using two bioassays. J Basic Microbiol 43:218\u0026ndash;229. https://doi.org/10.1002/jobm.200390025\u003c/li\u003e\n \u003cli\u003eNousiainen P, Kontro J, Manner H, et al (2014) Phenolic mediators enhance the manganese peroxidase catalyzed oxidation of recalcitrant lignin model compounds and synthetic lignin. Fungal Genet Biol 72:137\u0026ndash;149. https://doi.org/10.1016/j.fgb.2014.07.008\u003c/li\u003e\n \u003cli\u003eOllikka P, Alhonm\u0026auml;ki K, Lepp\u0026auml;nen V-M, et al (1993) Decolorization of Azo, triphenyl methane, heterocyclic, and polymeric dyes by lignin peroxidase isoenzymes from \u003cem\u003ePhanerochaete\u003c/em\u003e \u003cem\u003echrysosporium\u003c/em\u003e. Appl Environ Microbiol 59:4010\u003c/li\u003e\n \u003cli\u003ePatil S. (2014) Production and purification of lignin peroxidase from \u003cem\u003eBacillus megaterium\u003c/em\u003e and its application in bioremidation. J Microbiol 2:22\u0026ndash;28\u003c/li\u003e\n \u003cli\u003ePokharia A. ASS. (2015) Toxicological effect of textile dyes and their metabolites: A review. . 1:\u003c/li\u003e\n \u003cli\u003ePreethi PS, Vickram S, Das R, Hariharan NM, Rameshpathy M, Subbaiya R, Karmegam N, Kim W, Govarthanan M 2023 Bioprospecting of novel peroxidase from \u003cem\u003eStreptomyces coelicolor\u0026nbsp;\u003c/em\u003estrain SPR7 for carcinogenic azo dyes decolorization. Chemosphere. 1;310:136836.\u003c/li\u003e\n \u003cli\u003eRathour RK, Sharma V, Rana N, et al (2020) Bioremediation of simulated textile effluent by an efficient bio-catalyst purified from a novel \u003cem\u003ePseudomonas fluorescence\u003c/em\u003e LiP-RL5. Curr Chem Biol 14:128\u0026ndash;139. https://doi.org/10.2174/2212796814666200406100247\u003c/li\u003e\n \u003cli\u003eRoy DC, Biswas SK, Saha AK, et al (2018) Biodegradation of Crystal Violet dye by bacteria isolated from textile industry effluents. PeerJ 2018:e5015. https://doi.org/10.7717/peerj.5015\u003c/li\u003e\n \u003cli\u003eSadek MB. HJ. SHB. BA. SS (2016) Experiment Findingspdf available toxic effect of textile dyeing effluents on germination, growth, yield and nutritional quality of Okra (\u003cem\u003eAbelmoschus esculentus\u003c/em\u003e). Int J Ecotoxicol Ecobiol \u0026nbsp;1:82\u0026ndash;87\u003c/li\u003e\n \u003cli\u003eSainsbury PD, Hardiman EM, Ahmad M, et al (2013) Breaking Down lignin to high-value chemicals: the conversion of lignocellulose to vanillin in a gene deletion mutant of \u003cem\u003eRhodococcus jostii\u003c/em\u003e RHA1. ACS Chem Biol 8:2151\u0026ndash;2156. https://doi.org/10.1021/cb400505a\u003c/li\u003e\n \u003cli\u003eSamuel R, Foston M, Jiang N, et al (2011) Structural changes in switchgrass lignin and hemicelluloses during pretreatments by NMR analysis. Polym Degrad Stab. https://doi.org/10.1016/j.polymdegradstab.2011.08.015\u003c/li\u003e\n \u003cli\u003eSaratale RG, Saratale GD, Chang JS, Govindwar SP (2011a) Bacterial decolorization and degradation of azo dyes: A review. J. Taiwan Inst. Chem. Eng. 42:138\u0026ndash;157\u003c/li\u003e\n \u003cli\u003eSaratale RG, Saratale GD, Chang JS, Govindwar SP (2011b) Bacterial decolorization and degradation of azo dyes: A review. J Taiwan Inst Chem Eng 42:138\u0026ndash;157. https://doi.org/10.1016/j.jtice.2010.06.006\u003c/li\u003e\n \u003cli\u003eSasidhara R. TT (2014) . Lignolytic and lignocellulosic enzymes of \u003cem\u003eGanoderma lucidum\u003c/em\u003e in liquid medium. . Eur jouranal Exp Biol 4:375\u0026ndash;379\u003c/li\u003e\n \u003cli\u003eSelvam K, Swaminathan K, Chae K-S (2003) Decolourization of azo dyes and a dye industry effluent by a white rot fungus \u003cem\u003eThelephora\u003c/em\u003e sp. Bioresour Technol 88:115\u0026ndash;119. https://doi.org/10.1016/S0960-8524(02)00280-8\u003c/li\u003e\n \u003cli\u003eSheng L (2017) Market Size of the Global Textile and Apparel Industry: 2015 to 2020.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eShoiful A, Kambara H, Cao LTT, et al (2020) Mn(II) oxidation and manganese-oxide reduction on the decolorization of an azo dye. Int Biodeterior Biodegradation 146:104820. https://doi.org/10.1016/j.ibiod.2019.104820\u003c/li\u003e\n \u003cli\u003eStewart P, Kersten P, Vanden Wymelenberg A, et al (1992) Lignin peroxidase gene family of Phanerochaete chrysosporium: Complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome. J Bacteriol 174:5036\u0026ndash;5042. https://doi.org/10.1128/jb.174.15.5036-5042.1992\u003c/li\u003e\n \u003cli\u003eThiry MC Staying alive: Making textiles sustainable. AATCC Review . 2011\u003c/li\u003e\n \u003cli\u003eTien M, Kirk TK (1988) Lignin peroxidase of \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e. pp 238\u0026ndash;249\u003c/li\u003e\n \u003cli\u003eTiquia SM (2010) Reduction of compost phytotoxicity during the process of decomposition. Chemosphere 79:506\u0026ndash;512. https://doi.org/10.1016/j.chemosphere.2010.02.040\u003c/li\u003e\n \u003cli\u003eTuncer G, Tekkaya C, Sungur S, et al (2009) Assessing pre-service teachers\u0026rsquo; environmental literacy in Turkey as a mean to develop teacher education programs. Int J Educ Dev 29:426\u0026ndash;436. https://doi.org/10.1016/j.ijedudev.2008.10.003\u003c/li\u003e\n \u003cli\u003eUmesh JU, Rhushikesh DN, Vishal D V, et al (2016) phytotoxic effect of synthetic textile dye effluent on growth of five plant species\u0026nbsp;Trends Biotech. Res. 5(2), 1-6.\u003c/li\u003e\n \u003cli\u003eVandana TG, Rao R, Ashish Kumar S, et al (2018) Enhancing production of lignin peroxidase from white rot fungi employing statistical optimization and evaluation of its potential in delignification of crop residues. Int J Curr Microbiol Appl Sci 7:2599\u0026ndash;2621. https://doi.org/10.20546/ijcmas.2018.701.312\u003c/li\u003e\n \u003cli\u003eWang H, Tucker M, Ji Y (2013) Recent development in chemical depolymerization of lignin: A Review. J Appl Chem 2013:1\u0026ndash;9. https://doi.org/10.1155/2013/838645\u003c/li\u003e\n \u003cli\u003eWong DWS (2009) Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 157:174\u0026ndash;209. https://doi.org/10.1007/s12010-008-8279-z\u003c/li\u003e\n \u003cli\u003eWu K, Shi M, Pan X, et al (2022) Decolourization and biodegradation of methylene blue dye by a ligninolytic enzyme-producing \u003cem\u003eBacillus thuringiensi\u003c/em\u003es: Degradation products and pathway. Enzyme Microb Technol 156:109999. https://doi.org/10.1016/J.ENZMICTEC.2022.109999\u003c/li\u003e\n \u003cli\u003eZucconi F, Bertoldi DE 1981) Biological evaluation of compost maturity. Biocycle 22:27\u0026ndash;29\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Lignin peroxidase, Pseudomonas fluorescence, Synthetic dyes, Detoxification, Phytotoxicity, Microbial toxicity","lastPublishedDoi":"10.21203/rs.3.rs-3958055/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3958055/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUntreated disposal of toxic synthetic dyes is a serious threat to the environment. Every year, several thousand gallons of dyes are being disposed into the water resources without any sustainable detoxification. The accumulation of hazardous dyes in the environment poses a severe threat to the human health, flora, fauna, and microflora. Therefore, in the present study, a lignin peroxidase enzyme from \u003cem\u003ePseudomonas fluorescence\u003c/em\u003e LiP-RL5 has been employed for the maximal detoxification of selected commercially used dyes. The enzyme production from the microorganism was enhanced\u0026thinsp;~\u0026thinsp;20 folds using statistical optimization tool response surface methodology. Four different combinations (pH, production time, seed age, and inoculum size) were found to be crucial for the higher production of LiP. The crude enzyme showed decolorization action on commonly used commercial dyes such as Crystal violet, Congo red, Malachite green, and Coomassie brilliant blue. Successful toxicity mitigation of these dyes culminated in the improved seed germination in three plant species, \u003cem\u003eVigna radiate\u003c/em\u003e (20\u0026ndash;60%), \u003cem\u003eCicer arietinum\u003c/em\u003e (20\u0026ndash;40%), and \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e (10\u0026ndash;25%). The LiP treated dyes also exhibit reduced bactericidal effects against four common resident microbial species, \u003cem\u003eEscherichia coli\u003c/em\u003e (2\u0026ndash;10 mm), \u003cem\u003eBacillus\u003c/em\u003e sp. (4\u0026ndash;8 mm), \u003cem\u003ePseudomonas\u003c/em\u003e sp. (2\u0026ndash;8 mm), and \u003cem\u003eLactobacillus\u003c/em\u003e sp. (2\u0026ndash;10 mm). Therefore, apart from the tremendous industrial applications, the LiP from \u003cem\u003ePseudomonas fluorescence\u003c/em\u003e LiP-RL5 could be a potential biocatalyst for the detoxification of synthetic dyes.\u003c/p\u003e","manuscriptTitle":"Combatting synthetic dye toxicity through exploring the potential of lignin peroxidase from Pseudomonas fluorescence LiP RL5","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 13:17:42","doi":"10.21203/rs.3.rs-3958055/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2024-05-12T09:48:57+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-25T03:45:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-25T02:17:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-03-19T17:02:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-01T06:11:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-02-26T23:38:57+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":"f3c613c9-9200-4fc6-8782-a1ee157e136d","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:13:13+00:00","versionOfRecord":{"articleIdentity":"rs-3958055","link":"https://doi.org/10.1007/s11356-024-34400-9","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-08-05 15:57:49","publishedOnDateReadable":"August 5th, 2024"},"versionCreatedAt":"2024-03-27 13:17:42","video":"","vorDoi":"10.1007/s11356-024-34400-9","vorDoiUrl":"https://doi.org/10.1007/s11356-024-34400-9","workflowStages":[]},"version":"v1","identity":"rs-3958055","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3958055","identity":"rs-3958055","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}