Heavy metals degradation by Serratia marcescens immobilized into novel modified loofah sponges

Heavy metals degradation by Serratia marcescens immobilized into novel modified loofah sponges | 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 Heavy metals degradation by Serratia marcescens immobilized into novel modified loofah sponges Santhiya Jayakumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6388463/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract The intensive production of heavy metals by tannery industry is consequential to our environment. The present study involves novel modified loofah sponges immobilised indigenous Serratia marcescens, a new sustainable approach for bioremediation. The antibiotic resistance test showed, susceptibility of specific strains to 19 antibiotics. The morphological, biochemical and phylogenetic analysis were done and sangers DNA sequencing revealed the specific organism to be Serratia marcescens , which showed gram negative colonies with pink or bright red or purple colour colony with distinctive round, elevated structure. The Serratia marcescens R1 SANTH strain were submitted to NCBI [accession number: PQ069772]. The SEM images confirmed the immobilisation of Serratia marcescens R1 SANTH and heavy metals in novel modified loofah sponges and SEM-EDX analysis were done to confirm the atomic percentage and weight percentage of copper, chromium, nickel on the loofah sponges. The amount of degradation was 92.05%, 87.8%, and 80.92% for chromium, copper and nickel, respectively was confirmed with ICP-OES analysis. This study shows novel modification of loofah sponges can act as biocarrier as well as heavy metals sequestrant, which can improve the degradation process of conventional method, decreases the cost of treatment process and also supports sustainable development goal 6, by using green technology. Agricultural Engineering Loofah sponges chromium copper nickel natural fibres Serratia tannery effluent treatment wastewater Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights Chromium, nickel and copper contamination and its treatment is a major environmental challenge. Serratia marcescens R1 SANTH exhibits high adsorption and tolerance potential of chromium, copper and nickel. The novel modified loofah sponges for heavy metals mitigation and as the local and affordable biocarrier. This study provides a basis, to address heavy metals pollution. INTRODUCTION The world population gets exposed to industrial effluents with radioactive heavy metals, chemicals, dyes, infectious biological organism and causes various infection and water pollution. The brans, pith, sawdust and more from food and agriculture-based industry, which gets dumped in landfills or incinerated which greatly impacts environment by causing air pollution. Nanofiltration, reverse osmosis, chemical precipitation, electrochemical process, ion exchange, coagulation, flocculation, membrane filtration, electro-flocculation, and adsorption holds many disadvantages such as pollution, pH sensitivity, high cost, slow precipitation, etc., (Zhao et al., 2023 ), so new approach must be designed to reduce the disadvantages. With regard to facile functionalization and environmental friendliness, biomass has been considered the most promising adsorbent for the metal ions collection. The preference of natural fiber over other available sources was increased in past decade as it is toxicity free or low toxic, available in abundance, biodegradable, bio-compatible and economically cheap compared to other sources. The detoxification by loofah sponges were less explored than other fibres for effluent, heavy metals, and other toxicity (Santhiya & Sharmila, 2024a; Narayanan et al., 2016 ). Loofah sponges composed of cellulose, hemicellulose, Xylan, mannan, galactan, and lignin (Zeng et al., 2019 ). Many researchers carried out chemical modification of loofah sponges to increase its toxicity adsorption property and its mechanical properties (Santhiya Jayakumar, Sharmila, 2024 b). Previous reports were reported applications of various natural fiber such as sugarcane bagasse (Sharma & Forster, 1994 ), banana fiber (Begum et al., 2020 ), agave bagasse (Bernardo et al., 2009 ), coconut fiber (Franguelli et al., 2019 ), peat and coconut fiber(Kaszycki et al., 2004 ) adsorbed about 13.4, 30.5, 11.4, 25, 8.02, and 9.54 mg/g chromium respectively. However, no research reported the employment of novel chemical method in loofah sponges for heavy metals biodegradation. Whence, the novel loofah sponges containing Serratia marcescens R1 SANTH, isolated from tannery effluent was developed, compared with free cells, and evaluated for their application in heavy metals bioremediation. Materials and Methods Reagents, Solvents, and Culture Media The 1000 ppm stock solution of chromium (2.8 g/L of potassium dichromate (K 2 Cr 2 O 7 )), copper (3.9 g/L of copper sulphate (CuSO 4, ), and nickel (3.6 g of nickel sulphate (NiSO 4 .6H 2 O)) was prepared with analytical grade in HI-media Laboratories (Mumbai, India). The loofah sponges were bought from Perambur market, Chennai, Tamil Nadu. The Luria-Bertani broth (M1151, HI Media), Mueller-Hinton agar (M391, HI Media). Isolation, Molecular and Phylogenetic Identification The tannery effluent samples collected from Madhavaram (latitude 13.1345N and longitude 80.2401E) and Chrompet (latitude 12°57′51.8″N and longitude 80°07′58.1″E), Chennai, Tamil Nadu, in plastic container, transferred to laboratory in cooling condition, and stored at 4℃ for further use. The micro-organisms were isolated by 50 ppm multi-metals supplemented agar dilution method (Cervantes & Ohtake, 1988 ), and the heavy metals degrading organism was identified by the 16S rRNA sanger’s gene sequencing method using 968F (5′-AACGCGAAGAACCTTAC-3’) and 1401R (5′-CGGTGTGTACAAGACCC-3′) forward and reverse primers amplified PCR product. The sequence was compared with the GenBank using the BLAST/NCBI software and submitted to GenBank. The phylogenetic tree was constructed in MEGA v.11.0 using p-distances, complete deletion, and bootstrapping with 1000 replications. The Bureau of Indian Standards (BIS) methods was used to determine the physicochemical parameters of raw tannery effluents. Growth curve and Minimum Inhibitory Concentration The growth curve was determined using broth inoculation method for native and heavy metals (150, 200 ppm) containing Luria Bertani media at OD 600 nm, using UV- Visible spectrophotometer (Shimadzu model 1780). The MIC was determined by viable count method was at varying dosages of heavy metals between 50 and 1000 ppm (Mamay et al., 2024 ). ANTIBIOTIC SUSCEPTIBILITY The Mueller Hinton agar cultured R1 strain was impregnated with antibiotic-discs (6 mm, dia. HIMEDIA) as per Bauer-Kirby Method. Antibiotic susceptibility and multiple antibiotic resistance index of the isolate was determined (Kolář et al., 2001 ; Kwon et al., 2022 ). The PW096 scale calibrated inhibition zones and classified organisms as per National Committee for Clinical Laboratory Standards (NCCLS) (Survey, 2022 ). About 30 antibiotic discs was tested, discs containing Netillin (NET-30µg), Cefoperazone (CS-75 µg), Gentamycin (gen-10µg), Ceftazidime (CAZ-30µg), Imipenem (IPM-10µg), Cefazolin (CZ-30µg), Doripenem (DOR-10 µg), Tetracycline (TE-30µg), Norfloxacin (NX-10µg), Vancomycin (VA-30µg), Cefdinir (CDN-5µg), Aztroneum (A0-30µg), Ticarcillin/clavulanate (TCC-75/10µg), AmphotericinB (AP-50µg), Nystatin (NS-50µg), Levofloxacin (LE-5µg), Cephoxotin (CN-30 µg), Ceftizoxime (CX-30µg), Chloramphenicol (C-30µg), Ampicillin (AMP-10µg), Colistin (CL-10µg), Cefepime (CPM-30µg), Piperacillin/tazobactam (PIT100/10µg), Amoxillin/clavulanic acid (AMC-30µg), Cefotaxime (CTX-30 µg), Cefuroxime (CXM-30µg), Cefotaxime/clavulanic acid (CEC30/10- µg), Cephoxitin (CX-30µg), Ciproflaxacin (CIP-5 µg), Ceftriaxone (CI-30µg), Fosfomycin (F-200µg), Ciproflaxacin (CF-30 µg), Gatifloxacin (GF-30 µg). Serratia marcescens immobilized loofah sponges-a batch approach The dust, impurities, and seeds of loofah sponges was removed by washing repeatedly with distilled water and dried in hot air oven at 160℃ for 4 hours. The loofah sponges were chemically improved for its property with 4% sodium hydroxide, 2% ethanoic acid, 0.6% potassium permanganate, 2% Hexa-sodium hexametaphosphate and 30% 1, 2, 3- Trihydroxy-propane, which has been proven to adsorb heavy metal (Santhiya Jayakumar, Sharmila, 2024 ), is further considered as a biocarriers for indigenous organisms in this study. The free cells and modified loofah sponges immobilized cells, were tested in sucrose (2g) and loofah sponges (1g), supplemented Luria Bertani broth at various pH, contact time, temperature, metal dosages using a shaking incubator (Scigenics Biotech, India) and analysis was carried out in triplicates at 180 RPM (Srivastava & Thakur, 2007 ). different pH (6, 7, 8), contact time (1, 3, 6 days), temperature (28℃, 37℃, 42℃) and initial metal dosage (150, 200 ppm) Characterization of immobilized loofah sponges Scanning Electron Microscopy The fabricated loofah sponges were gold sputtered at 15KV, under vacuum precipitated with aluminium, and analysed with 2 µM imaging modes with a 20 mm distance were visualized by scanning electron microscopy (SEM, AMETEK, US) Scanning Electron Microscopy-Electron dispersive X-Ray analysis The presence of chromium, copper and nickel and its characteristic structure were noted. The SEM-EDX analysis carried out, atomic percentages and the weight percentages were noted for chromium, copper, and nickel. Inductively coupled plasma atomic emission spectroscopy The concentration percentage unit of copper, chromium, copper, and nickel in cell free filtrate was analysed at wavelength 324.754 nm, 267.716 nm and, 231.604 nm respectively using Inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies model 5800) (Kotelnikova et al., 2024 ) and analysed with data software ICP-EXPERT, and chromium, copper and nickel biodegradation was calculated (Srivastava & Thakur, 2007 ). The heavy metals adsorption percentage was calculated using percentage difference formula RESULTS Sample collection and analysis The Madhavaram and Chrompet collected tannery effluent samples were serial diluted in 0.9% sodium chloride solution (w/v) and cultured at 37℃ for 2 days. The morphologically different colonies were selected, and CFU about 9 × 10 − 1 CFU/ml was noted. The red colour pigmented colony (R1) were taken into account. The selected colony was positive for multi-metal resistance, as it showed good growth in 10% effluent enriched nutrient agar plates supplemented with 50 ppm of heavy metal, so red colour pigmented colony (R1) were taken into account. The bacterial isolates were morphologically and biochemically characterized as per Berge’s manual of determinative bacteriology, the colony showed gram negative behaviour, colonies showed bright red, pink and violet colour colonies. The biochemical test showed positive for catalase, negative for indole, methyl red, Voges Proskauer, oxidase, respectively. MICROBIAL AND RAW EFFLUENT CHARACTERIZATION The raw effluents were physiochemically characterized by Bureau of Indian Standards (BIS, under 3025, part 16–54) methods (table :1). The R1 strain was biochemical analysed, showed negative for indole, oxidase, methyl red and, positive for Voges Proskauer, Simmons citrate, catalase, urea test. The morphological analysis showed purple, red and pink colonies with circular, raised, round end morphology, and colony was gram-negative rod shaped in microscopic analysis. TABLE.1 RAW TANNERY EFFLUENTS CHARACTERIZATION PHYSICOCHEMICAL PARAMETERS (raw effluents) RESULTS (mg/l) Chromium 0.011 Copper 0.1 Nickel 0.011 Biological oxygen demand 1177.3 Chemical oxygen demand 3673.5 Total dissolved solids 6665 Total suspended solids 2667.1 Total alkalinity 893.4 Sulphate 2472.3 Nitrate 88 Phosphate 1.04 MINIMUM INHIBITORY CONCENTRATION INFLUENCED PIGMENTATION AND GROWTH CURVE Though certain metals are essential for Microbial survival and growth depends on essential metals, but higher dosages increase cell toxicity by accumulating in cell through permeases or ATP-binding cassette-type transport systems and inhibits proteins selectivity by incorporating into microbial enzymes and, damages cell function and DNA, which ultimately cause death of the cells, so metal detoxification genes like PGPR get activated and growth retards, which is visualized by plate method (Khoso et al., 2024 ). The Serratia marcescens performance on heavy metal adsorption was tested for 3 metals such as Ni, Cu, Cr in Luria-Bertani (LB) broth at 100 and 5000 ppm dosages. The MIC of R1 strain was 3000 ppm for copper, chromium and 5000 ppm for nickel, but pigmentation was affected, projected as white colonies. The pigmentation variation was observed in 15 days stored sample at -20℃, aseptically wrapped plates, such as red to pink colony for 100 ppm, pink to purple for 200 ppm, purple to pink centred white colonies at 400 ppm then to white colonies, for all metals. The change in colour was observed due to metal toxicity, the increase in metal dosage also influences the pigmentation genes of Serratia marcescens (Agostinho A Lima de Silva, 2012). About 50 ml cultures with or without heavy metals at 150 or 200 ppm cultured in 250 ml Erlenmeyer flasks were aerated at 200 RPM, 28℃ using Scigenics shaking incubator and growth curve was measured at OD 600 nm using Shimadzu UV-Visible spectrophotometer. The chromium, copper, and nickel presence retarded and delayed the microbial growth, but heavy metals detoxification gets triggered at log phase, but the death phase of the bacteria was achieved fast than native cells, due to cell toxicity conditions. The Figure.1 demonstrates the growth curve, shown in Figure.1, the microbial bio-stasis or mortality with respect to metal toxicity was noted. ANTIBIOTIC SUSCEPTIBILITY The R1 strain was cultured in Mueller Hinton agar with various antibiotic discs, to determine their antimicrobial activity, and HI-MEDIA calibrated scale was used to measure the zone of inhibition after 16–24 hours of incubation, according to Clinical & Laboratory Standards Institute. The R1 strain was susceptible to DOR10, NX10, IPM10, CAZ30, CEC30/10, PIT100/10, TCC75/10, CIP5, AK30, C30, GF30, LE5, FO200, AT30, AC30, K10, COX200, AO30, CDn5 and, resisted AP-50, NS-50, CL-10, CPM-30, CXM-30, CX30, AMC-30, CN30, CZ30, CEC30/10, CTX30, CIP5, CS75, TB10, NET30, CK10, and organisms showed multiple antibiotic resistance index of 0.4, which is more than 0.2, so the organisms is high risk infection causing agent, so further gene modification study must be considered for threat prevention in real time usage, however the efficiency of the strain was tested in this study (Kwon et al., 2022 ). DNA SEQUENCING AND PHYLOGENETIC ANALYSIS The commercial Qiagen kit extracted DNA sample of R1 strain was quantified with nanodrop microvolume UV-Visible spectrophotometer, and the amount of the DNA yield was 470µg/500µl at OD 260 nm. The DNA analysis was done by, 16s rRNA sangers sequencing, and the nucleotide data was analysed using BLAST tool. The R1 strain was analysed to be Serratia marcescens and submitted to NCBI. The R1 strain was submitted with accession number PQ069772 with strain name Serratia marcescens strain R1SANTH. The evolutionary history of Serratia marcescens inferred using MEGA12 software. The maximum likelihood method was used for computing evolutionary distance with optimal tree branch length of 0.063 (Saitou & Nei, 1987 ). The ambiguous position of each sequence applied with the pairwise deletion option which comprises 1473 position (Kumar et al., 2024 ; Tamura et al., 2004 ). HEAVY METALS DETOXIFICATION BY SERRATIA MARCESCENS IMMOBILIZED FABRICATED LOOFAH SPONGES-A BATCH STUDY The loofah sponges modified with sodium hydroxide, sodium hematophosphate, glycerol, potassium permanganate and acetic acid according to previous study (Santhiya Jayakumar & Sharmila, 2024 ). In short loofah sponges were washed with distilled water and then treated with 4% NaOH, 2%acetic acid, 0.6% sodium hematophosphate, 2% sodium hematophosphate, 30% glycerol at 4 hours, 30 minutes, 5 minutes, 3 hours, and 30 minutes respectively, which is used to sequester chromium. In this study the loofah sponges were analysed in real time for Serratia marcescens immobilizing biocarrier, and chromium, copper and nickel adsorbing property at 150 and 200 ppm dosages. The batch study was performed in shaker incubator at 180 RPM at various pH 6, 7, 8, and temperature 28, 37, 42℃ for 1–6 days of contact time. The R1 strain showed optimised heavy metals degrading capacity at pH 6 for chromium and pH 7 for copper and nickel, at temperature 28 ℃, within 6 days contact time. The R1 strain showed about 127.05, 116.7, 106.82 mg/l for 150 ppm, and 180.5, 172.53, 157.99 mg/l for 200 ppm of chromium, copper and nickel respectively (Kwon et al., 2022 ). SCANNING ELECTRON MICROSCOPY -ENERGY DISPERSIVE X-RAY SPECTROSCOPY ANALYSIS The modified loofah sponges act as a good biocarrier for immobilizing Serratia marcescens [PQ069772] and also sequesters copper, chromium, and nickel. The sample was analysed at 5 µm imaging mode. The sodium hydroxide treatment elongated the loofah fiber (Mwaikambo & Ansell, 2002 ), and as the result of modification the fiber showed increased tensile strength and increased sequesters metals and contaminants. Increased level of adsorption was noted after removal of dirt, waxes and more. The Serratia marcescens R1 SANTH strain was immobilized with exopolysaccharides production in the novel modified loofah sponges which was noted by SEM analysis (Figure.4). The chromium, nickel and copper adsorption on surface of loofah sponges and bacteria were confirmed with SEM-EDX as represented in Figure (3). MICROBIAL BIOASSAY-ICPOES The Serratia marcescens performance on heavy metal adsorption was tested for three heavy metals such as Ni, Cu, Cr in Luria-Bertani (LB) broth at 150 and 200 ppm dosages. The immobilized bacteria and free cells were analysed for its metal degradation property at 200 rpm under the optimised bacterial growth conditions. Serratia marcescens adsorbed about 122.43, 116.7, and 106.82 mg/g of chromium, copper and nickel respectively in 150 ppm dosages, while adsorption was increased with the increase in heavy metals dosages up to 200 ppm. At 200 ppm, the adsorption was noted to be 180.5, 172.53, 157.99 mg/g for chromium, copper and nickel, respectively. The bacteria were immobilized in modified loofah sponges for the support, which further increased the heavy metals adsorption up to 184.1, 175.6, and 161.85 mg/g for chromium, copper and nickel, respectively and the experiments were carried out for 200 ppm dosages at optimized pH 6 for chromium adsorption and pH 7 for copper and nickel, maintained at 28℃ temperatures with 6 days contact time. DISCUSSION This study also reported the strength and reusability of novel modified loofah sponges as there is no presence of visual degradation in experimentation time. The loofah sponges show no visible degradation during experiment time, which shows the durability of the treated loofah sponges, however further study must analyse the full degradation time of modified fiber. The Serratia marcescenes R1 SANTH has high tolerance up to 400 ppm chromium, 800 ppm of copper and nickel. The SEM analysis showed the clear surface of loofah sponges without impurities which increases the surface area and water adsorbing property of the sponges, and the indigenous cells grouping and adhesion to the surface of the loofah sponges was noted, and the membrane of the bacteria showed distorted shape, as increased heavy metals concentration damaged the rod shape and resulted in extended spherical or shrinked or damaged membrane layer. The characteristic crystal lattice structure of chromium, copper and nickel was observed in pristine and treated loofah sponges, which proved the heavy metal adsorbing property of the loofah sponges. The biofilm concentration increased to certain percentage initially by adding heavy metals as it creates stress, and adsorption also increases with increase in biofilm concentration as the heavy metals involves that biomagnification implications (Akram et al., 2018 ). The heavy metals listed to be human carcinogen and food contaminant by International Agency for Research, and the World Health Organization (Guodong Zhu et al., 2022 ). The usage of natural fibers is cost effective, supports circular economy and sustainability, but the problems arose with its fast degradation property. The fast degradability property of the loofah sponges was enhanced by chemical modification. The modification with ethanoic acid, sodium hydroxide, potassium permanganate and glycerol, removes the impurities and lignin on its layer to increase the loofah sponges tensile strength (Santhiya Jayakumar, Sharmila, 2024 ). This, excellent modification of loofah sponges has improved the heavy metals adsorption, detoxification property and acts as the immobilizer for tannery effluent indigenous microbes than free cells (Kalaiarasu et al., 2024 ). The microbe-immobilized loofah sponge adsorbed up to 90.25 % of chromium, 8627 % of copper, and 79 % of nickel, respectively. According to the study conducted by Kraer et al., the natie and citric acid teated sisal fiber adsorbed about 52 and 61.3 mg/g of nickel, respectively. The study by Hanif et al., showed Cassia fistula adsorbed 99.72 mg/g of nickel, whereas this study proved the efficiency of natural biomaterial, loofah sponges highly efficient for chromium, copper and nickel adsorption up to 184.1, 175.6, and 161.85 mg/g for chromium, copper and nickel, than previous studies. This is the first study to report the efficiency of the novel modified loofah sponges as metal sequestrant as well as biocarrier for Serratia marcescens, that has showed very fruitful result than previously reported studies, so this study could help industrialist and government pollution control persons for improving conventional waste water treatment without any new or additional treatment set-up. CONCLUSION The Serratia marcescens, an indigenous heavy metal sequestering bacteria isolated from raw tannery effluent, was immobilized in the novel modified loofah sponges, has an efficient heavy metal degradation property with heavy metal adsorption up to 184.1, 175.6, and 161.85 mg/g for chromium, copper and nickel, was observed. Though the organism’s multiple antibiotic indices are high, it can modify using gene mutation or manipulation in future and can be used for real time wastewater treatment, however modified loofah sponges can be used for sequestering heavy metals in real time usage in any climatic conditions or modification protocol can be tested and used as low-cost natural fibers for various part of the world. Though many fibres reported, this modification has given fruitful output than other reported studies. To utilize the efficiency of the organisms without impacting the nature, the organisms must be manipulated genetically by adding more copy of pollutant degrading genes and cutting the infectious gene in future research. Statements and Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Acknowledgment: The author (SANTHIYA JAYAKUMAR) orcid id:0009-0003-4124-3973 gratefully acknowledges supervisor Dr K J SHARMILA orcid id:0000-003-0685-9900 and Dr. M.G.R Educational and Research Institute, Maduravoyal, Chennai for immense support for completing my paper. Author contribution The authors confirm their contribution to the paper as follows: study conception and design, data collection, analysis and interpretation of data, draft manuscript preparation: Santhiya Jayakumar, supervision: Dr K J Sharmila, data drafting: Suganya Kalaiarasu. All authors reviewed the results and approved the final version of the manuscript. Competing interest The authors, Mrs. Santhiya Jayakumar, Dr K J Sharmila, Suganya Kalaiarasu, declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. LIFE SCIENCE REPORTING No life science threat was practiced in this research. References Agostinho A Lima de Silva, M. A. R. de C. S. A. L. de S. P. M. T. D. R. G. da S. F. C. S. de M. S. C. A. de M. B. E. H. (2012). Heavy metal tolerance (Cr, Ag AND Hg) in bacteria isolated from sewage. Brazilian Journal of Microbiology , 43 (4), 1620–1631. Akram, R., Turan, V., Hammad, H. M., Ahmad, S., Hussain, S., Hasnain, A., Maqbool, M. M., Rehmani, M. I. A., Rasool, A., Masood, N., Mahmood, F., Mubeen, M., Sultana, S. R., Fahad, S., Amanet, K., Saleem, M., Abbas, Y., Akhtar, H. M., Hussain, S., … Nasim, W. (2018). Fate of Organic and Inorganic Pollutants in Paddy Soils . 197–214. https://doi.org/10.1007/978-3-319-93671-0_13 Begum, H. A., Haque, A. K. M. M., Islam, Md. D., Hasan, M. M., Ahmed, S., Razzak, Md., & Khan, R. A. (2020). Analysis of the Adsorption of Toxic Chromium (VI) by Untreated and Chitosan Treated Banana and Areca Fiber. Journal of Textile Science and Technology , 06 (02), 81–106. https://doi.org/10.4236/jtst.2020.62007 Bernardo, G. R. R., Rene, R. M. J., & Alfaro-De la, A. D. la T. (2009). Chromium (III) uptake by agro-waste biosorbents: Chemical characterization, sorption-desorption studies, and mechanism. Journal of Hazardous Materials , 170 (2–3), 845–854. https://doi.org/10.1016/j.jhazmat.2009.05.046 Cervantes, C., & Ohtake, H. (1988). Plasmid-determined resistance to chromate in Pseudomonas aeruginosa. FEMS Microbiology Letters , 56 (2), 173–176. https://doi.org/10.1111/j.1574-6968.1988.tb03172.x Franguelli, F. P., Tannous, K., & Cione Coppi, C. (2019). Biosorption of hexavalent chromium from aqueous solutions using raw coconut fiber as a natural adsorbent. Chemical Engineering Communications , 206 (11), 1437–1450. https://doi.org/10.1080/00986445.2018.1557154 Guodong Zhu, Liguo Xie, Wenzhang Tan, Chunlan Ma, & Yunlin Wei. (2022). Cd2+ tolerance and removal mechanisms of Serratia marcescens KMR-3 - ScienceDirect . https://doi.org/https://doi.org/10.1016/j.jbiotec.2022.09.019 Jayakumar, S., & Sharmila, D. K. J. (2024). Applicability of Agricultural Wastes for Heavy Metals Bio Remediation-A Low-Cost Approach. In Tuijin Jishu/Journal of Propulsion Technology (Vol. 45, Issue 4). Kalaiarasu, S., Sharmila, K. J., & Jayakumar, S. (2024). Immobilization of Microbes and Iron Oxide Nanoparticles in Sisal Fiber for Crude oil Biodegradation – A Micro-Scale Study . 27 (3). Kaszycki, P., Fedorovych, D., Ksheminska, H., Babyak, L., Wójcik, D., & Koloczek, H. (2004). Chromium accumulation by living yeast at various environmental conditions. Microbiological Research , 159 (1), 11–17. https://doi.org/10.1016/j.micres.2003.12.002 Khoso, M. A., Wagan, S., Alam, I., Hussain, A., Ali, Q., Saha, S., Poudel, T. R., Manghwar, H., & Liu, F. (2024). Impact of plant growth-promoting rhizobacteria (PGPR) on plant nutrition and root characteristics: Current perspective. Plant Stress , 11 , 100341. https://doi.org/10.1016/J.STRESS.2023.100341 Kolář, M., Urbánek, K., & Látal, T. (2001). Antibiotic selective pressure and development of bacterial resistance. International Journal of Antimicrobial Agents , 17 (5), 357–363. https://doi.org/10.1016/S0924-8579(01)00317-X Kotelnikova, A. D., Borisochkina, T. I., Kolchanova, K. A., Shishkin, M. A., Egorov, F. S., Okorkov, V. V., & Rogova, O. B. (2024). Dataset on elemental composition of soils and plants under long-term application of mineral and organic fertilizers on gray forest soils in Vladimir region, Russia. Data in Brief , 53 , 110057. https://doi.org/10.1016/J.DIB.2024.110057 Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., & Tamura, K. (2024). MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Molecular Biology and Evolution , 41 (12). https://doi.org/10.1093/molbev/msae263 Kwon, J., Yang, M. H., Ko, H. J., Kim, S. G., Park, C., & Park, S. C. (2022). Antimicrobial Resistance and Virulence Factors of Proteus mirabilis Isolated from Dog with Chronic Otitis Externa. Pathogens , 11 (10), 1–9. https://doi.org/10.3390/pathogens11101215 Mamay, M., Mar’atiningsih, L., Atoilah, E. M., & Mutia, S. R. (2024). Minimal inhibitory concentration (MIC) of sodium hypochlorit against bacterial isolate from equipment in the microbiology laboratory (pp. 6(2), 585–590). Journal of Midwifery and Nursing. https://doi.org/https://doi.org/10.35335/jmn.v6i2.5171 Mwaikambo, L. Y., & Ansell, M. P. (2002). Chemical modification of hemp, sisal, jute, and kapok fibers by alkalization. Journal of Applied Polymer Science , 84 (12), 2222–2234. https://doi.org/10.1002/app.10460 Narayanan, R. M., Sharmila, K. J., & Dharanirajan, K. (2016). Evaluation of marine water quality – a case study between Cuddalore and Pondicherry coast, India. Indian Journal of Geo-Marine Sciences , 45 (4), 517–532. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution , 4 (4), 406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 Santhiya Jayakumar, Sharmila, K. J. (2024). Fabrication Of Loofah Sponge as An Effective Natural Copper Sequestrant-The Inexpensive Approach. African Journal of Biomedical Research , 27 (3S) (october), 2269–2276. https://doi.org/10.53555/AJBR.v27i3S.2603 Sharma, D. C., & Forster, C. F. (1994). A preliminary examination into the adsorption of hexavalent chromium using low-cost adsorbents. Bioresource Technology , 47 (3), 257–264. https://doi.org/10.1016/0960-8524(94)90189-9 Srivastava, S., & Thakur, I. S. (2007). Evaluation of biosorption potency of Acinetobacter sp. for removal of hexavalent chromium from tannery effluent. Biodegradation , 18 (5), 637–646. https://doi.org/10.1007/s10532-006-9096-0 Survey, N. H. (2022). M Pl E M Pl E Ly. Performance Standards for Antimicrobial Susceptibility Testing . https://clsi.org/standards/products/microbiology/documents/m100/ Tamura, K., Nei, M., & Kumar, S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences of the United States of America , 101 (30), 11030–11035. https://doi.org/10.1073/pnas.0404206101 Wang, W., Yu, L., Hao, W., Zhang, F., Jiang, M., Zhao, S., & Wang, F. (2021). Multi-Locus Sequence Typing and Drug Resistance Analysis of Swine Origin Escherichia coli in Shandong of China and Its Potential Risk on Public Health. Frontiers in Public Health , 9 . https://doi.org/10.3389/FPUBH.2021.780700 Zeng, L., Liu, Q., Lu, M., Liang, E., Wang, G., & Xu, W. (2019). Modified natural loofah sponge as an effective heavy metal ion adsorbent: Amidoxime functionalized poly(acrylonitrile-g-loofah). Chemical Engineering Research and Design , 150 , 26–32. https://doi.org/10.1016/j.cherd.2019.07.021 Zhao, Y., Chen, T., Liang, T., Yang, J. Y., Song, X. F., Yang, X. J., Li, Y. S., & Liu, Y. (2023). Radiation synthesis of IDAA functionalized loofah sponge for enhanced removal of Pb(II) and Cd(II): Behavior and mechanism investigation. Journal of Molecular Liquids , 392 , 123428. https://doi.org/10.1016/J.MOLLIQ.2023.123428 Additional Declarations The authors declare no competing interests. Supplementary Files copper.png GraphicalAbstract.png Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-6388463","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439982487,"identity":"0a5761bc-5176-4ba3-87be-ca87b7ec0be1","order_by":0,"name":"Santhiya Jayakumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYJACZgYDBgbGBhCzAiaCHzA2I7ScgYkQ1AJnthGhRb797PHHBQWH5Zkb2J9u+DmvTt7geDNQpIZBnl/sAFYtBmfyEptnGBw2bGzgMbvZu+2w4YYzx4AixxgMZ85OwK6FIcewmcfgMCNQC9ttxm0HGGfOAImwMSQY3MauRb7/DViLfWMD+7PbjHPq7GfOf/+xmecfbi0MNyC2JDY2MJjdZmxgTuyX4GFs5m3DrcXgxhvD2TwG6cmNzUC/9Bw7nNzPk2Y4m7dPAqdf5PtzDD7z/LG23dje/uzGj5o62zb2ww8+83yzkeeXxuEwCGhmMESLCwl8ykGgjkGekJJRMApGwSgYuQAA3w9hGAHZPV0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0003-4124-3973","institution":"Saveetha Medical College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University , Chennai-602105, Tamil Nadu, India","correspondingAuthor":true,"prefix":"","firstName":"Santhiya","middleName":"","lastName":"Jayakumar","suffix":""}],"badges":[],"createdAt":"2025-04-06 19:50:13","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6388463/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-6388463/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109123378,"identity":"ff5f1998-7419-45ce-872f-afed6870e486","added_by":"auto","created_at":"2026-05-12 17:55:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerratia marcescens growth dependency to heavy metals\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/24ac845475dc3999c2a14a66.png"},{"id":109123395,"identity":"0a0c0da5-4ef3-444b-855e-49208ef1d3ef","added_by":"auto","created_at":"2026-05-12 17:55:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePHYLOGENETIC TREE OF ISOLATES, the strain isolated mentioned as Serratia marcescens PQ069772.1 (this work).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/c29022d631ce27a8e39f2954.png"},{"id":109123396,"identity":"1e392a2c-0e01-4607-aea9-4cc3e94ffd71","added_by":"auto","created_at":"2026-05-12 17:55:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":557917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) SEM-EDX graph (b) heavy metals weight and atomic percentage (c) crystal lattice of heavy metals.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/f38c791f50a87559c0d5db2d.png"},{"id":109123430,"identity":"bf6868d1-9e77-4655-b835-60bb1437438b","added_by":"auto","created_at":"2026-05-12 17:55:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":800294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Treated loofah sponges with polysaccharide layer formed by Serratia marcescens (b) distorted or warped bacteria due to heavy metals stress (c) heavy metals lattice presence.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/fbc0f4050b220c7a2e0ed6b8.png"},{"id":109123456,"identity":"fe4294b7-170a-4ea6-b3e6-ab54f3c89040","added_by":"auto","created_at":"2026-05-12 17:56:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1767911,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/46b77d39-3c28-4bc8-b11a-841c3f15e4e8.pdf"},{"id":109123426,"identity":"a79b2955-a7c3-4597-93d3-7546e5714de1","added_by":"auto","created_at":"2026-05-12 17:55:49","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":341727,"visible":true,"origin":"","legend":"","description":"","filename":"copper.png","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/8e16dfc3d54850795cbd96c9.png"},{"id":109123397,"identity":"3a53d48f-86f7-4ed4-9651-66be0fb67332","added_by":"auto","created_at":"2026-05-12 17:55:46","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":414643,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6388463/v2/1b7cd2b6990a44d2ec17b50a.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Heavy metals degradation by Serratia marcescens immobilized into novel modified loofah sponges","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eChromium, nickel and copper contamination and its treatment is a major environmental challenge.\u003c/li\u003e\n \u003cli\u003eSerratia marcescens R1 SANTH exhibits high adsorption and tolerance potential of chromium, copper and nickel.\u003c/li\u003e\n \u003cli\u003eThe novel modified loofah sponges for heavy metals mitigation and as the local and affordable biocarrier.\u003c/li\u003e\n \u003cli\u003eThis study provides a basis, to address heavy metals pollution.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":" INTRODUCTION","content":"\u003cp\u003eThe world population gets exposed to industrial effluents with radioactive heavy metals, chemicals, dyes, infectious biological organism and causes various infection and water pollution. The brans, pith, sawdust and more from food and agriculture-based industry, which gets dumped in landfills or incinerated which greatly impacts environment by causing air pollution. Nanofiltration, reverse osmosis, chemical precipitation, electrochemical process, ion exchange, coagulation, flocculation, membrane filtration, electro-flocculation, and adsorption holds many disadvantages such as pollution, pH sensitivity, high cost, slow precipitation, etc., (Zhao et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), so new approach must be designed to reduce the disadvantages. With regard to facile functionalization and environmental friendliness, biomass has been considered the most promising adsorbent for the metal ions collection. The preference of natural fiber over other available sources was increased in past decade as it is toxicity free or low toxic, available in abundance, biodegradable, bio-compatible and economically cheap compared to other sources. The detoxification by loofah sponges were less explored than other fibres for effluent, heavy metals, and other toxicity (Santhiya \u0026amp; Sharmila, 2024a; Narayanan et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Loofah sponges composed of cellulose, hemicellulose, Xylan, mannan, galactan, and lignin (Zeng et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Many researchers carried out chemical modification of loofah sponges to increase its toxicity adsorption property and its mechanical properties (Santhiya Jayakumar, Sharmila, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003eb). Previous reports were reported applications of various natural fiber such as sugarcane bagasse (Sharma \u0026amp; Forster, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), banana fiber (Begum et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), agave bagasse (Bernardo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), coconut fiber (Franguelli et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), peat and coconut fiber(Kaszycki et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) adsorbed about 13.4, 30.5, 11.4, 25, 8.02, and 9.54 mg/g chromium respectively. However, no research reported the employment of novel chemical method in loofah sponges for heavy metals biodegradation. Whence, the novel loofah sponges containing Serratia marcescens R1 SANTH, isolated from tannery effluent was developed, compared with free cells, and evaluated for their application in heavy metals bioremediation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents, Solvents, and Culture Media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 1000 ppm stock solution of chromium (2.8 g/L of potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e)), copper (3.9 g/L of copper sulphate (CuSO\u003csub\u003e4,\u003c/sub\u003e), and nickel (3.6 g of nickel sulphate (NiSO\u003csub\u003e4\u003c/sub\u003e .6H\u003csub\u003e2\u003c/sub\u003eO)) was prepared with analytical grade in HI-media Laboratories (Mumbai, India). The loofah sponges were bought from Perambur market, Chennai, Tamil Nadu. The Luria-Bertani broth (M1151, HI Media), Mueller-Hinton agar (M391, HI Media).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, Molecular and Phylogenetic Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tannery effluent samples collected from Madhavaram (latitude 13.1345N and longitude 80.2401E) and Chrompet (latitude 12\u0026deg;57\u0026prime;51.8\u0026Prime;N and longitude 80\u0026deg;07\u0026prime;58.1\u0026Prime;E), Chennai, Tamil Nadu, in plastic container, transferred to laboratory in cooling condition, and stored at 4℃ for further use. The micro-organisms were isolated by 50 ppm multi-metals supplemented agar dilution method (Cervantes \u0026amp; Ohtake, \u003cspan class=\"CitationRef\"\u003e1988\u003c/span\u003e), and the heavy metals degrading organism was identified by the 16S rRNA sanger\u0026rsquo;s gene sequencing method using 968F (5\u0026prime;-AACGCGAAGAACCTTAC-3\u0026rsquo;) and 1401R (5\u0026prime;-CGGTGTGTACAAGACCC-3\u0026prime;) forward and reverse primers amplified PCR product. The sequence was compared with the GenBank using the BLAST/NCBI software and submitted to GenBank. The phylogenetic tree was constructed in MEGA v.11.0 using p-distances, complete deletion, and bootstrapping with 1000 replications. The Bureau of Indian Standards (BIS) methods was used to determine the physicochemical parameters of raw tannery effluents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth curve and Minimum Inhibitory Concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth curve was determined using broth inoculation method for native and heavy metals (150, 200 ppm) containing Luria Bertani media at OD 600 nm, using UV- Visible spectrophotometer (Shimadzu model 1780). The MIC was determined by viable count method was at varying dosages of heavy metals between 50 and 1000 ppm (Mamay et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANTIBIOTIC SUSCEPTIBILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Mueller Hinton agar cultured R1 strain was impregnated with antibiotic-discs (6 mm, dia. HIMEDIA) as per Bauer-Kirby Method. Antibiotic susceptibility and multiple antibiotic resistance index of the isolate was determined (Kol\u0026aacute;ř et al., \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e; Kwon et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). The PW096 scale calibrated inhibition zones and classified organisms as per National Committee for Clinical Laboratory Standards (NCCLS) (Survey, \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). About 30 antibiotic discs was tested, discs containing Netillin (NET-30\u0026micro;g), Cefoperazone (CS-75 \u0026micro;g), Gentamycin (gen-10\u0026micro;g), Ceftazidime (CAZ-30\u0026micro;g), Imipenem (IPM-10\u0026micro;g), Cefazolin (CZ-30\u0026micro;g), Doripenem (DOR-10 \u0026micro;g), Tetracycline (TE-30\u0026micro;g), Norfloxacin (NX-10\u0026micro;g), Vancomycin (VA-30\u0026micro;g), Cefdinir (CDN-5\u0026micro;g), Aztroneum (A0-30\u0026micro;g), Ticarcillin/clavulanate (TCC-75/10\u0026micro;g), AmphotericinB (AP-50\u0026micro;g), Nystatin (NS-50\u0026micro;g), Levofloxacin (LE-5\u0026micro;g), Cephoxotin (CN-30 \u0026micro;g), Ceftizoxime (CX-30\u0026micro;g), Chloramphenicol (C-30\u0026micro;g), Ampicillin (AMP-10\u0026micro;g), Colistin (CL-10\u0026micro;g), Cefepime (CPM-30\u0026micro;g), Piperacillin/tazobactam (PIT100/10\u0026micro;g), Amoxillin/clavulanic acid (AMC-30\u0026micro;g), Cefotaxime (CTX-30 \u0026micro;g), Cefuroxime (CXM-30\u0026micro;g), Cefotaxime/clavulanic acid (CEC30/10- \u0026micro;g), Cephoxitin (CX-30\u0026micro;g), Ciproflaxacin (CIP-5 \u0026micro;g), Ceftriaxone (CI-30\u0026micro;g), Fosfomycin (F-200\u0026micro;g), Ciproflaxacin (CF-30 \u0026micro;g), Gatifloxacin (GF-30 \u0026micro;g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerratia marcescens immobilized loofah sponges-a batch approach\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dust, impurities, and seeds of loofah sponges was removed by washing repeatedly with distilled water and dried in hot air oven at 160℃ for 4 hours. The loofah sponges were chemically improved for its property with 4% sodium hydroxide, 2% ethanoic acid, 0.6% potassium permanganate, 2% Hexa-sodium hexametaphosphate and 30% 1, 2, 3- Trihydroxy-propane, which has been proven to adsorb heavy metal (Santhiya Jayakumar, Sharmila, \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e), is further considered as a biocarriers for indigenous organisms in this study. The free cells and modified loofah sponges immobilized cells, were tested in sucrose (2g) and loofah sponges (1g), supplemented Luria Bertani broth at various pH, contact time, temperature, metal dosages using a shaking incubator (Scigenics Biotech, India) and analysis was carried out in triplicates at 180 RPM (Srivastava \u0026amp; Thakur, \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003edifferent pH (6, 7, 8), contact time (1, 3, 6 days), temperature (28℃, 37℃, 42℃) and initial metal dosage (150, 200 ppm)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of immobilized loofah sponges\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning Electron Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fabricated loofah sponges were gold sputtered at 15KV, under vacuum precipitated with aluminium, and analysed with 2 \u0026micro;M imaging modes with a 20 mm distance were visualized by scanning electron microscopy (SEM, AMETEK, US)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning Electron Microscopy-Electron dispersive X-Ray analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of chromium, copper and nickel and its characteristic structure were noted. The SEM-EDX analysis carried out, atomic percentages and the weight percentages were noted for chromium, copper, and nickel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInductively coupled plasma atomic emission spectroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration percentage unit of copper, chromium, copper, and nickel in cell free filtrate was analysed at wavelength 324.754 nm, 267.716 nm and, 231.604 nm respectively using Inductively coupled plasma atomic emission spectroscopy (ICP-AES, Agilent Technologies model 5800) (Kotelnikova et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) and analysed with data software ICP-EXPERT, and chromium, copper and nickel biodegradation was calculated (Srivastava \u0026amp; Thakur, \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). The heavy metals adsorption percentage was calculated using percentage difference formula\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\u003cb\u003eSample collection and analysis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe Madhavaram and Chrompet collected tannery effluent samples were serial diluted in 0.9% sodium chloride solution (w/v) and cultured at 37℃ for 2 days. The morphologically different colonies were selected, and CFU about 9 × 10\u003csup\u003e− 1\u003c/sup\u003e CFU/ml was noted. The red colour pigmented colony (R1) were taken into account. The selected colony was positive for multi-metal resistance, as it showed good growth in 10% effluent enriched nutrient agar plates supplemented with 50 ppm of heavy metal, so red colour pigmented colony (R1) were taken into account. The bacterial isolates were morphologically and biochemically characterized as per Berge’s manual of determinative bacteriology, the colony showed gram negative behaviour, colonies showed bright red, pink and violet colour colonies. The biochemical test showed positive for catalase, negative for indole, methyl red, Voges Proskauer, oxidase, respectively.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMICROBIAL AND RAW EFFLUENT CHARACTERIZATION\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe raw effluents were physiochemically characterized by Bureau of Indian Standards (BIS, under 3025, part 16–54) methods (table :1). The R1 strain was biochemical analysed, showed negative for indole, oxidase, methyl red and, positive for Voges Proskauer, Simmons citrate, catalase, urea test. The morphological analysis showed purple, red and pink colonies with circular, raised, round end morphology, and colony was gram-negative rod shaped in microscopic analysis.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTABLE.1 RAW TANNERY EFFLUENTS CHARACTERIZATION\u003c/b\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePHYSICOCHEMICAL PARAMETERS (raw effluents)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRESULTS (mg/l)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChromium\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCopper\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNickel\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiological oxygen demand\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1177.3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical oxygen demand\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3673.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal dissolved solids\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6665\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal suspended solids\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2667.1\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal alkalinity\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e893.4\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSulphate\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2472.3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhosphate\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e \u003cb\u003eMINIMUM INHIBITORY CONCENTRATION INFLUENCED PIGMENTATION AND GROWTH CURVE\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThough certain metals are essential for Microbial survival and growth depends on essential metals, but higher dosages increase cell toxicity by accumulating in cell through permeases or ATP-binding cassette-type transport systems and inhibits proteins selectivity by incorporating into microbial enzymes and, damages cell function and DNA, which ultimately cause death of the cells, so metal detoxification genes like PGPR get activated and growth retards, which is visualized by plate method (Khoso et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The Serratia marcescens performance on heavy metal adsorption was tested for 3 metals such as Ni, Cu, Cr in Luria-Bertani (LB) broth at 100 and 5000 ppm dosages. The MIC of R1 strain was 3000 ppm for copper, chromium and 5000 ppm for nickel, but pigmentation was affected, projected as white colonies. The pigmentation variation was observed in 15 days stored sample at -20℃, aseptically wrapped plates, such as red to pink colony for 100 ppm, pink to purple for 200 ppm, purple to pink centred white colonies at 400 ppm then to white colonies, for all metals. The change in colour was observed due to metal toxicity, the increase in metal dosage also influences the pigmentation genes of Serratia marcescens (Agostinho A Lima de Silva, 2012). About 50 ml cultures with or without heavy metals at 150 or 200 ppm cultured in 250 ml Erlenmeyer flasks were aerated at 200 RPM, 28℃ using Scigenics shaking incubator and growth curve was measured at OD 600 nm using Shimadzu UV-Visible spectrophotometer. The chromium, copper, and nickel presence retarded and delayed the microbial growth, but heavy metals detoxification gets triggered at log phase, but the death phase of the bacteria was achieved fast than native cells, due to cell toxicity conditions. The Figure.1 demonstrates the growth curve, shown in Figure.1, the microbial bio-stasis or mortality with respect to metal toxicity was noted.\u003c/p\u003e\u003cp\u003e \u003cb\u003eANTIBIOTIC SUSCEPTIBILITY\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe R1 strain was cultured in Mueller Hinton agar with various antibiotic discs, to determine their antimicrobial activity, and HI-MEDIA calibrated scale was used to measure the zone of inhibition after 16–24 hours of incubation, according to Clinical \u0026amp; Laboratory Standards Institute. The R1 strain was susceptible to DOR10, NX10, IPM10, CAZ30, CEC30/10, PIT100/10, TCC75/10, CIP5, AK30, C30, GF30, LE5, FO200, AT30, AC30, K10, COX200, AO30, CDn5 and, resisted AP-50, NS-50, CL-10, CPM-30, CXM-30, CX30, AMC-30, CN30, CZ30, CEC30/10, CTX30, CIP5, CS75, TB10, NET30, CK10, and organisms showed multiple antibiotic resistance index of 0.4, which is more than 0.2, so the organisms is high risk infection causing agent, so further gene modification study must be considered for threat prevention in real time usage, however the efficiency of the strain was tested in this study (Kwon et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003eDNA SEQUENCING AND PHYLOGENETIC ANALYSIS\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe commercial Qiagen kit extracted DNA sample of R1 strain was quantified with nanodrop microvolume UV-Visible spectrophotometer, and the amount of the DNA yield was 470µg/500µl at OD 260 nm. The DNA analysis was done by, 16s rRNA sangers sequencing, and the nucleotide data was analysed using BLAST tool. The R1 strain was analysed to be Serratia marcescens and submitted to NCBI. The R1 strain was submitted with accession number PQ069772 with strain name Serratia marcescens strain R1SANTH. The evolutionary history of Serratia marcescens inferred using MEGA12 software. The maximum likelihood method was used for computing evolutionary distance with optimal tree branch length of 0.063 (Saitou \u0026amp; Nei, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The ambiguous position of each sequence applied with the pairwise deletion option which comprises 1473 position (Kumar et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tamura et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003eHEAVY METALS DETOXIFICATION BY SERRATIA MARCESCENS IMMOBILIZED FABRICATED LOOFAH SPONGES-A BATCH STUDY\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe loofah sponges modified with sodium hydroxide, sodium hematophosphate, glycerol, potassium permanganate and acetic acid according to previous study (Santhiya Jayakumar \u0026amp; Sharmila, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In short loofah sponges were washed with distilled water and then treated with 4% NaOH, 2%acetic acid, 0.6% sodium hematophosphate, 2% sodium hematophosphate, 30% glycerol at 4 hours, 30 minutes, 5 minutes, 3 hours, and 30 minutes respectively, which is used to sequester chromium. In this study the loofah sponges were analysed in real time for Serratia marcescens immobilizing biocarrier, and chromium, copper and nickel adsorbing property at 150 and 200 ppm dosages. The batch study was performed in shaker incubator at 180 RPM at various pH 6, 7, 8, and temperature 28, 37, 42℃ for 1–6 days of contact time. The R1 strain showed optimised heavy metals degrading capacity at pH 6 for chromium and pH 7 for copper and nickel, at temperature 28 ℃, within 6 days contact time. The R1 strain showed about 127.05, 116.7, 106.82 mg/l for 150 ppm, and 180.5, 172.53, 157.99 mg/l for 200 ppm of chromium, copper and nickel respectively (Kwon et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003eSCANNING ELECTRON MICROSCOPY -ENERGY DISPERSIVE X-RAY SPECTROSCOPY ANALYSIS\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe modified loofah sponges act as a good biocarrier for immobilizing Serratia marcescens [PQ069772] and also sequesters copper, chromium, and nickel.\u003c/p\u003e\u003cp\u003eThe sample was analysed at 5 µm imaging mode. The sodium hydroxide treatment elongated the loofah fiber (Mwaikambo \u0026amp; Ansell, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and as the result of modification the fiber showed increased tensile strength and increased sequesters metals and contaminants. Increased level of adsorption was noted after removal of dirt, waxes and more. The Serratia marcescens R1 SANTH strain was immobilized with exopolysaccharides production in the novel modified loofah sponges which was noted by SEM analysis (Figure.4). The chromium, nickel and copper adsorption on surface of loofah sponges and bacteria were confirmed with SEM-EDX as represented in Figure (3).\u003c/p\u003e\u003cp\u003e \u003cb\u003eMICROBIAL BIOASSAY-ICPOES\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe Serratia marcescens performance on heavy metal adsorption was tested for three heavy metals such as Ni, Cu, Cr in Luria-Bertani (LB) broth at 150 and 200 ppm dosages. The immobilized bacteria and free cells were analysed for its metal degradation property at 200 rpm under the optimised bacterial growth conditions. Serratia marcescens adsorbed about 122.43, 116.7, and 106.82 mg/g of chromium, copper and nickel respectively in 150 ppm dosages, while adsorption was increased with the increase in heavy metals dosages up to 200 ppm. At 200 ppm, the adsorption was noted to be 180.5, 172.53, 157.99 mg/g for chromium, copper and nickel, respectively. The bacteria were immobilized in modified loofah sponges for the support, which further increased the heavy metals adsorption up to 184.1, 175.6, and 161.85 mg/g for chromium, copper and nickel, respectively and the experiments were carried out for 200 ppm dosages at optimized pH 6 for chromium adsorption and pH 7 for copper and nickel, maintained at 28℃ temperatures with 6 days contact time.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study also reported the strength and reusability of novel modified loofah sponges as there is no presence of visual degradation in experimentation time. The loofah sponges show no visible degradation during experiment time, which shows the durability of the treated loofah sponges, however further study must analyse the full degradation time of modified fiber. The Serratia marcescenes R1 SANTH has high tolerance up to 400 ppm chromium, 800 ppm of copper and nickel. The SEM analysis showed the clear surface of loofah sponges without impurities which increases the surface area and water adsorbing property of the sponges, and the indigenous cells grouping and adhesion to the surface of the loofah sponges was noted, and the membrane of the bacteria showed distorted shape, as increased heavy metals concentration damaged the rod shape and resulted in extended spherical or shrinked or damaged membrane layer. The characteristic crystal lattice structure of chromium, copper and nickel was observed in pristine and treated loofah sponges, which proved the heavy metal adsorbing property of the loofah sponges. The biofilm concentration increased to certain percentage initially by adding heavy metals as it creates stress, and adsorption also increases with increase in biofilm concentration as the heavy metals involves that biomagnification implications (Akram et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The heavy metals listed to be human carcinogen and food contaminant by International Agency for Research, and the World Health Organization (Guodong Zhu et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The usage of natural fibers is cost effective, supports circular economy and sustainability, but the problems arose with its fast degradation property. The fast degradability property of the loofah sponges was enhanced by chemical modification. The modification with ethanoic acid, sodium hydroxide, potassium permanganate and glycerol, removes the impurities and lignin on its layer to increase the loofah sponges tensile strength (Santhiya Jayakumar, Sharmila, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This, excellent modification of loofah sponges has improved the heavy metals adsorption, detoxification property and acts as the immobilizer for tannery effluent indigenous microbes than free cells (Kalaiarasu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The microbe-immobilized loofah sponge adsorbed up to 90.25 % of chromium, 8627 % of copper, and 79 % of nickel, respectively. According to the study conducted by Kraer et al., the natie and citric acid teated sisal fiber adsorbed about 52 and 61.3 mg/g of nickel, respectively. The study by Hanif et al., showed Cassia fistula adsorbed 99.72 mg/g of nickel, whereas this study proved the efficiency of natural biomaterial, loofah sponges highly efficient for chromium, copper and nickel adsorption up to 184.1, 175.6, and 161.85 mg/g for chromium, copper and nickel, than previous studies. This is the first study to report the efficiency of the novel modified loofah sponges as metal sequestrant as well as biocarrier for Serratia marcescens, that has showed very fruitful result than previously reported studies, so this study could help industrialist and government pollution control persons for improving conventional waste water treatment without any new or additional treatment set-up.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe Serratia marcescens, an indigenous heavy metal sequestering bacteria isolated from raw tannery effluent, was immobilized in the novel modified loofah sponges, has an efficient heavy metal degradation property with heavy metal adsorption up to 184.1, 175.6, and 161.85 mg/g for chromium, copper and nickel, was observed. Though the organism’s multiple antibiotic indices are high, it can modify using gene mutation or manipulation in future and can be used for real time wastewater treatment, however modified loofah sponges can be used for sequestering heavy metals in real time usage in any climatic conditions or modification protocol can be tested and used as low-cost natural fibers for various part of the world. Though many fibres reported, this modification has given fruitful output than other reported studies. To utilize the efficiency of the organisms without impacting the nature, the organisms must be manipulated genetically by adding more copy of pollutant degrading genes and cutting the infectious gene in future research.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author (SANTHIYA JAYAKUMAR) orcid id:0009-0003-4124-3973 gratefully acknowledges supervisor Dr K J SHARMILA orcid id:0000-003-0685-9900 and Dr. M.G.R Educational and Research Institute, Maduravoyal, Chennai for immense support for completing my paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm their contribution to the paper as follows: study conception and design, data collection, analysis and interpretation of data, draft manuscript preparation: Santhiya Jayakumar, supervision: Dr K J Sharmila, data drafting: Suganya Kalaiarasu. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors, Mrs. Santhiya Jayakumar, Dr K J Sharmila, Suganya Kalaiarasu, declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLIFE SCIENCE REPORTING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo life science threat was practiced in this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAgostinho A Lima de Silva, M. A. R. de C. S. A. L. de S. P. M. T. D. R. G. da S. F. C. S. de M. S. C. A. de M. B. E. H. (2012). Heavy metal tolerance (Cr, Ag AND Hg) in bacteria isolated from sewage. \u003cem\u003eBrazilian Journal of Microbiology\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e(4), 1620\u0026ndash;1631.\u003c/li\u003e\n \u003cli\u003eAkram, R., Turan, V., Hammad, H. M., Ahmad, S., Hussain, S., Hasnain, A., Maqbool, M. M., Rehmani, M. I. A., Rasool, A., Masood, N., Mahmood, F., Mubeen, M., Sultana, S. R., Fahad, S., Amanet, K., Saleem, M., Abbas, Y., Akhtar, H. M., Hussain, S., \u0026hellip; Nasim, W. (2018). \u003cem\u003eFate of Organic and Inorganic Pollutants in Paddy Soils\u003c/em\u003e. 197\u0026ndash;214. https://doi.org/10.1007/978-3-319-93671-0_13\u003c/li\u003e\n \u003cli\u003eBegum, H. A., Haque, A. K. M. M., Islam, Md. D., Hasan, M. M., Ahmed, S., Razzak, Md., \u0026amp; Khan, R. A. (2020). Analysis of the Adsorption of Toxic Chromium (VI) by Untreated and Chitosan Treated Banana and Areca Fiber. \u003cem\u003eJournal of Textile Science and Technology\u003c/em\u003e, \u003cem\u003e06\u003c/em\u003e(02), 81\u0026ndash;106. https://doi.org/10.4236/jtst.2020.62007\u003c/li\u003e\n \u003cli\u003eBernardo, G. R. R., Rene, R. M. J., \u0026amp; Alfaro-De la, A. D. la T. (2009). Chromium (III) uptake by agro-waste biosorbents: Chemical characterization, sorption-desorption studies, and mechanism. \u003cem\u003eJournal of Hazardous Materials\u003c/em\u003e, \u003cem\u003e170\u003c/em\u003e(2\u0026ndash;3), 845\u0026ndash;854. https://doi.org/10.1016/j.jhazmat.2009.05.046\u003c/li\u003e\n \u003cli\u003eCervantes, C., \u0026amp; Ohtake, H. (1988). Plasmid-determined resistance to chromate in Pseudomonas aeruginosa. \u003cem\u003eFEMS Microbiology Letters\u003c/em\u003e, \u003cem\u003e56\u003c/em\u003e(2), 173\u0026ndash;176. https://doi.org/10.1111/j.1574-6968.1988.tb03172.x\u003c/li\u003e\n \u003cli\u003eFranguelli, F. P., Tannous, K., \u0026amp; Cione Coppi, C. (2019). Biosorption of hexavalent chromium from aqueous solutions using raw coconut fiber as a natural adsorbent. \u003cem\u003eChemical Engineering Communications\u003c/em\u003e, \u003cem\u003e206\u003c/em\u003e(11), 1437\u0026ndash;1450. https://doi.org/10.1080/00986445.2018.1557154\u003c/li\u003e\n \u003cli\u003eGuodong Zhu, Liguo Xie, Wenzhang Tan, Chunlan Ma, \u0026amp; Yunlin Wei. (2022). \u003cem\u003eCd2+ tolerance and removal mechanisms of Serratia marcescens KMR-3 - ScienceDirect\u003c/em\u003e. https://doi.org/https://doi.org/10.1016/j.jbiotec.2022.09.019\u003c/li\u003e\n \u003cli\u003eJayakumar, S., \u0026amp; Sharmila, D. K. J. (2024). Applicability of Agricultural Wastes for Heavy Metals Bio Remediation-A Low-Cost Approach. In \u003cem\u003eTuijin Jishu/Journal of Propulsion Technology\u003c/em\u003e (Vol. 45, Issue 4).\u003c/li\u003e\n \u003cli\u003eKalaiarasu, S., Sharmila, K. J., \u0026amp; Jayakumar, S. (2024). \u003cem\u003eImmobilization of Microbes and Iron Oxide Nanoparticles in Sisal Fiber for Crude oil Biodegradation \u0026ndash; A Micro-Scale Study\u003c/em\u003e. \u003cem\u003e27\u003c/em\u003e(3).\u003c/li\u003e\n \u003cli\u003eKaszycki, P., Fedorovych, D., Ksheminska, H., Babyak, L., W\u0026oacute;jcik, D., \u0026amp; Koloczek, H. (2004). Chromium accumulation by living yeast at various environmental conditions. \u003cem\u003eMicrobiological Research\u003c/em\u003e, \u003cem\u003e159\u003c/em\u003e(1), 11\u0026ndash;17. https://doi.org/10.1016/j.micres.2003.12.002\u003c/li\u003e\n \u003cli\u003eKhoso, M. A., Wagan, S., Alam, I., Hussain, A., Ali, Q., Saha, S., Poudel, T. R., Manghwar, H., \u0026amp; Liu, F. (2024). Impact of plant growth-promoting rhizobacteria (PGPR) on plant nutrition and root characteristics: Current perspective. \u003cem\u003ePlant Stress\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, 100341. https://doi.org/10.1016/J.STRESS.2023.100341\u003c/li\u003e\n \u003cli\u003eKol\u0026aacute;ř, M., Urb\u0026aacute;nek, K., \u0026amp; L\u0026aacute;tal, T. (2001). Antibiotic selective pressure and development of bacterial resistance. \u003cem\u003eInternational Journal of Antimicrobial Agents\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(5), 357\u0026ndash;363. https://doi.org/10.1016/S0924-8579(01)00317-X\u003c/li\u003e\n \u003cli\u003eKotelnikova, A. D., Borisochkina, T. I., Kolchanova, K. A., Shishkin, M. A., Egorov, F. S., Okorkov, V. V., \u0026amp; Rogova, O. B. (2024). Dataset on elemental composition of soils and plants under long-term application of mineral and organic fertilizers on gray forest soils in Vladimir region, Russia. \u003cem\u003eData in Brief\u003c/em\u003e, \u003cem\u003e53\u003c/em\u003e, 110057. https://doi.org/10.1016/J.DIB.2024.110057\u003c/li\u003e\n \u003cli\u003eKumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., \u0026amp; Tamura, K. (2024). MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e, \u003cem\u003e41\u003c/em\u003e(12). https://doi.org/10.1093/molbev/msae263\u003c/li\u003e\n \u003cli\u003eKwon, J., Yang, M. H., Ko, H. J., Kim, S. G., Park, C., \u0026amp; Park, S. C. (2022). Antimicrobial Resistance and Virulence Factors of Proteus mirabilis Isolated from Dog with Chronic Otitis Externa. \u003cem\u003ePathogens\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(10), 1\u0026ndash;9. https://doi.org/10.3390/pathogens11101215\u003c/li\u003e\n \u003cli\u003eMamay, M., Mar\u0026rsquo;atiningsih, L., Atoilah, E. M., \u0026amp; Mutia, S. R. (2024). \u003cem\u003eMinimal inhibitory concentration (MIC) of sodium hypochlorit against bacterial isolate from equipment in the microbiology laboratory\u003c/em\u003e (pp. 6(2), 585\u0026ndash;590). Journal of Midwifery and Nursing. https://doi.org/https://doi.org/10.35335/jmn.v6i2.5171\u003c/li\u003e\n \u003cli\u003eMwaikambo, L. Y., \u0026amp; Ansell, M. P. (2002). Chemical modification of hemp, sisal, jute, and kapok fibers by alkalization. \u003cem\u003eJournal of Applied Polymer Science\u003c/em\u003e, \u003cem\u003e84\u003c/em\u003e(12), 2222\u0026ndash;2234. https://doi.org/10.1002/app.10460\u003c/li\u003e\n \u003cli\u003eNarayanan, R. M., Sharmila, K. J., \u0026amp; Dharanirajan, K. (2016). Evaluation of marine water quality \u0026ndash; a case study between Cuddalore and Pondicherry coast, India. \u003cem\u003eIndian Journal of Geo-Marine Sciences\u003c/em\u003e, \u003cem\u003e45\u003c/em\u003e(4), 517\u0026ndash;532.\u003c/li\u003e\n \u003cli\u003eSaitou, N., \u0026amp; Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. \u003cem\u003eMolecular Biology and Evolution\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(4), 406\u0026ndash;425. https://doi.org/10.1093/oxfordjournals.molbev.a040454\u003c/li\u003e\n \u003cli\u003eSanthiya Jayakumar, Sharmila, K. J. (2024). Fabrication Of Loofah Sponge as An Effective Natural Copper Sequestrant-The Inexpensive Approach. \u003cem\u003eAfrican Journal of Biomedical Research\u003c/em\u003e, \u003cem\u003e27 (3S)\u003c/em\u003e(october), 2269\u0026ndash;2276. https://doi.org/10.53555/AJBR.v27i3S.2603\u003c/li\u003e\n \u003cli\u003eSharma, D. C., \u0026amp; Forster, C. F. (1994). A preliminary examination into the adsorption of hexavalent chromium using low-cost adsorbents. \u003cem\u003eBioresource Technology\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(3), 257\u0026ndash;264. https://doi.org/10.1016/0960-8524(94)90189-9\u003c/li\u003e\n \u003cli\u003eSrivastava, S., \u0026amp; Thakur, I. S. (2007). Evaluation of biosorption potency of Acinetobacter sp. for removal of hexavalent chromium from tannery effluent. \u003cem\u003eBiodegradation\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(5), 637\u0026ndash;646. https://doi.org/10.1007/s10532-006-9096-0\u003c/li\u003e\n \u003cli\u003eSurvey, N. H. (2022). M Pl E M Pl E Ly. \u003cem\u003ePerformance Standards for Antimicrobial Susceptibility Testing\u003c/em\u003e. https://clsi.org/standards/products/microbiology/documents/m100/\u003c/li\u003e\n \u003cli\u003eTamura, K., Nei, M., \u0026amp; Kumar, S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e, \u003cem\u003e101\u003c/em\u003e(30), 11030\u0026ndash;11035. https://doi.org/10.1073/pnas.0404206101\u003c/li\u003e\n \u003cli\u003eWang, W., Yu, L., Hao, W., Zhang, F., Jiang, M., Zhao, S., \u0026amp; Wang, F. (2021). Multi-Locus Sequence Typing and Drug Resistance Analysis of Swine Origin Escherichia coli in Shandong of China and Its Potential Risk on Public Health. \u003cem\u003eFrontiers in Public Health\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e. https://doi.org/10.3389/FPUBH.2021.780700\u003c/li\u003e\n \u003cli\u003eZeng, L., Liu, Q., Lu, M., Liang, E., Wang, G., \u0026amp; Xu, W. (2019). Modified natural loofah sponge as an effective heavy metal ion adsorbent: Amidoxime functionalized poly(acrylonitrile-g-loofah). \u003cem\u003eChemical Engineering Research and Design\u003c/em\u003e, \u003cem\u003e150\u003c/em\u003e, 26\u0026ndash;32. https://doi.org/10.1016/j.cherd.2019.07.021\u003c/li\u003e\n \u003cli\u003eZhao, Y., Chen, T., Liang, T., Yang, J. Y., Song, X. F., Yang, X. J., Li, Y. S., \u0026amp; Liu, Y. (2023). Radiation synthesis of IDAA functionalized loofah sponge for enhanced removal of Pb(II) and Cd(II): Behavior and mechanism investigation. \u003cem\u003eJournal of Molecular Liquids\u003c/em\u003e, \u003cem\u003e392\u003c/em\u003e, 123428. https://doi.org/10.1016/J.MOLLIQ.2023.123428\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Dr. M.G.R. Educational and Research Institute","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Loofah sponges, chromium, copper, nickel, natural fibres, Serratia, tannery, effluent treatment, wastewater","lastPublishedDoi":"10.21203/rs.3.rs-6388463/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6388463/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe intensive production of heavy metals by tannery industry is consequential to our environment. The present study involves novel modified loofah sponges immobilised indigenous \u003cem\u003eSerratia marcescens, \u003c/em\u003ea new sustainable approach for\u003cem\u003e \u003c/em\u003ebioremediation. The antibiotic resistance test showed, susceptibility of specific strains to 19 antibiotics. The morphological, biochemical and phylogenetic analysis were done and sangers DNA sequencing revealed the specific organism to be \u003cem\u003eSerratia marcescens\u003c/em\u003e, which showed gram negative colonies with pink or bright red or purple colour colony with distinctive round, elevated structure. The Serratia marcescens R1 SANTH strain were submitted to NCBI [accession number: PQ069772]. The SEM images confirmed the immobilisation of Serratia marcescens R1 SANTH and heavy metals in novel modified loofah sponges and SEM-EDX analysis were done to confirm the atomic percentage and weight percentage of copper, chromium, nickel on the loofah sponges. The amount of degradation was 92.05%, 87.8%, and 80.92% for chromium, copper and nickel, respectively was confirmed with ICP-OES analysis. This study shows novel modification of loofah sponges can act as biocarrier as well as heavy metals sequestrant, which can improve the degradation process of conventional method, decreases the cost of treatment process and also supports sustainable development goal 6, by using green technology.\u003c/p\u003e","manuscriptTitle":"Heavy metals degradation by Serratia marcescens immobilized into novel modified loofah sponges","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-05-12 17:54:17","doi":"10.21203/rs.3.rs-6388463/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2025-04-08 09:00:18","doi":"10.21203/rs.3.rs-6388463/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd796a1d-a890-4312-9234-81eaef04abd2","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46838416,"name":"Agricultural Engineering"}],"tags":[],"updatedAt":"2025-04-08T09:00:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 17:54:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-6388463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6388463","identity":"rs-6388463","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}