Biodecolorization of Methylene Blue by Using Bacillus subtilis Immobilized into SA-PVA-Bentonite Matrix in Mineral Salt Medium and Non-nutritious Medium

Alya Awinatul Rohmah(1), Adi Setyo Purnomo(2*), Widiya Nur Safitri(3)

(1) Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya 60111, Indonesia
(2) Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya 60111, Indonesia
(3) Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember (ITS), Kampus ITS Sukolilo, Surabaya 60111, Indonesia
(*) Corresponding Author


Industrial dye wastewater can potentially cause significant harm to organisms and the environment across the world. Methylene blue (MB) is a synthetic dye that can be found in wastewater. Immobilizing clay material and degradative bacteria into a carrier is the best way to remove MB. Therefore, this study aimed to immobilize Bacillus subtilis in sodium alginate (SA)-polyvinyl alcohol (PVA)-Bentonite for adsorption and degrading MB in nutritious mineral salt medium (MSM) and non-nutritious media. The result showed that SA-PVA-Bentonite-non-living B. subtilis beads (SPB-nBS) had the highest result in non-nutritious medium, approximately 88.89%. While in nutritious MSM, living B. subtilis addition into beads (SPB-BS) reached the highest MB removal, which was 94.31%. Nutritious MSM had a role as the sole carbon and energy for living B. subtilis. So, it could adsorb and degrade MB by its enzymatic system. The degradation products were predicted as C7H10N2O4S, C8H10N2O2 and C6H8N2O3S. Hence, this study indicated that a nutritious medium was the suitable medium for MB degradation. According to the SEM result, the bacteria spread and covered the beads. Furthermore, the adsorption kinetics and isotherms were also analyzed; SPB beads followed the pseudo-second order kinetic model and Langmuir isotherm.


adsorption; immobilization; Bacillus subtilis; methylene blue

Full Text:

Full Text PDF


[1] Gao, T., Guan, G., Wang, X., and Lou, T., 2022, Electrospun molecularly imprinted sodium alginate/polyethylene oxide nanofibrous membranes for selective adsorption of methylene blue, Int. J. Biol. Macromol., 207, 62–71.

[2] Boumediene, M., Benaïssa, H., George, B., Molina, S., and Merlin, A., 2018, Effects of pH and ionic strength on methylene blue removal from synthetic aqueous solutions by sorption onto orange peel and desorption study, J. Mater. Environ. Sci., 9 (6), 1700–1711.

[3] Rajabi, M., Mahanpoor, K., and Moradi, O., 2017, Removal of dye molecules from aqueous solution by carbon nanotubes and carbon nanotube functional groups: Critical review, RSC Adv., 7 (74), 47083–47090.

[4] Zhi, S., Tian, L., Li, N., and Zhang, K., 2018, A novel system of MnO2-mullite-cordierite composite particle with NaClO for Methylene blue decolorization, J. Environ. Manage., 213, 392–399.

[5] Naushad, M., Alqadami, A.A., AlOthman, Z.A., Alsohaimi, I.H., Algamdi, M.S., and Aldawsari, A.M., 2019, Adsorption kinetics, isotherm and reusability studies for the removal of cationic dye from aqueous medium using arginine modified activated carbon, J. Mol. Liq., 293, 111442.

[6] Rizqi, H.D., and Purnomo, A.S., 2017, The ability of brown-rot fungus Daedalea dickinsii to decolorize and transform methylene blue dye, World J. Microbiol. Biotechnol., 33 (5), 92.

[7] Purnomo, A.S., Asranudin, A., Prasetyoko, D., and Azizah, Y.D.N., 2021, The biotransformation and biodecolorization of methylene blue by xenobiotic bacterium Ralstonia pickettii, Indones. J. Chem., 21 (6), 1418–1430.

[8] Purnomo, A.S., Rizqi, H.D., and Harmelia, L., 2020, Culture of bacterium Bacillus subtilis as degradation agent in attempt of sea water remediation contaminated by petroleum, J. Indones. Chem. Soc., 3 (1), 53–58.

[9] Upendar, G., Dutta, S., Chakraborty, J., and Bhattacharyya, P., 2016, Removal of methylene blue dye using immobilized Bacillus subtilis in batch & column reactor, Mater. Today: Proc., 3 (10), 3467–3472.

[10] Boelan, E.G., and Purnomo, A.S., 2018, Abilities of co-cultures of white-rot fungus Ganoderma lingzhi and bacteria Bacillus subtilis on biodegradation DDT, J. Phys.: Conf. Ser., 1095, 012015.

[11] Barathi, S., Aruljothi, K.N., Karthik, C., and Padikasan, I.A., 2020, Optimization for enhanced ecofriendly decolorization and detoxification of Reactive Blue160 textile dye by Bacillus subtilis, Biotechnol. Rep., 28, e00522.

[12] Mahmood, F., Shahid, M., Hussain, S., Shahzad, T., Tahir, M., Ijaz, M., Hussain, A., Mahmood, K., Imran, M., and Babar, S.A.K., 2017, Potential plant growth-promoting strain Bacillus sp. SR-2-1/1 decolorized azo dyes through NADH-ubiquinone:oxidoreductase activity, Bioresour. Technol., 235, 176–184.

[13] Zuorro, A., Maffei, G., and Lavecchia, R., 2017, Kinetic modeling of azo dye adsorption on non-living cells of Nannochloropsis oceanica, J. Environ. Chem. Eng., 5 (4), 4121–4127.

[14] Michalak, I., Chojnacka, K., and Witek-Krowiak, A., 2013, State of the art for the biosorption process - A review, Appl. Biochem. Biotechnol., 170 (6), 1389–1416.

[15] Zhang, Y., Yu, Z., Hu, Y., Song, C., Li, F., He, W., Wang, X., Li, Z., and Lin, H., 2021, Immobilization of nitrifying bacteria in magnetic PVA–SA-diatomite carrier for efficient removal of NH4+-N from effluents, Environ. Technol. Innovation, 22, 101407.

[16] Ruan, B., Wu, P., Chen, M., Lai, X., Chen, L., Yu, L., Gong, B., Kang, C., Dang, Z., Shi, Z., and Liu, Z., 2018, Immobilization of Sphingomonas sp. GY2B in polyvinyl alcohol–alginate–kaolin beads for efficient degradation of phenol against unfavorable environmental factors, Ecotoxicol. Environ. Saf., 162, 103–111.

[17] Xue, J., Wu, Y., Shi, K., Xiao, X., Gao, Y., Li, L., and Qiao, Y., 2019, Study on the degradation performance and kinetics of immobilized cells in straw-alginate beads in marine environment, Bioresour. Technol., 280, 88–94.

[18] Purnaningtyas, M.A.K., Sudiono, S., and Siswanta, D., 2020, Synthesis of activated carbon/chitosan/alginate beads powder as an adsorbent for methylene blue and methyl violet 2B dyes, Indones. J. Chem., 20 (5), 1119–1130.

[19] Rezvani, M.A., Oveisi, M., and Nia Asli, M.A., 2015, Phosphotungestovanadate immobilized on PVA as an efficient and reusable nano catalyst for oxidative desulphurization of gasoline, J. Mol. Catal. A: Chem., 410, 121–132.

[20] Vezentsev, A.I., Thuy, D.M., Goldovskaya-Peristaya, L.F., and Glukhareva, N.A., 2018, Adsorption of methylene blue on the composite sorbent based on bentonite-like clay and hydroxyapatite, Indones. J. Chem., 18 (4), 733–741.

[21] Belhouchat, N., Zaghouane-Boudiaf, H., and Viseras, C., 2017, Removal of anionic and cationic dyes from aqueous solution with activated organo-bentonite/sodium alginate encapsulated beads, Appl. Clay Sci., 135, 9–15.

[22] Ravi, R., and Pandey, L.M., 2019, Enhanced adsorption capacity of designed bentonite and alginate beads for the effective removal of methylene blue, Appl. Clay Sci., 169, 102–111.

[23] Su, C., Sun, X., Mu, Y., Li, P., Li, J., Fan, P., Zhang, M., Wang, M., Chen, X., and Feng, C., 2021, Multilayer calcium alginate beads containing diatom biosilica and Bacillus subtilis as microecologics for sewage treatment, Carbohydr. Polym., 256, 117603.

[24] Purnomo, A.S., Andyani, N.E.A., Nawfa, R., and Putra, S.R., 2020, Fenton reaction involvement on methyl orange biodegradation by brown-rot fungus Gloeophyllum trabeum, AIP Conf. Proc., 2237, 020002.

[25] Purnomo, A.A., Rizqi, H.D., Fatmawati, S., Putro, H.S., and Kamei, I., 2018, Effects of bacterium Ralstonia pickettii addition on DDT biodegradation by Daedalea dickinsii, Res. J. Chem. Environ., 22, 151–156.

[26] Chen, W., Zhang, H., Zhang, M., Shen, X., Zhang, X., Wu, F., Hu, J., Wang, B., and Wang, X., 2021, Removal of PAHs at high concentrations in a soil washing solution containing TX-100 via simultaneous sorption and biodegradation processes by immobilized degrading bacteria in PVA-SA hydrogel beads, J. Hazard. Mater., 410, 124533.

[27] Yuliana, M., Ismadji, S., Lie, J., Santoso, S.P., Soetaredjo, F.E., Waworuntu, G., Putro, J.N., and Wijaya, C.J., 2021, Low-cost structured alginate-immobilized bentonite beads designed for an effective removal of persistent antibiotics from aqueous solution, Environ. Res., 207, 112162.

[28] Hasan, R., Ying, W.J., Cheng, C.C., Jaafar, N.F., Jusoh, R., Abdul Jalil, A., and Setiabudi, H.D., 2020, Methylene blue adsorption onto cockle shells-treated banana pith: Optimization, isotherm, kinetic, and thermodynamic studies, Indones. J. Chem., 20 (2), 368–378.

[29] Kishor, R., Purchase, D., Saratale, G.D., Saratale, R.G., Ferreira, L.F.R., Bilal, M., Chandra, R., and Bharagava, R.N., 2021, Ecotoxicological and health concerns of persistent coloring pollutants of textile industry wastewater and treatment approaches for environmental safety, J. Environ. Chem. Eng., 9 (2), 105012.

[30] Obase, K., 2019, Extending the hyphal area of the ectomycorrhizal fungus Laccaria parva co-cultured with ectomycorrhizosphere bacteria on nutrient agar plate, Mycoscience, 60 (2), 95–101.

[31] Purnomo, A.S., Rohmah, A.A., Rizqi, H.D., Putro, H.S., and Nawfa, R., 2021, Biodecolorization of methylene blue by mixed cultures of brown-rot fungus Gloeophyllum trabeum and bacterium Bacillus subtilis, AIP Conf. Proc., 2370, 040006.

[32] Tural, B., Ertaş, E., Enez, B., Fincan, S.A., and Tural, S., 2017, Preparation and characterization of a novel magnetic biosorbent functionalized with biomass of Bacillus subtilis: Kinetic and isotherm studies of biosorption processes in the removal of methylene blue, J. Environ. Chem. Eng., 5 (5), 4795–4802.

[33] Mohsin, M.Z., Omer, R., Huang, J., Mohsin, A., Guo, M., Qian, J., and Zhuang, Y., 2021, Advances in engineered Bacillus subtilis biofilms and spores, and their applications in bioremediation, biocatalysis, and biomaterials, Synth. Syst. Biotechnol., 6 (3), 180–191.

[34] Kishor, R., Saratale, G.D., Saratale, R.G., Romanholo Ferreira, L.F., Bilal, M., Iqbal, H.M.N., and Bharagava, R.N., 2021, Efficient degradation and detoxification of methylene blue dye by a newly isolated ligninolytic enzyme producing bacterium Bacillus albus MW407057, Colloids Surf., B, 206, 111947.

[35] Maniyam, M.N., Hari, M., and Yaacob, N.S., 2020, Enhanced methylene blue decolourization by Rhodococcus strain UCC 0003 grown in banana peel agricultural waste through response surface methodology, Biocatal. Agric. Biotechnol., 23, 101486.

[36] Aljar, M.A.A., Rashdan, S., and El-Fattah, A.A., 2021, Environmentally friendly polyvinyl alcohol−alginate/bentonite semi-interpenetrating polymer network nanocomposite hydrogel beads as an efficient adsorbent for the removal of methylene blue from aqueous solution, Polymers, 13 (22), 4000.

[37] Cho, E.A., Seo, J., Lee, D.W., and Pan, J.G., 2011, Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores, Enzyme Microb. Technol., 49 (1), 100–104.

[38] Krithika, A., Gayathri, K.V., Kumar, D.T., and Doss, C.G.P., 2021, Mixed azo dyes degradation by an intracellular azoreductase enzyme from alkaliphilic Bacillus subtilis: A molecular docking study, Arch. Microbiol., 203 (6), 3033–3044.

[39] Houas, A., Lachheb, H., Ksibi, M., Elaloui, E., Guillard, C., and Herrmann, J.M., 2001, Photocatalytic degradation pathway of methylene blue in water, Appl. Catal., B, 31 (2), 145–157.

[40] Jia, P., Tan, H., Liu, K., and Gao, W., 2018, Synthesis, characterization and photocatalytic property of novel ZnO/bone char composite, Mater. Res. Bull., 102, 45–50.

[41] Alshehri, A.A., and Malik, M.A., 2019, Biogenic fabrication of ZnO nanoparticles using Trigonella foenum-graecum (Fenugreek) for proficient photocatalytic degradation of methylene blue under UV irradiation, J. Mater. Sci.: Mater. Electron., 30 (17), 16156–16173.

[42] Hong, E., Jeong, M.S., Kim, T.H., Lee, J.H., Cho, J.H., and Lee, K.S., 2019, Development of coupled biokinetic and thermal model to optimize cold-water microbial enhanced oil recovery (MEOR) in homogenous reservoir, Sustainability, 11 (6), 1652.

[43] Bhattacharyya, K.G., and Sharma, A., 2004, Azadirachta indica leaf powder as an effective biosorbent for dyes: A case study with aqueous Congo red solutions, J. Environ. Manage., 71 (3), 217–229.

[44] Kooh, M.R.R., Dahri, M.K., Lim, L.B.L., Lim, L.H., and Malik, O.A., 2016, Batch adsorption studies of the removal of methyl violet 2B by soya bean waste: Isotherm, kinetics and artificial neural network modelling, Environ. Earth Sci., 75 (9), 783.

[45] Aljeboree, A.M., Alshirifi, A.N., and Alkaim, A.F., 2017, Kinetics and equilibrium study for the adsorption of textile dyes on coconut shell activated carbon, Arabian J. Chem., 10, S3381–S3393.

[46] Sun, Y., Cheng, S., Lin, Z., Yang, J., Li, C., and Gu, R., 2020, Combination of plasma oxidation process with microbial fuel cell for mineralizing methylene blue with high energy efficiency, J. Hazard. Mater., 384, 121307.

[47] Das, L., Saha, N., Ganguli, A., Das, P., Bhowal, A., and Bhattacharjee, C., 2021, Calcium alginate–bentonite/activated biochar composite beads for removal of dye and Biodegradation of dye-loaded composite after use: Synthesis, removal, mathematical modeling and biodegradation kinetics, Environ. Technol. Innovation, 24, 101955.

[48] Langmuir, I., 1918, The adsorption of gases on plane surfaces of glass, mica and platinum, J. Am. Chem. Soc., 40 (9), 1361–1403.

[49] Freundlich, H., and Heller, W., 1939, The adsorption of cis- and trans-azobenzene, J. Am. Chem. Soc., 61 (8), 2228–2230.

[50] Sanchez, L.M., Ollier, R.P., and Alvarez, V.A., 2019, Sorption behavior of polyvinyl alcohol/bentonite hydrogels for dyes removal, J. Polym. Res., 26 (6), 142.

[51] Ahmad, A., Singh, A.P., Khan, N., Chowdhary, P., Giri, B.S., Varjani, S., and Chaturvedi, P., 2021, Bio-composite of Fe-sludge biochar immobilized with Bacillus sp. in packed column for bio-adsorption of methylene blue in a hybrid treatment system: Isotherm and kinetic evaluation, Environ. Technol. Innovation, 23, 101734.

[52] Wan Ibrahim, W.N., Zainal Abidin, N.H., Mohamad Hanapi, N.S., Ab Malek, N.F., Wan Sudin, W.N.A., Saim, N., and Rusmin, R., 2019, Adsorption studies of methylene blue by encapsulated nano-carbonaceous alginate beads, Int. J. Eng. Technol., 7 (4.14), 210–215.

[53] Yadav, S., Asthana, A., Chakraborty, R., Jain, B., Singh, A.K., Carabineiro, S.A.C., and Susan, M.A.B.H., 2020, Cationic dye removal using novel magnetic/activated charcoal/β-cyclodextrin/alginate polymer nanocomposite, Nanomaterials, 10 (1), 170.


Article Metrics

Abstract views : 1615 | views : 743

Copyright (c) 2022 Indonesian Journal of Chemistry

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.


Indonesian Journal of Chemistry (ISSN 1411-9420 / 2460-1578) - Chemistry Department, Universitas Gadjah Mada, Indonesia.

Analytics View The Statistics of Indones. J. Chem.