BIODIVERSITAS ISSN: 1412-033X Volume 21, Number 3, March 2020 E-ISSN: 2085-4722 Pages: 1136-1141 DOI: 10.13057/biodiv/d210338 Methylene blue decolorizing bacteria isolated from water sewage in Yogyakarta, Indonesia MICHELLE, RACHEL ARVY NABASA SIREGAR, ASTIA SANJAYA, JAP LUCY, REINHARD PINONTOAN Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan. Jl. M.H. Thamrin Boulevard 1100, Lippo Karawaci, Tangerang 15811, Banten, Indonesia. Tel./Fax. +62-21-5460901, email: [email protected] Manuscript received: 11 December 2019. Revision accepted: 20 February 2020. Abstract. Michelle, Siregar RAN, Sanjaya A, Jap L, Pinontoan R. 2020. Methylene blue decolorizing bacteria isolated from water sewage in Yogyakarta, Indonesia. Biodiversitas 21: 1136-1141. The textile industry contributes to water pollution issues all over the world. One of the most commonly applied cationic dye in the textile industry is methylene blue. This study aimed to isolate bacteria with the potential to decolorize methylene blue from dye contaminated sewage water located in Kulon Progo District, Yogyakarta, where several textile industries within the proximity, are located. Characterizations of bacterial candidates were done morphologically and biochemically. Molecular identification was conducted by 16S rRNA sequencing. The ability of isolates to decolorize methylene blue was observed by the reduction of methylene blue’s maximum absorption at the wavelength of 665 nm. The results showed that isolates were identified as Comamonas aquatica and Ralstonia mannitolilytica. C. aquatica PMB-1 and R. mannitolilytica PMB-2 isolates were able to decolorize methylene blue with decolorization percentage of 67.9% and 60.3%, respectively when incubated for 96 hours at 37°C. These findings present information on the capability of the genus Ralstonia and Comamonas to decolorize methylene blue cationic dye. Keywords: Comamonas aquatica, decolorization, methylene blue, Ralstonia mannitolilytica, Yogyakarta INTRODUCTION complexity, low economic feasibility, and disposal problems by some of these methods hinder their broad The textile industries are known as the major source of application, especially in a developing country (Zhou et al. water pollution all over the world (Ito et al. 2016). Textile 2019). Wastewater treatment by biological approach offers industrial wastes contain dyes which are often disposed a more cost-efficient and sustainable alternative solution is into the nature aggravating pollution problems due to their contrary to the huge expenditure specified by the toxicity. Most of these dyes are not readily degradable physicochemical approach. In the biological method, under natural conditions and resistant to conventional microbes were utilized for the degradation of the toxic wastewater treatment systems (Yaseen and Scholz 2019). textile dyes. Microorganisms that have been studied and The emitted textile effluents impose substantial adverse effects demonstrated effective removal of methylene blue include on water quality, soil fertility, and marine life, causing Stenotrophomonas maltophilia and Pseudomonas ecosystem disruptions (Croce et al. 2017). For those living aeruginosa isolated from contaminated soil, Sphingomonas adjacent to contaminated riverbanks or other water sources, paucimobilis isolated from the drainage system, and short-term exposure to textile dyes can lead to skin and eye Bacillus thuringiensis (Noraini et al. 2012; Chen et al. irritation as well as allergic reactions, while continued 2015; Eslami et al. 2017; Kilany 2017). exposure could even lead to cancer due to mutagenicity and Although Indonesia owns high biological diversity, carcinogenicity of these dyes (Lellis et al. 2019). there are only limited bacteria species being reported as a One of the most common cationic dyes that are often bioremediation agent of a textile dye in contaminated applied in textile, leather, paper, plastic, and craft industries water. Therefore, this study aimed to isolate potential is methylene blue (Rafatullah et al. 2010). Methylene blue methylene blue-decolorizing bacteria from dye contaminated waste was reported to impose health risks contaminated sewage water located in Kulon Progo such as nausea, dizziness, and chest pain to the exposed District, Yogyakarta, where several textile industries are population and reduce the biodiversity of the affected located within the proximity. ecosystems (Zhou et al. 2019). Many technologies have been employed to eliminate the contamination of methylene blue in the environment. Recently, MATERIALS AND METHODS physicochemical approaches, including photocatalysis, physiochemical adsorption, and non-thermal plasma Study area technology, have garnered popularity due to its high Kulon Progo is one of the regencies located in the efficiency and degradation percentage (Das et al. 2019; Yogyakarta Special Region, Indonesia, named after its Myung et al. 2019; Wu et al. 2019). However, high location from the Progo River. Being one area that MICHELLE et al. – Methylene blue decolorizing bacteria 1137 specialized in the textile industry businesses, the sewers in conducted as described by Bergey's Manual of Systematic this area receive a heavy load of textile dyes-contaminated Bacteriology (Guerrero 2001). wastewater from the surrounding home textile industries. To isolate bacteria with the ability to decolorize methylene Molecular identification of bacterial isolates blue, sewage water from a large sewer at Ngentakrejo, The 16S rRNA gene was amplified and sequenced to Kulon Progo, Special Region of Yogyakarta, Indonesia, identify bacteria isolates. Pure isolates were cultured on was taken as a sample. The sampling location was GYP broth media incubated at 37°C overnight. Three ml of presented in Figure 1. the broth culture of each isolate was centrifuged at 16,000 x g for 3 min and the pellet was used for DNA extraction. Procedures The extraction of bacterial genomic DNA was conducted Isolation of bacteria from Yogyakarta sewage water using the Wizard® Genomic DNA Purification Kit Collected water samples were diluted (10-1) with sterile according to manufacturer instructions (Promega, USA). water and plated (200 μl) on GYP (glucose yeast peptone) The quality and yield of extracted genomic DNA were agar (1% glucose, 0.5% yeast, 0.5% peptone, 2% agar) assessed by a UV/Vis spectrophotometer (BioDrop, UK) supplemented with 50 ppm methylene blue (Merck, and used as a template for 16S rRNA gene amplification by Germany) and incubated at 37°C for 96 hours. Bacteria PCR reactions. All PCR reactions were conducted by isolates with clear zones around their colonies were selected, employing universal primers 27F (5′-AGA GTT TGA purified, and maintained on GYP agar supplemented with 50 ppm methylene blue for further analysis. TCM TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) to amplify 16S rRNA gene using Characterization of bacteria from Yogyakarta sewage water Cell and colony morphology were observed to isolated genomic DNA from each isolate. KAPA HiFi™ characterize potential bacteria. Isolated candidates for Taq Polymerase, dNTP and fidelity buffer (Kapa methylene blue decolorizer were streak-plated on GYP Biosystems, USA) were mixed in 50 μL total volume per agar supplemented with 50 ppm methylene blue, incubated reaction under PCR conditions as follow, initial at 37°C for 96 hours. Cell appearances observed through denaturation at 95°C for 3 min, twenty-five cycles of Gram-staining examined using a light microscope with a amplification consisted of denaturation at 98°C for 20 sec, 1000x total magnification. Colony appearances include annealing at 55°C for 15 sec and extension at 72°C for 60 shape, color, margin, and elevations. Biochemical sec. A final extension phase was performed at 72°C for 2 min. characterization through Voges-Proskauer (VP) assay, indole assay, catalase assay, and starch hydrolysis were 1138 BIODIVERSITAS 21 (3): 1136-1141, March 2020 Figure 1. Sewerage in Kulon Progo District, Special Region of Yogyakarta, Indonesia as a water sampling location for bacterial isolation (geographic coordinate: -7.907293,110.264097). Source: Google Inc, 2019 Table 1. Cell, morphological colony and biochemical characterization of PMB-1, PMB-2 and PMB-3 isolates Isolates Characteristics PMB-1 PMB-2 PMB-3 Cellular morphology Shape Rod Rod Rod Gram staining Negative Negative Negative Colony morphology Shape Circular Circular Circular Color Blue Yellow Blue Margin Lobate Lobate Lobate Elevation Flat Flat Flat Biochemical characterization Voges-Proskauer (VP) - - - Indole - - - Catalase - - - Starch Hydrolysis - - - Note: (-) represents negative results PCR products were visualized on a 0.8% agarose gel 2010). Statistical analysis of the decolorization activity was stained with ethidium bromide under UV light to confirm done using one-way ANOVA with posthoc Tukey HSD the presence of an approximate 1.4 kbp band. The 16S test in GraphPad Prism software to determine the statistical rRNA PCR products were sent to First BASE Laboratories differences between treated samples and control. Pte. Ltd., Malaysia, for DNA sequencing. Measurement of decolorization activity RESULTS AND DISCUSSION Bacteria isolates were inoculated in GYP broth media supplemented with 50 ppm methylene blue and statically Isolation and identification of methylene blue incubated at 37°C for 96 hours under aerobic conditions. decolorizing bacteria Dye degradation was indicated by the reduction of the Three isolates, PMB-1,