Study suggests increased biotoxicity due to anaerobic decolorization of Reactive Black 5 dye by constructed wetland-derived Clostridium

Reactive Black 5 is a widely used textile dye that is difficult to degrade and commonly found in industrial wastewater. Researchers investigated the treatment potential of a constructed wetland-derived Clostridium sp. strain T4 by combining metabolite identification with ecotoxicological assessment. Although the bacterium removed up to 97% of the dye, toxic aromatic amines accumulated during treatment, increasing aquatic toxicity and demonstrating that decolorization does not necessarily mean detoxification.

Untreated synthetic dye effluents severely threaten aquatic ecosystems by blocking sunlight, depleting dissolved oxygen, and releasing compounds with potential ecological toxicity. Among the various treatment strategies, biological treatment is considered a cost-effective and environmentally friendly alternative to chemical methods.

Reactive Black 5 (RB5), one of the world's most widely used diazo (azo) dyes in the textile industry, is known for its stability, water solubility, and resistance to natural degradation. While certain bacterial genera demonstrated the ability to decolorize RB5 via anaerobic metabolism, understanding of the formation and ecotoxicological effects of the resulting intermediate metabolites remains limited.

To address this gap, Professor Satoshi Soda from the College of Science and Engineering, Ritsumeikan University, Japan, together with Dr. Kazuko Sawada, Research Assistant Professor at Ritsumeikan University, and Mr. Abd. Aziz Amin, a lecturer at Universitas Brawijaya in Indonesia and a doctoral student at Ritsumeikan University, investigated RB5 decolorization, metabolite formation, and the biotransformation pathway by a constructed wetland (CW)-derived Clostridium sp. strain T4. Along with evaluating the wastewater treatment potential of Clostridium sp. strain, the study also assessed the environmental implications of anaerobic RB5 decolorization. Their findings were made available online on June 11, 2026, and will be published in Volume 43 of the journal Environmental Technology & Innovation on September 01, 2026.

Prof. Soda explains the motivation behind this study, “We are involved in the Indonesia Linkage Program (Professional Human Resources Development Project Phase III), which enrolls Indonesian working professionals in our graduate program. Discussions with these students provided us insights on environmental problems related to textile and batik wastewater treatment in Indonesia. Their insights prompted us to ask if dye decolorization actually leads to environmental detoxification.”

The researchers isolated the Clostridium sp. strain T4 from the effluent of a laboratory-scale CW. Across concentrations ranging from 50 to 200 mg/L, under anaerobic conditions, the bacterium achieved up to 97% decolorization within 3 days. As no dye removal was observed in uninoculated controls, it was confirmed that the process was biologically mediated.

Spectral analysis confirmed that the dye's azo chromophore was disrupted. The researchers suggest that flavin-dependent NADH-linked azoreductase activity is the most plausible mechanism underlying this process. Additionally, two aromatic amine metabolites, 4-(ethylsulfonyl)aniline and 1,7-diamino-8-hydroxynaphthalene, were identified.

However, acute toxicity increased as decolorization progressed. Using Daphnia magna for acute toxicity assessment, the researchers found that the 48-hour effective concentration (EC₅₀) decreased from 64.4% before treatment to 20.9% after 5 days of anaerobic incubation. As lower EC₅₀ values indicate higher toxicity, the findings suggest that "decolorization" does not equal "detoxification."

This inverse relationship between decolorization and detoxification highlights a key limitation of anaerobic azo dye treatment. Under anaerobic conditions, bacteria can effectively cleave azo bonds and eliminate visible color, but they lack the downstream oxidative capacity needed to further degrade aromatic amines, allowing these toxic intermediates to accumulate.

The study does not propose a new RB5 degradation pathway, since anaerobic azo bond cleavage and aromatic amine formation are well established. Instead, its novelty lies in linking efficient single-strain anaerobic decolorization with time-dependent metabolite accumulation and increasing ecotoxicity.

Prof. Soda highlights, "Anaerobic treatment should be regarded as a reductive first stage for decolorization rather than a complete remediation process. To ensure safer effluent quality, integration with subsequent aerobic or oxidative treatment is necessary to achieve further detoxification in azo dye-containing wastewater management systems.”

Indonesia's batik industry, an internationally recognized cultural heritage, supports millions of livelihoods but poses severe environmental challenges. Untreated wastewater from dyeing and wax-removal processes, laden with synthetic dyes, heavy metals, and silicates, frequently pollutes local waterways. While wastewater management and dye removal are necessary, this study highlights the importance to ensure minimization and management of toxic intermediate byproducts.

Overall, the study findings demonstrate that wastewater treatment should be evaluated not only by color removal but also by ecotoxicological safety. The research supports routine toxicity monitoring, improved wastewater treatment regulations, and the development of safer treatment technologies to promote sustainable "green batik" production in Indonesia and other textile-producing countries while better protecting aquatic ecosystems and human health.

Title of original paper: Anaerobic decolorization of reactive black 5 by Clostridium sp. strain T4 and associated toxicity increase due to aromatic amine formation
Journal: Environmental Technology & Innovation
DOI: 10.1016/j.eti.2026.105043

About Ritsumeikan University, Japan

Ritsumeikan University is one of the most prestigious private universities in Japan. With an unwavering objective to generate social symbiotic values and emergent talents, it aims to emerge as a next-generation research-intensive university. It will enhance researcher potential by providing support best suited to the needs of young and leading researchers, according to their career stage. Ritsumeikan University also endeavors to build a global research network as a “knowledge node” and disseminate achievements internationally, thereby contributing to the resolution of social/humanistic issues through interdisciplinary research and social implementation.

Website: http://en.ritsumei.ac.jp/
Ritsumeikan University Research Report: https://www.ritsumei.ac.jp/research/radiant/eng/

About Professor Satoshi Soda from Ritsumeikan University, Japan

Dr. Satoshi Soda is a Professor in the College of Science and Engineering, Ritsumeikan University, Japan, and an Associate Fellow of the Ritsumeikan Advanced Research Academy (RARA). He also serves as the Director of the Research Center for Lake BIWA & Environmental Innovation at the university. He earned his Ph.D. in Engineering from the Graduate School of Engineering, Osaka University, Japan, in 1999. His research focuses on the removal of organic matter, nitrogen, and metals through biological wastewater treatment using microorganisms and aquatic plants. He has published over 100 peer-reviewed journal articles.

Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Media contact:

Chiaki Osuga
osuga-ch@st.ritsumei.ac.jp