Metagenomic profiling of cyanide-degrading microbial communities in steel industry wastewater with an implication for bioremediation

Abstract

Extremely toxic cyanide-contaminated wastewater discharged from steel industries poses serious environmental and health risks. Cyanide removal through physical and chemical treatments has cost-intensive operational challenges. Microbial bioremediation is cost-effective and environment friendly. As microorganisms can degrade cyanide by utilizing it as a source of nitrogen and converting it into less toxic compounds such as ammonia, this study was planned to get metagenomic data first and then potential microbial strains from the contaminated samples. In this study, wastewater samples from the equalizer and the sludge thickener tank of a steel wastewater treatment plant showed high cyanide concentration (mg/L) of 21.6 and 27.59, respectively. The free form of cyanide was predominant as the wastewater pH was ≥ 9. The contents of anions (F^–, Cl^–, NO₂^–, Br^–, NO₃^–, SO₄^2–) and elements (Na, Al, K, Fe) were also on the higher side. The metagenomic study revealed microbial community structure and taxonomic abundance with dominance of cyanide-degrading bacterial genera in the wastewaters, viz., Bordetella, Achromobacter, Pseudomonas, and Burkholderia, providing an opportunity to screen and obtain the efficient CN-degrading strains. Bacteria belonging to the phylum Deinococcus and the genus Mesorhizobium were reported for the first time from cyanide-contaminated water. Also, functional analysis showed high prevalence of genes encoding enzymes critical to cyanide degradation pathways e.g., rhodanese, nitrilase, nitrile hydratase, amidase, cyanide-insensitive terminal oxidase, and malate:quinone oxidoreductase. Eight distinct alkaliphilic cyanotropic microorganisms were successfully isolated and shown to degrade cyanide effectively at pH 9.5, indicating their metabolic adaptation to cyanide toxicity and alkaline stress. These findings can lead to a microbial technology against cyanide contamination.

Graphical abstract

Keywords

Wastewater / Cyanide / Microbiota / Metagenomics / Bioremediation

Highlight

	● Metagenomic profiling of CN-contaminated wastewater samples was studied.
	● Bordetella, Achromobacter, Pseudomonas and Burkholderia were dominant genera.
	● Phylum Deinococcus and Genus Mesorhizobium reported first-time in CN wastewater.
	● Rhodanese, nitrilase, malate-oxidoreductase are dominant in functional profiling.
	● Alkaliphilic microbes isolated from CN-wastewater degraded CN efficiently.

References

[1] AbdelMageed M F, ElRakaiby M T. (2025). Algal–bacterial bioremediation of cyanide-containing wastewater in a continuous stirred photobioreactor. World Journal of Microbiology and Biotechnology, 41(2): 26

[2] Abilaji S, Sathishkumar K, Narenkumar J, Alsalhi M S, Devanesan S, Parthipan P, Muthuraj B, Rajasekar A. (2023). Sequential photo electro oxidation and biodegradation of textile effluent: elucidation of degradation mechanism and bacterial diversity. Chemosphere, 331: 138816

[3] Ademakinwa A N, Agunbiade M O, Fagbohun O. (2021). Biodegradation of cyanide in cassava wastewater using a novel thermodynamically-stable immobilized rhodanese. Preparative Biochemistry & Biotechnology, 51(6): 607–617

[4] Adjei M D, Ohta Y. (2000). Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3. World Journal of Microbiology and Biotechnology, 16(2): 171–175

[5] Akinpelu E A, Adetunji A T, Ntwampe S K O, Nchu F, Mekuto L. (2018). Performance of Fusarium oxysporum EKT01/02 isolate in cyanide biodegradation system. Environmental Engineering Research, 23(2): 223–227

[6] AllenA RBooker LRockwoodG A (2015). Acute cyanide toxicity. In: Hall A H, Isom G E, Rockwood G A, eds. Toxicology of Cyanides and Cyanogens: Experimental, Applied and Clinical Aspects. Chichester: Wiley Blackwell, 1–20

[7] Alvillo-Rivera A J, Garrido-Hoyos S E, Buitrón G. (2022). Cyanide treatment of mining tailings using suspended biomass and moving bed biomass reactors. Environmental Science and Pollution Research, 29(25): 37458–37470

[8] APHA(1995). Standard Methods for the Examination of Water and Wastewater. 19th ed. Washington DC: American Public Health Association Inc

[9] APHA(2005). Standard Methods for the Examination of Water and Wastewater. 21st ed. Washington DC: American Public Health Association Press

[10] Behera B K, Chakraborty H J, Patra B, Rout A K, Dehury B, Das B K, Sarkar D J, Parida P K, Raman R K, Rao A R. . (2020a). Metagenomic analysis reveals bacterial and fungal diversity and their bioremediation potential from sediments of river Ganga and Yamuna in India. Frontiers in Microbiology, 11: 556136

[11] Behera B K, Patra B, Chakraborty H J, Sahu P, Rout A K, Sarkar D J, Parida P K, Raman R K, Rao A R, Rai A. . (2020b). Metagenome analysis from the sediment of river Ganga and Yamuna: in search of beneficial microbiome. PLoS One, 15(10): e0239594

[12] Belay G, Suarez C, Simachew A, Paul C J. (2025). A systematic evaluation of alkaliphilic microbial consortia from a soda lake for the biodegradation of cyanide-rich wastewater. International Biodeterioration & Biodegradation, 196: 105937

[13] Bhatt P, Bhatt K, Sharma A, Zhang W P, Mishra S, Chen S H. (2021). Biotechnological basis of microbial consortia for the removal of pesticides from the environment. Critical Reviews in Biotechnology, 41(3): 317–338

[14] Blanco-Vieites M, Suárez-Montes D, Delgado F, Álvarez-Gil M, Battez A H, Rodríguez E. (2022). Removal of heavy metals and hydrocarbons by microalgae from wastewater in the steel industry. Algal Research, 64: 102700

[15] Breen N E, Dwyer J, Olsen M, Sanford E, Rhyne A L. (2025). The half-life of cyanide in the blood of the marine fish, Amphiprion clarkii after cyanide exposure. Bulletin of Marine Science, 101(1): 271–281

[16] Brum J R, Ignacio-Espinoza J C, Roux S, Doulcier G, Acinas S G, Alberti A, Chaffron S, Cruaud C, De Vargas C, Gasol J M. . (2015). Patterns and ecological drivers of ocean viral communities. Science, 348(6237): 1261498

[17] Cáceda Quiroz C J, Fora Quispe G D L, Carpio Mamani M, Maraza Choque G J, Sacari Sacari E J. (2023). Cyanide bioremediation by Bacillus subtilis under alkaline conditions. Water, 15(20): 3645

[18] Choudhury A R, Singh N, Veeraraghavan A, Gupta A, Palani S G, Mehdizadeh M, Omidi A, Al-Taey D K A. (2023). Ascertaining and optimizing the water footprint and sludge management practice in steel industries. Water, 15(12): 2177

[19] Cooling F B, Fager S K, Fallon R D, Folsom P W, Gallagher F G, Gavagan J E, Hann E C, Herkes F E, Phillips R L, Sigmund A. . (2001). Chemoenzymatic production of 1,5-dimethyl-2-piperidone. Journal of Molecular Catalysis B: Enzymatic, 11(4−6): 295–306

[20] Cosmos A, Erdenekhuyag B O, Yao G, Li H J, Zhao J G, Wang L J, Lyu X J. (2020). Principles and methods of bio detoxification of cyanide contaminants. Journal of Material Cycles and Waste Management, 22(4): 939–954

[21] Cydzik-Kwiatkowska A, Zielińska M. (2016). Bacterial communities in full-scale wastewater treatment systems. World Journal of Microbiology and Biotechnology, 32(4): 66

[22] D’Argenio V, Casaburi G, Precone V, Salvatore F. (2014). Comparative metagenomic analysis of human gut microbiome composition using two different bioinformatic pipelines. BioMed Research International, 2014(1): 325340

[23] Das P P, Anweshan P, Mondal A, Sinha P, Biswas S, Sarkar M K. (2021). Integrated ozonation assisted electrocoagulation process for the removal of cyanide from steel industry wastewater. Chemosphere, 263: 128370

[24] Dey S, Kumar Rout A, Ghosh K, Dixit S, Kumar V, Kumar Das B, Behera B K. (2024). Unveiling the plastic degrading potential of the beneficial microbiome through plastisphere community diversity and predictive functionality analysis in waste disposal sites in the adjoining areas of Kolkata, West Bengal, India. Current Research in Biotechnology, 8: 100237

[25] Dhar K, Subashchandrabose S R, Venkateswarlu K, Megharaj M. (2020). Mesorhizobium tamadayense MM3441: a novel methylotroph with a great potential in degrading N,N′-dimethylformamide. International Biodeterioration & Biodegradation, 153: 105045

[26] Dumestre A, Chone T, Portal J, Gerard M, Berthelin J. (1997). Cyanide degradation under alkaline conditions by a strain of Fusarium solani isolated from contaminated soils. Applied and Environmental Microbiology, 63(7): 2729–2734

[27] Fisher F B, Brown J S. (1952). Colorimetric determination of cyanide in stack gas and waste water. Analytical Chemistry, 24(9): 1440–1444

[28] Gavagan J E, DiCosimo R, Eisenberg A, Fager S K, Folsom P W, Hann E C, Schneider K J, Fallon R D. (1999). A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles. Applied Microbiology and Biotechnology, 52(5): 654–659

[29] Gupta N, Balomajumder C, Agarwal V K. (2010). Enzymatic mechanism and biochemistry for cyanide degradation: a review. Journal of Hazardous Materials, 176(1−3): 1–13

[30] Hotta Y, Wakayama M, Niwa K, Hosoda A, Tamura H. (2012). Application of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry for the identification of alkylphenol polyethoxylate-degrading bacteria in the environment. Journal of Pesticide Science, 37(4): 305–311

[31] Huertas M J, Sáez L P, Roldán M D, Luque-Almagro V M, Martínez-Luque M, Blasco R, Castillo F, Moreno-Vivián C, García-García I. (2010). Alkaline cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Influence of pH. Journal of Hazardous Materials, 179(1−3): 72–78

[32] Igeño M I, Macías D, Guijo M I, Sánchez-Clemente R, Población A G, Merchán F, Blasco R. (2018). Bacterial consortiums able to use metal-cyanide complexes as a nitrogen source. Proceedings, 2(20): 1284

[33] Itakorode B O, Itakorode D I, Torimiro N, Okonji R E. (2024). Kinetic and thermodynamic investigation of Rhodanese synthesized by enhanced Klebsiella oxytoca JCM 1665 strain: a comparative between the free and immobilized enzyme entrapped in alginate beads. Preparative Biochemistry & Biotechnology, 54(10): 1275–1284

[34] Itakorode B O, Okonji R E, Torimiro N. (2023). Cyanide bioremediation potential of Klebsiella oxytoca JCM 1665 rhodanese immobilized on alginate-glutaraldehyde beads. Biocatalysis and Biotransformation, 41(4): 299–308

[35] Jaszczak E, Polkowska Ż, Narkowicz S, Namieśnik J. (2017). Cyanides in the environment-analysis-problems and challenges. Environmental Science and Pollution Research, 24(19): 15929–15948

[36] Kantor R S, van Zyl A W, van Hille R P, Thomas B C, Harrison S T L, Banfield J F. (2015). Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome‐resolved metagenomics. Environmental Microbiology, 17(12): 4929–4941

[37] Lawal O T, Sanni D M, Olajuyigbe F M. (2023). Characterization of thermotolerant and thermostable rhodanese from high cyanide-degrading and agricultural wastes-utilizing Aspergillus welwitschiae LOT1 isolated from battery-effluent contaminated soil. Biocatalysis and Agricultural Biotechnology, 52: 102807

[38] Lin Z Q, Pang S M, Zhou Z, Wu X Z, Li J Y, Huang Y H, Zhang W P, Lei Q Q, Bhatt P, Mishra S. . (2022). Novel pathway of acephate degradation by the microbial consortium ZQ01 and its potential for environmental bioremediation. Journal of Hazardous Materials, 426: 127841

[39] Liu Y, Liu P, Lin L, Zhao Y Q, Zhong W J, Wu L J, Zhou Z M, Sun W F. (2016). Activity enhancement based on the chemical equilibrium of multiple-subunit nitrile hydratase from Bordetella petrii. Applied Biochemistry and Biotechnology, 180(1): 3–9

[40] Lü H X, Wei J L, Tang G X, Chen Y S, Huang Y H, Hu R W, Mo C H, Zhao H M, Xiang L, Li Y W. . (2024). Microbial consortium degrading of organic pollutants: source, degradation efficiency, pathway, mechanism and application. Journal of Cleaner Production, 451: 141913

[41] Luque-Almagro V M, Merchán F, Blasco R, Igeño M I, Martínez-Luque M, Moreno-Vivián C, Castillo F, Roldán M D. (2011). Cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 involves a malate: quinone oxidoreductase and an associated cyanide-insensitive electron transfer chain. Microbiology, 157(3): 739–746

[42] Ma X J, Wu C D, Huang J, Zhou R Q, Shi B. (2018). Microbial community of tannery wastewater involved in nitrification revealed by Illumina MiSeq sequencing. Journal of Microbiology and Biotechnology, 28(7): 1168–1177

[43] Malmir N, Zamani M, Motallebi M, Fard N A, Mekuto L. (2022). Cyanide biodegradation by Trichoderma harzianum and cyanide hydratase network analysis. Molecules, 27(10): 3336

[44] Mekuto L, Ntwampe S K O, Akcil A. (2016a). An integrated biological approach for treatment of cyanidation wastewater. Science of the Total Environment, 571: 711–720

[45] MekutoLNtwampe S K OKenaMGolelaM TAmoduO S (2016b). Free cyanide and thiocyanate biodegradation by Pseudomonas aeruginosa STK 03 capable of heterotrophic nitrification under alkaline conditions. 3 Biotech, 6(1): 6

[46] MekutoLNtwampe S K OMudumbiJ B N (2018). Microbial communities associated with the co-metabolism of free cyanide and thiocyanate under alkaline conditions. 3 Biotech, 8(2): 93

[47] Mondal A, Sarkar S, Nair U G. (2021). Comparative characterization of cyanide-containing steel industrial wastewater. Water Science and Technology, 83(2): 322–330

[48] Mondal M, Mukherjee R, Sinha A, Sarkar S, De S. (2019). Removal of cyanide from steel plant effluent using coke breeze, a waste product of steel industry. Journal of Water Process Engineering, 28: 135–143

[49] Novak D, Franke-Whittle I H, Pirc E T, Jerman V, Insam H, Logar R M, Stres B. (2013). Biotic and abiotic processes contribute to successful anaerobic degradation of cyanide by UASB reactor biomass treating brewery waste water. Water Research, 47(11): 3644–3653

[50] Obidah J S, Owuama C I. (2017). Xanthan yield and conversion efficiency of pre-treated rice husk, sweet potato and cassava flours from Xanthomonas campestris fermentation. International Journal of Engineering Trends and Technology, 49(8): 477–481

[51] Pang S M, Lin Z Q, Chen W J, Chen S F, Huang Y H, Lei Q Q, Bhatt P, Mishra S, Chen S H, Wang H S. (2023). High-efficiency degradation of methomyl by the novel bacterial consortium MF0904: performance, structural analysis, metabolic pathways, and environmental bioremediation. Journal of Hazardous Materials, 452: 131287

[52] Paszti S, Biner O, Liu Y L, Bolli K, Jeggli S D, Pessi G, Eberl L. (2025). Insights into the diverse roles of the terminal oxidases in Burkholderia cenocepacia H111. Scientific Reports, 15(1): 2390

[53] Quan Z X, Rhee S K, Bae J W, Baek J H, Park Y H, Lee S T. (2006). Bacterial community structure in activated sludge reactors treating free or metal-complexed cyanides. Journal of Microbiology and Biotechnology, 16(2): 232–239

[54] Rishi S, Kaur I, Naseem M, Gaur V K, Mishra S, Srivastava S, Saini H S, Srivastava P K. (2023). Development of immobilized novel fungal consortium for the efficient remediation of cyanide-contaminated wastewaters. Bioresource Technology, 373: 128750

[55] Rosario C G A, Vallenas-Arévalo A T, Arévalo S J, Espinosa D C R, Tenório J A S. (2023). Biodegradation of cyanide using a Bacillus subtilis strain isolated from artisanal gold mining tailings. Brazilian Journal of Chemical Engineering, 40(1): 129–136

[56] Rout A K, Dehury B, Parida P K, Sarkar D J, Behera B, Das B K, Rai A, Behera B K. (2022). Taxonomic profiling and functional gene annotation of microbial communities in sediment of river Ganga at Kanpur, India: insights from whole-genome metagenomics study. Environmental Science and Pollution Research, 29(54): 82309–82323

[57] Rout A K, Tripathy P S, Dixit S, Behera D U, Behera B, Das B K, Behera B K. (2024). Metagenomics analysis of sediments of river Ganga, India for bacterial diversity, functional genomics, antibiotic resistant genes and virulence factors. Current Research in Biotechnology, 7: 100187

[58] Sabatini L, Ferrini C, Micheloni M, Pianetti A, Citterio B, Parlani C, Bruscolini F. (2012). Isolation of a strain of Aspergillus fumigatus able to grow in minimal medium added with an industrial cyanide waste. World Journal of Microbiology and Biotechnology, 28: 165–173

[59] Sáez L P, Rodríguez-Caballero G, Olaya-Abril A, Cabello P, Moreno-Vivián C, Roldán M D, Luque-Almagro V M. (2024). Genomic insights into cyanide biodegradation in the pseudomonas genus. International Journal of Molecular Sciences, 25(8): 4456

[60] Scher J U, Sczesnak A, Longman R S, Segata N, Ubeda C, Bielski C, Rostron T, Cerundolo V, Pamer E G, Abramson S B. . (2013). Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife, 2: e01202

[61] Shin D, Park J, Park H, Lee J C, Kim M S, Lee J. (2020). Key microbes and metabolic potentials contributing to cyanide biodegradation in stirred-tank bioreactors treating gold mining effluent. Mineral Processing and Extractive Metallurgy Review, 41(2): 85–95

[62] Shu W S, Huang L N. (2022). Microbial diversity in extreme environments. Nature Reviews Microbiology, 20(4): 219–235

[63] Singh N, Balomajumder C. (2016). Simultaneous biosorption and bioaccumulation of phenol and cyanide using coconut shell activated carbon immobilized Pseudomonas putida (MTCC 1194). Journal of Environmental Chemical Engineering, 4(2): 1604–1614

[64] Singh U, Arora N K, Sachan P. (2018). Simultaneous biodegradation of phenol and cyanide present in coke-oven effluent using immobilized Pseudomonas putida and Pseudomonas stutzeri. Brazilian Journal of Microbiology, 49(1): 38–44

[65] Tamir-Ariel D, Navon N, Burdman S. (2007). Identification of genes in Xanthomonas campestris pv. vesicatoria induced during its interaction with tomato. Journal of Bacteriology, 189(17): 6359–6371

[66] Terada A, Komatsu D, Ogawa T, Flamandita D, Sahlan M, Nishimura M, Yohda M. (2022). Isolation of cyanide-degrading bacteria and molecular characterization of its cyanide-degrading nitrilase. Biotechnology and Applied Biochemistry, 69(1): 183–189

[67] U.S EPA(2007). Method 3015A (SW-846): Microwave Assisted Acid Digestion of Aqueous Samples and Extracts,” Revision 1. Washington, DC: United States Environmental Protection Agency

[68] Vandenbergh P A, Bawdon R E, Berk R S. (1979). Rapid test for determining the intracellullar rhodanese activity of various bacteria. International Journal of Systematic and Evolutionary Microbiology, 29(4): 339–344

[69] Wang C C, Lee C M, Chen L J. (2004). Removal of nitriles from synthetic wastewater by acrylonitrile utilizing bacteria. Journal of Environmental Science and Health, Part A, 39(7): 1767–1779

[70] Wang Z P, Liu L L, Guo F, Zhang T. (2015). Deciphering cyanide-degrading potential of bacterial community associated with the coking wastewater treatment plant with a novel draft genome. Microbial Ecology, 70(3): 701–709

[71] Welman-Purchase M D, Castillo J, Gomez-Arias A, Matu A, Hansen R N. (2024). First insight into the natural biodegradation of cyanide in a gold tailings environment enriched in cyanide compounds. Science of the Total Environment, 906: 167174

[72] Xiao K Q, Tang R, Wei J, Wu J, Shao Y J, Ma Z H, Wang L B, Hu Z H, Zhou Z. (2025). Achieving stable partial nitrification through synergistic inhibition of free ammonia and salinity on nitrite-oxidizing bacteria. Frontiers of Environmental Science & Engineering, 19(4): 42

[73] Zhang C Y, Zhu Z P, Yuan L M, Yang D X, Xu Q, Ge S J. (2025). Insights into seasonal temperature shifts on a robust composite-substrate autotrophic denitrification reactor for treating municipal secondary effluent. Frontiers of Environmental Science & Engineering, 19(6): 82

[74] Zhang H, Shen Z, Fang W, Gao G.. (2019). Composition of bacterial communities in municipal wastewater treatment plant. Science of The Total Environment, 689: 1181–1191

[75] Zhang W P, Chen W J, Chen S F, Liu M Q, Ghorab M A, Mishra S, Bhatt P, Chen S H. (2024). Complete biodegradation of glyphosate with microbial consortium YS622: structural analysis, biochemical pathways, and environmental bioremediation. Journal of Environmental Chemical Engineering, 12(6): 114344

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