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Monod Kinetics for EM4-Driven Ammonia Biodegradation Under Molasses Variation and Microbial Acclimatization

Program Studi Teknologi Proses Industri Petrokimia, Politeknik Industri Petrokimia Banten, Kabupaten Serang, Banten 42166, Indonesia

Received: 26 May 2026; Revised: 9 Jul 2026; Accepted: 14 Jul 2026; Published: 31 Aug 2026.
Open Access Copyright 2026 Jurnal Kimia Sains dan Aplikasi under http://creativecommons.org/licenses/by-sa/4.0.

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Abstract

Ammonia is a toxic pollutant parameter in petrochemical wastewater and can impair the quality of aquatic environments. This experimental study evaluated the kinetics of ammonia biodegradation by an Effective Microorganisms-4 (EM4) consortium using the Monod model approach, focusing on molasses concentration as an external carbon source and the effect of microbial acclimatization. This experiment was conducted in aerobic batch systems with initial ammonia concentrations of 10, 30, and 50 ppm and the addition of 5% and 10% molasses. Mixed Liquor Suspended Solids (MLSS) and pH were monitored to evaluate environmental dynamics and biomass growth. The results showed that increasing the molasses concentration from 5% to 10% increased the maximum specific growth rate (μmax) from 6.29 to 7.17 day−1 and decreased the half-saturation constant (Ks) from 23.22 to 14.01 mg/L; therefore, a lower Ks indicates higher apparent substrate affinity. The acclimatization further improved the kinetic response, yielding higher μmax and lower Ks than the non-acclimated system, alongside higher MLSS and a faster pH decrease consistent with more intensive biodegradation activity. Overall, these results confirm that the combination of microbial acclimatization and sufficient molasses addition can enhance the kinetic performance of ammonia biodegradation by EM4.

Keywords: Microbial Acclimatization; Ammonia Biodegradation; Effective Microorganisms-4 (EM4); Monod Kinetics; Molasses

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  1. Tae-Jin Park, Jong-Hyeon Lee, Myung-Sung Lee, Chang-Hee Park, Chang-Hoon Lee, Seong-Dae Moon, Jiwoong Chung, Rongxue Cui, Youn-Joo An, Dong-Hyuk Yeom, Soo-Hyung Lee, Jae-Kwan Lee, Kyung-Duk Zoh, Development of water quality criteria of ammonia for protecting aquatic life in freshwater using species sensitivity distribution method, Science of The Total Environment, 634, (2018), 934-940 https://doi.org/10.1016/j.scitotenv.2018.04.018
  2. Fachtur Rahman, Analisis Kadar Amonia dan pH pada Limbah Cair Kanal 32 (K-32) PT Pusri Palembang, ALKIMIA: Jurnal Ilmu Kimia dan Terapan, 3, 1, (2019), 10-15 https://doi.org/10.19109/alkimia.v3i1.3137
  3. Iwona Beata Paśmionka, Karol Bulski, Piotr Herbut, Elżbieta Boligłowa, Frederico Márcio C. Vieira, Gabriela Bonassa, Marcelo Bortoli, Marina Celant de Prá, Toxic Effect of Ammonium Nitrogen on the Nitrification Process and Acclimatisation of Nitrifying Bacteria to High Concentrations of NH4-N in Wastewater, Energies, 14, 17, (2021), 5329 https://doi.org/10.3390/en14175329
  4. Pitriania Pitriania, Muh. Fajaruddin Natsir, Anwar Daud, The Effectiveness of EM4 Addition into Anaerob-Aerob Biofilter in the Processing of Wastewater at Hasanuddin University Hospital, Makassar Indonesia, International Journal of Sciences: Basic and Applied Research (IJSBAR), 22, 1, (2015), 178-187
  5. Icha Ayoe Bilbina, M. Mirwan, Efektivitas Free Floating Wetland Tanaman Kiambang (Salvinia molesta) dengan Penambahan EM4 dalam Pengolahan Limbah Laundry pada Kondisi Anoxic–Oxic, Departement of Environmental Engineering, Universitas Pembangunan Nasional "Veteran" Jawa Timur, 2025
  6. Abd Ziad Mohamad, Herlina Jusuf, Moh. Rivai Nakoe, Efektifitas EM-4 (Effective Microorganisms-4) dalam Menurunkan Kadar COD pada Air Limbah Industri Tempe, Jurnal Kolaboratif Sains, 8, 7, (2025), 4830-4837
  7. Annisa Maysabila, Rika Heryanti, Rega Permana, Zahidah Hasan, Bioremediation of shrimp pond wastewater using Effective Microorganism-4 (EM4), Aceh Journal of Animal Science, 8, 3, (2023), 72-77 https://doi.org/10.13170/ajas.8.3.28971
  8. Flávio Silva, Helena Nadais, António Prates, Luís Arroja, Isabel Capela, Molasses as an external carbon source for anaerobic treatment of sulphite evaporator condensate, Bioresource Technology, 100, 6, (2009), 1943-1950 https://doi.org/10.1016/j.biortech.2008.10.029
  9. Diah Ratnaningrum, Een Sri Endah, Puspita Lisdiyanti, Sri Priatni, Vienna Saraswaty, Harnessing molasses as a low-cost carbon source for production of poly-hydroxybutyrate (PHB) using Burkholderia sp. B73 bacteria, Menara Perkebunan, 89, 2, (2021), 146-155 https://doi.org/10.22302/iribb.jur.mp.v89i2.452
  10. Mengru Zhang, Wenhao Zhang, Qingyuan Jiang, Chuanfu Zhao, Fei Han, Hao Chen, Yuke Li, Yuping Zhuge, Weizhi Zhou, Assimilation overwhelms nitrification in saline wastewater nitrogen removal: From heterotrophic nitrification and aerobic denitrification strains to microbiomes, Water Research, 288, (2026), 124694 https://doi.org/10.1016/j.watres.2025.124694
  11. Qi Li, Yifeng Xu, Chuanzhou Liang, Lai Peng, Yan Zhou, Nitrogen removal by algal-bacterial consortium during mainstream wastewater treatment: Transformation mechanisms and potential N2O mitigation, Water Research, 235, (2023), 119890 https://doi.org/10.1016/j.watres.2023.119890
  12. M. Meraz, V. Sanchez-Vazquez, F. Martinez-Martinez, A systematic derivation of the Monod equation for multi-substrate conditions, Revista Mexicana de Ingeniería Química, 21, 2, (2022), 1-8 https://doi.org/10.24275/rmiq/Bio2798
  13. Hailin Tian, Ioannis A. Fotidis, Enrico Mancini, Laura Treu, Ahmed Mahdy, Mercedes Ballesteros, Cristina González-Fernández, Irini Angelidaki, Acclimation to extremely high ammonia levels in continuous biomethanation process and the associated microbial community dynamics, Bioresource Technology, 247, (2018), 616-623 https://doi.org/10.1016/j.biortech.2017.09.148
  14. Miao Yan, Ioannis A. Fotidis, Hailin Tian, Benyamin Khoshnevisan, Laura Treu, Panagiotis Tsapekos, Irini Angelidaki, Acclimatization contributes to stable anaerobic digestion of organic fraction of municipal solid waste under extreme ammonia levels: Focusing on microbial community dynamics, Bioresource Technology, 286, (2019), 121376 https://doi.org/10.1016/j.biortech.2019.121376
  15. Xin Zou, Mengjiao Gao, Yiduo Yao, Yihui Zhang, Hengbo Guo, Yang Liu, Efficient nitrogen removal from ammonia rich wastewater using aerobic granular sludge (AGS) reactor: Selection and enrichment of effective microbial community, Environmental Research, 251, (2024), 118573 https://doi.org/10.1016/j.envres.2024.118573
  16. Benfu Luo, Yuhang Liu, Yiqiang Huang, Weiwei Huang, Qiang Chen, Yujing Yan, Haixing He, Xiang Zhou, Jie Yu, Study on high-concentration activated sludge system for energy-efficient nitrogen removal in wastewater, Sustainable Environment Research, 35, (2025), 16 https://doi.org/10.1186/s42834-025-00254-0
  17. Xiangyi Deng, Shengnan Huang, Liqi Chai, Chunqiao Xiao, Wanqiang Dong, Zhenyue Zhang, Denitrification kinetics and microbial evolution of anaerobic ammonia oxidation for low-acidity ammonia nitrogen wastewater leached from rare earth tailings, Environmental Technology, 47, 11, (2026), 1687-1704 https://doi.org/10.1080/09593330.2026.2628262
  18. Yongqi Ma, Jiali Wang, Yindi Zhang, Wenping Guan, Wenrui Qi, Xisheng Tai, Dong Lin, Rong He, Likun Sun, Aiwen Zhang, Research on nitrogen transformation pathways of a thermophilic heterotrophic nitrifying bacterial consortium GW7, Frontiers in Microbiology, 16, (2025), 1578865 https://doi.org/10.3389/fmicb.2025.1578865
  19. Zhijie Wang, Kai Yang, Subhamoy Mahajan, Ying Li, Mohan Qin, Understanding and modelling ammonia partitioning and transport across reverse osmosis membrane, Nature Communications, 17, (2026), 4649 https://doi.org/10.1038/s41467-026-71260-y
  20. Shirin Shahgodari, Joan Llorens, Jordi Labanda, Study of Total Ammoniacal Nitrogen Recovery Using Polymeric Thin-Film Composite Membranes for Continuous Operation of a Hybrid Membrane System, Polymers, 17, 12, (2025), 1696 https://doi.org/10.3390/polym17121696
  21. Jiyan Lu, Yue Tan, Shanghong Tian, Yuxiao Qin, Meng Zhou, Hao Hu, Xiaohong Zhao, Zhoufeng Wang, Bo Hu, Effect of carbon source on carbon and nitrogen metabolism of common heterotrophic nitrification-aerobic denitrification pathway, Chemosphere, 361, (2024), 142525 https://doi.org/10.1016/j.chemosphere.2024.142525
  22. Marina Arnaldos, Youri Amerlinck, Usman Rehman, Thomas Maere, Stijn Van Hoey, Wouter Naessens, Ingmar Nopens, From the affinity constant to the half-saturation index: Understanding conventional modeling concepts in novel wastewater treatment processes, Water Research, 70, (2015), 458-470 https://doi.org/10.1016/j.watres.2014.11.046
  23. Janis E. Baeten, Mark C. M. van Loosdrecht, Eveline I. P. Volcke, Modelling aerobic granular sludge reactors through apparent half-saturation coefficients, Water Research, 146, (2018), 134-145 https://doi.org/10.1016/j.watres.2018.09.025
  24. Yuyu Li, Shuai Du, Lin Sun, Qiming Cheng, Junfeng Hao, Qiang Lu, Gentu Ge, ZhiJun Wang, Yushan Jia, Effects of Lactic Acid Bacteria and Molasses Additives on Dynamic Fermentation Quality and Microbial Community of Native Grass Silage, Frontiers in Microbiology, 13, (2022), 830121 https://doi.org/10.3389/fmicb.2022.830121
  25. Roumi Bhattacharya, Debabrata Mazumder, Evaluation of nitrification kinetics for treating ammonium nitrogen enriched wastewater in moving bed hybrid bioreactor, Journal of Environmental Chemical Engineering, 9, 1, (2021), 104589 https://doi.org/10.1016/j.jece.2020.104589
  26. Rima Avetisyan, Karen Trchounian, Anna Poladyan, Fermentation of Molasses by Clostridium spp. for Biohydrogen and Biomass Production: A Step Toward Renewable Energy and Circular Bioeconomy, Journal of Innovative Solutions for Eco-Environmental Sustainability, Special Issue No. 1, (2025), 124 https://doi.org/10.46991/jisees.2025.si1.124
  27. Roumi Bhattacharya, Debabrata Mazumder, Kinetic study on nitrification of ammonium nitrogen-enriched synthetic wastewater using activated sludge, Water Science and Technology, 81, 1, (2020), 62-70 https://doi.org/10.2166/wst.2020.080
  28. Ahmed Elsayed, Taeho Lee, Younggy Kim, Maximizing the efficiency of single-stage partial nitrification/Anammox granule processes and balancing microbial competition using insights of a numerical model study, Water Environment Research, 97, 3, (2025), e70059 https://doi.org/10.1002/wer.70059
  29. Qian Chen, Jinren Ni, Heterotrophic nitrification-aerobic denitrification by novel isolated bacteria, Journal of Industrial Microbiology and Biotechnology, 38, 9, (2011), 1305-1310 https://doi.org/10.1007/s10295-010-0911-6
  30. Yen-Keong Cheah, Carme Vidal-Antich, Joan Dosta, Joan Mata-Álvarez, Volatile fatty acid production from mesophilic acidogenic fermentation of organic fraction of municipal solid waste and food waste under acidic and alkaline pH, Environmental Science and Pollution Research, 26, (2019), 35509-35522 https://doi.org/10.1007/s11356-019-05394-6
  31. G. A. Pámanes-Carrasco, E. Y. García-Piña, E. E. Araiza-Rosales, E. Herrera-Torres, Inclusion of molasses to garlic foliage silage and its effect on in vitro ruminal fermentation and gas production, Journal of Animal and Feed Sciences, 32, 3, (2023), 316-323 https://doi.org/10.22358/jafs/161808/2023
  32. M. J. A. Gonçalves, C. González-Fernández, S. Greses, Unravelling the effects of short-term starvation and pH disturbances in stable volatile fatty acids production, Journal of Cleaner Production, 476, (2024), 143747 https://doi.org/10.1016/j.jclepro.2024.143747
  33. Yogananda Maspolim, Yan Zhou, Chenghong Guo, Keke Xiao, Wun Jern Ng, The effect of pH on solubilization of organic matter and microbial community structures in sludge fermentation, Bioresource Technology, 190, (2015), 289-298 https://doi.org/10.1016/j.biortech.2015.04.087
  34. Kalyani Naik, Snehasish Mishra, Haragobinda Srichandan, Puneet Kumar Singh, Abhishek Choudhary, Microbial formulation and growth of cereals, pulses, oilseeds and vegetable crops, Sustainable Environment Research, 30, 10, (2020), https://doi.org/10.1186/s42834-020-00051-x
  35. Nan Lv, Guanjing Cai, Xiaofang Pan, Yanlin Li, Ruming Wang, Junjie Li, Chunxing Li, Gefu Zhu, pH and hydraulic retention time regulation for anaerobic fermentation: Focus on volatile fatty acids production/distribution, microbial community succession and interactive correlation, Bioresource Technology, 347, (2021), 126310 https://doi.org/10.1016/j.biortech.2021.126310
  36. R. J. Zeng, Z. Yuan, J. Keller, Effects of solids concentration, pH and carbon addition on the production rate and composition of volatile fatty acids in prefermenters using primary sewage sludge, Water Science and Technology, 53, 8, (2006), 263-269 https://doi.org/10.2166/wst.2006.257
  37. Jiahui Chen, Yi Xue, Dongli Yang, Sijia Ma, Yuan Lin, Haiyue Wang, Yanru Wang, Hongqiang Ren, Ke Xu, Optimizing waste molasses utilization to enhance electron transfer via micromagnetic carriers: Mechanisms and high-nitrate wastewater denitrification performance, Environmental Research, 242, (2024), 117709 https://doi.org/10.1016/j.envres.2023.117709
  38. Chang Liu, Kai Cheng, Molasses fermentation to produce low-cost carbon source for denitrification, Biotechnology and Biotechnological Equipment, 36, 1, (2022), 878-890 https://doi.org/10.1080/13102818.2022.2138781
  39. Hamid Zentou, Zurina Zainal Abidin, Robiah Yunus, Dayang Radiah Awang Biak, Mustapha Zouanti, Abdelkader Hassani, Modelling of Molasses Fermentation for Bioethanol Production: A Comparative Investigation of Monod and Andrews Models Accuracy Assessment, Biomolecules, 9, 8, (2019), 308 https://doi.org/10.3390/biom9080308
  40. Nor I. Faekah, S. Fatihah, Zawawi Samba Mohamed, Kinetic evaluation of a partially packed upflow anaerobic fixed film reactor treating low-strength synthetic rubber wastewater, Heliyon, 6, 3, (2020), e03594 https://doi.org/10.1016/j.heliyon.2020.e03594
  41. Jiale Wang, Yanbing Pan, Xin Wen, Pei Gao, Jian Zhou, Rapid start-up of nitrogen and organic matter removal in sequencing batch biofilm reactors treating hypersaline mustard tuber wastewater with autochthonous microorganisms, Journal of Environmental Management, 383, (2025), 125490 https://doi.org/10.1016/j.jenvman.2025.125490
  42. Yi Ren, Huu Hao Ngo, Wenshan Guo, Dongbo Wang, Lai Peng, Bing-Jie Ni, Wei Wei, Yiwen Liu, New perspectives on microbial communities and biological nitrogen removal processes in wastewater treatment systems, Bioresource Technology, 297, (2020), 122491 https://doi.org/10.1016/j.biortech.2019.122491
  43. Guogen Su, Jianping Xu, Yishuai Du, Hexiang Wang, Huiqin Tian, Li Zhou, Yanfeng Wang, Jianming Sun, Tianlong Qiu, Research on the Optimization of Key Parameters for Heterotrophic Bacteria Assimilation Nitrogen Removal Technology in Aquaculture Tailwater, Sustainability, 17, 11, (2025), 5069 https://doi.org/10.3390/su17115069
  44. Fabiane P. Serra, Carlos A. P. Gaona, Plínio S. Furtado, Luis H. Poersch, Wilson Wasielesky Jr., Use of different carbon sources for the biofloc system adopted during the nursery and grow-out culture of Litopenaeus vannamei, Aquaculture International, 23, (2015), 1325-1339 https://doi.org/10.1007/s10499-015-9887-6
  45. Luchiandini Ika Pamaharyani, Ilham Ilham, Azam B. Zaidy, The use of different carbon sources to maintain water quality and growth performance of sangkuriang catfish (Clarias gariepinus) in the biofloc aquaculture system, Journal of Applied Aquaculture, 37, 1, (2024), 1-18 https://doi.org/10.1080/10454438.2024.2326953
  46. Wen Peng, Liyuan Zhang, Manlin Wei, Baiyila Wu, Ming Xiao, Runze Zhang, Ji Ju, Chenyang Dong, Liu Du, Yongjie Zheng, Meili Bao, Hailin Bao, Xiaoping Bao, Effects of Lactobacillus plantarum (L) and molasses (M) on nutrient composition, aerobic stability, and microflora of alfalfa silage in sandy grasslands, Frontiers in Microbiology, 15, (2024), 1358085 https://doi.org/10.3389/fmicb.2024.1358085
  47. Junfeng Wan, Xueting Wang, Yiping Ren, Xiaoyi Yu, Kewei Chen, Zhiguo Li, Jialu Tang, Qiong Guo, Physiological adaptations of anammox bacteria to cold stress in wastewater treatment: Bioaugmentation and process intensification strategies, Journal of Environmental Chemical Engineering, 13, 5, (2025), 118550 https://doi.org/10.1016/j.jece.2025.118550
  48. Miao Yan, Laura Treu, Xinyu Zhu, Hailin Tian, Arianna Basile, Ioannis A. Fotidis, Stefano Campanaro, Irini Angelidaki, Insights into Ammonia Adaptation and Methanogenic Precursor Oxidation by Genome-Centric Analysis, Environmental Science and Technology, 54, 19, (2020), 12568-12582 https://doi.org/10.1021/acs.est.0c01945
  49. S. Kalamaras, M. L. Christou, C. Tzenos, S. Vasileiadis, D. Karpouzas, T. Kotsopoulos, Investigation of the Critical Biomass of Acclimated Microbial Communities to High Ammonia Concentrations for a Successful Bioaugmentation of Biogas Anaerobic Reactors with Ammonia Inhibition, Microorganisms, 11, 7, (2023), 1710 https://doi.org/10.3390/microorganisms11071710
  50. Feng Gao, Yan Zeng, Hui-Zhong Wang, Ya-Ting Chen, Yue-Qin Tang, Proteiniphilum and Methanothrix harundinacea became dominant acetate utilizers in a methanogenic reactor operated under strong ammonia stress, Frontiers in Microbiology, 13, (2023), 1098814 https://doi.org/10.3389/fmicb.2022.1098814
  51. Sharif Hossain, Christopher W. K. Chow, David Cook, Emma Sawade, Guna A. Hewa, Review of Nitrification Monitoring and Control Strategies in Drinking Water System, International Journal of Environmental Research and Public Health, 19, 7, (2022), 4003 https://doi.org/10.3390/ijerph19074003
  52. Zhi-Cheng Wu, Chun-Yu Lai, He-Ping Zhao, Salinity acclimation of nitrifying microorganisms: Nitrification performance, microbial community, osmotic adaptation strategies, Journal of Hazardous Materials Advances, 15, (2024), 100448 https://doi.org/10.1016/j.hazadv.2024.100448
  53. Daisuke Hira, Nobuyuki Aiko, Yoshinori Yabuki, Takao Fujii, Impact of aerobic acclimation on the nitrification performance and microbial community of landfill leachate sludge, Journal of Environmental Management, 209, (2018), 188-194 https://doi.org/10.1016/j.jenvman.2017.12.056
  54. Hui Wang, Bin Wang, Wenwen Dong, Xiaoke Hu, Co-acclimation of bacterial communities under stresses of hydrocarbons with different structures, Scientific Reports, 6, (2016), 34588 https://doi.org/10.1038/srep34588
  55. Hager Galal Elsayed Elboghdady, Elisa Clagnan, Veronica De Franceschi, Mirko Cucina, Marta Dell’Orto, Patrizia De Nisi, Andrea Goglio, Fabrizio Adani, Microbial acclimation of thermophilic anaerobic digestate enhances biogas production and biodegradation of polylactic acid in combination with the organic fraction of municipal solid waste (OFMSW), Waste Management, 203, (2025), 114895 https://doi.org/10.1016/j.wasman.2025.114895
  56. Luca Bucci, Gabriele Ghiotto, Guido Zampieri, Roberto Raga, Lorenzo Favaro, Laura Treu, Stefano Campanaro, Adaptation of Anaerobic Digestion Microbial Communities to High Ammonium Levels: Insights from Strain-Resolved Metagenomics, Environmental Science and Technology, 58, 1, (2023), 580-590 https://doi.org/10.1021/acs.est.3c07737
  57. Salina Alias, Megawati Omar, Noor Hana Hussain, Nor Amani Filzah Mohd-Kamil, Suhaimi Abdul-Talib, Kinetics of Benzo(a)pyrene biodegradation and bacterial growth in sandy soil by Sphingobacterium spiritovorum, Heliyon, 8, 10, (2022), e10799 https://doi.org/10.1016/j.heliyon.2022.e10799
  58. Kabiru Ibrahim Karamba, Siti Aqlima Ahmad, Azham Zulkharnain, Nur Adeela Yasid, Salihu Ibrahim, Mohd Yunus Shukor, Batch growth kinetic studies of locally isolated cyanide-degrading Serratia marcescens strain AQ07, 3 Biotech, 8, (2017), 11 https://doi.org/10.1007/s13205-017-1025-x

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