Valorization of Dairy Sludge as a Low-Cost Substrate for Enhanced Biomass and Exopolysaccharide Production by Enterococcus durans K48

Articles in Press

Document Type : Research Article-en

Authors

Nazanin Taheri Nasab ¹

Mohammad Reza Edalatian Dovom ¹

Marzieh Moeenfard ¹

Leila Roozbeh Nasiraei ²

¹ Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

² Department of Food Science and Technology, No.C., Islamic Azad University, Nour, Iran

https://doi.org/10.22067/ifstrj.2026.98652.1575

Abstract

Dairy industry waste, particularly organic-rich dairy sludge, represents a promising and sustainable resource for biotechnological applications. This study evaluated the potential of dairy sludge as a cost-effective medium for the co-production of biomass and exopolysaccharides (EPS) by selected Enterococcus strains. The physicochemical parameters of the dairy sludge were characterized, revealing a composition of 32.70% fat, 29.32% protein, 9.56% ash, and 30.17% carbohydrates (which adjusted to 28.50% after spray-drying). Five strains of Enterococcus were screened through a quantitative culture-dependent approach to evaluate their growth and EPS production. Among the tested isolates, Enterococcus durans K48 exhibited the highest production potential and was selected for further study. Response Surface Methodology (RSM) was employed to optimize the concentrations of key medium components: dairy sludge, sucrose, and yeast extract. Under the optimized conditions (15% dairy sludge, 5% sucrose, and 2.5% yeast extract), a maximum biomass yield of 8,440 mg/L and an EPS yield of 394.2 mg/L were achieved. These findings demonstrate that dairy sludge can serve as a valuable source and sustainable substrate for Enterococcus cultivation, facilitating efficient biomass and EPS production while contributing to the valorization of dairy processing waste.

Keywords

Biomass

Dairy sludge

Lactic acid bacteria

Medium optimization

Valorization

Subjects

• Food Biotechnology

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Authors retain the copyright. This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0)

References

1.         Ahmed, Z., Mehmood, T., Ferheen, I., Noori, A. W., Almansouri, M., & Waseem, M. (2023). Optimization of exopolysaccharide produced by L. Kefiranofaciens ZW3 using response surface methodology. International Journal of Food Properties, 26(1), 2285–2293. https://doi.org/10.1080/10942912.2023.2245577

2.         Ashekuzzaman, S., Forrestal, P., Richards, K., & Fenton, O. (2019). Dairy industry derived wastewater treatment sludge: Generation, type and characterization of nutrients and metals for agricultural reuse. Journal of Cleaner Production, 230, 1266–1275. https://doi.org/10.1016/j.spc.2020.11.017

3.         Che, J., Fan, Z., Bijl, E., Thomsen, J.P.S., Mijakovic, I., Hettinga, K., Poulsen, N.A., & Larsen, L.B. (2025). Unravelling the dominant role of phosphorylation degree in governing the functionality of reassembled casein micelles: Implications for future dairy production through precision fermentation. Food Hydrocolloids, 159, 110615. https://doi.org/10.1016/j.foodhyd.2024.110615

4.         Choi, G.H., Lee, N.K., & Paik, H.D. (2021). Optimization of medium composition for biomass production of Lactobacillus plantarum 200655 using response surface methodology. Journal of Microbiology and Biotechnology, 31(5), 717. https://doi.org/10.4014/jmb.2103.03018

5.         Cui, Y., Jiang, X., Hao, M., Qu, X., & Hu, T. (2017). New advances in exopolysaccharides production of Streptococcus thermophilus. Archives of Microbiology, 199(6), 799–809. https://doi.org/10.1007/s00203-017-1366-1

6.         Esmaeilnejad-Moghadam, B., Mokarram, R.R., Hejazi, M.A., Khiabani, M.S., & Keivaninahr, F. (2019). Low molecular weight dextran production by Leuconostoc mesenteroides strains: Optimization of a new culture medium and the rheological assessments. Bioactive Carbohydrates and Dietary Fibre, 18, 100181 . https://doi.org/10.1016/j.bcdf.2019.100181

7.         Falah, F., Mortazavi, S.A., Danesh, A., Yazdi, F.T., & Ramezani, M. (2024). Production of cyclosporin A by Tolypocladium inflatum using dairy waste medium: optimization and investigation of the effect of ultrasound, high hydrostatic pressure, and pulsed electric field treatments on the morphology of fungus. Biomass Conversion and Biorefinery, 14(22), 29101–29113. https://doi.org/10.1007/s13399-023-03899-2

8.         Farkhondeh, T., Yavarmanesh, M., Tabatabaei Yazdi, F., & Edalatiane, M.R. (2023). Evaluation of pathogenicity indicators and technological properties of Enterococcus faecium isolates obtained from traditional Iranian cheeses. Journal of Food Science and Technology (Iran), 20(135), 69-87. https://doi.org/10.22034/FSCT.19.135.69

9.         Fusi, L., Primicerio, M., & Monti, A. (2015). A model for calcium carbonate neutralization in the presence of armoring. Applied Mathematical Modelling, 39(1), 348–362. https://doi.org/10.1016/j.apm.2014.05.037

10.     Ganta, A., Bashir, Y., & Das, S. (2022). Dairy wastewater as a potential feedstock for valuable production with concurrent wastewater treatment through microbial electrochemical technologies. Energies, 15(23), 9084. https://doi.org/10.3390/en15239084

11.     Gao, L., Wang, Y., Zhang, F., Li, S., Zhao, J., Zhang, Q., Ye, J., Ma, Y., Wang, Z., & Chen, W. (2022). A standardized method for the quantification of polysaccharides: An example of polysaccharides from Tremella fuciformis. Lwt, 167, 113860. https://doi.org/10.1016/j.lwt.2022.113860

12.     Gaur, S.S., & Annapure, U.S. (2025). Optimization of exopolysaccharide production from the novel Enterococcus species, using statistical design of experiment. Preparative Biochemistry & Biotechnology, 55(3), 297-308. https://doi.org/10.1080/10826068.2024.2402337

13.     Hu, S.-M., Zhou, J.-M., Zhou, Q.-Q., Li, P., Xie, Y.-Y., Zhou, T., & Gu, Q. (2021). Purification, characterization and biological activities of exopolysaccharides from Lactobacillus rhamnosus ZFM231 isolated from milk. Lwt, 147, 111561. https://doi.org/10.1016/j.lwt.2021.111561

14.     Iranian National Standardization Organization. (INSO). Milk and milk products—Determination of nitrogen content—Part 1: Calculation of crude protein content by the Kjeldahl method. Iranian National Standard No. 9188-1. Tehran, Iran; 2015.

15.     Iranian National Standardization Organization. (INSO). Milk—Determination of fat content—Acid butyrometric (Gerber) method. Iranian National Standard No. 23626. Tehran, Iran; 2024.

16.     Jia, K., Tao, X., Liu, Z., Zhan, H., He, W., Zhang, Z., Zeng, Z., & Wei, H. (2019). Characterization of novel exopolysaccharide of Enterococcus faecium WEFA23 from infant and demonstration of its in vitro biological properties. International Journal of Biological Macromolecules, 128, 710–717. https://doi.org/10.1016/j.ijbiomac.2018.12.245

17.     Kavitake, D., Tiwari, S., Devi, P.B., Shah, I.A., Reddy, G.B., & Shetty, P.H. (2024). Production, purification, and functional characterization of glucan exopolysaccharide produced by Enterococcus hirae strain OL616073 of fermented food origin. International Journal of Biological Macromolecules, 259, 129105. https://doi.org/10.1016/j.ijbiomac.2023.129105

18.     Kowsalya, M., Velmurugan, T., Mythili, R., Kim, W., Sudha, K.G., Ali, S., Kalpana, B., Ramalingam, S., & Rajeshkumar, M.P. (2023). Extraction and characterization of exopolysaccharides from Lactiplantibacillus plantarum strain PRK7 and PRK 11, and evaluation of their antioxidant, emulsion, and antibiofilm activities. International Journal of Biological Macromolecules, 242, 124842. https://doi.org/10.1016/j.ijbiomac.2023.124842

19.     Kwapinska, M., Pisano, I., & Leahy, J.J. (2023). Preliminary assessment of pyrolysis biochar derived from milk/dairy processing sludge as a potential component of fertilizers. ACS Sustainable Chemistry & Engineering, 11(6), 2345–2353. https://doi.org/10.1021/acssuschemeng.2c06093

20.     Liang, S., Wang, X., Li, C., & Liu, L. (2024). Biological activity of lactic acid bacteria exopolysaccharides and their applications in the food and pharmaceutical industries. Foods, 13(11), 1621. https://doi.org/10.3390/foods13111621

21.     McCarthy, O. (2011). Plant and Equipment| Centrifuges and Separators: Applications in the Dairy Industry.  https://doi.org/10.1016/b978-0-12-374407-4.00463-5

22.     Naykodi, A., Doriya, K., & Thorat, B.N. (2025). Optimization and characterization of exopolysaccharide production from Alkali-tolerant Alkalihalobacillus sp. using response surface methodology for cr (VI) biosorption. Biocatalysis and Agricultural Biotechnology, 64, 103522. https://doi.org/10.1016/j.bcab.2025.103522

23.     Netrusov, A.I., Liyaskina, E.V., Kurgaeva, I.V., Liyaskina, A.U., Yang, G., & Revin, V.V. (2023). Exopolysaccharides producing bacteria: a review. Microorganisms, 11(6), 1541. https://doi.org/10.3390/microorganisms11061541

24.     Oleksy-Sobczak, M., Klewicka, E., & Piekarska-Radzik, L. (2020). Exopolysaccharides production by Lactobacillus rhamnosus strains–Optimization of synthesis and extraction conditions. Lwt, 122, 109055. https://doi.org/10.1016/j.lwt.2020.109055

25.     Prazeres, A.R., Carvalho, F., & Rivas, J. (2012). Cheese whey management: A review. Journal of Environmental Management, 110, 48-68. https://doi.org/10.1016/j.jenvman.2012.05.018

26.     Quirino, D.F., Palma, M.N., Franco, M.O., & Detmann, E. (2023). Variations in methods for quantification of crude ash in animal feeds. Journal of AOAC International, 106(1), 6–13. https://doi.org/10.1093/jaoacint/qsac100

27.     Rahnama Vosough, P.R., Edalatian Dovom, M.R.E., Habibi Najafi, M.B., Javadmanesh, A., & Mayo, B. (2022). Biodiversity of exopolysaccharide-producing lactic acid bacteria from Iranian traditional Kishk and optimization of EPS yield by Enterococcus spp. Food Bioscience, 49, 101869. https://doi.org/10.1016/j.fbio.2022.101869

28.     Ryan, M.P., & Walsh, G. (2016). The characterisation of dairy waste and the potential of whey for industrial fermentation. Limerick: University of Limerick. Report. (173).

29.     Sakr, E.A., Massoud, M.I., & Ragaee, S. (2021). Food wastes as natural sources of lactic acid bacterial exopolysaccharides for the functional food industry: A review. International Journal of Biological Macromolecules, 189, 232–241. https://doi.org/10.1016/j.ijbiomac.2021.08.135

30.     Shete, B.S., & Shinkar, N. (2013). Dairy industry wastewater sources, characteristics & its effects on environment. International Journal of Current Engineering and Technology, 3(5), 1611–1615.

31.     Surayot, U., Wang, J., Seesuriyachan, P., Kuntiya, A., Tabarsa, M., Lee, Y., Kim, J.-K., Park, W., & You, S. (2014). Exopolysaccharides from lactic acid bacteria: structural analysis, molecular weight effect on immunomodulation. International Journal of Biological Macromolecules, 68, 233–240. https://doi.org/10.1016/j.ijbiomac.2014.05.005

32.     Wang, J., Zhang, J., Guo, H., Cheng, Q., Abbas, Z., Tong, Y., Yang, T., Zhou, Y., Zhang, H., & Wei, X. (2023). Optimization of exopolysaccharide produced by Lactobacillus plantarum R301 and its antioxidant and anti-inflammatory activities. Foods, 12(13), 2481. https://doi.org/10.3390/foods12132481

33.     Wu, J., Wu, Z., Pan, Y., Luo, D., & Zhong, Q. (2024). Effects of different stress conditions on the production, bioactivities, physicochemical and structural characteristics of exopolysaccharides synthetized by Schleiferilactobacillus harbinensis Z171. International Journal of Biological Macromolecules, 257, 128675. https://doi.org/10.1016/j.ijbiomac.2023.128675 

34.     Zanzan, M., Ezzaky, Y., Achemchem, F., Elmoslih, A., Hamadi, F., Hasnaoui, A., & Ait Ali, M. (2023). Optimisation of thermostable exopolysaccharide production from Enterococcus mundtii A2 isolated from camel milk and its structural characterisation. International Dairy Journal, 147, 105718. https://doi.org/10.1016/j.idairyj.2023.105718

35.     Ziaee, A., O'Connor, E.M., Murphy, E., & O'Reilly, E. (2023). Spray drying of food: principles and applications. In Drying Technology in Food Processing (pp. 123–155). Elsevier. https://doi.org/10.1016/b978-0-12-819895-7.00010-9