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Journals / Afyon Kocatepe Üniversitesi Fen ve Mühendislik Bilimleri Dergisi / 2026 / Cilt: 25 Sayı: 5

Lyophilization and Preservation Techniques for Extended Storage of Terephthalic acid Degrading Pseudomonas sp: Procedural Steps and Protective Agents

Tereftalik Asit Parçalayan Pseudomonas sp.’nin Depolanması için Liyofilizasyon ve Saklama Teknikleri: Prosedür ve Koruyucu Ajanlar

Basri Aksu(EGE UNIVERSITY, İZMİR APPLICATION AND RESEARCH CENTER)0000-0002-8983-4524
Didem Aksu(Ege Üniversitesi, Merkezi Araştırma Test ve Analiz Laboratuvarı Uygulama ve Araştırma Merkezi)0000-0002-7184-4011
Burçin Karabey(İZMİR KAVRAM MESLEK YÜKSEKOKULU)0000-0003-4834-6740
Güven Özdemir(EGE UNIVERSITY)0000-0002-7577-4233
Pages
1008–1017
Publication Date
2025-10-01
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AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

Results indicated that the highest viability after 30 weeks was observed in samples treated with skim milk.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:The uncontrolled release of industrial waste into nature leads to increased environmental pollution, posing serious threats to ecosystem balance.
📊
Key Findings & Data:The lyophilized products were evaluated for their ability to degrade TA and for microorganism viability after 1, 15, and 30 weeks.
💡
Conclusion & Impact:Results indicated that the highest viability after 30 weeks was observed in samples treated with skim milk.
🔬Design:Modeling & Simulation
📍Scope:Comprehensive Sample
⚙️Variables:Biyodegradasyon · Arıtım bakterileri · Kriyoprotektanlar

Abstract

The uncontrolled release of industrial waste into nature leads to increased environmental pollution, posing serious threats to ecosystem balance. Terephthalic acid (TA) is a fundamental component used in the production of polyethylene terephthalate (PET), which finds applications in a wide range of industrial products, including plastic bottles, polyester fibers, and packaging materials. Effectively removing TA from wastewater systems can be achieved through various biotechnological methods. In this context, bioremediation bacteria play a critical role in environmental protection by providing sustainable solutions for wastewater management. The aim of this study is to investigate the effects of different protective and lyophilization methods on the biodegradation activity and bacterial viability of Pseudomonas sp. over specific storage periods. Various cryoprotectant agents (CPAs), including skim milk, sucrose, silica gel, and nutrient broth, were tested in different concentrations and combinations to improve storage conditions. Furthermore, optimization of freeze-drying (FD) and liquid drying (SD) techniques was conducted. The lyophilized products were evaluated for their ability to degrade TA and for microorganism viability after 1, 15, and 30 weeks. Results indicated that the highest viability after 30 weeks was observed in samples treated with skim milk. Additionally, the freeze-drying method demonstrated superior performance compared to liquid-drying method for all cryoprotectant types.

Özet

Endüstriyel atıkların kontrolsüz bir şekilde doğaya salınması, çevre kirliliğini artırarak ekosistem dengesini tehdit eden ciddi sorunlara yol açmaktadır. Tereftalik asit (TA), polietilen tereftalat (PET) üretiminde temel bir bileşen olup, plastik şişeler, poliester lifler ve ambalaj malzemeleri gibi birçok endüstriyel uygulamada kullanılmaktadır. TA'nın atık su sistemlerinden etkin bir şekilde uzaklaştırılması, çeşitli biyoteknolojik yöntemlerle mümkün olmaktadır. Bu bağlamda, arıtım bakterileri, atık su yönetiminde sürdürülebilir çözümler sunarak çevre koruma açısından kritik bir rol oynamaktadır. Bu çalışmanın amacı, Pseudomonas sp. türünün farklı koruyucu ve liyofilizasyon yöntemleri ile belirli sürelerde saklanmasının biyodegradasyon aktivitesine ve bakteri canlılığına olan etkilerini incelemektir. Çalışmada, saklama koşullarını iyileştirmek amacıyla süt tozu, sakaroz, silika jel ve ortam sıvısı gibi çeşitli kriyoprotektan ajanların (KPA'lar) farklı konsantrasyonları ve kombinasyonları test edilmiştir. Ayrıca, dondurarak kurutma (D-kurutma) ve sıvı kurutma (S-kurutma) tekniklerinin optimizasyonu gerçekleştirilmiştir. Liyofilize edilmiş ürünler, 1, 15 ve 30 hafta sonra TA'yı parçalama yeteneği ve mikroorganizma canlılığı açısından değerlendirilmiştir. Sonuçlar, 30 hafta sonunda en yüksek canlılığın skim milk ile muamele edilen örneklerde gözlemlendiğini ortaya koymaktadır. Ayrıca, D-kurutma yöntemi, tüm kriyoprotektan türleri için S-kurutma yöntemine kıyasla üstün performans sergilemiştir.

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Cite This Article

Aksu, B., Aksu, D., Karabey, B., Özdemir, G. (2025). Lyophilization and Preservation Techniques for Extended Storage of Terephthalic acid Degrading Pseudomonas sp: Procedural Steps and Protective Agents. Afyon Kocatepe Üniversitesi Fen ve Mühendislik Bilimleri Dergisi, Cilt: 25 Sayı: 5, 1008–1017. https://doi.org/10.35414/akufemubid.1581597

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References (55)

  1. Abadias, M., Benabarre, A., Teixidó, N., Usall, J. and Vinas, I., 2001. Effect of Freeze Drying and Protectants on Viability of the Biocontrol Yeast Candida Sake. International Journal of Food Microbiology, 65, 173-182. https://doi.org/10.1016/s0168-1605(00)00513-4
  2. Aksu, B. 2018. Effect of Lyophilization on Pseudomonas sp. Viability and Biodegradation Activity of Terephthalic Acid. Master Thesis, Ege University Graduate School of Natural and Applied Sciences, İzmir,69.
  3. Aksu, D., Vural, C., Karabey, B. and Özdemir, G. 2021. Biodegradation of Terephthalic Acid by Isolated Active Sludge Microorganisms and Monitoring of Bacteria in a Continuous Stirred Tank Reactor. Brazilian Archives of Biology and Technology, 64, 1-10. https://doi.org/10.1590/1678-4324-2021200002
  4. Aragón-Rojas, S., Yolanda, R.P.R., Hernández-Álvarez, A.J. and Quintanilla-Carvajal, M.X. 2019. Sublimation Conditions as Critical Factors during Freeze-Dried Probiotic Powder Production. Drying Technology, 8(3), 333–349. https://doi.org/10.1080/07373937.2019.1570248
  5. Bellali, S., Bou Khalil, J., Fontanini, A., Raoult, D. and Lagier, J. C. 2020. A new protectant medium preserving bacterial viability after freeze drying. Microbiological research, 236, 126454. https://doi.org/10.1016/j.micres.2020.126454
  6. Bigdeli, A., Khorasheh, F., Tourani, S., Khoshgard, A. and Bidaroni, H. 2020. Removal of terephthalic acid from aqueous solution using metal-organic frameworks; A molecular simulation study, Journal of Solid State Chemistry, 282, 121059,ISSN 0022-4596. https://doi.org/10.1016/j.jssc.2019.121059
  7. Broeckx, G., Vandenheuvel, D., Claes, I. J., Lebeer, S. and Kiekens, F. 2016. Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics. International journal of pharmaceutics, 505(1-2), 303–318. https://doi.org/10.1016/j.ijpharm.2016.04.002
  8. Bodzen, A., Jossier, A., Dupont, S., Mousset, P.Y., Beney, L., Lafay, S. and Gervais, P. 2021. Increased Survival of Lactococcus lactis Strains Subjected to Freeze-Drying after Cultivation in an Acid Medium: Involvement of Membrane Fluidity Cultivation in Acid Medium to Improve Bacterial Survival of Freeze-Drying.Food Technology and Biotechnology, 59, 443–45
  9. Bushnell, L. D. and Haas, H. F. 1941. The Utilization of Certain Hydrocarbons by Microorganisms. Journal of Bacteriology, 41(5), 653–673. https://doi.org/10.1128/jb.41.5.653-673.1941
  10. Calaf, G.M., Ponce-Cusi, R., Aguayo, F., Muñoz, J.P. and Bleak, T.C. 2020. Endocrine disruptors from the environment affecting breast cancer. Oncology Letters. 20(1):19-32. https://doi.org/10.3892/ol.2020.11566
  11. Carr, C. M., Clarke, D. J. and Dobson, A. D. W. 2020. Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives. Frontiers in microbiology, 11, 571265. https://doi.org/10.3389/fmicb.2020.571265
  12. Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X. and Gibbs, P. 2004. Relevant factors for the preparation of freeze-dried lactic acid bacteria. International Dairy of Journal, 14, 835–847. https://doi.org/10.1016/j.idairyj.2004.02.001
  13. Cody, W.L, Wilson, J.W., Hendrixson, D.R, McIver, KS, Hagman K.E. Ott CM., Nickerson, C.A. and Schurr, M.J. 2008. Skim milk enhances the preservation of thawed -80 degrees C bacterial stocks. Journal of Microbiology Methods. 75(1):135-8. https://doi.org/10.1016/j.mimet.2008.05.006
  14. Dzulkapli, N.F., Abdul Talib, S., Ramasamy, K. and Yin, C.Y. 2007. Identification of Bacterial Strains Capable of Degrading Malaysian Petroleum Sludge. Scientific Research Journal. 4, 1, 49-55. https://doi.org/10.24191/srj.v4i1.5665.
  15. Farfan Pajuelo, D. G., Carpio Mamani, M., Maraza Choque, G. J., Chachaque Callo, D. M. and Cáceda Quiroz, C. J. (2023). Effect of Lyoprotective Agents on the Preservation of Survival of a Bacillus cereus Strain PBG in the Freeze-Drying Process. Microorganisms, 11(11), 2705. https://doi.org/10.3390/microorganisms11112705
  16. Garg, K.K. and Prasad, B. 2017. Treatment of toxic pollutants of purified terephthalic acid wastewater: A review. Environmental Technology & Innovation, 8, 191-217. https://doi.org/10.1016/j.eti.2017.07.001
  17. He, S., Ni, Y., Lu, L., Chai, Q., Liu, H. and Yang, C. 2019. Enhanced biodegradation of n-hexane by Pseudomonas sp. strain NEE2. Scientific reports, 9(1), 16615. https://doi.org/10.1038/s41598-019-52661-0
  18. Hubálek Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology, 46(3), 205–229. https://doi.org/10.1016/s0011-2240(03)00046-4
  19. Jing, J., Wang, T., Guo, X., Huang, P., Li, C. and Qu, Y. 2024. Construction and application of petroleum-degrading bacterial agents: Community composition, lyophilization technology, and degradation mechanism. Journal of Environmental Chemical Engineering, 12 (6), 114904. Juhasz, A.L., Britz, M.L. and Stanley, G.A. 2000. Evaluation of a Creosote-Based Medium for the Growth and Preparation of a PAH-Degrading Bacterial Community for Bioaugmentation. Journal of Industrial Microbiology and Biotechnology, 24, 277-284. https://doi.org/10.1038/sj.jim.2900819
  20. Kaymak Ertekin, F., Köprüalan Aydın, Ö., Altay, Ö. 2024.Enhancing Viability of Lactobacillus plantarumBG24 Through Optimized Spray Drying: Insights into Process Parameters, Carrier Agents, Comparative Analysis with Freeze Drying, and Storage Condition Influences. Food Science & Nutrition.7;12(12):10330-10346 https://doi.org/10.1002/fsn3.4572
  21. Keskintepe, L. and Eroglu, A. 2015. Freeze-drying of mammalian sperm. Methods in Moleculer Biology. 1257:489- 4 97. https://doi.org/10.1007/978-1-4939-2193-5_25
  22. Kupletskaya, M.B. and Netrusov, A.I. 2011. Viability of lyophilized microorganisms after 50-year storage. Microbiology, 80, 850–853. https://doi.org/10.1134/S0026261711060129
  23. Larena, I., De Cal, A., Liñán, M. and Melgarejo, P. 2003. Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease. Journal of Applied Microbiology, 94(3), 508–514. https://doi.org/10.1046/j.1365-2672.2003.01860.x
  24. Li, S., Zhao Y., Zhang L., Zhang X, Huang L, Li D, Niu C, Yang Z. And Wang, Q.2012. Antioxidant Activity of Lactobacillus plantarum Strains Isolated From Traditional Chinese Fermented Foods. Food Chemistry, 135: 3, 1914–1919. https://doi.org/10.1016/j.foodchem.2012.06.048
  25. Li, X.M., Che, L., Wu, Y., Li, C. and Xu, B.C. 2024. An effective strategy for improving the freeze-drying survival rate of Lactobacillus curvatus and its potential protective mechanism. Food Bioscience, 58, 103794. https://doi.org/10.1016/j.fbio.2024.103794
  26. Liu, J., Viverette, T., Virgin, M., Anderson, M. and Paresh, D. 2005. A study of the impact of freezing on the lyophilization of a concentrated formulation with a high fill depth. Pharmaceutical development and technology, 10 (2), 261–272. https://doi.org/10.1081/pdt-54452
  27. Luciani-Torres, M. G., Moore, D. H., Goodson, W. H., 3rd and Dairkee, S. H. 2015. Exposure to the polyester PET precursor--terephthalic acid induces and perpetuates DNA damage-harboring non-malignant human breast cells. Carcinogenesis, 36 (1), 168–176. https://doi.org/10.1093/carcin/bgu234
  28. Malik, K.A. 1990. A Simplified liquid-drying method for the preservation of microorganisms sensitive to freezing and freeze-drying. Journal of Microbiological Methods, 12, 125-132. https://doi.org/10.1016/0167-7012(90)90022-X
  29. Martos, G. I., Minahk, C. J., de Valdez, G. F. and Morero, R. 2007. Effects of protective agents on membrane fluidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus. Letters in applied microbiology, 45(3), 282–288. https://doi.org/10.1111/j.1472-765x.2007.02188.x
  30. Meena, K. K., Taneja N. K., Ojha A., and Meena S. 2023. “Application of Spray‐Drying and Freeze‐Drying for Microencapsulation of Lactic Acid Bacteria: A Review. Annals of Phytomedicine an International Journal 12:1, 706–716. http://dx.doi.org/10.54085/ap.2023.12.1.76
  31. Molonia, M. S., Muscarà, C., Speciale, A., Salamone, F. L., Toscano, G., Saija, A. and Cimino, F. 2022. The p-Phthalates Terephthalic Acid and Dimethyl Terephthalate Used in the Manufacture of PET Induce In Vitro Adipocytes Dysfunction by Altering Adipogenesis and Thermogenesis Mechanisms. Molecules, 27(21), 7645. https://doi.org/10.3390/molecules27217645
  32. Nasran, H. S., Mohd Yusof, H., Halim, M. and Abdul Rahman, N. 2020. Optimization of Protective Agents for The Freeze-Drying of Paenibacillus polymyxa Kp10 as a Potential Biofungicide. Molecules, 25(11), 2618. https://doi.org/10.3390/molecules25112618
  33. Ojo, O.A. 2006. Petroleum-Hydrocarbon Utilization by Native Bacterial Population from a Wastewater Canal Southwest Nigeria. African Journal of Biotechnology, 5 (4), 333-337. Ong, J.W., Song, Z., Abid, H.A., Lin, E.S., Liew, O.W,. and Ng, T.Q.W.2022. Cryoprotectant-free preservation of bacteria using semi-spherical drops, Cryobiology,104,98-101. https://doi.org/10.1016/j.cryobiol.2021.11.179
  34. Palmfeldt, J., Rådström, P. and Hahn-Hägerdal, B. 2003. Optimisation of Initial Cell Concentration Enhances Freeze-Drying Tolerance of Pseudomonas chlororaphis. Cryobiology, 47(1), 21–29. https://doi.org/10.1016/s0011-2240(03)00065-8
  35. Peiren, J., Buyse, J. and De Vos, P. 2015. Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections. Applied Microbiology and Biotechnolology, 99, 3559–3571. https://doi.org/10.1007/s00253-015-6476-6
  36. Perdigão, R., Almeida, C. M. R., Magalhães, C., Ramos, S., Carolas, A. L., Ferreira, B. S., Carvalho, M. F., & Mucha, A. P. 2021. Bioremediation of Petroleum Hydrocarbons in Seawater: Prospects of Using Lyophilized Native Hydrocarbon-Degrading Bacteria. Microorganisms, 9(11), 2285. https://doi.org/10.3390/microorganisms9112285
  37. Singh, C. and Lin, J. 2008. Isolation and characterization of diesel oil degrading indigenous microrganisms in Kwazulu-Natal, South Africa. African journal of Biotechnology, 7, 12. https://doi.org/10.5897/AJB07.728
  38. Song, T., Xu, Y., Ye, Chen, Y. And Shen, S. 2009. Electricity Generation from Terephthalic Acid Using a Microbial Fuel Cell. Journal of Chemical Technology and Biotechnology, 84(3), 356–360. https://doi.org/10.1002/jctb.2047
  39. Stacey, G. N. and Day, J. G. 2007. Long-term ex situ conservation of biological resources and the role of biological resource centers. Methods in molecular biology (Clifton, N.J.), 368, 1–14. https://doi.org/10.1007/978-1-59745-362-2_1
  40. Stephan, D., Matos Da Silva, A. and Bisutti, L.I. 2016. Optimization of a freeze-drying process for the biocontrol agent Pseudomonas spp. and its influence on viability, storability and efficacy. Biological Control, 94, 74-81. https://doi.org/10.1016/j.biocontrol.2015.12.004
  41. Tan, G.H. and Mustapha, N.A. 2014. Comparative Analysis of Preservation of Functional Food Cultures by Freeze- Drying, Liquid-Drying and Freezing Methods. Direct Research Journal of Agriculture and Food Science (DRJAFS), 2(2), 13–18.
  42. Trowbridge, J., Goin, D.E., Abrahamsson, D., Sklar, R. and Woodruff, T.J. 2023. Fossil fuel is the common denominator between climate change and petrochemical exposures, and effects on women and children’s health. International Journal of Gynecology & Obstetrics;160:368-371 https://doi.org/10.1002/ijgo.14408
  43. Tyagi, N., Gidlöf, Z., Osanlóo, D.T., Collier, E.S., Kadekar, S., Ringstad, L., Fureby, A.M. and Roos, S. 2023. The Impact of Formulation and Freeze Drying on the Properties and Performance of Freeze-Dried Limosilactobacillus reuteri R2LC. Applied Microbiology, 3(4), 1370-1387. https://doi.org/10.3390/applmicrobiol3040092
  44. Wang, Z.J., Teng, L.H., Zhang, J., Huang, X.L., & Zhang, J.F. (2011). Study on Optimal Biodegradation of Terephthalic Acid by an Isolated Pseudomonas sp. African Journal of Biotechnology, 10 (16), 3143–3148. https://doi.org/10.5897/AJB10.2045
  45. Woodruff, T.J. 2024. Health Effects of Fossil Fuel-Derived Endocrine Disruptors. The New England Journal of Medicine.390(10):922-933. https://doi.org/10.1056/NEJMra2300476
  46. Zhan, Y., Xu, Q., Yang, M. M., Yang, H. T., Liu, H. X., Wang, Y. P. and Guo, J. H. 2012. Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40. Letters in applied microbiology, 54(1), 10–17. https://doi.org/10.1111/j.1472765X.2011.03165.x
  47. Zhang, X. X., Sun, S. L., Zhang, Y., Wu, B., Zhang, Z. Y., Liu, B., Yang, L. Y. and Cheng, S. P. 2010. Toxicity of purified terephthalic acid manufacturing wastewater on reproductive system of male mice (Mus musculus). Journal of hazardous materials, 176 (1-3), 300–305. https://doi.org/10.1016/j.jhazmat.2009.11.028
  48. Zhang, Y.M., Sun, Y.Q., Wang, Z.J. and Zhang, J. 2013. Degradation of Terephthalic Acid by a Newly Isolated Strain of Arthrobacter sp. 0574. S. African Journal of Science, 109 (7/8), 1–4. https://doi.org/10.1590/sajs.2013/20120019
  49. Zeng, C., Ding, F., Zhou, J., Dong, W., Cui, Z. and Yan, X. 2023. Biodegradation of Poly(ethylene terephthalate) by Bacillus safensis YX8. International Journal of Molecular Sciences, 24(22), 16434. https://doi.org/10.3390/ijms242216434
  50. Zheng, X., Fu, N., Duan, M., Woo, M.W., Selomulya, C. and Chen, X.D. 2015. The mechanisms of the protective effects of reconstituted skim milk during convective droplet drying of lactic acid bacteria, Food Research International. 76:3,478-488, https://doi.org/10.1016/j.foodres.2015.07.045
  51. Dzulkapli, N.F., Abdul Talib, S., Ramasamy, K. and Yin, C.Y. 2007. Identification of Bacterial Strains Capable of Degrading Malaysian Petroleum Sludge. Scientific Research Journal. 4, 1, 49-55. https://doi.org/10.24191/srj.v4i1.5665.
  52. Keskintepe, L. and Eroglu, A. 2015. Freeze-drying of mammalian sperm. Methods in Moleculer Biology. 1257:489- 4 97. https://doi.org/10.1007/978-1-4939-2193-5_25
  53. Martos, G. I., Minahk, C. J., de Valdez, G. F. and Morero, R. 2007. Effects of protective agents on membrane fluidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus. Letters in applied microbiology, 45(3), 282–288. https://doi.org/10.1111/j.1472-765x.2007.02188.x
  54. Meena, K. K., Taneja N. K., Ojha A., and Meena S. 2023. “Application of Spray‐Drying and Freeze‐Drying for Microencapsulation of Lactic Acid Bacteria: A Review. Annals of Phytomedicine an International Journal 12:1, 706–716. http://dx.doi.org/10.54085/ap.2023.12.1.76
  55. Trowbridge, J., Goin, D.E., Abrahamsson, D., Sklar, R. and Woodruff, T.J. 2023. Fossil fuel is the common denominator between climate change and petrochemical exposures, and effects on women and children’s health. International Journal of Gynecology & Obstetrics;160:368-371 https://doi.org/10.1002/ijgo.14408