TY - JOUR T1 - Lyophilization and Preservation Techniques for Extended Storage of Terephthalic acid Degrading Pseudomonas sp: Procedural Steps and Protective Agents TT - Tereftalik Asit Parçalayan Pseudomonas sp.’nin Depolanması için Liyofilizasyon ve Saklama Teknikleri: Prosedür ve Koruyucu Ajanlar AU - Özdemir, Güven AU - Aksu, Basri AU - Aksu, Didem AU - Karabey, Burçin PY - 2025 DA - October Y2 - 2025 DO - 10.35414/akufemubid.1581597 JF - Afyon Kocatepe Üniversitesi Fen Ve Mühendislik Bilimleri Dergisi PB - Afyon Kocatepe Üniversitesi WT - DergiPark SN - 2149-3367 SP - 1008 EP - 1017 VL - 25 IS - 5 LA - en AB - 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. KW - Biodegradation KW - Freeze-drying KW - Liquid-drying KW - Lyophilization KW - Protective agent KW - Terephthalic acid N2 - 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. CR - 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 CR - 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. CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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. CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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. CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 CR - 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 UR - https://doi.org/10.35414/akufemubid.1581597 L1 - https://dergipark.org.tr/tr/download/article-file/4349808 ER -