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Investigation of microbial degradation of postharvest fungicides applied in fruit processing industry in bioreactor systems (Doctoral thesis)

Μαυριού, Ζωγραφίνα/ Mavriou, Zografina

This Ph.D. thesis investigated the microbial degradation of postharvest fungicides applied in the fruit processing industry in bioreactor systems. The research focused on the biodegradation of the most used postharvest fungicides, namely fludioxonil, imazalil and ortho-phenylphenol, in various types of bioreactor systems (immobilized cell bioreactors, batch bioreactors, sequencing batch reactors). The effectiveness of bioreactor systems in the removal of the above-mentioned postharvest fungicides was explored using high-performance liquid chromatography analysis and monitoring concurrently their physicochemical and biological traits. The microbial community structure during the degradation of the above-mentioned postharvest fungicides in bioreactor systems was also identified using high-throughput amplicon sequencing to link biosystems performance under various operating conditions with the proliferation of certain microbial groups. Firstly, fludioxonil-rich wastewater (250 mg/L) was treated in an aerobic immobilized cell bioreactor inoculated with activated sludge under the HRT of 10 days (Chapter 4). Fludioxonil was removed in a percentage greater than 95%, noting mineralization of the organic-F moiety of fludioxonil (94.0 ± 5.2%). COD removal efficiencies also overcame 80%. A notable increase in the proportion of Empedobacter, Sphingopyxis and Rhodopseudomonas was recorded throughout the biotreatment process. Moreover, a microaerophilic immobilized cell bioreactor was installed and operated to biotreat fludioxonil-rich wastewater (200 mg/L) under various hydraulic retention times (HRTs) (Chapter 5). In this bioreactor system, fludioxonil removal efficiency exceeded 96%, even at a low HRT of 3.9 days. Twelve (12) metabolites were identified by Quadrupole Time-Of-Flight Liquid Chromatography-Mass Spectrometry, indicating the hydroxylation, carbonylation, decyanation and deamination of the fludioxonil molecule. An increase in the relative abundances of the genera Pseudomonas, Clostridium and Terrimonas under shortened HRT was noted. The biotreatment of fludioxonil-rich wastewater (200 mg/L) was also examined in a Sequencing Batch Reactor (SBR) operating under intermittent aeration conditions (Chapter 6). Fludioxonil removal was higher than 96%, while COD removal efficiency was near 70%. Illumina sequencing revealed the predominance of Acidovorax and Fimbriimonadaceae spp., whereas nitrification was mainly carried out by members of the genera Nitrosomonas and Nitrospira. Further, a novel fludioxonil-degrading bacterial strain, named FLX-12, was isolated, which was able to effectively reduce high fludioxonil concentrations (even 250 mg/L)(Chapter 7). Phylogenetic analysis placed the bacterial strain FLX-12 in the genus Ochrobactrum. During the performance of Whole Genome Sequencing (WGS) and in vitro substrate utilization tests, it was found that the protocatechuic pathway was encoded by FLX-12. The optimal pH and temperature for the growth of strain FLX-12 were 7 and 37 oC, respectively. During the examination of fludioxonil degradation in aerobic batch bioreactors inoculated with strain FLX-12, high fludioxonil concentration (250 mg/L) was significantly reduced (by 88%) within an incubation period of 5 days. Fludioxonil degradation rate constants were increased in the case of co-metabolism, especially when amino acids L-asparagine, L-histidine, L-isoleucine, and L-proline were added. The ability of strain FLX-12 to effectively biotreat fludioxonil-rich wastewater was further examined in an aerobic immobilized cell bioreactor operated under various HRTs (Chapter 8). The immobilized cell bioreactor bioaugmented with strain FLX-12 resulted in high fludioxonil reduction throughout the whole experimental period, reaching removal efficiencies of 98%, even under an HRT of 1 day, whereas COD removal efficiency was steadily above 84%. In particular, Aridibacter (16.54 ± 3.89%) followed by Rhodococcus (12.05 ± 0.74%) were the most predominant genera under operation of the bioaugmented system at an HRT of 1 day. Furthermore, the bioprocessing of imazalil-rich wastewater (250 mg/L) was examined in an aerobic immobilized cell bioreactor inoculated with activated sludge and operated under an HRT of 10 days (Chapter 9). The immobilized cell bioreactor was capable of reducing imazalil concentration by 95%. After an acclimatization period of 15 days, COD removal efficiencies exceeded 76%, whereas a gradual increase in the electrical conductivity of the effluent was noted as a consequence of imazalil biotreatment. A predominance of bacterial genera Pseudomonas, Ochrobactrum, Stenotrophomonas, Cupriavidus, Rhodopseudomonas, Bordetella and Bellilinea was recorded, indicating thus their important role in the imazalil removal process. Moreover, the biotreatment of fungicide-based wastewater consisting of both imazalil and fludioxonil was performed in a horizontal immobilized cell bioreactor under various hydraulic retention times (Chapter 10). During the biotreatment of this fludioxonil and imazalil-based wastewater, fludioxonil concentration significantly decreased from 100 mg/L to 5.37 ± 0.17 mg/L, corresponding to removal efficiency greater than 94% throughout the entire experimental period. On the other hand, a limited decrease in imazalil concentration was observed, indicating a preference of immobilized microbiota for fludioxonil over imazalil. Pseudomonas and Azospirillum were the predominant bacterial genera throughout the entire experimental period. Lastly, ortho-phenylphenol-containing wastewater (250 mg/L) was also treated in an aerobic immobilized cell bioreactor under various hydraulic retention times (Chapter 11). A remarkable decrease in ortho-phenylphenol concentration from 250 mg/L to 0.09 ± 0.01 mg/L was denoted throughout the whole experimental period, resulting in a removal efficiency of 99.97 ± 0.01%. In addition, COD removal efficiencies greater than 90% were recorded, even under an HRT of 1.6 days. Illumina sequencing showed the predominance of the genera Dokdonella, Terrimonas, Mesorhizobium, Aridibacter, Reyranella, Nitrospira and Sphingopyxis during the biotreatment under the HRT range tested. Ιt is concluded that the examined bioreactor systems operating under various aeration conditions and hydraulic retention times were sufficient to deplete high concentrations of postharvest fungicides. These biological systems constitute a sustainable biological solution for the depuration of fungicide-rich wastewater from the fruit packaging industry.