Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L-1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L-1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

Biotreatment and bacterial succession in an upflow immobilized cell bioreactor fed with fludioxonil wastewater.

The large quantities and the persistent nature of fungicide wastewaters have increased the efforts towards a sustainable technological solution. In this context, fludioxonil-contaminated wastewater was treated in an upflow immobilized cell bioreactor, resulting in chemical oxygen demand (COD) removal efficiency even higher than 80%, whereas the electrical conductivity (EC) of the effluent was gradually increased. Organic-F was mineralized by 94.0 ± 5.2%, which was in accordance with the high fludioxonil removal efficiency (95.4 ± 4.0%). In addition, effluent total Kjeldahl nitrogen (TKN) concentration reduced significantly during bioprocessing. A strong relationship among COD removal, TKN/total nitrogen removal, and effluent EC increase (p < 0.01) was identified. Despite the adequate aeration provided, effluent nitrite and nitrate concentrations were negligible. Illumina sequencing revealed a reduction in the relative abundances of Betaproteobacteria, Chloroflexi, Planctomycetes, and Firmicutes and an increase in the proportion of Alphaproteobacteria and Actinobacteria. A shift in bacterial communities occurred during fludioxonil treatment, resulting in the significant increase of the relative abundances of Empedobacter, Sphingopyxis, and Rhodopseudomonas (from 0.67 ± 0.13% at the start-up to 34.34 ± 1.60% at the end of biotreatment). In conclusion, the immobilized cell bioreactor permitted the proliferation of specialized activated sludge microbiota with an active role in the depuration of postharvest fungicides.