PINTO IBIETA, FERNANDA EMILIA
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Flow cytometry: a tool for understanding the behaviour of polyhydroxyalkanoate accumulators
Abstract: The use of mixed microbial cultures (MMCs) is seen as an attractive strategy for polyhydroxyalkanoate (PHA) production. In order to optimize the MMC-PHA production process, tools are required to improve our understanding of the physiological state of the PHA-storing microorganisms within the MMC. In the present study, we explored the use of flow cytometry to analyse the metabolic state and polyhydroxybutyrate (PHB) content of the microorganisms from an MMC-PHA production process. A sequencing batch reactor under a feast and famine regime was used to enrich an MMC with PHB-storing microorganisms. Interestingly, once the PHB-storing microorganisms are selected, the level of PHB accumulation depends largely on the metabolic state of these microorganisms and not exclusively on the consortium composition. These results demonstrate that flow cytometry is a powerful tool to help to understand the PHA storage response of an MMC-PHA production process. Key points: Flow cytometry allows to measure PHB content and metabolic activity over time. Microorganisms showing high PHB content also have high metabolic activity. PHB producers with low metabolic activity show low PHB content. © 2023 Elsevier B.V., All rights reserved.
Use of Anthracophyllum Discolor and Stereum Hirsutum as a Suitable Strategy for Delignification and Phenolic Removal of Olive Mill Solid Waste
This study evaluated the use of the white-rot fungi (WRF) Anthracophyllum discolor and Stereum hirsutum as a biological pretreatment for olive mill solid mill waste (OMSW). The WRF strains proposed were added directly to OMSW. The assays consisted of determining the need to add supplementary nutrients, an exogenous carbon source or use agitation systems, and evaluating WRF growth, enzyme activity, phenolic compound removal and lignin degradation. The highest ligninolytic enzyme activity was found at day 10, reaching 176.7 U/L of manganese-independent peroxidase (MniP) produced by A. discolor, and the highest phenolic removal (more than 80% with both strains) was reached after 24 days of incubation. The confocal laser scanning microscopy analysis (CLSM) confirmed lignin degradation through the drop in lignin relative fluorescence units (RFU) from 3967 for untreated OMSW to 235 and 221 RFU, showing a lignin relative degradation of 94.1% and 94.4% after 24 days of treatment by A. discolor and S. hirsutum, respectively. The results demonstrate for the first time that A. discolor and S. hirsutum were able to degrade lignin and remove phenolic compounds from OMSW using this as the sole substrate without adding other nutrients or using agitation systems. This work indicates that it could be possible to design an in situ pretreatment of the valorization of OMSW, avoiding complex systems or transportation. In this sense, future research under non-sterile conditions is needed to evaluate the competition of WRF with other microorganisms present in the OMSW. The main drawbacks of this work are associated with both the low reaction time and the water addition. However, OMSW is seasonal waste produced in one season per year, being stored for a long time. In terms of water addition, the necessary optimization will be addressed in future research. © 2022 Elsevier B.V., All rights reserved.