Periodic Reporting for period 1 - REMIPLASWAS (Removal of microplastics from the environment using autochthonous wastewater-derived microbial consortia)
Reporting period: 2023-03-07 to 2025-03-06
The accumulation of microplastics (MPs) in the environment poses a global health threat to the biosphere. Known and demonstrated conventional methodologies used in the treatment of MPs in wastewater have several limitations. Therefore, there is a need to develop new eco-friendly, cost-effective biobased MPs (Polyethylene terephthalate-PET) removal strategies. Microorganisms can transform organic matter as well as remove pollutants through a chain of different catabolic reactions energized by the induction of non-stereoselective enzymes. We would have accomplished most of the work packages as intended, but the decision to conclude the project before its full duration and completion affected this. While we completed WPs 1 and 2, WPs 3-6 were not adequately addressed due to time constraints. We were able to isolate and characterize microplastics in the wastewater in Granada, Spain, as well as the different fungal strains from the sludge. These activities comprised most of the tasks in WPs 1 and 2. In effect, the identification of key genes in the catabolic process, selection of microbial consortium with the ability to remove MPs, scaling up the process into a bench-scale bioreactor to develop a bioaugmentation technology with a microbial native consortium based on their ability to degrade MPs. and assessment of the toxicity of the resulting effluent after MPs process were part of the objectives not achieved. The training and transfer of knowledge were achieved basically due to the cross-fertilization of ideas and the improvised techniques/protocol developed during the isolation of MPs from sludge/wastewater. I benefitted immensely from the professional training and seminar presentation attended at the Institute of Water Research, University of Granada, Spain.
1.0 Analysis of MPs in wastewater
The sludge/wastewater was procured from Edar Sur, a wastewater treatment plant located in Granada, Spain. The types and nature of different polymers making up the microplastics were thereafter analyzed following digestion, gravity floatation, filtration, and post-oxidation. A total of filters containing MPs were recovered after extraction and isolation processes. The work package was fully completed.
1.1. Qualitative and Quantitative analyses of MPs from sludge samples
The weight, fiber counts, shapes, colour, and size of the recovered MPs were determined. The average weight of the filters containing MPs ranges from 0.150 to 01872 mg/g of sludge. The shapes are mostly irregular tiny MP particles such as trapezoids, triangles, circular, and stone-like. Additionally, the colour differentiation was used to characterize further the MPs observed on the filters as follows:
Table 1. Morphological characterization of MPs based on colour
Colour Filter 1 Filter 2 Filter 3 Filter 4 Filter 5
Red 3 5 1 9 2
Yellow 10 7 4 3 2
Brown 4 11 3 9 8
Pink 2 0 4 0 1
Grey 4 8 7 9 5
Green 3 7 7 1 6
Blue 7 2 1 3 3
Black 3 2 0 3 4
Total 36 42 27 37 31
The MPs were further classified based on polymer types using small-field magnifying glass. The following polymer types were identified from the total of 63 fragments analyzed:
Table 2. Polymer types based on the fragment in the recovered MPs
Polymer type Number of fragments
Polyamide (PA) 17
Polyethylene (PE) 14
Polyethylene terephthalate (PET) 5
Polypropylene (PP) 16
Polystyrene (PS) 1
Polyvinylchloride (PVC) 10
The Attenuated total reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy analysis carried out revealed the spectra of the polymer types. The results on the microplastic concentrations and distribution in the sludge are currently under processing to be communicated to Junta de Andalusia (the Regional Government of Andalusia). We aim to work with the government to disseminate the findings to the general public through outreach events.
1.2. Work Package 2 - [Isolation of microbial community of pollutant degraders]
Fungi were isolated from the sludge following standard fungi isolation procedures. The fungi isolated were characterized by morphological features under the microscope and identified as Mucor sp., Aspergillus niger, Aspergillus flavus, Penicillium spp., and Trichoderma spp. The strains were submitted for molecular identification. Limited progress was attained with this work package because bacteria isolation and identification were currently ongoing before the project ended. Additionally, the metatranscriptomic analyses proposed were not achieved due to the exigencies of time and the non-completion of all microbial isolation processes.
This project does not involve any gender considerations, secondment, or non-academic placements. During the project, I attended several monthly workshops, meetings, and presentations at the Institute of Water Research, University of Granada, Spain. These presentations were done by researchers from all over the world who have come to visit Spain. The discourse has further enlightened my scope of open science and data management skills. While delivering the project, I learned technical skills and gained extra knowledge and valuable insights into microplastic standard extraction procedures. I also devised a unique modification that enhanced the recovery of MPs from sludge which involves post-oxidation. We aim to communicate the discoveries and findings gathered so far to a suitable journal in the field of environmental microbiology. I benefitted immensely from training and workshops organized for staff and researchers. Additionally, I have been able to present my findings at two (2) different conferences namely the 2024 International Union of Biochemistry and Molecular Biology (IUBMB) Focused Meeting at the University of Ljubljana, Slovenia, and 2024 Young Environmental Scientists (YES) Conference organized by the Society of Experimental Toxicology and Chemistry (SETAC) at the Institute of Environmental Sciences, Rheinland-Pfälzische Technische Universität (RPTU) Kaiserslautern-Landau, Germany. Additionally, the results obtained during this time and my collaboration with other members of the team have been presented in Spanish congresses such as IX Congreso Nacional de Microbiología Industrial y Biotecnología Microbiana (2024) and the 8th Jornadas de la Red de Compostaje (2024). I served as a Grant Reviewer on project submitted for funding to Polish National Science Centre (Narodowe Centrum Nauki-NCN).
The sludge/wastewater was procured from Edar Sur, a wastewater treatment plant located in Granada, Spain. The types and nature of different polymers making up the microplastics were thereafter analyzed following digestion, gravity floatation, filtration, and post-oxidation. A total of filters containing MPs were recovered after extraction and isolation processes. The work package was fully completed.
1.1. Qualitative and Quantitative analyses of MPs from sludge samples
The weight, fiber counts, shapes, colour, and size of the recovered MPs were determined. The average weight of the filters containing MPs ranges from 0.150 to 01872 mg/g of sludge. The shapes are mostly irregular tiny MP particles such as trapezoids, triangles, circular, and stone-like. Additionally, the colour differentiation was used to characterize further the MPs observed on the filters as follows:
Table 1. Morphological characterization of MPs based on colour
Colour Filter 1 Filter 2 Filter 3 Filter 4 Filter 5
Red 3 5 1 9 2
Yellow 10 7 4 3 2
Brown 4 11 3 9 8
Pink 2 0 4 0 1
Grey 4 8 7 9 5
Green 3 7 7 1 6
Blue 7 2 1 3 3
Black 3 2 0 3 4
Total 36 42 27 37 31
The MPs were further classified based on polymer types using small-field magnifying glass. The following polymer types were identified from the total of 63 fragments analyzed:
Table 2. Polymer types based on the fragment in the recovered MPs
Polymer type Number of fragments
Polyamide (PA) 17
Polyethylene (PE) 14
Polyethylene terephthalate (PET) 5
Polypropylene (PP) 16
Polystyrene (PS) 1
Polyvinylchloride (PVC) 10
The Attenuated total reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy analysis carried out revealed the spectra of the polymer types. The results on the microplastic concentrations and distribution in the sludge are currently under processing to be communicated to Junta de Andalusia (the Regional Government of Andalusia). We aim to work with the government to disseminate the findings to the general public through outreach events.
1.2. Work Package 2 - [Isolation of microbial community of pollutant degraders]
Fungi were isolated from the sludge following standard fungi isolation procedures. The fungi isolated were characterized by morphological features under the microscope and identified as Mucor sp., Aspergillus niger, Aspergillus flavus, Penicillium spp., and Trichoderma spp. The strains were submitted for molecular identification. Limited progress was attained with this work package because bacteria isolation and identification were currently ongoing before the project ended. Additionally, the metatranscriptomic analyses proposed were not achieved due to the exigencies of time and the non-completion of all microbial isolation processes.
This project does not involve any gender considerations, secondment, or non-academic placements. During the project, I attended several monthly workshops, meetings, and presentations at the Institute of Water Research, University of Granada, Spain. These presentations were done by researchers from all over the world who have come to visit Spain. The discourse has further enlightened my scope of open science and data management skills. While delivering the project, I learned technical skills and gained extra knowledge and valuable insights into microplastic standard extraction procedures. I also devised a unique modification that enhanced the recovery of MPs from sludge which involves post-oxidation. We aim to communicate the discoveries and findings gathered so far to a suitable journal in the field of environmental microbiology. I benefitted immensely from training and workshops organized for staff and researchers. Additionally, I have been able to present my findings at two (2) different conferences namely the 2024 International Union of Biochemistry and Molecular Biology (IUBMB) Focused Meeting at the University of Ljubljana, Slovenia, and 2024 Young Environmental Scientists (YES) Conference organized by the Society of Experimental Toxicology and Chemistry (SETAC) at the Institute of Environmental Sciences, Rheinland-Pfälzische Technische Universität (RPTU) Kaiserslautern-Landau, Germany. Additionally, the results obtained during this time and my collaboration with other members of the team have been presented in Spanish congresses such as IX Congreso Nacional de Microbiología Industrial y Biotecnología Microbiana (2024) and the 8th Jornadas de la Red de Compostaje (2024). I served as a Grant Reviewer on project submitted for funding to Polish National Science Centre (Narodowe Centrum Nauki-NCN).
Through this project, we have been able to document the polymer types in the MPs that are abundant in the wastewater in Granada, Spain. Additionally, fungal strains that showed capacity to remove MPs were also isolated and characterized