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Exploring MIcrobial networking in pesticides biodeGRAdaTion: novel inocula and biocatalysts for biodepuration of agro-industrial Effluents

Periodic Reporting for period 1 - EMIGRATE (Exploring MIcrobial networking in pesticides biodeGRAdaTion: novel inocula and biocatalysts for biodepuration of agro-industrial Effluents)

Reporting period: 2017-07-01 to 2019-06-30

Pesticides are a double-edged sword: on the one hand, they support productivity and quality of agricultural crop systems and, on the other hand, their use has undesirable consequences for human well-being and the environment. Their handling and disposal is currently subjected to a strict EU regulatory framework for reducing associated ecotoxicological and human health risks . Thiabendazole (TBZ), a benzimidazole commonly used protectively against postharvest fungal diseases and as anthelminthic in livestock farming, is highly stable (DT50 in soil of > 1 year ) in the environment without previously known potent biodegraders. TBZ-containing wastewaters produced by fruit-packaging plants should be treated, according to the relevant EC regulation, on site. Despite that there are no methods implemented for the treatment of these effluents leading to their land spreading which result in soil contamination at levels reaching up to 12 mg g-1 soil. Hence there is a need for methods to treat TBZ containing effluents to avert its environmental impact. In this context we have previously isolated the first bacterial consortium able to carry out a complete and rapid degradation of TBZ and foressee their use as microbial inocula in biological wastewater treatment facilities receiving effluents from wastewater treatment plants.

Societal impact
The project is expected to optimize the use of a microbial consortium able to degrade a persistent fungicide like Thiabendazole as inoculum in the implementation of tailored-made biological wastewater treatment units in fruit producing areas in Europe and beyond. This will have a major societal impact since (a) it will avert the environmental impact of a dynamic agro-industrial sector like fruit-packaging industry on the environment and the health of the rural population in the fruit producing areas where fruit packaging plants are located and (b) will create opportunities for environmental engineerign companies to uptake and implement novel technologies based on tailored made microbial inocula lke the one studied in the current project creating new work opportunities and opening new positions of work in the private sector

Aim and Objectives

The project aims to disentagle the interactions between consortium members at both nutritional and metabolic level, identify the transformation pathway of thiabendazole and the key players participating in each reaction (microbial, enzymes) and finally verify the efficacy of the consortium against thiabendazole-containing wastewaters under engineering conditions in bioreactors.

Achieving our aim relies on:
- Assessing the stability/composition of the microbial consortium under different degradation stages and conditions.
- Elucidating the roles of the consortium members concerning TBZ degradation
- Understanding the underlying chemical signalling and horizontal gene transfer interplay during and after TBZ biodegradation
- Identifying and validating the degradation pathway of the currently unknown TBZ biodegradation
- Testing/validating the degradation capacity of heterologously expressed enzymes and/or the microbial consortium members in real-world systems like membrane bioreactors
Overview of results
a) TBZ degradation by the microbial consortium was rapid with a DT90 of 70.5 h
b) stable isotope probing (SIP) analysis showed that a Sphingomonas was the sole TBZ degrader assimilating the 13C-labelled phenyl moiety of TBZ
c) Non target MS analysis showed the production of a single transformation product identified as thiazole carboxamidine which was not further transformed . These results suggest that TBZ is cleaved at the benzimidazole ring leading to the production of thiazole carboxamidine as a dead end product and catechol which is furtehr consumed by Sphingomonas
d) Shotgun metagenomics and genome binning generated a ~98 Mbp metagenome assembly with 18 main bins (putative genomes) of high completeness (>80%). Among these 6 were the dominant ones including Sphingomonas 3X21F, γ-Proteobacterium 34A, Bradyrhizobiaceae 9B and three Hydrogenophaga 19A, 13A, and 23F.
e) Meta-trancriptomic and -proteomic analysis suggested that Sphingomonas mobilizes a carbazole dioxygenase (car) operon during the initial cleavage of TBZ to thiazole-4-caroxamidine and catechol, the latter is further transformed by a catechol ortho-cleavage (cat) operon; both operons being up-regulated during TBZ degradation. The car operon components plus a ferredoxin reductase found not be contained in the car operon but being essential for carbazole dioxygenase operation are isolated, overexpressed and their in vitro activity at cell-free extracts is currently investigated
f) Gene expression network analysis revealed strong interactions between Sphingomonas MAG 3X12F and Hydrogenophaga MAG 23F, with Hydrogenophaga activating its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway along TBZ degradation.
g) The consortium was effective in removing 100% of TBZ contained in the wastewater applied to a bench top bioreactor. Amplicon sequencing analysis along the treatment process showed that the original members of the consortium consitute minor members of the community colonizing the bioreactor with Sphingomonas being present at low relative abundance

Dissemination and exploitation: Seveveral dissemination activities both at scientific and industrial level facilitated the maximum communication of the project findings to the scientific community (presentations in conferences, one paper is submitted, two under preparation) and relevant stakeholders (discussion with environmental engineering companies and fruit-packaging plants). This has led to the submission of a follow up project for scaling up the application of the microbial consortium to a fruit-packaging plant in Greece and has just started with a final goal the biological treatment of TBZ-containing wastewaters based on the thiabendazole-degrading consortium
Current status beyond the state of the art: We have provided through the use of a combination of omic tools and SIP analysis how a wild type stable consortium that degrades a recalcitrant fungicde operates, remains stable and operative, identified feeding interdependencies and new genes/enzymes with activity against the recalcitrant fungicide thiabendazole, firstly reported.

Ongoing research activity: Further tests will provide insights into the role of the other consortium members competing the puzzle of interactions between the consortium members

Potential impacts: The project is estimated to help improving current knowledge concerning microbial interactions and biocatalysts in the degradation of recalcitrant organic compounds. Furthermore, the use of the consortium either per se or as a synthetic biology model system for the depuration of post-harvest treatment wastewaters is an environmentally friendly and economically sound prospect that can be easily exploited in the fruit-packaging industry given the performance of the enriched consortium and its optimization based on the results of the current project.
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