Better harnessing of Trichoderma biotechnological potential for biorefinery and as plant biostimulants by controlled development and biosynthesis

(TOTAL)

Project code: PN-III-P4-ID-PCE-2020-2780

Project coordinator – National Institute for Research & Development in Chemistry and Petrochemistry – ICECHIM, Bucharest

Principal Investigator: Dr. Chim. Diana CONSTANTINESCU-ARUXANDEI

Period of project implementation: 2021 – 2023

Total funding of the contract: 1.198.032 RON

Funding from the Public Budget: 1.198.032 RON

The general objective of Project TOTAL is to investigate and integrate abiotic modulation and epigenetic control of Trichoderma strains within the context of Trichoderma interactions with biomass and living matter, for better harnessing of Trichoderma biotechnological potential.

The specific objectives of the project are: (i) To establish and optimize the combined effects of light and nutrients on expression of proteins acting on plant cell walls and spore formation by Trichoderma strains; (ii) To optimize the deconstruction of plant cell walls by Trichoderma secreted proteins; (iii) To synergize the plant biostimulants effects of Trichoderma chlamydospores.

Project phases:

Phase 1 (2021). Design and testing of experimental models (EMs) for the amplification of the Trichoderma potential as plant biostimulant and in biorefinery

Phase 2 (2022). Optimization of EMs for the amplification of the Trichoderma potential as plant biostimulant and in biorefinery

Phase 3 (2023). Demonstration of EM functionality for the amplification of the Trichoderma potential as plant biostimulant and in biorefinery

Phase 1 / 2021

Phase 1 objective: design and testing of EMs

The activities corresponding to the objectives of phase 1 are:

A1.1 Design and testing of EM for determination of the light and nutrient effects on the chlamydospore formation and enzymatic activities of Trichoderma

A1.2 Design and testing of EM for biomass deconstruction by the enzymes produced by Trichoderma

A1.3 Design and testing of EM for synergic formulation of Trichoderma chlamydospores for optimal effects as microbial biostimulants

A1.4 Communication, dissemination, and management

Results of phase 1

We designed and tested 3 EM for the 3 work packages (WPs), respectively, each including a series of designed and tested protocols, biotechnologies and products. The effect of laser light was tested on Trichoderma species grown on either silicious or non-silicious substrate. All estimated results were obtained. The results were disseminated by the participation with a scientific communication to the international conference PRIOCHEM, XVIIth edition/10.2021, Bucharest – abstract published in Chemistry Proceedings, MDPI, and a scientific article sent for publication. The project is in the stage of optimization of EM, this activity representing the objective of phase 2/2022.

Phase 2 / 2022

Phase 2 objective: optimization of EMs

The activities corresponding to the objectives of phase 2 are:

A2.1 Optimization of EM for determination of the light and nutrient effects on the chlamydospore formation and enzymatic activities of Trichoderma

A2.2 Optimization of EM for biomass deconstruction by the enzymes produced by Trichoderma

A2.3 Optimization of EM for synergic formulation of Trichoderma chlamydospores for optimal effects as microbial biostimulants

A2.4 Communication, dissemination, and management

Results of phase 2

The 3 EMs and protocols for the 3 WPs were optimized: isolation of Trichoderma strains, new strain identification, strain irradiation with various light sources, selenium nanoparticle formation, quantification of gene expression with RT-qPCR, optimization of enzyme and chlamydospore production, optimization of biomass degradation, formulation of microbial biostimulants for foliar and radicular application, biotests on model plants (Arabidopsis thaliana). The results are complex, depending on the tested Trichoderma strains, substrate, and incubation time. The results were disseminated by 2 articles submitted for publication and 2 scientific communications submitted to the NeXT-Chem workshop, IVth edition/2022, Bucharest. The next step will be the demonstration of functionality of the optimized EM, within phase 3/2023.

Phase 3

Phase 3 objective: demonstration of EM functionality

The activities corresponding to the objectives of phase 3 are:

A2.1 Demonstration of the EM functionality for the biomass deconstruction by the Trichoderma proteins

A2.2 Demonstration of the EM functionality for the synergic formulation of Trichoderma chlamydospores for optimal effects as microbial biostimulants

A2.3 Communication, dissemination, and management

Results of phase 3

The functionality of EMs was demonstrated, by selecting two Trichoderma strains that gave optimal results within phase 2, T. atroviride and T. harzianum. We compared laser irradiation at 400 nm with LED illumination. The experiments confirmed the previous results. Both the laser source and LED source stimulated the enzymatic activities and sporulation, but the variation differed in some cases. We made correlations between the analyzed parameters. The supernatant was tested on mung bean seedlings (Vicia fabia) and the irradiated samples showed increased biostimulant effect compared with non-irradiated samples. The results included 2 patent applications, 2 articles submitted for publication, 1 scientific communication at the NeXT-Chem workshop, Vth Edition/2023. Phase 3 was successfully implemented.

Results of project TOTAL

Trichoderma is a plant-beneficial filamentous fungus, used in agriculture and biotechnologically important due to a high capacity to produce industrially relevant enzymes. The project TOTAL had the scope to integrate the fundamental aspects of Trichoderma growth (light and nutrient effects on metabolism) and the growth optimization to be applied for the biomass valorization and formulations of plant biostimulants. Three EMs were developed based on the application of laser-type light or LED light and cultivation on various subproducts from bioeconomy:

  1. EM for the optimization of sporulation and enzyme expression by Trichoderma spp.
  2. EM for the optimization of the deconstruction of non-siliceous and siliceous biomass by Trichoderma spp.
  3. EM of Trichoderma chlamydospore formulation with supernatant metabolites.

Following these EMs, an amplification of the enzymatic activities (cellulase, protease, alpha-amylase) and various forms of sporulation (conidia, chlamydospores) of Trichoderma spp., with increased capacity of biomass deconstruction. The biotests on model plants demonstrated an amplified activity of the sterile filtered supernatant and of the formulations rich in chlamydospores.

Considering the knowledge advancement, the results of project TOTAL have an impact on the understanding of life cycle of Trichoderma species. From the practical application point of view, the project offers new technical solutions for simultaneous, amplified, and efficient production of enzymes and chlamydospores. The project results have an impact in both agriculture and biorefinery.

I. Patent applications:

  1. Foliar biostimulant based on Trichoderma spores and metabolites and preparation process, patent application a00839 from 29.12.2022 published as RO137398 A
  2. Process for increasing the efficiency of Trichoderma fungi cultivation, patent application a00794 from 05.12.2023

II. Scientific communications:

  1. N. Tritean et al., Optimization of enzyme production of Trichoderma atroviride using response surface methodology, PRIOCHEM, XVIIth edition/10.2021, Bucharest, doi.org/10.3390/chemproc2022007054
  2. I.-A. Bala et al., Stimulation of Trichoderma sporulation by physicochemical factors, NeXT-Chem, IVth Edition/2022, Bucharest
  3. V. Bînzari et al., Light effects on supernatant properties of Trichoderma atroviride, NeXT-Chem, IVth Edition/2022, Bucharest
  4. I.-A. Bala et al., Blue-light laser effects on Trichoderma and on its plant biostimulant formulations, NeXT-Chem, Vth Edition/2023, Bucharest

III. Articles:

  1. Ioana-Alexandra Bala et al., Influence of Foliar Treatment with Suspensions Rich in Trichoderma Chlamydospores on Momordica charantia Physiology, Yield and Quality, Agriculture (Q1), https://doi.org/10.3390/horticulturae10040371
  2. Livia Teodora Ciobanu et al., The Clothes Matter – Trichogenic Selenium Nanoparticles Share Activities with the Biological Systems wherein (were) Formed, Agronomy (Q1), https://doi.org/10.3390/agronomy14010190
  3. Naomi Tritean et al., Mechanistic insights into the biostimulant activity of a novel formulation based on rice husk nanobiosilica embedded in a seed coating alginate film, Frontiers in Plant Science (Q1), DOI: 10.3389/fpls.2024.134957
  4. I.-A. Bala et al., Upscaling of an integrated technology for in situ production of Trichoderma enzymes and chlamydospores, Journal of Biotechnology (Q2), submitted
  5. Diana Constantinescu-Aruxandei et al., Enhancement of Trichoderma bioactivity by cultivation on biosilica-rich side streams, Journal of Fungi (Q1), submitted

IV. Articles in preparation:

  1. N. Tritean et al., Optimization of Trichoderma cultivation on whey medium by laser light illumination, Journal of Applied Microbiology (Q2)