The project objective is to develop an innovative technology, based on enzymatic processes and green solvents (natural deep eutectic solvents), for multi-stage cascading use and valorisation of lignocellulose biomass involving the following specific objectives: enzymatic extraction of ferulic acid from biomass; cellulose valorisation into nanocellulose; subsequent enzymatic saccharification of the separated polysaccharides.

The expected results are: (i) selected NADESs with low (eco)toxicity, for lignocellulose pre-treatment and enzymatic extraction of ferulic acid; (ii) functional model of eco-biotechnological concept for the valorisation of lignocellulosic biomass, producing ferulic acid, nanocellulose and bioethanol; 1 national (OSIM) Patent application and/or 1 international Patent application (EPO/WIPO), 3 ISI papers submitted for publication.

Phase 1

Title: Elaboration of experimental models and technologies

Period: 02.05.2018 – 31.12.2018

The Objective of phase 1 was the elaboration and testing of experimental models (EMs). The activities corresponding to the objective of phase 1 were the following:

A1.1 Elaboration and testing of EMs for the preparation, characterization and selection of binary and ternary Natural Deep Eutectic Solvents (NADESs) compatible with enzymes, used for cascade processing of lignocellulose from brewer‘s spent grains (BSG)

A1.2 Elaboration and testing of EMs for cascade processing of lignocellulose from BSG

A1.3 Intellectual property rights – Patent application

The estimated results were:

• Functional website
• 1 kick-off meeting report
• 3 elaborated and tested EMs
• List of NADESs including toxicity tests
• 1 OSIM or EPO/WPO Patent
• Intermediate report

Summary of the scientific and technical report for phase 1

Within phase 1 of project INTELIGRENCE, three experimental models (EM) were elaborated and tested: (1) preparation and characterization of natural deep eutectic solvents (NADESs); (2) NaDESs selection for the enzymatic extraction of ferulic acid (FA); (3) valorisation of BSG by enzymatic extraction of ferulic acid in the presence of NADES.

Within EM 1 we have prepared 8 NADESs, which were characterized by Nuclear Magnetic Resonance (NMR) and Fourier-Transform Infrared Spectroscopy (FT-IR). The toxicity of these NADESs and 3 commercial ones, against beneficial microorganisms, was tested on agar plates resulting in a list with toxicity characteristics from which the ones that did not present toxicity under the conditions tested were selected.

Within EM 2, the solubilization of FA in the NADESs that presented low toxicity was tested (by quantification with HPLC) and the NADES with the highest capacity to solubilise FA was selected.

EM 3 consisted of the enzymatic extraction of FA from BSG, using feruloyl esterase in the presence of several NADES concentrations (30%, 50%, 70%, 90%). The optimal concentration obtained was 30% NADES.

The kick-off meeting report was drawn up, the project website was initiated and one National (OSIM) Patent application was initiated. The objectives and results of phase 1 were fulfilled.

Phase 2

Title: Implementation and optimization of experimental models and technologies

Period: 01.01.2019 – 31.12.2019

The Objective of phase 2 was the implementation and optimization of experimental models (EMs).  The activities corresponding to the objective of phase 2 were the following:

A2.1 Implementation and optimization of EMs for the preparation, characterization and selection of binary and ternary NADESs compatible with enzymes, used for cascade processing of lignocellulose from BSG

A2.2 Implementation and optimization of EMs for cascade processing of lignocellulose from BSG

A2.3 Dissemination and communication of results

The estimated results were:

• Up-to-date website
• Implemented and optimized EMs
• Updated list of NADESs including the toxicity report
• Minimum 1 participation at international conferences
• 1 paper submitted for publication in ISI Journal
• Intermediate report

Summary of the scientific and technical report for phase 2

Within phase 2 of project INTELIGRENCE, three EM were implemented and optimized: (1) preparation and characterization of NADESs; (2) NADESs selection for enzymatic extraction of FA and valorization of polysaccharides; (3) optimization of BSG valorisation by enzymatic extraction of FA in the presence of NADES.

Within EM 1, we have prepared 3 new NADESs that were characterized by NMR and FTIR. Moreover, pH values and the toxicity profiles against beneficial microorganisms were investigated, resulting in an up-to-date list of NADES toxicity. The ones showing low toxicity were selected for further studies.

Within EM 2, the solubility of FA and activity of FAE in different NADESs were analysed and optimized. The solubility of polysaccharides, in the absence and presence of polysaccharides was also investigated.

Within EM 3, we have partially optimized the enzymatic extraction of ferulic acid from BSG by varying the enzyme and NADES concentration. The samples were analysed by HPLC.

The results were disseminated by two communications at an international conference and one manuscript was sent for publication to an ISI Journal:

1. Carmen-Valentina Nicolae, Bogdan Trica, Sanda-Maria Doncea, Ioana Cristina Marinas, Florin Oancea, Diana Constantinescu-Aruxandei. Evaluation and optimization of polysaccharides and ferulic acid solubility in NaDES using response surface methodology, International Simpozium “Priorities of Chemistry for a Sustainable Development” (PRIOCHEM), XV-edition, ICECHIM-Bucharest, 30.10-1.11.2019, Book of Abstracts, ISSN 2285 8334, MDPI Proceedings 2019, 29(1), 96, https://doi.org/10.3390/proceedings2019029096 (poster)
2. Ioana-Cristina Marinas, Bogdan Trica, Carmen-Valentina Nicolae, Florin Oancea, Diana Constantinescu-Aruxandei. The Ethaline Effect on Feruloyl Esterase Activity. International Simpozium “Priorities of Chemistry for a Sustainable Development” (PRIOCHEM), XV-edition, ICECHIM-Bucharest, 30.10-1.11.2019, Book of Abstracts, ISSN 2285 8334, MDPI Proceedings, 29(1), 107, https://doi.org/10.3390/proceedings2019029107 (poster)
3. Diana Constantinescu-Aruxandei, Ioana-Cristina Marinas, Carmen-Valentina Nicolae, Luminita Dimitriu, Florin Oancea. Profitable Breakage – Enzymatic Assisted Extraction of Bioactive Compounds Trapped Inside Plant Food By-Products (submitted)

Phase 3

Title: Demonstration of functionality of experimental models and technologies

Period: 01.01.2020 – 30.06.2020

The Objective of phase 3 was to demonstrate the functionality of experimental models (EMs). The activities corresponding to the objective of phase 3 were the following:

A3.1 Demonstration of functionality of EMs for the preparation, characterization and selection of binary and ternary NADESs compatible with enzymes, used for cascade processing of lignocellulose from BSG

A3.2 Demonstration of functionality of EMs for cascade processing of lignocellulose from BSG

A3.3 Dissemination and communication of results

The estimated results were:

• Up-to-date website
• Functional EMs
• Eco-biotechnological concept demonstrated at experimental level for lignocellulose valorisation
• 2 papers submitted for publication in ISI Journals
• Intermediate and final report

Summary of the scientific and technical report for phase 3

Within phase 3 of project INTELIGRENCE the functionality of 3 EMs was demonstrated: (1) preparation, characterization and selection of new NADESs compatible with feruloyl esterase (FAE) and having the ability to extract ferulic acid from biomass; (2) preparation, characterization and selection of new NADESs for cellulose valorization from biomass; (3) NADESs utilization for cascade processing of lignocelluloses substrate from BSG.

Within EM 1, some NADESs reported in previous phases to significantly solubilise ferulic acid and to be compatible with FAE were prepared and characterized again, confirming the reproducibility of the method of preparation. These NADESs were mainly based on alcohols. Moreover, the list was extended with other NADESs based on choline chloride and alcohols, in order to understand the mechanism of ferulic acid solubilisation better and to find the best NADES. NADES based on choline chloride and 1,4 butandiol was chosen for the enzymatic extraction of ferulic acid from BSG.

Within EM 2, the reproducibility of lignin solubilisation in chosen NADESs was tested. This was based on previous observations that lignin is highly soluble in certain NADES while cellulose is poorly soluble. Therefore, these NADESs can separate lignin from cellulose (or holocellulose) and cellulose can be further purified. The best NADES was chosen for lignin extraction from BSG

Within EM 3, the reproducibility of using NADESs for ferulic acid and cellulose extraction from BSG was tested in a cascade approach. In order to improve the FAE activity, the enzyme was combined with other enzymes, such as cellulases, hemicellulases and lytic polysaccharide monooxygenases (LPMOs). The substrate was further delignified in an optimized process, using Response Surface Methodology (RSM) and the optimal condition was used before cellulose purification. The BSG substrate, lignin and purified cellulose were characterized by Scanning Electron Microscopy (SEM), FT-IR and X-ray Difrraction (XRD), which showed morphological changes in the substrate during processing and confirmed the structure of extracted lignin and cellulose. All extractions were performed in triplicate and the standard deviations were calculated. The yield and product quality of the proposed cascade process were compared with classical processes for cellulose extraction and the yield was comparable. The obtained cellulose was transformed to nanocellulose using the high pressure from a Microfluidizer, and the formation of nanocellulose was confirmed by electron microscopy.

The objective of phase 3 was achieved. The functionality of the following EMs was demonstrated: (EM1) for the preparation of NADESs compatible with FAE and high capacity to solubilise ferulic acid in order to enzymatically extract it from the lignocellulose matrix; (EM2) for the preparation of NADESs with high degree of lignin solubilisation, efficient for lignin separation from cellulose; (EM3) cascade extraction of ferulic acid, lignin and cellulose using enzymes (FAE, cellulases, etc) and NADES.

Moreover, the website was updated, the results were disseminated by 2 oral presentations at a National Exploratory Workshop and two scientific papers were submitted for publication:

1. Alina Negoi, Carmen Valentina Nicolae, Ioana-Cristina Marinas, Marius Ghiurea, Alina Nicolescu, Sanda-Maria Doncea, Diana Constantinescu-Aruxandei, Florin Oancea.  Ecofriendly extraction of polysaccharides from brewer’s spent grain with deep eutectic solvents. Exploratory Workshop “Innovative Trans-Sectorial Technologies” (NeXT-Chem)-2^nd edition, ICECHIM-Bucharest, 18.-19.06.2020, (oral presentation)
2. Daniel Preda, Alina Negoi, Ioana-Cristina Marinas, Diana Constantinescu-Aruxandei, Florin Oancea, Adina Raducanu. Utilization of laccases in the presence of deep eutectic solvents for degradation of lignocellulosic biomass. . Exploratory Workshop “Innovative Trans-Sectorial Technologies” (NeXT-Chem)-2^nd edition, ICECHIM-Bucharest, 18.-19.06.2020, (oral presentation)
3. Ioana-Cristina Marinas, Alina Negoi, Daniel Preda, Alina Nicolescu, Diana Constantinescu-Aruxandei, Florin Oancea. Mechanistic insight into ferulic acid solubilization by polyol- choline chloride deep eutectic solvents and their aqueous mixtures (submitted)
4. Alina Negoi, Bogdan Trica, Ioana-Cristina Marinas, Marius Ghiurea, Daniel Preda, Alina Nicolescu, Diana Constantinescu-Aruxandei, Florin Oancea. Cascading conversion of spent brewery grains into antioxidants, oligosaccharides, proteins and nanocellulose (submitted).

Conclusions

      The project INTELIGRENCE managed the development of an efficient extraction biotechnology of some bioproducts of economic importance (ferulic acid, lignin, cellulose) from BSG and proposed an eco-biotechnological concept, demonstrated at experimental level, for lignocellulose valorisation in general, based on enzymes and NADES. The project estimated results, e.g. website, functional EMs, 1 Patent application, 3 papers submitted, intermediate and final scientific reports were fulfilled.