PN-III-P1-1.1-TE-2019-1179
PROJECT CODE: PN-III-P1-1.1-TE-2019-1179, contract number TE 94 ⁄ 2020
PROJECT TITLE:
New sol-gel-magnetic biocatalysts used for the enzimatic hydrolysis of lignocellulosic biomass
Noi biocatalizatori sol-gel-magnetic utilizati pentru hidroliza enzimatică a biomasei lignocelulozice
ACRONYM: BIOCATLIGNOCELL
PROJECT COORDINATOR: Politehnica University Timişoara
PROJECT DIRECTOR: Lecturer dr. eng. Ana Cristina Paul

BUDGET: 431.900 RON (Public budget)
START TIME: 15/09/2020
END TIME: 14/09/2022

PROJECT LAYOUT
The major cause of environmental pollution is due to emissions generated by burning of fossil fuels. The known crude oil reserves are going to disappear in short time and the oil crisis in recent years, together with the rising of air pollution levels has shown the need for the replacement of fossil fuels with cleaner biofuels, obtained by processing a range of organic renewable raw materials. The first step in conversion of lignocellulosic biomass to bioethanol is the pretreatment for the release of cellulose from the network formed with lignin and to increase the yield of fermentable sugars. There are many methods of pretreatment, but they are energy consumable and pollute the environment.
In this sense the project proposes an innovative approach on studies regarding biomass pretreatment and enzymatic hydrolysis of cellulose in an integrated system that can improve the exploitation of biomass components and the reuse of the biocatalyst. It is desired to provide novel biocatalysts, immobilized cellulases customized by new sol-gel entrapment techniques, used in the hydrolysis of certain types of lignocellulosic biomass. By immobilization, stability and reusability of cellulases are significantly improved, a key issue for increasing the amount of fermentable sugars and to reduce process costs.

PROJECT TEAM:
Project leader: Lecturer dr. eng. Ana Cristina Paul
Postdoctoral researchers:
Lecturer dr. eng. Gerlinde Rusu
Lecturer DVM Simona Marc
RA chem. Corina Vasilescu (PhD student)

OBJECTIVE:
The main objective of the project is the obtaining of new immobilized enzymatic biocatalysts, customized by new sol-gel entrapment techniques, used for the hydrolysis of certain types of lignocellulosic biomass.

STAGES
Stage 1 (2020, 4 months)- Pretreatment of lignocellulosic biomass using unconventional methods.

Stage 2 (2021, 12 months)- Immobilization of cellulases using new sol-gel entrapment technics and structural and operational characterization of the obtained biocatalysts.

Stage 3 (2022, 8 months)- Enzymatic hydrolysis of the cellulose from biomass and biocatalyst reuse.

DELIVERABLES:
Stage 1
Lignocellulosic biomass pretreatment protocol.
Optimized lignocellulosic biomass pretreatment protocol.
Stage 1 report.

Stage 2
Immobilization protocol for cellulases.
Optimized immobilization protocol for cellulases using sol-gel technique.
Multigram quantities of biocatalysts with well-defined characteristics.
Stage 2 report.
Abstract for one presentation at scientific conference.
A scientific article submitted for publication / accepted in the journal with impact factor indexed in the Web of Science database.

Stage 3
Optimized protocol for enzymatic hydrolysis of lignocellulosic materials.
Technological laboratory process for enzymatic hydrolysis of cellulose from pre-treated biomass.
Stage 3 report.
Final report.
Abstract for one presentation at scientific conference.
A scientific article submitted for publication / accepted in the journal with impact factor indexed in the Web of Science database.

RESULTS 2020-2022

Abstract Stage 1 (2020) - Pretreatment of lignocellulosic biomass using unconventional methods.

At this stage, a series of research was carried out regarding: (Activity 1.1.) comparative evaluation of different pretreatment techniques; the optimization of the enzymatic hydrolysis conditions of Avicel microcrystalline cellulose with various commercial cellulases (experimental design) and (Activity 1.2.) the establishment of the optimal conditions for the selected pretreatment technique (experimental design).

For this purpose, five types of lignocellulosic biomass were chosen (hardwood - oak, softwood - fir, wheat straw, cardboard and mixture in equal parts of the four types of biomass), which were subjected to pretreatment methods: physical (hot water, ultrasound, hot water combined with ultrasound), chemical (acetic acid 50 %) and unconventional solvents (ionic liquid - 1-ethyl-3-methylimidazolium acetate [Emim]Ac). As a result, five pretreatment protocols applied to lignocellulosic biomass were developed.

The pretreated biomass was subjected to enzymatic hydrolysis, and in order to find the optimal conditions, an experimental design of the Avicel microcrystalline cellulose enzymatic hydrolysis reaction was was carried out with two commercial cellulase preparations (Accellerase 1000 and Cellic CTec2), using as a method: Response Surface (RSM, Response Surface Method) and the full factorial design, Experiment Central Composite Design (CCD, Central Composite Design).

The research regarding the establishment of optimal pretreatment conditions involved an experimental design in order to optimize the pretreatment conditions using the ultrasound method applied to lignocellulosic biomass from wheat straw. The response surface method (RSM, Response Surface Method) and the Behnken Box design were used, and thus the optimal ultrasound pretreatment parameters were established to be: 25°C, 5% biomass (w/v) and 48 min. Under the optimal conditions of ultrasound pretreatment of wheat straw, a hydrolysis yield of 88.93% was obtained by enzymatic hydrolysis with native cellulase Cellic CTec2. Based on these results, an optimized ultrasound pretreatment protocol of wheat straw biomass was developed.

Overall, the objectives of this stage have been achieved.

Abstract Stage 2 (2021) - Immobilization of cellulases using new sol-gel entrapment techniques and structural and operational characterization of the obtained biocatalysts.

In the second stage, the main scientific objectives were: (Activity 2.1.) obtaining immobilized cellulases using simple and combined sol-gel entrapment methods, (Activity 2.2.) optimization of the parameters that influence the immobilization, the structural characterization, and the operational stability study of the immobilized cellulases, (Activity 2.3.) structural characterization of immobilized cellulases, and (Activity 2.4.) operational stability study of immobilized cellulases.

To fulfill the objectives of this stage, a series of research aimed at the immobilization of the commercial cellulase Cellic CTec2 through entrapment techniques in sol-gel matrices, which provides an improvement in the enzyme's catalytic performance, operational stability and an increase in the quantity of fermentable sugars from the biomass, has been carried out. The immobilization methodology was developed through new simple sol-gel entrapment techniques (Method I – sol-gel and Method II – prepolymer sol) and combined with adsorption on magnetic supports (Method III – magnetic sol-gel), being achieved sol-gel matrices for binary and ternary systems of silane precursors. The activity of the obtained biocatalysts was evaluated in the hydrolysis reaction of a standard substrate, obtaining promising results in the perspective of using these enzymatic preparations for the hydrolysis of lignocellulosic biomass.

Research on the establishment of optimal immobilization conditions by sol-gel entrapment techniques involved the study of the influence of the nature of the silane precursors, the immobilization additives, and the immobilization technique used on the catalytic efficiency of the immobilized cellulase. The biocatalyst obtained was also tested in the enzymatic hydrolysis of Avicel microcrystalline cellulose Avicel.

The characterization of the selected immonilized biovatalyst was carried out by scanning electron microscopy, fluorescence microscopy FM, FT-IR spectroscopy, and TG/DTG thermal analysis, and a correlation of the enzymatic activity of the obtained biocatalysts with the morphology of their sol-gel matrix was also carried out. The presence and distribution of the enzyme in the sol-gel matrix, as well as the mass loss profile as a function of temperature, were highlighted.

The operational stability study of immobilized cellulases (thermal stability, stability at different pH values as well as stability in solvents), was also carried out, the immobilized biocatalysts proving their superiority compared to the native, non-immobilized cellulase. Based on the results, two optimized cellulase immobilization protocols were developed.

The dissemination of the results was achieved by preparing a paper for publication in a journal with a high impact factor and participating in four scientific conferences in the field. Overall, we appreciate that all the objectives of this stage have been accomplished.

Since the scientific results from this stage have not yet been fully published, they cannot be made public!

Abstract Stage 3 (2022) - Enzymatic hydrolysis of the cellulose from biomass and biocatalyst reuse.

In this stage, studies were made regarding: (Activity 3.1.) determination of optimal conditions for enzymatic hydrolysis of lignocellulosic material and (Activity 3.2.) increasing the efficiency of enzymatic hydrolysis by reusing immobilized cellulases .

Research on the establishment of optimal conditions for the enzymatic hydrolysis of cellulose and wheat straw biomass involved the study of the influence of the nature of the biocatalyst, the study of the time evolution of the enzymatic hydrolysis of microcrystalline cellulose, the thermal and pH influence on the enzymatic hydrolysis of cellulose by immobilized Cellic CTec2 biocatalysts by entrapment in sol-gel and magnetic sol-gel. On the basis of these results, a protocol for the enzymatic hydrolysis of pretreated wheat straw biomass under optimal conditions was developed.

Studies on the reuse of the new magnetic sol-gel biocatalysts in the enzymatic hydrolysis reaction of carboxymethylcellulose, microcrystalline cellulose, and pretreated lignocellulosic biomass from wheat straw have also been carried out. The new sol-gel magnetic biocatalysts were reused in seven successive batch hydrolysis cycles of pretreated wheat straw biomass with good values of remanent activity and sugar productivity, thus obtaining promising results for scaling up the process. On the basis of these results, a Laboratory Technological Process was developed for the enzymatic hydrolysis pretreated wheat straw biomass.

Dissemination of the results was achieved by preparing a paper for publication in a journal with a high impact factor and participating in three scientific conferences in the field. Consequently, we appreciate that all the objectives of this stage have been accomplished.

Since the scientific results from this stage have not yet been fully published, they cannot be made public!

Publications:

1. C. Vasilescu, C. Paul, S. Marc, I. Hulka, F. Péter, Development of a tailored sol-gel immobilized biocatalyst for sustainable synthesis of the food aroma ester n-amyl caproate in continuous solventless system,  Foods, 2022, 11(16), 2485, https://doi.org/10.3390/foods11162485, WOS:000846142000001, eISSN 2304-8158, Impact factor 5.561 (2021), JCR - Q1.
2. Vasilescu, S. Marc, I. Hulka, C. Paul, Enhancement of the catalytic performance and operational stability of sol-gel entrapped cellulase by tailoring the matrix structure and properties, Gels, 2022, 8(10), 626, https://doi.org/10.3390/gels8100626, WOS:000875190400001, eISSN 2310-2861 Impact factor 4.432 (2021), JCR - Q1.

Conferences:

1. Vasilescu, S. Marc, S. Liga, C. Paul, Comparative study and optimization of lignocellulosic biomass pretreatment methods, 15^th International Symposium on Biocatalysis and Biotransformations (Biotrans 2021 - Online), July 19-22, 2021, Graz, Austria.
2. Vasilescu, C. Paul, F. Péter, Tailoring the sol-gel entrapment method of enzymes by design of experiments for solvent-free ester synthesis, 15^th International Symposium on Biocatalysis and Biotransformations (Biotrans 2021 - Online), July 19-22, 2021, Graz, Austria.
3. Vasilescu, S. Marc, G. Scheuleac, C. Paul, Sol-gel entrapment of cellulases for the enzymatic hydrolysis of lignocellulosic biomass to fermentable sugars, 13^th European Congress of Chemical Engineering and 6^th European Congress of Applied Biotechnology (ECCE&ECAB 2021, virtual event), September 20-23, 2021, Berlin, Germany.
4. Miloș. M. Gheorghe, C. Vasilescu, F. Péter, C. Paul, Batch and continuous flow aroma ester synthesis by sol-gel entrapped Candida antarctica lipase B, The 13^th online edition of symposium with international participation, New trends and strategies in the chemistry of advanced materials with relevance in biological systems, technique and environmental protection, October 7-8, 2021, Timisoara, Romania.
5. Paul, S. Marc, C. Vasilescu, Structural characterization and catalytic activity of sol-gel entrapped cellulase Cellic CTec2, 18^th International Conference on Renewable Resources & Biorefineries (RRB-18), June 1-3, 2022 Bruge, Belgium.
6. Vasilescu, R. Subulescu, S. Marc, F. Péter, C. Paul, High stability sol-gel entrapped enzymes for biocatalysis, Sol-Gel Conference, July 24-29, 2022, Lyon, France.
7. Vasilescu, S. Marc, F. Péter, C. Paul, Optimized continuous-flow aroma ester synthesis by entrapped Candida antarctica lipase B in novel sol-gels with epoxy functional groups, 12^th Edition of Global Conference on Catalysis, Chemical Engineering & Technology (CAT 2022 – Online Event), September 5-7, 2022, Paris, France.

Summary of the project results

The project “New sol-gel-magnetic biocatalysts used for the enzymatic hydrolysis of lignocellulosic biomass” targeted as main objective the development of new immobilized enzyme biocatalysts, by sol-gel entrapment techniques, customized for the hydrolysis of certain types of lignocellulosic biomass.

The first step in the conversion of lignocellulosic biomass to valuable industrial products (as bioethanol) is  pretreatment for the release of cellulose from the network formed with lignin and to increase the yield of fermentable sugars. Thus, a series of research was carried out regarding the application of different non-conventional pretreatment methods on five types of lignocellulosic biomass, the optimization of the enzymatic hydrolysis conditions with commercial cellulases (experimental design), and the establishment of the optimal conditions for the ultrasound pretreatment technique applied to lignocellulosic biomass from wheat straw (experimental design).

To fulfill the main objective of the project, commercial cellulase Cellic CTec2 was immobilized by the entrapment technique in sol-gel matrices, and sol-gel entrapment with deposition onto magnetic nanoparticles, using binary or ternary systems of silane precursors with alkyl- or aryl-trimethoxysilanes, at different molar ratios. Tailoring of the immobilization parameters, such as the nature and ratio of the silane precursors, the nature of the immobilization additives, and the protein loading, allowed the optimization of the catalytic properties of the sol-gel and magnetic sol-gel biocatalysts. The best immobilized biocatalysts were obtained with a ternary silane precursor system consisting of PhTMOS:VTMOS:TMOS at 1.6:0.4:1 molar ratio, and also with deposition onto magnetic particles.

A correlation of the catalytic activity with the properties of the sol-gel matrix of the nanobiocatalysts was carried out using several characterization methods: scanning electron microscopy (SEM), fluorescence microscopy (FM), Fourier-transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA/DTA). The homogeneous distribution of the enzyme in the sol-gel matrix and the mass loss profile as a function of temperature were highlighted.

The influence of temperature and pH of the reaction medium on the catalytic performance of the nanobiocatalysts as well as the operational stability under optimized reaction conditions were also investigated, the immobilized biocatalysts proving their superiority compared to the native cellulase.

The experimental studies were carried out in the Biocatalysis Laboratory at the Politehnica University of Timișoara, and the characterization studies of the new enzymatic biocatalysts were carried out at: the Research Institute for Renewable Energies (ICER) from Politehnica University of Timișoara, the Center for Fundamental and Advanced Technical Research, Laboratory of Magnetic Fluids from the Romanian Academy Timișoara Branch and the Horia Cernescu Complex of Research Laboratories within the University of Life Sciences ‘’King Mihai I’’ from Timișoara.

The new sol-gel magnetic cellulase biocatalysts with the highest efficiency were reused in seven successive batch hydrolysis cycles of pretreated wheat straw biomass with good values of remanent activity and sugar productivity, thus obtaining promising results for scaling up the process.