PROJECT CODE: PN-III-P4-ID-PCE-2020-2177, contract number PCE 157/ 16.02.2021
Sustainable routes for carbohydrate-based biosurfactants in green reaction media
Biosurfactanți pe bază de hidrați de carbon obținuți prin căi de sinteză sustenabile în medii de reacție ecologice
ACRONYM:                              GreenBioSurfactant
PROJECT COORDINATOR:     Polytehnic University of Timişoara
PROJECT DIRECTOR:             Dr. Eng. Carmen Gabriela Boeriu
BUDGET:                                 1,198,032.00 RON (Public budget)
START DATE:                          15-02-2021
END DATE:                              14-02-2024


There is continuously growing interest in the development of biodegradable, renewable, and less toxic biosurfactants. Sugar fatty acid esters (SFAEs) are carbohydrate derivatives demonstrating excellent emulsifying properties and foaming ability, which can be used in the food industry, in cosmetics, detergents, pharmaceuticals and agriculture. The aim of this project is to develop and optimise an innovative and sustainable process and solvent system for the synthesis of new glycolipids. For this reason, the biocatalytic synthesis of novel fatty esters of oligomeric sugars will be carried out in natural deep electric solvents (NADES) and the optimal solvent composition, reaction conditions, enzyme recovery possibilities and downstream processing parameters will be determined. The biobased glycolipid surfactants will be characterized in detail by several analytical techniques for structure confirmation, assessment of the physico-chemical and surfactant properties, in view of possible applications in food and other sectors.


Project leader: Dr. Eng. Carmen G. Boeriu
Prof. Dr. Eng. Francisc Peter
Dr. Eng. Ramona Buzatu
Ș.l. Dr. Eng. Cristina Paul
Ș.l. Dr. Eng. Iulia Maria Păușescu
Ș.l. Dr. Eng. Valentin Badea
Asist. Dr. Chem. Diana Maria Dreavă
Eng. Ioan Bîtcan

Project objectives: The key objective of the project is to develop an innovative and sustainable process and solvent system for the synthesis of new sugar ester biosurfactants with possible food applications. Therefore, the project activities will be focused on two main directions: (i) reaction engineering of the biocatalytic synthesis a selected sugar ester using different NADES, to determine the optimal solvent composition, reaction conditions, enzyme recovery possibilities and downstream processing parameters; (ii) synthesis of at least three different novel biosurfactants using oligomeric sugars and fatty acid derivatives

Project phases:
Phase 1 (2021, 10.5 months) - Development of suitable natural deep eutectic solvent (NADES) systems and obtainment of sustainable and robust biocatalysts with catalytic activity in NADES
Phase 2 (2022, 12 months) – Synthesis of fatty acid esters of carbohydrate oligomers (SFAE) and engineering of the biocatalytic reaction
Phase 3 (2023, 12 months) – Characterization of biosurfactants
Phase 3 (2024, 1.5 months) – Determination of biosurfactant properties of the SFAEs obtained


Project Results:

Phase 1/2021: Development of suitable natural deep eutectic solvent (NADES) systems and obtainment of sustainable and robust biocatalysts with catalytic activity in NADES

Summary of results

In the first year of the project, we have studied the preparation of a large number of binary and ternary natural deep eutectic solvents (NADES) consisting of selected natural, biobased compounds. An operational protocol was developed for the preparation of NADES derived from mono- and disaccharides. The thermal properties of nine of the obtained NADES systems were determined using thermal analytical methods, such as TGA and DSC. Next studies focused on the screening and selection of biocatalysts for esterification reactions in NADES. Four most active lipases were selected from a set of thirteen commercial native lipases, based on their efficiency in the model esterification reaction between lauric acid with 1-propanol. The four most active lipases were utilized as biocatalysts for the synthesis of esters of lauric acid with glucose in various solvent systems, such as mixtures of  organic solvents and NADES. TLC, HPLC, MALDI-TOF-MS were used to monitor the progress of the reactions and to identify and characterize the products of the reactions. Sugar esters were also obtained using an commercial immobilized lipase as catalyst. The effect of reactions conditions (e.g. pressure and  stirring) on product formation was investigated. Next, the immobilization of two most efficient lipases by entrapment in sol-gel matrices was investigated. Nine catalytically-active immobilized lipase preparations were obtained using various silane precursors in different combinations. The operational stability of all 13 biocatalysts (i.e. four native and nine immobilized) was evaluated in six successive reaction  cycles; two of them showed high activity and stability in the NADES prepared in the project. Results of the project were disseminated in an scientific article (ISI) and two communications (one oral presentation and one poster) presented at two major international conferences. In conclusion, all objectives of this first phase of the project are achieved.


1. Todea, A.; Dreavă, D.M.; Benea, I.C.; Bîtcan, I.; Peter, F.; Boeriu, C.G. Achievements and trends in biocatalytic synthesis of specialty polymers from biomass-derived monomers using lipases. Processes 2021, 9, 646.

2. Buzatu, A.R.; Frissen, A. E.; van den Broek, L. A. M.; Todea, A.; Motoc, M.; Peter F.; Boeriu C. G. Biocatalytic synthesis of phenolic sugar esters. 2021. Abstract and poster presented at the 7th International Polysaccharide Conference, 11-15 October, Nantes, France

3. Boeriu, C.G., Van der Vlist, V., Aparaschivei, D.,  Frissen, A.E., Todea, A., Peter, F. Enzymatic synthesis of novel biobased aliphatic-aromatic copolyesters. 2021. Abstract and Oral Presentation presented at the 6th International Conference on Biocatalysis in Non-Conventional Media (BNCM 2021), 6-8 May 2021, Milan, Italy

Phase 2 /2022: – Synthesis of fatty acid esters of carbohydrate oligomers (SFAE) and engineering of the biocatalytic reaction

Summary of results

In the second phase of the project, we continued the studies on the stability of native and immobilized lipases when suspended in natural deep eutectic solvents (NADES).  For each NADES prepared, we determined the thermal properties by thermographic analysis (TG) and differential scanning calorimetry (DSC) and we measured their viscosity in a temperature ranging between 40-80°C. Thermogravimetric analysis showed a high thermostability of all NADES tested. Differences in thermostability were observed based on the composition of the eutectic mixtures. Explicitly, the decomposition temperature was the highest, e. g. 250°C for carbohydrate polyols-based NADES and decreased to 200°C for carbohydrate-based NADES, and to 150°C for the NADES containing urea and methylurea, respectively. 

From the 13 native lipases tested, four of the most active were selected for further characterization. The four enzymes displayed a relatively high thermostability in NADES and followed different models of thermal inactivation in time upon incubation at 70°C. We observed that at 24 h incubation lipase A from Candida antarctica (CalA) and the lipase from Aspergillus oryzae (A. oryzae) maintained 90% of the activity, while the lipase B from C. antarctica (CalB) and the lipase from Pseudomonas stutzeri (Ps. stutzeri) showed only 60% of the initial activity.

Further studies addressed the possibility to obtain esters of carbohydrates and carbohydrate polyols with fatty acids and fatty acid derivatives. During these studies, eight different NADES containing carbohydrates or carbohydrate polyols-based were treated with lauric acid in the presence of selected lipase catalysts. HPLC analysis showed conversion of lauric acid in the range of 10-23 mol%. In the case of glucose lauric acid esters, the highest conversion was obtained in reactions using the ternary glucose based ChCl:glucose:water 2:1:1 NADES.

Significant from the point of view of the engineering of the reactions studied, we investigated the effect of reaction parameters on the synthesis of lauryl esters of arabitol with the use of design of experiments (DOE). The results showed temperature (°C) and the quantity of enzymes are decisive parameters for the increase of substrate conversion, while the molar substrate ratio, e.g. lauric acid/arabitol is not significant. The model was validated with a preparative synthesis reaction carried out at the optimal conditions predicted by the model to achieve a conversion of lauric acid of 80 mol%, after 24 hours incubation. The product of the reaction was isolated by extraction in tetrahydrofuran and the major product was identified as 1,5-dilauryl-D-arabitol (1,5-DLDA).

The results were disseminated by publication of one scientific paper in an ISI journal, and by presentation at 5 international conferences, of which three were held abroad and two in Romania.



  1. Bîtcan, I., Petrovici, A., Pellis, A., Klébert, S., Károly, Z., L. Bereczki, L., Péter, F., Todea, A. Enzymatic route for selective glycerol oxidation using covalently immobilized laccases, Enzyme and Microbial Technology 163 (2023) 110168 


  1. Buzatu, A.R., Bîtcan, I., Dreavă, D.M., Todea, A., Peter, F., Boeriu, C.G. Reactive natural deep eutectic solvents for lipase catalyzed synthesis of fatty acid esters of carbohydrates and polyols. The 13th International Symposium of the Romanian Catalysis Society, RomCat 2022, 22 – 24 June 2022, Băile Govora, Romania. Oral presentation.
  2. Boeriu, C.G. A short history of biocatalysis: from dreams to industrial reality. The 13th International Symposium of the Romanian Catalysis Society, Summer School „Catalysis for Promising Future”, June 20-21, 2022, Băile Govora, Romania. Invited lecture.
  3. Buzatu, A.R., Bîtcan, I., Dreavă, D.M., Todea, A., Peter, F., Boeriu, C.G. Sustainable synthesis of carbohydrate-based surfactants in reactive natural deep eutectic solvents. 5th International Conference on Green Chemistry and Sustainable Engineering, 20 – 22 July 2022, Rome, Italy, pp. 066, ISBN 978-84-09-42560-0. Poster.
  4. Buzatu, A.R., Bîtcan, I., Dreavă, D.M., Todea, A., Peter, F., Boeriu, C.G. Enzymatic esterification of carbohydrates in reactive natural deep eutectic solvents. 10th International Congress on Biocatalysis 2022, Biocat 2022, 28 August – 1 September 2022, P3-12, pp 120, Hamburg, Germany. Poster + Lightening talk.
  5. Boeriu, C.G., Buzatu, A.R., Bîtcan, I., Dreavă, D.M., Todea, A., Peter, F., . Reactive natural deep eutectic solvents as essential reaction media for lipase catalyzed carbohydrate esterification, 9th IUPAC International Congress on Green Chemistry, 9th ICGC, 5 – 9 September 2022, Athens, Greece, O-168. Oral presentation.
  6. Buzatu, A.R., Bîtcan, I., Dreavă, D.M., Todea, A., Peter, F., Boeriu, C.G. Lipase catalyzed production of sugar fatty acid esters in reactive natural deep eutectic solvents, The 14th Edition of the Symposium with International Participation - New Trends And Strategies In The Chemistry Of Advanced Materials With Relevance In Biological Systems, Technique and Environmental Protection, 20-21 October 2022, Timișoara, România. Oral presentation.

Phase 3/2023: Characterization of the newly synthesized biosurfactants

Summary of results

In the third phase of the project, we continued with the studies initated during the 2nd phase addressing: (a) testing the suitability of selected oligosaccahrides as substrates for enzymatic esterification with fatty acids and fatty acid derivatives; (b) determination of the optimal reaction conditions for the synthesis of fatty acid esters of carbohydrates, including glucose, sucrose and low moelcular weight inulin oligomers in carbohydrate-based reactive deep eutectic solvents (RNADES). A broad range of binary and ternary RNADES containing choline chloride (HBA) and glucose, sucrose and inulin as hydrogen bond donor, respectively, with or without added water, have been prepared and characterized. Three glucose- and nine sucrose-based RNADES showed low viscosity and were compatible with CalB and TL-immobilized lipases, which maintaned high catalytic activity and operational stability in these solvents. The lipase-catalyzed esterification of glucose and sucrose with lauric acid in selected RNADES proceeded with conversion of lauric acid of  5% and 13%, respectively. HPLC, mass spectrometry (Maldi-Tof-MS, ESI-MS) and spectroscopic analysis (i.e. FTIR, 1H-RMN, 13C-RMN, 2D-RMN, HMQC, HMBC), confirmed the formation of mono- and di-lauryl esters of the carbohydrates, next to small amounts of choline monolaurate. The lauryl esters of glucose, sucrose and inulin, with values of the hydrophylic - lypophylic balance (HLB) in the range 8-10, are ideally suited to application as oil/water (O/W) emulsifiers. They also form very stable foams and emulsions, that was also

The esterification of carbohydrate polyols like D-arabitol, xylitol and D-sorbitol with lauric acid, palmitic acid and stearic acid, catalyzed by immobilized CalB (LAR) in the corresponding RNADES reaction mixtures produced exclusively the 1,5- and 1,6-diesters, as confirmed from structural analysis, carried out by mass spectrometry, FTIR and NMR spectroscopy, as detailed above. The reactions proceeded with high fatty acid conversion, ranging from 56 mol% for 1,5-dilauryl-xylitol, to 87 mol% for 1,5-dipalmityl-D-arabitol. The purified polyol esters are non-dispersible in water and have low HLB values, ranging from 4.4 for 1,5-distearyl-D-arabitol and 6.6 for 1,6-dilauryl sorbitol, and therefore are suited as water/oil (W/O) emulsifiers.


The results obtained were disseminated by (a)  two publications in high impact scientific journals (quartal Q1 at publication time); and (b) participation with two oral presentations and one poster at three renowned dedicated international conferences, all organized with physical presence.



  1. Buzatu, A.R., Soler, M.A., Fortuna, S., Ozkilinc, O., Dreavă, D.M., Bîtcan, I., Badea, V., Giannozzi, P., Fogolari, F., Gardossi, L., Peter, L., Todea, A., Boeriu, C.G. Reactive natural deep eutectic solvents as essential reaction media for lipase catalyzed esterification of carbohydrate polyols. Catalysis Today 426, 2024, 114373.
  2. Buzatu, A.R., Todea, A., Peter, F., Boeriu, C.G. Reactive Natural Deep Eutectic Solvents and their Multifunctional Positive Impact on Biocatalytic Syntheses. ChemCatChem, 2024, e202301597.


  1. Buzatu, A.R., A. Todea, D. M. Dreavă, V. Badea, F. Peter, C. G. Boeriu. Effective lipase catalyzed synthesis of lauryl esters of carbohydrate polyols in reactive natural deep eutectic solvents. 19th International Conference on Renewable Resources and Biorefineries, Riga, Latvia, 31 May-2 June 2023, oral presentation.
  2. F. Peter, A. R. Buzatu, A. Todea, D. M. Dreavă, V. Badea, C. G. Boeriu, Efficient lipase catalyzed esterification of carbohydrate polyols in reactive natural deep eutectic solvents. 16th International Symposium on Biocatalysis & Biotransformations (BIOTRANS), La Rochelle, France, 25-29 June 2023, poster.
  3. Boeriu, C.G., Buzatu, A.R., Todea,A., Dreavă, D.M., Bȋtcan, I., Badea, V., Peter.F. Efficient lipase catalyzed synthesis of biosurfactants in reactive natural deep eutectic solvents. 6th EuChemS Conference on Green and Sustainable Chemistry (EuGSC), Salerno, Italy, 3-6 september 2023, oral presentation.


Phase 3/2024: Characterization of the newly synthesized biosurfactants

Summary of results

In the fourth phase of the project, with a duration of six weeks, we focused on completing the functional characterization of the new biosurfactants produced in the project using the novel reactive deep eutectic solvents obtained and the innovative synthetic method developed. In this context, we evaluated the efficiency of the di-lauryl esters of arabitol (DLA) for stabilization of water/oil emulsions. The results showed that mixing of a fine dispersion of 0.1% DLA in sunflower oil in different ratios, stable emulsions are formed. Microscope evaluation showed formation of small droplets and stable emulsions. These results suggest that the polyol esters could find applications in the food and material industry.  

In this period, we also paid attention to dissemination of our work by publication. Results obtained in the last months are the topic of two manuscripts, that are currently in the final stage of preparation, and will be submitted before April 2024 for publications in two Q1 journals. 

Manuscripts for submission:

M1. Buzatu, A.R., Todea, A., Dreavă, D.M., Paul, C., Bîtcan, I., Motoc, M., Peter, P., Boeriu, C.G. Design reactive natural deep eutectic solvents enhance esterification activity and thermal stability of lipases. International Journal of Molecular Sciences, 2024, to be submitted

M2. Buzatu, A.R., Bîtcan, I., Soler, M.A., Fortuna, S., Ozkilinc, O., Giannozzi, P., Fogolari, F., Peter, F., Todea, A., Boeriu, C.G. Choline ester is the main product of lipase catalyzed esterification of a choline chloride/glucose reactive NADES, and not the anticipated glucose ester. Green Chemistry (with invitation), 2024. To be submitted.


Summary of the project results

The project “Sustainable routes for carbohydrate-based biosurfactants in green reaction media” developed an innovative and sustainable route and green and renewable solvent systems to produce sugar fatty acid esters (SFAEs), an important class of green biosurfactants. SFAEs have excellent emulsifying properties and foaming ability, and can be used in food ingredients, in cosmetics, detergents, pharmaceuticals and agrochemicals. Using renewable, natural raw materials and enzymes as catalysts, the current requirements and standards of the circular bioeconomy are met: the synthesis pathways are green, the products are nature-like and biodegradable, and have the same properties as the currently used chemically SFAEs. The experimental work was carried out in the Laboratory of Biocatalysis at the Politehnica University of Timişoara.

The green solvents developed were reactive natural deep eutectic mixtures (R-NADES(, that have a double function, as reaction medium and source of the substrate reagent. 32 binary and ternary choline chloride-based were prepared and characterized, most of them being new eutectics, not reported before.  The enzyme used were microbial lipases, native and immobilized. Both native and immobilized lipase showed catalytic activity in R-NADES, however the immobilized enzymes performed better, showing extremely high thermal and operational performance. After optimization of process parameters by experimental design (DOE), several esters of C12, C16 and C18 fatty acids, with polyols (i.e. xylitol, D-arabitol and D-sorbitol) and carbohydrates (i.e. glucose, sucrose, and inulin oligosaccharides DP 2-9) were obtained and characterized. All synthesized products were characterized by 1D NMR and 2D NMR, MALDI-TOF-MS and ESI-MS spectrometry, differential scanning calorimetry (DSC) and HPLC, to determine their structure, purity and physical and properties. For the first time, it was demonstrated the high regioselectivity of the lipase catalyst for the end-hydroxyl groups of polyols and the exclusive formation of α, ω-diesters of polyols. Functional characterization showed that fatty acid esters of polyols and carbohydrates are stabilizers of water/oil and oil/water emulsion, respectively. Fatty acid esters of carbohydrates are good foaming agents.

A graphic illustration of the scope and results of this project is given in the figure below.


Rezumatul proiectului

Proiectul “Căi sustenabile pentru sinteza biosurfactanţilor pe bază de carbohidraţi în medii de reacţie verzi” a dezvoltat o metodă inovativă şi sustenabilă pentru obţinerea unor esteri ai acizilor graşi cu zaharide şi polioli, ce aparţin unei clase importante de agenţi activi de suprafaţă cunoscută sub numele de “sugar fatty acid esters SFAEs”. SFAEs sunt emulgatori şi spumanţi excelenţi şi sunt folosiţi ca ingredienţi în industria alimentară, cosmetică,şi farmaceutică, în detergenţi şi produse agrochimice. Utilizând exclusiv materiale naturale şi regenerabile şi enzyme ca şi catalizator, acest proiect a respectat toate criteriile standard ale bioeconomiei circulare: procesele de sinteză sunt verzi, iar produşii de reacţie sunt naturali şi biodegradabili şi au aceleaşi proprietăţi ca şi produşii chimici industriali conscraţi.  Partea experimentală a proiectului a fost efectuată în Laboratorul de Biocataliză al Universităţii Politehnica din Timişoara.

Solvenţii verzi obţinuţi au fost amestecuri eutectice naturale reactive (R-NADES) ce îndeplinesc o funcţie duală, ca (a) mediu de reacţie şi sursă de reactanţi, cănd cel puţin unul din componenţi este substratul reacţiei enzimatice. S-au preparate şi caracterizat 32 de amestecuri eutectice naturale reactive binare şi ternare pe bază de clorură de colină, zaharide şi polioli, majoritatea fiind solvenţi eutectici noi, neraportaţi în literature de specialitate. Biocatalizatorii folosiţi au fost lipaze microbiene, atât în stare nativă cât şi imobilizate. Toate lipazele testate au prezentat activitate catalitică de esterificare în solvenţii eutctici naturali reactivi preparaţi. Totuşi, lipazele imobilizate au avut atât performanţă operaţională cât  şi stabilitate termică foarte ridicată. Optimizarea parametrilor de reacţie prin design expreimental (DOE) a permis sinteza cu randamente ridicate a esterilor acizilor graşi (C12, C16, C18) cu polioli (xilitol, D-arabitol, D-sorbitol) şi zaharide (glucoză, zaharoză şi ologomeri de fructoză cu DP 2-9. Toţi produşii noi sintetizaţi au fost caracterizaţi prin spectroscopie de infraroşu (FTIR), rezonanţă magnetică nucleară (1D-NMR, 2D-NMR) spectrometrie de masă (MALDI-TOF-MS şi ESI-MS), calorimetrie (DDC, differential scanning calorimetry) şi termogravimnetrie (TG), pentru a determina structura, puritatea  şi prorietăţile fizice. Pentru prima dată, s-a demonstrat în acest proiect, regiselectivitatea ridicată a lipazei B din Candida antarctica, imobilizată, pentru grupările hidroxil terminale ale ale poliolilor, şi formarea exclusivă a diesterilor α, ω- ai acizilor graşi cu poliolii. Caracterizarea funcţională a arătat că esterii acizilor graşi cu polioli şi zaharide sunt agenţi emulgatori şi stabilizează emulsii apă/ulei, şi respectiv ulei/apă. Esterii carbohidraţilor sunt agenţi de spumare.




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