Subscriber access provided by UNIV EST PAULISTA UNESP Article Protic ionic liquids as cell disrupting agents for the recovery of intracellular carotenoids from yeast Rhodotorula glutinis CCT-2186 Cassamo U Mussagy, Valéria de Carvalho Santos Ebinuma, Maria Gonzalez-Miquel, Joao A.P. Coutinho, and Jorge Fernando Brandão Pereira ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b04247 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 9, 2019 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 38 ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 Protic ionic liquids as cell disrupting agents for the recovery of intracellular 8 9 carotenoids from yeast Rhodotorula glutinis CCT-2186 10 11 12 † † ‡ 13 Cassamo U. Mussagy, Valéria C. Santos-Ebinuma, Maria Gonzalez-Miquel, 14 João A. P. Coutinho,§ and Jorge F. B. Pereira*† 15 16 17 18 †Department of Bioprocesses and Biotechnology, School of Pharmaceutical Sciences, Sao 19 20 Paulo State University (UNESP), Rodovia Araraquara Jaú, Km 01, Campos Ville – 14800- 21 22 903 - Araraquara/SP - Brazil 23 24 ‡Departamento de Ingeniería Química Industrial y del Medio Ambiente, ETS Ingenieros 25 26 Industriales, Universidad Politécnica de Madrid, C/José Gutiérrez Abascal 2, Madrid 27 28006, Spain 28 29 § 30 CICECO−Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 31 3810-193 Aveiro, Portugal 32 33 *Corresponding author: [email protected] 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering Page 2 of 38 1 2 3 4 5 6 ABSTRACT 7 8 Rhodotorula glutinis 9 yeasts are natural sources of intracellular carotenoids such as 10 β-carotene, torularhodin and torulene. Since these yeasts are constituted by a rigid 11 12 cell-wall structure, the use of energy-saving and high-efficiency cell disruption 13 14 procedures is critical for carotenoids recovery. A new technology using protic ionic 15 liquids (PILs) was here evaluated as alternative platform to permeabilize the R. 16 17 glutinis cells and to improve the extraction of β-carotene, torularhodin and torulene. 18 19 The cell disruption ability of twelve highly concentrated aqueous solutions of 20 ammonium-based PILs was determined, evaluating the influence of the relative ions 21 22 hydrophobicity, solid-liquid ratio (SLR), water content, and temperature. Carotenoid 23 24 extraction yields increased with the hydrophobicity of the PILs (i.e. increase of alkyl 25 chain length of anion or cation), temperature (from 25 ºC to 65 ºC) and PIL 26 27 concentration (from 75% to 90% v/v). Additionally, to demonstrate the potential of 28 29 PILs in carotenoids recovery, solvent recycling and carotenoids polishing were 30 carried out using a three-phase partitioning (TPP) system. The results demonstrate 31 32 that the use of PILs as cell disrupting agents can be a simple, efficient, sustainable 33 34 and feasible method to recover intracellular carotenoids from microbial biomass. 35 36 37 38 Keywords: Rhodotorula glutinis; carotenoids; extraction, protic ionic liquids; β- 39 40 carotene; torularhodin; torulene. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 3 of 38 ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 INTRODUCTION 7 8 Carotenoids are a group of yellow, orange and red polyisoprenoid pigments, 9 10 that can be naturally synthesized by different microorganisms, including microalgae, 11 12 bacteria, yeasts and filamentous fungi 1,2. These biopigments exhibit a plethora of 13 biological attractive properties, such as antioxidant, antiobesity, antidiabetic, 14 15 anticancer (particularly those that results from cardiovascular diseases and macular 16 17 degeneration) and antimicrobial activities 3–5. As a result of their interesting biological 18 19 properties, natural carotenoids have been attracting great interest from academic 20 and industrial partners for applications in pharmaceuticals, cosmetics and functional 21 22 foods formulations, as well as the most common uses in food industries 6–8. 23 9 24 Rhodotorula glutinis are aerobic yeasts able to synthesize, in an easy and 25 natural way, several industrial high-added value metabolites, e.g. lipids, carotenoids, 26 27 and enzymes. Particularly, these yeasts can produce and accumulate approximately 28 10,11 29 50% of their dried cellular biomass as fractions of carotenoids and lipids . Among 30 the carotenoids, Rhodotorula glutinis are able to synthesize β-carotene, torulene, 31 32 and torularhodin, exhibiting variable production yields according to the specific 33 11 12 34 cultivation conditions and fast grow . 35 However, since the carotenoids from R. glutinis yeast are produced 36 37 intracellularly, appropriate cell-disrupting methodologies are always required for their 38 13 39 recovery . A large number of cell disruption techniques, including conventional and 40 non-conventional procedures, have been reported in literature 2,14. Traditionally, 41 42 Rhodotorula glutinis yeasts are disrupted by using conventional solid-liquid 43 44 extraction procedures with volatile organic solvents (VOCs), such as petroleum 45 ether, dimethyl sulfoxide (DMSO), acetone, chloroform and hexane 15. Among the 46 47 common VOCs, the use of DMSO, in general, lead to the highest release rates of 48 49 intracellular carotenoids, but their use in industrial processes is limited by health and 50 environmental concerns 16,17. 51 52 Therefore, the search for more biocompatible and environmentally friendly 53 54 solvents, as for example, biosolvents, ionic liquids (ILs) and deep eutectic solvents, 55 is of great importance. Among these, ILs, commonly defined as salts with a melting 56 57 point lower than 100 °C 18, have been suggested as one of the most interesting 58 59 60 ACS Paragon Plus Environment ACS Sustainable Chemistry & Engineering Page 4 of 38 1 2 3 4 5 6 classes of alternative solvents, mainly due to their unique features like high hydrolytic 7 8 activity, low volatility, high solvation capacity, and excellent thermal and chemical 9 stabilities 19. 10 11 Some studies evaluated the capability of different ILs for the extraction of 12 19–21 19 13 carotenoids . Choi and collaborators extracted astaxanthin and lipids from 14 Haematococcus pluvialis cyst cells using ten 1-ethyl-3-methylimidazolium ([Emim]+)- 15 16 based ILs, obtaining complete astaxanthin recoveries (> 99%) and high lipid 17 18 extraction yields (∼82%) at optimum operating conditions, i.e. 6.7% (v/v) of IL in 19 water, 30 °C of temperature and 60 min of extraction time. Desai and collaborators 20 21 20 recovered more than 70% of intracellular astaxanthin from intact H. pluvialis cells 22 23 using an aqueous solution of 1-ethyl-3-methylimidazolium di-butyl-phosphate 24 21 25 ([Emim][DBP]) (40 wt%). Vieira and collaborators recovered fucoxanthin from 26 Sargassum muticum cells using aqueous solutions of different surface-active ILs. 27 28 These examples demonstrate that ILs are effective solvents to extract carotenoids 29 30 from natural sources; however, most of these works have been performed using 31 “classic” aprotic ILs (AILs). Recent studies have indicated that AILs are not the most 32 33 adequate for biological-based applications due to their cytotoxicity 22, which led to a 34 35 growing interest in the third-generation of more benign and biocompatible ILs, i.e. 36 protic ionic liquids (PILs) 23. 37 38 PILs are a subset of ILs prepared through the stoichiometric neutralization 39 40 reaction of certain Brønsted acids and Brønsted bases. They have a proton available 41 for hydrogen bonding, and differ from the classic AILs in having both cationic and 42 43 anionic counterparts formed by low molecular weight organic compounds, usually 44 23 45 substituted (or poly-substituted) amines as cations, and organic acids as anions . 46 PILs are more favourable than AILs in terms of toxicity and biodegradability 47 48 24. This fact, coupled with its low production cost, easier synthesis and more benign 49 50 nature (i.e. can be obtained fully or partly from natural raw materials) make them 51 attractive solvents for wider use in industrial bioprocesses 25. 52 53 Considering these assumptions, the aim of this study was to evaluate the 54 55 capability of PILs to disrupt (permeabilize) the cell wall of Rhodotorula glutinis yeasts 56 and to increase the recoveries of intracellular carotenoids.