Aqueous two-phase systems: Towards novel and more disruptive applications

Aqueous two-phase systems (ATPS) have been mainly proposed as powerful platforms for the separation and purification of high-value biomolecules. However, after more than seven decades of research, ATPS are still a major academic curiosity, without their wide acceptance and implementation by industry, leading to the question whether ATPS should be mainly considered in downstream bioprocessing. Recently, due to their versatility and expansion of the Biotechnology and Material Science fields, these systems have been investigated in novel applications, such as in cellular micropatterning and bioprinting, microencapsulation, to mimic cells conditions, among others. This perspective aims to be a reflection on the current status of ATPS as separation platforms, while overviewing their applications, strengths and limitations. Novel applications, advantages and bottlenecks of ATPS are further discussed, indicating some directions on their use to create innovative industrial processes and commercial products.publishe

Understanding the role of the hydrogen bond donor of the deep eutectic solvents in the formation of the aqueous biphasic systems

Deep eutectic solvents (DES) have been proposed as phase-forming compounds of aqueous biphasic systems (ABS). However, due to DES nature and the high water content of the ABS, their nature and behavior remains controversial. To foster the understanding of DES-based ABS, the present work uses the relative hydrophilicity of the hydrogen bond donor (HBD) as a descriptor to clarify its role on ABS for- mation and phase properties. Dipotassium phosphate (K2HPO4)-based ABS phase diagrams composed of cholinium chloride ([N111(2OH)]Cl), as hydrogen bond acceptor (HBA), and several HBD (alcohols and sugars) were compared in molality units and the saturation solubility point for each binodal curve determined. The results here reported establish the HBD role as function of its relative hydrophilicity: very hydrophilic HBD act only as an adjuvant in the formation of ABS; HBD of intermediate hydrophilicity influence the ABS formation; while the most hydrophobic HBD tend to form organic-aqueous two-phase systems, where the HBA acts as an adjuvant to the system.publishe

Separation and purification of curcumin using novel aqueous two-phase micellar systems composed of amphiphilic copolymer and cholinium ionic liquids

Novel aqueous two-phase micellar systems (ATPMS) composed of Pluronic F68, a triblock amphiphilic copolymer, and cholinium-based ionic liquids (ILs) were formulated and applied for separation/purification of curcumin (CCM). CCM stability in the presence of ATPMS components was also evaluated. CCM is stable up to 24 h in copolymer (1.0 10.0 wt%) and ILs (0.1 3.0 M) aqueous solutions. Very mild phase separation conditions (close to room temperature) were achieved by adding cholinium ILs to the Pluronic F68 + McIlvaine buffer at pH 6.0 solution. The decrease of cloud-point temperature is dependent on the relative hydrophobicity of IL anion, [Hex] > [But] > [Prop] > [Ac] > Cl. ATPMS composed of more hydrophobic ILs ([Ch][Hex] > [Ch][But] > [Ch][Prop]) are most efficient in the partition of commercial CCM into polymeric micelles-rich phase. The best ATPMS (0.70 M [Ch][But] and 0.60 M [Ch][Hex]-based ATPMS) were then used to purify CCM from a crude extract of Curcuma longa L. Both systems were very selective to separate CCM from protein-based contaminants (selectivity values 25; purification yields 12-fold). Pluronic F68-based ATPMS are promising for selective separation of hydrophobic biomolecules by using cholinium-based ILs as adjuvants to adjust phase separation temperatures and biomolecules partition.This study was funded by the Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil) and the State of São Paulo Research Foundation (FAPESP/Brazil, processes #2014/16424-7, #2017/10789-1, #2018/10799-0, #2018/05111-9; #2019/05624-9, and #2019/08549-8). A.M. Lopes and J.F.B. Pereira are grateful for the language revision of native speaker H.S. Pacheco Neto.info:eu-repo/semantics/publishedVersio

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans – anteaters, sloths, and armadillos – have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with 24 domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, ten anteaters, and six sloths. Our dataset includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data-paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the south of the USA, Mexico, and Caribbean countries at the northern portion of the Neotropics, to its austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n=5,941), and Cyclopes sp. has the fewest (n=240). The armadillo species with the most data is Dasypus novemcinctus (n=11,588), and the least recorded for Calyptophractus retusus (n=33). With regards to sloth species, Bradypus variegatus has the most records (n=962), and Bradypus pygmaeus has the fewest (n=12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other datasets of Neotropical Series which will become available very soon (i.e. Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans dataset

Rhodotorula sp.–based biorefinery: a source of valuable biomolecules

Abstract: The development of an effective, realistic, and sustainable microbial biorefinery depends on several factors, including as one of the key aspects an adequate selection of microbial strain. The oleaginous red yeast Rhodotorula sp. has been studied as one powerful source for a plethora of high added-value biomolecules, such as carotenoids, lipids, and enzymes. Although known for over a century, the use of Rhodotorula sp. as resource for valuable products has not yet commercialized. Current interests for Rhodotorula sp. yeast have sparked from its high nutritional versatility and ability to convert agro-food residues into added-value biomolecules, two attractive characteristics for designing new biorefineries. In addition, as for other yeast-based bioprocesses, the overall process sustainability can be maximized by a proper integration with subsequent downstream processing stages, for example, by using eco-friendly solvents for the recovery of intracellular products from yeast biomass. This review intends to reflect on the current state of the art of microbial bioprocesses using Rhodotorula species. Therefore, we will provide an analysis of bioproduction performance with some insights regarding downstream separation steps for the extraction of high added-value biomolecules (specifically using efficient and sustainable platforms), providing information regarding the potential applications of biomolecules produced by Rhodotorula sp, as well as detailing the strengths and limitations of yeast-based biorefinery approaches. Novel genetic engineering technologies are further discussed, indicating some directions on their possible use for maximizing the potential of Rhodotorula sp. as cell factories. Key points: • Rhodotorula sp. are valuable source of high value-added compounds. • Potential of employing Rhodotorula sp. in a multiple product biorefinery. • Future perspectives in the biorefining of Rhodotorula sp. were discussed

Purification of clavulanic acid produced by Streptomyces clavuligerus via submerged fermentation using polyethylene glycol/cholinium chloride aqueous two-phase systems

Clavulanic acid (CA) is an important pharmaceutical compound produced by batch fermentation of Streptomyces clavuligerus. Since, CA is chemically unstable, its downstream processing should be studied to develop more efficient and resolute techniques. Herein, the use of aqueous two-phase systems (ATPS) composed of cholinium chloride, [Ch]Cl, was proposed as a novel platform for the recovery and purification of CA. Thus, the stability of CA in presence of different [Ch]Cl concentrations was initially studied, and the high biocompatibility of this salt was demonstrated by the low CA degradation levels. Then, the partitioning of CA using two types of polymeric ATPS has been investigated. Two ATPS formed by the combination of [Ch]Cl and two polyethylene glycol (PEG) polymers, PEG 600 g mol 1 (PEG-600) and polyethylene glycol methyl ether 550 g mol 1 (PEG-500-OMe), were used to assess the influence of the PEG nature, in addition to the concentration of the phase forming agents on the CA partitioning. It has shown that CA is almost equally distributed between the two-phases in equilibrium (0.6 < KCA < 1.6). Nevertheless, the selective extraction of CA for the [Ch]Cl-rich or PEG-rich phase by the proper adjustment of ATPS composition was attained. In the search for higher extraction efficiencies [EE (%)] and partition coefficients (KCA), a second polymeric ATPS platform composed of PEG-600 and sodium polyacrylate 8000 g mol 1 (NaPA-8000), applying [Ch]Cl as adjuvant was tested. The main results suggest the recovery of CA towards the PEG-rich phase (KCA 5.6 ± 0.6 and EE 85.5± 1.4%). The higher migration levels of CA have mainly resulted from the electronegative repulsion of NaPA-8000 over CA molecules. The ATPS with best performance for the CA extraction were selected for the recovery of CA directly from fermented broth of Streptomyces clavuligerus. In this set of experiments, the highest values of CA recovery yield and purification factor (respectively, 64.91± 1.99% and 22.70 ± 0.87) were attained for the systems PEG-600/NaPA-8000 and PEG-600/[Ch]Cl, respectivelypublishe

Economic analysis of the production and recovery of green fluorescent protein using ATPS-based bioprocesses

Green fluorescent protein (GFP) is a useful biomolecule in biotechnology; however, its price makes its widespread application prohibitive. To overcome this issue, recently, the use of aqueous two-phase systems (ATPS) for GFP purification was proposed as an alternative platform to reduce processing costs. Aligned with this goal, this study performed bioprocess modelling coupled with economic analysis using the software Biosolve to evaluate the potential and commercial applicability of ATPS for GFP purification. This work analysed a collection of fourteen ATPS to discriminate through production costs while also incorporating the concept of product purity into the calculations. The two best systems (a PEG-based and an ionic liquid (IL)-based ATPS) were placed in a full bioprocess at different scale models (1 to 100 L) to elucidate the viability of applying ATPS at large scale. Although the results showed that the PEG-based ATPS exhibit the lowest costs (between USD 3.5x103.g−1 at 1 L and USD 0.33x103.g−1 at 100 L), for further developments, the inclusion of an ATPS granting a higher purity is desired for the development of simpler bioprocesses. Therefore, as a third approach in this work, a sensitivity analysis was performed to determine the impact of varying different model parameters (recovery yield, material costs discount and production titre), to elucidate the circumstances under which the IL-based system can overcome the production costs of the traditional PEG-based ATPS. The results indicate that the best cost-effectiveness approach is to improve the production titre (although it can affect all ATPS studied), as an increase from 1.33 to 3.8 g/L is enough for the IL-based ATPS to be less expensive than the traditional system at all analysed scales. This study demonstrates that ATPS can greatly reduce GFP manufacturing costs, which can potentially help to popularize new applications of fluorescent proteins that are currently mostly restricted to research kits due to their high prices.publishe