The biotechnology revolution in textile dyeing.

Biotechnology holds the potential to revolutionize textile dyeing by utilizing biopigments, biodegradable dyes, and fermentative dyeing methods as alternatives to synthetic options. While some challenges exist, these biotechnological approaches offer innovative solutions to minimize environmental impact and foster sustainable practices, leading toward a greener, circular and efficient textile bioindustry.

Unravelling the molecular interactions behind the formation of PEG/PPG aqueous two-phase systems.

The understanding of molecular interactions that control phase separation in polymer/polymer aqueous two-phase systems (ATPS) has been a subject of debate up to this day. In light of this, we set out to investigate the molecular interactions occurring in ternary mixtures composed of polyethylene glycol (PEG600), polypropylene glycol (PPG400) and water. The ternary phase diagram was plotted at two temperatures (298 K and 323 K), revealing a transition from a type 0 to a type I diagram. Molecular dynamics (MD) simulations were performed to elucidate the polymer-polymer and polymer-water interactions occurring at different temperatures and water concentrations. COnductor-like Screening Model for Realistic Solvents (COSMO-RS) was used to assess the thermodynamic properties of the polymer-water binary mixtures and their correlation with ATPS formation. The MD simulations clearly demonstrate the effect of segregation/separation with increasing water content and temperature, highlighting a significant reduction in PPG-water interactions compared to PEG-water counterparts. Polymer-water interactions were identified as those controlling the phase separation mechanism, and the thermodynamic properties determined with COSMO-RS for the polymer-water binary systems further support this view.

Tailor-made solvents for microbial carotenoids recovery.

In recent years, microbial carotenoids have emerged as a promising alternative for the pharmaceutical and food industries, particularly in promoting human health due to their potent antioxidant and antimicrobial properties. Microbial carotenoids, particularly those produced by yeast, bacteria, and microalgae, are synthesized intracellularly, requiring the use of solvents for their effective extraction and recovery. The conventional use of toxic volatile organic solvents (VOCs) like hexane, petroleum ether, and dimethyl sulfoxide in the extraction of microbial carotenoids has been common. However, ongoing research is introducing innovative, non-toxic, environmentally friendly tailor-made solvents, such as ionic liquids (IL) and deep eutectic solvents (DES), indicating a new era of cleaner and biocompatible technologies. This review aims to highlight recent advancements in utilizing IL and DES for obtaining carotenoids from microorganisms. Additionally, we explore the utilization of in silico tools designed to determine the solubilities of microbial carotenoids in tailor-made DES and ILs. This presents a promising alternative for the scientific community, potentially reducing the need for extensive experimental screening of solvents for the recovery of microbial carotenoids in the separation processing. According to our expert perspective, both IL and DES exhibit a plethora of exceptional attributes for the recovery of microbial carotenoids. Nevertheless, the current employment of these solvents for recovery of carotenoids is restricted to scientific exploration, as their feasibility for practical application in industrial settings has yet to be conclusively demonstrated. KEY POINTS: • ILs and DES share many tailoring properties for the recovery of microbial carotenoids • The use of ILs and DES for microbial carotenoid extraction remains driven by scientific curiosity. • The economic feasibility of ILs and DES is yet to be demonstrated in industrial applications.

Is the carotenoid production from Phaffia rhodozyma yeast genuinely sustainable? a comprehensive analysis of biocompatibility, environmental assessment, and techno-economic constraints.

Microorganisms, such as yeasts, filamentous fungi, bacteria, and microalgae, have gained significant attention due to their potential in producing commercially valuable natural carotenoids. In recent years, Phaffia rhodozyma yeasts have emerged as intriguing non-conventional sources of carotenoids, particularly astaxanthin and ß-carotene. However, the shift from academic exploration to effective industrial implementation has been challenging to achieve. This study aims to bridge this gap by assessing various scenarios for carotenoid production and recovery. It explores the use of ionic liquids (ILs) and bio-based solvents (ethanol) to ensure safe extraction. The evaluation includes a comprehensive analysis involving Life Cycle Assessment (LCA), biocompatibility assessment, and Techno-Economic Analysis (TEA) of two integrated technologies that utilize choline-based ILs and ethanol (EtOH) for astaxanthin (+ß-carotene) recovery from P. rhodozyma cells. This work evaluates the potential sustainability of integrating these alternative solvents within a yeast-based bioeconomy.

Microbial products for space nutrition.

Sustainably producing nutrients beyond Earth is one of the biggest technical challenges for future extended human space missions. Microorganisms such as microalgae and cyanobacteria can provide astronauts with nutrients, pharmaceuticals, pure oxygen, and bio-based polymers, making them an interesting resource for constructing a circular bioregenerative life support system in space.

Structure, aggregation dynamics and crystallization of superfolder green fluorescent protein: Effect of long alkyl chain imidazolium ionic liquids.

Green fluorescent protein (GFP) and its variants are widely used in medical and biological research, especially acting as indicators of protein structural integrity, protein-protein interactions and as biosensors. This study employs superfolder GFP (sfGFP) to investigate the impact of varying alkyl chain length of 1-Cn-3-methylimidazolium chloride ionic liquid (IL) series ([Cnmim]Cl, n = 2, 4, 6, 8, 10, 12) on the protein fluorescence, structure, hydration, aggregation dynamics and crystallization behaviour. The results revealed a concentration-dependent decrease in the sfGFP chromophore fluorescence, particularly in long alkyl chain ILs ([C10mim]Cl and [C12mim]Cl). Tryptophan (Trp) fluorescence showed the quenching rate increased with longer alkyl chains indicating a nonpolar interaction between Trp57 and the alkyl chain. Secondary structural changes were observed at the high IL concentration of 1.5 M in [C10mim]Cl and [C12mim]Cl. Small-angle X-ray scattering (SAXS) indicated relatively stable protein sizes, but with IL aggregates present in [C10mim]Cl and [C12mim]Cl solutions. Dynamic light scattering (DLS) data showed increased protein size and aggregation with longer alkyl chain ILs. Notably, ILs and salts, excluding [C2mim]Cl, promoted sfGFP crystallization. This study emphasizes the influence of the cation alkyl chain length and concentration on protein stability and aggregation, providing insights into utilizing IL solvents for protein stabilization and crystallization purposes.

Rhodotorula sp. as a cell factory for production of valuable biomolecules.

Rhodotorula sp. are well-known for their ability to biosynthesize a diverse range of valuable biomolecules, including carotenoids, lipids, enzymes, and polysaccharides. Despite the high number of studies conducted using Rhodotorula sp. at the laboratory scale, most of these do not address all processual aspects necessary for scaling up these processes for industrial applications. This chapter explores the potential of Rhodotorula sp. as a cell factory for the production of distinct biomolecules, with a particular emphasis on exploring their use from a biorefinery perspective. Through in-depth discussions of the latest research and insights into non-conventional applications, we aim to provide a comprehensive understanding of Rhodotorula sp.'s ability to produce biofuels, bioplastics, pharmaceuticals, and other valuable biochemicals. This book chapter also examines the fundamentals and challenges associated with the optimizing upstream and downstream processing of Rhodotorula sp-based processes. We believe that through this chapter, readers with different levels of expertise will gain insights into strategies for enhancing the sustainability, efficiency, and effectiveness of producing biomolecules using Rhodotorula sp.

Microbial astaxanthin: from bioprocessing to the market recognition.

The attractive biological properties and health benefits of natural astaxanthin (AXT), including its antioxidant and anti-carcinogenic properties, have garnered significant attention from academia and industry seeking natural alternatives to synthetic products. AXT, a red ketocarotenoid, is mainly produced by yeast, microalgae, wild or genetically engineered bacteria. Unfortunately, the large fraction of AXT available in the global market is still obtained using non-environmentally friendly petrochemical-based products. Due to the consumers concerns about synthetic AXT, the market of microbial-AXT is expected to grow exponentially in succeeding years. This review provides a detailed discussion of AXT's bioprocessing technologies and applications as a natural alternative to synthetic counterparts. Additionally, we present, for the first time, a very comprehensive segmentation of the global AXT market and suggest research directions to improve microbial production using sustainable and environmentally friendly practices. KEY POINTS: • Unlock the power of microorganisms for high value AXT production. • Discover the secrets to cost-effective microbial AXT processing. • Uncover the future opportunities in the AXT market.

Astaxanthin production using Paracoccus carotinifaciens: a way forward?

Paracoccus carotinifaciens could be considered a key microbial factory for obtaining healthier natural products such as astaxanthin (AXT), thus contributing to a bioeconomy. Short cultivation time, high production titers, and thin cell wall are the main advantages that make this bacterium promising in the development of sustainable third-generation biorefineries.

Insights into using green and unconventional technologies to recover natural astaxanthin from microbial biomass.

Microorganisms such as bacteria, microalgae and fungi, are natural and rich sources of several valuable bioactive antioxidant's compounds, including carotenoids. Among the carotenoids with antioxidant properties, astaxanthin can be highlighted due to its pharmaceutical, feed, food, cosmetic and biotechnological applications. The best-known producers of astaxanthin are yeast and microalgae cells that biosynthesize this pigment intracellularly, requiring efficient and sustainable downstream procedures for its recovery. Conventional multi-step procedures usually involve the consumption of large amounts of volatile organic compounds (VOCs), which are regarded as toxic and hazardous chemicals. Considering these environmental issues, this review is focused on revealing the potential of unconventional extraction procedures [viz., Supercritical Fluid Extraction (SFE), Ultrasound-Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE), High-Pressure Homogenization (HPH)] combined with alternative green solvents (biosolvents, eutectic solvents and ionic liquids) for the recovery of microbial-based astaxanthin from microalgae (such as Haematococcus pluvialis) and yeast (such as Phaffia rhodozyma) cells. The principal advances in the area, process bottlenecks, solvent selection and strategies to improve the recovery of microbial astaxanthin are emphasized. The promising recovery yields using these environmentally friendly procedures in lab-scale are good indications and directions for their effective use in biotechnological processes for the production of commercial feed and food ingredients like astaxanthin.

Advances and trends in biotechnological production of natural astaxanthin by Phaffia rhodozyma yeast.

Astaxanthin (AXT) is a natural xanthophyll with strong antioxidant, anticancer and antimicrobial activities, widely used in the food, feed, pharmaceutical and nutraceutical industries. So far, 95% of the AXT global market is produced by chemical synthesis, but growing customer preferences for natural products are currently changing the market for natural AXT, highlighting the production from microbially-based sources such as the yeast Phaffia rhodozyma. The AXT production by P. rhodozyma has been studied for a long time at a laboratory scale, but its use in industrial-scale processes is still very scarce. The optimization of growing conditions as well as an effective integration of upstream-downstream operations into P. rhodozyma-based AXT processes has not yet been fully achieved. With this critical review, we scrutinized the main approaches for producing AXT using P. rhodozyma strains, highlighting the impact of using conventional and non-conventional procedures for the extraction of AXT from yeast cells. In addition, we also pinpointed research directions, for example, the use of low-cost residues to improve the economic and environmental sustainability of the bioprocess, the use of environmentally/friendly and low-energetic integrative operations for the extraction and purification of AXT, as well as the need of further human clinical trials using yeast-based AXT.

Microbial torularhodin - a comprehensive review.

The demand for food, feed, cosmeceutical, and nutraceutical supplements/additives from natural sources has been rapidly increasing, with expectations for a faster expansion than the growth of the global markets in the coming years. In this framework, a particular interest is given to carotenoids due to their outstanding antioxidant activities, particularly the xanthophylls class. Torularhodin is one of these carotenoids that stands out for its multifunctional role as: antioxidant, anticancer and antimicrobial, yet its commercial potential is still unexplored. Although most xanthophylls can be naturally found in: microbial, plant and animal sources, torularhodin is only produced by microbial species, especially red oleaginous yeast. The microbial production of xanthophylls has many advantages as compared to other natural sources, such as: the need for low production area, easier extraction, high yields (at optimum operating conditions), and low (or no) seasonal, climatic, and geographic variation dependency. Due to the importance of natural products and their relevance to the market, this review provides a comprehensive overview of the: properties, characteristics and potential health benefits of torularhodin. Moreover, the most promising developments in both upstream and downstream processing to obtain this colorant from microbial sources are considered. For this purpose, the main microorganisms used for torularhodin production are firstly reviewed, including biosynthesis pathway and torularhodin properties. Following, an overall analysis of the processing aspects related with its: extraction, separation and purification is provided. Lastly, current status and future trends of torularhodin-based processes and products such as therapeutic agents or biomaterials are discussed, indicating promising directions toward biorefinery and circular economy.

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

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.

Protic Ionic Liquid Cation Alkyl Chain Length Effect on Lysozyme Structure.

Solvents that stabilize protein structures can improve and expand their biochemical applications, particularly with the growing interest in biocatalytic-based processes. Aiming to select novel solvents for protein stabilization, we explored the effect of alkylammonium nitrate protic ionic liquids (PILs)-water mixtures with increasing cation alkyl chain length on lysozyme conformational stability. Four PILs were studied, that is, ethylammonium nitrate (EAN), butylammonium nitrate (BAN), hexylammonium nitrate (HAN), and octylammonium nitrate (OAN). The surface tension, viscosity, and density of PIL-water mixtures at low to high concentrations were firstly determined, which showed that an increasing cation alkyl chain length caused a decrease in the surface tension and density as well as an increase in viscosity for all PIL solutions. Small-angle X-ray scattering (SAXS) was used to investigate the liquid nanostructure of the PIL solutions, as well as the overall size, conformational flexibility and changes to lysozyme structure. The concentrated PILs with longer alkyl chain lengths, i.e., over 10 mol% butyl-, 5 mol% hexyl- and 1 mol% octylammonium cations, possessed liquid nanostructures. This detrimentally interfered with solvent subtraction, and the more structured PIL solutions prevented quantitative SAXS analysis of lysozyme structure. The radius of gyration (Rg) of lysozyme in the less structured aqueous PIL solutions showed little change with up to 10 mol% of PIL. Kratky plots, SREFLEX models, and FTIR data showed that the protein conformation was maintained at a low PIL concentration of 1 mol% and lower when compared with the buffer solution. However, 50 mol% EAN and 5 mol% HAN significantly increased the Rg of lysozyme, indicating unfolding and aggregation of lysozyme. The hydrophobic interaction and liquid nanostructure resulting from the increased cation alkyl chain length in HAN likely becomes critical. The impact of HAN and OAN, particularly at high concentrations, on lysozyme structure was further revealed by FTIR. This work highlights the negative effect of a long alkyl chain length and high concentration of PILs on lysozyme structural stability.

Opposite Effects Induced by Cholinium-Based Ionic Liquid Electrolytes in the Formation of Aqueous Biphasic Systems Comprising Polyethylene Glycol and Sodium Polyacrylate.

Cholinium-based ionic liquids ([Ch]-based ILs) were investigated as electrolytes in the formation of aqueous biphasic systems (ABS) composed of polyethylene glycol (PEG) and sodium polyacrylate (NaPA) polymers. Both enhancement and decrease in the liquid-liquid demixing ability induced by electrolytes in PEG-NaPA aqueous biphasic systems were observed. It is shown that the ILs that most extensively partition to the PEG-rich phase tend to act as inorganic salts enhancing the two-phase formation ability, while those that display a more significant partition to the NaPA-rich phase decrease the ABS formation capacity. The gathered results allowed us to confirm the tailoring ability of ILs and to identify, for the first time, opposite effects induced by electrolytes on the PEG-NaPA ABS formation ability. The distribution of the electrolyte ions between the coexisting phases and the polyelectrolyte ion compartmentalization are key factors behind the formation of PEG-NaPA-based ABS.

Using coarse-grained molecular dynamics to understand the effect of ionic liquids on the aggregation of Pluronic copolymer solutions.

This study is aimed to enhance the understanding of the interaction between ionic liquids (ILs) and non-ionic Pluronic triblock copolymers in aqueous two-phase micellar systems (ATPMS) used for the selective separation/purification of hydrophobic biomolecules. The ILs allow a precise control of the cloud point phase separation temperature (CPT), particularly important when the stability of the molecule is highly dependent on temperature. The effect of choline-based ILs, with two different counter-anions, chloride and hexanoate, was evaluated using molecular dynamics simulations (MD) for F-68 and L-35 Pluronic aqueous solutions. The simulations revealed the role played by the anions during the Pluronic self-assembly, with choline chloride hindering Pluronic aggregation and the choline hexanoate favouring micelle formation and coalescence, in agreement with the experimental data. A detailed study of the accessible surface area of Pluronic showed a progressive dehydration of the Pluronic hydrophilic micelle corona in choline hexanoate mixtures promoting inter-micelle interactions and, consequently, micelle coalescence. With the addition of choline hexanoate, it was observed that the hydrophilic segments, which form the micelle corona, twisted towards the Pluronic micelle core. The electrostatic interaction is also shown to play a key role in this IL-Pluronic aqueous solution, as the hexanoate anions are accommodated in the Pluronic micelle core, while the choline cations are hosted by the Pluronic micelle corona, with the ions interacting with each other during the self-assembly process. In addition, a comparison study of F-68 and L-35 aqueous solutions shows that the IL impact depends on the length of the Pluronic hydrophilic segment. This work provides a realistic microscopic scenario of the complex interactions between Pluronic copolymers and ILs.

Polymeric micelles using cholinium-based ionic liquids for the encapsulation and release of hydrophobic drug molecules.

We generated stable amphiphilic copolymer-based polymeric micelles (PMs) with temperature-responsive properties utilizing Pluronic® L35 and a variety of ionic liquids (ILs) to generate different aqueous two-phase micellar systems (ATPMSs). The partitioning of the hydrophobic model compound curcumin (CCM) into the PM-rich phase and the drug delivery capabilities of the PMs were investigated. ATPMSs formed using more hydrophobic ILs (i.e., [Ch][Hex] ≈ [Ch][But] > [Ch][Pro] > [Ch][Ac] ≈ [Ch]Cl) were the most effective in partitioning (KCCM) and recovering (RECRich) CCM into the PM-rich phase (15.2 < KCCM < 22.0 and 90% < RECRich < 95%, respectively). Moreover, using 1.2 M [Ch][But] and 0.2 M [Ch][Hex] ILs yielded higher encapsulation efficiency (EE) (94.1 and 96.0%, respectively) and drug loading (DL) capacity (14.8 and 16.2%, respectively), together with an increase in the average hydrodynamic diameter of the PMs (DH) (42.5 and 45.6 nm, respectively). The CCM-PM formulations were stable at 4.0, 25.0, and 37.0 °C and the release of CCM was faster with the less hydrophobic ILs (i.e., [Ch]Cl and [Ch][Ac]). Furthermore, due to the lower critical solution temperature properties of Pluronic® L35, the PMs exhibit temperature responsiveness at 37.0 °C. In vitro cytotoxicity assays were also performed to determine the potency of CCM-PM formulations, and a 1.8-fold decrease in IC50 values was observed between the CCM-PMs/[Ch][Hex] and CCM-PMs/[Ch]Cl formulations for PC3 cells. The lower IC50 value for the [Ch][Hex] version corresponded to a greater potency compared to the [Ch]Cl version, since a lower concentration of CCM was required to achieve the same therapeutic effect. The ATPMSs investigated in this study serve as a novel platform for Pluronic® L35/PBS buffer (pH 7.4) + IL-based ATPMS development. The unique properties reported here may be useful in applications such as controlled-release drug delivery systems (DDS), encapsulation, and bioseparations.

Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors.

Enhanced Green Fluorescent Protein (EGFP) is a biomolecule with intense and natural fluorescence, with biological and medical applications. Although widely used as a biomarker in research, its application as a biosensor is limited by the lack of in-depth knowledge regarding its structure and behavior in adverse conditions. This study is focused on addressing this need by evaluating EGFP activity and structure at different pH using three-dimensional fluorescence, circular dichroism and small-angle X-ray scattering. The focus was on the reversibility of the process to gain insights for the development of biocompatible pH-biosensors. EGFP was highly stable at alkaline pH and quenched from neutral-to-acidic pH. Above pH 6.0, the fluorescence loss was almost completely reversible on return to neutral pH, but only partially reversible from pH 5.0 to 2.0. This work updates the knowledge regarding EGFP behavior in pH by accounting for the recent data on its structure. Hence, it is evident that EGFP presents the required properties for use as natural, biocompatible and environmentally friendly neutral to acidic pH-biosensors.

Subacute toxicity assessment of biobased ionic liquids in rats.

Ionic liquids (ILs) derived from compounds obtained from natural sources, such as fatty acids (FAs) have attracted the interest of the scientific and industrial communities because of their sustainable appeal and possible low toxic effects or nontoxicity. These aspects open new perspective of applications in other fields, which demands a better comprehension of their toxicity. This work evaluated the subacute toxicity of bis(2-hydroxyethyl)ammonium carboxylates in Wistar rats, considering the alkyl chain length of FAs (capric and oleic acids), and the concentration (0.16%, 1.6% or 3.2%, wIL/wOil) of ILs added in diets. The blood serum of the rats was evaluated in relation to total cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transferase. Lipid peroxidation was determined in plasma, liver and kidney tissues by determining the level of thiobarbituric acid reactive substances. Histological analyses of the liver and kidney tissues were performed in order to evaluate morphological changes. No signal of toxicity was observed according to lipid peroxidation. Triglycerides increased with the increasing of the concentration and alkyl chain length of the IL, but no difference in serum level of lipid peroxidation was observed. This behavior may be attributed to the amphiphilic nature of FAs based ILs, which might facilitate lipid digestion. However, more studies are necessary in order to understand such behavior. Therefore, the synthesis of ILs from FAs, has been evaluated as a strategy to produce compounds with low or without toxicity for the agro-food, pharmaceutical or cosmetic industries.