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.

Recovery Techniques Enabling Circular Chemistry from Wastewater.

In an era where it becomes less and less accepted to just send waste to landfills and release wastewater into the environment without treatment, numerous initiatives are pursued to facilitate chemical production from waste. This includes microbial conversions of waste in digesters, and with this type of approach, a variety of chemicals can be produced. Typical for digestion systems is that the products are present only in (very) dilute amounts. For such productions to be technically and economically interesting to pursue, it is of key importance that effective product recovery strategies are being developed. In this review, we focus on the recovery of biologically produced carboxylic acids, including volatile fatty acids (VFAs), medium-chain carboxylic acids (MCCAs), long-chain dicarboxylic acids (LCDAs) being directly produced by microorganisms, and indirectly produced unsaturated short-chain acids (USCA), as well as polymers. Key recovery techniques for carboxylic acids in solution include liquid-liquid extraction, adsorption, and membrane separations. The route toward USCA is discussed, including their production by thermal treatment of intracellular polyhydroxyalkanoates (PHA) polymers and the downstream separations. Polymers included in this review are extracellular polymeric substances (EPS). Strategies for fractionation of the different fractions of EPS are discussed, aiming at the valorization of both polysaccharides and proteins. It is concluded that several separation strategies have the potential to further develop the wastewater valorization chains.

Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020.

The last 5 years have seen a series of advances in the application of isothermal titration microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invaluable role in understanding multiprotein complex formation including proteolysis-targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction with LC3 and GABARAP. It has also helped elucidate complex allosteric communication in protein complexes like trp RNA-binding attenuation protein (TRAP) complex. Advances in kinetics analysis have enabled the calculation of kinetic rate constants from pre-existing ITC data sets. Diverse strategies have also been developed to study enzyme kinetics and enzyme-inhibitor interactions. ITC has also been applied to study small molecule solvent and solute interactions involved in extraction, separation, and purification applications including liquid-liquid separation and extractive distillation. Diverse applications of ITC have been developed from the analysis of protein instability at different temperatures, determination of enzyme kinetics in suspensions of living cells to the adsorption of uremic toxins from aqueous streams.

Selector screening for enantioseparation of DL-α-methyl phenylglycine amide by liquid-liquid extraction.

Enantioseparation through liquid extraction technology is an emerging field, e.g., enantioseparations of amino acids (and derivatives thereof), amino alcohols, amines, and carboxylic acids have been reported. Often, when a new selector is developed, the versatility of substrate scope is investigated. From an industrial point of view, the problem is typically approached the other way around, and for a target racemate, a selector needs to be found in order to accomplish the desired enantioseparation. This study presents such a screening approach for the separation of the enantiomers of DL-α-methyl phenylglycine amide (DL-α-MPGA), a model amide racemate with high industrial relevance. Chiral selectors that were reported for other classes of racemates were investigated, i.e., several macrocyclic selectors and Pd-BINAP complexes. It appeared very challenging to obtain both high extraction yields and good enantioselectivity for most selectors, but Pd-BINAP-based selectors performed well, with enantioselectivities up to 7.4 with an extraction yield of the desired enantiomer of 95.8%. These high enantioselectivities were obtained using dichloromethane as solvent. Using less volatile chlorobenzene or 1-chloropentane, reasonable selectivities of up to 1.7 were measured, making these the best alternative solvents for dichloromethane.

Fractionation of Extracellular Polymeric Substances by Aqueous Three-Phase Partitioning Systems.

Extracellular polymeric substances (EPS) are natural polymers secreted by microorganisms and represent a key chemical for the development of a range of circular economy applications. The production of EPS comes with notable challenges such as downstream processing. In this work, a three-phase partitioning (TPP) system was investigated as a fractionation technique for the separation of polysaccharides and proteins, both present in the EPS culture broth. The effect of the type of phase-forming compounds (alcohol, polymer, or ionic liquid, in combination with salt) and its concentration were evaluated and compared to the results previously obtained with model systems. The recyclability of phase-forming compounds used to form the fractionation platform was assessed by ultrafiltration. The best fractionation of EPS was achieved using a TPP system composed of 23 wt % ethanol and 25% K3C6H5O7 as 82% EPS-PS partitioned to the salt-rich/bottom phase, and 76% EPS-PN was recovered as an interfacial precipitate, which could be readily resolubilized in water. This represented an increase of 1.24 and 2.83-fold in the purity of EPS-PS and EPS-PN, respectively, in relation to the initial feed concentration. Finally, high recovery yields of phase-forming compounds (>99%) and fractionated EPS (>80%) were obtained using ultrafiltration/diafiltration (UF/DF) as the regeneration technique. The substantial fractionation yields, selectivity, and recyclability of the phase-forming compounds confirm the potential of TPP systems in combination with UF/DF as the separation method for real biopolymer mixtures. Key contributions of this study include the demonstration of the applicability of a readily scalable and cost-effective separation technique for the fractionation of EPS from real EPS-containing broths, while also the limitations of prescreening with model systems became clear through the observed deviating trends between model system studies and real broth studies.

High-throughput Soxhlet extraction method applied for analysis of leaf lignocellulose and non-structural substances.

The traditional Soxhlet extraction method is commonly employed to extract soluble components from non-soluble components in a solid matrix, for example, non-structural substances in biomass samples that can be separated from structural lignocellulosic compounds in biomass samples. Conventional laboratory procedures for such extractions typically involve a low sample throughput, with each run being performed individually, resulting in time-consuming and labour-intensive processes, making them impractical for analysing large sample sets. In research fields such as Earth Observation in Forest Ecosystems, extensive fieldwork sampling is required across large study areas, resulting in a substantial number of leaf samples, each with limited mass. In this study, an innovative adaptation of the conventional National Renewable Energy Laboratory (NREL) Soxhlet method is developed to create a high-throughput mini-Soxhlet apparatus that enables the simultaneous extraction of up to nineteen samples, each with a mass of 0.3 g per sample. With this adaptation, we measured the lignocellulose and extractive in 343 leaf samples collected from four temperate forest tree species. This modified approach enhances versatility and can be applied to all solid-liquid extractions and various types of vegetation tissues, such as tree leaves, shrubs, crops, feedstock, and other non-woody samples.•The solid-liquid extraction method has been implemented in a heating block facilitating 19 small flasks to measure multiple samples simultaneously while requiring only a small sample mass.•The apparatus set-up was constructed using an alumina heating block mounted on a standard laboratory heating plate. Boiling flask tubes were placed in the heating block and equipped with condenser caps and filters on glass rods on which the solid samples were placed.•The adjustments made the method suitable for application to diverse vegetation tissues and non-woody sample types. It holds particular appeal for research areas that necessitate a high sample number.

Chiral separation by enantioselective liquid-liquid extraction.

The literature on enantioselective liquid-liquid extraction (ELLE) spans more than half a century of research. Nonetheless, a comprehensive overview has not appeared during the past few decades. Enantioselective liquid-liquid extraction is a technology of interest for a wide range of chemists and chemical engineers in the fields of fine chemicals, pharmaceuticals, agrochemicals, fragrances and foods. In this review the principles and advances of resolution through enantioselective liquid-liquid extraction are discussed, starting with an introduction on the principles of enantioselective liquid-liquid extraction including host-guest chemistry, extraction and phase transfer mechanisms, and multistage liquid-liquid extraction processing. Then the literature on enantioselective liquid-liquid extraction systems is reviewed, structured on extractant classes. The following extractant classes are considered: crown ether based extractants, metal complexes and metalloids, extractants based on tartrates, and a final section with all other types of chiral extractants.

Recent Developments in Solvent-Based Fluid Separations.

The most important developments in solvent-based fluid separations, separations involving at least one fluid phase, are reviewed. After a brief introduction and discussion on general solvent trends observed in all fields of application, several specific fields are discussed. Important solvent trends include replacement of traditional molecular solvents by ionic liquids and deep eutectic solvents and, more recently, increasing discussion around bio-based solvents in some application fields. Furthermore, stimuli-responsive systems are discussed; the most significant developments in this field are seen for CO2-switchable and redox-responsive solvents. Discussed fields of application include hydrocarbons separations, carbon capture, biorefineries, and metals separations. For all but the hydrocarbons separations, newly reported electrochemically mediated separations seem to offer exciting new windows of opportunities.

Delignification of Low-Energy Mechanical Pulp (Asplund Fibers) in a Deep Eutectic Solvent System of Choline Chloride and Lactic Acid.

Deep eutectic solvents (DESs) are considered as a green and environmentally benign solvent class for various applications, including delignification of biomass. One of the major challenges in the delignification of biomass by DES is attributed to the limitations in mass transfer. By subjecting wood chips to a low-energy mechanical refining, i.e., the Asplund process, the accessible surface area increases greatly, which in turn improves the mass transfer and increases the reaction rate. In this research, the DES delignification of Asplund fibers made of Norway spruce was studied as a strategy to produce papermaking fibers under mild conditions. A DES consisting of lactic acid and choline chloride was used due to its proven performance in delignification. Various operational conditions, such as temperature, time, DES-to-wood ratio, and the type of stirring were studied. A novel parameter, Q, allowed to evaluate the impact of the operational conditions on the quality of the pulp in terms of delignification degree and fiber length. The results showed that cooking temperature had the most significant effect on the pulp quality. Additionally, it was observed that cooking times between 30 and 45 min result in a pulp yield of about 50%, while fibers have a lignin content of about 14% and a fiber length of 0.6 mm. These results demonstrate that it is possible to obtain fibers of relatively good quality from DES delignification using Asplund fibers as the starting material.

Complex coacervates as extraction media.

Various solvents such as ionic liquids, deep eutectic solvents, and aqueous two phase systems have been suggested as greener alternatives to existing extraction processes. We propose to add macroscopic complex coacervates to this list. Complex coacervates are liquid-like forms of polyion condensates and consist of a complex of oppositely charged polyions and water. Previous research focussing on the biological significance of these polyion-rich phases has shown that polyion condensates have the ability to extract certain solutes from water and back-extract them by changing parameters such as ionic strength and pH. In this study, we present the distribution coefficients of five commonly used industrial chemicals, namely lactic acid, butanol, and three types of lipase enzymes in poly(ethylenimine)/poly(acrylic acid) complex coacervates. It was found that the distribution coefficients can vary strongly upon variation of tunable parameters such as polyion ratio, ionic strength, polyion and compound concentrations, and temperature. Distribution coefficients ranged from approximately 2 to 50 depending on the tuning of the system parameters. It was also demonstrated that a temperature-swing extraction is possible, with back-extraction of butanol from complex coacervates with a recovery of 21.1%, demonstrating their potential as extraction media.

Chiral separation of substituted phenylalanine analogues using chiral palladium phosphine complexes with enantioselective liquid-liquid extraction.

Chiral palladium phosphine complexes have been employed in the chiral separation of amino acids and phenylalanine analogues in particular. The use of (S)-xylyl-BINAP as a ligand for the palladium complex in enantioselective liquid-liquid extraction allowed the separation of the phenylalanine analogues with the highest operational selectivity reported to date. (31)P NMR, FTIR, FIR, UV-Vis, CD and Raman spectroscopy methods have been applied to gain insight into the binding mechanism of the amino acid substrates with the chiral palladium phosphine complexes. A complexation in a bidentate fashion is proposed.

Process evaluation data supporting studies on swing strategies to recover N-ethylbutylamine after wet lipid extraction from microalgae.

In this paper, we publish information that has not been published before, but is needed to evaluate processes for wet lipid extraction from microalgae and recover the solvent N-ethylbutylamine (EBA), for example as presented in [1], the article entitled "Process evaluation of swing strategies to recover N-ethylbutylamine after wet lipid extraction from microalgae" in which we evaluate and interpret temperature swing and CO2-swing approaches. This includes selection of microalgae slurry concentration used in the extraction process, information on switching of EBA with CO2, data on the amount of EBA in solid residue after extraction, recoverability from the solid residue, and on recoverability of the solvent from the aqueous raffinate by liquid-liquid extraction and distillation of the solvent and EBA after the liquid-liquid extraction. Also information on phase behavior of binary mixtures of EBA and water is presented. Finally, detailed information on all flows in the process flow diagrams that are given in the article [1] is presented.

Thermal Activity in Affinity Separation Techniques Such as Liquid-Liquid Extraction Analyzed by Isothermal Titration Calorimetry and Accuracy Analysis of the Technique in the Molar Concentration Domain.

The applicability and accuracy of isothermal titration calorimetry (ITC) to investigate intermolecular interactions in a high concentration domain applicable to liquid-liquid extraction (LLX) was studied for acid-base interactions. More accurate fits can be obtained using a sequential binding mechanism compared to a single reaction model, at the risk of finding a local minimum. Experiments with 0.24 M tri-n-octylamine (TOA) resulted in a residue of fit of 4.3% for the single reaction model, with a standard deviation σ of 1.6% in the stoichiometry parameter n, 12% in the complexation constant K n,1, and 2.5% in the enthalpy ΔH n,1. For the sequential model, σ was higher: 11% in K 1,1, 26% in K n+1,1, and 12% in ΔH n+1,1. This study clearly showed that, at higher concentrations (order of moles per liter), accurate parameter estimation is possible and parameter values are concentration dependent. It is thus important to do ITC at the application concentration.

CO2-Switchable Solvents as Entrainer in Fluid Separations.

CO2-switchable solvents, typically neutral solvents that switch with CO2 into ionic species, were investigated for use as entrainer in fluid separations such as extractive distillation. Their switchable nature was investigated, which may facilitate liquid-liquid extraction or extractive distillation as ionic liquid (IL), whereas during regeneration their decarboxylation into the amine form prevents temperature shoot-up. Studied elements included a property screening and detailed mechanistic and kinetic studies on the switching of 2-ethylhexylamine and N,N-benzyl methylamine. Decarboxylation of a 50 vol % switchable solvent, 25 vol % heptane, and 25 vol % toluene mixture at 1.00 × 104 Pa showed a 40% CO2 release before reaching the operational pressure, and total decarboxylation took over 2 h. An effective increase of the relative volatility of heptane/toluene was found, showing that, indeed, CO2-switchable solvents can be applied for extractive distillation. However, low pressure in combination with elevated temperature will lead to quick decarboxylation, limiting the operational window of this class of solvents. Their use in low temperature application such as C4-distillations or liquid-liquid extraction appears more suited.

Maximizing Lipid Yield in Neochloris oleoabundans Algae Extraction by Stressing and Using Multiple Extraction Stages with N-Ethylbutylamine as Switchable Solvent.

The extraction yield of lipids from nonbroken Neochloris oleoabundans was maximized by using multiple extraction stages and using stressed algae. Experimental parameters that affect the extraction were investigated. The study showed that with wet algae (at least) 18 h extraction time was required for maximum yield at room temperature and a solvent/feed ratio of 1:1 (w/w). For fresh water (FW), nonstressed, nonbroken Neochloris oleoabundans, 13.1 wt % of lipid extraction yield (based on dry algae mass) was achieved, which could be improved to 61.3 wt % for FW stressed algae after four extractions, illustrating that a combination of stressing the algae and applying the solvent N-ethylbutylamine in multiple stages of extraction results in almost 5 times higher yield and is very promising for further development of energy-efficient lipid extraction technology targeting nonbroken wet microalgae.

Recovery of Volatile Fatty Acids from Fermented Wastewater by Adsorption.

Separation of volatile fatty acids (VFAs) from fermented wastewater is challenging, due to low VFA concentrations in mineral-rich streams. As a result, separation capacity and selectivity with traditional solvents and adsorbents are both compromised. In this study, using a complex artificial model solution mimicking real fermented wastewaters, it is shown that a simple and robust adsorption-based separation technique can retain a remarkable capacity and selectivity for VFAs. Four types of polystyrene-divinylbenzene-based resins (primary, secondary, and tertiary amine-functionalized, and nonfunctionalized) were examined as the adsorbents. The presence of chloride, sulfate, and phosphate salts resulted in coadsorption of their acidic forms HCl, H2SO4, and H3PO4 on amine-functionalized adsorbents, and severely reduced the VFA capacity. With the nonfunctionalized adsorbent, almost no mineral acid coadsorption was observed. This together with a high total VFA capacity of up to 76 g/kg in equilibrium with the model solution containing a total VFA concentration of 1 wt % resulted in a very high selectivity for the VFAs. Nitrogen-stripping with various temperature profiles was applied to regenerate the adsorbent, and study the potential for fractionation of the VFAs during regeneration. Butyric acid (HBu) was obtained in mole fractions of up to 0.8 using a stepwise increase in the stripping temperature from 25 °C via 120 to 200 °C. During four successive adsorption-regeneration cycles, no reduction in the adsorption capacity was observed.

Aromatics extraction from pyrolytic sugars using ionic liquid to enhance sugar fermentability.

Fermentative bioethanol production from pyrolytic sugars was improved via aromatics removal by liquid-liquid extraction. As solvents, the ionic liquid (IL) trihexyltetradecylphosphonium dicyanamide (P666,14[N(CN)2]) and ethyl acetate (EA) were compared. Two pyrolytic sugar solutions were created from acid-leached and untreated pinewood, with levoglucosan contents (most abundant sugar) of 29.0% and 8.3% (w/w), respectively. In a single stage extraction, 70% of the aromatics were effectively removed by P666,14[N(CN)2] and 50% by EA, while no levoglucosan was extracted. The IL was regenerated by vacuum evaporation (100mbar) at 220°C, followed by extraction of aromatics from fresh pyrolytic sugar solutions. Regenerated IL extracted aromatics with similar extraction efficiency as the fresh IL, and the purified sugar fraction from pretreated pinewood was hydrolyzed to glucose and fermented to ethanol, yielding 0.46g ethanol/(g glucose), close to the theoretical maximum yield.

A synthetic strategy for novel nonsymmetrical bola amphiphiles based on carbohydrates.

A number of novel nonionic bolaform amphiphiles with nonidentical aldityl head groups, 1-(1-deoxy-D-galactitol-1-ylamino)-6-(1-deoxy-D-glucitol-1-ylamino)hexane, 1-(1-deoxy-D-mannitol-1-ylamino)-6-(1-deoxy-D-glucitol-1-ylamino)hexane, and 1-(1-deoxy-D-galactitol-1-ylamino)-6-(1-deoxy-D-mannitol-1-ylamino)hexane were synthesized by two successive reductive aminations involving 1,6-diaminohexane (1) and the appropriate D-aldohexoses (D-glucose, D-mannose, and D-galactose) using 5% Pd on carbon as the catalyst. Typical reaction conditions were 40 degrees C, 4MPa hydrogen and a reaction time of 4.5 h. The compounds were isolated as white solids in yields ranging from 39% to 72%. The intermediate aminoalditols, 1-(1-deoxy-D-glucitol-1-ylamino)-6-aminohexane and 1-(1-deoxy-D-galactitol-1-ylamino)-6-aminohexane were obtained as off-white solids in 80-85% yield. The bolaform amphiphiles containing 1-deoxy-D-glucitol head group(s) showed markedly lower melting points than the compounds with the 1-deoxy-D-mannitol and 1-deoxy-D-galactitol head groups, due to the presence of 1,3-syn interactions within the carbohydrate moiety. The novel bolaform compounds are potential starting materials for the synthesis of a broad range of gemini surfactants with nonidentical, carbohydrate-based head groups.

Multi-responsive ionic liquid emulsions stabilized by microgels.

We present a complete toolbox to use responsive ionic liquid (IL) emulsions for extraction purposes. IL emulsions stabilized by responsive microgels are shown to allow rapid extraction and reversible breaking and re-emulsification. Moreover, by using a paramagnetic ionic liquid, droplets can be easily collected in low magnetic fields.