Potential of stable isotope analysis to deduce anaerobic biodegradation of ethyl tert-butyl ether (ETBE) and tert-butyl alcohol (TBA) in groundwater: a review.

Understanding anaerobic biodegradation of ether oxygenates beyond MTBE in groundwater is important, given that it is replaced by ETBE as a gasoline additive in several regions. The lack of studies demonstrating anaerobic biodegradation of ETBE, and its product TBA, reflects the relative resistance of ethers and alcohols with a tertiary carbon atom to enzymatic attack under anoxic conditions. Anaerobic ETBE- or TBA-degrading microorganisms have not been characterized. Only one field study suggested anaerobic ETBE biodegradation. Anaerobic (co)metabolism of ETBE or TBA was reported in anoxic microcosms, indicating their biodegradation potential in anoxic groundwater systems. Non-isotopic methods, such as the detection of contaminant loss, metabolites, or ETBE- and TBA-degrading bacteria are not sufficiently sensitive to track anaerobic biodegradation in situ. Compound- and position-specific stable isotope analysis provides a means to study MTBE biodegradation, but isotopic fractionation of ETBE has only been studied with a few aerobic bacteria (εC -0.7 to -1.7‰, εH -11 to -73‰) and at one anoxic field site (δ2H-ETBE +14‰). Similarly, stable carbon isotope enrichment (δ13C-TBA +6.5‰) indicated TBA biodegradation at an anoxic field site. CSIA and PSIA are promising methods to detect anaerobic ETBE and TBA biodegradation but need to be investigated further to assess their full potential at field scale.

Efficient three phase partitioning of actinidin from kiwifruit (Actinidia deliciosa) and its characterization.

Three phase partitioning (TPP) method was effectively utilized for the extraction and purification of milk clotting protease (actinidin) from the kiwifruit pulp. The different purification parameters of TPP such as ammonium sulfate saturation, ratio of the crude kiwifruit extract to tert-butanol, and the pH value of extract were optimized. The 40% (w/v) salt saturation having 1.0:0.75 (v/v) ratio of crude kiwifruit extract to tert-butanol at 6.0 pH value exhibited 3.14 purification fold along with 142.27% recovery, and the protease was concentrated exclusively at intermediate phase (IP). This fraction showed milk-clotting activity (MCA), but there was no such activity in lower aqueous phase (AP). The enzyme molecular weight was found to be 24 kDa from Tricine SDS-PAGE analysis. Recovered protease demonstrated greater stability at pH 7.0 and temperature 50 °C. The Vmax and Km values were 121.9 U/ml and 3.2 mg/ml respectively. Its cysteine nature was demonstrated by inhibition studies. This study highlighted that the TPP is an economic and effective method for extraction and purification of actinidin from kiwifruit, and it could be used as a vegetable coagulant for cheesemaking.

Non-salt based co-amorphous formulation produced by freeze-drying.

Amino acids-based co-amorphous system (CAM) has shown to be a promising approach to overcome the dissolution challenge of biopharmaceutics classification system class II drugs. To date, most CAM formulations are based on salt formation at a 1:1 M ratio and are prepared by mechanical activation. However, its use in medicinal products is still limited due to the lack of in-depth understanding of non-ionic based molecular interactions. There are also limited studies on the effect of drug-to-co-former ratio, the development of more scalable, less aggressive, manufacturing processes such as freeze drying and its dissolution benefits. This work aims to investigate the effect of the ratio of tryptophan (a model non-ionic amino acid) to indomethacin (a model drug) on a non-salt-based CAM prepared via freeze-drying with the tert-butyl alcohol-water cosolvent system. The CAM material was systemically characterized at various stages of the freeze-drying process using DSC, UV-Vis, FT-IR, NMR, TGA and XRPD. Dissolution performance and physical stability upon storage were also investigated. Freeze-drying using the cosolvent system has been successfully shown to produce CAMs. The molecular interactions involving H-bonding, H/π and π-π between compounds have been confirmed by FT-IR and NMR. The drug release rate for formulations with a 1.5:1 drug: amino acid molar ratio (or 1:0.42 wt ratio) or below is found to be significantly improved compared to the pure crystalline drug. Furthermore, formulation with a 2.3:1 drug:amino acid molar ratio (or 1:0.25 wt ratio) or below have shown to be physically stable for at least 9 months when stored at dry condition (5% relative humidity, 25 °C) compared to the pure amorphous indomethacin. We have demonstrated the potential of freeze-drying using tert-butyl alcohol-water cosolvent system to produce an optimal non-salt-based class II drug-amino acid CAM.

Mechanistic Investigation of tert-Butanol's Impact on Biopharmaceutical Formulations: When Experiments Meet Molecular Dynamics.

The use of tert-butyl alcohol for the lyophilization of pharmaceuticals has seen an uptick over the past years. Its advantages include increased solubility of hydrophobic drugs, enhanced product stability, shorter reconstitution time, and decreased processing time. While the mechanisms of protein stabilization exerted by cryo- and lyo-protectants are well known when water is the solvent of choice, little is known for organic solvents. This work investigates the interactions between two model proteins, namely, lactate dehydrogenase and myoglobin, and various excipients (mannitol, sucrose, 2-hydroxypropyl-ß-cyclodextrin and Tween 80) in the presence of tert-butyl alcohol. We thermally characterized mixtures of these components by differential scanning calorimetry and freeze-drying microscopy. We also spectroscopically evaluated the protein recovery after freezing and freeze-drying. We additionally performed molecular dynamics simulations to elucidate the interactions in ternary mixtures of the herein-investigated excipients, tert-butyl alcohol and the proteins. Both experiments and simulations revealed that tert-butyl alcohol had a detrimental impact on the recovery of the two investigated proteins, and no combination of excipients yielded a satisfactory recovery when the organic solvent was present within the formulation. Simulations suggested that the denaturing effect of tert-butyl alcohol was related to its propensity to accumulate in the proximity of the peptide surface, especially near positively charged residues.

Importance of Chain Length in Propagation Reaction on •OH Formation during Ozonation of Wastewater Effluent.

During the ozonation of wastewater, hydroxyl radicals (•OH) induced by the reactions of ozone (O3) with effluent organic matters (EfOMs) play an essential role in degrading ozone-refractory micropollutants. The •OH yield provides the absolute •OH formation during ozonation. However, the conventional "tert-Butanol (t-BuOH) assay" cannot accurately determine the •OH yield since the propagation reactions are inhibited, and there have been few studies on •OH production induced by EfOM fractions during ozonation. Alternatively, a "competitive method", which added trace amounts of the •OH probe compound to compete with the water matrix and took initiation reactions and propagation reactions into account, was used to determine the actual •OH yields (Φ) compared with that obtained by the "t-BuOH assay" (φ). The Φ were significantly higher than φ, indicating that the propagation reactions played important roles in •OH formation. The chain propagation reactions facilitation of EfOMs and fractions can be expressed by the chain length (n). The study found significant differences in Φ for EfOMs and fractions, precisely because they have different n. The actual •OH yield can be calculated by n and φ as Φ = φ (1 + n)/(nφ + 1), which can be used to accurately predict the removal of micropollutants during ozonation of wastewater.

Collaborative Purification of Tert-Butanol and N2O over Fe/Co-Zeolite Catalysts.

N2O is a greenhouse gas and a candidate oxidant. Volatile organic pollutants (VOCs) have caused great harm to the atmospheric ecological environment. Developing the technique utilizing N2O as the oxidant to oxidize VOCs to realize the collaborative purification has significant importance and practical value for N2O emission control and VOC abatement. Therefore, the study of N2O catalytic oxidation of tert-butanol based on zeolite catalysts was carried out. A series of molecular sieves, including FER, MOR, ZSM-5, Y, and BEA, were selected as the catalyst objects, and the 1.5% wt Fe and Co were, respectively, loaded on the zeolite catalysts via the impregnation method. It was found that the catalytic performance of BEA was the best among the molecular sieves. Comparing the catalytic performance of Fe-BEA under different load gradients (0.25~2%), it was found that 1.5% Fe-BEA possessed the best catalytic activity. A series of characterization methods showed that Fe3+ content in 1.5% Fe-BEA was the highest, and more active sites formed to promote the catalytic reaction. The α-O in the reaction eventually oxidized tert-butanol to CO2 over the active site. The Co mainly existed in the form of Co2+ cations over Co-BEA samples; the 2% Co-BEA possessing higher amounts of Co2+ exhibited the highest activity among the prepared Co-BEA samples.

Experimental study on the treatment of dye wastewater by plasma coupled biotechnology.

In this experiment, a gas-liquid two-phase discharge water treatment inverse device was designed independently to treat the actual workshop intermediate dye wastewater from a chemical plant. Firstly, the effects of initial concentration of wastewater, initial pH, circulation flow rate of solution, content of Fe2+, content of H2O2, and addition of tert-butanol on the organic removal rate and decolorization rate of dye wastewater treatment were investigated. The results showed that Fe2+ and tert-butanol would react with the active particles (H2O2, ·OH) and inhibit the degradation of the dye wastewater, resulting in the decrease of both organic matter degradation rate and decolorization rate. The experimentally degraded dye wastewater mainly contained benzoic acid and its derivatives in addition to dye molecules, thus the degradation mechanism of benzoic acid was mainly analyzed. Then, the actual dye wastewater treated by low-temperature plasma was combined with the traditional biological treatment technology. The biochemical properties of the wastewater treated by low-temperature plasma technology were greatly improved, and the B/C was increased from the initial 0.17 to 0.33. The effluent after the combined biological method could meet the effluent discharge standard, and the final CODcr reached 198 mg/L, BOD5 reached 65 mg/L, and pH and chromaticity reached 6.39 and 50, respectively.

Extraction and purification of grape seed polysaccharides using pH-switchable deep eutectic solvents-based three-phase partitioning.

T-butanol is widely used in three-phase partitioning (TPP), which is harmful to the environment. pH-switchable deep eutectic solvents (DESs) can be used as recyclable alternatives to t-butanol. This study aimed to construct DES-based TPP for extracting and purifying grape seed polysaccharides (GSP). The main influence factors were investigated in single-factor experiments. DES-1 (dodecanoic acid: octanoic acid = 1:1)-based extraction was screened, and the extraction yield reached the maximum of 98.04 mg/g under the optimal conditions. Furthermore, DES can be recycled, only suffering a small loss capacity in extraction yield after 25 cycles. Most importantly, the extractability of DES could be completely recovered after switching and regeneration. The molecular weight of obtained GSP was 60 kDa, and the main monosaccharides of GSP included mannose, glucose, galactose, and arabinose. This study provides an efficient and sustainable method for the extraction of bioactive substances.

Structure and dynamics of the hyaluronan oligosaccharides and their solvation shell in water: organic mixed solvents.

Hyaluronan (HA) is a natural polysaccharide occurring ubiquitously in the connective tissues of vertebrates widely used in the cosmetic and pharmaceutic industries. In numerous applications HA oligosaccharides are being chemically modified using reactions incompatible with aqueous solutions, often carried out in water:organic mixed solvents. We carry out molecular-dynamics (MD) simulations of HA oligosaccharides in water:1,4-dioxane and water:tert-butanol mixtures of different compositions. HA molecule causes a separation of the solvent components in its surroundings, especially in tert-butanol containing solutions, constituting thus a solvation shell enriched by water. Furthermore, interactions with ions are stronger than in pure water and depend on the solvent composition. Consequently, the dynamics of the HA chain varies with the solvent composition and causes observable conformational changes of the HA oligosaccharide. Composition of mixed solvents thus enables us to modify the interaction of HA with other molecules as well as its reactivity.

Acyl migration of 2-monoacylglycerols rich in DHA: Effect of temperature and solvent medium.

Acyl migration of 2-monoacylglycerols (2-MAGs) rich in DHA is a universal reaction occurring during storage and structural lipid synthesis, and affects their nutritional value. In this study, their acyl migration was investigated under different systems and temperatures. The enhanced temperature promoted acyl migration, leading to a 5.6-fold increase from 20 °C to 50 °C. The kinetic study indicated rate constants followed the order: hexane > solvent-free > dichloromethane > ethanol ≈ acetone ≈ acetonitrile > t-butanol, and positively correlated with log P of solvent. During acyl migration in ethanol, acetone, acetonitrile and t-butanol at 40 °C, DHA content in 2-MAGs was higher than in 1-MAGs, indicating slow acyl migration of DHA; while at 50 °C, the difference of DHA distribution was small, due to increasing acyl migration rate. The results suggest that acyl migration of different fatty acids can be regulated by changing conditions to enrich DHA at sn-2 position.

Three-phase partitioning, a recyclable method for the purification of R-phycoerythrin from a red algae Porphyra yezoensis.

In this study, R-phycoerythrin (R-PE) was isolated and characterized from Porphyra yezoensis by three-phase partitioning (TPP) method. The effects of temperature, time, pH, salt saturation, and volume ratio on the purity and recovery rate of R-PE were studied. The optimum extraction conditions were determined as follows: salt saturation of 70%, temperature of 25 °C, time of 45 min, pH of 7.0, and volume ratio of 1:1. Under the optimal extraction conditions, the purity of R-PE was 3.90. The results of SDS-PAGE showed that R-PE has three bands at 23 kDa, 22 kDa, and 18 kDa, corresponding to its α, ß, γ subunits. The structure and optical activity of R-PE did not change before and after purification based on ultraviolet, infrared, and fluorescence spectra. In addition, the purity and recovery rate of R-PE extracted by tert-butanol were evaluated. The results showed that the extraction performance of tert-butanol for R-PE remained unchanged in three recoveries. These show that TPP is an efficient, green, and recyclable extraction technology.

Liquid-liquid equilibrium experiment and mechanism analysis of menthol-based deep eutectic solvents extraction for separation of fuel additive tert-butanol.

Deep eutectic solvents (DESs) were synthesized using menthol as hydrogen bond acceptor (HBA) and different carbon chain carboxylic acids as hydrogen bond donors (HBD). The liquid equilibrium (LLE) experiment was used to determine the distribution coefficient (ß) and slectivity (S) at standard atmospheric pressure and temperature. The effect of DESs on the separation efficiency was discussed by changing the proportion. Non-random two fluid (NRTL) model was used to correlate the experimental data. The molecular dynamics (MD) simulation method was used to investigate the micro mechanism of the extraction process. The results show van der Waals force plays a leading role in the interaction between solvents and tert-butyl alcohol (TBA) and week force with water. Compared with experimental and simulation results, the interaction between DESs and TBA would also be affected by the change of the number of HBD carbon chains, and DESs with decanoic acid as HBD has the best separation effect, which verifies the feasibility of separating high alcohol compounds from water by DESs and then treating them by DESs.

Estimating the mineral density of trabecular bone using Compton scattering.

This study proposes a technique based on Compton scattering to estimate trabecular bone mineral density (TBMD), which is important for understanding bone strength, and hence, is pivotal for estimating the condition of the bone. Bone phantoms (a mixture of paraffin wax and bone powder) with various concentrations of bone ash were prepared to simulate the trabecular bone. These samples were exposed to primary gamma photon flux from a137Cs (222 GBq) radioisotope source one after the other, and the scattered photon flux was detected using an NaI(Tl) detector. The presence of the cortical bone (using aluminum sheets) and fat (tertiary butyl alcohol) around the trabecular bone was also studied to determine whether the TBMD measurements had been affected. The correlation between bone ash contents and the intensity of Compton scattering was high with a coefficient of 0.97. The outcomes suggest that TBMD is independent of the presence of the cortical bone and overlying fat, with a statistical uncertainty of ±0.3% in the count rate. The intensity of Compton scattering increased by only 1.5% when the thickness of the aluminum sheet (simulating the cortical bone) becomes was increased by four times, and by less than 5% when the bone phantom was surrounded by tertiary butyl alcohol.

Electronic fluctuation difference between trimethylamine N-oxide and tert-butyl alcohol in water.

Although small organic molecules in cells have been considered important to control the functions of proteins, their electronic fluctuation and the intermolecular interaction, which is physicochemical origin of the molecular functions, under physiological conditions, i.e., dilute aqueous solutions (0.18 mol L-1), has never been clarified due to the lack of observation methods with both accuracy and efficiency. Herein, the time evolutions of the interactions in dilute aqueous trimethylamine N-oxide (TMAO) and tert-butyl alcohol (TBA) solutions were analyzed via ab initio molecular dynamics simulations accelerated with the fragment molecular theory. It has been known that TMAO and TBA have similar structures, but opposite physiological functions to stabilize and destabilize proteins. It was clarified that TMAO induced stable polarization and charge-transfer interactions with water molecules near the hydrophilic group, and water molecules were caught even near the CH3- group. Those should affect protein stabilization. Understanding the solution dynamics will contribute to artificial chaperone design in next generation medicine.

Deciphering the binding mechanism of inhibitors of the SARS-CoV-2 main protease through multiple replica accelerated molecular dynamics simulations and free energy landscapes.

The pneumonia outbreak caused by the SARS-CoV-2 virus poses a serious threat to human health and the world economy. The development of safe and highly effective antiviral drugs is of great significance for the treatment of COVID-19. The main protease (Mpro) of SARS-CoV-2 is a key enzyme for viral replication and transcription and has no homolog in humans. Therefore, the Mpro is an ideal target for the design of drugs against COVID-19. Insights into the inhibitor-Mpro binding mechanism and conformational changes of the Mpro are essential for the design of potent drugs that target the Mpro. In this study, we analyzed the conformational changes of the Mpro that are induced by the binding of three inhibitors, YTV, YSP and YU4, using multiple replica accelerated molecular dynamics (MR-aMD) simulations, dynamic cross-correlation map (DCCM) calculations, principal component analysis (PCA), and free energy landscape (FEL) analysis. The results from DCCM calculations and PCA show that the binding of inhibitors significantly affects the kinetic behavior of the Mpro and induces a conformational rearrangement of the Mpro. The binding ability and binding mechanism of YTV, YSP and YU4 to the Mpro were investigated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The results indicate that substitution of the tert-butanol group by methylbenzene and trifluoromethyl groups enhances the binding ability of YSP and YU4 to the Mpro compared with YTV; moreover, massive hydrophobic interactions are detected between the inhibitors and the Mpro. Meanwhile, T25, L27, H41, M49, N142, G143, C145, M165, E166 and Q189 are identified as the key residues for inhibitor-Mpro interactions using residue-based free energy decomposition calculations, which can be employed as efficient targets in the design of drugs that inhibit the activity of the Mpro.

Improved Synthesis of the Amine Fragment of FR901464 and Thailanstatins through the Development of a Convenient N-Detosylation Method.

FR901464 and thailanstatins are potent cytotoxic natural products that share an amine-containing tetrahydropyran ring. We previously reported the synthesis of the tetrahydropyran component. Here, we changed the protecting group for the amine from Boc to tosyl, improving yields and the time economy. A highlight of the revised synthetic scheme is the use of lithium, t-butanol, and ethylenediamine in THF (nontraditional Birch reduction conditions) for the N-detosylation.

Insight into the performance of UV/chlorine/TiO2 on carbamazepine degradation: The crucial role of chlorine oxide radical (ClO•).

The UV/chlorine (UC) system is a homogeneous advanced oxidation process with increasing attention in water decontamination. The addition of TiO2 is a newly found strategy to enhance the generation of hydroxyl radical (HO•) and chlorine radical (Cl•) in the UC system. However, the crucial role of chlorine oxide radical (ClO•, generated by the reactions of HO• and Cl• with chlorine) on pollutant degradation, has not been noticed in UV/chlorine/TiO2 (UCT), the heterogeneous photocatalytic system for chlorine activation. Herein, the role of ClO• in UCT was clarified through quenching experiments combined with model simulations during carbamazepine degradation. Tert-butyl alcohol completely inhibited while bicarbonate only partly suppressed carbamazepine degradation in UCT, indicating the important role of ClO•. The second-order reaction rate constant between ClO• and carbamazepine (kClO•,carbamazepine) was fitted to be (1.21 ± 0.08) × 107 M-1 s-1 by the kinetic model, which avoided the influence of carbonate radical (CO3•-), whose contribution couldn't be excluded during kClO•,carbamazepine determination in commonly used competitive kinetic methods with bicarbonate. With the obtained kClO•,carbamazepine, model simulation suggested that ClO• contributed about 50 % to carbamazepine degradation in UCT, and its concentration was less affected under varied conditions (solution pH, chlorine, bicarbonate, and chloride concentration) to keep an efficient carbamazepine degradation. On the contrary, pollutant degradation dominated by HO• in UCT was largely inhibited with the increase of pH, chlorine, and bicarbonate concentration. In addition to the promotion of degradation efficiency, less disinfection byproducts and lower energy requirement were found in UCT compared with UC. Furthermore, UCT could maintain satisfactory degradation efficiency and energy saving in ground water and surface water samples. Results of this study unraveled the crucial role of ClO• for pollutant degradation in UCT, and showed bright prospects and great potentials of the system in water treatment.

Nano- and micro-scale zerovalent iron-activated peroxydisulfate for methyl phenyl sulfoxide probe transformation in aerobic water: Quantifying the relative roles of SO4·-, Fe(IV), and ·OH.

Multiple reactive intermediates have been proposed to be involved in peroxydisulfate (PDS) activation by zerovalent iron (ZVI), including sulfate radical (SO4·-) produced via iron-oxide shell mediated electron transfer, ferryl ion species (Fe(IV)) formed from Fe(II)-PDS interaction, and hydroxyl radical (·OH) generated by ZVI aerobic oxygenation. In this study, evolution of the relative role of these intermediates in microscale and nanoscale ZVI (mZVI vs. nZVI) activated PDS processes is comparatively investigated by using a methyl phenyl sulfoxide (PMSO) probe that selectively reacts with Fe(IV) to produce methyl phenyl sulfone (PMSO2). Interestingly, during PMSO transformation by mZVI/PDS process, yields of PMSO2 (η(PMSO2)) exhibit three-stage behavior that they first increase to a maximum (∼80% but lower than 100%) (Stage I) and then plateau for a period (Stage II) followed by a decrease phase (Stage III). Accordingly, the relative role of Fe(IV) in PMSO transformation is unceasingly improved in Stage I and subsequently reaches equilibrium with that of free radicals in Stage II, while it finally decreases in Stage III. Similar η(PMSO2) evolution trends are obtained in nZVI/PDS process, except that the η(PMSO2) increase in Stage I is negligible, possibly due to the exceptional fast nZVI dissolution. It was further clarified by tert-butyl alcohol scavenging assay that, in addition to Fe(IV), the free radical involved in Stages I and II is SO4·-, while ·OH was dominant in Stage III. Moreover, studies on O2 effect reveal that ZVI aerobic oxygenation participates in mZVI corrosion during the entire process, while it is only involved in nZVI corrosion when PDS content is reduced to a low concentration, indicating that the reactivities of PDS and O2 are similar in mZVI corrosion, but differ greatly in nZVI corrosion. Additionally, effects of reactant dose and pH on η(PMSO2) evolution are also explored. Dynamics of the relative role of different reactive oxidants should be taken into account in further applications of ZVI/PDS in situ chemical remediation technology considering their different chemistries.

Fabrication, characterization, and purification of nutraceutical diacylglycerol components from Camellia oil.

Converting triacylgycerols (TAGs) from edible oils and fats into structured diacylglycerols (DAGs) is meaningful for reducing obesity. Camellia oil, rich in linoleic acid, has the potential to form structured linoleic acid-1,3-diacylglycerol (LA-1,3-DAG) nutrients in the industry. In this research, the physicochemical properties of modified Camellia oil (MCO) by enzymatic esterification were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS), Differential Scanning Calorimetry (DSC), High Performance Liquid Chromatography-Evaporative Light Scattering Detection (HPLC-ELSD), and Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). The relationship between reaction conditions and the DAG compositions is disclosed using multiple factors. It is found that high constituents of DAG increase the melting and crystallization temperature of MCO, lipase Novozym 435 gives the best yield of targeted nutrients (DAG, 1,3-DAG, LA-DAG), and the mixture of lipases, Lipozyme TL IM and Lipozyme RM IM, shows a synergistic effect in the synthetic process of DAG. Subsequently, MCO containing 65.4% DAG, 54.7% LA-DAG, and 47.6% 1,3-DAG content at optimal conditions (2% enzyme dosage, 4 h reaction time, 2.4:1 substrate molar ratio, 25.8% t-butanol as solvent, 60°C temperature) has been obtained and purified using silica column to obtain the final DAG oil containing 96.1% DAG, 64.7% 1,3-DAG, and 78.4% LA-DAG. High constituents of structured DAG oil rich in LA-1,3-DAG can be obtained by enzymatic esterification for industrial production.