High Concentrations Of Glycerol Research Articles

Kinetic Modelling of Ralstonia eutropha H16 Growth on Different Substrates

Due to environmental pollution and the depletion of fossil fuels, there is growing interest in the development and use of biofuels as environmentally friendly alternatives. One of the most promising biofuels is biohydrogen, hydrogen produced through sustainable processes using microorganisms such as bacteria and algae. One of the most interesting bacteria for hydrogen production is Ralstonia eutropha H16, known for its ability to produce oxygen-tolerant hydrogenases. These enzymes play a crucial role in biohydrogen metabolism and production. The aim of this work was to determine the optimal conditions (reactor type and synthetic medium composition) for the cultivation of R. eutropha H16. The culture media contained different concentrations of fructose and glycerol (mono- or double-substrate cultivation) and the experiments were carried out in a batch reactor. The initial experiments were carried out with 4 g/L fructose or glycerol in the culture medium at pH 7, T = 30 °C, and 120 rpm. The mathematical model, consisting of the growth kinetics (described by the Monod’s model) and the corresponding mass balances, was proposed. The developed model was validated using two independent experiments with different initial substrate concentrations: 2 g/L glycerol and fructose in one medium and 4 g/L fructose and 1 g/L glycerol in the second. In order to propose the optimal cultivation procedure for future research, the mathematical model simulations were performed for different reactor types (batch, fed-batch, and continuous stirred tank reactors) and different initial substrate concentrations. The most successful experiment was the one with 4 g/L glycerol, where γX = 0.485 ± 0.001 g/L of biomass was achieved. Further calculations showed that the most biomass would be produced at higher glycerol concentrations (at γG = 6.358 g/L, γX = 1.311 g/L should be achieved after 200 h of cultivation) and when using a fed-batch reactor (γX = 0.944 g/L after 200 h of cultivation).

Oral Bacteria Counter Using Dielectrophoretic Impedance Measurement: Usefulness and Usage Considerations.

The oral cavity hosts numerous bacteria that are associated with various systemic diseases. The Oral Bacteria Counter (PHC Corporation, Tokyo, Japan), a microorganism quantitative analyzer that utilizes dielectrophoretic impedance measurements, enables rapid bacterial counting and is widely used in dental practice in Japan. However, it may also detect nonviable bacteria. This study aimed to assess the impact of disinfectants, electrolytes, and viscosity on the accuracy of the Oral Bacteria Counter and to determine whether it measures non-viable bacteria similarly to viable bacteria. To evaluate the effect of the disinfectants, samples of 7% povidone-iodine (PV-I), 0.2% benzethonium chloride, 5% chlorhexidine (CHX), 0.2% CHX, 0.05% CHX, sterile water, and saline were measured using the Oral Bacteria Counter. The effect of viscosity was assessed by mixing sterile water with glycerol in various ratios and measuring the dielectrophoretic impedance of the bacterial counts at different viscosities. For the electrolyte effects, samples of Staphylococcus aureus diluted in sterile water or saline were measured using the Oral Bacteria Counter. Additionally, samples of 7% PV-I or 5% CHX diluted in sterile water or saline were measured. Bacterial counts were then measured and compared using the Oral Bacteria Counter, our developed delayed real-time polymerase chain reaction (DR-PCR) method (which quantifies only viable bacteria), and culture methods. Disinfectants such as 5% CHX and 7% PV-I produced high readings on the Oral Bacteria Counter, even when no viable bacteria were present. Higher glycerol concentrations, which increased the viscosity, resulted in lower bacterial counts. The presence of electrolytes, particularly saline, led to higher readings on the Oral Bacteria Counter, which detected both viable and non-viable bacteria, whereas DR-PCR and culture methods did not detect non-viable bacteria. The Oral Bacteria Counter may be influenced by disinfectants, viscosity, and electrolytes, leading to potential inaccuracies in bacterial quantification. For accurate bacterial measurements, it is essential to consider these factors and ideally combine the results from the Oral Bacteria Counter with methods such as DR-PCR for more reliable assessment.

Синтез биоразлагаемого пластика из банановой кожуры с глицерином в качестве пластификатора

Biodegradable plastic from banana peel is durable and transparent. It breaks down naturally in the environment and can substitute traditional petroleum plastic, which is a source of pollution due to its slow degradation. This research is intended to improve the physical properties of biodegradable film obtained by the casting solution method from an Aceh variety of wak banana peel starch with glycerol as a plasticizer. The authors relied on a factorial completely randomized design with two replications. The variables included the concentrations of wak banana peel starch (6, 8, and 10%) and glycerol (2, 5, and 8%). The data were subjected to the analysis of variance (ANOVA). The physical tests covered tensile strength, elongation, water absorption, and biodegradation. The functional groups of biodegradable films were analyzed using Fourier-transform infrared spectroscopy (FTIR). The morphological structure was studied by scanning electron microscopy (SEM). The biodegradation test lasted for two and four days. The sample with less banana peel starch (6–8%) degraded faster. Higher glycerol concentrations (5–15%) affected the weight of the samples. The plastic samples with 15% glycerol degraded faster than the samples with minimal glycerol amount. A greater concentration of wak banana peel starch significantly affected tensile strength and elongation while the effect on water content and water absorption capacity was insignificant. Glycerol concentration affected water content and tensile strength, but had no significant effect on water absorption capacity and elongation. The ratio between the concentrations of wak banana peel starch and glycerol had a significant effect on tensile strength and water absorption capacity. The best results belonged to the sample with 8% wak banana peel starch and 2% glycerol. The research provided new options for utilizing banana peels as biodegradable packaging and an alternative to traditional plastic. The commercialization and scalability of this ecologically friendly plastic require furth er research.

Production of Defragmentable Bioplastic using Starch Extracted from Annona Muricata (Soursoup) Seed Reinforced with Bentonite Nanoclay

Abstract Seeds of Annona Muricata, often called soursop or guyabano were used as a source of starch for the production of defragmentable bioplastic. The researchers used mixture of water and starch content of 10 and 20 grams respectively from Annona Muricata (soursop) seeds with various concentration (20%, 25%, 30%) of glycerol (plasticizer) and reinforced with three different concentrations (0%, 3%, 6%) of bentonite nanoclay (filler). The characterization of the bioplastic was carried out by determining the effect of variation of the ratio between the quantity of starch from soursop seeds, bentonite nanoclay, and glycerol. The starch-based defragmentable bioplastic was evaluated with its water retention, degradation, and mechanical properties. The result showed that the tensile strength improved significantly with the addition of bentonite nanoclay and the elongation at break with the addition of glycerol. Same way in water retention test, where the presence of the bentonite nanoclay increased the strength of the bioplastic with its water solubility. In soil burial test, a higher level of glycerol concentration increased the rate of the bioplastic degradation. By adding the bentonite nanoclay as a filler, the rate of bioplastic degradation decreased compared to the bioplastic without filler. In shelf-life test, adding glycerol as plasticizer improved the shelf life of bioplastics. A high concentration of glycerol served as an anti-fungal for bioplastics.

BioMOF@cellulose Glycerogel Scaffold with Multifold Bioactivity: Perspective in Bone Tissue Repair.

The development of new biomaterials for musculoskeletal tissue repair is currently an important branch in biomedicine research. The approach presented here is centered around the development of a prototypic synthetic glycerogel scaffold for bone regeneration, which simultaneously features therapeutic activity. The main novelty of this work lies in the combination of an open meso and macroporous nanocrystalline cellulose (NCC)-based glycerogel with a fully biocompatible microporous bioMOF system (CaSyr-1) composed of calcium ions and syringic acid. The bioMOF framework is further impregnated with a third bioactive component, i.e., ibuprofen (ibu), to generate a multifold bioactive system. The integrated CaSyr-1(ibu) serves as a reservoir for bioactive compounds delivery, while the NCC scaffold is the proposed matrix for cell ingrowth, proliferation and differentiation. The measured drug delivery profiles, studied in a phosphate-buffered saline solution at 310 K, indicate that the bioactive components are released concurrently with bioMOF dissolution after ca. 30 min following a pseudo-first-order kinetic model. Furthermore, according to the semi-empirical Korsmeyer-Peppas kinetic model, this release is governed by a case-II mechanism, suggesting that the molecular transport is influenced by the relaxation of the NCC matrix. Preliminary in vitro results denote that the initial high concentration of glycerol in the NCC scaffold can be toxic in direct contact with human osteoblasts (HObs). However, when the excess of glycerol is diluted in the system (after the second day of the experiment), the direct and indirect assays confirm full biocompatibility and suitability for HOb proliferation.

Fast-track adaptive laboratory evolution of Cupriavidus necator H16 with divalent metal cations.

Microbial strain improvement through adaptive laboratory evolution (ALE) has been a key strategy in biotechnology for enhancing desired phenotypic traits. In this Biotech Method paper, we present an accelerated ALE (aALE) workflow and its successful implementation in evolving Cupriavidus necator H16 for enhanced tolerance toward elevated glycerol concentrations. The method involves the deliberate induction of genetic diversity through controlled exposure to divalent metal cations, enabling the rapid identification of improved variants. Through this approach, we observed the emergence of robust variants capable of growing in high glycerol concentration environments, demonstrating the efficacy of our aALE workflow. When cultivated in 10% v/v glycerol, the adapted variant Mn-C2-B11, selected through aALE, achieved a final OD600 value of 56.0 and a dry cell weight of 15.2gL-1, compared to the wild type (WT) strain's final OD600 of 39.1 and dry cell weight of 8.4gL-1. At an even higher glycerol concentration of 15% v/v, Mn-C2-B11 reached a final OD600 of 48.9 and a dry cell weight of 12.7gL-1, in contrast to the WT strain's final OD600 of 9.0 and dry cell weight of 3.1gL-1. Higher glycerol consumption by Mn-C2-B11 was also confirmed by high-performance liquid chromatography (HPLC) analysis. This adapted variant consumed 34.5 times more glycerol compared to the WT strain at 10% v/v glycerol. Our method offers several advantages over other reported ALE approaches, including its independence from genetically modified strains, specialized genetic tools, and potentially carcinogenic DNA-modifying agents. By utilizing divalent metal cations as mutagens, we offer a safer, more efficient, and cost-effective alternative for expansion of genetic diversity. With its ability to foster rapid microbial evolution, aALE serves as a valuable addition to the ALE toolbox, holding significant promise for the advancement of microbial strain engineering and bioprocess optimization.

Experimental evolution of extremotolerant and extremophilic fungi under osmotic stress.

Experimental evolution was carried out to investigate the adaptive responses of extremotolerant fungi to a stressful environment. For 12 cultivation cycles, the halotolerant black yeasts Aureobasidium pullulans and Aureobasidium subglaciale were grown at high NaCl or glycerol concentrations, and the halophilic basidiomycete Wallemia ichthyophaga was grown close to its lower NaCl growth limit. All evolved Aureobasidium spp. accelerated their growth at low water activity. Whole genomes of the evolved strains were sequenced. No aneuploidies were detected in any of the genomes, contrary to previous studies on experimental evolution at high salinity with other species. However, several hundred single-nucleotide polymorphisms were identified compared with the genomes of the progenitor strains. Two functional groups of genes were overrepresented among the genes presumably affected by single-nucleotide polymorphisms: voltage-gated potassium channels in A. pullulans at high NaCl concentration, and hydrophobins in W. ichthyophaga at low NaCl concentration. Both groups of genes were previously associated with adaptation to high salinity. Finally, most evolved Aureobasidium spp. strains were found to have increased intracellular and decreased extracellular glycerol concentrations at high salinity, suggesting that the strains have optimised their management of glycerol, their most important compatible solute. Experimental evolution therefore not only confirmed the role of potassium transport, glycerol management, and cell wall in survival at low water activity, but also demonstrated that fungi from extreme environments can further improve their growth rates under constant extreme conditions in a relatively short time and without large scale genomic rearrangements.

A comparative study of CARE 2D and N2V 2D for tissue-specific denoising in second harmonic generation imaging.

This study explored the application of deep learning in second harmonic generation (SHG) microscopy, a rapidly growing area. This study focuses on the impact of glycerol concentration on image noise in SHG microscopy and compares two image restoration techniques: Noise-to-Void 2D (N2V 2D, no reference image restoration) and content-aware image restoration (CARE 2D, full reference image restoration). We demonstrated that N2V 2D effectively restored the images affected by high glycerol concentrations. To reduce sample exposure and damage, this study further addresses low-power SHG imaging by reducing the laser power by 70% using deep learning techniques. CARE 2D excels in preserving detailed structures, whereas N2V 2D maintains natural muscle structure. This study highlights the strengths and limitations of these models in specific SHG microscopy applications, offering valuable insights and potential advancements in the field .

Beauveria bassiana submerged spores for control of two-spotted spider mite (Tetranychus urticae Koch (Acari: Tetranychidae)): production, stability, and virulence.

In this study, IBCB 66, IBCB 868, and CBMAI 1306 isolates of Beauveria bassiana were grown in liquid culture for 4days, leading to elevated submerged spores (SS) levels. The influence of the addition of different glycerol concentrations (0, 3, and 6%) (v/v) in the liquid culture was investigated regarding the stability (at 4 and 27°C) of dried formulations. The virulence of SS was compared with aerial spores (AS) against Tetranychus urticae (Koch) (Acari: Tetranychidae). The results demonstrate the potential of using SS to control T. urticae. CBMAI 1306 and IBCB 868 isolates caused T. urticae mortality rates of 91.11% and 88.89% 5days after treatment, respectively, when applied at concentrations of 1 × 108 SS mL-1. The median Lethal Time (LT50) values for these strains were 2.64 and 2.61days, respectively. The dried formulations showed potential acaricidal activity. Higher glycerol concentrations in the liquid culture medium reduced formulation stability at 27°C.

Macroscopic modelling of the growth and substrate consumption of wild type and genetically modified Pichia pastoris.

Pichia pastoris is a popular yeast platform to generate several industrially relevant products which have applications in a wide range of sectors. The complexities in the processes due to the addition of a foreign gene are not widely explored. Since these complexities can be dependent on the strain characteristics, promoter, and type of protein produced, it is vital to investigate the growth and substrate consumption patterns of the host to facilitate customised process optimisation. In this study, the growth rates of P. pastoris GS115 wild type and genetically modified strains grown on glycerol and methanol in batch cultivation mode were estimated and the model providing the best representation of the true growth kinetics based on substrate consumption was identified. It was observed that the growth of P. pastoris exhibits Haldane kinetics on glycerol rather than the most commonly used Monod kinetics due to the inability of the latter to describe growth inhibition at high concentrations of glycerol. Whereas, the Cardinal Parameter Model, a newly proposed model for this application, was found to be the best fitting to describe the growth of P. pastoris on methanol due to its ability to describe methanol toxicity. Interestingly, the findings from this study concluded that in both substrates, the genetically engineered strain exhibited a higher growth rate compared to the wild type strain. Such an observation has not been established yet in other published works, indicating an opportunity to further optimise the carbon source feeding strategies when the host is grown in fed-batch mode. This article is protected by copyright. All rights reserved.

Osmolytes and Lipids of the Conidia of the Xerohalophilic Micromycete <i>Aspergillus penicillioides</i>

Abstract—Investigation of the lipid and osmolyte composition in conidia of the xerohalophile Aspergillus penicilloides grown on media with high concentrations of NaCl and glycerol is important for the understanding of the functions of these compounds in the adaptation of the extremophile. Conidia of the fungus grown on a medium with glycerol contained up to 15% of the dry mass of carbohydrates and polyols (CaP) of the cytosol, with predominance of glycerol (60% of the total) in their composition, while on a medium with salt, the proportion of glycerol did not exceed 20%, and the amount of CaP was 10%. On both media, the proportion (20%) and amount (2.5%) of trehalose were the same; the proportion of polyols was thus about 80%, although their composition was different, indicating a possible synergy between trehalose and polyols in adaptation. Both variants of conidia were characterized by a low content of both membrane and storage lipids, represented mainly by triacylglycerides. Phosphatidylcholines and sterols were the predominant membrane lipids of the fungal conidia, regardless of the composition of the medium, which indicated high stability and conservatism of the lipid bilayer. The constancy of the composition of membrane and storage lipids and their low content, the high level of CaP, and significant differences in their composition indicate the key role of osmolytes in the adaptation of fungal conidia to various environmental conditions.

Ethanologenesis from glycerol by the gut acetogen Blautia schinkii.

The human gut is an anoxic environment that harbours a multitude of microorganisms that not only contribute to food digestion. The microbiome is also involved in malfunctions such as diseases, inflammation processes or development of obesity, but it is also involved in processes that increase the human well-being. Both, the good and the bad, are mediated by fermentation end products of bacterial metabolism, among others. However, despite a steadily growing knowledge of 'who lives out there', little in known of 'what do they do out there'. The genus Blautia is commonly found in the gut and associated with human well-being, but the exploration of their metabolic potential has just started. We demonstrate that B. schinkii grows on glycerol by producing acetate and ethanol. Transcriptome studies and biochemical analyses revealed a glycerol dehydrogenase and dihydroxyacetone kinase that funnel the substrate into glycolysis. Consequently, cells also grew on dihydroxyacetone. Cells could be adapted to grow at high (up to 1.5 M) glycerol concentrations but then only ethanol was formed. Ethanol production from glycerol is not only of relevance for the human host but also for potential bioindustrial production of bioethanol from waste glycerol.

A Discursive Review of Recent Development and Patents on Glycerosomes.

To achieve a target-based drug delivery with minimal side effects, novel drug delivery systems are being continuously explored. Vesicular systems are one such system that can ameliorate the bioavailability of the encapsulated drug by delivering the drug at the targeted site and can minimize the side effect. The objective of this patent review is to provide a vivid description of glycerosomes and their applications. Glycerosomes are sphere-shaped versatile vesicles consisting of one or more phospholipid bilayers similar to liposomes but contain a high concentration of glycerol, which modifies the liposome bilayer fluidity. Glycerosomes can encapsulate both hydrophobic and hydrophilic drugs, which makes them the promising vehicle in the field of drug delivery. Most of the glycerosome formulations prepared were targeted for topical delivery and in particular, a cutaneous route where they have shown promising results. These vesicles are biocompatible and due to the high glycerol concentration, they have improved spreadability and penetrability. It is therefore imperative to explore the other topical routes such as ocular, vaginal, nasal, and rectal for delivery of drugs.

Kinetic study of glycerol hydrodeoxygenation on Al2O3 and NiMo2C/Al2O3 catalysts

A kinetic study of glycerol hydrodeoxygenation (HDO) on NiMo2C/Al2O3 and Al2O3, prepared by sol-gel technique, was carried out in a batch reactor, from 250 °C to 285 °C, at high glycerol concentration (95wt%). Structural and surface properties of the catalysts were determined by XRD, nitrogen physisorption, TEM and NH3-TPD. Impregnation of alumina with Ni and Mo and subsequent carburization at 730 °C increased crystallite sizes and reduced total acidity of the catalyst to 1/3 that of pure alumina. All reactions were well fitted to a zeroth order model. Estimation of reagents mass transport rates suggests no diffusion limitation. Main products were propanediols, ethylene glycol and long-chain molecules from oligomerization reactions, as well as methane, ethane and propane. At this temperature range, long-chain compounds have the highest yields, mainly on alumina.

EVALUATION OF ETHANOL AND BIOMASS PRODUCTION RATE BY DIFFERENT $SACCHAROMYCES~CEREVISIAE$ STRAINS DEPENDING ON EXTERNAL pH AND TEMPERATURE

Bioethanol production is in high demand due to its potential to replace transportation fuel and its ability to be renewable and long-lasting. Therefore, the bioconversion of fermentable sugars to ethanol is of central importance. The work aimed to explore yeast properties changes during fermentation at different pHs and temperature conditions. Biomass production, specific growth rate (SGR), fermentation products, and metabolite composition and quantity changes were studied at 25℃, 30℃, and 37℃ as well as pH 5 and pH 6.5 conditions. SGR data shows that at 25℃ and 30℃ pH 5 is optimal for yeast biomass production. At pH 5.0–6.5, the biomass production of the ATCC 9804 strain almost 2 times exceeds the same parameter for another strain. The highest biomass production was detected at 30℃ and pH 5. Ethanol production by yeast increases as the growth temperature decreases at pH 5 in contrast to pH 6.5, where the temperature changes within 30–37℃ range have no significant effect on it in both strains. At 30℃ ethanol concent­ra­tion in the extracellular medium reached ~117 mM in the case of ATCC 13007 strain, whereas for ATCC 9804 the same parameter was 1.4-fold lower. Both strains had the same substrate assimilation rate. Glycerol production reduces with increasing growth temperature and pH; the highest glycerol concentration (6.1 mM) was observed within 32 h growth of ATCC 9804 strain at 25℃, pH 5 and reaches 5.5 mM within 24 h growth of ATCC 13007 strain under the same conditions. At pH 5 and 6.5, the free energy for glycerol production was 2.3-fold and 4-fold higher compared to that of ethanol and acetate production processes in both strains. Gibbs free energy of ethanol production reaches the lowest value compared to the same parameter of acetate and glycerol production at pH 5, suggesting a preference for the alcoholic fermentation metabolic pathway under these conditions. The lowest acetate production was observed after 24 hours of growth of the ATCC 13007 strain at 37°C and pH 5, with ΔG = 173.9 kJ/mol. Obtained data highlights temperature stress mechanisms regulation of yeast and can be used for improving ethanol production processes and yeast genetic modification tools.

Cryopreservation of local black rabbit spermatozoa in the Mekong delta region of Vietnam: Evaluating cryoprotectant effectiveness.

Rabbit production is well developed in the Mekong Delta and Vietnam, with increased consumption of rabbit meat in both urban and rural areas. Consequently, meat rabbit breeds have become popular in almost all provinces in Vietnam, resulting in a decline in the population of Local Black rabbits over the years. To support breeding programs, there is a crucial need to develop a cryopreservation method. The objective of this study was to develop sperm cryopreservation protocols and determine the optimal cryoprotectant for Local Black rabbit spermatozoa. Rabbit spermatozoa were obtained and diluted with TCG-EggYolk freezing medium containing 5%, 8%, and 10% glycerol or DMSO in a 1:1 ratio. Glycerol and DMSO-free mediums served as the controls. Samples were put into 0.5-mL straws, cooled to 15°C and 5°C, then placed on nitrogen vapour for 15 min and directly immersed in liquid nitrogen at -196°C. Samples were thawed at 37°C for 60 s after being frozen for 72 h Sperm quality measures, such as viability, motility, and membrane integrity, were assessed both before and after freezing. The results showed that the average viability, average motility, and average membrane integrity were 61.9%, 53.9%, and 42.9%, respectively, after thawing with 5% glycerol, which was higher than the two higher concentrations of glycerol (8% and 10%) (p < .05). Similarly, with 8% DMSO, the average viability, average motility, and membrane integrity were 59.2%, 51.0%, and 41.8%, respectively, which were higher than the two concentrations of DMSO (5% and 10%) (p < 0.05). Therefore, the cryopreservation of local black rabbit sperm with 5% glycerol or DMSO 8% resulted in high sperm quality, but considering the practical aspect of minimizing the volume of the preservative, a concentration of 5% glycerol may be the best option.

Study of the photocatalytic reforming and oxidation of Glycerol over Ag–Pd/TiO2

The photocatalytic reaction of glycerol was conducted over 0.1 at. % Ag – 0.3 at.% Pd/TiO2 catalyst in order to monitor the switch over between the photo-reforming to hydrogen and CO2 and photo-oxidation to H2O and CO2. This alloy of Ag–Pd has shown to be more active than Pd for hydrogen ion reduction previously. Up to a partial pressure of about 0.2 atm. of molecular oxygen the rates of hydrogen production were mostly unaffected. There seems to be a threshold for the effect of O2 on the inhibition of hydrogen production. This is found when the ratio of dissolved molecular oxygen to surface Ti cations is about 2. Also, the order of reaction with respect to PO2 was found to be −2. Hydrogen production was found to be largely insensitive to glycerol concentrations (above 0.05 vol %) as expected from a zero order reaction kinetics in the liquid phase, where the rate is determined by surface coverage. The considerable amounts of dissolved CO2 found indicate that monitoring gas phase CO2 is not a true measurement of the photo-reaction rates. The dissolved CO2 concentration was 15–20% of the gas phase concentration of CO2 at any reaction time investigated. The fast decrease of the pH of the aqueous solution within the first 30 min points to the build up of oxidation products of glycerol. Liquid phase analysis showed the presence of formaldehyde, hydroxy acetone and ethylene glycol as the main reaction products. At high glycerol concentration the main product in the liquid phase was hydroxy acetone while at low concentrations it was formaldehyde. At low glycerol concentrations, the rise of formaldehyde concertation in the liquid phase with time was concomitant with hydrogen production, its decay with time, however, was not accompanied by further increase in hydrogen production. This is most likely because of the competition between decomposition in the presence of water (to H2 and CO2) and oxidation (to formates).

Qualitative Analysis of Polyphenols in Glycerol Plant Extracts Using Untargeted Metabolomics.

Glycerol is a reliable solvent for extracting polyphenols from food and waste products. There has been an increase in the application of glycerol over benchmark alcoholic solvents such as ethanol and methanol for natural product generation because of its non-toxic nature and high extraction efficiency. However, plant extracts containing a high glycerol concentration are unsuitable for mass spectrometry-based investigation utilising electrospray ionization, inhibiting the ability to analyse compounds of interest. In this investigation, a solid phase extraction protocol is outlined for removing glycerol from plant extracts containing a high concentration of glycerol and their subsequent analysis of polyphenols using ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry. Using this method, glycerol-based extracts of Queen Garnet Plum (Prunus salicina) were investigated and compared to ethanolic extracts. Anthocyanins and flavonoids in high abundance were found in both glycerol and ethanol extracts. The polyphenol metabolome of Queen Garnet Plum was 53% polyphenol glycoside derivatives and 47% polyphenols in their aglycone forms. Furthermore, 56% of the flavonoid derivates were found to be flavonoid glycosides, and 44% were flavonoid aglycones. In addition, two flavonoid glycosides not previously found in Queen Garnet Plum were putatively identified: Quercetin-3-O-xyloside and Quercetin-3-O-rhamnoside.

Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol

BackgroundYarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance.ResultsY. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9–20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation.ConclusionALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable “chassis” for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

Acquired thermotolerance, membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.