Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Insights into the Binding Mode of Lipid A to the Anti-lipopolysaccharide Factor ALFPm3 from Penaeus monodon: An In Silico Study through MD Simulations.

The globally expanding threat of antibiotic resistance calls for the development of new strategies for abating Gram-negative bacterial infections. The use of extracorporeal blood cleansing devices with affinity sorbents to selectively capture bacterial lipopolysaccharide (LPS), which is the major constituent of Gram-negative bacterial outer membranes and the responsible agent for eliciting an exacerbated innate immune response in the host during infection, has received outstanding interest. For that purpose, molecules that bind tightly to LPS are required to functionalize the affinity sorbents. Particularly, anti-LPS factors (ALFs) are promising LPS-sequestrating molecules. Hence, in this work, molecular dynamics (MD) simulations are used to investigate the interaction mechanism and binding pose of the ALF isoform 3 from Penaeus monodon (ALFPm3), which is referred to as "AL3" for the sake of simplicity, and lipid A (LA, the component of LPS that represents its endotoxic principle). We concluded that hydrophobic interactions are responsible for AL3-LA binding and that LA binds to AL3 within the protein cavity, where it buries its aliphatic tails, whereas the negatively charged phosphate groups are exposed to the medium. AL3 residues that are key for its interaction with LA were identified, and their conservation in other ALFs (specifically Lys39 and Tyr49) was also analyzed. Additionally, based on the MD-derived results, we provide a picture of the possible AL3-LA interaction mechanism. Finally, an in vitro validation of the in silico predictions was performed. Overall, the insights gained from this work can guide the design of novel therapeutics for treating sepsis, since they may be significantly valuable for designing LPS-sequestrating molecules that could functionalize affinity sorbents to be used for extracorporeal blood detoxification.

Hydraulic Retention Time as an Operational Tool for the Production of Short-Chain Carboxylates via Anaerobic Fermentation of Carbohydrate-Rich Waste.

The carboxylate platform is a sustainable and cost-effective way to valorize wastes into biochemicals that replace those of fossil origin. Short-chain fatty acids (SCFAs) are intermediates generated during anaerobic fermentation (AF) and are considered high-value-added biochemicals among carboxylates. This investigation aimed to produce SCFAs through the AF of sugar beet molasses at 25 °C and semi-continuous feeding mode in completely stirred tank reactors. A particular focus was devoted to the role of hydraulic retention time (HRT) variation in SCFAs production and distribution profile. The highest SCFAs concentration (44.1 ± 2.3 gCOD/L) was reached at the HRT of 30 days. Caproic acid accounted for 32.5-35.5% (COD-concentration basis) at the long HRTs of 20 and 30 days due to the carbon chain elongation of shorter carboxylic acids. The findings of this study proved that HRT could be used to steer the anaerobic process toward the targeted SCFAs for specific uses. Furthermore, the successful operation at low-temperature conditions (i.e., 25 °C) makes the process economically promising.

Statistical correlation between waste macromolecular composition and anaerobic fermentation temperature for specific short-chain fatty acid production.

To properly exploit short-chain fatty acids (SCFAs) in the chemical industry, it is of foremost importance to ensure stable SCFA profile production via anaerobic fermentation (AF). The different macromolecular distribution of food wastes (FWs) used as feedstock might be crucial for process outcome. Targeting at a specific SCFAs profile and yield, this study explored the statistical correlation between the macromolecular composition of FWs and the produced SCFAs in batch-AFs at 25 °C and 55 °C. Principal Component Analysis (PCA) revealed that the carbohydrates fraction was directly related with butyric acid accumulation, regardless of process temperature. Nevertheless, operational temperature resulted in a pH change, which ultimately affected the process fate. PCA of 25 °C-batch-AF showed a positive correlation between high carbohydrate content and longer-chain acids accumulation. By contrast, 55 °C-AF resulted in higher product specificity than at 25 °C, mainly due to butyrate-type fermentation of carbohydrates. Batch results were further validated in a semicontinuous reactor. Prevailing SCFAs and high bioconversion efficiencies relied on 3 main FWs characteristics: high carbohydrate content (>77% w/w), high carbohydrate/protein ratio (≥10) and high soluble organic matter content. Results obtained herein allowed predicting a specific SCFAs profile based on FWs composition, which is relevant for setting proper downstream technologies.

Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica.

In recent years, there has been a growing interest in the use of renewable sources for bio-based production aiming at developing sustainable and feasible approaches towards a circular economy. Among these renewable sources, organic wastes (OWs) can be anaerobically digested to generate carboxylates like volatile fatty acids (VFAs), lactic acid, and longer-chain fatty acids that are regarded as novel building blocks for the synthesis of value-added compounds by yeasts. This review discusses on the processes that can be used to create valuable molecules from OW-derived VFAs; the pathways employed by the oleaginous yeast Yarrowia lipolytica to directly metabolize such molecules; and the relationship between OW composition, anaerobic digestion, and VFA profiles. The review also summarizes the current knowledge about VFA toxicity, the pathways by which VFAs are metabolized and the metabolic engineering strategies that can be employed in Y. lipolytica to produce value-added biobased compounds from VFAs.

Fighting Against Bacterial Lipopolysaccharide-Caused Infections through Molecular Dynamics Simulations: A Review.

Lipopolysaccharide (LPS) is the primary component of the outer leaflet of Gram-negative bacterial outer membranes. LPS elicits an overwhelming immune response during infection, which can lead to life-threatening sepsis or septic shock for which no suitable treatment is available so far. As a result of the worldwide expanding multidrug-resistant bacteria, the occurrence and frequency of sepsis are expected to increase; thus, there is an urge to develop novel strategies for treating bacterial infections. In this regard, gaining an in-depth understanding about the ability of LPS to both stimulate the host immune system and interact with several molecules is crucial for fighting against LPS-caused infections and allowing for the rational design of novel antisepsis drugs, vaccines and LPS sequestration and detection methods. Molecular dynamics (MD) simulations, which are understood as being a computational microscope, have proven to be of significant value to understand LPS-related phenomena, driving and optimizing experimental research studies. In this work, a comprehensive review on the methods that can be combined with MD simulations, recently applied in LPS research, is provided. We focus especially on both enhanced sampling methods, which enable the exploration of more complex systems and access to larger time scales, and free energy calculation approaches. Thereby, apart from outlining several strategies for surmounting LPS-caused infections, this work reports the current state-of-the-art of the methods applied with MD simulations for moving a step forward in the development of such strategies.

Biochemical interactions between LPS and LPS-binding molecules.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, often pose a serious risk not only when delivered in the bloodstream but also in air, the environment and several industrial fields such as pharmaceutics or food. LPS is constituted of three regions; the O-specific chain, the core region and the lipid A, which is the responsible segment of the toxicity. Previous literature dealt with the study of lipid A, its potential ligands as well as the mechanisms of Lipid A interactions that, among other applications, establish the basis for detection methods such as Limulus Amebocyte Lysate (LAL) assays and emerging biosensoring techniques. However, quantifying LPS binding affinity is an urgent need that still requires thorough studies. In this context, this work reviews the molecules that bind LPS, highlighting quantitative affinity parameters. Moreover, state of the art methods to analyze the affinity and kinetics of lipid-ligand interactions are also reviewed and different techniques have been briefly described. Thus, first, we review existing information on LPS ligands, classifying them into three main groups and targeting the comparison of molecules in terms of their interaction affinities and, second, we establish the basis for further research aimed at the development of effective methods for LPS detection and removal.

Volatile fatty acids as novel building blocks for oil-based chemistry via oleaginous yeast fermentation.

Microbial oils are proposed as a suitable alternative to petroleum-based chemistry in terms of environmental preservation. These oils have traditionally been studied using sugar-based feedstock, which implies high costs, substrate limitation, and high contamination risks. In this sense, low-cost carbon sources such as volatile fatty acids (VFAs) are envisaged as promising building blocks for lipid biosynthesis to produce oil-based bioproducts. VFAs can be generated from a wide variety of organic wastes through anaerobic digestion and further converted into lipids by oleaginous yeasts (OYs) in a fermentation process. These microorganisms can accumulate in the form of lipid bodies, lipids of up to 60% wt/wt of their biomass. In this context, OY is a promising biotechnological tool for biofuel and bioproduct generation using low-cost VFA media as substrates. This review covers recent advances in microbial oil production from VFAs. Production of VFAs via anaerobic digestion processes and the involved metabolic pathways are reviewed. The main challenges as well as recent approaches for lipid overproduction are also discussed.

Formation and manipulation of ferrofluid droplets with magnetic fields in a microdevice: a numerical parametric study.

We present a numerical model that describes the microfluidic generation and manipulation of ferrofluid droplets under an external magnetic field. We developed a numerical Computational Fluid Dynamics (CFD) analysis for predicting and optimizing continuous flow generation and processing of ferrofluid droplets with and without the presence of a permanent magnet. More specifically, we explore the dynamics of oil-based ferrofluid droplets within an aqueous continuous phase under an external inhomogeneous magnetic field. The developed model determines the effect of the magnetic field on the droplet generation, which is carried out in a flow-focusing geometry, and its sorting in T-junction channels. Three-channel depths (25 µm, 30 µm, and 40 µm) were investigated to study droplet deformation under magnetic forces. Among the three, the 30 µm channel depth showed the most consistent droplet production for the studied range of flow rates. Ferrofluids with different loadings of magnetic nanoparticles were used to observe the behavior for different ratios of magnetic and hydrodynamic forces. Our results show that the effect of these factors on droplet size and generation rate can be tuned and optimized to produce consistent droplet generation and sorting. This approach involves fully coupled magnetic-fluid mechanics models and can predict critical details of the process including droplet size, shape, trajectory, dispensing rate, and the perturbation of the fluid co-flow for different flow rates. The model enables better understanding of the physical phenomena involved in continuous droplet processing and allows efficient parametric analysis and optimization.

Continuous-Flow Separation of Magnetic Particles from Biofluids: How Does the Microdevice Geometry Determine the Separation Performance?

The use of functionalized magnetic particles for the detection or separation of multiple chemicals and biomolecules from biofluids continues to attract significant attention. After their incubation with the targeted substances, the beads can be magnetically recovered to perform analysis or diagnostic tests. Particle recovery with permanent magnets in continuous-flow microdevices has gathered great attention in the last decade due to the multiple advantages of microfluidics. As such, great efforts have been made to determine the magnetic and fluidic conditions for achieving complete particle capture; however, less attention has been paid to the effect of the channel geometry on the system performance, although it is key for designing systems that simultaneously provide high particle recovery and flow rates. Herein, we address the optimization of Y-Y-shaped microchannels, where magnetic beads are separated from blood and collected into a buffer stream by applying an external magnetic field. The influence of several geometrical features (namely cross section shape, thickness, length, and volume) on both bead recovery and system throughput is studied. For that purpose, we employ an experimentally validated Computational Fluid Dynamics (CFD) numerical model that considers the dominant forces acting on the beads during separation. Our results indicate that rectangular, long devices display the best performance as they deliver high particle recovery and high throughput. Thus, this methodology could be applied to the rational design of lab-on-a-chip devices for any magnetically driven purification, enrichment or isolation.

Anaerobic degradation of protein-rich biomass in an UASB reactor: Organic loading rate effect on product output and microbial communities dynamics.

Anaerobic degradation of enzymatically pretreated Chlorella vulgaris was aimed in an upflow anaerobic sludge blanket reactor (UASB) to evaluate the organic loading rate (OLR) effect on biomass valorization. Low OLRs resulted in high methane yields (171 mL CH4/g CODin) at low hydraulic retention time (HRT of 6 days). Firmicutes (35-43%), Bacteroidetes (17-18%) and Euryarchaeota (11%) dominated at low OLRs, promoting methanogenic activity. On the contrary, the highest OLRs resulted in low methane yield (86 mL CH4/gCODin) with a concomitant short-chain fatty acids (SCFAs) accumulation of 37% SCFAs-COD/CODin. The highest OLR decreased UASB reactor biodiversity, hampering Euryarchaeota population development (2.5%) and boosting Firmicutes (55%) and Proteobacteria (14%). These results demonstrated the suitability of UASB reactor configuration to reach high bioprocess efficiency for both, biogas and SCFAs production, with lower energetic and area requirements than those normally needed in continuous stirred tank reactors.

Microalgae Biomass as a Potential Feedstock for the Carboxylate Platform.

Volatile fatty acids (VFAs) are chemical building blocks for industries, and are mainly produced via the petrochemical pathway. However, the anaerobic fermentation (AF) process gives a potential alternative to produce these organic acids using renewable resources. For this purpose, waste streams, such as microalgae biomass, might constitute a cost-effective feedstock to obtain VFAs. The present review is intended to summarize the inherent potential of microalgae biomass for VFA production. Different strategies, such as the use of pretreatments to the inoculum and the manipulation of operational conditions (pH, temperature, organic loading rate or hydraulic retention time) to promote VFA production from different microalgae strains, are discussed. Microbial structure analysis using microalgae biomass as a substrate is pointed out in order to further comprehend the roles of bacteria and archaea in the AF process. Finally, VFA applications in different industry fields are reviewed.

Volatile Fatty Acids Production from Microalgae Biomass: Anaerobic Digester Performance and Population Dynamics during Stable Conditions, Starvation, and Process Recovery.

Disturbances in anaerobic digestion (AD) negatively impact the overall reactor performance. These adverse effects have been widely investigated for methane generation. However, AD recently appeared as a potential technology to obtain volatile fatty acids (VFAs) and thus, the impact of process disturbances must be evaluated. In this sense, microbial response towards a starvation period of two weeks was investigated resulting in a conversion of organic matter into VFAs of 0.39 ± 0.03 COD-VFAs/CODin. However, the lack of feeding reduced the yield to 0.30 ± 0.02 COD-VFAs/CODin. Microbial analysis revealed that the starvation period favored the syntrophic acetate-oxidizing bacteria coupled with hydrogenotrophic methanogens. Finally, the system was fed at 9 g COD/Ld resulting in process recovery (0.39 ± 0.04 COD-VFAs/CODin). The different microbiome obtained at the end of the process was proved to be functionally redundant, highlighting the AD robustness for VFAs production.

[Use of contraception with only progestogens in primary care: Study GESTAGAP]. / Uso de la anticoncepción con solo gestágenos en atención primaria: estudio GESTAGAP.

AIM: To estimate the prevalence of use of progestin-only contraceptive among women who request reversible contraception in Primary Care (PC). DESIGN: Multicentre cross-sectional study. SETTING: Primary Care Health Care Centres (Madrid). PARTICIPANTS: Women aged 16-50 years old, users of reversible contraception, who speak Spanish, and had attended the Primary Care Centre in the last year. MAIN MEASUREMENTS: Primary outcome: contraceptive method used: Contraception with progestins-only (yes/no). Age, parity, country of origin, type of contraceptive method used, reason for choice, source of information, satisfaction with the contraceptive method. Telephone survey. RESULTS: A total of 417 women were interviewed. The median age was 30.3years (SD: 7.7). Spanish 69%, and 82% of participants had secondary or university studies. More than half (57%) were nulliparous. The type of contraceptive used included: progestins only: 14%, combined hormonal contraceptive: 74%, copper IUD: 2%, and condom 10%. The prevalence of use of "progestins-only" was 13.9% (95%CI: 10.6-17.2). Medroxyprogesterone acetate injection was the most progestin-only method used (4.6%), desogestrel oral pill (4.1%), IUD-levonorgestrel IUD (3.9%), and etonogestrel subdermal implant (1.9%). The family doctor was the prescriber in 71% of the women. Satisfaction: high (range 9-10). Using only progestogens was associated with older age, being non-Spanish, breastfeeding, and having a medical contraindication for combined contraception (P<0.05). CONCLUSIONS: The prevalence of use for progestins was 14%, satisfaction was very high for all contraceptive methods. The user profile for the only progestins-only corresponds to older, and non-Spanish women with conditions such as breastfeeding or contraindications for other contraceptives.

Efficient Anaerobic Digestion of Microalgae Biomass: Proteins as a Key Macromolecule.

Biogas generation is the least complex technology to transform microalgae biomass into bioenergy. Since hydrolysis has been pointed out as the rate limiting stage of anaerobic digestion, the main challenge for an efficient biogas production is the optimization of cell wall disruption/hydrolysis. Among all tested pretreatments, enzymatic treatments were demonstrated not only very effective in disruption/hydrolysis but they also revealed the impact of microalgae macromolecular composition in the anaerobic process. Although carbohydrates have been traditionally recognized as the polymers responsible for the low microalgae digestibility, protease addition resulted in the highest organic matter solubilization and the highest methane production. However, protein solubilization during the pretreatment can result in anaerobic digestion inhibition due to the release of large amounts of ammonium nitrogen. The possible solutions to overcome these negative effects include the reduction of protein biomass levels by culturing the microalgae in low nitrogen media and the use of ammonia tolerant anaerobic inocula. Overall, this review is intended to evidence the relevance of microalgae proteins in different stages of anaerobic digestion, namely hydrolysis and methanogenesis.

Comparison of Chlorella vulgaris and cyanobacterial biomass: cultivation in urban wastewater and methane production.

Anaerobic digestion of microalgae is hampered by its complex cell wall. Against this background, cyanobacteria cell walls render this biomass as an ideal substrate for overcoming this drawback. The aim of the present study was to compare the growth of two cyanobacteria (Aphanizomenon ovalisporum and Anabaena planctonica) and a microalga (Chlorella vulgaris) in urban wastewater when varying the temperature (22, 27 and 32 °C). Cyanobacterial optimal growth for both strains was attained at 22 °C, while C. vulgaris did not show remarkable differences among temperatures. For all the microorganisms, ammonium removal was higher than phosphate. Biomass collected was subjected to anaerobic digestion. Methane yield of C. vulgaris was 184.8 mL CH4 g COD in(-1) while with A. ovalisporum and A. planctonica the methane production was 1.2- and 1.4-fold higher. This study showed that cyanobacteria growth rates could be comparable to microalgae while presenting the additional benefit of an increased anaerobic digestibility.

Enzymatic cell disruption of microalgae biomass in biorefinery processes.

When employing biotechnological processes for the procurement of biofuels and bio-products from microalgae, one of the most critical steps affecting economy and yields is the "cell disruption" stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed.

Modelling of anaerobic digestion of microalgae biomass: Effect of overloading perturbation.

Anaerobic digestion (AD) of microalgae is an intriguing approach for bioenergy production. The scaling-up of AD presents a significant challenge due to the systematic efficiency losses related to process instabilities. To gain a comprehensive understanding of AD behavior, this study assessed a modified version of the anaerobic digestion model No1 (ADM1) + Contois kinetics to represent microalgae AD impacted by overloading. To this end, two new inhibition functions were implemented: inhibition by acetate for acidogenesis/acetogenesis and total volatile fatty acids for hydrolysis. This proposed ADM1 modification (including Contois kinetics) simulated AD behavior during the stable, disturbed and recovery periods, showing that the inhibition functions described in the original ADM1 cannot explain the AD performance under one of the most common perturbations at industrial scale (overloading). The findings underscore the importance of refining the inhibitions present in original ADM1 to better capture and predict the complexities of microalgae AD against overloading.

Impact of Different Red Blood Cell Storage Solutions and Conditions on Cell Function and Viability: A Systematic Review.

Red blood cell (RBC) storage solutions have evolved significantly over the past decades to optimize the preservation of cell viability and functionality during hypothermic storage. This comprehensive review provides an in-depth analysis of the effects of various storage solutions and conditions on critical RBC parameters during refrigerated preservation. A wide range of solutions, from basic formulations such as phosphate-buffered saline (PBS), to advanced additive solutions (ASs), like AS-7 and phosphate, adenine, glucose, guanosine, saline, and mannitol (PAGGSM), are systematically compared in terms of their ability to maintain key indicators of RBC integrity, including adenosine triphosphate (ATP) levels, morphology, and hemolysis. Optimal RBC storage requires a delicate balance of pH buffering, metabolic support, oxidative damage prevention, and osmotic regulation. While the latest alkaline solutions enable up to 8 weeks of storage, some degree of metabolic and morphological deterioration remains inevitable. The impacts of critical storage conditions, such as the holding temperature, oxygenation, anticoagulants, irradiation, and processing methods, on the accumulation of storage lesions are also thoroughly investigated. Personalized RBC storage solutions, tailored to individual donor characteristics, represent a promising avenue for minimizing storage lesions and enhancing transfusion outcomes. Further research integrating omics profiling with customized preservation media is necessary to maximize post-transfusion RBC survival and functions. The continued optimization of RBC storage practices will not only enhance transfusion efficacy but also enable blood banking to better meet evolving clinical needs.

High-throughput screening of non-conventional yeasts for conversion of organic waste to microbial oils via carboxylate platform.

Converting waste into high-value products promotes sustainability by reducing waste and creating new revenue streams. This study investigates the potential of diverse yeasts for microbial oil production by utilizing short-chain fatty acids (SCFAs) that can be produced from organic waste and focuses on identifying strains with the best SCFA utilisation, tolerance and lipid production. A collection of 1434 yeast strains was cultivated with SCFAs as the sole carbon source. Eleven strains emerged as candidates with promising growth rates and high lipid accumulation. Subsequent fermentation experiments in liquid SCFA-rich media, which focused on optimizing lipid accumulation by adjusting the carbon to nitrogen (C/N) ratio, showed an increase in lipid content at a C/N ratio of 200:1, but with a concurrent reduction in biomass. Two strains were characterized by their superior ability to produce lipids compared to the reference strain Yarrowia lipolytica CECT124: Y. lipolytica EXF-17398 and Pichia manshurica EXF-7849. Characterization of these two strains indicated that they exhibit a biotechnologically relevant balance between maximizing lipid yield and maintaining growth at high SCFA concentrations. These results emphasize the potential of using SCFAs as a sustainable feedstock for oleochemical production, offering a dual benefit of waste valorisation and microbial oil production.