Dyer Method Research Articles

Enhanced Lipidomics Analysis of Breast Cancer Cells Using Three-phase Liquid Extraction and Ultra High-performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Lipid extraction of complex biological samples is essential for high-quality data in liquid chromatography-mass spectrometry (LC-MS)-based lipidomics. This study introduces a three-phase liquid extraction (3PLE)-ultra-high-performance LC coupled with quadrupole time-of-flight tandem MS method. This method was successfully applied to lipidomics analysis of breast cancer cells, including highly metastatic MDA-MB-231 and slightly metastatic MCF7 cells. The 3PLE method employed an n-hexane/methyl tert-butyl ether/acetonitrile/water solvent system that formed one aqueous and two organic phases. Neutral and polar lipids were enriched in the upper and middle organic phases, respectively, and combined for detection, thereby reducing analysis time. Compared with the Bligh and Dyer method, 3PLE achieved higher sensitivity and detected more features, with over a 50% increase in the relative abundance of nearly 50% of the differential lipids. In total, 21 differential lipids were identified in the MDA-MB-231 group and 22 in the MCF7 group compared to normal breast epithelial cells (MCF10A). Pathway analysis suggested that lipid changes in breast cancer cells were associated with glycerophospholipid metabolism, arachidonic acid metabolism, sphingolipid metabolism, and linoleic acid metabolism. The study presents a highly efficient lipidomics method, providing a scientific foundation for understanding breast cancer pathogenesis and aiding in diagnosis.

Valorization of Pig Brains for Prime Quality Oil: A Comparative Evaluation of Organic-Solvent-Based and Solvent-Free Extractions.

Pig processing industries have produced large quantities of by-products, which have either been discarded or used to make low-value products. This study aimed to provide recommendations for manufacturing edible oil from pig brains, thereby increasing the value of pork by-products. The experiment compared non-solvent extraction methods, specifically wet rendering and aqueous saline, to a standard solvent extraction method, the Bligh and Dyer method, for extracting oil from pig brains. The yield, color, fatty acid profile, a number of lipid classes, and lipid stability against lipolysis and oxidation of the pig brain oil were comprehensively compared, and the results revealed that these parameters varied depending on the extraction method. The wet rendering process provided the highest extracted oil yield (~13%), followed by the Bligh and Dyer method (~7%) and the aqueous saline method (~2.5%). The Bligh and Dyer method and wet rendering techniques produced a translucent yellow oil; however, an opaque light-brown-red oil was found in the aqueous saline method. The Bligh and Dyer method yielded the oil with the highest phospholipid, cholesterol, carotenoid, tocopherol, and free fatty acid contents (p < 0.05). Although the Bligh and Dyer method recovered the most unsaturated fatty acids, it also recovered more trans-fatty acids. Aqueous saline and wet rendering procedures yielded oil with low FFA levels (<1 g/100 g). The PV of the oil extracted using all methods was <1 meq/kg; however, the Bligh and Dyer method had a significant TBARS content (7.85 mg MDA equivalent/kg) compared to aqueous saline (1.75 mg MDA equivalent/kg) and wet rendering (1.14 mg MDA equivalent/kg) (p < 0.05). FTIR spectra of the pig brain oil revealed the presence of multiple components in varying quantities, as determined by chemical analysis experiments. Given the higher yield and lipid stability and the lower cholesterol and trans-fatty acid content, wet rendering can be regarded as a simple and environmentally friendly method for safely extracting quality edible oil from pig brains, which may play an important role in obtaining financial benefits, nutrition, the zero-waste approach, and increasing the utilization of by-products in the meat industry.

Extraction of Omega-3 Fatty Acids from Atlantic Sea Cucumber (Cucumaria frondosa) Viscera Using Supercritical Carbon Dioxide.

This study explores the potential of Cucumaria frondosa (C. frondosa) viscera as a natural source of omega-3 FAs using supercritical carbon dioxide (scCO2) extraction. The extraction conditions were optimized using a response surface design, and the optimal parameters were identified as 75 °C and 45 MPa, with a 20 min static and a 30 min dynamic extraction, and a 2:1 ethanol to feedstock mass ratio. Under these conditions, the scCO2 extraction yielded higher FAs than the solvent-based Bligh and Dyer method. The comparative analysis demonstrated that scCO2 extraction (16.30 g of FAs/100 g of dried samples) yielded more fatty acids than the conventional Bligh and Dyer method (9.02 g, or 13.59 g of FAs/100 g of dried samples with ultrasonic assistance), indicating that scCO2 extraction is a viable, green alternative to traditional solvent-based techniques for recovering fatty acids. The pre-treatment effects, including drying methods and ethanol-soaking, were investigated. Freeze-drying significantly enhanced FA yields to almost 100% recovery, while ethanol-soaked viscera tripled the FA yields compared to fresh samples, achieving similar EPA and DHA levels to hot-air-dried samples. These findings highlight the potential of sea cucumber viscera as an efficient source of omega-3 FA extraction and offer an alternative to traditional extraction procedures.

Optimization of Lipid Production of Rhodosporidium toruloides from Various Substrates

Background and Aim: In recent years, bioenergy has received a lot of attention from a technical point of view due to its ability to use reversible resources and its applicability worldwide. Biodiesel is a non-toxic, safe, renewable, and degradable fuel that is obtained from natural sources, such as vegetable oils, food waste oils, animal fats, algae, and microorganisms. This study aimed to optimize the lipid production of Rhodosporidium toruloides as a biofuel from different substrates. Materials and Methods: In this experimental laboratory study, after lipid extraction from Rhodosporidium toruloides CECT1137, cell biomass, nitrogen, and sugar consumption were measured according to the Bly and Dyer method. Then, using gas chromatography, the produced lipid compounds were identified, and lipid production was optimized by the one-factor method (type of nitrogen source and KH2PO4) and the Taguchi method (carbon and nitrogen source, temperature, pH, centrifuge, and time). Finally, the feasibility of mutation was investigated using UV to optimize lipid production. Results: The amount of lipid produced by Rhodosporidium toruloides was 4.95 g/l, which, after optimization by the Taguchi method, reached 8.54 g/l. Cell biomass, residual nitrogen, and consumed sugar were measured as 14.05, 0.19, and 28.85 g/l, respectively. Optimal production conditions were estimated by Taguchi, including 80 g/l carbon source, 1 g/l nitrogen source, 25 ° C, pH = 5.5, centrifuge at 180 rpm, and 96 hours. Most lipids produced were oleic acid and palmitic acid. Lipid production increased after UV exposure. Conclusion: The results showed that Rhodosporidium toruloides can accumulate a significant amount of lipid in its cells and can be a good alternative to plants and other sources of fuel production.

Characterizing lipid constituents of B. moojeni snake venom: a comparative approach for chemical and biological investigations.

Snake venoms are complex mixtures majorly composed of proteins with well-studied biological effects. However, the exploration of non-protein components, especially lipids, remains limited despite their potential for discovering bioactive molecules. This study compares three liquid-liquid lipid extraction methods for both chemical and biological analyses of Bothrops moojeni snake venom. The methods evaluated include the Bligh and Dyer method (methanol, chloroform, water), considered standard; the Acunha method, a modification of the Bligh and Dyer protocol; and the Matyash method (MTBE/methanol/water), featuring an organic phase less dense than the aqueous phase. Lipidomic analysis using liquid chromatography with high-resolution mass spectrometry (LC-HRMS) system revealed comparable values of lipid constituents' peak intensity across different extraction methods. Our results show that all methods effectively extracted a similar quantity of lipid species, yielding approximately 17-18 subclasses per method. However, the Matyash and Acunha methods exhibited notably higher proportions of biologically active lipids compared to the Bligh and Dyer method, particularly in extracting lipid species crucial for cellular structure and function, such as sphingomyelins and phosphatidylinositol-phosphate. In conclusion, when selecting a lipid extraction method, it is essential to consider the study's objectives. For a biological approach, it is crucial to evaluate not only the total quantity of extracted lipids but also their quality and biological activity. The Matyash and Acunha methods show promise in this regard, potentially offering a superior option for extracting biologically active lipids compared to the Bligh and Dyer method.

Bioactive lipids derived from red wine, beers, and their dealcoholized variants inhibit platelet-activating factor (PAF) induced platelet activation in vitro

This study assesses the in vitro antiplatelet properties of polar lipid extracts of two popular beers, a red wine and their 0% equivalents against platelet aggregation. Total lipids (TL) were isolated using the Bligh and Dyer method from each beverage and the total polar lipid (TPL) and total neutral lipid (TNL) fractions were further isolated using counter-current distribution. The lipid profile of each TPL fraction was analysed using LC-MS. Platelet aggregometry was performed to evaluate the TL, TNL, and TPL extracts of each beverage against platelet-activating factor (PAF) induced platelet aggregation. TPL and TL lipid fractions significantly inhibit platelet aggregation, with TPL fractions showing the highest inhibition as evidenced by generally low IC50 values, ranging from 9 μg to 553 μg. These results indicate that antiplatelet lipid microconstituents are present in beer, wine, and their 0% alcohol counterparts, in low but bioactive amounts. These lipids are characterized by predominance of saturated fatty acids (SFA) and a relatively high ratio of n-6 to n-3 polyunsaturated fatty acids (PUFA) in the TPL composition. The extracts also contained notable levels of phenolic compounds ranging from 0.05 to 0.31 mg/mL. These data provide evidence for the presence of antiplatelet lipid microconstituents that may contribute to the observed epidemiological cardioprotective effects of moderate beer and wine consumption. Furthermore, 0% alcoholic beverages may also provide comparable antiplatelet cardioprotective microconstituents devoid of the risk of ethanol ingestion.

Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Optimation of the cell density, biomass production, lipid, and carbohydrate content of Nannochloropsis oculata with fluorescein and pH manipulation

Nannochloropsis oculata is a potential marine microalga. However, the mass production of N. oculata encounters several challenges, such as the high costs of resources, contamination, and light absorption optimization. These problems can be solved by pH manipulation and fluorescein induction on culture media. Various studies have shown that pH values manipulation can cause fluctuations in cell density, dry biomass, and carbohydrate and lipid production of microalgae. On the other hand, fluorescein can act as a molecular antenna that improves light absorption. This research is novel because of its focus on its unique attempt to utilize pH manipulation combined with fluorescein induction to enhance the growth and metabolite production of N. oculata. The implications of this research may provide cost-effective, viable, and sustainable development of the algal industry in general. In this research, cultivation was carried out for seven days with four levels of pH (7 – 10) combined with three variations of fluorescein addition (0, 0.15, and 0.3 mL). Every treatment combination had three replications. Cell density and biomass as growth analysis parameters were calculated during the cultivation using the Haemocytometer and gravimetric methods. Bligh &amp; Dyer and phenol sulfate methods were used to analyze lipid and carbohydrate content. Based on the result, the best treatment combination, pH 9 with 0.15 mL of fluorescein, increases cell density and carbohydrate content of N. oculata by 31.45 and 4.4%, albeit not statistically significant.

ЭКСПЕРИМЕНТАЛЬНОЕ ОБОСНОВАНИЕ РАЦИОНАЛЬНОГО СПОСОБА ВЫДЕЛЕНИЯ ЖИРА ИЗ ОТХОДОВ ИКОРНОГО ПРОИЗВОДСТВА

The purpose of the study is to substantiate a rational method for isolating fat from caviar production waste to obtain a biologically active lipid composition. Objectives: to conduct an experimental substantiation of a rational method for isolating lipids from caviar production waste; to develop an experimental installation for the extraction of the lipid part from the studied secondary fish raw materials. The research material is caviar production waste generated and collected during the punching of chum salmon shells at a fish processing plant located in the Khabarovsk Region. Sampling of raw materials and their preparation for analysis were carried out according to standard methods (GOST 31339-2006, GOST 7631-2008). Protein content was carried out according to GOST 7636-85 “Fish, marine mammals, marine invertebrates and their processed products. Methods of analysis". The mass fraction of lipids was determined using the Bly and Dyer method. Fatty acids were determined on a capillary gas-liquid chromatograph Shimadzu GC-16A (Supelcowax-10). The choice of an extraction method for separating fat from biologically valuable secondary raw materials of caviar production, which is waste after punching Pacific salmon caviar, is justified. The created experimental setup, operating according to the Soxhlet method, made it possible to obtain a lipid fraction from secondary raw materials of salmon using an extraction method using two solvents (polar and non-polar). The quality of the resulting fat was assessed by fatty acid and fractional composition. Analysis of the results obtained showed some differences between the fat of the initial secondary raw materials and the fat obtained using the extraction method in an experimental installation. In the isolated fat, the content of physiologically valuable extremely unsaturated fatty acids and triglycerides is lower than in the original raw material. In general, the quality of the resulting lipid fraction did not change significantly as a result of long-term extraction processing of secondary raw materials. In the future, ways to improve the developed and presented method for separating fat from caviar production waste are proposed.

An integrated approach for wastewater treatment and algae cultivation: Nutrient removal analysis, biodiesel extraction, and energy and environmental metrics enhancement

Human urine is rich in nitrogen and phosphorus, and the presence of these elements in wastewater significantly disrupts the biogeochemical cycle. Meanwhile, green algal biomass cultivation is unfeasible without these nutrients. Hence, the present study integrates wastewater treatment and algae cultivation to extract biodiesel and improve its performance through fuel modification. Chlorella vulgaris algae was cultivated in different dilution ratios of water and urine, and the nutrient removal rate was analyzed. Chlorella vulgaris algae biodiesel (CAB) was derived through Bligh and Dyer's method followed by transesterification, and its functional and elemental groups were analyzed. The various volume concentrations of CAB were blended with regular diesel fuel (RDF), and 10% water was added to a 30% CAB blended RDF to evaluate the combustion performance and environmental impacts. The results of the experiments demonstrated that the algae cultivation effectively removed the wastewater nutrients. The functional and elemental groups of CAB are identical to those of RDF. The engine characteristics of test fuels report that the CAB-blend RDF fuel mixtures generate low carbon footprints, whereas negative impacts have been drawn for performance metrics and oxides of nitrogen emissions. The water-emulsified fuel outweighed the unfavorable effects and promoted more efficient and cleaner combustion.

RAPID DETECTION OF RAT MEAT ADULTERATION IN BEEF SAUSAGES USING FTIR‑ATR SPECTROSCOPY AND CHEMOMETRICS FOR HALAL AUTHENTICATION

Objective: The objective of this study was to employ Fourier Transform Infrared-Attenuated Total Reflectance (FTIR‑ATR) spectroscopy in combination with chemometrics for the analysis of rat meat adulteration in beef sausages.&#x0D; Methods: Lipid components in sausages were extracted using three extraction methods, namely Bligh and Dyer, Folch, and Soxhlet methods. The lipid components extracted were then analysed using FTIR‑ATR spectroscopy, and their spectra obtained were used as variables during chemometrics modeling. Samples were prepared by mixing beef with adulterant of rat meat in the concentration range of 0-100% of rat meat. Each sample was scanned using FTIR-Attenuated Total Reflectance (ATR) spectroscopy in three replicates at 4000-650 cm-1 wavenumber region.&#x0D; Results: The absorbance values at wavenumbers regions of 3100-700 cm-1 were used to discriminate lipid components extracted by the Bligh Dyer, Folch, and Soxhlet Method with an accuracy level of 100%. The prediction of rat sausages was successfully determined using multivariate calibrations of Partial Least Square (PLS) and Principle Component Regression (PCR) using optimised conditions.&#x0D; Conclusion: FTIR-ATR spectroscopy coupled with chemometrics is a rapid and accurate method for detecting and quantifying rat meat in beef sausages for halal authentication.

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies.

Interconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.

Supercritical CO2 Extraction of Fish Roe

Abstract Seafood is rich in omega-3 polyunsaturated fatty acids, and the good emulsifying properties of phospholipids make them suitable for use in the food, pharmaceutical and cosmetic industries. This study attempted to evaluate different fish species obtained using supercritical carbon dioxide without and with and ethanol as co-solvent extraction and organic solvent (ethanol) extraction. Supercritical CO2 extraction was performed at 30–50 °C and at different pressures (17.7–33.0 MPa), maintaining a CO2 flow rate of 0.6 mL/min for 3 hours. Gas chromatography and Bligh &amp; Dyer methods were used to analyze the extracts. The results showed that only one species of fish roe (rainbow trout) produced oil, but it was concluded that improvements were needed to be able to extract oil from African catfish roe using supercritical CO2 extraction. Rainbow trout roe contains the most monounsaturated fatty acids (44 %).

Adapting lipidomic sample processing methods for boars housed in commercial settings.

Multiple reaction monitoring (MRM) profiling is a sensitive method of lipid screening that has the capability to distinguish between different fertility phenotypes in gilts. However, MRM profiling has not yet been utilized to evaluate fertility phenotypes in boars. Markers indicative of fertility status in boars would be valuable as inclusion of subfertile boars in breeding programs results in a loss of efficiency and negative economic consequences. In addition, semen samples for lipidomic analysis are transported in liquid nitrogen or on dry ice to suspend metabolic activity within the sperm cells, however, these cryopreservation techniques are not commonly available at commercial boar studs. Therefore, the objective of this study was to develop a method of sample processing for MRM profiling that suspends metabolic activity within semen without freezing the sample. Five, sexually mature boars of similar genetics enrolled in a commercial breeding program were collected for the study. Following collection, ejaculates were aliquoted into methanol to suspend metabolic activity and shipped to Purdue University overnight for lipid extraction. Lipids were extracted using the Bligh and Dyer method and MRM profiling was used for lipid screening. A total of 329 ion transitions (MRMs) related to lipids were detected with most lipids being characterized as plasma membrane lipids (74%) which were comprised of phosphatidylcholines (40%), ceramides (16%), phosphatidylethanolamines (11%), and phosphatidylserines (7%). acylcarnitines (AC) represented approximately 8% of the ejaculate lipidome. Hierarchical cluster and principal component analysis revealed that boars have a distinct ejaculate lipidome profile based on major plasma membrane lipid classes. In addition, we observed that one boar was unique in his abundance of AC which are related to progressive motility and sperm cell metabolism. These results indicate that this method of sample processing for MRM profiling is suitable to be used to evaluate the lipidome of ejaculates from commercial boars and has the potential for broader applications across different livestock species in commercial environments.

Physicochemical Characteristics and Nutritional and Biological Properties of Fish Oils in Cameroon: An Overview

Due to their significant health benefits, fish oils have garnered increasing interest in recent decades. However, Cameroon’s fish oil market remains insignificant, and the few available products are imported, despite the country’s abundant marine resources. Additionally, research on Cameroonian fish oils is relatively recent and scarce. Therefore, this manuscript provides an overview of research on fish oils in Cameroon, focusing on their physicochemical characteristics, as well as their nutritional and biological properties. As of March 2023, 26 studies on fish oils in Cameroon have been published, with a focus on 23 species collected in the littoral, far-north, and west regions of Cameroon. Filets were the main parts used, and the Bligh Dyer and Soxhlet methods were the primary oil extraction techniques. Depending on the species, tissues, and extraction methods, oil contents ranged from 4.57% to 32.10% dry matter or yielded 0.36% to 66.83% wet weight. These oils generally meet recommended standards for markers of acidity and oxidation. Fatty acid profiles from 16 species showed a total of 48 fatty acids, including those that are beneficial to human health. Oils from eight species were found to significantly reduce weight, hyperlipidemia, hyperglycemia, hepatomegaly, and adipomegaly, while four species showed activity against bacteria responsible for food poisoning. Future work should include all fish species found in Cameroon, with a focus on by-products, and explore the physicochemical, functional, nutritional, and biological properties of these oils.

Isolation and Characterization of Bacterial Lipids and its Application as Antioxidant Agent

The study was aimed to isolate the oleaginous bacteria from various soil samples and characterize the bacterial lipids to study its suitable application. The study was carried out from June 2023 to October 2023 in the microbiology lab at Changu Kana Thakur Arts, Commerce &amp; Science College, New Panvel (Autonomous). The soil samples were collected from the garden, dumping, and compost area at New Panvel. The bacterial isolation was carried out by simple streak plate method, followed by screening by Sudan black B staining method. Production of oleaginous bacteria was done, followed by extraction of lipids by Bligh and dyer method. The extracted lipids were characterized by TLC and FTIR. The antioxidant potential of extracted lipids was assessed by DPPH method. The different oleaginous bacteria were isolated from soil sample and screened by Sudan black B staining method. The results indicate phospholipids, TAG, esters of fatty acids were mainly present. The extracted lipid samples S1C2, S2C4, S2C2, S1C1, S3C4, S2C1, S3C2 have 83.30%, 80%, 36.60%, 23.30%, 26.60%, 16.6%, 6.6% antioxidant potential respectively.

Study of sample preparation influence on bacterial lipids profile in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Lipids are one of the cell components therefore it is important to be able to accurately assess them. One of the analytical techniques used to study lipid profiles is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The present study attempted to select optimal conditions for sample preparation and MALDI MS analysis of bacterial lipidome in both positive and negative ion modes using different extraction protocols-Folch, Matyash, and Bligh & Dyer, solvents used to apply samples, and matrices such as 9-aminoacridine (9-AA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 2-mercaptobenzothiazole (MBT), and 2,4,6-trihydroxyacetophenone (THAP). The obtained results allowed concluding that DHB or CHCA matrices are suitable for lipid analysis in the positive mode, and in the negative mode THAP or 9-AA. The most appropriate protocol for extracting lipids from bacterial cells was the Bligh & Dyer method in both ionization modes. The use of the solvent TA30, which was a mixture of acetonitrile and 0.1% trifluoroacetic acid in water, provided on the spectra a significant number of signals from lipids in all groups analyzed, such as fatty acyls, glycerolipids, and glycerophospholipids.

Exploring Biomarker Signatures in Glaciovolcanic Environments: Implications for the Search for Life on Mars

Glaciovolcanic systems, where hydrothermal heat interacts with ice, offer favorable conditions for life by providing liquid water, nutrients, and physicochemical gradients (Cousins and Crawford 2011). Despite significant climate change, evidence for glaciovolcanism is widespread through Mars’ history. Such sites may have offered refugia for life after Mars lost much of its surface water, representing some of the most recent habitable areas and promising sites to recover biomarkers. We examined a terrestrial glaciovolcanic site to study the indigenous biological community structure, the supporting physicochemical parameters, and the distribution of biomarkers within the geologic context. The insights will help refine Mars exploration of analogous sites. Method: We studied a partially subglacial hydrothermal area at the summit of the active Kverkfjöll volcano, Iceland. The heated ground has created a large ice-damned meltwater lake with shoreline hot springs, thermal streams, and mud pots of variable activity. We collected water and sediment samples aseptically across the breadth of features, including the lake (surface &amp; depth). Samples were kept at -4°C. Environmental parameters were measured at each sample site. Sediment samples were split for mineralogical and organic analysis. Mineralogy was measured by X-ray Diffraction. Organic samples were freeze-dried and extracted with a Bligh &amp; Dyer method (Bligh and Dyer 1959). Extracts were divided into hydrocarbons, neutral lipids, glycolipids, and phospholipid fatty acids (PLFA) with hexane, dichloromethane, acetone, and methanol. Gas Chromatography/Mass Spectrometry was used to analyze hydrocarbons and PLFA as fatty acid methyl esters. Results: Environmental: Fluids ranged from acidic to alkaline (pH 3-9), low to high temperature (8-87°C), and severely dysoxic to oxic (0.5-5 mg/L dissolved O). Mineralogy comprised a dioctahedral swelling clay, heulandite, and minor quartz, anatase, and pyrite. The assemblage suggests argillic-grade alteration at 100-140°C (Fulignati 2020), confirming that the glaciovolcanic conditions were within theoretical boundaries for life (&amp;lt;150°C (Merino et al. 2019)), and formed minerals capable of protecting organic matter (e.g., swelling clay). Lipids: PLFA are essential components of cell membranes and degrade rapidly upon cell death. PLFA profiles thus provide insight into the composition and distribution of the viable community. Results revealed a diversity of PLFA with low molecular weights and several bacterial-diagnostic structures, indicating an active prokaryote-dominated biosphere. Molecular patterns correlated (p&amp;lt;0.05) with pH, temperature, and oxygen, suggesting homeoviscous adaptations or community composition variations. In either case, microbes demonstrate adaptability to extreme conditions in glaciovolcanic settings. Hydrocarbons are inert and used as molecular fossils. We detected multiple patterns attributable to microbial biosynthesis, including abundant C17 (straight and branched) diagnostic of cyanobacteria; high concentrations of short-chain n-alkane/alkene doublets (&amp;lt;C22) with an even carbon preference (CPI&amp;lt;1) indicating diverse microbial input; and a narrow unresolved complex mixture with elution parameters characteristic of microbial heterotrophy (Finkel et al. 2023). The pattern of even-numbered alkane/alkene pairs has been scarcely reported and offers insight into rare hydrocarbon distributions. Interestingly, the hydrocarbon and PLFA patterns do not align. Thus, PLFA are not a significant source of hydrocarbons here and we suggest direct microbial synthesis dominates. Implications of this hypothesis will be discussed in the context of palaeobiological reconstruction. Conclusion: Our site provides an array of physicochemical niches for diverse life modes and a mineral assemblage amenable to organic preservation. Analogous sites on Mars should be explored, but more work is needed to understand preservation biases of hydrocarbon sources and the implications for interpretation.

Optimisation of Bioflocculation Using Anabaena sp. and Navicula sp. for Harvesting of Glagah Microalgae Consortium

One of the problems in microalgae is harvesting. Currently, many chemical methods are used that impact the environment. Not all of them can be used as a filter, so bioflocculation is used because there is no need to change the medium. This method is an environmentally friendly and efficient alternative to chemical flocculants that usually cause contamination of biomass and health. Previous studies have shown that different ratios of auto-flocculated microalgae in cocultures affect the flocculation rate. This research was carried out by the Glagah Consortium bioflocculation using Anabaena sp. and Navicula sp., which had never been done before. The study aims to study the effect of the mixing ratio on the flocculation rate, carbohydrates, and lipid content of the Glagah Consortium. The consortium uses Anabaena sp. and Navicula sp. as bioflocculants. Glagah and Anabaena sp. consortium was cultured in Bold Basal Medium, while Navicula sp. was cultured in F/2 medium. Cell density was measured every 24 hr for 8 days with a hemocytometer. The cultures were harvested in the stationary phase, then mixed between non-flocculated microalgae (Glagah Consortium) and flocculated microalgae (Anabaena sp., Navicula sp.) in a ratio of 1:1, 1:0.5, and 1:0.25 for 24 hr. Bioflocculation was measured by spectrophotometer at 750 nm 0 and 24 hr after mixing. Carbohydrate levels were measured using the phenol sulfuric acid method, while lipid measurements were performed using the Bligh and Dyer method. The addition of Anabaena sp. and Navicula sp. as bioflocculant in Glagah Consortium culture results in an increase in flocculation rate with an effective ratio of 1:0.25 for Anabaena sp. (81%) and 1:1 for Navicula sp. (95%). Mixing of Anabaena sp. and Glagah Consortium results in carbon source competition, reducing carbohydrate content at higher mixing ratios (0.172, 0.364, and 0.500 mg/ml on 1, 1:0.5, and 1:0.25) while increasing lipid content as a result of lipid production in stationary phase (highest on ratio 1:1 = 0.011 mg/ml). Navicula sp. and Glagah Consortium mixture caused no significant changes to carbohydrate content but showed an increased amount of lipid at all ratios as a result of osmotic stress on Glagah Consortium from saline F/2 medium (highest on ratio 1:1 = 0.162 mg/ml).

Enhancing the Biodiesel Production by Improving the Yield of Lipids in Wild Strain by Inducing Nitrogen Ion Mutation in Rhodotorula mucilaginosa

The overconsumption of energy results in the depletion of fossil fuels. Generally, biodiesels are produced from wastes of animal fats and vegetable oils. In this study, we have tried to produce biodiesel from both the wild strain and ion beam mutated strain and compared the concentration of lipids produced from both the strains and their properties. Lipids were extracted from microbes using the Bligh and Dyer method and analyzed using gas chromatography and mass spectrophotometry (GCMS) and Fourier-transform infrared (FTIR) spectroscopy. Extracted lipids (free fatty acids) were converted into biodiesel (fatty acid methyl esters) using a base catalyst. The end product biodiesel was characterized and analyzed based on ASTM standards.