Salt Concentration Research Articles

Culturable Diversity of Halophilic Bacteria from Soil and Saline Water Samples of Arabian Sea, Karachi Port, Pakistan

Microorganisms capable of survival in extremely high salt concentration are termed as halophiles and a significant source of various enzymes such as hydrolases. Many hydrolases e.g., proteases, amylases and lipases are widely employed in industrial applications. In the current study, we isolated, screened and characterized halophilic bacteria from the shoreline of Arabian Sea, Karachi Port, Pakistan. The water and soil samples collected from Karachi Port; Pakistan were tested for the presence of halophilic bacteria using increasing salt concentrations. Isolated strains were described using various biochemical assays i.e., KOH production, ammonia production, catalase activity, and oxidase activity. For amylase and protease activity, formation of clearance zones – around bacterial growth – was observed, on starch and gelatin agar plates, respectively. Two bacterial strains, N20 (PQ460959) and M3 (PQ460960), with notable amylase and protease activities, were identified via 16S rRNA sequencing as Halobacillus salinus and Halobacillus fulvus, respectively. Further exploration of saline environments is crucial for discovering microbiota with potential applications in biotechnology and environmental remediation.

Inorganic-Dominated Interphase Enabled by Tuning Solvation Configuration for 4.8 V Lithium-Ion Batteries.

Constructing a dense inorganic component-dominated cathode electrolyte interphase (CEI) to meet the long-term cycling requirements of ultrahigh voltage cathodes has been a crucial challenge. Nevertheless, this goal is difficult to achieve in traditional electrolyte compositions due to the inevitable decomposition of organic solvents. Herein, by utilizing the localized mismatch between the strongly coordinating hexafluorophosphate anion (PF6-) and the weakly coordinating solvent 1,1,1-trifluoro-N,N-dimethylmethanesulfonamide (TFDMSA), abundant aggregates (AGGs) emerged under a regular Li salt concentration of 1 m lithium bis(fluorosulfonyl)imide (LiFSI) + 0.1 m LiPF6 in TFDMSA. This anion-rich Li+ solvation structure results in an inorganic-dominated LiF-rich CEI to suppress phase transitions of lithium-rich manganese-based cathode materials (LLMO). Consequently, the prepared LLMO||Li half-cells demonstrate a capacity retention of 80.7% after 350 cycles at 4.8 V. This work advances the practical application of new electrolyte systems by proposing a new approach to construct anion-dominated Li+ solvation structures in local environments.

Designing and Optimizing Electrode Materials for Energy Harvesting in CAPMIX Cells.

The growing demand for clean, decentralized energy has increased interest in blue energy, which generates power from water with different salt concentrations. Despite its potential as a renewable, low-cost energy source, optimizing electrode materials remains a challenge. This work presents a nanomaterial developed via microwave-assisted sol-gel methodology for blue energy applications, where ion diffusion and charge storage are critical. AX-7 carbon, designed for this study, features wide pores, enhancing ion diffusion. Compared to commercial NORIT carbon, AX-7 has a higher mesopore volume and external surface area, improving its overall performance. The synthesis process has been optimized and scaled up for evaluation in CAPMIX electrochemical cell stacks. Moreover, the lower series resistance (Rs) significantly boosts energy recovery, with AX-7 demonstrating superior performance. This advantage is especially evident during fresh-water cycles, where this material achieves significantly lower Rs compared to the commercial one.

Cyano and Fluorine-Enhanced Copolymer Electrolytes: Synergistically Boosting High-Voltage Lithium Metal Batteries.

In high-voltage lithium metal batteries, designing electrolytes with low salt concentrations to achieve stable electrode interfaces presents a formidable challenge. High-concentration electrolytes stabilize the interface through an anion-derived LiF-rich interphase; however, their anion-rich solvation structures compromise the ionic conductivity. This study introduces a polymer-derived interphase that maintains interface stability at low lithium salt concentrations (∼1 M). This strategy enables copolymer electrolytes to sustain the Li|Li cell for over 2500 h at 0.1 mA/cm2, even with a water content of 1000 ppm. Moreover, this research addresses the weak solvation effects in fluorinated polymer electrolytes by modulating the strongly solvating cyano groups, resulting in electrolytes with a high ionic conductivity of 4 × 10-5 S/cm at 30 °C. A 143.8 Wh/kg Li|LiNi0.8Co0.1Mn0.1O2 pouch cell, with a lean electrolyte ratio of 5 g/Ah and a low negative/positive capacity ratio of 4, maintains a capacity retention of 90.5% after 29 cycles.

Effects of salt concentration, temperature, and blanching time on insect protein extraction yield: optimisation by response surface methodology

Abstract This study aimed to optimise the protein extraction yield from black soldier fly larvae by employing a blanching process to enhance protein solubility and mitigate oxidation. Through response surface methodology, we determined the optimal blanching conditions, with the temperature set at 85.152 °C, a duration of 12.411 s, and a sodium chloride concentration of 0.46 M. These parameters were selected to maximise protein solubility while minimising oxidation. The results of this study reveal significant improvements in protein functionality after blanching, particularly with respect to increased emulsification stability. Notably, no significant effect on foam formation was observed. These findings underscore the efficacy of blanching as a pre-processing step to enhance the quality of proteins extracted from black soldier fly larvae. Moreover, these optimised conditions have promising applications in various food and feed formulations, contributing to sustainable protein sourcing and processing methods. By addressing key challenges related to food security and environmental sustainability, this study provides valuable insights into the utilisation of insect proteins as an alternative protein source in the face of global challenges, such as climate change and dwindling natural resources.

Concentrated, Gradient Electrolyte Design for Superior Low-Temperature Li-Metal Batteries

Improving the low-temperature performance of lithium-ion batteries is critical for their widespread adoption in cold environments. In this study, we designed a novel LHCE featuring a solvent polarity gradient, designed to maximize both room- and low-temperature ion mobility. Extremely polar fluoroethylene carbonate (FEC) and low-freezing-point, −135 °C, non-polar nonaflurobutyl methyl ether (NONA) were supplemented by two intermediate solvents with incremental step-downs in polarity. The intermediate solvents consist of methyl (2,2,2-triflooethyl) carbonate (FEMC) and either diethylene carbonate (DEC), ethyl methyl carbonate (EMC), or dibutyl carbonate (DBC). The four solvents were combined with 1 M lithium bis(fluorosulfonyl)amide (LiFSI) salt and were able to accommodate 37.5% diluent volume, resulting in ultra-low electrolyte freezing points below −120 °C. This contrasts with our previously investigated three-solvent LHCE, which only allowed for a 14% diluent volume and a −85 °C freezing point. Localized high salt concentrations were shown by less than 3% of FSI- anions being free in solution. The gradient LHCEs also showed room-temperature ionic conductivities above 10–3 S/cm and maintained high ion mobility below −40 °C. Lithium metal coin cells with LiFePO4 (LFP) cathodes featuring the gradient LHCEs, a reference three-solvent LHCE, and commercial (1 M LiPF6 in 1:1 EC:DEC) electrolyte were constructed. All gradient LHCEs outperformed both the three-solvent and commercial electrolytes at all temperatures, with the DEC-based gradient LHCE showing the best performance of 159.7 mAh/g at 25 °C and 109.2 mAh/g at −50 °C, corresponding to a 68% capacity retention. These findings highlight the potential of LHCE systems to improve battery performance in low-temperature environments and propose a new gradient design strategy for electrolytes to yield advantages of both polar and weakly polar solvents.

Optimizing in vitro spherulation cues in the fungal pathogen Coccidioides.

Coccidioides spp. are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The Coccidioides host form, the spherule, is unique and highly undercharacterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal medium to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.IMPORTANCECoccidioides spp. are thermally dimorphic fungal pathogens found in the Southwest United States, Mexico, Central America, and South America. Coccidioides can infect both immunocompetent and immunocompromised people and can cause a devastating disseminated infection, including meningitis, with 30% mortality despite all currently available treatments. In this work, we tackle one of the current largest technical barriers to studying the fungus Coccidioides: reliably growing its host form in vitro. Our work is impactful because we have created a set of foundational tools for the burgeoning field of Coccidioides pathogenesis research. We have carefully optimized conditions that allow the development of Coccidioides in vitro into its pathogenic form. This work will open up many lines of investigation into the molecules that underlie Coccidioides pathogenesis.

Enhancing Salty Taste Perception in Stroke Patients via Anodal Electrical Stimulation to the Chin.

A lower salt intake is an effective management strategy for hypertension and ultimately stroke. However, this strategy compromises the taste of food. To overcome this, a taste manipulation strategey using electronic taste simulation (ETS) has been established, but this has only been studied in healthy individuals. Therefore, this study aimed to demonstrate and quantitatively evaluate the taste enhancement effect of ETS in patients admitted to a hospital due to stroke. Twenty patients (mean = 67.8 ± 13.6 years) underwent two psychophysical experiments to assess the effects of ETS on salt taste perception using salt-impregnated filter paper. The patients' stroke types included twelve ischemic and eight hemorrhagic strokes. The median salt taste thresholds without ETS and with ETS were 0.7% and 0.6%, respectively (p = 0.083). The perceived concentration for the 0.8% concentration increased from 0.8% to 1.0% with the ETS (p = 0.041), and for the 1.0% concentration, from 1.0% to 1.2% (p < 0.001). The findings suggest that ETS significantly enhances salty taste perception in patients who have experienced a stroke without altering salt concentration, potentially aiding in reducing daily salt intake. Further research is necessary to explore its broad applicability in dietary management and blood pressure control.

Development of a Sealed Rechargeable Li-SO2 Battery.

Rechargeable Li-SO2 batteries offer low-cost, high-energy density benefits and can leverage manufacturing processes for the existing primary version at a commercial scale. However, they have so far only been demonstrated in an "open-system" with continuous gas supply, preventing practical application. Here, the utilization and reversibility of SO2 along with the lithium stability are addressed, all essential for long-life, high-energy batteries. The study discovers that high SO2 utilization is achievable only from SO2 dissolved in electrolytes between the lithium anode and carbon cathode. This results from a unique osmosis phenomenon where SO2 consumption increases salt concentration, driving the influx of organic solvents rather than SO2 from outside the current path. This insight leads to configure a bobbin-cell with all electrolytes between the electrodes, realizing nearly 70% of SO2 utilization, >12x greater than in conventional coin cells. To improve reaction rate and SO2 reversibility, triphenylamine is employed to the electrolyte, creating an electron-rich environment that alleviates the disproportionation of discharge products. Incorporating this additive into a bobbin-cell with a lithium protective layer yields a cell with a projected energy density exceeding 183.2Wh kg-1. The work highlights the potential of Li-SO2 batteries as affordable, sustainable energy storage options.

Phytoremediation Potential of Melia azedarach and Ailanthus altissima for Pb, Zn, and Cu from Aqueous Solution

Growing concerns over metal pollution highlight the need for effective remediation strategies. This study evaluates the accumulation capacity and tolerance of Melia azedarach and Ailanthus altissima for trace elements (Pb, Zn, and Cu), aiming to assess their phytoremediation potential. Three-month-old seedlings of both species, grown from seeds collected at the Touissit mine site, were cultivated in perlite and irrigated with Hoagland nutrient solution. Plants were exposed to various concentrations of metal salts—Pb(NO3)2 (8, 40, and 80 mg.L−1), ZnSO4 (8, 40, and 80 mg.L−1), and CuSO4 (2, 10, and 20 mg.L−1)—over a 90-day period. Growth, biomass, metal accumulation, chlorophyll, and carotenoid contents were measured. Results indicate that M. azedarach exhibited enhanced biomass under Pb exposure, suggesting notable tolerance and potential for phytoremediation. Conversely, A. altissima showed an initial increase in biomass at low Pb levels, followed by a significant reduction at higher concentrations. Both species demonstrated decreased biomass under Zn and Cu treatments, with varying degrees of sensitivity. Notably, A. altissima accumulated significant levels of Pb, Zn, and Cu, particularly in the roots, indicating high phytoremediation potential. While M. azedarach also accumulated metals, levels were comparatively lower. Both species maintained chlorophyll content under metal stress, indicating resilience. Overall, this hydroponic screening highlights the considerable capacities of M. azedarach and A. altissima for Pb, Zn, and Cu tolerance, with A. altissima showing particularly high potential for Pb phytostabilization.

Performance of Corrosion Behavior of Commercial Magnesium Alloy Anode Electrode in Seawater-Powered Lamps

In modern times, alternative technologies that are efficient and environmentally friendly continue to be developed, such as lamps fueled by seawater. By reacting between a magnesium alloy anode electrode and an electrolyte solution of salt, a difference in electrical potential is generated, which can power electronic equipment. In order to achieve optimal performance with the magnesium alloy anode electrode, an initial assessment of this component is necessary. This research discusses the corrosion behavior of the magnesium alloy anode electrode caused by variations in salinity in seawater batteries. In this study, five samples of commercial product anode electrodes were prepared. Each sample was immersed in a salt solution with different salt concentrations. The salt solution was prepared by mixing 380 ml of distilled water with salt masses of 14g, 16g, 18g, 20g, and 22g respectively. Potentiodynamic testing was conducted in this research. The results showed that the corrosion behavior produced from the five samples yielded the highest corrosion potential, electrical current, and corrosion rate values of (Ecorr = 1.5419 Volts), (Icorr = 0.0010 Amps/cm2), and 12.5850 mm/a, respectively. These results indicate an increase in corrosion rate with increasing salinity. It is hoped that in future research, additional treatments will be applied to the electrode to achieve better performance.

Salt Stress and its Implications in Vegetable Crops with Special Reference to the Cucurbitaceae Family

Salt stress is a significant abiotic factor that constrains agricultural productivity by impairing plant growth, particularly in arid and semi-arid regions. Vegetables, ranging from sensitive to moderately tolerant to salinity, experience adverse effects such as disruptions in seed germination, growth, flowering, and fruit development. Salinity hampers water uptake from the soil, as higher salt concentrations in the root zone increase the energy required by plants to absorb water. Sodium salts, in particular, interfere with the uptake of essential nutrients like nitrogen, phosphorus, and potassium, leading to nutritional imbalances. Furthermore, salinity induces oxidative and osmotic stress, ion toxicity, and hormonal disturbances, while also heightening plants‘ susceptibility to diseases. Crops in the Cucurbitaceae family, such as Cucumis sativus (cucumber) and Citrullus lanatus (watermelon), are known to exhibit diverse physiological and biochemical strategies to cope with salinity, including efficient ion transport regulation, osmolyte production, and antioxidant activity. Crops in the Cucurbitaceae family, such as Cucumis sativus (cucumber) and Citrullus lanatus (watermelon), exhibit diverse physiological strategies to cope with salinity. These traits are critical due to their economic significance in global agriculture. Understanding these mechanisms is crucial due to the economic significance of this family in global agriculture. This review examines the effects of salt stress on plant growth and development, explores tolerance mechanisms, and highlights the potential of crops from the Cucurbitaceae family to contribute to sustainable agricultural practices.

How sensitive are protein hydration shells to electrolyte concentration and protein composition?

Proteins of obligate halophilic organisms have an unusually high number of acidic amino acids, thought to enable them to function in multimolar KCl environments. Clarifying the molecular scale mechanisms by which this occurs is relevant for biotechnology, to enable enzymatic synthesis of economically important small molecules in salty environments and other environments with low water activity. Previous studies have suggested that acidic amino acids are necessary at high salt concentration to keep the proteins hydrated by competing with the ions in solution for available water (the "solvent-only" model). We use a combination of solvation shell spectroscopy and molecular dynamics simulations for in total 13 proteins, at high and low KCl concentration, to investigate this scenario. We show that the solvation shells of halophilic and mesophilic proteins of widely different amino acid compositions, net charges, sizes, and structure respond similarly, in terms of composition and of hydrogen bond network, to changes in KCl concentration. The results do not support the solvent-only model, and point to other mechanisms behind the acidity of halophilic proteins. Excess acidic amino acids may ensure protein solubility by the combined effects of having particularly favorable electrostatic interactions with the solvent, ensuring very short range protein-protein repulsion, and having smaller hydrophobic solvent accessible surface area than other charged amino acids. Also possible is that highly acidic proteins are well-tolerated-but not necessarily indispensable-in terms of stability and solubility.

Spatiotemporal characterization of water diffusion anomalies in saline solutions using machine learning force field.

Understanding water behavior in salt solutions remains a notable challenge in computational chemistry. Conventional force fields have shown limitations in accurately representing water's properties across different salt types (chaotropes and kosmotropes) and concentrations, demonstrating the need for better methods. Machine learning force field applications in computational chemistry, especially through deep potential molecular dynamics (DPMD), offer a promising alternative that closely aligns with the accuracy of first-principles methods. Our research used DPMD to study how salts affect water by comparing its results with ab initio molecular dynamics, SPC/Fw, AMOEBA, and MB-Pol models. We studied water's behavior in salt solutions by examining its spatiotemporally correlated movement. Our findings showed that each model's accuracy in depicting water's behavior in salt solutions is strongly connected to spatiotemporal correlation. This study demonstrates both DPMD's advanced abilities in studying water-salt interactions and contributes to our understanding of the basic mechanisms that control these interactions.

Fast Seawater Desalination Integrated with Electrochemical CO2 Reduction

AbstractCoupling desalination with electrocatalytic reactions is an emerging approach to simultaneously addressing freshwater scarcity and greenhouse gas emissions. However, the salt removal rate in such processes is slow, and the applicable water sources are often limited to those with high salt concentrations. Herein, we show high‐performance electrocatalytic desalination by coupling with electrochemical CO2 reduction using a carbon catalyst. A ZIF‐8‐derived carbon catalyst embedded with Cu nanoparticles delivers a high Faradaic efficiency of 94.3 % for CO production at 288 μmol cm−2 h−1. The efficient CO2 electroreduction generates high current densities, which drive fast salt ion transfer across ion exchange membranes. The integrated device enables one of the highest salt removal rates of 1043.49 μg cm−2 min−1 among various desalination methods. Drinking water can be obtained with an ion removal rate of 99 % when natural seawater is used as the water source.

Tailoring the Tribomechanical and Corrosion Behavior of Reverse Pulse Deposited Ni–Co–P Coating Through Cobalt Content Modulation

Abstract This study explores the influence of cobalt content on the tribomechanical and corrosion properties of Ni–Co–P coatings deposited through reverse pulse electroplating. The ratio of Co salt concentration to the total Ni and Co salt concentration was varied in the electrolyte to achieve different Co levels within relatively low cobalt (≤36 wt%) regime. X-ray diffraction revealed crystallite sizes of 1–3 nm in the deposited layers suggesting a mixture of amorphous and nanocrystalline structure. Critical loads in scratch adhesion tests are found to be in between 10.5 N and 15.1 N implying adequate adhesive bonding between the deposits and the substrates. Microhardness and pin-on-disc tribo test results demonstrate a positive correlation between Co content and both hardness and wear resistance of the coatings. However, incorporating small amounts of Co (≤6.2 wt%) exhibited minimal impact on these properties. Interestingly, corrosion resistance in saline medium peaked at the second-highest Co level (26.2 wt% of Co), followed by a decrease with further increase of Co. These findings indicate an optimal Co concentration for achieving a balance between wear resistance and corrosion protection in Ni–Co–P coatings.

Interaction between the Polyelectrolytes Unfractionated Heparin and Universal Heparin Reversal Agents.

The interaction of unfractionated heparin (UFH) with universal heparin reversal agent 7 (UHRA-7) is investigated. UHRA-7 is composed of a hyperbranched polyglycerol core onto which an array of methylated tris(2-aminoethylamine) (Me-TREN) charged groups is grafted, which in turn are shielded with a layer of small chain poly(ethylene glycol) methyl ether (mPEG) chains. This system has previously been shown to be biocompatible and to be effective at neutralizing heparin. The binding constant Kb was determined from isothermal titration calorimetry experiments, at temperatures ranging from 278 to 323 K and salt concentrations ranging from 0.06 to 0.20 M NaCl. The data were analyzed in terms of a number of different theoretical models to determine the contribution of counterion release and water release to driving the interaction between UFH and UHRA-7. With the support of NMR and molecular dynamics simulation data, a model of the interaction between UFH and UHRA-7 is proposed. The binding of heparin and universal heparin reversal agent 7 is mainly due to charge-charge interactions between the negatively charged units on UFH with positively charged Me-TREN and mPEG chains.

Synergistic enhancing of micellization and thermodynamic properties of some Gemini cationic surfactants related to benzo[d]thiazol-3-ium bromide

Herrin, three Gemini cationic surfactants related to benzo[d]thiazol-3-ium bromide with variable hydrocarbon chain lengths (TBC n = 6, 12, and 18) were synthesized successfully and confirmed by using IR and 1HNMR spectroscopies. Critical micelle concentration and different thermodynamic properties of all surfactants under study were measured using conductivity, density, molal volume, and refractive index techniques. The Critical micelle concentration of TBC 6, TBC 12, and TBC 18 surfactants measured from the different techniques shows an acceptable agreement. The molecular weight of the investigated surfactants was decreased with the order: TBC 18 > TBC 12 > TBC 6. An increase in the magnitudes of the association constant, Gibbs free energy of micellization, molar refraction, polarizability, and binding constant proved the effect of hydrocarbon chain length on increasing surfactant’s micellization as follows: TBC 18 < TBC 12 < TBC 6. The enhancement in surfactant properties was also indicated under the effect of different concentrations of inorganic salts (NaI, NaBr, NaCl, MnCl2, CuCl2, and CoCl2). This effect was measured using conductivity and refractive index measurements. Different salts were indicated to adsorb on head groups of micelles, leading to an increase in the degree of ionization of the surfactant solution and improved aggregation of the surfactant at lower concentrations. The increase in the negative value of Gibbs free energy of association in the presence of salts proved an increase in the stability of micelles formed in a 15% DMSO-water solvent at 298.15 K.

Evolution of Water State in the "Ionic Liquid EAN/Al(NO3)3/H2O" System Depending on Temperature and Aluminum Salt Concentration.

Although lithium salts are the most successful materials for electrochemical devices so far, there is growing interest in electrolytes containing divalent and trivalent salts, which are characterized by a much higher energy density than lithium systems. For this purpose, the physical and chemical properties of solutions of aluminum nitrate in ethylammonium nitrate (EAN) were studied in a number of works. However, it was demonstrated by the 1H NMR method that solutions contain water (from aluminum crystallohydrates) in the first solvated shell of the Al3+ cation even after usual drying procedures, and this solvated water cannot be directly detected by the standard Karl Fischer analysis. The goal of this work is to check the possibility/impossibility of removing water from the "Ionic Liquid EAN/Al(NO3)3/H2O" system and to investigate the evolution of water state in the system depending on temperature and aluminum salt concentration. To solve the problem, the NMR spectroscopy of 1H, 27Al, and 14N nuclei was used. To interpret the NMR spectra, quantum-chemical calculations of electron shielding constants for the 1H and 27Al nuclei in mixed cation-anion complexes were involved.

Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation.

Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.