Effect of gradual increase of salt on performance and microbial community during granulation process.

Salinity was considered to have effects on the characteristics, performance microbial communities of aerobic granular sludge. This study investigated granulation process with gradual increase of salt under different gradients. Two identical sequencing batch reactors were operated, while the influent of Ra and Rb was subjected to stepwise increments of NaCl concentrations (0-4 g/L and 0-10 g/L). The presence of filamentous bacteria may contribute to granules formed under lower salinity conditions, potentially leading to granules fragmentation. Excellent removal efficiency achieved in both reactors although there was a small accumulation of nitrite in Rb at later stages. The removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) in Ra were 95.31%, 93.70% and 88.66%, while the corresponding removal efficiencies in Rb were 94.19%, 89.79% and 80.74%. Salinity stimulated extracellular polymeric substances (EPS) secretion and enriched EPS producing bacteria to help maintain the integrity and stability of the aerobic granules. Heterotrophic nitrifying bacteria were responsible for NH4+-N and NO2--N oxidation of salinity systems and large number of denitrifying bacteria were detected, which ensure the high removal efficiency of TN in the systems.

Probing the structural evolution on the surface of cardiolipin vesicles with an amphiphilic second harmonic generation and fluorescence probe.

Investigating the influence of the ambient chemical environment on molecular behaviors in liposomes is crucial for understanding and manipulating cellular vitality as well as the capabilities of lipid drug carriers in various environments. Here, we designed and synthesized a second harmonic generation (SHG) and fluorescence probe molecule called Pyr-Py+-N+ (PPN), which possesses membrane-targeting capability. We employed PPN to investigate the response of lipid vesicles composed of cardiolipin to the presence of exogenous salt. The kinetic behaviors, including the adsorption and embedding of PPN on the surface of small unilamellar vesicles (SUVs) composed of cardiolipin, were analyzed. The response of the SUVs to the addition of NaCl was also monitored. A rapid decrease in vesicle size can be evidenced through the rapid drop in SHG emission originating from PPN located on the vesicle surface.

Characterization of Exopolysaccharides Having Potential Antiviral Properties from Priestia Aryabattai Strain MK1 and Bacillus sp. Strain MK2.

Viral diseases are a serious threat to humans while the most antiviral drugs have low efficiency and side effects on human health. Therefore, using microbial biopolymers as the drugs alternate to treat viral infections seems cost-effective and human friendly option. In the present study, thirty-four exopolysaccharides (EPSs) producing bacteria were isolated, and EPSs production capacity of five salt-tolerant isolates was determined under 0, 100 and 150 mM NaCl. Among these, two isolates exhibiting high anti-coliphage activity were identified through 16S rRNA gene analysis. Moreover, the EPSs were characterized by Fourier-transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis, and their composition was determined. Five salt-tolerant bacteria (MK1, MK2, MK10, MK22 and MK29) exhibited higher production of EPSs at 100 mM NaCl compared to that under non-saline control. At 100 mM NaCl, the yield of EPSs ranged between 105 and 330 mg 100 mL-1 broth. The EPSs produced by the isolates MK1 and MK2 exhibited higher anti-coliphage activity (plaque forming unit decreased from 43 × 106 mL-1 to 3 × 106 and 4 × 106 mL-1, respectively), and were comprised of glucose, fructose, galactose, sucrose, lactose and xylose sugars. FTIR spectroscopy depicted that EPSs are mainly composed of hydroxyl, aliphatic, carboxyl, sulfate and phosphate functional groups, which could have bound coliphage and thus conferred higher anti-coliphage activities to the EPSs. Phylogenetic analysis revealed that MK1 and MK2 isolates formed clades within genus Priestia and Bacillus sequences, respectively. High EPSs production capacity of bacterial isolates under saline condition and high anti-coliphage activity of the EPSs implies that bacterial biopolymers could be useful in antiviral drugs therapy.

Contribution to energy conservation and quality improvement of frozen pork via contact/contactless immersion freezing in NaCl solution.

High energy consumption and quality deterioration are major challenges in the meat freezing process. In this study, the energy consumption and qualities of frozen pork were investigated using three freezing methods: nonpackaged pork air freezing (NAF), contactless immersion freezing (PIF) and contact immersion freezing (NIF) with NaCl solution as a refrigerant. The results indicated that NIF could improve the energy conservation and freezing efficiency in >4 freezing treatment-times by increasing the unfrozen water content, decreasing the frozen heat load, shortening the freezing time and reducing evaporation loss. NIF could also increase the a* value of the pork and improve the water-holding capacity by facilitating the conversion of free water to immobilized-water. The two immersion freezing methods could reduce freezing-thawing loss and protein loss by alleviating muscle tissue freezing damage. These results provide a suitable application of immersion freezing with energy conservation, high efficiency and good quality of frozen-pork.

Salt tolerance in a neotropical orchid in the absence of local adaptation to salt spray.

PREMISE: Salt tolerance has rarely been investigated regionally in the neotropics and even more rarely in Orchidaceae, one of the largest families. Therefore, investigating local adaptation to salt spray and its physiological basis in Epidendrum fulgens, a neotropical orchid species, brings important new insights. METHODS: We assessed the degree of salt tolerance in E. fulgens by testing whether coastal populations are more tolerant to salt, which could point to local adaptation. To understand the physiological basis of such salt tolerance, we exposed wild-collected individuals to salt spray for 60 days, then measured leaf expansion, osmotic potential, sodium leaf concentration, chlorophyll leaf index, chlorophyll fluorescence, relative growth rate, and pressure-volume curves. RESULTS: There is no local adaptation to salt spray since both inland and coastal plants have a high tolerance to salt stress. This tolerance is explained by the ability to tolerate high concentrations of salt in leaf tissues, which is related to the high succulence displayed by this species. CONCLUSIONS: We showed an unprecedented salt tolerance level for an orchid species, highlighting our limited knowledge of that trait beyond the traditional studied groups. Another interesting finding is that salt tolerance in E. fulgens is linked to succulence, is widespread, and is not the result of local adaptation. We suggest that E. fulgens and its allied species could be an interesting group to explore the evolution of important traits related to tolerance to salt stress, like succulence.

From Sea Water to Salt Crystals: An Onsite Investigation of Microplastics in a Conventional Sea Salt Farming System.

This study investigated microplastic (MP) contamination in conventional sea salt farming systems. Various crude sea salt samples (n = 22) that were traditionally produced were collected from salt farms and local vendors. Salt water (n = 15), macroalgae (n = 6), and clay of pond floors (n = 6) were collected from ponds subjected to different production (stabilization, evaporation, and concentration and crystallization concentration) processes. All samples were analyzed for MP abundance and characteristics. The potential sources of MP contamination in the salt were also investigated. The mean abundance of MPs in the salt water and clay of pond floor increased progressively throughout the production process and reached its highest level in the concentration and crystallization ponds (7400 MP particles/m3 in salt water and 19,336 MP particles/m2 in the clay of the pond floor). A maximum of 26,500 MP particles/kg of macroalgal material indicated the potential sink of MPs on the surface of the algae. Approximately 34-2377 MP particles/kg salt were found in the crude sea salt samples. However, the mean abundance (378 MP particles/kg of salt) indicated nonsignificant impacts of different harvesting processes on MP contamination. Most MP size distributions, shapes and polymer types in the salts were similar to those found in the salt water, macroalgae and clay of the pond floor. Approximately 99% of the MPs were fragments that were suspected to be decomposed from larger plastic debris and plastic machinery and tools used at the salt farm. Similar patterns of polymer distribution, in which PP > PE > PET > PS, were found for all samples studied.

Combined use of chemical dust suppressant and herbaceous plants for tailings dust control.

Tailings dust can negatively affect the surrounding environment and communities because the tailings are vulnerable to wind erosion. In this study, the effects of halides (sodium chloride [NaCl], calcium chloride [CaCl2], and magnesium chloride hexahydrate [MgCl2·6H2O]), and polymer materials (polyacrylamide [PAM], polyvinyl alcohol [PVA], and calcium lignosulfonate [LS]) were investigated for the stabilization of tailings for dust control. Erect milkvetch (Astragalus adsurgens), ryegrass (Lolium perenne L.), and Bermuda grass (Cynodon dactylon) were planted in the tailings and sprayed with chemical dust suppressants. The growth status of the plants and their effects on the mechanical properties of tailings were also studied. The results show that the weight loss of tailings was stabilized by halides and polymers, and decreased with increasing concentration and spraying amount of the solutions. The penetration resistance of tailings stabilized by halides and polymers increased with increasing concentration and spraying amount of the solutions. Among the halides and polymers tested, the use of CaCl2 and PAM resulted in the best control of tailings dust, respectively. CaCl2 solution reduces the adaptability of plants and therefore makes it difficult for grass seeds to germinate normally. PAM solutions are beneficial for the development of herbaceous plants. Among the three herbaceous species, ryegrass exhibited the best degree of development and was more suitable for growth in the tailings. The ryegrass plants planted in the tailings sprayed with PAM grew the best, and the root-soil complex that formed increased the shear strength of the tailings.

Automated Imaging to Evaluate the Exogenous Gibberellin (Ga3) Impact on Seedlings from Salt-Stressed Lettuce Seeds.

Salinity stress is a common challenge in plant growth, impacting seed quality, germination, and general plant health. Sodium chloride (NaCl) ions disrupt membranes, causing ion leakage and reducing seed viability. Gibberellic acid (GA3) treatments have been found to promote germination and mitigate salinity stress on germination and plant growth. 'Bauer' and 'Muir' lettuce (Lactuca sativa) seeds were soaked in distilled water (control), 100 mM NaCl, 100 mM NaCl + 50 mg/L GA3, and 100 mM NaCl + 150 mg/L GA3 in Petri dishes and kept in a dark growth chamber at 25 °C for 24 h. After germination, seedlings were monitored using embedded cameras, capturing red, green, and blue (RGB) images from seeding to final harvest. Despite consistent germination rates, 'Bauer' seeds treated with NaCl showed reduced germination. Surprisingly, the 'Muir' cultivar's final dry weight differed across treatments, with the NaCl and high GA3 concentration combination yielding the poorest results (p < 0.05). This study highlights the efficacy of GA3 applications in improving germination rates. However, at elevated concentrations, it induced excessive hypocotyl elongation and pale seedlings, posing challenges for two-dimensional imaging. Nonetheless, a sigmoidal regression model using projected canopy size accurately predicted dry weight across growth stages and cultivars, emphasizing its reliability despite treatment variations (R2 = 0.96, RMSE = 0.11, p < 0.001).

Salt stress alters the cell wall components and structure in Miscanthus sinensis stems.

Miscanthus is a perennial grass suitable for the production of lignocellulosic biomass on marginal lands. The effects of salt stress on Miscanthus cell wall composition and its consequences on biomass quality have nonetheless received relatively little attention. In this study, we investigated how exposure to moderate (100 mM NaCl) or severe (200 mM NaCl) saline growing conditions altered the composition of both primary and secondary cell wall components in the stems of 15 Miscanthus sinensis genotypes. The exposure to stress drastically impacted biomass yield and cell wall composition in terms of content and structural features. In general, the observed compositional changes were more pronounced under severe stress conditions and were more apparent in genotypes with a higher sensitivity towards stress. Besides a severely reduced cellulose content, salt stress led to increased pectin content, presumably in the form of highly branched rhamnogalacturonan type I. Although salt stress had a limited effect on the total lignin content, the acid-soluble lignin content was strongly increased in the most sensitive genotypes. This effect was also reflected in substantially altered lignin structures and led to a markedly reduced incorporation of syringyl subunits and p-coumaric acid moieties. Interestingly, plants that were allowed a recovery period after stress ultimately had a reduced lignin content compared to those continuously grown under control conditions. In addition, the salt stress-induced cell wall alterations contributed to an improved enzymatic saccharification efficiency.

Elucidating the roles of electrolytes and hydrogen bonding in the dewetting dynamics of the tear film.

This study explores the impact of electrostatic interactions and hydrogen bonding on tear film stability, a crucial factor for ocular surface health. While mucosal and meibomian layers have been extensively studied, the role of electrolytes in the aqueous phase remains unclear. Dry eye syndrome, characterized by insufficient tear quantity or quality, is associated with hyperosmolality, making electrolyte composition an important factor that might impact tear stability. Using a model buffer solution on a silica glass dome, we simulated physiologically relevant tear film conditions. Sodium chloride alone induced premature dewetting through salt crystal nucleation. In contrast, trace amounts of solutes with hydroxyl groups (sodium phosphate dibasic, potassium phosphate monobasic, and glucose) exhibited intriguing phenomena: quasi-stable films, solutal Marangoni-driven fluid influx increasing film thickness, and viscous fingering due to Saffman-Taylor instability. These observations are rationalized by the association of salt solutions with increased surface tension and the propensity of hydroxyl-group-containing solutes to engage in significant hydrogen bonding, altering local viscosity. This creates a viscosity contrast between the bulk buffer solution and the film region. Moreover, these solutes shield the glass dome, counteracting sodium chloride crystallization. These insights not only advance our understanding of tear film mechanics but also pave the way for predictive diagnostics in dry eye syndrome, offering a robust platform for personalized medical interventions based on individual tear film composition.

A hyperstable, low-salt adapted protease from halophilic archaeon with potential applications in salt-fermented foods.

Salt-tolerant proteases with remarkable stability are highly desirable biocatalysts in the salt-fermented food industry. In this study, the undigested autocleavage product of HlyA (halolysin A), a low-salt adapted halolysin from halophilic archaeon Halococcus salifodinae, was investigated. HlyA underwent autocleavage of its C-terminal extension (CTE) at temperatures over 40 °C or NaCl concentrations below 2 M to yield HlyAΔCTE. HlyAΔCTE demonstrated robust stability over a wide range of -20-60 °C, 0.5-4 M NaCl, and pH 6.0-10.0 for at least 72 h. Notably, HlyAΔCTE is the first reported halolysin with such exceptional stability. Compared with HlyA, HlyAΔCTE preferred high temperatures (50-75 °C), low salinities (0.5-2.5 M NaCl), and near-neutral (pH 6.5-8.0) conditions to achieve high activity, consistently with its production conditions. HlyAΔCTE displayed a higher Vmax value against azocasein than HlyA. During fish sauce fermentation, HlyAΔCTE significantly enhanced fish protein hydrolysis, indicating its potential as a robust biocatalyst in the salt-fermented food industry.

Growth and no-growth boundary of Clostridium perfringens in cooked cured meats - A logistic analysis and development of critical control surfaces using a solid growth medium.

This study was conducted to evaluate the effect of sodium nitrite (NaNO2, 100-200 ppm), sodium erythorbate (SE, 0-547 ppm), sodium tripolyphosphate (STPP, 0-0.5 %), and sodium chloride (NaCl, 2-3 %) on growth of C. perfringens using a solid growth medium and to develop a growth/no-growth boundary (critical control surface, or CCS) to prevent its growth in cooked cured meat under the optimal temperature condition. Melted Shahidi Ferguson Perfringens (SFP) agar, inoculated with a 3-strain spore cocktail and mixed with NaNO2, SE, STPP, and NaCl according to a Box-Behnken response surface experimental design, was dispersed in 96-well microplates and incubated anaerobically in an incubator programmed to remain at 4 °C for 24 h, heat to 80 °C in 1.75 h, quickly (0.5 h) cool to 46 °C (optimum temperature), and then maintain at 46 °C overnight. The plates were examined optically and visually for colony formation. Any well free of growth was designated as no-growth. Logistic regression was used to analyze the growth probability (P) as affected by NaNO2, SE, STPP, and NaCl and define a CSS as meeting the criterion of P < 1/96. The results showed that STPP and the interactions of SE with NaNO2 and NaCl could reduce the growth probability of C. perfringens in SFP agar. The validation of CCS with ground beef showed an accuracy of 96.3 % for no growth of C. perfringens in the inoculated samples. The results of this study proved that cured meat can be formulated with proper combinations of NaNO2, SE, STPP, and NaCl to prevent the growth of C. perfringens even under the optimum temperature condition, thus preventing food poisoning caused by the growth of this microorganism.

Advancing HIC method development: Retention-time modeling and tuning selectivity with ternary mobile-phase systems.

The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k0) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k¯0) in aqueous buffer for ternary systems overcomes this bottleneck. Gradient retention factors were derived based on isocratic scouting runs after numerical integration of the isocratic Jandera model, leading to retention-time prediction errors below 11 % for linear gradients. Furthermore, an analytical expression was formulated to predict HIC retention for both linear and segmented linear gradients, considering the linear solvent-strength (LSS) model within ternary salt systems, relying on a fixed k0. The approach involved conducting two gradient scouting runs for each of the two binary salt systems to determine model parameters. Retention-time prediction errors for linear gradients were below 12 % for lysozyme and 3 % for trypsinogen and α-chymotrypsinogen A. Finally, the analytical expression for a ternary mobile-phase system was used in combination with a genetic algorithm to tune the HIC selectivity. With an optimized segmented ternary gradient, a critical-pair separation for a mixture of 7 proteins was achieved within 15 min with retention-time prediction errors ranging between 0.7 and 15.7 %.

Deficiency in Opu Systems Imparts Salt-Sensitivity to Weizmannia coagulans.

Weizmannia coagulans can be used as a starter strain in fermented foods or as a probiotic. However, it is salt-sensitive. Here, W. coagulans genomes were compared with the genomes of strains of Bacillus species (B. licheniformis, B. siamensis, B. subtilis, and B. velezensis) that were isolated from fermented foods and show salt tolerance, to identify the basis for the salt-sensitivity of W. coagulans. Osmoprotectant uptake (Opu) systems transport compatible solutes into cells to help them tolerate osmotic stress. B. siamensis, B. subtilis, and B. velezensis each possess five Opu systems (OpuA, OpuB, OpuC, OpuD, and OpuE); B. licheniformis has all except OpuB. However, W. coagulans only has the OpuC system. Based on these findings, the opuA and opuB operons, and the opuD and opuE genes, were amplified from B. velezensis. Expression of each of these systems, respectively, in W. coagulans increased salt-tolerance. W. coagulans expressing B. velezensis opuA, opuD, or opuE grew in 10.5% NaCl (w/v), whereas wild-type W. coagulans could not grow in 3.5% NaCl. The salt resistance of B. subtilis was also increased by overexpression of B. velezensis opuA, opuB, opuD, or opuE. These results indicate that the salt-susceptibility of W. coagulans arises because it is deficient in Opu systems.

Impact of Air Bubbles on the Saltiness Perception of NaCl-Loaded Oleogel-Stabilized Water-in-Oil Emulsions.

Reducing salt intake without affecting the saltiness perception remains a great challenge for the food industry. Herein, the demulsification of water droplets and air bubbles was controlled to modulate the release of sodium from oleogel-stabilized water-in-oil emulsions (OGEs) stabilized by monoglyceride crystals. The effect of monoglycerides with carbon chain length (glycerol monolaurate-GML, glyceryl monostearate-GMS, and glycerol monopalmitate-GMP) and homogenization methods (hand-shaking or high-speed blender) on sodium release and saltiness was investigated by in vitro and in vivo oral processing tests. Milky-white stable emulsions were formed with both water droplets and air bubbles dispersing in the oil phase, regardless of the selected homogenization methods. Air bubbles were more unstable than water droplets during oral digestion. GML OGEs with more and larger air bubbles and the lowest hardness exhibited the highest sodium release rate and the strongest saltiness, independent of homogenization methods. The balance between air bubbles and water droplets in the GMS and GMP OGEs caused slower sodium release and lower saltiness. Overall, the presence of air bubbles in NaCl-loaded W/O oleogel-based emulsions was shown to have important implications for tailoring their sodium release and saltiness.

Effect of salt-alkali stress on seed germination of the halophyte Halostachys caspica.

The increasing global phenomenon of soil salinization has prompted heightened interest in the physiological ecology of plant salt and alkali tolerance. Halostachys caspica belonging to Amaranthaceae, an exceptionally salt-tolerant halophyte, is widely distributed in the arid and saline-alkali regions of Xinjiang, in Northwest China. Soil salinization and alkalinization frequently co-occur in nature, but very few studies focus on the interactive effects of various salt and alkali stress on plants. In this study, the impacts on the H. caspica seed germination, germination recovery and seedling growth were investigated under the salt and alkali stress. The results showed that the seed germination percentage was not significantly reduced at low salinity at pH 5.30-9.60, but decreased with elevated salt concentration and pH. Immediately after, salt was removed, ungerminated seeds under high salt concentration treatment exhibited a higher recovery germination percentage, indicating seed germination of H. caspica was inhibited under the condition of high salt-alkali stress. Stepwise regression analysis indicated that, at the same salt concentrations, alkaline salts exerted a more severe inhibition on seed germination, compared to neutral salts. The detrimental effects of salinity or high pH alone were less serious than their combination. Salt concentration, pH value, and their interactions had inhibitory effects on seed germination, with salinity being the decisive factor, while pH played a secondary role in salt-alkali mixed stress.

Mitigating NaCl stress in Vigna radiata L. cultivars using Bacillus pseudomycoides.

Salt stress is one of the significant abiotic stress factors that exert harmful effects on plant growth and yield. In this study, five cultivars of mung bean (Vigna radiata L.) were treated with different concentrations of NaCl and also inoculated with a salt-tolerant bacterial strain to assess their growth and yield. The bacterial strain was isolated from the saline soil of Sahiwal District, Punjab, Pakistan and identified as Bacillus pseudomycoides. Plant growth was monitored at 15-days interval and finally harvested after 120 days at seed set. Both sodium and potassium uptake in above and below-ground parts were assessed using a flame photometer. Fresh and dry mass, number of pods, seeds per plant, weight of seeds per plant and weight of 100 seeds reduced significantly as the concentration of NaCl increased from 3 to 15 dSm-1. There was a significant reduction in the growth and yield of plants exposed to NaCl stress without bacterial inoculum compared to the plants with bacterial inoculum. The latter plants showed a significant increase in the studied parameters. It was found that the cultivar Inqelab mung showed the least reduction in growth and yield traits among the studied cultivars, while Ramzan mung showed the maximum reduction. Among all the cultivars, maximum Na+ uptake occurred in roots, while the least uptake was observed in seeds. The study concludes that NaCl stress significantly reduces the growth and yield of mung bean cultivars, but Bacillus pseudomycoides inoculum alleviates salt stress. These findings will be helpful to cultivate the selected cultivars in soils with varying concentrations of NaCl.

Isoptericola haloaureus sp. nov., a dimorphic actinobacterium isolated from mangrove sediments of southeast India, implicating biosaline agricultural significance through nitrogen fixation and salt tolerance genes.

Strain MP-1014T, an obligate halophilic actinobacterium, was isolated from the mangrove soil of Thandavarayancholanganpettai, Tamil Nadu, India. A polyphasic approach was utilized to explore its phylogenetic position completely. The isolate was Gram-positive, filamentous, non-motile, and coccoid in older cultures. Ideal growth conditions were seen at 30 °C and pH 7.0, with 5% NaCl (W/V), and the DNA G + C content was 73.3%. The phylogenic analysis of this strain based upon 16S rRNA gene sequence revealed 97-99.8% similarity to the recognized species of the genus Isoptericola. Strain MP-1014T exhibits the highest similarity to I. sediminis JC619T (99.7%), I. chiayiensis KCTC19740T (98.9%), and subsequently to I. halotolerans KCTC19646T (98.6%), when compared with other members within the Isoptericola genus (< 98%). ANI scores of strain MP-1014T are 86.4%, 84.2%, and 81.5% and dDDH values are 59.7%, 53.6%, and 34.8% with I. sediminis JC619T, I. chiayiensis KCTC19740T and I. halotolerans KCTC19646T respectively. The major polar lipids of the strain MP-1014T were phosphatidylinositol, phosphatidylglycerol, diphosphotidylglycerol, two unknown phospholipids, and glycolipids. The predominant respiratory menaquinones were MK9 (H4) and MK9 (H2). The major fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C14:0, C15:0, and C16:0. Also, initial genome analysis of the organism suggests it as a biostimulant for enhancing agriculture in saline environments. Based on phenotypic and genetic distinctiveness, the strain MP-1014 T represents the novel species of the genus Isoptericola assigned Isoptericola haloaureus sp. nov., is addressed by the strain MP-1014 T, given its phenotypic, phylogenetic, and hereditary uniqueness. The type strain is MP-1014T [(NCBI = OP672482.1 = GCA_036689775.1) ATCC = BAA 2646T; DSMZ = 29325T; MTCC = 13246T].

Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 µg/mL) as compared to controls (0.37 µg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.