Concentration Of Ectoine Research Articles

Production of ectoine by Vreelandella boliviensis using non-aseptic repeated-batch and continuous cultivations in an air-lift bioreactor.

Ectoine, an osmolyte produced by various microorganisms, has numerous commercial applications. Vreelandella boliviensis (formerly called Halomonas boliviensis) generates high ectoine concentrations, i.e., 78.6g/L. This study investigated three cultivation strategies for ectoine production in a non-aseptic air-lift bioreactor. The first strategy was performed in a repeated-batch mode with 5% (w/v) NaCl to induce cell growth, followed by the addition of solid NaCl to a final concentration of 12.5% (w/v) to prompt ectoine production. A maximum dry cell weight of 13.8g/L at 46.5h, a maximum ectoine concentration of 1.37g/L at 37.5h, and a maximum volumetric productivity of 0.93g/L/d at 34.5h were reached. The second strategy employed a three-step repeated-batch cultivation method. In the first step, cells were grown at the optimum salt concentration, harvested by centrifugation, and cultivated in a replenished medium for the second step. In the third step, the cells were harvested again and grown in a fresh medium containing 12.5% (w/v) NaCl. This strategy improved dry cell weight to 32g/L, ectoine concentration to 4.37g/L, and productivity to 1.76g/L/day at 60h of cultivation. The third strategy consisted of continuous cultivations that were investigated using different NaCl concentrations. The highest ectoine concentration of 2.83g/L and productivity of 3.49g/L/d were obtained with 8.5% (w/v) NaCl at a dilution rate of 0.05 (1/h). This study is the first to report ectoine production by V. boliviensis in continuous air-lift bioreactors under non-aseptic conditions.

Methylotuvimicrobium alcaliphilum 20Z based ectoine production from various industrial CH4 sources: Cultivation optimization and comprehensive analysis

Methanotroph-based CH4 conversion technology is being widely studied due to its advantages of mild operating conditions and environmental friendliness. In this study, operating strategies were investigated for industrial CH4 sources in order to widely disseminate methanotroph-based CH4 conversion technology using the promising strain, Methylotuvimicrobium alcaliphilum 20Z. The optimal O2/CH4 ratio for extremely different CH4 concentrations was explored based on kinetic-based simulation for biological reaction, and experimentally proven using fermenter-scale experiments. As a result, when high CH4 concentration is used, ectoine production rate can be maximized (33.8 mg/L·h) at an O2/CH4 ratio of 1 while favoring the “production of metabolites”. On the other hand, in the low CH4 concentration case, when changing from the general O2/CH4 ratio of 4 to the optimal O2/CH4 ratio (i.e., 1), although the improvement in ectoine concentration was not noticeable (3.9 to 7.5 mg/L·h), the CH4 conversion rate rapidly decreased (50 to 20 %), suggesting that the focus in the low concentration case should be on “removal of CH4”. In the case of biogas, the rapid pH drop caused by the large amount of CO2 in the feed is offset by a buffer solution, and the concentration of Na+ ions increases during the overall reaction time. Therefore, the production of metaboiltes can be maximized while simultaneously controlling pH and Na+ ion through continuous operation with a high dilution rate (19.8 mg/L·h). Furthermore, major performance indicators for each industrial gas were derived through comprehensive comparative analysis, through which the “dual-functions” of methanotrophs could be emphasized. This study provides a practical and strategic approach for methanotrophs that can flexibly respond to diverse industrial CH4 sources.

The synthesis of ectoine enhance the assimilation of ammonia nitrogen in hypersaline wastewater by the salt-tolerant assimilation bacteria sludge

In contrast to nitrification-denitrification microorganisms that convert ammonia nitrogen in hypersaline wastewater into nitrogen for discharge, this research utilizes sludge enriched with salt-tolerant assimilation bacteria (STAB) to assimilate organic matter and ammonia nitrogen in hypersaline wastewater into ectoine - a biomass with high economic value and resistance to external osmotic pressure. The study investigates the relationship between the synthesis of ectoine and nitrogen removal efficiency of STAB sludge in three sequencing batch reactors (SBR) operated at different salinities (50, 75, and 100 g/L) and organic matter concentrations. The research reveals that, under low concentration carbon sources (TOC/N = 4, NH4+-N = 60 mg/L), the ammonia nitrogen removal efficiency of SBR reactors increased by 14.51 % and 17.25 % within 5 d and 2 d, respectively, when salinity increased from 50 g/L to 75 g/L and 100 g/L. Under high concentration carbon sources (TOC/N = 8, NH4+-N = 60 mg/L), the ammonia nitrogen removal efficiency of STAB sludge in the three reactors stabilized at 80.20 %, 76.71 %, and 72.87 %, and the total nitrogen removal efficiency was finally stabilized at 80.47 %, 73.15 %, and 65.53 %, respectively. The nitrogen removal performance by ammonium-assimilating of STAB sludge is more sustainable under low salinity, while it is more short-term explosive under high salinity. Moreover, the intracellular ectoine concentration of STAB sludge was found to be related to this behavior. Empirical formulas confirm that STAB sludge synthesizes ectoine from nutrients in wastewater through assimilation, and intracellular ectoine has a threshold defect (150 mg/gVss). The ectoine metabolism pathways of STAB sludge was constructed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The ammonia nitrogen in sewage is converted into glutamic acid under the action of assimilation genes. It then undergoes a tricarboxylic acid cycle to synthesize the crucial precursor of ectoine - aspartic acid. Subsequently, ectoine is produced through ectoine synthase. The findings suggest that when the synthesis of intracellular ectoine reaches saturation, it inhibits the continuous nitrogen removal performance of STAB sludge under high salinity. STAB sludge does not actively release ectoine through channels under stable external osmotic pressure.

Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches.

The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

Adsorption and Desorption Behavior of Ectoine Using Dowex® HCR-S Ion-Exchange Resin

Dowex® HCR-S ion-exchange resin was used to adsorb ectoine in a batch system under varying operation conditions in terms of contact time, temperature, pH value, initial concentration of ectoine, and type of salt. Six adsorption isotherm models (Langmuir, Freundlich, Temkin, Dubinin–Radushkevich, Sips, and Redlich–Peterson) and three kinetic models (pseudo-first-order, pseudo-second-order, and intraparticle diffusion) were used to investigate the ectoine adsorption mechanism of ion-exchange resin. According to the experimental results, the mechanism of ectoine adsorption using an ion exchanger includes the ion-exchange reaction and physisorption. Both the Langmuir and Freundlich models were found to have a high fitting. For the kinetic analysis, the pseudo-second-order and intraparticle diffusion models were suitable to describe the ectoine adsorption. Dowex® HCR-S resin has an average saturated adsorption capacity of 0.57 g/g and 93.6% of ectoine adsorption at 25~65 °C, with an initial concentration of 125 g/L. By changing the pH of the environment using NaOH solution, the adsorbed ectoine on the ion-exchange resin can be desorbed to 87.7%.

Co-culturing Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 allows sustainable denitrifying activities under marine conditions.

BackgroundHyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities of a methanol-fed, fluidized-bed marine denitrification system. Strain NL23 possesses the complete denitrification pathway, but cannot grow under marine conditions in pure cultures. Strain JAM1 is a marine bacterium that lacks genes encoding a dissimilatory nitrite (NO2−) reductase and therefore cannot reduce NO2−. Here, we report the characterization of some of their physiological traits that could influence their co-habitation. We also perform co-cultures to assess the potential synergy between the two strains under marine and denitrifying conditions.MethodologyAnoxic planktonic pure cultures of both strains were grown with different concentrations of nitrate (NO3−). Anoxic planktonic co-cultures could only be cultured on low NaCl concentrations for strain NL23 to grow. Biofilm co-cultures were achieved in a 500-mL bioreactor, and operated under denitrifying conditions with increasing concentrations of NaCl. NO3− and NO2− concentrations and the protein content were measured to derive the denitrification rates. The concentrations of both strains in co-cultures were determined by quantitative PCR (qPCR). Ectoine concentration was measured by mass spectrometry in the biofilm co-culture. The biofilm was visualized by fluorescence in situ hybridization. Reverse-transcription-qPCR and RNA-seq approaches were used to assess changes in the expression profiles of genes involved in the nitrogen pathways in the biofilm cultures.ResultsPlanktonic pure cultures of strain JAM1 had a readiness to reduce NO3− with no lag phase for growth in contrast to pure cultures of strain NL23, which had a 2-3 days lag phase before NO3− starts to be consumed and growth to occur. Compared to strain NL23, strain JAM1 has a higher µmax for growth and higher specific NO3− reduction rates. Denitrification rates were twice higher in the planktonic co-cultures than those measured in strain NL23 pure cultures. The biofilm co-cultures showed sustained denitrifying activities and surface colonization by both strains under marine conditions. Increase in ectoine concentrations was observed in the biofilm co-culture with the increase of NaCl concentrations. Changes in the relative transcript levels were observed in the biofilm culture with genes encoding NapA and NapGH in strain NL23. The type of medium had a great impact on the expression of genes involved in the N-assimilation pathways in both strains.ConclusionsThese results illustrate the capacity of both strains to act together in performing sustainable denitrifying activities under marine conditions. Although strain JAM1 did not contribute in better specific denitrifying activities in the biofilm co-cultures, its presence helped strain NL23 to acclimate to medium with NaCl concentrations >1.0%.

Ectoine Production Using Novel Heterologous EctABCS. salarius from Marine Bacterium Salinicola salarius

Ectoine, a heterocyclic amino acid produced by various bacteria, was widely used in the fields of cosmetics and medicine. In this study, a novel ectoine synthesis cluster from marine bacterium Salinicola salarius 1A01339 was firstly introduced into Escherichia coli BL21(DE3) for heterologous production of ectoine. The bioinformatic analysis proved the function of these ectoine synthesis enzymes, and showed the highest identities of 83.3–87.7% with enzymes from other microorganisms. Using the whole-cell biocatalytic method, 3.28 g/L ectoine was synthesized and excreted into the medium with the substrate of 200 mM sodium aspartate at 25 °C, pH 6.5 in flask-level. Further bioconversion was performed in the fermentor system at the high cell density of 20 OD/mL, and the concentration of extracellular ectoine was increased to 22.5 g/L in 24 h (equivalent to the specific productivity of 0.94 g/L·h), achieving over 6 times of production compared with that in flasks. Significantly, the recombinant strain demonstrated a lower catalytic temperature with the optimum of 25 °C, and a stronger tolerance to the substrate aspartate of 300 mM. These results might provide a compelling case for ectoine synthesis as well as potential applications in large-scale industrial production.

Accumulation of Ectoines By Halophilic Bacteria Isolated from Fermented Shrimp Paste: An Adaptation Mechanism to Salinity, Temperature, and pH Stress.

Shrimp paste is a traditional fermented food produced by many Asian countries. Bacteria play important roles in the shrimp paste fermentation process. In order to survive under the low water activity (Aw) conditions caused by the high salt concentration, the bacteria need to employ a special adaptation strategy. This study found that most halophilic bacteria isolated from shrimp paste accumulated ectoines (ectoine and hydroxyectoine) as protective osmotic agents. Five isolated bacteria, including three high ectoine producers and two high hydroxyectoine producers, were selected for further study. Based on their morphological and biochemical characteristics and 16S rRNA gene sequences, the five strains were classified into three genera: Salinivibrio (strains M7 and M316), Salimicrobium (strains M31 and M69), and Vibrio (strain M92). The accumulation of ectoines by Salimicrobium species is reported here for the first time. The effects of salinity, incubation temperature, and initial pH on the growth rate and accumulation of ectoines by the five strains were investigated. The results revealed that the bacterial growth rate was inhibited while the accumulation of ectoines by the five selected strains was triggered by an increase in the external salinity, incubation temperature, or initial pH. In addition, a high concentration of ectoine only (21.2 wt%) was produced by strain M316 at the optimum salinity and temperature, and under pressure of a high initial pH value. To the best of our knowledge, this is the first report demonstrating that the production of ectoines by bacterial strains can be enhanced by increasing the pH of the culture medium to induce pH stress. This finding suggests a new ectoine producer and fermentation strategy that may help to improve the production of ectoines in the future.

Ability of Ectoine to Stabilize Lipase against Elevated Temperatures and Methanol Concentrations

Ectoine is one of the compatible organic molecules that can protect the protein from heating, freezing, and chemicals contact. This study aims to investigate the ability of ectoine to stabilize lipase on heating and in methanol treatments as an effort to provide a stable biocatalyst for the production of biodiesel. Various ectoine concentrations were added to lipase solutions, then the mixture was heated, and the residual activity of the lipase was determined. Similar steps were also conducted for methanol treatment. The results showed that ectoine maintained and even improved the catalytic activity of lipase after treatment with either heat or methanol. The addition of ectoine to a final concentration of 110 to 150 mM could maintain lipase activity up to 80% when heating to approximately 95 °C. Additionally, more than 20% of lipase activity increased on heating to temperatures below 75 °C in the presence of ectoine at a final concentration of 25 to 120 mM. Meanwhile, after incubation in methanol at a level of around 84% (v/v), the activity of lipase containing 40–90 mM ectoine was maintained. These results demonstrated that ectoine was highly effective in protecting lipase from heat and methanol.

Production and Solubility of Ectoine: Biochemical and Molecular Dynamics Simulation Studies

In this study, ectoine is produced by Streptomyces. sp IBRC-M PTCC 10615. Fermentation parameters such as flow regime, gas hold up, mass transfer coefficient, and mixing time were optimized by statistical analysis. Streptomyces. sp produced a maximal ectoine concentration of 270 mmol/kg at optimal conditions of ectoine and L-aspartic acid. Also, the amount of mass transfer, gas hold up, and mixing time were determined 0,41/s ,0.3, and 40 s, respectively. The amount of ectoine was measured by HPLC. Furthermore, Molecular Dynamics (MD) simulation was used for studying the solubility of ectoine in aqueous media. Equilibrium data such as temperature, potential energy, and volume graphs showed that the solubility of ectoine is 25%more than glycerol. Also, all the achieving graphs from the equilibrium of simulation were confirmed the appropriate structure of the system.

Metabolic Pathway Construction and Optimization of Escherichia coli for High-Level Ectoine Production.

Ectoine is widely produced by various bacteria as a natural cell protectant against environment stress, e.g., osmotic and temperature stress. Its protective properties therefore exhibit high commercial value, especially in agriculture, medicine, cosmetics, and biotechnology. Here, we successfully constructed an engineered Escherichia coli for the heterologous production of ectoine. Firstly, the ectABC genes from Halomonas elongata were introduced into E. coli MG1655 to produce ectoine without high osmolarity. Subsequently, lysA gene was deleted to weaken the competitive L-lysine biosynthesis pathway and ectoine bioconversion was further optimized, leading to an increase of ectoine titer by 16.85-fold. Finally, at the low cell density of 5 OD600/mL in Erlenmeyer flask, the concentration of extracellular ectoine was increased to 3.05mg/mL. At the high cell density of 15 OD600/mL, 12.7g/L of ectoine was achieved in 24h and the overall yield is 1.27g/g glycerol and sodium aspartate. Our study herein provides a feasible and valuable biosynthesis pathway of ectoine with a potential for large-scale industrial production using simple and cheap feedstocks.

Intracellular Metabolites in Marine Microorganisms during an Experiment Evaluating Microbial Mortality.

Metabolomics is a tool with immense potential for providing insight into the impact of biological processes on the environment. Here, we used metabolomics methods to characterize intracellular metabolites within marine microorganisms during a manipulation experiment that was designed to test the impact of two sources of microbial mortality, protozoan grazing and viral lysis. Intracellular metabolites were analyzed with targeted and untargeted mass spectrometry methods. The treatment with reduced viral mortality showed the largest changes in metabolite concentrations, although there were organic compounds that shifted when the impact of protozoan grazers was reduced. Intracellular concentrations of guanine, phenylalanine, glutamic acid, and ectoine presented significant responses to changes in the source of mortality. Unexpectedly, variability in metabolite concentrations were not accompanied by increases in microbial abundance which indicates that marine microorganisms altered their internal organic carbon stores without changes in biomass or microbial growth. We used Weighted Correlation Network Analysis (WGCNA) to identify correlations between the targeted and untargeted mass spectrometry data. This analysis revealed multiple unknown organic compounds were correlated with compatible solutes, also called osmolytes or chemical chaperones, which emphasizes the dominant role of compatible solutes in marine microorganisms.

The Skin-Whitening Effects of Ectoine via the Suppression of α-MSH-Stimulated Melanogenesis and the Activation of Antioxidant Nrf2 Pathways in UVA-Irradiated Keratinocytes.

Ultraviolet A (UVA)-irradiation induced reactive oxygen species (ROS) production mediates excessive melanogenesis in skin cells leading to pigmentation. We demonstrated the depigmenting and anti-melanogenic effects of Ectoine, a natural bacterial osmolyte, in UVA-irradiated human (HaCaT) keratinocytes, and the underlying molecular mechanisms were elucidated. HaCaT cells were pre-treated with low concentrations of Ectoine (0.5–1.5 μM) and assayed for various depigmenting and anti-melanogenic parameters. This pre-treatment significantly downregulated ROS generation, α-melanocyte-stimulating hormone (α-MSH) production, and proopiomelanocortin (POMC) expression in UVA-irradiated HaCaT cells. Also, antioxidant heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase [quinone 1] (NQO-1), and γ-glutamate-cysteine ligase catalytic subunit (γ-GCLC) protein expressions were mediated via the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) whose knockdown indeed impaired this effect signifying the importance of the Nrf2 pathway. Ectoine was mediating the activation of Nrf2 via the p38, protein kinase B (also known as AKT), protein kinase C (PKC), and casein kinase II protein kinase (CKII) pathways. The conditioned medium obtained from the Ectoine pre-treated and UVA-irradiated HaCaT cells downregulated the tyrosinase, tyrosinase-related protein-1 and -2 (TRP-1/-2), cyclic AMP (c-AMP) protein kinase, c-AMP response element-binding protein (CREB), and microphthalmia-associated transcription factor (MITF) expressions leading to melanoma B16F10 cells having inhibited melanin synthesis. Interestingly, this anti-melanogenic effect in α-MSH-stimulated B16F10 cells was observable only at 50–400 μM concentrations of Ectoine, signifying the key role played by Ectoine (0.5–1 μM)-treated keratinocytes in skin whitening effects. We concluded that Ectoine could be used as an effective topical natural cosmetic agent with depigmenting and anti-melanogenic efficacy.

Ectoine from Bacterial and Algal Origin Is a Compatible Solute in Microalgae.

Osmoregulation in phytoplankton is attributed to several highly polar low-molecular-weight metabolites. A widely accepted model considers dimethylsulfoniopropionate (DMSP) as the most important and abundant osmotically active metabolite. Using an optimized procedure for the extraction and detection of highly polar metabolites, we expand the group of phytoplankton osmolytes by identifying ectoine in several microalgae. Ectoine is known as a bacterial compatible solute, but, to the best of our knowledge, was never considered as a phytoplankton-derived product. Given the ability of microalgae to take up zwitterions, such as DMSP, we tested the hypothesis that the algal ectoine is derived from associated bacteria. We therefore analyzed methanol extracts of xenic and axenic cultures of two different species of microalgae and could detect elevated concentrations of ectoine in those that harbor associated bacteria. However, also microalgae without an associated microbiome contain ectoine in smaller amounts, pointing towards a dual origin of this metabolite in the algae from their own biosynthesis as well as from uptake. We also tested the role of ectoine in the osmoadaptation of microalgae. In the model diatoms Thalassiosira weissflogii and Phaeodactylum tricornutum, elevated amounts of ectoine were found when cultivated in seawater with salinities of 50 PSU compared to the standard culture conditions of 35 PSU. Therefore, we add ectoine to the family of osmoadaptive metabolites in phytoplankton and prove a new, potentially synergistic metabolic interplay of bacteria and algae.

Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

PurposeAt present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater.MethodsThe response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N.ResultHalomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%.ConclusionIn addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.

Betaine accumulation suppresses the de-novo synthesis of ectoine at a low osmotic concentration in Halomonas sp SBS 10, a bacterium with broad salinity tolerance.

The study aims to find out osmoadaptive mechanism used to overcome the salinity stress by Halomonas sp SBS 10 isolated from the saltern crystallizer ponds of the Sambhar Salt Lake and its taxonomic position using neighbor-joining algorithm. The strain SBS 10 was tested for accumulation of two major compatable solutes betaine and ectoine and was observed that osmoprotection in the strain SBS 10 is achieved by the accumulation of betaine or by the de-novo synthesis of betaine or ectoine. Amount of endogenous content of the betaine and ectoine per milligram of cell biomass was estimated to be 581µg, 587µg, 588µg, 617µg, and 761µg for betaine and 1.52µg, 2.74µg, 3.14µg, 3.50µg, and 52.67µg for ectoine, when exposed to 5, 10, 15, 20 and 25% of NaCl concentration. Results obtained from HPLC analysis showed that the betaine accumulation suppresses the de-novo synthesis of ectoine partially at low NaCl concentration in the growth medium. However, at a high NaCl concentration, the ectoine concentration increases abruptly as compared to the betaine. This indicates that the ectoine accumulation is transcriptionally up-regulated by the salinity stress. Phylogenetic analysis based on the neighbor-joining algorithm included the strain SBS 10 in the genus Halomonas of the family Halomonadaceae belonging to the class Gammaproteobacteria. Most closely related type strain was found to be Halomonas gudaonensis SL014B-69T (98.2% similarity). Ultrastructure characteristics showed the strain to be non-spore forming rod, 0.3-0.4 × 0.75-1.65 μm in size and motile with the help of peritrichous flagella.

The promotion and suppression of DNA charge neutralization by the cosolute ectoine.

Ectoine, a cosolute and osmolyte, is used by extremophilic microorganisms to maintain an osmotic equilibrium of cells with their surrounding medium under conditions of extreme salinity or thermal and pressure stresses. It is also considered a protectant of biomolecules such as protein and DNA in cells. In the present study, we investigate its influence on DNA charge neutralization and compaction through dynamic light scattering (DLS), atomic force microscopy (AFM) and single molecular magnetic tweezers (MT). We found that ectoine can promote DNA charge neutralization induced by multivalent cations at mild cosolute concentration in solution. When the concentration of ectoine is high enough, however, a mixed effect of promotion and suppression can be found under the same ionic conditions. In this case, the electrophoretic mobility (EM) of DNA is promoted in the region of low cation concentration, while suppressed in the region of high counterionic concentration. The charge neutralization of DNA by ectoine is also related to DNA compaction. The promotion and suppression of DNA compaction by ectoine was observed by AFM imaging. The condensed structure of DNA becomes more compact and then loose once more with the increasing concentration of ectoine. Meanwhile, the condensing forces of DNA measured by magnetic tweezers shows the same trend as does the DNA EM. We explained the experimental findings through the combined effect of two intrinsic features of ectoine, preferential exclusion and enhancement of the dielectric constant of the medium.

Protective effects of compatible solute ectoine against ethanol-induced toxic alterations in Daphnia magna

Ectoine (ECT) is a compatible solute synthesized mostly by halophilic microorganisms subjected to various stressful factors. Its protective properties in bacteria and some populations of isolated cells subjected to different stressors are reported; however, little is known on its effects against a commonly used compound, ethanol (ETH). The purpose of our study was to determine the effects of ETH alone (at 20 and 60 g/L) and in the combination with various concentrations of ECT (5, 10, and 25 mg/L) at various times of exposure on behavioural, physiological, and biochemical parameters of a model invertebrate Daphnia magna. In the present study, we determined the following parameters: immobilisation, heart rate, thoracic limb movement, catalase (CAT) activity, and nitric oxide species (NOx) level. Our study revealed that both concentrations of ETH alone induced immobilisation and decrease of swimming velocity, heart rate, and thoracic limb activity; however, catalase activity and NOx levels were increased. On the other hand, the animals exposed to the combinations of ETH + ECT showed a reduced immobilisation and alleviated inhibition of heart rate and thoracic limb activity, lower increase of CAT activity, and NOx level when compared to the crustaceans subjected to ETH alone. The most distinct alleviation of toxic effects was noted in the combinations in which the highest concentration of ECT were used. The results suggest that ETH may induce oxidative stress in daphnids and attenuating effects of ECT probably result from its antioxidative properties.

Stabilization of bovine lactoperoxidase in the presence of ectoine

Lactoperoxidase (LPO) is a heme peroxidase with various applications in industry and medicine. In this study, the effects of ectoine, as a compatible solute, on the structure, thermal stability, thermodynamic parameters, activity, and stability of LPO have been investigated. The results showed that the catalytic activity of LPO was improved by increasing ectoine concentration. The UV–visible absorption spectroscopy and FTIR spectra studies indicated that ectoine could bind to the LPO spontaneously. Moreover, ectoine increased the enzyme Tm and Gibbs free energy. The fluorescence measurements showed that LPO fluorescence was quenched in the presence of ectoine. The quenching mechanism was probably a static quenching by forming a ground state complex. The thermodynamic parameters indicated that hydrogen bonding and Vander Waals forces played a key role in the LPO-ectoine interaction process. The findings suggest that ectoine could be used as a lactoperoxidase stabilizing agent for industrial or medical purposes.

Ectoine bio-milking in methanotrophs: A step further towards methane-based bio-refineries into high added-value products

This communication showed for the first time that the methanotrophic strain Methylomicrobium alcaliphilum 20Z (M. alcaliphilum 20Z) can efficiently synthesize and excrete (through a tailored bio-milking process) ectoine under continuous mode using methane (CH4) as the sole energy and carbon source. First, three consecutive 50h fed batch fermentations consisting of alternating high salinity (6% NaCl for 24h) and low salinity (0% NaCl for 24h) cultivation stages were carried out in triplicate to determine the influence of sudden modifications in media salinity on ectoine synthesis and excretion. The results demonstrated that M. alcaliphilum 20Z exhibited a rapid response to osmotic shocks, which resulted in the release of the accumulated ectoine under hyposmotic shocks and the immediate uptake of the previously excreted ectoine during hyperosmotic shocks. A second experiment was carried out under continuous cultivation mode in two sequential stirred tank reactors operated at NaCl concentrations of 0 and 6%. Cells exhibited a constant intra-cellular ectoine concentration of 70.4±14.3mggbiomass−1 along the entire operation period when cultivated at a NaCl concentration of 6%. The centrifugation of the cultivation broth followed by a hyposmotic shock resulted in the excretion of ∼70% of the total intra-cellular ectoine. In brief, this research shows the feasibility of the continuous bioconversion of diluted CH4 emissions into high added-value products such as ectoine, which can turn greenhouse gas (GHG) abatement into a sustainable and profitable process.