High Acetate Concentrations Research Articles

Transplantation of Fecal Microbiota Shaped by Diet.

Alterations in diet and gut microbial ecology underlie the pathogenesis of type 1 diabetes (T1D). In the non-obese diabetic (NOD) mouse, we found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces correlated with protection from disease. We reconstituted germ free (GF) NOD mice with fecal bacteria from protected NOD mice fed with high acetate- and butyrate-yielding diets, to test whether the transferred gut microbiota protect against the development of T1D. GF NOD mice that received a microbiota shaped by high acetate- but not butyrate-yielding diet showed a marked protection against diabetes. This fecal transplantation assay demonstrated the potential for a dietary technology to reshape the gut microbiota that enables specific bacteria to transfer protection against T1D.

Phytochemical Compositions and Antidiabetic Potentials of Salvia sclarea L. Essential Oils.

Salvia sclarea (SS) is characterized by its valuable essential oils (Eos) and potent biological activities. This study aimed at investigating the phytochemical composition of SS Eos collected in within the same week, from two different regions in Lebanon, Beirut (SS-Bt) and Taanayel (SS-Tl), utilizing GC-MS methods, and to explore their acute and subchronic antidiabetic potentials. Moreover, studying the phytochemical diversity of twenty SS Eos established on our work and literature descriptions in order to recognize the origin of the Lebanese active chemotype(s). The Eos have been obtained by hydro-distillation and identified via GC-MS analyses. Five chemotypes of SS Eos have been identified. The Lebanese Eos, SS-Bt and SS-Tl, studied here have shown evidence to belong to two different chemotypes 1 and 5, respectively. SS-Bt has shown to belong to chemotype 1, which is characterized by high linalool (LL) concentration (average 40.2%). On the other hand, SS-Tl has shown to belong to chemotype 5, which is characterized by high linalyl acetate (LA) concentration (average 50.4%). The acute and subchronic antidiabetic activities of these EOs have been monitored along with LL and LA, in order to find the most active chemotype. Chemotypes 1 (owned to high LL content), present at low altitude places of Lebanon and Poland, has shown significantly higher acute and subchronic antidiabetic activities than that of chemotype 5 (owned to high LA content). In conclusion, Salvia sclarea Eos have shown potential antidiabetic activities, and their Eos might be used in the future as a complementary or an alternative medicine in the management of diabetes and related complications.

Improvement of the bioelectrochemical hydrogen production from food waste fermentation effluent using a novel start‐up strategy

AbstractBACKGROUNDFood waste is a valuable source of hydrogen by dark fermentation. Dark fermentation effluent contains volatile fatty acids that can be further converted into more hydrogen using microbial electrolysis cells (MECs). In this process, the anodic potential (Ean) has a significant influence on the MEC performance as well as the effluent composition. The objective of this study was to evaluate the effects of variation of the anode potential and substrate composition (food waste fermentation effluent) on the performance of hydrogen production using two‐chamber MECs.RESULTSColonization was conducted using an Ean of 0.5 V vs Ag/AgCl. After 38 days, the Ean had decreased to 0.3 V, resulting in an increase in the hydrogen production rate (from 287 to 482 mL H2 L‐1cat d‐1). A maximum hydrogen production rate of 685 mL H2 L‐1cat d‐1 was observed when effluent that contained the highest acetate concentration was utilized. Cathodic hydrogen recovery was higher than 93%, and hydrogen yield was greater than 873 mL H2 g‐1 COD.CONCLUSIONThe start‐up strategy in which Ean is decreased after the formation of an electroactive biofilm resulted in increased hydrogen production. The composition of the food waste fermented effluent influences the hydrogen production rate. © 2017 Society of Chemical Industry

Controls on groundwater quality and dug-well asphyxiation hazard in Dakoro area of Niger

In the Dakoro area of Niger, hand-dug wells are commonly used for village water supply. Several cases of asphyxiation during well digging have occurred, but the source and composition of the gas (suspected to be CO2, H2S, or CH4) remain uncertain. In addition, data on water quality in the region are sparse. We sampled ten dug wells and five deeper boreholes for analyses of solute chemistry and core samples from one borehole for sediment chemistry. With respect to inorganic solutes, groundwater quality was generally good, although one dug-well sample exceeded the drinking-water standard for NO3-. Solute chemistry tended to vary between dug wells (commonly Ca-HCO3 or Ca-SO4 waters, with elevated Si and circumneutral pH) and boreholes (typically Na-HCO3 waters, with lower Si and alkaline pH). These compositions variously appear to reflect dissolution of calcite, gypsum, and silicates; pyrite oxidation; and cation exchange. Some dug wells and boreholes had high acetate concentrations, low SO4-S relative to total S, and/or a rotten-egg odor, all of which are consistent with reduction of SO42- to H2S. This reaction is likely in Marine Series shales that are relatively high in S and organic C. Consequently, monitoring for H2S and other gases during well digging is warranted.

Effect of operation temperature on anaerobic digestion of food waste: Performance and microbial analysis

Temperature is one of the crucial parameters on anaerobic digestion (AD); however, key differences in the microbial community found in AD remain poorly understood. In this work, thermophilic and mesophilic AD fed with food waste were compared examining the relationship between the structure of the microbial community and methane (CH4) production. The maximum CH4 yield, 0.23LCH4·gCODadded−1, was observed under mesophilic condition at an organic loading rate (OLR) of 6.7gCOD·L−1·d−1 (HRT 30d) using step feeding, while it failed under thermophilic condition at an OLR of 5.0gCOD·L−1·d−1 (HRT 40d). A low concentration of propionate and acetate in the mesophilic condition was owing to the efficient acetogenesis and methanogenesis, while syntrophic acetogenesis was found to be the rate-limiting step in a sample collected on the day 110 under thermophilic condition (T1). In addition, hydrogenotrophic methanogenesis coupled with syntrophic acetate oxidation was mainly carried out in the sample collected on day 160 under mesophilic condition (M1) and in the sample T1. However, an increased OLR caused the accumulation of acetate resulting in the methanogenesis inhibition. The higher acetate concentration in the sample taken on day 240 under mesophilic condition (M2) was due to a more diverse and abundant syntrophic acetogenic bacteria indicating that methanogenesis became the rate-limiting step. Meanwhile, acetogenesis was found to be the rate-limiting step in the sample collected on day 130 under thermophilic condition (T2), resulting from that more lactate accumulation with shifting the microbial community to be favorable for lactate production.

Temporal Microbial Community Dynamics in Microbial Electrolysis Cells - Influence of Acetate and Propionate Concentration.

Microbial electrolysis cells (MECs) are widely considered as a next generation wastewater treatment system. However, fundamental insight on the temporal dynamics of microbial communities associated with MEC performance under different organic types with varied loading concentrations is still unknown, nevertheless this knowledge is essential for optimizing this technology for real-scale applications. Here, the temporal dynamics of anodic microbial communities associated with MEC performance was examined at low (0.5 g COD/L) and high (4 g COD/L) concentrations of acetate or propionate, which are important intermediates of fermentation of municipal wastewaters and sludge. The results showed that acetate-fed reactors exhibited higher performance in terms of maximum current density (I: 4.25 ± 0.23 A/m2), coulombic efficiency (CE: 95 ± 8%), and substrate degradation rate (98.8 ± 1.2%) than propionate-fed reactors (I: 2.7 ± 0.28 A/m2; CE: 68 ± 9.5%; substrate degradation rate: 84 ± 13%) irrespective of the concentrations tested. Despite of the repeated sampling of the anodic biofilm over time, the high-concentration reactors demonstrated lower and stable performance in terms of current density (I: 1.1 ± 0.14 to 4.2 ± 0.21 A/m2), coulombic efficiency (CE: 44 ± 4.1 to 103 ± 7.2%) and substrate degradation rate (64.9 ± 6.3 to 99.7 ± 0.5%), while the low-concentration reactors produced higher and dynamic performance (I: 1.1 ± 0.12 to 4.6 ± 0.1 A/m2; CE: 52 ± 2.5 to 105 ± 2.7%; substrate degradation rate: 87.2 ± 0.2 to 99.9 ± 0.06%) with the different substrates tested. Correlating reactor’s performance with temporal dynamics of microbial communities showed that relatively similar anodic microbial community composition but with varying relative abundances was observed in all the reactors despite differences in the substrate and concentrations tested. Particularly, Geobacter was the predominant bacteria on the anode biofilm of all MECs over time suggesting its possible role in maintaining functional stability of MECs fed with low and high concentrations of acetate and propionate. Taken together, these results provide new insights on the microbial community dynamics and its correlation to performance in MECs fed with different concentrations of acetate and propionate, which are important volatile fatty acids in wastewater.

Electrodeposition and characterization of metallic molybdenum from aqueous electrolytes containing high acetate concentrations

Metallic molybdenum coating was deposited from an aqueous electrolyte containing a high concentration of acetate and molybdate ions in pH of 6.8±0.2. It is complicated to deposit molybdenum from an aqueous electrolyte because of its high reactivity with oxygen. The mole ratios of water to acetate were in the range of (2.4±0.2):1. It was found that the change in bath stirring rate can result in the deposition of molybdenum oxide insisted of Mo coating. Also the morphological differences of metallic Mo and molybdenum oxide layer were investigated. In the present study, a successful attempt was made metallic Mo coating with a thickness of 3–7μm that was dependent on the DC current density.The XRD investigation indicated that the crystal structure of Mo coating was amorphous. The surface composition of deposits was ascertained by energy dispersive X-ray microanalysis (EDS) that showed the presence of high Mo content (90.7%) and low content of oxygen (9.3%). In addition, different EDS analysis was obtained from the top surface of coating and subsurface section. The surface morphology of coating was studied by scanning electron microscopy (SEM). Also, XPS analysis confirmed deposition of Mo(0) metal.

Autocatalytic and Non-Catalytic Oxidation of Manganese(II) on a Platinum Electrode in Acetic Buffer

Abstract The influence of acetate on Mn2+ oxidation by a platinum electrode has been investigated in aqueous solution with cyclic voltammetry. It is found that acetate at concentrations below 0.5 M gives rise to autocatalytic oxidation of Mn2+ with MnO2 serving as the catalyst, while in 0.5–1 M acetate (as well as in the absence of acetate) Mn2+ is oxidized non-catalytically. The autocatalytic oxidation is revealed by a separate voltammetric wave which has a much sharper rise and a lower potential compared to non-catalytic waves, by a sigmoid kinetics during chronoamperometry and by an enhanced Mn2+ uptake with electrodeposited MnO2. The increase in temperature and Mn2+ concentration promotes autocatalysis while acidification and the increase in sweep rate inhibits it that is thought to be due to the acceleration or delay of MnO2 formation from Mn3+. It is suggested that acetate acts in two ways: (1) thermodynamically stabilizes Mn3+ by complexation to enable the autocatalytic reaction Mn2++MnO2+OAc−+H+↔Mn3+−OAc+ MnIIIOOH and (2) delays the formation of MnO2 (presumably on the stage of Mn(III) crystallization) which leads to the inhibition of autocatalysis at high acetate concentrations.

Draft Genome Sequencing of Ascomycetes Yeast Pichia membranifaciens KS47-1, Which Shows High Acetate Resistance in Lignocellulosic Feedstock Hydrolysate.

ABSTRACTPichia membranifaciens KS47-1 is capable of growing on hydrolysate containing high concentrations of acetate and other growth inhibitors. To reveal the acetate-resistant associate genes of strain KS47-1, we present the 11.4-Mb draft genome sequence.

Assessment of the synbiotic properites of human milk oligosaccharides and Bifidobacterium longum subsp. infantis in vitro and in humanised mice.

The mode of delivery plays a crucial role in infant gastrointestinal tract colonisation, which in the case of caesarean section is characterised by the presence of clostridia and low bifidobacterial counts. Gut colonisation can be modified by probiotics, prebiotics or synbiotics. Human milk oligosaccharides (HMOs) are infant prebiotics that show a bifidogenic effect. Moreover, genome sequencing of Bifidobacterium longum subsp. infantis within the infant microbiome revealed adaptations for milk utilisation. This study aimed to evaluate the synbiotic effect of B. longum subsp. infantis, HMOs and human milk (HM) both in vitro and in vivo (in a humanised mouse model) in the presence of faecal microbiota from infants born by caesarean section. The combination of B. longum and HMOs or HM reduced the clostridia and G-bacteria counts both in vitro and in vivo. The bifidobacterial population in vitro significantly increased and produce high concentrations of acetate and lactate. In vitro competition assays confirmed that the tested bifidobacterial strain is a potential probiotic for infants and, together with HMOs or HM, acts as a synbiotic. It is also able to inhibit potentially pathogenic bacteria. The synbiotic effects identified in vitro were not observed in vivo. However, there was a significant reduction in clostridia counts in both experimental animal groups (HMOs + B. longum and HM + B. longum), and a specific immune response via increased interleukin (IL)-10 and IL-6 production. Animal models do not perfectly mimic human conditions; however, they are essential for testing the safety of functional foods.

Enhancement of acetate productivity in a thermophilic (55 °C) hollow-fiber membrane biofilm reactor with mixed culture syngas (H2/CO2) fermentation

Conversion of organic wastes to syngas is an attractive way to utilize wastes. The produced syngas can be further used to produce a variety of chemicals. In this study, a hollow-fiber membrane biofilm reactor with mix cultures was operated at 55°C to convert syngas (H2/CO2) into acetate. A high concentration of acetate (42.4g/L) was reached in batch experiment while a maximum acetate production rate of 10.5g/L/day was achieved in the continuous-flow mode at hydraulic retention time (HRT) of 1day. Acetate was the main product in both batch and continuous-flow experiments. n-Butyrate was the other byproduct in the reactor. Acetate accounted for more than 98.5 and 99.1% of total volatile fatty acids in batch and continuous modes, respectively. Illumina Miseq high-throughput sequencing results showed that microorganisms were highly purified and enriched in the reactor. The main genus was Thermoanaerobacterium (66% of relative abundance), which was usually considered as H2 producer in the literature, however, likely played a role as a H2 consumer in this study. This study provides a new method to generate the high producing rate and purity of acetate from syngas.

Urinary metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of Orthosiphon stamineus leaves elucidated by 1H NMR spectroscopy

Orthosiphon stamineus (OS) is a popular medicinal herb used in traditional Chinese medicine as a diuretic agent and for renal system disorders. This study employed 1H NMR based metabolomics approach to investigate the possible protective activity of OS in cisplatin induced nephrotoxicity owing to its diuretic and antioxidant activities. Aqueous (OSAE) and 50% aqueous ethanolic (OSFE) extracts of OS leaves were orally administered at 400mg/kg BW doses to rats which were then intraperitoneally injected with cisplatin at 5mg/kg BW dose. The 1H NMR profile of the urine samples collected on day 5 after cisplatin administration were analyzed by multivariate pattern recognition techniques, whereby 19 marker metabolites suggestive in the involvement of TCA cycle, disturbed energy metabolism, altered gut microflora and BCAA metabolism pathways were identified. It was observed that OSFE caused significant changes (p<0.05) in the levels of 8 markers namely leucine, acetate, hippurate, lysine, valine, 2-oxoglutarate, 3-HBT and acetoacetate resulting in a moderate ameliorative effect, however, it did not completely protect from nephrotoxicity. OSAE did not demonstrate significant down regulatory effects on any markers, albeit, it potentiated the cisplatin nephrotoxicity by inducing significant increase in glucose, glycine, creatinine, citrate, TMAO, acetate and creatine levels. A Principal Component Analysis (PCA) of the 1H NMR spectra of OS extracts identified that OSFE had higher concentrations of the secondary metabolites such as caffeic acid, chlorogenic acid, protocatechuic acid and orthosiphol, among others. Whereas, OSAE was characterized by higher concentrations of acetate, lactate, succinic acid, valine and phosphatidylcholine. This research denotes the first comprehensive analysis to identify the effects of OS extracts on cisplatin nephrotoxicity.

Impact of light oil toxicity on sulfide production by acetate-oxidizing, sulfate-reducing bacteria

Souring, the reduction of sulfate to sulfide by sulfate-reducing bacteria (SRB) is a serious problem for the oil and gas industry. Produced waters from oil fields, injected with seawater, often contain high concentrations of acetate and sulfate, indicating a lack of activity of SRB using acetate as electron donor for sulfate reduction. Experiments with an enrichment of lactate- and sulfate-utilizing SRB from a shale oil field, indicated oxidation of lactate to acetate and subsequent oxidation of acetate in the absence of added shale oil. However, acetate was not oxidized in the presence of shale oil. In view of these results we hypothesized that acetate-utilizing SRB were inhibited by low molecular weight components present in the light shale oil, which had an American Petroleum Institute gravity (API_G) of 38°. Low molecular weight components decrease in concentration with decreasing API_G of the oil. We therefore tested the effects of diluent (83° API_G, consisting predominantly of C5-C7 alkanes), light oils (38, 41 and 46° API_G) and heavy oil (16° API_G), as well as of selected light oil components (toluene, pentane and heptane). Acetate-dependent sulfate reduction by a pure culture of Desulfobacter postgatei at 30 °C was inhibited by diluent and light oils, but not by heavy oil. Similar results were obtained with a shale gas field source water enrichment (SWE), which contained Desulfotomaculum and Desulfoarculus as the most prominent acetate-utilizing SRB. Use of dilutions of oils in heptamethylnonane indicated that diluent inhibited acetate-utilizing SRB in SWE more strongly than light oils. Use of pure oil components indicated toluene to be more inhibitory than either pentane or heptane. GC-MS analysis confirmed that diluent had the highest concentrations of these low molecular weight components, explaining its strong inhibitory effect. Inhibition of acetate-utilizing SRB by light oil components, causing acetate accumulation in produced waters, may contribute to souring control and should be incorporated in souring control models.

Unexpected competitiveness of Methanosaeta populations at elevated acetate concentrations in methanogenic treatment of animal wastewater.

Acetoclastic methanogenesis is a key metabolic process in anaerobic digestion, a technology with broad applications in biogas production and waste treatment. Acetoclastic methanogenesis is known to be performed by two archaeal genera, Methanosaeta and Methanosarcina. The conventional model posits that Methanosaeta populations are more competitive at low acetate levels (<1mM) than Methanosarcina and vice versa at higher acetate concentrations. While this model is supported by an extensive body of studies, reports of inconsistency have grown that Methanosaeta were observed to outnumber Methanosarcina at elevated acetate levels. In this study, monitoring of anaerobic digesters treating animal wastewater unexpectedly identified Methanosaeta as the dominant acetoclastic methanogen population at both low and high acetate levels during organic overloading. The surprising competitiveness of Methanosaeta at elevated acetate was further supported by the enrichment of Methanosaeta with high concentrations of acetate (20mM). The dominance of Methanosaeta in the methanogen community could be reproduced in anaerobic digesters with the direct addition of acetate to above 20mM, again supporting the competitiveness of Methanosaeta over Methanosarcina at elevated acetate levels. This study for the first time systematically demonstrated that the dominance of Methanosaeta populations in anaerobic digestion could be linked to the competitiveness of Methanosaeta at elevated acetate concentrations. Given the importance of acetoclastic methanogenesis in biological methane production, findings from this study could have major implications for developing strategies for more effective control of methanogenic treatment processes.

Genomic characterization of a fructophilic bee symbiont Lactobacillus kunkeei reveals its niche-specific adaptation

Lactobacillus kunkeei is classified as a sole obligate fructophilic lactic acid bacterium that is found in fructose-rich niches, including the guts of honeybees. The species is differentiated from other lactobacilli based on its poor growth with glucose, enhanced growth in the presence of oxygen and other electron acceptors, and production of high concentrations of acetate from the metabolism of glucose. These characteristics are similar to phylogenetically distant Fructobacillus spp. In the present study, the genomic structure of L. kunkeei was characterized by using 16 different strains, and it had significantly less genes and smaller genomes when compared with other lactobacilli. Functional gene classification revealed that L. kunkeei had lost genes specifically involved in carbohydrate transport and metabolism. The species also lacked most of the genes for respiration, although growth was enhanced in the presence of oxygen. The adhE gene of L. kunkeei, encoding a bifunctional alcohol dehydrogenase (ADH)/aldehyde dehydrogenase (ALDH) protein, lacked the part encoding the ADH domain, which is reported here for the first time in lactic acid bacteria. The deletion resulted in the lack of ADH activity, implying a requirement for electron acceptors in glucose assimilation. These results clearly indicated that L. kunkeei had undergone a specific reductive evolution in order to adapt to fructose-rich environments. The reduction characteristics were similar to those of Fructobacillus spp., but distinct from other lactobacilli with small genomes, such as Lactobacillus gasseri and Lactobacillus vaginalis. Fructose-richness thus induced an environment-specific gene reduction in phylogenetically distant microorganisms.

A β-carotene ketolase gene (bkt1) promoter regulated by sodium acetate and light in a model green microalga Chlamydomonas reinhardtii

Many microalgae could accumulate high level of astaxanthin, especially a green microalga Haematococcus pluvialis. β-carotene ketolase gene (bkt) is a key enzyme for astaxanthin biosynthesis in Haematococcus pluvialis, and astaxanthin accumulation could be induced effectively by abiotic stresses such as high concentration of sodium acetate and high light. In Haematococcus there are 3 bkt gene copies, among them, the transcription level of bkt1 was obviously correlated with high light and sodium acetate. Thus, the function of bkt1 promoter played a key role in understanding the regulatory mechanisms of astaxanthin biosynthesis. A TATA-box and cis-acting elements associated with high light and stress-related responses were detected in bkt1 promoter in previously studies. However, detailed information about cis-acting regulatory elements of this important promoter is still unavailable since a reliable genetic manipulation technique is not available yet. To identify the functions of cis-elements in bkt1 promoter, 8 constructs contained different length of bkt1 promoter were successfully introduced into a model microalga Chlamydomonas reinhardtii and different transformants were obtained with zeomycin resistant selection. Exposed to high light or sodium acetate, differentially increased ble transcripts and protein production in transformants indicated that these cis-elements respond to high light and sodium acetate. Moreover, we found that TATA-box in bkt1 promoter is essential for the promoter function and ble expression was correlated with cis-acting elements in bkt1 promoter. Our results shed lights on regulation mechanisms of the key genes from Haematococcus for astaxanthin biosynthesis in Chlamydomonas reinhardtii.

Effect of in-situ immobilized anode on performance of the microbial fuel cell with high concentration of sodium acetate

To improve performance of the microbial fuel cell (MFC) with high concentration of organics, a procedure for in-situ immobilized anode with agarose gel was proposed in this study. The performance of power generation, electrochemical activity, and microbial community of the MFC with immobilized anode (i-MFC) was investigated using different concentrations (i.e., 1, 5, 10, and 20g/L) of sodium acetate. The i-MFC could generate electricity within one hour after refreshing substrate. With 5g/L acetate, the maximum voltages were 560 and 460mV in the i-MFC and controlled MFC (c-MFC), respectively. With 10g/L acetate, the maximum voltage of the i-MFC was much higher than that of the c-MFC (500 vs. 300mV). With the acetate concentrations of 5 and 10g/L, the maximum power densities in the i-MFC were 610±50 and 370±40mW/m2, respectively, while the maximum power densities in the c-MFC were 343±30 and 240±20mW/m2, respectively. Cyclic voltammetry measurements indicated that microbial activity of the i-MFC anode was higher than that of the c-MFC anode under the high acetate concentration (3.00 vs. 2.19mA). The relative abundance of Geobacter on the i-MFC anode was much higher than that on the c-MFC anode (62% vs. 40%). The in-situ immobilization strategy provided an easily performed and efficient way to keep the activity of exoelectrogens, resulting in the improvement of MFC performance.

Improvement of Malvar Wine Quality by Use of Locally-Selected Saccharomyces cerevisiae Strains

Malvar grape juice offers relatively little in the way of a sensory experience. Our interest lies in the use of locally-selected yeast strains in experimental fermentations to improve the sensory characteristics of Malvar wines. Two locally-selected strains of Saccharomyces cerevisiae were used as starter cultures in vinifications and compared with spontaneous fermentations of the same cultivar musts. Wine quality was investigated by their principal oenological parameters, analysis of the volatile aroma components, and corroborated by an experienced taster panel. The most salient chemical attributes were its high concentrations of isoamyl acetate and hexyl acetate and the high acidity, which have been detected to be key constituents in setting the fruity and fresh character of Malvar wines. Winemakers of winegrowing areas where this grape variety is cultivated will have improved options to elaborate new white wines styles, using selected yeast strains that enhance its aromatic properties.

Heavy metal chelator TPEN attenuates fura-2 fluorescence changes induced by cadmium, mercury and methylmercury.

Stimulation with heavy metals is known to induce calcium (Ca2+) mobilization in many cell types. Interference with the measurement of intracellular Ca2+ concentration by the heavy metals in cells loaded with Ca2+ indicator fura-2 is an ongoing problem. In this study, we analyzed the effect of heavy metals on the fura-2 fluorescence ratio in human SH-SY5Y neuroblastoma cells by using TPEN, a specific cell-permeable heavy metal chelator. Manganese chloride (30–300 µM) did not cause significant changes in the fura-2 fluorescence ratio. A high concentration (300 µM) of lead acetate induced a slight elevation in the fura-2 fluorescence ratio. In contrast, stimulation with cadmium chloride, mercury chloride or MeHg (3–30 µM) elicited an apparent elevation of the fura-2 fluorescence ratio in a dose-dependent manner. In cells stimulated with 10 or 30 µM cadmium chloride, the addition of TPEN decreased the elevated fura-2 fluorescence ratio to basal levels. In cells stimulated with mercury or MeHg, the addition of TPEN significantly decreased the elevation of the fura-2 fluorescence ratio induced by lower concentrations (10 µM) of mercury or MeHg, but not by higher concentrations (30 µM). Pretreatment with Ca2+ channel blockers, such as verapamil, 2-APB or lanthanum chloride, resulted in different effects on the fura-2 fluorescence ratio. Our study provides a characterization of the effects of several heavy metals on the mobilization of divalent cations and the toxicity of heavy metals to neuronal cells.

Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress.

Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days.