Haloferax mediterranei produces C50 carotenoids that have strong antioxidant properties. The response surface methodology (RSM) tool helps to accurately analyze the most suitable conditions to maximize C50 carotenoids production by haloarchaea. The effects of temperature (15–50 °C), pH (4−10), and salinity (5–28% NaCl (w/v)) on the growth and carotenoid content of H. mediterranei were analyzed using the RSM approach. Growth was determined by measuring the turbidity at 600 nm. To determine the carotenoid content, harvested cells were lysed by freeze/thawing, then re-suspended in acetone and the total carotenoid content determined by measuring the absorbance at 494 nm. The analysis of carotenoids was performed by an HPLC system coupled with mass spectrometry. The results indicated the theoretical optimal conditions of 36.51 or 36.81 °C, pH of 8.20 or 8.96, and 15.01% or 12.03% (w/v) salinity for the growth of haloarchaea (OD600 = 12.5 ± 0.64) and production of total carotenoids (3.34 ± 0.29 mg/L), respectively. These conditions were validated experimentally for growth (OD600 = 13.72 ± 0.98) and carotenoid production (3.74 ± 0.20 mg/L). The carotenoid profile showed four isomers of bacterioruberin (89.13%). Our findings suggest that the RSM approach is highly useful for determining optimal conditions for large-scale production of bacterioruberin by haloarchaea.

Microalgae have been widely recognized as a valuable source of natural, bioactive molecules that can benefit human health. Some molecules of commercial value synthesized by the microalgal metabolism have been proven to display anti-inflammatory activity, including the carotenoids lutein and astaxanthin, the fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), and sulphated polysaccharides. These molecules can accumulate to a certain extent in a diversity of microalgae species. A production process could become commercially feasible if the productivity is high and the overall production process costs are minimized. The productivity of anti-inflammatory molecules depends on each algal species and the cultivation conditions, the latter being mostly related to nutrient starvation and/or extremes of temperature and/or light intensity. Furthermore, novel bioprocess tools have been reported which might improve the biosynthesis yields and productivity of those target molecules and reduce production costs simultaneously. Such novel tools include the use of chemical triggers or enhancers to improve algal growth and/or accumulation of bioactive molecules, the algal growth in foam and the surfactant-mediated extraction of valuable compounds. Taken together, the recent findings suggest that the combined use of novel bioprocess strategies could improve the technical efficiency and commercial feasibility of valuable microalgal bioproducts production, particularly anti-inflammatory compounds, in large scale processes.

and finally, group simultaneously exposed to As and Cd (As/Cd 229 GROUP). Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the effects of metal interactions in mammals. Application to ..., J Prot (2014) hydrogen peroxide (4:1 v/v) was added. After 10 min, the PTFE 281 vessels were closed and introduced into the microwave oven. 282The mineralization was carried out at 400 W from room increased from 60 to 140°C at a rate of 7°C/min for 4 min. Then, peaks from cytosolic fractions of liver (Fig. 1). 448In Fig. 1 can be observed the presence of low molecular 449 mass As species (<300 Da) in liver cytosolic extracts analyzed by SEC-ICP-MS. The higher intensity of signals was obtained 451 from liver of mice exposed to As/Cd during 6 days (Fig. 1A), in 452 which As concentration is the highest (Table 2). In Fig. 1A Se concentrations determined by IDA-ICP-ORS-MS after acid 496 digestion (Table 3). 497The effect of mice independent exposure to As or Cd on plasma under Cd exposure (Table 3). However, the depletion 507 of SeP concentration is reversed under As/Cd exposure. Since Table 2 t2:1 -Quantification of arsenic and cadmium in plasma, liver and kidney of mice under As/Cd exposure by ICP-ORS-MS. Fig. 2 -Score plots of PLS-DA for ESI+ and ESI− ionization modes of polar and lipophilic metabolites from mice plasma. Black squares: control group; red circles: mice exposed to As during 12 days; green asterisk: mice exposed to Cd during 12 days; blue diamonds: mice exposed to As/Cd mice during 12 days. Finally, the increased levels of eGPx in plasma (Table 3) prediction. In addition, the complementarity of using both 542 ionization modes for polar and lipophilic metabolites is 543 remarkable (see Table 4). 544In addition, GC-MS was applied as complementary Table 5. 552These metabolic changes in GROUP As/Cd can be related to and increasing concentration in lactic acid under Cd exposure. 556However, As exposure produces a decrease of lactic acid levels 557 and As/Cd does not provoke any alteration in this metabolite 558 (Table 5). Further perturbations of energy metabolism interme-559 diates, such as citric acid, isocitric acid, α-ketoglutarate and 560 glutamic acid were also observed (Tables 4 and 5). Under As 561 exposure, increased concentrations of intermediate energy Please cite this article as: García-Sevillano MÁ, et al, A combination of metallomics and metabolomics studies to evaluate the effects of metal interactions in mammals. Application to ..., J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.02.011metabolites were obtained (Tables 4 and 5). This fact has been 563 previously reported in M. musculus mice exposed to arsenic [4] 564 and has been related with the synthesis of glutathione (GSH) [4]. 565Under As/Cd exposure, similar effects were observed. 566Perturbations of amino acid concentrations related to toxic 567 exposure [30] can also be seen in Table 4, which shows decreasing 568 levels of taurine and alanine. (Ta...

Haloarchaea produce C50 carotenoids such as bacterioruberin, which are of biotechnological in-terest. This study aimed to analyze the effect of different environmental and nutritional conditions on the cellular growth and dynamics of carotenoids accumulation in Haloferax mediterranei. The maximum production of carotenoids (40 µg·mL−1) was obtained during the stationary phase of growth, probably due to nutrient-limiting conditions (one-step culture). By seven days of culture, 1 mL culture produced 22.4 mg of dry weight biomass containing 0.18 % (w/w) of carotenoids. On the other hand, carbon-deficient cultures (low C/N ratio) were observed to be optimum for C50 bacterioruberin production by Hfx. mediterranei, but negatively affected the growth of cells. Thus, a two-steps process was evaluated for optimum carotenoids yield. In the first step, a nutri-ent-repleted culture medium enabled the haloarchaea to produce biomass, while in the second step, the biomass was incubated under osmotic stress and in a carbon-deficient medium. Under the conditions used, the obtained biomass contained 0.27% (w/w) of carotenoids after seven days, which accounts for 58.49 µg·mL−1 of carotenoids for a culture with turbidity 14.0.

Mass spectrometry (MS)-based toxicometabolomics requires analytical approaches for obtaining unbiased metabolic profiles. The present work explores the general application of direct infusion MS using a high mass resolution analyzer (a hybrid systems triple quadrupole-time-of-flight) and a complementary gas chromatography-MS analysis to mitochondria extracts from mouse hepatic cells, emphasizing on mitochondria isolation from hepatic cells with a commercial kit, sample treatment after cell lysis, comprehensive metabolomic analysis and pattern recognition from metabolic profiles. Finally, the metabolomic platform was successfully checked on a case-study based on the exposure experiment of mice Mus musculus to inorganic arsenic during 12 days. Endogenous metabolites alterations were recognized by partial least squares-discriminant analysis. Subsequently, metabolites were identified by combining MS/MS analysis and metabolomics databases. This work reports for the first time the effects of As-exposure on hepatic mitochondria metabolic pathways based on MS, and reveals disturbances in Krebs cycle, β-oxidation pathway, amino acids degradation and perturbations in creatine levels. This non-target analysis provides extensive metabolic information from mitochondrial organelle, which could be applied to toxicology, pharmacology and clinical studies.