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Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides

Abstract Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize. For large-scale hydrogen production, an integrated view of the overall metabolism is necessary in order to interpret results properly and facilitate experimental design. In this study, a summary of the hydrogen production metabolism of the photosynthetic purple non-sulfur (PNS) bacteria will be presented. Practically all hydrogen production by PNS bacteria occurs under a photoheterotrophic mode of metabolism. Yet results show that under certain conditions, alternative modes of metabolism—e.g. fermentation under light deficiency—are also possible and should be considered in experimental design. Two enzymes are especially critical for hydrogen production. Nitrogenase promotes hydrogen production and uptake hydrogenase consumes hydrogen. Though a wide variety of substrates can be used for growth, only a portion of these is suitable for hydrogen production. The efficiency of a certain substrate depends on factors such as the activity of the TCA cycle, the carbon-to-nitrogen ratio, the reduction-state of that material and the conversion potential of the substrate into alternative metabolites such as PHB. All these individual components of the hydrogen production interact and are subject to strict regulatory controls. An overall scheme for the hydrogen production metabolism is presented.

Kinetics of biological hydrogen production by the photosynthetic bacterium Rhodobacter sphaeroides O.U. 001

The kinetics and the effects of various parameters on hydrogen production by Rhodobacter sphaeroides O.U. 001 were investigated in a batch column photobioreactor. In particular, the effect of the inoculum age and the implementation of a light–dark cycle illumination scheme for emulating natural sunlight have been investigated in detail. The possibility of using yeast extract to replace the rather expensive vitamin mixture in the medium was also studied. The results show that hydrogen production is decreased when the initially inoculated bacteria have a high culture age. Exposure of the bacterial culture to light–dark cycles increased the amount of hydrogen compared to continuous illumination, all other parameters remaining the same. Similarly, the use of yeast extract to replace the vitamins increased the growth and hydrogen production rates, however, with a slight reduction in the total amount of gas produced and the hydrogen fraction in the evolved gas.

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed ∼2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C18-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C18-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C18-ceramide levels ∼6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C18-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis.

Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor

The effect of l-malic acid and sodium glutamate, which serve as the carbon and nitrogen source, respectively, on hydrogen production by Rhodobacter sphaeroides O.U.001 has been investigated in a batch water jacketed glass column photobioreactor (PBR), which has an inner volume of 400 ml. The PBR was operated at different carbon to nitrogen ratios at 32°C with a tungsten lamp at a light intensity of 200 W m−2. Carbon to nitrogen ratio was found to be an important parameter for bio-hydrogen production. Moreover, hydrogen gas production was dependent on certain threshold concentrations of sodium glutamate. l-malic acid consumption was found to be first order with respect to l-malic acid concentration, whereas sodium glutamate consumption was found to be second order with respect to glutamate concentration. It was concluded that there is a close relationship between the hydrogen production rate and substrate consumption rates. A kinetic model is developed, which relates hydrogen gas production per amount of biomass, l-malic acid, and sodium glutamate concentrations.

Photobiological hydrogen production by using olive mill wastewater as a sole substrate source

Abstract In the present work olive mill wastewater (OMW) collected from West Anatolia—Turkey during 2001, containing 36.02 g carbon, 5.26 g hydrogen, and 0.96 g nitrogen in 100 g suspended solid was used as a sole substrate for the production of hydrogen gas by Rhodobacter sphaeroides O.U.001 in 400 ml glass, column-photobioreactors. Hydrogen production studies on diluted-OMW were investigated in the range of 20% (v/v) and 1% (v/v) OMW containing media. Below 5% OMW containing media, bacterial growth rate fitted well to the logistic model where hydrogen production was observed for the ones below 4% OMW. A maximum hydrogen production potential (HPP) of 13.9 l H 2 / l OMW was obtained at 2% OMW. During the biological hydrogen production process, chemical oxygen demand (COD) of the diluted wastewater decreased from 1100 to 720 mg / l ; biochemical oxygen demand (BOD) decreased from 475 to 200 mg / l , and the total recoverable phenol content (ortho- and meta-substitutions) decreased from 2.32 to 0.93 mg / l . In addition, valuable by-products such as carotenoid (40 mg / l OMW ) and polyhydroxybutyrate (PHB) (60 mg / l OMW ) were obtained. According to these results, OMW was concluded to be a very promising substrate source for biohydrogen production process, with additional benefits of its utilization with regard to environmental and economical aspects.

Synthesis of Doxorubicin loaded magnetic chitosan nanoparticles for pH responsive targeted drug delivery

Targeted drug delivery is a promising alternative to overcome the limitations of classical chemotherapy. In an ideal targeted drug delivery system carrier nanoparticles would be directed to the tumor tissue and selectively release therapeutic molecules. As a novel approach, chitosan coated magnetic nanoparticles (CS MNPs) maintain a pH dependent drug delivery which provides targeting of drugs to the tumor site under a magnetic field. Among various materials, chitosan has a great importance as a pH sensitive, natural, biodegradable, biocompatible and bioadhesive polymer. The aim of this study was to obtain an effective targeted delivery system for Doxorubicin, using chitosan coated MNPs. Different sized CS MNPs were produced by in situ synthesis method. The anti-cancer agent Doxorubicin was loaded onto CS MNPs which were characterized previously. Doxorubicin loading was confirmed by FTIR. Drug loading and release characteristics, and stability of the nanoparticles were investigated. Our results showed that the CS MNPs have pH responsive release characteristics. The cellular internalization of Doxorubicin loaded CS MNPs were visualized by fluorescent microscopy. Doxorubicin loaded CS MNPs are efficiently taken up by MCF-7 (MCF-7/S) and Doxorubicin resistant MCF-7 (MCF-7/1 μM) breast cancer cells, which increases the efficacy of drug and also maintains overcoming the resistance of Doxorubicin in MCF-7/Dox cells. Consequently, CS MNPs synthesized at various sizes can be effectively used for the pH dependent release of Doxorubicin in cancer cells. Results of this study can provide new insights in the development of pH responsive targeted drug delivery systems to overcome the side effects of conventional chemotherapy.

Photoproduction of hydrogen from sugar refinery wastewater by Rhodobacter sphaeroides O.U. 001

Pretreated sugar refinery wastewater (SRWW) was used for the production of hydrogen by Rhodobacter sphaeroides O.U.001 in a 0.4 l column photobioreactor. Hydrogen was produced at a rate of 0.001 l hydrogen/h/l culture in 20% dilution of SRWW. To adjust the carbon concentration to 70 mM and nitrogen concentration to 2 mM, sucrose or L-malic acid was added as carbon source and sodium glutamate was added as nitrogen source to the 20% dilution of SRWW. By these adjustments, hydrogen production rate was increased to 0.005 l hydrogen/h/l culture. Continuous hydrogen production was achieved in the same medium for 100 days at three different dilution rates. The maximum hydrogen produced was 2.67 l (in 100 days) at a dilution rate of 0.0013 h−1.

Effect of clay pretreatment on photofermentative hydrogen production from olive mill wastewater

The aim of this paper was to gain further insight into the effect of the clay pretreatment process on photofermentative hydrogen production. This two-stage process involved a clay pretreatment step followed by photofermentation which was performed under anaerobic conditions with the illumination by Tungsten lamps. Rhodobacter sphaeroides O.U.001 was used for photofermentation. Higher amounts of color (65%), total phenol (81%) and chemical oxygen demand (31%) removal efficiencies were achieved after clay pretreatment process. During photofermentative hydrogen production with the effluent of clay pretreatment process, the main organic compounds resulting higher hydrogen production rates were found to be acetic, lactic, propionic, and butyric acids. Compared to photofermentation using raw olive mill wastewater ( 16LH2/LOMW), the amount of photofermentative hydrogen production was doubled by using the effluent of the clay pretreatment process (31.5LH2/LOMW). The reasons for the improvement of hydrogen production by clay treatment can be attributed to the high removal of the hardly biodegradable compounds such as phenols; minor removal of organic acids, sugars and amino acids that are known to enhance photofermentative hydrogen production; and the color depletion of raw OMW which might cause a shadowing effect on the photosynthetic bacteria.

Identification of by-products in hydrogen producing bacteria; Rhodobacter sphaeroides O.U. 001 grown in the waste water of a sugar refinery

Rhodobacter sphaeroides O.U. 001 is able to produce hydrogen anaerobically upon illumination. The cells were screened for the presence of valuable by-products such as poly-β-hydroxy (PHB) butyric acid aiming to improve the feasibility of the system. Also waste water from a sugar refinery was used for bacterial growth to further increase the feasibility. Under aerobic conditions the standard growth media containing l-malic acid and sodium glutamate in 7.5/10 and 15/2 molar ratios and a medium containing 30% waste water from sugar refinery were used. In this case the maximum concentration of PHB produced were approximately 0.2 g l− 1 in both of the standard media whereas it was 0.3 g l− 1 in medium containing 30% waste water. By using the medium containing 30% waste water, PHB and hydrogen productions were determined under anaerobic conditions. The maximum concentration of PHB produced was around 0.5 g l− 1 and the amount of gas collected was 35 ml in 108 h. From these results it can be concluded that PHB can be collected during hydrogen production. The use of waste water from sugar refinery increased the yield.

Effect of MDR modulators verapamil and promethazine on gene expression levels of MDR1 and MRP1 in doxorubicin-resistant MCF-7 cells

PurposeOne of the major problems of cancer chemotherapy is the development of multidrug resistance (MDR) phenotype. Among the numerous mechanisms of MDR, a prominent one is the increased expression of membrane transporter proteins, the action of which leads to decreased intracellular drug concentration and cytotoxicity of drugs. Among them, P-gp and MRP1, encoded by MDR1 and MRP1 genes, respectively, have been associated with MDR phenotype. Chemical modulators can be used to reverse MDR. These chemicals can either modulate MDR due to their substrate analogy (such as calcium channel blocker verapamil) or interact with phospholipid membranes (such as antihistaminic drug promethazine). This study focuses on the effect of verapamil and promethazine on the expression levels of MDR1 and MRP1 genes and the drug transport activity in doxorubicin-resistant MCF-7 breast carcinoma cell line.MethodsDoxorubicin-resistant MCF-7 (MCF-7/Dox) cells were incubated with either verapamil or promethazine, and total RNA was isolated. Real-time PCR (qPCR) was carried out by using specific primers for MDR1, MRP1, and ß-actin genes. Intracellular doxorubicin accumulation was also examined by confocal laser scanning microscopy in treated cells.ResultsResults demonstrated a significant decrease in both MDR1 and MRP1 expression levels after promethazine applications. It has also been shown that treatment of the cells with verapamil results in significant decrease in MDR1 mRNA levels. Confocal laser scanning microscopy images demonstrated that the intracellular accumulation of doxorubicin was increased after verapamil treatment in MCF-7/Dox cells.ConclusionsThe present study gives an idea about the efficiency of verapamil and promethazine on MDR reversal both in gene expression and in transport activity levels.