Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.     

Kinetics of growth of the hydrogen-oxidizing bacterium Alcaligenes eutrophus (ATCC 17707) in chemostat culture

The hydrogen-oxidizing bacterium, Alcaligenes eutrophus (ATCC 17707), was grown in chemostat culture with gas-phase (hydrogen, oxygen, and carbon dioxide) and liquid-phase (mineral nutrients) feedstreams; data were used to generate an analytical form for the specific growth rate equation. Model parameters obtained include Monod rate parameters for dissolved hydrogen and oxygen gases, yield coefficients, and specific maintenance rates under conditions of hydrogen or oxygen limitations. These values are similar to some obtained previously by Ohi et al. for another hydrogen bacterium. The observed increase in specific maintenance rates under hydrogen-versus-oxygen-limited culture may be associated with hydrogenase deactivation by oxygen.     

Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices

A dialysis culture device was used for growth of syntrophic fatty acid-oxidizing and ethanol-oxidizing anaerobic bacteria. A pure culture of the fatty acid oxidizer Clostridium bryantii was grown inside dialysis tubing which was surrounded by a pure culture of Desulfovibrio vulgaris. The same apparatus was used for the syntrophic cultivation of Pelobacter acetylenicus and Acetobacterium woodii with ethanol as substrate. In both cases, substrate degradation and product formation were about half as fast as with the homogeneously mixed control cultures. In the compartment of the hydrogen producer, the concentration of free hydrogen during syntrophic ethanol degradation was about 10 times as high as in that of the hydrogen utilizer, whereas the homogeneously mixed culture exhibited an intermediate hydrogen partial pressure.     

Kinetic study of pH effects on biological hydrogen production by a mixed culture

The effect of pH on anaerobic hydrogen production was investigated under various pH conditions ranging from pH 3 to 10. When the modified Gompertz equation was applied to the statistical analysis of the experimental data, the hydrogen production potential and specific hydrogen production rate at pH 5 were 1,182 ml and 112.5 ml/g biomass-h, respectively. In this experiment, the maximum theoretical hydrogen conversion ratio was 22.56%. The Haldane equation model was used to find the optimum pH for hydrogen production and the maximum specific hydrogen production rate. The optimum pH predicted by this model is 5.5 and the maximum specific hydrogen production rate is 119.6 ml/g VSS-h. These data fit well with the experimental data (r2=0.98).     

Evaluation of the oxidative burst in suspension cell culture of Phaseolus vulgaris

Plants respond to the attack of pathogens with the oxidative burst, a production of reactive oxygen species (ROS). In this work a cell culture suspension of Phaseolus vulgaris was used to investigate the oxidative burst triggered by a conidia suspension of different races of Colletotrichum lindemuthianum. As a defence response of the cells a two-phase peak was observed with all used races of Colletotrichum lindemuthianum, varying only in the produced amounts of hydrogen peroxide. Findings with additives such as superoxide dismutase (SOD), diphenyleneiodonium (DPI) and catalase gave rise to the conclusion that more superoxide radicals were produced than be detectable with Amplex Red as hydrogen peroxide. It is assumed that the conversion of the superoxide radical is spontaneous and not driven via a cell-derived superoxide dismutase. The addition of low-molecular cell wall components (ergosterol, glucosamine, galactosamine) showed clearly that compounds like this act as elicitors and thus are involved in triggering the burst. Furthermore, an evaluation of the metabolizing capacities of hydrogen peroxide of the suspension culture cells revealed the enormous capacity of the cells to detoxify this ROS.     

Characteristics of anaerobic ammonia removal by a mixed culture of hydrogen producing photosynthetic bacteria

It is known that the presence of ammonia inhibits hydrogen production by photosynthetic bacteria. In order to avoid it, a two-step process containing ammonia removal and hydrogen production was investigated in this study. Firstly, the effects of carbonate presence on ammonia removal by photosynthetic bacteria were investigated by the vial tests because it is known that the uptake of volatile fatty acids (VFAs) sometimes requires carbonate. The results of them showed that the presence of carbonate promoted the uptake of VFAs and ammonia. Especially, the uptake of propionate and/or butyrate required the presence of carbonate. The results of the batch experiments of two-step hydrogen production showed that the depletion of ammonia triggered hydrogen evolution. Herein, the presence of albumin did not inhibit hydrogen evolution and preferably it increased the hydrogen production rate. And the VFA-C/NH4-N ratio in substrate fed into two-step hydrogen production process should be more than 6.0.     

Fedbatch operation using Clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production

We demonstrated the feasibility of fedbatch operation using Clostridium acetobutylicum suspension culture as a biocatalyst for the continuous production of hydrogen. The optimum operating pH and temperature of the current cultivation system for hydrogen production were pH 6.0 and 37 degrees C, respectively. The volumetric loading of the bioreactor for hydrogen production can be as high as 650 mmol hydrogen/L culture with a yield at approximately 2.0 mol hydrogen/mol glucose. Acetate and butyrate made up approximately 80% of the total metabolites. The inhibitory effect from the two metabolites on the hydrogen production process was investigated. Butyrate at a concentration higher than 13 g/L significantly inhibited not only cell growth but also hydrogen production (i.e., specific hydrogen production rate). Acetate appears to be less toxic than butyrate to the hydrogen production process. While significantly inhibiting cell growth, acetate hardly affected hydrogen production. Finally, the factors limiting cultivation performance were discussed and possible strategies for enhancing the production of hydrogen were proposed.     

谷氨酸废液培养莱茵衣藻的产氢研究

通过在一般培养基中添加尿素、谷氨酸、葡萄糖等有机物,考察了菜茵衣藻混合营养培养的产氢效果。结果表明,含尿素混合营养培养菜茵衣藻产氢是含铵氮的培养基培养的6倍,无硫含尿素的混合营养培养具有最佳的产氢效果,是有硫含铵氮的培养基的10倍。谷氨酸及葡萄糖的添加对其生长和产氢也有促进作用,对菜茵衣藻生长和产氢促进作用的尿素的最佳添加量为0．25g/L,谷氨酸的最佳添加量为0．7g/L,葡萄糖的添加量为0．2g/L。     

Continuous culture of Methanococcus maripaludis under defined nutrient conditions

To study global regulation in the methanogenic archaeon Methanococcus maripaludis, we devised a system for steady-state growth in chemostats. New Brunswick Bioflo 110 bioreactors were equipped with controlled delivery of hydrogen, nitrogen, carbon dioxide, hydrogen sulfide, and anaerobic medium. We determined conditions and media compositions for growth with three different limiting nutrients, hydrogen, phosphate, and leucine. To investigate leucine limitation we constructed and characterized a mutant in the leuA gene for 2-isopropylmalate synthase, demonstrating for the first time the function of this gene in the Archaea. Steady state specific growth rates in these studies ranged from 0.042 to 0.24 h(-1). Plots of culture density vs. growth rate for each condition showed the behavior predicted by growth modeling. The results show that growth behavior is normal and reproducible and validate the use of the chemostat system for metabolic and global regulation studies in M. maripaludis.     

Effect of culture conditions on producing and uptake hydrogen flux of biohydrogen fermentation by metabolic flux analysis method

In this work, metabolic flux analysis (MFA) method was used to estimate the effects of the culture conditions on both the producing and uptake hydrogen flux inside the cell of Klebsiella pneumoniae ECU-15. The results indicated that higher temperature could reduce the amount of the uptake hydrogen and enhance the hydrogen production from the NADH pathway. Moreover, both the producing hydrogen flux from formate and the uptake hydrogen flux were attained to the maximum at pH 7.0-7.5. The producing hydrogen flux was higher at 5 g/L initial glucose than that of the other concentrations, and the uptake hydrogen flux showed the minimum value under the same condition. The apparent hydrogen generation was caused by the combined action of producing hydrogenase, uptake hydrogenase and bidirectional hydrogenase. These results were helpful to deeply understand the mechanism of the biohydrogen evolving process and establish the suitable molecular strategies for improving hydrogen production.     

Anaerobic biohydrogen production from monosaccharides by a mixed microbial community culture

Monosaccharides (e.g. glucose and fructose) are produced from the hydrolyzation of macromolecules, such as starch, cellulose, hemicellulose and lignin, which are abundant in various industrial wastewaters. The elucidation of anaerobic activated sludge microbial community utilizing monosaccharides will lay an important foundation for the industrialization of biohydrogen production. In this study, the hydrogen production by a mixed microbial culture on four monosaccharides (glucose, fructose, galactose and arabinose) was investigated in a batch cultures. The mixed microbial culture was obtained from anaerobic activated sludge in a continuous stirred-tank reactor (CSTR) after 29 days of acclimatization. The results indicated that glucose had the highest specific hydrogen production rate of 358 mL/g.g mixed liquid volatile suspended solid (MLVSS), while arabinose had the lowest hydrogen production rate of 28 mL/g.gMLVSS. Glucose also possessed the highest specific conversion rate to hydrogen of 82 mL/g glucose, while fructose had the highest specific conversion rate to liquid product of 443 mg/g fructose. Arabinose had the lowest conversion rates to both liquid products and hydrogen. Metabolic pathways and fermentation products were the major reasons for the difference in hydrogen production from these four monosaccharides. The complex fermentation pathways of arabinose reduced its hydrogen production efficiency and a long acclimation period (over 68 h) was required before the anaerobic activated sludge could effectively utilize arabinose in batch cultures.     

Efficacy of common laboratory disinfectants on the infectivity of Cryptosporidium parvum oocysts in cell culture

Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts.     

Ultrastructure of the cyanobacterium<Emphasis Type="Italic">Nostoc muscorum</Emphasis> and exploitation of the culture for hydrogen production

An effect of various physico-chemical parameters on nitrogenase-catalyzed oxygen-free hydrogen production by Nostoc muscorum was demonstrated. More hydrogen was produced in the light than in the dark. Optimum temperature was 40 degrees C Various sugars increased hydrogen production whereas on easily metabolized nitrogen sources it was inhibited. The production was sensitive to salinity and Fe3+, Cu2+, Zn2+ and Ni2+ ions. Ultrastructural study revealed many electron-dense layers outside the cell-wall area that have not been observed earlier.     

Hydrogen peroxide induces microvilli on human retinal pigment epithelial cells in culture.

We have found that hydrogen peroxide (10^-4 - 10^-2 M) rapidly induces microvilli on separate cells and confluent sheets of human retinal pigment epithelium in culture. t-butyl hydroperoxide and sodium arsenite do not induce microvilli. A role for hydrogen peroxide as an intercellular messenger has previously been proposed in the inflammatory response, in which hydrogen peroxide from phagocytes may signal to vascular endothelial cells. Our observations thus provide a second example of the induction of what may be a physiological response by this potentially toxic agent. In the retina, hydrogen peroxide released from illuminated photoreceptors may elongate the microvilli which extend into the spaces between them. Increased numbers of microvilli and their protrusion further into the photoreceptor layer may enhance various interactions between the two cell types, including the antioxidant functions of the epithelium.     

Oxidative stress on the astrocytes in culture derived from a senescence accelerated mouse strain

Astrocytes are one of the predominant glial cell types in the adult central nervous system functioning as both supportive and metabolic cells for the brain. Our objective in this experiment is to study the direct effects of hydrogen peroxide induced oxidative stress on astrocytes in culture. These astrocytes were derived from both an aged mouse strain (P8) and a matched control strain (R1). The astrocytes for both the P8 and R1 strains were treated with increasing concentrations of hydrogen peroxide. Our results showed that the oxidative stress had a similar effect in both strains of astrocytes; decreases in 3-(4,5-dimethylthiazol-2-yl)-2,2-diphenyltetrazolium bromide (MTT) and glial fibrillary acidic protein (GFAP) levels, and increases in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, lactate dehydrogenase (LDH) staining, and superoxide dismutase (SOD), caspase-3 and B-cell lymphoma 2-associated protein X (bax) levels. At a hydrogen peroxide concentration of 400 microM , the differences of the above parameters between P8 cultures and R1 cultures were statistically significant (p<0.05). This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a hydrogen peroxide concentration of 400 microM. The inability of the P8 astrocytes to counteract the oxidative stress might lead to inadequate protection from neuronal loss possibly resulting in significantly more astrocytic death. Our results suggested that the changes of astrocytes in peroxide detoxification may play a role in aging of the central nervous system, and further aging studies should examine the oxidative status of the samples.     

Hydrogen regulation of acetogenesis from glucose by freely suspended and immobilised acidogenic cells in continuous culture

Summary The pattern of end product formation from glucose was examined in an acidogenic culture where the hydrogen partial pressure was controlled by headspace gas recirculation and nitrogen purging. The results demonstrate that physical control of hydrogen displacement can shift the equilibrium toward acetate production with a concomitant fall in reduced end product formation in the freely-suspended cell system. Immobilised acidogenic cell did not show the same pattern of end product formation when subject to reduction in hydrogen partial pressure which has implications for control of methanogenic end product in two stage anaerobic digester systems using immobilised cell technology.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

Hydrogen as an electron donor for dechlorination of tetrachloroethene by an anaerobic mixed culture.

Hydrogen served as an electron donor in the reductive dechlorination of tetrachloroethene to vinyl chloride and ethene over periods of 14 to 40 days in anaerobic enrichment cultures; however, sustained dechlorination for more extended periods required the addition of filtered supernatant from a methanol-fed culture. This result suggests a nutritional dependency of hydrogen-utilizing dechlorinators on the metabolic products of other organisms in the more diverse, methanol-fed system. Vancomycin, an inhibitor of cell wall synthesis in eubacteria, was found to inhibit acetogenesis when added at 100 mg/liter to both methanol-fed and hydrogen-fed cultures. The effect of vancomycin on dechlorination was more complex. Methanol could not sustain dechlorination when vancomycin inhibited acetogenesis, while hydrogen could. These results are consistent with a model in which hydrogen is the electron donor directly used for dechlorination by organisms resistant to vancomycin and with the hypothesis that the role of acetogens in methanol-fed cultures is to metabolize a portion of the methanol to hydrogen. Methanol and other substrates shown to support dechlorination in pure and mixed cultures may merely serve as precursors for the formation of an intermediate hydrogen pool. This hypothesis suggests that, for bioremediation of high levels of tetrachloroethene, electron donors that cause the production of a large hydrogen pool should be selected or methods that directly use H2 should be devised.     

Effect of hydrogen acceptors on D-xylose fermentation by anaerobic culture of immobilized Pachysolen tannophilus cells

The effect of hydrogen acceptors on the kinetic parameters of D-xylose fermentation under anaerobic conditions was studied in a transient culture of immobilized Pachysolen tannophilus cells. Addition of oxygen to a steady-state culture resulted in a rapid increase (up to fivefold) in the rates of ethanol production and D-xylose uptake, but the rate of xylitol production was unaffected. Furthermore, the molar ethanol yield increased from 0.97 to 1.43 in the presence of oxygen. The moles of ethanol produced per moles of oxygen utilized were considerably greater than would be predicted from the stoichiometry of D-xylose fermentation, which suggests that the organism required oxygen for other functions in addition to its role as a hydrogen acceptor in D-xylose metabolism. When the artificial hydrogen acceptors acetone, acetaldehyde, and acetoin were added to the culture, the rate of ethanol production increased while the xylitol production rate decreased but the rate of xylose uptake was unaffected. The molar ethanol yields increased from 1.03 to 1.63, 1.43, and 1.24 upon addition of acetaldehyde, acetone, and acetoin, respectively, at the expense of the molar xylitol yields. The hydrogen acceptors sodium acetate, methylene blue, benzyl viologen, phenazine methosulfate, indigo carmine, and tetrazolium chloride had no effect on ethanol production.     

Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal

The difficulties associated with the growth of Legionella species in common laboratory media may be due to the sensitivity of these organisms to low levels of hydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) broth to fluorescent light generated superoxide radicals (3 microM/h) and hydrogen peroxide (16 microM/h). Autoclaved YE medium was more prone to photochemical oxidation than YE medium sterilized by filtration. Activated charcoals and, to a lesser extent, graphite, but not starch, prevented photochemical oxidation of YE medium, decomposed hydrogen peroxide and superoxide radicals, and prevented light-accelerated autooxidation of cysteine. Also, suspensions of charcoal in phosphate buffer and in charcoal yeast extract medium readily decomposed exogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations of bovine superoxide dismutase and catalase also decreased the rate of photooxidation of YE medium. Medium protected from light did not accumulate appreciable levels of hydrogen peroxide, and autoclaved YE medium protected from light supported good growth of Legionella micdadei. Various species of Legionella (10(4) cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide (26.5 microM) in challenge experiments. The level of hydrogen peroxide that accumulated in YE medium over a period of several hours (greater than 50 microM) was in excess of the level tolerated by Legionella pneumophila, which contained no measurable catalase activity. Strains of L. micdadei, Legionella dumoffi, and Legionella bozmanii contained this enzyme, but the presence of catalase did not appear to confer appreciable tolerance to exogenously generated hydrogen peroxide.