Nutrient availability affects the response of the calcifying chlorophyte Halimeda opuntia (L.) J.V. Lamouroux to low pH

Atmospheric carbon dioxide emissions cause a decrease in the pH and aragonite saturation state of surface ocean water. As a result, calcifying organisms are expected to suffer under future ocean conditions, but their physiological responses may depend on their nutrient status. Because many coral reefs experience high inorganic nutrient loads or seasonal changes in nutrient availability, reef organisms in localized areas will have to cope with elevated carbon dioxide and changes in inorganic nutrients. Halimeda opuntia is a dominant calcifying primary producer on coral reefs that contributes to coral reef accretion. Therefore, we investigated the carbon and nutrient balance of H. opuntia exposed to elevated carbon dioxide and inorganic nutrients. We measured tissue nitrogen, phosphorus and carbon content as well as the activity of enzymes involved in inorganic carbon uptake and nitrogen assimilation (external carbonic anhydrase and nitrate reductase, respectively). Inorganic carbon content was lower in algae exposed to high CO2, but calcification rates were not significantly affected by CO2 or inorganic nutrients. Organic carbon was positively correlated to external carbonic anhydrase activity, while inorganic carbon showed the opposite correlation. Carbon dioxide had a significant effect on tissue nitrogen and organic carbon content, while inorganic nutrients affected tissue phosphorus and N:P ratios. Nitrate reductase activity was highest in algae grown under elevated CO2 and inorganic nutrient conditions and lowest when phosphate was limiting. In general, we found that enzymatic responses were strongly influenced by nutrient availability, indicating its important role in dictating the local responses of the calcifying primary producer H. opuntia to ocean acidification.

Proteomic analysis of Rhizobium freirei PRF 81 responses to low pH.

Rhizobium freirei PRF 81 is employed in common bean commercial inoculants in Brazil, due to its outstanding efficiency in fixing nitrogen, competitiveness and tolerance to abiotic stresses. Among the environmental conditions faced by rhizobia in soils, acidity is perhaps the encountered most, especially in Brazil. So, we used proteomics based approaches to study the responses of PRF 81 to a low pH condition. R. freirei PRF 81 was grown in TY medium until exponential phase in two treatments: pH 6,8 and pH 4,8. Whole-cell proteins were extracted and separated by two-dimensional gel electrophoresis, using IPG-strips with pH range 4-7 and 12% polyacrilamide gels. The experiment was performed in triplicate. Protein spots were detected in the high-resolution digitized gel images and analyzed by Image Master 2D Platinum v 5.0 software. Relative volumes (%vol) of compared between the two conditions tested and were statistically evaluated (p ≤ 0.05). Even knowing that R. freirei PRF 81 can still grow in more acid conditions, pH 4.8 was chosen because didn´t affect significantly the bacterial growth kinetics, a factor that could compromise the analysis. Using a narrow pH range, the gel profiles displayed a better resolution and reprodutibility than using broader pH range. Spots were mostly concentrated between pH 5-7 and molecular masses between 17-95 kDa. From the six hundred well-defined spots analyzed, one hundred and sixty-three spots presented a significant change in % vol, indicating that the pH led to expressive changes in the proteome of R. freirei PRF 81. Of these, sixty-one were up-regulated and one hundred two was downregulated in pH 4.8 condition. Also, fourteen spots were only identified in the acid condition, while seven spots was exclusively detected in pH 6.8. Ninety-five differentially expressed spots and two exclusively detected in pH 4,8 were selected for Maldi-Tof identification. Together with the genome sequencing and the proteome analysis of heat stress, we will search for molecular determinants of PRF 81 related to capacity to adapt to stressful tropical conditions.

Effect of low extracellular pH on NF-κB activation in macrophages

Objective: Many diseases, including atherosclerosis, involve chronic inflammation. The master transcription factor for inflammation is NF-κB. Inflammatory sites have a low extracellular pH. Our objective was to demonstrate the effect of pH on NF-κB activation and cytokine secretion. Methods: Mouse J774 macrophages or human THP-1 or monocyte-derived macrophages were incubated at pH 7.0–7.4 and inflammatory cytokine secretion and NF-κB activity were measured. Results: A pH of 7.0 greatly decreased pro-inflammatory cytokine secretion (TNF or IL-6) by J774 macrophages, but not THP-1 or human monocyte-derived macrophages. Upon stimulation of mouse macrophages, the levels of IκBα, which inhibits NF-κB, fell but low pH prevented its later increase, which normally restores the baseline activity of NF-κB, even though the levels of mRNA for IκBα were increased. pH 7.0 greatly increased and prolonged NF-κB binding to its consensus promoter sequence, especially the anti-inflammatory p50:p50 homodimers. Human p50 was overexpressed using adenovirus in THP-1 macrophages and monocyte-derived macrophages to see if it would confer pH sensitivity to NF-κB activity in human cells. Overexpression of p50 increased p50:p50 DNA-binding and in THP-1 macrophages inhibited considerably TNF and IL-6 secretion, but there was still no effect of pH on p50:p50 DNA binding or cytokine secretion. Conclusion: A modest decrease in pH can sometimes have marked effects on NF-κB activation and cytokine secretion and might be one reason to explain why mice normally develop less atherosclerosis than do humans.

Photosynthate translocation increases in response to low seawater pH in a coral-dinoflagellate symbiosis

This study has examined the effect of low seawater pH values (induced by an increased CO2 partial pressure) on the rates of photosynthesis, as well as on the carbon budget and carbon translocation in the scleractinian coral species Stylophora pistillata, using a new model based on 13C labelling of the photosynthetic products. Symbiont photosynthesis contributes to a large part of the carbon acquisition in tropical coral species, and it is thus important to know how environmental changes affect this carbon acquisition and allocation. For this purpose, nubbins of S. pistillata were maintained for six months at two pHTs (8.1 and 7.2, by bubbling seawater with CO2). The lowest pH value was used to tackle how seawater pH impacts the carbon budget of a scleractinian coral. Rates of photosynthesis and respiration of the symbiotic association and of isolated symbionts were assessed at each pH. The fate of 13C photosynthates was then followed in the symbionts and the coral host for 48 h. Nubbins maintained at pHT 7.2 presented a lower areal symbiont concentration, and lower areal rates of gross photosynthesis and carbon incorporation compared to nubbins maintained at pHT 8.1. The total carbon acquisition was thus lower under low pH. However, the total percentage of carbon translocated to the host as well as the amount of carbon translocated per symbiont cell were significantly higher under pHT 7.2 than under pHT 8.1 (70% at pHT 7.2 vs. 60% at pHT 8.1), such that the total amount of photosynthetic carbon received by the coral host was equivalent under both pHs (5.5 to 6.1 µg C/cm**2/h). Although the carbon budget of the host was unchanged, symbionts acquired less carbon for their own needs (0.6 compared to 1.8 µg C/cm**2/h), explaining the overall decrease in symbiont concentration at low pH. In the long term, such decrease in symbiont concentration might severely affect the carbon budget of the symbiotic association.

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal+) or galactose-non-metabolising (Gal-) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). Sc. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal+ or Gal-). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that Sc. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E-MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E-MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E-MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb Delta whiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Last, Mtb Delta whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Lux-biosensor assessment of pH effects on microbial sorption and toxicity of chlorophenols

Lux-marked bacterial biosensors and a commercial toxicity testing bacterial strain (Microtox) were exposed to 2,4-dichlorophenol (DCP) and the light output response measured. Increasing DCP concentrations caused a decrease in light output in all three biosensors with an order of sensitivity (in terms of luminescence decrease over the DCP concentration range) of Pseudomonas fluorescens <Escherichia coli <Microtox. Adsorption of DCP to E. coli was measured using uniformly ring labelled [14C]DCP and found to be very rapid. The effect of pH on toxicity and adsorption was also investigated. Low pH values increased the amount of DCP adsorbed to the cell and increased the toxicity of DCP.

Arsenic biogeochemistry as affected by phosphorus fertilizer addition, redox potential and pH in a west Bengal (India) soil

This laboratory experiment systematically examines arsenic, iron, and phosphorus solubilities in soil suspensions as affected by addition of phosphorus fertilizer under different redox potential (Eh) and pH conditions. Under aerobic conditions, As solubility was low, however, under moderately reducing conditions (0, -150 mV), As solubility significantly increased due to dissolution of iron oxy-hydroxides. Upon reduction to -250 mV, As solubility was controlled by the formation of insoluble sulfides, and as a result soluble As contents significantly decreased. Soluble Fe concentration increased from moderate to highly anaerobic conditions; however, it decreased under aerobic conditions likely due to formation of insoluble oxy-hydroxides. A low pH, 5.5, led to increased soluble concentrations of As, Fe, and P. Finally, addition of P-fertilizers resulted in higher soluble P and As, even though the concentration of As did not increased after an addition rate of 600 mg P kg(-1) soil. (c) 2006 Elsevier B.V. All rights reserved.

Electrochemical methodology to study labile trace metal/natural organic matter complexation at low concentration levels in natural waters

A new electrochemical methodology to study labile trace metal/natural organic matter complexation at low concentration levels in natural waters is presented. This methodology consists of three steps: (i) an estimation of the complex diffusion coefficient (DML), (ii) determination at low pH of the total metal concentration initially present in the sample, (iii) a metal titration at the desired pH. The free and bound metal concentrations are determined for each point of the titration and modeled with the non-ideal competitive adsorption (NICA-Donnan) model in order to obtain the binding parameters. In this methodology, it is recommended to determine the hydrodynamic transport parameter, α, for each set of hydrodynamic conditions used in the voltammetric measurements. The methodology was tested using two fractions of natural organic matter (NOM) isolated from the Loire river, namely the hydrophobic organic matter (HPO) and the transphilic organic matter (TPI), and a well characterized fulvic acid (Laurentian fulvic acid, LFA). The complex diffusion coefficients obtained at pH 5 were 0.4 ± 0.2 for Pb and Cu/HPO, 1.8 ± 0.2 for Pb/TPI and (0.612 ± 0.009) × 10−10 m2 s−1 for Pb/LFA. NICA-Donnan parameters for lead binding were obtained for the HPO and TPI fractions. The new lead/LFA results were successfully predicted using parameters derived in our previous work.

Problems in the use of benzoic acid for estimating the internal pH of yeasts

Some yeasts have the peculiar ability to grow in the presence of weak acids at rather low pH. These conditions are predominant in preserved foods and beverages such as fruit concentrates, juices, wine, where these yeasts are responsible for spoilage. The main preservatives currently utilized by food industries are sorbic, propionic, benzoic acids and SO2. It is usually assumed that weak acids diffuse through the plasma membrane in the undissociated form. In the cytoplasm, where the pH is higher, dissociation occurs resulting in accumulation of the lipid-insoluble anion and internal acidification. This is probably a very general mechanism of preventing microbial growth in foods.

Effect of pH on non-photochemical quenching in spinach photsystem 2 particles as measured by picosecond fluorescence decay kinetics

Single photon timing was used to study picosecond chlorophyll a fluorescence decay kinetics of pH induced non-photochemical quenching in spinach photosystem 2 particles. The characteristics of this quenching are a decrease in chlorophyll a fluorescence yield as well as a decrease in photochemistry at low pH. Picosecond kinetics of room temperature fluorescence temporally resolve the individual components of the steady state fluorescence yield into components that are related to primary energy conversion processes in photosystem 2. Four components were resolved for dark adapted (Fo), light saturated (Fm), and chemically reduced (Nadithionite) photosystem 2 reaction centres. The fastest and slowest components, indicative of energy transfer to and energy capture by the photosystem 2 reaction centre and uncoupled ("dead") chlorophyll, respectively, were not affected by changing pH from 6.5 to 4.0. The two intermediate components, indicative of electron transfer processes within the reaction centre of photosystem 2, were affected by the pH change. Results indicate that the decrease in the steady state fluorescence yield at low pH was primarily due to the decrease in lifetime and amplitude of the slower of the intermediate components. These results imply that the decrease in steady state fluorescence yield at low pH is not due to changes in energy transfer to and energy capture by the photosystem 2 reaction centre, but is related to changes in charge stabilization and charge recombination in the photosystem 2 reaction centre.

Photocatalytic degradation of humic acid in saline waters. Part 1. Artificial seawater: influence of TiO2, temperature, pH, and air-flow

We report the first systematic study on the photocatalytic oxidation of humic acid (HA) in artificial seawater (ASW). TiO2 (Degussa P25) dispersions were used as the catalyst with irradiation from a medium-pressure mercury lamp. The optimum quantity of catalyst was found to be between 2 and 2.5 g l(-1); whiled the decomposition was fastest at low pH values (pH 4.5 in the range examined), and the optimum air-flow, using an immersion well reactor with a capacity of 400 ml, was 850 ml min(-1). Reactivity increased with air-flow up to this figure, above which foaming prevented operation of the reactor. Using pure. oxygen, an optimal flow rate was observed at 300 nil min(-1), above which reactivity remains essentially constant. Following treatment for 1 h, low-salinity water (2700 mg l(-1)) was completely mineralised, whereas ASW (46000 mg l(-1)) had traces of HA remaining. These effects are interpreted and kinetic data presented. To avoid problems of precipitation due to change of ionic strength humic substances were prepared directly in ASW, and the effects of ASW on catalyst suspension and precipitation have been taken into account. The Langmuir-Hinshelwood kinetic model has been shown to be followed only approximately for the catalytic oxidation of HA in ASW. The activation energy for the reaction derived from an Arrhenius treatment was 17 ( +/-0.6) kJ mol(-1). (C) 2003 Elsevier Science Ltd. All rights reserved.

CLSM method for the dynamic observation of pH change within polymer matrices for oral delivery

If acid-sensitive drugs or cells are administered orally, there is often a reduction in efficacy associated with gastric passage. Formulation into a polymer matrix is a potential method to improve their stability. The visualization of pH within these materials may help better understand the action of these polymer systems and allow comparison of different formulations. We herein describe the development of a novel confocal laser-scanning microscopy (CLSM) method for visualizing pH changes within polymer matrices and demonstrate its applicability to an enteric formulation based on chitosan-coated alginate gels. The system in question is first shown to protect an acid-sensitive bacterial strain to low pH, before being studied by our technique. Prior to this study, it has been claimed that protection by these materials is a result of buffering, but this has not been demonstrated. The visualization of pH within these matrices during exposure to a pH 2.0 simulated gastric solution showed an encroachment of acid from the periphery of the capsule, and a persistence of pHs above 2.0 within the matrix. This implies that the protective effect of the alginate-chitosan matrices is most likely due to a combination of buffering of acid as it enters the polymer matrix and the slowing of acid penetration.

Influence of pH on the phototransformation process of PhotogemA (R)

PhotogemA (R) is a hematoporphyrin derivative that has been used as a photosensitizer in experimental and clinical Photodynamic Therapy (PDT) in Brazil. Photosensitizers are degraded under illumination. This process, usually called photobleaching, can be monitored by decreasing in fluorescence intensities and includes the following photoprocesses: photodegradation, phototransformation, and photorelocalization. Photobleaching of hematoporphyrin-type sensitizers during illumination in aqueous solution is related not only to photodegradation but is also followed by the formation of photoproducts with a new fluorescence band at around 640-650 nm and with increased light absorption in the red spectral region at 640 nm. In this study, the influence of pH on the phototransformation process was investigated. PhotogemA (R) solutions, 40 mu g/ml, were irradiated at 514 nm with intensity of 100 mW/cm(2) for 20 min with different pH environments. The controls were performed with the samples in the absence of light. The PhotogemA (R) photodegradation is dependent on the pH. The behavior of photodegradation and photoproducts formation (monitored at 640 nm) is distinct and depends on the photosensitizer concentration. The processes of degradation and photoproducts formation were monitored with Photogemin the concentration of 40 mu g/mL since that demonstrated the best visualization of both processes. While below pH 5 the photodegradation occurred, there was no detectable presence of photoproducts. The increase of pH led to increase of photoproducts formation rate with photodegradation reaching the highest value at pH 10. The increase of photoproducts formation and instability of PhotogemA (R) from pH 6 to pH 10 are in agreement with the desired properties of an ideal photosensitizer since there are significant differences in pH between normal (7.0 < pH < 8.6) and tumor (5.8 < pH < 7.9) tissues. It is important to know the effect of pH in the process of phototransformation (degradation and photoproduct formation) of the molecule since low pH values promotes increase in the proportion of aggregates species in solution and high pH values promotes increase in the proportion of monomeric species. There must be an ideal pH interval which favors the phototransformation process that is correlated with the singlet oxygen formation responsible by the photodynamic effect. These differences in pH between normal and tumor cells can explain the presence of photosensitizers in target tumor cells, making PDT a selective therapy.