Fungi of a forest soil nitrifying at low pH values

Abstract No autotrophic nitrifying organisms were found in a podzolic brown earth forming nitrate. 350 fungi and aerobic heterotrophic bacteria were isolated from this soil and examined for their nitrifying abilities. About one quarter of the isolates produced 0.05–0.90 mg N·1⁻¹ nitrite or nitrate in peptone solution, soil extract mixture or sterilised soil. The nitrification rate of the most active fungus, Verticillium lecanii , was highest at pH 3.5 in defined media. The results support the significance of heterotrophic nitrification in acid soils.     

Influence of pH on phosphatidic acid multilayers a rippled structure at high pH values

The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5–14 (2.6 M K⁺), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.     

Letter: Electron microscopic observation of DNA condensates at low pH values

Coliphage T2 DNA in sufficiently low concentration forms a monomolecular conderisate in acidic (pH 1.9) saline (0.3 m) solution. Electron microscopic observations of acidic monolayered DNA-cytochrome c films confirm previous indirect evidence concerning the dimensions of the in vitro condensation products and their ability to increase and compose confluent aggregates. The phenomena described here suggest that hydrophobic interactions may play a role in both the condensation and aggregation processes.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Dodecyl sulphate/polyacrylamide-gel electrophoresis at low pH values and low temperatures.

A simple method is described for dodecyl sulphate/polyacrylamide-gel electrophoresis of pH- and temperature-labile biological intermediates. The method is based on a catalyst system that works at temperatures of 2--4 degrees C and pH values of 2--4 and an appropriate buffer system containing Li+ or Tris [CH2OH--C(CH2OH)2--NH3+] instead of Na+. This system does not lead to the precipitation of 1% dodecyl sulphate.     

Growth and toxin formation by Clostridium botulinum at low pH values

Spores of Clostridium botulinum were found to initiate growth and to produce toxin in aqueous suspensions of soya protein at pH values as low as 4-2 and in skimmed milk at pH 4.4. Most of the experiments were done with mixed cultures of CI. botulinum types A and B in the presence of two strains of Bacillus subtilis. The role of the latter organism was concluded to be to lower the oxygen content and the E_h of the suspensions. Toxin was produced at pH 4-4 after 4 weeks of incubation at 30^oC when either hydrochloric or citric acids were used as the acidulant and after 12 and 14 weeks when, respectively, lactic and acetic acids were used. Thus, amongst other factors the nature of the acid and not solely the pH value is an important factor in controlling the growth of Cl. botulinum at low pH. Pure cultures of Cl botulinum type A grew at 30^oC under strictly anaerobic conditions and produced toxin at pH 4-3 in the presence of hydrochloric acid.     

Mushroom strains able to grow at high temperatures and low pH values

Seven strains of edible mushrooms were studied with regard to mycelial growth on different growth media and culture conditions. Medium WDA (wheat/dextrose/agar) promoted higher rates of mycelial growth for all the mushrooms investigated. The majority of the strains presented higher growth rates at 30°C, but only Lentinus edodes kept maximum rates at low pH (pH 4.0), followed by Stropharia rugosoannulata and Pleurotus ostreatus (pH 5.0). Absence of light favoured rapid mycelium development in all the strains tested.     

High-Rate Nitrification at Low pH in Suspended- and Attached-Biomass Reactors

This article reports on high-rate nitrification at low pH in biofilm and suspended-biomass reactors by known chemolithotrophic bacteria. In the biofilm reactor, at low pH (4.3 ± 0.1) and low bulk ammonium concentrations (9.3 ± 3.3 mg·liter⁻¹), a very high nitrification rate of 5.6 g of N oxidized·liter⁻¹·day⁻¹ was achieved. The specific nitrification rate (0.55 g of N·g of biomass⁻¹·day⁻¹) was similar to values reported for nitrifying reactors at optimal pH. In the suspended-biomass reactor, the average pH was significantly lower than that in the biofilm reactor (pH 3.8 ± 0.3), and values as low as pH 3.2 were found. In addition, measurements in the suspended-biomass reactor, using isotope-labeled ammonium (¹⁵N), showed that in spite of the very low pH, biomass growth occurred with a yield of 0.1 g of biomass·g of N oxidized⁻¹. Fluorescence in situ hybridization using existing rRNA-targeted oligonucleotide probes showed that the nitrifying bacteria were from the monophyletic genus Nitrosomonas, suggesting that autotrophic nitrification at low pH is more widespread than previously thought. The results presented in this paper clearly show that autotrophic nitrifying bacteria have the ability to nitrify at a high rate at low pH and in the presence of only a negligible free ammonia concentration, suggesting the presence of an efficient ammonium uptake system and the means to cope with low pH.     

Increased binding of chlorin e6 to lipoproteins at low pH values

It is well known that the extracellular pH in tumors is lower than that of normal tissue. This has been proposed to be one of the reasons for the tumor selective uptake of several photosensitizers. Photosensitizers like chlorin e(6) are bound to blood components and delivered to different sites in the organism. Thus, the effect of pH on their interaction with human plasma needs to be studied in order to understand a possible role of the acidic microenvironment in tumors for the drug distribution. Increasing amounts of human plasma in the sample resulted in a gradual red shift of the fluorescence emission maxima of chlorin e(6), indicating binding of the drug to some of the plasma components. Titration showed that the drug-plasma interaction was pH-dependent. The titration curve had an inflection point at 7.4+/-0.1. The relative distribution of the drug among plasma components, as found after ultracentrifugation of chlorin e(6)-doped plasma in a salt gradient, showed more binding of the drug to nonlipoproteins than to lipoprotein classes at both pH values studied (6.5 and 7.4). A decrease in the pH was connected with a significant increase in drug-lipoprotein binding. The pH of the environment affects chlorin e(6)-plasma interaction and the distribution of the drug among different plasma components. The results of this study indicate a possible role of the acidic microenvironment in tumors for the preferential uptake and retention of several photosensitiziers.     

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetone-butanol production by Clostridium acetobutylicum in an ammonium-limited chemostat at low pH values

Clostridium acetobutylicum was grown in continuous culture under ammonium limitation (15.15 mM NH₄ ⁺). At a pH of 6.0 and at various dilution rates only acetate, butyrate and ethanol were formed as non-gaseous products. A decrease of the pH to values between 5.2 and 4.3 resulted in a shift of the fermentation towards acetone-butanol formation.     

Disruption of sulfur cycling and acid neutralization in lakes at low pH

Experimental acidification of a softwater lake to below pH 5 fundamentally changed the sulfur cycle and lowered internal alkalinity generation (IAG). Prior to reaching pH 4.5, the balance of sulfur reduction and oxidation reactions within the lake was in favour of reduction, and the lake was a net sink for sulfate. In the four years at pH 4.5 the balance of reduction and oxidation reactions was in favour of oxidation, and there was a net production of sulfate (SO₄ ^2–) within the lake. Evidence indicating a decrease in net SO₄ ^2– reduction at pH 4.5 was also obtained in an anthropogenically acidified lake that had been acidified for many decades. In both lakes, the decrease in net SO₄ ^2– reduction appeared to be linked not to a simple inhibition of SO₄ ^2– reduction but rather to changes in benthic ecosystem structure, especially the development of metaphytic filamentous green algae, which altered the balance between SO₄ ^2– reduction and sulfur oxidation.At pH's above 4.5, net SO₄ ^2– reduction was the major contributor to IAG in the experimental lake, as it is in many previously studied lakes at pH 5 and above. At pH 4.5, the change in net annual SO₄ ^2– reduction (a decrease of 110%) resulted in a 38% decrease in total IAG. Because of the important role of net SO₄ ^2– reduction in acid neutralization in softwater lakes, models for predicting acidification and recovery of lakes may need to be modified for lakes acidified to pH <5.     

Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH₃ and HNO₂) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low K_ms for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.     

Activation of tumor tissue glycolysis by inorganic phosphate at low pH values]

It was shown that the activating effect of inorganic phosphorus on glucose utilization by the tumour tissue in vitro was retained at low pH of the incubation medium. The 0.15M Na2HPO4 in tris-buffer solutions infusion at the moment of cessation of the tumour selfacidity under glucose infusion led to further decrease of the tumour tissue pH. The tumour tissue pH values of about 4.5--4.6 were obtained. It is recommended to use the solutions with inorganic phosphorus for acidifying the tumour tissue in optimisation of the complex therapy schemes.     

Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation

Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.     

Influence of pH on phosphatidic acid multilayers. A rippled structure at high pH values.

The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5--14 (2.6 M K+), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.     

pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

The limitation of pH inside electrode‐respiring biofilms is a well‐known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode‐respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm. Biotechnol. Bioeng. 2012; 109: 2651–2662. © 2012 Wiley Periodicals, Inc.     

Induction of acid tolerance at neutral pH in log-phase Escherichia coli by medium filtrates from organisms grown at acidic pH

Escherichia coli grown at pH 5·0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5·0 (pH 5·0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7·0. In contrast, filtrates from pH 7·0-grown cultures were ineffective. The pH 5·0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 °C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5·0 plus phosphate or adenosine 3':5'-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7·0-grown log-phase cells to acid tolerance by pH 5·0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response (acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components ; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.     

Microbial sulphate reduction at a low pH

It is now well established that microbial sulphate-reduction can proceed in environments with a pH<5. This review summarizes existing reports on sulphate reduction at low pH and discusses possible pH effects on sulphate-reducing bacteria. Microbial sulphate reduction has been observed in acidic lakes, wetlands, mesocosms, acidic sulphate soils and bioreactors. Possible inhibitory factors include the metabolites H(2)S and organic acids, which can be toxic depending on pH. Metal sulphide precipitation and competition with other bacteria, namely iron-reducing bacteria, can inhibit sulphate reduction. Theoretical considerations show that normal sulphate reduction rates are too low to maintain a neutral micro niche in an acidic environment. The first acidotolerant sulphate-reducing bacteria have been isolated recently.