Nitrification at Low pH by Aggregated Chemolithotrophic Bacteria

A study was performed to gain insight into the mechanism of acid-tolerant, chemolithotrophic nitrification. Microorganisms that nitrified at pH 4 were enriched from two Dutch acid soils. Nitrate production in the enrichment cultures was indicated to be of a chemolithoautotrophic nature as it was (i) completely inhibited by acetylene at a concentration as low as 1 μmol/liter and (ii) strongly retarded under conditions of carbon dioxide limitation. Electron microscopy of the enrichment cultures showed the presence of bacteria that were morphologically similar to strains of known chemolithotrophic nitrifying genera. Many of the enriched bacteria, in particular those that were identified as ammonium oxidizers, were aggregated. Filtration experiments indicated that aggregated cells were able to nitrify at low pH, whereas single cells were not. It is hypothesized that cells inside the aggregates are protected against the toxicity of nitrous acid. Nitrification by aggregated chemolithoautotrophic bacteria may be the dominating process of nitrate formation in many acid soils as it does not appear to depend on the existence of microsites of high pH (acid-sensitive autotrophic nitrification) or on the availability of organic carbon (heterotrophic nitrification).     

High-Rate Anaerobic Treatment of Wastewater at Low Temperatures

Anaerobic treatment of a volatile fatty acid (VFA) mixture was investigated under psychrophilic (3 to 8°C) conditions in two laboratory-scale expanded granular sludge bed reactor stages in series. The reactor system was seeded with mesophilic methanogenic granular sludge and fed with a mixture of VFAs. Good removal of fatty acids was achieved in the two-stage system. Relative high levels of propionate were present in the effluent of the first stage, but propionate was efficiently removed in the second stage, where a low hydrogen partial pressure and a low acetate concentration were advantageous for propionate oxidation. The specific VFA-degrading activities of the sludge in each of the modules doubled during system operation for 150 days, indicating a good enrichment of methanogens and proton-reducing acetogenic bacteria at such low temperatures. The specific degradation rates of butyrate, propionate, and the VFA mixture amounted to 0.139, 0.110, and 0.214 g of chemical oxygen demand g of volatile suspended solids⁻¹ day⁻¹, respectively. The biomass which was obtained after 1.5 years still had a temperature optimum of between 30 and 40°C.     

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.     

Direct Characterization of Methanogens in Two High-Rate Anaerobic Biological Reactors

The methanogenic flora from two types of turbulent, high-rate reactors was studied by immunologic methods as well as by phase-contrast, fluorescence, and scanning electron microscopy. The reactors were a fluidized sand-bed biofilm ANITRON reactor and an ultrafiltration membrane-associated suspended growth MARS reactor (both trademarks of Air Products and Chemicals, Inc., Allentown, Pa.). Conventional microscopic methods revealed complex mixtures of microbes of a range of sizes and shapes, among which morphotypes resembling Methanothrix spp. and Methanosarcina spp. were noticed. Precise identification of these and other methanogens was accomplished by antigenic fingerprinting with a comprehensive panel of calibrated antibody probes of predefined specificity spectra. The methanogens identified showed morphotypes and antigenic fingerprints indicating their close similarity with the following reference organisms: Methanobacterium formicicum MF and Methanosarcina barkeri W in the ANITRON reactor only; Methanosarcina barkeri R1M3, M. mazei S6, Methanogenium cariaci JR1, and Methanobrevibacter arboriphilus AZ in the MARS reactor only; and Methanobrevibacter smithii ALI and Methanothrix soehngenii Opfikon in both reactors. Species diversity and distribution appeared to be, at least in part, dependent on the degree of turbulence inside the reactor.     

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 Km s 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.     

Lipid Metabolism of Caddisfly Larvae at Low pH

The influence of low pH (5.0 and 4.0) on lipid metabolism of caddisfly larvae Hydropsyche contubernalis L. (Trichoptera) was studied in 48 h toxicity experiments. The results were correlated with lipid composition of caddisfly larvae directly isolated from natural water. Phospholipids, cholesterol, mono-, di-, triacylglycerols, and fatty acids were detected by thin-layer and liquid chromatography. Minimal environmental changes were shown to initiate the biochemical adaptation mechanisms strengthening the cellular membranes through their condensation due to additional phospholipid and cholesterol synthesis. In the natural medium the adaptation processes are more active than in the artificial medium. More serious changes, such as pH decrease to 4.0, suppress the adaptation processes in the first medium and terminate them in the second one.     

Engineering of Phytase for Improved Activity at Low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Autotrophic Ammonia Oxidation at Low pH through Urea Hydrolysis

Ammonia oxidation in laboratory liquid batch cultures of autotrophic ammonia oxidizers rarely occurs at pH values less than 7, due to ionization of ammonia and the requirement for ammonium transport rather than diffusion of ammonia. Nevertheless, there is strong evidence for autotrophic nitrification in acid soils, which may be carried out by ammonia oxidizers capable of using urea as a source of ammonia. To determine the mechanism of urea-linked ammonia oxidation, a ureolytic autotrophic ammonia oxidizer, Nitrosospira sp. strain NPAV, was grown in liquid batch culture at a range of pH values with either ammonium or urea as the sole nitrogen source. Growth and nitrite production from ammonium did not occur at pH values below 7. Growth on urea occurred at pH values in the range 4 to 7.5 but ceased when urea hydrolysis was complete, even though ammonia, released during urea hydrolysis, remained in the medium. The results support a mechanism whereby urea enters the cells by diffusion and intracellular urea hydrolysis and ammonia oxidation occur independently of extracellular pH in the range 4 to 7.5. A proportion of the ammonia produced during this process diffuses from the cell and is not subsequently available for growth if the extracellular pH is less than 7. Ureolysis therefore provides a mechanism for nitrification in acid soils, but a proportion of the ammonium produced is likely to be released from the cell and may be used by other soil organisms.     

Ionic Conductance Changes in Voltage Clamped Crayfish Axons at Low pH

Giant axons from the crayfish have been voltage clamped with an axial wire system. General characterististics of observed ionic currents under normal conditions are similar to those measured in other giant axons and in nodes of Ranvier. As the pH of the external bath is lowered below 7, a marked, reversible slowing of potassium currents is seen with little effect on sodium currents. The steady-state potassium conductance-voltage curve is shifted along the voltage axis in a manner consistent with the development of a hyperpolarizing surface charge. Results suggest that this potential shift accounts for part, though not all, of the observed increase in τ_n. From the behavior of the kinetics of the delayed current with external pH these alterations in potassium conductance are attributed to the titration of a histidine imidazole residue of a membrane protein. Chemical modification of histidine by carbethoxylation at pH 6 slows and strongly depresses potassium currents. The results suggest that in addition to the introduction of electrostatic forces, possibly resulting from a hyperpolarizing surface charge, protonation of a histidine group at low pH also alters the nonelectrostatic chemical interactions determining the ease with which potassium gates open and close. The evidence indicates that the modified histidine residue is closely associated with the membrane components involved in the control of potassium conductance.     

低pH孵放、干热处理冻干静脉注射人免疫球蛋白的实验研究

主要介绍冻干静脉注射人免疫球蛋白 (IVIG)分别通过 60℃ 72h,80℃ 72h ,低pH孵放 2 1d处理后 ,IVIG的各项理化及生物活性的变化 ,以及IVIG经过低pH孵放的灭活病毒情况。实验结果表明 ,处理后的IVIG其IgG各组份相对含量、抗补体活性 (ACA)、前激肽释放酶激活剂 (PKA)、白喉抗体、抗 HBs等结果 ,除ACA ,PKA略有下降 ,其他指标无明显变化 ,各项指标均符合 2 0 0 0版《中国生物制品规程》 ;加热后没有新抗原产生。低pH孵放 2 1d通过加指示病毒的实验 ,病毒灭活效果达到 7.0 0logTCID50 / 0 .1mL以上 ,灭活病毒有效。     

Acetoin Synthesis Acquisition Favors Escherichia coli Growth at Low pH

Some members of the family Enterobacteriaceae ferment sugars via the mixed-acid fermentation pathway. This yields large amounts of acids, causing strong and sometimes even lethal acidification of the environment. Other family members employ the 2,3-butanediol fermentation pathway, which generates comparatively less acidic and more neutral end products, such as acetoin and 2,3-butanediol. In this work, we equipped Escherichia coli MG1655 with the budAB operon, encoding the acetoin pathway, from Serratia plymuthica RVH1 and investigated how this affected the ability of E. coli to cope with acid stress during growth. Acetoin fermentation prevented lethal medium acidification by E. coli in lysogeny broth (LB) supplemented with glucose. It also supported growth and higher stationary-phase cell densities in acidified LB broth with glucose (pH 4.10 to 4.50) and in tomato juice (pH 4.40 to 5.00) and reduced the minimal pH at which growth could be initiated. On the other hand, the acetoin-producing strain was outcompeted by the nonproducer in a mixed-culture experiment at low pH, suggesting a fitness cost associated with acetoin production. Finally, we showed that acetoin production profoundly changes the appearance of E. coli on several diagnostic culture media. Natural E. coli strains that have laterally acquired budAB genes may therefore have escaped detection thus far. This study demonstrates the potential importance of acetoin fermentation in the ecology of E. coli in the food chain and contributes to a better understanding of the microbiological stability and safety of acidic foods.     

Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Isolation of Bacteria Which Can Grow at Both High pH and Low Temperature

Fifty bacterial strains able to grow at pH 10 and 0°C were isolated from soils, and growth characteristics of three selected strains were investigated. Strain 207, which showed the best growth rate of all the isolates at the conditions described above, could grow at a temperature of −5 to 39°C at pH 8.5. The optimum pH for this strain changed from 9.5 at 10°C to 9.0 at 20°C.     

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx .     

A Partially Folded State of Ovalbumin at Low pH Tends to Aggregate

At pH 2, ovalbumin retains native-like secondary structure as seen by far-UV CD and FTIR, but lacks well-defined tertiary structure as seen by the fluorescence and near-UV CD spectra. Addition of 20 mM Trifluoroacetic acid (TFA) or 30 mM Trichloroacetic acid (TCA) on acid-induced state results in protein aggregation. This aggregated state possesses extensive β-sheet structure as revealed by far-UV CD and FTIR spectroscopy. Furthermore, the aggregates exhibit decreased ANS fluorescence and increased thioflavin T fluorescence. The presence of aggregates was confirmed by size exclusion chromatography. Such a formation of β-sheet structure is found in the amyloid of a number of neurological diseases such as Alzheimer’s and scrapie. Ovalbumin at low pH, in the presence of K₂SO₄, exists in partially folded state characterized by native-like secondary structure and tertiary folds.     

Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.     

Low pH myoglobin photoproducts.

Recently, there has been interest in determining the conditions under which the iron-histidine bond ruptures in myoglobin at low pH, so that the effect of proximal heme ligation can be studied. A 220-cm-1 Raman mode, assigned to iron-histidine stretching, is clearly visible after photolysis of aqueous MbCO samples below pH4 at room temperature (Sage et al. Biochemistry. 30:1237-1247). In contrast, Iben et al. (Biophys. J. 59:908-919) do not observe this mode upon photolysis of a pH3 MbCO sample in a glycerol/water glass at low temperature. In order to account for both the low temperature and the room temperature experiments, Iben et al. suggest a scheme involving an unusual protonation state of the proximal histidine. Here, we discuss some inconsistencies in their explanation of the room temperature results and offer instead a simple modification of an earlier model. In addition, circular dichroism data are presented that indicate partial unfolding of MbCO in aqueous solution below pH4, and raise questions about the claim of Iben et al. that MbCO remains folded in 75% glycerol at pH3.     

静注丙球低pH孵放灭活病毒效果验证

以水泡性口炎病毒（ＶＳＶ）为指示病毒,考察了低ｐＨ孵放不同时间对低温乙醇法生产的静注丙球（ＩＶＩＧ）中ＶＳＶ的灭活效果,并对不同厂家及不同批号ＩＶＩＧ中病毒灭活情况进行了比较。结果表明,液体ＩＶＩＧ在ＰＨ４．１±０．３,２０－２５℃,孵放２１天可灭活ＶＳＶ达６Ｌｏｇｓ以上（即低于实验检测限）,但不同厂家及不同批号的ＩＶＩＧ在病毒灭活的发生上有所不同     

Multiple Intermediate Conformations of Jack Bean Urease at Low pH: Anion-induced Refolding

Structural and functional characteristics of jack bean urease (JBU), a hexameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using CD, fluorescence, ANS binding and enzyme activity measurements. At low pH and low ionic strength, JBU exists in a partially unfolded state (U_A-state), having predominantly β structure and no tertiary interactions along with a strong ANS binding. Addition of salts like NaCl, KCl and Na₂SO₄ to the U_A-state induces refolding resulting in structural propensities similar to that of native hexamer. Moreover, at low concentrations, GuHCl behaves like an anion by inducing refolding of the U_A-state. The anion-induced refolded state (I_A-state) is more stable than U_A-state and the stability is nearly equal to that of the native protein against chemical-induced and thermal denaturation. Overall, these observations support a model of protein folding for a multimeric protein where certain conformations (ensembles of substates) of low energy prevail and populated under non-native conditions with different stability.