Buffer requirements for enhanced hydrogen production in acidogenic digestion of food wastes

The requirements for pH buffer addition for hydrogen production and acidogenesis in batch acidogenic digestion of a food waste (FW) feedstock with limited alkalinity was studied at various initial pH conditions (6.0–8.0). The results showed that, without buffer addition, hydrogen production from this feedstock was insignificant regardless of the initial pH. With buffer addition, hydrogen production improved significantly if the initial pH was greater than 6.0. Substantial hydrogen production occurred when the pH at the end of the batch digestion was higher than 5.5. The maximum hydrogen production was found to be 120 mL/g VS added when the initial pH was 6.5 and buffer addition was in the range of 15–20 mmol/g VS. The effect of pH buffering on the formation of volatile fatty acids (acetic acid, propionic acid and butyric acid) was similar to its effect on hydrogen production. The results of this study clearly indicated shifts in the metabolic pathways with the pH of fermentation. The changes in metabolic pathways impacted upon the dosage of buffer that was required to achieve maximum hydrogen generation.     

Choice of buffer anion for the assay of adenosine 5′-triphosphate using firefly luciferase

Anions inhibit firefly luciferase. We have compared the extent of inhibition of luciferase by the anions from various acids used to adjust Tris buffer solutions to pH 7.75, the optimum pH for enzyme activity. Acetate and succinate were the least inhibitory of the anions tested. Tris-acetate buffers are recommended for maximum sensitivity of ATP assays with firefly luciferase.     

Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae

Abstract Fluorescence ratio imaging microscopy using pH-sensitive fluorescent dyes makes it possible to evaluate statistical distribution of intracellular pH in a population of the yeast S. cerevisiae examined in a thin layer of suspension in a Petri dish. The distribution appears to fit a Gaussian curve with a half-width around the 0.4 pH unit. The curve became slightly narrower after resuspension in a strong buffer; the mean values shifted with the pH of the buffer. The shape of the distribution curves of both resting and growing cells in various phases of growth does not change significantly. Likewise, addition of 1% of glucose, 50 μM suloctidil or 100 μM diethylstilbestrol brings about no alteration. The only value which clearly changes is the average cytoplasmic pH.     

pH Transitions in ion-exchange systems: role in the development of a cation-exchange process for a recombinant protein

Unexpected transient changes in effluent pH can occur during ion-exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation-exchange resin. Incomplete column equilibration was eliminated as a possible cause of these effects. Various buffering species and various salt ions were studied at different solution concentrations to investigate pH transitions on strong cation-exchange resins. A further comparison was made between cation-exchange resins with different backbone chemistries. On the basis of these studies, a mechanism is proposed for these phenomena based on competitive equilibria between ions from the buffer salts and H(+)/OH(-) ions. In addition to the equilibria between these ions and the functional groups on the resins, charged groups on the resin backbone were also found to contribute to transient pH changes. The results from this study were applied to the cation-exchange step for a recombinant protein that was sensitive to pH excursions to help maintain activity of the protein during the purification process.     

Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH

We described a novel approach to directly amplify genomic DNA from whole blood and dried blood spotted on filter paper without any DNA isolation by using the PCR buffer with a higher pH, which was optimized as pH 9.1-9.6. Direct PCR on blood treated with various anticoagulants showed that the buffer worked well with the blood treated by citrate, EDTA, or heparinate. DNA fragments with different lengths could be efficiently amplified directly from various forms of blood samples. By coupling the buffer with tetra-PCR, a "true" single-tube genotyping was realized by using whole blood or paper-dried blood as starting material.     

Bioluminescence characteristics of the fruiting body of Mycena chlorophos

Bioluminescent fungi are widely distributed on land and most belong to the class Basidomycetes. Light of about 530 nm wavelength maximum is emitted continuously. The molecular basis for the light‐emitting process remains unclear. We investigated the characteristics of the bioluminescence using cultivated fruiting bodies of M. chlorophos. Only fresh fruiting bodies exhibited long‐lasting light emission; rapid decay of light emission was observed with frozen and freeze‐dried samples. Freeze‐dried samples can be stored at room temperature under dry conditions and may be useful for the isolation of luciferin. The light emission of the fresh fruiting bodies was maintained in various buffers at varying pH; it could be stopped with pH 4 acetate buffer and could be recovered at pH 6. The isolation of luciferin from the fresh fruiting bodies might be possible by the control of buffer pH. The effect of temperature on the light emission of fruiting bodies indicated that bioluminescence in M. chlorophos may involve enzymatic reaction(s). The solubilization of bioluminescent components from the fruiting bodies could not be achieved with various surfactants. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of pH and Light on the Storage Stability of the Purple Pigment,Hordeumin, from Uncooked Barley Bran Fermented Broth

The pigment retention rate of hordeumin was higher than that of two standard anthocyanidins, cyanidin and delphinidin, when hordeumin and anthocyanidins were dissolved in Walpole buffer (pH 1.0) and stored. Moreover, when pigment solutions were stored at 15°C under light irradiation, the pigment retention rate of the hordeumin solution became higher than those of standard anthocyanidins (2 to 10 times) as the storage period increased. Comparing various pH buffers (MacIlvaine buffer, pH 2.2 to 7.0), the pigment retention rate of hordeumin at pH 5.0 was highest. Furthermore, the half-life of hordeumin at pH 5.0 was increased from 9 days to 17.5 days when nitrogen gas was bubbled into the hordeumin solution. We considered that the storage stability of hordeumin is higher than standard anthocyanidins because hordeumin is a complex with anthocyanin, tannin, and protein.     

Effects of pH and light on the storage stability of the purple pigment, hordeumin, from uncooked barley bran fermented broth

The pigment retention rate of hordeumin was higher than that of two standard anthocyanidins, cyanidin and delphinidin, when hordeumin and anthocyanidins were dissolved in Walpole buffer (pH 1.0) and stored. Moreover, when pigment solutions were stored at 15 degrees C under light irradiation, the pigment retention rate of the hordeumin solution became higher than those of standard anthocyanidins (2 to 10 times) as the storage period increased. Comparing various pH buffers (MacIlvaine buffer, pH 2.2 to 7.0), the pigment retention rate of hordeumin at pH 5.0 was highest. Furthermore, the half-life of hordeumin at pH 5.0 was increased from 9 days to 17.5 days when nitrogen gas was bubbled into the hordeumin solution. We considered that the storage stability of hordeumin is higher than standard anthocyanidins because hordeumin is a complex with anthocyanin, tannin, and protein.     

Structural studies of staphylococcal protease. I. Spin labelling of the active site and a comparison with other proteases.

Staphylococcus aureus protease has been spin-labelled at the active-site serine residue with the monocyclic-phosphorus spin label (MSL), 1-oxyl-2,2,6,6-tetramethyl-4-peperi-dinylethylphosphorofluoridate. The electron paramagnetic resonance (E.P.R.) sbectra of the protease in different buffers at various pH's have been analyzed and compared with those of trypsin, subtilisin BPN', and alpha-chymotrypsin under identical conditions. In a given buffer, the shape of E.P.R. signals of spin-labelled staphylococcal protease is unaffected by pH changes except below pH 4.0, at which a gradual loss of conformational integrity of the active site occurs. In bicarbonate buffer and particularly in acetate buffer, the mobility of the label is much more restricted than in phosphate buffer or in potassium chloride solution. The implications of this finding are discussed in terms of a model whereby the label is able to orient towards two different but adjacent regions of the active site. The relative population of the label in each of these orientations is believed to be buffer-dependent. An attempt to correlate the shape of the te.p.r. signals with the pH values of maximal proteolytic avtivity of the enzyme is also presented. These results show that to obtain meaningful information from a comparative spin label study of the geometry of the active site of serine proteases, particular care should be exercised to assure that the different proteases experience identical conditions of pH, buffer, and temperature.     

Factors of the shape change of human erythrocytes induced with lidocaine

We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lidocaine when ATP was present. But, the shape of resealed cells which were prepared with 10 mM Tris-HCl buffer (pH 7.4) containing 2 mM ATP-MgCl2 and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HCl buffer (pH 7.4) were incubated with various concentrations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These membranous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.     

Evaluation of Antigen Retrieval Buffer Systems

The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (pH 8.0) and Tris buffer (pH 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (pH 8.0) or Tris (pH 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use.     

Effect of control of pH on the excretion of acid phosphatase by Rhodotorula glutinis

Summary The excretion of an acid phosphatase by Rhodotorula glutinis is related to the pH of the medium. During growth, the phosphatase excretion into the medium at a constant pH of 4.5 was 5 times higher than that observed at variable pH. After cultivation at a constant pH of 4.5 or at variable pH, cells were incubated at various pH values between pH 2 and 7. During this second incubation acid phosphatase release occured at pH 4.5 to 6.5 only. There was no release at pH 3.0; but when resting cells incubated at this pH were placed in a buffer solution at pH 5.5 a high activity was released. Extensive washing did not eliminate residual intrinsic acid phosphatase activity. These two types of acid phosphatase were phosphomonoesterases with an identical specificity for different substrates.     

Impact of reaction conditions on the laccase-catalyzed conversion of bisphenol A

The oxidative conversion of aqueous BPA catalyzed by laccase from Trametes versicolor was conducted in a closed, temperature-controlled system containing buffer for pH control. The effects of medium pH, buffer concentration, temperature and mediators and the impacts of dissolved wastewater constituents on BPA conversion were investigated. The optimal pH for BPA conversion was approximately 5, with greater than half maximal conversion and good enzyme stability in the range of 4-7. The stability of the enzyme was not impacted by buffer concentration, nor was BPA conversion. Despite the observation that the enzyme tended to be inactivated at elevated temperatures, enhanced conversion of BPA was observed up until a reaction temperature of 45 degrees C. Of the mediators studied, ABTS was most successful at enhancing the conversion of BPA. Dissolved wastewater constituents that were studied included various inorganic salts, organic compounds and heavy metal ions. BPA conversion was inhibited in the presence of anions such as sulfite, thiosulfate, sulfide, nitrite and cyanide. The metal ions Fe(III) and Cu(II) and the halogens chloride and fluoride substantially suppressed BPA conversion, but the presence of selected organic compounds did not significantly reduce the conversion of BPA.     

Rat albumin inhibits the formation of apoceruloplasmin during storage

Purified rat ceruloplasmin is extraordinarily unstable in storage at –70 °C. In a 20 mM phosphate buffer, pH 7.0, the ferroxidase and amine oxidase of ceruloplasmin are over 90% inactivated within two weeks. Holoceruloplasmin stored for three months in a 20 mM barbital buffer (or acetate buffer), pH 7.0 (or pH 5.5) was transformed into an apo-protein and amine (o-dianisidine) oxidase of ceruloplasmin was inactivated by 50–55%. The patterns of ferroxidase activity loss were similar to those of amine oxidase activity loss. On the contrary, when holoceruloplasmin was mixed with rat serum albumin, transformation into apoceruloplasmin was significantly prevented in a 20 mM barbital buffer, pH 7.0 (or 20 mM acetate buffer, pH 5.5). Consequently, ferroxidase and amine oxidase activities of ceruloplasmin were not inactivated and the immunochemical reactivity was not changed. These results can be applied for laboratorial and clinical purposes.     

Unusual Behavior of Membrane Somatic Angiotensin-Converting Enzyme in a Reversed Micelle System

Properties of the membrane and soluble forms of somatic angiotensin-converting enzyme (ACE) were studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. The membrane enzyme with a hydrophobic peptide anchor was more sensitive to anions and to changes in pH and composition of the medium than the soluble enzyme without anchor. The activity of both forms of the enzyme in the reversed micelles significantly depended on the molarity of the buffer added to the medium (Mes-Tris-buffer, 50 mM NaCl). The maximum activity of the soluble ACE was recorded at buffer concentration of 20-50 mM, whereas the membrane enzyme was most active at 2-10 mM buffer. At buffer concentrations above 20 mM, the rate of hydrolysis of the substrate furylacryloyl-L-phenylalanyl-glycylglycine by both ACE forms was maximal at pH 7.5 both in the reversed micelles and in aqueous solutions. However, at lower concentrations of the buffer (2-10 mM), the membrane enzyme had activity optimum at pH 5.5. Therefore, it is suggested that two conformers of the membrane ACE with differing pH optima for activity and limiting values of catalytic constants should exist in the reversed micelle system with various medium compositions. The data suggest that the activity of the membrane-bound somatic ACE can be regulated by changes in the microenvironment.     

Development of acetaminophen proline prodrug

In this research work, proline ester prodrug of acetaminophen (Pro-APAP) was synthesized and evaluated for its stability in PBS buffer at various pH and Caco-2 cell homogenate. The Pro-APAP is more stable at lower pH than higher pH, with half-life of 120 min in PBS buffer at pH 2.0, half-life of 65 min at pH 5.0, and half life of 3.5 min at pH 7.4, respectively. The half-life of Pro-APAP in Caco-2 cell homogenate is about 1 min, much shorter than the half-life in PBS buffer at pH 7.4, indicating enzymes in the cell homogenate contribute to the hydrolysis of the ester bond. Carboxypeptidase A was incubated with Pro-APAP at pH 7.4 with half-life of 3.8 min which is very close to the half life in buffer itself. This clearly indicates carboxypeptidase A is not one of the enzymes contributing to the hydrolysis of the prodrug. Physicochemical characteristics such as melting point and stability of newly synthesized prodrug were determined by MDSC technique.     

Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

Coating efficiency of a 1 -acid glycoprotein in competitive enzyme immunosorbent assays with antigen immobilized on the solid phase

Coating efficiency of rat and human serum a 1 -acid glycoprotein (a 1 -AGP) was investigated for competitive enzyme immunosorbent assays with antigen immobilized on the solid phase by using different pHs and buffers. Blocking materials and pH of coating buffer had a marked influence on the amount of a 1 -AGP that binds to plate. Usually, carbonate buffer is used at pH 9.6 or 9.0, but phosphate buffered saline (PBS) at pH 7.2 can be used for an effective coating. At pH 7.2, coating of a 1 -AGP in Tris buffered saline was five - tenfold as effective as in PBS and phosphate buffer. Blocking of uncoated surface with casein was ten - twenty times as effective as with fetal bovine serum albumin for coating of a 1 -AGP.