Decolorization of reactive dyes by mixed cultures isolated from textile effluent under anaerobic conditions

In the present study mixed cultures that could grew in the molasses media were isolated from textile dye effluent and its decolorization activity was studied in a batch system under anaerobic conditions, in order to determine the optimal conditions required for the highest decolorization activity. The optimum pH value for decolorization was determined as 8 for all the dyes tested. In the experiment with pH 8 dye decolorizations by mixed cultures were investigated at about 96.2–1031.3 mg l⁻¹ initial dye concentrations. The highest dye removal rates of mixed cultures were 94.9% for Reactive Red RB, 91.0% for Reactive Black B and 63.6% for Remazol Blue at 953.2, 864.9 and 1031.3 mg l⁻¹ initial dye concentrations respectively within 24 h incubation period. When the Reactive Red RB was used, approximately 82–98% total color removal was obtained at between 96.2 and 953.2 mg l⁻¹ initial dye concentrations after 12 h of incubation at 35 °C. These results show that our enriched mixed cultures have the potential to serve as an excellent biomass for the use in reactive dye removal from wastewaters under anaerobic conditions.     

Optimisation for enhanced decolourization and degradation of Reactive Red BS C.I. 111 by Pseudomonas aeruginosa NGKCTS

Soil samples collected from dye contaminated sites of Vatva, Gujarat, India were studied for the screening and isolation of organisms capable of decolourizing textile dyes. The most efficient isolate, which showed decolourization zone of 48 mm on 300 ppm Reactive Red BS (C.I.111) containing plate, was identified as Pseudomonas aeruginosa. Reactive Red BS (C.I.111) was used as a model dye for the study. The isolated culture exhibited 91% decolourization of 300 ppm dye within 5.5 h over a wide pH range from 5.0 to 10.5 and temperature ranging from 30 to 40°C. The culture was able to decolourize more than 91% of Reactive Red BS under static conditions in presence of either glucose, peptone or yeast extract. Addition of 300 ppm of Reactive Red BS, in each step, in ongoing dye decolourization flask, gave more than 90% decolourization within 2 h corresponding to 136 mg l⁻¹ h⁻¹ dye removal rate. The isolate had the ability to decolourize six different reactive dyes tested as well as the actual dye manufacturing industry’s effluent. The degradation of the dye was confirmed by HPTLC.     

Adsorption and decolorization of dyes using solid residues from Pleurotus ostreatus mushroom production

We investigated the use of solid residues from Pleurotus ostreatus mushroom production in adsorbing and decolorizing different dyes. The solid residue used in this study was composed of hemicellulose and cellulose (52.81 %), acid-insoluble lignin (25.42%), chitin (6.5%), and water extractives (14.82%). After incubating 14% (wt/vol) solid residue in distilled water for 4 h, laccase and manganese peroxidase (MnP) activities were 0.5 U/g and 12 mU/g, respectively. Enzymatic decolorization percentages were up to 100 for azure B (heterocyclic dye) and indigo carmine (indigoid dye), 74.5 for malachite green (MG) (triphenylmethane dye), and zero for xylidine (azoic dye). The optimum temperature for decolorization was in the range of 26 ∼ 36°C for all dyes. Data obtained on adsorption (enzymatic decolorization was prevented with sodium azide) at different dye concentrations and in a pH range of 3 ∼ 7 were used to plot Freundlich isotherms. The spent fungal substrate (SFS) displayed large differences in adsorption capacity, depending on the dye tested. The highest adsorption capacity was observed at pH 3 for MG, while xylidine was slightly adsorbed at pH 3 and 4 and not adsorbed at higher pH values. Laccase and MnP production were affected by the presence of the dyes. The highest enzyme levels were observed in the presence of MG, when laccase and MnP increased 1.39- and 2.13-fold, respectively. Decolorization and adsorption to SFS are both important processes in removing dyes from aqueous solutions. The application of this spent substrate for wastewater treatment will be able to take advantage of both of these dye removal processes. An important problem in bioremediation processes involving microorganisms is the amount of time required for their growth. In this report, we used the spent substrates from mushroom cultivation in wastewater treatment, thus solving the problem of waiting for microorganisms to grow.     

The flocculation performance of Tamarindus mucilage in relation to removal of vat and direct dyes

A food grade natural mucilage, extracted from the seeds of Tamarindus indica pods, is used as a flocculant for removal of solubilised vat (golden yellow) and direct dye (direct fast scarlet) in aqueous solutions. The maximum removal obtained was 60% for golden yellow after 2 h and was 25% for direct fast scarlet after 1 h. The optimum mucilage dose was 10 mg/l and 15 mg/l for golden yellow and direct fast scarlet, respectively. The pH values also seem to affect the percent removal of both the dyes significantly. In case of vat dye, the pH value of the test samples affected the percent removal significantly. The change was highly significant between neutral and alkaline pH. In case of direct dye, there was no significant change in percent removal at pH 7 and pH 4 whereas a significant change in percent removal was observed between pH 7 and pH 9.2. The plausible mucilage-dye interaction and flocculation mechanism has been discussed. This new flocculant works better in the case of vat dye removal compared with the direct dye.     

DNA interaction, cytotoxicity, and radiosensitization with PtCl4(Nile Blue)2 and PtCl4(Neutral Red)2

Complexes of the positively charged, nuclear staining, quinone-imine dyes Nile Blue and Neutral Red with negatively charged tetrachloroplatinum (II) have been prepared in an effort to form neutral drugs which could gain ready access to the cellular nucleus and deliver significant quantities of the reactive tetrachloroplatinum anion to the vicinity of the DNA. Elemental analysis showed that both the Nile Blue and Neutral Red complexes with tetrachloroplatinum (II) comprised 2 mol of dye and 1 mol of tetrachloroplatinum, forming Pt(Nile Blue)2 and Pt(Neutral Red)2. Exposure of superhelical pBR322 DNA to the complexes or the dyes for 24 h followed by agarose gel electrophoresis showed that Neutral Red and Pt(Neutral Red)2 had little effect on DNA conformation, but that both Nile Blue and Pt(Nile Blue)2 could produce single-strand DNA breaks in a dose-dependent fashion. Studies in exponentially growing asynchronous, hypoxic, and normally oxygenated EMT6 cells at normal pH (7.40) and pH 6.45 demonstrated that neither dye was highly toxic, but that both complexes were capable of producing significant cytotoxicity. Both complexes killed normally oxygenated cells more efficiently than hypoxic cells, but Pt(Neutral Red)2 was more cytotoxic at pH 6.45, while Pt(Nile Blue)2 killed significantly more cells at normal pH. Both complexes decreased the survival of hypoxic EMT6 cells as indicated by the slope of the radiation survival curve [dose modifying factor (DMF) 2.90 for Pt(Nile Blue)2 and 1.45 for Pt(Neutral Red)2]. Studies with the FSaIIC murine tumor showed that both complexes were active radiosensitizing agents in vivo [DMF 1.76 for Pt(Nile Blue)2 and 1.25 for Pt(Neutral Red)2]. These results indicate that these new platinum complexes have characteristics which may make them and similar complexes effective radiosensitizing agents in humans.     

Accumulation of Acid Orange 7, Acid Red 18 and Reactive Black 5 by growing Schizophyllum commune

The effect of Acid Orange 7, Acid Red 18 and Reactive Black 5 on the growth and decolorization properties of Schizophyllum commune was studied with respect to the initial pH varying from 1 to 6 and initial dye concentration (10-100 mg/L). The optimum pH value was found to be 2 for both growth and color removal of these azo dyes. Increasing the concentration of azo dyes inhibited the growth of S. commune. It was observed that S. commune was capable of removing Acid Orange 7, Acid Red 18 and Reactive Black 5 with a maximum specific uptake capacity of 44.23, 127.53 and 180.17 (mg/g) respectively for an initial concentration of 100 mg/L of the dye. Higher decolorization was observed at lower concentrations for all the dyes. Finally it was found that the percentage decolorization was more in the case of Reactive Black 5 dye compared to the other two dyes used in the present investigation.     

Biodegradation of C.I. Reactive Red 195 by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> strain YZ66

Synthetic dyes are extensively used in textile dyeing, paper, printing, colour photography, pharmaceutics, cosmetics and other industries. Among these, azodyes represents the largest and most versatile class of synthetic dyes. As high as 50% of the dyes are released into the environment during manufacture and usage. Traditional methods of treatment are found to be expensive and have operational problems. Biological decolourization has been investigated as a method to transform, degrade or mineralize azo dyes. In the present studies bacteria from soil from dye waste area, dye waste, sewage and dung were subjected to acclimatization with C.I. Reactive Red 195 an azo dye, in the basal nutrient media. The most promising bacterial isolate was used for further dye degradation studies. The 16s rRNA gene sequencing and biochemical characteristics revealed the isolated organism as Enterococcus faecalis strain YZ66. The strain showed 99.5% decolourization of the selected dye (Reactive Red 195–50 mg/l) within one and half hour in static anoxic condition. The optimum pH and temperature for the decolourization was 5.0 and 40°C respectively. The biodegradation was monitored by UV–Vis, FTIR, TLC and HPLC. The final products were characterized by Gas chromatography and Mass Spectrophotometry. Toxicity study demonstrated no toxicity of the biodegradation product. The results suggest that the isolated organism E. faecalis strain YZ 66 can be used as a useful tool to treat waste water containing reactive dyes.     

Removal of basic and reactive dyes using ethylenediamine modified rice hull

Wastewaters from textile industries may contain a variety of dyes that have to be removed before their discharge into waterways. Rice hull, an agricultural by-product, was modified using ethylenediamine to introduce active sites on its surface to enable it to function as a sorbent for both basic and reactive dyes. The sorption characteristics of Basic Blue 3 (BB3) and Reactive Orange 16 (RO16) by ethylenediamine modified rice hull (MRH) were studied under various experimental conditions. Sorption was pH and concentration dependent. Simultaneous removal of BB3 and RO16 occurred at pH greater than 4. The kinetics of dye sorption fitted a pseudo-second order rate expression. Increase in agitation rate had no effect on the sorption of BB3 but increased uptake of RO16 on MRH. Decreasing particle size increased the uptake of dyes in binary dye solutions. Equilibrium data could be fitted into both the Langmuir and Freundlich isotherms. Maximum sorption capacities calculated from the Langmuir model are 14.68 and 60.24 mg/g for BB3 and RO16, respectively in binary dye solutions. This corresponds to an enhancement of 4.5 and 2.4 fold, respectively, compared to single dye solutions. MRH therefore has the potential of being used as an efficient sorbent for the removal of both dyes in textile wastewaters.     

Biodegradation of reactive textile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1

A novel bacterial strain capable of decolorizing reactive textile dye Red BLI is isolated from the soil sample collected from contaminated sites of textile industry from Solapur, India. The bacterial isolate was identified as Pseudomonas sp. SUK1 on the basis of 16S rDNA analysis. The Pseudomonas sp. SUK1 decolorized Red BLI (50 mg l(-1)) 99.28% within 1h under static anoxic condition at pH range from 6.5 to 7.0 and 30 degrees C. This strain has ability to decolorize various reactive textile dyes. UV-Vis spectroscopy, FTIR and TLC analysis of samples before and after dye decolorization in culture medium confirmed decolorization of Red BLI. A significant increase in the activities of aminopyrine N-demethylase and NADH-DCIP reductase in cells obtained after decolorization indicates involvement of these enzymes in the decolorization process. Phytotoxicity testing with the seeds of Sorghum vulgare and Phaseolus mungo, showed more sensitivity towards the dye, while the products obtained after dye decolorization does not have any inhibitory effects.     

Studies on desorption of individual textile dyes and a synthetic dye effluent from dye-adsorbed agricultural residues using solvents

Two solvents, A and B (A: methanol, chloroform, water in the ratio 1:1:1; B: 50% methanol), were used to extract textile dyes adsorbed onto substrates for the purpose of future analyses of the amount of dyes degraded through solid state fermentation (SSF) using white rot fungi. Barley husk, apple pommace and corncob were separately soaked in five different dye solutions and a synthetic textile effluent. A maximum value of 93% desorption of Cibacron Red from corncob was achieved using solvent A. Barley husk was the only substrate from which the synthetic textile effluent could be desorbed, with 82% being recovered using solvent A.     

Decolorization of reactive dyes by <Emphasis Type="Italic">Clostridium bifermentans</Emphasis> SL186 isolated from contaminated soil

Decolorization of textile reactive azo dyes by a strain of bacteria (SL186) isolated from a contaminated site was investigated. SL186 was identified as Clostridium bifermentans by phenotypic characterization and 16S rDNA sequence comparison. Under anaerobic conditions, SL186 had decolorized the dyes Reactive Red 3B-A, Reactive Black 5, and Reactive Yellow 3G-P by over 90% after 36 h post-inoculation. The bacterium retained decolorizing activity over a wide range of pH values (6–12), with peak activity at pH 10. Additionally, SL186 decolorized a relatively high concentration of Reactive Red 3B-A dye (1,000 ppm) by over 80% and raw industrial effluent effectively. The addition of glucose increased the decolorization rate a little. Spectrophotometric analyses of the reactive dyes showed no distinct peak indicating aromatic amines. However, a new peak was detected between 300 and 450 nm from the decolorized raw industrial effluent. These results suggest that C. bifermentans SL186 is a suitable bacterium for the biological processing of dye-containing wastewater.     

Degradation of reactive dyes by ozonation and oxalic acid-assimilating bacteria isolated from soil

Ozonation and treatment of wastewaters with oxalic acid-assimilating bacterium was attempted for the complete degradation of reactive dyes. Oxalic acid-assimilating bacterium, Pandoraea sp. strain EBR-01, was newly isolated from soil under bamboo grove and was identified to be a member of the genus Pandoraea by physicochemical and biochemical tests including 16S rDNA sequence analysis. The bacterium was grown optimally at pH 7 and temperature of 30 degrees C under the laboratory conditions. Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Black 5 (RB5) and Remazol Brilliant Blue R (RBBR) were used in degradation experiments. At the initial reactive dye concentrations of 500 mg/l and the ozonation time of 80 min, it was confirmed that 75-90 mg/l oxalic acid was generated from reactive dyes by ozonation. Microbial treatment using EBR-01 greatly decreased the amount of oxalic acid in the mixture after 48 h, but it was not removed completely. TOC/TOC(0) of reactive dye solutions was also decreased to 80-90% and 20-40% by ozonation and microbial treatment using EBR-01, respectively. The study confirmed that consecutive treatments by ozone and microorganisms are efficient methods to mineralize reactive dyes.     

Biosorption of reactive dyes by the mycelium pellets of a new isolate of Penicillium oxalicum

Three reactive dyes were rapidly adsorbed by the mycelium pellets of Penicillium oxalicum. Dye removal of Reactive Blue 19 was up to 60% in 10 min and 91% in 80 min. Dye adsorption isotherms fitted Langmuir model well and the maximum adsorption capacities at 20 °C were calculated to be 160 mg g^–1 for Reactive Blue 19, 122 mg g^–1 for Reactive Red 241 and 137 mg g^–1 for Reactive Yellow 145, respectively. The pellets exhibited a high dye adsorption capacity (80–180 mg g^–1) for all of the 3 dyes over a wide pH range (pH 2–10), and the maximum adsorption was obtained at pH 2. The adsorption capacity was mildly increased by increasing salinity.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

Chitosan-coated Lentinus polychrous Lév.: Integrated biosorption and biodegradation systems for decolorization of anionic reactive dyes

Research and development of an effective color removal system is needed to reduce the severity of water pollution caused by effluent that contains dyes. In this study, the integrated biosorption and biodegradation system of chitosan coated Lentinus polychrous Lév. was developed and evaluated for its decolorization efficiency with regard to anionic reactive dye mixtures of Reactive Blue 19, 160, and 198. The fungi were coated with 0.1, 0.5, and 1.0% w/v of low molecular weight chitosan. The scanning electron micrographs confirmed that chitosan was successfully coated on the surface of the fungi. Studies of changes in UV–visible absorption spectra, dye desorption, ligninolytic enzyme activity, and Fourier transform infrared spectroscopy showed that within 6 h, the biosorption was the control mechanism and the dyes were reduced to 91.50, 77.66, 37.39, and 26.93% by the fungi coated with 0, 0.1, 0.5, and 1.0% w/v chitosan, respectively. From the 36th hour to the end of colorization at the 72nd hour, the fungal biodegradation by laccase and manganese peroxidase was dominant and all treatments had 5–8% of the dye remaining. Therefore, the chitosan coat acted as an efficient biosorbent for the anionic reactive dyes, thereby effectively improving the decolorization efficiency of the white rot fungus.     

绒毛栓孔菌菌丝体在无营养条件下对偶氮染料刚果红的脱色作用

【目的】在无营养条件下,利用白腐真菌绒毛栓孔菌(Trametes pubescens)菌丝体对染料进行脱色可减少试验成本,提高染料处理的实用性。【方法】将该菌株液体培养的菌丝体在无营养条件下对染料进行脱色,并对其中脱色效果较好的偶氮染料刚果红的脱色过程进行分析。在此过程中,测定了该菌株分泌的胞外胞内酶活力,优化影响因子如初始pH值、温度、染料浓度和盐度,同时利用气相色谱-质谱联用技术分析无营养条件下偶氮染料刚果红的降解产物。植物毒性试验测定刚果红经绒毛栓孔菌菌丝体脱色前后的毒性变化。【结果】菌丝体对偶氮染料刚果红有较好的脱色效果,在初始pH值为2.0,温度为30°C,染料浓度为80 mg/L,盐度为2.5%(质量体积比)时,150 r/min转速下培养7 d后脱色率可达80.52%。在此过程中,菌丝体可被连续使用2次,且其所分泌的酶系可降解染料。此外,通过气相色谱-质谱联用分析得到刚果红的降解产物为萘胺、联苯胺和叠氮萘。植物毒性试验显示在无营养条件下的绒毛栓孔菌菌丝体对染料有明显的脱毒作用。【结论】研究发现绒毛栓孔菌菌丝体在无营养条件下的偶氮染料废水处理中具有广阔的应用前景。     

Effective bioremoval of reactive dye and heavy metals by Aspergillus versicolor

In this study, bioaccumulation of heavy metal and dye by Aspergillus versicolor was investigated. Optimum pH values of the maximum heavy metal bioaccumulation was found as 6 for 50 mg/L Cr(VI), Ni(II) and 5 for Cu(II) ions with the 99.89%, 30.05% and 29.06% removal yield, respectively. The bioremoval of the dye up to 800 mg/L at pH 5 and 6 was investigated and 27.15% and 28.95% removal rates were measured respectively. The presence of Cr(VI) with dye, decreased the uptake yield for both pollutants. In the medium with Cu(II) and dye, dye removal was not affected by Cu(II), but Cu(II) removal rate increased from 29.06% to 37.91% by the existence of the dye. When Ni(II) and dye were combined, neither pollutant affected the other’s removal yield. These results indicate that the isolated A. versicolor strain deserves attention as a promising bioaccumulator of heavy metal ions and reactive dyes in wastewater effluents.     

Decolourization and degradation of C.I.Reactive Red 195 by Georgenia sp.CC-NMPT-T3

Azo dye Reactive Red 195 was selected for decolourization and degradation studies by Georgenia sp.CC-NMPT-T3. Optimization of parameters for dye decolourization was studied under static anoxic condition. Under optimized condition decolourization of Reactive Red 195 by Georgenia sp.CC-NMPT-T3 was found to be 95.93 % at 50 mg/L within five hours in static anoxic condition. The optimum pH and temperature for the decolourization was 7.0 and 40 degrees C respectively. The biodegradation was monitored by UV-Vis, and TLC and HPLC. Toxicity study demonstrated no toxicity of the biodegraded product. The results suggest that the isolated organism Georgenia sp.CC-NMPT-T3 as a useful tool to treat wastewater containing reactive dyes.     

Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces.

An indicator dye attached to an electrostatically charged macromolecular surface generally has a pK value (pKb') different from that of uncombined dye (pKf'). The question if changes in (pKb' - pKf'), designated as increment incrementpK, records changes in the electrostatic potential at the binding site has been examined in spectrophotometric and binding experiments, using the interaction of Chlorophenol Red and Phenol Red with human serum albumin and cationic micelles as examples. (1) In serum albumin solutions increment incrementpK is decreased by a reduction of pH. The decrease is correlated with the increase in positive charges on the protein molecule, and the response is attenuated by high ionic strength in accordance with electrostatic theory. (2) Opposite changes in binding affinity to serum albumin and increment incrementpK as a function of pH are observed; the binding of basic (bivalent anion) dye is more susceptible to a change in pH than in the acidic (univalent anion) form. (3) Preferential uptake of the basic as compared to the acidic form of dye is observed by binding to cetyltrimethylammonium chloride and cetylpyridinium chloride micelles (mu equals 0.033, [Cl-] equals 0.033 M). An increase in the ionic strength is accompanied by a positive value of increment incrementpK. The results are consonant with the view that the observed increment incrementpK values reflect changes in the electrostatic potential at the binding site with consequently little, if any, effect on the intrinsic pK. The extension of the method to measure changes in the electrostatic potential at binding sites on cell membranes is briefly discussed.