Evidence for in situ methanogenic oil degradation in the Dagang oil field

In situ biotransformation of oil to methane was investigated in a reservoir in Dagang, China using chemical fingerprinting, isotopic analyzes and molecular and biological methods. The reservoir is highly methanogenic despite chemical indications of advanced oil degradation, such as depletion of n-alkanes, alkylbenzenes and light polycyclic aromatic hydrocarbon (PAH) fractions or changes in the distribution of several alkylated polycyclic aromatic hydrocarbons. The degree of degradation strongly varied between different parts of the reservoir, ranging from severely degraded to nearly undegraded oil compositions. Geochemical data from oil, water and gas samples taken from the reservoir are consistent with in situ biogenic methane production linked to aliphatic and aromatic hydrocarbon degradation. Microcosms were inoculated with production and injection waters in order to characterize these processes in vitro. Subsequent degradation experiments revealed that autochthonous microbiota are capable of producing methane from ¹³C labelled n-hexadecane or 2-methylnaphthalene and suggest that further methanogenesis may occur from the aromatic and polyaromatic fractions of Dagang reservoir fluids. The microbial communities from produced oil–water samples were composed of high numbers of microorganisms (on the order to 10⁷), including methane producing Archaea within the same order of magnitude. In summary, the investigated sections of the Dagang reservoir may have significant potential for testing the viability of in situ conversion of oil to methane as an enhanced recovery method and biodegradation of the aromatic fractions of the oil may be an important methane source.     

<Emphasis Type="Italic">Trichoderma viride</Emphasis> (MTCC 800): a potential candidate for agri-horti waste utilization by solid state fermentation

Waste management is one of the major environmental concerns globally. Meaningful utilization of various types of wastes for the production of useful products not only provides added economic benefits through variety of products but is also helpful in minimizing the environmental pollution. Trichoderma viride, a well-known fungus, has been used in the past for production of enzymes like proteases, cellulases and chitinase besides various beneficial biological activities, i.e., mycoremediation, mycoparasitism and for increasing soil fertility. In the present investigation, biodegradation potential of T. viride (MTCC-800) was evaluated by solid state fermentation using wastes like pomace, floral waste, plant litter, vegetable refuse, sawdust and sugarcane bagasse. The fungus degraded all the waste materials including sawdust that contain toxic compounds. Among all agri-horti wastes used, maximum growth of T. viride was recorded in vegetable refuse (58.16%) followed by sugarcane bagasse (48.76%) and others. The results of the present investigation, indicate that T. viride can be a potential candidate for meaningful utilization of industrial and other wastes and if. Successful at large scale it can not only help in reduction of environmental pollution but can also be a better substitute for chemical fertilizers and pesticides besides economic gains.     

Biological markers from Green River kerogen decomposition

Isoprenoid and other carbon skeletons that are formed in living organisms and preserved essentially intact in ancient sediments are often called biological markers. The purpose of this paper is to develop improved methods of using isoprenoid hydrocarbons to relate petroleum or shale oil to its source rock. It is demonstrated that most, but not all, of the isoprenoid hydrocarbon structures are chemically bonded in kerogen (or to minerals) in Green River oil shale. The rate constant for thermally producing isoprenoid, cyclic, and aromatic hydrocarbons is substantially greater than for the bulk of shale oil. This may be related to the substantial quantity of CO₂ which is evolved coincident with the isoprenoid hydrocarbons but prior to substantial oil evolution. Although formation of isoprenoid alkenes is enhanced by rapid heating and high pyrolysis temperatures, the ratio of isoprenoid alkenes plus alkanes to normal alkenes plus alkanes is independent of heating rate. High-temperature laboratory pyrolysis experiments can thus be used to predict the distribution of aliphatic hydrocarbons in low temperature processes such as in situ shale oil production and perhaps petroleum formation. Finally, we demonstrate that significant variation in biological marker ratios occurs as a function of stratigraphy in the Green River formation. This information, combined with methods for measuring process yield from oil composition, enables one to relate time-dependent processing conditions to the corresponding time-dependent oil yield in a vertical modified-in situ retort even if there is a substantial and previously undetermined delay in drainage of shale oil from the retort.     

Evaluation of the benthic macroinvertebrate distribution in a stream environment during summer using biotic index

The purpose of this study is to evaluate the benthic macroinvertebrate distribution in the Ke?ap Stream (in the Eastern Black Sea Region of Turkey) for the summer period using biotic index. The water quality was assessed through the application of the Belgian biotic index. For this purpose, benthic macroinvertebrate sampling was performed in six sites along the Ke?ap Stream in the summer period, in July 2007. The distribution of dominant genera of the sites was evaluated according to water quality. According to the Belgian biotic index, II, III and IV water quality classes (slightly, moderately and heavily polluted, respectively) were determined in the stream. In addition, diversities of benthic macroinvertebrates in six sites were calculated. The biotic index values are found also consistent with the diversity values. Especially, in areas of the stream after industrial facilities water quality decreased. However, very heavy pollution was not observed accord to the biotic index values. It was found that Ephemeroptera was the dominant major taxon in all sampling sites. As a result of this biological evaluation, Ephemeroptera were concluded to be the most common taxa in the stream and the ephemeropterans, Potamanthus, Baetis and Ephemerella were dominant genera in the conditions of slightly, moderately and heavily polluted.     

Characterization of fresh and weathered petroleum for potential impacts to soil fertility

In many petroleum-producing regions, there are not adequate controls to prevent pipeline breaks and spills, and thus soil is frequently contaminated with petroleum hydrocarbons. Different petroleum oil compounds may produce negative impacts on soil fertility. In this study, four fresh crudes, a weathered petroleum, and oils from bioremediated and burned sites were investigated (specific gravities 0.83–1.27). Fourier transform infrared spectroscopy revealed three predominant polar functional groups to be more plentiful in the heavier crudes. The relative abundance of these groups was used to calculate an index that was directly correlated with specific gravity (R ² = 0.9960) and the percent of asphaltene plus (polars + resins) fractions in the oil (R ² = 0.9643). This index correlated exponentially to the water repellency caused by petroleum in an alluvial soil (R ² = 0.9928). Furthermore, extra-heavy oil at a concentration of 10,000 ppm, the maximum allowable oil concentration in the soil that is within regulatory norms in many US states and other countries, and with a specific gravity >1.002, showed severe water repellency. This study presents an alternative for determining soil remediation criteria based on the API gravity of the oil rather than the C-range of the hydrocarbon mixtures, simplifying analytical methods and systematically studying the interaction between the kinds of petroleum mixtures and potential impacts to soil fertility.     

Chemical compatibility testing of the entomopathogenic fungus Lecanicillium muscarium to control Bemisia tabaci in glasshouse environment

The potential for using the entomopathogenic fungus Lecanicillium muscarium to control the sweetpotato whitefly, Bemisia tabaci has been well established in previous studies under both laboratory and glasshouse conditions. In the current study, five chemicals were assessed for their compatibility with L. muscarium for control of B. tabaci under glasshouse conditions. On treatments following the sequential application of chemical product and fungus high mortality of second instar larvae was obtained (the known most susceptible B. tabaci life-stage to fungal infection). Sequential treatment of Savona and Certis spraying oil with L. muscarium produced 95 % and 96 % larval mortality, respectively. Commercially, unacceptable poinsettia foliage damage was recorded seven days post application of Agri-50E. Other plant foliage may prove more tolerant to this product. Incorporation of these chemicals with L. muscarium into integrated control programmes for B. tabaci control in glasshouses is discussed. Further information has been added to the knowledge base for the combined use of chemicals and fungi for the control of B. tabaci.     

Treatment of an ECF bleaching effluent with white-rot fungi in an air-lift bioreactor

Pulp and paper mills utilise huge amount of natural resources, inorganic and organic materials along with large volume of water in different stages of paper manufacturing, resulting in a significant volume of effluents. The aim of this work was to investigate the treatment of a bleaching effluent [effluent chlorine free (ECF)] from the cellulose industry with white-rot fungi in an air-lift bioreactor. This effluent was submitted to the biological treatment with three white-rot fungi, and, every 24 h for 120 h, analytical tests were performed to analyse the quality parameters of treatment (COD, phenols, colour, pH). Before and after treatment, the effluent was analysed as its molecular mass distribution and absorptivity in the UV/VIS. Lentinus edodes, Trametes versicolor and Trametes villosa promoted similarly significant reductions in the following characteristics: (a) effluent colour (40–44 %), (b) total phenol (30–51 %) and (c) COD (37–43 %). The UV/visible spectrometry reading of the effluent after the fungi treatment showed a reduction in the absorbance of all wavelengths between 260 and 500 nm. The size exclusion HPLC profile of the effluent was modified, and the treatment promoted changes in the intensity of the peaks associated with compounds of high and low molecular weight. Phenoloxidases were produced during the treatment. T. versicolor produced the highest levels of laccase, and L. edodes was the only fungus that produced peroxidases.     

Intrinsic biodegradation of diesel fuel in an interval of separate phase hydrocarbons

Emerging acceptance of the limitations of separate phase product recovery has spawned interest in the intrinsic alteration of residual separate phase petroleum products. In this study the geochemical changes in a continuous core through soil containing a separate phase diesel fuel #2 (SPD) in contact with groundwater are investigated. Chemical heterogeneities are shown to exist which can be attributed to weathering, particularly intrinsic biodegradation. The results show that the aliphatic hydrocarbon content is reduced and the δ¹³C ratio of the aliphatic hydrocarbons increased from top to bottom in the core. Both changes are thought to be due to preferential biodegradation of (isotopically lighter) n-alkanes. A slight increase in the relative abundance of shorter chain n-alkanes (<n-C₁₇) was also observed. The distribution of the dominant aromatic hydrocarbons (C₀–C₃ alkyl-naphthalenes) is remarkably consistent throughout the core, although naphthalene is depleted below the oil–water interface. In spite of low oil saturation (S₀), little or no evidence of biodegradation is noted at the uppermost boundary of the SPD. However, intrinsic biodegradation is evident approximately 0.3 m above the oil–water interface in spite of higher S₀. The extent of the chemical changes attributable to biodegradation (described above) gradually increases below the oil–water interface, eventually reaching a maximum at the bottom of the SPD profile (∼1.2 m below the interface) where S₀ is again reduced. The relatively higher level of biodegradation observed at and below the oil–water interface may be attributed to the reduced S₀ in this zone. An estimate of the mass reduction in diesel fuel between the uppermost and bottommost parts of the core is calculated to be 23% (by weight), due predominantly to the biodegradation of n-alkanes.     

Application of fluid-rock reaction studies to in situ recovery from oil sand deposits,Alberta, Canada—I. Aqueous phase results for an experimental-statistical study of water-bitumen-shale reactions

In situ recovery operations in oil sand deposits effectively represent man-imposed low to intermediate temperature metamorphism of the sediments in the deposit. In order to evaluate some of the reactions which occur, a factorial experiment was earned out in which a shale from the Lower Cretaceous McMurray Formation in the Athabasca oil sand deposit of Alberta, in the presence or absence of bitumen, was subjected to hydrothermal treatment with aqueous fluids of varying pH and salinity, at two different temperatures, for periods up to 92 hours. The aqueous fluid was analyzed and the analytical data subjected to statistical factor analysis and analysis of variance, which enabled identification of the main processes, namely, cation exchange, the production of two types of colloidal material, and the dissolution of quartz There is also saturation of the aqueous phase by. as yet unidentified, “total organic carbon” and complete conversion and removal of all nitrogen in the shale to the aqueous phase. These reactions have implications with regards to the economics of the in situ recovery process, specifically with respect to the reuse and/or disposal of the produced water and the plugging of the pore space and hence of reduction of permeability between the injection and production wells. As a result of these experiments it is suggested that monitoring of the composition of the produced water from in situ recovery operations in oil sand deposits would be advisable.     

Vanadyl porphyrins in exploration: maturity indicators for source rocks and oils

The Porphyrin Maturity Parameter (PMP), which is derived from the vanadyl porphyrin distribution, is an excellent parameter for: (1) identifying the zone of hydrocarbon generation from marine source rock extracts; and (2) determining from oils the thermal maturity of their source rocks at expulsion.The PMP is measured using a methodology which is inexpensive, reliable and faster than earlier methods, allowing it to be used as a routine exploration tool. The PMP may be a more reliable maturity indicator for marine organic matter than some conventional methods such as vitrinite reflectance. Unlike most conventional maturity parameters guided by processes other than kerogen conversion, the reactions causing PMP evolution directly monitor the generation of bitumen and the concurrent thermal degradation of kerogen.Measurements on hydrous pyrolyzates from the Monterey Formation (offshore California), source rock bitumens from the Devonian-Mississippian Bakken Shale (Williston Basin), and Miocene Monterey equivalent source strata (San Joaquin Basin, California) illustrate the method. In all cases reviewed so far, PMP begins increasing at the onset of hydrocarbon generation and increases systematically and predictably as kerogen decomposition proceeds.In oils generated from high-S marine kerogens, PMP reflects the maturity of the source rock at the time of oil expulsion, provided that the oil does not undergo subsequent reservoior maturation or mixing with in-situ bitumen.     

Log-derived petrophysical characteristics and oil potentiality of the Upper Qishn Clastic Member,Masila Basin,Yemen

The Lower Cretaceous Qishn Formation at Masila Basin, Yemen, contains substantial amounts of proven crude oil. It is divided into a lower shaly silisiclastic member and an upper carbonate member. In the present work, detailed formation evaluation of the Upper Qishn Clastic Member was carried out using the digitized well logging data from 12 wells, via computer-assisted analysis, to accomplish its reservoir characteristics and oil potentiality. Accordingly, the true resistivity (R _t), porosity (total φ _t and effective φ _e), shale content (V _sh), permeability (K), water saturation (S _w), hydrocarbon saturation (S _h) (movable “S _hm” and residual “S _hr”) and lithologic composition in each well were estimated, analyzed, and illustrated versus depth on litho-saturation cross-plots. Likewise, the areal distribution of such parameters alongside the member in the investigated area and their relations to geological setting were defined from the constructed isoparametric contour maps. Based on the obtained results, the components of Upper Qishn Clastic Member of Masila Basin are mainly shaly sandstone and minor calcareous sandstone with considerable amount of shale. Regionally, the Member was deposited on an inner neritic to shallow-marine platform setting. It is interpreted as a good-quality reservoir rock which has been established from high total porosity (15–22 %), good effective porosity (8–13 %), and variable shale volume (24–31 %). A number of high hydrocarbon saturation zones (exceeding 55 %) are detected through intervals having S _w?<?60 % and S _hm?>?20 % which have been considered as economic oil producers. The storage and pay capacities of the reservoir intervals have been resolved. The obtained results were confirmed from the available data of core analyses and production tests. The Upper Qishn Clastic Member reveals promising reservoir characteristics which should be taken into consideration during future development of the oilfields in the area.     

Bacterial degradation of crude oil: Comparison of field and experimental data

It has been reported that the composition of crude oils in the subsurface may be altered by bacterial action to the extent that oil correlations (Winters and Williams, 1969) and the value of the crude (Evans et al., 1971) are severely affected. Experimental documentation of these effects is provided by this study.A crude oil was degraded in a 21-day laboratory experiment by a culture of four aerobic bacteria isolated from an oil-contaminated soil. The progress of the experiment was measured by the changes induced in the chemical composition of the oil fraction boiling above 270°C. These changes were similar to the variations in composition found in the MC5 oils of Saskatchewan, Canada.Normal paraffins through to at least nC₃₄ were severely depleted although the attack was temporarily blocked at nC₂₅ (Jobson et al., 1972). The position of this blockage is a function of the isolate employed. The isoprenoids, pristane and phytane, were metabolised after the disappearance of the n-paraffins. Lower-ring naphthenes and aromatics were attacked at the same time as the lighter normal paraffins and before the heavier ones.The more condensed cyclic hydrocarbons were apparently unaffected. Additional non-hydrocarbon NSO, and particularly asphaltene (both defined under section “Methods”), compounds were formed by the metabolism of the hydrocarbons.The residual oil after attack was heavier by approximately 30° API than the initial crude oil.     

Improved understanding of the petroleum systems of the Niger Delta Basin,Nigeria

Biomarker and n-alkane compound specific stable carbon isotope analyses (CSIA) were carried out on 58 crude oil samples from shallow water and deepwater fields of the Niger Delta in order to predict the depositional environment and organic matter characteristics of their potential source rocks. Using a source organofacies prediction approach from oil geochemistry, the presence in the western deepwater oils relatively abundant C₂₇ steranes, C₃₀ 24-n-propyl cholestane, low oleanane index, relatively low pr/ph ratios, gammacerane, and positive to nearly flat C₁₂–C₃₀ n-alkane compound specific stable carbon isotope profiles, suggests that the source facies that expelled these oils contain significant marine derived organic matter deposited under sub-oxic and stratified water column conditions. This contrasts with the terrigenous organic matter dominated source rocks accepted for shallow water Niger Delta oils. Oils in the shallow water accumulations can be separated into terrigenous and mixed marine-terrigenous families. The terrigenous family indicates expulsion from source rock(s) containing overwhelmingly higher plant source organic matter (average oleanane index = 0.48, high C₂₉ steranes) as well as having negative sloping n-alkane isotope profiles. Oxic source depositional conditions (pr/ph > 2.5) and non-stratified conditions (absence to low gammacerane content) are inferred for the terrigenous family. The mixed marine-terrigenous family has biomarker properties that are a combination of the deepwater and terrigenous shallow water oils. Bitumen extracts of the sub-delta Late Cretaceous Araromi Formation shale in the Dahomey Basin are comparable both molecularly and isotopically to the studied western deepwater oil set, but with an over all poor geochemical correlation. This poor geochemical match between Araromi shale and the western deepwater oils does not downgrade the potential of sub-delta Cretaceous source rock contribution to the regional oil charge in the deepwater Niger Delta.     

Isolation,biodegradation ability and molecular detection of hydrocarbon degrading bacteria in petroleum samples from a Brazilian offshore basin

The detection of microorganisms with potential for biodeterioration and biodegradation in petroleum fields is of great relevance, since these organisms may be related to a decrease in petroleum quality in the reservoirs or damage in the production facilities. In this sense, petroleum formation water and oil samples were collected from the Campos Basin, Brazil, with the aim of isolating microorganisms and evaluating their ability to degrade distinct classes of hydrocarbon biomarkers (9,10-dihydrophenanthrene, phytane, nonadecanoic acid and 5α-cholestane). Twenty eight bacterial isolates were recovered and identified by sequencing their 16S rRNA genes. Biodegradation assays revealed that bacterial metabolism of hydrocarbons occurred through reactions based on oxidation, carbon–carbon bond cleavage and generation of new bonds or by the physical incorporation of hydrocarbons into microbial cell walls. Based on the biodegradation results, selective PCR-based systems were developed for direct detection in petroleum samples of bacterial groups of interest, namely Bacillus spp., Micrococcus spp., Achromobacter xylosoxidans, Dietzia spp. and Bacillus pumilus. Primer sets targeting 16S rRNA genes were designed and their specificity was confirmed in silico (i.e. computational analysis) and in PCR reactions using DNA from reference strains as positive and negative controls. Total DNA from oil was purified and the amplification tests revealed the presence of the target bacteria in the samples, unraveling a significant potential for petroleum deterioration in the reservoirs sampled, once proper conditions are present for hydrocarbon degradation. The application of molecular methods for rapid detection of specific microorganisms in environmental samples would be valuable as a supporting tool for the evaluation of oil quality in production reservoirs.     

Impact of various concentrations of crude oil on fungal populations of soil

The effect of various concentrations of crude oil on fungal populations of soil was investigated for a period of 18 weeks using standard methods. Total fungal counts ranged from 26 to 143 χ 10² cfu/gsoil while counts of petroleum-utilizing fungi ranged from 2 to 102 χ 10² cfu/g soil. Analysis of variance and randomized complete block design of counts of total fungi and petroleum-utilizers showed high significant difference between the control and the oil treated soils at p? 0.05 level. Counts of petroleum-utilizers expressed as a percentage of the corresponding total fungal count of the soils ranged from 4.7 % to 58.8 %. Species of fourteen fungal genera were isolated from the soils. These include Alternaria,Aspergillus, Candida, Cephalosporium, Cladosporium, Fusarium, Geotrichum, Mucor, Penicillium, Rhizopus, Rhodotolura, Saccharomyces, Torulopsis and Trichoderma. Of these, ten were petroleum-utilizers and were all isolated from the control and 1 % oil treated soils. Only seven genera were isolated from the 5 % oil treated soil out of which five genera were petroleum-utilizers in both the 3 % and 5 % treatment. The decreasing order of occurrence of a variety of fungal genera (fungal diversity) of both total fungi and petroleum-utilizers is 0 % (control) > 0.5 % > 1 % > 3 % > 5 %. This order of fungal diversity is a reverse of the decreasing order of fungal counts of these same soils. This showed that higher concentrations of crude oil has adverse effect on fungal diversity while enhancing the population of a fewer fungi.     

Water lily (<Emphasis Type="Italic">Nymphaea</Emphasis> sp.): an alternative organic amendment for treatment of hydrocarbon-contaminated soil by chemical–biological stabilization

The chemical–biological stabilization technology has been employed in several successful studies using sugarcane cachasse as the organic amendment. However, in some petroleum-producing areas, there are no sugar mills nearby (which is the source of this material), and the cost of transport to the contaminated site is prohibitive. Therefore, water lily, which is considered a weedy plant in many tropical and subtropical areas, was evaluated as an alternative. In 3-month experiment, water lily was compared (with and without addition of molasses) with cachasse for the treatment of clayey sediment contaminated with > 6% extra-heavy crude oil. All treatments resulted in a reduction in the hydrocarbon concentration of 15–23%, without significant differences (P > 0.05). During this process, the pH was reduced to the 7–7.5 range and water repellency (molarity ethanol drop) to 3.5–3.6 M. Also, field capacity increased to 36.3–38.5% humidity, establishing adequate conditions for the development of vegetation at this site. Likewise, toxicity was reduced to practically null (Vibrio fischeri bioassay), and hydrocarbons in leachates were reduced to 3.4–4.3 mg/l, conditions adequate for the protection of groundwater and human health in rural areas. This study confirms that water lily is an adequate substitute for the application of this treatment method for hydrocarbon-contaminated sites that are far from sugar production areas.     

Phenolics occurrence in surface water of the Dniester river basin (West Ukraine): natural background and industrial pollution

Phenolics’ occurrence in surface water of the Dniester river basin (West Ukraine) with the definition of the natural background is studied. The main attention is given to the Upper Dniester basin and its tributary Stryj as the parts of the Sub-Carpathian oil- and gas province with the numerous objects of oil industry. The total amount of phenolics in water is studied. Phenolics’ concentrations from the first micrograms to the first milligrams per litre have been found in the surface water of the region. The natural background is defined as 0.012 mg l⁻¹ for the areas out of the industrial influence. The anthropogenic part of phenolics is caused mainly by oil industry. The oil-producing objects provide the main phenolics’ releases in the region, due to the low protection level of mechanical facilities as well as to breach of technological norms on the oil-extracting objects. A man-made pollution of the basin water has a regional character and the natural self-purification processes seem to be insufficient for its neutralisation on the plains in particular.     

Leaf dipping as an environmental screening measure to test chemical efficacy against Bemisia tabaci on poinsettia plants

The screening of potential chemicals for control of Bemisia tabaci on poinsettia (Euphorbia pulcherrima c.v. Lilo Pink) plants using a leaf dipping technique was investigated. All relevant B. tabaci lifestages (eggs, larvae and adults) were investigated. In leaf dipping tests, Certis spraying oil was the only compound to show potential to be used as a control agent against B. tabaci eggs, with 81% obtained mortality. Oberon resulted in no mortality of B. tabaci eggs in the reported experiments. Leaf dipping against second instar and adult B. tabaci proved more effective for all chemical products. Dipping techniques using Majestik, Certis spraying oil and Agri- 50 E all resulted in high second instar larval mortality (93, 87 and 85.5 %, respectively). Certis spraying oil again resulted in a high efficacy against adult B. tabaci with 100 % mortality obtained. The potential of the various chemicals to be incorporated into integrated pest management strategies for the control of B. tabaci is discussed.     

The operational nature of acidic functional group analyses and its impact on mathematical descriptions of acid-base equilibria in humic substances

Calorimetric and potentiometric titrations have been used to evaluate direct and indirect methods of analysis for the carboxyl content of aquatic humic substances. The effects of cation binding, base strength, and removal of humic reaction products on carboxyl content values have been examined. The results suggest that the most appropriate analytical method for determination of an operationally defined carboxyl content would utilize a weak base that does not contain a polyvalent cation in an indirect titration on a reaction mixture from which all humic reaction products have been removed prior to titration. Titration data have been analyzed in terms of a formation function (δn_OH) and in terms of operationally defined ‘mass action quotients’ ($\text{K}\text{?}$). The dependence of $\text{K}\text{?}$ on pH is more readily and realistically described if no artificial differentiation of carboxyl groups into two or more sub-groups is imposed upon the data.