Interactions between clay minerals and siderophores affect the respiration of Histoplasma capsulatum.

The reduction in the respiration of Histoplasma capsulatum in broth culture caused by montmorillonite appeared to be the result, in part, of the interference by the clay with the iron nutrition of the fungus. This interference was apparently the result of the adsorption by the clay of the iron-transporting siderophore (deferricoprogen B) produced by the fungus, as the reduction in respiration was partially alleviated by the addition of foreign siderophores. Neither kaolinite nor attapulgite (palygorskite) appeared to adsorb significant amounts of the siderophores, probably because of the low cation exchange capacity and specific surface area of kaolinite and the inaccessibility of adsorption sites in the fibrous attapulgite. These observations, in addition to the adhesion of montmorillonite to the hyphae, suggest mechanisms that may explain the discrete geographic distribution of this fungus, which is pathogenic to humans and which has been isolated essentially only from soils that do not contain montmorillonite.     

Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of Histoplasma capsulatum.

The respiration of three phenotypes of Histoplasma capsulatum, the causal agent of histoplasmosis in humans, was markedly reduced by low concentrations of montmorillonite but was reduced less by even higher concentrations of kaolinite or attapulgite (palygorskite). The reduction in respiration followed a pattern that suggested saturation-type kinetics: an initial sharp reduction that occurred with low concentrations of clay (0.01 to 0.5% [wt/vol]), followed by a more gradual reduction with higher concentrations (1 to 8%). Increases in viscosity (which could impair the movement of O2) caused by the clays were not responsible for the reduction in respiration, and the clays did not interfere with the availability of nutrients. Scanning electron microscopy after extensive washing showed that the clay particles were tightly bound to the hyphae, suggesting that the clays reduced the rate of respiration of H. capsulatum by adhering to the mycelial surface and, thereby, interfered with the movement of nutrients, metabolites, and gases across the mycelial wall.     

Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of Histoplasma capsulatum

The respiration of three phenotypes of Histoplasma capsulatum, the causal agent of histoplasmosis in humans, was markedly reduced by low concentrations of montmorillonite but was reduced less by even higher concentrations of kaolinite or attapulgite (palygorskite). The reduction in respiration followed a pattern that suggested saturation-type kinetics: an initial sharp reduction that occurred with low concentrations of clay (0.01 to 0.5% [wt/vol]), followed by a more gradual reduction with higher concentrations (1 to 8%). Increases in viscosity (which could impair the movement of O2) caused by the clays were not responsible for the reduction in respiration, and the clays did not interfere with the availability of nutrients. Scanning electron microscopy after extensive washing showed that the clay particles were tightly bound to the hyphae, suggesting that the clays reduced the rate of respiration of H. capsulatum by adhering to the mycelial surface and, thereby, interfered with the movement of nutrients, metabolites, and gases across the mycelial wall.     

Specificity of virus adsorption to clay minerals

Competitive adsorption studies indicated that reovirus type 3 and coliphage T1 did not share common adsorption sites on kaolinite and montmorillonite. Compounds in the minimal essential medium (e.g., fetal bovine serum, amino acids) in which the reovirus was maintained blocked adsorption of coliphage T1 to kaolinite and partially to montmorillonite in synthetic estuarine water, but they had no effect on coliphage adsorption to montmorillonite in distilled water or on the adsorption of the reovirus to either clay. The blockage of positively charged sites on kaolinite or montmorillonite by treatment of the clays with sodium metaphosphate or with the supernatants from montmorillonite or kaolinite, respectively, had no effect on adsorption of the reovirus. These data indicate that there was a specificity in adsorption sites for mixed populations of reovirus type 3 and coliphage T1 and emphasize the importance of using more than one type of virus, especially in combination, to predict virus behavior (e.g., adsorption, loss of infectivity) in soils and sediments containing clay minerals.     

Effect of proteins on reovirus adsorption to clay minerals

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Effect of proteins on reovirus adsorption to clay minerals.

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area.

The adsorption of reovirus to clay minerals has been reported by several investigators, but the mechanisms defining this association have been studied only minimally. The purpose of this investigation was to elucidate the mechanisms involved with this interaction. More reovirus type 3 was adsorbed, in both distilled and synthetic estuarine water, by low concentrations of montmorillonite than by comparable concentrations of kaolinite containing a mixed complement of cations on the exchange complex. Adsorption to the clays was essentially immediate and was correlated with the cation-exchange capacity of the clays, indicating that adsorption was primarily to negatively charged sites on the clays. Adsorption was greater with low concentrations of clays in estuarine water than in distilled water, as the higher ionic strength of the estuarine water reduced the electrokinetic potential of both clay and virus particles. The addition of cations (as chloride salts) to distilled water enhanced adsorption, with divalent cations being more effective than monovalent cations and 10(-2) M resulting in more adsorption than 10(-3) M. Potassium ions suppressed reovirus adsorption to montmorillonite, probably by collapsing the clay lattices and preventing the expression of the interlayer-derived cation-exchange capacity. More virus was adsorbed by montmorillonite made homoionic to various mono-, di-, and trivalent cations (except by montmorillonite homoionic to potassium) than by comparable concentrations of kaolinite homoionic to the same cations. The sequence of the amount of adsorption to homoionic montmorillonite was Al greater than Ca greater than Mg greater than Na greater than K; the sequence of adsorption to kaolinite was Na greater than Al greater than Ca greater than Mg greater than K. The constant partition-type adsorption isotherms obtained when the clay concentration was maintained constant and the virus concentration was varied indicated that a fixed proportion of the added virus population was adsorbed, regardless of the concentration of infectious particles. A heterogeneity within the reovirus population was indicated.     

Adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area

The adsorption of reovirus to clay minerals has been reported by several investigators, but the mechanisms defining this association have been studied only minimally. The purpose of this investigation was to elucidate the mechanisms involved with this interaction. More reovirus type 3 was adsorbed, in both distilled and synthetic estuarine water, by low concentrations of montmorillonite than by comparable concentrations of kaolinite containing a mixed complement of cations on the exchange complex. Adsorption to the clays was essentially immediate and was correlated with the cation-exchange capacity of the clays, indicating that adsorption was primarily to negatively charged sites on the clays. Adsorption was greater with low concentrations of clays in estuarine water than in distilled water, as the higher ionic strength of the estuarine water reduced the electrokinetic potential of both clay and virus particles. The addition of cations (as chloride salts) to distilled water enhanced adsorption, with divalent cations being more effective than monovalent cations and 10(-2) M resulting in more adsorption than 10(-3) M. Potassium ions suppressed reovirus adsorption to montmorillonite, probably by collapsing the clay lattices and preventing the expression of the interlayer-derived cation-exchange capacity. More virus was adsorbed by montmorillonite made homoionic to various mono-, di-, and trivalent cations (except by montmorillonite homoionic to potassium) than by comparable concentrations of kaolinite homoionic to the same cations. The sequence of the amount of adsorption to homoionic montmorillonite was Al greater than Ca greater than Mg greater than Na greater than K; the sequence of adsorption to kaolinite was Na greater than Al greater than Ca greater than Mg greater than K. The constant partition-type adsorption isotherms obtained when the clay concentration was maintained constant and the virus concentration was varied indicated that a fixed proportion of the added virus population was adsorbed, regardless of the concentration of infectious particles. A heterogeneity within the reovirus population was indicated.     

Binding of the protoxin and toxin proteins ofBacillus thuringiensis subsp.kurstaki on clay minerals

The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (M_r=132 kDa) and toxins (M_r=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na₂CO₃, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni.     

Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.     

Adsorption/Desorption of toluene on clay minerals

Adsorption/desorption of toluene on montmorillonite, illite, and kaolinite was studied using the batch equilibrium method. The isotherms measured fit the Freundlich equation (r² >0.95). Montmorillonite adsorbed more toluene than illite or kaolinite; the adsorption of toluene on illite and kaolinite was not significantly different. Adsorption of toluene by montmorillonite showed an exponential increase as the ratio of toluene to clay was increased from 5 to 100. The rate studies showed that 62% of the adsorption was completed within 6 h. A rapid desorption was observed initially, followed by slow desorption after 1 h. The desorption rate decreased as the time of adsorption was increased. Almost all of the adsorbed toluene was extracted with water from the clay when the adsorption time was 0.1 h, but only 61% of the toluene could be desorbed when the adsorption time was 24 h.     

Abiotic factors affecting the toxicity of lead to fungi.

The toxicity of lead (Pb) to fungi in pure culture was influenced by several abiotic factors: pH, inorganic anions, clay minerals, and particulate (humic acid) and soluble organic matter. The toxicity of Pb was potentiated under acidic conditions (pH 5 and 6), and phosphate or carbonate anions reduced the toxicity, apparently as a result of the formation of sparingly soluble Pb salts. Clay minerals (montmorillonite greater than attapulgite greater than kaolinite) and particulate humic acid protected against the toxicity of Pb, presumably as the result of sorption, by cation exchange of the Pb to the exchange complexes, which reduced its availability for uptake by the fungi. Soluble organics, such as tryptone, yeast extract, cysteine, succinic acid, and increasing concentrations of neopeptone, also reduced the toxicity of Pb.     

Abiotic factors affecting the toxicity of lead to fungi.

The toxicity of lead (Pb) to fungi in pure culture was influenced by several abiotic factors: pH, inorganic anions, clay minerals, and particulate (humic acid) and soluble organic matter. The toxicity of Pb was potentiated under acidic conditions (pH 5 and 6), and phosphate or carbonate anions reduced the toxicity, apparently as a result of the formation of sparingly soluble Pb salts. Clay minerals (montmorillonite greater than attapulgite greater than kaolinite) and particulate humic acid protected against the toxicity of Pb, presumably as the result of sorption, by cation exchange of the Pb to the exchange complexes, which reduced its availability for uptake by the fungi. Soluble organics, such as tryptone, yeast extract, cysteine, succinic acid, and increasing concentrations of neopeptone, also reduced the toxicity of Pb.     

Interaction between bacteriophage PBS1 and clay minerals and transduction of Bacillus subtilis by clay-phage complexes

Bacteriophage PBS1 of Bacillus subtilis was rapidly adsorbed on montmorillonite (M) and kaolinite (K), and adsorption was maximal after 30 min on both clays. There was no correlation between adsorption and the cation exchange capacity of the clays. Studies with sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) indicated that positively charged sites on K were primarily responsible for the adsorption of the phage, whereas other mechanisms appeared to be involved in adsorption of the phage on M. X-ray diffraction and electron microscopic analyses showed that the phage partially intercalated M. Survival of the phage was increased by adsorption on the clays, and adsorbed phage maintained its ability to transduce bacterial cells for at least 30 days (the longest time studied) after the preparation of the clay–phage complexes. Electron microscopic observations indicated that transduction by the clay–phage complexes was primarily the result of the phage detaching from the clays in the presence of host cells.     

Interaction of Rhizobium sp. with Toxin-Producing Fungus in Culture Medium and in a Tropical Soil

Experiments were conducted to determine the influence of a toxin-producing fungus on a rhizobial population in yeast-mannitol medium and in a tropical soil. The fungus, which was isolated from a highly weathered soil (Tropeptic Eutrustox), was identified as a Metarhizum sp. The density of rhizobial populations established in yeast-mannitol medium in the absence of the fungus was 10⁵ times higher than that established in its presence. However, the fungus did not exert similar antagonistic influence on the rhizobial population incubated with it in the sterilized test soil. Rhizobial growth activity in yeast-mannitol medium was also insensitive to the presence of the fungus when the medium was amended with 1% (wt/vol) kaolinite or montmorillonite. The results suggest that clay minerals may be responsible for protecting rhizobia against toxin-producing fungi in soil.     

Studies on Plant Viruses-soil Colloids Interactions

Stability and infectivity of cucumber mosaic virus, strain D (CMV-D), associations with kaolinite and montmorillonite were determined, as affected by: i) nature of clay minerals; ii) nature of clay saturating cations; iii) exposure to dissociating salt solutions (2 M LiCl). Infectivity experiments carried out with sediments following centrifugation of the virus-clay mixtures (sd fractions), showed that, in absence of LiCl, the highest values were obtained with kaolinite, in the order Li⁺= K⁺ > NH₄⁺= Mg⁺⁺ > Na⁺ > Ca⁺⁺ clay saturating cations, ranging between 91 and 30 % of the untreated control, whereas comparable montmorillonite fractions gave infectivity values with all cations about 10–15 % of the control. In presence of 2 M LiCl, montmorillonite preserved infectivity of the same fraction (Lsd fraction), which, in the case of Li⁺- or Ca⁺⁺ -saturated samples, was higher when compared with the corresponding sd values, thus revealing for these cations an amplifying effect on infection. This did not occur with kaolinite which, however, gave a Lsd fraction more infectious than the other clay. The results confirmed that clay minerals preserve infectivity of virus preparations exposed to critical conditions, thus providing an explanation for the persistence in soil of infectivity of viruses which are normally not soil-borne. Under appropriate soil conditions these viruses may form complexes with clay minerals thus retaining an infectivity which may be enhanced by addition of cations as those contained in fertilizers.     

Biodegradation of crude oil saturated fraction supported on clays

The role of clay minerals in crude oil saturated hydrocarbon removal during biodegradation was investigated in aqueous clay/saturated hydrocarbon microcosm experiments with a hydrocarbon degrading microorganism community. The clay minerals used for this study were montmorillonite, palygorskite, saponite and kaolinite. The clay mineral samples were treated with hydrochloric acid and didecyldimethylammonium bromide to produce acid activated- and organoclays respectively which were used in this study. The production of organoclay was restricted to only montmorillonite and saponite because of their relative high CEC. The study indicated that acid activated clays, organoclays and unmodified kaolinite, were inhibitory to biodegradation of the hydrocarbon saturates. Unmodified saponite was neutral to biodegradation of the hydrocarbon saturates. However, unmodified palygorskite and montmorillonite were stimulatory to biodegradation of the hydrocarbon saturated fraction and appears to do so as a result of the clays’ ability to provide high surface area for the accumulation of microbes and nutrients such that the nutrients were within the ‘vicinity’ of the microbes. Adsorption of the saturated hydrocarbons was not significant during biodegradation.     

Adsorption of coliphages T1 and T7 to clay minerals.

Coliphages T1 and T7 of Escherichia coli were absorbed by kaolinite (K) and montmorillonite (M). Maximum adsorption of T7 (96%) to M was greater than that of T1 (84%), but the adsorption of both coliphages to K was the same (99%). Positively charged sites (i.e., anion exchange sites) on the clays appeared to be primarily responsible for the adsorption of T1 to K but only partially responsible for the adsorption of T1 to M; equilibrium adsorption isotherms of T1 to K and M did not show a correlation between adsorption and the cation exchange capacity of the clays, and the reduction in adsorption caused by sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) was more pronounced with K than with M. The equilibrium adsorption isotherms of T7 to K and M suggested a correlation between adsorption and the cation exchange capacity of the clays. However, studies with sodium metaphosphate indicated that T7 also adsorbed to positively charged sites on the clays, especially on K. Adsorption of the coliphages to positively charged sites was greater with K than with M, probably because the ratio of positively charged sites to negatively charged sites was greater on K than on M.     

Clay-Nucleic Acid Complexes: Characteristics and Implications for the Preservation of Genetic Material in Primeval Habitats

The equilibrium adsorption of three nucleic acids: chromosomal DNA, supercoiled plasmid DNA, and 25S rRNA, on the clay minerals, montmorillonite (M) and kaolinite (K), were studied. Adsorption of the nucleic acid on the clays was rapid and maximal after 90 min of contact time. Chromosomal DNA was adsorbed to a greater extent than plasmid DNA and RNA, and the adsorption was also greater on M than on K. Adsorption isotherms were of the L type, and a plateau was reached with all the complexes, with the exception of chromosomal DNA adsorbed on M. To determine where nucleic acids are adsorbed on clay minerals and the nature of the interaction, complexes were studied by X-ray diffraction (X-RD), electron microscopy, and Fourier transform infrared (FT-IR) spectroscopy. X-RD showed that nucleic acids did not penetrate the clay, indicating that the adsorption occurred primarily on the external surfaces of clay particles, as also suggested by electron microscopy observations. FT-IR spectra of clay-tightly bound nucleic acid complexes showed absorption bands that indicate a variation of the nucleic acids status as a consequence of their adsorption on clay. Data obtained suggested that the formation of clay-nucleic acid complex could have an important role in the preservation of genetic material in primeval habitats.     

Genetic transformation of Bacillus subtilis cells in the presence of clay minerals montmorillonite and kaolinite

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competent Bacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.