The document summarizes research into what makes certain natural clays antibacterial. Key points:
- Antibacterial clays contain nano-scale illite-smectite and reduced iron phases that buffer water pH and oxidation state, promoting Fe2+ solubility.
- E. coli exposed to an antibacterial clay's leachate accumulate high intracellular concentrations of Fe and P, supporting a role for polyphosphate or phospholipids in regulating Fe2+.
- Excess Fe2+ overwhelms cell membrane regulatory proteins, then oxidizes inside cells to Fe3+, producing lethal hydroxyl radicals through the Fenton reaction.
- High-resolution SEM images show the nano-scale crystals and Fe
Poly-훽-hydroxybutyrate degradation by Aspergillus fumigates isolated from soi...IOSRJPBS
Poly-β-hydroxybutyrate acid (PHB) is thermoplastic biopolymers, synthesized by some bacterial genera under stress conditions. The degradation of PHB was detected in Petri dish by formation of a clear zone around the fungal colonies due to production of depolymerase enzyme which has interesting role in PHB degradation process. The most active PHB degrader fungi was selected and identified as Aspergillus fumigates using morphological characters. The highest PHB degradation in Petri dish by A. fumigates was at pH 5, 30ᵒC and 7days. In liquid medium, degradation by A. fumigates was studied using enzyme assay method (U/ml). All the experiments were performed enzyme activities were monitored. After 3 days of incubation, maximum PHB depolymerase production was at pH 5 and 30°C. In conclusion, PHB can be degraded in solid and liquid medium using fungal depolymerase enzyme.
An Investigation Into The Mechanisms Underlying Enhanced Biosulphidogenesis I...iosrjce
Anthropogenic activities like mining, processes of metallurgy and other chemical industries lead to
the discharge of a high amount of sulphate into the environment that causes serious problems to human health.
This paper illustrates the employment of thermophilic sulphate reducing bacteria for biosulphidogenesis. Two
different species have been isolated from hot water spring of Vajreshwari and Ganeshpuri,Thane, Maharashtra,
INDIA.The mechanism involved in biosulphidogenesis includes production of specific protein as well as
liberation of some extracellular polymeric compound (EPS) e.g. proteins, carbohydrate, acids etc. that are
produced during the microbial cell metabolism. These compounds plays an important role in the faster
reduction of sulphate and decrease in production rate of sulphide.The isolate was found to be of genus
Bacillusand type strain was found to be subtilis Zankar and licheniformis Sonali. The strain sequence were
deposited in NCBI database with accession number KJ939324 and KJ939325 respectively. The result highlights
the potential use of these organism in biosulphidogenesis.
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Phylogenetic Analysis of the Potential Microorganism for Remediation of Heavy...CSCJournals
The present research work has been carried out to study the waste disposal contaminated site for its physico chemical and microbial characterization and identification of potential microorganism capable of bioaccumulation and biodegradation of heavy metals. The ambient conditions present in the metal contaminated environment shows the values: pH(5.4),temperature(30°C), moisture(11.71%), nutrients; Nitrogen(0.2mg/l), phosphorus(22.65mg/l) and sulphur(559.3mg/l) respectively. The biological parameters studied indicate Dissolved oxygen (7.4mg/l), Biological oxygen demand (3.8 mg/l), Chemical oxygen demand (64.6 mg/l). The microbial consortium identified was found to survive and multiply in the present environmental conditions. Microbial consortium was sequenced and compared using Bioinformatics tools like BLAST, ClustalW and PHYLIP. In order to identify potential microorganism, microbial consortium was exposed to increasing concentrations of heavy metals viz 5mg/l, 25mg/l, 50mg/l, 100mg/l up to 800mg/l with special reference to Iron. At a concentration of 500mg/l, only one microorganism was found survived and multiplied. This shows that potential microorganism was only survived at higher concentration of iron. The 16SrRNA sequence and phylogenetic tree characterized the organism as Klebsiella pneumoniae, which was also confirmed by biochemical tests. The potential microorganism identified by BLAST technique can be used for remediation of the heavy metal from contaminated environment.
Poly-훽-hydroxybutyrate degradation by Aspergillus fumigates isolated from soi...IOSRJPBS
Poly-β-hydroxybutyrate acid (PHB) is thermoplastic biopolymers, synthesized by some bacterial genera under stress conditions. The degradation of PHB was detected in Petri dish by formation of a clear zone around the fungal colonies due to production of depolymerase enzyme which has interesting role in PHB degradation process. The most active PHB degrader fungi was selected and identified as Aspergillus fumigates using morphological characters. The highest PHB degradation in Petri dish by A. fumigates was at pH 5, 30ᵒC and 7days. In liquid medium, degradation by A. fumigates was studied using enzyme assay method (U/ml). All the experiments were performed enzyme activities were monitored. After 3 days of incubation, maximum PHB depolymerase production was at pH 5 and 30°C. In conclusion, PHB can be degraded in solid and liquid medium using fungal depolymerase enzyme.
An Investigation Into The Mechanisms Underlying Enhanced Biosulphidogenesis I...iosrjce
Anthropogenic activities like mining, processes of metallurgy and other chemical industries lead to
the discharge of a high amount of sulphate into the environment that causes serious problems to human health.
This paper illustrates the employment of thermophilic sulphate reducing bacteria for biosulphidogenesis. Two
different species have been isolated from hot water spring of Vajreshwari and Ganeshpuri,Thane, Maharashtra,
INDIA.The mechanism involved in biosulphidogenesis includes production of specific protein as well as
liberation of some extracellular polymeric compound (EPS) e.g. proteins, carbohydrate, acids etc. that are
produced during the microbial cell metabolism. These compounds plays an important role in the faster
reduction of sulphate and decrease in production rate of sulphide.The isolate was found to be of genus
Bacillusand type strain was found to be subtilis Zankar and licheniformis Sonali. The strain sequence were
deposited in NCBI database with accession number KJ939324 and KJ939325 respectively. The result highlights
the potential use of these organism in biosulphidogenesis.
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Phylogenetic Analysis of the Potential Microorganism for Remediation of Heavy...CSCJournals
The present research work has been carried out to study the waste disposal contaminated site for its physico chemical and microbial characterization and identification of potential microorganism capable of bioaccumulation and biodegradation of heavy metals. The ambient conditions present in the metal contaminated environment shows the values: pH(5.4),temperature(30°C), moisture(11.71%), nutrients; Nitrogen(0.2mg/l), phosphorus(22.65mg/l) and sulphur(559.3mg/l) respectively. The biological parameters studied indicate Dissolved oxygen (7.4mg/l), Biological oxygen demand (3.8 mg/l), Chemical oxygen demand (64.6 mg/l). The microbial consortium identified was found to survive and multiply in the present environmental conditions. Microbial consortium was sequenced and compared using Bioinformatics tools like BLAST, ClustalW and PHYLIP. In order to identify potential microorganism, microbial consortium was exposed to increasing concentrations of heavy metals viz 5mg/l, 25mg/l, 50mg/l, 100mg/l up to 800mg/l with special reference to Iron. At a concentration of 500mg/l, only one microorganism was found survived and multiplied. This shows that potential microorganism was only survived at higher concentration of iron. The 16SrRNA sequence and phylogenetic tree characterized the organism as Klebsiella pneumoniae, which was also confirmed by biochemical tests. The potential microorganism identified by BLAST technique can be used for remediation of the heavy metal from contaminated environment.
Metals accumulation and As releasing during interaction of clay and iron mine...Agriculture Journal IJOEAR
Abstract— The soil and sediment samples with different content of metals and clay minerals were investigated during bioleaching. The increasing of clay and metal concentrations with decreasing particle size were found both in contaminated soil and sediment. Heterotrophic bioleaching of the iron rich clay fractions from the soil and the sediment were evaluated for his effectiveness in the cycling of iron bound As by consuming organic nutrients. The treatment involved the use of the indigenous bacteria, whose activity was combined with the chelating strength of EDDS, SDS, Na4P2O7 and fertilizers. Heterotrophic bacteria caused decomposition of iron binding deposition as is adsorption on clay with followed dissolving of Fe mainly by the sediment bioleaching. The concentration of iron decreased by precipitation with As sorption after 19 days of sediment bioleaching. The Cu and Zn extraction was inhibited by bioleaching during the iron and arsenic dissolution and precipitation. By contrast, the additives 3mM Cu and 3mM Zn were applied into medium and thus affected the activity of soil resistant heterotrophic bacteria with followed increasing of the iron and arsenic extraction by the soil clay bioleaching. Therefore, this study confirmed the soil and sediment bioleaching in Fe or As releasing efficiency under different conditions regulated by indigenous bacteria. The bioleaching can be a suitable technology for As removal from the untreated soil and sediment by stimulation of the resistant bacteria activity. The separation of clays from the soil and sediment samples did not decreased of toxic element limits because clay and iron minerals coated on coarse silicate particles and the clay fraction is bearers of metals which contaminate the soil and sediment environment.
Antioxidant and Antimicrobial Activities Of Algerian Populus Nigra L. Buds Ex...bioejjournal
This study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of
extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against
eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit
antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC=
90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The
antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALGERIAN POPULUS NIGRA L. BUDS EX...bioejjournal
his study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC= 90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
Plant growth promoting characterization of soil bacteria isolated from petrol...Agriculture Journal IJOEAR
Abstract— Contaminant-degrading bacteria can be included among the plant-growth promoting bacteria; because the presence of contaminants, in general produce negatively effects on plant’s growth; thus, the elimination of the inhibiting contaminants will benefit them. Although contaminant-degrading strains have been traditionally isolated from various environments; the number of studies that reported the isolation and identification of soil bacteria with contaminant- degrading abilities have increased. The aim of this study was to characterized microbial strains isolated from petroleum contaminated soil by plant growth promotion traits to recommend them as potential bioinoculants. In this work, five of the six soil isolates were classified as Indole Acetic Acid higher producers and only one of them as lower producer. Sporosarcina aquimarina strain -Q3 and Bacillus cereus strain +F2 tested in Axonopus affinis plantlets bioassay, showed that these isolates were the most effective promoters of this plant species; therefore, these soil bacteria with possible hydrocarbon degradation ability could be considered as potential bioinoculants and can be recommended with a practical importance for the rhizoremediation of petroleum contaminated sites and plant growth promotion.
Comparative Study on Antimicrobial Activity and Microbial Load of Alternanthe...Premier Publishers
The present study analyses the antimicrobial activity and the microbial load of an edible plant Alternanthera philoxeroides (Mart.) Griseb growing in polluted and unpolluted site. The plants were collected and tested against various Gram positive, Gram negative bacteria and fungi. Antimicrobial activity was performed with acetone, aqueous, chloroform, ethanol and petroleum ether extracts of aerial parts A. philoxeroides collected from polluted and unpolluted site that showed significant antimicrobial activity against tested bacterial and fungal organisms. The extracts were compared with standards like Amoxicillin for antibacterial activity and Ketoconazole for antifungal activity. The extracts showed remarkable antimicrobial activity as measured from the zone of inhibition and results were comparable with that of standard drugs against the organisms tested. The microbial load is also enumerated in the cooked and cooked refrigerated samples from polluted and unpolluted site. In conclusion, plant extract of A. philoxeroides collected from polluted site showed less antimicrobial activity and higher antimicrobial activity in unpolluted site. The ethanol extract showed higher activity when compared to other extracts. The microbial load is higher in cooked refrigerated sample when compared to cooked sample.
Application of rapid bioassay method for assessing its water purification by ...inventionjournals
Integral toxicity of four water samples taken from various sources, urban and rural environment was evaluated, and some of the properties of potassium ferrate K2FeO4 as the reagent for chemical purification of water were explored. These data allow to suggest bacterial luminescence based test system for practical use. Thetest system is suitable for rapid evaluation of toxicity of the used for water purification chemical agents, as well as for selection of their effective concentrations and for optimization of treatment time.
Metals accumulation and As releasing during interaction of clay and iron mine...Agriculture Journal IJOEAR
Abstract— The soil and sediment samples with different content of metals and clay minerals were investigated during bioleaching. The increasing of clay and metal concentrations with decreasing particle size were found both in contaminated soil and sediment. Heterotrophic bioleaching of the iron rich clay fractions from the soil and the sediment were evaluated for his effectiveness in the cycling of iron bound As by consuming organic nutrients. The treatment involved the use of the indigenous bacteria, whose activity was combined with the chelating strength of EDDS, SDS, Na4P2O7 and fertilizers. Heterotrophic bacteria caused decomposition of iron binding deposition as is adsorption on clay with followed dissolving of Fe mainly by the sediment bioleaching. The concentration of iron decreased by precipitation with As sorption after 19 days of sediment bioleaching. The Cu and Zn extraction was inhibited by bioleaching during the iron and arsenic dissolution and precipitation. By contrast, the additives 3mM Cu and 3mM Zn were applied into medium and thus affected the activity of soil resistant heterotrophic bacteria with followed increasing of the iron and arsenic extraction by the soil clay bioleaching. Therefore, this study confirmed the soil and sediment bioleaching in Fe or As releasing efficiency under different conditions regulated by indigenous bacteria. The bioleaching can be a suitable technology for As removal from the untreated soil and sediment by stimulation of the resistant bacteria activity. The separation of clays from the soil and sediment samples did not decreased of toxic element limits because clay and iron minerals coated on coarse silicate particles and the clay fraction is bearers of metals which contaminate the soil and sediment environment.
Antioxidant and Antimicrobial Activities Of Algerian Populus Nigra L. Buds Ex...bioejjournal
This study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of
extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against
eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit
antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC=
90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The
antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ALGERIAN POPULUS NIGRA L. BUDS EX...bioejjournal
his study is part of a goal to investigate chemical composition, antibacterial, antifungal and antioxidant activities of the flower buds extracts from the Algerian Polulus nigra L., which were collected from Djarifet - mansourah at Tlemcen city in the West Northern of Algeria. In organic extracts, tanins, flavonoïds, coumarins, alkaloids and terpenoïds were the principals secondary metabolites identified from the flower buds of black poplar. Antibacterial and antifungal activities of extracts were tested using agar-well diffusion method and micro-well determination of MIC assay against eleven bacteria and two Candida species. It was found that extracts of black poplar buds exhibit antibacterial and anticandidal activities with agar disk diffusion (7 to 43mm) and MIC methods (MIC= 90.33 µg/ml against several strains of bacteria and MIC=45.16 µg/ml against Candida albicans). The antioxidant effect of hydroalcoholic extract was evaluated using DPPH and FRAP assays. It was showed good and similar activity than ascorbic acid and BHA by DPPH method: IC50= 220µg/mL for hydroethanol extract.
Plant growth promoting characterization of soil bacteria isolated from petrol...Agriculture Journal IJOEAR
Abstract— Contaminant-degrading bacteria can be included among the plant-growth promoting bacteria; because the presence of contaminants, in general produce negatively effects on plant’s growth; thus, the elimination of the inhibiting contaminants will benefit them. Although contaminant-degrading strains have been traditionally isolated from various environments; the number of studies that reported the isolation and identification of soil bacteria with contaminant- degrading abilities have increased. The aim of this study was to characterized microbial strains isolated from petroleum contaminated soil by plant growth promotion traits to recommend them as potential bioinoculants. In this work, five of the six soil isolates were classified as Indole Acetic Acid higher producers and only one of them as lower producer. Sporosarcina aquimarina strain -Q3 and Bacillus cereus strain +F2 tested in Axonopus affinis plantlets bioassay, showed that these isolates were the most effective promoters of this plant species; therefore, these soil bacteria with possible hydrocarbon degradation ability could be considered as potential bioinoculants and can be recommended with a practical importance for the rhizoremediation of petroleum contaminated sites and plant growth promotion.
Comparative Study on Antimicrobial Activity and Microbial Load of Alternanthe...Premier Publishers
The present study analyses the antimicrobial activity and the microbial load of an edible plant Alternanthera philoxeroides (Mart.) Griseb growing in polluted and unpolluted site. The plants were collected and tested against various Gram positive, Gram negative bacteria and fungi. Antimicrobial activity was performed with acetone, aqueous, chloroform, ethanol and petroleum ether extracts of aerial parts A. philoxeroides collected from polluted and unpolluted site that showed significant antimicrobial activity against tested bacterial and fungal organisms. The extracts were compared with standards like Amoxicillin for antibacterial activity and Ketoconazole for antifungal activity. The extracts showed remarkable antimicrobial activity as measured from the zone of inhibition and results were comparable with that of standard drugs against the organisms tested. The microbial load is also enumerated in the cooked and cooked refrigerated samples from polluted and unpolluted site. In conclusion, plant extract of A. philoxeroides collected from polluted site showed less antimicrobial activity and higher antimicrobial activity in unpolluted site. The ethanol extract showed higher activity when compared to other extracts. The microbial load is higher in cooked refrigerated sample when compared to cooked sample.
Application of rapid bioassay method for assessing its water purification by ...inventionjournals
Integral toxicity of four water samples taken from various sources, urban and rural environment was evaluated, and some of the properties of potassium ferrate K2FeO4 as the reagent for chemical purification of water were explored. These data allow to suggest bacterial luminescence based test system for practical use. Thetest system is suitable for rapid evaluation of toxicity of the used for water purification chemical agents, as well as for selection of their effective concentrations and for optimization of treatment time.
In recent years, nanoparticles that have size of 1-100 nm is widely used for textile, pharmacy,
cosmetic and treatment of industrial wastewater. Producing and using of nanoparticles widely, causes
important accumulation in nature and toxicity on ecosystem. Knowledge of potential toxicity of nanoparticles is
limited. In this study, six different nanoparticles nano-zinc oxide, nano-silicon dioxide, nano-cerium oxide,
nano-aluminum oxide, nano-hafnium oxide, and nano-tantalum oxide which used commonly, were studied to
investigate toxic impacts on organisms. We studied nine different acute toxicity test (bacteria – Escherichia coli
(gram negative bacteria) ; bacteria – Bacillus cereus (gram positive bacteria) ; bacteria – Vibrio fischeri
(bioluminescences bacteria) ; methane Archae Bacteria ; yeast – Candida albicans ; mold – Aspergillus niger ;
algae – Chlorella sp. ; Crustacea – Daphnia magna ; lepistes - Poecillia reticula) for the effect of
nanoparticles to different trophic levels. In general, the most toxic nanoparticle is nano-zinc oxide and the least
toxic nanoparticle is nano-hafnium oxide. Among the used organisms in acute toxicity test; the most sensitive
organism is algae - Chlorella sp ;the most resistant organism is fish- Poecillia reticula.
Characterization of Bacteria Isolated from Tropical Soils of Puerto Rico
Oregon Blue clay ASU report
1. 1
What makes a clay antibacterial?
Lynda B. Williams t, *, David W. Metge tt, Dennis D. Eberl tt, Ronald W. Harvey tt,
Amanda G. Turner t, Panjai Prapaipong t, Amisha T. Poret-Peterson t
t School of Earth & Space Exploration, Arizona State University, Tempe, AZ 85287
tt U.S. Geological Survey, 3215 Marine St., Suite E127, Boulder, CO, 80303
Keywords: biogeochemistry, antibacterial clay, illite-smectite, Fenton reaction
Corresponding author:
Lynda B. Williams, School of Earth and Space Exploration, Arizona State University, Tempe,
AZ 85282
2. 2
ABSTRACT
Natural clays have been used in ancient and modern medicine, but the mechanism(s) that
make certain clays lethal against bacterial pathogens has not been identified. We have compared
the depositional environments, mineralogy’s and chemistries of clays that exhibit antibacterial
effects on a broad spectrum of human pathogens including antibiotic resistant strains. Natural
antibacterial clays contain nano-scale (<200nm), illite-smectite and reduced iron phases. The role
of clay minerals in the bactericidal process is to buffer the aqueous pH and oxidation state to
conditions that promote Fe2+ solubility.
Chemical analyses of E. coli killed by aqueous leachates of an antibacterial clay show that
intracellular concentrations of Fe and P are elevated relative to controls. Phosphorus uptake by
the cells supports a regulatory role of polyphosphate or phospholipids in controlling Fe2+. Fenton
reaction products can degrade critical cell components, but we deduce that extracellular
processes do not cause cell death. Rather, Fe2+ overwhelms outer membrane regulatory proteins
and is oxidized when it enters the cell, precipitating Fe3+ and producing lethal hydroxyl radicals.
3. 3
Introduction
Overuse of antibiotics in healthcare is a major concern because of the consequential
proliferation of antimicrobial resistance. Our studies of natural antibacterial minerals were
initiated to investigate alternative antimicrobial mechanisms. Indigenous people worldwide have
used clays for healing throughout history. Recently, French green clay poultices were
documented for healing Buruli ulcer (1), a necrotizing fasciitis caused by Mycobacterium
ulcerans. However, only one of the French clays used for healing proved to be antibacterial (2).
Other sources of French green clay increased bacteria growth relative to controls (3).
Continued testing of over 50 clays worldwide, used for healing, revealed only a few
deposits that are truly antibacterial. Each deposit is mineralogically different in detail, but the
depositional environments are similar. All of the deposits are from hydrothermally altered
volcaniclastic environments, either altered pyroclastic material or bentonite (volcanic ash).
This study examines the geochemical characteristics of the most effective antibacterial clay
that we have found to date. It is mined by Oregon Mineral Technologies (Grants Pass, Oregon,
USA), hereafter referred to as Oregon blue clay. It was shown to completely eliminate
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium
and antibiotic resistant extended-spectrum beta lactamase (ESBL) E. coli and methicillin
resistant S. aureus (MRSA) within 24 hrs (4). The source of this material is an open pit mine in
hydrothermally altered, pyroclastic material associated with volcanic activity in the Cascade
Mountains (OR, USA). We present evidence for the natural antibacterial components in this clay
and deduce the chemical conditions required for its antibacterial effect.
Antibacterial clay mechanisms
A variety of physical and/or chemical processes can make clays antibacterial. Physical
bactericide can occur by surface attraction between clay minerals and bacteria, which can
hamper passive and active uptake of essential nutrients, disrupt cell envelopes or impair efflux of
metabolites (5). The natural antibacterial clays we have studied do not kill by physical
associations between the clay and bacterial cells (3). The Oregon blue clay shows no zone of
inhibition when applied dry to bacterial colonies growing in Petri dishes. However, the clays are
antibacterial when hydrated and incubated with bacteria. Furthermore, when an aqueous
suspension of Oregon blue clay (50 mg/ml water) was placed in dialysis tubing (25000 MDCO)
and submerged into a beaker of log phase E. coli suspended in sterile Tryptic soy broth, the
bacteria were killed over the course of 24 hrs. This indicates exchange of soluble constituents
(either toxins liberated from the clay or sequestration of essential nutrients required for bacterial
growth) across the membrane (6).
Other clays, e.g., allophane and imogolite, have been made antibacterial by chemical
sorption of known bactericidal elements (Ag, Cu, Co, Zn) onto certain crystallographic sites of
the mineral surfaces (7-9). Nanoparticulate metal oxides and ceramics can also be
antibacterial;(10) releasing soluble toxic compounds in proportion to their high specific surface
area (11, 12). Therefore, we evaluated what soluble elements E. coli assimilate from the naturally
antibacterial Oregon blue clay.
Mineralogy of antibacterial clays
Each antibacterial clay deposit is mineralogically different (Table 1) but they have in
common the presence of expandable clay minerals (smectite) and Fe-rich phases (e.g., biotite,
jarosite, pyrite, magnetite, hematite, goethite, and amphibole). The presence of pyrite in some
samples may be important for bactericidal action (13), but not all of the antibacterial clays
4. 4
contain pyrite. The average crystal diameter of the antibacterial clays (<200nm) is an order of
magnitude smaller than standard clay reference materials (www.clays.org). The nano-minerals in
these deposits may enhance solubility of toxic elements (14). Additionally, the smectite may
sequester toxic ions in the interlayer sites, which could be released upon rehydration in a clay
poultice.
Because the Oregon blue clay is only antibacterial when hydrated, we studied the soluble
elements in aqueous leachates of the clay (Supporting Information). Table 2 presents the
chemistry of aqueous leachates from the Oregon blue clay compared to the French antibacterial
clay (3) and a non-antibacterial clay (NAB) (15) used medicinally. The elemental concentrations
are all below minimum inhibitory concentrations (MIC) published for E. coli at circum-neutral
pH (16-18). However, under the pH, oxidation state and aqueous chemistry conditions created
within a poultice, the MIC may be quite different due to stabilization of different soluble species.
Experimental Methods
Aqueous leachates were prepared using sterilized clay and water (50mg/ml). The mixture
was ultra-sonified (Branson Sonifier 450) then shaken 24h to equilibrate. The suspension of
minerals in water was centrifuged (20Krpm) to remove solids.
E. coli (JM109) was grown to log phase and a cell density of ~109 cfu/ml in LB. The cell
population produced ~30mg of cells (dry wt.) per experiment and each experiment was repeated
in triplicate. E. coli was incubated in 1:1 ratio with clay leachate. All cultures were grown at
37˚C in a shaker-incubator for 24 hrs. Bacterial growth was evaluated after incubation, by
standard plate counts. Bacteria (live or dead) from experiments were centrifuged at 7000rpm for
5 minutes to pellet. Bacterial cells were then rinsed with DDI water (25mls) in triplicate. Finally,
one aliquot was rinsed in 25 ml of 50mM EDTA:100mM oxalic acid with 0.85% NaCl (pH
6.95). This solution removes metals from the exterior cell envelope (19). Test tubes containing
bacteria were dried at room temperature, ground and accurately weighed. Twelve milliliters of
10% nitric acid (Omnitrace Ultra, EMD chemicals) was used to digest cells, and then diluted to
40mls.
Leachates, whole cells, and cells with exterior metals removed, were analyzed for
elemental compositions with a Thermo Fisher Element 2 single-collector, double-focusing
magnetic sector inductively coupled plasma mass spectrometer (ICP-MS) in low-, medium- and
high-resolution modes, depending on spectral interferences. The samples, blanks and standards
were acidified with nitric acid and spiked with 1 ppb indium solution to correct for instrumental
response affected by solution matrix. Analyses of river water standard reference materials NIST
1640, NIST 1643e, and NRC SLRS4 determined analytical accuracy and precision. The
measurement uncertainties were <5% (1s). Major anion concentrations were measured by ion
chromatography using a Dionex DX 600 Dual IC System with IonPac AS11-HC column.
Results
E.coli (JM109) was used as a model species to evaluate mass transfer from the clay to
bacteria. Incubation of the antibacterial Oregon blue clay leachate 1:1 with a population of 109
cfu/ml E. coli grown in Luria Broth (LB) to log phase, showed no surviving bacteria in three
independent experiments, compared to non-antibacterial (NAB) clay leachate and control (E.coli
in LB without leachate). The control group produced on average 7.9±0.4 x109 cfu/ml, and the
NAB clay leachate produced 9.0±0.8 x109 cfu/ml. Elements taken up by the Oregon blue clay-
amended E. coli suspensions were analyzed using direct injection ICP-MS (20) (Table 3). Only
elements showing significant differences in concentrations from the controls are presented.
5. 5
Whole cells were rinsed in triplicate with filter-sterilized, distilled-deionized (DDI) water before
elemental measurements. Intracellular elemental abundances were determined on EDTA-oxalic
acid treated samples (19); which removes extracellular metals, particularly iron.
High-resolution scanning electron microscope (SEM) images of the Oregon blue clay (Fig.
1) show a) the nano-scale crystals; b) the clay matrix encompassing sub-micron spherical Fe-S
particles; and c) a lath-shaped mineral, rich in Ca, Al, S and O but no Si. This sulfate crystal
might be jarosite, identified by XRD, or gypsum with a metal oxide coating. Co-existence of
sulfate and pyrite in the sample, places the chemical stability of the clay on the equal activity line
for S-SO4 (21).
Important energy-requiring processes, for nutrient uptake and metabolism, motility, and
cell division are localized at the cell membrane. Transmission electron microscope (TEM)
imaging of E.coli treated with the Oregon blue leachate (Fig. 4) shows initial development of
“hairy” vesicles on the cell membrane in response to acidic conditions (22). These dark, electron-
opaque particles are initially evenly distributed on the cell walls. However, after 6 hrs of
incubation, the black particles are concentrated at polar ends of the cells, which indicates that the
cells are metabolically active, as bacteria regulate uptake of metals through polar attractions (23).
The greatest cell damage is observed after 24 hrs when the black particles appear on the interior
of the E. coli cells, near large voids or vacuoles in the cytoplasm.
Discussion
Significantly higher concentrations of Al, P, V, Fe Cu and Pb are associated with the whole
bacteria killed by the Oregon blue leachate (Table 3), than in the live bacteria (controls). In
contrast, the concentrations of Mg and Ca are much lower in the Oregon blue clay leachate-
treated whole bacteria. This is consistent with results of Borrok et al. (24) who showed that
protons irreversibly exchange with Mg and Ca on the surface of bacteria exposed to acidic
solutions and increase adsorption of other metals. Nonetheless, the interior of the Oregon blue
clay treated bacteria shows Mg and Ca concentrations similar to the NAB sample that did not kill
E. coli, indicating that limitation of these nutrients was not the cause for bacterial cell death.
On the cell interior of the Oregon blue clay killed bacteria, Al, P, Fe and Cu have elevated
concentrations relative to controls. Whereas Cu can be toxic to bacteria, its abundance is not
statistically different than the control or NAB samples. However, concentrations of P, Fe and Al
in the cell interior of Oregon blue clay killed bacteria are significantly higher than controls.
The high concentration of Al in the Oregon blue clay leachate is of interest because the
antibacterial clay leachates have extreme pH (≤4 or ≥10; Table 2). Aluminum is soluble in
extreme pH conditions, but precipitates in circum-neutral fluids (25). Notably, whole-cell
concentrations of Al in Oregon blue clay leachate-treated samples are four times higher than
those in the controls, even though the cytoplasmic Al contents in treated and control samples
were similar. Apparently, Al has limited transport through cell membranes, as tri- and tetra-
valent metals are often precluded by cell pore diameters (26). Aluminum precipitated on the cell
envelope might inhibit influx of nutrients or efflux of waste. However, the elevated intracellular
P and Fe suggest that Al did not block their influx channels.
The P-content of the E. coli killed by Oregon blue clay leachate is also four times greater
than the control, and the interior cell concentration is twice as high. This indicates significant
precipitation of P on the cell wall that does not preclude uptake into the cell. Phosphorus is an
important part of ATP, DNA, polyphosphates and phospholipids in cells, and phosphate anions
have been found essential for the regulation of cation transport across the cell membrane (27).
The negatively charged phosphate group in the lipid molecules of the cell membrane
6. 6
counterbalance positively charged arginine in a voltage-controlled gate across pore channels. The
Oregon blue clay leachate treated E. coli accumulated P, possibly in response to chemical
stresses (28), whereas it was not required in excess for the normal cell function of the controls.
The enhancement of P in the E. coli cell interior may represent an attempt by the bacteria to
control the influx of Fe2+ across the cell membrane, or to remove the metal.
Whole cell Fe concentrations in the Oregon blue clay leachate-treated E. coli are twenty
times greater than in the controls. The intracellular Fe contents are eight times higher. Thus
excess Fe was transported through the cell wall and is implicated as the primary reactant in the
bactericidal process. The elevated concentrations of P and Fe in the E. coli interior imply that
extracellular metals did not block transport channels through the cell envelope at least not
initially.
Images depicting the progression of dying E. coli incubated with Oregon blue clay leachate
(Fig. 4) are similar to images of E. coli treated with FeSO4 (14), where black particles were
oxides of Fe3+. The vacuoles formed may result from a) polyphosphate granules that regulate
intracellular metals, b) metabolic byproducts (e.g., NO), c) destruction of DNA or other cell
components, d) leakage of cytoplasm after cell death. Bacteria require Fe for many metabolic
processes, and use ferritin protein to regulate intracellular Fe levels and storage capacities.
Ferritins can keep some Fe in solution, but excess Fe can form toxic precipitates (29). E. coli
exposed to Oregon blue clay leachate may have accumulated P as a stress response (28) and may
employ P to gate the cation flux across the cell wall (27), but eventually the bacteria were
overwhelmed by the high Fe2+ concentration of the Oregon blue clay leachate.
Evaluating the antibacterial process
The bioavailability of metals to bacteria depends on the aqueous metal speciation in the
clay poultice. The process of transferring elements from a clay surface through water to a cell
membrane involves numerous chemical reactions and the formation of rapidly reactive
intermediates (radicals) that are affected by clay mineralogy and by surface complexation on the
bacteria. The pH and oxidation state of the water added to clay to make a poultice is most
influenced by the buffering capacity of the clay minerals with relative surface areas >100 m2/g
(30).
Clays that buffer water to circum-neutral pH values are not antibacterial.(31) Therefore, we
tested the tolerance of E. coli to low pH fluids. Adjusting the pH of sodium phosphate buffer to
acidic conditions similar to that of Oregon blue clay leachate (pH 3.5) reduced the population of
E. coli by two orders of magnitude (107.5 to 105.5 cfu/ml) over 2 hrs. However, the Oregon blue
clay leachate (CB07-L) (4) completely killed the bacteria in less than 1hr, indicating that it is not
pH alone that killed the E. coli but that metals soluble at low pH play an important antibacterial
role.
The oxidation state of the clay poultice is also critical to the antibacterial process. A low
oxidation state (log fO2 < –74) is inferred from the presence of sulfide and sulfate in the Oregon
blue clay (Fig 2) where Cu+ or Fe2+ are dominant soluble cations (21). Experiments with Oregon
blue leachates show loss of antibacterial capacity after oxidation. It is common for clay leachate
to precipitate iron oxide coincident with the loss of bactericidal capacity. However, when small
amounts (50mg/ml) of clay are suspended in the aqueous solution, it is stabilized and remains
antibacterial. A rapid decrease of dissolved oxygen (D.O.), ORP and pH occurs when the Oregon
blue clay is added to DDI water (Fig. 5). The pH stabilizes within the first hour, but the D.O.
declines over 6 hrs, coincident with E.coli death. It is unlikely that decline in D.O. was
responsible for the E. coli death, because this bacterium is fully capable of anaerobic
7. 7
respiration(32). However, E. coli may take up Fe2+ under the anaerobic conditions induced by
the clay. The Oregon blue clay contains up to 8% pyrite, which is known to be bactericidal. In
the presence of water, pyrite produces reactive oxygen species (ROS) such as hydrogen peroxide
and hydroxyl radicals that degrade nucleic acids in RNA and DNA via the Fenton reaction (33).
Chelation of Fe2+ in solution by EDTA, or hydroxyl radicals by catalase or other hydroxyl
scavengers can prevent their destruction of DNA (34). Therefore, EDTA, thiourea, and bi-pyridal
were used to chelate Fe and ROS from Oregon blue clay aqueous leachates (4). The bactericidal
effect on E. coli was reduced (compared to controls), but was not eliminated. Bacteria have
developed mechanisms for tolerating external oxidative stress, (35) therefore ROS formed in the
extracellular leachate are not the key antibacterial components. The Fe-chelation experiments
may have failed due to excess supply Fe2+ from minerals in suspension.
Park and Imlay (36) showed that significant damage to cellular DNA occurs when
hydrogen peroxide reacts with Fe2+ to form hydroxyl radicals in vivo. Exogenous hydrogen
peroxide is normally chelated in the cell envelope (37), but penetration of Fe2+ into the cell will
catalyze the Fenton reaction. The rapid influx of reduced Fe2+ and possibly Cu+ produced by the
Oregon blue clay may overwhelm the metal resistance mechanisms of pathogenic bacteria.
Oxidation of Fe2+ occurs within the bacteria and produces destructive hydroxyl radical reactions
precipitating Fe3+ near the intracellular voids (Fig. 3d). Hydroxyl radical production may be
enhanced through cellular biochemical reduction of the Fe3+ by cysteine; reported as the rate-
limiting step for oxidative DNA damage (36). Kohanski et al. (38) showed that production of
intracellular hydroxyl radicals is a common underlying mechanism for cellular death by synthetic
antibiotics. Evidence presented here suggests that antibacterial clays can provide an external
stimulant of the same bactericidal reaction.
Development of protective biochemical mechanisms by bacteria (17) may not be possible
on the time scale of a clay poultice application. Because of this, the external application of
antibacterial clays may remain more effective for wound care than systemic antibiotics. The
growth of human tissue, coincident with the antibacterial action of clays (1) remains
unexplained. However, nanoparticles of Fe-oxide (magnetite) were recently found to be
bactericidal against S. aureus, while increasing growth of human bone cells (39). The differential
effect of Fe on eukaryotic versus prokaryotic cells is consistent with a role of host defense
mechanisms that target bacterial Fe utilization, (40) and may be key to understanding the
observed clay healing process(1).
Acknowledgements
We thank Oregon Mineral Technologies, Inc. for access to their mineral deposit. T
Cunningham, and D. Lowry provided TEM images; S. Haydel assisted with microbiology pilot
studies. NIH grant R21 AT003618 partially supported this research. We appreciate support from
the ASU School of Life Sciences, Center for Solid State Science, and NASA Astrobiology
Institute. The author(s) declare that they have no competing interests.
Brief : Certain volcanic environments produce antibacterial clays with soluble reduced metals in
concentrations that overwhelm bacterial defense mechanisms, causing lethal intracellular
hydroxyl radical attack of biomolecules.
8. 8
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Table Captions
Table 1. Mineralogical comparison of antibacterial and non-antibacterial clays discussed,
determined by quantitative X-ray diffraction (41) using CuKa radiation.
Table 2. Chemical analyses of aqueous clay leachates, A) ICP-MS analyses of antibacterial
French clay (ARG), Oregon blue clay and a non-antibacterial (NAB) clay leachate (23), B)
Ion chromatography analysis of anions in the Oregon blue clay leachate.
Table 3. Metals assimilated by whole bacterial cells compared to intracellular concentrations.
Figure Captions
Fig, 1. SEM of the Oregon blue clay using 30kV, 2.1nA current, A) Clay particles on graphite
(dark substrate). EDS (right) shows elements over a rastered area, B) Close up of Fe-S
spherules in aluminosilicate matrix. EDS spectrum is rastered over spherules, C) Lath shaped
particle with high Ca, S and O (black dot) interpreted as gypsum.
Fig. 2. Time series TEM images of pressure frozen, fixed (glutaraldehyde and Os vapor), resin
embedded, sectioned E. coli after incubation with Oregon blue clay leachate. A) initial
incubation shows uniform black precipitate of electron opaque metal, B) after 1 h metal
migrates toward cell poles, C) metals are concentrated at cell poles after 6h, D) after 24 h
metal has penetrated the cell interior and voids form accompanying cell death (images by T.
Cunningham).
Fig. 3. Plot showing changes in pH, ORP and dissolved O2 content of DDI water over 24 h after
addition of Oregon blue clay (50mg/ml).