Sequestration of metal and metalloid ions by thermophilic bacteria

This Ph. D. thesis presents results and conclusions from studies 1) investigating the interaction between metal and metalloid ions and thermophilic bacteria, and 2) characterizing microbial populations in a geothermally active habitat with relatively high concentrations of metalloid ions and compounds. In initial cadmium ion toxicity assays, the minimal inhibition concentration for 46 thermophilic bacteria of the genera Aneurinibacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, and Thermus were determined. The highest tolerances to cadmium ions (Cd2+) in the range of 400 to 3200 micro;M were observed for species belonging to the genus Geobacillus. The thermophilic Gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected to describe further biosorption reactions between cadmium ions and chemically reactive functional groups (potential ligands) within and onto the bacterial cell walls. Data obtained from electrophoretic mobility, potentiometric titration and cadmium ion adsorption experiments were used to quantify the number and concentrations of ligands and to determine the thermodynamic stability constants for the ligand-cation complexes. The first reported surface complexation models (SCMs) quantifying metal ion adsorption by thermophilic microorganisms predicted cadmium adsorption and desorption by both studied Geobacillus strains over a range of pH values and for different biomasses. The results indicated the functional group, with a deprotonation constant pK value of approximately 3.8, to be more dominant in cation biosorption accounting for 66 and 80% of all titrable groups for G. thermocatenulatus and G. stearothermophilus, respectively. The generated SCMs are different from model parameters obtained from mesophilic species that have been studied to date and might indicate a different biosorption behavior for both studied Geobacillus strains. Another objective of this thesis was to characterize microbial populations in the hot spring Champagne Pool, located in Waiotapu, New Zealand. The thermal spring is approximately 65 m in diameter and discharges water at 75eg; C and pH 5.5, which is oversaturated with arsenic and antimony compounds that precipitate and form orange deposits. Recovered nucleic acids and adenosine 5'-triphosphate (ATP) concentrations obtained for Champagne Pool water samples indicated low microbial density and were in good agreement with relatively low cell numbers of 5.6 plusmn; 0.5 x10^6 cells per ml. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analyses revealed the abundance of Sulfurihydrogenibium, Sulfolobus and Thermofilum-like populations in Champagne Pool. Two novel bacteria and one novel archaeon were successfully isolated with a distant phylogenetic relationship to Sulfurihydrogenibium, Thermoanaerobacter, and Thermococcus, respectively. Genotypic and metabolic characteristics differentiated isolate CP.B2 from described species of the genus Sulfurihydrogenibium. CP.B2 represents a novel genus within the Aquificales order, for which the name Venenivibrio stagnispumantis gen. nov., sp. nov. is proposed. V. stagnispumantis is a thermophilic, chemolithothrophic bacterium, that utilizes molecular hydrogen as electron donor and oxygen as electron acceptor and displayed growth in the presence of up to 8 mM NaAsO2 (As3+) and more than 20 mM Na2HAsO4.7H2O (As5+). However, growth was not observed when Na2HAsO4.7H2O and NaAsO2 were provided as the sole electron acceptor and donor pair. Arsenic resistance was conferred by the genes arsA and ars

Proton and cadmium adsorption by the archaeon Thermococcus zilligii: Generalising the contrast between thermophiles and mesophiles as sorbents

Adsorption by microorganisms can play a significant role in the fate and transport of metals in natural systems. Surface complexation models (SCMs) have been applied extensively to describe metal adsorption by mesophilic bacteria, and several recent studies have extended this framework to thermophilic bacteria. We conduct acid-base titrations and batch experiments to characterise proton and Cd adsorption onto the thermophilic archaeon Thermococcus zilligii. The experimental data and the derived SCMs indicate that the archaeon displays significantly lower overall sorption site density compared to previously studied thermophilic bacteria such Anoxybacillus flavithermus, Geobacillus stearothermophilus, G. thermocatenulatus, and Thermus thermophilus. The thermophilic bacteria and archaea display lower sorption site densities than the mesophilic microorganisms that have been studied to date, which points to a general pattern of total concentration of cell wall adsorption sites per unit biomass being inversely correlated to growth temperature

Cadmium Ion Biosorption by the Thermophilic Bacteria Geobacillus stearothermophilus and G. thermocatenulatus

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl(2) (as high as 400 to 3,200 μM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd(2+) adsorption experiments to characterize Cd(2+) complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd(2+) adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria

Quantitative Molecular Detection of Putative Periodontal Pathogens in Clinically Healthy and Periodontally Diseased Subjects

<div><p>Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of <i>Bacteria</i> and <i>Archaea</i>. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of <i>P. gingivalis</i> and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high <i>P. gingivalis</i> and total bacterial abundance. Interestingly, we found that moderate abundances of <i>A. actinomycetemcomitans</i> were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.</p></div

Subject characteristics.

<p>Data are presented as numbers (percentages) or mean ± SD.</p>1<p>paired t-test or McNemar test.</p><p>CAL, clinical attachment loss; PD, probing depth.</p

Adjusted Odds Ratios quantifying chance of being periodontally diseased depending on detection (yes/no) or abundance of different pathogens in tongue scrapings in the overall study population (N = 88 pairs).

<p>Conditional logistic regression modeling periodontal status (cases versus controls, dependent variable) on detection (yes/no) or abundances adjusting for age (cont.), school education, smoking status and BMI.</p>◊<p>Abundances were categorized as tertiles (T1–T3). Numbers within tertiles were: <i>P. gingivalis</i>: 75-43-58, <i>A. actinomycetemcomitans</i>: 92-26-58, <i>F. nucleatum</i>: 93-25-58, <i>S. sanguinis</i>: 75-43-58, Archaea 85-33-58, %Archaea: 85-33-58, Bacteria: 59-59-58.</p>1<p>proportion of 16S rRNA gene copies per ng extracted DNA; N, number of matched pairs.</p>2<p>percent of archaeal 16S rRNA gene copies per prokaryotic 16S rRNA gene copies (Archaea+Bacteria)*100.</p><p>*p<0.05, ** p<0.01, *** p<0.001 versus reference group.</p

Comparison of prokaryotic abundances between periodontally healthy (controls) and periodontally diseased subjects (cases). N = 88 pairs.

<p>Data are presented as median (25%; 75% quantile).</p>1<p>Wilcoxon matched-pairs signed-ranks test.</p>2<p>proportion of 16S rRNA gene copies per ng extracted DNA; N, number of matched pairs.</p>3<p>percent of archaeal 16S rRNA gene copies per prokaryotic 16S rRNA gene copies (Archaea+Bacteria)*100.</p

Crude associations between periodontal status (periodontally healthy (controls) versus periodontally diseased subjects (cases)) and detection of different pathogens (yes/no) in tongue scrapings.

1<p>Exact McNemar Test.</p><p>NA, not annotated; OR, Odds Ratio.</p

Sequence information, specificity, amplicon size and molarity of oligonucleotide primers and probes used in the qPCR assays.

a)<p>F, forward primer; R, reverse primer; P, probe; FAM, 6-carboxyfluorescein; BHQ1, Black Hole Quencher 1.</p>b)<p>Published oligonucleotide sequences showing mismatches were modified by using the nucleotides which are underlined at these positions.</p>c)<p>In their study, Morillo and colleagues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099244#pone.0099244-Morillo1" target="_blank">[28]</a> designated the forward primer as reverse and the other way around. Here, the correct designation is given.</p