Danielle Fortin

The effect of growth phase on proton and metal adsorption by Bacillus subtilis

Several recent studies have applied surface complexation models to quantify metal adsorption by bacterial surfaces. Although these models can account for the effects of many abiotic variables (such as pH and ionic strength), to date, the effects of biotic variables (such as growth phase) have not been investigated. In this study, we quantify the effect of growth phase on surface site concentrations, deprotonation constants, and metal-binding constants by performing acid-base titrations and Cd and Fe(III) batch adsorption experi- ments using suspensions containing Bacillus subtilis cultured to exponential, stationary, and sporulated phase. For each type of surface site, concentrations and pKa values describing deprotonation decrease as the cells move from exponential to stationary phase, but remain constant from stationary to sporulated phase. Due to the variations in site concentrations and deprotonation constants, Cd and Fe(III) binding constants are largest for stationary-phase cells and smallest for sporulated cells, even though cells in stationary phase adsorb roughly 5% to 10% less metal (per unit weight) than exponential-phase cells, and roughly 10% to 20% more metal than sporulated cells. These variations in surface complexation model parameters indicate that any attempt to predict proton or metal adsorption by bacteria must consider the growth phase of the population. Copyright

Role of sediment composition in trace metal distribution in lake sediments

Sediment cores were collected from 20 lakes from the Muskoka region of Ontario, Canada, to study vertical changes in trace metal concentrations with depth and the distribution of metals amongst humic material, amorphous and crystalline Fe and Mn oxides, insoluble organics/sulphides, and silicates. Based on their total concentrations, trace elements displayed different degrees of affinity for the organic fraction (represented by organic C) and the mineral fraction (represented by Al). Certain elements (Hg, As, Sb, Pb, Cd, and Zn) displayed a positive correlation with organic C, a negative correlation with Al, and enrichment in surface sediments (with enrichment factors ranging from 2 to 24). Detailed speciation studies revealed that these elements were associated mainly with humic material and to a lesser extent with oxides in surface sediments. Other elements (Al, Cr, Co, Fe, and Mn) displayed a negative correlation with organic C, a positive correlation with Al, and no consistent enrichment in their total concentration at the surface. The speciation study revealed that metals of the latter group were mainly associated with the silicate fraction in both surface and deep sediments. This study shows that relative affinities for organic and mineral fractions play an important role in the distribution of trace metals during burial and diagenesis, and hence in the shape of their vertical profiles.

Mineralogy of a natural As-rich hydrous ferric oxide coprecipitate formed by mixing of hydrothermal fluid and seawater: Implications regarding surface complexation and color banding in ferrihydrite deposits

Abstract We characterized the most As-rich natural hydrous ferric oxide (HFO) material ever reported using powder X-ray diffraction (pXRD), transmission electron microscopy (TEM), X-ray fluorescence spectroscopy (XRF), light element analysis using gas chromatography (GC), visible-infrared (vis-IR) diffuse reflectivity, 57Fe Mössbauer spectroscopy, and superconducting quantum interference device (SQUID) magnetometry. We find that the natural As-HFO material is very similar to synthetic coprecipitated As-HFO materials, but is significantly different from all known natural and synthetic As-free HFO materials and ferrihydrite samples. The pXRD patterns show systematic differences with patterns for 2-line ferrihydrite, that are interpreted as evidence for significant populations of oxygen-coordinated Fe-As pairs. Observations by TEM, combined with energy dispersive spectroscopy (EDS) microanalysis, show agglomerations of nanophase primary particles and no evidence for other Fe- or As-bearing phases. Mössbauer spectroscopy shows octahedrally coordinated Fe3+, with a large fraction (~20%) of the octahedral Fe environments that are significantly distorted by the presence of As, compared to the Fe local environments in As-free ferrihydrite and HFO samples. The loss on ignition (LOI) is quantitatively consistent with OH + H2O, measured by GC, which, in turn, is consistent with ~1 nm diameter primary particles having all their surface cations (Fe3+, As5+, Si4+, C4+) coordinated on the free surface side by OH- and OH2. The banding into adjacent yellowish and reddish layers that occurs in the As-HFO deposits was studied by performing mineralogical analyses of the separated adjacent layers of two couplets of yellowish and reddish material. The yellowish samples were found not to contain secondary crystalline phases (as did the reddish samples, in small amounts) and to be relatively As-rich, C- and Si-poor. The observed anticorrelations between As and Si and between As and inorganic C suggest that natural HFOs, which usually contain significant molar amounts of Si, may not be as efficient at surface complexing As (and P) as their Si and C-free synthetic counterparts, unless formed by co-precipitation with the As (or P). The yellowish and reddish layers were also clearly resolved by both Mössbauer spectroscopy and magnetometry. Complexation of arsenate onto the HFO core was found to significantly increase the average quadrupole splitting (QS) obtained from Mössbauer spectroscopy by an amount that could not be explained by other chemical differences and that is consistent with an ~1 nm diameter particle size and somewhat smaller HFO core. The Munsell hue YR index (5-10 YR) was found to be strongly correlated to the average QS, thereby establishing that the color differences, corresponding to the measured shifts of the main visible band edge, are due to the local distortions in the [6]Fe3+ environments that are induced by As complexation, via their influence on the relevant ligand field transitions. SQUID magnetometry allows the following observations. (1) The superparamagnetic to superferromagnetic transitions occur at 25 K and lower in As-HFO, compared to 55 K in synthetic 2-line ferrihydrite, suggesting a smaller magnetic primary particle (or core) size for As-HFO and inter-particle magnetic interaction reduction by surface complexed As, Si, and C. (2) The ratio of supermoment magnitude to magnetic particle size (m2/n, where m is the net number of Fe3+ atomic moments per supermoment and n is the number of Fe3+ cations per particle or HFO core) decreases with increasing As content in the sequence synthetic-HFO > reddish-As-HFO > yellowish-As-HFO. (3) The magnetic susceptibility magnitudes for As-HFO and synthetic 2-line ferrihydrite differ by a factor of 10 and suggest different supermoment formation mechanisms (m2/n < 1 vs. m2/n > 1, respectively) related to differences in intra-particle cationic and anionic disorder and magnetic particle size.

Characterization of Iron-Oxides Formed by Oxidation of Ferrous Ions in the Presence of Various Bacterial Species and Inorganic Ligands

The oxidation of ferrous ions in the presence of an excess of dissolved oxygen at neutral pH generally leads to the formation of lepidocrocite. The effect of inorganic ligands (PO4, SO4, or Si(OH)4) in concentrations typical of those in sediment pore waters, and of microorganisms (Escherichia coli K12, Pseudomonas aeruginosa PA01, Bacillus subtilis or Bacillus licheniformis) on the mineralogy, chemical composition, morphology and spatial distribution of the iron-oxides were examined using various complementary techniques, including TEM, XRD, and EXAFS. The presence of inorganic ligands during the oxidation can affect the mineralogy as well as the size and structure of the Fe-oxide particles. While the presence of sulfate (SO4/Fe = 0.5) had little effect on the outcome of the Fe-oxide synthesis, low quantities of phosphate (PO4/Fe = 0.05) inhibited lepidocrocite and large quantities of aqueous silica (Si/Fe = 5) favored the formation of 2-line ferrihydrite. The presence of any of the four representative species of bacterial cells in the various systems did not modify the mineralogy of the Fe-oxides. However, the size of the Fe-oxide particles tended to be reduced, and the presence of the cells also affected the spatial organization and the morphology of the particles. In addition, in some systems, some of the iron remains adsorbed on the cells and does not contribute to the formation of mineral phases.

Biogeochemical factors influencing net mercury methylation in contaminated freshwater sediments from the St. Lawrence River in Cornwall, Ontario, Canada

The activity of various anaerobic microbes, including sulfate reducers (SRB), iron reducers (FeRP) and methanogens (MPA) has been linked to mercury methylation in aquatic systems, although the relative importance of each microbial group in the overall process is poorly understood in natural sediments. The present study focused on the biogeochemical factors (i.e. the relative importance of various groups of anaerobic microbes (FeRP, SRB, and MPA) that affect net monomethylmercury (MMHg) formation in contaminated sediments of the St. Lawrence River (SRL) near Cornwall (Zone 1), Ontario, Canada. Methylation and demethylation potentials were measured separately by using isotope-enriched mercury species ((200)Hg(2+) and MM(199)Hg(+)) in sediment microcosms treated with specific microbial inhibitors. Sediments were sampled and incubated in the dark at room temperature in an anaerobic chamber for 96h. The potential methylation rate constants (K(m)) and demethylation rates (K(d)) were found to differ significantly between microcosms. The MPA-inhibited microcosm had the highest potential methylation rate constant (0.016d(-1)), whereas the two SRB-inhibited microcosms had comparable potential methylation rate constants (0.003d(-1) and 0.002d(-1), respectively). The inhibition of methanogens stimulated net methylation by inhibiting demethylationand by stimulating methylation along with SRB activity. The inhibition of both methanogens and SRB was found to enhance the iron reduction rates but did not completely stop MMHg production. The strong positive correlation between K(m) and Sulfate Reduction Rates (SRR) and between K(d) and Methane Production Rates (MPR) supports the involvement of SRB in Hg methylation and MPA in MMHg demethylation in the sediments. In contrast, the strong negative correlation between K(d) and Iron Reduction Rates (FeRR) shows that the increase in FeRR corresponds to a decrease in demethylation, indicating that iron reduction may influence net methylation in the SLR sediments by decreasing demethylation rather than favouring methylation.

Impact of Cell Wall Structure on the Behavior of Bacterial Cells as Sorbents of Cadmium and Lead

Batch metal sorption studies were conducted to compare the behavior of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli as sorbents of Cd 2+ and Pb 2+ . A pH range from 3.0 to 6.5 was investigated at total metal concentrations of 1 2 10 -4.0 and 3.2 2 10 -5 M. Concentration apparent equilibrium sorption constants (K s n M ) and sorption capacity (S max n ) values were determined for the bacteria by fitting experimental data to one- ( n = 1) and two-site ( n = 2) Langmuir sorption isotherms. The sorption data for each of the bacteria were described well by a one-site model (r 2 > 0.9), Cd 2+ exhibited somewhat lower sorption affinities (log K s M =- 1.5 for B. subtilis , and -0.7 for E. coli ) than Pb 2+ (log K s M =-0.6 for B. subtilis and -0.8 for E. coli ). Corresponding S max values for Cd 2+ and Pb 2+ on B. subtilis were 0.36 mmole/g and 0.27 mmole/g, respectively. For E. coli Cd 2+ and Pb 2+ S max values were lower at 0.10 mmole/g and 0.21 mmole/g. A two-site sorption model yielded an improved fit for only the E. coli data with several orders of magnitude difference evident between high (Cd 2+ log K s1 M = 0.9; Pb 2+ log K s1 M = 1.5) and low (Cd 2+ log K s2 M =- 1.1; Pb 2+ log K s2 M = -1.6) affinity sorption sites. In addition, allowing for the presence of low affinity sorption (i.e., S max2 ) sites further increased the total E. coli metal sorption capacity closer to that of B. subtilis . As expected, the sorption of Cd 2+ and Pb 2+ by the bacteria exhibited a strong dependence on pH with sorption edges in the range of pH 4.2 to 5.6. The results of this study show that, despite differences in cell wall structure and composition, B. subtilis and E. coli exhibit remarkably similar sorption behavior toward Cd 2+ and Pb 2+ , respectively. These similarities can be attributed to the specific chemical reactivity of acidic functional groups (e.g., carboxyl, phosphoryl) that occur in the cell walls of both bacteria.

Effect of the presence of bacterial surfaces during the synthesis of Fe oxides by oxidation of ferrous ions

Natural iron-oxides are often found in close association with bacterial cells in aquatic environments, but the effect of bacteria on their formation is still under investigation. The present study was undertaken to assess the effect of two common bacteria, Bacillus subtilis and Escherichia coli , on the morphology and mineralogy of Fe oxides. All Fe oxides were synthesised by oxidation of Fe(II) (2 × 10 −4 M) at pH = 7. Three systems were studied, i.e. , abiotic Fe oxides, Fe oxides formed in the presence of bacteria (which we call “biogenic” Fe oxides) and abiotic Fe oxides mixed with bacterial cells. Samples were analysed by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Fe oxide particles in all systems showed a needle-like morphology, with many needles seeming to be attached to a sheet, and were identified as lepidocrocite. However, the biogenic lepidocrocite crystals were generally shorter than the abiotic ones, and the crystals were found in association with the bacterial cell-wall, especially with B. subtilis , a Gram-positive bacterium. Biogenic lepidocrocite crystals also displayed an attenuation of the XRD 120 line, which is indicative of a low crystallinity. Growth limitation and poor crystalline order are then likely to affect the surface area of Fe oxides and indirectly, their sorptive capacity.

Prevalence of Anaerobic Ammonium-Oxidizing Bacteria in Contaminated Groundwater

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.

Preliminary characterization and biological reduction of putative biogenic iron oxides (BIOS) from the Tonga-Kermadec Arc, southwest Pacific Ocean

Sediment samples were obtained from areas of diffuse hydrothermal venting along the seabed in the Tonga sector of the Tonga-Kermadec Arc, southwest Pacific Ocean. Sediments from Volcano 1 and Volcano 19 were analyzed by X-ray diffraction (XRD) and found to be composed primarily of the iron oxyhydroxide mineral, two-line ferrihydrite. XRD also suggested the possible presence of minor amounts of more ordered iron (hydr)oxides (including six-line ferrihydrite, goethite/lepidocrocite and magnetite) in the biogenic iron oxides (BIOS) from Volcano 1; however, Mössbauer spectroscopy failed to detect any mineral phases more crystalline than two-line ferrihydrite. The minerals were precipitated on the surfaces of abundant filamentous microbial structures. Morphologically, some of these structures were similar in appearance to the known iron-oxidizing genus Mariprofundus spp., suggesting that the sediments are composed of biogenic iron oxides. At Volcano 19, an areally extensive, active vent field, the microbial cells appeared to be responsible for the formation of cohesive chimney-like structures of iron oxyhydroxide, 2-3 m in height, whereas at Volcano 1, an older vent field, no chimney-like structures were apparent. Iron reduction of the sediment material (i.e. BIOS) by Shewanella putrefaciens CN32 was measured, in vitro, as the ratio of [total Fe(II)]:[total Fe]. From this parameter, reduction rates were calculated for Volcano 1 BIOS (0.0521 day(-1)), Volcano 19 BIOS (0.0473 day(-1)), and hydrous ferric oxide, a synthetic two-line ferrihydrite (0.0224 day(-1)). Sediments from both BIOS sites were more easily reduced than synthetic ferrihydrite, which suggests that the decrease in effective surface area of the minerals within the sediments (due to the presence of the organic component) does not inhibit subsequent microbial reduction. These results indicate that natural, marine BIOS are easily reduced in the presence of dissimilatory iron-reducing bacteria, and that the use of common synthetic iron minerals to model their reduction may lead to a significant underestimation of their biological reactivity.

Sorption of Cadmium and Lead by Bacteria–Ferrihydrite Composites

The sorptive behavior of bacteria—iron oxide composites was investigated in batch metal sorption assays using ferrihydrite in isolation (0.13 and 0.14 g/L ferrihydrite in cadmium and lead systems, respectively) as well as in combination with Bacillus subtilis (0.25 g/L adsorbent mixture) and Escherichia coli (0.27 g/L adsorbent mixture). A pH range from 3.0 to 6.5 was studied using total metal concentrations of 1.0 × 10 − 4.0 and 3.2 × 10 − 5 M with adsorbent mixtures proportioned on a 1:1 mass/volume basis. The log of the apparent surface complex formation constants (log K S M ) and sorption capacity (S max ) values were determined by fitting the experimental data to one-site Langmuir sorption isotherms. The one-site model effectively described the sorption data (r 2 > 0.9), where Cd 2 + exhibited somewhat lower sorption affinities (log K S M = −3 for ferrihydrite, −1.7 for B. subtilis–ferrihydrite, and −1.1 for E. coli–ferrihydrite) than Pb 2 + (log K S M = −0.9 for ferrihydrite, − 0.2 forB. subtilis–ferrihydrite, and –0.1 for E. coli–ferrihydrite). The corresponding S max values for Cd 2 + and Pb 2 + on ferrihydrite were 0.78 mmole/g and 1.34 mmole/g, respectively. For the B. subtilis–ferrihydrite composites, Cd 2 + and Pb 2 + S max values were lower at 0.29 mmole/g and 0.5 mmole/g, respectively. Similar values were determined for the E. coli–ferrihydrite composites (0.15 mmole/g and 0.68 mmole/g for Cd 2 + and Pb 2 + , respectively). The sorption of Cd 2 + and Pb 2 + by each of the sorbent systems exhibited a strong dependence on pH with sorption edges in the range of pH 4.0 to 7.3. The observed S max of the composites were lower than values predicted upon available site additivity (Cd 2 + B. subtilis −ferrihydrite : 0.29 mmole/g (observed) < 0.57 mmole/g (calculated); Cd 2 + E. coli −ferrihydrite : 0.15 mmole/g (observed) < 0.44 mmole/ g (calculated); Pb 2 + B. subtilis −ferrihydrite : 0.5 mmole/g (observed) < 0.805 mmole/g (calculated); Pb 2 + E. coli –ferrihydrite : 0.68 mmole/g (observed) < 0.775 mmole/g (calculated)), implying that a masking of reactive surface sites by attachment had occurred between the bacteria and ferrihydrite. Electrophoretic mobility analysis indicated that the ferrihydrite surface properties dominate the net surface charge for each composite system with lesser contributions from the bacteria.