Larry E. Hersman

SIDEROPHORE-PROMOTED DISSOLUTION OF HEMATITE

Siderophores are highly Fe( III)-specific bidentate ligands excreted by aerobic and facultative anaerobic microorganisms to facilitate Fe uptake in oxic environments. These compounds are thought to influence mineral weathering and the biogeochemical cycling of Fe, but quantitative information concerning this possible influence is nonexistent. Preparative quantities of a soil bacterium siderophore were extracted and purified for use in batch dissolution experiments performed with synthetic hematite particles suspended in 1 mmol dm−3 NaNO3 at pH 3 under exclusion of light. The initial siderophore concentration used, 0.24 mmol dm−3 was representative of microniche environments. Soluble Fe per unit mass of hematite was linear with time over an observational period between 2 and 24 h, leading to an area-based dissolution rate of 10−8 mol m−2 h−1 . Comparative dissolution experiments, performed with oxalate and ascorbate ligands at the 2–3 mmol dm−3 initial concentration typical of soil environments (otherwise identical conditions to the siderophore experiments), led to dissolution rates of 5 X 10−8 mol m−2 h−1 , in agreement with literature values. The comparability of dissolution rates for a soil bacterium siderophore and two terrestrial organic ligands, despite an order-of-magnitude difference in their initial concentrations, suggests that siderophores may indeed figure significantly in Fe(Ill) -mineral weathering reactions under natural conditions.

Enhanced Exopolymer Production and Chromium Stabilization in Pseudomonas putida Unsaturated Biofilms

ABSTRACT Chromium-contaminated soils threaten surface and groundwater quality at many industrial sites. In vadose zones, indigenous bacteria can reduce Cr(VI) to Cr(III), but the subsequent fate of Cr(III) and the roles of bacterial biofilms are relatively unknown. To investigate, we cultured Pseudomonas putida, a model organism for vadose zone bioremediation, as unsaturated biofilms on membranes overlaying iron-deficient solid media either containing molecular dichromate from potassium dichromate (Cr-only treatment) or with deposits of solid, dichromate-coated hematite (Fe+Cr treatment) to simulate vadose zone conditions. Controls included iron-deficient solid medium and an Fe-only treatment using solid hematite deposits. Under iron-deficient conditions, chromium exposure resulted in lower cell yield and lower amounts of cellular protein and carbohydrate, but providing iron in the form of hematite overcame these toxic effects of Cr. For the Cr and Fe+Cr treatments, Cr(VI) was completely reduced to Cr(III) that accumulated on biofilm cells and extracellular polymeric substances (EPSs). Chromium exposure resulted in elevated extracellular carbohydrates, protein, DNA, and EPS sugars that were relatively enriched in N-acetyl-glucosamine, rhamnose, glucose, and mannose. The proportions of EPS protein and carbohydrate relative to intracellular pools suggested Cr toxicity-mediated cell lysis as the origin. However, DNA accumulated extracellularly in amounts far greater than expected from cell lysis, and Cr was liberated when extracted EPS was treated with DNase. These results demonstrate that Cr accumulation in unsaturated biofilms occurs with enzymatic reduction of Cr(VI), cellular lysis, cellular association, and extracellular DNA binding of Cr(III), which altogether can facilitate localized biotic stabilization of Cr in contaminated vadose zones.

Petrobactin is the primary siderophore synthesized by Bacillus anthracis str. Sterne under conditions of iron starvation

The siderophores of Bacillus anthracis are critical for the pathogen’s proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of 1H and 13C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.

Plutonium(V/VI) Reduction by the Metal-Reducing Bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1.

ABSTRACT We examined the ability of the metal-reducing bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1 to reduce Pu(VI) and Pu(V). Cell suspensions of both bacteria reduced oxidized Pu [a mixture of Pu(VI) and Pu(V)] to Pu(IV). The rate of plutonium reduction was similar to the rate of U(VI) reduction obtained under similar conditions for each bacteria. The rates of Pu(VI) and U(VI) reduction by cell suspensions of S. oneidensis were slightly higher than the rates observed with G. metallireducens. The reduced form of Pu was characterized as aggregates of nanoparticulates of Pu(IV). Transmission electron microscopy images of the solids obtained from the cultures after the reduction of Pu(VI) and Pu(V) by S. oneidensis show that the Pu precipitates have a crystalline structure. The nanoparticulates of Pu(IV) were precipitated on the surface of or within the cell walls of the bacteria. The production of Pu(III) was not observed, which indicates that Pu(IV) was the stable form of reduced Pu under these experimental conditions. Experiments examining the ability of these bacteria to use Pu(VI) as a terminal electron acceptor for growth were inconclusive. A slight increase in cell density was observed for both G. metallireducens and S. oneidensis when Pu(VI) was provided as the sole electron acceptor; however, Pu(VI) concentrations decreased similarly in both the experimental and control cultures.

Iron acquisition from hydrous Fe(III)-oxides by an aerobic Pseudomonas sp.

Iron is a metabolic requirement of living systems, yet iron is very insoluble in aerobic, neutral environments. Therefore, the amount of iron in solution under these conditions is not sufficient to meet the nutrient requirements of microorganisms. It has been assumed that microorganisms acquire iron in these environments through the use of specific iron chelating compounds called siderophores. Interestingly, there is little quantitative data in the literature to support this hypothesis. Our studies were initiated to investigate the mechanism(s) used by an aerobic microorganism to acquire iron from a relatively insoluble iron oxide. When iron was supplied in the form of hematite, a Pseudomonas sp. was able to achieve moderate growth, as compared to growth on FeEDTA. Analysis using high-performance liquid chromatography (HPLC) of the metabolic products of this species showed significant differences between growth on hematite as compared to FeEDTA. The results of these experiments will be discussed in terms of our current knowledge of microbial enhanced iron dissolution, and compared to abiotic dissolution rates.

Enhancement of Kaolinite Dissolution by an Aerobic Pseudomonas mendocina Bacterium

This research focused on whether bacteria living in aerobic environments where Fe is often a limiting nutrient could access Fe associated with the clay mineral kaolinite. Kaolinite is one of the most abundant clays at the Earths surface, and it often contains trace quantities of Fe as surface precipitates, accessory minerals, and structural substitutions. We hypothesized that aerobic bacteria may enhance kaolinite dissolution as a means of obtaining associated Fe. To test this hypothesis, we conducted microbial growth experiments in the presence of an aerobic Pseudomonas mendocinaThis research focused on whether bacteria living in aerobic environments where Fe is often a limiting nutrient could access Fe associated with the clay mineral kaolinite. Kaolinite is one of the most abundant clays at the Earths surface, and it often contains trace quantities of Fe as surface precipitates, accessory minerals, and structural substitutions. We hypothesized that aerobic bacteria may enhance kaolinite dissolution as a means of obtaining associated Fe. To test this hypothesis, we conducted microbial growth experiments in the presence of an aerobic Pseudomonas mendocina

Dissolution of Al-substituted goethites by an aerobic Pseudomonas mendocina var. bacteria

Abstract Goethite particles in soil environments often contain Al 3+ substituted for Fe 3+ in octahedrally coordinated sites. Al substitution has been shown to alter mineral stability and abiotic dissolution rates. This study focused on the effects of Al substitution (to 8.8 mol%) on synthetic goethite dissolution by an aerobic Pseudomonas mendocina var. bacteria. In contrast to dissimilatory iron reducing bacteria (DIRB), this bacteria is not capable of using Fe as a terminal electron acceptor for oxidative phosphorylation, and hence only requires μM concentrations of Fe for metabolism. Pure and substituted goethites were reacted with microorganisms in Fe-limited growth media wherein the only source of Fe was the solid phase, so that microbial populations could only grow by obtaining Fe through mineral dissolution. Because at least some Fe was taken up by the bacteria, we could not measure Fe release rates directly from dissolved Fe concentrations. Rather, we relied upon microbial growth measurements as indirect indicators of mineral dissolution. Increasing Al substitution resulted in particles with progressively decreasing mean particle length and aspect ratios, as well as fewer domains, as measured by atomic-force microscopy (AFM); but with increasing structural order as determined by XRD line widths. Experiments conducted in the dark at 22°C, exposed to the atmosphere, showed that maximum microbial population did not correlate with particle specific surface area, which is in contrast with previous studies using DIRB. Maximum microbial population increased a small amount with increasing Al content of the goethites, in contrast with several previous investigations of abiotic dissolution. Because dense biofilms formed, we were unable to use AFM to observe mineral dissolution features. AFM imaging suggested that more highly substituted goethites formed denser aggregates, and previous investigations have shown that aggregate structure is important for microbial attachment, which is prerequisite for dissolution. Hence, effects of Al substitution on aggregate structure is a focus of ongoing research.

Siderophore Production and Iron Reduction by Pseudomonas mendocina in Response to Iron Deprivation

In aerobic environments microorganisms are faced with a discrepancy of ~10 orders of magnitude between the available Fe (~10-17M) and their metabolic requirement for it (~10-7M). In contrast to facultative anaerobic environments, where dissimilatory iron-reducing bacteria (DIRB) are often abundant, few studies have detailed microbial interactions with Fe(III) (hydr)oxides in aerobic environments. To better understand acquisition of Fe from Fe(III) (hydr)oxides, we investigated the production of siderophore and Fe(III) reduction by a strict aerobe in the presence of synthetic hematite as a source of Fe. Pseudomonas mendocina grew best when Fewas supplied as FeEDTA (~1.8x108 colony-forming units [CFU] ml-1), grew abundantly when Fe was supplied as hematite (~1.2x108 CFU ml-1), and grew poorly when Fe was withheld from the medium (~5.5x107 CFU ml-1). As expected, negligible siderophore was produced per cell when Fe was supplied as FeEDTA and more siderophore was produced in the hematite flasks than in the co...In aerobic environments microorganisms are faced with a discrepancy of ~10 orders of magnitude between the available Fe (~10-17M) and their metabolic requirement for it (~10-7M). In contrast to facultative anaerobic environments, where dissimilatory iron-reducing bacteria (DIRB) are often abundant, few studies have detailed microbial interactions with Fe(III) (hydr)oxides in aerobic environments. To better understand acquisition of Fe from Fe(III) (hydr)oxides, we investigated the production of siderophore and Fe(III) reduction by a strict aerobe in the presence of synthetic hematite as a source of Fe. Pseudomonas mendocina grew best when Fewas supplied as FeEDTA (~1.8x108 colony-forming units [CFU] ml-1), grew abundantly when Fe was supplied as hematite (~1.2x108 CFU ml-1), and grew poorly when Fe was withheld from the medium (~5.5x107 CFU ml-1). As expected, negligible siderophore was produced per cell when Fe was supplied as FeEDTA and more siderophore was produced in the hematite flasks than in the controls. Thus, growth of P. mendocina and the production of siderophore in the presence of hematite present compelling evidence that siderophore was produced as a mechanism to acquire Fe from hematite. For the Fe reduction experiments, Fe reduction by components of the supernatant fluid was induced weakly when Fe was supplied as hematite or as FeEDTA, but much more when the cells were cultured under extreme Fe deprivation. In fact, 16 times as much Fe reduction occurred in the controls as in the presence of either of the FeEDTA or hematite amendments. Our results, which contravene the long-held assumptions that Fe acquisition was facilitated solely by siderophores, provides a new perspective regarding microbial interactions with Fe bearing minerals.

Dissolution of well and poorly ordered kaolinites by an aerobic bacterium

Abstract Previous research by our group (e.g., [Chem. Geol. 132 (1996) 25; Geochim. Cosmochim. Acta 64 (2000) 1363]) has shown that an aerobic Pseudomonas mendocina bacterium enhances Fe(hydr)oxide dissolution in order to obtain Fe under Fe-limited conditions. The P. mendocina is incapable of utilizing Fe as a terminal electron acceptor and requires several orders of magnitude lower Fe concentrations than do dissimilatory Fe reducing bacteria. The research reported here compared the effects of the P. mendocina on dissolution of well and poorly ordered Clay Minerals Society Source Clay kaolinites KGa-1b and KGa-2, respectively, under Fe-limited conditions. KGa-1b and KGa-2 contain 0.04 and 0.94 bulk wt.% Fe, respectively, and their surface Fe/Si atomic ratios=0.008 and 0.012. Following strong cleaning of the kaolinites in 5.8 M HCl at 85 °C, the surface Fe/Si atomic ratios decreased to 0.004 and 0.008, respectively. Both kaolinites also developed a Si-enriched surface precipitate upon strong cleaning. Because the P. mendocina take up Fe, we could not measure Fe release from the kaolinite directly, but rather had to monitor it indirectly by comparing microbial populations sizes under Fe-limited growth conditions. We found that microbial growth on uncleaned, weakly cleaned, and strongly cleaned kaolinites increased with the amount of Fe readily available to organic ligands as estimated by dissolution in 0.001 M oxalate (pH 3). This suggests that it is the amount of readily accessible Fe that controls Fe acquisition and hence microbial growth. The trend is based on only a relatively small range of kaolinite Fe contents, and the research thus needs to be expanded to include kaolinites with a broader range of bulk and surface Fe concentrations. Significant enhancement of Al release was observed in the presence of the bacteria, along with generally some enhancement of Si release. This enhancement of kaolinite dissolution could be related to an observed pH increase from ∼7–8 to ∼9 in the presence of the bacteria and/or to production of Al chelating agents. The P. mendocina produce a variety of organic exudates, including siderophores [Chem. Geol. 132 (1996) 25; Geomicrobiology (2001b)], and further studies into the effects of the siderophores on Al complexation and on kaolinite dissolution are ongoing.

Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides

ABSTRACT Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe,Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe.P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth ofP. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the minerals surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.