Scale-up of the production of highly reactive biogenic magnetite nanoparticles using Geobacter sulfurreducens.

Although there are numerous examples of large-scale commercial microbial synthesis routes for organic bioproducts, few studies have addressed the obvious potential for microbial systems to produce inorganic functional biomaterials at scale. Here we address this by focusing on the production of nanoscale biomagnetite particles by the Fe(III)-reducing bacterium Geobacter sulfurreducens, which was scaled up successfully from laboratory- to pilot plant-scale production, while maintaining the surface reactivity and magnetic properties which make this material well suited to commercial exploitation. At the largest scale tested, the bacterium was grown in a 50 l bioreactor, harvested and then inoculated into a buffer solution containing Fe(III)-oxyhydroxide and an electron donor and mediator, which promoted the formation of magnetite in under 24 h. This procedure was capable of producing up to 120 g of biomagnetite. The particle size distribution was maintained between 10 and 15 nm during scale-up of this second step from 10 ml to 10 l, with conserved magnetic properties and surface reactivity; the latter demonstrated by the reduction of Cr(VI). The process presented provides an environmentally benign route to magnetite production and serves as an alternative to harsher synthetic techniques, with the clear potential to be used to produce kilogram to tonne quantities.

Cr(VI) and azo dye removal using a hollow-fibre membrane system functionalized with a biogenic Pd-magnetite catalyst.

This study investigates the application of a hybrid system combining hollow-fibre membrane technology with the reductive abilities of magnetic nanoparticles for the remediation of toxic Cr(VI) and the azo dye, Remazol Black B. Nano-scale biogenic magnetite (Fe3O4), formed by microbial reduction of the mineral ferrihydrite, has a high reductive capacity due to the presence of Fe(II) in the mineral structure. The magnetic nanoparticles (approximately 20 nm) can be arrayed with Pd0 nanoparticles (approximately 5 nm) making a catalytically active nanomaterial. Membrane units, with and without nanoparticles, were challenged with either Cr(VI) or azo dye and some were supplemented with sodium formate, as an electron donor for contaminant reduction promoted by the Pd. The combination of Pd-magnetite with formate resulted in the most effective remediation strategy for both contaminants and the lifetime of the membrane unit was also increased, with 55% (19 days) and 70% (23 days) removal of the azo dye and Cr(VI), respectively. Low flow rates of 0.1 ml/min resulted in improved efficiencies due to increased contact time with the membrane/nanoparticle unit, with 70-75% removal of each contaminant. Chemical analyses of the nanoparticles post-exposure to Cr(VI) in the membrane modules indicated Pd to be more oxidized when Cr removal was maximized, and that the Cr was partially reduced to Cr(III) at the surface of the magnetite. These results have demonstrated that hollow-fibre membrane units can be enhanced for the removal of soluble, redox sensitive contaminants by incorporation of a layer of palladized biogenic nanoparticulate magnetite.

Controlled cobalt doping in biogenic magnetite nanoparticles.

Cobalt-doped magnetite (CoxFe3 -xO4) nanoparticles have been produced through the microbial reduction of cobalt-iron oxyhydroxide by the bacterium Geobacter sulfurreducens. The materials produced, as measured by superconducting quantum interference device magnetometry, X-ray magnetic circular dichroism, Mössbauer spectroscopy, etc., show dramatic increases in coercivity with increasing cobalt content without a major decrease in overall saturation magnetization. Structural and magnetization analyses reveal a reduction in particle size to less than 4 nm at the highest Co content, combined with an increase in the effective anisotropy of the magnetic nanoparticles. The potential use of these biogenic nanoparticles in aqueous suspensions for magnetic hyperthermia applications is demonstrated. Further analysis of the distribution of cations within the ferrite spinel indicates that the cobalt is predominantly incorporated in octahedral coordination, achieved by the substitution of Fe(2+) site with Co(2+), with up to 17 per cent Co substituted into tetrahedral sites.

Ex situ formation of metal selenide quantum dots using bacterially derived selenide precursors.

Luminescent quantum dots were synthesized using bacterially derived selenide (Se(II-)) as the precursor. Biogenic Se(II-) was produced by the reduction of Se(IV) by Veillonella atypica and compared directly against borohydride-reduced Se(IV) for the production of glutathione-stabilized CdSe and ß-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological Se(II-) formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic Se(II-) included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic Se(II-) is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, 'green' synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams.

Characterisation of the dissimilatory reduction of Fe(III)-oxyhydroxide at the microbe-mineral interface: the application of STXM-XMCD.

A combination of scanning transmission X-ray microscopy and X-ray magnetic circular dichroism was used to spatially resolve the distribution of different carbon and iron species associated with Shewanella oneidensis MR-1 cells. S. oneidensis MR-1 couples the reduction of Fe(III)-oxyhydroxides to the oxidation of organic matter in order to conserve energy for growth. Several potential mechanisms may be used by S. oneidensis MR-1 to facilitate Fe(III)-reduction. These include direct contact between the cell and mineral surface, secretion of either exogenous electron shuttles or Fe-chelating agents and the production of conductive 'nanowires'. In this study, the protein/lipid signature of the bacterial cells was associated with areas of magnetite (Fe3O4), the product of dissimilatory Fe(III) reduction, which was oversaturated with Fe(II) (compared to stoichiometric magnetite). However, areas of the sample rich in polysaccharides, most likely associated with extracellular polymeric matrix and not in direct contact with the cell surface, were undersaturated with Fe(II), forming maghemite-like (γ-Fe2O3) phases compared to stoichiometric magnetite. The reduced form of magnetite will be much more effective in environmental remediation such as the immobilisation of toxic metals. These findings suggest a dominant role for surface contact-mediated electron transfer in this study and also the inhomogeneity of magnetite species on the submicron scale present in microbial reactions. This study also illustrates the applicability of this new synchrotron-based technique for high-resolution characterisation of the microbe-mineral interface, which is pivotal in controlling the chemistry of the Earth's critical zone.

Control of nanoparticle size, reactivity and magnetic properties during the bioproduction of magnetite by Geobacter sulfurreducens.

The bioproduction of nanoscale magnetite by Fe(III)-reducing bacteria offers a potentially tunable, environmentally benign route to magnetic nanoparticle synthesis. Here, we demonstrate that it is possible to control the size of magnetite nanoparticles produced by Geobacter sulfurreducens by adjusting the total biomass introduced at the start of the process. The particles have a narrow size distribution and can be controlled within the range of 10-50 nm. X-ray diffraction analysis indicates that controlled production of a number of different biominerals is possible via this method including goethite, magnetite and siderite, but their formation is strongly dependent upon the rate of Fe(III) reduction and total concentration and rate of Fe(II) produced by the bacteria during the reduction process. Relative cation distributions within the structure of the nanoparticles have been investigated by x-ray magnetic circular dichroism and indicate the presence of a highly reduced surface layer which is not observed when magnetite is produced through abiotic methods. The enhanced Fe(II)-rich surface, combined with small particle size, has important environmental applications such as in the reductive bioremediation of organics, radionuclides and metals. In the case of Cr(VI), as a model high-valence toxic metal, optimized biogenic magnetite is able to reduce and sequester the toxic hexavalent chromium very efficiently to the less harmful trivalent form.

Use of biogenic and abiotic elemental selenium nanospheres to sequester elemental mercury released from mercury contaminated museum specimens.

Mercuric chloride solutions have historically been used as pesticides to prevent bacterial, fungal and insect degradation of herbarium specimens. The University of Manchester museum herbarium contains over a million specimens from numerous collections, many preserved using HgCl(2) and its transformation to Hg(v)(0) represents a health risk to herbarium staff. Elevated mercury concentrations in work areas (∼ 1.7 µg m(-3)) are below advised safe levels (<25 µg m(-3)) but up to 90 µg m(-3) mercury vapour was measured in specimen boxes, representing a risk when accessing the samples. Mercury vapour release correlated strongly with temperature. Mercury salts were observed on botanical specimens at concentrations up to 2.85 wt% (bulk); XPS, SEM-EDS and XANES suggest the presence of residual HgCl(2) as well as cubic HgS and HgO. Bacterially derived, amorphous nanospheres of elemental selenium effectively sequestered the mercury vapour in the specimen boxes (up to 19 wt%), and analysis demonstrated that the Hg(v)(0) was oxidised by the selenium to form stable HgSe on the surface of the nanospheres. Biogenic Se(0) can be used to reduce Hg(v)(0) in long term, slow release environments.

Arsenic release and attenuation in low organic carbon aquifer sediments from West Bengal.

High arsenic concentrations in groundwater are causing a humanitarian disaster in Southeast Asia. It is generally accepted that microbial activities play a critical role in the mobilization of arsenic from the sediments, with metal-reducing bacteria stimulated by organic carbon implicated. However, the detailed mechanisms underpinning these processes remain poorly understood. Of particular importance is the nature of the organic carbon driving the reduction of sorbed As(V) to the more mobile As(III), and the interplay between iron and sulphide minerals that can potentially immobilize both oxidation states of arsenic. Using a multidisciplinary approach, we identified the critical factors leading to arsenic release from West Bengal sediments. The results show that a cascade of redox processes was supported in the absence of high loadings of labile organic matter. Arsenic release was associated with As(V) and Fe(III) reduction, while the removal of arsenic was concomitant with sulphate reduction. The microbial populations potentially catalysing arsenic and sulphate reduction were identified by targeting the genes arrA and dsrB, and the total bacterial and archaeal communities by 16S rRNA gene analysis. Results suggest that very low concentrations of organic matter are able to support microbial arsenic mobilization via metal reduction, and subsequent arsenic mitigation through sulphate reduction. It may therefore be possible to enhance sulphate reduction through subtle manipulations to the carbon loading in such aquifers, to minimize the concentrations of arsenic in groundwaters.

The role of indigenous microorganisms in the biodegradation of naturally occurring petroleum, the reduction of iron, and the mobilization of arsenite from west bengal aquifer sediments.

High levels of naturally occurring arsenic are found in the shallow reducing aquifers of West Bengal, Bangladesh, and other areas of Southeast Asia. These aquifers are used extensively for drinking water and irrigation by the local population. Mechanisms for its release are unclear, although increasing evidence points to a microbial control. The type of organic matter present is of vital importance because it has a direct impact on the rate of microbial activity and on the amount of arsenic released into the ground water. The discovery of naturally occurring hydrocarbons in an arsenic-rich aquifer from West Bengal provides a source of potential electron donors for this process. Using microcosm-based techniques, seven sediments from a site containing naturally occurring hydrocarbons in West Bengal were incubated with synthetic ground water for 28 d under anaerobic conditions without the addition of an external electron donor. Arsenic release and Fe(III) reduction appeared to be microbially mediated, with variable rates of arsenic mobilization in comparison to Fe(III) reduction, suggesting that multiple processes are involved. All sediments showed a preferential loss of petroleum-sourced n-alkanes over terrestrially sourced sedimentary hydrocarbons n-alkanes during the incubation, implying that the use of petroleum-sourced n-alkanes could support, directly or indirectly, microbial Fe(III) reduction. Samples undergoing maximal release of As(III) contained a significant population of Sulfurospirillum sp., a known As(V)-reducing bacterium, providing the first evidence that such organisms may mediate arsenic release from West Bengali aquifers.

Biomineralization: linking the fossil record to the production of high value functional materials.

The microbial cell offers a highly efficient template for the formation of nanoparticles with interesting properties including high catalytic, magnetic and light-emitting activities. Thus biomineralization products are not only important in global biogeochemical cycles, but they also have considerable commercial potential, offering new methods for material synthesis that eliminate toxic organic solvents and minimize expensive high-temperature and pressure processing steps. In this review we describe a range of bacterial processes that can be harnessed to make precious metal catalysts from waste streams, ferrite spinels for biomedicine and catalysis, metal phosphates for environmental remediation and biomedical applications, and biogenic selenides for a range of optical devices. Recent molecular-scale studies have shown that the structure and properties of bionanominerals can be fine-tuned by subtle manipulations to the starting materials and to the genetic makeup of the cell. This review is dedicated to the late Terry Beveridge who contributed much to the field of biomineralization, and provided early models to rationalize the mechanisms of biomineral synthesis, including those of geological and commercial potential.

Probing the biogeochemistry of arsenic: response of two contrasting aquifer sediments from Cambodia to stimulation by arsenate and ferric iron.

Many millions of people worldwide are at risk of severe poisoning through exposure to groundwater contaminated with sediment-derived arsenic. An ever-increasing body of work is reinforcing the link between microbially-mediated redox cycling in aquifer sediments and the mobilisation of sorbed As(V) into groundwaters as the potentially more mobile and toxic As(III) anion. However, to date, few studies have examined the biogeochemical cycling of Fe and As species by microbes indigenous to Cambodian sediments. In this study two contrasting sediments, taken from a shallow As-rich reducing aquifer in the Kien Svay district of Cambodia, were used in a laboratory microcosm study. We present evidence to show that microbes present in these sediments are able to reduce Fe(III) and As(V) when provided with an electron donor, and that the two sediments respond differently to stimulation with Fe(III) and As(V). Shifts in the community composition of the two sediments after stimulation with As(V) suggest a potential role for members of the beta-Proteobacteria in As(V) reduction, a phylogenetic grouping known to contain microorganisms capable of As(III) oxidation, but not previously implicated in As(V) reduction. PCR-based analysis of the sediment microbial DNA using primers specific to the arrA gene, (a gene essential for microbial As(V) respiration), indicates the presence of microorganisms capable of dissimilatory As(V) reduction.

Molecular analysis of arsenate-reducing bacteria within Cambodian sediments following amendment with acetate.

The health of millions is threatened by the use of groundwater contaminated with sediment-derived arsenic for drinking water and irrigation purposes in Southeast Asia. The microbial reduction of sorbed As(V) to the potentially more mobile As(III) has been implicated in release of arsenic into groundwater, but to date there have been few studies of the microorganisms that can mediate this transformation in aquifers. With the use of stable isotope probing of nucleic acids, we present evidence that the introduction of a proxy for organic matter ((13)C-labeled acetate) stimulated As(V) reduction in sediments collected from a Cambodian aquifer that hosts arsenic-rich groundwater. This was accompanied by an increase in the proportion of prokaryotes closely related to the dissimilatory As(V)-reducing bacteria Sulfurospirillum strain NP-4 and Desulfotomaculum auripigmentum. As(V) respiratory reductase genes (arrA) closely associated with those found in Sulfurospirillum barnesii and Geobacter uraniumreducens were also detected in active bacterial communities utilizing (13)C-labeled acetate in microcosms. This study suggests a direct link between inputs of organic matter and the increased prevalence and activity of organisms which transform As(V) to the potentially more mobile and thus hazardous As(III) via dissimilatory As(V) reduction.

Molecular analysis of a sulphate-reducing consortium used to treat metal-containing effluents.

A sulphate-reducing consortium used in a bioprocess to remove toxic metals from solution as insoluble sulphides, was characterised using molecular (PCR-based) and traditional culturing techniques. After prolonged cultivation under anoxic biofilm-forming conditions, the mixed culture contained a low diversity of sulphate-reducing bacteria, dominated by one strain closely related to Desulfomicrobium norvegicum, identified by three independent PCR-based analyses. The genetic targets used were the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region and the disulfite reductase (dsr) gene, which is conserved amongst all known sulphate-reducing bacteria. This organism was also isolated by conventional anaerobic techniques, confirming its presence in the mixed culture. A surprising diversity of other non-sulphate-reducing facultative and obligate anaerobes were detected, supporting a model of the symbiotic/commensal nature of carbon and energy fluxes in such a mixed culture while suggesting the physiological capacity for a wide range of biotransformations by this stable microbial consortium.

XAS and XMCD evidence for species-dependent partitioning of arsenic during microbial reduction of ferrihydrite to magnetite.

Poorly crystalline Fe(III) oxyhydroxides, ubiquitously distributed as mineral coatings and discrete particles in aquifer sediments, are well-known hosts of sedimentary As. Microbial reduction of these phases is widely thought to be responsible for the genesis of As-rich reducing groundwaters found in many parts of the world, most notably in Bangladesh and West Bengal, India. As such, it is important to understand the behavior of As associated with ferric oxyhydroxides during the early stages of Fe(lll) reduction. We have used X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) to elucidate the changes in the bonding mechanism of As(III) and As(V) as their host Fe(III) oxyhydroxide undergoes bacterially induced reductive transformation to magnetite. Two-line ferrihydrite, with adsorbed As(III) or As(V), was incubated under anaerobic conditions in the presence of acetate as an electron donor, and Geobacter sulfurreducens, a subsurface bacterium capable of respiring on Fe(lll), but not As(V). In both experiments, no increase in dissolved As was observed during reduction to magnetite (complete upon 5 days incubation), consistent with our earlier observation of As sequestration by the formation of biogenic Fe(III)-bearing minerals. XAS data suggested that the As bonding environment of the As(III)-magnetite product is indistinguishable from that obtained from simple adsorption of As(lll) on the surface of biogenic magnetite. In contrast, reduction of As(V)-sorbed ferrihydrite to magnetite caused incorporation of As5+ within the magnetite structure. XMCD analysis provided further evidence of structural partitioning of As5+ as the small size of the As5+ cation caused a distortion of the spinel structure compared to standard biogenic magnetite. These results may have implications regarding the species-dependent mobility of As undergoing anoxic biogeochemical transformations, e.g., during early sedimentary diagenesis.

Interactions between the Fe(III)-reducing bacterium Geobacter sulfurreducens and arsenate, and capture of the metalloid by biogenic Fe(II).

Previous work has shown that microbial communities in As-mobilizing sediments from West Bengal were dominated by Geobacter species. Thus, the potential of Geobacter sulfurreducens to mobilize arsenic via direct enzymatic reduction and indirect mechanisms linked to Fe(III) reduction was analyzed. G. sulfurreducens was unable to conserve energy for growth via the dissimilatory reduction of As(V), although it was able to grow in medium containing fumarate as the terminal electron acceptor in the presence of 500 muM As(V). There was also no evidence of As(III) in culture supernatants, suggesting that resistance to 500 muM As(V) was not mediated by a classical arsenic resistance operon, which would rely on the intracellular reduction of As(V) and the efflux of As(III). When the cells were grown using soluble Fe(III) as an electron acceptor in the presence of As(V), the Fe(II)-bearing mineral vivianite was formed. This was accompanied by the removal of As, predominantly as As(V), from solution. Biogenic siderite (ferrous carbonate) was also able to remove As from solution. When the organism was grown using insoluble ferrihydrite as an electron acceptor, Fe(III) reduction resulted in the formation of magnetite, again accompanied by the nearly quantitative sorption of As(V). These results demonstrate that G. sulfurreducens, a model Fe(III)-reducing bacterium, did not reduce As(V) enzymatically, despite the apparent genetic potential to mediate this transformation. However, the reduction of Fe(III) led to the formation of Fe(II)-bearing phases that are able to capture arsenic species and could act as sinks for arsenic in sediments.

Reduction of uranium(VI) phosphate during growth of the thermophilic bacterium Thermoterrabacterium ferrireducens.

The thermophilic, gram-positive bacterium Thermoterrabacterium ferrireducens coupled organotrophic growth to the reduction of sparingly soluble U(VI) phosphate. X-ray powder diffraction and X-ray absorption spectroscopy analysis identified the electron acceptor in a defined medium as U(VI) phosphate [uramphite; (NH4)(UO2)(PO4) . 3H2O], while the U(IV)-containing precipitate formed during bacterial growth was identified as ningyoite [CaU(PO4)2 . H2O]. This is the first report of microbial reduction of a largely insoluble U(VI) compound.

Stimulation of microbial sulphate reduction in a constructed wetland: microbiological and geochemical analysis.

Microbial sulphate reduction was stimulated successfully in enclosures installed in a constructed wetland. When sucrose (2.4mM) and NH(4)Cl (600 microM) were added to water in the test enclosures, the indigenous microbial community was able to remove over 90% of the sulphate, present as a contaminant from nearby mining activity at a concentration of 384 mg x l(-1) (4mM), over 50 days. Over 90% of the sucrose was also removed. Sulphate was not reduced in control enclosures containing no added sucrose or NH(4)Cl. Fermentation of sucrose by obligate anaerobes including Clostridium sp. and Bacteriodes sp. preceded sulphate reduction in the test enclosures. Sulphate reduction was biphasic, with maximum rates noted between 2-5 and 23-27 days after the addition of the growth substrates. Relatively unbiased 16S rDNA analysis suggested that nitrogen-fixing bacteria were important constituents of the microbial community in the test enclosures at day 23, suggesting that soluble nitrogen was limiting in the amended test enclosures during the experiment.

Alzheimer's disease and related dementias increase costs of comorbidities in managed Medicare.

OBJECTIVES: To analyze the relationship between comorbid conditions and costs for patients with AD and related dementias (ADRD) in a Medicare managed care organization (MCO). To derive implications for improving management of patients with ADRD. METHODS: Retrospective analysis was carried out on administrative data for 3,934 patients with ADRD and 19,300 age/sex-matched control subjects enrolled in a large Medicare MCO. Patients with ADRD were identified from diagnoses on medical claims and encounter data for a 2-year period. Control subjects were selected from health plan members without dementia. Comorbid conditions were based on the diagnostic classifications from the Charlson comorbidity index. Health care costs and utilization for MCO-covered services for cases were compared with those of control subjects. RESULTS: Prevalence of ADRD was 4.4%, substantially higher than reported in previous studies of Medicare managed care and similar to population-based estimates. After controlling for comorbid conditions, age, and sex, annual costs were $4,134 higher for ADRD patients, resulting in excess costs of $16 million to the MCO. For the 10 most prevalent comorbidities in ADRD patients, adjusted costs were higher for ADRD patients compared with control subjects with the same condition. Higher costs were attributable to higher inpatient and skilled nursing facility utilization. CONCLUSIONS: In this study, prevalence rates for ADRD mirrored population estimates. Costs for patients with ADRD in this Medicare MCO varied considerably by comorbid condition and were substantially higher for patients with both AD and comorbid diseases commonly targeted for disease management, indicating that AD increases costs through effects on the management of comorbid illnesses. These findings indicate that better treatment and care management of AD could reduce the costs of comorbid illnesses commonly experienced by the frail elderly.

Prevalence, costs, and treatment of Alzheimer's disease and related dementia: a managed care perspective.

BACKGROUND: The number of patients with Alzheimer's disease (AD) and related dementia treated in managed care organizations (MCOs) is increasing, and this trend is expected to continue. Therefore, it is critical that MCOs develop disease management strategies for this population. OBJECTIVE: To review the literature on the prevalence, costs, and treatment of AD and related dementia. STUDY DESIGN: Review of published articles from MEDLINE and peer-reviewed journals. RESULTS: Prevalence of AD and related dementia is approximately 5.7% among those aged 65 and older. Prevalence data from claims-based studies of AD in managed care are lower, ranging from 0.55% to 0.83%. Costs for formal care average $27,672 per patient annually, with long-term care being the most costly component. Annual costs for informal care are estimated to be $10,400 to $34,517 per patient. Additional costs associated with AD include lost wages and productivity of patients and caregivers and costs associated with increased morbidity of caregivers. Donepezil treatment is well tolerated and has been extensively tested and evaluated in clinical settings. Early diagnosis and treatment of AD with donepezil has been shown to slow cognitive decline in AD. Although study findings regarding the cost offsets of donepezil-treated patients to date are mixed, there is a growing body of evidence to support the inclusion of this and other therapies into an MCO's AD treatment armamentarium. CONCLUSIONS: It is unlikely that MCOs will escape the increased prevalence and costs associated with AD. Opportunities exist through patient management programs targeted toward early diagnosis, effective use of medications, control of comorbidities, and patient and family support to partially offset these costs while providing quality patient care.