The present disclosure relates generally to semiconducting nanofibers, and more particularly, to a process of producing macroscopic quantities of arsenic sulfide nanofibers within one to three days using the Shewanella sp. strain ANA-3.
The synthesis of nanomaterials by biological or biomimetic means in physiological conditions offers multiple advantages over traditional physical and chemical strategies that typically require more extreme environments (temperature, pressure, and pH). In addition to the promise of cheaper and greener synthesis processes, the resulting biogenic materials can exhibit unique morphologies and physical/chemical properties stemming from the tight control organisms exert over the composition, nucleation, crystallography, and desired function of these materials
U.S. Patent Application Publication No. 2009/0155876 A1 to Hor-Gil Hur et al. (“Hur”) discloses a biological method for preparing arsenic sulfide (As—S) compounds. More particularly, Hur provides a method for production of nanotubes based on As—S compounds including As2S3 by reacting thiosulfate S2O32− with arsenate As(V) through mediation of Shewanella sp. strain. However, the process of Hur is slow, and relies on the bacterium Shewanella sp. strain HN-41. Further, it is unclear if the structures produced by the described process are semiconductors. There is thus a need for a process to rapidly produce As—S compounds for applications such as semiconductors.
The process disclosed herein produces macroscopic quantities of semiconducting arsenic sulfide nanofibers within one to three days. The process is biotically influenced by the bacteria Shewanella sp. strain ANA-3. The resulting nanofibers are semiconductors with bandgaps between 2.2 and 2.5 eV.
The measured semiconducting and bandgap properties from the resulting nanofibers may lead to applications in the semiconductor, transistor, and solar energy industries, or similar industries. A faster manufactured and more robust biological component makes the overall process of nanofiber production more commercially feasible than it would have been otherwise. The faster rate of growth allows for easier research and development as well as larger yield per day of the As—S compounds.
A rapid biological synthesis process to produce semiconducting chalcogenide nanostructures for transistor or solar cell applications is described. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the exemplary embodiments. It is apparent to one skilled in the art, however, that embodiments can be practiced without these specific details or with an equivalent arrangement. In some instances, well-known structures and devices are shown in block diagram form in order to avoid unnecessarily obscuring the embodiments.
Electron flow is fundamental to biology: organisms extract electrons from a wide array of electron donors (fuels) and transfer them to electron acceptors (oxidants). These redox reactions between electron donors and electron acceptors power the production of ATP.
Many prokaryotes can use multiple electron acceptors. Shewanella species, for instance, respire O2, Mn(IV)/Mn(III)/Fe(III) oxides, NO3−/NO2−, U(VI), Cr(VI), As(V), S2O32−, SeO32− and TeO32−. Shewanella's ability to reduce these chalcogenide compounds with the reduced products precipitating in solution, offers a low temperature route to synthesizing chalcogenide nanomaterials in mild environmental conditions (pressure, temperature, and pH), while taking advantage of the exceptional control organisms exert over the structures and functional properties of biogenic materials.
Chalcogenides (materials containing S, Se or Te) are used extensively for their photonic and electrical properties. CdTe and CdSe are important semiconductors used in solar cells and luminescent quantum dots. Photodeformation has been studied in As20S80 and Sb2S3 films, photodarkening in As50Se50 films, and As2Se3 has exhibited various photomechanical properties. Applications for chalcogenide glasses include phase change memory, solar cells and infrared waveguides and sensors. Biotic fabrication of such materials can produce vast quantities of nanostructures in a scalable low temperature process.
PbS can be produced with Torulopsis sp., CdS with E. coli, Fusarium oxysporum and Rhododpseudomonas palustris, ZnS from Rhodobacter spaeroides, and As2Se3 from Shewanella HN-41. Work using Shewanella strain HN-41 and Shewanella sp. Strain ANA-3 demonstrates the concomitant reduction of As(V) and S2O32− in solution to synthesize macroscopic quantities of inorganic AsxSy nanofibers. The resulting nanomaterials exhibit novel morphologies as shown in
Bacterial Growth Methods
In one example, all biological cultures were prepared in lysogeny broth (LB), which served as a nutrient rich medium, until the optical density (OD) was between 1.5 and 2 absorption units (which correspond to 1.2E10 to 1.6E10 cells/ml). Cells were then inoculated into a chemically defined anaerobic minimal medium (MM) for experimentation.
The inocula were grown aerobically in 20 mL of LB medium from a frozen (−80° C.) stock, up to an optical density at 600 nm (OD600) of 1.5±0.15. These aerobic pre-cultures were inoculated at 0.1% (vol/vol) into anaerobic serum bottles each containing 80 mL HEPES-buffered (30 mM) medium consisting of: 20 mM sodium DL-lactate, as electron donor; 28 mM ammonium chloride; 1.34 mM potassium chloride; 4.35 mM sodium phosphate monobasic; 20 mM sodium hydroxide; 10 mM thiosulfate, as Na2S2O3.5H2O; 5 mM arsenate, as Na2HAsO4.7H2O. Vitamins, amino acids, and trace mineral stock solutions were used to supplement the medium. The medium was adjusted to an initial pH of 7.25, and anaerobic conditions were reached by purging with 100% N2. The anaerobic serum bottles, sealed with butyl stoppers and aluminum seals, were sterilized by autoclaving at 120° C. for 15 minutes. Arsenate, thiosulfate, and vitamins were added after autoclaving. All cultures were grown at 30° C. and agitated at a rate of 150 rpm.
A general process for producing nanofibers includes inoculating a Shewanella sp. strain ANA-3 with a carbon and energy source (acting as an electron donor), a sulfur source and an arsenic source (acting as electron acceptors). The Shewanella sp. strain ANA-3 is grown. The resulting AsxSy nanofibers are harvested after a predetermined time.
The Shewanella sp. strain ANA-3 may grow on various carbon sources and electron donors. For example, the ANA-3 strain is able to grow on Lactate, Acetate (only anaerobically), and Pyruvate acting as carbon sources and electron donors while 5 mM arsenate may be used as the terminal electron acceptor. Other electron donors may include Citrate, Ethanol, Formate, Fumarate, Glucose, Glycerol, Malate, Propionate, and Succinate. Other electron acceptors may include Oxygen, Fumerate, Selenate, Nitrate, MnO2, Fe(OH)3, AQDS, Sulfate, Thiosulfate, Sulfite and DMSO.
Specifically, a Shewanella sp. strain ANA-3 was grown anaerobically in MM containing 20 mM lactate, 10 mM thiosulfate, and 5 mM arsenate. During incubation, within 18 to 20 hours after inoculation, macroscopic quantities of AsxSy nanofibers began to form.
Morphological, Compositional and Crystallographic Properties of Biogenic Fibers
Precipitated AsxSy observed by scanning electron microscope (SEM) showed the material to be polydisperse fibers ranging from 20 to 600 nm in diameter and from 0.1 to 100 μm in length. Energy-dispersive X-ray Spectroscopy (EDS) confirmed the fibers to be made of arsenic and sulfur and Electron Microprobe Analysis (EMPA) found the average ratio to be 38:62 which corresponds within 5% to that of As2S3.
Some fiber morphologies suggest that the material possesses a crystalline structure as illustrated by
Optical Properties
Average band gap of the fibers was calculated fitting UV-Vis spectra to the equation:
(ahv)n=A(hv−Eg)
where a is the absorption (in arbitrary units), hv is the energy of the incident photon, n is an exponential constant that depends on the type of the band gap (n=2 or ½ for direct and indirect band gaps, respectively), A is a scaling factor, and Eg is the band gap energy. Fitting the linear region of the UV-Vis data to the above equation and extrapolating the fit to the abscissa axis gives the band gap energy Eg=hv.
Identifying the Arsenic Reduction Pathway: Arsenic Operons of ANA-3
Shewanella sp. strain ANA-3 was used because it is a prodigious sulfur reducer and has two well understood arsenic related operons.
Four strains of ANA-3 were used to investigate the arsenic reduction pathway responsible for AsxSy production: ANA-3 wild type (ANA-3 WT), ANA-3 arrA mutant missing the respiratory reductase ArrA (ARRA3), ANA-3 arsB mutant missing the detoxification pump ArsB (ARSB1) and ANA-3 arsC and arrA mutant missing both reductases ArsC and ArrA (ARM1). When inoculated into MM with initial concentrations of 20 mM lactate, 10 mM thiosulfate and 5 mM arsenate, they all produced macroscopic quantities of arsenic sulfide material within 18 and 85 hours. Arsenic sulfide material made by each mutant was imaged using SEM showing all were in the form of nanofibers of similar dimensions (data not shown).
Table 1 below shows a list of strains used in this research alongside the delay between inoculation and the production of macroscopically visible quantities of AsxSy.
ARM1′s ability to produce AsxSy suggests that either arsenic is being reduced abiotically or there is an additional pathway by which the cells can reduce arsenic.
Arsenic Reduction is Achieved Biotically
To test for abiotic arsenic reduction, ANA-3 WT was inoculated into MM with 20 mM lactate and 10 mM thiosulfate, but no arsenic. The culture developed for five days providing the bottles with reduced sulfur. The cultures were then heat killed (by heating to 55° C. oven for two hours) and injected with 5 mM of an either unreduced arsenic, As(V), or reduced arsenic, As(III). Thus, both bottles contained dead cells and reduced sulfur when injected with arsenic precursors.
The inability of the As(V) bottles to produce coloration resembling the arsenite bottles over long time spans strongly indicates that dead cells (and the remnants thereof) cannot reduce As(V) to As(III). This data in conjunction with the mutant screening strongly suggests that there is an alternate biotic pathway to arsenic reduction that is independent of the ArrA and ArsC reductases. One hypothesis is that ARM1 utilizes outer cytochromes, such as ones similar to OmcA and MtrC, to reduce As(V) extracellularly similar to how Shewanella MR-1 reduces Se(IV) and Fe(III). For instance, CymA, the protein at the base of the direct electron transfer pathway ending with OmcA/MtrC, is needed in addition to ArrA and ArrB for As(V) reduction. Such extracellular reduction would not necessitate toxic As(V) entering the cell where it may do damage. This pathway may explain why MR-1 can produce As—S fibers at all, despite not having any genes that match the As reductases ArrA or ArsC to any high degree of similarity.
Another possibility is that As(V) is being reduced similarly to several proposed S0 reduction pathways revolving around an intraspecies sulfur cycle. In this system, thiosulfate S2O32− is reduced to HS− and SO32−. One, or both, of the products then reacts with As(V) to produce As(III). The resulting oxidized sulfur is then able to be reduced again by cellular metabolism. This cycle, or one similar, may be responsible for the apparent As(V) reduction by the double deletion mutant, ARM1.
Nanofiber Growth Investigation: Demonstration of Abiotic Nanofiber Extension
To understand the role of the bacteria in fiber extension, a typical ANA-3 experiment was interrupted while the fibers were still extending. Two cultures were heat killed after 42 hours of normal cell growth and fiber production. The OD continued to rise after death indicating an increase in particulate AsxSy. Thus, once formation has begun, fiber extension can proceed abiotically.
In Vivo Measurements of Fiber Growth
Direct in vivo observation of fiber growth was done using an inverted Nikon Ti light microscope taking time-lapse images every five minutes. “Perfect focus” feature allowed videos lasting several days to be collected. Analysis of the time-lapse images determined the average rate of fiber extension at room temperature to be 3.8 nm/min, starting as high as 10 nm/min and slowing over time as shown in
Several videos demonstrated that fiber extension is occurring at the tip of the fiber rather than the base as shown in
A non-specific protein stain (NanoOrange) was added to check for proteins along the length of the fibers that may act as scaffolds or leads as shown in
Determination of Nucleation/Seed Sites
In vivo data has shown fibers growing from ANA-3, inanimate particulate matter, and empty sections of glass. This suggests that the nucleation sites are not restricted to the cells themselves. To verify, ANA-3 was inoculated into two MM bottles with 20 mM lactate and 10 mM thiosulfate but no arsenic. After 27 hours an estimated 99% of the original cell density was removed via filtration with a 220 nm filter. Remaining cells in both cultures were killed with kanamycin (50 and 100 μg/mL in filtered and unfiltered, respectively). The resulting bottles shown in
Nanofiber Field Effect Transistor
The Field Effect Transistor (FET) is an important device, both for its applications in electronics and as a tool to probe the properties of semiconductors. A MOSFET is a FET characterized by a meta-oxide-semiconductor (MOS) structure consistent of a metallic acting gate (usually highly doped polysilicon), an insulating oxide layer (usually SiO2), and the underlying semiconductor as shown in
The above As—S nanofiber material may be applied to different semiconductor devices such as the MOSFET described above. The versatility of the As—S nanofiber material in relation to light leads to a wide range of tunable functionalities in various components including sensors, waveguides, photonic crystals, infrared and photoactive devices. In such devices, the nanofiber material may be used advantageously for the semiconductor material.
For example, fabrication and testing of an inverted MOSFET structure may be done where a biotically influenced AsxSy nanofiber fabricated according to the above mentioned process serves as the semiconducting material as shown in
Fibers were drop deposited onto polydoped silicon chips with an SiO2 layer topped with gold contacts as shown in
Nonlinear ISD−VSD curves shown in
Nonlinear ISD−VSD curves shown in
62% of fabricated devices were P-type, 23% were N-type, and 15% were too complex to fit clearly as one type. For example,
The higher percentage of P-type fibers is thought to be due to carbon acting as a dopant among the dominant As2S3 glass. Deviations from (2,3) stoichiometry may also lead to P-type fibers (As2+XS3) or N-type fibers (As2S3+X). The effect of the present crystalline β-As4S4 is unknown, though from a purely stoichiometric standpoint β-As4S4 would increase the ratio of arsenic, suggesting P-type behavior.
Regardless of physical geometry, the devices have similar properties to traditional MOSFETs, such as the threshold voltage and linear mobility. More AsxSy nanofiber FET devices can be constructed and characterized to provide average values of these properties.
Measure the Optomechanical Effect in Nanostructures
The optomechanical properties of As40Se60 and As40S60 amorphous films have been reported. No work, as of yet, has been published exploring the optomechanical properties of As2S3 when formed as a quai-1D nanostructure. In the optical microscope, several fibers can be observed exhibiting plastic deformation upon exposure to high intensity non-filtered light. The characterization of this effect for biotically influenced nanofibers is new information on this property.
Conclusion
The biological route proposed is scalable via large-volume bioreactors and offers a unique approach to combinatorial materials science by taking advantage of comparative genomic analyses to discover microorganisms capable of processing different metals and chalcogens. The targeted materials have the potential to serve energy-conversion needs in photovoltaic solar cells and micro-opto-mechanical systems. The optomechanical effect, in particular, can be used for all-optical actuation in flammable and aqueous environments where the use of high voltages (e.g., piezoelectric devices) would be hazardous.
For the disclosed model system of ANA-3 and AsxSy, it has been demonstrated that arsenic reduction is being done by the cell, but not by using either of the two known reductases. The inventors have verified reported properties such as composition and diverse general morphology. The inventors have also measured several new properties of the fibers, such as growth rate, primary crystalline phase, band gap, and semiconducting type. The nature of nanofiber nucleation points and single fiber crystal structure will continue to be investigated. Fabrication of nanofiber FET devices and optomechanical studies hold the potential to be both illuminating and applicable to the nanotech industry.
Embodiments have been described in relation to particular examples, which are intended in all respects to be illustrative rather than restrictive. Further, while embodiments have been described in connection with a number of examples and implementations, it is understood that various modifications and equivalent arrangements can be made to the examples while remaining within the scope of the inventive embodiments.
Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the embodiments disclosed herein. Various aspects and/or components of the described embodiments may be used singly or in any combination. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.
The present disclosure claims priority to U.S. Provisional Application 61/770,483, filed on Feb. 28, 2013, which is hereby incorporated by reference in its entirety.
Number | Date | Country | |
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61770483 | Feb 2013 | US |