Bacterial spores as environmentally friendly and efficient bio-inorganic hybrid phosphors

Abstract

Rare earth elements (REEs) are crucial raw materials for modern technologies such as light-emitting diodes (LEDs), solar panels, and solar cells. Issues associated with these materials include geopolitical supply risks, environmental concerns, and recycling challenges. To address these challenges, enhancing the performance efficiency of lanthanide-based phosphors, simplifying their manufacturing processes, and reusing them are key priorities. In this study, we demonstrate the remarkable ability of Bacillus and Clostridium spores as biomaterials to sequester lanthanides and function as phosphors. Adding antenna molecules such as thenoyltrifluoroacetone (TTA) can increase the quantum yield of the phosphors and specifically increase the amount of lanthanides sequestered by Clostridium spores. Furthermore, adding strontium as a co-dopant to the spores can double the luminescence output from the spores. Furthermore, various colors can be obtained by varying the lanthanide composition. UV LEDs coated with spore phosphors have significantly higher conversion efficiencies than control phosphors. In addition to the improved performance provided by the use of spores, the inert properties of spores facilitate easy production and reuse, making them a promising material candidate in line with our goal of minimizing environmental impact by maximizing the utilization of precious and competitive lanthanide resources.

Description

[Technical Field]

[0001] The present invention relates to phosphor materials, methods for forming said phosphor materials, composite materials including said phosphor materials, and improved light emitting diodes (LEDs) including said composite materials. [Background technology]

[0002] The listing of previously published documents cited in this specification and the discussion contained therein need not necessarily be acknowledged as forming part of the state of the art or common general knowledge.

[0003] Fluorescent materials are materials that emit light in a very specific way called phosphorescence. Phosphorescence means that the fluorescent material can emit light for a long period of time (ranging from microseconds to milliseconds or longer) after receiving energy and becoming excited. Generally, fluorescent materials are composed of a host material or compound that can absorb energy and transfer it to an activator, which is the material that actually emits light. When the activator receives energy from the host, it becomes excited and transitions to a higher-energy singlet or triplet state. When the activator ion returns to a lower-energy state, the excess energy is released in the form of photons, which are perceived as light. The host's role is to provide an environment in which the absorbed energy can be efficiently transferred to the activator ion. The exact characteristics of the emitted phosphorescence depend on the energy transfer between the host and activator materials.

[0004] Phosphors are constructed by doping a host material with an activator. Most commercially available phosphors are doped with transition metal or rare-earth ions as activators. Rare-earth ions (also known as lanthanide ions) contain many unpaired electrons in their 4f orbitals compared to transition metals, shielding the 6s valence electrons. This shielding effect minimizes vibrational losses to the environment, resulting in sharp, linear emission spectra. Furthermore, emission from electron transitions between 4f-4f orbitals is parity-forbidden, resulting in long-duration phosphorescence. Another important property of lanthanide ions is their significantly longer Stokes shift, which reduces self-absorption / quenching effects and improves luminescence efficiency. These advantageous luminescence properties have revolutionized the majority of photon-output applications (e.g., lighting, display technology, biological assays, and lasers) as well as photon-input applications (e.g., medical imaging and photovoltaic cells).

[0005] However, there are several challenges that could affect the market for these materials. These challenges include: 1. Resource scarcity and geopolitical risks: The majority of the world's rare earth elements are supplied by only a few countries, and in most other countries, a stable supply of rare earth elements is highly dependent on geopolitical stability. 2. Environmental Issues: Extraction and processing of rare earth elements often involves environmentally harmful processes, including the release of radioactive waste. As a result, environmental regulators are increasing their scrutiny, and the costs associated with complying with stricter environmental standards may impact the market. 3. Recycling challenges: Rare earth elements are difficult to recycle and are expensive to recycle, which contributes to supply problems. While improving the efficiency of recycling processes could alleviate the problems associated with recycling, developing such technologies takes time and requires huge investments. 4. Color Quality: While LED technology has made great strides, many consumers prefer the warm light of incandescent bulbs, and recreating this quality remains challenging. Advances in phosphor technology are helping to address this issue, but it remains a concern. 5. Supply Chain Issues: Many of the materials used in LED manufacturing, such as rare earth elements, are subject to price fluctuations and supply chain disruptions, especially during times of geopolitical tension or global economic instability.

[0006] For rare-earth phosphors to function efficiently with high quantum yields in various applications, they require suitable host materials that maximize energy absorption. A good host material must satisfy several characteristics (Zhuo, Y. et al., Nat. Commun. 2018, 9, 4377; Hermus, M. & Brgoch, J. The Electrochemical Society Interface, 2015, 24, 55). In addition to compatibility with the dopant ions, a good host material must possess low phonon energy, high transparency, high chemical stability, high mechanical strength, preferably a high refractive index, and an appropriate energy gap to match the energy levels of the dopant ions and promote efficient energy transfer. Given these numerous critical parameters, it is not surprising that the most common host materials are naturally occurring inorganic compounds, such as garnets, perovskites, nitrides, silicates, and borates. The phosphorescent properties of inorganic lattices can be tuned by adjusting the lattice composition (Zhao, M. et al., Sci. Adv. 2019, 5, eaav0363; Wang, S.-S. et al., J. Am. Chem. Soc., 2013, 135, 12504) and rare earth dopant concentration (Malik, C. et al., Radiat. Phys. Chem., 2020, 168, 108561; Zhang, J. et al., Inorg. Chem., 2020, 59, 2241).However, obtaining the desired lattice composition requires complex and tedious manufacturing conditions, such as high temperature (Lavat, A. et al., Cryst. Res. Technol., 2004, 39, 840; Upadhyay, K. et al., Superlattices Microstruct., 2015, 78, 116; Chen, X. et al., Minerals, 2017, 7, 44), pressure (Panda, DP et al., J. Mater. Chem., 2022, 10, 16723; Atuchin, VV et al., ACS Appl. Mater. Interfaces, 2015, 7, 26235), and the use of corrosive agents such as ammonium hydroxide and nitric acid (Gao, W. et al., J. Rare Earths, 2009, 27, 886). In some cases, the manufacturing process of the host materials generates large amounts of heat and toxic gases, which have a negative impact on the environment (Deyneko, DV et al., J. Alloys Compd., 2021, 887, 161340). Other drawbacks of inorganic host materials include limited tunability, difficulty in incorporating them into flexible or thin film forms, re-absorption of light emitted from dopant ions, and, in the case of nanoscale inorganic phosphors, difficulty in controlling the size and shape (Chiriu, D. et al., Phys. Status Solidi C, 2016, 13, 989).

[0007] Given these challenges, organic materials have been developed that offer improved tunability, flexibility, and photonic properties, potentially compensating for some of the shortcomings of inorganic materials (Zhao, Y.-W. et al., ACS Appl. Mater. Interfaces, 2016, 8, 24123; Song, B. et al., ACS Appl. Mater. Interfaces, 2020, 12, 6137; Lu, Y. & Yan, B., Chem. Commun., 2014, 50, 15443; Zhang, N.-N. et al., Chem. Commun., 2017, 53, 9269). However, organic materials themselves present challenges related to their host. Not surprisingly, organic materials generally have poorer thermal and chemical stability than inorganic materials. Organic materials are often sensitive to oxygen and moisture, which can quench phosphorescence and lead to material decomposition. Therefore, organic phosphors must be encapsulated to prevent exposure to air. Silent energy dissipation due to vibrations or swings leads to extremely fast intersystem crossing transitions, resulting in short phosphorescence lifetimes in many organic fluorophores.

[0008] Bacterial spores are dormant, hardy structures constructed by nature to withstand harsh environmental conditions.

[0009] Previous research on bacteria and lanthanides has focused solely on the ability of bacteria to sequester lanthanides for bioremediation purposes. Specifically, to the best of our knowledge, there are few publications addressing the accumulation of lanthanides in spores in the context of mining / extraction or bioremediation (Dong, W. et al. Minerals, 2022, 12, 866; Dong, W. et al., Appl. Environ. Microbiol., 2019, 85, e00956-19; Cockell, C.S. et al., Nat. Commun., 2020, 11, 5523).

[0010] The present inventors are not aware of any prior art that describes the combination of spores and lanthanides to obtain luminescence. Although many studies have been reported on the use of bacterial spores to recycle lanthanides, none of these studies have actually demonstrated the functionality of the phosphor. Summary of the Invention [Means for solving the problem]

[0011] Surprisingly, it has been found that incorporating small amounts of phosphors into bacterial spores can produce results similar to current phosphor technology. These results are achieved by improving the phosphorescence efficiency of rare earth elements, potentially optimizing scarce rare earth element resources and mitigating issues related to the environmental and recycling challenges associated with rare earth metals. Furthermore, the manufacturing processes required to produce such phosphors are easy to implement and energy-saving, making them environmentally friendly.

[0012] Aspects and embodiments of the present invention are described below with reference to the following numbered sections: 1. A phosphor material comprising: Bacterial spores as host material; a lanthanide dopant within and / or on the surface of said host material; Including, the bacterial spores are dormant, the bacterial spore is selected from one or more bacterial spores selected from one or more of the genera Bacillus and Clostridium; Phosphor material. 2. The lanthanide dopant is Eu 3+ , Sm 3+ , Tb 3+ and Dy 3+ and Eu 3+ , Sm 3+ and Tb 3+ may be selected from one or more of the group consisting of (e.g., Eu 3+ Item 1. The phosphor material according to Item 1. 3. The phosphor material according to item 1 or 2, wherein the bacterial spores are formed from one or more of Bacillus megaterium, Bacillus cereus, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium botulinum, Clostridium difficile, Clostridium novyi-NT, and Clostridium septicum CS11, and may be formed from one or more of Bacillus megaterium, Clostridium novyi-NT, and Clostridium septicum (for example, Clostridium novyi-NT, Clostridium septicum, or both). 4. The phosphor material of any one of the preceding claims, further comprising an antenna molecule complexed with the lanthanide-based dopant. 5. The phosphor material according to item 4, wherein the antenna molecule is selected from one or more of the group consisting of β-diketones and triphenylphosphine oxides. 6. The antenna molecule is an aromatic β-diketone, and the aromatic β-diketone may be selected from the group consisting of thenoyltrifluoroacetone (TTA), 1-(-2-naphthoyl)-3,3,3-trifluoroacetone (NTFA), and 4,4,4-trifluoro-1-phenyl-1,3-butanedione (BTFA). The phosphor material according to item 4. 7. The antenna molecule is complexed with the lanthanide dopant, and the complex has the formula Ln[ANT] n (wherein Ln is a member of the lanthanoid series, ANT is an antenna molecule, and n is an integer, which may be 3 to 300, for example, 30). 8. The above Ln[ANT] nis selected from one or more of the group consisting of Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, Tb[TTA]3, Eu[BTFA]3, and Eu[NTFA]3, and may be selected from one or more of the group consisting of Eu[TTA]3, Sm[TTA]3, and Tb[TTA]3 (e.g., Eu[TTA]3 and Tb[TTA]3, e.g., Eu[TTA]3). 9. The phosphor material according to any one of items 4 to 8, wherein the molar ratio of the lanthanoid dopant to the antenna molecule is 1:300 to 1:3, for example, 1:200 to 1:10, for example, 1:100 to 1:20, or for example, approximately 1:30. 10. The method further comprising the step of: 2+ and Ca 2+ Item 10. The phosphor material of any one of the preceding items, wherein the phosphor material is selected from one or more of the group consisting of: 11. The phosphor material according to any one of the preceding paragraphs, wherein the content of the lanthanoid dopant is 20 to 200 nmol per 1 mg of dry spore biomass. 12. A composite material comprising: Item 12. The phosphor material according to any one of items 1 to 11, Bulk materials and Including, the bulk material is selected from one or more of the group consisting of a resin, a coating, an adhesive, and an encapsulating material; the bulk material is optically transparent or optically translucent to the excitation and emission wavelengths of the phosphor material; Composite material. 13. The bulk material includes an adhesive, the adhesive being optically transparent to the excitation wavelength and emission wavelength of the phosphor material, Norland TM Item 13. The composite material according to item 12, which may be 88. 14. An improved light emitting diode, comprising: a light emitting diode having a light emitting diode bulb portion; A cured composite material containing the phosphor material according to any one of items 1 to 11, an optically transparent adhesive that covers a surface of the light emitting diode bulb portion so that light from the light emitting diode passes through the cured composite material; Improved light emitting diodes, including: 15. The improved light emitting diode according to paragraph 14, wherein the light emitting diode is an ultraviolet light emitting diode. 16. A method for forming the phosphor material according to any one of items 1 to 11, (a) providing a plurality of bacterial spores of the genera Bacillus and Clostridium; and (b) adding a lanthanide material to the plurality of bacterial spores to obtain a phosphor material. Including, the lanthanide material is a lanthanide salt or a lanthanide complexed with an antenna molecule; method. 17. The method of claim 16, wherein the lanthanide material is added to the bacterial spores in a solvent. 18. The method according to item 17, wherein the solvent comprises water and ethanol, and the ethanol may be 1 to 50% vol / vol, for example, about 3% vol / vol. 19. The method according to item 17 or 18, wherein the concentration of the lanthanide material in the solvent is 0.005 mM to 10 mM, for example, 0.01 mM to 3 mM, for example, 0.015 mM to 1.5 mM, for example, 0.15 mM to 0.75 mM. 20. The method according to any one of items 17 to 19, wherein, when the antenna molecule is contained, the content of the antenna molecule is 0.1 mM to 10 mM, for example, about 4.5 mM. 21. The method according to any one of items 17 to 20, further comprising the step of substantially removing the solvent from the phosphor material. [Brief explanation of the drawings]

[0013] [Figure 1] 1 illustrates the overall conceptual concept of the present invention. [Figure 2]This shows that bacterial spores sequester lanthanides in the presence of β-diketones. Cultures of dormant spores, germinated spores, and overnight vegetative cultures of each bacteria were supplemented with 0.14 mM europium (Eu) and 4.2 mM TTA, or 0.14 mM Eu alone, and the Eu content in the supernatant was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). Each graph shows the Eu content (nmol / mg) per biomass weight in spore and vegetative forms of B. subtilis (A), B. thuringiensis (B), C. novyi-NT (C), or C. septicum-CS11 (D) in the presence (circles) or absence (squares) of TTA. Data are presented as mean ± SD, with two biological replicates. [Figure 3] This figure shows that Clostridium spore fluorophores exhibit high phosphorescence in the presence of the β-diketone TTA. Spores loaded with Eu or Eu-TTA were dried under high vacuum in a UV-transparent 384-well microplate to prepare spore fluorophores, and their fluorescence was measured. Images of dried spore fluorophores of (A) B. subtilis (column 1), B. thuringiensis (column 3), C. novyi-NT (column 5), and C. septicum CS11 (column 7) doped with 1.4 mM Eu (rows 1 and 2), 0.14 mM Eu (rows 3 and 4), or Eu-TTA (4.2 mM) and a control without spore fluorophores (column 9) were excited at 365 nm using a UV transilluminator. The solvent was 3% ethanol. Images were taken using a Samsung S10 Plus smartphone. Fluorescence intensity values ​​of all bacterial spore fluorophores were measured at 620 nm and plotted against fluorescence delay time in the presence of (B) TTA (4.2 mM) or (C) various Eu concentrations (1.4 mM (triangles), 0.14 mM (circles), and 0.014 mM (squares)) in the absence of TTA. All samples were excited at 370 nm. Data are shown as mean ± SEM, with n = 4 biological replicates. [Figure 4]Among Bacillus spore fluorophores, B. megaterium exhibits the highest phosphorescence in the presence of the β-diketone TTA. Spores loaded with Eu or Eu-TTA were dried under high vacuum in a UV-transparent 384-well microplate to prepare spore fluorophores, and their fluorescence was measured. Images of dried spore fluorophores of (A) B. subtilis (column 1), B. thuringiensis (column 4), B. cereus (column 7), B. megaterium (column 10), C. novyi-NT (column 13), and C. septicum CS11 (column 16) doped with 0.14 mM Eu or Eu-TTA (4.2 mM), as well as a control without spore fluorophores (column 19), excited at 365 nm using a UV transilluminator, are shown. The solvent used was 3% ethanol. Images were taken using a Google Pixel 6a smartphone. (B) Fluorescence intensity values ​​of all bacterial spore fluorophores loaded with 0.14 mM Eu were measured at 620 nm and plotted against fluorescence delay time in the presence (circles) or absence (triangles) of TTA (4.2 mM). All samples were excited at 370 nm. Data are shown as mean ± SEM, with n = 2 biological replicates. [Figure 5] Excitation-emission matrix (EEM) of europium spore fluorophores is shown. Spore fluorophores were prepared by drying Eu or Eu-TTA-loaded spores under high vacuum in a UV-transparent 384-well microplate, or by using the respective solutions before drying. (A) EEM contour maps of C. novyi-NT fluorophores, C. septicum CS11 fluorophores, and a control fluorophor without spores loaded with Eu (1.4 mM)-TTA (4.2 mM) are shown in the solid (left panel) or liquid (right panel) states. (B) EEM contour maps of C. novyi-NT fluorophores, C. septicum CS11 fluorophores, and a control fluorophor without spores loaded with Eu (1.4 mM) are shown in the solid (left panel) or liquid (right panel) states. The intensity scale indicates the fluorescence intensity on a log10 scale. [Figure 6]This demonstrates that the clostridial spore fluorescent material can be stored for several months. Spore fluorescent material was prepared by drying Eu- or Eu-TTA-loaded spores under vacuum in a UV-transparent 384-well microplate, and fluorescence was monitored at specific time points. Fluorescence intensity values ​​of all bacterial spore fluorescent material were measured at 620 nm with a 20 μs delay time in the presence or absence of TTA (4.2 mM) and various Eu concentrations (1.4 mM or 0.14 mM). The values ​​are plotted against the number of weeks measured. All samples were excited at 370 nm. Data are shown as mean ± SEM, with n = 4 biological replicates. [Figure 7] This demonstrates that various lanthanides can be doped to produce spore fluorophores of various colors. These spore fluorophores were prepared by drying spores loaded with europium (Eu), samarium (Sm), terbium (Tb), or dysprosium (Dy), or Eu-TTA, Sm-TTA, Tb-TTA, or Dy-TTA, in polymerase chain reaction (PCR) tubes. (A) Images of dried C. novyi-NT spore fluorophores doped with 0.14 mM of each lanthanide (Ln) or Ln-TTA (4.2 mM) were shown using a UV transilluminator at 254 nm (upper panel) or 365 nm (lower panel). (B) Images of dried C. septicum CS11 spores doped with 0.14 mM of each lanthanide (Ln) or 4.2 mM of Ln-TTA were taken using a Google Pixel 6a smartphone, excited at 254 nm (upper panel) or 365 nm (lower panel) under a UV transilluminator. [Figure 8]Excitation-emission matrix (EEM) of samarium spore fluorophores. Spore fluorophores loaded with Sm or Sm-TTA were prepared by drying under high vacuum in a UV-transparent 384-well microplate, or prepared in the respective solution forms before drying. EEM measurements were performed on these fluorophores. (A) EEM contour maps of C. novyi-NT fluorophores, C. septicum CS11 fluorophores, and a control fluorophor without spores loaded with Sm (1.4 mM)-TTA (4.2 mM) are shown in the solid (left panel) or liquid (right panel) states. (B) EEM contour maps of C. novyi-NT fluorophores, C. septicum CS11 fluorophores, and a control fluorophor without spores loaded with Sm (1.4 mM) are shown in the solid (left panel) or liquid (right panel) states. The intensity scale indicates the fluorescence intensity on a log10 scale. [Figure 9] Excitation-emission matrix (EEM) of terbium spore fluorophores is shown. Spore fluorophores were prepared by drying Tb- or Tb-TTA-loaded spores under high vacuum in a UV-transparent 384-well microplate, or by using the respective solutions before drying. EEM measurements were performed on these fluorophores. (A) EEM contour maps of C. novyi-NT fluorophores, C. septicum CS11 fluorophores, and a control fluorophor without spores loaded with Tb (1.4 mM)-TTA (4.2 mM) are shown in the solid (left panel) or liquid (right panel) states. (B) EEM contour maps of C. novyi-NT fluorophores, C. septicum CS11 fluorophores, and a control fluorophor without spores loaded with Tb (1.4 mM) are shown in the solid (left panel) or liquid (right panel) states. The intensity scale indicates the log10 scale of fluorescence intensity. [Figure 10]Co-doping of strontium into Eu-TTA spore fluorophores increases fluorescence. Eu-Sr-TTA or Eu-TTA-loaded spores were dried under high vacuum in PCR tubes or UV-transparent 384-well microplates to prepare spore fluorophores, and their fluorescence was measured. (A) Images of dried C. novyi-NT spore fluorophores doped with 0.06 mM or 1.9 mM Eu-Sr (476 mM)-TTA (5.4 mM) or 0.06 mM or 1.9 mM Eu-TTA (5.4 mM) in PCR tubes using a UV transilluminator with excitation at 365 nm, shown with (lower panel) and without (upper panel) a long-pass filter. Images were taken using a Samsung S10 Plus smartphone. Fluorescence intensity values ​​of C. novyi-NT spores loaded with 1.9 mM Eu (B) or 0.06 mM Eu (C) and TTA (5.4 mM) in the presence (circles) or absence (triangles) of strontium (476 mM) were measured at 620 nm and plotted against the fluorescence delay time. All samples were excited at 370 nm. Mean ± SD, n = 6 biological replicates. [Figure 11]This figure shows a comparison of Eu-TTA spore phosphors with commercially available red yttrium oxide phosphors. Spores doped with 0.14 mM Eu-TTA (4.2 mM) and yttrium oxide suspensions (containing 0.14 mM Eu) were dried under high vacuum in a UV-transparent 384-well microplate to prepare solid spore phosphors and yttrium oxide phosphors, respectively, and their fluorescence was measured. (A) Images of solid C. novyi-NT spore phosphors doped with 0.14 mM Eu-TTA (4.2 mM) (solid, column 2; liquid, column 6) and yttrium oxide phosphors containing 0.14 mM Eu (solid, column 4; liquid, column 8) excited at 254 nm using a UV transilluminator are shown. Images were taken using a Google Pixel 6 Pro smartphone. (B) Fluorescence intensity values ​​of spore fluorophores and yttrium oxide fluorophores in liquid and solid states when excited at 250 nm. (C) Fluorescence intensity values ​​of spore fluorophores and yttrium oxide fluorophores in liquid and solid states when excited at 370 nm. Fluorescence intensity values ​​for (B) and (C) were measured at 620 nm with no delay. Data are shown as mean ± SD, with n = 3 biological replicates. [Figure 12]We demonstrate that different color LEDs can be obtained by coating a UV LED with Ln-TTA spore phosphor. Spores loaded with Eu(0.14 mM)-TTA(4.2 mM) or Tb(0.14 mM)-TTA(4.2 mM) were dried under reduced pressure to prepare the spore phosphor, which was then mixed with a UV-transparent adhesive, Norland 88, and coated onto a UV LED. (A) Images are shown of a UV LED coated with dried Eu-TTA and a UV LED coated with 0.14 mM Eu-TTA (4.2 mM)-doped C. novyi-NT spore phosphor, excited at 365 nm using a 9 V battery. (B) Images of UV LEDs coated with Eu or Eu-TTA and dried, and UV LEDs coated with 0.14 mM Eu-TTA (4.2 mM) or 0.14 mM Eu-doped C. novyi-NT spore phosphors, excited at 365 nm using a 9 V battery in 3% ethanol. (C) Images of UV LEDs coated with Tb or Tb-TTA and dried, and UV LEDs coated with 0.14 mM Tb-TTA (4.2 mM) or 0.14 mM Tb-doped C. novyi-NT spore phosphors, excited at 275 nm using multiple 9 V batteries connected in series. The solvent is 50% ethanol. Images were taken with a Google Pixel 6 Pro smartphone (A) or a Nikon COOLPIX L120 DSLR camera with (B) a long-pass filter or (C) without a long-pass filter. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure describes the subject matter encompassed by the present disclosure. Accordingly, the subject matter of the present disclosure is not limited to the forms or embodiments set forth herein.

[0015] Surprisingly, we have found that bacterial spores can function as host materials for rare-earth metals, providing surprisingly excellent phosphors. Bacillus and Clostridium spores were found to efficiently sequester lanthanides and function as efficient phosphors. Furthermore, we found that these phosphors are very easy to prepare, simply adding lanthanides to the spores in solution. Phosphorescence can potentially be further enhanced by adding antenna molecules and codopants. Based on current results, Clostridium strains appear to be superior to Bacillus strains in terms of phosphorescence output and shelf life. As a basic application, we have demonstrated that ultraviolet (UV) light can be efficiently converted to red light by coating spore phosphors on ultraviolet (UV) LEDs.

[0016] Thus, in a first aspect of the present invention, there is provided a phosphor material comprising: Bacterial spores as host material; a lanthanide dopant within and / or on the surface of said host material; Including, the bacterial spores are dormant, the bacterial spore is selected from one or more bacterial spores selected from one or more of the genera Bacillus and Clostridium; A phosphor material is provided.

[0017] In the embodiments described herein, the term "comprising" may be interpreted to require the features described herein, but not to limit the presence of other features. Alternatively, the term "comprising" may relate to a situation in which only the components / features described herein are present (e.g., the term "comprising" may be replaced with the terms "consisting of" or "essentially consisting of"). It is expressly intended that both such broad and narrow interpretations are applicable to all aspects and embodiments of the present invention. In other words, the term "comprising" and its equivalents may be replaced with the terms "consisting of" or "substantially consisting of," and vice versa.

[0018] As used herein, the term "substantially consisting of" and synonyms thereof may be interpreted to mean a material that may contain trace amounts of impurities. For example, the purity of this material may be 90% or more, such as greater than 95%, such as greater than 97%, such as greater than 99%, such as greater than 99.9%, such as greater than 99.99%, such as greater than 99.999%, or such as 100% purity.

[0019] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "composition" includes a mixture of two or more compositions, and the like.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although the present specification describes methods and materials used in the present invention, other suitable methods and materials known in the art can also be used. The materials, methods, and examples described herein are for illustrative purposes only and are not intended to limit the present invention. All published documents, patent applications, patents, sequences, databases, and other references mentioned herein are incorporated by reference in their entirety. If the definitions and other descriptions do not match the descriptions in the cited documents, the descriptions in the present specification shall control.

[0021] Other features and advantages of the invention will become apparent from the following detailed description of the invention, the drawings and the claims.

[0022] Concentrations, amounts, and other numerical data may be expressed or described in range format herein. Such ranges are used solely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​expressly recited as the upper and lower limits of the range, but also to include any individual numerical values ​​or subranges within the range, as if those numerical values ​​or subranges were expressly recited. As an example, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly recited numerical value of about 0.01 to about 2.0, but also each individual numerical value and subrange within that range. Thus, this numerical range also includes individual numerical values ​​such as 0.5, 0.7, and 1.5, as well as subranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5. Such interpretation should apply regardless of the breadth of the range or the characteristics described. Furthermore, unless otherwise noted, all percentages are by weight.

[0023] In this specification, multiple compounds, components, or steps may be presented in a general list for convenience. However, such a list should be construed as individually recognizing each member of the list as a separate and unique member. Accordingly, the individual members of such a list should not be construed as being in fact equivalent to other members in the same list solely based on being listed in a common group, unless otherwise specified.

[0024] As described above, it has been discovered that bacterial spores may be used as a suitable host material for enhancing the phosphorescent properties of lanthanide-based dopant materials. The lanthanide-based dopant material may be present inside the bacterial spore, on the surface of the bacterial spore, or both inside and on the surface of the bacterial spore. In the present invention, the bacterial spores are provided in a dormant state because this state facilitates their integration into displays and consumer electronic devices. Any suitable bacterial spore may be used in the present invention. For example, the bacterial spore may be selected from one or more bacterial spores selected from one or more of the genera Bacillus and Clostridium.

[0025] The lanthanide dopant material can be any suitable material. For example, the lanthanide dopant can be Eu 3+ , Sm 3+ , Tb 3+ and Dy 3+ In certain embodiments of the present invention, the lanthanide dopant may be selected from one or more of the group consisting of Eu 3+ , Sm 3+ and Tb 3+ may be selected from one or more of the group consisting of (e.g., Eu 3+ may be).

[0026] Bacterial spores include Bacillus megaterium, Bacillus cereus, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium botulinum, Clostridium difficile, Clostridium novyi-NT, and Clostridium septicum. The bacterial spores may be formed from one or more of Bacillus megaterium, Clostridium novyi-NT, and Clostridium septicum (e.g., Clostridium novyi-NT or Clostridium septicum, or both).

[0027] In certain embodiments of the invention that may be mentioned herein, the bacterial spore may be selected from the genus Clostridium. Thus, the bacterial spore may be selected from one or more of Clostridium acetobutylicum, Clostridium butyricum, Clostridium botulinum, Clostridium difficile, Clostridium novyi-NT, and Clostridium septicum CS11 (e.g., Clostridium novyi-NT, Clostridium septicum, or both).

[0028] In certain embodiments of the present invention, the phosphor material may further include an antenna molecule that forms a complex with the lanthanide-based dopant. In other embodiments of the present invention that may be mentioned herein, the phosphor material may be a phosphor material that does not include an antenna molecule that can form a complex with the lanthanide-based dopant. In embodiments that include an antenna molecule, the antenna molecule may be selected from one or more of the group consisting of β-diketones and triphenylphosphine oxides. In even more specific embodiments that include an antenna molecule, the antenna molecule may be an aromatic β-diketone.

[0029] Any suitable aromatic β-diketone capable of acting as an antenna molecule may be used in the present invention, including, but not limited to, thenoyltrifluoroacetone (TTA), 1-(-2-naphthoyl)-3,3,3-trifluoroacetone (NTFA), 4,4,4-trifluoro-1-phenyl-1,3-butanedione (BTFA), and combinations thereof.

[0030] In embodiments where an antenna molecule is included, the antenna molecule is complexed with a lanthanide dopant, and the complex has the formula Ln[ANT] n wherein Ln is a member of the lanthanide series, ANT is an antenna molecule, and n is an integer between 3 and 300, e.g., 30. In certain embodiments of the invention, Ln[ANT] n may be selected from one or more of the group consisting of Eu[TTA]3, Sm[TTA]3, Dy[TTA]3, Tb[TTA]3, Eu[BTFA]3, and Eu[NTFA]3. In more specific embodiments of the present invention, Ln[ANT] n may be selected from one or more of the group consisting of Eu[TTA]3, Sm[TTA]3, and Tb[TTA]3 (e.g., may be Eu[TTA]3 and Tb[TTA]3, e.g., may be Eu[TTA]3).

[0031] In embodiments including an antenna molecule, the antenna molecule is complexed with a lanthanide-based dopant, and the two components may be used in any suitable molar ratio, such as 1:300 to 1:3, such as 1:200 to 1:10, such as 1:100 to 1:20, or about 1:30.

[0032] In certain embodiments, the phosphor materials disclosed herein may further comprise a co-dopant material. Any suitable co-dopant material may be used. For example, the co-dopant material may be Sr 2+ Or Ca 2+ Or it may be both.

[0033] The lanthanoid dopant may be contained in a suitable amount, for example, 20 to 200 nmol per 1 mg of dry spore biomass.

[0034] As will be readily appreciated, the phosphor materials disclosed herein may be suitable for use in making materials that can be used in a variety of light emitting applications. Thus, in a second aspect of the present invention, there is provided a composite material comprising: a phosphor material as previously described herein; Bulk materials and Including, the bulk material is selected from one or more of the group consisting of a resin, a coating, an adhesive, and an encapsulating material; the bulk material is optically transparent or optically translucent to the excitation and emission wavelengths of the phosphor material; A composite material is provided.

[0035] As previously mentioned, the bulk material may be any of resins, coatings, adhesives, and encapsulating materials. As can be readily understood, these materials are intended to be permanently solid in their final state at the operating temperature of the appropriate system (e.g., an LED), and therefore the operating temperature of these materials may be at or near ambient temperature, or even slightly above ambient temperature. Therefore, the bulk material may be utilized to apply and hold phosphors in place for use in a particular device, such as an LED or LED display. It can also be readily understood that the bulk material may be any suitable material, but should not affect the dormancy of bacterial spores. Therefore, the bulk material preferably does not contain water.

[0036] The term "optically transparent" refers to the property of a material that allows light to pass through it without any perceptible scattering (i.e., no perceptible scattering of light at the level of the naked eye, and photons obey Snell's law). The material may therefore be a single component or multiple components with a uniform refractive index. In effect, a transparent material allows light to pass through it, allowing objects behind it to be clearly seen.

[0037] The term "optically translucent" refers to a material's property of allowing light to pass through but causing noticeable scattering of light (i.e., scattering of light is noticeable to the naked eye and photons do not obey Snell's law). Thus, the material may be formed from a single component with different refractive indices or multiple components with different refractive indices. In effect, a translucent material allows light to pass through, but objects behind the material may not be clearly visible.

[0038] As will be readily understood, if a bulk material is a single optically transparent material, then the bulk material is optically transparent, whereas if the bulk material is formed of two or more optically transparent materials, then the bulk material may be optically transparent (if the two or more materials have a uniform refractive index) or optically translucent (if the two or more materials have different refractive indices).

[0039] As noted herein, the bulk material or materials may be selected to be optically transparent or optically translucent to the excitation and emission wavelengths of the phosphor material. For example, the phosphor materials used in the examples have excitation and emission wavelengths in the UV spectrum, and therefore, Norland 88 adhesive, which is UV transparent, was selected for use in the Examples section of this specification.

[0040] As used herein, the term "encapsulant" may refer to a material intended to completely or partially encapsulate the phosphor material and thus serve to physically protect the phosphor material. Any suitable encapsulant may be used in the present invention. For example, the encapsulant may be selected from one or more of the following groups: epoxy, epoxy / silicon hybrid, silicone, and glass, but is not limited thereto. In addition to its physical protection function, the encapsulant may also reduce light coupling loss and allow light (e.g., LED light) to be incident at a specific viewing angle.

[0041] As previously mentioned, the composite materials (ie, phosphor materials) of the present invention may be particularly useful in LED devices. Accordingly, in a third aspect of the present invention there is provided an improved light emitting diode comprising: a light emitting diode having a light emitting diode bulb portion; a cured composite material comprising the phosphor material described herein; an optically transparent adhesive that covers a surface of the light emitting diode bulb portion so that light from the light emitting diode passes through the cured composite material; An improved light emitting diode is provided, including:

[0042] The light emitting diode may be any suitable light emitting diode, for example, the light emitting diode may be an ultraviolet light emitting diode.

[0043] In a fourth aspect of the present invention, there is provided a method of forming the phosphor material previously described herein, comprising the steps of: (a) providing a plurality of bacterial spores of the genera Bacillus and Clostridium; and (b) adding a lanthanide material to the plurality of bacterial spores to obtain a phosphor material. Including, A method is provided wherein the lanthanide material is a lanthanide salt or a lanthanide complexed with an antenna molecule.

[0044] These components have already been described in connection with the phosphor materials earlier in this specification and, for the sake of brevity, will not be described in detail here.

[0045] In the method for forming the phosphor material of the present invention, the lanthanoid material may be provided in any suitable manner. For example, the lanthanoid material may be provided to the bacterial spores in a solvent. Any suitable solvent may be used to provide the lanthanoid material. For example, the solvent may include water and ethanol (e.g., the solvent may have a total solvent concentration (relative to water) of 1 to 50% vol / vol, e.g., about 3% vol / vol). In such an embodiment, the lanthanoid material may be provided at a suitable concentration. For example, the lanthanoid material may be provided in the solvent at a concentration of 0.005 mM to 10 mM, e.g., 0.01 mM to 3 mM, e.g., 0.015 mM to 1.5 mM, e.g., 0.15 mM to 0.75 mM.

[0046] In an embodiment of the phosphor material of the present invention, when an antenna molecule is included, the antenna molecule may be provided in a solvent of the lanthanide at a suitable concentration. For example, when an antenna molecule is included, the content of the antenna molecule may be 0.1 mM to 10 mM, for example, about 4.5 mM.

[0047] As will be readily understood, the method of the present invention aims to provide a phosphor having dormant bacterial spores. Therefore, the method of the present invention may further include a step of substantially removing the solvent from the phosphor material. As used herein, the term "substantially removing" may mean removing the solvent to a degree sufficient to prevent the bacterial spores from escaping dormancy. For example, the residual solvent may be 5 wt% or less of the total spores, such as 2 wt% or less, such as 1 wt% or less, such as 0.5 wt% or less, such as 0.1 wt% or less, such as 0.01 wt% or less, such as 0.001 wt% or less.

[0048] As described herein, the present invention surprisingly discloses that lanthanides, either alone or as lanthanide-antenna complexes, can be accumulated within spores to form phosphors, and further discloses that such phosphors are significantly more efficient at converting UV excitation light into phosphorescence than either the lanthanide alone or the lanthanide-antenna complex alone. Briefly, binding of a lanthanide to a spore increases the phosphorescence output for the same excitation input, regardless of whether the lanthanide is complexed with an antenna molecule. Figure 1 illustrates the overall conceptual scheme. It is highly surprising that the phosphorescence output of the phosphor material obtained according to the present disclosure (with or without an antenna molecule) is greater than that obtained when the lanthanide is excited alone without the spore.

[0049] There are many advantages associated with the present invention, some of which are set forth below. (1) Lanthanides may have an impact on the environment and may be ecotoxic. Therefore, by improving the conversion efficiency of lanthanides, it is thought that it may be possible to obtain phosphorescence of the same level as conventional methods with a small amount of lanthanides. (2) The method for producing the spore phosphor of the present invention is simple, requiring only a bioreactor for spore production. Instant results can be obtained by simply mixing the spores with lanthanides in the presence or absence of antenna molecules. In contrast, doping a semiconductor host material with a lanthanide requires cleanroom facilities and complex techniques. For example, the core of every LED contains multiple semiconductor layers, each with a different composition and doping concentration. Each LED layer is deposited using a technique called organometallic vapor phase epitaxy (OMVPE). The epitaxy method aligns the axial orientation of atoms at every location on the wafer, avoiding defects that reduce conversion efficiency. Using spores instead of semiconductors is expected to significantly reduce manufacturing costs and facilitate scalability. (3) Because spores are easy to handle and manipulate, they can be easily incorporated into many materials, such as resins, coatings, adhesives, composites, and casting materials.

[0050] Apart from their luminescence, one of the main advantages of fluorescent spores is their ease of production. Spores can be produced in bioreactors in a scalable manner without the need for cleanrooms or chemical synthesis facilities (Tzeng, Y.-M. et al., J. Appl. Microbiol., 2008, 104, 1275; Zhao, S. et al., World J. Microbiol. Biotechnol., 2008, 24, 2859). Furthermore, spores are highly monodisperse in terms of size and structure, as these parameters are hard-coded into the genetic program of each spore. In a sense, the quality control of spore production has been optimized in nature over more than a billion years. Furthermore, spores have been naturally selected to survive extreme conditions, resisting heat, radiation, and ultraviolet light for decades, with the longest-lived bacterial spores having been found to be over 100 million years old (Vreeland, RH et al., Nature, 2000, 407, 897). Simply put, spores are nature's most flexible solution for chemical stability and preservation. The utility of spores as a material is perhaps best demonstrated in their application to self-healing concrete.

[0051] Another major advantage of spore fluorophores is their adaptability. Mixtures of different lanthanides, as well as compatible blends with other antenna molecules and co-dopants, are extremely easy to optimize and implement, especially since spore fluorophores can be prepared by simply incubating the spores with the supporting material for a short time in aqueous solution. Furthermore, the microparticle nature of the spores and their compatibility with adhesives means they can be easily incorporated into flexible substrates as needed. The combinatorial possibilities are endless.

[0052] Various lanthanides and dopants can be freely incorporated into spores by simply incubating them with a spore suspension. This incorporation occurs instantaneously. Dopants such as strontium can further enhance the luminescence output of lanthanides.

[0053] Further aspects of the invention are described below. (1) A luminescent material, a. Contains host material that is a bacterial spore; b. Incorporating at least one dopant selected from the lanthanide series (e.g., europium); c. The dopant may be complexed with an antenna molecule (e.g., thenoyltrifluoroacetone (TTA)); Luminescent material. (2) The composition according to (1) above, further comprising a co-dopant (e.g., strontium) added to enhance the light output. (3) The composition according to (1) above, wherein the molar ratio of the lanthanoid to the antenna molecule is precisely adjusted to about 1:3 (lanthanoid:antenna).

[0054] Exemplary embodiments of the present invention may utilize Clostridium spp. Additionally, certain embodiments of the present invention (e.g., spores + Eu + TTA) may be particularly efficient at converting UV light to red light, thereby providing color-converting phosphors that may be suitable for use in numerous applications where ultraviolet (UV) and blue light should be minimized for aesthetic or health reasons.

[0055] In embodiments of the present invention that may be mentioned herein, the phosphor material may be a phosphor material that does not include antenna molecules.

[0056] Further aspects and embodiments of the present invention will be described with reference to the following embodiments, but the present invention is not limited to these embodiments. [Example]

[0057] material Europium(III) chloride hexahydrate (212881), terbium(III) chloride hexahydrate (212903), dysprosium(III) chloride hexahydrate (289272), and samarium(III) chloride hexahydrate (204277); TTA (2-thenoyltrifluoroacetone) (T27006), strontium chloride hexahydrate (255521), sodium glycocholate (G7132), maltose (M5895), disodium phosphate (S7907), D-cycloserine (C6880), L-alanine (A7469), L-cysteine ​​(C7352), and hypoxanthine (H9377); and yttrium oxide phosphor (756490) were purchased from Sigma. DMSO (dimethyl sulfoxide) (D12345) was purchased from Life Technologies. Brain Heart Infusion (BHI) broth (237500), BBL Polypeptone Peptone (211910), and Dehydrated Cooked Meat Medium (226730) were purchased from BD Difco. Broth oxylase was purchased from Oxyrase and Sigma (SAE0013). Fetal bovine serum (FBS) (S1810) was obtained from iDNA Biotechnology. Percoll (17089109) was purchased from GE Healthcare.

[0058] Bacterial strains The ATCC strains used were C. novyi (19402), C. septicum (11424), and B. cereus (10987). For C. novyi and C. septicum, avirulent variants with genetically inactivated alpha-toxins were used. These variants were C. novyi-NT (a gift from the laboratory of Professor Bert Vogelstein at Johns Hopkins University, Baltimore, Maryland, USA) and C. septicum CS11 (ATCC 11424 strain with its lethal alpha-toxin inactivated for safe handling). Wild-type B. subtilis 168 (P1A1), B. megaterium (7A16, i.e., QM B1551), and B. thuringiensis (4AJ1) were purchased from the Bacillus Genetic Stock Center (BGSC) in the United States. Stock solutions were prepared in LB medium-glycerol mixture (30% glycerol) and stored at -80°C.

[0059] Example 1. Sequestration of the lanthanide element europium by spores To develop efficient spore-based host lattices for lanthanide-based phosphors, we first attempted to identify the optimal spore system by quantifying the uptake of lanthanides by various bacterial spores. To identify the optimal spore system, we selected two nonpathogenic species each from the spore-forming bacteria Bacillus (Bacillus subtilis and Bacillus thuringiensis) and Clostridium (Clostridium novyi-NT and C. septicum-CS11) and screened them. Because the majority of bioremediation studies use vegetative bacteria (Moriwaki, H. et al., Appl. Microbiol. Biotechnol., 2013, 97, 3721; Fischer, C.B. et al., RSC Adv., 2019, 9, 32581; Moriwaki, H. & Yamamoto, H., Appl. Microbiol. Biotechnol., 2013, 97, 1), both spore forms (dormant and germinated) and vegetative forms were tested for each bacterium. Europium (Eu), a lanthanide model, was tested alone or in combination with the β-diketone thenoyltrifluoroacetone (TTA). TTA was included in this study because our previous Raman spectroscopy study showed that it enhanced the transfer of Eu into C. novyi-NT spores (Singapore Patent Publication No. 10202260603W). Eu uptake was estimated by measuring the amount of Eu remaining in solution after incubation with spores or vegetative forms using ICP-OES. The Eu concentration (0.14 mM) was chosen to be within the typical range used in other bioremediation and lanthanide uptake studies. The TTA concentration (4.2 mM) was set at 30-fold higher than that of Eu.

[0060] Clostridium novyi-NT sporulation Spores of Clostridium novyi strain NT were prepared according to the method reported by Cheong et al. (Cheong, I. et al., Science, 2006, 314, 1308). TMIn an anaerobic jar, 5 × 10 cells were cultured in 1 L of medium containing 45 g of NaHPO, 30 g of polypeptone peptone, 0.5 g of L-cysteine, 10 g of maltose, 50 g of dried cooked meat powder (Difco), and 10% v / v FBS. 9 Two hundred microliters of C. novyi-NT spores (200 μL, CFU / mL) were inoculated and cultured anaerobically at 37°C and pH 7.4. After 3 weeks of culture in this medium, the spores were purified from contaminating vegetative forms by centrifuging a discontinuous density gradient of Percoll (90%) at 15,000 rcf for 30 minutes in a Beckman Avanti J-20 XP high-speed centrifuge. Spore quality was confirmed under a phase-contrast microscope, revealing that over 99% of the spores were observed as white, glowing spores. The spore concentration was determined to be approximately 5 × 10 by diluting appropriately with 1 × phosphate-buffered saline (PBS) using a calibration curve constructed from the correlation between absorbance at OD600 and cell number. 9 The results were adjusted to the order of CFU / mL.

[0061] Sporulation of Bacillus spp. Sporulation of B. subtilis, B. cereus, B. megaterium, and B. thuringiensis was performed according to the protocol by Nicholson and Setlow (Nicholson WL and Setlow, P. Sporulation, germination, and outgrowth. In Molecular Biology Methods for Bacillus (eds. Harwood, CR & Cutting, SM) 391-450 (John Wiley & Sons, 1990)). Each Bacillus strain was inoculated onto an LB agar plate and grown overnight at 37°C. The next day, for Bacillus species, except for B. megaterium, single colonies were picked and inoculated into 30 mL of 2x SG medium (Nicholson, W. L., and Setlow, P. Sporulation, germination, and outgrowth. in Molecular Biology Methods for Bacillus (eds. Harwood, C. R. & Cutting, S. M.) 391–450 (John Wiley & Sons, 1990)) at pH 7. For B. megaterium, a single colony was inoculated into supplemented nutrient broth (SNB) medium at pH 7.2. The strains were then grown on a MaxQ8000 orbital shaker at 200 rpm and 37°C until an OD600 of 0.3–0.5 was reached. Next, this culture (25 mL) was reinoculated into a 2 L Erlenmeyer flask containing 225 mL of 2x SG medium or 225 mL of SNB medium and capped with a gas-permeable membrane (Breathe Easy®) to ensure adequate aeration. The flask was then placed on a Gerhardt orbital shaker (130 rpm, 37°C) and cultured for 92 hours. The recovered spores were washed with 1x PBS and then subjected to isopycnic centrifugation using a Beckman Avanti J-20 XP high-speed centrifuge (JS 13.1 rotor, 15,000 rcf, 30 minutes, 4°C) using a self-density gradient formed with Percoll (90%). The majority of the shiny white spores of Bacillus strains, except for B. cereus, were obtained in the lower fraction, while those of B. cereus were obtained in the upper fraction.These fractions were washed repeatedly with 1x phosphate-buffered saline (PBS) and stored at 4°C until further use. Spore quality was confirmed by phase-contrast microscopy, and more than 99% of the spores were observed as white, glowing spores. The spore concentrations of all strains were determined by counting them using a Neubauer counting chamber (Petroff-Marenfield type) at a predetermined depth, and each strain was approximately 10 9 The results were adjusted to the order of CFU / mL.

[0062] Clostridium septicum sporulation C. septicum CS11 spores were purified using a modified protocol from Dang et al. (Dang, LH et al., Proc. Natl Acad. Sci. USA, 2001, 98, 15155). All steps were performed in a Plas Labs anaerobic chamber. Briefly, an overnight culture of C. septicum was diluted 50-fold with 100 mL of BHI-S medium containing 0.05% L-cysteine ​​and grown until the OD600 reached 1.5–3. The entire culture was added to 900 mL of sporulation medium (0.05% L-cysteine, 3% Bacto polypeptone, 5% dried cooked meat medium, and 10% fetal bovine serum) and incubated at 37°C for 5 days. Spores were purified from vegetative cells using an 80% Percoll discontinuous density gradient by centrifugation at 15,000 rcf for 30 minutes in a Beckman Avanti J-20 XP high-speed centrifuge. The resulting spores were washed twice, resuspended in water, and stored at 4°C until further use. Spore quality was confirmed using a phase-contrast microscope, revealing that over 99% of the spores were white and shiny. The spore concentrations of all strains were determined by counting using a Neubauer counting chamber (Petroff Marenfield type) at a predetermined depth. Each strain was counted at approximately 10 9 The results were adjusted to the order of CFU / mL.

[0063] Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) Sample Preparation 10 dormant spores suspended in 1x PBS 9 They were used on the order of CFU / mL, slightly concentrated, and then resuspended in saline (150 mM NaCl).

[0064] Spore germination was performed as follows: C. novyi-NT spores were germinated in L-cysteine ​​(100 mM), hypoxanthine (0.1 mM), and an oxyrase mixture (1:50). C. septicum spores were germinated in sodium glycocholate (61 mM) and an oxyrase mixture (1:14). B. subtilis spores were activated by heating at 70°C (1 h) in 1x PBS and then germinated in L-alanine (74 mM). B. thuringiensis spores were activated by heating at 70°C (30 min) in D-cycloserine solution (7.3 mM) and then germinated in L-alanine (74 mM). All germination experiments were performed at 37°C for 30 min except for C. septicum, which was performed at 37°C for 3 h. Each spore was then resuspended in saline (150 mM NaCl).

[0065] For vegetative bacterial samples, overnight cultures of B. subtilis and B. thuringiensis were grown in LB broth at 37°C and 220 rpm. C. novyi-NT was grown in BHI broth containing 10% FBS at 37°C in the presence of oxyrase, and C. septicum was grown in BHI broth containing 10% FBS at 37°C in a Plas Labs anaerobic chamber. Vegetative bacteria were harvested after approximately 20 hours and resuspended in saline (150 mM NaCl).

[0066] Equal volumes of europium chloride (0.15 mM) and TTA (4.5 mM) were mixed in 3% ethanol and incubated at room temperature for 30 minutes before use.

[0067] To each 35 μL of spore-forming or vegetative bacteria, 500 μL of Eu or Eu-TTA was added to give a final Eu concentration of 0.14 mM and a final TTA concentration of 4.2 mM. Each sample was immediately centrifuged at 3900 rcf for 5 minutes, followed by centrifugation at 15,000 rcf for 5 minutes in a Thermo Fisher Scientific Sorvall Legend Micro 17 centrifuge. The supernatant (500 μL) of each solution was diluted to 10 mL with 3% ethanol, and 221 μL of nitric acid was added. Each sample was then analyzed using a PerkinElmer Avio 500 ICP-OES instrument to determine the residual Eu concentration (ppm) in the supernatant. The amount of Eu uptake by spore-forming or vegetative bacteria was determined by subtracting the Eu concentration in the supernatant from the initial Eu concentration of 0.14 mM.

[0068] To measure biomass, equal volumes (35 μL each) of spore- or vegetative bacterial suspensions were added to empty 1.5 mL Eppendorf tubes that had been previously heated (60°C for 4 hours) and dried overnight in a Sanyo MOV 112 dryer set at 60°C. The weights of spore- and vegetative bacterial biomass were determined using a Mettler-Toledo AT261 DeltaRange balance by measuring the difference between the weight of the empty Eppendorf tube before biomass addition and the weight of the Eppendorf tube after adding the dried biomass. The final biomass values ​​were calculated as the average of three independent samples for each bacterial form.

[0069] Results and Discussion Significant differences in Eu uptake were observed between bacterial species, bacterial morphologies, and whether or not TTA was added to Eu (Fig. 2). Overall, dormant and germinated spores showed similar Eu uptake abilities to vegetative forms, but in B. thuringiensis, vegetative forms showed significantly lower Eu uptake than vegetative forms without TTA. These results suggest that dormant and germinated spores are effective substitutes for vegetative forms in Eu uptake.

[0070] Focusing only on spore types, Bacillus spores outperformed Clostridium spores in uptake of Eu alone. All Bacillus spores, with or without TTA, showed similar uptake rates of approximately 100–140 nmol / mg spore (Figures 2A and 2B). These uptake rates were in the same range as previous studies examining the uptake of other lanthanides, such as terbium and dysprosium, by dormant B. subtilis spores (Fischer, CB et al., RSC Adv., 2019, 9, 32581; Dong, W. et al., Appl. Environ. Microbiol., 2019, 85, e00956-19). In contrast, Eu uptake by Clostridium spores was significantly lower (<50 nmol / mg) (Figures 2C and 2D). However, the addition of TTA improved the Eu uptake capacity of Clostridium spores to a level almost equivalent to that of Bacillus spores. The addition of TTA increased Eu uptake by spores, particularly C. novyi-NT, by threefold. We speculate that the difference in the effect of TTA on Eu uptake by spores is due to the structural differences between Bacillus and Clostridium spores. Except for C. septicum, only slight differences were observed between dormant and germinated spores with respect to Eu uptake. In C. septicum, germinated spores showed approximately twice the Eu uptake of dormant spores.

[0071] We concluded that both B. subtilis and B. thuringiensis spores can sequester lanthanides with high efficiency, regardless of the presence or absence of TTA, but that TTA is required to achieve comparable levels of lanthanide uptake in Clostridium spores. Based on these results, we used dormant spores in subsequent experiments (described in the Examples below). Dormant spores are structurally more robust than germinated spores or vegetative forms and perform equally well in lanthanide sequestration. Furthermore, once dormant spores are prepared by sporulation, they can be stored in a ready-to-use state without further processing.

[0072] Example 2. Phosphorescence properties exhibited by europium-loaded spores To investigate whether lanthanoid-incorporated spores emit light when used as fluorescent materials, dormant spores of Bacillus and Clostridium species were prepared using the protocol described in Example 1.

[0073] Eu (0.14 mM or 1.4 mM) was loaded onto resting spores of Bacillus and Clostridium in the presence or absence of TTA, and the spores were placed in a 384-microwell plate and dried overnight under reduced pressure. Luminescence was then observed and photographed.

[0074] Preparation of spore fluorophores To prepare the Eu-based phosphors, equal amounts of europium chloride (3 mM, 0.3 mM, or 0.03 mM) and TTA (9 mM) were mixed in 3% ethanol and incubated at room temperature for 30 minutes before use. 500 μL of Eu or Eu-TTA was added to 35 μL of dormant spores or saline to give final Eu concentrations of 1.4 mM, 0.14 mM, or 0.014 mM, and TTA concentrations of 4.2 mM.

[0075] For experiments with other lanthanide phosphors, equal amounts of samarium chloride (Sm), terbium chloride (Tb), or dysprosium chloride (Dy) (3 mM or 0.3 mM) and TTA (9 mM) were mixed in 50% ethanol and incubated at room temperature for 30 minutes before use. To each 35 μL of dormant spores or saline, 500 μL of Sm, Tb, or Dy, or Sm-TTA, Tb-TTA, or Dy-TTA was added to give final concentrations of Sm, Tb, or Dy of 1.4 mM or 0.14 mM, and a final TTA concentration of 4.2 mM.

[0076] Each sample was immediately centrifuged at 3900 rcf for 5 minutes, followed by centrifugation at 15,000 rcf for 5 minutes in a Thermo Fisher Scientific Sorvall Legend Micro 17 centrifuge. The supernatant (500 μL) of each solution was discarded, and the remaining 35 μL pellet was transferred to a PCR tube or a Greiner Bio-One 384-well UV-star microplate. Each pellet was then dried overnight under high vacuum.

[0077] Visualization of spore fluorophore fluorescence Fluorescence of spore fluorophores was visualized with a Major Science MUV26 series UV transilluminator (excitation wavelength: 254 nm or 365 nm) or measured using a Tecan Spark® multimode fluorescence microplate reader with a delay time of 20 μs, a gain of 50, and an integration time of 1000 μs, at an excitation wavelength of 370 nm and an emission wavelength of 620 nm (for Eu-based spore fluorophores) unless otherwise noted.

[0078] Excitation Emission Matrix (EEM) measurements For EEM measurements, a delay time of 20 μs, a gain of 50, and an integration time of 1000 μs were used, with an excitation wavelength range of 250–400 nm and an emission wavelength range of 450–800 nm. EEM data were analyzed in Microsoft Excel using logarithmic regression. 10 Scales were converted (negative and zero intensity values ​​were converted to 1 before logarithmic transformation), and contour plots were created using the Plotly package in Rstudio (2022.07.2, build 576).

[0079] All other graphs were generated using GraphPad Prism 9.

[0080] Results and Discussion In this study, we loaded various bacterial spores with the same Eu + TTA activator and obtained highly variable results. When illuminated at 365 nm using a UV transilluminator, all four bacterial spores emitted visible luminescence compared to spore-free controls (Eu concentrations of 0.14 mM or 1.4 mM in the presence or absence of TTA) (Figure 3A). No visible difference in luminescence brightness was observed between Eu concentrations of 0.14 mM and 1.4 mM. Interestingly, Bacillus spores emitted significantly less luminescence than Clostridium spores. This result indicates that increased Eu uptake does not necessarily result in higher luminescence output. This result also suggests that differences in spore composition and structure are as important as Eu uptake. Among Bacillus spores, B. subtilis exhibited brighter fluorescence than B. thuringiensis. The luminescence of Clostridium spores was so bright that it was difficult to distinguish between C. novyi-NT and C. septicum. The Bacillus spores were not as bright as Clostridium spores, even though they had higher Eu uptake than Clostridium spores.

[0081] To quantify the luminescence of these spore fluorophores, we loaded them with three concentrations of Eu (0.014 mM, 0.14 mM, and 1.4 mM) in the presence or absence of TTA, dried them under reduced pressure, and measured the luminescence output (excitation wavelength: 370 nm, emission wavelength: 620 nm) with an extended delay (Figure 3B). All spore fluorophores exhibited significantly greater luminescence output than the no-spore control. To determine the best-performing luminescence curve for each spore fluorophore, it was also possible to measure the luminescence output beyond 500 μs. This long-term transient decay was consistent with the luminescence output, and this luminescence exhibited phosphorescence rather than fluorescence characteristics. As expected, the 0.014 mM Eu concentration produced the lowest luminescence for all samples tested. However, for all spore fluorophores except B. thuringiensis, 0.14 mM Eu produced stronger luminescence than the highest concentration, 1.4 mM Eu. This decrease in luminescence is thought to reflect quenching caused by the high concentration of Eu within the spores. The addition of TTA increased luminescence in all spore fluorophores except for B. thuringiensis, but the increase in luminescence was small in B. subtilis. This quantitative data on luminescence decay confirmed the results of the visual examination of spore fluorophores in the initial experiments and emphasized that luminescence output levels are highly dependent on the type of bacteria used.

[0082] Because the two Bacillus spores tested (B. subtilis and B. thuringiensis) did not exhibit significant luminescence, we further expanded the screening panel by adding two more Bacillus strains to test whether the observed spore luminescence was restricted to Clostridium spores. To test this, we prepared fluorescent B. cereus and B. megaterium spores loaded with Eu (0.14 mM) and compared their luminescence with that of the other spores used in the previous experiment (Figure 4A). Surprisingly, both B. cereus and B. megaterium spores exhibited significant luminescence in the presence of TTA compared with B. subtilis and B. thuringiensis spores. Notably, B. megaterium spores exhibited bright red fluorescence, although this was slightly weaker than that of Clostridium spores. B. megaterium spores also exhibited luminescence comparable to that of Clostridium spores, even in the absence of TTA. Quantitative measurements showed that B. megaterium spores exhibited a 15-fold increase in luminescence in the presence of TTA compared to B. subtilis and B. thuringiensis spores (Figure 4B). Furthermore, B. megaterium spores, like other spores, exhibited measurable phosphorescence even after 500 μs. These results demonstrate that spore fluorescent materials can be prepared using both Clostridium and Bacillus species.

[0083] Lanthanide phosphorescence is known to be affected by vibrational coupling in aqueous environments (Werts, MHV Sci. Prog., 2005, 88, 101; Meshkova, SB et al., J. Appl. Spectrosc., 1997, 64, 229). Therefore, the presence or absence of water could theoretically affect the absorption and emission profiles of spore fluorophores. C. novyi-NT and C. septicum exhibited the highest luminescence output and were therefore used in this study to investigate this question. First, spores were loaded with Eu (1.4 mM) in the presence or absence of TTA (4.2 mM) and then either dried to a solid state or maintained in aqueous suspension. Spectroscopic scans were performed to obtain excitation-emission matrix (EEM) profiles of these spore fluorophores (Figures 5A and 5B). These data reveal that for all spore fluorescent materials tested, the dried spore fluorescent materials exhibited EEM profiles that were visually different from those in aqueous suspension. 5 D0→ 7 Two maxima at 610 nm and 620 nm correspond to the F2 transition and Eu 5 D0→ 7 The emission spectrum consisted mainly of a small peak at 700 nm, corresponding to the F4 transition. The emission peak in the solid state was stronger than that in the liquid state, likely due to the elimination of solvent effects in the solid state. The most notable difference between the solid and liquid profiles was observed in the excitation spectra. While the liquid-state spores were excitable only in the wavelength range of 360–380 nm, the solid-state spore fluorophore was excitable over a much broader wavelength range, from 300–380 nm (Figures 5A and 5B). These results suggest that spores can function as fluorophores even in aqueous suspensions, but removing water can broaden the UV spectral range that the spore fluorophore can capture, resulting in a further increase in luminescence output. The addition of TTA did not qualitatively change the EEM spectra in either the solid or liquid states; only a quantitative enhancement of luminescence was observed.

[0084] Example 3. Long-term stability of spore fluorescent material Next, the long-term stability of the spore fluorescent material was evaluated. The spore fluorescent material was prepared according to the protocols in Examples 1 and 2. Spores were loaded with Eu (1.4 mM or 0.14 mM) in the presence or absence of TTA (4.2 mM) and dried under vacuum until a solid was obtained in a 384-well microplate. The plate was covered with a non-airtight lid and stored on a laboratory bench in ambient air and at room temperature. Time-resolved luminescence (excitation wavelength: 370 nm, emission wavelength: 620 nm, delay time: 0.02 ms) was measured for 24 weeks.

[0085] Results and Discussion B. thuringiensis showed a dramatic drop in luminescence immediately after the first week. Meanwhile, the other three bacteria produced significantly higher luminescence over 24 weeks than the lanthanide-only control without spores (Figure 6). These other three bacteria also appeared to stabilize their luminescence output after the first seven weeks, when luminescence output fluctuated significantly. Of the bacteria tested, C. novyi-NT exhibited the best luminescence output profile, with high and stable luminescence over 24 weeks. Long-term stability may be further improved by incorporating spore fluorophores into materials that exclude moisture and oxygen.

[0086] Example 4. Varying lanthanides to create different colors Using Eu as an activator resulted in a spore phosphor that emitted orange-red light. However, other colors can be achieved by varying the lanthanide loaded onto the spores; therefore, the use of Eu is not limited to this. To confirm this feasibility, we loaded C. novyi-NT or C. septicum spores with samarium (Sm), terbium (Tb), and dysprosium (Dy) in addition to Eu, in the presence or absence of TTA, according to the protocol disclosed in Example 2. Each spore was then dried to produce a spore phosphor panel, which was visualized using a UV transilluminator set at a wavelength of 254 nm or 365 nm (Figures 7A and 7B).

[0087] Results and Discussion Consistent with the predicted emission spectra of Sm and Tb, differences in luminescence output were observed; the Sm spore fluorophore exhibited a red color, while the Tb spore fluorophore exhibited a green color. However, the emission from the Dy spore fluorophore was not visible in any bacterial species, regardless of the excitation wavelength or the presence or absence of TTA. Further optimization is considered necessary to observe luminescence in Dy-loaded spores.

[0088] As with Eu, the addition of TTA dramatically altered the spore fluorescence of Sm (Figures 7A and 7B). While luminescence was only slightly observed in Sm-loaded spores, the addition of TTA improved the luminescence output of both bacterial species (except for C. septicum spores excited at 254 nm, which showed no luminescence in either Sm-loaded or Sm + TTA-loaded spores). The emission profile of Tb was distinct from that of Sm and Eu. The addition of TTA did not improve the green luminescence output of Tb in C. novyi-NT, regardless of the excitation wavelength. Similarly, the addition of TTA significantly reduced luminescence in C. septicum spores excited at 365 nm, and TTA did not improve luminescence at 254 nm. These data further emphasize that the choice of bacteria and antenna molecule significantly influences the luminescence output of spore fluorescence.

[0089] Further investigation of the Sm and Tb fluorophores was carried out by creating excitation-emission matrices (EEM). Sm and TTA-loaded spores showed red fluorescence, 4 G 5 / 2 → 6 H 5 / 2 560 nm, which corresponds to the transition 4 G 5 / 2 → 6 H 7 / 2 600 nm, which corresponds to the transition 4 G 5 / 2 → 6 H 9 / 2 650 nm, which corresponds to the transition4 G 5 / 2 → 6 H 11 / 2 The emission maximum was at 700 nm, which corresponds to the transition of Tb and Eu. In the solid state, the excitation wavelength range was shown to be extended to the UV-B region, resulting in 10-100 times higher emission intensity compared to the liquid state (Fig. 8A). Without TTA loading, the emission of Sm spores was significantly weaker (Fig. 8B). As shown in the image data, the performance of Tb was completely different from that of Sm and Eu (Fig. 9A and Fig. 9B). For Tb-loaded spores, a wide excitation wavelength range was observed overall, regardless of bacterial species, state (solid or liquid), and the presence or absence of TTA. Tb-loaded spores exhibited green fluorescence, 5 D4 → 7 480 nm, which corresponds to the F6 transition; 5 D4 → 7 545 nm, which corresponds to the F5 transition; 5 D4 → 7 580 nm, which corresponds to the F4 transition; 5 D4 → 7 The maximum emission was observed at 620 nm, which corresponds to the F3 transition. An important observation was that TTA enhanced the emission of Eu, but not necessarily Tb. This result was particularly evident in C. septicum spores, where Tb alone exhibited stronger emission than Tb + TTA.

[0090] The high conversion efficiency from UV light to red light indicates that the present invention can convert not only lanthanides but also various types of UV light emitted from LED semiconductor elements. Currently, there is a problem that UV / blue light leakage from LEDs affects human circadian rhythms (i.e., sleep cycles). The present invention can address this problem by potentially being used as a phosphor that can convert light color.

[0091] Example 5. Strontium co-doping to further enhance luminescence In many rare-earth phosphors, the luminescence is often enhanced by co-doping with other metal ions (Lin, Y. et al., J. Eur. Ceram. Soc., 2003, 23, 175; Santos, P.C.L. et al., Phys. Status Solidi B Basic Res., 2019, 256, 1900024). Based on this, we investigated whether a similar enhancement could be observed in spore phosphors. In this study, C. novyi-NT spores were co-doped with strontium chloride (476 mM), TTA (5.4 mM), and Eu (0.06 mM or 1.9 mM). The spores were obtained according to the protocol described in Example 1.

[0092] Strontium doping of spore phosphors Appropriate amounts of europium chloride (3.9 mM or 0.12 mM in deionized water (DIW)), strontium chloride (1 M in DIW), and TTA (225 mM in DMSO) were mixed with C. novyi-NT dormant spores in 1x PBS to achieve final concentrations of 1.9 mM or 0.06 mM (Eu), 476 mM (strontium chloride), or 5.4 mM (TTA). In control samples, strontium chloride was replaced with DIW. Each sample was immediately centrifuged at 3900 rcf for 5 minutes, followed by centrifugation at 15,000 rcf for 5 minutes, using a Thermo Fisher Scientific Sorvall Legend Micro 17 centrifuge. The supernatant (1 mL) of each solution was discarded, and the remaining 50 μL pellet was transferred to a PCR tube or a 384-well Greiner Bio-One UV-star microplate. Each pellet was then dried under high vacuum overnight.

[0093] Fluorescence of the strontium-doped spore fluorophores was visualized using a Major Science MUV26 series UV transilluminator (excitation wavelength: 365 nm) or measured using a Tecan Spark® multimode fluorescence microplate reader at an excitation wavelength of 370 nm and an emission wavelength of 620 nm with a gain of 40, an integration time of 1000 μs, and various delay times.

[0094] Results and Discussion Visualization by UV transmission at 365 nm confirmed that the addition of strontium increased the luminescence of the Eu+TTA spore fluorophore (Figure 10A). This increase in luminescence intensity was more pronounced at higher Eu concentrations (1.9 mM). At this high concentration, the luminescence intensity of the Eu-Sr-TTA-doped spore fluorophore increased by approximately fivefold compared to an otherwise similar spore fluorophore without Sr co-doping (Figure 10B). At a significantly lower Eu concentration of 0.06 mM, Sr doping did not appreciably change the luminescence (Figure 10C). The ability to co-do the spore fluorophore with various metals with a short incubation period allows for the creation of countless combinations for tuning the spore fluorophore's performance.

[0095] Example 6. Comparison of spore phosphor with commercially available yttrium oxide phosphor In order to perform a benchmark test of the spore phosphor of the present invention, the emission of the spore phosphor of the present invention was compared with that of a commercially available red phosphor, Y 1.98 EU 0.08 The emission of yttrium (Y) phosphor was compared with that of O3. Yttrium (Y) phosphor is the most commonly used red phosphor, and is often used in television screens due to its narrow-band emission around 610 nm (Rao, RP, Solid State Commun., 1996, 99, 439). C. novyi-NT spores loaded with Eu and TTA and Y phosphors containing an equal amount of Eu were prepared. The spores were obtained according to the protocol described in Example 1.

[0096] Comparison with commercially available yttrium oxide phosphors Yttrium oxide powder was suspended in DIW containing 0.14 mM Eu. This suspension was added to spores to prepare a Eu-based spore phosphor. The suspension was then dried overnight under high vacuum to obtain a solid yttrium oxide phosphor. A water suspension of yttrium oxide was also used as a liquid sample. Furthermore, C. novyi-NT spore phosphors doped with Eu (0.14 mM) and TTA (4.2 mM) were used as both liquid and solid spore phosphors. Equal amounts of yttrium oxide and spore phosphor were used in dry and liquid forms. Fluorescence of each fluorophore was visualized using a Major Science MUV26 series UV transilluminator (excitation wavelength: 254 nm or 365 nm) or measured using a Tecan Spark fluorescence microplate reader with no delay, a gain of 50, and an integration time of 1000 μs at an excitation wavelength of 370 nm or 250 nm and an emission wavelength of 620 nm.

[0097] Results and Discussion Under UV light (254 nm), the spore fluorophore exhibited brighter fluorescence than the Y fluorophore in both the dry solid and liquid suspension (Figure 11A). Relatively speaking, the spore fluorophore was brighter in the dry solid than in the liquid suspension, whereas the Y fluorophore exhibited the opposite tendency, being brighter in the liquid suspension than in the solid. To quantify these observations, we measured fluorescence at the excitation maximum of the Y fluorophore, 250 nm, and at the excitation maximum of the spore fluorophore, 370 nm. Consistent with the visual observations, the Y fluorophore excited at the optimal excitation wavelength (250 nm) was less bright than the spore fluorophore excited at the same wavelength (Figure 11B). Using the optimal excitation wavelength for the spore fluorophore (370 nm), the difference was further accentuated, with the spore fluorophore becoming 3 log-fold brighter than the Y fluorophore (Figure 11C). The spore fluorophore exhibited a 1000-fold increase in fluorescence intensity compared to the commercially available yttrium oxide fluorophore. These results indicate that the spore fluorophore may have a superior quantum efficiency to the commercially available Y fluorophore.

[0098] The present invention has high quantum efficiency and low manufacturing costs, allowing for more efficient utilization of these rare earth element resources while minimizing adverse environmental impacts.

[0099] Example 7. LEDs coated with spore phosphors to demonstrate color tunability One of the most important applications of phosphors is their use in LEDs. One of the key challenges in this application is how to efficiently convert the color of the phosphor (Xia, Z. & Liu, Q., Prog. Mater. Sci., 2016, 84, 59). In addition to the aesthetic goal of achieving a desired emission profile, there are concerns that UV and blue light may unduly affect human circadian rhythms. For example, melatonin is produced by the pineal gland in the brain and is responsible for regulating sleep. Melatonin production increases in the dark but is suppressed by exposure to light, especially UV and blue light, which can disrupt sleep patterns.

[0100] We investigated how effectively the spore phosphor could convert UV light from an LED into red light. Two types of UV LEDs were used. One UV LED was a single LED bulb emitting light with a wavelength of 365 nm. This LED bulb was suitable for exciting Eu. The other UV LED was a string of LED bulbs emitting light with a wavelength of 275 nm. This LED bulb was more suitable for exciting Tb. C. novyi-NT spore phosphor was mixed with a UV-transparent adhesive (Norland 88) and coated onto these two types of UV LEDs.

[0101] Fabrication of LEDs coated with spore phosphors The spore phosphor was prepared according to the protocol in Example 2. The solid spore phosphor (lanthanide concentration: 0.14 mM, TTA concentration: 4.2 mM) prepared in a PCR tube was mixed with a drop of Norland 88 adhesive and then coated onto a commercially available UV LED bulb using a pipette tip. The coated LED was cured for 10–15 minutes using a UV transilluminator at 365 nm wavelength. The coated LED bulb was then powered using a single GP Ultra Plus alkaline 9V battery or multiple 9V batteries connected in series on a breadboard, and images were captured using a Nikon COOLPIX L120 SLR camera. For Eu-based spore fluorophores, a UV-A LED bulb with an excitation wavelength of 365 nm (EOLD-365-525 LED, element14) was used, and for Tb-based spore fluorophores, a UV-C LED bulb with an excitation wavelength of 275 nm (ILS-OV12-O275-VL004-SC201-W2, element14) was used.

[0102] Results and Discussion The results were clear: a single UV LED coated with Eu+TTA spore phosphor emitted a reddish-orange light that was visually much brighter than the UV LED coated with Eu+TTA alone or any of the other controls (Figures 12A and 12B). A string of UV LED bulbs coated with Tb+TTA spore phosphor produced less discernible results, exhibiting a slight greenish hue compared to the control, which was pale blue upon excitation with UV light (Figure 12C).

Claims

1. A phosphor material, Bacterial spores as host material; a lanthanide dopant within and / or on the surface of said host material; Including, the bacterial spores are dormant, the bacterial spore is selected from one or more bacterial spores selected from one or more of the genera Bacillus and Clostridium; Phosphor material.

2. The lanthanide dopant is Eu 3+ , Sm 3+ , Tb 3+ and Dy 3+ and Eu 3+ , Sm 3+ and Tb 3+ may be selected from one or more of the group consisting of (e.g., Eu 3+ 2. The phosphor material of claim 1 .

3. The phosphor material according to claim 1 or 2, wherein the bacterial spores are formed from one or more of Bacillus megaterium, Bacillus cereus, Bacillus subtilis, Clostridium acetobutylicum, Clostridium butyricum, Clostridium botulinum, Clostridium difficile, Clostridium novyi-NT, and Clostridium septicum CS11, and may be formed from one or more of Bacillus megaterium, Clostridium novyi-NT, and Clostridium septicum (e.g., Clostridium novyi-NT, Clostridium septicum, or both).

4. 10. The phosphor material of any one of the preceding claims, further comprising an antenna molecule complexed with the lanthanide-based dopant.

5. 5. The phosphor material of claim 4, wherein the antenna molecule is selected from one or more of the group consisting of β-diketones and triphenylphosphine oxides.

6. 5. The phosphor material of claim 4, wherein the antenna molecule is an aromatic β-diketone, and the aromatic β-diketone may be selected from the group consisting of thenoyltrifluoroacetone (TTA), 1-(-2-naphthoyl)-3,3,3-trifluoroacetone (NTFA), and 4,4,4-trifluoro-1-phenyl-1,3-butanedione (BTFA).

7. The antenna molecule is complexed with the lanthanide dopant, the complex having the formula Ln[ANT] n 7. The phosphor material of claim 4, having the formula: wherein Ln is a member of the lanthanide series of elements, ANT is an antenna molecule, and n is an integer, which may be from 3 to 300, e.g., 30.

8. Ln[ANT] n However, Eu[TTA] 3 , Sm[TTA] 3 , Dy[TTA] 3 , Tb[TTA] 3 , Eu[BTFA] 3 , and Eu[NTFA] 3 and Eu[TTA] 3 , Sm[TTA] 3 , and Tb[TTA] 3 (e.g., Eu[TTA] 3 and Tb[TTA] 3 For example, Eu[TTA] 3 8. The phosphor material according to claim 7, wherein

9. 9. The phosphor material according to claim 4, wherein the molar ratio of the lanthanide dopant to the antenna molecule is from 1:300 to 1:3, for example, from 1:200 to 1:10, for example, from 1:100 to 1:20, for example, about 1:

30.

10. The method further comprises the step of adding a co-dopant material, the co-dopant material being Sr 2+ and Ca 2+ 10. The phosphor material of any one of the preceding claims, wherein the phosphor material may be selected from one or more of the group consisting of:

11. 10. The phosphor material according to any one of the preceding claims, wherein the content of the lanthanide dopant is 20 to 200 nmol per mg of dry spore biomass.

12. A composite material comprising: A phosphor material according to any one of claims 1 to 11, Bulk materials and Including, the bulk material is selected from one or more of the group consisting of a resin, a coating, an adhesive, and an encapsulating material; the bulk material is optically transparent or optically translucent to the excitation and emission wavelengths of the phosphor material; Composite material.

13. The bulk material includes an adhesive, the adhesive being optically transparent to the excitation wavelength and the emission wavelength of the phosphor material; TM 88. The composite material of claim 12.

14. 1. An improved light emitting diode, comprising: a light emitting diode having a light emitting diode bulb portion; A cured composite material comprising the phosphor material of any one of claims 1 to 11; an optically transparent adhesive that covers a surface of the light emitting diode bulb portion so that light from the light emitting diode passes through the cured composite material; Improved light emitting diodes, including:

15. 15. The improved light emitting diode of claim 14, wherein said light emitting diode is an ultraviolet light emitting diode.

16. A method for forming the phosphor material according to any one of claims 1 to 11, comprising: (a) providing a plurality of bacterial spores of the genera Bacillus and Clostridium; and (b) adding a lanthanide material to the plurality of bacterial spores to obtain a phosphor material. Including, the lanthanide material is a lanthanide salt or a lanthanide complexed with an antenna molecule; method.

17. 17. The method of claim 16, wherein the lanthanide material is added to the bacterial spores in a solvent.

18. 18. The method of claim 17, wherein the solvent comprises water and ethanol, which may be 1-50% vol / vol, for example about 3% vol / vol.

19. 19. The method of claim 17 or 18, wherein the concentration of the lanthanide material in the solvent is from 0.005 mM to 10 mM, such as from 0.01 mM to 3 mM, such as from 0.015 mM to 1.5 mM, such as from 0.15 mM to 0.75 mM.

20. 20. The method of any one of claims 17 to 19, wherein the content of the antenna molecule, if present, is between 0.1 mM and 10 mM, for example about 4.5 mM.

21. The method of any one of claims 17 to 20, further comprising the step of substantially removing the solvent from the phosphor material.

Images