Seed compositions for improved germination

Abstract

Composition are provided that include a plurality of viable seeds; and a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN), wherein a. ratio between the number of seeds and the number of prokaryotic cells is between about 1 : 1x102 and about 1 : 1x1010, and their use for improving seed germination.

Description

[0001] Seed Compositions for Improved Germination

[0002] Federal Funding Statement

[0003] This invention was made with Government support under Grant No. W911NF-22-1-0106 awarded by the Army Research Office, and Grant Numbers 1DP2GM154015, P42-ES027707, and P30-ES002109 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0004] Background

[0005] Chemical fertilizers have been essential to feed the current global population, supplementing overused farmland to support consistent food production. But these chemicals are unsustainable; they damage the environment and contribute to greenhouse gas emissions. Further, agricultural runoff containing chemical fertilizers can enter surface and groundwater and damage native ecosystems. In addition to the environmental concerns, chemical fertilizer is costly, representing between 20% and 36% of a farm’s operating budget. In the past two years, chemical fertilizer prices have tripled, climbing from $94 to $232 per acre of com. This cost impacts consumer food prices and can lead to decreased yields due to sub-optimal fertilizer application. These environmental and economic impacts necessitate alternative agricultural solutions; land for agriculture is a finite resource, and its long-term sustainable use is essential for food stability.

[0006] Summary

[0007] In one aspect, the disclosure provides compositions comprising:

[0008] (a) a plurality of viable seeds; and

[0009] (b) a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN);

[0010] wherein a ratio between the number of seeds and the number of prokaryotic cells is between about 1:1x102and about 1:1x1010.

[0011] In various embodiments, tire ratio between the number of seeds and the number of prokaryotic cells is between about 1:1x103and about 1:1x1010, or between about 1:1x104and about 1:1x1010, or between about 1:1x103and about 1:1x1010, or between about 1:1x103and about 1:1x109, or between about 1:1x104and about 1:1x109, or between about 1:1x105and about 1:1x109, or between about 1:1x103and about 1:1x108, or between about 1:1x104and about 1:1x108, or between about 1:1x105and about 1:1x10s.

[0012] In one embodiment, the coating comprises a complete coating over the entire cell surface. In another embodiment, the coating is between 10 nm and 500 nm in thickness. In a further embodiment, the coating comprises 2-4 MPN layers, or comprises 2 MPN layers.

[0013] In various embodiments, the metal ion component in tire MPN comprises one or more cations of aluminum (Al), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), zirconium (Zr), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), cadmium (Cd), cerium (Ce), europium (Eu), gadolinium (Gd), terbium (Tb), or combinations thereof, including but. not limited to one or more of he:. Cr24\ CT'. Cr6 i, Cu+, Cu24; Cu3', Cu44; Mn2’, Mn5. Mn4 i, Mn6+, Mn7+, Mo2+, Mo34; Mo'. Mo64, Co2+, Ni2+, Cd2+, Al3’, V3’, Rii3. Ru3+, Zi4+, Eu3+, Gd3+, Tb3’. Zn2+, or a combination thereof. In one embodiment, the metal ion component in the MPN comprises Fe3+or Mn2’.

[0014] In another embodiment, the polyphenol component, of the MPN comprises tannic acid (TA), gallic acid (GA), epigallocatechin gallate (EGCG), or combinations thereof. In one embodiment, the metal ion component in the MPN comprises Mn2+or Mn3+, and the polyphenol component of the MPN comprises EGCG.

[0015] In various embodiments, the prokaryotic cells are selected from Pseudomonas including Pseudomonas fluorescens, P. protegens P. brassicacearum, P aeruginosa, P. putida, Pseudomonas stutzeri, P. cepacian, P. viscularis, P. paudmobilis, Rhizobium, Azotobacter, Bacillus, Enterobacter, Streptomyces, Frankia, Microbacterium, Flavobacteriu, Glomus, Trichoderma, Penicillum, Aspergillus, Beuvaria, Metarhizium, Fusarium. Rhizopus, Actinomyces, Methanobacterium, Azosporilium, Bradyrhizobium, Burkholderia, Glucoacetobacter, Herbaspirllum, Methylobacterium, Clostridium. Nitrobacter, Xanthobacter, Sinorhizobium, Klebsiella pneumonia, Pantoea agglomerans, or a combination thereof. In one embodiment, the prokaryotic cells comprise P. chlororaphis. In another embodiment, the prokaryotic cells are lyophilized.

[0016] In another aspect, the disclosure provides methods of preparing a seed composition, comprising:

[0017] (a) providing a plurality of viable seeds: and

[0018] (b) contacting the plurality of viable seeds with a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN) and having a ratio of a number of seeds to a number of prokaryotic cells of between 1: 1x102and 1: 1x105° thereby preparing a seed composition. In one embodiment, the seed composition comprises the composition of any embodiment or combination of embodiments of the first aspect of the disclosure.

[0019] In another aspect, the disclosure provides methods of germinating a plurality’ of seeds, comprising:

[0020] (a) providing the composition of any embodiment or combination of embodiments of the first aspect of the disclosure: and

[0021] (b) exposing the composition to germination conditions, thereby germinating a portion of the plurality of seeds.

[0022] Description of the Figures

[0023] Figure 1. Metal-phenolic network (MPN) assembly on P. chlororaphis and its fluorescence images, (a) Cells were coated with a mixture of polyphenols (TA, EGCG, and GA) and metal ions (Fe and Mn). (b) Cells were treated with a live-dead staining kit. Shown are fluorescence images of uncoated and FeIII-TA-coated P. chlororaphis in the FITC channel (SYTO-9) and TxRed channel (propidium iodide) and as merged images. Scale bars are 20 pm. Tire viabilities of P. chlororaphis were 98.3% (uncoated), 98.4% (FeIII-TA-coated), and 79.6% (FeIII-TA-coated but without MOPS addition)

[0024] Figure 2. MPN protection of lyophilized P. chlororaphis under extreme temperatures, (a) Overview of workflow for microbial protection with MPNs, followed by exposure to stressors, (b) Photo of 96-well plate containing P. chlororaphis coated in MPNs composed of diverse metal ions and polyphenols, (c) OD600of lyophilized P. chlororaphis following 48 h of growth in the presence and absence of tannic-acid-based MPNs, The lyophilized cells were stored at various temperature (20, 25, 30, 35, and 40°C) and 43% relative humidity (RH) tor ten days before inoculations, (d) CFU counts from serial dilution plating of MPN-coated lyophilized P. chlororaphis after storage under 50°C and 48% RH over five days (The coating thickness was controlled by repeating the coating step multiple times). Error bars represent standard deviations of n = 3 replicates, (e) CFU counts from serial dilution plating of lyophilized P. chlororaphis under extreme conditions (top 37°C, bottom 50°C, relative humidity varies) using a mixture of polyphenols (TA, EGCG, and G A ) and metal ions (Fe and Mn). (h = hours, d = days, II = 43% RH under 37°C or 48% RH under 50°C, and D = dry' condition.) Error bars represent standard deviations of n = 50 replicates.

[0025] Figure 3. Impact of MPN -coated P. chlororaphis on seed germination, (a) Images of seeds treated either with MPN-coated, lyophilized P. chlororaphis or with water only, (b) Percentages of seed germination after applying water-only, fresh live, lyophilized, Mn2+-EGCG-coated lyophilized P. chlororaphis in agar plates for five days. Error bars represent standard deviations of n = 3 replicates. Percentages of (c) bok choy seed and (d) soy bean germination after applying water-only, fresh live cells only, fresh live cells with Mn2+, fresh live cells with EGCG, lyophilized cells only, lyophilized cells with Mn2+, lyophilized cells with EGCG and Mn2+-EGCG-coated lyophilized P. chlororaphis in agar plate for five or ten days. The lyophilized cells (both coated and uncoated) were stored at 50°C and 48% RH for five days before incubation with seeds.

[0026] Figure 4. Growth curves monitored at OD600 of TA-Felll -coated and uncoated P. chlororaphis following lyophilization with or without cryoprotectants. Left: PB with 0.1M trehalose; center: PC with 0.1M trehalose, and right: PC only (PB: phosphate-buffered saline; PC: phosphate-citrate buffer; cry oprotectant: 0.1 M trehalose). (Shaded region represents error bars represent SD forn=3 replicates).

[0027] Figure 5. CFU counts from serial dilution plating of P. chlororaphis following lyophilization and storage for variable lengths of time either with or without humidity. Dark bars are EGCG-Mn2+-coated P. chlororaphis, and light bars are uncoated cells. Tinies include 48 hours, 5 days, and 10 days. “D” indicates dry storage, and “H” indicates humid storage (43% RH at 37 °C and 48% RH at 50 °C). The two temperatures evaluated are 37 °C and 50 °C. Error bars represent SD for n = 3 biological replicates.

[0028] Figure 6. Percent germination data from Figure 4 of the main text presented with error bars. Error bars represent SD for n=8 biological replicates. Under each condition, the bars are from left to right: bok choy 5 days, sprouts 5 days, bok choy 10 days, and sprouts 10 days.

[0029] Figure 7. Percent germination data extended from Figure 4 of the main text to include alginate beads coated in MPNs for evaluation. Data are presented with error bars. Error bars represent SD for n=8 biological replicates.

[0030] Detailed Description

[0031] As used herein and unless otherwise indicated, the terms “a” and "an" are taken to mean ‘"one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0032] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of ‘‘including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the words “herein,” “above” and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0033] All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

[0034] As used herein, “about” means w'- 5% of the recited value.

[0035] In a first aspect, the disclosure provides compositions comprising

[0036] (a) a plurality of viable seeds; and

[0037] (b) a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN),

[0038] wherein the ratio between the number of seeds and the number of prokaryotic cells is between about 1:1x102and about 1:1x1010

[0039] As shown in the examples, the compositions of the present disclosure provide, for example, significantly improved seed germination relative to other conditions (including fresh seeds and lyophilization). The compositions can be used, for example, in regenerative agriculture applications.

[0040] In various embodiments, the ratio between the number of seeds and the number of prokaryotic cells is between about wherein the ratio between the number of seeds and the number of prokaryotic cells is between about 1:1x103and about 1:1x1010, or between about 1:1x104and about 1:1x1010, or between about 1:1x105and about 1:1x1010, or between about 1:1x103and about 1:1x109, or between about 1:1x104and about 1:1x109, or between about 1:1x105and about 1:1x109, or between about 1:1x103and about 1:1x108, or between about 1:1x104and about 1:1x108, or between about 1:1x105and about 1:1x108.

[0041] As used herein, metal-phenolic networks (MPNs) are non-covalent coordination complexes of metal ions and polyphenols. MPNs adsorb to surfaces through noncovalent interactions. Each cell is coated (sometimes referred to as “encapsulated”) with an assembled metal-phenolic network.

[0042] As used herein, “phenol” is an aromatic organic compound with the molecular formula C6H5OH. Phenol as used herein also includes benzenediol (molecular formula C6H4(OH)2) and benzenetriol (molecular formula C6H3(OH)3) groups.

[0043] As used herein, polyphenols are organic compounds characterized by one or more phenol units, provided at least 2, 3, or more hydroxyl groups are present. In some embodiments, the polyphenol comprises two or more phenol groups, which can independently be phenol, benzenediol, or benzenetriol groups. For example, in certain embodiments, polyphenol independently includes two or more benzenediol and / or benzenetri ol groups.

[0044] In one embodiment, the coating comprises a complete coating over the entire cell surface (also referred to as contiguous herein). In other embodiments, the coating is between about 10 nm and about 500 nm in thickness. In various other embodiments, the coating is between about 10 nm and about 450 nm, about 10 nm and about 400 nm, about 10 nm and about 350 nm, about 10 nm and about 300 nm, about 10 nm and about 250 nm, about 10 nm and about 200 nm, about 10 nm and about 150 nm, about 10 nm and about 100 nm, about 10 nm and about 100 nm, about 10 nm and about 75 nm, about 10 nm and about 50 nm, about 10 nm and about 25 nm, about 20 nm and about 500 nm, about 20 nm and about 450 nm, about 20 nm and about 400 nm, about 20 nm and about 350 nm, about 20 nm and about 300 nm, about 20 nm and about 250 nm, about 20 nm and about 200 nm, about 20 nm and about 150 nm, about 20 nm and about 100 nm, about 20 nm and about 75 nm, about 20 nm and about 50 nm, or about 20 nm and about 40 nm in thickness.

[0045] In one embodiment, the coating may comprise a single MPN layer. In other embodiments, the coating comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more MPN layers. In one embodiment, the coating comprises 2-4 MPN layers. In a further embodiment, the coating comprises 2 MPN layers.

[0046] A given MPN coating layer may comprise a single metal ion component and a single phenolic component, or may comprise combinations of metal ion and / or phenolic components. In embodiments where 2 or more MPN layers are present, each layer may be identical, or may include different metal ion and / or phenolic components than other layers in the MPN.

[0047] In various embodiments, the metal ion component in the MPN comprises one or more cations of aluminum (Al), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), zirconium (Zr), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), cadmium (Cd), cerium (Ce), europium ( Eu ), gadolinium (Gd), terbium (Tb), or combinations thereof, including but not limited to one or more of Fe3i, Cr2+, Cr3+, ( V’. Cu. Cu2+, Cu3+, Cu '. Mn2\ n5. Mn4+, Mn6+, Mti' t Mo2+, Mo3+, Mo'. Mo6+, Co 'A Ni2+, Cd2+, Al3+, V3+, Rh3+, Ru3+, Zr4*, Eu3+, Gd3+, Tb3+. Zn2+, or a combination thereof. In specific embodiments, the metal ion component in the MPN comprises Fe3+or Mn2+.

[0048] Any polyphenol may be incorporated into the MPN coatings as deemed appropriate for an intended use. In some embodiments, the MPNs comprises one or more of flavonoids (including isoflavonoids and neoflavonoids), tannins, condensed tannins, phenolic acids, catechols, lignans, and stilbenes. In some embodiments, the MPNs comprises one or more of flavonoids, tannins, and phenolic acids. In one embodiment, the polyphenol component of the MPN comprises tannic acid (TA), gallic acid (GA), epigallocatechin gallate (EGCG), or combinations thereof.

[0049] In a specific embodiment, the metal ion component in the MPN comprises Mn2+or Mn3+, and the polyphenol component of the MPN comprises EGCG.

[0050] In various embodiments, the prokaryotic cells are selected from Pseudomonas including P. fluorescens, P. chlororaphis, P. protegens P. brassicacearum, P aeruginosa, P. putida, P. stutzeri, P. cepacian, P. viscularis, P. paucimobilis, Rhizobium, Azotobacter, Bacillus, Enterobacter, Streptomyces, Frankia, Microbacterium, Flavobacteriu, Glomus, Trichoderma, Penicillum, Aspergillus, Beuvaria, Metarhizium, Fusarium, Rhizopus, Actinomyces, Methanobacterium, Azosporilium, Bradyrhizobium, Burkholderia, Glucoacetobacter, Herbaspirllum, Methylobacterium, Clostridium, Nitrobacter, Xanthobacter, Sinorhizobium, Klebsiella pneumonia, Pantoea agglomerans, or a combination thereof. The prokaryotic cells in the composition may comprise a plurality of a single coated prokaryotic cell type, or may comprise a plurality of a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different coated prokaryotic cell types. In one specific embodiment, the prokaryotic cells comprise P. chlororaphis.

[0051] In one embodiment, the prokaryotic cells are lyophilized. As described herein, tire prokaryotic cells of the disclosure can be lyophilized, even in the absence of canonical cryoprotectants, while retaining viability. Tire coatings are shown to protect the cells from multiple manufacturing and storage stresses, including heat and humidity, without requiring additives (e.g. cryoprotectants or ROS scavengers).

[0052] The prokaryotic cells may be present in the composition in any amount appropriate for an intended use. In one non-limiting embodiment, the prokaryotic cells are present in the composition at between about 104and 1011colony forming units (cfu).

[0053] The viable seeds may be of any type as deemed suitable for an intended purpose. The examples include data using exemplary' seed including com, radish, dill, bok choy, and sprout viable seeds. Those of skill in the art will understand that the compositions may comprise any other viable seed type.

[0054] The composition may comprise any other suitable components as appropriate for an intended agricultural use, including but not limited to seed coatings, soil, fertilizer, soil additive, a soil stabilizer, soil adj uvant, and plant biostimulant. In a second aspect, the disclosure provides methods of preparing a seed composition, comprising:

[0055] (a) providing a plurality of viable seeds; and

[0056] (b) contacting the plurality of viable seeds with a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN) and having a ratio of a number of seeds to a number of prokaryotic cells of between 1:1x102and 1:1x1010thereby preparing a seed composition.

[0057] The methods of this aspect can utilize any embodiment of combination of embodiments of the compositions and MPN-coated prokaryotic cells as described in the first aspect of the disclosure.

[0058] In another aspect, the disclosure provides methods germinating a plurality of seeds, comprising:

[0059] (a) providing the composition of any embodiment or combination of embodiments of the first aspect of the disclosure; and

[0060] (b) exposing the composition to germination conditions, thereby germinating a portion of the plurality of seeds.

[0061] As disclosed in the examples that follow, tire inventors have shown that the compositions of the invention lead to significantly increased seed germination than if the germinating occurred in the absence of the plurality of MPN-coated prokaryotic cells. In some embodiments, seed germination is increased at least 10%, 20%, 30%, 40%, 50%, or more than if the germinating occurred in the absence of the plurality of MPN-coated prokaryotic cells. The methods of this aspect can utilize any embodiment of combination of embodiments of the compositions and MPN-coated prokaryotic cells as described in the first aspect of the disclosure.

[0062] Those of skill in the art are familiar with appropriate germination conditions, which may include, but are not limited to, adequate water, oxygen, light, temperature, and appropriate soil conditions.

[0063] Examples

[0064] Abstract

[0065] Chemical fertilizers have been crucial for sustaining the current global population by supplementing overused farmland to support consistent food production, but their use is unsustainable. Pseudomonas chlororaphis is a nitrogen-fixing bacterium that could be used as a fertilizer replacement, but this microbe is delicate. It is sensitive to stressors, such as freeze-drying and high temperatures. Here, we demonstrate protection of P. chlororaphis from freeze-drying, high temperatures (50°C), and high humidity using selfassembling metal -phenolic network (MPN) coatings. The composition of the MPN is found to significantly impact its protective efficacy, and with optimized compositions, no viability loss is observed for MPN-coated microbes under conditions where uncoated cells do not survive. Further, we demonstrate that MPN-coated microbes improve germination of seeds by 150% as compared to those treated with fresh P. chlororaphis.

[0066] Introduction

[0067] Microbes offer sustainable alternatives to chemical fertilizers, especially due to their native role in plant growth. Microbes can efficiently process essential nutrients (i.e., nitrogen, phosphorus, and sulfur) to render them bioavailable for plants, they can manipulate plant hormone signaling, and they can stop invasive pathogens. Because of their importance, researchers have worked to deliver microbes to soil, but critical species are difficult to produce and transport because of their sensitivity to temperature and humidity. Accordingly, a platform to stabilize delicate microbes against stressors during processing, storage, and transport would enable widespread agricultural adoption. Here, we establish the ability of metal-phenolic network (MPN) coatings to protect the agriculturally-important nitrogen-fixing bacteria Pseudomonas chlororaphis. MPNs are composed of polyphenols and metal ions that assemble into two-dimensional networks. Importantly for agricultural applications, the components of these materials are generally recognized as safe (GRAS) by the U. S. Food and Drug Administration (FDA).

[0068] P. chlororaphis natively interacts with plant roots and fixes nitrogen at them. It can also protect plants against phytopathogenic fungi and pests. However, P. chlororaphis is highly unstable at elevated temperatures, and prior efforts to protect this species have failed. We demonstrate the protection ofP. chlororaphis with MPNs. We demonstrate that the MPN composition plays a critical role in protective efficacy, based on both the valency of the metal as well as the size and number of chelation sites on the polyphenol. Upon determining the most suitable MPN composition for P. chlororaphis protection, such compositions were shown to enable these microbes to survive harsh environmental exposure including lyophilization, storage at temperatures as high as 50 °C and humidity of 48% (relative humidity)- We further demonstrate that our formulated P. chlororaphis is more beneficial to seed germination than even fresh microbes. Together, these results establish that MPN composition directly impacts protective behavior. Results and Discussion

[0069] We have shown that MPNs readily form on diverse prokaryotic microbes, including Bacillus subtilis and Bacteroides thetaiotaomicron (J. Mater. Chem. B. 2022, 10, 7600- 7606; J Am. Chem. Soc. 2022, 144 (6), 2438- 2443; ACS Appl. Bio. Mater. 2022, 5, 4687- 4695). P. chlororaphis is a nitrogen-fixing species that is natively found in soil ecosystems and has been used as a bioinoculant for agriculture because of its beneficial properties, making it an important target for protection. Additionally, P. chlororaphis is especially useful for studying the MPN protective efficacy because of its well-known temperature instability’. In previous work, we have shown that MPNs can significantly improve protection of living biotherapeutics and probiotics to enable their delivery?. (J. Mater. Chem. B. 2022, 10, 7600- 7606; J Am. Chem. Soc. 2022, 144 (6), 2438- 2443; ACS Appl. Bio. Mater. 2022, 5, 4687- 4695). Here, we find that the components of the MPN significantly impact their protective efficacy against freeze-drying, high temperatures, and high humidity.

[0070] MPN Formation on P. chlororaphis and Lyophilization Protection

[0071] MPNs are higher-dimensional coordination complexes of metal ions and polyphenols that form through self-assembly. Though a diverse array of polyphenols and metal ions can be used for MPN formation, we have focused on three well-studied polyphenols that are GRAS approved by the FDA (Figure l a). These three polyphenols, tannic acid (TA), epigallocatechin gallate (EGCG), and gallic acid (GA), span the range of molecular diameters and number of chelation sites, with TA being both the largest with the most catechols and GA being the smallest with the fewest catechol groups. In the presence of a di- or trivalent cation, the polyphenols chelate and generate an extended coordination network. We have generally observed more structurally rigid assemblies with trivalent cations, and we want to focus on metals that are beneficial and have low risk of cytotoxicity. We therefore focused on Fe3+, Mn3+, Al3+and Zn2+as metal components of the MPN. Upon transition of the MPN formation solution to a pH above 8.0, the metal ions chelate the phenolic groups of the polyphenols, forming well-de fined coordination centers.

[0072] Through UV-vis spectroscopy, we readily established the formation of MPNs on P. chlororaphis through the appearance of a well-defined ligand-to-metal charge transfer (LMCT) band upon full deprotonation and coordination of the network. The appearance of this LMCT band corresponds to the formation of contiguous core-shell coatings on microbial substrates (Figure 2b). In addition to the UV-vis verification of MPN formation, we imaged ceils using a fluorophore-BSA complex that specifically interacts with MPNs, and not uncoated cells.. We confirmed through these images that the MPNs form a contiguous coating on the microbes that is not present on the uncoated cells. The consistent fluorescence across the surface of the coated cells confirms that uniform core-shell structures are formed on these microbes. Moreover, the viability of MPN -coated P. chlororaphis (98.4%) is comparable to that of the uncoated controls (98.3%) immediately following coating. This was assessed by live and dead staining with fluorescence microscopy (Figure lb). We also found that the addition of a MOPS buffer helped maintain the viability of MPN-coated P. chlororaphis. In contrast, loosely formed MPNs and low pH were found to negatively affect cell viability. Both of these results are critical, as the components of the MPNs on their own are associated with toxicity, which we observe when they are not set by using a high pH buffer. Thus, we demonstrate that setting tire MPN coating with a basic MOPS buffer is necessary’ to observe the MPN protective effects.

[0073] Following confirmation of MPN formation on the bacteria and validation of viability maintenance, we evaluated MPN protection against key stressors that P. chlororaphis would encounter during formulation and production. After growth, the first step in processing microbes is to enable their effective transport and delivery' via dry ing. However, such processes cause significant stress and damage to ceils, leading to decreases in viability of between three and six orders of magnitude. Thus, we evaluated the impact of the MPNs on protecting P. chlororaphis from the freeze-drying (lyophilization) process. Ly ophilization generally requires freezing a sample in liquid nitrogen, followed by sublimation of the ice under vacuum. Both steps cause significant stress to microbes. We observed that MPNs significantly improve survival following lyophilization, even without additional cryoprotectants.

[0074] For evaluation of stress tolerance, we coated P. chlororaphis in either two or four layers of MPNs composed of the aforementioned components. Two and four layers of MPNs were selected because we find these to be the best balance between protection and unhindered microbial growth. Two layers are the minimum required to observe protection with many strains, and four layers provide additional protection in many cases but do not significantly hinder microbial division. Additional coatings beyond 4 layers significantly inhibit microbial division and often do not offer additional protective benefits.

[0075] Following coating and dry ing the microbes, we evaluated their viability using both optical density for growth analysis at 600 nm (OD«oo) and serial dilution plating for colony- forming unit (CFU) counting. We additionally evaluated P. chlororaphis growth following coating, lyophilization, and rehydration using a viability stain. The MPN -coated P. chlororaphis reached exponential phase after incubation for 15 h, whereas exponential growth of the uncoated control was delayed to 35 h (Figure 4). This improvement in growth lag indicates that MPN-coated. chlororaphis samples have more viable cells remaining than their uncoated counterparts. Because of the success of MPN protection against these initial stressors forP. chlororaphis, we investigated additional critical stressors.

[0076] Temperature Stability of MPN-Coated P. chlororaphis

[0077] A major challenge with P. chlororaphis, among many other living microbial agriculture products, is their long-term stability at temperatures above refrigeration. Cold storage and transport are the standard for biologies, but these processes are costly and energy-intensive and require centralized facilities for storage. Additionally, over one billion people globally live farther than 20 km from an all-weather road. This limitation prevents access to large transportation vehicles required for most cold transport, which is currently essential for transport of agricultural products. Because of the temperature sensitivity ofP. chlororaphis combined with the need for methods of storage and transport that do not require a cold chain or temperature control, we next evaluated the efficacy of diverse MPN coatings to protect these microbes from high temperatures and humidity.

[0078] At 4 °C, the temperature of refrigerated cold-chain transport, even uncoated bacteria survive. However, the advantage of MPNs lies in their ability to protect microbes at temperatures at which uncoated cells do not survive. We screened different MPN compositions composed of rnetal ions and polyphenols for / ’, chlororaphis stability. As can be seen in Figure 2a, at low temperatures, there is no statistical significance between the survival of microbes coated with the different MPNs. However, at the transition temperature of 35 °C, no uncoated cells survive, while some of the coated microbes do. This difference is statistically significant (p<0.005) and demonstrates the large difference in survival afforded by optimized MPNs. The marked survivability of. chlororaphis at 35 °C with only certain MPNs further confirms that the MPN composition remains an essential factor in protection.

[0079] The lack of a difference in the protective efficiency of MPNs across metal ions evaluated with TA motivated further screening of polyphenols (Figure 2d). In prior w ork, the rigidity and thickness of the MPN coating has been determined for a variety’ of polyphenols and metal ions. Both the number of chelation sites (the number of catechols in the molecule) and the cation valency impact the thickness and morphology of coatings, ranging from somewhat rigid coatings 10 nm thick (usually found with TA and atrivalent cation) to very rigid and thin coatings only about 2 nm thick (usually observed with GA and a trivalent cation such as Fe3+and Mn3+. Thus, we expected the physical properties of the MPNs to differ more significantly upon variation of the polyphenol.

[0080] As expected, much larger differences in protective efficacy were observed upon variation of the polyphenol as compared to the metal (Figure 2d). In fact, these results highlight the critical importance of evaluating each of the three variables tested: metal ion used in the MPN, polyphenol used in the MPN, and the number of coating layers used. As is evidenced from the two-coating data for EGCG and Mn, this coating offers significantly more effective protection than the other coatings, with statistical significance of p < 0.05 for the other coatings. In contrast, this same coating yielded no viable cells if it was applied four times to cells (Figure 2d, right). We attribute this to the rigidity of this coating, which provided protection at two layers but inhibited growth to tire point where no cells grew' with four coatings. While these experiments demonstrated that some MPN combinations provided little to no protection of cells, the ability of some MPNs to protect against the initial exposure challenges demonstrated the need for further investigation.

[0081] Following the initial MPN testing, a screen of post-lyophilization storage conditions was performed. The variables selected for screening are based on the harsh conditions to which these microbes could be exposed during transport and storage to farms where they would be distributed. Critically, many of these farms are not accessible by paved roads and do not have temperature-controlled storage for their seeds and fertilizers, so necessitating cold storage would be a non-starter. Thus, we focused on temperature and humidity preservation to ensure that the microbes survive under conditions under which they could be stored in farmland in South America and South Asia. As we previously identified differences in MPN efficacy based on the polyphenol and metal ions used for assembly on these microbes, we anticipated the observation of similar trends here in the more expansive study. Variables for stress testing of the cells included high temperatures and humidity' for incubation times on the order of production and transport times to potentially challenging agricultural locations. We therefore evaluated incubation times ranging from 48 hours to 10 days. The upper temperature limit for the experiment was set at the estimated temperature a storage shed in Brazil would reach during a warm day m the planting season, where outdoor temperatures reach 43 °C and 70-80% humidity. Because of the differences in recovery observed with two and four coatings in our initial experiments (Figure 2d), for our larger evaluation of stressors, we evaluated only two coatings. As seen from CFU data (Figure 2e), MPNs are capable of protecting P. chlororaphis under the harshest transport and storage conditions, even enabling growth following multiple days at the highest temperature and humidity (50 °C, 48% RH for ten days, Figures 2e and 5). Importantly, the uncoated microbes demonstrated little to no survival under similar conditions (Figure 2e). In fact, it is critical to emphasize that the uncoated microbes did survive some conditions at 37 °C storage, but in every' case, 1-2 orders of magnitude fewer uncoated cells survived as compared to the coated cells. Additionally, the uncoated cells fared significantly worse under humid conditions. At 37 °C storage with humidity, no uncoated cells survived. Similarly, beyond 48 h of exposure to 50 °C, no uncoated cells survived, while many combinations of MPNs effectively protected cells. Finally, we observed significantly improved survival of coated microbes at elevated temperatures (30 °C) for extended lengths of time (two months) due to MPN coatings (). These resul ts emphasize the importance of MPNs for microbial protection.

[0082] Interestingly, for the majority of the stressor studies, no specific MPN significantly outperforms the rest. Based on the optimal coatings, which are GA and Fe, GA and Mn, and EGCG and Mn, we anticipate that protection against heat and humidity-’ is dependent on the rigidity of the coatings, as the thinner, more rigid coatings formed from GA and EGCG were favored for protection in these cases.

[0083] Impact of Microbes on Seed Germination

[0084] Though the protection of microbes is important, we additionally sought to investigate whether the impressive recovery' results would translate to seed germination rates, the ultimate goal ofP. chlororaphis use. We applied fresh live, lyophilized and MPN-coated, or just lyophilized P. chlororaphis to a variety of seeds, including com, bok choy, sprouts, two types of radishes, and dill (Figure 3b). The MPN employed for seed germination studies was the one found to be best in the initial studies (Figure 2d) and continued to be one of the best coatings across the expanded stressor experiments (Figure 2e), which was EGCG and Mn. Initial studies on a variety of seeds were important to establish whether any observed germination effects were seed -dependent. As can be seen, MPN-coated cells, despite their harsh storage conditions at 50°C and 48% RH, yielded higher rates of germination than any other condition, including seeds treated with the fresh cells. Within each type of seed, statistically significant differences were observed between the conditions P < 0.05). These initial studies provided support for our hypothesis that seeds growing in nutrient-depleted environments would benefit from the presence of nitrogen-fixing bacteria, such as P. chlororaphis. However, these initial studies did not enable clarification of why the MPN-coated cells -were superior even than fresh cells.

[0085] This lack of clarity as to whether improvements were due to the cells themselves, the components of the MPNs, or a combination of the two led to further investigation of the germination rate that incorporated additional controls. To further investigate the observed improvement, we studied germination with bok choy and sprout seeds because they are relatively rapid germinators (Figure 3c, d). For bok choy, after five days, the fresh cells improved germination over the water-only cells by 16.7%, while the lyophilized bacteria exhibited slightly smaller improvements (13.7%). Importantly, MPN-coated cells improved germination by 58.3%, significantly more than under any other conditions, including fresh cells with the individual MPN components added (Figures 3c, 6).

[0086] After ten days, overall germination increased, but the relative percentages of germinated cells remained consistent. Similar results were observed with sprout seeds. After five days, the fresh cells had 8.3% improved germination over water-only, while applying lyophilized cells had slightly lower germination than the water alone, although this difference is not statistically significant (2.1% less) (Figure 3d, 6). In contrast, MPN-coated cells had 47.9% higher germination than that of water only. Additionally, combining freeze-dried uncoated P. chlororaphis with alginate beads (500 nm diameter) coated in the MPN did not show' statistically significant differences relative to the seeds treated with only freeze-dried cells (Figure 7). These improvements confirm that it is the combination of the MPN coatings with the microbes that leads to improvements, even when the microbes are stored under exceptionally harsh conditions. The results further demonstrate the immense power of MPNs to maintain microbial viability and directly translate that to improved seed germination.

[0087] Conclusions

[0088] Taken together, these results demonstrate the power of some MPN assemblies to protect temperature-sensitive microbes from high temperatures and humidity. Critically, the microbes evaluated are capable of fixing nitrogen in soil and could be used in regenerative agriculture applications. MPNs protected P. chlororaphis from lyophilization. Importantly, though, upon screening combinations of polyphenols and metal ions, we found MPN combinations that protect microbes from exceptional heat and humidity. Importantly, these are not the types of MPNs that we have previously identified as most effective, as they are assembled with smaller polyphenols that form thinner, more compact shells on microbes that seem to be more rigid than some of the larger polyphenols that we have tested. Our ability to protect P. chlororaphis for 10 days at extremely high temperatures (50 °C) and humidity as well as slightly lower temperatures (30 °C) for months pushes the protective ability of our coatings into the realm of utility for regenerative agriculture by negating the need for either microbial growth in bioreactors on-site or employing cold chain transport to enable the microbes to reach their destination. Further, we demonstrated that the microbes protected by MPN coatings yielded significantly higher germination rates for multiple types of seeds and, more specifically, bok choy and sprouts, as compared to seeds treated with fresh microbes. These results are promising for the application of these microbes as replacements for chemical fertilizers at scale. Long-term, we anticipate these coatings will enable the storage and transport of critical microbial species without cold chain transport and enable microbes to be viable, sustainable alternatives to chemically-produced fertilizer.

[0089] Methods

[0090] Encapsulation of Bacteria with MPNs

[0091] P. chlororaphis were coated in MPNs. Briefly, 125 pL of aqueous solution of phenol (TA-1,6 mg ml / 1; GA-1.5 mg ml / 1; EGCG-2.7 mg ml / 1, 50% from tea extract) and 125 JL of aqueous solution of the cation to be used (iron chloride (0.24 mg mL ), zinc sulfate heptahydrate (0.42 mg mL’1), aluminum chloride (0.19 mg mL’1), or manganese sulfate monohydrate (0.72 mg ml’1)) were added sequentially to an aqueous suspension of cells (250 pL, ODeoo of 3.0). The resulting suspension was mixed vigorously for 10 s. A volume of 0.5 mL of MOPS buffer (20 mM, pH 7.4) was then added to form a stable MPN shell. The resulting encapsulated cells were washed with ultrapure water three times to remove any residual starting materials. The coating process can be repeated as many times as desired; here, we either used two total coatings or four total coatings.

[0092] Germination Studies

[0093] Seed germination studies were performed in 0.5% agar plates made with ultrapure water. Seeds were inserted in the agar to halfway down the seed, and 10 pL of the relevant microbial sample was added to each seed. Samples were either ultrapure water or microbes and MPN components in uitrapure water. For samples containing cells, I O7bacteria per seed were added. To add seeds, P. chlororaphis was diluted to concentrations of 1 x

[0094] 109bacteria / mL in uitrapure water. Seeds were allowed to incubate for five days with the Petri dish cover on at ambient temperature under grow lights. Germinated seeds were then counted. For ten-day studies, the lids of the dishes were removed for the second half of the incubation time (the final five days). Following ten days of incubation, the germinated seeds were counted.

[0095] Supporting Information

[0096] Methods and Materials

[0097] Reagents. All chemicals were reagent grade and used without further purification unless noted. Tannic acid (99%), gallic acid monohydrate (98%), and epigallocatechin gallate (95%) were purchased from TCI Chemicals. Iron (III) chloride (FeC13, 97%), aluminum chloride (99.9%), zinc sulfate heptahydrate (99%), manganese sulfate monohydrate (99%), 3-(N-morpholino) propanesulfonic acid (MOPS, 99.5%), sodium hydroxide (98%), hydrochloride acid (37%), acetone (99.5%), D-(+)-trehalose dihydrate (99%), sodium phosphate dibasic (99%), sodium phosphate monobasic (99%), and citric acid (99.5%) were purchased from Millipore Sigma, Ethanol (100%, molecular biology grade) and sodium chloride (NaCl) were purchased from Fisher BioReagents, and agar (Bacteriological grade) and nutrient broth (NB, nncrobiologically tested) powder were purchased from Apex Bioresearch products. Type I uitrapure water was generated with an ELGA PURELAB Quest UV. LIVE / DEAD™ BacLight™ bacterial stain was purchased from ThermoFisher. NB media was prepared with 8 g of NB powder per L of uitrapure water with autoclaving (20 min, 121° C). Nutrient agar plates for colony selection and culture inoculation were prepared with 20 mL of nutrient agar solution (8 g NB powder, 15 g of agar per L) in 150 mm round petri dishes. Plates for serial dilution CFU preparation were prepared with the nutrient agar solution described above. 50 mL of the solution was added to a 10 cm square petri dish. Germination agar plates were prepared with 20 mL of uitrapure water agar solution (0.5% agar; 5 g of agar per L).

[0098] Instruments and Measurements. Cuvette optical density and absorbance measurements were made with a NanoDrop™ One Micro volume UV-Vis Spectrophotometer (Thermo Scientific, USA). 96 well plate measurements were made on a Biotek synergy mx microplate reader (BioTek Instruments, USA). Bacteria were imaged with a Revolve Fluorescence Microscope (Echo, USA). Bacterial Culture. Pseudomonas chlororaphis (Guignard and Sauvageau) (ATCC 9446) was used in this study. Bacteria were prepared for coating with MPNs as follows: P. chlororaphis freezer stocks (25% glycerol) were stored at -80 °C. Cells from these stocks were freshly streaked onto nutrient agar plates and incubated aerobically for — 18h at 25 °C. Single colonies were selected and used to inoculate 5 mL NB cultures. Liquid cultures were maintained for 36 h at 25 °C with shaking at 200 RI’M. Stationary-phase cultures were washed by transferring cells to centrifuge tubes and spinning at 9000 x g for 3 min.

[0099] Supernatant was exchanged with ultrapure water or buffer as necessary, with three washes performed. Following the final wash, cells were concentrated to a 3x stock (OD600 of 3.0) and were used immediately.

[0100] Bacterial Lyophilization and Storage. Both uncoated and MPN-coated P. chlororaphis were lyophilized according to previous-reported protocols.! The lyophilized bacteria were stored under reported conditions for lengths of time up to 10 d prior to reconstitution for viability assessment. Cells were then stored at variable temperatures and with or without humidity. Temperature was maintained in a sealed lab oven, and humidity was maintained using saturated potassium carbonate solutions. Prior to use, the lyophilized cells were reconstituted in nutrient broth or ultrapure water as necessary.

[0101] Bacterial Growth / V lability Assessment. OD600 growth assays were performed in sterile 96- well plates, with OD600 monitored by plate reader. Colony forming unit (CFU) counts were determined from serial dilution assays. Bacterial viability was also determined by live / dead viability staining. Independent images were captured on a fluorescence microscope for live (green fluorescence: kex= 470 / 40 nm; Xem= 525 / 50 nm) and dead cells (red fluorescence: / .ex:::560 / 40 nm, kern:::630 / 75 nm) with a 60X magnification oil objective. Live and dead cells were quantified by Image) from five randomly -chosen views to obtain a percentage of viable cells. For serial dilution assays, lyophilized cells were diluted in NB to an initial concentration of OD600=1.0. Cells were then diluted by five-fold (40 pL NB with 10 pL of cells) with seven dilutions for a total of eight cell concentrations. To a 10 cm square plate, 5 pL of each sample was then plated. Plates were allowed to grow at 25 °C for 24 h. Cells were quantified through colony counting of the most concentrated sample in which colonies were distinguishable.

Claims

1. We claim:

1. A composition comprising:3.(a) a plurality of viable seeds; and4.(b) a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN);5.wherein a ratio between the number of seeds and the number of prokaryotic cells is between about 1: IxlO2and about 1: IxlO10.

2. The composition of claim 1, wherein the ratio between the number of seeds and the number of prokaryotic cells is between about 1: IxlO3and about 1: 1 x 1010, or between about 1: 1x104and about l:lx!0!0, or between about 1: Ixl O5and about 1: Ixl O10, or between about klxlO3and about 1: IxlO9, or between about 1: IxlO4and about LlxlO9, or between about 1: IxlO5and about 1: IxlO9, or between about 1: IxlO3and about 1: IxlO8, or between about 1:lxl04and about 1:1x10s, or between about l:lxl05and about 1: IxlO8,3. Hie composition of claim 1 or 2, wherein the coating comprises a complete coating over the entire ceil surface.

4. The composition of any one of claims 1-3, wherein the coating is between 10 nm and 500 nm in thickness.

5. The composition of any one of claims 1-4, wherein the coating comprises 2-4 MPN layers, or comprises 2 MPN layers.

6. The composition of any one of claims 1 -5, wherein the metal ion component in the MPN comprises one or more cations of aluminum (Al), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), zirconium (Zr), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), cadmium (Cd), cerium (Ce), europium (Eu), gadolinium (Gd), terbium (Tb), or combinations thereof, including but not limited to one or more of Fe3*, Cr2*, Cr3*, Cr6*, Cu+, Cu2*, Cu3*, Cu4*, Mn2*, Mn3*, Mn4*, Mn6*, Mn7*, Mo'. Mo3*. Mo4*, Mo6*, Co2*, Ni2*. Cd2*, Al3*, V3*, Rh3*, Ru3*, Zr4*, Eu3*, Gd3*, Tb3. Zn2*, or a combination thereof.

7. Hie composition of any one of claims 1-5, wherein the metal ion component in the MPN comprises Fe3+or Mir.

8. The composition of any one of claims 1 -7, wherein the polyphenol component of the MPN comprises tannic acid (TA), gallic acid (GA), epigallocatechin gallate (EGCG), or combinations thereof.

9. The composition of any one of claims 1-8, wherein the metal ion component in the MPN comprises Mrr or Mn3+, and the polyphenol component of the MPN comprises EGCG.

10. The composition of any one of claims 1 -9, wherein the prokaryotic cells are selected from Pseudomonas including Pseudomonas fluorescens, P. protegens P. brassicacearum, P aeruginosa, P. putida, Pseudomonas stutzeri, P. cepacian, P. viscularis, P. paucimobilis, Rhizobium, Azotobacter, Bacillus, Enterobacter, Streptomyces, Frankia, Microbacterium, Flavobacteriu, Glomus, Trichoderma, Peni cilium, Aspergillus, Beuvaria, Metarhizium, Fusarium, Rhizopus, Actinomyces, Methanobacterium, Azosporilium, Bradyrhizobium, Burkholderia, Glucoacetobacter, Herbaspirllum, Methylobacterium, Clostridium, Nitrobacter, Xanthobacter, Sinorhizobium, Klebsiella pneumonia, Pantoea agglomerans, or a combination thereof.

11. The composition of any one of claims 1-10, wherein the prokaryotic cells comprise P. chlororaphis.

12. The composition of any one of claims 1-11, wherein the prokaryotic cells are lyophilized.

13. A method of preparing a seed composition, comprising:17.(a) providing a plurality of viable seeds; and18.(b) contacting the plurality of viable seeds with a plurality of prokaryotic cells having a coating comprising metal-phenolic networks (MPN) and having a ratio of a number of seeds to a number of prokaryotic cells of between 1: 1x102and 1: 1x1010thereby preparing a seed composition.

14. The method of claim 13, wherein the seed composition comprises the composition of any one of claims 1-12.

15. A method of germinating a plurality of seeds, comprising:20.(a) providing the composition of any one of claims 1-12; and21.(b) exposing the composition to germination conditions, thereby germinating a portion of the plurality of seeds.

16. The method of any one of claims 13-15, carried out as detailed herein.

17. The composition of any one of claims 12-17, comprising components as recited in any embodiment of combination of embodiments disclosed herein.

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